Ectopic Endometrial Tissue Research Articles

Caulis Sargentodoxae Prescription Plays a Therapeutic Role with Decreased Inflammatory Cytokines in Peritoneal Fluid in the Rat Endometriosis Model.

Caulis Sargentodoxae prescription has been confirmed by the gynecological clinical observation to be effective in the treatment of endometriosis (EMs), and inflammatory cytokines were involved in EMs. In this paper, animal experiments were designed to explain anti-inflammatory mechanisms of Caulis Sargentodoxae prescription on endometriosis. The EMs model was established by autoplastic transplantation, and rats were randomly divided into seven groups: normal control group, model group, ovariectomized group, gestrinone (Western medicine) group, Caulis Sargentodoxae prescription (Chinese medicine) group, celecoxib (inhibitor) group, and combination (Chinese medicine + inhibitor) group. After oral administration for 21 days, the growth inhibitory rates of the ectopic endometria in treatment groups were evaluated, and the levels of inflammatory cytokines in the serum and peritoneal fluid were determined by ELISA, as well as the expression of prostaglandin endoperoxide synthase 2 (PTGS2) in the ectopic endometrial tissues was detected by real-time polymerase chain reaction, immunohistochemistry, and western blotting. The growth inhibitory rates of the ectopic endometria were significantly higher in the Caulis Sargentodoxae prescription group and gestrinone group, in comparison with the model group (p < 0.05). In the Caulis Sargentodoxae prescription group, the levels of inflammatory cytokines including IL-1, IL-2, IL-6, TNF-α, and PGE2 were all reduced in the serum and peritoneal fluid (p < 0.05). In addition, the specific expression of PTGS2 in the ectopic endometrial tissues significantly decreased in the Caulis Sargentodoxae prescription group and PTGS2 inhibitor celecoxib group both at mRNA and protein levels, but in the steroid hormone drug gestrinone group not at the mRNA level. So, Caulis Sargentodoxae prescription has a reliable therapeutic effect on the EMs by its comprehensive anti-inflammatory roles, possibly in a way different from gestrinone.

Endometriosis – the importance of early diagnosis

Endometriosis is a progressive disease, characterized by the presence of ectopic endometrial tissue, with various locations. In most cases, it occurs in women of re­pro­duc­tive age, affecting both the physical and men­tal health of the patients. Today, the etiology and path­o­ge­ne­sis of en­do­metriosis are not completely elucidated, the phe­no­me­non of retrograde menstruation being the most accepted theory. Clinically, the patients may be asymp­to­matic, but in most cases there are symptoms such as dys­me­nor­rhea, dyspareunia, pelvic pain and infertility. Due to the fact that the clinical manifestations of endometriosis are so variable and nonspecific, and because the diagnosis can only be con­­firmed through laparoscopy and histopathological exa­mi­nation, the positive diagnosis is difficult to sustain, and in most cases this happens late, when the chances of complications, such as infertility and chronic pelvic pain, are greater. It is extremely important that the doctor who has the first contact with the patients – in most cases, being the general practitioner – to know the symp­toms, the evolution, respectively the possible com­pli­ca­tions of endometriosis. A family doctor who possesses this know­ledge could suspect endometriosis in the early stages of the disease and refer patients to the gynecologist, thus play­ing a key role in early diagnosis, and in preventing the oc­cur­rence of complications with serious and irreversible con­sequences.

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.

