The ever growing complexity of placental epigenetics – Role in adverse pregnancy outcomes and fetal programming

Placenta 33 (2012) 959e970

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The ever growing complexity of placental epigenetics e Role in adverse pregnancy outcomes and fetal programming B. Novakovic a, b, *, R. Saffery a, b a b

Cancer and Disease Epigenetics, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, VIC 3052, Australia Department of Paediatrics, The University of Melbourne, Parkville, VIC 3052, Australia

a r t i c l e i n f o

a b s t r a c t

Article history: Accepted 6 October 2012

As the primary interface between maternal and fetal circulations, the placenta is subject to a myriad of environmental exposures with the capacity to alter placental function and fetal development. Many of these effects are likely to be mediated by epigenetic (‘above DNA’) change, which is also in turn regulated by maternal and fetal genetic factors. Linking specific environmental exposures, genetic, and epigenetic variation to maternal and fetal outcomes may provide valuable mechanistic insights into the role of placental dysfunction in pregnancy-associated disease and later health. The complexities are manifold but are rapidly being overcome by technological advances and emerging analytical approaches. Although focussing on recent genome-scale and gene-specific DNA methylation studies in the human placenta, this review also discusses the potential of a future broader exploration of combined environmental, genetic and epigenomic approaches, encompassing higher order epigenetic modifications, for unravelling the molecular mechanisms underlying gene-environment interaction at the fetomaternal interface. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Epigenetics DNA methylation Environment Diet Placenta Preeclampsia Birth-weight

1. Introduction The term ‘epigenetics’ literally means ‘above DNA’ and refers to the study of molecular modifications that influence gene activity and chromosome structure [1]. The two most commonly studied epigenetic processes are DNA methylation and histone modification, each of which is subject to developmental and environmental regulation and potentially modifiable with appropriate interventions [1]. Histone modifications are primarily added to carboxy and amino terminal tails resulting in changes to DNA accessibility and gene expression via altered nucleosome positioning [2]. These changes are relatively dynamic and play a major role in short term gene regulation. In contrast, the more stable DNA methylation in mammals is primarily found at CpG dinucleotides, which are underrepresented in the genome, generally at low density, but may be enriched at the promoter regions of genes [3,4]. Elevated DNA methylation at such regions is generally associated with gene inactivity, whereas a lack of methylation indicates the potential for gene expression. A third class of epigenetic regulation is specified

* Corresponding author. Cancer and Disease Epigenetics, Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville, VIC 3052, Australia. Tel.: þ61 3 8341 6205. E-mail address: [email protected] (B. Novakovic). 0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.placenta.2012.10.003

by non-coding RNAs, which can also modulate gene expression and chromatin structure, potentially in a sequence-specific manner [5]. Following fertilisation, global DNA methylation levels are progressively erased, such that by the blastocyst stage (implantation), the genome is in a state of hypomethylation. After blastocyst hatching, DNA methylation levels are re-established in a lineage specific manner such that trophectoderm-derived cells remain relatively hypomethylated compared to inner-cell mass-derived cell counterparts [6,7]. This manifests as measurable differences in DNA methylation patterns in the extra-embryonic tissue (including placenta) relative to somatic human tissues [8e10]. Interestingly, the DNA methylation patterns associated with imprinted genes, which are established in the germ cell [1], are shielded from this early methylation remodelling. Genomic imprinting refers to the phenomenon of allele-specific silencing of one copy of a gene in a parent-of-origin manner. As this parent-of-origin pattern is thought to be required for proper development, DNA methylation marks, associated with maintenance of an imprinted phenotype, escape erasure during the methylation remodelling that takes place in the embryo very early post fertilisation. The role of DNA methylation in placenta development and function has been studied extensively at imprinted gene loci, (reviewed in Ref. [11]). However, recent years have seen an increase in the number of non-imprinting epigenetic studies in the human (reviewed in Refs. [11,12]), and mouse placenta (reviewed in Refs. [13,14]) that are providing valuable insights into how epigenetic

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Fig. 1. Influences and consequences of placental epigenetic disruption. Several factors are known to influence placental epigenetic profile, including environmental exposures, stochastic events and genetic and sex effects. Disruption of DNA methylation patterns can lead to aberrant gene expression and placental development and function. In turn, placental dysfunction can lead to pregnancy-associated disease (PE or IUGR) or more subtle effects, such as low birth-weight and fetal maladaptation. Disruptions in normal fetal development can lead to predisposition to adult onset disease and health problems.

marks are established, their role in trophoblast specification and function, sensitivity to environmental and genetic influence, potential role in adverse pregnancy outcomes, and the phenomenon of ‘fetal programming’ of adult disease Fig. 1. In the current review we discuss recent genome-scale and gene-specific placental epigenetic studies, with a focus on environmental influences on DNA methylation patterns in the human. 2. Human placenta-specific DNA methylation 2.1. Genomic imprinting The most widely studied epigenetic phenomenon in the placenta is genomic imprinting, which refers to the monoallelic expression of a gene in a parent-of-origin manner. Where tested, this is universally controlled by DNA methylation [15]. There is strong evidence that genomic imprinting evolved with placentation and viviparity, with several imprinted genes implicated in fetal growth [16]. For detailed reviews on genomic imprinting in the placenta and the parental-conflict theory see Refs. [17e19]. Significantly more imprinted placental genes have been identified in mice relative to humans [15,20] and interestingly, all mousespecific imprinted genes described thus far appear placentaspecific [21]. Coupled with recent reports of high rates of loss-ofimprinting (LOI) in the human placenta, especially in the first trimester [22e24], this has led to a view that genomic imprinting is less strictly regulated in the human relative to mouse placenta. However, the recent identification of novel imprinted differentially methylated regions in human placenta [25e27], and speculation that several previously reported mouse-specific imprinted genes may be false positives, are reshaping the field [28,29]. Therefore,

the role of imprinted genes in human placental development and function, and the effect of the environment on their expression, remains hotly debated [30]. Genomic imprinting in the placenta has been reviewed extensively elsewhere [16e18,31e33]. 2.2. Unique general features of the human placental methylome The human placenta has a strikingly different DNA methylation profile relative to all somatic tissues [34]. This includes low global DNA methylation (total 5-methylcytosine content) of about 2.5e3%, compared to 4e5% in healthy somatic tissue [9,35]. Most of this hypomethylation appears to be located at repetitive DNA elements such as long interspersed elements (LINE1) [36]. However, the functional role (if any) of lower methylation in the human placenta is not completely understood, but may include the regulation of genome ‘plasticity’ and/or gene regulation, as evidenced by the identification of several cryptic retrotransposon-derived promoters that are specifically active in placental tissues [37e41]. Macaulay et al. [41] used genome-scale methods to detect hypomethylated promoters in the human placenta. Their analysis identified several active SINE retrotransposon-derived alternative promoters that result in expression of placenta-specific transcripts [41]. Similarly, Cohen et al. [37] identified placenta-specific expression of IL-2 receptor beta, through loss of methylation at an LTR-derived promoter. The placenta also shows lower methylation on the inactive X chromosome [8], as well as extensive intra-placental mosaicism in X chromosome inactivation (XCI) pattern relative to somatic tissues [42]. This mosaicism was utilised to determine the clonal origin of villous tree regions and the origin of epigenetic variation within the same placenta [43]. Closely correlated XCI patterns along a villous tree, from the fetal to the maternal side of

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the placenta, but higher variation between sites horizontally across the placenta, suggests that each tree arises from a precursor stem cell population. DNA methylation analysis of different genomic regions revealed that some epigenetic variation occurs early in development, such as at LINE1 elements, while some occurs following villous tree formation, such as the H19/IGF2 locus [43]. Therefore, depending on the genomic loci studied, one or more sites within a placenta are required to accurately reflect the methylation patterns across the whole tissue. 2.3. Cell type, gestational age and genomic context-dependent DNA methylation in the human placenta The placenta is a heterogeneous tissue comprising many cell types, each with the potential to display unique epigenetic features. As such, analysis of chorionic villous sampling (CVS) tissue generally reflects the average methylation state across several different cellular compartments. Purification of specific cells is necessary for defining the epigenetic state of any one cell type. A recent genomewide expression study identified over 3000 transcripts that are differentially expressed between villous cytotrophoblasts (VCTs) and extravillous cytotrophoblasts (EVTs), many of them involved in cell migration and invasion, and immune modulation [44]. DNA methylation comparison between VCT and EVTs is lacking, however Grigoriu et al. [45] identified 442 CpG sites that showed differences between VCT and placental fibroblasts. Interestingly, comparison between VCT and placental fibroblasts to whole placenta identified only 61 and 315 CpG sites with >20% difference in methylation, respectively [45], suggesting that placental methylation as a whole reflects methylation levels of the cytotrophoblast cell population. Analysis of VCT and the inner cell mass (ICM) derived extraembryonic mesoderm identified methylation differences in genes involved in protein transport and transcription factor activity [46]. Interestingly, a recent analysis of mouse trophectoderm stem (TS) and embryonic stem (ES) cells and E3.5 and E6.5 trophoblasts and ICM derived cells found that extra-embryonic specific DNA methylation is established immediately following the separation of the ICM and TS cell lineages [47]. This suggests that DNA methylation is not involved in the specification of stem cells that will give rise to the trophectoderm, and that DNA methylation levels are established following the establishment of histone marks [47,48]. Although clear evidence of a placenta-specific DNA methylation profile exists [49], there is also evidence for inter-placental variation and a programmed, reproducible change in DNA methylation in response to gestational age [50]. Genes involved in immune regulation are overrepresented in those subject to changes in methylation over gestation [50]. This is an interesting observation, given that promoter methylation was found to play a major role in specifying fetal from maternal immune cells at the fetoematernal interface [51]. The gestational change in promoter methylation profile is reflected in changes in gene expression over time [52,53], and changes in global DNA methylation level [54]. The overall pattern at both the promoter-specific and global level is an increase in methylation as pregnancy progresses [50,54]. These dynamic changes may help explain the time-of-exposure-dependent effects of certain environments or stresses on fetal health [55,56], but could equally reflect a change in cell composition of placental tissue. Determining which genomic loci to focus on is an important issue when undertaking DNA methylation profiling, because there is generally little concordance for methylation across different genomic regions, and the degree of inter-individual variation and susceptibility to the environment is gene or genomic-region specific [57]. Current affordable array-based platforms generally target gene promoter regions and CpG Islands only, whereas whole

