Association between Maternal Perinatal Stress and Depression on Infant DNA Methylation in the First Year of Life

Maternal stress and depression during pregnancy and the first year of the infant’s life affect a large percentage of mothers. Maternal stress and depression have been associated with adverse fetal and childhood outcomes as well as differential child DNA methylation (DNAm). However, the biological mechanisms connecting maternal stress and depression to poor health outcomes in children are still largely unknown. Here we aim to determine whether prenatal stress and depression are associated with changes in cord blood mononuclear cell DNAm (CBMC-DNAm) in newborns (n = 119) and whether postnatal stress and depression are associated with changes in peripheral blood mononuclear cell DNAm (PBMC-DNAm) in children of 12 months of age (n = 113) from the Canadian Healthy Infant Longitudinal Development (CHILD) cohort. Stress was measured using the 10-item Perceived Stress Scale (PSS) and depression was measured using the Center for Epidemiologic Studies Depression Questionnaire (CESD). Both stress and depression were measured at 18 weeks and 36 weeks of pregnancy and six months and 12 months postpartum. We conducted epigenome-wide association studies (EWAS) using robust linear regression followed by a sensitivity analysis in which we bias-adjusted for inflation and unmeasured confounding using the bacon and cate methods. To investigate the cumulative effect of maternal stress and depression, we created composite prenatal and postnatal adversity scores. We identified a significant association between prenatal stress and differential CBMC-DNAm at 8 CpG sites and between prenatal depression and differential CBMC-DNAm at 2 CpG sites. Additionally, we identified a significant association between postnatal stress and differential PBMC-DNAm at 8 CpG sites and between postnatal depression and differential PBMC-DNAm at 11 CpG sites. Using our composite scores, we further identified 2 CpG sites significantly associated with prenatal adversity and 7 CpG sites significantly associated with postnatal adversity. Several of the associated genes, including PLAGL1, HYMAI, BRD2, and ERC2 have been implicated in adverse fetal outcomes and neuropsychiatric disorders. This suggested that differential DNAm may play a role in the relationship between maternal mental health and child health.


