Eutopic Endometrium Research Articles

Bioinformatics Analysis Reveals Novel Differentially Expressed Genes Between Ectopic and Eutopic Endometrium in Women with Endometriosis.

Endometriosis is one of the chronic and prevalent diseases among women. There is limited knowledge about its pathophysiology at the cellular and molecular levels, causing a lack of a definite cure for this disease. In this study, differentially expressed genes (DEGs) between ectopic and paired eutopic endometrium in women with endometriosis were analyzed through bioinformatics analysis for better understanding of the molecular pathogenesis of endometriosis. Gene expression data of ectopic and paired eutopic endometrium were taken from the Gene Expression Omnibus database. DEGs were screened by the Limma package in R with considering specific criteria. Then, the protein-protein interaction network was reconstructed between DEGs. The fast unfolding clustering algorithm was used to find sub-networks (modules). Finally, the three most relevant modules were selected and the functional and pathway enrichment analyses were performed for the selected modules. A total of 380 DEGs (245 up-regulated and 135 down-regulated) were identified in the ectopic endometrium and compared with paired eutopic endometrium. The DEGs were predominantly enriched in an ensemble of genes encoding the extracellular matrix and associated proteins, metabolic pathways, cell adhesions and the innate immune system. Importantly, DPT, ASPN, CHRDL1, CSTA, HGD, MPZ, PED1A, and CLEC10A were identified as novel DEGs between the human ectopic tissue of endometrium and its paired eutopic endometrium. The results of this study can open up a new window to better understanding of the molecular pathogenesis of endometriosis and can be considered for designing new treatment modalities.

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.

Expression of ZEB1 in different forms of endometriosis: A pilot study

ObjectivesEpithelial-Mesenchymal Transition (EMT), a cellular process in which epithelial cells lose epithelial characteristics while acquire mesenchymal features, is believed to contribute to migration and invasion abilities of the endometriotic cells. Studies on gene expression of the transcription factor ZEB1, a crucial transcription factor of EMT, show that there is probably a modified expression in the endometriotic lesions.The aim of the study was to compare the expression levels of ZEB1 in types of endometriotic lesions with different biological behavior such as endometriomas and deep infiltrating endometriotic nodules. Study DesignWe have studied 19 patients with endometriosis and 8 patients with benign gynecological lesions without endometriosis.The endometriosis patient group included 9 women with only endometriotic cysts without deep infiltrating endometriotic lesion (DIE) and 10 women with DIE who had developed concurrent endometriotic cysts.The technique applied to investigate ZEB1 expression levels is Real-Time PCR. The results of the reaction were normalized by simultaneously investigating the expression of the house-keeping gene G6PD. ResultsAnalysis of the samples showed underexpression of ZEB1 in the eutopic endometrium of women with only endometriotic cysts when compared to normal endometrium. A tendency of higher ZEB1 expression, without reaching significant difference, was found between the endometriotic cysts and their paired eutopic endometrium.In women with DIE, no significant difference was found between their eutopic and normal endometrium. No significant difference was found between the endometriomas and DIE lesions.ZEB1 shows different expression profile in the endometriotic cysts of women with and without DIE when the cyst is compared to their paired eutopic endometrium. ConclusionsIt therefore appears that ZEB1 expression differs between different types of endometriosis. The expression levels of ZEB1 in the eutopic endometrium could affect the development of infiltrating lesions or not. However, the most important observation is the different ZEB1 expression profile of endometriomas between women with and without DIE. Although, they both share the same histologic characteristics, they show different ZEB1 expression indicating different pathogenetic mechanisms of endometriomas in cases with and without DIE. Therefore, future research on endometriosis should consider DIE and ovarian endometriosis as different diseases.

