Summary Background Ageing is a sex-specific process characterised by a progressive decline in physiological integrity. DNA methylation represents a primary epigenetic hallmark of ageing, yet sex-specific patterns of epigenetic ageing within and across tissues remain poorly understood. This study aims to address these gaps through an integrated analysis of sex-moderated epigenetic ageing across eight human tissues. Methods A total of 137 DNA methylation datasets comprising over 36,000 individuals aged 10–114 years were analysed using a meta-analytic workflow to identify age-associated differentially methylated positions (aDMPs) and regions (aDMRs), meta-regression to assess sex moderation, and pathway enrichment analyses to interpret functional relevance. Findings Individual tissues displayed distinct age-related methylation trajectories, but some DMP sites showed consistent hyper- or hypomethylation across tissues. Across tissues, we identified 68,630 aDMPs (10%) robustly associated with ageing. Age-associated changes at the regional level were less common, with only 80 robust age-associated aDMRs detected across tissues, representing 0.09% of analysed regions. Sex moderation was observed for only 16 aDMPs (0.002%), indicating that sex effects on age-associated DNA methylation are largely tissue-specific rather than shared across tissues. Interpretation Our findings indicate that age-associated DNA methylation changes predominantly occur at isolated CpG sites rather than extended genomic regions and are strongly dependent on tissue and genomic context. The minimal overlap of sex-moderated methylation signals across tissues suggests that age-related sex differences at the epigenetic level are more likely attributable to tissue- and cell-type–specific variation rather than to broadly conserved epigenetic mechanisms shared across tissues. Funding This study was funded by an Australian Research Council (ARC) Discovery project (DP200101830). Séverine Lamon was funded by an ARC Future Fellowship (FT210100278). Nir Eynon was funded by NHMRC Investigator Grant (APP1194159), and a Hevolution/AFAR New Investigator Award in Aging Biology and Geroscience Research. Mandhri D. Abeysooryia was supported by an Australian Government Research Training Program (RTP) Scholarship. Research in context Evidence before this study DNA methylation is widely recognised as a central epigenetic hallmark of ageing. Previous research has demonstrated that some age-related methylation changes are conserved across tissues, forming the basis of pan-tissue epigenetic clocks. Most studies to date have primarily examined age effects in isolation. Although biological sex influences ageing trajectories and susceptibility to nearly all age-related diseases, sex-moderated epigenetic ageing has received limited investigation. Specifically, pan-tissue clocks, including GrimAge and PhenoAge, are “sex-aware” but were trained and validated in mixed-sex cohorts, limiting their capacity to disentangle tissue-specific sex effects. Consequently, it remains unclear whether sex-moderated epigenetic ageing signals are shared across tissues or are tissue-specific. Added value of this study This study provides a large-scale, comprehensive multi-tissue analysis of sex-moderated epigenetic ageing, integrating 137 DNA methylation datasets across eight human tissues and more than 36,000 male and female individuals spanning the lifespan. Our findings show that while age-associated methylation changes are widespread at the CpG level, sex-moderated effects are rare and largely tissue-specific, with minimal overlap across tissues. Implications of all the available evidence Together, the available evidence indicates that epigenetic ageing is predominantly driven by shared, conserved age-related methylation changes, whereas sex differences in epigenetic ageing are modest and context dependent. These sex-related effects are more likely to reflect tissue- and cell-type–specific variation rather than widespread, shared mechanisms. This underscores the need to develop sex-specific epigenetic clocks and to conduct longitudinal cohort and intervention studies to more precisely characterise sex-specific dynamics of epigenetic ageing across tissues.

