Eutopic Tissue Research Articles

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.

P–341 Epigenetic role of the H2BK5ac histone modification on the expression of ALX1 and PDHX genes in the endometriotic tissues and normal endometrium

Abstract Study question Evolution of the ALX1 and PDHX genes expression and incorporation of H2BK5ac mark on the promoter of these genes in endometriotic tissues versus normal endometrium Summary answer Lower incorporation of H2BK5ac mark in ALX1 and PDHX promoters can because to downregulation of these genes in endometriotic tissues compared to normal endometrium. What is known already Endometriosis is considered as multifactorial disease affected by genetic, hormonal, and environmental factors. Recent evidences suggest the role of epigenetic mechanisms in this disease. Aristaless-like homeobox1 (ALX1) and Pyruvate Dehydrogenase Protein X (PDHX) genes are considered in this study. Studies show that upregulation of the ALX1 gene cause cell proliferation, migration, and invasion in cancer cells. PDHX is involved in cellular metabolism and acts as a tumor suppressor gene while maintaining normal homeostasis. It is Hypothesized that H2BK5ac which is known as a dynamic marker in promoter regions of active genes, may be involved in regulation of these gene expression. Study design, size, duration Ten eutopic and ectopic endometrium tissue, as well as ten normal endometrium, were collected. Ectopic biopsies were obtained using diagnostic laparoscopy, while the endometrial control samples and eutopic samples were collected via pipelle. Participants/materials, setting, methods RNA extraction and cDNA synthesis were done then the expression of ALX1 and PDHX genes evaluated by quantitative real-time PCR. Promoter regions of mentioned genes investigated for the incorporation of the epigenetic mark of H2BK5ac using Chromatin immunoprecipitation (ChIP) followed by real-time PCR. Data analysis performed using One-way ANOVA analysis (SPSS software) considered the significant level of P < 0.05. Main results and the role of chance Results showed that the expression of ALX1 was significantly decreased in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, there was a significant reduction in PDHX mRNA level in the eutopic and ectopic samples vs. normal endometrium (p = .017 and p =.021, respectively). The chromatin immunoprecipitation real-time PCR (ChIP PCR) analyses showed significantly lower incorporation of H2BK5ac epigenetic mark in ALX1 promoter in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, reduced incorporation of H2BK5ac at the PDHX promoter region was observed in both eutopic and ectopic endometrial samples compared to normal endometrium (p = 0.004 and p = 0.003, respectively). Limitations, reasons for caution The main limitation of our study is the low number of samples. Wider implications of the findings: Our results suggest that the marked lower levels of H2BK5ac in regulatory regions of ALX1and PDHX might lead to deregulation of these genes in tissue endometriotic samples. Trial registration number ‘not applicable’ for non-clinical trials

Effect of amber powder on endometrial ultrastructure and MAPK pathway in endometriosis model rats

Purpose: To explore the therapeutic role of amber powder in endometriosis by investigating its effect on endometrial ultrastructure, ERK1/2, p38MAPK, and NF-κB mRNA pathways and CSRC/EFR/ERK1/2 proteins.
 Methods: Sprague Dawley (SD) rats were randomly divided into blank group, disease model group (untreated), amber powder high-dose group, amber powder medium-dose group, amber powder lowdose group and danazol group. Morphological changes in endometrial cells were studied using transmission electron microscopy. The expression of ERK1/2, p38MAPK, and NF-κB mRNA in endometrial tissues of each group was determined using quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemistry was utilized for the measurement of C-SRC/EFR/ERK1/2 pathway protein expression.
 Results: The endometriosis rats treated with a high-, medium- and low-dose amber powder showed a decrease in the volume of ectopic lesions, compared with the untreated disease model group. The expressions of ERK1/2, p38MAPK, NF-κB mRNA, and C-SRC/EFR/ERK1/2 protein were higher in the eutopic and ectopic endometrial tissues in untreated disease group than those in normal control group. Moreover, treatment of endometriosis rats with amber powder revealed a reduction in the expressions of ERK1/2, p38MAPK, NF-κB mRNA and C-SRC/EFR/ERK1/2 proteins in eutopic and ectopic endometrium tissues.
 Conclusion: Amber powder reduces ectopic lesions and slows down the development of endometriosis, probably via inhibition of MAPK pathway genes in eutopic and ectopic endometrial tissues.

