Eutopic Endometrial Cells Research Articles

Extracellular vesicle-encapsulated miR-25-3p promotes epithelial-mesenchymal transition and migration of endometrial epithelial cells by inducing macrophage polarization.

The pathogenesis of adenomyosis is closely related to the epithelial-mesenchymal transition and macrophages. MicroRNAs have been extensively investigated in relation to the epithelial-mesenchymal transition in a range of malignancies. However, there is a paucity of research on extracellular vesicles derived from the eutopic endometrium of adenomyosis and their encapsulated microRNAs. In this study, we investigated the role of microRNA-25-3p derived from extracellular vesicles in inducing macrophage polarization and promoting the epithelial-mesenchymal transition in endometrial epithelial cells of patients with adenomyosis and controls. We obtained eutopic endometrial samples and isolated extracellular vesicles from the culture supernatant of primary endometrial cells. Real-time quantitative PCR analysis demonstrated that microRNA-25-3p was highly expressed in extracellular vesicles, as well as in macrophages stimulated by extracellular vesicles from eutopic endometrium of adenomyosis; and macrophages transfected with microRNA-25-3p exhibited elevated levels of M2 markers, while displaying reduced levels of M1 markers. After co-culture with the above polarized macrophages, endometrial epithelial cells expressed higher levels of N-cadherin and Vimentin, and lower protein levels of E-cadherin and Cytokeratin 7. It was revealed that microRNA-25-3p encapsulated in extracellular vesicles from eutopic endometrial cells could induce macrophage polarization toward M2, and the polarized macrophages promote epithelial-mesenchymal transition in epithelial cells. However, in vitro experiments revealed no significant disparity in the migratory capacity of endometrial epithelial cells between the adenomyosis group and the control group. Furthermore, it was observed that microRNA-25-3p-stimulated polarized macrophages also facilitated the epithelial-mesenchymal transition and migration of endometrial epithelial cells within the control group. Thus, the significance of microRNA-25-3p-induced polarized macrophages in promoting the development of adenomyosis is unclear, and macrophage infiltration alone may be adequate for this process. We emphasize the specificity of the local eutopic endometrial microenvironment and postulate its potential significance in the pathogenesis of adenomyosis.

Endometrial cell‑derived exosomes facilitate the development of adenomyosis via the IL‑6/JAK2/STAT3 pathway.

Interleukin (IL)-6 upregulation is involved in the pathogenesis of adenomyosis, but the underlying mechanism remains to be elucidated. Exosomes mediate intercellular communication, therefore the present study investigated whether endometrial cell-derived exosomes mediated the crosstalk between the endometrium and the myometrium via IL-6 signaling. Primary adenomyotic myometrial (AM) cells and eutopic endometrial cells were isolated from patients with adenomyosis. Exosomes were obtained from endometrial cells and incubated with AM cells in the presence or absence of tocilizumab (an IL-6 inhibitor). MTT, flow cytometry and wound-healing assays were performed to examine AM cell proliferation, apoptosis, cell cycle distribution and migration. Western blotting and reverse transcription-quantitative PCR were conducted to determine the expression of the IL-6/Janus kinase 2 (JAK2)/STAT3 pathway proteins. Incubation with endometrial cell exosomes suppressed cell apoptosis of AM cells compared with controls, accompanied by increases in IL-6 production and JAK2/STAT3 phosphorylation. Endometrial cell exosomes promoted cell proliferation, increased the percentage of S-phase cells and enhanced the migration of AM cells. These effects were completely reversed by tocilizumab, along with substantial decreases in IL-6 production and JAK2/STAT3 phosphorylation. Endometrial cell-derived exosomes promote cell proliferation, migration and cell cycle transition of AM cells through IL-6/JAK2/STAT3 activation, facilitating the development of adenomyosis by mediating the crosstalk between the endometrium and the myometrium, and IL-6 targeted therapy could be a complementary approach against adenomyosis.

P-316 Involvement of collective cell migration in endometrial invasiveness in uterine adenomyosis and the role of M2 macrophages

