Human placental methylome in the interplay of adverse placental health, environmental exposure, and pregnancy outcome

The placenta is the interface between maternal and fetal circulations, integrating maternal and fetal signals to selectively regulate nutrient, gas, and waste exchange, as well as secrete hormones. In turn, the placenta helps create the in utero environment and control fetal growth and development. The unique epigenetic profile of the human placenta likely reflects its early developmental separation from the fetus proper and its role in mediating maternal–fetal exchange that leaves it open to a range of exogenous exposures in the maternal circulation. In this review, we cover recent advances in DNA methylation in the context of placental function and development, as well as the interaction between the pregnancy and the environment.


Introduction
Establishing a successful pregnancy depends on a complex sequence of interactions between maternal and fetal cells, and their interlocutory signals [1]. This interaction takes place on many levels, from systematic endocrine factors to direct paracrine and juxtacrine contact between the placenta and maternal decidual cells. The placenta is a major transient endocrine organ that plays a key role in enabling many of these complex interactions. Placental trophoblasts extensively remodel the maternal decidua and uterine vasculature to ensure appropriate blood flow for the exchange of nutrients and waste between fetal and maternal circulations. In turn, this creates a unique environment at the fetal-maternal interface [2].
The extent to which the placenta invades the maternal decidua and interacts with the maternal circulation depends on the structure of the placenta. Evolutionarily, the placenta is one of the most diverse organs, evolving independently in multiple lineages and with different placental patterning [3]. The hemochorial human placenta is unique from other primates and model organisms like mice in that trophoblast invasion is more extensive and implantation is interstitial [4]. The placenta differentiates from the extraembryonic trophectoderm, giving rise to villous cytotrophoblasts (VCTs) that can proliferate or fuse to form the multinuclear syncytiotrophoblast (ST) layer, which acts as the barrier between maternal and fetal circulations, and the extra-villous trophoblasts (EVTs), which invade the decidua and remodel maternal a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 spiral arteries [5]. Placental cell composition and function are dynamic over gestation, with a major change being the increased flow of maternal blood into the intravillous space around the transition from first to second trimester, which results in a change in oxygen concentration and extensive remodelling of villous structure [6,7]. This interaction of maternal and fetal cells is facilitated by considerable adaptation of the maternal immune response, ensuring tolerance of the developing fetus while retaining capability for host cell defence and contributing to healthy development [8].
Compelling evidence from human epidemiological studies suggests that adverse exposures in utero can alter the intrauterine environment, with potential implications for fetal development and long-term offspring health [9]. Due to its role as interface between maternal and fetal circulations, the placenta is constantly exposed to exogenous factors that are present in the maternal circulation, with the potential for these exposures to leave a lasting molecular 'footprint' on the placenta. In keeping with this, it is not surprising that the extraembryonic origin, relatively short life span, and ongoing direct exposure to all exogenous signals are reflected in a unique epigenetic profile [10]. Here, we highlight and discuss emerging knowledge on the unique features of the human placental methylome (total DNA methylation profile) and summarise research linking in utero exposures, adverse placental health, and fetal outcomes to altered DNA methylation in the human placenta. We go on to discuss the potential impact of single-cell techniques on understanding the placental methylome and how this will pave the way for future use of DNA methylation in the placenta as a biomarker to diagnose placental syndromes and predict fetal outcomes.

DNA methylation remodelling in extraembryonic cells
Epigenetics refers to the molecular interactions that influence DNA packaging into chromatin, thereby regulating DNA accessibility and gene expression. Epigenetic modifications are added either directly to DNA (as in DNA methylation) or to the amino acid tails of histones that constitute the nucleosome around which DNA is coiled.
