Endometriosis Endometrium Research Articles

Cytotoxic T Lymphocytes, Tc17 Cells, Th1 Cells, and ThGM Cells are Increased in the Blood and Ectopic Endometrium of Patients With Adenomyosis.

Adenomyosis (AM) is associated with immune response and inflammation. However, the role of T cell subsets in AM development has not been thoroughly understood. Patients with focal or diffuse AM were recruited. Serum cytokines were quantified by enzyme-linked immunosorbent assay (ELISA). Different T cell subsets in the blood and ectopic endometrium were determined by flow cytometry. Serum interleukin-6 (IL-6) and macrophage-colony-stimulating factor (GM-CSF) were increased in patients with focal or diffuse AM before focused ultrasound ablation surgery(FUAS), but not after FUAS. Compared with the healthy control, the frequencies of CD8+ interferon-gamma (IFN-γ)-expressing cytotoxic T lymphocytes (CTLs), interleukin-17A (IL-17A)-expressing Tc17 cells, CD4+ T helper 1 (Th1) cells, and GM-CSF-expressing T helper (ThGM) cells were up-regulated in the blood of patients with AM, especially those with diffuse AM. However, these changes were eradicated after FUAS. Meanwhile, the frequencies of these T cell subsets were positively correlated with the CA-125 level. Furthermore, these T cell subsets were also increased in ectopic endometrium. Our study delineates for the first time the presence of CTLs, Tc17 cells, Th1, and ThGM cells in the blood and ectopic endometrium in AM. The results imply that T cell response might impact AM development.

Are Adenomyosis and Endometriosis Phenotypes of the Same Disease Process?

In recent literature reviews, we concluded that the possibility that endometrial molecular aberrations are the sole or a necessary determinant of endometriosis and the Tissue Injury and Repair (TIAR) theory are yet to be convincingly proven. Here, we critically examine the theory that adenomyosis and endometriosis represent different phenotypes of a single disease. A common etiopathology for adenomyosis and endometriosis has been suggested because both conditions entail the presence of endometrial tissue at locations other than the lining of the uterus. There are wide differences in reported disease incidence and prevalence and, consequently, in estimates of the coexistence of both conditions. There are some similarities but also differences in their clinical features and predisposing factors. Each condition has a range of subtypes. These differences alone pose the question of whether subtypes of endometriosis and adenomyosis have different etiopathologies, and, in turn, this raises the question of whether they all share a common etiology. It is debatable whether the recognized differences between the eutopic endometrium in adenomyosis and endometriosis compared to those in unaffected women are the cause or the effect of the disease. The finding of common mutations, particularly of KRAS, lend support to the notion of shared predisposing factors, but this alone is insufficient evidence of causation.

P-321 increased expression of TGF-β1 contributes to the downregulation of progesterone receptor expression in the eutopic endometrium of infertile women with minimal/mild endometriosis

