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The purinergic receptor P2X ligand-gated ion channel 3 (P2X3) is crucially involved in peripheral nociceptive processes of somatic and visceral pain. Endometriosis pain is considered as a kind of inflammatory and neuropathic pain. However, whether P2X3 is involved in endometriosis pain has not been reported up to date. Here, we aimed to determine whether P2X3 expression in endometriotic lesions is involved in endometriosis pain, which is regulated by inflammatory mediators through extracellular regulated protein kinases (ERK) signalling pathway. We found that P2X3 expressions in endometriosis endometrium and endometriotic lesions were both significantly higher as compared with control endometrium (P<0.05), and both positively correlated with pain (P<0.05). The expression levels of phosphorylated –ERK (p-ERK), phosphorylated-cAMP-response element binding protein (p-CREB), and P2X3 in endometriotic stromal cells (ESCs) were all significantly increased in comparison to the initial levels after treated with interleukin (IL)-1β (P<0.05) or adenosine triphosphate (ATP) (P<0.05), respectively, and did not increase after the ESCs were pre-treated with ERK1/2 inhibitor. Additionally, P2X3 and calcitonin gene related peptide (CGRP) were co-expressed in endometriotic lesions. These obtained results suggest that P2X3 might be involved in endometriosis pain signal transduction via ERK signal pathway. [ABSTRACT FROM AUTHOR]

Background: Many factors can affect the recurrence of endometriosis after surgery, however, whether endometriotic cyst fluid contributes to endometriosis recurrence after surgical excision of ovarian endometriomas remains unclear. The objective of this study was to determine the presence of endometrial cells in ovarian endometriosis cyst fluid and the potential differences between these cells and those in the cyst wall. Methods: Samples of cyst fluid (n = 39) and drainage fluid (n = 14) were collected from patients with ovarian endometriomas undergoing laparoscopic surgery. Drainage fluid from 14 patients without endometriosis was used as a control. The presence of endometrial cells in cyst fluid and drainage fluid was determined by cell culture in vitro and immunostaining. In addition, cyst fluid endometrial fragments and viscosity were analysed by transcriptome sequencing analysis and apparent diffusion coefficients, respectively. An animal model was used to confirm the ability of endometrial cells in cyst fluid to form new lesions. Results: We found endometrium-like tissues in 71.8% (28/39) of cyst fluid and 71.4% (10/14) of drainage fluid samples by histopathological examination, and the presence of endometrioid tissue in cyst fluid was related to the viscosity of the cyst fluid. The living endometrial cells in cyst fluid and drainage fluid were confirmed by cell culture in vitro and immunostaining. Moreover, the adhesion ability of endometrial fragments in cyst fluid was significantly higher than that of ectopic tissues in the cyst wall (P < 0.05). In addition, living endometrial cells in the cyst fluid were able to adhere and alive in the animal model. Conclusions: The existence of living endometrial cells with high adhesion ability in ovarian endometriotic cyst fluid may contribute to the recurrence of endometriosis after surgical excision of endometriomas due to cyst fluid outflow during the surgical procedure. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is a refractory and recurrent disease and it affects nearly 10% of reproductive-aged women and 40% of infertile patients. The commonly accepted theory for endometriosis is retrograde menstruation where endometrial tissues invade into peritoneal cavity and fail to be cleared due to immune dysfunction. Therefore, the comprehensive understanding of immunologic microenvironment of peritoneal cavity deserves further investigation for the previous studies mainly focus on one or several immune cells. Results: High-quality transcriptomes were from peritoneal fluid samples of patients with endometriosis and control, and firstly subjected to 10 × genomics single-cell RNA-sequencing. We acquired the single-cell transcriptomes of 10,280 cells from endometriosis sample and 7250 cells from control sample with an average of approximately 63,000 reads per cell. A comprehensive map of overall