Knockdown of circ_0075503 suppresses cell migration and invasion by regulating miR-15a-5p and KLF12 in endometriosis.

Endometriosis is an estrogen-dependent disease. Several researches have reported the dysregulated circular RNAs (circRNAs) in endometriosis, whereas the functions of circRNAs are largely unknown. This study aims to explore the role and mechanism of circ_0075503 in migration and invasion of eutopic endometrial stromal cells. 30 paired ectopic and eutopic endometrium tissues were collected from patients with endometriosis. And primary endometrial stromal cells (ESCs) were stimulated with estradiol (E2) to establish the in vitro cellular model of endometriosis. The levels of circ_0075503, miR-15a-5p and Krüppel-like factor 12 (KLF12) were measured by quantitative reverse transcription polymerase chain reaction or western blot assays. Cell viability, migration and invasion were examined via 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide, transwell assay or western blot assays. The target relationship between miR-15a-5p and circ_0075503 or KLF12 was analyzed by dual-luciferase reporter assay and RNA Immunoprecipitation (RIP) assay. Circ_0075503 expression was elevated in ectopic endometrium and ectopic ESCs. Down-regulation of circ_0075503 suppressed E2-induced promotion of cell viability, migration and invasion in eutopic ESCs. Circ_0075503 could act as a sponge for miR-15a-5p, and KLF12 was targeted by miR-15a-5p. Inhibition of miR-15a-5p reversed the effects of circ_0075503 knockdown on E2-treated ESCs migration and invasion. Besides, miR-15a-5p repressed E2-induced promotion effects on cell migration and invasion via targeting KLF12. Circ_0075503 could regulate KLF12 expression by sponging miR-15a-5p. Knockdown of circ_0075503 inhibited E2-induced enhancement of cell migration and invasion in eutopic ESCs by regulating miR-15a-5p/KLF12 axis, indicating a novel target for the treatment of endometriosis.

Alterations in clock genes expression in eutopic and ectopic endometrial tissue

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

HMGB1 Mediated Inflammation and Autophagy Contribute to Endometriosis.

AimHigh mobility group box (HMGB)-1 has been implicated in endometriosis due to the important regulatory roles of inflammation in endometriosis. The aim of the present study was to explore the roles of HMGB-1 in endometriosis and to elucidate the underlying mechanism.MethodsEndometrial specimens were collected from women with endometriosis and healthy volunteers. Immunohistochemistry staining was used to determine the expression patterns and localization of HMGB-1 in the normal, eutopic and ectopic endometrial tissues. Western blotting and qRT-PCR were used to determine the mRNA and protein levels of inflammatory cytokines [interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-1β], autophagy-related markers [beclin-1, autophagy-related (atg)13, microtubule-associated protein light chain (LC)3-I, LC-II and p62] and HMGB-1, respectively. Spearman’s rank correlation analysis was employed to investigate the correlation between HMGB-1 with inflammatory cytokines and beclin-1. Besides, human endometrial stromal cells (HESCs) were isolated from ectopic endometrium and subsequently transfected with shRNA against HMGB-1. After the transfected cells were subjected to hypoxia, ELISA was used to determine the levels of HMGB-1 and inflammatory cytokines in the cell supernatant. Western blotting was used to determine the expression levels of autophagy-related markers in the cells.ResultsPositive correlations were observed between HMGB-1 and the inflammatory cytokines. In addition, a positive correlation was also identified between HMGB-1 and beclin-1 in the ectopic endometrium. Further results demonstrated that autophagy-related markers beclin-1, atg13 and p62 were significantly upregulated in the ectopic endometrium. In addition, HMGB-1 knockdown suppressed the levels of inflammatory cytokines IL-6, TNF-α and IL-1β and autophagy-related markers beclin-1 and atg13, while upregulated p62 in HESCs under hypoxic condition.ConclusionKnockdown of HMGB-1 under hypoxic condition regulated inflammatory cytokines and autophagy-related markers. HMGB-1 might contribute to the development of endometriosis in part through regulating inflammatory response and autophagy.

