Article Examination of frozen cycles with replacement of a single thawed blastocyst

Size: px

Start display at page:

Download "Article Examination of frozen cycles with replacement of a single thawed blastocyst"

Job Jordan
5 years ago
Views:

RBMOnline - Vol 11. No 3. 2005 349-354 Reproductive BioMedicine Online; www.rbmonline.

1 RBMOnline - Vol 11. No Reproductive BioMedicine Online; on web 5 July 2005 Article Examination of frozen cycles with replacement of a single thawed blastocyst Dr Nina Desai Dr Nina Desai received her doctorate in microbiology/cell biology from the University of Medicine and Dentistry of New Jersey, USA, in She completed a post-doctoral fellowship in infertility at The Ohio State University, Columbus. Dr Desai joined the faculty of Case Western Reserve University, Cleveland, Ohio in 1993 and was subsequently appointed Director of the IVF/Andrology Laboratories at University MacDonald Women s Hospital. In 2000, she was invited to join the Cleveland Clinic Foundation as Director of IVF and Clinical Research and to design a new state-of-the-art IVF laboratory. Her research has focused on growth factors, extracellular matrices, co-culture systems and the study of embryonic apoptosis. Other on-going projects in her laboratory include embryo vitrification, and testicular sperm isolation and cryopreservation. Nina Desai 1, James Goldfarb Cleveland Clinic Fertility Centre, Cedar Road, Suite 220 South, Beachwood, OH 44122, USA 1 Correspondence: desain@ccf.org Abstract This investigation examined frozen cycle outcomes with the transfer of a single thawed blastocyst. It also aimed to determine if the suggested model could aid in achieving a better understanding of factors that influence success with frozen blastocyst transfer. A retrospective analysis was conducted of single frozen embryo transfer cycles carried out at the Cleveland Clinic Fertility Centre in Beachwood, OH. Between January 2001 and March 2004, 88 thaw procedures were initiated that resulted in 75 frozen cycles in which only a single thawed blastocyst was transferred. In 66 of these 88 thaw procedures, only a single embryo was available for thawing. The post-thaw survival rate of a single frozen blastocyst was 85% (56/66). These 56 frozen transfers with a single thawed blastocyst resulted in a clinical pregnancy rate per transfer of 27% (15/56) and a live birth rate of 18%. Blastocyst post-thaw morphology at transfer was found to be an important prognostic factor associated with success. The ability to re-expand within a few hours of the thaw appeared to be a strong indicator of blastocyst potential. Blastocyst age at cryopreservation did not impact outcomes. The single frozen embryo transfer model can yield valuable information and help gauge the effectiveness of a laboratory s cryopreservation protocol. Keywords: blastocyst, cryopreservation, culture media, embryo morphology, implantation, pregnancy Introduction Culture and transfer of human embryos at the blastocyst stage on day 5 has many potential advantages, such as allowing better embryo selection, increasing the embryo implantation rate and reducing high order multiple pregnancies through transfer of only one or two embryos. Although the benefit of this approach has yet to be proven and may require further refinement in embryo culture methodology/media, there is certainly a trend amongst laboratories to attempt blastocyst culture (Gardner et al., 2004; Desai et al., unpublished work). Culture of supernumerary embryos to the blastocyst stage has been a stepping stone used by many laboratories to learn how to culture embryos effectively to the blastocyst stage. To avoid embryo wastage, laboratories need to simultaneously devise methodology and embryo parameters for successful cryopreservation and thaw at the blastocyst stage. Glycerol-based cryoprotectants combined with programmed slow freeze have been to date the most widely used methodology (Hartshorne, 1991; Menezo et al., 1992; Kaufman et al., 1995; Behr et al., 2002; Gardner et al., 2003). Several protocols have evolved, differing mostly in the in the step-wise removal of cryoprotectant after blastocyst thaw. Pregnancy rates of 15 35% have been reported. More recently, vitrification techniques have been introduced into the clinical laboratory as an alternative for human blastocyst cryopreservation (Mukaida et al., 2001, 2002). Frozen transfer cycle outcomes with vitrification have been comparable with that achieved using slow freeze protocols. This study aimed to use single frozen blastocyst transfers as models to better understand the relationship between post-thaw morphology of human blastocysts and pregnancy outcome, and to give consideration to other parameters that might influence outcomes. 349

