Developmental rate and ultrastructure of vitrified human pronuclear oocytes after step-wise versus direct rehydration

BACKGROUND: This study compared the viability of human pronuclear oocytes subjected to vitrification followed by post-thaw step-wise removal of cryoprotectants versus direct rehydration, in terms of their subsequent in vitro survival and ultrastructural features. METHODS: A total of 115 three-pronuclei stage oocytes were cryopreserved in super-open-pulled straws by vitrification in 40% ethylene glycol + 0.75 mol/l sucrose for either 1 min or 10 s at 38 degrees C, followed by direct plunging into liquid nitrogen. After thawing, oocytes vitrified for 1 min (group 1) or 10 s (group 2) were expelled into a graded series of sucrose solutions (1.0, 0.75, 0.5, 0.25 and 0.12 mol/l) for removal of the cryoprotectant in five 2.5 min steps. A second batch of oocytes vitrified for either 1 min (group 3) or 10 s (group 4) were directly expelled into culture medium at 38 degrees C after thawing. Finally, the ultrastructural changes occurring in oocytes in each of the treatment groups were evaluated. RESULTS: Oocyte development (division to two-blastomere stage) rates after in vitro culture were 82, 83, 0 and 0% for groups 1, 2, 3 and 4, respectively. The harsh osmotic process involved in direct rehydration provoked ultrastructural changes, including the disruption of cytoplasmic and pronuclear membranes as well as intracellular organelles. CONCLUSION: The direct post-thaw rehydration of human pronuclear oocytes has lethal osmotic effects, such that protocols for vitrifying human pronuclear oocytes should include the step-wise removal of the cryoprotectant.