Continued on Page 3 Microanalysis of Rapid Temperature Fluctuations in IVF Culture Systems [Or Musings of a Cold Embryologist Longing for Hot Summer to Return] Lynette Scott, Ph.D., HCLD, The Fertility Center of New England, Reading, MA -- [email protected]DEDICATED TO THE DISSEMINATION OF SHARED BEST PRACTICES AND ISSUES OF INTEREST FOR PROFESSIONALS IN THE FIELD OF HUMAN ASSISTED REPRODUCTION Volume 7, Issue 4 (Formerly The Embryologists’ Newsletter) WINTER, 2004 PRSRT STD U.S. Postage PAID West Palm Bch, FL Permit #611 MEDICAL MARKETS CONSULTANTS, INC. 115 N.W. 84 Way / Coral Springs, FL 33071 ❖ (954) 341-0623 ❖ Fax (954) 341-0698 E-mail: [email protected]❖ www.embryologists.com The Clinical Embryologist M E D I C A L M A R K E T S C O N S U L T A N T S , I N C . T emperature control in IVF laboratories is an ongoing controversial issue. There is a whole, lucrative industry around helping us keep oocytes and embryos warm. There are papers show- ing negative affects of cooling on outcome but many of these are not well controlled. There are also some major laboratories in the country that do not believe in keeping everything at 37°C all the time and it does not appear to affect their results. Another factor that is hardly ever taken into account is the ambient temperature. In the cold northern states, where we are seemingly forever under harsh conditions of snow and ice, we may be psychologically tempted to keep our labs very warm (!) whereas labs in the balmy climes of Florida and the muggy states of the South will resort to cooler air-conditioned temperatures. Some labs have a rule of keeping the ambient temperature at a strict set temperature; others do not. Does this play a role in the affects of cooling? Can a difference between an ambient temperature of 22 and 26°C be that important? In any paper where temperature is a critical issue, there is almost never a reference to the ambient temperature in the laboratory. It is well known that changes both within the oocyte and the embryo do occur with a decrease in the temperature. But are these really that detrimental -- especially considering that we can cool and freeze both oocytes and embryos fairly effectively? If cooling does affect them, when and over what time period does this occur and is it due to a sudden single temperature dip -- a “one off” -- or is it due to continual abuse or to very rapid temperature perturbations? Temperature can affect both metabolism and microtubules. During cooling, metabolism will be decreased and
Transcript
Continued on Page 3
Microanalysis of Rapid TemperatureFluctuations in IVF Culture Systems
[Or Musings of a Cold Embryologist Longing for Hot Summer to Return]
Lynette Scott, Ph.D., HCLD, The Fertility Center of New England,Reading, MA -- [email protected]
D E D I C A T E D T O T H E D I S S E M I N A T I O N O F S H A R E D B E S T P R A C T I C E S A N D I S S U E S O F I N T E R E S T F O R P R O F E S S I O N A L S I N T H E F I E L D O F H U M A N A S S I S T E D R E P R O D U C T I O N
Volume 7, Issue 4 (Formerly The Embryologists’ Newsletter) WINTER, 2004
PRSRT STDU.S. Postage
PAIDWest Palm Bch, FL
Permit #611
M E D I C A L M A R K E T S C O N S U L T A N T S , I N C . 115 N.W. 84 Way / Coral Spr ings , FL 33071 ❖ (954) 341-0623 ❖ Fax (954) 341-0698
E-mai l : embphi l@bel l south.net ❖ w w w. e m b r y o l o g i s t s . c o m
TheClinical EmbryologistM E D I C A L M A R K E T S C O N S U L T A N T S , I N C .
Temperature control in IVF laboratories is an ongoing controversial issue. There is a whole, lucrative industry around helping us keep
oocytes and embryos warm. There are papers show-ing negative affects of cooling on outcome but many of these are not well controlled. There are also some major laboratories in the country that do not believe in keeping everything at 37°C all the time and it does not appear to affect their results.
Another factor that is hardly ever taken into account is the ambient temperature. In the cold northern states, where we are seemingly forever under harsh conditions of snow and ice, we may be psychologically tempted to keep our labs very warm (!) whereas labs in the balmy climes of Florida and the muggy states of the South will resort to cooler air-conditioned temperatures. Some labs have a rule of keeping the ambient temperature
at a strict set temperature; others do not. Does this play a role in the affects of cooling? Can a difference between an ambient temperature of 22 and 26°C be that important? In any paper where temperature is a critical issue, there is almost never a reference to the ambient temperature in the laboratory.
It is well known that changes both within the oocyte and the embryo do occur with a decrease in the temperature. But are these really that detrimental -- especially considering that we can cool and freeze both oocytes and embryos fairly effectively? If cooling
does affect them, when and over what time period does this occur and is it due to a sudden single temperature dip -- a “one off” -- or is it due to continual abuse or to very rapid temperature perturbations?
Temperature can affect both metabolism and microtubules. During cooling, metabolism will be decreased and
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The Clinical Embryologist Volume 7, Issue 4
3
THE CLINICAL EMBRYOLOGIST
EDITORIAL BOARD
Christopher De Jonge, Ph.D., HCLDReproductive Medicine CenterUniversity of MinnesotaMinneapolis, MN • 612-627-4807E-mail: [email protected]
Kenneth C. Drury, Ph.D., HCLDDepartment of Obstetrics and GynecologyUniversity of Florida College of MedicineGainesville, FL • 352-392-5680E-mail: [email protected]
Kathryn J. Go, Ph.D., HCLDPennsylvania Reproductive AssociatesThe Women’s InstitutePhiladelphia, PA • 215-922-3173, #335E-mail: [email protected]
David L. Hill, Ph.D., HCLDART Reproductive CenterBeverly Hills, CA • 310-246-2417E-mail: [email protected]
Thomas B. Pool, Ph.D., HCLDFertility Center of San AntonioSan Antonio, TX • 210-614 3232E-mail: [email protected]
Microanalysis of Rapid Temperature Fluctuations in IVF Culture Systems .............................. 1
Bovine Oocytes and Embryos as Models for Human ART ................. 9
Improvements in Blastocyst Cryopreservation: Modifications of the Common Two-Step Protocol ... 17
American Association of Bioanalysts .................................... 20
Continued from Page 1
mircrotubules will be disrupted. Reduced metabolism will lead to decreased ATP production, which if sustained will result in slowed or arrested embryo development. Microtubules are composed of tubulin, which is temperature sensitive and will begin to depolymerize with decreasing temperature. If the depolymerizistion is prolonged or the reassembly is abnormal, oocyte spindles will be abnormal and/or disrupted, either of which may lead
to reduced developmental potential (Battaglia, 1996; Park, 1997, Zenes, 2001).
