Comparing the viability and in vitro maturation of cumulus germinal vesicle break down (GVBD) oocyte complexes using two vitrification techniques in mice

Background: Vitrification is assumed to be a promising method to cryopreserve human oocytes but still needs optimizing. Objective: The aim of this study was to improve the single step and step-wise vitrification effects on maturing mouse GVBD oocytes, by ethylene glycol (EG) in conventional straws. Materials and Methods: Oocytes with compact cumulus cells were cultured for 3hr in TCM199 supplemented with 10% fetal bovine serum (FBS) in 5% CO2 in air. GVBD oocytes were randomly allocated into three groups. (1) Control (non-vitrified group), (2) exposed to single-step vitrification (contained of EG 20%+0.5M sucrose), (3) exposed to step-wise vitrification (2%, 5%, 10%, 20%EG +0.5M sucrose). In vitrification groups,oocytes were thawed and underwent additional 21 hr maturation. Viability of oocytes and maturation to MII stage were analyzed using inverted microscope and additionally by staining of propidium iodide and Hoechst 33342. Results: All non-vitrified oocytes were viable after 24 hr; however, viability of vitrified samples in single-step group was significantly lower than that of the step-wise and control Groups. Also, the maturation rate in the step-wise group was significantly higher (p < 0.05) compared to single-step. Conclusion: These results suggest that step-wise vitrification of GVBD oocytes as compared to single step vitrification was better in the rate of survival and in vitro maturation of oocytes.