Impaired secretion of C-X-C motif chemokine ligand 10 by stimulation with a Toll-like receptor 4 ligand in endometrial epithelium of infertile patients with minimal-to-mild endometriosis.

Successful embryo implantation requires transient, well-controlled inflammation in decidualizing cells. In mice, Toll-like receptor (TLR) 4 signaling in endometrial epithelial cells (EECs) by stimulation with factors present in seminal fluids has been shown to be a key upstream driver of a controlled inflammatory response. Clinical evidence supports that exposure of the female reproductive tract to seminal plasma promotes implantation success. We investigated the response of EECs to TLR2 (Pam3Csk4), TLR 3 (Poly I:C), and TLR4 (lipopolysaccharides [LPS]) ligands with respect to secretion of C-X-C motif chemokine ligand (CXCL) 10 (CXCL10) and interleukin-6 (IL-6) in infertile patients with minimal-to-mild endometriosis (EECs-endo) (n = 38) and those of healthy, fertile women (EECs-healthy) (n = 30). Stimulation with either Pam3Csk4, Poly I:C or LPS, significantly induced CXCL10 and IL-6 in EECs-healthy (p < 0.05). In EECs-endo, either Pam3Csk4 or Poly I:C significantly induced CXCL10 (p < 0.05), whereas no significant response was observed after stimulation with LPS. Neither LPS, Poly I:C, nor Pam3Csk4 significantly induced IL-6 secretion in EECs-endo. Secretion of CXCL10 in EECs-healthy after stimulation with LPS was significantly higher (p < 0.05) than that in EECs-endo. CXCL10 decreased cell proliferation of EECs from both groups. Activation of nuclear factor kappa light chain enhancer of activated B cells and signal transducer and activator of transcription 3 signalings was not impaired, but activation of p38 mitogen-activated protein kinases signaling by LPS stimulation was impaired in EECs-endo. The present findings suggested that an insufficient response of EECs to a TLR4 ligand may be involved in molecular mechanisms of endometriosis-associated infertility.

IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis: IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression.

STUDY QUESTION: Is interleukin-10 (IL-10) anti-fibrotic in endometriosis? SUMMARY ANSWER: IL-10 is not anti-fibrotic but pro-fibrotic in endometriosis, because IL-10 treatment of endometriotic stromal cells in vitro promotes myofibroblast proliferation and collagen type I protein expression. WHAT IS KNOWN ALREADY: We previously showed that persistent activation of signal transducer and activator of transcription 3 (STAT3) via IL-6 trans-signaling promotes fibrosis of endometriosis. Studies showed marked anti-fibrotic effects of IL-10 via the STAT3 signaling pathway, which is generally considered to be anti-inflammatory, in various organs. STUDY DESIGN, SIZE, DURATION: Endometrial and/or endometriotic samples of 54 patients who had histological evidence of deep endometriosis, and endometrial samples from 30 healthy fertile women were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of IL-10/STAT3 signaling as well as inhibition of STAT3 activation by knockdown of STAT3 gene on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. Then, the effects of various time points of IL-10 treatment in combination with transforming growth factor (TGF)-ß1 and/or IL-6/soluble IL-6 receptor (sIL-6R) on the profibrotic phenotype of endometrial and endometriotic stromal cells were investigated. MAIN RESULTS AND THE ROLE OF CHANCE: IL-10 induced pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers and collagen gel contraction) of endometriotic stromal cells. Knockdown of STAT3 gene decreased the IL-10 induced pro-fibrotic phenotype of endometriotic stromal cells. In contrast, IL-10 had no significant effects on pro-fibrotic phenotype of endometrial stromal cells of healthy women. Sequential IL-10 treatment with or without TGF-ß1 and/or IL-6/sIL-6R induced persistent activation of STAT3 and significantly increased proliferation of myofibroblasts (cells with α-smooth muscle actin positive stress fibers) and protein expression of collagen type I in endometriotic stromal cells. TGF-ß1 and/or IL-6/sIL6RIL-6/sIL6R treatment significantly increased tissue inhibitor of metalloproteinase 1 (TIMP1) protein expression, whereas IL-10 had no significant effects. Knockdown of STAT3 gene significantly decreased the TGF-ß1 and/or IL-6/sIL6R induced TIMP1 protein expression. In contrast, pre-treatment with IL-10 before TGF-ß1 and/or IL-6/sIL-6R treatment and sequential IL-10 treatment with or without TGF-ß1 and/or IL-6/sIL-6R significantly decreased proliferation of fibroblasts (cells without α-smooth muscle actin positive stress fibers) and collagen type I protein expression in endometrial stromal cells of healthy women. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Given the large number of complex interactions and signaling pathways of pro- and anti-inflammatory mediators that are involved in the pathophysiology of endometriosis, the present study investigated only a very small portion of the whole. Further in vivo studies are required to validate the present findings. WIDER IMPLICATIONS OF THE FINDINGS: Inflammatory mediators in the pathophysiology of endometriosis have been extensively investigated as potential therapeutic targets. However, the present study showed that anti-inflammatory signals of IL-10 and IL-6 through persistent STAT3 activation may promote endometriosis fibrosis. Therapeutic strategies, such as suppression of 'inflammation', might dysregulate the cross-regulation of 'pro- and anti-inflammatory mediators', leading to detrimental effects in patients with endometriosis, such as fibrosis. To develop new, but not deleterious, therapeutic strategies, studies are required to investigate whether, how and what 'anti-inflammatory mediators' along with pro-inflammatory mediators are involved in individual patients with endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by KARL STORZ SE & Co. KG (Tuttlingen, Germany). The authors have no conflict of interest to disclose.

