The research, published in the open-access journal Epigenetics Communications, represents the first systematic characterization of DNA methylation age in cerebrospinal fluid during the acute recovery period after aneurysmal subarachnoid hemorrhage. DNA methylation age, often called epigenetic age, is estimated from chemical tags called methyl groups that attach to specific positions in the genome, known as CpG sites. Over the past decade, scientists have developed several so-called epigenetic clocks, mathematical models that convert methylation patterns into an age estimate. When a person’s epigenetic age exceeds their chronological age, a phenomenon called age acceleration, it has been linked to cancer, Parkinson’s disease, cardiovascular disease, and all-cause mortality. What remained unknown was whether these clocks, most of which were trained on blood or a mixture of tissues, could produce meaningful readings in cerebrospinal fluid, a tissue that normally contains fewer than five cells per milliliter.

Lead author Lacey W. Heinsberg of the University of Pittsburgh and colleagues tackled this question using an extraordinary dataset: serial cerebrospinal fluid samples from 273 patients recovering from aneurysmal subarachnoid hemorrhage, yielding 850 individual measurements collected over the first 14 days after the hemorrhage. The samples came from external ventricular drains placed as part of standard clinical care to relieve pressure inside the skull, meaning the researchers could track the molecular environment of the injured central nervous system without any additional invasive procedures. For a subset of 72 participants, the team also had blood samples collected during the same early window, allowing a direct comparison between the epigenetic age signatures of the brain’s local environment and the peripheral circulation.

The researchers applied four different epigenetic clocks to their data: the Horvath clock, built from 353 CpG sites and designed as a pan-tissue estimator; the Hannum clock, built from 71 sites and trained on blood; the Levine clock, built from 513 sites and designed to estimate a broader biological metric called phenotypic age; and the Zhang clock, also known as the Improved Precision clock, built from 514 sites and trained on 13,661 blood and saliva samples, by far the largest training dataset of the four. Across all cerebrospinal fluid samples, DNA methylation age correlated moderately to strongly with chronological age, with Pearson correlation coefficients ranging from 0.66 for the Levine clock to a striking 0.97 for the Zhang clock. In blood, the correlations were similarly strong, ranging from 0.83 to 0.97, confirming that even in the chaotic aftermath of a brain hemorrhage, the fundamental relationship between methylation patterns and age remains intact.

The comparison between tissues proved especially revealing. For the 72 participants with both cerebrospinal fluid and blood data, the two tissues produced correlated but clearly distinct age estimates in the Horvath, Hannum, and Levine clocks, with correlations ranging from 0.69 to 0.87. The Zhang clock, however, produced a near-perfect correlation of 0.98 between the two tissues, suggesting that its estimates are essentially interchangeable regardless of whether the DNA comes from the fluid surrounding the brain or from the bloodstream. This robustness extended to the thorniest technical challenge in the study: cell-type heterogeneity. After an aneurysm ruptures, cerebrospinal fluid becomes heavily contaminated with blood cells that gradually clear during recovery, while cells originating in the brain and from the ruptured vessel appear in the mix. Because methylation patterns differ across cell types, these shifting proportions can distort age estimates. No reference-based deconvolution method exists for cerebrospinal fluid, so the team used a reference-free approach to estimate cell-type proportions and repeated their analyses with and without this adjustment.

The results of that sensitivity analysis were decisive. The Zhang clock’s data distributions looked essentially the same whether the estimates came from cerebrospinal fluid or blood, and whether or not cell-type heterogeneity was accounted for. The other clocks showed greater sensitivity to tissue source and cell composition. The authors conclude that, of the four clocks examined, the Zhang clock is the most robust to cell-type heterogeneity and is the recommended choice for complex tissues such as cerebrospinal fluid. This finding is particularly notable because the Zhang clock, like the others, was never trained on cerebrospinal fluid data at all. Its apparent resilience likely stems from the sheer scale of its training dataset, which taught the model to distinguish age-related methylation signals from tissue-specific noise more effectively than its predecessors.

To assess whether epigenetic age shifts during recovery, the team used group-based trajectory analysis, a statistical method that infers distinct subgroups of patients following different temporal patterns. For the Horvath clock, both adjusted and unadjusted for cell-type heterogeneity, the analysis identified four distinct but flat trajectory groups, meaning age acceleration did not change over the 14-day window. The Zhang clock, unadjusted, showed the same pattern of four stable groups. The Hannum clock showed modest temporal variation in its unadjusted form, but once cell-type heterogeneity was controlled, that variation washed out into flat trajectories as well. The Levine clock’s trajectory models failed the study’s posterior quality-control checks due to uncertainty in classifying middle groups, underscoring that this clock is the least well suited to this tissue. Taken together, the trajectories suggest that epigenetic age, as measured by these clocks, is not materially perturbed by the acute pathological environment after aneurysmal subarachnoid hemorrhage.

