Embryo thawing
The cleavage-stage embryo was placed on the tip of the Cryotop (Kitazato
Corporation, Tokyo, Japan). All cryotop were separated from the liquid nitrogen
48 hours before the planed transfer and were quickly immersed into the thawing
solution and left there for 1 minute at room temperature then the embryo was
aspirated using a pipette and gently placed on the bottom of the dilution
solution for 3 minutes and at the end, it was placed in washing solution for 5
minutes in 2 step. The 5-day-old embryo was cultured in Blastocyst-media
(Origio, Denmark). Blastocyst embryos were assessed according to Gardner’s
criteria based on inner cell mass, blastocyst expansion, and trophectoderm
development. One or two embryos of top quality under Phillips transabdominal
ultrasound (affinity 70) were transferred by Cook catheter (Cook, Medical, and
Eight Mile Plains, Queensland, Australia). When there was no blastocyst on day
5, the most advanced embryo was transferred.