VOLUME 15
IVF and embryo transfer: historicalorigin and developmentby John D. Biggers, DSc, PhD
The Importance of pH MeasurementWithin the IVF Laboratoryby Jason Swain, PhD, HCLD The Patient's Corner
The Use of global® forTime-lapse Videographic Analysis
of Human Embryo Developmentby Don Rieger, PhD
ESHRE 2013, London, UK
Remembering Bob EdwardsNobel Laureate and Pioneer of IVF
Effect of Coda® Air Filtration onChemical and Clinical Pregnancy Rates
(Battaglia et al., Fertil Steril 75, Suppl. 1, 6S, 2001)
ECOTM 1500 ECOTM 1200 CodaAir® 800CodaAir® 900
253390
Effect of Follicular Phase Particulate AirPollution on Pregnancy Loss
(Perin et al., Fertil. Steril., 2009, in press)
Q4 (High Air Pollution)Q1-3 (Low Air Pollution)
P = 0.001
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30
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Carbon-activated gas filtration during in vitroculture increased pregnancy rate following
transfer of in vitro-produced bovine embryos
(Merton et. al., Theriogenology 67, 1233-8, 2007)
0
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Coda
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Aero® 700 Aero® 500 CodaAir® Positive
Pressure Unit
Coda® Unit II Coda® Filters Coda® Xtra
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Fertility Magazine The First Magazine In FertilityTM
The Use of global®
Embryo Development by Don Rieger, PhD ............................................... 5
Pioneer of IVF ............................................................................14
DON RIEGER, PHD
IVF and embryo transfer: historical origin and development by John D Biggers, DSc, PhD ...........................................................................................................................16
by Jason E. Swain, PhD, HCLD .......................................................................................................................28
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The Use of global® for Time-lapse Videographic Analysis of Human Embryo Developmentby Don Rieger, PhDVice President, Research and Development, LifeGlobal, [email protected]
DON RIEGER, PHD
THE FEATURED ARTICLE
1. The Research
“As a matter of fact we can safely say that the motion picture
originated in the biological laboratory.” (Rosenberger 1929)
et al.
et al. et al. 2012).1926).
“Marey also indicated the use of time-lapse photography in the study of phenomena which, because of their extreme slowness, are difficult to appreciate by direct observation: attention wearies, the eye tires, and the changes are imperceptible. …Sometimes the movement is too rapid, sometimes it is too slow. …With cinematographic projection, the movement can be accelerated and rendered
perceptible to the eye.”
THE FEATURED ARTICLE
2. Time-lapse Development
Table 1.
Reference Species Observation(s)
et al. Cattle
et al. Cattle
et al.
et al.
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et al.
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et al. Cattle
et al. Cattle
et al. Cattle
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et al.
et al. Cattle
et al. Cattle
Wale
et al.
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t al. 2011; Barrie et al.
et al.
et al.
THE FEATURED ARTICLE
a temporally demonstrable change in shape or form
in vitro
et al. et al. et al. et al.
et al. et al.
et al.
et al.
et al.
et al. et al.
et al.
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et al.
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THE FEATURED ARTICLE
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THE FEATURED ARTICLE
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THE FEATURED ARTICLE
5.
1.
2.
4. ®
5. ®
References
Brit. Med. J. 1910, 598.
Hum Reprod 27Barrie
J Bacteriol 14,
Hum Reprod 28Biggers
Fertil. Steril. 90Bin
Arch Biol (Liege) 91
Cochrane Database Syst Rev 7, CD008950.
Proceedings of the Royal Netherlands Academy of Arts and Sciences 14
Reprod Biomed Online In press.
J Exp Med 15
J Exp Med 13
J Exp Med 44
Cassini Bolletino di zoologia 28
259.
J Assist Reprod Genet In press.
Nat Commun 3, 1251.
Cohen
Fertil Steril 49
J Embryol Exp Morphol 17
Comptes rendus hebdomadaires des séances de l’Académie des sciences 149
Journ. de Physiol. Path. gén
J Clin Invest. 2
J Clin Invest. 2
J Assist Reprod Genet 28
THE FEATURED ARTICLE
Reprod Biomed Online 25
Reprod Biomed Online In Press
J Bacteriol 109
Fertil Steril In press.
