Endometriosis Tissue Research Articles

A transcriptomic analysis of the interplay of ferroptosis and immune filtration in endometriosis and identification of novel therapeutic targets.

Endometriosis is an inflammatory disease, involving immune cell infiltration and production of inflammatory mediators. Ferroptosis has recently been recognized as a mode of controlled cell death and the iron overload and peroxidative environment prevailing in the ectopic endometrium facilitates the occurrence of ferroptosis. In the current investigation, gene expression data was obtained from the dataset GSE7305.The variation in infiltration of immune cells amongst the samples with endometriosis and normal tissue was analysed using the CIBERSORTx tool which revealed higher infiltration of T cells gamma delta, macrophages M2, B cells naïve, T cells CD4 memory resting cells, plasma cells, T cells CD8 and mast cells activated in the tissue samples with endometriosis. An overlap of the differentially expressed genes (DEGs) and ferroptosis related genes revealed 32 ferroptosis related DEGs (FR-DEGs). GO and KEGG pathway analysis showed the FR-DEGs to be enriched in ferroptosis. The PPI network of the FR-DEGs was constructed and TP53, HMOX1, CAV1, CDKN1A, CD44, EPAS1, SLC2A1, MAP3K5, GCLC and FANCD2 were identified as the hub genes. Pearson correlation revealed significant correlation between the hub genes and infiltrating immune cells in endometriosis, thereby suggesting existence of a regulatory crosstalk between immune responses and ferroptosis in endometriosis. Hub gene- miRNA network analysis revealed that 7 of the 10 hub genes were targets of 3 miRNAs -hsa-miR-20a-5p, hsa-miR-16-5p and hsa-miR-17-5p, thereby providing further insight into the regulatory mechanisms underlying disease progression. Predictive analysis and cross validation studies revealed TP53 and CDKN1A as common targets of hsa-miR-16-5p, hsa-miR-17-5p, and hsa-miR-20a-5p, thereby revealing their regulatory roles in ferroptosis and immune modulatory pathways relevant to endometriosis. The present study indicates an important role of both immune dysregulation and ferroptosis in the pathogenesis of endometriosis and identifies ferroptosis related hub genes and their miRNA regulators as favourable novel targets for further studies and therapeutic interventions.

Glycosaminoglycan Adenogenesis Factors: Immunohistochemical Expression in Endometriosis Tissues Compared with the Endometrium.

Endometriosis is a chronic inflammatory pathology estrogen-dependent. It is a condition affecting 5%-10% of women of reproductive age worldwide. Recent evidence indicating an embryological origin of endometriosis has provided new insights into its pathogenesis and potential therapeutic approaches. In this study, we compared the immunohistochemical expression of extracellular matrix molecules involved in the interaction between epithelium and stroma in endometriotic lesions and normal endometrial tissue. A total of 41 cases were analyzed. We examined the immunohistochemical expression of chondroitin sulfate proteoglycan 4 (CSPG4), keratan sulfate, chondroitin sulfate (CS-56), hyaluronic acid, and heparan sulfate (HEP). Our results showed higher expression of CSPG4 and CS-56 in epithelial endometriosis samples compared with normal endometrial tissue, while HEP, keratan sulfate, and hyaluronic acid showed decreased expression in epithelial endometriosis samples relative to normal endometrial tissue. Additionally, endometriotic stroma exhibited more frequent low intensity of hyaluronic acid and HEP compared with normal endometrial stroma. Investigating the levels of these molecules in eutopic and ectopic endometrial tissues enables the identification of potential therapeutic targets, and the development of novel treatments aimed at disrupting the adhesive and invasive properties of endometriotic lesions.

Living with acuteness in chronic illness: The temporal underpinnings of endometriosis.

This article explores how acuteness is experienced by people with endometriosis in Finland. Drawing on in-depth interviews as well as anonymous written endometriosis stories, we trace instances when the sense of chronicity and cyclicality of endometriosis is disrupted by a possibility of risk to life. These instances include when endometriosis tissue grows in unanticipated and aggressive ways, when medical interventions lead to unexpected complications or medications raise concerns about a gradually developing risk, and when endometriosis diagnosis becomes a catch-all category that could mask the onset of a life-threatening condition. Our analysis of illness experiences suggests that, while risk to life is an unlikely outcome in chronic conditions such as endometriosis, concerns about risk shape how the chronicity and cyclicality of endometriosis are felt and managed in everyday life.

