Endometriotic Stromal Cells Research Articles

Lipopolysaccharide promoted proliferation and invasion of endometriotic stromal cells via induction of cyclooxygenase-2

OBJECTIVE: An important recent general concept is that of endometriosis as a local pelvic inflammatory process with altered function of immune-related cells and of endometriotic cells in the peritoneal environment. A number of studies suggest that the peritoneal fluid (PF) of women with endometriosis contains increased levels of a variety of local products, such as growth factors, cytokines and prostaglandins. We conducted this study to evaluate the effect of lipopolysaccharide (LPS) on the expression of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in endometriotic stromal cells (ESC) and on their proliferation and invasion.

Involvement of nuclear factor-κB in macrophage migration inhibitory factor gene transcription up-regulation induced by interleukin-1β in ectopic endometrial cells

To investigate the involvement of the nuclear factor (NF)-kappaB in the interleukin (IL)-1 beta-mediated macrophage migration inhibitory factor (MIF) gene activation. Prospective study. Human reproduction research laboratory. Nine women with endometriotic lesions. Endometriotic lesions were obtained during laparoscopic surgery. The MIF protein secretion was analyzed by ELISA, MIF mRNA expression by quantitative real-time polymerase chain reaction (PCR), NF-kappaB translocation into the nucleus by electrophoresis mobility shift assay, I kappaB phosphorylation and degradation by Western blot, and human MIF promoter activity by transient cell transfection. This study showed a significant dose-dependent increase of MIF protein secretion and mRNA expression, the NF-kappaB translocation into the nucleus, I kappaB phosphorylation, I kappaB degradation, and human MIF promoter activity in endometriotic stromal cells in response to IL-1 beta. Curcumin (NF-kappaB inhibitor) significantly inhibited all these IL-1 beta-mediated effects. Analysis of the activity of deletion constructs of the human MIF promoter and a computer search localized two putative regulatory elements corresponding to NF-kappaB binding sites at positions -2538/-2528 bp and -1389/-1380 bp. This study suggests the involvement of the nuclear transcription factor NF-kappaB in MIF gene activation in ectopic endometrial cells in response to IL-1 beta and identifies a possible pathway of endometriosis-associated inflammation and ectopic cell growth.

Interleukin-10 attenuates TNF-α–induced interleukin-6 production in endometriotic stromal cells

To determine whether high levels of interleukin (IL)-10 can attenuate the production of tumor necrosis factor (TNF)-alpha-induced proinflammatory cytokines in endometriotic stromal cells. Prospective study. Department of Ob/Gyn, Tottori University, Japan. Thirty-five patients with ovarian endometrioma and ten patients with uterine myoma. Endometriotic stromal cells were obtained from chocolate cyst linings of ovaries. Endometrial stromal cells obtained from patient with uterine myoma. Expression of IL-10 gene in endometriotic or endometrial stromal cells was determined by real-time reverse-transcriptase polymerase chain reaction (RT-PCR). We performed immunohistochemical staining to find the presence of IL-10 and IL-10 receptors 1 and 2. We examined the effects of TNF-alpha and IL-10 on the expression of IL-6 or IL-8 by real-time RT-PCR and ELISA. We examined the activation of intracellular signal transduction molecules in endometriotic stromal cells by Western blotting. Addition of IL-10 suppressed the expressions of IL-6 induced by TNF-alpha and IL-10 induced the phosphorylation of STAT3 in endometriotic stromal cells. TNF-alpha induced the expression of phosphorylated ERK1/2, JNK1/2, and I kappaB. Adding IL-10 suppressed the phosphorylation of these signal molecules. Interleukin-10 attenuates TNF-alpha-induced IL-6 synthesis via NF-kappaB and MAPK pathways in endometriotic cells.. Interleukin-10 may play a significant role in the pathogenesis of endometriosis.

