Fertility preservation in cancer patients: the artificial ovary. Allografting of murine follicles in immunocompromised mice

Thousands of women suffering from malignant diseases can now be treated by chemotherapy, radiotherapy or bone marrow transplantation, but these treatments are known to impair fertility (Larsen et al., 2003). Different techniques can, however, be used to preserve fertility in these women. Cryopreservation and transplantation of ovarian tissue is the only strategy that can be applied in prepubertal patients and women who need to start their cancer treatment immediately. Despite increasingly successful results, this technique cannot be implemented if the patient was affected by a disease that carries a high risk of transferring malignant cells while grafting the ovarian tissue (Dolmans et al., 2013). To avoid this risk, the Gynecology Research Unit is working on a new concept to restore fertility in cancer patients, involving isolation of ovarian follicles from their environment and hence from malignant cells, and grafting inside a matrix – essentially an ‘artificial ovary’.

The first step in creating an artificial ovary is development of a 3D matrix to graft isolated preantral follicles. To this end, different polymers have been tested, with fibrin yielding the best results so far in terms of follicle recovery rate, and matrix vascularization and degradation. However, some puzzling findings need to be addressed, such as the lower recovery rate of primordial-primary follicles compared to secondary follicles (Luyckx et al., 2014; Vanacker et al., 2014).

The goal of this study was to confirm these results and investigate why secondary follicles appear to be more resistant than primordial-primary follicles. Ovaries from NMRI mice were therefore used for isolation of preantral follicles. Primordial-primary follicles were separated from secondary follicles, forming two follicle groups, and then grafted to each side of the abdominal wall of SCID mice inside a fibrin matrix (F12.5/T1). The grafts were left in place for 2 days in 5 mice and 7 days in 6 mice. The follicle recovery rate was 16% in the primordial-primary group and 40% in the secondary group after 2 days of grafting, and 6% in the primordial-primary group and 28% in the secondary group after 7 days of grafting, confirming previous studies. Indeed, we found a higher recovery rate of secondary follicles than primordial-primary follicles after both periods of grafting (23%, p value <0.001), despite follicles from each group showing good viability prior to grafting. Follicle apoptosis after grafting was analyzed by TUNEL, which revealed no difference between the two groups. Follicle growth was assessed using the immunomarker Ki67, which indicated that grafted follicles from both groups were able to grow. Larger numbers of new vessels were identified by CD34 immunostaining in the secondary group after 7 days of grafting. These results are discussed and future prospects described in this dissertation.