Fertility and Sterility Volume 88, Issue 4, Pages 771-1020 (October 2007) 1. Masthead Page A13 2. TOC Pages A27, A28, A34, A42, A44, A46, A48 Modern trends 3. Erectile dysfunction: does insulin resistance play a part? Pages 771-778 J.C. Trussell and Richard S. Legro Editor's corner 4. Limiting access to letrozole—is it justified? Pages 779-780 Togas Tulandi and Alan H. DeCherney 5. Substandard application of preimplantation genetic screening may interfere with its clinical success Pages 781-784 Santiago Munné, Luca Gianaroli, Ilan Tur-Kaspa, Cristina Magli, Mireia Sandalinas, Jamie Grifo, David Cram, Semra Kahraman, Yury Verlinsky and Joe L. Simpson Endometriosis 6. Estrogen receptor β gene +1730 G/A polymorphism in women with endometriosis Pages 785-788 Gyoung Hoon Lee, Sung Hoon Kim, Young Min Choi, Chang Suk Suh, Jung Gu Kim and Shin Yong Moon
Transcript
Fertility and Sterility
Volume 88, Issue 4, Pages 771-1020 (October 2007)
1. Masthead Page A13
2. TOC Pages A27, A28, A34, A42, A44, A46, A48
Modern trends
3. Erectile dysfunction: does insulin resistance play a part? Pages 771-778 J.C. Trussell and Richard S. Legro
Editor's corner
4. Limiting access to letrozole—is it justified? Pages 779-780 Togas Tulandi and Alan H. DeCherney
5. Substandard application of preimplantation genetic screening may interfere with its clinical success Pages 781-784 Santiago Munné, Luca Gianaroli, Ilan Tur-Kaspa, Cristina Magli, Mireia Sandalinas, Jamie Grifo, David Cram, Semra Kahraman, Yury Verlinsky and Joe L. Simpson
Endometriosis
6. Estrogen receptor β gene +1730 G/A polymorphism in women with endometriosis Pages 785-788 Gyoung Hoon Lee, Sung Hoon Kim, Young Min Choi, Chang Suk Suh, Jung Gu Kim and Shin Yong Moon
7. Efficacy of vaginal danazol treatment in women with recurrent deeply infiltrating endometriosis Pages 789-794 Sandro Razzi, Stefano Luisi, Francesco Calonaci, Aldo Altomare, Caterina Bocchi and Felice Petraglia
8. Different types of small nerve fibers in eutopic endometrium and myometrium in women with endometriosis Pages 795-803 Natsuko Tokushige, Robert Markham, Peter Russell and Ian S. Fraser
Genetics
9. Preimplantation genetic screening as an alternative to prenatal testing for Down syndrome: preferences of women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment Pages 804-810 Moniek Twisk, Maaike L. Haadsma, Fulco van der Veen, Sjoerd Repping, Sebastiaan Mastenbroek, Maas-Jan Heineman, Patrick M.M. Bossuyt and Johanna C. Korevaar
10. Oocyte dysmorphism is not associated with aneuploidy in the developing embryo Pages 811-816 Kayhan Yakin, Basak Balaban, Aycan Isiklar and Bulent Urman
In vitro fertilization
11. Social concerns of women undergoing infertility treatment Pages 817-821 Peter S. Finamore, David B. Seifer, Cande V. Ananth and Sandra R. Leiblum
12. Aspirin in women undergoing in vitro fertilization treatment: a systematic review and meta-analysis Pages 822-831 Mohammed Khairy, Kaberi Banerjee, Tarek El-Toukhy, Arri Coomarasamy and Yakoub Khalaf
13. GnRH agonist and antagonist protocols for stage I–II endometriosis and endometrioma in in vitro fertilization/intracytoplasmic sperm injection cycles Pages 832-839 Recai Pabuccu, Gogsen Onalan and Cemil Kaya
Male factor
14. Markov modeling of vasectomy reversal and ART for infertility: how do obstructive interval and female partner age influence cost effectiveness? Pages 840-846 Michael H. Hsieh, Maxwell V. Meng and Paul J. Turek
15. The beneficial effects of toremifene administration on the hypothalamic-pituitary-testicular axis and sperm parameters in men with idiopathic oligozoospermia Pages 847-853 Dimitrios Farmakiotis, Christos Farmakis, David Rousso, Anargyros Kourtis, Ilias Katsikis and Dimitrios Panidis
16. The contribution of genetic variations of aryl hydrocarbon receptor pathway genes to male factor infertility Pages 854-859 Ave Merisalu, Margus Punab, Signe Altmäe, Kadri Haller, Tarmo Tiido, Maire Peters and Andres Salumets
17. Alterations in sperm motility after acute oral administration of sildenafil or tadalafil in young, infertile men Pages 860-865 Giorgio Pomara, Girolamo Morelli, Domenico Canale, Paolo Turchi, Carolina Caglieresi, Cecilia Moschini, Giovanni Liguori, Cesare Selli, Enrico Macchia, Enio Martino and Francesco Francesca
Menopause
18. Tibolone histology of the endometrium and breast endpoints study: design of the trial and endometrial histology at baseline in postmenopausal women Pages 866-878 David F. Archer, Susan Hendrix, Alex Ferenczy, Juan Felix, J. Chris Gallagher, Janice Rymer, Sven O. Skouby, Wil den Hollander, Victoria Stathopoulos and Frans A. Helmond
Ovulation induction
19. A comparison of letrozole to gonadotropins for ovulation induction, in subjects who failed to conceive with clomiphene citrate Pages 879-885 Rudolfo B. Quintero, Renata Urban, Ruth B. Lathi, Lynn M. Westphal and Michael H. Dahan
Polycystic ovary syndrome
20. Role of insulin in the hyperandrogenemia of lean women with polycystic ovary syndrome and normal insulin sensitivity Pages 886-893 Jean-Patrice Baillargeon and André Carpentier
21. Assessment of three-dimensional sonographic features of polycystic ovaries after laparoscopic ovarian electrocautery Pages 894-899 Miklós Vizer, Ludwig Kiesel, István Szabó, Antal Arany, Péter Tamás and András Szilágyi
22. Lack of association between polycystic ovary syndrome and embryonic aneuploidy Pages 900-905 Andrea Weghofer, Santiago Munne, Serena Chen, David Barad and Norbert Gleicher
Reproductive endocrinology
23. Stage I ovarian carcinoma: different clinical pathologic patterns Pages 906-910 Liane Deligdisch, Frédérique Pénault-Llorca, Peter Schlosshauer, Albert Altchek, Michele Peiretti and Farr Nezhat
24. Anxiety and sexual stress in men and women undergoing infertility treatment Pages 911-914 Brennan D. Peterson, Christopher R. Newton and Tal Feingold
25. Bayesian selection of optimal rules for timing intercourse to conceive by using calendar and mucus Pages 915-924 Bruno Scarpa, David B. Dunson and Elena Giacchi
26. Detection of antizona pellucida antibodies in the sera from premature ovarian failure patients by a highly specific test Pages 925-932 Satoru Takamizawa, Hiroaki Shibahara, Tamaho Shibayama and Mitsuaki Suzuki
Reproductive surgery
27. A multicenter randomized, controlled study comparing laparoscopic versus minilaparotomic myomectomy: reproductive outcomes Pages 933-941 Stefano Palomba, Errico Zupi, Angela Falbo, Tiziana Russo, Daniela Marconi, Achille Tolino, Francesco Manguso, Alberto Mattei and Fulvio Zullo
Techniques and instrumentation
28. A multicenter randomized, controlled study comparing laparoscopic versus minilaparotomic myomectomy: short-term outcomes Pages 942-951 Stefano Palomba, Errico Zupi, Tiziana Russo, Angela Falbo, Daniela Marconi, Achille Tolino, Francesco Manguso, Alberto Mattei and Fulvio Zullo
29. Survival rate of human oocytes and pregnancy outcome after vitrification using slush nitrogen in assisted reproductive technologies Pages 952-956 Tae Ki Yoon, Dong Ryul Lee, Soo Kyung Cha, Hyung Min Chung, Woo Sik Lee and Kwang Yul Cha
Lessons from the lab
30. Effects of cellular phone emissions on sperm motility in rats Pages 957-964 Ji-Geng Yan, Michael Agresti, Tim Bruce, Yu Hui Yan, Amy Granlund and Hani S. Matloub
Images in reproductive medicine
31. Single large cystic adenomyoma of the uterus after cornual pregnancy and curettage Pages 965-967 Jian-Hua Wang, Rui-Jin Wu, Kai-Hong Xu and Jun Lin
Case report summaries
32. Maternal derivative chromosome 9 and recurrent pregnancy loss Pages 968.e1-968.e3 Ling-Wei Chang, I.-Wen Lee, Pao-Lin Kuo and Long-Ching Kuan
33. Spontaneous heterotopic triplets: a case report Pages 968.e5-968.e7 Aarathi Cholkeri-Singh and Ann LaBarge
34. Massive ascites and hydrothorax after leuprolide acetate administration in a down-regulated woman undergoing assisted reproduction Pages 968.e9-968.e11 Bruno Ferrari, Antonio Pezzuto and Francesco Coppola
35. Torsion causing interruption of the ampullary portion of the fallopian tube Pages 968.e13-968.e14 Sonia Grover
36. Extremely elevated serum CA-125 level as a result of unruptured unilateral endometrioma: the highest value reported Pages 968.e15-968.e17 Korhan Kahraman, Irem Ozguven, Mete Gungor and Cem Somer Atabekoglu
37. Peri-implantation pelvic inflammatory disease with normal pregnancy outcome Pages 969.e1-969.e2 Gad Liberty, Jordana Hadassah Hyman and Ehud J. Margalioth
38. Treatment of heterotopic cervical pregnancy after in vitro fertilization–embryo transfer by using transvaginal ultrasound–guided aspiration and instillation of hypertonic solution of sodium chloride Pages 969.e3-969.e5 Milenko Prorocic and Mladenko Vasiljevic
39. Intestinal endometriosis complicated by ileal perforation after initiation of gonadotropin-releasing hormone agonist therapy Pages 969.e7-969.e9 Sayaka Saito, Takashi Murakami, Kichiya Suzuki, Yukihiro Terada, Kouhei Fukushima and Takuya Moriya
40. Identification of a “cryptic mosaicism” involving at least four different small supernumerary marker chromosomes derived from chromosome 9 in a woman without reproductive success Pages 969.e11-969.e17 Mònica Santos, Kristin Mrasek, Maria Àngels Rigola, Heike Starke, Thomas Liehr and Carme Fuster
41. High risk of temporary alteration of semen parameters after recent acute febrile illness Pages 970.e1-970.e7 Martin Sergerie, Roger Mieusset, Françoise Croute, Myriam Daudin and Louis Bujan
Correspondence
42. Severity of energy-related menstrual disturbances increases in proportion to indices of energy conservation in exercising women Pages 971-975 Mary Jane De Souza, Daniel K. Lee, Jaci L. VanHeest, Jennifer L. Scheid, Sarah L. West and Nancy I. Williams
43. Pharmacokinetics of a single oral dose of 1.5-mg levonorgestrel when administered as 750-μg tablets or as 30-μg minipills Pages 976-977 Luigi Devoto, Alejandra Espinoza, Alex Muñoz, Ariel Fuentes and Helena von Hertzen
44. Ten-year experience with preimplantation genetic diagnosis (PGD) at the New York University School of Medicine Fertility Center Pages 978-981 J. Grifo, S. Talebian, D. Keegan, L. Krey, A. Adler and A. Berkeley
45. Human albumin does not prevent ovarian hyperstimulation syndrome in assisted reproductive technology program: a prospective randomized placebo-controlled double blind study Pages 982-985 Mete Isikoglu, Murat Berkkanoglu, Zeliha Senturk and Kemal Ozgur
46. Pelvic pain after gonadotropin administration as a potential sign of endometriosis Pages 986-987 Sunny Hee Jun and Ruth B. Lathi
47. Mutations of SYCP3 are rare in infertile Spanish men with meiotic arrest Pages 988-989 Juanjo Martínez, Sandra Bonache, Alejandro Carvajal, Lluís Bassas and Sara Larriba
48. Normal percentage of CD56bright natural killer cells in young patients with a history of repeated unexplained implantation failure after in vitro fertilization cycles Pages 990-993 Maria G. Matteo, Pantaleo Greco, Piergiorgio Rosenberg, Anna Mestice, Domenico Baldini, Teresa Falagario, Vincenzo Martino, Michele Santodirocco, Francesca Massenzio, Laura Castellana, Giorgina Specchia and Arcangelo Liso
49. In vitro sildenafil citrate use as a sperm motility stimulant Pages 994-996 Taymour Mostafa
50. Relative contribution of ovarian stimulation versus in vitro fertilization and intracytoplasmic sperm injection to multifetal pregnancies requiring reduction to twins Pages 997-999 Willem Ombelet, Michel Camus and Luc de Catte
51. Korean ginseng induces spermatogenesis in rats through the activation of cAMP-responsive element modulator (CREM) Pages 1000-1002 Wan Su Park, Dong Youp Shin, Do Rim Kim, Woong Mo Yang, Mun Seog Chang and Seong Kyu Park
52. Differential effects of aging on activin A and its binding protein, follistatin, across the menopause transition Pages 1003-1005 Nancy E. Reame, Jane L. Lukacs, Pamela Olton, Rudi Ansbacher and Vasantha Padmanabhan
53. Preferences of subfertile women regarding elective single embryo transfer: additional in vitro fertilization cycles are acceptable, lower pregnancy rates are not Pages 1006-1009 Moniek Twisk, Fulco van der Veen, Sjoerd Repping, Maas-Jan Heineman, Johanna C. Korevaar and Patrick M.M. Bossuyt
54. Recombinant follicle-stimulating hormone (rFSH) supplemented with low-dose human chorionic gonadotropin compared with rFSH alone for ovarian stimulation for in vitro fertilization Pages 1010-1013 Anne K. Van Horne, G. Wright Bates Jr., Randal D. Robinson, Nancy J. Arthur and Anthony M. Propst
55. Role of embryo transfer in fellowship training Pages 1014-1015 Michael D. Wittenberger, William H. Catherino and Alicia Y. Armstrong
Letters to the editor
56. Ethical issues surrounding the cryopreservation of human oocytes Page 1016 Jason Barritt, Martha Luna, Marlena Duke and Alan Copperman
57. Reply: Ethical issues surrounding the crypreservation of human oocytes Pages 1016-1017 Inmaculada de Melo-Martin and Ina N. Cholst
58. An expert forum for the histology of endometriomas Pages 1017-1018 Camran Nezhat, Ceana Nezhat, Daniel Seidman, Bulent Berker and Farr Nezhat
59. Reply: “An expert forum for the histologic of endometriomas” Pages 1018-1019 Ludovico Muzii, Antonella Bianchi, Emanuela Cristi, Marzio A. Zullo, Roberto Angioli, Filippo Bellati, Milena Pernice and Pierluigi Benedetti Panici
60. Perspectives on oocyte research Pages 1019-1020 Jason Barritt, Martha Luna, Marlena Duke and Alan Copperman
61. Reply: “Perspectives on oocyte research” Page 1020 Sharon E. Moayeri, Barry Behr, Ruth B. Lathi, Lynn M. Westphal and Amin A. Milki
62. Erratum Page 1020
OFFICIAL JOURNAL OF THE AMERICAN SOCIETY FORREPRODUCTIVE MEDICINE, Society for Reproductive Endo-crinology and Infertility, Society of Reproductive Surgeons,Society for Assisted Reproductive Technology, Society forMale Reproduction and Urology, and the Pacific Coast Re-productive Society.
Editor-in-ChiefAlan H. DeCherney, M.D.Bethesda, Maryland
Deputy EditorsGautam Chaudhuri, M.D., Ph.D.Los Angeles, California
Richard J. Paulson, M.D.Los Angeles, California(Images in Reproductive Medicine)
Associate EditorsPaul G. McDonough, M.D.Augusta, Georgia(Letters-to-the-Editor)
Edward E. Wallach, M.D.Lutherville, Maryland(Modern Trends)
Managing EditorEric SteinmehlBirmingham, Alabama
Editorial BoardKurt Barnhart, M.D., M.S.C.E.Philadelphia, Pennsylvania(Society for Reproductive Endocrinology andInfertility)
Susan H. Benoff, Ph.D.Manhasset, New York
Robert G. Brzyski, M.D., Ph.D.San Antonio, Texas
John E. Buster, M.D.Houston, Texas (Book Review Editor)
Charles C. Coddington, M.D.Denver, Colorado
Bryan D. Cowan, M.D.Jackson, Mississippi
Owen K. Davis, M.D.New York, New York(Society for Assisted ReproductiveTechnology)
Christopher DeJonge, Ph.D., H.C.L.D.Minneapolis, Minnesota
Esther Eisenberg, M.D.Nashville, Tennessee
Tommaso Falcone, M.D.Cleveland, Ohio
William E. Gibbons, M.D.Baton Rouge, Louisiana
William W. Hurd, M.D.Dayton, Ohio
Keith Isaacson, M.D.Newton, Massachusetts(Society of Reproductive Surgeons)
William H. Kutteh, M.D., Ph.D.Memphis, Tennessee
Stanley P. Leibo, Ph.D.New Orleans, Louisiana
William R. Meyer, M.D.Chapel Hill, North Carolina
Camran Nezhat, M.D.Stanford, California
Steven J. Ory, M.D.Margate, Florida
Nanette Santoro, M.D.Bronx, New York
William D. Schlaff, M.D.Aurora, Colorado
Mark Sigman, M.D.Providence, Rhode Island(Society for Male Reproduction andUrology)
Ronald C. Strickler, M.D.Detroit, Michigan (CME Editor)
Eric S. Surrey, M.D.Englewood, Colorado
Hugh Taylor, M.D.New Haven, Connecticut
International Editorial BoardPaul Devroey, M.D., Ph.D.Brussels, Belgium
David Healy, M.D., Ph.D.Melbourne, Victoria, Australia
Neri Laufer, M.D.Jerusalem, Israel
Guillermo Marconi, M.D.Buenos Aires, Argentina
Francois Olivennes, M.D., Ph.D.Paris, France
Antonio Pellicer, M.D.Valencia, Spain
Basil C. Tarlatzis, M.D., Ph.D.Thessaloniki, Greece
Former EditorsPendleton Tompkins, M.D.Editor 1950–1952
M. Edward Davis, M.D.Editor 1953–1969
Luigi Mastroianni, Jr., M.D.Editor 1970–1975
Roger D. Kempers, M.D.Editor 1976–1997
Fertility and Sterility� (ISSN 0015-0282) is a registered trademark of the American Society of Reproductive Medicine and is published monthly in two indexed volumes by Elsevier Inc., 360 Park Avenue
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OCTOBER 2007
VOLUME 88
NUMBER 4
COVERThe cover shows a subscenM-FISH scheme and a normalchromosome 9 depicted in pseudocolors (see page 969.e13).
MODERN TRENDS
771 Erectile dysfunction: does insulin resistance playa part?
J. C. Trussell and R. S. LegroHershey, Pennsylvania
Insulin resistant states are characterized by defectivevascular nitric oxide production and impaired insulin-induced vasodilation, both of which are likely to causeerectile dysfunction.
EDITOR’S CORNER
779 Limiting access to letrozole—is it justified?
T. Tulandi and A. H. DeCherneyMontreal, Quebec, Canada; and Bethesda, Maryland
Letrozole is an effective drug for ovulation inductionand superovulation. Limiting its access is a disserviceto our infertile patients and to the advancement ofovulation-related research.
781 Substandard application of preimplantation geneticscreening may interface with its clinical success
S. Munn�e, L. Gianaroli, I. Tur-Kaspa, C. Magli,M. Sandalinas, J. Grifo, D. Cram, S. Kahraman,Y. Verlinsky, and J. L. SimpsonLivingston, New Jersey; Bologna, Italy; Chicago,Illinois; Barcelona, Spain; New York, New York;Victoria, Australia; Istanbul, Turkey; and Miami, Florida
A closer look at a recent randomized clinical trial eval-uating the effect of preimplantation genetic screeningreveals significant lack of expertise in biopsy, cell fix-ation, and genetic analysis.
Copyright ª2007 American Society for Reproductive Medicine
ENDOMETRIOSIS
785 Estrogen receptor b gene D1730 G/A polymorphismin women with endometriosis
G. H. Lee, S. H. Kim, Y. M. Choi, C. S. Suh, J. G. Kim,and S. Y. MoonSeoul, Korea
In a case–control study, theþ1730 G/A polymorphismof the estrogen receptor b gene was not associatedwith the risk of endometriosis in a Korean population.
789 Efficacy of vaginal danazol treatment in women withrecurrent deeply infiltrating endometriosis
S. Razzi, S. Luisi, F. Calonaci, A. Altomare, C. Bocchi,and F. PetragliaSiena, Italy
Vaginal danazol is an effective medical treatment forthe various painful symptoms in women with recurrentdeeply infiltrating endometriosis.
795 Different types of small nerve fibers in eutopicendometrium and myometrium in women withendometriosis
N. Tokushige, R. Markham, P. Russell, and I.S. FraserSydney, Australia
Endometrium appears to be mainly innervated bysensory Ad, sensory C, and adrenergic fibers, and my-ometrium by sensory Ad, sensory C, adrenergic andcholinergic fibers in women with endometriosis.
GENETICS
804 Preimplantation genetic screening as an alternativeto prenatal testing for Down syndrome: preferencesof women undergoing in vitro fertilization/intracytoplasmic sperm injection treatment
M. Twisk, M. L. Haadsma, F. van der Veen, S. Repping,S. Mastenbroek, M-J. Heineman, P. M. M. Bossuyt,and J. C. KorevaarAmsterdam and Groningen, the Netherlands
Most women favor preimplantation genetic screeningfor Down syndrome as an alternative to prenatal test-ing, even if it is not 100% sensitive.
811 Oocyte dysmorphism is not associated withaneuploidy in the developing embryo
K. Yakin, B. Balaban, A. Isiklar, and B. Urman_Istanbul, Turkey
Blastocyst formation rate was significantly lower forembryos derived from oocytes with cytoplasmic ormultiple abnormalities. Oocyte dysmorphism was notassociated with a higher risk of aneuploidy in the de-veloping embryo.
IN VITRO FERTILIZATION
817 Social concerns of women undergoing infertilitytreatment
P. S. Finamore, D. B. Seifer, C. V. Ananth,and S. R. LeiblumNew Brunswick, New Jersey; and Brooklyn, New York
In a survey of 267 women undergoing evaluation and ortreatment for infertility we found that women who diddisclose their infertility status to their employer weredifferent from those who did not disclose with regardto level of education, race/ethnicity, and number ofvisits per month to the doctor. The decision to disclosedid not seem to have a significant impact on stress.
822 Aspirin in women undergoing in vitro fertilizationtreatment: a systematic review and meta-analysis
M. Khairy, K. Banerjee, T. El-Toukhy, A. Coomarasamy,and Y. KhalafLondon and Birmingham, United Kingdom
Aspirin in IVF treatment does not improve clinical preg-nancy or live birth rates. An improvement in uterine ar-tery pulsatility index was noted with aspirin use.
832 GnRH agonist and antagonist protocols for stage I–IIendometriosis and endometrioma in in vitrofertilization/intracytoplasmic sperm injection cycles
R. Pabuccu, G. Onalan, and C. KayaAnkara, Turkey
The controlled ovarian hyperstimulationprotocols includ-ing GnRH agonist and GnRH antagonist may be equallyeffective among patients having stage I–II endometriosisand endometrioma with or without ovarian surgery.
MALE FACTOR
840 Markov modeling of vasectomy reversal and ART forinfertility: how do obstructive interval and femalepartner age influence cost effectiveness?
M. H. Hsieh, M. V. Meng, and P. J. TurekSan Francisco, California
Markov modeling of vasectomy reversal and assistedreproduction treatment provides quantitative evidencethat female partner age influences cost effectivenessof infertility treatment more than vasectomy obstruc-tive interval.
847 The beneficial effects of toremifene administrationon the hypothalamic-pituitary-testicular axis andsperm parameters in men with idiopathicoligozoospermia
D. Farmakiotis, C. Farmakis, D. Rousso, A. Kourtis,I. Katsikis, and D. PanidisThessaloniki, Greece
Toremifene administration for a period of 3 months inmen with idiopathic oligozoospermia results in signifi-cant improvements of sperm count, motility, and mor-phology.
854 The contribution of genetic variations of arylhydrocarbon receptor pathway genes to male factorinfertility
A. Merisalu, M. Punab, S. Altmae, K. Haller, T. Tiido,M. Peters, and A. SalumetsTartu, Estonia; and Malm€o, Sweden
This case-control study demonstrated that a poly-morphism at codon 185 of the aryl hydrocarbonreceptor repressor gene was associated withsusceptibility to male factor infertility, with greaterprevalence of the Ala/Ala genotype among infertilemale patients.
860 Alterations in sperm motility after acute oraladministration of sildenafil or tadalafil in young,infertile men
G. Pomara, G. Morelli, D. Canale, P. Turchi,C. Caglieresi, C. Moschini, G. Liguori, C. Selli,E. Macchia, E. Martino, and F. FrancescaPisa, Prato, and Trieste, Italy
In a randomized, double-blind, crossover study on theacute effect of sildenafil and tadalafil on several semi-nal parameters, sperm motility significantly increasedwith sildenafil and decreased with tadalafil, respec-tively.
MENOPAUSE
866 Tibolone histology of the endometrium and breastendpoints study: design of the trial and endometrialhistology at baseline in postmenopausal women
D. F. Archer, S. Hendrix, A. Ferenczy, J. Felix, J.ChrisGallagher, J. Rymer, S. O.Skouby, W. Hollander,V. Stathopoulos, and F. A. HelmondNorfolk, Virginia; Detroit, Michigan; Quebec, Canada;Los Angeles, California; Omaha, Nebraska; London,United Kingdom; Copenhagen, Denmark;and Roseland, New Jersey
THEBES is a prospective, randomized, double-blindclinical trial of tibolone vs. conjugated E and medroxy-progesterone acetate protocol and histologic interpre-tation of the screening endometrial biopsies arepresented.
OVULATION INDUCTION
879 A comparison of letrozole to gonadotropins forovulation induction, in subjects who failed toconceive with clomiphene citrate
R. B. Quintero, R. Urban, R. B. Lathi, L. M. Westphal,and M. H. DahanStanford, California
The pregnancy rate with letrozole may justify its use asa second-line treatment for ovulation induction inwomen who did not conceive with CC.
POLYCYSTIC OVARY SYNDROME
886 Role of insulin in the hyperandrogenemia of leanwomen with polycystic ovary syndrome and normalinsulin sensitivity
J-P. Baillargeon and A. CarpentierQuebec, Canada
We studied nine lean normoinsulinemic women withpolycystic ovary syndrome (PCOS) and normal insulinsensitivity compared with 17 lean control subjects. Inthe PCOS women, free testosterone decreased andSHBG increased only after insulin lowering withdiazoxide.
894 Assessment of three-dimensional sonographicfeatures of polycystic ovaries after laparoscopicovarian electrocautery
Miklo. Vizer, L. Kiesel, Istva. Szabo, A. Arany,P�e. Tamas, and Andra. SzilagyiP�ecs, Hungary; and Munster, Germany
The impact of laparoscopic electrocautery on ovarianvolume and vascular flow patterns assessed bythree-dimensional sonography was studied and com-pared with the hormonal effects of surgery in patientswith polycystic ovary syndrome.
900 Lack of association between polycystic ovarysyndrome and embryonic aneuploidy
A. Weghofer, S. Munne, S. Chen, D. Barad, andN. GleicherNew Haven, Connecticut; Vienna, Austria; West Orangeand Livingston, New Jersey; and New York, New York
Women with polycystic ovary syndrome (PCOS) arenot at increased risk for embryonic aneuploidy inthe course of in vitro fertilization treatment andeven produce higher absolute numbers of euploidembryos. High-responder patients with PCOS (>20eggs), however, demonstrate lower pregnancy rateswhen compared with high-responder controlwomen.
REPRODUCTIVE ENDOCRINOLOGY
906 Stage I ovarian carcinoma: different clinicalpathologic patterns
L. Deligdisch, F. P�enault-Llorca, P. Schlosshauer,A. Altchek, M. Peiretti, and F. NezhatNew York, New York; Clermont-Ferrand, France; andCagliari, Italy
Stage I ovarian cancers have different clinicopathologicfeatures than all stage ovarian cancers. They are diag-nosed early due to associated symptomatic pathologysuch as endometriosis and endometrial neoplasia.
911 Anxiety and sexual stress in men and womenundergoing infertility treatment
B. D. Peterson, C. R. Newton, and T. FeingoldOrange, California; and London, Ontario, Canada
Although women report greater amounts of anxietyand sexual stress, the relationship between the twomay be stronger for men. Gender differences are high-lighted and discussed.
915 Bayesian selection of optimal rules for timingintercourse to conceive by using calendar and mucus
B. Scarpa, D. B. Dunson, and E. GiacchiPada and Rome, Italy; and Research Triangle Park,North Carolina
For couples attempting conception, simple rules fortiming intercourse based on a combination of calendarinterval and the most fertile type of mucus symptomobserved at the vulva can shorten time to conceptionsubstantially.
925 Detection of antizona pellucida antibodies in the serafrom premature ovarian failure patients by a highlyspecific test
S. Takamizawa, H. Shibahara, T. Shibayama,and M. SuzukiTochigi, Japan
Some idiopathic POF patients have anti-ZP antibodiesin their sera, which were detected by a newly devel-oped highly specific test using a very small amountof human ZP.
REPRODUCTIVE SURGERY
933 A multicenter randomized, controlled studycomparing laparoscopic versus minilaparotomicmyomectomy: reproductive outcomes
S. Palomba, E. Zupi, A. Falbo, T. Russo, D. Marconi,A. Tolino, F. Manguso, A. Mattei, and F. ZulloCatanzaro, Rome, Naples, and Florence, Italy
Compared with the minilaparotomic approach, laparo-scopic myomectomy provides better reproductiveoutcomes in fertile patients with symptomatic leio-myomas, but not in women with unexplained infertility.
TECHNIQUES AND INSTRUMENTATION
942 A multicenter randomized, controlled studycomparing laparoscopic versus minilaparotomicmyomectomy: short-term outcomes
S. Palomba, E. Zupi, T. Russo, A. Falbo, D. Marconi,A. Tolino, F. Manguso, A. Mattei, and F. ZulloCatanzaro, Rome, Naples, and Florence, Italy
Minilaparotomic and laparoscopic approaches touterine leiomyomas: two safe and minimally invasiveprocedures with different limits and indications.
952 Survival rate of human oocytes and pregnancyoutcome after vitrification using slush nitrogen inassisted reproductive technologies
T. K. Yoon, D. R. Lee, S. K. Cha, H. M. Chung, W. S. Lee,and K. Y. ChaSeoul, Korea
A successful pregnancy outcome was obtained fromvitrified/warmed human oocytes using slush nitrogenfor super-rapid cooling when combined with intracyto-plasmic sperm injection for fertilization.
LESSONS FROM THE LAB
957 Effects of cellular phone emissions on spermmotility in rats
J-G. Yan, M. Agresti, T. Bruce, Y. H. Yan, A. Granlund,and H. S. MatloubMilwaukee, Wisconsin
Rats exposed to cellular phone emissions exhibiteda significantly higher incidence of sperm cell deathand abnormal clumping of sperm cells than controlgroup rats.
IMAGES IN REPRODUCTIVE MEDICINE
965 Single large cystic adenomyoma of the uterus aftercornual pregnancy and curettage
J-H. Wang, R-J. Wu, K-H. Xu, and J. LinHangzhou, Zhejiang, People’s Republic of China
A case of single large cystic adenomyoma of theuterus (anechoic area 1.6 cm in diameter) was diag-nosed by surgery and histopathology more than 3years after transcervical curettage for an early rightcornual pregnancy.
CASE REPORT SUMMARIES
968 Maternal derivative chromosome 9 and recurrentpregnancy loss
L-W. Chang, I-W. Lee, P-L. Kuo, and L-C. KuanTainan, Taiwan
Maternal derivative chromosome 9 may cause recur-rent pregnancy loss.
968 Spontaneous heterotopic triplets: a case report
A. Cholkeri-Singh and A. LaBargePark Ridge, Illinois
The rarity of heterotopic pregnancies can lead toa missed diagnosis, as in this case, of a spontaneoustriplet heterotopic pregnancy thought to be appendicitis.
968 Massive ascites and hydrothorax after leuprolideacetate administration in a down-regulated womanundergoing assisted reproduction
B. Ferrari, A. Pezzuto, and F. CoppolaParma, Italy
Massive ascites and hydrothorax developed after leu-prolide acetate administration in a down-regulatedwoman undergoing assisted reproduction.
968 Torsion causing interruption of the ampullary portionof the fallopian tube
S. GroverMelbourne, Australia
A girl with a presentation consistent with adnexal tor-sion was found to have a torsion of paratubal cystand midsegment of tube, with fimbrial and proximalends of tube unaffected.
968 Extremely elevated serum CA-125 level as a result ofunruptured unilateral endometrioma: the highestvalue reported
K. Kahraman, I. Ozguven, M. Gungor, andC. S. AtabekogluAnkara, Turkey
Ovarian endometrioma should be considered with re-spect to differential diagnosis of reproductive-agewomen presenting with ovarian mass–associated ex-tremely elevated serum CA-125 levels even if it resem-bles an ovarian malignancy.
G. Liberty, J. H. Hyman, and E. J. MargaliothJerusalem, Israel
Pelvic inflammatory disease (PID) may rarely complicateearly pregnancy and usually is associated with poor out-come.Wereport the first case of peri-implantation PID ina pregnancy with favorable obstetric outcome.
969 Treatment of heterotopic cervical pregnancy after invitro fertilization–embryo transfer by usingtransvaginal ultrasound–guided aspiration andinstillation of hypertonic solution of sodium chloride
M. Prorocic and M. VasiljevicBelgrade, Serbia, and Montenegro
Transvaginal ultrasound-guided aspiration and instilla-tion hypertonic solution of sodium chloride may beused in treatment of heterotopic cervical pregnancy.
969 Intestinal endometriosis complicated by ilealperforation after initiation of gonadotropin-releasinghormone agonist therapy
S. Saito, T. Murakami, K. Suzuki, Y. Terada,K. Fukushima, and T. MoriyaSendai, Japan
We treated a rare case of small intestinal perforationdue to the transitory exacerbation of ileal endometri-osis after initiation of GnRH agonist treatment.
969 Identification of a ‘‘cryptic mosaicism’’ involving atleast four different small supernumerary markerchromosomes derived from chromosome 9 ina woman without reproductive success
M. Santos, K. Mrasek, M. A. Rigola, H. Starke, T. Liehr,and C. FusterBarcelona, Spain; and Jena, Germany
Various small supernumerary marker chromosomes(sSMCs) derived from chromosome 9 are character-ized in a woman who is the infertile member ofa couple who has no further clinical symptoms. Weconclude that the sSMCs present in the womancould influence the couple’s infertility and that the9p12 chromosomal band is a euchromatic variantwithout any phenotypic impact apart from its possi-ble influence on infertility.
970 High risk of temporary alteration of semenparameters after recent acute febrile illness
M. Sergerie, R. Mieusset, F. Croute, M. Daudin, andL. BujanToulouse, France
Fever can have marked effects on semen quality andsperm DNA integrity, most particularly on the protami-nation state, measured by sperm chromatin structureassay, than on the apoptotic activities, measured byterminal uridine nick-end labeling assay.
CORRESPONDENCE
971 Severity of energy-related menstrual disturbancesincreases in proportion to indices of energyconservation in exercising women
M. J. De Souza, D. K. Lee, J. L. VanHeest, J. L. Scheid,S. L. West, and N. I. WilliamsToronto, Ontario, Canada; Storrs, Connecticut; andUniversity Park, Pennsylvania
Energy-related menstrual disturbances in exercisingwomen increase in severity proportionally to the mag-nitude of increase in energy conservation.
976 Pharmacokinetics of a single oral dose of 1.5-mglevonorgestrel when administered as 750-mg tabletsor as 30-mg minipills
L. Devoto, A. Espinoza, A. Mu~noz, A. Fuentes, andH. von HertzenSantiago, Chile; and Geneva, Switzerland
Two levonorgestrel 0.75 mg tablets taken orally and 50levonorgestrel 30 mg minipills taken orally have com-parable bioavailability.
978 Ten-year experience with preimplantation geneticdiagnosis (PGD) at the New York University School ofMedicine Fertility Center
J. Grifo, S. Talebian, D. Keegan, L. Krey, A. Adler,and A. Berkeley
New York, New York
We describe our ten-year experience performing pre-implantation genetic diagnosis. Respectable preg-nancy rates support using this technology to screenfor genetic disorders and chromosomal abnormalitiesin high-risk patients.
982 Human albumin does not prevent ovarianhyperstimulation syndrome in assisted reproductivetechnology program: a prospective randomizedplacebo-controlled double blind study
M. Isikoglu, M. Berkkanoglu, Z. Senturk, and K. OzgurAntalya, Turkey
In this prospective randomized placebo-controlleddouble blind study, 20% human albumin solution IVdid not seem to either prevent or reduce the incidenceof severe ovarian hyperstimulation syndrome in highrisk patients undergoing intracytoplasmic sperminjection.
986 Pelvic pain after gonadotropin administration asa potential sign of endometriosis
S. H. Jun and R. B. LathiStanford, California
Women who experience significant pelvic pain duringcontrolled ovarian hyperstimulation may have undiag-nosed endometriosis or a recurrence of previously di-agnosed and treated disease.
988 Mutations of SYCP3 are rare in infertile Spanish menwith meiotic arrest
J. Martınez, S. Bonache, A. Carvajal, L. Bassas,and S. LarribaBarcelona, Spain
No association between SYCP3 mutations and meio-sis arrest was observed in Spanish patients with mei-osis blockade in primary spermatocyte.
990 Normal percentage of CD56bright natural killer cellsin young patients with a history of repeatedunexplained implantation failure after in vitrofertilization cycles
M. L. Matteo, P. Greco, P. Rosenberg, A. Mestice,D. Baldini, T. Falagario, V. Martino, M. Santodirocco,F. Massenzio, L. Castellana, G. Specchia, and A. LisoFoggia and Bari, Italy
The endometrium of young patients with repeated unex-plained implantation failure after IVF was analyzed byflow cytometry. We observed no difference in the per-centage of CD56bright natural-killer cells and of theother lymphocyte subpopulations of patients enrolled,when compared with normal fertile young women.
994 In vitro sildenafil citrate use as a sperm motilitystimulant
T. MostafaCairo, Egypt
Sildenafil citrate solution has a concentration-related,in vitro stimulatory effect on ejaculated weak spermmotility.
997 Relative contribution of ovarian stimulation versus invitro fertilization and intracytoplasmic sperminjection to multifetal pregnancies requiringreduction to twins
W. Ombelet, M. Camus, and L. de CatteGenk and Brussels, Belgium
Multiple pregnancy reduction of higher-order multiplesto twins is seen more often after hormone stimulationwhen compared with in vitro fertilization/intracytoplas-mic sperm injection treatment, which can be importantwhen assessing the perinatal risk of these twin preg-nancies.
1000 Korean ginseng induces spermatogenesis in ratsthrough the activation of cAMP-responsive elementmodulator (CREM)
W. S. Park, D. Y. Shin, D. R. Kim, W. M. Yang,M. S. Chang, and S. K. ParkSeongnam and Seoul, Korea
Korean ginseng may enhance spermatogenesisthrough cAMP-responsive element modulator(CREM) activation in rat testes.
1003 Differential effects of aging on activin A and itsbinding protein, follistatin, across the menopausetransition
N. E. Reame, J. L. Lukacs, P. Olton, R. Ansbacher, andV. PadmanabhanAnn Arbor, Michigan; and New York, New York
The sustained presence of activin A across the meno-pause transition coupled with an aging-related declinein its binding protein, follistatin 288, suggest an increasein activin bioavailability during reproductive aging.
1006 Preferences of subfertile women regarding electivesingle embryo transfer: additional in vitrofertilization cycles are acceptable, lower pregnancyrates are not
M. Twisk, F. Veen, S. Repping, M-J. Heineman,J. C. Korevaar, and P. M. M. BossuytAmsterdam, Groningen, The Netherlands
The primary goal of treatment for women undergoingin vitro fertilization or intracytoplasmic sperm injectionis pregnancy, and women are not willing to trade offthat goal to avoid a multiple pregnancy.
1010 Recombinant follicle-stimulating hormone (rFSH)supplemented with low-dose human chorionicgonadotropin compared with rFSH alone for ovarianstimulation for in vitro fertilization
A. K. Van Horne, G. W. Bates, R. D. Robinson,N. J. Arthur, and A. M. PropstSan Antonio, Texas
Low-dose human chorionic gonadotropin supplemen-tation administered at the start of ovarian stimulationresulted in similar implantation and pregnancy rateswhile significantly reducing the amount of recombinantfollicle-stimulating hormone required and the cost percycle.
1014 Role of embryo transfer in fellowship training
M. D. Wittenberger, W. H. Catherino, andA. Y. ArmstrongBethesda, Maryland
Although all recent reproductive endocrinology gradu-ates and 98% of fellows believe training in embryotransfer is important, only 44% received such training.It is noteworthy that 38% of graduates reported thatthis training positively impacted their employmentprospects.
LETTERS TO THE EDITOR
1016 Ethical issues surrounding the cryopreservation ofhuman oocytes
J. Barritt, M. Luna, M. Duke, and A. CoppermanNew York, New York
-Reply: I. de Mello-Martin and I. N. Cholst
1017 An expert forum for the histology of endometriums
C. Nezhat, C. Nezhat, D. Seidman, B. Berker, andF. NezhatStanford, California; Tel Aviv, Israel; and Ankara,Turkey
-Reply: L. Muzii, A. Bianchi, E. Cristi, M. A. Zullo, R.Angioli, F. Bellati, M. Pernice, and P. Bennedetti Panici
1019 Perspectives on oocyte research
J. Barritt, M. Luna, M. Duke, and A. CoppermanNew York, New York
-Reply: S. E. Moayeri, B. Behr, R. B. Lathi, L. M. West-phal, and A. A. Milki
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MODERN TRENDSEdward E. Wallach, M.D.Associate Editor
Erectile dysfunction: does insulin resistance playa part?J. C. Trussell, M.D.,a and Richard S. Legro, M.D.b
a Division of Urology, and b Department of Obstetrics and Gynecology, Penn State Milton S. Hershey Medical Center and Penn
State College of Medicine, Hershey, Pennsylvania
Objective: To review MEDLINE literature for correlations between insulin resistance and erectile dysfunction(ED).Design: MEDLINE literature review (1966 to present).Setting: Academic medical center.Patient(s): None.Intervention(s): None.Main Outcome Measure(s): None.Result(s): Erectile dysfunction affects more than half of men over the age of 40. Fortunately, most men with EDcan be successfully treated with phosphodiesterase 5A (PDE-5) inhibitors, which up-regulate the vasodilatory ef-fects of nitric oxide (NO). Insulin resistance affects 25% of U.S. adults and increases to a 60% occurrence in in-dividuals who are overweight. Endothelial dysfunction, which is associated with insulin resistance states, can causedisturbances in the subcellular signaling pathways required for NO production. Because endothelial production ofNO and insulin sensitivity are positively related in healthy humans, the relationships among insulin resistance, NO,and ED are the target of this review of MEDLINE literature.Conclusion(s): Insulin resistance states are characterized by defective vascular NO production and impaired insu-lin-induced vasodilation, both of which are likely to cause ED. Diagnosing and treating insulin resistance should bepart of the initial management plan for ED. Future studies concerning the cause and effect relationship of insulinresistance and ED should be implemented. (Fertil Steril� 2007;88:771–8. �2007 by American Society for Repro-ductive Medicine.)
Erectile dysfunction (ED) affects more than half of menbetween the ages of 40 and 70 years (1) and is associatedwith a significant decline in quality of life (1–3). Moreover,ED in an otherwise asymptomatic man should be considereda sentinel event for endothelial dysfunction and cardiovascu-lar disease. Such a person should be carefully evaluated forundiagnosed risk factors predisposing to endothelial dysfunc-tion such as hypertension, diabetes, lipid disorders, and obe-sity (4, 5). With obesity being an epidemic in America, half ofpeople over age 60 meet the criteria for the metabolicsyndrome (Table 1) (6, 7). These disorders also cluster withinsulin resistance. Insulin resistance affects 25% of U.S.adults (8) and increases to a 60% occurrence in individualswho are overweight (9). Endothelial dysfunction is associ-
Received October 19, 2006; revised and accepted January 17, 2007.
Supported by NIH grant K24 HD01476 (RSL).
Reprint requests: J. C. Trussell, M.D., Assistant Professor. Division of
Urology, Penn State Milton S. Hershey Medical Center, 500 University
Drive, Hershey, PA 17033-0850 (FAX: 717-531-4475; E-mail:
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2007.01.116 Copyright ª2007 America
ated with insulin-resistance states and can cause disturbancesin the subcellular signaling pathways required for nitric oxide(NO) production (9). Because endothelial production of NOand insulin sensitivity are positively related in healthyhumans (10), the relationships between insulin resistance,NO, and ED are the target of this review of MEDLINE liter-ature. Understanding this relationship may serve to expandtreatment options for both insulin resistance and ED.
OVERVIEW OF ERECTILE DYSFUNCTION
Erectile dysfunction was defined in 1993 by the NationalInstitutes of Health as ‘‘the consistent inability to attain ormaintain a penile erection satisfactory for sexual function’’(11). A consensus panel on ED convened in 2003 to offerclinical suggestions and outline future research directions(12). Erectile dysfunction is a common condition affectingaround 30 million U.S. men, most of whom have an organiccause (as opposed to a psychogenic cause) for their dysfunc-tion. Increasing age is clearly associated with ED (13), but
Fertility and Sterility� Vol. 88, No. 4, October 2007 771n Society for Reproductive Medicine, Published by Elsevier Inc.
other treatable factors that predispose men to organic EDinclude hypertension, depression, hormonal abnormalities,diabetes, and surgically induced neurologic disruption. Thesedisorders can lead to two interrelated forms of organic ED,vasculogenic and neurogenic, both of which rely on NO pro-duction via nitric oxide synthetase (NOS) to facilitate propererectile function (14).
Tumescence requires a complex series of events to occur,starting with the release of NO from either nonadrenergicnoncholinergic nerve terminals or endothelial cells. Produc-tion of NO is accomplished by one of three NOS isoforms.NOS converts L-arginine into NO, which then acts on gua-nylyl cyclase to convert guanosine triphosphate (GTP) intoits active form, cyclic guanosine monophosphate (cGMP).Specific to an erection, phosphorylated cGMP results in ac-tive removal of cytoplasmic calcium (15), allowing for penilecavernosal smooth muscle relaxation and subsequent tumes-cence. As long as NO is available to maintain adequate levelsof cGMP, a penile erection is possible.
Detumescence occurs when there is a breakdown of cGMPcaused by phosphodiesterase 5A (PDE-5) enzyme. The oralPDE-5 inhibitors (currently sildenafil, tadalafil, and vardena-fil) work by inhibiting this breakdown of cGMP. Inhibition ofPDE-5 permits NO-related cGMP levels to remain robust,thereby allowing for enhanced tumescence.
Erectile dysfunction has both neurogenic and vasculogeniccauses. Neurogenic ED occurs when there is a lack of nona-drenergic noncholinergic nerve function within the penilecorpora cavernosa. This neurologic dysfunction may be dueto surgery, trauma, aging, and/or diabetes (16). VasculogenicED, on the other hand, is caused by an impairment of corporalsmooth muscle relaxation. The latter is the most commoncause of organic ED, affecting nearly 80% of ED patients.Potential causes include NOS, endothelial, corporal venooc-clusive, and NO dysfunctions, along with corporal fibrosis.
TABLE 1Metabolic syndrome.
Metabolic syndrome is diagnosed whena minimum of 3 of 5 possible criteria are met:
� Waist circumference greater than 40 inches� Triglycerides greater than 150 mg/dL� Blood pressure higher than 130/85 mm Hg� Fasting glucose greater than 110 mg/dL� High-density lipoprotein less than 40 mg/dL
Source: Expert Panel on Detection, Evaluation, andTreatment of High Blood Cholesterol in Adults. Exec-utive summary of the third report of the National Cho-lesterol Education Program (NCEP) Expert Panel onDetection, Evaluation, And Treatment of High BloodCholesterol In Adults (Adult Treatment Panel III).JAMA 2001;285:2486–97.
Trussell. Insulin resistance and erectile dysfunction. Fertil Steril 2007.
772 Trussell and Legro Insulin resistance and erectile dy
Gonzalez-Cadavid and Rajfer (3) summarized four maincauses of ED based on the following molecular or cellularevents (Table 2):
1. ‘‘Insufficient production of NO at nerve terminals dur-ing sexual stimulation. Most neurogenic NO is releasedat penile nerve terminals, with a much smaller contribu-tion coming from the brain or spinal cord. Impaired NOrelease can be due to either the loss of neuronal bodies/connections to and from the penis, or to a down-regula-tion of NOS activity/expression.’’
2. ‘‘An ancillary reduction in NO levels from eNOS down-regulation—due to endothelial damage in patients witheither diabetes or vascular disease.’’
3. ‘‘An excessive release of adrenergic compounds thatincrease corpora cavernosal smooth muscle tone, mainlythrough endothelin and rho kinase activation.’’
4. ‘‘Impairment in the relaxation of target smooth muscleby endogenous factors. This results mainly from eithera putative excessive cGMP degradation by PDE-5A, orfrom a relative loss of penile smooth muscle cells sec-ondary to replacement with collagen fibers/fibrosis.’’
Realizing that NO is the essential mediator of corporacavernosa relaxation (17, 18) provides a better understanding ofNOS function and its potential for modulation. For instance,one potential transcellular point for NOS regulation includesthe insulin receptor and its subsequent signal transductionpathway. It is surprising that little written work is availableconcerning the effects of NOS dysfunction stemming fromeither insulin resistance or diabetes.
INSULIN RESISTANCE
Insulin resistance was described by Wheatcroft as ‘‘a state ofdysregulation of glucose-insulin homeostasis in which the
TABLE 2Molecular/cellular causes for erectiledysfunction.
1. Loss of nitric oxide production:a. From nerve terminalsb. Endothelial dysfunction resulting in
down-regulation of eNOS activity2. Excessive adrenergic compounds resulting
in increased corporal cavernosal smoothmuscle tone.
3. Impaired relaxation of corporal smoothmuscles:a. Excessive cGMP degradation
by PDE-5A enzyme activity.b. Muscle replacement by collagen/fibrosis.
Source: Gonzolez-Cadavid NF, Rajfer J. Molecularpathophysiology and gene therapy of aging-relatederectile dysfunction. Exp Gerontol 2004;39:1705–12.
Trussell. Insulin resistance and erectile dysfunction. Fertil Steril 2007.
sfunction Vol. 88, No. 4, October 2007
ability of insulin to stimulate glucose uptake in peripheraltissues (such as skeletal muscle and adipose tissue) is reduced’’(9). It affects 25% of U.S. adults and increases to a 60% prev-alence in individuals who are overweight (8, 9). Endothelialdysfunction is found in cases with insulin resistance and isassociated with disturbances in the subcellular signalingpathways required for NO production (9). Overt diabetesmellitus (DM) is thought to be preceded by a long period ofinsulin resistance, which is initially compensated for by excessinsulin production by the pancreatic beta cells. During thattime, this hyperinsulinemic state allows for adequate periph-eral glucose uptake to maintain stable serum glucose levels,despite declining insulin responsiveness. Endothelial produc-tion of NO and insulin sensitivity are positively related inhealthy humans (10).
Insulin-resistance states are characterized by defectivevascular NO synthesis and impaired insulin-induced vasodi-lation (19). There is also a corresponding decline in basalNO production (10). Both in vitro and in vivo studies haveshown that insulin will increase the production of NO by in-ducing both the expression and the activation of endothelialnitric oxide synthetase (eNOS) (19–21). Insulin acts throughits receptor to promote NOS-related NO release along thephosphatidylinosital 3-kinase (PI3K)–dependent signalingpathway (9, 20). Insulin sensitizers allow for enhanced insu-lin-receptor interaction. Metformin, an insulin sensitizerused to reduce insulin resistance, has been shown to improveendothelial function in both animals and humans (22–23).Unfortunately, the mechanism by which metformin improvesinsulin sensitivity is not completely understood, but it is be-lieved to involve an adenosine monophosphate (AMP) kinasepathway (24).
The endothelium plays a vital role in vascular homeostasis.These cells not only act as a physical barrier for blood prod-ucts, but also secrete a number of mediators that regulateplatelet activity, coagulation, and blood vessel tone (25). En-dothelial cells secrete both vasoconstrictor and vasodilatorsubstances, with NO secretion considered to be the majorcontributor to endothelium-dependent arterial relaxation(9). This fact is successfully exploited by the currently avail-able oral PDE-5 inhibitors (sildenafil, tadalafil, and vardena-fil), all of which act to up-regulate the vasodilatory effects ofNO. This process effectively increases NO levels within thecorpora cavernosa, allowing for vasodilation and, as a result,improved erections.
NITRIC OXIDE
Nitric oxide is produced from L-arginine by one of threeNOS. Three distinct NOS isoforms have been isolated andare named after the cells from which they were initiallypurified (Table 3). [1] Neuronal NOS (nNOS) was purifiedfrom rat/porcine cerebellum and is the most abundant iso-form. [2] Inducible NOS (iNOS) was purified from macro-phages and has variable levels of activity based on thespecies and degree of ‘‘disease state.’’ [3] Endothelial NOS(eNOS) is found both in the endothelium and skeletal muscle
Fertility and Sterility�
cells (19), and is regulated by several mechanisms includingacylation, phosphorylation, interaction with other proteins,shear stress, and insulin. In insulin-resistance states,calcium-dependent phosphorylation is down-regulated.Down-regulated phosphorylation can be reversed, however,by improved insulin function, thereby stimulating eNOSactivity (9, 26).
Neuronal NOS
Neuronal NOS (nNOS) was purified from rat/porcinecerebellum and is the most abundant isoform. It is found ex-tensively in skeletal muscle tissue (19); nNOS is preferen-tially located at neuromuscular junctions and seems to beessential for the initiation of a penile erection. AlthoughNO from nonadrenergic noncholinergic (NANC) nerve ter-minals can initiate an erection, NO from another isoform(such as eNOS) is essential for maintaining an erection.This is especially true, and important, during the rigid phaseof an erection.
Endothelial NOS
Endothelial NOS (eNOS) was originally detected on chromo-some 7 within endothelial cells (19). Since then, it has alsobeen located on the mitochondria of skeletal myocytes. Theskeletal muscle location can help to explain how eNOS playsa major role in the regulation of insulin sensitivity—by con-trolling the insulin-induced increase in muscular blood flow(through vasodilation) in response to increased substrate(glucose) delivery to the skeletal muscle cells (19). Insulinpromotes NO release via the PI3K-dependent signaling path-way (20–21), and it is believed that the PI3K-related activa-tion of eNOS mediates the sustained phase of a rigid penis.As mentioned earlier, nNOS is essential for initiating an erec-tion, and eNOS is essential for the rigid and sustained phasesof an erection (27).
Through exposure to insulin, eNOS can be up-regulated.Insulin causes an increase in NO production by both up-regu-lating eNOS expression and increasing activation of existingeNOS (19). Insulin stimulates eNOS by a calcium-dependentphosphorylation, which is down-regulated in insulin-resis-tance states. Other ways to up-regulate eNOS expression in-clude administering lysophosphatidylcholine (28), cyclicstraining (29), exercise training (30), introducing vascular en-dothelial growth factor (31), lowering concentrations of oxi-dized low-density lipoprotein (LDL) (32), increasingprotein phosphorylation, enhancing interactions with otherproteins, or shearing stress. Conversely, eNOS expression isreduced by tumor necrosis factor alpha (TNF-a) (33), hypoxia(34), and high concentrations of oxidized LDL (Table 4) (35).Increased levels of TNF-a and oxidized LDL cholesterol areassociated with insulin-resistant states.
It is clear that eNOS and insulin sensitivity are positivelyrelated in healthy humans (10). What is not so easily under-stood is why patients with insulin resistance have a corre-sponding decrease in basal eNOS production. Further study
773
TABLE 3Three known nitric oxide synthetase (NOS) isoforms.
iNOS (inducible) Chromosome 17 Macrophages Activity controlledat transcription level,but not fast enough toaffect spontaneouserections
� Variable activity� Negative feedback
effect on eNOS� Higher activity
associated withinsulin resistance
� Proapoptoticeffects
Trussell. Insulin resistance and erectile dysfunction. Fertil Steril 2007.
is required to determine which event causes the other. Forinstance, does reduced eNOS production cause a decline ininsulin sensitivity? Or does reduced insulin sensitivity causea reduction in eNOS activity? Finally, we must questionwhether there may be a common environmental or geneticcause for both insulin resistance and reduced activity levelsof eNOS (10).
Elevated LDLCyclic strainingLowering LDLIncreasing protein
phosphorylationVascular endothelial
growth factor (VEGF)Lysophosphatidylcholine
Note: LDL: low-density lipoprotein.
Trussell. Insulin resistance and erectile dysfunction. Fertil Steril 2007.
774 Trussell and Legro Insulin resistance and erectile dy
Inducible NOS
Inducible NOS (iNOS) was first purified from macrophagesand has been localized to chromosome number 17 (3). As‘‘inducible’’ implies, iNOS activity can vary, not only be-tween species but also among different disease states (19).Factors related to heightened iNOS activity include increas-ing age, acute and chronic inflammation (such as psoriasis),and cytokine activation. In addition, up-regulation is foundin human penile tissues in association with diabetes (36)and cytokine activation—TNF-a, interleukin beta (IL-b),and interferon (IF). The combination of diabetes and cytokineactivation synergistically increases iNOS activity. Sustainedlevels of NO, produced by iNOS, give rise to the proapoptoticcompound peroxynitrite, which is both good and bad. For in-stance, if age-related increases in iNOS occur in the brain, theapoptosis could cause neuronal loss and memory failure. Onthe other hand, if this were to occur in the penis, the increasedapoptotic rate would act as a possible mechanism to combatfibrosis, such as Peyronie disease. In fact, increased levels ofPeyronie scarring have been seen in the rat model when iNOSactivity was blocked.
Because iNOS responses are controlled at the transcrip-tional level, it is doubtful that iNOS is a vital part of a spon-taneous erection. The process of transcriptional proteinproduction is relatively slow; therefore, it is unlikely thatsuch a response would be fast enough to modulate erectionsrelated to sexual stimulation. As discussed, both acute andchronic inflammatory states are associated with increased
sfunction Vol. 88, No. 4, October 2007
iNOS activity. In addition, chronic inflammation is associatedwith insulin resistance. A study involving mice demonstratedthat if iNOS activity was inhibited obese mice did not de-velop high-fat diet-induced insulin resistance—implyingthat increased iNOS activity is associated with insulin resis-tance. To reinforce this finding, up-regulation of iNOS activ-ity is found in diabetic patients with ED. In addition, highiNOS activity is associated with both hyperglycemia and in-sulin sensitivity (36). Research shows that iNOS can havea negative feedback effect on eNOS activity, which can resultin reduced corporal eNOS activity (36).
MODULATORS OF NOS PRODUCTION
Effects of Insulin on NOS
Insulin acts in target cells by binding with receptor proteinslocated on the plasma membrane. The insulin receptor isa glycoprotein consisting of two extracellular alpha subunits(which bind insulin) and two transmembrane beta subunits(which enable tyrosine kinase activity).
When normal insulin regulation is disrupted (as in statesof insulin resistance), abnormalities in endothelial function,blood pressure regulation, and coagulation can occur. Theseabnormalities can then cause atherosclerosis and chronic in-flammation (37). Insulin-resistance states are more oftenfound in patients with abdominal obesity in comparisonwith those who have peripheral obesity (38). These insulin-resistance states are characterized by defective vascularNO synthesis and impaired insulin-induced vasodilation(39–42). Experimentally induced diabetes in rats has demon-strated reduction in penile expression of both nNOS andeNOS (43, 44). It appears that the underlying pathophysiologyof ED in diabetes relates to a defect in NO productionby eNOS and nNOS within the penile cavernosal tissue(45, 46). This eNOS-specific decline in function has twopotential sources: [1] by way of the insulin receptor, viathe PI3K/Akt pathway, and [2] by iNOS-induction, whichinhibits eNOS-related vasodilation by negative feedbackpathways.
Effect of PI3K/Akt pathway Insulin receptors cross the cellmembrane and interact with a signal transduction pathway,leading to eNOS activation. This pathway involves at leasttwo steps: PI3K and then Akt. The PI3K/Akt-dependentphosphorylation and activation of eNOS are involved in thesustained phase of maximal penile erection (27). Accord-ingly, reduced Akt (and eNOS) activity, as seen in insulin-resistant rats, plays an important role in diabetes-relatedED (36). Besides involvement in eNOS-related vasodilation,the Akt-signaling pathway also provides for continued healthof endothelial cells, in part by offering protection from apo-ptotic stress (36). Of note, vascular endothelial growth factor(VEGF) also relies on an intact PI3K/Akt pathway to inhibitapoptotic stress, while increasing endothelial eNOS expres-sion (47).
Fertility and Sterility�
Low-Density Lipoproteins
In the metabolic syndrome, LDL production is associatedwith a decline in NOS activity (48, 49). Insulin resistanceis associated with elevated LDL levels—which down-regu-late eNOS expression and may contribute to the endothelialdysfunction seen in hyperlipidemic patients (50). The useof statins may help to restore erectile function in men withboth ED and dyslipidemia (51). Low testosterone levelshave an inverse correlation with LDL levels (37).
Sex Steroids
Morbidly obese, insulin-resistant men frequently have lowserum testosterone and increased estradiol levels (52, 53),both of which can be reversed with weight loss (54, 55). Itis theorized that peripheral conversion of androgen to estro-gen by adipose tissue that contains aromatase is responsiblefor this abnormality. In males, circulating estradiol (E2) levelsare typically low. However, levels increase noticeably in obesemen who convert more testosterone into E2 via adipocyte aro-matase. High levels of E2 appear to contribute to the develop-ment of insulin resistance by diminishing cellular insulinresponsiveness in terms of their survival, DNA synthesis, glu-cose transport, and glucose oxidation—which probably in-volves phosphatidylinosital 3-kinase (PI3-kinase) activity.Lowering adipose-related production of E2 levels by exerciseand weight loss may translate into less insulin resistance, betterPI3-kinase activity, and overall improved erectile vasodilation.
Male hypogonadism is associated with insulin resistanceand diabetes (37). Low levels of both testosterone and freetestosterone correlate strongly with insulin resistance andthe metabolic syndrome (56). It appears that low testosteroneis a component of the multidimensional metabolic syndromecharacterized by obesity, diabetes mellitus, hypertension,dyslipidemia, and a procoagulant/antifibrinolytic state (37).In fact, hypogonadism is a predictor for subsequent develop-ment of diabetes in middle-aged men (57). Other studiesdemonstrate an inverse relationship between plasma testos-terone and serum insulin levels (53). Experimental ratshave shown marked insulin resistance after orchiectomy,with insulin sensitivity restored by testosterone replacement(58). Nawras et al. (37) suggest that with further clarificationof the epidemiological association between hypogonadismand metabolic syndrome, testosterone replacement therapywill be an effective option for treating hypogonadism aswell as a novel treatment option for metabolic syndrome.
C-reactive Protein
C-reactive protein at concentrations known to cause diversevascular insult will profoundly inhibit NO synthesis. It alsois associated with proinflammatory and proatheroscleroticchanges with the endothelium (59).
Beta Blockers
Beta-adrenoceptor blocking agents (beta blockers) are es-tablished therapeutic options for treating patients with
775
hypertension, heart disease, arrhythmias, and glaucoma.This class of medication is associated with worsening erec-tile function (60). Benefits of taking this medication, in pa-tients with heart disease, far outweigh such side effects asED, making their continued use nonnegotiable. Alterna-tively, there is now a ‘‘third-generation’’ beta-blocker classthat has vasodilating properties, minimal side effects, andexpanded benefits such as decreased LDL levels and im-proved HDL cholesterol levels. Of interest, this class ofbeta blockers is also associated with reduction in insulin re-sistance (61). Mechanisms underlying the vasodilating ac-tion, responsible for the beneficial therapeutic effects ofthe third-generation beta blockers, are believed to be sec-ondary to release of NO, antioxidant action, b2-adrenocep-tor activation, and calcium entry blockade (61). Switchingpatients to third-generation beta blockers should improveboth insulin resistance and erectile function while main-taining the cardioprotective benefits (62).
IMPLICATIONS FOR TREATMENT
More than half of men over age 40 experience some degree ofED. Unfortunately, only a small percentage of affected menseek diagnosis and treatment. The emphasis of treatmenthas moved away from invasive monitoring to focus moreon outcome-based therapies. Questionnaires, such as the In-ternational Index of Erectile Function (IIEF) (63) and theSexual Encounter Profile (SEP) (64), allow for the earlieridentification of men at risk for ED so that treatments canstart earlier. Non-medicinal recommendations include [1] in-corporating a sexual history into the normal medical work-upfor every patient; [2] reducing risk factors for ED such assmoking, alcohol consumption, and a sedentary lifestyle;[3] aggressively managing comorbid disease states such ashypertension, diabetes, depression, hyperlipidemia, coronaryartery disease, and peripheral vascular disease; and [4] con-sidering changing to medications that have less impact onsexual function (65).
Thereafter, treating ED involves a three-tiered algorithmproposed by the Process of Care Consensus Panel (Table 5).Insulin-sensitizing therapy currently has no role in treatmentschemes for ED. First-line treatment options for ED includeoral agents (using PDE-5 inhibitors), vacuum-constriction de-vices, and psychosexual therapy. Fortunately, up to 70% ofmen using oral agents will be adequately treated. Thosewho do not benefit will need to consider second-line therapiessuch as penile injections, intraurethral prostaglandin (MUSE), orpossibly a combination of the above. Finally, third-lineoptions include several surgical interventions (66).
Effect on Erectile Dysfunction of Improving InsulinSensitivity
A variety of methods to improve insulin sensitivity havebeen adapted for diabetes prevention studies, including in-creased physical activity, dietary changes, and pharmaceuticinterventions (67, 68). Most current recommended treat-
776 Trussell and Legro Insulin resistance and erectile d
ments for ED limit its progression, but only exercise hasbeen proven to reverse it. Cross-sectional studies have sug-gested links between both obesity (69) and a sedentary life-style (70). Regular exercise increases eNOS proteinexpression (71) and has been shown to have a strong inverseassociation with the risk of ED, especially among obesemen (72).
To date, no randomized trials have been performed of insu-lin sensitizers in patients with ED, although theoretically theymay benefit. Patients with both insulin resistance and diabe-tes have impaired vasodilation and a corresponding decline inactivation of eNOS. In vitro and in vivo studies have shownthat enhanced insulin responses will increase production ofNO, by both induced expression and activation of eNOS(19–21). Insulin sensitizers enhance insulin-receptor interac-tions and their subsequent effects (23). Through its receptor,insulin acts to promote eNOS-related NO release, along thePI3K-dependent signaling pathway. Insulin sensitizers,such as metformin, improve endothelial function in both hu-mans and animals (22) and may someday be a part of combi-nation treatment options for men with ED recalcitrant to oralagent therapies.
SUMMARY
Insulin-resistant states are characterized by both defectivevascular NOS and impaired insulin-induced vasodilation.Insulin acts via the PI3K system to increase NO productionby activating eNOS and, in addition, by enhancing eNOSexpression and release of vasodilatory NO. Future studiesshould attempt to exploit the relationship between insulinreceptors, the PI3K pathway, and NO–related vasodilation tosynergistically improve erectile function, particularly amongmen with insulin resistance.
TABLE 5Erectile dysfunction treatment algorithm.
First line� Oral therapy, such as PDE-5 inhibitors� Psychosexual therapy� Vacuum-constriction devices
Second line� Injection therapy� Intraurethral therapy� Combination therapy
Third line� Surgery
Source: The process of care model for evaluation andtreatment of erectile dysfunction. The Process ofCare Consensus Panel. Int J Impot Res 1999;11:59–74.
Trussell. Insulin resistance and erectile dysfunction. Fertil Steril 2007.
ysfunction Vol. 88, No. 4, October 2007
REFERENCES1. Laumann EO, Paik A, Rosen RC. Sexual dysfunction in the United
States: prevalence and predictors. JAMA 1999;281:537–44.
2. Litwin MS, Nied RJ, Dhanani N. Health related quality of life in men
with erectile dysfunction. J Gen Intern Med 1998;13:159–66.
3. Gonzolez-Cadavid NF, Rajfer J. Molecular pathophysiology and gene
therapy of aging-related erectile dysfunction. Exp Gerontol 2004;39:
1705–12.
4. Esposito K, Giugliano F, Martedi E, Giovanni F, Marfella R,
D’Armiento M, et al. High proportions of erectile dysfunction in men
with the metabolic syndrome. Diabetes Care 2005;28:1201–3.
5. Blumentals WA, Gomez-Caminero, Joo S, Vannappagari V. Should erec-
tile dysfunction be considered as a marker for acute myocardial infarc-
tion? Int J Impot Res 2004;16:350–3.
6. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome
among US adults: finding from the third National Health and Nutrition
Examination Survey. JAMA 2002;287:356–9.
7. Makhsida N, Shah J, Yan G, Fisch H, Shabsigh R. Hypogonadism and
metabolic syndrome: implications for testosterone therapy. J Urol
2005;174:827–34.
8. Ford ES, Giles WH, Dietz WH. Prevalence of the metabolic syndrome
among US adults: findings from the third national health and nutrition
examination survey. JAMA 2002;187:356–9.
9. Wheatcroft SB, Williams IL, Shah AM, Kearney MT. Pathophysiologi-
cal implications of insulin resistance on vascular endothelial function.
Diabetes 2003;20:255–68.
10. Petrie JR, Shinichiro U, Webb DJ, Elliott HL, Connell JM. Endothelial
nitric oxide production and insulin sensitivity: a physiological link
with implications for pathogenesis of cardiovascular disease. Circulation
Nankin HR, et al, for the American Association of Clinical Endocrinol-
ogists Male Sexual Dysfunction Task Force. American Association of
Clinical Endocrinologists medical guidelines for clinical practice for
the evaluation and treatment of male sexual dysfunction: a couple’s
problem—2003 update. Endocr Pract 2003;9:77–95.
66. The process of care model for evaluation and treatment of erectile dys-
function. The Process of Care Consensus Panel. Int J Impot Res 1999;
11:59–74.
67. Knowler WC, Barrett-Connor E, Fowler SE, Hamman RF,
Ladim JM, Walker EA, et al. Reduction in the incidence of type 2
diabetes with lifestyle intervention or metformin. N Engl J Med
2002;346:393–403.
68. Lindstrom J, Louheranta A, Mannelin M, Rastas M, Salminen V,
Eriksson J, et al. The Finnish Diabetes Prevention Study (DPS): lifestyle
intervention and 3-year results on diet and physical activity. Diabetes
Care 2003;26:3230–6.
69. Giuliano FA, Leriche A, Jaudinot EO, de Gendre AS. Prevalence of erec-
tile dysfunction among 7689 patients with diabetes or hypertension, or
both. Urology 2004;64:1196–201.
70. Ferrannini E, Hafner SM, Mitchell BD, Stern MP. Hyperinsulinemia: the
key feature of a cardiovascular and metabolic syndrome. Diabetologia
1991;34:416–8.
71. Kaiser DR, Billups K, Mason C, Wetterling R, Lundberg JL, Bank AJ.
Impaired brachial artery endothelium-dependent and -independent vaso-
dilation in men with erectile dysfunction and no other clinical cardiovas-
cular disease. J Am Coll Cardiol 2004;43:179–84.
72. Bacon CG, Murray AM, Kawachi I, Giovannucci E, Glasser DB,
Rimm EB. A prospective study of risk factors for erectile dysfunction.
J Urol 2006;176:217–21.
sfunction Vol. 88, No. 4, October 2007
EDITOR’S CORNER
Limiting access to letrozole—is it justified?Togas Tulandi, M.D., M.H.C.M.,a and Alan H. DeCherney, M.D.b
a Department of Obstetrics and Gynecology, McGill University, Montreal, Quebec, Canada; and b Reproductive Biology and
Medicine Branch, National Institute of Health and Development, Bethesda, Maryland
Letrozole is an effective drug for ovulation induction and superovulation. Limiting its access is a disservice to ourinfertile patients and to the advancement in ovulation-related research. (Fertil Steril� 2007;88:779–80. �2007 byAmerican Society for Reproductive Medicine.)
Well, it’s all over. At the 2005 annual meeting of the Ameri-can Society for Reproductive Medicine, an abstract presenta-tion examined a relatively small number of letrozolepregnancies compared with a large control group of sponta-neous conceptions (1). The presenter suggested that the useof letrozole for infertility treatment might be associatedwith a higher risk of congenital cardiac malformation in new-borns.
We are left with mixed feelings. Did we miss anything? Wehave been studying letrozole for ovulation stimulation fora few years, and we have not seen the side effects describedin that presentation (1). Instead, we have found that letrozoleis an effective oral ovulation-inducing agent with minimalside effects (2). Letrozole has been associated with goodpregnancy rates, and it can be used alone or in conjunctionwith gonadotropins.
Bad news travels fast. The results in the presentation re-ceived a lot of attention from medical professionals, theindustry, and the media. On November 17, 2005, the manufac-turer, Novartis Pharmaceutical, issued a statement to physi-cians in Canada, the United States, and countries worldwideadvising that letrozole use in premenopausal women—andspecifically its use for ovulation induction—is contraindi-cated (3). As a result, many physicians have stopped usingit for infertile women, and investigators have canceled re-search programs related to letrozole and infertility.
Not so fast. We then examined their study carefully. The in-vestigators reported the outcome of 170 infants, of which 20were lost to follow-up evaluation. As a result, 150 babiesfrom 130 pregnancies were compared with a control group
Received November 15, 2006; revised and accepted January 22, 2007.
Reprint requests: Togas Tulandi, M.D., M.H.C.M., Department of Obstet-
rics and Gynecology, McGill University, Women’s Pavilion, 687 Pine Av-
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2007.01.115 Copyright ª2007 American
of over 36,000 infants born to low-risk pregnant women ina community hospital. The control population was youngerthan the letrozole group (1).
The investigators reported that the incidence of cardiacanomalies was higher in the letrozole group than in the con-trol group. The cardiac anomalies comprised two cases ofaortic stenosis among the 150 babies, which the authors cal-culated to be statistically higher than the rate of cardiacanomalies in the 36,050 babies born from the low-risk preg-nancies. However, they did not provide the number of babieswith cardiac anomalies in their control group. If the incidenceof congenital cardiac malformation in the general populationis 5 per 1000 babies, we can assume that, in this study, 180babies had congenital heart malformation. Compared withthe incidence in the letrozole group, this is not statisticallydifferent (95% CI, 0.3–10.0).
In addition, their control group was composed predomi-nantly of spontaneous conceptions, which are known to be as-sociated with a lower risk of pregnancy complications andcongenital malformations than pregnancies in the infertilitypopulation. Cardiac and possibly skeletal abnormalities arelikely to be diagnosed prepartum by ultrasound and trans-ferred to a tertiary care hospital for delivery; thus, these ab-normalities could be underrepresented among the 36,000deliveries from one low-risk hospital, making the calculationof relative risk for the letrozole group impossible.
Biological plausibility. The half-life of letrozole, approxi-mately 30 to 60 hours, is shorter than that of clomiphene cit-rate (5 to 7 days). Letrozole is eliminated as an inactivecarbinol metabolite mainly via the kidneys. Letrozole shouldbe cleared from the body completely by the time of embryoimplantation, which means that exposure to the drug predatesthe critical fetal development period. This casts doubt on thebiological plausibility of teratogenicity in the use of the drugfor ovulation induction. For a drug to have a teratogenic ef-fect, it must be present at the time of organogenesis (4).
Fertility and Sterility� Vol. 88, No. 4, October 2007 779Society for Reproductive Medicine, Published by Elsevier Inc.
There is light at the end of the tunnel. Tulandi et al. (5) sub-sequently evaluated the incidence and type of congenital mal-formation among 911 newborns from mothers who hadconceived with letrozole compared with a control group ofinfertile women who had conceived with clomiphene citrate.Their study, which was published in Fertility and Sterility,demonstrated no difference in the overall rates of malforma-tions or chromosomal abnormalities among the newbornsfrom mothers who had conceived after letrozole or after clo-miphene citrate treatments. Congenital cardiac anomalies intheir study were statistically significantly less frequent in theletrozole group than in the clomiphene citrate group. Basedon their data, the concern that letrozole use for ovulation in-duction could be teratogenic is unfounded.
Despite these recent results, the manufacturer and the reg-ulatory agencies have never issued a further statement on thesubject. Ironically, they have paid more attention to a nega-tive, sensational 10-minute presentation than to a publicationin a peer-reviewed journal. Although it is understandable thata statement favoring the use of the drug for infertile womenwould have considerable medico-legal implications, theyalso might want to wait for results from a larger study.Such a study would require approximately 3500 newbornsfrom mothers who conceived with and without letrozole—and in the case of clomiphene acetate, to the best of ourknowledge, such a study was never conducted.
780 Tulandi and DeCherney Letrozole as an ovulation-indu
Let’s keep an open mind. Letrozole is arguably an effectivedrug for ovulation induction and superovulation. Severalstudies have demonstrated that letrozole is as effective as go-nadotropin (6) but is easy to use and less costly. Limiting ac-cess to this substance is a disservice to our infertile patientsand to the advancement in ovulation-related research. Atthe very least, the use of letrozole in a research context orin a registry should not be restricted. This would ensuregood, thorough monitoring.
REFERENCES1. Biljan MM, Hemmings R, Brassard N. The outcome of 150 babies follow-
ing the treatment with letrozole or letrozole and gonadotropins [abstract].
Substandard application of preimplantation geneticscreening may interfere with its clinical successSantiago Munn�e, Ph.D.,a Luca Gianaroli, M.D.,b Ilan Tur-Kaspa, M.D.,c Cristina Magli, Ph.D.,b
a Reprogenetics, Livingston, New Jersey; b S.I.S.Me.R., Bologna, Italy; c Reproductive Genetics Institute, Chicago, Illinois;d Reprogenetics-Spain, Barcelona, Spain; e New York University, New York, New York; f Monash IVF, Clayton, Victoria,
Australia; g Istanbul Memorial Hospital, Istanbul, Turkey; and h Florida International University College of Medicine, Miami,
Florida
The intent of this study was to evaluate a recent randomized clinical trial evaluating the effect of preimplantationgenetic screening (PGS) that reports a negative effect on pregnancy outcome. This article reviews appropriate PGStechniques and how they differ from the trial in question. A closer look at the clinical trial in question reveals sig-nificant lack of expertise in biopsy, cell fixation, genetic analysis, and patient selection. At most, this trial demon-strates that in inexperienced hands, PGS can be detrimental. No other conclusions concerning the effect of PGSon pregnancy results can be drawn from the trial. (Fertil Steril� 2007;88:781–4. �2007 by American Societyfor Reproductive Medicine.)
Pregnancy rates after IVF steadily decline with maternal age,and pregnancy loss increases. It is widely accepted that re-duced fertility with age is mostly egg related and is notcaused by reduced uterine receptivity (1). Chromosome ab-normalities arising from spindle disorders in aging eggs areone of the major reasons for the reduced egg quality. Evenin young patients after follicular stimulation, close to 50%of preimplantation embryos are abnormal; the figure in-creases to nearly 80% for patients R40 years of age (2, 3).It should be possible to mitigate this high incidence ofchromosome abnormalities by aneuploidy testing throughpreimplantation genetic diagnosis, often described as preim-plantation genetic screening (PGS) and hereafter referred toas PGS. This consists of biopsying usually one cell fromeach IVF-generated embryo and testing this biopsied cellwith molecular techniques or fluorescence in situ hybridiza-tion before embryo transfer to the patient.
Given this unassailable rationale, PGS was expected to im-prove IVF outcome by selecting for transfer only embryosthat are chromosomally normal (4). To date, >20,000 PGSprocedures have been performed worldwide for that purpose(personal communication) (5). However, controversy still ex-ists regardin the procedure’s efficacy. The likely reason for
Received July 29, 2007; revised and accepted August 3, 2007.
S.M. is Director of Reprogenetics, a laboratory specializing in preimplan-
tation genetic diagnosis. I.T.-K. has received research funding from Se-
rono and is on the speaker’s bureau for Organon. M.S. has a financial
interest in Reprogenetics. Y.V. is owner and chief executive officer of
Reproductive Genetics Institute, which operates clinics that provide
preimplantation genetic diagnosis. J.L.S. is a member of the medical
advisory board for Biocept and is a member of the scientific advisory
board for IMETRIKUS.
Reprint requests: Santiago Munn�e, Ph.D., Reprogenetics, 3 Regent
Street, Suite 301, Livingston, New Jersey 07039 (FAX: 973-992-1423;
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2007.08.002 Copyright ª2007 American
this is that realizing a benefit from PGS requires optimalapplication. Low error rate and minimal embryo damageare absolutely critical to the success of the procedure.
Without a low error rate and without expert biopsy, benefitsare not necessarily realized, and the effort could even becounterproductive. Thus, PGS is no different than any surgi-cal procedure.
Initial and subsequent comparative studies from certainlarge centers showed that PGS increased implantation rates,decreased pregnancy loss, and reduced trisomic conceptions(6–11). However, whereas safety has been demonstrated inexperienced hands, none of these studies were randomized,nor did they have sufficient case numbers to detect a signifi-cant increase in live-birth rates. Attempts to entice patientsinto randomized clinical trials (RCTs) in the United States(and many other countries) proved difficult because of thehigh cost associated and the self-pay nature of IVF. Thelack of federal funds for human embryo research has hin-dered all sound applications of preimplantation geneticdiagnosis and PGS in the United States.
Several randomized, controlled trials (RCTs) recently havebeen performed in countries in which IVF costs are coveredby national health services. One well-cited study from arespected center demonstrated no difference in outcome be-tween control and PGS-treated cases (12). In that study,two cells were biopsied per embryo, a procedure that isavoided by most other experienced centers because of itspossible negative impact on the embryos. Nevertheless,even with two-cell biopsies, the mean implantation rate ofthe PGS embryos was 17.1%, compared with 11.5% of thenon-PGS group. These results demonstrated again that inexperienced hands, biopsy for preimplantation genetic diag-nosis is safe for embryos’ viability. Another well-designed,
Fertility and Sterility� Vol. 88, No. 4, October 2007 781Society for Reproductive Medicine, Published by Elsevier Inc.
randomized study recently was published by Mastenbroeket al. (13). Their sample size was large enough to determineongoing pregnancy rate changes, and only one cell per em-bryo was removed. The results appear to indicate that PGSfor advanced maternal age in those investigators’ handswas not helpful and actually was detrimental to pregnancyoutcome. Thus, results of two RCTs, especially Mastenbroeket al. (13), are at odds with those of comparative studies.This contribution explores potential explanations for thesediscrepancies.
BIOPSY TECHNIQUES
Removal of a blastomere is not logically salutary to embryodevelopment. Even if performed impeccably, there are fewercells at a stage when numbers of cells must increase. In addi-tion, the means used to breach the zona pellucida could bedeleterious to the embryo, and the environmental changes oc-curring while the embryo is being micromanipulated outsidethe incubator also can pose a risk. Thus, an inevitable effectof the biopsy must be compensated for by improvement asa result of better selection, that is, by the transfer of a euploidembryo after PGS.
Studies assessing the effects of biopsy and its repercussionon the implantation potential of the embryo have not been at-tempted without including PGS selection. The true effect ofbiopsy therefore is still unknown. However, in experiencedcenters, polar body, blastomere, or a combination of polarbody and biopsy does not affect pregnancy rates negatively(14–16). In addition, indirect evidence concerning the effectof biopsy on embryo viability was obtained in studies assess-ing the loss of cells from 5- to 10-cell embryos after freezingand thawing. With loss of two cells, the implantation poten-tial of the embryo decreased by >50%; the loss of one celldecreased its potential by 10% (17). In a similar study, theloss of implantation potential proved proportional to thenumber of cells lost (18); a single cell loss decreased implan-tation rate by approximately 10%, and that of two, by 20%.The impact of embryo biopsy after PGS can be modeled onthe cryopreservation data, because the condition is similarto the case of removal of cells for PGS. The loss of develop-ment potential helps explain the well-known decrease in suc-cess rate after transfer of previously cryopreserved embryos.What is unknown is the additional effect of the zona-breach-ing methods on the implantation potential of the embryo, andthe extent to which differences in technique and experienceplay roles. These effects may limit the potential for develop-ment further but also may improve development.
It follows that in PGS, pregnancy rates after removal ofa single blastomere must be improved by some measure ofimplantation potential to overcome the initial loss of thecell and to return to baseline (unbiopsied embryos). Theremust then be additional benefit if the procedure is to bejustified. Thus, biopsy must be done with consummate skill.Lacking this, PGS should not be performed. If more than onecell is removed, it becomes very difficult to show a benefit of
782 Munn�e et al. Substandard PGS interferes with clinica
PGS; an increase over baseline of R30% would be needed. If
a center’s optimal rate is lower, a positive result for PGS be-
comes nearly impossible. Thus, Staessen et al. (12) may ac-
tually have shown a scientifically beneficial effect of PGS,
because the estimated damage caused by two-cell biopsy
(>50%) was similar to the potential of selecting against ab-
normal embryos by using PGS (>50%). That is, the preg-
nancy rate in the biopsied group would have been much
lower if it were not compensated for by the benefit of embryo
selection.
Even if only one blastomere is removed, difficulties with
embryo biopsy represent an explanation for why Masten-
broek at al. (13) failed to show a benefit of PGS in their hands.
Experienced programs with positive outcomes after PGS
for any indication have several characteristics in common.
Biopsies are done rapidly (<2 min per embryo) by experi-
enced embryologists, the method for breaching the zona
(acid, laser, mechanical) does not significantly change the
pH or the temperature in the vicinity of the opening, and
very few embryos or cells are damaged during biopsy. Other
important factors include the use of Ca- and Mg-free media
containing amino acids to prevent culture shocks and the
use of sucrose to diminish cell volume. In experienced labo-
ratories in which large numbers of PGS cycles have been
performed, low rates of failed analyses (no results in <5%)
have been encountered (7, 8).
By contrast, the frequency of biopsied embryos without re-
sults in Mastenbroek et al. (13) was 20%. Moreover, the im-
plantation rate in their study was only 6% in cycles in which
biopsy was performed, diagnosis failed, and so-called unde-
termined (no-result) embryos were replaced. The implanta-
tion rate in a control group of patients who did not have
PGS was 14.7%. Simple calculation reveals that in the Mas-
tenbroek et al. (13) experience, there was an estimated 59%
reduction in implantation potential as a result of the biopsy
procedure. When the biopsied embryos with normal results
were replaced, the implantation rate was 16.8% (again, com-
pared with 14.7% in the control group). Overall, it is hardly
surprising that with a 59% reduction in implantation poten-
tial, the results of this RCT were not salutary. Had there
been only the expected (10%) proportional reduction of
implantation potential and had only euploid embryos been
replaced, the result might have been positive, with a larger
sample size.
One can only speculate what problems led to the untoward
losses during biopsy and/or transfer procedures. One clue lies
in table 4 of Mastenbroek et al. (13), in which it is alluded that
laser malfunction and other logistic problems occurred. It iswell known that slight misalignment of lasers will still allowholes to be made in the zona pellucida, yet this problem willseriously compromise embryo survival (19). A 2.8%frequency of biopsy failure is also mentioned, which is quiteunusual in experienced centers.
l success Vol. 88, No. 4, October 2007
GENETIC ANALYSIS
Once the cell is biopsied, fixation is a critical step to obtain-ing good genetic analysis and to minimizing cases in whichthere are no results or are errors. As noted frequently in theliterature, some fixation methods are better than others(20), and failure to use the best method could also render di-agnosis less accurate. For instance, if fixative is added afterthe cytoplasm already has been breached, DNA fibers and mi-cronuclei can become lost, producing false-positive results.Once a nucleus has been appropriately fixed, several techni-cal requirements regarding PGS analysis should be taken intoaccount. First, key chromosomes that disproportionately im-pact the rate of aneuploidy in cleavage-stage embryos andspontaneous abortions (chromosomes 22, 16, 21, and 15,respectively) should be included (21, 22). These four,combined with other chromosomes at risk for producingliveborn trisomies (chromosomes XY, 13, and 18), are thestandard cocktail of probes that is used in most PGSlaboratories. To this end, the Mastenbroek et al. (13) studyfailed to use DNA probes for chromosomes 15 and 22, whichalone account for >10% of abnormalities in cleavage-stagehuman embryos. This further reduces the selection potentialof the technique.
Personnel analyzing the cell slides need to be highly expe-rienced. The proper way to validate the prowess of a PGS labis through their published error rate, which is calculated byreanalyzing abnormal embryos that were not replaced andthen comparing this and the PGS result. Error rates rangefrom as low as 4% (3) to as high as 50% (23). The higherthe error rate, the less selection potential. A 50% error rateis akin to selecting embryos at random. The error rate forany important study should thus be known. Unfortunately,this was not reported by Mastenbroek et al. (13). The authorsappear to have no prior publications on the topic, and hence,accuracy and quality-control measures of their laboratoriesare unclear.
Other standard procedures involve the use of two observersto analyze results. This minimizes error rate and number ofcells with no results. Using this approach, Colls et al. (11),showed a significant improvement in pregnancy outcome.
PATIENT SELECTION
Preimplantation genetic screening for infertility is merelya selection tool. The underlying principle is that the smallerthe pool of embryos available, the less improvement in resultscan be expected. It follows that there must exist a certain min-imum number of embryos for biopsy and potential replace-ment to allow detection of an increase in live birth rateafter PGS. This number is generally taken to be six or eight,given that 50%–80% will be abnormal (8). In Mastenbroeket al. (13), the average number of embryos biopsied was4.8; thus, many patients must have had only 2 or 3 embryosbiopsied. Even if biopsy and diagnosis had been done opti-mally, there would have been little beneficial effect on preg-nancy rates, although a decreased risk of miscarriage andtrisomic conceptions should have resulted.
Fertility and Sterility�
RESPONSIBILITIES OF THE PGS COMMUNITY
Notwithstanding our concern over the technical performanceor technique used in certain RCTs, the authors’ ability to con-duct an RCT needs to be praised. We realize that the onus ison established centers and experienced PGS laboratories toconduct randomized studies to provide sound data on theefficacy of PGS for infertility. The PGS community, in col-laboration with the American Society for Reproductive Med-icine, the Society for Assisted Reproductive Technology, andthe Genetics and Public Policy Center, is proceeding with de-velopment of a North American PGS registry to provide forlongitudinal data on accuracy and safety. Restrictive US gov-ernmental policies on embryo research have precluded a ma-jor, funded RCT in the United States. We are hopeful that thiswill change, but regardless, we will redouble efforts to con-duct RCTs with private funds. In the meantime, it is crucialthat centers offering PGS demonstrate experience and qualitycontrol in biopsy and genetic testing. Training courses inbiopsy are provided through the Preimplantation GeneticDiagnosis International Society and the European Societyof Human Reproduction and Embryology.
In summary, conclusions, even of RCTs, concerning effi-cacy of PGS for aneuploidy in women of advanced maternalage should not necessarily lead to the conclusion that the pro-cedure is without benefit. Such a conclusion is appropriateonly if all biologic and technical requisites are met. It is crit-ical to properly perform the biopsy procedure, to use appro-priate chromosome probes, and to identify the patients thatmay benefit from it. Published RCTs do not appear to havemet these requirements.
REFERENCES1. Abdalla HI, Wren ME, Thomas A, Korea L. Age of the uterus does not
affect pregnancy or implantation rates; a study of egg donation in women
of different ages sharing oocytes from the same donor. Hum Reprod
1997;12:827–9.
2. Munn�e S, Serena C, Colls P, Garrisi J, Zheng X, Cekleniak N, et al. Ma-
ternal age, morphology, development and chromosome abnormalities in
over 6000 cleavage-stage embryos. Reprod Biomed Online 2007;14:
628–34.
3. Magli MC, Gianaroli L, Ferrareti AP, Lappi M, Ruberti A, Farfalli V.
Embryo morphology and development are dependent on the chromo-
some complement. Fertil Steril 2007;87:534–41.
4. Munn�e S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major
chromosome aneuploidies in human preimplantation embryos.
The combination of polar body and embryo biopsy does not affect
embryo viability. Hum Reprod 2004;19:1163–9.
17. Cohen J, Wells D, Munn�e S. Removal of two cells from cleavage stage
embryos is likely to reduce the efficacy of chromosomal tests employed
to enhance implantation rates. Fertil Steril 2007;87:496–503.
18. Edgar DH, Bourne H, Jericho H, McBain JC. The developmental poten-
tial of cryopreserved human embryos. Mol Cell Endocrinol 2000;27;
169:69–72.
19. Li L, Munn�e S, Licciardi F, Noev Y, Tadik Y, Godke R, et al. Effect of UV
laser on the development of mouse embryos assessed by microinjection
of FITC-Dextran. Zygote 1993;1:43–8.
20. Velilla E, Escudero T, Munne S. Blastomere fixation techniques and risk
of misdiagnosis for preimplantation genetic diagnosis of aneuploidy.
Reprod Biomed Online 2002;4:210–7.
21. Munn�e S, Bahcxe M, Sandalinas M, Escudero T, M�arquez C, Velilla E,
et al. Differences in chromosome susceptibility to aneuploidy and sur-
vival to first trimester. Reprod Biomed Online 2004;8:81–90.
22. Simpson JL. Incidence and timing of pregnancy losses: relevance to eval-
uating safety of early prenatal diagnosis. Am J Med Genet 1990;35:
165–73.
23. Baart E, Van Opstal D, Los FJ, Fauser BCJM, Martini E. Fluorescence in
situ hybridization analysis of two blastomeres from day 3 frozen-thawed
embryos followed by analysis of the remaining embryo on day 5.
Hum Reprod 2004;19:685–93.
l success Vol. 88, No. 4, October 2007
ENDOMETRIOSIS
Estrogen receptor b gene D1730 G/A polymorphismin women with endometriosisGyoung Hoon Lee, M.D.,a Sung Hoon Kim, M.D.,c Young Min Choi, M.D.,a,b Chang Suk Suh, M.D.,a,b
Jung Gu Kim, M.D.,a and Shin Yong Moon, M.D.a,b
a Department of Obstetrics and Gynecology, and b The Institute of Reproductive Medicine and Population, Medical Research
Center, Seoul National University College of Medicine, Seoul; and c Department of Obstetrics and Gynecology, College of
Medicine, University of Ulsan, Asan Medical Center, Seoul, Korea
Objective: To investigate whether the þ1730 G/A polymorphism of the estrogen receptor b (ER-b) gene is asso-ciated with the risk of endometriosis in a Korean population.Design: Case–control study.Setting: University Department of Obstetrics and Gynecology.Patient(s): Women with (n ¼ 239) or without (n ¼ 287) endometriosis.Intervention(s): The þ1730 G/A polymorphism of 30-UTR of exon 8 in the ER-b gene was assessed by polymer-ase chain reaction-restriction fragment-length polymorphism analysis utilizing digestion with AluI restrictionenzyme.Main Outcome Measure(s): Genotype distribution and allele frequency of the þ1730 G/A polymorphism in theER-b gene.Results: The genotype distribution of the þ1730 G/A polymorphism in the ER-b gene was not different betweenthe endometriosis patients and the controls (G/G of 74.9% vs. 72.5%, G/A of 25.1% vs. 26.1%, and A/A of 0.0% vs.1.4%, respectively). There was also no difference in the G and A allele frequencies between the two groups (87.4%vs. 85.5%, and 12.6% vs. 14.5%, respectively). Even when the endometriosis cases were subdivided into AmericanSociety for Reproductive Medicine stage I–II, III, IV, and III–IV, no differences were found at all in the genotypedistribution or allele frequencies between the two groups.Conclusion(s): Our results suggest that the þ1730 G/A polymorphism of the ER-b gene may not be associatedwith the risk of endometriosis in the Korean population, which was not the case in the Japanese population. (FertilSteril� 2007;88:785–8. �2007 by American Society for Reproductive Medicine.)
Key Words: Endometriosis, estrogen receptor b gene, þ1730 G/A polymorphism
Endometriosis (MIM 131200) is defined as the presence ofendometrial tissue outside the uterus, causing diverse clinicalmanifestations such as infertility, pelvic pain, and dysmenor-rhea. Although the mechanisms responsible for its pathogen-esis and progression remain poorly understood, it is wellestablished that endometriosis grows and regresses in an es-trogen-dependent fashion. Utilizing various laboratorymethods, several investigators have demonstrated that ec-topic endometriotic implants contain estrogen receptors(ERs) (1–5). In addition, these implants have been shownto express aromatase cytochrome P450, suggesting that localestrogen production can stimulate the growth of endometri-otic lesions (6, 7).
Received July 22, 2006; revised and accepted December 5, 2006.
Supported by grant (01-PJ10-PG6-01GN13-0002) from the Korea Health
21 R&D Project, Ministry of Health & Welfare, Republic of Korea.
Reprint requests: Young Min Choi, M.D., Department of Obstetrics and
Gynecology, The Institute of Reproductive Medicine and Population,
Medical Research Center, Seoul National University College of Medi-
cine, 28 Yungun-dong, Chongno-ku, Seoul 110-744, Korea (FAX:
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.032 Copyright ª2007 American S
Endometriosis shows heritable tendencies, with five- toeightfold increased recurrence risks for first-degree relatives,indicating that polygenic and multifactorial etiologies are farmore likely to be the cause than Mendelian inheritance (8).Based upon the expression of ERs in the endometriotic le-sions and the marked familial tendencies, several associationstudies have shown that a number of ER-a gene polymor-phisms may be associated with susceptibility to endometri-osis. Especially the PP genotype in ER-a, assessed byPvuII restriction fragment-length polymorphism assay, hasbeen reported to be less frequently observed in the endome-triosis patients compared with the controls in Greek and Jap-anese populations (9, 10). However, our study did not find anassociation of PvuII polymorphism with endometriosis, andour study showed that fewer thymine-adenine (TA) repeatpolymorphisms (12–15 repeats) in the ER-a gene was morefrequently found in the stage I–II of endometriosis than con-trol subjects in a Korean population (11). Despite such activeassociation studies in the ER-a gene, there has been only onestudy exploring the association between the ER-b gene poly-morphisms and the risk of endometriosis. Wang et al. (12)
Fertility and Sterility� Vol. 88, No. 4, October 2007 785ociety for Reproductive Medicine, Published by Elsevier Inc.
reported that the þ1730 G/A polymorphism in ER-b genewas associated with the risk of stage IV endometriosis ina Japanese population with no difference in the genotype dis-tribution and allele frequency of the another polymorphism inER-b using RsaI restriction enzyme.
Considering that inconsistent results have been reportedfor a few polymorphisms in the ER-a gene and that therehave been no further supporting evidence published forknown polymorphisms in the ER-b gene, it is necessary toclarify whether polymorphisms in the ER-b are associatedwith the risk of endometriosis in another ethnic group. Weperformed this study to evaluate whether a þ1730 G/A poly-morphism of the ER-b gene is associated with the risk of en-dometriosis in a Korean population, which is the closestethnic group to Japanese.
MATERIALS AND METHODS
Subjects
Peripheral blood was obtained from a total of 526 patients.All subjects were of Korean origin, which is made up ofa single ethnic trait. A total of 239 patients had surgicaland histologic evidence of endometriosis, while another287 patients without the disease served as controls. In theendometriosis group, the extent of disease was staged ac-cording to the American Society for Reproductive Medicine(13). None of the subjects had received hormone therapyduring the previous 12 months. Endometriosis status wasconfirmed during diagnostic laparoscopy, pelviscopic sur-gery, exploratory laparotomy, or transabdominal hysterec-tomy in both groups. Approval for this study was obtainedby the institutional review board at the Seoul National Uni-versity Hospital, and informed consent was obtained fromeach woman.
Genomic DNA Analysis
Genomic DNA was extracted from the peripheral blood withthe Wizard DNA purification kit (Promega, Madison, WI).Genotyping of ER-b þ1730 G/A polymorphism wasperformed using a polymerase chain reaction-restrictionfragment-length polymorphism analysis, as previously de-scribed (12). Briefly, genomic DNA was amplified usingthe following oligonucleotide primers: primers ERb-F, 50-TTTTTGTCCCCATAGTAACA-30 (upstream) and ERb-R,50-AATGAGGGACCACAGCA -30 (downstream) to amplify307 bp of exon 8. A 0.1-mg sample of genomic DNA wasadded to a PCR mixture containing 1.5 mM MgCl2, 0.2mM dNTPs, 0.4 mM each primer, and 1.25 U of Taq polymer-ase (Takara, Shiga, Japan). The PCR cycling conditions wereas follows: an initial denaturation step at 94�C for 5 minutes,amplification for 35 cycles at 94�C for 30 seconds, 52�C for60 seconds and 72�C for 60 seconds, followed by a final ex-tension step at 72�C for 10 minutes (MJ Research, Waltham,MA). The PCR products were digested using 2 IU of AluI(New England Biolabs, Beverly, MA) at 37�C overnightand separated by 2% agarose gel electrophoresis, and identi-fied using ethidium bromide staining.
786 Lee et al. Estrogen receptor b gene in endometriosis
An amplification product of 307 bp was obtained usingprimer ERb-F and ERb-R. The ERb-F þ ERb-R PCR prod-uct contained one recognition site for AluI. So wild-type ofER-b with two þ1730 G genotypes (GG) has no recognitionsite of AluI, yielding 307 bp and mutant type with twoþ1730A genotypes (AA) has a recognition site of AluI product inPCR product, yielding 240 bp and 67 bp and heterozygotewith one þ1730 G and one þ1730 A (GA) gives all frag-ments, 307 bp, 240 bp, and 67 bp.
Statistical Analysis
The ages of each group were normally distributed as tested byKolmogorov-Smirnov test, and thus were compared by Stu-dent’s t test. Genotype distributions were examined for sig-nificant departure from the Hardy-Weinberg equilibrium bya goodness-of-fit chi-square test with 1 df, and the chi-squaretest was used to examine difference in the proportions of ge-notypes between endometriosis cases and the controls. Whenthe assumption of the chi-square test was violated and>20%of the cells had an expected count of <5, a binomial test wasused. P<.05 was considered statistically significant.
RESULTS
Genotyping was successfully achieved in all subjects. Themean age (�SD) of all endometriosis patients and controlswas 33.8 � 6.7 and 41.6 � 10.7, respectively. The differencebetween cases and controls was statistically significant(P<.05).
Genotypic distribution of the þ1730 G/A SNP in the con-trol group was in a Hardy-Weinberg equilibrium.
The distribution of genotypes was not different betweenthe endometriosis group and the control group (GG/non-GG; 74.9%/25.1% and 72.5%/27.5%, respectively;P¼.531). There was also no difference in the G and A allelefrequencies between the two groups (87.4% vs. 85.5%, and12.6% vs. 14.5%, respectively). For further analysis, the en-dometriosis group was divided according to the AmericanSociety for Reproductive Medicine classification. We foundno difference in the frequency ofþ1730 G/A polymorphismsbetween each of the endometriosis subgroup and the controlgroup (Table 1). The minor allele, allele A, frequency ob-served for þ1730 G/A polymorphisms of ER b gene in thecontrol group was similar to those reported in other Asianor Asian-derived populations (12, 14).
DISCUSSION
Recent studies have demonstrated a possible association be-tween ER-b polymorphism and various diseases. Westberget al. (15) have shown that patients with an early age of onsetof Parkinson disease was more associated with the G allele ofthe þ1730 G/A polymorphism than those with a late age ofonset (P¼.006). Investigating the association of five SNPs lo-cated at introns of ER-b, Pirskanen et al. (16) reported thatvariations in the ER-b gene were linked with a risk of Alz-heimer disease in eastern Finland women, not in men.
Vol. 88, No. 4, October 2007
TAB
LE1
Ge
no
typ
ea
nd
alle
lefr
eq
ue
nc
ies
of
ER
-bD
1730
G/A
po
lym
orp
his
min
en
do
me
trio
sis
pa
tie
nts
an
dc
on
tro
ls.
Pa
tie
nt
No
.G
GN
on
GG
(GA
/AA
)P
va
lue
aT
ota
la
lle
leG
AP
va
lue
a
End
om
etr
iosis
Sta
ge
I-II
50
38
(76%
)12
(12/0
)(2
4%
).6
04
100
88
(88.0
%)
12
(12.0
%)
.514
Sta
ge
III-
IV189
141
(74.6
%)
48
(48/0
)(2
5.4
%)
.607
378
330
(87.3
%)
48
(12.7
%)
.440
III
35
24
(68.6
%)
11
(11/0
)(3
1.4
%)
.627
70
59
(84.3
%)
11
(15.7
%)
.779
IV154
117
(76.0
%)
37
(37/0
)(2
4.0
%)
.426
308
271
(88.0
%)
37
(12.0
%)
.312
Co
ntr
ol
287
208
(72.5
%)
79
(75/4
)(2
7.5
%)
574
491
(85.5
%)
83
(14.5
%)
aV
ers
us
co
ntr
ol.
Lee
.E
stro
gen
rece
pto
rb
gen
ein
endom
etri
osi
s.F
erti
lSte
ril
200
7.
Fertility and Sterility�
Aschim et al. (17) investigated the two polymorphisms—RsaI (þ1082 G/A) and AluI (þ1730 G/A)—and reportedthat the infertile men (sperm concentration below 5 � 106
spermatozoa/mL) were three times more frequently associ-ated with the þ1082 G/A polymorphism (P¼.01), whichwas linked with an about 20% decrease in LH concentrationcompared with wide-type GG genotype. Sundarrajan et al.(14) also investigated the ER-b þ1730 G/A and þ1082 G/Apolymorphisms in patients with ovulatory dysfunction and re-ported that mutant type of ER-bþ1082 G/A andþ1730 G/Apolymorphisms were found more frequently in the case groupwith idiopathic ovulatory dysfunction than normal controls.
We performed the present study in a Korean population toevaluate whether we can replicate the originally reported as-sociation of the ER-b þ1730 G/A polymorphism with therisk of endometriosis in a Japanese population, which is theclosest ethnic group to Korean. However, contrary to originalWang et al.’s report (12), the genotype distribution or theallele frequency of the þ1730 G/A polymorphism was notsignificantly different between women with and without en-dometriosis. This inconsistency between the two studiescould have resulted from the difference of ethnic makeup.Despite striking similarity, genetic background is still differ-ent between the two close ethnic groups, and the subtle ge-netic difference can make it possible that genetic factorsinvolved in the pathogenesis of endometriosis vary in thesepopulations. Therefore, the putative gene involved inthe pathogenesis of endometriosis may be distinct fromER-b þ1730 G/A locus in the Korean population, while itis closely linked in the Japanese population.
For association study, it appears logical to limit case defi-nition to more severe stages (18), and it is hard to accept thatthe genotype is different between patients with stage III andIV, because the difference between stage III and stage IV en-dometriosis seems to be score dependent and operator depen-dent. However, we also compared the genotype between thepatients with stage IV endometriosis and the control groupto ascertain whether there is any association between theER-bþ 1730 G/A and the most severe class of endometriosis,and found no significant difference at all. Salanti et al. (19)have put special emphasis on the sample size by suggestingthat researchers should design a genetic association studywith enough sample size to overcome the lack of power in ge-netic studies and to find a real odds ratio. Based on the find-ings that the genotype distribution of the control groups inWang et al.’s report was not different from ours, and thatour study had a nearly double sample size (154 vs. 78) in pa-tients with stage IV endometriosis with histologic evidence,our results are likely to be more reliable findings.
The mean age of the healthy female subjects in the presentstudy was higher than the endometriosis patients. However,this may play a favorable role in our result, as described byHadfield et al. (20). Recruiting women from the older controlgroup has a merit of maximizing the probability that theywere unaffected by endometriosis, that is, to avoid includingyounger women who might develop the disease in later life.
787
In conclusion, we have shown that the þ1730 G/A poly-morphism of ER-b is not associated with any stage of endo-metriosis in a Korean population, which was not the case inthe Japanese population. Further studies are necessary to elu-cidate the role of ER-b in the pathogenesis of endometriosis,as well as to confirm the possible association of the ER-b genepolymorphism with susceptibility to endometriosis in anotherethnic group.
REFERENCES1. Bergqvist A, Ferno M. Oestrogen and progesterone receptors in endo-
metriotic tissue and endometrium: comparison of different cycle phases
and ages. Hum Reprod 1993;8:2211–7.
2. Howell RJ, Dowsett M, Edmonds DK. Oestrogen and progesterone re-
ceptors in endometriosis: heterogeneity of different sites. Hum Reprod
1994;9:1752–8.
3. Jones RK, Bulmer JN, Searle RF. Immunohistochemical characterization
of proliferation, oestrogen receptor and progesterone receptor expression
in endometriosis: comparison of eutopic and ectopic endometrium with
normal cycling endometrium. Hum Reprod 1995;10:3272–9.
4. Rey JM, Pujol P, Dechaud H, Edouard E, Hedon B, Maudelonde T. Ex-
pression of oestrogen receptor-alpha splicing variants and oestrogen
receptor-beta in endometrium of infertile patients. Mol Hum Reprod
1998;4:641–7.
5. Fujishita A, Nakane PK, Koji T, Masuzaki H, Chavez RO, Yamabe T,
et al. Expression of estrogen and progesterone receptors in endometrium
and peritoneal endometriosis: an immunohistochemical and in situ hy-
Kennedy SH, OXEGENE Collaborative Group. Linkage and association
studies of the relationship between endometriosis and genes encoding the
detoxification enzymes GSTM1, GSTT1 and CYP1A1. Mol Hum Re-
prod 2001;7:1073–8.
Vol. 88, No. 4, October 2007
0d
Efficacy of vaginal danazol treatment in womenwith recurrent deeply infiltrating endometriosisSandro Razzi, M.D., Stefano Luisi, M.D., Francesco Calonaci, M.D., Aldo Altomare, M.D.,Caterina Bocchi, M.D., and Felice Petraglia, M.D.
Division of Obstetrics and Gynecology, Department of Pediatrics, Gynecology and Reproductive Medicine, University of Siena,
Siena, Italy
Objective: To describe a safe long-term medical treatment for deeply infiltrating endometriosis, a critical conditioncharacterized by multiple painful symptoms and a high recurrence rate after surgical treatment.Design: Prospective study.Setting: University of Siena.Patient(s): Twenty-one women with deeply infiltrating endometriosis.Intervention(s): In a nonrandomized prospective study a low dose of vaginal danazol (200 mg/d) was self-admin-istered for 12 months. After a previous laparoscopic surgery, these patients had reported recurrent severe dyspar-eunia, dysmenorrhea, and pelvic pain (in five cases also painful defecation).Main Outcome Measure(s): Before and every 3 months during the treatment a visual analogue pain scale was used.Transvaginal and transrectal ultrasound examinations were performed before and after 6 and 12 months of treat-ment. Adverse effects were registered, and serum concentration of cholesterol, triglycerides, aspartate aminotrans-ferase, alanine aminotransferase, glycemia, protein S, protein C, antithrombin III, and homocysteine was evaluatedbefore and after 12 months.Result(s): Dysmenorrhea, dyspareunia, and pelvic pain significantly decreased within 3 months and disappearedafter 6 months of treatment, with a persistent effect during the 12 months of treatment. A relief of painful defecationwas also shown. Ultrasound examination showed a reduction of the nodularity in the rectovaginal septum within 6months. The medical treatment did not affect metabolic or thrombophilic parameters; few local vaginal adverseeffects were reported.Conclusion(s): Vaginal danazol resulted in effective medical treatment for the various painful symptoms in womenwith recurrent deeply infiltrating endometriosis, and because of the lack of significant adverse effects it may beproposed as an alternative to repeated surgery. (Fertil Steril� 2007;88:789–94. �2007 by American Society forReproductive Medicine.)
Deeply infiltrating endometriosis is a particular form of en-dometriosis that penetrates >5 mm under the peritoneal sur-face, typically associated with marked proliferation ofsmooth muscle cells and fibrosis and strongly associatedwith pelvic pain (1). In particular, the deeply infiltrating en-dometriosis lesions are classified according to the invadedorgans: [1] bladder, with an infiltration of the muscularispropria; [2] uterosacral ligaments, when the uterosacral lig-aments are infiltrated; [3] vagina, when the deeply infiltrat-ing endometriosis invades the anterior rectovaginal pouch,the posterior vaginal fornix, and the retroperitoneal area inbetween (rectovaginal septum); and [4] intestine, with an in-filtration of the muscularis propria (2). Diagnosis of deeplyinfiltrating endometriosis is made by clinical signs, by trans-vaginal/transrectal ultrasound examination, and by biopsyduring surgery (3).
Received June 28, 2006; revised December 21, 2006; accepted Decem-
ber 28, 2006.
Reprint requests: Felice Petraglia, M.D., Division of Obstetrics and Gyne-
cology, University of Siena, Policlinico Le Scotte, 53100 Siena, Italy
015-0282/07/$32.00oi:10.1016/j.fertnstert.2006.12.077 Copyright ª2007 American
Dysmenorrhea, dyspareunia, and chronic pelvic pain arethe most frequent symptoms in women with deeply infiltrat-ing endometriosis. In several cases pain is mechanical and isprovoked by the mobilization of the organ affected by thedeeply infiltrating endometriosis lesions. Dyspareunia anddysmenorrhea are associated with involvement of the utero-sacral ligaments, pelvic pain, and painful defecation duringmenses with involvement of the rectovaginal septum, and/or of bowel and functional urinary tract signs when the blad-der and/or ureter are involved (4).
Deeply infiltrating endometriosis is a critical condition thatis improved by surgical treatments; both laparoscopic andlaparotomic consecutive operations are associated with painrelief even though associated with the risk of major compli-cations. However, the recurrence rate is very high in these pa-tients, and they want to postpone reoperation or do not acceptthe risk of additional morbidity or the results of surgery (hys-terectomy, bilateral oophorectomy, deafferentation ofnerves). The development of new drugs and alternative routesof administration is the object of several research efforts, aswell as the attempts to prolong the beneficial effects of theseagents (5).
Fertility and Sterility� Vol. 88, No. 4, October 2007 789Society for Reproductive Medicine, Published by Elsevier Inc.
In particular, vaginal or intrauterine methods of drug re-lease represent an appealing option. A danazol-loaded vagi-nal ring has been tested with encouraging results in patientswith deeply infiltrating endometriosis (6), and pain reliefhas been reported after use of a danazol-loaded intrauterinedevice in women with endometriosis (7). In addition, reducedside effects and proved safety suggest that the levonorgestrel-releasing intrauterine device may play an important role inlong-term treatment of pain associated with endometriosisof the rectovaginal septum in patients with no immediate de-sire for pregnancy (8). We prospectively assessed the efficacyof a vaginal danazol treatment in painful symptoms in womenwith recurrent deeply infiltrating endometriosis.
MATERIALS AND METHODS
The study was approved by the Institutional Review Board ofthe Academic Health Center of Siena, and informed consentwas obtained from each participant. Included in the studywere women (N ¼ 21) (median age 32.6 years; age range,28–37 years) regularly menstruating, not wanting pregnancy,with a diagnosis of deeply infiltrating endometriosis involvingthe uterosacral ligaments and the rectovaginal septum. Thebaseline clinical characteristics of the women enrolled inthe study are shown in Table 1 (9). The diagnosis had beenmade by biopsy during conservative laparoscopic surgery.The patients had been treated after surgery with various drugs(estrogenic-progestogenic, GnRH analogues, progestins) fordifferent period of times. They were referred to our center be-cause of pain recurrence (in all women dysmenorrhea, dyspar-eunia, and chronic pelvic pain were registered; five womenalso reported painful defecation). An involvement of the rec-tovaginal septum was confirmed by vaginal and rectal exam-ination and by transvaginal and transrectal ultrasonographicevaluation (MyLab70; Esaote SpA, Genoa, Italy). The sizeof the endometriotic lesions was calculated by using the for-mula for the volume of an ovoid (D1 � D2 � D3 � 0.5222).
Exclusion criteria were obstructive uropathy or bowel ste-nosis; evidence of complex adnexal cysts or an ovarian endo-metrioma of diameter R3 cm at vaginal ultrasonography;therapies for endometriosis other than nonsteroidal anti-in-flammatory drugs in the 3 months before study entry; theusual contraindications to estrogens and progestogens; a diag-nosis of concomitant pelvic inflammatory disease; or knowngastrointestinal, urologic, and orthopedic diseases.
Each patient was asked to complete a questionnaire on thepresence and severity of dysmenorrhea, nonmenstrual pelvicpain, and deep dyspareunia, graded by using a 0- to 3-pointmultidimensional categorical rating scale modified fromthat devised by Biberoglu and Behrman (10). This scale de-fines dysmenorrhea according to loss of work efficiencyand need for bed rest, nonmenstrual pain according to variousdegrees of discomfort and use of analgesics, and deep dyspar-eunia according to limitation of sexual activity. With respectto the original scheme, ratings of ‘‘pelvic tenderness’’ and‘‘induration’’ were excluded to avoid the physician subjectiv-ity inherent in an open-label trial (Table 2) (10).
790 Razzi et al. Vaginal danazol and endometriosis
Vaginal danazol (200 mg/d) was self-administered for 12months. Dysmenorrhea, dyspareunia, and chronic pelvicpain intensity were assessed before and every 3 months onthe first day of the menses for 12 months, with use of an in-terview comprising a 10-point visual analogue pain scale(11). A score of 6 or greater was considered indicative ofmoderate or severe pain, whereas a score of 4 or less indi-cated mild or negligible pain. When enrolled all patients eli-gible for the study had a score consistent with moderate orsevere pain. Transvaginal and transrectal ultrasound evalua-tion was done before and after 12 months of treatment. Serumconcentrations of cholesterol, triglycerides, aspartate amino-transferase, alanine aminotransferase, glycemia, protein S,protein C, antithrombin III, and homocysteine were evaluatedbefore and after 6 and 12 months. Menstrual cycle durationwas monitored, and patients were required to follow naturalcontraceptive methods. All other possible adverse effectswere monitored at each observation.
Statistical analysis included ANOVA for repeated mea-sures by post hoc test with significance as a two-tailed P<.05.
RESULTS
The entire group of patients completed the 12-month studyand remained regularly menstruating throughout the entireperiod of observation. Dysmenorrhea, dyspareunia, andpelvic pain significantly decreased after 3 months of treat-ment (P<.01), with a persistent effect until 12 months
TABLE 1Distribution of study patients accordingto age, parity, body mass index, andendometriosis stage at previous surgery.
Variable No. of patients %
Age (y)<30 9 43>30 12 57
Parity0 17 81R1 4 19
Body mass indexa
<21 3 1421–22 9 4323–24 7 33>25 2 10
Disease stageb
I 0 0II 0 0III 13 62IV 8 38
a Body mass index ¼ Weight (kg)/Height (m2).b According to the revised American Fertility Society
classification (9).
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
TABLE 2Verbal rating scale to score pain symptoms associated with rectovaginal endometriosis.
Symptom Description Degree Score
Dysmenorrheaa No discomfort Absent 0Some loss of work efficiency Mild 1In bed part of 1 day, occasional loss of work Moderate 2In bed for one or more days, incapacitation Severe 3
Pelvic paina No discomfort Absent 0Occasional pelvic discomfort Mild 1Noticeable discomfort for most of the cycle Moderate 2Pain persisting during the cycle or requiring
strong analgesicsSevere 3
Dyspareuniaa No discomfort Absent 0Tolerated discomfort Mild 1Intercourse painful to the point of
interruptionModerate 2
Intercourse avoided because of pain Severe 3
a Modified from Biberoglu and Behrman (10).
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
(P<.01) (Table 3 and Fig. 1). In the five cases of painfuldefecation the treatment was effective in reducing thesymptoms.
Transvaginal and transrectal ultrasonography showed adecrease of the mean � SD volume of rectovaginal plaquesfrom a baseline value of 3.1� 1.2 mL to 1.9� 1.2 mL within6 months (P<.01) (Fig. 2). A reduced volume (1.2� 0.8 mL)was confirmed at the end of the treatment (P<.05) (Fig. 3).
No significant change of serum metabolic and thrombo-philic parameters measured was registered after 12 months
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of treatment (Table 4). A vaginal irritation during the firstmonth of treatment in four cases was the major side effectbut did not cause treatment withdrawal.
DISCUSSION
Our results showed that the use of low-dose vaginal danazolis highly effective in the treatment of the painful symptomsassociated with recurrent deeply infiltrating endometriosis,reduces the size of endometriotic lesions, and has no systemicside effects.
TABLE 3Grading of pain symptoms based on the verbal rating scale in patients with rectovaginal endometriosisbefore and after 12 months of medical therapy.
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
791
The subjective assessment of pain intensity by a visual an-alogue pain scale provided an adequate assessment of dys-menorrhea, dyspareunia, and pelvic pain, associated with
FIGURE 1
Variant of intensity of pain symptoms as assessed ona visual analogue scale during the study period.
0
2
4
6
8
10
before 3 months 6 months 9 months 12 months
dysmenorrhea dyspareunia
pelvic pain
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
FIGURE 2
A transvaginal scan of a woman affectedby deep infiltrating endometriosis (expression ofa rectovaginal septum localization of endometriosis)before (A) and after 12 months of therapy (B).
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
792 Razzi et al. Vaginal danazol and endometriosis
an improved quality of life in these patients with deeply infil-trating endometriosis (11). The painful activity of endometri-otic lesions is related to the vascularity of the lesions, to theinflammation, or to the adhesions (12–14). Indeed, similarto tumor metastasis, endometriotic implants require neovas-cularization to establish, grow, and invade. The effect on or-ganic symptoms may be due not only to the volumetricreduction of rectovaginal plaques but also most probably toreduced intralesional and perilesional inflammation and to re-duced production of prostaglandins and cytokines, whichstimulate pain fibers. Major cytokines, growth factors, steroidhormones, and eicosanoids are responsible for angiogenesisand inflammation in endometriosis, and angiogenic factorsare increased in the peritoneal fluid of patients with endome-triosis, in the peritoneal endometric implants, and in ovarianendometriomas (15).
Other treatments are also effective in pain control inwomen with deeply infiltrating endometriosis. In fact, theintrauterine device releasing levonorgestrel decreases pelvicpain symptoms caused by peritoneal and rectovaginal endo-metriosis and reduces the risk of recurrence of dysmenorrheaafter conservative surgery, associated with local drug concen-trations greater than plasma levels and limited adverse effects(16).
Some oral progestogens (danazol, levonorgestrel, medrox-yprogesterone acetate) may be effective in the control of painsymptoms in women with pelvic endometriosis also becauseof their anti-inflammatory in vitro and in vivo effects, butwith adverse effects (17). Recently, low-dose norethindroneacetate treatment resulted in an effective, tolerable, and inex-pensive medical alternative to repeated surgery for treatingsymptomatic rectovaginal endometriotic lesions in patientswho did not seek conception (18).
The present ultrasound data confirmed the role of transva-ginal and transrectal sonography as a first-line imaging
FIGURE 3
Mean � SD volume of rectovaginal plaques froma baseline value to the end of the treatment (P< .05).
0
1
2
3
4
before 6 months 12 months
*
volume (mL)
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
TABLE 4Serum metabolic and coagulative parameters measured before and after 12 months of treatment in allpatients (N [ 21).
Note: Values are expressed as mean � SD. All parameters, not significant (P>.05).
Razzi. Vaginal danazol and endometriosis. Fertil Steril 2007.
technique for suspected deeply infiltrating endometriosis. So-novaginography is a reliable method for the assessment of therectovaginal septum and for the vaginal involvementassociated with other sites of deeply infiltrating endometriosis(19). Transvaginal ultrasound is able to detect the rectovaginalseptum because it is apposed to the peritoneum of the pouch ofDouglas, which is directly in contact with the top of the recto-vaginal septum (20). Nevertheless, the addition of transrectalsonography is a good option for identifying rectovaginal anduterosacral involvement (21).
Our study showed that the use of vaginal danazol does notaffect the menstrual cycle and has few side effects. This isprobably due to the negligible systemic absorption leadingto undetectable serum levels, as previously demonstrated(6). The low dose of danazol does not affect the pituitaryovarian axis and does not modify the endometrial thicknessinduced by estrogens and progesterone. Oral danazol athigh doses affects endometrium (22, 23). The evidence on un-affected serum concentrations of metabolic or thrombophilicparameters supports a safe treatment in contrast with the clas-sical danazol treatment, which is associated with an increaseof protein S, protein C, and antithrombin (24, 25). The pres-ent data on the safety and high compliance of vaginal admin-istration of danazol agree with previous reports referring tovaginal or uterine administration and support a limited sys-temic absorption (6, 7). Conservative surgical treatmentalso obtains persistent improvement of the painful symptomsassociated with endometriosis in large numbers of the pa-tients. Presacral neurectomy markedly reduces the midlinecomponent of menstrual pain, but it is not effective in deepdyspareunia (26). The limits of conservative surgery are thefrequent recurrences, also in the presence of classical medicaltreatments. Therefore, it is critical to have medical therapy toobtain meaningful improvement of the painful symptoms as-sociated with limited side effects and good compliance, andour present data suggest that vaginal danazol administration
Fertility and Sterility�
may be one of the new alternatives to repeated surgery inthe medical treatment of recurrent deeply infiltrating endo-metriosis. The issue of tolerability is of major importancewhen evaluating the overall benefits of long-term treatmentsfor chronic conditions such as symptomatic endometriosis. Itmay be misleading to focus only on pain relief because drugsmay well alleviate pain but also be associated with importantdisadvantages. The present study showed the efficacy of vag-inal danazol in women with symptomatic deeply infiltratingendometriosis who want to avoid further surgery.
REFERENCES1. Chapron C, Fauconnier A, Dubuisson JB, Barakat H, Vieira M, Br�eart G.
Deep infiltrating endometriosis: relation between severity of dysmenor-
rhea and extent of disease. Hum Reprod 2003;18:760–6.
2. Chapron C, Fauconnier A, Vieira M, Barakat H, Dousset B, Pansini V,
et al. Anatomical distribution of deeply infiltrating endometriosis: surgi-
cal implications and proposition for a classification. Hum Reprod
2003;8:157–61.
3. Konickx PR, Martin D. Treatment of deeply infiltrating endometriosis.
Curr Opin Obstet Gynecol 1994;6:231–41.
4. Fauconnier A, Chapron C. Endometriosis and pelvic pain: epidemiolog-
ical evidence of the relationship and implications. Hum Reprod Update
Different types of small nerve fibers in eutopicendometrium and myometrium in women withendometriosisNatsuko Tokushige, Ph.D.,a Robert Markham, Ph.D.,a Peter Russell, M.D.,b and Ian S. Fraser, M.D.a
a Department of Obstetrics and Gynaecology, Queen Elizabeth II Research Institute for Mothers and Infants; andb Department of Pathology, University of Sydney, Sydney, Australia
Objective: To investigate types of nerve fibers in endometrium and myometrium in women with endometriosis.Design: Laboratory study using human tissue.Setting: University-based laboratory.Patient(s): Women with and without endometriosis undergoing hysterectomy.Intervention(s): Histologic sections of contiguous endometrial and myometrial tissues were prepared from hyster-ectomies performed on women with and without endometriosis.Main Outcome Measure(s): Types and density of nerve fibers in endometrium and myometrium in women withand without endometriosis were determined using a series of specific markers for neuronal structure and function:PGP9.5, NF, SP, CGRP, TH, VAChT, VIP, and NPY.Result(s): Nerve fibers stained with PGP9.5 and NF in endometrium and myometrium were significantly in-creased in women with endometriosis compared with women without endometriosis. Nerve fibers in the functionallayer of endometrium in women with endometriosis were likely to be sensory C, a mixture of sensory Ad, sensoryC, and adrenergic fibers in the basal layer of the endometrium, a mixture of sensory Ad, sensory C, adrenergic andcholinergic fibers in the myometrium.Conclusion(s): Increased nerve fiber density in endometrium and myometrium, and sensory C fibers and adrener-gic nerve fibers in the endometrium in women with endometriosis may play an important role in the mechanisms ofpain generation in this condition. (Fertil Steril� 2007;88:795–803. �2007 by American Society for ReproductiveMedicine.)