Reproduction 140
Biol Reprod 52,
Hum Reprod 11
J Reprod Fertil 105
Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character 107
Br Med J 1
Fertil Steril 97Herrero
Fertil Steril 99
Reproduction 123
Theriogenology 50
Fertil Steril ., S88
J Fertiliz In Vitro 2, e112.
Hum Reprod 27
J Assist Reprod Genet 29
Hum Reprod 27
Fertil Steril 99
Biol Reprod 69,
Lewis Science 69
Hum Reprod 25
California State Journal of Medicine 10
Br Med J 1
J. Phys. Theor. Appl. 3
Nature (Lond.) 19
J Reprod Fertil 58
J Anat 134405.
Vet Rec 112
Reproduction 129
THE FEATURED ARTICLE
Fertil Steril 97
Fertil Steril 98
J Assist Reprod Genet 29
Am J Obstet Gynecol 199
Am J Public Health (N Y) 6
J Exp Med 40
Reprod Biomed Online 25
Eur J Obstet Gynecol Reprod Biol In press.
Nature (Lond.) 25, 605.Payne
Hum Reprod 12
Zygote 9
Fertil Steril 98
Fertil Steril 92
J. Clin. Embryol. 13
Rieger
Rogers J Bacteriol 19
Rosenberger J. Dental Res 9
Fertil Steril 98
Mol Reprod Dev 26
Fertil Steril
Reprod Biomed Online
Singh
Fertil Steril ., S106
J Bone Joint Surg Am s2-16
Molec. Reprod. Devel. 26
Hum Reprod 16
Reprod Biomed Online 21
JAMA 59
Reprod Biomed Online 26
Nat Biotechnol 2821.
Fertil Steril .,
Fertil Steril
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Remembering Bob Edwards
Nobel Laureate and Pioneer of IVF
Bob Edwards, you will be sadly missed.
from the IVFonline/LifeGlobal Team
ESHRE, Lausanne, Switzerland 2001Michael Cecchi, Bob Edwards
ASRM, Philadelphia, Pennsylvania 2004Elena Chuy, Jean Rossiar, Mirela Pino, Bob Edwards, Monica Mezezi, Renata Gruosso, Amy Rossiar
ASRM, Philadelphia, Pennsylvania 2004Don Rieger, Bob Edwards, Monica Mezezi
ESHRE, Bologna, Italy 2000Yury Verlinsky, Monica Mezezi, Bob Edwards
ESHRE, Lausanne, Switzerland 2001Bob Edwards, Michael Cecchi, Pamela Madsen
ASRM, Orlando, Florida 2001Glenn Kroecker, Bob Edwards, Fiona Bennett
ARTICLES
IVF and embryo transfer: historical origin and developmentJohn D Biggers
Department of Cell Biology, 240 Longwood Avenue, Harvard Medical School, Boston, MA 02115, USAE-mail address: [email protected]
John Biggers, DSc, PhD is professor of cell biology at Harvard Medical School. His current research interests are evaporative drying of spermatozoa, vitrification, embryo culture, embryo assessment and the biography of Walter Heape. He is a former Commonwealth Fellow of St John’s College, Cambridge, past President of the Society of Reproduction, former Editor in Chief Biology of Reproduction, Chief Scientific Advisor to the Ethics Committee, US Department of Health, Education and Welfare that made recommendations on IVF and embryo transfer, Fellow of the American Association for the Advancement of Science, Hartman Award of the Society of Reproduction, Pioneer Award of the International Embryo Transfer Society, Marshall Medal of the Society for the Study of Fertility and a Life Member of the New England Fertility Society and the Society for the Study of Reproduction.
Abstract IVF and embryo transfer for the treatment of human infertility has now resulted in the birth of over 4 million babies. The technique did not arise as a quantum event but was built on the efforts of many earlier workers in the fields of reproductive endocrinology and development. One should remember the famous saying of Isaac Newton: ‘If I have seen further than most, it is because I have stood on the shoulder’s of giants’. Ethical and moral issues have always arisen when investigators study early mammalian development, particularly human development. This paper documents these earlier studies and also draws attention to the ethical and moral arguments that inevitably arose.
©2012, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.KEYWORDS: ethical issues, history, in-vitro fertilization, IVF, Gregory Pincus, John Rock
This article was published in Reproductive BioMedicine Online, Vol 25, 2012, p118-127, ‘IVF and embryo transfer: historical origin and development’. Copyright Elsevier. It is reprinted here with permission.
