Morphology and fertilizability of frozen human oocytes

Human oocytes were frozen and thawed by four methods previously used for cryopreservation of human embryos. Most of these oocytes were inseminated after thawing to assess their capacity to fertilize and form pronuclear ova. Their morphology was assessed by phase-contrast microscopy used in routine IVF. Twenty-three oocytes were examined by electron microscopy to critically evaluate the effects of cooling and cryopreservation and to confirm fertilization. Morphological survival was observed in more than 60% of the oocytes examined after freeze-thawing. The main features of cryoinjury were cracks in the zona pellucida, disruption of the plasma membrane and extensive disorganization of the ooplasm. Subtle changes in the cytosol of cumulus cells was also observed. Cooling to 0 degrees C or -6 degrees C had little effect on cytoplasmic structure. Spindles were damaged in two frozen oocytes. Cumulus cell activity, sperm binding to the zona, sperm penetration of the zona seem to be largely unaffected by freeze-thawing. Fertilization was observed in eight oocytes after postthaw insemination and three embryos (8-cell to morula stages) were developed from pronuclear ova on further culture. Both monospermic and polyspermic fertilization were confirmed by electron microscopy and micronuclei were detected in three pronuclear ova. The genetic implications of these nuclear aberrations are discussed. These preliminary studies indicate that oocyte freezing needs to be integrated cautiously with clinical IVF by further assessment of embryos developed from frozen oocytes.