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genome bisulphite sequencing approaches are unbiased but are not cost effective for most laboratories. This is likely to change in coming years. Proxy measures of DNA methylation should also be interpreted with caution. For example, Price et al. (2012) correlated placental DNA methylation across different types of CpG islands (low to high density CG) and repetitive DNA (LINE1 and Alu elements), and found platform-dependent differences in methylation at repetitive DNA, as well as differences in correlation between repetitive DNA and specific CpG island features. Further, Chu et al. [58] used a CpG tiling array to compare placental methylation to blood and discovered that most tissue-specific methylation is located outside of CpG islands. Therefore those studies carried out to date, targeting only CpG islands, are unlikely to have identified the majority of placenta-specific methylation events [58]. 2.4. The utility of placental methylation as a diagnostic tool Non-invasive prenatal diagnosis (NIPD) has the potential to minimise harm to both the mother and fetus associated with invasive procedures. The presence of fetal (trophoblast) DNA in maternal blood was first reported in 1997 [59] and it has been estimated that this accounts for about 3e5% of total DNA in maternal blood (reviewed in Ref. [60]). Increasing levels of fetal DNA in maternal plasma is associated with trisomy 21 [61] and preeclampsia [62], presumably due to a higher rate of breakdown of placental tissue in these diseases [60]. However, the detection of fetal aneuploidies in maternal plasma using fetal DNA is cumbersome, due to the technical difficulty of measuring SNP frequency in a small proportion of DNA. Despite this, recent advances have made it possible to detect trisomy 13, 18, and 21 directly from fetal DNA by massively parallelled sequencing of maternal plasma-derived DNA [63e65]. In addition to placental DNA, mRNA can also be detected in maternal serum and has utility for detecting fetal aneuploidies (reviewed in Ref. [66]). One of the first successful applications of this technique resulted in the non-invasive detection of trisomy 21 by measuring the level of PLAC4 expression [67]. The identification of placenta-specific DNA methylation marks has raised the exciting prospect of non-invasive tests for fetal aneuploidies and placenta-associated diseases [60,68]. By using high-resolution tiling array for chromosomes 21, 13, 18, X and Y, Papageorgiou and colleagues identified differences in methylation between placenta and maternal blood [69]. This has subsequently led to the development of a non-invasive methylation test for trisomy 21 [70]. A similar strategy, using methylation-specific immunoprecipitation (MeDIP) has identified methylation marks useful for the detection of trisomy 18 [71]. Beyond aneuploidy, the identification of methylation marks associated with placental disease (such as preeclampsia), has also been a focus for several groups. In order for a DNA methylation biomarker to have clinical utility, it must show a difference of sufficient magnitude between normal and disease pregnancy to be detected in the small amount of fetal DNA present in maternal plasma. Such hurdles have yet to be overcome in the race for non-invasive diagnostic tests in this area [72e76]. 3. The role of DNA methylation in placental physiology and function The human placenta executes a complex repertoire of specific functions in response to specific maternal and fetal cues and other environmental signals. It is highly likely that the placental epigenome is intimately linked to placental development and function and this is best illustrated by the demonstrated link between imprinting status in the placenta and fetal growth [77]. Evidence

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for a role of DNA methylation, at non-imprinted genes, in placental function is emerging at a rapid pace. 3.1. The pseudo-malignant nature of the human placenta is reflected in the DNA methylation profile The stem cell like nature of cytotrophoblasts and the ability of the extravillous trophoblasts to migrate, invade and remodel the maternal decidua have been likened to ‘controlled’ malignancy [78,79]. These physiological similarities are reflected in the molecular networks shared between placental and some cancer cells [80]. Gene ontologies with similar expression patterns include proto-oncogenes, growth factor receptors, tumour-associated antigens, as well as telomerase [80e82]. Recently, several studies have identified epigenetic similarities between placenta and human tumours (reviewed in Ref. [83]). One of the major similarities is the demonstrated low global methylation levels in both systems ([9,10,84]; discussed above) and methylationmediated silencing of several tumour-suppressor genes, including several negative regulators of Wnt/b-catenin signalling [85e88]. This is particularly interesting in light of recent data linking b-catenin induced up-regulation of TERT expression and longer telomere length in mouse cancer and stem cells [89], and the reported longer telomere lengths of human placenta [90]. Tumour-suppressor methylation appears to be limited to the trophoblast compartment, with no evidence of this in placental fibroblasts [45]. Most recently, natural variation in the methylation levels at the promoter regions of PRKCDBP and MMP2, two cancer-related genes, has been linked to the outgrowth potential of trophoblast explants [91]. The other major epigenetic similarity between malignancy and placentation is the wide-ranging loss-of-imprinting in both instances, especially in the first trimester placenta [23,24,92,93]. Loss-of-imprinting refers to the aberrant expression of an allele that is normally imprinted or silenced [22]. LOI is one of the earliest and most common epigenetic aberrations in cancers [94] and loss of Dnmt1 in chimeric mice leads to global LOI and predisposition to a range of different tumours [95]. It is possible that frequent LOI in human placenta is due to DNA-methylation-mediated silencing of DNMT1 (possibly maternally imprinted) in human placenta [10,25]. On the other hand, the mouse placenta is less invasive, with little similarity to an invasive tumour. In this system the Dnmt1 promoter is not methylated. It would be interesting to test if LOI is common in baboon and marmoset placenta, which are also more invasive, and in which the DNMT1 promoter is methylated [96]. 3.2. Inherent complexity in ascribing functional roles to placentaspecific DNA marks Due to its role in gene expression, tissue-specific DNA methylation patterns are assumed to reflect the function of a specific gene in a particular tissue. DNA methylation studies in placental disease have the potential to identify new genes associated with placental function. However, identification of such functional roles requires empirical testing on a case-by-case basis. Evidence that proper DNA methylation establishment is necessary for normal placental function comes from mouse knockdown studies. Placentas of Dnmt3L KO mice show several morphological defects, including hyperproliferation of trophoblast giant cells and defects in labyrinth formation [97,98]. Treatment of pregnant rats with the demethylating agent 5-aza-dC at different stages of gestation results in a range of phenotypic abnormalities, including low trophoblast proliferation, smaller placental weight, and altered placental structure [99]. The same group further showed that 5-aza-dC treatment of rat trophoblasts in culture affected their differentiation and invasive ability [100]. These studies suggest that disruption of

DNA methylation patterns in the rat placenta can have profound effects on function. Interestingly, the fact that phenotypic outcomes are different depending on time of treatment suggests that DNA methylation patterns in the placenta are dynamic and control different genes at different developmental stages. The link between promoter methylation and trophoblast development has also been investigated in the mouse, with methylation of Oct4 and Elf5 genes implicated in trophoblast lineage determination [101,102]. We have previously demonstrated placenta-specific methylation of CYP24A1 (encoding 24-a-hydroxylase, the major catabolic enzyme of vitamin D) and demonstrated attenuation of the capacity for up-regulation of this gene in response to active vitamin D in human placental cells [103]. We speculated that this may represent a mechanism for maximising transfer of active vitamin D from maternal to fetal circulations during pregnancy [104]. Surprisingly however, a direct examination of the relationship between placental CYP24A1 methylation with neonatal and maternal circulating vitamin D levels from 86 twin pregnancies, failed do identify such a relationship [105], raising the possibility of a placenta-specific function of this methylation, or a ‘functional window’ in earlier gestation. Similar functional roles remain to be identified for a large number of placenta-specific DNA methylation events. 4. Environmental influence on placental DNA methylation External environmental influence on fetal growth and development has been firmly established in humans and animal models. Many studies have linked maternal malnutrition to low birthweight, high fat diet to diabetes, exposure to teratogens to congenital birth defects, as well as maternal stress to infant neurodevelopmental disorders [106e110]. The timing and level of exposure to a particular environmental factor or insult are important, and may have differing effects on fetal health [55,56]. Furthermore, the Developmental Origins of Health and Disease (DOHaD) hypothesis (derived from the earlier Barker hypothesis [111]), that links adult onset disease to early environmental exposures, is now widely accepted, being supported by a growing body of evidence. Epigenetic variation represents the strongest candidate mechanism linking environmental factors to specific pregnancy outcome [112]. As the tissue most exposed to environmental factors during pregnancy, the placenta is likely to show the strongest evidence of any environmental ‘footprint’ or ‘memory’ of environmental insult during pregnancy. Therefore, mapping placental epigenetic profile provides a unique opportunity to understand placental responsiveness to the environment, including the identification of genes that are sensitive to environmental perturbation. A wide range of gene-environment studies involving measurement of DNA methylation has been carried out in the human (Table 1) and mouse placenta [reviewed in Refs. [32,113e115]]. Generally the data being accumulated suggests that genetics and the environment together modulate placental DNA methylation, development and function. Furthermore, evidence for an association between specific environmental factors and placental DNA methylation patterns is emerging. However, it is difficult to draw strong conclusions from such studies due to either conflicting results (e.g. assisted reproductive technology studies e see below), or general lack of data replication. In either case, it is becoming evident that these studies require larger sample sizes in order to identify meaningful associations. 4.1. Epigenetic drift in the human placenta? Epigenetic drift refers to an increase in inter-individual variation at specific genomic sites in response to genetic, environmental and/ or stochastic factors over time [116]. Conflicting data exist in relation to epigenetic drift in somatic tissues and few studies have

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Table 1 Association between environment and DNA methylation in human placenta. Environment

Genomic-region assayed

Major finding

Reference

Maternal alcohol, smoking, vitamin supplementation Maternal and neonatal 25-OH-D level

LINE1, AluYb8 and genome-scale promoter regions 25(OH)D-24-hydroxylase (CYP24A1)

[124]

Maternal choline intake

Cortisol regulating genes

Maternal blood glucose

Adiponectin (ADIPOQ)

ART

KvDMR imprinted region

ART

H19/IGF2

ART ART

Genome-scale promoter regions (800 genes) H19/IGF2

ART

10 imprinted DMRs

Maternal smoking

CYP1A1

Maternal smoking

Genome-scale promoter regions (14,000 genes)