Abstract Study question Do immune cells regulate progesterone resistance and endometrial receptivity of eutopic endometrial stromal cells (ESCs) by transforming growth factor-β1 (TGF-β1)? Summary answer By activating TGF-β/Smad signaling in eutopic ESCs, elevated TGF-β1 from CD45+ immune cells could attenuate expression of progesterone receptor (PR), and further decrease endometrial receptivity. What is known already Endometriosis is a hormone-dependent disease related to impaired immunoregulation. We characterized the trascriptomic transformation of eutopic endometrium from patients with minimal/mild endometriosis and controls across the menstrual cycle. However, the regulatory mechanism of altered immune microenvironment on eutopic ESCs remains unclear. Study design, size, duration Single-cell RNA transcriptomes were profiled using 10x Genomics platform. Primary culture of eutopic ESCs was performed to explore the effects of TGF-β1 on the expression of Smad / PR, and the in vitro decidualization. Additionally, co-immunoprecipitation (Co-IP) was used to explore the direct interaction between Smad and PR. Participants/materials, setting, methods Endometrial tissue samples were obtained from twenty-four women who were laparoscopically confirmed minimal/mild endometriosis according to the revised American Fertility Society classification. Additionally, four patients who were laparoscopically endometriosis-free were enrolled as controls. All samples were collected in DMEM/F12 medium and processed in one of two ways, either for single-cell sequencing (n = 10) or primary cell culture (n = 14). Main results and the role of chance The results of single-cell RNA sequencing revealed that the percentage of CD45+ cells in eutopic endometrium was increase in both proliferative (26% vs 24.1%) and secretory (19.3% vs 8.3%) stages. Overexpression of TGF-β1 of CD45+ cells was observed in eutopic endometrium of endometriosis in both proliferative and secretory stages. We found attenuate expression of PRB (p = 0.026) and PRA (p = 0.678) after using TGF-β1 in eutopic ESCs by western blot. Similarly, qRT-PCR results showed that the mRNA level of PR (p＜0.001), PRB (p = 0.003) and HOXA10 (p＜0.001) decreased significantly after TGF-β1 treatment, but that increased (p＜0.01) after SB431542 treatment in the eutopic ESCs. Moreover, TGF-β1 has a negative effect on the in vitro decidualization of eutopic ESCs (p = 0.003). And the group with treatment of both TGF-β1 and SB435142 in eutopic ESCs showed significant decidual-like changes with increased prolactin level (p = 0.01). Co-IP showed no physical interaction between the PR and p-Smad3/Smad3 proteins. Limitations, reasons for caution This study focused on CD45+ immune cells and the cytokine, TGF-β1. Further in vivo and in vitro studies are needed to elucidate the specific types of immune cell and the mechanisms that behind endometrial receptivity decrease in endometriosis. Wider implications of the findings Our results demonstrate abnormal activation of CD45+ cells in eutopic endometrium with minimal/mild endometriosis. And overexpression of TGF-β1 was found by CD45+ cells. We found that TGF-β1 suppresses PR expression and decidualization in eutpoic ESCs. These results provide a novel insight into the mechanisms behind progesterone resistance in endometriosis. Trial registration number not applicable

Novel in vivo endometriotic models associated eutopic endometrium by implanting menstrual blood-derived stromal cells from patients with endometriosis

The eutopic endometrium provides novel insights into endometriotic pathophysiology and treatment. However, no in vivo models currently available are suitable for eutopic endometrium in endometriosis. In this study, we present new endometriotic in vivo models associated with eutopic endometrium using menstrual blood-derived stromal cells (MenSCs). First, we isolated endometriotic MenSCs (E-MenSCs) and healthy MenSCs (H-MenSCs) from the menstrual blood of patients with endometriosis (n = 6) and healthy volunteers (n = 6). Then, we identified MenSCs’ endometrial stromal cell properties using adipogenic and osteogenic differentiation. A cell counting kit-8 and wound healing assay were used to compare the proliferation and migration capability between E-MenSCs and H-MenSCs. Seventy female nude mice were used to prepare endometriotic models related to eutopic endometrium by implanting E-MenSCs relying on three approaches, including surgical implantation using scaffolds seeded with MenSCs, and subcutaneous injection of MenSCs in the abdomen and the back (n = 10). H-MenSCs or scaffolds only were implanted in control groups (n = 10). One month after the surgical implantation and 1 week after the subcutaneous injection, we evaluated modeling by hematoxylin–eosin (H&E) and immunofluorescent staining of human leukocyte antigen α (HLAA). Fibroblast morphology, lipid droplets, and calcium nodules in E-MenSCs and H-MenSCs identified their endometrial stromal cell properties. We noticed that the proliferation and migration of E-MenSCs were considerably enhanced compared to H-MenSCs (P < 0.05). E-MenSCs implanted in nude mice formed ectopic lesions using three approaches (n = 10; lesions formation rate: 90%, 115%, and 80%; average volumes: 123.60, 27.37, and 29.56 mm3), while H-MenSCs in the nude mice shaped nothing at the implantation sites. Endometrial glands, stroma, and HLAA expression in these lesions further verified the success and applicability of the proposed endometriotic modeling. Findings provide in vitro and in vivo models and paired controls associated with eutopic endometrium in women with endometriosis using E-MenSCs and H-MenSCs. The approach of subcutaneous injection of MenSCs in the abdomen is highlighted due to non-invasive, simple, and safe steps, a short modeling period (1 week), and an excellent modeling success rate (115%), which could improve the repeats and success of endometriotic nude mice model and shorten the modeling period. These novel models could nearly intimate human eutopic endometrial mesenchymal stromal cells in the progress of endometriosis, opening a new path for disease pathology and treatment.