Abdominal Wall Endometriosis: Purpose of a Case and Review of Literature

Endometriosis of the abdominal wall is defined as the presence of superficial ectopic endometrial tissue to the parietal peritoneum, whose origin may be associated with previous gynecological surgical procedures. Its prevalence is low, around 0.03%, being the first isolated case in our institution, its report and bibliographic review was necessary. The following paper is a case report and a brief bibliographic literature review. Endometriosis is defined as the presence of endometrial glands and stroma outside the uterine cavity. The definition includes injuries that may or may not be related to previous surgical procedures. It is associated with cesarean section in 57% and hysterectomy in 17%. We present the case of a 37 years old female patient, with a history of three previous cesarean sections 3, 6 and 10 years ago; who presented a clinical picture of a sensation of a mass in the abdominal wall, accompanied by cyclical pain and mass growth related to menstrual periods. Abdominal ultrasound reported a heterogeneous vascularized 4x5cm mass. She underwent surgery at our institution where an endometrial mass was evidenced that infiltrated the rectus abdominis, later the diagnosis was confirmed with the histopathological study. Abdominal endometriosis is a rare entity in medical practice. A high index of suspicion should be considered in the case of a woman who presents with disabling abdominal pain located in the abdominal wall, with a history of previous gynecological surgical procedures. This pathology can be confused with many other surgical entities; for this reason, resorting to paraclinical studies can be essential in the diagnostic certainty.

Endometriosis MRI lexicon: consensus statement from the society of abdominal radiology endometriosis disease-focused panel.

Endometriosis is a common gynecologic disorder characterized by the presence of ectopic endometrial tissue outside the endometrial cavity. Magnetic Resonance Imaging (MRI) has become a mainstay for diagnosis and staging of this disease. In the literature, significant heterogeneity exists in the descriptions of imaging findings and anatomic sites of involvement. The Society of Abdominal Radiology's Endometriosis Disease-Focused Panel presents this consensus document to establish an MRI lexicon for endometriosis MRI evaluation and anatomic localization.

Impaired Expression of Ectonucleotidases in Ectopic and Eutopic Endometrial Tissue Is in Favor of ATP Accumulation in the Tissue Microenvironment in Endometriosis.

Endometriosis is a prevalent disease defined by the presence of endometrial tissue outside the uterus. Adenosine triphosphate (ATP), as a proinflammatory molecule, promotes and helps maintain the inflammatory state of endometriosis. Moreover, ATP has a direct influence on the two main symptoms of endometriosis: infertility and pain. Purinergic signaling, the group of biological responses to extracellular nucleotides such as ATP and nucleosides such as adenosine, is involved in the biology of reproduction and is impaired in pathologies with an inflammatory component such as endometriosis. We have previously demonstrated that ectonucleotidases, the enzymes regulating extracellular ATP levels, are active in non-pathological endometria, with hormone-dependent changes in expression throughout the cycle. In the present study we have focused on the expression of ectonucleotidases by means of immunohistochemistry and in situ activity in eutopic and ectopic endometrial tissue of women with endometriosis, and we compared the results with endometria of women without the disease. We have demonstrated that the axis CD39-CD73 is altered in endometriosis, with loss of CD39 and CD73 expression in deep infiltrating endometriosis, the most severe, and most recurring, endometriosis subtype. Our results indicate that this altered expression of ectonucleotidases in endometriosis boosts ATP accumulation in the tissue microenvironment. An important finding is the identification of the nucleotide pyrophophatase/phosphodiesterase 3 (NPP3) as a new histopathological marker of the disease since we have demonstrated its expression in the stroma only in endometriosis, in both eutopic and ectopic tissue. Therefore, targeting the proteins directly involved in ATP breakdown could be an appropriate approach to consider in the treatment of endometriosis.

Thalidomide Reduces Cell Proliferation in Endometriosis Experimentally Induced in Rats.

