Slow Freezing Versus Vitrification of Mouse Ovaries: from Ex Vivo Analyses to Successful Pregnancies after Auto-Transplantation

Slow freezing (SF) is the reference method for ovarian tissue cryopreservation. Vitrification (VT) constitutes an alternative but controversial method. This study compares SF and VT (open [VTo] and closed [VTc] systems) in terms of freezing damage and fertility restoration ability. In vitro analyses of C57Bl/6 SF or VTo-ovaries, immediately after thawing/warming or after culture (cult), revealed that event though follicular density was similar between all groups, nuclear density was decreased in VTo-ovaries compared to CT-ovaries (CT = 0.50 ± 0.012, SF = 0.41 ± 0.03 and VTo = 0.29 ± 0.044, p < 0.01). Apoptosis was higher in VTo-cult ovaries compared to SF-cult ovaries (p < 0.001) whereas follicular Bmp15 and Amh gene expression levels were decreased in the ovaries after culture, mostly after VTo (p < 0.001). Natural mating after auto-transplantation of SF, VTo and VTc-ovaries revealed that most mice recovered their oestrous cycle. Fertility was only restored with SF and VTo ovaries (SF: 68%; VTo: 63%; VTc: 0%; p < 0.001). Mice auto-transplanted with SF and VTo-ovaries achieved the highest number of pregnancies. In conclusion, in vitro, no differences between SF and VTo were evident immediately after thawing/warming but VTo ovaries displayed alterations in apoptosis and follicular specific proteins after culture. In vivo, SF and VTo ovary auto-transplantation fully restored fertility whereas with VTc-ovary auto-transplantation no pregnancies were achieved.