Endometriosis Endometrium Research Articles

Molecular profile of eutopic and ectopic endometrium in endometriosis

Molecular profile of eutopic and ectopic endometrium in endometriosis

Transcriptome sequencing of adenomyosis eutopic endometrium: A new insight into its pathophysiology.

The eutopic endometrium has been suggested to play a crucial role in the pathogenesis of adenomyosis. However, the specific genes in eutopic endometrium responsible for the pathogenesis of adenomyosis still remain to be elucidated. We aim to identify differentially expressed genes (DEGs) and molecular pathways/networks in eutopic endometrium from adenomyosis patients and provide a new insight into disease mechanisms at transcriptome level. RNA sequencing (RNA‐Seq) was performed with 12 eutopic endometrium from adenomyosis and control groups. Differentially expressed genes in adenomyosis were validated by quantitative real‐time PCR (qPCR) and immunochemistry. Functional annotations of the DEGs were analysed with Ingenuity Pathway Analysis (IPA). Quantitative DNA methylation analysis of CEBPB was performed with MassArray system. A total of 373 differentially expressed genes were identified in the adenomyosis eutopic endometrium compared to matched controls. Bioinformatic analysis predicted that IL‐6 signalling and ERK/MAPK signalling were activated in adenomyosis endometrium. We also found that the increased expression and DNA hypomethylation of CEBPB were associated with adenomyosis. Our results revealed key pathways and networks in eutopic endometrium of adenomyosis. The study is the first to propose the association between C/EBPβ and adenomyosis and can improve the understanding of the pathogenesis of adenomyosis.

ARID1A, Prostaglandin E2, and Its Receptor as Possible Predictors of Malignant Transformation of the Endometrium in Endometriosis.

We studied the expression of ARID1A, prostaglandin E2 synthase, and prostaglandin E2 receptor in the endometrium and ovarian, peritoneal, and intestinal endometrioid heterotopies in women with endometriosis of young and middle reproductive age. ARID1A protein is a tumor suppressor, its expression reduced in different types of cancer. Prostaglandin E2 synthase and prostaglandin E2 receptor are involved in the signaling cascade of inflammatory reactions presumably underlying the development of endometriosis. In endometrioid heterotopies, expression of ARID1A was reduced in 1.2-4.0 times, the expression of prostaglandin E2 synthase and prostaglandin E2 receptor was reduced in 2.9-5.2 times These findings suggest that ARID1A, prostaglandin E2 synthase, and prostaglandin E2 receptor can be used as predictors of malignant transformation of endometrioid heterotopies in women with endometriosis.

Expression of PROK 1 and its receptor PROKR 1 in endometriosis and its clinical significance

To investigate the role of prokineticin (PROK) 1 and prokineticin-receptor (PROKR) 1 in the pathogenesis of endometriosis and its clinical signifaicance. Methods: Quantitative real-time PCR (qPCR) and Western bloting were used to detect the expression of PROK 1 and PROKR 1 in eutopic and ectopic endometrium of endometriosis (n=22) and normal control endometrium (n=18). Endometrial stromal cells were isolated and cultured in 6 normal controls. The expression of PROK 1 mRNA was detected by qPCR after treated with estradiol (E2) or TNF-α. Results: PROK 1 and PROKR 1 mRNA were expressed in eutopic and ectopic endometrium of endometriosis and normal control endometrium, and the expression level gradually declined (P<0.05). The expression of PROKR-1 protein in eutopic and ectopic endometrium of endometriosis and normal control endometrium gradually declined (P<0.05). The expression of PROK-1 protein in normal control endometrial cells and eutopic endometrium cell was higher in secretory phase than in proliferative phase (P<0. 05). E2 did not change the expression of PROK 1, whereas TNF-α up-regulated the expression of PROK 1. Conclusion: PROK-1 and its receptors are involved in the pathogenesis and development of endometriosis. TNF-α can promote angiogenesis via up-regulating the expression of PROK 1.

