Fracture damage of embryos and its prevention during vitrification and warming

The frequency of fracture damage in mouse blastocysts was examined by repeated cycles of vitrification and warming. Mouse blastocysts suspended in a solution of ethylene glycol, Ficoll, and sucrose in a straw were plunged into liquid nitrogen either directly (rapidly) or after holding them in liquid nitrogen vapor for 3 min or more (moderately). Vitrified samples were warmed by plunging them into 25 degrees C water either immediately (rapidly) or after holding in air for 5-30 s (moderately). Warmed straws were recooled and rewarmed up to 9 times, to exaggerate the effect of cooling and warming. When embryos were cooled and warmed rapidly once, the incidence of the zona damage was only 1.2%, and 91% of recovered embryos reexpanded in culture. However, with repeated rapid cooling and warming, the incidence of zona damage increased, reaching 75% after 10 vitrifications; survival also dropped. When embryos were subjected to 10 cycles of moderate cooling and moderate warming with 15 or 30 s of suspension in air, 100% of the embryos had intact zonae. On the other hand, with moderate cooling followed by rapid warming or with rapid cooling followed by moderate warming, 41 and 16% of embryos had damaged zona, respectively, after 10 vitrifications. Therefore, fracture damage occurs during both cooling and warming, but it can be prevented completely by employing somewhat slower rates of cooling and warming. Furthermore, a high survival rate (88%) after 10 cycles of moderate cooling and moderate warming with 15 s of suspension in air indicates that vitrification, melting, or temperature fluctuation per se do not affect embryonic survival.