2 350 Materials and methods Frozen transfer cycles carried out at the Cleveland Clinic Fertility Centre from January 2001 to March 2004 were retrospectively analysed. No patient selection was involved. Following selection of the best embryos for the fresh transfer on day 3, surplus embryos were cultured for an additional 2 3 days until blastocyst formation. The embryo culture regimen consisted of a simple human tubal fluid medium until day 3, followed by a more complex medium (α-mem) containing both essential and non-essential amino acids until blastocyst formation. This type of sequential approach to human blastocyst culture using α-mem has been very successful in the laboratory, yielding high quality blastocysts. Details of this culture protocol have been previously described (Desai et al., 1997). A small number (n = 3) of single blastocysts frozen and thawed in this study were derived from commercially available blastocyst media. Criteria for blastocyst cryopreservation were independent of culture medium. Only blastocysts of good morphology were cryopreserved. The current study examines outcomes with these frozen thawed blastocysts. Embryo replacement Patients were prepared for frozen embryo transfer using hormone replacement therapy consisting of increasing doses of Estrace (from 2 to 6 mg p.o. daily; Barr Laboratory, Pomona, NY, USA). Progesterone in oil (micronized in cottonseed oil; Paddock Laboratory, NY, USA) was given intramuscularly starting on day 13, at a dose of 50 mg for patients under 38 and 100 mg for all others. The only monitoring in the replacement cycle was the evaluation of the endometrium prior to progesterone administration. Frozen cycles were cancelled if the endometrial thickness was less than 7 mm. Thawed blastocysts were replaced on either day 5 or 6 of progesterone treatment. Daily progesterone and Estrace were continued until the pregnancy test 2 weeks after transfer. If the pregnancy test was positive, both medications were continued until 10 weeks gestation. Cryopreservation/thaw procedure MEMα supplemented with 10% SSS was the basal medium used for preparation of blastocyst cryopreservation and thaw solutions. All steps were performed at room temperature under constant gassing with 5% CO 2. A two-step glycerol freeze protocol was used for human blastocyst cryopreservation (Menezo et al., 1992). In the first step, blastocysts were incubated in a solution containing 5% glycerol for 10 min. This was followed by a second 10-min incubation in a solution containing 9% glycerol and 0.2 mol/l sucrose. One to two embryos were loaded into round bottom Nunc cryovials containing 0.3 ml of cryoprotectant solution (9% glycerol/0.2 mol/l sucrose). A controlled rate Planer freezer was used for embryo cryopreservation. Vials were cooled at a rate of 2 C/min until 6 C. After a 5-min hold, vials were seeded. Following seeding, vials were further cooled at a rate of 0.3 C/min until 36 C and then plunged into liquid nitrogen to complete the freezing process. Blastocyst thaw was performed using a seven-step rehydration protocol. The rehydration ladder was as follows: (i) 5% glycerol/0.2 mol/l sucrose for 5 min; (ii) 4% glycerol/0.1 mol/l sucrose for 6 min; (iii) 3% glycerol/0.1 mol/l sucrose for 7 min; (iv) 2% glycerol/0.1 mol/l sucrose for 7 min; (v) 1% glycerol/0.1 mol/l sucrose for 6 min; (vi) 0% glycerol/0.1 mol/l sucrose for 2 min; and finally (vii) 0% glycerol/0.0 mol/l sucrose for 2 min. Thawed blastocysts were cultured for 3 4 h prior to transfer to allow re-expansion. Blastocyst morphology and evaluation The blastocyst grading system described in this paper was initially set up in 1995 to monitor blastocyst quality and morphology (Desai et al., 1996, 1997). Blastocyst grade was assigned based on the following criteria: (i) day of blastocyst formation, (ii) blastocyst maturity, (iii) inner cell mass development, (iv) trophectoderm appearance and cell number and finally (v) degree of necrosis (Figure 1a, b). Blastocysts were photographed and graded pre- and post-thaw in the manner described, and tracked by computer database. Video micrographs at several different focal planes were also taken of the blastocysts to ensure uniform grading and assessment by all embryologists. Blastocysts were re-evaluated just prior to transfer and graded for blastocoel cavity (maturity) and morphological attributes of inner cell mass (ICM) and trophectoderm. Data analysis Pregnancy outcomes and implantation rates were compared with transfer of different grades of blastocysts. Clinical pregnancy was determined by the presence of a fetal heartbeat on ultrasound at approximately 7 weeks gestation (5 weeks after the transfer). Other parameters examined were post-thaw survival, day of blastocyst formation, degree of post-thaw re-expansion and day of freezing. Statistical analysis was performed using nonparametric tests (chi-squared and Fisher s exact tests) and also Student s t-test where appropriate. Results Between January 2001 and March 2004, 88 thaw procedures were initiated in an unselected patient population that resulted in 75 frozen cycles in which only a single thawed blastocyst was transferred. In 66 of these 88 thaw procedures, only a single embryo was available for thawing. The data presented here describe these 66 thaw cycles (Table 1). Ten of the thaw procedures resulted in no embryo survival. The postthaw survival rate of a single frozen blastocyst was 85% (56/66). Thirty-four per cent of patients had a positive human chorionic gonadotrophin (HCG) after embryo replacement, and this resulted in a clinical pregnancy rate per transfer of 27% (15/56). The resultant live birth rate with a single frozen Table 1. Outcomes with transfer of a single thawed blastocyst. No. thaws 66 No. transfers 56 Mean age (years) 35.6 ± 6.3 Positive pregnancy test (%) 34 Clinical pregnancy rate (%) 27

