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

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.

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.

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.

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.