Epigenetic landscape of 5-hydroxymethylcytosine and associations with gene expression in placenta

5-hydroxymethylcystosine (5hmC), is an intermediate product in the DNA demethylation pathway, but may act as a functional epigenetic modification. We have conducted the largest study of site-specific 5hmC in placenta to date using parallel bisulphite and oxidative bisulphite modification with array-based assessment. Incorporating parallel RNA-sequencing data allowed us to assess associations between 5hmC and gene expression, using expression quantitative trait hydroxymethylation (eQTHM) analysis. We identified ~ 47,000 loci with consistently elevated (systematic) 5hmC proportions. Systematic 5hmC was significantly depleted (<i>p</i> &lt; 0.0001) at CpG islands (CGI), and enriched (<i>p</i> &lt; 0.0001) in ‘open sea’ regions (CpG &gt;4 kb from CGI). 5hmC was most and least abundant at CpGs in enhancers and active transcription start sites (TSS), respectively (<i>p</i> &lt; 0.05). We identified 499 significant (empirical-p &lt;0.05) eQTHMs within 1 MB of the assayed gene. At most (75.4%) eQTHMs, the proportion of 5hmC was positively correlated with transcript abundance. eQTHMs were significantly enriched among enhancer CpGs and depleted among CpGs in active TSS (<i>p</i> &lt; 0.05 for both). Finally, we identified 107 differentially hydroxymethylated regions (DHMRs, <i>p</i> &lt; 0.05) across 100 genes. Our study provides insight into placental distribution of 5hmC, and sheds light on the functional capacity of this epigenetic modification in placenta.