Proliferative characteristics of eutopic and ectopic endometrium in adenomyosis using AgNOR technology

Adenomyosis is a benign lesion of the uterus, which is characterized by the presence and pathological growth of endometrial glands and stroma in the myometrium. During adenomyosis, endometrial tissue abnormally grown in the myometrium is called ectopic endometrium, and endometrial tissue in the uterine cavity is called eutopic endometrium. Women with adenomyosis may experience abnormal uterine bleeding, dysmenorrhea, dyspareunia, and infertility, although, in one-third of women, the lesion is asymptomatic. Adenomyosis is the most frequently diagnosed pathology after hysterectomy in perimenopausal women due to heavy bleeding or pain. AgNOR is used as an aid for evaluating the proliferative activity of cells. Certain types of studies have demonstrated their effectiveness in differentiating various benign or malignant processes, as well as in determining the histological grade of intraepithelial neoplastic processes. Within the framework of this study, the proliferative activity of the eutopic and ectopic endometrium, as well as their stroma and normal myometrium, was evaluated on archival blocks fixed in formalin and embedded in paraffin (FFPE), diagnosed in cases of adenomyosis, using AgNOR technology. In the studied system, eutopic endometrium - basal endometrium - in both the glandular and stromal components of ectopic endometrium, the highest proliferative activity is manifested in ectopic endometrium, which is why it can probably represent a risk for the development of neoplasms.

Deubiquitinase and unconjugated ubiquitin activate microenvironment inflammation and pain in eutopic and ectopic endometrium in endometriosis

Deubiquitinase and unconjugated ubiquitin activate microenvironment inflammation and pain in eutopic and ectopic endometrium in endometriosis

Circulating CD200 is increased in the secretory phase of women with endometriosis as is endometrial mRNA, and endometrial stromal cell CD200R1 is increased in spite of reduced mRNA.

Estrogen-dependent extrauterine implantation and growth of menstrual endometrial tissue affects roughly 10% of reproductive age women and depends on suppression of local innate immune defenses to prevent ectopic tissue rejection. Immunohistochemistry has shown the immune check-point inhibitor CD200 which can suppress rejection is expressed in eutopic endometrium and in ectopic deposits. Soluble CD200 accumulated in venules draining eutopic and ectopic endometrium of endometriosis cases in the secretory phase but not proliferative phase of the menstrual cycle, and should be increased in the circulation. Sera from endometriosis and non-endometriosis controls were tested by ELISA for CD200. Endometrial CD200, CD200R1 and CD200R2 mRNA in eutopic was quantified by RT-PCR and localized by in situ hybridization. CD200R1 protein was quantified by immunohistochemistry. Secretory phase serum CD200 was elevated in women with endometriosis compared to controls. Serum CD200 correlated with matched endometrial CD200 mRNA levels. Expression of mRNA for CD200R1 which signals immune suppression was decreased whereas mRNA for the CD200R2 activating receptor was increased. In situ staining of CD200R1 and CD200R2 mRNA showed both receptors were expressed and the fraction of CD200R that is CD200R1 was reduced in secretory and menstrual phase endometriosis endometrium consistent with the RT-PCR result. By contrast, CD200R1 protein and CD200R1 fraction of total CD200R protein were increased in endometriosis. Failure to suppress circulating CD200 levels in the secretory phase had an 87% specificity and 90% sensitivity for endometriosis. CD200 and increased CD200R1 expression may facilitate development of ectopic deposits by suppressing rejection mechanisms.

O-031 Activation of NLRP3 inflammasome enhances neutrophil extracellular traps in women with stage III/IV endometriosis: convergence of multiple signaling portray association between genetics and lesion type