Neurotrophins and Their Receptors, Novel Therapeutic Targets for Pelvic Pain in Endometriosis, Are Coordinately Regulated by IL-1β via the JNK Signaling Pathway

Pelvic pain in women with endometriosis is attributed to neuroinflammation and afferent nociceptor nerves in ectopic and eutopic endometrium. The hypothesis that uterine nociception is activated by IL-1β, a prominent cytokine in endometriosis, was tested herein. Immunofluorescence histochemistry confirmed the presence of neurons in human endometrial tissue. Expression of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) and their receptors in endometrial tissue and cells was validated by immunohistochemistry and Western blotting. Isolated endometrial stromal cells (ESCs) were subjected to dose-response and time-course experiments with IL-1β and kinase inhibitors to characterize invitro biomarkers. Neural biomarkers were co-localized in endometrial nerve fibers. NGF, BDNF, and their receptors tropomyosin receptor kinase (Trk) A, TrkB, and p75 neurotrophin receptor were all expressed in primary ESCs. IL-1β stimulated higher TrkA/B expression in ESCs derived from endometriosis cases (2.8-±0.2-fold) than cells from controls (1.5-±0.3-fold, t-test, P<0.01), effects that were mediated via the c-Jun N-terminal kinase (JNK) pathway. BDNF concentrations trended higher in peritoneal fluid of endometriosis cases but were not statistically different from controls (P=0.16). The results support the hypothesis that NGF and BDNF and their corresponding receptors orchestrate innervation of the endometrium, which is augmented by IL-1β. We postulate that JNK inhibitors, such as SP600125, have the potential to reduce neuroinflammation in women with endometriosis.

Bushen Wenyang Huayu Decoction inhibits autophagy by regulating the SIRT1-FoXO-1 pathway in endometriosis rats.

Bushen Wenyang Huayu Decoction (BWHD) is a traditional Chinese medicine for tonifying kidney and warming Yang, thereby resolving blood stasis and relieving pain. BWHD can significantly improve the clinical symptoms of patients with endometriosis (EMs), but its mechanism is still unclear. We evaluated the expression and role of the SIRT1-FoxO-1 pathway and autophagy levels in EMs rats. The therapeutic effects and potential therapeutic mechanisms of BWHD were also investigated. Twenty rats were randomized into the sham group and eighty rats were used for model establishment by autologous transplantation. After successful modeling, they were randomized into the model, BWHD, EX527+BWHD and EX527 groups, with 20 rats in each group. All rats were intragastrically administered with for 3 weeks. Localization of Sirtuin 1 (SIRT1), Forkhead boxO-1 (FoXO-1), Beclin-1, autophagy-related 5 (Atg5) and autophagy-related 7 (Atg7) was determined by immunohistochemical staining. The expression of the above proteins was determined by Western blot and their messenger RNA (mRNA) levels were detected by Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). The protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in the model group were markedly increased, while that of SIRT1 was markedly decreased relative to the sham group (p<0.05 and p<0.01, respectively). Results showed that the protein and mRNA expressions of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of BWHD group were lower, while SIRT1 expression was higher than in the model group (p<0.05 and p<0.01, respectively). Furthermore, protein and mRNA expression levels of FoXO-1, Beclin-1, Atg5 and Atg7 in eutopic and ectopic endometrium of EX527 group were higher, while SIRT1 level was significantly lower than in the model group (p<0.05 and p<0.01, respectively). The EX527-induced changes in protein and mRNA expressions were reversed in the EX527+BWHD group (p<0.05 and p<0.01, respectively). BWHD inhibits autophagy by up-regulating SIRT1 and down-regulating FoXO-1 expression in EMs via the SIRT1-FoXO-1 signaling pathway. Therefore, it is a potential treatment for EMs.

Single-Cell Profiling Uncovers the Roles of Endometrial Fibrosis and Microenvironmental Changes in Adenomyosis.

