According to the “Developmental Origins of Health and Disease” (DOHaD) concept, the early-life environment is a critical period for fetal programming. Given the epidemiological evidence that air pollution exposure during pregnancy adversely affects newborn outcomes such as birth weight and preterm birth, there is a need to pay attention to underlying modes of action to better understand not only these air pollution-induced early health effects but also its later-life consequences. In this review, we give an overview of air pollution-induced placental molecular alterations observed in the ENVIRONAGE birth cohort and evaluate the existing evidence. In general, we showed that prenatal exposure to air pollution is associated with nitrosative stress and epigenetic alterations in the placenta. Adversely affected CpG targets were involved in cellular processes including DNA repair, circadian rhythm, and energy metabolism. For miRNA expression, specific air pollution exposure windows were associated with altered miR-20a, miR-21, miR-146a, and miR-222 expression. Early-life aging markers including telomere length and mitochondrial DNA content are associated with air pollution exposure during pregnancy. Previously, we proposed the air pollution-induced telomere-mitochondrial aging hypothesis with a direct link between telomeres and mitochondria. Here, we extend this view with a potential co-interaction of different biological mechanisms on the level of placental oxidative stress, epigenetics, aging, and energy metabolism. Investigating the placenta is an opportunity for future research as it may help to understand the fundamental biology underpinning the DOHaD concept through the interactions between the underlying modes of action, prenatal environment, and disease risk in later life. To prevent lasting consequences from early-life exposures of air pollution, policy makers should get a basic understanding of biomolecular consequences and transgenerational risks.

Background: There is evidence that altered DNA methylation is an important epigenetic mechanism in prenatal programming and that developmental periods are sensitive to environmental stressors. We hypothesized that exposure to fine particles (PM_2.5) during pregnancy could influence DNA methylation patterns of the placenta. Methods: In the ENVIRON AGE birth cohort, levels of 5’-methyl-deoxycytidine (5-mdC) and deoxycytidine (dC) were quantified in placental DNA from 240 newborns. Multiple regression models were used to study placental global DNA methylation and in utero exposure to PM_2.5 over various time windows during pregnancy. Results: PM_2.5 exposure during pregnancy averaged (25^th-75^th percentile) 17.4 (15.4-19.3) μg/m³. Placental global DNA methylation was inversely associated with PM_2.5 exposures during whole pregnancy and relatively decreased by 2.19% (95% confidence interval [CI]: -3.65, -0.73%, p = 0.004) for each 5 μg/m³ increase in exposure to PM_2.5. In a multi-lag model in which all three trimester exposures were fitted as independent variables in the same regression model, only exposure to PM_2.5 during trimester 1 was significantly associated with lower global DNA methylation (-2.13% per 5 μg/m³ increase, 95% CI: -3.71, -0.54%, p = 0.009). When we analyzed shorter time windows of exposure within trimester 1, we observed a lower placental DNA methylation at birth during all implantation stages but exposure during the implantation range (6-21d) was strongest associated (-1.08% per 5 μg/m³ increase, 95% CI: -1.80, -0.36%, p = 0.004). Conclusions: We observed a lower degree of placental global DNA methylation in association with exposure to particulate air pollution in early pregnancy, including the critical stages of implantation. Future studies should elucidate genome-wide and gene-specific methylation patterns in placental tissue that could link particulate exposure during in utero life and early epigenetic modulations.

Studies emphasize the importance of particulate matter (PM) in the formation of reactive oxygen species and inflammation. We hypothesized that PM exposure during different time windows in pregnancy influences mitochondrial 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, which is an established biomarker for oxidative stress, in both maternal and foetal blood. We investigated maternal (n = 224) and cord blood (n = 293) from mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. We determined mitochondrial 8-OHdG by quantitative polymerase chain reaction (qPCR). Multivariable regression models were used to assess the association between mitochondrial 8-OHdG with PM10 and PM2.5 exposure over various time windows during pregnancy. In multivariable analysis, PM10 exposure during the entire pregnancy was positively associated with levels of mitochondrial 8-OHdG in maternal blood. For an IQR increment in PM10 exposure an increase of 18.3 % (95 % confidence interval (CI): 5.6 to 33.4 %, p = 0.004) in 8-OHdG was observed. PM10 exposure during the last trimester of pregnancy was positively associated with levels of 8-OHdG (28.1, 95 % CI: 8.6 to 51.2 %, p = 0.004, for an IQR increment in PM10). In a similar way, PM2.5 exposure was significantly associated with an increase of mitochondrial 8-OHdG levels in maternal blood during the entire pregnancy (13.9, 95 % CI: 0.4 to 29.4 %, p = 0.04 for an IQR increment in PM2.5 exposure) and third trimester of pregnancy (28.1, 95 % CI: 3.6 to 58.4 %, p = 0.02 for an IQR increment in PM2.5 exposure). In umbilical cord blood, 8-OHdG levels were significantly associated with PM10 exposure during first and second trimester of pregnancy with respectively an increase of 23.0 % (95 % CI: 5.9 to 42.8 %, p = 0.007) and 16.6 % (95 % CI: 1.8 to 33.5 %, p = 0.03) for an IQR increment in PM10 exposure. We found PM-associated increased mitochondrial oxidative DNA damage during pregnancy in both mothers and their newborns. Accordingly, our study showed that particulate air pollution exposure in early life plays a role in increasing systemic oxidative stress, at the level of the mitochondria, both in mother and foetus.

