Single Step Protocol and Multi-step Warming Protocol for Blastocyst FET

Potentially eligible patients' vitrified blastocysts will be thawed by a single-step thawing protocol. For the warming phase, vitrified blastocysts are exposed to the thawing solution of a commercial embryo thawing kit (Irvine Scientific Inc., USA) at 37°C for one minute. Immediately following this, embryos will be rinsed in a 35mm diameter dish of 2ml of pre-equilibrated thawing solution before being placed in culture media in the incubator for at least 2 hours before transfer.

For the MS protocol, thawing kits were equilibrated overnight in a 37°C incubator. Warming procedures utilized the kits (Cryotech RtU, Japan). To remove the cryoprotectants, blastocysts were warmed, and cryoprotectants were diluted in a three-step process. The warming process starts with the exposure of blastocysts to thaw solution (TS) with 1M trehalose for one minute at 37°C. Subsequently, the blastocyst will be transferred to a second well containing a dilution solution (DS) of 0.5M trehalose for a two-minute rinse at room temperature. This is followed by two additional three-minute and 30-second rinses in the wash solution (WS) at room temperature. The timeline for standard warming of blastocysts requires a total of 6.5 min. After thawing, embryo will be placed in the incubator at least 2 hours before transfer.