Endometriosis is a gynecological disease defined by thehistologic presence of endometrial-like glands and stromaoutside the uterine cavity, most commonly implanted overvisceral and peritoneal surfaces within the female pelvis.Endometriosis is usually associated with pelvic pain suchas chronic dysmenorrhea and deep dyspareunia; however,the relationship between pain and endometriosis still remainsunclear.
Several researchers have demonstrated nerve fibers inendometriotic lesions, and several neurotransmitters andother neural proteins such as neurofilament, transforminggrowth factor b1 (TGF-b1), calcitonin gene-related peptide(CGRP), substance P (SP), and vesicular monoamine trans-porter (VMAT) immunoreactive nerve fibers have beendescribed in endometriotic lesions (1–3). We recently dem-onstrated that sensory, adrenergic, and cholinergic nervefibers are all present in peritoneal endometriotic lesions inwomen with endometriosis (4).
Received October 9, 2006; revised December 14, 2006 and accepted De-
cember 22, 2006.
Supported by research funding from the Department of Obstetrics and
Gynaecology, University of Sydney, Sydney, Australia.
Reprint requests: N. Tokushige, Ph.D., Department of Obstetrics and Gy-
naecology, Queen Elizabeth II Research Institute for Mothers and In-
fants, University of Sydney, Sydney, NSW, 2006, Australia (FAX:
015-0282/07/$32.00oi:10.1016/j.fertnstert.2006.12.078 Copyright ª2007 American
There is now considerable evidence that eutopic endome-trium from women with endometriosis exhibits manybiochemical differences compared with that of women with-out endometriosis (5, 6) in terms of structure, proliferation,apoptosis, immune components, cell adhesion molecules,proteases and their inhibitors, steroid and cytokine produc-tion and responsiveness, gene expression, protein synthesis,and angiogenesis. We have also demonstrated multiple smallunmyelinated nerve fibers in the eutopic endometrium ofwomen with laparoscopically confirmed endometriosis butnone in the endometrium of women without endometriosis(7). These abnormalities suggest that the eutopic endome-trium itself may play a role in the genesis of pain symptomsin women with endometriosis.
Increased density of nerve fibers was also demonstrated inthe myometrium in the lower half of the uterus in women withadvanced endometriosis compared with women withoutendometriosis (8). In this study, more collateral sproutingwith microneuromas formation and ingrowth around bloodvessels (perivascular nerve fiber proliferation) in the myome-trium in women with endometriosis and chronic pelvic painwere observed. However, nerve fibers in the endometriumand the types of nerve fibers in the myometrium were notinvestigated.
We studied innervation with different types of specific im-munohistochemical neural markers in endometrial and
Fertility and Sterility� Vol. 88, No. 4, October 2007 795Society for Reproductive Medicine, Published by Elsevier Inc.
myometrial specimens from women with and without visu-ally and biopsy-proven endometriosis. We used markers formyelinated, unmyelinated, sensory Ad, sensory C, adrenergicand cholinergic fibers.
MATERIALS AND METHODS
Tissue Collection
This study was approved by the Human Ethics Committees ofthe Central Sydney Area Health Service and the University ofSydney and all women gave their informed consent for par-ticipation.
One full-thickness uterine block, which included endome-trium and contiguous myometrium, was selected from eachof 10 women with endometriosis (mean age, 44.0 years;range, 42–46 years) and 35 women without endometriosis(mean age, 46.0 years; range, 38–54 years) who had under-gone hysterectomy. None of them were postmenopausal.The indications for hysterectomy in the 35 women withoutendometriosis included multiple small uterine fibroids,uterine prolapse, abdominal adhesions, or abnormally heavymenstrual bleeding without recognizable pathology.
The basal layer of the endometrium was defined as 300 mmabove the endometrial and myometrial interface (%300 mm)(9) and the functional layer of endometrium was defined asthe area from the epithelial surface to the basal layer. Thebasal and functional layers were further arbitrarily dividedinto two equal depth layers, namely superficial and deeperportions.
The patients with endometriosis all complained of dysmen-orrhea and a range of other related pain symptoms. The sever-ity of pain was not assessed systematically and prospectivelyin this study, but detailed clinical information was recorded ina standard format. All patients with endometriosis werestaged according to the revised American Fertility Society(AFS) score (10), which ranged from I to IV.
Menstrual cycle staging was blindly determined on allsamples of endometrium by a single experienced gynecolog-ical histopathologist (P.R.), and all phases of the cycle wererepresented in both groups: menstrual (days 1–7 of the cycle),proliferative (days 8–14), and secretory (days 15–28) of a nor-malized 28- day cycle. Of the 10 women with endometriosis,1 was assessed as being in the menstrual phase, 2 prolifera-tive, and 7 secretory, whereas of the 35 women without endo-metriosis, 5 were assessed as being in the menstrual phase, 16proliferative, and 14 secretory. None of the women hadreceived medical therapy for endometriosis in the 3 monthsbefore endometrial or hysterectomy sampling.
Immunohistochemistry
After surgical removal, thin slices of endometrium andmyometrium were immediately fixed in 10% neutral bufferedformalin for approximately 18–24 hours, processed, andembedded in paraffin wax according to a standard protocol.Each section was cut at 4 mm and routinely stained with
796 Tokushige et al. Endometrial nerve fibers in women
hematoxylin and eosin (H & E). Markers included polyclonalrabbit anti-protein gene product 9.5 (PGP9.5) (11), monoclo-nal mouse antihuman neurofilament (NF) (12), polyclonalrabbit anti-SP (3), rabbit anti-CGRP (3), polyclonal rabbitanti-vesicular acetylcholine transporter (VAChT) (13),monoclonal antityrosine hydroxylase (TH) (14), polyclonalrabbit anti-vasoactive intestinal polypeptide (VIP) (15), andpolyclonal rabbit anti-neuropeptide Y (NPY) (16). ThePGP9.5 is a highly specific pan-neuronal marker and NF isa highly specific marker for myelinated nerve fibers. TheSP and CGRP are sensory fiber markers, and they can be pres-ent in both Ad and C nerve fibers. The VAChT is present insympathetic preganglionic neurons, parasympathetic pregan-glionic and postganglionic neurons, and it is a specific markerfor cholinergic fibers. The TH is present in sympathetic post-ganglionic neurons, and it is a specific marker for adrenergicfibers. The VIP is a specific marker for parasympathetic neu-rons and can be present in both sensory and cholinergic nervefibers. The NPY is a specific marker for sympathetic neuronsand can be present in both sensory and adrenergic nervefibers.
Antigen retrieval techniques for PGP9.5 and NF were usedas described previously (7). Serial sections, cut at 4 mm, wereimmunostained using antibodies for polyclonal rabbit anti-protein gene product 9.5 (dilution 1:700; Dako Australia,Sydney, Australia), a highly specific pan-neuronal marker(11) and monoclonal mouse antihuman neurofilament protein(dilution 1:200; Dako), a highly specific marker for myelin-ated nerve fibers (12) individually on different slides for 30minutes at room temperature. Serial sections were alsoimmunostained with rabbit anti-SP (dilution 1:2,000; Sero-tec, Raleigh, NC) (3), polyclonal rabbit anti-CGRP (dilution1:10,000; Sigma, St. Louis, MO) (3), polyclonal rabbit anti-VAChT (dilution 1:6,000; Sigma) (13), monoclonal anti-TH(dilution 1:1,200; Sigma) (14), polyclonal rabbit anti-VIP(dilution 1: 3,000; Chemicon, Temecula, CA) (15), and poly-clonal rabbit anti-NPY (dilution: 1:2,500, Chemicon) (16)overnight at 4�C. Sections stained with PGP9.5, NF, SP,CGRP, TH, VAChT, VIP, and NPY were washed in Trisbuffer, incubated with REAL Detection System, AlkalinePhosphatase/RED, Link, Biotinylated Secondary Antibodies(AB2) (Dako) for 15 minutes and REAL Detection System,Alkaline Phosphatase/RED, Streptavidin Alkaline Phospha-tase (AP) (Dako) for 15 minutes, and stained with REAL De-tection System, chromogen (RED) (Dako) for 10 minutes.
All immunostaining was carried out on a Dako AutostainerModel S3400 (Dako, Carpinteria, CA). Images of the sec-tions were captured using Olympus microscope BX51 anddigital camera DP70 (Olympus, Tokyo, Japan). We used nor-mal skin as a positive control as it reliably contains myelin-ated and unmyelinated nerve fibers expressing PGP9.5, NF,SP, CGRP, TH, VAChT, VIP, and NPY. Eutopic endometrium(the functional layer) from women without endometriosiswas used as a negative control as it does not contain any nervefibers expressing PGP9.5, NF, SP, CGRP, TH, VAChT, VIP,and NPY antibodies.
with endometriosis Vol. 88, No. 4, October 2007
FIGURE 1
Nerve fibers in eutopic endometrium in women with and without endometriosis. (A) Eutopic endometrium froma woman without endometriosis stained with PGP9.5 and permanent fast red chromogen (magnification,�200).No nerve fibers were stained. (B) Eutopic endometrium from a woman with endometriosis stained with SP andpermanent fast red chromogen (magnification, �400). Arrows denote tiny SP-positive nerve fibers. (C) Eutopicendometrium from a woman with endometriosis stained with CGRP and permanent fast red chromogen(magnification, �400). Arrow denotes tiny CGRP-positive nerve fibers. (D) Eutopic endometrium from a womanwith endometriosis stained with NPY and permanent fast red chromogen (magnification, �200). Arrows denotetiny NPY-positive nerve fibers. (E) Eutopic endometrium from a woman with endometriosis stained with VIP andpermanent fast red (magnification, �200). Arrows denote tiny VIP-positive nerve fibers. Bars ¼ 50 mm.
Tokushige. Endometrial nerve fibers in women with endometriosis. Fertil Steril 2007.
Fertility and Sterility� 797
TABLE 1A quantitative assessment of the density of nerve fibers stained with PGP9.5 and NF in eutopicendometrium and myometrium in women with and without endometriosis.
Women withoutendometriosis
Women withendometriosis
Area Functional layer of endometriumNumber of specimens 35 10Total nerve fiber density (mean � SD/mm2)
stained with PGP9.50 11� 5 (P< .001)
Total nerve fiber density (mean � SD/mm2)stained with NF
0 0
Area Basal layer of endometriumNumber of specimens 35 10Total nerve fiber density (mean � SD/mm2)
stained with PGP9.50 18 � 8 (P< .001)
Total nerve fiber density (mean � SD/mm2)stained with NF
0 2 � 2 (P< .001)
Area MyometriumNumber of specimens 35 10Total nerve fiber density (mean � SD/mm2)
stained with PGP9.50.9 � 0.8 3.3 � 1.2 (P< .001)
Total nerve fiber density (mean � SD/mm2)stained with NF
0 0.7 � 0.5 (P< .001)
Tokushige. Endometrial nerve fibers in women with endometriosis. Fertil Steril 2007.
Statistical Analysis
The images were captured by using an Olympus microscopeBX51 and digital camera DP70 and an assessment of nervefiber density was performed by Image Pro Plus Discovery(MediaCybernetics, Silver Springs, MD). Once the images’features were controlled, an orthogonal grid mask wassketched above the original images. The sections of thegrid were 250 mm per side. Once the grid was in position,nerve fibers in the endometrial–myometrial sections, andthe total number of squares covering the endometrial–myo-metrial sections were counted. The total number of nerve
798 Tokushige et al. Endometrial nerve fibers in wome
fibers was divided by the total number of squares to obtainan average of nerve fibers per square (each square of 250 x250 mm). The results were expressed as the mean (� SD)number of nerve fibers per mm2 in each specimen from all en-dometrial–myometrial sections, stained with the polyclonalantibody against PGP9.5, as well as monoclonal antibodyagainst NF. The counting procedure was carried out twiceby two independent observers (each blinded to the other)without any knowledge of the clinical parameters or otherprognostic factors. The concordance rate was more than95% between the observers. Nerve fiber density of eutopic
TABLE 2A quantitative assessment of the density of SP, CGRP, VIP, and NPY nerve fibers in the functional layerof endometrium in women with endometriosis.
Functional layer ofendometrium in women with
endometriosis
Total nerve fiber density (mean � SD/mm2) stained with PGP 9.5 11 � 5Total nerve fiber density (mean � SD/mm2) stained with SP 1.0 � 0.2Total nerve fiber density (mean � SD/mm2) stained with CGRP 1.7 � 0.4Total nerve fiber density (mean � SD/mm2) stained with VIP 8.5 � 2.3Total nerve fiber density (mean � SD/mm2) stained with NPY 9.6 � 2.8
Tokushige. Endometrial nerve fibers in women with endometriosis. Fertil Steril 2007.
n with endometriosis Vol. 88, No. 4, October 2007
endometrium and myometrium from women with and with-out endometriosis as well as SP, CGRP, VIP, and NPY inthe functional layer of endometrium in women with endome-triosis was compared using the Mann-Whitney test. Differ-ences were considered to be significant at P<.05.
RESULTS
No nerve fibers were detected in the functional layer of endo-metrium in women without endometriosis (Fig. 1A). Nervefibers were demonstrated in the functional layer in all womenwith endometriosis stained with PGP9.5 as demonstrated inour previous study (mean density � SD, 11 � 5/mm2; range,5–23; P<.001; Table 1) (7), but not NF (a highly specificmarker for myelinated nerve fibers), suggesting that all nervefibers in the functional layer were unmyelinated. Whenstained with six neurotransmitter markers, nerve fibers inthe functional layer were stained with SP (a marker forsensory Ad and sensory C nerve fibers), CGRP (a markerfor sensory Ad and sensory C nerve fibers), VIP (a markerfor sensory Ad, sensory C, and cholinergic nerve fibers),and NPY (a marker for sensory Ad, sensory C, and adrenergicnerve fibers), but not with TH (a marker for adrenergic nervefibers) or VAChT (a marker for cholinergic nerve fibers).There were greater nerve fiber densities of VIP (mean density� SD, 8.5� 2.3/mm2; range, 5.3–12.8) and NPY (mean den-sity � SD, 9.6 � 2.8/mm2; range, 6.1–14.9) than SP (meandensity � SD, 1.0 � 0.2/mm2; range, 0.8–1.3) and CGRP(mean density � SD, 1.7 � 0.4/mm2; range, 1.3–2.4) in thefunctional layer of endometrium in women with endometri-osis (P<.001; Table 2). These results indicate that nervefibers in the functional layer of endometrium in womenwith endometriosis were most likely to be sensory C nervefibers and probably of different sensory types (Fig. 1B–E).
In 35 women without endometriosis, small numbers ofPGP9.5-stained nerve fibers were only demonstrated in thedeeper portion of the basal layer of endometrium in fourendometrial and myometrial specimens (mean density �SD, 0/mm2; range, 0–1; Table 1) (7) and never in the func-tional layer. These nerve fibers were stained with SP,CGRP, VIP, and NPY but not TH, VAChT, and NF, exceptfor one patient (mean density � SD, 0/mm2; range, 0–1;Table 1) (7). Therefore, nerve fibers in the basal layer of en-dometrium in women without endometriosis were mostlikely to be sensory C fibers and probably of different types.
Nerve fibers were present in the basal layer of endome-trium in all women with endometriosis and those nerve fiberswere stained with both PGP9.5 (mean density � SD, 18 � 8/mm2; range, 9–80; P<.001; Table 1) (7) and NF (meandensity � SD, 2 � 2/mm2; range, 1–7; P<.001; Table 1)(7). These nerve fibers were also stained with SP, CGRP,TH, VIP, and NPY but not with VAChT. Therefore, nervefibers in the basal layer of endometrium in women with endo-metriosis were most likely to be a mixture of sensory Ad,sensory C, and adrenergic nerve fibers.
Nerve fibers were present in the myometrium in allwomen with and without endometriosis were stained with
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PGP9.5 (mean � SD, 3.3 � 1.2/mm2; range, 2.2–6.2 andmean � SD, 0.9 � 0.8/mm2; range, 0.2–3.1, respectively,P<.001; Table 1) (7) and NF (mean density � SD, 0.7 �0.5/mm2; range, 0–1.0 and mean density � SD, 0/mm2;range, 0–0.1 , respectively, P<.001; Table 1) (7). Nerve fi-bers in myometrium stained with all six of the markers forSP, CGRP, TH, VAChT, VIP, and NPY. Therefore, nerve fi-bers in the myometrium in women with and without endo-metriosis were most likely to be a mixture of sensory Ad,sensory C, adrenergic and cholinergic fibers, but densitieswere much higher in women with endometriosis. Choliner-gic fibers were present only in the myometrium and not inendometrium (Fig. 2).
When serial sections of myometrium from women with en-dometriosis were stained with SP, CGRP, TH, VAChT, VIP,and NPY, several nerve fiber bundles expressed all six ofthe markers, suggesting that those nerve fibers were co-expressed with sensory C, sensory Ad, adrenergic and cholin-ergic nerve fibers (Fig. 3).
DISCUSSION
This study demonstrated highly significantly increasedmyelinated and unmyelinated nerve fiber densities and
FIGURE 2
Nerve fibers in myometrium in women with andwithout endometriosis. (A) Myometrium froma woman without endometriosis stained with TH andpermanent fast red chromogen (magnification,�100). (B) Myometrium from a woman withendometriosis stained with TH and permanent fastred chromogen (magnification, �100). Arrowsdenote TH-positive nerve fibers. There was a greaternerve fiber density with much thicker and moreobvious nerve trunks in women with endometriosisthan in women without endometriosis.
Tokushige. Endometrial nerve fibers in women with endometriosis. Fertil
Steril 2007.
799
FIGURE 3
Co-localization of different types of a large nerve trunk in myometrium from a woman with endometriosis (serialsections). (A) Myometrium from a woman with endometriosis stained with SP (magnification,�200). Nerve fiberswere stained with permanent fast red. (B) Myometrium from a woman with endometriosis stained with CGRP(magnification, �200). Nerve fibers were stained with permanent fast red. (C) Myometrium from a woman withendometriosis stained with TH (magnification, �200). Nerve fibers were stained with permanent fast redchromogen. (D) Myometrium from a woman with endometriosis stained with VAChT (magnification,�200). Nervefibers were stained with permanent fast red chromogen. (E) Myometrium from a woman with endometriosisstained with NPY (magnification, �200). Nerve fibers were stained with permanent fast red chromogen. (F)Myometrium from a woman with endometriosis stained with VIP (magnification,�200). Nerve fibers were stainedwith permanent fast red chromogen. Bars ¼ 50 mm.
Tokushige. Endometrial nerve fibers in women with endometriosis. Fertil Steril 2007.
800 Tokushige et al. Endometrial nerve fibers in women with endometriosis Vol. 88, No. 4, October 2007
different types of nerve fiber expressions in endometrium andmyometrium from women with endometriosis comparedwith women without endometriosis.
Although VIP and NPY are expressed in cholinergic andadrenergic nerve fibers, respectively, we believe that theappearance of these unmyelinated nerve fibers in endome-trium must be sensory C fibers, as specific markers for cholin-ergic and adrenergic fibers were never expressed in thefunctional layer of endometrium in women with endometri-osis. These findings are therefore consistent with these fourmarkers (SP, CGRP, VIP, and NPY) being expressed in sen-sory nerve fibers in the functional layer of endometrium inwomen with endometriosis.
Sensory C fibers (mainly VIP and NPY) in the functionallayer of endometrium and adrenergic fibers in the basal layerof endometrium were detected only in women with endome-triosis, and never detected in women without endometriosis.The different types of nerve fibers in women with and withoutendometriosis in the endometrium may play an importantrole in the mechanisms of pain generation in women with en-dometriosis.
Several researchers have identified types of nerve fibersin normally sited endometrium and myometrium in womenwithout endometriosis. Neuropeptide Y (17, 18), SP (17),neurotensin (17), and VIP (17, 19, 20) immunoreactivenerve fibers were detected in the basal layer of the endome-trium and myometrium in nonpregnant women, and SP andCGRP immunoreactive nerve fibers were also demonstratedin myometrium in regularly menstruating women and in-fants (3–6 months postnatally) (21). We have also demon-strated NPY, SP, CGRP, and VIP immunoreactive nervefibers in the basal layer of endometrium and myometriumin women without endometriosis, but not in the functionallayer.
The functions of individual neurotransmitters and neuro-peptides in eutopic endometrium and myometrium are notunderstood. Substance P can induce contraction in the my-ometrium (21), and CGRP can inhibit the breakdown of SPby a specific endopeptidase (22) and is involved in inhib-iting spontaneous myometrial contractions in the humanuterus (21). Neuropeptide Y co-exists with noradrenalinein most sympathetic vasoconstrictor nerve fibers (23) andis considered to regulate vascular tone and exert inhibitoryeffect on myometrial contractility (21). Vasoactive intesti-nal polypeptide is a potent vasodilator of the uterine artery,and can be involved in the relaxation of nonvascular andvascular smooth muscle, myometrial blood flow increase,ovarian steroidogenesis and ovulation, vaginal lubricant,and endometrial glandular secretion (24, 25). Acetylcho-line is associated with myometrial and vascular smoothmuscle (26), and evokes contraction of the myometrium(27). Vasoactive intestinal polypeptide often co-existswith ACh (28) and promotes relaxation of the myometriumthrough inhibition of the excitatory action of ACh (29).
Fertility and Sterility�
Neurotensin has been shown to exert excitatory effectson human myometrium (30) and induce contractions ofrat uterus (31).
Several studies have reported increased density of nervefibers in the myometrium in women with pelvic pain (8, 32,33). Widespread nerve fiber proliferation with extensive peri-vascular nerve fiber proliferation around both arteries andveins in the myometrium has been reported in a patientwith endometriosis and dysmenorrhea and dyspareunia(32). Microneuroma formation and perivascular nerve fiberproliferation in the myometrium has been reported in 16women with endometriosis and chronic pelvic pain (8), andincreased small diameter nerve fiber proliferation in themyometrium of 17 women with chronic pelvic pain (33).There was also proliferation and hypertrophy of bundles ofunmyelinated nerve fibers in a woman with chronic pelvicpain (34), suggesting a relationship between an increasednerve fiber density in the myometrium and pelvic painsymptoms. However, nerve fibers were not studied in theendometrium of these subjects.
It is not known what conditions or molecular stimuli mayattract nerve fibers to grow into the eutopic endometrium inwomen with endometriosis. Several molecules are presentin eutopic endometrium in women with endometriosis thatcould have effects on nerve fiber growth. They include nervegrowth factor (NGF), insulin-like growth factor I (IGF-1),Bcl-2, hepatocyte growth factor, and heat shock protein 27.Insulin-like growth factor I can induce sensory fiber growthand potentiate NGF-induced neuritogenesis in adult rat dor-sal root ganglion neurons (35, 36). Bcl-2 is expressed in neu-rons (37) and can promote axonal growth in mouseembryonic sensory fibers (38), and also has been shown to in-hibit neuronal death (37, 39). Hepatocyte growth factor alonehad no outgrowth-promoting activity; however, it cooperatedwith NGF in enhancing axonal growth of sensory fibers andalso enhanced the neurotrophic activities of NGF in cultureddorsal root ganglion neurons (40). Heat shock protein 27 isexpressed by sensory fibers (41, 42) and significantly in-creased survival for rat sensory and sympathetic fibers afteraxotomy or NGF withdrawal in adult rat dorsal root ganglionneurons (43).
Oophorectomy significantly decreased NGF mRNA andbrain-derived neurotrophic factor mRNA levels, whereasestrogen (E) treatment increased these levels (44, 45), andE also up-regulated NGF and brain-derived neurotrophicfactor expressions in endometrium (46–48). Several studieshave shown that neuroprotective effects of E were blockedby E receptor (ER) antagonists (49–51).
Estrogen stimulated IGF-I gene and protein expression inthe rat uterus, and regulated the expression of IGF-I receptorin the rat uterus (52, 53). Estrogen treatment up-regulated theexpression of Bcl-2 in the rat brain (54), and increased thenumber of Bcl-2 immunoreactive nerve fibers (55). Estrogenalso increased the production of hepatocyte growth factor byperitoneal macrophages in women with endometriosis (56),
801
and heat shock protein expression was stimulated by E in theendometrium (57).
Because endometriosis is an E-dependent disease, E mayprovide trophic support to nerve fibers by regulating the pro-duction of neurotropins and other molecules that are associ-ated with nerve fiber growth in endometrium andmyometrium in women with endometriosis.
Sensory C fibers in the functional layer of the endometriumand adrenergic fibers in the basal layer of the endometriumcould play important roles in pain generation in womenwith endometriosis. Inflammatory mediators released fromendometrium can activate or sensitize sensory C fiber endingsto evoke neurogenic inflammation (58, 59), and adrenergic fi-ber endings can release prostaglandin (PG) E2, PGI2 (60), andnorepinephrine, which can sensitize sensory C fibers (61).Recent studies reported that VIP caused allodynia, secondaryhyperalgesia, and peripheral sensitization, but they wereblocked by the VIP receptor antagonist VIP6-28 in a rat modelof osteoarthritis (62, 63). Neuropeptide Y also exacerbatedhyperalgesia, but it was attenuated by the NPY antagonista-trinositol in a rat model (64, 65).
In summary, there were increased nerve fiber densities inendometrium and myometrium in women with endometriosiscompared with that of women without endometriosis. Inwomen with endometriosis, the functional layer of the endo-metrium was innervated by sensory C fibers expressing largeamounts of VIP and NPY, and smaller amounts of SP andCGRP. The basal layer of the endometrium was innervatedby a mixture of sensory Ad, sensory C, and adrenergic fibers.In myometrium, sensory Ad, sensory C, adrenergic and cho-linergic fibers were all present in women with and withoutendometriosis. Sensory C in the functional layer of endome-trium and adrenergic fibers in the basal layer of endometriumwere detected only in women with endometriosis. The func-tions and stimuli for sensory C and adrenergic fibers in endo-metrium in women with endometriosis should be highpriority targets for further research.
REFERENCES1. Tulandi T, Felemban A, Chen MF. Nerve fibers and histopathology of en-
dometriosis-harboring peritoneum. J Am Assoc Gynecol Laparosc
2001;8:95–8.
2. Tamburro S, Canis M, Albuisson E, Dechelotte P, Darcha C, Mage G. Ex-
pression of transforming growth factor beta1 in nerve fibers is related to
dysmenorrhea and laparoscopic appearance of endometriotic implants.
Fertil Steril 2003;80:1131–6.
3. Berkley KJ, Dmitrieva N, Curtis KS, Papka RE. Innervation of ectopic
endometrium in a rat model of endometriosis. Proc Natl Acad Sci
U S A 2004;101:11094–8.
4. Tokushige N, Markham R, Russell P, Fraser IS. Nerve fibers in peritoneal
endometriosis. Hum Reprod 2006;21:3001–7.
5. Healy DL, Rogers PA, Hii L, Wingfield M. Angiogenesis: a new theory
for endometriosis. Hum Reprod Update 1998;4:736–40.
6. Sharpe-Timms K. Endometrial anomalies in women with endometriosis.
Ann N Y Acad Sci 2001;943:131–47.
7. Tokushige N, Markham R, Russell P, Fraser IS. High density of small
nerve fibers in the functional layer of the endometrium in women with
endometriosis. Hum Reprod 2006;21:782–7.
802 Tokushige et al. Endometrial nerve fibers in women
8. Atwal G, du Plessis D, Armstrong G, Slade R, Quinn M. Uterine inner-
vation after hysterectomy for chronic pelvic pain with, and without, en-
45. Simpkins JW, Green PS, Gridley KE, Singh M, de Fiebre NC,
Rajakumar G. Role of estrogen replacement therapy in memory enhance-
ment and the prevention of neuronal loss associated with Alzheimer’s
disease. Am J Med 1997;103:19S–25.
46. Sohrabji F, Miranda RC, Toran-Allerand CD. Identification of a putative
estrogen response element in the gene encoding brain-derived neurotro-
phic factor. Proc Natl Acad Sci U S A 1995;92:11110–4.
47. Gibbs RB. Treatment with estrogen and progesterone affects relative
levels of brain-derived neurotrophic factor mRNA and protein in differ-
ent regions of the adult rat brain. Brain Res 1999;844:20–7.
Fertility and Sterility�
48. Krizsan-Agbas D, Pedchenko T, Hasan W, Smith PG. Oestrogen regu-
lates sympathetic neurite outgrowth by modulating brain derived neuro-
trophic factor synthesis and release by the rodent uterus. Eur J Neurosci
2003;18:2760–8.
49. Singer CA, Figueroa-Masot XA, Batchelor RH, Dorsa DM. The mito-
gen-activated protein kinase pathway mediates estrogen neuroprotection
after glutamate toxicity in primary cortical neurons. J Neurosci 1999;19:
2455–63.
50. Patrone C, Andersson S, Korhonen L, Lindholm D. Estrogen receptor-
dependent regulation of sensory neuron survival in developing dorsal
root ganglion. Proc Natl Acad Sci U S A 1999;96:10905–10.
51. Wilson ME, Dubal DB, Wise PM. Estradiol protects against injury-in-
duced cell death in cortical explant cultures: a role for estrogen receptors.
Brain Res 2000;873:235–42.
52. Ghahary A, Murphy LJ. Uterine insulin-like growth factor-I receptors:
regulation by estrogen and variation throughout the estrous cycle. Endo-
crinology 1989;125:597–604.
53. Moyano P, Rotwein P. Estrogen action in the uterus and insulin-like
growth factor-I. Growth Hormone & Igf Res 2004;14:431–5.
54. Fernandez AM, Gonzalez de la Vega AG, Planas B, Torres-Aleman I.
Neuroprotective actions of peripherally administered insulin-like growth
factor I in the injured olivo-cerebellar pathway. Eur J Neurosci 1999;11:
2019–30.
55. Cardona-Gomez GP, Mendez P, DonCarlos LL, Azcoitia I, Garcia-
Segura LM. Interactions of estrogens and insulin-like growth factor-I
in the brain: implications for neuroprotection. Brain Res Brain Res
Rev 2001;37:320–34.
56. Khan KN, Masuzaki H, Fujishita A, Kitajima M, Sekine I, Matsuyama T,
et al. Estrogen and progesterone receptor expression in macrophages and
regulation of hepatocyte growth factor by ovarian steroids in women with
endometriosis. Hum Reprod 2005;20:2004–13.
57. Tang PZ, Gannon MJ, Andrew A, Miller D. Evidence for oestrogenic
regulation of heat shock protein expression in human endometrium
and steroid-responsive cell lines. Eur J Endocrinol 1995;133:
598–605.
58. Mantyh PW. Neurobiology of substance P and the NK1 receptor. J Clin
Psychiatry 2002;63(Suppl 11):6–10.
59. Schaible HG, Ebersberger A, Von Banchet GS. Mechanisms of pain in
arthritis. Ann N Y Acad Sci 2002;966:343–54.
60. Gonzales R, Goldyne ME, Taiwo YO, Levine JD. Production of hyper-
algesic prostaglandins by sympathetic postganglionic neurons. J Neuro-
chem 1989;53:1595–8.
61. Banik RK, Sato J, Yajima H, Mizumura K. Differences between the
Lewis and Sprague-Dawley rats in chronic inflammation induced norepi-
nephrine sensitivity of cutaneous C-fiber nociceptors. Neurosci Lett
2001;299:21–4.
62. McDougall JJ, Watkins L, Li Z. Vasoactive intestinal peptide (VIP) is
a modulator of joint pain in a rat model of osteoarthritis. Pain
2006;123:98–105.
63. Schuelert N, McDougall JJ. Electrophysiological evidence that the vaso-
active intestinal peptide receptor antagonist VIP6-28 reduces nocicep-
tion in an animal model of osteoarthritis. Osteoarthritis Cartilage
2006;14:1155–62.
64. Tracey DJ, Romm MA, Yao NN. Peripheral hyperalgesia in experimental
neuropathy: exacerbation by neuropeptide Y. Brain Res 1995;669:
245–54.
65. White DM. Intrathecal neuropeptide Y exacerbates nerve injury-induced
mechanical hyperalgesia. Brain Res 1997;750:141–6.
803
GENETICS
Preimplantation genetic screening as an alternative toprenatal testing for Down syndrome: preferences ofwomen undergoing in vitro fertilization/intracytoplasmicsperm injection treatmentMoniek Twisk, M.D.,a,b Maaike L. Haadsma, M.D.,b Fulco van der Veen, Ph.D.,a
Patrick M. M. Bossuyt, Ph.D.,c and Johanna C. Korevaar, Ph.D.c
a Center for Reproductive Medicine, Department of Obstetrics and Gynaecology, Academic Medical Center, Amsterdam;b Department of Obstetrics and Gynaecology, University Medical Center Groningen, Groningen; and c Department of Clinical
Epidemiology & Biostatistics, Academic Medical Center, Amsterdam, the Netherlands
Objective: Although the primary goal of preimplantation genetic screening (PGS) is to increase pregnancy rates inwomen undergoing IVF/intracytoplasmic sperm injection treatment, it has been suggested that it may also be usedas an alternative to prenatal testing for Down syndrome.Design: Trade-off questionnaires.Setting: Two university centers for reproductive medicine.Patient(s): Two hundred forty-four subfertile women.Intervention(s): Scenarios with different pregnancy chances after PGS and with different risk reductions ofa Down syndrome pregnancy were presented.Main Outcome Measure(s): Willingness to have PGS performed in the various scenarios.Result(s): In case PGS would discover all Down syndrome embryos without affecting pregnancy chances, 83% ofthe women would have PGS performed. If PGS lowered pregnancy chances from one in five to one in seven, 36% ofthe women preferred to have PGS performed. If PGS reduced the chance of a Down syndrome pregnancy with 80%without affecting pregnancy chances, 75% of the women would have PGS performed, and 31% of them wouldrefrain from prenatal testing afterward.Conclusion(s): Most women favor PGS for Down syndrome screening, even if it is not 100% sensitive. Theacceptability depends on the effect PGS has on pregnancy chances, and, to a lower extent on its sensitivity todetect Down syndrome embryos. (Fertil Steril� 2007;88:804–10. �2007 by American Society for ReproductiveMedicine.)
Key Words: Down syndrome, in vitro fertilization, patient preferences, preimplantation genetic screening (PGS),prenatal diagnosis
The chance of having a child with Down syndrome increaseswith maternal age (1). To diagnose trisomy 21 antenatally,chromosomal analysis can be performed after chorionicvillus sampling or amniocentesis. These invasive tests arelabor-intensive, expensive, and carry a procedure-relatedrisk of fetal loss of 0.5–1% (2–4). Therefore, not all pregnant
Received July 21, 2006; revised and accepted December 22, 2006.
There were no conflict of interest in this article.
Sponsored by the Netherlands Organisation for Health Research and
Development, The Hague, the Netherlands (grant 945-03-013).
This study was presented at the ASRM annual meeting in New Orleans,
2006.
Reprint requests: Moniek Twisk, M.D., Center for Reproductive Medicine,
Academic Medical Center, Meibergdreef 9, room H4-205, 1105 AZ
Amsterdam, the Netherlands (FAX: þ31-20-5669206; E-mail: M.
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicin
804
women accept chromosomal analysis. Noninvasive screeningtests, like sonographic measurement of nuchal translucencythickness or first and second trimester maternal serum screen-ing, have been developed as alternatives (5–8). By usingthese noninvasive screening tests, it is possible to selectwomen with a high probability of a Down syndrome preg-nancy who can then be offered invasive testing.
In case a Down syndrome pregnancy is detected, the cou-ple has to decide whether or not to terminate the pregnancy.For many parents terminating a Down syndrome pregnancyis a very difficult decision to make. The grief following termi-nation for a fetal abnormality can be similar to the grief fol-lowing neonatal death (9).
With IVF and intracytoplasmic sperm injection (ICSI)fertilization occurs in the laboratory. This provides the
0015-0282/07/$32.00e, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.033
possibility to examine embryos before implantation. In pre-implantation genetic screening (PGS) one or two blastomeresare removed from an early cleavage stage embryo and usedfor aneuploidy screening. Only embryos scored as euploidfor the chromosomes tested are transferred.
Although the primary goal of this technique is to increasepregnancy rates in women undergoing IVF/ICSI, it has beensuggested that it may also be used as an alternative to prenataltesting for Down syndrome (10). The thought underlying theuse of PGS in these cases is that with PGS couples do nothave to decide whether they want to undergo invasive prena-tal procedures with the risk of miscarriage, nor do they haveto make any decisions about terminating a Down syndromepregnancy. A disadvantage is that embryos can be misdiag-nosed, as the chromosomal content of blastomeres from thesame embryo can differ, a phenomenon known as mosaicism(11, 12). Indeed, Down syndrome fetuses have been diag-nosed with invasive prenatal procedures after IVF/ICSIwith PGS (13, 14), and births of children with Down syn-drome have been reported after PGS (14–17).
Another disadvantage of PGS is that the effect it has onpregnancy chances is yet unknown. Although PGS has beendeveloped to improve pregnancy rates, a recent Cochrane re-view (18) reported no significant difference between ongoingpregnancy rates after IVF/ICSI with PGS compared withIVF/ICSI without PGS. The ongoing pregnancy rate perpatient after PGS was 15%, and the ongoing pregnancy rateafter IVF/ICSI without PGS was 20% (odds ratio [OR]0.64, 95% confidence interval [CI] 0.37 to 1.09).
By applying PGS, treatment costs are increased. Thesecosts are usually not covered by insurance. Therefore, itmight not be affordable for all patients.
Before PGS can be introduced as an alternative to conven-tional prenatal testing, it is important to know women’s atti-tudes toward both methods of testing for Down syndrome, aswell as their preferences. To date, only one study has assessedwomen’s preferences for PGS as an alternative to prenataltesting (19). In this study, 74 infertile couples undergoingIVF/ICSI were asked for their opinion on the acceptabilityof PGS. However, only information on invasive prenatal test-ing was provided, and couples were not informed on the pos-sibility of noninvasive prenatal testing. In this study PGS wasmore acceptable than prenatal testing for 82% of the couples,and 96% of the couples were willing to undergo PGS.Whether women would refrain from prenatal tests afterPGS, and whether women would still opt for PGS if it didnot detect all embryos with Down syndrome, was not as-sessed.
Furthermore, it is unknown whether couples undergoingIVF/ICSI would still consider PGS if it would negativelyaffect pregnancy chances.
To assess the attitudes of women undergoing IVF/ICSItreatment toward PGS as an alternative to prenatal testing,we designed a questionnaire. In this questionnaire we as-sessed whether women would opt for PGS if this screening
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method would be ‘‘ideal’’ and when it would not be ideal,that is, would lower pregnancy chances or would not detectall Down syndrome embryos.
MATERIALS AND METHODS
Eligible for the study were women in the stimulation phase ofan IVF/ICSI cycle, attending the Centers for ReproductiveMedicine of the Academic Medical Center and the UniversityMedical Center Groningen between May and December2005. Consenting women were handed out a questionnaire.They were asked to complete the questionnaire at homeand return it on their next visit to the clinic. Institutional re-view board approval was obtained for this study.
Demographic characteristics and information on reproduc-tive history including age, parity, duration of infertility, num-ber of previous IVF/ICSI cycles, previous experiences withprenatal diagnosis, and education level were collected. Writ-ten general information about the various possibilities of pre-natal tests for Down syndrome pregnancies with theiradvantages and disadvantages was provided in the question-naire. Women were then asked whether they would considerprenatal testing for Down syndrome if they were pregnant,and if so, what type of testing: invasive or noninvasive. Ifa woman would not consider prenatal testing, she was askedto indicate her main reason for not wanting prenatal testing:‘‘every child is welcome,’’ ‘‘I don’t want to decide about ter-mination of the pregnancy,’’ or ‘‘other reason, being .’’
Subsequently, information on PGS as a possible alternativewas presented. Three scenarios were presented in which preg-nancy chances and sensitivity in detecting Down syndromeembryos varied. After each scenario women were askedwhether they would opt for PGS in that particular situation.
In the first scenario PGS prevented all Down syndromepregnancies, without any negative effects on pregnancy chan-ces. One out of five women was expected to become pregnantafter one cycle of IVF/ICSI.
In the second scenario PGS still prevented all Down syn-drome pregnancies, but with a lower chance to become preg-nant with IVF/ICSI treatment: one out of seven.
In the third scenario PGS lowered the chance of havinga child with Down syndrome from 1 in 200—the risk ofa 37-year-old woman—to 1 in 1,000, without having any neg-ative effect on pregnancy chances. This implied a risk reduc-tion of 80% on having a Down syndrome child after PGS. Ifwomen answered ‘‘yes’’ to PGS in this situation, they wereasked if they would subsequently undergo prenatal testingfor Down syndrome.
Addendum 1 contains a translated version of the informa-tion provided on PGS in the questionnaire and the three pre-sented scenarios.
Statistical Analysis
For data analysis SPSS for Windows version 11.5.1 was used.Statistical significance was defined as P<.05. To determine
805
factors associated with accepting a lower pregnancy changeafter PGS and determine factors associated with still wantingPGS when its detection possibility is not 100%, we per-formed univariable and multivariable logistic regressionanalyses.
RESULTS
A total of 343 questionnaires were handed out, of which 244were returned (response rate 71%). Baseline characteristicsof the respondents are provided in Table 1. Mean age was34.2 years (SD 4.12). Most women (77%) had no children,and only 8% had previous experience with prenatal testingfor Down syndrome.
Sixty-eight percent of the respondents wanted to have pre-natal testing for Down syndrome if they became pregnant(Table 2). Of these women, 92% preferred to undergo nonin-vasive diagnostic tests and 78% of them also considered
TABLE 1Baseline characteristics of respondents(n [ 244).
Age (y) [mean (SD)] 34.2 (4.12)Women with children with current
or previous partner (%)None 77One or more 23
Previous experience with prenataltesting (%)
None 92Noninvasive prenatal
diagnostic test2
Invasive prenatal diagnostic test 2Both invasive and noninvasive
diagnostic test4
Women with acquaintances withDown syndrome child (%)
27
Infertility duration (months)[mean (SD)]
50.2 (26.8)
No. of previous IVF/ICSI cycles (%)0 331 282 or more 39
Educational level (%)Low 3Medium 54High 43
Educational level of partner (%)Low 1Medium 59High 40
Twisk. PGS versus prenatal testing. Fertil Steril 2007.
806 Twisk et al. PGS versus prenatal testing
invasive diagnostic tests, depending on the outcome of thenoninvasive test. Eight percent of the women would immedi-ately go for an invasive procedure. Most women who did notconsider prenatal testing gave as the reason for this decision‘‘every child is welcome’’ (38 women, 53%). Fifteen (21%)women did not want prenatal testing because they did notwant to have to decide about termination of a pregnancy.Nine (12%) women gave various reasons (e.g., ‘‘I am young,therefore I am not at risk,’’ ‘‘with normal ultrasound you canalso see enough’’), and 10 (14%) women gave no reason.
Willingness of women to have PGS performed is shown inTable 3. In case PGS discovered Down syndrome embryoswith 100% certainty without affecting pregnancy chances,201 (83%) women favor PGS.
If PGS was 100% effective in detecting Down syndromeembryos, but lowered pregnancy chances from one out offive to one out of seven, 89 (36%) women would opt for PGS.
If PGS would reduce the risk of a Down syndrome preg-nancy by 80% (i.e., PGS lowered the chance of having a childwith Down syndrome from 1 in 200 to 1 in 1,000) without af-fecting pregnancy chances, 183 (75%) women would preferto have PGS, and 57 (31%) of them would refrain from pre-natal testing afterwards. Of the women who did chose prena-tal testing afterward, 23% would only consider noninvasivetests, 4% would immediately opt for an invasive test, and73% would start with a noninvasive test possibly followedby an invasive test depending on the outcome of the noninva-sive test.
Seventy-two (30%) women answered that they would nothave any form of prenatal testing in case of a pregnancy. Asignificant proportion of them (42 [58%] women) was willingto undergo PGS if PGS would not negatively affect preg-nancy chances (Table 3). The 15 women that did not wantprenatal testing because they did not to want to decide on
TABLE 2Preferences for Down syndrome testing (%).
If you were pregnant at this moment, wouldyou consider testing for Down syndrome?Yes 165 (68%)
Only noninvasive testing 23 (14%)Noninvasive testing, possibly
followed by invasive testing128 (78%)
Invasive testing 14 (8%)No 72 (30%)
Every child is welcome 38 (53%)I don’t want to have to decide
about termination of pregnancy15 (21%)
Other reason 9 (12%)Unknown 10 (14%)
Unknown/unclear 7 (3%)
Twisk. PGS versus prenatal testing. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
TAW g after PGS, for different scenarios.
rformed?)
Refrain from prenatal testing?N (%)
A %) n.a.a
%) n.a.
%) 57 (31%)
W %) n.a.
%) n.a.
%) n.a.
W %) n.a.
%) n.a.
%) 23 (16%)
Na
b
T
Fertilityand
Sterility
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BLE 3omen’s willingness to have PGS performed and willingness to refrain from prenatal testin
ote: PGS ¼ preimplant genetic screening.n.a.: not asked.That is, lowers the chance of having a child with Down syndrome from 1 in 200 to 1 in 1,000.
wisk. PGS versus prenatal testing. Fertil Steril 2007.
termination of a pregnancy, were significantly more willingto undergo PGS than the 57 women that do not want prenataldiagnostic tests (PND) for other reasons (14 [93%] vs. 28[49%], OR 14.5, 95% CI 1.79 to 117.7).
Of the 165 women that answered they would have anyform of prenatal testing in case of a pregnancy, 154 (93%)were willing to undergo PGS if it would not negatively affectpregnancy chances and would detect all Down syndrome em-bryos. If PGS would lower their pregnancy chance 72 (44%)were willing to undergo PGS. If PGS would not detect allDown syndrome embryos 144 (87%) women were still will-ing to undergo PGS, and 23 (16%) of them would refrainfrom prenatal testing afterwards (Table 3).
As presented above, out of a total of 201 (44%) womenwanting PGS, 89 were willing to accept a lower pregnancychance after PGS while 112 (56%) were not willing to acceptthis. Subgroup analysis showed that women who had alreadyundergone one or more IVF treatments in the past were sig-nificantly less inclined to accept a negative effect on theirpregnancy chance (40% vs. 58%, OR 0.45, 95% CI 0.2 to0.85). There were no significant differences between agegroups (above or below 36 years) or between women withand without children (Table 4).
808 Twisk et al. PGS versus prenatal testing
A second subgroup analysis showed no significant differ-ences between age groups (above or below 36 years), bet-ween women with and without children, and betweenwomen with and without IVF/ICSI treatments in the past inwomen who were still in favor of PGS, even when PGS isnot able to prevent all Down syndrome pregnancies, com-pared with women who only want PGS if this prevents allDown syndrome pregnancies (Table 4).
DISCUSSION
The results of this study show that most women opt for pre-implantation genetic screening as an alternative to prenataltesting for Down syndrome if this would have no negative ef-fect on their pregnancy chances. Women are still favorabletoward PGS if this would not detect all Down syndrome em-bryos but would reduce their risk of having a child with Downsyndrome with 80% (i.e., PGS lowered the chance of havinga child with Down syndrome from 1 in 200 to 1 in 1,000).Thirty-six percent of the women would even accept a lowerpregnancy chance, provided that PGS would prevent allDown syndrome pregnancies. Almost half (42%) of thewomen who would refrain from any form of prenatal testingfor Down syndrome, would choose PGS if this was offered.
TABLE 4Results of univariable and multivariable logistic regression analysis on willingness of PGS in womenthat are in favor of PGS (n [ 201).
Would you still want PGS if this lowersyour pregnancy chance? OR 95%CI P value OR 95%CI P value
Clinical parameter Answer ‘‘yes’’Age <36 years 43% 1.00 1.00Age R36 years 49% 1.28 0.73–2.26 .39 1.21 0.67–2.21 .53Nulliparous women 42% 1.00 1.00Multiparous women 58% 1.93 0.97–3.83 .06 1.88 0.92–3.84 .08No IVF treatments in the past 58% 1.00 1.001 or more IVF treatments in the past 40% 0.47 0.25–0.87 .02 .45 0.24–0.85 .01
Would you still want PGS if this wouldgive a risk reduction of 80% of havinga Down syndrome child? OR 95%CI P value OR 95%CI P value
Clinical parameter Answer ‘yes’Age <36 years 93% 1.00 1.00Age R36 years 91% 0.76 0.27–2.11 .60 0.66 0.23–1.91 .45Nulliparous women 92% 1.00 1.00Multiparous women 93% 1.21 0.33–4.46 .77 1.36 0.35–5.22 .67No IVF treatments in the past 87% 1.00 1.001 or more IVF treatments in the past 94% 2.45 0.88–6.88 .09 2.55 0.90–7.21 .08
Note: OR ¼ odds ratio; CI ¼ 95% conficence intervals; PGS, preimplant genetic screening.
Twisk. PGS versus prenatal testing. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
A possible limitation of our study is that 29% of the womendid not return the questionnaire. Women who did not respondcould differ systematically in their preferences from thosewho did respond.
Another limitation of our study could be the fact thatwomen were not informed about the cost of PGS. Womenmight change their attitude to PGS once they have to paythe cost of the procedure.
The 83% of women wanting PGS if this would prevent allDown syndrome pregnancies without a negative influence ontheir pregnancy chances is slightly lower than the 96% re-ported by Chamayou et al. (19). In the latter study, the proba-bility of lowering pregnancy chances and the chance ofmisdiagnosis was not mentioned. The women in our studycould be slightly less favorable toward PGS because we didaddress these topics. Another explanation for the differencecould be that in the study by Chamayou et al. (19) womenwere not given information on the possibility of noninvasiveprenatal testing. Because invasive prenatal testing carriesthe risk of inducing a miscarriage, it could be women weremore favorable to PGS because this alternative does not jeop-ardize their pregnancy.
Similar studies have been performed on patients’ prefer-ences for preimplantation genetic diagnosis (PGD) as an al-ternative to prenatal diagnosis. PGD uses a nearly identicaltechnology as PGS, with embryos being screened for a spe-cific genetic disorder. It is used to prevent the birth of af-fected children in couples with a high risk of transmittinggenetic disorders. These risks are much higher than therisk of having a child with Down syndrome. However,women in these studies have to take the similar pros andcons into consideration of testing before versus during theirpregnancy. The studies performed differ in study populationand the way preferences were asked for, resulting in a largevariation in preferences for PGD in these studies, rangingfrom 28% to 77% (19, 20–25). PGD is performed mostlyin fertile couples, and the main reason for not preferringPGD is the low pregnancy chance after IVF/ICSI. If onlywomen with an IVF/ICSI indication were asked, 70% ofthe women expressed a preference for PGD (21), which islower than the results found in our study.
Most women would not want PGS if this lowered their preg-nancy chance, even if PGS discovered all Down syndromeembryos. This is not surprising, because most women withan IVF/ICSI indication suffer from a long period of infertility.In case of a pregnancy, they are more cautious with any pro-cedure that might jeopardize their pregnancy. It has been re-ported that women who became pregnant after IVF/ICSI areless willing to undergo invasive testing because of the riskof a procedural related miscarriage. Instead, they prefer to un-dergo noninvasive tests, although these tests provide themwith a risk assessment instead of a definite diagnosis (26, 27).
Fifty-eight percent of the women who did not want prenataltesting in case of a pregnancy would want PGS. The reason forthis seems to be that with PGS a woman does not have to decide
Fertility and Sterility�
about termination of her pregnancy. The women who did notwant PND for the reason ‘‘I don’t want to have to decide abouttermination of pregnancy’’ were more willing to undergo PGSthan women that did not want PND for other reasons.
If PGS would reduce the risk of a Down syndrome preg-nancy with 80%, 75% of the women would prefer to undergoPGS, and 69% of them would opt for prenatal testing after-wards. However, women should be advised that the positivepredictive value of the prenatal test is lowered with this se-quential testing (28, 29). Because the risk of carrying a childwith Down syndrome is lower after PGS, the percentage offalse-positive results with noninvasive testing increases.
In conclusion, we have demonstrated a preference for PGSas an alternative to prenatal testing for Down syndromeamong subfertile women with an IVF/ICSI indication. Theacceptability of PGS depends largely on its effect on preg-nancy chances and, to a lower extent, on its effectiveness indetecting Down syndrome embryos. Before PGS can be of-fered as an alternative to prenatal testing, trials need to be per-formed to assess the sensitivity of PGS in detecting Downsyndrome embryos and its effect on pregnancy rates.
Acknowledgment: The authors wish to thank Moira Muller for her helpful
comments.
REFERENCES1. Penrose LS. The relative effects of paternal and maternal age in mongo-
lism. J Genet 1933;27:219.
2. Kuliev A, Jackson L, Froster U, Brambati B, Simpson JL, Verlinsky Y,
et al. Chorionic villus sampling safety. Report of World Health Organi-
zation/EURO meeting in association with the Seventh International Con-
ference on Early Prenatal Diagnosis of Genetic Diseases, Tel-Aviv,
Israel, May 21, 1994. Am J Obstet Gynecol 1996;174:807–11.
3. Leschot NJ, Verjaal M, Treffers PE. Risks of midtrimester amniocentesis;
assessment in 3000 pregnancies. Br J Obstet Gynaecol 1985;92:804–7.
4. Tabor A, Philip J, Madsen M, Bang J, Obel EB, Norgaard-Pedersen B.
Randomised controlled trial of genetic amniocentesis in 4606 low-risk
27. Geipel A, Berg C, Katalinic A, Ludwig M, Germer U, Diedrich K, et al.
Different preferences for prenatal diagnosis in pregnancies following
assisted reproduction versus spontaneous conception. Reprod Biomed
Online 2004;8:119–24.
28. Thilaganathan B, Slack A, Wathen NC. Effect of first-trimester nu-
chal translucency on second-trimester maternal serum biochemical
screening for Down’s syndrome. Ultrasound Obstet Gynecol 1997;10:
261–4.
29. Hackshaw AK, Wald NJ. Inaccurate estimation of risk in second trimes-
ter serum screening for Down syndrome among women who have
already had first trimester screening. Prenat Diagn 2001;21:741–6.
Vol. 88, No. 4, October 2007
Oocyte dysmorphism is not associated with aneuploidyin the developing embryoKayhan Yakin, M.D., Basak Balaban, B.Sc., Aycan Isiklar, B.Sc., and Bulent Urman, M.D.
Assisted Reproduction Unit, American Hospital of Istanbul, Turkey
Objective: Controlled ovarian hyperstimulation yields numerous oocytes with different morphologic features. Thepurpose of this study was to assess the effect of oocyte dysmorphism on the development and ploidy status of thederived embryo.Design: Retrospective study.Setting: Private assisted reproduction unit.Patient(s): A retrospective analysis was performed on 616 oocytes and 266 embryos of 65 infertile couples whohad undergone preimplantation genetic diagnosis and aneuploidy screening for repeated implantation failures.Intervention(s): Oocytes with normal and abnormal morphology and the derived embryos were compared in termsof fertilization, cleavage, and blastocyst formation rates as well as the ploidy status.Result(s): Oocyte dysmorphism did not have an adverse effect on fertilization and cleavage rates and quality ofday 3 embryos. Single cytoplasmic and multiple abnormalities of the oocyte significantly decreased the blastocystformation rate of the cleaving embryo. Oocyte dysmorphism did not have a significant association with embryonicaneuploidy.Conclusions(s): Oocyte dysmorphism was not associated with a higher risk of aneuploidy in the developingembryo. Cytoplasmic dysmorphism or multiple morphologic abnormalities of the oocyte adversely affects theblastocyst formation potential of the derived embryo. (Fertil Steril� 2007;88:811–6. �2007 by American Societyfor Reproductive Medicine.)
Gamete quality has been regarded as a variable influencingthe implantation potential of the derived human embryo.Male and female gametes are assessed using subjective mor-phologic criteria that are often crude and inefficient.
To date, there have been numerous reports on the impact ofoocyte morphology on embryo development. Earlier reportssuggested that oocyte defects were associated with poorfertilization and clinical outcome in intracytoplasmic sperminjection (ICSI) cycles (1). Alikani et al. (2) showed thatwomen with dysmorphic oocytes have a higher rate of mis-carriage. On the contrary, several studies reported that oocytemorphology was insignificant in terms of fertilization andembryo quality (3, 4). Various morphologic deviations ofthe oocyte have been studied, and only severe cytoplasmic in-clusions and central granulation were found to affect embry-onic quality and the ploidy status. Cytoplasmic abnormalitiesin particular were questioned about their effects on fertiliza-tion, cleavage, and blastocyst formation. De Sutter et al. (3)studied the developmental outcome of oocytes with cytoplas-mic abnormalities such as dark cytoplasm, refractile bodies,dark incorporations, spots, and single or multiple vacuoliza-tion in the cytoplasm, and found that none of these cytoplas-mic abnormalities were related to fertilization or embryoquality. Although there was an increased degeneration rate
Received July 21, 2006; revised and accepted December 5, 2006.
Reprint requests: Kayhan Yakin, M.D., Guzelbahce sok No. 20, Nisantasi,
Istanbul (FAX: þ90 212 3112339; E-mail: kayhany@amerikanhastanesi.
com.tr).
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.031 Copyright ª2007 American
after ICSI of oocytes with multiple vacuoles, the differencewas not significant. Serhal et al. (5) showed normal fertiliza-tion and embryo development but lower implantationpotential when oocytes showed cytoplasmic granulationsor inclusions. Xia (6) also showed significantly lower fertil-ization rates, embryo cleavage rates, and lower embryo qual-ity for the group of oocytes with cytoplasmic inclusions whencompared with the group of oocytes with normal cytoplasm.However, there is limited data on the relationship betweenoocyte dysmorphism and the chromosomal status of the de-rived embryos (7–10).
The purpose of this study was to compare fertilization,cleavage, and blastocyst formation rates as well as the ploidystatus of embryos derived form oocytes with normal and ab-normal morphology in couples who had preimplantationgenetic diagnosis and aneuploidy screening (PGD-AS) forrepeated implantation failures.
MATERIAL AND METHODS
Study Group
A retrospective evaluation of oocyte morphology, laboratoryperformance, and ploidy status in the resultant embryos wasundertaken in 65 couples that scheduled to undergo PGD-ASfor recurrent implantation failures in assisted reproductivetechniques. Recurrent implantation failure has been de-scribed in couples with three or more failures following em-bryo transfer. The female age was warranted to be <38 years
Fertility and Sterility� Vol. 88, No. 4, October 2007 811Society for Reproductive Medicine, Published by Elsevier Inc.
to exclude advanced maternal age as a confounding variable.Cases with surgical sperm extraction were also excludedfrom the study group. Pretreatment cytogenetic analyses ofall couples were normal.
Controlled Ovarian Hyperstimulation
Controlled ovarian hyperstimulation was undertaken follow-ing down-regulation with a midluteal GnRH analogue proto-col (Lucrin, Abbott, France) combined with 150–450 IUrecombinant follicle stimulating hormone (Gonal F, Serono,Bari, Italy). Final maturation of the oocytes was triggeredwith 10,000 IU human chorionic gonadotropin (hCG)(Pregnyl, Serono, Italy) when the leading follicle reached20 mm with two or more accompanying follicles largerthan 16 mm in mean diameter. Oocytes were retrieved36 hours after the injection of hCG.
Oocyte Morphology Assessment
Oocytes were dissected mechanically from the surroundingcumulus cells, and following incubation for 2–4 hours theywere treated with 10 IU/mL hyaluronidase (Hyase, Vitrolife,Gothenburg, Sweden). Oocyte morphology was assessed us-ing an Olympus inverted microscope IX-70 with Hoffmannmodulation contrast system (Olympus Optical Co., Tokyo,Japan), under 400� magnification. The oocytes were classi-fied as normal and abnormal according to their morphologicappearance. Normal oocytes showed clear cytoplasm withuniform texture and homogenous fine granularity, a roundor ovoid first polar body with a smooth surface, and perivitel-line space of normal size (11).
Oocyte dysmorphism was classified as: [1] extracytoplas-mic abnormalities: dark zona and large perivitelline space,[2] cytoplasmic abnormalities: dark, granular or vacuolar cy-toplasm, and refractile bodies, [3] shape abnormalities, [4]multiple abnormalities.
Intracytoplasmic sperm injection was performed in all cy-cles. In vitro culture of embryos was undertaken as previouslydescribed by Balaban et al. (4). Sequential media system (G1
and G2 media, Vitrolife, Gothenburg, Sweden) designed forfurther embryonic development was used.
Preimplantation Genetic Diagnosis and AneuploidyScreening
On the third day of culture, embryos suitable for blastomerebiopsy were determined according to their blastomere num-ber and quality. Only embryos with five or more blastomereswere biopsied following zonal opening using a 1.48-mm in-frared diode laser (Hamilton-Thorne Biosciences, Beverly,MA). Blastomere biopsy was performed within calcium–magnesium-free medium (EB-10, Vitrolife). A single blasto-mere containing a clearly visible nucleus was removed fromeach embryo. Multicolor fluorescence in situ hybridization(FISH) analysis was performed using appropriate probes(Vysis, Abbott Molecular Inc., Des Plaines, IL) for the
812 Yakin et al. Oocyte dysmorphism and aneuploidy
simultaneous detection of chromosomes 13, 18, 21, X, andY. A fluorescence microscope equipped with recommendedfilter set for configuration of PGD was used for observationand the signals were determined under 1,000� using fluores-cence immersion oil. Following blastomere biopsy, theembryos were cultured in vitro until the blastocyst stageand transferred on day 5.
Data Analysis
During the period from fertilization to embryo transfer,cleavage rate, embryo quality, blastocyst formation, andFISH results of the biopsied blastomeres were recorded. Allthese parameters were compared with oocyte morphology.Statistical analysis was performed using SPSS version 11.5.Chi-square and Fisher’s exact tests were applied where ap-propriate. A value of P < .05 was accepted as significant.
The study was approved by the institutional review boardof the American Hospital.
RESULTS
Only one treatment cycle for each couple was analyzed for 65couples. The mean female age in the study group was 33.5years (27–37 years). A total of 616 oocytes were retrievedand ICSI was performed on 456 metaphase II oocytes. Thecharacteristics of the study group are summarized in Table 1.A mean number of 1.9 embryos were transferred yielding 15pregnancies (23.1%). The clinical pregnancy rate was 18.5%and the implantation rate was 9.9%.
Fertilization Rate
Fertilization rates were similar for morphologically normaland abnormal oocytes (76.3% vs. 70.8%, respectively,
TABLE 1Characteristics of the study group.
No. of patients 65Female age (y) 33.5 � 3.0Total oocytes
Note: Numbers in parenthesis are mean � standarddeviations.
Yakin. Oocyte dysmorphism and aneuploidy. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
P>.05). Although statistically not significant, oocytes withcytoplasmic abnormalities showed the lowest fertilizationrate (64.7%) (Table 2). There was also no significant differ-ence between fertilization rates of oocytes with single or mul-tiple abnormalities.
Embryo Cleavage and Quality
Although cleavage rates on day 3 were lower in embryos de-rived from oocytes with cytoplasmic and multiple abnormal-ities, there was no statistically significant difference betweenembryos derived from morphologically normal and abnormaloocytes (98.2% vs. 93.7%, respectively) (Table 2).
Morphologically normal oocytes developed into grade Iand II embryos at a higher rate (66.7%) than oocytes withcytoplasmic (42.8%) or multiple abnormalities (51.3%);however, the difference was not statistically significant.Overall, abnormal oocyte morphology did not have a signifi-cant effect on cleavage rates or quality of day 3 embryos.
Blastocyst Formation
Blastocyst formation rates were similar for embryos derivedfrom morphologically normal and abnormal oocytes (35.2%vs. 29.8%, respectively, P>.05). Blastocyst formation rate forembryos derived from oocytes with cytoplasmic or multipleabnormalities were significantly lower compared with othergroups (normal vs. cytoplasmic, P¼.025; normal vs. multi-ple, P¼.04) (Table 2). In contrast, there was no statisticallysignificant difference between embryos developed from oo-cytes with normal morphology, shape abnormality, or extrac-
Fertility and Sterility�
ytoplasmic abnormalities. The rate of grade 1–2 blastocysts(73.7% vs. 86.8%) and hatching (10.5% vs. 5.3%) blastocystswere also similar for embryos derived from morphologicallynormal and abnormal oocytes.
Cytoplasmic and multiple abnormalities significantly im-paired blastocyst formation.
Aneuploidy Rate
A total of 266 embryos were biopsied, and successful FISHresults were obtained in 248 (93.2%). Total aneuploidy ratein all diagnosed embryos was 50%. Embryos that developedfrom oocytes with normal morphology showed a 41.9% (18of 43) aneuploidy rate. Embryos that developed from oocyteswith different types of morphologic abnormalities showeda higher rate of aneuploidy as 51.7% (106 of 205). However,this was not statistically significant.
When embryos were categorized according to the morpho-logic abnormalities of the oocytes which they had beenderived from, the lowest aneuploidy rate was seen in embryosderived from oocytes with only shape abnormalities (Table 3).Except the cytoplasmic abnormality group, all the othergroups showed similar rates of aneuploidy. Embryos de-rived from oocytes with cytoplasmic and multiple abnor-malities showed the highest rate of aneuploidy (60.0%and 61.8%, respectively); however, the difference was notstatistically significant (normal vs. cytoplasmic, P¼.074;normal vs. multiple, P¼.083) (Table 3).
Therefore, abnormal oocyte morphology did not have a sig-nificant effect on aneuploidy rate in the cleaving embryo.
TABLE 2Fertilization rates, embryo quality, and blastocyst formation of embryos derived from oocytes withnormal and abnormal morphology.
Note: Blastocyst formation rate for embryos derived from oocytes with cytoplasmic or multiple abnormalities weresignificantly lower compared to other groups (anormal vs. cytoplasmic, P¼ .025; bnormal vs. multiple, P ¼ .04).
Yakin. Oocyte dysmorphism and aneuploidy. Fertil Steril 2007.
813
TABLE 3FISH results of the embryos derived from oocytes with normal and abnormal morphology.
Yakin. Oocyte dysmorphism and aneuploidy. Fertil Steril 2007.
Blastocyst Formation and Aneuploidy
Of the 124 euploid embryos, 70 (56.5%) developed into blas-tocysts. On the other hand, out of 124 aneuploid embryos,only 25 (20.2%) developed into blastocysts. Blastocyst for-mation rate was significantly higher for euploid embryos(P¼.0001). Blastocyst formation rate of euploid and aneu-ploidy embryos according to the morphology of the oocytesthey had been derived from is presented in Table 4.
Although the blastocyst formation rate was lower ineuploid embryos derived from oocytes with cytoplasmicand multiple abnormalities, there was no statistically signifi-cant difference between embryos derived from oocytes withnormal or abnormal morphology of all types. This was alsotrue for aneuploid embryos.
DISCUSSION
In this study we showed that oocyte dysmorphism did nothave an adverse effect on fertilization and cleavage ratesand quality of day 3 embryos. Single cytoplasmic and multi-
814 Yakin et al. Oocyte dysmorphism and aneuploidy
ple abnormalities of the oocyte decreased the blastocyst for-mation rate of the cleaving embryo. This cannot be explainedby the abnormal genetic composition of the resultant embryobecause oocyte dysmorphism did not have a significant asso-ciation with embryonic aneuploidy. We may speculate thatchromosomal constitution may be normal for these embryos,but other defects like gene expression alterations may lead tofailure in blastocyst formation. Wells et al. (12) demonstratedan association between certain forms of abnormal oocyte andzygote morphology and disturbances of gene activity.
We previously reported that in couples undergoing ICSI,abnormal oocyte morphology (both extracytoplasmic and cy-toplasmic) was not associated with decreased fertilizationrates or unfavorable embryo quality (4). Furthermore, trans-fer of embryos derived from dysmorphic oocytes yieldedsimilar clinical pregnancy and implantation rates comparedwith embryos derived from normal oocytes. However, oo-cytes with severe cytoplasmic defects such as different typesof vacuolization and excessive central or whole granulationswere not studied.
TABLE 4Blastocyst formation rate of the derived euploid and aneuploidy embryos according to the oocytemorphology.
Yakin. Oocyte dysmorphism and aneuploidy. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
Ebner et al. (13) showed that cytoplasmic abnormalities aswell as the extracytoplasmic abnormalities did not affect thefertilization rate, mean number of blastomeres, and frag-ments on day 2. However, in a recent study, the same groupshowed that the presence of multiple and large vacuoleswas associated with poor embryo development (14). Otsukiet al. (15) also showed that the presence of vacuoles in oocytecytoplasm, which represents smooth endoplasmic reticulumclusters, significantly impaired embryo quality and was asso-ciated with a low chance of pregnancy.
Loutradis et al. (16) suggested that it is not the type but theseverity of the cytoplasmic defect that affects fertilizationrates, embryo quality, and its developmental potential. Al-though oocytes with dark cytoplasm, or many vacuoles, orfragments in the cytoplasm showed similar fertilization ca-pacity and yielded embryos of similar quality, when triple cy-toplasmic defects such as dark cytoplasm, many vacuoles andfragments in the cytoplasm were combined in the same oo-cyte, embryo quality was significantly impaired.
Kahraman et al. (17) reported significantly decreased preg-nancy and implantation rates following the transfer of em-bryos developed from oocytes with centrally locatedgranular cytoplasm. The investigators also reported a higherrisk of aneuploidy in these embryos.
In a prospective study of 831 oocytes, Verlinsky et al. (18)showed that the first polar body morphology was not a reliablepredictor of the development potential of the resulting em-bryo in terms of embryo quality or cleavage rates. The inves-tigators also reported that the first polar body morphologywas not associated with the chromosomally normality ofthe developing embryo.
Meriano et al. (19) showed that oocyte dysmorphism wasa negative predictor for pregnancy and implantation onlywhen it was a repetitive finding. Intracytoplasmic organelleclustering was reported to be the only significant repetitiveabnormality. Although fertilization rate, embryo cleavagerate, and quality were not affected, implantation rates weresignificantly lower for that particular group of patients.
It is not clear how cytoplasmic abnormalities compromiseembryo development. The presence of a normal nuclear ge-netic material will not guarantee the potential of the oocyteto develop into a healthy embryo and fetus. While resumptionof the first meiotic division takes place and the nucleus entersmetaphase II stage, synchronously cytoplasmic maturationgoes on achieving zonal ability to release calcium and corti-cal granules, mitochondrial changes, protein synthesis, andcytoskeletal changes (20). However, nuclear or cytoplasmicmaturation can proceed one without the other, resulting incompromised embryos (21–23). Defects in the cytoplasmicmachinery may block gene activation, cleavage, and cell de-terminations, which also take place in the oocyte cytoplasm.In humans, successful male pronuclear assembly and fertil-ization are largely determined by the quality of the oocyte cy-toplasm (24). Sperm chromosome decondensation, release ofprotamines, DNA repair, chromosome remodeling, assembly
Fertility and Sterility�
of organelles, and the nuclear envelope around the reprog-rammed haploid chromosomes are all accomplished byspecific proteins accumulated in the cytoplasm (25, 26). Oo-plasmic factors may play important roles in the continued de-velopment of the zygote, particularly during early cleavage,when transcription of the embryonic genome is minimal (27).
There are two major drawbacks of this study. First, as wedid not perform polar body analysis, we cannot state forsure that embryonic aneuploidy was related to the oocyte. Ex-clusion of men with azoospermia may have minimized butnot totally refused the impact of spermatozoa-related embry-onic aneuploidy. Second, it was not possible to compare theclinical outcome in terms of implantation or pregnancy ratesbecause more than one embryo was transferred, thereforemaking it impossible to identify which embryo had suc-ceeded to implant.
Another point that may be questioned can be the singleblastomere biopsy for the genetic analysis of the cleavagestage embryo. The major concern in single blastomere biopsytechnique is the risk of mosaicism. However, owing to therisk of decreased implantation rates and because so far thereis no convincing data demonstrating the safety of double oversingle blastomere biopsy, we perform single blastomerebiopsy in our center (28–30).
In conclusion, we showed that oocyte dysmorphism wasnot associated with a higher risk of aneuploidy in the devel-oping embryo. Although statistically insignificant, oocyteswith cytoplasmic dysmorphism and oocytes showing multi-ple morphologic abnormalities were associated with lowerfertilization and cleavage rates, as well as higher risk of aneu-ploidy in the derived embryos. Blastocyst formation rate wassignificantly lower for embryos derived from oocytes withcytoplasmic or multiple abnormalities.
REFERENCES1. Sousa M, Tesarik J. Ultrastructural analysis of fertilization failure after
trates the rate of ovarian follicular development. Proc Natl Acad Sci USA
2002;99:2890–4.
24. Banerjee S, Lamond S, McMahon A, Campbell S, Nargung G. Does blas-
tocyst culture eliminate paternal chromosomal defects and select good
embryos?: inheritance of an abnormal paternal genome following
ICSI. Hum Reprod 2000;15:2455–9.
25. Collas P. Cytoplasmic control of nuclear assembly. Reprod Fertil Dev
1998;10:581–92.
26. Collas P, Poccia D. Remodelling the sperm nucleus into a male pronu-
cleus at fertilisation. Theriogenology 1998;49:67–81.
27. Cohen J, Scott R, Alikani M, Schimmel T, Munne S, Levron J, et al. Oo-
plasmic transfer in mature human oocytes. Mol Hum Reprod 1998;4:
269–80.
28. Munne S, Sable D, Cohen J. ‘‘Floating denominators’’—effect of verifi-
cation of bias on accuracy estimates for preimplantation genetic diagno-
sis. Reply of the authors. Fertil Steril 2003;79:1042.
29. Van de Velde H, De Vos A, Sermon K, Staessen C, De Rycke M, Van
Assche E, et al. Embryo implantation after biopsy of one or two cells
from cleavage stage embryos with a view to preimplantaion genetic
diagnosis. Prenat Diagn 2000;20:1030–7.
30. Edwards RG, Hansis C. Initial differentiation of blastomeres in 4-cell
human embryos and its significance for early embryogenesis and implan-
tation. Reprod Biomed Online 2005;11:206–18.
Vol. 88, No. 4, October 2007
IN VITRO FERTILIZATION
Social concerns of women undergoing infertilitytreatmentPeter S. Finamore, M.A., M.D.,a David B. Seifer, M.D.,b Cande V. Ananth, Ph.D., M.P.H.,c andSandra R. Leiblum, Ph.D.d
a Department of Obstetrics, Gynecology and Reproductive Sciences, c Division of Epidemiology and Biostatistics, andd Department of Psychiatry, UMDNJ–Robert Wood Johnson Medical School, New Brunswick, New Jersey; and b Genesis
Fertility and Reproductive Medicine Maimonides Medical Center, Brooklyn, New York
Objective: Our study was undertaken to determine [1] what women are disclosing to their employer with regard totheir infertility, [2] what demographic characteristics are associated with women who are more likely to disclose,and [3] if there is an association between disclosure and lowering one’s stress. We hypothesize that, in certainwomen, disclosure may lower stress, and therefore increase success rate of in vitro fertilization.Design: Cross-sectional questionnaire.Setting: University Infertility Treatment Center.Patient(s): We handed out a questionnaire to patients being evaluated and treated for infertility over a 6-monthperiod. A total of 267 questionnaires were handed out and all were collected.Main Outcome Measure(s): We collected demographic data as well as information regarding privacy, disclosure,and stress. We then compared women who disclose to their employer that they are being seen by an infertility spe-cialist to those women who do not disclose. We also measured stress and determined if higher stress level wasassociated with disclosure or nondisclosure.Result(s): Most women who did disclose did so because they needed a reason to leave work for frequent doctorvisits. Among women who did not disclose, the main reason for nondisclosure was to protect their privacy. Womenwith a high school education were more likely to disclose compared with those with a college and postgraduateeducation. African American/Caribbean American women were least likely to disclose. Those who were out ofwork more often because of their infertility were more likely to disclose. There was not an association withdisclosure and decreasing stress level.Conclusion(s): Women who did or did not disclose their infertility status to their employer were different with re-gard to level of education, race/ethnicity, and number of visits per month to the doctor. The decision to disclosedoes not seem to have a significant impact on stress. (Fertil Steril� 2007;88:817–21. �2007 by American Societyfor Reproductive Medicine.)
In 1998, the Supreme Court of the United States of Americaruled that reproduction is a ‘‘major life activity’’ covered un-der the Americans with Disabilities Act (1). Since then, theFamily Building Act was put into legislation (2), which pro-vides insurance coverage for women undergoing treatmentfor infertility. The state of New Jersey is one of 13 states inthe United States that currently provides mandatory insur-ance coverage for infertility treatment. In another two statesinsurance coverage is not mandatory; however, companiesare required to offer coverage for infertility treatment. Themandate includes coverage for up to four egg retrievals inwomen younger than 45 years of age. These mandates verify
Received August 5, 2006; revised and accepted December 29, 2006.
Reprint requests to: Peter S. Finamore, M.A., M.D., 3 Cooper Plaza,
Cooper University Hospital, Camden, NJ 08103 (E-mail: pfinamore@
hotmail.com).
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2007.01.009 Copyright ª2007 America
that federal and local governments are supporting womenundergoing treatment for infertility.
Women being treated or evaluated for infertility must havea flexible work schedule and be willing and able to go forfrequent office visits. This can lead to hours and sometimesdays missed at work. If an employer is not supportive of thesefrequent absences, the situation can lead to increased stresslevels, which in turn, can affect fertility. Research suggeststhat stress, depression, anxiety, and other negative psycho-logic feelings result in poorer outcomes for patients undergo-ing IVF (3–6).
Women treated for infertility have many social concernswith regard to their infertility. There also can be profoundemotional stress as a result of infertility (7). One of themore controversial issues for many couples concerns privacyand disclosure with respect to their infertility and/or
Fertility and Sterility� Vol. 88, No. 4, October 2007 817n Society for Reproductive Medicine, Published by Elsevier Inc.
infertility treatments. This study was designed to investigatesome of these social concerns. Our objectives were as fol-lows: [1] to determine what women disclose to their em-ployers, [2] to compare women who do disclose theirinfertility to their employer with women who do not disclose,and [3] to evaluate if there is an association between reportedstress and the decision to either disclose or not to disclose in-fertility treatment. We hypothesize that certain women couldlower their stress by disclosing that they are currently beingtreated for infertility, and by doing so, they may improve theiroutcomes of infertility treatment.
MATERIALS AND METHODS
Internal review board approval was obtained for this study.All patients who presented to the University Center for Re-productive Endocrinology and Fertility at UMDNJ–RobertWood Johnson Medical School, New Jersey, for a 6-monthperiod (April to September 2004) were invited to participateand complete our questionnaire. The questionnaire consistedof 38 questions, and was designed to take approximately 10minutes. Patients were asked to complete the questionnairewhile waiting for the doctor. All surveys handed out were col-lected from each patient as they were called into the doctor’soffice. The survey was given to women who sought first-timeevaluation for infertility as well as those who were currentlyundergoing treatment.
The survey contained questions regarding demographicdata, including age, years with partner, education (highschool, college, postgraduate), race/ethnicity (Caucasian, Af-rican American/Caribbean American, Asian, other), and reli-gion (Christian, Hindu/Buddhist, Jewish, other). Questionsaddressing employment satisfaction, freedom to leave workfor frequent doctor visits, reproductive history, infertilitytreatments, as well as questions on disclosure and perceivedlevel of support were included in the questionnaire. Finally,the questionnaire included questions regarding stress leveland privacy considerations.
Included in the survey was a validated global measure ofstress known as the Perceived Stress Scale (8). The originalscale is a 14-item instrument; however Cohen et al (8) provedthe reliability and validity of a four-item version, which wasused in this study.
Satisfaction with employment as well as freedom to comeand go from work was assessed. Scores were assigned ona 1 to 7 scale, with 1 being equivalent to ‘‘not satisfied,’’ 3to ‘‘slightly satisfied,’’ 5 to ‘‘moderately satisfied,’’ and 7 to‘‘very satisfied.’’ A similar scoring pattern was assigned tofreedom to come and go from work, with 1 being equivalentto ‘‘no freedom,’’ 3 to ‘‘some freedom,’’ 5 to ‘‘moderate free-dom,’’ and 7 to ‘‘total freedom.’’
Continuous variables such as patient’s age, PerceivedStress Scale scores, and so on, were analyzed using meanand SD. The Student’s t test was used to examine differencesin characteristics between women who did and did not dis-
818 Finamore et al. Social concerns of women undergoin
close their infertility status. Interval data such as gravidity,parity, and number of people disclosed were analyzed as pro-portion of total subjects who responded to the survey. A chi-square test was applied to compare differences in proportionsbetween women who did and did not disclose their infertilitystatus, and evaluated associations based on odds ratio (OR)with a 95% confidence interval (CI). All tests of hypothesiswere two tailed, with a type 1 error rate set at 5%. Incompletesurveys were not analyzed.
RESULTS
A total of 267 questionnaires were handed out and collected.They were divided into groups based on whether the patientdid or did not disclose to their employer that they were under-going treatment for infertility. Forty-three percent (114 of267) of the women surveyed did not disclose, 32% (85 of267) did disclose, 12% (33 of 267) surveyed stated disclosurewas not applicable (i.e., they were either unemployed, home-makers, or were self-employed), and 12% (35 of 267) wereincomplete surveys. Therefore, there were 199 subjects inwhom analysis could be conducted to compare women whodisclose and women who do not disclose, excluding thosein which disclosure was not applicable and the incompletesurveys.
Subjects were asked for their reasons for disclosure or non-disclosure, and were permitted to give more than one reason.Analysis of the 114 subjects who did not disclose revealedthat over four fifths (82%; 94 of 114) indicated the reasonwas because they were protecting their privacy, 25% (28 of114) were protecting the privacy of their spouse, 18% (20of 114) were worried disclosing would hurt their chance ofpromotion, 10% (11 of 114) did not want special treatment,and 7% (8 of 114) were embarrassed. Among women that dis-closed to their employer, 79% (67 of 85) did so because theyneeded to leave work for infertility-related appointments,45% (38 of 85) disclosed because they had a close relation-ship with their employer, 41% (35 of 85) had nothing tohide, and 8% (7 of 85) desired additional support.
We considered whether there was a correlation betweeneducation and disclosure. Of the 199 surveys analyzed 1subject did not answer the question regarding level of ed-ucation, 18 were high school educated, 106 were collegeeducated, and 74 were postgraduate educated. For thewomen in whom the highest level of education was highschool, 72% (13 of 18) disclosed, for those respondentswho attended college, 44% (47 of 106) disclosed, and forthose who completed postgraduate education, 32% (24 of74) disclosed. This difference was statistically significant(P¼.008) (Table 1).
The difference in race/ethnicity between those who dis-close and those who did not disclose revealed that amongCaucasian women, 46% (48 of 105) did disclose, whereasamong African/Caribbean American women, the disclosurerate was 24% (8 of 33) (Table 1). Two subjects did not answerthis question.
g infertility treatment Vol. 88, No. 4, October 2007
TABLE 1Disclosure versus nondisclosure in each group and the correlation.
Disclose Nondisclose P value
Education (nonresponders N ¼ 1)High school 13/18 (72%) 5/18 (28%)College 47/106 (44%) 59/106 (56%)Postgraduate 24/74 (32%) 50/74 (68%) .008
Race/ethnicity (nonresponders N ¼ 2)Caucasian 48/105 (46%) 57/105 (54%)African/CaribbeanAmerican
8/33 (24%) 25/33 (76%)
Asian 16/36 (44%) 20/36 (56%)Other 12/23 (52%) 11/23 (48%) .1
Pregnancy loss (nonresponders N ¼ 0)Previous loss 57/139 (41%) 82/139 (59%)No loss 28/60 (47%) 32/60 (53%) .5
First visit to infertility specialist (nonresponders N ¼ 1)No 54/106 (51%) 52/106 (49%)Yes 30/92 (33%) 62/92 (67%) .009
Days out of work because of infertility evaluation or treatment (nonresponders N ¼ 37)0–3 days/month 45/115 (39%) 70/115 (61%)4–6 days/month 16/27 (59%) 11/27 (41%)>7 days/month 12/20 (60%) 8/20 (40%) .06
Sex of supervisor (nonresponders N ¼ 4)Female 52/105 (50%) 53/105 (50%)Male 33/90 (37%) 57/90 (63%) .07
Stress about infertility (nonresponders N ¼ 18)Not at all 4/13 (31%) 9/13 (69%)Mildly 17/44 (39%) 27/44 (61%)Moderately 37/86 (43%) 49/86 (57%)Very stressed 21/38 (55%) 17/38 (45%) .3
Finamore. Social concerns of women undergoing infertility treatment. Fertil Steril 2007.
Women who were seeing the fertility doctor for the firsttime were less likely to disclose to their employer comparedwith those women who had been to the fertility specialistmore than once (P¼.009) (Table 1). Analysis of the respon-dents’ answer to a question regarding the number of daysmissed from work per month revealed that 39% (45 of115) of those who missed 0 to 3 days per month did dis-close, 59% (16 of 27) of those who missed 4 to 6 daysper month did disclose, and 60% (12 of 20) of those whomissed 7 or more days per month did disclose. This differ-ence approached statistical significance (P¼.06) (Table 1).There were 37 respondents who did not answer this ques-tion.
We analyzed our data for ORs and found high school edu-cated women were over nine times as likely to disclose com-
Fertility and Sterility�
pared with those with a postgraduate level of education (OR9.7, 95% CI 2.59, 36.25). Compared with African/CaribbeanAmerican women, Caucasian women were 3.8 times as likelyto disclose (95% CI 1.31, 11.08), and Asian/Indian womenwere almost five times as likely to disclose (95% CI 1.35,16.35). Women who were out of work for>7 days per monthwere 4.2 times as likely to disclose when compared withthose who were out of work for 0 to 3 days per month(95% CI 1.17, 14.84). Finally, respondents with a female su-pervisor were two times as likely to disclose compared withthose with a male supervisor; however, this was not statisti-cally significant (95% CI 0.27, 1.06).
We used two different methods to analyze stress in thosewho disclose and those who do not disclose. We comparedthese groups with regard to their own assessment of stress
819
TABLE 2Comparison of age, satisfaction at work, freedom at work, and global stress scale for those whodisclose versus those who do not disclose.
Finamore. Social concerns of women undergoing infertility treatment. Fertil Steril 2007.
level and found there to be no differences between stresslevels and disclosure (Table 1). We also found there to beno difference between those who disclose and those who donot disclose when the comparison was based on the resultsof the Perceived Stress Scale (Table 2).
DISCUSSION
The demands on infertile couples can be great. Currenttreatment regiments can be very time consuming,and frequent office visits are mandatory. For working women,this necessitates understanding, flexibility, and cooperationat the workplace from co-workers and supervisors, whichwould require in some instances disclosing ones infertility.Stress has been shown to affect the outcomes of infertilitytreatment, and it was our hypothesis that some patientsmay lower their stress by disclosing their problems with fer-tility, whereas others may benefit from not disclosing. How-ever, results of this survey suggest that disclosure of one’sinfertility status is not a significant factor in either increas-ing or diminishing personal stress. Hence, the decision todisclose must reflect the values and decisions of eachwomen and her partner; it appears to have little implicationsfor treatment.
There was an association between disclosure and numberof days away from work. The more time spent away fromwork, the more likely a woman was to disclose that shewas undergoing treatment and or evaluation for infertility.This is not surprising, considering that most of women whoelected to disclose their infertility did so because they neededto leave work for appointments. There were a large number ofrespondents who did not answer this question, possible be-cause of the fact that for many of these women it may havebeen the first time being evaluated for infertility and theyfelt the question was not applicable. When we analyzedthis data including the nonresponders we got a P value of.05, indicating that there still was an association betweendisclosure and number of days away from work.
The overwhelming majority of women who did not dis-close were protecting their privacy. Of the women who chosenot to disclose, almost 20% indicated they were worriedtheir chances of promotion would be hindered by the knowl-edge that they were being treated for infertility. This findingis particularly significant because fears regarding career
820 Finamore et al. Social concerns of women undergoing
advancement can only lead to increased stress. Thesewomen may believe that the knowledge of their infertilitymay be viewed as a weakness; they may believe their super-visor would realize how demanding these treatments are andtherefore not consider them for a promotion; or they may be-lieve their supervisor would think ahead at the prospect ofa successful pregnancy and extended leave of absence dur-ing and after the pregnancy and therefore not considerthem for promotion.
There was no difference between subjects who disclosedand those who did not disclose with regard to age, length oftime with partner, satisfaction at work, freedom to comeand go from work, if they had a previous pregnancy loss ornot, and religion. Some of the religious categories containedvery few women; however, when looking at each group therewere no differences with regard to disclosure versus nondis-closure.
There was a significant correlation between education anddisclosure. Women with a higher educational status were lesslikely to disclose their infertility status. In part, this may bea function of the fact that these women had higher positionsthat permitted more flexible scheduling, and therefore theydid not need to disclose in order to leave work for doctorvisits. It would be interesting to further investigate this dataand its association with the aforementioned findings thatone out of five women did not disclose because they are wor-ried that it would hurt their chance of promotion. In otherwords, are the women with higher education less likely to dis-close because they are the ones worried about being penal-ized at work for their infertility status? There are manyconfounding factors regarding level of education, and wewould be hesitant to draw many conclusions from this data;however, it is an interesting finding that we would like topursue with further research.
With respect to ethnicity, African America/CaribbeanAmerican women were least likely to disclose their infertilitystatus. There may be confounding factors, such as level of ed-ucation, and location where this study was conducted. Theremay also be cultural differences with respect to maintainingprivacy.
An interesting finding of our study was that women weremore likely to disclose their infertility status to their
infertility treatment Vol. 88, No. 4, October 2007
supervisor if their supervisor was a female. This may be be-cause women have a greater comfort level with other womenwhen discussing infertility. This difference approachedstatistical significance, and we believe that further studyin this area is warranted.
There was no difference in stress scores and disclosure.Although there are many sources of stress for women under-going infertility treatment, the decision to disclose or keepprivate one’s infertility status and treatments from one’semployer does not appear to be a significant stress-enhancing consideration.
We are not able to determine how many patients refused toparticipate in our study. We attempted to limit this bias by ourstudy design in that every questionnaire given out was col-lected before seeing the doctor; however, there were manyquestionnaires that were incomplete. We do not know if thesewere incomplete because subjects refused to participate inthe study, or they did not have enough time to complete thesurvey. Because we are unable to determine who refused toparticipate versus those who did not have enough time, weare not able to identify any differences between those whodid and did not want to participate in our study.
There are many factors that could confound this data,and in a survey it is particularly difficult to control for the
Fertility and Sterility�
psychologic, behavioral, and social differences betweenthese groups. However, some of the differences noted inthis study may help to direct future work in this area.
REFERENCES1. U.S Supreme Court Ruling: Bragdon v. Abbott, 118 S. Ct. 2196 (1998).
2. Family Building Act of 2001. New Jersey Permanent Statutes: 17B:27-
46.1X Group Health Insurance Policies; 17:48A-7W Medical Service
Corporations; 17:48–6X Hospital Service Corporations; 17:48E-35.22
Health Service Corporations; 26:2J-4.23 Health Maintenance
Organizations.
3. Boivin J, Schmidt L. Infertility-related stress in men and women predicts
treatment outcomes 1 year later. Fertil Steril 2005;83:1745–52.
4. Gallinelli A, Roncaglia R, Matteo ML, Ciaccio I, Volpe A, Facchinetti F.
Immunological changes and stress associated with different implantation
rates in patients undergoing in vitro fertilization-embryo transfer. Fertil
The influence of psychological factors on the outcome of the fertilization
step of in vitro fertilization. J Psychosom Obstet Gynaecol 1997;18:
189–202.
6. Demyttenaere K, Nijis P, Evers-Kiembooms G, Koninckx PR. Personality
characteristics, psychoendocrinological stress and outcome of IVF depend
upon the etiology of infertility. Gynecol Endocrinol 1994;8:233–40.
7. Menning B. The emotional needs of infertile couples. Fertil Steril
1980;34:313–9.
8. Cohen S, Kamarck T, Mermelstein R. A global measure of perceived
stress. J Health Soc Behav 1983;24:385–96.
821
Aspirin in women undergoing in vitro fertilizationtreatment: a systematic review and meta-analysisMohammed Khairy, M.B.Ch.B.,a Kaberi Banerjee, M.B.Ch.B., M.D.,a Tarek El-Toukhy, M.B.Ch.B.,M.D.,a Arri Coomarasamy, M.D.,a,b and Yakoub Khalaf, M.B.Ch.B., M.D.a
a Assisted Conception Unit, Guys Hospital, London; and b Department of Public Health & Epidemiology, University of
Birmingham, Edgbaston, Birmingham, United Kingdom
Objective: Many trials have evaluated the effects of aspirin in women undergoing IVF or intracytoplasmic sperminjection (ICSI) treatment. These trials have generally shown inconclusive or inconsistent findings. We conducteda systematic review of trials of aspirin during IVF or ICSI treatment to generate more precise estimates of effectsand attempt to explore the reasons for the inconsistencies.Design: A systematic review and meta-analysis.Setting: Assisted conception units in different countries.Patient(s): Seven trials including 1,241 women undergoing controlled ovarian hyperstimulation (COH), IVF, orICSI and day 3 embryo transfer.Intervention(s): Low-dose aspirin supplementation versus placebo or no supplementation.Main Outcome Measure(s): Clinical pregnancy and live birth.Result(s): Searches were conducted in MEDLINE, EMBASE, Cochrane Library, ISI Proceedings, and SCI-SEARCH, and all randomized controlled trials that evaluated the effectiveness of aspirin compared to placeboor no treatment in women undergoing IVF–ICSI treatment were included. Study selection, quality appraisal,and data extractions were performed independently and in duplicate. Seven relevant trials were identified.Meta-analysis of these studies did not show a significant benefit of aspirin therapy in improving clinical pregnancyrate (relative risk [RR] 1.11, 95% confidence interval [CI] 0.95, 1.31) or live birth rate (RR 0.94, 95% CI 0.64,1.39). There was no significant difference in miscarriage rate (RR 1.06, 95% CI 0.53, 2.11) or ectopic pregnancyrate (RR 2.24, 95% CI 0.70, 7.24). An improvement was noted in uterine artery pulsatility index (weighted meandifference: –0.78, 95% CI –0.87, –0.69) in women taking low-dose aspirin. The evidence regarding other outcomeswas either not significant or contradictory.Conclusion(s): Currently available evidence does not support the use of aspirin in IVF or ICSI treatment. However,the noted trend of improvement in clinical pregnancy, and the lack of power even when the studies were pooledhighlight the need for a definitive trial.(Fertil Steril� 2007;88:822–31. �2007 by American Society for Reproduc-tive Medicine.)
The success of IVF treatment is generally reported to varybetween 20% and 40% per cycle started (1, 2). In an effortto improve IVF outcome, numerous pharmacologic interven-tions have been studied as adjuvant therapy over the years(3–5). Aspirin (acetylsalicylic acid) is a widely used vasoac-tive substance that exerts its effects by inhibiting the enzymecyclo-oxygenase in platelets. In low doses, it inhibits synthe-sis of thromboxane A2 (a vasoconstrictor and promoter ofplatelet aggregation) more than that of prostacyclin (a vaso-dilator) (6–8). Due to these antithrombotic and vasodilatoryeffects, aspirin has been one of the agents studied in severaltrials to evaluate its potential role in increasing IVF successrate through improving either ovarian blood flow, folliculo-genesis, and ovarian responsiveness (9) or uterine vascularityand receptiveness, or both (10). If aspirin were effective, it
Received August 17, 2006; revised December 9, 2006; accepted
December 13, 2006.
Reprint requests: A. Coomarasamy, M.D., 4th Floor, Assisted Conception
Unit, Thomas Guy House, Guys Hospital, London, SE1 9RT, UK (FAX:
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
822
would be a simple and affordable intervention to improveIVF outcome.
At present, the trials conducted to evaluate the effects ofaspirin in women undergoing IVF or intracytoplasmic sperminjection (ICSI) have yielded inconclusive or conflicting re-sults and there is still no consensus on the value of usingaspirin to enhance IVF outcome. We therefore conducteda systematic review of trials of low-dose aspirin during IVFor ICSI treatment to generate a more precise estimate ofthe effects of aspirin in IVF or ICSI treatment, and attemptedto explore the reasons for the inconsistencies currently pres-ent in the literature.
MATERIALS AND METHODS
We searched MEDLINE (1966–2006), EMBASE (1974–2006), Cochrane Library (2006:3), and SCISEARCH(1974–2006) for relevant studies. A combination of MedicalSubject Headings (MeSH) and text words were used to gen-erate two subsets of citations, one including studies of aspirin
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.080
(‘‘aspirin,’’ ‘‘antiplatelet(*),’’ ‘‘acetylsaly(*),’’ ‘‘acetylsali(*),’’‘‘platelet aggregation inhibitors’’) and the other studies ofIVF and ICSI (‘‘invitro fertilization,’’ ‘‘Fertilization-in-vitro,’’ ‘‘intracytoplasmic-sperm-injection,’’ ‘‘sperm-injec-tions-intracytoplasmic,’’ ‘‘reproductive techniques assisted,’’‘‘embryo transfer,’’ and ‘‘embryo implantation’’). These sub-sets were combined using ‘‘AND’’ to generate a subset ofcitations relevant to our research question. We also searchedISI Proceedings for conference abstracts, and ISRCTN Regis-ter and Meta-register for RCTs (mRCT) for ongoing and ar-chived randomized controlled trials. The reference lists of allknown primary and review articles were examined to identifycited articles not captured by electronic searches. Articlesfrequently cited were used in the Science Citation Index toidentify additional citations. We also made enquiries about un-published studies from researchers investigating in this field.No language restrictions were placed in any of our searches.
Studies were selected if the target population was womenundergoing IVF or ICSI treatment, the therapeutic interven-tion was low-dose aspirin (75–150 mg/day) compared to pla-
Fertility and Sterility�
cebo or no drug treatment. Studies addressing the use of otheradjuvant therapies such as heparin or steroids were excluded.Studies were included only if they were of randomized de-sign. The primary outcomes were clinical pregnancy andlive births. The secondary outcomes were ectopic pregnancyand miscarriage rates. We also obtained data on surrogateoutcomes such as the dose of gonadotropins used for stimu-lation, duration of treatment, number of oocytes retrieved,and uterine artery pulsatility index.
Studies were selected in a two-stage process. First, the ti-tles and abstracts from the electronic searches were scruti-nized by two reviewers independently (M.K. and K.B.) andfull manuscripts of all citations that were likely to meet thepredefined selection criteria were obtained. Second, finalinclusion or exclusion decisions were made on examinationof the full manuscripts. In cases of duplicate publication,the most recent and complete versions were selected. The as-sessment of English language manuscripts was performed in-dependently by two reviewers (M.K. and K.B.) and otherlanguage manuscripts by people who had command of the
FIGURE 1
Study selection process for systematic review of the effects of aspirin in patients undergoingIVF–intracytoplasmic sperm injection (ICSI) treatment.
Primary articles included in the systematic review: n= 7(See Tables 1 and 2 for further details)
Full manuscripts retrieved for detailed evaluation: n= 17
Citations excluded after screening titlesand/or abstracts: n= 219
Total number of citations from electronic searches and from examinationof reference lists of primary and review articles: n= 236
Articles excluded with reasons: • Retrospective study=4 • Observational study=1 • Before and After study=1 • Inappropriate study design=2 • Duplication of data =1 • Quasi randomized = 1
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
823
language. Any disagreements about inclusion were resolvedby consensus or arbitration by a third reviewer (A.C.). Per-centage agreement and kappa statistic were used for analysisof agreement between the two reviewers (M.K. and K.B.).
The selected studies were assessed for methodologicalquality by using the components of study design that are re-lated to internal validity (11). Information on the adequacy ofrandomization, concealment, blinding, intention to treat, andfollow-up rates was sought by examining the full text articlesand by contacting the authors if clarification was needed.
Study characteristics such as population features and inter-ventions (for example, dose, time of commencement, and du-
824 Khairy et al. Aspirin in IVF-ICSI: systematic review
ration of treatment) were extracted from each study. Fromeach study outcome data were extracted in 2x2 tables (for dis-crete variables) or as means or medians with a measure of var-iance (such as standard deviation) for continuous variables.Weighted mean difference was calculated for continuous var-iables using means and standard deviations from individualstudies. For discrete variables, we performed meta-analysisusing a fixed effects model initially, with the random effectsmodel being used subsequently as part of a sensitivity analy-sis. Because there is no clear consensus on the most appropri-ate method for meta-analysis, we have used both fixed andrandom effects models to explore the robustness of our find-ings (12, 13). Heterogeneity of treatment effects was evalu-ated graphically using forest plot and statistically using c2
TABLE 1Quality of studies included in the review of aspirin use in IVF–ISCI.
TrialMethod of randomization& allocation concealment Blinding
Intentionto treatanalysis Follow-up
Wecksteinet al., 1997
Method of randomizationnot given
None Unreported >95% untildelivery
Adequate concealment bysealed envelopes
Check et al.,1998
Method of randomizationnot given
Unreported Yes >95% untilclinicalpregnancy
Allocation Concealment notgiven
Rubinsteinet al., 1999
Method of randomizationnot given
Doubleblinded
Unreported >95% untilpregnancy
Adequate concealment bydrawing sealedenvelopes
Urman et al.,2000
Randomization bycomputer generatedrandom numbers
Doubleblinded
Unreported >95 % untilclinicalpregnancy
Adequate concealment bythird party
Lok et al., 2004 Randomization bycomputer generatedrandom numbers
Doubleblinded
Yes >95% untilclinicalpregnancy
Adequate concealment bythird party
Van Doreenet al., 2004
Method of randomizationnot given
Doubleblinded
Yes >95% till morethan 12 weeks
Allocation Concealment notgiven
Pakkila et al.,2005
Block randomization butmethod of randomizationnot given
Doubleblinded
Yes >95% untildelivery
Adequate concealment bysealed envelopes
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
826K
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test (13, 14). Exploration of clinical heterogeneity was plannedusing variation in features of the population, intervention, out-come, and study quality. To assess for publication bias, we per-formed a funnel plot analysis, using Egger’s test (15).
Because the meta-analysis did not involve subjectingpatients to an intervention, and data were extracted from pre-existing literature, there was no need for obtaining approvalby local research ethics committee. All statistical analyses
FIGURE 2
Meta-analysis of studies evaluating the effect of aspirin on clinical pregnancy rate in women undergoing IVF–ICSIper patient randomized.
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
FIGURE 3
Meta-analysis of studies evaluating the effect of aspirin on live birth rate in women undergoing IVF–ICSI perpatient randomized.
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
Fertility and Sterility� 827
were performed using Stata 8.0 (STATA Corp LP, CollegeStation, TX) and RevMan 4.2 (Cochrane Collaboration,Oxford, UK).
RESULTS
Figure 1 summarizes the process of literature identificationand selection. Of the 236 citations identified, 17 were se-lected during the initial screening, and on examination oftheir full manuscripts, seven articles (3, 9, 10, 16–19), includ-ing a total of 1,241 women, were identified to satisfy the se-lection criteria for our review. The search also resulted in theidentification of three non-English citations (Japanese, Ital-ian, and Taiwanese). We obtained the full manuscripts forthese articles, and after translation, none of the studies metthe inclusion criteria, as all were found to be nonrandomizedobservational studies.
The quality and the main characteristics of the includedstudies are presented in Tables 1 and 2. There was no evi-dence of allocation concealment in two of the studies (2/7,29%).
Primary Outcomes
Meta-analysis of the included studies did not show a signifi-cant benefit of aspirin therapy in improving clinical preg-nancy rate (PR) (7 studies; relative risk [RR] 1.11, 95%confidence interval [CI] 0.95, 1.31; Fig. 2) or live birth rate(2 studies, RR 0.94, 95% CI 0.64, 1.39; Fig. 3) in patients un-dergoing IVF or ICSI treatment. The results for both live birthand clinical pregnancy were not statistically heterogeneous(P¼.31, P¼.07, respectively). The analysis for heterogeneity,particularly for live birth rate, is, however, unreliable giventhe small number of studies included in the analysis. Onlyone study (18) reported on ongoing PR, and this study didnot find any difference between the aspirin and placebogroups (24/85, 28.3% and 25/85, 29.4%, respectively).
828 Khairy et al. Aspirin in IVF-ICSI: systematic review
Sensitivity analysis in which the studies with no evidenceof allocation concealment were excluded from the analysiswas done, and this did not change these findings (clinicalpregnancy: RR 1.24, 95% CI 0.95, 1.62).
Secondary Outcomes
There was no difference in miscarriage rate (RR 1.06, 95% CI(0.52, 2.11) (Table 3) or ectopic PR (RR 2.24, 95% CI 0.70,7.24) (Table 3) in patients taking aspirin compared to thosetaking placebo or no treatment.
Surrogate Outcomes
There was an improvement in uterine artery pulsatility indexin patients taking low-dose aspirin (weighted mean differ-ence: –0.78, 95% CI: –0.87, –0.69) (Table 4). The evidenceregarding other surrogate outcomes (such as number of oo-cytes retrieved endometrial thickness, number of FSH am-pules used, duration of stimulation, number of follicles)was either not significant or contradictory (Table 4).
Funnel plot analysis for publication and related biases didnot suggest evidence of bias (Fig. 4) (Begg’s test P¼.76, andEgger’s test P¼ .62). The analysis was again limited due tothe small number of studies identified.
Analysis for agreement between the two reviewers (M.K.and K.B.) showed a percentage agreement of 95% and kappastatistic of 0.86, P<.0001, indicating high level of agreement.
DISCUSSION
Although early studies suggested a beneficial effect of low-dose aspirin on the outcome of IVF–ICSI cycles (3, 10),subsequent randomized controlled trials showed conflictingresults (17–20). To our knowledge this is the first systematicreview examining the evidence of the effect of low-dose
TABLE 3Effects of aspirin on secondary outcomes in women undergoing IVF–ICSI.
Outcome
Events inaspiringroup
Events incontrolgroup
Relative risk(95% CI)
Pooled relativerisk (95% CI)
Miscarriage per patientrandomizedUrman et al., 2000 8/139 7/136 1.13 (0.40, 3.19) 1.06 (0.53, 2.11)Pakkila et al., 2005 8/186 8/188 1.01 (0.37, 2.75)
Duration of stimulationUrman et al., 2000 10.7 (1.6) 10.9 (1.6) �0.2 (�0.58, 0.18) __Lok et al., 2004 9.5 [8.75, 12.0] 11 [9.75, 12]
No. of folliclesRubinstein et al., 1999 19.8 (7.2) 10.2 (5.3) 9.6 (8.2, 11.0) __Lok et al., 2004 3.0 [2.0, 6.0] 3.5 [1.75, 5.0]Van Doreen et al., 2004a 10.0 (0–50) 9.0 (0–36)
Note: SD ¼ standard deviation; median [IQR ¼ interquartile range]; CI ¼ confidence interval; RR ¼ relative risk; hCG ¼human chorionic gonadotrophin.
a Data provided in median and range.b Data not pooled due to highly significant statistical heterogeneity.
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
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aspirin on IVF–ICSI outcome. This review shows that there isinsufficient evidence to support the routine use of low-doseaspirin to improve clinical pregnancy and live birth rates.The review also shows that there is no effect on either miscar-riage or ectopic PRs. Although there was an improvement inuterine artery pulsatility index with the use of aspirin, this didnot result in better clinical outcomes. This finding concurswith previous reports showing that uterine artery Dopplerindices do not correlate significantly with the outcome ofIVF cycles as they may reflect enhanced blood flow to theuterus but not necessarily the endometrium (21–23).
The validity of our findings depends on the methodologicalrigor of our review and of the component primary studies. We
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used a prospective protocol and made a concerted effort tofind all the evidence. Two independent reviewers assessedstudy quality and extracted data and the agreement betweenthe two reviewers (M.K. and K.B.) was high. Where appro-priate, we carried out sensitivity analysis to explore the influ-ence of various factors on the results.
One major problem usually encountered on synthesizingthe evidence in meta-analyses is clinical and methodologicalheterogeneity between the studies, namely the differencesbetween the studies in the populations they evaluated, theaspirin dosage, time of commencement and duration of treat-ment, variations in IVF–ICSI treatment protocols, differ-ences in the way outcomes were defined and reported, as
well as important differences in the quality features betweenthe studies. A rigorous exploration of effects of these factorswith techniques such as meta-regression was not feasible dueto the limited number of studies identified. However, the de-gree of clinical heterogeneity was judged as not high enoughto invalidate the pooling of data. This judgment gained fur-ther credibility by the observation that there was no statisticalheterogeneity.
The largest study to date reporting on the use of low-doseaspirin in IVF was a quasi-randomized study (20). This studyreported a trend of beneficial effect of low-dose aspirin onboth clinical pregnancy and live birth rates (OR 1.3, 95% CI1.0, 1.6 and OR 1.2, 95% CI 1.0, 1.6), respectively. In thisstudy, however, the researchers allocated subjects to aspirinor ‘‘no treatment’’ arms on alternate days. This study was,therefore, not only nonrandomized, but also lacked conceal-ment of allocation (which can seriously bias the results, by ex-aggerating treatment effects by up to 41%) (24). Furthermore,the study reported on 1,380 cycles in 1,022 patients, whichmeans that some patients must have contributed to morethan one cycle of treatment. Thus, observations from thisstudy are not independent, and pooling of results using suchdata would invalidate the meta-analysis. For all these reasons,this study was excluded from our review.
A closer examination of the studies that suggested benefitin using low-dose aspirin (10, 20) in comparison with otherstudies (3, 9, 16–19) did not show any obvious factors thatcould be related to a better outcome with aspirin. For exam-ple, there were no obvious or consistent differences in factorssuch as the time of commencement, duration of treatment,and the dose of aspirin between the studies that suggestedbenefit and those that did not, to explain why aspirin mayor may not have been effective. The implication of this is
FIGURE 4
Funnel plot to assess publication and related biasesin the systematic review of aspirin in IVF or ICSIcycles for the outcome of clinical pregnancy.
Begg's funnel plot with pseudo 95% confidence limits
logo
r
s.e. of: logor0 .5 1 1.5
-2
0
2
4
Khairy. Aspirin in IVF-ICSI: systematic review. Fertil Steril 2007.
830 Khairy et al. Aspirin in IVF-ICSI: systematic review
that it is not possible to identify any subgroups in whom ben-efit with aspirin could be expected.
The large 95% confidence intervals around the relativerisks for clinical PRs (RR 1.11, 95% CI 0.95, 1.31) and livebirths (RR 0.94, 95% CI 0.64, 1.39) suggest imprecise resultsin which the clinical PRs with aspirin may have been up to5% less, or as much as 31% higher than the rates of clinicalpregnancy in the control group, and similarly the live birthrates may have been up to 36% less or as much as 39%more than the control group, within a 95% confidence limit.This imprecision in the results is due to the relatively smallsample sizes of the studies in the review.
For example, sample size calculations show that for a studyto have 80% power (at an a of 0.05) to detect a clinically min-imal important difference of 5% (25% vs. 30%) in live births,an individual trial will need to randomize more than 2,500women (1,251 in each arm of the trial). Thus, the interpreta-tion of our review, in which we found only seven studies in-cluding a total of 1,241, is likely to suffer from the possibilityof a false-negative finding (type II error).
There have been concerns regarding the safety of aspirin. Aliterature search for adverse events associated with the use oflow-dose aspirin identified three large cohort studies and onelarge case-control study—these observational studies, withmore than 96,000 women between them, did not provide anyevidence of teratogenicity or long-term adverse effects of aspi-rin use (25). The only evidence of harm came from a case-control study that showed evidence of increased risk ofmiscarriage when nonsteroidal anti-inflammatory drugs werestarted early in gestation (26). However, no adjustment wasmade in this study for the indication of nonsteroidal anti-in-flammatory use. This would be expected to introduce substan-tial bias because women with conditions such as systemiclupus erythematosus and rheumatoid arthritis are more likelyto be users of nonsteroidal anti-inflammatory drugs duringpregnancy but have an inherently higher risk of miscarriageas a result of the underlying condition rather than the drugitself. Thus, we regard this evidence of association betweennonsteroidal drugs and miscarriage as unreliable (25).
In conclusion, based on the findings of our review, aspirintherapy cannot currently be recommended for routine clinicaluse, outside of the context of a clinical trial. However, giventhe paucity of the existing evidence, the lack of power in theavailable trials to detect minimally important treatment effectin clinically meaningful outcomes, and the trend of benefitdemonstrable in the point estimate of clinical pregnancy inthis review, a double-blinded, placebo-controlled random-ized trial with adequate sample size may be warranted toreach a definitive answer on the effect of low-dose aspirinon the important clinical outcomes as PRs or live birth ratesin the IVF setting.
REFERENCES1. Society for Assisted Reproductive Technology, The American Society
for Reproductive Medicine, assisted reproductive technology in the
Vol. 88, No. 4, October 2007
United States and Canada: 2000 results generated from the American So-
ciety for Reproductive Medicine/Society for Assisted Reproductive
Rudigoz RC. Preliminary report of an ultrasonography and color Doppler
uterine score to predict uterine receptivity in an in-vitro fertilization
program. Hum Reprod 1998;13:1669–73.
24. Schulz KF, Chalmers I, Hayes RJ, Altman DJ. Empirical evidence of
bias: dimensions of methodological quality associated with estimates
of treatment effects in controlled trials. JAMA 1995;273:408–12.
25. Coomarasamy A, Honest H, Papaioannou S, Gee H, Khan KS. Aspirin
for prevention of pre-eclampsia in women with historical risk factors:
a systematic review. Obstet Gynecol 2003;101:1319–32.
26. Nielsen GL, Sorensen HT, Larsen H, Pedersen L. Risk of adverse birth
outcome and miscarriage in pregnant users of non-steroidal anti-inflam-
matory drugs: population based observational study and case-control
study. BMJ 2001;322:266–70.
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GnRH agonist and antagonist protocols for stage I–IIendometriosis and endometrioma in in vitro fertilization/intracytoplasmic sperm injection cyclesRecai Pabuccu, M.D.,a Gogsen Onalan, M.D.,b and Cemil Kaya, M.D.a
a Ufuk University School of Medicine, Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and
Infertility and b Centrum Clinic, Ankara, Turkey
Objective: To investigate the outcomes of intracytoplasmic sperm injection (ICSI) cycles after controlled ovarianhyperstimulation (COH) with GnRH antagonist or GnRH agonist (GnRH-a) in mild-to-moderate endometriosisand endometrioma.Design: Prospective randomize trial.Setting: A private IVF center.Patient(s): A total of 246 ICSI cycles in 246 patients were divided into three groups: women with mild-to-mod-erate endometriosis (n ¼ 98); women who had ovarian surgery for endometrioma (n ¼ 81); women with endome-trioma and no history of previous surgery (n ¼ 67).Intervention(s): Patients in each group were randomized to COH with either triptrolein or cetrorelix.Main Outcome Measure(s): Clinical parameters, characteristics of COH, and ICSI results were analyzed.Result(s): Outcomes of COH with both GnRH antagonist and GnRH-a were similar in patients with mild-to-mod-erate endometriosis. Implantation rates were 15.9% vs. 22.6% and clinical pregnancy rates were 27.5% vs. 39%with GnRH antagonist and GnRH-a protocols, respectively, in patients who had ovarian surgery for endometrioma.Implantation rates were 12.5% vs. 14.8% and clinical pregnancy rates were 20.5% vs. 24.2% with GnRH antag-onist and GnRH-a protocols, respectively, in patients with endometrioma and no history of ovarian surgery.Conclusion(s): Considering the implantation and clinical pregnancy rates, COH with both GnRH antagonist andGnRH-a protocols may be equally effective in patients with mild-to-moderate endometriosis and endometriomawho did and did not undergo ovarian surgery. (Fertil Steril� 2007;88:832–9. �2007 by American Society for Re-productive Medicine.)
Endometriosis is a challenging disease observed in 20%–40%subfertile women (1). Alterations of the immunologic milieuwithin the peritoneal cavity create a ‘‘hostile’’ environment inendometriosis that may impair gamete interaction and earlyembryo development (2, 3). Expectant management, surgicalresection, medical therapy, and controlled ovarian hyperstim-ulation (COH) with assisted reproductive technologies (ART)may be applied for conception in these patients (4, 5).
In vitro fertilization may improve conception rates in endo-metriosis-associated infertility, although some studies reportequal pregnancy rates (PR) but lower fertilization or implan-tation rates in endometriosis compared to tubal or unex-plained infertility (4, 6–8). Decreased numbers of retrievedoocytes are observed in patients with endometriosis undergo-ing intracytoplasmic sperm injection (ICSI) due to severemale infertility with similar fertilization rates, implantation
Received June 8, 2006; revised December 25, 2006 and accepted De-
cember 29, 2006.
Presented in part at the 61st Annual Meeting of the American Society for
Reproductive Medicine, Montreal, Quebec, Canada, October 15–19,
2005.
Reprint requests: Gogsen Onalan, MD, Centrum Clinic, Nenehatun,
No. 59, Ankara, Turkey 06520-8063 (FAX: 90-312-447-1818; E-mail:
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
832
rates, and PRs in comparison to those without endometriosis(9, 10).
Gonadotropin-releasing hormone agonists (GnRH-a) inovarian stimulation protocols are used to prevent possibledeleterious effects of premature LH surges in ART (11).The IVF–embryo transfer outcomes after a long protocolwith GnRH-a and stimulation with hMG/hCG in patientswith stage I–II endometriosis demonstrate similar outcomesof IVF–embryo transfer as in those of the patients with tubalinfertility (12). Recently developed GnRH antagonists causeimmediate suppression of gonadotropin secretion by compet-itively blocking pituitary GnRH receptors and prevent pre-mature LH surges during ovarian stimulation (13). Thesefeatures constitute the GnRH antagonist as a reasonablechoice for poor responder patients in IVF cycles (13, 14).The use of GnRH antagonist may be rational for IVF cyclesin patients with decreased ovarian reserve due to ovarian en-dometrioma and after its surgical treatment (15, 16).
Individualized IVF protocols, according to various stagesof endometriosis and according to management of endome-trioma, may improve clinical outcomes of IVF in patientswith endometriosis. To the best of our knowledge, there areno prospective studies in literature comparing the outcomesof COH with GnRH-a and GnRH antagonist protocols in
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.046
patients with different stages of endometriosis. The aim of thecurrent study is to compare the efficiency of COH protocolsincluding GnRH-a and GnRH antagonist among patients hav-ing stage I–II endometriosis and endometrioma with or with-out ovarian surgery and to optimize the appropriate protocolfor each group of patients.
MATERIALS AND METHODS
A total of 246 ICSI cycles in 246 patients who were admittedto Centrum IVF Clinic between November 2002 and February2006 were included in the current study. Informed consent inwriting was obtained from each patient. Consent forms andprotocols were approved by the Local Ethics Committee.
Patients were divided into three groups: women with stageI–II endometriosis confirmed by laparoscopy (n¼ 98) (groupI); women with history of ovarian surgery for endometriomaand without active endometrioma at the commencement ofthe current cycle (n ¼ 81) (group II); women with unilateralor bilateral endometrioma and no history of previous ovariansurgery (n ¼ 67) (group III). Patients were randomized intoCOH with either GnRH-a long protocol or multiple dosesGnRH antagonist protocol according to the randomizationlist generated by the computer.
Endometrioma was confirmed by the aspirated materialsent for cytopathologic examination during oocyte retrieval(17, 18).
All patients in group I had no endometrioma. Patients whohad endometrioma recurrences or with advanced endometri-osis (stage III–IV) without endometrioma were excluded.
Patients in group II had a history for resection of endome-trioma by laparoscopy or laparotomy, no more than 4 yearsbefore the study (each patient in this group had an operativereport consistent with biopsy-proven endometriosis; bilateral[n ¼ 36] and unilateral [n ¼ 45]). Endometriomas were re-sected either once (n ¼ 57) or twice (n ¼ 24). Accordingto the revised American Fertility Sterility classification(19), 51 women had stage III and the remainder had stage IVendometriosis.
Patients with hydrosalpinx, documented tuberculosis,male factor infertility, and thaw cycles were excluded fromthe study. All patients had a normal uterine cavity docu-mented by either hysterosalpingography or hysteroscopydone no longer than 1 year before the study. All couples un-derwent the first ICSI cycle with ejaculated sperm.
Pituitary desensitization was performed with 100 mg oftriptrolein (decapeptyl 0.1 mg/day injected SC initiated onday 21 of the previous cycle) in the long protocol withGnRH-a. Daily injections of cetrorelix (Cetrotide; Serono,Geneva, Switzerland; 0.25 mg, SC) in the multiple doses ofGnRH antagonist protocol was initiated when the leading fol-licle was 14 mm in diameter with serum levels of E2 exceeding600 pg/mL and was maintained until the day of hCG injection.The COH with 225–300 IU of recombinant FSH (Gonal F75 IU ampules; Serono) was initiated on the third day of the
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menstrual cycle. The dose of recombinant FSH was decreasedas a step-down regimen in the presence of at least three folli-cles larger than 11 mm and serum E2 levels more than 450 pg/mL. After the development of at least three follicles largerthan 17 mm and serum E2 levels more than 500 pg/mL,10,000 IU of hCG (Profasi ampules, 5,000 IU; Serono) was in-jected. Oocyte retrieval was performed at hour 35 after hCGinjection.
An oocyte pick-up was done with a 17-guage needle foroocyte retrieval under local anesthesia. The oocyte –coronacomplexes (OCC) were denuded, and ICSI was performed af-ter 2 hours of incubation and embryos were transferred onday 3 or 5. All cycles included fresh transfers. The embryotransfer policy largely depended on the woman’s age, thenumber and quality of embryos developed, and history of pre-vious assisted conception attempts. Overall, up to two orthree high quality embryos were transferred when the womanwas <30 and >30 years, respectively. The transfer protocolwas individualized for poor grade embryos and history of pre-vious unsuccessful assisted conception attempts.
Methylprednisolone (16 mg/day) and doxycycline (100 mgtwice a day) (20) were administered for 4 days, starting on theday of oocyte retrieval, to support implantation. All patientshad luteal phase support with micronized vaginal P after em-bryo transfer. Clinical pregnancy was defined as the presenceof at least one gestational sac with detectable fetal cardiac ac-tivity by transvaginal ultrasonography. Implantation rate wascalculated as total number of gestational sacs divided by totalnumber of embryos transferred for each group analyzed.
Outcomes of IVF/ICSI cycles with COH protocols includ-ing GnRH antagonist and GnRH-a were analyzed among thestudy groups. Baseline parameters compared among the studygroups included the following: serum levels of FSH, LH onday 3 of the menstrual cycle, number of antral follicles, dura-tion of COH, total unit of drugs, serum levels of E2 on the dayof hCG injection, and total number of oocytes>17 mm in di-ameter, eggs retrieved, metaphase I and II oocytes, rate of fer-tilization, number of available embryos, number of embryostransferred, and rates of biochemical and clinical pregnancies.
Statistical Analysis
One sample Kolmogorov-Simirnov test was used to test nor-mality. One-way ANOVA test was used for the analysis ofnormally distributed variables. In the presence of statisticaldifferences among groups, data were analyzed by post-hocTuckey HSD test (mean � SD). For nonparametric distribu-tion, Kruskal Wallis test was used. Mann Whitney U testwith Bonferoni correction was performed (median, mini-mum–maximum) for differences among the study groups.Categorical variables were analyzed with c2. P value < .05was considered to be statistically significant.
RESULTS
A total of 266 eligible patients were enrolled initially and in-cluded in the study. Patients were randomized into three
833
groups: patients with stage I–II endometriosis (n ¼ 103;GnRH-a, n ¼ 51 and GnRH antagonist, n ¼ 52), history ofovarian surgery for endometrioma (n ¼ 88; GnRH-a, n ¼44 and GnRH antagonist, n ¼ 44), unilateral or bilateral en-dometrioma and no history of previous ovarian surgery (n ¼75; GnRH-a, n ¼ 37 and GnRH antagonist, n ¼ 38). Afterrandomization and starting the treatment, 20 cases were can-celed because of insufficient ovarian response and risk of hy-perstimulation (cycle cancellation) (group I: n ¼ 5; group II:n¼ 7; group III: n¼ 8). Finally, a total of 246 cycles with 246patients completed the study as three groups; patients withstage I–II endometriosis (n ¼ 98; GnRH-a, n ¼ 48 andGnRH antagonist, n ¼ 50), history of ovarian surgery for en-dometrioma (n¼ 81; GnRH-a, n¼ 41 and GnRH antagonist,n ¼ 40), unilateral or bilateral endometrioma and no historyof previous ovarian surgery (n ¼ 67; GnRH-a, n ¼ 33 andGnRH antagonist, n ¼ 34).
As far as we know no studies have been performed com-paring groups similar to ours. Because no data exist for theantagonist treatment in IVF-ICSI cycle in advancedendometriosis also with previous surgery, we could not calcu-late the required sample size for pretest power estimation. Asa result we decided to enroll all patients in a predeterminedtime interval. Our study may be accepted as a feasibilitystudy. The modest aim of the current study was to providean estimate of effect of antagonist to the IVF-ICSI outcomein patients’ severe endometriosis, resected endometriomaon a relatively large patient population, and to be includedin a future meta-analysis focusing on GnRH antagonist usein patient with endometriosis in IVF-ICSI cycles.
The mean age of the patients was 30.9 � 4.1 years. Con-trolled ovarian stimulation with both GnRH-a and GnRH an-tagonist protocols resulted in similar IVF outcomes inpatients with stage I–II endometriosis. Implantation rateswere 15.4% vs. 18.2% and clinical PRs were 30% vs.31.2% with GnRH antagonist and GnRH-a protocols, respec-tively (P¼.9 and P ¼ 1) (Table 1).
The COH with GnRH antagonist resulted in significantlylower number of MII oocytes (4.3 � 2.6 vs. 8.8 � 4.6,P¼.0001), lower number of available embryos (2.3 � 0.8vs. 5.4 � 4, P¼.01), and decreased fertilization rate (63.9� 21.1 vs. 71.2 � 22.4, P¼.001) compared to those withthe GnRH-a protocol in patients who had ovarian surgeryfor endometrioma. Implantation rates were 15.9% vs.22.6% and clinical PRs were 27.5% vs. 39% with GnRH an-tagonist and GnRH-a protocols, respectively (P¼.6 andP¼.38) (Table 1).
Controlled ovarian stimulation including GnRH antagonistresulted in significantly lower levels of E2 on the day of hCGinjection (1,512 � 448 vs. 1,968.5 � 1,211 pg/mL, P¼.02),lower number of follicles R17 mm (3.5 � 0.9 vs. 5.1 � 1.9,P¼.0001), total number of retrieved oocytes (6.7 � 2.6 vs.8.2 � 5.5, P¼.002), number of available embryos (2.9 � 1vs. 4.2 � 2.6, P¼.01), and MII oocytes (4.9 � 1.6 vs. 6.5 �4.2, P¼.01) compared to those with the GnRH-a protocol inpatients with ovarian endometrioma and no history of
834 Pabuccu et al. GnRH protocols for endometriosis in I
previous surgery. Implantation rates were 12.5% vs. 14.8%and clinical PRs were 20.5% vs. 24.2% with GnRH antagonistand agonist protocols, respectively (P¼.7 and P¼.9)(Table 1). There were no differences between the GnRH-aand GnRH antagonist groups according to diameter andbilaterality of endometrioma (25.8 � 10.3 mm vs. 27.3 �10.7 mm, P¼.6 and 34% � 36% vs. 42% � 49%, P¼.5,respectively).