Maternal smoking

MicroRNA

The relationship varied between environment and genomic-region. A sex effect was also observed. No association between placental CYP24A1 methylation level and maternal or neonatal vitamin D concentrations. Higher maternal choline intake increased promoter methylation of several genes involved in cortisol regulation. Higher ADIPOQ methylation in placenta was associated with higher level of maternal glucose following glucose tolerance test. Hypomethylation of KvDMR was observed in w15% of clinically normal children conceived through IVF or ICSI. Differential expression, but not methylation in placenta from the in vitro group. Overall hypomethylation in IVF group, however the changes are slight. No difference in placental methylation between IVF and natural conception groups. No evidence for increased DMR methylation variability in amnion/chorion or cord blood between children conceived by natural conception and IVF or ICSI. Lower methylation and higher expression of CYP1A1 in smoking mothers. Over 1000 genes show significant differences in methylation. Of these 38 showed differences greater than 10%. Smoking causes downregulation of miR-16, miR-21, and miR-146a.

attempted to identify the potential source of such variation. At present, there are no currently available methodologies for ‘teasing out’ the relative effects of environment and stochastic variation to epigenetic variation. However, the twin study design is amenable to identifying the level of drift in humans due to genetics and these factors in combination. Any variation in monozygotic twins is unlikely to have a genetic basis, instead arising through combined non-shared environmental and stochastic factors [117]. Emerging evidence suggests that epigenetic drift begins in utero, possibly from conception, and that monozygotic twins are epigenetically divergent even at birth [105,118,119]. These data involved measuring DNA methylation discordance at specific genomic loci (for example the H19/IGF2 imprinted locus [118] or CYP24A1 gene [105]) and on a genome-scale using array-based methods, in a range of birth tissues including placenta [119]. Placentas from monozygotic twins (both monochorionic and dichorionic) showed differences in DNA methylation profile, although the degree of discordance differed between pairs, suggesting differential in utero exposures for each placenta during pregnancy. The existence of CpG sites exhibiting highly variable methylation in the 3rd trimester placenta was first reported using a relatively low resolution methylation array platform [120]. In that study, it was proposed that highly variable CpG sites (epipolymorphisms) were especially sensitive to environmental influence and may be associated with placental disease. Subsequently it was shown that the number of variable CpG sites in the placenta increases from first trimester to term, suggesting an accumulation of environmentally mediated or stochastic effects over time [50].

[105]

[121]

[194]

[135]

[133] [136] [132] [134]

[126] [125]

[83]

increased choline uptake increases methionine and S-adenosyl methionine in human placenta, and is associated with higher methylation of several genes [121], while prenatal alcohol exposure is generally associated with reduced DNA methylation in both the placenta and fetal tissue in mice [122,123]. A recent study in humans found slightly elevated LINE-1 repeat methylation in association with alcohol intake during pregnancy [124]. Therefore, the effect of alcohol on DNA methylation levels may be dependent on genomicregion or may also be time and dose dependent. Further, adequately powered, studies are required in this important area. The effects of maternal smoking on placental epigenetics have also been a focus of research. Smoking during pregnancy has been associated with minor alterations in genome-scale promoter DNA methylation in the placenta, with at least 1000 (w3% of analysed) CpG sites affected [125]. One of those genes subject to change was CYP1A1 (cytochrome P450 1A1), which codes for a protein that catabolises harmful nicotine compounds into less active metabolites [126]. Up-regulation of CYP1A1 may represent a placental response to elevated levels of toxins [126]. Furthermore, maternal smoking has been shown to alter the expression of several developmentally important microRNAs in human placenta [83]. Of four candidate miRNAs tested in one study, three (miR-16, miR-21 and miR-146a) were significantly reduced in smoke-exposed placentas [83]. Data on the effect of maternal illicit drug use is not currently available but is likely to be of relevance to placental epigenetics given that many such compounds have now been shown to modify epigenetic profile (reviewed in Ref. [127]). Similarly, environmental pollutants and toxins have the potential to alter the developing placental epigenome during pregnancy.

4.2. Maternal diet, alcohol intake and smoking 4.3. Assisted reproductive technology A range of common environmental exposures and nutritional factors, previously linked to adverse pregnancy outcomes, has also been shown to alter DNA methylation in the placenta. For example

The effect of assisted reproductive technology (ART) on DNA methylation has been hotly debated for several years. Interestingly,

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evidence in mice suggests that in vitro fertilisation can alter imprinted gene methylation in the placenta, while the fetus may be spared such disruption [128]. This finding supports a placental epigenetic response to IVF culture, possibly to shield the developing embryo from an adverse environmental exposure. Studies in humans have linked ART to higher incidence of imprinting disorders [129,130], however data is mixed [131]. Studies in humans have generally reported no effect on placental imprinting following IVF [132e134], although a single study identified methylation differences at the KvDMR locus in placentas of about 15% (3/18) of clinically healthy babies [135]. A low resolution genome-scale DNA methylation profile of full term placenta identified widespread hypomethylation in association with IVF, with 246 of 1536 CpG sites examined, showing significantly lower methylation in the IVF group [136]. The same study also failed to identify a link between IVF and altered DNA methylation at imprinted genes. Similarly, imprinting aberrations and placental defects have been reported in cloned animals [137,138], implicating these technologies in the disruption of epigenetic profile. 5. Role of DNA methylation in pregnancy-associated disease Placental function is directly linked to fetal development and health, and as such placental dysfunction is implicated in many pregnancy-associated diseases [139]. Accordingly, aberrant placental gene expression has been linked to preeclampsia, intrauterine growth restriction, gestational diabetes mellitus and gestational trophoblastic disease [140e146]. Given the link between gene expression and promoter methylation, it is reasonable to assume that aberrant expression in placental disease is associated with disrupted DNA methylation profile or other epigenetic change [120]. Furthermore, epigenetics may provide a link between environmental factors that have previously been linked to poor pregnancy outcome and fetal programming [147,148]. A number of recent studies support such a link (Table 2) with the potential to provide mechanistic insights into the origin

and progression outcomes.

of

placenta-associated

adverse

pregnancy

5.1. Preeclampsia Preeclampsia (PE) alone affects up to 5% of all pregnancies, and is the biggest cause of maternal and neonatal morbidity and mortality [149]. It is usually diagnosed in the second half of pregnancy, and is thought to be a ‘two-stage’ disease, with a placental phenotype followed by a maternal phenotype. The only treatment is delivery. The placentas of PE pregnancies are characterised by shallow EVT invasion, abnormal remodelling of spiral arteries and hypoxia [149]. Several environmental factors, known to influence DNA methylation, have been implicated in preeclampsia aetiology, including elevated maternal homocysteine and decreased vitamin D levels [150e152]. Furthermore, several studies have provided a link between genome-wide and imprinted gene DNA methylation aberrations and preeclampsia [23,120,153,154] (Table 2). Higher maternal homocysteine and an increase in global DNA methylation were reported in preeclampsia, with no difference in maternal folate levels [151,155]. These findings were supported by a study that found increased LINE1 methylation in early-onset PE (EOPE) [156]. In their study, Gao et al. [156] also reported increased expression of the maintenance DNA methyltransferase (DNMT1) in EOPE compared to late onset PE (LOPE), raising the possibility that these distinct types of PE may involve different epigenetic aberrations. This is supported by a study by Yuen et al. [157], which reported hypomethylation at 34 genes in EOPE, but only 4 in LOPE, relative to disease free matched control tissue. Specific genes included matrix metalloproteinase-9 (MMP9), involved in the degradation of the extracellular matrix, and its inhibitor, TIMP3 [157,158] and homoeobox genes SOX7 and CDX1 (Table 2) [159]. In addition to DNA methylation studies, differential expression of several miRNAs has also been associated with preeclampsia [160e162], including overexpression of the hypoxia inducible miR-200 [163].

Table 2 Association between pregnancy-associated disease and DNA methylation in human placenta. Disease

Genomic-region assayed

Major finding

Reference

Preeclampsia

Global

[155]

Preeclampsia

LINE1 and H19

Preeclampsia Preeclampsia Preeclampsia Preeclampsia Preeclampsia

CpG island array Genome-scale promoter regions (w800 genes) COMT MMP9 gene RASSF1A and SERPINB5

Preeclampsia/IUGR

SERPINA3

Preeclampsia

MicroRNA

Preeclampsia

Genome-scale microRNA

IUGR IUGR

Genome-scale promoter regions (w14,000 genes) Imprinted genes

IUGR

H19/IGF2, LINE1, miRNA

IUGR IUGR Recurrent miscarriage

IGF2 H19 CGB5

Higher global methylation in term and preterm preeclampsia compared to healthy placenta. Higher LINE-1 methylation in EOPE compared to controls. Higher expression of DNMT1 in EOPE compared to LOPE Hypermethylation and lower expression of H19 in EOPE. Significantly aberrant methylation of 296 genes in preeclampsia Hypomethylation of 34 loci in EOPE and 5 in LOPE. Top ranked gene was TIMP3. No association between COMT methylation and preeclampsia. Lower methylation and higher expression of MMP9 in preeclampsia. No association between methylation level at either gene and preeclampsia Hypomethylation of the promoter region and higher expression observed in disease placenta. mir-210 is up-regulated in patients with pre-eclampsia and trophoblast cells cultured under hypoxic conditions. Up-regulation of miRNA involved in angiogenesis. Targets include genes involved in early placental development. DNA methylation profiling can distinguish IUGR and SGA placentas from AGA placentas. Frequent LOI in normal and IUGR placentas, however there was no overlap between LOI and gene expression level. No difference between IUGR and normal placenta in H19 and LINE1 methylation or miRNA expression. Loss-of-imprinting of IGF2 in association with IUGR. Hypomethylation and higher expression of H19 is associated with IUGR. Hemimethylation and monoallelic expression in recurrent miscarriage.

[156]

[159] [157] [195] [158] [76] [170] [163] [161] [167] [23] [36] [169] [168] [196]

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Gene expression aberrations in the first trimester CVS, preceding the onset of PE symptoms, have been reported [164]. Such analysis has the potential to provide predictive markers of PE, and improve the ability to monitor and treat women at high risk of PE. A recent comparison between first trimester genome-wide expression levels in pregnancies destined for preeclampsia [145] and known candidate gene susceptibility loci identified by several linkage studies, showed that about 40% of gene expression changes associated with PE occur at these previously identified susceptibility loci [165]. This study highlights the link between placental gene expression aberrations in the first trimester and the development of preeclampsia [165].