Research progress on biomarkers for endometriosis based on lipidomics

The pathogenesis of endometriosis is not well understood at the moment, and the lack of effective biomarkers often leads to delayed diagnosis of the disease. Lipidomics provides a new approach for the diagnosis and prediction of endometriosis. Sphingomyelin, phosphatidylcholine and phosphatidylserine in peripheral blood, endometrial fluid, peritoneal fluid and follicular fluid have good diagnostic value for endometriosis and disease classification; the lipid metabolites in the eutopic endometrium tissue are expected to be biomarkers of early endometriosis; and the lipid metabolites in peripheral blood are also of great value for predicting endometriosis-related infertility. The development of lipidomics technique will further advance the progress on the pathogenesis, prediction, diagnosis and treatment of endometriosis.

Investigating the association of matrix metalloproteinase-2 gene variants with endometriosis in an Iranian population

Objectivesmatrix metalloproteinases including matrix metalloproteinase-2 play a key role in endometrial extra cellular matrix breakdown in endometriosis. Aberrant expression of matrix metalloproteinase-2 has been reported in eutopic and ectopic endometrial tissue of endometriosis patients so altered expression of matrix metalloproteinase-2 due to polymorphisms may lead to establishment and progression of endometriosis. In this study the association between -735 C/T (rs2285053) and −1575 G/A (rs243866) variants of matrix metalloproteinase-2 gene with presence of endometriosis in an Iranian population were investigated for the first time. Study designA case-control association study was conducted to investigate the role of MMP-2−735 C/T and _1575 G/A variants in development of endometriosis. Polymerase chain reaction-restriction fragment length polymorphism method was used to determine genotype frequencies of these variants in 100 endometriosis patients and 200 normal samples. Total genomic DNA was extracted from blood samples and single-nucleotide polymorphism flanking regions were amplified using designed specific primers. Enzymatic digestion was performed using Pag I and Hinf I restriction enzymes for rs2285053 and rs243866 variants, respectively. Statistical analysis was ascertained using statistical package for social science version 16 and “SHEsis” software. ResultsThere were no significant differences in genotype frequencies of rs2285035 (-735C/T) variant between case and control groups (CC + CT vs. TT p = 0.40; OR = 0.50, 95 % CI 0.100–2.551). There were also no significant differences for C allele frequencies in both case and control groups (p = 0.9). For variant rs243866 (−1575 G/A) the differences in genotype frequencies between case and controls group were determined to be significant (GG + GA vs. AA p = 0.041; OR = 6.46, 95 % CI 0.82–50.43). The frequency of G allele was significantly different in case and control groups (p = 0.037). ConclusionIn conclusion, existence of rs243866 variant in promoter region of matrix metalloproteinase-2 gene can increase the risk of endometriosis in Iranian women.

Cellular Phenotyping of Endometriosis at the Single Cell Level Highlights Complex Heterogeneity

Study Objective To understand how endometriosis cells work and interact with the surrounding cells by looking at cellular components and biomarkers. Design In this DoD funded study, both eutopic and peritoneal ectopic tissue were collected from patients with stage III-IV endometriosis. An unbiased single cell transcriptomic (scRNAseq) approach was used to comprehensively identify all the cellular components of endometriosis lesions and their microenvironment. Setting Tissue samples were obtained in the operating room and brought to the Laboratory for its fresh processing RNA sequencing Patients or Participants Women between 18-50 who were preoperatively diagnosed with stage III or IV endometriosis were consented prior to their scheduled laparoscopic surgery. Tissue banked samples of eutopic endometrium from “non-endometriosis patients” were used for comparison. Interventions Peritoneal endometriosis lesions and eutopic endometrium were collected during surgery from patients with stage III and IV endometriosis. These tissue samples were then used to generate scRNAseq data using a droplet based RNAseq platform (10x Genomics). Matched eutopic endometrium was also analyzed for comparison. Measurements and Main Results Our unbiased single cell approach allowed us to capture multiple cell types from ectopic, adjacent ectopic, and eutopic endometrium. We identified the major cell types composing the ectopic endometrium and its microenvironment, as well as in the adjacent peritoneum. Cell types identified within the microenvironment included T cells, B cells, macrophages, endothelial, fibroblast, muscle cells and pericytes. We identified endometrial epithelial cells, in both ectopic and matched eutopic tissues, and their single-cell differential expression analysis revealed substantial differences in their gene expression and main differences emerged from the macrophage populations. Conclusion Precise determination of the heterogeneous cellular composition of endometriosis lesion allows for characterization of important key players, such as epithelial cells and macrophages among others, for lesion formation and evolution. With this detailed approach, we hope to establish a list of potential biomarkers, a much needed effort for endometriosis diagnosis and treatment.