To analyze the effect of thalidomide on the progression of endometriotic lesions experimentally induced in rats and to characterize the pattern of cell proliferation by immunohistochemical Proliferating Cell Nuclear Antigen (PCNA) labeling of eutopic and ectopic endometrium. Fifteen female Wistar rats underwent laparotomy for endometriosis induction by resection of one uterine horn, isolation of the endometrium and fixation of a tissue segment to the pelvic peritoneum. Four weeks after, the animals were divided into 3 groups: control (I), 10mg/kg/day (II) and 1mg/kg/day (III) intraperitoneal thalidomide for 10 days. The lesion was excised together with the opposite uterine horn for endometrial gland and stroma analysis. Eutopic and ectopic endometrial tissue was submitted to immunohistochemistry for analysis of cell proliferation by PCNA labeling and the cell proliferation index (CPI) was calculated as the number of labeled cells per 1,000 cells. Group I showed a mean CPI of 0.248 ± 0.0513 in the gland and of 0.178 ± 0.046 in the stroma. In contrast, Groups II and III showed a significantly lower CPI, that is, 0.088 ± 0.009 and 0.080 ± 0.021 for the gland (p < 0.001) and 0.0945 ± 0.0066 and 0.075 ± 0.018 for the stroma (p < 0.001), respectively. Also, the mean lesion area of Group I was 69.2 mm2, a significantly higher value compared with Group II (49.4 mm2, p = 0.023) and Group III (48.6 mm2, p = 0.006). No significant difference was observed between Groups II and III. Thalidomide proved to be effective in reducing the lesion area and CPI of the experimental endometriosis implants both at the dose of 1 mg/kg/day and at the dose of 10 mg/kg/day.

Molecular Imaging of Endometriosis Tissues using Desorption Electrospray Ionization Mass Spectrometry

Endometriosis is a pathologic condition affecting approximately 10% of women in their reproductive years. Characterized by abnormal growth of uterine endometrial tissue in other body areas, endometriosis can cause severe abdominal pain and/or infertility. Despite devastating consequences to patients’ quality of life, the causes of endometriosis are not fully understood and validated diagnostic markers for endometriosis have not been identified. Molecular analyses of ectopic and eutopic endometrial tissues could lead to enhanced understanding of the disease. Here, we apply desorption electrospray ionization (DESI) mass spectrometry (MS) imaging to chemically and spatially characterize the molecular profiles of 231 eutopic and ectopic endometrial tissues from 89 endometriosis patients. DESI-MS imaging allowed clear visualization of endometrial glandular and stromal regions within tissue samples. Statistical models built from DESI-MS imaging data allowed classification of endometriosis lesions with overall accuracies of 89.4%, 98.4%, and 98.8% on training, validation, and test sample sets, respectively. Further, molecular markers that are significantly altered in ectopic endometrial tissues when compared to eutopic tissues were identified, including fatty acids and glycerophosphoserines. Our study showcases the value of MS imaging to investigate the molecular composition of endometriosis lesions and pinpoints metabolic markers that may provide new knowledge on disease pathogenesis.

Profiling of Mirna and Mrna in Eutopic and Ectopic Endometrial Tissues in Patients with Endometrioma

Study Objective Profiling of miRNA and mRNA in eutopic and ectopic endometrial tissues in patients with endometrioma. Design A prospective cohort study. Setting This study was conducted at the Department of Operative Gynecology of V.I. Kulakov National Medical Center for Obstetrics, Gynecology, and Perinatology. Patients or Participants This study included 136 women of reproductive age from 18 to 38 years with endometriomas (OE). Interventions Determination of the differences in the expression of miRNA and mRNA in the tissues of OE (EcE) and eutopic endometrium (EuE); bioinformatic analysis to evaluate the role of differentially expressed RNA in the pathogenesis of OE; analysis of microRNA transcriptome (TRA) performed by the sequencing method. Measurements and Main Results TRA revealed significant differences in the expression of miRNA and mRNA in EcE and EuE. It was found that the expression of miRNA and mRNA depends on the type of ovarian reserve (OVR). Lists of target genes were obtained for identified miRNAs were involvement of regulated target genes in the processes of proliferation, in migration and invasion, and in the key cascades of intracellular signaling and inflammatory processes and in the processes of DNA repair and regulation of the mitotic cycle, which indicates the genotoxic effect of pathogenetic factors acting in the tissues of OE and on adjacent ovarian tissues. Conclusion The data obtained suggest that the change in the expression of microRNA in the tissues of the ectopic endometrium can make a significant contribution to the development of endometriosis and the associated reduction of the OVR.