Persistent activation of signal transducer and activator of transcription 3 via interleukin-6 trans-signaling is involved in fibrosis of endometriosis.

STUDY QUESTION: Is activation of signal transducer and activator of transcription 3 (STAT3) via interleukin-6 (IL-6) trans-signaling involved in fibrosis of endometriosis? SUMMARY ANSWER: Persistent activation of STAT3 via IL-6 trans-signaling is involved in fibrosis of endometriosis. WHAT IS KNOWN ALREADY: Our previous study showed that sustained low-grade inflammation promotes a fibrotic phenotype in endometriotic stromal cells. However, the underlying mechanisms of the establishment of non-resolving, low-grade inflammation in endometriosis remain to be clarified. STUDY DESIGN, SIZE, DURATION: Endometrial and/or endometriotic samples of 60 patients who had histological evidence of deep endometriosis and endometrial samples from 32 healthy fertile women were analyzed. PARTICIPANTS/MATERIALS, SETTING, METHODS: The effects of priming with ligands of Toll-like receptors (TLRs) 2, 3 and 4 on secretion of inflammatory mediators (tumor necrosis factor-α, C-X-C motif chemokine ligand-10 [CXCL-10], IL6 and IL-10) after a second challenge with TLR ligands in endometrial and endometriotic stromal cells were investigated. Then, the effects of IL-6/soluble (s) IL-6 receptor (R)/STAT3 signaling, as well as inhibition of STAT3 activation by knockdown of STAT3 or pharmacological inhibition (S3I-201), on the pro-fibrotic phenotype in endometrial and endometriotic stromal cells in vitro were investigated. MAIN RESULTS AND THE ROLE OF CHANCE: Priming with TLR ligands for 4 h had no significant effects, whereas 24 h of priming significantly decreased secretion of IL-6, after a second challenge in endometrial stromal cells of healthy women. In endometriotic stromal cells, whereas 24 h of priming had no significant effects, priming with TLR ligands for 4 h significantly increased secretion of IL-6 after a second challenge. IL-6/soluble IL-6 receptor (sIL-6R) induced a pro-fibrotic phenotype (cell proliferation, collagen type I synthesis, α-smooth muscle actin positive stress fibers, cell migration and collagen gel contraction) as well as nuclear factor-kappa B (NF-κB) activation of endometriotic stromal cells. In contrast, IL-6/sIL-6R had no significant effects on either a pro-fibrotic phenotype or NF-κB activation of endometrial stromal cells of healthy women. Stimulation with transforming growth factor (TGF)-ß1 and/or IL-6/sIL-6R for 1 h and 48 h activated STAT3, but induced very low or no suppressor of cytokine signaling (SOCS) 1 and 3 protein expression in endometriotic stromal cells. In endometrial stromal cells of healthy women, IL-6/sIL-6R-induced STAT3 and SOCS1/3 expression at 1 h, whereas no STAT3 activation was detected at 48 h. Knockdown of STAT3 gene or S3I-201 (a STAT3 inhibitor) decreased the IL-6/sIL-6R-induced pro-fibrotic phenotype as well as NF-κB activation and TGF-ß1-induced cell proliferation of endometriotic stromal cells. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In vivo studies are required to confirm the present in vitro results. However, it remains challenging to mimic non-resolving chronic inflammation in animal models, as active inflammation can resolve spontaneously. WIDER IMPLICATIONS OF THE FINDINGS: Dysfunction of negative regulators of IL-6/sIL-6R/STAT3 signaling may cause persistent activation of STAT3 in endometriosis. Since STAT3 activation in the endometrium is essential for successful embryo implantation, treatment with STAT3 inhibitors would not be appropriate for women wishing to conceive. However, targeting impaired negative regulation of IL-6/sIL-6R/STAT3 signaling may still represent a promising avenue for the treatment of endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This study was supported in part by the KARL STORZ SE & Co. KG (Tuttlingen, Germany). There are no conflicts of interest.