Insight into epigenetics of human endometriosis organoids: DNA methylation analysis of HOX genes and their cofactors

To evaluate and compare the methylation pattern of Human Homeobox (HOX) clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial tissues with ectopic and eutopic endometriosis organoids as advanced preclinical research models. A chromatin immunoprecipitation (ChIP) array containing 84 genes was used to analyze methylation levels of HOX clusters (A-D) and HOX cofactors in normal, eutopic, and ectopic endometrial biopsy specimens as well as ectopic and eutopic endometriosis organoids. Reproductive biomedicine and cell science research centers. Nine healthy women without endometriosis (control) and 16 women diagnosed with endometriosis. Ectopic endometrial lesions were obtained using a laparoscopic procedure, and eutopic and control endometrium biopsy specimens were obtained using pipelle sampling. Methylation levels of HOX clusters (A-D) and HOX cofactors in eutopic and ectopic endometrial biopsy specimens, as well as eutopic and ectopic endometriosis organoids and normal endometrium. Most HOX clusters (A-D) and HOX cofactors showed methylation alterations in ectopic/eutopic endometrial tissues and ectopic/eutopic endometriosis organoids compared with normal endometrium. These methylation alterations had the same pattern in ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids in most genes. A contrariwise methylation pattern was observed in 28 of 84 genes in the ectopic/eutopic tissue biopsy specimens and ectopic/eutopic endometriosis organoids. Because a conserved pattern of methylation alterations in endometriosis tissues and organoids was observed for most of the investigated genes (56 of 84), it can be concluded that endometriosis organoids maintain epigenetic changes. Therefore, our study suggests endometriosis organoids as a novel preclinical model to determine the epigenetic mechanisms that underlie endometriosis.

MicroRNA‑202 inhibits endometrial stromal cell migration and invasion by suppressing the K‑Ras/Raf1/MEK/ERK signaling pathway.

The enhanced migratory ability of endometrial stromal cells (ESCs) is a key factor in the formation of functional endometrium-like tissues outside the uterine cavity during endometriosis (EMS). Although accumulating evidence has suggested the importance of microRNAs (miRNAs) in the pathogenesis of EMS, the role of particular miRNAs in the invasiveness of ESCs remain poorly understood. In the present study, the function of miRNAs in the invasiveness of ESCs, along with the associated underlying mechanism involved, were investigated. Initially, the expression patterns of miRNAs in the ectopic and eutopic endometrium isolated from patients with EMS were analyzed using microarray. MicroRNA-202-5p (miR-202) was selected for further study due to its previously reported suppressive effects on the invasion in various types of cancers. The expression of miR-202 and K-Ras in eutopic and ectopic endometrioma tissues were detected using reverse transcription-quantitative PCR, immunohistochemistry and western blotting. The migration and invasion ability of ESCs was determined using wound healing and Transwell invasion assays, respectively. Compared with that from healthy individuals, miR-202 expression was demonstrated to be lower in the eutopic endometrium from patients with EMS, which was even lower in ectopic endometrium. Functional experiments in primary ESCs revealed that enhanced miR-202 expression suppressed the cell invasion and migration abilities, which was also accompanied with increased E-cadherin and reduced N-cadherin expression in ESCs, suggesting its potentially suppressive role in epithelial-mesenchymal transition. K-Ras is a well-known regulator of the ERK signaling pathway that was shown to be directly targeted and negatively regulated by miR-202. In addition, K-Ras expression was found to be upregulated in the ectopic endometrium, where it correlated negatively with that of miR-202. Knocking down K-Ras expression mimicked the anti-invasive effects of miR-202 overexpression on ESCs, whilst K-Ras overexpression attenuated the inhibitory role of miR-202 overexpression in ESC invasion. The K-Ras/Raf1/MEK/ERK signaling pathway was also blocked by miR-202 overexpression. These findings suggested that miR-202 inhibited ESC migration and invasion by inhibiting the K-Ras/Raf1/MEK/ERK signaling pathway, rendering miR-202 a candidate for being a therapeutic target for EMS.

