Conditional loss of Brca1 in oocytes causes reduced litter size, ovarian reserve depletion and impaired oocyte in vitro maturation with advanced reproductive age in mice.

BACKGROUND: An estimated 1 in 350 women carry germline BRCA1/2 mutations, which confer an increased risk of developing breast and ovarian cancer, and may also contribute to subfertility. All mature, sex steroid-producing ovarian follicles are drawn from the pool of non-renewable primordial follicles, termed the 'ovarian reserve'. The clinical implications of early ovarian reserve exhaustion extend beyond infertility, to include the long-term adverse health consequences of loss of endocrine function and premature menopause. We aimed to determine whether conditional loss of Brca1 in oocytes impacts ovarian follicle numbers, oocyte quality and fertility in mice with advancing maternal age. We also aimed to determine the utility of AMH as a marker of ovarian function, by assessing circulating AMH levels in mice and women with BRCA1/2 mutations, and correlating this with ovarian follicle counts. METHODS: In this study, we addressed a longstanding question in the field regarding the functional consequences of BRCA1 inactivation in oocytes. To recapitulate loss of BRCA1 protein function in oocytes, we generated mice with conditional gene deletion of Brca1 in oocytes using Gdf9-Cre recombinase (WT: Brca1fl/flGdf9+/+; cKO: Brca1fl/flGdf9cre/+). FINDINGS: While the length of the fertile lifespan was not altered between groups after a comprehensive breeding trial, conditional loss of Brca1 in oocytes led to reduced litter size in female mice. Brca1 cKO animals had a reduced ovarian reserve and oocyte maturation was impaired with advanced maternal age at postnatal day (PN)300, compared to WT animals. Serum anti-Müllerian hormone (AMH) concentrations (the gold-standard indirect marker of the ovarian reserve used in clinical practice) were not predictive of reduced primordial follicle number in Brca1 cKO mice versus WT. Furthermore, we found no correlation between follicle number or density and serum AMH concentrations in matched samples from a small cohort of premenopausal women with BRCA1/2 mutations. INTERPRETATION: Together, our data demonstrate that BRCA1 is a key regulator of oocyte number and quality in females and suggest that caution should be used in relying on AMH as a reliable marker of the ovarian reserve in this context. FUNDING: This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC IRIISS. This work was supported by funding from the Australian Research Council (ALW - DE21010037 and KJH - FT190100265), as well as the National Breast Cancer Foundation (IIRS-22-092) awarded to ALW and KJH. LRA, YML, LT, EOKS and MG were supported by Australian Government Research Training Program Scholarships. LRA, YML and LT were also supported by a Monash Graduate Excellence Scholarship. YC, SG and XC were supported by Monash Biomedicine Discovery Institute PhD Scholarships. LRA was also supported by a Monash University ECPF24-6809920940 Fellowship. JMS was supported by NHMRC funding (2011299). MH was supported by an NHMRC Investigator Grant (1193838).

Methods for Studying Uterine Contributions to Pregnancy Establishment in an Ovariectomized Mouse Model.

For pregnancy to be established, a viable blastocyst must successfully interact with a receptive uterine lining (endometrium) to facilitate implantation and placenta formation and enable ongoing pregnancy. The limitations to pregnancy success caused by embryonic defects are well known and have been largely overcome in recent decades with the rise of in vitro fertilization (IVF) and assisted reproductive technologies. As yet, however, the field has not overcome the limitations caused by an inadequately receptive endometrium, thus resulting in stagnating IVF success rates. Ovarian and endometrial functions are closely intertwined, as hormones produced by the ovary are responsible for the endometrium's menstrual cyclicity. As such, when using rodent models of pregnancy, it can be difficult to ascertain whether an observed result is due to an ovarian or uterine deficit. To overcome this, an ovariectomized mouse model was developed with embryo transfer or artificial decidualization to allow the study of uterine-specific contributions to pregnancy. This article will provide instructions on how to perform ovariectomy and offer insights into various techniques for supplying exogenous hormones to support successful artificial decidualization or pregnancy following embryo transfer from healthy donors. These techniques include subcutaneous injection, slow-release pellets, and osmotic mini pumps. The key advantages and disadvantages of each method will be discussed, enabling researchers to choose the best study design for their specific research question.