The metaphase spindle is made of tubulin, which is bundled -- ordered in arrays of long and short microtubules in a bi-polar structure. Chromosomes are attached to the spindle through the kinetachore, which is a very stable structure. During normal spindle formation, polymers of tubulin are added to each elongating microtubule at its kinetochore. Tubulin is very temperature dependent. It begins to depolymerizes at about 35°C, but conversely, begins to reassemble spontaneously if the temperature rises.
The kinetics of irreversible reduction in normal oocyte developmental potential due to temperature reduction has yet
to be systematically studied. Therefore, only qualitative inferences about this parameter and process can be offered below.
However, at least we can now ask, how much does the temperature really drop in a dish while oocytes and embryos are being handled in the laboratory? A series of very simple but strictly controlled experiments, in which a micro-temperature probe was utilized under routine IVF conditions, were performed in order to investigate this problem.
Standard Falcon 3002 dishes were utilized with 8ml light mineral oil and 10 ul drops of culture medium. The microprobe was placed in the drop and temperature readings were taken every 15 seconds for 3 minutes of cooling and 5 minutes of warming. Finally, the time taken for drops to re-equilibrate to 37°C after cooling was recorded.
Microanalysis of Rapid Temperature Fluctuations in IVF Culture Systems
Continued on Page 5INSTRUCTIONS FOR CONTRIBUTORS
E-mail as Word documents, double-
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The Clinical Embryologist Volume 7, Issue 4
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Five groups of dishes were read.Group A: These were set up to test the
decrease in temperature in ambient air of a drop of medium in a basic culture dish with ambient temperature set at 18, 22 or 25°C.
a: After cooling the dishes were returned to either a flat surface heated to 37°C, as any normal work surface would be, or
b: into an incubator where both the surface and the surrounding air would be at 37°C.
Group B: Drop temperatures were assayed when dishes were removed form an incubator and placed on a warmed surface at 37°C in different ambient air temperatures. After 3 minutes of cooling they were returned to an incubator setting and the rise to 37°C was measured.
Group C: Dishes were placed on an inverted microscope stage to mimick an ICSI set-up, in which the stage was warmed but the ambient temperature was 22°C.
Group D: Dishes were placed on an inverted microscope stage to mimick an ICSI set-up, in which the stage was warmed and the whole set up was in an environmental chamber at 37°C (Nikon).
Group E: Dishes were moved from one incubator to another -- both at 37°C.
The results are presented in Table 1 and related graph.
These data reveal that the cooling rates are affected by the ambient temperature, both in air and on an open heated surface. The warming rates were affected by the lowest temperature reached and the environment in which the dishes were rewarmed to. What was most interesting was that the lowest cooling point occurred after the return to 37°C, which indicates that the oil and medium are acting as a heat sink and that the drops take time to re warm. This may in fact be a very good thing in that it may buffer
System
Dishes at 37°C in Hoffman chamber then moved:
Cooling DegC/Min
Warming DegC/Min
Low temp after 3 min
High after 5 min
Time to reach 37°C
Ambient 18°C, to slide warmer
3.2 1.52 27.3 34.9 14m
Ambient 22°C, to slide warmer
1.56 0.6 32.3 35.3 13m
Ambient 24°C, to slide warmer
1.61 0.6 32.8 35.2 12m
Ambient 18°C, to Hoffman Chamber
3.3 1.9 27.6 36.4 8m
Ambient 22°C, to Hoffman Chamber
1.58 0.82 32.2 36.5 7.6m
Ambient 24°C, to Hoffman Chamber
1.57 0.9 32.5 36.8 8m
To slide warmer at Ambient 18°C
1.2 0.48 33.4 35.8 7m
To slide warmer at Ambient 22°C
0.73 0.38 34.8 36.7 5m
To slide warmer at Ambient 24°C
0.53 0.28 35.4 36.8 5m
To inverted stage heater at 37°C
0.87 0.06 35.0 36.7 7m
To inverted stage in Nikon chamber
0.07 0.04 36.8 37.0 2m
Hoffman chamber to Hoffman
0.07 0.04 36.8 37.0 2m
Representative Cooling Curves
Table 1.Continued from Page 3
The Clinical Embryologist Volume 7, Issue 4
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the temperatures of systems and allow slow, gradual reassembly of microtubules and the re-establishment of metabolism. What may be most detrimental is a repeated abuse of this cooling on a frequent basis or any system that increases the rate of warming, such that tubulin that has depolymerized can not reassemble correctly.
Thus, although cooling does occur in most systems that are employed in the IVF lab, how rapidly the cooling occurs and more importantly how the heat sinks buffer the oocytes and embryos by promoting gradual cooling and rewarming may be the most important factor for us to consider. Some cooling will always occur, and the idea of providing heat sinks to buffer and slow down the process into smooth curves
may be more important than trying to keep everything exactly at 37°C. This may well be what has in the past saved us when inevitable cooling occurs during specimen examinations and manipulations.
It could be that those labs that are effective in ICSI, scoring and all such manipulations have temperature drops of no more than 3-4 degrees and if they are in a good buffers of oil - so that they will cool and re-warm smoothly and thus the oocyte or embryo will not be too adversely affected. If they cool too far or if the abuse is repeated over short time periods, the tubulin and metabolism might become “confused” and “decide” to throw up its hands and go south (looking for warm temperatures, of course!!) ■
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The Clinical Embryologist Volume 7, Issue 4
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This article was first published in Fertility World 2, 20-21, 2004. It is reproduced here with permission from IVFonline, LLC.
