Embryo Slow Freezing Protocol (Zygote/Cleavage/Blastocyst)

Until the advent of vitrification, slow freezing has played the most prominent role in human embryo cryopreservation and is still nowadays employed by some IVF centers. The aim of this chapter is to provide a detailed protocol for slow freezing of zygotes, cleavage stage embryos, and blastocysts. The preparatory steps of the procedure include the preparation of freezing solutions, the record of cryopreservation data, the labelling of straws and canes, and the setting of the freezing machine. The following steps involve the equilibration of embryos in the freezing solution, the loading of embryos into straws, and the slow cooling by using a freezing machine. Finally, the embryos are plunged in the liquid nitrogen at −196 °C and transferred to dewar tanks. Manual seeding is a crucial step of the protocol as it initiates ice formation in a controlled temperature. Two different protocols are described, the zygote and cleavage stage protocol with PROH and sucrose as cryoprotectants and the blastocyst protocol with glycerol and sucrose.