Introduction
Psychosocial factors such as maternal stress and depression during pregnancy and the rst year of the infants' life affect a large percentage of mothers (pre-and postnatal depression: ~12%; pre-and postnatal stress: ~25% 1,2 ).Gestation and the rst years of life are critical and particularly sensitive periods for child development 3 .Psychosocial stress during this time can affect the developmental trajectory of the child and increase susceptibility to adverse fetal and childhood outcomes [4][5][6][7] .Maternal stress and depression during pregnancy have been associated with adverse fetal outcomes such as low birth weight and preterm birth 8,9 as well as adverse childhood outcomes [10][11][12][13][14][15] .Prenatal stress has been previously associated with neurodevelopmental disorders in children, including an increased risk of attention de cit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), cognitive delay, and schizophrenia [10][11][12] .
Prenatal depression has been associated with variations in white matter integrity, with implications for emotional and behavioral function, cognitive development, language development, and motor development in children [13][14][15] .Mother-infant interactions during the rst year of life play a major role in the behavioral and cognitive development of the child [16][17][18] .Maternal postpartum stress or depression during this time has been associated with behavioral dysfunction (anger, withdrawal) and delayed cognitive development in infants 19,20 .Additionally, infants may express abnormal attachment patterns in response to maternal disengagement 21 .
While maternal stress and depression during the perinatal period have been linked to the etiology of a range of health outcomes in children, the biological mechanisms are still unknown.In the prenatal period, it has been hypothesized that maternal stress and depression affect the fetus through the mother's hypothalamic-pituitary-adrenal (HPA) axis, which produces cortisol in response to stressors 22 .Cortisol, a glucocorticoid (GC) hormone colloquially known as the "stress hormone", can cross the placenta and shape fetal development 23 .While physiological concentrations of GCs are critical for fetal brain development, excess exposure to GCs can be neurotoxic and negatively affect fetal development 24 .Other biological pathways have also been proposed, including catecholamines, oxidative stress, proin ammatory cytokines, serotonin, and the maternal gut-brain axis 6 .Postnatally, it has been hypothesized that cortisol may be passed to the child via breastmilk which can in turn affect child development 25 .However, postpartum maternal stress and depression seem to manifest in the child as behavioral and cognitive dysfunction even after accounting for breastfeeding status 26,27 .At the biological level, high stress and depression levels may induce epigenetic modi cations which in turn can increase the production of maternal cortisol and other potential biological mediators 6,28, 29 .Epigenetic modi cations, such as differential DNA methylation (DNAm) are malleable and sensitive to psychosocial factors 30 .
Epigenetic regulation plays an important role in cell differentiation and development, as well as in mediating adaptive responses to psychosocial in uences.Additionally, DNAm is potentially reversible, suggesting that the methylome could be a therapeutic target for disease treatment and prevention 31 .
Despite widespread interest in the role of epigenetics as a potential nexus between the mother and the child, the literature on maternal psychosocial stress and child DNA methylation is incomplete and contradictory.Several epigenome-wide association studies (EWAS) have identi ed associations between prenatal depression and differential cord blood DNAm [32][33][34][35] ,while only a few EWAS have investigated the associations between prenatal stress and DNA methylation, and the associations they report are inconsistent.For example, one study found null associations between prenatal perceived stress and cord blood DNAm 36 .Another study did nd an association between prenatal perceived stress and neonatal saliva DNAm using a prenatal distress questionnaire and cortisol as measures of prenatal stress 37 .Metaanalyses of large-scale EWAS have also been inconsistent in their ndings regarding the nature and strength of the associations between prenatal stress, depression, and variations in DNAm [38][39][40][41] .In contrast, several candidate gene studies have investigated differential DNAm in genes such as 11β-HSD2, FKBP5, and NR3C1 as potential mediating factors 40,42,43 .For example, differential DNAm in 11β-HSD2 and NR3C1 and their respective interaction with prenatal depression have been associated with adverse neurodevelopmental outcomes in children 42 .During the postnatal period, associations between postpartum stress and depression and child DNA methylation have not been extensively studied despite the established relationship between postpartum maternal mental health and child development.While evidence of an association between postpartum depressive symptoms and numerous differentially methylated gene regions have been identi ed in two studies using next-generation sequencing 44 and the Illumina EPIC array 45 respectively, more EWASs, candidate gene studies, and large-scale meta-analyses have yet to be conducted.
Here, we conducted a prospective EWAS of maternal psychosocial status and child DNAm in a study of 131 children from the Canadian Healthy Infant Longitudinal Development (CHILD) cohort, using wellestablished measures of stress and depression 46 .Speci cally, we investigated whether prenatal stress and depression are associated with variations in cord-blood mononuclear cell DNAm (CBMC-DNAm) in newborns and whether postnatal stress and depression are associated with changes in peripheral blood mononuclear cell DNAm (PBMC-DNAm) among one-year-olds.Stress and depression were analyzed separately, as well as in combination using composite adversity scores to explore the cumulative effect of stress and depression on DNAm.Additionally, to validate the robustness of our ndings across exposure time points, we 1) investigated the associations between prenatal stress and depression and infant DNA methylation (PBMC-DNAm) at 12 months of age and 2) adjusted for prenatal stress in the postnatal stress model and for prenatal depression in the postnatal depression model to control for possible confounding by prenatal exposure.

Study population
The CHILD cohort is a Canadian population-based birth cohort 46,47 .Mothers (N = 3624) were enrolled during their second trimester of pregnancy between 2008 and 2012 and followed through pregnancy at four sites in Canada (Vancouver, Edmonton, Winnipeg, and Toronto).Mother-child pairs were then followed from birth through at least ve years of age.Our analysis sample consisted of 131 mother-child pairs from an atopy-enriched subset of CHILD participants with DNAm data from cord blood and PBMCs, genotype data, prenatal and postnatal mental health measures, and important covariates.Of these, there were 119 infants with complete information for the prenatal models and 113 infants with complete information for the postnatal models.Ethical approval for human subjects research was given by the research ethics board at each study site: McMaster University, University of British Columbia, University of Manitoba, University of Alberta, and The Hospital for Sick Children.Written consent for participation was obtained from the mother at enrollment on behalf of herself and the infant.