Abstract Study question How do endometrial cells manage to invade surrounding myometrium to establish adenomyotic lesions and what is the role of macrophages in this process? Summary answer M2 macrophage-mediated invasiveness and resistance of endometrial cells to physiological cell death appear to contribute to the pathogenesis of adenomyosis. What is known already Despite the high prevalence and debilitating symptoms of uterine adenomyosis, its pathogenesis has not yet been elucidated. To date, the most acceptable theory to explain disease development suggests invasion of the myometrium by eutopic endometrial cells. Recent data suggest aberrant infiltration of immune cells but not platelets in adenomyosis, eventually leading to enhanced cell motility via collective cell migration (CCM) and resistance to physiological cell death during disease pathogenesis. Study design, size, duration A retrospective immunohistochemistry-based study was conducted on formalin-fixed paraffin-embedded uterine tissue from 17 women (8 adenomyosis patients and 9 healthy subjects). Fresh endometrial biopsies were retrieved from 16 women (6 adenomyosis patients and 10 healthy subjects) for in vitro culture of epithelial and stromal cells, and co-culture with THP-1 monocyte-derived M2 macrophages. Participants/materials, setting, methods Double immunofluorescence was performed to detect macrophages of M1 and M2 phenotypes. Invasiveness of endometrial cells upon interaction with macrophages was assessed by in vitro invasion assays and quantitative PCR for genes involved in cell motility and epithelial-mesenchymal transition (EMT). Mechanisms of disease progression were investigated by immunohistochemistry against E-cadherin, N-cadherin and matrix metalloproteinase 9 (MMP9). Caspase 3 and microtubule-associated protein light chain 3 beta (LC3B) were studied as markers of apoptosis and autophagy respectively. Main results and the role of chance Only M2 macrophages were found to accumulate in adenomyosis, in higher numbers in both endometrium and ectopic lesions from adenomyosis patients than healthy controls. Co-culture with M2 macrophages significantly increased invasion capacity in endometrial epithelial and stromal cells from both adenomyosis patients and healthy controls. Epithelial cells from adenomyosis patients were nonetheless more invasive than their healthy counterparts in the presence of M2 macrophages. No gene expression differences pointing to EMT were noted, either between co-cultured and control cells, or between adenomyotic and healthy cells, refuting the implication of this mechanism in adenomyosis invasiveness. E- and N-cadherin protein expression did not differ significantly between eutopic endometrium from adenomyosis subjects and healthy tissue, but N-cadherin expression was stronger in ectopic lesions. In adenomyosis, both E- and N-cadherin were more extensively expressed in basal (also known as invasive) glands than functional glands, suggesting maintenance of intercellular junctions and subsequent CCM. Extracellular matrix-degrading enzyme MMP9 was more abundantly expressed in eutopic stroma from adenomyosis patients compared to healthy tissue. Lower levels of caspase 3 were detected in both endometrium and lesions from adenomyosis patients, while LC3B was found significantly decreased in adenomyotic stroma compared to that of healthy subjects. Limitations, reasons for caution A limitation of the study is the 2D nature of in vitro cultures, which cannot fully reconstitute in vivo cell arrangement and cell-cell interactions, nor allow to observe CCM in real time. Wider implications of the findings M2 macrophages accumulating in endometrium and ectopic lesions from adenomyosis patients boost invasion capacity of endometrial epithelial and stromal cells, as shown by in vitro co-culture experiments. These findings indicate that M2 macrophages play a key role in disease pathogenesis and could be targeted to develop novel therapeutic options. Trial registration number not applicable

Targeting YAP1 ameliorates progesterone resistance in endometriosis

Does YAP1 inhibition alleviate progesterone resistance in endometriosis? YAP1 inhibition reduces progesterone resistance in vitro and in vivo. Progesterone resistance not only causes treatment failure for endometriosis but also inhibits eutopic endometrial cell proliferation, dysregulates decidualization, and reduces the success rates of pregnancy. Hippo/yes-associated protein 1 (YAP1) signaling pathway plays an important role in the pathogenesis of endometriosis. Paraffin-embedded tissues containing paired endometriotic and endometrial specimens (n = 42) and serum samples isolated from normal controls (n = 15) or endometriotic patients with (n = 25) or without (n = 21) prior dienogest treatment were analyzed. A mouse model of endometriosis was also used to evaluate the effects of YAP1 inhibition on progesterone resistance. Primary endometriotic and endometrial stromal cells treated with YAP1 inhibitor or miR-21 mimic/inhibitor were used for the in vitro studies including decidualization induction, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation. Tissue specimens and serum from human and mouse were used for immunohistochemistry staining, exosome isolation, and microRNA (miRNA) quantification, respectively. Herein, we report, by using ChIP-PCR and RNA-IP, that YAP1 inhibits progesterone receptor (PGR) expression through upregulation of miR-21-5p. Upregulation of miR-21-5p not only reduces PGR expression but also inhibits endometrial stromal cell decidualization. Indeed, levels of YAP1 and miR-21-5p are inversely correlated with the level of PGR in human endometrial samples. In contrast, knockdown of YAP1 or treatment with verteporfin (VP), a YAP1 inhibitor, reduces miR-21-5p expression, thus leading to an increase in PGR expression in ectopic endometriotic stromal cells. In the mouse model of endometriosis, treatment with VP increases PGR expression and enhances decidualization. More importantly, VP synergistically increases the treatment effect of progestin in causing the regression of endometriotic lesions and improves the decidualization capability of the endometrium. Interestingly, treatment with dienogest, a synthetic progestin, reduces YAP1 and miR-21-5p expression in human cells and in the mouse model of endometriosis. Patients who received dienogest treatment for 6 months show a significant decrease in serum extracellular vesicle-associated miR-21-5p level. A public dataset (GSE51981) containing a large cohort of endometriotic tissues is available from the Gene Expression Omnibus (GEO). A large cohort of clinical samples is needed to verify the current diagnostic value of miR-21-5p in future studies. The reciprocal regulation of YAP1 and PGR suggests that combined YAP1 inhibitor and progestin may be a better therapeutic approach for treating endometriosis. This study was supported by the Ministry of Science and Technology, Taiwan (MOST-111-2636-B-006-012, MOST-111-2314-B-006-075-MY3, and MOST-106-2320-B-006-072-MY3). The authors have no conflict of interest to disclose.