Postmenopausal women represent the fastest-growing demographic at risk of sarcopenia and cardiometabolic disease, yet exercise biology research remains disproportionately derived from male or hormone-replete phenotypes. Menopause constitutes a chronic endocrine perturbation characterized by sustained reductions in estrogen and progesterone, and altered androgen balance, superimposed on the acute and chronic perturbations induced by exercise. This hormonal shift modifies substrate metabolism, inflammation, redox balance, and recovery capacity, factors that shape molecular responses to exercise across tissues and time. Here, we synthesize current evidence on exercise responses in postmenopausal females across genomics, epigenomics, transcriptomics, proteomics, and metabolomics/lipidomics. Across omics layers, direct data in postmenopausal cohorts remain limited, with frequent underreporting of menopausal status, hormone therapy exposure, circulating hormone concentrations, medication use, and biosampling timing relative to exercise and hormone dosing. We outline a menopause-aware framework for exercise-omics that prioritizes endocrine stratification, repeated sampling across exercise and recovery, and integrative multi-omics approaches linking molecular responses to functional outcomes. We also outline minimum reporting standards to improve reproducibility, inclusivity, and translational relevance. Advancing menopause-aware exercise-omics will be essential for developing precision exercise strategies that improve health span and functional independence in later life.

Proteomic ageing clocks are emerging tools that estimate biological age from protein abundance patterns. Most models are developed using blood capturing inflammatory, metabolic, and extracellular proteins, including secreted signalling factors. Large-scale plasma studies now predict chronological age, disease risk and mortality, but muscle-related models remain indirect, relying on organ-enriched circulating proteins rather than biopsy-derived skeletal muscle clocks. We review current proteomic clock frameworks, computational approaches, platform considerations and validation strategies, highlighting the potential of muscle-based clocks to capture tissue and subcellular-specific ageing processes. We discuss future integration of spatial, single-cell, and multi-omic data to enhance mechanistic insight and translational relevance.

Epigenetic changes, in particular DNA methylation, accumulate with age across different tissues, but whether these changes follow consistent patterns across different organs remains poorly understood. Here we show, through a meta-analysis of more than 15,000 human methylation profiles spanning 17 tissues, that aging produces both conserved and tissue-specific epigenetic signatures. We identify systemic shifts in methylation levels, increases in methylation variability, and growing molecular disorder across tissues. Network analysis revealed tightly connected gene clusters that are not modified by beneficial interventions, alongside a more modifiable cluster linked to NAD+ metabolism, supporting NAD+ as a potential therapeutic target in aging. A gene encoding a cell-adhesion protein, PCDHGA1, emerged as a conserved hub across tissues, implicating cell-to-cell communication pathways in aging across multiple organs. Our methylation atlas therefore provides a resource for dissecting the molecular basis of human aging and for identifying potential biomarkers and translational therapies.

Aging is accompanied by a decline in physiological function and increased vulnerability to disease, with mitochondrial dysfunction and epigenetic alterations recognized as key hallmarks. Nicotinamide riboside (NR), a vitamin B3 precursor to NAD+, and high-intensity interval training (HIIT) have both been proposed to ameliorate aging-related mitochondrial decline, but their effects on skeletal muscle epigenetic aging are not fully elucidated. Here, we assessed the impact of 5-month NR supplementation and 4-6 weeks HIIT on epigenetic age acceleration (EAA, via seven epigenetic clocks) in human skeletal muscle across three independent studies. NR supplementation was associated with reduced muscle EAA, particularly when measured with the PCHannum, MEAT, and DunedinPACE clocks, while HIIT produced opposite effects in some clocks, notably increasing pace of aging by DunedinPACE. Correlation analyses revealed that changes in skeletal muscle mitochondrial content correlated with changes in MEAT-derived EAA after NR and 6 weeks of HIIT. Together, these findings indicate that skeletal muscle epigenetic aging can be modulated by NR and HIIT interventions but in opposing directions, highlighting a potential link between mitochondrial abundance and epigenetic clocks. Further studies are warranted to clarify how NR and exercise regulate epigenetic aging. These results offer new insights into development of strategies for promoting epigenetic outcomes and healthy aging.