Interleukin-4 Stimulates Proliferation of Endometriotic Stromal Cells

Several lines of evidence indicate that the Th2 immune response is associated with endometriosis. Although an increased concentration of interleukin (IL)-4, a typical Th2 cytokine, has been reported in endometriotic tissues, the implication of this for endometriosis has not been determined. To investigate a possible role of IL-4 in the development of endometriosis, we examined the presence of IL-4-producing cells in endometriotic tissues and the effect of IL-4 on proliferation of endometriotic stromal cells. Endometriotic stromal cells were isolated from endometriotic tissues obtained from women undergoing surgery for endometrioma. Immunohistochemistry of endometriotic tissues revealed that IL-4-positive cells were abundant in the stroma. The effect of IL-4 on proliferation of endometriotic stromal cells was studied using cell counting and BrdU incorporation assays. IL-4 (0.1 to 10 ng/ml) significantly increased cell number and BrdU incorporation in a dose-dependent manner, and the proliferative effect of IL-4 was inhibited by anti-IL-4 receptor antibody. IL-4-induced activation of mitogen-activated protein kinases in endometriotic stromal cells was examined by Western blotting. IL-4 induced phosphorylation of p38 mitogen-activated protein kinase, stress-activated protein kinase/c-Jun kinase, and p42/44 mitogen-activated protein kinase and inhibitors of these kinases suppressed IL-4-induced proliferation of endometriotic stromal cells. These findings suggest that proliferation of endometriotic stromal cells induced by locally produced IL-4 is involved in the development of endometriosis.

Cyclic Adenosine 3′,5′-Monophosphate Response Element-Binding Protein and CCAAT/Enhancer-Binding Protein Mediate Prostaglandin E 2-Induced Steroidogenic Acute Regulatory Protein Expression in Endometriotic Stromal Cells

Aberrant expression of the steroidogenic acute regulatory (StAR) protein in human endometriotic stromal cells plays an important role in the development of endometriosis. Prostaglandin E(2) (PGE(2)) is a potent inducer of StAR expression in these cells; however, the mechanisms responsible for the transcriptional regulation of StAR remain to be elucidated. Herein we report that PGE(2)-induced StAR expression is independent of the transcriptional suppressor DAX-1 but is regulated by the transcriptional activator cyclic adenosine 3',5'-monophosphate (cAMP) response element-binding protein (CREB). A promoter activity assay revealed that the cis-element needed for the binding of the CCAAT/enhancer-binding protein (C/EBP) was critical for PGE(2)-induced StAR expression. Electrophoretic mobility shift assay demonstrated that this region of the StAR promoter was bound by C/EBPalpha, C/EBPbeta, and CREB. Forced expression of either C/EBPalpha or C/EBPbeta alone was sufficient to up-regulate StAR promoter activity whereas PGE(2) was needed to induce StAR promoter activity in CREB-overexpressed cells. Results from a chromatin immunoprecipitation assay demonstrated that the binding of C/EBPbeta to the StAR promoter was increased whereas CREB binding was unchanged after PGE(2) treatment. Taken together, PGE(2)-induced StAR promoter activity appears to be regulated by CREB and C/EBPbeta in a cooperative manner in ectopic human endometriotic stromal cells, providing a molecular framework for the etiology of endometriosis.