Identification of key modules and candidate genes associated with endometriosis based on transcriptome data via bioinformatics analysis

BackgroundsEndometriosis is a common disease in women, but the signaling pathways and genes involved remain unclear. This study screened genes that were differentially expressed in ectopic endometrium (EC) and eutopic endometrium (EU) in endometriosis and provided clues for subsequent experimental verification. MethodsEndometriosis samples were harvested from inpatients that underwent surgery from 2017 to 2019 with pathological evidence of endometriosis. We assessed the mRNA expression profiles in endometriosis and further conducted gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, Gene Set Enrichment Analysis (GSEA) and weighted gene co-expression network analysis (WGCNA) to identify potential biomarkers in endometriosis. Finally, we further validated hub genes using public databases and immunohistochemistry assays. ResultsThe upregulated DEGs of ectopic endometrium from endometriosis patients were mainly involved in cell adhesion, MAPK signaling, PI3K-Akt signaling pathways, cytokine receptor interactions, and epithelial-mesenchymal transformation (EMT)-associated signaling pathways. The downregulated DEGs between ectopic endometrium and eutopic endometrium were related to decidualization-associated genes in endometriosis. The correlated gene modules in eutopic endometrial cells were mainly enriched in cell adhesion, embryo implantation and inflammation. The eutopic and ectopic endometrial lesions in endometriosis were involved in the EMT process. Furthermore, we identified 18 co-expression modules during WGCNA analysis. Hub genes in the pale turquoise module were FOSB, JUNB, ATF3, CXCL2, FOS, etc. Significantly enriched KEGG pathways included the TNF, MAPK, foxO, oxytocin, and p53 signaling pathways. Enrichment pathways were directly related to immune surveillance, stem cell self-renewal, and epithelial-mesenchymal transformation. Several pathways and modules of endometriosis are related to cancer-associated pathways, which substantiates the correlation between endometriosis and various gynecological tumors. ConclusionsEndometriosis was tightly correlated with EMT and fibrosis mediated by inflammatory immunity, cytokines, estrogen, kinases and protooncogene through transcriptomics. Overall, our findings lay the groundwork for understanding the pathogenesis of endometriosis and its relationship with malignant transformation.

Influence of the ectopic location on the antigen expression and functional characteristics of endometrioma stromal cells

Are the alterations observed in the endometriotic cells, such as progesterone resistance, already present in the eutopic endometrium or acquired in the ectopic location? The response to decidualization with progesterone and cyclic AMP for up to 28 days was compared in different endometrial stromal cell (EnSC) lines established from samples of endometriomas (eEnSC), eutopic endometrium from women with endometriosis (eBEnSC), endometrial tissue from healthy women (BEnSC) and menstrual blood from healthy donors (mEnSC). Usual features of decidualized cells, such as changes in cell morphology and expression of prolactin, were similarly observed in the three types of eutopic EnSC studied, but not in the ectopic cells upon decidualization. Among the phenotypic markers analysed, CD105 was down-regulated under decidualization in all cell types (mEnSC, P = 0.005; BEnSC, P = 0.029; eBEnSC, P = 0.022) except eEnSC. mEnSC and BEnSC underwent apoptosis during decidualization, whereas eBEnSC and eEnSC were resistant to the induction of cell death. Lastly, migration studies revealed that mEnSC secreted undetermined factors during decidualization that inhibited cell motility, whereas eEnSC showed a significantly lower ability to produce those migration-regulating factors (P < 0.0001, P < 0.001 and P = 0.0013 for the migration of mEnSC at 24, 48 and 72 h, respectively; P < 0.0001 for the migration of eEnSC at all times studied). This study provides novel insights into the differences between endometriotic and eutopic endometrial cells and reinforces the idea that the microenvironment in the ectopic location plays additional roles in the acquisition of the alterations that characterize the cells of the endometriotic foci.

