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Background: T cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) initially discovered on the surface of Th1 cells, negatively regulates immune responses and mediates apoptosis of Th1 cells. An increasing number of studies have since shown that TIM-3 is crucial in the genesis and development of immune diseases, cancers, and chronic infectious illnesses. However, the effect of TIM-3 on endometriosis is still unknown., Methods: Quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry were used to measure TIM-3 levels in endometriosis. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine, colony-forming, Transwell® migration, Matrigel® invasion, and flow cytometry assays were used to explore the function of TIM-3 in vitro, and xenograft experiments in nude mice were used to assess its role in vivo. According to the RNA seq, brain-derived neurotrophic factor (BDNF) was screened. The involvement of specific proliferation-related signaling molecules was determined by transfecting a plasmid and adding an inhibitor in vivo and in vitro., Results: TIM-3 mRNA and protein expression levels were significantly higher in eutopic and ectopic endometrial tissues than in normal endometrial tissues. By examining the effects of TIM-3 overexpression and knockdown on cell proliferation, migration, and invasion in vitro, and lesions formation in vivo, we found that the expression of TIM-3 was positively correlated with cell proliferation and clone formation in vitro, as well as lesions growth in nude mice. By adding the phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT) pathway inhibitor LY294002 and knocking down PI3K, we further verified that TIM-3 promotes proliferation in vivo and in vitro via the PI3K pathway. By transfecting the plasmid into ESC cells and gave inhibitors to endometriotic rats models, we tested that TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis., Conclusion: TIM-3 can promote the proliferation of endometriosis by BDNF-mediated PI3K/AKT axis in vivo and in vitro, which may provide a new therapeutic target for the treatment of endometriosis., (© 2023. The Author(s).)

Endometriosis is a common estrogen-dependent chronic inflammatory disease that leads to infertility in women of reproductive age. Perhaps infertility reflects the reduced expression of integrin αvβ3 and HOXA10 in endometriosis. Previous studies have shown that administration of letrozole, a non-steroidal aromatase inhibitor for cancer treatment, increased the clinical pregnancy rate in women with endometriosis, but the mechanisms remain to be determined. In this communication, a rat model of endometriosis was established. Animals were treated with letrozole at 2ug/kg of body weight, intragastric administration for 15 consecutive days. Letrozole increased the expression of αvβ3 and HOXA10 in the endometriosis model and endometrial receptivity. Abbreviations: WOI: window of implantation; RGD: Arg-Gly-Asp; HOX: homeobox; E2: estradiol; SPF: specific pathogen-free.

Aim: Study the effect of low-dose aspirin on the endometrial receptivity in endometriosis rat models., Materials and Methods: This study is to explore the expressions of progesterone receptor and LIF among three groups of endometriosis rat models: control group ( n  = 12), EMs group ( n  = 15), and aspirin group ( n  = 17). The expressions of progesterone receptor (PR), PRA, PRB, and leukemia inhibitory factor receptor (LIFR) in eutopic endometrium were determined using immunohistochemistry technology, western blot, and qRT-PCR. The levels of LIF in eutopic endometrium and serum were detected by western blot, qRT-PCR, and ELISA., Results: The expressions of PR, PRA, and PRB protein were significantly increased in the eutopic endometrium after low-dose aspirin treatment, and the level of PRB mRNA was also increased while the ratio of PRA/PRB mRNA was decreased in the eutopic endometrium. The levels of LIF in eutopic endometrium and serum were increased compared with the untreated endometriosis rats. However, the expression of LIFR was not statistically different among the three groups., Conclusions: The results suggest that the low-dose aspirin treatment could downregulate progesterone resistance and increase the expression of LIF of endometriosis rats during the implantation window, which could improve endometrial receptivity and enhance the pregnant rate of endometriosis. It may provide a potential treatment method for endometriosis-related infertility.