Abstract Study question Do NOD-like receptor pyrin-containing 3 (NLRP3) signaling affect neutrophil extracellular traps (NET)-osis in women with stage III/IV endometriosis? Summary answer Inflammasome activation whether due to pathogen (disease itself) or damage-associated molecular pattern (through NET) might be one mechanism involved in establishing perturbed endometrium in endometriosis. What is known already Endometriosis is characterized by number of processes like vascularization, hypernociception, and fibrosis, cardinal cause being inflammation. Recently, expression of inflammasome components, including NLRP3 and apoptosis signal-regulating kinase has been demonstrated in human endometrium cueing involvement in uterine innate immunity. Moreover, a novel extracellular killing mechanism, NET, is documented to reflect an inflammatory status in deep infiltrating endometriosis. Since, endometriosis demonstrates similarities with chronic inflammatory and autoimmune disorders; we postulated inflammatory responses in endometriosis may become modulated through a feed-forward loop of NET-induced specific cytokine production thus providing insights against potentiality of endometriotic cells to limit progression of the disease. Study design, size, duration Twenty-two consented women (24-39 years) with endometriosis (Group A) (Stages III–IV) (based on ASRM-2018 guidelines) and age-matched counterpart/s of male sub-fertility (Group B; control), free of uterine abnormalities (n = 18) were recruited between January to December 2021 from Institute of Reproductive Medicine, Kolkata. Eutopic endometrium, were collected from women undergoing diagnostic laparoscopy (Group A) or by curettage from women undergoing endometrial ablation (Group B). Serum was collected during window-of-implantation (LH + 7) for both the group/s. Participants/materials, setting, methods Pro-(IFN-g, TNF-a, IL-6, TGF-b) and anti-inflammatory (IL-10, IL-13, IL-4, IL-5) cytokines was evaluated by western blot. Expression level of inflammasome-related proteins (NLRP3, ASC, CASPASE1, PYCARD, IL-1-b) and mRNA expression was estimated by western-blot and quantitative-real-time PCR (qRT-PCR) respectively from tissue biopsies in Group A and B. SYTOX® green assay by flow-cytometry and neutrophil-elastase activity by immunofluorescence was done to quantify and characterize NET production in blood monocytes. P &amp;lt; 0.05 was considered statistically significant. Main results and the role of chance The mean (±SD) age of study population was 31.6±5.2 years. Hyperestrogenic milieu possibly stimulated (p &amp;lt; 0.001) pro-inflammatory molecules (IFN-g, TNF-a, IL-6, TGF-b) in endometriosis as observed by western-blot and qRT-PCR. A significant up-regulation (p &amp;lt; 0.001) was observed in relative mRNA expression of NLRP3 and PYCARD gene in Group A. However, caspase 1 expression documented non-significant variation in biopsies from endometriosis patients. Western blot corroborated the finding/s. The outcome of NLRP3 activation was supported by increased (p &amp;lt; 0.002) mRNA expression of IL-1b. NETs were detected significantly higher (p &amp;lt; 0.01) in 54.54% (12/22) patients in group A compared to control (16.66%; (3/18)). Moreover, quantification of NETs showed a significantly higher amount in endometriosis compared to group B (0.097 vs. 0.02, p &amp;lt; 0.03). Spermann-rank correlation by SPSS version22 revealed positive correlation between IL-1b with NLRP3 (r = 0.56, p&amp;lt; 0.001), PYCARD (r = 0.11, p&amp;lt; 0.01); and caspase1 (r = 0.16, p&amp;lt; 0.01) and IL-1b (r = 0.47, p&amp;lt; 0.01) with NET-positive cell/s in endometriosis. Moreover, posterior cul-de-sac lesions correlated positively with NET-positive cell/s (r = 0.29; p &amp;lt; 0.01) cueing to possible prognostic marker/s. In summary, a coordinated-fashioned action of components of NLRP3 inflammasome machinery may regulate production of NET/s forming a complex network allowing communication between cell-types in order to maintain viability and development of endometrial lesion/s. Limitations, reasons for caution Our findings need to be replicated in larger study cohort/s, especially stratified by severity of endometriosis sub-groups and in women of different ethnicities. Another limitation is the missing information about NETs in normal functioning human endometrium throughout the menstrual cycle. Wider implications of the findings The proposed study aims to understand potential role of NLRP3 inflammasome complex as a “double-edged sword” in the development and pathophysiology of endometriosis. Therapeutic alternatives that aim to re-balance the pro- to anti-inflammatory milieu in the endometrium should consider the inflammasome as part of the equation. Trial registration number Not applicable

Epigenomic Analysis Reveals the KCNK9 Potassium Channel as a Potential Therapeutic Target for Adenomyosis.