Expression of annexin A2 in adenomyosis and dysmenorrhea

PurposeTo investigate the expression of annexin A2 (ANXA2) in ectopic and eutopic endometrium and serum of women with adenomyosis, and their relationships with adenomyosis-associated dysmenorrhea.MethodsThe expression of ANXA2 was detected by immunohistochemical S-P method in ectopic and eutopic endometrium tissues from 30 patients with adenomyosis who underwent hysterectomy. The correlation between ANXA2 expression and dysmenorrhea degree was evaluated. The endometrium tissues from 15 patients with uterine fibroids which underwent hysterectomy were used as controls. The preoperative serum level of ANXA2 was measured by enzyme-linked immunosorbent assay in 30 patients with adenomyosis and 15 patients with hysteromyoma.ResultThe expression of ANXA2 in eutopic and ectopic endometrium of adenomyosis was higher than in normal endometrium (P < 0.05), but no significant difference of ANXA2 expression was observed between the eutopic endometrium and the ectopic endometrium (P > 0.05). In the ectopic endometrium, but not in the eutopic endometrium, of women with adenomyosis, ANXA2 expression was positively correlated with the severity of dysmenorrhea (R = 0.831, P = 0.000). The preoperative serum level of ANXA2 was markedly higher in patients with adenomyosis compared with the patients with hysteromyoma (P < 0.05).ConclusionThe increased ANXA2 may contribute to the occurrence and development of adenomyosis, and may play a important role in the dysmenorrhea. The present study may provide a new idea of diagnosis and treatment to adenomyosis-associated dysmenorrhea.

SAT-023 Cyclic Regulation of 11 Beta-Hydroxysteroid Dehydrogenase Type 1 in Endometriosis in Rhesus Macaques

Endometriosis is an estrogen-dependent disorder defined by endometrium-like tissue outside the uterine cavity that results in pelvic inflammation. Pro-inflammatory cytokines can increase 11 β -hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts inactive cortisone into active cortisol. We hypothesized that cyclic changes in estrogen (estradiol; E2) and progesterone (P4) could regulate endometriotic 11β-HSD1. To test this, we assessed 11β-HSD1 expression in rhesus macaques (Macaca mulatta) with advanced endometriosis. Animal care was provided by our Department of Comparative Medicine and all work was reviewed and approved by the Institutional Animal Care and Use Committee. Endometrium and lesions were collected in the proliferative and secretory phase of the cycle (n=5 each). Control endometrium (endometriosis-free animals) was also collected in the proliferative (n=5) and secretory (n=4) phase. Mean (+SE) hormone levels were: 103+10 pg E2/ml and 0.2 + 0.04 ng P/ml in the proliferative phase; 43.8 + 9 pg E2/ml and 4.41 + 0.98 ng P/ml in the secretory phase. Tissues were frozen in liquid nitrogen for RNA isolation or fixed for histology. RNA was isolated in TRIzol and purified on Qiagen Quick columns with DNase digestion. Total RNA (1 µg) was reverse transcribed in the presence of random hexamer primers. TaqMan® quantitative real-time PCR (q-RT-PCR) was conducted for 11β-HSD1, estrogen receptor 1 (ESR1), ESR2 and progesterone receptor (PGR). Results are presented as ratio of target/ribosomal S10 RNA. Normal cyclic regulation of endometrial histology was observed. Histology confirmed the presence of endometrium-like tissue in all lesions. The highest expression of 11β-HSD1 was in proliferative phase endometriotic lesions (0.64 + 0.28). Levels were reduced in the secretory phase (0.13 + 0.02; P<0.01). 11β-HSD1 was minimal in the endometrium of control and endometriosis animals (proliferative phase control = 0.11 + 0.03; versus endometriotic animals = 0.11+ 0.06). There was a non-significant trend toward higher levels in the secretory phase (control = 0.24+ 0.06; endometriosis = 0.16 + 0.03). Normal cyclic levels of ESR1 and PGR were observed. For instance, levels of ESR1 (proliferative versus secretory) in the control endometrium, endometriosis endometrium, and lesions were as follows: 2.36 + 0.39 versus 0.43 + 0.21 (P<0.01), 1.41 + 0.22 versus 0.74 + 0.34 (P<0.01), and 1.05 + 0.35 versus 0.48 + 0.11(P<0.05); respectively. Levels of PGR followed the pattern of ESR1. In contrast, ESR2 transcript was higher in lesions compared to endometrium, and menstrual cycle-specific regulation was observed. We conclude that 11β-HSD1 is elevated in the proliferative phase resulting in increased intracellular cortisol. Elevated cortisol could promote estrogen dependent lesion growth by modulating inflammation. This work was supported by NIH/NICHD.