The future of fertility preservation for women treated with chemotherapy.

Cytotoxic chemotherapies have been a mainstay of cancer treatment, but are associated with numerous systemic adverse effects, including impacts to fertility and endocrine health. Irreversible ovarian damage and follicle depletion are side-effects of chemotherapy that can lead to infertility and premature menopause, both being major concerns of young cancer patients. Notably, many women will proceed with fertility preservation, but unfortunately existing strategies don't entirely solve the problem. Most significantly, oocyte and embryo freezing do not prevent cancer treatment-induced ovarian damage from occurring, which may result in the impairment of long-term hormone production. Unfortunately, loss of endogenous endocrine function is not fully restored by hormone replacement therapy. Additionally, while GnRH agonists are standard care for patients receiving alkylating chemotherapy to lessen the risk of premature menopause, their efficacy is incomplete. The lack of more broadly effective options stems, in part, from our poor understanding of how different treatments damage the ovary. Here, we summarise the impacts of two commonly utilised chemotherapies - cyclophosphamide and cisplatin - on ovarian function and fertility, and discuss the mechanisms underpinning this damage. Additionally, we critically analyse current research avenues in the development of novel fertility preservation strategies, with a focus on fertoprotective agents.

Radiotherapy exposure directly damages the uterus and causes pregnancy loss.

Female cancer survivors are significantly more likely to experience infertility than the general population. It is well established that chemotherapy and radiotherapy can damage the ovary and compromise fertility, yet the ability of cancer treatments to induce uterine damage, and the underlying mechanisms, have been understudied. Here, we show that in mice total-body γ-irradiation (TBI) induced extensive DNA damage and apoptosis in uterine cells. We then transferred healthy donor embryos into ovariectomized adolescent female mice that were previously exposed to TBI to study the impacts of radiotherapy on the uterus independent from effects to ovarian endocrine function. Following TBI, embryo attachment and implantation were unaffected, but fetal resorption was evident at midgestation in 100% of dams, suggesting failed placental development. Consistent with this hypothesis, TBI impaired the decidual response in mice and primary human endometrial stromal cells. TBI also caused uterine artery endothelial dysfunction, likely preventing adequate blood vessel remodeling in early pregnancy. Notably, when pro-apoptotic protein Puma-deficient (Puma-/-) mice were exposed to TBI, apoptosis within the uterus was prevented, and decidualization, vascular function, and pregnancy were restored, identifying PUMA-mediated apoptosis as a key mechanism. Collectively, these data show that TBI damages the uterus and compromises pregnancy success, suggesting that optimal fertility preservation during radiotherapy may require protection of both the ovaries and uterus. In this regard, inhibition of PUMA may represent a potential fertility preservation strategy.

Checkpoint inhibitor immunotherapy diminishes oocyte number and quality in mice.

Loss of fertility is a major concern for female reproductive-age cancer survivors, since a common side-effect of conventional cytotoxic cancer therapies is permanent damage to the ovary. While immunotherapies are increasingly becoming a standard of care for many cancers-including in the curative setting-their impacts on ovarian function and fertility are unknown. We evaluated the effect of immune checkpoint inhibitors blocking programmed cell death protein ligand 1 and cytotoxic T lymphocyte-associated antigen 4 on the ovary using tumor-bearing and tumor-free mouse models. We find that immune checkpoint inhibition increases immune cell infiltration and tumor necrosis factor-α expression within the ovary, diminishes the ovarian follicular reserve and impairs the ability of oocytes to mature and ovulate. These data demonstrate that immune checkpoint inhibitors have the potential to impair both immediate and future fertility, and studies in women should be prioritized. Additionally, fertility preservation should be strongly considered for women receiving these immunotherapies, and preventative strategies should be investigated in future studies.