DNA methylation profiles showed the ovarian cysts were closely grouped with normal ovarian but not endometrial tissues

Total of 12 samples from 4 patients, each with endometriosis, ovarian and endometrial tissues were profiled with illumina 850k methylation array. Principal component analysis clearly showed that endometriosis samples were tightly clustered with samples of ovarian tissue (Fig. 1). One of endometrial sample was located further away from other samples as an outlier, and this sample also showed a slightly different normalized beta distribution compared with other samples (Fig. 2). Then we plotted the heatmap of DNA methylation beta value matrix after normalization, and clustered the samples by hierarchical clustering. Again, 1B_E exhibited a hybrid DNA methylation profile.(Fig. 3) Therefore, we removed this sample for the downstream analyses. To confirm this result, we also performed PCA on our samples as well as publically available DNA methylation data on healthy endometrial biopsies [11], and as expected, healthy endometrial samples from public data were closer to our endometrial samples (Fig. 4).

Fig. 1

PCA plot of DNA methylation data from endometriosis, normal ovarian and endometrial tissues

Fig. 2

One of endometrial sample was located further away from other samples as an outlier, and this sample also showed a slightly different normalized beta distribution compared with other samples

Fig. 3

Hierarchical clustering analysis of 12 samples based on methylation levels. Top 1000 CpGs with the highest variance among 12 samples were included. Color mapping from blue to red indicates methylation level from low to high. Group C: ovarian endometrial cysts; N: normal_ovarian; E: eutopic endometriosis

Fig. 4

Endometriosis samples closely resembled normal ovarian tissues, but not endometrial tissues

As for DMRs analysis, we used the software ChAMP to explore the DMRs and the methylation beta value of each DMR was calculated using the mean value of all the probes that included in each DMR. The heatmap was generated using the beta value of DMRs. And from the heatmap we can see the ovarian endometrial cysts (group C) and normal_ovarian (group N) are clusted together, suggesting that the methylation profile of the cyst is more similar to ovarian tissues compared with endometrial tissue (Fig. 5 ).

Fig. 5

Hierarchical clustering analysis of the three groups based on DMRs. Color mapping from blue to red indicates methylation level from low to high. Group C: ovarian endometrial cysts; N: normal_ovarian; E: eutopic endometriosis

We finally got 431 DMRs (with p-value < 0.05) using the champ.DMR function. But the counts of DMRs with |log2(FoldChange)| > 0.5 and adjusted p-value < 0.05 was 55 (Fig. 6). Figure 7 shows the GO analysis of Genes with lower methylation levels in eutopic endometriosis (group E) compared with in ovarian endometrial cysts (group C). And Fig. 8 shows the GO analysis of Genes with higher methylation levels in eutopic endometriosis (group E) compared with ovarian endometrial cysts (group C).

Fig. 6

Volcano plot of log2(fold change) against ‑log10(p.adj ) of DMRs. The red blue spots stand for DMRs with adjusted p-value < 0.05 and log2FC > 0.5, and blue spots stand for DMRs with adjusted p-value < 0.05 and log2FC < -0.5, grey spots stand for non‑significant DMRs. The horizontal dash line denotes adjusted p-value < 0.05; the vertical dash lines denote |log2FC|>0.5. Group C: ovarian endometrial cysts; E: eutopic endometriosis

Fig. 7

Significantly enriched term by the genes with higher methylation levels in ovarian endometrial cysts (group C). Genes with higher methylated levels in eutopic endometriosis (group E) compared with ovarian endometrial cysts (group C) GO analysis

Fig. 8

Significantly enriched term by the genes with lower methylation levels in ovarian endometrial cysts (group C)

Abnormal epigenetic profiles of genes involving receptors, signaling pathways and immune responses

Most of previous studies compared the endometriosis with endometrial tissues; therefore we analyzed differential DNA methylation between endometriosis and endometrial tissues and identified the differentially methylated regions (Table S1). In comparison with eutopic endometriosis, many genes in ovarian endometrial cysts (choESC) had different degrees of methylation, high and low (Tables 1 and 2),With acquired methylation profiles data, we identified the detailed features of aberrantly methylated genes in choESC using gene cards (the humor gene database) analyses. Depending on pathway analysis of screened genes, there are abnormalities of relevant signal transduction pathways involving endometriosis onset and progression, developmental processes, human early embryonic development, regulation of caspase, tyrosine, and mRNA metabolic processes et al. It indicated an abnormal expression pattern of choESC in peritoneal environment.

Table 1 Hypomethylation in endometrial tissues compared the endometriosisTable 2 Hypermethylation in endometrial tissues compared the endometriosis

GO term enrichment analysis showed that the hypomethylation genes in ovarian endometrial cysts were primarily engaged in embryonic organ development and embryonic organ development, stem cell population maintenance, et al. And the hypermethylation genes in ovarian endometrial cysts were primarily engaged in mRNA destabilization, female uterus and genitalia development, et al. The function of genes related to steroid binding, transcription factor activity and receptor activity.(Figures 7 and 8 ).

We compared our differentially methylated sites and only found consistent results on hyper-methylation on ESR1 [1, 12].Consistently, downregulation of ESR1 mRNA level in endometriosis was also reported in few studies [12, 13]. Then we searched for the literature for the differentially methylated genes we identified and examined whether the associated expression patterns were observed. Indeed, we observed consistent results on 5 genes: TNFSF13B, FOXP1, TCF21, BST2 and STRA6 [14,15,16,17,18,19,20].Taken together, despite little overlap with previously characterized genes, the characterized methylated genes in endometriosis were consistent with the reported expression changes in endometriosis(Table 3).RT-PCR results showed that ER1,STAR6 and PEMT were significantly downregulated in ectopic tissues, and BST2,TCF21 and FOXP1 were significantly upregulated in ectopic tissues (Fig. 9 ). In summary, we validated the top candidate genes in endometriosis which might be regulated by DNA methylation.

Table 3 The differentially methylated genes were consistent with the reported expression changes in endometriosisFig. 9

qRT-PCR validation of the gene expression of the candidate genes. ER1,STAR6 and PEMTwere significantly downregulated in ectopic tissues, and BST2,TCF21 and FOXP1 were significantly upregulated in ectopic tissues. ***P < 0.001, **P < 0.01, *P < 0.05, ns, not significant