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inducibility of SERPINA3, in a SNP-dependent manner [171]. Furthermore, a second SNP was identified in the first exon, and was associated with a predisposition to PE, suggesting that SERPINA3 plays a mechanistic role in PE development [171]. Cumulatively, these findings suggest that placental pathologies are characterised by specific DNA methylation profiles, which can differ between EOPE, LOPE, IUGR and ‘normal’ pregnancies that lead to small or large for gestational age infants. Notably, the condition with the highest morbidity, EOPE, appears to have the most disrupted epigenetic profile [156,157]. 6. Placental DNA methylation and neonatal outcome

5.2. Intrauterine growth restriction

6.1. Birth-weight

An IUGR fetus is diagnosed as having a birth-weight of below the 10th percentile for its gestational age and whose abdominal circumference is below the 2.5th percentile, using ultrasound measurements [166]. IUGR placentas are smaller and display vascular abnormalities and hormonal aberrations compared to placentas from healthy pregnancies (reviewed in Ref. [147]). Methylation BeadChip array analysis of 206 term placentas identified 22 methylation loci that could predict SGA and IUGR pregnancies with some confidence [167]. Analysis of a larger number of placentas would enable more accurate prediction of normal and disease placentas [167]. In a related study, LINE1 methylation was quantified in 60 normal and 60 IUGR placentas, using pyrosequencing [36]. Unlike EOPE placentas, which showed higher LINE1 methylation [156], IUGR placentas did not display difference in LINE1 methylation pattern compared to controls [36]. Methylation and expression analysis of the H19/IGF2 locus in IUGR identified lower methylation and increased expression of H19 and decreased expression of IGF2 in disease relative to control placentas [168,169]. Furthermore, LOI was identified to occur in IUGR at the same rate as normal placentas, but the subset of imprinted genes showing LOI differed [23]. SERPINA3 promoter hypomethylation was also observed in PE and IUGR placentas, however the effect on gene expression differed between the two diseases [170]. Further analysis indicated that SERPINA3 expression was controlled by a combination of genetic (SNPs) and epigenetic mechanisms, and transcription factor binding. The change in promoter methylation level was found to influence the

In addition to a potential role in adverse pregnancy outcomes, the association between placental methylation and other neonatal outcomes, such as birth-weight, may provide a functional link between the previously described minor variations in placental function and fetal growth. Whereas DNA methylation changes associated with disease may reflect aberrant signalling pathways and cell death, changes associated with the spectrum of phenotypes in ‘normal’ pregnancies, may reflect the placental adaptation to specific environments and fetal or maternal needs. Several recent studies have examined the link between placental methylation change and neonatal outcome (Table 3). Reported associations between altered placental DNA methylation and SGA include lower methylation of HERWE1 in the smaller of two discordant twins [38], and elevated methylation at WNT2 [172] and HSD11B2 [173], the primary regulator of cortisol levels, in SGA infants. The promoter region of the glucocorticoid receptor gene (NR3C1) showed higher methylation in large for gestational age (LGA) infants, compared to SGA and control infants [174]. Methylation array analysis of 48 placentas identified 23 differentially methylated genes that explain up to 80% of birth-weight variance [175]. However, another study using the same platform, failed to identify significant associations between placenta methylation and birth-weight discordance in twins [119]. Furthermore, variations in HSD11B2 and NR3C1 promoter methylation and differential expression of imprinted genes have recently been associated with infant neurobehaviour [173,176,177]. Interestingly, similar elevated methylation at the HSD11B2 gene in the placenta

Table 3 Association between pregnancy outcome and DNA methylation in human placenta. Outcome

Genomic-region assayed

Major finding

Reference

LGA

Glucocorticoid receptor

[174]

SGA Birth-weight Birth-weight

WNT2 Genome-scale promoter regions (w14,000 genes) LINE1

Birth-weight discordance SGA

Genome-scale promoter regions (w14,000 genes) HERWE1

SGA

H19/IGF2 and KvDMR

Allergy

CD14

Birth-weight and neuro-behavioural outcomes Neuro-behavioural outcomes

HSD11B2

Differential methylation associated with LGA. Link between environment, placenta epigenetics and infant outcome. Higher methylation and lower expression of WNT2 in SGA placenta. Identified 23 genes, for which DNA methylation levels explain up to 80% of birth-weight variance. In SGA infants, placenta and cord blood showed higher and lower LINE-1 methylation compared to AGA infants, respectively. No CpG sites showed significant association with birth-weight discordance, after correction for multiple testing. Top gene HLA-B. Loss of methylation and higher expression of HERWE1 in smaller fetuses. Differential expression of DNMT3B isoforms was also detected. Loss of H19 methylation in 1/20 SGA placentas. Lower IGF2 expression in all SGA. Living on a farm results in lower methylation and higher expression of CD14, as well as lower allergy rates. Higher methylation at the HSD11B2 promoter is associated with lower birth-weight and reduced scores in neuro-behavioural tests in infants. In genetically predisposed individuals, placental methylation at the NR3C1 promoter is associated with infant movement and attention.

NR3C1

[172] [175] [197] [119] [38] [198] [180] [173]

[177]

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has also been linked to maternal stress during pregnancy in rats [178]. Although requiring further investigation in larger sample sizes, these cumulative data support a role of placental methylation in neonatal outcomes beyond just fetal growth.

7.3. Beyond DNA methylation

Specific environmental exposures during intrauterine development can predispose the fetus to the development of allergic disease [179]. Evidence is starting to suggest that this programming involves an epigenetics [180]. This is very interesting, given recent evidence of the role of DNA methylation in T cell specification [181] and immune compartment development postnatally [182]. The potential link between placental epigenetic profile and allergy risk is now being investigated. Living on a farm (an indicator of higher microbial burden) is one of the strongest protective exposures to allergy development [183] and is associated with lower methylation (and higher expression) of CD14 in the placenta [180]. Up-regulation of CD14 could result in skewing away from the Th2 immune response, which is associated with asthma and allergy development [180]. Similarly farm exposure has been linked to elevated FOXP3 levels with associated decreasing methylation in T cells [184]. Interestingly FOXP3 expression was reduced in placentas from children who later developed allergy [185].

As with any tissue, specification and function of placental cells undoubtedly involves a complex interplay between many different types of epigenetic modification to regulated gene expression and cell function. DNA methylation can be considered a primary epigenetic modification, being directly added to underlying DNA sequence [3]. Higher order modifications include (i) packaging of DNA into nucleosomes containing variant histones, (ii) a wide range of histone modifications, (iii) association of non-coding RNAs (both short and long) and (iv) association of a diverse range of chromatin modifying proteins and complexes. Each of these has been examined to some extent in the human placenta [191e193]. However, the technologies for studying such factors are more challenging and prone to sample quality issues than DNA methylation analysis. Only when a concerted effort to profile additional epigenetic regulators is undertaken in a range of purified placenta-derived cell types, in association with specific exposures and phenotypes, will we begin to fully understand the complexities and role of epigenetics in placental function in humans. Nevertheless, the presence of placental DNA in the maternal circulation provides the tantalising possibility that one day it will be possible to non-invasively monitor the effect of certain diets and harmful environmental exposures on placental DNA methylation, and to extrapolate that data to predict pregnancy outcome.

7. Challenges and future directions

Acknowledgements

7.1. Placental heterogeneity

BN is supported by a National Health and Medical Research Council (Australia) Dora Lush Postgraduate Research scholarship and RS is supported by an NHMRC:EU grant. The Murdoch Childrens Research Institute is supported by the Victorian Government’s Operational Infrastructure Support Program.

6.2. Infant allergy

The placenta is a heterogeneous tissue with a chimeric pattern of gene expression and DNA methylation. Differences in methylation across different sites, and at different depths, within the same placenta have been identified in several studies, however these differences appear smaller than those between different individuals [186]. Nevertheless, depending on the genomic-region assayed, inter-individual differences in methylation may be due to sampling different cellular populations [186]. This is supported by an analysis of X inactivation (associated with DNA methylation) that showed variable skewing across placental sites, suggesting that each villous tree was clonally derived from a small population of precursor cells [43]. Further data suggest that such effects are likely to be locus specific, as an examination of methylation at LINE1 and stress-related genes between sampling sites within the same placenta did not reveal sampling site effects [187]. 7.2. Association vs causation Very few studies have yet examined the direct functional consequences of altered DNA methylation in the human placenta. Although epigenetic association studies provide prima facie evidence for a link with altered phenotype, it is equally plausible that changes in DNA methylation are a consequence of the measured phenotype rather than part of the causal pathway [188]. All candidate associations require further testing in appropriate model systems before any causative link can be established. In humans, this is complicated by the fact that trophoblast cells show limited survival in culture [189], and show vastly different genomescale DNA methylation patterns and functional properties relative to trophoblast-derived cell lines [190] and to animal model counterparts. A recent example of a study linking methylation and function is by van Dijk and colleagues, who showed a link between PRKCDBP and MMP2 methylation and trophoblast invasion potential [91].

References [1] Reik W, Dean W. DNA methylation and mammalian epigenetics. Electrophoresis 2001;22(14):2838e43. [2] Strahl BD, Allis CD. The language of covalent histone modifications. Nature 2000;403(6765):41e5. [3] Bird A. DNA methylation patterns and epigenetic memory. Genes Dev 2002; 16(1):6e21. [4] Bird AP. CpG-rich islands and the function of DNA methylation. Nature 1986; 321(6067):209e13. [5] Mattick JS, Makunin IV. Non-coding RNA. Hum Mol Genet 2006;15(1):R17e29. [6] Monk M, Boubelik M, Lehnert S. Temporal and regional changes in DNA methylation in the embryonic, extraembryonic and germ cell lineages during mouse embryo development. Development (Cambridge, England) 1987; 99(3):371e82. [7] Santos F, Hendrich B, Reik W, Dean W. Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 2002;241(1):172e82. [8] Cotton AM, Avila L, Penaherrera MS, Affleck JG, Robinson WP, Brown CJ. Inactive X chromosome-specific reduction in placental DNA methylation. Hum Mol Genet 2009;18(19):3544e52. [9] Gama-Sosa MA, Wang RY, Kuo KC, Gehrke CW, Ehrlich M. The 5-methylcytosine content of highly repeated sequences in human DNA. Nucleic Acids Res 1983; 11(10):3087e95. [10] Novakovic B, Wong NC, Sibson M, Ng HK, Morley R, Manuelpillai U, et al. DNA methylation-mediated down-regulation of DNA methyltransferase-1 (DNMT1) is coincident with, but not essential for, global hypomethylation in human placenta. J Biol Chem 2010;285(13):9583e93. [11] Nelissen EC, van Montfoort AP, Dumoulin JC, Evers JL. Epigenetics and the placenta. Hum Reprod Update 2011;17(3):397e417. [12] Maccani MA, Marsit CJ. Epigenetics in the placenta. Am J Reprod Immunol 2009;62(2):78e89. [13] Rugg-Gunn PJ. Epigenetic features of the mouse trophoblast. Reprod Biomed Online 2012;25(1):21e30. [14] Serman L, Dodig D. Impact of DNA methylation on trophoblast function. Clin Epigenetics 2011;3:7. [15] Monk D, Arnaud P, Apostolidou S, Hills FA, Kelsey G, Stanier P, et al. Limited evolutionary conservation of imprinting in the human placenta. Proc Natl Acad Sci U S A 2006;103(17):6623e8.

