We examined PTB-related DNA methylation changes using genome-wide methylation analysis in maternal blood. Although we found no differential methylation of these CpGs after correcting for multiple tests, seven VTRNA2-1 CpGs in the promoters were differently methylated in blood samples of term compared with preterm delivered women. In addition, VTRNA2-1 hypermethylation was associated with increased risk of PTB (through pyrosequencing), and negatively correlated with gene expression. This result may help elucidate the underlying mechanism of PTB and affect future efforts by systems biologists to predict PTB.
PTB remains the leading cause of childhood morbidity and death. Its aetiology remains unclear; however, significant advancements have been made in the identification of biomarkers to predict high-risk pregnancies resulting in PTB. Indeed, in the last decade, DNA methylation has emerged as an important player of both physiological and pathophysiological responses. Studies have suggested that maternal factors, including pre-pregnancy BMI [21], nutrition [22], smoking during pregnancy [27], and socioeconomic status [11] increase the risk of PTB, and DNA methylation has partially explained many of these factors [17–23]. In this study, we found an association among maternal BMI, delivery season, and WBC count with PTB; however, these factors did not influence the DNA methylation levels in maternal blood.
VTRNA2-1 was determined to have significant DM sites in various regions and is associated with several diseases [28–30]. VTRNA2-1 is a putative tumour suppressor and modulator of innate immunity [28]. Although few studies have studied the relationship between VTRNA2-1 and PTB, its hypermethylation is associated with poor outcomes in patients with human small cell lung cancer and acute myeloid leukaemia [29, 30]. The expression of VTRNA2-1 negatively correlated with methylation levels in this study. A recent study showed that the inhibition of VTRNA2-1 led to the activation of the cellular antiviral response pathway involving protein kinase-R (PKR) [31, 32]. PKR activation can be induced by various stressful stimuli, such as cytotoxic cytokines, growth factor deprivation, and DNA damage [33]. Therefore, we hypothesise that the association between VTRNA2-1 methylation and PTB will result in PKR activation due to stressful stimuli.
One study reported that the methylation level of VTRNA2-1 was associated with the season of conception and maternal nutrition in rural Gambia [28]. Specifically, the authors reported that women had differing nutrition in the dry and rainy seasons during pre-conception. However, although our VTRNA2-1 methylation data were associated with delivery season, it is difficult to explain the relationship between PTB and methylation changes by season. In our study, the methylation of VTRNA2-1 was divided into two levels: low (hypomethylation, 0–12.5%) and high (hypermethylation, 31.7–54.2%). Younger maternal age was associated with PTB in the VTRNA2-1 hypomethylation group. Moreover, in the hypermethylation group, low BMI, higher WBC counts, and delivery season were associated with PTB.
We acknowledge several limitations of our study, including the paucity of the clinical and demographic data of our samples, such as smoking, alcohol consumption, and the use of other drugs or medications. We were only able to demonstrate a correlation between methylation and expression at a small subset of CpG sites and in a small sample size due to the limited availability of RNA from maternal blood samples. Finally, when we analysed the VTRNA2-1 methylation level using a simple linear regression model that included the cell composition percentages as covariates, the VTRNA2-1 methylation level was not affected by cell composition (P > 0.05). However, we did not consider DNA methylation status according to cellular heterogeneity because we did not sort blood cell type at the time of blood collection.