Peritoneal Fluid Of Endometriosis Patients Research Articles

Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis

BackgroundAs a dual-function metabolite, succinate has emerged in cell function and plays a key signaling role in linking mitochondrial function to other cellular functions. Succinate accumulation in the cytoplasm is commonly associated with hypoxia in the microenvironment and immune cell activation. Extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, which boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate plays an important role in the development of inflammatory diseases. Whether succinate facilitates the progression of endometriosis (EMs), characterized by chronic inflammation and peritoneal adhesion, is worth exploring.ObjectiveWe mimicked the ectopic milieu in vitro and in vivo to evaluate the main source and potential role of succinate in endometriosis. We assessed the molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity. The effect of succinate/SUCNR1 signaling on ectopic endometrial stromal cells (ESCs) was further explored in this study.MethodsIn this study, we used targeted organic acid metabolomics analysis and in vitro assays to assess the potential accumulation of succinate in the peritoneal fluid of EMs patients. We examined its correlation with disease severity, Visual Analogue Scale, and the Endometriosis Fertility Index. Flow cytometry, enzyme linked immunosorbent assay, western blot assay, quantitative real-time PCR, and other molecular biology techniques were used to explore the potential mechanisms.ResultsBy mimicking the ectopic milieu, we constructed an in vitro co-culture system and found that M1 polarized macrophages and that the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarized the macrophages and significantly enhanced the invasive survival of ESCs, and the adhesion to the peritoneum. We further investigated the pathological effects of extracellular succinate in vivo using a xenograft mouse models of endometriosis.ConclusionsSuccinate-SUCNR1 signaling facilitates the creation of inflammatory cells and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanisms underlying succinate accumulation and function will help elucidate the phenotypic mysteries of pain and infertility in EMs.BkiB34NQBDvkGHWZYD1GB2Video

Expression of Gal-9 on Dendritic Cells and Soluble Forms of TIM-3/Gal-9 in Patients Suffering from Endometriosis.

Immune system dysregulation is clinically evident in the pathogenesis of endometriosis (EMS). Changes in the dendritic cells (DCs) activity or phenotype may be involved in the implantation and growth of endometrial tissue outside the uterus in the disease. The TIM-3/Gal-9 axis is implicated in the development of immune tolerance. However, the knowledge about the exact role of this pathway in the EMS is extremely poor. In the present study, we evaluated the expression of Gal-9 on myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the peripheral blood (PB) and peritoneal fluid (PF) of both EMS patients (n = 82) and healthy subjects (n = 10) via flow cytometry. We also investigated the concentrations of soluble Gal-9 and TIM-3 in the plasma and PF of EMS patients and the control group using ELISA. We showed significantly elevated percentages of mDCs-Gal-9+ and pDCs-Gal-9+, and significantly higher concentrations of the soluble form of Gal-9 and TIM-3 in the PF of EMS patients than in circulation. Our results led us to conclude that the accumulation of Gal-9 expressing mDCs and pDCs in the PF and high sTIM-3/Gal-9 production in the peritoneal cavity could represent the hallmark of immune regulation in EMS patients, which may augment the inflammatory process and development/maintenance of local immunosuppression.

Does the Use of the “Proseek® Multiplex Inflammation I Panel” Demonstrate a Difference in Local and Systemic Immune Responses in Endometriosis Patients with or without Deep-Infiltrating Lesions?

