Scanning electron microscopic analysis of the zona pellucida in mouse blastcysts cryopreserved by vitrification

Advances in embryo freeze-thawing techniques can be cited as one factor in the development and widespread use of assisted reproductive medicine. It has been reported, however, that freezing causes hardening of the zona pellucida in embryos. Cryopreservation was initially carried out by slow freezing, but recently, vitrification has become a widely used, well-established method. In this study, we used vitrification to freeze-thaw mouse blastcysts obtained by in vitro fertilization, and compared fresh blastcysts with blastcysts directly after freeze-thawing and after 2, 4, 6, 12, and 24 h of recovery culture using scanning electron microscopy (SEM). The reticulate structure seen in fresh blastcysts was observed to be in a closed state after freeze-thawing, suggesting hardening of the zona pellucida. During recovery culture, the structure of the zona pellucida gradually approached that of fresh blastcysts, but was not fully restored even after extending the recovery culture period. These findings suggest that freezing affects the structure of the zona pellucida, which could not be fully restored despite some degree of recovery after recovery culture.