CD33+CD14+CD11b+HLA‐DR− monocytic myeloid‐derived suppressor cells recruited and activated by CCR9/CCL25 are crucial for the pathogenic progression of endometriosis

Endometriosis (EM) is a chronic immunoinflammatory disease associated with an abnormal immunotolerant microenvironment. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that play a major role in immunosuppression in cancer, inflammation and other diseases. This paper aims to elucidate whether or not MDSCs are involved in regulating this microenvironment in EM and how this regulation occurs. Immunochemistry (IHC) and qPCR were conducted to measure CD11b and ARG1 expression in the ectopic endometrium samples from EM patients. CCL25 levels in EM PF and the expression of CCR9 on M-MDSCs were measured by ELISA. M-MDSC migration was determined towards rhCCL25, α-CCR9, α-CCL25 and EM PF through in vitro chemotaxis assay. CD33+ CD14+ CD11b+ HLA-DR- M-MDSCs isolated from EM PBMCs were added to CD8+ T cells stimulated with α-CD3/α-CD28 antibody. After 72hours of co-culture, proliferation was measured to rate the immunosuppressive function of M-MDSCs. Finally, levels of IL-10, GM-CSF and arginase activity in the cultured supernatants were detected. IHC and qPCR results revealed higher CD11b and ARG1 expression in EM endometrium than normal endometrium. MDSCs accumulated in the EM microenvironment, in which M-MDSCs were the predominant type. CD33+ CD14+ CD11b+ HLA-DR- M-MDSCs expressed high CCR9 levels and were recruited through CCL25. M-MDSCs from EM PBMCs inhibited proliferation and activity in autologous T cells. rhCCL25 promoted IL-10 and GM-CSF secretion and arginase enzymatic activity in CD33+ CD14+ CD11b+ HLA-DR- M-MDSCs. CD33+ CD14+ CD11b+ HLA-DR- M-MDSCs recruited and activated by CCR9/CCL25 play a crucial role in the pathogenic progression of endometriosis, thus providing a potential target for EM treatment.

Transcriptome Sequencing of Adenomyosis Eutopic Endometrium: A New Insight into Its Pathophysiology