Comparisons of ICSI cycle outcome among three distinctclinical endometriosis groups—each were maintained bycombining the analogue and antagonist groups—are shownin Table 2. The COH in patients with resected endometriomahad significantly higher levels of day 2 FSH (8.8 � 1.6 vs.5.4 � 1 IU/mL, P¼.001), number of recombinant FSH am-pules (31 � 8.9 vs. 28� 8.7, P¼.02), lower number of antralfollicles (3.1 � 0.9 vs. 5.2 � 1, P¼.004), lower levels of E2
on the day of hCG injection (1,716.7� 1,163.5 vs. 2,473.5�1,057.5 pg/mL, P¼.006), lower number of follicles R17 mm(3.8 � 1.6 vs. 5.6 � 1.7, P¼.001), total number of retrievedoocytes (9.3 � 5.2 vs. 12.7 � 6.1, P¼.01), MII oocytes (6.5� 3.6 vs. 9.2 � 4.4, P¼.008), available embryos (3.8 � 2.3vs. 6.3 � 4.6, P¼.001), and lower fertilization rate (67% �21.7% vs. 75% � 20.8%, P¼.01) compared with thosewith stage I–II endometriosis group (Table 2).
The COH in patients with active endometrioma had signif-icantly lower levels of E2 on the day of hCG injection(1,740.3 � 829.5 vs. 2,473.5 � 1,057.5 pg/mL, P¼.008),lower number of follicles R17 mm (4.3 � 1.4 vs. 5.6 �1.7, P¼.005), total number of retrieved oocytes (7.4 � 4vs. 12.7 � 6.1, P¼.003), MII oocytes (5.7 � 2.9 vs. 9.2 �4.4, P¼.001), and available embryos (3.6 � 1.8 vs. 6.3 �4.6, P¼.001) compared with those with stage I–II endometri-osis group (Table 2).
In addition, COH in patients with resected endometriomahad significantly higher levels of day 2 FSH (8.8 � 1.6 vs.6.7 � 2.8 IU/mL, P¼.009), compared with those with activeendometrioma (Table 2).
In all endometriosis groups, patients treated with the antag-onist protocol had significantly lower numbers of MII oocytes(6� 2.9 vs. 8.3� 4.4, P¼.005) and available embryos (3.7�2 vs. 5.4� 3.8, P¼.003) compared to patients treated with theanalogue protocol. Implantation rates were 14.6% vs. 18.5%,clinical PRs were 26.6% vs. 31.9%, abortion rate were 3.2%vs. 2.,5% with the GnRH antagonist and GnRH-a protocols,respectively (P¼.35 and P¼.6) (Table 3).
DISCUSSION
Endometriosis may have adverse effects on various aspects ofreproductive physiology including folliculogenesis, ovula-tion, sperm, embryo quality, and fertilization (21–24). Arecent meta-analysis demonstrates that patients with endo-metriosis-associated infertility undergoing IVF have a 36%reduction of PR compared to women with other indicationsfor IVF (25). Moreover, the severity of endometriosis is likelyto affect the outcome of assisted reproduction, women with
Note: BMI ¼ body mass index.P< .05, statistically significant differences among stage I–II, active endometrioma and resected endometrioma.a P< .05, stage I–II group vs. active and resected endometrioma groups.b P%.001, stage I–II group vs. active endometrioma and resected endometrioma groups.c P < .05, active endometrioma group vs. resected endometrioma group.
Pabuccu. GnRH protocols for endometriosis in ICSI. Fertil Steril 2007.
stage III and IV disease having fewer numbers of oocytes re-trieved, lower peaks of E2, lower fertilization and implanta-tion rates compared with those in stage I and II. Impairedfertilization and implantation in endometriosis may be traceddirectly to the defects in the oocyte (26). Some altered factorsinvolved in the process of oocyte activation in endometriosispatients could be overcome by the ICSI procedure and similarfertilization, implantation, and clinical PRs could be obtainedin patients with endometrioma (10). Such data indirectly sug-gest that ICSI may be a useful technique to overcome fertil-ization defects in selected women with endometriosis (26).
Direct visualization of the pelvis through a laparoscopicexamination is essential for the diagnosis of peritoneal im-plants and adhesions. However, endometriotic ovarian cystscan be reliably identified by transvaginal ultrasound. Atrained sonographer can easily distinguish endometriomasfrom other ovarian cysts according to their characteristicechogenic appearance (27). Sensitivity and specificity of thetransvaginal ultrasound for the detection of endometriomaare 84%–100% and 90%–100%, respectively (28–33). Thepossibility of identifying endometriotic ovarian cyst withoutlaparoscopic removal and histologic confirmation and the
836 Pabuccu et al. GnRH protocols for endometriosis in
development of IVF techniques have led to new therapeuticscenarios (28). Hence, aspiration of endometrioma beforeIVF-ICSI cycles offers a nonsurgical approach (34).
Although the depressing effect of endometriosis on IVFoutcome was demonstrated, IVF still offers the highest PRsfor endometriosis, especially in early stage disease (25). InIVF cycles, in addition to the observed negative effects of en-dometriosis on developing follicles, oocytes, and embryos,advanced endometriosis as well as the presence of the cystper se or inadvertent removal of consistent amount of ovariantissue during cystectomy may pose further risks to the ovarianreserve and response (27). A debated and still unsolved topicis whether endometriomas should be treated before undergo-ing IVF cycles because of these risks (34, 35). According tothe studies opposing the surgical management of endome-trioma before IVF, excision of endometriotic ovarian cystsis associated with a significant reduction in ovarian reserveand nonsurgical treatment is a better option to avoid reduc-tion of the ovarian response in infertile patients (15, 16).Studies on the behalf of surgical management report similarnumber of oocytes and embryos obtained after laparoscopiccystectomy, suggesting that this procedure done by
ICSI Vol. 88, No. 4, October 2007
TABLE 3Controlled ovarian hyperstimulation (COH) parameters and ICSI outcomes in the study groups.
Note: BMI ¼ body mass index.P< .05, statistically significant differences between GnRH analogue and antagonist groups.
Pabuccu. GnRH protocols for endometriosis in ICSI. Fertil Steril 2007.
experienced surgeons is a valuable surgical tool for the treat-ment of large ovarian endometriomas without compromisingovarian reserve (36). Hence, in the current IVF-ICSI practiceovarian reserve and follicular health have been the center ofthe debate on the infertility–endometriosis relationship, in es-pecially active and resected endometrioma groups. Newmedical therapies, such as use of GnRH antagonists, are po-tential candidate drugs for improving IVF outcome in endo-metriosis because their mechanism of action may bebeneficial for a limited cohort of follicles in patients with ad-vanced stages of endometriosis and patients who have under-gone resection of endometrioma (15, 16).
Although the GnRH antagonist protocol in the currentstudy resulted in significantly lower number of MII oocytes,number of available embryos, and decreased fertilizationrates—surrogacy of oocyte quality and follicular health—compared to those patients undergoing the GnRH-a protocolwho had ovarian surgery for endometrioma, these findingsdid not reach a statistically significant decrease in pregnancyand implantation rates. Nevertheless, there was a trend forhigher implantation and PRs with GnRH-a compared to theGnRH antagonist protocol in these patients (29% vs. 37%).The COH with GnRH antagonists in the current study re-sulted in significantly lower levels of E2 on the day of hCGinjection, lower number of follicles R17 mm, total number
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of retrieved oocytes, MII oocytes, and available embryoscompared to those on the GnRH-a protocol in patients with-out a history of previous surgery for ovarian endometrioma.Nevertheless, these results did not alter the overall implanta-tion and clinical PRs between the two protocols. Similarimplantation rate between the analogue and antagonistgroups may imply a negative effect of endometriosis on endo-metrial receptivity. Further in vitro and in vivo experimentsinvestigating the role of GnRH-a and GnRH antagonists onendometrial receptivity and ovarian steriodogenesis of gran-ulosa/theca cells are required for the explanation of thesefindings.
Cryopreserved embryos offer another chance at pregnancyto patients who either did not conceive during the fresh IVFcycle or want another pregnancy. It was suggested that the ad-vantage of cryopreserved embryo transfer in women withendometriosis was even more apparent, because ovarianhyperstimulation, which might activate the endometriosis,was not required (37). In our study the resected and active en-dometrioma groups treated with agonist protocols had signif-icantly higher number of available embryos that can becryopreserved. Moreover, in all endometriosis groups, pa-tients treated with the agonist protocol have significantlyhigher number of MII oocytes and available embryos com-pared to patients treated with the analogue protocol. When
837
cryopreserved embryos are considered, cumulative fecundityrate may be higher with the GnRH-a protocol.
The newly published review evaluating results of three ran-domized controlled trials confirms the fact that pretreatmentof women with endometriosis with a GnRH-a for at least 3months (and up to 6 months) before IVF or ICSI increasesthe odds of clinical pregnancy by at least fourfold. Neverthe-less, and based on currently available evidence, women withendometriosis treated with IVF or ICSI should receiveGnRH-a therapy for a minimum of 3 months before the pro-cedure as this will increase the odds of clinical pregnancy byfourfold, bearing in mind that the data regarding adverse ef-fects of this therapy on the mother or fetus are not available atpresent (38).
We observed similar IVF outcomes with GnRH-a andGnRH antagonist protocols in mild-to-moderate endometri-osis suggesting that GnRH antagonist is an alternative toGnRH-a. In the literature, the data are controversial. Amongtwo recent meta-analyses, Ludwig et al. (39) do not reportany significant difference in the PRs between GnRH antago-nist and GnRH-a protocols, whereas Al-Inany and Aboulghar(40) present lower PRs with GnRH antagonists. Merviel et al.(41) have also reported lower PRs with the GnRH antagonistprotocols.
In conclusion, COH with both GnRH-a and GnRH antag-onist protocols has similar IVF outcomes in patients withmild-to-moderate endometriosis. Considering the implanta-tion and clinical PRs, COH with both GnRH antagonist andGnRH-a protocols may be equally effective in patients withand without ovarian surgery for endometrioma. Similar im-plantation rates between the analogue and antagonist groupsmay point toward the endometrium rather than the oocyte be-ing the cause of a lower trend for PR in women with advancedend-stage endometriosis. In all endometriosis groups, pa-tients treated with the agonist protocol have a significantlyhigher number of MII oocytes and available embryos thatcan be cryopreserved compared to patients treated with theantagonist protocol. When the outcome of subsequentfreeze–thaw cycles are considered, cumulative fecundityrate will be higher in the agonist protocol.
To our knowledge this is the first prospective study inves-tigating GnRH antagonist treatment in patients with endo-metriosis. Modifications in GnRH antagonist schemes,including day of initiation, adjustment of doses, criteria forhCG injection, and strategies for endometrium quality arestill investigated. Studies, especially individualizing GnRHantagonist therapies according to the type of infertility, mayimprove the data about these recently introduced regimens.
REFERENCES1. Hart R. Unexplained infertility, endometriosis, and fibroids. BMJ
2003;27:721–4.
2. Surrey ES, Halme J. Effect of peritoneal fluid from endometriosis pa-
tients on endometrial stromal cell proliferation in vitro. Obstet Gynecol
1990;76:792–7.
838 Pabuccu et al. GnRH protocols for endometriosis in I
3. Ryan IP, Taylor RN. Endometriosis and infertility: new concepts. Obstet
Gynecol Surv 1997;52:365–71.
4. Kodama H, Fukuda J, Karube H, Matsui T, Shimizu Y, Tanaka T. Benefit
of in vitro fertilization treatment for endometriosis-associated infertility.
Fertil Steril 1996;66:974–9.
5. Zikopoulos K, Kolibianakis EM, Devroey P. Ovarian stimulation for in
vitro fertilization in patients with endometriosis. Acta Obstet Gynecol
Scand 2004;83:651–5.
6. Arici A, Oral E, Bukulmez O, Duleba A, Olive DL, Jones EE. The effect
of endometriosis on implantation: results from the Yale University invitro
fertilization and embryo transfer program. Fertil Steril 1996;65:603–7.
7. Pal L, Shifren JL, Isaacson KB, Chang Y, Leykin L, Toth TL. Impact of
varying stages of endometriosis on the outcome of in vitro fertilization–
39. Ludwig M, Albano C, Olivennes F, et al. Plasma and follicular fluid
concentrations of LHRH antagonist cetrorelix (Cetrotide) in con-
trolled ovarian stimulation for IVF. Arch Gynecol Obstet 2002;266:
12–7.
40. Al-Inany H, Aboulghar M. GnRH antagonist in assisted reproduction:
a Cochrane review. Hum Reprod 2002;17:874–85.
41. Merviel P, Antoine JM, Mathieu E, Millot F, Mandelbaum J, Uzan S.
Luteinizing hormone concentrations after gonadotropin-releasing hor-
mone antagonist administration do not influence pregnancy rates in in vi-
tro fertilization–embryo transfer. Fertil Steril 2004;82:119–25.
839
MALE FACTOR
Markov modeling of vasectomy reversal and ARTfor infertility: how do obstructive interval and femalepartner age influence cost effectiveness?Michael H. Hsieh, M.D., Ph.D., Maxwell V. Meng, M.D., and Paul J. Turek, M.D.
University of California San Francisco, Department of Urology, San Francisco, California
Objective: To apply Markov models to assess the cost effectiveness of the relative impact of obstructive intervaland female partner age on fertility using either assisted reproductive technology (ART) or vasectomy reversal, andelucidate the impact of these variables on fertility.Design: Markov models based on review of published literature and available ART outcome data.Setting: University-based clinical practice.Patient(s): Simulation runs of 50,000 patients for each analysis.Intervention(s): Varying vasectomy obstructive interval and maternal age.Main Outcome Measure(s): Cost effectiveness, willingness to pay (WTP), and net health benefit.Result(s): Base case analysis showed ART yields a higher pregnancy rate and higher cost than vasectomy reversal.Sensitivity analysis showed female age has a greater effect on cost effectiveness than obstructive interval. Ata WTP < $65,000, vasectomy reversal is more cost effective than ART. With increasing WTP, ART is morecost effective over wider windows of female age.Conclusion(s): Markov modeling of fertility after vasectomy suggests female age has more impact than vasectomyobstructive interval on cost effectiveness. (Fertil Steril� 2007;88:840–6. �2007 by American Society for Repro-ductive Medicine.)
It is estimated that over 800,000 vasectomies are performedannually in the United States (1). In addition, 3% to 8% ofmen seek future fertility after the procedure (1).Vasectomyreversal and assisted reproductive technology (ART) areboth used to treat male infertility resulting from vasectomy.In 2004, over 110,000 ART procedures were reported tothe Centers for Disease Control and Prevention, some ofwhich were performed for vasectomy-induced infertility(2). With an average cost of $12,400 per in vitro fertilization(IVF) cycle (3), ART is clearly a costly option. Although va-sectomy reversal can be less costly in many instances, manyreproductive endocrinologists routinely recommend ART tocouples with vasectomy-associated infertility.
Randomized, controlled clinical trials to determine theoptimal treatment for vasectomy-associated infertility havenot been undertaken, nor are they considered feasible. Inaddition, few cost-effectiveness studies exist to help physi-cians and patients make appropriate decisions regarding in-fertility treatment after vasectomy (4–6). In such instances,decision modeling can be very helpful in dissecting out
Received August 24, 2006; revised and accepted November 29, 2006.
Reprint requests: Paul J. Turek, M.D., 1600 Divisadero Street, Room A633,
San Francisco, CA 94143-1695 (FAX: 415-885-7443; E-mail: pturek@
urology.ucsf.edu).
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine,
840
relevant and significant variables that impact a clinicalcondition.
Decision analytic models are methods of estimating andcalculating outcomes by identifying the clinical question, dis-aggregating the problem into discrete units to include all rea-sonable choices and consequences, and assigning probabilitiesand costs to the various events and outcomes (7). Based on ourprior work with decision analysis modeling of ART versus va-sectomy reversal, we observed that vasectomy reversal is oftenmore cost effective than ART, but the variable of vasectomyreversal patency, and indirectly vasectomy obstructive inter-val, is an important determinant of cost effectiveness (6). Inaddition, we assumed that female fecundity, and hence femaleage, was independent of treatment modality. Clinically, the de-cision to choose vasectomy reversal or ART is more complexin the setting of the ‘‘older’’ vasectomy (obstructive interval>14 years) or with advanced maternal age (>38 years old). In-deed, the relative impact of these clinical variables is very realto the many couples affected by them.
To our knowledge, the literature has not yet examined thecombined effects of obstructive interval and female partnerage with time on the success of fertility treatment optionsafter vasectomy. Thus, we felt an analysis of these twoclinical variables over time, through Markov modeling,
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.11.199
would provide important additional information regardingthe cost effectiveness of vasectomy reversal versus ART aftervasectomy.
Markov modeling is a form of decision analysis in whichhypothetical patients proceed through health states overtimebased on pre-defined probabilities and costs (7). As patientsare cycled, outcomes, including incurred costs and events,are tracked. Male infertility resulting from vasectomy is suit-able for this form of analysis because the condition entailsdiscrete states (i.e., obstruction or patency) which can changewith time as interventions are performed.
Although decision modeling has suggested that vasectomypatency rate and maternal age are relevant variables that drivethe cost effectiveness of treatments for vasectomy-induced in-fertility (6), how these variables interact to influence outcomesover time is unclear. Therefore, to better understand the rela-tive impact of obstructive interval and female partner age onfertility after vasectomy, we applied Markov models to com-pare the cost effectiveness of ART versus vasectomy reversal.
MATERIALS AND METHODS
Model Design
For this study, Markov models were created for infertile menseeking paternity with postvasectomy obstruction (Fig. 1).The decision tree compared initial ART versus vasectomy re-versal. After two failed vasectomy reversals, the latter armcrossed over to ART. The interval between a second failed va-sectomy reversal attempt and cross-over to ART was 1 year.Failed ART led to further ART attempts, with cycles per-formed every 3 months as needed. The interval for a secondvasectomy reversal attempt, if needed, was 1 year.
Markov model design and cost-effectiveness analyseswere performed using the TreeAge Pro software suite withthe Healthcare module (www.treeage.com).
Cohort Size and Time Horizon
We simulated 50,000 hypothetical patients in each treatmentarm until pregnancy was achieved or for 3 years, whichevercame first.
Fertility and Sterility�
Model Assumptions
Although there is no literature that specifically addresses thisissue, we made the conservative assumption that pregnancyrates after vasectomy reversals with obstructive intervals<15 years are 10% higher than those for obstructive intervals>15 years (8). We assumed that infertility in men was duesolely to obstruction, and that failure of pregnancy afterreversal was not the result of nonazoospermic failure. Forpatients who crossed over from vasectomy reversal to ART,costs for ART were assumed to be the same whether or notthe vasa were patent after vasectomy reversal. We also as-sumed that pregnancy rates after subsequent IVF cyclesand vasectomy reversals were the same as those after initialIVF cycles and vasectomy reversals (i.e., no decrementwith successive treatments).
Probability Values
Outcome probabilities applied to the model were derivedfrom published sources whenever possible. Other outcomeswere procured from institutional sources. A list of all relevantoutcomes is given in Table 1.
Base Case Values and Analysis
‘‘Base case’’ analysis refers to running Markov models witha set of chosen baseline values for variables.
Cost-effectiveness Analysis
‘‘Costs’’ were equated with out-of-pocket costs for patients.Costs of interventions were calculated from national averagesand institutional costs (see Table 1). ‘‘Effectiveness’’ was de-fined as clinical pregnancy. Each hypothetical patient achiev-ing pregnancy received an effectiveness score of 1, whereaspatients failing to achieve pregnancy received an effective-ness score of 0. Hence, ‘‘cost effectiveness’’ was defined asthe ratio of out-of-pocket costs to pregnancy rate, with lowerratios being more desirable.
By definition, if one treatment option is more effective butmore costly than another option, both options are equally costeffective. Subsequently, cost effectiveness was referenced
FIGURE 1
Markov model for immediate assisted reproduction versus vasectomy reversal protocols for male infertility aftervasectomy.
Hsieh. Markov models of vasectomy reversal and ART. Fertil Steril 2007.
0-$20,000 <$14,0000-$16,000 >$7,0000-20 years None25-50 years 33.0 to 33.66-24 months >7.8
1-3 None
1-6 months None12-60 months <52.2
0-100% >60%b n/ab n/ab n/a
b n/a
0-100% <40%
inion b n/a
inion b n/a
c n/a
c n/a
c n/a
c n/a
-pay of $100,000.
.
ity analysis was not performed.
TABLE 1Costs, values, and outcome probabilities for base case analysis, and clinical parameter value ranges and thresh
Row VariableBase case
valueSource of evidencefor base case value
1 Costs per IVF cycle $12,400 2004 SART National Summary2 Costs per vasectomy reversal $8,500 2006 UCSF institutional costs3 Obstructive interval 14 years 2006 UCSF institutional average4 Female partner age 34 years 2006 UCSF institutional average5 Interval for vasectomy
reversal or cross-over to ART12 months 2006 UCSF institutional practice
6 Maximum number ofvasectomy reversals
2 2006 UCSF institutional practice
7 Interval for IVF cycles 3 months 2006 UCSF institutional practice8 Time limit for pregnancy to occur 36 months 2006 UCSF institutional average
Probability of pregnancy, per IVF cycle,9 female partner age <35 years 42.2% 2004 SART National Summary10 35–37 years 35.4% 2004 SART National Summary11 38–40 years 26.4% 2004 SART National Summary12 >40 years 17.1% 2004 SART National Summary
Probability of pregnancyafter vasectomy reversal,
13 obstructive interval <15years, female age <30 years
74% Large retrospective reviews(8, 17) and expert opinion
14 obstructive interval <15years, female age 30-35 years
59% Large retrospectivereviews (8, 17) and expert opinion
15 obstructive interval <15years, female age 36-40 years
42% Large retrospective reviews (8, 17) and expert op
16 obstructive interval <15years, female age >40 years
38% Large retrospective reviews (8, 17) and expert op
17 obstructive interval >15years, female age <30 years
64% Review of 127 patients (17)
18 obstructive interval >15 years, femaleage 30–35 years
49% Review of 127 patients (17)
19 obstructive interval >15 years, femaleage 36–40 years
32% Review of 127 patients (17)
20 obstructive interval >15 years, femaleage >40 years
28% Review of 127 patients (17)
aThreshold indicates variable value(s) at which the immediate ART treatment protocol becomes more cost-effective at a willingness-toFor example, ‘‘<$10,000’’ indicates that at less than $10,000 for a given cost, the immediate ART protocol is more cost-effective.
bAge ranges for parameter (see second column) do not include base case age, hence one-way sensitivity analysis was not performed
c Obstructive interval ranges for parameter (see second column) do not include base case obstructive interval, hence one-way sensitiv
Hsieh. Markov models of vasectomy reversal and ART. Fertil Steril 2007.
842H
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against willingness-to-pay (WTP), which represents thehighest increase in costs that patients are willing to pay perunit increase in pregnancy rate.
Net Health Benefits
Net health benefit (NHB) is calculated for a treatment proto-col using associated costs and effectiveness as well as WTP,under a given set of values for clinical parameters. The for-mula is as follows:
Net health benefit ¼ Effectiveness
� ðCosts=Willingness to payÞ
Thus, a higher NHB for a treatment option indicates highercost effectiveness. For this Markov model, effectiveness isequated with clinical pregnancy rate.
Sensitivity Analysis
Sensitivity analysis refers to the analytic process by whichone or more variables are altered while keeping other vari-ables unchanged (i.e., at base case values) to determine whichvariables impact the Markov model. One-way sensitivityanalyses were performed by calculating the thresholds atwhich a given value for a variable would change the treatmentprotocol that was deemed most cost effective (less costly fora higher pregnancy rate). Two-way sensitivity analysis wasalso performed, where two variables are varied simulta-neously and thresholds are measured.
RESULTS
Base Case Example
A hypothetical patient who underwent vasectomy 14 yearsago (see Table 1, row 3) presents for infertility treatment.His female partner is 34 years old (see Table 1, row 4).The couple starts in the vasectomy reversal treatment arm,and the man undergoes vasectomy reversal (see Table 1:row 14: 59% probability of success; row 2: cost of $8,500).
The vasectomy reversal fails, and he undergoes a secondvasectomy reversal attempt 1 year later. The second attempt(59% probability of success, cost $8,500) also fails. One yearlater, the couple crosses over to ART. The first IVF cycle (seeTable 1, row 10: 35.4% probability of success because fe-male partner is now 36 years old; row 1: cost of $12,400)fails to result in pregnancy, and 3 months later, a second cy-cle (35.4% probability of success, cost $12,400) results inpregnancy. The couple incurs a total cost of $41,800 for thesetreatments, and receives an effectiveness score of 1.
Base Case Analysis
Base case analysis showed that immediate ART results ina higher overall pregnancy rate (99.4 % vs 95.3%) but incurshigher costs than vasectomy reversal (Table 2). Hence, at basecase values and assuming an unlimited WTP, immediate ARTand vasectomy reversal are both cost effective.
Fertility and Sterility�
Sensitivity Analysis
One-way sensitivity analysis was performed on all variablesused in the Markov model. Sensitivity analysis showed thatfemale partner age affected cost effectiveness more pro-foundly than obstructive interval. This is illustrated in Fig-ure 2 (tornado diagram).
At a defined WTP of $100,000, these two variables were var-ied independently to examinewhich one more strongly affectedNHB. When female partner age was varied from 25 to 50 years,the NHB for the most cost-effective protocol, vasectomy rever-sal, varied from 0.82 to 0.38. When female age was held con-stant and obstructive interval varied from 0 to 20 years, theNHB for vasectomy reversal varied much less, from 0.681 to0.683. Because female partner age influenced cost effective-ness much more than obstructive interval, we performed subse-quent NHB analyses using female partner age.
Other clinical parameters also influenced cost effective-ness of vasectomy reversal versus ART (see Fig. 2, Table1). At a WTP of $100,000, IVF pregnancy rates influencedNHB more than any other clinical parameter. For example,NHB ranged from -0.09 to 0.88 when the IVF pregnancyrate was varied from 0 to 100%. When IVF pregnancy rateswere >60%, ART became more cost effective than vasec-tomy reversal.
Additional clinical parameters with significant impact oncost effectiveness included costs per IVF cycle and vasec-tomy reversal, pregnancy rates after vasectomy reversal, thelength of time that hypothetical couples spent on infertilitytreatment, and the interval between IVF cycles. Of these clin-ical parameters, all featured threshold values (see Table 1 forexact values) except for the interval between IVF cycles. It isinteresting that, for a WTP of $100,000, varying the maxi-mum number of reversal attempts from one to three (two
TABLE 2What it takes to get pregnant: base caseincurred costs and outcomes after 3 years oftreatment.
ImmediateART
Vasectomyreversal
first
Mean costs$31,399 $29,274
Cumulativepregnancy rate 99.4% 95.3%
Mean numberof IVF cycles 2.5 1.6
Mean numberof vasectomyreversals
0 1.17
Hsieh. Markov models of vasectomy reversal and ART. Fertil Steril 2007.
843
attempts were used for base case analysis) did not make theIVF arm more cost effective than the vasectomy reversal arm.
We performed additional sensitivity analyses by testinglower vasectomy reversal success rates for obstructive inter-vals of <14 years (younger) when compared with >14 years(older) (data not shown). Our original assumption was that‘‘older’’ vasectomy reversals had a 10% lower associatedpregnancy rate than ‘‘younger’’ vasectomy reversals. How-ever, when this difference in success rate was increased to36% (the maximum possible value given that the pregnancyrate after ‘‘younger’’ vasectomy reversals with female part-ners<30 years is 64% [see Table 1]), female partner age con-tinued to exert a greater impact on cost effectiveness thanobstructive interval.
At a WTP of up to $65,000, assuming that patients werewilling to pay up to $65,000 to obtain a pregnancy, vasec-tomy reversal was more cost effective than ART (Fig. 3).With increasing WTP, assuming that couples were willingto spend more than $65,000 for a pregnancy, immediateART became more cost effective; this was true over widerwindows of female partner age. For example, at a WTP of$100,000, vasectomy reversal was more cost effective whenthe female partner age ranged between 33.1 and 38.0 years.When WTP is increased to $150,000, vasectomy reversalwas more cost effective when the female partner age rangedbetween 32.6 and 39.3 years.
Two-sensitivity analysis showed that particular combina-tions of clinical parameters interacted to influence cost effec-tiveness. For instance, female partner age interacted withcosts of IVF and vasectomy reversal (data not shown). Like-wise, IVF pregnancy rates interacted with these same clinical
FIGURE 2
Tornado diagram of female partner age, obstructiveinterval, and other clinical parameters. Widths ofhorizontal bars indicate magnitude of impact ofvarying a parameter on net health benefits (NHB).The wider the bar, the greater the impact on NHB.Thick vertical bars indicate threshold values at whichvasectomy reversal and assisted reproductionswitch being more cost effective. (Exact thresholdvalues are shown in Table 1.)
Hsieh. Markov models of vasectomy reversal and ART. Fertil Steril 2007.
844 Hsieh et al. Markov models of vasectomy reversal an
parameters to determine cost effectiveness (data not shown).Importantly, vasectomy reversal was more cost effective overmost combinations of parameter values.
DISCUSSION
The success of ART for treating female infertility has led to itsempirical use in cases of surgically treatable forms of male in-fertility, including vasectomy-associated infertility. Despiteits wide acceptance, it has become clear that ART treatmentis costly (9–11) and carries risks of associated complicationsfor potential mothers as well as offspring (12, 13). In addition,there is little hard evidence to guide clinical decision-makingregarding vasectomy reversal versus ART in cases of older(>14 years) vasectomies with advanced (>38 years) maternalage. Given the lack of randomized controlled trials addressingthis issue, we have applied the tools of decision science to in-vestigate the relevant issues on this topic (6).
In this study, we extend our prior work with decision mod-eling to further examine the cost-effectiveness issues that sur-round the management options for vasectomy-associatedinfertility. Markov modeling, unlike decision analysis, pro-vides the added benefit of analyzing cost effectiveness of in-fertility treatments over time. This type of modeling providesa more realistic framework for clinicians to counsel patientsusing clinically apparent, patient-specific information such asfemale partner age, obstructive interval, and WTP. In general,we observed that when cost effectiveness is considered overtime, female age (and indirectly female reproductive poten-tial) has a greater influence than vasectomy obstructive inter-val on outcomes. Many infertility clinicians have longsuspected this empirically, but this study provides quantita-tive support for this hypothesis.
Our findings concerning patient willingness to pay are alsoinformative. It is well recognized that patients have ceilingvalues for WTP for elective medical procedures, includingemotionally sensitive therapies such as infertility treatment(14, 15). In 1995, Granberg et al. (15) surveyed infertile cou-ples at two Swedish IVF clinics, and 55% of couples reporteda WTP of 10,000 pounds, the equivalent of US$14,757. Fromthis work, it was also clear that WTP for infertility treatmentsvaried widely, and socioeconomic factors that contributed tothose findings in 1995 may not apply today.
Neumann and Johannesson (14) showed that, dependingon the methodology used for calculations, WTP for infertilitytreatment among surveyed Americans ranged from $40,640to $63,896 for a hypothetical pregnancy rate of 100%. Apply-ing these findings to the current study, it is interesting to notethat previously reported WTP are lower than the WTP($65,000) needed for the vasectomy reversal protocol to bemore cost effective than immediate IVF. This implies thatfor most infertile couples, vasectomy reversal may be morecost effective.
The one-way sensitivity analysis of the variables used inour Markov models showed that clinical parameters such as
d ART Vol. 88, No. 4, October 2007
female partner age, IVF and vasectomy reversal costs andpregnancy rates, the interval between IVF cycles, and thelength of time spent on infertility treatments all profoundlyaffect cost effectiveness. Furthermore, two-way sensitivityanalysis showed that female partner age interacted withIVF and vasectomy reversal costs to determine cost effective-
FIGURE 3
Net health benefits for initial ART versus vasectomyreversal as a function of female partner age ata willingness-to-pay of (A) $65,000, (B) $100,000,and (C) $150,000. The vertical dotted lines indicatethe female age at which the most cost-effectivetreatment changes from one treatment to another.
Hsieh. Markov models of vasectomy reversal and ART. Fertil Steril 2007.
Fertility and Sterility�
ness. Of these clinical parameters, only female partner ageand length of time spent on treatment are strictly patient spe-cific. It is interesting that obstructive interval had little effecton cost effectiveness. However, it must be recognized thatcosts and pregnancy rates vary greatly among individualpractices and geographic locations, and although we didnot examine costs as a primary end point in this study, ourmodels can be customized to determine optimal treatment al-gorithms for individual infertility centers.
Decision analysis models have intrinsic limitations. Wechose pregnancy rates rather than live birth rates for as ouroutcome measure largely because the former has been re-ported more widely in the vasectomy reversal literature. Mul-tiple births can significantly increase costs associated withIVF. However, to simplify the model, we did not includethe consideration of multiple gestations/births. We believethat because [1] the economic perspective of our analysiswas out-of-pocket costs for patients, [2] multiple births arecovered by insurance, and [3] the costs of raising multiplechildren are postprocedural issues, it was reasonable to ex-clude multiple gestations from our models.
We assumed that infertility in these men was due solely toobstruction, and that failure of pregnancy after reversal wasnot the result of nonazoospermic failure. Furthermore, wedid not factor in costs of sperm retrieval at time of reversalor for IVF/intracytoplasmic sperm injection, because we at-tempted to use national level data whenever possible andcosts and success rates vary widely according to clinical prac-tices. We assumed that, after a first reversal attempt, subse-quent attempts would have the same success rates. Whenwe tested the effects of a 10% decrement in success of subse-quent reversal attempts (8), the cumulative pregnancy rate forthe vasectomy reversal arm dropped from 95.3% to 94.1% inbase case analysis (data not shown). The windows of femaleage in which IVF was more cost effective remained essen-tially unchanged despite a 10% decrement in success of sub-sequent reversal attempts. Hence, a realistic decrease insubsequent reversal attempt success rates did not alter cost ef-fectiveness to any significant degree.
Because there is no consensus in the cost-effectiveness lit-erature regarding whether and how productivity costs (i.e., in-direct costs) should be incorporated into cost-effectivenessanalyses (16), this study focused on patient out-of-pocketcosts for infertility treatment and did not include indirectcosts. However, a comparison of indirect costs betweenART and vasectomy reversal would likely benefit vasectomyreversal, given the more intense monitoring and ongoing pa-tient care involved with IVF cycles.
Cost-effectiveness analyses of long-term outcomes mustalso consider inflation and changing costs and success rates.We chose to exclude these factors in our modeling becauseof the short time horizon (<3 years) used for the study. De-spite these caveats, it is our hope that this study providesimportant information for physicians, patients, and insur-ance carriers regarding the various clinical parameters
845
linked with the treatment of vasectomy-associated maleinfertility.
Markov models, incorporating important variables such assurgeon or ART experience, female partner age, obstructiveinterval, and cost effectiveness, can guide individual couples’decision making in cases of infertility due to vasectomy. Thistype of model could be used by both physicians and patientsin the clinical decision process. We are currently creatinga Web-based tool that permits the use of individual parame-ters (e.g., costs, reversal results) to determine outcomes forspecific situations. Our findings support the opinion that fe-male partner age is particularly critical in determining themost cost-effective treatments for male infertility.
REFERENCES1. Schiff J, Li PS, Goldstein M. Toward a sutureless vasovasostomy: use of
biomaterials and surgical sealants in a rodent vasovasostomy model.
J Urol 2004;172:1192–5.
2. Society for Assisted Reproductive Technology. SART National
Summary, 2004. Available at: https://www.sartcorsonline.com/rptCSR_
PublicMultYear.aspx?ClinicPKID¼0. Accessed January 2007.
3. American Society of Reproductive Medicine. Frequently asked ques-
tions about infertility. Available at: http://www.asrm.org/Patients/faqs.
html. Accessed January 2007.
4. Pavlovich CP, Schlegel PN. Fertility options after vasectomy: a cost-
The beneficial effects of toremifene administration onthe hypothalamic-pituitary-testicular axis and spermparameters in men with idiopathic oligozoospermiaDimitrios Farmakiotis, M.D., Christos Farmakis, M.D., David Rousso, M.D., Anargyros Kourtis, M.D.,Ilias Katsikis, M.D., and Dimitrios Panidis, M.D., Ph.D.
Division of Endocrinology and Human Reproduction, Second Department of Obstetrics and Gynecology, Aristotle University of
Thessaloniki, Thessaloniki, Greece
Objective: To evaluate whether toremifene, a selective estrogen receptor modulator (SERM), has a beneficiaryeffect on all three main sperm parameters.Design: Prospective interventional clinical study.Setting: University hospital.Patient(s): One-hundred subfertile men with idiopathic oligozospermia.Intervention(s): Toremifene (60 mg daily) was administered to all men for 3 months. At baseline and at the end ofeach month, serum concentrations of follicle-stimulating hormone (FSH), testosterone, inhibin B, and sex hor-mone–binding globulin (SHBG) were measured. At baseline and at the end, semen analysis was performed andsperm concentration, spermatozoal motility and normal sperm forms were determined.Main Outcome Measure(s): Gonadotropin, testosterone, inhibin-B levels, total sperm count, sperm morphologyand motility.Result(s): Toremifene administration resulted in a significant increase in FSH, testosterone, SHBG, and inhibin Blevels, as well as in sperm concentration, percentage motility and normal sperm forms. Twenty-two men’s partnersachieved pregnancy within 2 months of the end of treatment. At the end of the third month, serum FSH levels weresignificantly higher in the men whose partners did not achieve pregnancy, and total sperm count and normal spermforms were significantly lower compared with the group of men whose partners achieved pregnancy.Conclusion(s): Toremifene administration for a period of 3 months in men with idiopathic oligozoospermia is as-sociated with significant improvements of sperm count, motility, and morphology, mediated by increased gonad-otropin secretion and possibly a direct beneficial effect of toremifene on the testes. The above findings are alsoindicative of a better testicular exocrine (improved sperm parameters) response to treatment in men whose partnersachieved pregnancy compared with those who did not. Further randomized, placebo-controlled trials shouldbe conducted to determine whether this particular selective estrogen receptor modulator can be useful as aninitial approach in men with oligozoospermia. (Fertil Steril� 2007;88:847–53. �2007 by American Society forReproductive Medicine.)
Key Words: Toremifene, oligozoospermia, male infertility, SERM
A selective estrogen receptor modulator (SERM) is a com-pound that can act as an estrogen agonist or antagonist,depending on the specific target tissue (1). At present, fourSERMs are approved for clinical use: clomiphene, raloxifene,tamoxifen, and toremifene. Three of these compounds belongto the triphenylethylene family: clomifene, tamoxifen, andtoremifene. Raloxifene belongs to the benzothiophene family(2).
Most of the unique pharmacology of SERMs can be ex-plained by three interactive mechanisms. The first is differen-tial estrogen-receptor expression in a given target tissue.The second consists of the differential estrogen-receptorconformation on ligand binding. The third is the differential
Received August 8, 2006; revised and accepted December 22, 2006.
Reprint requests: Anargyros Kourtis, M.D., 2nd Department of Obstetrics
and Gynecology, 45 Aristotelous Street, 55236, Panorama, Greece
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.038 Copyright ª2007 American
expression and binding to the estrogen receptor of coregula-tor proteins (3).
In women, at least three SERMs have been shown toincrease circulating levels of gonadotropin. The exactmechanism for this effect is based on the estrogen antagonisticproperties of SERMs at the hypothalamic and pituitary level(4). In addition, SERMs have been shown to increase sex hor-mone–binding globulin (SHBG) levels, which can be attrib-uted to the estrogen agonist activity of SERMs in the liver(2, 3).
There have been relatively few studies of SERMs in males.Clomiphene citrate and tamoxifen have been proposed for themanagement of male factor infertility (1). Tamoxifen citratewas introduced 30 years ago as an empiric treatment foridiopathic oligozoospermia because of its stimulatory actionon gonadotropin secretion and its postulated direct effects onLeydig cell function and 5a-dihydrotestosterone productionin seminiferous tubules and epididymis (5). The main effect
Fertility and Sterility� Vol. 88, No. 4, October 2007 847Society for Reproductive Medicine, Published by Elsevier Inc.
of tamoxifen on spermatogenesis is stimulatory, resulting ina twofold increase in spermatozoa concentration. Such anincrease in spermatozoa concentration may be importantbecause a change of this magnitude at the low range of sper-matozoa concentrations found in oligozoospermic men hasbeen associated with disproportionately higher fecundity.However, this particular SERM has not been shown to induceany marked changes in motility and morphology (6, 7).
The effect of treatment with tamoxifen citrate on cumula-tive achievement of pregnancy over a long period of time issimilar to that of assisted reproductive techniques (ART)(8, 9). On the basis of these results (6, 10, 11), tamoxifen cit-rate was proposed by a World Health Organization workingcommittee as the first line of treatment for idiopathic oligo-zoospermia (12).
Because tamoxifen increases spermatozoa concentrationbut has no marked effect on spermatozoa motility and mor-phology, this study was designed to evaluate whether anotherSERM, toremifene, has a beneficiary effect on all three mainsemen parameters. To the best of our knowledge, no previousdata have been reported concerning the effect of this specificSERM on the hypothalamic-pituitary-testicular axis and se-men parameters in men with idiopathic abnormalities inone or more of the three main semen parameters.
MATERIALS AND METHODS
Patients
One hundred subfertile men with idiopathic oligozoosper-mia, mean (� SEM) age 20 to 47 years (33.53 � 0.5 years),were consecutively recruited from the fertility center of ourdepartment. All of the men were characterized as subfertilebecause they had been unsuccessful in achieving pregnancywith their partners for 12 months, although their partnersdid not show any of the known causes of female subfertility.
Idiopathic oligozoospermia was defined as quantitativeand/or qualitative aberrations of sperm variables accordingto World Health Organization criteria (12). Men with knownor demonstrable causes of oligozoospermia (varicocele, in-fections, autoimmunity, stress, chromosomal abnormalities,environmental factors, or epididymitis) were excluded.
Careful clinical examination showed that all of the menhad complete development of the secondary sex characteris-tics, with a mean (� SEM) right testicular volume of 18.45�0.39 cm3 and mean (� SEM) left testicular volume 17.80 �0.41 cm3 (mean testicular volume 18.14 � 0.39 cm3). Totaltesticular volume was assessed by comparison with a standardvalue on orchidometry. None of the men had received anymedication during the 6-month period preceding the study.
Study Protocol
All of the men received toremifene as monotherapy at a doseof 60 mg daily for a period of 3 months. At baseline, and atthe end of the first, second, and third month of treatment,blood samples were collected at 9 AM after an overnight fast.
848 Farmakiotis et al. Toremifene in male infertility
All samples were centrifuged immediately, and serum wasstored at �70�C until assayed for FSH, testosterone, inhibinB, and SHBG. Sperm was examined at baseline and at the endof the third month of treatment. All participants were prop-erly informed about the purpose of the study and gave in-formed written consent. The study was approved by theethics committee of the hospital.
Hormonal Measurements
We measured FSH (IU/L) using the FSH IRMA kits fromBiosource Technologies (Vacaville, CA). Total testosterone(ng/dL) was measured by enzyme-linked immunosorbentassay (ELISA; testosterone enzyme immunoassay test kit,LI7603; Linear Chemicals). The serum levels of SHBG(nmol/L) were measured by ELISA (SHBG ELISA, MX520 11; IBL, Hamburg, Germany). Inhibin B levels were mea-sured using ELISA kits from Oxford Bio Innovation DSL Ltd(Upper Heyford, Bicester, Oxfordshire, United Kingdom).
Semen Analysis
Semen was collected by masturbation into sterilized glasscontainers after a 3- to 6-day abstinence. After evaluation ofliquefaction and measurement of viscosity and volume, motil-ity was measured, at room temperature (22� to 25�C), 1 hourafter ejaculation, as previously described elsewhere (12–15).
Sperm morphology was evaluated from Papanicolaou-stained smears, and the classification of abnormal spermforms was made according to the guidelines of the WHO(12). One hundred spermatozoa were studied from each se-men specimen, and the same individual (D.P.) evaluated allsmears. The percentage of motile spermatozoa was measuredby the subjective method at room temperature (22� to 25�C),and sperm concentration was determined in an undiluted se-men specimen with the use of Makler’s Counting Chamber(16, 17).
Statistical Analysis
Statistical analysis was performed with SPSS statistical soft-ware, v. 13.0 (SPSS Inc, Chicago, IL). Two-tailed statisticalsignificance was set at 5%. Categorical parameters (smokingstatus) were compared with Fischer’s exact test. The normal-ity of distribution was assessed with the Kolmogorov-Smir-nov test (K-S) test. Values that did not fit the normaldistribution were log-transformed.
Mean was compared at baseline with Student’s t-test andduring treatment with general linear model–based two-wayrepeated measures of analysis of variance (ANOVA); time(treatment) was set as the within-groups factor and achieve-ment of pregnancy as the between-subjects factor. Within-groups post hoc analysis was performed after Bonferroniadjustment for multiple comparisons. Between groups posthoc analysis was based on model parameter estimates.
For those parameters where statistically significant or bor-derline differences or interactions between the men whose
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partners achieved pregnancy and those who failed were ob-served, binary logistic regression analysis was also per-formed to assess prognostic value.
RESULTS
The basal epidemiologic and anthropometric features of themen studied are presented in Table 1. Twenty-two men hadpartners who achieved pregnancy within 2 months from theend of treatment (22%; 95% CI, 15.0–31.1%). The hormonalfeatures before and during treatment with toremifene of themen whose partners eventually achieved pregnancy (group1) and those whose partners did not (group 2) are summarizedin Table 2, and their semen parameters are presented inTable 3. Men whose partners achieved pregnancy did not dif-fer significantly in epidemiologic and somatotopic featuresfrom the other group (see Table 1).
Effect of Treatment
During treatment with toremifene, a significant increase incirculating levels of FSH (Fig. 1), testosterone, and inhibinB levels was observed (P<.001 for FSH, and T, P<.05 forinhibin-B; Table 2). SHBG levels (P<.001) and free andro-gen index (FAI) values (P<.05) (Fig. 2) levels was observed.The number of spermatozoa per mL, as well as per ejacula-tion, and the percentage of spermatozoa with normal motilityand morphology were also significantly increased at 3 months(P<.001; Table 3).
The increase in FSH levels was basically observed duringthe first (P<.001) and second (P<.05) months of treatment,whereas the change during the last month was not significant.Testosterone (T) and SHBG levels (P<.001), as well as FAIvalues (P<.05), were significantly increased only duringthe first month (P<.001), while the increase in SHBG levelswas significant during both the first and second month(P<.001); no significant change in T levels, SHBG concen-tration, or FAI was observed during the second and thirdmonth of treatment.
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Differences between Men Whose Partners AchievedPregnancy and Those Whose Partners Did Not
Overall, FSH levels were borderline higher in those menwhose partners did not achieve pregnancy compared withthose whose partners did (P¼.067; see Table 2, Fig. 1). Thedifference between the two groups was statistically signifi-cant at the end of treatment (P<.05; see Fig. 1). Likewise,the total number of spermatozoa per ejaculation was, overall,statistically significantly higher in men whose partnersachieved pregnancy (P<.05; see Table 2, Fig. 3). This differ-ence was, again, statistically significant at 3 months (P<.05)but not at baseline (see Fig. 3).
A borderline interaction between time and treatment out-come (pregnancy or no pregnancy) was observed with respectto the total number of spermatozoa per ejaculation (P¼.058;see Table 2, Fig. 3) and the percentage of spermatozoa withnormal morphology (P¼.064; see Table 2). Men whose part-ners achieved pregnancy had a statistically significantlyhigher percentage of spermatozoa with normal morphologyat the end of treatment (P<.05).
In univariate logistic regression analysis, FSH levels(B ¼ �1.894 � 0.959), sperm count per ejaculation(B ¼ 0.006 � 0.003), and the percentage of spermatozoawith normal morphology (B ¼ 0.04 � 0.02) after treat-ment had a statistically significant prognostic value forthe achievement of pregnancy (P<.05). In multivariate lo-gistic regression, FSH levels at 3 months were the moststatistically significant independent parameter of the three(B ¼ �1.73 � 1.02, P¼.089).
DISCUSSION
Tamoxifen citrate, a selective estrogen receptor modulator(SERM), has been proposed as an empiric treatment for idi-opathic oligozoospermia because of its stimulatory action ongonadotropin secretion and its postulated direct effects onLeydig cell function and 5a-dihydrotestosterone productionin seminiferous tubules and epididymis (5, 6, 10–12). Based
TABLE 1Epidemiologic and anthropometric features of the men in couples who eventually achieved pregnancy(group 1) and those who did not (group 2).
Note: Baseline is mean � SEM. Statistical significance determined by Student’s t-test after log-transformation, Fischer’sexact test for smoking status.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
849
TABLE 2Basic hormonal features before and during treatment with toremifene of the men in couples whoeventually achieved pregnancy (group 1) and those who did not (group 2).
FSH (mIU/mL) T (mmol/L) SHBG (nmol/L) FAI INH (pg/mL)
Note: Mean � SEM. Statistical significance by two-way repeated measures analysis of variance after log-transformation.FAI, free androgen index; FSH, follicle-stimulating hormone; INH, inhibin B; T, testosterone; SHBG, sex hormone–bind-ing globulin.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
on the above evidence, the present study was designed toinvestigate whether toremifene, another compound from thesame category (13), has a beneficiary effect on the mainsemen parameters as well.
In the present study, a statistically significant increase inserum FSH was observed after administration of toremifeneat a dose of 60 mg daily. This increase was more marked atthe end of the first month of treatment; FSH levels werealso statistically significantly increased after one additionalmonth of treatment, reaching a plateau after the end of thesecond month with no statistically significant increase duringthe last month of toremifene administration (see Table 2,Fig. 1). This increase should be attributed to the well-knownanti-estrogenic properties of SERMs in general (3) andtoremifene in specific (18, 19). The stimulatory effect ofSERMs on hypophyseal gonadotropin secretion has beenproposed as a possible mechanism for its beneficial effecton semen quality (5).
Toremifene administration also induced a statisticallysignificant increase in total testosterone levels, which wasagain significant only during the first month of treatment,reaching a plateau afterward (see Table 2). The increasedgonadotropin secretion could be the reason for this observedincrease in total testosterone levels (1, 3). However, it should
850 Farmakiotis et al. Toremifene in male infertility
be noted that SERMs have also been reported to havedirect stimulating effects on Leydig cell function (20) and5a-dihydrotestosterone production in seminiferous tubulesand epididymis (5, 21).
Despite the increase in testosterone levels, which would beexpected to suppress SHBG production by the liver (22), thelevels of this globulin were statistically significantly in-creased during the first and the second months of toremifeneadministration (see Table 2). These results are indicative ofthe estrogenic activity of toremifene at the liver, which is con-sistent with previous reports on SERM properties (23).
An interesting finding of this study, which, to the best ofour knowledge, has not been previously reported, was thestatistically significant increase in circulating inhibin B aftertoremifene administration (see Table 2, Fig. 2). Inhibin pro-duction by the testes is stimulated by FSH; moreover, inhibinsecretion has been proposed as a reliable index of Sertoli cellfunction. Therefore, the increase of inhibin levels observed inthe present study could be attributed to both increased FSHsecretion (see Table 2, Fig. 1) and the potential beneficiaryeffect of SERMs on testicular function (5, 20, 21).
Toremifene administration for a period of 3 monthsresulted in a statistically significant improvement of all three
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TABLE 3Semen parameters before and during treatment with toremifene of the men in couples who eventuallyachieved pregnancy (group 1) and those who did not (group 2).
Group �TimeF 2.123 3.674 .046 3.510P .148 .058 .831 .064
Note: Mean � SEM. Statistical significance by two-way repeated measures analysis of variance after log-transformation.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
FIGURE 1
Follicle-stimulating hormone levels (mean � SEM) ofthe men whose partners eventually achievedpregnancy and those whose partners did not, beforeand during treatment with toremifene. *P< .05 versusprevious value, yP < .05 between the men whosepartners became pregnant and those whosepartners did not.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
Fertility and Sterility�
FIGURE 2
Inhibin B (INH) levels (mean � SEM) of the menwhose partners eventually achieved pregnancy andthose whose partners did not, before and duringtreatment with toremifene.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
851
main semen parameters, namely, sperm count (see Table 3,Fig. 3), motility, and morphology (see Table 3). It shouldbe noted that, although tamoxifen has also been shown to in-duce a statistically significant increase in spermatozoa count,this particular SERM has not been shown to induce anymarked changes in motility and morphology (6, 7).
The partners of 22 men achieved pregnancy within 2months from the end of treatment. At baseline, these men pre-sented with lower serum FSH levels compared with thosewhose partners did not achieve pregnancy but not statisticallysignificantly so. Although treatment with toremifene induceda statistically significant increase in FSH levels in bothgroups, FSH levels were statistically significantly lower(P<.05) in men whose partners achieved pregnancy at theend of the third month of treatment (see Fig. 1). We postulatethat lower FSH levels in the men whose partners achievedpregnancy are due to a more functional negative feedbackfrom the testes, thus reflecting better testicular response totreatment. This trend was also apparent in the higher levelsof inhibin observed in the group of men whose partners even-tually became pregnancy, although a level of statistical sig-nificance was not reached.
At baseline, no statistically significant differences in thethree main semen parameters of sperm count, motility, andmorphology were observed between men whose partners
FIGURE 3
Total number of spermatozoa (SZ) per ejaculation(mean� SEM) of the men whose partners eventuallyachieved pregnancy and those whose partners didnot, before and after 3-month-treatment withtoremifene. *P < .05 versus previous value. yP < .05between the men whose partners became pregnantand those whose partners did not.
Farmakiotis. Toremifene in male infertility. Fertil Steril 2007.
852 Farmakiotis et al. Toremifene in male infertility
achieved pregnancy and those whose partners did not, al-though there was a trend for increased values in the formergroup. Notably, total sperm count as well as spermatozoamorphology was improved with toremifene administration;this change was more marked in the group of men whosepartners achieved pregnancy (see Table 3, Fig. 3). Moreover,total sperm count and spermatozoa morphology at the end oftreatment had statistically significant prognostic value for theachievement of pregnancy within the subsequent 2 months.This finding is again consistent with better testicularfunctional response in the subgroup of men whose partnersachieved pregnancy.
Conclusively, the present study shows that toremifene ad-ministration for a period of 3 months in men with idiopathicoligozoospermia results in statistically significant improve-ments in all three main semen parameters of sperm count,motility, and morphology, mediated by increased gonadotro-pin secretion and possibly a direct beneficial effect of toremi-fene on the testes. It is possible, therefore, that this particularSERM could be useful as an initial approach in the treatmentof subfertile men with idiopathic oligozoospermia. Neverthe-less, the precise standards of treatment need to be further in-vestigated by randomized, placebo-controlled trials.
REFERENCES1. Plouffe L Jr, Siddhanti S. The effect of selective estrogen receptor mod-
ulators on parameters of the hypothalamic-pituitary-gonadal axis. Ann
NY Acad Sci 2001;949:251–8.
2. Plouffe L Jr. Selective estrogen receptor modulators (SERMs) in clinical
tropin-induced 16-alpha-hydroxy-progesterone accumulation in normal
men. J Clin Endocrinol Metab 1980;51:1026–9.
22. de Ronde W, van der Schouw YT, Muller M, Grobbee DE, Gooren LJ,
Pols HA, de Jong FH. Associations of sex-hormone-binding globulin
(SHBG) with non-SHBG-bound levels of testosterone and estradiol in in-
dependently living men. J Clin Endocrinol Metab 2005;90:157–62.
23. Duschek EJ, Gooren LJ, Netelenbos C. Effects of raloxifene on gonado-
trophins, sex hormones, bone turnover and lipids in healthy elderly men.
Eur J Endocrinol 2004;150:539–46.
853
The contribution of genetic variations of arylhydrocarbon receptor pathway genes to malefactor infertilityAve Merisalu, B.S.,a Margus Punab, M.D.,b Signe Altmae, M.Sc.,a Kadri Haller, M.D.,c
Tarmo Tiido, M.Sc.,d Maire Peters, Ph.D.,c and Andres Salumets, Ph.D.a,c
a Department of Biotechnology, Institute of Molecular and Cell Biology, Tartu University; b Andrology Unit, Tartu University
Clinicum, and c Department of Obstetrics and Gynecology, Tartu University, Tartu, Estonia; and d Molecular Reproductive
Medicine Research Unit, Department of Clinical Sciences, Malmo, Lund University, Sweden
Objective: To determine whether the polymorphisms in aryl hydrocarbon receptor (AHR), aryl hydrocarbon recep-tor repressor (AHRR), and aryl hydrocarbon receptor nuclear translocator (ARNT) genes are associated with malefactor infertility.Design: An association study.Setting: University research laboratory and andrology clinic.Patient(s): The subjects were infertile Estonian men (n¼ 112) with azoospermia or oligozoospermia and controls(n ¼ 212) with normal sperm parameters.Intervention(s): Blood samples were obtained for DNA extraction and genotyping.Main Outcome Measure(s): AHR (Arg554Lys), AHRR (Pro185Ala), and ARNT (G/C allele) polymorphisms weregenotyped using allele-specific polymerase chain reaction. Allele and genotype frequencies were comparedbetween infertile men and controls and separately in the normozoospermia, oligozoospermia, and azoospermiagroups.Result(s): The AHRR Ala185Ala genotype was implicated in susceptibility to male factor infertility. Ala/Alagenotype frequency increased in the following order: normozoospermia (18.0%), oligozoospermia (26.0%),azoospermia (42.1%). Allele and genotype frequencies of AHR and ARNT polymorphisms were similar betweencases and controls.Conclusion(s): We demonstrated that the AHRR Pro185Ala polymorphism contributed to a predisposition to malefactor infertility in the Estonian population. A greater prevalence of the Ala/Ala genotype was found among infer-tile patients. (Fertil Steril� 2007;88:854–9. �2007 by American Society for Reproductive Medicine.)
Key Words: AHR pathway, male infertility, single nucleotide polymorphisms
Approximately one-third of unexplained human infertilityhas been attributed to adverse environmental factors, includ-ing exposure to hazardous organohalogen pollutants such asdioxins (1, 2). The biological effects of dioxins result fromtheir binding to aryl hydrocarbon receptor (AHR) and subse-quent activation of the AHR-mediated signal transductionpathway (3). In the absence of ligand, AHR is predominantlypresent in the cytoplasm in a latent conformation and boundwith several cellular proteins (4). Upon ligand binding, thenuclear localization signal of the receptor is exposed, andAHR translocates to the nucleus. Nuclear AHR forms a tran-scriptionally active heterodimer with the aryl hydrocarbonreceptor nuclear translocator (ARNT) (5).
AHR-ARNT heterodimer binds specifically to the xenobi-otic-responsive element (XRE) within the promoter region of
Received October 3, 2006; revised and accepted December 5, 2006.
This study was supported by the Estonian Science Foundation, grant nos.
6498 and 6585, and the Estonian Ministry of Education and Science,
core grant no. 0182582Cs03.
Reprint requests: Andres Salumets, Ph.D., Department of Obstetrics and
Gynecology, University of Tartu, Lossi 36, 51003 Tartu, Estonia (FAX:
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicin
854
AHR-regulated genes, resulting in their transcription (6, 7).Target genes for the upregulation of AHR-mediated tran-scription involve predominantly phase I and II xenobioticmetabolizing enzymes. Additionally, the ligand-activatedAHR induces transcription of the aryl hydrocarbon receptorrepressor (AHRR) gene. AHRR is capable of inhibiting theAHR pathway by competing with AHR for dimerizationwith ARNTand binding to XRE sequences without subsequenttranscription activation (8). Therefore, AHRR constitutesa component of negative feedback in this ligand-triggeredpathway.
Although AHR signaling plays a prominent toxicologicalrole, it has also been implicated in several physiological func-tions independent of exogenous chemical exposure (9–12).Among others, the AHR pathway is thought to be necessaryfor mammalian fertility (1, 11, 13, 14). Involvement of theAHR pathway in normal female and male fertility rendersthe reproductive system particularly vulnerable to the toxiceffects of exogenous pollutants (10). The AHR pathwaymay interact with the steroid hormone receptor function.For example, AHR-ARNT heterodimers are capable of inhib-iting the estrogen signaling by binding to estrogen response
0015-0282/07/$32.00e, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.041
elements adjacent to or overlapping with XRE sequences(15) or by increasing estrogen receptor degradation (16).Similar mechanisms may also be responsible for cross talkbetween AHR and androgen receptor pathways (17, 18). Ad-ditionally, the activated AHR pathway induced and enhancedapoptosis of testicular and ovarian germ cells (19, 20).
The wide distribution of AHR pathway proteins in humantesticular tissue might explain the mechanism of how variousorganic environmental pollutants interfere with spermatogen-esis and male fertility, resulting in elevated cell death andreduced sperm count (20). As AHR pathway genes harborseveral functional polymorphisms that possibly modify theindividual susceptibility to hazardous organohalogen pollut-ants (21–24), the aim of the current study was to explore thecorrelation between genetic variance in AHR, AHRR, andARNT genes and male factor infertility. We investigated threesingle-nucleotide polymorphisms (SNPs): nonsynonymouschanges at codon 554 (Arg to Lys) in exon 10 of the AHRgene and at codon 185 (Pro to Ala) in exon 5 of the AHRRgene as well as a synonymous change (Val to Val) at codon189 in exon 7 of the ARNT gene.
MATERIALS AND METHODS
Study Participants
The study included 112 patients from the Andrology Unit ofTartu University Clinicum during 2005. All study subjectsexperienced infertility for a period lasting more than 12months. Patients underwent a detailed medical history andgeneral physical examination. Fertility status was evaluatedby assessment of the testicular size using a Prader orchidom-eter and semen sample analysis following World HealthOrganization guidelines (25). Infertile males with nonob-structive azoospermia (n ¼ 39) and oligozoospermia (%20� 106 sperm cells/mL of ejaculate) (n¼ 73) were consideredeligible for the study (25). Patients with known causes ofmale factor infertility such as chromosomal abnormalities,Y chromosome microdeletions, and the pathologies ofthe epididymis and/or vas deferens were excluded from thestudy. Study controls (n ¼ 212) were selected based on thebest sperm parameters (sperm count between 75 and 716 �106/mL) from the young men who underwent compulsorymedical examination for possible military service (26). How-ever, no fertility data concerning their previous conceptionsand children were known. Ethical approval for the studywas obtained from the Tallinn Medical Research EthicsCommittee.
Genotyping
All patients and controls were genotyped for the followingSNPs: G / A (rs2066853) Arg554Lys in AHR, C / G(rs2292596) Pro185Ala in AHRR, and G / C (rs2228099)in ARNT (Table 1). Genomic DNA was extracted fromperipheral EDTA blood using the salting-out method (27)and amplified by allele-specific polymerase chain reaction(ASPCR). Primers for AHR, AHRR, and ARNT polymor-
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phisms are listed in Table 1. Amplification of genomicDNA (50 ng) was performed in a total volume of 15 mL con-taining 0.25 mM each of dNTP-s (MBI Fermentas, Vilnius,Lithuania), 2.5 mM MgCl2, 1 � PCR buffer (Solis BioDyne,Tartu, Estonia), 0.06 mM of allele-specific primers, 0.5 mM ofboth control primers (Invitrogen, Paisley, UK), and 1 U HotStart thermostable DNA polymerase HOT FIREPol (SolisBioDyne). Amplifications were performed with the PTC-200 thermal cycler (MJ Research, Watertown, MA). Reac-tions were initiated with denaturation (96�C, 3 minutes),followed by 32 (AHR), 35 (AHRR), and 32 (ARNT) cyclesof denaturation (96�C, 1 minute), annealing at 55�C (AHR),63�C (AHRR), and 57�C (ARNT) for 30 sec, elongation(72�C, 1 minute), and a final extension step (72�C, 5 min-utes). All products were visualized under UV light with ethid-ium bromide after separation by agarose gel electrophoresis.
A total of 32 randomly selected DNA samples (10, 12, and10 samples for AHR, AHRR, and ARNT, respectively) weresequenced to verify the results obtained by ASPCR. Beforesequencing, unincorporated PCR primers and mononucleo-tides were removed, and PCR products were treated with 1U exonuclease I (MBI Fermentas) and 1.5 U shrimp alkalinephosphatase (MBI Fermentas) (37�C, 20 minutes) and thensubjected to enzyme inactivation (80�C, 15 minutes). PurifiedPCR products (1.5–3 mL) served as templates in sequencingreactions (10 mL) with sequencing primer (5 pmol) and DYE-namic ET Terminator Cycle Sequencing Kit reagent premixas recommended by the supplier (GE Healthcare, Piscataway,NJ). All samples were sequenced from both strands. Se-quencing reactions (1.5 mL) were run on an ABI 377 Prismautomated DNA sequencer (Applied Biosystems, FosterCity, CA) using ReproGel 377 gels (GE Healthcare). The re-sults were analyzed using BioEdit version 7.0.5.3 software(Isis Pharmaceuticals, Carlsbad, CA).
Statistical Analysis
The R2.3.1A Language and Environment software (Free Soft-ware Foundation, Boston) was used for statistical analysis.The fertility parameters are provided as medians, with theminimum and maximum values given. Comparison of fertilitycharacteristics between patients and controls was performedusing the Mann-Whitney U-test and linear regression analy-sis. Allele frequencies were evaluated using the c2 test. Logis-tic and Poisson regressions were used to analyze theassociation between the polymorphisms studied and the riskfor male factor infertility, and the odds ratios (ORs) wereprovided. P<.05 was considered statistically significant.
RESULTS
Clinical Data
The fertility characteristics of the study participants areshown in Table 2. As expected, infertile patients were older,had lower mean testicular volume, and showed significantlycompromised sperm quality parameters (sperm concentrationand progressive motility) compared with controls.
TABLE 1Primers used for genotyping of AHR, AHRR, and ARNT variations using allele-specific polymerasechain reaction.
Gene SNP (rs number) Primer Primer sequence 5’ - 3’
AHR Arg554Lys (G / A) (rs2066853) Control Fa GAAATTGGCAAGATAATACTGCACCGControl Rb AAGGCACGAATTGGTTAGAGTTCCASOc-G GAAAAATTTTTCATTCTGCATGTGTCASO-A GAAAAATTTTTCATTCTGCATGTGTT
AHRR Pro185Ala (C / G) (rs2292596) Control F TTACTCGTCGGTGGAATAAAGTGTCTControl R CAGGACTGCAGCTCGTGGTASO-C AGGTGGTGTTTGGGCAGCASO-G AGGTGGTGTTTGGGCAGG
ARNT Val189Val (G / C) (rs2228099) Control F TCTTATCGATGAACTACATTTAGGControl R GAAGCCTTGCCAGAGTCAACASO-G CAGGCAGGGTGGTGTATGTGASO-C CAGGCAGGGTGGTGTATGTC
a Control F marks for control forward primer.b Control R marks for control reverse primer.c ASO marks for allele-specific oligonucleotide primer.
Merisalu. AHR pathway and male factor infertility. Fertil Steril 2007.
Genetic Variations in AHR, AHRR, and ARNT Genes
Comparisons of AHR (Arg554Lys), AHRR (Pro185Ala), and
ARNT (Val189Val) allele and genotype frequencies between
cases and controls are shown in Table 3. Genotype distribu-
tions were found to be in Hardy-Weinberg equilibrium.
Allele frequency evaluation suggested a significantly
higher incidence of the AHRR Ala185 allele among infertile
men (0.51) compared with controls (0.42, c2 test, P¼.026).
Similarly, genotype frequency estimations suggested a mark-
edly higher prevalence for the AHRR Ala/Ala genotype when
compared with combined genotypes of Pro/Pro and Pro/Ala
among infertile men (31.5% vs. 68.5%) compared with
controls (18.0% vs. 82.0%; OR ¼ 2.10, P¼.006, logistic
856 Merisalu et al. AHR pathway and male factor infertili
regression analysis), which is in agreement with the hypoth-esis of an AHRR Ala/Ala genotype being the susceptibilitylocus for male factor infertility (Table 3). The value of theAHRR Pro185Ala locus on male fertility was further evalu-ated by comparing the prevalence of the AHRR Ala/Ala geno-type in controls and in patients with azoospermia andoligozoospermia. The AHRR Ala/Ala genotype showed theassociation with the diminished sperm count, as the frequencyof the AHRR Ala/Ala genotype increased along with thedecrease in sperm count in the following order: controls withnormozoospermia (18.0%), patients with oligozoospermia(26.0%), patients with azoospermia (42.1%) (OR ¼ 1.73,P<0.001, Poisson regression analysis). As the age of patientsand controls differed, we analyzed the role of age on the
TABLE 2Comparisons of fertility characteristics between the patients and controls using the Mann-WhitneyU-test.
Patients (n [ 112) Controls (n [ 212)
Characteristic Median Minimum Maximum Median Minimum Maximum P
Note: Allele frequencies of infertile patients and controls were evaluated using the c2 test. The logistic regression analysiswas used to analyze the associations between the genotypes and the risk for male factor infertility, with the odds ratiosprovided. AHR ¼ aryl hydrocarbon receptor gene; AHRR ¼ aryl hydrocarbon receptor repressor gene; ARNT ¼ arylhydrocarbon receptor nuclear translocator gene.
a Reference group.
Merisalu. AHR pathway and male factor infertility. Fertil Steril 2007.
857
sperm concentration. The age of man was not associated withthe sperm concentration in controls or patients (r ¼ �0.57,P¼.881; and r ¼ �0.03, P¼.517, respectively, linear regres-sion analysis). In addition, the age of man did not correlatewith the diminished sperm count among controls or patientswho were carrying the AHRR Ala/Ala risk genotype(r ¼ 2.08, P¼.851; and r ¼ �0.08, P¼0.355, respectively,linear regression analysis stratified by the AHRR Ala/Alagenotype).
No significant differences were noted in allele frequen-cies of AHR Arg554 and Lys554 and ARNT Val189
Fertility and Sterility�
(G allele) and Val189 (C allele). Similar to the allelic distri-butions, there were no significant differences in genotypefrequencies of AHR Arg554Lys and ARNT Val189Val(G/C allele) loci between male factor infertility patientsand controls (Table 3).
DISCUSSION
Recent research has focused on the links between humanfertility and polymorphisms in genes of the AHR pathway(21–23), as it is the main mediator by which dioxins actand alter cellular signaling. Additionally, functional
variations in related genes may be responsible for differentsusceptibility to these environmental pollutants. Few poly-morphisms have been identified in the genes involved inthe AHR pathway, some of which are population-specific(28). We analyzed the selected polymorphisms from the cod-ing regions of major component genes of the AHR pathway:nonsynonymous changes at codon 554 (Arg to Lys) in theAHR gene and at codon 185 (Pro to Ala) in the AHRR geneas well as a synonymous change (Val to Val) at codon 189in the ARNT gene.
Previous studies have failed to associate AHRR Pro185Alapolymorphism with the etiology of endometriosis (21) andlung cancer (29). We found that this nonsynonymous changein the AHRR gene may serve as a susceptibility locus for malefactor infertility. Recently, the AHRR Pro185Ala polymor-phism was shown to contribute significantly to a genetic pre-disposition to reduced male fertility (22) and the presence ofmicropenis (30, 31) in the Japanese population. In these stud-ies, the presence of the Pro185 allele was associated with anincreased incidence of male factor infertility and abnormalexternal genital development. On the contrary, we demon-strated that the Ala185 allele of AHRR, rather than thePro185 allele, was more prevalent among patients withmale factor infertility. In addition, we showed a significantnegative association between the sperm count and the inci-dence of the AHRR Ala/Ala genotype. Our data are in agree-ment with a recent study by Tiido et al. (32), where theAla185 allele was found to contribute to the elevated spermY:X chromosome ratio after prolonged exposure to organo-halogen pollutants.
Further research is needed to clarify the reasons for the dis-crepancy between our results and those of Watanabe et al.(22). The disparity may be due to differences in study partic-ipants. Although the inclusion criteria for infertile males weresimilar, the criteria for control selection were slightly diver-gent between the studies. In the current study, the controlswere selected based only on the best sperm parameters, withno information available about their fertility history. On thecontrary, the two-fold smaller control group of the Watanabestudy consisted of men with proven fertility, but no preciseinformation was known about their semen sample quality.
Because AHRR Pro185Ala polymorphism could be associ-ated with the toxic effects of environmental pollutants, anage-dependent cumulative impairment of semen qualitycould be suspected. However, we were unable to show an in-fluence of age on the sperm concentration among all men (in-fertile and fertile) carrying the infertility-prone AHRR Ala/Ala genotype. At the same time, the age-dependent impair-ment of male factor fertility associated with the AHRRAla/Ala genotype cannot be excluded either. Half of ourinfertile patients were between 28 and 31 years. The 3 years’variability may not be enough to detect changes caused byenvironmental factors. Instead of a case-control study, thishypothesis needs further cohort studies with many years offollow-up.
858 Merisalu et al. AHR pathway and male factor infertili
To date, studies associating the AHRR Pro185Ala poly-morphism and male fertility potential and external genitaldevelopment have been descriptive in nature, with no reportsevaluating the possible functional relevance of this variation.As the Pro185Ala polymorphism resides in the heterodimeri-zation domain of AHRR, it could alter the function of the re-pressor, leading to weaker negative feedback and exacerbatedtoxic effects of environmental pollutants (31). Alternatively,the AHRR Pro185Ala variation may be linked to another, yetunknown, functional polymorphism in the AHRR gene.
The nonsynonymous Arg554Lys change in the AHR gene islocated in the transactivation domain of the receptor proteinthat is responsible for the initiation of transcription from thetarget genes (33). The Arg554Lys polymorphism of AHRwas shown to correlate with altered expression of theCYP1A1 detoxification enzyme, one of the main target genesof the activated AHR pathway (34). However, other investiga-tors have found no association between the AHR Arg554Lyspolymorphism and CYP1A1 induction (35, 36). The impactof the AHR Arg554Lys polymorphism has been studied in sev-eral pathological conditions, such as defective spermatogene-sis (22), endometriosis (21), lung cancer (35), and dementia(37); however, no associations have been found. In the currentstudy, allele and genotype frequencies of the AHR Arg554Lyspolymorphism were comparable among male infertilepatients and controls in the Estonian population.
The Val189Val (G/C allele) polymorphism resides in thedomain of the ARNT protein, which is necessary for dimer-ization with AHR. Although this silent mutation has no ef-fect on the amino acid content of the ARNT protein, itsoverall impact is largely unknown. The Val189Val polymor-phism in the ARNT gene has previously been studied in re-lation to nonsyndromic oral cleft (24). Those data suggestthat the Val189Val (C allele) polymorphism might be ina linkage disequilibrium with another as yet unidentifiedfunctional polymorphism of the ARNT gene related to oralcleft formation (24). However, we found no associationbetween the ARNT Val189Val polymorphism and malefactor infertility.
In conclusion, this is, to our knowledge, the first associa-tion study investigating the contribution of AHR pathwaygenetic variations (AHR Arg554Lys, AHRR Pro185Ala, andARNT Val189Val) to male factor infertility in a Caucasianpopulation. While we demonstrated that the AHR Arg554Lysand ARNT Val189Val genotypes were not predictive of malefertility potential, our findings provide strong evidence thatimplicates the AHRR Ala/Ala genotype in a susceptibilityto male factor infertility with impaired semen quality in theEstonian population.
REFERENCES1. Valdez KE, Petroff BK. Potential roles of the aryl hydrocarbon receptor
in female reproductive senescence. Reprod Biol 2004;4:243–58.
2. Kogevinas M. Human health effects of dioxins: cancer, reproductive and
endocrine system effects. Hum Reprod Update 2001;7:331–9.
ty Vol. 88, No. 4, October 2007
3. Dolwick KM, Swanson HI, Bradfield CA. In vitro analysis of Ah receptor
domains involved in ligand-activated DNA recognition. Proc Natl Acad
Sci USA 1993;90:8566–70.
4. Perdew GH. Association of the Ah receptor with the 90-kDa heat shock
protein. J Biol Chem 1988;263:13802–5.
5. McGuire J, Whitelaw ML, Pongratz I, Gustafsson JA, Poellinger L. A
cellular factor stimulates ligand-dependent release of hsp90 from the
et al. Expression of aryl hydrocarbon receptor and aryl hydrocarbon
receptor nuclear translocator messenger ribonucleic acids and proteins
in rat and human testis. Endocrinology 2003;144:767–76.
Fertility and Sterility�
21. Watanabe T, Imoto I, Kosugi Y, Fukuda Y, Mimura J, Fujii Y, et al.
Human arylhydrocarbon receptor repressor (AHRR) gene: genomic
structure and analysis of polymorphism in endometriosis. J Hum Genet
2001;46:342–6.
22. Watanabe M, Sueoka K, Sasagawa I, Nakabayashi A, Yoshimura Y,
Ogata T. Association of male infertility with Pro185Ala polymor-
phism in the aryl hydrocarbon receptor repressor gene: implication
for the susceptibility to dioxins. Fertil Steril 2004;82(Suppl 3):
1067–71.
23. Guo SW. The association of endometriosis risk and genetic polymor-
phisms involving dioxin detoxification enzymes: a systematic review.
Eur J Obstet Gynecol Reprod Biol 2006;124:134–43.
24. Kayano S, Suzuki Y, Kanno K, Aoki Y, Kure S, Yamada A, et al. Signif-
icant association between nonsyndromic oral clefts and arylhydrocarbon
receptor nuclear translocator (ARNT). Am J Med Genet A 2004;130:
40–4.
25. World Health Organization. WHO laboratory manual for the examina-
tion of human semen and sperm-cervical mucus interaction 1999.
26. Jensen TK, Jorgensen N, Punab M, Haugen TB, Suominen J,
Zilaitiene B, et al. Association of in utero exposure to maternal smoking
with reduced semen quality and testis size in adulthood: a cross-sectional
study of 1,770 young men from the general population in five European
countries. Am J Epidemiol 2004;159:49–58.
27. Aljanabi SM, Martinez I. Universal and rapid salt-extraction of high
quality genomic DNA for PCR-based techniques. Nucleic Acids Res
1997;25:4692–3.
28. Wong JM, Okey AB, Harper PA. Human aryl hydrocarbon receptor poly-
morphisms that result in loss of CYP1A1 induction. Biochem Biophys
Res Commun 2001;288:990–6.
29. Cauchi S, Stucker I, Cenee S, Kremers P, Beaune P, Massaad-Massade L.
Structure and polymorphisms of human aryl hydrocarbon receptor repres-
sor (AhRR) gene in a French population: relationship with CYP1A1
inducibility and lung cancer. Pharmacogenetics 2003;13:339–47.
30. Soneda S, Fukami M, Fujimoto M, Hasegawa T, Koitabashi Y, Ogata T.
Association of micropenis with Pro185Ala polymorphism of the gene for
aryl hydrocarbon receptor repressor involved in dioxin signaling. Endo-
crinol J 2005;52:83–8.
31. Fujita H, Kosaki R, Yoshihashi H, Ogata T, Tomita M, Hasegawa T, et al.
Characterization of the aryl hydrocarbon receptor repressor gene and as-
sociation of its Pro185Ala polymorphism with micropenis. Teratology
2002;65:10–8.
32. Tiido T, Rignell-Hydbom A, Jonsson BA, Rylander L, Giwercman A,
Giwercman YL. Modifying effect of the AR gene trinucleotide repeats
and SNPs in the AHR and AHRR genes on the association between per-
sistent organohalogen pollutant exposure and human sperm Y:X ratio.
Mol Hum Reprod 2007;13:223–9.
33. Harper PA, Wong JY, Lam MS, Okey AB. Polymorphisms in the human
AH receptor. Chem Biol Interact 2002;141:161–87.
34. Smart J, Daly AK. Variation in induced CYP1A1 levels: relationship to
CYP1A1, Ah receptor and GSTM1 polymorphisms. Pharmacogenetics
2000;10:11–24.
35. Cauchi S, Stucker I, Solas C, Laurent-Puig P, Cenee S, Hemon D, et al.
Polymorphisms of human aryl hydrocarbon receptor (AhR) gene in
a French population: relationship with CYP1A1 inducibility and lung
cancer. Carcinogenesis 2001;22:1819–24.
36. Anttila S, Lei XD, Elovaara E, Karjalainen A, Sun W, Vainio H, et al. An
uncommon phenotype of poor inducibility of CYP1A1 in human lung is
not ascribable to polymorphisms in the AHR, ARNT, or CYP1A1 genes.
Pharmacogenetics 2000;10:741–51.
37. Lin GF, Ma QW, Zhang DS, Zha YL, Lou KJ, Shen JH. Polymorphism of
alpha-estrogen receptor and aryl hydrocarbon receptor genes in dementia
patients in Shanghai suburb. Acta Pharmacol Sin 2003;24:651–6.
859
Alterations in sperm motility after acute oraladministration of sildenafil or tadalafil in young,infertile menGiorgio Pomara, M.D.,a Girolamo Morelli, M.D.,a Domenico Canale, M.D.,b Paolo Turchi, M.D.,c
Enrico Macchia, M.D.,b Enio Martino, M.D.,b and Francesco Francesca, M.D.a
a Urology Unit and b Department of Endocrinology, Azienda Ospedaliera Pisana, University of Pisa, Pisa; c Andrology Unit,
Department of Urology, Prato Hospital, Prato; and d Department of Urology, Cattinara Hospital, University of Trieste,
Trieste, Italy
Objective: To evaluate the acute effect of sildenafil and tadalafil on seminal parameters in young, infertile patients.Design: Prospective, randomized, double-blind, crossover clinical investigation on semen parameters after theadministration of a single dose of sildenafil (50 mg) or tadalafil (20 mg).Setting: An academic hospital as well as a male infertility center and clinical andrology laboratories.Patient(s): Eighteen young, infertile men.Intervention(s): Oral administration of a single dose of sildenafil (50 mg) or tadalafil (20 mg) in a blind, random-ized order. The semen samples were collected 1 or 2 hours after each treatment.Main Outcome Measure(s): Changes in sperm parameters after sildenafil and tadalafil administration, comparedwith the basal conditions.Result(s): A significant increase in sperm progressive motility (median value, 37.0% vs. 28.5%) was observed af-ter sildenafil administration as compared with baseline; in contrast, a significant decreased value was observed aftertadalafil (median value, 21.5% vs. 28.5%).Conclusion(s): These preliminary results indicate that sperm motility appears to be acutely affected in young,infertile patients by a single dose of sildenafil and tadalafil, with opposite effects: stimulatory by the former andinhibitory by the latter. (Fertil Steril� 2007;88:860–5. �2007 by American Society for Reproductive Medicine.)
Key Words: Sildenafil, tadalafil, male fertility, PDE11, PDE5 inhibitors
Erectile dysfunction (ED) is defined as the inability to achieveand maintain an erection adequate for sexual intercourse. Apanel of experts at the first International Consultation onErectile Dysfunction recommended oral agents as the first-line treatment of this condition, regardless of etiology (1).
Sildenafil, an inhibitor of the enzyme phosphodiesterasetype 5 (PDE5), is nowadays the most prescribed oral agent.A considerable number of clinical trials have been conductedworldwide in several patient categories, establishing its effi-cacy and safety in the treatment of ED.
Nitric oxide synthase (2) and two distinct PDE isoforms(PDE1 and PDE4) are present in human sperm cells (3). Itis known that after acute oral administration of sildenafil(100 mg), the drug reaches a concentration of 0.1–0.3mmol/L in the ejaculate (4); this concentration is consistentwith a possible inhibitory interaction of sildenafil with spermPDE isoforms (5).
Tadalafil is a potent PDE5 and PDE11 inhibitor. Phospho-diesterase type 11 has been found in the smooth muscles ofinternal organs, cardiac and skeletal muscles, and pituitarygland. In the male reproductive tract, PDE11 is highly
Received July 2, 2006; revised and accepted December 22, 2006.
Reprint requests: Giorgio Pomara, M.D., Urology Unit, Azienda Ospeda-
liera Pisana, University of Pisa, Via Roma, 67, 56126, Pisa, Italy (FAX:
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
860
expressed in the prostate gland, Leydig cells, and developinggerm cells in the testes (6–8). The physiological significanceof the enzyme and the consequences of its inhibition have notyet been established.
Although some studies reported no unfavorable effect onspermatogenesis or sex hormones for both drugs (4, 9, 10),researchers from Belfast (Glenn D, unpublished data) re-cently have shown that sildenafil may affect fertility. More-over, we have already underlined our concern in the articleby Hellstrom et al. (10), and we have mentioned that to thisdate, no sufficient data are available that confirm the safetyof tadalafil administered on a daily basis, particularly inhigh-risk patients (11).
No data comparing sildenafil and tadalafil effect on spermparameters have been published so far. Furthermore, theeffect of both drugs in high-risk infertile patients has notbeen ever investigated. We present here our preliminary ex-perience on the acute effect of sildenafil (50 mg) and tadalafil(20 mg) on seminal parameters in young, infertile patients.
MATERIALS AND METHODS
Study Design and Subjects
The study design consisted of a prospective, randomized,double-blind, crossover clinical investigation.
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.019
Inclusion criteria were as follows: normal erectile func-tion, stable relationship, couple infertility lasting R2 years,proven abnormal seminal parameters, and no prior or con-comitant serious illness or drug assumption during the3-month period before the enrollment.
Exclusion criteria were as follows: major illnesses (hyper-tension, poorly controlled diabetes mellitus or associatedwith untreated proliferative diabetic retinopathy, previoushistory of stroke or myocardial infarction, life-threateningarrhythmia, untreated endocrine diseases), treatment withnitrates or anticoagulants, a known history of retinitis pig-mentosa, genital anatomical deformities that would impairerection, elevated PRL level, and low serum T level.
Among all patients referred to us for infertility, 18 menwho were<40 years of age and in a stable relationship agreedto participate in the study. Two were current smokers (20 cig-arettes per day for R2 y) and moderate consumers of alcohol,and 16 were nonsmokers. Baseline visits included hematol-ogy, serum chemistry, semen analysis, and reproductive-hormone assay. All subjects were asked not to ejaculate forR3 days before providing a semen sample and to avoid pro-ducts containing alcohol, caffeine, or cigarette smoking fora period of 3 days before each study session.
At the time of the baseline visit, each patient was recom-mended to accurately report the actual abstinence periodand to strictly follow the protocol, to avoid being dismissedfrom the study. As baseline evaluation, each patient providedtwo semen samples spaced 2 weeks apart before any treat-ment, according to World Health Organization guidelines(12). Because the results of both semen assessments foreach patient were not remarkably different, additional semensamples were not necessary.
In a double-blind fashion, patients were randomly assignedto receive one of the two drugs: sildenafil (50 mg) or tadalafil(20 mg). These drugs were encapsulated to preserve thedouble-blind study design and administered to the fastingpatients by a designated investigator.
Taking into account the different median time to achievethe maximum plasma concentration (Cmax), patients wereasked to collect semen samples 1 hour or 2 hours after silde-nafil or tadalafil administration, respectively. After a 2-weekwash-out period, each patient was crossed over to receive theother drug. The patient did not take any PDE 5 inhibitors orother medications during these weeks.
All subjects were enrolled in the study after giving in-formed consent. The institutional review board approved thestudy.
Semen Collection, Processing, and Examination
The sample was ejaculated into a clean, wide-mouthedplastic container, allowed to liquefy at 37�C, and thenanalyzed without any information about the drug given tothe patient, according to World Health Organization guide-lines (12). The same biologists (C.C. and C.M.) performed
Fertility and Sterility�
all seminal analyses. Parallel visual sperm motility, concen-tration and morphology assessments were made by the twobiologists. Our internal quality control scheme and analysisof actual data showed highly significant correlations betweenbiologists in the determination of all seminal parameters(P<.001, with variation of <10% for all parameters tested).During the initial microscopic investigation of the sample,concentration, motility, agglutination of spermatozoa, andpresence of cellular elements other than spermatozoa wereassessed. Motility was graded as a (rapid-progressive motil-ity), b (slow progressive motility), c (nonprogressive motil-ity), and d (immotility).
Statistical Analysis
Data are expressed as mean � SD for population data and asmedian value and 25th and 75th percentiles in box plot forstudy groups. Differences between groups (basal, sildenafil,and tadalafil) were assessed by Friedman test for nonpara-metric data. If significant, the difference between two groupswas tested by Wilcoxon test.
All statistical analysis was performed by using commercialsoftware (Statview; SAS Institute Inc., Cary, NC).
RESULTS
Eighteen Caucasian male infertile volunteers, aged between32 and 39 years, were enrolled in the study. Basal seminalparameters are reported in Table 1.
Six patients (33.3%), after sildenafil administration, andeight patients (44.4%), after tadalafil administration, reportedadverse effects that possibly were related to the treatment.The most frequently reported side effects were flushing,headache, nasal congestion, and back pain. They were self-limited and presented a mild to moderate severity. Hematol-ogy, serum chemistry, and reproductive hormones were in thenormal range.
Significant changes in sperm rapid-progressive (class a)and total-progressive (classes aþb) motility compared withbaseline were observed after sildenafil and tadalafil adminis-tration: Friedman test c2 data were 19.1, P<.0001 and 16.3,P¼.0003, respectively (Figs. 1 and 2). In particular, a statisti-cally significant increase in sperm rapid-progressive (class a)and total-progressive (classes aþb) motility compared withbaseline was recorded after sildenafil: respective medianvalues, 18.5% vs. 10.5% (Wilcoxon z-value, –3.42;P¼.0006) and 37.0% vs. 28.5% (Wilcoxon z-value, –2.60;P¼.009), respectively.
On the contrary, a significant decrease in sperm rapid-progressive (class a) and total-progressive (classes aþb) mo-tility was found after tadalafil: median value, 6.0% vs. 10.5%(Wilcoxon z-value, �2.76; P¼.006), and 21.5% vs. 28.5%(Wilcoxon z-value, �2.41; P¼.015), respectively.
Accordingly, sperm rapid-progressive and total-progres-sive motility after sildenafil and tadalafil were significantly
861
TABLE 1Seminal parameters in basal conditions and after either sildenafil or tadalafil.
Note: All data are mean � SE (median).a Class a (rapid) þ b (slow) progressive motility.
Pomara. Sildenafil, tadalafil, and seminal parameters. Fertil Steril 2007.
statistically different (Wilcoxon z-value, –3.68; P¼.0002 andz-value, –3.48; P¼.0005, respectively). No significant differ-ences were recorded for the other seminal parameters.
DISCUSSION
To the best of our knowledge, this is the first prospective, dou-ble-blind, randomized, crossover study, describing the acuteeffect of both sildenafil (50 mg) and tadalafil (20 mg) inyoung, infertile men. To maximize the practical impact ofour study, the most popular dosage of each of the two drugsworldwide was deliberately chosen, even if the two dosageswere not equivalent in potency.
However, in a limited number of patients, our results showa correlation between acute sildenafil administration and anincrease of sperm motility in infertile patients; conversely,tadalafil administration correlates with a decrease in motility.Total progressive motility was 37.0% after sildenafil and21.5% after tadalafil administration, vs. 28.5% at the baseline(Friedman c2, 16.3; P¼.0003).
Why Is There an Acute Transient Effect on Sperm Motility?
Because the total study duration was <70 days, the spermcells examined arose from the same spermatogenetic cycle.This implies that the effects described might be due topost-spermatogenetic processes, such as epididymal matura-tion, passage into the vas deferens, and prostate and seminal-vesicle secretion, as well as due to a direct effect on spermcell mitochondria or calcium channels. Because the effecton sperm motility took place 1 to 2 hours after the assumptionof the drug, the epididymal maturation and passage into thevas deferens of the spermatozoa reasonably may be excluded.Thus, this effect appears to be generated in the sperm that arepresent in the vas ampulla or in the seminal componentsreleased by the prostate or the seminal vesicles.
The different selectivity of the two drugs for the varioushuman PDE isoenzymes and the different localization ofthese isoenzymes in the seminal pathways or in the spermsubcellular components may give an explanation for our
862 Pomara et al. Sildenafil, tadalafil, and seminal param
results. Tadalafil actually is the only PDE5 inhibitor thatalso inhibits PDE11 at dosages still in the therapeutic range.The isoenzyme PDE11 has been found in the smooth musclesof internal organs, cardiac and skeletal muscles, and pituitarygland. In the male reproductive tract, PDE11 is highly ex-pressed in the prostate gland, in the Leydig cells, and in thedeveloping germ cells of the testis (6–8). The physiologicalsignificance of this enzyme and the consequences of its inhi-bition have not been established yet. Dogs given tadalafildaily for 6 and 12 months showed alterations of the seminif-erous epithelium and subsequent effects on spermatogenesis(8, 13). These findings are consistent with the recently pub-lished data on knockout mouse for the PDE11 gene that sug-gest a role for PDE11 in spermatogenesis and fertilizationpotential (14). Sperm forward progression from PDE11�/�
mice was reduced by 24%, compared with that of PDE11þ/þ
sperm. A similar reduction was observed in the concentrationof ejaculated sperm and in the percentage of living spermfrom PDE11�/� compared with PDE11þ/þ mice. These ani-mal studies provide support for the assumption that PDE11may play a role also in human sperm quality (13, 14).
To date, only one study has evaluated the long-term effectsof tadalafil administration on spermatogenesis and reproduc-tive hormones in healthy volunteers (10). The conclusionsreached by those investigators suggested that tadalafil is gen-erally harmless in men >45 years of age. However, it shouldbe reminded that the study included men with high spermconcentrations (70–80 million per milliliter) (10). It appearsreasonable not to expect serious adverse effects on spermquality of healthy patients with proven fertility, because theirsperm parameters almost certainly can remain in the normalrange (11).
The double novelty of our study is the inclusion of young(<40 y of age) infertile patients and the evaluation of acutedrug effects. The results invite a more cautious attitude to-ward tadalafil use in young men who may have fertility prob-lems, because a decrease in sperm motility in such cases mayaffect fertilization potential. This issue is even more relevantin high-risk infertile patients who are undergoing assisted
eters Vol. 88, No. 4, October 2007
reproductive technique procedure when a temporary erectiledysfunction may be present (5, 15).
More consistent data have been published about sildenafil.Since its introduction, various reports have addressed the invitro effects of sildenafil on sperm parameters. Lefievreet al. (16) have shown that sildenafil stimulates human spermmotility and capacitation but not acrosome reaction. Burgeret al. (17) performed a similar study in a group of infertile pa-tients and found that sildenafil did not significantly alter themotility, viability, sperm integrity, or sperm penetration char-acteristics. A recent report on sea urchin spermatozoa has
FIGURE 1
Sperm rapid-progressive motility (class a) in basalconditions, after sildenafil, and after tadalafil: thedifference is statistically significant (Friedman c2,19.1, P< .0001). Sildenafil is correlated witha statistically significant increase, comparedwith baseline (median value, 18.5% vs. 10.5%;Wilcoxon z-value, –3.42; P¼ .0006), whereas tadalafilis correlated with a significant decrease (medianvalue, 6.0% vs. 10.5%; Wilcoxon z-value, �2.76;P¼ .006). Correspondingly, the difference betweensildenafil and tadalafil is even more statisticallysignificant (Wilcoxon z-value, –3.68; P¼ .0002). (A)Individual data. (B) Box plot with median value andwith 25th and 75th percentiles.
Pomara. Sildenafil, tadalafil, and seminal parameters. Fertil Steril 2007.
Fertility and Sterility�
shown that PDE5 localizes mainly to sperm flagella, regu-lates inner cyclic guanosine monophosphate levels, and mod-ulates sperm motility; its inhibition by sildenafil increasessperm motility (18).
The acute effect of sildenafil has been investigated by var-ious investigators only in healthy volunteers (4, 9, 19). Aversaet al. (9) demonstrated that acute sildenafil treatment (100mg) did not modify semen characteristics in healthy patientsand further emphasized the potential use of the drug duringassisted reproductive techniques when a temporary erectiledysfunction may occur. This was initially suggested byTur-Kaspa et al. (15), who reported his experience on theuse of sildenafil in men who had to produce sperm on demand
FIGURE 2
Sperm total-progressive motility (classes aþb) inbasal conditions, after sildenafil, and after tadalafil:the difference is statistically significant (Friedman c2,16.3, P¼ .0003). Sildenafil is correlated witha statistically significant increase, compared withbaseline (median value, 37.0% vs. 28.5%; Wilcoxonz-value, –2.60; P¼ .009), whereas tadalafil iscorrelated with a significant decrease (median value,21.5% vs. 28.5%; Wilcoxon z-value, �2.41;P¼ .015). Accordingly, the difference betweensildenafil and tadalafil was even more statisticallysignificant (Wilcoxon z-value, –3.48; P¼ .0005). (A)Individual data. (B) Box plot with median value andwith 25th and 75th percentiles.
Pomara. Sildenafil, tadalafil, and seminal parameters. Fertil Steril 2007.
863
for an assisted reproductive technique procedure. DuPlessiset al. (4) and Jannini et al. (20) published preliminary reportson the in vivo effect of sildenafil administration on spermfunction of infertile men. Our study confirms in a double-blind crossover fashion these experimental and clinical find-ings demonstrating a positive effect on sperm progressivemotility in infertile patients after an acute administration ofsildenafil.
Although safety of sildenafil concerning semen parametersappears to be well established by this and previous reports (9,19, 20), this scientific evidence is still missing for the newerPDE5-Is, like tadalafil and vardenafil. It also should be re-membered that all these drugs, even being selective PDE5-Is, have an effect on other PDE isoenzymes (21). The roleof these isoenzymes is not fully understood.
Does Inhibition of PDE5 or PDE11 Have an Effecton the Milieu of the Seminal Vesicle or of the Prostateor on Sperm Cell Mitochondria or Calcium Channels?
Recently there has been a growing interest worldwide inunderstanding cross-reaction of PDE5 inhibitors with otherPDEs and particularly with PDE11. Weeks et al. (22), com-paring the potencies of tadalafil, vardenafil, and sildenafilfor PDE11A4 and the fold-selectivity of these inhibitors forPDE5A1 over PDE11A4, provided several important infor-mations for safety issues. Whereas the selectivity for PDE5over PDE11A4 for sildenafil (1,000-fold selectivity) and var-denafil (9,000-fold selectivity) found in this study revealedthat these drugs are very unlikely to cross-react withPDE11A4, in contrast, the 40-fold selectivity ratio of tadalafilfor PDE5 over PDE11A4 is not high enough to excludea cross-reaction (23). Certainly, it reduces the likelihood ofsuch an effect when compared with the fivefold tadalafilselectivity that has been reported for the shortest isoform,PDE11A1 (24).
Loughney et al. (25) published another interesting studythat considered the pattern of expression of PDE11 in humantissues. Their results, demonstrating the presence ofPDE11A4 protein in human prostate, pituitary, heart, andliver, are partially consistent with the published reports onthe distribution of PDE11A mRNA (6, 26, 27) but differ sub-stantially from the previous reports on the distribution ofPDE11A protein (6, 7, 28). It appears reasonable that thesedifferences could be due to differences in the specificityand selectivity of the PDE11A antibodies used for thePDE11A protein analysis. All these reports, published closetogether, underline that PDE11, the newest discovered mem-ber of the PDE family, has become the center of controversy,and obviously the controversial issue is the inhibition ofPDE11 by tadalafil. The recent article by Francis (29) givesfurther light on this hot topic and suggests the necessity ofmore comprehensive clinical studies focused on the effectof tadalafil on testicular function, germ cell viability, andcharacteristics of prostatic fluid.
864 Pomara et al. Sildenafil, tadalafil, and seminal param
In conclusion, this first study in high-risk patients indicatesthat sildenafil acutely correlates with an improvement insperm motility. On the contrary, the median sperm motilityacutely decreased after tadalafil. The most important limita-tion of this study is the relatively low number of patients in-vestigated; hence, an attempt to make definitive conclusionswould not be appropriate at the moment. However, to ourknowledge, we report the first prospective, double-blind, ran-domized, crossover study, on the acute effect of sildenafil andtadalafil in young, infertile males. The use of PDE-5 inhibi-tors is becoming a routine procedure in IVF centers, becausethe percentage of patients having temporary ED during thesemen collection is relatively high. In this context, the acuteeffect of these drugs on semen parameters could be relevant.
Larger studies and a longer follow-up period are necessaryto confirm the hypothesis of a detrimental effect of tadalafilon some sperm parameters, such as motility. However, thepresent study suggests caution with tadalafil prescription ininfertile patients, especially in those collecting specimensfor intrauterine insemination, considering the paramountimportance of the motile sperm fraction in these procedures.
REFERENCES1. First International Consultation on Erectile Dysfunction: Sponsored by
World Health Organization (WHO), International Consultation on Uro-
logical Disease (ICUD), and Soci�et�e Internationale d’Urologie (SIU).
Paris, 1–3 July 1999.
2. Lewis SE, Donnelly ET, Sterling ES, Kennedy MS, Thompson W,
Chakravarthy U. Nitric oxide synthase and nitric production in human
spermatozoa: evidence that endogenous nitric oxide is beneficial to
sperm motility. Mol Hum Reprod 1996;2:873–8.
3. Fisch JD, Behr B, Conti M. Enhancement of motility and acrosome
reaction in human spermatozoa: differential activation by type-specific
tion and characterization of two novel phosphodiesterase PDE11A vari-
ants showing unique structure and tissue-specific expression. J Biol
Chem 2000;275:31469–79.
27. Yuasa K, Kanoh Y, Okumura K, Omori K. Genomic organization of the
human phosphodiesterase PDE11A gene: evolutionary relatedness with
other PDEs containing GAF domains. Eur J Biochem 2001;268:168–78.
28. Ueckert S, Hedlund P, Oelke M, Steif C. Immunohistochemical presence
of phosphodiesterase (PDE)11A in the human prostate [abstract]. Eur
Urol 2004;3(Suppl):18.
29. Francis SH. Phosphodiesterase 11 (PDE11): is it a player in human
testicular function? Int J Impot Res 2005;17:467–8.
865
MENOPAUSE
Tibolone histology of the endometrium and breastendpoints study: design of the trial and endometrialhistology at baseline in postmenopausal womenDavid F. Archer, M.D.,a Susan Hendrix, D.O.,b Alex Ferenczy, M.D.,c Juan Felix, M.D.,d J. ChrisGallagher, M.D.,e Janice Rymer, M.D., Ph.D.,f Sven O. Skouby, M.D.,g Wil den Hollander, Ph.D.,h
Victoria Stathopoulos, Ph.D.,i and Frans A. Helmond, Ph.D.,i for the THEBES Study Groupa Department of Obstetrics and Gynecology, Contraceptive Research and Development Program Clinical Research Center,
Eastern Virginia Medical School, Norfolk, Virginia; b Wayne State University/Hutzel Women’s Hospital, Detroit, Michigan;c McGill University and Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada; d Department of
Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California; e Bone Metabolism Section,
Creighton University Medical Center, Omaha, Nebraska; f King’s College School of Medicine at Guy’s and St. Thomas’
Hospitals, Division of Obstetrics and Gynaecology, London, United Kingdom; g Department of Obstetrics and Gynecology,
Frederiksberg Hospital, Copenhagen, Denmark; h NV Organon, Oss, the Netherlands; and i Organon International, Roseland,
New Jersey
Objective: To address the endometrial safety of tibolone.Design: The Tibolone Histology of the Endometrium and Breast Endpoints Study (THEBES) is a randomized,double-blind, parallel-group trial of tibolone compared with continuous combined conjugated equine estrogen(CEE) and medroxyprogesterone acetate (MPA).Setting: Multi-country, multi-center ambulatory care setting.Patient(s): A total of 5,185 subjects were screened, and biopsies were obtained from 4,446 women.Intervention(s): Participants were randomized in a 1:1:2 ratio, to tibolone (1.25 or 2.5 mg/d) or CEE-MPA.Main Outcome Measure(s): The one-sided 95% confidence intervals for the incidence of hyperplasia or cancerwere evaluated for tibolone compared with CEE-MPA.Result(s): Endometrial biopsy results at baseline: atrophic (87.29%), inactive (0.25%), proliferative (6.12%), se-cretory (2.86%), menstrual type (0.40%), and hyperplasia (0.18%). Only subjects with atrophic or inactive endo-metrium were eligible for this study, and 3% of the women at screening either had no tissue (0.18%) or had anamount of tissue that was insufficient for diagnosis (2.72%). Three thousand two hundred forty postmenopausalwomen with a mean (�SD) age of 54.4 � 4.4 years and a mean time since menopause of 4.5 � 3.6 years wererandomized.Conclusion(s): The Tibolone Histology of the Endometrium and Breast Endpoints Study is a prospective, random-ized clinical trial, designed to provide evidence of the endometrial safety of tibolone compared with estrogen andprogestogen. Screening endometrial histology shows a low prevalence of endometrial hyperplasia (0.18%) and nocarcinoma. (Fertil Steril� 2007;88:866–78. �2007 by American Society for Reproductive Medicine.)
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
866
Estrogen therapy (ET) is well known to relieve vasomotorsymptoms and prevent osteoporosis in postmenopausalwomen. Unopposed Es promote endometrial proliferationand are associated with an increased risk of endometrialhyperplasia and carcinoma in women with an intact uterus(1–5). The addition of a progestogen, either sequentially orcontinuously (estrogen plus progestin [EPT]), offsets thisrisk (1, 3–6) but may cause adverse effects such as vaginalbleeding or spotting (1, 7), changes in mood (7–9), and anincreased risk of breast cancer (10–14).
Tibolone (NV Organon, Oss, the Netherlands) has specificeffects on different tissues as a result of its tissue-selectivemetabolism, enzyme regulation, and/or receptor binding
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.052
and activation. It is therefore referred to as a selective tissueestrogenic activity regulator (STEAR) (15). Conversion of ti-bolone to 3a-OH and 3b-OH tibolone metabolites results inE-like effects on bone, vagina, mood, and climacteric symp-toms. Tibolone itself and the third metabolite, the D4 isomer,have progestogen-like (and androgen-like) properties andprevent the E-induced stimulation of the endometrium.
In vitro studies using endometrial cell lines have shownthat tibolone is selectively converted to the progestogenicD4 metabolite (which cannot be reduced by 5a-reductase)(16–18). Tibolone, through regulation of the sulfotransfer-ase–sulfatase system, reduces endometrial (and breast)E-like activity by converting the active estrogenic metabo-lites into sulfated metabolites (19). A lack of endometrialstimulation by tibolone was observed in a comparison withconjugated equine estrogens (CEE), with or without medrox-yprogesterone acetate (MPA), in ovariectomized femalecynomolgus monkeys (20).
Clinical trials investigating the changes in endometrialhistology during tibolone treatment have found mainlya high level of atrophic endometrium with no increased inci-dence of proliferation, hyperplasia, and/or cancer (21–23).No significant differences in ultrasound measures of endome-trial thickness between tibolone-treated women and controlshave been observed in studies conducted over periods of %10years (23, 24). When compared with EPT, tibolone had a sim-ilar (continuous combined EPT) or less marked (sequentialEPT) effect on endometrial thickness (25, 26).
Two observational trials from the United Kingdom (27, 28)have suggested an increased risk of endometrial cancer fortibolone users when compared with sequential EPT and non-users, respectively. These findings may reflect preferentialprescribing behavior in the United Kingdom (29), furtherstressing the need for a prospective randomized controlledtrial. To resolve the discrepancy in endometrial histologicalresults between the biological data and the earlier clinicaltrials (and the recently published observational United King-dom data), a prospective randomized controlled clinical trialwas designed to investigate the endometrial safety of tibo-lone.
The Tibolone Histology of the Endometrium and BreastEndpoints Study (THEBES) was designed to confirm andto extend the knowledge about the endometrial safety of tibo-lone. Two doses of tibolone, 1.25 and 2.5 mg/d, were used.The 2.5-mg dose of tibolone is the one recommended forthe treatment of moderate to severe vasomotor symptoms,whereas the lower dose has also proven to be effective inthe prevention of postmenopausal bone loss (30, 31). Conju-gated equine estrogen–MPA was chosen as the comparatorbecause it is the most frequently prescribed EPT in theUnited States, and its endometrial safety has been con-firmed in a number of large pivotal studies (32–35). The de-sign and rationale of the THEBES trial, together with thebaseline characteristics of the subjects are described inthis report.
Fertility and Sterility�
MATERIALS AND METHODS
General
The THEBES is a multicountry, multisite, prospective, ran-domized, active-controlled, double-blind, parallel-group trialof oral tibolone (1.25 or 2.5 mg/d) and continuous combinedoral CEE-MPA (0.625 mg þ 2.5 mg/d) in postmenopausalwomen. A summary of the protocol has been posted online(http://www.organon-trials.com). A total of 5,185 womenwere screened, and 3,240 were randomized into the clinicaltrial. Participants were recruited from 77 US centers, 70European centers (Belgium, 6 centers; Czech Republic, 9centers; Denmark, 4 centers; Finland, 5 centers; France, 3centers; Germany, 1 center; Hungary, 11 centers; Italy, 1 cen-ter; the Netherlands, 6 centers; Norway, 5 centers; Poland, 2centers; the Slovak Republic, 7 centers; Spain, 2 centers;Sweden, 6 centers; and the United Kingdom, 2 centers),and 4 centers in Chile. Endometrial biopsies were obtainedat baseline and repeated after 1 and 2 years of treatment.
The primary objective of this 2-year trial was to determinethe endometrial safety of tibolone by evaluating the one-sided 95% confidence intervals for the incidence of abnormalendometrium (hyperplasia or cancer) and for hyperplasia andcancer separately, for each of the two tibolone treatmentgroups (and for the tibolone treatment groups combined)after 1 and 2 years of treatment with tibolone. The continuouscombined preparation of 0.625 mg of CEE and 2.5 mg ofMPA has been used as an active comparator in this trial.
Secondary aims were to compare the effects of both treat-ments on incidence rate of abnormal endometrial histology,double-layer endometrial thickness as measured by transva-ginal ultrasound (TVUS), vaginal bleeding patterns, the inci-dence of breast pain or tenderness and vaginal dryness, bodyweight, mammographic density (in a subgroup), and health-related quality of life and sexual functioning.
Approval for the conduct of the THEBES was obtainedfrom the independent ethics committee or the institutionalreview board at each participating center. Written informedconsent was obtained from all study participants before anytrial assessment.
Eligibility and Exclusion Criteria
The inclusion and exclusion criteria are summarized inTable 1. The study was designed so that the subjects reflecteda large proportion of women who might potentially benefitfrom tibolone or EPT. The exclusion criteria therefore werelargely limited to medical contraindications for the use oftibolone or EPT and a high risk of failing to complete thestudy or of developing serious side effects.
Recruitment
Potential participants were recruited from the investigators’practices, referrals from clinical staff, existing ongoing pop-ulation-based cohorts, and advertisements in local print andbroadcast media. A total of 5,185 women were screened, of
TABLE 1Inclusion and exclusion criteria in the THEBES.
Inclusion criteria� Intact uterus� 45–65 y old� Healthy� Postmenopausal, defined as follows: [1] amenorrheic for R1 y; in cases of previous HT use, appropriate
washout periodsa required before screening and [2] amenorrheic for R6 mo with serum E2 of %20 pg/mLand FSH of R40 IU/L; in cases of previous HT use, appropriate washout periodsa required before drawingblood for E2 and FSH determinations� Postmenopausal for <15 y� Screening endometrial biopsy (atrophic or inactive)� Body mass index of R18 and %32 kg/m2
Exclusion criteria� Screening endometrial biopsy: ‘‘no tissue’’ or ‘‘tissue insufficient for diagnosis’’ as determined by both
hyperplasia, cancer, or any other histologic abnormality, as determined by at least one pathologist� Double-layer endometrial thickness of >6 mm, as assessed by TVUS� Any serious disease or disorder and any endocrine disorder (controlled hypothyroidism or
hyperthyroidism and diabetes mellitus type 2 allowed)� Prior or current use of unopposed E; prior use of E pellets or tamoxifen citrate; prior use of continuous
combined CEE-MPA (0.625 and 2.5 mg) for >2 y; sequential combination of P with E must have been forR10 d per 28-d cycle� Unexplained vaginal bleeding after the menopause� Diseases for which exogenous hormonal steroids are contraindicated� History or presence of malignancy (except successfully treated non-melanoma skin cancer)� History or presence of cardiovascular or cerebrovascular conditions: thrombophlebitis, thrombosis,
or thromboembolic disorders� History or presence of liver, gallbladder, familial hyperlipidemia, or renal disease; epilepsy; or classical
migraine headaches� History or presence of clinically significant depression or other psychiatric disorders that might
compromise or confound participation� Uncontrolled hypertension (systolic, >170 mm Hg and/or diastolic, >105 mm Hg)�Clinically significant abnormal hematological or biochemical parameters, or other abnormalities that might
lead to adverse events� Abnormal cervical Papanicolaou smear result (corresponding to PAP IIb or higher, including LSIL, HSIL,
ASCUS, and AGCUS)� Clinically significant abnormal mammography� Use of R1 of the following in the previous 2 mo: hepatic microsomal enzyme-inducing anticonvulsants
or drugs known to affect the pharmacokinetics of steroids; any investigational drug; or raloxifenehydrochloride� Previous use of raloxifene hydrochloride for >1 mo� Alcohol and/or drug abuse within the previous 12 mo� Current participation in other investigational drug studies� Clinically significant findings on physical and/or pelvic examination and/or on TVUS� Hypersensitivity to tibolone and/or CEE-MPA or any of their components
a Washout periods are as follows: 4 weeks for transdermal HT or local E; 8 weeks for phytoestrogens, tibolone, intrauterineor oral P, and oral E-P therapy; and 6 mo for P implants or injections and E-P injectable therapy.
Archer. THEBES: design and endometrial histology. Fertil Steril 2007.
868 Archer et al. THEBES: design and endometrial histology Vol. 88, No. 4, October 2007
whom 3,240 were eligible for randomization. The recruit-ment phase lasted 20 months, starting in November 2001and ending in July 2003. The baseline characteristics of thestudy population are shown in Table 2.
Randomization and Sample Size
Subjects who fulfilled all the inclusion criteria and violatednone of the exclusion criteria were enrolled in the trial,randomly assigned to one of the three treatment groups,and given a subject code number and a medication number.
TABLE 2Population characteristics in the THEBESat baseline (N [ 3,240).
Past hormonal contraceptiveor HT use (%)Yes 67.8No 32.2Missing 0.1
Note: Data are n (%) unless otherwise indicated.HT ¼ hormone therapy.
Archer. THEBES: design and endometrial histology. Fertil Steril 2007.
Fertility and Sterility�
The randomization ratio for 1.25 mg of tibolone, 2.5 mgof tibolone, and CEE-MPA was 1:1:2. Randomization wasperformed by using an interactive voice response system.The interactive voice response system randomization callprovided the investigator with the subject and medicationnumbers. Treatment was started on the day of randomizationand within 2 months of screening.
The projected sample size of 650 subjects in each tibolonearm was determined after discussions with the US Food andDrug Administration. In anticipation of premature discontin-uation, an additional 100 subjects were included in eachtreatment group. To keep the number of subjects assignedto the CEE-MPA arm equivalent to the total number of sub-jects in the tibolone arms, 1,500 subjects were assigned tothe CEE-MPA arm.
Screening and Follow-Up Visits
The screening process began after subjects had signed theinformed consent form and had been registered witha screening number by using an interactive voice responsesystem. During the screening process, the women had aninterview at the local clinic, during which data werecollected on medical and gynecological history, demo-graphics, current medications, and previous use of oralcontraceptives and hormone therapy. Height and weightwere measured, and body mass index was calculated; sit-ting systolic and diastolic blood pressure and heart ratealso were determined. A physical examination (includingbreast examination), pelvic examination, cervical Papa-nicolaou smear, TVUS scan, and an endometrial biopsy(after the TVUS scan) were performed. A mammogramwas obtained unless a normal mammogram had been takenwithin 3 months before screening or if local regulationsprohibited frequent mammogram evaluations (non–UnitedStates centers), where formal documented evidence wasrequired and the local regulations were followed. Bloodsamples were drawn for routine hematology and biochem-istry, and all blood samples were shipped to one centrallaboratory for analysis.
At the randomization (baseline) visit, vital signs, pretreat-ment signs and symptoms, and concomitant medicationswere documented, and the vaginal bleeding pattern wasrecorded in a bleeding episode log. Health-related qualityof life was measured by using the 37-item Women’s HealthQuestionnaire (WHQ-37) and the McCoy Female SexualityQuestionnaire–Short Form (MFSQ-SF) at sites in the UnitedStates, the United Kingdom, Germany, France, and Italy.
Follow-up visits were scheduled every 13 weeks afterrandomization, for a total of 104 weeks. At each follow-upvisit, vital signs, concomitant medication, vaginal bleedingepisodes, and adverse events were documented. Drug ac-countability (compliance) was determined every 6 months.A physical examination, pelvic examination, cervical Papani-colaou smear, TVUS scan, endometrial biopsy, mammogra-phy, and routine laboratory determinations were performedannually. Health-related quality of life was assessed after
869
6 months, 1 year, and 2 years at sites in the United States, theUnited Kingdom, Germany, France, and Italy.
The follow-up visit, performed within 30 days of the finalstudy medication intake, evaluated adverse events and use ofconcomitant medications.
Baseline and Follow-Up Endometrial Biopsiesand Histological Examination
The primary outcome was the upper one-sided 95% CI for theincidence of abnormal endometrial histology (hyperplasiaand cancer) and for hyperplasia and cancer separately after1 and 2 years of treatment.
Biopsy Procedures
Endometrial biopsies were obtained by gynecologists with anendometrial suction curette at screening and after 1 and 2years of treatment. All biopsy specimens were collected informalin and shipped to a central pathology laboratory forprocessing.
Processing of biopsy samples Tissue blocks were cut attwo levels from which two serial sections were made. A ran-domization number was allocated to each hematoxylin andeosin–stained slide at the central laboratory before dispatchto the two independent gynecological pathologists.
Evaluation of endometrium The endometrial tissues wereevaluated in compliance with the guidance from the USFood and Drug Administration (36) and the EuropeanAgency for the Evaluation of Medicinal Products (37), in-cluding the use of histological criteria presented in the latestedition of Blaustein’s Pathology of the Female Genital Tract(38, 39). A set of positive-control endometrial tissues (disor-dered proliferative endometrium, hyperplasia, and cancer)were intermixed with the subjects’ slides for internal qualitycontrol. These control tissues had been independently re-viewed by the two pathologists, before study start, andtheir diagnoses had been agreed upon before their randominclusion with the study tissues.
The final endometrial tissue diagnosis was based on theexamination by two independent pathologists. Their final his-tologic interpretation of the endometrial tissue was cate-gorized by using a numbered category for specific tissuediagnosis (Table 3). Categories 0 and/or 1, ‘‘no tissue ob-tained’’ or ‘‘tissue insufficient for diagnosis’’, were consid-ered not evaluable, and the TVUS result determined thefinal diagnosis. Participants with these findings on screeningwere excluded from the study.
Normal or benign endometrial histology (atrophic, inac-tive, proliferative, secretory, and menstrual) were reflectedin categories 2, 3, 4, 5, and/or 6, respectively (38).
The diagnoses of simple, complex, and atypical hyperplasiaof the endometrium were categories 7, 8, and/or 9, respec-tively, and ‘‘endometrial carcinoma’’ was category 10 (39).
Any subject whose biopsy was categorized as 7–10 (i.e.,abnormal) during or before enrollment in the study was
870 Archer et al. THEBES: design and endometrial histolog
excluded and was managed according to local, currentpractice guidelines.
At screening, the two independent gynecological patholo-gists, blinded to treatment and each other’s diagnosis, exam-ined one slide from each level of tissue cut to determineeligibility for the study. Classification as category 0 or 1,as determined by both pathologists, excluded the subject.Women with biopsies classified as category 2 or 3 were in-cluded in the study. Women classified in diagnostic categories4 to 10, including endometrial polyps, metaplasia, stromalor myometrial pathology; or abnormal exo-endocervicalgrowths, endocervical polyps, cervical squamous metaplasia,and epithelial atypia of repair also were excluded.
The highest diagnostic category was assigned in the case ofdiscrepancies in the interpretation by the pathologists. Thesame approach was used for subsequent discrepancies ininterpretation of normal tissue (categories 0–6). The diagno-sis of an endometrial polyp was based on the findings in thesurrounding endometrial tissue. However, if the polyp wascancerous, the final diagnosis for the biopsy was category10 (carcinoma). Any instance of hyperplasia or carcinomadiagnosed by any of the pathologists required a follow-upsafety procedure. During the study, endometrial polypswere evaluated by two readers, and in case of a discrepancybetween these two readers, the worst-case scenario waschosen.
If there was disagreement between the two pathologists,either between normal and abnormal or between categories
TABLE 3Classification of endometrial histologicinterpretation.
Classification Histologic diagnosis
0 No tissue obtained1 Tissue insufficient for diagnosis2 Atrophic3 Inactive4 Proliferative
4a. Weakly proliferative4b. Active proliferative4c. Disordered proliferative
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y Vol. 88, No. 4, October 2007
for hyperplasia and cancer, a third independent pathologist(also blinded) reviewed the same slides.
The third pathologist to ensure quality control also re-viewed a random sample of biopsies for which the diagnoseswere not previously in dispute. Concordance between two ofthe three pathologists was used as the final diagnosis withinthe categories normal, hyperplasia, or cancer. In the case oflack of agreement (i.e., cancer or normal, simple, complex,or atypical hyperplasia), the category with the most severemorphology was taken as the final diagnosis.
During the study, in cases of category 0 or 1 biopsies,a double-wall endometrial thickness of %4 mm was inter-preted as supportive of a normal endometrium. The TVUSwas repeated in cases of an endometrial strip thickness of>4 mm. The endometrial biopsy was repeated if the repeatTVUS result was %4 mm. If the second biopsy failed toprovide an evaluable sample, the last TVUS result was con-sidered to be indicative of normal endometrium. If the repeatTVUS result was >4 mm, a hysteroscopy-directed endome-trial biopsy or curettage was performed. If the endometrialtissue obtained was still not evaluable, the biopsy was de-scribed as missing. If this sequence of events occurred afterthe 1st year of the study, the subject was allowed to continueat the investigator’s discretion (Fig. 1).
Quality control of endometrial biopsy interpretations Toassess the consistency of readings by individual patholo-gists over time and the consistency between pathologistsinitially and over time, a number of control (nonstudy) en-dometrial tissues with known diagnosis were evaluated by
Fertility and Sterility�
all pathologists before study start. These tissues were theninterspersed randomly among the study slides, by the cen-tral laboratory, during the study as an internal quality con-trol to assess consistency in the interpretation of thetissues.
The following three conditions were compared at differenttime points during the study by using kappa statistics: [1]readings of reader A vs. readings of reader B, [2] baselinereadings of reader A vs. follow-up readings of reader A,and [3] baseline readings of reader B vs. follow-up readingsof reader B.
Secondary Outcome Measures
Incidence rate of abnormal endometrium Tibolone wascompared with CEE-MPA in terms of the incidence rate ofabnormal endometrium by using histology diagnosis.
Double-layer endometrial thickness assessed by TVUSDouble-layer endometrial thickness was measured byTVUS at screening and then annually. When a TVUS wasperformed with an endometrial biopsy, the TVUS wasalways performed first. A TVUS of >6 mm or any otherclinically significant abnormalities at screening meant thatthe subject was excluded from the study. To ensure the qualityof the ultrasound scans, a quality assurance program wasdeveloped that included a certification procedure for eachultrasonographer involved in the study.
Vaginal bleeding The participants were provided witha bleeding episode log to record the days on which vaginal
FIGURE 1
Endometrial evaluation procedure after 1 and 2 years of study treatment. TIFD ¼ Tissue insignificant fordiagnosis.
TVUS > 4mm Discontinue and treat TVUS =< 4mm Normal
TVUS & Endometrial biopsy
Inactive/AtrophicProliferative/Secretory
Menstrual Hyperplasia / Cancer
No tissue
TIFD
Repeat TVUS Normal
TVUS > 4mm
Hysteroscopy &
biopsy or D & C
TVUS =< 4mm
Aspiration biopsy
No tissue
TIFD
Inactive / AtrophicProliferative/Secretory
Menstrual Hyperplasia / Cancer Hyperplasia / Cancer
Inactive/AtrophicProliferative/Secretory
Menstrual
No tissue
TIFD
No diagnosis:Investigator call
Normal Discontinue and treat Discontinue and treat Normal Normal
Archer. THEBES: design and endometrial histology. Fertil Steril 2007.
871
spotting and/or bleeding occurred. The data were collectedby the investigator at randomization and at each subsequentvisit during the study.
Any bloody vaginal discharge was classified as spotting ifit required no more than one sanitary pad or tampon per dayand was classified as bleeding if it required two or moresanitary pads or tampons per day. Any bleeding patternsthat required further investigation, in the opinion of theinvestigator, were evaluated by TVUS and, if necessary, byendometrial biopsy.
Breast pain or tenderness and vaginal dryness The inci-dence and severity of breast pain or tenderness and vaginaldryness were documented on the pretreatment signs andsymptoms form at randomization as well as on the adverseevent form at each visit during the study.
Weight changes Body weight was recorded at each visitduring the study. The subject was required to remove herclothes and shoes and to don an examination gown.
Mammographic density Changes in mammographic den-sity during the 1st year of treatment were studied in a sub-set of 200 participants from selected US centers only. Amammogram was performed at screening and after 1year, using the same screening technique each time. Allmammograms were assessed for safety evaluation andalso sent to a central laboratory for digitization and assess-ment of breast density. Mammographic density was as-sessed by the Breast Imaging Reporting and Datasystemsfour-score classification and a computer-assisted quantita-tive method (40, 41). Participants in this subgroup alsohad a mammogram after 2 years in the study, but thesedata were used for safety evaluation only and not to assessmammographic density.
Health-related quality of life Health-related quality of lifewas assessed only at sites in the United States, the UnitedKingdom, Germany, France, and Italy. Measurement ofhealth-related quality of life, using reliable and well-validatedquestionnaires, is accepted as an appropriate means ofsupplementing conventional efficacy evaluations to providea comprehensive picture of the impact of symptoms and to as-sess the potential benefits of treatment (42). The question-naires used for the THEBES were the WHQ-37 (43), whichis designed to assess mood and physical symptoms in perime-nopausal and postmenopausal women experiencing concur-rent hormonal and psychosocial changes, and the MFSQ-SF(44, 45), which assesses sexual functioning in postmeno-pausal women with respect to sexual problems, sexualsatisfaction, and partner satisfaction.
Adverse Events
Participants were questioned about possible adverse events ateach visit during the study and at the follow-up visits. Thenature of the adverse event was recorded, together with itsduration and intensity, any remedial action taken, the relation-
872 Archer et al. THEBES: design and endometrial histolo
ship to study treatment, and the outcome. Serious adverseevents were defined as any event that resulted in death, waslife-threatening, required inpatient hospitalization or prolon-gation of existing hospitalization, resulted in persistent or sig-nificant disability or incapacity, or was a congenital anomalyor birth defect. All serious adverse events were to be reportedto Organon within 24 hours of awareness of the event by theinvestigator.
Independent Data and Safety Monitoring Board
The data and safety monitoring board was comprised of threephysician-scientists and was independent of the study path-ologists and endpoint adjudication committees. This boardreviewed all serious adverse events every 6 months, com-mencing after the last participant was entered into the trial.
Data Quality Assurance
Monitoring visits by an Organon clinical research associatewere scheduled at intervals of approximately 4–6 weeks,upon request of the investigator, or whenever deemed neces-sary by the clinical research scientist or the clinical researchassociate. A final monitoring visit was made after all partic-ipants had finished the trial and follow-up period, and allqueries and data issues were resolved. To ensure accuracyand completeness of data, the clinical research associatescompared data from the interactive voice response systemdatabase and clinical report forms with the participants’medical records. Clinical report forms and electronic datareceived from the central laboratory were also reviewed bythe responsible clinical data analyst at Organon for in-housequality control of data. All data were entered and verified byusing an in-house data management system. The clinicalquality assurance department at Organon is independent ofthe clinical development departments. The clinical qualityassurance audit strategy includes evaluation of systems ineffect at Organon, at clinical trial locations, and at third-partyorganizations (e.g., technical facilities).
Data Analysis
Analyses were based on the all-subjects-treated (23) group(i.e., all randomized subjects who received R1 dose of studymedication). The primary parameter, histological classifi-cation of the endometrial biopsy (normal, hyperplasia, orcarcinoma), was based on an Intent-to-Treat (1) approach(i.e., all subjects from the All Subjects Treated (AST) groupwith a follow-up biopsy that could be classified as normal orabnormal). In addition to an intention-to-treat analysis, a per-protocol analysis also was performed. The per-protocol groupconsisted of all subjects from the intention-to-treat groupwho had no protocol violations.