DNA methylation is the most widely studied and well-characterised epigenetic modification. It involves the addition of a methyl group to a cytoside within a cytoside-guanine dinucleotide (CpG site) [11]. CpG sites often cluster together in CpG islands close to the promoter and regulatory regions of genes, and within this context increased DNA methylation blocks access to the underlying DNA sequence, leading to reduced gene expression. During human preimplantation development, DNA methylation is highly dynamic (Fig 1A). Notably, at the blastocyst stage, the inner cell mass and trophectoderm undergo a wave of de novo DNA methylation, but to different extents, leading to differences in final global DNA methylation levels [12].
DNA methylation continues to be dynamic in trophoblast cells after implantation and throughout gestation, with the process of VCTs' differentiation to ST and the acquisition of an invasive phenotype in EVT, both of which involving widespread DNA methylation changes [13]. Interestingly, culture in a low oxygen concentration induced opposing DNA methylation change at certain differentiation-associated differentially methylated regions (DMRs) and was associated with lower differentiation potential, an effect potentially modulated by the activator protein 1 (AP-1) transcription factor complex [14]. These findings highlight the intimate relationship between DNA methylation patterns, trophoblast differentiation, and the sensing of environmental signals.

Placental global hypomethylation is not specific to repetitive elements
The aforementioned differences in DNA methylation re-establishment result in the placenta exhibiting a unique, globally hypomethylated DNA methylation profile compared with normal After fertilisation, the sperm and egg undergo a wave of hypomethylation, except at imprinted genes. Around implantation, the inner cell mass of the blastocyst, which develops into the epiblast, and the trophoblast, which develops into the placenta, undergo de novo methylation to differing extents. (B) Unique features of the placenta methylome arising as a result of the remethylation differences in A. B (i) Global hypomethylation [15,16], (ii) polymorphic imprinting [17], (iii) PMDs [18,19], (iv) trophoblast differentiation dynamics [13], (v) epigenetic regulation of HLA-G [20] [14], (xii) hypomethylated retrotransposons used as alternative somatic tissue (Fig 1B) [15]. High-performance liquid chromatography (HPLC) analysis of the total 5-methylcytosine content estimates that human placental tissue is on average 2.5%-3% methylated compared to approximately 4% in human cord blood (Fig 1B(i)). Repetitive elements, which cover approximately 35% of the human genome, are supressed with elevated levels of DNA methylation [28,29]. Earlier studies of the placental methylome found that some repetitive elements, such as long interspersed element-1 (LINE-1) [30, 31]) and human endogenous reterovirus (HERV) [32], are key DMRs in placental tissue. Placental hypomethylation of these repetitive elements is thought to regulate placenta-specific functions. For example, some retrotransposons function as alternative promoters for placental-specific transcripts, such as potassium voltage-gated channel subfamily H member 5 (KCNH5) and interleukin 2 receptor, beta 1 subunit (IL2RB) (Fig 1B(xii)) [26, 27, 32]. Expression of an alternative form of KCNH5 may contribute to trophoblast invasiveness, a conclusion that is supported by the finding that the placenta-specific transcript and associated hypomethylated promoter occur frequently in melanoma cells, but never in healthy somatic cell types [33]. IL2RB is a subunit of the IL2 receptor and is expressed primarily by lymphocytes, and the function of the trophoblast specific-transcript is currently unknown [27]. In fact, despite the widespread potential for retrotransposon-driven novel transcription in the placenta, relatively little is known about its potential role in correct placental functioning.
Recent findings have dispelled the notion that retrotransposons are specifically hypomethylated in the placenta. In a landmark study, Schroeder and colleagues [18] showed that placental DNA hypomethylation mainly occurs within partially methylated domains (PMDs)-that is, large stretches (up to 100 kb) of gene-poor regions with reduced DNA methylation that are interspersed with regions of higher DNA methylation (Fig 1B(iii)). Using Methyl-C sequencing, it was estimated that PMDs covered approximately 37% of the placental genome and that genes within placental PMDs have placental-specific functions. An important observation was that LINE-1 elements located in a PMD showed the same level of methylation as the surrounding region (approximately 40%), while LINE-1 elements outside PMDs were not hypomethylated (approximately 80% methylation). Likewise, another study used reduced representation bisulfide sequencing (RRBS) to show that placental hypomethylation is enriched at CpG-poor intergenic regions and gene bodies, and that retrotransposons in these regions were, in fact, slightly more methylated than non-retrotransposon elements [34]. Taken together, this suggests that while the placenta may utilise unmethylated retrotransposon elements as alternative promoters, these regions do not solely explain the function global hypomethylation in the placenta. Further research into PMDs may reveal new insights into their function, although role of PMDs in placenta is yet to be determined.