Adenomyosis (AM) is a common benign uterine disorder that has deleterious effects on women's health. However, the pathogenesis of AM is not clearly understood. We aimed to investigate the pathophysiological changes and molecular mechanism in AM. Single-cell RNA sequencing (scRNA-seq) was employed to construct a transcriptomic atlas of various cell subsets from the ectopic endometrium (EC) and eutopic endometrium (EM) of one AM patient and evaluate differential expression. The Cell Ranger software pipeline (version 4.0.0) was applied to conduct sample demultiplexing, barcode processing and mapping reads to the reference genome (human GRCh38). Different cell types were classified with markers with the "FindAllMarkers" function, and differential gene expression analysis was performed with Seurat software in R. The findings were confirmed by Reverse Transcription Real-Time PCR using samples from three AM patients. We identified nine cell types: endothelial cells, epithelial cells, myoepithelial cells, smooth muscle cells, fibroblasts, lymphocytes, mast cells, macrophages and unknown cells. A number of differentially expressed genes, including CLO4A1, MMP1, TPM2 and CXCL8, were identified from all cell types. Functional enrichment showed that aberrant gene expression in fibroblasts and immune cells was related to fibrosis-associated terms, such as extracellular matrix dysregulation, focal adhesion and the PI3K-Akt signaling pathway. We also identified fibroblast subtypes and determined a potential developmental trajectory related to AM. In addition, we identified increased cell-cell communication patterns in EC, highlighting the imbalanced microenvironment in AM progression. Our results support the theory of endometrial-myometrial interface disruption for AM, and repeated tissue injury and repair could lead to increased fibrosis in the endometrium. Therefore, the present study reveals the association between fibrosis, the microenvironment, and AM pathogenesis. This study provides insight into the molecular mechanisms regulating AM progression.

Evaluation of protein expression of microchimeric Y-chromosome in ovarian endometriosis

Introduction: Endometriosis is a benign chronic gynaecological disorder with the presence of endometrium-like tissue outside the uterus. It affects 10% of the reproductive age group of women. The symptoms include infertility 30 – 50%, and dysmenorrhea 40 – 60%. Thus, endometriosis is a global burden with a wide range of symptomatology and hence causes difficulty for women who suffer from the disease. But the pathogenesis of the disease is yet to be understood. The presence of fetal Y-chromosome microchimerism in endometriosis is first reported using the FISH (Fluorescence In-Situ Hybridization) technique. Later, with a similar technique, conflicting results have been reported. The detection probes used were Y-specific chromosomes Yp11.1 – q11.1-Alpha Satellite DNA and Yq12 Satellite III DNA. The genomic evidence of Y chromosome microchimerism is evaluated using microarray expression followed by RNA-seq and quantitative RT-PCR experiments of target inserts for selected targets for further validation. This study reported the presence of 21 non-coding, and 33 protein-coding genes present in the Male Specific region of Y-chromosome (MSY) in ovarian endometriosis. Genomic studies have been reported in the literature but protein expression from Y-chromosome genes in ovarian endometriosis has not been reported. So, we aimed to evaluate protein expressions of EIF1aY, NLGN4Y, and ZFY in the eutopic endometrium of ovarian endometriosis. Method: The 2 study groups are allotted; Group A (Control) and Group B (Endometriosis). The tissue samples collected were annotated, processed, and performed western blot for identification of EIF1aY, ZFY, and NLGN4Y gene expression. The blots were scanned and analyzed using Bio-Rad Pharos FX TM Plus Molecular Imager and NIH Image J software ( http://imagej.nih.gov/ij/ ) respectively. Result: There was a significant increase in the gene expression of EIF1aY in the endometriosis group when compared to the control group (p &lt; 0.01). The Spearman correlation scores for NLGN4Y and ZFY are strongly associated (r = 0.617). Conclusion: The ectopic site promotes the niche and eutopic tissue has an error which enhances the histogenesis. It appears from the present study that proteins coded from Y-chromosome genes, namely EIF1aY, NLGN4Y and ZFY are involved in the pathogenesis of ovarian endometriosis, which could be a potential therapeutic target in future. To the best of our knowledge, this is the first report suggesting the involvement of Y-microchimerism at the protein level in endometriosis. Further studies with larger sample sizes and different stages and phenotypes of endometriosis may bring forth more conclusive knowledge in this regard that seems to promote the histogenesis of endometriosis at ectopic sites. Department of Physiology, AIIMS, New Delhi. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genomic Insults and their Redressal in the Eutopic Endometrium of Women with Endometriosis