ObjectivesTo evaluate the scientific validity of the International Olympic Committee's (IOC) 2021 framework principle of “No Presumption of Performance Advantage,” which suggests that circulating testosterone levels alone does not confer a competitive advantage.DesignA critical review of existing scientific literature concerning the physiological effects of testosterone and male puberty on athletic performance, complemented by a forward-looking proposal for integrating multi-layered biological and performance data.MethodRelevant peer-reviewed studies were examined, focusing on the role of endogenous testosterone, the long-term effects of male puberty, and performance outcomes among transgender women and athletes with differences in sexual development (DSD). Emphasis was placed on strength, power, and endurance metrics. In addition, we outline the emerging potential of combining wearable sensor monitoring with omics profiling to generate a comprehensive, dynamic evidence base.ResultsEvidence consistently shows that male puberty is associated with lasting increases in muscle mass, strength, speed, and endurance due to elevated testosterone. Current evidence also suggests such physiological adaptations may endure even after testosterone suppression, potentially mediated by muscle memory mechanisms such as myonuclei permeance and/or epigenetic changes. If verified, transgender women who experienced male puberty and later reduced testosterone levels retain physical advantages, particularly in strength-based sports. Similarly, athletes with DSD exhibiting male-range testosterone levels also show performance advantages in select events.ConclusionsThe IOC's principle of “No Presumption of Performance Advantage” contradicts a substantial body of scientific evidence. While inclusion and fairness are important goals, the framework's rejection of testosterone as a key determinant of athletic performance risks undermining competitive fairness and athlete safety in sex-segregated sports, particularly where strength and power are critical. Acknowledging testosterone as a key determinant of performance, our study proposes a novel multi-layered evaluation framework that integrates real-time sensor data with omics analyses. This innovative approach supports the development of adaptive, context-specific, and ethically grounded policies that promote fair, inclusive, and safe competition while offering a forward-looking roadmap for future policy and research.

DNA methylation research has vastly expanded over the past decade, producing a wealth of epigenome-wide association studies, biomarker algorithms such as epigenetic clocks, technical performance analyses, and functional annotations for CpG sites. However, these resources remain fragmented across dozens of databases and supplementary files within manuscripts, forcing researchers to spend time and effort on data cleaning and integration prior to meaningful analyses. No single resource currently unifies this information into a centralized, easy-to-query framework. Here, we present CpG Atlas, a curated relational database that integrates 18 distinct annotation layers encompassing over 1.2 million CpG sites across all four generations of Illumina methylation arrays (HM450K, EPIC v1, EPIC v2, and MSA). Built on a snowflake schema with a canonical probe identifier hub implemented in SQL, CpG Atlas consolidates over 800,000 CpG-trait associations, results from Mendelian randomization analyses, CpG membership across 81 epigenetic clocks, array manifest information, and probe reliability data. It further includes specialized layers such as solo-WCGW, CoRSIVs, PRC2 binding, transposon and retroelement annotations, tissue-specific differentially methylated positions across 17 tissues, and hallmarks of aging and cancer. To maximize utility and ease of use, the database is paired with an interactive web tool and a natural language-to-SQL query interface, enabling users to quickly perform complex multi-dimensional queries. Detailed documentation about every data source and table is also provided, facilitating the identification and interpretation of relevant studies. We demonstrate the utility of CpG Atlas through two case studies: a systematic enrichment analysis revealing distinct functional signatures across 16 epigenetic clocks, and an iterative biomarker discovery workflow for IBD that leverages cross-layer integration. Because it is readily scalable simply by adding or updating tables in the database, CpG Atlas provides a continuously evolving and extensible infrastructure for the epigenetics community that supports collaborative research, interpretable biomarker development, and integrative analyses across the growing landscape of epigenetic data.

