Quantitative real-time PCR (qRT-PCR), western blotting, and immunohistochemical (IHC) staining were used to assess the expression of NLRC3 in tissues and cells. The effects of NLRC3 on the proliferation, apoptosis, migration, and invasion of endometrial cells were investigated via Cell Counting Kit-8 (CCK-8), colony formation, 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, cell scratch and transwell assays, respectively. The mouse model of adenomyosis was constructed. The regulation mechanisms by which NLRC3 acts were further verified in vivo study. The study revealed epithelial-mesenchymal transition (EMT) related protein expression was upregulated and NLRC3 was downregulated in endometria of patients with adenomyosis. Upregulation of NLRC3 expression reduced endometrial cell growth, migration, invasion, and promoted cell apoptosis rate. Mechanistically, upregulation of NLRC3 expression inhibited the level of EMT and blocked the PI3K/AKT/mTOR pathway activation in endometrial cells. In vivo, increased the expression of NLRC3 decreased the levels of cytokines (IL-6 and IL-8), inhibited the levels of PI3K/AKT/mTOR pathway related genes and mitigated disease severity. Our findings indicate that NLRC3 inhibits migration and invasion of adenomyosis by modulating PI3K/AKT/mTOR pathway in endometrial cells. Consequently, NLRC3 holds promise as a potential therapeutic target for adenomyosis management.

RESEARCH QUESTION:Does the NOD-like receptor protein 3 (NLRP3) inflammasome have an effect in adenomyosis? DESIGN:Fresh-frozen endometrial tissues and paraffin specimens were obtained from endometrial tissues from patients with adenomyosis and controls. Western blot, quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry (IHC) were applied to assess expression of the NLRP3 inflammasome components. Primary eutopic endometrial stromal cells were isolated from the uteri of patients with adenomyosis. After NLRP3 was knocked down using small interfering RNA, proliferation, invasion and epithelial-mesenchymal transition (EMT) were evaluated using EdU, CCK8, transwell assays and western blot. Importantly, a mouse model of adenomyosis was established to evaluate the effects of the NLRP3 inhibitor MCC950 on the formation of adenomyosis. RESULTS:Expression of the NLRP3 inflammasome components was elevated in the ectopic or eutopic endometrium of patients with adenomyosis. NLRP3 knockdown inhibited migration, invasion and EMT in endometrial cells and primary endometrial cells (P < 0.0001). MCC950, which blocks the NLRP3 inflammasome, reduced migration and invasion of endometrial cells (P < 0.01) and primary endometrial cells (P < 0.0001) considerably. Importantly, in the mouse model of adenomyosis, MCC950 had a mitigating effect on the severity of adenomyosis (P < 0.01). CONCLUSIONS:NLRP3 was found to enhance migration, invasion and EMT of human endometrial cells in adenomyosis. Notably, the NLRP3 inhibitor MCC950 reduced migration and invasion of endometrial cells effectively. Furthermore, in the mouse model of adenomyosis, MCC950 exhibited a therapeutic effect by alleviating the severity of adenomyosis.

Background: Multi-center research has demonstrated that adopting Silva's pattern-based classification system (SPBC) enhances the clinical prognosis and facilitates hierarchical management of patients with endocervical adenocarcinomas (EAC). However, inconsistencies in SPBC can arise due to variations in pathologists' experience levels. Thus, the implementation of standardized decision-making tools becomes crucial to enhance the practicality of SPBC in clinical diagnosis and treatment. Methods: We enrolled a total of 90 patients with EAC in this study, of which 63 were assigned to the training group, and the remaining 27 were allocated to the validation group. To create and validate the prediction models for SPBC, we utilized a deep learning system (DLS) and calculated the area under the receiver operating characteristic curve (AUC). Results: In Silva pattern classification, ResNet50 achieved an average accuracy of 74.36% (63.64% for pattern A, 55.56% for pattern B, and 89.47% for pattern C respectively). Moreover, in test set, ResNet50 achieved an AUC of 0.69 for pattern A, 0.58 for pattern B, and 0.91 for pattern C. Conclusions: We successfully established a DLS for SPBC, which holds the potential to aid pathologists in accurately classifying patients with EAC.

