Stepwise Vitrification and Warming Protocol (Standard Operation Procedure) of Embryo

Vitrification of blastocysts needs to be undertaken utilizing a “closed system” (HSV: High Security Vitrification Kit; CryoBioSystem, L’Aigle, France; FDA 510(k) clearance for cleavage-stage embryos in blastocysts) after a two-step loading with cryoprotectant agents at 24 °C. If assisted collapsing is done prior to vitrification, then the blastocyst needs to be put on an inverted microscope equipped with a laser system (ZILOS-tk, or Lykos, Hamilton Thorne); the junction of two trophectoderm cells in each blastocyst needs to be located, and one shot (100% power, 500 μs pulse length) was applied. Then the blastocysts can be moved back in the incubators for 5–10 min. Briefly, blastocysts must be placed in equilibration solution, which is the base medium [M199 with 20% serum supplement substitution (SSS) containing 7.5% (v/v) ethylene glycol (EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO)]. After 8 min, the blastocysts need to be washed quickly in vitrification solution, which is the base medium containing 15% (v/v) DMSO, 15% (v/v) EG, and 0.5 M sucrose, for 60 s and transferred onto the HSV using a micropipette. Immediately after the loading of one or not more than two blastocysts in a 1 μL drop on the HSV, the straws can be heat sealed, then plunged in LN2, and secondarily stored inside 5 mL liquid nitrogen-prefilled canes (Visotube Rond, IMV, France). Each single step is described in detail below. The timeline for vitrifying blastocysts requires a total of 9 min (Fig. 56.1):