DNA methylation measurements
DNA methylation was measured from cord blood mononuclear cells at birth and from peripheral blood mononuclear cells at 12 months of age using the Illumina In nium HumanMethylation450 BeadChip array.Background subtraction and color correction were performed with Illumina GenomeStudio software before data was imported to R for preprocessing.All subsequent preprocessing was performed using R version 3.5.1.
Sixty-ve single nucleotide polymorphism probes were used to check concordances between paired samples and the corresponding probes were removed from the dataset.Next, probes not detected above the background or with fewer than three beads contributing to the signal in at least one sample (n = 5464), X/Y chromosome probes (n = 11,186), and poorly designed probes (n = 31,076) were removed 48 .
Invariant probes as identi ed from a meta-analysis by Edgar et al. were also removed (n = 111,193) 49 .
Samples were determined to be outliers if detected using the detectOutlier function from the lumi package 50 .One CBMC-DNAm sample was detected as an outlier and it and its corresponding PBMC-DNAm sample were removed.The probe design bias was removed using beta-mixture quantile normalization (BMIQ) 51 and batch effects (sentrix ID, sentrix position, and run) were removed using the ComBat function from the sva package 52 .After sampling and probe ltering, 131 samples (overlap of 113 CBMC samples and 119 PBMC samples), 307,566 CBMC probes, and 309,620 PBMC probes remained for analysis.Cell type composition estimates were calculated using the most recent reference data for cord blood 53 and whole blood 54 .Genetic principal components were calculated to adjust for population strati cation.

Maternal stress and depression measurements
Prenatal and postnatal stress were measured using the 10-item Perceived Stress Scale (PSS) 55 .Prenatal and postnatal depression were measured using the Center for Epidemiologic Studies Depression Questionnaire (CESD) 56 .Prenatal stress and depression were measured at 18 weeks and 36 weeks of pregnancy and postnatal stress and depression were measured at six months and 12 months postpartum.For prenatal stress and depression, measurements at 18 weeks and 36 weeks were ztransformed and collapsed by addition to create composite prenatal stress and prenatal depression variables.For postnatal stress and depression, measurements at 6 months and 12 months were ztransformed and collapsed by addition to create composite postnatal stress and postnatal depression variables.

Statistical Analysis
Our analysis pipeline consisted of a primary analysis, bias and in ation adjustment, and a series of sensitivity analyses (Fig. 1).In our primary analyses, we investigated the associations between prenatal stress (z-score) and CBMC-DNAm, prenatal depression (z-score) and CBMC-DNAm, postnatal stress and PBMC-DNAm, and postnatal depression and PBMC-DNAm.Next, we adjusted the estimates from the primary analysis for bias and in ation (e.g., due to unmeasured confounding) to assess the robustness of our primary ndings.Additionally, we further explored the relationship between maternal stress and depression on infant DNA methylation at birth and the rst year of life using a composite adversity score.Finally, we conducted functional enrichment analyses and methylation quantitative trait loci (mQTL) mapping as secondary analyses for all signi cant CpG sites to support our ndings.