[Avenues of reflection for endometriosis research in France]. / Des pistes de réflexion pour la recherche sur l'endométriose en France.

Endometriosis is a chronic disease in which lesions resembling endometrial tissue are found outside the uterus, mainly in the pelvis or abdomen. It may affect 10% of women of childbearing age. It is the cause of a significant alteration in quality of life and a major cost to the health system. Few research teams are working on this subject, and its pathophysiology is still poorly understood. This article proposes avenues of reflection for research on endometriosis in France, notably based on the mobilization of related scientific communities (involved in cancer, development, epigenetics, and neurosciences research studies).

Title: Des pistes de réflexion pour la recherche sur l'endométriose en France. Abstract: L'endométriose est une maladie chronique dans laquelle des lésions ressemblant à du tissu endométrial se retrouvent hors de l'utérus, principalement dans la cavité abdomino-pelvienne. Cette maladie pourrait toucher 10 % des femmes en âge de procréer. Elle est à l'origine d'une importante altération de la qualité de vie et d'un coût majeur pour le système de santé. Peu d'équipes de recherche sont mobilisées sur ce sujet, et la physiopathologie de la maladie reste mal comprise. Nous proposons dans cet article des pistes de réflexion pour la recherche sur l'endométriose en France, fondées notamment sur la mobilisation de communautés scientifiques connexes (notamment celles impliquées dans la recherche sur le cancer, la biologie du développement, l'épigénétique, les neurosciences).

Mechanobiology of the female reproductive system.

BACKGROUND: Mechanobiology in the field of human female reproduction has been extremely challenging technically and ethically. METHODS: The present review provides the current knowledge on mechanobiology of the female reproductive system. This review focuses on the early phases of reproduction from oocyte development to early embryonic development, with an emphasis on current progress. MAIN FINDINGS RESULTS: Optimal, well-controlled mechanical cues are required for female reproductive system physiology. Many important questions remain unanswered; whether and how mechanical imbalances among the embryo, decidua, and uterine muscle contractions affect early human embryonic development, whether the biomechanical properties of oocytes/embryos are potential biomarkers for selecting high-quality oocytes/embryos, whether mechanical properties differ between the two major compartments of the ovary (cortex and medulla) in normally ovulating human ovaries, whether durotaxis is involved in several processes in addition to embryonic development. Progress in mechanobiology is dependent on development of technologies that enable precise physical measurements. CONCLUSION: More studies are needed to understand the roles of forces and changes in the mechanical properties of female reproductive system physiology. Recent and future technological advancements in mechanobiology research will help us understand the role of mechanical forces in female reproductive system disorders/diseases.

