Reproduction predicts shorter telomeres and epigenetic age acceleration among young adult women

Evolutionary theory predicts that reproduction entails costs that detract from somatic maintenance, accelerating biological aging. Despite support from studies in human and non-human animals, mechanisms linking ‘costs of reproduction’ (CoR) to aging are poorly understood. Human pregnancy is characterized by major alterations in metabolic regulation, oxidative stress, and immune cell proliferation. We hypothesized that these adaptations could accelerate blood-derived cellular aging. To test this hypothesis, we examined gravidity in relation to telomere length (TL, n = 821) and DNA-methylation age (DNAmAge, n = 397) in a cohort of young (20–22 year-old) Filipino women. Age-corrected TL and accelerated DNAmAge both predict age-related morbidity and mortality, and provide markers of mitotic and non-mitotic cellular aging, respectively. Consistent with theoretical predictions, TL decreased (p = 0.031) and DNAmAge increased (p = 0.007) with gravidity, a relationship that was not contingent upon resource availability. Neither biomarker was associated with subsequent fertility (both p > 0.3), broadly consistent with a causal effect of gravidity on cellular aging. Our findings provide evidence that reproduction in women carries costs in the form of accelerated aging through two independent cellular pathways.

shortening of lifespan [12][13][14][15][16] , but see 17 and that these costs are exacerbated when resources are limited [18][19][20] . However, most studies of CoR in humans are restricted to modeling mortality as the sole outcome, and are therefore unable to address the underlying biological processes through which CoR might translate into senescence and functional decline.
Among women, CoR likely accumulate predominantly during lactation and pregnancy 21,22 . Lactation is energetically taxing, while the highly invasive hemochorial placentation of human pregnancy places substantial physiological and immunological demands on the female body [23][24][25] . At the cellular level, pregnancy-induced senescence may be mediated through mitotic or non-mitotic pathways, or both. Mitotic -or replicative -cellular aging can be measured using telomere length (TL). Telomeres are non-coding DNA sequences that cap chromosomes, and are required for cell division and survival 26,27 . Telomere length shortens with cell division and chronological age, placing a limit on the number of cell divisions [28][29][30] . At a critical threshold, TL attrition leads to the exhaustion of a cell's proliferative potential, a process referred to as 'cellular senescence' 31,32 . Shorter TL controlling for age in turn predicts higher morbidity and mortality rates [33][34][35][36] .
Pregnancy may also affect cellular aging through pathways operating independently from TL. A powerful emerging marker of non-mitotic cellular aging is epigenetic age (DNAmAge) 37,38 . DNAmAge in human 39 and non-human genomes [40][41][42] is calculated from methylation at a species-specific subset of cytosine-guanine dyads (CpGs), and is strongly correlated with chronological age 38,43 . Independent of a host of associated risk factors in humans, accelerated DNAmAge relative to chronological age is associated with elevated risks for morbidity and mortality [44][45][46] . Vital to capitalizing on epigenetic age as a marker of non-mitotic cellular aging, accelerated DNAmAge predicts senescence and mortality independently of TL in living humans 47,48 , and independently of both TL and the DNA damage response in vitro 37,39 .
Human pregnancy could generate costs to female health and lifespan by shortening TL (mitotic age), accelerating DNAmAge (non-mitotic age), or both. During pregnancy, blood cells proliferate to compensate for fluid volume expansion 49,50 , and women experience a shift towards innate immunity and an increased sensitivity to infection [51][52][53][54] . Data from cell culture, rodent based experiments, and clinical studies show that inflammation and infection increase cell proliferation and DNA damage, both expected to accelerate the pace of telomere shortening [55][56][57][58][59][60][61][62] . Accelerated DNAmAge relative to chronological age has been observed in other pro-inflammatory contexts 63,64 , and with menopause 65 , an important physiological and life-history transition in human females. DNAmAge acceleration arising from menopause, whether naturally-occurring or surgically-induced is attenuated by hormone therapy 65 , suggesting that physiological and hormonal changes like those accompanying pregnancy could have effects on DNAmAge. While recent studies examining TL or DNA damage and pregnancy have yielded mixed results [66][67][68][69] , none have attempted to test for CoR in humans using mitotic and non-mitotic measures of cellular aging simultaneously.
