Steroidogenic enzyme and key decidualization marker dysregulation in endometrial stromal cells from women with versus without endometriosis

Identification of mechanisms underlying endometriosis pathogenesis will facilitate understanding and treatment of infertility and pain associated with this disorder. Herein, we investigated the expression of steroidogenic pathway enzymes and key decidualization biomarkers in endometrial tissue and in eutopic endometrial stromal fibroblasts (hESFs) from women with vs. those without endometriosis, and subsequently treated in vitro with 8-bromo-cAMP (8-Br-cAMP) or progesterone (P4). Real-time quantitative PCR, immunohistochemistry, ELISA, and radiometric aromatase activity assay were used. The results demonstrate significantly increased (14.5-fold; P=0.037) expression of aromatase in eutopic endometrium of women with disease. In 8-Br-cAMP-treated hESF from eutopic endometrium of women with endometriosis, the balance in estradiol (E2) and P4 biosynthetic and metabolizing enzymes is disturbed (decreased HSD3B1 and HSD17B2, and increased HSD17B1 and aromatase), with the equilibrium being shifted towards an E2-enriched milieu. However, hESF from the same group of women treated with P4 did not demonstrate such responsiveness. Lower expression of IGFBP1 and prolactin mRNA and protein was observed in hESF from women with vs. those without endometriosis in response to 8-Br-cAMP, but not P4, suggesting a blunted response of these decidual biomarkers to activation of the PKA pathway in eutopic endometrium in women with disease. The dichotomy of 8-Br-cAMP regulation of select steroidogenic enzymes leading to an enriched E2 milieu within the endometrium and a blunted response of decidual biomarkers to this decidualizing agent of hESF from women with endometriosis suggests resistance to full decidualization of the stromal fibroblasts and mechanisms underlying implantation failure and the pathophysiology of this disorder.     

Estrogen receptor-alpha (ER-alpha) and defects in uterine receptivity in women

Endometriosis is a disorder that affects 5% of the normal population but is present in up to 40% of women with pelvic pain and/or infertility. Recent evidence suggests that the endometrium of women with endometriosis exhibits progesterone insensitivity. One endometrial protein that fluctuates in response to progesterone is the estrogen receptor-alpha (ER alpha), being down-regulated at the time of peak progesterone secretion during the window of implantation. Here we demonstrate that the biomarker of uterine receptivity, beta 3 integrin subunit, is reduced or absent in some women with endometriosis and that such defects are accompanied by inappropriate over-expression of ER alpha during the mid-secretory phase. Using a well-differentiated endometrial cell line we showed that the beta 3 integrin protein is negatively regulated by estrogen and positively regulated by epidermal growth factor (EGF). By competing against estrogen with various selective estrogen receptor modulators (SERMs) and estrogen receptor agonists and antagonists, inhibition of expression of the beta 3 integrin by estrogen can be mitigated. In conclusion, we hypothesize that certain types of uterine receptivity defects may be caused by the loss of appropriate ER alpha down-regulation in the mid-secretory phase, leading to defects in uterine receptivity. Such changes might be effectively treated by timely administration of the appropriate anti-estrogens to artificially block ER alpha and restore normal patterns of gene expression. Such treatments will require further clinical studies.     

Characterization of periostin expression in human endometrium and endometriotic lesions

Objective(s): To investigate the expression of periostin in the eutopic and ectopic endometrium of women diagnosed as endometriosis and evaluate the role of periostin in the pathogenesis of endometriosis. Study design: In this study, the expression of periostin was evaluated in the endometrial specimens from 35 women diagnosed as endometriosis and from 30 healthy women. To assess the presence and localization of periostin throughout the menstrual cycle in both eutopic and ectopic endometrium of women with endometriosis, microscopic evaluation was conducted. It was also subsequently compared with normal endometrium. Results: In the eutopic and ectopic endometrium of women with endometriosis, immunoreactivities of periostin increased compared with those of normal endometrium. We also observed a cyclic variation in the eutopic stromal periostin immunoreactivity throughout their menstrual cycle because higher H score values were observed in the proliferative phase than those in the secretory phase. Conclusion(s): These findings indicated that periostin may be involved in the pathophysiology of endometriosis.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

The estrogen early response gene FOS is altered in a baboon model of endometriosis