Exosomal Long Noncoding RNA-NONHAT076754 Faciliates Endometriosis Invasion and Predicts Endometriosis Recurrence

Study Objective Exosome-mediated transfer of long noncoding RNAs (lncRNAs) to influence recipient cells is emerging as a novel mechanism for disease progression. NONHAT076754 is a newly identified metastasis-related lncRNA involved in cancer. Since endometriosis exhibits malignancy-like prometastasis behaviour similar to those observed in cancer, we aimed to investigate whether NONHAT076754 is involved in endometriosis and, if so, whether the exosome-mediated transfer of NONHAT076754 contributes to endometriosis migration/invasion. Additionally, we aimed to investigate the clinical value of serum exosomal NONHAT076754 in endometriosis. Design A molecular and clinical retrospective study. Setting Obstetrics and Gynecology Hospital of Fudan University. Patients or Participants Ninety two patients with ovarian endometriotic cysts recruited from 2014 to 2016. Interventions N/A. Measurements and Main Results The distribution and expression of NONHAT076754 in ectopic, eutopic and normal endometria was evaluated using fluorescence in situ hybridization and real-time polymerase chain reaction. Isolation, characterization, labeling and tracking of exosomes were performed. Migration/invasion assays were conducted. We found that NONHAT076754 was highly expressed in ectopic endometrial stromal tissues than in the paired eutopic and normal endometria. In vitro, using NONHAT076754-high-expression ectopic endometrial stromal cells (Ec-ESCs) as exosome-generating cells and NONHAT076754-low-expression eutopic endometrial stromal cells (Eu-ESCs) as recipient cells, we observed that the PKH67-labeled Ec-ESCs derived exosomes were effectively internalized by recipient Eu-ESCs. Exosome-shuttled NONHAT076754 was transferred from Ec-ESCs to Eu-ESCs which in turn elicited the migratory/invasive ability of Eu-ESCs partially by regulating ZO-1, E-cadherin and N-cadherin, eventually facilitating endometriosis migration/invasion. Notably, elevated serum exosomal NONHAT076754 expression was associated with clinical stages and recurrence of endometriosis (P Conclusion Our study suggests a novel mechanism for the malignancy-like prometastasis behaviour of endometriosis from the perspective of the “exosomal transfer of lncRNAs” and highlights the potential of serum exosomal NONHAT076754 as a biomarker for endometriosis recurrence.

CTHRC1 overexpression promotes ectopic endometrial stromal cell proliferation, migration and invasion via activation of the Wnt/β-catenin pathway.

Endometriosis is characterized by the occurrence of endometrial-like tissue outside the uterus. Collagen triple helix repeat containing-1 (CTHRC1) is known as a tumour-promoting factor in several neoplasms. This study aimed to examine the roles of CTHRC1 in the development and progression of endometriosis, and to unravel the underlying mechanisms. Quantitative real-time PCR, western blot analyses and enzyme-linked immunosorbent assay were performed to determine the expression levels of CTHRC1 in tissues and serum. In addition, CTHRC1 expression levels were knocked down by small-interfering RNA in ectopic endometrial stromal cells (EESC). Cell Counting Kit-8, fluorescence-activated cell sorting, Transwell and wound scratch assays were carried out to assess the underlying biological behaviours, and western blot analyses were performed to reveal the molecular mechanisms. mRNA and protein expression levels of CTHRC1 were markedly higher in ectopic endometrial tissues than in eutopic and control endometrial tissues. In addition, the serum concentration of CTHRC1 was apparently higher in the endometriosis group than the control group. Small interfering RNA knockdown of CTHRC1 suppressed the proliferation, migration, invasion and healing abilities of EESC. Furthermore, the protein expressions of key molecules in the Wnt/β-catenin pathway showed an obvious down-regulated expression after siRNA transfection. These findings suggest that CTHRC1 may be partly responsible for the development and progression of endometriosis by increasing EESC proliferation, migration and invasion via the Wnt/β-catenin pathway. CTHRC1 may thus serve as a diagnostic and therapeutic target for endometriosis.

