A comparison of post-thaw survival and pregnancy rates in day 5 and day 6 frozen-thawed human blastocysts

Abstract

Objective: It is generally assumed that human preembryos (PE) and blastocysts (BL) that develop in a timely manner in vitro are of better quality than conceptuses that develop more slowly. The objective of the present study was to determine if the cryopreservation of human blastocysts on Day 5 post-retrieval resulted in better post-thaw survival and pregnancy rates when compared to Day 6 frozen-thawed blastocysts. Design: A retrospective analysis of 97 Day 5 and 73 Day 6 blastocysts frozen and thawed between July 2000 and March 2002 was performed. Endpoints included: post-thaw survival, clinical and ongoing/delivered pregnancy rates, and rate of implantation. Only thaws involving Day 5 or Day 6 frozen-thawed blastocysts exclusively (not mixed) were included in this study. Materials/Methods: Human preembryos were cultured in C1/C2 sequential media under standard laboratory conditions. Transfers were performed on either Day 3 or D5 post-retrieval. After Day 3 transfers, remaining PE were cultured in fresh C2 media and any resulting good quality BL were cryopreserved on Day 5. Those BL that were of insufficient freezing quality on Day 5 or that lagged in development were frozen on Day 6 if appropriate. After fresh Day 5 transfers, any leftover good quality BL were frozen the same day. BL or PE of subtoptimal quality on Day 5 were further cultured to Day 6 and frozen if of sufficient quality. Freezing criteria included BL expansion with a well defined inner cell mass and trophectoderm. BL were frozen using a glycerol/sucrose protocol and a programmable rate freezer and stored in liquid nitrogen until thawed. BL thawing involved an initial warming at 30°C followed by a stepwise removal of both glycerol and sucrose. Day 5 frozen BL were thawed the day before transfer, while Day 6 BL were thawed the morning of the transfer. Frozen-thawed BL were replaced in either a natural or programmed cycle. Data were analyzed using the Fisher’s Exact Test or a 2-sample T-test where appropriate. Results: A total of 58 patients were included in this study. Thirty patients received a transfer from Day 5 frozen-thawed BL and Day 6 BL were transferred in 28 patients. There were no significant group differences in patient age, average number of blastocysts transferred, or grade of the best BL after thawing. No significant differences were found in the post-thaw survival rate of BL cryopreserved on Day 5 (74.2%) or Day 6 (79.4%). In addition, the clinical and ongoing/delivered pregnancy rates were not different between Day 5 (60 and 50%) and Day 6 (64 and 61%) patients. No significant differences in implantation rate (sacs/BL transferred) were observed between the two groups (30.4% Day 5; 42.3% Day 6). Conclusions: No differences were found in the rate of survival, pregnancy or implantation when human BL were frozen on either Day 5 or Day 6. While it seems logical to assume that those PE reaching the BL stage faster (Day 5) would be “healthier” than their Day 6 counterparts, these data suggest that rate of development to the BL stage is not crucial to subsequent post-thaw success. Supported by: The Center for Reproductive Medicine and Infertility, Weill Medical College of Cornell University.