The role of unfolded protein response in the pathogenesis of endometriosis: contribution of peritoneal fluid

Research questionEndoplasmic reticulum stress (ERS) is caused by the accumulation of the misfolded or unfolded proteins in the endoplasmic reticulum and induces the unfolded protein response (UPR). Peritoneal fluid is important in the pathogenesis of endometriosis. In this study, the role of UPR associated with ERS in endometriosis, and peritoneal fluid, were investigated. DesignNormal, eutopic and ectopic endometrium tissues were divided into menstrual cycle phases, and endometrial stromal cells (ESC) were treated with 10–20% concentration of control peritoneal fluid and peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min, and 24 and 48 h. The UPR signalling proteins were analysed immunohistochemically and immunocytochemically. Data were compared statistically. Resultsp-IRE1 was increased in ectopic glandular and stromal cells in the early proliferative phase compared with normal and eutopic endometrium. p-PERK increased in ectopic glandular and stromal cells in the late proliferative phase compared with normal endometrium. ATF6 was increased in ectopic glandular epithelium compared with normal endometrium in the proliferative phases, versus eutopic endometrium in the late secretory phase. p-IRE1 and p-PERK were increased in high concentrations of ESC treated with peritoneal fluid obtained from women with endometriosis for 10, 30 and 60 min compared with controls. In ESC treated with peritoneal fluid from women with endometriosis, p-IRE1 decreased at 24–48 h compared with 30 min. ConclusionsIn endometriosis, UPR pathways are activated as highly dependent on cell type and phase. Also, p-PERK and p-IRE1 increased because of exposure to high-dose peritoneal fluid from women with endometriosis in stromal cells. Our findings provide a basis for further studies searching for a potential biomarker for the diagnosis of endometriosis.

CircATRNL1 promotes epithelial\u2013mesenchymal transition in endometriosis by upregulating Yes-associated protein 1 in vitro

Endometriosis is a common and benign gynecological disorder but exhibits malignant features. However, the underlying pathogenesis and pathophysiology of endometriosis remain unclear. Circular RNAs have been demonstrated to participate in the occurrence and progression of multiple diseases. This study was aimed to explore the roles of circATRNL1 in endometriosis in vitro. Based on the results of reverse transcription-quantitative polymerase chain reaction analysis, we found significant upregulation of circATRNL1 and Yes-associated protein 1 (YAP1), while downregulation of miR-141-3p and miR-200a-3p in ectopic tissues compared to eutopic tissues. The immunohistochemistry and western blot analysis showed differentially expressed epithelial–mesenchymal transition (EMT) markers between EuEM and EcEM tissues. The in vitro assays indicated that overexpression of circATRNL1 could promote the proliferation, migration, and invasion of Ishikawa cells, and induce EMT process, while circATRNL1 silencing showed the opposite effect. The mechanical investigation indicated that circATRNL1 upregulated YAP1 by sponging miR-141-3p and miR-200a-3p. Gain-of-function assays validated the inhibitory function of miR-141-3p and miR-200a-3p in endometriosis. The results of rescue assays confirmed the function of circATRNL1–miR-141-3p/miR-200a-3p–YAP1 axis on Ishikawa cells. Our findings demonstrate that abnormal upregulation of circATRNL1 regulates cell proliferation and motility and promotes EMT process via the miR-141-3p/miR-200a-3p–YAP1 axis in vitro, which could contribute to the progression of endometriosis.

