In vitro viability of sheep whole ovaries and cortical fragments after cryopreservation with different techniques

Cryopreservation of ovarian tissue is a promising technique for preserving fertility in young female cancer patients. At present two options are available: cryopreservation of ovarian cortical fragments or of the whole ovary. We compared ovarian tissue viability, cryopreserved as fragments or whole organs using a conventional (CF) or directional freezing apparatus (DF). Cortical fragments (10x5x1 mm) were immersed into Leibovitz L-15 medium, 10% FCS and 1,5 M DMSO, while whole ovaries were perfused with the same solution. CF was performed at 0.5°C/min in a Kryo 560M (Planer, UK). DF was performed at 0.01 mm/sec, resulting in cooling rates of 0.3°C/min with a Multi-Thermal-Gradient (IMT, Israel). In both cases freezing was arrested at -40°C, before plunging the samples into liquid nitrogen. After thawing, whole ovaries and cortical fragments were cut in 2x2x1 mm pieces and cultured for 7 days in α-MEM medium supplemented with ITS, glutamine, pyruvate, hypoxantine, BSA, FSH and bFGF. After culture, the percentage of primordial follicles developed to the primary stage in DF whole ovaries, was comparable to controls. A lower rate (P<0.05) of growing follicles was observed in all other groups, and CF whole ovaries were unable to support any development. DNA double-strand breaks formation and repair was analyzed by immunofluorescent expression of γ-H2AX and of RAD51. At the beginning of the culture, DNA damage in frozen samples was higher than in fresh controls. However after 7 days, DNA damage significantly decrease in DF groups and in CF cortex samples concomitantly with an increase of DNA repair. Conversely, in CF whole ovaries DNA repair signal was absent leaving the rate of DNA damage substantially unchanged. We conclude that DF allows a better preservation than CF of whole ovaries and cortical fragments and eliminates any disadvantage related to the bigger volume of whole organs.