cells in peritoneal fluid was first exhibited. We unveiled the heterogeneity of immune cells and discovered new cell subtypes including T cell receptor positive (TCR+) macrophages, proliferating macrophages and natural killer dendritic cells in peritoneal fluid, which was further verified by double immunofluorescence staining and flow cytometry. Pseudo-time analysis showed that the response of macrophages to the menstrual debris might follow the certain differentiation trajectory after endometrial tissues invaded into the peritoneal cavity, that is, from antigen presentation to pro-inflammation, then to chemotaxis and phagocytosis. Our analyses also mirrored the dysfunctions of immune cells including decreased phagocytosis and cytotoxic activity and elevated pro-inflammatory and chemotactic effects in endometriosis. Conclusion: TCR+ macrophages, proliferating macrophages and natural killer dendritic cells are firstly reported in human peritoneal fluid. Our results also revealed that immune dysfunction happens in peritoneal fluid of endometriosis, which may be responsible for the residues of invaded menstrual debris. It provided a large-scale and high-dimensional characterization of peritoneal microenvironment and offered a useful resource for future development of immunotherapy. [ABSTRACT FROM AUTHOR]

Study Question: Does P2X ligand-gated ion channel 3 (P2X3) play a role in endometriosis pain?Summary Answer: Upregulation of P2X3 in dorsal root ganglia (DRG) tissues via the activating transcription factor 3 (ATF3)/activator protein (AP)-1 pathway contributed to endometriosis-associated hyperalgesia, which could be attenuated by the chitosan oligosaccharide stearic acid (CSOSA)/liposomes (LPs)/SP600125 delivery system.What Is Known Already: Infiltrating nerve fibers and elevated nociceptors in endometriotic lesions are associated with endometriosis pain. P2X3 has been demonstrated to play an important role in neuropathic pain.Study Design, Size, Duration: A rat model of endometriosis was used to investigate the signaling pathways involved in P2X3-induced pain.Participants/materials, Setting, Methods: Degrees of hyperalgesia, endogenous adenosine 5'-triphosphate (ATP) contents and P2X3 expression levels in endometriotic lesions and DRG tissues were detected in a rat model of endometriosis. The expression levels of ATF3 and P2X3 were measured using qRT-PCR, western blot analysis and immunofluorescence analysis after adenosine 5'-diphosphate (ADP) exposure in DRG cells. Plasmids encoding ATF3 and its siRNA were used to investigate the role of ATF3 on ADP-induced P2X3 upregulation. The activity of ATF binding to the P2X3 promoter was evaluated by using chromatin immunoprecipitation (CHIP) and luciferase assays. SP600125, an inhibitor of c-JUN N-terminal kinase, was wrapped in CSOSA/LPs delivery system and its inhibitory effects on ADP-induced upregulation of P2X3 in DRG cells and endometriosis-induced hyperalgesia in rats were tested.Main Results and the Role Of Chance: The concentrations of endogenous ATP and expression levels of P2X3 were significantly increased in both endometriotic lesions and DRG tissues in endometriosis rat models and were found to be positively correlated with the severity of hyperalgesia. In DRG cells, P2X3 expression levels were elevated by ADP stimulation, but dramatically inhibited by blocking ATF3 with its siRNA and SP600125. CHIP and luciferase assay showed that ADP increased the binding of ATF3 to the P2X3 promoter, resulting in an increase in P2X3 expression levels. In the CSOSA/LPs/SP600125 delivery system, the drug could be effectively concentrated in endometriotic lesions, and it could alleviate endometriosis-induced hyperalgesia, reduce the size of endometriotic lesions and attenuate upregulated P2X3 expression levels in endometriosis rat models.Large Scale Data: N/A.Limitations, Reasons For Caution: Changes in the sensitivity and function of P2X3 caused by endometriosis need to be further investigated.Wider Implications Of the Findings: This study indicates that ATP and the P2X3 receptor are involved in endometriosis pain, thus providing a novel therapeutic approach for the treatment of endometriosis pain by targeting the P2X3 receptor.Study Funding/competing Interest(s): This work was funded by National Key R&D Program of China (Grant No. 2017YFC1001202) and National Natural Science Foundation of China (Grant Nos. 81974225, 81671429 and 81471433). There are no competing interests. [ABSTRACT FROM AUTHOR]