Ureaplasma Urealyticum Infection Contributes to the Development of Pelvic Endometriosis Through Toll-Like Receptor 2.

Endometriosis is a chronic gynecological disorder, characterized by the presence of ectopic endometrial tissue outside the uterine cavity. Among several hypotheses, Sampson's theory of retrograde menstruation is still applicable. Recent studies have reported the importance of inflammation among endometrial tissue, the peritoneum, and immune cells. However, less is known regarding the role of bacterial infection in the pathophysiology of endometriosis. We hypothesized that Ureaplasma urealyticum infection might contribute to the development of endometriosis by inducing the production of inflammatory mediators by peritoneal mesothelial cells (PMCs), possibly through TLR2. Hence, our objective was to reveal whether PMC infection by U. urealyticum is associated with endometriosis. Moreover, we aimed to demonstrate the molecular mechanism involved in this relationship. We developed a new infection-induced mouse model of endometriosis with wild type and Tlr2-deficient mice. Based on the in vivo mouse model, U. urealyticum-infected mice showed significantly increased numbers and sizes of ectopic endometriotic lesions. U. urealyticum upregulated not only the production of IL-6, CXCL1, and CCL2, but also the expression of ICAM-1, VCAM-1, and MMP2 in murine PMCs. Similarly, endometrial stromal cells dose-dependently produced IL-6, CXCL1, and CCL2 in response to U. urealyticum infection. The series of inflammatory responses in PMCs was mediated mainly through TLR2. The phosphorylation of ERK and JNK was observed when U. urealyticum was added to PMCs and knock out of Tlr2 inhibited these MAPKs phosphorylation. Based on our co-culture study, U. urealyticum-infected PMCs exhibited significantly increased attachment to ESCs compared with uninfected PMCs. Collectively, U. urealyticum infection promotes the development of endometriosis by increasing inflammatory mediators, adhesion molecules, and MMP-2 expression in PMCs through TLR2 signaling. Through our results, we present a theory that infection-induced pelvic inflammation contributes to the initiation and progression of endometriosis. Appropriate treatment of reproductive tract infection may decrease the prevalence of endometriosis.