The primary objective of this 2-year randomized, activecontrolled trial was to confirm the endometrial safety oftibolone by evaluating the upper one-sided 95% confidenceintervals for the incidence of abnormal endometrial histology(hyperplasia or cancer), and hyperplasia and cancer sepa-rately, for each of the two tibolone treatment groups (and
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for the tibolone treatment groups combined) after 1 and 2years of treatment with tibolone.
For the secondary outcome measures, comparisonsbetween the tibolone and CEE-MPA groups were performedto assess and compare the vaginal bleeding profiles of thethree treatment groups during the study period. The propor-tion of subjects with breast pain or tenderness and vaginaldryness was compared by using Fisher’s exact test; the per-centage of subjects with a change in body weight of R10%from baseline was compared by using the Cochran-Mantel-Haenszel test, adjusted for center. The percentage of subjectswith an increase from baseline in breast density based on theBreast Imaging Reporting and Datasystems four-score sys-tem (40) was compared by using a c2 test, and the changein breast density values from baseline based on digitizedreadings (41) was compared by using an overall one-wayanalysis of variance with treatment as the factor in the model;if the overall test was statistically significant, then an inde-pendent-samples t-test was applied. Change from baselinein the scores on the WHQ-37 and the MFSQ-SF was analyzedby using the Kruskal-Wallis test, and in case of statisticaldifference among the three treatment groups, pairwise com-parison was performed with nonparametric Wilcoxon rank-sum test. No formal statistical analyses were performed onthe vaginal bleeding data. Descriptive statistics are providedfor the double-layer endometrial thickness for each treatmentgroup and for the combined tibolone group.
Fertility and Sterility�
RESULTS
The histological nomenclature used by the pathologists isfeatured in Table 3. A total of 5,185 postmenopausalwomen in US, South American, and European centerswere screened, and endometrial biopsies were obtainedfrom 4,446 women. A total of 3,240 postmenopausalwomen met the entry criteria and were randomized. Themean age of the randomized group was 54.4 � 4.4 years,and the mean time since menopause was 4.5 � 3.6 years.Almost all subjects were Caucasian (96.7%). More detailsare provided in Table 2.
The endometrial histology obtained from the 4,446 womenat screening is presented in Table 4. The majority of partici-pants had atrophic endometrium (87.29%), followed by pro-liferative (6.12%), secretory (2.86%), and inactive (0.25%).Hyperplasia was encountered in only 0.18% of the partici-pants, and none of the participants had carcinoma. In about3% of the women, either no tissue could be obtained(0.18%) or the amount of tissue was insufficient for diagno-sis. Further details are in Table 4. A total of 649 (54%)women with eligible biopsies (atrophic or inactive) failedscreening because they didn’t meet one or more of the otherentry criteria, as described in Table 1. Baseline endometriumhistology of the 3,240 randomized women is presented inTable 5 and shows that 99.8% of the biopsies were atrophicand 0.2% inactive, with equal distribution over the treatmentgroups.
TABLE 4Endometrial histology results at screening.
ClassificationScreening
total with biopsiesScreen failureswith biopsies Randomized
N 4,446 (100) 1,206 3,240No tissue obtained 8 (0.18) 8 0Tissue insufficient for
diagnosis121 (2.72) 121 0
Atrophic 3,881 (87.29) 649 3,232Inactive 11 (0.25) 3 8Proliferative, total 272 (6.12) 272 0Weakly proliferative 94 94 0Active proliferative 132 132 0Disordered proliferative 46 46 0Secretory, total 127 (2.86) 127 0Cyclic type 8 8 0Progestational type 92 92 0Mixed type 27 27 0Menstrual type 18 (0.40) 18 0Hyperplasia, total 8 (0.18) 8 0Simple hyperplasia 3 3 0Complex hyperplasia 3 3 0Atypical hyperplasia 2 2 0Carcinoma 0 0 0
Note: Data are given as n, with percentages in parentheses.
Archer. THEBES: design and endometrial histology. Fertil Steril 2007.
873
TABLE 5Endometrial histology results at baseline in all randomized subjects.
Classification (%) 1.25 mg of tibolone 2.5 mg of tibolone CEE-MPA Total
N 807 816 1,617 3,240Atrophic, n (%) 806 (99.9) 814 (99.8) 1,612 (99.7) 3,232 (99.8)Inactive, n (%) 1 (0.1) 2 (0.2) 5 (0.3) 8 (0.2)
Archer. THEBES: design and endometrial histology. Fertil Steril 2007.
The consistency of readings by individual pathologists overtime was evaluated by means of a number of control slides.By using kappa statistics, the following three conditionswere compared at different time points: [1] readings of readerAvs. readings of reader B; [2] baseline readings of reader Avs.follow-up readings of reader A; and [3] baseline readings ofreader B vs. follow-up readings of reader B.
Weighted kappa coefficients were 0.86, 0.87, and 0.89 forconditions 1, 2, and 3, respectively.
DISCUSSION
The THEBES is a large prospective, randomized activecontrolled clinical trial conducted to confirm the endometrialsafety of tibolone in postmenopausal women and to compareit with continuous combined CEE-MPA. It is the largest trialconducted to date to assess the endometrial safety of tibolone,involving a total of 3,240 women. The trial is multinational,involving women from Europe, South America, and theUnited States. The primary parameter is the incidence ofabnormal endometrial histology (hyperplasia or carcinoma)after 1 and 2 years of treatment, as assessed by histologicalexamination of endometrial tissue specimens obtained byPipelle suction curette.
Endometrial tissues were evaluated in compliance with theguidance from the US Food and Drug Administration (36)and the European Agency for the Evaluation of MedicinalProducts (37). Histological examination and classificationwere conducted by two independent, blinded, gynecologicalpathologists, with a third independent pathologist who re-viewed the histology in the case of disagreement on abnormalvs. normal results. A set of positive-control endometrialhistology slides was randomly added in a blinded mannerto the endometrial histology slides obtained from the studysubjects to ensure quality control.
The reason for excluding the 649 women with an atrophicendometrium before randomization was that they failed tomeet one or more of the entry criteria listed in Table 1.There were very few nonevaluable endometrial biopsies(tissue insufficient for diagnosis or no endometrial tissueseen), that is, 3%.
The pathologists in this study had extensive experience ingynecological pathology, were from different medical cen-ters, and had different rating backgrounds; these factors
874 Archer et al. THEBES: design and endometrial histolo
were intended to reduce as much as possible the morpholog-ical interpretation of the tissue. The consistency of readingsby individual pathologists over time was assessed by a num-ber of control slides. Kappa coefficients for the various con-ditions were close to 0.9, pointing to a very good agreementbetween readers. The consistency between readers appears tobe at the same level or better as that reported in other trials,such as the Women’s Health, Osteoporosis, Progestin, Estro-gen (35) and the Postmenopausal Estrogen-Progestin Inter-ventions (46) trials. In those two trials, consistency wasrelatively low in the CEE-only groups and very high in theplacebo and CEE plus progestogen groups.
The endometrial biopsy data collected during screeningprovided unique data about the prevalence of the differenttypes of endometrial histology in a large population of post-menopausal women who might be seeking menopausal ther-apy. In>90% of the women, the endometrium was not active,as would be expected from a population of only postmeno-pausal women with an average time since menopause of4.5 years. Archer et al. reported data from 801 women andfound atrophy in 46.9%, proliferation in 16.7%, and hyper-plasia in 5.2% of the women (47). Korhonen et al. (48)screened 2,964 women and reported 68.7% atrophy, 23.5%proliferation, 0.6% hyperplasia, and 0.07% adenocarci-nomas. Both studies contained a mixture of perimenopausaland postmenopausal women, which could at least partlyexplain the lower prevalence of atrophic endometrium intheir screening biopsies. Also, the morphological definitionof endometrial atrophy and proliferative endometrial lesionsmay vary among pathologists.
The reason for choosing oral continuous combinedCEE-MPA as a comparator to tibolone was its previouslydocumented endometrial safety. The Women’s Health,Osteoporosis, Progestin, Estrogen study was a multicenter,randomized, double-blind, placebo-controlled trial involving2,673 healthy postmenopausal women (aged 40–65 y) withan intact uterus who received CEE (0.625 mg/d), CEE-MPA (0.625 and 2.5 mg/d), CEE (0.45 mg/d), CEE-MPA(0.45 and 2.5 mg/d), CEE-MPA (0.45 and 1.5 mg/d), CEE(0.3 mg/d), CEE-MPA (0.3 and 1.5 mg/d), or placebo for1 year (33). Endometrial biopsies were performed, mainlywith a Pipelle suction curette (88%), at baseline and after 6months and 1 year of treatment. A centralized protocol wasused for the histopathology readings to reduce variabilityand eliminate biases. The endometrial histology was
gy Vol. 88, No. 4, October 2007
reviewed by two pathologists, with a third being consulted inthe event of disagreement. The primary endpoint wasthe incidence of endometrial hyperplasia at year 1 in an effi-cacy-evaluable population. Among the 2,153 women whowere evaluable after 1 year, 30 developed endometrial hyper-plasia on CEE alone, and 2, on CEE-MPA. All CEE-MPAgroups had a low incidence of hyperplasia (%0.37%). Ina subset of 822 women enrolled in the Women’s Health, Os-teoporosis, Progestin, Estrogen study who were treated for 2years, no cases of endometrial hyperplasia were observed inany of the CEE-MPA groups (35).
The Women’s Health Initiative (5) multicenter, random-ized, double-blind, placebo-controlled study of 16,608 healthypostmenopausal women with an intact uterus confirmed theendometrial safety of continuous combined CEE-MPA (0.625and 2.5 mg/d) (6), based on the collected number of endome-trial cancer events. After an average follow-up of 5.2 years, thehazard ratio for endometrial cancer in women assigned toCEE-MPA compared with placebo was 0.81 (95% CI, 0.48–1.36). There were no differences between the CEE-MPA andthe placebo groups with respect to the distribution of histolog-ical class, morphological grade, or stage at diagnosis.
The observational United Kingdom Million Women Studyrecently reported follow-up data on 716,738 postmenopausalwomen who used daily combined E-progestin (22%), sequen-tial E-progestin (45%), E only (4%), and tibolone (9%) (28).Data about hormone therapy use and other personal detailswere collected by means of questionnaires at recruitment,and the date and type of cancer were provided by the UnitedKingdom National Health Service Central Registers. Duringthe follow-up period of an average 3.4 years, there were1,320 cases of invasive endometrial cancer. The relative risk(11) of endometrial cancer was 0.71 (95% CI, 0.56–0.90)with the use of continuous, combined E-progestin. Inthe same study, the relative risk for E only was 1.45 (95%CI, 1.02–2.06); and for tibolone, it was 1.79 (95% CI,1.43–2.25). The excess endometrial carcinoma risk in partic-ipants on tibolone was surprising, because it has a knownprogestogenic effect on the endometrium (19).
Estrogenic activity in the endometrium during tibolonetreatment is reduced and counteracted by the following twomechanisms: tibolone deactivates estrogenic compounds,such as the two estrogenic 3-OH tibolone metabolites, bystimulating the formation of biological inactive sulfatedcompounds locally in the endometrium (19). The two estro-genic 3-OH tibolone metabolites are also converted in theendometrium into tibolone and, subsequently, into the D-4isomer (16, 17). Both tibolone and the D-4 isomer bind andactivate the P receptor and protect the endometrium fromthe agonist effects of any remaining 3-OH metabolites (18).
A number of studies have demonstrated a lack of endome-trial stimulation during tibolone treatment of various dura-tions (%10 y). These findings are discussed in more detailbelow. Tibolone did not stimulate the endometrium as mea-sured by Ki67 proliferation marker and histological assess-
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ments after 2 years of treatment in a nonhuman primatestudy (20). The incidence of endometrial hyperplasia wassimilar in tibolone-treated women with an intact uterus (3 of600), compared with placebo-treated subjects (1 of 146)during a 2-year, prevention of osteoporosis, dose-findingstudy in postmenopausal women. Three cases of endometrialcancer were documented in this study, but in each case, evi-dence of pre-existing carcinoma was detected when the base-line biopsy samples were further sectioned and examinedmore extensively (31). In another 2-year study, 150 postmen-opausal women were treated with 2.5 mg of tibolone, and theendometrium remained atrophic in the majority of women(92%), and one subject developed hyperplasia (22). Lastly, ti-bolone was found to be effective in maintaining an inactive en-dometrium over a period of 10 years in a cohort of 110 women,of whom 55 received tibolone, and 55, no treatment (24).
All these results are at odds with the data from the obser-vational Million Women Study, which may reflect preferen-tial prescribing of tibolone. Indeed, data from the UnitedKingdom MediPlus primary care database indicate that clini-cians often prescribed tibolone to women who were at in-creased risks for breast and endometrial cancer (29).Women prescribed tibolone in the United Kingdom moreoften had chronic breast disease, personal history of breastcancer, previous dysfunctional uterine bleeding, hyperten-sion, and previous uterine operations, compared with usersof other forms of postmenopausal hormone therapy. Most im-portant, more women who were prescribed tibolone had ahistory of long-term treatment with unopposed E (29).
THEBES, by virtue of its design, provides important infor-mation regarding the endometrial safety of tibolone in com-parison to E-progestogen therapy. These data complementand add to the existing information relative to the effect oftibolone on the endometrium. The confirmation of endome-trial safety with tibolone by a large, prospective, double-blind,randomized multinational trial in postmenopausal women isimportant to provide level I, evidence-based endometrialsafety. The THEBES also provides information on double-layer endometrial thickness as measured by TVUS, vaginalbleeding patterns, the incidence of breast pain or tenderness,vaginal dryness, changes in body weight, mammographicdensity, and health-related quality of life and sexual function-ing in postmenopausal women who were using tibolone,compared with standard E plus progestin therapy.
In summary, this article provides the prevalence of differ-ent endometrial histology findings in a large population ofuntreated postmenopausal women who are potential candi-dates for menopausal therapy. The THEBES study design,outcomes evaluated, and statistical evaluation have beenpresented in detail.
Acknowledgments: The authors thank the THEBES investigators for their
assistance in conducting this study and thank the subjects and their families
for their willing participation in this research. The authors also thank Lev
Sverdlov, Ph.D. (Biometrics Department, Organon USA Inc., Roseland,
NJ), for the statistical analyses of the baseline data.
logic classification and pathologic findings for endometrial biopsy
specimens obtained from 2964 perimenopausal and postmenopausal
women undergoing screening for continuous hormones as replacement
therapy (CHART 2 Study). Am J Obstet Gynecol 1997;176:377–80.
APPENDIXThe THEBES Study Group
The THEBES Publication Committee includes the followingmembers: D. F. Archer (Jones Institute of Medicine, Norfolk,VA)—Chair, S. Hendrix (Wayne State University HutzelHospital, Detroit, MI), C. Gallagher (St. Joseph Hospital,Omaha, NE), J. Rymer (Guy’s Hospital, London, UnitedKingdom), S. Skouby (Frederiksberg Hospital, Frederiks-berg, Denmark), V. Stathopoulos and F. A. Helmond (Orga-non International, Roseland, NJ), and W. den Hollander(Organon International, Oss, the Netherlands).
The THEBES Data Safety and Monitoring Board includesthe following members: D. A. Grimes (Family HealthInternational, Durham, NC), K. F. Schulz (Family HealthInternational, Durham, NC), and J. E. Wheeler (Universityof Pennsylvania, Pennsylvania, PA).
The THEBES gynecological pathologists are as follows:A. Ferenczy (McGill University and SMBD-Jewish GeneralHospital, Montreal, Quebec, Canada), J. Felix (Universityof South California, Los Angeles, CA), and R. Zaino (PennState Milton S. Hershey Medical Center, Hershey, PA).
The following are the THEBES investigators.
Belgium: M. Dhont (University Hospital, Ghent), J.-M.Foidart (Hopital de la Citadelle, Liege), U. Gaspard (CentreHospitalier Universitaire de Liege Sart Tilman, Liege),H. De Gezelle (private practice, Ghent), D. Janssens (privatepractice, Turnhout), and P. Simon (Hopital UniversitaireErasme, Bruxelles)
Chile: S. Cheviakoff (Instituto Chileno de MedicinaReproductiva, Santiago), J. C. Montero (Hospital San Juande Dios, Santiago), M. Villarroel (Clinica Alemana,Santiago), and A. Glavic (Hospital Barros Luco Trudeau,Santiago)
Czech Republic: T. Reslova (Charles University, HradecKralove), P. Strnad (Charles University, Prague), J. Jenicek(Centre for Climacteric Medicine Lekarsky dum Praha,Prague), P. Bartos (Homolka Hospital, Prague), J. Matlo-
Fertility and Sterility�
chova (Porodnicko–Gynekologicka Ordinace, Olomouc),D. Makalova (private practice, Prague), I. Blstak (SupermedCentre, Usti nad Labem), P. Sak (Hospital C. Budejovice,Budejovice)
Denmark: S. O. Skouby (Frederiksberg Hospital, Freder-iksberg), G. Kramshøi-Larsen (Hillerød Hospital, Hillerød),M. Dueholm (Skejbey Hospital, Arhus), and L. Nilas(Hvidovre Hospital, Hvidovre)
Finland: A. Kivela (Doctor’s Centre Gyneko, Oulu),H. Rautiainen (Doctor’s Centre Gyneko, Oulu), M. Ruuska-nen (Doctor’s Centre Materna, Kajaani), A-M. Heikkinen(Sairaala Lasaretti, Kuopio), and S. Korhonen (SPR Laakar-iasema, Mikkeli)
France: A. Beck (Institute de Medecine et de PhysiologieSpatiales, Toulouse), D. Chassard (ASTER, Paris), andP. Reboud (Laboratoire de Recherche pour l’Industrie duMedicament, Lagord)
Germany: M. Doren (Free University of Berlin, Berlin)
Hungary: J. Urbancsek (Semmelwis University, Budapest),A. Pal (University of Szeged, Szeged), I. Marton (MAV Hos-pitals, Budapest), I. Kis Csitari (Affiliation, Salgotarjan), E.Huszar (County Hospital, Miskolc), A. Borsos (Universityof Debrecen, Debrecen), L. Rokay (Selye Janos Hospital,Komarom), F. Racz (Regional Hospital of Csongrad County,Szentes), A. Rucz (Reti Pal Hospital, Bekescsaba), G. Apro(Hospital of Hodmezovasarhely, Hodmezovasarhely), andK. Pap (Josa Andras Hospital, Nyiregyhaza)
Italy: D. De Aloysio (University of Bologna, Bologna)
The Netherlands: J. Jonker (ANDROMED, Groningen,Rotterdam, Zoetermeer), D. van Duren (Menox MenopauseResearch, Nijmegen), and V. van de Walle (PreCare Trialand Recuitment, Geleen)
Norway: I. Fjaerestad (Sentralsykehus, Kristiansand),N. Sørheim (Gyn Tromsø Søndre, Tromsø), K. Dalaker (Vol-vat Medisinske Senter, Oslo), M. H. Moen (GynekologiskPoliklinik, Trondheim), and T. Sørdal (MEDICUS, Trond-heim)
Poland: R. Debski (private gynecological practice, War-saw) and J. Tomaszewski (private gynecological practice,Bialystok)
Slovak Republic: J. Stencl (Derer’s University Hospital,Bratislava), S. Lukacin (Faculty Hospital, Kosice), Z. Petro-vicova (Cesta ku nemocnici, Banska Bystrica), V. Cupanik(Sanatorium KOCH Mediline, Bratislava), M. Borovsky(Faculty Hospital of Cyril and Metod, Bratislava), D. Milly(private outpatient clinic, Bratislava), and E. Lanyi (HospitalBojnice, Bojnice)
Spain: S. Palacios (Instituto Palacios, Madrid) andF. Nohales (Hospital Francesc Borja de Gandia, Gandia)
Sweden: M. Hurtig (Centralllasarettett, Vaxjo), L. Wil-helmsson (Avenyklinikken, Goteborg), A. Linden Hirschberg
877
(Karolinska Hospital, Stockholm), M. Olovsson (AcademicHospital, Uppsala), A. F. Jonasson (Huddinge Universityhospital, Stockholm), and H. Wennerstrom (WennerstromGynecologcentrum, Goteborg)
United Kingdom: J. Rymer (Guy’s Hospital, London) andN. Panay (Queen Charlotte and Chelsea Hospital, London)
United States: S. G. Swanson (Women’s Clinic of Lincoln,Lincoln, NE), S. A. Funk (Radiant Research, Atlanta, GA),R. E. Hedrick (Salem Research Group, Winston-Salem,NC), S. R. Drosman (Genesis Center for Clinical Research,San Diego, CA), M. McDermott (Radiant Research, Austin,TX), C. Gallagher (St. Joseph Hospital, Omaha, NE), W. D.Koltun (Medical Center for Clinical Research, San Diego,CA), L. S. Seidman (Philadelphia Women’s Research, Phila-delphia, PA), A. S. Waldbaum (Downtown Women’s Health-care, Denver, CO), H. A. Soper (Piedmont Medical Research,Winston-Salem, NC), J. Jensen (Oregon Health and ScienceUniversity, Portland, OR), I. Kerber (University of Texas,Dallas, TX), D. W. Baldwin (Life Span Research, PaloAlto, CA), B. Kalro (Magee Women’s Research Center,Pittsburgh, PA), T. McCarthy (Radiant Research, SantaRosa, CA), J. C. Millin, Jr. (Vision Clinical Research, Sara-sota, FL), M. DeFrancesco (Women’s Health USA, Water-bury, CT), N. P. DeLahunty (Radiant Research, Greer, SC),S. England (Miami Valley Clinical Trial Resources, Franklin,OH), T. P. Hutchens (Clinical Research Services, Bismarck,ND), M. I. Keller (SD Arthritis and Osteoporosis MedicalClinic, San Diego, CA), S. Barbier (Women’s ClinicalResearch Center, Seattle, WA), B. Miller (Radiant Research,Scottsdale, AZ), A. H. Moffett (Ob-Gyn Associates ofMid-Florida, Leesburg, FL), M. J. Noss (Radiant Research,Cincinnati, OH), M. Nunez (CNS Research, St. Petersburg,FL), T. C. Olson (Deaconess Billings Clinical Research Divi-sion, Billings, MT), A. K. Parsons (USF Gynecology, Ultra-sound & Research Services, Tampa, FL), J. G. Herrmann(Radiant Research, St. Louis, MO), D. J. Portman (ColumbusCenter for Women’s Research, Columbus, OH), M. Saidel(Women’s Health USA, Avon, CT), M. T. Saunders
878 Archer et al. THEBES: design and endometrial histolo
(Advanced Clinical Research, Salt Lake City, UT), W. Larson(Radiant Research, Lakewood, WA), R. Brenner (privatepractice, Venice, FL), B. D. Geary (Laurek Creek ResearchAssociates, Moorestown, NJ), C. Touey (Women’s HealthCare PC, Pottstown, PA), W. D. Schlaff (University ofColorado, Aurora, CO), R. D. Burlison (Highland ResearchCenter, Shreveport, LA), G. A. Bachmann (Women’s HealthInstitute, New Brunswick, NJ), P. Chervinsky (New EnglandClinical Studies, North Dartmouth, MA), D. G. Young(Northern California Research Corporation, Fair Oaks,CA), R. Hines (University of Mississippi Medical Center,Jackson, MS), B. A. Soltes (Center for Women’s Research,Chicago, IL), F. Fingerhut (Estrella Women’s Health Center,Phoenix, AZ), J. Hume (University of Kansas Medical Cen-ter, Kansas City, KS), D. W. Marden (TriCities Medical Re-search, Bristol, TN), S. L. Hendrix (Wayne State UniversityHutzel Hospital, Detroit, MI), S. Kreis (private practice,Waco, TX), W. H. Utian (Rapid Medical Research, Cleve-land, OH), J. Gersten (private practice, Miami, FL),B. Horowitz (Women’s Health USA, Hartford, CT), D. A.Tomlinson (Medford Women’s Clinic, Medford, OR), J. K.Jain (University of Southern California Women’s and Chil-dren’s Hospital, Los Angeles, CA), P. Zedler (VirginiaWomen’s Center, Richmond, VA), M. Singh (Weill MedicalCollege of Cornell University, New York, NY), C. L. Barker(Research Solutions LLC, Jonesboro, AR), C. K. Skokos(Research Solutions LLC, Little Rock, AR), M. A. Ziboh(nTouch Research, South Bend, IN), P. Troia (Cape Cod Clin-ical Trials, Hyannis, MA), A. Dahdul (FutureCare Studies,Springfield, MA), E. Eisenman (Innovative Health Research,Las Vegas, NV), C. M. Zador-Silverman (Bock ClinicalResearch, Levittown, PA), N. J. Kakos (Quest Research,Bingham Farms, MI), S. K. Khairi (Physician’s ResearchGroup, Indianapolis, IN), R. A. Beyerlein (Advanced ClinicalTrials, Eugene, OR), K. Aqua (Visions Clinical Research,Palm Springs, FL), T. L. Anderson (Clinical Research Asso-ciates, Nashville, TN), W. S. McKenzie (Radiant Research,Birmingham, AL), and R. S. Margolis (Ntouch ResearchCorporation, Washington, DC)
gy Vol. 88, No. 4, October 2007
OVULATION INDUCTION
A comparison of letrozole to gonadotropinsfor ovulation induction, in subjects who failedto conceive with clomiphene citrateRudolfo B. Quintero, M.D., Renata Urban, M.D., Ruth B. Lathi, M.D., Lynn M. Westphal, M.D.,and Michael H. Dahan, M.D.
Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University,
Stanford, California
Objective: To compare pregnancy rates (PR) for letrozole and gonadotropins in individuals who failed to conceivewith clomiphene citrate (CC).Design: Retrospective cohort study.Setting: University reproductive center.Patient(s): Individuals treated with letrozole or gonadotropins who failed to conceive with CC.Intervention(s): Controlled ovarian hyperstimulation (COH), transvaginal ultrasound, ovulation induction, IUI.Main Outcome Measure(s): Pregnancy rates per cycle.Result(s): Among patients who failed to conceive with at least three cycles of CC, gonadotropins had a higher PRper cycle than letrozole. Among individuals who failed to conceive with less than three cycles of CC and whosemedications were changed because of thin uterine lining or intolerable side effects, average PR per cycle for letro-zole and gonadotropin treatments were equivalent. All patients conceived within three stimulation cyles with eithergonadotropins or letrozole.Conclusion(s): In patients who failed to conceive with CC, gonadotropins have higher PR for ovulation inductionthan letrozole. However, PR were high enough with letrozole to justify its use in this population of patients.Letrozole and gonadotropins should not be used for more than three cycles without a conception. (Fertil Steril�
2007;88:879–85. �2007 by American Society for Reproductive Medicine.)
Letrozole is an aromatase inhibitor that, when given in theearly follicular phase, releases the pituitary/hypothalamicaxis from estrogenic negative feedback (1). This results in anincrease of gonadotropin secretion and augmented stimulationof ovarian folliculogenesis (1). Transient inhibition of aroma-tase has been shown to stimulate the ovary with similar efficacyas clomiphene citrate (CC) without any apparent adverse effecton the endometrium or midcycle LH surge (2). In the murinemodel, aromatase inhibition resulted in favorable embryo de-velopment and an increase in preimplantation receptivity andimplantation while supporting the release of implantationmarkers (3), when compared to unstimulated cycles.
Received August 7, 2006; revised November 29, 2006 and accepted
November 30, 2006.
Supported by Women’s Reproductive Health Research (WRHR) Scholars
program.
Presented at the 53rd Annual Meeting of the Pacific Coast Reproductive
Society, Indian Wells, CA, April 26–30, 2006, and WRHR Scholars’
Symposium, Detroit, MI, June 5–6, 2006.
Reprint requests: Michael H. Dahan, M.D., WRHR Scholar, Department
of Obstetrics and Gynecology, Stanford University, 300 Pasteur Drive,
Stanford, CA 94305-5317 (FAX: 650-723-7737; E-mail: dahanhaim@
hotmail.com).
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.11.166 Copyright ª2007 American S
The early menstrual cycle reduction of estrogen (E) levelsby letrozole may be one of its advantages as an ovarian stim-ulation medication. In the early proliferative phase of thecycle, the intrafollicular hyperandrogenic state, which resultsfrom decreased aromatization of androgens to E, may yieldimproved oocyte quality (4, 5). When women with polycysticovary syndrome (PCOS) are treated with metformin, the ad-dition of letrozole has superior pregnancy rates (PR) whencompared to CC (6).
These data suggest that letrozole may be a superior ovula-tion-inducing agent when compared to CC (7). Letrozole hasalso been demonstrated to induce ovulation in patients whofailed to ovulate with CC (1). In studies comparing differentoral agents given with FSH, letrozole produces a higher PRwhen compared to CC (8, 9).
Letrozole’s increased PR when compared to CC as well asits ability to induce ovulation in individuals who remainedanovulatory after treatment with CC, may provide rationalefor letrozole’s use as a second-line agent. Traditionally go-nadotropin is the second-line treatment for patients whofail to conceive with CC. However, gonadotropins (FSH)
Fertility and Sterility� Vol. 88, No. 4, October 2007 879ociety for Reproductive Medicine, Published by Elsevier Inc.
are associated with a 30%– 40% risk of multiple gestationand other negative outcomes linked to assisted reproductivetechnologies (ART), including premature birth and the asso-ciated high costs of neonatal intensive care (10).
Clomiphene citrate is associated with only a 5%–8% riskof multiple gestation (11). The multiple gestation rate of le-trozole may be lower than that of CC and thus, should bemuch lower than that of gonadotropins (12). A lower rateof multiple gestation would favor the use of letrozole overgonadotropins after failure to conceive with CC. However,it is unclear whether letrozole yields comparable PR togonadotropins after failure to conceive with CC. Therefore,we sought to compare letrozole to gonadotropins as a treat-ment option for patients who failed to conceive with CC.
MATERIALS AND METHODS
Inclusion and Exclusion Criteria
We performed a retrospective chart analysis of patients whofailed to conceive with CC and were then treated with letro-zole (Femara, Novartis, East Hanover, NJ), 5 mg orally,from days 3–7 of the menstrual cycle or FSH starting onday 3 of the menstrual cycle. We examined charts from No-vember 1, 2003 to November 30, 2005. We evaluated billinginformation to verify that all possible patients taking letrozolewere identified. Patients were considered to have failed CC if[1] they did not conceive with at least three cycles of CC; [2]they failed to ovulated with three cycles of CC increased to150 mg daily, taken on days 3–7 of the menstrual cycle; [3]they had an endometrial lining less than 6 mm at the timeof their last ultrasound that cycle; or [4] if they hadintolerable symptoms when treated with CC. Intolerablesymptoms were severe hot flashes affecting daily function,debilitating headaches, vision changes, and depression.
Subjects studied had either PCOS-associated anovulation,unexplained infertility, or mild male factor infertility. Thewomen with PCOS were oligo-ovulatory (menstrual cyclesless frequent than every 35 days) or anovulatory and had clin-ical (Ferryman-Gallway score R8) or biochemical evidence(total or free T more than female assay maximum) of hyper-androgenism. As indicated, subjects with PCOS were evalu-ated for thyroid abnormalities (0.39<TSH>4.0 mIU/mL);hyperprolactinemia (morning fasting PRL <26 ng/mL); hy-pothalamic pituitary dysfunction and ovarian failure (1.4<FSH>20 mIU/mL and E2>20 pg/mL); ovarian and adrenalandrogen-secreting tumors (total T<200 ng/mL and DHEAS<800 mg/dL); and nonclassic congenital adrenal hyperplasia(morning fasting 17-hydroxy-P <3 ng/mL).
Subjects defined as having unexplained infertility hada spouse with normal semen analysis as per the World HealthOrganization (WHO) criteria and a strict Kruger analysisR4%, patent tubes on hysterosalpingogram, and had ovula-tory menstrual cycles every 21–35 days with premenstrualmolimina.
Subjects with mild male factor infertility had R5 milliontotal motile sperm count in an IUI specimen and a Kruger
880 Quintero et al. Letrozole vs. FSH in clomiphene failur
strict morphology analysis R4% at semen analysis. How-ever, on two semen analysis, they demonstrated persistentabnormalities in count, motility, or morphology when com-pared to WHO parameters. These individuals had not demon-strated any normal semen analyses by WHO parameters.
We excluded subjects from evaluation if: [1] they had notpreviously failed to conceive with CC; [2] they had previouslybeen treated with gonadotropins or IVF; [3] they had a Krugerstrict analysis %4% on two semen analysis; [4] total motilesperm count was <5 million at the time of IUI; [5] they hadtubal factor infertility; [6] hypothalamic amenorrhea; or [7]serum-to-TSH (0.4<TSH or TSH>3.9 mIU/mL); [8] elevatedserum PRL (morning fasting PRL R26 ng/mL); or [9]>3 fol-licles, 15–18 mm in mean diameter and serum E2 R2,500pg/mL on the day of b-hCG injection. Hyperandrogenic sub-jects did not have serum-to-total T >200 ng/dL, DHEAS>800 mg/dL or morning fasting 17-hydroxy-P >2.9 ng/mL.
Timing of Stimulation Relative to Clomiphene Citrate
Many studies exclude patients who are treated less than 2months after receiving the last dose of CC because of itslong half-life. However, subjects in this study were treated30 days after the last dose of CC with either gonadotropinsor letrozole. Although limiting evaluation to subjects withat least 2 months between CC and subsequent treatmentswould eliminate any possible lingering effects of CC, itdoes not represent a realistic clinical scenario. Patients whofail to conceive often undergo subsequent treatments assoon as possible to expedite conception. Patients treatedwith either letrozole or gonadotropins after failing to con-ceive with CC do not usually have a 2-month washout periodbetween medications. Therefore, we included patients whofailed to conceive with CC citrate in the previous cycle, andwere subsequently treated with letrozole or gonadotropins,to make the results translate to patients in a clinic setting.
Stimulation Monitoring
Transvaginal ultrasounds were performed on day 3 of themenstrual cycle. Patients were not stimulated if they hadovarian cysts >20 mm in mean diameter or <20 mm inmean diameter with a day 3 serum E2 level>60 pg/mL. Sub-sequently, transvaginal ultrasounds were performed to mon-itor folliculogenesis on days 10–12 of the menstrual cyclein the letrozole group and on day 7 in the gonadotropin group.Ultrasounds were then performed as indicated by maximumfollicular mean diameter in both groups where growth wasanticipated to be 2 mm in diameter daily. The b-hCG trigger-ing of ovulation (Pregnyl 10,000 IU, Organon, Inc., WestOrange, NJ; Novarel 10,000 IU, Ferring Pharmaceuticals,Inc., Tarrytown, NY) or Ovidrel 250 mg (Serono Laborato-ries, Rockland, MD) was performed when transvaginal ultra-sounds revealed a largest follicle of mean diameter ofR18 mm. Subjects underwent intrauterine insemination36 hours after b-hCG injection or sexual intercourse beforeb-hCG injection, and every 2 days until 4 days after b-hCG.
es Vol. 88, No. 4, October 2007
Letrozole Treatment
The protocol at our institution is that most subjects undergo-ing letrozole ovulation induction receive 5 mg, daily, cycledays 3–7. In a prospective randomized trial, Fadhli et al.(13) demonstrated that when compared to 2.5 mg, 5 mg of le-trozole resulted in a higher PR as well as a greater number ofovarian follicles without affecting endometrial development.The data from the study by Fadhli et al. suggest that 5 mgof letrozole should be the preferred dose for ovulationinduction.
Gonadotropin Medication
The FSH-treated subjects received either Gonal-f (Serono),Follistim (Organon), Menopur, or Bravelle (Ferring) andwere monitored as described previously. The injection dosewas modified to prevent more than three follicles, 15–18mm in mean diameter, and serum E2 R2,500 pg/mL on theday of b-hCG injection.
Statistical Analysis
All statistical analyses were done using the statistical packagefor social sciences 11.0 (SPSS Inc., Chicago, IL). Continuousvariables were evaluated for normal distribution using theKolmogorov-Smirnov test. Results are reported as meanvalue � standard error of the mean (SE). Student’s t-testwas used for comparison of nominal data. Levene’s test forequality of variances was applied to the data, and the corre-sponding t-test and P values were accepted depending onwhether the variances were equal. The c2 and Fisher’s exacttests were used to compare noncontinuous variables. Correc-tions were applied when cell size was too small. Life tableanalysis was used to analyze pregnancy results over time. Sta-tistical significance was accepted as a two-sided P%.05. Ap-
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proval of the study was obtained from the Stanford UniversityCommittee for the Protection of Human Research Subjects.
RESULTS
All continuous variables were evenly distributed.
Study Population
We identified 52 subjects who underwent 99 letrozole-controlled ovarian hyperstimulation cycles after failing toconceive with CC. All subjects treated with letrozole received5 mg from days 3–7 of the menstrual cycle. We identifieda control group of 33 subjects who were treated for 65 gonad-otropin-controlled ovarian hyperstimulation cycles after fail-ing to conceive with CC. The mean dose of gonadotopin usedwas 1,122 IU, with a standard deviation of 419 IU, and a rangeof 450–2,700 IU per cycle of stimulation.
Letrozole Versus Gonadotropin Stimulation
Baseline characteristics Table 1 compares characteristics ofsubjects receiving letrozole or gonadotropins. These charac-teristics are represented as the mean � SE of the mean orpercent of subjects affected in each group. There was nodifference in any of the characteristics when both groupswere compared, including age, duration of infertility, andnumber of failed CC cycles, or frequency of diagnosis. Itshould be noted that the maximum day 3 FSH level forboth treatment groups was in the normal range for the clinicassay (<12 mIU/mL). There was no statistically significantdifference between the groups in the number of true CC fail-ures, defined as failure to conceive after three or more cycles(letrozole 53% vs. gonadotropin 51%) and those who hadless than three CC cycles before initiating letrozole (47%)or gonadotropin (49%, P¼.75).
TABLE 1Comparison of baseline characteristic in subjects who had failed treatment with clomiphene citrateand were subsequently treated with either letrozole or gonadotropins.
Quintero. Letrozole vs. FSH in clomiphene failures. Fertil Steril 2007.
881
TABLE 2Comparison of pregnancy outcomes per treatment cycle among subjects treated with letrozole orgonadotropins after failing to conceive with clomiphene citrate.
Letrozole Gonadotropin P value
Positive pregnancy test 9% 28% .002a
Fetal heart beat 7% 18% .03a
Miscarriage rate among pregnancies 44% 44% 1.0
a Represents statistical significance.
Quintero. Letrozole vs. FSH in clomiphene failures. Fertil Steril 2007.
Stimulation parameters The percent of subjects undergoingintercourse as opposed to IUI was comparable between theletrozole and gonadotropin groups (10% vs. 5%, P¼.25),and in all other cycles subjects were treated with IUI. Therewas no difference in the number of follicles R12 mm in di-ameter on the day of ovulation triggering between the letro-zole and gonadotropin groups (1.7 � 0.1 vs. 2.0 � 0.1,P¼.13). However, endometrial thickness was greater in thegonadotropin group when compared to the letrozole groups(8.6 � 0.3 vs. 7.5 � 0.2 mm, P¼.001).
Pregnancy outcomes in all patients Table 2 represents preg-nancy outcomes per stimulation cycle in groups treated withletrozole or gonadotropins after failing to conceive with CC.The rate of positive serum b-hCG level per stimulation cycle2 weeks after ovulation and the percent of cycles where fetalheart motion at 6–7 weeks gestational age was present, weregreater among the gonadotropin group than in the letrozolegroup. There was no difference in the miscarriage rate be-tween the two groups. There were two multiple gestationsamong gonadotropin-treated subjects, but no multiple gesta-tions in the letrozole group. However, this was not statisti-cally different, particularly in view of the small sample size.
Percent of conception with subsequent treatment did notdiffer whether the subjects failed to conceive after at least
882 Quintero et al. Letrozole vs. FSH in clomiphene fail
three CC cycles (9%) or with less than three CC cycles(9%), P¼1.0. Likelihood of clinical pregnancy with subse-quent treatment also did not differ whether the subjects failedto conceive after at least three CC cycles (8%) or with lessthan three CC cycles (7%), P¼1.0.
Pregnancy Outcomes Among Subjects Failing to ConceiveAfter at Least Three Versus Those Who Underwent FewerThan Three Clomiphene Cycles
Table 3A compares the response per cycle among subjectswho failed to conceive after at least three cycles of CC. Table3B compares the response among those who underwent lessthan three CC cycles before treatment with either letrozoleor gonadotropins. Among subjects who failed to conceivewith at least three CC cycles, those treated with gonadotropinswere more likely to have positive pregnancy tests and fetalheart beats on ultrasound than those treated with letrozole,which is consistent with the cumulative data presented inTable 2. However, among those who failed to conceive aftertwo or less CC cycles, there was no difference in the rateof positive pregnancy tests per cycle and rate of fetal heartbeats noted on ultrasound between subjects treated withletrozole or gonadotropins. This is attributed to a decreasedPR per cycle among the FSH-treated subjects. There was nochange in the PR per cycle for the letrozole-treated subjects.
TABLE 3Comparison of pregnancy outcomes per treatment cycle among subjects treated with letrozole orgonadotropins after failing to conceive with at least three cycles (A) and with less than three cycles (B)of clomiphene citrate.
Letrozole Gonadotropin P value
APositive pregnancy test 9% 33% .006a
Fetal heart beat 7% 26% .02a
Miscarriage rate among pregnancies 22% 45% .56B
Positive pregnancy test 9% 22% .18Fetal heart beat 7% 16% .26Miscarriage rate among pregnancies 75% 42% .55
a Represents statistical significance.
Quintero. Letrozole vs. FSH in clomiphene failures. Fertil Steril 2007.
ures Vol. 88, No. 4, October 2007
TABLE 4Life table analysis of clinical pregnancy rate per gonadotropin cycle (A) and letrozole cycle (B) insubjects who did not conceive with less than three clomiphene citrate cycles.
Cycle numberPercent clinical
pregnancy per cycleCumulative clinical
pregnancy rateSE for cumulative
pregnancy rate
A1 12 12 62 18 27 113 0 27 114þ 0 27 11
B1 3 3 32 6 9 63 11 19 114þ 0 19 11
Quintero. Letrozole vs. FSH in clomiphene failures. Fertil Steril 2007.
Response to Letrozole and Gonadotropins Over Time
To evaluate the response to letrozole or gonadotropins perstimulation cycle, a life table analysis was performed in thetwo groups, individuals who failed to conceive with at leastthree CC cycles and those who failed to conceive with lessthan three CC cycles. The data are presented in Tables 4 and5, respectively. These data confirm that the PR per cyclewith letrozole in individuals who had failed less than or atleast three cycles of CC without conception was similar at3%–11% per cycle. The PR per cycle with gonadotropinswas 10%– 19%. However, patients who did not conceivewith at least three cycles of CC had a cumulative PR of37%, which was greater than the 27% if they did not conceivewith less than three cycles of CC. The lower PR per cycle seenwith gonadotropins, shown in the previous section, in individ-uals who had less than three CC cycles, was because none
Fertility and Sterility�
conceived after two treatment cycles. The PR per cyclewhen conception occurred was still 12%–18%. It should benoted that no patients conceived after undergoing more thanthree cycles of gonadotropins or letrozole in any of the groups.
DISCUSSION
Several studies have shown letrozole to be superior to CCwhen comparing side effects, ovulation, and PR in a generalinfertile population (1, 2, 14). Among anovulatory patientswith PCOS, letrozole has also been shown to induce ovula-tion and attain a 25% PR (15). Letrozole’s use for ovulationinduction has become more acceptable as it has a lower riskof birth defects than CC (16).
After failing to conceive with three to six cycles of CC,subjects traditionally have proceeded to treatment with
TABLE 5Life table analysis of clinical pregnancy rate per gonadotropin cycle (A) and letrozole cycle (B) insubjects who did not conceive with at least three clomiphene citrate cycles.
Cycle numberPercent clinical
pregnancy per cycleCumulative clinical
pregnancy rateSE for cumulative
pregnancy rate
A1 19 19 72 8 24 93 17 37 144þ 0 37 14
B1 3 3 22 10 12 73 11 22 114þ 0 22 11
Quintero. Letrozole vs. FSH in clomiphene failures. Fertil Steril 2007.
883
gonadotropins (11). The ease of letrozole’s orally inducedovulation and its low cost make it an attractive alternativeto gonadotropins. Multiple gestation rates are lower withletrozole than gonadotropins (12). However, the successrate of treatment with letrozole compared to gonadotropinin patients previously treated with CC without conceptionhas not previously been studied.
Our data suggest that gonadotropins are more potent ovu-lation-inducting agents than letrozole among individuals whohave failed to conceive with CC. The mean clinical PR percycle in the first three cycles of treatment is better with go-nadotropins than with letrozole (16% vs. 7%) in these pa-tients. For the purpose of counseling patients who havepreviously failed to conceive with CC, they can expecta 3%–10% PR per cycle with letrozole versus a 10%– 20%PR per cycle with gonadotropins.
Medication costs with letrozole should be significantly lessthan that of gonadotropins per month of stimulation. Letrozolerequires less ultrasound monitoring and possibly less bloodassay measurements per cycle of treatment when comparedto gonadotropins, which should also reduce treatment costs.However, data on cost per pregnancy is lacking when compar-ing letrozole to gonadotropins for ovulation induction. Giventhe significantly lower PR with letrozole, data are needed todetermine which is more cost effective per live birth.
In subjects who stopped CC before three cycles due to in-tolerable side effects or a thin uterine lining, no differencewas documented in average PR per cycle between treatmentwith letrozole or gonadotropins during three cycles. Thesedata suggest that letrozole should be the next treatment inthis patient population given the expected lower cost perstimulation cycle when compared to gonadotropins. Al-though patients are hypoestrogenic during their 5-day courseof letrozole, their E levels quickly return to normal, amelio-rating some of the deleterious side effects of CC (4). Letro-zole should not negatively impact the endometrial lining,unlike CC (1, 2, 17). However, no difference was seen inthe group who stopped CC before three cycles, not becauseof an improvement in PR per cycle with letrozole, but be-cause of a decrease in cumulative PR among the gonadotro-pin-treated subjects.
There were no multiple gestations in the letrozole-treatedsubjects, and two multiple gestations in the gonadotropin-treated subjects. Although these rates were not statisticallysignificant, the relatively small number of individuals studiedand the relatively low multiple gestation rates compared tohistoric data make comparisons related to multiple gestationsinaccurate from our data.
Our miscarriage rate appears to be high at 44%. However,early pregnancy detection was performed with serum quanti-tative pregnancy hormone levels 14 days after ovulation trig-gering. Studies evaluating early miscarriage rates havedocumented the frequency of pregnancy wastage to be be-tween 30% and 66% (18). Therefore, the miscarriage ratein this study may not differ from baseline levels.
884 Quintero et al. Letrozole vs. FSH in clomiphene failur
One of the weaknesses of this study is that subanalysis ofthe PR in letrozole-treated individuals with different diagno-ses cannot be undertaken due to small subset sample sizes.Nevertheless, as far as we know this is the first study to com-pare the use of letrozole and gonadotropins in CC failures.This study has demonstrated that letrozole has use as a sec-ond-line agent after failing CC for many patients.
Pregnancy rates of 10% per cycle are often considered ac-ceptable for infertility treatments. Our findings suggest that le-trozole is a possible second-line treatment after failure toconceive with CC. There are few reports of multiple births inthe letrozole group (12). The decreased risk of multiple gesta-tion associated with letrozole ovulation induction when com-pared to treatment with gonadotropins would also havedecreased societal and hospital costs during the infant’s first5 years (10, 19). Gonadotropin/IUI cycles remain risky formultiple births, with reported rates of twin gestations of30%– 40% (10) and a high order multiple PR of 4/1,000 cycles(20). Because letrozole is taken orally, requires less monitoringthan gonadotropins, and has a low rate of multiple gestation, itmay represent a reasonable alternative to CC. However, trueCC failures should be counseled as to the significantly higherPR noted with gonadotropins when compared to letrozole.
REFERENCES1. Mitwally MF, Casper RF. Use of an aromatase inhibitor for induction of
ovulation in patients with an inadequate response to clomiphene citrate.
Fertil Steril 2001;75:305–9.
2. Fisher SA, Reid RL, Van Vugt DA, Casper RF. A randomized double-
blind comparison of the effects of clomiphene citrate and the aromatase
inhibitor letrozole on ovulatory function in normal women. Fertil Steril
2002;78:280–5.
3. Karaer O, Vatansever HS, Oruc S, Ozbilgin K, Cilaker S, Koyuncu MF.
The aromatase inhibitor anastrozole is associated with favorable embryo
development and implantation markers in mice ovarian stimulation cy-
cles. Fertil Steril 2005;83:1797–806.
4. Mitwally MF, Casper RF, Diamond MP. The role of aromatase inhibitors
in ameliorating deleterious effects of ovarian stimulation on outcome of
Role of insulin in the hyperandrogenemia of leanwomen with polycystic ovary syndrome and normalinsulin sensitivityJean-Patrice Baillargeon, M.D., M.Sc., and Andr�e Carpentier, M.D.
Department of Medicine, Division of Endocrinology, Universit�e de Sherbrooke, Sherbrooke, Quebec, Canada
Objective: To determine the effect of reducing insulin secretion on hyperandrogenemia in lean normoinsulinemicwomen with polycystic ovary syndrome (PCOS) and normal metabolic insulin sensitivity.Design: Transversal assessment at baseline and prospective follow-up of lean PCOS group after 8 days of diazo-xide, which reduces insulin secretion, and 1 month of leuprolide, which suppresses LH.Setting: Clinical research center of an academic hospital.Patient(s): Nine lean women (body mass index %25 kg/m2) with PCOS and normal insulin levels, as well as 17lean healthy women.Intervention(s): Lean PCOS women were reassessed after 8 days of diazoxide and after 1 month of leuprolide,which suppresses LH.Main Outcome Measure(s): Androgen levels and insulin-stimulated glucose disposal (metabolic insulin sensiti-vity), determined by euglycemic-hyperinsulinemic clamp (M-value).Result(s): Mean M-value of lean PCOS women (48.5 mmol/kg$min) was similar to lean control subjects (52.9mmol/kg$min). They also had comparable anthropometric measures, lipids, fibrinogen, and plasminogen activatorinhibitor 1. The LH did not change significantly after diazoxide, but was almost suppressed after leuprolide in thePCOS group. Androstenedione decreased significantly after diazoxide and even more after leuprolide. However,free T significantly decreased only after diazoxide in lean PCOS women. Diazoxide also increased SHBG signifi-cantly in this group.Conclusion(s): In women with typical PCOS and normal insulin levels and metabolic insulin sensitivity, reducinginsulin secretion significantly decreased androgen and increased SHBG levels. These results suggest that insulincontributes to hyperandrogenemia even in PCOS women with normal metabolic insulin sensitivity, which mightbe due to increased sensitivity of their androgenic insulin pathway. (Fertil Steril� 2007;88:886–93. �2007 byAmerican Society for Reproductive Medicine.)
Polycystic ovary syndrome (PCOS) affects 6%–10% ofwomen of childbearing age (1) and is defined by hyperandro-genism, chronic anovulation, and/or polycystic ovaries (2 cri-teria out of 3) (2). However, it has become apparent thatinsulin resistance and hyperinsulinemia play a critical rolein the syndrome’s pathogenesis (1). Despite advances overthe past decade, many questions remain regarding the mech-anism by which insulin resistance or insulin produces hyper-androgenemia.
Received November 3, 2006; revised and accepted December 28, 2006.
This work was supported by the Canadian Institutes of Health Research
(Operating Grant Program MOP-62946) and the Fonds de la Recherche
en Sant�e du Qu�ebec (Young Clinical Investigator Establishment Grant
#2834). Dr. Baillargeon is a Junior 1 Clinical Investigator of the Fonds
de la Recherche en Sant�e du Qu�ebec (#3158) and Dr. Carpentier is
a New Investigator of the Canadian Institutes of Health Research.
Reprint requests: Jean-Patrice Baillargeon, M.D., M.Sc., Division of Endo-
crinology, Universit�e de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine,
886
Several studies have demonstrated that insulin stimulatesovarian steroidogenesis in vitro. At physiologic concentra-tions, insulin stimulates androgen production by culturedovarian cells to a greater extent in women with PCOS com-pared with control subjects (3). Moreover, combined stimula-tion with LH and insulin at physiologic concentrations havebeen shown to synergistically increase androgen biosynthesisby ovarian tissues from normal and PCOS women (4).
Increased androgen response to LH stimulation in womenwith PCOS has also been demonstrated in vivo (5). Chronicstimulation by both LH and insulin have been implicated inthis ovarian androgen hyper-responsiveness, because mostwomen with PCOS have increased LH and/or insulin levels.To control for chronic stimulation by LH, normal and PCOSwomen were challenged with hCG before and 4 weeks afterLH suppression with a long-acting analog of GnRH (5). Sup-pression of LH did not alter the typical exaggerated plasma17a-hydroxyprogesterone response to hCG. Conversely,
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.055
serum total and nonSHBG-bound testosterone (T) levels de-creased significantly after direct suppression of pancreatic in-sulin release for 10 days with diazoxide in obese PCOSwomen (6). Moreover, reduction of insulinemia with acar-bose, which slows down intestinal absorption of carbohy-drates, also reduced serum T levels in PCOS (7). Thesestudies all highlight the importance of insulin in the patho-genesis of PCOS.
Numerous studies have demonstrated that any treatmentaimed at improving metabolic insulin resistance in womenwith PCOS lowers androgen levels and improves ovulatoryfunction (1). The exaggerated steroidogenic response to LHstimulation tests also improves with these treatments (8, 9),suggesting normalization of ovarian androgen hyper-respon-siveness. We previously conducted a study using insulin-sensitizing drugs (namely metformin, a biguanine, androsiglitazone, a peroxisome proliferator-activated receptorg (PPARg) agonist) in nonobese women with PCOS and nor-mal insulin levels (10). The results demonstrated a normaliza-tion of serum T levels and ovulation in actively treated groupscompared with the placebo-treated group. These findingssuggest that insulin-sensitizing drugs improve hyperandroge-nemia even in nonobese women with PCOS who appear tohave normal metabolic insulin sensitivity. Whether it is thecorrection of abnormal insulin action per se or the reductionof plasma insulin levels that was responsible for these bene-ficial effects of insulin sensitizers is currently unclear.
Therefore, we hypothesized that insulin contributes to an-drogen levels even in lean women with PCOS and normalinsulin levels owing to hypersensitivity to the androgenic ac-tions of insulin. We sought to determine if a reduction in in-sulin levels with diazoxide, an agent devoid of any influenceon insulin-signaling pathways (11), can reduce androgen pro-duction in lean normoinsulinemic women with PCOS andnormal metabolic sensitivity to insulin, as demonstrated bythe insulin-glucose clamp. Indeed, studied women exhibitednormalization of their serum free T levels after treatment withdiazoxide, which was more pronounced than after LH sup-pression with a long-acting agonist of GnRH. These resultsfurther support the notion that insulin contributes to hyperan-drogenemia even in women with PCOS and normal meta-bolic sensitivity to insulin.
MATERIALS AND METHODS
Subjects
We recruited women with PCOS and normal control womenwho were both lean (body mass index [BMI] %25 kg/m2) andnormally insulin sensitive. For practical reasons, a directmeasure of metabolic insulin sensitivity could not be per-formed as a screening procedure before enrollment into thestudy. Therefore, to increase the probability of enrollingPCOS women without insulin resistance, the inclusion crite-ria for this group were: PCOS; absence of hypertension oracanthosis nigricans; and normal insulin levels, i.e., fastingserum insulin<15 mIU/mL (104 pmol/L), peak serum insulin
Fertility and Sterility�
during oral glucose tolerance test (OGTT) <100 mIU/mL(695 pmol/L) (12), and fasting serum glucose-to-insulin ratio>4.5 mg$mL/mIU$dL (36 mmol/mmol) (13). The lean con-trol group consisted of normally cycling women with normalweight and normal T levels (as described in the following).
Polycystic ovary syndrome was defined by oligomenorrhea(%8 menstrual periods in the preceding year) or confirmed an-ovulation, and hyperandrogenemia (serum total T >75 ng/dL[2.6 nmol/L] and calculated free T >1.4 ng/dL [50 pmol/L]).These T normal values were determined by the clinical labora-tory of the Centre hospitalier universitaire de Sherbrooke(CHUS). All participants were between 18 and 40 years ofage and had normal serum PRL, thyroid-function tests, andglucose tolerance after a 75-g OGTT. Late-onset adrenal hyper-plasia was excluded by a serum 17a-OHP of <3.0 mg/L (10nmol/L). None of the participants ever used insulin-sensitizingdrugs, and none were using oral contraceptives or medicationsthat may affect insulin sensitivity. The study protocol was ap-proved by the institutional review board of CHUS, and eachwoman gave written informed consent.
Experimental Protocol
The PCOS women were studied at least 35 days after theirlast menstrual period to reduce the possibility that a spontane-ous ovulation occurred during the last month, as this maytransiently normalize many features of the syndrome.Healthy control women were studied during the midfollicularphase of the menstrual cycle (days 5–10), which most closelymimics the hormonal milieu of anovulatory women withPCOS. Subjects were instructed to use nonhormonal contra-ceptive methods throughout the period of the study and toremain fasting after 8:00 p.m. the night before each visit.
For the first study visit, subjects from the two groups cameto the Metabolic Unit of our Clinical Research Center (CRC)in the morning and weight, height, waist-to-hip ratio (WHR),and supine blood pressure were measured, as well as leanbody mass by standing electrical bioimpedance (Tanitaweight scale model TBF-300A). Then, fasting blood andurine samples were drawn. At 9:00 a.m. an OGTT was per-formed by administrating 75 g dextrose orally and collectingblood samples for determination of serum glucose and insulinat 0, 15, 30, 45, 60, 90, and 120 min. Subjects were instructedon following a 300 g/day carbohydrate diet in preparation forthe insulin-glucose clamp. At least 2 days later, after a 12-hour overnight fast, an euglycemic-hyperinsulinemic clampwas performed as described by DeFronzo et al. (14) (insulindose of 40 mU/m2$min). From this clamp, insulin-stimulatedglucose disposal (M-value) was calculated as follows: glu-cose infusion rate during the last 30 minutes of the clamp(mmol/min) divided by the weight of the subject (kg). Be-cause sensitivity to insulin may differ for each of its actions,it is important to note that insulin-stimulated glucose disposalis a measure of metabolic insulin sensitivity.
The following procedures were performed only in the PCOSgroup. Twenty days after the euglycemic-hyperinsulinemic
887
clamp, the PCOS women began the diazoxide treatment ata dose of 100 mg every 8 hours for 8 days. This duration oftherapy was chosen because it was found sufficient to de-crease T levels in obese PCOS women by Nestler JE et al.(personal communication) and because the incidence ofside effects with diazoxide, essentially swelling, was foundto be higher in our lean population. At the end of this treat-ment period, the PCOS women were admitted to the Meta-bolic Unit of the CRC to collect fasting blood samples forthe determination of plasma insulin, glucose, C-peptide,FSH, LH, sex steroids, and SHBG. The participants then re-ceived an IM injection of the long-acting GnRH agonist leu-prolide acetate (3.75 mg) and were instructed to return to theCRC 28 days later. This dose and such delay were found toappropriately suppress LH levels and decrease basal T levelsin obese PCOS women (5). During this last study visit, thesame fasting blood tests were repeated and the weight andwaist-to-hip ratio were measured again. C-Peptide was as-sayed a posteriori on all frozen samples that were still avail-able, i.e., in 14 control and 7 lean PCOS women at baselineand 6 lean PCOS women during interventions.
Assays
Blood samples were assayed at the biochemistry core labora-tory of CHUS, except for fasting C-peptide which wasassayed in the laboratory of Dr. Baillargeon. Total T, andros-tenedione, and 17a-OHP levels were assayed by radioimmu-noassay (RIA; Diagnostic Systems Laboratories, Webster,TX). The SHBG was assayed by immunoradiometric assay,and DHEAS and insulin by RIA (Diagnostic ProductsCorp., Los Angeles, CA). Serum free T was calculated bythe method of Sodergard et al. (15) using a serum albuminconcentration of 4.0 g/dL (40 g/L). Estradiol, P, FSH, LH,TSH, and PRL were measured by chemiluminescent immu-noassay on an automated ADVIA Centaur analyzer (BayerHealthCare, Toronto, Canada). Glucose, total cholesterol,triglycerides, and high-density lipoprotein–associatedcholesterol (HDL-C) were measured by chemiluminescentimmunoassay on an automated Vitros analyzer (Ortho-Clin-ical Diagnostics, Missisauga, Canada). Low-density lipopro-tein–associated cholesterol (LDL-C) was calculated usingthe Friedewald equation (16). Fibrinogen was assayed bythe modified Clauss technique using BCS kits (Behring Co-agulation System, Newark, DE). Plasminogen activatorinhibitor 1 (PAI-1) was determined by ELISA (DiagnosticaStago, Asnieres-sur-Seine, France). C-Peptide was measuredby RIA (Linco Research, St. Charles, MO). Inter- and intra-assay coefficients of variation were less than 7.5% for insulin,less than 10% for C-peptide and total T, and less than 8.5%for other steroid hormones. The lower limit of detectionwas 4 mIU/mL (30 pmol/L) for insulin.
Statistical Analysis
Results not normally distributed were log transformed to nor-malize their distribution for statistical analysis and are re-ported back-transformed in their original units (geometric
888 Baillargeon and Carpentier Role of insulin in PCOS hy
mean with 95% confidence intervals). Other results are re-ported as mean� SEM. Unpaired t tests were used at baselineto compare variables between the two groups. Diazoxide andleuprolide acetate treatment effects were assessed usingpaired t tests to compare variables after intervention to base-line within the two PCOS groups.
Two-tailed P values of %.05 were considered to be signif-icant for all analyses, which were performed using JMP 4.0software (SAS Institute, Cary, NC). Even if the number ofsubjects was relatively small in each group, paired analysesincreased significantly the power of the study. This methodalso decreases the variability of differences, because eachsubject is compared to herself, which factors out interindivid-ual variability.
RESULTS
Clinical Characteristics of the Subjects (Table 1)
We studied 17 lean normal control subjects and 9 lean PCOSwomen with normal insulin levels. Our selected group of leanwomen with PCOS were younger than lean control subjects(P¼.02) but had comparable BMI, WHR, and percentageof fat mass. The BMI and WHR did not change significantlyin the lean PCOS group throughout the study. Finally, systolicand diastolic blood pressures also were similar betweengroups.
Metabolic Measures and Insulin Sensitivity
At baseline, fasting insulin (Table 2) and glucose (Fig. 1)levels were comparable between lean PCOS women and con-trol subjects (P¼.78 and P¼.45, respectively). Fasting levelsof triglycerides, HDL-C, LDL-C, fibrinogen, and PAI-1, aswell as the total cholesterol–to–HDL-C ratio, also were allcomparable between groups.
Metabolic insulin sensitivity (M-value), directly measuredby the euglycemic-hyperinsulinemic clamp, was 52.9 mmol/kg$min in lean normal women, with 95% of the results rang-ing between 24.9 mmol/kg$min and 105.9 mmol/kg$min. Themean M-value of lean women with PCOS and normal insulinlevels (mean 48.5 mmol/kg$min) was very close to that ofcontrol women, and all M-values (range 29.5–73.3 mmol/kg$min) in lean PCOS women were within the 95% intervalrange of controls.
Effects of Reduction of Insulin or LH Secretion(Table 2 and Fig. 1)
Fasting insulin levels did not change significantly with inter-ventions in lean PCOS women (Table 2). However, fasting C-peptide levels reduced significantly after diazoxide in leanPCOS women (P¼.02) but not after leuprolide (P¼.36).The FSH and 17b-E2 levels were comparable between thetwo groups at baseline and were not affected by diazoxide,but they were significantly reduced by leuprolide acetate inthe lean PCOS group (P¼.003 and P¼.001, respectively).However, progesterone levels, which were comparable
perandrogenemia Vol. 88, No. 4, October 2007
TABLE 1Baseline clinical characteristics and metabolic measurements.
CharacteristicControl, lean
(n [ 17)PCOS, lean
(n [ 9) P value
Age (yr) 31.3 � 1.6 24.3 � 2.2 .02BMI (kg/m2)
Baseline 22.0 � 0.5 22.6 � 0.6 .45End of study 22.2 � 0.5
Waist-to-hip ratioBaseline 0.77 � 0.01 0.79 � 0.01 .38End of study 0.78 � 0.02
Note: Plus-minus values are mean � SEM. BMI¼ body mass index; BP¼ blood pressure; HDL-C ¼ high-density lipopro-tein–associated cholesterol; LDL-C¼ low-density lipoprotein–associated cholesterol; Chol./HDL-C ratio¼ total choles-terol–to–HDL-C ratio; PAI-1 ¼ plasminogen activator inhibitor 1 (by immunoenzymatic assay). To convert values fortriglycerides to mmol/L, multiply by 0.0259; and for cholesterol to mmol/L, multiply by 0.0113.
a Geometric means with 95% confidence interval.
Baillargeon. Role of insulin in PCOS hyperandrogenemia. Fertil Steril 2007.
between groups at baseline, did not change with diazoxide orleuprolide. The 17a-OHP and DHEAS levels were signifi-cantly higher in lean PCOS women than in control subjectsat baseline (P¼.02 and P<.001, respectively) and were notsignificantly altered by treatment with diazoxide or leupro-lide acetate.
At baseline, total T levels were significantly higher in leanPCOS women than in control women (P<.001), as expected(Fig. 1). Total T levels decreased after both diazoxide andleuprolide acetate in PCOS women, but these effects werenot significant (P¼.18 and P¼.14, respectively). TheSHBG concentrations were slightly lower in lean PCOSwomen at baseline, although this was not significant(P¼.06). Treatment with diazoxide increased significantlythe levels of SHBG in lean PCOS women (P¼.04), butthey were reduced back to baseline levels after leuprolide ac-etate (P¼.78 vs. baseline; P¼.02 vs. diazoxide). At baseline,free T was significantly higher in lean PCOS women than incontrol women (P<.001), and treatment with diazoxide sig-nificantly normalized free T levels in lean PCOS women(P¼.03). However, treatment with leuprolide acetate led to el-evation of free T back to baseline levels (P¼.57 vs. baseline).
At baseline, androstenedione levels were significantlyhigher in lean PCOS women than in control women(P<.001). In this group, androstenedione levels significantly
Fertility and Sterility�
decreased after diazoxide (P¼.05) and decreased further afterleuprolide acetate (P¼.002 vs. baseline; P¼.002 vs. diazo-xide treatment), to levels within the normal range. At base-line, LH levels were significantly higher in lean PCOSwomen than in control women (P<.001). They tended to de-crease after diazoxide (P¼.17) and were almost completelysuppressed after leuprolide acetate (P<.001 vs. baseline;P<.001 vs. diazoxide). Finally, treatment with diazoxidetended to increase fasting glucose levels in lean PCOSwomen compared with baseline, though not significantly(P¼.06). Fasting glucose did not change after treatmentwith leuprolide acetate (P¼.84).
DISCUSSION
This study assessed for the first time the in vivo effect of in-sulin in lean PCOS women with normal metabolic insulinsensitivity and insulin levels. The results demonstrated thatreduction of insulin secretion with diazoxide in these womensignificantly decreased levels of free T and androstenedioneand significantly increased SHBG (Fig. 1). Importantly, ithas been earlier determined that suppression of serum insulinsecretion by diazoxide does not alter serum T or SHBG levelsin healthy nonobese women (11). The significant improve-ment of free T and SHBG levels observed after treatmentwith diazoxide were in contrast to the absence of change
889
TABLE 2Laboratory results at baseline, after diazoxide, and after leuprolide acetate in lean control (n [ 17) andlean PCOS women with normal insulin levels (n [ 9).
Characteristic Group Baseline After diazoxide Pa After leuprolide Pa
Fasting insulin(mU/mL)b
Control,lean
5.8 (4.3–7.5)
PCOS,lean
6.1 (4.2–9.0) 5.8 (4.6–7.1) .60 5.8 (4.3–7.8) .68
Fasting C-peptide(ng/mL)b
Control,lean
3.4 (2.6–4.4)(n ¼ 14)
PCOS,lean
3.0 (2.1–4.3)(n ¼ 7)
2.0 (1.5–2.8)(n ¼ 6)
.02 2.5 (2.1–3.0)(n ¼ 6)
.36
FSH (IU/L) Control,lean
4.2 � 0.3
PCOS,lean
4.7 � 0.5 4.1 � 0.5 .24 2.8 � 0.4d .003
17b-E2 (pg/mL) Control,lean
115 � 22
PCOS,lean
65 � 30 91 � 27 .33 18 � 2d .001
Progesterone(ng/mL)
Control,lean
0.6 � 0.8
PCOS,lean
3.7 � 1.2 4.5 � 1.3 .71 1.2 � 0.1d .26
17-OHP (mg/L) Control,lean
1.7 � 0.3(n ¼ 13)
PCOS,lean
2.9 � 0.4c 2.5 � 0.3 .48 1.9 � 0.2 .10
DHEAS (mg/dL) Control,lean
130 � 15
PCOS,lean
315 � 19c 326 � 22 .53 367 � 44 .29
Note: Plus-minus values are mean� SEM. To convert values for insulin to pmol/L, multiply by 6.9; for C-peptide to nmol/L,multiply by 0.333; for 17b-E2 to pmol/L, multiply by 3.671; for progesterone to pmol/L, multiply by 3.18; for 17-OHP tonmol/L, multiply by 3.026; and for DHEAS to mmol/L, multiply by 0.027. The normal ranges for ovulatory women are asfollows: insulin 5–20 mIU/mL; FSH 1.5 to 7.6 mIU/mL; 17b-E2 19 to 247 pg/mL; progesterone 0.2 to 1.4 ng/mL; 17-OHP0.3 to 1.0 mg/L; and DHEAS 40 to 340 mg/dL.
a P value vs. baseline (by two-tailed paired t test).b Geometric mean with 95% confidence interval.c P%.05 vs. control at baseline (by two-tailed unpaired t test).d P%.05 vs. diazoxide (by two-tailed paired t test).
Baillargeon. Role of insulin in PCOS hyperandrogenemia. Fertil Steril 2007.
observed after treatment with the long-acting GnRH agonistleuprolide acetate, despite near total suppression of LHlevels.
The present results underscore the preponderant role ofinsulin action over LH on the hyperandrogenemia of nor-moinsulinemic lean PCOS women. Furthermore, these re-sults are not solely explained by increased SHBG levelsin the PCOS women, because insulin reduction with diazo-xide also significantly decreased androstenedione levels,which is not bound to SHBG. Based on our results, it ap-pears that LH suppression is more effective than insulin
890 Baillargeon and Carpentier Role of insulin in PCOS hy
reduction to decrease androgen biosynthesis, as assessedby androstenedione levels; but insulin lowering is more ef-fective to reduce hyperandrogenemia, as assessed by free Tlevels, because it improves both androgenesis and SHBGlevels.
We succeeded in recruiting nine women with typicalPCOS, normal insulin levels during both fasting and afteran OGTT, and normal sensitivity to insulin-stimulated glu-cose metabolism, as assessed by the euglycemic-hyperinsuli-nemic clamp technique. Indeed, the M-values of these leanPCOS women were all within the 95% interval range of the
perandrogenemia Vol. 88, No. 4, October 2007
FIGURE 1
Selected laboratory results at baseline and after treatment with diazoxide and leuprolide acetate in lean control(solid bars; n ¼ 17) and lean PCOS (clear bars; n ¼ 9) women. Results are presented as mean with SEM. Toconvert values for total T to nmol/L, multiply by 0.0347; for SHBG to nmol/L, multiply by 34.7; for free T to pmol/L,multiply by 34.7; and for androstenedione to nmol/L, multiply by 3.492; and for glucose to mmol/L, multiply by0.0556. Free T was calculated by the method of Sodergard et al. (15). The normal ranges for ovulatory women are:total T, 20–80 ng/dL; SHBG, 0.6–3.0 mg/dL; free T,<1.4 ng/dL; androstenedione, 0.7–3.1 mg/L; LH, 0.5–9.8; andfasting glucose, 60–110 mg/dL. *P%.05 vs. control at baseline (by two-tailed paired t test); yP%.05 vs. baseline(by two-tailed paired t-test); zP%.05 vs. diazoxide (by two-tailed paired t test).
Baillargeon. Role of insulin in PCOS hyperandrogenemia. Fertil Steril 2007.
17 lean healthy controls (Table 1) and even within or abovethe normal range for lean normal women published by Dunaifat al. (whole range 29.1–46.8 mmol/kg$min; n¼ 8) (17). Ad-justing M-value for fat-free mass instead of weight in thepresent population resulted in the same conclusion (PCOS:
Fertility and Sterility�
66.2 mmol/kg$min [range 36.2–98.0]; control: 71.6 mmol/kg$min [range 37.4–147.1]). Furthermore, the present leanPCOS group was characterized by normal anthropometricand metabolic parameters which were comparable to thosein the lean healthy control group (Table 1).
891
Fasting insulin levels were only slightly reduced by diazo-xide in the present study. This finding is consistent with re-sults from Nestler et al. (18), who did not find significantchange of fasting insulin levels in obese PCOS women after10 days of diazoxide but demonstrated significantly bluntedinsulin response to glucose during OGTT. However, diazo-xide significantly reduced fasting C-peptide levels in thepresent group of PCOS women, demonstrating that insulinsecretion was indeed reduced by this intervention. C-Peptideis cosecreted with insulin, minimally extracted by the liver,and excreted mainly unchanged in urine. Therefore, multiplestudies have determined that it is a better index of insulin se-cretion than fasting insulin (19, 20). Furthermore, fasting glu-cose levels were increased with diazoxide, and maintained inthe normal range, which is consistent with clinically signifi-cant insulin reduction and with earlier findings (18, 21).
The lack of suppression of total and free T levels aftertreatment with a GnRH agonist in the present study, despitenear suppression of LH, was in contrast to previous studies(5, 22, 23). This discrepancy might be explained by shortertreatment period (4 weeks) as compared with Dunaif et al.(22) and Lasco et al. (23) (12 weeks). However, althoughGilling-Smith et al. (5) found some reduction of T levels af-ter 4 weeks, androgen responses to hCG stimulation werenot decreased. Another important difference is the selectionof predominantly obese women in those earlier studies (5,22, 23). It is therefore possible that GnRH contribution tohyperandrogenemia differs in lean insulin-sensitive PCOSwomen. Even if such a conclusion cannot be ascertainedin the present study, the post-GnRH results are importantto conclude with certitude that the improvement of andro-gen levels after diazoxide is not explained by the small de-crease in LH levels (Fig. 1). Finally, because the duration ofaction of diazoxide is short (8 hours) and was used for only8 days, we do not believe that it interfered with leuprolideaction.
A possible weakness of the present study is the small numberof lean insulin-sensitive PCOS women. This drawback reducesthe power of the study to determine the absence of differencebetween groups or after versus before treatments. However,we have used robust nonparametric statistical tests that are in-dependent from the number of subjects to show statistically sig-nificant differences. Thus, significant results are internallyvalid even if the number of subjects is relatively small.
In summary, the present results suggest that some womenwith typical PCOS are insulin sensitive to glucose metabo-lism and normoinsulinemic and that insulin contributessignificantly to the hyperandrogenemia of these women.Therefore, women with PCOS might develop hyperandroge-nemia because of increased sensitivity of their androgenicpathway to insulin. Because SHBG was also significantlyaffected by diazoxide-induced insulin reduction in thesewomen, it is probable that insulin actions on liver productionof SHBG are also increased. A pituitary effect is not excludedeither, because LH tended to decrease after treatment withdiazoxide.
892 Baillargeon and Carpentier Role of insulin in PCOS
Therefore, we hypothesize that women develop PCOS inpart because of a selective and tissue-specific hypersensitiv-ity to insulin (24). In a minority of women, this defect issufficiently severe to cause typical PCOS without insulin re-sistance. However, it is important to recognize that in mostwomen with PCOS the severity of this androgenic hypersen-sitivity to insulin is such that concomitant development ofinsulin resistance and hyperinsulinemia is necessary forphenotypic expression of the syndrome.
Our hypothesis probably explains the results of our previ-ous clinical trial (10). Subjects in that trial were recruited us-ing the same inclusion criteria as in the present study, whichhighly suggests that they were normally insulin sensitive aswell (for glucose metabolism). Even if insulin sensitive, thosePCOS women significantly improved T levels and ovulationafter treatment with insulin-sensitizing drugs metformin orrosiglitazone. Metformin, but not rosiglitazone, significantlyreduced insulin levels during that trial. We therefore proposethat metformin decreased hyperandrogenemia mainly by re-ducing insulin levels, as did diazoxide in the present study,and that rosiglitazone may have been beneficial by directlyimproving the androgenic hypersensitivity to insulin.
A recent study (25) highlighted a selective defect in insulinactivity in granulosa cells from women with PCOS, i.e., resis-tance in the metabolic pathway associated with an increase inmitogenic activity. Moreover, this study demonstrated thattroglitazone, a PPARg agonist, can correct this insulin hyper-sensitivity of the mitogenic pathway along with a significantimprovement of the insulin-resistant metabolic pathway.Therefore, these results support the hypothesis that PPARg
may directly improve the androgenic hypersensitivity to insu-lin in PCOS. The observation that insulin-signaling pathwaysmay express differential, and even divergent, activity levelsunder various circumstances also supports the possibility ofa selective defect of the insulin androgenic pathway inwomen with PCOS.
In conclusion, reducing insulin secretion in women withtypical PCOS, normal insulin levels and normal metabolic in-sulin sensitivity significantly decreased androgen and signifi-cantly increased SHBG levels. These results suggest thatinsulin contributes to hyperandrogenemia even in PCOSwomen with normal metabolic insulin sensitivity, which maybe due to increased sensitivity of their androgenic insulin path-way. Liver insulin pathways may also be implicated throughreduced secretion of SHBG. The characterization of thispotential defect could have important implications for the de-velopment of specific and more effective treatments for PCOS.
Acknowledgments: The authors thank John E. Nestler, M.D., Professor and
Chair, Division of Endocrinology and Metabolism, Medical College of
Virginia, Virginia Commonwealth University, for his critical review of the
20. Faber OK, Binder C. C-Peptide: an index of insulin secretion. Diabetes
Metab Rev 1986;2:331–45.
21. Nestler JE, Barlascini CO, Matt DW, Steingold KA, Plymate SR,
Clore JN, et al. Suppression of serum insulin by diazoxide reduces serum
testosterone levels in obese women with polycystic ovary syndrome.
J Clin Endocrinol Metab 1989;68:1027–32.
22. Dunaif A, Green G, Futterweit W, Dobrjansky A. Suppression of hyper-
androgenism does not improve peripheral or hepatic insulin resistance in
the polycystic ovary syndrome. J Clin Endocrinol Metab 1990;70:
699–704.
23. Lasco A, Cucinotta D, Gigante A, Denuzzo G, Pedulla M, Trifiletti A,
et al. No changes of peripheral insulin resistance in polycystic ovary syn-
drome after long-term reduction of endogenous androgens with leupro-
lide. Eur J Endocrinol 1995;133:718–22.
24. Baillargeon JP, Nestler JE. Polycystic ovary syndrome: a syndrome of
ovarian hypersensitivity to insulin? J Clin Endocrinol Metab 2006;91:
22–4.
25. Wu XK, Zhou SY, Liu JX, Pollanen P, Sallinen K, Makinen M, et al.
Selective ovary resistance to insulin signaling in women with polycystic
ovary syndrome. Fertil Steril 2003;80:954–65.
893
Assessment of three-dimensional sonographicfeatures of polycystic ovaries after laparoscopicovarian electrocauteryMiklos Vizer, M.D.,a Ludwig Kiesel, M.D.,b Istvan Szabo, M.D., D.Sc.,a Antal Arany, M.D.,a
P�eter Tamas, M.D., Ph.D.,a and Andras Szilagyi, M.D., Ph.D.a
a Department of Obstetrics and Gynecology, Health Science Center, University of P�ecs, P�ecs, Hungary; and b Department
of Gynecology and Obstetrics, University of Munster, Munster, Germany
Background: Treatment of polycystic ovary syndrome–related infertility includes laparoscopic ovarian electrocau-tery. Three-dimensional (3-D) sonographic characterization of polycystic ovaries has been performed recently,including the study of the effect of laparoscopic ovarian drilling on ovarian volume. The impact of laparoscopictreatment on ovarian volume and vascular flow-patterns assessed by 3-D color power angiography (CPA), however,has not yet been elucidated.Objective: To measure ovarian volume, to evaluate and quantify intraovarian blood flow with 3-D CPA histogramanalysis before and after laparoscopic ovarian electrocautery, and to compare the hormonal effects of surgery with3-D sonographic findings.Setting: University hospital.Patient(s): Ten patients, aged 18–34 years, with polycystic ovary syndrome.Intervention(s): Evaluation of serum and urinary hormone profiles as well as transvaginal 3-D ultrasonographywere performed before and after laparoscopic ovarian surgery.Result(s): Ovarian volume decreased, and 3-D CPA showed increased intraovarian flow intensity after laparo-scopic electrocautery. Serum LH and T levels, ratios of urinary steroids reflecting 5a-reductase enzyme activity,and androgen to cortisol metabolites decreased; serum FSH levels increased 1 week after laparoscopy and corre-lated well with changes of 3-D sonographic features. Seven patients ovulated regularly after surgery, and five preg-nancies were conceived within 1 year.Conclusion(s): Three-dimensional ultrasonography may be a useful adjunct and noninvasive method for correlat-ing clinical parameters with the blood flow alterations in polycystic ovary syndrome patients. (Fertil Steril�
2007;88:894–9. �2007 by American Society for Reproductive Medicine.)
Polycystic ovary syndrome (PCOS) is a heterogeneous en-docrinopathy affecting women of reproductive age. It isone of the most common causes of anovulatory infertility(1). The syndrome is characterized by PCO morphologyand by clinical and biochemical features of hyperandro-genism and chronic anovulation (2). The pathophysiologyof PCOS appears to be both multifactorial and polygenic(2–6).
Polycystic ovary syndrome is well defined clinically, bio-chemically, and sonographically. Key features include men-strual cycle disturbance, hirsutism, obesity, infertility, andbiochemical evidence of hyperandrogenemic anovulation.At a recent American Society of Reproductive Medici-ne–European Society of Human Reproduction and Em-bryology consensus meeting (7), a refined ultrasonographicdefinition of PCOS was agreed upon: either the presence of
Received June 22, 2005; revised and accepted August 27, 2006.
Reprint requests: Miklos Vizer, M.D., Department of Obstetrics and Gyne-
cology, Health Science Center, University of P�ecs, 17 Edesanyak
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
894
R12 follicles measuring 2–9 mm in diameter or the presenceof increased ovarian volume (>10 cm3) at a time of ovarianquiescence. The presence of a single PCO is sufficient forthe diagnosis; however, the distribution of follicles and a de-scription of the stromal echogenicity are not required. In con-trast, a woman with PCO in the absence of anovulation orhyperandrogenism should not be considered to have PCOS(7, 8).
There is no effective single strategy for the treatment ofPCOS. For the treatment of infertility associated withPCOS, ovulation induction with clomiphene citrate and/or in-sulin sensitizers (9) appears to be the simpliest method. In theevent of clomiphene citrate failures, the next step may bestimulation of the ovaries by exogenous gonadotropins,according to the conventional step-up regimen (10), or maybe surgical manipulation of the ovaries. Surgical treatmentby laparoscopic approaches (unipolar electrocautery, laserphotodiathermy, or laser drilling) was found to be effectivein restoring ovulation for R6 months, but the benefit ofmonopolar current may last for several years (11, 12). Asa consequence of profound changes in gonadotropin secre-tion in response to ovarian surgery, resumption of normal
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.08.117
menstrual cyclicity with subsequent ovulation has beenobserved (13, 14).
As Findlay (15) postulated in 1986, vascular and flow pat-terns of angiogenesis in reproductive tissues clearly representmorphology and function. Thus, alterations in ovarian vascu-larity and perfusion may correlate with changes in consecu-tive cyclic events involving follicular growth and hormoneproduction.
The availability of endovaginal power Doppler–colorpower angiography (CPA) has made the study of pelvic organperfusion possible. This technique uses the amplitude com-ponent of the Doppler shift. Frequency filtering is not needed;therefore, the sensitivity of CPA to low-flow signals is high, isfree from aliasing, and is angle independent, although CPAcannot give information on the direction or velocity of flow(16, 17).
Three-dimensional (3-D) sonographic characterization(volume, stromal vascularity, and flow) of PCOs have beenperformed recently (18–27), including the study of the effectof laparoscopic ovarian drilling on ovarian volume (28). Theimpact of laparoscopic treatment on both ovarian volume andvascular flow patterns assessed by 3-D CPA, however, havenot yet been elucidated, although the effect of laparoscopicovarian diathermy on serum and urinary hormone levels,and on resumption of menstrual cyclicity and pregnancyrates, has already been thoroughly investigated and reported(11–14, 29).
The aim of the present study was to measure ovarian vol-ume, to evaluate and quantify intraovarian blood flow with3-D CPA histogram analysis before and after laparoscopicovarian electrocautery, and to compare the hormonal (serumand urinary) and clinical effects of laparoscopic electrocau-tery with 3-D sonographic findings. The rationale and thenovelty of the study is that the mechanism of action of ovar-ian diathermy on resumption of fertility is still not completelyclarified. The data on ovarian volume and blood flow changesafter ovarian diathermy as measured by 3-D sonographycould contribute greatly to our knowledge on ovarian physi-ology and could help understanding about the resumption ofmenstrual cyclicity and fertility after the commonly usedovarian surgery.
MATERIALS AND METHODS
Subjects
The study was approved by the local ethics committee of themedical school. Patients provided written informed consent.Ten infertile patients (age range, 18–34 y) with PCOS wererecruited from the infertility clinic of University of P�ecs.The diagnosis of PCOS was based on sonographic appear-ance of the ovaries, clinical signs of chronic hyperandrogen-ism (oligo-amenorrhea, hirsutism, and infertility), andcharacteristic endocrinological features of hyperandrogene-mia according to the Rotterdam consensus of 2003 (7). Allpatients had failed to ovulate in response to clomiphene cit-
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rate (100 mg/d for 5 d per week) that had been administeredfor 3 months and were counseled to proceed to laparoscopicovarian electrocautery.
The laparoscopic ovarian electrocautery was applied onboth ovaries (40W monopolar current), and 15–20 cauteriza-tion points were performed at a depth of 5–7 mm. The samesurgeon (A.S.) performed all laparoscopic surgeries.
Serum LH, FSH, T, and E2 levels were determined withcommercially available kits before and after surgery. To eval-uate 5a-reductase enzyme activity in the skin and liver, spe-cific ratios of androsterone to etiocholanolone (An-Et) and of5a-tetrahydrocortisol to tetrahydrocortisol (aTHF-THF)were calculated from 24-hour urine samples by column gaschromatography according to the methods of Shackleton(30) and Homoki et al. (31), before and after laparoscopicelectrocautery. Changes in the ratio of androgen metabolitesto cortisol metabolites (AM-CM) before and after surgerywere also evaluated (AM measured were the following: An,Et, 11-keto-An, 11-hydroxy-An, 11-hydroxy-Et, DHEA,16a-hydroxy-DHEA, 16b-hydroxy DHEA, androsten-3b,16a,17b-triol, and pregnentriol-3b-hydroxy-5-en; andCM measured were tetrahydrocortisone, THF, and aTHF).
Three-Dimensional CPA and Histogram Analysisof Intraovarian Circulation
Examinations were performed with a Kretz Voluson 730D ul-trasound scanner (Kretztechnik, Zipf, Austria) equipped witha transvaginal 3- to 7-MHz volume transducer (100� field ofview). In all patients, identical preinstalled instrument set-tings were used. After visualizing the ovary in 2-D B-mode, the mobile sector for angio mode was switched onand set up to cover the region of interest, then the 3-D volumemode was engaged. The volume sector angle was preset to90�, and the fast volume acquisition setting was selected toavoid motion artifacts.
The acquired 3-D volumes were stored immediately inthe ultrasound machine. All the stored volumes were ana-lyzed later by the same observer (M.V.) by using the VO-CAL imaging program (Virtual Organ Computer-aidedAnaLysis), which is integrated into the Voluson 730D ultra-sound system. The contour mode in the VOCAL programwas set to manual. The longitudinal view was used as a ref-erence image, and the rotation step was selected as 30�, re-sulting in the definition of six contours for each ovary. Oncea contour was defined in all image planes, the volume of theovary was obtained.
After definition of the contour, the VOCAL program auto-matically calculates indices for gray-scale and color-scalevoxels (voxel¼ smallest unit of volume). According to thesevalues, four indices are calculated: the vascularization index(VI), flow index (FI), vascularization flow index (VFI), andmean grayness. Vascularization index is thought to representthe presence of blood vessels in the tissue region of interest(vascularization) and is expressed as a percentage value.
895
Flow index (scale, 0–100), the mean value of the color voxels,is thought to express the average intensity of flow in the ves-sels. Vascularization flow index measures the ratio of themean values of color voxels and all the voxels in the definedcontour and is a feature of both vascularization and flow (32).Mean grayness (scale, 0–100) expresses the gray-scalebrightness or echogenicity of the tissue.
Three-dimensional CPA and histogram analysis of intrao-varian circulation was performed in all patients before lapa-roscopic electrocautery as well as at 1 week and at 1 and 4months after the procedure (if menstrual cycle had resumed,this was performed on cycle day 3). All quantitative measure-ments were performed by the same author (M.V.). Dopplersettings were not changed during examination. The regionof interest covered the whole ovary. Ovaries were examinedbilaterally, and the average value from both sides was usedas a single parameter.
For statistical analysis of the ovarian volume and flowparameters, Student’s t-test was applied, and data were ex-pressed as mean � SD. Serum and urinary hormone valuesare represented as mean� SEM. Because normal distributionof the data could not be assumed, the nonparametric tests forpaired (Wilcoxon test) and unpaired (Mann-Whitney U-test)samples were used. P values of < .05 were considered to bestatistically significant.
Clinical outcome was also evaluated in terms of resump-tion of menstrual cyclicity, ovulation, and pregnancy ratesduring a 1-year follow up.
RESULTS
The mean (� SD) body mass index of the patients was 27.0�3.1 kg/m2. All patients had normal serum PRL levels and hadnormal thyroid and adrenal function (data are not shown). Se-rum LH and T levels decreased significantly 1 week after lap-aroscopy (from 9.9� 2.1 IU/L to 7.9� 1.8 IU/L and from 3.4� 0.7 nmol/L to 2.8 � 0.6 nmol/L, respectively, P<.05; Fig.1). Both serum LH and T concentrations remained low at 1and 4 months after ovarian surgery.
Specific ratios of urinary steroids reflecting 5a-reductaseenzyme activity significantly decreased at 1 week afterovarian drilling (An-Et range, 1.7 � 0.7 to 1.4 � 0.6;aTHF-THF range, 0.8 � 0.2 to 0.6 � 0.1; P<.05), as didthe PCOS-characteristic elevated AM-CM ratio (AM-CMrange, 1.0 � 0.3 to 0.8 � 0.2, P<.05). Nevertheless, theAn-Et and AM-CM ratios still remained considerablyhigher than those in healthy females. Serum FSH levelsincreased significantly 7 days after laparoscopic ovarianelectrocautery (from 4.7 � 1.5 IU/L to 12.9 � 2.7 IU/L,P<.05; Fig. 1) and remained high 1 and 4 months after sur-gical treatment.
Seven (70%) of 10 women had documented biochemicalevidence of ovulation (serum P, >20 nmol/L on cycle day21) and clinical resumption of menstruation within the next
896 Vizer et al. Three-dimensional sonography in PCOS
4 months after laparoscopic surgery. Five pregnancies wereconceived within 1 year after ovarian diathermy.
Mean ovarian volume was significantly decreased alreadyat 1 week after surgery (from 13.436 � 2.874 cm3 to 10.164� 2.013 cm3, P<.05; Fig. 2). We detected a mild continuousincrease of VI throughout the 4-month follow-up period afterlaparoscopy, but it did not reach statistical significance (meanVI before and 4 mo after surgery: 0.412% � 0.226% and0.787% � 0.398%, respectively, P>.05; Fig. 3). Both FI
FIGURE 1
Serum FSH, LH, and T levels (mean � SEM) beforeand after laparoscopic ovarian electrocautery(n¼ 10). Red bars, FSH (IU/L); cream bars, LH (IU/L);yellow bars, T (nmol/L).
Vizer. Three-dimensional sonography in PCOS. Fertil Steril 2007.
FIGURE 2
Ovarian volume (in mL; mean � SD) before and afterlaparoscopic ovarian electrocautery (n ¼ 10).
Vizer. Three-dimensional sonography in PCOS. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
and VFI increased significantly 7 days after surgical interven-tion (mean FI before and 1 wk after electrocautery: 14.249�2.357 and 18.248 � 2.811, respectively, P<.05; mean VFIbefore and 1 wk after electrocautery: 0.114 � 0.046 and0.207� 0.048, respectively, P<.05). The difference betweenmean initial and 4-month FI and VFI values were even morepronounced (Fig. 4).
Serum LH, T concentrations, and specific ratios of uri-nary steroids, reflecting 5a-reductase enzyme activity,have changed in parallel with ovarian volume and havechanged inversely with FI and VFI values after laparoscopicelectrocautery. In contrast, serum FSH levels and the sub-sequent chance for resumption of menstrual cyclicity within4 months have changed in parallel with ovarian FI and VFIvalues after surgery. An inverse correlation was found
FIGURE 3
Vascularization index (%; mean � SD), before andafter laparoscopic ovarian electrocautery (n ¼ 10).NS ¼ not significant (P>.05).
Vizer. Three-dimensional sonography in PCOS. Fertil Steril 2007.
FIGURE 4
Flow index (cream bars) and VFI (red bars; range forboth indices, 0–100; mean � SD) before and afterlaparoscopic ovarian electrocautery.
Vizer. Three-dimensional sonography in PCOS. Fertil Steril 2007.
Fertility and Sterility�
between serum FSH levels and ovarian volume afterlaparoscopy.
DISCUSSION
To our best knowledge, this is the first report on the study ofthe effects and correlation of laparoscopic ovarian electro-cautery on serum gonadotropin, androgen, and urinarysteroid hormone concentrations and on ovarian blood flowand vascularity as assessed by 3-D CPA in patients withPCOS.
Effects of laparoscopic surgical approach for the treatmentof PCOS on serum hormone levels and clinical findings havebeen studied intensively. Laparoscopic surgical methods inclomiphene citrate–resistant patients with PCOS were foundto be effective for restoring fertility (11, 12). A further advan-tage of surgical treatment of PCOS-related infertility is thatstimulation with gonadotropins after ovarian electrocauterygives an enhanced ovarian response compared with the pre-diathermy response (29). However, surgical manipulationsof the ovaries are not really effective in reducing hirsutism(11).
With the introduction of 3-D power Doppler technique,a multiplanar display of the total vascularity and blood flowwithin a tissue region of interest can be studied interactively,and the rendered 3-D image resembles one created by usingangiography. Reports on the feasibility of 3-D CPA for the so-nographic evaluation of PCOs have been performed recently(18–27). The results of these reports all were in agreementthat the volume of ovaries in patients with PCOS wasincreased.
Eight of 10 of these studies demonstrated that the PCOstroma was more vascularized than was the stroma in the nor-mal ovaries. Jarvela et al. (25) could not confirm this phe-nomenon, even if cortical and stromal vascular flow wereexamined separately. In another Jarvela et al. study (26), itwas found that differences observed between dominant andnondominant ovaries appear to disappear after pituitarydown-regulation; however, neither stromal brightness norvascularity of PCOs changed after the administration ofGnRH agonist therapy.
Pan et al. (23) have shown that flow intensity decreasesalong with the aging process in the ovarian stroma. Nardoet al. (27) demonstrated that only total ovarian volume, follic-ular volume, and preantral follicle number correlated posi-tively with serum FSH and LH, but not T, concentrationsand that there was no association between ovarian stromalvolume and serum FSH, LH, or T levels in women withPCOS.
Tulandi et al. (28) studied the effect of laparoscopic ovar-ian drilling of PCOs on the ovarian volume in women withclomiphene citrate–resistant PCOS and observed a transientincrease, followed by a significant reduction, in ovarianvolume 1 week after surgery.
897
In the present study, we measured the ovarian volume,evaluated and quantified ovarian blood flow with 3-D CPAhistogram analysis before and after laparoscopic ovarianelectrocautery, and at the same time compared the hormonal(serum and urinary) effects of laparoscopic electrocauterywith 3-D sonographic findings in patients with PCOS. Weagree with Pan et al. (18) in that quantification of Doppler sig-nals within the entire ovary by 3-D CPA histogram analysisappears to be superior to the conventional 2-D Dopplermethod, although early Doppler ultrasound studies (33, 34)could also demonstrate the increased stromal vascularity inPCOs. Blood flow (FI) and vascularization (VI), as wellas both parameters together (VFI), can be studied only with3-D CPA.
In our study, PCO volume decreased significantly, and 3-DCPA showed increased intraovarian flow intensity after lapa-roscopic ovarian electrocautery. Hormonal changes (serumand urinary) after laparoscopic surgery are in accordancewith changes in 3-D sonographic features; in other words,they may correlate with alterations of measurable 3-D vol-ume and vascular flow data of the PCOs.
Ovarian blood flow parameters, as quantified by the nonin-vasive 3-D CPA histogram analysis, are probably some of themost accurate variables reflecting ovarian function or quies-cence. Decreased ovarian volume is in accordance with thedecreased androgen production, whereas increased ovarianblood flow (FI[, VFI[) and partial neovascularization(VI[) around the developing follicles make ovarian tissue ac-cessible to higher circulating levels of FSH. Subsequently,follicular development is triggered, and resumption of men-strual cyclicity and fertility may be achieved.
The size of the group studied does not allow a correlationto be drawn between the clinical results (ovulation and preg-nancy rates) and the changes in blood flow and neovasculari-zation. Such correlation would require further study.
There were four main outcome measures (serum gonado-tropin and androgen hormone concentrations, as well as ovar-ian volume and 3-D CPA flow) to be compared at fourdifferent time points (before surgery and at 1 wk, 1 mo, and4 mo after surgery). In dealing with this multiple-comparisonsstatistical problem, applying the Bonferroni correction for thealpha level would have been more reliable, but the aim of ourstudy with this small sample size was just to highlight possibletendencies of clinical interest. Furthermore, for us, these mul-tiple comparisons appeared reasonable on their own merit.
On the basis of the present data, it appears to be evidentthat changes in ovarian volume and blood flow parametersafter laparoscopic ovarian electrocauterization contribute toand partly explain hormonal alterations and the consequentclinical results.
In summary, we can conclude that transvaginal 3-D ultra-sonography, equipped with a color Doppler angiography-based, computer-aided histogram analysis option, may bea useful adjunct and a noninvasive method for correlating
898 Vizer et al. Three-dimensional sonography in PCOS
clinical parameters with the blood-flow alterations in patientswith PCOS.
REFERENCES1. Adams J, Polson DW, Franks S. Prevalence of polycystic ovaries in
woman with anovulation and idiopathic hirsutism. Br Med J 1986;293:
355–9.
2. Homburg R. Polycystic ovary syndrome—from gynaecological curiosity
to multisystem endocrinopathy. Hum Reprod 1996;11:29–39.
3. Ehrmann DA, Barnes RB, Rosenfield RL. Polycystic ovary syndrome as
a form of functional ovarian hyperandrogenism due to dysregulation of
androgen secretion. Endocr Rev 1995;16:322–53.
4. Dunaif A. Insulin resistance in polycystic ovary syndrome. Ann NYAcad
Sci 1993;687:60–4.
5. Szilagyi A, Rossmanith WG. Polyzystisches Ovar-Syndrom: Zentrale
oder periphere Regulationsstorung? Zentralbl Gynakol 1991;113:851–6.
The role of colour Doppler imaging in the diagnosis of polycystic ovary
syndrome. Am J Obstet Gynecol 1995;72:108–13.
34. Zaidi J, Campbell S, Pittrof R, Kyei-Mensah A, Shaker A, Jacobs HS,
et al. Ovarian stromal blood flow in women with polycystic ovaries—
a possible new marker for diagnosis? Hum Reprod 1995;10:1992–6.
899
Lack of association between polycystic ovarysyndrome and embryonic aneuploidyAndrea Weghofer, M.D., Ph.D.,a,b Santiago Munne, Ph.D.,c Serena Chen, M.D.,d
David Barad, M.D., M.Sc.,e and Norbert Gleicher, M.D.a,e
a Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine,
New Haven, Connecticut; b Department of Obstetrics and Gynecology, Medical University Vienna,
Vienna, Austria; c Reprogenetics, LLC, West Orange, New Jersey; d The Institute for Reproductive Medicine
and Science, Saint Barnabas Medical Center, Livingston, New Jersey; and e The Center for Human Reproduction,
New York, New York
Objective: To determine whether women with polycystic ovary syndrome (PCOS) are at increased risk for embry-onic aneuploidy.Design: Retrospective cohort study.Setting: Academic teaching department, privately owned preimplantation genetics laboratory, and academicallyaffiliated, private infertility center.Patient(s): The study included 174 women, ages 27–45 years, who underwent a single cycle of controlled ovarianhyperstimulation with gonadotropins for in vitro fertilization. Amongst those, 74 were proven patients with PCOS,and 100 were control women with proven absence of PCOS.Intervention(s): Preimplantation genetic diagnosis for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22.Main Outcome Measure(s): Embryonic morphology, in vitro fertilization cycle outcome parameters, and euploidyand aneuploidy rates.Result(s): Women with PCOS demonstrated similar overall percentages of euploid embryos (49.1%� 28.1) whencompared with control women (51.8%� 30.1). However, a statistically significantly higher oocyte yield in patientswith PCOS (22.8 � 9.8 vs. 16.5 � 7.6) resulted in statistically significantly higher absolute numbers of euploidembryos (3.3 � 2.1 vs. 2.4 � 2.0). When stratified for age (<38 y and R38 y) and egg numbers (10–20 and>20), euploidy rates still did not vary between study and control patients. High-responder patients with PCOSshowed, however, statistically significantly reduced clinical- (42.9% vs. 69.0%) and ongoing-pregnancy rates(40.5% vs. 65.5%) compared with high-responder control women.Conclusion(s): Women with PCOS are not at increased risk for embryonic aneuploidy in the course of in vitro fer-tilization treatment. Indeed, because of their larger oocyte numbers, they produce more euploid embryos but havelower pregnancy rates after high oocyte yields. This lower pregnancy rate is, thus, not genetically caused andrequires further investigation. (Fertil Steril� 2007;88:900–5. �2007 by American Society for Reproductive Med-icine.)
Polycystic ovary syndrome (PCOS) is the most commoncause of ovulatory dysfunction, and concomitant infertility,in women of childbearing age, although only a small cohortof patients with PCOS ultimately requires in vitro fertiliza-tion to achieve pregnancy (1, 2). After stimulation with go-nadotropins, PCOS is associated with higher oocyte yield,poorer oocyte quality, low fertilization rate, and an increasedrisk of spontaneous pregnancy loss in comparison to womenwith normal ovarian function (3–5). A widely held percep-tion, therefore, suggests that oocytes, and embryos, from pa-tients with PCOS are of poorer overall quality (6).
Received June 28, 2006; revised and accepted December 22, 2006.
Reprint requests: Andrea Weghofer, M.D., Ph.D., Department of Obstet-
rics and Gynecology, Medical University Vienna, Waehringer Guertel
18-20, 1090 Vienna, Austria (FAX: 431404002861; E-mail: andrea.
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine,
900
Poor oocyte and embryo quality has been statisticallycorrelated to increased aneuploidy rates (7–9). There is, how-ever, little information on whether PCOS results in increasedaneuploidy rates. This study, therefore, compared the aneu-ploidy rate in women with PCOS with that in infertile controlwomen with normal ovarian function.
MATERIALS AND METHODS
Study Design and Patients
The study involved the retrospective analysis of 174 consec-utive infertile women aged 27 to 45 years who, between theyears of 2003 and 2005, had undergone a single in vitro fer-tilization cycle at The Institute for Reproductive Medicineand Science, St. Barnabas Medical Center, including preim-plantation genetic diagnosis at Reprogenetics Laboratories(West Orange, NJ). Exclusion criteria from the study wereabnormal baseline FSH levels (R10 mIU/mL) on days 2/3
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.018
of the menstrual cycle, carrier status for a translocation, a his-tory of prior aneuploid conception, a history of spontaneousabortions (defined as R3 consecutive pregnancy losses), andbeing an oocyte donor.
These 174 women were then, on the basis of patient char-acteristics, divided into 74 patients with PCOS and 100 con-trol women with normal ovarian function. The diagnosis ofPCOS was reached according to the Rotterdam consensus(10). Women classified as having PCOS had hyperandrogen-ism and oligomenorrhea, whereas patients with diagnoses ofeither tubal factor or unexplained infertility lacked any fea-tures of PCOS and served as control women. In both groups,the diagnosis indicated was generally the only infertilitydiagnosis.
In study and control groups, the indications for the perfor-mance of preimplantation genetic diagnosis were statisticallyidentical: in older patients, preimplantation genetic diagnosismainly was performed for advanced female age, whereas inyounger women the primary indications were prior implanta-tion failure, patient concerns about an aneuploid offspring, orattempts at embryo selection.
Controls and patients with PCOS were compared for thefollowing parameters: female chronological age, baseline(day 2/3) FSH and E2 levels, peak E2 levels, total ampulesof gonadotropins used, number of oocytes retrieved and fer-tilized, embryologic development, such as fragmentation andmean embryonic cell numbers, euploidy rates, day of embryotransfer, number of embryos transferred, and miscarriage andclinical- as well as ongoing-pregnancy rates.
Ovarian Stimulation and Fertilization
All patients received GnRH agonist treatment and ovarianstimulation with gonadotropins. Three patients with PCOSand none of the control women received metformin co-treat-ment. After menses, baseline ultrasounds and E2 measure-ments were performed. Gonadotropin treatment wasinitiated in the presence of E2 values of %50 pg/mL and inthe absence of follicular structures of size >10 mm, witha daily dose of 150–450 IU of recombinant FSH (Gonal F[Serono, Rockland, MA] or Follistim [Organon, Roseland,NJ]) and/or human menopausal gonadotropin (Repronex orMenopur; Ferring, Suffern, NY), and were adjusted accord-ing to follicular response. Cycle monitoring, oocyte retrieval,and embryo transfer were performed in a routine fashion. Aclinical pregnancy was defined as the presence of a gesta-tional sac on ultrasound. Miscarriage was diagnosed in theabsence of fetal heartbeat, with or without concomitantbleeding, after the confirmation of a clinical pregnancy.
Embryo Biopsy, Fixation, and Fluorescence In SituHybridization
Acidified Tyrode’s solution, placed in a calcium- and magne-sium-free biopsy medium to loosen blastomere attachments,was used for zona drilling and consecutive cleavage-stage
Fertility and Sterility�
embryo biopsy on day 3 after fertilization. Biopsied single-cell blastomeres were then transferred into hypotonic solu-tion and fixed on slides, following a protocol to minimizesignal overlap and loss of micronuclei (11). In three subjects(2 control women and 1 patient with PCOS), as a result oftechnical difficulties, a second blastomere was biopsied inone of their embryos.
Preimplantation genetic diagnosis analysis was performedby fluorescence in situ hybridization with probes specific forchromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. As reportedelsewhere, the first hybridization cycle included probes forchromosomes 13, 16, 18, 21, and 22 (Multivysion PB; Vysis,Downer’s Grove, IL), followed by a second round of hybrid-ization with probes for chromosomes 15, 17, X, and Y (12).Because of changing laboratory policies, a smaller number ofchromosomes was analyzed in a small number of cases.
Embryos diagnosed as euploid contained two distinct sig-nals for each of the chromosomal pairs tested. Polyploidy wasdiagnosed in the presence of three or more signals for eachchromosome, whereas blastomeres with one or fewer copiesof each chromosome were classified as haploid. Aneuploidywas consistent with an abnormal number of signals for one ortwo chromosomes. Embryos with abnormal copy numbersfor three or more chromosomes and that did not fit in anyalready mentioned classification were considered to be com-plex abnormal (13). In some cases, a third round of hybridiza-tion, using probes with different locus-binding capacities,was performed to confirm diagnostic results (14).
Reanalysis of all blastomeres by fluorescence in situ hy-bridization that was performed as part of our internal qualitycontrol revealed a false-normal rate (chromosomallyabnormal embryo classified as chromosomally normal) of4.1% (15).
Data Generation and Statistical Analysis
The data analysis was based on computer-generated data-bases at Saint Barnabas Medical Center and Reprogenetics.This means that initial entry errors into the database, at theinitial time of cycle performance, cannot be completely ruledout. However, the data were validated at least once after ini-tial entry, showing an error rate of data entry of 0.3%.
Data analysis was performed by using the Statistical Pack-age for Social Sciences (SPSS, Chicago, IL). Descriptive datafor baseline characteristics and outcome measures werecalculated by one-way analysis of variance. Results are pre-sented as means with SDs. P<.05 was considered statisticallysignificant.
The reduction of the follicle pool starts to accelerate at age37.5 years and results in a significant decline in ovarian func-tion (16). To account for this change in ovarian function thathas a significant influence on aneuploidy rates, the cutoff forage stratification was set at age 38 years.
A mean number of 2.5 euploid embryos per study subjectwas expected. Consequently, a 25% difference in percentage
901
of euploid embryos was assumed to result in a mean numberof less than two euploid embryos available for transfer (i.e.,1.87 euploid embryos per patient). Because single-embryotransfer is known to result in comparable pregnancy ratesin young, good-prognosis patients but may impair pregnancyrates in others, the 25% difference in euploidy rates was con-sidered clinically significant (17, 18). The number of patientsrequired to confirm the test hypothesis with a power of 90%,at a significance level of 5%, was calculated to be 70 pergroup.
Institutional review board approval was obtained from theethics committee at Saint Barnabas Medical Center.
RESULTS
Patients’ baseline characteristics are presented in Table 1.The mean ages were 36.4 � 4.2 years for patients withPCOS and 36.6 � 4.0 years for control women. Their meanbaseline (day 2/3) FSH levels were 5.0 � 2.0 mIU/mL and5.6 � 2.0 mIU/mL, respectively. Study patients required sig-nificantly lower medication dosages (2,146.1 � 1,269.2 IU)than did control women (2,596.1 � 1,498.0 IU; P<.05) andproduced significantly higher oocyte yields (22.8 � 9.8 vs.16.5 � 7.6; P<.01).
Cleavage stage development resulted in comparable meanday 3 cell numbers and fragmentation percentages of 6.3%�
902 Weghofer et al. Polycystic ovary syndrome and embry
1.1 and 13.3% � 8.1, respectively, in women with PCOS,compared with 6.3% � 1.0% and 14.4% � 10.3% in controlwomen. Preimplantation genetic diagnosis revealed a meannumber of 3.3 � 2.1 euploid, 2.7 � 3.2 aneuploid, and 2.7� 2.7 complex abnormal embryos in women with polycysticovaries, whereas a mean number of 2.4 � 2.0 euploid, 1.7 �2.1 aneuploid, and 1.7 � 1.8 complex abnormal embryoswere found in control women. These numbers resulted insimilar overall percentages of euploid embryos in patientswith PCOS (49.1% � 28.1) and in control women (51.8%� 30.1). Women with PCOS produced, however, sig-nificantly higher absolute numbers of euploid embryos(P<.01).
The transfer of a mean number of 3.0 � 1.0 embryos inthe study group and of 2.9 � 1.3 in control women resultedin comparable clinical-pregnancy rates per embryo transferof 48.6% in patients with PCOS and of 53.2% in controlwomen. Miscarriages occurred in 14.3% of study patientsand in 8.0% of control women, leading to comparable on-going-pregnancy rates per transfer of 41.7% and 48.9%, re-spectively (Table 1).
After stratification by age, young patients with PCOS (age,<38 y) demonstrated again significantly higher oocyte num-bers but similar euploidy rates compared with controlwomen: 55.6% in women with PCOS and 60.4% in controlwomen. There was also no significant difference observed
TABLE 1Patients’ baseline characteristics and cycle parameters.
Note: Data are mean � SD or are n (%). NS ¼ not statistically significant; ET ¼ embryo transfer.
Weghofer. Polycystic ovary syndrome and embryonic aneuploidy. Fertil Steril 2007.
onic aneuploidy Vol. 88, No. 4, October 2007
in morphology, absolute number of chromosomally normaland aneuploid embryos, and other cycle outcome parameters.
The same was true for patients of advanced reproductiveage (age R38 y). They demonstrated similar embryoniceuploidy rates (38.3% in PCOS vs. 43.7% in control women)but a significantly higher absolute number of euploid andaneuploid embryos in study patients (P<.01). Cleavagedata and outcome parameters were, however, comparable(Table 2).
When patients were stratified according to oocyte num-bers, women with a moderate oocyte yield (10–20 eggs)revealed comparable oocyte numbers and similar gonadotro-pin amounts, with similar treatment durations. Morphologi-cal cleavage criteria and embryonic euploidy rates also didnot differ significantly between patients with PCOS and con-trol women, and even the absolute numbers of euploid em-bryos were comparable. All of this resulted in similar ratesof implantation, clinical pregnancy, miscarriage, and ongoingpregnancy.
When only high responders (R21 oocytes) were com-pared, women with PCOS were comparable in terms ofeuploidy rates and absolute euploid embryo numbers. Al-though both groups demonstrated similar embryologic cleav-age and morphology, in vitro fertilization in patients withPCOS resulted in significantly reduced clinical- (42.9% vs.69.0%, P<.05) and ongoing-pregnancy rates (40.5% vs.65.5%, P<.05) per embryo transfer, when compared withhigh-responding control patients (Table 3).
DISCUSSION
The number of oocytes retrieved in the course of in vitro fer-tilization cycles is deemed to be a crucial factor in fertilitytreatment, determining pregnancy chances in the current cy-cle and even in subsequent cycles (19, 20). Despite signifi-
Fertility and Sterility�
cantly higher oocyte yields, cumulative pregnancy rates inwomen with PCOS are not superior to those in controlwomen (21).
Elevated tonic LH levels and hyperandrogenemia appearto play a possibly causative role in reduced fertilization ratesand increased pregnancy wastage that are seen in PCOS, be-cause LH suppression by GnRH agonists results in improvedfertilization and lower miscarriage (6, 22). Though the under-lying pathophysiology is still poorly understood, it has beenpostulated that LH induces premature meiotic resumptionand may cause detrimental prolongation of the interval be-tween completion of meiosis I and fertilization. The disrup-tion of the endocrine control of meiosis, resulting in theimpaired extrusion of the first polar body, may compromisethe chromosomal normality of oocytes in patients with poly-cystic ovary syndrome (23). Embryonic aneuploidy, ratherthan oocyte immaturity, has, therefore, been suggested asa possible cause for the higher incidence of spontaneousabortions associated with PCOS (24).
The here-presented results, however, challenge this con-cept. As a result of significantly higher oocyte yields,PCOS women had more euploid and aneuploid embryoswhen compared with control women, yet their embryonic eu-ploidy rates were similar. Even when stratified by age and oo-cyte numbers, no difference in euploidy rates was detectedbetween PCOS and control women.
Our findings are supported by those of Sengoku and collab-orators (23), who also reported comparable aneuploidy, dip-loidy and prematurely condensed sperm chromosomes inunfertilized oocytes in women with PCOS and controlwomen. Those oocytes analyzed were, however, not repre-sentative of the whole cohort of eggs, whereas the presentstudy includes all day 3 embryos produced in the cycle. Itis yet to be determined whether the fact that all patientswere exposed to LH suppression and, consequently, produced
TABLE 2PGD results and cycle outcomes according to female age.
Note: Data are mean � SD or are n (%). NS ¼ not statistically significant; ET ¼ embryo transfer.
Weghofer. Polycystic ovary syndrome and embryonic aneuploidy. Fertil Steril 2007.
embryos of comparable morphology, may partially accountfor these findings.
Fertility treatment in the study cohort and in controlwomen resulted in equivalent clinical- and ongoing-preg-nancy rates. This is especially interesting, considering thatsignificantly higher embryo numbers were obtained in olderpatients with PCOS compared with in their age-stratified con-trol women (3.5 � 2.3 vs. 2.0 � 1.5).
When stratified for age, there was a trend toward highermiscarriage rates in young patients with PCOS comparedwith in control women, although this difference failed toreach statistical significance (12.5% vs. 2.7%). Consideringthe false-normal fluorescence in situ hybridization rate (i.e.,an abnormal embryo incorrectly classified as normal) of4.1% in our program (15), there is a rare but slight risk thatan abnormal embryo that was incorrectly diagnosed as nor-mal may have been transferred. However, potential misdiag-nosis can be assumed to occur to an equal extent in studysubjects and control women.
The fact that patients with PCOS had significantly moreeuploid embryos available for transfer, however, reducesthe likelihood that an embryo incorrectly diagnosed as nor-mal may be transferred, thus favoring patients with PCOS.If, indeed, one of the rare occasions of transfer of such an ab-normal embryo occurred, it is therefore more appropriate toassume that this transfer happened in a patient in the controlgroup. As a consequence, young patients with PCOS shouldrather demonstrate lower miscarriage rates than their age-stratified controls. We, however, observed a trend towardhigher miscarriage rates in young women with PCOS.
This possible difference in miscarriage risk in women withPCOS may be particularly obvious in young patients, who aregenerally at lower risk for miscarriage because of medicalcauses, such as gestational diabetes, compared with women
904 Weghofer et al. Polycystic ovary syndrome and embry
of advanced reproductive age. This may serve as an explana-tion for the trend toward increased miscarriage rates in youngpatients with PCOS compared with age-stratified controlwomen, whereas this observation could not be made in olderstudy subjects when compared with age-stratified controlwomen.
When only high responders, producing >20 eggs, wereconsidered for analysis, women with PCOS demonstratedcomparable miscarriage rates but significantly reduced clini-cal- and ongoing-pregnancy rates, despite comparable em-bryo morphology.
These findings correspond to earlier reports in the litera-ture. For example, Homburg (2) reported that despite largeroocyte or embryo numbers, cumulative pregnancy rates in pa-tients with PCOS are not superior to those of control women.Those findings are also supported by those of Pellicer et al.(25), who reported significantly reduced implantation rates,despite comparable embryo morphology, in high-responderpatients.
The here-reported observation that chromosomallyscreened PCOS embryos, with similar cleavage morphology,were significantly less likely to implant raises the question ofwhy this should be the case. The here-presented data suggestthat factors other than chromosomal and/or morphologiccharacteristics may be responsible. What these may be has re-mained subject to speculation, though the literature may offersuggestions.
Patients with PCOS may have an implantation problem:Cermik et al. (26) demonstrated that women with PCOS ex-hibit decreased amounts of HOXA-10, a marker of endome-trial receptivity. Insulin-like growth factor I has been claimedto facilitate implantation and the survival of pregnancy. De-creased local insulin-like growth factor levels in womenwith PCOS have been associated with implantation failure
onic aneuploidy Vol. 88, No. 4, October 2007
and spontaneous pregnancy loss (27). Tuckerman and collab-orators (28) reported that androstenedione, often elevated inpatients with PCOS, inhibits human endometrial cell growthand secretory activity. The presence of hyperandrogenemia,as well as increased androgen receptor expression, detectedin endometrial epithelial cells of patients with PCOS, maytherefore further contribute to endometrial dysfunction andconcomitant implantation failure or miscarriage (29, 30).
Alternatively, the problem may be immunologic in nature:PCOS is characterized by an increased prevalence of antio-varian autoantibodies (31). Increased miscarriage rates havebeen observed in autoimmune-mediated thyroid disease,and autoimmune-mediated mechanisms in PCOS may be re-sponsible for the impaired pregnancy chances observed inhigh-responder patients with PCOS, as suggested by van Gel-deren and Gomes dos Santos (31). However, further studiesare needed to confirm these assumptions.
In conclusion, our data provide support for the assumptionthat women with PCOS are not at increased risk for embry-onic chromosomal abnormalities. Therefore, factors otherthan chromosomal ones are likely to be responsible for thesignificantly reduced pregnancy chances observed in high-responder patients with PCOS. Further investigations areneeded to elucidate the underlying pathophysiologic mecha-nisms for poorer pregnancy chances in patients with PCOSwith high oocyte yields.
Acknowledgments: The authors acknowledge the support of Giles Tomkin
and Jill Fisher, M.Sc., in data collection. Moreover, the authors are indebted
to their colleagues from St. Barnabas Medical Center and Reprogenetics
Laboratories.
REFERENCES1. Balen A. The pathophysiology of polycystic ovary syndrome: trying to
understand PCOS and its endocrinology. Best Pract Res Clin Obstet Gy-
naecol 2004;18:685–706.
2. Homburg R. The management of infertility associated with polycystic
dometrial androgen receptor expression in women with polycystic ovar-
ian syndrome. Biol Reprod 2002;66:297–304.
30. Maliqueo M, Clementi M, Gabler F, Johnson MC, Palomino A, Sir-
Petermann T, et al. Expression of steroid receptors and proteins related
to apoptosis in endometria of women with polycystic ovary syndrome.
Fertil Steril 2003;80(Suppl 2):812–9.
31. van Gelderen CJ, Gomes dos Santos ML. Polycystic ovarian syndrome.
Evidence for an autoimmune mechanism in some cases. J Reprod Med
1993;38:381–6.
905
REPRODUCTIVE ENDOCRINOLOGY
Stage I ovarian carcinoma: different clinical pathologicpatternsLiane Deligdisch, M.D.,a Fr�ed�erique P�enault-Llorca, M.D., Ph.D.,b Peter Schlosshauer, MD.,a
Albert Altchek, M.D.,c Michele Peiretti, M.D.,d and Farr Nezhat, M.D.c
a The Mount Sinai School of Medicine, Department of Pathology, New York, New York; b Centre de Lutte Contre le Cancer de la
R�egion Auvergne, Department of Pathology, Clermont-Ferrand, France; c The Mount Sinai School of Medicine, Department of
Obstetrics, Gynecology and Reproductive Science, New York, New York; and d University of Cagliari, Department of Obstetrics
and Gynecology, Cagliari, Italy
Objective: To analyze clinicopathologic patterns of early ovarian carcinoma.Design: Retrospective chart and histopathology review.Setting: Mount Sinai School of Medicine, New York and the Centre Jean Perrin, Clermont Ferrand, France.Patient(s): Seventy-six consecutive cases of F�ed�eration Internationale de Gyn�ecologie et d’Obst�etrique stage Iovarian carcinoma.Intervention(s): Surgical staging.Main Outcome Measure(s): Symptomatology, pathology, and histology analysis.Result(s): Twenty-two cases (29%) were serous papillary carcinomas and 54 were nonserous carcinomas (71%)(40 endometrioid, 10 clear cell, and 4 mixed endometrioid and clear cell carcinomas). Ninety-eight percent of ovar-ian endometriosis, 95% of endometrial carcinomas, and 83% of endometrial polyps and hyperplasias were asso-ciated with nonserous carcinomas. Most patients with serous papillary carcinoma presented with asymptomaticpelvic masses; patients with nonserous carcinomas presented with pelvic pain or abnormal vaginal bleedingwith or without pelvic mass.Conclusion(s): Over two thirds of stage I ovarian carcinomas were nonserous, and were diagnosed because of as-sociated symptoms: pelvic pain with endometriosis and/or adnexal masses, or vaginal bleeding from endometrialpathology. Serous papillary carcinomas were often asymptomatic and diagnosed during follow-up evaluations inbreast cancer patients. Stage I ovarian carcinoma has different clinical and pathologic patterns than advanced ovar-ian carcinoma. The risk of ovarian and endometrial malignancy should be taken into consideration during evalu-ation of patients with endometriosis and breast cancer histories. (Fertil Steril� 2007;88:906–10. �2007 byAmerican Society for Reproductive Medicine.)
Key Words: Stage I ovarian carcinoma, endometriosis, endometrial neoplasia
Ovarian neoplasms of epithelial origin represent the mostcommon group of malignant ovarian tumors, among whichserous carcinomas are the most prevalent. Often there is a de-lay in diagnosis such that the majority of cases are diagnosedin F�ed�eration Internationale de Gyn�ecologie et d’Obst�etrique(FIGO) stages III and IV. Only approximately 24% of allovarian carcinomas are diagnosed in FIGO stage I (1). Thisstudy of FIGO stage I ovarian carcinoma aims to correlatehistopathologic patterns with the associated symptomaticpathology likely to have an impact on early diagnosis.
MATERIALS AND METHODS
The study included 76 consecutive patients diagnosed from1998 to 2004 with FIGO stage I ovarian carcinoma (OC) at
Received October 21, 2006; revised and accepted December 22, 2006.
Correspondence to: Farr Nezhat, M.D., Department of Obstetrics, Gyne-
cology and Reproductive Science, The Mount Sinai School of Medicine,
1176 5th Avenue, 9th floor, Box 1170, New York, NY 10029 (FAX:
5%, 5% to 50%, or more than 50% solid tumor, respectively).Clear cell carcinomas were considered high grade (G3).Ovarian tumors of borderline malignancy were excludedfrom the review. Mucinous tumors were excluded from thereview because most displayed features of borderline malig-nancy, and, in some of the reviewed cases, a coexisting or pri-mary intestinal tumor could not be ruled out. In cases ofsynchronous endometrial and ovarian tumors, their indepen-dence was determined based on accepted criteria, includinghistologic dissimilarity, no or superficial myometrialinvasion, absence of lymphvascular involvement, and noevidence of tumor spread elsewhere (1). Mixed carcinomaswere defined as tumors exhibiting two or more differenthistologic subtypes with each component comprising at least10% of the total tumor volume.
Additional pathologic findings such as endometriosis, inparticular ovarian endometriosis adjacent to the ovarian tu-mor and endometrial pathology including endometrial carci-noma, endometrial polyps and/or hyperplasia, and associatedbenign tumors (adenofibroma, fibrothecoma, adenomyomas)were also analyzed.
Histologic findings were correlated with the followingclinical data: symptomatic and asymptomatic pelvic masses,vaginal bleeding, gastrointestinal symptoms, ascites, historyof breast cancer, BRCA germline mutations (whenavailable), and age of the patients. For statistical analyses,chi-square tests were used. A P-value of < .5 was consideredstatistically significant. This study was approved by theMount Sinai Institutional review board.
Fertility and Sterility�
RESULTS
The histopathologic findings are summarized in Table 1.Among a total 76 cases of stage I ovarian carcinoma, 22(29%) were OSPC and 54 (71%) were nonserous carcinomas(NSC), consisting of 40 OEC, 10 OCCC, and four MCsubtypes. Twenty-three carcinomas were well differentiated(G1), 34 moderately differentiated (G2), and 19 poorlydifferentiated (G3).
Ovarian endometriosis was found in 40 cases, of which 39were associated with NSC (P<.0000001). Sixteen of 17 casesof extraovarian pelvic endometriosis were present in NSC(P¼.038). Endometrioid type endometrial carcinoma wasdiagnosed in 19 cases, of which 13 were synchronous tumors;the 18 cases with NSC (P¼.0086) comprised 17 OEC and 1OCCC. Most associated endometrial carcinomas were G2; noinformation was available on the degree of differentiation offour of six cases diagnosed prior to the ovarian carcinoma.Bilateral ovarian tumors (stage IB) were present in 11OSPC and five NSC (P¼.00027). Endometrial polyps and/or endometrial hyperplasia were found in 18 cases, of whichthree were OSPC and 15 NSC. Ovarian adenofibroma wasdiagnosed in eight patients, seven with NSC.
The clinical data are summarized in Table 2. The averageage of the 22 OSPC patients was 61 years. Thirteen wereasymptomatic, and pelvic masses were diagnosed on routineexaminations; eight patients had a history of previous breastcancer and had been under medical surveillance with transva-ginal sonograms. Two patients presented with painful pelvicmasses, one with vaginal bleeding, two with increased
a Incidence in OSPC compared to nonserous papillary carcinomas.
Deligdisch. Stage I ovarian carcinoma. Fertil Steril 2007.
907
TABLE 2Stage I ovarian carcinoma—clinical features.
