Epigenetic clocks of female reproductive system aging: Current application and future prospects

Abstract

A progressive decline in fertility is a well-documented aspect of female aging and is associated with a range of cellular and molecular alterations, including genomic instability and modifications in epigenetic regulation. Epigenetic clocks, which estimate biological age based on DNA methylation patterns, have been extensively utilized to evaluate general health status and the risk of various diseases. Despite their broad application, the utility of epigenetic clocks in assessing female reproductive health remains only partially characterized. This minireview consolidates recent advancements in the application of epigenetic clocks to evaluate the functional status of the female reproductive system. The objective is to investigate their potential for quantifying and predicting the biological age of reproductive tissues, thereby establishing a theoretical basis for clinical applications in reproductive medicine. To date, no comprehensive minireview has systematically examined multi-tissue epigenetic clock models in the context of female reproductive aging, positioning this minireview as a novel contribution to the field.

Key Words: Endometrium; Epigenetic clock; Granulosa cell; Leukocyte; Methylation; Ovarian aging; Uterine aging

Core Tip: The aim of this minireview is to examine the application of epigenetic clocks in evaluating the functional status of the female reproductive system. Through the assessment of DNA methylation patterns, epigenetic clocks offer a means of estimating biological age and identifying age-related changes in reproductive tissues. Models derived from blood and reproductive-specific tissues demonstrate potential utility in assessing female reproductive health, particularly in quantifying and predicting the biological age of the reproductive system. Epigenetic clocks developed using endometrial tissue, follicular fluid, leukocytes, and granulosa cells exhibit varying capacities for predicting reproductive aging in females.

INTRODUCTION

Female fertility undergoes a natural decline with increasing age. While this decline progresses gradually in individuals aged 30-35 years, it tends to accelerate after 35 years of age, largely due to a reduction in ovarian reserve and a decline in oocyte quality[1]. Subfertility frequently emerges as one of the earliest clinical indicators of reproductive aging in females. At the cellular and molecular levels, this aging process is characterized by several hallmarks, including age-associated aneuploidy, compromised genomic stability, alterations in epigenetic regulation, telomere attrition, reduced levels of nicotinamide adenine dinucleotide, mitochondrial DNA mutations and dysfunction, enhanced inflammatory responses, and increased apoptosis[2].

Chronological age, which reflects the time elapsed since birth, does not always align with biological aging. In contrast, biological age provides a more accurate reflection of an individual's physiological condition and results from the interplay of intrinsic genetic determinants and external environmental factors. The estimation of biological age through epigenetic biomarkers, particularly DNA methylation, is referred to as the 'epigenetic clock'[2]. When biological age exceeds chronological age, a state referred to as age acceleration, it is often associated with adverse health outcomes. Conversely, when biological age is lower than chronological age, known as age deceleration, it typically corresponds with more favorable health conditions. Among the various methods for estimating biological age, DNA methylation-based epigenetic clocks represent a promising tool for evaluating reproductive health in females. Epigenetic mechanisms, such as DNA methylation, regulate gene expression and influence the function of hormones within the reproductive system. These mechanisms play crucial roles in modulating ovarian activity, the uterine environment, ovulation cycles, and embryonic development processes[3-5]. Age-related variations in DNA methylation provide a quantitative approach to estimate biological age reliably[6]. Moreover, DNA methylation patterns are tissue-specific, and those observed in reproductive cells, such as granulosa cells and oocytes, offer important insights into reproductive system aging[6]. Notably, the reversible nature of DNA methylation creates opportunities for developing individualized therapeutic interventions tailored to an individual's unique epigenetic profile, thereby supporting precise strategies for optimizing reproductive health care[7].

This minireview presents the first comprehensive analysis of the potential applications of epigenetic clocks derived from various tissue sources in the assessment of female reproductive system function. Emphasis is placed on their clinical relevance in quantifying and predicting the biological age of the reproductive system. The aim of this minireview is to clarify the association between DNA methylation-based epigenetic clocks and female reproductive health, thereby contributing to a foundational understanding for future clinical practices. By incorporating multi-tissue epigenetic clock models, the minireview addresses a significant gap in the current literature on reproductive aging and supports future directions in precision reproductive medicine. Table 1 provides a summary of the key studies related to epigenetic clocks in the context of female reproductive aging discussed herein[8-20].