Characteristics of endometrial hormonal homeostasis and receptor apparatus in women with adenomyosus who had papillary thyroid carcinoma

Objectives: to evaluate the hormonal status and receptor apparatus of the eutopic endometrium in patients with adenomyosis who had a history of papillary thyroid carcinoma.Materials and methods. 63 women were examined: group I consisted of 31 patients with adenomyosis and papillary carcinoma of the thyroid gland in history, group II consisted of 32 patients with adenomyosis and unencumbered thyroid status. The severity of pelvic pain was assessed using a visual analog scale. The level of luteinizing and follicle-stimulating hormones, estradiol, prolactin, thyroid-stimulating hormone and progesterone was determined in the peripheral blood serum. The material for the morphological study was obtained using endometrial pipelle biopsy. Morphological research was performed on 30 biopsies of eutopic endometrium (15 samples from patients of group I and 15 samples from patients of group II). Immunohistochemical study was performed on 20 paraffin sections (10 samples from patients of group I and 10 samples from patients of group II) using monoclonal antibodies.Results. High ER-α expression was detected in the endometrial glandular epithelial cells (EGECs) in 80 and 50% of samples of patients from groups I and II, respectively (р &lt; 0.05), no significant difference in the number of positive cells was found between groups. High ER-α expression in endometrial stromal cells (ESCs) was detected in 50% of samples in patients from both groups, the number of positive cells was significantly higher in the endometrium specimens from I group (84.0 (10.5%) in group I versus 62.2 (12.3%) in group II, р &lt; 0.05). High PgR expression in the EGECs was detected in 90 and 75% of samples in groups I and II respectively (р &lt; 0.05), ESCs expressed PgR in 100% of samples of patients from both groups. Significant difference in the number of positive cells was found between groups – 96.0 (8.4%) and 84.9 (12.6%) in groups I and II respectively, р &lt; 0.05.Conclusions. Our results suggest that the ectopic endometrium in female thyroid cancer survivors with adenomyosis has high expression of ER and PgR, that may have important implications for the survival and proliferation of the eutopic endometrial cells. Further research is needed to optimise prevention and treatment algorithms for this group of patients.

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.

SINGLE CELL ANALYSIS REVEALS HETEROGENEITY IN THE DISEASE RELEVANT CELL TYPES OF ADENOMYOSIS

To identify cellular composition and gene expression profiles of the cell types in adenomyotic tissue, eutopic endometrium, and normal myometrium of the women with diffuse adenomyosis. Adenomyotic tissue (n=3), eutopic endometrium (n=3), and myometrium (n=3) were obtained from premenopausal women who underwent hysterectomy for diffuse adenomyosis and confirmed by frozen section histologic evaluation. Following digestion, the stromal and smooth muscle cells were collected, and the epithelial cell pellets were further digested. After checking the viability, which was at least 75%, samples subjected the 10x Genomics® Single Cell Protocol. Processed data from the Cell Ranger pipeline was analyzed using the R package Seurat (v3.2). Uniform Manifold Approximation and Projection was generated to visualize cell clusters. The Wilcoxon rank sum test was used to compare the gene expression profile for each cluster which were annotated by marker genes. We identified ciliated and unciliated epithelial cells, fibroblast-like cells, smooth muscle cells (SMC), endothelial cells, pericytes, vascular SMCs, progenitor cells, T cells, NK cells, B cells, MQ, and monocytes, neurons and myeloid progenitors in the adenomyotic uterus. Notably, comparative transcriptome analysis of epithelial cells in eutopic endometrium and adenomyotic tissue revealed a small subset of differentially expressed genes, indicating the stability of the transcriptional program of these cells. On the other hand, we identified a high degree of heterogeneity among the fibroblast populations. Intriguingly, fibroblast-like cells in adenomyotic vs eutopic endometrial tissue showed higher expression of progesterone-responsive genes. In contrast, eutopic endometrial epithelial cells displayed higher expression of progesterone-responsive genes. Pseudo-time analysis in eutopic endometrium have demonstrated evidence of both epithelial-mesenchymal and mesenchymal-epithelial transition originating from a progenitor cell population and pericytes, respectively. Our analyses revealed 15 distinct cell populations in adenomyotic tissue. Fibroblast-like cells constituted the majority of all cell types which showed a high degree of heterogeneity, which may be due to different transcriptional states linked to the intense inflammatory processes in adenomyosis. Our results suggest that progesterone response may vary between cell types in adenomyotic tissue. The lack of a substantial difference between epithelial cells in adenomyosis and adjacent endometrium supports the postulated mechanism whereby invagination of eutopic endometrial tissue is responsible for adenomyosis.