Background: The eutopic endometrium has been suggested to play a crucial role in the pathogenesis of adenomyosis. However, the specific genes in eutopic endometrium responsible for the pathogenesis of adenomyosis still remain to be elucidated. We aim to identify differentially expressed genes (DEGs) and molecular pathways/networks in eutopic endometrium from adenomyosis patients, and provide a new insight into disease mechanisms at a transcriptome level. Methods: RNA sequencing (RNA-Seq) was performed with 12 eutopic endometrium from adenomyosis and control groups. DEGs in adenomyosis were validated by quantitative real-time PCR (qPCR) and immunochemistry. Functional annotations of the DEGs were analyzed with Ingenuity Pathway Analysis (IPA). Quantitative DNA methylation analysis of CEBPB was performed with MassArray system. Findings: Totally, 373 genes were differentially expressed in the adenomyosis eutopic endometrium compared to matched controls. Bioinformatic analysis indicated that IL-6 signaling and ERK/MAPK signaling was activated in adenomyosis endometrium. We also found that the increased expression and DNA hypomethylation of C/EBPI² were associated with adenomyosis. Interpretation: Our results revealed key pathways and networks in eutopic endometrium of adenomyosis. The study is the first to propose the link between C/EBPI² and adenomyosis and can improve the understanding of the pathogenesis of adenomyosis. Funding Statement: This work is supported by the National Natural Science Foundation of China [NO. 81771551, 81501276 and 81401219], the Shanghai Municipal Commission of Science and Technology Program [NO. 17411972800 and 15411966700], the Shanghai Municipal Commission of Health and Family Planning [NO. 20154Y0039], the Municipal Human Resources Development Program for Outstanding Young Talents in Medical and Health Sciences in Shanghai [NO. 2018YQ39], the Shanghai Shenkang Hospital Development Center Clinical Science and Technology Innovation Project [NO. SHDC12017123], and the Combined Engineering and Medical Project of Shanghai Jiao Tong University [NO. YG2015MS41 and YG2017MS39]. Declaration of Interests: The authors have declared that no competing interests exist. Ethics Approval Statement: The study protocols were approved by the Ethics Review Committee of IPMCH and conducted according to the Declaration of Helsinki Principles.

Curcumin attenuates proangiogenic and proinflammatory factors in human eutopic endometrial stromal cells through the NF-κB signaling pathway.

Endometriosis is a chronic gynecological inflammatory disorder in which immune system dysregulation is thought to play a role in its initiation and progression. Due to altered sex steroid receptor concentrations and other signaling defects, eutopic endometriotic tissues have an attenuated response to progesterone. This progesterone‐resistance contributes to lesion survival, proliferation, pain, and infertility. The current agency‐approved hormonal therapies, including synthetic progestins, GnRH agonists, and danazol are often of limited efficacy and counterproductive to fertility and cause systemic side effects due to suppression of endogenous steroid hormone levels. In the current study, we examined the effects of curcumin (CUR, diferuloylmethane), which has long been used as an anti‐inflammatory folk medicine in Asian countries for this condition. The basal levels of proinflammatory and proangiogenic chemokines and cytokines expression were higher in primary cultures of stromal cells derived from eutopic endometrium of endometriosis (EESC) subjects compared with normal endometrial stromal cells (NESC). The treatment of EESC and NESC with CUR significantly and dose‐dependently reduced chemokine and cytokine secretion over the time course. Notably, CUR treatment significantly decreased phosphorylation of the IKKα/β, NF‐κB, STAT3, and JNK signaling pathways under these experimental conditions. Taken together, our findings suggest that CUR has therapeutic potential to abrogate aberrant activation of chemokines and cytokines, and IKKα/β, NF‐κB, STAT3, and JNK signaling pathways to reduce inflammation associated with endometriosis.

Endometrium metabolomic profiling reveals potential biomarkers for diagnosis of endometriosis at minimal-mild stages

Sensitivity and specificity of non-invasive diagnostic methods for endometriosis, especially at early stage, is not optimal. Clinical diagnostic indicator of cancer antigen 125 (CA125) performs poorly in diagnosing minimal endometriosis with a sensitivity of 24%. Therefore, it is urgent to explore novel diagnostic biomarkers. We evaluated metabolomic profile variation of eutopic endometrium between minimal-mild endometriosis patients and healthy women by ultra high performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS). Our study comprised 29 patients with laparoscopically confirmed endometriosis at stage I-II and 37 infertile women who underwent diagnostic laparoscopy combined with hysteroscopy from January 2014 to January 2015. Eutopic endometrium was collected by pipelle endometrial biopsy. The metabolites were quantified by UHPLC-ESI-HRMS. The best combination of biomarkers was then selected by performing step-wise logistic regression analysis with backward elimination. 12 metabolites were identified as endometriosis-associated biomarkers. The eutopic endometrium metabolomic profile of endometriosis patients was characterized by significant increase in concentration of hypoxanthine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lysophosphatidylethanolamine and asparagine. In contrast, concentration of uric acid was decreased. Metabolites were filtered by step-wise logistic regression with backward elimination and a model containing uric acid, hypoxanthine, and lysophosphatidylethanolamine were constructed. Receiver-operating characteristic (ROC) analysis confirmed the prognostic value of these parameters in diagnosing minimal/mild endometriosis with a sensitivity of 66.7%, a specificity of 90.0%. Metabolomics analysis of eutopic endometrium in endometriosis was effectively characterized by UHPLC-ESI-HRMS based metabolomics. Our study supports the importance of purine and amino acids metabolites in the pathophysiology of endometriosis and provides the potential biomarkers for semi-invasive diagnose of endometriosis at early stage.