Table 1 Overview of key studies related to the female reproductive epigenetic clock.

Clock	Tissue	Target population	Main findings	Ref.
Ovarian aging
Horvath clock	Leukocytes, cumulus cells	77 infertile women	Horvath clock accurately predicted age based on WBCs but not on cumulus cells. It was not associated with the response to ovarian stimulation	Morin et al[9]
Horvath clock	Leukocytes, cumulus cells	175 infertile women	Epigenetic age acceleration for WBCs samples was associated with a poor ovarian response in women < 38 years	Hanson et al[10]
Horvath, with 500 additional targeted intergenic loci	Leukocytes	39 infertile women	Epigenetic age acceleration was associated with a lower AMH and lower oocyte yield	Monseur et al[8]
Horvath clock, PhenoAge clock, Hannum clock, DunedinPoAm clock	Leukocytes	1000 spontaneous couples, 894 ART pregnant couples	DunedinPoAm epigenetic age acceleration was faster in ART mothers compared to non-ART mothers. Epigenetic age acceleration was not associated with intracytoplasmic sperm injection fathers	Lee et al[11]
Skin-Blood clock, PhenoAge clock	Leukocytes	107 infertile women	Both Skin-Blood and PhenoAge epigenetic age acceleration in WBCs not associated with young women with idiopathic early ovarian aging	Christensen et al[12]
Zbieć-Piekarska clock	Leukocytes	181 infertile women	Epigenetic age was lower in women with live birth	Li et al[13]
Horvath clock, Skin-Blood clock, Granulosa cell clock	Mural granulosa cells, leukocytes	119 infertile women	Leukocytes but not MGCs Horvath age predictor was associated with chronological age. Granulosa cell clock successfully predicted the age of both MGCs and leukocytes	Olsen et al[14]
Granulosa cell clock, Skin-Blood clock	Mural granulosa cells, leukocytes	119 infertile women	Both granulosa cell clock and Skin-Blood epigenetic age acceleration in MGCs and leukocytes were not associated with ovarian reserve	Olsen et al[15]
Horvath clock, Granulosa cell clock, PhenoAge clock, GrimAge clock	Follicular fluid	70 infertile women	GrimAge age acceleration was negatively associated with AMH levels, antral follicle count, the total number of retrieved oocytes, mature oocytes, and fertilized or two-pronuclear oocytes	Knight et al[16]
Horvath clock, Granulosa cell clock, GrimAge clock	Follicular fluid	61 infertile women	Horvath age acceleration of follicular fluid was associated with lower peak estradiol levels and decreased number of total and mature oocytes	Hood et al[17]
Uterine aging
Horvath clock	Endometrium, blood	9 infertile women	Significant correlation between chronological age and Horvath-biological age in endometrium	Olesen et al[18]
Horvath clock	Endometrium	26 women with endometriosis, 17 women without endometriosis	Horvath epigenetic age strongly correlates with chronological age in the endometrium. Horvath age deceleration in endometrial tissue from women with endometriosis	Leap et al[19]
Horvath clock, Hannum clock, GrimAge clock, PhenoAge clock	Myometrial, blood	27 women undergoing cesarean	Horvath, Hannum, and GrimAge epigenetic age were significant correlation with maternal chronological age, for both myometrium and blood samples	Erickson et al[20]

AMH: Anti-Müllerian hormone; ART: Assisted reproductive technology; MGCs: Mural granulosa cells; WBCs: White blood cells.

EPIGENETIC CLOCKS

First-generation epigenetic clocks estimate biological age by exploiting the observation that DNA methylation levels at specific genomic sites change progressively with chronological age[21,22]. Among these, the Horvath and Hannum clocks are widely recognized as representative models[23-25]. In 2013, Horvath[23,24] developed the first multi-tissue epigenetic clock using approximately 8000 samples from 51 tissue and cell types, analyzed via the Illumina 27K and 450K array platforms. This model identified 353 CpG sites with methylation patterns closely associated with aging, enabling accurate age prediction across diverse human tissues and cell types. The Hannum clock was developed from Illumina 450K array data obtained from 656 individuals aged 19-101 years and comprises 71 methylation sites, demonstrating a strong correlation with chronological age (r = 0.96) and a mean absolute error of 3.9 years in validation datasets[25].

