In this prospective cohort, we identified significant associations between maternal psychosocial stress and differential DNA methylation in the first 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 identified eight CpGs significantly associated with prenatal stress and 11 CpGs associated with prenatal depression. Additionally, eight CpGs were significantly 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 findings 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 find 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 five stress domains) and cord blood DNAm (450K and EPIC) did find evidence of an association at five CpG sites (N = 5496)38. However, none of the identified CpG sites were significant 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 identified significant associations, however, none of these associations were replicated in our study32,33,35(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 identified one significant CpG site after bias-adjustment, however, this was not replicated in our study33. To the best of our knowledge, this is one of the first EWAS investigating the associations between postpartum stress and depression and DNAm and therefore we are unable to compare our results to previous findings 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 axis6,22,43. 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 findings 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 significant 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 significant 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 remodeling61. 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 epilepsy62,63. Additionally, an epigenome-wide meta-analysis from the PACE consortium (N = 2190) identified differential DNAm at this gene in association with general psychopathology in school-age children64. 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 identified ERC2 as a locus that may induce neuronal excitability in association with febrile seizures66. Additionally, differential cord-blood DNAm at ERC2 was associated with ADHD symptoms in school-age children67. 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 TLK-regulated 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.
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 adhesion70. It is primarily expressed in the epidermis and pancreatic endocrine cells and has been associated with diseases such as atopic dermatitis and pancreatic cancer71,72. 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 disease73. 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 first year of life and may recur in adolescence or adulthood74,75. 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 gametogenesis76. 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 development77–79. 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 significantly 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 find evidence of mediation or an association with maternal stress at this gene81. 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 findings 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 brain82. 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 exposures83. However, even small changes in DNAm could cause differences in transcriptional regulation of gene expression83. 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-course83. 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 significant CpG sites were associated with at least one known mQTL, an indicator of the genetic influence on DNAm60. 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 alone84,85.
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 first 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 first year of life, a critical window for development.
In summary, these findings suggest that prenatal and postnatal stress and depression are associated with epigenomic changes in the fetus and the first year of life. Larger studies need to be conducted to replicate our findings and to investigate whether changes in DNAm in response to maternal mental health problems increase the risk of adverse birth and childhood outcomes.