New and improved techniques for assisted reproduction in humans must be tested for efficacy and safety before they can be intro-
duced into clinical practice. For practical, ethical, and legal reasons, basic experiments can not be done on human gametes or embryos, and consequently the gametes and embryos of other species must be used as models. The mouse embryo has been extensively used as a general model for early development and is rou-tinely used for quality control testing of human ART media and materials. It is widely available, relatively inexpensive, and there is a wealth of information about its developmental genetics, morphology, and physiol-ogy. However, many aspects of the developmental biol-ogy of the mouse embryo differ significantly from those of the human embryo and consequently embryos of other species are, in many cases, better models.
Clearly, non-human primates (NHP) are phyllogenetically closest to humans and would therefore be the best models for human embryo development (Bavister 2004). Unfortunately, as noted by Bavister, most of the studies of non-human primate ART have been done in the rhesus macaque, but there is now an intense demand for these animals for AIDS research. Moreover, human ART has progressed much more quickly than that for non-human primates. For these and other reasons, it is doubtful that non-human primates will be practical as a model for human ART in the immediate future.
For both biological and practical reasons, bovine oocytes and embryos are very useful models for human ART. In a recent review, Menezo and Herubel (2002) have argued that the bovine embryo is a
better model than the mouse embryo for studies of oocyte maturation, fertilization, extended culture, and cryopreservation. As well, the development and application of assisted reproductive technologies in cattle have usually preceded, and been the basis for, their practical application in humans. Examples are embryo transfer, sperm and embryo cryopreservation, embryo biopsy for cytogenetic evaluation, embryo culture, and in-vitro oocyte maturation (IVM) (Betteridge and Rieger 1993). These techniques are widely used in the field (sometimes literally).
Bovine Oocytes and Embryos as Models for Human ART
Don Rieger, Ph.D.,Scientific DirectorLifeGlobal, LLC, The ART Media Company • www.LifeGlobal.com
[The Clinical Embryologist Editor’s Note: Many of us customarily peruse studies of mouse and primate oocytes and embryos. This commentary contends that bovine oocytes and embryos may also be useful animal models for human development; so perhaps these should also be included in our routine personal surveys of ART literature. H.M.P.]
The Clinical Embryologist Volume 7, Issue 4
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According to a survey by the International Embryo Transfer Society, more than 500,000 in-vivo developed cattle embryos, and more than 83,000 IVF cattle embryos were transferred in 2002, world-wide (Thibier 2003). Studies of bovine IVM and extended embryo culture are two areas of particular significance to human ART.
Edwards (1965) observed that oocytes from a variety of species will undergo spontaneous nuclear maturation when removed from the follicle. However, many subsequent studies showed that the in-vitro fertilization and development of such oocytes is very limited, and this was attributed to the lack of cytoplasmic maturation. Consequently, human IVF has relied on the collection of in-vivo matured oocytes, collected after the LH surge. The first successful IVF in cattle similarly relied on in-vivo oocyte maturation (Brackett et al. 1982), but the extensive hormone monitoring required for this approach is impractical for routine application in cattle. A great many studies, notably by Leibfried-Rutledge and others in Neal First’s laboratory in Madison, Wisconsin, led to the birth of the first calf from an in-vitro matured oocyte (Leibfried-Rutledge et al. 1986). Based largely on these studies, Trounson et al. (1994) successfully matured human oocytes taken from small follicles of PCOS patients. For the most part, serum and pharmacological concentrations of gonadotrophins are used in bovine IVM, but many studies have shown that physiological concentrations of EGF, IGF-1 and other growth factors are also effective (Smitz et al. 2001). It seems likely that the use of growth factors will ultimately supplant the use of serum and gonadotrophins in bovine and human IVM.
Extended culture of human embryos for transplant at the blastocyst stage has been suggested as an approach to better evaluation of developmental potential. Fewer embryos (ideally one) can be transferred and yield good pregnancy rates with a much reduced chance of multiples (Gardner and Lane 2003). With very few exceptions, bovine IVF embryos are cultured to the morula or blastocyst stage because, unlike in the human, cleavage stage bovine embryos will not survive in the uterus. Initially, this problem was circumvented by temporary culture in the ligated sheep oviduct, and later
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The Clinical Embryologist Volume 7, Issue 4
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in co-culture with oviductal or other cells. Except in special situations, these techniques have been replaced by culture in defined culture media, containing serum albumin, but no somatic cells or serum (Thompson 2000). In one example (Gandhi et al. 2000), approximately 25% of the original immature bovine oocytes developed to the blastocyst stage following IVM and culture in defined media.
In cattle, transfer of one embryo is the norm, because of the risk of freemartinism. As for human embryos, morphology is used to predict the viability of bovine embryos. Although this works on a population basis, it is notoriously unreliable for an individual embryo, and efforts have been made to predict viability based on metabolism for more than 25 years (Renard et al. 1978; Rieger 1984). Recently, highly sensitive probes have been used to measure oxygen uptake by individual bovine embryos (Shiku et al. 2001; Lopes et al. 2004) and could be applied to human embryos.
The bovine embryo is also an important model for the possible negative effects of embryo culture, originating from the observations in two landmark papers. In the first, Iannaccone (1984) showed that mouse blastocysts survived exposure to low concentrations of methylnitrosylurea, but the mortality rate of the pups originating from these blastocysts was significantly increased in the first year following birth. In the second, Willadsen et al. (1991) showed that there was a significant incidence of high birth weight, birth defects and perinatal death in calves produced by cloning of early bovine embryos. This was later dubbed the large offspring syndrome (LOS) and was shown to occur in cattle and sheep fetuses produced by IVM/IVF as well as by cloning (Kruip and den Daas 1997). Together, these observations indicated that the environment to which an early embryo is exposed can have significant and deleterious effects on development at a much later stage. In humans, low birth weight of IVF babies, even among singletons, is the usual concern, but DeBaun et al. (2003) have suggested that Beckwith-Wiedemann syndrome (BWS) may be related to in-vitro culture. It has yet to be determined whether these LOS and BWS are truly analogous, but
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The Clinical Embryologist Volume 7, Issue 4
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understanding LOS has enormous implications for human developmental biology.
Several recent developments will facilitate the study of the developmental biology of the bovine embryo, and its use as a model for the human embryo. A $53 million (US) project to sequence the bovine genome is being undertaken through a collaboration of U.S., Australian, New Zealand and Canadian researchers, and a microarray of over 23,000 bovine transcripts is commercially available. It has been shown that mRNA expression differs between in-vivo and in-vitro produced bovine embryos (Wrenzycki et al. 2001), and complete sequencing of the bovine genome and the availability of microarrays will undoubtedly reveal further differences. As well, bovine oocytes and early embryos up to the blastocyst stage are now commercially available, making this model available to many more research laboratories.
