Cryopreservation of immature mouse oocytes

Mouse oocytes enclosed in cumulus cells were isolated from antral follicles at the germinal vesicle (GV) stage. They were stored in straws at -196 degrees C by a conventional mouse embryo freezing method using dimethylsulphoxide (1.5 M) as the cryoprotectant. Overall survival assessed after removal of the cumulus cells was 93% (299/320). A significantly greater proportion of fresh oocytes remained arrested at the GV stage during culture (11 versus 1%), but the rate of maturation to metaphase II was not significantly different between frozen and fresh oocytes (83 versus 74%). The rate of fertilization in vitro was similar for frozen and fresh oocytes matured in vitro (70 versus 81%) but significantly less than with mature ovulated oocytes (96%). Fertilization of frozen and fresh oocytes arrested after germinal vesicle breakdown was similar (77 versus 95%). No evidence of parthenogenetic activation was found in the different groups after overnight incubation of metaphase II oocytes. Implantation was similar for embryos derived from fresh and frozen GV-stage oocytes matured in vitro and mature ovulated oocytes, but the loss of embryos after implantation was significantly higher in the in-vitro matured groups (frozen, 40% and fresh, 46% versus 24%). The overall survival of oocytes frozen at the GV stage was 27%. This compares favourably with the estimated overall survival of mature oocytes cryopreserved by a similar procedure. We conclude that the increased post-implantation loss is due to suboptimal conditions for maturation in vitro rather than freezing injury.