The placenta plays a pivotal role in nutrient transfer, growth, and organ development of the embryo. Epigenetic modification may provide a plausible link between particulate air pollution and alteration in gene expression that might lead to disease phenotypes related to fetal programming. The key finding of our study is that exposure to particulate air pollution from fertilization up to and including embryo implantation was associated with lower global DNA methylation levels in placental tissue at birth. This observation persisted after adjustment for newborn’s gender, maternal age, gestational age, parity, smoking, maternal education, prenatal exposure to acetaminophen, season at conception, trimester-specific apparent temperature or any other covariate studied.
DNA methylation patterns are established in two developmental periods (germ cells and early embryos) and are likely needed to generate cells with a broad developmental potential and correct initiation of embryonic gene expression . In this regard, epigenetic reprogramming of imprinted genes in germ cells and early embryos appear to be particularly important for the regulation of embryonic growth and placental development . It has been hypothesized that regulation of imprinted gene expression is less stable in the placenta than in the fetus itself which may aid the placenta in adapting to changing physiological conditions [25, 26]. This leads to speculation that perturbations in DNA methylation patterns or sporadic loss-of-imprinting in the early stages of development lie at the basis of altered gene expression and contribute to abnormal placental or fetal development . Indeed, research suggests that transplacental exposure to environmental toxicants during critical developmental periods lead to disease pathogenesis in later life [12, 14, 27, 28]. Both in animal and human cells, there is direct evidence for the role of hypomethylation for inducing genomic and chromosomal instability [29–31].
The sensitivity of the epigenetic system to environmental factors occurs primarily during the period of developmental plasticity because this is the time when epigenetic marks undergo critical modifications . After fertilization and prior to implantation, DNA methylation patterns are largely erased but are reestablished by de novo DNA methyltransferases (DNMTs) in the blastocyst stage . The placenta develops from the outer layer of the blastocyst upon implantation into the maternal endometrium . Our results show that exposure to particulate air pollution during the implantation window is associated with the methylation profile of placental tissue. The finding of lower methylation levels from the beginning of placental formation is of critical interest in development considering that disturbance of maintenance DNA methylation in placental tissue is associated with abnormal embryonic development in the mouse model  and genetic inactivation of DNMTs is lethal to developing mouse embryos . Experimental evidence showed that oxidative DNA damage could interfere with the capability of methyltransferases to interact with DNA resulting in lower methylation of cytosine residues at CpG sites . Since trophoblast differentiation is most important early in pregnancy when the placenta is initially being constructed  and maternal air pollution exposure may influence markers of placental growth and function , it could well be that altered global DNA methylation during early pregnancy influences placental development. Maternal tobacco smoke, a personalized form of air pollution, has shown to alter placental methylation levels [22, 40] and underlie changes to placental function that may lead to altered fetal development and programming  or pregnancy pathologies such as impaired fetal growth  and preterm delivery [42, 43]. Our relative estimates of lower global DNA methylation levels for an increase of 5 μg/m3 in the first trimester is associated with a decrease of 2.13% (p = 0.009) in global DNA methylation, compared with -2.17% (p = 0.13) in active smokers and -2.84% (p = 0.05) in past smokers. Our observations in smokers are much smaller compared with the estimates in cord blood assessed by ELISA in the study of Guerrero-Preston and colleagues showing a -48.5% (p < 0.01) lower global DNA methylation among newborns with smoking mothers compared with their nonsmoking counterparts . However, differences in tissue and techniques make direct comparison of methylation status difficult. The mechanisms of air pollution-induced health effects involve oxidative stress and inflammation [45, 46]. The associations we observed in our current study may be part of the systemic consequences of induced inflammatory conditions both in mother lungs as well as in placental tissue. An alternative hypothesis is that inhaled particles may translocate directly from the lung into the blood stream where these fine particles induce oxidative stress in blood cells and potentially in placental tissue [47, 48].
Although this is the first study investigating the effect of PM2.5 on DNA methylation in early life, several other studies have examined the role of environmental factors on DNA methylation levels in adults. Baccarelli and colleagues showed that blood DNA methylation in the LINE-1 repetitive element was decreased in elderly individuals of the Normative Aging Study with recent exposure to higher levels of traffic particles including PM2.5, whereas no association was observed between methylation of the Alu repetitive element and particle levels . Another study within the same elderly cohort found that prolonged exposure to black carbon and sulfate particles is associated with hypomethylation of Alu and LINE-1 in leukocytes respectively . Besides surrogate markers of global DNA methylation, several studies also report associations of gene-specific DNA methylation in leukocytes and exposure to airborne polycyclic aromatic hydrocarbons and PM [19, 27]. In contrast to particulate exposure, arsenic was positively associated with DNA methylation in LINE-1 repeated element in both maternal and fetal leukocytes .