JOHN D. BIGGERS, DSC, PHD
IVF/ET in mythology
ARTICLES
technologies
in vitro;
in vitro
in vitro. His
in vitro
It was 1951 that Dupont and Schwartz produced the first ectogenetic child . . . France was the first country to adopt ectogenesis officially, and by 1968 was producing 60,000 children annually by this method. In most countries the opposition was far stronger, and was intensified by the Papal Bull ‘Nunquam prius audito’, and by the similar ‘fetwa’ of the Khalif, both of which
appeared in 1960. (Haldane, 1923)
Brave New World
ARTICLES
in vitro
Proceedings of the National Academy of Sciences, USA
in vitro
New York Times
As rabbits and men belong to the mammalian group, the work is viewed as pointing toward the possibility of human children being brought into the world by a ‘host-mother’ not related by blood to the child.
It is reasoned that eventually women capable of having children whose health does not permit them to do so may ‘hire’ other women to bear their children for them, children actually their own flesh and blood.
To one who desires to speculate at this point the Harvard experiment offers another possibility. Theoretically, at least, it may become possible for a woman so inclined, particularly in a country influenced by eugenic
considerations, to bring into the world twelve children a year by ‘hiring’ twelve ‘host-mothers’ to bear their test-tube-conceived children for them.
Advocates of ‘race betterment’ might urge such procedures for men and women of special aptitudes, physical, mental or spiritual (Laurence, 1936).
New York Times
Collier’s Magazine
In the resulting world man’s value would shrink. It is conceivable that the process would not even produce males. The mythical land of the Amazons would then come to life. A world where woman would be self-
sufficient; man’s value precisely zero.
New England Journal of Medicine
Conception in a watch glass
Contemplating this new discovery, one’s mind travels much farther. Lewis and Hartman have isolated a fertilized monkey ovum and photographed its early cleavage in vitro. Pincus and Enzmann have started one step earlier with the rabbit, isolating an ovum, fertilizing it in a watch glass, and re-implanting it in a doe other than the one that furnished the egg, and have thus successfully inaugurated pregnancy in an unmated animal. If such an accomplishment with rabbits were to be duplicated in human beings, we should, in the words of ‘flaming youth’, be ‘going places’. The difficulty with human ova has been that those recovered from tubes have regressed beyond the possibility of fertilization in vitro. But by utilizing the electrical sign we may be able to obtain them from
ARTICLES
the follicle at the peak of their maturity. If the new peritoneoscope can be developed along the lines of the operating cystoscope, laparotomy may even be dispensed with. What a boon for the barren woman
with closed tubes!
New England Journal of Medicine
in vitro
in vitro
in vitro
in vitro
I don’t believe you ever got in vitro fertilization . . . Have a dozen reasons to question your conclusions, chief of which is the simultaneous and independent discovery by Chang, Austin and Blandau [Braden?] that ‘raw’ sperms won’t fertilize any egg even in vivo! Sperms must be ‘capacitated’ (Austin) in the female tract, either in
the uterus or the tube.
Now, I want you to go back to the problem and clean it up and really immortalize yourself. Inject 50,000,000 sperm into a woman’s uterus. In 2 h take out the sperms and add to the ovarian egg (but only from a 16–18 mm. follicle, eggs in lesser ones are N.G.). I’m betting
heavy odds on the outcome of this experiment.
The time may be rapidly approaching when the poor woman whose tubes had been excised, yet who still wants a baby, will rejoice that Dr Shettles will be able to extract an ovum from her ovary, probably not by laparotomy, but through an operating telescope (which can be done – we have done it); then fertilize the egg in vitro by the husband’s spermatozoa; and finally put it back in the uterus. Thus will he impregnate the woman in spite of the fact that she has no tubes.
(Shettles, 1958)
in vitro
ARTICLES
in vivo
eggs.
in vitro
in vitro
The Eggs of Mammals
in vitro
Daily Telegraph
Discovery
It is inevitable that the thoughts of anyone who has worked on the subjects outlined in this article should turn to Aldous Huxley’s fantasy ‘Brave New World’, where he describes completely artificial fertilization and development of human embryos. Fortunately we are far removed from this frightening prospect. The study of the cultivation and transfer of embryos is none the less of the greatest interest, both from the point of view of pure science, and because the techniques associated with it are potentially of immense value in the investigation of many biological problems in medicine and agriculture. (Biggers and McLaren,
1958)
ARTICLES
in vitro
New England Journal of
Medicine
(1) ‘Maturation in vitro of human ovarian oocytes’ in The Lancet (Edwards, 1965b).
