Cryovial monolayer vitrification for ovarian tissue cryopreservation

The cryopreservation of ovarian tissue has been proved to be effective for fertility preservation. To find a better cryopreservation method, we tested the efficacy of cryovial monolayer vitrification method in comparison with that of needle immersed vitrification (NIV). Ovaries from 10 female kunming mice aged 6-8 weeks were cut into pieces and allocated into group A (cryovial monolayer vitrification method), group B (NIV method) and group C (fresh control). In group A, pieces of ovarian tissue were layered around the inner wall of cryovial as a monolayer; and in group B, pieces of ovarian tissue were pierced with a needle. Other than the difference in the carrier for ovarian tissue, the cryoprotectants and the protocols in group A and B were the same. The viability and in vitro growth potential of the follicles after warming in groups A and B were evaluated respectively and compared with those of fresh control. The results showed that the viabilities of the follicles were not statistically different among three groups. The average diameter of follicles did not show statistically significant difference among three groups before culture and between group A and group B after culture (p > 0.05), but demonstrated statistically significant difference between group A and group C (p < 0.01), group B and group C (p < 0.01), respectively. In the procedure of loading ovarian tissue onto carriers, group A took less time compared with group B. In addition, the small pieces and debris which were harder or impossible to be pierced with needle in group B could be easily layered onto the inner wall of the cryovial in group A. Hence the follicles existed within the small pieces and debris of the ovarian tissue could also be cryopreserved. It is concluded that, in cryopreserving both viability and growth potential of follicles, the cryovial monolayer vitrification method is comparable with NIV method but with higher efficiency.