Aging is a multi-modal process, leaving distinct molecular signatures across the epigenome. DNA methylation is among the most robust biomarkers of biological aging, yet most studies assume linear age relationships and analyze mixed-sex cohorts, overlooking known sex differences. Such approaches risk obscuring critical nonlinear transitions and sex-specific trajectories. We develop SNITCH, a computational framework to detect complex nonlinear methylation trajectories and disentangle shared from sex-divergent patterns. Applied to the array-derived whole-blood methylomes from 252 females and 246 males (ages 19–90 years), SNITCH reveals convergent and divergent epigenetic aging pathways independent of immune cell composition. Nonlinear trajectories are enriched for developmental transcription factor motifs, including NF1/CTF and REST, with known oncogenic roles. Importantly, a female-specific nonlinear cluster is prospectively associated with cancer onset and systemic inflammation in an independent cohort, nominating clinically relevant biomarkers. We replicate the analysis in an additional cohort and highlight consistent nonlinear trajectories. Our results uncover sex-specific, nonlinear aging programs that capture the dynamics of epigenetic change beyond linear models. These findings provide potential candidate biomarkers for early disease risk and advance understanding of how aging trajectories diverge between sexes.

The ageing process is often portrayed as a steady linear decline, as reflected by the methodological frameworks used to study it. Although linear models successfully capture relevant age-related mechanisms, they may fail to identify key transition states occurring during the lifespan. Indeed, ageing entails a series of transitions - from early development, through adolescence, to older age - that are each characterized by distinct biological remodelling events. The study of these underlying patterns, through an appropriate framework, is essential because interventions may only be effective during specific windows when the system transitions to a new state. This Perspective highlights the evidence for non-linearity in ageing, overviews current analytical approaches to capture non-linear processes and then discusses important considerations for ageing research going forward.

BACKGROUND:Skeletal muscle is an important organ for health and movement, largely driven by specific muscle fibres. However, the comparison of fibre-type-specific DNA methylation and protein abundance from the same sample presents challenges. By combining previous methodological approaches we were able to directly compare the methylome and proteome in Type I and Type II human skeletal muscle fibres in males and females. METHODS:We assessed the methylome using the EPICv2 Infinium array and the proteome using liquid chromatography tandem mass spectrometry (LC-MS/MS) from Type I and Type II fibre pools from both males ( n = 7 ) and females ( n = 5 ). RESULTS:We identified 5,689 robust differentially methylated regions (Fisher P-value < 0.001 ) and found strong relationships between methylation and protein abundance in key contractile and metabolic genes. Further, we generated a reference matrix of Type I and Type II fibres and leveraged deconvolution algorithms to accurately estimate fibre-type proportions using whole-muscle DNA methylation data, providing a method to correct for fibre-type in future studies. These results are presented primarily as a resource for others to utilise. CONCLUSION:We provide integrated methylome and proteome profiles of human muscle fibre-types generalisable to both male and females as a freely accessible interactive repository, MyoMETH ( https://myometh.net ), allowing further investigation into fibre regulation. Data are available via ProteomeXchange with identifier PXD066393 and the Gene Expression Omnibus at GSE304045 .

Aging involves widespread epigenetic remodeling across tissues, yet the nature and consistency of these changes remain unclear. We conducted a meta-analysis of more than 15,000 human methylomes spanning 17 tissues, identifying both conserved and tissue-specific aging signatures. We examined linear changes via differentially methylated positions, variability shifts via variably methylated positions, and Shannon-entropy to capture methylation disorder. Network analysis revealed fragile co-methylation modules largely resistant to beneficial perturbation. Key disruptors, including PCDHGA1, MEST, HDAC4, and HOX genes, exacerbated aging signals across tissues. Notably, a resilient module enriched for NAD□ salvage metabolism supports therapeutic targeting of NAD□ in aging. PCDHGA1 emerged as a conserved cross-tissue driver, suggesting protocadherin-mediated adhesion plays a broader role in maintaining structural and signaling stability in multiple organ systems. Our open-access atlas provides a foundational resource for dissecting the molecular architecture of human aging and identifying testable targets for intervention, biomarkers, and translational epigenetic therapies. ### Competing Interest Statement The Regents of the University of California are the sole owner of patents and patent applications directed at epigenetic biomarkers for which Steve Horvath is a named inventor; SH is a founder and paid consultant of the non-profit Epigenetic Clock Development Foundation that licenses these patents. SH is a Principal Investigator at the Altos Labs, Cambridge Institute of Science, a biomedical company that works on rejuvenation. The other authors declare no conflict of interests. National Institute on Aging, https://ror.org/049v75w11 Hevolution Grant National Health and Medical Research Council, https://ror.org/011kf5r70 Australian Research Council, https://ror.org/05mmh0f86

