Comparing the effects of a commercial and a prototype vitrification medium on meiotic spindle morphology and survival rate of mouse oocytes

To compare oocyte survival and meiotic spindle normality between vitrified-warmed oocytes in a mouse embryo assay using Tvitri-4 or Ingámed vitrification media. C57BL/6 female mice aged 8-12 weeks were submitted to superovulation with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG) for obtaining of in vivo matured oocytes. The oocytes were randomly distributed into one of the following three groups: CTR - control (fresh oocytes); ING - oocytes vitrified-warmed in a standard commercial kit supplied by Ingámed, and T4 - oocytes vitrified-warmed in the novel prototype Tvitri-4 medium. After warming and recovery culture, oocytes were assessed with respect to survival rate (SR) and both meiotic spindle morphology and chromosome alignment of each oocyte fixed in the sagittal position after immunostaining and analysis by confocal microscopy. A total of 354 mature oocytes were vitrified in ING (n=178) and T4 (n=176), out of which 299 (85%) survived after warming. Oocyte survival rates were not statistically different (p=0.08) between ING (145/178=81.5%) and T4 (154/176=87.5%). Regarding meiotic normality, there were no significant changes in the proportion of oocytes with normal meiotic spindle morphology and chromosome structure between ING (52,2%) and T4 (63.4%) after warming (RR: 0.95, 95% CI: 0.92-1.607). When the meiotic normality was assessed using the CTR group as a reference in the analysis of relative risk, no significant differences were observed between T4 (63.4%) and CTR (70.5%) (RR: 0.95, 95% CI: 0.72-1.12). On the other hand, the percentage of oocytes retaining normal meiotic spindle morphology and chromosome configuration in ING (52.2%) was lower than in the CTR group (RR: 0.95, 95% CI: 0.57-0.97). The novel prototype Tvitri-4 medium was efficient in preserve survival rate and meiotic spindle normality of oocytes and, with further verification, may be able to replace commercially available media in future clinical applications.