Clinical Features OSPC (n [ 22) OEC (n [ 40) OCCC (n [ 10) MC (n [ 4)
Average age 61.05 52.9 58.6 52.2Asymptomatic pelvic mass 13 3 – –Symptomatic pelvic mass 2 19 10 4Vaginal bleeding 1 19 1 –Hx of breast carcinoma 8 – 1 –BRCA mutations, tested 4 – – –Ascites 2 – 1 –Pleural effusion 1 – – –Upper gastrointestinal symptoms 2 1 1 –Abnormal Pap smear 2 – – –
Deligdisch. Stage I ovarian carcinoma. Fertil Steril 2007.
abdominal girth due to benign ascites, and one benign pleuraleffusion. Two complained of upper gastrointestinal symptoms(bloating or nausea), and two had abnormal Pap smears, al-though thesewere unrelated to the ovarian tumor. Four patientshad BRCA germline mutations, and four were not tested.
The average age of the NSC patients were as follows: 52.9years for OEC, 58.6 years for OCCC, and 52.2 years for MC.Out of 54 total patients diagnosed with NSC, 33 had pre-sented with painful pelvic masses, all of whom had associ-ated ovarian endometriosis. Twenty-one patients presentedwith vaginal bleeding, 16 due to endometrial endometrioidcarcinoma, and four with endometrial polyps and hyperpla-sia. An additional 16 had pelvic endometriosis, seven withovarian adenofibromas and three with asymptomatic pelvicmasses. One patient with OCCC had a history of breast can-cer, one had upper gastrointestinal tract symptoms and a pain-ful pelvic mass, and one patient had ascites. In five patients,ovarian endometrioid carcinoma was diagnosed incidentallyat surgery performed for endometrial pathology.
DISCUSSION
The histologic pattern distribution in the reviewed ovariancarcinomas diagnosed in stage I was markedly differentfrom the overall histologic distribution of ovarian epithelialtumors, where serous carcinomas are the most frequent (1).About half of all the malignant ovarian tumors of epithelialorigin are serous carcinomas, whereas endometrioid carcino-mas represent about 15% and clear cell carcinomas 6% (4). Inthe Mount Sinai School of Medicine records, over a 10-yearperiod there were 345 cases of ovarian carcinoma, compris-ing 65% OSPC, 14.5% OEC, 5.2% OCCC, and 15% mucin-ous and others. In the present study, OSPC representsa minority of 22 of 76 (29%) cases. Nonserous carcinomaswere 71% of cases, with endometrioid carcinomas beingthe most common (52.6%).
Ovarian serous papillary carcinomas are often advanced attime of diagnosis due to the paucity of clinical symptoms in
908 Deligdisch et al. Stage I ovarian carcinoma
their early stages. The ‘‘symptomless’’ nature of serous ovar-ian carcinoma has been challenged recently by reports men-tioning gastrointestinal symptoms and other manifestationsof paraneoplastic syndromes (5). In our series, eight patientswith stage I OSPC had been previously diagnosed with breastcancer, and four also had BRCA germline mutations. Thesepatients were under close surveillance, leading to the diagno-ses of asymptomatic pelvic masses. Only one patient withOSPC presented with vaginal bleeding due to endometrialadenocarcinoma, after tamoxifen therapy for breast cancer.
Relatively few patients presented with gastrointestinalsymptoms and ascites. About half of the OSPC were unilat-eral, probably due to their early diagnosis, as most OSPCare bilateral.
Over two thirds of the tumors diagnosed in stage I werenonserous; endometrioid carcinomas represented the major-ity and were frequently associated with endometrioticovarian cysts. Their early diagnosis was based on the findingof symptomatic cystic or solid pelvic masses representing en-dometriosis rather than tumor tissue, which in some caseswas small. Another frequent presenting symptom of thenonserous tumors was vaginal bleeding due to endometrialpathology, often associated with the ovarian malignancy. Itis known that ovarian endometrioid carcinomas are often as-sociated with primary endometrial carcinomas (6–8), and itshould be emphasized that in these cases the earlier diagnosisis based on the more often symptomatic malignancy, which isthat of the endometrium. This is also the case for endometrialhyperplasia/polyps, which are more frequently seen in pa-tients with OEC, being associated with hyperestrogenismand pelvic endometriosis. These patients are usually youngerand sometimes present with infertility problems (9, 10). A re-cent article reported a high percentage of women aged 25 to45 undergoing hysterectomy for endometrial carcinoma werefound to have coexisting primary ovarian cancers, mostlyendometrioid (11). In our series, the ovarian tumor wasdiscovered during surgery performed in five patients for
Vol. 88, No. 4, October 2007
endometrial pathology, which obviously had been moresymptomatic than the ovarian tumor.
The question remains of why only certain ovarian carcino-mas are diagnosed in early stages. Associated symptomaticlesions such as endometriosis and endometrial pathologybring patients to medical attention sooner than clinicallysilent ovarian malignancies. The question also arises ofwhether the minority of ovarian cancers diagnosed in stageI have a different natural history than the majority diagnosedin stage III or IV. The NSC are found in different clinical set-tings and in younger patients, often presenting with hyperes-trogenic lesions like endometriosis and endometrialpathologies. Although ovarian endometriosis is a frequentdisorder, its association with ovarian cancer is relativelyrare. However, there are reports asserting ovarian endometri-osis as a potential cancer precursor, with identification ofatypical endometrioid lesions at the transition between endo-metriosis and endometrioid and clear cell carcinoma (12–14)and abnormal immunoreactivity of this tissue (15, 16). Lap-aroscopic findings of ovarian endometriosis may raise thesuspicion of a possible associated ovarian malignancy. Theprevalence of endometriosis in premenopausal women,including those presenting with infertility problems, necessi-tates directed counseling before surgery as well as thoroughinspection and frozen section specimens during surgery.
Most nonserous ovarian carcinomas were better differenti-ated histologically. This could be because they are less viru-lent in their growth pattern or they were diagnosed andremoved at an early stage and thus did not progress toa more aggressive phenotype. It is possible that differenthistogenetic pathways exist. Genetic analysis of early versuslate stages of ovarian cancer indicated that there are profoundalterations in gene expression in early stage tumors, provid-ing support for a progression model for ovarian cancer devel-opment (17). Certain genetic alterations such as beta-cateninmutations, PTEN mutations, and microsatellite instabilitythat are frequently seen in ovarian endometrioid carcinomaare associated with low-grade, low-stage tumors and a favor-able prognosis (18–21). Other studies indicate that early stageinvasive serous carcinoma shares genetic expression patternswith long-term survivors of late stage OSPC (22). Geneticanalysis and protein expression studies may contribute tothe identification of basic biologic and/or molecular differ-ences, in addition to the histopathologic and clinical differ-ences between the malignant nature of ovarian serous andnonserous carcinomas.
Our study revealed a different distribution of histologictypes of ovarian carcinoma diagnosed in stage I comparedwith the all stage distribution. Over two thirds of stage Iovarian carcinomas included in this study were nonserous,predominantly endometrioid carcinomas, diagnosed at anearly stage because of the associated symptomatic lesions,such as ovarian endometriosis manifested clinically as symp-tomatic pelvic masses, and endometrial neoplasia manifestedclinically as vaginal bleeding, The serous carcinomas repre-sented less than one third of the cases and were diagnosed
Fertility and Sterility�
rather fortuitously, often in patients with prior breast cancerwho were under close medical surveillance.
Some patients with endometriosis, including those treatedfor infertility and those with history of breast cancer, may beat higher risk for ovarian cancer. The possibility of ovarianand endometrial malignancy should be taken into consider-ation during the evaluation of patients with endometriosisand breast cancer histories. Furthermore, there appear to beessential clinical, pathological, and biological differences be-tween ovarian serous and nonserous carcinomas that need tobe explored in the future by molecular genetic methods.
REFERENCES1. Scully RE, Young RH, Clement PB. Tumors of the ovary, maldeveloped
gonads, fallopian tube, and broad ligament. Washington, DC: Armed
Forced Institute of Pathology, 1998.
2. Scully RE. Histologic typing of ovarian tumors. World Health Organiza-
tion international histologic classification of tumors. 2nd ed. New York:
Springer, 1999.
3. Greene FL, Page DL, Fleming ID, Fritz AG, Balch CM, Haller DG, et al.
AJCC cancer staging handbook. 6th ed. New York: Springer-Verlag,
Thomas EJ, et al. Frequent PTEN/MMAC mutations in endometrioid
909
but not serous or mucinous epithelial ovarian tumors. Cancer Res
1998;58:2095–7.
19. Irving JA, Catasus L, Gallardo A, Bussaglia E, Romero M, Matias-
Guiu X, et al. Synchronous endometrioid carcinomas of the uterine
corpus and ovary: alterations in the beta-catenin (CTNNB1) pathway
are associated with independent primary tumors and favorable prognosis.
Hum Pathol 2005;36:605–19.
20. Catasus L, Bussaglia E, Rodriguez I, Gallardo A, Pons C, Irving JA, et al.
Molecular genetic alterations in endometrioid carcinomas of the ovary:
similar frequency of beta-catenin abnormalities but lower rate of micro-
910 Deligdisch et al. Stage I ovarian carcinoma
satellite instability and PTEN alterations than in uterine endometrioid
carcinomas. Hum Pathol 2004;35:1360–8.
21. Gamallo C, Palacios J, Moreno G, Calvo de Mora J, Suarez A, Armas A.
Beta-catenin expression pattern in stage I and II ovarian carcinomas:
relationship with beta-catenin gene mutations, clinicopathological fea-
tures, and clinical outcome. Am J Pathol 1999;155:527–36.
22. Berchuck A, Iversen ES, Lancaster JM, Pittman J, Luo J, Lee P, et al.
Patterns of gene expression that characterize long-term survival in
advanced stage serous ovarian cancers. Clin Cancer Res 2005;11:
3686–90.
Vol. 88, No. 4, October 2007
Anxiety and sexual stress in men and womenundergoing infertility treatmentBrennan D. Peterson, Ph.D.,a Christopher R. Newton, Ph.D.,b and Tal Feingold, M.A.a
a Marriage and Family Therapy Program, Department of Psychology, Chapman University, Orange, California; andb Reproductive Endocrinology and Infertility, University Hospital, London Health Sciences Centre, London, Ontario, Canada
Objective: To better understand the specific nature of the relationship between anxiety and sexual infertility-related stress in men and women.Design: Prospective study.Setting: University-affiliated teaching hospital.Patient(s): Consecutively referred patients referred for in vitro fertilization and intrauterine insemination(306 women, 295 men).Intervention(s): None.Main Outcome Measure(s): Fertility Problem Inventory (FPI), Beck Anxiety Inventory (BAI).Result(s): Women reported greater anxiety and sexual infertility stress than men. However, men and womenshowed a similar pattern in the way anxiety symptoms were related to sexual infertility stress, with subjective anx-iety and autonomic anxiety having the strongest relationship. Anxiety symptoms accounted for a significant pro-portion of the variance in sexual infertility stress for both sexes and predicted sexual stress to a considerable degreein men.Conclusion(s): Although this study found that there is more similarity than difference in how men and womenexperience anxiety and sexual infertility stress, the strong linkage between anxiety and sexual stress in men wassurprising, because men tend to report less sexual stress and also less anxiety. Sexual stress among infertile menmay be more closely tied to performance anxiety rather than to a more general deterioration in sexual satisfactionassociated with infertility. (Fertil Steril� 2007;88:911–4. �2007 by American Society for Reproductive Medicine.)
Men and women undergoing assisted reproductive treatment(ART) experience a number of stresses related to both the ex-perience of infertility and participation in associated medicaltreatments. One of the most commonly reported forms of in-fertility-related stress is that of stress in the couple’s sexualrelationship. For example, sexual infertility stress has beendefined as loss of enjoyment of sexual relations, feelings ofpressure to schedule sexual relations, and loss of sexualself-esteem (1). Although a recent study demonstrated thatoverall levels of stress are related to ART treatment success,it also found that certain forms of infertility-related stress(e.g., stress on the sexual relationship) were more stronglylinked to treatment outcome than others (2).
There has also been a substantial amount of research exam-ining the impact of psychological and emotional factors onthe infertility experience. The role of patient anxiety is onefactor that has been studied (3). Research has found thatlevels of anxiety are higher in men and women experiencinginfertility compared with fertile control groups (4). Further-more, the presence of ‘‘state anxiety’’ (anxiety triggered bya particular problem or situation) in men and women pursu-ing ARTs has been shown to be a predictor of treatmentsuccess (3).
Received July 11, 2006; revised and accepted December 5, 2006.
Reprint requests: Brennan Peterson, Ph.D., Department of Psychology,
Chapman University, One University Drive, Orange, California 92866
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.023 Copyright ª2007 American S
Past research has also shown that infertility has a negativeimpact on sexual self-esteem in both men and women. For ex-ample, among men, sexual infertility stress is often linkedwith feelings of reduced masculinity and a perceived threatto the male identity (5). In addition, sexual infertility stresscan interfere with early medical interventions (e.g., medica-tion coupled with timed intercourse) and with infertilityevaluations associated with the use of more advanced tech-nologies (6). Overall levels of psychological stress also havebeen linked with decreased semen quality in men undergoingin vitro fertilization (IVF) (7).
Although it is clear that anxiety and sexual infertility stressare commonly found in men and women experiencing infertil-ity, relatively little is known about the specific nature of the re-lationship between these variables and how they may presentdifferently for men and for women. The purpose of the presentstudy was to more clearly describe the nature of anxiety andsexual infertility stress in men and women seeking ART.The study sought to answer the following research questions:1) To what extent is anxiety related to sexual infertility stressin men and women?; and 2) if a relationship exists, is therea difference in how men and women experience it?
MATERIALS AND METHODS
Procedures
The sample for this study was composed of men and womendiagnosed with infertility and referred to a university-affiliated
Fertility and Sterility� Vol. 88, No. 4, October 2007 911ociety for Reproductive Medicine, Published by Elsevier Inc.
teaching hospital for IVF and intrauterine insemination (IUI).The study was approved by a university review board that pro-tects human subjects and ensures ethical procedures in datacollection and analysis. Informed consent was also obtainedfrom all study participants before data collection.
Study participants were mailed a packet of treatment mea-sures approximately 2 months before treatment and wereasked to separately complete and return the forms by mail be-fore making a pretreatment appointment with the programstaff. A total of 787 consecutively referred patients were in-cluded in the original sample, of which 601 (76%) had com-plete data on both study measures and constituted the finalsample.
Measures
Infertility stress The Fertility Problem Inventory (FPI) (1) isa 46-item questionnaire that measures an individual’s globallevel of infertility stress. The FPI includes five subscaleswhich measure more specific areas of infertility stress, in-cluding the experience of sexual infertility stress. Higherscores indicate increased levels of infertility stress. The FPIdemonstrates high reliability and good discriminant validity(the degree to which each subscale measures something dif-ferent) and convergent validity (the degree to which thescales demonstrates moderate and expected associationswith other variables) (1). Norms on the FPI for sexual infer-tility stress are derived from the responses of a large sampleof men (n ¼ 1,149) and women (n ¼ 1,153). High levels ofinfertility stress were indicated by scores that were one stan-dard deviation above the mean (i.e., the 84th percentile) forboth genders. For women, mean scores greater than 27 indi-cated high levels of sexual infertility stress, and for menscores of 20 or more indicated high levels of sexual infertilitystress. The different cut-off scores are the result of higher re-ports of overall sexual infertility stress in women than in men.
Anxiety The Beck Anxiety Inventory (BAI) was used to as-sess the levels of anxiety of the men and women who partic-ipated in the study. Comprising 21 items, the BAI asks therespondent to rate how much he or she has experienced anx-iety symptoms over the preceding week (8). The BAI includesfour subscales: neurophysiologic anxiety (e.g., dizziness,shakiness), autonomic anxiety (e.g., sweating, indigestion),subjective anxiety (e.g., inability to relax, fearing the worst,nervous), and panic symptoms (e.g., heart racing, difficultybreathing). Respondents rate each item on a four-point Likertscale ranging from 0 (not at all) to 3 (severely—I couldbarely stand it), and responses are summed so that totalBAI scores range from 0 to 63. Total scores of 8 or more in-dicate the presence of mild to severe anxiety symptoms. TheBAI is a widely accepted self-report instrument with highinternal consistency (alpha ¼ 0.92) and test-retest reliability(r ¼ 0.75 after 1 week) and has been shown to have goodvalidity (8).
Data analysis and sample characteristics Independent sam-ples t tests, Pearson correlation coefficients, and standardized
912 Peterson et al. Anxiety and sexual stress
betas from linear regression analyses were used for statisticalanalyses. A total of 306 women and 295 men participated inthe study. Men were slightly older than women, with a mean(M) age (�SD) of 34.5 � 5.7 compared with 32.4 � 4.2 forwomen (t¼�5.1; P<.001). Three hundred forty-eight (58%)of the participants were scheduled to undergo IVF treatments,and 253 (42%) participants were scheduled to undergo IUItreatment using either clomiphene citrate or gonadotropins.Participants were heterosexual couples, and although specificracial/ethnic data were not collected, the majority of partici-pants were Caucasian, which represented the regional popu-lation at that time.
RESULTS
Results from the study indicated that women (M¼ 5.0; SD¼5.4) reported greater total anxiety symptoms than men(M¼ 2.5; SD¼ 3.7) (t¼ 6.5; P<.001). Women also reportedsignificantly greater symptoms of anxiety on each of the foursubscales. A total of 24% of women reported mild to severesymptoms of anxiety (BAI R8), whereas 7% of men reportedscores in this range.
In terms of sexual infertility stress, women reported greaterstress (M ¼ 18.2; SD ¼ 8.1) than men (M ¼ 14.9; SD ¼ 6.8)(t ¼ 5.4; P<.001). Twenty-one percent of men and 17% ofwomen reported high levels of sexual infertility stress.
Men and women showed similar patterns in the way anxi-ety symptoms were related to sexual infertility stress. Each ofthe four anxiety subscales and total anxiety had a positive re-lationship with sexual infertility stress, meaning that as anx-iety symptoms increased, sexual infertility stress alsoincreased. However, this pattern varied slightly betweenmen and women (Table 1). For men, the strongest relation-ships were between sexual infertility stress and autonomicanxiety (r ¼ 0.38; P<.01) and subjective anxiety (r ¼ 0.34;P<.01). For women, sexual infertility stress was most
TABLE 1Bivariate correlations between sexualinfertility stress and anxiety in womenand men.
Anxietysymptoms
Women’ssexualstress
(n [ 306)
Men’ssexualstress
(n [ 295)
Neurophysiologic 0.24b 0.17b
Subjective 0.33b 0.34b
Panic 0.13a 0.20b
Autonomic 0.29b 0.38b
Global anxiety 0.35b 0.38b
a Correlation is significant at the .05 level (one-tailed).b Correlation is significant at the .01 level (one-tailed).
Peterson. Anxiety and sexual stress. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
strongly related to subjective anxiety (r ¼ 0.33; P<.01) andautonomic anxiety (r ¼ 0.29; P<.01).
A simple linear multiple regression was performed to as-sess the predictive nature of anxiety on sexual infertilitystress. The results of the regression analysis are shown inTable 2. When sexual infertility stress was used as the depen-dent variable, anxiety symptoms combined explained 21% ofthe variance in sexual infertility stress scores for men and 14%for women. Standardized beta values, which illustrate theseparate contribution of each type of anxiety, showed thatautonomic anxiety and subjective anxiety significantly con-tributed to the model for both men and women, but did so indifferent ways. For men, autonomic anxiety (b ¼ 0.33;P<.01) had the greatest contribution of unique variance, fol-lowed by subjective anxiety (b ¼ 0.29; P<.01). For women,subjective anxiety (b¼ 0.26; P<.01) contributed the greatestamount of unique variance, followed by autonomic anxiety(b ¼ 0.18; P<.01). Neither neurophysiologic nor panicsymptoms of anxiety significantly contributed to the modelfor men or women.
DISCUSSION
The experience of infertility can be accompanied by anxietyand infertility-related stressors in men and women. The pres-ent study attempted to examine the nature of the relationshipbetween anxiety and sexual infertility stress in men andwomen seeking assisted reproductive treatment (ART). Al-though many studies over the past two decades have focusedon female response to infertility, the present study helps shedlight on the experience of both women and men, particularlyhow anxiety and sexual infertility stress are uniquely relatedfor each gender.
One of the key findings in this study was that subjectiveanxiety (inability to relax, feeling nervous) and autonomicanxiety (feeling hot, sweating, feelings of indigestion) weresignificantly related to sexual infertility stress for both menand women, whereas neurophysiologic anxiety (hands trem-bling, feeling faint) and panic symptoms (heart racing, diffi-
Fertility and Sterility�
culty breathing) were not. These findings highlight theinteraction between subjective perceptions of anxiety, auto-nomic anxiety responses, and sexual infertility stress. It is un-clear why men and women who engage in subjective worryand who react with autonomic arousal are more likely to ex-perience strain in their sexual relationship than men andwomen experiencing neurophysiologic anxiety and panicsymptoms. It may be that neurophysiologic anxiety and par-ticularly panic symptoms represent a more severe anxietyreaction that may be largely independent of and less a conse-quence of the infertility experience. In contrast the mildersubjective and autonomic anxiety symptoms may be com-pounded and heightened as a result of the infertility.
One of the most surprising findings in this study was thestrength of the relationship between anxiety and sexual infer-tility stress for men. It was unexpected that the regressionmodel would explain more of the variance in sexual infertilitystress for men, because men tend to report less sexual infer-tility stress and also less anxiety. Although men report feelingless anxiety than women and less sexual infertility stress,these two variables appear to have a stronger connectionfor men than they do for women.
There are several reasons why the relationship betweenanxiety and sexual infertility stress may be more closely re-lated for men than for women. First, anxiety in men may bemore directly related to sexual performance. Quite possibly,mild subjective worry and autonomic arousal may create ini-tial difficulties in sexual performance. Initial sexual difficul-ties then lead to elevated worry and autonomic arousal andhence to greater sexual difficulties in a self-perpetuatingcycle. Second, although both men and women experiencethreats to their identity because of infertility, a man’s identityand sense of masculinity may be more closely tied to his sex-ual performance rather than his social role (i.e., fatherhood)(9). And third, anxiety related to external expectations andpressures (e.g., a spouse’s insistence that the couple have in-tercourse at the peak of ovulation), could result in decreasedsexual performance and increased sexual stress, whereas forwomen anxiety may be more related to the achievement of
TABLE 2Stepwise multiple regression analyses examining anxiety symptoms as predictors of sexual infertilitystress.
Anxietysymptom
Women’s sexualinfertility stress (n [ 306),
standardized beta
Men’s sexualinfertility stress (n [ 295),
standardized beta
Neurophysiologic 0.05 �0.05Subjective 0.26a 0.29a
Panic �0.08 �0.05Autonomic 0.18a 0.33a
R2 0.14a 0.21a
a Significant at the .01 level.
Peterson. Anxiety and sexual stress. Fertil Steril 2007.
913
a successful outcome of sexual relations (pregnancy) ratherthan the successful completion of sexual intercourse.
Findings from this study may also be important when ex-amining the relational factors that are linked with increasesin stress due to the infertility experience. Although thereare few studies which specifically examine the link betweenrelationship factors and sexual infertility stress, a prospectivelongitudinal study found that 6 months after unsuccessfulIVF treatments, men and women showed greater dissatisfac-tion in their sexual relationship than men and women whoachieved a pregnancy (10). Results from the present studyshow that men and women who report greater subjectiveand autonomic anxiety at the onset of treatment may have in-creased levels of sexual infertility stress. For couples whosetreatments are unsuccessful, the increases in anxiety and sex-ual infertility stress may be exacerbated. Physicians can assistpatients by providing basic education regarding the relation-ship between anxiety and sexual infertility stress and byreferring couples reporting heightened levels of stress tomental health professionals who are knowledgeable about in-fertility-specific stress.
The findings from this study may also have implications toART treatment retention. A recent study found that after con-trolling for financial reasons, psychological stress and poorprognosis were the largest barriers to continued treatment(11). Physicians might use the findings from this study to in-crease their sensitivity to patients experiencing anxiety andsexual infertility stress. Physicians can normalize anxiety re-lated to the sexual relationship that likely occurred duringprevious treatment phases and may consider counseling cou-ples to take a break from scheduled sexual relations as part ofthe ART process. For couples whose sexual relationshipserves as a continued reminder of infertility and their failureto conceive, this break could serve to decrease the sexualstress that is related to infertility.
Finally, the findings from this study must be interpretedwith an awareness of the study’s limitations. First, the rela-tionship between anxiety and sexual infertility stress is beingexamined before ART, and success or failure of the proce-dures may impact future findings. Second, the study did notobtain data on which couples had a male factor versus a fe-male factor diagnosis of infertility. It may be that these find-ings would be more pronounced depending on the type of
914 Peterson et al. Anxiety and sexual stress
diagnosis received, and future research examining this issueis warranted. Third, the directionality of the study findingsmust be interpreted with caution, because increases in sexualinfertility stress may lead to more anxiety in some partici-pants. Future studies using more controlled designs areneeded to explore this relationship further. Fourth, it is impor-tant to note that although the findings from this study showa strong relationship between anxiety and sexual infertilitystress, the majority of men and women in the study reportedclinically normal levels of anxiety and sexual infertility stress(although over one in four women and one in five menreported high levels of sexual infertility stress). And fifth,although the sample was primarily Caucasian, specificdemographic data were lacking, and the relationship betweenanxiety and sexual infertility stress may differ across racialand cultural groups.
REFERENCES1. Newton CR, Sherrard MA, Glavac I. The fertility problem inventory:
9. Dudgeon MR, Inhorn MC. Gender, masculinity, and reproduction: an-
thropological perspectives. Int J Mens Health 2003;2:31–56.
10. Slade P, Emery J, Lieberman BA. A prospective, longitudinal study
of emotions and relationships in in-vitro fertilization treatment. Hum
Reprod 1997;12:183–90.
11. Olivius C, Friden B, Borg G, Bergh C. Why do couples discontinue in
vitro fertilization treatment? A cohort study. Fertil Steril 2004;81:
258–61.
Vol. 88, No. 4, October 2007
Bayesian selection of optimal rules for timingintercourse to conceive by using calendar and mucusBruno Scarpa, Ph.D.,a David B. Dunson, Ph.D.,b and Elena Giacchi, M.D.c
a Department of Statistical Sciences, University of Padua, Padua, Italy; b Biostatistics Branch, National Institute of Environmental
Health Sciences, Research Triangle Park, North Carolina; and c Centre of Study and Research on Natural Fertility Regulation,
Catholic University of The Sacred Heart, Rome, Italy
Objective: To find optimal clinical rules that maximize the probability of conception while limiting the number ofintercourse days required.Design: Multicenter prospective study. Women were followed prospectively while they kept daily records of men-strual bleeding, intercourse, and mucus symptom characteristics. In some cycles, women sought to conceive,whereas in other cycles, they sought to avoid pregnancy.Setting: Four centers providing services on fertility awareness.Patient(s): One hundred ninety-one healthy women using the Billings Ovulation Method. Women were invited toenroll by their instructors if they satisfied the entry criteria. We excluded cycles in which mucus was not recordedon a day with intercourse.Intervention(s): None.Main Outcome Measure(s): Clinically identified pregnancies. There were 161 clinically identified pregnancies in2,536 menstrual cycles from 191 women.Result(s): Our approach relies on a statistical model that relates daily predictors, such as type of mucus symptom,to the day-specific probabilities of conception. By using Bayesian methods to search over a large set of possibleclinical rules, focusing on rules based on calendar and mucus, we found that simple rules that are based ondays within the midcycle calendar interval that also have the most fertile-type mucus symptom present havehigh utility.Conclusion(s): Couples can shorten their time to pregnancy efficiently by timing intercourse on days that the mostfertile-type mucus symptom is observed at the vulva. (Fertil Steril� 2007;88:915–24. �2007 by American Societyfor Reproductive Medicine.)
Key Words: Bayesian analysis, cervical mucus, conception, decision theory, fertility awareness, natural familyplanning, time to pregnancy
Couples in Western countries are postponing childbirth tolater ages (1), with the reasons for delaying including educa-tion, career choice and development, and having a stable re-lationship. A recent Swedish study showed an intentionamong college students to delay marriage (2). However, ascouples age into their late 30s, there is an increasing concernthat they may have difficulty in procreating (1). Couples areoften diagnosed as clinically infertile if they fail to conceivewithin a year of starting an attempt, and many couples worryabout their chances of achieving pregnancy even after a fewmonths of attempting.
To shorten the time to pregnancy (TTP) and reduce the riskof being diagnosed as clinically infertile, couples can attemptto prospectively identify the fertile days of the cycle and time
Received June 28, 2006; revised and accepted December 13, 2006.
Supported in part by the Intramural Research Program of the National
Institutes of Health (Bethesda, Maryland) and by the National Institute
of Environmental Health Sciences (Research Triangle Park, North
Carolina).
Reprint requests: David B. Dunson, Biostatistics Branch, National Institute
of Environmental Health Sciences, Research Triangle Park, North Caro-
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.017 Copyright ª2007 American
intercourse on these days. Because intercourse occurring out-side of the 6-day fertile interval that ends on the day of ovu-lation is unlikely to result in conception (3, 4), coupleswishing to time intercourse on highly fecund days must usea method of identifying days occurring before ovulation. Po-tentially, women may attempt to identify the day of ovulationby using urinary LH surge kits, though these kits can be ex-pensive and may result in both false positives and false neg-atives, each in %9% of cycles (5, 6). In fact, although therehas not been a study in which couples using ovulation testkits based on urinary LH are followed prospectively to assessthe impact of intentional timing using such an approach onthe TTP, there have been studies (e.g., Wilcox et al. [3]) inwhich urinary LH later is assayed from urine that has beencollected from women attempting pregnancy. Dunson et al.(7) show that the day-specific conception probability on dif-ferent days relative to the urinary LH surge peaked with inter-course occurring 1–2 days before ovulation, with theprobability substantially lower on the day of ovulation.Hence, even in cycles in which the true LH surge is accuratelydetected in urine, kits may miss the most fecund day, whichtypically precedes ovulation. There are also various devices
Fertility and Sterility� Vol. 88, No. 4, October 2007 915Society for Reproductive Medicine, Published by Elsevier Inc.
available, such as the ClearPlan Easy Fertility Monitor (Uni-path Diagnostics Company [8]), which identifies the fertileinterval by monitoring of estrogen and LH in the urine.This monitor is promising but remains to be empiricallyvalidated by conception rates, and its expense may be a dis-advantage (8, 9).
An alternative to kits and devices is to use a simple rulebased on self-monitoring of the menstrual cycle and estab-lished symptoms of the fertile days, such as vulvar observa-tions of cervical mucus symptom. The use of such rules toidentify potentially fecund days forms the basis for naturalfamily planning (NFP) methods to avoid pregnancy (10,11) and can similarly be used to identify fertile days by cou-ples attempting pregnancy (12). Widely used means of iden-tifying the day of ovulation and the fertile window includecalendar calculations (13–17) and fertility charting of mucussymptom observed at the vagina (18–21). A recent Europeanstudy on symptothermal methods (22) found that rules com-bining calendar and mucus are somewhat more effective thanmucus alone in identifying the beginning of the fertile inter-val for the purposes of avoiding pregnancy.
Recently, Fehring et al. (23) demonstrated that a particularrule for identifying fertile days prospectively based on mucuscharacteristics compared favorably with the hormonallyidentified fertile days that were predicted by the ClearPlanEasy Fertility Monitor. However, data are not yet availablefor a large sample of couples who use fertility monitors whilealso collecting daily records of mucus and intercourse. Forthese reasons, we focus in this article on choosing the bestclinical rules for conception based on mucus symptom andcalendar.
Wilcox et al. (3) proposed that having intercourse consis-tently two or three times per week will likely result in one ortwo acts of intercourse occurring during the fertile window,and this should be sufficient for couples of normal fertility toconceive. However, this approach may be unsatisfactory forcouples for whom it is difficult to maintain a schedule of reg-ular intercourse constantly throughout the cycle, or for cou-ples who wish to be certain of timing intercourse correctly;for such couples, it would be better to ensure a highfrequency of intercourse during the likely-fertile centralwindow of the cycle close to ovulation. Although datasuggest that some regular pattern of intercourse will resultin a high per-year probability of conception, the highfrequency of intercourse days required consistently through-out the entire cycle over multiple cycles may be unrealisticfor many couples. There potentially may be rules identifyingfertile days that are yet to be found that require fewer inter-course days without decreasing the pregnancy rate.
There has been recent concern (24) about the generalityof results obtained in studies of couples using NFP methods(25, 26). Certainly, without randomization or a comparisongroup, it is not possible to definitively show that intentionaltiming of intercourse with NFP methods causes a reductionin TTP. However, our focus is on using the availableobservational data to search for good rules for timing
916 Scarpa et al. Optimal rules for timing intercourse
intercourse to achieve conception. One hopes that such rulescan later be validated in well-designed clinical trials.
Our analysis relies on new data from a multicenter pro-spective study of Italian users of the Billings OvulationMethod (27), a widely used NFP method based on vulvar ob-servation of mucus symptom. Recent studies of mucus andconception probabilities (21, 28) have used data from theMultinational Fecundability Study (29). Unfortunately, thisearlier study only collected mucus information during daysin the midcycle, as accepted by the symptothermal method(30). Hence, for most of the cycles, there are many dayswith missing mucus information early and late in the cycle.In contrast, the new Italian database has complete informa-tion on mucus symptom on each day of the cycle for a largenumber of cycles (2,755 from 193 women, with 177 cyclesending in conceptions that result in clinically detected preg-nancies). Women also kept daily records of intercourse andmenstrual bleeding. The availability of such complete re-cords for women at risk of pregnancy is a necessity for properevaluation and comparison of different possible rules.
To select optimal rules, we apply a Bayesian decision the-oretic approach in which a utility function is chosen to rewardpregnancies and penalize high frequency of intercourse. Torelate the cycle day and mucus characteristics to the probabil-ity of conception, we use a recently proposed statisticalmodel (31), which generalizes the Barrett and Marshall(32) model to allow for variability among couples in their fer-tility and predictors of fecundability.
As noted by Barrett and Marshall (32) and by Wilcox et al.(3), it is necessary to use a statistical model to predict theprobability of conception in menstrual cycles with multipleacts of intercourse occurring during a potentially fertile phaseof the cycle. Earlier investigators have evaluated the theoret-ical effectiveness of existing rules to avoid conception (e.g.,Arevalo et al. [16], Dunson et al. [21]), relying on combiningestimates of conception probabilities with data on menstrualcycle characteristics. Colombo (33) compared a list of widelyused NFP rules in terms of applicability, acceptability, reli-ability, and effectiveness, whereas Stanford et al. (12) com-pared the physiologic basis of a list of different approachesfor timing intercourses to achieve pregnancy.
Our goal is not to evaluate a particular rule or set of exist-ing rules but instead to search for new rules that are based ex-clusively on calendar or on calendar and mucus observations,selecting the best from among the very large number of pos-sible candidates by using innovative Bayesian statisticalmethods.
MATERIALS AND METHODS
Description of Study Design and Data
These data are drawn from a large study (27) that enrolled193 women, during 1993–1997, from four Italian centers pro-viding services on fertility awareness using the Billings Ovu-lation Method (17). Information on the study was provided at
Vol. 88, No. 4, October 2007
the participating centers, and women were invited to enroll bytheir instructors if they satisfied the entry criteria, which in-cluded the following: experienced in use of the BillingsMethod, married or in a stable relationship, between 18 and40 years of age, had at least one menses after cessation ofbreastfeeding or after delivery, not taking hormonal medica-tion or drugs affecting fertility, and no known permanent in-fertility or illnesses that might cause subfertility in neitherpartner. The women in the study were selected to not be cur-rently using condoms or other methods of contraception. Ifthey started using such methods during the study, the relevantcycles were excluded. In fact, only 33 of 193 women had usedhormonal contraception before entering the study, with only 1woman stopping use within 3 months of the first cycle in thestudy. The protocol was approved by the institutional reviewboard of Fondazione Lanza (Padua, Italy).
At enrollment, the women were given a questionnaire toobtain demographic and reproductive history information.The women then were followed prospectively as they col-lected detailed daily records of vulvar observations of themucus symptom and recorded the days during which inter-course and menstrual bleeding occurred. Because women en-rolled were followed for an arbitrary portion of theirreproductive lives, they included a mixture of avoiders andachievers, with intention status possibly changing across cy-cles from the same woman, but no direct information wascollected on pregnancy intention in each cycle.
The Ovulation Method is based on self-observation ofcharacteristic changes of vulvar sensation and cervical mucusduring the cycle. The estrogen rise from a ripening follicle ineach menstrual cycle stimulates cervical production of fluidmucus and indicates the beginning of fertility. Vulvar obser-vations by women correlate well with the hydration and re-lated biophysical characteristics of cervical mucus, anda fertile type of mucus is necessary for sperm survival andtransport (34–36). Further, changes in mucus hydration in-crease as ovulation approaches and immediately decreasethereafter (17, 37). The peak of the mucus symptom (thatis, the last day of fluid mucus and/or slippery sensation) cor-relates closely with the day of ovulation and allows recogni-tion of the end of the fertile window (23, 38).
The women had received training at the study centers onhow to identify different types of sensation and mucus andwere experienced in the use of the method. Teachers classi-fied each day of the cycle according to a five-point scale ac-cording to the type of mucus symptom described by thewomen. As discussed in Colombo et al. (27), the two mostfertile types of mucus score are very similar clinically. There-fore, we collapsed these into one category, resulting in thefollowing four-point scale: [1] dry; [2] a humid or damp feel-ing; [3] thick, creamy, elastic, whitish moist mucus; and [4]slippery, stretchy, watery, clear mucus. Higher scores indicatehigher levels of estrogenic-type mucus and hence conditionsmore conducive to sperm survival and transport.
It is difficult to formally evaluate the reliability of the re-ported mucus classification and intercourse records. In fact,
Fertility and Sterility�
there have been some previous NFP studies in which inter-course was underreported because only the intercourse actsclosest to the mucus peak were recorded (e.g., World HealthOrganization [39], Trussel and Grummer-Strawn [40]). How-ever, the women enrolled in our study were in the habit ofroutinely recording mucus symptoms and intercourse ona daily chart. In addition, women were informed about theaims of the study and about the importance of recording everyintercourse act. They understood that carefully recording thisinformation would lead to more reliable study results, whichwould potentially help them personally. In addition, a teacherwas responsible for the women, monitoring their data collec-tion and checking in to verify that they had recorded the dailyinformation.
Day 1 of the menstrual cycle was defined as the 1st day offresh red bleeding, excluding any previous days with spot-ting. A conception was assumed in the presence of a preg-nancy ongoing at 60 days from the onset of the last menses,or when, before that term, a miscarriage was clinicallydetected. A detailed description of the study protocol isavailable (27).
Bayesian Statistical Analysis
Most statistical models for relating daily records of inter-course to the probability of conception require a conventionalindicator of ovulation day, such as rise of temperature, mucuspeak, or hormonal observations, for each cycle under consid-eration (31, 41–46). Unfortunately, such models require an-ovulatory cycles and cycles with no clear indicator ofovulation to be discarded, possibly biasing estimation. In ad-dition, there can be problems with measurement error in in-dicators of ovulation day (21, 46, 47). To allow a missingovulation day in the model, Dominik et al. (48) extendedthe day-specific conception model of Zhou and Weinberg(41). Dominik and Chen (49) later modified this approachto model a per-cycle day pregnancy curve, avoiding relianceon potentially error-prone imputation of the unknown ovula-tion day. A drawback of these models is that they do not ac-count explicitly for heterogeneity among couples in theirpregnancy probabilities, instead using a variance adjustmentmethod. Hence, because less-fecund couples may contributemore cycles to a data set, the resulting pregnancy probabili-ties will be biased downward. For these reasons, we use a dif-ferent model that does not require information on timing ofintercourse relative to ovulation, instead relying only on thecycle day and mucus characteristics, but that introducesa woman-specific random effect to accommodate between-cycle dependency that is caused by women who contributemultiple cycles of data.
This model generalizes the model applied by Scarpa et al.(50) to incorporate information on cycle day and mucus ondays other than the intercourse day. In particular, consideringthat intercourse has a significant probability of resulting inconception only if it occurs in a midcycle 6-day fertile interval(3, 4), we divide the cycle into three temporal windows: [1] anearly infertile window during which mucus score is not
917
considered; [2] a midcycle, potentially fecund window duringwhich a daily 1–4 categorization of mucus can have a time-varying effect on the probability of conception; and [3]a late infertile window during which mucus score is againnot considered. In focusing on simple rules that prospectivelyidentify days of the cycle with high fecundability, we rely ona daily mucus score on days within a known midcycle interval.Peak mucus may sometimes occur outside this interval, orthere may be other mucus score 4 days early or late in the cyclethat is informative of fertility. However, using such informa-tion adds to the complexity of the rules considered and isunlikely to improve the prediction of highly fecund days.
Our statistical model does not include an abstinence effect.Although sperm concentration and motility increase in the 1–2 days after ejaculation (e.g., Lunenfeld et al. [51]), the mag-nitude of the improvement in semen parameters caused by anadditional day of abstinence does not appear sufficient to re-sult in a significant increase in the day-specific conceptionprobability (41).
The model that we use allows day-specific conceptionprobabilities to depend nonparametrically on the 1–4 dailycategorization of mucus score type. In Bayesian models, pa-rameters are considered to be random variables, which are as-signed prior distributions that quantify the state of knowledgeabout the parameters before examination of the current data.These prior distributions are then updated by using data fromthe study to obtain posterior distributions quantifying the cur-rent uncertainty in the parameters. The process of updatingrelies on Markov chain Monte Carlo algorithms to implementthe high-dimensional integration, which is required. OurMarkov chain Monte Carlo algorithm is based on a simplemodification of the approach of Dunson and Stanford (31)and is closely related to stochastic search algorithms for sub-set selection of genes in microarray experiments and otherapplications.
Identifying an Optimal Rule
We do not rely on conception probabilities obtained by plug-ging in point estimates of fecundability parameters into a sta-tistical model (as in Arevalo et al. [16]), because this strategypotentially can give misleading results by ignoring estima-tion uncertainty. Instead, we follow a formal Bayesian deci-sion-theoretic approach, relying on Markov chain MonteCarlo methods to remove the unknown parameters by inte-grating them out. This is performed by estimating the poste-rior probabilities associated with different parameters’ valuesand using these probabilities as weights in estimating the ex-pected utility of a rule, as presented in detail by Scarpa andDunson (52). By using computer simulations, we estimate ex-pected utilities for a wide class of rules, assuming perfect useto assess theoretical effectiveness. Rules are then comparedon the basis of their expected utilities, incorporating a rewardfor pregnancy probability and a penalty for intercoursefrequency.
The rule utility function is the sum of two components: [1]the predictive probability of conceiving in a cycle conditional
918 Scarpa et al. Optimal rules for timing intercourse
on the pattern of intercourse allowed by the rule and [2] a pen-alty for the number of prescribed intercourse days. We con-sider a range of values for this penalty and focus on rulesprescribing intercourse on certain days within a midcycle in-terval, including [1] every day, [2] on days with mucus score>1, [3] on days with mucus score>2, [4] on days with mucusscore> 3, [5] on days with mucus score>1 on that day or theday before, [6] on days with mucus score >2 on that day orthe day before, and [7] on days with mucus score >3 onthat day or the day before.
We allowed the last day of the first interval of the cycle tovary between 5 and 12, and the last day of the second interval,to vary between 17 and 25, resulting in 504 different rules.For each rule, we estimated the probability of conception.To apply each rule and to define the intercourse days, the ob-served mucus patterns of the data from the Italian study areused for the rules based on mucus. Couples often do notstrictly follow a rule, so in evaluating rules, we considereddifferent patterns of intercourse acts for prescribed days(for instance, we allow intercourse every prescribed day oronly on half of them), and we assume that intercourse occurson either 1/7 or 0/7 of the days outside of the midcycle inter-val. For each rule, the average number of extra days of inter-course required by the rule also was estimated.
We did not stratify by a woman’s usual cycle length, age, orreproductive history. Although the statistical method easilycan accommodate such information in obtaining individual-ized optimal rules for women having particular characteris-tics, it becomes much more complicated to present anddisseminate this information to couples. Hence, we focusedon optimal selection of rules for couples overall.
RESULTS
When one focuses on menstrual cycles with complete recordsof mucus score and excludes cycles in which mucus was notrecorded on a day with intercourse, in our entire data set(2,755 cycles of 193 women with 177 pregnancies), thereare 2,536 menstrual cycles from 191 women, with 161 ofthese cycles (from 132 women) ending in a clinical preg-nancy. Table 1 presents some simple summaries of the demo-graphic characteristics of the 191 women who produced thecycles of data analyzed, and Table 2 shows the average num-ber of days per cycle with each type of mucus for women indifferent age groups. Although the number of days with themost fertile-type mucus (mucus score of 4) declines some-what with age (all women in the study are between 18 and40 y of age), this trend is modest. There are, however, cleardifferences in mucus according to a woman’s reproductivehistory. As shown in Table 3, as the number of prior pregnan-cies increases, the number of days with no secretions (mucusscore of 1) decreases. The median length of the observednonconception cycles is 28 days, with range between 18and 76 days (first quantile, 27 d; third quantile, 31 d; mean,29.33 d; SD, 4.98 d).
Vol. 88, No. 4, October 2007
TABLE 1Descriptive statistics of the couples in the study.
Parameter Mean Median Interquartile range SD
Female partner’s age 29.95 30 6 4.15Male partner’s age 32.64 32 6 4.76Previous pregnancies 0.99 1 2 1.17No. of cycles for
woman13.28 9 14 12.66
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
We first estimated the timing of the midcycle interval. Infitting the Bayesian model, priors were chosen to assignhigh probability to a wide range of plausible values, withequal probability assigned to each model. The posteriormean (Bayesian estimate updating prior information with in-formation in the data) for the last day of the first interval is 6,and the 95% credible interval (Bayesian equivalent of a con-fidence interval) ranges between days 5 and 8. For the last dayof the second interval, the posterior mean is 21, and the 95%credible interval is between days 19 and 23. These results areconsistent with the interval between days 6 and 21 being con-sidered to involve a high probability of fertile days, as pre-sented by Wilcox et al. (53), and also are similar to thefixed fertile window of days 8–19 that were chosen for a fixedcalendar calculation method (16) that was suggested towomen with cycle lengths between 26 and 32 days. Wealso obtained posterior distributions for the day-specificprobabilities of conception given a single act of intercoursein the cycle, occurring in one of the three phases. For inter-course acts in the second phase, we stratify by the type ofmucus on the intercourse day.
Consistent with earlier analysis of these data and two otherdata sets (28, 50, 54), mucus score does an excellent job ofpredicting the conception probabilities. In the midcycle inter-val, the probability is quite low for days with no secretions(0.01) and with a mucus score of 2 (0.038) or 3 (0.064) butthen increases dramatically to 0.41 on days with most fer-tile-type mucus. These differences are all statistically signif-
Fertility and Sterility�
icant, having posterior probabilities for no difference of
< .05. On days in the first and third phase, the probability
of conception is essentially zero (0.002 and 0.0004). Given
these estimates, mucus characteristics early and late in the
menstrual cycle are unlikely to be very informative about
conception probabilities.
Table 4 presents the results of the optimal rules, assumingthat a normal couple has intercourse only when required bythe rule in the midcycle interval and never within the otherintervals. The first line presents the optimal rule and the cor-responding integrated utility function, initially considered tobe simply the probability of conception without an explicitpenalty for intercourse days. This figure allows one to iden-tify the pregnancy probability and the relative number ofintercourse days associated with each rule. To choose anoptimal rule, it is necessary to weigh the pregnancy probabil-ity against the number of intercourse days required. To for-malize this tradeoff, we estimated the integrated utilityfunction with different penalties, resulting in progressivelyreduced numbers of days on which intercourse is prescribed,while still maximizing the probability of conception.
The remaining rows of Table 4 present results for progres-sively reduced numbers of prescribed intercourse days. Fora very limited number of prescribed intercourse days(mean, 2.42 d per cycle), the optimal rule suggests inter-course between cycle days 13 and 17 on days with a mucusscore of 4. For an intermediate number of 9 prescribed days
TABLE 2Descriptive statistics of the number of days per menstrual cycle with each type of mucus for womenin different age groups (means, with SD in parentheses).
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
919
TABLE 3Descriptive statistics of the number of days per menstrual cycle with each type of mucus according tothe number of previous pregnancies reported by the woman (means, with SD in parentheses).
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
per cycle, the optimal rule suggests intercourse between cycle
days 10 and 18 on days with any fertile type of mucus. Fi-
nally, for couples desiring an intermediate number of pre-
scribed intercourse days, an average of 3.9 days per cycle,
the optimal rule suggests intercourse in the interval between
cycle days 13 to 17 on days with a mucus score of 3 or 4.
By using the probabilities of conception estimated by themodel, we simulated a distribution of the TTP (the numberof cycles of attempts needed by a couple to conceive) forcouples following each of the optimal rules obtained withdifferent penalties. Table 4 also presents some estimatedpercentiles of these distributions.
Table 5 presents results for couples that have intercourseonce per week during the first and third intervals (mostly non-fertile) of the cycle, in addition to the intercourse prescribedby the rules to try to conceive. For high and low levels of pre-scribed intercourse frequency, the choice of the optimal ruleis the same as in the previous case (with no intercourse in the
920 Scarpa et al. Optimal rules for timing intercourse
mostly nonfertile first and third intervals), but the results fora medium frequency of intercourse are slightly different. Inparticular, for a prescribed intercourse frequency of an aver-age of 4.45 days per cycle, the optimal rule recommends in-tercourse within the cycle day 13 to 17 interval on days witha mucus score of >1, whereas for couples not having any in-tercourse outside the prescribed window, a slightly highernumber of prescribed intercourse days (5 d) recommends in-tercourse in the same cycle day 13 to 17 interval without re-gard to mucus score. Overall, having additional intercourseoccasionally in the mostly nonfertile first and third intervalsraises the cycle probability of conception slightly, all else be-ing equal. We estimated that the median waiting time fora couple having intercourse on one seventh of the days of a cy-cle without regard for timing or mucus characteristics isabout four cycles. For couples using the optimal rule pro-posed (Table 5), this time is decreased to one to two cycles.
We also considered the case in which couples do notstrictly follow the rule but instead have intercourse on half
TABLE 4Optimal rules, utility function, and probabilities of conception for couples who strictly follow the rules:intercourse on each day identified by each rule in the midcycle interval and never in the other intervals.
Note: Each row is related to a different penalty, expressed in terms of the decrease in pregnancy probability that one iswilling to face in exchange for each additional abstinence day. The last three columns describe some relevant percentileof the distribution (obtained by simulation) of the number of cycles until conception for couples who use each optimalrule.
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
TABLE 5Optimal rules, utility function, and probabilities of conception for couples who strictly follow theproposed rules: intercourse on each day identified by each rule in the midcycle interval and on1/7th of days in the other intervals.
Note: Each row is related to a different penalty, expressed in terms of the decrease in pregnancy probability that one iswilling to face in exchange for each additional abstinence day. The last three columns describe some relevant percentileof the distribution (obtained by simulation) of the number of cycles until conception for couples who use each optimalrule.
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
of the recommended days. Table 6 presents the optimal rulesfor different penalties, along with percentiles of the TTP dis-tribution. Here, mucus is less important in choosing the opti-mal rule, and the midcycle interval is enlarged.
DISCUSSION
This article used a Bayesian decision-theoretic approach tosearch for optimal rules for timing intercourse to achieveconception. We first considered a biologically based model ofthe day-specific probabilities of conception in the menstrualcycle, incorporating information on timing and vulvar obser-vation of cervical mucus score. Estimating the parametersin this model by using Bayesian methods applied to a newItalian database, we found that conception probabilities wereeffectively 0 outside of a midcycle interval starting on day 7and ending on day 20. Within this midcycle interval, therewas a dramatic increase in the probability of conceptionwith increases in a 1–4 mucus score.
The day-specific probability of conception on a day withmost-fertile type mucus is 40 times higher than that ona day with no noticeable mucus score. Although these initialresults clearly implied that rules based on both calendar andmucus may perform well in predicting fertile days, we pro-ceeded to conduct a systematic search to choose from amongthe enormous number of specific rules. By using a Bayesianapproach, we found that simple rules that are based on in-creasing the frequency of intercourse on days within a mid-cycle interval having a mucus score at or above a thresholdon a 1–4 scale have high theoretical effectiveness.
The optimal width of the midcycle interval and choice ofthreshold depend on the extent to which a couple is willing
Fertility and Sterility�
to face a modest decrease in conception probability in ex-change for a lower frequency of intercourse. In particular,for couples strictly following the rule, placing a high penaltyon additional intercourse days, the optimal rule prescribes in-tercourse between cycle days 13 and 17, only when there ismost-fertile type mucus (mucus score, 4). The average num-ber of required intercourse days is 2.42, and the predictedconception probability in a cycle for couples followingsuch a rule is 0.35, so the median waiting time for conceptionis about three cycles. For couples placing a smaller penalty onintercourse days, the optimal rule prescribes intercourse ondays with mucus type 3 or 4, in the midcycle interval betweendays 13 and 17. The average number of intercourse days is3.92, and the predicted conception probability for couplesfollowing this rule is 0.47, which gives a median waitingtime of about two cycles.
We estimate, using the same data and the same model,that a couple having intercourse on one seventh of thedays of a cycle without regard for timing or mucus char-acteristics has a median waiting time of about four cycles.Optimally utilizing information on calendar and mucus,while assuming an intercourse frequency of one seventhof days not recommended by the rule, the median TTPdecreases to one to two cycles, with a waiting time ofone cycle obtained if a low penalty on the number of in-tercourse days is assumed. These results are for coupleswho have biologic fecundity comparable to that of thesample of women in this study. The impact on TTP of in-tentionally timing intercourse using optimal rules will beless for more fecund couples (couples who have a higherthan average probability of conception in a generic cyclewhen following the same pattern of intercourse) but sub-stantially higher for low-fecundity couples.
921
TABLE 6Optimal rules, utility function, and probability of conception for couples who have intercourse 1/2of the days identified by each rule in the midcycle interval, and either never or 1/7 of the daysin the other intervals.
Rule parametersCycles to pregnancy, by
percentile (n)
Penalty
Midcycleinterval
start
Midcycleinterval
endMucus
type
Cycleprobability
ofconception
Mean no.of prescribedintercourse
days 50% 75% 90%
Never 0 6 23 No 0.630 13.00 1 2 40.01 9 18 No 0.524 7.00 1 3 50.05 13 17 No 0.504 5.00 2 4 70.1 12 17 3,4 0.365 2.41 3 7 15
1/7th of days 0 6 22 No 0.642 13.00 1 2 40.01 9 18 No 0.603 7.00 1 3 50.05 13 17 No 0.525 4.00 2 5 130.1 11 17 No 0.417 2.00 3 9 18
Note: Each row is related to a different penalty, expressed in terms of the decrease in pregnancy probability that one iswilling to face in exchange for each additional abstinence day. The last three columns describe some relevant percentileof the distribution (obtained by simulation) of the number of cycles until conception for couples who use each optimalrule.
Scarpa. Optimal rules for timing intercourse. Fertil Steril 2007.
The identification of wet, slippery, stretchy or clear mu-cus, characterizing most-fertile type mucus, is very usefulfor couples wanting to shorten their TTP, achieving con-ception without requiring a high intercourse frequency.However, for couples who have intercourse on average ev-ery other day during the fertile window and occasionallyduring the rest of the cycle, calendar rules are sufficient,and mucus identification does not give any additionalbenefit.
Given the high degree of heterogeneity in biological fe-cundability and the degree of control that couples exerciseover their procreation, it is impossible to obtain a truly repre-sentative sample of a modern population. The womencurrently using an NFP method are not necessarily represen-tative of the general population of women attempting preg-nancy. However, this is also true of other designs, whichrecruit a convenience sample of couples interested in con-ceiving. It is reassuring that the day-specific conception prob-ability estimates follow a similar pattern to those reported inprevious studies, including Wilcox et al. (55).
Given the nature of our sample, our results apply di-rectly to couples with normal or high-normal fecundityand to cycles of median length of 28–29 days. However,Stanford et al. (12) argue that charting of vaginal dischargeshould be taken as a first step for couples having some dif-ficulty achieving in a timely manner and for women with
922 Scarpa et al. Optimal rules for timing intercourse
a history of irregular or infrequent cycles. This chartingcan serve to identify days with high conception probabili-ties, while also being diagnostic of sources of reproductivedysfunction. Correct classification of mucus requires someeducation of the women; at the beginning of our study,a teacher followed each woman, with the training then al-lowing women to identify easily the most-fertile type ofmucus.
Our short median waiting time (around 1 or 2 cycles)for healthy couples using optimal rules suggests thatone can potentially shorten the time required to diagnoseinfertility by having women chart discharge while usingoptimal rules for timing intercourse (calendar or mucus,depending on frequency of intercourse) to achieve concep-tion. Hilgers (56) suggested that with timed intercourse,a diagnosis of infertility can be established in 6 months,and Gnoth et al. (57) suggested that after 6 months of in-tercourse with fertility-focused rules, a medical evaluationis appropriate.
Unsuccessful timed intercourse is diagnostic of the needfor additional medical evaluation and intervention. The ac-cepted approach is to allow younger couples with no histori-cal risks for infertility %1 year before recommending aninitial evaluation for infertility (58). Our results appear toconfirm what already has been suggested by others (56,57), which is that this interval could be shortened with
Vol. 88, No. 4, October 2007
documentation of appropriately timed intercourse basedexclusively on calendar, or on calendar and mucus, indepen-dently of the peak day.
In future work, it will be useful to consider personalizedrules that incorporate information such as age of the woman,hormone data obtained from fertility monitors, lengths of pre-vious menstrual cycles, or even number of cycles attempting.
Potentially, there may be certain rules that work well forsome couples but not for others. For example, the optimalmidcycle interval may vary depending on whether a womanhas long or short cycles and how regular they are. Also, mu-cus types can have different effects on the probability of con-ception in different women and in different cycles of the samewoman. Incorporating such woman-specific information intothe rule selection process should improve the performance ofthe rule, and potentially software could be developed thatoutputs the optimal rule when a user inputs personal cyclehistory, age, and other characteristics, such as desired inter-course frequency.
Acknowledgments: The authors thank Medua Boioni, R.N., Lorella Miretti,
R.N., and Erika Bucher, M.D., the principal investigators at the three Billings
Teaching Centres of Milano, Saluzzo, and Parma, which jointly with the
Centre of Rome generously provided the data; and they thank Bernardo
Colombo, M.S., Joe Stanford, Ph.D., and Rene Ecochard, M.D., Ph.D., for
their suggestions and comments on a first draft of the article.
REFERENCES1. Pinnelli A, Di Cesare M. Human fertility: socio-demographic aspects.
Contraception 2005;72:303–7.
2. Stobel-Richter Y, Beutel MF, Finck C, Brahler E. The wish to have
a child, childlessness, and infertility in Germany. Hum Reprod 2005;
20:2850–7.
3. Wilcox AJ, Weinberg CR, Baird DD. Timing of sexual intercourse in re-
lation to ovulation: effects on the probability of conception, survival of
the pregnancy and sex of the baby. N Engl J Med 1995;333:1517–21.
of conception with a single act of intercourse: providing benchmark rates
for assessment of post-coital contraceptives. Contraception 2001;63:
211–5.
56. Hilgers TW. The medical applications of natural family planning: a con-
temporary approach to women’s health care. Omaha, NE: Pope Paul VI
Institute Press, 1991.
57. Gnoth C, Godehardt E, Frank-Herrmann P, Friol K, Tigges J, Freundl G.
Definition and prevalence of subfertility and infertility. Hum Reprod
2005;20:1144–7.
58. Practice Committee of the American Society for Reproductive Medicine.
Birmingham, AL: American Society for Reproductive Medicine, 2000.
Vol. 88, No. 4, October 2007
Detection of antizona pellucida antibodiesin the sera from premature ovarian failurepatients by a highly specific testSatoru Takamizawa, M.D., Hiroaki Shibahara, M.D., Ph.D., Tamaho Shibayama, M.D.,and Mitsuaki Suzuki, M.D., Ph.D.
Department of Obstetrics and Gynecology, School of Medicine, Jichi Medical University, Tochigi, Japan
Objective: To develop a highly specific test for the detection of antizona pellucida (ZP) antibodies in the sera frompremature ovarian failure (POF) patients.Design: Laboratory study.Setting: University hospital.Patient(s): Twenty-seven idiopathic POF patients, 30 control women, and 30 healthy males.Intervention(s): Anti-ZP antibodies were detected by the microdot assay using a very small amount of human ZPor porcine ZP. The effect of anti-ZP antibodies on sperm-ZP binding was examined by hemizona assay.Main outcome measure(s): Results from the microdot assay and hemizona assay.Result(s): By the microdot assay using human ZP, the sera from POF patients reacted significantly stronger thanthose of control women and healthy males. However, no obvious difference could be found by the same assay usingporcine ZP among these three groups. Anti-ZP antibodies against sera from some POF patients showed significantblocking effects on sperm-ZP binding assessed by hemizona assay. Anti-ZP antibodies were detected in 7 of 27POF patients, while none were detected in control women and healthy males.Conclusion(s): Some idiopathic POF patients have anti-ZP antibodies in their sera, which were detected withhigh specificity by a newly developed microdot assay using a very small amount of human ZP. (Fertil Steril�
2007;88:925–32. �2007 by American Society for Reproductive Medicine.)
Premature ovarian failure (POF) is a syndrome characterizedby development of amenorrhea before the age of 40 yearswith elevated serum gonadotropin levels and low serum es-trogen levels (1). It occurs in 1% of women, in 10%–28%of women with primary amenorrhea, and in 4%–18% of thosewith secondary amenorrhea (2, 3).
The etiology of POF is thought to involve a wide spectrumof pathogenic mechanisms including chromosomal, genetic,environmental (radiation or medicine), metabolic, and auto-immune factors (4). In the past 3 decades, some investigatorshave reported the clinical association of POF with other auto-immune disorders. Approximately 20%–40% of patientswith POF have associated autoimmune disorders (4–7). Ina large proportion of cases no cause has been identified,and these cases are classified as idiopathic POF. Autoimmunemechanisms are involved in the pathogenesis of up to 30% ofcases in idiopathic POF (8, 9).
The presence of organ-specific autoimmune antibodiesmay support the role of autoimmune mechanisms in endo-crine diseases. For example, antiovarian antibodies inpatients with POF were detected by different methods, themost common being indirect immunofluorescence (IF) and
Received July 21, 2006; revised and accepted December 22, 2006.
Reprint requests: Hiroaki Shibahara, M.D., Ph.D., Department of Obstet-
rics and Gynecology, School of Medicine, Jichi Medical University,
Yakushiji 3311-1, Shimotsuke, Tochigi 329-0498, Japan (FAX: 81-
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.029 Copyright ª2007 American
enzyme-linked immunosorbent assay (ELISA). Numerousinvestigators have tested POF patient groups using IF,ELISA, and other methods. The prevalence of antiovarian an-tibodies ranges widely, between 2.2% and 69% of patients(10–16). Considering these variable results, the pathogenicroles of antiovarian antibodies in POF are still uncertain interms of their specificity. Such conflicting results could be ex-plained by the methodologic differences, relatively smallnumber of patients, the different stages of the disease whentested, the differences of antigen sources, and the multiplicityof potential immune targets that comprised various steroido-genic enzymes, gonadotropins, and their receptors: the corpusluteum, oocyte, and zona pellucida (ZP) (9, 17).
Zona pellucida consists of glycoproteins that have strongantigenic potency. Therefore, anti-ZP antibodies may beone of the causes of autoimmune POF. Evidence showingthat antibodies directed to ZP could cause POF has been dem-onstrated. Ovarian failure could be induced in rabbits immu-nized with porcine ZP proteins (18). Ovarian autoimmunedisease was induced in B6AF1 mice by 15-amino acid pep-tides from mouse ZP3 (19). Because ZP proteins are con-served among mammals (mouse and human ZP3 proteinsare 67% identical), those animal models may lead to a betterunderstanding of the pathogenesis of human autoimmune oo-phoritis. Circulating antibodies against ZP were consistentlydetected by immunohistochemical assay in mice with auto-immune oophoritis that could be induced with high incidenceby thymectomy at 3 days of age and caused great oocyte loss
Fertility and Sterility� Vol. 88, No. 4, October 2007 925Society for Reproductive Medicine, Published by Elsevier Inc.
(20, 21). Smith and Hosid (22) reported two cases of POFassociated with antibodies directed against ZP .
After antigenic crossreactivity between human and porcineZP was revealed (23, 24), porcine ovaries and oocytes havebeen generally used to detect anti-ZP antibodies becausethey are easy to obtain in large quantities. Anti-ZP antibodieshave also been discussed as a possible cause of infertility inwomen for the blocking effect on sperm-ZP binding. Somestudies reported that anti-ZP antibodies were detected withhigh incidence in infertile women by IF using porcine oo-cytes and ZP (24–26). However, the specificity of immune re-action with porcine oocytes has been questioned because ofthe nonspecific reaction of porcine ZP. To have better speci-ficity in the detection of anti-ZP antibodies using porcine ZP,passive hemagglutination reaction or absorptive treatmenttechniques were developed (27–29). Although such passivehemagglutination reaction or absorption techniques contrib-uted to better specificity and reliability for detecting anti-ZP antibodies, the significance of these antibodies remainsunclear because of possible nonspecific reactions.
Therefore, we concluded that it is necessary to developa highly specific test for the detection of anti-ZP antibodiesusing human ZP. However, one limitation is that it is still dif-ficult to obtain human ZP in large quantities, even in the eraof modern assisted reproductive technology. In the presentstudy, a microdot assay using a very small amount of humanZP was developed. Moreover, sera containing anti-ZP anti-bodies from patients with idiopathic POF patients wereused to investigate the blocking effect on sperm-ZP binding.
MATERIALS AND METHODS
Approval for the study was obtained from the InstitutionalEthics Committee of Jichi Medical University Hospital, andinformed consent was obtained from all patients.
Serum Samples
Serum samples were collected from 27 women diagnosed withidiopathic POF. Criteria for POF included secondary amenor-rhea, age <40 years at the onset of ovarian failure, persistenthigh serum gonadotropin levels, and low serum estrogenlevels (1). In addition, to classify as idiopathic POF, patientswith abnormal karyotype, previous pelvic irradiation, opera-tive castration, and previous cytotoxic chemotherapy were ex-cluded, and patients with no cause identified were selected (8).
As the control, sera from 30 fertile females without disor-der on ovulation and fertilization were obtained. Women withregular ovulatory cycle and experience of conception, whohad undergone at least two cycles of IVF treatment with totalnumber of oocytes retrieved >10 and 100% fertilization ratein their IVF cycles, were defined as the control women. Serafrom 30 normally healthy volunteer males were also obtainedas the negative control. Because anti-ZP antibodies wereautoimmune antibodies, there was a possibility that normalcycling women had these antibodies. On the other hand,healthy males were not expected to have these antibodieslogically and suitable for the true negative control.
926 Takamizawa et al. Detection of anti-ZP antibodies in P
Whole serum was diluted 1:4 in phosphate-buffered saline(PBS) without calcium containing 3% bovine serum albuminas the test serum sample.
Preparation of Human and Porcine ZP
Under informed consent, human oocytes that failed to fertil-ize in vitro or that were not used because of being immaturewere obtained from infertile couples that had undergone as-sisted reproductive technology. They were stored until useat 4�C in a solution of 0.5 M ammonium sulfate with 1 Mmagnesium chloride and 0.1% dextran. After hundreds of hu-man oocytes were stored, they were put to use.
Porcine ovaries obtained from a local slaughterhouse werefrozen and stored. The porcine oocytes were collected fromthawed porcine ovaries by aspiration using syringes andneedles. Several sets of ten oocytes from one ovary couldbe collected at one time.
Human and porcine ZP were individually separated fromtheir cytoplasm mechanically by pipetting with a narrowglass pipette with a caliber smaller than the oocyte diameter.They were then placed in PBS (100 mg/mL ¼ 100 ZP/30 mLPBS) at 75�C for 30 minutes.
Microdot Assay
The microdot assay is an immunostaining method, and anti-ZP antibodies in the sera can be detected when combinedwith soluble ZP proteins used for antigen.
A small piece of nitrocellulose membrane measuring 1.5cm in width and 1.0 cm in length was divided into six areaswith two vertical lines and one horizontal line. On each ofthe upper panels, a microdot was made with serial dilution(�1, �3, �9) of 0.2 mL soluble human ZP, while on that ofthe lower panels, it was made with soluble porcine ZP. Ap-proximately 0.7 ZP, 0.2 ZP, and 0.07 ZP were contained inone dot of 0.2 mL soluble human and porcine ZP with serialdilution �1, �3, and �9, respectively. After drying, theywere blocked in PBS containing 3% bovine serum albumin(Sigma Chemical Co., St. Louis, MO) for 10 minutes. Asa primary antibody reaction, 180 mL of the test serum wasadded on the nitrocellulose membrane. After incubation ofthe nitrocellulose membranes with patients’ sera in a moistchamber at 4�C overnight, they were washed three times inPBS containing 0.02% Tween 20 (Kanto Chemical Co.,Inc., Tokyo, Japan, 40350-32, polyoxyethylenesorbitanmonolaurate, nonionic detergent) for 5 minutes. The excessPBS containing 0.02% Tween 20 was aspirated off, thenthe nitrocellulose membranes were incubated in horseradishperoxidase-conjugated antihuman IgG (Sigma ChemicalCo.) diluted 1:1,000 in PBS as a second reaction at room tem-perature for 1 hour. Then, they were washed three times inPBS containing 0.005% Tween 20 for 5 minutes. The dotson the nitrocellulose membranes were colored and visualizedby chloronaphthol, 3 mg of which was dissolved in 1 mL ofmethanol with 5 mL of PBS and 2 mL of H2O2 (Kanto
OF patients Vol. 88, No. 4, October 2007
Chemical Co., Inc.). The nitrocellulose membranes werewashed in distilled water and dried.
Analysis
The color development of each dot was evaluated by com-puter-assisted image analysis, and expressed the existenceof anti-ZP antibody in the sera. After scanning the nitrocellu-lose membranes using a flat bed scanner in 256 grayscales,the density of staining was measured by NIH image (a publicdomain image processing and analysis program for the Mac-intosh, developed at the National Institutes of Health [NIH],USA). NIH image number (NIN) was calculated by subtract-ing the density of the background stain from that of the dotstain. All serum samples were assayed, and then the NIN ofeach dot was calculated and compared.
Hemizona Assay
The hemizona assay (HZA) was developed originally to pre-dict the fertilizing potential of spermatozoa (30). The effect ofanti-ZP antibodies in the sera from POF patients on sperm-ZPinteraction was investigated by the HZA as follows. A pair ofhemizona (HZ) was made of one oocyte cut into halves byhand using biocut blade (Feather Safety Razor Co., Ltd.,Osaka, Japan), a microblade for biotechnology bisection, ona phase contrast microscope. One HZ was placed in a 50 mLdrop of each test serum from a POF patient under mineraloil for 1 hour at 37�C in 5% CO2 in air, while the othermatched HZ was placed in a drop of the serum from a healthymale selected as a control. After incubation, each HZ waswashed five times in the culture medium and inseminatedby swim-up sperm obtained from proven fertile healthymales. Semen was centrifuged and washed twice in sperm-washing medium after liquefaction. The motile spermatozoawere collected by a standard swim-up technique and resus-pended in the culture medium. Each preincubated HZ wasexposed to 50 mL of sperm suspension (2.5 � 105/mL) andcoincubated for 1 hour at 37�C in 5% CO2 in air. After insem-ination, each HZ was removed and rinsed vigorously to detachloosely associated spermatozoa. The number of spermatozoatightly bound to the outer HZ surface was counted.
The hemizona index (HZI) was calculated as follows: (thenumber of spermatozoa bound to HZ preincubated with POFpatient’s serum) divided by (the number of spermatozoabound to HZ preincubated with control serum) � 100. Serafrom POF patients that blocked sperm-ZP tight binding atleast 50% (HZI <50) using HZA were considered to havea blocking effect on fertilization (31). Those sera were alsoconsidered to have the anti-ZP antibodies.
Sera from representative 10 of 27 POF patients were se-lected for sampling and used for the analysis by HZA.
Statistical Methods
Student’s t test and chi-square test were used for data analy-sis, and P<.05 was considered statistically significant. Statis-tical analysis was performed with Excel statistical softwarefor Macintosh.
Fertility and Sterility�
RESULTS
Characteristics of Idiopathic POF Patients
The characteristics of 27 idiopathic POF patients were asfollows. In the POF patients, serum FSH levels were 95.6 �38.0 mIU/mL, and 10 of them showed serum FSH levels>100 mIU/mL. On the other hand, serum estradiol levelswere as low as those in postmenopausal women. Notably,19 patients had serum estradiol levels under the lowest limitof examination (<10.0 pg/mL). The age at the onset of amen-orrhea of the POF patients was 23–38 years old (mean 30.0years).
Other autoimmune antibodies: antinuclear antibodies,lupus anticoagulant antibodies, antithyroid antibodies, anti-double-stranded DNA antibodies, rheumatoid factors, andanticardiolipin and anti-beta2-glycoprotein-1 antibodies,were also examined (data not shown). There was no relationamong these autoimmune antibodies. In final, about theseautoimmune antibodies, there was no characteristic in thePOF patients with anti-ZP antibodies compared with otherswithout anti-ZP antibodies.
Results of the Microdot Assay
The nitrocellulose membrane after the microdot assay of theserum from an idiopathic POF patient is shown in Figure 1.The dots on the upper panels demonstrated reaction to solublehuman ZP, and those on the lower panels demonstrated reac-tion to soluble porcine ZP. The density of dots were staineddose dependently in both panels.
NIN after the microdot assay using soluble human ZP andsoluble porcine ZP are presented in Figure 2. The NIN(14.0 � 14.2; mean � SD) of the sera from POF patientswas significantly higher (P<.01) than both that (5.6 � 5.9)of control women and that (0.2 � 1.4) of healthy males bythe microdot assay using soluble human ZP. There was alsosignificant difference (P<.01) between the NIN of controlwomen and that of healthy males (Fig. 2A). In contrast, therewas no significant difference among the NIN (36.2� 23.9) ofPOF patients, that (23.9 � 21.1) of control women, and that(25.8 � 22.1) of healthy males by the microdot assay usingsoluble porcine ZP (Fig. 2B). These results represent thatthe microdot assay using soluble human ZP is superior inspecificity to the assay using soluble porcine ZP to detectanti-ZP antibodies in the sera.
NIN of the sera from POF patients, control women, andhealthy males by the microdot assay using soluble humanZP were compared (Fig. 3). None of the sera from healthymales reacted with soluble human ZP except one with verylow NIN (7.4). Sera from 12 of 30 (40%) control womendid not react (NIN ¼ 0); all the other 18 sera reacted withlow NIN (<18), and the highest NIN of them was 17.0. Fewer(7 of 27, 26%) sera from POF patients did not react, whileseven other POF patients strongly reacted with high NIN(>18). The NIN of control women was 5.6 � 5.9 (mean �SD) and the NIN of meanþ2SD of them was 17.5. Therefore,the sera showed their NIN above 17.5 by the microdot assay
927
using soluble human ZP would be decided as positive for themicrodot assay.
Results of HZA and Criterion of Value Point of NIN
All HZI and NIN of sera from representative 10 (Fig. 3; Band C) of 27 POF patients are presented in Table 1.
For example, the sera from a POF patient (Table 1; code5593017) with anti-ZP antibodies demonstrated a blockingeffect on sperm-ZP tight binding and the test result is shownin Figure 4. The number of spermatozoa bound to the HZ (A)treated with the patient’s serum and HZ (B) treated with thecontrol serum were 49 and 104, respectively. The HZI (hemi-zona index) was calculated as follows: 49/104� 100¼ 47.1.
The value point of NIN that presented anti-ZP antibodiespositive was defined as follows. Sera from three (Fig. 3; B)of the representative 10 patients inhibited sperm-ZP tightbinding (HZI<50), and they were considered to have anti-ZPantibodies. The other seven patients (Fig. 3; C) had HZI>50, and they were not considered to have anti-ZP anti-bodies. The NIN of the sera from these three POF patientswas 39.6 � 26.4, while that of the other seven POF patientswas 8.8 � 6.7. There was a significant difference in NIN be-tween the two groups (P¼.01) (Fig. 5). The lowest NIN ofthese three POF patients considered to have anti-ZP anti-
FIGURE 1
NC membrane after microdot assay. Microdots weremade with a serial dilution (�1, �3, �9) of 0.2 mLsoluble human ZP on each area of the upper panelsof the nitrocellulose membrane (1–3), while they weremade with soluble porcine ZP on that of the lowerpanels (4–6). Approximately 0.7 ZP, 0.2 ZP, and 0.07ZP were contained in one dot of 0.2 mL solublehuman and porcine ZP with a serial dilution of �1,�3, and �9, respectively. This figure was reactedwith the serum from a POF patient. The density ofdots were stained dose dependently in both panels.
Takamizawa. Detection of anti-ZP antibodies in POF patients. Fertil Steril 2007.
928 Takamizawa et al. Detection of anti-ZP antibodies in P
bodies was 18.3, and the highest NIN of these seven POFpatients considered not to have anti-ZP antibodies was 16.9.On the other hand, as mentioned above, the NIN of controlwomen was 5.6 � 5.9 (mean � SD) (0�17.0), and the NINof meanþ2SD was 17.5. Based on these results of the micro-dot assay and HZA, the sera from POF patients, whichshowed their NIN above 18 by the microdot assay using sol-uble human ZP, were decided as positive for anti-ZP anti-bodies, which had obvious blocking effects on sperm-ZPbinding. Therefore, the other 4 of 17 sera (Fig. 3; > andA), that showed their NIN above 18 by the microdot assay,were considered as anti-ZP antibodies positive according tothe established criteria. Taken together, 7 of 27 POF patientswere considered to be anti-ZP antibodies positive (Fig. 3; Band >).
DISCUSSION
Numerous studies have reported that autoimmunity was asso-ciated with POF, especially with idiopathic POF (4–16).Antiovarian antibodies were considered one of the causesof idiopathic POF, and they were detected using variablemethods (5–14). Although the pathology and mechanism ofantiovarian antibodies on POF is still unclear, it is likelythat autoimmunity and these antibodies contribute to theetiology of idiopathic POF. In those antiovarian antibodies,anti-ZP antibodies most likely cause POF, and some mammalexperimental models support this hypothesis (18–20). Somestudies have reported the existence of antibodies directedagainst ZP in human POF patients (22, 32). Being associatedwith autoimmune disease, POF patients might have produc-tive ability of any autoimmune antibodies. Anti-ZP anti-bodies might also be one of the autoimmune antibodies thatwere produced by POF patients. We suppose anti-ZP anti-bodies are mainly the cause, and also might be the result ofPOF.
However, to detect anti-ZP antibodies, a commercial kitwas used with monkey ovaries and porcine ZP, not humanZP as the antigen. Questions of reliability still remained. Be-cause the microdot assay that we developed used human ZPas the antigen, the anti-ZP antibodies detected by microdotassay in the sera from idiopathic POF patients were reliable.It is certain that anti-ZP antibodies are related to the causeand result of POF.
In some infertile women, anti-ZP antibodies are also con-sidered to take part in infertility because of their blocking ef-fects on sperm-ZP binding. Zona pellucida is composed ofsome biochemical distinct proteins and surrounds the mam-malian oocyte. Zona pellucida proteins are considered toact as the sperm receptor in fertilization. Therefore, anti-ZPantibodies are considered to act as the inhibitor to the recep-tor. Monoclonal antibodies to a glycoprotein family of por-cine ZP showed a significant blocking effect on humansperm binding and penetration of human ZP (33). Antibodiesagainst bonnet monkey ZP proteins significantly inhibitedthe binding of human spermatozoa to ZP in HZA (34).
OF patients Vol. 88, No. 4, October 2007
FIGURE 2
Comparison of NIN of POF patients, control women, and healthy males by the microdot assay using solublehuman and porcine ZP. The NIN (NIH image number: 14.0� 14.2; mean� SD) of the sera from POF patients wassignificantly higher (P< .01) than both that (5.6 � 5.9) of control women and that (0.2 � 1.4) of healthy males bythe microdot assay using soluble human ZP. There was also a significant difference between the NIN of controlwomen and that of healthy males (A). In contrast, there was no significant difference among the NIN (36.2� 23.9)of POF patients, that (23.9 � 21.1) of control women, and that (25.8 � 22.1) of healthy males by the microdotassay using soluble porcine ZP (B). These results indicate that the microdot assay using human ZP is specific,while that using porcine ZP is not specific.
Takamizawa. Detection of anti-ZP antibodies in POF patients. Fertil Steril 2007.
Preexposure of human ZP to the sera showing positive IFgreatly diminished the number of spermatozoa of normalquality that bound to and penetrated across human ZP (28).In our study, sera from some idiopathic POF patients withanti-ZP antibodies detected by the microdot assay using sol-uble human ZP showed strong blocking effects on sperm-ZPbinding by the HZA (Table 1). These findings suggest that hu-man anti-ZP antibodies also had inhibitory effects on sperm-ZP binding, and might be one of causes of infertility.
Papale et al. (35) showed low fertilization rates in patientswith anti-ZP antibodies in IVF treatment cycles. Mardesicet al. (36) reported that fertilization failure after standardIVF occurred in most cases with anti-ZP antibodies, and in-tracytoplasmic sperm injection was the method of choice inanti-ZP antibody-positive infertile couples. Although theydetected anti-ZP antibodies by the hemagglutination test,the passive hemagglutination test, or ELISA, as far as theyused porcine antigenic fractions and porcine ZP as the anti-gen, their results likely had problems in reliability and accu-racy. In our study, anti-ZP antibodies were detected in thesera from idiopathic POF patients, control women, andhealthy males by the microdot assay using both soluble hu-man and porcine ZP. No obvious difference could be found
Fertility and Sterility�
among the three groups in their reactions to porcine ZP. Therewas no difference among their NIN by the microdot assay us-ing soluble porcine ZP (Fig. 2B). However, by the microdotassay using soluble human ZP, the NIN of sera from idio-pathic POF patients was significantly higher than that of con-trol women and that of healthy males (Fig. 2A). No sera fromhealthy males reacted (NIN¼ 0) to human ZP proteins exceptonly one with very low NIN (7.4) (Fig. 3). Sera from 12 of 30(40%) control women did not react (NIN ¼ 0); all the other18 sera reacted with low NIN (<18). Fewer (7 of 27, 26%)sera from POF patients did not react, while seven otherPOF patients strongly reacted with high NIN (>18). Theseresults clearly indicate that the microdot assay using solublehuman ZP is specific, while that using soluble porcine ZP isnot specific for the detection of anti-ZP antibodies. The scien-tific basis of the specificity of reactivity to human ZP and thenonspecificity of reactivity to porcine ZP is not certain yet. Itwould be expected to be clear by further molecule biologicalstudies in the future.
Zona pellucida is an extracellular matrix surrounding themammalian oocyte, and it plays an important role in attach-ment of sperm, fertilization, inhibition of polyspermic fertil-ization, and protection of early embryos. It is very important
929
FIGURE 3
NIN of POF patients, control women, and healthymales and the value point of NIN by the microdotassay using soluble human ZP. NIN (NIH imagenumber) of the sera from POF patients (B, C, >,A), control women (-), and healthy males (:) bythe microdot assay using soluble human ZP werecompared. None of the sera from healthy malesreacted with soluble human ZP, except only one withvery low NIN (7.4). Sera from 12 of 30 control womendid not react (NIN¼ 0); the other 18 sera reacted withlow NIN (<18), and the highest NIN was 17.0. TheNIN of control women was 5.6 � 5.9 (mean � SD),and the NIN of mean þ2SD was 17.5. There werefewer (7 of 27) sera from POF patients who did notreact, while some other POF patients stronglyreacted with high NIN (>18). Hemizona assay wasperformed using sera from a representative 10 (Band C) of 27 POF patients, and the HZI wascalculated. Sera from 3 (B; NIN: 69.1, 31.3, 18.3) ofa representative 10 patients inhibited sperm-ZP tightbinding (HZI<50), and they were considered to haveanti-ZP antibodies. The other seven patients (C;NIN: 16.9, 15.5, 10.6, 10.1, 8.7, 0, 0) had HZI >50,and they were not considered to have anti-ZPantibodies. The lowest NIN of these three POFpatients was 18.3, and the highest NIN of these sevenPOF patients was 16.9. According to these results,the value point of NIN was defined as 18.0,represented by the dotted line in the figure. The serafrom POF patients who showed their NIN above 18by the microdot assay using soluble human ZP weredecided as positive for anti-ZP antibodies, which hadobvious blocking effects on sperm-ZP binding. Theother 17 POF patients (> and A) were divided intotwo groups of anti-ZP antibodies positive (>) andnegative (A) according to the established criteria.
Takamizawa. Detection of anti-ZP antibodies in POF patients. Fertil Steril 2007.
930 Takamizawa et al. Detection of anti-ZP antibodies in P
to investigate anti-ZP antibodies that might have inhibitoryeffects on these ZP functions. All the past reported studies in-cluded great methodologic heterogeneity, such as the type ofantiovarian or anti-ZP antibodies, the timing of test and bloodsample collection, the selection of control groups, and so on.Therefore, no definitive conclusions regarding the incidenceand the role of anti-ZP antibodies in POF and infertile womencould have been drawn yet. Such indefinite and conflicting re-sults are attributed to the use of porcine or other mammalianZP for antigens, involving problems with specificity, insteadof human ZP. To resolve these problems, it is necessary to de-velop a new method for the detection of anti-ZP antibodiesusing human ZP as the real antigen. As human oocytes orZP are difficult to obtain in a large quantity, we developeda microdot assay that uses a very small amount of ZP. Inthis microdot assay, approximately 1.5 serum samples perone ZP could be tested on calculation. Less soluble ZP wasused to make the microdots, more serum samples could betested. By using this method, large amounts of homogenousZP antigen was available, and soluble ZP antigen in equalcondition was supplied to the microdot assay wheneveranti-ZP antibodies were detected.
Some idiopathic POF patients demonstrated strong reac-tions to human ZP by the microdot assay, suggesting thatthe anti-ZP antibodies detected by the microdot assay mightbe a cause of POF. There was no difference in age, serum LH,and FSH levels between the two groups of idiopathic POF pa-tients with or without anti-ZP antibodies. There was no cor-relation between other autoimmune antibodies and anti-ZPantibodies (data not shown). It is impossible to predictwhether anti-ZP antibodies are positive or not in the serafrom idiopathic POF patients by their characteristics.
TABLE 1HZI and NIN of the sera from 10 POF patients.
Patient code HZIa NINb
With inhibitory effect on zona bindingc
6060420 35.7 31.36160428 42.9 18.35593017 47.1 69.1Without inhibitory effect on zona bindingd
Takamizawa. Detection of anti-ZPantibodies in POF patients. Fertil Steril 2007.
OF patients Vol. 88, No. 4, October 2007
FIGURE 4
A pair of HZ after HZA using the sera from POF patients with anti-ZP antibodies positive. A pair of HZ after HZAthat demonstrated a blocking effect of serum from a POF patient (Table 1, code 5593017) with anti-ZP antibodieson sperm-ZP tight binding is shown. Before HZA, HZ (A) was preincubated with the serum from a POF patient(Table 1, code 5593017) with high NIN and was considered to be anti-ZP antibodies positive. The other matchedHZ (B) was preincubated with control serum from one healthy male selected as being anti-ZP antibodiesnegative. There were fewer spermatozoa bound to HZ (A) preincubated with the sera from the POF patient withanti-ZP antibodies than HZ (B) preincubated with control serum. The number of spermatozoa bound to HZ (A)and HZ (B) were 49 and 104, respectively. The HZI was calculated as follows: 49/104 � 100 ¼ 47.1.
Takamizawa. Detection of anti-ZP antibodies in POF patients. Fertil Steril 2007.
FIGURE 5
Comparison of NIN of POF patients with HZI <50and with HZI R50. The NIN (NIH image number) ofthe sera from three POF patients with HZI <50 was39.6 � 26.4 (mean � SD), while that of the otherseven POF patients with HZI R50 was 8.8 � 6.7(mean � SD). There was a significant difference ofNIN between the two groups (P¼ .01).
Takamizawa. Detection of anti-ZP antibodies in POF patients. Fertil Steril 2007.
Fertility and Sterility�
There were some idiopathic POF patients whose serashowed increased NIN by the microdot assay using solublehuman ZP and strong blocking effect on sperm-ZP bindingby HZA simultaneously (Fig. 5). These idiopathic POF pa-tients were considered to have anti-ZP antibodies in theirsera. The antibodies could be detected specifically by the mi-crodot assay using soluble human ZP, and might be one of thecauses of infertility with blocking effects on sperm-ZP bind-ing. The microdot assay using soluble human ZP seems to beuseful for detecting anti-ZP antibodies that are associated notonly with the cause of idiopathic POF but also with sperm-ZPinteraction. Further studies are required to show whether themicrodot assay using soluble human ZP is useful for indicat-ing that some unexplained infertility in women with a historyof fertilization failure in IVF treatment cycles might haveanti-ZP antibodies.
Acknowledgements: We thank Prof. Koji Koyama and Dr. Minoru Shigeta for
helpful comments, and Ms. Akiko Hasegaswa for technical advise.
REFERENCES1. de Moraes-Ruehsen M, Jones GS. Premature ovarian failure. Fertil Steril
Mikova M, et al. The influence of different types of antibodies on in vitro
fertilization results. Am J Reprod Immunol 2000;43:1–5.
POF patients Vol. 88, No. 4, October 2007
REPRODUCTIVE SURGERY
A multicenter randomized, controlled study comparinglaparoscopic versus minilaparotomic myomectomy:reproductive outcomesStefano Palomba, M.D.,a Errico Zupi, M.D.,b Angela Falbo, M.D.,a Tiziana Russo, M.D.,a
Daniela Marconi, M.D.,b Achille Tolino, M.D.,c Francesco Manguso, M.D.,d Alberto Mattei, M.D.,e
and Fulvio Zullo, M.D.a
a Department of Obstetrics & Gynecology, University ‘‘Magna Graecia’’ of Catanzaro, Catanzaro; b Department of Obstetrics &
Gynecology, University ‘‘Tor Vergata’’ of Rome, Rome; c Department of Obstetrics & Gynecology, and d Department of Clinical
and Experimental Medicine, University ‘‘Federico II’’ of Naples, Naples; and e Department of Obstetrics & Gynecology,
University of Florence, Florence, Italy
Objective: To assess the reproductive outcomes after minilaparotomic and laparoscopic myomectomy in patientswishing to conceive.Design: Randomized controlled trial.Setting: Departments of obstetrics and gynecology of the universities of Catanzaro, Rome, and Florence, Italy.Patient(s): One hundred thirty-six women with symptomatic uterine leiomyomas or unexplained infertility.Intervention(s): Laparoscopic and minilaparotomic myomectomy.Main Outcome Measure(s): Pregnancy, abortion, and live-birth rates.Result(s): Between the laparoscopic and minilaparotomic groups no difference was observed in cumulative preg-nancy, live-birth, and abortion rates, whereas pregnancy and live-birth rates per cycle, and time to first pregnancyand live-birth were significantly higher in the laparoscopic than in the minilaparotomic group. Categorizing thepatients according to surgical indication for myomectomy, cumulative pregnancy rate, pregnancy, and live-birthrates per cycle, and time to first pregnancy and live-birth were significantly better after laparoscopic myomectomyin symptomatic patients, whereas all reproductive outcomes were similar between the two groups in patients withunexplained infertility.Conclusion(s): Minilaparotomic and laparoscopic myomectomy improves in a similar manner the reproductiveoutcomes in patients with unexplained infertility, whereas the laparoscopic approach provides the best benefitsin fertile patients with symptomatic leiomyomas. (Fertil Steril� 2007;88:933–41. �2007 by American Societyfor Reproductive Medicine.)
Myomectomy is the best and, probably, the one and only ap-proach for treating uterine fibroids in reproductive-aged womendesiring to preserve childbearing potential, even if varioustreatments have been proposed and experimented (1–5).
Several consensus statements and guidelines agree thatmyomectomy is the best therapeutic indication in patientswho desire a pregnancy and in patients with unexplained in-fertility (6–8). Furthermore, very little is suggested regardingthe surgical approach to use in these cases (6–8). The reasonis probably due to a limited number of well-designed pro-spective studies evaluating the subsequent fertility in patientswho undergo myomectomy through different approaches. Inparticular, to date, only one randomized controlled trial
Received July 3, 2006; revised December 20, 2006; accepted December
27, 2006.
Reprint requests: StefanoPalomba, M.D., Via Mario Greco 10, vicoXI, 88100
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.047 Copyright ª2007 American
(RCT) assessed the reproductive outcomes after laparoscopicor laparotomic myomectomy in a sample of infertile patientswith large leiomyomas (9). On the contrary, no RCT wasdesigned to assess fertility after myomectomy in symptom-atic patients without infertility or subfertility factors, al-though the uterine leiomyoma excision is closely related topostoperative pelvic adhesion formation (10).
Recently, the minilaparotomic access was proposed asa minimally invasive alternative procedure to laparotomyfor myomectomy (11–14). Minilaparotomic myomectomy,in fact, seems associated with the advantages of classic lapa-rotomy (i.e., easy to perform and learn) and with those of min-imal access (i.e., little blood loss, short hospitalization, littlepostoperative pain, rapid return to normal activities) (11–14).
To date, only one RCT (14) compared laparoscopic andminilaparotomic surgical approaches for myomectomy dem-onstrating that both are minimally invasive and safe
Fertility and Sterility� Vol. 88, No. 4, October 2007 933Society for Reproductive Medicine, Published by Elsevier Inc.
procedures. Presently, no data are available in the literatureregarding their long-term effects on future fertility.
Based on these considerations, the aim of the present RCTwas to evaluate the effects of the laparoscopic and minila-parotomic approaches on reproductive outcomes in patientsundergoing myomectomy.
MATERIALS AND METHODS
The procedures used in the present study were in accordancewith the guidelines of the Helsinki Declaration on human ex-perimentation. The study was approved by the InstitutionalReview Board (IRB) of the University ‘‘Magna Graecia’’ ofCatanzaro, Italy. Before entering the study, the protocolwas explained to the patients, and their written consent wasobtained.
Subjects
Between January 2002 and March 2003, 162 ambulatory pre-menopausal women with uterine leiomyomas and candidatesfor myomectomy were consecutively screened in the threeuniversity departments of obstetrics and gynecology (Cata-nzaro, Rome, and Florence). The indications for myomec-tomy were leiomyoma-related symptoms or unexplainedinfertility (6). The diagnosis of unexplained infertility wasmade after exclusion of endocrine abnormalities, and tubaland male infertility factors with a complete hormonal assay,a hysterosalpingogram, and a semen analysis. In each patient,the ovulation was retrospectively confirmed by plasma P as-say >10 ng/mL assessed 7 days before the expected menses.
The following were all considered exclusion criteria: majormedical conditions, endocrine diseases (including a basalFSH >10 UI/L), psychiatric disorders, current or past historyof acute or chronic physical illness, premenstrual syndrome(15, 16), current or past (a washout period of at least 6 monthswas considered appropriate before enrolment) use of hormonaldrugs or drugs influencing cognition, vigilance, or mood, inabil-ity to complete the daily diary, and a history of alcohol abuse.
In addition, specific exclusion criteria were considered: nodesire to conceive, presence of more than three uterine leio-myomas and of leiomyomas with a main diameter less than 3cm or more than 10 cm, hypoechoic or calcified leiomyomasdiagnosed at ultrasound (17), presence of submucosal leio-myomas or alterations of the uterine cavity screened by hys-teroscopy and of other uterine or adnexal abnormalities atultrasound, pattern of hyperplasia with cytologic atypia inthe endometrial biopsy performed for abnormal uterinebleeding (AUB), an abnormal Papanicolau smear, and apositive urine pregnancy test.
Lastly, symptomatic women who did not have a previousconception of a live baby or with a tubal/male factor subfer-tility were also excluded.
Protocol
At study entry, age, parity, body mass index (BMI), work andsocioeconomic status, leiomyoma-related symptoms, quality
934 Palomba et al. Reproductive outcomes after laparosc
of life (QoL), previous laparotomies, and associated medicalconditions were assessed in each patient by the same clinicianfor each investigational center (18). Each patient underwenttransvaginal ultrasound examinations performed by three ex-perienced operators (one for each center) who assessed thepresence/absence of associated pelvic diseases and recordeduterine dimensions, number, size, and location of uterineleiomyomas (18). A sample of venous blood was obtainedfrom each patient between 8 and 9 AM, after an overnightfast and bed rest during the early proliferative phase (secondto third day of the cycle) to evaluate a complete hormonal as-say and blood count.
The subjects were then randomly allocated into two surgi-cal treatment groups of 68 women each (laparoscopic andminilaparotomic groups) using online software (www.randomization.it) that generated a random allocation se-quence in single blocks as method of restriction. The randomallocation sequence was concealed in a closed and dark-col-ored envelope until the surgeries were assigned, and specifi-cally just before entering in the operating room.
For each group, the duration of surgery, intraoperativeblood loss, intraoperative and postoperative complications,and degree of surgical difficulty were recorded during eachsurgical procedure.
The surgical steps were similar in both groups (18). Briefly,the uterine incision was always performed using the sameelectrosurgical device (Erbotom ACC 450, Erbe GmbH, Tu-bingen, Germany) with a cutting current of 70 W. After cleav-age plane identification, the leiomyoma was enucleated withstrong tractions, the myometrial edges were reapproximatedin one or two layers, according to the depth of the uterinedefect, with Vicryl CT 2-0 interrupted figure-eight sutures(intracorporeal knots for laparoscopic group). A carefulcoagulation of significant bleeding was performed usingbipolar forceps. Finally, at the end of each intervention, thepelvis was washed with saline solution, and no adhesionbarrier or saline dextrane macromolecular solutions wereleft in the peritoneal cavity.
The number of vials of drugs used for pain control duringthe hospital stay, duration of postoperative ileus, and hospitalstay were also recorded in both treatment groups. All patientsunderwent gynecological and ultrasonographic examinationsto assess leiomyomas recurrence at 1 and 3 months after sur-gical intervention. Postoperative complications, time to re-turn to full activity/work, leiomyoma-related symptoms,and QoL were also evaluated. These data will be reportedelsewhere (18).
Each subject was instructed to use barrier contraceptionmethods during the first 3 months after surgical intervention.After this period, a P assay in the secretive phase, a hystero-salpingogram, and a semen analysis were again performed inall patients to exclude organic causes of infertility. Then, eachcouple was instructed to have free intercourses and to recordon a personal diary the characteristics of their menstrualbleedings and the number of sexual intercourses.
opic and minilaparotomic myomectomy Vol. 88, No. 4, October 2007
Each patient underwent a monthly telephone interview; se-rial evaluations of the personal daily diary were performedevery 3 months. In absence of menstrual bleeding, a b-hCGassay was performed 7 days after the expected menses.
Patients underwent follow-up for 12 months, and thoseobtaining pregnancy for another 9 months to evaluate live-birth rate and obstetrics outcomes. Throughout the study,all reproductive events were recorded, and for each group cu-mulative pregnancy (PR) and live-birth rates, PR and live-birth rate per cycle, and abortion rate were calculated. An in-creasing b-hCG level and sonographic evidence of an intra-uterine gestational sac were both considered confirmation forpregnancy. Cumulative PR was calculated as the ratio be-tween number of pregnant women and total number of pa-tients studied. Cumulative live-birth rate was calculated aswomen with a baby alive over the total number of pregnantwomen.
Pregnancy and live-birth rates per cycle were defined as theratio between number of pregnancies and live-births, respec-tively, and the total number of cycles studied. Cumulative PRwas also calculated according to time to the first event (preg-nancy). Abortion rate was defined as the ratio between num-ber of miscarriages during the first 12 weeks of gestation andtotal pregnancies.
At the end of the study, an analysis of reproductive out-comes was performed, categorizing patients according to in-dication for myomectomy (i.e., symptomatic leiomyomas) orunexplained infertility.
Statistical Analysis
At the study design, we consider as primary end point of thepresent pilot clinical trial the effect of the two procedures onthe cumulative live-birth rate. After 15 months of follow-upfrom surgery and an interim analysis of our initial data, thedifferences between the groups on cumulative live-birthrate and PR were considered too little or clinically irrelevantto continue the study to obtain an adequate power. Thus, ourpatient enrolment was stopped.
For categorical variables the c2 test or the exact test wasapplied as appropriate. The normal distribution of continuousvariables data was evaluated with the use of Kolmogrov-Smirnov test and expressed as median and interquartile range(IQR) with minimum–maximum values. Thus, the differ-ences between groups were analyzed using Mann-WhitneyU test.
Cumulative event (pregnancy) rate was calculated by theKaplan-Meier method, using the time to a first event as theoutcome variable, and the differences between groups weretested with the use of log-rank test. In addition, Cox propor-tional hazards model was used to calculate the hazard rate(HR) for pregnancy in patients treated with the laparoscopicor minilaparotomic approach. The HR represented also therelative risk (RR), calculated for dichotomous variable inwhich there are two levels.
Fertility and Sterility�
Data were analyzed using the intention-to-treat method(ITT) on the basis of treatment assignment and not on treat-ment receipt. The Statistics Package for Social Science(SPSS 14.0.1; SPSS Inc., Chicago, IL) was used for statisticalanalyses. Statistical significance was set at P<.05.
RESULTS
Figure 1 illustrates the flow diagram of the present clinicalstudy according to the consolidated standard of reporting oftrials (CONSORT) guidelines (19).
After randomization, the two treatment groups had similarmain demographic and clinical characteristics (Table 1). Af-ter surgical interventions, only the rate of laparotomic con-versions was significantly (P¼.012) different between thetwo groups (Table 1).
No difference between groups was detected in uterine andleiomyoma characteristics (Table 2), and in number of sexualintercourses (data not shown). No difference in associatedsubfertility factors was observed at the third month follow-up visit. In only one case per treatment group (both for symp-tomatic women), a mild male factor was detected.
During the 12-month follow-up, the patients of the laparo-scopic and minilaparotomic groups were studied for a total of556 and 669 cycles, respectively.
At the end of the study, the cumulative PR (P¼.090) andlive-birth rate (P¼.620) were similar between the two groups,whereas the PR and live-birth rate per cycle were signifi-cantly (P<.05) higher in the laparoscopic group than in theminilaparotomic one (Table 3). The time to first pregnancyand live-birth was also significantly (P<.01) lower in the lap-aroscopic than in the minilaparotomic group (Table 3). Theprobability of first pregnancy was different between groups
FIGURE 1
Flow diagram of the clinical trial.
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomec-
tomy. Fertil Steril 2007.
935
TABLE 1Main characteristics of the laparoscropic and minilaparotomic groups.
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
(P¼.025), also in the Kaplan-Meier survival analysis(Fig. 2). Compared with the minilaparotomic group, the lap-aroscopic group had a RR of pregnancy of 1.733 (95% con-fidence interval [CI], 1.046–2.871; P¼ .025). No differencein other reproductive outcomes was observed betweengroups (Table 3).
936 Palomba et al. Reproductive outcomes after laparosc
Table 4 shows the main demographic and clinical charac-teristics of the groups obtained categorizing our populationaccording to indications for myomectomy. Age and paritywere significantly (P<.05) lower and higher, respectively,in patients with symptomatic leiomyomas in comparisonwith those with unexplained infertility, without differences
TABLE 2Uterine and leiomyoma characteristics in the laparoscropic and minilaparotomic groups.
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
937
FIGURE 2
Cumulative probability of first pregnancy by theKaplan-Meier survival analysis in the laparoscopicand minilapartomic groups.
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomec-
tomy. Fertil Steril 2007.
Fertility and Sterility�
between laparoscopic and minilaparotomic groups. In pa-tients with symptomatic leiomyomas alone, a significant(P<.05) difference in laparotomic conversion rate was de-tected between laparoscopic and minilaparotomic groups(Table 4).
No other difference was observed between and withingroups (Tables 4 and 5).
At the end of the study, patients with symptomatic leio-myomas were studied for a total of 253 and 336 cycles forthe laparoscopic and minilaparotomic groups, respectively,whereas patients with unexplained infertility were studiedfor a total of 303 and 333 cycles for the laparoscopic and min-ilaparotomic groups, respectively.
In patients with symptomatic leiomyomas, cumulative PR,and the PR and live-birth rate per cycle were significantly(P<.05) higher, whereas the times to first pregnancy andlive-birth were significantly (P<.05) lower after laparoscopicthan minilaparotomic myomectomy (Table 6). No differencebetween treatment groups was detected in abortion rate and inobstetric outcomes in patients who received myomectomy forsymptomatic leiomyomas (Table 6).
In patients with unexplained infertility, no difference inany reproductive outcomes assessed was observed betweenlaparoscopic and minilaparotomic groups (Table 6).
TABLE 4Main characteristics of the two groups.
GroupLaparoscopy
(n [ 68)Minilaparotomy
(n [ 68)
Age (y)Symptomatic leiomyomas 26 (2.5), 21–31a 26.5 (3.5), 22–36a
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).a P< .05 vs. unexplained infertility.b P< .05 vs. minilaparotomic group.
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
DISCUSSION
At present, surgical treatment of uterine leiomyomas is rec-ommended in symptomatic women or in patients with unex-plained infertility (6). Myomectomy, regardless of surgicaltechnique, represents the gold standard in women wishingto preserve their reproductive potential (20). Furthermore, al-though this procedure is efficient in reducing leiomyoma-related symptoms in women with symptomatic leiomyomas(20), its effects on reproductive outcomes are not well known,and few data are available (9).
938 Palomba et al. Reproductive outcomes after laparosc
The primary end point of the current multicenter RCTwas to investigate the effects on reproduction of two mini-mally invasive approaches to myomectomy (i.e., laparos-copy and minilaparotomy). Specifically, we evaluatedfertility and obstetric outcomes, after a long-term follow-up, in women candidates for myomectomy for symptomaticleiomyomas or unexplained infertility. After 12 months offree intercourses and another 9 months to obtain obstetricdata, reproductive outcomes were similar between the twogroups studied.
opic and minilaparotomic myomectomy Vol. 88, No. 4, October 2007
TABLE 5Uterine and leiomyoma characteristics of the two groups.
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
In this regard, our study was underpowered to demonstratea significant difference in cumulative PR or live-birth rate, andit would have been necessary to enroll a much larger popula-tion sample to obtain significant differences. For this reason,patient enrolment was stopped after 15 months after surgery.
However, significant differences were observed betweenlaparoscopic and minilaparomic groups in PR and live-birthrate per cycle, and in the time to first pregnancy and live-birth.
Fertility and Sterility�
Specifically, PR and live-birth rate per cycle were signifi-
cantly higher after laparoscopic myomectomy, whereas the
time to first pregnancy and live-birth was significantly lower
in women who had undergone laparoscopic myomectomy.
In addition, the probability of first pregnancy using the
Kaplan-Meier survival analysis was higher in the laparo-
scopic than in the minilaparotomic group, with a relative
risk of pregnancy of 1.7.
939
TABLE 6Reproductive and obstetrics outcomes of the two groups.
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).a P< .05 vs. minilaparotomic group.b P< .05 vs. unexplained infertility.
Palomba. Reproductive outcomes after laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
The data analysis, performed categorizing our populationaccording to indication to myomectomy, demonstrated thatthe benefits of the laparoscopic and minilaparotomicmyomectomy vary according to the patients’ characteristics,and, specifically, to the indication for surgery. In particular,in patients with unexplained infertility, no difference inreproductive outcomes was observed between two surgicalapproaches, whereas in patients with symptomatic leiomyo-
940 Palomba et al. Reproductive outcomes after laparosco
mas the laparoscopy showed to be the best approach in termsof fertility.
Our data on the population with unexplained infertility arein agreement with a recent RCT (9), which found no differ-ence in fertility and obstetric outcomes in infertile womenaffected by large leiomyomas who received laparoscopic orlaparotomic myomectomy. Specifically, the cumulative PR
pic and minilaparotomic myomectomy Vol. 88, No. 4, October 2007
at long-term follow-up, evaluated using the Kaplan-Meieranalysis, was similar between women treated with laparo-scopic and laparotomic myomectomy (9).
In our study, as well as in the study by Seracchioli et al. (9),myomectomy, independently from the surgical route, wasmost effective in patients with unexplained infertility as toeliminate other minimal differences due to different tech-nique. Unfortunately, in that study (9) no power calculationwas provided and it is not known whether the ITT principlewas applied.
To the contrary, in symptomatic women, the PR and live-birth rate per cycle was higher after laparoscopic than mini-laparotomic myomectomy, demonstrating the beneficialeffects of minimally invasive laparoscopy over minilaparot-omy in terms of reproductive outcomes.
These findings could be explained with the lower pelvicadhesion rate due to the laparoscopic approach (21). Infact, myomectomy is a surgical intervention with a higherrate of postoperative pelvic adhesions (10). Unfortunately,no clear data are available in the literature regarding the rela-tionship between adhesions and infertility, and the real effi-cacy of adhesiolysis in infertile patients (22). In addition,no laparoscopic second-look was performed in the currentstudy to evaluate the de novo formation of adhesions afterboth laparoscopic and minilaparotomic myomectomy.
Because this clinical trial is the first evaluating, in a ran-domized manner, the reproductive outcomes in a populationof women with symptomatic leiomyomas and without subfer-tility factors, any further comment on our results or compar-ison with other studies is not possible.
In conclusion, both minilaparotomic and laparoscopicmyomectomies are related to similar reproductive outcomesin patients with unexplained infertility, whereas fertile butsymptomatic patients with uterine leiomyomas have thebest reproductive benefits from the laparoscopic approach.
REFERENCES1. Wallach EE, Vlahos NF. Uterine myomas: an overview of development,
clinical features, and management. Obstet Gynecol 2004;104:393–406.
2. Bachmann G. Expanding treatment options for women with symptom-
atic uterine leiomyomas: timely medical breakthroughs. Fertil Steril
2006;85:46–7.
3. Pron G. New uterine-preserving therapies raise questions about interdis-
ciplinary management and the role of surgery for symptomatic fibroids.
Fertil Steril 2006;85:44–5.
4. Munro MG. Management of leiomyomas: is there a panacea in Pandora’s
box? Fertil Steril 2006;85:40–3.
Fertility and Sterility�
5. Manyonda I, Sinthamoney E, Belli AM. Controversies and challenges in
the modern management of uterine fibroids. BJOG 2004;111:95–102.
6. ACOG Committee on Practice Bulletins-Gynecology. ACOG practice
bulletin. Surgical alternatives to hysterectomy in the management of
leiomyomas. Number 16, May 2000. Int J Gynaecol Obstet 2001;73:
285–93.
7. Farquhar C, Arroll B, Ekeroma A, Fentiman G, Lethaby A,
Rademaker L, et al. Working Party of the New Zealand Guidelines
Group. An evidence-based guideline for the management of uterine
fibroids. Aust N Z J Obstet Gynaecol 2001;41:125–40.
21. Miller CE. Myomectomy. Comparison of open and laparoscopic
techniques. Obstet Gynecol Clin North Am 2000;27:407–20.
22. Diamond MP, Freeman ML. Clinical implications of postsurgical
adhesions. Hum Reprod Update 2001;7:567–76.
941
TECHNIQUES AND INSTRUMENTATION
A multicenter randomized, controlled study comparinglaparoscopic versus minilaparotomic myomectomy:short-term outcomesStefano Palomba, M.D.,a Errico Zupi, M.D.,b Tiziana Russo, M.D.,a Angela Falbo, M.D.,a
Daniela Marconi, M.D.,b Achille Tolino, M.D.,c Francesco Manguso, M.D.,d Alberto Mattei, M.D.,e
and Fulvio Zullo, M.D.a
a Department of Obstetrics & Gynecology, University ‘‘Magna Graecia’’ of Catanzaro, Catanzaro; b Department of Obstetrics &
Gynecology, University ‘‘Tor Vergata’’ of Rome, Rome; c Department of Obstetrics & Gynecology, and d Department of Clinical
and Experimental Medicine, University ‘‘Federico II’’ of Naples, Naples; and e Department of Obstetrics & Gynecology,
University of Florence, Florence, Italy
Objective: To compare the laparoscopic and minilaparotomic approaches for symptomatic uterine leiomyomastreatment in terms of safety and feasibility.Design: Randomized controlled trial.Setting: Three university departments of obstetrics and gynecology of Catanzaro, Rome, and Florence, Italy.Patient(s): One hundred thirty-six women wishing to conceive and candidate for myomectomy due to symptom-atic uterine leiomyomas or unexplained infertility.Intervention(s): Myomectomy through laparoscopic or minilaparotomic access.Main Outcome Measure(s): Surgical outcomes.Result(s): Leiomyoma enucleation and hysterotomy suturing times were significantly shorter after minilaparo-tomic myomectomy, whereas the degree of surgical difficulty was significantly higher for the laparoscopic myo-mectomy. Intraoperative blood loss, variation in hemoglobin levels, quantity of pain control drugs usedpostoperatively, and hospitalization were significantly lower in the laparoscopic group than in the minilaparotomicone. Our surgical outcomes were significantly influenced by specific investigational centers involved, and by leio-myoma dimensions and localizations. This last variable is the strongest predictor of surgical outcome.Conclusion(s): Laparoscopic and minilaparotomic approaches to myomectomy are two safe and minimallyinvasive surgical procedures. A careful evaluation of the dimensions and localizations of fibroids are needed toaddress to the right choice to the best approach. (Fertil Steril� 2007;88:942–51. �2007 by American Societyfor Reproductive Medicine.)
Key Words: Fibroids, laparoscopy, leiomyomas, minilaparotomy, myomectomy, surgery
Uterine leiomyomas are the most frequent benign neoplasmsof the female genital apparatus (1). During the past years, var-ious options have been proposed and experimented for thetreatment of these tumors (2–5). Moreover, the best approachfor reproductive-age women, desiring to preserve childbear-ing potential, has been myomectomy (2–5).
Although the laparotomic approach is still consideredby some investigators the gold standard surgical procedure(5–8), laparoscopic myomectomy seems to provide severaladvantages over laparotomy (9). Unfortunately, the laparo-scopic approach is a complex surgical procedure witha long learning curve, especially in the case of myomectomy(10). A recent survey made in the United Kingdom has, infact, showed that a very high percentage of myomectomies
Received June 22, 2006; revised December 22, 2006; accepted
December 28, 2006.
Reprint requests: Stefano Palomba,M.D., Via MarioGreco 10, vicoXI, 88100
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
942
are still performed through laparotomy (8). Recently, minila-parotomic access was proposed as an alternative procedure toreduce the rate of laparotomies in the treatment of severalmalignant (11, 12) and benign gynecologic diseases(13–17). In particular, minilaparotomic myomectomy seemsto associate the advantages of classic laparotomy with that ofa minimal access (i.e., easy learning and performance, shortoperating time, minimal blood loss, short hospitalization, lit-tle postoperative pain, rapid return to normal activities) (18).
To date, one randomized controlled trial (RCT) (19) com-pared both laparoscopic and minilaparotomic approaches formyomectomy. Operative time was shorter during minilaparo-tomic myomectomy, whereas a significant advantage was ob-served using the laparoscopic access in terms of hemoglobindecline, pain intensity, and duration of postoperative ileus andhospital discharge. Moreover, no clinical relevant conclusionor suggestion was provided in respect the predictors to be usedregarding the choice of the different surgical approaches.
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.048
Because the myomectomy is the only fertility-sparingsurgical treatment for symptomatic patients desiring to con-ceive and for women with unexplained infertility related touterine leiomyomas, we designed a pilot RCT comparing lap-aroscopic and minilaparotomic myomectomies, and the re-productive data were considered as primary end point.
The current study was aimed to assess the effects of the twoprocedures on surgical outcomes, studying how our results varyaccording to dependent variables in defining potential predic-tors for the best surgical approach to uterine leiomyomas.
MATERIALS AND METHODS
The procedures used in the present study were in accordancewith the Helsinki Declaration guidelines on human experi-mentation. The study was approved by the InstitutionalReview Board (IRB) of the University ‘‘Magna Graecia’’ ofCatanzaro, Italy. Before entering the study, the protocolwas explained to the patients, including the possibility ofconversion to traditional laparotomic surgery, and a writtenconsent was obtained from each patient.
Patient Selection
Between January 2002 and March 2003, 162 ambulatory pre-menopausal women with uterine leiomyomas were chosen ascandidates for myomectomy. They were consecutivelyscreened in the three university departments of obstetricsand gynecology (Catanzaro, Rome, and Florence). The indi-cations for myomectomy were leiomyoma-related symptomsor unexplained infertility (20). Unexplained infertility wasdiagnosed after exclusion of endocrine and tubal abnormali-ties and male factor infertility, with a complete hormonalassay, a hysterosalpingogram, and a semen analysis, respec-tively. In each patient, the ovulation was retrospectivelyconfirmed by plasma P assay >10 ng/mL assessed 7 daysbefore the expected menses.
Major medical conditions, endocrine disease (includinga basal FSH >10 UI/L), psychiatric disorders, current or pasthistory of acute or chronic physical illness, premenstrual syn-drome (21, 22), current or past (a washout period of at least 6months was considered appropriate before enrolment) use ofhormonal drugs or drugs influencing cognition, vigilance, ormood, inability to complete the daily diary, and a history of al-cohol abuse were all considered as exclusion criteria.
In addition, the following specific exclusion criteria wereincluded: no desire to conceive, presence of more than threeuterine leiomyomas and of leiomyomas with a main diameterless than 3 cm or more than 10 cm, hypoechoic or calcifiedleiomyomas diagnosed at ultrasound (23), presence of sub-mucosal leiomyomas or alterations of the uterine cavityscreened by hysteroscopy and of other uterine or adnexal ab-normalities at ultrasound (e.g., adenomyosis, abnormal endo-metrial thickness), pattern of hyperplasia with cytologicalatypia in the endometrial biopsy performed for abnormaluterine bleedings under hysteroscopy on suspected areas,an abnormal Papanicolau smear, and a positive urine preg-nancy test.
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Lastly, symptomatic women who did not have a previousconception resulting in a live baby or with a tubal/male factorsubfertility were also excluded.
Study Protocol
At study entry, age, parity, and body mass index (BMI), workand socioeconomic status, leiomyoma-related symptoms,quality of life (QoL), previous laparotomies, and associatedmedical conditions were assessed in each patient by thesame clinician for each investigational center.
Leiomyoma-related symptoms, such as menorrhagia, pel-vic pressure, and pain, were assessed using a visual analoguescale (VAS) (24). During the same visit, QoL was alsoassessed in each patient using the Italian version of theShort-Form Healthy Survey (SF-36), a valid tool for QoLmeasurement (25). Both evaluations were self-administeredwithout or with minimal operator’s influence.
All ultrasound examinations were performed transvagi-nally by an experienced operator in each center, who assessedthe presence/absence of associated pelvic diseases, evaluateduterine dimensions, and leiomyoma number, dimension andlocation. Uterine and leiomyoma dimensions were calculatedmeasuring the main diameters (D1, D2, D3) and applying theformula of the ellipsoid (D1 � D2 � D3 � 0.52). In presenceof more than one leiomyoma, an arithmetic mean of the sizeswas used.
At study entry, a sample of venous blood was obtainedfrom each patient between 8 and 9 AM, after an overnightfast and bed rest during the early proliferative phase (secondto third day of the cycle) to evaluate a complete hormonalassay and blood count.
Randomization was carried out using online software ofrandomization (www.randomization.it) to generate a randomallocation sequence in single blocks as method of restriction.The random allocation sequence was concealed in closed anddark-colored envelope until surgeries were assigned (beforeentering in the operating room). Thus, patients were assignedto one of the two surgical procedure groups (i.e., laparoscopicor minilaparotomic).
During each surgical procedure, total operative time, enu-cleation time of each leiomyoma, and repairing time of theuterine defect after hysterotomy were recorded. Specifically,the total duration time of the surgical procedure was calcu-lated during the laparoscopic procedure beginning from ab-dominal skin incision, before Verres needle insertion, toskin suture, and during the minilaparotomic procedure fromabdominal skin incision to suture.
Intraoperative blood loss, intraoperative and postoperativecomplications, and degree of surgical difficulty were alsoevaluated and recorded in both groups. The intraoperativeblood loss was differentially expressed as the difference be-tween aspirated and irrigated liquid, and as hemoglobin leveldecrease (DHb). Intraoperative complications were defined
as laparotomic conversions, and as bowel, bladder, ureteral,or vascular injuries. Laparotomic conversions were definedas open abdominal access through a more than 7-cm longskin incision. Postoperative complications were defined asadverse events resulting from surgery within 30 days andwere considered major if they resulted in an unplanned read-mission, blood transfusion, or secondary surgical procedure.
At the end of each surgical procedure, the degree of surgi-cal difficulty was evaluated by the operators using VAS vary-ing from 1 (low difficulty) to 10 (high difficulty) as describedby Vassiliou et al. (26).
After intervention all patients received an IV bolus of tra-madol (100 mg), followed by patient-controlled analgesia(tramadol 200 mg [2 vials] in 500 mL of saline solution)(27). The postoperative analgesia was administered duringthe first 12 hours after surgery. The number of vials of drugused during the hospitalization was carefully recorded. Atthe end of the surgical procedures the postoperative painwas also evaluated using VAS, and the severity of the painwas expressed as a score ranging from 0 to 10 (26). Postop-erative pain was specifically assessed in each group every 2hours for the first 12 hours after surgery, after which, at 24and 48 hours after the surgical procedure, and at hospital dis-charge.
Women were allowed to eat and drink the morning aftersurgery, and to ambulate as soon as they felt comfortable.The urinary catheter was removed the evening of the surgery.A blood sample was obtained and DHb was calculated foreach woman 24 hours after surgery.
Duration of postoperative ileus and hospitalization wereevaluated in both groups. Hospitalization time was definedfrom entry (the day of surgery) to discharge. In particular,each patient was routinely discharged 2 days after surgery,whereas hospitalization continued if a postoperative compli-cation was detected.
Each subject was instructed to use barrier contraceptionmethods during the first 3 months after surgical intervention,successively followed by free intercourse. All patients werealso instructed to record on a personal diary the length andthe quantity of menstrual bleeding, the number of sexual in-tercourses, and eventual stress factors influencing QoL.
All subjects underwent a follow-up visit after 1 and 3months from surgery. At each visit, all patients underwent gy-necological and ultrasonographic examinations to assessleiomyoma recurrence, postoperative complications, time toreturn to full activity/work, leiomyoma-related symptoms,and QoL. These two last parameters were assessed by useof self-administered tests and the relative scores wererecorded.
Surgical Procedures
The surgeons, one from each investigational center, wereskilled in both surgical approaches. In fact, at the beginning
944 Palomba et al. Laparoscopic and minilaparotomic m
of this study, they each had performed more than 100 surgicalprocedures for each procedure in the previous 2 years.
The operator was informed on the type of intervention toperform (if laparoscopic or minilaparotomic) just before en-tering the operating room. A drug for thrombosis prophylaxiswas not administered to any patient. After anesthesia admin-istration, each patient was placed in a modified lithotomic po-sition. Immediately before surgery, each patient received 2 gof IV cephalosporin as antibiotic prophylaxis, and if neces-sary, a same dose was repeated if the intervention lastedmore than 2 hours.
Laparoscopic myomectomy In the laparoscopic group, theprocedures were performed using a 10-mm scope with twoor three ancillary ports. A longitudinal incision, using an uni-polar hook scissor, was performed close to the midline. Aftercleavage plane identification, by choice of the operator, theleiomyoma was enucleated by means of either adequatetraction with a myoma drill, or a strong grasper and counter-traction maneuvers with Manhes forceps, or scissors, ora hydrodissection technique.
Coagulation of significant bleeding was performed usingbipolar forceps, and the myometrial edges were reapproxi-mated in one or two layers, according to the depth of the uter-ine defect, with Vicryl CT 2-0 interrupted figure-eight suturesusing intracorporeal knots.
In all cases leiomyomas were removed with a 12-mmSteiner automatic morcellator positioned through the first10-mm laparoscopic access, specifically at umbilical (in themost of cases) or in a higher position (for selected cases ofvery large uterus or fibroid). During leiomyoma morcella-ment a 5-mm laparoscope was used in a lateral port.
Minilaparotomic myomectomy In the minilaparotomicgroup, all procedures were performed without laparoscopicassistance. A 4- to 7-cm transverse incision was performedfor minilaparotomic access. To avoid accidental extensionof the minilaparotomies two double sutures were made atthe end of the skin incisions. The length and the distancefrom pubic symphysis of the skin incision varied accordingto leiomyoma dimension and localization.
The anterior rectus fascia was incised vertically (18) andthe muscular fibers were dissected. The parietal peritoneumwas exposed and incised vertically. A self-retaining retractor(Bookwalter retractor system; Codman Italy, Pratica di Mare,Rome, Italy) with two Richardson’s retractors were used dur-ing all surgical minilaparotomic procedures. In no case, intes-tinal gauzes were inserted into the abdomen.
The hysterotomy was performed with a monopolar knifeon the most prominent part of each tumor, and after identifi-cation of the pseudocapsule, two small retractors were placedbetween the leiomyoma and myometrium inside the pseudo-capsule, and the leiomyomas excision was obtained applyinga strong traction with a grasper. Vicryl CT 2-0 interrupted fig-ure-eight sutures in one or two layers, according to the depthof the uterine defect, were used to repair the uterus. A
yomectomy Vol. 88, No. 4, October 2007
progressive morcellament of the leiomyomas was performedusing a cold knife scalpel if the tumor size was more than theskin incision.
In both groups (laparoscopic and minilaparotomic), in caseof infraligamentary fibroids the broad ligament was opened,the leiomyoma was excised by adequate traction, and a verycareful hemostasis by bipolar coagulation of the tissue andvessels around the fibroids without any peritoneal reconstruc-tion was performed. In addition, a manipulator probe wasplaced into the cervix to place the uterus in an optimal posi-tion during enucleation and suturing. A careful inspection ofthe uterus, adnexa, and pelvic cavity was performed after sur-gical access. An infiltration of the serosa or myometriumoverlying the leiomyoma was performed before uterine inci-sion with a solution of 50 mL of bupivacaine cloridrate 0.25%and 0.5 mL of epinephrine (0.5 vial of 1 mg/mL) (23). Theuterine incision was performed using the same electrosurgi-cal device (Erbotom ACC 450, Erbe GmbH, Tubingen, Ger-many) with a cutting current of 70 W. At the end of eachintervention, the pelvis was washed with saline solution.No adhesion barrier or saline dextrane macromolecular solu-tions were left in the peritoneal cavity. All operative sampleswere submitted for pathologic examination.
Statistical Analysis
At study design, we considered as primary end point of thepresent pilot clinical trial the effect of the two procedureson fertility and, specifically, on the cumulative live-birthrate. After 15 months of follow-up from surgery and an in-terim analysis of our initial data, the differences betweengroups on reproductive outcome were considered too littleor clinically irrelevant to continue the study in order to obtainan adequate power (28). Thus, our patient enrolment wasstopped.
For categorical variables the c2 test or the Exact test wasapplied as appropriate. The normal distribution of continuousvariables data was evaluated with the use of Kolmogrov-Smirnov test and expressed as median and interquartile range(IQR) with minimum–maximum values. Thus, the differ-ences between groups were analyzed using Mann-WhitneyU test.
The Friedman test was used to analyze the QoL assessmentat baseline, and after surgical intervention (1 and 3 months)with Dunn test for multiple comparisons. The Mann-WhitneyU test was used to analyze the difference between the twogroups at each follow-up visit.