M1 Macrophage-Derived Nanovesicles Repolarize M2 Macrophages for Inhibiting the Development of Endometriosis.

BackgroundEndometriosis is a common nonmalignant gynecological disorder that affects 10–15% women of reproductive age and causes several symptoms that result in decreased quality of life and a huge social burden. In recent decades, extracellular vesicles (EVs) have gained attention as a potential therapeutic tool; however, the therapeutic effects of EVs against endometriosis have not been reported. Accordingly, in this study, we investigated the feasibility of nanovesicles (NVs) derived from M1 macrophages (M1NVs) in treating endometriosis.MethodsM1NVs were prepared by serial extrusion. Co-culture assays were performed to investigate changes in tube formation and migration/invasion of eutopic endometrial stroma cells (ESCs) obtained from patients with endometriosis (EM-ESCs). A mouse model of endometriosis was established, and mice were treated with phosphate-buffered saline, M0NVs, or M1NVs to evaluate the efficacy and safety of M1NV for treating endometriosis.ResultsM1NVs directly or indirectly inhibited the migration and invasion of EM-ESCs and reduced tube formation. In the mouse model, M1NVs suppressed the development of endometriosis through reprogramming of M2 macrophages, without causing damage to the organs.ConclusionsM1NVs inhibit the development of endometriosis directly, or through repolarizing macrophages from M2 to M1 phenotype. Hence, administration of M1NVs may represent a novel method for the treatment of endometriosis.

Endometriosis: New Perspective for the Diagnosis of Certain Cytokines in Women and Adolescent Girls, as Well as the Progression of Disease Outgrowth: A Systematic Review.

Endometriosis is a common chronic gynecological disorder that undoubtedly impacts on quality of life, and is one of the more complex and mysterious illnesses of our century, which is associated with the improper growth of endometrial tissue outside of the uterine cavity. This pathologically implanted tissue can be found most frequently in the minor pelvis, but also in the peritoneal cavity, and can affect many organs, leading to chronic pelvic pain syndrome, infertility, and dysmenorrhea. Endometrial tissue is a particularly dynamic tissue that has a direct impact on the progression of the disease, with altered immunity, as well as cytokine storms within the metaplastic endometriotic site, as possible key factors. Currently, diagnosis of this mysterious chronic illness relies on performing a laparoscopic procedure with tissue sampling. One of the most troublesome outcomes of this unintended progression is that we lack any specific, sensitive, non-invasive diagnostic tools. Currently, the vast majority of regime stewardship options rely on anti-contraceptive drugs, or other remedies that suppress the release of estrogen through the gonads—although in most clinical trials, endometriosis is a chronic progressive disorder that depends mostly on the high concentration of estrogen. Moreover, many specific trials have demonstrated that the eutopic endometrial cells in individuals with endometriosis remain much more resistant to the immunological annihilation process caused by certain elements of the immune system. Nevertheless, eutopic endometrial cells have the potential to similarly escalate the expression of aromatase receptors on the surface of the pathological cells, which in the final cascade cause an increase in the concentration of estrogen, as well as other inflammatory proteins that contribute to pathological outgrowth. Data reveal occurrence among first-degree relatives, suggesting that the specific cascade could be related to inherited as well as epigenetic (acquired) mechanisms. In women with the disease, confirmed by laparoscopic procedures, diagnosis of endometriosis can be established also via detection by gene polymorphism in the genes which are responsible for responsible for the detoxification phase of estrogen receptors and other immunomodulator components. A recent publication aims to reveal a new prospect for the non-invasive diagnosis, detection, and estimation of certain biomarkers for much more specific investigation of the disease’s progression.

Analysis of differences in the transcriptomic profiles of eutopic and ectopic endometriums in women with ovarian endometriosis.