B. Novakovic, R. Saffery / Placenta 33 (2012) 959e970 [16] Renfree MB, Hore TA, Shaw G, Graves JA, Pask AJ. Evolution of genomic imprinting: insights from marsupials and monotremes. Annu Rev Genomics Hum Genet 2009;10:241e62. [17] Moore GE, Oakey R. The role of imprinted genes in humans. Genome Biol 2011;12(3):106. [18] Moore T. Review: parent-offspring conflict and the control of placental function. Placenta 2012;33(Suppl.):S33e6. [19] Wagschal A, Feil R. Genomic imprinting in the placenta. Cytogenet Genome Res 2006;113(1e4):90e8. [20] Frost JM, Moore GE. The importance of imprinting in the human placenta. PLoS Genet 2010;6(7):e1001015. [21] Proudhon C, Bourc’his D. Identification and resolution of artifacts in the interpretation of imprinted gene expression. Brief Funct Genomics 2010; 9(5e6):374e84. [22] Lambertini L, Diplas AI, Lee MJ, Sperling R, Chen J, Wetmur J. A sensitive functional assay reveals frequent loss of genomic imprinting in human placenta. Epigenetics 2008;3(5):261e9. [23] Diplas AI, Lambertini L, Lee MJ, Sperling R, Lee YL, Wetmur J, et al. Differential expression of imprinted genes in normal and IUGR human placentas. Epigenetics 2009;4(4):235e40. [24] Pozharny Y, Lambertini L, Ma Y, Ferrara L, Litton CG, Diplas A, et al. Genomic loss of imprinting in first-trimester human placenta. Am J Obstet Gynecol 2010;202(4):391. e391e398. [25] Yuen RK, Jiang R, Penaherrera MS, McFadden DE, Robinson WP. Genomewide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies. Epigenetics Chromatin 2011;4(1):10. [26] Choufani S, Shapiro JS, Susiarjo M, Butcher DT, Grafodatskaya D, Lou Y, et al. A novel approach identifies new differentially methylated regions (DMRs) associated with imprinted genes. Genome Res 2011;21(3):465e76. [27] Barbaux S, Gascoin-Lachambre G, Buffat C, Monnier P, Mondon F, Tonanny MB, et al. A genome-wide approach reveals novel imprinted genes expressed in the human placenta. Epigenetics Off J DNA Methylation Soc 2012;7(9):1079e90. [28] Proudhon C, Bourc’his D. Evolution of genomic imprinting in mammals: what a zoo! Med Sci (Paris) 2010;26(5):497e503. [29] Okae H, Hiura H, Nishida Y, Funayama R, Tanaka S, Chiba H, et al. Reinvestigation and RNA sequencing-based identification of genes with placenta-specific imprinted expression. Hum Mol Genet 2012;21(3):548e58. [30] Frost JM, Moore GE. The importance of imprinting in the human placenta. PLoS Genet 2010;6:e1001015. [31] Piedrahita JA. The role of imprinted genes in fetal growth abnormalities. Birth Defects Res A Clin Mol Teratol 2011;91(8):682e92. [32] Fowden AL, Coan PM, Angiolini E, Burton GJ, Constancia M. Imprinted genes and the epigenetic regulation of placental phenotype. Prog Biophys Mol Biol 2011;106(1):281e8. [33] Barlow DP. Genomic imprinting: a mammalian epigenetic discovery model. Annu Rev Genet 2011;45:379e403. [34] Christensen BC, Houseman EA, Marsit CJ, Zheng S, Wrensch MR, Wiemels JL, et al. Aging and environmental exposures alter tissue-specific DNA methylation dependent upon CpG island context. PLoS Genet 2009;5(8): e1000602. [35] Perrin D, Ballestar E, Fraga MF, Frappart L, Esteller M, Guerin JF, et al. Specific hypermethylation of LINE-1 elements during abnormal overgrowth and differentiation of human placenta. Oncogene 2007;26(17):2518e24. [36] Tabano S, Colapietro P, Cetin I, Grati FR, Zanutto S, Mando C, et al. Epigenetic modulation of the IGF2/H19 imprinted domain in human embryonic and extra-embryonic compartments and its possible role in fetal growth restriction. Epigenetics Off J DNA Methylation Soc 2010;5(4):313e24. [37] Cohen CJ, Rebollo R, Babovic S, Dai EL, Robinson WP, Mager DL. Placentaspecific expression of the interleukin-2 (IL-2) receptor beta subunit from an endogenous retroviral promoter. J Biol Chem 2011;286(41):35543e52. [38] Gao Y, He Z, Wang Z, Luo Y, Sun H, Zhou Y, et al. Increased expression and altered methylation of HERVWE1 in the human placentas of smaller fetuses from monozygotic, dichorionic, discordant twins. PloS One 2012;7(3): e33503. [39] Gimenez J, Montgiraud C, Oriol G, Pichon JP, Ruel K, Tsatsaris V, et al. Comparative methylation of ERVWE1/syncytin-1 and other human endogenous retrovirus LTRs in placenta tissues. DNA Res 2009;16(4):195e211. [40] Knerr I, Beinder E, Rascher W. Syncytin, a novel human endogenous retroviral gene in human placenta: evidence for its dysregulation in preeclampsia and HELLP syndrome. Am J Obstet Gynecol 2002;186(2):210e3. [41] Macaulay EC, Weeks RJ, Andrews S, Morison IM. Hypomethylation of functional retrotransposon-derived genes in the human placenta. Mamm Genome 2011;22(11e12):722e35. [42] Moreira de Mello JC, de Araujo ES, Stabellini R, Fraga AM, de Souza JE, Sumita DR, et al. Random X inactivation and extensive mosaicism in human placenta revealed by analysis of allele-specific gene expression along the X chromosome. PloS One 2010;5(6):e10947. [43] Penaherrera MS, Jiang R, Avila L, Yuen RK, Brown CJ, Robinson WP. Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation. Hum Reprod 2012;27(6):1745e53. [44] Apps R, Sharkey A, Gardner L, Male V, Trotter M, Miller N, et al. Genomewide expression profile of first trimester villous and extravillous human trophoblast cells. Placenta 2011;32(1):33e43.

967

[45] Grigoriu A, Ferreira JC, Choufani S, Baczyk D, Kingdom J, Weksberg R. Cell specific patterns of methylation in the human placenta. Epigenetics Off J DNA Methylation Soc 2011;6(3):368e79. [46] Tolmacheva EN, Kashevarova AA, Skriabin NA, Lebedev IN. DNA methylation profile in human placental tissues. Mol Biol (Mosk) 2011;45(3):538e45. [47] Nakanishi MO, Hayakawa K, Nakabayashi K, Hata K, Shiota K, Tanaka S. Trophoblast-specific DNA methylation occurs after the segregation of the trophectoderm and inner cell mass in the mouse periimplantation embryo. Epigenetics Off J DNA Methylation Soc 2012;7(2):173e82. [48] O’Neill LP, VerMilyea MD, Turner BM. Epigenetic characterization of the early embryo with a chromatin immunoprecipitation protocol applicable to small cell populations. Nat Genet 2006;38(7):835e41. [49] Rakyan VK, Down TA, Thorne NP, Flicek P, Kulesha E, Graf S, et al. An integrated resource for genome-wide identification and analysis of human tissue-specific differentially methylated regions (tDMRs). Genome Res 2008; 18(9):1518e29. [50] Novakovic B, Yuen RK, Gordon L, Penaherrera MS, Sharkey A, Moffett A, et al. Evidence for widespread changes in promoter methylation profile in human placenta in response to increasing gestational age and environmental/ stochastic factors. BMC Genomics 2011;12:529. [51] Kim SY, Romero R, Tarca AL, Bhatti G, Kim CJ, Lee J, et al. Methylome of fetal and maternal monocytes and macrophages at the feto-maternal interface. Am J Reprod Immunol 2012;68(1):8e27. [52] Winn VD, Haimov-Kochman R, Paquet AC, Yang YJ, Madhusudhan MS, Gormley M, et al. Gene expression profiling of the human maternal-fetal interface reveals dramatic changes between midgestation and term. Endocrinology 2007;148(3):1059e79. [53] Mikheev AM, Nabekura T, Kaddoumi A, Bammler TK, Govindarajan R, Hebert MF, et al. Profiling gene expression in human placentae of different gestational ages: an OPRU Network and UW SCOR study. Reprod Sci 2008; 15(9):866e77. [54] Chavan-Gautam P, Sundrani D, Pisal H, Nimbargi V, Mehendale S, Joshi S. Gestation-dependent changes in human placental global DNA methylation levels. Mol Reprod Dev 2011;78(3):150. [55] Laplante DP, Barr RG, Brunet A, Galbaud du Fort G, Meaney ML, Saucier JF, et al. Stress during pregnancy affects general intellectual and language functioning in human toddlers. Pediatr Res 2004;56(3):400e10. [56] Dancause KN, Laplante DP, Oremus C, Fraser S, Brunet A, King S. Disasterrelated prenatal maternal stress influences birth outcomes: project ice storm. Early Hum Dev 2011;87(12):813e20. [57] Price EM, Cotton AM, Penaherrera MS, McFadden DE, Kobor MS, Robinson W. Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues. Epigenetics Off J DNA Methylation Soc 2012;7(6):1272e80. [58] Chu T, Handley D, Bunce K, Surti U, Hogge WA, Peters DG. Structural and regulatory characterization of the placental epigenome at its maternal interface. PloS One 2011;6(2):e14723. [59] Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350(9076):485e7. [60] Litton C, Stone J, Eddleman K, Lee MJ. Noninvasive prenatal diagnosis: past, present, and future. Mt Sinai J Med New York 2009;76(6):521e8. [61] Lo YM, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45(10):1747e51. [62] Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45(2):184e8. [63] Chiu RW, Akolekar R, Zheng YW, Leung TY, Sun H, Chan KC, et al. Noninvasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. BMJ 2011;342:c7401. [64] Chen EZ, Chiu RW, Sun H, Akolekar R, Chan KC, Leung TY, et al. Noninvasive prenatal diagnosis of fetal trisomy 18 and trisomy 13 by maternal plasma DNA sequencing. PloS One 2011;6(7):e21791. [65] Kitzman JO, Snyder MW, Ventura M, Lewis AP, Qiu R, Simmons LE, et al. Noninvasive whole-genome sequencing of a human fetus. Sci Transl Med 2012;4(137):ra76. [66] Go AT, van Vugt JM, Oudejans CB. Non-invasive aneuploidy detection using free fetal DNA and RNA in maternal plasma: recent progress and future possibilities. Hum Reprod Update 2011;17(3):372e82. [67] Lo YM, Tsui NB, Chiu RW, Lau TK, Leung TN, Heung MM, et al. Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection. Nat Med 2007;13(2):218e23. [68] Yuen RK, Manokhina I, Robinson WP. Are we ready for DNA methylationbased prenatal testing? Epigenomics 2011;3(4):387e90. [69] Papageorgiou EA, Fiegler H, Rakyan V, Beck S, Hulten M, Lamnissou K, et al. Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies. Am J Pathol 2009;174(5):1609e18. [70] Papageorgiou EA, Karagrigoriou A, Tsaliki E, Velissariou V, Carter NP, Patsalis PC. Fetal-specific DNA methylation ratio permits noninvasive prenatal diagnosis of trisomy 21. Nat Med 2011;17(4):510e3. [71] Tsui DW, Lam YM, Lee WS, Leung TY, Lau TK, Lau ET, et al. Systematic identification of placental epigenetic signatures for the noninvasive prenatal detection of Edwards syndrome. PloS One 2010;5(11):e15069.