Endometriotic lesions are able to infiltrate surrounding tissue. This is made possible partly by an altered local and systemic immune response that helps achieve neoangiogenesis, cell proliferation and immune escape. Deep-infiltrating endometriosis (DIE) differs from other subtypes through the invasion of its lesions over 5 mm into affected tissue. Despite the invasive nature of these lesions and the wider range of symptoms they can trigger, DIE is described as a stable disease. This elicits the need for a better understanding of the underlying pathogenesis. We used the "Proseek® Multiplex Inflammation I Panel" in order to simultaneously detect 92 inflammatory proteins in plasma and peritoneal fluid (PF) of controls and patients with endometriosis, as well as in particular patients with DIE, in order to gain a better insight into the systemically and locally involved immune response. Extracellular newly identified receptor for advanced gycation end-products binding protein (EN-RAGE), C-C motif Chemokine ligand 23 (CCL23), Eukaryotic translation initiation factor 4-binding protein 1 (4E-BP1) and human glial cell-line derived neurotrophic factor (hGDNF) were significantly increased in plasma of endometriosis patients compared to controls, whereas Hepatocyte Growth factor (HGF) and TNF-related apoptosis inducing ligand (TRAIL) were decreased. In PF of endometriosis patients, we found Interleukin 18 (IL-18) to be decreased, yet Interleukin 8 (IL-8) and Interleukin 6 (IL-6) to be increased. TNF-related activation-induced cytokine (TRANCE) and C-C motif Chemokine ligand 11 (CCL11) were significantly decreased in plasma, whereas C-C motif Chemokine ligand 23 (CCL23), Stem Cell Factor (SCF) and C-X-C motif chemokine 5 (CXCL5) were significantly increased in PF of patients with DIE compared to endometriosis patients without DIE. Although DIE lesions are characterized by increased angiogenetic and pro-inflammatory properties, our current study seems to support the theory that the systemic immune system does not play a major role in the pathogenesis of these lesions.

Clinical Value of the PD-1/PD-L1/PD-L2 Pathway in Patients Suffering from Endometriosis.

The interaction between dendritic cells (DCs) and T cells mediated by the programmed cell death 1 (PD-1)/programmed cell death ligand 1 (PD-L1)/programmed cell death ligand 2 (PD-L2) pathway is the most important point in regulating immunological tolerance and autoimmunity. Disturbances in the quantity, maturity, and activity of DCs may be involved in the implantation and growth of endometrial tissue outside the uterus in endometriosis (EMS). However, little is known about the role of the immune checkpoint pathways in EMS. In our study, we examined the expression of PD-L1/PD-L2 on myeloid DCs (mDCs) and plasmacytoid DCs (pDCs) in the peripheral blood (PB) and peritoneal fluid (PF) of both EMS patients (n = 72) and healthy subjects (n = 20) via flow cytometry. The concentration of soluble PD-L1 and PD-L2 in the plasma and PF of EMS patients and the control group were determined using ELISA. We demonstrated an elevated percentage of mDCs, mDCs and pDCs with the PD-L1or PD-L2 expression, and a higher concentration of the soluble forms of PD-L1 and PD-L2 in the PF than in the plasma of EMS patients. We conclude that the peritoneal cavity environment and the PD-1/PD-L1/PD-L2 axis may play an important role in the modulation of immune response and the development and/or progression of EMS.

Oxidative stress-related effects on various aspects of endometriosis.

Endometriosis is a chronic and relatively common disease in women of childbearing age. Complications of this disease include a wide range of disorders. The cause of this disease is not known for sure, but several hypotheses have been proposed for it. In this review, an attempt has been made to discuss the effects of oxidative stress on various complications of endometriosis. In endometriosis, the entry of endometrial tissues into the peritoneal cavity causes oxidative stress through the Fenton reaction and inflammation in this site. Fenton reaction can produce reactive oxygen species through a catalytic form of iron. This process can provoke inflammatory responses and oxidative injury. As a result, the activity of macrophages and expression of nuclear factor-kappa B increase. Oxidative stress can be associated with many complications of endometriosis. It has been reported that in the peritoneal fluid of endometriosis patients, there are activated macrophages, growth factors, and high concentrations of cytokines. These conditions act as a toxic to embryo survival and sperm function. Novel therapeutic strategies must regulate intracellular ROS signaling to inhibit the adverse effects of ROS-induced endometriosis-promoting events. According to features of antioxidants, they may inhibit early events of the development of endometriosis.