BackgroundEndometriosis is a common gynecological disease among women in their reproductive years. Although much effort has been made, the pathogenesis of this disease and the detailed differences between eutopic endometrial cells and ectopic endometrial cells are still unclear.MethodsIn this study, eutopic and ectopic endometrial cells were collected from patients with and without endometriosis and RNA sequencing was performed. The gene expression patterns and differentially expressed genes (DEGs) in eutopic and ectopic endometrial cells, as well as control endometrial cells, were analyzed using a weighted gene co-expression network analysis (WGCNA) and the DESeq2 package. The functions of significant genes were detected using Gene ontology (GO) enrichment analysis, and qRT-PCR validation was performed.ResultsThe results indicated that eight gene modules were found among these three groups. They also indicated that the gene module, which is highly related to eutopic endometrial cells, was mainly enriched in cell adhesion, embryo implantation, etc., while the gene module related to ectopic endometrial cells was mainly enriched in cell migration, etc. The results of differential expression analysis were generally consistent with the WGCNA results through identified significant DEGs between different groups. These DEGs may play an important role in the occurrence of endometriosis, including the infertility associated gene ARNTL and PIWIL2, tissue remodeling gene MMP11, cell survival and migration gene FLT1, inflammatory response gene GNLY, the tumor suppressor genes PLCD1, etc. Further analysis suggested the function of adhesion is stronger in ectopic endometrial cells than in eutopic endometrial cells, while the ectopic endometrium may have a higher potential risk of malignant transformation than eutopic endometrium.ConclusionsOverall, these data provide a reference for understanding the pathogenesis of endometriosis and its relationship with malignant transformation.

Antioxidant ameliorative effect of caffeic acid on the ectopic endometrial cells separated from patients with endometriosis

ObjectiveConsidering the role of oxidative stress in the development and progression of endometriosis, the ameliorative effect of caffeic acid treatment on ectopic, eutopic endometrial cells enzyme activities was investigated. We also determined the underlying cellular mechanisms. Materials and methodsEctopic endometrial specimens were collected from women with confirmed cases of endometriosis (n = 10) and eutopic specimens from (n = 10) controls. Following endometrial cell isolation and culture, eutopic and ectopic endometrial cells were treated with caffeic acid. Then, reactive oxygen species (ROS) level, NAD(P)H quinone oxidoreductase 1 (NQO1), and Heme oxygenase 1 (HO-1) enzyme activities, nuclear factor erythroid 2-related factor 2 (Nrf-2) gene expression were measured. ResultsIn ectopic endometrial cells, caffeic acid caused a significant elevation in Nrf-2 gene expression level, NQO1, and HO-1 enzyme activities. In addition, reduced ROS level was observed in caffeic acid-treated ectopic endometrial cells in comparison with the control. On the contrary, we did not observe any significant changes in caffeic acid-treated eutopic endometrial ones. ConclusionCaffeic acid can protect the endometrial cells against oxidative stress and might be able to prevent the progression of endometriosis and its related complications, such as pain and infertility.

Elevated Circular RNA PVT1 Promotes Eutopic Endometrial Cell Proliferation and Invasion of Adenomyosis via miR-145/Talin1 Axis

Several theories on the origin of adenomyosis (ADS) have been proposed, of which the most widely accepted is the fundamental pathogenic role of uterine eutopic endometrium. Emerging evidence suggests that circular RNAs participate in the multiple tumorgenesis. The vital importance of circular RNA PVT1 (circPVT1) in the pathological progress like malignancies has been well documented. Nevertheless, its underlying correlation with ADS remains elusive yet. The purpose of this study was to investigate the expression pattern, regulatory effect, and internal mechanism of circPVT1 in ADS. qRT-PCR was performed to detect the relative mRNA expression of circPVT1, miR-145, and Talin1 in ADS endometrial tissue and cells. The protein level of Talin1 was measured by Western blot and immunochemistry. Immunofluorescence was used to identify the primary endometrial epithelial and stromal cells. circPVT1 knockdown in vitro was achieved by transfecting with specific lentivirus vector CCK-8, and colony formation assays were utilized to assess cell proliferation; meanwhile, the transwell assay was employed for evaluating cell invasion ability. By conducting bioinformatics, dual-luciferase reporter assay, or RNA immunoprecipitation (RIP) experiment, the interaction between miR-145 and circPVT1 or Talin1 was verified. Rescue experiments further determined the regulatory effect of circPVT1/miR-145/Talin1 axis. We found both circPVT1 and Talin1 were markedly upregulated in ADS endometrial tissue and cells, whereas miR-145 was decreased. Elevated expression of circPVT1 was closely related to the severity of dysmenorrhea, menorrhagia, and uterine enlargement of patients with ADS. Knockdown of circPVT1 inhibited adenomyotic epithelial and stromal cell proliferation and invasion. Further mechanistic experiments revealed that circPVT1 negatively regulated miR-145 through serving as a molecular sponge. And the facilitating effect of circPVT1 was partially reversed by miR-145. Talin1 was demonstrated to be a down target of miR-145 and indirectly affected by circPVT1. Our findings unveiled that enhanced circPVT1 may be involved in the pathogenesis of ADS via stimulating endometrial cell proliferation and invasion. The establishment of circPVT1/miR-145/Talin1 pathway might present a novel therapeutic insight for ADS.