Effects of Low Intraperitoneal Pressure on Quality of Postoperative Recovery after Laparoscopic Surgery for Genital Prolapse in Elderly Patients Aged 75 Years or Older.

STUDY OBJECTIVE: Previous clinical trials for laparoscopic surgery have included few elderly patients aged ≥75 years. We aimed to evaluate the quality of postoperative recovery after laparoscopic surgery using low intraperitoneal pressure (IPP) (6 mm Hg) and warmed, humidified carbon dioxide gas for genital prolapse in elderly patients aged ≥75 years. DESIGN: Prospective consecutive case series. SETTING: University hospital. PATIENTS: Consecutive patients (n = 30) aged ≥75 years planning to undergo laparoscopic surgery for genital prolapse by the same surgeon were recruited from October 2016 through December 2019. INTERVENTIONS: Laparoscopic promontofixation for the treatment of genital prolapse was performed using low IPP and warmed, humidified carbon dioxide gas. When a promontory could not be easily identified, laparoscopic pectopexy was alternatively performed. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the Quality of Recovery-40 (QoR-40) score at 24 hours postoperatively. The secondary outcomes were postoperative pain using a 100-mm visual analog scale and the length of hospital stay after surgery (LHSS). For the global QoR-40 score and for 4 dimensions of the QoR-40, "emotional state," "physical comfort," "psychologic support," and "pain," no differences were observed between the baseline score and the score at 24 hours. The score for the "physical independence" dimension at 24 hours was significantly lower than the baseline score (p <.001). No patient had visual analog scale pain scores >30 out of 100 at 12 hours or later. LHSS was <48 hours in 22 patients (73.3%) and <72 hours in 8 patients (26.7%). Multivariable analysis showed that the odds of an LHSS >48 hours were more than 8 times higher in patients who were discharged from the operating room in the afternoon compared with those with a morning discharge. CONCLUSION: The use of a low IPP is feasible, safe, and has clinical benefits for elderly patients aged ≥75 years who undergo laparoscopic surgery for genital prolapse.

Hyperactivation of dormant primordial follicles in ovarian endometrioma patients.

Serum anti-Müllerian hormone (AMH) levels decrease after surgical treatment of ovarian endometrioma. This is the main reason that surgery for ovarian endometrioma endometriosis is not recommended before in vitro fertilization, unless the patient has severe pain or suspected malignant cysts. Furthermore, it has been suggested that ovarian endometrioma itself damages ovarian reserve. This raises two important challenges: (1) determining how to prevent surgical damage to the ovarian reserve in women with ovarian endometrioma and severe pain requiring surgical treatment and (2) deciding the best treatment for women with ovarian endometrioma without pain, who do not wish to conceive immediately. The mechanisms underlying the decline in ovarian reserve are potentially induced by both ovarian endometrioma and surgical injury but the relative contribution of each process has not been determined. Data obtained from various animal models and human studies suggest that hyperactivation of dormant primordial follicles caused by the local microenvironment of ovarian endometrioma (mechanical and/or chemical cues) is the main factor responsible for the decreased primordial follicle numbers in women with ovarian endometrioma. However, surgical injury also induces hyperactivation of dormant primordial follicles, which may further reduce ovarian reserve after removal of the endometriosis. Although further studies are required to elucidate the mechanisms underlying diminished ovarian reserve in women with ovarian endometrioma, the available data strongly suggests the need to prevent/minimize hyperactivation of dormant primordial follicles, regardless of whether surgery is performed, for better clinical management of ovarian endometrioma.

Impaired pathogen-induced autophagy and increased IL-1ß and TNFα release in response to pathogenic triggers in secretory phase endometrial stromal cells of endometriosis patients.