CSF-1-induced DC-SIGN+ macrophages are present in the ovarian endometriosis

BackgroundResearchers have found that macrophages are the predominant cells in the peritoneal fluid (PF) of endometriosis patients. CSF-1 has been found to accumulate in the lesions and PF of endometriosis patients, and CSF-1 induces THP-1-derived macrophages to polarize toward a CD169+ DC-SIGN+ phenotype. Does the cytokine CSF-1 induce monocytes to differentiate into macrophages with a DC-SIGN+ phenotype in endometriosis?MethodsThe level of CSF-1 in the endometrium of control subjects, and the eutopic, and ectopic endometrium of endometriosis patients was evaluated by real-time polymerase chain reaction (qRT–PCR) and was determined by enzyme-linked immunosorbent assay (ELISA) in the PF of control and endometriosis patients. CSF-1 expression was examined with a MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. DC-SIGN+ macrophages were detected by immunohistochemical staining of tissues and flow cytometric analysis of the PF of control subjects (N = 25) and endometriosis (N = 35) patients. The phenotypes and biological activities of CSF-1 -induced macrophages were compared in an in vitro coculture system with peripheral blood lymphocytes from control subjects.ResultsIn this study, we found that the proportion of DC-SIGN+ CD169+ macrophages was higher in the abdominal immune microenvironment of endometriosis patients. CSF-1 was primarily secreted from ectopic lesions and peritoneum in mice with endometriosis. In addition, CSF-1 induced the polarization of macrophages toward a DC-SIGN+ CD169+ phenotype; this effect was abolished by the addition of an anti-CSF-1R antibody. CSF-1 induced the generation of DC-SIGN+ macrophages, leading to a depressed status of peripheral blood lymphocytes, including a high percentage of Treg cells and a low percentage of CD8+ T cells. Similarly, blockade with the anti-CSF-1R antibody abrogated this biological effect.ConclusionsThis is the first study on the role of DC-SIGN+ macrophages in the immune microenvironment of endometriosis. Further study of the mechanism and biological activities of CSF-1-induced DC-SIGN+ macrophages will enhance our understanding of the physiology of endometriosis.Graphical

The colonized microbiota composition in the peritoneal fluid in women with endometriosis.

The imbalance of microbiome in vivo is believed to be involved in the pathogenicity of endometriosis. This study aimed to investigate and analyze the composition of bacterial communities in the peritoneal fluid of women with endometriosis. To collect peritoneal fluid samples from women with (N = 36) and without (N = 25) endometriosis in a generalized hospital in Hunan, China during January to December of 2019. Genomic DNA was extracted from peritoneal fluid samples, and targeted amplified for the V4 region of 16S ribosomal RNA gene followed by amplicon sequencing. Non-parametric Wilcoxon rank-sum test and chi-squared test were used to compare and analysis the difference between groups. Analysis showed that microbiota diversity was similar in the peritoneal fluid of women with or without endometriosis. Ralstonia mainly dominated in the peritoneal fluid of patients in both groups, with an overall relative abundance of 11.15% (95% CI: 10.51-11.80%) in endometriosis patients, followed by Acinetobacter, Pseudomonas, Asticcacaulis, and Methyloversatilis, with no significant difference between endometriosis patients and the control group. Nevertheless, there were microbes with different abundance in peritoneal fluid of the two groups, and the relative abundance was less than 0.5%. Acidovorax (P = 0.01), Devosia (P = 0.03), Methylobacterium (P = 0.03), Phascolarctobacterium (P = 0.03), and Streptococcus (P = 0.04) were more abundant in the peritoneal fluid of endometriosis patients than the controls, while Brevundimonas (P = 0.01) and Stenotrophomonas (P = 0.04) were less abundant. The composition of minority microbiota including Acidovorax, Devosia, Methylobacterium, Phascolarctobacterium, and Streptococcus in peritoneal fluid were found to change among women with endometriosis. Further research is needed to explore the mechanisms of these microorganisms in the pathophysiology of endometriosis.

SCM-198 Alleviates Endometriosis by Suppressing Estrogen-ERα mediated Differentiation and Function of CD4+CD25+ Regulatory T Cells.