Background: Endometriosis is defined as a chronic inflammatory disease. Recent studies have shown that increased coagulation parameters including fibrinogen and platelets are associated with endometriosis. The objective of this study was to determine the levels of inflammatory markers and coagulation parameters and their correlations in women with endometriomas compared to those with benign ovarian cysts or normal pelvic anatomy.Methods: Between June 2015 and June 2017, a total of 548 women who underwent laparoscopic/laparotomic surgery for ovarian endometriomas (OMA group, n = 226), non-endometriosis benign ovarian cysts (Cyst group, n = 210) and tubal reanastomosis (Control group, n = 112) were recruited in this study. Inflammatory markers including c-reactive protein (CRP), neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR) and coagulation parameters including platelet count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time, and plasma fibrinogen as well as CA-125 were determined.Results: Compared with Cyst group and Control group, TT and PT in OMA group were significantly shorter and plasma fibrinogen levels were significantly higher (P < 0.05). Moreover, the levels of plasma fibrinogen were positively correlated with CRP, NLR and PLR (P < 0.05). In addition, the confidence intervals for the area under the curve (AUC) for CA-125 × fibrinogen were significantly higher than those for CA-125 (0.904-0.952 vs. 0.899-0.949) in the diagnosis of endometrioma.Conclusions: These results indicate that women with endometriomas demonstrate a hypercoagulable status due to the inflammatory nature of endometriosis. The combined determination for CA-125 and fibrinogen demonstrate a higher area under the curve than the single detection of CA-125 in those with endometriomas compared to these with benign ovarian cysts.Trial Registration: This study was approved by the Human Ethics Committee of the Women's Hospital, School of Medicine, Zhejiang University (No.20170174) and all women provided written informed consent. [ABSTRACT FROM AUTHOR]

This study demonstrates that the crosstalk between endometrial cells and mast cells promotes the migratory ability of endometrial epithelial and stromal cells and angiogenesis via CCL8/CCR1, thereby contributing to the development of endometriosis. • Endometrial cells promote the secretion of CCL8 from mast cells by their crosstalk. • CCL8 promotes the migratory ability of endometrial cells and novel angiogenesis. • CCL8 takes its effects via CCL8/CCR1. • CCR1 antagonist suppresses the development of endometriosis. The density and the activity of mast cells are associated with endometriosis. However, the role of mast cells on the pathogenesis of endometriosis remains unclear. Our study aims to investigate whether endometrial cells interact with mast cells and the involvement of their crosstalk in the development of endometriosis. The transwell assay was applied to investigate the effect of mast cells on the migratory ability of human primary endometrial cells. Mast cells were cocultured with endometrial epithelial and stromal cells respectively and total RNAs were isolated and subjected to mRNA sequencing. Next, the transwell assay, CCK-8, and tube formation were applied to study the role of CCL8 on the endometrial and endothelial cells in vitro. The mouse model was also established to confirm the role of CCL8 in the development and angiogenesis of endometriosis. CCL8 was up-regulated in mast cells when cocultured with endometrial cells. CCL8 was highly expressed in the ectopic endometrium and the serum of patients with endometriosis. CCL8 promoted the migratory ability of endometrial epithelial and stromal cells and increased the proliferation, migration, and tube formation of endothelial cells. CCR1, the receptor of CCL8, was over-expressed in the ectopic endometrium and colocalized with blood vessels in ovarian endometriomas. The inhibition of CCR1 suppressed the development and angiogenesis of endometriosis in vivo. The crosstalk between endometrial cells and mast cells in the development of endometriosis via CCL8/CCR1 was demonstrated, thereby providing a new treatment strategy for endometriosis. [ABSTRACT FROM AUTHOR]