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REPRODUCTIONREVIEW

Mechanisms of ovarian aging

Selena U Park1,3, Leann Walsh1 and Karen M Berkowitz 1,2 1Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA, 2Department of Obstetrics and Gynecology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA and 3Department of Obstetrics, Gynecology, and Reproductive Sciences, Rutgers University Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA Correspondence should be addressed to K M Berkowitz; Email: [email protected]

Abstract

Ovarian aging in women correlates with the progressive loss of both the number and quality of oocytes. When these processes occur early or are accelerated, their clinical correlates are diminished ovarian reserve and/or premature ovarian insufficiency. Both these conditions have important consequences for the reproductive and general health of women, including infertility. Although there are many contributing factors, the molecular mechanisms underlying many of the processes associated with ovarian aging have not been fully elucidated. In this review, we highlight some of the most critical factors that impact oocyte quantity and quality with advancing age. We discuss chromosomal factors including cohesion deterioration and mis-segregation, errors in meiotic recombination, and decreased stringency of the spindle assembly checkpoint. DNA damage, telomere changes, reactive oxygen species and mitochondrial dysfunction as they relate to ovarian aging, and well-known gene mutations associated with primary ovarian insufficiency and diminished ovarian reserve are also discussed. Additionally, studies investigating recently acknowledged cytoplasmic factors associated with ovarian aging including protein metabolic dysregulation and microenvironmental alterations in the ovary are presented. We use both mouse and human studies to support the roles these factors play in physiologic and expedited ovarian aging, and we propose directions for future studies. A better understanding of the molecular basis of ovarian aging will ultimately lead to diagnostic and therapeutic advancements that would provide women with information to make earlier choices about their reproductive health. Reproduction (2021) 162 R19–R33

Introduction this peak, there is a stage of pronounced oocyte atresia that decreases the oocyte number to approximately Delayed childbearing brings forth a unique problem 2 million at birth. The process of atresia gradually in women. Unlike males who possess a renewing leads to a decline in the follicle pool, and by puberty, population of spermatogonial cells, females begin life approximately 400,000 primordial follicles remain. with ultimately a finite number of oocytes. Oocytes Ultimately, the number of primordial follicles decreases eventually comprise a pool of primordial follicles that to approximately 1000 at menopause. Newer models of diminish in number throughout a woman’s lifetime reproductive aging in women, which take into account (McGee & Hsueh 2000). Although, the quantity of data from older studies, propose that attrition of the non- this discrete population of oocytes in the follicle pool growing follicle pool continually increases with age and constitutes the ovarian reserve, both the number does not abruptly change (Hansen et al. 2008). The and quality of oocytes impact reproductive potential changes that occur in this dynamic cohort of oocytes and aging (Tal & Seifer 2017, ASRM 2020). Despite correlate with the changes in fertility over the course the widely accepted dogma that the oocyte pool is of a woman’s reproductive life, which become most determinate, some reports suggest that new oocytes clinically apparent when a woman reaches her mid can form and contribute to the ovarian reserve (Johnson to late 30s and beyond (Wallace & Kelsey 2010, Tal & et al. 2004). Such studies may hold potential promise Seifer 2017). for the field of reproductive medicine. However, it is Multiple factors, including chromosomal, genetic, unclear whether these new cells can function as oocytes mitochondrial, and cytoplasmic, impact the quantity (Wood & Rajkovic 2013). Gametogenesis begins during and quality of oocytes in the ovarian reserve (Fig. 1). fetal development in females, and by mid-gestation, The effects of these factors on the ovarian reserve and the number of oocytes reaches a maximum number of fertility also vary among different women of similar approximately 6–7 million (Allen 2010). After reaching ages (te Velde & Pearson 2002, Hansen et al. 2008).

© 2021 Society for Reproduction and Fertility https://doi.org/10.1530/REP -21-0022 ISSN 1470–1626 (paper) 1741–7899 (online) Online version via https://rep.bioscientifica.com Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access

-21-0022 R20 S Park and others

From embryonic development to menopause During human embryonic development, by the seventh week of gestation, primordial germ cells (PGCs) migrate from the yolk sac endoderm to the gonadal ridge. PGCs synchronize their entrance into mitotic divisions throughout their migration. Upon reaching the gonads, PGCs differentiate into oogonia and continue mitotic proliferation. At about 11–12 weeks of gestation, oogonia enter meiosis and develop into primary oocytes. Primary oocytes then individually become encased by pregranulosa cells to form primordial follicles – a process that begins around the 20th week of Figure 1 A multitude of physiological factors can diminish the gestation and continues until birth (AlAsiri et al. 2015) quantity and quality of the ovarian reserve and lead to ovarian aging. Oocytes in the primordial follicles remain arrested in Each factor is explained further under subheadings in the text. the diplotene stage of prophase I during meiosis until the onset of puberty (Picton 2001, Hansen et al. 2008). Current research has been focused on determining These primordial follicles are the origin of the ovarian the factors that are most important to ovarian health. reserve (Fig. 2). Ultimately, follicles containing oocytes One goal of these studies is to elucidate the molecular arrested in prophase I have one of three following mechanisms underlying the changes in ovarian reserve, outcomes: they undergo atresia, remain quiescent, or oocyte viability, and oocyte health. Although current become recruited to grow. A majority of the follicles diagnostic tools exist, their accuracy in assessing ovarian undergo atresia, a mechanism of apoptotic cell death. reserve remains a significant area of debate. In addition, Atresia reduces the follicle population significantly from once the ovarian reserve has diminished markedly, approximately 7 million to 2 million at birth (Baker few treatments other than assisted reproductive 1963, Picton 2001). Other follicles remain dormant and technologies are effective in treating resulting infertility. are recruited throughout a woman’s reproductive life. Thus, there is potential for the development of better Finally, primordial follicles that enter the growth phase, diagnostic tools and improved treatments in the field of in a process known as initial recruitment, develop reproductive medicine. In this survey of the literature, into secondary follicles and, eventually, antral follicles we broadly review molecular mechanisms of ovarian (McGee & Hsueh 2000). Most antral follicles undergo aging, recent developments in the reproductive field, atresia. However, some are spared by recruitment and and prospective directions. activation, processes that are cyclically regulated under

Figure 2 Schematic of folliculogenesis. During the stages of follicle development, granulosa cells (depicted by dashed lines) proliferate in layers around the growing oocyte. Many primordial and antral follicles undergo atresia during this process. Ovulation occurs when the egg is released at the time of follicle rupture during the antral follicle stage (antral cavity is depicted in blue). Following ovulation, a corpus luteum forms from the remaining antral follicle. If the egg is not fertilized, the follicle will undergo luteal regression and ultimately be degraded.