Here, we test for human CoR using mitotic (TL) and non-mitotic (Horvath's DNAmAge 39 ) measures of cellular aging. We test three inter-related hypotheses in a relatively young cohort (age 20-22) of women in the Philippines. First, we ask whether pregnancy history increases mitotic or non-mitotic measures of cellular aging, or both (H1). We consider whether any associations between reproductive history and cell aging are stronger among women of lower socioeconomic status, for whom resource constraints are expected to be highest (H2). Finally, we evaluate the potential for reverse causation by examining the effect of both TL and DNAmAge on the number of pregnancies over the subsequent 4 years (H3).

Results
The relatively young women in our sample (21.7 ± 0.4 years old) displayed a range of reproductive histories. While women who had never been pregnant formed the largest group (n = 507; 61.7%), women having experienced one (n = 174; 21.2%), two (n = 102; 12.4%), and three (n = 28; 3.4%) pregnancies were also well represented. A small subset of women had experienced four (n = 7; 0.8%) or five (n = 3; 0.4%) pregnancies. Although the women in our sample fell into a relatively narrow age range, age-adjusted measures of DNAmAge and TL were themselves uncorrelated (p = 0.64; n = 396), consistent with their independent roles in cellular aging.
Reproductive History and Cellular Aging. TL decreased and DNAmAge acceleration increased with the number of pregnancies in a woman's reproductive history ( Fig. 1 and Table 1). The relationship between gravidity and both measures were also relatively robust -in nested models controlling for a range of potential confounders, effects sizes for pregnancy number remained stable or increased (Table 1) (Table 2).

Discussion
TL and DNAmAge, measures of mitotic and non-mitotic cellular aging, respectively, were both associated with reproductive history in our sample of young women. The relationship between gravidity and cellular aging was relatively robust to a number of potential confounders, and did not appear to be mediated by socioeconomic status, a measure of resource availability. Moreover, neither measure was associated with gravidity over the subsequent 4 years, consistent with a causal effect of the number of pregnancy on cellular aging. Although consistent with theoretical predictions and non-human animal work, this is the first study to our knowledge to examine CoR using both mitotic and non-mitotic measures of cellular aging. Gravidity predicted age-related changes in both TL and DNAmAge in our study, yet several recent studies of CoR in women using TL alone did not find the predicted relationship. The first, conducted among 75 Guatamalan Maya women, reported a positive association between TL and number of surviving offspring over a 13-year period 66 . TL in that study was determined using a combination of saliva-and buccal-derived DNA samples, which unfortunately have not been consistently associated with chronological age [70][71][72] . Furthermore, two separate measures of TL in that study were uncorrelated within individuals between the two timepoints, making comparisons between these findings and our own blood-derived TL findings difficult.
Contrasting with our findings, a study among 620 participants of the US-based CARDIA study did not find evidence for any relationship between parity and TL 69 . Why this study found no evidence for an effect of parity on TL while our findings support CoR is unclear, but could relate to pronounced differences in the age ranges and socio-ecological conditions in the two populations. Notably, markers of oxidative stress appear to be affected  Table 2, Model 7, and statistics from same model. Graphs are labeled and dots are colored by relative aging for each marker (blue, low; red, high) and best fit lines are drawn with 95% CI of beta value. by parity in some socio-ecological contexts but not others 67,68 . Furthermore, TL attrition occurs more rapidly at younger ages 73 , suggesting that any impacts of reproduction on TL shortening could be most pronounced among young women, especially if reproduction begins in adolescence and overlaps with late stages of the mother's own somatic growth 1,74 . Whether or not the relationship between TL and DNAmAge will persist, or if women with accelerated cellular aging will 'recover' and return to more age-typical levels remains an open question.
We found evidence for CoR using both TL and DNAmAge, yet these two measures of cellular aging appear to reflect different biological pathways linking reproductive effort with senescence. Congruent with this interpretation is the observation that TL and DNAmAge measured in the same individuals have been independently associated with aging and mortality in prior studies 48,75 , and capture distinct dimensions of cellular aging 37,38,76 . Accordingly, TL and DNAmAge acceleration were not associated with each other in this study. Accelerated TL attrition -a measure of 'mitotic age' that is modified directly by cellular division -could stem from factors that modify cellular proliferation rates, such as the elevated inflammation, blood cell production, and cell-turnover rates that characterize pregnancy in this and other samples 25,77 .