Additional first-generation clocks include the Bocklandt clock, Skin and Blood clock, and other DNA methylation-based age predictors[26-28]. In contrast to first-generation clocks, which focus solely on age-associated methylation changes, second-generation epigenetic clocks incorporate additional parameters such as health status, disease risk, and lifestyle factors to provide a more nuanced and personalized estimate of biological age[21,22,29]. Examples of second-generation clocks include PhenoAge and GrimAge[30,31].

The PhenoAge clock was developed using 513 CpG sites to construct a phenotypic model that effectively predicts a variety of age-related outcomes, including all-cause mortality, cancer incidence, health span, and physical function decline[30]. GrimAge, developed using CpG sites present on the Illumina Infinium 450K and Human Methylation EPIC Bead Chip (EPIC) methylation arrays, integrates 1030 CpG sites along with smoking history and serum protein biomarkers related to aging, providing robust predictions of mortality risk and multiple age-related diseases[31].

DunedinPoAm represents a third-generation epigenetic clock[21,22]. This model tracks longitudinal changes in 18 biomarkers associated with the functional health of blood and various organ systems, based on Illumina 450K and EPIC array data collected from individuals within a single birth cohort. By combining DNA methylation data with a wide array of multidimensional health indicators, DunedinPoAm offers a comprehensive and individualized assessment of biological aging rate and health status[32].

EPIGENETIC CLOCKS AND OVARIAN AGING

Ovarian aging is a natural physiological process linked to advancing age and characterized by a decline in both the quantity and quality of oocytes, reflected in the depletion of the ovarian follicle pool[33,34]. This process constitutes a prevalent cause of female infertility and is clinically marked by a reduction in ovarian reserve and diminished responsiveness to ovulation stimulation[35]. In clinical settings, serum anti-Müllerian hormone (AMH) concentrations and antral follicle count (AFC) assessed via ultrasound are established markers for evaluating ovarian reserve function[35,36]. Furthermore, serum AMH levels and AFC serve as indicators of ovarian responsiveness during controlled ovarian hyperstimulation[8]. However, these markers predominantly quantify oocyte numbers and may not reliably reflect oocyte quality[8]. Recently, epigenetic clocks have emerged as promising tools for assessing ovarian aging, with growing interest in their ability to provide novel predictive insights. Application of various epigenetic clock models to leukocytes or granulosa cells may offer new biomarkers for evaluating ovarian aging.

Leukocyte epigenetic clock: A potential tool for assessing ovarian aging

Morin et al[9] applied the Horvath epigenetic clock to evaluate age acceleration risk in infertile women exhibiting either normal or poor ovarian stimulation responses. Initial results indicated that the Horvath epigenetic clock in white blood cells accurately predicted chronological age[9]. Subsequently, a second phase of recruitment was conducted. In this prospective cohort, subgroup analysis of individuals younger than 38 years revealed a significant association between poor ovarian response (POR) and leukocyte-based Horvath clock-predicted age acceleration (P = 0.017)[10]. It is important to consider that varying definitions of ovarian reserve across the two study phases may have influenced the Horvath clock results. Another pilot study expanded the Horvath clock by incorporating an additional 500 target intergenic loci[8]. Findings indicated that women with epigenetic age acceleration exhibited lower AMH levels (P = 0.053) and reduced oocyte yield (P = 0.0020)[8]. These results indicate that biological age, particularly age acceleration predicted by the leukocyte-based Horvath clock, holds potential as an epigenetic biomarker to improve the precision of ovarian reserve assessment models.