Artificial intelligence-based tissue segmentation and cell identification in multiplex-stained histological endometriosis sections.

How can we best achieve tissue segmentation and cell counting of multichannel-stained endometriosis sections to understand tissue composition? A combination of a machine learning-based tissue analysis software for tissue segmentation and a deep learning-based algorithm for segmentation-independent cell identification shows strong performance on the automated histological analysis of endometriosis sections. Endometriosis is characterized by the complex interplay of various cell types and exhibits great variation between patients and endometriosis subtypes. Endometriosis tissue samples of eight patients of different subtypes were obtained during surgery. Endometriosis tissue was formalin-fixed and paraffin-embedded before sectioning and staining by (multiplex) immunohistochemistry. A 6-plex immunofluorescence panel in combination with a nuclear stain was established following a standardized protocol. This panel enabled the distinction of different tissue structures and dividing cells. Artificial intelligence-based tissue and cell phenotyping were employed to automatically segment the various tissue structures and extract quantitative features. An endometriosis-specific multiplex panel comprised of PanCK, CD10, α-SMA, calretinin, CD45, Ki67, and DAPI enabled the distinction of tissue structures in endometriosis. Whereas a machine learning approach enabled a reliable segmentation of tissue substructure, for cell identification, the segmentation-free deep learning-based algorithm was superior. The present analysis was conducted on a limited number of samples for method establishment. For further refinement, quantification of collagen-rich cell-free areas should be included which could further enhance the assessment of the extent of fibrotic changes. Moreover, the method should be applied to a larger number of samples to delineate subtype-specific differences. We demonstrate the great potential of combining multiplex staining and cell phenotyping for endometriosis research. The optimization procedure of the multiplex panel was transferred from a cancer-related project, demonstrating the robustness of the procedure beyond the cancer context. This panel can be employed for larger batch analyses. Furthermore, we demonstrate that the deep learning-based approach is capable of performing cell phenotyping on tissue types that were not part of the training set underlining the potential of the method for heterogenous endometriosis samples. All funding was provided through departmental funds. The authors declare no competing interests. N/A.

A narrative review about the intricate crosstalk among endometrium, adipose tissue, and neurons in endometriosis. The multifaceted role of leptin.

Endometriosis is a highly prevalent gynecological disease characterized by the presence of endometrium-like tissue outside the uterus, whose etiopathology is far from being elucidated. The most frequent complains of patients are pelvic pain and infertility. Increasing evidence supports the systemic impact of endometriosis suggesting that an intricate crosstalk among distinct organs underlies the development of the disease. In this setting, endometriosis patients present an increased risk for developing other diseases, such as cancer, cardiovascular pathologies, and autoimmune diseases, and manifest neurologic disturbances, including neuropathic hyperalgesia. Whilst the ovary-secreted estrogen dependency of ectopic endometrium growth is well established, we conjecture that adipose tissue-secreted molecules also intervene in endometriosis development and pain manifestation. In fact, women with endometriosis present a peculiar pattern of adipokine secretion that ensues the disease onset. Unexpectedly, the levels of adipose tissue-secreted molecules in those women present similarities with those found in patients with obesity, despite the recognized association of low body mass index with endometriosis. Taking this evidence into consideration, we hypothesize that endometriosis patients present a dysfunctional adipose tissue, which is associated with enhanced metabolism and unregulated browning that not only intervene in the control of body weight but also in peculiar pain processing pathways.

Morphology of mitochondrial network in disseminated endometriosis cells in spontaneous pneumothorax diagnostic process.