Note added to the original article: There are several studies underway using Global medium, supplemented with additional glucose, to successfully develop bovine embryos to the blastocyst stage. This is important in that it allows us to perform tests on bovine embryos and then to readily transfer our findings to the human embryo, utilizing the commonality and control of Global medium. In addition, it allows us to build a data bank of results to complete a ‘full’ bovine model, and to allow further studies of various areas, relying on this track record of success. ■
Don Rieger, Ph.D.Scientific DirectorLifeGlobal, LLC
ReferencesBavister BD (2004) ARTs in action in nonhuman primates:
symposium summary--advances and remaining issues. Reprod Biol Endocrinol 2, 43.
Betteridge KJ, Rieger D (1993) Embryo transfer and related techniques in domestic animals, and their implications for human medicine. Hum Reprod 8, 147-167.
Brackett BG, Bousquet D, Boice ML, Donawick WJ, Evans JF, Dressel MA (1982) Normal development following in vitro fertilization in the cow. Biol Reprod 27, 147-158
DeBaun MR, Niemitz EL, Feinberg AP (2003) Association of in vitro fertilization with Beckwith-Wiedemann syndrome and epigenetic alterations of LIT1 and H19. Am J Hum Genet 72, 156-60.
Edwards RG (1965) Maturation in vitro of mouse, sheep, cow, pig, rhesus monkey and human ovarian oocytes. Nature 208, 349-51.
Gandhi AP, Lane M, Gardner DK, Krisher RL (2000) A single medium supports development of bovine embryos throughout maturation, fertilization and culture. Hum Reprod 15, 395-401.
Gardner DK, Lane M (2003) Towards a single embryo transfer. Reprod Biomed Online 6, 470-81.
Iannaccone PM (1984) Long-term effects of exposure to methynitrosourea on blastocysts following transfer to surrogate female mice. Cancer Res 44, 2785-2789.
Kruip TAM, den Daas JHG (1997) In vitro produced and cloned embryos: effects on pregnancy, parturition and offspring. Theriogenology 47, 43-52.
Leibfried-Rutledge ML, Critser ES, First NL (1986) Effects of fetal calf serum and bovine serum albumin on in vitro maturation and fertilization of bovine and hamster cumulus-oocyte complexes. Biol Reprod 35, 850-7.
Lopes A, Ottosen LDM, Greve T, Callesen H (2004) Oxygen measurements as indicators of culture conditions and viability of cattle embryos. Reprod Fertil Devel 16, 199 (Abstract).
Menezo YJ, Herubel F (2002) Mouse and bovine models for human IVF. Reprod Biomed Online 4, 170-5.
Renard J-P, Ménézo Y, Saumande J, Heyman Y (1978) Attempts to predict the viability of cattle embryos produced by superovulation. In ‘Control of Reproduction in the Cow’. (Ed. JR Sreenan) pp. 398-417.
Rieger D (1984) The measurement of metabolic activity as an approach to evaluating viability and diagnosing sex in early embryos. Theriogenology 21, 138-149.
Shiku H, Shiraishi T, Ohya H, Matsue T, Abe H, Hoshi H, Kobayashi M (2001) Oxygen consumption of single bovine embryos probed by scanning electrochemical microscopy. Anal Chem 73, 3751-8.
Smitz J, Nogueira D, Albano C, Cortvrindt R, Devroey P (2001) Improving in vitro maturation of oocytes in the human: taking lessons from experiences in animal species. Reprod Domest Anim 36, 11-7.
Thibier M (2003) IETS Data Retrieval Committee Annual Report: More than half a million bovine embryos transferred in 2002. IETS Newslett, 21 (4). 12-19.
Thompson JG (2000) In vitro culture and embryo metabolism of cattle and sheep embryos - a decade of achievement. Anim Reprod Sci 61, 263-75.
Trounson A, Wood C, Kausche A (1994) In vitro maturation and the fertilization and developmental competence of oocytes recovered from untreated polycystic ovarian patients. Fertil Steril 62, 353-62.
Willadsen SM, Jansen RE, McAlister RJ, Shea BF, Hamilton G, McDermand D (1991) The viability of late morulae and blastocysts produced by nuclear transplantation in cattle. Theriogenology 35, 161-170.
Wrenzycki C, Herrmann D, Keskintepe L, Martins A, Jr., Sirisathien S, Brackett B, Niemann H (2001) Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos. Hum Reprod 16, 893-901.
This article was first published in Fertility World 2, 20-21. It is reproduced here with permission from IVFonline, LLC. For further information, contact the editor, Monica Mezezi, M.B.A., [email protected].
Continued from Page 11
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The Clinical Embryologist Volume 7, Issue 4
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Abstract: We have compared the efficacy for a better treatment outcome of a modified protocol versus the commonly used two-step
protocol for human blastocyst freezing and thawing. A total of 92 blastocyst frozen thaw transfer (FET) cycles were retrospectively analyzed in this study. Of these, 24 FET were completed using the two- step method as described by Menezo et al., 1992. A total of 68 FET were performed using a modified protocol.
For both groups the freeze and thaw base medium was mHTF. However, for the modified protocol, human serum albumin (HSA) concentration was raised from 12 mg/ml to 20 mg/ml and also 0.1 mM ascorbate was added to the freezing medium. Blastocysts in the modified protocol were incubated at room temperature (RT) for 10 minutes in 5% Glycerol and for 10 minutes in 10% glycerol and 0.2 M sucrose, then loaded into straws and frozen using the same slow freezing protocol as utilized in the two-step protocol.
For thawing, blastocysts treated according to the modified protocol were subjected to a multi-step protocol as opposed to the two-step protocol. They were removed from the straws after thawing and cryoprotectants were removed in several serial dilutions of glycerol with sucrose, then in sucrose alone, as described in our Materials and Methods section below.
