Embryo culture, freezing and thawing protocols
Fertilization and embryo culture were carried out in Quinn’s IVF sequential medium suite (Quinn’s, SAGE, USA) with adding 10% human serum substitute (Quinn’s, SAGE, USA) in 5% O2, 5% CO2, 90% N2 and saturated humidity. Follicles were aspirated transvaginally under ultrasound guidance at 36 h post hCG injected. Cumulus-oocyte complexes and sperm were mixed at 39-40 h post hCG injected. For ICSI, the cumulus cells were removed enzymatically and the sperm was injected at about 42 h after hCG injected. The fertilization results were evaluated 16–18 h after fertilization and embryos were cultured individually in 25 μl microdroplet of cleavage medium covered with oil. Cleavage-stage embryos were scored and graded every day for blastomere number, cleavage plane and degree of fragmentation. Available embryos were vitrified using vitrification media (Kitazato Biopharma, Shizuoka, Japan) on day 3 after egg collection (D3). Briefly, embryos were transferred from the culture medium to the center of the equilibration solution surface and remained in the media for about 7 minutes at room temperature. Then, the embryos were moved into vitrification solution and blown and inhaled at different positions. After 40-50 s in this solution, the embryos were placed on the Cryotop (Kitazato Biopharma, Shizuoka, Japan) and rapidly placed into liquid nitrogen. Embryos were thawed using vitrification media (Kitazato Biopharma, Shizuoka, Japan). Briefly, Cryotop was taken rapidly from liquid nitrogen and immersed in a 300 μl droplet of thawing solution (TS) at 37℃. At the end of 1 minute, the embryos were transferred to a 300 μl droplet of diluent solution (DS) at room temperature for 3 minutes. Then, the embryos were shifted to 300 μl droplets of washing solution 1 (WS1) and washing solution 2 (WS2) at room temperature for 5 minutes, respectively. Finally, the embryos were transferred to a 30 μl droplet of blastocyst medium and cultured for 2-3 h or 18-20 h for transfer.