Problems in the cryopreservation of unfertilized eggs by slow cooling in dimethyl sulfoxide

The survival, fertilization, development, and viability in vitro and in vivo of unfertilized mouse eggs frozen by slow cooling to -36 degrees C or -80 degrees C in 1.5 M dimethyl sulphoxide (DMSO) was examined in a series of experiments which explored some of the problems in freezing the egg. DMSO was added to the eggs at either room temperature or at 0 degrees C. Maximum success rate (42% of frozen eggs developing to two cells) was obtained when DMSO was added at 0 degrees C and the eggs slow cooled to -80 degrees C. Removal of cumulus failed to improve freezing success rates. Addition of DMSO at temperatures above 0 degrees C significantly reduced the fertilizing capacity of eggs. Excessive exposure of eggs to temperatures around 15 degrees C also caused a significant reduction in fertilization rates. The effects of DMSO and cooling on fertilization are likely to be due to zona hardening by cortical granule release and to disorganization of the egg cytoskeleton and plasma membrane. These problems will be difficult to overcome if cryopreservation of the unfertilized human egg is preferred to the fertilized egg or early cleavage stage embryo in clinical in vitro fertilization.