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Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access Mechanisms of ovarian aging R21 the hormonal influence at the onset of puberty (McGee reserve. Women with DOR have regular menses but & Hsueh 2000, Picton 2001, te Velde & Pearson 2002). exhibit decreased fecundity and responsiveness to Beginning at menarche, with each menstrual cycle, exogenous ovarian hormone stimulation compared to the non-dominant antral follicles become atretic and women of similar ages (ASRM 2020). Ten percent of are degraded to nourish the dominant follicle, which women who present to an infertility clinic in the United will ovulate (Baker 1963). The process of atresia leads States are diagnosed with DOR, and these women to a significant loss of oocytes as women age and is have considerably lower success rates with assisted not well understood. Atresia may occur as a protective reproductive technologies (ART) (Tal & Seifer 2017, mechanism to eliminate poor-quality oocytes that Pastore et al. 2018). More recent data reveal that up to cannot be fertilized. The earliest studies of follicular 32% of women in the United States who undergo in atresia estimate that 99% of human oocyte loss occurs by vitro fertilization are diagnosed with DOR. The age at this mechanism (McGee 2006, Hunt & Hassold 2008). which DOR becomes clinically apparent varies among Though the cause of this increased apoptosis has not yet women, making it difficult to predict if and when a been elucidated, it is certainly a contributing factor to the woman will be affected. This is unlike primary ovarian loss of oocytes during a woman’s reproductive life. Pools insufficiency (POI), a condition that becomes clinically of follicles that are not degraded become recruited and apparent in women younger than 40. Although less activated to grow. Protein kinase B (AKT), mammalian common, POI, formerly called premature ovarian target of rapamycin (mTOR), and signaling pathways failure (POF) or premature menopause, also results in a downstream of these initial activators contribute to decline in oocyte number (Collins et al. 2017). POI has the growth of follicles with each menstrual cycle been proposed to exist as a continuum of changes in (reviewed in Hsueh et al. 2015). Activation also induces ovarian function that includes an 'occult' state in which other morphological changes of follicles that result in women have reduced fecundity but normal FSH levels maturation of part of the oocyte pool; a portion of the and regular menses, a 'biochemical' state with reduced pool remains quiescent. The mTOR serine/threonine fecundity, elevated FSH levels but regular menses, and kinase, in particular, is crucial to help maintain female finally, an 'overt' state with reduced fecundity, elevated fertility, as it signals this selective activation process FSH, and irregular or lack of menses (Welt 2008, Pastore (McGee & Hsueh 2000, Picton 2001). After activation et al. 2018). Women with POI have a failure of ovarian and growth, the granulosa cells of the secondary follicle function and infertility, too. Unlike women with DOR, secrete estrogens, which lead to ovarian follicle growth women with POI exhibit cessation of menses for at least and ultimately ovulation. Following the release of the 3 months to 4 months with postmenopausal levels of egg, the follicle transitions to become the corpus luteum. FSH (>40 IU/L) (Collins et al. 2017). Although POI and If the ovulated egg is fertilized, the corpus luteum will DOR are different in onset and clinical presentations, persist into early pregnancy; if not, the follicle will they share some of the same genetic mutations that are undergo luteal regression. Each month, progression believed to contribute to ovarian aging (Bodega et al. through this cycle is mainly controlled by varying 2006, Greene et al. 2014). Additionally, some women concentrations of estrogen, progesterone, follicle- with DOR still reach menopause at an earlier age of less stimulating hormone (FSH), and luteinizing hormone than 45 years, suggesting clinical overlap between the (LH) (McGee & Loucks 1993). two conditions. These conditions have also been studied By menopause, the primordial follicle pool decreases in mice as a model organism, and quantification of the to about 1000. These changes in follicle number ovarian follicle pool, litter size, and oocyte quality are result in increased activation, leading to a significant parameters commonly used to evaluate DOR and POI in increase in the rate of loss of the remaining follicles, comparison to these disorders in women. For example likely through atresia (Hansen et al. 2008). At the end in mice, CHTF18 (chromosome transmission fidelity of the menopausal transition, there is a complete loss factor 18) has been demonstrated to play a crucial role of follicles. The majority of women reach menopause in female fertility and assuring the quantity and quality around the ages of 49–52 (te Velde et al. 1998, Takahashi of the ovarian reserve (Holton et al. 2020). Chtf18−/− & Johnson 2015). The decline in ovarian reserve, ovarian female mice exhibit age-dependent subfertility with aging, and the onset of menopause are inevitable and fewer offspring beginning at 6 months of age compared occur with age in all women. However, the rate at which to WT females. Ovaries of Chtf18−/− females also have these events ensue and primordial follicles are lost varies fewer follicles at all stages of folliculogenesis, and (Tal & Seifer 2017). Though much of menopause is well the follicle pools are nearly depleted by 6 months of characterized, the molecular basis of ovarian aging is age. Oocytes of poor quality result in aneuploid eggs. far less understood. It is clear that reproductive ability in Thus, the Chtf18−/− female phenotypes resemble that of women ultimately ceases with age, but the mechanism(s) DOR in women (Holton et al. 2020). Utilizing oocyte that underly this process remains uncertain. and DNA samples from patients with POI or DOR, Diminished ovarian reserve (DOR) is a term used and model organisms as investigative tools, has helped to describe a clinically significant decline in ovarian elucidate mechanisms of ovarian aging. https://rep.bioscientifica.com Reproduction (2021) 162 R19–R33