Circulating endometrial cells (CECs) have emerged as a new biomarker of advanced disease in women with endometriosis. The identification of several subtypes of CECs (e.g., stem cell-like, epithelial, glandular, stromal) has opened the way for characterization of endometriosis-associated CECs. This study focused on the isolation and characterization of CECs and disseminated endometrial cells (DECs) in patients with spontaneous pneumothorax (SP). The primary objective was to differentiate between cancer and non-cancer cells in patients with no previous cancer diagnosis. The MetaCell® size-based separation protocol was used to enrich CECs/DECs. Evaluation of the captured cells by 3D microscopy was performed using a NANOLIVE™ microscope using a holographic approach. Based on gene expression analysis (GEA), we can conclude that mitochondria are much more active in primary tumors compared to endometriosis tissue (e.g. MT-ND1, MT-ATP6 genes). The culture of DECs is made of stromal, stem and immune cells. In vitro culture of DECs is characterized by an increase in the epithelial marker KRT18. Similarly, NFE2L2, a proerythroid factor, is also elevated. Further, a significant decrease in the amount of stem and immune cells was observed in the cell culture of DECs. The data presented here show how morphologically plastic the changes in the mitochondrial network can be and how cells can reflect them at the level of gene expression. The markers identified could help in the accompanying diagnostic process of the spontaneous pneumothorax in women of reproductive age.

Elevated MMP-9, Survivin, TGB1 and Downregulated Tissue Inhibitor of TIMP-1, Caspase-3 Activities are Independent of the Low Levels miR-183 in Endometriosis.

This study aimed to measure the correlation between miR-183 and gene expression that regulates apoptosis and adhesion mechanism that may be linked to the pathogenesis of endometriosis. Forty-four subjects, including 22 control subjects, participated in this study. We collected ectopic endometriosis and endometrial samples. For the control, the sample was taken from endometrial tissue through pipelle biopsy. RNA was extracted from all tissues using RNA mini kit, and the expression was assessed using quantitative-real time PCR. Relative mRNA and miRNA expression were presented using the formula of the Livak method. The data were statistically analyzed using GraphPad Prism 8. The expression of Caspase-3, Survivin, Integrin β1 (ITGB1), matrix metalloproteinase-9 (MMP-9), and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) (adhesion- and apoptosis-related gene) were calculated using the relative expression method. We found significant differences in Caspase-3, Survivin, ITGB1, MMP-9, and TIMP-1 expression between ectopic endometriosis tissues of women with endometriosis compared to healthy endometrium. MMP-9, Survivin, and ITGB1 was significantly increased in the endometriosis group, while Caspase-3, TIMP-1, and miR-183 were significantly reduced in the endometriosis group. No correlation was found between the expression level of miR-183 and Caspase3, Survivin, ITGB1, and Cadherin in both tissue types. Despite the difference in expression levels of miR-183 and associated adhesion- and apoptosis-related genes, there was no significant association between miR-183 with specific adhesion and apoptosis genes in endometriosis tissue.

Modulation of Long Non-coding RNA FAS-AS1/FAS/Caspase3 Axis in Endometriosis: A Cross-sectional Study

ABSTRACT Background: An increasing number of studies have demonstrated that excessive proliferation and apoptosis play a pivotal role in the development of endometriosis. Aim: The aim of the study was to evaluate the expression of long non-coding RNA (lncRNA) FAS-AS1, FAS, soluble Fas (sFas) and caspase-3 in patients with different stages of endometriosis. Setting and Design: The design of the study was a cross-sectional study. Materials and Methods: The relative expression of lncRNA FAS-AS1 and FAs gene was evaluated by the quantitative real-time polymerase chain reaction in 60 ectopic endometrial samples from women with endometriosis in relation to 85 normal endometrial tissues from healthy women, whereas the protein level of sFAs in the peritoneal fluid samples and cleaved caspase-3 in ectopic and normal endometrial tissue samples were determined using the enzyme-linked immunosorbent assay and western blot, respectively. Furthermore, in silico analyses were performed to investigate protein–protein interactions as well as molecular function and cellular location of selected proteins. Statistical Analysis Used: The student’s t-test was used to analyse the difference between the means of the two groups. Results: The expression of FAS and sFas increased in endometriosis tissues as compared to the control group (P < 0.05). However, lncRNA FAS-AS1 and cleaved caspase-3 decreased in ectopic endometrial tissues compared to normal endometrial tissues and low lncRNA FAS-AS1 expression was correlated with disease stages. In addition, the in silico analysis revealed the importance of FAS/caspase3 in the biological processes involved in the development of endometriosis. Conclusion: The current study suggests that lncRNA FAS-AS1 may function as an ectopic endometriotic suppressor. Moreover, the results showed that severity of endometriosis is also closely correlated with the expression of lncRNA FAS-AS1 and sFAS.