By the time of transfer, 78% of the thawed blastocysts had either partially or completely re-expanded and this appeared to be strongly related to pregnancy outcome.

3 blastocyst was 18%. No difference in age or diagnosis was noted between pregnant and non-pregnant patients. The mean patient age was 35.6 ± 6.3. The laboratory protocol has been to incubate thawed blastocysts for 3 4 h to allow for re-expansion prior to transfer. By the time of transfer, 78% of the thawed blastocysts had either partially or completely re-expanded and this appeared to be strongly related to pregnancy outcome. In high quality blastocysts, re-expansion was often visible within 1 h of thawing. Transfers with a single re-expanded blastocyst with a grade C or D blastocoel resulted in a clinical pregnancy rate of 44% (12/27; Figure 2). This was significantly higher (P < 0.05) than outcomes in transfer cycles with one early blastocyst (15%, 3/20; with blastocoel grade A or B) No pregnancies were achieved with the transfer of a single nonre-expanded blastocyst. Almost all of these non-re-expanded blastocysts showed major areas of necrosis (N2), but enough viable looking cells to warrant transfer. Figures 3 and 4 describe ICM and trophectoderm morphology in these single thawed blastocysts and relationship to pregnancy outcome. A trend was observed towards better clinical outcomes in blastocysts with a well differentiated trophectoderm (TE grade 2). Analysis of a larger number of single blastocyst transfer cycles will be necessary to further assess the impact of TE and ICM grade on outcome. The majority of blastocysts in this study group had been cryopreserved on culture day 6 (n = 45). Clinical outcomes were, however, similar between blastocysts frozen on day 6 (26.7%; 12/45) versus day 5 (27.2%; 3/11). This study also looked at the day of thawed blastocyst replacement to the patient s uterus. Sixty-five per cent of transfers were performed on day 5 of progesterone therapy. Clinical pregnancy results were, however, similar regardless of the day of embryo replacement (Figure 5). Figure 1. Blastocyst evaluation based on grading of blastocyst maturity, trophectoderm development, inner cell mass, degree of necrosis and day of blastocyst formation. The degree of embryonic cavity and expansion were used to categorize blastocyst maturity: A = early blastocyst, cavity just starting to form; B = early blastocyst, cavity less than half the volume of embryo; C = expanded with cavity greater than half embryo volume; D = fully expanded; E = hatching. Grading of trophectodermal and ICM components and derivation of final score shown in (a) and (b). (a) Grading of the trophectoderm. The three scores (1, 2, 3) for trophectoderm development require the embryologist to judge whether the cell number is adequate for the maturity of the blastocyst. A poorly developed trophectoderm in an expanded blastocyst is identified by observation of cells that appear to stretch to line the blastocoel (score 1). Embryos that have started to cavitate without adequate cell number, are also graded as having a poor trophectoderm (TE1; score 1). In contrast, cells in the well-developed trophectoderm (TE2 and TE3; scores 2 and 3) line the blastocoel and give a scalloped appearance. TE3 blastocysts occur infrequently and were not observed in this study group of embryos (score 3). (b) Grading of the inner cell mass. In early blastocysts, the inner cell mass was often not yet visible (score 0). In expanded blastocysts, assessment of ICM was critical in determining whether or not to freeze the embryo. The degree of necrosis was graded as either score 1 = minor, isolated sites, or else score 2 = severe necrosis throughout the embryo. Blastocyst morphological attributes were combined with age of the blastocyst (in days) to yield a single descriptive score. 351