Adenomyosis is linked to dysmenorrhea and infertility. The pathogenesis of adenomyosis remains unclear, and little is known of the genetic and epigenetic changes in the eutopic endometrium in adenomyosis, which may predispose patients to the invasion and migration of endometrial tissues into the myometrium. Transcriptome studies have identified genes related to various cell behaviors but no targets for therapeutic intervention. The epigenetics of the eutopic endometrium in adenomyosis have rarely been investigated. Endometrial tissue was obtained from premenopausal women with (n = 32) or without adenomyosis (n = 17) who underwent hysterectomy aged 34–57 years at a tertiary hospital. The methylome and transcriptome were assessed by using a Methylation 450 K BeadChip array and Affymetrix expression microarray. Protein expression was examined by immunohistochemistry. Differential methylation analysis revealed 53 lowly methylated genes and 176 highly methylated genes with consistent gene expression in adenomyosis, including three genes encoding potassium ion channels. High expression of KCNK9 in the eutopic and ectopic endometria in patients with adenomyosis but not in normal controls was observed. Hormone-free, antibody-based KCNK9 targeting is a potential therapeutic strategy for adenomyosis-related dysmenorrhea, menorrhagia, and infertility.

Endometrial galectin‐3 causes endometriosis by supporting eutopic endometrial cell survival and engraftment in the peritoneal cavity

The pathogenesis of endometriosis remains unclear. Endometrial cells in retrograde menstruation are considered the source of endometriosis; therefore, we hypothesized that the eutopic endometrium may provide clues regarding the pathogenesis. We aimed to clarify the role of eutopic endometrial cells in endometriosis development. Eutopic endometrial tissues were obtained from patients with or without endometriosis, and expression of cell surface molecules in eutopic endometrial stromal cells (ESCs) was evaluated via iTRAQ-based proteomic analysis. Based on the results, we focused on galectin-3. Galectin-3 expression in clinical samples was confirmed by immunohistochemistry and Western blot analysis. The concentration of secreted galectin-3 was measured using enzyme-linked immunosorbent assays. Adhesion and migration of ESCs were evaluated by in vitro adhesion and wound healing assays. The cytotoxicity of natural killer cells was measured via calcein release assays. Cell proliferation was measured using the CyQUANT Cell Proliferation Assay Kit. iTRAQ analysis revealed that galectin-3 expression was specifically elevated in the ESCs from endometriosis patients. Immunohistochemistry confirmed galectin-3 overexpression in the eutopic endometrium of endometriosis, irrespective of the menstrual phase. Galectin-3 was overexpressed and secreted by the eutopic ESCs from patients with endometriosis compared to that from patients without endometriosis. Galectin-3 expression in ESCs increased adhesion and migration, whereas galectin-3 inhibitors impaired these processes. Galectin-3 reduced the cytotoxicity of natural killer cells toward ESCs, while not affecting cell proliferation. Galectin-3 promotes peritoneal engraftment of ESCs due to impaired immune surveillance in the peritoneal cavity and increases ESCs adhesion and migration to the peritoneum.

Comparative Proteomic Analysis Reveals Metformin Improves the Expression of Biomarkers of Endometrial Receptivity in Infertile Women with Minimal/Mild Endometriosis.

The prevalence of endometriosis is approximately 10% in women of reproductive age, and 30-50% of women with endometriosis are infertile. Metformin has been reported to inhibit the growth of ectopic lesions in endometriosis. However, its effect on the eutopic endometrium of endometriosis is unknown. This study aimed to identify whether metformin affects endometrial receptivity in infertile women with minimal/mild endometriosis. We enrolled 10 infertile women who were diagnosed with minimal/mild endometriosis through laparoscopy. Paired endometrial tissues of the secretory phase from participants were collected during surgery and after 2months of metformin treatment (n = 5) or no medical treatment (n = 5). Protein expression profiles of the paired endometrium were detected by proteomics and compared using the self-control method (2months later vs. in surgery). Proteomics data revealed six proteins associated with endometrial receptivity among the significantly upregulated proteins after metformin treatment (fold change > 1.5, P < 0.05). Insulin-like growth factor binding protein 7 (IGFBP-7) showed the most robust increase in these six endometrial receptivity-related proteins (fold change: 8.668, P < 0.05), while there was no significant change in the controls (fold change: 1.906, P > 0.05). The upregulation of IGFBP-7 has been validated through target proteomics, immunohistochemistry, and further demonstrated in endometriosis mouse models induced by autotransplantation. This study revealed that metformin upregulated the expression of IGFBP-7 in the endometrium of human and mouse models of endometriosis. Metformin potentially affects endometrial receptivity of minimal/mild endometriosis by improving the expression of the endometrial receptivity marker IGFBP-7.

Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment

BackgroundAdenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood.MethodsEutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry.ResultsA total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis.ConclusionThis study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

Letrozole promotes the expression of integrin αvβ3 and HOXA10 in endometrium of endometriosis

ABSTRACT Endometriosis is a common estrogen-dependent chronic inflammatory disease that leads to infertility in women of reproductive age. Perhaps infertility reflects the reduced expression of integrin αvβ3 and HOXA10 in endometriosis. Previous studies have shown that administration of letrozole, a non-steroidal aromatase inhibitor for cancer treatment, increased the clinical pregnancy rate in women with endometriosis, but the mechanisms remain to be determined. In this communication, a rat model of endometriosis was established. Animals were treated with letrozole at 2ug/kg of body weight, intragastric administration for 15 consecutive days. Letrozole increased the expression of αvβ3 and HOXA10 in the endometriosis model and endometrial receptivity. Abbreviations: WOI: window of implantation; RGD: Arg-Gly-Asp; HOX: homeobox; E2: estradiol; SPF: specific pathogen-free.

Transcriptomic Analysis of Endometrium in Patients with Adenomyosis and Endometriosis

<h3>Study Objective</h3> to analyze specific gene expression patterns and signaling pathways activation level in endometrial samples of patients with adenomyosis and endometriosis. <h3>Design</h3> Retrospective cohort study. <h3>Setting</h3> University Clinic. <h3>Patients or Participants</h3> 50 women with endometriosis, 20 women with adenomyosis, 35 women with tubal factor infertility (control group). <h3>Interventions</h3> Laparoscopic excision of endometriotic foci and adenomyomas, hysteroscopy with endometrial sampling. RNA was isolated from all samples and stored in RNA Later. Following histologic analysis of all samples, RNA sequencing was performed using Illumina HiSeq 3000 equipment for single-end sequencing. We normalized all gene expression and pathway activation profiles of studied samples on endometrial samples of women from the control group, and then proceeded with identification of strongly differentially expressed genes and activated signaling pathways. <h3>Measurements and Main Results</h3> We performed transcriptomic analysis of 50 endometrial samples of patients with endometriosis, 20 endometrial samples of patients with adenomyosis, and 35 endometrial samples from patients of control group. Statistically significant difference in transcriptomic profiles of endometrium of women with endometriosis and adenomyosis was found. Top-10 most strongly similarly and differentially expressed genes and up- and down-activated signaling pathways were identified. <h3>Conclusion</h3> Our findings suggest that both adenomyosis and endometriosis are associated with specific molecular changes in endometrium. Such data could be potentially used for early diagnosis of adenomyosis, even when it is missed on ultrasonography. Moreover, identification of molecular differences between endometrium in adenomyosis and endometriosis can have important implications on understanding of the etiological aspects and can help in identifying novel drug targets.

Oestrogen up-regulates DNMT1 and leads to the hypermethylation of RUNX3 in the malignant transformation of ovarian endometriosis

Research questionWhat is the mechanism of hypermethylation of runt-related transcription factor 3 (RUNX3) in the eutopic endometrium of endometriosis as biomarker in the malignant transformation of endometriosis? DesignMethylation-specific polymerase chain reaction was used to analyse the methylation status of RUNX3 in endometriosis-associated ovarian cancer (EAOC). Primary eutopic endometrial stromal cells (ESC) were isolated from the uteri of patients with ovarian endometriosis. After RUNX3 knockdown by RNA interference technology or ESC treated with oestradiol, the proliferation and invasion ability were evaluated in ESC by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and transwell assays. ResultsThe frequency of methylation of RUNX3 in neoplastic tissue in the EAOC group was significantly higher than that in the ectopic endometrium of the endometriosis group (P < 0.001), and the frequency of methylation of RUNX3 in the eutopic endometrium of the EAOC group was significantly higher than that in the endometriosis group (P < 0.001). However, there was no significant difference in the eutopic endometrium when compared between the endometriosis group and the control endometrium group (P = 0.233). Silencing RUNX3 promoted the proliferation and invasion of ESC (P < 0.001 and P < 0.001). Following intervention with oestrogen, it was observed that the oestradiol group showed higher levels of RUNX3 methylation (P < 0.001) and DNA methyltransferase 1 (DNMT1) mRNA and protein expression (P < 0.001 and P < 0.001), and lower RUNX3 mRNA and protein expression when compared with the ESC group (P < 0.001 and P < 0.001). ConclusionThis study demonstrated that hypermethylation of the RUNX3 was related to the malignant transformation of endometriosis and that this process was related to corresponding changes in the eutopic endometrium. Furthermore, the ‘oestrogen-DNMT1’ signalling pathway may induce the hypermethylation of RUNX3 to promote the malignant transformation of endometriosis.

