The effects of vitrification on spindle apparatus of in vitro matured germinal vesicle in mice

Background: Routine oocytes cryopreservation remained an elusive technique in the wide ranges of available assisted reproductive technologies. The microtubules of oocytes are vulnerable to cryoprotectants and thermal change during cryopreservation. Objective: The effects of a vitrification protocol were investigated on the spindle and chromosome configurations of mice oocytes cryopreserved at the germinal vesicle stage. Materials and Methods: Germinal vesicle with cumulus cells were transferred to vitrification solution which was composed of 30% (v/v) ethylene glycol, 18% Ficoll-70 and 0.3 M Sucrose either by single step or in step-wise way. Following vitrification and in vitro maturation (MII), the matured oocytes were immonostained for meiotic spindles and chromosomes, before visualization using fluorescent microscopy. Results: A statistically significant increase was observed in the survival and maturation rate in step-wise vitrification (88.96% and 71.23% respectively) compared to single step vitrification (70.6% and 62.42% respectively) (p < 0.05). Normal spindle morphology after vitrification-thawing in step-wise vitrification group (77.26%) was higher than single step vitrification group (64.24%) but lower than control group (94.75%) (p < 0.05). Conclusion: The results suggest that vitrification with step-wise procedure on mice germinal vesicle oocytes has positive effects on survival and maturation rate and normal spindle configuration compare with single step vitrification procedure.