Serum interleukin-8 levels are elevated in patients with ovarian endometrioma

To determine whether serum interleukin (IL)-8 concentration can be measured in patients with ovarian endometrioma and whether this measurement is a useful tool in diagnosing this disease. A controlled clinical study and an in vitro study. Department of Obstetrics and Gynecology, Tottori University, Japan. Seventy patients with ovarian endometrioma and 21 patients with benign ovarian cyst. Laparoscopic surgery or laparotomy for ovarian endometriomas or benign ovarian cyst was performed. Preoperative blood samples were obtained. Endometriotic stromal cells obtained from nine patients with endometrioma were cultured. Interleukin-8 concentration in the serum or supernatant of the cell culture was measured with use of ELISA. The serum concentration of IL-8 in patients with endometrioma was significantly higher than in patients with benign ovarian cyst. The serum IL-8 threshold (25 pg/mL) had a higher sensitivity (71.4%) for diagnosing ovarian endometrioma than did serum CA-125 level. The increased rates of IL-8 concentration in the culture supernatants after adding tumor necrosis factor alpha were significantly higher in patients whose serum IL-8 levels were >or=25 pg/mL than in those with levels <25 pg/mL. Measuring of serum IL-8 concentration may be a valuable tool in diagnosing endometriosis.

Extracellularly Signal-Regulated Kinase Activity in the Human Endometrium: Possible Roles in the Pathogenesis of Endometriosis

Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial tissue outside of the uterine cavity, causing pelvic pain and infertility in 10% of reproductive-aged women. It is unclear why ectopic endometrium remains viable in only a subset of women. ERK1/2 plays key intracellular roles in activating cellular survival and differentiation processes. We sought to determine ERK1/2 activity in patients with endometriosis and its possible roles in regulating endometrial cell survival. ERK1/2 phosphorylation and expression throughout the menstrual cycle were evaluated in vivo in normal and endometriotic human endometrium, and in vitro techniques assessed the steroidal regulation of ERK1/2 and its effect on endometrial cell survival. Total ERK1/2 remained constant in normal and endometriotic endometrium throughout the menstrual cycle. Phospho-ERK1/2 was high in the late proliferative and secretory phases in normal endometrium (P < 0.05). In endometriotic glandular cells, there was no cyclical variation in phospho-ERK1/2. In endometriotic stromal cells, there was also a reduction in phospho-ERK1/2 variation, with higher levels in the early-mid secretory phase (P < 0.05). In cultured endometrial stromal cells (ESCs), estrogen plus progesterone increased ERK1/2 phosphorylation within 15 min (P < 0.05). Although estrogen alone did not induce ERK1/2 phosphorylation in normal ESCs, there was a significant response to estrogen in ESCs isolated from eutopic endometriotic endometrium (P < 0.05). ERK1/2 inhibition in ESCs reduced proliferation and increased apoptosis (P < 0.05). Abnormally high levels of ERK1/2 activity may be involved in endometriosis, possibly by stimulating endometrial cell survival.

Hypoxia potentiates the accessibility of cyclooxygenase‐2 gene promoter to transcription factors

Aberrant expression of cyclooxygenase‐2 (COX‐2) has been observed in many human diseases including cancer and endometriosis. Overproduction of COX‐2 derived eicosnoids, especially prostaglandin (PG) E2 not only enhances disease progression but also results in poor prognosis. However, mechanisms responsible for overexpression of COX‐2 in these tissues are largely unknown. Herein, we provide evidence to demonstrate that aberrant expression of COX‐2 in these tissues may be mediated by predisposition to hypoxia. Pre‐exposure of cells to hypoxic stress result in increased sensitivity of COX‐2 gene to IL‐1β stimulation even after 48 h of reoxygenation. Promoter activity assay revealed that induction of COX‐2 expression was mediated by upregulation of cox‐2 gene by hypoxia‐inducible factor‐1α (HIF‐1α). Similarly, treatment of cells with DFO, a chemical that will induce HIF‐1α accumulation, also increased promoter sensitivity of cox‐2 to IL‐1β. The increase in cox‐2 gene sensitivity by pre‐exposure to hypoxia is also evident when other cox‐2 stimulators such as EGF and PGE2 were used to treat the cells. Together, we have shown for the first time that overexpression of COX‐2 in endometriotic stromal cells is likely due to hypoxia‐mediated epigenomic modification of chromatin structure surrounding COX‐2 promoter.

