Embryo culture, freezing and thawing protocols
Embryo culture was carried out in Quinn’s IVF sequential medium suite (Quinn’s, SAGE, USA) after adding 10% human serum substitute (Quinn’s, SAGE, USA) in 5% O2, 5% CO2, 90% N2 and saturated humidity. Embryos were cultured individually in 25 μl microdroplet of cleavage medium covered with oil and then transferred from the cleavage medium to a blastocyst medium covered with oil on day 3. About 3 embryos were cultured in each 30 μl microdroplet of blastocyst medium. Blastocysts were scored according to the criteria proposed by Gardner on the morning of day 5 or 6.8Good-quality blastocysts were given a numerical score from 3 to 6 according to their degree of expansion and hatching status, and the inner cell mass and trophectoderm were assessed as A or B, while other types of blastocysts were poor-quality.
Available blastocysts were vitrified using vitrification media (Kitazato Biopharma, Shizuoka, Japan). Briefly, blastocysts were transferred from the culture medium to the center of the equilibration solution and remained in the media for about 9 minutes at room temperature. Then, blastocysts were transferred 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.
Blastocysts were thawed using vitrification media (Kitazato Biopharma, Shizuoka, Japan). Briefly, Cryotop was transferred rapidly from liquid nitrogen and immersed in a 300 μl droplet of thawing solution (TS) at 37℃. After 1 minute, the blastocysts were transferred to a 300 μl droplet of diluent solution (DS) at room temperature for 3 minutes. Then, the blastocysts were transferred to 300 μl droplets of washing solution 1 (WS1) and washing solution 2 (WS2) at room temperature for 5 minutes, respectively. Finally, the blastocysts were transferred to a 30 μl droplet of blastocyst medium and regarded before transfer.