Beyond the Horvath clock, other leukocyte-based epigenetic clocks have been investigated. A cohort study employing four epigenetic clocks—Horvath, PhenoAge, Hannum, and DunedinPoAm—examined associations between assisted reproductive technology (ART) usage among mothers and epigenetic age acceleration[11]. After adjusting for confounding factors, mothers who conceived via ART exhibited a 0.021-year faster aging rate as measured by the DunedinPoAm clock compared to non-ART mothers, indicating that DunedinPoAm may outperform other clocks in evaluating biological aging in reproductive-aged women[11]. In contrast, a prospective cohort study using the Skin and Blood and PhenoAge clocks found no evidence of accelerated aging in whole blood samples from young women with idiopathic premature ovarian failure, indicating possible limitations of tissue-specific clocks when applied across different tissues[12]. Additionally, the Zbieć-Piekarska clock, which is based on methylation at five CpG sites, has been associated with live birth outcomes following in vitro fertilization (IVF)[13,37]. Women who achieved live births demonstrated significantly lower epigenetic age compared to those who did not (P = 0.04)[13]. Although a universally accepted epigenetic clock for reproductive function has not yet been established, existing evidence supports a role for epigenetic clocks beyond quantifying ovarian reserve, potentially serving as predictors of broader reproductive health outcomes.

In summary, these studies indicate that epigenetic clocks, particularly those derived from leukocytes, exhibit promise for predicting biological age and evaluating ovarian function and reserve, with some clocks offering greater predictive utility than others. Future research should explore how leukocyte epigenetic modifications, as components of peripheral blood, reflect the ovarian microenvironment, thereby advancing the development of clinically applicable leukocyte-based epigenetic clocks.

Granulosa cell epigenetic clock: A targeted insight into ovarian biological aging

The epigenetic age of follicular somatic cells, including mural granulosa cells (MGCs) and cumulus cells (CCs), as well as follicular fluid, in relation to infertility and POR were examined in several studies.

Studies found that the biological age of CCs, as predicted by the Horvath epigenetic clock, is significantly younger than chronological age and demonstrates no association with ovarian response[9,10]. A notable methodological limitation was the low initial DNA quantity in CC samples. Similarly, MGCs derived from women with low ovarian reserve and from those with normal or high ovarian reserve predicted an epigenetic age significantly lower than chronological age, with an average predicted age difference of approximately 6.8 years[14]. In contrast, the Skin and Blood clock applied to MGCs demonstrated a modest correlation with chronological age (P = 0.02)[14].

The weak correlations observed between the epigenetic age of follicular somatic cells and ovarian aging may stem from the fact that these epigenetic clocks were originally designed to predict outcomes related to chronic diseases or mortality rather than female reproductive health[9,10,14,15]. To overcome this limitation, Olsen et al[14] developed an epigenetic clock specifically tailored for MGCs. This clock was constructed by integrating 27 MGC samples with normal AMH levels into the Skin and Blood clock training dataset (n = 621) and applying elastic net regression analysis to 296 CpG sites, resulting in the Granulosa cell clock. This specialized clock significantly improved the correlation between biological and chronological age in MGCs (P = 0.006), with an average predicted biological age of 32.4 years[14]. Nonetheless, the Granulosa cell clock did not demonstrate an association between age acceleration and ovarian reserve[15].

Follicular fluid, which surrounds the developing oocyte, can be readily aspirated during oocyte retrieval without compromising oocyte viability[38]. Follicular fluid contains a complex mixture of proteins and diverse cell types, including granulosa cells, theca cells, ovarian surface epithelial cells, and non-steroidogenic cells[39,40].

Knight et al[16] analyzed epigenetic data from cell pellets obtained via centrifugation of follicular fluid collected from 70 women undergoing IVF. The Horvath and PhenoAge clocks showed no significant correlation with chronological age, whereas the Granulosa cell clock and GrimAge clock exhibited significant correlations with chronological age[16]. Moreover, GrimAge acceleration was negatively associated with ovarian reserve[16]. Conversely, Hood et al[17] reported that Horvath clock age acceleration in follicular fluid correlated with ovarian stimulation response, characterized by lower peak estradiol levels and fewer total and mature oocytes retrieved. However, neither GrimAge nor Granulosa cell clock acceleration was significantly associated with ovarian stimulation response[17]. While centrifugation of follicular fluid is intended to isolate granulosa cells, these divergent findings highlight the complexity of follicular fluid compared to direct follicular somatic cell sampling.