Protein arginine methyltransferase 5 mediates THP‑1‑derived macrophage activation dependent on NF‑κB in endometriosis

Macrophage-induced inflammation is a major factor in the pathogenesis of endometriosis. The underlying mechanisms, however, remain largely unknown. TNF-α, IL-6, IL-10 and C-C motif chemokine 20 (CCL20) levels in endometrial extracts were determined using Luminex cytokine kits. Additionally, protein arginine methyltransferase 5 (PRMT5) levels were measured using reverse transcription-quantitative PCR and western blotting. IL-6 and IP-10 levels in cells were measured using ELISA kits. In the present study, it was revealed that PRMT5 expression at both the mRNA and protein levels in THP-1-derived macrophages was significantly decreased following treatment with serum or extracts of endometrium from patients with endometriosis in the presence of lipopolysaccharide, compared with that in control cells, suggesting a possible role for macrophage-derived PRMT5 in mediating the interaction between macrophages and endometrium in endometriosis. Mechanistically, macrophage PRMT5 expression was regulated in an NF-κB-dependent and Smad2/3-independent manner, indicating that PRMT5 is a downstream target of NF-κB. Importantly, macrophage-derived PRMT5 was required for macrophage activation in endometriosis, as evidenced by the PRMT5-dependent secretion of IL-6 and IFN-γ-induced protein 10 from THP-1-derived macrophages. The present study identified NF-κB-dependent PRMT5 as a novel regulator of macrophage activation in endometriosis. Targeting PRMT5 in macrophages may be a potential therapeutic strategy against endometriosis.

E3 ubiquitin ligase TRIM24 deficiency promotes NLRP3/caspase-1/IL-1β-mediated pyroptosis in endometriosis.

Endometriosis is an inflammation-dependent disease that shares similarities with malignant tumors including attachment and infiltration. Tripartite motif-containing 24 (TRIM24) has been illustrated in inflammatory responses and gynecological tumors, and Nod-like receptor protein 3 (NLRP3) inflammasome has been implicated in endometriosis. However, the involvement of TRIM24 and the role of NLRP3/caspase-1/interleukin-1β (IL-1β)-mediated pyroptosis in endometriosis remain obscure. In this study, we originally detected the decreased expression of TRIM24 in the ectopic endometrium of endometriosiscompared with the normal endometrium. Then we measured the promoted protein expression of pyroptotic biomarkers (NLRP3, procaspase-1, caspase-1, pro-IL-1β, and IL-1β) using Western blot analysisand the stimulated secretion of IL-1β and IL-18 by enzyme-linked immunosorbent assayin ectopic human endometrial stromal cells (hESC) compared with normal hESC. TRIM24-small-interfering RNA (siTRIM24) was used to silence TRIM24, whereas TRIM24-pcDNA3.1 was used for overexpressing TRIM24. The migration of hESC was determined by a Transwell migration assay. Coimmunoprecipitation and ubiquitination analyses were conducted to explore the interaction between TRIM24 and NLRP3. Subsequently, we found that TRIM24 negatively regulated NLRP3/caspase-1/IL-1β-mediated pyroptosis and cell migration of hESC, and CY-09, the specific inhibitor of NLRP3, could reverse the promoted pyroptosis and cell migration induced by siTRIM24. Furthermore, TRIM24 interacted with NLRP3 and the upregulation of TRIM24 facilitated the ubiquitination of NLRP3 in ectopic hESC. Our findings suggest that TRIM24 may participate in the progression of endometriosis through the NLRP3/caspase-1/IL-1β-mediated pyroptotic pathway via ubiquitination of NLRP3, which reveals the significant molecular mechanism underlying endometriosis.

