A number of studies have begun to examine the association between growth restriction and epigenetic regulation in fetal tissues including placenta.16–18,21–23
Much of the recent research on placental epigenetics has focused on the role of genomic imprinting and new technologies have been developed to more efficiently identify imprinted genes.24
From this we have learned much about the timing of epigenetic remodeling25
and the role of specific genes, including imprinted tumor-suppressor genes in the human placenta.26
Specific genes, such as H19/IGF2
, have been studied in the context of growth restriction, demonstrating differential methylation of the imprinting control regions in placentas from growth restricted infants.17,18
A number of other imprinted loci, such as PHLDA2, ILK2, NNAT, CCDC86, PEG10, PLAGL1, DHCR24, ZNF331
have been shown to demonstrate differential expression between growth restricted and non-restricted infant placentas.10
Animal models have described the functional significance of alterations in imprinting; for example, loss of the maternal allele of Grb10
limits placental size and efficiency.27
Our goal in this study was to build on this knowledge and to expand the examination of epigenetic alterations to encompass a larger genome-wide profile of genes, beyond those genes subjected to regulation by genomic imprinting, and thus to identify if the intrauterine environment can be represented by gross alteration to the epigenetic landscape of the placenta.
We focused our investigation on methylation patterning due to the highly stable nature of DNA methylation marks. Previous studies have shown that labor induces altered expression of genes in the human placenta28
and, while the methylation status is stable, the corresponding gene expression may not be.29
Therefore, we believe that examination of methylation may reflect changes occurring over the course of in utero development and not only at the final moments of pregnancy and delivery. We used a relatively unbiased, genome-wide method to identify loci whose methylation status in the term placenta was most associated with infant birth weight as a marker of growth. The genome-wide analysis was driven by our hypothesis that the complex and multifactorial outcome of infant growth in the relatively non-pathologic context from which our population is drawn, results from the interplay of various genes, and thus, profiling their regulation by DNA methylation can provide insight into the global regulation of the genome. In order to avoid the confounding effects of premature birth, we only included placentas from patients with gestational age >36 weeks. Our analysis, using a novel validated statistical strategy aimed not at identifying a single locus whose methylation is associated with growth, but more importantly, a pattern of alterations, identified that the pattern of methylation of 22 critical loci is highly predictive of SGA or IUGR diagnosis. The identified pattern is potentially indicative of altered cellular processes leading to targeted DNA methylation alterations that are linked to infant growth restriction. The GSEA examining transcription factor binding sites is aimed at better characterizing genomic similarities amongst loci whose methylation was associated with growth status. The CCAAT/enhancer binding protein β (C/EBPβ) is a downstream effector of estrogen-mediated implantation and decidualization,30
and controls target genes such as PLAC1
, which are involved in the maternal-placental interface.31
Thus, DNA methylation of CEBPβ target genes may affect the maternal-fetal interface, resulting in growth restriction. The GSEA also identified over-representation of FOXO4 TFBS as targets of methylation alteration. FOXO4, a homeobox transcription factor, has been localized to differentiated syncytiotrophoblasts32
and has been shown to be involved in cellular stress responses.33
It may likewise play a role in integrating environmental signals, resulting in altered placenta function and infant growth.
The GSEA of KEGG pathways revealed two neurological disease pathways: Huntington disease and Alzheimer disease. While it is unlikely that the methylation patterns we identified in the placenta are directly linked to the development of these diseases later in life, it is known that the placenta plays a critical role in neuropeptide homeostasis for the developing fetus.34–36
The placenta has been postulated to represent the “third brain” that links the developed (maternal) and developing (fetal) brains,35
playing a critical role in the pathophysiology of intrauterine insults on the developing nervous system.37
Placental production of the corticotropin-releasing hormone (CRH) and thyrotropin-releasing hormone (TRH) subserves intrauterine hypothalamic control of fetal pituitary development throughout most of gestation.38,39
Thus, our findings may represent alterations to such pathways related to growth restriction and potentially linking growth restriction with neurodevelopmental and mental health outcomes later in life. Additional studies are warranted to expand on these examinations and these later life endpoints to clarify the biological mechanisms at play.
It is important to note that we cannot definitively determine if these altered profiles of DNA methylation are a response of the placenta to the intrauterine environment and/or growth restriction, or are extant, such that they have led to the growth phenotypes observed. Nonetheless, by oversampling for infants who were small for gestational age, we generated a robust interrogation of the effects of intrauterine growth on placental DNA methylation and identified strong, significant and independent associations between these specific profiles of DNA methylation and infant growth. An additional limitation of this study lies in the overrepresentation of CpG island-associated loci found on the Illumina Infinium Human Methylation27 BeadArray. Ongoing research is now revealing that gene regulatory methylation events may occur in regions outside of CpG islands, such as on CpG shores found up to 2 kb upstream of the regulated gene.40
More comprehensive approaches including more inclusive arrays and genome-wide sequencing will be needed to fully identify all regions contributing to the regulation of infant growth.
The biological basis and implications of these altered profiles remains unclear. One possibility is that these different profiles represent changes in the population of cells present in the placenta. It is clear that individual tissues and cells demonstrate unique patterns of DNA methylation41
and that altered DNA methylation can identify specific sub-populations of cells in a highly sensitive manner.42
Thus, within our placenta tissues we may be detecting, changes in the distribution of mature trophoblasts, immune cells and stromal cells, or even changes to sub-populations of these cells within these placenta tissue samples. Alternatively, these profiles may reflect phenotypic differences in the maturity or differentiation of cells in the placenta, as it is clear that epigenetic mechanisms play critical roles in cellular differentiation.43
The difficulty in obtaining placental tissue from uncomplicated pregnancies at various time points throughout pregnancy limits available data that could be used to examine this hypothesis more definitively.
In summary, we have demonstrated the methylation profile of 22 genes from human term placentas yielded five different classes, and that these classes differed significantly by SGA or IUGR diagnosis. This work serves as a proof of principle that variation in the DNA methylation profile of human term placenta can serve as a marker of growth. Further analysis is warranted to elucidate additional covariates, including environmental factors and exposures that may be affecting the methylation profiles that distinguish these classes. As prospective associations have already been demonstrated for peripheral blood-based methylation profiles and diseases such as acute myeloid leukemia, longitudinal follow-up on these subjects may demonstrate the prospective utility of these placenta-specific methylation profiles.44
This would strengthen our hypothesis that epigenetic alterations in the placenta are acting functionally to program the health of an individual far beyond the intrauterine environment.