Abstract. Introduction. RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 15 December 2003

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RBMOnline - Vol 8. No 2. 207-211 Reproductive BioMedicine Online; www.rbmonline.

1 RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 15 December 2003 Article Determining the most optimal stage for embryo cryopreservation Anthony Anderson received his BSc in Animal Science from Oklahoma State University in He recently received his MSc in Clinical Embryology from the University of Leeds School of Medicine. Mr Anderson started his career in bovine nuclear embryo transfer in Texas and California. He has been a human embryologist since 1992 and has been working at the Institute for Assisted Reproduction since Current research interests involve embryo metabolism and optimizing pregnancy outcome by morphological characterization of embryos to predict viability. Dr Anthony Anderson Anthony R Anderson 1, 3, Margaret L Weikert 1, Jack L Crain 2 1 Presbyterian Hospital Institute for Assisted Reproduction, 200 Hawthorne Lane, 6A-IVF, Charlotte, North Carolina, 28204, USA 2 Reproductive Endocrine Associates of Charlotte, Charlotte, NC 28204, USA 3 Correspondence: Tranderson@novanthealth.org Abstract The objective of this study was to compare cleaved embryo and blastocyst freezing to determine the optimal time for embryo cryopreservation. A retrospective analysis was carried out for all frozen embryo transfer cycles where transfer occurred on either day 3 or day 5. Blastocyst freezing increased the ongoing pregnancy, implantation and survival rates post-thaw as compared with cleaved embryo thaws. There was no difference in ongoing pregnancies for fresh and frozen thawed blastocyst transfers (59% versus 62%) respectively. Additionally, fresh blastocyst transfers had a higher proportion of supernumerary embryos cryopreserved than did day 3 transfers (38% versus 26%). Blastocyst freezing is a viable option for handling patient s supernumerary embryos while optimizing pregnancy outcomes per oocyte retrieval. Keywords: blastocyst, cryopreservation, embryo, frozen embryo transfer, pregnancy Introduction Prior to the development of sequential media, human embryos were primarily cultured in simple balanced salt solutions. Historically, the mouse has been the model for developing media for human use. Implementation of co-culture methods has been shown to improve the development rates for early embryos as well as blastocysts (Menézo et al., 1992; Wiemer et al., 1995). However, none of these early culture environments provided the metabolic requirements for optimal embryonic development. The first successful pregnancy arising from a frozen thawed embryo was reported by Trounson and Mohr (1983). Shortly after this, Cohen et al. (1985) reported the first successful frozen thawed blastocyst pregnancy. These reported pregnancies were derived from culturing embryos in simple media. Blastocyst cryopreservation was virtually abandoned due to the unreliable survival and poor pregnancy rates. Menézo and Veiga (1997) showed that blastocysts could be frozen and thawed with fewer dehydration and rehydration steps. The shorter protocol was easier to use, however pregnancy rates were still lower than for early embryo cryopreservation. Recently, more emphasis has been placed on extended embryo culture with sequential media to the blastocyst stage of development. Blastocyst transfer provides the ability to reduce the incidence of high order multiples while optimizing pregnancy rates (Gardner et al., 1998). Today the efficacy of blastocyst culture and cryopreservation is still controversial, primarily due to the high degree of variability in the actual outcomes reported. Regardless of the stage of development when embryos are cryopreserved, lower outcomes are still an acceptable quality measure for success in most cryopreservation programmes. In the following study, data will show that blastocyst freezing is the preferred stage over cleavage-stage embryos, for storing embryos for later subsequent cycles. 207

Materials and methods Stimulation The Presbyterian Hospital Institutional Review Board approved the following study. All completed frozen thawed embryo transfers were included during the study period.

Patients received a programmed cycle endometrial preparation. During the mid-luteal phase of the menstrual cycle, 10 mg of leuprolide acetate (Lupron; TAP Pharmaceutical Inc.