Previous studies report that fibroblast growth factor 2 (FGF2) modulates Sproutys (SPRYs)/dual specificity phosphatase 6 (DUSP6)/extracellular signal-regulated kinase (ERK) signaling pathway in endometrial glandular epithelial cells. However, its role in endometriosis remains unclear. The expression patterns and localization of related proteins in endometrium patients' samples were determined using quantitative reverse transcription PCR, Western blotting, and immunohistochemistry, respectively. Human endometrial stromal cells (HESCs) were isolated and transfected with small interfering RNA (siRNA) targeting FGF2 (FGF2-siRNA). Cell viability was determined using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It was found that FGF2 mRNA and protein levels were increased in the ectopic endometrium, whilst the mRNA and protein levels of SPRYs/DUSP6/ERK signaling pathway related-genes were dysregulated. Spearman's rank correlation analysis revealed a negative correlation between FGF2 and SPRYs/DUSP6 signaling pathway-related proteins. In vitro study demonstrated that FGF2 silencing suppressed cell proliferation. Our results suggest that FGF2 upregulation might contribute to endometriosis via the regulation of the SPRYs/DUSP6/ERK signaling pathway., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The programmed death-1 (PD-1)/PD-L1 pathway plays important roles in immune responses and peripheral tolerance. Under pathological conditions, this pathway can suppress protective T cell responses and induce immune system disorders. Endometriosis (EM) is an estrogen-dependent, immune-associated disease. The expression of PD-1 and PD-L1 has been studied in a variety of cancers and autoimmune diseases. Few studies, however, have attempted to explore their expression and the possibility that they are regulated by estrogen in EM. Eutopic and ectopic endometria and blood samples were collected from 15 women with EM and 15 women without EM. PD-1/PD-L1 expression in endometrial tissues was detected using immunohistochemistry and western blot analysis. Their expression in blood was detected using flow cytometry. In addition, their expression was evaluated in endometrial cells after estrogen and cytokine treatment. We observed PD-1/PD-L1 expression in both eutopic and ectopic endometria, with elevated expression in EM. Their expression was also higher in CD4 + /CD8 + T cells in blood from EM patients. In addition, treatment with 17β-estradiol upregulated PD-L1 expression in eutopic epithelial cells in EM. These data suggest that the PD-1/PD-L1 pathway may be involved in EM immune dysfunction and be regulated by estrogen.

Background: Melatonin is a potential therapeutic agent for endometriosis, but its molecular mechanism is unclear. Here, we investigated the effect of melatonin on the epithelial-mesenchymal transition (EMT) in endometriotic endometrial epithelial cells and explored the pathway that might be involved., Methods: This hospital-based study included 60 women of reproductive age using the endometrium for immunohistochemistry, 6 women of reproductive age undergoing bilateral tubal ligation and 6 patients with endometriosis for isolation of endometrial epithelial cells or subsequent analysis, respectively. We examined the expression of Notch1/Numb signaling and EMT markers by immunohistochemistry analysis and western blot analysis, the invasion and migration of endometrial epithelial cells by transwell assays, and the cell proliferation by CCK8 assays., Results: Compared with normal endometrium, the endometriotic eutopic endometrium showed increased expression of Notch1, Slug, Snail, and N-cadherin, and decreased expression of E-cadherin and Numb. Melatonin or Notch inhibition by specific inhibitor blocked 17β-estradiol-induced cell proliferation, invasion, migration and EMT-related markers in both normal and endometriotic epithelial cells., Conclusions: Our data suggest that aberrant expression of Notch1/Numb signaling and the EMT is present in endometriotic endometrium. Melatonin may block 17β-estradiol-induced migration, invasion and EMT in normal and endometriotic epithelial cells by upregulating Numb expression and decreasing the activity of the Notch signaling pathway.

In previous studies, we found that endometriotic stromal cells lose the ability to regulate cell survival signaling in endometriotic epithelial cells. Here, we invested the effect of Metformin on the stromal-epithelial cells crosstalk in endometriosis and explored the pathway that might be involved. We found that ectopic endometriotic stromal cells (ESC) expressed and secreted higher Wnt2 protein compared with normal endometrial stromal cells (NSC). Conditioned medium (CM) from ESC supplemented with Wnt2 antibody significantly inhibited the growth of normal endometrial epithelial cells (NEC), while CM from ESC per se showed no significant effect on the growth of NEC. Metformin decreased the expression and secretion of Wnt2 in ESC. CM from Metformin-pretreated ESC significantly inhibited the growth of NEC. In conclusion, Wnt2/β-catenin signaling was involved in stromal-epithelial cells interaction in endometriosis. Metformin might regulate the stroma-epithelium communication via Wnt2-mediated signaling in endometriosis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Objective: To explore the stroma-epithelium interactions in endometriosis and to identify the possible signalling pathways involved in this cross-talk., Design: Laboratory study via primary cultured endometrial stromal and epithelial cells., Setting: University Hospital., Patients: Fifteen patients with endometriosis confirmed by histopathology were recruited in the study, and 12 women free of endometriosis were used as control group., Intervention(s): Specific NFkappaB inhibitor 1-Pyrrolidinecarbodithioic acid ammonium salt (PDTC) was used in cell cultures., Main Outcome Measure(s): The expression and secretion of MMP-2, MMP-9, TIMP-1, TIMP-2 and the DNA-binding activity of NFkappaB in normal endometrial stromal cells or in co-cultures with normal or endometriotic epithelial cells from patients with endometriosis., Result(s): Endometrial epithelial cells induced MMP-9 and MMP-2 expression in normal stromal cells in vitro. In co-cultures with endometriotic epithelial cells, normal endometrial stromal cells expressed and secreted higher MMP-2 (p < 0.05) and MMP-9 (p < 0.05). Specific inhibition of NFkappaB pathway in stromal cells abolished this induction effect by epithelial cells., Conclusion(s): Endometriotic epithelial cells induce MMPs expression and secretion in normal endometrial stromal cells via an NFkappaB-dependent pathway in vitro. This cross-talk between epithelial cells and stromal cells may facilitate the implantation and extension of the ectopic foci and favour the development of the disease.