THORACIC ENDOMETRIOSIS SYNDROME PRESENTING AS RECURRENT CATAMENIAL PNEUMOTHORAX

SESSION TITLE: Tuesday Medical Student/Resident Case Report Posters SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM INTRODUCTION: Catamenial pneumothorax (CP) is a rare spontaneous pneumothorax (PTX) that occurs cyclically with menstrual periods. It is part of the spectrum of thoracic endometriosis syndrome (TES) in which ectopic endometrial tissue is implanted in thoracic structures. CASE PRESENTATION: A 36-year-old G0P0 female nurse, presented with worsening right-sided pleuritic chest pain for 6 days. She had a history of stage 4 endometriosis associated with recurrent CP. Her first episode was 4 years ago when she had a large right-sided PTX in association with biopsy proven diaphragmatic endometriosis, treated with a right middle lobe wedge resection and chemical pleurodesis. She had multiple recurrences of PTX requiring repeat pleurodesis and subsequent long-term medical therapy with leuprolide and combination oral contraceptive pills. On examination, she was hemodynamically stable with decreased breath sounds in the right lower lung field. CT chest revealed a moderate volume right PTX, a sub-pleural right lower lobe mass and pleural thickening. She underwent a video-assisted thoracoscopic surgery (VATS) with combination (mechanical and chemical) pleurodesis. Pleural biopsy was negative for endometriosis. Postoperative chest x-rays revealed a resolving pneumothorax. Three days after surgery, the patient was discharged home. DISCUSSION: CP is characterized by collapsed lung, which occurs within 3 days of menstruation onset. The pathophysiology involves deposition of focal endometrial pleural implants migrating during leakage of air from the peritoneal cavity, which subsequently break down during menstruation or hypertrophy in response to hormonal changes. Another plausible theory is vasoconstriction and bronchospasm induced by prostaglandin F2 release during menses leading to alveolar rupture and PTX. Due to the vague symptomatology and limited disease awareness, the diagnosis is often missed or delayed leading to life-threatening complications. Histologically proven thoracic endometriosis is frequently but not always associated with CP. Our patient is unique as she previously had a diaphragmatic endometriosis associated CP and later had a non-TES associated bout of CP. Recent evidence suggests that combination surgical (VATS) followed by medical (GnRH agonist) therapy effectively suppresses CP recurrence. However, our patient suffered multiple recurrences even after both modalities. A combination of mechanical and chemical pleurodesis was attempted in an effort to prevent further recurrences. CONCLUSIONS: TES remains an exceedingly rare condition and very few cases have been reported. Our case is unique given her multiple recurrences and unresponsiveness to guideline directed therapies. Physicians should keep a high level of clinical suspicion for thoracic endometriosis in non-smoking women over 31 years, with a history of pelvic endometriosis, and presenting with recurrent pneumothorax. Reference #1: Junejo SZ, Singh Lubana S, Shina SS, Tuli SS.A Case of Thoracic Endometriosis Syndrome Presenting with Recurrent Catamenial Pneumothorax. Am J Case Rep. 2018 May 17;19:573-576. DISCLOSURES: No relevant relationships by Prasanta Basak, source=Web Response No relevant relationships by Stephen Jesmajian, source=Web Response No relevant relationships by Ahmed Qavi, source=Web Response

ENDOMETRIOSIS OF THE LUNG PRESENTING AS INNUMERABLE NODULES

ENDOMETRIOSIS OF THE LUNG PRESENTING AS INNUMERABLE NODULES

A patient with catamenial pneumothorax confirmed to have ectopic endometrial tissue in multiple holes of the diaphragm

A patient with catamenial pneumothorax confirmed to have ectopic endometrial tissue in multiple holes of the diaphragm

Diagnosis Of Endometriosis In The Light Of Prevalent Theories

Endometriosis is a gynecological condition recognized by the existence of ectopic endometrial tissue outside the uterus. It is predominantly present in females of reproductive age group and is one of the main causes of infertility. Even with a predictable prevalence of 11% in females and considerable historical explanations adopted from the seventeenth century, the diagnosis of endometriosis still remains doubtful. The conventional concepts on histological basis of endometriosis are explained by a number of theories. Medical signs of endometriosis contain prolonged pelvic ache, dyspareunia, repeated menstrual discomfort and chronic pelvic pain which can severely affect the excellence of life and health of the patient. In this review we will discuss the prevalent theories for the diagnosis of endometriosis and suggestions to identify the condition well in time for better control and management