968

B. Novakovic, R. Saffery / Placenta 33 (2012) 959e970

[72] Zejskova L, Jancuskova T, Kotlabova K, Doucha J, Hromadnikova I. Feasibility of fetal-derived hypermethylated RASSF1A sequence quantification in maternal plasma e next step toward reliable non-invasive prenatal diagnostics. Exp Mol Pathol 2010;89(3):241e7. [73] Tsui DW, Chan KC, Chim SS, Chan LW, Leung TY, Lau TK, et al. Quantitative aberrations of hypermethylated RASSF1A gene sequences in maternal plasma in pre-eclampsia. Prenat Diagn 2007;27(13):1212e8. [74] Chim SS, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, et al. Detection of the placental epigenetic signature of the maspin gene in maternal plasma. Proc Natl Acad Sci U S A 2005;102(41):14753e8. [75] Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al. Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52(12):2211e8. [76] Bellido ML, Radpour R, Lapaire O, De Bie I, Hosli I, Bitzer J, et al. MALDI-TOF mass array analysis of RASSF1A and SERPINB5 methylation patterns in human placenta and plasma. Biol Reprod 2010;82(4):745e50. [77] Lambertini L, Marsit CJ, Sharma P, Maccani M, Ma Y, Hu J, et al. Imprinted gene expression in fetal growth and development. Placenta 2012;33(6): 480e6. [78] Soundararajan R, Rao AJ. Trophoblast ‘pseudo-tumorigenesis’: significance and contributory factors. Reprod Biol Endocrinol 2004;2:15. [79] Perry JK, Lins RJ, Lobie PE, Mitchell MD. Regulation of invasive growth: similar epigenetic mechanisms underpin tumour progression and implantation in human pregnancy. Clin Sci (Lond) 2010;118(7):451e7. [80] Ferretti C, Bruni L, Dangles-Marie V, Pecking AP, Bellet D. Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts. Hum Reprod Update 2007;13(2):121e41. [81] Rama S, Suresh Y, Rao AJ. Regulation of telomerase during human placental differentiation: a role for TGFbeta1. Mol Cell Endocrinol 2001;182(2):233e48. [82] Bamberger AM, Bamberger CM, Aupers S, Milde-Langosch K, Loning T, Makrigiannakis A. Expression pattern of the activating protein-1 family of transcription factors in the human placenta. Mol Hum Reprod 2004;10(4): 223e8. [83] Maccani MA, Avissar-Whiting M, Banister CE, McGonnigal B, Padbury JF, Marsit CJ. Maternal cigarette smoking during pregnancy is associated with downregulation of miR-16, miR-21, and miR-146a in the placenta. Epigenetics Off J DNA Methylation Soc 2010;5(7):583e9. [84] Gama-Sosa MA, Slagel VA, Trewyn RW, Oxenhandler R, Kuo KC, Gehrke CW, et al. The 5-methylcytosine content of DNA from human tumors. Nucleic Acids Res 1983;11(19):6883e94. [85] Wong NC, Novakovic B, Weinrich B, Dewi C, Andronikos R, Sibson M, et al. Methylation of the adenomatous polyposis coli (APC) gene in human placenta and hypermethylation in choriocarcinoma cells. Cancer Lett 2008; 268(1):56e62. [86] Novakovic B, Rakyan V, Ng HK, Manuelpillai U, Dewi C, Wong NC, et al. Specific tumour-associated methylation in normal human term placenta and first-trimester cytotrophoblasts. Mol Hum Reprod 2008;14(9):547e54. [87] Guilleret I, Osterheld MC, Braunschweig R, Gastineau V, Taillens S, Benhattar J. Imprinting of tumor-suppressor genes in human placenta. Epigenetics 2009;4(1):62e8. [88] Chiu RW, Chim SS, Wong IH, Wong CS, Lee WS, To KF, et al. Hypermethylation of RASSF1A in human and rhesus placentas. Am J Pathol 2007; 170(3):941e50. [89] Hoffmeyer K, Raggioli A, Rudloff S, Anton R, Hierholzer A, Del Valle I, et al. Wnt/beta-catenin signaling regulates telomerase in stem cells and cancer cells. Science 2012;336(6088):1549e54. [90] Allsopp R, Shimoda J, Easa D, Ward K. Long telomeres in the mature human placenta. Placenta 2007;28(4):324e7. [91] van Dijk M, Visser A, Posthuma J, Poutsma A, Oudejans CB. Naturally occurring variation in trophoblast invasion as a source of novel (epigenetic) biomarkers. Front Genet 2012;3:22. [92] Lambertini L, Diplas AI, Lee MJ, Sperling R, Chen J, Wetmur J. A sensitive functional assay reveals frequent loss of genomic imprinting in human placenta. Epigenetics Off J DNA Methylation Soc 2008;3(5):261e9. [93] Diplas AI, Hu J, Lee MJ, Ma YY, Lee YL, Lambertini L, et al. Demonstration of all-or-none loss of imprinting in mRNA expression in single cells. Nucleic Acids Res 2009;37(21):7039e46. [94] Jelinic P, Shaw P. Loss of imprinting and cancer. J Pathol 2007;211(3):261e8. [95] Holm TM, Jackson-Grusby L, Brambrink T, Yamada Y, Rideout 3rd WM, Jaenisch R. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell 2005;8(4):275e85. [96] Ng HK, Novakovic B, Hiendleder S, Craig JM, Roberts CT, Saffery R. Distinct patterns of gene-specific methylation in mammalian placentas: implications for placental evolution and function. Placenta 2010;31(4):259e68. [97] Arima T, Hata K, Tanaka S, Kusumi M, Li E, Kato K, et al. Loss of the maternal imprint in Dnmt3Lmat/ mice leads to a differentiation defect in the extraembryonic tissue. Dev Biol 2006;297(2):361e73. [98] Bourc’his D, Xu GL, Lin CS, Bollman B, Bestor TH. Dnmt3L and the establishment of maternal genomic imprints. Science 2001;294(5551):2536e9. [99] Serman L, Vlahovic M, Sijan M, Bulic-Jakus F, Serman A, Sincic N, et al. The impact of 5-azacytidine on placental weight, glycoprotein pattern and proliferating cell nuclear antigen expression in rat placenta. Placenta 2007; 28(8e9):803e11.

