Seng H. Liew

Reproductive ageing in females is defined by a progressive decline in follicle number and oocyte quality. This is a natural process that leads to the loss of fertility and ovarian function, cycle irregularity and eventually menopause or reproductive senescence. The factors that underlie the natural depletion of follicles throughout reproductive life are poorly characterised. It has been proposed that inflammatory processes and fibrosis might contribute to ovarian ageing. To further investigate this possibility, we evaluated key markers of inflammation and immune cell populations in the ovaries of 2, 6, 12 and 18-month-old C57BL/6 female mice. We report that the decrease in follicle numbers over the reproductive lifespan was associated with an increase in the intra-ovarian percentage of CD4 + T cells, B cells and macrophages. Serum concentration and intra-ovarian mRNA levels of several pro-inflammatory cytokines, including IL-1α/β, TNF-α, IL-6, and inflammasome genes ASC and NLRP3, were significantly increased with age. Fibrosis levels, as determined by picrosirius red staining for collagen I and III, were unchanged up to 18 months of age. Collectively, these data suggest that inflammation could be one of the mechanisms responsible for the age-related regulation of follicle number, but the role of fibrosis is unclear. Further studies are now required to determine if there is a causative relationship between inflammation and follicle depletion as females age.

BACKGROUND: Within the ovary, oocytes are stored in long-lived structures called primordial follicles, each comprising a meiotically arrested oocyte, surrounded by somatic granulosa cells. It is essential that their genetic integrity is maintained throughout life to ensure that high quality oocytes are available for ovulation. Of all the possible types of DNA damage, DNA double-strand breaks (DSBs) are considered to be the most severe. Recent studies have shown that DNA DSBs can accumulate in oocytes in primordial follicles during reproductive ageing, and are readily induced by exogenous factors such as γ-irradiation, chemotherapy and environmental toxicants. DSBs can induce oocyte death or, alternatively, activate a program of DNA repair in order to restore genetic integrity and promote survival. The repair of DSBs has been intensively studied in the context of meiotic recombination, and in recent years more detail is becoming available regarding the repair capabilities of primordial follicle oocytes. OBJECTIVE AND RATIONALE: This review discusses the induction and repair of DNA DSBs in primordial follicle oocytes. SEARCH METHODS: PubMed (Medline) and Google Scholar searches were performed using the key words: primordial follicle oocyte, DNA repair, double-strand break, DNA damage, chemotherapy, radiotherapy, ageing, environmental toxicant. The literature was restricted to papers in the English language and limited to reports in animals and humans dated from 1964 until 2017. The references within these articles were also manually searched. OUTCOMES: Recent experiments in animal models and humans have provided compelling evidence that primordial follicle oocytes can efficiently repair DNA DSBs arising from diverse origins, but this capacity may decline with increasing age. WIDER IMPLICATIONS: Primordial follicle oocytes are vulnerable to DNA DSBs emanating from endogenous and exogenous sources. The ability to repair this damage is essential for female fertility. In the long term, augmenting DNA repair in primordial follicle oocytes has implications for the development of novel fertility preservation agents for female cancer patients and for the management of maternal ageing. However, further work is required to fully characterize the specific proteins involved and to develop strategies to bolster their activity.

It is well established that DNA-damaging chemotherapies can cause infertility and ovarian endocrine failure by depleting the ovarian reserve of primordial follicles. Currently, no effective pharmacological therapies exist for the preservation of long-term fertility and ovarian function in female cancer patients, due to a limited understanding of the mechanisms of chemotherapy-induced follicle depletion. This study investigated the cellular targets, molecular mechanisms, and temporal course of ovarian reserve depletion following treatment with commonly used chemotherapeutic drugs. Adult female C57BL/6 mice were injected i.p. with saline, cisplatin (5mg/kg), or cyclophosphamide (300mg/kg); ovaries were harvested after 8 or 24 hours. Follicle counts showed depletion of all follicular stages 24 hours after administration of cisplatin or cyclophosphamide. Eight hours post-treatment, H2A histone family member X (γH2AX) immunofluorescence showed DNA double-stranded breaks at all follicular stages, including within primordial follicle oocytes. This staining was resolving by 24 hours, indicating that primordial follicle oocytes begin to undergo either apoptosis or repair in this timeframe. γH2AX-positive follicles were further examined to identify the specific cell types damaged. In primordial, transitional, and primary follicles, only oocytes sustained DNA damage, whereas in secondary and antral follicles, only somatic cells were affected. TUNEL staining confirmed that apoptosis occurs in these targeted cell types. Whilst multi-drug and multi-dose regimens were not examined, this study conclusively shows that cyclophosphamide and cisplatin cause direct damage to primordial follicle oocytes, which then undergo apoptosis. Therefore, future pharmacological strategies to prevent chemotherapy-induced infertility in females must specifically prevent primordial follicle oocyte death.

Abstract Female gametes are stored in the ovary in structures called primordial follicles, the supply of which is non-renewable. It is well established that DNA-damaging cancer treatments can deplete the ovarian reserve of primordial follicles, causing premature ovarian failure and infertility. The precise mechanisms underlying this chemotherapy-driven follicle loss are unclear, and this has limited the development of targeted ovarian-protective agents. To address this fundamental knowledge gap, we used gene deletion mouse models to examine the role of the DNA damage-induced pro-apoptotic protein, PUMA, and its transcriptional activator TAp63, in primordial follicle depletion caused by treatment with cyclophosphamide or cisplatin. Cyclophosphamide caused almost complete destruction of the primordial follicle pool in adult wild-type (WT) mice, and a significant destructive effect was also observed for cisplatin. In striking contrast, Puma −/− mice retained 100% of their primordial follicles following either genotoxic treatment. Furthermore, elimination of PUMA alone completely preserved fertility in cyclophosphamide-treated mice, indicating that oocytes rescued from DNA damage-induced death can repair themselves sufficiently to support reproductive function and offspring health. Primordial follicles were also protected in TAp63 −/− mice following cisplatin treatment, but not cyclophosphamide, suggesting mechanistic differences in the induction of apoptosis and depletion of the ovarian reserve in response to these different chemotherapies. These studies identify PUMA as a crucial effector of apoptosis responsible for depletion of primordial follicles following exposure to cyclophosphamide or cisplatin, and this indicates that inhibition of PUMA may be an effective ovarian-protective strategy during cancer treatment in women.