4 Discussion

Ovarian tissue transplantation can be handled as autologous transplantation, allogeneic transplantation or xenotransplantation. Autologous ovarian transplantation is the most commonly used clinically because it rarely leads to immune rejection or raises ethical concerns. After ovarian tissue transplantation, the ovarian tissue should undergo ischemia-reperfusion. The ischemia during the formation of new blood vessels can last for 3–7 days. This process will result in the ischemic injury of ovarian tissue and lead to the loss of most of the original follicles and almost all of the growth follicles.⁶ Therefore, the rapid generation of neovascularization is particularly important, and it is very important to select graft sites containing a rich blood supply.

The graft sites of autologous ovarian tissue transplantation may be orthotopic or heterotopic. Orthotopic transplantation mainly involves implanting thawed frozen ovarian cortical tissues into ovary remnants or their surrounding tissues through abdominal or laparoscopic surgery with nonvascular anastomosis.⁷ After orthotopic transplantation, natural conception may be achieved. Donnez et al. reported that >130 infants were successfully born after freezing and transplantation of ovarian tissue, most of which resulted from orthotopic transplantation. More than 95% of the cases reported after orthotopic transplantation restored the ovarian endocrine function, and more than 40% of the patients regained their fertility.⁸ The parts of the body with rich blood supply, such as the abdominal subcutaneous tissue, forearm, breast tissue, the renal capsule, peritoneum and omentum are often chosen in the case of heterotopic transplantations, which are more affected by the local environment. These factors include temperature, pressure, paracrine factor and blood supply factors. Despite the need to develop in vitro assisted reproduction technologies in order to achieve pregnancy, the relatively simple operation procedure and graft monitoring make this a popular technique. Successful reproduction resulting from the heterotopic transplantation of ovarian tissue has also been reported. In 2013, Stern et al. reported, for the first time, a patient with granulosa cell tumor that successfully gave birth to a pair of twins after bilateral oophorectomy by autologous subcutaneous heterotopic transplantation of cryopreserved ovarian tissue in the abdomen.⁹

At present, ovarian tissue freezing and transplantation essentially remain in the experimental stage, and a recognized standardized protocol is currently lacking. Hence, it is necessary to conduct animal experiments before clinical trials to explore and master feasible technical strategies. Dath et al.¹⁰ compared the short-term (3 weeks) effects of human ovarian tissue transplantation in the peritoneum, ovary, subcutaneous tissue and muscle of mice. The authors found that, of these four graft sites, the morphology and structure of follicles in the ovarian tissue implant at the muscle were more complete, with significantly less graft fibrosis. Compared with the commonly used small experimental animals, such as mice and non-human primates (e.g., monkeys), sheep have very similar ovaries to humans, in particular regarding structure and size. These characteristics have made sheep ovaries a commonly used model in ovarian cryopreserved transplantation.¹¹ In our previous study, we explored different freezing methods of the sheep ovarian tissue, and successfully xenografted it under the skin of immunodeficient mice.⁵ In this study, we further explored the site of autologous transplantation and the transplantation method using fresh sheep ovarian tissue. Considering the influence of factors such as blood supply richness, temperature and pressure, as well as the possible locations that are convenient for natural pregnancy and surgical operation and observation, we selected three transplantation sites: the ovarian mesangial latum (orthotopic transplantation), the greater omentum and the groin subcutaneous tissue (heterotopic transplantation). Our results showed that, two months after transplantation, multiple primitive follicles were able to survive. Furthermore, primary, secondary and cystic follicles developed in the grafts placed in the ovarian mesangial latum, the greater omentum, and the subcutaneous groin. These observations indicate that all of these sites are feasible for autologous graft transplantation in sheep, and that ovarian tissue can survive and follicles grow and develop in good condition.