In contrast to TL, Horvath's DNAmAge is not considered a marker of mitotic age. In vitro DNAmAge is associated with cell passage number, but only in conjunction with the expression of the Telomerase Reverse Transcriptase (TERT) gene 76 , and DNAmAge tracks chronological age even in immortal, non-dividing, and non-proliferative tissues and cells 39 . Although the biological significance of DNAmAge is unknown, it is hypothesized to reflect the integrity of an epigenetic maintenance system, itself responsible for maintaining dynamic regulatory stability within cells 39 . In light of the hypotheses about the functional underpinnings of DNAmAge, our findings are consistent with the prediction that reproduction comes at a cost of 'maintenance' -in this case at the scale of cellular regulatory integrity. Exactly how gravidity might lead to DNAmAge acceleration is unclear, but tradeoffs between protein homeostasis and epigenetic control arising from immune activation or the buffering of oxidative stress are plausible pathways [78][79][80][81] . Indeed, cumulative changes in immune cell composition during pregnancy likely contribute to DNAmAge acceleration with gravidity, although the measure of DNAmAge used here is remarkably robust across tissue types 39 . Nevertheless, the fact that the functionally-distinct measures of TL and DNAmAge show similar associations with gravidity provides strong support for our prediction that reproduction accelerates cellular aging and organismal senescence, at least among the young adult women represented by our sample. Contrary to our prediction that the costs of reproduction would be greatest among individuals with limited resources 18-20 , we found no evidence for an interaction between gravidity and SES in models predicting either TL or DNAmAge acceleration. While women in low SES conditions in our study very likely experience constraints in energy or nutrient availability, it is still unclear to what extent our measure of SES adequately captures limitations in the resources most relevant to CoR. Given the relatively young age of the participants, however, it is possible that the moderating effect of resource limitation will emerge at more advanced ages. SES in this population may also index factors other than resource availability that contribute to accelerated aging, such as less healthful diets or decreases in physical activity. This does not appear to be a major confounding factor, however, as neither TL or DNAmAge were significantly associated with SES in our models.
Importantly, neither measure of cellular aging obtained in 2005 predicted parity over the subsequent 4 years (2005-2009). This suggests that the women in our study are not altering their reproductive output based on their future prospects of health and survival, nor in response to separate physiological or environmental factors also responsible for accelerating cellular aging. This runs counter to a life-history framework whereby 'pace-of-living' as captured by TL and DNAmAge is itself predictive of future fecundity 5,82 .
Intriguingly, currently pregnant women exhibited significantly 'younger' DNAmAge. This finding could reflect the suite of immunological and physiological shifts that occur during pregnancy, including changes in immune cell composition and elevated estrogen levels. At least in some contexts, estrogen can lower oxidative stress 83 , and elevated estrogen is protective for both TL and DNAmAge 65,84 . Pregnancy status and accompanying changes in cell composition may therefore be an important confounder to include in future studies investigating the costs of reproduction in women.
Our findings should be considered in the context of several limitations. First, while we attempt to control for socio-ecological factors that could affect both gravidity and our markers of cellular aging, residual confounding arising from differences in health and/or resources remains a possibility. Although the effects were modest, confounding could help explain the slight decrease in effect size of gravidity after adjusting for SES in models 2 and 6. Future studies employing longitudinal measures of TL and DNAmAge acceleration would minimize the potential effects of such confounders 85 , while modeling lactation and other indices for reproductive effort will be necessary for a more complete estimate of the CoR 86,87 . Finally, the women in this study all fall within a relatively narrow age range in young adulthood (20-22 years old). Because both TL and DNA-methylation change more rapidly early in adulthood 39,73 , it is possible that both measures are particularly sensitive to reproduction at this time. This leaves open the possibility that the relationship between gravidity and cellular aging is transient -and measurements of TL and DNAmAge later in life will prove important for resolving this question.