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Cohesion, meiotic recombination, and the spindle FitzHarris 2017). Altered spindle dynamics increased assembly checkpoint susceptibility to transient multipolar spindles, leading to erroneous K-MT attachments and mis-segregation even Chromosomal cohesion in intact sister chromatid pairs (Nakagawa & FitzHarris Mechanisms that ensure accurate meiotic division and 2017). Live time-lapse imaging of spindle assembly in chromosome segregation are essential during oocyte young and old mouse oocytes revealed that initially development to prevent aneuploidy. Aneuploidy 90% of young oocytes formed normal bipolar spindles resulting from chromosome mis-segregation can lead compared to 50% of old oocytes (Nakagawa & FitzHarris to miscarriage, congenital anomalies, and infertility. 2017). Instead, older mouse oocytes transiently Therefore, it is critical that sister chromatids remain developed multipolar spindles consisting of unstable together until anaphase. Cohesins are multi-protein microtubules in a disorganized manner. Multipolar complexes that mediate cohesion between sister spindles had significantly fewer correct kinetochore chromatid arms and at centromeres during both mitosis attachments compared to bipolar spindles, and this led and meiosis (reviewed in Brooker & Berkowitz 2014). to mis-segregation and oocyte aneuploidy (Nakagawa & Studies in mice have shown that with increasing FitzHarris 2017). Although the duration of meiosis I was female age, chromosome cohesion in oocytes naturally not significantly different between older and younger deteriorates (Chiang et al. 2010, 2011). Cohesins in oocytes, there was a delay in establishing correct older oocytes are more susceptible to removal by K-MT attachments in older oocytes. Thus, cohesion separase, a cysteine protease that cleaves the cohesin loss or altered MT dynamics each cause erroneous subunit REC8 (Uhlmann et al. 2000, Waizenegger et al. K-MT attachments, mis-segregation, and ultimately 2000). Decreased cohesion leads to a greater frequency aneuploidy. Errors such as these contribute to pregnancy of chromosome mis-segregation, premature chromatid loss and are consistent with the reported increased separation, and aneuploidy (Chiang et al. 2010,2011, frequency of Trisomy 21 in offspring of older women Holton et al. 2020). Meiotic cohesins have also been (Handel & Schimenti 2010, Nagaoka et al. 2012, Jones shown to decrease in human oocytes with age (Duncan & Lane 2013). Recent studies have suggested that K-MT et al. 2012, Tsutsumi et al. 2014). defects in aged oocytes are more extreme in human Shugoshin-like 2 (SGO2, formerly SGOL2) is a oocytes than those observed in mouse oocytes (reviewed conserved protein that protects centromeric cohesion in Mihajlović & FitzHarris 2018). In human oocytes, the from degrading during mitosis and meiosis (Kitajima et al. distance between sister kinetochores was increased with 2004, Salic et al. 2004). SGO2 localizes to centromeres age in both meiosis I and meiosis II. In some instances, and prevents separase from cleaving cohesin until the the distance led to a 90-degree rotation of homologous metaphase to anaphase transition. Notably, SGO2 chromosomes which resulted in mis-segregation expression decreases in oocytes with advancing age (Zielinska et al. 2015, Mihajlović & FitzHarris 2018). (Lister et al. 2010, Rattani et al. 2013). Depletion of SGO2 The authors speculate that unlike in mouse oocytes, in in mouse oocytes causes loss of centromeric cohesion human oocytes, interkinetochore distances, in addition during anaphase I, leading to premature separation to multiple other factors, contribute to aneuploidy of sister chromatids; depletion also affects proper seen with ovarian aging (Cimini et al. 2001, Zielinska kinetochore-microtubule (K-MT) attachments, as well as et al. 2015). spindle assembly checkpoint (SAC) silencing (discussed further subsequently) (Rattani et al. 2013). SGO2 is Meiotic recombination vital in maintaining proper chromosomal alignment before division and protecting cohesion at centromeres One of the distinguishing and critical features of to ensure accurate chromosome segregation (Rattani meiosis is the process of homologous recombination. et al. 2013). This process, including the formation of DNA double- Deterioration of cohesion in older mouse oocytes strand breaks (DSBs) and repair as crossovers between also results in erroneous K-MT attachments during homologous chromosomes, gives rise to the genetic meiosis (Shomper et al. 2014, Nakagawa & FitzHarris diversity of the gametes and ultimately offspring (Handel 2017). Oocytes from aged mice had significantly more & Schimenti 2010). Meiotic crossovers, cytologically erroneous K-MT attachments than those from younger seen as chiasmata, form direct physical contacts between mice during meiosis I and meiosis II (Shomper et al. nonsister chromatids of homologous chromosomes, 2014). The impact of cohesion loss on attachments allowing the exchange of genetic information. However, was especially significant during meiosis II (Shomper when crossovers occur too close to the centromere, there et al. 2014). Loss of cohesion during meiosis II in older is an increased risk of mis-segregation. In fact, kinetochore mouse oocytes caused K-MT misattachments, leading to proteins that suppress the occurrence of crossovers chromosome lagging and mis-segregation. In aged MI too proximal to the centromere have been identified oocytes, changes in spindle dynamics were the main in lower eukaryotes (Vincenten et al. 2015). Studies cause of erroneous K-MT attachments (Nakagawa & have also reported the dramatic increase in aneuploidy

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Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access Mechanisms of ovarian aging R23 secondary to recombination errors with increased et al. 2011, 2012, Marangos et al. 2015, Vincenten et al. maternal age. Studies of recombination frequencies 2015, Lagirand-Cantaloube et al. 2017, Wang et al. of chromosome 21 have shown that younger women 2017a). have an increase in proximal and distal recombination Other components of the SAC when impaired also events. However, older women have more exchanges contribute to increased aneuploidy with advancing closer to the centromere, resulting in mis-segregation female age. Budding-uninhibited-by-benzimidazole and aneuploidy (Lamb et al. 2005, Ghosh et al. 2009). (BUB1) and BUB1-related (BUBR1) protein kinases Both human and mouse studies suggest that errors in the localize to kinetochores upon activation of the position or frequency of recombination events impact SAC (Lagirand-Cantaloube et al. 2017). Decreased aneuploidy rates (Chiang et al. 2012). As a result of mis- localization of BUB1 and BUBR1 have been shown segregation, near the end of a woman’s reproductive life, to correlate with aneuploidy in aged human oocytes at least 50% of pregnancies are trisomic or monosomic (Lagirand-Cantaloube et al. 2017). The SAC, BUB1, (Wang et al. 2017a). BUBR1, and MPS1 all aid in monitoring proper Based on data from human oocytes, meiotic spindle and chromosome attachment. Theoretically, segregation errors and aneuploidy have been postulated manipulating different components of the SAC could to result from crossover maturation inefficiency (CMI) lead to new therapeutic approaches for age-related (Wang et al. 2017a). CMI is proposed to occur when female aneuploidy. recombination intermediates fail to become mature Breast cancer-associated 1 (BRCA1), a tumor crossovers. Interestingly, it is inferred to occur uniquely suppressor gene, has also been implicated in spindle in 25% of recombination intermediates in human assembly and the SAC. Earlier studies revealed that oocytes and may be a major contributing factor to BRCA1 colocalizes with γ-tubulin at spindle poles chromosome mis-segregation (Wang et al. 2017a). At during meiosis in mouse oocytes (Pan et al. 2008, Xiong least 10% of pregnancies are trisomic or monosomic et al. 2008). Decreasing or depleting BRCA1 in mouse usually due to oocyte aneuploidy (Nagaoka et al. oocytes leads to severely impaired spindle formation and 2012). Meanwhile, mis-segregation in male germ cells chromosome mis-alignment, resulting in chromosome is more rare, occurring only about 2–5% of the time. mis-segregation and aneuploidy (Pan et al. 2008, Xiong In fact, the more frequent occurrence of aneuploidy et al. 2008). Similarly, aged mouse oocytes demonstrate in females than males is likely due, in part, to female- decreased BRCA1 expression, leading to aberrant specific maturation inefficiencyWang ( et al. 2017a). spindle formation and chromosome misalignment. Additionally, CMI in females is hypothesized to affect These findings are also consistent with age-related crossover recombination patterns, leading to increased differences demonstrated in global gene expression aneuploidy, especially with increased age. of oocytes and eggs (Pan et al. 2008). Interestingly, differences in expression of genes involved in spindle assembly, the SAC, and chromosome alignment were Spindle assembly checkpoint also shown (Pan et al. 2008), Although the SAC appears During cell division, the spindle assembly checkpoint to be an important safeguard against aneuploidy even (SAC) monitors attachments of chromosomes to the with advancing maternal age, studies in mice indicate spindle. The SAC prevents the onset of anaphase until that defective SAC function is not a primary cause of all chromosomes are correctly attached to the spindle. female age-related aneuploidy (Duncan et al. 2009). Errors in attachment activate the SAC, causing the cell However, impaired function of the SAC with age is likely cycle to arrest. In oocytes of young mice, inhibition an important contributing factor. of the SAC and monopolar spindle 1 (MPS1), a kinase and component of the SAC, results in reduced securin levels, a phenotype typical of oocytes of old mice (Nabti DNA damage, telomeres, reactive oxygen species, and 2017). Securin is a protein that interacts with separase mitochondrial dysfunction to inhibit cohesin cleavage. Ultimately, inhibition of DNA damage MPS1 in mice leads to early sister chromatid separation. On the contrary, overexpression of MPS1 decreases the As organisms age, cellular mechanisms that repair DNA frequency of premature sister chromatid separation. damage become less effective. Decreased efficacy In human oocytes, the SAC can be inactivated by loss of DNA repair mechanisms leads to DNA damage, of centromeric cohesion (Lagirand-Cantaloube et al. altered DNA repair, and accumulation of mutations 2017). With advancing age, as centromeric cohesion (Gorbunova et al. 2007). In oocytes, this could result decreases, the activity and the stringency of the SAC in poor quality, apoptosis, and ultimately infertility and decrease as well. The compromised SAC is less able to miscarriage. In the presence of DNA damage, cells respond to DNA damage, resulting in premature sister activate a coordinated mechanism called the DNA chromatid separation and aneuploidy (discussed below damage response (DDR), which activates different repair in DNA damage) (Chiang et al. 2010,2012 Nagaoka processes to correct the damage. Activation of the DDR https://rep.bioscientifica.com Reproduction (2021) 162 R19–R33

Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access R24 S Park and others during meiosis can lead to the elimination of oocytes cycle in mitosis and meiosis. CHK2 is activated by ataxia- with unrepaired meiotic DSBs above a threshold level; telangiectasia mutated (ATM) kinase in the presence of this has been shown to occur in mice via the TRP53 DSBs and plays an important role in culling the ovarian and TAp63 pathways (Rinaldi et al. 2020). The DDR follicle pool, especially oocytes with unrepaired or also coordinates with the SAC so that both of these cell induced DSBs or those with unsynapsed homologous cycle checkpoints are able to identify most of the DNA chromosomes (Bolcun-Filas et al. 2014, Rinaldi et al. damage that occurs (Collins & Jones 2016). Among the 2017). Errors detected by CHK2 activate the TRP53/ types of DNA damage, DSBs are the most detrimental TAp63 pathway to eliminate defective oocytes (Bolcun- to cells, often leading to cell death if left unrepaired Filas et al. 2014, Rinaldi et al. 2017). Chk2-null mice (Collins & Jones 2016). Both physically and chemically are fertile and possess similar numbers of oocytes as WT induced DSBs activate the SAC and arrest oocytes in mice. Mice that are doubly deficient in Chk2 and other metaphase I. The arrest blocks APC/C activity and genes necessary for DSB repair showed partial rescue of stabilizes securin. Thus, the SAC is critical in preventing oocyte elimination compared to mice singly deficient DNA damage including DSBs in oocytes (Collins & in these repair genes. Since not all oocytes are rescued Jones 2016). in these double mutants, CHK2 is not essential for the In addition to roles related to spindle assembly and elimination of all defective oocytes; this suggests that the SAC, BRCA genes have functions in DNA repair. other mechanisms are also necessary to repair DSBs in BRCA1, and its closely related tumor suppression oocytes. Nevertheless, CHK2 plays an important role in gene BRCA2, are both involved in the repair of ataxia- DNA damage repair and in ensuring the development telangiectasia-mutated (ATM)-mediated DSBs in oocytes of undamaged healthy oocytes. Thus, as oocytes age, (Yoshida & Miki 2004, Titus et al. 2013, Lin et al. 2017b). surveillance mechanisms of DNA damage are crucial in They do so by regulating the transcription of DNA maintaining the quality of oocytes and female fertility. repair genes. Through hyperphosphorylation events by a multitude of kinases, the proteins encoded by BRCA1 Telomere length and telomerase activity and BRCA2 repair DSBs via homologous recombination (HR) (Yoshida & Miki 2004). Studies have scrutinized the Telomeres, present at chromosome ends in eukaryotes, role of BRCA1 and BRCA2 in ovarian aging and have consist of tandem DNA repeat sequences. They act in found that gene mutations in both mice and humans are concert with proteins and RNA molecules to preserve associated with an age-related decline in ovarian reserve chromosome integrity (Venkatesan et al. 2017). In (Titus et al. 2013, Lin et al. 2017b). Brca1-deficient most cell types in the human body, DNA is lost with female mice had decreased reproductive potential, each cycle of DNA replication as a result of telomere lower numbers of primordial follicles compared to WT shortening. Some cells, including oocytes, possess the females. Follicles of older Brca1-deficient ovaries also enzyme telomerase to help extend telomeric DNA had increased DSBs compared to WT controls (Titus et al. (Keefe et al. 2015). Telomerase is an RNA-dependent 2013). In women with BRCA1-mutations, mean serum DNA polymerase (also known as reverse transcriptase) anti-mullerian hormone (AMH) levels were significantly that ensures the proper DNA replication of the highly lower, consistent with diminished ovarian reserve (Titus repetitive sequences of telomeres (Venkatesan et al. et al. 2013). BRCA1 mRNA and protein expression were 2017). Telomere length is known to directly correlate also decreased in oocytes of older women compared to with both reproductive life span and life expectancy ( younger women (Titus et al. 2013). The impact of BRCA1 Keefe et al. 2007, Kalmbach et al. 2013). and BRCA2 mutations on ovarian reserve has also been Comparison of telomere lengths in the leukocytes studied in women (Lin et al. 2017b). Unaffected carriers of women revealed that telomeres are shorter in of BRCA1 and BRCA2 mutations were shown to have postmenopausal women than those of a similar age but an age-related decline in the number of primordial still menstruating (Gray et al. 2014). In fact, women with follicles consistent with decreased ovarian reserve. This longer telomeres were found to enter menopause up to rate of decline was much faster than that observed in 3 years later than in those with shorter telomeres (Gray the control group without BRCA1 or BRCA2 mutations et al. 2014). This suggests that menopause may occur (Lin et al. 2017b). Interestingly, oocytes of carriers after telomere shortening bypasses or reaches a specific with only BRCA1 mutations showed increased DSBs length. As such, quantification of telomere length in (Lin et al. 2017b). These studies suggest and highlight leukocytes could potentially be used as a biomarker of the importance of DNA repair and BRCA1 and BRCA2 reproductive aging (Gray et al. 2014). function in delaying ovarian aging (Titus et al. 2013, Lin A study of women undergoing IVF suggested that et al. 2017b). telomere length may also predict oocyte quality and be Another component essential to the repair of DSBs is associated with aneuploidy in human embryos (Treff checkpoint kinase 2 (CHK2) (Bolcun-Filas et al. 2014). et al. 2011). Aneuploid embryonic cells were found to CHK2 is a serine/threonine-protein kinase that aids in possess significantly less telomere DNA than euploid checkpoint signaling during the progression of the cell embryonic cells. This highlights the significant role of