2 Materials and methods Stimulation The Presbyterian Hospital Institutional Review Board approved the following study. All completed frozen thawed embryo transfers were included during the study period. Cleaved embryo patients included all cycles where zygotes and cleaved embryos were thawed and transferred on day 3. There were no exclusions for age as well as donor oocyte cycles. Patients received a programmed cycle endometrial preparation. During the mid-luteal phase of the menstrual cycle, 10 mg of leuprolide acetate (Lupron; TAP Pharmaceutical Inc., Deerfield, IL, USA) was administered daily. On the third day of the menstrual period, leuprolide acetate was reduced to 5 mg at the time Vivelle oestrogen patches (Novartis Pharmaceuticals, East Hanover, NJ, USA) were started until cycle day 13. Vivelle patches were started with a 0.1 mg patch and changed every other day. Patches were increased to 0.2 mg on day 5, increasing to 0.3 mg on day 7 and 0.4 mg on day 9. Patients remained on 0.4 mg patches every other day until pregnancy test. On day 12 or 13 when the endometrial lining was 7 mm or greater, oestradiol levels were above 200 pg/ml and progesterone was below 2.0 ng/ml, leuprolide acetate was discontinued and 50 mg progesterone in oil was administered daily. For 1861 retrievals in the study group, the average age of patients was 33.7 years and an average of 14.7 oocytes were recovered, 12.1 being mature. Cleaved embryos were transferred on the fourth day of progesterone support. Similarly, blastocysts were transferred on the sixth day of progesterone administration. Cleavage stage culture conditions Prior to genome activation, embryos were cultured in either a simple human tubal fluid (HTF) or G1 (Vitrolife, Sweden) microdroplets under oil. Early embryos need 6 cells and 20% fragmentation to be considered for cryopreservation. These embryos were frozen in 1.5 mol/l 1,2-propanediol (PROH; Sigma) with 0.1 mol/l sucrose (Sigma) as described by Lassalle et al. (1985). Cleaved embryos were air thawed for 30 s then placed in a 30 C water bath for an additional 30 s. Embryos were rehydrated through decreasing concentrations of 1.0 mol/l, 0.75 mol/l, 0.5 mol/l, 0.25 mol/l, 0.0 mol/l PROH with 0.2 mol/l sucrose. Survival for cleaved embryos was defined as at least 50% cell survival for each embryo. Cleavage stage embryos were hatched with acidic Tyrode s solution and necrotic cells were removed. Surviving embryos were evaluated and transferred using a soft Wallace catheter under ultrasound guidance. All freezing and thawing procedures were performed at ambient room temperature. Blastocyst culture conditions From day 3 to day 5, embryos were cultured in G2 (Vitrolife, Sweden) or a potassium simplex optimized media with amino acids (KSOM AA ). A full blastocoele cavity with a defined inner cell mass and a multicellular trophectoderm was required in order for blastocysts to be frozen. Blastocysts were frozen in 9% glycerol with 0.2 mol/l sucrose as described by Menezo and Viega (1997). Blastocysts were air thawed for 2 min and rehydrated using 6% glycerol with 0.4 mol/l sucrose followed by decreasing concentrations of 4%, 3%, 2%, 1%, and 0% glycerol with 0.2 mol/l sucrose. Figure 1 shows the reexpansion of a blastocyst after 2 4 h culture before transfer. Cleaved embryos and blastocysts were frozen in a planer freezer starting at 20 C. Embryos were loaded into a 0.25 ml straw and placed into the freezing unit. Embryos were cooled at a rate of 2 C/min to 7 C, where embryos were manually seeded. Following a 10 min hold, embryos were cooled at 0.3 C/min to 35 C and plunged into liquid nitrogen for longterm storage. Outcome variables Clinical characteristics as well as pregnancy outcomes were evaluated using chi-squared analysis. Embryo survival was defined as 50% cell survival for the cleaved subgroup and reexpansion of blastocysts post-thaw. Ongoing pregnancy was defined as the visualization of an intrauterine sac with cardiac activity as confirmed by ultrasound. Implantation rates were characterized by the number of gestational sacs with fetal hearts per embryo transferred. 208 Figure 1. Re-expansion of frozen thawed blastocysts after 4 h culture: post-thaw (a); at 4 h post-thaw (b).

