Background
Preterm birth (PTB) refers to all births occurring before 37 weeks of gestation, as defined by the World Health Organization [
1]. PTB occurs in approximately 11% of live births worldwide, although there is substantial variability in rates on a per-country basis [
2]. Children who are born preterm are at a higher risk for life-long health complications, including chronic lung disease, cerebral palsy, mental retardation, attention-deficit hyperactivity disorder, and learning disabilities [
3,
4]. Clinically, PTB can be categorized into spontaneous PTB (sPTB) or medically-indicated PTB (iatrogenic) [
1]. sPTB, making up the majority of PTBs, typically results from a dysregulation of inflammatory signalling pathways [
5]. This often presents as acute chorioamnionitis (aCA), which is characterized by an infiltration of maternal neutrophils into the chorioamniotic membranes, typically in response to an ascending microbial infection from the genital tract. This acute placental inflammation can also be triggered by non-microbial “danger signals” including cellular stress and/or cell death [
6,
7].
Genetic susceptibility for aCA can be hypothesized based on: i) high heritability estimates of PTB (15–30%) [
8,
9], ii) evidence supporting familial segregation of PTB [
10,
11], (iii) association of placental histopathological inflammatory lesions with recurrent PTB [
12,
13], and (iv) ethnic disparities in PTB [
14‐
16] and chorioamnionitis rates [
17]. Inherited differences in immune system genes also influence susceptibility to microbial infection [
18‐
21], which is a well-known cause of aCA. In addition, a strong genetic predisposition underlies many infectious and inflammatory diseases, particularly in early childhood [
22‐
24]; this may also hold true for in utero susceptibility for aCA.
Studies investigating candidate genes have reported that maternal and fetal genetic variation in Toll-like receptors (
TLRs) is associated with sPTB [
25‐
27]. Allelic variation in
TLR genes has been shown to modulate immune responses during parturition, and thus confer an altered risk of preterm delivery [
28]. Genetic variants in cytokine genes such as Interleukin-6 (
IL6) have also been associated with intrauterine infection and/or inflammation in sPTB [
29‐
31]. Furthermore, elevated concentration of
IL6 in maternal serum, cervical secretions and amniotic fluid are associated with sPTB [
32‐
35]. Recently, a genome-wide association study investigating > 40,000 women of European ancestry identified several genetic variants associated with sPTB [
36]. Although variants in
EBF1, EEFSEC, and
AGTR2 genes were replicated in an independent cohort of > 8000 women, none of the identified genes had been previously identified in sPTB or known to have a direct role in inflammatory mechanisms [
36]. While these studies have provided some insight on genetic variation linked to sPTB, rarely are the same loci reported with sPTB risk. sPTB is heterogeneous in etiology [
37], thus inconsistent phenotyping of sPTB cases and differences in population structure may explain the discrepancies across these studies.
Genetic variants within coding regions may directly affect protein function, while those in regulatory regions may affect molecular processes such as DNA methylation [
38‐
40] that are involved in regulating gene expression [
41,
42]. Alternatively, genetic variants can alter the binding site of transcription factors and affect gene expression, which then influences DNA methylation levels, suggesting DNA methylation as a consequence of gene regulation [
43]. Irrespective of the underlying mechanism, these effects can in turn affect susceptibility to inflammatory diseases. For example in rheumatoid arthritis, DNA methylation at an
IL6-related CpG was altered in affected patients, and a negative relationship between DNA methylation and
IL6 mRNA levels was observed, suggesting a DNA methylation-dependent regulation of
IL6 transcription [
44]. While increased serum levels of
IL6 have been previously reported in aCA [
45‐
47], these studies did not take into account the genotype at the
IL6 locus and/or the DNA methylation status of the
IL6-related CpGs. Furthermore, the maternal genotype is often investigated although the placental genotype may be more relevant in terms of mediating pregnancy-related inflammation. Elucidating these complex relationships between genotype, DNA methylation and gene expression is important to improve our understanding of the genetic regulation of placental inflammation.
In this study, we investigated the association between 16 candidate SNPs in 12 innate immune system genes and the presence of aCA. These SNPs were chosen based on published reports of an association with chorioamnionitis [
48‐
50], placental inflammation [
51,
52] or neonatal sepsis/infection [
53‐
55]. We validated these associations in a population of 269 placentas, of which 72 were affected with aCA and 197 were unaffected (non-aCA). Further, we investigated whether aCA-associated SNPs showed also a correlation with altered DNA methylation of the associated gene, and determined whether DNA methylation levels correlated with gene expression.
Discussion
In this study, we sought to validate associations of SNPs in innate immune system genes with aCA status in the placenta. As the placenta and fetus are genetically identical, risk-conferring genetic variants in innate immune system genes may impact inflammation-response pathways in the placenta and fetus similarly. Additionally, the placenta employs a number of mechanisms to protect the developing fetus from inflammation and/or infection [
79,
80]. Few studies have reported an association between genetic variants in inflammatory genes, alterations in immune function and risk for aCA. The majority of these studies suggest a genetic predisposition of the mother to aCA [
48,
49], but the contribution of fetal (placental) genetic variants are significantly understudied.
In our study sample, we were able to confirm that the C allele in the IL6 SNP rs1800796 was associated with aCA status (p = 0.04). This allele was linked to increased DNA methylation, at both an upstream regulatory region and within the gene body, and associated with decreased expression of IL6.
Interleukin-6 is a pleiotropic cytokine with a wide range of biological functions [
81]. Primarily, IL6 facilitates neutrophil recruitment and their subsequent clearance from the sites of inflammation [
82]. In addition to eliciting innate immune responses, IL6 also regulates adaptive immunity by influencing proliferation and maturation of T cells and B cells [
81]. As such, increased IL6 has been shown to be protective against bacterial infection [
83]. Therefore, decreased expression observed in the placenta in association with rs1800796 may lead to increased risk for inflammation and aCA.