Abnormal activation of the sonic hedgehog signaling pathway in endometriosis and its diagnostic potency

To investigate the abnormal expression of sonic hedgehog (SHH) signaling molecules in 52 eutopic endometrial tissues and its diagnostic potency in endometriosis. Retrospective study. University hospital. Twenty-six women with histologically confirmed endometriosis and 26 women with histologically normal endometria who were undergoing curettage or hysterectomy were selected. None. The mRNA and protein levels of molecules in the SHH signaling pathway. The levels of SHH, smoothened, GLI family zinc finger 3, and its downstream signaling transcription factor (GLI1) not only were upregulated in the eutopic endometrium of endometriosis compared with the control endometrium, but also independently predicted the onset and severity of the disease. This study is the first to reveal differences in the activation of the SHH signaling pathway between women with and without endometriosis and suggests that the SHH signaling pathway has potential in the diagnosis of endometriosis.

Endometrium metabolomic profiling reveals potential biomarkers for diagnosis of endometriosis at minimal-mild stages

BackgroundThe sensitivity and specificity of non-invasive diagnostic methods for endometriosis, especially at early stages, are not optimal. The clinical diagnostic indicator cancer antigen 125 (CA125) performs poorly in the diagnosis of minimal endometriosis, with a sensitivity of 24%. Therefore, it is urgent to explore novel diagnostic biomarkers. We evaluated the metabolomic profile variation of the eutopic endometrium between minimal-mild endometriosis patients and healthy women by ultra-high-performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS).MethodsOur study comprised 29 patients with laparoscopically confirmed endometriosis at stages I-II and 37 infertile women who underwent diagnostic laparoscopy combined with hysteroscopy from January 2014 to January 2015. Eutopic endometrium samples were collected by pipelle endometrial biopsy. The metabolites were quantified by UHPLC-ESI-HRMS. The best combination of biomarkers was then selected by performing step-wise logistic regression analysis with backward elimination.ResultsTwelve metabolites were identified as endometriosis-associated biomarkers. The eutopic endometrium metabolomic profile of the endometriosis patients was characterized by a significant increase in the concentration of hypoxanthine, L-arginine, L-tyrosine, leucine, lysine, inosine, omega-3 arachidonic acid, guanosine, xanthosine, lysophosphatidylethanolamine and asparagine. In contrast, the concentration of uric acid was decreased. Metabolites were filtered by step-wise logistic regression with backward elimination, and a model containing uric acid, hypoxanthine, and lysophosphatidylethanolamine was constructed. Receiver-operating characteristic (ROC) analysis confirmed the prognostic value of these parameters for the diagnosis of minimal/mild endometriosis with a sensitivity of 66.7% and a specificity of 90.0%.ConclusionsMetabolomics analysis of the eutopic endometrium in endometriosis was effectively characterized by UHPLC-ESI-HRMS-based metabolomics. Our study supports the importance of purine and amino acid metabolites in the pathophysiology of endometriosis and provides potential biomarkers for semi-invasive diagnosis of early-stage endometriosis.

Discovery of Phosphatidic Acid, Phosphatidylcholine, and Phosphatidylserine as Biomarkers for Early Diagnosis of Endometriosis.