Different Effect of Dienogest on Endometrium Mesenchymal Stem Cells Derived from Healthy and Endometriosis Tissues

Endometriosis (EM) is an inflammatory condition in which the endometrium is observed to develop outside the uterine cavity. Endometrium has conventionally been recognized as a rich source of endometrial mesenchymal stem cells (E-MSCs). The influence of dienogest, a medication frequently prescribed for EM, on E-MSCs has not been extensively investigated. To explore effects of dienogest on the E-MSCs derived from healthy (E-MSCs-control) and diseased (E-MSCs-endometriosis) endometrial tissue samples in vitro. In vitro study. We collected samples from healthy and diseased endometrial tissues. E-MSCs were derived from both healthy and EM tissues. The effect of dienogest (VISANNE) on E-MSCs was assessed by examining cell proliferation, telomerase activity, cell migration, and estrogen secretion levels after the isolation and characterization of E-MSCs. We discovered that cellular proliferation rate was higher in the E-MSCs derived from EM tissues compared to those derived from healthy tissue. The proliferation rate and telomerase activity were both suppressed by dienogest treatment, particularly in E-MSCs-endometriosis. The drug treatment also resulted in a decrease in the migration capacity of E-MSCs-endometriosis, from 60.4% to 59.2%. The expression of CXCL12, Ki67, and beta-catenin was analyzed in both E-MSCs-endometriosis and E-MSCs-control. The CXCL12 and Ki67 expressions were quite elevated in the E-MSCs-endometriosis without drug treatment compared to the E-MSCs-control. Following the treatment, these levels declined drastically to the levels close to E-MSCs-control. Similarly, this decrease in gene expression was accompanied by a decrease in estrogen secretion into the medium. This research demonstrates that dienogest exerts a substantial impact on both stromal and stem cells, as it effectively controls the disease by reversing EM markers, despite the absence of progesterone receptors on endometrial stem cells.

Origin of Peritoneal Cancer With Features of High-grade Serous Carcinoma: A Detailed Molecular Analysis.

Primary peritoneal cancer has characteristics similar to high-grade serous carcinomas of ovarian and fallopian tube origin. However, the relationship between endometriosis and primary peritoneal cancer is not well understood. This study focuses on a case of peritoneal cancer in a patient who had undergone hysterectomy and bilateral salpingo-oophorectomy 5yr before. In addition to morphology, there was a positive for TP53 in immunohistochemistry and homologous recombination deficiency test, which were similar to high-grade serous carcinomas. However, WT1 was negative in the tumor, and extensive endometriosis coexisted. To reveal the clonal relationship between tumor and endometriosis, we dissected 3 sites each from the tumor and endometriosis and performed whole-exome sequencing analysis. As a result, we found that the tumors were of identical origin. Contrarily, no shared mutations were found in the 3 endometriosis sites. Furthermore, several shared mutations were found between the tumor and 1 endometriosis tissue, showing that the tumor and 1 ectopic endometrial gland originated from the same clone. This study indicates that several peritoneal cancers may be derived from endometriosis. We should consider the possibility of more diverse origins of peritoneal cancer than we speculated before.

Progressively Diminished Prostaglandin E2 Signaling in Concordance with Increasing Fibrosis in Ectopic Endometrium.

The prostaglandin E2 (PGE2) signaling has traditionally been viewed to play a pivotal role in endometriosis, linking inflammation and hyperestrogenism. We have previously reported that asectopic endometrium becomes more fibrotic, the expression of both COX-2 and PGE2 receptors (EP2 and EP4) are reduced. This study further investigatedwhether the expression levels of genes involved in the biosynthesis and metabolism of PGE2in ectopic endometrium diminish in concordance with increasing lesional fibrosis. We performed immunohistochemistry analyses of COX-2, mPGES-1, mPGES-2, cPGES, 15-PGDH, EP2 and EP4 and Masson trichrome staining for ovarian endometrioma (OE), adenomyosis (AD), and deep endometriosis (DE) tissue samples and control endometrial tissue samples (CT). Gene and protein expression analyses were performed by real-time RT-PCR and Western blotting, respectively. We found that as the extent of lesional fibrosis increased, immunoexpression of COX-2, mPGES-1/2, cPGES, EP2 and EP4 in OE lesions was increased but no change in these genes/proteins in DE lesions as compared with CT. Immunoexpression of COX-2 was found to be reduced while that of 15-PGDH was found to be elevated in DE lesions. In AD lesions, only EP2 and COX-2 were overexpressed. Thus, our data indicate that when the extent of lesional fibrosis is high, the PGE2 signaling pathway is depressed, manifesting as reduced COX-2 expression and elevated expression of 15-PGDH. They underscore the fact that not all ectopic endometria are the same and equal, and highlight the importance of the extracellular matrix in shaping the lesional behavior and response to drug treatment.