Endometrium, a highly dynamic tissue, is known for its remarkable ability to regenerate, differentiate, and degenerate in a non-conception cycle and transform into a specialized tissue to nurture and protect the embryo in a conception cycle. This plasticity of the endometrium endows the uterus to execute its major function, i.e., embryo implantation. However, this boon becomes a bane, when endometrium- or endometrium-like cells adhere, grow, and invade extrauterine sites, leading to endometriosis. Endometrial deposits at the extrauterine site lead to severe pelvic pain, painful menstruation, and infertility in endometriosis. Although benign, endometriotic lesions share several traits with cancerous cells, excessive proliferation, adhesion, invasion, and angiogenesis make endometriotic lesions analogous to cancer cells in certain aspects. There exists evidence to support that, akin to the cancer cell, endometriotic lesions harbor somatic mutations. These lesions are known to experience higher proliferative stress, oxidative stress, and inflammation, which may contribute to somatic mutations. However, it would be of more interest to establish whether in the eutopic endometriosis also, the mutational burden is higher or whether the DNA Damage Response (DDR) is compromised in the eutopic endometrium, in endometriosis. Such investigations may provide more insights into the pathobiology of endometriosis and may also unravel cellular events associated with the origin of the disease. This review compiles inferences from the studies conducted to assess DNA damage and DDR in endometriosis.

Assessment of BMP7, SMAD4, and CDH1 Expression Profile and Regulatory miRNA-542-3p in Eutopic and Ectopic Endometrium of Women with Endometriosis

Alterations in the expression of numerous genes and the miRNAs that are recognized as their regulators in the endometrial cells of women with endometriosis may disrupt the intracellular signaling pathways associated with epithelial-mesenchymal transition (EMT). So far, the functional role of BMP7 in endometrial physiology has been confirmed, especially in the context of fertility, but the role of the activation of a specific mechanism operating through the BMP-SMAD-CDH1 axis in the formation of endometrial lesions remains unexplored. The aim of this study was to evaluate the expression profile of miR-542-3p and the EMT markers (BMP7, SMAD4, CDH1) in matched eutopic endometrium (EUE) and ectopic endometrium (ECE) samples from women with endometriosis in relation to healthy women. The levels of expression of the studied genes and miRNA in peripheral blood mononuclear cells (PBMCs) obtained from women diagnosed with endometriosis and those without the disease were also evaluated. Fifty-four patients (n = 54: with endometriosis-n = 29 and without endometriosis-n = 25) were included in the study. A comparative analysis of the relative mean expression values (RQ) of the studied mRNA and miRNA assessed by RT-qPCR demonstrated downregulation of BMP7, SMAD4, and CDH1 expression in ectopic lesions and upregulation in the eutopic endometrium compared with the control group. In the eutopic tissue of women with endometriosis, miR-542-3p expression was similar to that of the control but significantly lower than in endometrial lesions. We also confirmed a trend towards a negative correlation between miR-542-3p and BMP7 in ectopic tissue, and in PBMC, a significant negative correlation of miR-542-3p with further BMP signaling genes, i.e., SMAD4 and CDH1, was observed. These results indicate that the miRNA selected by us may be a potential negative regulator of BMP7-SMAD4-CDH1 signaling associated with EMT. The different patterns of BMP7, SMAD4, and CDH1 gene expression in ECE, EUE, and the control endometrium observed by us suggests the loss of the endometrial epithelium phenotype in women with endometriosis and demonstrates their involvement in the pathogenesis and pathomechanism of this disease.

The role of &lt;i&gt;WNT&lt;/i&gt; and &lt;i&gt;HOXA&lt;/i&gt; signaling cascades in the pathogenesis of adenomyosis