Endometriosis, the presence of a functional endometrium outside of the uterine cavity, is associated with infertility. In our simulated model of pregnancy in baboons with experimental endometriosis, hCG infusion fails to induce expression of the immunoregulatory protein glycodelin. To test the hypothesis that the development of endometriosis is associated with an aberrant endometrial immunological environment, we examined the expression of a series of immunoregulatory genes in endometrium from baboons with and without endometriosis. Six months following intraperitoneal inoculation with menstrual endometrium, eutopic endometrium was surgically collected between Days 9 and 11 postovulation. Control endometrium was similarly collected from disease-free animals. Total RNA was extracted, and biotinylated cDNA probes were hybridized to the SuperArray GEArray Q series Th1/Th2/Th3 cDNA array, representing 96 genes. Gene expression levels were determined using ScanAlyze and GEArray Analyzer software. Seven genes were upregulated, including JUND, FOS, CCL11, NFKB1 and others, in the endometrium from baboons with endometriosis compared with the endometrium from disease-free animals; one gene, IL1R1, was downregulated. Quantitative RT-PCR confirmed upregulation of FOS and CCL11 in endometriotic eutopic endometrium. Immunohistochemical analysis revealed altered levels and distribution of FOS protein in the eutopic endometrium of baboons with induced endometriosis. These data suggest that in an induced model of endometriosis an aberrant eutopic immunological environment results in a decreased apoptotic potential and in rapid alterations in endometrial gene expression. We propose that the reduced fecundity associated with endometriosis has a multifold etiology in spontaneous and induced disease.     

Role of aromatase in endometrial disease

Aromatase is the key enzyme for estrogen biosynthesis. It is normally expressed in the human ovary, skin, adipose tissue and brain. Aromatase activity is not detectable in normal endometrium. In contrast, aromatase is expressed aberrantly in endometriosis and is stimulated by PGE₂. This results in local production of estrogen, which induces PGE₂ formation and establishes a positive feedback cycle. Another abnormality in endometriosis, i.e. deficient 17β-hyroxysteroid dehydrogenase (17β-HSD) type 2 expression, impairs the inactivation of estradiol to estrone. These molecular aberrations collectively favor accumulation of increasing quantities of estradiol and PGE₂ in endometriosis. The clinical relevance of these findings was exemplified by the successful treatment of an unusually aggressive case of post-menopausal endometriosis using an aromatase inhibitor.     

Aromatase and cyclooxygenases: enzymes in breast cancer

Aromatase (estrogen synthase) is the cytochrome P450 enzyme complex that converts C₁₉ androgens to C₁₈ estrogens. Aromatase activity has been demonstrated in breast tissue in vitro, and expression of aromatase is highest in or near breast tumor sites. Thus, local regulation of aromatase by both endogenous factors as well as exogenous medicinal agents will influence the levels of estrogen available for breast cancer growth. The prostaglandin PGE₂ increases intracellular cAMP levels and stimulates estrogen biosynthesis, and previous studies in our laboratories have shown a strong linear association between aromatase (CYP19) expression and expression of the cyclooxygenases (COX-1 and COX-2) in breast cancer specimens. To further investigate the pathways regulating COX and CYP19 gene expression, studies were performed in normal breast stromal cells, in breast cancer cells from patients, and in breast cancer cell lines using selective pharmacological agents. Enhanced COX enzyme levels results in increased production of prostaglandins, such as PGE₂. This prostaglandin increased aromatase activity in breast stromal cells, and studies with selective agonists and antagonists showed that this regulation of signaling pathways occurs through the EP₁ and EP₂ receptor subtypes. COX-2 gene expression was enhanced in breast cancer cell lines by ligands for the various peroxisome proliferator-activated receptors (PPARs), and differential regulation was observed between hormone-dependent and -independent breast cancer cells. Thus, the regulation of both enzymes in breast cancer involves complex paracrine interactions, resulting in significant consequences on the pathogenesis of breast cancer.     

