Thawing solution

Embryos were thawed on the day of ET. The vitrified embryo was warmed in Thawing Solution (Thawing Media; Kitazato Corp.) at 37°C for 1 min. After thawing, the embryo was placed into Diluent Solution (Thawing Media; Kitazato Corp.) for 3 min at room temperature to dilute the cryoprotectant, followed by stepwise washing in Washing Solution (Thawing Media; Kitazato Corp.) as follows: the first wash was performed for 5 min at room temperature, followed by a second wash for 1 min at room temperature. Embryos were then placed in Embryo Glue (Vitrolife AB; Västra Frölunda, Sweden) and cultured in a CO₂ incubator at 37°C under 6% CO₂ and 5% O₂ until ET. Due to undetectable E2 levels in these patients, ET was performed under HR therapy using transdermal E2 (Estrana tape^®; Hisamitsu Pharmaceutical, Tokyo, Japan) at a starting dose of 1.44 mg/48 h. The dose of transdermal E2 was increased every 48 h until serum E2 levels were >350 pg/mL and the endometrial thickness as measured by ultrasound was >12 mm. ET was then performed.