Coculture of monkey ovarian tissue increases survival after vitrification and slow-rate freezing

Objective: To assess whether coculture of monkey ovarian tissue after low-temperature storage enhances follicular viability. To assess a novel method of vitrifying ovarian tissue. Design: Prospective in vitro study. Setting: University-affiliated national research center. Animal(s): Ovaries from 15 cynomolgus or rhesus macaques (1-11 years). Intervention(s): Vitrification using a containerless liquid nitrogen emersion system that involves dropping thin cortical pieces suspended in cyroprotectant directly into liquid nitrogen with outcome compared with slow-rate-controlled freezing. Before analysis, some of the thawed tissue was cocultured on mitotically inactivated mouse fetal fibroblast monolayers supplemented with FSH, insulin, transferrin, and selenium. Main Outcome Measure(s): Percentage of oocytes viable using live-dead fluorescent staining with carboxyfluorescein diacetate succinimidyl ester and propidium iodide. Result(s): Postthaw survival rates were 70.4% ± 4.8% of 1,705 follicles after vitrification and 67.3% ± 1.9% of 1,895 follicles after slow-rate freeze in six trials with each method. Coculture of the thawed tissue increased the viability, respectively, to 89% ± 2.1% of 2,833 follicles previously vitrified and to 90.3% ± 1.9% of 2,109 follicles after a slow-rate freeze (P<.01). Primordial follicles (30- to 50-μm diameter) were the vast majority of surviving follicles after thaw and coculture. Follicular viability in control fresh tissue (eight trials) was 76.0% ± 4.1%, suggesting negligible loss in follicular viability after cryopreservation. Conclusion(s): Coculture of thawed ovarian tissue on mouse fetal fibroblasts and FSH increases the percentage of viable follicles. A novel method of vitrifying ovarian tissue is as effective as slow-rate freezing. These approaches may improve graft survival and function when used to treat chemotherapy-induced sterility.