In sum, our study suggests that gravidity predicts shorter telomeres and epigenetic age acceleration, measures of mitotic and non-mitotic aging, respectively, among the young women in our sample. The consistency in relationships between gravidity and aging in two distinct pathways-one reflecting cellular turnover, and the second a putative marker of epigenomic regulation-support a cost of reproduction from pregnancy in humans. Telomere length. TLs were measured using a modified form of the monochrome multiplex quantitative polymerase chain reaction assay that was externally validated. Details of the protocol and external validity can be found in 89 and since the coefficient of variation has recently been recognized to be an invalid statistic to assess TL measurement reliability 90,91 , intraclass correlation coefficient statistics of measurement error can be found in 92 . Epigenetic age (DNAmAge). 160 ng of sodium bisulfite converted DNA (Zymo AZDNA methylation kit, Zymo Research, Irvine, CA, USA) was applied to the Illumina HumanMethylation450 Bead Chip using manufacturer's standard conditions. Standard methods for background subtraction and color correction were carried out using default parameters in Illumina Genome Studio and exported into R for further analyses. Quality control involved first confirming participant sex and replicate status. This was followed by quantile normalization using lumi 93 on all probes including SNP-associated and XY multiple binding probes. To maximize the number of sites available for the epigenetic age calculator, probes with detection p-values above 0.01 were called NA for poor performing samples only, and were otherwise retained. Horvath's DNAmAge was calculated using an online calculator (http://labs.genetics.ucla.edu/horvath/dnamage/), designed to be generally robust to cell-type differences associated with age 39 . Background-corrected beta values were pre-processed using the calculator's internal normalization algorithms. Socioeconomic status (SES). SES is measured as a combination of income, education, and assets.

Methods
Participants reported their annual income from all sources, including in-kind services, and the sale of livestock or other products by household members during the prior year, which were summed to determine total household income. Incomes were deflated to 1983 levels, and log-transformed. Maternal education (in years) was also reported. Participants also reported on nine assets (coded 0, 1) that were selected to capture population-relevant aspects of social class, including electricity, televisions, refrigerators, air conditioners, tape recorder, electric fans, jeepneys, cars, and their residence. In addition, house construction type (i.e., light, mixed, permanent structure) was coded as 0, 1, and 2, respectively. Thus, asset scores ranged from 0 to 11. A principal components analysis was run on log income and assets at birth (1983) and at sample collection (2005) along with maternal education in Stata (v. 14.1). The first component of variation accounted for 49% of the variation and individual scores for this component of variation were used as our measure of SES.

Statistical methods.
The key predictor variable was gravidity (the number of pregnancies including stillbirths, miscarriages and live births, but not current pregnancies) the respondent reported having had in 2005 (at the time of blood sampling). Control variables included chronological age in 2005 (the time of blood collection), the measure of socioeconomic status (SES) described above, average urbanicity score between 1983 and 2005 94 , and whether the respondent was pregnant at the time of blood collection. Pregnancy status was reported at the time of sampling, and through back-calculation based on parturition within 9 months of the original interview (maternal and infant measures are recorded with each pregnancy as part of ongoing tracking process). DNAmAge acceleration refers to DNAmAge residualized on chronological age. Principal components (PCs) of genome-wide genetic variation were included to control for potential population genetic structure. The derivation of these principal components has been described previously [95][96][97] . As in previous analyses 92,98 , the bivariate association between the first ten principal components and TL were tested. The top principal components up to and including the last one showing a significant bivariate association with TL (10 total) were retained as control variables, with the same 10 principal components used for DNAmAge models.
Linear regression was used for analyses predicting TL and DNAmAge (both normally distributed continuous outcome variables), while generalized linear models with a Poisson family and log-link were used to test for reverse association -that TL/DNAmAge would predict gravidity (a discrete integer) over the subsequent 4 years. The negative effect of time between 2005-2009 surveys and number of pregnancies during this time is an artifact tied to household visit schedules and urbanicity (less urban participants tend to have more pregnancies, and were visited later in the data collection wave). All models were two-tailed with α = 0.05 and were followed by standard model diagnostics 99 . For all linear regressions, the absence of collinearity in predictor variables was confirmed with variance inflation factors (VIFs) for all models falling below 1.1, while Poisson GLMs showed no signs of under-or over-dispersion 100 . Despite the large number of nulliparous women and relatively small number of women with 3 or more pregnancies, all model assumptions were met, and there was no evidence of heteroscedasticity, outliers, or high leverage data points confounding our analyses. All analyses were run in R 101 with ggplot2 102 and stargazer 103 for figures and tables.