The
IL6 SNPs rs1800796 and rs1800795 were previously associated with increased incidence of aCA and development of sepsis in children in a study undertaken in Finland [
30]. In the cases of rs1800796, the heterozygous “CG” genotype was found to be associated with sepsis, and in fact the “CC” genotype was absent from their study population. These same genetic variants have also been investigated in association with other inflammatory disorders including chronic periodontitis, systemic onset juvenile chronic arthritis, and distal interphalangeal osteoarthritis [
84‐
87]; however, results from these studies are conflicting. Inconsistencies across these studies may be explained by ancestry differences in the study populations as the CC genotype at rs1800796 is common in East Asians and rare in individuals of European ancestry [
85,
88,
89]. Though a small sample, we also found no CC genotypes in the placentas of individuals of European ancestry and the C allele was more common in individuals of East Asian ancestry (Fig.
2). Further, we found that the allele frequencies of rs1800796 were associated with aCA status only in individuals of East Asian ancestry, however there was poor power to detect an effect in Europeans as the CC genotype was lacking and heterozygotes are expected to have less of an effect. Similarly, variants at the
PGR locus, specifically the
PGR SNP rs11224580, that significantly modulates PGR expression in the ovary has been shown to be common in East Asians compared to individuals of European and African ancestry [
90], but in this case the Asian-specific variant is linked with decreased incidence of early sPTBs [
90]. Because polymorphisms such as rs1800796 (
IL6) and rs11224580 (
PGR) exhibit extreme population-specific allelic variation, these genomic loci are likely to have undergone positive selection that is specific to Asian populations [
90,
91].
In addition to genetic variation, circulating levels of IL6 in the serum and amniotic fluid may influence the risk for aCA. Although placental trophoblast cells have the capacity to synthesize IL6 [
92,
93], the source of elevated IL6 plasma levels observed in pregnancy complications such as PE is primarily attributed to maternal leukocytes and/or endothelial cells [
94]. Elevated IL6 levels in the mother may occur as part of a pro-inflammatory response to infection. In contrast, we found that the placental genotype (rs1800796) associated with aCA is linked with decreased
IL6 expression in the placenta
. As
IL6 mediates a protective immune response against microbial infections [
83,
95‐
98], reduced
IL6 expression observed in the placenta may predispose an individual to infection, by preventing an appropriate innate immune response to microbes.
Although the role of
IL6 in modulating innate immune response has been well-elucidated, molecular mechanisms such as DNA methylation underlying the genetic regulation of
IL6 transcription have rarely been investigated [
99]. To our knowledge, this is the first study to show the role of
IL6 genetic variants in modulating DNA methylation patterns in aCA-affected placentas. We identified that carriers of the rs1800796 C allele had increased placental DNA methylation levels at multiple IL6
-related CpGs, most significantly at cg01770232, which is located at an upstream enhancer, compared to individuals with
IL6 G allele. Although this CpG has been described as linked to an mQTL in blood, we showed that this relationship also exists in the placenta. Further, we observed that aCA cases were more methylated than the non-aCA cases at cg01770232, cg07998387, and cg02335517 (Additional file
1: Figure S4).
The DNA methylation patterns in the placenta may imply a primary phenomenon where increased DNA methylation at cg01770232 is associated with an increased risk of developing aCA in individuals carrying the
IL6 C allele. Alternatively, some of these subtle DNA methylation changes could be secondary to the disease itself. Although we were not able to measure circulating levels of IL6, using publicly available matched placental DNA methylation and gene expression data, we observed that DNA methylation levels at cg01770232 negatively correlated with
IL6 gene expression. Dandrea et al. (2009) [
99] demonstrated that
IL6 repression in pancreatic adenocarcinoma cell lines is facilitated by binding of methyl-CpG-binding protein (MeCP2) and H3meK9 to the methylated CpGs spanning from positions − 666 to − 426 relative from the transcription start site of
IL6. Interestingly, rs1800796 and cg01770232 is located at position 572 and 662 respectively, and it is therefore possible that rs1800796 alters the binding of MeCP2 and H3meK9 to cg01770232, thereby affecting IL6 expression and DNA methylation, though this has not been tested in placental tissue. Further, the
IL6 upstream region contains several (A/T)
> 4 motifs adjacent to the methylated CpGs including cg01770232, shown to mediate high-affinity MeCP2 binding [
100]. Understanding these mechanisms will provide insights into how genetic variation in
IL6 may contribute towards disease pathogenesis in aCA.
Overall, the present study has limitations. Our sample size was relatively small, especially among the genetically homogenous subpopulations; therefore the findings of this study should be evaluated in larger subpopulations of different ancestries. In particular, the lack of an association between the other genetic variants we investigated and aCA might be a result of our small sample size. Although we utilized publicly available datasets to investigate functional consequences of the
IL6 rs1800796 polymorphism, and confirmed that DNA methylation changes correlated with changes in
IL6 gene expression, we could not examine whether IL6 protein levels in maternal blood would reflect placental DNA methylation and expression. Further, potential confounding factors for our study, such as socio-economic status, maternal smoking status, maternal alcohol use, and PPROM status, were not documented for all the cases in our study cohort and thus not accounted for in statistical analyses. Finally, our results only highlight the biological significance of placental (fetal) genetic variants in aCA, but this does not exclude the role of maternal genetic factors in altering disease risk to aCA, given that maternal genetic effects have been shown as important contributors to PTB [
10].