Confounding Assessment
Confounding was assessed by constructing directed acyclic graphs (DAGs).DAGs were created for each time point: prenatal stress and depression -CBMC-DNAm and postnatal stress and depression -PBMC-DNAm (Figure S1).Potential confounders were selected based on existing literature.All models were adjusted for cell-type proportions and population strati cation as represented by the rst ve genetic principal components, which explained > 90% of the variation.Prenatal models were additionally adjusted for prenatal smoking, household income, child sex, maternal age, and study center.Postnatal models were additionally adjusted for postnatal tobacco exposure, prenatal tobacco exposure, household income, child sex, birth weight, maternal age, and study center.Prenatal tobacco exposure was ascertained at the 18th week of pregnancy and was dichotomized into a binary variable (0 = zero cigarettes smoked per day, 1 = at least 1 cigarette smoked per day).Postnatal tobacco exposure was ascertained at 3, 6, and 12 months postpartum using the average number of cigarettes smoked in the household per day (Table S1).Total postnatal tobacco exposure scores were calculated as the weighted average across the three time points.

Bias and In ation Adjustment
To adjust for bias due to unmeasured and residual confounding, we implemented the bacon and cate methods for bias adjustment.Bacon adjusts for in ation by utilizing a Bayesian method to estimate the empirical null distribution 57 .Cate adjusts for unmeasured confounding by rst estimating the number of unmeasured confounders using bi-cross validation factor analysis and then correcting for this bias 58 .

Sensitivity Analyses
We conducted three sensitivity analyses to assess the robustness of our primary analysis.First, to validate the robustness of our ndings across exposure time points, we investigated the associations between prenatal stress and depression and infant DNA methylation (PBMC-DNAm) at 12 months of age.Second, we adjusted for prenatal stress in the postnatal stress model and for prenatal depression in the postnatal depression model to control for possible confounding by prenatal exposure.Multicollinearity between covariates was evaluated by calculating the variance in ation factors for each predictor (Table S2).Finally, to investigate the cumulative effects of stress and depression, we created a composite adversity score for each time point.For the prenatal adversity score, prenatal stress and depression were z-transformed and collapsed.Similarly, for the postnatal adversity score, postnatal stress and depression were z-transformed and collapsed.

Functional Enrichment Analysis
To support our ndings, we conducted follow-up analyses for all signi cant CpG sites.To identify potential biological pathways that may be altered by differential DNAm, we conducted gene ontology functional enrichment analysis using the gometh function of the missMethyl package 59 .We utilized both the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set collections available from the R package.Gometh identi es GO terms and KEGG pathways that are overrepresented among genes containing differentially methylated CpG sites

Methylation Quantitative Trait Loci (mQTL) Mapping
To quantify the potential genetic in uence on DNAm levels at signi cant CpG sites, we identi ed mQTLs using the GoDMC API 60 .GoDMC is a database comprising of mQTLs from over 32,000 participants from 36 cohorts using the 450K BeadChip.We ltered the results by a stringent p-value threshold of 1e-14, as the authors recommended.

Study population characteristics
This analysis sample included 131 infants with DNAm data at birth and 12 months, genotype data, and other relevant covariates, with a subset of 119 infants and 113 infants that had complete cases for the prenatal and postnatal models, respectively (Table 1).This cohort is comprised primarily of children from higher socioeconomic backgrounds, with 45% of the population having a household income greater than $100,000 (CAD).Less than 4% of mothers smoked during pregnancy.The median maternal depression and stress scaled scores were generally low at both antenatal and postpartum time points.The median stress scaled score increased postpartum (-0.1 to 0.03), while the median depression scaled score decreased postpartum (-0.4 to -0.6).  1 Weighted average of postnatal smoking across all three timepoints.Categorical data of postnatal smoke exposure across the three timepoints can be found in Table S1. 2 For the whole blood reference, only neutrophil cell type estimates are provided.  Weighted average of postnatal smoking across all three timepoints.Categorical data of postnatal smoke exposure across the three timepoints can be found in Table S1. 2 For the whole blood reference, only neutrophil cell type estimates are provided. 1Weighted average of postnatal smoking across all three timepoints.Categorical data of postnatal smoke exposure across the three timepoints can be found in Table S1. 2 For the whole blood reference, only neutrophil cell type estimates are provided.