(2) ‘Early stages of fertilization in vitro of human oocytes matured in vitro’ in Nature (Edwards et al., 1969).
(3) ‘Fertilization and cleavage in vitro of preovular human oocytes’ in Nature (Edwards et al., 1970).
(4) ‘Laparoscopic recovery of preovulatory human oocytes after priming of ovaries with gonadotrophins’ in The Lancet (Steptoe and
Edwards, 1970).
in vitro
in vitro
in vitro
ARTICLES
Nature
1981).
ARTICLES
Sunday
in vitro
ARTICLES
Presentation
References
ARTICLES
Press.
ARTICLES
682.
Declaration: The author is a consultant to IVFOnline, Guelph, Ontario, Canada.
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The Importance of pH Measurement Within the IVF Laboratory
by Jason E. Swain, PhD, HCLD, Assistant Professor, Department of Obstetrics & Gynecology, Scientific Director ART Laboratories, University of Michigan, Ann Arbor, MI, USA
You can contact Jason Swain at [email protected]
JASON E. SWAIN, PHD, HCLD
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2
2
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2
2
2
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2
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Hum Reprod Update
Figure 1. CO
1 2 3 4 5 6 7 8 9 10 11 12 13
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pH
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O2
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pH
Day
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Figure 3.
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7
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7.2
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Me
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Working to eliminate multiple pregnancies: asuccess story in QuébecF Bissonnette a,*, SJ Phillips a, J Gunby b, H Holzer c, N Mahutte d,P St-Michel e, IJ Kadoch a
a OVO Fertility, 8000 Boulevard Decarie #100, Montréal, Québec, Canada H4P 2S4; b Canadian ART Register, 2534 Cavendish Drive, Burlington, Ontario, Canada L7P 4E4; c McGill Reproductive Centre, 687 Pine Avenue West, Montréal, Québec, Canada H3A 1A1; d The Montreal Fertility Centre, 5252 de Maisonneuve Blvd West, Suite 220, Montréal, Québec, Canada H4A 3S5; e Procrea Cliniques, 1361, avenue Beaumont, #301, Mont-Royal, Québec, Canada H3P 2W3
* Corresponding author. E-mail addresses: [email protected],[email protected] (F Bissonnette).
Dr. François Bissonnette is the medical director of OVO, a fertility clinic in Montreal, and a professor in the department of Obstetrics-Gynecology at the University of Montreal. He completed his speciality training in obstetrics and gynecology in 1980 and was certified by the Royal College of Physicians and Surgeons of Canada in 1985. He was subsequently trained in reproductive endocrinology and infertility at the University of Louisville, Kentucky. He is currently the director of the Canadian ART Registry and has been President of the Canadian Fertility and Andrology Society in 2009.
Abstract In August 2010, the provincial government of Québec, Canada introduced funding of assisted reproduction treatment through the provincial health programme. Alongside this benefit, legislation was introduced to control assisted reproduction treatment activities in the province, including restrictions on the number of embryos that could be transferred in any one cycle. The aim of the programme was to transfer a single embryo in every cycle; multiple embryos could be transferred under suboptimal conditions but required physician justification. In the first 3 months of this programme, 1353 cycles of IVF were performed in five Québec assisted reproduction centres, with an overall clinical pregnancy rate of 32% per embryo transfer and 50% of transfers used elective single-embryo transfer (eSET). The multiple-pregnancy rate was only 3.7% per clinical pregnancy. In 2009, prior to the introduction of the programme, eSET was used in only 1.6% of embryo transfers, resulting in a multiple-pregnancy rate of 25.6%. These data demonstrate that providing provincially funded assisted reproduction treatment created an environment in which the aggressive use of eSET was not only possible, but also rapidly implemented. The result was a dramatic drop in multiple-pregnancy rates, approaching those for natural pregnancies.