The sensitivity and specificity of clinical diagnostic indicators and non-invasive diagnostic methods for endometriosis at early stage is not optimal. Previous studies demonstrated that abnormal lipid metabolism was involved in the pathological development of endometriosis. Our cross-sectional study included 21 patients with laparoscopically confirmed endometriosis at stage I–II and 20 infertile women who underwent diagnostic laparoscopy combined with hysteroscopy from January 2014 to January 2015. Eutopic endometrium was collected by pipelle endometrial biopsy. Lipid metabolites were quantified by ultra-high performance liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry (UHPLC-ESI-HRMS). Lipid profiles of endometriosis patients at early stage (I–II) was characterized by a decreased concentration of phosphatidylcholine (18:1/22:6), (20:1/14:1), (20:3/20:4), and phosphatidylserine (20:3/23:1) and an increased concentration of phosphatidic acid (25:5/22:6) compared with control. The synthesized predicting strategy with 5 biomarkers has a specificity of 75.0% and a sensitivity of 90.5%. Lipid profile of eutopic endometrium in endometriosis was effectively characterized by UHPLC-ESI-HRMS-based metabolomics. Our study demonstrated the alteration of phosphatidic acid, phosphatidylcholine, phosphatidylserine metabolites in endometriosis and provided potential biomarkers for semi-invasive diagnose of endometriosis at early stage.

The expression of moesin, p21-activated kinase 4 (PAK 4), matrix metalloproteinases (MMP 2, MMP 9), and CD34 in the eutopic and ectopic endometrium in adenomyosis

To investigate the expression of moesin, p21-activated kinase 4 (PAK 4), matrix metalloproteinases (MMP 2, MMP 9), and CD34 in the eutopic and ectopic endometrium in different forms of adenomyosis. Fifty uteri removed for diffuse adenomyosis and for adenomyomas were examined in reproductive-aged (n=25) and premenopausal (n=25) women. A comparison group included 20 uteri removed for intramural and subserosal fibroids in reproductive-aged and premenopausal women. The investigators performed histological and immunohistochemical (using antibodies to moesin, PAK 4, MMP 2, MMP 9, and CD34) examinations of the eutopic and ectopic endometrium. Different forms of adenomyosis were characterized by the irregular border of the endometrium and myometrium due to that there were multiple foci of ingrowth of the basal layer of the endometrium through the terminal plate into the myometrium. In both diffuse adenomyosis and adenomyomas, the basal layer of the eutopic and ectopic endometrium differed many (3-8.5) times, showing the higher expression of the enzymes in the epithelial and stromal cells, which affected their invasive activity (moesin, PAK 4, MMP 2 and MMP 9), and the increased number of CD34 cells in its stroma. At the same time, there were no statistically significant differences in their expression in the basal layer of the eutopic and ectopic endometrium in diffuse adenomyosis and adenomyomas. The findings favor the theory of the pathogenesis of adenomyosis due to the invasion of the eutopic endometrium into the myometrium.

Accents of molecular markers expression profiling in eutopic and ectopic endometrium in endometriosis

Accents of molecular markers expression profiling in eutopic and ectopic endometrium in endometriosis

Effect of Mst1 on Endometriosis Apoptosis and Migration: Role of Drp1-Related Mitochondrial Fission and Parkin-Required Mitophagy