Unlabelled: The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition., Conclusion(s): Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

In normal endometrium, stromal factors regulate the growth of epithelial cells. However, epithelial cells in endometriotic lesions display increased proliferation and decreased apoptosis. This work tested the hypothesis that in endometriosis stromal cells lose the ability to regulate survival signaling and cell growth in epithelial cells. Primary normal, endometriotic eutopic and ectopic epithelial cells were cultured in the presence of medium conditioned by normal, eutopic and ectopic endometriotic endometrial stromal cells. Endometriotic epithelial cells showed higher Survivin expression than normal epithelial cells. Conditioned medium (CM) from normal or eutopic endometriotic stromal cells significantly inhibited the Survivin expression and AKt phosphorylation in normal or eutopic endometriotic epithelial cells. However, CM from ectopic endometriotic stromal cells did not have an inhibitory effect on normal or ectopic endometriotic epithelial cells. Inhibition of AKt phosphorylation and Survivin expression in normal or eutopic endometriotic epithelial cells in the presence of stromal factors from normal or eutopic endometriotic stromal cells was enhanced by progesterone, whereas progesterone had little effect in the presence of stromal factors from ectopic endometriotic stromal cells. The inability of ectopic endometriotic stromal cells to regulated PI3K/AKt/Survivin signaling and mediate the progesterone response in endometriotic epithelial cells may facilitate epithelial cell proliferation in endometriosis and promote the survival of endometriotic lesions.

2Immunoexpression and comparison of B7-H3 in normal, eutopic and ectopic endometrium of adenomyosis treated with and without mifepristone. a Ectopic endometrium of proliferative phase in patient with untreated adenomyosis (n = 35); b Ectopic endometrium of secretory phase in patient with untreated adenomyosis (n = 23); c Eutopic endometrium of proliferative phase in patient with untreated adenomyosis (n = 35); d Eutopic endometrium of secretory phase in patient with untreated adenomyosis (n = 23); e Normal endometrium of proliferative phase in patients without adenomyosis (n = 47); f Normal endometrium of secretory phase in patients without adenomyosis (n = 27); g Ectopic endometrium in patient with mifepristone-treated adenomyosis (n = 11); h and i eutopic endometrium in patient with mifepristone-treated adenomyosis (n = 11); j Immunoscore comparation of B7-H3 between each groups, * P < 0.05, ** P < 0.01. a- i magnification: × 100 The original article [[1]] has been updated. Reference 1 Qin X, Sun W, Wang C. Mifepristone inhibited the expression of B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis. [Extracted from the article]

Background: The immune mechanism was shown to be involved in the development of adenomyosis. The aim of the current study was to evaluate the expression of the immune checkpoints B7-H2, B7-H3, B7-H4 and PD-L2 in adenomyosis and to explore the effect of mifepristone on the expression of these immune checkpoints. Methods: The expression of B7-H2, B7-H3, B7-H4 and PD-L2 in normal endometria and adenomyosis patient samples treated with or without mifepristone was determined by immunohistochemistry analysis. Results: In adenomyosis patient samples, the expression of B7-H2, B7-H3 and B7-H4 was increased in the eutopic and ectopic endometria compared with normal endometria, both in the proliferative and secretory phases. Moreover, the expression of B7-H2 and B7-H3 was higher in adenomyotic lesions than in the corresponding eutopic endometria, both in the proliferative and secretory phases. The expression of PD-L2 was higher in adenomyotic lesions than in normal endometria in both the proliferative and secretory phases. In the secretory phase but not the proliferative phase, the expression of B7-H4 and PD-L2 in adenomyotic lesions was significantly higher than that in the corresponding eutopic endometria. In normal endometria and eutopic endometria, the expression of B7-H4 was elevated in the proliferative phase compared with that in the secretory phase, while in the ectopic endometria, B7-H4 expression was decreased in the proliferative phase compared with the secretory phase. In addition, the expression of B7-H2, B7-H3, B7-H4 and PD-L2 was significantly decreased in adenomyosis tissues after treatment with mifepristone. Conclusions: The expression of the immune checkpoint proteins B7-H2, B7-H3, B7-H4 and PD-L2 is upregulated in adenomyosis tissues and is downregulated with mifepristone treatment. The data suggest that B7 immunomodulatory molecules are involved in the pathophysiology of adenomyosis. [ABSTRACT FROM AUTHOR]

Abstract: The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFκB) signaling overactivities coexisted in endometriosis. In vitro, 17β-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17β-estradiol-induced cell proliferation. 17β-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFκB inhibition. Conclusion(s): Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFκB signal overactivities coexist in endometriosis. In vitro, 17β-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFκB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17β-estradiol→high NFκB→low PTEN→high PI3K→high NFκB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease. [Copyright &y& Elsevier]