Aging is the greatest risk factor for a multitude of age-related diseases including sarcopenia-the loss of skeletal muscle mass and strength-which occurs at remarkable rates each year. There is an unmet need not only to understand the mechanisms that drive sarcopenia but also to identify novel therapeutic strategies. Given the ease and affordability of husbandry, along with advances in genomics, genome editing technologies, and imaging capabilities, teleost models are increasingly used for aging and sarcopenia research. Here, we explain how teleost species such as zebrafish, African turquoise killifish, and medaka recapitulate many of the classical hallmarks of sarcopenia, and discuss the various dietary, pharmacological, and genetic approaches that have been used in teleosts to understand the mechanistic basis of sarcopenia.

Lipocalin-2 (LCN2), a hormone produced by adipocytes, osteoblasts, and renal tubular cells, is implicated in age-related diseases, including cardio-metabolic disease. To understand the role LCN2 may play in pathological states, we first need to elucidate the relationship between circulating LCN2 with indices of cardio-metabolic health during "normal" aging. This study examined the relationship between serum levels of LCN2, age, and cardio-metabolic measures across the adult lifespan in males and females. We conducted a pooled cohort analysis including 124 community-dwelling males (n = 52) and females (n = 72) (age 20-87 yr, median BMI 25.92 [23.04, 29.81] kg/m2). Serum LCN2 was analyzed using a two-step chemiluminescent microparticle monoclonal immunoassay. The relationship between LCN2 and age was evaluated by linear regression and cubic spline. Simple linear regressions were performed to investigate the relationship between LCN2 and the following variables: BMI, VO2peak, serum glucose, and body composition (DXA). For every 1 yr increase in age, LCN2 levels were 0.26 mg/L higher (P = .007, 95% CI [0.07, 0.45]). Each 1 unit increase in BMI (kg/m2) was associated with 0.88 mg/L higher LCN2 levels (P = .027, [0.10, 1.66]) and each 1 unit increase in VO2peak (mL/kg/min) was associated with 0.38 mg/L lower LCN2 (p = .003, [-0.63, -0.13]).There was no significant relationship between LCN2 and sex, glucose levels or body composition (all p > .05). LCN2 increased linearly across the adult lifespan while it decreased as fitness level increased. Future research should build on these findings to determine whether LCN2 can be used as a biomarker for chronic disease and if exercise can mitigate age-related disease associated with LCN2 changes.

Aging biomarkers are essential tools for quantifying biological aging, but systematic validation has been hindered by methodological inconsistencies and fragmented datasets. Here we show that the ability of traditional aging clocks to predict chronological age does not correlate with mortality prediction capacity (R = 0.12, P = 0.67), suggesting that these metrics capture distinct biological processes. We developed Biolearn, an open-source framework enabling standardized evaluation of 39 biomarkers across over 20,000 individuals from diverse cohorts. The Horvath skin and blood clock achieved the highest chronological age accuracy (R2 = 0.88), while GrimAge2 demonstrated the strongest mortality association (hazard ratio = 2.57) and healthspan prediction (hazard ratio = 2.00). Our systematic evaluation reveals considerable heterogeneity in biomarker performance across different clinical outcomes, with optimal biomarkers varying according to specific application. Biolearn provides unified data processing pipelines with quality control and cell-type deconvolution capabilities, establishing a foundation for reproducible aging research and facilitating development of robust aging biomarkers. Ying, Paulson and collagues have developed an open-source framework, Biolearn, to harmonize and systematically evaluate 39 aging biomarkers across diverse populations, enabling standardized validation and facilitating development of robust aging biomarkers.