BACKGROUND: Adenomyosis is a common gynecological disease with unknown pathogenesis. The HOXA10, HOXA11 and WNT4 genes may play an important role in the pathogenesis of adenomyosis both at the stage of embryonic development and in the postnatal period. The study of their expression in the endometrium of patients with adenomyosis can expand the understanding of the pathogenesis of this disease.&#x0D; AIM: The aim of this work was to study the peculiarity of the WNT4, HOXA10 and HOXA11 gene expression in the eutopic endometrium of patients with isolated adenomyosis.&#x0D; MATERIALS AND METHODS: The study included 38 women: the main group involved patients with isolated adenomyosis established by ultrasound / magnetic resonance imaging (n = 20) and the control group consisted of healthy patients (n = 18). Endometrial sampling was obtained during surgery or by aspiration biopsy at 512 day of the menstrual cycle (proliferative phase) or 2024 day of the menstrual cycle (secretory phase). The expression of the WNT4, HOXA10 and HOXA11 genes in endometrial samples was assessed by a real-time reverse transcription polymerase chain reaction.&#x0D; RESULTS: In the proliferative phase endometrial samples of patients with adenomyosis, a significant increase in the WNT4 (of almost two times), HOXA10 and HOXA11 (of one and a half to two times) gene expression levels was shown compared to the control group. In 88% of patients with adenomyosis, there is a significant increase (up to the level of fourth quartile) in the expression of at least one of these genes, such changes being not typical for the endometrium of women in the control group. In the secretory phase endometrial samples, the expression of the studied genes did not differ from the level characteristic of the corresponding groups in the proliferative phase of the cycle.&#x0D; CONCLUSIONS: The aberrant expression of the WNT4, HOXA10 and HOXA11 genes in the endometrium of patients with adenomyosis indicates a significant role of these genes in the development of the disease and infertility associated with adenomyosis.

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.

The Association between Deoxyribonucleic Acid Hypermethylation in Intron VII and Human Leukocyte Antigen-C∗07 Expression in Patients with Endometriosis.

Endometriosis, which is a common disease affecting approximately 10% of women of reproductive age, usually causes dysmenorrhea and infertility, thus seriously harming the patients' physical and mental health. However, there is a mean delay of 6.7 years between the onset of the symptoms and the surgical diagnosis of endometriosis. There is an increasing amount of evidence that suggests that epigenetic aberrations, including deoxyribonucleic acid (DNA) methylation, play a definite role in the pathogenesis of endometriosis. This study aimed to explore the noninvasive or minimally invasive biomarkers of this disease. Six patients with surgically confirmed ovarian endometriosis and six patients who received IUD implantation for contraception without endometriosis were recruited in the East Hospital of Tongji University in 2018. The genome methylation profiling of the eutopic and ectopic endometrium of ovarian endometriosis patients and normal endometrial specimens from healthy women was determined using a methylation microarray test. The test screened methylation-differentiated 5'-C-phosphate-G-3' (CpG) sites and then located the target genes affected by these sites following sequence alignment. Then, an additional 22 patients and 26 healthy controls were enrolled to further verify the difference in the selected genes between endometriosis patients and healthy women. The differential DNA methylation of the selected genes was validated via direct bisulfite sequencing and analysis of their messenger ribonucleic acid (mRNA) levels using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Fifteen differentially methylated CpG sites were found among the patients and healthy women, and five CpG sites were mapped to the introns of the human leukocyte antigen-C (HLA-C) gene; these were highly polymorphic between different HLA-C alleles and were HLA-C∗07 specific. The results indicated that the HLA-C∗07 carrier patients exhibited significantly higher DNA methylation levels at the intron VII of HLA-C compared to the HLA-C∗07 carrier healthy controls. High HLA-C∗07 mRNA levels were also observed using qRT-PCR with HLA-C∗07-specific primers, which indicated that the hypermethylation of CpG in intron VII might suppress a silencer that regulates HLA-C∗07 expressions. Deoxyribonucleic acid hypermethylation in the intron VII of the HLA-C∗07 gene appears to regulate the expression of HLA-C∗07. The aberrant DNA methylation in this region was positively correlated with the occurrence of endometriosis.

Establishment of a novel glycolysis-immune-related diagnosis gene signature for endometriosis by machine learning

PurposeThe objective of this study was to investigate the key glycolysis-related genes linked to immune cell infiltration in endometriosis and to develop a new endometriosis (EMS) predictive model.MethodsA training set and a test set were created from the Gene Expression Omnibus (GEO) public database. We identified five glycolysis-related genes using least absolute shrinkage and selection operator (LASSO) regression and the random forest method. Then, we developed and tested a prediction model for EMS diagnosis. The CIBERSORT method was used to compare the infiltration of 22 different immune cells. We examined the relationship between key glycolysis-related genes and immune factors in the eutopic endometrium of women with endometriosis. In addition, Gene Ontology (GO)-based semantic similarity and logistic regression model analyses were used to investigate core genes. Reverse real-time quantitative PCR (RT-qPCR) of 5 target genes was analysed.ResultsThe five glycolysis-related hub genes (CHPF, CITED2, GPC3, PDK3, ADH6) were used to establish a predictive model for EMS. In the training and test sets, the area under the curve (AUC) of the receiver operating characteristic curve (ROC) prediction model was 0.777, 0.824, and 0.774. Additionally, there was a remarkable difference in the immune environment between the EMS and control groups. Eventually, the five target genes were verified by RT-qPCR.ConclusionThe glycolysis-immune-based predictive model was established to forecast EMS patients’ diagnosis, and a detailed comprehension of the interactions between endometriosis, glycolysis, and the immune system may be vital for the recognition of potential novel therapeutic approaches and targets for EMS patients.