A total of 325 blastocysts were thawed in 92 frozen thaw transfer cycles. Twenty-four frozen transfers were performed using the two-step protocol. This resulted in a (fetal-heart-beat) clinical pregnancy rate of 16%. By switching to the modified protocol in 68 frozen transfers, we achieved a clinical pregnancy rate of 52%. One of the most notable observations seen by using the modified method was the higher rate of survival of blastocysts -- 71% as compared to 46%
using the two-step method. More blastocysts were reexpanded in modified method groups -- 89% as opposed to 65% in the two-step group. All of these differences are statistically significant (p<0.05).
Only expanding, expanded, hatching or hatched blastocysts with defined inner cell mass and trophectoderm from day 5-7 cultures were cryopreserved. Most of these blastocysts were cryopreserved on day 6 of culture (82%). There was no difference in clinical pregnancy rates of the blastocysts cryopreserved on either day 5 or day 6.
From this study we conclude that our modified protocol of freeze and thaw for human blastocysts has significantly enhanced the clinical pregnancy outcome in blastocyst freeze thaw cycles as compared to the earlier two-step method. Our ongoing analysis reveals that these improved results are now comparable to our results with fresh blastocyst transfers. It is possible that – acting individually and/or in concert --the antioxidant properties of ascorbate in the freezing and thawing media, the higher protein concentrations therein and the multi-step (rather than just two-step) removal of cryoprotectant may each and/or all facilitate this enhanced clinical outcome achieved with our modified culture medium and protocol for blastocyst cryopreservation.
IntroductionSince the development of sequential media
culturing, pre-embryos at the blastocyst stage have become immensely popular. This is due to the high rate of implantation at the blastocyst stage that allows fewer pre-embryos to be replaced without compromising the chance of pregnancy. At the same time, prolonged culture reduces the risk of high order pregnancies (Gardner et al., 1998). This has obviously necessitated the development of an optimal protocol
Improvements in Blastocyst Cryopreservation:Modifications of the Common Two-Step Protocol
Rajesh K. Srivastava, Ph.D, Peter Wieckowski, B.S. & Julio E. Pabon, MDFertility Center and Applied Genetics of Florida, Inc., Sarasota, Florida
The Clinical Embryologist Volume 7, Issue 4
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for the cryopreservation of supernumerary blastocysts in order to gain maximum benefit from each IVF cycle. Early reports of human blastocyst cryopreservation included Cohen et al., 1985; and Fehilly et al., 1985. More recent reports of successful blastocyst freezing and thawing included Kaufman et al., 1995; and Freeman, 1998.
One of the most popular slow-freezing and thaw protocols for blastocysts was described by Menezo et al., 1992, used mainly for cyopreserving cocultured blastocysts. Some clinics have used a modification of this protocol that has yielded better outcomes in frozen thaw blastocysts transfers (Veeck, 2000; 2003) and Tucker, MJ (personal communication). Here we have evaluated the efficacy of the Menezo protocol (Menezo et al., 1992) as compared to a modified version of this protocol as described by Veeck (Veeck, 2000).
Materials and MethodsGroup A: Base medium for freezing and thawing blastocysts
in this group was Hepes-HTF (mHTF) with 12 mg/ml Human Serum Albumin (HSA). Blastocysts in this group were washed in base medium at room temperature (RT) and then transferred to 5% glycerol in mHTF + 12 mg/ml HSA and incubated for 10 min at RT. Afterwards they were transferred to a solution of 9% glycerol + 0.2 M sucrose in mHTF + 12 mg/ml HSA for 10 min, during which time they were loaded to cryostraws and cooled in a Cryologic freezer (Biogenics) from 20oC to –7oC at the rate of 2oC/min, held at –7oC for 5 min, manually seeded and, after holding for another 10 min, cooled at the rate of 0.3oC/min until –38oC, then plunged in liquid nitrogen (LN2).
For thawing, cryostraws were removed from LN2, exposed to RT for 30 seconds and to 30oC for another 30-40 seconds. Blastocysts were expelled in 0.5 M sucrose solution in mHTF + 12 mg/ml HSA
and incubated for 10 min at RT. Thereafter, they were transferred to 0.2 M sucrose sol.in mHTF + 12 mg/ml HSA for another 10 min, washed through several drops of mHTF and incubated at 37 degrees C in blastocyst culture medium for approximately 4 h prior to intrauterine transfer.
Group B: Base medium for this group consisted of mHTF +
20 mg/ml HSA and 0.1 mM ascorbate. Blastocysts in this group were washed in base medium at 37oC, cooled to RT and then transferred to 5% glycerol in base medium for 10 min at RT. Next they were transferred to 10% glycerol + 0.2 M sucrose in base medium for another 10 min, loaded to cryostraws in the same medium and frozen following the same protocol as described for Group A.
Blastocysts were thawed by a multi step protocol. After thawing the cryostraws, contents of the straws were expelled in 10% glycerol + 0.4 M sucrose for 30-40 seconds at RT until blastocysts were located, transferred to 5% glycerol + 0.4 M sucrose for 3 min at RT, 0.4 M sucrose for 3 min at RT and 0.2 M sucrose for 3 min at RT and then to 0.1 M sucrose for 1 min at near 37oC, washed through several droplets of base medium at 37oC and incubated in the blastocyst culture medium containing 20 mg/ml HSA for approximately 4 h before intrauterine transfer. All thawing solutions were made in the base medium containing 20 mg/ml HSA. Blastocysts were replaced after estradiol and progesterone supplementation.
ResultsA total of 325 blastocysts were thawed in 92 frozen
thaw transfer cycles. Clinical pregnancies were defined as the presence of a fetal heart beat by ultrasound detection. Twenty-four frozen transfers were performed using the two-step protocol. This resulted in clinical pregnancy rate of 16% (4/24) – see Table
Table 1: Comparison of the results obtained after frozen-thawed blastocysts using two protocols
Clinical pregnancy per blastocyst transfer
Blastocystssurvival
Blastocyst expansion in culture after thaw
Group A 16% (4/24) 46% (39/85) 65% (55/85)
Group B 52% (35/68)* 71% (170/240)* 89% (151/170)*
The Clinical Embryologist Volume 7, Issue 4
19
l. After switching to the modified protocol in 68 frozen transfers, we attained a statistically significant (p < 0/.05) improvement – a clinical pregnancy rate of 52% (35/68).