Exploring the Influence of IL-8, IL-10, Patient-Reported Pain, and Physical Activity on Endometriosis Severity.

Endometriosis is known to be a chronic, debilitating disease. The pathophysiological mechanisms of endometriosis development include local chronic inflammation and a certain degree of local immune deficit. We investigated the relationship between the endometriosis severity, IL-8, IL-10, BDNF, VEGF-A serum and tissue levels, patient-related pain, and physical activity in a cohort of 46 patients diagnosed with endometriosis who underwent surgery. The same panel of biomarkers was investigated in a control group of 44 reproductive-aged patients with non-endometriotic gynecological pathology who underwent surgical intervention. Our data show a high statistical significance between tissue expression of IL-8, IL-10, patient-related pain, and the severity of endometriosis. No relationship was identified between serum or tissue levels of VEGF-A and BDNF and the severity of endometriosis. These results validate the presence of local chronic inflammation and immune deficit, thereby creating, alongside other studies in the field, an opportunity for the development of innovative and personalized treatment approaches in endometriosis.

Endometriosis and Sjögren's syndrome: Bidirectional associations in population-based 15-year retrospective cohorts.

Primary Sjögren's syndrome (pSS) is a chronic autoimmune disorder affecting salivary and lacrimal glands, while endometriosis involves uterine-like tissue growth outside the uterus, causing pelvic pain and infertility. Investigating their intricate relationship using real-world data is crucial due to limited research on their connection. This population-based cohort study included patients with endometriosis and controls without endometriosis. Propensity score matching was used to balance baseline differences in demographic and clinic characteristics between the two groups. Cox proportional hazards model were used to estimate the effect of endometriosis on the risk of new-onset pSS over time. A symmetrical cohort study, including patients with pSS and propensity score-matched controls without pSS, was conducted to investigate the effect of pSS on the risk of endometriosis over time. To elaborate on the mechanisms linking endometriosis and pSS, Ingenuity Pathway Analysis was performed to identify activated pathways in eutopic endometrium from patients with endometriosis and parotid tissues from patients with pSS. A total of 15 947 patients with endometriosis and 15 947 propensity score-matched controls without endometriosis were included. Patients with endometriosis presented a significantly greater risk of pSS compared to non-endometriosis controls (adjusted hazard ratio, aHR = 1.57, 95% CI = 1.29-1.91, p < 0.001). In the symmetrical cohort study, which included 4906 pSS patients and 4,906 propensity score-matched controls without pSS, patients with pSS were found to be at a significantly higher risk of endometriosis compared to non-pSS controls (aHR = 1.51, 95% CI = 1.12-2.04, p = 0.012). Ingenuity Pathway Analysis showed that the underlying cellular mechanisms involved autoimmune-related pathways, including activation of dendritic cell maturation, and chronic inflammatory pathways,including the fibrosis signaling pathway. These findings support a bidirectional association between endometriosis and pSS, which may be driven by dendritic cell maturation and fibrosis signaling pathways.

Assessment of the Ferroptosis Regulators: Glutathione Peroxidase 4, Acyl-Coenzyme A Synthetase Long-Chain Family Member 4, and Transferrin Receptor 1 in Patient-Derived Endometriosis Tissue.