3 Results In the two groups the average age for day 3 and day 5 frozen embryo transfers was 34.2 and 33.5 days respectively. Table 1 describes the 224 completed transfers for cleavage stage frozen embryo transfers and 39 frozen thawed blastocyst transfers. Blastocysts had a higher proportion (P < 0.05) of post-thaw survival than cleaved embryos. Blastocysts survived at a rate of 164 of 202 thawed (81%) as compared with 931 of 1275 cleaved embryos thawed (73%). An average of 2.4 blastocysts was transferred compared with 3.2 for cleavage stage embryos (not significant; Table 1). Table 2 describes the pregnancy outcomes for the 39 blastocysts as well as the 224 cleaved frozen thawed embryo transfers. Frozen thawed blastocyst transfers resulted in a significantly (P < 0.05) higher proportion of ongoing pregnancies than for cleaved embryos. The ongoing pregnancy rate for blastocyst transfers was 62% (24/39) as compared with 41% (91/224) for cleaved transfers. Similarly, the implantation outcomes for the two groups were significantly different (P < 0.05). Blastocyst implantation indicated a 43% implantation rate compared with 18% for cleaved embryo transfers (Table 2). Table 3 compares fresh blastocyst transfers to frozen thawed blastocyst transfers. Although not significant for the two groups, the ongoing pregnancy rates for frozen thawed blastocysts in comparison to fresh blastocyst transfers were 62% and 59% respectively. Frozen thawed blastocysts had a 43% implantation rate, which compares to 41% for fresh transferred blastocyst transfers (not significant). Data showed that an average of 2.4 frozen thawed blastocysts were transferred. This was similar to the average of 2.2 embryos for fresh blastocyst transfers (Table 3). Table 4 describes the same data for fresh cleaved embryo transfers in comparison to frozen thawed cleaved embryo transfers. The ongoing pregnancy outcomes for frozen thawed cleaved embryo transfers was significantly (P < 0.01) lower than for fresh cleaved transfers. Ongoing pregnancy rates for frozen thawed cleaved embryo transfers were 41% in comparison to 50% for fresh embryo transfers. Implantation outcomes and average number of embryos transferred in both groups were significantly (P < 0.05) lower for frozen thawed cleaved embryo transfers. Table 4 illustrates an 18% implantation rate for frozen thawed cleaved transfers when compared with 23% for fresh cleaved embryo transfers. The average number of embryos transferred was considerably (P < 0.05) higher for fresh transfers (3.8) compared with thawed cleaved transfers (3.2). Results described in Table 5 show the number of patients that were able to have supernumerary embryos frozen for cleaved embryo transfers and blastocyst transfers. A significantly (P < 0.05) higher proportion of patients receiving a blastocyst transfer (38%) subsequently had embryos frozen than did patients with day 3 transfers (26%) (Table 5). Patients with fresh blastocyst transfers had an average of 5.0 embryos frozen compared with 5.8 embryos for day 3 embryo transfers (not significant). Table 1. Survival outcomes comparing cleaved embryos to blastocyst thaws. Cleaved embryos Blastocysts Average age (days) Total thawed (n) Total no. survived (%) 931 (73) a 164 (81) a Embryos transferred (n) Transfers completed (n) Average no. transferred 3.2 b 2.4 b Table 2. Pregnancy outcome comparing cleaved embryos to blastocyst thaws. Cleaved Blastocyst embryos Clinical pregnancies (%) 94/224 (42) a 27/39 (69) a Ongoing pregnancies (%) 91/224 (41) a 24/39 (62) a FHB/embryo transferred (%) 129/726 (18) a 42/97 (43) a b Not significant. Table 3. Pregnancy outcome comparing fresh to frozen thawed blastocyst transfers. Fresh blastocyst Thawed blastocyst Table 4. Pregnancy outcome comparing fresh to frozen thawed day 3 transfers. Fresh Thawed day 3 day 3 Transfers completed Ongoing pregnancies (%) 142/240 (59) a 24/39 (62) a FHB/embryo transferred (%) 217/532 (41) a 42/97 (43) a Average no. transferred 2.2 a 2.4 a a Not significant. Transfers completed Ongoing pregnancies (%) 757 (50) a 91 (41) a FHB/embryo transferred (%) 1248/ /726 (23) a (18) a Average no. transferred 3.8 a 3.2 a 209