Upregulated Circular RNA hsa_circ_0008433 Regulates Pathogenesis in Endometriosis Via miRNA.

circRNAs (circular RNAs) play important roles in the development of endometriosis. This study aimed to explore the functions of circRNAs on endometriosis. Two ectopic, two paired eutopic, and two normal endometrial tissue samples were collected for RNA-seq to obtain circRNA profiles and construct a circRNA-miRNA-mRNA network. The validation of 9 circRNAs in 15 patients was assessed by qRT-PCR. We selected hsa_circ_0008433 as the potential biomarker, followed by examining cell proliferation, colony formation, migration, angiopoiesis, cell cycle, and apoptosis. Furthermore, the expression of apoptosis-related proteins was detected using immunofluorescence (IF) and Western blotting. Bioinformatic analysis was used to select the potential target miRNA and genes of hsa_circ_0008433. A total of 209 upregulated and 117 downregulated differentially expressed circRNAs were identified from the eutopic and ectopic endometrial tissue samples. Eight circRNA levels were significantly increased in ectopic endometrial tissue sample compared with eutopic endometrial tissue. The hsa_circ_0008433 knockdown inhibited endometrial stromal cell proliferation, migration, colony formation, and angiopoiesis; promoted cell apoptosis; and downregulated Ki67 and PCNA expression levels. Moreover, the hsa_circ_0008433 knockdown increased Bax and E-CAD expression and decreased Bcl2, CDKN1B, and CyclinD1 levels. Ten potential target miRNAs of hsa_circ_0008433 were selected, and six of them occur significantly aberrant in hsa_circ_0008433-expressing cells. Increased hsa_circ_0008433 levels regulate epithelial mesenchymal transition (EMT) in endometriosis through the circRNA-miRNA-mRNA axis.

TIPE2 inhibits the migration and invasion of endometrial cells by targeting β-catenin to reverse epithelial-mesenchymal transition.

Do changes in tumor necrosis factor-α-induced protein 8 (TNFAIP8)-like 2 (TIPE2) levels in endometrium of patients with adenomyosis alter the proliferation, migration and invasion ability of endometrial cells? TIPE2 expression levels were low in eutopic and ectopic endometrium of adenomyosis patients, and TIPE2 inhibited the migration and invasion of endometrial cells, mainly by targeting β-catenin, to reverse the epithelial-mesenchymal transition (EMT). Adenomyosis is a benign disease, but it has some pathophysiological characteristics similar to the malignant tumor. TIPE2 is a novel negative immune regulatory molecule, and it also participates in the development of malignant tumors. Control endometrium (n = 48 women with non-endometrial diseases) and eutopic/ectopic endometrium from patients with adenomyosis (n = 50), human endometrial cancer cell lines, and primary endometrial cells from the eutopic endometrium of adenomyosis patients were used in the study. The expression level of TIPE2 mRNA and protein in the eutopic/ectopic endometrial tissues of adenomyosis patients and control endometrium was determined by quantitative RT-PCR (qRT-PCR), western blot and immunohistochemistry. The effects of TIPE2 overexpression and knockdown on the proliferation, migration and invasion of endometrial cell lines and primary adenomyotic endometrial cells were determined using a cell counting kit-8, 5-ethynyl-2'-deoxyuridine assay, colony-forming assay, transwell migration assay and matrigel invasion assay. The expression of EMT-related markers and signal molecules was detected by western blot. The interaction between TIPE2 and β-catenin was detected by co-immunoprecipitation and laser confocal microscopy. The mRNA and protein expression levels of TIPE2 in the eutopic and ectopic endometrial tissues of adenomyosis patients were significantly downregulated compared with the control endometrium (P˂0.01). TIPE2 could bind to β-catenin and inhibit the nuclear translocation of β-catenin, downregulate the expression of stromal cell markers, upregulate the expression of glandular epithelial cell markers, decrease the occurrence of epithelial-mesenchymal transition (EMT) and suppress the migration and invasion of endometrial cells (P˂0.01). N/A. In this study, the experiments were performed only in eutopic and ectopic endometrial tissues, endometrial cancer cell lines and primary adenomyotic endometrial cells. A mouse model of adenomyosis will be constructed to detect the effects of TIPE2 in vivo. These results suggest that TIPE2 is involved in the development of adenomyosis, which provides a potential new diagnostic and therapeutic strategy for the treatment of adenomyosis. This present study was supported by grants from the National Natural Science Foundation of China (81471437, 81771554), Natural Science Foundation of Shandong (ZR2018MH013), Science and technology development plan provided by Health and Family Planning Committee in Shandong (2014-25). The authors declare that they have no conflicts of interest.