Background: Endometriosis (EMS), a typical endocrine immune disorder, associates with dramatically increased estrogen production and disorganized immune response in ectopic focus. Peritoneal regulatory T cells (Tregs) expansion in women with EMS and their pathogenic role attributable to endometriotic immunotolerance has been reported. Whether local high estrogen promotes EMS by discipling Tregs needs to be further explored. Up to date, there is no effective medicine for the treatment of EMS. SCM-198 is a synthetic leonurine with multiple physiological activities. Whether SCM-198 could regulate Tregs via estrogen and facilitate the radical cure of EMS has not yet been reported.Methods: Proportion of Tregs in peritoneal fluid of patients with EMS was firstly analyzed via flow cytometry. Peritoneal estrogen concentration and the mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ) of Tregs were detected by ELISA and RT-PCR, respectively. Grouped in vitro induction assays were performed to explore the effects of SCM-198 and estrogen signaling on Tregs. Cell invasion and viability assays were utilized to detect the crosstalk between Tregs and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Furthermore, EMS mice models were established to verify the therapeutic effects of SCM-198.Results: Increased Tregs were found in peritoneal fluid of EMS patients, accompanied with estrogen-ERα overactivation. Estrogen-ERα triggered the expansion of Tregs and their cytokine production (IL-10 and TGF-β1), which could be reversed by SCM-198 treatment. Moreover, SCM-198 abated the invasion and viability of eESCs enhanced by Tregs. In vivo experiments confirmed that SCM-198 obviously retarded the growth of ectopic lesions and downregulated the functions of Tregs via estrogen-ERα inactivation.Conclusions: These data suggest that SCM-198 attenuates Tregs expansion via the inhibition of estrogen-ERα signaling in EMS and offer a promising therapy for such a refractory disease.

The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

Metabolite profiles in the peritoneal cavity of endometriosis patients and mouse models

Research questionWhich metabolites are altered in the peritoneal cavity of women with endometriosis? Could the mouse endometriosis model simulate these alterations? DesignThirteen women with endometriosis and seven women with other benign gynaecological diseases, who underwent laparoscopic surgery, were included in this study. None had received hormonal therapy for 3 months before surgery. For the animal experiments, six and five mice were included in the endometriosis and control groups, respectively. Peritoneal fluid from the patients and peritoneal lavage fluid from the mice was collected and analysed. Non-targeted metabolomics via liquid chromatography with tandem mass spectrometry was used to identify the altered metabolites in the peritoneal fluid of endometriosis patients and mouse models. MetaboAnalyst 4.0 was used to visualize the data. ResultsSeveral metabolites in the peritoneal cavity were significantly altered in both humans and mice with endometriosis. Concentrations of lysophosphatidylcholine (LysopC) (P=0.017 in patients and P=0.041 in the mouse model) and derivatives of phosphoethanolamine (1-arachidonoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.027; 1-oleoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.0086; and phosphorylethanolamine in the mouse model, P=0.0027) were significantly up-regulated in both, whereas concentrations of acylcarnitines (l-palmitoylcarnitine, P=0.047; and stearoylcarnitine, P=0.029) and kynurenine (P=0.045) were significantly increased only in humans. The human and mouse samples shared three altered enriched metabolite sets. ConclusionsWomen with endometriosis show an altered metabolic state in the abdominal cavity. The endometriosis mouse model shared half of the significantly altered metabolite sets found in the abdominal cavity of humans.

IL-33 activates ILC2 expansion and modulates endometriosis

Abstract Introduction: Chronic inflammation drives endometriosis (EM) and its associated symptoms. Interleukin (IL)-33, has gained therapeutic interest as it is elevated in the plasma, peritoneal fluid (PF), and lesions of EM patients. However, how IL-33 contributes to EM is largely unknown. For the first time, we demonstrate, using both human samples and murine models, that IL-33 expands local group 2 innate lymphoid cells (ILC2s) and that IL-33 activated ILC2s modulate hallmark features of EM. Methods: We identified ILC2s (CD45+Lin−CRTH2+CD127+ST2+) in the PF of EM patients using flow cytometry. Using immunofluorescence, we determine whether IL-33 forms epithelial or stromal cell niches within the EM lesion. Then, using our well established EM model, we induced EM in female C57Bl/6 mice and treated with PBS or IL-33. Hallmark features of EM were assessed including inflammation (using multiplex cytokine analysis), immune cell profiling (using flow cytometry), and lesion architecture (using immunohistochemistry and Nanostring transcriptomic profiling). To establish cause and effect, we treated ILC2 deficient mice with PBS (n=5) or IL-33 (n=5). Unpaired t-tests and one-way ANOVAs were done when appropriate. Results/Discussion: In humans, ILC2s are present and elevated in EM patients (n=5) compared to healthy donors (n=3) and IL-33 colocalized with both epithelial and stromal cells within the EM lesion. Using murine models, IL-33 drove hallmark features of EM including increased inflammation, alterations in PF immune cells (including eosinophils, T cells, macrophages and ILC2s), as well as increased lesion proliferation, neurogenesis and fibrosis. Using ILC2 deficient mice, we show that IL-33 induced pathology is ILC2 dependent.