RhoA/ROCK pathway mediates the effect of oestrogen on regulating epithelial-mesenchymal transition and proliferation in endometriosis.

Endometriosis is a benign gynaecological disease appearing with pelvic pain, rising dysmenorrhoea and infertility seriously impacting on 10% of reproductive‐age females. This research attempts to demonstrate the function and molecular mechanism of RhoA/ROCK pathway on epithelial‐mesenchymal transition (EMT) and proliferation in endometriosis. The expression of Rho family was abnormally changed in endometriotic lesions; in particular, RhoA and ROCK1/2 were significantly elevated. Overexpression of RhoA in human eutopic endometrial epithelial cells (eutopic EECs) enhanced the cell mobility, epithelial‐mesenchymal transition (EMT) and proliferation, and RhoA knockdown exhibited the opposite function. Oestrogen up‐regulated the RhoA activity and expression of RhoA and ROCK1/2. RhoA overexpression reinforced the effect of oestrogen on promoting EMT and proliferation, and RhoA knockdown impaired the effect of oestrogen. oestrogen receptor α (ERα) was involved with the regulation of oestrogen on EMT and proliferation and up‐regulated RhoA activity and expression of RhoA and ROCK1/2. The function of ERα was modulated by the change in RhoA expression. Furthermore, phosphorylated ERK that was enhanced by oestrogen and ERα promoted the protein expression of RhoA/ROCK pathway. Endometriosis mouse model revealed that oestrogen enhanced the size and weight of endometriotic lesions. The expression of RhoA and phosphorylated ERK in mouse endometriotic lesions was significantly elevated by oestrogen. We conclude that abnormal activated RhoA/ROCK pathway in endometriosis is responsible for the function of oestrogen/ERα/ERK signalling, which promoted EMT and proliferation and resulted in the development of endometriosis.

Endometriosis-Associated Ovarian Cancer: The Origin and Targeted Therapy.

Endometrial cysts (ECs) are thought to be the origin of endometriosis-associated ovarian cancer (EAOC). A hypothesis that the oxidative stress of iron in cysts causes “malignant transformation of ECs” has been proposed, but this has not been verified. Several population-based studies showed that endometriosis was a risk factor but did not reflect the “malignant transformation of ECs”. A review showed that most patients were diagnosed with EAOC early in monitoring following detection of ECs, and that these cases might have been cancer from the start. Epidemiologically, EAOC was reduced by hysterectomy rather than by cystectomy of ECs. Gene mutation analyses identified oncogenic mutations in endometriosis and normal endometrium and revealed that the same mutations were present at different endometriotic lesions. It was also shown that most of the gene mutations found in endometriosis occurred in normal endometrium. Taking together, EAOC might be caused by eutopic endometrial glandular epithelial cells with oncogenic mutations that have undergone menstrual blood reflux and engrafted in the ovary, rather than by low-risk ECs acquiring oncogenic mutations and becoming malignant. This review discusses the mechanisms of EAOC development and targeted therapy based on genetic variation in EAOC with a focus on eutopic endometrium.

Effects of human umbilical cord mesenchymal stem cells derived from exosomes on migration ability of endometrial glandular epithelial cells.

The present study aimed to investigate the effects of human umbilical cord mesenchymal stem cells (Huc-MSCs)-derived exosomes on the migratory abilities of endometrial glandular epithelial cells, and to evaluate the underlying mechanism from the perspective of epithelial-mesenchymal transition (EMT). Huc-MSCs were prepared from human umbilical cord, and eutopic endometrial glandular epithelial cells were isolated from patients with endometriosis. The exosomes derived from Huc-MSCs (Huc-MSCs-exo) were prepared using an exosome extraction kit. The endometrial glandular epithelial cells were randomly divided into two groups: Huc-MSCs-exo and control. Cell migratory ability was assessed and western blotting was used to detect the expression levels of EMT. The results of the present study demonstrated that Huc-MSCs-exo treatment significantly enhanced the migration of endometrial glandular epithelial cells from patients with endometriosis (P<0.05). The present study also demonstrated that treatment with Huc-MSCs-exo inhibited the expression levels of E-cadherin and promoted the expression levels of Vimentin and N-cadherin at both the mRNA and protein level. The results of the current study indicate that Huc-MSCs-exo enhance the migratory ability of endometrial glandular epithelial cells via promotion of EMT.

Focal Adhesion Kinase-Mediated Sequences, Including Cell Adhesion, Inflammatory Response, and Fibrosis, as a Therapeutic Target in Endometriosis.