Prenatal stress and depression and DNAm in newborns
Our analysis pipeline consisted of a primary analysis, bias and in ation adjustment, and a series of sensitivity analyses (Fig. 1).We identi ed a signi cant association between prenatal stress and differential newborn CBMC-DNAm at eight CpG sites and between prenatal depression and differential PBMC-DNAm at two CpG sites (Fig. 2; Table 2; Figures S2-S3).One CpG site (cg12390344; LAMA3) exhibited signi cantly differential DNAm for both prenatal stress (β=-9.64E-03,P = 1.98E-08) and prenatal depression (β=-9.61E-03,P = 1.06E-07).Beta estimates for prenatal stress and depression were correlated for CpG sites with p-values less than 5.0E-04 (Figure S4).After adjusting effect estimates for in ation with bacon¸ none of the eight CpG sites in association with prenatal stress remained signi cant and only one of 11 CpG sites remained signi cant in association with prenatal depression (cg02121104 (EIF2B2)).
The remaining CpG sites exhibited suggestive p-values (suggestive threshold = 1E-05) (Table S3; Figure S5).Using cate to adjust for in ation, no CpG sites remained signi cant or exhibited suggestive p-values for either prenatal stress or depression (Table S3; Figure S6).  1 The mean effect size represents the difference in DNA methylation according to the range of the distribution of β values which are on a 0 to 1 scale.

Postnatal stress and depression and DNAm in infants
We identi ed a signi cant association between postnatal stress and differential child PBMC-DNAm at 12 months of age at eight CpG sites and between postnatal depression and differential PBMC-DNAm at 11 CpG sites (Fig. 3; Table 3; Figures S7-S8).There was no overlap between postnatal stress and depression CpG sites.Beta estimates for postnatal stress and depression were correlated for CpG sites with p-values less than 5.0E-04 (Figure S9).After adjusting with bacon, six of the eight CpG sites remained signi cant in association with postnatal stress and the other two CpG sites exhibited suggestive p-values.For postnatal depression, ve of the 11 CpG sites remained signi cant in association with postnatal depression and the other 6 CpG sites exhibited suggestive p-values (Table S3; Figure S10).With cate, two CpG sites (cg10770652 (BRD2); cg25301180 (ERC2)) were suggestive in association with postnatal stress and one CpG site (cg05051393 (ASF1A)) was signi cant in association with postnatal depression (Table S3; Figure S11).  1 The mean effect size represents the difference in DNA methylation according to the range of the distribution of β values which are on a 0 to 1 scale.

Robustness across different exposure timepoints
After investigating prenatal stress and depression were associated with CBMC-DNAm in newborns, we additionally assessed whether prenatal stress and depression were also associated with PBMC-DNAm at 12 months of age and whether there was overlap between both timepoints.A signi cant association between prenatal stress and differential PBMC-DNAm was found at four CpG sites and between prenatal depression and differential PBMC-DNAm at eight CpG sites (Table S4; Figure S12).For one PBMC-DNAm CpG site, we found robust associations across both exposure time windows (i.e., pregnancy/prenatal and 1st year of life).CpG site cg09730369 (HYMAI; PLAGL1) exhibited signi cantly differential PBMC-DNAm for both prenatal depression (β = 7.33E-03, P = 1.79E-12) and postnatal depression (β = 5.29E-03, P = 1.34E-07).Beta estimates across prenatal CBMC-DNAm-and postnatal PBMC-DNAm-associated CpG sites were also correlated (Figure S13; Figure S14).Additionally, we identi ed robust associations across prenatal stress and depression and PBMC-DNAm at one CpG site.