4 Figure 2. Relationship between blastocyst maturity and pregnancy outcome. Thawed blastocysts were assessed for maturity just prior to transfer. Pregnancy outcomes with nonre-expanded blastocysts (NR) and early blastocysts (maturity A and B) were compared with outcomes with late mature blastocysts (C, D). *Significantly different from other groups P < Figure 3. Inner cell mass morphology and clinical outcome. Figure 4. Trophectodermal appearance and clinical outcome. Figure 5. Day of embryo replacement and clinical outcome. 352

5 Discussion The availability of commercial media systems for human blastocyst culture has generated new interest in blastocyst culture as a means of reducing multiple pregnancies. Cryopreservation of supernumerary blastocysts becomes especially important as programmes considered moving towards blastocyst stage transfer in fresh IVF cycles (Gardner and Lane, 2003). Effective cryotechnology has been advocated as a tool to reduce multiple pregnancies and increase the reproductive potential of a single IVF procedure (DeNeubourg and Gerris, 2003; Gerris et al., 2003). To date, a very limited number of trials with frozen thawed human blastocysts have been reported (Hartshorne et al., 1990; Menezo et al., 1992, 1993; Kaufman et al., 1995; Shoukir et al., 1998; Lane et al., 1999; Mukaida et al., 2001; Behr et al., 2002). In the largest of these trials (n = 516), pregnancy and implantation rates of 22 and 13% respectively were reported with frozen blastocysts arising from co-culture (Kaufmann et al., 1995). Pregnancy and implantation rates of 36 and 16% respectively were recently reported in a smaller trial (n = 64) with blastocysts derived from sequential media (Behr et al., 2002). The present work describes an approach to understanding the features that may be critical for establishing a successful blastocyst cryopreservation programme. The single embryo transfer model allows monitoring of individual parameters and their influence on outcomes. Blastocyst post-thaw morphology at transfer was found to be an important prognostic factor associated with success. The ability to re-expand within a few hours of the thaw was a strong indicator of blastocyst potential. Figure 4 suggests that trophectoderm grade may also be a vital factor in successful implantation. The stretched, scanty appearance of the trophectodermal cells lining the blastocoel in trophectoderm grade 1 blastocysts may be a reflection of lower overall blastomere number at freezing, making such blastocysts more vulnerable to the freeze thaw process and possibly reducing pregnancy potential. Monitoring trophectoderm grades with different culture formulations may aid in improving results with cryopreserved embryos. The nature of the culture system from which blastocysts are derived may play an important role in timing of morulation, blastocyst formation, and optimum day for freezing and ultimately post-thaw outcome in individual laboratories. In our laboratory, blastocysts cryopreserved on both day 5 and 6 had equivalent pregnancy potential. Other published studies with frozen transfers have reported similar results (Shoukir et al., 1998; Behr et al., 2002). In contrast, investigators examining fresh blastocyst transfer cycles noted an increase in pregnancy and implantation rates with day 5 versus day 6 blastocysts (Shapiro et al., 2001). Growth factor supplementation of thaw media is an avenue that needs further exploration. There is much data to suggest that growth factors, both autocrine and paracrine, are especially necessary for advanced stage embryos and may play a role in preventing apoptosis or programmed cell death (Brison and Schultz, 1997; O Neill, 1997). Cryopreserved mouse morula show significant enhancement in post-thaw development when cultivated on cell monolayers or after addition of specific growth factors to the culture milieu (Desai et al., 2000). Growth factors and hormones have similarly been shown to improve post-thaw survival of vitrified bovine blastocysts (Mitango et al. 2002). Such factors may help jump start the thawed embryo, accelerating post-thaw re-expansion and promoting continued proliferation of cells of the ICM and trophectoderm. Cryopreservation of a single embryo is often not considered to be time- and cost-effective. Depending on laboratory staffing, many IVF programmes will only freeze if there are two or more embryos available for cryopreservation. The authors are not aware of any published studies that have in fact examined clinical outcomes with cryopreservation and transfer of a single thawed blastocyst. Based on current experience and the data presented in this study, it is clear that cryopreservation of even a single embryo at the blastocyst can augment the overall pregnancy potential of a single egg harvest. It also provides a unique opportunity to define more clearly factors that predispose to successful outcomes. In conclusion, the single frozen embryo transfer model can yield valuable information and help gauge the effectiveness of a laboratory s blastocyst culture and cryopreservation protocols. The tremendous progress being made in understanding the metabolic requirements of human embryos and designing appropriate culture environments, along with advances in cryopreservation technology, should lead to even better outcomes with cryopreserved embryos in the near future. References Behr B, Gephardt J, Lyon J et al Factors relating to a successful cryopreserved blastocyst transfer program. Fertility and Sterility 77, Brison D, Schultze R 1997 Apoptosis during mouse blastocyst formation: evidence for a role for survival factors including transforming growth factor alpha. Biology of Reproduction 56, Desai N, Lawson J, Goldfarb J 2000 Assessment of growth factor effects on post-thaw development of cryopreserved mouse morulae to the blastocyst stage. Human Reproduction 15, Desai N, Kinzer D, Loeb A et al Use of synthetic serum substitute and alpha-minimum essential medium for the extended culture of human embryos to the blastocyst stage. Human Reproduction 12, Desai N, Kinzer D, Loeb A, Goldfarb J 1996 Use of synthetic serum substitute and alpha-minimum essential medium for the extended culture of human embryos to the blastocyst stage. Annual Meeting of the American Society of Reproductive Medicine, Nov 2 6, 1996, Boston, MA, USA. Oral presentation. De Neubourg D, Gerris J 2003 Single embryo transfer-state of the art. Reproductive BioMedicine Online 7, Gardner D, Lane M 2003 Towards a single embryo transfer. Reproductive BioMedicine Online 6, Gardner DK, Surrey E, Minjarez D et al Single blastocyst transfer: a prospective randomized trial. Fertility and Sterility 81, Gardner DK, Lane M, Stevens J, Schoolcraft WB 2003 Changing the start temperature and cooling rate in a slow-freezing protocol increases human blastocyst viability. Fertility and Sterility 79, Gerris J, De Neubourg D, De Sutter P et al Cryopreservation as a tool to reduce multiple birth Reproductive BioMedicine Online 7, Hartshorn G, Elder K, Crow J et al The influence of in 353