A first limitation of this study is that placental tissue is composed of a complex population of cells (syncytiotrophoblasts/cytotrophoblasts, mesenchymal cells, Hofbauer cells, fibroblasts). Also maternal blood is a major constituent of placental tissue which makes that this organ shows high variability in overall DNA methylation compared to other tissues . However, within-placenta variability for several genes showed generally less sample-to-sample variation for DNA methylation than gene expression levels and different placental sites and depths show consistent methylation patterns [21, 50]. In our study, the coefficient of variation of global methylation between sample spots from different quadrants of the placenta was 4.5% with an intraclass correlation coefficient (ICC) of 0.25. To minimize the impact of regional differences in methylation patterns within a mother’s placenta, we standardized our method and chose one spot. Most of the methylation variation is not due to sample location but rather cell composition differences between samples. Heterogeneity in cell types in placental tissue may also contribute to inter-individual variation . Global methylation status measured by quantifying 5-mdC and dC using ultra-pressure liquid chromatography in combination with tandem mass spectrometry, gives a good estimate of global methylation because it averages total methylation of all cell types. Although most genes present in the two main cell types of the placenta (cytotrophoblasts and fibroblasts) exhibit similar promoter methylation patterns, some specific genes show differential promoter methylation . Methylation status of placental villi reflects mainly the profile of the cytotropoblast cells. We did not observe any obvious differences in the histology or cell type composition between the fetal samples taken at four standardized sites across the middle region of the placenta (approximately 4 cm away from the umbilical cord) nor between placentas. Regardless of the limitations, the placenta can be used as a proxy for methylation changes in the fetus as it is derived from the outer layer of the blastocyst. The organ has a great plasticity to a range of intrauterine conditions/exposures and the question remains if the fetus is affected in a direct manner or indirectly by adaptations in its function. Variables interfering with placental integrity may predispose to placenta-related gestational complications such as preeclampsia, fetal growth restriction and abruption . Fetuses adapt their mitochondrial structure and metabolism when the supply of nutrients is limited. Changes in mitochondrial DNA content, may represent a biological effect along the path linking air pollution to effects on the unborn. Recently we showed that mitochondrial DNA content in placental tissue, but not cord blood, was influenced by PM10 exposure during the last trimester of pregnancy. The effects of these molecular changes must be further elucidated . Secondly, although our results were consistent after multiple adjustments, we cannot exclude the possibility of residual confounding by some unknown factor that is associated with both placental methylation levels and ambient air pollution. Ambient exposure does not account for indoor exposure, but we obtained information on environmental tobacco smoke. Season and apparent temperature were taken into account as epigenetic adaptive changes to season have been reported in aquatic species . We found the highest methylation levels in placental tissue for conceptions at spring and the lowest in fall, which corresponds with observations in blood from adults by Baccarelli and colleagues . Thirdly, the resolution of our interpolation model (4 x 4 km) may not represent perfect PM2.5 exposure at the individual level, however our exposure model has good validation statistics with an explained variance higher than 80%  and also validation regarding to personal exposure by measuring carbon load in lung macrophages . Our study was not designed to evaluate temporal changes of DNA methylation during pregnancy and may be hampered by the fact that assays of term placentas may not reflect in vivo methylation patterns occurring earlier at critical points of development. Nevertheless, our associations were robust and strong in the context of environmental epidemiology.
Generally, two approaches can be performed to analyze DNA methylation, either gene-specific or global analysis. In our study, we chose to measure global DNA methylation instead of surrogate markers of global DNA methylation. Gene-specific assays are crucial for integrating information about DNA methylation patterns with gene expression at promoter level but do not provide a global picture of DNA methylation changes within the genome . Genome-wide methylation assays and gene expression analysis are needed to complement our findings of lower global methylation levels. For example, investigating DNMTs should give more insight into possible mechanisms that control epigenetic programming and thus placental development. Additional studies should also elucidate gene-specific methylation patterns since there is evidence that altered DNA methylation at the human H19/IGF2 imprinting control region , genes such as TIMP3
 and disruption of imprinted genes in mouse models may be associated with abnormal placental outcomes and fetal development . Our findings give mechanistic plausibility to the hypothesis that air pollution is linked to fetal programming. Indeed, there is an increasing awareness that the placenta responds to and modulates perturbations in the maternal environment, thereby playing a key role in transmitting the programming stimuli to the fetus .
The current study was performed in an European hotspot regarding particulate air pollution  with 33 days in 2011 exceeding the European legislation of 50 μg/m3. Thanks to legislation, levels of urban air pollution have generally decreased over the course of the latter half of the 20th century in the United States and Western Europe. However, no such trend has taken place in many cities and megacities of developing countries.