Adjusting for prenatal stress or depression in the postnatal models
Most of the signi cant CpG sites from the main analysis were still either signi cant (cg10601057 (CAB39) and cg10770652 (BRD2) for postnatal stress; cg11284959 (intergenic) and cg11452653 (PIP5K1C) for postnatal depression) or at least suggestive (P < 0.05 for all but two CpG sites) after adjusting for prenatal stress or depression in the postnatal models (Table S5).The VIFs were not indicative of problematic multicollinearity (Table S2), but the results should still be interpreted with caution given the moderately high Pearson correlations between the two time points (prenatal and postnatal stress: 0.56; prenatal and postnatal depression: 0.65).
Using a combined adversity score to explore the cumulative effects of stress and depression on DNAm The correlation between prenatal stress and depression (r = 0.82) and postnatal stress and depression (r = 0.86) was quite high.There was substantial overlap between the combined adversity score models and the separate stress and depression models from the main analysis.Two CpG sites were signi cantly associated with prenatal adversity and these sites were also signi cantly associated with prenatal depression (cg12390344 (LAMA3)) and prenatal stress (cg12390344 (LAMA3) and cg21544975 (GPR133)) (Figure S15-S16, Table S6a).The remaining six CpG sites signi cantly associated with prenatal stress and one CpG site signi cantly associated with prenatal depression were at least suggestive in the prenatal adversity score model (all p-values < 2.85E-05).Seven CpG sites were signi cantly associated with postnatal adversity and six of these sites were also signi cantly associated with either postnatal stress (three CpG sites) or postnatal depression (three CpG sites) (Figure S17-S18, Table S6b).The CpG site that was not signi cantly associated with postnatal stress or depression was suggestive of an association with postnatal stress and depression (Table S7; cg23102197 (TRIM49); stress: p-value = 4.64E-06; depression: p-value = 1.02E-06).The remaining CpG sites were all at least suggestive of an association in the postnatal adversity score model (all p-values < 7.78E-05).Among overlapping CpG sites, the magnitude of effect was consistently stronger with the combined adversity score in comparison to the individual stress and depression models (Table S6a-S6b).

Secondary Analyses Functional Analysis
After correction for multiple testing (FDR < 0.05), we did not identify any GO terms or KEGG pathways with an overrepresentation of genes containing signi cantly, differentially methylated CpGs that would indicate an enriched biological pathway.The top GO terms and KEGG pathways for each model are included in the supplement (Table S8a and Table S8b).