Galectin-1 Overexpression in Endometriosis and Its Regulation by Neuropeptides (CRH,UCN) Indicating Its Important Role in Reproduction and Inflammation

Endometriosis is an inflammatory disease of women of reproductive age featured by the presence of ectopic endometrium and is strongly related to infertility. Galectins, carbonhydrate-binding proteins, have been found to have pro- or anti-inflammatory roles in the reproductive tract and in pathological conditions concerning infertility. Galectin-1, which is expressed at endometrium and decidua, plays a major role in implantation and trophoblast invasion. Also, the neuropeptides, corticotropin releasing hormone (CRH) and urocortin (UCN) and their receptors are expressed in eutopic and ectopic endometrium showing a differential expression pattern in endometriotic women compared to healthy ones. The aim of this study was to examine the galectin-1 expression in endometriotic lesions and compare its expression in eutopic endometrium of endometriotic and healthy women. Furthermore, we examined the effect of CRH and UCN in galectin-1 expression in Ishikawa cell line and macrophages and investigated the implication of CRHR1 in these responses. Eutopic and ectopic endometrium specimens, Ishikawa cell line and mice macrophages were used. Immunohistochemistry and western blot analysis were performed in order to identify galectin-1 expression in ectopic and eutopic endometrium of women with and without endometriosis and the regulatory effect of CRH and UCN on galectin-1 expression. This study presents for the first time that galectin-1 is overexpressed in endometriotic lesions compared to eutopic endometrium of endometriotic women and is more abundantly expressed in eutopic endometrium of disease women compared to healthy ones. Furthermore, it is shown that CRH and UCN upregulate galectin-1 expression in Ishikawa cell line and macrophages and this effect is mediated through CRHR1. These results suggest that galectin-1 might play an important role in endometriosis pathology and infertility profile of women suffering from endometriosis by being at the same time regulated by CRH and UCN interfering in the immune disequilibrium which characterizes this pathological condition.     

Aromatase in endometriosis and uterine leiomyomata

Endometrial tissue from uterine disease-free women does not exhibit aromatase activity. In contrast, aromatase enzyme activity and mRNA levels are readily detectable in endometriosis. PGE₂ stimulates both aromatase expression and activity in endometriotic stromal cells via promoter II region of the aromatase gene. This results in local production of estradiol, which induces PGE₂ formation and establishes a positive feedback cycle. This mechanism seems to contribute to continuous production of estradiol and PGE₂. Aromatase mRNA levels and enzyme activity are also present in uterine leiomyomata that are estrogen-dependent benign tumors of the myometrium. Successful treatment of endometriosis and uterine leiomyomata using aromatase inhibitors by recent pilot trials underscores the clinical significance of these molecular studies.     

The effects of dietary phytoestrogens on aromatase activity in human endometrial stromal cells

Dietary phytoestrogens have been reported to inhibit aromatase activity in placental microsomes, but the effects in the human endometrium are unknown. Aromatase, the rate-limiting enzyme in the conversion of androgens to estrogens, has recently been shown to be expressed in the endometrium of women with endometriosis and is thought to play a role in the pathophysiology of this disease. Therefore, the objective of this study was to screen dietary phytoestrogens for their ability to inhibit aromatase activity in human endometrial stromal cells (ESC) and identify potential novel therapeutic agents for the treatment of endometriosis. The inhibition of aromatase activity by direct interaction with the dietary phytoestrogens genistein, daidzein, chrysin, and naringenin was tested in a cell free assay. Furthermore, test compound effects on aromatase activity in ESC cultures were also examined. Genistein and daidzein were inactive in the human recombinant aromatase assay whereas naringenin and chrysin inhibited aromatase activity. However, genistein (1 nM to 1 mM) stimulated aromatase activity in ESC whereas other phytoestrogens had no effect. Immunopositive aromatase cells were demonstrated in genistein-treated ESC but not in untreated control cultures. Taken together, our data suggest that genistein can increase aromatase activity in ESC likely via increased enzyme expression.     

Aromatase in the normal breast and breast cancer

Adipose tissue and muscle constitute the larger proportion of body mass, and therefore aromatization in these tissues is the major source of circulating estrogens in postmenopausal women. Although plasma estrogen concentrations are very low, levels in breast cancers from postmenopausal patients are reported to be 10-fold higher than in plasma and normal tissue. Whereas studies on aromatase activity in the tumor suggest that estrogen may be produced locally, the significance of this contribution has been questioned. Using immunocytochemistry (ICC) to an anti-aromatase antibody, a relatively strong immunoreaction was detected in tumor epithelial cells as well as in the terminal ductal lobular units (TDLUs) of the normal breast. Aromatase expression was detected in the cytoplasm of tumor epithelial cells and the surrounding stromal cells of over 50% of tumors in a series of 19 breast cancers. In situ hybridization (ISH) to aromatase mRNA confirmed the immunocytochemical result that the epithelial cells are the primary site of estrogen synthesis in the breast and breast cancers. In the 10 tumors which showed immunoreaction to aromatase, the average aromatase activity measured in cryosections was 286.5 ± 18.6 fmol estrogen/mg protein/h (SE), whereas in nine tumors with weak aromatase immunoreaction, the enzyme activity was 154.7 ± 19.3 fmol estrogen/mg protein/h (P < 0.05) (SE). The functional significance of tumor aromatase and locally produced estrogens on the growth of tumors was suggested by the correlation between aromatase activity and proliferating cell nuclear antigen (PCNA), a marker of cell proliferation (P < 0.005). Although intratumoral aromatase activity did not correlate with steroid receptors significantly, there was a trend for estrogen receptor (ER)-positive tumors to express aromatase. In addition, proliferation ([³H]-thymidine incorporation into DNA) during histoculture, was increased by both estradiol and testosterone in tumors with high aromatase activity. Our results suggest that some tumors synthesize sufficient estrogen to stimulate their proliferation. It may thus be important to inhibit tumor aromatase as well as to reduce circulating levels of estrogen for effective breast cancer treatment.     

