Culturing cryopreserved cleavage stage embryos to blastocysts is a cost-effective method of utilizing frozen embryos

Veeck. Weill Medical College of Cornell University, New York, NY. OBJECTIVE: One or more viable preembryos (PE) often remain following transfer on Day 3 (D3) post-harvest. In our laboratory, these extra PE are cultured in sequential media for the purpose of cryopreserving any good quality blastocysts (BL) formed on Day 5 or Day 6 (D5/6). This has resulted in an augmentation of fresh clinical pregnancy rates for many patients. It is not known if the pregnancy status following D3 transfer is indicative of a patient’s chances of becoming pregnant in a subsequent thaw cycle using sibling BL. The objective of the current study was to determine if the pregnancy and implantation potential of thawed human BL derived from patients who had attained pregnancy in their D3 fresh cycle differed from those patients with a negative D3 pregnancy result. DESIGN: A retrospective analysis of 173 BL thaw transfers from 2002– 2004 was performed. Patients included in the analysis had one or more sibling BL cryopreserved following extended culture after D3 transfer. Thaw cycles were categorized into two groups based on the pregnancy status of their D3 fresh cycle: Group 1 included patients (N⫽73) who achieved pregnancy in their D3 cycle, while Group 2 included patients (N⫽100) who had a negative pregnancy result on D3. Endpoints included post-thaw blastocyst survival rate, clinical and ongoing pregnancy rates, and implantation rate. MATERIALS AND METHODS: Stimulation protocols, sperm preparation techniques, PE and extended culture methods, and BL freezing and thawing methods used in the Cornell IVF laboratory have been described in detail elsewhere. Following the transfer of fresh PE on D3 post-harvest, any remaining PE were placed in extended culture using C2 medium and evaluated daily. Blastocysts of good quality were subsequently cryopreserved on D5 or D6. The transfer of thawed BL was performed in either a natural or programmed cycle. Day 5 cryopreserved BL were thawed and cultured overnight in C2 medium prior to transfer, while D6 cryopreserved BL were thawed on the day of transfer. Data were analyzed by chi-square or t-test where appropriate. RESULTS: The overall clinical pregnancy rate of BL thaw patients in this analysis was high (87/173; 50%). There were no significant differences between Group 1 and Group 2 patients when comparing post-thaw blastocyst survival rates (74% and 79%). Similarly, clinical pregnancy rates (52% and 49%), ongoing/delivered pregnancy rates (44% and 37%) and implantation rates (39% and 35%) were not different between the two groups. No significant group differences were found in mean patient age and the number of blastocysts replaced at transfer. CONCLUSION: Blastocysts resulting from the extended culture of sibling PE following D3 transfer can be cryopreserved on D5/6 and subsequently thawed, resulting in high rates of implantation and pregnancy. The pregnancy status of the D3 fresh cycle has no effect on the potential success of subsequent thaw cycles using sibling blastocysts. Supported by: None

P-57 Culturing cryopreserved cleavage stage embryos to blastocysts is a cost-effective method of utilizing frozen embryos. M. Feinman, R. Boostanfar, A. Le, D. Potter, C. Khoury, B. Behr. Huntington Reproductive Center, Westlake Village, CA; Huntington Reproductive Center, Laguna Hills, CA. OBJECTIVE: Success with cryopreserved embryos varies greatly and depend heavily on the quality of the frozen embryos. Repeated frozen embryo transfers can be expensive. Furthermore, after achieving a pregnancy some patients are left with unwanted embryos in the freezer. This study compares our experience of trasferring cleavage stage embryos and allowing these embryos to develop into blastocysts before transfer. DESIGN: Retrospective Chart Review. MATERIALS AND METHODS: We reviewed the records of 93 women who underwent a frozen embryo transfer over a two year period in our two labs. Thirty-four women chose to allow their embryos to be cultured for two extra days in Blast Media (Irvine Scientific, Irvine, CA). Embryo transfers were performed in hormone replacement cycles. Patients received twice weekly injections of estradiol valerate (5–10mg) and daily combinations of progesterone in oil, 50mg, and 200mg vaginal suppositories. Ongoing pregnancy rates and implantation rates were compared in both groups. Chi-square was used for statistical analysis. RESULTS: The ongoing pregancy rate in the cleavage stage transfers was