Linc-ROR Promotes EMT by Targeting miR-204-5p/SMAD4 in Endometriosis.

Endometriosis (EMs) is a systemic and chronic disease with cancer-like feature, namely, distant implantation, which caused heavy healthy burden of nearly 200 million females. LncRNAs have been proved as new modulators in epithelial-mesenchymal transition (EMT) and EMs. Quantitative real-time PCR was conducted to measure the expression level of long intergenic non-protein coding RNA, regulator of reprogramming (Linc-ROR), and miR-204-5p in ectopic endometrium (n = 25), eutopic endometrium (n = 20), and natural control endometrium (n = 22). Overexpression of Linc-ROR, knockdown or overexpression of miR-204-5p in End1/E6E7 and Ishikawa cells, was conducted to detect the function of Linc-ROR and miR-204-5p in EMs. Furthermore, luciferase reports were used to confirm the combination of Linc-ROR and miR-204-5p and the combination between miR-204-5p and SMAD4. Cell-Counting Kit-8, EdU assay, transwell assays, and Western blotting were used to detect the function of Linc-ROR and miR-204-5p in EMs cancer-like behaviors and EMT process. Linc-ROR was up-regulated in ectopic endometrium. Overexpressed Linc-ROR promotes cell proliferation, invasion, and EMT process. Linc-ROR regulated the EMT process, cellular proliferation, and invasion of EMs via binding to miR-204-5p. In addition, overexpression of Linc-ROR up-regulated SMAD4, a target protein of miR-204-5p, with which regulated EMT process and cancer-like behaviors in EMs together. Linc-ROR/miR-204-5p/SMAD4 axis plays a vital role in regulation EMT process in EMs, which might become a novel therapeutic targets and powerful biomarkers in EMs therapy.

Comparative Study of BAX/BcL2 Expression Ratio in Menstrual Blood between Endometriosis Women and Women without Endometriosis

Introduction: Endometriosis is characterized by the presence of endometrial tissue outside the uterus and is commonly associated with chronic pelvic pain and infertility. One of the endometrial changes that appear in the eutopic and ectopic endometrium of women with endometriosis is the regulation of apoptosis. The proportion value of the BAX/BcL-2 ratio is a crucial factor in determining the ability of cells to stimulate apoptosis. Higher apoptotic activity is linked to an increased BAX/BcL-2 ratio. The aim of this study was to compare the BAX/BcL-2 ratios in menstrual blood of endometriosis and non-endometriosis patients. Material and Methods: This research is a quantitative observational study with a cross-sectional method at dr. Saiful Anwar Malang. The case group was postoperative women with ovarian cysts with anatomical pathology examination results indicating endometriosis. While the control group was patients without endometriosis. Data analysis was carried out by using the Independent T Test with SPSS for Windows 19.0 software. Results: Each group consisted of 17 patients. The normality test for each variable shows p&gt; 0.05. BAX average in patients with endometriosis 4556.29 ± 2338.33 and 6488.06 ± 2649.08 in patients without endometriosis (p = 0.031). The average BcL-2 in endometriosis patients was 6483.82±2489.36 and 4877.18±2023.82 in patients without endometriosis (p=0.047). The average BAX/BcL-2 ratio in patients with endometriosis was 0.698±0.19 and 1.367±0.26 in patients without endometriosis (p=0.000). Conclusion: The BAX/BcL-2 ratio in women with endometriosis cysts has a lower average rate than women without endometriosis cysts.