Finally, after checking assumptions, data were also ana-lyzed with the use of GLM univariate procedure that providesregression analysis and ANOVA for one dependent variableby one or more factors or variables. Analysis of variance isrobust to departures from normality, although the data shouldbe symmetric (29). The model included as dependent variablethe total operative time, considering the main limiting factorin laparoscopic surgery, and as between-subjects factors,
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which divided the population into groups as fixed factors,the following variables: BMI (<25/>25, coded 0/1), surgicalprocedure (laparoscopic/minilaparotomic, coded 0/1), leio-myomas localization (posterior/intraligamentary, coded 0/1),investigational center involved (Catanzaro, Rome, and Flor-ence, coded 0/1/2), leiomyomas dimensions (>7 cm/<7 cm,coded 0/1), and the interaction between surgical procedurex leiomyomas localization, surgical procedure x inves-tigational center, and surgical procedure x leiomyomasdimension.
Data were analyzed using the intention-to-treat (ITT) prin-ciple on the basis of treatment assignment and not on treat-ment receipt. The Statistics Package for Social Science(SPSS 14.0.2; SPSS Inc., Chicago, IL) was used for statisticalanalyses. Statistical significance was set at P<.05.
RESULTS
Figure 1 illustrates the flow diagram of the present clinicalstudy according to the consolidated standard of reporting oftrials (CONSORT) guidelines (30). The two groups of 68 pa-tients each were obtained from randomization of 136 patients(Fig. 1).
Demographic and Clinical Data
In Table 1 the main demographic and clinical characteristicsof the laparoscopic and minilaparotomic groups are shown.After randomization, the two groups had similar characteris-tics (Table 1).
The leiomyoma-related symptoms consisted of menorrha-gia (36/68 [52.9%] vs. 35/68 [51.5%]), pelvic pressures (23/68 [33.8%] vs. 26/68 [38.2%]), pelvic pain (12/68 [17.6%]vs. 15/68 [22.1%]), constipation (3/68 [4.4%] vs. 1/68 [1.5%]),and urinary symptoms (4/68 [5.9%] vs. 3/68 [4.4%] for laparo-scopic and minilaparotomic groups, respectively). No
FIGURE 1
Flow diagram of the clinical trial according toCONSORT guidelines (30).
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
945
TABLE 1Main characteristics of the two study groups.
Previous major abdominal surgery (n, %) .906None 58 (85.3) 55 (80.9)One operation 5 (7.4) 7 (10.3)Two operations 3 (4.4) 4 (5.9)More than two operations 2 (2.9) 2 (2.9)
Work status (n, %) .734Full time 24 (35.3) 19 (27.9)Part time 22 (32.4) 26 (38.2)Retired 1 (1.5) 0 (0.0)Housewife 9 (13.2) 10 (14.7)Unemployed 12 (17.6) 13 (19.1)
Socioeconomic status (n, %) .903<15,000 e 21 (30.9) 22 (32.4)15,000–30,000 e 26 (38.2) 24 (35.3)30,000–45,000 e 18 (26.5) 17 (25.0)>45,000 e 3 (4.4) 5 (7.4)
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
significant difference in leiomyoma-related symptoms distri-bution was detected between the two groups.
No difference was observed between the two groups inuterine size, dimension of leiomyomas, main dimension ofthe largest leiomyoma, and number of leiomyomas (Table2). The localization of leiomyomas was also similarly distrib-uted in the two groups (Table 2). In all cases the estimatedsize and the histologic leiomyomas diagnosis, suspected be-fore surgery through ultrasound, were confirmed, respec-tively, by macroscopic and microscopic examination of theenucleated tumors.
In 12 and 9 cases for the laparoscopic and minilaparotomicgroups, respectively, peritoneal adhesions (8/68 [11.8%] and6/68 [8.8%]) and mild endometriosis (4/68 [5.9%] and 3/68[4.4%]) were observed. The distribution of these associatedpelvic diseases was not different (P¼ .477) in the two groups.
Total operative timewas similar (P¼.227) in the two groups,even if the enucleation time of leiomyomas (P¼.005) and thesuturing time of hysterotomies (P¼.020) were statisticallylonger in the laparoscopic than in the minilaparotomic group(Table 3). The intraoperative blood loss (P¼.001) and the DHb(P<.001) were significantly lower in the laparoscopic than in
946 Palomba et al. Laparoscopic and minilaparotomic m
the minilaparotomic group (Table 3). In no case a blood trans-fusion was required.
The degree of surgical difficulty was significantly (P¼.003)higher in the laparoscopic group than in the minilaparotomicone (Table 3). No difference (P¼.457) in postoperativepain was detected at the end of the surgical proceduresand throughout the study between the laparoscopic andminilaparotomic groups (Fig. 2), whereas the number of vialsof analgesic used during hospitalization was significantly(P<.001) lower in the laparoscopic group than in the minila-parotomic group (Table 3).
Hospitalization was significantly (P<.001) lower in thelaparoscopic than in the minilaparotomic group, whereasno difference in duration of postoperative ileus (P¼.061),and in time necessary to return to full activity/work (P¼.502)was detected between the two groups (Table 3).
No intraoperative complication was observed during lapa-roscopy, whereas six cases of laparotomic conversions wererecorded during the minilaparotomic procedures (0/68 [0%]vs. 6/68 [8.8%]; P¼.012). The laparotomic conversions,equally distributed in all three investigational centers, weredue to posterior isthmic (four cases) and infraligamentary
yomectomy Vol. 88, No. 4, October 2007
TABLE 2Uterine and leiomyoma characteristics in the two treatment groups.
Note: Data are expressed as median (interquartile range) and minimum–maximum values or as number (n) and percent (%).
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
leiomyomas (two cases). In particular, in these convertedcases, the mean diameters of the largest leiomyomas were7.4 cm (5.8–8.2) and 8.6 cm (7.5–9.7) (median [minimum–maximum values]), for posterior and infraligamentary leio-myomas, respectively. Two and five postoperative complica-tions were observed in the laparoscopic and minilaparotomicgroups, respectively (2/68 [2.9%] vs. 5/68 [7.4%]; P¼ .261).Specifically, one case (1/68 [1.5%]) of urinary tract infectionand one case (1/68 [1.5%]) of fever >38�C were detected inthe laparoscopic group, whereas two cases (2/68 [2.9%]) offever >38�C, 2 cases (2/68 [2.9%]) of wound infection,and one case (1/68 [1.5%]) of wound dehiscence were re-corded in the minilaparotomic group. The total complicationrate was significantly higher in the minilaparotomic than inthe laparoscopic group (2/68 [2.9%] vs. 11/68 [16.2%];P¼.009).
In Table 4 the SF-36 scores for each domain assessed areshown. No difference in any domain was detected betweengroups at baseline (Table 4). After 1 and 3 months from sur-gery, a significant (P<.05) improvement in all domains wasobserved in both groups, without any difference betweenthem (Table 4).
Using the univariate ANOVA, total operative time wassignificantly influenced by leiomyoma dimensions (>7 cm)(P¼.001) and localizations (posterior/intraligamentary)(P<.001), independent from surgical procedure. On the con-trary, the investigational center alone did not influence(P¼.351) the total operative time. Also, the BMI did not in-fluence (P¼ .906), both alone and after interaction with otherdependent variables, the total operative time. Moreover, the
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total operative time was significantly influenced by the inter-action of the surgical procedure with the investigational cen-ter (P ¼ .001), and by the interaction of surgical procedurewith leiomyoma localization (P ¼ .003), whereas it was notsignificantly (P¼ .857) influenced by the interaction betweensurgical procedure and leiomyoma dimensions.
Surgical Data Analysis
Considering the main independent variable influencing theresults (i.e., leiomyoma localization) and evaluating the rea-sons for laparotomic conversions, an analysis of our data wasperformed according to presence/absence of the main leio-myoma at the posterior or intraligamentary site.
Our data are summarized in Table 5 according to the local-ization of the main leiomyomas.
In both groups, total operative time, time of hysterotomysuturing, and degree of surgical difficulty were significantly(P<.05) higher for the posterior/intraligamentary leiomyo-mas than for leiomyomas located at other sites. In the mini-laparotomic group alone, time of leiomyoma enucleation,intraoperative blood loss, DHb, postoperative ileus, and hos-pitalization were significantly (P<.05) increased for poste-rior/intraligamentary leiomyomas versus those located atother sites (Table 5).
Considering both leiomyomas located at posterior/infrali-gamentary sites and those located at other sites, a significant(P<.05) difference between treatment groups was detectedin total operative time. Time of leiomyoma enucleation and
947
TABLE 3Main parameters evaluated during and after surgery in the laparoscopic and minilaparotomic groups.
GroupLaparoscopy
(n [ 68)Minilaparotomy
(n [ 68) P value
Total operative time (min) 108 (28); 69–150 95 (53); 62–174 .227Time of enucleation for each leiomyoma (min) 12 (3); 8–16 10 (7); 4–16 .005Time of suturing hysterotomy (min) 18 (4); 13–26 16.5 (12); 7–26 .020Intraoperative blood loss (mL) 130 (78); 90–200 160.0 (65); 90–280 .001DHb 0.8 (0.7); 0.2–2.1 1.3 (0.8); 0.2–2.5 < .001Degree of surgical difficulty 7.5 (2); 4–9 6 (3); 3–9 .003Vials of analgesic used (n) 3 (2); 1–8 7 (2); 2–10 < .001Postoperative ileus (days) 1 (0); 1–3 1 (1); 1–3 .061Hospitalization (days) 2 (0); 2–5 3 (0); 3–5 < .001Time to return to full activity (days) 5 (2); 3–11 5 (2); 3–12 .502
Note: Data are expressed as median (interquartile range) and minimum–maximum values.
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
time of hysterotomy suturing were significantly (P<.05)higher in the laparoscopic group in comparison with the mini-laparotomic group for leiomyomas located at other sites,whereas no difference between treatment groups was detectedfor posterior/infraligamentary leiomyomas. Intraoperativeblood loss, DHb, and postoperative ileus were significantly(P<.05) lower in the laparoscopic group in comparison withthe minilaparotomic group for posterior/infraligamentaryleiomyomas, whereas no difference between treatmentgroups was detected when leiomyomas were located at othersites. The degree of surgical difficulty was not different be-tween groups for posterior/infraligamentary leiomyomas,whereas it was significantly (P<.05) higher in the laparo-
FIGURE 2
Visual analogue scale (VAS) scores for postoperativepain evaluated in two surgical groups every 2 hoursduring the first 12 hours, and 24 and 48 hours afterthe surgical procedure.
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
948 Palomba et al. Laparoscopic and minilaparotomic my
scopic versus the minilaparotomic group for leiomyomas lo-cated at other sites. The number of vials of analgesic usedand the duration of hospitalization were significantly(P<.05) lower in the laparoscopic versus the minilaparotomicgroup regardless of leiomyomas localization. No differencebetween treatment groups was detected in time necessary toreturn to full activity according to leiomyomas localization(Table 5).
DISCUSSION
The minilaparotomic approach was developed as a minimallyinvasive procedure to avoid the traditional open abdominalaccess in several gynecological diseases (11–18). In fact, be-cause laparoscopic surgery is more complex than laparo-tomic surgery, particularly for uterine leiomyoma excision,the minilaparotomic approach was recently experimentedfor myomectomy.
Cagnacci et al. (14) first studied in a randomized controlledfashion, the short-term outcomes of minilaparotomic myo-mectomy, with and without laparoscopic assistance, versusthe standard abdominal myomectomy. The minilaparotomicapproach had significantly lower DHb, duration of ileusand hospitalization in comparison with the classic laparo-tomic approach.
More recently, Alessandri et al. (19) compared in anotherRCT the minilaparotomic approach to the laparoscopic pro-cedure for uterine leiomyomas treatment. The global opera-tive time was significantly lower in the minilaparotomicgroup, whereas DHb, duration of ileus and hospitalizationwere significantly lower in the laparoscopic group.
The current study confirms the study by Alessandri et al.(19) that the laparoscopic myomectomy has several advan-tages over the minilaparotomy, although it is a complex sur-gical procedure. In fact, the subjective degree of surgicaldifficulty was significantly higher in the laparoscopic group
omectomy Vol. 88, No. 4, October 2007
TABLE 4QoL assessment (SF-36 scores) in women treated with laparoscopic or minilaparotomic myomectomyat baseline, and after 1 and 3 months after surgical intervention.
Note: Data are expressed as median (interquartile range) and minimum–maximum values.a P< .05 vs. baseline.
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
than in the minilaparotomic group. In this regard, our studypopulation was composed of women with characteristics ofhigh homogeneity. In particular, to obtain a homogeneous se-ries of cases with comparable surgical difficulty, we selectedonly cases having no more than three leiomyomas, in whichthe largest leiomyomas were intramural and reconstruction ofthe uterine walls with sutures was necessary, and we excludedwomen pretreated with GnRH agonist (GnRH-a) and withhypoechoic or calcified leiomyomas because, in these cases,the operating time is significantly higher due to the difficultyin grasping the tumor (23).
The present RCT demonstrates a significant influence ofthe dimension and localization of the largest leiomyoma. Al-though laparoscopic myomectomy for leiomyomas equal ormore than 7 cm has been already indicated as a very complexand difficult procedure and closely related to very long oper-ative time (31), in the current study, leiomyoma localizationwas the most significant variable influencing our findings.
In particular, our data demonstrate that when the main leio-myoma is anterior, fundal, or lateral, minilaparotomy can beconsidered a valid alternative to laparoscopy. In fact, in thesecases minilaparotomic myomectomy was simpler and faster
Fertility and Sterility�
than laparoscopy, and intraoperative blood loss, duration ofileus and hospitalization were similar. On the other hand, inpresence of the main leiomyoma located at posterior or infra-ligamentary sites, the minilaparotomic approach looses all itsadvantages over laparoscopy, and the degree of surgicaldifficulty was similar to that of laparoscopy.
Thus, the exact knowledge regarding leiomyoma charac-teristics is crucial to understand results of previous studies.
Unfortunately, in the study by Alessandri et al. (19), no in-formation was provided regarding the number of leiomyomasand leiomyoma localization for each patient, and the datawere analyzed without the application of the ITT principle.The specific localization of the leiomyomas was also not re-ported in two other studies available in literature comparingminilaparotomic and laparoscopic myomectomy (14, 17).
In a recent nonrandomized prospective study (17) compar-ing minilaparotomic and laparoscopic myomectomies, thechoice to use one of the two procedures was made accordingto the patient and leiomyoma characteristics by a senior phy-sician skilled in both surgical techniques. Because the onlydifference between the two groups was the number of
949
TABLE 5Surgical data according to main leiomyoma localization in laparoscopic and minilaparotomic groups.
Group Laparoscopy (n [ 68) Minilaparotomy (n [ 68)
Total operative time (min)Posterior/intraligamentary 119 (20); 88–150a,b 125 (36); 88–174b
Other sites 95.5 (29); 69–125a 74 (16); 62–105Time of leiomyoma enucleation (min)
Note: Data are expressed as median (interquartile range) and minimum–maximum values.a P< .05 vs. minilaparotomic group.b P< .05 vs. other sites.
Palomba. Laparoscopic and minilaparotomic myomectomy. Fertil Steril 2007.
leiomyomas removed, we consequently supposed that theonly factor regarding the choice of surgical access was thenumber of leiomyomas (32).
Another important point of discussion, poorly addressedin almost all studies comparing surgical techniques, is theinfluence of the different investigational centers on the re-sults. Although the surgeons involved in the current protocolwere skilled in both procedures, it was very difficult, or evenimpossible, that an operator had the same experience or hadnot a personal preference for a specific approach, especiallyin cases of large or particularly located uterine leiomyomas.
This finding demonstrates that not only randomization iscrucial but also multicentricity of trials to obtain a good gen-eralization of the results. Our data were obtained in a random-ized controlled fashion from three different investigationalcenters. On the other hand, all RCTs studying safety and ef-
950 Palomba et al. Laparoscopic and minilaparotomic m
ficacy of myomectomy have been performed in single surgi-cal setting, and, probably, reflect the experience of only onesurgeon (14, 19, 33, 34).
There are two limitations in the present RCT. First, no in-fluence of BMI on the total operative time was detected. Theexplanation for this finding is probably related to the homo-geneous patient characteristics of our sample. We can sup-pose that in a large population including obese and leanpatients, BMI could significantly influence the surgical re-sults. Second, our population was composed, as required byinitial protocol study, of patients with three uterine leiomyo-mas in a very low proportion.
Finally, our study confirms that minilaparotomic myo-mectomy is related to a lower global degree of surgical dif-ficulty, and that it is a highly feasible and safe procedure,even if the laparoscopic approach remains, in expert hands,
yomectomy Vol. 88, No. 4, October 2007
the procedure better related to the best short-term outcomes.In addition, our analysis demonstrates that the surgical out-comes of the two approaches are strongly influenced by op-erator, dimension, and localization of the main leiomyoma.This last parameter should be the main criteria in thesurgeon’s decision for the choice of the best approach tomyomectomy.
REFERENCES1. Wallach EE, Vlahos NF. Uterine myomas: an overview of development,
clinical features, and management. Obstet Gynecol 2004;104:393–406.
2. Bachmann G. Expanding treatment options for women with symptom-
atic uterine leiomyomas: timely medical breakthroughs. Fertil Steril
2006;85:46–7.
3. Pron G. New uterine-preserving therapies raise questions about interdis-
ciplinary management and the role of surgery for symptomatic fibroids.
Fertil Steril 2006;85:44–5.
4. Munro MG. Management of leiomyomas: is there a panacea in Pandora’s
box? Fertil Steril 2006;85:40–3.
5. Manyonda I, Sinthamoney E, Belli AM. Controversies and challenges in
the modern management of uterine fibroids. BJOG 2004;111:95–102.
6. West S, Ruiz R, Parker WH. Abdominal myomectomy in women with
very large uterine size. Fertil Steril 2006;85:36–9.
7. McIlveen M, Li TC. Myomectomy: a review of surgical technique. Hum
Fertil (Camb) 2005;8:27–33.
8. Taylor A, Sharma M, Tsirkas P, Arora R, Di Spiezio Sardo A,
Mastrogamvrakis G, et al. Surgical and radiological management of uter-
ine fibroids—a UK survey of current consultant practice. Acta Obstet
Gynecol Scand 2005;84:478–82.
9. Hurst BS, Matthews ML, Marshburn PB. Laparoscopic myomectomy for
29. SPSS Advanced Models 12.0. SPSS Inc. Chicago 2003.
30. Egger M, Juni P, Bartlett C, CONSORT Group (Consolidated Standards
of Reporting of Trials). Value of flow diagrams in reports of randomized
controlled trials. JAMA 2001;285:1996–9.
31. Dubuisson JB, Fauconnier A, Babaki- Fard K, Chapron C. Laparoscopic
myomectomy: a current view. Hum Reprod Update 2000;6:588–94.
32. Palomba S, Zupi E, Zullo F. Laparoscopy or minilaparotomy as mini-
mally invasive surgical approach for uterine leiomyomas? An unsolved
question. J Minim Invasive Gynecol 2006;13:253.
33. Mais V, Ajossa S, Guerriero S, Mascia M, Solla E, Melis GB. Laparo-
scopic versus laparotomic myomectomy: a prospective randomized trial
to evaluate benefits in early outcome. Am J Obstet Gyneol 1996;174:
654–8.
34. Seracchioli R, Rossi S, Govoni F, Rossi E, Venturoli S, Bulletti C, et al.
Fertility and obstetric outcome after laparoscopic myomectomy of large
myomata: a randomized comparison with abdominal myomectomy.
Hum Reprod 2000;15:2663–8.
951
Survival rate of human oocytes and pregnancyoutcome after vitrification using slush nitrogenin assisted reproductive technologiesTae Ki Yoon, M.D.,a Dong Ryul Lee, Ph.D.,a Soo Kyung Cha, M.Sc.,a Hyung Min Chung, Ph.D.,a,b
Woo Sik Lee, M.D., Ph.D.,a and Kwang Yul Cha, M.D.a
a Fertility Center of CHA General Hospital, CHA Research Institute, Pochon CHA University, College of Medicine; andb Chabiotech Co., Seoul, Korea
Objective: To report the survival rate of oocytes and the rate of successful pregnancies obtained from super-rapidcooling of oocytes using slush nitrogen (SN2).Design: Prospective clinical research.Setting: A university-affiliated hospital.Patient(s): Twenty-eight infertile women who underwent 30 cycles of IVF-ET using previously vitrified oocytes.Intervention(s): Oocytes were vitrified by super-rapid cooling using SN2.Main Outcome Measure(s): Morphological normality of thawed oocytes and clinical outcome.Result(s): In 30 cycles of ovarian stimulation for IVF, 364 surplus oocytes from 28 patients were vitrified usingSN2. Three hundred two (85.1% � 2.9%) of the oocytes survived after warming. Fertilization and cleavage rateswere 77.4% � 3.5% (168/218) and 94.3% � 2.1% (158/168), respectively. Thirteen pregnancies (43.3%) resultedfrom 30 uterine transfers of 120 embryos with an implantation rate of 14.2% (17/120). There were no differencesbetween the pregnancy rate after vitrification/warming and that obtained from routine noncryopreserved oocytes.Conclusion(s): The present report suggests that super-rapid cooling may improve the clinical efficacy of humanoocyte vitrification and may be a valuable tool for human assisted reproductive technologies. (Fertil Steril�
2007;88:952–6. �2007 by American Society for Reproductive Medicine.)
Since the first reported pregnancy from the use of frozen ma-ture human oocytes (1), numerous studies have been carriedout with the goal of developing an ideal method for oocytecryopreservation. These studies have used surplus matureoocytes from patients undergoing IVF-ET, which were storedfor future use by slow cooling or vitrification methods (2–9).When the patients failed to become pregnant in their freshIVF-ET cycles, the stored oocytes were then used for IVF-ET. In addition, several centers have offered oocyte bankingto patients who were at risk of losing gonadal function be-cause of chemotherapy, radiation treatment, or extirpativetherapy for pelvic pathology and pain.
Although cryopreservation of human oocytes has beensuccessfully carried out, clinical outcome has been limitedbecause of the decreased viability of thawed oocytes. Toimprove the viability and quality of oocytes after thawing,several changes have been introduced. Recently, a modified
Received March 4, 2006; revised and accepted December 29, 2006.
T. K. Yoon and D. R. Lee contributed equally to this study.
Supported by grant no. SC2190 of the Stem Cell Research Center, which
is funded by the Korea Ministry of Science and Technology, Republic of
Korea (to HMC).
Reprint requests: Tae Ki Yoon, M.D., Fertility Center of CHA General
Hospital, CHA Research Institute, Pochon CHA University College of
Medicine, 606-5 Yeoksam-dong, Gangnam-gu, Seoul 135-081, Korea
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
952
protocol using slow cooling has been suggested to improvesurvival rates as a result of changes involving an increasein sucrose concentration or the replacement of sodium withcholine in the freezing media (10–13). Our group has devel-oped a vitrification method for the cryopreservation of humanoocytes, and previous results have been encouraging (6, 7, 14,15). To optimize our vitrification method, we have recentlyintroduced a new method, super-rapid cooling using slushnitrogen (SN2). Boiling of liquid nitrogen (LN2) occurswhen a sample is immersed in it and results in gas bubblesaround the specimen, which, in turn, results in poor heattransfer. By applying negative pressure with a vacuum, LN2
will freeze and will be transformed into a slush state (SN2)with a lower internal temperature of�210�C and without va-porization (16). In 2001, Isachenko and coworkers reportedthat they obtained a higher maturation rate of ovine immatureoocytes using SN2 rather than LN2 (17). In this paper, we re-port a high survival of vitrified human oocytes with SN2,which resulted in good morphology and an excellent preg-nancy rate using this new super-rapid cooling technique.
MATERIALS AND METHODS
The Institutional Review Board of CHA General Hospital,Seoul, Korea, approved this clinical application in January2003. All of the study participants gave their written in-formed consents.
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.071
Controlled Ovarian Hyperstimulation for Standard IVF-ET
Starting on the third day of the cycle, controlled ovarian hy-perstimulation was carried out with recombinant FSH (rFSH)or rFSH/hMG. To prevent a premature LH surge, we usedGnRH agonist (Lucrin; Abbott, Seoul, Korea) in the long pro-tocol or the GnRH antagonist cetrorelix (Cetrotide; Serono,Halle, Germany) after 5–6 days of stimulation. Ovulationwas triggered with 10,000 IU of hCG (Profasi; Serono)when at least two follicles were over 18 mm in diameter.Transvaginal oocyte retrieval was carried out 34–36 hours af-ter hCG administration.
Vitrification and Warming of Oocytes
Recovered cumulus-oocytes complexes (COCs) were brieflyincubated for 10 seconds with 80 IU/mL of hyaluronidase(Sigma, St. Louis) to remove excess cumulus cells (CCs)and were then pre-equilibrated for 2.5 minutes in 2 mL of Dul-becco’s phosphate-buffered saline (DPBS; Gibco BRL,Grand Island, NY) supplemented with 1.5 M of ethyleneglycol (EG; E-9129; Sigma) and 10% (vol/vol) fetal bovineserum (FBS; Gibco BRL) at 37�C. COCs with two to threelayers of CCs were then placed for the final equilibration inthe same volume of DPBS supplemented with 5.5 M EG,1.0 M sucrose, and 10% FBS for 20 seconds. Two to fiveCOCs that were partially denuded were mounted on an elec-tron microscope gold grid (Gilder, Westchester, PA) usinga fine glass pipette. Excess cryoprotectant solution wasremoved with sterilized paper (Kimwipes, Yuhan-Kimberly,Gunpo, Korea). The gold grids containing COCs were imme-diately plunged into either LN2 or SN2 as described below.For long-term storage, a cryovial cap and goblet were usedfor placement of the grid. For thawing, the gold grids weresequentially transferred to culture dishes containing 2 mL ofDPBS supplemented with 1.0, 0.5, 0.25, 0.125, or 0 M sucroseand 10 % (vol/vol) FBS at intervals of 2.5 minutes at 37�C.
Fertility and Sterility�
Generation of SN2
SN2 was generated in a rapid-cooling LN2 chamber by apply-ing negative pressure (Vit-Master, IMT, Ness Ziona, Israel).To make SN2, three-quarters of the chamber of this machinewas filled with LN2 and the vacuum pump was switched on.After approximately 10–20 minutes, the temperature in thechamber declined to a maximum of�210�C, and the pressurewas then reduced to approximately 0 bars. LN2 in the cham-ber was then transformed into a slush state (SN2). SN2 hasa lower internal temperature without vaporization (Figs.1A–1C) and results in high rates of cooling (18).
Oocyte Freezing
From December 2003 to August 2005, 76 patients agreed toparticipate in an oocyte cryopreservation protocol using theSN2 vitrification method for surplus oocytes while theywere undergoing IVF. Twenty-eight patients who failed toconceive with their fresh IVF-ET cycles returned for ETusing vitrified-warmed oocytes (30 cycles). The mean(�SD) age and duration of infertility for the patients were33.7 � 4.6 years and 4.5 � 2.8 years, respectively. Reasonsfor IVF-ET were as follows: tubal factor (n¼ 10), male factor(n ¼ 8), unexplained (n ¼ 4), ovum donation (n ¼ 2), poly-cystic ovary syndrome (n ¼ 3), and endometriosis (n ¼ 1).The average number of IVF attempts was 2.0 � 1.7.
Patients with more than 15 oocytes retrieved were giventhe option to freeze their supernumerary oocytes for futureuse. Oocytes were stored for 1–17 months before thawing;the mean interval between the fresh and vitrified cycles was4.3 months.
Fertilization, Embryo Culture, and ET after Warming
After being washed four to six times, CCs were removed bymechanical pipetting and then transferred into the preimplan-tation-1 (P-1) medium (Irvine Scientific, Irvine, CA) with
FIGURE 1
Photographs of slush nitrogen (SN2). (A) Gas bubbles were formed when the grid was immersed into LN2. (B) SN2
was formed in the chamber when negative pressure was applied. (C) No bubble formation occurred in SN2.
Yoon. Human oocyte vitrification using SN2. Fertil Steril 2007.
953
10% synthetic serum substitute (SSS; Irvine Scientific). In-tracytoplasmic sperm injection (ICSI) was performed 3–6hours after the warming of oocytes (day 0). The pronucleiin the cytoplasm were counted to verify normal fertilizationof vitrified/warmed oocytes at 16–19 hours after ICSI. Theembryos from 2PN zygotes were cultured in the P-1 mediumwith 10% SSS.
For the artificial preparation of the endometrium, E2 valer-ate (4–6 mg/day Progynova; Schering, Seoul, Korea) was ad-ministrated and P was injected (100 mg/day IM; SamilPharmaceutical, Seoul, Korea) after the endometriumreached 8 mm in thickness. In natural cycles, P supplementa-tion (50 mg/day) had begun after ovulation (day 0). We trans-ferred embryos to the uterus 3 days after the ICSI procedure.When endometrial thickness as assessed before starting P ad-ministration was less than 7 mm or>12 mm, the thawing wascanceled. Pregnancy was identified by serum b-hCG 12 daysafter ET. Once a clinical pregnancy was established, maternalblood testing and amniocentesis were carried out to excludefetal anomalies.
Statistical Analysis
Unless otherwise specified, data were expressed as mean �SEM. For statistical comparisons, clinical outcomes were an-alyzed using the c2-test and Student’s t-test. P<.05 was con-sidered statistically significant.
RESULTS
Twenty-eight patients agreed to undergo transfer of embryosderived from cryopreserved oocytes after failing a fresh IVF-ET cycle. In fresh cycles, rFSH or rFSH/hMG was adminis-trated at 225–300 IU/day for 3–5 days, and then dosages wereadjusted according to the ovarian response. The cumulativedose and duration of rFSH or rFSH/hMG were 2,215.2 �129.1 IU and 9.3 � 2.0 days, respectively. E2 concentrationand numbers of follicles (R14 mm on the day of hCG injec-tion) were 3,650 � 330 pg/mL and 29.9 � 2.3.
Of the 784 COCs (26.1 � 2.5) retrieved from 28 patients,358 oocytes were used for fresh IVF-ET cycles, and theremaining 426 COCs were cryopreserved by our vitrificationmethod using electron microscopy (EM) gold grids and SN2.Two patients had two different cycles of ET.
The morphological survival rate of oocytes was 85.1% �2.9% (302/364). Of those, 72.2% (218/302) were metaphaseII oocytes suitable for ICSI. The fertilization rate of these vit-rified/warmed oocytes was 77.4%� 3.5% (168/218), and thecleavage rate on day 2 was 94.3% � 2.1% (158/168).
Three to five developed embryos were selected on the basisof their morphology and cell stage and transferred into theuteri of the patients. The average number of embryos trans-ferred was 4.0 � 0.2. All patients underwent ET procedureswithout any cancellation. A total of 120 cleaving embryosfrom vitrified oocytes were transferred during the 30 cyclesin 28 patients, and 13 patients achieved clinical pregnancies.
954 Yoon et al. Human oocyte vitrification using SN2
The pregnancy rate per ET and implantation rate for SN2-vitrified IVF-ET were 43.3% (13/30) and 14.2% (17/120),respectively. Three multiple pregnancies (23.1 % [3/13])were obtained from this procedure. Two pregnancies miscar-ried at 8 weeks and 9 weeks, respectively. One abortus wastetraploid (88þXXYY), and the other was normal(44þXY). Four pregnancies delivered five normal babies(three singletons [male/male/female] and one twin [male/male]), and the remaining seven pregnancies are ongoing(Table 1).
Although the numbers are small, we did not detect any sig-nificant difference in clinical outcomes after vitrification/warming and the various ovarian hyperstimulation protocols(57.1% [8/14] with GnRH agonist-rFSH, 44.4% [4/9] withGnRH agonist-rFSH/hMG, and 14.3% [1/7] with GnRHantagonist-rFSH).
Eleven patients did not have an ET in their fresh cycles forthe following reasons: two patients had failed fertilization,five were at risk for ovarian hyperstimulation syndrome(OHSS), two were not well synchronized with ovum donors,and two were for personal reasons. In these patients, the preg-nancy and implantation rates after using SN2 vitrificationwere much higher than in the other patients (n ¼ 17) whohad failed to become pregnant in their fresh IVF-ET cycles(72.7% [8/11], 22.2% [12/54] vs. 29.4% [5/17], 7.6% [5/66]; P<.05; Table 2). The number of retrieved oocytes wasnot different in these subgroups: 25.5 � 3.4 with no ET intheir fresh cycles vs. 26.6 � 1.7 with ET in their fresh cycle.There was also no difference in the indication for IVF-ET inthese subgroups.
DISCUSSION
Improving survival and fertilization rates remains a majorchallenge in oocyte cryopreservation. The cooling rate duringvitrification has proven to be one of the main factors influenc-ing the survival of embryos and oocytes. Recently, severalapproaches have been used to increase the cooling rate of em-bryos/oocytes. Otoi and coworkers vitrified bovine oocytes inconventional straws and achieved results better than thosewith the slow cooling method (19). Also, Vajta et al. devel-oped open-pulled straws to hold bovine oocytes in a smallamount of vitrification solution (20). The use of the EMgrid as an oocyte vehicle may help rapid heat conductionfrom the outside into the oocyte, and the relatively short pro-tocol prevents oocytes from possible damage due to the solu-tion effect of the cryoprotectant (14, 15). In fact, with use ofthe EM grid for high cooling rates of vitrification, chilling-sensitive Drosophila embryos and bovine oocytes havebeen successfully frozen (16, 21). The gold grid may alsoprovide low-toxicity with its extremely high heat conductiv-ity. In this study, we have tested the effects of super-rapidcooling by SN2 on human oocyte vitrification.
In this clinical trial in 30 cycles, survival and pregnancyrates were very high when SN2 was applied to oocyte vitrifi-cation (Table 1), and these clinical results were comparable to
Vol. 88, No. 4, October 2007
TABLE 1Clinical outcomes of IVF-ET programs using vitrified/warmed human mature oocytes vitrified by slushnitrogen (SN2) from stimulated cycles.
No. of patients 28No. of vitrified oocytes 426No. of warmed oocytes 364No. of survived oocytes (%)a 302 (85.1 � 2.9,b 79.2–90.9c)No. of microinjected oocytes 218No. of fertilized oocytes (%) 168 (77.4 � 3.5,b 70.2–84.7c)No. of cleaved embryos (%) 158 (94.3 � 2.1,b 90.1–98.5c)No. of patients who underwent ET (%) 30 (100)No. of pregnancies (%/ET) 13/30 (43.3)No. of pregnancies (%/patient) 13/28 (46.4)No. of multiple pregnancies 3/13 (23.1)No. of miscarriages (%) 2/13 (15.4)No. of deliveries/ongoing 4d/7No. of transferred embryos 120 (4.0 � 0.2,b 3.6–4.4c)Implantation rate (%) 17 (14.2)
a No. of intact oocytes after warming (%/vitrified oocytes).b Mean � SEM.c 95% confidence interval.d Three singletons (male/male/female), one twin (male/male).
Yoon. Human oocyte vitrification using SN2. Fertil Steril 2007.
those with fresh IVF-ET or with conventional embryo freez-ing. Moreover, seemingly higher pregnancy and implantationrates were also observed after SN2 vitrification in patientswho did not undergo ET in their fresh cycles for several rea-sons (Table 2), although there was no difference in the indi-cation for IVF-ET in these two groups. More importantly,
Fertility and Sterility�
we successfully performed ET in all patients, which resultedin a high pregnancy rate. These data therefore suggest thatour vitrification procedure using super-rapid cooling doesnot seem to be restricted in specific patients and may be anoption for fertility preservation, avoiding the ethical problemof embryo freezing or the risk of OHSS.
TABLE 2Comparison of clinical outcomes of slush nitrogen-vitrified human mature oocytes from stimulatedcycles in women with failed attempts in fresh cycles and in others who had all oocytes cryopreserved.
VariablesPregnancy, failedon fresh cyclesa
All cryopreservation,on fresh cyclesb
No. of patients 17 11No. of patients undergoing ET, % 18 12Pregnancies (%/ET) 5/18 (27.8) 8/12 (66.7)c
a IVF-ET program using vitrified/warmed human mature oocytes by slush nitrogen after failing to become pregnant usingfresh oocytes.
b IVF-ET program using vitrified/warmed human mature oocytes by slush nitrogen after failing to complete the fresh cycle.c P<.05d Mean � SEM.
Yoon. Human oocyte vitrification using SN2. Fertil Steril 2007.
955
The advantage of vitrification using super-rapid coolingmay still be controversial based on several recent reports us-ing experimental or domestic animals. Increasing the freez-ing rate by super-cooled LN2 may not be of any advantagefor cryopreservation of mouse pronuclear-stage embryos(22). Also, applying SN2 did not increase the fertilizationrate and further embryonic development of immature bovineoocytes, despite improving survival rates and morphology af-ter vitrifying/warming (23). These data suggest that the effectof the cooling rate in vitrification may be species-specificor dependent on the cell stage, showing differences amongoocytes, zygotes, embryos, or blastocysts. However, the sur-vival rate of cryopreserved oocytes has been a major obstacleto the successful use of this technology until now. Previouslarge-scale clinical studies have reported survival rates inthe range of 37%–74% (6, 7, 12, 13, 24, 25). Our data usingSN2 vitrification represents a highly improved survival rateby virtue of increasing the cooling rate and therefore maybe useful when applied clinically.
In summary, in this study, we have obtained successfulpregnancies from vitrified/warmed human oocytes by usingSN2 for super-rapid cooling when combined with ICSI forfertilization. To our knowledge, this is the first report of preg-nancies resulting from oocytes vitrified using SN2. Thismethodology may open the possibility for oocyte cryopreser-vation to be more readily applied in human assisted reproduc-tive technologies.
Acknowledgments: The authors thank Professor Rogerio Lobo, M.D. (De-
partment of Obstetrics and Gynecology, Columbia University College of
Physicians and Surgeons, New York), for his kind comments and review of
the manuscript.
REFERENCES1. Chen C. Pregnancy after human oocyte cryopreservation. Lancet 1986;1:
884–6.
2. Tucker M, Wright G, Morton P, Shanguo L, Massey J, Kort H. Prelimi-
nary experience with human oocyte cryopreservation using 1,2-propane-
diol and sucrose. Hum Reprod 1996;11:1513–5.
3. Porcu E, Fabbri R, Seracchioli R, Ciotti PM, Magrini O, Flamigni C.
Birth of healthy female after intracytoplasmic sperm injection of cryo-
preserved human oocytes. Fertil Steril 1997;68:724–6.
4. Kuleshova L, Gianoroli L, Magli C, Ferraretti A, Trounson A. Birth fol-
lowing vitrification of small number of human oocytes. Hum Reprod
1999;14:3077–9.
5. Porcu E, Fabbri R, Damiano G, Giunchi S, Fratto R, Ciotti PM, et al.
Clinical experience and applications of oocyte cryopreservation. Mol
Cell Endocrinol 2000;169:33–7.
6. Yoon TK, Chung HM, Lim JM, Han SY, Ko JJ, Cha KY. Pregnancy and
delivery of healthy infants developed from vitrified oocytes in a stimu-
956 Yoon et al. Human oocyte vitrification using SN2
lated in vitro fertilization–embryo transfer program. Fertil Steril
2000;74:180–1.
7. Yoon TK, Kim TJ, Park SE, Hong SW, Ko JJ, Chung HM, et al. Live birth
after vitrification of oocytes in a stimulated in vitro fertilization–embryo
transfer program. Fertil Steril 2003;79:1323–6.
8. Borini A, Bonu MA, Coticchio G, Bianchi V, Cattoli M, Flamigni C.
Pregnancies and births after oocyte cryopreservation. Fertil Steril
2004;82:601–5.
9. Chen ZJ, Li M, Li Y, Zhao LX, Tang R, Sheng Y, et al. Effects of sucrose
concentration on the developmental potential of human frozen-thawed
oocytes at different stages of maturity. Hum Reprod 2004;19:2345–9.
10. Fabbri R, Porcu E, Marsella T, Rocchetta G, Venturoli S, Flamigni C.
Human oocyte cryopreservation: new perspectives regarding oocyte
survival. Hum Reprod 2001;16:411–6.
11. Stachecki JJ, Cohen J, Willadsen S. Cryopreservation of unfertilized
mouse oocytes: the effect of replacing sodium with choline in the freez-
ing medium. Cryobiology 1998b;37:346–54.
12. Quintans CJ, Donaldson MJ, Bertolino MV. Birth of two babies using oo-
cytes that were cryopreserved in a choline-based freezing medium. Hum
Reprod 2002;17:3149–52.
13. Boldt J, Cline D, McLaughlin D. Human oocyte cryopreservation as an
adjunct to IVF-embryo transfer cycles. Hum Reprod 2003;18:1250–5.
14. Hong SW, Chung HM, Lim JM, Ko JJ, Yoon TK, Yee B, et al. Improved
human oocyte development after vitrification: a comparison of thawing
methods. Fertil Steril 1999;72:142–6.
15. Chung HM, Hong SW, Lim JM, Lee SH, Cha WT, Ko JJ, et al. In vitro
blastocyst formation of human oocytes obtained from unstimulated
and stimulated cycles after vitrification at various maturational stages.
Fertil Steril 2000;73:545–51.
16. Steponkus PL, Caldwell S. An optimized procedure for the cryopreserva-
tion of Drosophila melanogaster embryos. Cryo Lett 1993;14:375–80.
17. Isachenko V, Alabart JL, Nawroth F, Isachenko E, Vajta G, Folch J. The
open pulled straw vitrification of ovine GV-oocytes: positive effect of
rapid cooling or rapid cooling or rapid thawing or both? Cryo Lett
24. Chen C. Pregnancies after human oocytes cryopresrvation. Ann N Y
Acad Sci 1988;541:541–9.
25. Porcu E, Fabbri R, Ciotti PM, Marsella T, Balicchia B, Damiano G, et al.
Cycles of human oocyte cryopreservation and intracytoplasmic injec-
tion: results of 112 cycles. Fertil Steril 1999b;72(Suppl 1):S2.
Vol. 88, No. 4, October 2007
LESSONS FROM THE LAB
Effects of cellular phone emissions on sperm motilityin ratsJi-Geng Yan, M.D., Ph.D.,a Michael Agresti, M.S.,a Tim Bruce, B.S.,a Yu Hui Yan, M.D.,a
Amy Granlund, B.S.,b and Hani S. Matloub, M.D.a
a Department of Plastic and Reconstructive Surgery, Medical College of Wisconsin, Milwaukee; and b Reproductive Medicine
Clinic, Froedtert Memorial Lutheran Hospital, Milwaukee, Wisconsin
Objective: To evaluate the effects of cellular phone emissions on rat sperm cells.Design: Classic experimental.Setting: Animal research laboratory.Subjects: Sixteen 3-month-old male Sprague-Dawley rats, weighing 250–300 g.Intervention(s): Rats in the experimental group were exposed to two 3-hour periods of daily cellular phone emis-sions for 18 weeks; sperm samples were then collected for evaluation.Main Outcome Measure(s): Evaluation of sperm motility, sperm cell morphology, total sperm cell number, andmRNA levels for two cell surface adhesion proteins.Result(s): Rats exposed to 6 hours of daily cellular phone emissions for 18 weeks exhibited a significantly higherincidence of sperm cell death than control group rats through chi-squared analysis. In addition, abnormal clumpingof sperm cells was present in rats exposed to cellular phone emissions and was not present in control group rats.Conclusion(s): These results suggest that carrying cell phones near reproductive organs could negatively affectmale fertility. (Fertil Steril� 2007;88:957–64. �2007 by American Society for Reproductive Medicine.)
Cellular phone usage is increasing worldwide at an astonish-ing rate. The manufacturer of Nokia mobile phones estimatedthat more than 2 billion people would now be cell phone sub-scribers, based on 2004 growth rate trends (1). With this in-crease in popularity, concerns have arisen regarding humansafety related to radiation emissions from cellular phones.Large doses of this radiofrequency electromagnetic radiation(RFEMR) have been related in previous studies to genetic de-fects, such as changes in the integrity of epididymal mito-chondrial DNA (2), altered proto-oncogene c-fos (3) andprotein kinase C expression (4), increased micronuclei for-mations (5, 6), increased chromosomal instability (7, 8),and changes in morphology, gene expression, and prolifera-tion of fibroblasts (9). Research also suggests that RFEMRis related to sperm parameter deterioration (10) and to an in-crease in the risk of cancers through the changes in chromo-somal stability (7–9).
However, current research on the effects of cellular phoneson the human body is contradictory and inconclusive. Several
Received July 27, 2006; revised and accepted December 12, 2006.
Reprint requests: Hani S. Matloub, M.D., Department of Plastic and
Reconstructive Surgery, Medical College of Wisconsin, 8700 Water-
town Plank Road, Milwaukee, Wisconsin 53226 (FAX: 414-259-0901;
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.022 Copyright ª2007 American
researchers believe the source of many of the abnormalitiesfound in laboratory tests is the combination of RFEMR andheat (10–13). According to some investigators, the effectsof cellular phones are minimal (14–17) to nonexistent (18)when the factor of heat is eliminated (14–17) or not extreme(18).
Determining more definitively the effects of cellular phoneuse on male fertility is important, considering that men oftencarry cellular phones in their pockets, close to reproductiveorgans. The purpose of this study is, therefore, to clarifywhether cell phones negatively affect sperm fertility, throughevaluation of rats following exposure to phone emissions.
MATERIALS AND METHODS
Subjects
Sixteen 3-month-old male Sprague-Dawley rats, weighing250–300 g, were the subjects of this research. For the careand use of laboratory animals, this study used the guidelinesof the Biomedical Resource Center of the Medical Collegeof Wisconsin. The Institutional Animal Care and Use Com-mittee of the Medical College of Wisconsin approved the pro-tocol. The rats were divided into two groups of eight ratseach. One group received cell phone radiation exposure,and the other group acted as a control group.
Fertility and Sterility� Vol. 88, No. 4, October 2007 957Society for Reproductive Medicine, Published by Elsevier Inc.
The four cell phones used in the study were Nokia 3588i(Keilalahdentie, Finland), which have a personal communi-cations service code division multiple access (PCS CDMA)frequency band of 1.9 GHz (800 MHz digital and 800 MHzanalog). These cell phones have three different modes:AMPS mode, CELL mode, and PCS mode. The variousmodes can be used based on signal reception, antenna use,and other factors associated with reception of different typesof radiofrequency signals.
In AMPS mode, the specific absorbence rate (SAR) at a dis-tance of 2.2 cm was measured to be 1.80 W/kg, and the powerrange was 0.0063–0.607 W. The SAR at a distance of 2.2 cmin CELL mode was 0.9 W/kg, and the power range was0.00001–0.487 W. The SAR at 2.2 cm away in PCS modewas 1.18 W/kg, and the power range was 0.00001–0.335W. The frequencies and specific modes of this phone fallwithin the cell phone radiation parameters set by the U.S.Federal Communications Commission (FCC) (19). Eachcell phone was positioned 1 cm from the head of the rats,at equal distances between two rats in holding chambers.
Holding Units
Customized holding units (Fig. 1) and cell phone platformswere constructed for this study. The holding units for therats consisted of 5.1 cm � 15.2 cm PVC tubes with holesfor circulation, fitted with 0.59-liter clear plastic bottle topson one end and common 7.6-cm bolts with nuts at the otherend. As the rats grew larger during this study, new tubeswere fashioned using 8.9-cm-diameter PVC pipes with holesfor air circulation, 1-liter bottles, and 10.2-cm bolts with nuts.The holding units were plastic, because metal can absorbradiation energy.
The rats were acclimated to these holding units for 1 weekbefore the beginning of testing by placing the units in the ratcages to allow the rats to become familiar with their smell andfeel. After less than one day, the rats voluntarily entered theunits to rest and sleep in them. By the end of the week, therats would enter the holding units as soon as they sawthem. Owing to this acclimation process, anesthesia was
958 Yan et al. Cellular phones affect sperm motility
not required during the exposure time. Therefore, the ratsdid not have any ill effects or altered physiology from anes-thesia, rendering the comparison to humans more relevant.
Methods
The experimental rat group was exposed to 3 hours of cellphone radiation, followed by a 30-minute rest period outsideof the tubes and a second exposure for 3 more hours per day.During the 30-minute rest period, the rats were removed fromthe tubes and were free to walk around, eat and drink. The ratsreceived this daily cell phone exposure for 18 weeks. The 8rats in the control group were placed in identical tubes forthe same amount of time as the experimental rats but withoutcell phone exposure.
After week 18, the rats were killed for harvest of the tissuesof interest. Incisions were made in the rat scrotums to dissectout the testicles and the epididymides. After transecting theproximal vas deferens, the sperm of each rat was allowedto passively flow into a Petri dish at 37�C in 2 mL HBSSfor 10 minutes. Then 5-mL samples of sperm were movedfrom the dish into a microcell 50-mm chamber (ConceptionTechnologies, San Diego, California) for evaluation of spermmotility and morphology. Total sperm counts and a molecularstudy were also completed.
Evaluation
To address the concern that the harmful effects of cell phonesare due to heat given off by the phone rather than RFEMR, wetook temperatures from both groups during a standard day ofexposure. Temperatures of the rats were taken one day at theside of the face surface nearest the phone, using a Mini-Alarm thermometer with a probe (Fisher Scientific, Hamp-ton, NH). These readings were taken approximately every12 minutes during both of the 3-hour exposures. Final rectaltemperatures were taken at the end of each of the two expo-sure times with a Big-Digit thermometer (Fisher Scientific).Because the rectal measurements irritated the animals,repeated measurements were not practical.
FIGURE 1
Rat in plastic PVC holding tube. The tube has a clear plastic bottle top and numerous air holes for adequateventilation.
The slides on which the sperm cells were counted werewarmed to 37�C until the time of the analysis. The analysiswas carried out at room temperature. An embryologist fromthe Reproductive Medicine Clinic performed a blind analysisof sperm motility using one epididymis of each rat. Theembryologist was unaware of the purpose of the studywhen performing the counts.
The percentage of sperm motility was calculated using thenumber of live sperm cells over the total number of spermcells (both motile and nonmotile), from two samples fromone epididymis of each rat. All sperm cells that were notmoving at all were considered to be nonmotile, while therest, which displayed some movement, were considered tobe motile.
Morphologic Analysis
Using the same epididymis from each rat, the morphology ofthe sperm cells was evaluated from two sperm samples perrat. One drop from the dish containing the sperm was placedon each slide, and immediately smeared and stained withDiff-Quik stain (Allegiance Scientific Products, McGawPark, Illinois) to facilitate identification of morphologic dif-ferences between experimental and control groups.
The 32 slides (16 from the experimental group and 16 fromthe control group) were sent to the Department of VeterinaryPathobiology at the University of Missouri. There a professorspecializing in deformities/mutations completed a blindassay of the slides.
All sperm cells were counted from each slide. The totalnumber of cells per slide ranged from 70 to 128, with an av-erage of 99 cells. Sperm cells were considered deformed ifthey were definitely headless, broken, or had bent tails andbodies which coiled back on themselves. Sperm cells thatdid not have hook-shaped heads and/or were not elongatedwere considered abnormal.
Sperm counts were done on a Nikon microscope (Nikon,Japan) at �20 magnification using a 0.5-mm2 countingarea. Four locations on each slide were chosen for counting.The total number of sperm was counted first, followed by thenumber of abnormal sperm.
Total Sperm Counts
The other testicle and epididymis of each rat were flash fro-zen and a total sperm count was performed on the testicle.The testicles were thawed in saline, and the outer capsuleswere dissected away. The testicles were put into a glassDounce homogenizer with 1 mL 0.9% saline/1% TX-100and were homogenized in seven passes with the loosewand. An additional 1.5-mL buffer was added, and sevenmore passes were made with the tight wand. The volumewas brought up to 10 mL with buffer, and two 11-mL sampleswere counted in a bright-line Neubauer hemocytometer.
The total sperm count reflects the number of sperm cellsper mL of solution, as determined by the following method.
Fertility and Sterility�
Each sample was counted 4 times. Five diagonal squareswere counted, which is a total of 0.2 mm2 of the solution.We multiplied the result by 5 to get the total count in1 mm2 of solution and then multiplied that result by 10 to ac-count for the dilution factor (of 10 mL). We multiplied thatresult by 10,000, to obtain the results in sperm cells/mL(per the instructions for the hemocytometer). The final num-bers therefore reflect the first 5 diagonal square numbers mul-tiplied by 5 � 105 cells.
Molecular Study
The remaining epididymides of the two groups were used forreverse-transcription polymerase chain reaction (RT-PCR) toassess the messenger (m) RNA levels of beta-actin (control)and two cell surface adhesion proteins, cadherin-1 (CAD-1)and interstitial cell adhesion molecule 1 (ICAM-1). TotalRNA was extracted from each remaining epididymis usingan SV Total RNA Isolation Kit (Promega, Madison, Wiscon-sin). The RNAwas quantified by a 260/280 ratio using a Beck-man Spectrophotometer (Fullerton, California).
The RT-PCR was performed using a SuperScript III One-Step RT-PCR Kit (Invitrogen, Carlsbad, California). Each re-action had 50 ng RNA with 0.5 mL of each specific forwardand reverse primer (10 mmol/L). The primers used wereas follows: beta-actin forward 30-agccatgtacgtagccatcc-50
and reverse 30-ctctcagctgtggtggtgaa-50; CAD-1 forward 30-gggttgtctcagccaatgtt-50 and reverse 30-caccaacacacccagcatag-50; and ICAM-1 forward 30-aggtatccatccatcccaca-50 andreverse 30-gccacagttctcaaagcaca-50. The reactions ran at55�C for 30 minutes, 94�C for 2 minutes and 15 seconds,55�C for 30 seconds, and 72�C for 1 minute for 40 cycles.
The DNA was run on a 1% agarose-gel-containing ethid-ium bromide in TAE buffer. A photo of the gel was takenon a Fotodyne 21 UV box (Fotodyne, Hartland, WI) withan Electrophoresis Photo Documentation Camera and Hood(Fisher Scientific). The photo was then quantitated in a Multi-Image Light Cabinet with AlphImager 2000 software (AlphaInnotech Corporation, San Leandro, CA).
RESULTS
Temperature
During the exposure times, the facial temperatures did not de-viate by more than 1�C between experimental and controlgroups (Table 1). The mean initial temperature was 32.8�Cin the experimental group and 33�C in the control group. Dur-ing the first 3 hours, the average temperature was 33.3 �0.5�C in the experimental group, and 33.5� 1.1�C in the con-trol group. The mean temperature during the last 3 hours was32.6� 0.6�C in the experimental group, and 32.4� 0.8�C inthe control group.
The rectal temperatures were measured before testing andat the 3-hour and 6-hour points. The results were similar inthe two groups. After 3 hours, the mean experimental grouptemperature was 35.6�C, and the mean control group temper-ature was 36.4�C. After the full 6 hours of daily exposure, the
959
TABLE 1Comparison of mean facial surface temperatures (in �C).
First 3 hours Last 3 hours
Time (min) Control Experimental Time (min) Control Experimental
mean value in both the experimental group and the controlgroup was 35.8�C. None of the temperature differences be-tween the two groups were statistically significant throughpaired t tests (P>.05).
Sperm Motility
Sperm motility was significantly different between the twogroups in a chi-squared analysis (N ¼ 16; p<.05). In theexperimental group, a majority of the sperm cells were dead,with no motion and straight rigid tails (Fig. 2A). (Sperm celldeath was determined through edema in the tails of the sperm,clumping of red and white blood cells to the body of the sperm,rigidity, and complete lack of movement.) In the controlgroup, most of the sperm cells were alive, with constant activemovement (Fig. 2B). The average percentage of live sperm inthe experimental group was 44.88� 20.66%, versus a mean of70.93 � 12.94% for the control group (Table 2).
Morphologic Study
The percentage of deformities for the experimental groupwas 34.3%, and the percentage of deformities for the controlgroup was 32.1%. This difference in the occurrence of defor-mities between the two groups was not statistically signifi-cant (P>.05) through a paired t test.
However, whereas most of the live sperm cells in bothgroups appeared relatively normal, without severe abnormal-ities, sperm cells from the experimental samples frequently
960 Yan et al. Cellular phones affect sperm motility
stuck together in large clumps. Sperm cells were consideredto be forming a large cluster when 90% or more of the spermcells were stuck together in one field under the microscopewith �20 magnification (Figs. 2C through 2E). In the exper-imental group, more than 80% of the experimental slides (13of the 16 slides) had large clumps of sperm cells. The spermcells in these clumps were able to do little more than squirmabout, and they could not break free.
Three types of clumps were present. In type I, the spermheads were closely stuck together to form an umbrella shape(Fig. 2C). In type II, the sperm cells were stuck together toform a small clump, with many small clumps stuck togetherto form a big grass-bundle shape (Fig. 2D). Type III clumpscontained sperm cell tails stuck to the heads to form a ringshape (Fig. 2E). In addition to adhesion between sperm cells,many sperm cells in the experimental group were also limitedin their mobility by the bonding of blood cells to them. Largeclusters of sperm cells were not present in slides from thecontrol group (Fig. 2B).
Total Sperm Count
The total sperm counts in the testicles did not statistically sig-nificantly differ between the experimental and the controlgroups, through a paired t test (P>.05; Table 3). The experi-mental group had a mean of 7.45 � 107 � 1.03 � 107 spermcells/mL, and the control group had a mean of 7.7 � 107 �8.11 � 106 sperm cells/mL.
Vol. 88, No. 4, October 2007
FIGURE 2
Sperm samples at �20 magnification. (A) Sperm cells from the experimental group, which appear dead.These cells are rigid with edema in the tails, giving them a wider appearance. Many blood cells are also stickingto the dead sperm cells. (B) Sperm sample from the control group. These individual sperm cells are evenlydistributed across the slide without changes in gross morphology. These cells were actively moving. (C) Spermsample from the experimental group, with sperm cells stuck together in type I clumps: Sperm heads are closelystuck together to form an umbrella shape. (D) Sperm sample from the experimental group, with sperm cells stucktogether in type II clumps: Sperm cells are stuck together to form a small clump, and these small clumps arestuck together to form a big grass-bundle shape. (E) Sperm sample from the experimental group, with spermcells stuck together in type III clumps: Sperm tails are stuck to the heads to form a ring shape. Some sperm cellsin all types of clumps appeared to be alive, because there was some flagellar action, but they were unable tomove individually.
The PCR analysis of the epididymides revealed an up-regula-tion of mRNA levels for the two cell surface adhesion pro-
Fertility and Sterility�
teins tested, CAD-1 and ICAM-1 (Fig. 3). The results forboth cell surface adhesion proteins were statistically signifi-cant between experimental and control groups through pairedt tests (P<.001; Table 4).
961
TABLE 2Numbers and percentages of live and total sperm cells.
962 Yan et al. Cellular phones affect sperm motility
DISCUSSION
As the data show, no significant differences emerged in thenumber of structural sperm mutations between the experi-mental and the control groups. The total sperm counts fromthe testes also were not significantly different between thetwo groups. However, the relative motility and appearanceof the sperm from the epididymides in the experimental ratgroup differed from those of the control group.
The most striking abnormalities in the experimentalgroup were significantly fewer motile sperm cells andnumerous clumps of sperm cells. In these clumps, theheads of the sperm cells appeared to be sticking together.In the experimental group, an up-regulation was present inthe mRNA levels of cell surface adhesion proteins CAD-1and ICAM-1, which would create abnormal adhesion ofthe sperm cells. These proteins are normally present onthe heads of sperm cells to facilitate the egg/sperminteraction during fertilization (20, 21), but the experi-mental group mRNA levels of these proteins were signif-icantly higher (P<.001) than the control group levels.This up-regulation could explain the frequency of spermclumping and the reduced number of motile sperm cells,owing to increased relative stickiness to each other. Theadherent sperm cells would lose motility and eventuallydie. Further research should address this finding of spermcell death and limited mobility and its underlyingmechanism.
Vol. 88, No. 4, October 2007
FIGURE 3
Representative RT-PCR reactions in a control group rat (A) and an experimental group rat (B). The first lanes onthe left represent beta-actin for the internal control, the second lanes cadherin-1 (CAD-1), and the third lanesinterstitial cell adhesion molecule 1 (ICAM-1). These gels show greater product formation in the experimentallanes than in the control lanes. Also, because the level of beta-actin is greater in the control group than in theexperimental group, the CAD-1 and ICAM-1 differences are actually much greater when the beta-actin levels arenormalized to each other.
Mean for experimental group 84782.798 83407.545SD for experimental group 6899.862 10811.081
9 Control 107114 75771 69352 0.97 73497.87 67271.4410 Control 107103 70660 71546 0.97 68540.2 69399.6211 Control 105318 66178 77885 0.98 64854.44 76327.312 Control 97302 69723 57388 1.06 73906.38 60831.2813 Control 85456 57792 55567 1.21 69928.32 67236.0714 Control 96327 62236 63362 1.07 66592.52 67797.3415 Control 102334 59540 59759 1.01 60135.4 60356.5916 Control 88434 51140 47576 1.17 59833.8 55663.92Mean for all rats 103547.938 Mean for control group 67161.116 65610.445
SD for control group 5397.177 6422.835
Note: The mean of the 16 beta-actin values was used to normalize the raw values of CAD-1 and ICAM-1 for a proper com-parison between the experimental (Exp.) and control animals.
Higher temperatures affect sperm maturity and motility(10–14). To ensure that the cellular phones did not increasethe temperature of the rat through direct emissions from therunning phone for hours, we rigorously measured tempera-ture. Sensitive electronic temperature probes were placed ad-jacent to the rats’ faces in the plastic tubes used in this study.After 3 hours of cellular phone exposure, mean face temper-ature of the experimental group did not differ from that of thecontrol group, because of constant airflow through the tube.The rectal temperatures of both groups were virtually identi-cal, even after the full 6 hours of exposure. The plastic wallof the tube and the wood-chip padding insulated againstheat from cellular phone emissions. Furthermore, the epidid-ymides were located within 16–18 cm from the phone with-out receiving any heat from the running phone. These factorseliminated mechanical heat influence from the phone, so onlyRFEMR affected the epididymides.
Male infertility is an increasing problem around the world.To address this problem, evaluating the possible side effectsfrom use of new technology is critical. With over 2 billionpeople currently using cell phones, identifying the risks ofcellular phone use is particularly crucial. Men should beaware that carrying cell phones in their pants pockets placesthem at risk of exposure to harmful microwaves, which couldlater hinder their ability to produce children.
Further study is necessary regarding the effects of long-term cellular phone usage on other tissues in the body aswell, particularly the head and neck. Our current knowledge,combined with future experiments, will help to provide thegeneral public with an improved awareness of the hazardsof cellular phone use and the means for protecting itself.
Acknowledgments: The authors thank data analysts Lin-Ling Zhang, M.D.,
and Julie Weidner, B.S., as well as scientific advisor James Sanger, M.D.,
and freelance writer/reviser, Betsy Foss-Campbell, B.S.
REFERENCES1. Wearden G. Nokia: 2 billion cell phone users by 2006. Dec. 9, 2004. Avail-
able at: http://news.com.com/Nokiaþ2þbillionþcellþphoneþusersþbyþ2006/2100-1039_3-5485543.html. Accessed February 2, 2006.
2. Aitken R, Bennetts L, Sawyer D, Wiklendt A, King B. Impact of radio
frequency electromagnetic radiation on DNA integrity in the male germ-
IMAGES IN REPRODUCTIVE MEDICINERichard J. PaulsonDeputy Editor
Single large cystic adenomyoma of the uterus aftercornual pregnancy and curettageJian-Hua Wang, M.D., Rui-Jin Wu, M.D., Ph.D., Kai-Hong Xu, M.D., and Jun Lin, M.D.
Department of Obstetrics and Gynecology, Women’s Hospital, The School of Medicine, Zhejiang University, Hangzhou,
Zhejiang, People’s Republic of China
A case of single large cystic adenomyoma of the uterus (anechoic area 1.6 cm in diameter) was diagnosed by sur-gery and histopathologic analysis more than 3 years after a transcervical curettage for an early right-cornual preg-nancy. (Fertil Steril� 2007;88:965–7. �2007 by American Society for Reproductive Medicine.)
Adenomyosis or adenomyoma is a condition characterizedby benign invasion of the endometrium into the myometrium,associated with reactive hypertrophy of the surrounding mus-culature. The ectopic endometrium in adenomyosis or adeno-myoma is of the basalis type, and accompanying secretorychanges and menstrual bleeding are less common than infoci of endometriosis. The cystic spaces filled with choco-late-colored blood of uterine adenomyosis or adenomyomaare almost invariably small, usually less than 0.5 cm in diam-eter (1). Large adenomyotic cysts are rare (2). We report anunusual case of focal adenomyoma that had a single, large,active adenomyotic cyst after a transcervical curettage. Theresearch protocol was approved by the medical researchreview board of the Women’s Hospital, School of Medicine,Zhejiang University, Hangzhou, People’s Republic of China.
We present the case of a 26-year-old woman (gravida 2,para 1) with over 3-years’ history of progressive dysmenor-rhea during days 3 to 7 of her menstrual period. She had aregular menstrual cycle (for 30 to 32 days) and a regularmenstrual period (for 6 days). According to her medicalrecords, she had had an early right-cornual pregnancy (7weeks of gestation) and had undergone transcervical curet-tage (suction curettage and sharp curette) and methotrexatetreatment over 3 years ago. Before curettage, ultrasono-graphic examination had revealed a gestational sac, 21 mmin diameter, in the right uterine corner. Transcervical curet-tage was performed under ultrasound guidance, and embryoelements were found. Persistently slightly elevated serumbeta human chorionic gonadotropin (b-hCG) levels were
Received November 12, 2006; revised and accepted December 22, 2006.
Reprint requests: Jian-Hua Wang, M.D., Department of Obstetrics and
Gynecology, Women’s Hospital, School of Medicine, Zhejiang Univer-
sity, Hangzhou 310006, People’s Republic of China (FAX: 86-0571-
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.085 Copyright ª2007 American
found after the transcervical curettage, indicating that shehad possibly retained trophoblastic tissue; she was subse-quently treated with single-dose methotrexate (50 mg/m2).Fourteen days after methotrexate treatment, her serumb-hCG level returned to zero. Shortly afterward, she experi-enced dysmenorrhea during days 3 to 7 of her menstrual cy-cle, and the dysmenorrhea became progressive. A 4-months’postoperative ultrasonographic examination showed an intra-myometrial hypoechoic mass measuring 2.2 cm � 2.4 cm �2.0 cm in the inferior aspect of the right uterine cornu. Herserum b-hCG concentration was still zero.
The patient was referred to our hospital after she had expe-rienced the progressive and severe dysmenorrhea during days3 to 7 of her menstrual cycle for more than 3 years. Bimanualpelvic examination revealed a hard mass with smooth surfacenear the right uterine corpus. The mass was not mobile andwas not separable from the right uterine corpus. Ultrasono-graphic examination (Fig. 1) demonstrated a slight enlargeduterus, with an intramyometrial mass measuring 3.2 cm �2.7 cm � 2.4 cm in the inferior aspect of the right uterinecornu, which consisted of both solid and cystic components.The central cystic area exhibited an echolucent mass (choco-late-colored blood) measuring 1.6 cm � 1.6 cm � 1.6 cm,which was surrounded by hypoechoic rim (reactive hypertro-phy of the surrounding musculature). Vascular blood flowdistribution around the echolucent mass was observed.
Because of her intractable dysmenorrhea, the patient un-derwent an exploratory laparotomy. The intramyometrialcystic mass was located in the anterior uterine walls justunder the right cornua, and it bulged outward slightly fromthe myometrium. The cystic mass did not invade the uterineserosa. The cystic mass was removed through a sharp dissec-tion, and the uterine cavity was not entered during theoperation. The uterine incision was closed by interruptedsuture technique.
Fertility and Sterility� Vol. 88, No. 4, October 2007 965Society for Reproductive Medicine, Published by Elsevier Inc.
Transvaginal ultrasound of single large cystic adenomyoma of the uterus. (A) Intramyometrial mass (calipers) inthe inferior aspect of the right uterine cornu. (B) Sagittal hysterosonogram of the endometrium and uterine wallwith thickened anterior (ventral) myometrium of right uterine cornu. (C) Color Doppler ultrasound image of theecholucent mass showing feeding artery and surrounding vein distribution.
Wang. Single large cystic adenomyoma. Fertil Steril 2007.
The lesion consisted of a cyst of 1.6 cm in diameter filledwith chocolate-colored blood with surrounding focalhypertrophy of the myometrium of 1.5-cm thickness. Histo-pathologic examination (Fig. 2) revealed endometrial glandsaround the cyst wall. Hemosiderin-laden macrophages alsowere evident around the cyst wall. The operative findingand histopathologic examination confirmed the diagnosis ofsingle large cystic adenomyoma of the uterus. The patient’s
FIGURE 2
Histology of single large cystic adenomyoma of theuterus. (A) Hematoxylin and eosin stain. (B)Immunohistochemical localization of epithelialmembrane antigen (EMA). Original magnification,�200.
Wang. Single large cystic adenomyoma. Fertil Steril 2007.
966 Wang et al. Single large cystic adenomyoma
postoperative recovery was uneventful, and she was dis-charged on the fourth postoperative day. After excision ofthe intramyometrial cystic mass, the patient’s symptom ofdysmenorrhea disappeared completely.
The 10-month follow-up examination after the patient’slaparotomy revealed that the patient had selected condomsas her contraceptive method and had not had a subsequentpregnancy. Her symptom of dysmenorrhea did not recur.
DISCUSSION
The etiology of adenomyosis or adenomyoma remainsunclear. It is now generally believed that adenomyosis oradenomyoma develops as a result of down-growth and invag-ination of the basalis endometrium into the myometrium aftera disruption of the normal boundary between the basal endo-metrial layer and the myometrium (3). The cause of suchdisruptions is not clearly understood, but it may be relatedto pregnancy, cesarean delivery, postpartum endometritis,uterine surgery, or uterine trauma. A significantly increasedrisk of adenomyosis has been found to follow uterine surgery,such as cesarean delivery, myomectomy, endometrialablation, dilation and evacuation, and dilation and curettage(4). This supports the hypothesis that intrauterine manipula-tions are the main predisposing factor of endometrial cells inthe myometrium.
A MEDLINE search of English-language publications onthe search terms ‘‘adenomyotic cyst of the uterus,’’ ‘‘cysticadenomyosis,’’ or ‘‘cystic adenomyoma’’ during the past 3decades was performed. Only three cases of blood-filledadenomyotic cyst of the uterus were found (2). The threecases included one intramyometrial and two subserosalpedunculated adenomyotic cysts. To date, ours is the firstreport of a single large cystic adenomyoma of the uterus
Vol. 88, No. 4, October 2007
caused by early cornual pregnancy and transcervicalcurettage (suction curettage and sharp curette).
That cystic adenomyoma of the uterus as found in ourpatient was caused by the disruption of the normal boundarybetween the basal endometrial layer and the myometrium byright cornual pregnancy and curettage, and down-growth andinvagination of the basalis endometrium into the myome-trium, is supported by several medical observations. First,our patient began to experience dysmenorrhea just after shehad undergone transcervical curettage for an early right cor-nual pregnancy, and her dysmenorrhea afterward was pro-gressive. Second, the lesion of cystic adenomyoma of theuterus was located in the uterine myometrium near the rightcornua, the site of her pregnancy and curettage. Third, she ex-perienced dysmenorrhea during days 3 to 7 of her menstrualcycle, when the cyst-tension of the adenomyoma was highest(as a result of bleeding in the cyst) rather than on other days ofthe menstrual cycle. Dysmenorrhea in women with cystic ad-enomyoma of the uterus could be attributed to the progressiveincrease in the size of the cyst due to periodic intracysticsecretion and bleeding. The symptom is not specific butcan be a clue for diagnosis and differential diagnosis becausecystic degeneration of uterine leiomyoma rarely presents
Fertility and Sterility�
with dysmenorrhea; although a uterine malformation suchas a rudimentary uterine horn can present with dysmenorrhea,the dysmenorrhea usually is primary.
In conclusion, pregnancies terminated by curettage may bethe main predisposing factor for endometrial cells in the my-ometrium and may be involved in the pathogenesis and devel-opment of adenomyosis.
Acknowledgments: The authors thank Dr. XiaoDuan Chen, Dr. XiaoFei
Zhang, and Dr. HaiYan Shi (Department of Pathology) for their support.
REFERENCES1. Slezak P, Tillinger K. The incidence and clinical importance of hystero-
graphic evidence of cavities in the uterine wall. Radiology 1976;118:
581–6.
2. Kataoka ML, Togashi K, Konishi I, Hatabu H, Morikawa K, Kojima N,
et al. MRI of adenomyotic cyst of the uterus. J Comput Assist Tomogr
1998;22:555–9.
3. Su HY, Chen CH, Gao HW, Liu JY. A bicornuate uterus with a unilateral
4. Panganamamula UR, Harmanli OH, Isik-Akbay EF, Grotegut CA,
Dandolu V, Gaughan JP. Is prior uterine surgery a risk factor for adeno-
myosis? Obstet Gynecol 2004;104:1034–8.
967
CASE REPORT
Maternal derivative chromosome 9 and recurrentpregnancy lossLing-Wei Chang, M.D.,a I-Wen Lee, M.D.,a Pao-Lin Kuo, M.D.,a and Long-Ching Kuan, M.D.b
a Department of Obstetrics and Gynecology, National Cheng Kung University, College of Medicine; and b Department of
Obstetrics and Gynecology, Kuo General Hospital, Tainan, Taiwan
Objective: To disclose the relationship between a derivative chromosome 9 and recurrent pregnancy loss.Design: Case report.Setting: Referral from primary clinical care to tertiary hospital for further intervention.Patient(s): A 31-year-old woman who had four recurrent early spontaneous abortions.Intervention(s): Cytogenetic and fluorescence in situ hybridization analyses of the chromosome harvested fromperipheral blood sample.Main Outcome Measure(s): Karyotype of the patient.Result(s): The patient has a unique derivative chromosome 9, der(9)(9qter/9p24::9p24/9p11::9p13/9qter).ish der(9) (CEP9�1,43N6�1,D9Z3�1).Conclusion(s): Maternal derivative chromosome 9 may cause recurrent pregnancy loss. (Fertil Steril�
2007;88:968.e1–3. �2007 by American Society for Reproductive Medicine.)
Recurrent pregnancy loss (RPL) is a health problem involv-ing about 3% of couples. Despite progress during the pastdecades, there are still some controversies in diagnosisand management for couples who experience RPL. Amongthe potential etiologic factors, chromosomal abnormalities,anatomic anomalies of uterus, endocrine dysfunction, throm-bophilia, and autoimmune disorders are most well character-ized (1). To date, the standard protocols for investigation ofinfertile couples include hysterography, hysteroscopy, andthree-dimensional sonography of uterus; thyroid hormones;lupus anticoagulant; anticardiolipin antibodies; FactorV Leiden mutation; protein S; protein C; and karyotypingof the couple (1).
Despite progress during the past 2 decades, no discern-ible cause has been uncovered by standard evaluation pro-tocols in the majority of RPL cases. The clinicalevaluation of couples with a history of recurrent spontane-ous abortion regularly includes cytogenetic analyses, andthe incidence of chromosomal abnormalities is approxi-mately 6% in couples with a history of at least two RPLs(2). Previous reports have indicated a definite relationshipbetween balanced rearrangements (reciprocal translocations,Robertsonian translocations, and inversions) and recurrentspontaneous abortion. Here we present a 31-year-old woman
Received September 13, 2006; revised and accepted November 27, 2006.
Reprint requests: Long-Ching Kuan, M.D., Department of Obstetrics and
Gynecology, Kuo General Hospital, No. 22, Sec. 2, Ming-sheng Road,
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medic
968.e1
who had four recurrent early spontaneous abortions and aderivative chromosome 9.
The proband is a 31-year-old woman who had been mar-ried to her 36-year-old husband for 2 years and had fourrecurrent spontaneous abortions at early gestational age,without delivery of any liveborn baby. The medical and fam-ily histories of the patient and her husband were unremark-able. Genetic studies for the patients with RPL had beenapproved by the Institutional Review Board of NationalCheng Kung University Medical Center, and informed con-sent was obtained from the patients. Chromosomes fromthe peripheral blood lymphocytes of the couple were ana-lyzed by using the standard GTG method (G-banding byTrypsin-Giemsa technique). The husband had normal malekaryotype (46,XY), but the proband was found to have a de-rivative chromosome 9 for which the initial karyotypic inter-pretation was 45,XX, -9, psu dic (9)(9qter/9p24::9p24/9p13::9p13/9qter; Fig. 1A). The karyotypes of the pro-band’s parents were normal. Dual-color fluorescence in situhybridization was applied to investigate the nature of the de-rivative chromosome by using the following probes: 43N6,mapped to 9p24.3, labeled with Spectrum Orange (Vysis,Inc., Downers Grove, IL), was used to detect the subtelomericregion of the short arm of chromosome 9; clone D9Z3, map-ped to 9q12, labeled with Spectrum Green (Vysis, Inc.), wasused to detect the 9q heterochromatin. Another fluorescencein situ hybridization experiment was performed by usingprobe CEP9, which was mapped to the centromere(9p11�q11) and labeled with Spectrum Orange. At least 10
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The karyotype and fluorescence in situ hybridization (FISH) pictures of the proband. (A) Complete karyotypeof the proband. The breakpoints were located at 9p24 for both chromosomes 9. (B) Dual-color FISH analysiswith 43N6 probe (9q24.3, red) and D9Z3 probe (9q12, green) showed only one signal for both probes on theder(9). (C) Fluorescence in situ hybridization analysis with probe CEP9 (9p11�q11, red) also revealed onlyone signal on der(9). (D) Derivative chromosome 9 and the corresponding ideogram. There was loss ofchromatin from 9p11 to 9q12 for one chromosome 9. Both chromosomes 9 of her parents had an intactheterochromatic region (from 9p11to 9q12; data not shown).
Chang. Derivative chromosome 9 and recurrent miscarriages. Fertil Steril 2007.
metaphase cells and some interphase cells were analyzed foreach fluorescence in situ hybridization analysis, and all cellsshowed one 9p subtelomeric signal, one 9q heterochromatin(9q12) signal, and one signal for the centromere (9p11�q11)on the derivative chromosome 9 (Fig. 1B and C). Close exam-ination of the derivative chromosome 9 revealed that therewas indeed loss of chromatin from 9p11 to 9q12 for one chro-mosome 9 (Fig. 1D). Therefore, the proband’s karyotype wasreassigned as 45,XX, -9, der (9)(9qter/9p24:: 9p24/9p11:: 9p13/9qter). ish der(9) (CEP9�1,43N6�1,D9Z3�1).
The effect of chromosomal abnormalities on reproductiveperformance is different for males and females. In mamma-lian species, male germ cells that carry unpaired chromosome
Fertility and Sterility�
segments usually do not complete meiosis and are targeted
for removal by apoptosis. In contrast, there appears to be
no equivalent meiotic quality-control system at work in fe-
males during oogenesis (3). Therefore, the proband is fertile
but destined to have RPL. The chromosome rearrangement of
this proband was also unique. At first glance, the der(9) ap-
peared to be an isodicentric chromosome with one centromere
inactivated. However, fluorescence in situ hybridization anal-
ysis showed only one centromere, and there was absence of
heterochromatic material of 9p12. A reasonable hypothesis
is that the proband received only one chromosome 9 from
one of her parents as a result of a meiotic nondisjunction ep-isode, followed by duplication of nearly the whole
968.e2
chromosome 9 for complementation (4). It is possible that thecentromeric heterochromatin and heterochromatin on 9q12were not duplicated. Considering the normal phenotype ofthe proband, we hypothesize that duplication of chromosome9 was almost complete except for the centromeric heterochro-matin, heterochromatin on 9q12, and the subtelomeric area of9p. Uniparental disomy associated with an isochromosomehas been reported in cases with i(1p) plus i(1q), i(2p) plusi(2q), i(4p) plus i(4q), i(7p) plus i(7q), psu dic(8)(p23.3),i(9p) plus i(9q), i(13q), i(14q), i(15q), i(21q), and i(22q) (5).Should our scenario be true, the proband would have unipa-rental disomy for the whole chromosome 9. Considering theabsence of imprinting genes on chromosome 9, it is expectedthat there is no abnormal phenotype for cases of UPD (9) (6).Although it was not possible to check whether the proband in-herited both chromosomes 9 from a single parent because oflack of parental samples, both chromosomes 9 of her parentshad an intact heterochromatic region (from 9p11to 9q12; datanot shown). That observation provides evidence to support ourhypothesis that the proband inherited a single chromosome 9from one parent and that the derivative chromosome was de-rived from partial duplication of a single chromosome 9.
968.e3 Chang et al. Derivative chromosome 9 and recurr
To our knowledge, this is the first case addressing the rela-tionship between 45,XX,-9,der(9) and RPL. The mechanismof the derivative chromosome formation still remains to beelucidated by molecular studies. Considering the absenceof normal chromosome 9, the woman’s oocyte will be nullis-omy or disomy for chromosome 9. She was advised toundergo IVF cycle treatment with donated eggs.
REFERENCES1. Christiansen OB, Andersen AN, Bosch E, Daya S, Delves PJ, Hviid TV,
et al. Evidence-based investigations and treatments of recurrent pregnancy
and incidence. Clin Obstet Gynecol 1994;37:625–34.
3. Braun RE. Every sperm is sacred—or is it? Nat Genet 1998;18:202–4.
4. Berend SA, Horwitz J, McCaskill C, Shaffer LG. Identification of unipa-
rental disomy following prenatal detection of Robertsonian translocations
and isochromosomes. Am J Hum Genet 2000;66:1787–93.
5. Kotzot D. Complex and segmental uniparental disomy (UPD) review and
lesions from rare chromosomal complements. J Med Genet 2001;38:
497–507.
6. Morison IM, Ramsay JP, Spencer HG. A census of mammalian imprint-
ing. Trends Genet 2005;21:457–65.
ent miscarriages Vol. 88, No. 4, October 2007
CASE REPORT
Spontaneous heterotopic triplets: a case reportAarathi Cholkeri-Singh, M.D., and Ann LaBarge, M.D.
Department of Obstetrics and Gynecology, Advocate Lutheran General Hospital, Park Ridge, Illinois
Objective: To present a case report of a spontaneous, heterotopic, triplet conception of a patient without any riskfactors.Design: Case report.Setting: Community hospital in Park Ridge, Illinois.Patient(s): One patient, 30-year-old multigravida, sent to the emergency room for evaluation of right lower quad-rant pain after confirmed identification of an early, intrauterine pregnancy by an office ultrasound. The patient wastaken to the operating room for suspected appendicitis and found to have a right ruptured ectopic pregnancy witha normal appendix.Intervention(s): The patient underwent a laparoscopic right salpingostomy with hydrodissection of the ectopicpregnancy.Main Outcome Measure(s): Follow-up pelvic ultrasounds confirmed a viable, spontaneous twin intrauterinepregnancy.Result(s): The twin pregnancy continued without complications until 34 weeks’ gestation, when the patient beganpreterm labor and delivered healthy twins.Conclusion(s): Even in the absence of infertility treatments and/or risk factors, heterotopic pregnancy should beincluded in the differential diagnosis for lower quadrant abdominal or pelvic pain. (Fertil Steril� 2007;88:968.e5–7.�2007 by American Society for Reproductive Medicine.)
Heterotopic pregnancies are rare in natural occurrence. Ithas been documented that the frequency is approximately1/30,000 pregnancies (1–3). Duverny, who discovered thiscondition during an autopsy, documented the first heterotopicpregnancy in 1708 (4). This rare entity is increasing with theuse of assisted reproductive technologies to a frequency be-tween 1/100 and 1/500 for all pregnancies (3). The rate ofmultiple fetal pregnancies is also increasing with the use ofassisted reproductive technologies. There was a 5.9-fold in-crease in triplet conception between 1971–1977 and 1998 (5).
We report a case of a spontaneous conception resulting intriplets, found to have a simultaneous extrauterine pregnancyand an intrauterine twin pregnancy. The extrauterine preg-nancy was not found at her initial ultrasound, and the twinintrauterine pregnancy was identified after the surgical resec-tion of the ectopic. We present the case and then discuss theimportance of maintaining differential diagnoses of ectopicpregnancy in conjunction with an intrauterine pregnancyand management.
Received November 2, 2006; revised and accepted December 13, 2006.
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medic
968.e5
CASE
A 30-year-old G4 P1021, 5 weeks from her last menstrualperiod, presented to her primary obstetrician’s office withcomplaints of right lower quadrant pain for 1 day that hadcontinued throughout the night and persisted. She describedher pain as sharp and constant, which radiated to her rightleg. Nausea and vomiting were present, but she denied any fe-ver, chills, or changes in her bladder or bowel habits. She alsostated that she never experienced pain similar to this before.The patient was aware of being pregnant, but never had anultrasound prior to this visit.
A complete history revealed that she had a previous full-term vaginal delivery without complications and two sponta-neous miscarriages. She has desired future fertility, andtherefore has never been on birth control. The patient hasbeen in a long-term healthy, monogamous relationship, anddenied any history of sexually transmitted diseases. She de-nied any medical problems, previous surgeries, or use of to-bacco, alcohol, or illicit drugs. Her family history wasunremarkable for any pelvic diseases.
In the office, a transvaginal ultrasound was performed thatidentified an intrauterine gestational sac measuring approxi-mately 5.5 weeks of gestation. The adnexa were poorly visu-alized, and there was no documentation regarding free fluid in
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the pelvis. The patient was immediately sent to the emer-gency room for a surgical consultation and evaluation forpossible appendicitis.
In the emergency room, her vital signs were stable and shewas found to be afebrile. The general surgery team describedthe patient’s abdominal exam as soft, nondistended, withgood bowel sounds. Right lower quadrant tenderness wasnoted, but neither rebound nor guarding was observed. Thegeneral surgery team did not perform a pelvic exam. Labora-tory values revealed an elevated white blood cell count to15.3 thousand/mL. Because of an already documented intra-uterine pregnancy, a quantitative hCG was not obtained.The patient underwent a right lower quadrant ultrasound,which did not visualize the appendix but noted free fluid pres-ent in the right colonic gutter.
The patient was taken to the operating room and underwenta diagnostic laparoscopy for presumed appendicitis. Thefindings noted during the laparoscopy were approximately100 cc of blood in the cul-de-sac, normal appendix, normalleft fallopian tube, normal 9 weeks’ size uterus, normal ova-ries bilaterally, and a right fallopian tube enlarged and bloodfilled. No evidence of pelvic adhesive disease or postinflam-matory changes was noted. The gynecology service was con-sulted intraoperatively for a right ruptured ectopic pregnancy.A right salpingostomy with hydrodissection of the ectopicpregnancy and evacuation of the blood in the pelvis was per-formed with minimal manipulation of the uterus without anycomplications. The tissue was sent to pathology and wasnoted to have immature chorionic villi. The patient was trans-ferred to the recovery room in stable condition.
On postoperative day 1, a repeat transvaginal ultrasoundwas performed to evaluate the intrauterine pregnancy thatwas noted on initial evaluation. The ultrasound noted twofluid collections within the endometrial cavity. The superiorfluid collection had a yolk sac within the gestational sac,which measured 6.2 � 6.5 � 7.2 mm, equivalent to 5-4/7weeks’ gestational age. The other fluid collection measured11.9 � 8.3 � 9.4 mm, equivalent to 6-0/7 weeks’ gestationalage, but nonspecific in appearance with no yolk sac identi-fied. There was no fetal cardiac activity identified in eitherfluid collection.
The patient was discharged home on postoperative day 1 instable condition. On follow-up visits in the office, the patienthad an ultrasound to follow the fluid collection and yolk sac,and was found to have two viable fetuses with an estimateddue date consistent with her last menstrual period. She con-tinued routine antenatal care and underwent serial ultra-sounds with her primary obstetrician. All of the growthultrasounds noted a dividing membrane with appropriateand concordant growth of the twin fetuses.
At 34 weeks’ gestational age, the patient went into laborand underwent a primary low transverse cesarean section sec-ondary to fetal malpresentation of breech and transverse. Shedelivered a viable female infant weighing 1,865 g, with Ap-gars of 9 and 9, and a viable male infant weighing 2,096 g,
Fertility and Sterility�
with Apgars of 8 and 9. Both infants were stable and broughtto the newborn nursery. The patient had an unremarkable re-covery and was discharged home with both of her newbornson postoperative day 4.
COMMENT
The prevalence of heterotopic pregnancies is rising, mainlybecause of the increasing use of infertility medications andtechniques such as ovarian hyperstimulation medicationsand multiple embryo transfers. Risk factors also known to in-crease this prevalence are history of pelvic inflammatory dis-ease, previous abdominal or pelvic surgery, previous ectopicpregnancy, uterine malformations, and the use of intrauterinedevices (1, 4). It is reported that approximately 70% of het-erotopic pregnancies are diagnosed between 5 and 8 weeksof gestation, 20% are diagnosed between 9 and 10 weeks,and the remaining 10% are diagnosed after 11 weeks (3).
In the absence of infertility treatments and/or risk factors,the diagnosis of heterotopic pregnancy can easily be over-looked. An early diagnosis is usually difficult because ofthe lack of symptoms. Most likely the patient will presentwith abdominal pain, adnexal mass, enlarged uterus, perito-neal signs, and a positive pregnancy test (3). The presumeddiagnosis at the time is usually ectopic pregnancy. In ourcase, the patient was diagnosed with an intrauterine preg-nancy and immediately sent to the emergency room for pre-sumed appendicitis because of signs and symptoms of anacute abdomen. The differential diagnosis of an ectopic preg-nancy was not entertained because of the presence of an in-trauterine pregnancy and poor visualization of the adnexaon ultrasound. With advances in technology, reliance on im-aging techniques rather than physical diagnosis has becomeproblematic. The art of physical diagnosis may be gettinglost among the fast growth of technology. Emphasis shouldbe placed on the fact that technology at this point in timeshould still remain a supplement to physical findings andnot a replacement.
The gold standard treatment for ectopic pregnancy is sur-gery: either a salpingostomy or salpingectomy. Careful atten-tion should be paid to minimal handling of the uterus so as notto disrupt the intrauterine gestation. Another known treat-ment that has been used for stable patients with intact tubalpregnancies is the injection of potassium chloride into the ec-topic to abort the pregnancy. The body then reabsorbs the tis-sue. It is also common practice to use methotrexate, RU486,or prostaglandins to abort ectopic pregnancies in stablepatients, but this treatment in a patient with a heterotopicpregnancy would potentially compromise the intrauterinegestation. After treatment of the ectopic pregnancy, therehas been a reported survival rate of 35%–54% for the intra-uterine gestation, with a delivery rate of 66% in those patientstreated by surgery for the heterotopic pregnancy (3). How-ever, it has been documented that 8% of patients are foundto have a persistent ectopic after salpingostomy is performed(6). Although salpingostomy is the preferred method of
968.e6
removal of an ectopic in those patients desiring future preg-nancy, it would be difficult to follow serum b-hCG to a nega-tive range to assure complete resolution of the ectopicpregnancy because of a concurrent pregnancy altering thevalue. Therefore, perhaps a salpingectomy would be neces-sary to confirm complete removal of the ectopic tissue. Other-wise, the patient may be at risk for a subsequent surgery whilestill pregnant, carrying a small risk of miscarriage for the in-trauterine pregnancy. Serial ultrasounds could be used to de-tect any resolution of the ectopic pregnancy, but again, if it ispersistent, the patient will need a second surgery whilepregnant.
Our case differs from most cases of heterotopic pregnan-cies, not only because it was a naturally conceived tripletpregnancy, but also because there were no risk factors in-volved for an ectopic implantation. Heterotopic pregnanciesare rare, and this life-threatening diagnosis is easily missed. Itis important when evaluating a patient for acute abdominalpain with a positive pregnancy test that a complete evaluationis performed. Whether the differential diagnoses are rare ornot, each should be considered and fully worked up. It is ofteneasy to accept an intrauterine gestation and defer the likeli-
968.e7 Cholkeri-Singh and LaBarge Spontaneous heteroto
hood of a heterotopic pregnancy, especially when the patientpresents with minimal to no risk factors. Evaluation witha thorough history and physical along with a pelvic ultra-sound, visualizing the intrauterine cavity and both adnexaare important in establishing a diagnosis and preventingany compromise to the intrauterine gestation, which in ourcase resulted in two healthy newborn infants.
REFERENCES1. Duce M, Ozer C, Egilmez H, Apaydin F, Yildiz A, Kara E. Heterotopic
pregnancy: case report. Abdom Imaging 2002;27:677–9.
2. Stefanetti M, Comerci G, Bulletti C. Heterotopic pregnancy. J Am Assoc
Gyn Laparoscopists 2003;10:140.
3. Varras M, Akrivis C, Hadjopoulos G, Antoniou N. Heterotopic pregnancy
in a natural conception cycle presenting with tubal rupture: a case report
and review of the literature. Eur J Obstet Gynecol Reprod Biol 2003;106:
79–82.
4. Aneziokoro E. Heterotopic pregnancy with live twins. Afr J Reprod
Health 2002;6:117–9.
5. Kiely JL, Kiely M. Epidemiological trends in multiple births in the United
States, 1971–1998. Twin Res 2001;4:131–3.
6. Murray H, Baakdah H, Bardell T, Tulandi T. Diagnosis and treatment of
ectopic pregnancy. CMAJ 2005;173:905–12.
pic triplets Vol. 88, No. 4, October 2007
CASE REPORT
Massive ascites and hydrothorax after leuprolideacetate administration in a down-regulated womanundergoing assisted reproductionBruno Ferrari, M.D., Antonio Pezzuto, M.D., and Francesco Coppola, M.D.
Centre for Reproductive Medicine, Department of Obstetrics, Gynaecology, and Neonatology, University of Parma, Parma, Italy
Objective: To present an atypical case of massive ascites and hydrothorax after leuprolide acetate administration ina down-regulated woman undergoing assisted reproduction.Design: Case report.Setting: Centre for Reproductive Medicine, Department of Obstetrics, Gynaecology, and Neonatology, Universityof Parma, Parma, Italy.Patient(s): A 41-year-old, nulliparous, white woman who developed massive ascites and hydrothorax after admin-istration of 0.50 mg/day of subcutaneous leuprolide acetate, beginning at the midluteal phase.Intervention(s): Down-regulation with the gonadotropin-releasing hormone analogue was discontinued, and ther-apy was started with furosemide 50 mg/day for 10 days.Main Outcome Measure(s): Successful medical reduction of ascites and hydrothorax.Result(s): Resolution of symptoms.Conclusion(s): A comprehensive MEDLINE search revealed this to be the first reported case of massive ascitesand hydrothorax after leuprolide acetate administration (0.5 mg daily) in a down-regulated woman undergoing as-sisted reproduction. This case can be explained by an increase in capillary permeability, which resulted in a rapidfluid shift from the intravascular space into the third space. We believe that ascites in our patient resulted from anincrease in estradiol in the ovaries, due to a direct action of the gonadotropin-releasing hormone analogue on thecorresponding ovarian receptors in the first few days after the start of therapy. (Fertil Steril� 2007;88:968.e9–11.�2007 by American Society for Reproductive Medicine.)
Gonadotropin-releasing hormone (GnRH) analogues areused in long protocols as adjuvant therapy for infertilewomen undergoing controlled ovarian stimulation with go-nadotropins before in vitro fertilization (IVF) procedures.The rationale for use of GnRH analogues is to achieve pitui-tary suppression and prevent a premature luteinizing hor-mone (LH) surge during ovarian stimulation. We reporta case of an infertile woman who developed ascites with hy-drothorax soon after down-regulation with GnRH agonists.To our knowledge, this is the first such complication ever re-ported in the literature.
CASE REPORT
A 41-year-old, nulliparous, white woman came presented fortreatment the Centre for Reproductive Medicine in Parmawith a 3-year history of infertility. Her height and weight at
Received August 24, 2006; revised and accepted December 20, 2006.
Reprint requests: Bruno Ferrari, M.D., Centre for Reproductive Medicine,
Department of Obstetrics, Gynaecology, and Neonatology, University
of Parma, Via Gramsci 14, 43100 Parma, Italy (FAX: þ39 0521
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medi
968.e9
the time were 170 cm and 61 kg, respectively. The hysterosal-pingography examination showed that the woman’s fallopiantubes were occluded. Her uterus and ovaries were normal onultrasound. Her baseline serum hormonal levels on day 3 ofthe menstrual cycle were in the normal range: follicle-stimu-lating hormone (FSH) 4.78 mIU/mL, LH 4.16 mIU/mL, es-tradiol 68 pg/mL, and prolactin 9 ng/mL. The patient wasgiven a consent form to sign indicating her understandingand acceptance of the IVF procedure. The stimulation proto-col was approved by the hospital’s review board.
In April 2006, a long protocol of ovarian hyperstimulationwas started, with 0.50 mg/day of subcutaneous leuprolide ac-etate (Enantone die, Takeda Italia, Rome, Italy) administeredbeginning at the midluteal phase. Fourteen days later, beforeinitiating FSH therapy, the patient came to our hospital andreported increasing abdominal girth, abdominal pain, short-ness of breath, and rapid weight gain (10 kg). At that time,her physical examination revealed dyspnea with an edema-tous face, and a distended abdomen without fever. A transva-ginal ultrasound examination showed massive ascites in thepelvis, the abdomen, and the Morrison pouch. The uteruswas regular in size and shape, and the ovaries were not
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enlarged (measuring 24 � 22 � 20 mm on the right side and26 � 29 � 25 mm on the left).
A chest x-ray revealed a moderate bilateral pleural fluid ac-cumulation, more marked on the right side. Laboratory testsshowed a hemoglobin value of 16.3 g/dL, a hematocrit valueof 48.2%, and a white blood cell count of 4300 cells/mm3. Al-bumin, protein, electrolyte, kidney and liver function testswere normal. The coagulation profile showed a D-dimer con-centration of 1539 ng/mL. Tumor markers (alpha-fetopro-teins, carcinoembryonic antigen [CEA], cancer antigen[CA] 19-9, CA 125, and CA 15.3) were in the normal range.Infectious disease markers (hepatitis B surface antigen[HBsAg], hepatitis C virus antibody [HCV Ab], and Ep-stein-Barr virus [EBV]) were all negative.
An abdominal ultrasound examination ruled out the pres-ence of liver, kidney, spleen, or pancreas disorders. The elec-trocardiogram revealed a sinus rhythm with 65 ms frequencyand normal morphologic features. On echocardiography, theleft ventricle was unenlarged and normally contractile (ejec-tion fraction [EF] 0.60), the cardiac valves and the right heartwere normal, and the pericardium was undamaged. A com-puted tomography scan did not reveal any processes expand-ing into the peritoneum or retroperitoneum.
Down-regulation with the agonist was discontinued. Ther-apy was started with furosemide, 50 mg/day for 10 days, result-ing in a weight loss of 4 kg in 2 days and the disappearance ofperipheral edemas, with rapid regression of the respiratorysymptoms.
After 10 days of treatment, there were no more signs ofpleural fluid accumulation on the chest x-ray. Only a slight(50 � 30 mm) pelvic fluid accumulation in the Douglaspouch could still be found on transvaginal ultrasound. Thelaboratory tests were back in the normal range, with a hemo-globin value of 12.4 g/dL, hematocrit value of 37%, andwhite blood cell count of 6300 cells/mm3; electrolyte, liverfunction, and coagulation tests were also in the normal range.The course of the disease was otherwise benign with sponta-neous resolution, and the patient was able to go back to workafter 20 days. An adverse reaction (ADR) form was sent tothe drug surveillance manager of our hospital and to the med-ical director of the Italian branch of Takeda Corporation.
In October 2006, the patient underwent another stimulationprotocol with recombinant FSH and GnRH antagonists as partof her IVF procedure without developing any complications.
DISCUSSION
In over 90% of reported cases, ascites is caused by cirrhosis ofthe liver, tumors, heart failure, or tuberculosis (TBC). Themost common gynecologic causes of ascites in young womenare ovarian cancer, benign ovarian fibroma (Meigs syn-drome), endometriosis with ruptured endometrioma, largeuterine leiomyomas, and ovarian hyperstimulation syndrome.
A case of massive ascites after leuprolide acetate depot ad-ministration was previously described elsewhere following
Fertility and Sterility�
treatment of uterine leiomyomas. The pathogenesis of ascitesin that case was not clearly understood. It was postulated thatthe degeneration of leiomyomas may have caused the releaseof a substance that increased tissue permeability and resultedin the transudation of fluid from the myomas into the perito-neal cavity (1).
Our patient was the first case of ascites associated with hy-drothorax in a woman receiving leuprolide acetate. It alsowas the first case in which the symptom developed after ad-ministration of a 0.5 mg/day dose of subcutaneous leuprolideacetate for infertility treatment. There was no evidence ofprior rupture of endometrioma or uterine myomas that couldhave caused the ascites. Moreover, the computed tomographyscan ruled out the presence of tumors in the abdomen and ret-roperitoneum. The ascites and hydrothorax appeared to be re-lated to the administration of leuprolide acetate. The rapiddevelopment of ascites after only a few days of leuprolide ac-etate therapy as well as the rapid disappearance of both asci-tes and hydrothorax on therapy discontinuation stronglysuggests a causal relationship, although we cannot objec-tively prove it in this case.
Conflicting data exist about the presence of GnRH recep-tors in the human ovary, and their gene expression and func-tion. Receptors for GnRH also have been found in oocytes(2). Gatje (3) reported an inhibitory effect of GnRH-a onovarian steroidogenesis. Pellicer and Miro (4) found stimu-lation of estradiol production. It has also been demonstratedin vitro that GnRH-a may increase estradiol production inimmature follicles (5). Other studies have shown thatGnRH analogues do not have any effect on steroidogenesis(6, 7).
Our patient’s case could be explained by an increase incapillary permeability, which results in a rapid fluid shiftfrom the intravascular space into the third space. Evidencewas found of hemoconcentration with increased hematocritvalues. Estrogens can be implicated as a possible triggeringfactor for the increase in capillary permeability. Leuprolideacetate has been found to produce menopausal estradiollevels within 2 weeks (<20 pg/mL) after an initial rise in se-rum estradiol levels (flare-up). This stimulatory effect couldexplain our case report. We believe that ascites in our patientresulted from an increased concentration of estradiol in theovaries as a direct consequence of GnRH agonist action onthe corresponding ovarian receptors in the first few days afterthe start of therapy. This did not result either in cyst formationor ovary enlargement, as has been commonly found in ovar-ian hyperstimulation syndrome.
REFERENCES1. Lee MJ, Kazer RR. Massive ascites after leuprolide acetate administration
for the treatment of leiomyomata uteri. Fertil Steril 1992;58:416–8.
2. Dekel N, Lewysohn O, Ayalon D, Hazum E. Receptor for GnRH are pres-
ent in rat oocytes. Endocrinology 1988;123:1205–7.
3. Gaetje R. Influence of gonadotropin releasing hormone (GnRH) and
a GnRH-agonist on granulosa cell steroidogenesis. Clin Exp Obstet Gyne-
col 1994;21:164–9.
4. Pellicer A, Miro F. Steroidogenesis in vitro of human granulosa-luteal
cells pretreated in vivo with gonadotropin-releasing hormone analogs.
Fertil Steril 1990;54:590–6.
5. Mori H, Ohkawa T, Takada S, Morita T, Yago N, Arakawa S, Okinaga S.
Effects of gonadotropin-releasing hormone agonist on steroidogenesis in
the rat ovary. Horm Res 1994;41(Suppl 1):14–21.
968.e11 Ferrari et al. Ascites and hydrothorax after GnRH
6. Lanzone A, Panetta V, Di Simone N, Arno E, Fulghesu AM, Caruso A,
et al. Effect of gonadotrophin-releasing hormone and related analogue
on human luteal cell function in vitro. Hum Reprod 1989;4:906–9.
7. Casper RF, Erickson GF, Rebar RW, Yen SS. The effect of luteinizing hor-
mone-releasing factor and its agonist on cultured granulosa cells. Fertil
Steril 1982;37:406–9.
-a Vol. 88, No. 4, October 2007
CASE REPORT
Torsion causing interruption of the ampullary portionof the fallopian tubeSonia Grover, M.D.
Department of Paediatric and Adolescent Gynaecology, Royal Children’s Hospital, Melbourne, Australia
Objective: To present a description of a young girl who presented acutely with symptoms and findings that areconsistent with acute adnexal torsion, where resultant outcome would have led to perception of a congenitalanomaly.Design: Case report.Setting: Tertiary pediatric hospital.Patient(s): A young girl who presented acutely with symptoms consistent with adnexal torsion but who was foundto have a torsion affecting a paratubal cyst and the midsegment of her fallopian tube.Intervention(s): A salpingectomy was performed because of damage involving the ischemic paratubal cystand tubal segment that left too little residual tube to allow for a future anastomosis of the residual unaffectedcomponents.Main Outcome Measure(s): Operative findings, which give an explanation for midsegmental tubal absence.Result(s): This case challenges previous reports that absent midsegment of a tube is a rare congenital anomaly.Conclusion(s): Absent midsegment of a tube can be explained as an acquired anomaly, rather than proposingan unusual congenital anomaly. (Fertil Steril� 2007;88:968.e13–4. �2007 by American Society for ReproductiveMedicine.)
An 11 1/2 year old girl presented to the emergency depart-ment of the Royal Children’s Hospital with a 28-hour historyof dull right lower quadrant pain. Pain was in the right loin,radiating to the right iliac fossa, progressively worsening ina colicky pattern. She did not have any nausea, vomiting, orurinary or bowel symptoms and was afebrile. She also waspremenarchal. On examination, maximal tenderness wasright iliac fossa, with no loin tenderness. The initial impres-sion was renal colic, although there was no hematuria, witha gynecological cause also considered possible. She was ob-served overnight. An ultrasound demonstrated a thick-walledheterogeneous mass with hypoechoic and hyperechoic foci inthe right iliac fossa measuring 3 � 5 cm. The appendix andright ovary were not identified separately. The right iliac fossamass was thought to potentially represent a torted ovariancyst, hemorrhagic ovarian cyst, or appendix abscess.
At laparoscopy, a 5 � 5–cm right adnexal ischemic cysticstructure was noted that was initially thought to be a tortedright ovary with its accompanying tube. On closer examina-tion, the normal right ovary was located beneath this cysticstructure. The ischemic structure consisted of the midseg-
Received October 26, 2006; revised and accepted November 27, 2006.
Reprint requests: Sonia Grover, M.D., Department of Gynaecology, Royal
Children’s Hospital, Flemington Road, Parkville, Melbourne, Victoria
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medic
968.e13
ment of the right tube and a paratubal cyst that had undergone360� torsion. The fimbrial end of the tube and the uterinemedial or isthmic aspect of the tube were identified andwere not ischemic.
In view of the ischemic damage to the mid portion of thetube with the accompanying paratubal cyst, a right salpingec-tomy was performed. An inadequate length of tube to allowa future anastomosis resulted in the decision to undertakea complete salpingectomy.
DISCUSSION
Unusual anomalies of the genital tract are often considered tobe congenital, with absence of the midsegment of the fallo-pian tube no exception to this (1–5). The possibility of theseanomalies being acquired has also been documented (6–8).This case offered the opportunity to observe the acute processof torsion of the midsegment of a fallopian tube and thus re-inforces the possibility that many of the previously describedanomalies may also be acquired rather than congenital. Thelong-term outcome of our patient if no surgery had beenundertaken would have been an interruption of the ampullaryportion of the fallopian tube. Where there is absence ofthe ovary and tube, it is presumed to be the result of relativelyasymptomatic adnexal torsion during childhood, adoles-cence, or adulthood or similarly as an antenatal event (9).
0015-0282/07/$32.00ine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.11.159
Cases of amputation of the tube have been described with therecognition that these are likely to be the result of torsion (7,8, 10). One of the consistent features of these cases is thepresence of 1–2 cm of the proximal portion of the tube, oftena little distended (2, 11). Cases in the literature in which otheranomalies are present, such as unicornuate (4) and bicornuateuteri (3, 10), do not actually increase the likelihood of thetubal anomaly being congenital, because these uterine anom-alies are relatively common (12). There are some reportedcases that are more challenging when one attempts to under-stand the origin of the anomaly. Kozlowski and Luciano (13)describe a case in which there was bilateral atresia of theproximal ampule of both tubes, although on both sides therewas a normal proximal segment consistent with a torsionevent. Likewise, a case was reported in which the proximaltube was absent, and the distal end separated into three por-tions is more difficult to explain (14).
Great care needs to be taken when describing anomaliesand attributing them as congenital, when clearly some un-usual outcomes that occur as a consequence of events involv-ing torsion may adequately explain the finding. Trying tounderstand the true congenital anomalies of the genital tractis challenging enough (15) without muddying the waters withinappropriately attributed acquired anomalies (2).
REFERENCES1. Nawroth F, Nugent W, Ludwig M. Congenital partial atresia of the fallo-
pian tube. Reprod Biomed Online 2006;12:205–8.
Fertility and Sterility�
2. Dahan MH, Burney R, Lathi R. Congenital interruption of the ampullary
portion of the fallopian tube. Fertil Steril 2006;85:1820–1.
3. Szlachter N, Weiss G. Distal tubal pregnancy in a patient with a bicorn-
uate uterus and segmental absence of the fallopian tube. Fertil Steril
1979;32:602–3.
4. Richardson DA, Evans MI, Talerman A, Maroulis GB. Segmental
absence of the mid-portion of the fallopian tube. Fertil Steril 1982;37:
577–9.
5. Farber M, Noumoff J, Freedman M, Heyner S. Anomalous develop-
ment of the fallopian tubes. Semin Reprod Endocrinol 1986;4:
55–7.
6. Kanter E. Segmental absence of the mid-portion of the fallopian tube.
Fertil Steril 1983;40:551–2.
7. Anderson HF. Torsion of an apparently normal ovary and spontaneous
amputation of the fallopian tube during adolescence. Am J Obstet
Gynecol 1945;49:283.
8. Flickinger FM, Masson JC. Spontaneous amputation of the fallopian
tube. Am J Surg 1947;74:850.
9. Mares AJ, Mordechai Y, Grabarnik R, Barki Y, Bar-Ziv J. Antenatal
torsion of the uterine adnexa in a neonate. Z Kinderchir 1987;42:
381–3.
10. Farber M, Mitchell GW Jr. Bicornuate uterus and partial atresia of the
fallopian tube. Am J Obstet Gynecol 1979;134:881–4.
11. Silverman AY, Greenberg EI. Absence of a segment of the proximal
portion of a fallopian tube. Obstet Gynecol 1983;62:90s–1s.
12. Simon C, Martinez L, Pardo F, Tortajada M, Pellicer A. Mullerian defects
in women with normal reproductive outcome. Fertil Steril 1991;
56:1192–3.
13. Kozlowski D, Luciano AA. Bilateral atresia of the proximal ampullary
segment of the fallopian tubes. J Am Assoc Gynecol Laparosc 1995;3:
15. Acien P. Embryological observations on the female genital tract. Hum
Reprod 1992;7:437–45.
968.e14
CASE REPORT
Extremely elevated serum CA-125 level as a resultof unruptured unilateral endometrioma: the highestvalue reportedKorhan Kahraman, M.D., Irem Ozguven, M.D., Mete Gungor, M.D., andCem Somer Atabekoglu, M.D.
Department of Obstetrics and Gynecology, Ankara University School of Medicine, Ankara, Turkey
Objective: To present a case of a unilateral endometrioma with extremely elevated serum CA-125 levels.Design: Case report.Setting: University hospital.Patient(s): A 25-year-old woman with left adnexal mass and extremely elevated serum CA-125 level, 7,900 U/mL, underwent laparoscopy. There were unilateral endometrioma and stage IV endometriosis.Intervention(s): Laparoscopic excision of the endometrioma, unilateral salpingectomy, adhesiolysis, ablation ofendometriotic foci, and partial omentectomy were performed, and histopathologic results confirmed the diagnosisof endometriosis.Main Outcome Measure(s): Extremely elevated serum CA-125 levels and unruptured ovarian endometrioma.Result(s): On the second, sixth, and 13th days of the menstrual period, serum CA-125 levels were 7,900 U/mL,1,577 U/mL, and 627 U/mL, respectively. On the third postoperative day serum CA-125 level was 56 U/mL.Conclusion(s): Ovarian endometrioma and advanced endometriosis may be associated with extremely elevatedserum CA-125 levels. For this reason ovarian endometrioma should be considered with respect to differential di-agnosis of reproductive-age women presenting with an ovarian mass even if it resembles an ovarian malignancy.(Fertil Steril� 2007;88:968.e15–7. �2007 by American Society for Reproductive Medicine.)
Cancer antigen 125 (CA-125), a tumor-associated antigen, isused for the detection of persistence and recurrence of thedisease and monitoring of the response to treatment in pa-tients with epithelial ovarian carcinomas, but also CA-125could be used to discriminate between malignant and benignovarian masses as an adjuvant together with other diagnosticmethods. Many studies have demonstrated elevated serumCA-125 levels in patients with endometriosis (1). However,patients with endometriosis rarely have serum CA-125 levels>1,000 U/mL. The present case demonstrates that ovarianendometrioma and advanced-stage endometriosis may beassociated with extremely elevated serum CA-125 levels.
CASE REPORT
A 25-year-old nulligravida woman was referred to our clinicwith a left adnexal mass and cardiac problems includingmitral valve stenosis and pulmonary hypertension. On pelvic
Received October 22, 2006; revised December 12, 2006; accepted
December 13, 2006.
Reprint requests: Korhan Kahraman, M.D., Ankara Universitesi Tıp Fakul-
tesi Kadın Hastalıkları ve Dogum Anabilim Dalı, 06100, Cebeci, Ankara,
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medi
968.e15
examination, a mass that was fixed and elastic, extendingfrom the left adnexal region to the cul-de-sac, was found.An ultrasound examination of the pelvis revealed a 5 � 5 �4 cm cystic mass in the left adnexa, and the cyst had a regularsurface wall and a homogeneous semisolid internal compo-nent indicating a ‘‘chocolate cyst.’’ Magnetic resonance im-aging of the pelvis confirmed the ultrasound findings. Hermenstrual cycles were regular, and a mild degree of dysmen-orrhea was noted dating from puberty.
On the second day of her menstruation, serum CA-125level was 7,900 U/mL (reference range <35 U/mL). Mea-surement of serum CA-125 level was repeated on the sixthand 13th days of the menstruation; 1,577 U/mL and 627 U/mL were found, respectively. Serum CA-125 levels weremeasured by using appropriate chemiluminescent micro-particle immunoassay kits (Abbott Diagnostics, AbbottPark, IL).
At laparoscopy, a unilateral unruptured ovarian mass wasdetected. The mass was covered with small bowel loops, sig-moid bowel, the left fallopian tube, and the uterus (Fig. 1).The cul-de-sac was totally obliterated by the mass, withdense adhesions especially among the sigmoid bowel, leftfallopian tube, and uterus. In addition, the pelvic peritoneal
0015-0282/07/$32.00cine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.076
surfaces, distal omentum, uterus, and right adnexa were cov-ered with diffuse endometriotic foci. The diagnosis was stageIV endometriosis according to the revised American FertilitySociety classification. A peritoneal washing for cytologicexamination was performed. After lysing adhesions andfenestrating the ovarian mass, dark brown (chocolate-like)content was noticed and aspirated, then the capsule of thecyst was totally stripped with blunt and sharp dissection,and unilateral salpingectomy was performed because ofhydrosalpinx in the left fallopian tube. Electrocoagulationof endometriotic implants was carried out. On the third post-operative day serum CA-125 level was found to be 56 U/mL.The histologic examination of the specimen confirmed thediagnosis of endometrioma.
The patient recovered uneventfully and was discharged onthe third postoperative day in stable condition with combinedoral contraceptives for a 3-month period continuously.
DISCUSSION
An association between endometriosis and elevated serumCA-125 levels has been known for years. It has also been re-ported that serum CA-125 levels are higher during menses inwomen with or without endometriosis (2). On the other hand,elevation of serum CA-125 levels in patients with endometri-osis was significantly higher than in those without endometri-osis during menstruation (3).
The reported highest serum CA-125 level with surgicallyand histologically confirmed unruptured endometrioma hadbeen 3,890 U/mL in the English literature (4). The CA-125level in our case is the highest value reported so far for an un-ruptured endometrioma, with a level of 7,900 U/mL, approx-imately twice the highest level previously reported.
FIGURE 1
Laparoscopic appearance of the unruptured ovarianendometrioma (arrow). The mass is covered by theleft fallopian tube, uterus, small bowel loop, sigmoidbowel, and an endometriotic implant.
Kahraman. Endometrioma and elevated serum CA-125. Fertil Steril 2007.
Fertility and Sterility�
Many investigators have studied the value of CA-125 asa diagnostic tool for endometriosis with or without endome-trioma. Although a meta-analysis based on 23 articlesshowed a limited diagnostic performance of serum CA-125in detecting endometriosis (1), it may be more useful in diag-nosis of recurrent disease or assessing the success of treat-ment (6).
Several hypotheses have been postulated to explain thecause of extremely elevated serum CA-125 levels at thetime of menstruation in patients with endometriosis. Theseinclude higher CA-125 membrane concentration in ectopiccells than in eutopic endometrial cells (7), bleeding relatedto eutopic endometrium (8), increased transition CA-125from endometrial tissue to peritoneum related to retrogrademenses and ectopic endometrium (7, 8), an enlarged surfacearea of endometrial tissue (5), inflammatory reaction due topresence of the endometriotic foci, and blood and endome-trial shedding into the peritoneal cavity (5, 8).
Adhesions in the peritoneum, omentum, ovary, fallopiantube, colon, and cul-de-sac, size and rupture of endome-trioma, and stage of the endometriosis have been reportedas the cause of elevated serum CA-125 levels in patientswith endometriosis (9). Also, in the same study, it was sug-gested that ruptured endometrioma and omental adhesionshave been found to be the most important factors in havingserum CA-125 levels increased. In addition, it has beenknown that the content of ovarian endometrioma includesvery high levels of CA-125 antigens and it is covered witha thick capsule, which will not make an exaggerated elevationin systemic serum CA-125 levels without a rupture (10, 11).In the present case, existence of stage IV endometriosis,dense adhesions, and the patient being at menstruation all to-gether seem to be responsible for extremely elevated serumCA-125 levels. We want to emphasize the importance ofthe present case with such high levels being obtained in ourpatient without a ruptured endometrioma.
In conclusion, endometriosis should be taken into accountas a possible diagnosis in women with an adnexal mass at re-productive age with exaggerated elevation of the serum CA-125 level during menstruation, if a significant decrease of theserum CA-125 level is detected after menstruation and if thefindings related to malignancy are ruled out. Because of cy-clic variations of CA-125 levels throughout the menstrual cy-cles, new blood samples are needed after menses, especiallyduring the late luteal phase or premenstrual phase. Also, webelieve that laparoscopy as a minimally invasive surgery isan appropriate approach for premenopausal women with sus-pected adnexal mass associated with such high serum levelsof CA-125, which are typical of advanced ovarian carcinoma,after ruling out manifest malignancy.
REFERENCES1. Mol BW, Bayram N, Lijmer JG, Wiegerinck MA, Bongers MY, vander
Veen F, et al. The performance of CA-125 measurement in the detection
of endometriosis: a meta-analysis. Fertil Steril 1998;70:1101–8.
968.e16
2. Pittaway DE, Fayez JA. Serum CA-125 antigen levels increase during
menses. Am J Obstet Gynecol 1987;156:75–6.
3. Kafali H, Artuc H, Demir N. Use of CA125 fluctuation during the
menstrual cycle as a tool in the clinical diagnosis of endometriosis;
a preliminary report. Eur J Obstet Gynecol Reprod Biol 2004;116:
85–8.
4. Atabekoglu CS, Sonmezer M, Aydinuraz B, Dunder I. Extremely ele-
vated CA 125 level due to an unruptured large endometrioma. Eur J Ob-
stet Gynecol Reprod Biol 2003;110:105–6.
5. Johansson J, Santala M, Kauppila A. Explosive rise of serum CA 125 fol-
lowing the rupture of ovarian endometrioma. Hum Reprod 1998;13:
3503–4.
6. Chen FP, Soong YK, Lee N, Lo SK. The use of serum CA-125 as a marker
for endometriosis in patients with dysmenorrhea for monitoring therapy
and for recurrence of endometriosis. Acta Obstet Gynecol Scand
1998;77:665–70.
968.e17 Kahraman et al. Endometrioma and elevated seru
7. Niloff JM, Knapp RC, Schaetzl E, Reynolds C, Bast RC Jr. CA-125 an-
tigen levels in obstetric and gynecologic patients. Obstet Gynecol
Peri-implantation pelvic inflammatory diseasewith normal pregnancy outcomeGad Liberty, M.D., Jordana Hadassah Hyman, M.D., and Ehud J. Margalioth, M.D.
Department of Obstetrics and Gynecology, Shaare Zedek Medical Center, Jerusalem, Israel (affiliated with the Faculty of
Health Science, Ben-Gurion University of the Negev, Beer Sheba, Israel)
Objective: To report on the presentation, diagnostic assessment, treatment, and outcome of a case of peri-implan-tation pelvic inflammatory disease.Design: Case report.Setting: Emergency department and gynecology ward of a tertiary university hospital.Patient(s): A 25-year-old multiparous woman in her 1st month of pregnancy.Intervention(s): Diagnostic laparoscopy, antibiotic therapy, and pregnancy surveillance.Main Outcome Measure(s): Surveillance and outcome of pregnancy.Result(s): The patient presented with abdominal pain and fever 26 days after her last menstrual period. Examina-tion revealed peritoneal irritation and cervical tenderness, with free fluid in the pelvis. Diagnostic laparoscopy wasperformed, with no evidence of ectopic pregnancy. A significant quantity of yellow purulent fluid was drained fromthe pelvis, which cultured for Escherichia coli. Antibiotic therapy yielded excellent response. The pregnancy con-tinued, with no complications, to healthy term delivery.Conclusion(s): This rare diagnosis of peri-implantation pelvic inflammatory disease should be considered in thedifferential diagnosis of abdominal pain in early pregnancy. Timely, rational treatment, including early pelvicdrainage and appropriate antibiotic therapy, may save the pregnancy. (Fertil Steril� 2007;88:969.e1–2. �2007by American Society for Reproductive Medicine.)
We report a case of pelvic inflammatory disease in the peri-implantation period of a spontaneous pregnancy, the outcomeof which was a normal pregnancy. The few cases of peri-implantation pelvic inflammatory disease that have beendocumented in the literature report pregnancies subsequentto assisted reproductive technology, with no term pregnancyachieved (1–4).
CASE REPORT
A 25-year-old married woman presented to the hospital withfever and 12 hours of lower abdominal pain, with progressivegeneralization. There was no vaginal discharge or bleeding.Her last menstrual period was 26 days earlier. The patienthad had three prior pregnancies with three normal vaginaldeliveries, at term. She had had no previous gynecologicalcomplaints and had not been using contraception.
On admission the woman was in pain, febrile (38�C),tachycardic (104 bpm), and had a blood pressure of 115/48mmHg. She had diffuse abdominal tenderness, with signs
Received July 20, 2006; revised and accepted November 29, 2006.
Reprint requests: Gad Liberty, M.D., Department of Obstetrics and
Gynecology, Shaare Zedek Medical Center, P.O. Box 3235, Jerusalem,
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.11.154 Copyright ª2007 American Soci
of peritoneal irritation in the lower abdomen. On speculumexamination, there was no bleeding or discharge. Bimanualpalpation revealed a normal-sized, anteverted uterus, withexquisite tenderness on cervical motion. Laboratory ß-hCGwas 82 IU/L, with borderline leukocytosis (10,400 per micro-liter) with left shift (82% granulocytes) and with Hb of12.5 g/dL. Transvaginal sonography identified normal uteruswith homogenous endometrium of 15-mm thickness. Neitheran intrauterine nor extrauterine pregnancy sac was demon-strated. A small follicle, appropriate for corpus luteum, wasseen in the right ovary. Free fluid of moderate volume wasnoted in the pelvis, with no additional pathologic findings.
The patient underwent urgent diagnostic laparoscopy.Sixty microliters of yellow purulent fluid drained fromthe pelvis. The fallopian tubes were intact and reddenedwith edematous fimbriae, with no signs of ectopic preg-nancy. The appendix and gall bladder were normal. Postop-eratively, pelvic aspirate cultured positive for Escherichiacoli, whereas vaginal, urine, and blood cultures were ster-ile. A course of antibiotic therapy with cefuroxime (a sec-ond-generation cephalosporin, class B in pregnancy) wasinitiated, according to bacterial sensitivity. Two dayslater, the woman was discharged from hospital in goodcondition.
Fertility and Sterility� Vol. 88, No. 4, October 2007 969.e1ety for Reproductive Medicine, Published by Elsevier Inc.
At outpatient review, 11 days after her initial presentation,the quantitative ß-hCG was 7,861 IU/L. Transvaginal sonog-raphy at 9 and 12 weeks demonstrated a single viableembryo, compatible with gestational age. At 39 gestationalweeks, the woman had a spontaneous vaginal delivery ofa healthy female baby, weighing 3,280 g, with an Apgar scoreof 91 to 95. There were no maternal or neonatal complica-tions.
DISCUSSION
Our case implies acute pelvic inflammatory disease duringthe peri-implantation period, indicated by the ß-hCG of 82IU. Infection in spontaneous pregnancy may be secondaryto mechanical spread from the vagina or via infected sperma-tozoa (5), lymphatic or vascular spread, or reactivation ofchronic infection (6).
Pelvic infection after oocyte retrieval and embryo transferis a known complication of IVF treatment. Forty-two casesdocumenting IVF-associated pelvic inflammatory diseaseyielded a zero pregnancy rate (7–9). A single case of preg-nancy continuing to 7 weeks has been reported in a patientwith ovarian abscess after puncture of endometrioma duringoocyte retrieval; follow-up was not reported (10).
The rarity of implantation in the presence of acute infec-tion may be attributed to inflammatory mediators that inter-fere with the processes of normal embryonic growth andimplantation (11). Conception may be inhibited directly bythe E. coli endotoxin, via its stimulatory effect on uterinesmooth muscle and its luteolytic effect (12).
Our case shows that pregnancy may survive pelvic infec-tion, even in the peri-implantation period. It is likely thatthe resilience of the conceptus and its normal placentationwas a result of the early drainage of the infection and the ap-
969.e2 Liberty et al. Peri-implantation PID
propriate antibiotic treatment. If so, this intervention resultedin a viable pregnancy and a healthy baby, born at term.
REFERENCES1. Younis JS, Ezra Y, Laufer N, Ohel G. Late manifestation of pelvic ab-
scess following oocyte retrieval, for in vitro fertilization, in patients
with severe endometriosis and ovarian endometriomata. J Assist Reprod
et al. Effects of endotoxin infusion on circulating levels of eicosanoids,
progesterone, cortisol, glucose and lactic acid, and abortion in pregnant
cows. Vet Microbiol 1990;21:211–31.
Vol. 88, No. 4, October 2007
CASE REPORT
Treatment of heterotopic cervical pregnancy afterin vitro fertilization–embryo transfer by usingtransvaginal ultrasound–guided aspiration andinstillation of hypertonic solution of sodium chlorideMilenko Prorocic, M.D., Ph.D., and Mladenko Vasiljevic, M.D., Ph.D.
Clinic of Gynecology and Obstetrics ‘‘Narodni Front,’’ Belgrade Medical School, Belgrade, Serbia and Montenegro
Objective: To report a case of successful treatment of heterotopic cervical pregnancy resulting from IVF–embryotransfer.Design: Case report.Setting: Tertiary university clinical center.Patient(s): A 31-year-old patient, treated for heterotopic cervical pregnancy, diagnosed in the 6th gestationalweek.Intervention(s): Transvaginal ultrasound-guided aspiration of cervical pregnancy and instillation of hypertonic so-lution of sodium chloride, upon ligation of descending cervical branches of the uterine arteries.Main Outcome Measure(s): Patient recovery and further maintenance of intrauterine twin pregnancy.Result(s): The cervical pregnancy was successfully aborted, and the intrauterine twin pregnancy was successfullymaintained.Conclusion(s): The intervention applied may be used in treatment of heterotopic cervical pregnancy. (Fertil Steril�
2007;88:969.e3–5. �2007 by American Society for Reproductive Medicine.)