Ferroptosis, an iron-dependent form of non-apoptotic cell death, plays a pivotal role in various diseases and is gaining considerable attention in the realm of endometriosis. Considering the classical pathomechanism theories, we hypothesized that ferroptosis, potentially driven by increased iron content at ectopic sites, may contribute to the progression of endometriosis. This retrospective case-control study provides a comprehensive immunohistochemical assessment of the expression and tissue distribution of established ferroptosis markers: GPX4, ACSL4, and TfR1 in endometriosis patients. The case group consisted of 38 women with laparoscopically and histologically confirmed endometriosis and the control group consisted of 18 women with other gynecological conditions. Our study revealed a significant downregulation of GPX4 in stromal cells of endometriosis patients (M = 59.7% ± 42.4 versus 90.0% ± 17.5 in the control group, t (54) = -2.90, p = 0.005). This finding aligned with slightly, but not significantly, higher iron levels detected in the blood of endometriosis patients, using hemoglobin as an indirect predictor (Hb 12.8 (12.2-13.5) g/dL versus 12.5 (12.2-13.4) g/dL in the control group; t (54) = -0.897, p = 0.374). Interestingly, there was no concurrent upregulation of TfR1 (M = 0.7 ± 1.2 versus 0.2 ± 0.4 for EM, t (54) = 2.552, p = 0.014), responsible for iron uptake into cells. Our empirical findings provide support for the involvement of ferroptosis in the context of endometriosis. However, variances in expression patterns within stromal and epithelial cellular subsets call for further in-depth investigations.

Endometriotic Tissue-derived Exosomes Downregulate NKG2D-mediated Cytotoxicity and Promote Apoptosis: Mechanisms for Survival of Ectopic Endometrial Tissue in Endometriosis.

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a "protective shield" around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.

P-303 Calcitonin Gene-Related Protein as a key driver of myometrial fibrogenesis in adenomyosis pathology

Abstract Study question Does CGRP play a role in the fibrosis of adenomyosis lesions? Summary answer CGRP converts fibroblasts to myofibroblasts in adenomyosis lesions via the RAMP1 receptor and participates in the inflammatory activation processes. What is known already Adenomyosis is a prevalent and challenging disease in gynecology. It is characterized by the invasion of endometrial glands and mesenchyme into the myometrium, resulting in the development of limited or diffuse lesions. These lesions interact with in situ fibroblasts and recruit immune cells, leading to a chronic and persistent inflammatory microenvironment, which ultimately contributes to myometrial fibrosis. One of the typical clinical symptoms of adenomyosis is secondary progressive dysmenorrhea. Accumulated research suggests that sensory nerves, which are implicated in dysmenorrhea, and neuropeptides such as CGRP play a role in the development of ectopic lesions, thus establishing a feed-forward cycle. Study design, size, duration This study employed a laboratory-based experimental design. The in vitro experiment included a total of 67 clinical samples and the in vivo part included 57 ICR mice as subjects, conducted over 20 weeks, with data collection taking place between August 2023 and December 2023. Participants/materials, setting, methods Myometrium samples were collected from 37 women with adenomyosis, while control samples were obtained from 30 women without adenomyosis. Among them, the tissues from three adenomyosis patients and three control patients were subjected to scRNA-seq, and the other 61 samples were for validation. A mouse model of adenomyosis was established by administering tamoxifen. The mice were divided into three groups: a control group, a model group, and a treatment group that received Rimegepant via gavage. Main results and the role of chance In adenomyosis tissue, an enrichment of sensory nerve fibers, elevated expression of nerve-related proteins（NGF, PGP9.5）, and increased CGRP expression and its receptor RAMP1 were observed. Furthermore, the qRT-PCR results demonstrated that the adenomyosis lesions were in a chronic inflammatory state which is consistent with the scRNA-seq result. Moreover, the scRNA-seq data suggested a high expression of the CGRP receptor (RAMP1) on fibroblasts, which plays a critical role in causing fibrosis in adenomyosis. Additionally, it was found that CGRP prompted the transition of fibroblasts to myofibroblasts (FMT), and the activated fibroblasts, in turn, attracted macrophages and stimulated their shift towards an M1 phenotype. These significant findings were further confirmed through experimental animal studies. The fertility outcome indicated a positive trend, with the Rimegepant group showing an increased embryo implantation rate. Limitations, reasons for caution In the present experiment, while we have confirmed the involvement of CGRP in the fibrogenesis of adenomyosis, further investigation is needed to understand the specific biological processes occurring in fibroblasts following CGRP administration. Wider implications of the findings Limited research has been conducted on the association between adenomyosis and sensory nerves. This study adds the existing evidence by demonstrating the crucial role of sensory nerves and CGRP in promoting fibrosis. The findings of this study offer new perspectives on the treatment and understanding of the pathogenesis of adenomyosis. Trial registration number not applicable

