Viability and function of the cryopreserved whole ovary: in vitro studies in the sheep.

BACKGROUND: Cryopreservation of whole ovaries followed by vascular transplantation may improve long-term function in comparison to conventional cryopreservation of ovarian cortex and avascular transplantation. The aim of this study was to assess methods for the evaluation of viability and function of frozen-thawed whole ovaries.

METHODS: Ewe ovaries were flushed with either cryoprotectant (propandiol: FROZEN-PROH) or Ringer Acetate (FROZEN-RA) followed by slow freezing. Some ovaries were assessed fresh after flushing with Ringer Acetate (FRESH-RA). Assessment was done by light microscopy, biochemical response (cyclic adenosine 3',5'-monophosphate (cAMP) and steroids) during in vitro perfusion with forskolin, viability assay and cell culture.

RESULTS: Microscopy showed well-preserved morphology with the presence of small follicles in all groups before perfusion. Stromal oedema was seen after in vitro perfusion of FROZEN ovaries, and shrunken small follicles were seen only in FROZEN-RA at the end of perfusion. During in vitro perfusion, FRESH-RA ovaries responded with large increase in levels of cAMP after stimulation with forskolin. FROZEN-PROH and FROZEN-RA ovaries exhibited lower production of cAMP. Progesterone concentrations in cell cultures of dispersed ovarian cells were higher in FRESH-RA when compared with FROZEN groups. Addition of hCG to cell cultures resulted in higher progesterone levels in the FROZEN-PROH compared with FROZEN-RA. Cell viability assay showed overall viability of 60-75% with no significant difference between groups.

CONCLUSION: In vitro perfusion may prove to be a suitable method to test viability and function of frozen-thawed whole ovaries contributing to the optimization of current cryopreservation protocols.