FoxA2 represses ERβ-mediated pyroptosis in endometriosis by transcriptionally inhibiting IGF2BP1

BackgroundEndometriosis is a severe disease which is associated with excessive activation of pyroptosis. Our present research aimed to investigate the function of Forkhead Box A2 (FoxA2) in regulating pyroptosis in endometriosis. MethodsIL-1β and IL-18 concentrations were assessed using ELISA. Cell pyroptosis was analyzed using flow cytometry. TUNEL staining was performed to determine human endometrial stromal cells (HESC) death. Moreover, ERβ mRNA stability was assessed using RNA degradation assay. Finally, the binding relationships between FoxA2, IGF2BP1 and ERβ were verified by dual-luciferase reporter system, ChIP, RIP and RNA pull-down assays. ResultsOur results revealed that IGF2BP1 and ERβ were significantly upregulated in ectopic endometrium (EC) tissues of endometriosis patients compared to that in eutopic endometrium (EU) tissues as well as IL-18 and IL-1β levels. Loss-of-function experiments subsequently demonstrated that either IGF2BP1 knockdown or ERβ knockdown could repress HESC pyroptosis. In addition, IGF2BP1 upregulation promoted the pyroptosis in endometriosis by binding to ERβ and promoting ERβ mRNA stability. Our further research displayed that FoxA2 upregulation suppressed HESC pyroptosis by interacting with IGF2BP1 promoter. ConclusionOur research proved that FoxA2 upregulation downregulated ERβ by transcriptionally inhibiting IGF2BP1, thereby repressing pyroptosis in endometriosis.

Single-cell transcriptome analysis reveals endometrial immune microenvironment in minimal/mild endometriosis.

Endometriosis is a common inflammatory disorder in women of reproductive age due to an abnormal endometrial immune environment and is associated with infertility. This study aimed to systematically understand the endometrial leukocyte types, inflammatory environment, and impaired receptivity at single-cell resolution. We profiled single-cell RNA transcriptomes of 138 057 endometrial cells from endometriosis patients (n = 6) and control (n = 7), respectively, using 10x Genomics platform. We found that one cluster of epithelial cells that expressed PAEP and CXCL14 was mostly from the control during the window of implantation (WOI). This epithelial cell type is absent in the eutopic endometrium during the secretory phase. The proportion of endometrial immune cells decreased in the secretory phase in the control group, whereas the cycle variation of total immune cells, NK cells, and T cells was absent in endometriosis. Endometrial immune cells secreted more IL-10 in the secretory phase than in the proliferative phase in the control group; the opposite trend was observed in endometriosis. Proinflammatory cytokines levels in the endometrial immune cells were higher in endometriosis than in the control group. Trajectory analysis revealed that the secretory phase epithelial cells decreased in endometriosis. Ligand-receptor analysis revealed that 11 ligand-receptor pairs were upregulated between endometrial immune and epithelial cells during WOI. These results provide new insights into the endometrial immune microenvironment and impaired endometrial receptivity in infertile women with minimal/mild endometriosis.

Increased NKG2A+CD8+ T-cell exhaustion in patients with adenomyosis

Immune dysregulation has long been proposed to be associated with adenomyosis, but the underlying mediators and mechanisms remain largely unexplored. Here, we used flow cytometry to investigate the alterations in immune cell subsets in adenomyotic uteri and analyze the phenotype and function of abnormal immune cells. We found that an increase in cluster of differentiation (CD)8+ T-cell number was the predominant alteration in ectopic lesions in patients with adenomyosis and was significantly associated with the severity of adenomyosis. Importantly, we identified an exhausted natural killer group protein 2A (NKG2A)+CD8+ T-cell subset that was associated with the severity of adenomyosis and found that the number of these cells was significantly increased in the eutopic endometrium and ectopic lesions. In addition, the increases in the expression of NKG2A ligand histocompatibility leucocyte antigen E and interleukin-15 in glandular epithelial cells in the adenomyotic microenvironment might contribute to CD8+ T-cell exhaustion by promoting NKG2A expression on CD8+ T cells or inhibiting the effector function of these cells. In conclusion, our data revealed a previously unrecognized role for NKG2A+CD8+ T-cell exhaustion in the pathogenesis of adenomyosis, indicating that therapeutic interventions designed to target and reinvigorate exhausted CD8+ T cells may be beneficial for patients with adenomyosis.