Other statistically significant improvements in blastocyst cryopreservation were evident (Table 1). One of the most conspicuous observations apparent upon changing to the modified method was the increased rate of survival of thawed blastocysts -- 71% (170 /240) as compared to 46%(39 /85) using the two step method. More blastocysts were reexpanded in the modified method groups (89% - 151 /170) as opposed to 65% (55/85) in the two-step groups.
The age of patients for blastocyst freezing and thawing was not a significant criterion in this study. Only expanding, expanded, hatching or hatched blastocysts with defined inner cell mass and trophectoderm from day 5-7 were cryopreserved. Most of the blastocysts were cryopreserved on day 6 of culture (82%). There was no difference in clinical pregnancy rates of the blastocysts cryopreserved on either day 5 or 6.
Discussion:Our modification of the conventional two-step protocol
involved relatively minor – but nonethless noteworthy – freezing changes, as well as comparatively major and likewise notable thawing alterations. Compared with that conventional protocol, ours produced statistically significant improvements, not only in blastocyst survival and expansion, but also in clinical pregnancy rates. In fact, our freeze/thaw results with our modified protocol approximate our clinical pregnancy rates with fresh blastocyst transfers. Therefore, the multi-step freeze/thaw protocol investigated here may optimize embryo cryopreservation for our IVF culture system.
While this freezing protocol did not differ much from the freezing protocol as described by Menezo et al., (1992), instead of 9% Glycerol + 0.2 M sucrose in the second step, we used 10% Glycerol + 0.2 M sucrose. We consistently used 20 mg/ml HSA to the freeze and thaw medium and also added 0.1 mM ascorbate in the freezing medium. Blastocysts were cultured in a suitable blastocyst culture medium containing high glucose concentration post thaw and was supplemented with 20 mg/ml HSA in order to provide better osmotic protection.
The Clinical Embryologist Volume 7, Issue 4
20
Although blastocysts can be cultured successfully in 5 to 6 mg/ml HSA, survival rates following freeze and thaw at this concentration tend to suffer (Pool, 2003). It has been shown (Lane et al., 2002) that the presence of 0.1 mM ascorbate in the freezing medium resulted in slightly lower lactate dehydrogenase levels and increased rates of metabolism in the embryos after thaw as compared to the absence of ascorbate. Ascorbate may be beneficial in stimulating embryo development post thaw due to its antioxidant properties.
To achieve an optimum success, it also appears important to choose only those blastocysts that are expanded, hatching or hatched on either day 5, 6 or day 7 of culture with well-defined inner cell mass and cohesive tropectoderm. Indeed, if a blastocyst has a small cavity and if its zona has not thinned considerably, it seems better to prolong the culture before cryopreservation. ■
Rajesh K. Srivastava, Ph.DFertility Center and Applied Genetics of Florida,
Announces the offering of an EMBRYO BIOPSY & FIXATION COURSE
ABB Approved for 1.6 CEUsOffered 1 weekend/per month
in Beverly Hills, CARegistration limited
One on one instruction by Dr. David Hill & Dr. Man Li
Cost for Course $2500
E-mail Kim Monroe at [email protected] for dates & details or call 310/428-2100
The Clinical Embryologist Volume 7, Issue 4
22
Thank you to our Valued Readers and
Deeply Appreciated Authors and
Advertisers for your Procreative
and Prolific, Productive Support.
From
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The Clinical Embryologist
BRIEF SUMMARYPlease see package insert for full prescribing information.INDICATIONS AND USAGEFollistim® AQ Cartridge (follitropin beta injection) is indicated for the development of multiple follicles in ovulatory patientsparticipating in an Assisted Reproductive Technology (ART) program. Follistim® AQ Cartridge is also indicated for theinduction of ovulation and pregnancy in anovulatory infertile patients in whom the cause of infertility is functional and notdue to primary ovarian failure.Selection of PatientsBefore treatment with Follistim® AQ Cartridge (follitropin beta injection) is initiated:1) A thorough gynecologic and endocrinologic evaluation of the patient must be performed. The evaluation should
include a hysterosalpingogram (to rule out uterine and tubal pathology) and documentation of anovulation by meansof reviewing a patient’s history, performing a physical examination, determining serum hormonal levels as indicated,and optionally performing an endometrial biopsy. Patients with tubal pathology should receive Follistim® AQ Cartridgeonly if enrolled in an ART program.
2) Primary ovarian failure should be excluded by the determination of circulating gonadotropin levels.3) Careful examination should be made to rule out early pregnancy.4) Evaluation of the partner’s fertility potential should be included in the workup procedure.CONTRAINDICATIONSFollistim® AQ Cartridge (follitropin beta injection) is contraindicated in women who exhibit:1) Prior hypersensitivity to recombinant hFSH products2) A high circulating FSH level indicating primary ovarian failure3) Uncontrolled thyroid or adrenal dysfunction4) Tumor of the ovary, breast, uterus, hypothalamus, or pituitary gland5) Pregnancy6) Heavy or irregular vaginal bleeding of undetermined origin7) Ovarian cysts or enlargement not due to polycystic ovary syndrome (PCOS)8) Hypersensitivity reactions to streptomycin or neomycin. Follistim® AQ Cartridge may contain traces of these anti-