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Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access Mechanisms of ovarian aging R25 telomeres in genomic stability. This also suggests that quality without affecting oocyte quantity (Shi et al. decreased telomere length may be associated with 2016). Ovaries of ozone-exposed mice showed high aneuploidy in oocytes and early-stage embryos (Treff levels of ROS and oxidized metabolites. Consistent with et al. 2011). Therefore, oocytes and early embryos with these findings, ozone-exposed female mice had smaller shorter telomere lengths may be an indicator of improper and fewer litters compared to controls. However, serum chromosome segregation, leading to aneuploidy (Treff estrogen levels were no different between the two groups et al. 2011). Thus, in the future, it may be possible to while serum testosterone and progesterone levels were improve IVF success rates by selecting oocytes and decreased in ozone-exposed females. Although markers embryos based on telomere length (Treff et al. 2011). of ovarian reserve, including the size of the primordial However, further studies are needed to determine a follicle pool and expression levels of anti-mullerian possible correlation between telomere length and hormone (AMH), also did not change (Shi et al. 2016). oocyte and embryo quality. The authors propose that long-term moderate oxidative Telomere length and telomerase activity have also damage decreased the fertility and fecundity potential of been associated with specific age-related conditions. mice (Shi et al. 2016). In a separate study, mice that were Studies in human granulosa cells indicate that telomerase exposed to ozone and fed with the antioxidant N-acetyl- homeostasis, the combination of telomere loss and L-cysteine (NAC) had better quality oocytes than those repair, correlates with POI in young women (Butts et al. of ozone-exposed mice not given NAC (Liu et al. 2012). 2009). Specifically, in women 37 years old and younger, Specifically, NAC-treated mice ovulated greater numbers a lack of telomerase activity in human granulosa cells of intact oocytes and yielded significantly increased was associated with ovarian insufficiency. Women numbers of blastocysts compared to unexposed mice that with 'biochemical' POI, an intermediate stage of POI ovulated more fragmented or lysed oocytes. Interestingly, characterized by irregular but spontaneous menstrual NAC treatment also increased oocyte telomere length, periods and elevated serum FSH levels ranging from 10 telomerase activity, and expression of genes associated to 40 IU/L, had reduced telomerase activity and shorter with telomerase activity (Liu et al. 2012). Collectively, telomere lengths in their follicle granulosa cells as well these studies suggest that oxidative damage caused by as peripheral blood leukocytes (Butts et al. 2009, Xu ROS may impact reproductive potential by decreasing et al. 2017). Women with biochemical POI also had the quality of ovarian follicles and oocytes, and that about 50% less telomerase activity and telomere length antioxidants may prevent oxidative damage and delay in these cells compared to women without POI of the oocyte aging (Liu et al. 2012, Shi et al. 2016). same age (Xu et al. 2017). Thus, telomere length and Mitochondrial DNA (mtDNA) damage and telomerase activity may have a significant impact on dysfunction have also been associated with ovarian ovarian aging, and perhaps these parameters could be aging. Mitochondrial DNA is uniquely inherited by used to study changes and decline of ovarian function. the offspring directly from oocyte mitochondria. In females, the mtDNA copy number increases from approximately 200 to 100,000 as oogenesis progresses. ROS and mitochondrial dysfunction Each mitochondrion contains about 2–10 copies (Keefe Reactive oxygen species (ROS) and mitochondrial DNA et al. 2015, Wang et al. 2017b). The mitochondrial- (mtDNA) have been shown to impact cellular aging in free radical theory of aging puts forth the notion that the human body, including in the female reproductive high levels of ROS cause oxidative damage, leading to tract (Rizzo et al. 2012). Importantly, studies have mtDNA mutations in cells; this negatively affects the suggested that excess ROS may negatively impact production and function of electron transport chain ovarian aging. ROS are highly reactive compounds proteins (Harman 1956, Keefe et al. 2015, Wang et al. containing oxygen such as superoxide anions, 2017b). Since mitochondria have limited DNA repair hydrogen peroxide, and hydroxyl radicals. ROS form mechanisms, mitochondrial mutations accumulate in endogenously from the metabolism of oxygen during cells with age at an exponential rate, almost 25 times that cellular processes. Normally, cells are able to eliminate of nuclear DNA (Lynch et al. 2006). Accrual of mtDNA excess ROS (Remacle et al. 1995). When overproduced, mutations has been proposed to compromise oxidative these compounds cause oxidative stress and cellular phosphorylation and increase ROS, resulting in increased damage. High concentrations of ROS in cells lead to mtDNA copy number to offset these effects. Thus, mitochondrial and nuclear DNA damage and apoptosis mtDNA copy number and ATP content have been used (Rizzo et al. 2012). These types of damage have been as parameters to assess aging in oocytes, too. Although shown to adversely affect ovarian follicle development mtDNA mutations and oxidative damage resulting from and ovulation (Guerin et al. 2001, Rizzo et al. 2009, high levels of ROS have been thought to contribute to 2012). In fact, ROS may be lethal to cells but is a normal ovarian aging, studies have been conflicting. Previous part of ovarian aging (reviewed in Rizzo et al. 2012). studies that analyzed mitochondrial DNA deletions In mice, long-term oxidative stress caused by ROS has using nested PCR techniques found no relationship been associated with decreased follicle and oocyte between mtDNA mutations in oocytes or embryos and https://rep.bioscientifica.com Reproduction (2021) 162 R19–R33

Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access R26 S Park and others a woman’s age (Brenner et al. 1998). However, more et al. 2012). Surprisingly, in the same study, women with recent research suggests that an elevated mtDNA copy a poor response to exogenous ovarian stimulation were number in oocytes is more closely correlated with found to have intermediate levels of mtDNA. A separate ovarian aging (Keefe et al. 2007, 2015, Wang et al. study in mouse oocytes found that low copy numbers 2017b). Day 3 and day 5 preimplantation embryos from of mtDNA had minimal effect on oocyte and embryo older women undergoing IVF were found to have higher development until copy numbers were lower than that mtDNA copy numbers compared to those from younger found in human oocytes (Keefe et al. 2015). Although women when analyzed by array comparative genomic there is potential for utilizing mtDNA copy numbers in a hybridization, PCR, and next-generation sequencing clinical setting, further studies are needed to determine (Fragouli et al. 2015). In addition, aneuploid embryos the absolute impact on ovarian aging. from women, regardless of age, were found to have increased mtDNA copy numbers compared to euploid Genetic factors embryos (Fragouli et al. 2015). Consequently, women with increased mtDNA copy numbers of their embryos Genetic factors play an important role in ovarian had decreased implantation rates with IVF (Fragouli aging as indicated by studies suggesting that the ages et al. 2015, Seli 2016, Ravichandran et al. 2017). These of menarche and natural menopause are heritable findings suggest that increased mtDNA copy number is (Snieder et al. 1998, de Bruin et al. 2001, Murabito et al. associated with decreased embryo viability and quality 2005, He & Murabito 2014). A few of the most well- (Wang et al. 2017b). However, conflicting studies have studied genes are mentioned in this section (Table 1). found no significant correlation between mtDNA copy The earliest studies of genetic factors that demonstrate number and implantation rate (Treff et al. 2017, Victor a clear association between ovarian aging and genetic et al. 2017, Klimczak et al. 2018, Kim & Seli 2019). factors identified a premutation of FMR1 in women with Other studies demonstrate that low copy numbers of POI (Man et al. 2017). Currently, it is unclear whether mtDNA are associated with ovarian aging (Bonomi mutations in FMR1 decrease ovarian function due to et al. 2012, Keefe et al. 2015). In particular, women changes during the development of the primordial germ with POI were found to have lower copy numbers of cell population and/or attrition of the oocyte pool during mtDNA in peripheral blood cells compared to women a woman’s life (Reyniers et al. 1993, Man et al. 2017). A of the same age with normal ovarian reserve (Bonomi mutation in FMR1 is associated with fragile X syndrome,

Table 1 Well-studied genes that when mutated or deleted in humans and/or mice impair ovarian function and impact oocyte quantity and/or quality.

Gene Physiologic consequence Phenotype caused by (human) Function of mutation in humans deletion/mutation in mice References FMR1 RNA-binding Premutation POI Early decline in reproduction; Bodega et al. (2006), polysome-associated protein reduced litter size; reduced Pastore et al. (2012), follicle survival; ovarian Elizur et al. (2014), Gleicher mitochondria abnormalities et al. (2015), Banks et al. (2016), Conca Dioguardi et al. (2016), Hipp et al. (2016), Mok-Lin et al. (2018), MYADML Myeloid-associated differentiation POI No studies documented in mice. Schuh-Huerta et al. (2012a) marker like pseudogene function in oogenesis unknown MCM8 Mini chromosome maintenance Gonadal dysgenesis POI Defective meiotic recombination; Maiorano et al. (2005), complex- initiation of DNA subfertility; Impaired Lutzmann et al. (2012), replication, formation of gametogenesis Schuh-Huerta et al. (2012b), replication fork Stolk et al. (2012), Al Asiri et al. (2015) POLG Catalytic subunit of mtDNA Phenotype caused by mtDNA mutations accumulation; Luoma et al. (2004), polymerase deletion/mutation in mice age-related phenotypes; Trifunovic et al. (2004), subfertility Pagnamenta et al. (2006), Stolk et al. (2012) NOBOX Transcription factor for oogenesis POI Accelerated postnatal oocyte loss; Rajkovic et al. (2004), Lin arrest of ovarian follicle et al. (2017a) development at the primordial stage FIGLA Transcription factor in oocyte POI Sterility; small, underdeveloped Rankin & Dean (2000), Soyal gene expression ovaries; depletion of oocytes et al. (2000), Zhao et al. (2008), Tosh et al. (2015) POI, primary ovarian insufficiency.