RESEARCH QUESTION: It is not clear whether innate immunity along with autophagy is altered in endometrial cells of patients with endometriosis. DESIGN: This study evaluated the effects of lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (poly I:C) stimulation on autophagy induction, pro-IL-1ß expression, and secretion of interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα) in endometrial epithelial and/or stromal cells of patients with endometriosis (EE-endo, ES-endo, respectively), those of patients with hydrosalpinx (EE-hydro, ES-hydro, respectively) and those of healthy fertile women (EE-healthy, ES-healthy, respectively), with and without inhibition of autophagy by autophagy-related (ATG)13 gene small interfering RNA (siRNA). RESULTS: Stimulation with either LPS or poly I:C triggered autophagy in EE/ES-healthy, whereas no significant induction was observed in either EE/ES-endo or EE/ES-hydro. In EE- and/or ES-healthy, IL-1ß and/or TNFα secretion after stimulation with LPS or poly I:C was significantly higher in cells with ATG13 knockdown compared with those with siRNA control (P < 0.03), whereas no significant difference was observed in either EE/ES-endo or EE/ES-hydro. In the secretory phase ES-endo without autophagy inhibition, IL-1ß and TNFα secretion were significantly higher compared with those of ES-healthy after stimulation with either LPS or poly I:C for 4 h (P < 0.001) and for 24 h (P < 0.01). CONCLUSION: Pathogen-induced autophagy was impaired in EE/ES-endo. Increased IL-1ß and TNFα release in response to pathogenic triggers in the secretory phase ES-endo may result in the development of an inflammatory uterine microenvironment detrimental to successful embryo implantation.

Dose-dependent pro- or anti-fibrotic responses of endometriotic stromal cells to interleukin-1ß and tumor necrosis factor α.

Endometriosis are characterized by dense fibrous tissue. Numerous studies have investigated roles of inflammation on the pathophysiology of endometriosis. However, the interplay of inflammation and fibrosis remains to be clarified. Here we show that low levels of interleukin-1ß (IL-1ß) and tumor necrosis factor-alpha (TNFα) promoted a fibrotic phenotype, whereas high levels of IL-1ß and TNFα inactivated the fibrotic phenotype of endometriotic stromal cells (Ectopic-ES). IL-1ß 10 pg/mL and TNFα 100 and 1,000 pg/mL had minimal effects, whereas the highest dose of IL-1ß (100 pg/mL) significantly decreased collagen gel contraction in Ectopic-ES. Furthermore, in Ectopic-ES, low levels of IL-1ß (1 pg/mL) and/or TNFα 10 pg/mL significantly increased Col I mRNA expression, whereas higher doses of IL-1ß (10 and/or 100 pg/mL) and/or TNFα (100 and/or 1,000 pg/mL) significantly decreased Col I and/or αSMA mRNA expression and the percentage of cells with Col I + and/or αSMA + stress fibers. In contrast, in either menstrual endometrial stromal cells of patients with endometriosis or those of healthy women, varying doses of IL-1ß and/or TNFα had no significant effects on either Col I or αSMA mRNA/protein expression. The present findings bring into question whether we should still continue to attempt anti-inflammatory treatment strategies for endometriosis.

In vitro and in vivo effects of MK2206 and chloroquine combination therapy on endometriosis: autophagy may be required for regrowth of endometriosis.

BACKGROUND AND PURPOSE: A high recurrence rate after medical treatment is a major clinical problem for patients with endometriosis. Here, we have evaluated the in vitro effects of combined treatment with MK2206 (an AKT inhibitor) + chloroquine on cell growth and regrowth of endometriotic stromal cells and the in vivo effects on endometriotic implants in a mouse xenograft model of endometriosis. EXPERIMENTAL APPROACH: We evaluated the effects of autophagy inhibition by knockdown of the ATG13, Beclin-1 and ATG12 genes and pharmacological agents (chloroquine, bafilomycin A1 or 3-methyalanine) individually and in combination with MK2206 on cell growth and/or cell regrowth of endometriotic stromal cells in vitro. Furthermore, we evaluated treatment with MK2206 + chloroquine on endometriotic implants in a mouse xenograft model of endometriosis. KEY RESULTS: Combined treatment with MK2206 and chloroquine markedly reduced cell growth and regrowth after discontinuation of treatment in endometriotic stromal cells compared with cells treated with either drug alone. Autophagy inhibition by ATG13, Beclin-1 or ATG12 gene knockdown only affected regrowth of endometriotic stromal cells, but not endometrial stromal cells from the same patients, after a 72 h discontinuation of the combined treatment. Furthermore, combined treatment reduced the size of endometriotic implants, whereas no effects on endometriotic implants treated with either drug alone were observed in a mouse xenograft model of endometriosis. CONCLUSIONS AND IMPLICATIONS: The present findings suggest that a novel strategy for treatment of endometriosis may involve decreasing the number of endometriotic cells that can survive treatment and then preventing regrowth by autophagy inhibition.