Androgens act through androgen receptor (AR) to maintain muscle mass. Evidence suggests that this pathway is influenced by "the gene for speed," ACTN3 (α-actinin-3). Given that one in five people lack α-actinin-3, it is possible that they may respond to androgens differently. Here, we show that α-actinin-3 deficiency decreases AR in muscles of mice and humans (in males and females) and that AR positively correlates with α-actinin-3 expression in a dosage-dependent manner. α-Actinin-3 deficiency exacerbates gastrocnemius mass loss with androgen deprivation in male mice and stunts the muscle growth response to dihydrotestosterone in female mice at the onset of puberty. This is mediated by differential activation of pathways regulating amino acid metabolism, intracellular transport, autophagy, mitochondrial activity, MAPK, and calcineurin signaling, likely driven by seven key genes that are both androgen sensitive and α-actinin-3-dependent in expression. Our results highlight a role for ACTN3 as a regulator of muscle mass and a genetic modifier of androgen action in skeletal muscle.

Integrin complexes facilitate cell communication, playing a role in ligament homeostasis. ITGB2 rs2230528 (C/T) was implicated in anterior cruciate ligament rupture (ACL) risk in a South African cohort. Identifying biologically significant DNA signatures in the predisposition to ACL rupture risk remains important towards understanding mechanisms of ACL ruptures. ITGB2 is essential for the activation of important biological pathways regulated by structural components such as collagens and biomechanical components such as vasculo-endothelial growth factors. This study tested the association of (i) ITGB2 rs2230528 and (ii) allele-allele combinations of ITGB2's network partners (COL5A1 rs12722 C/T, VEGFA rs699947 C/A and VEGFA rs2010963 G/C) with ACL rupture risk. The genetic study was conducted in a combined cohort [n=1279: uninjured controls (CON), n=548; ACL ruptures (ACL), n=731; subgroup with non-contact mechanism of ACL ruptures (NON, n=425)] recruited from Australia, Poland, Sweden and South Africa. The combined cohort, rs2230528 TT (best fit model) was significantly over-represented in the ACL (p=8.00 × 10-8; OR:3.21; 95% CI:2.10-4.89, AIC=1549) and NON (p=1.59 × 10-6; OR:3.11; 95% CI:1.97-4.91, AIC=1191) groups compared to CON. ITGB2 rs2230528-COL5A1 rs12722-VEGFA rs699947-VEGFA rs2010963, the C-C-A-G and C-T-C-G combinations were significantly associated with reduced ACL risk. This study provided additional evidence highlighting ITGB2 as potentially being associated with ACL ruptures even though the gene-gene combinations had a small effect size. Integrins containing the b2 subunit together with its key extracellular matrix components (type V collagen and VEGFA) are potential therapeutic targets for ACL ruptures and potentially other connective tissue-related conditions.

Aging is a multi-modal process, leaving distinct signatures across molecular layers, including the epigenome. DNA methylation changes are among the most robust markers of biological aging. Yet, most studies rely on models assuming linear relationships with age and often analyze mixed-sex cohorts, overlooking well-known sex differences in the timing and nature of aging phases. Such approaches risk obscuring critical, non-linear transitions and sex-specific trajectories that may better capture the biology of aging. We developed a computational approach to detect complex, non-linear trajectories and disentangle shared from sex-divergent patterns. Applied to whole-blood deconvoluted methylomes from 252 females and 246 males spanning ages 19–90 years, this analysis revealed convergent and divergent epigenetic aging pathways independent of immune cell composition. These non-linear trajectories were enriched for developmental transcription factor binding motifs, including NF1/CTF and REST, which are known for their oncogenic potential. Strikingly, a female-specific non-linear cluster was robustly associated with cancer onset and systemic inflammation. Our results uncover sex-specific, non-linear aging programs that better capture the dynamics of epigenetic change than linear models. These findings nominate candidate biomarkers for early disease risk and offer mechanistic insight into how aging trajectories diverge between the sexes. ### Competing Interest Statement The Regents of the University of California are the sole owners of patents and patent applications directed at epigenetic biomarkers for which Steve Horvath is a named inventor; SH is a founder and paid consultant of the non-profit Epigenetic Clock Development Foundation that licenses these patents. SH is a Principal Investigator at Altos Labs, Cambridge Institute of Science. The other authors declare no conflict of interest.