Upstream Stimulatory Factor-2 Regulates Steroidogenic Factor-1 Expression in Endometriosis

Local estrogen biosynthesis is a major factor in the pathogenesis of endometriosis. Aberrant expression of steroidogenic acute regulatory protein (StAR) and aromatase in endometriotic tissue leads to an up-regulation of estrogen production. The transcription factor steroidogenic factor-1 (SF-1) activates the promoters of both StAR and aromatase in endometriotic tissue. We investigated differences in SF-1 expression in endometriotic tissue and normally located endometrium to elucidate the mechanism underlying increased StAR and aromatase activities in endometriosis. Serial deletion and site-directed mutants of the SF-1 promoter showed that an E-box sequence was critical for its activity in endometriotic stromal cells. EMSAs showed that the upstream stimulatory factor (USF) 1 and 2 in nuclear extracts from endometrial and endometriotic stromal cells bound to the E-box. Chromatin-immunoprecipitation-PCR assay, however, demonstrated in intact cells that binding activity of USF2 to the SF-1 promoter was strikingly higher than that of USF1 in endometriotic stromal cells and that USF1 or USF2 binding activity was hardly detectable in endometrial stromal cells. Moreover, knockdown of USF2 but not USF1 resulted in robust and consistent down-regulation of SF-1 and its target genes StAR and aromatase in endometriotic stromal cells. USF2 but not USF1 mRNA and protein levels were significantly higher in endometriotic vs. endometrial stromal cells. In vivo, USF2 mRNA and immunoreactive USF2 levels in endometriotic tissues were strikingly higher than those in endometrium. Taken together, the elevated levels of USF2 in endometriosis account for, in part, the aberrant expression of SF-1 and its target gene StAR and aromatase.

Interleukin (IL)-17A Stimulates IL-8 Secretion, Cyclooxygensase-2 Expression, and Cell Proliferation of Endometriotic Stromal Cells

IL-17A is secreted from Th17 cells, a discovery leading to revision of the mechanism underlying the role of Th1/Th2 in the immune response. Strong evidence suggests that immune responses associated with inflammation are involved in the pathogenesis of endometriosis. In the present study, we first demonstrated that the presence of Th17 cells in peritoneal fluid of endometriotic women by flow cytometric analysis and IL-17A-positive cells in endometriotic tissues by immunohistochemistry. To investigate the role of IL-17A in the development of endometriosis, we then studied the effect of IL-17A on IL-8 production, cyclooxygensase-2 expression, and cell proliferation of cultured endometriotic stromal cells (ESCs). IL-17A enhanced IL-8 secretion from ESCs in a dose-dependent manner. The IL-17A-induced secretion of IL-8 from ESCs was suppressed by anti-IL-17 receptor A antibodies or inhibitors of p38 MAPK, p42/44 MAPK, and stress-activated protein kinase/c-Jun N-terminal kinase. Addition of TNFalpha synergistically increased IL-17A-induced IL-8 secretion from ESCs. IL-17A also enhanced the expression of cyclooxygensase-2 mRNA and proliferation of ESCs. IL-17A may play a role in the development of endometriosis by stimulating inflammatory responses and proliferation of ESCs.

Cyclooxygenase-2 Regulates Survival, Migration, and Invasion of Human Endometriotic Cells through Multiple Mechanisms

