Pelvic endometriosis is a complex syndrome characterized by an estrogen-dependent chronic inflammatory process that affects primarily pelvic tissues, including the ovaries. It is caused when shed endometrial tissue travels retrograde into the lower abdominal cavity. Endometriosis is the most common cause of chronic pelvic pain in women and is associated with infertility. The underlying pathologic mechanisms in the intracavitary endometrium and extrauterine endometriotic tissue involve defectively programmed endometrial mesenchymal progenitor/stem cells. Although endometriotic stromal cells, which compose the bulk of endometriotic lesions, do not carry somatic mutations, they demonstrate specific epigenetic abnormalities that alter expression of key transcription factors. For example, GATA-binding factor-6 overexpression transforms an endometrial stromal cell to an endometriotic phenotype, and steroidogenic factor-1 overexpression causes excessive production of estrogen, which drives inflammation via pathologically high levels of estrogen receptor-β. Progesterone receptor deficiency causes progesterone resistance. Populations of endometrial and endometriotic epithelial cells also harbor multiple cancer driver mutations, such as KRAS, which may be associated with the establishment of pelvic endometriosis or ovarian cancer. It is not known how interactions between epigenomically defective stromal cells and the mutated genes in epithelial cells contribute to the pathogenesis of endometriosis. Endometriosis-associated pelvic pain is managed by suppression of ovulatory menses and estrogen production, cyclooxygenase inhibitors, and surgical removal of pelvic lesions, and in vitro fertilization is frequently used to overcome infertility. Although novel targeted treatments are becoming available, as endometriosis pathophysiology is better understood, preventive approaches such as long-term ovulation suppression may play a critical role in the future.

Endometriotic stromal cells synthesize estradiol via the steroidogenic pathway. Nuclear receptor subfamily 5, group A, member 1 (NR5A1) is critical, but alone not sufficient, in activating this cascade that involves at least 5 genes. To evaluate whether another transcription factor is required for the activation of this pathway, we examined whether GATA Binding Protein 6 (GATA6) can transform a normal endometrial stromal cell (NoEM) into an endometriotic-like cell by conferring an estrogen-producing phenotype. We ectopically expressed GATA6 alone or with NR5A1 in NoEM or silenced these transcription factors in endometriotic stromal cells (OSIS) and assessed the messenger RNAs or proteins encoded by the genes in the steroidogenic cascade. Functionally, we assessed the effects of GATA6 expression or silencing on estradiol formation. In OSIS, GATA6 was necessary for catalyzing the conversion of progesterone to androstenedione (CYP17A1; P < .05). In NoEM, ectopic expression of GATA6 was essential for converting pregnenolone to estrogen (HSD3B2, CYP17A1, and CYP19A1; P < .05). However, simultaneous ectopic expression of both GATA6 and NR5A1 was required and sufficient to confer induction of all 5 genes and their encoded proteins that convert cholesterol to estrogen. Functionally, only simultaneous knockdown of GATA6 and NR5A1 blocked estradiol formation in OSIS ( P < .05). The presence of both transcription factors was required and sufficient to transform endometrial stromal cells into endometriotic-like cells that produced estradiol in large quantities ( P < .05). In summary, GATA6 alone is essential but not sufficient for estrogen formation in endometriosis. However, simultaneous addition of GATA6 and NR5A1 to an endometrial stromal cell is sufficient to transform it into an endometriotic-like cell, manifested by the activation of the estradiol biosynthetic cascade.

The transcription factor GATA2 is important for endometrial stromal cell decidualization in early pregnancy. Progesterone receptor (PGR) is also critical during decidualization but its interaction with GATA2 in regulating genes and pathways necessary for decidualization in human endometrium are unclear. RNA-sequencing (RNA-seq) was performed to compare gene expression profiles (n = 3), and chromatin immunoprecipitation followed by sequencing (ChIP-seq) using an antibody against GATA2 (n = 2) was performed to examine binding to target genes in human endometrial stromal cells undergoing in vitro decidualization (IVD including estrogen, progestin, and 3′,5′-cyclic AMP analogue) or vehicle treatment. We identified 1232 differentially expressed genes (DEGs) in IVD vs vehicle. GATA2 cistrome in IVD-treated cells was enriched with motifs for GATA, ATF, and JUN, and gene ontology analysis of GATA2 cistrome revealed pathways that regulate cholesterol storage, p38 mitogen-activated protein kinase, and the c-Jun N-terminal kinase cascades. Integration of RNA-seq and ChIP-seq data revealed that the PGR motif is highly enriched at GATA2 binding regions surrounding upregulated genes in IVD-treated cells. The integration of a mined public PGR cistrome in IVD-treated human endometrial cells with our GATA2 cistrome showed that GATA2 binding was significantly enhanced at PGR-binding regions in IVD vs vehicle. Interrogating 2 separate ChIP-seq data sets together with RNA-seq revealed integration of GATA2 and PGR action to coregulate biologic processes during decidualization of human endometrial stromal cells, specifically via WNT activation and stem cell differentiation pathways. These findings reveal the key pathways that are coactivated by GATA2 and PGR that may be therapeutic targets for supporting implantation and early pregnancy.

Objective: To investigate the gene targets of estradiol (E2)-estrogen receptor-a (ESR1) in human endometrial stromal cells. Design: Basic science. Setting: University research center. Patient(s): Premenopausal women with or without endometriosis. Intervention(s): Primary cultures of human endometrial stromal cells from healthy endometrium, with or without small-interfering RNA (siRNA) knockdown of ESR1 expression, were treated with E2 or vehicle control. Main Outcome Measure(s): Genome-wide RNA expression by RNA sequencing was compared in endometrial stromal cells with or without siRNA knockdown of ESR1 in the presence or absence of E2. Genome-wide recruitment of ESR1 to chromatin was assessed by chromatin immunoprecipitation sequencing. Gene expression by real-time qualitative polymerase chain reaction of a potential E2-ESR1 target gene was determined in endometrial stromal cells and endometriotic stromal cells. Result(s): We identified several important pathways that are dependent on E2-ESR1 signaling in endometrial stromal cells, including progesterone signaling, cell-matrix adhesion, and cytoskeleton rearrangement, as well as paracrine signaling by members of the fibroblast growth factor family. We detected a total of 709 ESR1 target sites on chromatin. By integrating data on genome-wide transcriptomic changes and E2-ESR1 binding sites, we identified inositol polyphosphate phosphatase type II (INPP4B) as a candidate E2-mediated suppressor of proliferation in healthy endometrial cells. INPP4B was downregulated in endometriosisderived stromal cells. Conclusion(s): E2-ESR1 activates genes involved in human endometrial stromal cell cycle regulation, progesterone response, and production of stromal growth factors. Understanding the direct role of estrogen on the endometrial stroma and identifying downstream targets of E2-ESR1 can inform the development of targeted therapies for endometriosis and diminished endometrial receptivity.