Abstract Background Endometriosis is a common gynecological disease in women of childbearing age. Commonly used treatment methods, such as endocrine and surgical therapies, display poor therapeutic effects with a high relapse probability. Thus, novel treatments for endometriosis are required. Methods In our study, polydopamine (PDA), letrozole (LTZ), and agarose (AG) hydrogels were combined to construct an injectable hydrogel with near-infrared controlled drug delivery named LTZ-PDA@AG hydrogel for endometriosis treatment. The release of letrozole can be accurately controlled by the near-infrared light intensity, exposure duration, polydopamine concentration, and hydrogel composition. Meanwhile, we isolated endometrial stromal cells from endometrium in patients with endometriosis, and constructed the rats’ model of endometriosis to verify the biological effects of LTZ-PDA@AG hydrogel. Results Owing to the sufficiently deep penetration of near-infrared light, the LTZ-PDA@AG hydrogel displayed a high temperature increase for efficient photothermal therapy. In addition, high local temperatures can further enhance the diffusion and penetration of letrozole, thereby achieving excellent therapeutic effect in vivo. Importantly, the in vivo and vitro test demonstrated the capacity of the nanocomposite hydrogel for endocrine-photothermal synergistic therapy and the biocompatibility. Conclusion Our work proposes a novel concept for precision endometriosis therapy by photothermal-enhanced endocrine therapy for endometriosis, which is proposed for the first time for the treatment of endometriosis and demonstrates excellent potential for further clinical translation. Trial registration Not applicable. Graphical Abstract LTZ-PDA@AG hydrogels were synthesized and displayed a high temperature increase for efficient photothermal therapy under NIR. The present study shows the capacity of the nanocomposite hydrogel for endocrine-photothermal synergistic therapy and the biocompatibility.

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. 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. 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. 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.

Objectives: Melatonin (MLT) shows antitumor effects in various tumor types, including endometrial carcinoma. However, the molecular mechanism involved is unclear. In the current study, we investigated the effect of MLT on the estrogen-induced epithelial-mesenchymal transition (EMT) in endometrial adenocarcinoma cells and explored the pathway that might be involved. Design: Laboratory study was via cultured endometrial cancer cells. Design refers only to in vitro experiments. Methods: In cell culture experiments, cell growth was examined using CCK-8 assays. The expression of Numb and EMT markers in Ishikawa cells was examined using Western blot analysis and real-time PCR. Cell invasion was examined using transwell assays. Cell migration was examined using wound-healing assays and transwell assays. Using immunohistochemistry analysis, the expression of Numb in human endometrial cancers was examined. Results: In immunohistochemistry experiments, we found that 15.2% of atypical endometrial hyperplasia and 15.6% of endometrial carcinoma did not express Numb. In cell culture experiments, MLT inhibited cell proliferation, invasion, and migration induced by 17β-estradiol (E2) in endometrial cancer cells. MLT decreased the expression of vimentin and Slug and increased the expression of Numb and E-cadherin in Ishikawa cells. Numb knockdown in cancer cells significantly increased cell proliferation, invasion, and migration. Limitations: No animal experiments were performed. Conclusions: MLT blocked E2-induced cell growth and EMT in endometrial cancer cells via upregulating Numb expression.

Abstract 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.

Background: The presence of ectopic functional endometrial glands and stroma in the myometrium of the uterine cavity is considered as adenomyosis. Various inflammatory, vascular and mechanical factors are involved in the symptoms and evolution of this pathology. Lymphocyte-activation gene 3 (Lag-3) is an immune inhibitory receptor and fibrinogen-like protein 1 (Fgl-1) is a major functional ligand of Lag-3. The binding of Lag-3 and Fgl-1 leads to inhibition of T-cell immunity, which is an important target of immunotherapy. The objective of this study was to evaluate the expression of Lag-3 and Fgl-1 in normal endometrium and adenomyosis. Methods: The expression of the Lag-3 and Fgl-1 in normal endometrium (proliferative phase: n=15; secretory phase: n=15) and adenomyotic endometrium (proliferative phase: n=15; secretory phase: n=15) were determined using immunohistochemistry and immunofluorescence analysis. Results: In normal and adnomyotic endometrium, no significant difference of Fgl-1 expression was noted between proliferative and secretory phases. Compared with normal endometrium, eutopic and ectopic endometrium of adenomyosis showed increased expression of Fgl-1. Lag-3 was almost negative in endometrial glands of normal and adenomyosis. Compared with normal endometrium, Lag-3 positive T-lymphocytes were more common in the stroma of adenomyosis. Conclusions : Our data suggest that aberrant expression of Lag-3 and Fgl-1 is present in the eutopic and ectopic endometrium of adenomyosis. We conclude that Lag-3/Fgl-1 signaling may be involved in the pathogenesis and development of adenomyosis.