In addition to the global hypomethylation and presence of PMDs, earlier studies showed that promoters of common tumour suppressor genes are monoallelically methylated in the placenta and trophoblasts (Fig 1B(vi)) [21,22]. These promoters become completely methylated in choricarcinomas (trophoblast cancers), supporting the notion that these cancer-like programs are carefully regulated in the placenta. Similarly, cancer-like global DNA hypomethylation blocks in early gestation placenta are lost in later gestation, suggesting a necessary switch that limits placenta invasion and other tumour-like properties in later pregnancy [40].
Studying the establishment of these pseudomalignant methylomes during early implantation is difficult in humans. As such, we do not know how a completely hypomethylated human trophectoderm develops these features. An interesting mouse study that addressed this question comes from the Meissner lab, which showed that partially methylated promoter regions are established rapidly [19]. In embryonic stem cells, these regions are marked by a repressive histone modification, histone 3 lysine 27 trimethylation (H3K27me3), indicating that polycomb group of proteins are keeping these regions closed and thereby potentially allow DNA methyltransferases (DNMTs) to methylate DNA. An important question remains: are PMDs and tumour suppressor promoter methylation drivers of placental function or bystanders?

Novel imprinted regions in the placenta
During development, certain regions of DNA, called germline differentially methylated regions (gDMRs), maintain gamete DNA methylation during genome-wide reprogramming and throughout development [41]. These gDMRs can regulate expression at nearby genes, resulting in monoallelic parent-of-origin expression known as genomic imprinting [42]. There are multiple theories as to why genes evolved to be imprinted [43], but the prevailing hypothesis is that of the kinship theory [44]. In brief, imprinting allows for natural selection to maximise the fitness of the allele through maximising not just the health of the individual inheriting the allele but also the related individuals who will also inherit that allele from the same parent [42]. An example is a gene that regulates growth in utero, and consequently influences the fetal demand for maternal resources [45]. As multiple offspring from the same mother may have different fathers, maximising the kinship fitness bestowed by the maternally inherited allele is more likely to improve kinship fitness overall than the paternally inherited allele. As such, paternal alleles may evolve as silenced to avoid conflicts between maternal and paternal allelic expression [45]. Recent research indicates that the placenta has more gDMRs than previously thought [17,46], which is consistent with the importance of the placenta in regulating fetal growth and maternal resource demand. However, it remains unclear whether the majority of placenta-specific gDMRs are functional in pregnancy, or whether they simply reflect the methylation status of a gamete. Hanna and colleagues [17] identified 144 gDMRs in the human placenta, in which most appeared to maintain maternal DNA methylation patterns (mDMRs). Similarly, Hamada and colleagues [46] performed combined whole-genome bisulfide sequencing and RNA-seq, which identified approximately 1,800 mDMRs in the human placenta, some of which were associated with potential paternal and maternal imprinted genes such as TP53 induced glycolysis regulatory phosphatase (TIGAR), sodium bicarbonate cotransporter 3 (SLC4A7), and zinc finger protein (ZFP90). The function of these imprinted genes in placenta or fetal development, however, remains to be elucidated. An interesting observation requiring follow-up study is that the DNA methyltransferase 1 (DNMT1) promoter itself is maternally imprinted in the human placenta [47]. As with several other aspects of human placental methylation, this promoter methylation is evolutionary conserved only in primates, not in other mammals, and does not seem to be required for global hypomethylation in the placenta [48,49]. It is easy to speculate that maternal imprinting of DNMT1 is a mechanism to prevent the activation of growth-promoting genes, such as insulin-like growth factor 2 (IGF2), by DNMT1 overexpression [50]. In line with this, DNMT1 expression shows a modest positive correlation with placental growth [51]. However, there is no confirmed function for DNMT1 imprinting in the placenta. A limitation of using whole placental tissue to identify novel imprinted genes is that there may be variation between different cell types, which can lead to some imprinted regions being missed. Additionally, without identifying the specific cell types that show imprinting, it is hard to study the function of these novel imprinted regions.