©2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.KEYWORDS: cumulative pregnancy rate, eSET, funding, IVF, multiple pregnancies
This article was published in Reproductive BioMedicine Online, Vol 23, 2011, p500-504, ‘Working to eliminate multiple pregnancies: a success story in Québec’. Copyright Elsevier. It is reprinted here with permission.
DR. FRANÇOIS BISSONNETTE
ARTICLES
Table 1
Materials and methods
Figure 1 Figure 2
ARTICLES
Table 2
Table 3
Table 4).
Table 5.
ARTICLES
ARTICLES
References
ARTICLES
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A Unique Microsurgical Sperm Extraction Program in a Non-hospital Based IVF Clinic – a Report on our Experience
by Carole Lawrence, Laboratory Director, Pacific Centre for Reproductive Medicine, Burnaby, British Columbia, Canada
You can contact Carole Lawrence at [email protected]
CAROLE LAWRENCET
(1,2,5).
ARTICLES
ARTICLES
References
et al.
et al.
Cook CA et al.
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ARTICLES
Social egg freezing: for better, not for worseHeidi Mertes *, Guido Pennings
Bioethics Institute Ghent, Blandijnberg 2, 9000 Gent, Belgium* Corresponding author. E-mail address: [email protected] (H Mertes).
Heidi Mertes is a postdoctoral research fellow of the Research Foundation, Flanders and member of the Bioethics Institute, Ghent. Her current research focuses on the ethical implications of new techniques for fertility preservation. Other research interests include the ethics of embryonic stem cell research and reproductive ethics.
Abstract The possibility for healthy women to cryopreserve their oocytes in order to counter future infertility has gained momentum in recent years. However, women tend to cryopreserve oocytes at an age that is suboptimal from a clinical point of view – in their late thirties – when both oocyte quantity and quality have already considerably diminished and success rates for eventually establishing a pregnancy are thus limited. This also gives rise to ethical concerns, as the procedure is seen as giving false hope to (reproductively speaking) older women. This study evaluates which measures can be taken to turn social freezing into a procedure that is both clinically and ethically better than the current practice. The main objective of these measures is to convince those women who are most likely to (want to) reproduce at an above-average age to cryopreserve their oocytes at a time when this intervention is still likely to lead to a live birth and to discourage fertility clinics from specifically targeting women who have already surpassed the age at which good results can be expected.
©2011, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.KEYWORDS: cryostorage, fertility decline, fertility preservation, oocyte cryopreservation, reproductive aging, social egg freezing
This article was published in Reproductive BioMedicine Online, Vol 23, 2011, p824-829, ‘Social egg freezing: for better, not for worse’. Copyright Elsevier. It is reprinted here with permission.
HEIDI MERTES
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The divergence between the best-case scenario and the worst-case scenario
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2009).
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area.
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References
ARTICLES
S201.
Nekkebroeck, J., Stoop, D., Devroey, P., 2010. A preliminary pro"le of women opting for oocyte cryopreservation for non-medical reasons. Hum. Reprod. 25, i14–i17.
Noyes, N., Boldt, J., Nagy, Z.P., 2010. Oocyte cryopreservation: is it time to remove its experimental label? J. Assist. Reprod. Genet. 27, 69–74.
Ravin, A.J., Mahowald, M.B., Stocking, C.B., 1997. Genes or gestation? Attitudes of women and men about biologic ties to children. J. Womens Health 6, 639–647.
Rienzi, L., Ubaldi, F.M., 2011. Embryo development of fresh ‘versus’ vitri"ed metaphase II oocytes a#er ICSI: a prospective randomized sibling-oocyte study. Hum. Reprod. 26, i1.
Rienzi, L., Romano, S., Albricci, L., Maggiulli, R., Capalbo, A., Baroni, E., Colamaria, S., Sapienza, F., Ubaldi, F., 2010. Embryo development and gestation using fresh and vitri"ed oocytes. Hum. Reprod. 25, 1192–1198.
Rudick, B.J., Paulson, R., Bendikson, K., Chung, K., 2009. $e status of oocyte cryopreservation in the United States. Fertil. Steril. 92, S187.
Rybak, E.A., Lieman, H.J., 2009. Egg freezing, procreative liberty and ICSI: the double standards confronting elective selfdonation of oocytes. Fertil. Steril. 92, 1509–1512.
