Interrogating Altered DNA Methylation in Human Endometriotic Stromal Cells.

Abstract

Endometriosis is a benign gynecological disease causing infertility, adhesions and chronic pain in approximately 10% of reproductively-aged women due to the extrauterine growth of endometrial stromal and glandular cells. Endometriotic lesions resemble the eutopic endometrium both morphologically and in their response to estrogen, which potently stimulates stromal cell proliferation and gene expression. However, diseased cells are characterized by a number of cellular and molecular defects with unexplained etiology. Growing evidence suggests that abnormal DNA methylation is an underlying problem in the disease, since such epigenetic changes can be both mutable and heritable. Aberrant DNA methylation within the promoters of estrogen receptor beta, steroidogenic factor 1, aromatase, and HOXA10—all genes tied directly to the pathology of endometriosis—is now known to alter their expression in endometriotic cells. We hypothesize that deviations in DNA methylation may accrue in the stromal cells of endometriotic lesions, fundamentally altering patterns of gene expression and changing the response of these cells to steroid hormone signaling during the menstrual cycle. We used normal and endometriotic stromal cells to identify differences in genome-wide methylation and gene expression using Illumina's Infinium HumanMethylation450 and HumanHT-12v4 Expression BeadChips. Normal endometrial stromal cells (HESC) were obtained from six patients without endometriosis undergoing hysterectomy for benign complications. Endometriotic stromal cells (OSIS) were obtained from six patients having surgical removal of ovarian endometriotic cysts. All patients were premenopausal, and underwent surgery during the proliferative phase of their menstrual cycle having received no preoperative hormonal therapy. The gene expression results identified 982 differentially regulated genes between the two groups (515 upregulated genes, and 467 downregulated). The methylation array revealed 53,977 cytosine-guanine sites (CpGs) that were differentially methylated between HESC and OSIS (beta>0.1; fold change>1.5; p<0.05). Only 11% of these differences were observed in canonical CpG islands, whereas more than 55% were located in regions unassociated with any CpG island. Notably, 3817 of the differentially methylated CpGs were mapped to genes also showing altered expression between the two groups. Within this group, 2502 CpGs were hypermethylated in OSIS, and methylation was inversely correlated with gene expression. Furthermore, gene ontology analysis of the genes that were hypermethylated in OSIS identified an enrichment of biological process involved in urogenital and female sexual development, whereas hypomethylated genes were enriched in biological processes regulating cell migration and locomotion. We have uncovered global differences in methylation between normal and diseased cells that correlate with extensive changes in gene expression, and we predict that these epigenetic changes are involved in the establishment and progression of endometriosis.