Regulation of follicular activation signaling pathways by in vitro inhibition of YAP/TAZ activity in mouse ovaries.

The Hippo pathway plays a crucial role in the regulation of follicular activation, which constitutes the first step of the folliculogenesis process. Disruption of this pathway occurs in several non-physiological contexts, after fragmentation for ovarian tissue cryopreservation procedures or chemotherapy exposure, leading to massive follicular growth and depletion. This study aimed to investigate the effect of controlling the Hippo pathway using verteporfin (VERT) during in vitro ovarian culture and to evaluate its potential preventive effects on chemotherapy-induced follicle activation using a mouse model. After exposure of cut ovaries to different concentrations of VERT for 3 h, a dose-dependent effect of VERT was observed that reached significant inhibition of YAP activity at 3 µmol/L. To assess the potential effect of controlling chemotherapy-induced Hippo pathway disruption, whole mouse ovaries were exposed to VERT alone or as a co-treatment with 4-hydroperoxycylophosphamide (4HC). VERT co-treatment prevented chemotherapy-induced YAP activation but had a limited impact on downstream effector gene, Ccn2. Surprisingly, VERT co-treatment also prevented mTOR and survival signaling pathway alterations following chemotherapy exposure. These results suggest an interaction between the two main signaling pathways regulating follicle activation and a protective effect of VERT on 4HC-induced DNA damage.

Gene Expression Analyses in Human Follicles.

The ovary is a heterogeneous organ composed of different cell types. To study the molecular mechanisms occurring during folliculogenesis, the localization of proteins and gene expression can be performed on fixed tissue. However, to properly assess gene expression levels in a human follicle, this complex and delicate structure must be isolated. Hence, an adapted protocol previously described by Woodruff's laboratory has been developed to separate follicles (the oocyte and the granulosa cells) from their surrounding environment. The ovarian cortical tissue is first manually processed to obtain small fragments using two tools: a tissue slicer and a tissue chopper. The tissue is then enzymatically digested with 0.2% collagenase and 0.02% DNase for at least 40 min. This digestion step is performed at 37 °C and 5% CO2 and is accompanied by mechanical pipetting of the medium every 10 min. After incubation, the isolated follicles are collected manually using a calibrated microcapillary pipette under microscope magnification. If follicles are still present in the pieces of tissue, the procedure is completed with manual microdissection. The follicles are collected on ice in a culture medium and are rinsed twice in droplets of phosphate-buffered saline solution. This digestion procedure must be carefully controlled to avoid follicle deterioration. As soon as the structure of the follicles appears to be compromised or after a maximum of 90 min, the reaction is stopped with a 4 °C blocking solution containing 10% fetal bovine serum. A minimum of 20 isolated follicles (sized under 75 µm) should be collected to obtain an adequate amount of total RNA after RNA extraction for real-time quantitative polymerase chain reaction (RT-qPCR). After extraction, the quantification of total RNA from 20 follicles reaches a mean value of 5 ng/µL. The total RNA is then retrotranscribed into cDNA, and the genes of interest are further analyzed using RT-qPCR.

Impact of first chemotherapy exposure on follicle activation and survival in human cryopreserved ovarian tissue.

STUDY QUESTION: Does chemotherapy exposure prior to ovarian tissue cryopreservation (OTC) impact the signaling pathways governing follicle activation and survival for prepubertal and postpubertal patients? SUMMARY ANSWER: Chemotherapy exposure prior OTC increases follicle apoptosis rates but not follicular activation, although the PI3K/AKT/mTOR and Hippo signaling pathways were modified in the cortex. WHAT IS KNOWN ALREADY: OTC is currently the only available fertility preservation procedure for children and for patients who have already started their treatment. While previous studies have not observed harmful impacts of first chemotherapy exposure on OTC outcomes, the consequences of treatment on follicle activation and survival need to be further investigated. To address this question, we evaluated signaling pathway modifications induced by chemotherapy exposure according to pubertal status. STUDY DESIGN, SIZE, DURATION: Cryopreserved ovarian tissues from postpubertal (12-29 years old, n = 8) and prepubertal (3-10 years old, n = 8) cancer patients donated for research were thawed and cultured for 24 h. Analyses of the survival of the follicles and stroma, and of the PI3K/AKT/mTOR and Hippo signaling pathways, were conducted at thawing and after culture. Ovarian fragments exposed to chemotherapy before collection were compared to non-exposed controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Histological investigations were performed to assess the distribution of the follicles, stroma fibrosis, vessel integrity, and apoptosis levels. It included follicular counting, collagen staining, immunostaining on CD31 and gH2AX, as well as TUNEL staining. To explore follicle activation in the different groups, the PI3K/AKT/mTOR and Hippo signaling pathways were investigated by gene expression analyses of isolated follicles and protein analyses on whole fragments through western blots and immunostaining. MAIN RESULTS AND THE ROLE OF CHANCE: We first assessed the impact of a first exposure to chemotherapy on the collagen density and vessels in ovarian tissues at thawing and after culture. While no differences in collagen density were observed according to age or previous treatment, the vascularization area (CD31+) was significantly lower in tissue from previously exposed patients compared to non-treated ones. Apoptosis analyses (TUNEL) revealed an acute deleterious impact on follicle survival after chemotherapy exposure without affecting the follicular density. Surprisingly, leukemic patients had a significantly higher percentage of gH2AX-positive follicles, indicating a DNA damage response, compared to the other patients. The proportion of activated follicles appeared to decrease following exposure to chemotherapy, suggesting that it at least did not increase activation process. Stable KIT LIGAND gene and protein expression and cKIT protein levels were observed among the groups, confirming the absence of activation. Analysis of the PI3K pathway did not reveal a difference in the AKT phosphorylation level between the groups, but pRPS6 was significantly higher in tissue from patients previously exposed to chemotherapy compared to that from non-exposed patients. Finally, protein and gene analyses on Hippo pathway signaling showed a higher LATS1 protein level in the cortex after chemotherapy exposure. LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of the human fragments, and the limited number of patients included in the cohort have to be considered as important study limitations. Moreover, this study did not explore the long-term consequences of chemotherapy on follicular development. Therefore, the results should be interpreted with caution. WIDER IMPLICATIONS OF THE FINDINGS: These results underscore the deleterious effect of previous chemotherapeutic treatment on follicle survival. Although follicular density was not reduced, these data suggested that exposure to chemotherapy impacts follicular apoptosis and the DNA damage response. Chemotherapy-induced activation was not observed despite the impact on mTOR and Hippo signaling pathways in the whole cortex. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by an Excellence of Science (EOS) Grant (ID: 30443682) and was supported by Fonds Erasme. I.D. and M.-M.D. are associate researchers at Fonds National de la Recherche Scientifique de Belgique (FNRS). There are no competing interests. TRIAL REGISTRATION NUMBER: N/A.

