P-191: Optimizing a vitrification protocol for human embryo cryopreservation on day 2; assessing the quality of embryos confirmed with early cleavage and morphological grading

Abstract

The establishment of a vitrification protocol for any stage embryos with minimal invasion is an immediate need for positive clinical outcome. This study aimed to optimize a vitrification protocol for human day 2-embryo cryopreservation, confirming the quality of embryos with early cleavage and morphological grading. Retrospective clinical study. 263 patients with day 2 embryos that needed cryopreservation for either avoiding OHSS or as supernumerary embryos for subsequent transfer attempts, agreed to cryopreservation of the embryos by vitrification. Embryos were evaluated for early cleavage (EC) or late cleavage (LC) at 27 hours after insemination, and morphological scored on day 2 (poor: <=4 cells with >=50% frag., fair: 4 cells with <50% and >=20% frag., and good: >=4 cells with <20% frag.). A total of 1053 day 2 embryos were vitrified with equilibration solution, an equal mixture of 7.5% DMSO and ethylene glycol (EG) in HTF supplemented with 20% HSA and a vitrification solution, a mixture of 15% DMSO, 15% EG and 0.5 M sucrose in HTF/HSA. Initially, embryos were exposed to the equilibration solution for 5 to 10 min, and to the vitrification solution for 1 min at room temperature. The duration of the equilibration time was adjusted by morphological changes that indicated shrinkage for dehydration and re-expansion for cryoprotectant (CPA) permeation, and was individually recorded for further analysis. Embryos were then loaded onto a minute nylon sheet (cryotop), and plunged into LN2 immediately. For warming, vitrified embryos on the tip of a cryotop were dipped and kept in 1M of sucrose solution for 1 min. and then diluted in 0.5 M of sucrose solution for 3 min. Embryos with 70% or more intact blastomeres were considered as a survival and kept in culture until transfer the following day. The uterine endometrium of patients was prepared with transdermal estrogen and suppository of progesterone (200 mg/day) before embryo-transfer. Pregnancy was defined as the presence of gestational sac(s). Total survival and cleavage rates were 96.3% (1053/1094) and 69.0% (727/1053), respectively. The mean number of embryos transferred was 2.4±0.7, pregnancy rate was 26.1% (112/429), implantation rate was 12.7% (130/1024), and abortion rate was 29.5% (33/112). The cleavage (83%), pregnancy (30.1%) and implantation (15.4%) rates in the EC group were significantly higher than those in the LC group. Also, the equilibration time of vitrified embryos with poor morphological scores was statistically longer than that of vitrified embryos with poor and fair scores. Embryos in both the good score and EC groups statistically had the highest pregnancy rate (33.6%) and implantation rate (17.7%). Fifty-five healthy babies were born from 47 deliveries. We concluded that morphological score combined with EC/LC confirmation was significantly related to survival and clinical outcome of day 2-embryo vitrification. Vitrified embryos with poor morphological score had impaired dehydration and permeation ability because they required longer equilibration of CPA and had poor developmental ability. It is very important to adjust the equilibration time and assess the indications whether embryos are suitable for vitrification or not according to EC/LC and morphological scores. An acceptable pregnancy rate and healthy babies confirmed the usefulness and safety of this vitrification protocol.