INTRODUCTION:Menopause is associated with immunosenescence and altered immune profiles, potentially reducing immune competence. Physical exercise may counteract these changes by modulating DNA methylation in fitness-related genes. METHODS:This observational bioinformatics study analyzed genome-wide DNA methylation profiles derived from whole blood using two publicly available datasets from Brazilian cohorts (Illumina MethylationEPIC 850K). The analysis included pre- (PreM, n = 13; 34 ± 4.7 years) and postmenopausal (PostM, n = 49; 59.8 ± 4.7 years) women who completed supervised combined exercise training. Four linear models were applied to examine associations between DNA methylation, cardiorespiratory fitness (VO₂ peak), and exercise intervention (pre- vs. post-training), while adjusting for age, fat percentage, and estimated immune-cell proportions (EpiDISH). Interaction terms were tested to assess whether immune cell composition or menopausal status modulated the relationship between VO₂ peak, exercise response, and DNA methylation. RESULTS:After adjusting for baseline values using analysis of covariance (ANCOVA), no statistically significant between-group differences were observed in the estimated immune cell proportions (FDR > 0.05). In PostM, exercise-induced DNA methylation changes were significantly associated with baseline VO₂ peak and were modulated by B cells, CD4+ T cells, NK cells, and monocytes. Functional enrichment highlighted pathways involved in lipid kinase regulation, nucleobase metabolism, and membrane organization. CONCLUSION:Postmenopausal women showed distinct epigenetic patterns in response to exercise, although these differences must be interpreted cautiously given the small premenopausal sample size. These results suggest potential immune cell-associated DNA methylation markers to inform personalized exercise strategies supporting healthy aging in women.

During ageing, the human methylome exhibits both differential (i.e. change in mean) and variable (i.e. change in variance) shifts, along with a general rise in entropy. However, it remains unclear whether DNA methylation sites that increasingly diverge between people (i.e. variably methylated positions (VMPs)) are distinct from those undergoing changes in mean methylation levels (i.e. differentially methylated positions (DMPs)), which changes drive entropy, how they contribute to epigenetic age measured by epigenetic clocks, and whether cell type heterogeneity plays a role in these alterations. To address these questions, we conducted a comprehensive analysis using > 32,000 human blood methylomes from 56 datasets (age range = 6-101 years). Our findings revealed an unprecedented proportion of the blood methylome that is differentially methylated with age (48% DMPs; FDR< 0.005) and variably methylated with age (37% VMPs; FDR< 0.005), with many sites overlapping between the two groups (59% of DMPs are VMPs). We observed that bivalent and Polycomb regions become increasingly methylated and divergent between individuals, while quiescent regions lose methylation in a more homogeneous manner between individuals. Unexpectedly, both chronological and biological clocks, but not pace-of-aging clocks, show a strong enrichment for those CpGs that accrue both mean and variance changes during aging. Furthermore, we uncovered that it is the accumulation of DMPs shifting towards a methylation fraction of 50% that drive the increase in entropy, resulting in an overall smoothening of the epigenetic landscape. However, approximately a quarter of DMPs oppose this direction of change, exhibiting anti-entropic effects. While DMPs were mostly unaffected by changes in cell type composition, VMPs and entropy measurements showed moderate sensitivity to such alterations. This investigation represents the largest to date of genome-wide DNA methylation changes and ageing in a single tissue, offering valuable insights into primary molecular changes that hold meaning for chronological and biological ageing.