Cervical pregnancy is a rare form of ectopic pregnancy. Theincidence of cervical pregnancy varies from 1 per 1,000 to 1per 18,000 pregnancies (1). The application of high-resolu-tion ultrasound as well as color Doppler has enabled diagno-sis of early cervical pregnancy. Heterotopic pregnancy is, infact, a multiple pregnancy, in which one or more intrauterinepregnancies coexist with one ectopic pregnancy. The inci-dence of heterotopic pregnancies has risen with increasingapplication of assisted reproduction methods such as IVF–embryo transfer and ranges from 1% to 3%, in relation tothe percentage of clinical pregnancies that occur as a resultof this procedure (2). In the literature, there have been reportson medical treatment of heterotopic cervical pregnancy. Thefollowing medical therapeutic procedures have been per-formed: ultrasound-guided potassium chloride or methotrex-ate injections, hypogastric iliac artery ligation and arterialembolization, cervical cerclage, and hysteroscopic resection(3, 4).
Received August 15, 2006; revised and accepted November 27, 2006.
Reprint requests: Milenko Prorocic, M.D., Ph.D., University Clinic for
Gynaecology and Obstetrics, Department for Sterility and Fertility, Nar-
odnog Fronta 62 Street, 11000 Belgrade, Serbia and Montenegro (FAX:
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.11.194 Copyright ª2007 American Soc
This is a case report of an early heterotopic cervical preg-nancy that was treated successfully by ultrasound-guided as-piration and instillation of hypertonic solution of sodiumchloride after ligation of descending cervical branches ofthe uterine arteries.
CASE REPORT
The 31-year-old patient was admitted to the Clinic of Gynae-cology and Obstetrics ‘‘Narodni front’’ (Belgrade, Serbia andMontenegro) in mid-December 2005 as a result of hemor-rhage in the 6th gestational week of a desired pregnancy(clinic’s history of illness no. 14581). The patient’s preg-nancy occurred in the IVF–embryo transfer program. Con-trolled ovarian hyperstimulation was performed by usinga long protocol, applying follitropin-a (Gonal-F; Serono)and GnRH-agonist (Suprefact; Aventis Intercontinental).Embryo transfer was performed with three embryos. In vitrofertilization–embryo transfer was used because bilateral sal-pingectomy had been performed because of two tubal ectopicpregnancies.
Gynecological findings on admission were as follows:there was a bigger vaginal blood coagulum; fresh uterinehemorrhage; livid cervix; and external cervical ostium
Fertility and Sterility� Vol. 88, No. 4, October 2007 969.e3iety for Reproductive Medicine, Published by Elsevier Inc.
excentrically posed, closed. The established diagnosis wasthreatened abortion. Ultrasound examination was performedimmediately, with three gestation sacs diagnosed. Two gesta-tion sacs were in the uterine cavity. These were 19 mm and 17mm in diameter, respectively, with yolk sacs present. Embry-onic cardiac output was 124 beats per minute and 132 beatsper minute, respectively. The third gestation sac was locatedin the upper part of the cervix, 2 cm from the external cervicalostium. It was 19 mm in diameter, with a visible yolk sac andvital embryo with cardiac output of 129 beats per minute. Ul-trasound showed residual follicles and yellow bodies bilater-ally on the ovaries. Serum values were as follows: b-hCG,74.572 U/L; E2, 2.686 pg/mL; and P, 117 ng/mL. Blood pres-sure was 110/60 mm Hg, and the patient had prominent ane-mia. Laboratory findings on admission were the following:white blood cell count, 10.3 � 109 per liter; red blood cellcount, 2.33� 1012 per liter; hemoglobin, 77 g/L; hematocrit,0.231 L/L; mean cell volume, 98.9 fL; mean cell hemoglobin,33.1 pg; mean cell hemoglobin concentration, 335 g/L; plate-lets, 254 � 109 per liter; urea, 2.6 mmol/L; glycemia, 4.2mmol/L; total proteins, 58 g/L; acidum uricum, 144 mmol/L; serum creatinine, 60 mmol/L; cholesterol, 4.1 mmol/L;GOT, 26 U/L; and GPT, 36 U/L. The patient was immediatelysubjected to therapy, as follows: 17a-hydroxyprogesteronecaproate (250-mg ampule, 1 given IM every 3rd day), withvaginal P (600 mg daily) plus antibiotic therapy. The follow-ing day (the day after admission), the intervention wasperformed on the patient under general endotracheal anesthe-sia; it was aimed at cessation of the cervical pregnancy butmaintenance of the intrauterine twin pregnancy. The proce-dure was performed in the following way: upon cleaningand disinfection of the external genitalia, the vagina was re-tracted, and two DEXON sutures were placed bilaterally onthe cervix, high below the fornix vaginae. This significantlyreduced hemorrhage. By using transvaginal ultrasound, thegestation sac located in the cervical canal was puncturedthrough the anterior cervical lip with the 35-G needle, and ap-proximately 1.5 mL of liquor amnii was retrieved. Then, 2 mLof sodium chloride solution was instilled into the gestationsac. The intervention is depicted in Figure 1.
Upon completion of the intervention, it was ultrasono-graphically confirmed that twin pregnancy in the uterine cav-ity was intact and there was no embryonic cardiac actionregistered in the cervical canal. Once the intervention had be-gun, therapy was introduced before the procedure continued.Seven days after the intervention, the patient stopped bleed-ing. Her general condition was good. A cervical swab wastaken every 5 days and was sterile. The development oftwin pregnancy was monitored by weekly transvaginal ultra-sound examination. The pregnancy had a normal course. Atpresent, the pregnancy is at 12 weeks of gestational age,and the patient is feeling well.
DISCUSSION
In literature, the incidence of heterotopic cervical pregnan-cies upon IVF–embryo transfer ranges from 1% to 3%, in re-
969.e4 Prorocic and Vasiljevic Treatment of cervical preg
lation to the percentage of clinical pregnancies that occur asa result of this procedure (2). Before routine ultrasound wasintroduced in obstetrics, cervical pregnancy was rarely diag-nosed at its early stage. It used to be diagnosed during dilata-tion of the cervical canal and curettage and usually resulted inlife-threatening massive hemorrhage. As a result of this, mostpregnancies of this kind ended in hysterectomy to save thepatient’s life. The introduction of routine high-resolution ul-trasound as well as color Doppler in obstetrics enabled diag-nosis of early cervical pregnancy. Thus, various medicaltherapeutic methods could be successfully applied in treatingcervical pregnancy. In the last 2 decades, cervical pregnan-cies most commonly have been treated by application oftransvaginal ultrasound-guided injection of methotrexateand potassium chloride (4). Recently, ligation of the hypogas-tric artery and embolization of the uterine arteries, as well ascerclage of the cervix, have been performed to treat cervicalpregnancy. In 2003, Jozwiak et al. (3) reported a successfulhysteroscopic resection of heterotopic cervical pregnancy.In our case, we did not decide to apply local methotrexate be-cause of its systemic action, because, apart from the cervicalpregnancy, there was a vital intrauterine twin pregnancy to beconsidered. We did not decide to apply uterine artery embo-lization either, because the patient is exposed to x-rays duringthe procedure. By placing DEXON sutures laterally on thecervix, we managed to reduce bleeding almost completelyas a result of ligation of the descending branches of the uter-ine arteries. Ultrasound-guided aspiration and instillation ofhypertonic sodium chloride solution into the cervical gesta-tion sac has proven to be very successful, and there havebeen no side effects on the vital intrauterine twin pregnancy.
The technique applied has been shown to be one possiblesuccessful treatment for heterotopic cervical pregnancy,and its application does not threaten the intrauterinepregnancy.
FIGURE 1
Ultrasound view of the surgical intervention.
Prorocic. Treatment of cervical pregnancy. Fertil Steril 2007.
a Department of Obstetrics and Gynecology, b Department of Surgery, and c Department of Pathology, Tohoku University
Graduate School of Medicine, Sendai, Japan
Objective: To report a rare complication of GnRH agonist therapy for intestinal endometriosis.Design: Case report.Setting: University hospital.Patient(s): A 45-year-old nulliparous Japanese woman with catamenial digestive symptoms.Intervention(s): GnRH agonist therapy.Main Outcome Measure(s): Acute abdomenal crisis with free air in the abdominal X-ray.Result(s): An emergency laparotomy showed both an ileal constriction and perforation. An ileocecal enterectomywith an end-to-end anastomosis was performed. A pathological examination of the ileum revealed ileal endome-triosis.Conclusion(s): Flare-up of intestinal endometriosis induced by GnRH agonist has the potential to lead to intestinalperforation. Careful diagnosis and treatment are necessary for cyclic and periodic gastrointestinal manifestation.(Fertil Steril� 2007;88:969.e7–9. �2007 by American Society for Reproductive Medicine.)
A 1987 review article found that 87 patients had experiencedbowel obstruction during 204 cases of ileal endometriosis asof 1985 (1); however, perforation is an extremely rare com-plication of small intestinal endometriosis. A recent case re-port and a review of the literature indicated three previouscase reports of spontaneous perforation during ileal endome-triosis (2), but to the best of our knowledge, this is the firstreport of ileal perforation resulting from ileal endometriosisafter GnRH agonist therapy.
CASE REPORT
A 45-year-old woman (gravida 1, para 0) had presented withrepeated episodes of diffuse abdominal pain and distension,diarrhea, vomiting, and anorexia for 4–5 days during her peri-menstrual period for several months. Her family history wasnoncontributory, and she had previously been treated for anacoustic nerve tumor by radiotherapy. She reported histori-cally regular 24-day menstrual cycles without dysmenorrheaand had been diagnosed with myoma uteri 10 years earlier.Because multiple myomas had been identified previously
Received November 24, 2006; revised and accepted December 28, 2006.
Reprint requests: Takashi Murakami, Department of Obstetrics and
Gynaecology, Tohoku University Graduate School of Medicine, 1-1
Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan (FAX:
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.082 Copyright ª2007 American Soc
and the patient’s serum CA125 levels were shown to haveincreased to 103 U/mL at another clinic, she was referredto our department for further evaluation and treatment.Multiple myomas were identified after a pelvic examination,but no tenderness was detected in the gynecologic organs. Nointervention was performed at that time, and the patient wasfollowed closely as an outpatient.
Approximately 1 month later, a protruding lesion of thesigmoid colon was observed during a colonoscopy, and itwas diagnosed as intestinal endometriosis after biopsy andpathological examination. She was referred to our depart-ment for consultation and treatment. We administered 3.75mg of leuprolide acetate (Takeda Pharmaceutical, Osaka)SC on the patient’s third menstrual day. Fifteen days after ini-tiating GnRH agonist therapy, the patient experienced lowerabdominal pain with genital bleeding that resembled men-strual flow. On the 19th day, the abdominal pain worsenedquickly and the patient was admitted for acute abdomen.An abdominal X-ray showed the appearance of free air inthe peritoneal cavity, and an intestinal perforation with com-plicating general peritonitis was diagnosed. An emergencylaparotomy was performed.
During the laparotomy, a large amount of purulent and bil-ious fluid was found in the peritoneal cavity, as well as bothan ileal constriction and perforation. The constriction and
Fertility and Sterility� Vol. 88, No. 4, October 2007 969.e7iety for Reproductive Medicine, Published by Elsevier Inc.
perforation were 5 cm and 10 cm proximal to the ileocecalvalve, respectively. Surgeons performed an ileocecal enterec-tomy with an end-to-end anastomosis, and the peritonealcavity was extensively irrigated and drained. Multiple uterinemyomas were noted along with intact ovaries of normalappearance. Endometriosis was present throughout theserosal surface of the rectum, and no other sites of endome-triosis were visible within the pelvic cavity.
A pathological examination of the ileum revealed that en-dometriotic gland–associated proper stroma had reached intothe level of the submucosa, and the patient was diagnosedwith ileal endometriosis. Some of the glands were dilatedand filled by the blood contents.
After surgery, the patient remained febrile, and a computedtomography (CT) scan revealed the presence of a deep ve-nous embolism and a hepatic abscess 10 days after the firstoperation. Anticoagulant therapy was immediately initiated,and a second laparotomy to drain the abscess was performed1 week later. However, the patient remained febrile. As a CTscan suggested cholecystolithiasis, antibiotics and fastingtherapy were started. The patient subsequently went into re-mission and was released 6 weeks after the second operation.
After the resumption of menstruation after discharge fromthe hospital, the patient was restarted on GnRH agonist ther-apy. After six courses of GnRH agonist medication, endome-triosis had not recurred for 12 months.
DISCUSSION
The frequency of intestinal endometriosis ranges from 3% to37% in women with known endometriosis (3–5). The rectumand sigmoid colon are most commonly affected, representing71%–97% of cases; in addition, the appendix is involved in3%–20% of cases, and the small intestines are involved in5%–7% of cases (3–5).
969.e8 Saito et al. Ileal perforation with endometriosis
Rectal bleeding or dyschezia, especially during the men-strual period, is a specific symptom of rectal or sigmoid endo-metriosis, but symptoms of small intestinal endometriosis areusually nonexistent or nonspecific. Symptoms typically de-velop when lesions inhibit peristaltic activity or twist theintestinal lumen. Cardinal symptoms are cyclic, periodiccomplaints such as abdominal pain and/or distension, nausea,vomiting, diarrhea, and constipation. These symptoms ofsmall intestinal endometriosis may mimic those of severalchronic and acute intestinal diseases, including irritablebowel syndrome, Crohn’s disease, ulcerative colitis, gastro-enteritis, appendicitis, diverticulosis, and bowel cancer.Hence, small intestine endometriosis often goes unrecog-nized, and, when it is diagnosed, it is often after seriouscomplications have developed.
Exacerbation of the disease can potentially result in perfo-ration or obstruction of the small intestine, and an enterec-tomy is indicated if either of these occurs, although casesof complicated perforation are rarely reported. The obstruc-tion is usually owing to kinking and fibrous degenerationdue to endometriosis in the serosa and muscular coats (6).Previous reports have shown that spontaneous obstruction of-ten occurs during menstruation (6) and midcycle (7). A singlecase of ileocecal endometriosis complicated by bowel ob-struction after administration of GnRH agonist was reportedin 1995 (8). This patient received GnRH agonist on cycle day7, and abdominal pain became increasingly more severe andincluded episodes of intense cramping, vomiting, and a 9-kgweight loss over the following 2 weeks; the patient underwentexploratory laparotomy 3 weeks after beginning GnRH ago-nist therapy. Our patient was administered GnRH agonist oncycle day 3, and beginning 15 days later, she experiencedlower abdominal pain and genital bleeding; her pain becamemore severe by day 19. Both cases likely demonstrate that theestrogen surge caused by GnRH agonist exacerbated thepatients’ existing disease and caused ileal constriction and
FIGURE 1
(A) Endometriotic glands and stroma were seen in the wall of the ileum, through the submucosal level(hematoxylin and eosin, �40). (B) Glandular epithelium was not atypical, and blood contents were evidentwithin the dilated glands (hematoxylin and eosin, �100).
Saito. Ileal perforation with endometriosis. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
obstruction and that an increase in pressure proximal to thesite of constriction perforated the ileum of our patient.
GnRH agonist is often used and is effective as a treatmentfor endometriosis cases that include bowel lesions, althoughit does not usually completely cure endometriosis. One reportfound that 3 months of GnRH agonist therapy cleared themultilobulated polyp region of sigmoid endometriosis for atleast 2 years (9). However, the flare-up induced by GnRHagonist can transiently exacerbate the disease, such as withthe present case. GnRH antagonist or danazol may be saferwhen cases of intestinal endometriosis are suspected.
Surgery is a radical treatment, but the preoperative diagno-sis of small intestine endometriosis is not simple, as de-scribed above. Close collaboration and communicationamong gynecologists, general surgeons, and gastroenterolo-gists are needed to efficiently determine an early diagnosis.A careful history, with particular emphasis on perimenstrualsymptoms, is essential to an accurate diagnosis. Additionally,colonoscopy and magnetic resonance imaging or CT scanssometimes reveal an intestinal mass or external compressioncaused by intestinal endometriosis. In particular, radio-graphic contrast studies of the small intestine and colon candetect segmental strictures and mucosal lesions. In retro-spect, given the presence of the periodic obstructive symp-toms in this patient, an abdominal radiogram should havebeen obtained before beginning GnRH agonist therapy. Ifthe presence of intestinal constriction had been clearlydetected, an earlier surgical intervention might have beenchosen.
In summary, we treated a rare case of small intestinal per-foration with ileal endometriosis after initiation of GnRH
Fertility and Sterility�
agonist treatment. Catamenial digestive symptoms shouldbe considered carefully for patients with endometriosisbecause they may be the only clue that will allow an accurateearly diagnosis. The transitory exacerbation of disease in-duced by GnRH agonist treatment can cause perforation ofthe small intestine. The informed consent of this rare compli-cation should be obtained from such patients.
Acknowledgment: The authors thank the patient for permitting her case to be
reported.
REFERENCES1. Stahl C, Grimes EM. Endometriosis of the small bowel. Case report and
review of the literature. Obstet Gynecol Surv 1987;42:131–6.
2. Decker D, Konig J, Wardelmann E, Richter O, Popat S, Wolff M, et al. Ter-
minal ileitis with sealed perforation—a rare complication of intestinal
endometriosis: case report and short review of the literature. Arch Gynecol
Obstet 2004;269:294–8.
3. McAfee CH, Greer HL. Intestinal endometriosis: a report of 29 cases and
a review of the literature. Br J Obstet Gynaecol 1960;67:539–55.
4. Weed JC, Ray JE. Endometriosis of the bowel. Obstet Gynecol 1987;69:
727–30.
5. Williams TJ, Pratt JH. Endometriosis in 1,000 consecutive celiotomies:
incidence and management. Am J Obstet Gynecol 1977;129:245–50.
7. Ridha JR, Cassaro S. Acute small bowel obstruction secondary to ileal
endometriosis: report of a case. Surg Today 2003;74:234–6.
8. Hall LH, Malone JM, Ginsburg KA. Flare-up of endometriosis induced by
gonadotropin-releasing hormone agonist leading to bowel obstruction.
Fertil Steril 1995;64:1204–6.
9. Porpora MG, Pallante D, Ferro A, Crobu M, Cerenzia P, Panici PLB.
Intestinal endometriosis without evident pelvic foci treated with gonado-
tropin releasing hormone agonist. Eur J Obstet Gynecol Reprod Bio
2005;125:265–6.
969.e9
CASE REPORT
Identification of a ‘‘cryptic mosaicism’’ involvingat least four different small supernumerary markerchromosomes derived from chromosome 9 in awoman without reproductive successM�onica Santos, M.S.,a Kristin Mrasek, M.S.,b Maria �Angels Rigola, Ph.D.,a Heike Starke, Ph.D.,b
Thomas Liehr, Ph.D.,b and Carme Fuster, Ph.D.a
a Unitat de Biologia Cel$lular i Genetica Medica, Facultat de Medicina, Universitat Aut�onoma de Barcelona, Barcelona, Spain;
and b Institut fur Humangenetik und Anthropologie, Jena, Germany
Objective: To characterize the small supernumerary marker chromosomes (sSMCs) present in the female memberof an infertile couple who has no further clinical symptoms.Design: Case report.Setting(s): Faculty of medicine and institute of human genetics and anthropology.Patient(s): A young, healthy, nonconsanguineous couple asked for genetic evaluation for infertility.Intervention(s): Intracytoplasmic sperm injection, conventional and molecular cytogenetic analyses.Main Outcome Measure(s): We characterized the sSMCs present in a woman, who was a member of an infertilecouple, by molecular cytogenetic techniques.Result(s): The G-banding technique showed that a marker chromosome was present in some of the examined cellsdescribing the 47,XX,þmar[30]/46,XX[70] karyotype. Subsequently, using new fluorescence in situ hybridization(FISH) techniques, four distinguishable sSMCs (cryptic mosaicism), all derived from chromosome 9, were ob-served, including minute and ring chromosomes. This heterogeneity was impossible to detect by the conventionalG-banding technique or conventional FISH technique that were used before the new FISH techniques (subcentro-mere-specific multicolor-FISH [subcenM-FISH]) and specific probe for the 9q12 band. In each metaphase withsSMCs, only one or two markers were observed. On the basis of the FISH analyses, the patient’s karyotype wasdefined as 47,XX,þmin(9)(:p12/q12:)/47,XX,þmin(9)(:p12/q12::q12/p12:)/47,XX,þr(9)(::p12/q12::)/47,XX,þr(9)(::p12/q12::p12/q12::)x2/46,XX.Conclusion(s): The presence of sSMCs derived from chromosome 9 could influence the couple’s infertility. Thenew subcenM-FISH techniques are very useful in the characterization of cryptic mosaicisms of marker chromo-somes. Additionally, the hypothesis that the 9p12 chromosomal band is an euchromatic variant region withoutany phenotypic impact other than possible infertility is supported by this case study since the woman shows anormal phenotype. (Fertil Steril� 2007;88:969.e11–7. �2007 by American Society for Reproductive Medicine.)
Key Words: Small supernumerary marker chromosome (sSMC), multiplex-fluorescence in situ hybridization(M-FISH), subcentromere-specific multicolor-FISH (subcenM-FISH), infertility
Infertility and sterility are an extensive widespread problemin the general population. It has been suggested that infertilitymay be due to different causes such as systemic infections,endocrine and immunology disorders, or cytogenetic alter-ations. In fact, one important cause of infertility is the pres-ence of a chromosomal aberration in one member of the
Received October 20, 2006; revised and accepted December 20, 2006.
This work was supported by the Ministerio de Ciencia y Tecnologıa (SAF
2003-03894), CIRIT (SGR05-00495), Generalitat de Catalunya (grant
nos. 2002FI00281 and 2005BE00172), and the Evangelische Studien-
werk e.V. Villigst.
Reprint requests: Carme Fuster, Unitat de Biologia Cel$lular i Genetica
Medica, Facultat de Medicina, Edifici M., Universitat Autonoma de
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.073 Copyright ª2007 American Soc
couple. In the general population, there is a 1% frequencyof chromosomal aberration; however, it has been reportedthat in couples with repeated spontaneous abortions, thefrequency is 2.4%–6.8% (1).
Small supernumerary marker chromosomes (sSMCs) wererecently defined as structurally abnormal chromosomes thatcannot be identified or characterized unambiguously by con-ventional-banding cytogenetics alone and are generally equalin size to or smaller than chromosome 20 of the same meta-phase spread (2). The sSMCs are common findings in karyo-types of cancer, in constitutional genetic disorders ormentally retarded patients, in patients with reproductionproblems, and in prenatal diagnosis (3–5). In a review pub-lished by Liehr et al. (2), the sSMC frequency was described
Fertility and Sterility� Vol. 88, No. 4, October 2007 969.e11iety for Reproductive Medicine, Published by Elsevier Inc.
as 0.043% in newborns, 0.077% in prenatal cases, 0.433% inmentally retarded patients, and 0.171% in infertile people.
The genetic origin of these sSMCs usually remainsunknown by conventional cytogenetic techniques, but thedevelopment of technologies based on fluorescence in situhybridization (FISH) has allowed for important progress to-ward this goal. Comparative genomic hybridization (CGH)(6), multiplex-FISH (M-FISH) (7), spectral karyotyping(SKY) (8), centromere-specific multicolor FISH (cenM-FISH) (9), and subcentromeric multicolor FISH (subcenM-FISH) (10) are some of these methodologies.
Here we describe the combined use of G banding, CGH,M-FISH, and subcenM-FISH for the detection and character-ization of the sSMCs present in a woman who is the infertilemember of a couple with no other clinical symptoms.
CASE REPORT
A young, healthy, nonconsanguineous couple with no repro-ductive success over a period of 4 years asked for geneticevaluation due to their apparent infertility. Both membersof the couple had a normal phenotype. A gynecologicalexamination of the woman was normal, but the husband’sexam revealed teratoasthenozoospermia (22 million/mL,spermatozoids with normal morphology 5%, and motility3 þ 2 and 30%). Cytogenetic analysis revealed a normal(46,XY) karyotype in the man and the 47,XX,þmar denovo/46,XX karyotype in his wife.
The couple decided to undergo an in vitro fertilization(IVF) program, and they underwent two intracytoplasmicsperm injection (ICSI) treatments because with this tech-nique the teratoasthenozoospermia was circumvented.Fourteen eggs were retrieved, and three were fertilized. Allthree embryos were transferred. However, no pregnancywas achieved.
The study was approved by the Universitat Autonoma deBarcelona’s Institutional Ethics Committee. Informed con-sent was given in writing by the patient.
Cytogenetic and FISH Studies
A peripheral blood sample from the 31-year-old woman wascultured for 72 hours in RPMI-1640 medium. Metaphasicchromosomes were analyzed by Wright G-banding andFISH techniques. FISH was performed by M-FISH accordingto the methodology used by Speicher et al. (7). Moreover, thechromosome 9 centromeric probe (alpha satellite), the probefor 9q12, and the subcenM-FISH mixture for chromosome 9described by Starke et al. (10) were used for analysis. Sub-cenM-FISH is a new FISH technique that combines variousFISH probes to characterize the composition of sSMCs.
Slides were evaluated on a fluorescence microscopeequipped with a charge-coupled-device camera and an im-age-analysis system (MetaSystems; Altlussheim, Germany).For M-FISH analysis, digital fluorescence imaging was per-
969.e12 Santos et al. ‘‘Cryptic mosaicism’’ of marker chro
formed using an Olympus BX-60 epifluorescence micro-scope and SpectraVysion software (Vysis).
CGH Analysis
To determine gains and losses of genomic material, the DNAfrom the patient was compared with the control DNA usingthe CGH technique. DNA isolation and labeling, hybridiza-tion, and detection were performed essentially as describedby Kallioniemi et al. (11). Slides were analyzed using aCytovision Ultra workstation (Applied Imaging; Sunderland,United Kingdom) and high-resolution CGH (HR-CGH) soft-ware (Rigshospitalet; Copenhagen, Denmark). Aberrationswere detected by standard reference intervals as describedin Kirchhoff et al. (6).
RESULTS
G-banded chromosome preparations analysis revealed thepresence of normal cells and cells with an extra markerchromosome (mosaicism) that define karyotype 47,XX,þmar[30]/46,XX [70], in which the origin of the small chro-mosome marker was impossible to characterize by thistechnique. HR-CGH analysis detected no imbalances in thegenome. M-FISH showed that the marker chromosomestained in chromosome 9—a typical fluorochrome combina-tion, with no other abnormal findings being found. Thehybridization with the chromosome 9 centromeric probeconfirmed the origin of the marker as chromosome 9.
Further analysis with the subcenM-FISH mixture for chro-mosome 9 and the probe for 9q12 showed that the markerconsisted of chromosome 9 heterochromatic material andthat it also contained the 9p12 euchromatic region. Molecularcytogenetic analysis showed the presence of at least foursSMCs (Fig. 1), all derived from chromosome 9, whichconfirms a ‘‘cryptic mosaicism’’ (not detected previously)involving chromosome 9.
This heterogeneity was impossible to detect by the conven-tional G-banding or M-FISH techniques that were usedbefore the new FISH techniques (subcenM-FISH). In eachmetaphase with sSMCs, only one or two markers wereobserved.
Based on the FISH analyses, the patient’s karyotypewas defined as 47,XX,þmin(9)(:p12/q12:)/47,XX,þmin(9)(:p12/q12::q12/p12:)/47,XX,þr(9)(::p12/q12::)/47,XX,þr(9)(::p12/q12::p12/q12::)x2/46,XX. The abbreviation‘‘min’’ is used here according to the definition of Crolla (12),not according to ISCN 2005 (13).
DISCUSSION
The interpretation of the clinical significance of marker chro-mosomes is extremely problematic, as sSMCs have heteroge-neous phenotypic consequences and their effects seem todepend on their size, origin, presence or absence of euchro-matin, degree of mosaicism, affected tissues, and, in somecases, their parental origin (whenever the marker contains
mosomes Vol. 88, No. 4, October 2007
FIGURE 1
The subcenM-FISH scheme and a normal chromosome 9 depicted in pseudocolors are shown. The four differentsSMCs 9 from the present patient are shown (2-minute and two-ring chromosomes). Scheme of sSMCformation. A trisomy 9 was omitted by degradation of the superfluous chromosome (left part of figure).The resulting sSMCs 9 developed by further degradation and inverted duplication to the observed 2-minutechromosomes and/or by ring formation directly and ring duplication to the observed ring chromosomes.The not detected and postulated variants are marked with asterisks.
Santos. ‘‘Cryptic mosaicism’’ of marker chromosomes. Fertil Steril 2007.
imprinted genes). Our patient with at least four differentsSMCs, all derived from chromosome 9, in combinationwith a normal cell line, presents the possibility of infertilitywithout further clinical symptoms.
This report is a new case study that illustrates the useful-ness of FISH methodologies to detect and characterize thepresence of at least four different sSMCs, all derived fromchromosome 9, present in a phenotypically normal woman,who is a member of an infertile couple and who has a47,XX,þmar[30]/46,XX[70] karyotype.
The CGH technique, described by Kallioniemi et al. (11),has been used for marker chromosome identification to deter-mine the chromosome region involved in the composition ofthe marker chromosome. CGH determines gains and lossesof genomic material comparing DNA from the patient withcontrol DNA. High-resolution CGH, described by Kirchhoffet al. (6), defines aberrations using standard reference inter-vals by increasing resolution in the detection of imbalances
Fertility and Sterility�
of conventional CGH. However, mosaicism and the smallamount of euchromatin found in sSMCs < 3-5 Mb are twoimportant limitations (14). In our case, it is important to re-mark that the HR-CGH cannot detect the origin of the markerchromosome even when it contains euchromatic material.The explanation could be that the mosaicism degree is atthe limit of HR-CGH resolution and that the euchromaticgain is very near the centromere region and is blocked withCOT-1 DNA (DNA rich in repetitive sequences used toexclude from analysis polymorphic regions) in HR-CGHexperiments.
Mosaicism in association with sSMCs is well known, andgreat variations in mosaicism with no clinical consequenceshave been reported (15, 16). Our patient, phenotypically nor-mal, has at least four different sSMCs, all composed of chro-mosome 9 pericentromeric euchromatin and heterochromatin(p12/q12) and with a high degree of morphological hetero-geneity (ring/min and one/two centromeres). The sSMC
969.e13
otypeFertility
problems? Reference
ed — (5)
ed — (10) (case 13)
ed — (5)
ed — (5)
ffected ? (16) (case 3)
ed — (5)
ed — (9) (case 10)
ed — (5)
ffected Yes Present case
TABLE 1Cryptic mosaicism for sSMC described in the literature.
Case Age Origin Karyotype SSMC Phen
1m 1 Week De novo 47,XX,þmar[69%] min(5)(:p12�13.1/10:)[11]/min(5)(:p12�13.1/q10:q10/p12�13.1:)[2]/r(5)(:p12�13.1/q10:q10/p12�13.1:)[1]
Affect
2m 4 Months De novo 47,XY,þmar[65%] r(7)(:p11.1/q11.21:)[15]/r(7;7)(:p11.1/q11.21::p11.1/q11.21:)[4]/r(7;7;7;7)(:p11.1/q11.21::p11.1/q11.21::p11.1/q11.21::p11.1/q11.21:)[1]
Affect
3m 5 Years De novo 47,XX,þmar[50%] min(8)(:p21/q11.21:)[4]/r(8)(::p21/q11.21::)[8]/r(8)(::p21/q11.21::p21/q11.21::)[3]/min(8)(:p21/q11.21:)/min(8)(p21/q10::q10/p21)[1]
Affect
4m 3 Months De novo 47,XY,þmar[10%] min(8)(:p11.1/q11.21:)[11]/r(8)(::p11.1/q11.21::)[2]/r(8)(::p11.1/q11.21::p11.1/q11.21::)[1]/r(8)(::p11.1/q11.21::p11.1/q11.21::p11.1/q11.21:)[2]/dic(8)(:q11.21/p11.1::p11.1/q11.21:)[5]/dic(8)(:q11.21/p11.1::p11.1/q11.1:)[2]
Affect
5m 30 Years ? 47,XX,þmar[27%] r(8)(::p23.1/q1?1::)/r(8;8)(::p23.1/q1?1::p23.1/q1?1::)
Not a
6m Prenatal De novo 47,XX,þmar[69%] r(19)(::p13.11/q13.11�13.12::)[16]/r(19;19)(::p13.11/q13.11�13.12::p13.11/q13.11�13.12::)[2]/min(19)(:p13.11/q13.11�13.12:)[5]
Affect
7m 2 Years De novo 47,XX,þmar min(20)(:p11.1/q11.22:)[18]/min(20)(:q11.22/p11.1::p11.1/q11.22:)[7]/r(20)(::p11.1/q11.22::p11.1/q11.22::)[3]
Affect
8m 13 Years ? 47,,XY,þmar min(X)(:p11.21/q10:)[4]/min(X)(:p11.21/q10::q10/p11.21:)[8]/r(X)(::q10/p11.21::q10/p11.21::)[2]
14 1 week De novo 47,XY,þmar Alpha-, beta-satellite
satIII probes,
telomeric probe, all
wcp, YAC probes
inv dup(9)(pter/p21.1::p21.1/pter)
Affecte
15 1 week De novo 47,XY,del(9)(p12)
þmar
Alpha-, beta-satellite
satIII probes,
telomeric probe, all
wcp, YAC probes
inv dup(9)(qter/q22::
q22/qter)
Affecte
— 31 years De novo 47,XX,þmar[30]/
46,XX[70]
M-FISH; HR-CGH;
subcenM;
cep9; probe
for 9q12
min(9)(:p12/q12:)[1]/
min(9)(:p12/q12::
q12/p12:)[5]/
r(9)(::p12/q12::)[2]/r(9)(::p12/q12::
p12/q12::)x2[1]
Not aff
Note: ISH = in situ hybridization; cep = centromeric probe; YAC = yeast artificial chromsome.
Santos. ‘‘Cryptic mosaicism’’ of marker chromosomes. Fertil Steril 2007.
Fertilityand
Sterility
�969.e15
mosaicism observed in this woman points out a common or-igin of all marker chromosomes, but a high degree of rear-rangements, due to mitotic instability of the markers, led tothe ‘‘cryptic karyotype.’’
In recent years, several cases with cryptic mosaicism forsSMCs derived from different chromosomes (17–24) orfrom the same chromosome (5, 7, 8, 19, 20) or X chromo-somes (5, 9, 10, 16) have been published. To date, our patientis the first case reported with cryptic mosaicism for sSMCsderived from chromosome 9. Detections of all cryptic mosa-icisms derived from the same chromosome (Table 1), with theexception of the case reported by Daniel and Malafiej (16),have been made using subcenM-FISH, testing the utility ofthis methodology; moreover, in our study we also used theprobe for 9q12. It is interesting to note that 8 of the 9 cases(including the present report) with mosaicism for the samechromosome presented with infertility or phenotypic mani-festations (congenital malformations or/and dysmorphisms).These findings show that cryptic mosaicism of sSMCs isa new factor to take into account to establish the relationshipbetween genotype and phenotype.
To the best of our knowledge, there are three publishedcases with markers derived from chromosome 9 combinedwith an sSMC derived from another chromosome (20, 10,24) and 15 reported cases with markers only derived fromchromosome 9 (Table 2) (25–39) that have been determinedcytogenetically at the molecular level. In one patient (case55) reported by Starke et al. (25), the sSMC, without crypticmosaicism, also contained the 9p12 chromosome euchro-matic region; the patient was a healthy woman with a historyof three miscarriages. The rest of the cases with sSMC, con-taining only the 9p12 region, presented normal phenotypes,with the exception of a child who was affected with moderatemental retardation and speech delay (31). In general, when-ever other chromosome 9 bands are implied in the composi-tion of the sSMC, the phenotype seems to be affected.
The 9p12 euchromatic region has been described as a var-iant without clinical effects (40, 41). Starke et al. (25) sug-gested that this chromosome region is neither reallyeuchromatic nor really heterochromatic since this regionshows homology with short arms of acrocentric chromo-somes and many genes located at this band are pseudogenes.All data summarized in this report and the absence of clinicalsymptoms in our patient (apart from possible infertility) arein agreement with this hypothesis.
It has been reported that the presence of a marker chromo-some in infertile couples is four times more frequent com-pared with the overall population (2). Small SMCs havebeen described in 0.171% of the infertile population (5). Ingeneral, the presence of sSMCs in the male causes infertility,while females usually present normal fertility (42, 43). How-ever, to date, two reports about infertile females with sSMCshave been published (25, 42). Although the confirmation ofthe unusual karyotype in a second type of tissue was not pos-sible, it is probable that the woman had a germ-line mosai-
969.e16 Santos et al. ‘‘Cryptic mosaicism’’ of marker chr
cism. The heterochromatin excess present in the sSMCs inour patient could disturb correct chromosome pairing, andit could influence acquisition of unbalanced gametes. Itmay be the cause of the couple’s infertility. The present reportcould be a case in which the presence of sSMCs in a womanalso led to infertility. Nevertheless, it is not possible to con-firm or refute this fact because of the presence of teratoasthe-nozoospermia in the husband. At first sight, the low qualityof sperm in the husband could explain the infertility, butthe unsuccessful ICSI treatment and the published reportscorrelating cytogenetic anomalies with infertility suggestthat the presence of the sSMCs could influence in the couple’sreproductive problems.
We conclude that it is essential to perform karyotype anal-ysis in couples with fertility problems to discover the possiblereason for the infertility, such as the cytogenetic anomalies.The presence of various sSMCs could exert influence onthe reproductive problems in the couple. New FISH tech-niques like subcenM-FISH are very useful in the character-ization of cryptic mosaicism of marker chromosomes. And,finally, our findings support the hypothesis that the 9p12chromosomal band is a euchromatic variant region withoutphenotypic impact other than possible infertility since thewoman in this case presents a normal phenotype.
REFERENCES1. Clementini E, Palka C, Iezzi I, Stuppia L, Guanciali-Franchi P,
Tiboni GM. Prevalence of chromosomal abnormalities in 2078 infertile
couples referred for assisted reproductive techniques. Hum Reprod
2005;20:437–42.
2. Liehr T, Claussen U, Starke H. Small supernumerary marker chromo-
somes (sSMC) in humans. Cytogenet Genome Res 2004;107:55–67.
3. Li MM, Howard-Peebles PN, Killos LD, Fallon L, Listgarten E,
Stanley WS. Characterization and clinical implications of marker chro-
mosomes identified at prenatal diagnosis. Prenat Diagn 2000;20:138–43.
4. Eggermann K, Mau UA, Bujdoso G, Koltai E, Engels H, Schubert R,
et al. Supernumerary marker chromosomes derived from chromosome
15: analysis of 32 new cases. Clin Genet 2002;62:89–93.
5. Liehr T, Mrasek K, Weise A, Dufke A, Rodriguez L, Martinez
Guardia N, et al. Small supernumerary marker chromosomes—progress
towards a genotype-phenotype correlation. Cytogenet Genome Res
2006;112:23–34.
6. Kirchhoff M, Gerdes T, Rose H, Maahr J, Ottesen AM, Lundsteen C. De-
tection of chromosomal gains and losses in comparative genomic hybrid-
ization analysis based on standard reference intervals. Cytometry
with an additional ‘‘marker’’ chromosome. Hum Genet 1985;69:353–70.
43. Manenti E. Two extra inv dup(15) chromosomes and male infertility:
second case. Am J Med Genet 1992;42:402–3.
969.e17
CASE REPORT
High risk of temporary alteration of semen parametersafter recent acute febrile illnessMartin Sergerie, Ph.D., D.E.S.S.,a,b Roger Mieusset, M.D., Ph.D.,a,c Francoise Croute, Ph.D.,d
Myriam Daudin, M.D.,a,b and Louis Bujan, M.D.a,b
a Human Fertility Research Group, Universit�e Toulouse III Paul Sabatier (EA 3694), b CECOS Midi-Pyrenees, c Centre de
Sterilite Masculine, CHU Paule de Viguier, and d Laboratoire de Biologie Cellulaire et Pollution, Universit�e Toulouse III Paul
Sabatier, Facult�e de M�edecine Purpan, Toulouse, France
Objective: To report parameters in semen samples and sperm deoxyribonucleic acid integrity in a fertile volunteerpresenting a 2-day fever of 39�– 40�C.Design: Case report.Setting: University-affiliated teaching hospital.Intervention(s): None.Patient(s): Semen samples from a fertile volunteer of proven fertility were obtained and analyzed before the fe-brile illness episode and at days 15, 37, 58, 79, and >180 after the fever.Main Outcome Measure(s): Semen parameters (total sperm count, motility aþb, and vitality), sperm protamina-tion state, measured by sperm chromatin structure assay (SCSA) and apoptotic activities, measured by terminaluridine nick-end labeling (TUNEL) assay.Results: Total sperm count significantly decreased at days 15, 37, and 58 after the fever and returned to normal byday 79 after the fever. The percentage of motility significantly decreased at days 15 and 37 after the fever and re-turned to normal by day 58. Vitality score also showed a slight, although not statistically significant, decrease afterthe fever. The DNA fragmentation index (DFI, a SCSA parameter), which defines abnormal chromatin structure,significantly increased by 24% and 36% at days 15 and 37 after the fever, respectively, and decreased to 15% and8% when reaching days 58 and 79 after the fever. High DNA stainability (HDS, a SCSA parameter) also signifi-cantly increased at day 37 after the fever. On the other hand, sperm DNA fragmentation, as measured by TUNELassay, increased up to 23% by day 15 after the fever but this was not statistically significant.Conclusion(s): This report demonstrates that a febrile episode can have marked effects on semen parameters andsperm DNA integrity. These results are particularly important for the counseling of infertile couples and in relationto assisted reproductive techniques (ART). (Fertil Steril� 2007;88:970.e1–7. �2007 by American Society for Re-productive Medicine.)
Key Words: Sperm DNA integrity, SCSA, spermatogenesis, TUNEL, temperature
It is well known that testis exposure to mild or high temper-atures adversely affects testicular function, causing partial ortotal spermatogenic arrest (1). We have already demonstratedthat mild elevation (1�–2�C) in testicular temperature for 15hours/day, repeated daily in fertile men, leads to qualitativeand quantitative decline in spermatogenesis (2, 3).
Received June 13, 2006; revised December 21, 2006 and accepted
December 28, 2006.
Supported by a grant from the Centre d’Etude et de Conservation des
Oeufs et du Sperme Humain (CECOS), Toulouse, France. Martin Serg-
erie is supported by a postdoctoral fellowship from the Sidaction Asso-
ciation (no. A015-2). His arrival in France was facilitated by the
invaluable assistance of the Kastler Foundation.
Presented in part by Martin Sergerie at the 3rd International Congress on
Male-Mediated Developmental Toxicity, Bradford, United Kingdom,
July 31–August 3, 2005.
Reprint requests: Louis Bujan, M.D., Human Fertility Research Group,
CECOS Midi-Pyrenees, CHU Paule de Viguier, 330 Avenue de Grande
Bretagne, 31059 Toulouse Cedex 9, France (FAX: 33-(0)5-67-77-10-49;
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medi
970.e1
There are few reports on the impact of febrile episodes onsemen parameters (4, 5). Medical students who presenteda febrile illness (chickenpox and pneumonia) were followedby MacLeod (5) to study semen quality. Sperm count, motil-ity, and morphology parameters were decreased after thefebrile episode. Recovery of morphology and motility param-eters occurred 4 weeks after normalization of the tempera-ture. However, sperm count parameters only returned tonormal 8 weeks after normalization of the temperature.Buch and Havlovec (4) also found decreased sperm countin a case report of a semen donor who suffered an acute viralillness with fever (38.3�C for 24 hours). More interestingly,they demonstrated a variation in egg penetration ability atweeks 6 and 7 after the fever.
Few experimental research reports on the possible impactof testicular heating by ‘‘artificial fevers,’’ such as sauna orsteam baths, on human spermatogenesis or fertility have
0015-0282/07/$32.00cine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.045
been carried out in the past (6–8). However, their resultsclearly showed, between 2 and 4 weeks after exposure, a de-crease in total sperm count (>50%) of healthy volunteerswho had either one (6) or several sauna exposures (8), withcomplete recovery after 8 weeks.
Recently, Carlsen et al. (9) analyzed the effect of a historyof febrile illness on semen quality in 15 men who experiencedone or more episodes of fever. Sperm concentration, mor-phology, and motility were significantly affected by fever,particularly during the postmeiotic period of spermiogenesis.However, individual response in semen parameters to fevervaried considerably, with some men even presenting an in-crease in sperm count. This is probably related to absenceof measurement of body temperature and therefore inade-quate evaluation of febrile episodes. In fact, it is extremelydifficult to establish a causal relationship without objectivemeasurement of fever.
In a case report, Evenson et al. (10) studied human spermchromatin structure (SCSA) after an episode of influenza andhigh fever. They found that the effects of the influenza virusor a fever episode manifested as an increase of sperm DNAfragmentation index (DFI), an increase of high DNA stain-ability (HDS), a decreased number of free-SH groups, andan alteration in the nuclear protein composition of ejaculatedsperm. However, this study did not examine semen parame-ters and did not rule out the possible implication of apoptosisin the alteration of sperm DNA integrity.
The most commonly used techniques to assess sperm DNAintegrity are the SCSA and terminal uridine nick-end labeling(TUNEL) assays. Because SCSA can detect abnormalities inchromatin packaging (protamination state) and TUNEL as-say can detect single- and double-stranded DNA breaks (ap-optotic cells) in human sperm, it would be interesting to studythe effects of high fever on semen parameters and also onsperm DNA using these two biomarkers.
CASE REPORT
The present work describes semen parameters (total spermcount, motility aþb, and vitality) and sperm DNA integrity,as measured by SCSA and TUNEL assay, of a volunteer se-men donor presenting with a 2-day fever of 39�– 40�C.
Study Volunteer
Semen samples from a 47-year-old nonsmoker volunteer ofproven fertility who participated in our study programs (qual-ity control) and who developed, for 2 days, a high fever (rec-tal temperature 39�–40�C) due to influenza, were analyzedbefore the febrile illness episode and at days 15, 37, 58, 79,and >180 after the fever. During the febrile episode, no anti-biotherapy was taken. The volunteer gave his informed con-sent. Medical examination was normal except for fever. Theexclusion criteria for a healthy volunteer were occupationalexposure to heavy metals, any previous treatment affectingspermatogenesis (such as chemotherapy or radiotherapy),
Fertility and Sterility�
and the use of recreational drugs, including marijuana, co-caine, or narcotics. At each visit, the usual questionnairewas completed concerning such items as time since last ejac-ulation. In addition, for each sample, the volunteer was ques-tioned about any unusual events since his last visit to thelaboratory, such as disease episodes (other than fever), stress-ful conditions, intake of medications or dietary supplements,as well as any change in lifestyle habits.
The time lapse between ‘‘event and effect’’ was taken intoconsideration with respect to the timing of spermatogenesisand epididymal sperm maturation (11). Factors known tocontribute to variation in semen quantity and quality, suchas abstinence, collection in a laboratory, medium, and labora-tory technician, were minimized.
Sample Collection and Storage
Semen samples were collected by masturbation, after a rec-ommended period of 3–6 days of sexual abstinence, into ster-ile polypropylene containers at the Centre d’Etude et deConservation des Oeufs et du Sperme Humain (CECOS) lab-oratory. Conventional semen analysis was performed accord-ing to World Health Organization (WHO) criteria (12).Sperm count, total sperm count, motility aþb (rapidly pro-gressive: grade a, and slowly progressive: grade b), roundcells, and ratio of total round cells count/total sperm countwere assessed according to previously published methodsby a single technician (13). Sperm vitality was determinedin all samples by the eosin–nigrosin test (14).
From a clinical point of view, it is of particular importanceto distinguish between neutrophil leukocytes and cells ofspermatogenic origin when immature spermatozoa are pres-ent in sperm (15). To exclude the presence of neutrophil leu-kocytes (both eosinophilic and basophilic) in semen samples,we used the peroxidase stain procedure described by Politchet al. (16), with minor modifications. Briefly, 30% of hydro-gen peroxide (H2O2) was added to 2 mL of benzidine stocksolution. Thirty microliters of semen samples were mixedwith 30 mL of fresh benzidine–H2O2 solution. After 5 min-utes of incubation, 160 mL of phosphate-buffered saline(PBS) were added and peroxidase-positive cells (yellow tobrown stained round cells) and negative (unstained or pinkstained) were counted in a hemocytometer using a phase con-trast microscope. The number of peroxidase-positive cellsper mL of semen samples was calculated.
After sperm assessment, semen samples were cryopre-served within 1 hour of collection according to the standardprocedures used for sperm banking in our laboratory untillater pooled assessment of sperm DNA integrity, as previ-ously published (17).
One day before assessment of sperm DNA integrity, strawscontaining semen were removed from liquid nitrogen storageand thawed on ice. The stored sperm sample was suspendedin 4.7 mL of ice-cold TNE (0.01 M Tris-HCl, 0.15 M NaCl,and 1 mM EDTA, pH 7.4) and centrifuged at 600 � g for 10
minutes. The pellets (�10 � 106 spermatozoa) were then re-suspended in 1 mL of ice-cold TNE and fixed with 4 mL of1% formaldehyde (Prolabo, Paris, France) in TNE (pH 7.4)for at least 30 minutes at 4�C. After centrifugation, the pelletswere washed twice and each sample was divided into four al-iquots (2 for SCSA, 2 for TUNEL) of �2.5 million sperma-tozoa and then stored in ice-cold TNE with 0.1% sodiumazide (Sigma Chemical Co., St. Louis, MO) at 4�C.
Sperm Chromatin Structure Assay and Flow Cytometry
We used the SCSA procedure described by Evenson and Jostin 2000 (18), with minor modifications as previously pub-lished (17). Acridine orange (chromatographically purified;Polysciences, Warrington, PA) that intercalates into double-stranded DNA fluoresces green (515–530 nm ¼ nativeDNA), whereas acridine orange that associates with single-stranded DNA fluoresces red (>630 nm ¼ denatured DNA)after excitation by a 488-nm wavelength light source witha 15-mW argon laser. A total of 10,000 events were analyzedon a Beckman Coulter Epics XL-MCL flow cytometer (Coul-ter, Hialeah, FL) at a rate of �250 cells per second.
Sperm chromatin structure was analyzed in duplicate. Datafor each semen sample analyzed were saved, transferred toa personal computer, and analyzed by WinMDI 2.8 (PhoenixFlow Systems, San Diego, CA). Computer gating determinedthe DFI as being equal to red fluorescence divided by the sumof red and green fluorescence, and HDS as being sperm withhigh DNA stainability. One aliquot of quality control spermwas analyzed in pooled samples (results not shown). The an-alytical coefficient of variation was <5% as calculated fromvalues obtained from aliquots of a semen sample.
Measurement of DNA Fragmentation by TUNEL Assay
A detailed protocol for the TUNEL assay of human sperm haspreviously been described (19). Sperm DNA fragmentationand propidiun iodide (Sigma Chemical Co.) labeling weremeasured on a Beckman Coulter Epics XL-MCL flow cytom-eter (Coulter). Each analysis included a minimum of 10,000stained spermatozoa quantified simultaneously by green andred fluorescence. Light-scattering and fluorescence data wereobtained at a fixed gain setting in logarithmic mode. Greenfluorescence (from FITC) was detected in the FL1 sensorthrough a 550-nm dichromic long-pass filter and a 525-nmband-pass filter, whereas red fluorescence (from propidiumiodide) was collected in the FL3 sensor through a 645-nm di-chromic long-pass filter and a 620-nm band-pass filter. Debrisand leukocytes were gated out by establishing a regionaround the population of interest on the basis of propidiumiodide characteristics of the selected sperm. Sperm DNAfragmentation (TUNEL assay) was analyzed in duplicate.
The detailed protocol for TUNEL analysis has previouslybeen described (20). Briefly, TUNEL assay list mode fileswere converted to dotplot and histogram files with WinMDI2.8 (Phoenix Flow Systems) and analyzed by Multi2D soft-ware (Phoenix Flow Systems). TUNEL analysis consistedof subtracting control (no terminal desoxynucleotidyl trans-
970.e3 Sergerie et al. High risk of male infertility after fe
ferase [TdT] enzyme) green fluorescence histograms fromTdT-positive green fluorescence histograms, yielding the per-centage of cells showing DNA strand breaks. The number ofcells analyzed in both control and TdT-positive green fluores-cence histograms were equal for each sample pair.
Statistical Analyses
The analyses were conducted with Stata 6.0 software (StataCorporation, College Station, TX). Analysis of variancewas used to test for differences in time after onset of fever(before fever, after fever, and days 79 and >180 after the fe-ver). Semen parameters, TUNEL, and SCSA were tested forsignificant differences from control values. P values of %.05were considered statistically significant.
RESULTS
Total sperm count was significantly decreased at days 15, 37,and 58 after the fever and returned to normal values by day79after the fever (P<.001). Motility percentage was signifi-cantly decreased at days 15 and 37 after the fever and re-turned to normal values by day 58 after the fever (P<.05).Vitality slightly decreased after the fever, but this was not sta-tistically significant (Fig. 1A). Semen volume was quite sta-ble during the study period and no statistically significantdifference was observed (P¼.14).
The DFI (SCSA parameter), which defines abnormal chro-matin structure, significantly increased from 9% (before fe-ver) to 24% and 36% at days 15 and 37 after the fever,respectively (P<.05), and decreased to 15% and 8% whenreaching days 58 and 79 after the fever. The HDS also signif-icantly increased at day 37 after the fever (P<.05) (Fig. 1B).Sperm DNA fragmentation, as measured by TUNEL assay,increased from 17% (before fever) to 23% by day 15 afterthe fever, but this was not statistically significant comparedwith baseline values (Fig. 1B). By days 79 and >180 afterthe fever, semen parameters and also sperm DNA integrity re-turned to normal values (Fig. 1).
Based on the works of Bedford et al. (21) in which epididy-mal transport was estimated to be 12 days, the first postfeversample (day 15) represents epididymal spermatozoa and fewtesticular spermatozoa exposed to fever. The highest DFI value(36%) occurred at day 37 after the fever. This sample (day 37)represents spermatozoa produced by cells that were, during thefever episode, a few late primary spermatocytes and a largequantity of secondary spermatocytes and spermatids.
In samples from days 15 and 37, round cells were signifi-cantly more numerous and the total round cells count/totalsperm count ratio was significantly higher than before feverand than at days 58, 79, and >180 after fever (P<.001)(Table 1). This means that germinal cells are released in ejac-ulates without completion of testicular sperm division anddifferentiation. Propidium iodide stain with flow cytometry(Fig. 2) confirmed that these semen samples (days 15 and37) contained more round cells with highly intense red fluo-rescence (related to DNA quantity). Furthermore, histogramsat day 37 after the fever showed three important shape
brile episode Vol. 88, No. 4, October 2007
FIGURE 1
Impact of 2 days of fever on sperm parameters and DNA in relation to spermatogenesis and epididymal transit(E.T.). (A) Effect of fever on semen parameters, and (B) impact of fever on sperm DNA integrity as measured bysperm chromatin structure assay (SCSA) and terminal uridine nick-end labeling (TUNEL) assay. DFI ¼ DNAfragmentation index; HDS ¼ high DNA stainability. For (A) total sperm count and motility and (B) SCSAparameters, * ¼ P values indicate statistically significant differences compared with before fever(P< .05). The different stages of spermatogenesis are adapted from May et al., 2000 (11).
0
10
20
30
40
50
60
70
80
90
100
Before fever Before fever D15 post
fever
D37 post
fever
D58 post
fever
D79 post
fever
D>180 post
fever
0
50
100
150
200
250
300
350
400
450
500
Motility Vitality Total sperm count
Mo
tility
a
nd
V
ita
lity
(%
)
To
tal sp
erm
co
un
t (x10
6)
*
**
**
FE
VE
R (2
d
ay
s)
0
5
10
15
20
25
30
35
40
Before fever Before fever D15 post
fever
D37 post
fever
D58 post
fever
D79 post
fever
>D180 post
fever
SCSA-DFI SCSA-HDS TUNEL
Percen
tag
e(%
)
*
*
*
FE
VE
R (2
d
ay
s)
No DNA repair DNA repair until prophase of spermatocytes I
Stem
cells
Sp
erm
ato
go
nia
Sp
erm
ato
cytes II
Sp
erm
ato
cytes I
Sp
erm
atid
s
Sp
erm
ato
zo
a
86583534120Days to reach
ejaculate
Spermiogenesis
Meiosis
Mitosis
E.T.
A)
B)
Sergerie. High risk of male infertility after febrile episode. Fertil Steril 2007.
Fertility and Sterility� 970.e4
TABLE 1Semen parameters in a healthy fertile semen donor before and after an acute 2-day febrile episode.
D58 post fever 6.9 21.4 147.7 0.2 1.4 0.9D79 post fever 7.3 70.0 511.0 0.2 1.5 0.3>D180 post fever 6.5 48.5 315.3 0.2 1.3 0.4
TRC: total round cell count.TSC: total sperm count.* Leukocyte exclusion by peroxidase stain.a p<0.001 compared with before fever and D58, D79 and >D180 post fever.
Sergerie. High risk of male infertility after febrile episode. Fertil Steril 2007.
characteristics (haploid, diploid, and probably tetraploid)compared with data before fever and day 58 after fever. Itis also interesting to note the perfect compatibility of histo-grams before fever and day 58 after the fever, suggestingthat the effect of fever is completely reversible.
DISCUSSION
Influenza is one of the most common febrile illnesses, with asmany as one in five adults of reproductive age affected each
year (22). Because high fever is one of the principal symp-toms, the prevalence of influenza is an important factor to con-sider in particular in sperm assessment and also in relation toassisted reproductive techniques (ART). We examined the im-pact of an episode of 2 days of fever in a 47-year-old fertileman. The results demonstrated that a febrile episode of 39�–40�C markedly decreases conventional semen parametersand results in a fourfold increase in alteration of spermDNA integrity. To the best of our knowledge, this is the first
FIGURE 2
Histogram showing intensity of fluorescence after propidium iodide staining of spermatozoa and roundcells in semen samples. D ¼ day.
Sergerie. High risk of male infertility after febrile episode. Fertil Steril 2007.
970.e5 Sergerie et al. High risk of male infertility after febrile episode Vol. 88, No. 4, October 2007
970.e6
report that presents the impact of an episode of fever (objec-tively measured) and its ‘‘event and effect’’ relation on semenparameters and sperm DNA integrity, as measured by SCSAand TUNEL.
In the literature, a considerable body of work addresses theimpact of an increase in testis temperature on semen quality.These studies were mainly carried out on animal models (23)or assessed the results of occupational heat exposure (24).However, it is difficult to measure the relevance of many ofthese studies, as in most research on occupational heat expo-sure, body temperature was increased by about 1�C in relationto the high ambient temperature, but scrotal or testis temper-atures were not measured. On the other hand, in studies of an-imal models with local heating, testicular temperature wasincreased to supraphysiological levels. Nevertheless, all thedata presented in animals and humans clearly show that ele-vated or moderate testicular/scrotal heating markedly affectsspermatogenesis and fertility (25). They even suggested sig-nificant effects on spermatogenesis, with certain phases beingmore susceptible than others (26). In the present report, ourdata show that total sperm count and motility were signifi-cantly affected by fever occurring during meiosis (primaryand secondary spermatocytes) and the postmeiotic period(spermatids and epididymal transit of spermatozoa).
Because of the unique change of the DNA material withineach germ cell nucleus during the process of spermatogene-sis, DNA assessment with propidium iodide by flow cytome-try is a very suitable assay to estimate the number of cellswithin each stage of spermatogenesis (27). In Figure 2, pro-pidium iodide staining confirmed that semen samples atdays 15 and 37 after the fever contained more cells withhighly intense fluorescence (diploid and probably tetraploid)compared with findings before fever and day 58 after fever. Infact, those that are haploid correspond to spermatozoa andspermatids. The diploid peak could contain germ cells suchas spermatogonia in G1 phase, preleptotene primary sper-matocytes, and secondary spermatocytes. The tetraploidpeak is probably composed of spermatogonia in G2, lepto-tene, and preleptotene primary spermatocytes.
Our results concerning sperm chromatin packaging(SCSA) parameters showed significant four- and threefold in-creases in DFI and HDS, respectively, before a return to base-line values at days 58 and 79 after the fever. One case studyhas shown that fever temporarily decreased sperm DNA in-tegrity (10). The investigators showed that a febrile episodewas associated with an increase of DFI and HDS, a decreasednumber of free-SH groups, and an alteration in the nuclearprotein composition of ejaculated sperm. However, this studydid not rule out the possible implication of apoptosis in thealteration of sperm DNA integrity.
The TUNEL assay is a useful and sensitive method for as-sessing DNA strand breaks, as seen in apoptosis in humansperm (20, 28, 29). In the present study, TUNEL data slightlyincreased up to 23% by day 15 after the fever, but this was not
Fertility and Sterility�
statistically significant compared with baseline values. Thisobservation supposes that fever was not associated with a tes-tis temperature high enough to initiate germ cell apoptosis, asseen in an animal model at 42�– 43�C (30). It becomes obvi-ous that compensation mechanisms in the thermodependenceof spermatogenesis (3) had supported the ‘‘time effect’’ of thefever exposure (2 days fever at 39�– 40�C).
One of the best characterized responses by cell tissues toenvironmental stresses such as elevated temperature expo-sure is heat shock protein (Hsp) overexpression (31). Severalcellular antiapoptotic proteins have been described such asHsp 70 (32) and Hsp 27 (33). Hsp 70 interacts with BAG-1and inhibits apoptosis by preventing recruitment of procas-pases 9 to the Apaf-1 apoptosome complex (34). In thecase of Hsp 27, overexpression of this protein may reducein vivo apoptosis of tumor cells (35). In this study, we decidedto test whether the enhanced fluorescent labeling, as shownby TUNEL assay and induced by fever, truly representeda slight increase of DNA strand breaks (apoptosis activity)followed by a decrease of DNA fragmentation mediated byan overexpression of Hsp. For this purpose, the Hsp-27 andHsp-70 family was analyzed by Western blot. We did not ob-serve any increase of Hsp-27 (monomeric and nonphos-phorylated), or any increase or decrease in the activity ofHsp-70 after fever compared with baseline values (data notshown). Unfortunately, we could not further investigate theexpression of the Hsp family, as no more day 37 post feverstraws were available.
The exact role of the constitutive or inducible Hsp expres-sion during spermatogenesis in humans is not well established(31). In addition, because standard cryoprotectants (freezingmedium) do not contain a proteolytic enzyme inhibitor suchas phenyl-methyl-sulfonyl-fluoride (PMSF), it might be pos-sible that thawed straws were associated with deterioration ofHsp (36). In fact, it would be valuable to further investigatethe functions of the Hsp gene family in the spermatogeniccell cycle and cell death, particularly in human sperm.
The level of sperm DNA integrity reflects the quality of ge-netic material of the gamete. In addition, transmission of dam-aged DNA to the offspring, particularly at levels that exceedthe DNA repair capacity of the oocytes, could have seriousconsequences (37). Research has demonstrated that spermchromatin defects may influence the initiation and regulationof paternal gene activity during late stages of human embryodevelopment (38, 39). Mieusset et al. (40) studied the fertil-ization rate and embryonic mortality in normal female sheepinseminated with semen from rams submitted to moderate tes-ticular heating. Results had showed that increasing scrotaltemperature by z2�C for 16 hours a day induced an increasein embryonic loss without any modification in the fertilizationrate starting after 4 consecutive days of scrotal heating. Thesedata have been supported by other in vitro models (41, 42).
This report demonstrates that a febrile episode can havemarked effects on semen parameters and sperm DNA integ-rity. The results of this study are particularly important for
the counseling of infertile couples and whether ART tech-niques are used. Couples of reproductive age should be awarethat if a febrile episode occurs (an objectively documentedtemperature of R39�C for at least 1 day), natural conceptionmust be postponed until at least after one spermatogenic cy-cle. If ART programs are used, febrile episodes in the manmust be recorded before and during ovarian stimulationprocedures, as if such an episode occurs, the ART programshould be postponed for 3 months because of the high riskof infertility and the possible consequences on the offspring.
Acknowledgments: We thank Prof. Pierre Si�e and Dr. V�eronique Mansat-De
Mas for access to flow cytometry in the hematology laboratory, Dr. Nathalie
Moinard for histogram analysis, Francoise Cendres of the spermiology lab-
oratory for clinical semen analyses, Bourras Bengoudifa of the Human Fer-
tility Research Group, Universit�e Paul Sabatier, Toulouse, for his
collaboration in the statistical analysis, and Marie-Julie Garneau and Nina
Crowte for editing the text.
REFERENCES1. Waites GMH, Setchell BP. Physiology of the mammalian testis. In: Lam-
ming CE, ed. Marshall’s physiology of reproduction. Vol. 2. London:
Churchill Livingston; 1990:1–105.
2. Mieusset R, Grandjean H, Mansat A, Pontonnier F. Inhibiting effect of ar-
tificial cryptorchidism on spermatogenesis. Fertil Steril 1985;43:589–94.
3. Mieusset R, Bujan L. The potential of mild testicular heating as a safe,
effective and reversible contraceptive method for men. Int J Androl
1994;17:186–91.
4. Buch JP, Havlovec SK. Variation in sperm penetration assay related to
viral illness. Fertil Steril 1991;55:844–6.
5. MacLeod J. Effect of chickenpox and of pneumonia on semen quality.
Fertil Steril 1951;2:523–33.
6. Brown-Woodman PD, Post EJ, Gass GC, White IG. The effect of a single
sauna exposure on spermatozoa. Arch Androl 1984;12:9–15.
7. MacLeod J, Hotchkiss R. The effect of hyperpyrexia upon spermatozoa
counts in men. Endocrinology 1941;28:780–4.
8. Procope BJ. Effect of repeated increase of body temperature on human
sperm cells. Int J Fertil 1965;10:333–9.
9. Carlsen E, Andersson AM, Petersen JH, Skakkebaek NE. History of fe-
brile illness and variation in semen quality. Hum Reprod 2003;18:
2089–92.
10. Evenson DP, Jost LK, Corzett M, Balhorn R. Characteristics of human
sperm chromatin structure following an episode of influenza and high fe-
ver: a case study. J Androl 2000;21:739–46.
11. May CA, Tamaki K, Neumann R, Wilson G, Zagars G, Pollack A, et al.
Minisatellite mutation frequency in human sperm following radiother-
apy. Mutat Res 2000;453:67–75.
12. World Health Organization. WHO laboratory manual for the examina-
tion of human semen and sperm–cervical mucus interaction. 4th ed.
Cambridge: Cambridge University Press, 1999.
13. Bujan L, Mieusset R, Mansat A, Moatti JP, Mondinat C, Pontonnier F.
Testicular size in infertile men: relationship to semen characteristics
and hormonal blood levels. Br J Urol 1989;64:632–7.
14. Eliasson R, Treichl L. Supravital staining of human spermatozoa. Fertil
Steril 1971;22:134–7.
15. Johanisson E, Campana A, Luthi R, de Agostini A. Evaluation of ‘‘round
cells’’ in semen analysis: a comparative study. Hum Reprod Update
2000;6:404–12.
16. Politch JA, Wolff H, Hill JA, Anderson DJ. Comparison of methods to
enumerate white blood cells in semen. Fertil Steril 1993;60:372–5.
17. Sergerie M, Mieusset R, Daudin M, Thonneau P, Bujan L. Ten-year var-
iation in semen parameters and sperm deoxyribonucleic acid integrity in
a healthy fertile man. Fertil Steril 2006;86:1513.e11–8.
18. Evenson D, Jost L. Sperm chromatin structure assay is useful for fertility
assessment. Methods Cell Sci 2000;22:169–89.
970.e7 Sergerie et al. High risk of male infertility after fe
19. Sergerie M, Ouhilal S, Bissonnette F, Brodeur J, Bleau G. Lack of asso-
ciation between smoking and DNA fragmentation in the spermatozoa of
normal men. Hum Reprod 2000;15:1314–21.
20. Sergerie M, Laforest G, Bujan L, Bissonnette F, Bleau G. Sperm DNA frag-
mentation: threshold value in male fertility. Hum Reprod 2005;20:3446–51.
21. Bedford JM, Calvin H, Cooper GW. The maturation of spermatozoa in
the human epididymis. J Reprod Fertil Suppl 1973;18:199–213.
22. Thompson WW, Shay DK, Weintraub E, Brammer L, Cox N,
Anderson LJ, et al. Mortality associated with influenza and respiratory
syncytial virus in the United States. JAMA 2003;289:179–86.
23. Setchell BP. The Parkes Lecture. Heat and the testis. J Reprod Fertil
1998;114:179–94.
24. Kumar S. Occupational exposure associated with reproductive dysfunc-
tion. J Occup Health 2004;46:1–19.
25. Mieusset R, Bujan L. Testicular heating and its possible contributions to
male infertility: a review. Int J Androl 1995;18:169–84.
26. Lue YH, Hikim AP, Swerdloff RS, Im P, Taing KS, Bui T, et al. Single
exposure to heat induces stage-specific germ cell apoptosis in rats: role
of intratesticular testosterone on stage specificity. Endocrinology
bryo growth in normal female mice made pregnant by males whose testes
had been heated. Hum Reprod 1998;13:342–7.
42. Zhu B, Walker SK, Oakey H, Setchell BP, Maddocks S. Effect of paternal
heat stress on the development in vitro of preimplantation embryos in the
mouse. Andrologia 2004;36:384–94.
brile episode Vol. 88, No. 4, October 2007
CORRESPONDENCE
0d
Severity of energy-related menstrual disturbancesincreases in proportion to indices of energyconservation in exercising women
Alterations in resting energy expenditure and metabolic hormones (energy conservation) are evident inincreasing magnitude across a continuum of increasing severity of clinical menstrual disturbances, includingluteal-phase defects, anovulation, and amenorrhea in exercising women. These data provide further evidenceof the tight association between energy balance and reproduction and suggest that subtle declines in energyavailability can produce clinically recognized menstrual disturbances. (Fertil Steril� 2007;88:971–5. �2007by American Society for Reproductive Medicine.)
Alterations in nutritional status and energy metabolism canhave a profound modulatory effect on the reproductive axisand fertility (1). In humans, the suppression of reproductivefunction with chronic energy deficiency is more stronglypredicted by the magnitude of a daily energy deficit ratherthan by loss in body mass (2). These findings are consistentwith the hypothesis that acute fuel availability, not chronicenergy stores, is a key modulator of reproductive function(1). Sustained reductions in resting energy expenditure(REE), presumably in an attempt to conserve energy, havebeen observed in exercising women with amenorrhea com-pared with their normally cycling counterparts (3). Thesechanges have been observed in concert with other endocrineadaptations that reflect an energetically challenged condi-tion, including decreased concentrations of triiodothyronine(TT3), leptin, insulin, insulin-like growth factor-1 (4–7),and increased counterregulatory hormones such as growthhormone, ghrelin, and cortisol (4, 8, 9).
Although energy-related menstrual disturbances in ath-letes and physically active women have been described asexisting along a continuum, ranging from subtle perturba-tions like luteal phase defects (LPDs) and anovulatorycycles, to the more severe perturbations, oligomenorrheaand amenorrhea (10), the majority of metabolic and energystudies has been limited to amenorrheic athletes. To thisend, the purpose of this study was to determine whethermenstrual disturbances along a continuum of severity areassociated with a concomitant range of severity in indicesof energy conservation, defined as decreased REE per kilo-
Received July 1, 2006; revised and accepted November 30, 2006.
Supported in part by the Arthur Thornton Cardiopulmonary Fund of New
Britain General Hospital, New Britain, Connecticut.
Reprint requests: Mary Jane De Souza, Ph.D., Women’s Exercise and
Bone Health Laboratory, Graduate Department of Exercise Science,
University of Toronto, 55 Harbord Street, Toronto, Ontario, Canada
015-0282/07/$32.00oi:10.1016/j.fertnstert.2006.11.171 Copyright ª2007 American S
gram of fat-free mass (FFM) and alterations in metabolichormones consistent with adaptation to energy deficiency.We hypothesized that because menstrual disturbances existon a continuum, indices of energy conservation would beobservable even when changes in menstrual cyclicity aresubtle and that these indications of energy conservationshould increase in magnitude as the severity of the men-strual disturbance increases. If our hypothesis is correct,this finding would underscore the importance of adequateenergy for fertility and the maintenance of endocrinehomeostasis in exercising women.
Forty-nine volunteers participated in the study and wereretrospectively grouped according to exercise status andby menstrual status. Menstruating women were monitoredfor two to three menstrual cycles, and amenorrheic women,for two to three 30-day monitoring periods. All data pre-sented in this study represent the mean of the two to threerepeated time periods monitored. The study was approvedby the Ethics Review Board at the University of Toronto,and all volunteers signed an approved informed consentdocument.
Exercise status was defined as sedentary when purposefulexercise was<2 hours per week and as exercising when pur-poseful exercise was>2 hours per week (11, 12). Menstrualstatus was determined from daily first–morning void urinesamples assayed for LH, pregnanediol 3-glucuronide, andestrone 3-glucuronide (13). Menstrual categories from thesedaily urinary measures included ovulatory, LPD, anovula-tory, and amenorrheic. After consideration of exercise status(sedentary or exercising) and menstrual status, volunteerswere grouped as follows: [1] sedentary ovulatory group(SedOv, n ¼ 11) with consistently ovulatory cycles for theduration of the 2- to 3-month study period; [2] exercisingovulatory group (ExOv, n ¼ 9) with consistently ovulatorycycles for the duration of the study period; [3] exercisingwith inconsistent presentations of subtle menstrual distur-bances group (ExIncon, n ¼ 8), including various
Fertility and Sterility� Vol. 88, No. 4, October 2007 971ociety for Reproductive Medicine, Published by Elsevier Inc.
inconsistent combinations of ovulatory, LPD, and anovula-tory cycles from cycle to cycle for the duration of the studyperiod; [4] exercising anovulatory group (ExAnov, n ¼ 7),with consistently anovulatory cycles for the duration of thestudy period; and [5] exercising amenorrheic group(ExAmen, n ¼ 13) with no menses for the duration of thestudy period and for at least 3 months before study.
Menstrual cycle length, follicular and luteal phase length,ovulatory status, and the presences or absence of LPD wasdefined as reported elsewhere (12). Amenorrhea was de-fined as no menses for R100 days, confirmed by estrone3-glucuronide and pregnanediol 3-glucuronide profiles.
Total body mass, height, body mass index, and bodycomposition (Prodigy, GE Lunar, enCORE 2002 software,version 6.50.069) were measured. Peak oxygen uptake(VO2 peak) was measured once, as defined elsewhere (14).Resting energy expenditure was measured two to three timesby indirect calorimetry by using methods reported elsewhereusing the Weir equation (14, 15), and the average is reported(corrected for FFM).
Microtiter plate competitive enzyme immunoassays wereused to measure estrone 3-glucuronide and pregnanediol3-glucuronide by using polyclonal antibodies supplied byanother investigator (16, 17). Urine samples were correctedfor specific gravity (18). Urinary LH was determined byRIA (Diagnostics Products Corporation, Los Angeles, CA).Eight-hour–fasted blood samples were collected between7:30 AM and 10:00 AM for hormone assays two to three timesduring the study period, and we report the average of theserepeated measurements. Triiodothyronine (DPC), and lep-tin (Linco Research, St. Charles, MO) were measured byusing an immunoassay, and total ghrelin was measured byusing an RIA (Linco Research).
All data sets were tested for nonnormality, homogeneityof variance, and outliers before statistical hypothesis testswere performed. Outliers detected were rejected. Data wereexpressed as mean� SEM. Data for all variables were com-pared among the groups by using analysis of variance.When main effects were found, post hoc tests (least signif-icant difference) were used to detect where differences oc-curred. Forward stepwise regression was used to determinepredictors of REE. The criteria for entry into and removalfrom the model were P< .05 and P< .10, respectively.The number of volunteers included in this study was suffi-cient to detect significant differences caused by our group-ing variable (exercise and menstrual status) at a power levelof 0.80 by using a P level of .05. Significance level of < .05was chosen to identify all significant differences and wasadjusted for multiple comparisons. All data were analyzedby using SPSS for Windows (version 12.0; Chicago, IL).
Age (24.8 � 1.4 y) height (165.1 � 1.5 cm), body mass(58.0 � 1.9 kg), body mass index (21.3 � 0.8 kg/m2), andgynecological age (12.1 � 1.1 y) were not significantly dif-ferent among the groups. All exercising groups had a lower
972 De Souza et al. Correspondence
percentage of body fat (F ¼ 4.40, P¼ .005) than did theSedOv group (22.5 � 1.9 vs. 30.5 � 2.1%). Total exercisevolume (336.0 � 59.3 vs. 101.9 � 23.8 min/wk, F ¼ 4.091,P¼ .008) and VO2 peak (45.4 � 1.8 vs. 39.2 � 1.3 mL/kgper minute, F ¼ 4.918, P¼ .003) were higher in all exercis-ing groups compared with the SedOv group.
Composite graphs of the study group’s cycles are pre-sented in Figure 1. Average menstrual cycle length (28.9� 1.1 d), follicular phase length (17.1 � 1.7 d), and lutealphase length (12.2 � 0.6 d) were not significantly differentamong the menstruating sedentary and exercising groups.The average duration of amenorrhea before the study inthe ExAmen subjects was 215.8 � 40.1 days.
Resting energy expenditure and metabolic hormone dataare presented in Figure 1. All groups with menstrual distur-bances, including the ExAmen, ExAnov, and ExIncongroups, had a significantly lower REE/FFM comparedwith the SedOv group, and the ExAmen had a significantlylower REE/FFM than the ExOv group. Stepwise forwardregression was used to predict REE/FFM. Variables in themodel predicted 45.3% (R2¼ 0.453) of the variance inREE/FFM (F¼ 17.4; P< .0001). Menstrual status categorywas the most powerful predictor, accounting for 32.5% ofthe variance (R2 ¼ 0.325), whereas leptin accounted foran additional 12.8% of the variance at step 2 (R2 change¼ 0.128).
Serum TT3 levels were lower (F¼ 4.830, P¼ .003) in allgroups with menstrual disturbances, including the ExAmen,ExAnov, and ExIncon groups, compared with the SedOvgroup. Leptin levels were lower (F ¼ 3.57, P¼ .014) in allexercising groups compared with the SedOv group. Therewas a trend toward higher ghrelin levels in the ExAmengroup compared with the other groups (F ¼ 2.51,P¼ .092). The REE/FFM was correlated with TT3 (r ¼0.474, P< .001), ghrelin (r ¼ �0.410, P¼ .002), and leptin(r ¼ 0.533, P< .001).
This study examined indices of energy status, includingREE and metabolic hormones, TT3, ghrelin, and leptin,across the continuum of energy-related menstrual distur-bances in physically active women, and it included both rec-reational athletes and competitive athletes. We found that
alterations in REE and metabolic hormones were consistentwith adaptations to a chronic energy deficiency and thatthese alterations were distributed across the continuum ofclinical menstrual disturbances in accordance with the mag-nitude of severity of the menstrual dysfunction observed.This study illustrates the sensitivity of this association andprovides support for the existence of a dose–response rela-tionship between laboratory measures of energy status, i.e.,REE, TT3, leptin and ghrelin, and clinical categories ofmenstrual dysfunction, i.e., LPD, anovulation, and amenor-rhea. These data suggest that even subtle changes in energyavailability may be translated to the reproductive axis, i.e.,that energy deficiency is associated with delays in follicular
Vol. 88, No. 4, October 2007
FIGURE 1
(A) Bar graph of REE per kilogram of FFM (kcal/d of REE per kg of FFM), TT3 (nmol/L), ghrelin (pg/mL), and leptin(mg/L) in the sedentary and exercising women grouped by menstrual status. Values are mean � SEM. Significantdifferences are denoted as follows: *ExAmen, ExAnov, ExIncon vs. SedOv; yExAmen vs. ExOv; xExAmen vs.SedOv, ExOv, ExIncon, ExAnov; and �ExAmen, ExAnov, ExIncon, ExOv vs. SedOv. (B) Composite graph ofmenstrual status depicted by daily estrone 3-glucuronide (E1G) and pregnanediol 3-glucuronide (PdG)concentrations in the sedentary and exercising women grouped by menstrual status. The E1G and PdG data forSedOv, ExOv, and ExIncon groups are aligned by the day of the LH peak, defined as day 0. The anovulatory(ExAnov) and amenorrheic (ExAmen) subjects’ E1G and PdG data are aligned by chronological day of daily urinaryhormone collections. The number of days depicted for the amenorrheic subjects is the mean cycle length of themenstruating subjects. Values are mean � SEM. SedOv ¼ sedentary ovulatory; ExOv ¼ exercising ovulatory;ExIncon ¼ exercising ovulatory, luteal phase defect and anovulatory inconsistent cycles; ExAnov ¼ exercisinganovulatory; ExAmen ¼ exercising amenorrheic.
De Souza. Menstrual cycle, exercise, and energy conservation. Fertil Steril 2007.
Fertility and Sterility� 973
maturation and compromised luteal function, and as themagnitude of the energy deficiency increases, the failureto ovulate and sustain reproductive quiescence is observed.It is important to note that our findings are also more gener-alizable to the broader category of physically active womennot involved in competitive sport, because our sampleincluded 47% recreationally active women.
A reduction in REE is characteristic of energy conserva-tion in the face of a chronic energy deficiency in exercisingwomen who are not ingesting enough calories to support to-tal energy expenditure, an adaptation that is seminal to theenergy conservation hypothesis (10, 19). A sustained reduc-tion in serum TT3 and REE/FFM was observed in our data,not only in the exercising women with amenorrhea as ob-served elsewhere (20), but also in the exercising womenwith mild to moderate menstrual disturbances, i.e., LPDand anovulatory menstrual cycles. In our work publishedelsewhere (20) in a different group of exercising womenwith LPD, we also reported lower concentrations of TT3.One other study has reported a reduced REE/FFM in exer-cising women with either LPD or anovulatory cycles (21),but menstrual disturbances were categorized from salivarysamples of P that were collected every other day for onlyone menstrual cycle. In this study, we have very preciselycharacterized menstrual function by the quantificationof daily urinary excretion of the ovarian steroids estrone3-glucuronide and pregnanediol 3-glucuronide for at leasttwo menstrual cycles, and we have carefully discriminatedovulatory cycles from LPD and anovulatory cycles. Thispoint is important because clinical sequelae such as infertil-ity and bone loss become exacerbated in the face of an en-ergy deficit and increasingly severe estrogen deficiency (10).Whereas TT3 and REE/FFM decreased in magnitude withthe severity of the menstrual cycle defect, average ghrelinconcentrations increased with the severity of the defectsbut were significantly greater (P< .10) only in those womenwith amenorrhea compared with the women with ovulatory,LPD, and anovulatory menstrual cycles, as reported in ourstudy published elsewhere (4).
The findings from this study advance our understandingof energy deficiency and menstrual cycle disturbances inexercising women. The primary findings of this study dem-onstrate that [1] alterations in REE and metabolic hormoneswere distributed across the continuum of clinical menstrualdisturbances in accordance with the severity of the men-strual dysfunction observed, providing evidence that en-ergy-related menstrual disturbances increase in severityproportional to the magnitude of increase in energy conser-vation; [2] a reduction in serum TT3 concentration likelypromotes the energy conservation through the lowering ofREE, an effect that is coupled with a paradoxical elevationin total ghrelin; [3] this relationship was not exclusive to themost severe menstrual disturbance, amenorrhea, and thefact that it also was observed in the exercising womenwith consistent subtle and less severe menstrual dysfunction
974 De Souza et al. Correspondence
such as anovulation provides key evidence that a progressivemetabolic adjustment exists before the onset of amenor-rhea; and [4] this study illustrates the sensitivity of the asso-ciation between metabolic status and reproductive functionand provides support for the existence of a dose–responserelationship between laboratory measures of energy status,such as REE and metabolic hormones, and clinical cate-gories of menstrual dysfunction, that is, LPD and anovula-tion, suggesting that even subtle changes in energyavailability may be translated to the reproductive axis andassociated with delays in follicular maturation and compro-mised luteal function.
The observed shifts in metabolic hormones and REE ob-served in this study are not exclusive to high-performanceathletes; women engaged in physical activity for recrea-tional (or occupational) purposes are impacted also. Inde-pendent of the level of activity that exercising womenmay choose, it is ultimately the combination of inadequatenutritional intake relative to energy expenditure that initi-ates this metabolic cascade.
Acknowledgments: Polyclonal antibodies for microtiter plate competitive
enzyme immunoassays that were used to measure estrone 3-glucuronide
and pregnanediol 3-glucuronide were supplied by Coralie Munro, Ph.D.,
University of California, Davis, CA. The authors thank Tanya Burke,
Hon. B.Sc., Rayisa Hontscharuk, M.Sc., and Emma O’Donnell, M.Sc.,
for their important contributions to this research. The authors also appreci-
ate the extraordinary cooperation of the study volunteers.
Mary Jane De Souza, Ph.D.a
Daniel K. Lee, M.Sc.a
Jaci L. VanHeest, Ph.D.b
Jennifer L. Scheid, B.Sc.a
Sarah L. West, B.P.H.E.a
Nancy I. Williams, Sc.D.ca Women’s Exercise and Bone Health Laboratory,
Graduate Department of Exercise Science, Universityof Toronto, Toronto, Ontario, Canada; b Department ofKinesiology, University of Connecticut, Storrs,Connecticut; and c Exercise Endocrinology andMetabolism Laboratory, Noll Laboratory, Departmentof Kinesiology, Penn State University, University Park,Pennsylvania
REFERENCES1. Wade GN, Jones JE. Neuroendocrinology of nutritional infertility. Am
19. Chan JL, Mantzoros CS. Role of leptin in energy-deprivation states:
normal human physiology and clinical implications for hypothalamic
amenorrhoea and anorexia nervosa. Lancet 2005;366:74–85.
20. De Souza MJ, Van Heest J, Demers LM, Lasley BL. Luteal phase de-
ficiency in recreational runners: evidence for a hypometabolic state.
J Clin Endocrinol Metab 2003;88:337–46.
21. Lebenstedt M, Platte P, Pirke KM. Reduced resting metabolic rate in
athletes with menstrual disorders. Med Sci Sports Exerc 1999;31:
1250–6.
975
Pharmacokinetics of a single oral dose of 1.5-mglevonorgestrel when administered as 750-mg tabletsor as 30-mg minipills
We examined the pharmacokinetics of a single dose of 1.5 mg of levonorgestrel when administered orally in twodifferent formulations: two tablets of 0.75 mg or 50 minipills of 30 mg of levonorgestrel. Bioavailability of lev-onorgestrel with minipills was comparable to that with levonorgestrel tablets. These findings suggest that lev-onorgestrel-containing minipills can be considered as an alternative to standard levonorgestrel tablets for usein emergency contraception. (Fertil Steril� 2007;88:976–7. �2007 by American Society for ReproductiveMedicine.)
Levonorgestrel (D-norgestrel) is a progestin that has beenused for several decades in different contraceptive methods,such as regular contraceptive pills, ethynil-estradiol, mini-pills, implants, and intrauterine devices and more recentlyin emergency contraception (EC) pills.
When used for EC in a single dose of 1.5 mg, it is recom-mended that levonorgestrel be taken as soon as possiblewithin 72 hours after intercourse (1). Studies have shownthat ECs are more effective the sooner they are taken afterintercourse (2). Therefore, if women must wait for a clinicor a physician’s appointment to get access to the medica-tion, the efficacy of the treatment to prevent the unwantedpregnancy is compromised.
In many countries, access to EC has been improved byprovision of levonorgestrel tablets from the pharmacist ortablets that can be found on the pharmacy shelf (3, 4). Cer-tainly, the price of dedicated tablets can be an obstacle towomen in some countries because of their high cost. How-ever, progestin-only contraceptive pills containing 30 mg oflevonorgestrel are widely available at low cost in most phar-macies, even in rural areas.
We recently studied the pharmacokinetics of levonorges-trel after administration of a single 1.5-mg dose (two 0.75mg tablets) by oral and vaginal routes (5). Using the sameclinical and laboratory methodology, we extended the phar-macokinetic study of levonorgestrel administered as twotablets of 0.75 mg of levonorgestrel each (group A), the‘‘standard treatment,’’ or as 50 minipills containing 30 mgof levonorgestrel each (group B).
Received July 11, 2006; revised and accepted December 27, 2006.
Supported by UNDP/UNFPA/WHO/World Bank Special Programme of
Research, Development and Research Training in Human Reproduc-
tion, World Health Organization, Geneva (project A25167); and the
Chilean Research Council, CONICYT-FONDAP, Santiago, Chile (pro-
ject 15010006).
Reprint requests: Luigi Devoto, M.D., Instituto de Investigaciones Ma-
terno Infantil, Facultad de Medicina, Universidad de Chile, P.O. Box
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
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The subjects allocated into group B (n ¼ 6) were 33–38years old, with a body mass index ranging from 23 to 30 kg/m2. They had regular menstrual cycles and had undergonetubal ligation >6 months before participating in the studyto avoid the risk of pregnancy. None of them had receivedany hormone treatment for the last 3 months. The partici-pants of this study were healthy Caucasians and displayeda similar clinical and endocrine profile to those reportedelsewhere (5).
The study design encompassed vaginal ultrasound assess-ment of follicular development and plasma E2 determina-tion. When the leading follicle achieved a diameter of >16mm and after urinary LH was detected (Clearplant; Unipath,Bedford, UK), two tablets of levonorgestrel (0.75 mg each;Postinor II; Gedon Ritcher, Budapest, Hungary) wereadministered as a single dose orally (group A, n ¼ 13). Ingroup B, six women received two starch cookies containing25 minipills of 30 mg of levonorgestrel each (MicrovalWyeth, Kildare, Ireland). Blood samples were collectedfrom only three patients. Thus, the pharmacokinetics of lev-onorgestrel minipills was determined only in these individ-uals (Fig. 1).
The control group received placebo tablets (n¼ 5). Bloodsamples (5 mL) were taken before administration of the tab-lets or minipills (time 0) and then serially at 0.5, 1, 2, 4, 6, 8,24, and 48 hours after treatment (5). The concentration ofplasma levonorgestrel was determined by radioimmunoas-say according to the method described by Weiner and Jo-hansson (6). The experimental protocol was approved bythe Institutional Review Board of the Hospital San BorjaArriaran Santiago, Chile, and a signed informed consentwas obtained from each woman who agreed to participatein the study after it was completely explained to her.
PHARMACOKINETICS ANALYSIS
Each individual concentration time curve was fitted. Thearea under the concentration time curve (AUC), maximalconcentration (CMAX), time to reach maximal concentration
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(TMAX), and biological half-life were obtained for each oralformulation (group B). The pharmacokinetics values werenot different than those reported elsewhere (5) for the stan-dard treatment group A.
The starch cookies were swallowed without difficulty,and the women did not express any specific clinical com-plaint. Interestingly, the mean plasma concentrations of lev-onorgestrel, when given as a single oral dose of 1.5 mg,either as two tablets of 0.75 mg or as starch cookies contain-ing levonorgestrel minipills of 30 mg, were comparable dur-ing the study period. Despite the fact that the treatmentpresented different formulations, tablets or pills, the phar-macokinetic parameters of levonorgestrel minipills includ-ing (CMAX), (TMAX), biological half-life, and AUC for 24hours were not different from those of the standard treat-ment, which suggests that both formulations have similarsolubility and absorption rates.
Fewer intersubject variations of plasma levonorgestrelvalues were seen in the minipills group of three women.However, this observation should be taken with cautiongiven the small size of this study group. Despite the small
FIGURE 1
Levonorgestrel plasma levels in women after oraltablets (group A, n ¼ 13) and oral starch cookies(group B, n ¼ 3) of levonorgestrel (1.5 mg) duringthe periovulatory period of the menstrual cycle. Allplasma assays performed for levonorgestrel oncontrol women demonstrated undetectable levels.Individual concentrations are plotted versus time.
Devoto. Two formulations of levonorgestrel. Fertil Steril 2007.
Fertility and Sterility�
number of women in this study, the pharmacokinetics datathat we observed are encouraging. Because prompt accessto EC is critical for prevention of unwanted pregnancies,levonorgestrel-containing minipills could be considered asan alternative to the standard treatment because of theiravailability. Therefore, pharmacists could be trained sothey would be able to counsel patients about the use of lev-onorgestrel pills (30 mg) for EC in countries where access tostandard EC treatment is limited (7). Finally, the prelimi-nary data presented herein offer the opportunity for furtherinvestigations of EC treatments.
Acknowledgments: The authors thank Sirpa Ranta, M.Sc., Steroid Research
Laboratory, Institute of Biomedicine, University of Helsinki, and Oskari
Heikinheimo, M.D., Director of the Department of Obstetrics and Gynecol-
ogy, University of Helsinki, Helsinki, Finland, for the levonorgestrel assay.
Luigi Devoto, M.D.a
Alejandra Espinoza, B.S.a
Alex Mu~noz, B.S.a
Ariel Fuentes, M.D.a
Helena von Hertzen, M.D.ba Instituto de Investigaciones Materno Infantil and
Departamento de Obstetricia y Ginecologia,Universidad de Chile, Santiago, Chile; andb Department of Reproductive Health and Research,World Health Organization, Geneva, Switzerland
REFERENCES1. von Hertzen H, Piaggio G, Ding J, Chen J, Song S, Bartfai G, et al. Low
dose mifepristone and two regimens of levonorgestrel for emergency
contraception: a WHO multicentre randomised trial. Lancet 2002;360:
1803–10.
2. Task Force on Post-ovulatory Methods of Fertility Regulation. Random-
ized controlled trial of levonorgestrel versus the Yuzpe regimen of com-
bined oral contraceptives for emergency contraception. Lancet
1998;352:428–33.
3. Killick SR, Irving G. A national study examining the effect of making
emergency hormonal contraception available without prescription.
Hum Reprod 2004;19:553–7.
4. Gainer E, Blum J, Toverud E-L, Portugal N, Tyden T, Nesheim B-I, et al.
Bringing emergency contraception over the counter: experiences of non-
prescription users in France, Norway, Sweden and Portugal. Contracep-
tion 2003;68:117–24.
5. Devoto L, Fuentes A, Palomino A, Espinoza A, Kohen P, Ranta S, et al.
Pharmacokinetics and endometrial tissue levels of levonorgestrel after
administration of a single 1.5 mg dose by the oral and vaginal route. Fer-
til Steril 2005;84:46–51.
6. Weiner E, Johansson EDB. Plasma levels of d-norgestrel, estradiol and
progesterone during treatment with silastic implants containing
d-norgestrel. Contraception 1976;14:18–92.
7. Bacon L, Savage I, Cook S, Taylor B. Training and supporting pharma-
cists to supply progesterone-only emergency contraception. J Fam Plann
Reprod Health Care 2003;29:17–22.
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Ten-year experience with preimplantation geneticdiagnosis (PGD) at the New York University School ofMedicine Fertility Center
We describe our experience of over 300 cycles of preimplantation genetic diagnosis (PGD) and report clinicalpregnancy rates (35%–67%) that support using this technology to screen for genetic disorders and chromosomalabnormalities. In clinical practice for over ten years, PGD offers couples the earliest form of genetic screeningand may help improve ongoing pregnancy rates in poor-prognosis patients. (Fertil Steril� 2007;88:978–81.�2007 by American Society for Reproductive Medicine.)
Preimplantation genetic diagnosis (PGD) was first used onhuman embryos in the early 1990s to diagnose sex-linkedgenetic disorders (1–3). The clinical horizons for PGDhave since broadened to include diagnoses for more than100 different genetic disorders as well as for chromosomalaneuploidies (AS) and translocations (TS). As a result, over1,000 healthy children have been born worldwide fromPGD-IVF cycles (4, 5).
Originally, PGD was provided for couples at risk of hav-ing a child with a sex-linked disorder. Initially, single blas-tomeres were analyzed using multiplex polymerase chainreaction (PCR) to amplify X- and Y-chromosome–specificsequences. Now, fluorescence in situ hybridization (FISH)technology is routinely used for sex chromosome assess-ment (2, 6–8). For single gene defects (SGD), identificationof the specific mutations have led to the development of spe-cific PCR probes to identify affected, carrier, and normalembryos (9).
Other patients now routinely seek PGD to screen forchromosomal abnormalities. These include carriers of chro-mosomal translocations as well as women of advanced ma-ternal age and women with repeated failed IVF cycles andrecurrent miscarriage. For these patients, FISH technologyhas been applied to screen for AS or TS. Initially AS eval-uated chromosomes 13, 16, 18, 21, X, and Y; the list subse-quently broadened to include chromosomes 14, 15, and 22,allowing the evaluation of the most frequently observedchromosomal aneuploidies in human aborteses (10–12).
A final group seeking PGD includes patients with a childwith a genetic disease who wish to create an unaffectedHLA match for stem cell transplantation therapy. Preim-plantation genetic diagnosis has been used to obtain HLAmatches for children with Fanconi anemia, acute lymphoidleukemia, and acute myeloid leukemia (13, 14).
Received January 30, 2006; revised and accepted December 20, 2006.
Reprint requests: James A. Grifo M.D., Ph.D., NYU Fertility Center, 660
First Avenue, Fifth Floor, New York, New York 10016 (FAX 212-
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We have performed PGD at New York University (NYU)Fertility Center (formerly the Program for IVF, Reproduc-tive Surgery and Infertility) at the NYU School of Medicinesince 1995 and have completed over 300 PGD-IVF cyclesthrough 2005. Performing PGD in a successful IVF pro-gram offers a unique opportunity to assess the outcomesof these cycles in an optimal setting. We completed a retro-spective chart review for PGD patients from 1995 to 2005(Institutional Board of Research Associates study no. 05–156). Cycles were reviewed for indications, patient histo-ries, and outcomes.
All IVF, embryo biopsy, and transfer procedures wereperformed at our center as well as PCR and FISH analysesfrom 1995 to 2000. As genetic protocols became more spe-cialized, biopsied cells were sent to Genesis Genetics(Detroit, MI) for PCR analyses and Reprogenetics (WestOrange, NJ) for FISH analyses.
A patient’s ovarian stimulation protocol was individual-ized to achieve adequate numbers of mature oocytes at re-trieval. Most patients were down-regulated with a GnRHagonist and then treated with combinations of recombinanthuman FSH (rFSH) and/or hMG. Recent protocols usedGnRH antagonists. When lead follicles reached a mean di-ameter of 17–18 mm, hCG was given, and about 34 hourslater oocytes were collected by ultrasound-guided transva-ginal aspiration and placed in human tubal fluid media(HTF; Irvine Scientific, Irvine, CA) supplemented with6% Plasmanate (5% USP plasma protein fraction (human);Bayer, Elkhart, IN) overlaid with Sage mineral oil (CooperSurgical Co., Trumbull, CT). Partner’s sperm was collectedon day of retrieval and washed. Oocytes were fertilized withroutine insemination (4–6 hours after retrieval) or intracyto-plasmic sperm injection (ICSI) if PCR analysis was indi-cated for SGD. The ICSI was routinely performed forSGD cases to eliminate DNA contamination by stray spermcells at embryo biopsy. Fertilization was assessed 18 hoursafter insemination/ICSI by detection of two pronuclei(2PN).
Embryo biopsy was performed approximately 72 hoursafter retrieval in Caþþ/Mgþþ-free media, supplemented
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with HEPES, BSA, and sucrose (Sage Sigma, In Vitro Fer-tilization Inc, Trumbull, CT). This medium disrupts cell ad-hesion and causes osmotic shrinkage of the cells from thezona pellucida, facilitating the removal of a single cell(2). The embryo was stabilized by a holding pipette andthe zona pellucida breached using acidified Tyrode’s solu-tion. The single cell was gently removed by suction. Forboth PCR and FISH, biopsy was performed only on 2PNembryos that were R5 cells on day 3. If PCR was sched-uled, the biopsied cell was rinsed and placed in a DNA-free prelabeled microfuge tube containing 5 mL lysis buffer.If FISH was scheduled, the cell was rinsed and fixed toa glass slide as previously described (15, 16). Biopsy spec-imens were sent to the appropriate referral laboratory. In themajority of cases only a single blastomere was biopsied; ifa nucleus was not present, a second cell was biopsied.
After biopsy, the embryos were rinsed and placed inQuinn’s Blastocyst Media (Sage) supplemented with 10%Plasmanate and cultured under oil for an additional 24–72hours. Following review of either FISH or PCR results (1day after biopsy), the embryos were evaluated. The most ad-vanced embryos that had either a normal chromosomalcomplement or were unaffected by the genetic mutation,were selected for transfer to the uterus on day 4, 5, or 6 afterretrieval. In most cases, %2 embryos were suitable fortransfer on day 4 and extended culture for selection pur-poses was not needed. Additional unaffected good-quality(grade 4BB or better per Gardner’s criteria) embryos werecryopreserved on day 5 or 6 (17).
The methods used at our center for FISH were previouslydescribed for simultaneous enumeration of chromosomes13, 18, 21, X, and Y in interphase cells (11, 12). At thetime of this review, cells were routinely screened for ninechromosomes (13, 15, 16, 17, 18, 21, 22, X, and Y) at Re-progenetics. For many inheritable disorders for which thegenetic mutations were determined, PCR was used toamplify the genetic material contained in a single cell; pro-tocols for PCR on single cells have been described else-where (18).
Fertility and Sterility�
From 1995 to 2005, 304 PGD-IVF cycles were per-formed for 190 patients; 181 (60%) were performed forSGD, and 123 (40%) were performed for AS and TS. Em-bryos were transfered in 158 of 181 cycles (87%) forSGD, 87 of 111 cycles (78%) for AS, and 9 of 12 cycles(75%) for TS. Table 1 lists the number of PGD referralsand their outcomes. Implantation rate (IR) is the numberof gestational sacs divided by the number of embryos trans-fered. The IRs for SGD, AS, and TS were 24%, 27%, and47%, respectively. Clinical pregnancy rate (CPR) is thepresence of a fetal heartbeat divided by the number ofcycles resulting in an embryo transfer. The CPRs forSGD, AS, and TS were 35%, 37%, and 67%, respectively.
Clinical histories were reviewed for the 92 patients re-ferred for SGD. Many (35%) had a prior affected pregnancy,and half (50%) underwent a second-trimester termination.The additional patients had a maternal, paternal, and/orfamily history of a disorder. In most cases, both parentswere known carriers of cystic fibrosis, predominantly thedelta F508 mutation.
Embryos were not transfered in 23 cycles for SGD: inseven cycles with no HLA match, nine cycles with no unaf-fected embryo, and two cycles with unaffected embryos thatdid not divide properly. In five cycles, genetic analysisfailed, because no PCR signal was obtained.
When HLA matching was performed for Diamond-BlackFanconi anemia, adrenoleukodystrophy, and Bloom’s dis-ease for four patients, 8 of 16 cycles (50%) resulted in an em-bryo transfer, and one eventuated in a full-term delivery of anunaffected child that was not an HLA match. In seven cycles,no HLA match was obtained. In one cycle, an HLA matchwas found but the embryo stopped dividing before transfer.
Eighty-eight patients underwent 111 cycles of PGD forAS: 44 for recurrent (R2) miscarriage, 37 for advancedmaternal age (generally R38 years, all with a history of el-evated FSH), 5 for repeated IVF failures, 7 for couples withan aneuploid fetus/child, and 18 for a combination of theabove indications. Of note, 78 cycles (70%) were performed
TABLE 1PGD outcome 1995–2005 at New York University Fertility Center.
Note: AS ¼ aneuploidy screening; CPR ¼ clinical pregnancy rate; IR ¼ implantation rate; PGD ¼ preiimplantation geneticdiagnosis; SGD ¼ single gene defects; STD ¼ standard deviation; TS ¼ translocation screening.
Grifo. PGD at NYU Fertility Center. Fertil Steril 2007.
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in 2005, indicating a recent increase in demand for PGD forthis indication. There was no embryo transfer in 24 cycles ofPGD for AS; in all cases, no euploid embryos were identi-fied. Twelve cycles (in ten patients) used PGD for TS; threedid not have an embryo transfer, because of a lack of unaf-fected embryos.
Miscarriage rates, defined as a proportion of gestationalsacs not resulting in live births, were 22% for SGD, 29%for AS, and 14% for TS. When calculated as the proportionof fetal heartbeats aborted per fetal heartbeats detected, thenrates were much lower: 12% for SGD, 12.5% for AS, and14% for TS.
Congenital anomalies were rare: a female child with bi-lateral clubfoot after PGD for SGD and an aortic arch anom-aly in a premature twin born after PGD for AS for advancedmaternal age. Two cycles resulted in newborn carriers of ge-netic disorders: twin carriers of Tay Sachs disease and a sin-gleton carrier of cystic fibrosis. However, in both cases thecarrier status of the embryos was revealed by PGD and thepatients elected to continue with the transfer. Two cycles re-sulted in newborns who were affected by disease (one withhemophilia and one with familial dysautonomia) presum-ably owing to misdiagnoses by PCR.
Twenty cycles of PGD for SGD and two cycles for AS re-sulted in cryopreservation of unaffected embryos. Five fro-zen embryo transfer cycles in the SGD group resulted in anunaffected live birth; a patient who did not get pregnantfrom the PGD-AS cycle did get pregnant and deliveredfull term from her frozen cycle.
Our ten-year experience performing PGD illustrates thatthis procedure successfully provides couples with healthychildren unaffected by either genetic diseases or chromo-somal abnormalities. Over 33% of those who underwentPGD for SGD had an earlier affected pregnancy and ofthese, 50% terminated an earlier pregnancy for this reason.Performing PGD allowed these patients to initiate a preg-nancy with a reduced potential of undergoing a second-trimester termination.
Verlinsky et al. (19) recently published a multicenterreport on PGD, citing a pregnancy rate of 25.2% in 4,748cycles (23.3% for AS, 30.5% for SGD, and 34.6% forTS). The present data in conjunction with those publishedin this large multicenter study support the use of PGD forappropriate cases.
Data from 2005 indicate that more PGD cycles are beingconducted for AS in high-risk patients. Whether PGD forAS increases the implantation and clinical pregnancy ratesfor IVF patients is currently controversial. In a retrospectiveblinded control study, higher ongoing pregnancies and de-livered babies were reported in patients who underwentPGD for AS compared with those who underwent routineIVF (20). Another randomized controlled study reportedsimilar clinical pregnancy rates but higher ongoing preg-
980 Grifo et al.
nancy rates in older patients (R36 years) who underwentPGD for AS compared with IVF alone (21). Another pro-spectively randomized controlled trial did not find any sta-tistically significant difference in positive pregnancy tests orimplantation rates in older patients (R37 years) who under-went PGD for AS compared with patients who underwentIVF alone (22). However, in that study, two blastomereswere biopsied on average, which may compromise embryoquality.
The present data reflect several explanations of whypregnancy rates are not higher when PGD is added to IVFin poor-prognosis patients. Approximately 20% of the pa-tients who underwent PGD for AS failed to have geneticallynormal and viable embryos for transfer. Moreover, nearly20% of patients who sought PGD for AS were canceled be-fore retrieval owing to an insufficient number of follicles.Several investigators have clearly demonstrated that oncepregnancy is initiated with embryos identified as chromoso-mally normal, the incidence of miscarriage is significantlylower (23–26). As a result, the live birth rate per transferappears to be increased after PGD.
While there are potential pitfalls with PGD, as in the caseof the two misdiagnoses, all efforts are made to avoid them,and patients should be counseled about the risks involved.Confirmatory prenatal diagnosis with chorionic villus sam-pling or amniocentesis should be encouraged.
We anticipate that more PGD cycles will be performed toselect embryos that are not only morphologically attractivebut also free of potentially harmful genetic mutations andchromosomal aberrations. Although embryo biopsy addsto the cost of an IVF cycle, potentially improved ongoingpregnancy rates will likely offset the cost of repeatedlyfailed IVF cycles in patients at risk for AS. At the minimum,performing PGD for AS in poor-prognosis patients, by re-vealing the high number of abnormal embryos, may identifythe cause of their failed cycles.
To date we have performed PGD for over 30 different ge-netic disorders, for chromosomal aberrations, and for HLAmatching, resulting in the birth of over 90 healthy children.Our clinical pregnancy rates for PGD cycles are consistentwith those we have reported to the national CDC registry(27). We foresee a global expansion in the use of PGD asits application is realized to its fullest potential and morephysicians and embryologists are trained to perform thisprocedure.
Acknowledgments: The authors thank Mark Hughes, M.D. Ph.D. (Genesis
Genetics), and Santiago Munne, Ph.D. (Reprogenetics) for their invaluable
contributions to the practice of PGD as well as their specific roles in ana-
lyzing biopsied specimens from our center. The authors recognize as well
the great skill of our embryology staff, whose role in performing PGD at
our center was instrumental: Caroline McCaffrey, Patty LaBella, Patricia
Roy, Melicia Clarke-Williams, Emy Ampeloquio, and Yaxu Tang. Finally,
they must acknowledge the physicians at the NYU Fertility Center who
Vol. 88, No. 4, October 2007
F
have contributed to the art of IVF and PGD over the past ten years: Fred
Licciardi, M.D., Nicole Noyes, M.D., and Lisa Kump M.D.
J. Grifo, M.D. Ph.D.S. Talebian, M.D.D. Keegan, M.D.L. Krey, Ph.D.A. Adler, B.S.A. Berkeley, M.D.Division of Reproductive Endocrinology and Infertility,
New York University School of Medicine,New York, New York
Human albumin does not prevent ovarianhyperstimulation syndrome in assisted reproductivetechnology program: a prospective randomizedplacebo-controlled double blind study
We aimed to clarify the efficiency of IV human albumin in the prevention of ovarian hyperstimulation syndrome(OHSS). We found that human albumin at the described strength does not seem to either prevent or reduce theincidence of severe OHSS in high risk patients undergoing intracytoplasmic sperm injection (ICSI). (FertilSteril� 2007;88:982–5. �2007 by American Society for Reproductive Medicine.)
Ovarian hyperstimulation syndrome (OHSS) is a purely iat-rogenic condition and the most serious complication of con-trolled ovarian hyperstimulation (COH) with gonadotropinpreparations (1). Severe forms complicate�1% of all IVF cy-cles (2). The pathophysiology of the syndrome still remainsobscure. Prostaglandins (3), plasma renin activity, and aldo-sterone levels (4), and vascular endothelial growth factor (5)have been studied to elucidate the mechanism of OHSS. Al-though IV human albumin (HA) seems to have some effi-ciency as a preventive measure, basic comparative studieshave some methodological limitations. We designed a prop-erly randomized, placebo-controlled double blind study toclarify the efficiency of IV HA in the prevention of OHSS.
Patients entering the intracytoplasmic sperm injection(ICSI) program in Antalya IVF between January 2003 andDecember 2004 were evaluated for high risk factors of se-vere OHSS. High blood E2 level (>4,000 pg/mL) and>20 follicles R14 mm on the day of hCG administrationhave been defined as the basic risk factors for severeOHSS (6). Patients having one of these factors were in-cluded in the study. Patients were randomized by third-partysealed envelope entry to receive either a 20% HA solutionor an isotonic NaCl solution intravenously.
Approval for the study was obtained from the institu-tional review board. Signed informed consent was also ob-tained from the patients before enrolling in the study.
All patients underwent a down-regulation protocol withthe GnRH agonist triptorelin 0.05 mg/day (Decapeptyl Er-Kim, Istanbul, Turkey), beginning from the 21st day ofthe cycle. After the assessment of pituitary down-regulationon the third day of the cycle with a blood E2 level <50pg/mL, linear endometrium and suppressed ovaries, the
Received May 10, 2006; revised November 29, 2006; accepted
November 30, 2006.
Presented in part at the 60th ASRM meeting held in Philadelphia, PA,
October 16–20, 2004.
Reprint requests: Mete Isikoglu, M.D., Antalya IVF, Halide Edip Cad.
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
82
dosage of triptorelin was reduced to half and gonadotropinstimulation including 75 IU FSH (Gonal F; Serono, Istan-bul, Turkey) and 75 IU hMG (Pergonal; Organon, Istanbul,Turkey) was commenced. The dose was adjusted on day 6and thereafter in accordance with the E2 level and numberof developing follicles. Gonadotropin injections continueduntil at least two follicles of R18 mm were detected. ThehCG (10,000 IU) was then administered, followed 35 hourslater by oocte pickup. Embryo transfers were done 50 hoursafter oocte pickup. Progesterone in oil (50 mg/day) wasadministered intramuscularly for luteal phase support untilthe day of b-hCG assay. Clinically determined pregnancywas defined as the detection of an embryo with fetal cardiacactivity by ultrasound 3 weeks after the embryo transfer.
Immediately after oocte pickup, patients were admitted tothe recovery room where they received either 20% HSA(50 cc, Human Albumin, Eczacibasi Baxter, Istanbul, Tur-key) diluted in 100 mL 0.9% NaCl or 100 mL 0.9% NaCl.Solutions were infused IV during 1 hour, approximately36 hours after the administration of hCG. Because the infu-sions were done in a separate location by one of the staffnurses, the outcome assessors were blind to the interven-tions. The patients were also blind to the infusions as bothgroups received the infusions as isotonic NaCl solutions.Blood was drawn on the day of hCG administration, 5 daysand 12 days after embryo transfer for E2 assays, whole bloodcount, creatinine, and transaminases. On days 5 and 12 afterembryo transfer, ultrasound examinations for ovarian sizeand detection of ascites were done as well as the assessmentfor other symptoms of severe OHSS. Diagnosis of severeOHSS was made according to the criteria of Navot et al. (6).
Estradiol concentrations were determined from venousblood and measured by VIDAS 12 device (bioMerieuxVitek, Philadelphia, PA). Intra-assay and interassay coeffi-cients of variation were less than 7.5% and 9.5%, respectively.
Severe OHSS rate was the main outcome parameter andclinical pregnancy rates/embryo transfer and first trimestermiscarriage rates were the main secondary outcome param-eters. As previously reported, OHSS occurs as two distinct
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clinical entities depending on the timing of onset (7). In ac-cordance with this report, we classified OHSS as late onset(R10 days after oocte pickup) or early onset.
The c2 test, Fisher’s exact test, Mann Whitney U test, andStudent’s t-test were used for statistical analyses of the data.An a value <0.05 was considered significant.
A total of 75 patients with high risk factors for severeOHSS were enrolled in the study on day of hCG administra-tion. Group 1 patients (n ¼ 38) received HA and group 2patients (n ¼ 37) received isotonic NaCl. Both groupswere similar with regard to age of the women, the incidenceof polycystic ovary syndrome (PCOS), and the number ofpatients with a history of severe OHSS (Table 1). The distri-bution of the etiology of infertility of the two groups was notdifferent. Total units of gonadotropins used, length of stim-ulation, peak E2 levels, E2 levels on days 5 and 12 after em-bryo transfer, number of follicles R14 mm on the day ofhCG administration, number of MII oocytes, and numberof embryos replaced did not show any statistically signifi-cant difference (Table 1). The outcome variables are also
Fertility and Sterility�
shown in Table 1. Severe OHSS developed in eight womenfrom group 1 and in six women from group 2. All patientswith late onset severe OHSS (n ¼ 4) and both early andlate onset severe OHSS (n ¼ 6) had clinical pregnancies.Onlyonewomen with early onset severe OHSS had a negativepregnancy test result. Clinical pregnancy rates/embryo trans-fer and rates of miscarriage during the first trimester weresimilar for both groups. One patient in group 2 had an ectopicpregnancy and underwent laparoscopic salpingectomy.
None of the patients with severe OHSS had renal failureor any other life-threatening complications. Only one pa-tient in the albumin group underwent paracentesis. All ofthe patients in the albumin group tolerated the infusionsand we did not encounter any hypersensitivity reactions.
Although the key to controlling severe OHSS is in its pro-phylaxis, complete prevention is not as yet possible. Variouspreventive measures have been suggested for avoiding se-vere OHSS in assisted reproductive technology (ART) cy-cles, most of which have had debatable success. One ofthe first measures used, other than merely cancelling the
TABLE 1Demographic characteristics, stimulation, and outcome data.
Albumin Placebo P value
No. of cycles 38 37 NAAge of the patients (mean � SD) 29.3 � 3.9 29.1 � 4.1 .87a
PCOS 15/38 (39.5%) 8/37 (21.6%) .09b
History of severe OHSS 4/38 (10.5%) 4/37 (10.8%) .96b
First trimester miscarriage rate 1/20 (5%) 4/23 (17.3%) .35d
Note: NA ¼ not applicable; OHSS ¼ ovarian hyperstimulation syndrome; PCOS ¼ polycystic ovary syndrome.a Student’s t-test.b c2 test.c Mann Whitney U test.d Fisher’s exact test.
Isikoglu. Human albumin does not prevent OHSS in ART program. Fertil Steril 2007.
983
cycle, was the avoidance of hCG for luteal phase supple-mentation, as there has been virtually unanimous agreementthat severe OHSS is strongly associated with endogenous orexogenous hCG. Other measures have focused on hCG trig-ger substitutes. The use of GnRH agonist as the trigger is,however, limited to gonadotrophin-only or GnRH antago-nist cycles. Another substitute for the conventional hCGtrigger has been recombinant LH (8). Other measures thatalso have only gained limited acceptance as successful mea-sures for the prevention of OHSS are coasting, GnRH antag-onist protocols (9), reducing the trigger dose, follicularreduction (10–12), and freezing all cycles. The success ofthe latter measure is also highly dependent on the successof the laboratory’s cryopreservation program.
Histamine has been claimed to be mediator of OHSS inanimal model (13). However, studies failed to show any ef-fect of H1 and H2 blockage on the syndrome (14, 15).
Although, as with other measures described there has notbeen an absolute consensus, the IV administration of HA atthe time of oocyte retrieval has been widely accepted asa measure that may facilitate the prevention of OHSS(16). It has been suggested that the binding and transportproperties of HA play a major role in the prevention of se-vere OHSS through the ability to bind and inactivate the va-soactive intermediates responsible for the pathogenesis ofthe syndrome. The osmotic properties of HA may also beresponsible for maintaining the intravascular volume inthe event of capillary leakage (17).
Asch et al. (18) were the first to suggest that IV albuminmay prevent the development of severe OHSS. This was fol-lowed by several clinical studies with conflicting results. Atpresent there have been eight published randomized con-trolled trials exploring the use of HA in preventing severeOHSS (1, 19–24). A recent Cochrane review, based onfive of these randomized controlled studies in which 378patients were involved, showed definite benefit from the ad-ministration of IV albumin at the time of oocyte pickup inhigh risk patients (16). However, in all but one of these trials,the exception being the study by Ben-Cherit et al. (22), theoutcome assessors were not blind to the intervention (19–21). A potential for outcome bias therefore exists. Bellveret al. (24) published the largest prospective single-centeredstudy. They compared 40 g of HAwith no treatment in a totalof 976 patients and concluded that albumin infusion is notuseful in the prevention of moderate-to-severe OHSS. Inthis study, similarly, the outcome assessors were not blindto the intervention. In addition, two kinds of GnRH agonistwere used and only early OHSS was assessed. Placebo wasnot used in their control group, which eliminates the placeboeffect of albumin administration. Serum E2, level which waspreviously reported as an independent risk factor for severeOHSS, was not considered high risk factor in this study. Inour study, both the patients and the outcome assessorswere blind to the interventions and the results of our studyare in accordance with those of Bellver et al. (24).
984 Isikoglu et al.
Various HA concentrations between 10 g (19, 20) and50 g (21, 22) were also used in these randomized studies.In the present study we assessed the efficacy of 10 g ofHA and found that it does not seem to either prevent orreduce the risk of severe OHSS in high risk patients.
The incidence of severe OHSS in patients undergoingART during the study period in our center was 1.95%,which is consistent with previous reports (2, 25). The rela-tively high incidence of severe OHSS in our study popula-tion was mainly due to our sample selection criteria, as wellas our aggressive COH policy.
In our study, patients experiencing early severe OHSSand the resolution of the symptoms thereafter were all asso-ciated with a negative pregnancy result, whereas patientsexperiencing late severe OHSS with or without early severeOHSS were always associated with a positive pregnancytest result. The association between the late onset ofOHSS and pregnancy has been previously reported to bea result of the endogenous hCG from the initiated preg-nancy (6). Late OHSS is reported as being able to compli-cate a nonconception cycle only if additional hCG isgiven during the luteal phase (25).
It has been shown that once a clinical pregnancy has beenestablished in a patient with OHSS (both early and lateOHSS), there is a normal risk of abortion (25). Our findingsregarding first trimester abortion rates were consistent withprevious reports.
Although albumin is generally tolerated, hypersensitivityreactions such as urticaria (19) and even life-threateninganaphylactic reactions have been reported. The incidenceof urticaria, fever, chills, or hypotension ranges from0.47%–1.53% (26) and 0.011% for the incidence of allergicreactions (27). Despite the very low rate of life-threateningrisks of HA, it is mandatory to prove a definite clinical ben-efit that outweighs the unnecessary risks before its routineuse in clinical practice.
In conclusion, the administration of 10 g of HA to highrisk OHSS patients does not seem to either prevent or reducethe risk of severe OHSS. The efficacy of higher doses of HAneeds to be clarified by future randomized studies with largestudy samples. We believe the results of the present studywill assist other investigators in the decision to use HA asan OHSS preventative measure after COH for ART.
Acknowledgment: We are grateful to Kevin Coetzee for the English proof-
reading of the article. We also thank to Dr. Levent Donmez for his valuable
Steirteghem AV, et al. Early and late ovarian hyperstimulation syn-
drome: early pregnancy outcome and profile. Hum Reprod 2005;20:
636–41.
26. Tullis JL. Albumin. Background and use. J Am Med Assoc 1977;237:
355–60.
27. Roberts JS, Bratton SL. Colloid volume expanders: problems, pitfalls,
and possibilities. Drugs 1998;55:621–30.
985
Pelvic pain after gonadotropin administrationas a potential sign of endometriosis
We describe five patients who developed significant pelvic pain, requiring narcotics, during a controlled ovarianhyperstimulation cycle and who were surgically diagnosed with significant endometriosis. Severe pain, espe-cially if it requires narcotics, is unusual for patients undergoing controlled ovarian hyperstimulation andmay be an indicator of endometriosis. (Fertil Steril� 2007;88:986–7. �2007 by American Society for Repro-ductive Medicine.)
Endometriosis is a disease that is characterized by the pres-ence of endometrial glands and stroma outside the uterinecavity (1). Its incidence among women with infertilityranges from 20% to 50% (2). Medical treatments aimed atlowering circulating estrogen (E) levels and inducing endo-metrial suppression often are effective in treating paincaused by endometriosis (1, 3). Conversely, infertility treat-ments with controlled ovarian hyperstimulation temporarilyincrease circulating Es and may worsen endometriosis orsymptoms related to the disease.
Although women with endometriosis and infertility mayhave the classic symptoms of dysmenorrhea, dyspareunia,pelvic pain, and/or dyschezia, some of these women are oth-erwise asymptomatic (4). Here, we report several cases ofwomen who developed significant pelvic pain during a con-trolled ovarian hyperstimulation cycle and who either wereknown to have endometriosis or were later surgically con-firmed to have the disease. The purpose of this report is tobetter describe a symptom complex observed in our patientsthat may indicate the presence of endometriosis or the exac-erbation of a known condition and to gain information aboutthe behavior of this disease in a high-E environment.
CASE REPORTS
Patient 1
This woman was 36 years old and a gravida 0. She had a his-tory of unexplained infertility for 4 years and of moderatedysmenorrhea during the first 2 days of her menstrual cy-cles, for which she took ibuprofen. As she underwent IVF,she complained of increasing pelvic pain during the ovarianstimulation. The day after the retrieval, her pain worsened,requiring an IM injection of meperidine hydrochloride. Shecontinued to take oral narcotics off and on for the subse-quent 2 weeks. Because of high suspicion for possible endo-metriosis, she underwent a laparoscopy, whichdemonstrated that she had biopsy-proven stage III perito-neal disease.
Received October 10, 2006; revised and accepted December 27, 2006.
Reprint requests: Ruth Lathi, M.D., Reproductive Endocrinology and In-
fertility, Stanford University, 900 Welch Road, Suite 350, Stanford,
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
986
Patient 2
This woman was 31 years old and a gravida 0. She had a his-tory of infertility for 10 years and a known history of severeendometriosis. She had undergone multiple laparoscopicprocedures and at least three courses of leuprolide acetateto treat her recurrent disease, with adequate control of herpain. Her last 3-month course of leuprolide acetate therapywas 6 months before her first controlled ovarian hyperstim-ulation cycle. She tolerated oral ovulation induction agents(clomiphene citrate and letrozole) well but did not conceive.With injectable gonadotropins, she complained of severeabdominal cramping, which required a visit to the emer-gency room and intravenous narcotics. She discontinuedher treatment because of this pain. The decision wasmade to proceed with laparoscopy because recurrence ofher disease was suspected as a cause of her worseningpain. She was found to have biopsy-proven recurrence ofher stage IV peritoneal endometriosis, in addition to signif-icant disease involving both small and large intestines.
Patient 3
This woman was 37 years old and a gravida 0. She had a his-tory of infertility for almost 2 years as well as mild dysmen-orrhea, which was relieved with ibuprofen, and occasionaldyspareunia. Mild (stage I) endometriosis had been foundon diagnostic laparoscopy and was treated approximately1 year before her IVF cycle. During stimulation for IVF,she complained of pelvic pain that required pain medica-tions. After her transfer, she returned to clinic for severeright lower-quadrant abdominal pain and subsequently re-quired an injection of ketorolac and oral narcotics. Recur-rence of endometriosis was suspected, and the decisionwas made to proceed with a repeat laparoscopy. She wasfound to have biopsy-proven stage II peritoneal disease.
Patient 4
This woman was 40 years old and a gravida 0. She had a his-tory of infertility, severe dysmenorrhea, and occasional dys-pareunia and had had several laparoscopic surgeries fortreatment of endometriosis. She underwent leuprolide ace-tate with add-back therapy for 8 years. During her first IVF
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.054
cycle, she experienced a significant amount of abdominaland pelvic pain that required narcotics for relief. Even afterthe cycle, she continued to have persistent severe pain. Ul-timately, because of this persistent pain, she underwenta laparoscopic hysterectomy with bilateral salpingo-oopho-rectomy, after which she achieved complete pain relief.
Patient 5
This woman was 33 years old and a gravida 0. She had a his-tory of infertility and mild dysmenorrhea and had a laparo-scopic diagnosis of endometriosis. It was her first IVF cycle.During and after ovarian stimulation, she complained ofsignificant right lower-quadrant abdominal pain. She alsocomplained of severe pain and pressure in the rectum.Oral narcotics were insufficient to treat her pain, and she re-quired an IM injection of meperidine hydrochloride in addi-tion to indomethacin for pain control. Laparoscopy revealedstage I peritoneal endometriosis, which was confirmed bybiopsy.
In summary, all patients had new onset of or worseningpelvic pain while being treated with gonadotropins. Thispain was attributed to exacerbation of endometriosis. Onepatient who did not have a diagnosis of endometriosis be-fore her controlled ovarian hyperstimulation treatment cy-cle later was surgically confirmed to have this disease.Two of the women experiencing severe pain during stimu-lation subsequently underwent repeat treatment after sur-gery without pain, which suggested that endometriosiswas the cause of the pain.
DISCUSSION
The literature contains multiple case reports that suggesta possible correlation between ovarian stimulation and pro-gression of the disease in some patients. For example,ureteral endometriosis has been reported after ovarian hy-perstimulation in a patient previously diagnosed with endo-metriosis (5), and several cases of gastrointestinalcomplications have been reported as a result of rapid growthof bowel endometriosis in patients undergoing gonadotro-pin administration (6). In addition, there have been manystudies supporting the E-dependent nature of this disease.For example, men who received E therapy for their prostaticcarcinoma have been found to have biopsy-proven endome-triosis (7, 8).
The impact of gonadotropin stimulation and associatedhigh E2 levels on endometriosis still remains unclear.D’Hooghe et al. (9) were the first to retrospectively examinethe recurrence rate of endometriosis after surgical treatment
Fertility and Sterility�
for moderate to severe endometriosis in patients who under-went ovarian hyperstimulation for either an insemination oran IVF cycle. Those investigators were not able to concludethat higher levels of E exposure led to a higher recurrencerate of this disease. However, in this group of patients, noteveryone had laparoscopy after treatment, and there wasno mention of pain or other symptoms during or immedi-ately after stimulation.