biotics and may cause hypersensitivity reactions in susceptible persons.WARNINGSFollistim® AQ Cartridge (follitropin beta injection) should be used only by physicians who are experienced in infertil-ity treatment. Changes in brand (manufacturer), type (recombinant, urinary, etc.), and/or method of administration(Follistim Pen™, conventional syringe, etc.) may result in the need to adjust the dose. Follistim® AQ Cartridgeadministered with the Follistim Pen™ contains a potent gonadotropic substance and delivers on average an 18%higher amount of follitropin beta as compared to lyophilized preparations administered by conventional syringe.Accordingly, a lower starting dose for gonadotropin stimulation and dose adjustments during gonadotropin stimula-tion should be considered for each woman treated with Follistim® AQ Cartridge.Overstimulation of the Ovary During Treatment With Follistim® AQ Cartridge (follitropin beta injection)In order to minimize the hazards associated with the occasional abnormal ovarian enlargement that may occur withFollistim® AQ Cartridge therapy, the lowest effective dose should be used. Use of ultrasound monitoring of ovarianresponse and/or measurement of serum estradiol levels can further minimize the risk of overstimulation.If the ovaries are abnormally enlarged on the last day of treatment with Follistim® AQ Cartridge, hCG should not beadministered in this course of treatment, to reduce the chances of developing Ovarian Hyperstimulation Syndrome (OHSS).Ovarian Hyperstimulation Syndrome (OHSS): OHSS is a medical entity distinct from uncomplicated ovarian enlargementand may progress rapidly to become a serious medical event. OHSS is characterized by a dramatic increase in vascularpermeability, which can result in a rapid accumulation of fluid in the peritoneal cavity, thorax, and potentially, the peri-cardium. The early warning signs of OHSS developing are severe pelvic pain, nausea, vomiting, and weight gain. The following symptoms have been reported in cases of OHSS: abdominal pain, abdominal distension, gastrointestinal symp-toms including nausea, vomiting and diarrhea, severe ovarian enlargement, weight gain, dyspnea, and oliguria. Clinicalevaluation may reveal hypovolemia, hemoconcentration, electrolyte imbalances, ascites, hemoperitoneum, pleural effu-sions, hydrothorax, acute pulmonary distress, and thromboembolic events (see WARNINGS-Pulmonary and VascularComplications).During clinical trials with Follistim® and Follistim® AQ Cartridge therapy, OHSS occurred in 60 (5.3%) of the 1132 womentreated and of these 33 (2.9%) were hospitalized. Cases of OHSS are more common, more severe, and more protracted if pregnancy occurs; therefore, patients should be followed for at least two weeks after hCG administration. Most often,OHSS occurs after treatment has been discontinued and it can develop rapidly, reaching its maximum about seven to tendays following treatment. Usually, OHSS resolves spontaneously with the onset of menses. If there is evidence that OHSSmay be developing prior to hCG administration (see PRECAUTIONS-Laboratory Tests), the hCG must be withheld.If serious OHSS occurs, treatment should be stopped and the patient should be hospitalized. Treatment is primarily symp-tomatic and should consist of bed rest, fluid and electrolyte management, and analgesics (if needed). Hemoconcentrationassociated with fluid loss into the peritoneal cavity, pleural cavity, and the pericardial cavity may occur and should bethoroughly assessed in the following manner: 1) fluid intake and output; 2) weight; 3) hematocrit; 4) serum and urinaryelectrolytes; 5) urine specific gravity; 6) BUN and creatinine; 7) total proteins with albumin: globulin ratio; 8) coagulationstudies; 9) electrocardiogram to monitor for hyperkalemia and 10) abdominal girth. These determinations should be per-formed daily or more often based on clinical need.OHSS increases the risk of injury to the ovary. The ascitic, pleural, and pericardial fluid should not be removed unlessthere is the necessity to relieve symptoms such as pulmonary distress or cardiac tamponade. Pelvic examination maycause rupture of an ovarian cyst, which may result in hemoperitoneum, and should, therefore, be avoided. If bleedingoccurs and requires surgical intervention, the clinical objective should be to control the bleeding and retain as much ovar-ian tissue as possible. Intercourse should be prohibited in patients with significant ovarian enlargement after ovulationbecause of the danger of hemoperitoneum resulting from ruptured ovarian cysts.The management of OHSS may be divided into three phases: an acute, a chronic, and a resolution phase. Because the useof diuretics can accentuate the diminished intravascular volume, diuretics should be avoided except in the late phase ofresolution as described below.Acute Phase: Management during the acute phase should be directed at preventing hemoconcentration due to loss ofintravascular volume to the third space and minimizing the risk of thromboembolic phenomena and kidney damage. Treat-ment is intended to normalize electrolytes while maintaining an acceptable but somewhat reduced intravascular volume.Full correction of the intravascular volume deficit may lead to an unacceptable increase in the amount of third space fluidaccumulation.Management includes administration of limited intravenous fluids, electrolytes, human serum albumin, and strict monitor-ing of fluid intake and output. Monitoring for the development of hyperkalemia is recommended.Chronic Phase: After stabilizing the patient during the acute phase, excessive fluid accumulation in the third space shouldbe limited by instituting severe potassium, sodium, and fluid restriction.Resolution Phase: A fall in hematocrit and an increasing urinary output without an increased intake are observed due tothe return of the third space fluid to the intravascular compartment. Peripheral and/or pulmonary edema may result if thekidneys are unable to excrete third space fluid as rapidly as it is mobilized. Diuretics may be indicated during the resolu-tion phase, if necessary, to combat pulmonary edema.Pulmonary and Vascular ComplicationsSerious pulmonary conditions (e.g., atelectasis, acute respiratory distress syndrome) have been reported in womentreated with gonadotropins. In addition, thromboembolic events both in association with, and separate from, the OvarianHyperstimulation Syndrome have been reported following gonadotropin therapy. Intravascular thrombosis, which mayoriginate in venous or arterial vessels, can result in reduced blood flow to vital organs or the extremities. Sequelae ofsuch events have included venous thrombophlebitis, pulmonary embolism, pulmonary infarction, cerebral vascular occlu-sion (stroke), and arterial occlusion resulting in loss of limb. In rare cases, pulmonary complications and/or thrombo-embolic events have resulted in death.Multiple BirthsMultiple births have been reported for all FSH treatments including Follistim® (follitropin beta for injection) treatment. Thepatient and her partner should be advised of the potential risk of multiple births before starting treatment.PRECAUTIONSGeneralCareful attention should be given to the diagnosis of infertility and in the selection of candidates for treatment withFollistim® AQ Cartridge (follitropin beta injection) (see INDICATIONS AND USAGE-Selection of Patients).