α1D-adrenergic receptor expression in human endometrial stromal cells contributes to catecholestradiol-induced cell survival: implications for endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance generation of CCEs in women with endometriosis. During gestation, CCEs bind adrenergic receptors (ARs) to mediate uterine endothelial cell proliferation. Our recent data demonstrated that compared to the basalis layer of the endometrium, the functionalis layer displayed increased β2-AR expression in women with endometriosis. Moreover, binding of CCEs to β-ARs enhanced human endometrial stromal cell (HESC) viability in culture, suggesting a modulatory role for CCE-β-AR binding in retrograde menstruation that contributes to endometriosis development. It is unknown if, in addition to β-ARs, α-ARs also have a role in this process. We, therefore, tested the hypothesis that ectopic endometrial tissue expresses α1D-AR and that 2-OHE2 and 4-OHE2 will potentiate HESC viability via α-ARs. Immunohistochemistry was performed on paired eutopic/ectopic endometriotic tissue and XTT analysis was conducted on cultured CCE-treated HESCs derived from endometrial biopsies. Paired eutopic/ectopic endometrial sections from women with endometriosis in the proliferative (n=5) or secretory (n=4) phases were immunostained using a1D-AR antibody and evaluated semi-quantitatively by HSCORE. Confluent HESCs derived from endometrial biopsies at time of surgery for benign reasons were cultured in 96-well plates (5x103 cells/well) and treated with vehicle (control) or with 10-8 M E2 or 2-OHE2 or 4-OHE2 ± nonspecific α-AR inhibitor (phentolamine) for 48h. XTT assays measured cell viability. Experiments (n=4) were performed in duplicate and results were analyzed by One-way ANOVA and post hoc Tukey test. Immunohistochemistry analysis revealed overall weak to moderate α1D-AR staining in endometrial epithelial cells and weak staining in endometrial stromal cells with no significant difference between eutopic and ectopic endometrial tissues in either phase. Compared to the basalis layer, both stromal and epithelial cells in the functionalis layer of eutopic endometrium displayed stronger α1D-AR staining. In vitro XTT analyses revealed phentolamine partially inhibited (P<0.05 and P<0.001, respectively) 2-OHE2 and 4-OHE2-enhanced HESC survival versus control (P<0.05 and P<0.05, respectively). This inhibitory effect of phentolamine was specific to CCEs since E2-enhanced HESC survival was not inhibited by phentolamine. Immunostaining with α1D-AR showed similar expression patterns as β2-AR in that there was increased staining to the functionalis layer of the endometrium; albeit, α1D-AR staining was overall weaker in both ectopic and eutopic tissue. Our in vivo and in vitro results indicate that induction of HESC viability by CCE-α-AR binding may contribute to the propagation of endometriosis. Future studies into different α-AR isoform expressions in endometriotic lesions are warranted.

Propranolol inhibits catecholestrogen-induced human endometrial stromal cell survival mediated by p38 MAPK signaling: potential therapy for endometriosis

Catecholestrogens (CCEs), 2-Hydroxyestradiol (OHE2) and 4-OHE2, are biologically active metabolites of 17β-estradiol (E2). Studies indicate that local increases in E2 production as well as aberrant expression of E2 metabolizing enzymes enhance local generation of CCEs in women with endometriosis. CCEs have low binding affinity to estrogen receptors whereas CCEs display high binding affinity to β2-adrenergic receptor (AR), which induce uterine endothelial cell proliferation during gestation. Our recent data demonstrated β2-AR expression in eutopic and ectopic endometrial tissue. In addition, binding of CCEs to β-AR enhances human endometrial stromal cell (HESC) viability, suggesting contribution of CCEs to the pathogenesis of endometriosis. Thus, we tested the hypothesis that the mechanism of CCE-enhanced HESC viability involves alterations in either proliferation or apoptosis mediated by β-AR-induced common intracellular signaling pathways, i.e. AKT, MAPK and/or NFκB. BrdU, Apoptotic Cell Detection ELISA, q-PCR, Western blot and XTT analyses were performed on cultured HESCs derived from endometrial biopsies. Cultured HESCs treated with 10-8 M E2 or 2-OHE2 or 4-OHE2 were measured by BrdU for proliferation and ELISA for apoptosis (n=3 with quadruplicate). Total RNA from cultured HESCs was isolated and pro-apoptotic, anti-apoptotic, and proliferation markers were evaluated by q-PCR (n=5 with duplicate). Total and phosphorylated AKT, p38 and ERK1/2 MAPKs, and NFκB levels were detected in lysates of cultured HESCs (n=3 with triplicate) treated for 10 min with vehicle (control) or 10-8 M E2 or 2-OHE2 or 4-OHE2 ± 2x10-5 M non-specific β-antagonist (propranolol). Subsequently, XTT assays were conducted with p38 MAPK inhibitor to assess the role of p38 MAPK on CCE-induced HESC viability (n=4 with triplicate). Results were analyzed by One-way ANOVA and post hoc Tukey test. An increased HESC proliferation index by E2 and 4-OHE2 (P<0.05 and P<0.05, respectively) and decreased apoptosis were detected in HESCs treated with 2-OHE2 and 4-OHE2 vs. control (P<0.01 and P<0.01, respectively). Analysis of apoptotic markers by q-PCR revealed a significant decrease in Bax mRNA expression in response to 2-OHE2 treatment vs. control (P<0.01). Among the several intracellular signaling cascades analyzed, only phosphorylation levels of p38 MAPK were increased by either treatment with 2-OHE2 or 4-OHE2 (P<0.05 and P<0.05 vs. control, respectively), but not with E2. β-AR antagonism with propranolol mitigated this increased phosphorylation in p38 MAPK levels (P<0.05 and P<0.01, respectively) and inhibition of p38 MAPK by SB203580 blocked CCE-induced HESC survival (P<0.05 and P<0.001, respectively). These data indicate that induction of endometrial stromal cell viability by CCE-β-AR interactions results from an imbalance in both proliferation and anti-apoptotic mechanisms and is specifically mediated by the activation of p38 MAPK signaling. These data also suggest that inhibition of β2-ARs with propranolol may be a novel treatment option in endometriosis.