Endometriosis has several distinguishing features in the ectopic endometrium, including chronic inflammation and fibrosis. According to the retrograde menstruation theory, endometriotic cells are derived from eutopic endometrial cells, and adhesion of endometrial cells to the extracellular matrix can be the initial step in the development of endometriosis. Therefore, we hypothesized that cell adhesion, which mediates a sequence of events in the development of endometriosis triggering inflammatory responses and tissue fibrosis could be a possible therapeutic target for endometriosis. We found co-upregulation of focal adhesion kinase (FAK) and monocyte chemoattractant protein-1 (MCP-1) in the endometriotic tissues compared with that in the normal endometrium. MCP-1 secretion was significantly higher in the endometriotic stromal cells than in the eutopic endometrial stromal cells. Furthermore, co-culture of U937 cells and endometriotic stromal cells upregulated secretion of transforming growth factor-β1 (TGF-β1). A FAK inhibitor significantly inhibited the secretion of MCP-1 in the endometriotic stromal cells and TGF-β1 in the co-culture with macrophages. FAK inhibitor treatment in the murine endometriosis model demonstrated a decrease in the formation of endometriotic lesions as well as the expression of MCP-1 and TGF-β1. Our results suggest that the FAK-mediated sequential development of endometriosis, including inflammatory response and tissue fibrosis, can be a new therapeutic target in endometriosis.

Establishment and characterization of immortalized human eutopic endometrial stromal cells

The application of primary eutopic endometrial cells from endometriosis patients in research is restricted for short life span, dedifferentiation of hormone responsiveness. Human telomerase reverse transcriptase (hTERT)-induced immortalized cells (iheESCs) were infected by lentivirus. mRNA level was examined by qRT-PCR, and protein expression was quantified by Western blot. CCK-8 and EdU assay were assigned to assess the proliferation. The migration and invasion of cells were assessed by transwell assay. Clone formation assay and nude mouse tumorigenicity assay were used to evaluate colony-formation and tumorigenesis abilities. hTERT mRNA and protein were significantly expressed higher in iheESCs compared to primary cells. iheESCs grew without morphological change for 42 passages which is much longer than 18 passages of primary cells. There was no obvious difference between primary cells and iheESCs in growth, mobility, and chromosome karyotype. Furthermore, the expression of epithelial-mesenchymal transition (EMT) markers and estrogen/progesterone receptors remained unchanged. The decidualization of iheESCs could be induced by progesterone and cAMP. Estrogen increased the proliferation and mobility of iheESCs, and lipopolysaccharides (LPS) induced the IL-1β and IL-6 promoting inflammatory response. The colony-forming ability of iheESCs, like primary cells, was lower than Ishikawa cells. In addition, tumorigenicity assay indicated that iheESCs were unable to trigger tumor formation in BALB/c nude mouse. This study established and characterized iheESCs that kept the cellular physiology of primary cells and were not available with tumorigenic ability. Thus, iheESCs would be useful as in vitro cell model to investigate pathogenesis of endometriosis.

LIM Kinase 1 Mediates Estradiol Effects on the Phosphorylation of Cofilin1 in Eutopic Endometrial Stromal Cells During the Invasion and Proliferation of Endometriosis.

Endometriosis is an estrogen-dependent gynecological disease; however, the mechanism by which estradiol promotes the development of endometriosis, including invasion and proliferation, remains unclear. Estradiol is involved in cell invasion and proliferation by regulating the cytoskeleton. The abnormally high expression of cytoskeletal regulators (LIM kinase 1 [LIMK1] and cofilin1) is closely related to increased invasiveness and proliferation of eutopic endometrial stromal cells from endometriosis patients compared to normal eutopic endometrial cells. The aim of this study was to analyze the role of estradiol during invasion and proliferation through the LIMK1/cofilin1 pathway in the endometrium of women with endometriosis. To address this, primary eutopic endometrial stromal cells were isolated from the uteri of patients with endometriosis and cultured without estradiol. The phosphorylation of cofilin1 was analyzed by western blotting. Cell invasiveness and proliferation were evaluated following LIMK1 knockdown by RNA interference technology. We found that, before LIMK1silencing, the phosphorylation levels of cofilin1 and LIMK1 of eutopic endometrial stromal cells from endometriosis patients treated with estradiol were higher than cells not treated with estradiol (P < .05 and P < .01, respectively). The total levels of cofilin1 and LIMK1 protein did not change (P > .05 and P > .05, respectively). After LIMK1 silencing, the phosphorylation of cofilin1 by estradiol was significantly reduced, and invasiveness and proliferation were clearly and concurrently decreased (P < .05 and P < .05, respectively). Thus, the phosphorylation of cofilin1 by estradiol is mediated by LIMK1, and estradiol is involved in regulating cell invasion and proliferation in endometriotic patients through the LIMK1/cofilin1 pathway.