Because not all women with endometriosis experiencepain with gonadotropin stimulation, this observation high-lights the heterogeneity of the disease. These cases may rep-resent a variant of the disease, which is highly sensitive toacute changes in E2 levels. These five patients were selectedbecause of the substantial amount of pain medication thatthey required during treatment, which is not the norm inour patient population. Many women with the diagnosisof endometriosis tolerate gonadotropin stimulation welland therefore should not be discouraged from using this ef-fective treatment.
In conclusion, the symptom of significant pelvic pain in-duced by gonadotropin stimulation may help identify thosepatients who require surgery to diagnose new or recurrentendometriosis. Physicians should have a high suspicionfor this disease in patients with this complaint and shouldconsider further evaluation and treatment.
Sunny Hee Jun, M.D.Ruth B. Lathi, M.D.Department of Reproductive Endocrinology and
Infertility, Stanford University Medical Center,Stanford, California
REFERENCES1. Olive DL, Pritts EA. Treatment of endometriosis. N Engl J Med
2001;345:266–75.
2. Olive DL, Schwartz LB. Endometriosis. N Engl J Med 1993;328:1759.
3. Attar E, Bulun SE. Aromatase inhibitors: the next generation of thera-
peutics for endometriosis? Fertil Steril 2006;85:1307–18.
4. Berkley KJ, Rapkin AJ, Papka RE. The pains of endometriosis. Science
2005;308:1587–9.
5. Renier M, Verheyden B, Termote L. An unusual coincidence of endome-
triosis and ovarian hyperstimulation. Eur J Obstet Gynecol Reprod Biol
1995;63:187–9.
6. Anaf V, El Nakadi I, Simon P, Englert Y, Peny MO, Fayt I, et al. Sigmoid
endometriosis and ovarian stimulation. Hum Reprod 2000;15:790–4.
7. Pinkert TC, Catlow CE, Straus R. Endometriosis of the urinary bladder
in a man with prostatic carcinoma. Cancer 1979;43:1562–7.
8. Schrodt GR, Alcorn MO, Ibanez J. Endometriosis of the male urinary
system: a case report. J Urol 1980;124:722–3.
9. D’Hooghe TM, Denys B, Spiessens C, Meuleman C, Debrock S. Is the
endometriosis recurrence rate increased after ovarian hyperstimulation?
Fertil Steril 2006;86:283–90.
987
Mutations of SYCP3 are rare in infertile Spanish menwith meiotic arrest
No functional SYCP3 exonic SNP was found in infertile Spanish patients with meiosis arrest, suggesting thatSYCP3 mutations very likely are not relevant in Spain in genetic susceptibility to meiosis arrest, just as inother studied European populations. (Fertil Steril� 2007;88:988–9. �2007 by American Society for Repro-ductive Medicine.)
The SYCP3 gene, located on chromosome 12, encodes aDNA-binding protein as a component of the synaptonemalcomplex that regulates the synapsis between homologouschromosomes during meiosis of germ cells (1). Althoughthe SYCP3 involvement in male fertility has been well es-tablished by knockout mice (1, 2), the role of the presenceof SYCP3 variants in male infertility as a result of meioticarrest is still a matter of debate (3, 4). The goal of our studywas to evaluate the frequency of SYCP3 mutations in ourpopulation to better define the relationship betweenSYCP3 variants and defects of meiotic process that lead toinefficient spermatogenesis.
Twenty-two infertile men with a phenotype of azoosper-mia (n ¼ 14) or severe oligozoospermia (<5 million spermper mL; n ¼ 8) who underwent testicular biopsy showeda histological pattern of a complete or incomplete matura-tion arrest of spermatogenesis in primary spermatocyte andwere subsequently screened for sequence variants inSYCP3 gene. Men with a chromosomal aberration or witha Y-chromosome microdeletion had been previously ex-cluded from the study. Patients were recruited from the An-drology Unit of the Fundaci�o Puigvert (Barcelona, Spain)and gave their informed consent for the study, which wasapproved by the institutional ethical committee. All ofthem were Caucasians of Spanish (n ¼ 20) and Maghribian(n ¼ 2) origin.
SYCP3-coding and immediately flanking regions wereamplified in seven fragments (primer sequences are des-cribed in Table 1) that were analyzed by single-strand con-formation polymorphism (SSCP)-heteroduplex technique
Received July 14, 2006; revised and accepted November 30, 2006.
Supported by grants from Fondo de Investigaci�on Sanitaria/Fondos
Europeos de Desarrollo Regional (PI02/0120, PI05/0759), Red de
Centros de Gen�etica Cl�ınica y Molecular ISCiii (C03/07), the Gener-
alitat de Catalunya (2005SGR00018), and the Institut Catala de la
Salut. S.L. is supported by Fondo de Investigaci�on Sanitaria
(CP03/00088).
Reprint requests: Sara Larriba, Ph.D., Medical and Molecular Genetics
Center, Fundaci�o IDIBELL (Institut d’Investigaci�o de Bellvitge), Gran
V�ıa s/n km. 2.7, 08907 L’Hospitalet de Llobregat, Barcelona, Spain
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988
(Genephor Electrophoresis Unit; Amersham-PharmaciaBiotech, Buckinghamshire, United Kingdom) using12.5% polyacrylamide gels (ExcelGel, Amersham Biosci-ences, Uppsala, Sweden). The characterization of sequencealterations was performed by directly sequencing a newPCR product corresponding to the fragments displayingmobility shift.
We found only one azoospermic Spanish individual whowas heterozygous for the noncoding c.454-9_13delTTTATvariant. To check whether this variant affects splicing, theexon-skipping analysis on lymphocyte complementaryDNA samples was performed and evidenced no differencein exon splicing among individuals who were either pre-senting these variants or not (data not shown). No changewas observed in the coding regions, so that amino acid se-quence remained unchanged.
Our results add information about SYCP3 mutationalstudies in patients from different populations who have in-fertility caused by maturation arrest of spermatogenesis.An association was previously observed in North Americansubjects: 2 of 19 azoospermic patients, one Hispanic and theother Arab, presented a c.643delA mutation causing defec-tive SYCP3 expression (3); however, this was not presentin European populations, either in 36 azoospermic and 22severe oligozoospermic patients from Belgium (4) or inour study on a Mediterranean population, suggesting thatthe cause of this discordance may be related to the influenceof ethnicity differences. We conclude that SYCP3 mutationsvery likely are not relevant in genetic susceptibility to mei-osis arrest in European individuals and suggest that geneticbackground could explain the differences observed in thefrequency of the SYCP3 genetic defects from different pop-ulations. Because of the multiple components that partici-pate in meiotic chromosome pairing and synapsis (cohesincomplex subunits, recombination proteins, and other synap-tonemal complex subunits apart from SYCP3) (5), geneticalterations in one or several of these subunits could be re-sponsible for meiosis arrest.
Acknowledgments: The authors are indebted to the patients who partici-
pated in this study.
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Westphal H, et al. Azoospermia in patients heterozygous for a mutation
in SYCP3. Lancet 2003;362:1714–9.
4. Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. SYCP3 mu-
tations are uncommon in patients with azoospermia. Fertil Steril 2005;84:
1019–20.
5. Pelttari J, Hoja MR, Yuan L, Liu JG, Brundell E, Moens P, et al. A mei-
otic chromosomal core consisting of cohesin complex proteins recruits
DNA recombination proteins and promotes synapsis in the absence of an
axial element in mammalian meiotic cells. Mol Cell Biol 2001;21:
5667–77.
989
Normal percentage of CD56bright natural killer cellsin young patients with a history of repeatedunexplained implantation failure after in vitrofertilization cycles
Changes in endometrial leukocyte subpopulations and most of all in the percentage of uterine natural-killercells (uNK), during the menstrual cycles, may have a pivotal role in the implantation process. An increaseof activated NK cells (CD56dim CD16þ CD69þ) in the peripheral blood of patients with a reduced rate ofembryo implantation in IVF treatment has been reported elsewhere, but we found, by using flow cytometry,normal endometrial lymphocyte subpopulations in young patients with a history of repeated unexplained im-plantation failure who were undergoing IVF cycles for idiopathic infertility. (Fertil Steril� 2007;88:990–3.�2007 by American Society for Reproductive Medicine.)
Endometrial receptivity is a key determinant of the successof implantation. Changes in endometrial leukocyte subpop-ulations and most of all, in the percentage of uterine natural-killer cells (uNK), during menstrual cycles may havea pivotal role in the implantation process. Uterine NK cellsrepresent the largest leukocyte subpopulation in the endo-metrium (1). These cells, also named large granulatedlymphocytes, resemble phenotypically the dominant CD56-positive (CD56þ) population of peripheral blood NK cells,but it is important to note that they are also different fromthe phenotypic and functional points of view. More indetail, uNK are identified by the expression of the NKmarker CD56 at high concentrations (CD56bright) butlack the expression of other typical NK markers, such asCD16, that are responsible for NK-mediated, antibody-dependent cellular cytotoxicity, found on most peripheralblood NK cells (2, 3). During the normal menstrual cycle,there is a significant increase in the numbers of uNK cellsthroughout the secretory phase and in early pregnancy,when uNK comprise >70% of the endometrial leukocytesin the first-trimester decidua (4).
Therefore, it is generally believed that uNK cells arelikely to play a critical role in tolerance of the hemi-alloge-neic fetal graft (5, 6). Indeed, women with repeated preg-nancy loss were found to have a lower percentage ofendometrial CD56bright CD16� uNK cells and a higherpercentage of CD56dim CD16þ uNK cells, with a greatercytolytic activity, when compared with control patients(1, 7). An increase of activated NK cells (CD56dimCD16þ CD69þ) in the peripheral blood of patients witha reduced rate of embryo implantation in IVF treatment
Received August 22, 2006; revised and accepted January 5, 2007.
Supported by University of Foggia (Foggia, Italy) research grants.
Reprint requests: Maria Matteo, M.D., Operative Unit of Obstetric Gyne-
cology, Department of Surgical Sciences, University of Foggia, viale
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has been reported by several investigators (8–11), butwhether the percentage and phenotype of endometrial leu-kocytes are altered in those patients has not beenextensively explored. We evaluated, by flow cytometry, en-dometrial lymphocyte subpopulations in young patientswho were undergoing IVF cycles for idiopathic infertilitywith a history of repeated unexplained implantation failure.
Endometrium was obtained from 10 patients, aged 30–35years, with normal menstrual cycles and with a history ofrepeated unexplained implantation failure after ovarianstimulation and IVF-ET for idiopathic infertility. Repeatedunexplained implantation failure was defined by at leastthree IVF failures, despite good hormonal reserve (FSHof<8), after transfers of fresh embryo with at least two em-bryos of good quality. Written informed consent was ob-tained from all patients enrolled in the study, which wasapproved by the local ethical committee. Patients takingoral contraceptives or other medications that were knownto interfere with the normal menstrual cycle were excludedfrom the study. Patients were biopsed R6 months after thelast IVF cycle. On day 3 of the menstrual cycle, patients un-derwent FSH and estradiol serum determination as well asultrasound transvaginal examination to exclude any pelvicpathology.
A diagnostic hysteroscopy was performed in the earlyproliferative phase of the cycle to exclude any uterine orendometrial abnormality. In the late secretory phase (LS)of the next spontaneous menstrual cycle, on day 22–26, pa-tients underwent PRL and P serum evaluations, hystero-scopy, and multiple endometrial biopsies performed inseveral sites of the uterine cavity, under hysteroscopicview through a 5-Fr forceps introduced into the operativechannel of a 5-Fr office hysteroscope (Karl Storz, Tuttlin-gen, Germany). A fraction of the sample of the endome-trium obtained from each patient was embedded inparaffin for histological dating, according to Noyes criteria(12); the other part was prepared for flow cytometry.
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2007.01.028
Endometrial tissue was washed extensively in phosphate-buffered saline that was supplemented with 50 mg/mL ofgentamicin before mincing with sterile scissors. Endome-trial lymphocytes were released by digesting the tissuewith 0.1% collagenase type IV and 0.01% DNase I (bothfrom Sigma-Aldrich, Milano, Italy) in RPMI 1640 mediumfor 30 minutes at 37�C. Decidual stromal cells and macro-phages were allowed to adhere to tissue culture plates for2 hours at 37�C in a humidified 5% CO2 incubator.
Lymphocytes from the overlaying solution were subse-quently purified by density gradient centrifugation (Ficoll-Hypaque; Amersham Biosciences). To exclude any biason lymphocyte populations’ recovery deriving from thespecific sample-processing protocol in two cases, we alsotested in parallel (using fractions of the same sample) a dif-ferent cell isolation procedure (13).
Cells were stained with the following monoclonalantibodies, conjugated with peridinin chlorophyll protein,fluorescein isothiocyanate, or phycoerythrin: anti-CD45peridinin chlorophyll protein (clone 2D1; Becton, Dickin-son), anti-CD3/CD16þ56 fluorescein isothiocyanate–phycoerythrin (Simul Test; Becton, Dickinson), anti-CD16fluorescein isothiocyanate (clone 3G8; Beckman Coulter),anti-CD56 phycoerythrin (clone N901/NKH-1; BeckmanCoulter), and anti–CD4/CD8 fluorescein isothiocyanate–phycoerythrin (Dual Colour; Becton, Dickinson). Stainingof cells for flow cytometry was performed as described else-where (14). Briefly, 100 mL of cell suspension in RPMI-1640was added to 5 mL of the appropriate monoclonal antibody.After incubation for 15 minutes, cells were washed, resus-pended in phosphate-buffered saline, and acquired ona FACSCalibur (Becton, Dickinson); analysis was per-formed on R2,000 leukocytes (defined as CD45þ events)by using CellQuest software (Becton, Dickinson). Natu-ral-killer cells were defined as CD56bright when the meanfluorescence intensity was R102 above the background.
Results were expressed as means � SD. Natural-killerCD56, as well as the other lymphocyte subpopulations,was expressed as percentage of total endometrial leuko-cytes. Hysteroscopy was performed in all patients success-fully, and the endometrial biopsies resulted adequate in allcases. The mean weight of endometrial samples was 0.24 g.The mean age of patients was 34 years (range, 30–35 y).The mean number of IVF attempts before participationin the study was four (range, 3–6), with a mean of threeembryos transferred per patient.
The mean FSH level on day 3 of the menstrual cycle was7� 1.1 mIU/mL. Mean serum levels of P and PRL were 5�3.6 ng/mL and 18.7� 6.8 ng/mL, respectively. Histologicaldating, according to Noyes criteria (12), confirmed an LSpattern in all the endometrial samples. At flow cytometry,the mean percentages of CD56þ CD16– cells and ofCD56bright CD16– cells in endometrial samples were80.3% � 3.36% and 70.2% � 6.91%, respectively. Themean percentage of CD3þ cells was 8.8% � 2.7%.
ertility and Sterility�
Within the T-cell population, the proportion of CD4þand CD8þ cells was 50.8% � 2.5% and 49.2%� 2.5%, re-spectively. We observed no difference in the percentage ofendometrial CD56þ CD16– and of CD56bright CD16–cells, or in the percentage of CD3þ cells, or in the propor-tion of CD4þ and CD8þ, of the patients enrolled in thestudy when compared with the percentage of the samelymphocyte subpopulations reported in literature regardingnormal human endometrium (13). Our results are compara-ble with the ones presented in literature, because the differ-ence is not statistically significant either for CD3 (P¼.257)and for CD56þ/CD16� (P¼.616). Furthermore, poweranalysis demonstrated that the type 2 error is b¼0.54 forCD3 and b¼0.45 for CD56þ/CD16�, making the statisticwe used suitable for detection of possible false negatives.
We asked whether the endometrium of infertile womenwith repeated unexplained implantation of good-quality em-bryos after IVF cycles could harbor a decreased percentageof NK cells that were CD56bright CD16� when comparedwith normal human endometrium. Results demonstratedthat in these patients, the percentage of the uNK subpopula-tion of CD56þCD16– and CD56bright CD16– cells did notdiffer in the LS phase from those reported in the literature innormal human endometrium, in the same phase of the men-strual cycle (Fig. 1) (13). The endometrial lymphocyte
FIGURE 1
Typical figures obtained at flow cytometry: uNKCD56bright cells of an endometrial sample that wasobtained from a young infertile patient with a historyof repeated unexplained implantation failure afterIVF cycles. PE ¼ phycoerythrin; FITC ¼ fluoresceinisothiocyanate.
Matteo. Uterine NK cells in IVF implantation failure. Fertil Steril 2007.
991
subpopulation consists predominantly of NK cells and Tlymphocytes CD3þ, composed mainly of CD4þ andCD8þ phenotypes. Flynn et al. (13) demonstrated that inthe LS phase, the proportion of NK cells CD56þCD16–, ex-pressed as a percentage of CD45þ, was about 83.2%, from26.4% in the late proliferative (LP) phase, whereas the per-centage of T lymphocytes in the LS phase was found to be6.7% from 55% in the LP (15). The increase of the endome-trial NK population and the decrease of the CD3þ lympho-cyte proportion in the LS phase are likely to be offundamental importance to the success of implantation. Infact, experimental studies showed that uNK cells may be in-volved in the early steps of the implantation process, in partthrough their role in vascular remodeling (16, 17).
Moreover, uNK cells, but not peripheral blood NK cells,were found to selectively express genes of immunomodula-tory proteins, such as glycodelin and galectin-1 and multipleother genes, including tetraspanins, integrins, lectin-likereceptors, and inhibitory receptors in early pregnancy(KIRs), providing a locally immunosuppressive environ-ment at the maternal–fetal interface (1, 18, 19). Our re-sults showed, in the endometrium of infertile youngwomen, a mean percentage of NK cells CD56 þCD16–and CD56bright CD16–, as well as of CD3 þ cells,that was comparable with the percentage of the samelymphocytes reported by Flynn et al. (13) in normal hu-man endometrium. Within the T-cell population, CD4 andCD8 subsets were nearly equally represented, similar tothe figures obtained in normal fertile women. BecauseNK-cell recruitment is performed under the influence ofsex steroids, we choose to investigate only young womenwith normal menstrual cycles to exclude any influence onthe endometrial decidualization process from steroid-impaired ovarian secretion. Accordingly, in all casesincluded in the study, histological dating confirmed a se-cretory pattern of the endometrial samples.
The role of NK cells in women experiencing repeatedIVF failure already has been investigated in literature. Sev-eral studies reported an increase of activated NK cells(CD56dim CD16þ CD19þ) in the peripheral blood of pa-tients with a reduced rate of embryo implantation in IVFtreatment (8–11). Fukui et al. (20) reported a significant in-crease in the percentage of cytotoxic NK cells, on the day ofembryo transfer, in the peripheral blood of patients with im-plantation failure when compared with their implantedcounterparts. Those same investigators demonstrated a sig-nificant decrease of the percentage of NK cells CD56brightCD16– in the endometrial samples of the aborted groupwhen compared with the endometrium of the deliveredgroup after IVF cycles. A significant reduction in the num-ber of CD56bright cells in the endometrium of women withrepeated unexplained implantation failure after IVF cycleshas been reported recently (21). However, those investiga-tors performed only immunohistochemical analysis on en-dometrial samples obtained on day 21 of a natural cycle,
992 Matteo et al. Correspondence
and, notably, the age of women enrolled in that study rangedfrom 25 to 40 years. Indeed, our data are difficult to com-pare with those generated by Ledee-Bataille et al. (21) be-cause they report numbers of cells observed per field.Moreover, at immunohistochemistry, intensity of antigenexpression can be ill defined. Finally, the endometrium ofolder women, despite good hormonal reserve, could welldisplay a different pattern of lymphocyte subset comparedwith that of younger women, and this could explain at leastin part the discrepancy with our data. To the best of ourknowledge, our study is the first in the literature to befocused on the flow-cytometric analysis and on thephenotypic characterization of the LS endometrial lympho-cyte subpopulations of young patients with a history ofrepeated unexplained implantation failure after IVF cycles.
The repeated implantation failures of young patientsundergoing IVF cycles who were enrolled in our studycould not be related to an abnormal percentage of endome-trial lymphocyte subsets. Further investigations are neededto confirm these preliminary results and to evaluate whetherfunctional defects in uNK cells play a role in the pathogen-esis of repeated unexplained implantation failure after IVFcycles in young women.
Maria G. Matteo, M.D.a
Pantaleo Greco, M.D.a
Piergiorgio Rosenberg, M.D.a
Anna Mestice, Biol.Sc.b
Domenico Baldini, M.D.c
Teresa Falagario, M.D.c
Vincenzo Martino, M.D.c
Michele Santodirocco, M.D.d
Francesca Massenzio, Biol.Sc.d
Laura Castellana, Ph.D.e
Giorgina Specchia, M.D.b
Arcangelo Liso, M.D.da Operative Unit of Obstetrics and Gynecology,
Department of Surgical Sciences, University ofFoggia, Foggia; b Institute of Hematology, Universityof Bari, Bari; c Centre for Medically AssistedProcreation, Casa di Cura S. Maria, Bari; andd Hematology Unit, Department of Medical andOccupational Sciences, and e Department ofBiomedical Sciences, University of Foggia, Foggia,Italy
REFERENCES1. Dosiou C, Giudice LC. Natural killer cells in pregnancy and recurrent
pregnancy loss: endocrine and immunologic perspectives. Endocr Rev
2005;26:44–62.
2. King A. Uterine leukocytes and decidualization. Hum Reprod Update
2000;6:28–36.
3. Saito S. Cytokine network at the feto-maternal interface. J Reprod
Immunol 2000;47:87–103.
4. Bulmer JN, Morrison L, Longfellow M, Ritson A, Pace D. Granulated
lymphocytes in human endometrium: histochemical and immunohisto-
chemical studies. Hum Reprod 1991;6:791–8.
Vol. 88, No. 4, October 2007
5. Mincheva-Nilsson L, Kling M, Hammarstrom S, Nagaeva O,
Sundqvist KG, Hammarstrom ML, et al. Gamma delta T cells of hu-
man early pregnancy decidua: evidence for local proliferation, pheno-
typic heterogeneity, and extrathymic differentiation. J Immunol
1997;159:3266–77.
6. Streilein JW, Wegmann TG. Immunologic privilege in the eye and the
et al. Human decidual natural killer cells are a unique NK cell subset
with immunomodulatory potential. J Exp Med 2003;198:1201–12.
19. Ponte M, Cantoni C, Biassoni R, Tradori-Cappai A, Bentivoglio G,
Vitale C, et al. Inhibitory receptors sensing HLA-G1 molecules in
pregnancy: decidua-associated natural killer cells express LIR-1 and
CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proc
Natl Acad Sci USA 1999;96:5674–9.
20. Fukui A, Fujii S, Yamaguchi E, Kimura H, Sato S, Saito Y. Natural
killer cell subpopulations and citotoxicity for infertile patients under-
going in vitro fertilization. Am J Reprod Immunol 1999;41:413–22.
21. Ledee-Bataille N, Dubanchet S, Kadoch J, Castello-Branco A,
Frydman N, Chaouat G. Controlled natural in vitro fertilization
may be an alternative for patients with repeated unexplained implan-
tation failure and a high uterine natural killer cell count. Fertil Steril
2004;82:234–6.
993
In vitro sildenafil citrate use as a sperm motilitystimulant
The effect of different concentrations of sildenafil citrate (Viagra) solution in vitro on human sperm motility wasassessed in 85 asthenozoospermic semen specimens. Semen samples were divided equally into six tubes, one asa control and to the others, sildenafil dissolved solution was added (v/v) in vitro with five different concentra-tions (4.0, 2.0, 1.0, 0.5, 0.1 mg/mL). The tubes were incubated and were followed up for sperm motility changesat 0.5, 1, 2, and 3 hours. It is demonstrated that the in vitro use of sildenafil citrate solution has a concentration-related stimulatory effect on ejaculated sperm motility. (Fertil Steril� 2007;88:994–6. �2007 by AmericanSociety for Reproductive Medicine.)
Spermatozoa are highly specialized cells to accomplisha single objective: fertilization of an ovum. During theirtransport in either the male or the female reproductivetracts, spermatozoa encounter different environments inwhich they have to survive. Motility is one of the featuresthat has been investigated due to its importance during thefertilization process (1). Not only have new methods beendeveloped to evaluate motion characteristics, but manypharmacologic treatments for weak sperm motility suchas pentoxifylline, caffeine, and follicular fluid (FF) havealso been suggested (2).
Sildenafil citrate, the first introduced phosphodiesterase-5(PDE5) inhibitor, has proven effective in the treatment ofmale erectile dysfunction (3). In addition it has diversebeneficial, nonerectogenic actions in the management of pul-monary hypertension, female sexual dysfunction, enhancedfemale genital blood flow, endometrial thickening, some gas-trointestinal disorders, and Raynaud’s phenomenon (4).
The effect of sildenafil on human sperm motility is de-bated. Aversa et al. (5) indicated that in healthy subjects,acute oral sildenafil treatment does not modify semen char-acteristics. Lefievre et al. (6) demonstrated that sildenafiltriggers human sperm motility, probably by its inhibitoryaction on PDE activity other than type 5 with a resultant in-crease in cAMP. Burger et al. (7) indicated that sildenafil, atthe doses they used, did not alter the motility or viability ofhuman sperms. Andrade et al. (8) showed that 200 mg/mL invitro dose of sildenafil had no effect on sperm motility, buta dose of 2,000 mg/mL significantly reduced it. Purvis et al.(9) showed that 100 mg of sildenafil orally had no an ad-verse effect on ejaculate quality. du Plessis et al. (10)showed that oral sildenafil with in vitro 8-bromo-cGMPcan enhance sperm motility.
This study aimed to assess the effect of the in vitro use ofsildenafil citrate solution on human sperm motility.
Received May 9, 2006; revised November 29, 2006; accepted
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine,
994
Eighty-five asthenozoospermic semen specimens wereselected prospectively from the Andrology laboratory ofthe University Hospital. The study protocol was approvedby the Institutional Review Board (IRB) with signed in-formed consents. Cases with leukocytospermia or scrotalvaricocele were excluded. Medical history and physical ex-amination was performed for the studied cases (age range18–39 years). Ejaculates were obtained by masturbationinto a dry wide-mouthed sterile plastic container (7.00–9.30 AM) after 4–5 days of sexual abstinence. Twelve hoursbefore attendance, each subject was asked to refrain fromcaffeine or nicotine-containing agents. The samples wereexamined immediately after liquefaction by light micros-copy according to WHO guidelines (11) to estimate initialresults.
Semen samples were divided into six equal volumes into3-mL falcon tubes (Becton Dickinson, Franklin Lakes, NJ),one was kept as a control (semen þ vehicle v/v) and to theothers similar volume (v/v) of sildenafil solution withdifferent concentrations were added (4.0, 2.0, 1.0, 0.5,0.1 mg/mL). Then, all tubes were incubated at 37�C. Thesildenafil citrate solution was prepared by dissolving com-pletely a crushed tablet in distilled water. Sperm motilitypercent was assessed in the tested samples after 0.5, 1, 2,3 hours.
It was demonstrated that control samples showed declineof percent sperm motility with time. Sildenafil citrate solu-tions added to the asthenozoospermic semen samplesshowed immediate changes in their sperm motility. It wasfound that the concentration used played an importantrole in the degree of enhancement. Semen specimenstreated with 4 mg/mL sildenafil solution demonstratedsignificant decreases in sperm motility compared to the con-trols. Specimens treated with 2 mg/mL sildenafil solutiondemonstrated initial nonsignificant increases in sperm mo-tility. Specimens treated with 1.0 mg/mL sildenafil solutiondemonstrated significant increases in progressive forwardmotility. These effects were shown to be reproducible fordifferent semen samples at the same time or at differenttimes. Specimens treated with 0.5 and 0.1 mg/mL sildenafilsolutions demonstrated significant increases also in sperm
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.11.182
a significant, P< .05.b significant, P< .01.c significant, P< .001; other relations nonsignificant (P>.05).
Mostafa. In vitro sildenafil citrate use as a sperm motility stimulant. Fertil Steril 2007.
motility but lower than that with the 1 mg/mL concentration(Table 1, Fig. 1).
The results of the present study indicated that the in vitroaddition of sildenafil citrate solution to ejaculated semenspecimens can exhibit changes in sperm motility attributedto the concentration used, mostly due to the tolerable pH aschanges in pH were shown to affect sperm motility (12).Previous studies gave different observations, mostly dueto the methodologies or the doses used. Burger et al. (7)used a single Percoll (80%) gradient, suspended in Ham’sF-10 medium, and incubated with various doses of sildenafil(125, 250 and 750 ng/mL); pentoxifylline (3 mM) as a pos-itive control, and Ham’s F-10 as a reagent control. Theyfound that sildenafil did not affect sperm motility or viabil-ity as compared to the control or to pentoxifylline. Andradeet al. (8) found that 200 mg/mL in vitro doses had no effecton sperm motility, but a dose of 2,000 mg/mL significantlyreduced it by 50%, mostly due to reduced pH. This biphasicresponse of sperm motility to sildenafil action resembles
Fertility and Sterility�
nitric oxide action, which stimulates sperm motility at lowconcentrations and acts as an oxidant at higher one (13).
Sildenafil citrate is one of the PDE inhibitors that includespentoxifylline, 3- isobutyl-1-methyl xanthine, and caffeine.Levels of cAMP are regulated by two main metabolic reac-tions: synthesis from ATP through the action of adenylyl cy-clase and degradation by cyclic nucleotide (14). PDEhydrolyzes the 30,50-phosphodiester bond of cAMP to main-tain low levels of cAMP and regulate its effects at a rate 9- to600-fold faster than that of its formation in human sperma-tozoa (15), suggesting its importance in the control of cyclicnucleotide levels in sperms. Two signaling pathways haveemerged as central to normal mammalian sperm motility:cAMP/protein kinase-A (PK-A) pathway and calcium sig-naling (12). Cyclic AMP also may work through pathwaysindependent of PK-A providing an alternative pathwaysfor the PK-A-mediated regulation of flagella motility (16).
Two PDEs, the calcium-calmodulin-dependent (type 1)and the cAMP-specific (type 4), have been reported in
FIGURE 1
Time course study of in vitro effect of sildenafil on sperm motility percent.
0
10
20
30
40
50
60
70
0 hr 1/2 hr 1 hr 2 hrs 3 hrs
Control
Sild. 4 mg/ml
Sild. 2 mg/ml
Sild. 1 mg/ml
Sild. 0.5 mg/ml
Sild. 0.1 mg/ml
Mostafa. In vitro sildenafil citrate use as a sperm motility stimulant. Fertil Steril 2007.
995
spermatozoa (17). Moreover, messenger RNA transcripts ofsix PDEs have been found in the ejaculated human sperma-tozoa (18). Lefievre et al. (6) used a highly potent cGMP-specific PDE (type 5) inhibitor to investigate whether thisPDE is present in the human spermatozoa. They concludedthat PDE5 represents a small fraction of the whole PDEsperm activity because the IC50 of sildenafil obtained forPDE5 was much lower than that obtained with spermPDE. They showed also that sildenafil causes dose-depen-dent increases in sperm cAMP levels associated with in-creased levels of tyrosine phosphorylation of two fibroussheath proteins (p105/81). They observed that the sperm ve-locity, amplitude of lateral head displacement, and hyper-activation were increased at 30–180 minutes withouttriggering the acrosome reaction.
It is concluded that the in vitro use of sildenafil citrate so-lution has a concentration-related stimulatory effect onejaculated sperm motility. This adds for the beneficial ther-apeutic spectrum of the PDE5 inhibitors.
14. Bentley JK, Beavo JA. Regulation and function of cyclic nucleotides.
Curr Opin Cell Biol 1992;4:233–40.
15. Cheng CY, Boettcher B. Partial characterization of human spermato-
zoal phosphodiesterase and adenylate cyclase and the effects of ste-
roids on their activities. Int J Androl 1982;5:253–66.
16. Burton KA, Treash-Osio B, Muller CH, Dunphy EL, McKnight GS.
Deletion of type II-alpha regulatory subunit delocalizes protein kinase
A in mouse sperm without affecting motility or fertilization. J Biol
Chem 1999;274:24131–6.
17. Naro F, Zhang R, Conti M. Developmental regulation of unique aden-
osine 30,50-monophosphate-specific phosphodiesterase variants during
rat spermatogenesis. Endocrinol 1996;137:2464–72.
18. Richter W, Dettmer D, Glander HJ. Detection of mRNA transcripts of
cyclic nucleotide phosphodiesterase subtypes in ejaculated human
spermatozoa. Mol Hum Reprod 1999;5:732–6.
Vol. 88, No. 4, October 2007
Relative contribution of ovarian stimulation versusin vitro fertilization and intracytoplasmic sperminjection to multifetal pregnancies requiringreduction to twins
The proportion of twins resulting from multifetal pregnancy reduction of higher-order multiples is increased inpregnancies resulting from hormone stimulation when compared with twins following in vitro fertilization/in-tracytoplasmic sperm injection treatment. These reduced twin pregnancies may carry a higher perinatal riskcompared with other twin pregnancies, which should be taken into account when assessing the perinatal out-come of twin pregnancies after assisted reproduction. (Fertil Steril� 2007;88:997–9. �2007 by AmericanSociety for Reproductive Medicine.)
Over the past few decades, an epidemic of iatrogenic mul-tiple births has been observed in most Western countriesdue to the widespread use of assisted reproductive technol-ogies (ART). Pregnancies with multiples, associated withinfertility treatment, has to be regarded as an adverse out-come (1). In in vitro fertilization/intracytoplasmic sperm in-jection (IVF/ICSI) programs, prevention has becomemandatory by reducing the number of embryos transferred(2). Despite the changes in the embryo transfer practices,the incidence of multiple gestation remains high in theUnited States, mainly due to the increased twin rate associ-ated with the transfer of two embryos (3). For non-IVF hor-mone stimulation, which is responsible for more than onethird of all multiple pregnancies after ART, adherence toa strict ovarian stimulation (OS) protocol aiming at mono-ovulation is crucial (1, 4).
According to the 2004 report from the U.S. Centers forDisease Control and Prevention (CDC), the incidence ofmultifetal pregnancy in nondonor IVF patients was 36%in 2002 (5). An estimated 53% of multiple births in theUnited States are the result of ART (6). It was shown that,as of 2002, higher-order multiple gestations associatedwith ART were responsible for more than 40% of all tripletand higher multiple births in the United States and that thetotal number was still increasing (5). However, in terms ofabsolute figures, because of their higher incidence, twinpregnancies outweigh by far the effects of higher-ordermultiple pregnancies.
In 2005, based on the register of the Flanders, Belgium,Study Centre for Perinatal Epidemiology (SPE), we re-ported on the perinatal outcome of 26,656 ART pregnancies(IVF/ICSI and non-IVF hormonal stimulation) (1). Pretermdelivery occurred in 95.9% of triplet pregnancies (meangestational age: 32.7 weeks) and 53.8% of twin deliveries
Received November 8, 2006; revised and accepted December 20, 2006.
Reprint requests: Willem Ombelet, M.D., PH.D., Genk Institute for Fertil-
0015-0282/07/$32.00doi:10.1016/j.fertnstert.2006.12.036 Copyright ª2007 American
(mean gestational age: 35.6 weeks). The incidence of earlypreterm delivery (<32 weeks) and very low birth weight in-fants (less than 1500 grams) was almost three times higherin triplet pregnancies compared with twins after ART,whether or not IVF-related. As the number of fetuses in-creased, the duration of gestation decreased at a mean of3 weeks per additional fetus. Our results were very similarto the U.S. data reported in 1999 by Keith and Oleszczuk(7), which described the gestational age and birth weightcharacteristics of 3,603,971 singletons, 463,856 twins,and 18,843 triplets. Comparable figures were also reportedby Martin et al. (8). On the other hand, dramatic improve-ment in neonatal care for premature babies has resulted ina 95% and 92% take-home-baby rate for triplets and qua-druplets, respectively, in the United States (9–11).
However, even if the infants of multiple pregnancies sur-vive the early postnatal period, they continue to have an in-creased risk for long-term developmental and physicaldisabilities. Compared with singletons, triplets have a post-neonatal survivors’ relative risks for severe handicap and in-fant mortality of 1.7 and 19.4, respectively (12). The risk ofhandicap increases with the number of fetuses, and at leastone handicapped child is found in 7.4%, 21.6%, and 50% fortwin, triplet and quadruplet pregnancies, respectively (13).
Considering these figures, it is not surprising thatmultiple pregnancy reduction (MPR) has become a well-established part of infertility therapy to improve the chancesof survival and health of the remaining fetuses, especially intriplet and higher-order multiple pregnancies (4, 9–11).Multiple pregnancy reduction is performed either transab-dominally or transvaginally between 10 and 13 weeksof gestation. In triplet and higher-order multiple pregnan-cies, MPR increases survival rates and lessens obstetriccomplications at the expense of a small pregnancy lossrate (14–17). Reduction of triplet pregnancies to twins in-creases gestation by 2 to 3 weeks, and reduction to twinsfrom higher order multiplets prolongs the duration of gesta-tion even more (4).
Fertility and Sterility� Vol. 88, No. 4, October 2007 997Society for Reproductive Medicine, Published by Elsevier Inc.
It is believed that about 50% of triplet and quadrupletgestations undergo spontaneous fetal demise in the first tri-mester of pregnancy and will continue as viable singletonsor twins (18). Looking at these figures, MPR might be anoption to improve perinatal outcome in almost half of trip-lets and quadruplet gestations. The acceptability of MPR,even in higher-order multiple pregnancies, will dependboth on ethnic/cultural background and underlying beliefs(16); however, Bergh et al. (19) reported that, althoughwomen who have undergone MPR experience some feel-ings of sadness and guilt following the procedure, almostall couples were convinced that higher-order pregnanciesshould be avoided.
On the other hand, it is very important to realize thattwins resulting from MPR may have a higher risk for pre-term delivery and/or low birth weight compared with non-reduced twins, especially when MPR was performed inhigher-order multiples (20–26). Other studies have de-scribed comparable outcomes for reduced and unreducedtwins, especially when considering triplet pregnancies re-duced to twins (27–31).
Data from the United States have shown that multifetalpregnancies following ART were caused by OS and IVF/ICSI in 30% and 70% of cases, respectively (11). Surpris-ingly, there are no data available on the relative proportionof IVF/ICSI and OS pregnancies in MPR programs.
In this study, we present data of 475 multifetal pregnancyreductions to twins performed in Flanders (Belgium) duringthe period of 1993 to 2004. Data were collected by personalcommunication with the directors of all recognized infertil-ity and perinatal centers in Flanders. More than 95% (453out of 475) of all embryo reductions were done by two ex-perienced clinicians (L.D.C., M.C.). These data were pro-spectively registered during the study period by bothinvestigators. First, we studied the relative proportion ofIVF/ICSI and OS in the total number of multifetal pregnancy
998 Ombelet et al. Correspondence
reductions. Second, our data were compared with the SPEdata for the total number of twin pregnancies in Flandersduring the same study period. The SPE prospectively col-lects data on medical and obstetric history, and on perinatalevents for each hospital delivery in Flanders of more than 21weeks of gestational age and R500 grams at birth. Full co-operation of all 80 departments of obstetrics in Flanders hasbeen established since 1991. Each year, a complete analysisof these data is performed and published as a yearly globalreport and as a unique report for each participating obstetricunit. A small chance of underreporting MPR is present inthis study if a procedure was performed outside the recog-nized centers, but this is very unlikely under current obstetricpractice in Flanders. Also, undetected MPR would only re-sult in an underestimation of MPR secondary to non-IVFhormone stimulation as IVF procedures are only allowedin recognized centers. Therefore, all MPR following IVF/ICSI should be present in this study, although this may notbe true for OS pregnancies.
Table 1 shows the number of multifetal pregnancy reduc-tions to twins categorized according to origin. Of allMPR twins, the relative proportion was 5.7%, 44.6%, and49.6% for natural conception, IVF/ICSI, and OS pregnan-cies, respectively. Our study showed that, during the periodof 1993 to 2004, MPR was performed in 14.1% (236 out of1673) of OS twins compared with 6.9% (212 out of 3060)for IVF/ICSI twins (P<.001). The great majority (78.8%)of reductions of higher-order multiples to twins were per-formed in pregnancies following OS; IVF/ICSI was only re-sponsible for 18.8% of MPR in higher-order multiples(difference: P<.001). Out of 1673 OS twins, 40 (4%)were the result of a higher-order MPR compared withonly 0.5% (16 out of 3060) for IVF/ICSI (P<.001).
The results shown in Table 1 demonstrate that ART-related twins are more likely to be the result of MPRcompared with naturally conceived twins, and MPR ismore frequently carried out for higher-order multiples in
TABLE 1Multifetal pregnancy reduction (MPR) to twins in Flanders during the period of 1993 to 2004.
Note: To compare the relative proportion of higher-order and triplet MPR to twins for non-IVF OS and IVF/ICSI, we useda chi-square test for the comparison of two proportions from independent samples. IVF, in vitro fertilization; ICSI, intra-cytoplasmic sperm injection; NC, natural conception; OS, ovarian stimulation; SPE, Study Centre for Perinatal Epidemi-ology.
a Highly statistically significant, P< .001.b Not statistically significant.c Total number of twins in Flanders during the period of 1993 to 2004.
Ombelet. Multifetal pregnancy reduction to twins. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
the non-IVF hormone stimulation subgroup. As in mostcountries, registration of non-IVF OS pregnancies is notavailable in Flanders, so it is impossible to estimate the ex-act risk for triplets and higher-order multiples in this partic-ular subgroup.
This is the first study reporting on the relative contribu-tion of OS and IVF/ICSI when MPR to twins is performed.It is difficult to put our results in perspective because ofa lack of comparative reported data. However, becauseMPR can be responsible for a higher incidence of pretermdelivery and low birth weight, the type of ART, original fe-tal count, and/or application of MPR should be taken intoconsideration when assessing the perinatal outcome oftwin pregnancies following ART. According to our results,non-IVF OS is associated with an unexpected high rate ofhigher-order multiple gestations and thus needs to be mon-itored more carefully.
Acknowledgments: The authors thank Eric de Jonge, M.D., Ph. D., and Piet
Hinoul, M.D., for the critical revision of this article, and Ingrid Jossa for
technical support in preparing this manuscript. We also thank all of the
Flemish midwifes, obstetricians, and pediatricians who have completed
the SPE questionnaires for many years.
Willem Ombelet, M.D., Ph.D.a
Michel Camus, M.D.b
Luc de Catte, M.D., Ph.D.ca Genk Institute for Fertility Technology, Department of
Obstetrics and Gynaecology, Genk; bCenter forReproductive Medicine of the Vrije UniversiteitBrussel, Brussels; and c Department of Obstetrics andGynecology, University Hospital Vrije UniversiteitBrussel, Brussels, Belgium
REFERENCES1. Ombelet W, De Sutter P, Van der Elst J, Martens G. Multiple gestation
and infertility treatment: registration, reflection and reaction: the Bel-
gian project. Hum Reprod Update 2005;11:3–14.
2. Elster N. Less is more: the risk of multiple births. Fertil Steril 2000;74:
617–23.
3. Reynolds MA, Schieve LA. Trends in embryo transfer practices and
multiple gestation for IVF procedures in the USA, 1996–2002. Hum
Reprod 2006;21:694–700.
4. Stone J, Eddleman K, Lynch L, Berkowitz RL. A single center experi-
ence with 1000 consecutive cases of multifetal pregnancy reduction.
Am J Obstet Gynecol 2002;187:1163–7.
5. U.S. Centers for Disease Control and Prevention. 2002 Assisted repro-
ductive technology success rates: national summary and fertility clinic
25. Luke B, Brown MB, Nugent C, Gonzalez-Quintero VH, Witter FR,
Newman RB. Risk factors for adverse outcomes in spontaneous
versus assisted conception twin pregnancies. Fertil Steril 2004;81:315–9.
26. Multiple gestation: complicated twin, triplet, and high-order multifetal
pregnancy. ACOG Practice Bulletin no. 56. Obstet Gynecol 2004;104:
869–83.
27. Lipitz S, Uval J, Achiron R, Schiff E, Lusky A, Reichman B. Outcome
of twin pregnancies reduced from triplets compared to nonreduced
twin gestations. Obstet Gynecol 1996;87:511–4.
28. Antsaklis AJ, Drakakis P, Vlazakis GP, Michalas S. Reduction of multi-
fetal pregnancies to twins does not increase obstetric and perinatal
risks. Hum Reprod 1998;14:1338–40.
29. Selam B, Lembet A, Stone J, Lapinski R, Berkowitz RL. Pregnancy
complications and neonatal outcomes in multifetal pregnancies
reduced to twins compared with nonreduced twin pregnancies. Am J
Perinatol 1999;16:65–71.
30. Yaron Y, Bryant-Greenwood PK, Dave N, Moldenhauer JS,
Kramer RL, Johnson MP, Evans MI. Multifetal pregnancy reductions
of triplets to twins: comparison with nonreduced triplets and twins.
Am J Obstet Gynecol 1999;180:1268–71.
31. Barr S, Poggi S, Keszler M. Triplet morbidity and mortality in a large
case series. J Perinatol 2003;23:368–71.
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1
Korean ginseng induces spermatogenesis in ratsthrough the activation of cAMP-responsive elementmodulator (CREM)
The effect of Korean ginseng (ginseng) on spermatogenesis and cAMP-responsive element modulator (CREM)in rat testes was evaluated using sperm analysis, reverse transcription polymerase chain reaction, and Westernblot analysis. The ginseng-treated rats exhibited significantly increased sperm count and motility with enhancedlevels of CREM messenger RNA and protein. Ginseng appears to induce spermatogenesis via CREM activationin rat testes. (Fertil Steril� 2007;88:1000–2. �2007 by American Society for Reproductive Medicine.)
Infertility affects 15% of all couples; in 39% of these cou-ples, the male generates semen analyzed as abnormal (1).Spermatogenetic failure, including azoospermia and oligo-spermia, is one of the important causes of male infertility(2). The transcription factor cAMP-responsive elementmodulator (CREM) plays an essential role in primate sper-matid maturation (3). Male mice lacking a functionalCREM gene are infertile. Infertile men with arrested roundspermatid maturation exhibit substantially reduced levels ofCREM protein and CREM messenger (m) RNA or none atall (4). Combinations of genetic changes in the humanCREM gene can explain some forms of male infertility (5).
Traditionally, Korean ginseng (ginseng), the root ofPanax ginseng C. A. Meyer (Araliaceae), has been usedin herbal medicine as a tonic and in antiaging prescriptions(6). Its major compounds are the ginseng saponins of ginse-nosides (7). Earlier reports have shown that ginseng protectsagainst testicular toxicity (8). However, few studies haveused rat testes to examine the effects of ginseng on male re-productive functions. This study investigated the effect ofginseng on spermatogenesis and CREM expression in rattestes, using sperm analysis and a molecular biologicmethod.
Ginseng was purchased from Wonkwang Herb (Seoul,Korea). A 300-g sample of dried ginseng was boiled with6 L water for 2 hours at 100�C, and the suspension wasthen filtered and concentrated under reduced pressure.The filtrate was lyophilized, yielding 76.60 g (26%) of pow-der, which was stored at 4�C. Before each experiment, thedried extract was dissolved in distilled deionized waterand vortexed for 2 minutes at room temperature.
Male Wistar rats (8 weeks old) were purchased fromJapan SLC (Shizuoka, Japan). They were housed in a specific
Received April 28, 2006; revised and accepted December 28, 2006.
Reprint requests: Seong Kyu Park, M.D., Ph.D., Department of Prescrip-
tionology, College of Oriental Medicine, Kyung Hee University,
1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Korea (FAX: þ82-
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine,
000
pathogen-free environment with a 12/12-hour light/dark cy-cle and with free access to standard rodent pellets (Purina,Seongnam, Korea) and water. Animal care and experimentalprocedures followed the requirements in the ‘‘Guide for theCare and Use of Laboratory Animals’’ (Department ofHealth, Education, and Welfare, National Institutes ofHealth, 1996), which was approved by Institutional ReviewBoard of College of Oriental Medicine in Kyung HeeUniversity.
After 7 days of adaptation, the rats were blindly random-ized into two treatment groups, the control group (n ¼ 8)and the ginseng-treated group (n ¼ 8). The ginseng-treatedgroup was administered ginseng (1.0 g/kg/day orally) dis-solved in water for 56 days. The same volume of vehiclewas administered to the control rats. After the treatment pe-riod ended, the rats were anesthetized with pentobarbital so-dium (50 mg/kg IP). Their testes were removed, cleared ofadhering tissue, and weighed. Each epididymis was re-moved and used for sperm analysis. Testes samples werefrozen for reverse transcription polymerase chain reaction(RT-PCR) and Western blotting assays.
Epididymal sperm counts and motility were evaluatedwith minor modification as described elsewhere (9). Spermcounts were obtained by mincing an entire rat epididymis inM199 media containing 0.5% BSA and incubating it for 5minutes at 37�C. Sperm motility was assessed by recover-ing sperm from excised caudal epididymis and allowingthe sample to capacitate for 5 minutes at 37�C in thesame solution. Sperm were considered motile if they ex-hibited any movement. The numbers of total sperm andmotile sperm were determined using a hemocytometer(Neubauer, Wolfenbuettel, Germany).
Fenozol (Active & Motif, Carlsbad, CA) was used to iso-late total RNA from testis tissue samples, following themanufacturer’s instruction. First strand cDNA synthesiswith 2 mg total RNA was performed using MMLV reversetranscriptase and oligo dT primer for 1 hours at 42�C.The PCR amplification was performed in a reaction volumeof 25 mL containing 5 mL of the appropriate cDNA, 1 mL of
0015-0282/07/$32.00Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.014
Effect of Korean ginseng treatment on spermatogenesis and expressions of cAMP-responsive elementmodulator (CREM) mRNA and protein in rat testes. b-Actin was used as the internal control. Results arepresented as mean � SE. Sperm count (A) and motility (B) were significantly increased. A remarkable increasein expression of CREM mRNA (C) was detected, which was confirmed by the significant increase in expressionof CREM protein (D). *P<.05 compared with the control group. Control ¼ control group; GR ¼ Koreanginseng–treated (56 days) group.
Park. Ginseng enhances spermatogenesis in rats. Fertil Steril 2007.
each set of primers at concentration of 4 pmol/L, 2.5 mL of10-fold reaction buffer, 1 mL of 2.5 mmol/L dNTP, and1 unit of Taq DNA polymerase (Bioquest, Seoul, Korea).For CREM, the primer sequences were 50-GATTGAAGAAGAAAAATCAGA-30 as forward primer (exon B)and 50-TTGACATATTCTTTCTTCTT-30 as reverse primer(exon H). The rat b-actin was used as the internal control(10). The mixture was heated at 95�C for 5 minutes, followedby 35 sequential cycles of primer extension at 72�C for 1.5minutes, heat denature at 95�C for 1.5 minutes, and primerannealing at 52�C–56�C for 3 minutes. The PCR productswere separated on a 1.5% agarose gel, visualized by ethid-ium bromide using i-Max gel image analysis system (Core-BioSystem, Seoul, Korea), and analyzed using Alpha EasyFC software (Alpha Innotech, San Leandro, CA).
Proteins from homogenized testes were separated usinga nuclear extract kit (Active & Motif). The samples usedfor protein extraction were the halves of the testes thatremained from the RNA extraction. Fifty micrograms ofprotein were electrophoresed in SDS-polyacrylamide gelsand transfered onto nitrocellulose membranes; X-12CREM-1 (Santa Cruz Biotechnology, Santa Cruz, CA)was used as the primary antibody against CREM-t, and
Fertility and Sterility�
conjugated goat antirabbit IgG (Sigma, St. Louis, MO)was used as the secondary antibody. Band detection wasperformed using a SeePico CBB stain kit (Benebiosis,Seoul, Korea).
The data were expressed as mean � SE and assessed sta-tistically using the Student t test. All statistical analyseswere performed using SPSS v11.0 for Windows (SPSS,Chicago, IL). The significance level was .05.
No noticeable adverse effects, including animal death,were observed in any of the animals after administrationof the ginseng extract or vehicle. The weights of body andtestes, respectively, from the ginseng-treated group were104.33 � 1.00% and 102.73� 2.18% of the control values.No significant differences were observed. Figures 1A and1B show the sperm counts and motility for the two groups;the ginseng-treated group exhibited significantly increasedsperm count (161.53 � 16.04%) and motility (125.33 �10.50%) compared with the control group (P<.05). Thetestes from the ginseng-treated group show markedly in-creased CREM mRNA (Fig. 1C). The expression ofCREM protein from the ginseng-treated group was 108.41� 1.19% of the control value (Fig. 1D; P<.05).
1001
Recent research has indicated that ginseng improvessperm quality in guinea pigs exposed to dioxin (11). Al-though many clinical trials have attempted to improve thequality and number of sperm, the results have been limited(12). In the present study, rats treated with ginseng ex-hibited significantly increased epididymal sperm countcompared with those of the control group. Ginseng treat-ment also improved sperm motility but produced littlechange in body or testes weights.
cAMP-Responsive element modulator is a nuclear factorinvolved in the regulation of gene expression by cAMP andhas an important role in spermatogenesis (13). Germ celldifferentiation is dependent on CREM function (14). Thefunction of CREM in human spermatid development sug-gests that altered CREM expression could be associatedwith defects in spermatid maturation in some cases of idio-pathic male infertility (15). cAMP-Responsive elementmodulator was also reported to be related with genes impli-cated in sperm motility (16). Male mice deficient in anyCREM proteins are sterile (17). The present study foundthat rats treated with ginseng exhibit increased CREMmRNA and CREM protein expression.
These results indicate that ginseng may improve the mo-tility and total number of sperm, probably through CREMactivation, with no severe side effects. This suggests that gin-seng could be recommended as a treatment for spermatogen-esis-related male infertility. A molecular understanding ofhow ginseng enhances spermatogenesis would help broadenthe therapeutic categories for male factors of infertility.
Wan Su Park, M.D., Ph.D.a
Dong Youp Shin, M.D., Ph.D.b
Do Rim Kim, M.Sc.b
Woong Mo Yang, M.D.b
Mun Seog Chang, Ph.D.b
Seong Kyu Park, M.D., Ph.D.ba Department of Pathology, College of Oriental
Medicine, Kyungwon University, Seongnam;and b Department of Prescriptionology, College ofOriental Medicine, Kyung Hee University, Seoul,Korea
REFERENCES1. World Health Organization (WHO). Towards more objectivity in
diagnosis and management of male infertility. Results of a World Health
Organization multi-centre study. Int J Androl 1987;10(Suppl 7):1–35.
1002 Park et al. Correspondence
2. Shinka T, Sato Y, Chen G, Naroda T, Kinoshita K, Unemi Y, et al.
Molecular characterization of heat shock–like factor encoded on the
human Y chromosome, and implications for male infertility. Biol
Reprod 2004;71:297–306.
3. Behr R, Weinbauer GF. cAMP response element modulator (CREM):
an essential factor for spermatogenesis in primates. Int J Androl
2001;24:126–35.
4. Steger K, Behr R, Kleiner I, Weinbauer GF, Bergmann M. Expression
of activator of CREM in the testis (ACT) during normal and impaired
spermatogenesis: correlation with CREM expression. Mol Hum Re-
prod 2004;10:129–35.
5. Vouk K, Hudler P, Strmsnik L, Fink M, Majdic G, Zorn B, et al. Com-
binations of genetic changes in the human cAMP-responsive element
modulator gene: a clue towards understanding some forms of male
infertility? Mol Hum Reprod 2005;11:567–74.
6. Ji YC, Kim YB, Park SW, Hwang SN, Min BK, Hong HJ, et al. Neuro-
protective effect of ginseng total saponins in experimental traumatic
brain injury. J Korean Med Sci 2005;20:291–6.
7. Ali MB, Hahn EJ, Paek KY. CO(2)-induced total phenolics in suspen-
sion cultures of Panax ginseng C.A. Mayer roots: role of antioxidants
and enzymes. Plant Physiol Biochem 2005;43:449–57.
8. Kang J, Lee Y, No K, Jung E, Sung J, Kim Y, et al. Ginseng intestinal
metabolite-I (GIM-I) reduces doxorubicin toxicity in the mouse testis.
Reprod Toxicol 2002;16:291–8.
9. Connolly CM, Dearth AT, Braun RE. Disruption of murine Tenr
results in teratospermia and male infertility. Dev Biol 2005;278:
13–21.
10. Li H, Dunbar JC, Dhabuwala CB. Expression of cAMP-respon-
sive element modulator (CREM) in rat testes following chronic
cocaine administration. J Environ Pathol Toxicol Oncol 2003;22:
111–6.
11. Hwang SY, Kim WJ, Wee JJ, Choi JS, Kim SK. Panax ginseng im-
proves survival and sperm quality in guinea pigs exposed to 2,3,7,
8-tetrachlorodibenzo-p-dioxin. BJU Int 2004;94:663–8.
12. Charny CW. Clomiphene therapy in male infertility: a negative report.
Fertil Steril 1979;32:551–5.
13. Li H, Dunbar JC, Dhabuwala CB. Expression of cAMP-respon-
sive element modulator (CREM) in rat testes following chronic
cocaine administration. J Environ Pathol Toxicol Oncol 2003;22:
111–6.
14. Wistuba J, Schlatt S, Cantauw C, von Schonfeldt V, Nieschlag E,
Behr R. Transplantation of wild-type spermatogonia leads to complete
spermatogenesis in testes of cyclic 30,50-adenosine monophosphate re-
sponse element modulator–deficient mice. Biol Reprod 2002;67:
1052–7.
15. Weinbauer GF, Behr R, Bergmann M, Nieschlag E. Testicular cAMP
responsive element modulator (CREM) protein is expressed in round
spermatids but is absent or reduced in men with round spermatid
maturation arrest. Mol Hum Reprod 1998;4:9–15.
16. Horowitz E, Zhang Z, Jones BH, Moss SB, Ho C, Wood JR, et al. Pat-
terns of expression of sperm flagellar genes: early expression of genes
encoding axonemal proteins during the spermatogenic cycle and
shared features of promoters of genes encoding central apparatus
proteins. Mol Hum Reprod 2005;11:307–17.
17. Blendy JA, Kaestner KH, Weinbauer GF, Nieschlag E, Schutz G. Se-
vere impairment of spermatogenesis in mice lacking the CREM
gene. Nature 1996;380:162–5.
Vol. 88, No. 4, October 2007
0d
Differential effects of aging on activin A and its bindingprotein, follistatin, across the menopause transition
To assess the involvement of ovarian-derived regulatory proteins in FSH modulation, we compared FSH, in-hibin A, inhibin B, activin A, and follistatin (FS) in 79 women from the following five groups: young cycling,older cycling, perimenopause (PERI), spontaneous menopause (PM), and surgical menopause receiving estro-gen (OVXþET). Although inhibin B varied as expected by ovarian function, no group differences were ob-served in activin A, barring a tendency for an increase in PERI, while FS 288 was lower in the PERI, PM,and OVXþET groups and negatively correlated with advancing age. (Fertil Steril� 2007;88:1003–5. �2007by American Society for Reproductive Medicine.)
The monotropic rise in FSH in the late reproductive years be-fore menopause is accompanied by declining inhibin B (1–3)and increasing activin A (2, 3) in the presence of unchangedfollistatin (FS) (2), which suggests the potential for a net stim-ulatory effect on FSH regulation. To what extent suchchanges reflect ovarian aging is unknown because these pep-tides are also produced in multiple sites including the pitui-tary (4–7). To better assess the importance of ovarian agingin FSH modulation by these regulators, we compared circu-lating levels of inhibin A, inhibin B, activin A, and theneutralizing activin-binding protein FS in women withfunctioning ovaries invarying pre- and perimenopausal stateswith those in women of similar age after spontaneous meno-pause or ovariectomy while receiving estrogen therapy.
Before the initiation of the study, approval of the protocolwas obtained from the University of Michigan Hospital’sInstitutional Review Board for the Use of Human Subjects.All participants (n ¼ 79) provided written consent. Sixty-three healthy volunteers ages 40–50 years were recruitedinto the following four study groups: older cycling (OC;mean age ¼ 45.9 � 0.8 years, n ¼ 17), perimenopause(PERI; mean age ¼ 49.0 � 0.6 years, n ¼ 21), spontaneousmenopause (PM; mean age¼ 49.4� 0.6 years, n¼ 10), andsurgical menopause receiving estrogen (OVXþET meanage ¼ 49.0 � 1.6 years, n ¼ 15). A fifth group of 16 youngcycling women (YC; mean age¼ 23.0� .9 years) served ascontrols. All participants had a body mass index of 20–25and reported no current medical or psychiatric illness, nocurrent use of sex steroid therapy, no pregnancy or breast-feeding in the past 6 months, and no current history of diet-ing, excessive exercise, or smoking. Plasma values for PRL,T, and DHEAS, which were obtained during the screeningvisit, were within normal female ranges of the referencehospital laboratory. Criteria for the regular-cycling groups
Received December 20, 2005; revised and accepted December 20, 2006.
Supported by National Institutes of Health grant nos. U54 HD29184, NU
01373, 5MO RR00042, and AG15083 and Blue Cross-Blue Shield of
Michigan Foundation Award.
Reprint requests: Nancy King Reame, M.S.N., Ph.D., 630 168th Street,
Columbia University, New York, NY 10032 (FAX: 212-305-2139;
015-0282/07/$32.00oi:10.1016/j.fertnstert.2006.12.009 Copyright ª2007 American S
included a history of menses every 25–32 days with evi-dence of presumptive ovulation as determined by a midlu-teal serum P value above 9.5 nmol/L during the studycycle. For the perimenopausal group, subjects includedthose with a menstrual cycle in the last 3 months and withat least one menopause-related complaint. Postmenopausalsubjects had experienced their final spontaneous menstrualperiod at least 12 months before the study. Women in theovariectomized group had experienced an elective total hys-terectomy for benign causes in the last 5 years and were re-ceiving estrogen therapy. For study purposes, those womentaking equine estrogens were placed on the E2 patch (0.1 mgevery 3 days) for 8 weeks before the study. Ovarian statuswas confirmed by transvaginal ultrasound.
Participants were admitted to the General Clinical Re-search Center of the University of Michigan Hospital foran overnight study to undergo intensive blood sampling ev-ery 10 minutes for 8–24 hours as part of a study addressingchanges in LH pulse characteristics (data not presentedhere). Cycling subjects were studied on cycle day 5 � 1of the follicular phase. Concentrations of FSH, estrogen,and FSH regulatory proteins (inhibin A, inhibin B, activinA, FS 288) were measured from blood samples drawn at0900, 1500, 0100, and 0500 hours.
Plasma FSH from the first 52 subjects studied was mea-sured in duplicate by Delfia fluorometric immunoassay(Wallacoy, Turku, Finland, and Perkin Elmer Life Sciences,Sorton, OH). Plasma FSH from the remaining 27 subjectswas measured using an automated Chemiluminiscent Im-mulite system (Diagnostic Products, Los Angeles). The sen-sitivities of the Delfia and Immulite FSH assays were 0.05and 1 IU/L, respectively. Overall, there was excellent corre-lation of results measured by Delfia and Immulite for FSH(r ¼ 0.976, P<.001). The E2 levels were measured usingdouble antibody radioimmunoassay kits (Diagnostic Prod-ucts). The sensitivity of the E2 assay was 18 pmol/L. Theintra- and interassay coefficients of variation (CV) were<10%. Two-site assays for inhibin A (8), inhibin B (9),activin A (10), and FS 288 (11) were used. The FS 288 assaycross-reacts 9.9% with FS 315, an alternate spliced variant
Fertility and Sterility� Vol. 88, No. 4, October 2007 1003ociety for Reproductive Medicine, Published by Elsevier Inc.
of FS (11). The sensitivities of the inhibin A, inhibin B, ac-tivin A, and FS 288 assays were 3.9, 8.2, 40, and 20.1 pg/mL, respectively. The intra-assay CV for all four assays av-eraged less than 10%, with interassay CVs averaging be-tween 13.2% and 18.6%. Hormone values are expressedas international units per liter for FSH calibrated to the Del-fia assay, picomoles per liter for E2 (1 pg ¼ 3.6 pmol), andpg/mL for inhibin A, inhibin B, activin A, and FS 288.Group comparisons were conducted using two-tailed non-parametric tests for nonpaired observations (Kruskal-Wallisanalysis of variance and post hoc Mann-Whitney U-test).Data are reported as mean � standard error of the mean.
Mean FSH and E2 values were in the expected ranges forthe two postmenopausal groups. For cycling women, de-spite similar E2 concentrations, the mean FSH was higherin the OC and PERI women versus in the YC group(Fig. 1, top left). Endometrial thickness on cycle day 5was similar across the YC, OC, and PERI groups. In con-trast, ovarian volumes differed by study group (P%.001):volumes were larger in the YC versus the OC group(1,316 � 132 vs. 971 � 130 mm, P¼ .02), lowest in thePM group (514 � 82 mm), and highest in the PERI group(mean� standard error, 1,551� 149 mm) owing to the pres-ence of numerous cysts in seven of the 19 PERI subjects.
1004 Reame et al. Correspondence
Follicular phase levels of inhibin Awere low and near theassay detection limit in all groups (data not shown). InhibinB was highest in the YC and OC groups, reduced in thePERI, and near or below detection in the PM and OVXþETgroups (Fig. 1, top right). In contrast, there were no differ-ences in activin A between groups, barring a tendency forincreased activin A levels in the PERI group (Fig. 1, bottomleft). Circulating FS 288 levels were significantly lower inthe PERI, PM, and OVXþET groups versus in the YCgroup (P%.03) but did not differ from each other (Fig. 1,bottom right). A significant inverse correlation (�0.31;P%.01) was demonstrated between chronological ageand FS 288 but not for the other FSH regulators or forestrogen.
These findings of similar levels of activin in PM,OVXþE, and YC substantiate the view that the ovary, whileserving as the major source of inhibin B, contributes verylittle to the circulating pool of activin. This has been sug-gested by earlier work (12), although not stringently testedacross the menopause transition in tandem with its bindingneutralizer FS. In support of earlier findings of a modest in-crease in activin A across the menopausal transition (2, 3),circulating levels of activin A tended to be higher in thePERI group. Taken together, these findings suggest an
FIGURE 1
FSH, inhibin B, activin A, and FS 288 by study group. Data are mean� standard error of the mean. For inhibin B,mean concentrations in the PM and OVXþET groups were near the limits of assay detection, thus the standarderror bars for these groups are not shown. Mean E2 concentrations are indicated at the top of the FSHpanel. Significant group differences (*, **) were determined by Kruskal-Wallis analysis of variance andpost hoc Mann-Whitney U-test.
0
100
200
300
400
500
600
YC(n=16)
OC(n=17)
PERI(n=21)
PM(n=10)
OVX+ET(n=15)
YC(n=16)
OC(n=17)
PERI(n=21)
PM(n=10)
OVX+ET(n=15)
Act
ivin
A (
pg/m
l)
0
20
40
60
80
100
FSH
(IU
/L)
E2 96 114 228 25 297
*
**
*p < 0.01 vs all groups**p < 0.001 vs all groups
0
30
60
90
Inhi
bin
B (
pg/m
l)
*p < 0.001 vs PERI, PM, OVX **p < 0.001 vs PM, OVX
**
**
0
400
800
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1600
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tin
288
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Reame. Menopause and FSH peptide regulators. Fertil Steril 2007.
Vol. 88, No. 4, October 2007
inverse relationship between inhibin B and activin A duringthe menopausal transition.
In contrast to the age-related decline in FS observed hereusing the ultrasensitive FS 288 assay (11), we (2) and others(13) have found circulating levels of FS to increase acrossthe menopause transition (2) and with advancing age (13)when measured with an assay for total FS. These discrepantfindings are likely due to the much larger contribution of FS315, an alternate spliced variant of FS 288 of pituitary origin,to the overall total FS concentrations in human serum (14).However, as FS 288 levels were lower in the PERI, PM,and OVXþET groups compared with the YC group, an ovar-ian contribution of FS 288 to the circulating pool cannot beruled out. Until an FS 315–specific assay is developed, therole of ovarian-derived FS 288 in regulating FSH in relationto pituitary-derived FS 315 (14) will remain unclear.
Considering that both FS and inhibin regulation of FSHare mediated by altered activin signaling (15, 16) and theimportance of their relative equilibrium for FSH control(5), our findings of a decline in these negative regulatorsin concert with unchanging levels of activin A point to in-creased bioavailability of activin during reproductive aging.Although very little free activin has been demonstrated inthe circulation (17, 18), confirmation of this premise awaitsavailability of more sensitive assays.
In summary, this cross-sectional study, which for the firsttime examined all regulators of activin availability in unison,reports that the changes in FSH and inhibin B across the men-opause transition are accompanied by a sustained presence ofactivin A in concert with an age-dependent reduction in itsbinding protein FS. The differential patterns of secretion inthese regulatory proteins, which occur before the loss ofE2-negative feedback, are consistent with enhanced activinbioavailability and potentially contribute to the rise in FSH.Longitudinal studies of large cohorts of healthy women acrossthe menopause transition, such as the Study of Women acrossthe Nation (19), should help shed light on the role of bothovarian and nonovarian contributors to reproductive aging.
Acknowledgments: The authors thank Alice Rolfes-Curl (hormone assays),
Julie Chilimigras (data analysis), and the nursing staff of the General Clin-
ical Research Center. We are especially grateful to the women who served
as research participants. This manuscript is dedicated to the memory of Dr.
Mei-Yu Yu, a dedicated women’s health researcher and friend.
Nancy E. Reame, Ph.D.a,c,e
Jane L. Lukacs, Ph.D.a
Pamela Olton, B.S.b
Rudi Ansbacher, M.D.d
Vasantha Padmanabhan, Ph.D.b,c
a School of Nursing, b Pediatric Endocrinology,c Reproductive Sciences Program, and d Departmentof Obstetrics and Gynecology, University of Michigan,Ann Arbor, Michigan; and the e School of Nursing,Columbia University, New York, New York
Fertility and Sterility�
REFERENCES1. Soules MR, Battaglia DE, Klein NA. Inhibin and reproductive aging in
women. Maturitas 1998;30:193–204.
2. Reame NE, Wyman TL, Phillips DJ, de Kretser DM, Padmanabhan V.
Net increase in stimulatory input resulting from a decrease in inhibin B
and an increase in activin A may contribute in part to the rise in follic-
ular phase follicle-stimulating hormone of aging cycling women. J Clin
Endocrinol Metab 1998;83:3302–7.
3. Santoro N, Adel T, Skurnick JH. Decreased inhibin tone and increased
activin A secretion characterize reproductive aging in women. Fertil
Steril 1999;71:658–62.
4. Phillips DJ, de Kretser DM, Padmanabhan V. Follistatin: from puberty
to menopause. In: Muttukrishna S, Ledger W, eds. Inhibin, activin and
follistatin in human reproductive physiology. London, UK: Imperial
College Press, 2001:6141–64.
5. Padmanabhan V, Sharma TP. Neuroendocrine vs. paracrine control of
follicle-stimulating hormone. Arch Med Res 2001;32:533–43.
in peripheral serum levels of total activin A during the human menstrual
cycle and pregnancy. J Clin Endocrinol Metab 1996;81:3328–34.
19. Randolph Jr JF, Sowers M, Gold EB, Mohr BA, Luborsky J, Santoro N,
et al. Reproductive hormones in the early menopausal transition: rela-
tionship to ethnicity, body size, and menopausal status. J Clin Endocri-
nol Metab 2003;88:1516–22.
1005
1
Preferences of subfertile women regarding electivesingle embryo transfer: additional in vitro fertilizationcycles are acceptable, lower pregnancy rates are not
With identical pregnancy rates after elective single embryo transfer (ET) and double ET strategies consisting ofthree cycles of IVF or intracytoplasmic sperm injection (ICSI) plus transfers of thawed/frozen embryos if avail-able, 46% of the women undergoing IVF/ICSI favor elective single ET. If elective single ET lowers pregnancychances with 1%, 3%, or 5%, the percentage of women preferring elective single ET drops to 34%, 24%, and15%, respectively. If four, five, or six cycles with elective single ETare needed to match the success rate of threecycles with double ET, the percentage of women with a preference for elective single ET drops from 46% to40%, 36%, and 35% respectively. (Fertil Steril� 2007;88:1006–9. �2007 by American Society for Reproduc-tive Medicine.)
Twin pregnancies carry risks for both mother and child.Compared with singleton pregnancies there is an increasein obstetric complications (1–3) and in neonatal complica-tions (4, 5). The infant mortality rate in twins is about twiceas high as in singleton pregnancies (6).
The risks and complications of multiple pregnancies ledto a consensus meeting of the European Society of HumanReproduction and Embryology (ESHRE) in May 2002 todecide that ‘‘.the essential aim of IVF/ICSI is the birthof one single healthy child, with a twin pregnancy beingregarded as a complication’’ (7).
Multiple pregnancies after IVF or intracytoplasmicsperm injection (ICSI) arise mainly when more than oneembryo is transferred to the uterus. As a consequence, elec-tive single embryo transfer (ET) has been proposed as a wayto reduce multiple pregnancy rates (8). The importance ofpreventing multiple pregnancy through elective single ETincreasingly is emphasized by gynecologists, despite thefact that one cycle of elective single ET leads to lower preg-nancy rates than one cycle of double ET (9–11). Offeringmore IVF cycles may improve pregnancy rates after elec-tive single ET (12).
We designed a study to assess the attitudes and trade-offs toward elective single ET versus double ET in sce-narios with various pregnancy chances and with differenttreatment cycles needed in women undergoing IVF orICSI treatment. Institutional review board approval wasobtained for this study. All women in the stimulationphase of an IVF/ICSI treatment at the Centers for Repro-ductive Medicine of the Academic Medical Center
Received June 1, 2006; revised December 4, 2006; accepted December
5, 2006.
Supported by the Netherlands Organisation for Health Research and
Development (grant 945-03-013).
Reprint requests: Moniek Twisk, M.D., Center for Reproductive Medi-
cine, Academic Medical Center, Meibergdreef 9, Room H4-205,
1105 AZ Amsterdam, The Netherlands (FAX: 31-20-5669206;
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
006
(AMC), Amsterdam, and the University Medical CenterGroningen (UMCG) between May and December 2005were eligible for this study. Consenting eligible womenwere handed a questionnaire.
Demographic characteristics and reproductive history in-formation were collected including age, parity, duration ofinfertility, and number of previous IVF/ICSI cycles. Prefer-ence for a singleton or twin pregnancy was asked. Next,written information about elective single ET as a possiblealternative to double ET in preventing multiple pregnancieswas provided. All women were given information on therisks of twin pregnancies before starting with the IVF/ICSI treatment. In the AMC, double ET is standard proce-dure in IVF or ICSI treatment. In the UMCG, IVF/ICSI inthe modulated natural cycle is the standard procedure forwomen under 35 years of age (13, 14) and double ET forwomen aged 35 years or older. Generally speaking, in theNetherlands three IVF/ICSI treatments are covered byhealth care insurance.
To assess whether women are willing to accept a lowerpregnancy rate to prevent a twin pregnancy, we developedfour scenarios differing in pregnancy rates with doubleET and elective single ET. All scenarios consisted of threecycles of IVF/ICSI including the transfer of all availablefrozen embryos. After presentation of each scenario, theparticipant was asked whether she would prefer doubleET or elective single ET in that particular situation.
In the first scenario, the pregnancy rate was equal inwomen treated with either double ET or elective singleET: of 100 women receiving treatment, 50 were expectedto become pregnant with either approach. This number ofpregnancies was chosen because it is a percentage thatcan be expected after an IVF/ICSI treatment strategy con-sisting of three fresh cycles of IVF/ICSI including frozen/thawed ETs. We based the pregnancy rate after the doubleET strategy on the results in our own center. The scenariodescribed a twin pregnancy rate of 25% after double ETversus 1% after elective single ET.
0015-0282/07/$32.00, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.12.004
TABLE 1Women’s preferences for elective single ET or double ET.
ScenarioWomen preferring elective
single ET (%)Women preferring
double ET (%)
Different pregnancy ratesElective single ET ¼ double ET 46 54Elective single ET 1% less effective 36 66Elective single ET 3% less effective 24 76Elective single ET 5% less effective 15 85
Different treatment cyles4 Elective single ET ¼ 3 double ET 40 605 Elective single ET ¼ 3 double ET 36 646 Elective single ET ¼ 3 double ET 35 65
In the next three scenarios the pregnancy rate after doubleET was kept at 50 of 100, whereas the pregnancy rate afterelective single ET was reduced: of 100 women undergoingelective single ET 49, 47, or 45 were expected to becomepregnant. The rate of twin pregnancies was kept at 25%after double ET and 1% after elective single ET.
To assess whether women are willing to undergo addi-tional IVF/ICSI cycles to prevent a twin pregnancy, we pre-sented three more scenarios. As in the previous scenarios,25% of the pregnant women were expected to carrya twin pregnancy after double ET versus 1% after electivesingle ET. In the first scenario the number of pregnantwomen after three cycles with double ET was the same asafter four cycles with elective single ET. In the next two sce-narios we increased the number of elective single ET cyclesneeded to maintain the same number of pregnancies as withdouble ET to five and six cycles.
To assess whether participants understood the questionsand scenarios we proposed, we conducted a pilot study inApril 2005 among a similar target group. Twelve womenwere invited to participate; all of them consented. Theywere asked to comment on the questions and the proposedsituations and to describe each scenario in their own wordsto assess whether they clearly understood them.
In the pilot study we dropped pregnancy chances afterelective single ET with 5%, 10%, and 15%. All womenswitched directly to double ET if elective single ETdecreased their pregnancy chance with 5%, making thereductions of 10% and 15% redundant. To provide thequestionnaires with a true trade-off character, we subse-quently adjusted the scenarios. In the final questionnairepregnancy chances were reduced with 1%, 3%, and 5%for the three different scenarios.
For data analysis SPSS for Windows version 11.5.1 wasused. Statistical significance was defined as P<.05. Differ-
Fertility and Sterility�
ences between groups in frequencies were analyzed withlogistic regression analysis.
A total of 343 questionnaires were handed out, of which244 were returned (response rate 71%). Mean age of the re-spondents was 34.2 years (�4.12 SD). The vast majority(77%) had primary infertility. Two thirds of the womenhad undergone at least one previous IVF cycle. Whenwomen were asked for their preferences, 27% of the respon-dents preferred a singleton pregnancy when given thechoice, 24% a twin pregnancy, and 45% had no preference.
Women’s preferences for elective single ET or double ETin the various scenarios are shown in Table 1. In the scenariowhere elective single ET was as effective as double ET interms of pregnancy rates, 46% of the respondents wouldprefer elective single ET. When pregnancy rates were low-ered with 1%, 3%, and 5% after elective single ET thisnumber decreased to 34%, 24%, and 15%, respectively.Multivariable logistic regression analysis showed no signif-icant differences in preference in subgroups differing inage (above or below 38 years), parity, treatment center, andprevious IVF treatments.
When the number of IVF/ICSI cycles with elective singleET needed to achieve a pregnancy rate similar to three cyclesof double ET was set at four, five, and six cycles, 40%, 36%,and 35% of the respondents would be willing to undergothese additional cycles. Multivariable analysis showed thatwomen treated in the UMCG and women who had under-gone one or more IVF cycles in the past were significantlymore often willing to undergo additional cycles. No signifi-cant difference between age groups (above or below 38years) and parity subgroups could be found. With our samplesize of 244, we were able to detect variables with an odds ra-tio of 1.8 with an a of 5% and 80% power.
The results of our study show that in case of equal suc-cess rates about half of the women would prefer elective
1007
single ET and the other half would prefer double ET. Ifelective single ET reduces pregnancy rates only mildly,the preferences switch strongly to double ET. If electivesingle ET lowers pregnancy chances with just 1%, the per-centage of women preferring elective single ET dropswith 12%, from 46% to 34%. This emphasizes unambig-uously the overwhelming dominance of pregnancy as theprimary goal of treatment for women undergoing IVF/ICSI and the absence of willingness to trade off thatgoal to avoid a multiple pregnancy. Nearly half of thewomen are willing to undergo additional elective singleET cycles to prevent a multiple pregnancy, given thatwith this strategy similar pregnancy rates to those of dou-ble ET can be achieved.
Preferences for elective single ET or double ET could bedifferent when couples have to pay for the treatments. In ourstudy, preferences for elective single ET or double ET werenot confounded by economic issues in the scenarios pre-senting a reduced pregnancy chance after elective singleET. In the scenarios where the number of treatment cycleswith elective single ET was increased to match the successrates obtainable with three cycles of double ET, preferencescould have been influenced by the fact that women have topay for these extra treatment cycles. Because the percentageof women with a preference for elective single ET onlyslightly changed in these scenarios, economic issues donot seem to have an important role in the decision-makingprocess.
The percentage of the women preferring elective singleET in the absence of a difference in pregnancy rates inour study (46%) is higher than the percentages reportedin previous studies: 1% to 34% (15–17). It can be postu-lated that this percentage would have increased evenmore if more information on the risks of a multiple preg-nancy had been provided. An association between betterperception of risks of a multiple pregnancy and less de-sirability of a twin pregnancy indeed has been reportedpreviously (18, 19). However, whether better perceptionof risks also leads to a stronger preference for electivesingle ET is unclear. Two studies reported that manywomen prefer to have double ET despite good informa-tion about the risks for complications with multiple preg-nancies (15, 20).
In conclusion, to reduce the risk of twin pregnancies insubfertile women undergoing IVF/ICSI treatment it isessential to develop and offer an elective single ET–basedstrategy that does not affect pregnancy rates negatively. Itis only such an approach that will not jeopardize patientacceptance of elective single ET in an IVF/ICSI program.
Moniek Twisk, M.D.a,b
Fulco van der Veen, Ph.D.a
Sjoerd Repping, Ph.D.a
Maas-Jan Heineman, Ph.D.a,b
1008 Twisk et al.
Johanna C. Korevaar, Ph.D.c
Patrick M. M. Bossuyt, Ph.D.ca Center for Reproductive Medicine, Department of
Obstetrics and Gynecology, Academic Medical Center,Amsterdam; b Department of Obstetrics andGynecology, University Medical Center Groningen,Groningen; and the c Department of ClinicalEpidemiology and Biostatistics, Academic MedicalCenter, Amsterdam, The Netherlands
REFERENCES1. Campbell DM, Templeton A. Maternal complications of twin preg-
nancy. Int J Gynaecol Obstet 2004;84:71–3.
2. Pinborg A, Loft A, Schmidt L, Langhoff-Roos J, Andersen AN. Mater-
nal risks and perinatal outcome in a Danish national cohort of 1005
twin pregnancies: the role of in vitro fertilization. Acta Obstet Gynecol
Scand 2004;83:75–84.
3. ESHRE Capri Workshop Group. Multiple gestation pregnancy. The
multiple gestations: an assessment of knowledge and risk aversion.
Am J Obstet Gynecol 2001;185:920–4.
Fertility and Sterility�
19. Child TJ, Henderson AM, Tan SL. The desire for multiple preg-
nancy in male and female infertility patients. Hum Reprod
2004;19:558–61.
20. Blennborn M, Nilsson S, Hillervik C, Hellberg D. The couple’s
decision-making in IVF: one or two embryos at transfer? Hum Reprod
2005;20:1292–7.
1009
Recombinant follicle-stimulating hormone (rFSH)supplemented with low-dose human chorionicgonadotropin compared with rFSH alonefor ovarian stimulation for in vitro fertilization
Low-dose hCG supplementation was administered at the start of ovarian stimulation, concomitantly with re-combinant FSH (rFSH) in GnRH antagonist cycles, and these were compared with GnRH-a cycles that usedrFSH alone. The low-dose hCG group had similar implantation and pregnancy rates but had significantly re-duced rFSH requirements, allowing for an average cost savings of $600 per cycle. (Fertil Steril� 2007;88:1010–3. �2007 by American Society for Reproductive Medicine.)
Gonadotropin-releasing hormone antagonists now arewidely used in IVF stimulation protocols. They allow forimmediate suppression of pituitary gonadotropins andcause a rapid and profound inhibition of LH. Inhibition ofLH may lead to a suboptimal ovarian response. Althougha minimal threshold level for LH has not yet been estab-lished, there are studies to suggest that very low levels ofLH are associated with decreased fertilization and implan-tation rates and with increased rates of early pregnancyloss (1–3). This has led to the hypothesis that supplementa-tion of stimulation protocols with LH may improve out-comes.
A greater understanding of folliculogenesis and the two-cell, two-gonadotropin model has allowed for better optimi-zation of ovarian response in controlled ovarian stimulation.Luteinizing hormone stimulates the growth of follicles thatalready have been primed by FSH and have acquired LH re-ceptors (4, 5). Luteinizing hormone activity in the form ofrecombinant LH or low-dose hCG is capable of stimulatingfollicular maturation in the absence of continued FSH andpromotes the growth of large follicles while reducing thenumber of small follicles (6, 7), potentially reducing therisk of ovarian hyperstimulation syndrome (7). It also hasbeen shown that LH alone in the absence of continued ad-ministration of FSH is capable of maintaining ovarian estro-gen secretion (7). Supplementation of hCG also may shortenovulation induction and decrease the amount of FSH neededfor stimulation (8).
An optimal regimen for LH or hCG supplementation hasnot yet been established. Although the question of whether
Received April 27, 2006; revised and accepted December 29, 2006.
The opinions and conclusions in this paper are those of the authors and
are not intended to represent the official position of the Department
of Defense, United States Air Force, United States Army, or any other
government agency.
Reprint requests: Anthony M. Propst, M.D., 2200 Bergquist Drive,
MMNO, Lackland Air Force Base, Texas 78236 (FAX: 210-292-6084;
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
1010
supplementation is necessary is controversial (9), bothrLH and hCG appear to be effective in optimizing ovarianstimulation (6–8, 10). However, hCG has higher affinityfor the LH or hCG receptor and has a longer half-life, allow-ing for low-dose regimens. In addition, hCG is very inex-pensive, making it an attractive alternative to high-costrecombinant protocols (5).
The purpose of this study was to determine whether low-dose hCG supplementation would provide pregnancy ratescomparable to or improved from those of GnRH antagonistcycles that use only rFSH for ovarian stimulation. A second-ary outcome measure was by how much low-dose hCG sup-plementation would reduce rFSH requirements and cost percycle.
We performed a retrospective analysis of all patientsbetween 23 and 40 years of age who underwent IVF orIVF–intracytoplasmic sperm injection cycles by using aGnRH antagonist at Wilford Hall Medical Center. The pro-tocol was approved by our institutional review board. Pa-tients using GnRH agonists were excluded. Ninety-sixpatients underwent ovarian stimulation with rFSH alone be-tween 2002 and 2004. Ninety-four patients underwent ovar-ian stimulation with rFSH and hCG supplementationbetween October 2004 and May 2005.
Pituitary down-regulation was achieved with combinedoral contraceptives, which were started on day 5 of the cyclebefore ovarian stimulation. In the group using rFSH alone,rFSH (Gonal F [Serono, Geneva, Switzerland] or Follistim[Organon, Roseland, NJ]) was started at a dose of 150–600IU SC per day (divided between a morning and eveningdose), 5 days after discontinuation of combined oral contra-ceptives. In the group that was supplemented with low-dosehCG, rFSH was started at a fixed morning dose of 150 or 225IU. Low-dose hCG (Pregnyl, Organon) was started concom-itantly with rFSH and was given nightly in doses of either 50or 100 IU SC, with the higher dose corresponding to thehigher morning rFSH dose. After 4 days, both groups had
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a transvaginal ultrasound, and the dose of rFSH was adjustedbased on the number and size of follicles and the E2 level.
Ganirelix acetate was started when lead follicles were 13to 14 mm in mean diameter. Ganirelix acetate was started ona median of day 7 of stimulation and was administered fora median of 4 days in both groups. When at least two folli-cles were a mean diameter of R18 mm, with at least twoadditional follicles sized >10 mm, hCG was administered(5,000–10,000 IU IM). Oocyte retrieval was performed 36hours later, and embryos were transferred either 3 or 5days after retrieval depending on embryo number and qual-ity. Luteal-phase support was maintained with P (50 mg IMper day).
Serum LH and E2 levels were measured before stimula-tion with rFSH, before the start of the GnRH antagonist,and at several intervals after the start of ganirelix acetate, in-cluding the day of hCG administration for final oocyte mat-uration. Luteinizing hormone and E2 were measured byusing an electrochemiluminescence immunoassay (Modu-lar Analytics E170 module; Roche, Mannheim, Germany).The detection limits for LH and E2 were 0.10 mIU/mL and5.0 pg/mL, respectively. The intra-assay and interassay co-efficients of variation were 1.2% and 2.0%, respectively, atthe lowest mean dose of LH. The intra-assay and interassaycoefficients of variation for E2 were 2.0% and 2.2%, respec-tively, at a mean level of 1,276 pg/mol, and were 2.0% and2.2%, respectively, at a mean level of 3,715 pg/mol.
All analyses were performed by using SPSS 13 for Win-dows (SPSS, Chicago, IL). Exploratory data analysis was ini-
Fertility and Sterility�
tially performed to determine normality of the data. Theparametric continuous variables were analyzed by using Stu-dent’s t-test, and the results wereexpressed as mean�SD. Thenonparametric continuous or ordinal data were analyzed withthe Mann-Whitney U test. Percentages or rates were comparedby using either c2 or Fisher’s exact tests, as indicated.
The baseline patient characteristics of the two treatmentgroups are shown in Table 1. There was no statistical differ-ence in terms of age or use of intracytoplasmic sperm injec-tion. Four cycles were canceled before oocyte retrieval inthe group using rFSH alone, including one that was can-celed because of premature luteinization and three thatwere canceled because of poor ovarian response. Five cy-cles were canceled in the low-dose hCG group: four becauseof poor ovarian response and one because of a significantdecline in the level of E2. The patients in the low-dosehCG supplementation group were more likely to have un-dergone IVF before (65% vs. 42%, P<.01). Estradiol levelson the day of hCG were significantly higher in the low-dosehCG group (2,823 � 1,489 pg/mol vs. 2,131 � 1,253 pg/mol, P<.01). The FSH-alone stimulation group had signif-icantly more oocytes, mature oocytes, and fertilized em-bryos than did the hCG-supplemented group. Duringstimulation, the low-dose hCG group used significantlyless FSH (1,883 � 602 IU vs. 2,847 � 1,192 IU, P<.01).
The ongoing-pregnancy rates were similar in the rFSH-alone group and the low-dose hCG group (44.8% vs.48.9%, P¼.57). Although there was a slightly higher im-plantation rate in the rFSH group (38.6% vs. 33.8%,
TABLE 1Baseline and cycle characteristics between the rFSH-alone group and the low-dosehCG–supplemented group.
ParameterrFSH alone
(n [ 96)
Low-dose hCGsupplementation
(n [ 94) P value
Age in y 33.1 � 3.6 33.9 � 3.4 .09Prior IVF (%) 42 65 < .01ICSI (%) 40 45 .54FSH (IU) 2,847 � 1,192 1,883 � 602 < .01Lowest value of LH after start of GnRH-a 1.8 � 1.6 2.6 � 3.0 .12LH of %0.5 before oocyte retrieval (%) 50 50 .89Canceled cycles 4 5E2 on day of hCG (pg/mol) 2,131 � 1,253 2,823 � 1,489 < .01No. of oocytes 14.9 � 9.2 10.3 � 5.0 < .01No. of mature oocytes 12.7 � 7.8 9.0 � 4.5 < .01No. of embryos 8.8 � 6.0 6.2 � 3.5 < .01Implantation rate (%) 38.6 (76/197) 33.8 (66/195) .43Spontaneous-abortion rate (%) 17 (9/52) 6 (3/49) .15Ongoing-pregnancy rate per cycle (%) 44.8 (43/96) 48.9 (46/94) .57
Note: Values not specified in stub column as percentages are expressed as either mean � SD or n (%).
Van Horne. Low-dose hCG supplementation. Fertil Steril 2007.
1011
P¼.43), the rate of spontaneous abortion in this group washigher than that in the low-dose hCG group (17% vs. 6%,P¼.15). However, neither measurement was statisticallysignificant, and the study was not powered to detect a signif-icant difference in these outcomes.
This study shows that implantation and pregnancy ratesare similar between patients stimulated with rFSH aloneand those stimulated with rFSH that is supplemented withlow-dose hCG. The amount of rFSH needed to obtain sim-ilar results was significantly less in the low-dose hCG–supplemented cycles. Although it may not be necessary tosupplement cycles with LH (9) or hCG to achieve better out-comes, this study shows that supplementation with hCG cansignificantly reduce the cost of an IVF cycle without a neg-ative effect on outcome. The average cost of 1 ampule (75IU) of rFSH (Gonal F) at the Serono Fertility LifeLinespharmacy and the Freedom Fertility Pharmacy is $47.50.The low-dose hCG group used on average 1,000 IU lessof rFSH than did the rFSH-alone group, which translatesto an average cost savings of approximately $600 per IVFcycle. The additional cost for low-dose hCG supplementa-tion is minimal, at approximately $35 per cycle.
There may be other benefits of low-dose hCG in addition tocost savings. Low-dose hCG appears to be more effective instimulating ovarian estrogen secretion than is rFSH alone(5). Despite fewer numbers of oocytes in the low-dose hCGgroup, E2 levels were higher. Enhanced estrogen secretionmay lead to better quality oocytes and embryos (6), althoughoutcomes between the two groups in our study were the same.This supports the findings of a study in which higher levelsof E2 in supplemented cycles were not predictive of a betteroutcome (11).
Endogenous levels of LH and profoundly low levels ofLH after the start of the GnRH antagonist were similar inboth groups (Table 1). Levels of LH may not be predictiveof cycle outcomes, but higher levels of LH in cycles usingrFSH and a GnRH antagonist are associated with shorterovarian stimulation, lower FSH requirements, and higherserum E2 levels (12). Similarly, those with an initiallypoor ovarian response benefit more from supplementationwith LH rather than from increased dosages of rFSH (13).Perhaps hCG supplementation should be targeted specifi-cally to those patients with very low LH or with a poor ovar-ian response, in order to improve outcomes while alsodecreasing rFSH requirements.
The ideal regimen for administration of low-dose hCG stillremains to be optimized. In studies in which supplementationwith hCG or rLH is started later in the cycle, similar numbersof oocytes were obtained, but fertilization rates were im-proved with supplementation (8, 14). In our study, low-dose hCG supplementation was started simultaneously withrFSH, and there were significantly more mature oocytesand embryos in the group stimulated with rFSH alone, al-though implantation and pregnancy rates were similar. Re-
1012 Van Horne et al. Correspondence
ceptors for LH or hCG on the granulosa cells are capableof supporting continued growth of the follicle in the absenceof FSH administration once they reach a size of approxi-mately 10 mm (5). Because only these large follicles are sen-sitive to LH activity stimulation, there may not have beensufficient FSH priming to allow for the growth of as manyfollicles as in the rFSH-alone group. When started at thetime of stimulation with rFSH, low-dose hCG is not as effec-tive in stimulating follicles, and the morning daily rFSH dosemay need to be increased to compensate for this finding. Todate, we are unaware of any studies comparing different startdays for administration of low-dose hCG, and this is an areain which further research will lead to better optimizationof COH.
In summary, our study demonstrated that GnRH antago-nist cycles using rFSH and low-dose hCG supplementationhave similar implantation and pregnancy rates when com-pared with those cycles using rFSH alone. More impor-tantly, the reduction in rFSH requirements resulted in anaverage savings of $600 per cycle in pharmacy costs.
Anne K. Van Horne, M.D.G. Wright Bates, Jr., M.D.Randal D. Robinson, M.D.Nancy J. Arthur, R.N.Anthony M. Propst, M.D.Division of Reproductive Endocrinology and Infertility,
San Antonio Uniformed Services Health EducationConsortium, Wilford Hall Medical Center, LacklandAir Force Base, Texas
REFERENCES1. Esposito M, Barnhart K, Coutifaris C, Patrizio P. Role of periovulatory
luteinizing hormone concentrations during assisted reproductive
technology cycles stimulated exclusively with recombinant follicle-
of two doses of recombinant luteinizing hormone supplementation in
an unselected group of women undergoing follicular stimulation for
in vitro fertilization. Fertil Steril 2005;83:309–15.
11. Cedrin-Durnerin I, Grange-Dujardin D, Laffy A, Parneix I, Massin N,
Galey J, et al. Recombinant human LH supplementation during GnRH
antagonist administration in IVF/ICSI cycles: a prospective random-
ized study. Hum Reprod 2004;19:1979–84.
Fertility and Sterility�
12. Bosch E, Escudero E, Crespo J, Simon C, Remohi J, Pellicer A.
Serum luteinizing hormone in patients undergoing ovarian stimulation
with gonadotropin-releasing hormone antagonists and recombinant
follicle-stimulating hormone and its relationship with cycle outcome.
Fertil Steril 2005;84:1529–32.
13. De Placido G, Algviggi C, Perino A, Strina I, Lisi L, Fasolino A, et al.
Recombinant human LH supplementation versus recombinant human
FSH (rFSH) step-up protocol during controlled ovarian stimulation
in mormogonadotrophic women with initial inadequate ovarian re-
sponse to rFSH. A multicentre, prospective, randomized controlled
trial. Hum Reprod 2005;20:390–6.
14. Acevedo B, Sanchez M, Gomez J, Cadros J, Ricciarelli E,
Hernandez E. Luteinizing hormone supplementation increases preg-
nancy rates in gonadotropin-releasing hormone antagonist donor
cycles. Fertil Steril 2004;82:343–7.
1013
1
Role of embryo transfer in fellowship training
Our objective was to survey training in ET techniques among fellows, its perceived importance, and potentialbarriers to ET training during fellowship. Although ET training remains an important issue for most fellows andrecent graduates, 44% of respondents did not receive this training during their fellowship. (Fertil Steril� 2007;88:1014–5. �2007 by American Society for Reproductive Medicine.)
Approximately 12%–15% of couples in the United Statesexperience infertility, leading many to turn to assisted re-productive techniques for help in conceiving a desiredbaby (1). Assisted reproductive techniques rely on thewell-coordinated processes of ovarian stimulation, oocyteretrieval, embryo culture, and ET to achieve a high proba-bility of ongoing pregnancy. Although all of these processesare important, proper ET technique is particularly critical.Multiple studies have evaluated different variables thatare involved in successful ET (2–5). Some of these factorsare related to instrumentation, whereas others are directlyrelated to provider technique (6, 7). The nature and typeof training in ET techniques appears to influence the successrates of the individuals performing the technique, witha minimum number of transfers required for proficiency(8). Because training in ET techniques affects assisted re-productive technique success rates, it is important to exam-ine where this training occurs as well as potential barriers tothis training. Our objective was to survey reproductive en-docrinology fellows and recent graduates about ET trainingin fellowship and their perception of the relative importanceof this training.
To accomplish this undertaking, a cross-sectional surveywas performed of all the American Board of Obstetrics andGynecology–approved reproductive endocrinology and in-fertility fellowships. In 2005, surveys were distributed toall directors of approved reproductive endocrinology andinfertility fellowship programs, for distribution to currentfellows and to recent graduates of their programs. Recentgraduates included all fellows graduating within the previ-ous 2 years. All questionnaires were mailed with a self-addressed return envelope. Careful instructions were givenwith each survey to maintain the anonymity of both the in-dividuals and the institutions. Evaluation of fellow trainingin ET was assessed with questions to determine the partici-pant’s year in fellowship training, performance of ETs as
Received July 13, 2006; revised and accepted December 9, 2006.
Supported in part by the intramural research program of the Reproduc-
tive Biology and Medicine Branch of the National Institute of Child
Health and Human Development, National Institutes of Health (Be-
thesda, Maryland).
Reprint requests: Alicia Y. Armstrong, M.D., M.H.S.C.R., Reproductive
Biology and Medicine Branch, National Institute of Child Health and Hu-
man Development,1-E-3140, CRC 10Center Drive, Bethesda, Maryland
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
014
a fellow, and potential obstacles that the fellow encounteredwhile learning this skill. Likert scales were used to measurethe perceived importance of ET training in fellowship aswell as the impact of ET training on their future employ-ment opportunities. Results were listed as frequencies,and the c2 test was used for statistical comparisons.
Eighty-one of 209 surveys were returned (39% responserate). Response rates were 51% (62/122) for current fellowsand were 22% (19/87) for recent graduates. Eighty-ninepercent indicated that training in ET techniques was eitherextremely important or important, whereas 10% believed itwas somewhat important, and only 1% believed it was notimportant at all. Responses were similar between currentfellows and recent graduates, particularly in the fractionof respondents who believed that ET training was extremelyimportant (c2 test, P¼.51). Fifty-six percent of all fellowsreceived experience in ET during their fellowship. Mostperformed ETs in their 1st (67%) or 2nd (24%) years.Consistent with this, the majority of respondents with ETexperience reported >20 transfers during their fellowship(62%). Among fellows who did not perform transfers, themost commonly cited reasons were that only attending phy-sicians may perform transfers (83%) and that patients wereunwilling to let fellows perform ETs (33%). Of recent grad-uates who received training in ET, 38% agreed that thistraining positively impacted their ability to obtain a post-graduate position. However, of recent graduates who didnot receive ET training in fellowship, 44% did not believethat this deficit negatively impacted their ability to procurea postgraduate position.
Although ET training remains an important issue formost fellows and recent graduates, 44% of respondentsdid not perform any ETs during their fellowship training.There are probably multiple reasons for this, but the mostcommon reason cited on this survey was, ‘‘only attendingsmay perform transfers.’’ Historically, there is precedent forthis practice. Although many aspects of the IVF treatmentprocess—patient education, counseling, ultrasound folliclemonitoring, and early pregnancy verification—eventuallywere relegated to nurses, physicians uniformly retainedtheir roles in performing the supposedly critical ET proce-dure. However, in one study evaluating whether appropri-ately trained nurses could perform this task, the nurses’resulting pregnancy rates were the same as those achieved
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after physician transfer (9). Interestingly, the training ofthese nurses, aside from their previous experience in intra-uterine insemination, consisted only of observing at leastfive transfers and then performing at least five transfers un-der direct supervision. Therefore, because other clinicians(and presumably fellows) easily can be trained to performET, perhaps there are other factors governing the decisionto allow only the most senior physicians to perform theET. Success rates may be one such factor. From thepatient’s perspective, it appears reasonable that the mostexperienced providers should have the highest pregnancyrates. Because patients desire to maximize their chancesof having a baby, there may be spoken or unspoken assump-tions leading to this practice. However, in studies performedby our group, trained fellows had pregnancy rates similar tothose of experienced providers (8) and, in some instances,had greater pregnancy rates than did experienced providerswhen embryo quality was considered (7).
Because ET by fellows does not adversely affect the pro-gram’s pregnancy rate, the question of what constitutes ap-propriate training remains unanswered. Outside of actualET, other forms of training such as intrauterine insemina-tions or mock ETs are commonly used. However, the abilityof these measures to impart this essential skill needs to bevalidated. We have reported elsewhere that our programhas developed a model for training fellows in ET that dem-onstrated efficacy in pregnancy outcomes that was consis-tent with that of more experienced providers (8). Beforeparticipating in ET, fellows are required to document R30mock-ET or intrauterine insemination procedures with theWallace catheter to assure familiarity with the mechanismsof this devise. They are also required to view actual ETs.Only then are they allowed to participate in ET under anattending physician’s supervision. Frequent evaluation isperformed of the trainees’ transfer technique and pregnancyoutcomes. The first assessment occurs after three transferattempts. If blood is present on the catheter tip, possibly in-dicating a traumatic procedure, the fellow is required to per-form additional mock-transfer or intrauterine inseminationprocedures, carefully documenting the presence or absenceof blood. The fellow then performs ten day 3 transfers inpatients with high-grade embryos. If the pregnancy rate is<30%, 30 additional mock transfers are performed beforeresuming ETs. To further standardize ET between pro-viders, the embryo-afterloading technique is taught, as de-scribed elsewhere (10). This technique, in conjunctionwith embryo injection by the embryologist, is most likelyto simulate the mock transfers that are practiced in thepre-ET stage of training.
Last, although many fellows who performed ETs be-lieved that this enhanced their postgraduate job opportuni-ties, the exact impact of this training on employment
Fertility and Sterility�
remains unclear. Indeed, many who did not receive thistraining did not report a negative effect on their employmentopportunities. Whether a graduate’s decision to go intoacademic vs. private practice factored into this perceptionis unknown. It is conceivable that private practices havemore incentive than do academic practices to recruit fellowswho have experience in ET. Regardless of its impact onpostgraduate employment, if one of the goals of fellowshiptraining is to produce physicians who are fully trained inclinical assisted reproductive technique services, thenhands-on training in ET should be included in reproductiveendocrinology and infertility fellowship training.
Michael D. Wittenberger, M.D.a,b
William H. Catherino, M.D., Ph.D.a,b
Alicia Y. Armstrong, M.D., M.H.S.C.R.a,b
a Reproductive Biology Medicine Branch, NationalInstitute of Child Health and Human Development,National Institutes of Health; and b Department ofObstetrics and Gynecology, Uniformed ServicesUniversity of Health Sciences, Bethesda, Maryland
REFERENCES1. Chandra A, Martinez GM, Mosher WD, Abma JC, Jones J. Fertility,
family planning, and reproductive health of U.S. women: data from
the 2002 National Survey of Family Growth. Vital Health Stat 23
Segars JH. The presence of blood in the transfer catheter negatively in-
fluences outcome at embryo transfer. Hum Reprod 2003;18:1848–52.
5. Mansour R, Aboulghar M, Serour G. Dummy embryo transfer: a tech-
nique that minimizes the problems of embryo transfer and improves the
pregnancy rate in human in vitro fertilization. Fertil Steril 1990;54:
678–81.
6. Karande VC, Morris R, Chapman C, Rinehart J, Gleicher N. Impact of
the ‘‘physician factor’’ on pregnancy rates in a large assisted reproduc-
tive technology program: do too many cooks spoil the broth? Fertil
Steril 1999;71:1001–9.
7. Hearns-Stokes RM, Miller BT, Scott L, Creuss D, Chakraborty PK,
Segars JH. Pregnancy rates after embryo transfer depend on the pro-
vider at embryo transfer. Fertil Steril 2000;74:80–6.
8. Papageorgiou TC, Hearns-Stokes RM, Leondires MP, Miller BT,
Chakraborty P, Cruess D, et al. Training of providers in embryo trans-
fer: what is the minimum number of transfers required for proficiency?
Hum Reprod 2001;16:1415–9.
9. Sinclair L, Morgan C, Lashan H, Afnan M, Sharif K. Nurses perform-
ing embryo transfer: the development and results of the Birmingham
experience. Hum Reprod 1998;13:699–702.
10. Neithardt AB, Segars JH, Hennessy S, James AN, McKeeby JL.
Embryo afterloading: a refinement in embryo transfer technique that
may increase clinical pregnancy. Fertil Steril 2005;83:710–4.
1015
LETTERS TO THE EDITORPaul G. McDonough, M.D.Associate Editor
Reply of the Authors:
We thank Drs. Barritt, Luna, Duke, and Cooperman fortheir response to our discussion of some of the ethical issuesencountered in researching human oocyte cryopreservation(1).
Our discussion highlighted many of the pitfalls involved insuch investigation and suggested possible ways to conductsuch research ethically. Because our main intention in writingthe article was to use the issues raised by this experimentalprocedure as a way to broaden the conversation around thecomplex issue of informed consent, we are happy to seesuch a conversation taking place. Our intent was not to pro-vide a set of recommendations. Also, although the Barrittet al. (1) work was published after the submission of our
Ethical issues surrounding the cryopreservation of humanoocytes
To the Editor:
In response to the article by Drs. de Melo-Martin andCholst, we support and oblige the authors’ request for ethi-cally performed oocyte cryopreservation studies (1).
The authors conclude that the most ethical way to performthe research necessary to evaluate this new technology, oo-cyte cryopreservation, would be through the use of donor oo-cytes and recipient women with infertility. We completelyagree with their recommendations, as evidenced by thefact that we published an article elsewhere that used sucha protocol (2).
Our analysis of four oocyte cryopreservation cases usingdonor oocytes and recipient couples was performed afteryears of intense debate within our center about the ethicaland possible medical concerns with this type of human study.Only after applying for and receiving institutional reviewboard approval for a prospective study that used oocyte do-nors and IVF patient recipients; after spending years on lab-oratory research and development to technically master theprocedures of oocyte cryopreservation and thawing; and fi-nally, after appropriately counseling and providing informedconsent to the patients involved, did we at last, with great cau-tion, undertake this study.
In the de Melo-Martin article, the authors state, ‘‘current(pregnancy rates) still appear to be lower than those seenwith standard IVF procedures (1).’’ They cite Borini et al.(3) for the success rates with this new procedure; however,that study was severely restricted by Italian laws that onlyan extremely limited number of oocytes and embryos maybe fertilized and transferred, which clearly reduces the truepotential success that could be achieved with this technique.Although our series was small, the results were dramatic. Wedemonstrated that three of four patients conceived and deliv-ered after using frozen and thawed donor oocytes, which iscomparable to the pregnancy rate that we found in ourfresh-oocyte donor program. In addition, our implantationrate of 26% is five times greater than that in the results of Bor-ini et al. (3), demonstrating that both the number of embryostransferred and the quality of the embryos created after oo-cyte cryopreservation and thaw resulted in our high successrate. Finally, the authors suggest, ‘‘Also important to considerwhen designing research protocols for oocyte cryopreserva-tion is the issue of costs. Because oocyte cryopreservationis still an investigational procedure, and until a centerachieves some degree of success, it can be argued that thecosts of the procedure should not be charged to the patient.’’
Fertility and Sterility� Vol. 88, No. 4, October 2007Copyright ª2007 American Society for Reproductive Medicine
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In our study, the recipient did not pay donor compensation orfor health screening or cycle medications that were associ-ated with the donor.
Our study demonstrated that it is indeed possible to per-form oocyte cryopreservation research in an ethical, institu-tional review board–controlled, donor egg model. Wepublished our methods and findings in this respected peer-re-viewed journal to provide the reproductive community withthe knowledge that we gained. Human oocyte cryopreserva-tion, when performed in a safe and controlled manner, can bean effective technique that can be applied in clinical situa-tions and can show high oocyte survival and clinical preg-nancy rates, opening the door for this technique to be usedfor women choosing to preserve their fertility.
Jason Barritt, Ph.D.Martha Luna, M.D.Marlena Duke, M.Sc.Alan Copperman, M.D.Mount Sinai School of MedicineReproductive Medicine Associates of New YorkNew York, New York
June 29, 2007
REFERENCES1. de Melo-Martin I, Cholst IN. Researching human oocyte cryopreserva-
tion: ethical issues. Fertil Steril. Published online May 16, 2007.
2. Barritt J, Luna M, Duke M, Grunfeld L, Mukherjee T, Sandler B, et al. Re-
port of four donor-recipient oocyte cryopreservation cycles resulting in
high pregnancy and implantation rates. Fertil Steril 2007;87:189. e13–7.
3. Borini A, Sciajno R, Bianchi V, Sereni E, Flamigni C, Coticchio G. Clin-
ical outcome of oocyte cryopreservation after slow cooling with a protocol
utilizing a high sucrose concentration. Hum Reprod 2006;21:512–7.
doi:10.1016/j.fertnstert.2007.07.1365
0015-0282/07/$32.00, Published by Elsevier Inc.
article (and we therefore were unable to reference theirstudy), we are pleased to see that their group was attentiveto some of the ethical problems that are raised when subjectsare recruited for oocyte cryopreservation research studies (2).
We are, however, surprised that the correspondents take is-sue with our statement that ‘‘current (pregnancy rates) stillappear to be lower than those seen with standard IVF proce-dures,’’ on the basis that 75% (3 of 4) of their subjects con-ceived. These certainly are encouraging data, but wecaution against the overenthusiastic use of small series togeneralize about outcomes. In our article, we cite 10 studiesof outcomes (references 18–27), as well as a review of theliterature (reference 32). Taken together, and adding theexcellent recent review by Jain and Paulson (3), these studiessuggest that there still is considerable work to be done beforewe can consider this medical procedure comparable to onesthat presently are available, and thus, one that can be offeredconfidently to patients, both for medical indications as well aspossibly for nonmedical ones.
As we point out in our article, overenthusiastic responsesto clinical advances specifically may be related to some ofthe difficulties in evaluating new medical procedures andthe resulting obstacles to obtaining informed consent fromresearch participants.
Inmaculada de Melo-Martin, Ph.D.Ina N. Cholst, M.D.Weill Cornell Medical CollegeNew York, New York
July 17, 2007
REFERENCES1. de Melo-Martin I, Cholst IN. Researching human oocyte cryopreserva-
tion: ethical issues. Fertil Steril. Published online May 16, 2007.
2. Barritt J, Luna M, Duke M, Grunfeld L, Mukherjee T, Sandler B, et al. Re-
port of four donor-recipient oocyte cryopreservation cycles resulting in
high pregnancy and implantation rates. Fertil Steril 2007;87:189. e13–7.
An expert forum for the histology of endometriomas
To the Editor:
We read with interest the article by Muzii et al. (1) regard-ing the histologic analysis of endometriomas. However, wedo not agree with the conclusions reached in that study re-garding pathogenesis of endometriotic cysts. Because the au-thors have made important clinical recommendations on thebasis of their findings, we would like to express our concernswith certain aspects of their methodology.
Since we first reported on the laparoscopic management ofsevere endometriosis more than 2 decades ago, there has been
Fertility and Sterility�
a welcome burgeoning of clinical studies related to endome-
triosis (2). On the basis of our research and clinical experi-ence, we have established a classification method forendometriomas. Although this classification has been re-ferred to by Muzii et al. (1), it appears that they misunder-stood some of our assumptions. In brief, we classify the
endometriomas into two types: primary, or type I, endome-triomas, which originate from endometriotic lesions that ad-here to the ovary, bleed inside, and expand; and secondary, ortype II, endometriomas, which are what Sampson originallyreferred to (3) and which originate from functional cysts
that were invaded by plaques of endometriosis that bleedinside the cyst.
Type I endometriomas usually are <5 cm in size, containa dark fluid, and have capsules that are difficult to remove
because they are associated with dense fibrosis and adhe-sions. Histopathological examination always shows endome-trial glands and stroma in such cases. As for type IIendometriomas, we have shown elsewhere that the func-tional wall of the cyst may gradually change to an endome-
trioma (4).
It appears that the present study may have been affected tosome extent by a selection bias, because all of the cases in-
cluded were either type I or late-stage type II. Functionalcyst-related endometriomas are diagnosed most clearly intheir formative stages, whereas ovarian endometriomas of>3 cm were included in the present trial. Furthermore, therelatively small number of cases (n ¼ 59) investigated in
the present study limits the ability of the investigators toreach a conclusive observation in regard to the controversialpathogenesis of endometriomas. Muzii et al. (1) note that thepurpose of their study was to thoroughly examine the endo-metrioma wall to assess the presence, extent, and depth of
penetration of endometriosis in the cyst capsule. However,it is recognized that ovarian cystectomy by itself may pre-clude complete evaluation of the ovary. It appears that onlya study in which oophorectomy specimens were includedwould allow for more conclusive observations.
Admittedly, in the literature, only a few studies report indetail the histologic nature of the endometrioma wall. Inour article, we noted that the capsule of endometriomas couldbe up to 4 to 5 mm in thickness, so it must be removed to de-
crease the chances of recurrence (3). In the present study, themean value of maximal depth of endometriosis penetration inthe endometrioma wall was merely 0.6 mm. Yet Stratton et al.(5) reported in their study that endometriomas deeper than 10mm were always histologically confirmed as endometriosis.
This wide variability in the depth of invasion of endometri-osis into the cyst wall may often be the explanation for the ul-timate success of cystectomy. Supporting what we and othercolleagues reported in 1986 (2), on the basis of histopatho-logical findings, the latest systematic review of the Cochrane
Database concluded that excisional surgery for endometrio-mata provides for a more favorable outcome than does
1017
Our study was merely a thorough histological evaluation of70 endometrioma cyst walls that was performed by an unbi-ased pathologist who was not aware of the different theorieson the pathogenesis of endometriomas. The data set providedby our study gives us a detailed description of the histologicalnature of the endometrioma wall that may help in the clinicaldecision of which treatment may be best for the endome-trioma. In our discussion section, we do make some clinicalrecommendations, as Nezhat et al. state in their letter, andwe are happy that we and they, in this respect, follow exactlythe same lines, by suggesting cystectomy as the technique ofchoice (1). This clinical recommendation, although it still isdebated today, is supported by sound data from a meta-anal-ysis of randomized clinical trials (2).
In our study, the histologic confirmation rate for endome-triosis was 100%. A mean of 60% (ranging from 10% to98%) of the surface of the inner cyst wall was lined by en-dometrium. No other epithelial lining type was observed. Inthe areas of the inner wall that were not covered by endome-trium, only a fibrotic capsule could be observed. In 81% ofthe cases, beyond the fibrotic wall, ovarian tissue (stroma,follicles, or both) was observed. In no case was a transitionnoted from luteal tissue to the endometrial cyst. Only sucha finding would give support to the existence of type IIendometrioma, according to the classification by Nezhatet al (3). Admittedly, in their much larger series of 187women, Nezhat et al. (3) also were not able to detect thisfinding.
In a recent study by Scurry et al. (4), a thorough histolog-ical evaluation was performed in an attempt to classifyendometriomas into different pathogenetic types. The con-firmation rate for endometriosis at histology was 100%.The investigators were able to identify only two types ofcysts. The first, occurring in 10 (42%) of 24 evaluable spec-imens, was defined as ‘‘cortical invagination endome-trioma,’’ further defined as a cyst with a layer of ovariancortex interspersed between the endometrial lining and theovarian medulla. The second type, occurring in 14 (58%)of 24 evaluable specimens, was defined as ‘‘unclassified,’’because it did not fall into any of the three preset categories.This second type of cyst was described as endometrial lin-ing surrounded by fibrosis, without identifiable ovarian tis-sue between the endometriosis and the medulla. Fivespecimens (17%) of the 29 that were included could not
drainage and ablation, with regard to the recurrence of the en-dometrioma (6).
This study once again appears to highlight the need fora prospective study of ovarian endometriotic cysts, pre-ferably removed by oophorectomy, using a standardizedmethod for examination, sampling, and histological evalua-tion, to elucidate better the pathogenesis of endometriomasand to determine an accurate frequency of the various cysttypes.
Camran Nezhat, M.D.Ceana Nezhat, M.D.Stanford University Medical CenterStanford, California
Daniel Seidman, M.D.Tel Aviv UniversityTel Aviv, Israel
Bulent Berker, M.D.Ankara University School of MedicineAnkara, Turkey
Farr Nezhat, M.D.Mount Sinai School of MedicineNew York, New York
June 5, 2007
REFERENCES1. Muzii L, Bianchi A, Bellati F, Cristi E, Pernice M, Zullo MA, et al. His-
tologic analysis of endometriomas: what the surgeon needs to know. Fertil
Steril 2007;87:362–6.
2. Nezhat C, Crowgey SR, Garrison C. Surgical treatment of endometriosis
by laser laparoscopy. Fertil Steril 1986;45:778–83.
3. Sampson JA. Perforating hemorrhagic (chocolate) cysts of the ovary. Arch
Surg 1921;3:245–323.
4. Nezhat F, Nezhat C, Allan CJ, Metzger DA, Sears DL. Clinical and histo-
logical classification of endometriomas: implications for a mechanism of
pathogenesis. J Reprod Med 1992;37:771–6.
5. Stratton P, Winkel CA, Sinaii N, Merino MJ, Zimmer C, Nieman LK. Lo-
cation, color, size, depth, and volume may predict endometriosis in lesions
resected at surgery. Fertil Steril 2002;78:743–9.
6. Hart RJ, Hickey M, Maouris P, Buckett W, Garry R. Excisional surgery
versus ablative surgery for ovarian endometriomata. Cochrane Database
Syst Rev 2005;3:CD004992.
doi:10.1016/j.fertnstert.2007.07.1367
Reply of the Authors:
First, we thank Nezhat et al. for their appreciation of ourwork (1) and for their valuable comments that have givenus the opportunity to further clarify the findings of our study.
Those authors report, as their first comment, that they donot agree with the conclusions of our study regarding thepathogenesis of endometriotic cysts. We want to stress thefact that we did not come to any conclusion on the pathogen-esis of endometriomas.
be assessed, other than the diagnosis of endometriosis, be-cause of extensive destruction of the tissue. In no casewere a surface inclusion–related endometrioma (definedas a cyst with continuity between a surface-inclusion cystand endometrial lining) or a physiological cyst–related en-dometrioma (defined as a cyst with continuity betweena functional cyst and endometrial lining) observed. In sum-mary, Scurry et al. (4) were able to identify two types of en-dometriomas, one with only endometrial lining and fibrosis(58%) and one with endometrial lining, fibrosis, and ovariantissue (42%). These figures are consistent with those re-ported in our study published elsewhere on the histology
1018 Letters to the Editor Vol. 88, No. 4, October 2007
of the endometrioma wall (5), in which endometriosis andfibrosis were found in 54% of the specimens, whereas endo-metriosis, fibrosis, and ovarian tissue were present in 46%of the specimens. Scurry et al. (4) try to link their findingto existing pathogenetic theories, which we did notdo (1, 5).
Also, Hachisuga and Kawarabayashi (6) were not able to‘‘histopathologically demonstrate the secondary involvementof functional ovarian cysts in the process of endometriosis,’’because ‘‘no ovarian endometriotic cyst showed both lutei-nized and epithelial lining’’ in their series of 73 ovarian endo-metriomas.
As to the depth of penetration of endometriosis in the cystwall, we report a mean maximal depth of 0.6 mm. However,this is not the thickness of the entire cyst wall, which alsocontains fibrosis and ovarian tissue, if present. The meancyst wall thickness, in fact, is reported as 1.4 mm (1). Thecyst wall gets thicker (2.4 mm in our study) where the endo-metriosis penetrates more deeply into the tissue underneath.Therefore, the figure of 0.6 mm does not necessarily contrastwith the figure of 4 to 5 mm that was given by Nezhat et al. intheir letter.
In conclusion, it may well be that our study included onlytype I and late type II endometriomas, as described originallyby Nezhat et al. (3) and as subsequently modified with thesimplification into two types instead of three. However, iflate-stage type II means that a type II endometrioma haslost the initial functional lining, the existence of a true typeII, according to the existing literature on the histological anal-ysis of endometriomas, still is unproven. In our study, any-how, we did not intend to make endometriomas fall intoany preset categories or to fit our findings in any existingpathogenetic theory. We intended simply to describe the his-tology of the endometrioma wall that was excised with thestripping technique.
Ludovico Muzii, M.D.Antonella Bianchi, M.D.Emanuela Cristi, M.D.Marzio A. Zullo, M.D.Roberto Angioli, M.D.University Campus Bio-MedicoRome, Italy
REFERENCES1. Muzii L, Bianchi A, Bellati F, Cristi E, Pernice M, Zullo MA, et al. His-
tologic analysis of endometriomas: what the surgeon needs to know. Fertil
Steril 2007;87:362–6.
2. Hart R, Hickey M, Maouris P, Buckett W, Garry R. Excisional surgery ver-
sus ablative surgery for ovarian endometriomata: a Cochrane Review.
Hum Reprod 2005;20:3000–7.
3. Nezhat F, Nezhat C, Allan CJ, Metzger DA, Sears DL. Clinical and histo-
logical classification of endometriomas: implications for a mechanism of
pathogenesis. J Reprod Med 1992;37:771–6.
4. Scurry J, Whitehead J, Healey M. Classification of ovarian endometriotic
cysts. Int J Gynecol Pathol 2001;20:147–54.
5. Muzii L, Bianchi A, Croce’ C, Manci N, Benedetti Panici P. Laparoscopic
excision of ovarian cysts: is the stripping technique a tissue-sparing
procedure? Fertil Steril 2002;77:609–14.
6. Hachisuga T, Kawarabayashi T. Histopathological analysis of laparos-
copically treated ovarian endometriotic cysts with special reference to
loss of follicles. Hum Reprod 2002;17:432–5.
doi:10.1016/j.fertnstert.2007.07.1368
Fertility and Sterility�
Perspectives on oocyte research
To the Editor:
In response to the findings presented by Moayeri et al. (1),we oblige the authors’ request for a larger series of data toconfirm their conclusions.
Our analysis of 922 clinical pregnancies resulting fromblastocyst transfers over a 56-month period revealed a similardecline in the monozygotic (MZ) twinning rate with time. Asignificant drop in the rate of MZ twinning resulting from blas-tocyst transfers was demonstrated when comparing the 29-month period before 2005 (18/412, 4.4%) with the subsequent27 months beginning in 2005 (10/510, 2.0%; P<.05). An evenlarger decline was found in the MZ rate after, specifically, do-nor oocyte pregnancies during the same time periods (6/99[6.1%] vs. 3/122 [2.5%]), but this difference did not reachstatistical significance because of the limited sample size.
Our results occurred over a shorter time period, <5 years,with a larger number of cases (922), lending great support forthe authors’ hypothesis of an experience factor. There wereno obvious changes in laboratory technique, protocols, or cul-ture media during the experimental period, supporting the find-ings that experience, including very highly controlled cultureconditions, may play a larger role in the MZ rate drop. Wealso analyzed all the MZ twinning cases at our center over theentire study period and could not find a significant correlationwith mean oocyte age, method of fertilization (intracytoplasmicsperm injection vs. IVF), use of assisted hatching, or media lotnumber used for embryo culture to day 3 or blastocyst.
Our supportive finding that the rate of MZ twinning de-clines with time and experience also is difficult to explain.Although we do not have a cause to directly relate to the de-cline, we have eliminated a large number of proposed causa-tive factors in our lab, including ICSI and assisted hatching.General improvements in blastocyst culture systems (media-manufacturer related), the grading of blastocysts, and the pro-cess of embryo selection for transfers all may have improvedover the past 5 years, and that learning curve may helpaccount for our findings. Analysis of other potential causes
1019
those authors for obliging our request for other investigatorsto share their recent experience with blastocyst transfer tohelp confirm our findings. Their data provide valuable infor-mation to the IVF community by verifying our results in theirlarge series.
Sharon E. Moayeri, M.D.Barry Behr, Ph.D.Ruth B. Lathi, M.D.Lynn M. Westphal, M.D.Amin A. Milki, M.D.Stanford University of MedicineStanford, California
July 10, 2007
REFERENCES1. Moayeri SE, Behr B, Lathi RB, Westphal LM, Milki AA. Risk of mono-
zygotic twinning with blastocyst transfer decreases over time: an 8-year
experience. Fertil Steril 2007;87:1028–32.
doi:10.1016/j.fertnstert.2007.07.1370
for these rare pregnancies, such as epigenetic regulationchanges caused by in vitro culture, or possible growth factorsfound, or lacking, in media or protein supplements, will needto be investigated to determine why IVF increases the risk ofthese types of pregnancies over natural conception. We be-lieve that our findings, involving more cases in a shortertime period, verify these authors’ published results.
Jason Barritt, Ph.D.Martha Luna, M.D.Marlena Duke, M.Sc.Alan Copperman, M.D.Mount Sinai School of MedicineReproductive Medicine Associates of New YorkNew York, New York
May 8, 2007
REFERENCES1. Moayeri SE, Behr B, Lathi RB, Westphal LM, Milki AA. Risk of mono-
zygotic twinning with blastocyst transfer decreases over time: an 8-year
experience. Fertil Steril 2007;87:1028–32.
doi:10.1016/j.fertnstert.2007.07.1369
Reply of the Authors:
We appreciate the letter that Barritt et al. wrote in responseto our study (1). We found a significant decline in the inci-dence of monozygotic twinning with blastocyst transferwhen we compared our recent experience with our earlyexperience with this modality.
It is reassuring to us to learn that a similar trend was ob-served by Barritt et al. in New York, among a large numberof pregnancies after blastocyst transfer. We are grateful to
Erratum
In the article ‘‘Assisted reproductive technology in theUnited States: 2001 results generated from the AmericanSociety for Reproductive Medicine/Society for AssistedReproductive Technology registry’’ (June 2007;87(6):1253–66), the ‘‘Participants’’ section of the abstract should say thatdata were collated after November 2002 so that outcomesof all pregnancies would be known. A corrected versionappears online.
1020 Letters to the Editor Vol. 88, No. 4, October 2007