S156

21/42 (50%), compared to 19/34 (56%) in the blastocyst transfers. The implantation rate in the cleavage stage group was 31/134 (23%) vs. 29/91 (32%) in the blastocyst group. Neither difference was statistically significant. CONCLUSION: Culturing cryopreserved cleavage stage embryos to blastocyst is an efficient and cost-effective alternative for patients undergoing frozen embryo transfers. While the implantation rate was higher in the blastocyst transfers, this was not statistically significant. This is likely a reflection of the sample size. the monozygotic twin rate in our study is similar to that reported in the literature with fresh blastocyst transfers. Supported by: Huntington Reproductive Centers of California.

P-58 Improved outcome of cryopreserved blastocyst transfer after a modification of the common two step protocol. R. K. Srivastava, P. Wieckowski, J. E. Pabon. Fertility Center and Applied Genetics of Florida, Inc., Sarasota, FL. OBJECTIVE: To evaluate the efficacy of a modified protocol as compared to a commonly used two-step protocol for human blastocyst freezing and thawing for a better treatment outcome. DESIGN: Retrospective study on clinical pregnancy rates after freezing and thawing human blastocysts using two different protocols. MATERIALS AND METHODS: This study analyzed a total of 92 blastocysts frozen thaw transfer (FET) cycles. Of these, 24 FET were performed using the two step method as described by Menezo et al., 1992. A total of 68 FET used a modified protocol . For both groups 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, loaded into straws and frozen using the same slow freezing protocol as described for two step protocol. For thawing, blastocysts were subjected to a multistep protocol as opposed to two step protocol. Blastocysts were removed from the straws after thawing and cryoprotectant were removed in several steps as follows. Blastocysts were expelled in 10% Glycerol and 0.4 M sucrose until they are located and then immediately transferred to 5% Glycerol and 0.4 M sucrose for 3 min, 2.5% Glycerol and 0.4 M sucrose for 3 min, 0.4 M sucrose for 3 min, 0.2 M sucrose for 3 min and finally in 0.1 M sucrose for 3 min at RT, rinsed in mHTF and cultured for approximately 4 hour in blastocyst culture medium with 20 mg/ml HSA before intrauterine transfer. All blastocysts were replaced after estradiol and progesterone supplementation. RESULTS: A total of 325 blastocysts thawed in 92 frozen thaw transfer cycles. Twenty four frozen transfers were performed using the two-step protocol. This resulted in clinical pregnancy rate of 16%. By switching to the modified protocol in 68 frozen transfers we achieved a clinical pregnancy rate of 52%. Clinical pregnancy is defined as the presence of fetal heart beat on ultrasound. One of the most noticeable observation by using the modified method was the rate of survival of blastocysts which was 71% as compared to 46% using the two-step method. Most blastocysts were reexpanded in modified method groups (89%) as opposed to 65% in two step group. The age of patients for blastocyst freeze and thaw was not a 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 either on day 5 or day 6. CONCLUSION: Based on this study we conclude that a modified protocol of freeze and thaw for human blastocysts have significantly enhanced the clinical pregnancy outcome in blastocysts freeze-thaw cycles as compared to the earlier two step method. Our ongoing analysis reveals that these results are comparable to fresh blastocyst transfer. It is possible that the antioxidant properties of ascorbate in freezing and thawing medium, the higher protein concentration and the multistep removal of cryoprotectant all have a cumulative effect in enhancing this clinical outcome. Supported by: None

Vol. 82, Suppl. 2, September 2004