The ovarian mesangial ligament contains abundant blood supply, which is basically in the ovarian situ, making it an ideal candidate site for orthotopic transplantation. Donnez et al. suggested two schemes for orthotopic ovarian transplantation¹²: in the case of one ovary remaining, the ovarian cortex after cryopreservation was replanted on the ovarian medulla; in the absence of both ovaries, the ovarian cortex was transplanted to the anterior lobe of the broad ligament where the vascular network is visible. In this study, two months after the fresh sheep ovary cortex was transplanted into the broad ligament of the ovary mesangial, there were multiple surviving primitive follicles, developing primary or cystic follicles, and two secondary follicles, indicating that the broad ligament of the ovary mesangial was suitable for transplantation. However, it is also worth noting that follicles were abundant only on the left graft of the ovarian mesangial ligament, which was similar to previous observations in the subcutaneous neck of xenograft mice.⁵ Basically, in each transplanted mouse, only one of the two grafts had visible follicles. Hence, it is possible that one graft provides hormonal support for follicular survival and development in the other graft. The specific reasons are unknown, and similar reports could not be retrieved. However, this suggests that in the transplantation of the broad ligament of the ovarian mesangium, multiple tissue grafts should be performed at both sides or multiple points as far as possible.

The greater omentum has sufficient space for tissue transplantation, good flexibility and abundant blood supply, and is recommended as a good heterotopic ovarian tissue transplantation site. In the present study, we found that the graft in the greater omentum seemingly contained more surviving primitive follicles than those in the ovarian mesangial latum and the subcutaneous groin. The differences of survival follicles in grafts can’t completely reflect the effect of different transplantation site due to the heterogeneity of the original transplant tissues, which might have different primordial follicle reservation before transplantation. However, it is a good indication that ovarian tissue can survive and that the follicles can grow and develop in the omentum, which may thus constitute an alternative site for transplantation.

Although the majority of successful births result from orthotopic transplantation, heterotopic transplantation outside the pelvic cavity has been favored owing the simple operation procedure and low trauma. Considering the effects of temperature and skin tension on ovarian tissue transplantation, we chose the groin skin, instead of the commonly used rectus or abdominal subcutaneous, for transplantation. Our results showed that there were multiple graft primitive follicles and survival and development of primary or cystic follicles, illustrating that the groin skin can also be used as a suitable transplantation location.

Several studies have previously attempted different ways to promote the formation or regeneration of blood vessels soon after transplantation, in order to reduce the damage of ischemic hypoxia-reperfusion damage to the transplanted ovaries. At present, exogenous intervention factors, including extracellular matrix molecules, vascular epidermal growth factors, antioxidants, and hormone support, are commonly used for ovarian tissue,¹³ but are only used for in vitro studies. Mechanical stimulation, such as surgical trauma, promotes the formation of new blood vessels. After mechanical injury, the inflammatory phase is accompanied by the deposition of collagen and contributes to the formation of new blood vessels. Therefore, based on this principle, a mechanical injury at the graft site before tissue transplantation can induce the formation of new blood vessels. Donnez et al. suggested that the peritoneal windows should be created in the anterior lobe of the broad ligament, where the vascular network can be seen for ovarian orthotopic transplantation.¹² In this study, we used a sharp device to create mechanical damage at the implant sites, which may promote the formation of new blood vessels and the survival of the transplanted ovarian tissues. Two months after transplantation, we observed multiple surviving primitive follicles and several developed primary or cystic follicles. Furthermore, two secondary follicles were found in the grafts of the broad ligament of the ovary mesangial, suggesting that this transplantation method may be beneficial to the growth and development of transplanted ovarian tissue. We note that these observations are consistent with Donnez’s recommendation.

However, some limitations might affect our study. First, more samples are needed for transplantation after cryopreservation to provide a feasible technical solution for future clinical applications. Second, longer studies are needed after transplantation to assess the function of hormone recovery, follicular maturation, and successful pregnancy and reproduction, providing more evidence needed for clinical reference.