Talin1 Induces Epithelial-Mesenchymal Transition to Facilitate Endometrial Cell Migration and Invasion in Adenomyosis Under the Regulation of microRNA-145-5p.

Adenomyosis (ADS) is a commonly encountered benign gynecological disorder. Epithelial-mesenchymal transition (EMT) may serve a pivotal role in the pathogenesis of ADS. Talin1 has been identified to be implicated in multiple human carcinomas, probably through inducing EMT process. However, available data on the precise molecular mechanism of Talin1 in the pathogenesis of ADS remain extremely scanty. In the present study, we aim to investigate the clinical roles of Talin1 and its effects on uterine endometrial cell migration, invasion, and EMT in ADS. Relative mRNA expression of Talin1, microRNA-145-5p (miR-145-5p), and EMT-related markers was determined by qRT-PCR. Immunohistochemistry and immunofluorescence were performed to examine the distribution of Talin1 in ADS endometrium. Protein levels of Talin1, EMT-related markers, and wnt/β-catenin pathway were measured by western blot. Wound healing assay and transwell assay were utilized for evaluating cell migration and invasion respectively. Dual-luciferase reporter assay was performed to verify the relationship between Talin1 and miR-145-5p. We found Talin1 was markedly overexpressed in ADS endometrial tissue and cells, whereas miR-145-5p was downregulated. Elevated Talin1 mRNA level might be closely related to some clinicopathological features of ADS. Through functional experiments, we demonstrated that overexpression of Talin1 induced EMT and enhanced migration and invasion ability of ADS eutopic and ectopic endometrial epithelial cells (ADS_Eu_EEC and ADS_Ec_EEC) in vitro through activating the canonical wnt/β-catenin pathway. From a mechanistic perspective, Talin1 was inversely regulated by miR-145-5p as a direct target. Our findings unveiled that under the regulation of miR-145-5p, Talin1 might promote endometrial cell migration and invasion through inducing EMT, presenting a novel insight for elucidating the pathogenesis of ADS.

DIFFERENT PROTEOMICS ANALYSIS INDICATES METFORMIN AS A NOVEL THERAPY TO AMELIORATE ENDOMETRIAL RECEPTIVITY OF ENDOMETRIOSIS

Endometriosis reduces female fecundity, decreased endometrial receptivity should be responsible for the pathogenesis. Metformin inhibited the growth of ectopic lesions, while the effect of metformin on eutopic endometrium of endometriosis has not been reported. This study aims to identify whether metformin can ameliorate endometrial receptivity of women with endometriosis and endometriosis model mice.

Molecular signature of eutopic endometrium in endometriosis based on the multi-omics integrative synthesis

PurposeTo synthesise data from genome-wide studies reporting molecular signature of eutopic endometrium through the phases of the menstrual cycle in endometriosis.MethodsExtraction of data from publications reporting genetic signatures characterising endometrium associated with endometriosis. The nomenclature of extracted differentially expressed transcripts and proteins was adopted according to the HUGO Gene Nomenclature Committee (HGNC). Loci were further sorted according to the different phases of the menstrual cycle, i.e. menstrual (M), proliferative (P), secretory (S), early-secretory (ES), mid-secretory (MS), late-secretory (LS), and not specified (N/S) if the endometrial dating was not available. Enrichment analysis was performed using the DAVID bioinformatics tool.ResultsAltered molecular changes were reported by 21 studies, including 13 performed at the transcriptomic, 6 at proteomic, and 2 at epigenomic level. Extracted data resulted in a catalogue of total 670 genetic causes with available 591 official gene symbols, i.e. M = 3, P = 188, S = 81, ES = 82, MS = 173, LS = 36, and N/S = 28. Enriched pathways included oestrogen signalling pathway, extracellular matrix organization, and endothelial cell chemotaxis. Our study revealed that knowledge of endometrium biology in endometriosis is fragmented due to heterogeneity of published data. However, 15 genes reported as dysregulated by at least two studies within the same phase and 33 significantly enriched GO-BP terms/KEGG pathways associated with different phases of the menstrual cycle were identified.ConclusionsA multi-omics insight into molecular patterns underlying endometriosis could contribute towards identification of endometrial pathological mechanisms that impact fertility capacities of women with endometriosis.