STUDY QUESTIONIs it possible to restore ovarian function and natural fertility following the cryopreservation and autotransplantation of whole ovaries, complete with vascular pedicle, in adult females from a large monovulatory animal model species (i.e. sheep)?SUMMARY ANSWERFull (100%) restoration of acute ovarian function and high rates of natural fertility (pregnancy rate 64%; live birth rate 29%), with multiple live births, were obtained following whole ovary cryopreservation and autotransplantation (WOCP&TP) of adult sheep ovaries utilizing optimized cryopreservation and post-operative anti-coagulant regimes.WHAT IS KNOWN ALREADYFertility preservation by WOCP&TP requires successful cryopreservation of both the ovary and its vascular supply. Previous work has indicated detrimental effects of WOCP&TP on the ovarian follicle population. Recent experiments suggest that these deleterious effects can be attributed to an acute loss of vascular patency due to clot formation induced by damage to ovarian arterial endothelial cells.STUDY DESIGN, SIZE, DURATIONStudy 1 (2010–2011; N = 16) examined the effect of post-thaw perfusion of survival factors (angiogenic, antioxidant, anti-apoptotic; n = 7–8) and treatment with aspirin (pre-operative versus pre- and post-operative (n = 7–9)) on the restoration of ovarian function for 3 months after WOCP&TP. Study 2 (2011–2012; N = 16) examined the effect of cryoprotectant (CPA) perfusion time (10 versus 60 min; n = 16) and pre- and post-operative treatment with aspirin in combination with enoxaparine (Clexane®; n = 8) or eptifibatide (Integrilin®; n = 8) on ovarian function and fertility 11–23 months after WOCP&TP.PARTICIPANTS/MATERIALS, SETTING, METHODSBoth studies utilized mature, parous, Greyface ewes aged 3–6 years and weighing 50–75 kg. Restoration of ovarian function was monitored by bi-weekly blood sampling and display of behavioural oestrus. Blood samples were assayed for gonadotrophins, progesterone, anti-Müllerian Hormone and inhibin A. Fertility restoration in Study 2 was quantified by pregnancy rate after a 3 month fertile mating period and was confirmed by ultrasound, hormonal monitoring and live birth. Ovarian function was assessed at sacrifice by ovarian appearance and vascular patency (Doppler ultrasound) and by follicular histology.MAIN RESULTS AND THE ROLE OF CHANCEIn Study 1, survival factors were found to have no benefit, but the inclusion of pre-operative aspirin resulted in four ewes showing acute restoration of ovarian function within 3 weeks and a further six ewes showing partial restoration. The addition of post-operative aspirin alone had no clear benefit. In Study 2, combination of aspirin with additional post-operative anti-coagulants resulted in total acute restoration of ovarian function in 14/14 ewes within 3 weeks of WOCP&TP, with 9/14 ewes becoming pregnant and 4/14 giving birth to a total of seven normal lambs. There was no difference between anti-coagulants in terms of restoration of reproductive function and fertility. In contrast, the duration ...

These results are encouraging for whole ovary cryopreservation, demonstrating maintained cell viability, however, they do not support S-1-P inclusion at this concentration to improve tissue viability following cryopreservation.

In conclusion, these results demonstrate successful partial restoration of ovarian function following cryopreservation of the whole ovary and vascular pedicle in a large monovulatory species. The inability to restore full ovarian function was related to loss of primordial follicles rather than vascular patency in both frozen and fresh tissue, suggesting that factors associated with cannulation and perfusion may contribute to this depletion. Further work is therefore needed to elucidate these factors before the procedure could be considered a viable option for fertility preservation.