Endometriosis is a debilitating disease characterized by the presence of functional endometrial glandular epithelium and stroma outside the uterine cavity that affects up to 20% of women of child-bearing age. Cyclooxygenase-2 (COX-2), a rate-limiting enzyme in the biosynthesis of prostaglandin E(2) (PGE(2)), is highly expressed in endometriotic tissues and results in increased concentrations of peritoneal PGE(2) in women. In this study, we determined the expression of COX-2 protein in ectopic and eutopic endometria in humans and the role of COX-2 in endometriotic cell survival, migration, and invasion in humans. Our results indicate that COX-2 protein is abundantly expressed in ectopic endometria compared with eutopic endometria. Comparatively, expression of COX-2 protein is higher in eutopic endometria from women with endometriosis compared with women without endometriosis. Inhibition of COX-2 decreases survival, migration, and invasion of endometriotic cells that are associated with decreased production of PGE(2). Cell growth inhibitory effects of COX-2 inhibition/silencing are mediated through nuclear poly (ADP-ribose) polymerase-mediated apoptosis. Cell motility and invasion inhibitory effects of COX-2 inhibition/silencing are mediated through matrix metalloproteinase-2 and -9 activities. Interestingly, effects of COX-2 inhibition is more profound in endometriotic epithelial than in stromal cells. Furthermore, inhibition of COX-2 affects invasion rather than migration of endometriotic epithelial and stromal cells. It is the first evidence showing that inhibition of COX-2 decreases endometriotic epithelial and stromal cell survival, migration, and invasion in humans. Our results support the emerging concept that COX-2/PGE(2) promotes the pathophysiology and pathogenesis of endometriosis in humans.

Gene expression profiles and functional characterization of human immortalized endometriotic epithelial and stromal cells

To determine whether immortalized endometriotic cells can be used as potential models to unravel pathophysiology/pathogenesis of endometriosis in humans. In vitro study. Human immortalized endometriotic epithelial and stromal cells. College of Veterinary Medicine and Biomedical Sciences, Texas A&M University. Not applicable. None. Gene expression pattern and prostaglandin E(2) production and activity of matrix metalloproteinase 2 and matrix metalloproteinase 9. Several genes associated with estrogen and progesterone biosynthesis and signaling, cell cycle regulation, extracellular matrix degradation, angiogenesis, cell growth and survival, cytokine production, and prostaglandin E(2) biosynthesis, transport, and signaling were expressed in human immortalized endometriotic cells. These endometriotic epithelial and stromal cells exhibited migrating and invading potential, produced a high quantity of prostaglandin E(2), and showed higher matrix metalloproteinase 2 and matrix metalloproteinase 9 activity compared to normal endometrial, epithelial, and stromal cells. Human immortalized endometriotic epithelial and stromal cells could be used as ideal models to study the molecular and cellular aspects of endometriosis in humans.

ERβ regulates ERα expression and response to estradiol via specific promoters in endometrium and endometriosis

Gene expression in endometrium and its pathological counterpart endometriosis is highly regulated by estradiol (E2) and its nuclear receptors ERα and ERβ. The human ERα gene is regulated by a number of alternative promoters including 3 major ones - A, B and C, which are used in part in a tissue-specific manner. Although E2 regulates ERα expression in endometrium and endometriosis the underlying mechanism is unknown. ERα to ERβ ratio is strikingly higher in endometrial vs. endometriotic stromal cells due to elevated ERβ and lower ERα levels in endometriotic cells. We investigated how E2 and ERβ regulate ERα expression in endometrial and endometriotic stromal cells. We used primary stromal cells in culture from ovarian endometrioma walls (n = 3) and eutopic endometrium from disease-free woman (n = 3) to conduct experiments. Isolated endometrial and endometriotic stromal cells in primary culture were treated with E2 (10−7M) or vehicle for 15 min, 30 min, 1, 3, 6, 12 or 24 h. Real-time PCR employing exon-specific primers was used to quantify total and promoter (A, B, or C) - specific ERα mRNA levels. ERβ was selectively knocked down by siRNA. Westen analyses were used to determine ERα and ERβ protein levels. Real-time PCR employing exon-specific primers was used to quantify total and promoter (A, B, or C) – specific ERα mRNA levels in the presence of ERβ knockdown. Chromatin immunoprecipitation (ChIP) assays were performed to determine binding of ERβ to specific ERα promoter regions. Treatment with E2 regulated total ERα mRNA levels in a time-dependent manner in both endometrial and endometriotic stromal cells. Levels of ERα promoter A, B, or C-specific mRNA also showed comparable alterations in response to E2. ERβ knockdown gave rise to an induction of ERα mRNA by E2 in endometriosis that has high baseline ERβ levels. This was mediated primarily through promoters A and C of ERα gene. ChIP assays demonstrated ERβ binding to the promoters A and C but not promoter B of the ERα gene. E2 regulates ERα gene expression via its alternative promoters A, B and C in endometrial and endometriotic stromal cells. High ERβ levels in endometriotic cells might be responsible for the lack of E2-mediated induction of ERα expression via its promoters A and C. Based on the above results ERβ may serve as a potential target for medical treatment of endometriosis.