In utero exposures alter the placenta methylation, which may impact placental and fetal health DNA methylation is sensitive to environmental exposure but relatively stable once established. Consequently, studies have examined the influence of in utero exposures on DNA methylation in the placenta in the hope that these patterns could be used as potential biomarkers to diagnose placental disorders or even predict fetal outcomes. The overall level of placental tissue DNA methylation increases over gestation, as does the level of interindividual variation between placentas, potentially indicative of a response to a myriad of environmental exposures and stochastic events, or with a transition and variation in cell composition over time [24]. Many of these studies take the form of epigenome-wide association studies (EWAS), and generally use the Infinium HumanMethylation arrays [52,53]. Mounting evidence suggests that exposures in utero influence the human placental methylome. In Tables 1 and 2, we summarise key EWAS studies using the human placenta since 2016. For a summary of studies predating 2016, please see [54]. Table 1 summarises emerging research linking in utero exposures and the human placental methylome. Given the key role of the placenta in fetal programming, altered DNA methylation profiles of the placenta may cause aberrant placental development and function, which may in turn influence fetal outcomes. Table 2 summarises emerging research linking the human placental methylome and pregnancy outcomes.
Over the past 5 years, there has been a shift from looking at maternal consumption (smoking, adiposity, and nutrition) towards exposures related to the natural and social environment, like plastics, chemicals, air pollution, other pollutants, and war trauma, reflecting the emerging broader scientific and societal interest in the impact of climate change and social issues on human health. We anticipate that as these climate change and societal issues continue to emerge, increased multidisciplinary research will be required to examine the impact of such exposures on many aspects of pregnancy, including placental development, epigenetic profile, and subsequent impact on placental and fetal health. We must also note that the majority of current studies are underpowered due to small sample size, and we urge that researchers consider EWAS power calculations and other considerations of study design prior to conducting such studies [55,56]. It would be considerably beneficial to the field if the suppliers of the most commonly used platform made efforts to reduce the current excessive pricing of DNA methylation arrays relative to the current SNP-based genotyping equivalents.

Exploiting the unique placental epigenome for noninvasive tracking of pregnancy health and outcome
Circulating fetal cell-free DNA in maternal blood is placental in origin, and has been used to noninvasively test for aneuploidy and disease-causing mutations [57,58]. These tests depend Generally, promoter methylation across the genes was decreased in the BPA exposed group compared with control. BPA exposure also impaired cell viability and proliferation and was associated with decreased expression of genes associated with proliferation. on the detection of paternally inherited disease-causing SNPs that are not present in the mother. Early on, it was realised that placenta-specific DNA methylation patterns, such as the hypermethylation of the ras association domain-containing protein 1 (RASSF1A) promoter region [21], can be used as a positive control to rule out false negative results [59]. Since this pioneering work, Lo and colleagues used DNA methylation of fetal cell-free DNA for gestational dating, indicating that this technique has far-reaching potential [60]. However, FGR associated with 970 differential H3K27ac peaks (3.2% of total). As a group, genes near differential H3K27ac peaks showed differential expression.