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.

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.

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.

目的:探讨腹壁子宫内膜异位症(AWE)的临床特征、分型、治疗及预后.方法:回顾分析2010年1月1日至2019年12月31日就诊于山东大学附属省立医院的241例AWE患者的临床资料.并按病灶浸润深浅分为皮下型、鞘膜型及腹膜型,按是否合并盆腔子宫内膜异位症分为单纯型及复杂型,按病灶个数分为单发型及多发型,比较各组的临床特点.结果:241例AWE患者的平均发病年龄(30.6±4.6)岁.98.8％(238例)既往有剖宫产手术史.93.4％患者就诊时可触及明显腹壁包块,94.6％患者因刀口处或周围疼痛就诊,其中82.9％疼痛与月经周期有明显相关性.208例(86.7％)AWE患者术前做了超声检查,均提示明显包块.病灶大小与病程、手术时间、术中出血量、是否放置补片、单纯型和复杂型、皮下型鞘膜型和腹膜型及住院天数存在正相关,差异有统计学意义(P<0.05).皮下型、鞘膜型和腹膜型的血清CA125水平相比,差异有统计学意义(P<0.05);单纯型和复杂型血清CA125水平相比,差异有统计学意义(P<0.05).皮下型、鞘膜型和腹膜型三型的病灶大小及术中出血量相比,差异有统计学意义(P<0.05);病灶直径越大,侵及深度越深,差异有统计学意义(P<0.05).AWE患者的复发率8.8％,病灶个数为AWE复发的独立危险因素(P=0.033),与单发型相比,多发型AWE患者的复发风险远高于单发型(HR=3.152,95％CI为1.095～9.078).术后药物治疗的26例患者均未复发.结论:AWE应做到早发现、早诊断、早治疗,一旦确诊应立即手术治疗.超声检查和血清CA125对其诊断和分型有意义.多发型AWE术后易复发.术后药物治疗对预防复发有价值.

The T cell immunoglobulin mucin molecule 3 (TIM-3), as a negative regulatory immune checkpoint, plays an important role in cellular immunity by binding to its ligand galectin-9 (Gal-9). Under abnormal conditions, this pathway can regulate the depletion of T cells and suppress the immune response. Abnormal expression of TIM-3 and Gal-9 has been confirmed in a variety of cancers, recurrent miscarriages, chronic infectious diseases and autoimmune diseases. More and more studies have shown that immune factors play an important role in the pathogenesis of adenomyosis. However, few studies have attempted to explore their expression in adenomyosis. The ectopic and eutopic endometrium were collected from 15 women with adenomyosis and 13 women without adenomyosis. TIM-3/Gal-9 expression in endometrial tissue was detected using immunohistochemistry, western blot analysis and real-time PCR. We observed TIM-3/Gal-9 expression in both eutopic and ectopic endometria, with elevated expression in adenomyosis. These data suggest that TIM-3/Gal-9 may be involved in the development and progression of adenomyosis.

BACKGROUND:NUMB is an inhibitory regulator of NOTCH signaling, which is critical for the induction of epithelial-mesenchymal transition (EMT). Loss of NUMB expression is correlated with the genesis and development of multiple tumors. Recent studies reported that NUMB expression was upregulated in human ovarian cancer. However, the role of NUMB in ovarian cancer is still unclear. Here, we invested the effect of NUMB knockdown on the proliferation and EMT in ovarian cancer cells and explored the role of NUMB in the effect of cisplatin.METHODS:Two ovarian cancer cells (OVCAR-3 and SK-OV-3) were used in the experiments. The proliferation and apoptosis of ovarian cancer cells was examined using methyl thiazolyl tetrazolium (MTT) test and flow cytometry assays. The invasion and migration of ovarian cancer cells were examined using Transwell assays. The expression of EMT markers were examined using Simple Western analysis.RESULTS:NUMB knockdown inhibited cell proliferation, invasion, and migration in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell growth inhibiting and apoptosis in both ovarian cancer cells. NUMB knockdown enhanced cisplatin-induced cell invasion in SK-OV-3 cells. NUMB knockdown also decreased the expression of N-cadherin and Vimentin in SK-OV-3 cells.CONCLUSIONS:NUMB acted as an oncogene in ovarian cancer and NUMB knockdown enhanced the anti-tumor role of cisplatin on ovarian carcinoma cells by inhibiting cell proliferation and EMT.

