Genome-wide analysis of DNA methylation in human amnion

Abstract

The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

Figure 1

Principal component analysis (PCA) plot of DNA methylation profiles in term (non-labored and labored) and preterm amnion. Each colored dot represents a pooled DNA sample from term no labor (TNL), term labor (TL), or preterm labor (PTL) group. Note that the TNL sample is placed distantly from the TL or PTL samples, indicating that the TNL group displays distinctly different methylation patterns compared to the other two groups.

Figure 2

Schematic representation of CpG island regions of UCN (a) and OXTR (b) analyzed by bisulfite sequencing (BS). Black horizontal arrows denote BS PCR primer binding sites. Solid box: coding region; open box: untranslated region. The expected PCR product sizes and positions of the primer binding sites (chromosome and base count, NCBI Build GRCh37/hg19) are indicated. Further details on the PCR-amplified regions are provided in Section 2.

Figure 3

UCN and OXTR mRNA expression levels in term (non-labored and labored) and preterm amnion. Expression levels were normalized to that of beta-actin (ACTB). Experiments were performed in triplicate. Data presented are mean ± standard error of the mean (SEM). Asterisks represent statistically significant differences (P < 0.05, K-W one-way ANOVA by ranks followed by Dunn's post hoc test) between specified groups.

Figure 4

Methylation-specific PCR (MSP) analysis of SLC30A3. (a) Schematic representation of MSP primer binding sites. Black horizontal arrows: methylated-specific primer (MSPM) binding sites; gray horizontal arrows: unmethylated-specific primer (MSPU) binding sites. The expected PCR product sizes and positions of the primer annealing sites (chromosome and base count, NCBI Build GRCh37/hg19) are indicated. Solid box: coding region; open box: untranslated region. (b) Agarose gel electrophoresis of MSP products. M: product amplified with MSPM; U: product amplified with MSPU. PC: positive control, human methylated DNA standard; NC: negative control, human unmethylated DNA standard; W: water control (details can be found in Section 2). Note that the TL samples show only unmethylated PCR products, while many of the PTL samples show both methylated and unmethylated PCR products, indicative of partial methylation.