Background/Aims: Mitochondrial homeostasis is implicated in the development and progression of endometriosis through poorly defined mechanisms. Mst1 is the major growth suppressor related to cancer migration, apoptosis and proliferation. However, whether Mst1 is involved in endometriosis apoptosis and migration via regulating the mitochondrial function remains to be elucidated. Methods: Expression of Mst1 in endometriosis was examined via western blots. Cellular apoptosis was detected via MTT and TUNEL assay. Gain of function assay about Mst1 was conducted via adenovirus over-expression. Mitochondrial functions were evaluated via mitochondrial membrane potential JC-1 staining, ROS flow cytometry analysis, mPTP opening assessment and immunofluorescence of HtrA2/Omi. The mitophagy activity were examined via western blots and immunofluorescence. Results: First, we found that Mst1 was significantly downregulated in the ectopic endometrium of endometriosis compared to the normal endometrium. However, the recovery of Mst1 function was closely associated with the inability of endometrial stromal cells (ESCs) to migrate and survive. A functional study indicated that regaining Mst1 enhanced Drp1 post-transcriptional phosphorylation at Ser616 and repressed Parkin transcription activity via p53, leading to mitochondrial fission activation and mitophagy inhibition. Excessive Drp1-related fission forced the mitochondria to liberate HtrA2/Omi into the cytoplasm. Moreover, Mst1-induced defective mitophagy evoked cellular oxidative stress, energy metabolism and calcium overload. Through excessive mitochondrial fission and aberrant mitophagy, Mst1 launched caspase 9-related mitochondrial apoptosis and abrogated F-actin/lamellipodium-dependent cellular migration. Notably, we also defined NR4A/miR181c as the upstream signal for Mst1 dysfunction in endometriosis. Conclusion: Collectively, our results comprehensively described the important role of the NR4A-miR181c-Mst1 pathway in endometriosis, which handled mitochondrial apoptosis and F-actin/ lamellipodium-based migration via the regulation of Drp1-related mitochondrial fission and Parkin-required mitophagy, with a potential application in endometriosis therapy by limiting ESCs migration and promoting apoptosis.

Decreased expression of FOXA2 promotes eutopic endometrial cell proliferation and migration in patients with endometriosis.

Endometriosis is characterized by eutopic endometrial cell 'metastasis' to ectopic foci. FOXA2 is a member of the forkhead transcription factor family, which may participate in transcriptional regulation in endometrial cells and contribute to the aetiology of endometriosis. This study investigated the roles played by FOXA2 in eutopic endometrium using endometriosis samples. Western blotting showed that the relative expression of FOXA2 was significantly reduced in eutopic endometrium from patients with endometriosis (n = 14) compared with endometriosis-free controls (n = 16) (0.69 ± 0.07 versus 1.24 ± 0.06, P < 0.05). To mimic eutopic endometrium of endometriosis, primary eutopic endometrial stromal cells (ESC) of controls were harvested and transfected with FOXA2 siRNA. MTT assay showed that cell viability of ESC with transfected FOXA2 siRNA increased significantly, whereas the apoptosis rate decreased as indicated by flow cytometry experiments (both P < 0.05). Wound healing assays revealed that transfection of FOXA2 siRNA promoted ESC migration. Moreover, real-time PCR analysis showed progesterone-induced FOXA2 expression in ESC under physiological conditions. In conclusion, these findings indicate that FOXA2 might be a progesterone-induced gene, which may participate in the 'metastatic' process of eutopic endometrium to ectopic loci in patients with endometriosis.

Aberrant expression of epithelial leucine-rich repeat containing G protein–coupled receptor 5–positive cells in the eutopic endometrium in endometriosis and implications in deep-infiltrating endometriosis

To characterize leucine-rich repeat containing G protein-coupled receptor 5-positive (LGR5+) cells from the endometrium of women with endometriosis. Prospective experimental study. University hospital/fertility clinic. Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Obtaining of uterine aspirates by using a Cornier Pipelle. Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5+ cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Immunofluorescence showed that LGR5+ cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5+ cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5+ versus LGR5- cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5+ cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Our results revealed the presence of aberrantly located LGR5+ cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes.

P2X3 receptor involvement in endometriosis pain via ERK signaling pathway.

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

Stress-limiting and stress-realizing systems in endometrium of endometriosis: effects of treatment on the expression of stress proteins and angiogenesis

Stress-limiting and stress-realizing systems in endometrium of endometriosis: effects of treatment on the expression of stress proteins and angiogenesis