Sage, C.F.F., Kolb, B.M., Treiser, S.L., Silverberg, K.M., Barritt, J., Copperman, A., 2008. Oocyte cryopreservation in women seeking elective fertility preservation – a multicenter analysis. Obstet. Gynecol. 111, 20S.
Shkedi-Ra"d, S., Hashiloni-Dolev, Y., 2011. Egg freezing for age-related fertility decline: preventive medicine or a further medicalization of reproduction? Analyzing the new Israeli policy. Fertil. Steril. 96, 291–294.
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Declaration: "e authors report no #nancial or commercial con$icts of interest.
The availability of international donor oocyte banks offer Canadian couples renewed hope for achieving pregnancy despite poor or absent ovarian reserve.
by G. Scot Hamilton, MSc, PhDLaboratory Director, ISIS Regional Fertility CentreMississauga, ON, Canada, L5N 5S3
You can contact Scot Hamilton at [email protected]. SCOT HAMILTON, MSC, PHD
of Donor Gametes for Recipients and Donors
ARTICLES
The
References
Borini,
Hum. Reprod.
Cobo, Reprod. BioMed. Online
(2011).Hum.
Reprod. Update,
Fertil. Steril.
Fertil. Steril.
Reprod. Biomed. Online
Fertil. Steril.
ARTICLES
ARTICLES
Spermatozoal RNA profiling towards a clinical evaluation of sperm qualityToshio Hamatani
Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, JapanE-mail address: [email protected]
Abstract Human spermatozoal RNAs were recently profiled using microarrays and explored as clinical markers of male infertility. An appropriate study design with a considerable number of biological replicates (sperm samples) is necessary to validate the accuracy and reproducibility of these microarray data. If the genes identified as sperm quality markers by microarray studies are successfully attributed to the pathogenesis of male infertility, then the microarray strategy may be used as a clinical diagnostic tool for male infertility. On the other hand, spermatozoal RNAs may contain not only remnant RNAs after spermatogenesis, but also RNAs that may contribute extragenomically to early embryonic development. Therefore, spermatozoal RNA profiling may enable a better understanding of what is contributed to the oocyte by sperm, in addition to their genome, to facilitate early embryonic development.
©2010, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.KEYWORDS: ICSI, male fertility, RNA profiling, sperm, spermatozoa, transcriptome
This article was published in Reproductive BioMedicine Online, Vol 22, 2011, p103-105, ‘Spermatozoal RNA profiling towards a clinical evaluation of sperm quality’. Copyright Elsevier. It is reprinted here with permission.
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References
ARTICLES
455.
global® DMSO Blastocyst
Vitrification Warming Kit
global® DMSO Blastocyst
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Fertility Treatment of Aged Women By Laparoscopic Intra Ovarian Injection of Peripheral Blood Mononucler Cell (PBMNC) a New Modality
Ali Farid Mohammed Ali
Professor and Exchairman of Department of Obstetrics and Gynecology, Ein Shams University, Cairo Egypt. Head of Heliopolis Research Reproductive Center.13 El Montazah Street, Heliopolis Square, Cairo, EgyptEmail: [email protected]
Ibrahim Khalil, Ali Mohammed
Professor of Clinical Pathology Faculty of Medicine, Ein Shams University, Cairo, Egypt.Email: [email protected]
LailaFarid Ali
Lecturer Department of Obstetrics and Gynecology Faculty of Medicine, Cairo, Egypt.13 El Montazah Street, Heliopolis Square, Cairo, EgyptEmail: [email protected]
ALI FARID MOHAMMED ALI, MD
ARTICLES
Funding: Non
Details of ethical approval: The study was approved by local ethical committee of Heliopolis hospital and written informed consent was signed by the patient before being enrolled in the study.
Contribution to the authorship: Ali Farid conducted the study and analysed the data, Ibrahim Khalil prepared PBMNC, and Laila Farid did laparoscopy.
Design:
Main
Key words: The
ARTICLES
Patient and Methods
Patient: 49
Method
Preparation of autologous PBMNCsLaparoscopic intra ovarian injection of PBMNCs
The
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The
ARTICLES
The
A new
The
References
4. Skinner
6. Johnson
France.
ARTICLES
16. Ferrara
18. Larsen
19. Dong
France.
26. Chen
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Natural Health FertilityCharting Cycles, Lunaception, and a Naturopathic Health Plan
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KATHERINE PETTIT
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