Higher frequency of circulating, but not tissue regulatory T cells in patients with endometriosis

BackgroundEndometriosis is one of the most common chronic gynecological disorders affecting women at reproductive age. Dysregulation of immune cells, including regulatory T (Treg) cells has contributed to the growth of ectopic lesion in patients with endometriosis. ObjectiveThe present study investigated the frequency of Tregs in peripheral blood and the expression of Foxp3 in eutopic and ectopic endometriotic tissues in women with and without endometriosis. Materials and methodsPeripheral blood mononuclear cells (PBMCs) and eutopic and ectopic endometriotic tissues were obtained from 23 endometriotic and 20 non-endometriotic control women. The frequency of Treg cells in PBMCs was measured using flowcytometry and the expression of Foxp3 in eutopic and ectopic endometriotic tissues was determined by real-time PCR, western blotting and immunohistochemistry. ResultThe frequency of circulating Tregs was significantly higher in endometriotic patients compared with non-endometriotic controls (P < 0.01). The mRNA and protein expression of Foxp3 in eutopic and ectopic endometriotic tissues had no significant differences between the two study groups. ConclusionHigher frequency of circulating Tregs in patients with endometriosis compared with controls may be considered as a compensatory mechanism to regulate the inflammatory condition in this disease.

Immunoreactivity of Kisspeptin and Kisspeptin Receptor in Eutopic and Ectopic Endometrial Tissue of Women With and Without Endometriosis.

Endometriosis is characterized by the presence of ectopic endometrial tissues. Mechanisms of tissue dissemination in endometriosis may be similar to those involved in tumor metastasis. We hypothesize that dysregulation of kisspeptin (KISS1), a metastasis suppressor in endometrial carcinoma, may contribute to the pathogenesis of endometriosis. In this study, we characterized the immunoreactivity of kisspeptin and its receptor, KISS1R, in eutopic and ectopic endometrial tissue of women with and without endometriosis, in proliferative and secretory menstrual cycle phases. Immunohistochemistry was performed using KISS1 and KISS1R antibodies on samples from women with (n = 35) and without (n = 14) endometriosis. Samples from women with endometriosis included eutopic endometrium (n = 20) samples, superficial endometriotic implants (SUP, n = 10) deep infiltrating endometriotic implants (DIE, n = 15), and ovarian endometriomas (OMA, n = 15). Immunoreactivity was quantified using histoscores. KISS1 and KISS1R immunoreactivity was significantly lower in eutopic endometrial stroma of women with versus without endometriosis, regardless of the menstrual cycle phase (P = 0.001 and P = 0.015 respectively). In endometriotic implants, KISS1 levels were significantly lower in both glandular and stromal components of DIE (P < 0.01) and OMA (P < 0.01) compared to SUP. KISS1R immunoreactivity was lower in the glandular component of OMA (P = 0.035) compared to SUP. KISS1 and KISS1R levels are lower in eutopic endometrial stroma from women with versus without endometriosis, consistent with a role for decreased KISS1 expression in the pathogenesis of endometriosis. As deeply invasive lesions showed lower KISS1 levels than superficial lesions, downregulation of KISS1 levels may contribute to implant invasiveness.

