This is the first study to investigate synergistic effects of antenatal maternal PRS and PM2.5 exposure on newborns’ SLC6A4 DNAm levels at birth. Higher levels of methylation of the SLC6A4 gene at 6 CpG sites were found in newborns born to mothers reporting higher levels of PRS in pregnancy and greater PM2.5 exposure across gestation. Further, exploratory analyses provided preliminary evidence suggesting that these effects might become especially evident when exposure to elevated PM2.5 levels occurred during the second trimester of pregnancy. Of note, the observed associations were found soon after birth and were independent of infant’s sex.
Evidence from both animal and human studies suggests that pregnancy is an especially vulnerable period for exposure to environmental stressors on the developing fetus [54]. Both individual-level stressors, such as maternal symptoms of anxiety, depression and stress during pregnancy, and environmental-level stressors, such as exposure to air pollution or toxicants, occurring during gestation have been studied and found to be independently associated with behavioral and physiological outcomes in the offspring [3, 4]. However, literature concerning the effect of co-occurring exposures is still sparse.
We previously showed that variations in levels of maternal PRS reported by women who were pregnant during the COVID-19 outbreak and subsequent lockdown in Northern Italy were associated with the levels of newborns’ SLC6A4 methylation and further predicted infant’s temperament [9]. Our current findings extend those results by showing higher levels of SLC6A4 DNAm at 6 CpG sites in newborns co-exposed to both higher mean PM2.5 levels across gestation and higher levels of maternal antenatal PRS. In contrast, the association between maternal PRS during pregnancy and infant’s methylation was not significant at lower levels of PM2.5, raising important conceptual, methodological and clinical implications.
First, the findings of an interactive effect between antenatal exposure to maternal PRS and PM2.5 levels on newborns’ methylation are suggestive of a cumulative or joint effect of individual-level and environmental stressors and are in line with emerging literature on the effects of multiple antenatal exposures [41]. In particular, current findings might indicate that concurrent (and possibly prior) exposure to air pollution could exacerbate the effects of exposure to maternal PRS on infant methylation levels. This might occur through sensitization mechanisms whereby exposure to high levels of PM2.5 sets fetal biological systems to be more susceptible to the effect of concurrent or later adversities. It is important to underline that exposure to air pollution has itself been associated with increased levels of stress in non-pregnant individuals [55, 56]. However, we did not report any significant association between PM2.5 exposure and maternal PRS during pregnancy, possibly suggesting that these two factors act independently to affect infant methylation levels. The biological mechanisms underlying these results still need to be elucidated. It is noteworthy that several biological pathways are known to be affected by both air pollution exposure and psychological distress and may thus be activated by both exposures to determine epigenetic changes and impact offspring development. These include, but are not limited to, inflammation, oxidative stress and endocrine disruption [24, 57]. For example, increased maternal systemic oxidative stress and levels of pro-inflammatory cytokines related to air pollution and stress exposure may lead to placental dysfunction, increase fetal oxidative stress and affect fetal development.
Second, from a methodological account, the current findings emphasize the need for research into the effects of antenatal maternal stress to account for the influence of environmental stressors, as overlooking this aspect might lead to misguiding findings and possibly explain inconsistencies in the literature.
Third, as air pollution is a global health problem, with around 86% of urban population living in places exceeding air quality recommended limits [58] and exposure to PM2.5 being listed among the top five mortality risk factors worldwide [59], the current findings have important practical implications. In particular, while replication is needed, they suggest that being exposed to lower levels of air pollution might buffer the effects of higher levels of antenatal maternal PRS on infant methylation levels. In this sense, they might indicate that, in light of limited resources, greater priority to efforts to improve environmental air quality should be directed to disadvantaged communities that may be at greater risk of experiencing multiple psychosocial stressors as well. This might also be protective in the event of future major stressful experiences, such as a new pandemic. Ultimately, a multi-level approach that targets environmental stressors as well as individual stressors is likely to make the greatest impact on perinatal and infant outcomes.
Noteworthy, exploratory analyses indicate that the second trimester of pregnancy is the most sensitive window for a significant interactive effect between PM2.5 exposure and maternal PRS on infant SLC6A4 DNAm levels. While averaging PM2.5 exposure across pregnancy is thought to provide a robust marker of total PM2.5 exposure across gestation, previous studies indicate timing-related variability in the effects of air pollution exposure on neonatal methylation [60, 61]. Fetal brain development is characterized by a progression of sensitive time windows, during which specific anatomical and physiological structures undergo rapid development and are particularly vulnerable to environmental stressors [62]. Our findings align with emerging evidence suggesting timing-specific association between antenatal adverse exposures and pattern of neonatal DNA methylation. For example, maternal community deprivation during the second or third trimester of pregnancy was found to be significantly associated with greater infant DNA methylation in eight CpG sites of the SLC6A4 gene [28]. Likewise, exposure to the COVID-19 lockdown in the second and third trimester of pregnancy was associated with higher mother and infants’ SLC6A4 DNAm [31]. Mechanisms for a timing-specific effects are still poorly understood. Noteworthy, important processes in the fetal brain begin in mid-pregnancy, including myelination, starting from the subcortical regions, or synaptogenesis [63]. Furthermore, the fetal stress response system is not fully developed until the second trimester. Thus, stress-related exposures at this time might interfere with these important brain development processes [64]
Some study limitations warrant attention. First, while we employed outdoor PM2.5 exposure data based on mothers’ residential addresses during pregnancy, data concerning indoor air pollution exposure are missing. Secondly, newborns’ SLC6A4 DNAm was peripherally assessed in buccal cells. It is unclear how epigenetic variation in the peripheral tissue relates to epigenetic changes within the brain. Partial evidence exists on the cross-tissue consistency of DNA methylation measures in humans. It is plausible that the methylation status of genes that have widespread effects and actions across central and peripheral tissues, such as the SLC6A4 gene, may be tissue- and site-specific [65, 66]. Third, maternal antenatal PRS was assessed through an ad hoc questionnaire developed ad hoc for this study, prioritizing sensitivity to the specific and unprecedented nature of COVID-19 emergency over measure standardization. Fourth, it is important to underline that the current study is not able to address the functional and adaptive consequences of the observed patterns of methylation in infants exposed to high levels of antenatal PM2.5 and maternal PRS. Future studies are needed to investigate how these early mechanisms might predict later infant neurodevelopmental trajectories. Lastly, while newborns’ methylation was assessed soon after birth, thus limiting the effects of postnatal influences [67–69], we cannot rule out unmeasured confounding factors affecting neonatal levels of methylation and no causal conclusions should be drawn.