6 vitro development upon post-thaw survival and implantation of cryopreserved human blastocysts. Human Reproduction 6, Kaufman R, Menezo Y, Hazout A et al Cocultured blastocyst cryopreservation: Experience of more than 500 transfer cycles. Fertility and Sterility 64, Lane M, Schoolcraft WB, Gardner DK 1999 Vitrification of mouse and human blastocysts using a novel cryoloop container-less technique. Fertility and Sterility 72, Menezo Y, Nicollet B, Dumont M, Hazout A et al Factors affecting human blastocyst formation in vitro and freezing at the blastocyst stage. Acta Europa Fertility 24, Menezo Y, Nicollet B, Herbaut N et al Freezing co-cultured human blastocysts. Fertility and Sterility 58, Mitango NR, Varisanga MD, Dong YG et al Growth factors and growth hormone enhance in vitro embryo production and postthaw survival of vitrified bovine blastocysts. Theriogenology 59, Mukaida T, Takahashi K, Kasai M 2002 Blastocyst cryopreservation: ultrarapid vitrification using the cryoloop technique. Reproductive BioMedicine Online 6, Mukaida T, Nakamura S, Tomiyama T et al Successful birth after transfer of vitrified human blastocysts with use of a cryoloop containerless technique. Fertility and Sterility 76, O Neill C 1997 Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro. Biology of Reproduction 56, Shapiro B, Richte R K, Harris D et al A comparison of day 5 and day 6 blastocyst transfers. Fertility and Sterility 75, Shoukir Y, Chardonnens D, Campana A et al The rate of development and time of transfer play different roles in influencing the viability of human blastocysts. Human Reproduction 13, Paper based on contribution presented in part at the Annual Meeting of the American Society of Reproductive Medicine, Toronto, Ontario, Canada, September 25 30, Received 17 December 2004; refereed 11 January 2005; accepted 7 June

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

Abstract. Introduction. RBMOnline - Vol 8. No Reproductive BioMedicine Online; on web 15 December 2003 RBMOnline - Vol 8. No 2. 207-211 Reproductive BioMedicine Online; www.rbmonline.com/article/1023 on web 15 December 2003 Article Determining the most optimal stage for embryo cryopreservation Anthony Anderson