Effects of low intraperitoneal pressure and a warmed, humidified carbon dioxide gas in laparoscopic surgery: a randomized clinical trial.

Laparoscopic surgery technology continues to advance. However, much less attention has been focused on how alteration of the laparoscopic surgical environment might improve clinical outcomes. We conducted a randomized, 2 × 2 factorial trial to evaluate whether low intraperitoneal pressure (IPP) (8 mmHg) and/or warmed, humidified CO2 (WH) gas are better for minimizing the adverse impact of a CO2 pneumoperitoneum on the peritoneal environment during laparoscopic surgery and for improving clinical outcomes compared to the standard IPP (12 mmHg) and/or cool and dry CO2 (CD) gas. Herein we show that low IPP and WH gas may decrease inflammation in the laparoscopic surgical environment, resulting in better clinical outcomes. Low IPP and/or WH gas significantly lowered expression of inflammation-related genes in peritoneal tissues compared to the standard IPP and/or CD gas. The odds ratios of a visual analogue scale (VAS) pain score >30 in the ward was 0.18 (95% CI: 0.06, 0.52) at 12 hours and 0.06 (95% CI: 0.01, 0.26) at 24 hours in the low IPP group versus the standard IPP group, and 0.16 (95% CI: 0.05, 0.49) at 0 hours and 0.29 (95% CI: 0.10, 0.79) at 12 hours in the WH gas group versus the CD gas group.

Effects of matrix stiffness on epithelial to mesenchymal transition-like processes of endometrial epithelial cells: Implications for the pathogenesis of endometriosis.

Endometriosis is defined as the presence of endometrial glands and stroma within extrauterine sites. Our previous study revealed an epithelial to mesenchymal transition (EMT)-like process in red peritoneal endometriosis, whereas membrane localization of E-cadherin was well maintained in epithelial cells of deep infiltrating endometriosis (DIE). Here we show that endometrial epithelial cells (EEE) grown on polyacrylamide gel substrates (PGS) of 2 kilopascal (kPa), a soft matrix, initiate a partial EMT-like process with transforming growth factor-ß1 (TGF-ß1) stimulation. Increasing matrix stiffness with TGF-ß1 stimulation reduced the number of cell-cell contacts. Cells that retained cell-cell contacts showed decreased expression of E-cadherin and zonula occludens 1 (ZO-1) to cell-cell junctions. Few deep endometriotic epithelial cells (DEE) grown on 30-kPa PGS, which may mimic in vivo tissue compliance of DIE, retained localization of E-cadherin to cell-cell junctions with TGF-ß1 treatment. Immunohistochemical analysis showed no phosphorylated Smad 2/3 nuclear localization in E-cadherin+ epithelial cells of DIE. We hypothesize that EEE may undergo an EMT-like process after attachment of endometrium to peritoneum in a TGF-ß1-rich microenvironment. However, TGF-ß1 signaling may be absent in DIE, resulting in a more epithelial cell-like phenotype in a rigid microenvironment.

Effects of U0126 and MK2206 on cell growth and re-growth of endometriotic stromal cells grown on substrates of varying stiffness.