[100] Serman L, Serman A, Fabijanovic D. An in vivo rat model to study epigenetic control of cell invasion. Med Hypotheses 2011;76(3):407e9. [101] Zhang HJ, Siu MK, Wong ES, Wong KY, Li AS, Chan KY, et al. Oct4 is epigenetically regulated by methylation in normal placenta and gestational trophoblastic disease. Placenta 2008;29(6):549e54. [102] Hemberger M, Udayashankar R, Tesar P, Moore H, Burton GJ. ELF5-enforced transcriptional networks define an epigenetically regulated trophoblast stem cell compartment in the human placenta. Hum Mol Genet 2010;19(12): 2456e67. [103] Novakovic B, Sibson M, Ng HK, Manuelpillai U, Rakyan V, Down T, et al. Placenta-specific methylation of the vitamin D 24-hydroxylase gene: implications for feedback autoregulation of active vitamin D levels at the fetomaternal interface. J Biol Chem 2009;284(22):14838e48. [104] Saffery R, Ellis J, Morley R. A convergent model for placental dysfunction encompassing combined sub-optimal one-carbon donor and vitamin D bioavailability. Med Hypotheses 2009;73(6):1023e8. [105] Novakovic B, Galati JC, Chen A, Morley R, Craig JM, Saffery R. Maternal vitamin D predominates over genetic factors in determining neonatal circulating vitamin D concentrations. Am J Clin Nutr 2012;96(1):188e95. [106] Rothman KJ, Moore LL, Singer MR, Nguyen US, Mannino S, Milunsky A. Teratogenicity of high vitamin A intake. N Engl J Med 1995;333(21):1369e73. [107] Hernandez-Diaz S, Werler MM, Walker AM, Mitchell AA. Folic acid antagonists during pregnancy and the risk of birth defects. N Engl J Med 2000; 343(22):1608e14. [108] Godfrey K, Robinson S, Barker DJ, Osmond C, Cox V. Maternal nutrition in early and late pregnancy in relation to placental and fetal growth. BMJ 1996; 312(7028):410e4. [109] Crowther CA, Hiller JE, Moss JR, McPhee AJ, Jeffries WS, Robinson JS. Effect of treatment of gestational diabetes mellitus on pregnancy outcomes. N Engl J Med 2005;352(24):2477e86. [110] Talge NM, Neal C, Glover V. Antenatal maternal stress and long-term effects on child neurodevelopment: how and why? J Child Psychol Psychiatry 2007; 48(3e4):245e61. [111] Barker DJ. The fetal and infant origins of adult disease. BMJ 1990;301(6761): 1111. [112] Sundrani DP, Chavan Gautam PM, Mehendale SS. Joshi SR: altered metabolism of maternal micronutrients and omega 3 fatty acids epigenetically regulate matrix metalloproteinases in preterm pregnancy: a novel hypothesis. Med Hypotheses 2011;77(5):878e83. [113] Senner CE, Hemberger M. Regulation of early trophoblast differentiation e lessons from the mouse. Placenta 2010;31(11):944e50. [114] Maltepe E, Bakardjiev AI, Fisher SJ. The placenta: transcriptional, epigenetic, and physiological integration during development. J Clin Invest 2010;120(4): 1016e25. [115] Hemberger M. Genetic-epigenetic intersection in trophoblast differentiation: implications for extraembryonic tissue function. Epigenetics Off J DNA Methylation Soc 2010;5(1):24e9. [116] Poulsen P, Esteller M, Vaag A, Fraga MF. The epigenetic basis of twin discordance in age-related diseases. Pediatr Res 2007;61(5 Pt 2):38Re42R. [117] Bell JT, Saffery R. The value of twins in epigenetic epidemiology. Int J Epidemiol 2012;41(1):140e50. [118] Ollikainen M, Smith KR, Joo EJ, Ng HK, Andronikos R, Novakovic B, et al. DNA methylation analysis of multiple tissues from newborn twins reveals both genetic and intrauterine components to variation in the human neonatal epigenome. Hum Mol Genet 2010;19(21):4176e88. [119] Gordon L, Joo JE, Powell JE, Ollikainen M, Novakovic B, Li X, et al. Neonatal DNA methylation profile in human twins is specified by a complex interplay between intrauterine environmental and genetic factors, subject to tissuespecific influence. Genome Res 2012;22(8):1395e406. [120] Yuen RK, Avila L, Penaherrera MS, von Dadelszen P, Lefebvre L, Kobor MS, et al. Human placental-specific epipolymorphism and its association with adverse pregnancy outcomes. PloS One 2009;4(10):e7389. [121] Jiang X, Yan J, West AA, Perry CA, Malysheva OV, Devapatla S, et al. Maternal choline intake alters the epigenetic state of fetal cortisol-regulating genes in humans. Faseb J 2012;26(8):3563e74. [122] Downing C, Johnson TE, Larson C, Leakey TI, Siegfried RN, Rafferty TM, et al. Subtle decreases in DNA methylation and gene expression at the mouse Igf2 locus following prenatal alcohol exposure: effects of a methyl-supplemented diet. Alcohol (Fayetteville, NY) 2011;45(1):65e71. [123] Garro AJ, McBeth DL, Lima V, Lieber CS. Ethanol consumption inhibits fetal DNA methylation in mice: implications for the fetal alcohol syndrome. Alcohol Clin Exp Res 1991;15(3):395e8. [124] Wilhelm-Benartzi CS, Houseman EA, Maccani MA, Poage GM, Koestler DC, Langevin SM, et al. In utero exposures, infant growth, and DNA methylation of repetitive elements and developmentally related genes in human placenta. Environ Health Perspect 2012;120(2):296e302. [125] Suter M, Ma J, Harris AS, Patterson L, Brown KA, Shope C, et al. Maternal tobacco use modestly alters correlated epigenome-wide placental DNA methylation and gene expression. Epigenetics Off J DNA Methylation Soc 2011;6(11). [126] Suter M, Abramovici A, Showalter L, Hu M, Shope CD, Varner M, et al. In utero tobacco exposure epigenetically modifies placental CYP1A1 expression. Metabolism 2010;59(10):1481e90. [127] Kovatsi L, Fragou D, Samanidou V, Njau S, Kouidou S. Drugs of abuse: epigenetic mechanisms in toxicity and addiction. Curr Med Chem 2011; 18(12):1765e74.

B. Novakovic, R. Saffery / Placenta 33 (2012) 959e970 [128] Fauque P, Ripoche MA, Tost J, Journot L, Gabory A, Busato F, et al. Modulation of imprinted gene network in placenta results in normal development of in vitro manipulated mouse embryos. Hum Mol Genet 2010;19(9): 1779e90. [129] Orstavik KH, Eiklid K, van der Hagen CB, Spetalen S, Kierulf K, Skjeldal O, et al. Another case of imprinting defect in a girl with Angelman syndrome who was conceived by intracytoplasmic semen injection. Am J Hum Genet 2003;72(1):218e9. [130] Gicquel C, Gaston V, Mandelbaum J, Siffroi JP, Flahault A, Le Bouc Y. In vitro fertilization may increase the risk of Beckwith-Wiedemann syndrome related to the abnormal imprinting of the KCN1OT gene. Am J Hum Genet 2003;72(5):1338e41. [131] Bowdin S, Allen C, Kirby G, Brueton L, Afnan M, Barratt C, et al. A survey of assisted reproductive technology births and imprinting disorders. Hum Reprod 2007;22(12):3237e40. [132] Wong EC, Hatakeyama C, Robinson WP, Ma S. DNA methylation at H19/IGF2 ICR1 in the placenta of pregnancies conceived by in vitro fertilization and intracytoplasmic sperm injection. Fertil Steril 2011;95(8):2524e6. e2521e 2523. [133] Turan N, Katari S, Gerson LF, Chalian R, Foster MW, Gaughan JP, et al. Interand intra-individual variation In allele-specific DNA methylation and gene expression in children conceived using assisted reproductive technology. PLoS Genet 2010;6(7):e1001033. [134] Tierling S, Souren NY, Gries J, Loporto C, Groth M, Lutsik P, et al. Assisted reproductive technologies do not enhance the variability of DNA methylation imprints in human. J Med Genet 2010;47(6):371e6. [135] Gomes MV, Huber J, Ferriani RA, Amaral Neto AM, Ramos ES. Abnormal methylation at the KvDMR1 imprinting control region in clinically normal children conceived by assisted reproductive technologies. Mol Hum Reprod 2009;15(8):471e7. [136] Katari S, Turan N, Bibikova M, Erinle O, Chalian R, Foster M, et al. DNA methylation and gene expression differences in children conceived in vitro or in vivo. Hum Mol Genet 2009;18(20):3769e78. [137] Wei Y, Zhu J, Huan Y, Liu Z, Yang C, Zhang X, et al. Aberrant expression and methylation status of putatively imprinted genes in placenta of cloned piglets. Cell Reprogram 2010;12(2):213e22. [138] Su JM, Yang B, Wang YS, Li YY, Xiong XR, Wang LJ, et al. Expression and methylation status of imprinted genes in placentas of deceased and live cloned transgenic calves. Theriogenology 2011;75(7):1346e59. [139] Benerischke K, Kaufmann P. Pathology of the human placenta, vol. 6. New York: Springer; 2006. [140] Sobrevia L, Abarzua F, Nien JK, Salomon C, Westermeier F, Puebla C, et al. Review: differential placental macrovascular and microvascular endothelial dysfunction in gestational diabetes. Placenta 2011;32(Suppl. 2):S159e64. [141] Kang JH, Song H, Yoon JA, Park DY, Kim SH, Lee KJ, et al. Preeclampsia leads to dysregulation of various signaling pathways in placenta. J Hypertens 2011;29(5):928e36. [142] Holdsworth-Carson SJ, Lim R, Mitton A, Whitehead C, Rice GE, Permezel M, et al. Peroxisome proliferator-activated receptors are altered in pathologies of the human placenta: gestational diabetes mellitus, intrauterine growth restriction and preeclampsia. Placenta 2010;31(3):222e9. [143] Hoegh AM, Borup R, Nielsen FC, Sorensen S, Hviid TV. Gene expression profiling of placentas affected by pre-eclampsia. J Biomed Biotechnol 2010; 2010:787545. [144] Fulop V, Mok SC, Berkowitz RS. Molecular biology of gestational trophoblastic neoplasia: a review. J Reprod Med 2004;49(6):415e22. [145] Founds SA, Conley YP, Lyons-Weiler JF, Jeyabalan A, Hogge WA, Conrad KP. Altered global gene expression in first trimester placentas of women destined to develop preeclampsia. Placenta 2009;30(1):15e24. [146] Dieber-Rotheneder M, Beganovic S, Desoye G, Lang U, Cervar-Zivkovic M. Complex expression changes of the placental endothelin system in early and late onset preeclampsia, fetal growth restriction and gestational diabetes. Life Sci 2012;91(13e14):710e5. [147] Myatt L. Placental adaptive responses and fetal programming. J Physiol 2006; 572(Pt 1):25e30. [148] Burton GJ, Jauniaux E, Charnock-Jones DS. The influence of the intrauterine environment on human placental development. Int J Dev Biol 2010;54(2e3): 303e12. [149] Redman CW, Sargent IL. Latest advances in understanding preeclampsia. Science 2005;308(5728):1592e4. [150] Robinson CJ, Alanis MC, Wagner CL, Hollis BW, Johnson DD. Plasma 25hydroxyvitamin D levels in early-onset severe preeclampsia. Am J Obstet Gynecol 2010;203(4):366. e361e366. [151] Mao D, Che J, Li K, Han S, Yue Q, Zhu L, et al. Association of homocysteine, asymmetric dimethylarginine, and nitric oxide with preeclampsia. Arch Gynecol Obstet 2010;282(4):371e5. [152] Robinson CJ, Wagner CL, Hollis BW, Baatz JE, Johnson DD. Maternal vitamin D and fetal growth in early-onset severe preeclampsia. Am J Obstet Gynecol 2011;204(6):556. e551e554. [153] Bourque DK, Avila L, Penaherrera M, von Dadelszen P, Robinson WP. Decreased placental methylation at the H19/IGF2 imprinting control region is associated with normotensive intrauterine growth restriction but not preeclampsia. Placenta 2010;31(3):197e202. [154] Yu L, Chen M, Zhao D, Yi P, Lu L, Han J, et al. The H19 gene imprinting in normal pregnancy and pre-eclampsia. Placenta 2009;30(5):443e7.

