Purpose Our aim was to elucidate the mechanisms involved in follicle activation of the ovarian reserve after human ovarian tissue transplantation, with specific focus on the role of the effectors of the PI3K (mTOR and FOXO1) and Hippo (YAP) signaling pathways and whether they are somehow altered. Methods Frozen-thawed ovarian tissue was collected from six women (age 25-35 years) undergoing surgery for non-ovarian pathologies and divided into 4 fragments in each case: one for non-grafted controls and three for grafting to immunodeficient mice for 3, 7 and 21 days. The tissue was processed for hematoxylin and eosin staining, immunohistochemistry and immunofluorescence at different timepoints before and after grafting. Activation of the PI3K and Hippo signaling pathways was investigated by analysis of mTOR phosphorylation, FOXO1 cytoplasmic localization and YAP nuclear localization. Results No change in mTOR levels was observed in primordial follicles post-transplantation, but a significant upturn was recorded in growing follicles compared with primordial follicles, irrespective of grafting time. A higher percentage of primordial follicles was also found with FOXO1 in the cytoplasm after 3 days of transplantation than in non-grafted controls. Finally, a greater proportion of primordial follicles was detected with YAP in the nucleus at all timepoints after grafting. Conclusions This study supports the hypothesis that follicle activation may occur as an early event after transplantation, with follicle growth and death both contributing to the burnout phenomenon. This is the first time that the effectors of the PI3K and Hippo pathways have been investigated in grafted human ovarian tissue and their role in burnout documented.

No abstract

Chemotherapy, pelvic radiotherapy and ovarian surgery have known gonadotoxic effects that can lead to endocrine dysfunction, cessation of ovarian endocrine activity and early depletion of the ovarian reserve, causing a risk for future fertility problems, even in children. Important determinants of this risk are the patient’s age and ovarian reserve, type of treatment and dose. When the risk of premature ovarian insufficiency is high, fertility preservation strategies must be offered to the patient. Furthermore, fertility preservation may sometimes be needed in conditions other than cancer, such as in non-malignant diseases or in patients seeking fertility preservation for personal reasons. Oocyte and/or embryo vitrification and ovarian tissue cryopreservation are the two methods currently endorsed by the American Society for Reproductive Medicine, yielding encouraging results in terms of pregnancy and live birth rates. The choice of one technique above the other depends mostly on the age and pubertal status of the patient, and personal and medical circumstances. This review focuses on the available fertility preservation techniques, their appropriateness according to patient age and their efficacy in terms of pregnancy and live birth rates.

Ovarian tissue cryopreservation and transplantation is the only fertility preservation option that enables both restoration of fertility and resumption of ovarian endocrine function, avoiding the morbidity associated with premature menopause. It is also the only technique available to prepubertal patients and those whose treatment cannot be delayed for life-threatening reasons. Ovarian tissue cryopreservation can be carried out in two different ways, either as ovarian cortical fragments or as a whole organ with its vascular pedicle. Although use of cortical strips is the only procedure that has been approved by the American Society for Reproductive Medicine, it is fraught with drawbacks, the major one being serious follicle loss occurring after avascular transplantation due to prolonged warm ischemia. Whole ovary cryopreservation involves vascular transplantation, which could theoretically counteract the latter phenomenon and markedly improve follicle survival. In theory, this technique should maintain endocrine and reproductive functions much longer than grafting of ovarian cortical fragments. However, this procedure includes a number of critical steps related to (A) the level of surgical expertise required to accomplish retrieval of a whole ovary with its vascular pedicle, (B) the choice of cryopreservation technique for freezing of the intact organ, and (C) successful execution of functional vascular reanastomosis upon thawing. The aim of this systematic review is to shed light on these challenges and summarize solutions that have been proposed so far in animal experiments and humans in the field of whole ovary cryopreservation and transplantation.

Purpose To investigate whether adipose tissue-derived stem cells (ASCs) protect the primordial follicle pool, not only by decreasing direct follicle loss but also by modulating follicle activation pathways. Methods Twenty nude mice were grafted with frozen-thawed human ovarian tissue from 5 patients. Ten mice underwent standard ovarian tissue transplantation (OT group), while the remaining ten were transplanted with ASCs and ovarian tissue (2-step/ASCs+OT group). Ovarian grafts were retrieved on days 3 (n = 5) and 10 (n = 5). Analyses included histology (follicle count and classification), immunohistochemistry (c-caspase-3 for apoptosis and LC3B for autophagy), and immunofluorescence (FOXO1 for PI3K/Akt activation and YAP for Hippo pathway disruption). Subcellular localization was determined in primordial follicles on high-resolution images using structured illumination microscopy. Results The ASCs+OT group showed significantly higher follicle density than the OT group (p = 0.01). Significantly increased follicle atresia (p < 0.001) and apoptosis (p = 0.001) were observed only in the OT group. In primordial follicles, there was a significant shift in FOXO1 to a cytoplasmic localization in the OT group on days 3 (p = 0.01) and 10 (p = 0.03), indicating follicle activation, while the ASCs+OT group and non-grafted controls maintained nuclear localization, indicating quiescence. Hippo pathway disruption was encountered in primordial follicles irrespective of transplantation, with nuclear YAP localized in their granulosa cells. Conclusion We demonstrate that ASCs exert positive effects on the ovarian reserve, not only by protecting primordial follicles from direct death but also by maintaining their quiescence through modulation of the PI3K/Akt pathway.