Altered Differential Expression of Genes and microRNAs Related to Adhesion and Apoptosis Pathways in Patients with Different Phenotypes of Endometriosis.

We aim to investigate the expression of genes (MAPK1 and CAPN2) and microRNAs (miR-30a-5p, miR-7-5p, miR-143-3p, and miR-93-5p) involved in adhesion and apoptosis pathways in superficial peritoneal endometriosis (SE), deep infiltrating endometriosis (DE), and ovarian endometrioma (OE), and to evaluate whether these lesions share the same pathophysiological mechanisms. We used samples of SE (n = 10), DE (n = 10), and OE (n = 10), and endometrial biopsies of these respective patients affected with endometriosis under treatment at a tertiary University Hospital. Endometrial biopsies collected in the tubal ligation procedure from women without endometriosis comprised the control group (n = 10). Quantitative real-time polymerase chain reaction was performed. The expression of MAPK1 (p < 0.0001), miR-93-5p (p = 0.0168), and miR-7-5p (p = 0.0006) was significantly lower in the SE group than in the DE and OE groups. The expression of miR-30a (p = 0.0018) and miR-93 (p = 0.0052) was significantly upregulated in the eutopic endometrium of women with endometriosis compared to the controls. MiR-143 (p = 0.0225) expression also showed a statistical difference between the eutopic endometrium of women with endometriosis and the control group. In summary, SE showed lower pro-survival gene expression and miRNAs involved in this pathway, indicating that this phenotype has a different pathophysiological mechanism compared to DE and OE.

Epithelial-mesenchymal transition as a pathogenetic mechanism of development and progression of adenomyosis

BACKGROUND: In recent years, an important role in the pathogenesis of adenomyosis has been assigned to invasive properties of the cells of the basal layer of the endometrium, which provide them with the capacity to grow into the underlying layers of the myometrium.&#x0D; AIM: The aim of this study was to evaluate the invasive and migratory properties of the ectopic and heterotopic endometrium in patients with adenomyosis.&#x0D; MATERIALS AND METHODS: We performed clinical, morphological and immunohistochemical analyses of the surgical material of 98 patients with adenomyosis. Immunohistochemical study was carried out according to the standard avidin-biotin method, using mouse monoclonal antibodies to estrogen receptors, matrix metalloproteinase type 9, vimentin, and fibronectin (DAKO, Denmark) as primary immune sera.&#x0D; RESULTS: The maximum number of estrogen receptors in both the proliferation and secretion phases was found in the endometrial glands and superficially located endometrioid heterotopias. A weaker expression of this marker was also found in the cells of the cytogenic stroma. In the foci of adenomyosis, located in the deep layers of the myometrium, the expression of estrogen receptors in the epithelial and stromal components of heterotopias varied in a wide range from 0 to 100%. The most pronounced expression of matrix metalloproteinase type 9 was characteristic of the epithelial component of superficial endometrioid heterotopias. In the foci of adenomyosis, located in the deep parts of the myometrium, a pronounced expression of matrix metalloproteinase type 9 was preserved in the proliferation phase, and its significant decrease was found in the secretion phase. The glands of the eutopic endometrium were also characterized by a more significant level of matrix metalloproteinase type 9 expression in the proliferation phase in comparison with the secretion phase. A pronounced expression of vimentin was detected in the epithelium of the glands of both superficial and deep foci of adenomyosis, as well as in the eutopic endometrium (100%). In the cytogenic stroma, the largest area of vimentin expression was found in the eutopic endometrium and superficially located endometrioid heterotopias. However, its value significantly decreased in the foci of adenomyosis located in the deep parts of the myometrium. The largest area of fibronectin expression was characteristic of the cytogenic stroma of superficial adenomyosis foci.&#x0D; CONCLUSIONS: The displacement of elements of the eutopic endometrium into the thickness of the myometrium and further progression of adenomyosis is provided by two parallel pathogenetic mechanisms, namely, invasive growth due to matrix metalloproteinase type 9 activation and epithelial cell migration due to the epithelial-mesenchymal transition.