Endometriosis is a common gynecological disorder responsible for infertility and pelvic pain. A complete cure for patients with endometriosis awaits new targets and strategies. Here we show that U0126 (a MEK inhibitor) and MK2206 (an AKT inhibitor) synergistically inhibit cell growth of deep endometriotic stromal cells (DES) grown on polyacrylamide gel substrates (PGS) of varying stiffness (2 or 30 kilopascal [kPa]) or plastic in vitro. No significant differences in cell proliferation were observed among DES, endometrial stromal cells of patients with endometriosis (EES) from the proliferative phase (P), EES-S (secretory phase) and EES-M (menstrual phase) compared to cells grown on a substrate of the same stiffness at both higher (U0126 [30 µM] and MK2206 [9 µM]) and lower (U0126 [15 µM] and MK2206 [4.5 µM]) combined doses. However, cell re-growth of DES after drug discontinuation was higher than that of EES-P and EES-S when cells were grown on rigid substrates at both combined doses. Combination U0126 and MK2206 treatment is more effective than each drug alone in cell growth inhibition of DES. However, further studies are required to investigate the mechanisms underlying high cell survival and proliferation after drug discontinuation for developing target therapies that prevent recurrence.

Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of deep endometriotic stromal cells in vitro.

STUDY QUESTION: Can deep infiltrating endometriotic stromal cells (DES) sense changes in extracellular matrix (ECM) stiffness and respond to them? SUMMARY ANSWER: Soft matrices inhibit cell proliferation and inactivate the fibrotic phenotype of DES in vitro. WHAT IS KNOWN ALREADY: Deep infiltrating endometriosis (DIE) is characterized histologically by dense fibrous tissue. Tissue stiffening is a hallmark of fibrosis. Studies show that matrix stiffness is involved in the progression of numerous diseases, including cancer and fibrosis. However, no studies to date have investigated whether tissue stiffening could influence cell behavior in DIE. Previous in vitro studies typically analyzed cells grown on rigid plastic or glass substrates with stiffness in the gigapascal (gPa) range, which is much stiffer than that occurring in vivo. To investigate how changes in ECM stiffness affect the behavior of DES, it is critical to model in vivo tissue compliance conditions in vitro. STUDY DESIGN, SIZE, DURATION: For this laboratory study, paired endometrial and endometriotic samples from 40 patients who had histological evidence of DIE and endometrial samples from 23 patients without endometriosis were analyzed (uterine fibroma: n = 10, tubal infertility: n = 13). PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants were 20-37 years old and had regular menstrual cycles of 26-32 days. The abundance of F-actin, alpha smooth muscle actin (αSMA), Ki67, and procollagen type I in DES and endometrial stromal cells (EES) on polyacrylamide gel substrates of varying stiffness (2, 4, 8, 16 and/or 30 kPa) was determined by immunofluorescence confocal microscopy. mRNA level of type I collagen, matrix metalloproteinase-1 (MMP-1), MMP-14 and cyclin D1 was measured by real-time PCR. The cellular proliferation index (CPI), assessed as the percentage of Ki67-positive cells among the total number of nuclei stained by 4',6-diamidino-2-phenylindole (DAPI) was determined. MAIN RESULTS AND THE ROLE OF CHANCE: Increased matrix stiffness induced F-actin stress fiber formation in both EES and DES, whereas αSMA-containing stress fibers were induced only in DES. Furthermore, increased stiffness increased the CPI in both EES (16 or 30 kPa versus 2 kPa, P < 0.05) and DES (16 or 30 kPa versus 2, 4 or 8 kPa, P < 0.05). Increased stiffness increased the percentage of procollagen I-positive cells as well as mRNA levels of type I collagen in both EES and DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa) (P < 0.05). Increased stiffness also increased MMP-14 mRNA levels in EES (30 versus 2 kPa, P < 0.05), but decreased MMP-1 mRNA levels in DES in a matrix stiffness-dependent manner (2, 8 and 30 kPa; P < 0.05). Treatment with transforming growth factor (TGF)-ß1 further increased type I collagen mRNA levels in both EES and DES when compared with cells grown on a substrate of the same stiffness (2, 8 or 30 kPa, with versus without TGF-ß1, P < 0.05). Treatment with TGF-ß1 also increased MMP-1 (8 or 30 kPa, P < 0.05 versus no TGF-ß1) and MMP-14 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-ß1) in EES, but decreased MMP-1 mRNA levels (2, 8 or 30 kPa, P < 0.05 versus no TGF-ß1) in DES. On a soft substrate (2 kPa), both EES and DES exhibited a small rounded morphology with diffuse labeling for F-actin. No F-actin-positive stress fibers were observed in either EES or DES grown on 2 kPa substrates. There were more Ki67-positive EES when grown on 2, 4 or 8 kPa compared with Ki67-positive DES (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: A tremendous gap exists between the present in vitro model and in vivo deep endometriotic tissues. Cell culture systems that more closely mimic the cellular complexity typical of in vivo endometriotic tissues are required to develop novel strategies for treatment of DIE. A disadvantage of polyacrylamide is its cytotoxicity but in the two-dimensional culture models used here, where cells are seeded above the polyacrylamide gel, this should not have a major impact. Finally, the soft substrates we used in vitro (2 and 4 kPa) may represent the elasticity of the endometrium in vivo, however, currently there are no data regarding tissue stiffness in DIE in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Hormonal suppressive therapy is not usually effective for treating DIE. Interrupting the mechanical interactions between endometriotic fibroblasts and aberrant ECM may be a novel strategy for treatment of DIE. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Co-operation between the AKT and ERK signaling pathways may support growth of deep endometriosis in a fibrotic microenvironment in vitro.