New insights into molecular mechanisms underlying malignant transformation of endometriosis: BANCR promotes miR-612/CPNE3 pathway activity

Research questionDoes LncRNA BANCR promote the malignant transformation of endometriosis by activating the miR-612/CPNE3 pathway? DesignThe expression patterns of BANCR, miR-612 and CPNE3 in normal endometrium, eutopic endometrium from endometriosis, eutopic endometrium or malignant tissues from endometriosis-associated ovarian cancer. On the basis of primary normal endometrial stromal cells (NESC) and eutopic endometrial stromal cells (EESC), the regulatory relationships between BANCR, miR-612 and CPNE3, and the potential mechanisms that promote the malignant transformation of endometriosis, were elucidated in vitro and in vivo. ResultsThe expression levels of BANCR and CPNE3 were lowest in normal endometrium, significantly increased in eutopic endometrium (P < 0.05) and was significantly increased in eutopic endometrium (P < 0.05). During the malignant transformation of endometriosis, the expression levels of BANCR and CPNE3 were significantly upregulated (P < 0.05), whereas those of miR-612 were significantly downregulated (P < 0.05). miRNA-612 was found to target BANCR and CPNE3. The overexpression and knockdown of BANCR in NESC and EESC upregulated and downregulated the expression of CPNE3 and promoted or prevented cell proliferation and migration, respectively; these effects were reversed by miR-612 mimics and inhibitor. These changes were all statistically significant (P < 0.05). In-vivo experiments revealed that BANCR significantly increased the survival of subcutaneous endometrial cells by regulating miR-612/CPNE3 (P < 0.05). ConclusionThe expression of BANCR gradually increased with the progression of endometriosis during malignant transformation, and promoted the proliferation and migration of endometrial cells via the miR-612/CPNE3 pathway. BANCR may represent a novel target for monitoring the malignant transformation of endometriosis.

(042) IMMUNOSTAINING FOR MARKERS OF MAST CELLS AND NERVES IN BOTH EXCISED VESTIBULAR AND ENDOMETRIOSIS IMPLANT TISSUES IN A SINGLE PATIENT

Abstract Introduction Neuroproliferative vestibulodynia (NPV) is a chronic genital pain condition characterized by severe vestibular allodynia and hyperalgesia. The clinically suspected diagnosis of NPV is confirmed in excised vestibular tissue by immunohistochemical staining of &amp;gt; 8 cells per 100x magnification positively immunostained for CD 117, a mast cell marker, and 10 times the area compared to controls positively immunostained for PGP 9.5, a neuronal marker, Endometriosis, on the other hand, is a chronic pelvic pain condition associated with growth of ectopic endometrial-like tissue outside the uterus. The clinically suspected diagnosis is confirmed in excised endometriosis tissue revealing, in some patients, endometrial type glands and/or stroma. In patients with NPV and endometriosis, it is hypothesized that excess mast cells release cytokines and growth factors that promote neuroproliferation in their respective tissues, resulting in chronic genital and pelvic pain symptoms, respectively. In a study involving 65 patients with pathologically confirmed NPV, 63% reported having additional conditions associated with aberrant mast cell activity, including endometriosis. The conditions of NPV and endometriosis may both be focal to the vestibule and pelvis, respectively, but both may also share pathologies of aberrant mast cell activity with other systemic disorders. Objective To perform immunostaining for markers of mast cells and nerves in excised vestibular and endometriosis implant tissues from a single patient. Methods Fixed, paraffin-embedded vestibular tissue specimens from the 1:00-11:00 and 12:00 vestibule, and from excised endometriosis fibroadipose tissue on the rectosigmoid colon, were sectioned and immunostained for CD 117 (C-kit) and PGP 9.5. The immunostained slides were examined and digital photomicrographs taken. A minimum of 2 photomicrographs were examined at 100x magnification for each stained slide, identifying representative regions of the excised tissue. Manual counting of CD117 and PGP 9.5 immunopositive profiles, cells/nerves per 100x magnification, was performed for each photomicrograph. Results A 23-year-old woman presented with a history marked by painful and prolonged menstrual periods, inability to insert tampons, and discomfort with both entry and deep thrusting during intercourse. After excluding other forms of vestibulodynia, she underwent complete 1:00-11:00 and 12:00 subepithelial vestibulectomy with vaginal advancement flap reconstruction. Post-operatively, she reported “very much better” symptoms, with a reduction of entrance dyspareunia, but continued to experience deep thrusting pain with penetrative sexual activity. She subsequently underwent endometriosis excision surgery with positive lesions identified in 12 locations in the pelvis and retroperitoneum. Tissue stained for CD117 and PGP9.5 analyzed by manual counting exhibited &amp;gt; 20 immunopositive cells and nerves per 100x microscopic field in the epithelial basement membrane and adjacent subepithelial regions of the 1:00-11:00 and 12:00 vestibule, as well as in the endometriosis fibroadipose tissue on the rectosigmoid colon. Conclusions We report for the first time, in a single patient diagnosed with both NPV and endometriosis, immunostaining findings of high densities of mast cells and nerves in their respective tissues. These findings reinforce the hypothesis that NPV and endometriosis are linked to a broader condition associated with aberrant mast cell activity. Disclosure No.