4 210 Table 5. Summary of supernumerary embryos frozen for day 3 and blastocyst transfer. Discussion Day 3 transfer Blastocyst transfer Total fresh transfers Patients with cryopreservation Total embryos frozen Average frozen/patient 5.8 a 5.0 a Number with 387/ /240 cryopreservation (%) (26) b (38) b a Not significant. b P < From the data described in this study, blastocyst freezing is a viable option for handling patient s supernumerary embryos. Historically, cryopreserved embryos have a lower expected pregnancy outcome when compared with fresh embryo transfers. However, the data shown here indicate that frozen thawed blastocyst clinical outcomes are comparable to those for fresh blastocyst transfers. Additionally, these figures illustrate a dramatic difference between fresh and frozen thawed transfers for cleavage stage embryo transfers. Moreover, the number of patients with supernumerary embryos remaining for cryopreservation was proportionately higher where fresh blastocyst transfers were completed. Similar results for cryopreservation of supernumerary embryos have been reported by Wilson et al. (2002). Although not significant, a source of bias in this study is the decreased maternal age for the two groups. It is important to note that most high-quality supernumerary embryos in any cryopreservation programme are derived from the younger patient population. Initial experiences in this laboratory with blastocyst thaws were not as successful. The first five embryo transfers utilized a two-step protocol thaw (Menézo and Viega, 1997) and were not successful. Although the embryos appeared to have intact cells, no pregnancies were established. Shortly after this, the long protocol previously described was attempted and had immediate success. This may be primarily due to the multistep thaw protocol as well as the quality of embryos frozen (Cho et al., 2002). Embryos re-expanded and pregnancies finally came to fruition. Recently Behr et al. (2002) reported a 36% clinical pregnancy rate and a 16% implantation rate with frozen thawed blastocysts. In the same study, Behr showed that there was no significant difference between day 5 and day 6 frozen thawed transfers. Similarly, Menezo and Viega (1997) showed a 29% clinical pregnancy rate and 14.5% implantation rate. This would indicate that the technology has not improved much over the years. Recently Choi et al. (2000) and Yokota et al. (2001) reported a 23.7 and 21.9% implantation rate respectively, utilizing vitrification methods for freezing. Similarly, Mukaida et al. (2002) showed a 36% pregnancy rate with ultrarapid cryoloop vitrification. Vitrification is attractive as it takes less time and equipment to cryopreserve embryos. However, the reported outcomes still do not exceed those of the established slow freezing protocols. In the past, lower pregnancy outcomes with frozen thawed embryos have been an accepted measure of quality performance. Since the introduction of sequential media, blastocyst transfer has been instrumental in reducing the incidence of high-order multiples. The risk associated with high-order multiples requires a more proactive effort on the part of the laboratory and clinician to reduce the number of embryos transferred. In this programme, it was possible to dramatically reduce the average number of embryos from approximately 3.5 to 2.2 embryos per transfer since implementing blastocyst transfers. When the implantation rate for blastocyst transfers is more than double the implantation rate for cleavage embryo transfers, it is much easier to risk a negative outcome than to risk the potential for a triplet pregnancy. The decision to freeze embryos at the blastocyst stage or at an earlier stage continues to be a matter of debate. If the implantation rates are not improved when freezing blastocysts, then the argument for freezing only cleaved embryos is justified. Many laboratories do not advocate freezing blastocysts due to the potential of losing embryos that might have made a pregnancy if frozen on day 3. Naturally some early embryos will be lost with extended culture. However, these embryos may be lost due to suboptimal culture conditions or inadequate maternal genomic reserves to allow progression beyond genome activation. The data presented here show there is no difference in the average number of embryos frozen between the two groups. However, a significantly higher proportion of fresh blastocyst transfers subsequently had embryos frozen. The developmental potential for chromosomally abnormal embryos to develop to the blastocyst stage has been described by Sandalinas et al. (2001). These authors describe how 65% of abnormal embryos arrest at the compaction stage of development compared with only 28% of normal embryos. In a recent report by Veeck (2003), the cumulative pregnancy outcomes with blastocyst cryopreservation are described. In this study there was no difference between frozen blastocyst transfer results and fresh blastocyst transfer results. However, there was a significant difference between results obtained after transferring fresh compared with frozen cleaved embryos. One could conclude from the data presented that blastocyst freezing could optimize overall pregnancy outcomes and is the preferred stage for cryopreservation. References Behr B, Gebhardt J, Lyon J et al Factors relating to a successful cryopreserved blastocyst program. Fertility and Sterility 77, Cho H, Son W, Yoon S et al An improved protocol for dilution of cryoprotectants from vitrified human blastocysts. Human Reproduction 17, Choi DH, Chung HM, Lim JM et al Pregnancy and delivery of healthy infants developed from vitrified blastocysts in an IVF-ET program. Fertility and Sterility 74, Cohen J, Simons RF, Edwards RG et al Pregnancies following the storage of expanding human blastocysts. Journal of In Vitro Fertilization and Embryo Transfer 2, Gardner DK, Vella P, Lane M et al Culture and transfer of human blastocysts increases implantation rates and reduces the need for multiple embryo transfers. Fertility and Sterility 69,

5 Lassalle B, Testort B, Renard JP 1985 Human embryo features that influence the success of cryopreservation with the use of 1,2- propanediol. Fertility and Sterility 44, Menézo Y, Viega A 1997 Cryopreservation of blastocysts. Proceedings of the 10th World Congress on IVF and Assisted Reproduction, Vancouver, Canada, May 1997, pp Menézo Y, Nicollet B, Herbaut N et al Freezing co-cultured human blastocysts. Fertility and Sterility 58, Mukaida T, Takahashi K, Kasai M 2002 Blastocyst cryopreservation: ultrarapid vitrification using cryoloop technique. Reproductive BioMedicine Online 6, Sandalinas M, Sadowy S, Alikani M et al Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Human Reproduction 16, Trounson A, Mohr L 1983 Human pregnancy following cryopreservation, thawing and transfer of an eight-cell embryo. Nature 305, Veeck L 2003 Does the developmental stage at freeze impact clinical results post-thaw? Reproductive BioMedicine Online 6, Wiemer KE, Dale B, Hu Y et al Blastocyst development in coculture: development and morphological aspects. Human Reproduction 10, Wilson M, Hartke K, Kiehl M et al Integration of blastocyst transfer for all patients. Fertility and Sterility 77, Yokota Y, Sato S, Yokota M et al Birth of a healthy baby following vitrification of human blastocysts. Fertility and Sterility 75, Received 27 May 2003; refereed 11 June 2003; accepted 20 October

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