Interleukin-17 stimulates the proliferation and IL-8 secretion from endometriotic stromal cells

IL-17 has now been described in various immune responses and inflammation. A recent study have shown that IL-17 concentrations in peritoneal fluid correlated with the severity of endometriosis. The present study was undertaken to elucidate the effects of IL-17 on endometriotic stromal cells.

Tenascin is highly expressed in endometriosis and its expression is upregulated by estrogen

To investigate the localization of tenascin protein expression in the endometrium of women without endometriosis and in endometriotic implants, and to determine the in vitro regulation of tenascin by estradiol in these tissues. Experimental laboratory study. Proliferative (n = 14), and secretory (n = 14) endometrium from women without endometriosis and endometriosis implants (n = 14) were used for immunohistochemical analysis. Endometrial and endometriotic stromal cells were grown in culture and treated with estradiol, the estrogen receptor antagonist ICI 182 780 (ICI) alone, estradiol in combination with ICI, or with vehicle (control) for 24 h and tenascin expression was analyzed by Western blotting. Differences in histologic score values were analyzed using one-way ANOVA test. Statistical significance was defined as P<0.05. Tenascin immunostaining revealed an increasing intensity in the stromal cells starting from normal secretory endometrium (Figure 1B), then normal proliferative endometrium (Figure 1A), finally reaching the highest expression in endometriotic implants (Figure 1C–D). Western blot analysis showed that estradiol exerted a more stimulatory effect on tenascin expression in cells from the endometriotic implants than in cells from the proliferative and secretory phases (Figure 2A–D). Estradiol-induced tenascin expression was significantly abrogated by co-treatment with ICI (Figure 2B and D, P<0.05).Figure 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) The upregulation of tenascin could have several roles in the pathogenesis of endometriosis. One role would be by contributing to the aberrant attachment or proliferation of endometriotic cells. The modulation of tenascin as an extracellular matrix protein by estradiol in endometriotic stromal cells may be one of the factors playing a role in the development of endometriosis.

Comprehensive analysis of gene expression alteration in response to gonadotropin releasing hormone (GnRH) analogue in endometriotic stromal cells

Comprehensive analysis of gene expression alteration in response to gonadotropin releasing hormone (GnRH) analogue in endometriotic stromal cells

Curcumin suppresses angiogenesis, cell proliferation and induces apoptosis in an in vitro model of endometriosis

Curcumin suppresses angiogenesis, cell proliferation and induces apoptosis in an in vitro model of endometriosis

Tenascin is highly expressed in endometriosis and its expression is upregulated by estrogen

To investigate the localization of tenascin expression in the endometrium of women without endometriosis and in endometriotic implants, and to determine the in vitro regulation of tenascin by E(2) in these tissues. Experimental laboratory study. University medical center. Reproductive age women with or without endometriosis. Proliferative (n = 14), and secretory (n = 14) endometrium from women without endometriosis and endometriosis implants (n = 14) were used for immunohistochemical analysis. Endometrial and endometriotic stromal cells were grown in culture and treated with E(2), the estrogen receptor antagonist ICI 182 780 (ICI) alone, E(2) in combination with ICI, or vehicle (control) for 24 hours, and tenascin expression was analyzed by Western blotting. Expression levels of tenascin in normal endometrium and endometriotic implants and its regulation by E(2). Tenascin immunostaining revealed an increasing intensity in the stromal cells, starting from normal secretory endometrium, then normal proliferative endometrium, and reaching the highest expression in endometriotic implants. Estradiol induced a significant increase in tenascin protein levels in the endometriotic stromal cells in culture. The modulation of tenascin as an extracellular matrix protein by E(2) in endometriotic stromal cells may be one of the factors playing a role in the development of endometriosis.