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Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access Mechanisms of ovarian aging R27 an X-linked dominant genetic disorder characterized by earliest GWA studies to examine ovarian aging evaluated intellectual disabilities and physical abnormalities. The genetic variants associated with FSH and AMH, hormonal mutation is an expansion of the trinucleotide sequence biomarkers of ovarian reserve. This study, performed in a CGG beyond a normal number of approximately 5–40 population of Caucasian and African American women in the 5’ UTR of the FMR1 gene, and expansion of the aged 25–45, revealed a significant correlation between repeat occurs in successive generations within a family variants near and within the myeloid associated (Reyniers et al. 1993, Man et al. 2017). The mutation differentiation marker-like (MYADML) gene and serum is characterized by the number of expanded CGG FSH levels (Schuh-Huerta et al. 2012a); some variants repeats: intermediate corresponds to approximately of MYADML were associated with higher serum FSH, 40–55 CGG repeats, premutation about 55–200 while other variants were associated with lower serum repeats, and a full mutation greater than 200 repeats. A FSH (Schuh-Huerta et al. 2012a). Fewer genetic variants full mutation results in DNA hypermethylation, leading were found to correlate with AMH levels in both to silencing of FMR1 and loss of FMR protein (FMRP) Caucasian and African American women. In addition, expression (Cronister et al. 1991, Devys et al. 1993, both FSH and AMH levels were more variable and Tassone et al. 2000, Juncos et al. 2011). Less than 200 less strongly associated with age in African American CGG repeats confer low to normal FMRP expression. women compared to in Caucasian women (Schuh- Premutation of FMR1 is responsible for 2–11% of POI Huerta et al. 2012a). cases in the Caucasian population, a 20-fold increased A large meta-analysis of GWA studies by Stolk risk compared to women without a mutation (Hipp et al. found an association between the age of natural et al. 2016). Interestingly, the premutation of FMR1 was menopause and minichromosome maintenance more strongly associated with POI than intermediate complex component 8 (MCM8), a gene subsequently expansions of CGG repeats (Bodega et al. 2006). demonstrated to have functions in fertility and Approximately 75% of women were able to have at least folliculogenesis (Stolk et al. 2012). MCM8 encodes a one child with or without the help of infertility treatments. DNA helicase that has previously been shown to function A study of women with FMR1 premutations undergoing in homologous recombination in mice (Maiorano et al. IVF had a statistically significant, but weakly associated, 2005, Lutzmann et al. 2012). In a separate GWA study decreased responsiveness to ovarian stimulation and evaluating ovarian reserve and reproductive lifespan, lower fertilization rates compared to women with >100 women with a specific SNP inMCM8 (rs16991615) were CGG repeats (Banks et al. 2016). shown to reach menopause approximately 1 year later Although FMR1 has been most implicated with POI, than women without this DNA variant. These women other studies have revealed a more general role for this also had higher antral follicle counts by pelvic ultrasound gene in ovarian aging. Different ranges of CGG repeats (Schuh-Huerta et al. 2012b). Other studies utilizing have been associated with varying degrees of decline whole-exome sequencing revealed MCM8 mutations in ovarian reserve. While prior studies suggested in families of individuals with gonadal dysgenesis and that individuals without a mutation in FMR1 (CGG POI (Maiorano et al. 2005, Lutzmann et al. 2012, AlAsiri repeats < 40) show no clinical phenotype, more recent et al. 2015 ). In fact, these findings support prior studies studies suggest an association with diminished ovarian of MCM8 in mice. Targeted deletion of MCM8 in mice reserve (Pastore et al. 2012, Elizur et al. 2014, Gleicher caused defective meiotic recombination, subfertility, et al. 2015,). In fact, fewer than 26 CGG repeats in FMR1 and impaired gametogenesis in both female and male may have the most negative impact on ovarian aging and mice (Lutzmann et al. 2012). MCM8-deficient female infertility. Analysis of young oocyte donors demonstrated mice had smaller ovaries containing fewer, as well that women with CGG repeats of < 26 showed as apoptotic oocytes compared to WT controls. Adult accelerated reductions in serum AMH levels compared ovaries of MCM8-deficient female mice had aberrant- to women with greater copy numbers (Gleicher et al. appearing follicles that were arrested in the primary 2015). These results suggest the importance of FMR1 in stage of follicle development. Collectively, these findings ovarian aging even when CGG trinucleotide repeats are demonstrate the importance of MCM8 in ovarian considered to be in the 'normal' range of < 40 ( Elizur function and suggest a role in ovarian aging. et al. 2014, Gleicher et al. 2015, Banks et al. 2016). POLG, another novel gene identified by genetic Although the loss of FMRP expression leads to fragile studies in humans and mice, has also been associated X syndrome, the mechanisms by which lower levels of with ovarian aging (Trifunovic et al. 2004, Stolk et al. FMRP impact ovarian aging are not yet known. 2012). POLG encodes the alpha subunit of DNA Interestingly, genetic studies in humans and mice polymerase gamma that functions in the synthesis of have revealed genes with noncanonical functions mtDNA (Luoma et al. 2004, Pagnamenta et al. 2006). in the processes of folliculogenesis, ovarian follicle Previously, POLG mutations have been identified in pool maintenance, and ovarian aging. Genome-wide patients with an associated inherited mitochondrial association studies (GWAS) have identified candidate disorder (Luoma et al. 2004). A study examining this genes that are implicated in ovarian aging. One of the condition in individuals from seven families of different https://rep.bioscientifica.com Reproduction (2021) 162 R19–R33

Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access R28 S Park and others ethnic backgrounds found that women who possessed with POI (Tosh et al. 2015). In a separate study of a specific mutation in POLG (Y955C) also had POI Chinese women with POI, a missense mutation and a (Luoma et al. 2004). A subsequent study found the same deletion of FIGLA were present in 2% of women with POLG mutation in three generations of women with POI (Zhao et al. 2008). Mutations in FIGLA caused dominantly inherited POI (Pagnamenta et al. 2006). premature termination of translation and disrupted the Mice with a homozygous knock-in mutation expressing formation of primordial follicles (Zhao et al. 2008). a proof-reading-deficientPolgA , which encodes the Figla-null female mice are sterile, possessing small catalytic subunit of Polg, displayed accumulation ovaries that are depleted of oocytes. In the absence of of mtDNA mutations, age-related phenotypes, and Figla, oocytes cannot develop to maturity (Soyal et al. subfertility. Intriguingly, most mutant females had only 2000). Although gonadogenesis occurs normally during one or two litters of normal size each, and none had embryonic development in both Figla-null females litters after 20 weeks of age (Trifunovic et al. 2004). and males, primordial follicles fail to form in Figla-null The newborn ovary homeobox (NOBOX) is a ovaries following birth (Soyal et al. 2000). gene expressed in primordial germ cells, oocytes, and granulosa cells. It is essential for regulating the Protein metabolism dysregulation and transcription of genes that are critical in early oocyte microenvironmental alterations in the ovary differentiation (Rajkovic et al. 2004, Bouilly et al. 2015). Recent studies have also demonstrated the importance Dysregulation of protein metabolism in oocytes during of NOBOX in women with POI (Bouilly et al. 2015, the crucial period of oogenesis has been suggested to be 2016). Six percent of women with POI were found to deleterious to oocyte quality (Duncan & Gerton 2018). be heterozygous for a mutation in NOBOX (Bouilly In a novel study using RNA-Seq, single growing follicles et al. 2015). In another study of European women with from reproductively young and aged female mice were idiopathic POI, 6.5% of women were heterozygous for analyzed to investigate the effects of age on cellular NOBOX variants. Additionally, a truncating variant of processes in the oocyte. Follicles from older females NOBOX that causes a loss-of-function mutation on the had increased numbers of nucleoli and ribosomes and transcriptional activation of GDF9, a known target of disrupted protein metabolism compared to follicles NOBOX, was identified in women with POI (Lin et al. from younger mice (Duncan et al. 2017). Consequently, 2017a). This novel variant of NOBOX, when inherited older oocytes showed increased expression of specific in a homozygous manner, results in a compromised proteins such as fibrillarin, an rRNA methyltransferase DNA binding domain of the protein and impaired that alters ribosome function and is also known to be transcriptional activation. In addition, this truncation overexpressed in breast and prostate cancers in mice results in aberrant G2/M checkpoint control, which and humans (Duncan et al. 2017). Alterations during the suggests that NOBOX may be important for cell-cycle early oogenesis period have a clear impact on oocyte control (Lin et al. 2017a). Previous studies in Nobox- quality and reproductive potential. deficient mice have shown accelerated postnatal oocyte Fibrosis of ovarian stroma, caused by an accumulation loss and arrest of ovarian follicle development at the of extracellular matrix, has also been associated with primordial stage. At birth, histomorphometric analyses ovarian aging (Briley et al. 2016). When connective tissue showed Nobox-deficient mice had no loss of germ cells. from the ovaries of mice was stained with picrosirius However, by day 7, ovaries had fewer oocytes, fewer red (PSR), the ovaries from older mice aged 22 months primary follicles, and no secondary follicles. By day showed increased staining intensity compared to ovaries 14 postnatally, ovaries were almost devoid of oocytes from younger mice aged 6 weeks. This suggested that (Rajkovic et al. 2004). Consistent with these findings, with advancing reproductive age, there was increased accelerated loss of oocytes in Nobox-deficient females ovarian fibrosis Briley( et al. 2016). In this study, oocytes resulted in infertility (Rajkovic et al. 2004). of older mice had increased expression of genes Factor in the germline alpha (Figla, formerly FIGα) associated with chronic inflammation including more encodes a helix-loop-helix transcription factor that multinucleated macrophage giant cells (Briley et al. regulates oocyte-specific genes and folliculogenesis in 2016). These findings emphasize the importance of the mammals (Soyal et al. 2000). Figla regulates oocyte- stromal microenvironment in reproductive aging. specific gene expression postnatally, particularly the The presence of inflammation in the expression of Zona Pellucida (ZP)1, ZP2, and ZP3. microenvironment has also been shown to play a role These genes encode proteins that comprise the zona in ovarian aging. With age, there is increased low- pellucida, a specialized glycoprotein layer surrounding grade, chronic inflammation that contributes to the the developing oocyte and preimplanatation embryo development of age-related diseases (Foley et al. 2021). (Rankin & Dean 2000, Soyal et al. 2000). Studies have Inflammation in the ovary of mice has been suggested identified mutations of FIGLA in women with POI (Zhao to impact the number and quality of oocytes (Zhang et al. 2008, Tosh et al. 2015). Seven unique variants of et al. 2020, Foley et al. 2021, Lliberos et al. 2021b). FIGLA were identified in a study of 219 Indian women Examination of whole ovaries throughout the lifespan of