Collectively, these findings indicate that epigenetic age prediction in follicular somatic cells and follicular fluid relates to ovarian function and responsiveness. However, differential performance of various epigenetic clocks across tissue types underscores the need for developing specialized epigenetic clocks focused on female reproductive health to enhance the accuracy of ovarian function assessment and prediction of reproductive outcomes.

EPIGENETIC CLOCKS AND UTERINE AGING

Uterine aging is closely associated with reproductive and fertility disorders in women, potentially contributing to infertility, miscarriage, pregnancy complications, adverse pregnancy outcomes, and failure of ART[41,42]. Currently, clinical strategies to enhance embryo implantation rates in older women primarily focus on selecting high-quality embryos, utilizing donor oocytes, and encouraging younger women who plan delayed childbearing to consider elective oocyte cryopreservation, as endometrial age is not yet recognized as a clinical risk factor[43].

Emerging research indicates that assessing age acceleration or deceleration through epigenetic clocks may offer a promising approach to predicting risks related to various endometrial conditions, including recurrent implantation failure, cancer, and endometriosis, as well as providing deeper insights into their pathogenesis[44]. DNA methylation-based epigenetic clocks represent novel and powerful tools for advancing reproductive health, particularly among older women and those experiencing infertility.

Endometrium epigenetic clock: Correlation with chronological age

Female fertility decline with age is well established; however, the extent to which uterine aging contributes to this decline remains under debate[44]. Several studies have explored the relationship between uterine aging and chronological age using epigenetic clocks.

The Horvath and PhenoAge epigenetic clocks demonstrate relatively weak correlations with chronological age when applied to endometrial tissue analyzed from blood samples[23,30]. This may be influenced by variations across menstrual cycle stages or the presence of disease states. To examine this, Olesen et al[18] performed endometrial biopsies in two consecutive menstrual cycles in nine women, with biopsies synchronized to the seventh day after the luteinizing hormone (LH) surge (LH + 7). Their results indicated a significant correlation between the Horvath epigenetic clock and participants’ chronological age in endometrial tissue. Additionally, biological age measurements from the two biopsies remained consistent, indicating that the biopsy procedure did not affect subsequent biological age assessment. Nonetheless, the small sample size (n = 9) limits the generalizability of these findings.

Another investigation found that Horvath methylation age strongly correlates with chronological age in the endometrium, with neither menstrual phase nor endometriosis significantly influencing methylation age or epigenetic age acceleration[19]. However, this study relied on data from a public database, which may lack comprehensive details, representing a limitation.

In a separate study involving women undergoing full-term cesarean sections, Horvath, Hannum, and GrimAge epigenetic ages in myometrial tissue showed significant correlations with maternal age, although these correlations were weaker compared to those observed in blood samples[20]. The findings are limited to pregnant women undergoing cesarean delivery and may not be generalizable to a wider population.

Collectively, these studies highlight epigenetic features of endometrial tissue under various physiological conditions and their associations with chronological age, offering valuable insights into uterine aging mechanisms. However, current understanding remains limited, and further research is necessary.

CONCLUSION

This article provides a systematic minireview of the application of epigenetic clocks in evaluating female reproductive system function, focusing on ovarian and uterine aging. By analyzing DNA methylation patterns, epigenetic clocks enable the prediction of biological age and assessment of the aging status within the female reproductive system.

Evidence indicates that epigenetic clocks derived from blood and reproductive-specific tissues, including granulosa cells, follicular fluid, and endometrium, hold potential for assessing female reproductive health. These clocks are particularly valuable for quantifying and predicting the biological age of reproductive tissues. Future research should prioritize the development of tissue-specific epigenetic clocks that incorporate hormonal, environmental, and clinical factors to improve predictive accuracy regarding reproductive outcomes and facilitate personalized approaches to infertility management.

In summary, these findings contribute novel insights into the complex mechanisms underlying female reproductive health and provide scientific support for advancing more precise and individualized treatment strategies. Progress in this area is anticipated to significantly enhance reproductive health outcomes, especially for women of advanced age or those experiencing infertility.