Discussion
In this prospective cohort, we identi ed signi cant associations between maternal psychosocial stress and differential DNA methylation in the rst year of life.Maternal stress and depression were associated with variations in the CBMC and PBMC methylome for both prenatal and postnatal exposures.Additionally, cumulative stress and depression was associated with similar variations in CBMC-and PBMC-DNAm yet demonstrated larger effect estimates.In our primary analyses we identi ed eight CpGs signi cantly associated with prenatal stress and 11 CpGs associated with prenatal depression.Additionally, eight CpGs were signi cantly associated with postnatal stress and 11 CpGs were associated with postnatal depression.
While maternal mental health has been extensively studied in association with childhood outcomes, investigations of the potential epigenetic mechanisms remain sparse, and ndings have been inconsistent.For example, large-scale meta-analyses have not been able to identify replicable evidence of an association due to reasons such as population heterogeneity and differences in stress/depression measures that were used.One meta-analysis investigating the association between prenatal stress (composite stress score calculated from four stress domains) and cord blood DNAm (450K) did not nd any evidence of an association (N = 1740) 39 .In contrast, another meta-analysis from the Pregnancy and Childhood Epigenetics (PACE) consortium investigating the association between prenatal stress (composite stress score calculated from ve stress domains) and cord blood DNAm (450K and EPIC) did nd evidence of an association at ve CpG sites (N = 5496) 38 .However, none of the identi ed CpG sites were signi cant in our study (Table S10a).Large-scale meta-analyses have yet to be conducted for the associations between prenatal depression, postnatal stress, postnatal depression, and child DNA methylation.Several EWAS of prenatal depression and CBMC-DNAm have identi ed signi cant associations, however, none of these associations were replicated in our study 32,33,35 (Table S10b).For example, an EWAS that investigated the association between prenatal depression (Edinburgh Postnatal Depression Scale (EPDS) and Beck Depression Inventory (BDI-II)) and cord blood DNAm (N = 248) in a South African birth cohort identi ed one signi cant CpG site after bias-adjustment, however, this was not replicated in our study 33 .To the best of our knowledge, this is one of the rst EWAS investigating the associations between postpartum stress and depression and DNAm and therefore we are unable to compare our results to previous ndings for these analyses.
Maternal stress and depression have been hypothesized to exert their effects through multiple biological pathways such as oxidative stress, cortisol transmission through breastmilk, and the HPA axis 6, 22,43 .
There was substantial overlap between the combined adversity score models and the individual stress and depression models.The magnitude of effect for overlapping CpG sites was consistently larger among the combined adversity score models both prenatally and postnatally, which may indicate that cumulative stressors have a stronger effect on differential DNAm than either stress or depression alone.. Nevertheless, due to the small sample size and high correlation between the stress and depression measures in this cohort, future studies should test the replicatability of these ndings and further try to disentangle the biological mechanisms between stress and depression and their cumulative effects.
While the bias-adjustment with bacon was generally robust with our main analysis, adjusting with the cate method yielded no signi cant or suggestive results.However, with our relatively small sample size and comprehensive list of included covariates cate, which adds additional surrogate variables as covariates, may be slightly conservative with the potential of losing true positive signals.Three CpG sites exhibited robust suggestive or signi cant p-values across the unadjusted and bias-adjusted estimates for the postnatal models (postnatal stress: cg10770652 (BRD2); cg25301180 (ERC2); postnatal depression: cg05051393 (ASF1A)).BRD2 (bromodomain-containing protein 2) is a transcriptional regulator that plays a role in nucleosome assembly, DNA damage repair, and chromatin remodeling 61 .Mutations in BRD2 have been implicated in juvenile myoclonic epilepsy and there is some evidence from animal models that exposure to maternal stress in combination with other teratogens may be associated with comorbid ASD and epilepsy 62,63  A few CpG sites overlapped between time points and exposures.One CpG site exhibited differential CBMC-DNAm in association with both prenatal stress and depression (cg12390344 (LAMA3)).Additionally, cg03927037 (ARHGAP20) was differentially methylated in PBMC-DNAm in association with both prenatal stress and depression.Finally, cg09730369 (HYMAI; PLAGL1) was differentially methylated in PBMC-DNAm in association with both prenatal and postnatal depression.The gene LAMA3 (laminin subunit alpha 3) is involved in cell growth, motility, and adhesion 70 .It is primarily expressed in the epidermis and pancreatic endocrine cells and has been associated with diseases such as atopic dermatitis and pancreatic cancer 71,72 .ARHGAP20 (RHO GTPase Activating Protein 20) is involved in neurite outgrowth, differentiation, and maturation, however, not many studies have investigated this gene in association with disease 73 .Overexpression of HYMAI and PLAGL1, maternally imprinted genes, has been strongly associated with 6q24-related transient neonatal diabetes mellitus (6q24-TNDM), a rare type of diabetes that presents at birth, resolves after the rst year of life and may recur in adolescence or adulthood 74,75 .Maternally imprinted genes are genes in which the maternal allele is epigenetically suppressed, and the paternal allele is epigenetically expressed through DNA and histone methylation during gametogenesis 76 .Because only one allele is expressed, imprinted genes are especially vulnerable to mutations and epigenetic changes due to environmental perturbations and can subsequently affect fetal growth and development [77][78][79] .One study investigated the mediation of maternal prenatal depression and birth weight by cord blood DNAm of PLAGL1 (N = 922) 80 .While no evidence was found of mediation or an association with prenatal depression, they did identify signi cantly increased methylation (3.6%) at the PLAGL1 differentially methylated region among high birth weight infants.Furthermore, another candidate gene study that investigated the mediation of maternal prenatal stress and preterm birth by cord blood DNAm of PLAGL1 (N = 537) also did not nd evidence of mediation or an association with maternal stress at this gene 81 .Larger studies and mediation analyses should be conducted to further investigate potential associations between maternal mental health and differential methylation of HYMAI/PLAGL1 and its potential role in 6q24-TNDM.
Our study has a few limitations.First, we were unable to replicate any of our ndings in previous studies due to a lack of comparability across studies.Studies that have been conducted on prenatal depression and stress use various measures of depression and stress and therefore may capture different constructs.Additionally, using cord blood and peripheral blood mononuclear cell tissue which are largely missing granulocytes, rather than unmanipulated cord or peripheral whole blood which most studies typically use may also in part explain challenges in replicating our results.Additionally, while the stress response is physiologically systematic, DNAm changes that may be exerted by maternal stress and depression may not be fully captured by blood tissue alone and could also be exerting effects in another relevant tissue such as brain 82 .Next, this study had a small sample size for both the prenatal (n = 119) and postnatal (n = 113) analyses, which likely limited our statistical power to detect associations.. Furthermore, we did not adjust for maternal intake of anti-depressant and anti-anxiety medications, a potentially important confounder.Another potential limitation is the small-magnitude effect sizes, which is common and expected in studies of early-life exposures 83 .However, even small changes in DNAm could cause differences in transcriptional regulation of gene expression 83 .Additionally, many studies of early-life exposures, such as maternal smoking during pregnancy, have found consistently small yet robust effect sizes, suggesting that small changes in DNAm may persist across populations and throughout the life-course 83 .More studies of prenatal mental health exposures need to be conducted to determine the generalizability and biological relevance of these small effect sizes.Lastly, several of our signi cant CpG sites were associated with at least one known mQTL, an indicator of the genetic in uence on DNAm 60 .However, only a proportion of variation in DNAm is due to genetic effects.Further, the joint effect of environmental factors and single nucleotide polymorphisms (SNPs) may have a larger association with differential DNAm than SNPs alone 84,85 .
Despite these limitations, our study contributes to the sparse literature on maternal mental health and child DNAm.To our knowledge, this is one of the rst EWAS to investigate the association between postpartum stress and depression and child DNAm.Additionally, we conducted a bias analysis to assess the effects of unmeasured confounding.Finally, we were able to look at multiple time points of stress, depression, and DNAm, which allowed us to investigate the possible effects of maternal mental health on DNAm across the child's rst year of life, a critical window for development.