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Downloaded from Bioscientifica.com at 09/23/2021 10:09:57AM via free access Mechanisms of ovarian aging R29 mice revealed that specific macrophage markers were telomere shortening, genetic mutations, and alterations in increased in old ovaries compared to young ovaries protein metabolism and the stromal microenvironment. (Zhang et al. 2020). This is consistent with the presence These factors influence both the quantity and quality of of more proinflammatory cytokines, macrophages, and the ovarian reserve. With a greater number of women collagen, which may contribute to the inhospitable, seeking fertility counseling and infertility treatment, inflammatory microenvironment in aging ovaries Foley( methods to evaluate and protect the ovarian reserve, et al. 2021). Interestingly, when factors that contribute and ultimately, mitigate ovarian aging have become to an inflammation state in mice were suppressed, increasingly important to study. In fact, recently, there macrophage infiltration was reduced and follicle has been a substantial investigative effort to further reserves were maintained with aging (Lliberos et al. elucidate measures of oocyte quality over quantity, as 2021a). In humans, metabolic syndrome, particularly markers of the diminished ovarian reserve may not be that resulting from obesity, worsens with increasing an accurate representation of a woman’s reproductive age and has been shown to contribute to the increase potential. As the aging population continues to increase, in the extrinsic and intrinsic inflammatory state Kase( it is also critical to investigate further the complex et al. 2020). The consequence of this includes an relationship between somatic and ovarian aging and its increased risk for the development of cancer including consequences on the reproductive and general health of ovarian and endometrial cancers. Thus both follicles women. This review provides a broad survey and insight and the ovarian microenvironment likely contribute to into mechanisms of ovarian aging, as well as potential a decline in oocyte quality with aging. The extent of areas to explore further to gain a better understanding of this multifactorial impact on oocyte quality and ovarian the complexities of the ovarian reserve. aging warrants further investigation.

Declaration of interest Quality over quantity The authors declare that there is no conflict of interest Current measures of reproductive aging rely heavily on that could be perceived as prejudicing the impartiality of markers of follicle quantity. However, the potential use this review. of these biomarkers has come under scrutiny in a recent study of the association between hormonal biomarkers of ovarian reserve and infertility (Steiner et al. 2017). In Funding this study of reproductive-aged women without a history of infertility, diminished markers of follicle quantity This work was supported by the National Institutes of Health were, in fact, not associated with a lower probability of (NIH) R01 GM106262 grant and the American Society for conceiving. Women with markers of diminished ovarian Reproduction 2020 Research Grant to K.M.B. reserve had similar predicted probabilities of conceiving as women with normal hormonal values (Steiner et al. 2017). Another study found similar conclusions in Author contribution statement reproductive-aged women with a history of one or two K B and L W conceived of the work. L W, S P, and K B wrote miscarriages. AMH levels were not associated with and edited the manuscript. clinical pregnancy loss (Zarek et al. 2016). Women with low AMH levels had similar rates of pregnancy loss compared to women with normal or high AMH levels. These findings highlight the limitations of current References biomarkers that are being used to counsel women about AlAsiri S, Basit S, Wood-Trageser MA, Yatsenko SA, Jeffries EP, Surti U, their reproductive potential (Zarek et al. 2016). The Ketterer DM, Afzal S, Ramzan K, Faiyaz-Ul Haque M et al. 2015 Exome sequencing reveals MCM8 mutation underlies ovarian failure and importance of evaluating oocyte quality over quantity chromosomal instability. Journal of Clinical Investigation 125 258–262. and its effects on ovarian aging are an area of research (https://doi.org/10.1172/JCI78473) that must further be investigated. Allen JW 2010 Fertility and Pregnancy: an Epidemiologic Perspective:Oxford, NY: Oxford University Press. ASRM 2020 Testing and interpreting measures of ovarian reserve: a Conclusions committee opinion. Fertility and Sterility 98 1407–1415. (https://doi. org/10.1016/j.fertnstert.2020.09.134) The field of ovarian biology continues to evolve and Baker TG 1963 A quantitative and cytological study of germ cells in human ovaries. Proceedings of the Royal Society of London: Series B, Biological expand at a remarkable pace as new areas of research Sciences 158 417–433. (https://doi.org/10.1098/rspb.1963.0055) investigate female fertility and ovarian aging. Recent Banks N, Patounakis G, Devine K, DeCherney AH, Widra E, Levens ED, studies have provided a deeper understanding of the Whitcomb BW & Hill MJ 2016 Is FMR1 CGG repeat length a predictor of mechanisms of ovarian aging and the diverse factors in vitro fertilization stimulation response or outcome? Fertility and Sterility 105 1537.e8–1546.e8. (https://doi.org/10.1016/j.fertnstert.2016.02.011) that impact it including deterioration of chromosomal Bodega B, Bione S, Dalprà L, Toniolo D, Ornaghi F, Vegetti W, Ginelli E cohesion, DNA damage and mitochondrial dysfunction, & Marozzi A 2006 Influence of intermediate and uninterrupted FMR1 https://rep.bioscientifica.com Reproduction (2021) 162 R19–R33

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