Characterization of exosomes in peritoneal fluid of endometriosis patients

ObjectiveTo demonstrate the feasibility of studying exosomes directly from peritoneal fluid, we isolated exosomes from endometriosis patient samples and from controls, and characterized their cargo.DesignCase-control experimental study.SettingAcademic clinical center.Patient (s)Women with and without endometriosis who underwent laparoscopic surgery (n = 28 in total).Intervention (s)None.Main Outcome Measure (s)Concentration of exosomes within peritoneal fluid and protein content of the isolated exosomes.Result (s)Peritoneal fluid samples were pooled according to the cycle phase and disease stage to form six experimental groups, from which the exosomes were isolated. Exosomes were successfully isolated from peritoneal fluid in all the study groups. The concentration varied with cycle phase and disease stage. Proteomic analysis showed specific proteins in the exosomes derived from endometriosis patients that were absent in the controls. Five proteins were found exclusively in the endometriosis groups: PRDX1, H2A type 2-C, ANXA2, ITIH4, and the tubulin α-chain.Conclusion (s)Exosomes are present in peritoneal fluid. The characterization of endometriosis-specific exosomes opens up new avenues for the diagnosis and investigation of endometriosis.

Neutrophil recruitment and function in endometriosis patients and a syngeneic murine model.

Endometriosis is a chronic inflammatory, gynecological disease characterized by the presence of endometrial-like tissue lesions outside of the uterus. Neutrophils are elevated in the systemic circulation and peritoneal fluid of endometriosis patients; however, whether and how neutrophils contribute to endometriosis pathophysiology remain poorly understood. With emerging roles for neutrophils in chronic and sterile inflammatory conditions, we sought to provide in-depth characterization of neutrophil involvement in endometriosis. We demonstrate that neutrophils reside within patient endometriotic lesions and that patient lesions possess a microenvironment that may influence neutrophil recruitment and function. We also provide the first evidence that systemic circulating neutrophils from endometriosis patients display distinct transcriptomic differences compared neutrophils from healthy control subjects. Time course characterization of our syngeneic, immunocompetent mouse model of endometriosis revealed that neutrophils are rapidly recruited to the peritoneal environment early after endometriotic lesion establishment and remain present in murine lesions long term. In vivo neutrophil depletion altered the systemic and peritoneal immune microenvironment of mice with endometriosis as demonstrated by changes in pro-inflammatory and angiogenic mediators. Taken together, these findings highlight a novel role for neutrophils in early events such as angiogenesis and modulation of the local inflammatory environment associated with endometriosis pathogenesis.

1856 Circulating Exosomal Long Noncoding RNA-TC0101441 as a Non-Invasive Biomarker for the Prediction of Endometriosis Severity and Recurrence

Study Objective Exosomal long noncoding RNAs (lncRNAs) are emerging as novel non-invasive biomarkers for various diseases. This study focused on TC0101441, a new malignant-related lncRNA identified by our group, and aimed to investigate: (1) the involvement of TC0101441 in endometriosis, (2) the presence of TC0101441 in exosomes derived from serum and peritoneal fluid of endometriosis patients, (3) the potential of circulating serum exosomal TC0101441 as a biomarker for endometriosis. Design Clinical retrospective study (3 years follow-up). Setting Obstetrics and Gynecology Hospital of Fudan University. Patients or Participants Ninety two patients with ovarian endometriotic cysts and thirty control non-endometriotic patients were recruited from 2014 to 2016. Interventions N/A. Measurements and Main Results The distribution and expression of TC0101441 in ectopic, eutopic and normal endometria was evaluated using fluorescence in situ hybridization and real-time polymerase chain reaction. Exosomes were extracted from serum and peritoneal fluid of endometriosis patients and confirmed using transmission electron microscopy, nanoparticle tracking analysis and western blotting. We observed that TC0101441 was mainly located and highly expressed in ectopic stromal endometria than that in the paired eutopic and normal endometria (P Conclusion Circulating exosomal TC0101441 can serve as a promising biomarker for the prediction of endometriosis severity and recurrence.