Figures
Figure 1 Overview of the statistical analysis pipeline.

Table 1
Study population characteristics for the total population, prenatal sample, and postnatal sample.

Table 2
Effect sizes and p-values of signi cant CpG sites for prenatal stress and depression and CBMC-DNAm in newborns.

Table 3
Effect sizes and p-values of signi cant CpG sites for postnatal stress and depression and PBMC-DNAm at 12 months of age.
1The mean effect size represents the difference in DNA methylation according to the range of the distribution of β values which are on a 0 to 1 scale.Abbreviations: PBMC-DNAm, peripheral blood mononuclear cell DNAm; CBMC-DNAm, cord blood mononuclear cell DNAm . Additionally, an epigenome-wide meta-analysis from the PACE consortium (N = 2190) identi ed differential DNAm at this gene in association with general psychopathology in school-age children 64 .ERC2 (ELKS/RAB6-interacting/CAST family member 2) is a gene that is primarily expressed in the brain and plays a role in regulating neurotransmitter release65.A genome-wide association study identi ed ERC2 as a locus that may induce neuronal excitability in association with febrile seizures 66 .Additionally, differential cord-blood DNAm at ERC2 was associated with ADHD symptoms in school-age children 67 .ASF1A (anti-silencing function 1A histone chaperone) is a histone chaperone that is involved in chromatin assembly during DNA replication and repair.ASF1A is regulated by tousled-like kinases, a family of serine-threonine kinases that are involved in many regulatory functions including DNA replication and repair, chromatin structure, and genomic and epigenomic stability68.Dysfunction in TLKregulated pathways has been associated with neurodevelopmental disorders such as ASD69.Further research is necessary to determine whether differential DNAm at BRD2, ERC2, or ASF1A mediate the relationship between maternal stress and neuropsychiatric outcomes in children.