Expression Pattern of Mitogen-inducible Gene 6 in Relation to the Extracellular Signal-regulated Kinase Pathway and Redox Status in Patients with Endometriosis

Background: Endometriosis is a gynecological disorder characterized by the presence of ectopic endometrium outside the uterus. It is a multi-factorial disease, where different processes facilitate the implantation and survival of ectopic endometrial tissue. &#x0D; The Aim: This study aims to evaluate the levels of the expression patterns of mitogen-inducible gene 6 (Mig-6) in eutopic and ectopic endometrial tissues of endometriosis patients and their relevance to the levels of phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) and redox status.&#x0D; Methods: Relative gene expression levels of Mig-6 and the levels of pERK1/2 were assessed in endometrial tissues of control group and in both eutopic and ectopic endometrial tissues of endometriosis patients .Serum levels of total protein thiols and total protein carbonyls were also assessed in endometriosis and control groups.&#x0D; Results: Relative gene expression levels of Mig- 6 showed significant lower values in endometriosis group as compared to control group, and they were associated with significant increase in pERK 1/2 levels and altered redox status.&#x0D; Conclusion: These data, signified a significant role of downregulated Mig-6 expression, and activated ERK 1/2 signaling in the pathogenesis of endometriosis. Moreover, it suggested that oxidative stress indicated by high protein carbonyls level contributes to the pathogenesis of endometriosis and that protein thiols have a defensive role against oxidative stress.

Differential Expression of ANXA2 in Eutopic and Ectopic Endometrium

The purpose of this study was to compare the differences in expression of annexin A2, ANXA2 between eutopic and ectopic endometria and to explore the pathogenesis of adenomyosis. In this study, 22 adenomyosis patients who underwent uterine laparotomy in Heilongjiang Provincial Hospital from April 2016 to October 2018 were enrolled as the observation group. The eutopic and ectopic endometria were used for the experiment. In addition, 22 uterine fibroids patients with normal endometrium who underwent open hysterectomy were selected as the control group. The levels of ANXA2 in endometrial tissue and serum were compared between the two groups. The results showed that the ANXA2 level in normal endometrial tissue was significantly lower than that in the eutopic and ectopic endometrial tissue and the ANXA2 level in the ectopic endometrium was significantly higher than that in the eutopic endometrium (p<0.05). The ANXA2 level in the serum of the observation group was significantly (p<0.05) higher than that of the control group. Clinical monitoring of changes in ANXA2 level could be useful as auxiliary diagnostic marker to start treatment of adenomyosis, so as to project prognostic changes more accurately. However, the results of this study still need to be verified with multi-center samples.

60S acidic ribosomal protein P1 (RPLP1) is elevated in human endometriotic tissue and in a murine model of endometriosis and is essential for endometriotic epithelial cell survival in vitro.

Endometriosis is a female disease which is defined as the presence of ectopic endometrial tissue and is dependent on estrogen for its survival in these ectopic locations. Expression of the ribosomal protein large P1 (RPLP1) is associated with cell proliferation and invasion in several pathologies, but a role in the pathophysiology of endometriosis has not been explored. In this study, we aimed to evaluate the expression and function of RPLP1 with respect to endometriosis pathophysiology. RPLP1 protein was localised by immunohistochemistry (IHC) in eutopic and ectopic tissue from 28 subjects with confirmed endometriosis and from 20 women without signs or symptoms of the disease, while transcript levels were evaluated by qRT-PCR in 77 endometriotic lesions and 55 matched eutopic endometrial biopsies, and protein expression was evaluated using western blotting in 20 of these matched samples. To evaluate the mechanism for enhanced lesion expression of RPLP1, an experimental murine model of endometriosis was used and RPLP1 expression was localized using IHC. In vitro studies using an endometriosis cell line coupled with shRNA knockdown was used to demonstrate its role in cell survival. Expression of RPLP1 mRNA and protein were significantly higher in ectopic lesion tissue compared to paired eutopic endometrium and immunohistochemical localisation revealed predominant localisation to epithelial cells. This pattern of lesion RPLP1 was recapitulated in mice with experimentally induced endometriosis. Stable knockdown of RPLP1 protein resulted in a significant decrease in cell survival in vitro. These studies reveal that RPLP1 is associated with cell proliferation and/or survival and may play a role in the pathophysiology of endometriosis.