MicroRNA-142-3p suppresses endometriosis by regulating KLF9-mediated autophagy in vitro and in vivo

ABSTRACTThe detailed pathogenesis of endometriosis remains largely unclear despite decades of research. Recent studies have demonstrated that miRNAs plays an important role in endometriosis. The expression of miR-142-3p was decreased in ectopic endometrial tissues, while KLF9 and VEGFA expression levels were increased. Overexpression of miR-142-3p or knockdown of KLF9 significantly suppressed CRL-7566 cell proliferation and metastasis, induced cell apoptosis, and decreased both cell autophagy and vascularization. Additionally, KLF9 was confirmed to be a direct target of miR-142-3p and to directly bind to the promoter of the VEGFA gene, regulating its expression. Finally, intraperitoneal injection of miR-142-3p lentivirus significantly attenuated ectopic endometriotic lesions in vivo.miR-142-3p directly targeted KLF9, regulated VEGFA expression, and was protective against the growth of ectopic endometriotic lesions. Therefore, the miR-142-3p/KLF9/VEGFA signalling pathway may be a potential target in endometriosis treatment.

Fibrinogen alpha chain is up-regulated and affects the pathogenesis of endometriosis.

In the group's previous study, fibrinogen alpha chain (FGA) was identified as an up-regulated differential protein that was highly expressed in women with endometriosis. The current study investigated the expression and effects of FGA in endometriosis. It also evaluated the effects of FGA on human endometrial stromal cells and studied the possible mechanism. This was a cross-sectional analysis of FGA expression in plasma and endometrial tissue of matched eutopic and ectopic samples from women with endometriosis undergoing laparoscopic surgery and samples from women without endometriosis. Forty-four patients with endometriosis and 32 healthy control subjects who donated plasma for FGA analysis, including 26 matched cases of eutopic and ectopic endometria from endometriosis patients and 22 endometria from healthy control subjects, were analysed. The effects of FGA were studied in a human endometrial stromal cell line after transfection with FGA short interfering RNA (siRNA). FGA concentrations in serum and expression in eutopic and ectopic endometrial tissue were significantly higher in women with endometriosis than controls (P<0.05 and P<0.01 respectively), whereas FGA expression was not significantly different in eutopic compared with ectopic endometrial tissues from the same patients. High FGA concentrations in serum were related to disease stage and ovarian involvement, but were not affected by age and menstrual cycle. The knockdown of FGA expression by FGA siRNA inhibited hEM15A cellular adhesion, migration and invasion, and attenuated matrix metalloproteinase-2 (MMP-2) expression. High FGA expression in endometriosis was closely related to disease severity and affected cell adhesion, migration and invasion, which might play an important role in the pathogenesis of endometriosis.