Expression and significance of autophagy genes LC3, Beclin1 and MMP-2 in endometriosis.

Expression of autophagy-related proteins, microtubule-associated protein light chain 3 (LC3) and Beclin1, and matrix metalloproteinase-2 (MMP-2) was investigated in serum and peritoneal fluid of patients with endometriosis (EM). The messenger ribonucleic acid (mRNA) expression levels of MMP-2, LC3 and Beclin1 in endometrial tissues of EM patients and correlation of these genes with EM and their significance were evaluated. The serum, peritoneal fluid and endometrial tissues of 84 patients treated in The First Affiliated Hospital of Qiqihar Medical University (Qiqihar, China) from March 2016 to March 2017 were collected. The serum, peritoneal fluid and endometrial tissues of 42 EM patients were used as the experimental group, while those of 42 non-EM patients were used as the control group. The levels of LC3, Beclin1 and MMP-2 in serum and peritoneal fluid of EM patients and non-EM patients were quantitatively detected via enzyme-linked immunosorbent assay (ELISA), followed by comparative analysis based on data in both groups. In addition, mRNA expression of LC3, Beclin1 and MMP-2 in the endometrium in both groups were detected via reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and differences in expression of these genes between the groups were analyzed and evaluated. Correlation of LC3, Beclin1 and MMP-2 with EM was explored. Results of ELISA showed that levels of LC3 and Beclin1 in the EM group were significantly lower than those in the control group, while levels of MMP-2 in serum and peritoneal fluid of the EM group were significantly higher than those in the control group (P<0.05). Results of RT-qPCR revealed that mRNA expression of LC3 and Beclin1 in the endometrium of patients in the EM group were obviously decreased compared with those in the control group, while the expression of MMP-2 was high, and differences in expression were statistically significant (P<0.05). The expression of MMP-2 is high, and expression of LC3 and Beclin1 is low in serum, peritoneal fluid and endometrium of EM patients, and investigating the expression of MMP-2, LC3 and Beclin1 in EM is helpful to further clarify the pathogenesis of EM, and guide the clinical diagnosis and treatment.

The expressions and the roles of SDF-1/CXCR-4 and SDF-1/CXCR-4 in human endometriosis

Objective: To investigate the expressions and significances of SDF-1 and its receptor CXCR-4 in endometriosis. Methods: 50 hysteromyoma patients treated at Beijing Tongren Hospital, Capital Medical University between 1(st) January 2016 and 31(st) December 2017 were divided into control group, that is, non-endometriosis group, while another 50 endometriosis patients were divided into experimental group.The endometrial tissues, endometriosis lesions, and peritoneal fluid samples of hysteromyoma patients were collected by operation.RT-PCR, ELISA and immunohistochemistry were adopted to detect the expressions of SDF-1 and CXCR-4 in the two groups.Cell count was used to analyze the roles of SDF-1 and CXCR4 in the mitosis and proliferation of endometrial stromal cells. Results: The mean value of SDF-1 and CXCR-4 expressions in ascites or peritoneal fluid of endometriosis patients were (2.56±0.33) mg/L and (4.47±0.32) mg/L, respectively. The mean concentrations in ascites or peritoneal fluid in hysteromyoma patients were (1.39±0.36) mg/L and (3.16±0.32) mg/L, respectively.The expressions of SDF-1 and CXCR-4 in ascites or peritoneal fluid of endometriosis patients were both significantly higher than those of patients in non-endometriosis group (P<0.05). SDF-1 and CXCR-4 were expressed in both endometriosis lesions and the glandular epithelial cells and mesenchymal cells of normal endometrial tissue.Positive staining sites were located in the cytoplasm.A value was used to calculate and analyze the expression of immune staining.The mean A value of SDF-1 and CXCR-4 in endometriosis group were 0.21±0.13 and 0.21±0.13, respectively, and the mean A value in normal endometrial tissues were 0.15±0.13 and 0.14±0.13, respectively.The expressions of these two in endometrial tissues were significantly higher than that in normal endometrial tissues, and the differences were statistically significant (P<0.05). The expression of CXCR-4 mRNA was abundant in the mesenchymal cells of endometriosis cultured in vitro.SDF-1 promoted the mitosis and proliferation of endometrial stromal cells cultured in vitro in a dose-dependent manner.The neutralizing antibody against CXCR-4 was obviously inhibited. Conclusion: The high expressions of SDF-1 and CXCR-4 in endometriosis as well as SDF-1 through its specific receptor CXCR-4 promoted the mitosis and proliferation of endometrial stromal cells, suggesting that SDF-1 and CXCR-4 played important roles in the pathogenesis of endometriosis.