Information for PatientsPhysicians must instruct patients on the correct usage and dosing of Follistim® AQ Cartridge (follitropin beta injection) inconjunction with the Follistim Pen™.Patients should read and follow all instructions in the Follistim Pen™ Instructions for Use Manual/Treatment Diary prior toadministration of Follistim® AQ Cartridge.Prior to treatment with Follistim® AQ Cartridge, patients should be informed of the duration of treatment and monitoringprocedures that will be required. The risks of Ovarian Hyperstimulation Syndrome and multiple births (see WARNINGS),and other possible adverse reactions (see ADVERSE REACTIONS) should be discussed.Laboratory TestsIn most instances, treatment with Follistim® AQ Cartridge (follitropin beta injection) will result only in follicular growthand maturation. In order to complete the final phase of follicular maturation and to induce ovulation, hCG must be givenfollowing the administration of Follistim® AQ Cartridge or when clinical assessment of the patient indicates that sufficientfollicular maturation has occurred. This may be directly estimated by sonographic visualization of the ovaries and endo-metrial lining and/or measuring serum estradiol levels. The combination of both ultrasonography and measurement ofestradiol levels is useful for monitoring the growth and development of follicles, timing hCG administration, as well asminimizing the risk of OHSS and multiple gestations.The clinical evaluation of estrogenic activity (changes in vaginal cytology, changes in appearance and volume of cervicalmucus, spinnbarkeit, and ferning of the cervical mucus) provides an indirect estimate of the estrogenic effect upon thetarget organs, and therefore, it should only be used adjunctively with more direct estimates of follicular development(e.g., ultrasonography and serum estradiol determinations).The clinical confirmation of ovulation is obtained by direct and indirect indices of progesterone production. The indicesmost generally used are as follows:a) A rise in basal body temperatureb) Increase in serum progesteronec) Menstruation following the shift in basal body temperatureWhen used in conjunction with indices of progesterone production, sonographic visualization of the ovaries will assist indetermining if ovulation has occurred. Sonographic evidence of ovulation may include the following:a) Fluid in the cul-de-sacb) Follicle showing marked decrease in sizec) Collapsed follicleDrug InteractionsNo drug-drug interaction studies have been performed.Carcinogenesis and Mutagenesis, Impairment of FertilityLong-term toxicity studies in animals have not been performed with Follistim® AQ Cartridge (follitropin beta injection) toevaluate the carcinogenic potential of the drug. Follistim® (follitropin beta for injection) was not mutagenic in the Amestest using S. typhimurium and E. coli tester strains and did not produce chromosomal aberrations in an in vitro assayusing human lymphocytes.PregnancyPregnancy Category X: (See CONTRAINDICATIONS).Nursing MothersIt is not known whether this drug is excreted in human milk. Because many drugs are excreted in human milk andbecause of the potential for serious adverse reactions in the nursing infant from Follistim® AQ Cartridge (follitropin betainjection), a decision should be made whether to discontinue nursing or to discontinue the drug, taking into account theimportance of the drug to the mother.Pediatric UseSafety and effectiveness in pediatric patients have not been established.Geriatric UseClinical studies did not include subjects aged 65 and over.ADVERSE REACTIONSAssisted Reproductive Technologies (ART)Rates of adverse events from an open-label, non-controlled, multicenter study in 60 women undergoing COH for IVF or ICSIwith Follistim® AQ Cartridge (follitropin beta injection) administered with the Follistim Pen™ are summarized in table below.
Incidence of Adverse Clinical Experiences (≥5%)
Ovulation InductionRates of adverse events from an open-label, non-controlled, multicenter study in 43 clomiphene-resistant women withchronic anovulation (WHO group II) undergoing Ovulation Induction with Follistim® AQ Cartridge (follitropin beta injection) administered with the Follistim Pen™ are summarized in table below.
Incidence of Adverse Clinical Experiences (≥5%)
The following adverse events have been reported in women treated with gonadotropins: pulmonary and vascular compli-cations (see WARNINGS), hemoperitoneum, adnexal torsion (as a complication of ovarian enlargement), dizziness, tachy-cardia, dyspnea, tachypnea, febrile reactions, flu-like symptoms including fever, chills, musculoskeletal aches, joint pains,nausea, headache and malaise, breast tenderness, and dermatological symptoms (dry skin, erythema, body rash, hairloss and hives).There have been infrequent reports of ovarian neoplasms, both benign and malignant, in women who have undergonemultiple drug regimens for ovulation induction; however, a causal relationship has not been established.Congenital AnomaliesThe incidence of congenital malformations after Assisted Reproductive Technologies (ART) may be slightly higher thanafter spontaneous conception. This slightly higher incidence is thought to be related to differences in parental characteris-tics (e.g., maternal age, sperm characteristics) and to the higher incidence of multiple gestations after ART. There are noindications that the use of gonadotropins during ART is associated with an increased risk of congenital malformations.DRUG ABUSE AND DEPENDENCEThere have been no reports of abuse or dependence with Follistim® AQ Cartridge (follitropin beta injection).StorageStore refrigerated, 2–8°C (36–46°F) until dispensed. Upon dispensing, the product may be stored by the patient at 2–8°C(36–46°F) until the expiration date, or at 25°C (77°F) for 3 months or until expiration date, whichever occurs first. Oncethe rubber stopper of the Follistim® AQ Cartridge has been pierced by a needle, the product can only be stored for a maxi-mum of 28 days at 2–25°C (36–77°F). Protect from light. Do not freeze.For more information, call 1-866-836-5633
� only
Adverse Event Follistim® AQCartridge
n=43Ovarian hyperstimulation syndrome 9%Abdominal pain 5%Injection site reaction 5%
SAFETY INFORMATION• Follistim® AQ Cartridge administered with Follistim Pen™ delivers on average an 18% higher amount of follitropin
beta compared to lyophilized preparations. A lower dose should be considered when using Follistim® AQ Cartridge.• Follistim® AQ Cartridge, like all gonadotropins, is a potent substance capable of causing mild to severe side effects
including OHSS, with or without pulmonary or vascular complications.• Follistim® AQ Cartridge should be used only by physicians who are experienced in infertility treatment and should
advise their patients of treatment risks, including OHSS and multiple births.
*The product may be stored by the patient at 2-8°C (36-46°F) until the expiration date, or at 25°C (77°F) for 3 months, or until expirationdate, whichever occurs first. Once the rubber stopper of the cartridge has been pierced by a needle, the product can only be stored for amaximum of 28 days at 2-25°C (36-77°F). Protect from light. Do not freeze.
Simple todial andinject theexact dose.
Fingertip dosageadjustments provideprecise individualizeddosing from 50 IU to450 IU.
Accurate fine-tuneddosing in 25 IUincrements.
Premixed, multipledose cartridgesavailable in 300 IUand 600 IU.
BD Micro-Fine™ PenNeedle and micro-volumeresult in excellenttolerability.
Room temperature storage* does notcompromise work, social and travelschedules.
Please see brief summary of Prescribing Information on the following page.
For more information, please visit www.follistim.com
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