An epigenetic disorder may cause aberrant expression of aromatase gene in endometriotic stromal cells

To examine the molecular basis of aromatase expression in stromal cell culture from endometriotic chocolate cysts. Prospective study. Department of Obstetrics and Gynecology and Department of Biosignaling, Tottori University, Yonago Japan. Thirty women, selected randomly, who underwent laparoscopy (n = 18) or laparotomy (n = 12). Endometrial and endometriotic stromal cells were obtained from the uterus and chocolate cyst lining of the ovary. Estradiol concentrations in the culture media were measured by means of enzyme immunoassay. Aromatase expression was examined by quantitative real-time polymerase chain reaction. Promoter usage was examined using unique exon I (PII, I.1, I.3, I.4, I.5, and I.6) and exon II primers. To determine the effect of 5-aza-deoxycytidine on endometrial stromal cells, the cells were treated with the agent for 96 hours. Endometriotic cells secreted a marginal level of estradiol into the culture media, but adding testosterone to the culture produced a pronounced level of estradiol. In endometrial cells, estradiol production was far less efficient than in endometriotic cells even after adding testosterone. Real-time polymerase chain reaction analyses demonstrated the up-regulation of aromatase messenger RNA (mRNA) expression in endometriotic cells. Three proximal promoters, PII, 1.3, and 1.6, drove mRNA expression. In endometrial cells where a marginal level of aromatase mRNA expression was observed, the same promoters as those in the endometriotic cells were used. To determine the role of epigenetic modification of aromatase gene expression in endometriotic cells, endometrial cells were treated with 5-aza-deoxycytidine, which markedly enhanced aromatase mRNA expression, depending on the same proximal promoters as those in endometriotic cells. An epigenetic disorder may play a role in the pathophysiology of endometriosis.

Promoter Methylation Regulates Estrogen Receptor 2 in Human Endometrium and Endometriosis1

Steroid receptors in the stromal cells of endometrium and its disease counterpart tissue endometriosis play critical physiologic roles. We found that mRNA and protein levels of estrogen receptor 2 (ESR2) were strikingly higher, whereas levels of estrogen receptor 1 (ESR1), total progesterone receptor (PGR), and progesterone receptor B (PGR B) were significantly lower in endometriotic versus endometrial stromal cells. Because ESR2 displayed the most striking levels of differential expression between endometriotic and endometrial cells, and the mechanisms for this difference are unknown, we tested the hypothesis that alteration in DNA methylation is a mechanism responsible for severely increased ESR2 mRNA levels in endometriotic cells. We identified a CpG island occupying the promoter region (-197/+359) of the ESR2 gene. Bisulfite sequencing of this region showed significantly higher methylation in primary endometrial cells (n = 8 subjects) versus endometriotic cells (n = 8 subjects). The demethylating agent 5-aza-2'-deoxycytidine significantly increased ESR2 mRNA levels in endometrial cells. Mechanistically, we employed serial deletion mutants of the ESR2 promoter fused to the luciferase reporter gene and transiently transfected into both endometriotic and endometrial cells. We demonstrated that the critical region (-197/+372) that confers promoter activity also bears the CpG island, and the activity of the ESR2 promoter was strongly inactivated by in vitro methylation. Taken together, methylation of a CpG island at the ESR2 promoter region is a primary mechanism responsible for differential expression of ESR2 in endometriosis and endometrium. These findings may be applied to a number of areas ranging from diagnosis to the treatment of endometriosis.