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Objective To determine the extent of biochemical changes in the zona pellucida (ZP) and cortical granule release in eggs failing to fertilize in IVF. Design After insemination, unactivated eggs without two pronuclei (PN) were studied by high resolution microscopy and fluorescent probes to determine cortical granule density and meiotic stage. After ZP isolation, proteins from individual ZPs were biotinylated, electrophoresed, and visualized by western blots with avidin-chemiluminescence. Controls included mouse unfertilized and fertilized eggs and human germinal vesicle stage oocytes and ≥ 3PN eggs. Setting University medical center and hospital tertiary care IVF-ET program. Results Many of the unfertilized eggs were in metaphase and had relatively low cortical granule densities indicative of cortical granule loss. Approximately one half of the ZPs showed evidence of biochemical hardening with a modification of a 90 to 100 × 103 molecular (weight) ratio (Mr) ZP protein. The 3PN eggs had few cortical granules and their ZPs had a pronounced 90 to 100 × 103 Mr modification that was not detected in germinal vesicle stage ZP controls. Conclusion The observed changes in ZP biochemistry and cortical granule quantitation demonstrate that failed fertilization is frequently associated with spontaneous cytoplasmic, but not nuclear, activation. In affected eggs, these changes could prevent fertilization in routine IVF and in cases of reinsemination. The relationship of changes in the ZP and cortical granules to infertility and in vitro culture requires further investigation. The 90 to 100 × 103 Mr ZP protein modification can be detected biochemically with 1 10 of a human ZP. |
