Ultrastructure and intracellular calcium response during activation in vitrified and slow-frozen human oocytes

BACKGROUND: The sensitivity of human oocytes to cryodamage may compromise their developmental competence following cryopreservation. Herein, we compared the ultrastructure and the response to the calcium (Ca2+) ionophore A23187 of fresh, slow-frozen and vitrified metaphase II (MII) human oocytes. METHODS: Supernumerary fresh MII oocytes, donated under written informed consent, were cryopreserved through either a slow cooling procedure based on propane-1,2-diol and 0.3 M sucrose or a closed vitrification system based on dimethylsulphoxide (DMSO) and ethylene glycol (EG). Ultrastructure of fresh and cryopreserved oocytes was assessed by transmission electron microscopy and compared through morphometrical analysis; intracellular calcium ([Ca2+](i)) dynamics was studied by evaluating the response to the Ca2+ ionophore A23187. RESULTS: Morphometric analysis demonstrated a markedly higher proportion of oocytes with large vacuoles, inward displacement of organelles from the pericortical toward the deep cytoplasm, and mitochondrial damage in slow-frozen compared with both fresh and vitrified oocytes. A23187 increased the [Ca2+](i) in all oocyte groups and the peak average increase in slow-frozen oocytes was significantly higher than in both fresh and vitrified oocytes. Moreover, the ability of slow-frozen oocytes to recover [Ca2+](i) to basal levels was significantly reduced compared with both fresh and vitrified oocytes. CONCLUSIONS: Closed vitrification based on DMSO and EG preserves the ultrastructural features and the ability to respond to the Ca2+ ionophore A23187 significantly better than does slow freezing with 0.3 M sucrose. Damage to organelles involved in the [Ca2+](i) modulation might reduce the developmental competence of cryopreserved oocytes.