Inflammatory cytokines IL-6, IL-10, IL-13, TNF-α and peritoneal fluid flora were associated with infertility in patients with endometriosis.

To investigate the correlations of inflammatory factors, interleukin-6 (IL-6), IL-10, IL-13 and tumor necrosis factor-α (TNF-α), and composition of bacterial flora in the peritoneal fluid with infertility in endometriosis patients. A total of 55 patients diagnosed with endometriosis and infertility in the Gynecology Clinic of our hospital from June 2014 to July 2017 were selected as observation group, and another 30 non-endometriosis and non-infertility patients were enrolled as control group. The peritoneal fluid was extracted from patients in both groups, and the total white cell count and the percentage of leukocyte subset were determined. The total genome deoxyribonucleic acid (DNA) of microorganisms in peritoneal fluid was extracted, and the composition of microorganisms was analyzed using the Ion Torrent PGM platform (BGI). The levels of IL-6, IL-10, IL-13, and TNF-α in peritoneal fluid were detected via enzyme-linked immunosorbent assay (ELISA). Moreover, the correlations of inflammatory factors in the peritoneal fluid with endometriosis complicated with infertility were analyzed via Logistic regression analysis. The total white cell count, monocytes, neutrophils, eosinophils and basophils in endometriosis patients complicated with infertility were significantly higher than those in control group (p<0.05). Results of ELISA showed that the levels of IL-6, IL-10, IL-13, and TNF-α in peritoneal fluid of endometriosis patients complicated with infertility were significantly higher than those in control group (p<0.05). In peritoneal fluid of patients in both groups, Proteobacteria and Firmicutes were mainly dominated, followed by Actinobacillus, Bacteroidetes, Fusobacteria and Tenericutes, and there was no significant difference in the Eumycota between the two groups (p>0.05). Logistic regression analysis results showed that there were significant correlations of inflammatory factors (IL-6, IL-10, IL-13, and TNF-α) with endometriosis complicated with infertility. There are many kinds of Eumycota in the peritoneal fluid of endometriosis patients complicated with infertility, but they are not the main pathogenic factors. Inflammatory factors (IL-6, IL-10, IL-13, and TNF-α) can be used as important reference indexes for the diagnosis of endometriosis complicated with infertility.

Suppressive regulatory T cells and latent transforming growth factor-\u03b2-expressing macrophages are altered in the peritoneal fluid of patients with endometriosis

BackgroundEndometriosis is a known cause of infertility. Differences in immune tolerance caused by regulatory T cells (Tregs) and transforming growth factor-β (TGF-β) are thought to be involved in the pathology of endometriosis. Evidence has indicated that Tregs can be separated into three functionally and phenotypically distinct subpopulations and that activated TGF-β is released from latency-associated peptide (LAP) on the surfaces of specific cells. The aim of this study was to examine differences in Treg subpopulations and LAP in the peripheral blood (PB) and peritoneal fluid (PF) of patients with and without endometriosis.MethodsPB and PF were collected from 28 women with laparoscopically and histopathologically diagnosed endometriosis and 20 disease-free women who were subjected to laparoscopic surgery. Three subpopulations of CD4+ T lymphocytes (CD45RA+FoxP3low resting Tregs, CD45RA−FoxP3high effector Tregs, and CD45RA−FoxP3low non-Tregs) and CD11b+ mononuclear cells expressing LAP were analyzed by flow cytometry using specific monoclonal antibodies.ResultsProportions of suppressive Tregs (resting and effector Tregs) were significantly higher in the PF samples of patients with endometriosis than in those of control women (P = 0.02 and P < 0.01, respectively) but did not differ between the PB samples of patients and controls. The percentage of CD11b+LAP+ macrophages was significantly lower in PF samples of patients with endometriosis than in those of controls (P < 0.01) but was not altered in the PB samples.ConclusionProportions of suppressive Tregs and LAP+ macrophages are altered locally in the PF of endometriosis patients.