Maternal smoking during pregnancy results in an increased risk of low birth weight through perturbations in the utero-placental exchange. Epigenetics and mitochondrial function in fetal tissues might be molecular signatures responsive to in utero tobacco smoke exposure. In the framework of the ENVIRONAGE birth cohort, we investigated the effect of self-reported tobacco smoke exposure during pregnancy on birth weight and the relation with placental tissue markers such as, (1) relative mitochondrial DNA (mtDNA) content as determined by real-time quantitative PCR, (2) DNA methylation of specific loci of mtDNA (D-loop and MT-RNR1), and (3) DNA methylation of the biotransformation gene CYP1A1 (the last two determined by bisulfite-pyrosequencing). The total pregnant mother sample included 255 non-smokers, 65 former-smokers who had quit smoking before pregnancy, and 62 smokers who continued smoking during pregnancy. Smokers delivered newborns with a birth weight on average 208 g lower [95% confidence interval (CI) −318 to −99, p = 0.0002] than mothers who did not smoke during pregnancy. In the smoker group, the relative mtDNA content was lower (−21.6%, 95% CI −35.4 to −4.9%, p = 0.01) than in the non-smoker group; whereas, absolute mtDNA methylation levels of MT-RNR1 were higher (+0.62%, 95% CI 0.21 to 1.02%, p = 0.003). Lower CpG-specific methylation of CYP1A1 in placental tissue (−4.57%, 95% CI −7.15 to −1.98%, p < 0.0001) were observed in smokers compared with non-smokers. Nevertheless, no mediation of CYP1A1 methylation nor any other investigated molecular signature was observed for the association between tobacco smoke exposure and birth weight. mtDNA content, methylation of specific loci of mtDNA, and CYP1A1 methylation in placental tissue may serve as molecular signatures for the association between gestational tobacco smoke exposure and low birth weight.

While previous studies have demonstrated that prenatal exposure to environmental stressors is associated with mitochondrial DNA (mtDNA) methylation, more recent investigations are questioning the accuracy of the methylation assessment and its biological relevance. In this study, we investigated placental mtDNA methylation while accounting for methodological issues such as nuclear contamination, bisulphite conversion, and PCR bias. From the ENVIRONAGE birth cohort, we selected three groups of participants (n = 20/group). One group with mothers who smoked during pregnancy (average 13.2 cig/day), one group with high air pollutant exposure (PM_2.5: 16.0 ± 1.4 µg/m³, black carbon: 1.8 ± 0.3 µg/m³) and one control group (non-smokers, PM_2.5: 10.6 ± 1.7 µg/m³, black carbon: 0.9 ± 0.1 µg/m³) with low air pollutant exposure. DNA methylation levels were quantified in two regions of the displacement loop control region (D-loop and LDLR2) by bisulphite pyrosequencing. Additionally, we measured DNA methylation on nuclear genes involved in mitochondrial maintenance (PINK1, DNA2, and POLG1) and assessed mtDNA content using qPCR. Absolute D-loop methylation levels were higher for mothers that smoked extensively (+0.36%, 95% CI: 0.06% to 0.66%), and for mothers that were highly exposed to air pollutants (+0.47%, 95% CI: 0.20% to 0.73%). The relevance of our findings is further supported, as D-loop methylation levels were correlated with placental mtDNA content (r = −0.40, p = 0.002) and associated with birth weight (−106.98 g, 95% CI: −209.60 g to −4.36 g for an IQR increase in D-loop methylation). Most notably, our data demonstrates relevant levels of mtDNA methylation in placenta tissue, with significant associations between prenatal exposure to environmental stressors and D-loop methylation.

Air pollution exposure during pregnancy has been associated with adverse birth outcomes and health problems later in life. We investigated sex-specific transcriptomic responses to gestational long- and short-term exposure to particulate matter with a diameter < 2.5 μm (PM2.5) in order to elucidate potential underlying mechanisms of action. Whole genome gene expression was investigated in cord blood of 142 mother-newborn pairs that were enrolled in the ENVIRONAGE birth cohort. Daily PM2.5 exposure levels were calculated for each mother’s home address using a spatial-temporal interpolation model in combination with a dispersion model to estimate both long- (annual average before delivery) and short- (last month of pregnancy) term exposure. We explored the association between gene expression levels and PM2.5 exposure, and identified modulated pathways by overrepresentation analysis and gene set enrichment analysis. Some processes were altered in both sexes for long- (e.g. DNA damage) or short-term exposure (e.g. olfactory signaling). For long-term exposure in boys neurodevelopment and RhoA pathways were modulated, while in girls defensin expression was down-regulated. For short-term exposure we identified pathways related to synaptic transmission and mitochondrial function (boys) and immune response (girls). This is the first whole genome gene expression study in cord blood to identify sex-specific pathways altered by PM2.5. The identified transcriptome pathways could provide new molecular insights as to the interaction pattern of early life PM2.5 exposure with the biological development of the fetus.