Highly expressed lncRNA H19 in endometriosis promotes aerobic glycolysis and histone lactylation.

Abnormal glucose metabolism may be involved in the pathogenesis of endometriosis. The present study identifies that highly expressed H19 leads to increased aerobic glycolysis and histone lactylation levels in endometriosis. Previous studies from our group and others have shown increased IncRNA H19 expression in both the eutopic endometrium and the ectopic endometriosis tissue during endometriosis. In this study, we use immunofluorescence, immunohistochemistry, and protein quantification to determine that levels of aerobic glycolysis and histone lactylation are increased in endometriosis tissues. In human endometrial stromal cells, we found that high H19 expression resulted in abnormal glucose metabolism by examining the levels of glucose, lactate, and ATP and measuring protein levels of enzymes that participate in glycolysis. At the same time, immunofluorescence and western blotting demonstrated increased histone lactylation in H19 overexpressing cells. Altering aerobic glycolysis and histone lactylation levels through the addition of sodium lactate and 2-deoxy-d-glucose demonstrated that increased aerobic glycolysis and histone lactylation levels resulted in enhanced cell proliferation and cell migration, contributing to endometriosis. To validate these findings in vivo, we constructed an endometriosis mouse model, demonstrating similar changes in endometriosis tissues in vivo. Both aerobic glycolysis and histone lactylation levels were elevated in endometriotic lesions. Taken together, these data demonstrate elevated expression levels of H19 in endometriosis patients promote abnormal glucose metabolism and elevated histone lactylation levels in vivo, enhancing cell proliferation and migration and promoting the progression of endometriosis. Our study provides a functional link between H19 expression and histone lactylation and glucose metabolism in endometriosis, providing new insights into disease mechanisms that could result in novel therapeutic approaches.

Oestrogen promotes the progression of adenomyosis by inhibiting CITED2 through miR-145

Research questionIs the microRNA miR-145 involved in adenomyosis, and by what mechanisms does it affect disease development and is itself regulated? DesignFluorescence in-situ hybridization was used to observe the expression pattern of miR-145 in adenomyosis ectopic endometrium (n = 13), adenomyosis eutopic endometrium (n = 15) and non-adenomyosis eutopic endometrium (n = 14). RNA sequencing was used to screen target genes as well as downstream pathways of miR-145, which were validated by reporter gene assay, quantitative polymerase chain reaction and western blot, and further analysed using cell migration assay and chromatin immunoprecipitation assay. ResultsThe fluorescence in-situ hybridization assay revealed a noteworthy elevation in miR-145 expression in adenomyosis tissue compared with non-adenomyosis tissue. Furthermore, RNA sequencing analysis revealed that overexpression of miR-145 resulted in heightened expression of genes associated with the cytokine signalling pathway, nucleotide-binding and oligomerization domain-like pathway and adhesion pathway, including IL-1β and IL-6. Our study has identified CITED2 as a downstream direct target gene of miR-145, which is implicated in the inhibition of stromal cell migration induced by miR-145. Moreover, chromatin immunoprecipitation was used to validate the direct effect of oestradiol on the promoter region of miR-145, mediated by oestrogen receptor α, which facilitates the upregulation of miR-145 expression. ConclusionOur findings provide evidence supporting the role of oestradiol, acting through its receptor α, in modulating the discovered miR-145-CITED2 signalling axis, thereby promoting the progression of adenomyosis.