Genome‐wide analysis of DNA methylation in endometriosis using Illumina Human Methylation 450 K BeadChips

Endometriosis is a common chronic gynecologic disorder characterized by the presence and growth of endometrial‐like tissue outside of the uterine cavity. Although the exact etiology remains unclear, epigenetic modifications, such as DNA methylation, are thought to contribute to the pathogenesis of endometriosis. Here, we used the Illumina Human Methylation 450 K BeadChip Array to analyze the genome‐wide DNA methylation profiles of six endometriotic lesions and six eutopic endometria from patients with ovarian endometriosis and six endometria of women without endometriosis. Compared with the eutopic endometria of women with endometriosis, 12,159 differentially methylated CpG sites and 375 differentially methylated promoter regions were identified in endometriotic lesions. GO analyses showed that these putative differentially methylated genes were primarily associated with immune response, inflammatory response, response to steroid hormone stimulus, cell adhesion, negative regulation of apoptosis, and activation of the MAPK activity. In addition, the expression levels of DNMT1, DNMT3A, DNMT3B, and MBD2 in endometriotic lesions and eutopic endometria were significantly decreased compared with control endometria. Our findings suggest that aberrant DNA methylation status in endometriotic lesions may play a significant role in the pathogenesis and progression of endometriosis.     

Proteomics identification of annexin A2 as a key mediator in the metastasis and proangiogenesis of endometrial cells in human adenomyosis

Adenomyosis is a common estrogen-dependent disorder of females characterized by a downward extension of the endometrium into the uterine myometrium and neovascularization in ectopic lesions. It accounts for chronic pelvic pain, dysmenorrhea, menorrhagia, and infertility in 8.8-61.5% women worldwide. However, the molecular mechanisms for adenomyosis development remain poorly elucidated. Here, we utilized a two-dimensional polyacrylamide gel electrophoresis/MS-based proteomics analysis to compare and identify differentially expressed proteins in matched ectopic and eutopic endometrium of adenomyosis patients. A total of 93 significantly altered proteins were identified by tandem MS analysis. Further cluster analysis revealed a group of estrogen-responsive proteins as dysregulated in adenomyosis, among which annexin A2, a member of annexin family proteins, was found up-regulated most significantly in the ectopic endometrium of adenomyosis compared with its eutopic counterpart. Overexpression of ANXA2 was validated in ectopic lesions of human adenomyosis and was found to be tightly correlated with markers of epithelial to mesenchymal transition and dysmenorrhea severity of adenomyosis patients. Functional analysis demonstrated that estrogen could remarkably up-regulate ANXA2 and induce epithelial to mesenchymal transition in an in vitro adenomyosis model. Enforced expression of ANXA2 could mediate phenotypic mesenchymal-like cellular changes, with structural and functional alterations in a β-catenin/T-cell factor (Tcf) signaling-associated manner, which could be reversed by inhibition of ANXA2 expression. We also proved that enforced expression of ANXA2 enhanced the proangiogenic capacity of adenomyotic endometrial cells through HIF-1α/VEGF-A pathway. In vivo, we demonstrated that ANXA2 inhibition abrogated endometrial tissue growth, metastasis, and angiogenesis in an adenomyosis nude mice model and significantly alleviated hyperalgesia. Taken together, our data unraveled a dual role for ANXA2 in the pathogenesis of human adenomyosis through conferring endometrial cells both metastatic potential and proangiogenic capacity, which could serve as a potential therapeutic target for the treatment of adenomyosis patients.