The study also surfaced intriguing associations between epigenetic aging and patient characteristics. Sex was associated with trajectory group membership in the Hannum clock, with the proportion of women declining as age acceleration rose: the lowest-acceleration group was 93.3 percent female, while the highest-acceleration group was only 16.7 percent female. This aligns with a broader literature showing that men tend to have higher DNA methylation age than women. Independent of trajectory groups, sex was also associated with age acceleration in the Horvath and Hannum clocks, race was associated with acceleration in the Hannum and Levine clocks, and smoking was associated with Levine clock acceleration after cell-type adjustment. Notably, none of the trajectory groups differed meaningfully in injury severity, body mass index, or other clinical characteristics, a somewhat surprising result the authors highlight as deserving further study.

The practical implications cut in two directions. On one hand, the stability of cerebrospinal fluid epigenetic age during recovery suggests that researchers designing future studies could gain statistical power by collecting a single measurement from more participants rather than generating expensive longitudinal methylation data for each patient. On the other hand, the very stability that makes the measurement reliable also suggests that age acceleration from these particular clocks is unlikely to add predictive value for recovery outcomes beyond what chronological age already provides. The authors argue that a clock specifically trained on cerebrospinal fluid methylation data from the acute period after hemorrhage would have the greatest clinical potential, joining a growing movement toward tissue- and disease-specific epigenetic clocks, such as those developed for placental aging and for hippocampal and cortical tissue in Alzheimer’s models.

The study is not without limitations. Blood samples were processed on separate plates from cerebrospinal fluid samples, preventing adjustment for potential plate batch effects, though the authors note that correlation coefficients are invariant to changes in origin and scale, and the near-perfect Zhang clock concordance between tissues supports the validity of their comparisons. The blood subset was small, at 72 participants with a single time point, precluding trajectory comparisons in blood. And because no other cohort with serial cerebrospinal fluid methylation data exists, the findings could not be replicated in an independent sample. Still, as the first window into how the epigenetic clocks behave in the fluid that cushions an injured brain, the work establishes a methodological foundation: the Zhang clock delivers precise, stable, tissue-agnostic age estimates even amid the cellular turbulence of a bleeding stroke, while the field moves toward clocks tailored to the tissues and diseases that matter most.

Subject of Research: DNA methylation age in cerebrospinal fluid during recovery from aneurysmal subarachnoid hemorrhage

Article Title: Characterization of cerebrospinal fluid DNA methylation age during the acute recovery period following aneurysmal subarachnoid hemorrhage

Article References: Characterization of cerebrospinal fluid DNA methylation age during the acute recovery period following aneurysmal subarachnoid hemorrhage. (n.d.). https://doi.org/10.1186/s43682-021-00002-6

Image Credits: AI Generated

DOI: 10.1186/s43682-021-00002-6

Keywords: epigenetic clocks, DNA methylation age, cerebrospinal fluid, aneurysmal subarachnoid hemorrhage, biological aging, age acceleration, cell-type heterogeneity, Zhang clock, Horvath clock, stroke recovery, epigenetics, biomarkers

The study, led by Elise N. Erickson of Oregon Health and Science University together with colleagues at Emory University and Imperial College London, set out to answer two deceptively simple questions. First, do existing epigenetic clocks, mathematical models that estimate biological age from chemical tags on DNA, actually track maternal age in the myometrium at all? Second, and more importantly for future clinical use, can a simple blood draw stand in for a tissue that can only be obtained during surgery? The answer to both, the team reports, is a cautious yes, with one clock in particular standing out from the pack.

Epigenetic clocks rest on a phenomenon called DNA methylation, the attachment of methyl groups to specific positions in the genome that changes in reproducible patterns as cells age. Because methylation patterns shift predictably over the lifespan, researchers can compute a weighted average across hundreds or thousands of CpG sites, the cytosine-guanine sequences where these tags accumulate, to produce an age estimate in years. The team measured methylation across more than 850,000 CpG sites using the Illumina MethylationEPIC BeadChip, then calculated epigenetic age with four well-known clocks: the pan-tissue Horvath clock, the blood-derived Hannum clock, the PhenoAge clock, and GrimAge, which incorporates methylation surrogates of plasma proteins linked to disease and mortality.

The samples came from 27 pregnant individuals undergoing planned cesarean birth at term, of whom 21 had complete, quality-controlled matched specimens of myometrium and blood. Their chronological ages spanned an impressive range, from 20 to 50 years, with a median of 35.5. During each cesarean, surgeons excised a small full-thickness piece of myometrium from the edge of the uterine incision immediately after delivery, which was then flash-frozen in liquid nitrogen. Blood was collected by venipuncture one to two hours before delivery. This paired design, the first of its kind to compare epigenetic age across blood and myometrium in the same individuals, is what gives the findings their unusual value.