Fas-Related Apoptosis of Peritoneal Fluid Macrophages in Endometriosis Patients: Understanding the Disease.

Recent studies of the peritoneal cavity environment in endometriosis demonstrate quantitative and qualitative changes in the cells responsible for cell-mediated immunity. Such changes may have led to disturbances in the surveillance, recognition, and destruction of misplaced endometrial cells and might have, in fact, brought about the disease. The aim of the study was to assess CD95 (Fas) expression on (activated) peritoneal fluid (PF) macrophages, as well as to ascertain soluble Fas (sFas) concentration in the PF of endometriosis patients, as compared to the nonendometriotic group. The concentration of leukocytes in the PF, the percentage of cells expressing CD45+/CD14+, and the percentage of PF macrophages expressing the HLA-DR antigen were significantly higher in patients with stages I and II endometriosis. The percentage of Fas- (CD95+-) expressing macrophages was significantly higher in all stages of the disease, in comparison with controls. Moreover, the concentration of sFas in the PF of patients with moderate and severe endometriosis was significantly higher, as compared to the reference group. The high number of immune cells in PF in early stage endometriosis and their increased susceptibility to apoptosis confirm the role of the impaired peritoneal environment and immune defects in the development and progression of the disease.

Fractalkine/CX3CR1 is involved in the pathogenesis of endometriosis by regulating endometrial stromal cell proliferation and invasion.

Chemokines have been reported to play a sovereign role in the establishment and progression of endometriosis. Fractalkine is a chemokine that is upregulated in many inflammatory diseases including endometriosis. Fractalkine functions as a chemotactic role for lymphocytes and monocytes. In this study, we investigated the role of fractalkine/CX3CR1 in the pathogenesis of endometriosis. In this study, immunohistochemistry was performed on normal endometrium (taken from controls), eutopic endometrium (taken from patients with endometriosis), and ectopic tissues to analyze fractalkine/CX3CR1 expression. The levels of fractalkine in peritoneal fluid and the cell culture supernatant were examined by enzyme-linked immunosorbent assay (ELISA). Bromodeoxyuridine (BrdU) cell proliferation assay was applied to detect the proliferation of endometrial stromal cells (ESCs). The invasion of ESCs was measured by transwell invasion assay. The protein levels of Bcl2, MMP2, MMP9, p-AKT/AKT, p-p38/p38, p-JNK/JNK, and p-ERK/ERK were analyzed by Western blot. We found that the eutopic endometrium had significantly higher expression of fractalkine and CX3CR1 compared to normal endometrium, and the ectopic tissues had the highest expression. The concentrations of fractalkine in peritoneal fluid of endometriosis patients were obviously higher than that of the control and correlate very well with the severity of endometriosis. Fractalkine enhanced ESCs proliferation and invasion via activating AKT and p38 signal pathways. Moreover, high concentration of estradiol (10(-7) , 10(-6) molL(-1) ) induced fractalkine expression while high concentration of progesterone (10(-6) , 10(-5) molL(-1) ) inhibited fractalkine expression in ESCs. The results revealed that the high levels of fractalkine in ectopic milieu promoted proliferation and invasion of ESCs through activating AKT and p38 signal pathways. Estradiol has a stimulating effect on the expression of fractalkine. The present results increase our understanding of the significance of fractalkine in the progression of endometriosis and shed some lights on the targeted fractalkine/CX3CR1 therapies.