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.