To investigate the role of long noncoding RNA taurine-upregulated gene 1 (TUG1) on ovarian cancer-induced angiogenesis and to explore possible signaling pathways. Ovarian cancer cell line SKOV3 or CAOV3 was transfected with short hairpin-TUG1 to suppress TUG1 expression. Supernatant from cultured cancer cells was used as a condition medium to incubate endothelial cell line human umbilical vein endothelial cells, whose proliferation rate was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration and invasion of endothelial cells were examined by wound healing and Transwell assays, followed by in-vitro angiogenesis assay. One of the secretory factors mediating angiogenesis, leucine-rich α-2-glycoprotein-1 (LRG1), was measured in ovarian cancer cells. Signaling pathway mediating angiogenesis was further detected by western blotting. TUG1 was down-regulated in ovarian cancer cells by short hairpin RNA. Conditional medium originating from TUG1-knockdown cancer cells led to suppressed proliferation, migration, or invasion of endothelial cell line human umbilical vein endothelial cells. LRG1 expression and secretion was suppressed in ovarian cancer cells after TUG1 knockdown. Moreover, recombinant LRG1 rescued TUG1 knockdown-induced angiogenesis inhibition, and LRG1 probably mediated angiogenesis by tumor growth factor-β signaling pathway in endothelial cells. Long noncoding RNA-TUG1 mediates angiogenesis of endothelial cells by regulating LRG1 secretion from ovarian cancer cells partially through tumor growth factor-β pathway. Our results indicate the potency of TUG1 as a biomarker and therapeutic target for tumor-induced angiogenesis.

Background. Endometrial adenocarcinoma (EAC) is one of the most commonly diagnosed gynaecological malignancies among female population of the developed countries. DUSP6 is a negative regulator of ERK signaling, which is a molecular switch involved in MAPK signaling during the progress of malignancies. DUSP6 was previously found to inhibit tumorigenesis and EMT-associated properties in several cancers, however, its exact role in EAC remains unclear. Methods. The level of DUSP6, (E-cad) and (N-cad) in EAC cancerous tissues and respective adjacent non-cancerous tissues were examined by western-blot or immunohistochemistry. The cell growth, invasion and migration abilities were measured in Ishikawa 3H12 endometrial cancer cell lines with overexpressed or knock down DUSP6. Protein levels of EMT-associated markers E-cadherin, N-cadherin and Vimentin were also determined. The impacts of DUSP6 on ERK signaling was assessed by detection of ERK and p-ERK. Results. Down-regulation of DUSP6 was observed in EAC compared with the normal controls. The overexpression of DUSP6 significantly attenuated tumor cell growth, invasion, migration abilities and inhibited EMT-associated markers, while knock down of DUSP6 showed opposite trends. Overexpression of DUSP6 also down-regulated p-ERK and the knock down of DUSP6 inversely up-regulated p-ERK level. Conclusions. DUSP6 inhibited cell growth, invasion and migration abilities in Ishikawa 3H12 cells as well as attenuating EMT-associated properties. This tumor suppressive effect of DUSP6 in EAC is achieved by inhibiting ERK signaling pathway.

The epithelial-mesenchymal transition (EMT) is reported to have intimate crosstalk with androgen receptor (AR) signaling in prostate cancer (PCa) and is known to be responsible for castration resistance. Fibroblast growth factor/receptor (FGF/FGFR) signaling is also involved in tumor progression and EMT in multiple tissues. Several studies have investigated the role of Klotho, an FGF/FGFR signaling co-receptor in tumorigenesis. However, its role in PCa remains unknown. In the present study, the role of androgen in the EMT of PCa cells was examined by western blotting. The expression of Klotho was examined in prostate cells and PCa tissues by western blotting and immunohistochemistry, respectively. The biological role of Klotho was revealed by a series of functional in vitro and in vivo studies. We determined that Klotho expression was significantly decreased in PCa tissues compared with benign prostatic hyperplasia (BPH) tissues, and low Klotho expression was associated with a high Gleason score of PCa. Klotho overexpression inhibited the viability, migration, and androgen/AR-associated EMT of PCa cells through the inactivation of ERK1/2 signaling. Notably, Klotho overexpression inhibited prostate tumor growth and EMT in vivo. Knockdown of Klotho produced the opposite effects. In conclusion, Klotho inhibits EMT and plays a tumor-suppressive role in PCa, linking FGF/FGFR/Klotho signaling to the regulation of PCa progression.

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.