969

[155] Kulkarni A, Chavan-Gautam P, Mehendale S, Yadav H, Joshi S. Global DNA methylation patterns in placenta and its association with maternal hypertension in pre-eclampsia. DNA Cell Biol 2011;30(2):79e84. [156] Gao WL, Li D, Xiao ZX, Liao QP, Yang HX, Li YX, et al. Detection of global DNA methylation and paternally imprinted H19 gene methylation in preeclamptic placentas. Hypertens Res 2011;34(5):655e61. [157] Yuen RK, Penaherrera MS, von Dadelszen P, McFadden DE, Robinson WP. DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia. Eur J Hum Genet EJHG 2010;18(9):1006e12. [158] Wang Z, Lu S, Liu C, Zhao B, Pei K, Tian L, et al. Expressional and epigenetic alterations of placental matrix metalloproteinase 9 in preeclampsia. Gynecol Endocrinol 2010;26(2):96e102. [159] Jia RZ, Zhang X, Hu P, Liu XM, Hua XD, Wang X, et al. Screening for differential methylation status in human placenta in preeclampsia using a CpG island plus promoter microarray. Int J Mol Med 2012;30(1):133e41. [160] Zhu XM, Han T, Sargent IL, Yin GW, Yao YQ. Differential expression profile of microRNAs in human placentas from preeclamptic pregnancies vs normal pregnancies. Am J Obstet Gynecol 2009;200(6):661. e661e667. [161] Wang W, Feng L, Zhang H, Hachy S, Satohisa S, Laurent LC, et al. Preeclampsia up-regulates angiogenesis-associated microRNA (i.e., miR-17, e20a, and -20b) that target ephrin-B2 and EPHB4 in human placenta. J Clin Endocrinol Metab 2012;97(6):E1051e9. [162] Pineles BL, Romero R, Montenegro D, Tarca AL, Han YM, Kim YM, et al. Distinct subsets of microRNAs are expressed differentially in the human placentas of patients with preeclampsia. Am J Obstet Gynecol 2007;196:261. e261e266. [163] Zhang Y, Fei M, Xue G, Zhou Q, Jia Y, Li L, et al. Elevated levels of hypoxiainducible microRNA-210 in pre-eclampsia: new insights into molecular mechanisms for the disease. J Cell Mol Med 2012;16(2):249e59. [164] Farina A, Morano D, Arcelli D, De Sanctis P, Sekizawa A, Purwosunu Y, et al. Gene expression in chorionic villous samples at 11 weeks of gestation in women who develop preeclampsia later in pregnancy: implications for screening. Prenat Diagn 2009;29(11):1038e44. [165] Founds SA. Bridging global gene expression candidates in first trimester placentas with susceptibility loci from linkage studies of preeclampsia. J Perinat Med 2011;39(4):361e8. [166] Brodsky D, Christou H. Current concepts in intrauterine growth restriction. J Intensive Care Med 2004;19(6):307e19. [167] Banister CE, Koestler DC, Maccani MA, Padbury JF, Houseman EA, Marsit CJ. Infant growth restriction is associated with distinct patterns of DNA methylation in human placentas. Epigenetics Off J DNA Methylation Soc 2011; 6(7):920e7. [168] Koukoura O, Sifakis S, Zaravinos A, Apostolidou S, Jones A, Hajiioannou J, et al. Hypomethylation along with increased H19 expression in placentas from pregnancies complicated with fetal growth restriction. Placenta 2011; 32(1):51e7. [169] Koukoura O, Sifakis S, Soufla G, Zaravinos A, Apostolidou S, Jones A, et al. Loss of imprinting and aberrant methylation of IGF2 in placentas from pregnancies complicated with fetal growth restriction. Int J Mol Med 2011;28(4): 481e7. [170] Chelbi ST, Mondon F, Jammes H, Buffat C, Mignot TM, Tost J, et al. Expressional and epigenetic alterations of placental serine protease inhibitors: SERPINA3 is a potential marker of preeclampsia. Hypertension 2007;49(1): 76e83. [171] Chelbi ST, Wilson ML, Veillard AC, Ingles SA, Zhang J, Mondon F, et al. Genetic and epigenetic mechanisms collaborate to control SERPINA3 expression and its association with placental diseases. Hum Mol Genet 2012;21(9):1968e78. [172] Ferreira JC, Choufani S, Grafodatskaya D, Butcher DT, Zhao C, Chitayat D, et al. WNT2 promoter methylation in human placenta is associated with low birthweight percentile in the neonate. Epigenetics Off J DNA Methylation Soc 2011;6(4):440e9. [173] Marsit CJ, Maccani MA, Padbury JF, Lester BM. Placental 11-beta hydroxysteroid dehydrogenase methylation is associated with newborn growth and a measure of neurobehavioral outcome. PloS One 2012;7(3):e33794. [174] Filiberto AC, Maccani MA, Koestler D, Wilhelm-Benartzi C, AvissarWhiting M, Banister CE, et al. Birthweight is associated with DNA promoter methylation of the glucocorticoid receptor in human placenta. Epigenetics Off J DNA Methylation Soc 2011;6(5):566e72. [175] Turan N, Ghalwash MF, Katari S, Coutifaris C, Obradovic Z, Sapienza C. DNA methylation differences at growth related genes correlate with birth weight: a molecular signature linked to developmental origins of adult disease? BMC Med Genomics 2012;5(1):10. [176] Marsit CJ, Lambertini L, Maccani MA, Koestler DC, Houseman EA, Padbury JF, et al. Placenta-imprinted gene expression association of infant neurobehavior. J Pediatr 2012;160(5):854e60. e852. [177] Bromer C, Marsit CJ, Armstrong DA, Padbury JF, Lester B. Genetic and epigenetic variation of the glucocorticoid receptor (NR3C1) in placenta and infant neurobehavior. Dev Psychobiol 2012, http://dx.doi.org/10.1002/dev.21061. [178] Jensen Pena C, Monk C, Champagne FA. epigenetic effects of prenatal stress on 11beta-hydroxysteroid dehydrogenase-2 in the placenta and fetal brain. PloS One 2012;7(6):e39791. [179] Martino DJ, Prescott SL. Silent mysteries: epigenetic paradigms could hold the key to conquering the epidemic of allergy and immune disease. Allergy 2010;65(1):7e15.

970

B. Novakovic, R. Saffery / Placenta 33 (2012) 959e970

[180] Slaats GG, Reinius LE, Alm J, Kere J, Scheynius A, Joerink M. DNA methylation levels within the CD14 promoter region are lower in placentas of mothers living on a farm. Allergy 2012;67(7):895e903. [181] Martino DJ, Bosco A, McKenna KL, Hollams E, Mok D, Holt PG, et al. T-cell activation genes differentially expressed at birth in CD4þ T-cells from children who develop IgE food allergy. Allergy 2012;67(2):191e200. [182] Martino DJ, Tulic MK, Gordon L, Hodder M, Richman T, Metcalfe J, et al. Evidence for age-related and individual-specific changes in DNA methylation profile of mononuclear cells during early immune development in humans. Epigenetics Off J DNA Methylation Soc 2011;6(9). [183] von Mutius E, Vercelli D. Farm living: effects on childhood asthma and allergy. Nat Rev Immunol 2010;10(12):861e8. [184] Schaub B, Liu J, Hoppler S, Schleich I, Huehn J, Olek S, et al. Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells. J Allergy Clin Immunol 2009;123(4):774e82. e775. [185] Prescott SL, Tulic M, Kumah AO, Richman T, Crook M, Martino D, et al. Reduced placental FOXP3 associated with subsequent infant allergic disease. J Allergy Clin Immunol 2011;128(4):886e7. e885. [186] Avila L, Yuen RK, Diego-Alvarez D, Penaherrera MS, Jiang R, Robinson WP. Evaluating DNA methylation and gene expression variability in the human term placenta. Placenta 2010;31(12):1070e7. [187] Non AL, Binder AM, Barault L, Rancourt RC, Kubzansky LD, Michels KB. DNA methylation of stress-related genes and LINE-1 repetitive elements across the healthy human placenta. Placenta 2012;33(3):183e7. [188] Martin DI, Cropley JE, Suter CM. Epigenetics in disease: leader or follower? Epigenetics Off J DNA Methylation Soc 2011;6(7):843e8. [189] Hannan NJ, Paiva P, Dimitriadis E, Salamonsen LA. Models for study of human embryo implantation: choice of cell lines? Biol Reprod 2010;82(2):235e45.

[190] Novakovic B, Gordon L, Wong NC, Moffett A, Manuelpillai U, Craig JM, et al. Wide-ranging DNA methylation differences of primary trophoblast cell populations and derived cell lines: implications and opportunities for understanding trophoblast function. Mol Hum Reprod 2011;17(6):344e53. [191] Keniry A, Oxley D, Monnier P, Kyba M, Dandolo L, Smits G, et al. The H19 lincRNA is a developmental reservoir of miR-675 that suppresses growth and Igf1r. Nat Cell Biol 2012;14(7):659e65. [192] Kimura AP, Sizova D, Handwerger S, Cooke NE, Liebhaber SA. Epigenetic activation of the human growth hormone gene cluster during placental cytotrophoblast differentiation. Mol Cell Biol 2007;27(18):6555e68. [193] Rolfo A, Garcia J, Todros T, Post M, Caniggia I. The double life of MULE in preeclamptic and IUGR placentae. Cell Death Dis 2012;3:e305. [194] Bouchard L, Hivert MF, Guay SP, St-Pierre J, Perron P, Brisson D. Placental adiponectin gene DNA methylation levels are associated with mothers’ blood glucose concentration. Diabetes 2012;61(5):1272e80. [195] Zhao A, Cheng Y, Li X, Li Q, Wang L, Xu J, et al. Promoter hypomethylation of COMT in human placenta is not associated with the development of preeclampsia. Mol Hum Reprod 2011;17(3):199e206. [196] Uuskula L, Rull K, Nagirnaja L, Laan M. Methylation allelic polymorphism (MAP) in chorionic gonadotropin beta5 (CGB5) and its association with pregnancy success. J Clin Endocrinol Metab 2011;96(1):E199e207. [197] Michels KB, Harris HR, Barault L. Birthweight, maternal weight trajectories and global DNA methylation of LINE-1 repetitive elements. PloS One 2011; 6(9):e25254. [198] Guo L, Choufani S, Ferreira J, Smith A, Chitayat D, Shuman C, et al. Altered gene expression and methylation of the human chromosome 11 imprinted region in small for gestational age (SGA) placentae. Dev Biol 2008;320(1): 79e91.