Clinical outcome after transfer of human blastocysts (BLs) vitrified on day 5 and on day 6 with a cryoloop technique

Abstract

Objective: To optimize clinical outcomes of BL transfer, it is essential to establish a simple, fast and reliable procedure for the cryopreservation of BLs. We have reported the effectiveness of ultra-rapid vitrification using a cryoloop technique for human BLs (1). However, the timing of vitrification of BLs might affect the outcome. This study examined the post-warming survival and clinical outcome of human BLs vitrified on day 5 and on day 6. Design: Retrospective study on the vitrification human BLs. Materials/Methods: A total of 129 patients who had supernumerary BLs between Oct 1999 and Nov 2001 entered this study. A sequential culture system was used to obtain BLs. When embryos reached the BL stage 5 or 6 days after oocyte retrieval and patients agreed to cryopreserve the embryos by ultra-rapid vitrification, supernumerary BLs were cryopreserved using a cryoloop technique (1). Blastocysts were mounted on a minute nylon loop, which creates a thin, filmy layer of the solution by surface tension, and vitrified with the use of a two-step loading process with cryoprotectants at 37C. Initially, BLs were placed in cryoprotectant solution I, which is the base medium (Hepes-buffered modified HTF) containing 7.5% DMSO and 7.5% ethylene glycol (EG), for 2 min before being suspended in cryoprotectant solution II, which is the base medium containing 15% DMSO, 15% EG, 1% Ficoll 70 and 0.65 M sucrose, for 30 sec. Blastocysts were warmed and diluted in two steps with 0.33 M sucrose for 2 min and 0.2 M sucrose for 3 min. Blastocysts with morphologically intact inner cell mass and trophectoderm and a re-expanding blastocoel were judged to have survived and were transferred to the patient 5 days after progesterone administration, regardless of the timing of vitrification. Results: Of 301 day-5 BLs collected from 92 cycles, 274 (91%) survived after warming. Of 143 day-6 BLs collected from 45 cycles, however, only 77 (54%) survived (p <0.01). Pregnancy rates with day-5 and day-6 BLs were 39% (35/92) and 28% (10/45), respectively, and implantation rates with day-5 and day-6 BLs were 21% and 17%, respectively, both rates being not significantly different (p >0.05). Conclusions: Blastocysts on day 5 are more resistant to vitrification than those on day 6. Fast-developing embryos might be more viable than slow-developing ones. In addition, since more day-6 BLs had a larger blastocoel, BLs having a smaller blastocoelic cavity may be suitable for vitrification, probably with less risk of intracellular ice formation. However, once BLs survived vitrification, they seem to have similar ability to develop after transfer. Reference: (1) Mukaida T, Nakamura S, Tomiyama T, Wada S, Kasai M, Takahashi K (2001) Successful birth after transfer of vitrified human blastocysts using a cryoloop container-less technique. Fertil Steril 76: 618–620. Supported by: none.