Restoration of ovarian function and natural fertility following the cryopreservation and autotransplantation of whole adult sheep ovaries

Is 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)? Full (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. Fertility 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 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(A (R)); n = 8) or eptifibatide (Integrilin(A (R)); n = 8) on ovarian function and fertility 11-23 months after WOCP&TP. Both 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-Mullerian 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. In 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 of CPA perfusion was highly significant with a 60 min perfusion resulting in ovaries of normal appearance and function with high rates of primordial follicle survival (70%) and an abundant blood supply, whereas ovaries perfused for 10 min had either resorbed completely and were vestigial (7/14) or were markedly smaller (P < 0.01). It is concluded that both the degree of CPA penetration and the maintenance of post-operative vascular patency are critical determinants of the success of WOCP&TP. Before application of this technology to fertility preservation patients, it will be critical to optimize the CPA perfusion time for different sized human ovaries, determine the optimum period and level of anti-coagulant therapy, and confirm the normality of offspring derived from this procedure. This technology holds promise for the preservation of fertility in women. It could also potentially be applied to the cryopreservation of other reproductive or even major organs (kidneys) where there are considerable difficulties in storing donated tissue. Funding was received from the Medical Research Council, University of Nottingham. The authors confirm that they have no conflict of interest in relation to this work.