The results were strikingly tissue-specific. Three of the four clocks, Horvath, Hannum, and GrimAge, produced epigenetic ages that correlated significantly with chronological age in both tissues, meeting the researchers’ benchmark of a moderate Spearman correlation of at least 0.5. In blood, the correlations were strong, ranging from 0.72 to 0.87. In myometrium, they were somewhat weaker but still significant, between 0.62 and 0.70. The PhenoAge clock, however, failed to track age in uterine tissue at all, showing no significant correlation, and was dropped from subsequent analyses. The pattern suggests that clocks trained on blood or on broad tissue panels do not transfer equally well to the pregnant uterus.

Just as intriguing were the absolute age estimates, which diverged from chronological age in opposite directions depending on the clock. Horvath’s pan-tissue clock judged the myometrium to be dramatically older than the women themselves, estimating every single uterine sample at age 40 or older even though the youngest donor was 20, with a median myometrial estimate of 49.4 years. In blood, the same clock also overestimated age, yielding a median of 39.2 years. By contrast, the Hannum and GrimAge clocks returned estimates younger than chronological age in both tissues. The authors caution that these discrepancies likely reflect how each clock was built and validated, and that pregnancy itself, a state of profound hormonal and immunological change, may reshape methylation patterns in ways the clocks were never designed to capture.

The most clinically consequential finding concerned the relationship between the two tissues. If blood is to serve as a proxy for the uterus, epigenetic age measured in the two must agree. GrimAge delivered the strongest concordance by far, with a Spearman correlation of 0.81 between blood and myometrial epigenetic age, while Horvath and Hannum showed moderate correlations of 0.60 and 0.62. Even more telling, when the researchers calculated age acceleration, the gap between biological and chronological age that signals faster or slower aging, only GrimAge’s acceleration measure correlated significantly across tissues, with a coefficient of 0.49. In other words, if you want to know how quickly a woman’s uterus is aging, GrimAge applied to her blood currently offers the best window available.

Why does this matter? The myometrium is the engine of childbirth, and its function during labor, in the third stage of delivery, and during postpartum involution depends on coordinated smooth muscle contraction. Population studies consistently show that older parturients experience longer labors, need more oxytocin, and face higher rates of labor dystocia, unplanned cesarean, and postpartum hemorrhage. Yet laboratory studies of myometrial contractility by age have produced conflicting results, and many women over 35 deliver without any difficulty while some younger women experience severe dysfunction. Chronological age, in short, is a poor predictor of the biology that matters. A biomarker of uterine biological age could eventually identify, before labor begins, which pregnancies are at risk of myometrial failure.

The study also surfaced a provocative secondary observation: myometrial GrimAge acceleration was moderately correlated with pre-pregnancy body mass index, with a Spearman coefficient of 0.52. The association did not survive correction for multiple testing, and the authors are careful to flag it as potentially spurious, but it aligns with earlier work showing that obesity accelerates epigenetic aging in liver and adipose tissue. Given that higher body mass is independently linked to poor birth outcomes, future studies will need to disentangle the contributions of age and body composition to uterine aging, a task that will require far larger cohorts than the 21 participants analyzed here.

The limitations are real and the authors do not hide them. The sample was small, overwhelmingly of European ancestry, and composed entirely of women who never entered labor, since all samples came from planned cesareans. No in vitro contraction testing was possible with the preserved tissue, and limited clinical data prevented adjustment for confounders. But the consistency of the correlations across three clocks and two tissues, despite these constraints, gives the findings credibility. The study establishes proof of concept that blood-derived epigenetic age can plausibly stand in for the inaccessible uterus, opening the door to studies that follow vaginal births, sample earlier in pregnancy, and test whether epigenetic age acceleration predicts prolonged labor, hemorrhage, or even preeclampsia. It may also, the authors suggest, become a tool for measuring whether interventions such as diet, exercise, or stress reduction genuinely slow biological aging in pregnant women, and for examining how the cumulative wear of social disadvantage, which widens racial disparities in maternal outcomes with advancing age, is written into the genome of the uterus itself.

Subject of Research: Epigenetic clock-based estimation of biological age in maternal blood and uterine myometrium during pregnancy

Article Title: Advancing understanding of maternal age: correlating epigenetic clocks in blood and myometrium

Article References: Erickson, E. N., Knight, A. K., Smith, A. K., & Myatt, L. (2022). Advancing understanding of maternal age: correlating epigenetic clocks in blood and myometrium. Epigenetics Communications, 2(1), Article 3. https://doi.org/10.1186/s43682-022-00010-0

Image Credits: AI Generated

DOI: 10.1186/s43682-022-00010-0

Keywords: epigenetic clocks, DNA methylation, maternal age, myometrium, pregnancy, GrimAge, Horvath clock, biological aging, age acceleration, parturition, obstetrics, biomarkers