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To study the effect of freezing, in vitro culture (IVC) and grafting to chorioallantoic membrane (CAM) on follicle outcomes in human ovarian tissue. An experimental study. University-based research laboratory. Fresh and cryopreserved ovarian tissue from 10 patients was donated to research with their consent and institutional review board approval. Fresh and frozen-thawed ovarian cortical pieces were in vitro-cultured and compared (fresh-IVC vs FT-IVC). The FT-IVC fragments were then examined against fragments grafted to CAM (FT-CAM). After both IVC and CAM grafting, ovarian cortical pieces (4×2×1 mm) were analyzed on days 0, 1, and 6. Follicle analyses included histology (count and classification) and immunohistochemistry (Ki67 [proliferation], caspase-3 [apoptosis], 1A and 1B light chain 3B [autophagy], p-Akt, FOXO1, and p-rpS6 [PI3K activation]). Droplet digital polymerase chain reaction further explored expression of PI3K pathway- and oocyte-related genes in tissue sections. No major differences were detected between fresh-IVC and FT-IVC tissues in any conducted analyses. Although a significant drop was observed in primordial follicle (PF) proportions in the fresh-IVC and FT-IVC groups (d0 vs. d6, P.05). The PF rates were also significantly higher in the FT-CAM group than the FT-IVC group on d6 (P=.02). Importantly, avian erythrocytes were already present in 30% of implants from d1. Apoptotic and autophagic follicle rates increased during IVC (P

We report successful clinical outcomes after transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary in a patient diagnosed with stage IIIb rectal adenocarcinoma. Whole ovary cryopreservation was proposed as a fertility preservation strategy in 2006 prior to radiotherapy, chemotherapy and oncological surgery. To allow for minimal residual disease screening before ovarian reimplantation, the whole ovary was thawed and dissected into cortical strips. While awaiting the results, the majority of the cortical strips were refrozen. These refrozen-rethawed cortical strips were laparoscopically grafted to 2 sites: the previously irradiated pelvic cavity and the non-irradiated extrapelvic cavity. Ovarian function resumption was assessed by recovery of menses, hormone levels, ultrasound and oocyte pick-up following controlled ovarian stimulation (COS). Restoration of ovarian function occurred 6 months after reimplantation, with recovery of menses and estradiol secretion. A total of 12 cycles were followed by the IVF department. A second reimplantation was performed 1.5 years later, since the grafts were found to have stopped functioning for >3 consecutive months. Overall, 3 fertilizable oocytes were retrieved transabdominally from the extrapelvic graft following COS, yielding 2 embryos and culminating in one fresh embryo transfer, but no pregnancy. Concerning the reimplantation site, no ovarian activity was detected in the graft placed in the previously irradiated pelvic cavity. Indeed, only fibrotic-looking tissue was observed in the pelvic site at second laparoscopy 1.5 years later, while ovarian activity was noted in the extrapelvic graft, showing a large antral follicle. All in all, transplantation of refrozen-rethawed cortical strips from a cryopreserved whole ovary can lead to ovarian function resumption and embryo development if grafted to a non-irradiated field.

Purpose: The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure. Methods: Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21 days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson's green trichrome, and double CD34 immunostaining. Results: Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p < 0.0001) and refrozen-rethawed (p < 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization. Conclusion: We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.

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

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.

International audience; 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

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.

Research question The aim was to elucidate the delay in and mechanisms of follicle activation after ovarian tissue transplantation, with particular emphasis on the role of the Akt signalling pathway. Design Ovarian tissue was collected from six patients and divided into four fragments in each case: one for control purposes and three for grafting to immunodeficient mice for 3, 7 and 21 days. Follicle density, classification, growth and atresia, and the Akt pathway were analysed. Results A significant decrease in primordial follicles and significant increase in growing follicles were detected 3 days after transplantation (both P = 0.01). More than 50% of follicles were atretic after 3 days, and a further 50% after 7 days of grafting. Akt phosphorylation was significantly elevated in primordial follicles after 3 days of grafting (P = 0.048). Conclusions This study confirms that primordial follicle activation is an early event after transplantation, and significant follicle death also contributes to the burnout effect, eventually resulting in early depletion of the ovarian reserve. Increased Akt phosphorylation on day 3 post-grafting suggests it plays a role in follicle activation and subsequent burnout.