BRCA Mutations and Fertility Preservation.

Hereditary cancers mostly affect the adolescent and young adult population (AYA) at reproductive age. Mutations in BReast CAncer (BRCA) genes are responsible for the majority of cases of hereditary breast and ovarian cancer. BRCA1 and BRCA2 act as tumor suppressor genes as they are key regulators of DNA repair through homologous recombination. Evidence of the accumulation of DNA double-strand break has been reported in aging oocytes, while BRCA expression decreases, leading to the hypothesis that BRCA mutation may impact fertility. Moreover, patients exposed to anticancer treatments are at higher risk of fertility-related issues, and BRCA mutations could exacerbate the treatment-induced depletion of the ovarian reserve. In this review, we summarized the functions of both genes and reported the current knowledge on the impact of BRCA mutations on ovarian ageing, premature ovarian insufficiency, female fertility preservation strategies and insights about male infertility. Altogether, this review provides relevant up-to-date information on the impact of BRCA1/2 mutations on fertility. Notably, BRCA-mutated patients should be adequately counselled for fertility preservation strategies, considering their higher sensitivity to chemotherapy gonadotoxic effects.

Implications of Nonphysiological Ovarian Primordial Follicle Activation for Fertility Preservation.

In recent years, ovarian tissue cryopreservation has rapidly developed as a successful method for preserving the fertility of girls and young women with cancer or benign conditions requiring gonadotoxic therapy, and is now becoming widely recognized as an effective alternative to oocyte and embryo freezing when not feasible. Primordial follicles are the most abundant population of follicles in the ovary, and their relatively quiescent metabolism makes them more resistant to cryoinjury. This dormant pool represents a key target for fertility preservation strategies as a resource for generating high-quality oocytes. However, development of mature, competent oocytes derived from primordial follicles is challenging, particularly in larger mammals. One of the main barriers is the substantial knowledge gap regarding the regulation of the balance between dormancy and activation of primordial follicles to initiate their growing phase. In addition, experimental and clinical factors also affect dormant follicle demise, while the mechanisms involved remain largely to be elucidated. Moreover, most of our basic knowledge of these processes comes from rodent studies and should be extrapolated to humans with caution, considering the differences between species in the reproductive field. Overcoming these obstacles is essential to improving both the quantity and the quality of mature oocytes available for further fertilization, and may have valuable biological and clinical applications, especially in fertility preservation procedures. This review provides an update on current knowledge of mammalian primordial follicle activation under both physiological and nonphysiological conditions, and discusses implications for fertility preservation and priorities for future research.

Interaction between PI3K/AKT and Hippo pathways during in vitro follicular activation and response to fragmentation and chemotherapy exposure using a mouse immature ovary model.

Understanding and control of the massive and accelerated follicular growth that occurs during in vitro culture of ovarian tissue is a crucial step toward the development of efficient culture systems that offer an attractive alternative to ovarian tissue transplantation for fertility restoration in cancer survivors. One outstanding question focuses on processes that occur prior to cryopreservation, such as tissue sectioning or chemotherapeutic treatment, might exacerbate this follicular activation. Although the PI3K/AKT/mTOR pathway is well known as a major trigger of physiological and chemotherapy-induced follicular activation, studies have shown that disruption of Hippo pathway due to ovarian fragmentation acts as an additional stimulator. This study aimed to characterize the possible interactions between these pathways using post-natal day 3 mouse ovaries cultured for 4 or 48 h. Morphology, gene transcription, and protein levels were assessed to investigate the impact of sectioning or chemotherapy exposure (4-hydroperoxycyclophosphamide [4HC], 3 and 20 µM). The effect of an mTORC1 inhibitor, Everolimus, alone or as a 4HC co-treatment to prevent follicle activation was evaluated. The results showed that organ removal from its physiological environment was as effective as sectioning for disruption of Hippo pathway and induction of follicle activation. Both PI3K/AKT/mTOR and Hippo pathways were involved in chemotherapy-induced follicular activation and responded to fragmentation. Surprisingly, Everolimus was able to prevent the activation of both pathways during chemotherapy exposure, suggesting cross-talk between them. This study underscores the major involvement of PI3K/AKT/mTOR and Hippo pathways in in vitro follicle activation and provides evidence that both can be regulated using mTORC1 inhibitor.