[114] IUGR and LGA RRBS, EWAS 6 AGA 5 LGA 6 IUGR Placenta tissue More than 1,000 DMRs identified at FDR < 5%, DNA methylation change >15%. Two DMRs replicated in a previous Infinium HM27 dataset [115]. replication of this finding is needed to confirm its usefulness in the diagnosis of gestational age. Recent studies have suggested that preeclampsia-specific DNA methylation patterns in the placenta, e.g., TIMP metallopeptidase inhibitor 3 (TIMP3) and RASSF1A promoters, can be detected in maternal circulation [61,62]. However, no such test exists yet. It may also be possible to identify DNA methylation patterns associated with greater risk of preterm birth and small for gestational age (SGA) [63], or with their associated later morbidity [64,65]. Such approaches have potential predictive utility through the analysis of circulating cell-free placenta-derived DNA, and are currently in development in several laboratories internationally [66,67]. Key to this is identifying robust placenta methylation biomarkers associated with specific exposures and/or outcomes, which may not be feasible given the apparent low degree of methylation variation currently linked to most environmental exposures and outcomes (Table 1). It is now also possible to accurately reconstruct placental genome-wide DNA methylation by bisulfite sequencing of maternal plasma, which could allow the identification of epigenetic aberrations in the placenta without needing the placental sample itself [68].

Single-cell approaches may overcome issues associated with placenta heterogeneity
Placenta is a heterogeneous tissue comprised of many cell types [69], some with varying chromosome copy numbers [70]. These characteristics make it difficult to interpret DNA methylation analysis, as well as limit biomarker use and understanding of molecular pathways and networks. Furthermore, the composition of these cell populations and corresponding DNA methylation profile may change throughout pregnancy [24,71]. For the time being, it is likely that cohort-based EWAS will continue to use whole placental tissue because of the technical difficulties associated with isolating specific cell types on a large scale. Here, cell-specific DNA methylation profiles will aid research by providing signatures that can be used to deconvolute data and remove cell-specific signatures as variables in statistical analysis, similar to the way whole blood data are analysed [72]. However, deconvolution tools are currently limited to the number of different cell subtypes they include in their modelling, and further work is necessary to account for rare cell subtypes in analysis. Recent advances in genome sequencing and cell sorting technology have enabled the unbiased analysis of all cell types in the human placenta. This is highlighted by the publication of several papers reporting single-cell RNA-seq profiles of the human fetal-maternal interface at the first and third trimesters in the last 3 years [69,[73][74][75][76][77]. These studies detected new subsets of cytotrophoblasts and EVTs, as well as cell subtypes previously not observed in placental villi [69,73]. Continued application of single-cell RNA-seq to human placenta-derived cell populations, particularly over gestation, will greatly improve our understanding of how cell composition changes over time and how individual cells respond to changing environments, such as a shift in oxygen concentration. However, techniques for profiling epigenetic marks from low cell numbers are now available and in wide use [78,79], including single-cell techniques for DNA accessibility [80], protein expression/phosphorylation [81], and combinations of marks [82][83][84]. Given the relative ease of obtaining placental tissue, these techniques should be readily applied to purified placental cells. Finally, placental organoid models that recapitulate villi structure and can produce ST and EVT cells have now been successfully established [85,86], and unlike trophoblast cell lines [87], the organoids resemble first trimester trophoblasts in terms of their genome-wide DNA methylation patterns [86]. These novel models in combination with state-of-the-art molecular techniques represent a very powerful approach to provide hitherto unparalleled insights into human trophoblast differentiation and function.

Conclusion
In this review, we highlighted the recent advances across the placental epigenetics field, which is now dominated by studies examining environmental influences. Given the close interaction between trophoblasts and immune cells, which make up to 40% of the feto-maternal interface, we anticipate the next few years will see epigenetic profiling of these resident immune cells. It will be interesting to see what PMDs look like in different placental cell types and how individual cells control the expression of hypomethylated retrotransposon promoters. Multi-omics approaches also have the potential to give us insight into the drivers and passengers involved in the interaction between the unique placental methylome and the environment.