STUDY QUESTION: How can deep endometriotic stromal cells proliferate and persist in a fibrotic environment? SUMMARY ANSWER: The serine/threonine kinase AKT and extracellular regulated kinase (ERK) signaling pathways may co-operate to support growth of deep endometriotic lesions by enhancing endometriotic stromal cell proliferation and survival in a fibrotic microenvironment in vitro. WHAT IS KNOWN ALREADY: Endometriosis, particularly deep infiltrating endometriosis, is characterized histologically by dense fibrous tissue that is primarily composed of type I collagen. This tissue may cause pelvic pain and infertility, which are major clinical issues associated with endometriosis. Proliferation of normal fibroblasts is tightly regulated, and fibrillar, polymerized type I collagen inhibits normal fibroblast proliferation. However, no studies to date have investigated how deep endometriotic stromal cells can proliferate and persist in a fibrotic environment. STUDY DESIGN, SIZE, DURATION: Endometrial and/or endometriotic tissues from 104 patients (61 with and 43 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 25 nude mice received a single injection of endometrial fragments from a total of five samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: We evaluated cell proliferation, caspase 3/7 activity, and the AKT and ERK signaling pathways in endometrial and endometriotic stromal cells on three-dimensional (3D) polymerized collagen matrices in vitro. In addition, to determine whether aberrant activation of the AKT and ERK pathways is involved during progression of fibrosis in endometriosis in vivo, we evaluated the expression of phosphorylated AKT and ERK1/2 in endometriotic implants in a nude mouse model of endometriosis. Finally, we evaluated the effects of MK2206 (an AKT inhibitor) and U0126 (a MEK inhibitor) on cell proliferation, caspase 3/7 activity, and phosphorylation of AKT and ERK1/2 of endometriotic stromal cells on 3D polymerized collagen matrices. MAIN RESULTS AND THE ROLE OF CHANCE: Proliferation of endometriotic stromal cells was significantly less inhibited than that of endometrial stromal cells (P < 0.05) on 3D polymerized collagen. Levels of phosphorylated AKT, phosphorylated p70S6K and phosphorylated ERK1/2 were significantly higher in endometriotic stromal cells than in endometrial stromal cells at 24 h (P < 0.05) and at 72 h (P < 0.05) on 3D polymerized collagen. Phosphorylated AKT expression was significantly increased on Days 21 and 28 compared with those on Days 3 and 7 (all P < 0.05) in endometriotic implants during progression of fibrosis in a nude mouse model of endometriosis. Inhibition of AKT or ERK1/2 with MK2206 or U0126, respectively, did not significantly increase caspase 3/7 activity in endometriotic stromal cells on either two-dimensional or 3D collagen matrices. Western blot analysis showed that MK2206 alone decreased levels of phosphorylated AKT; however, it increased levels of phosphorylated ERK in endometriotic cells compared with vehicle-treated cells (both P < 0.05). In addition, U0126 treatment decreased levels of phosphorylated ERK; however, it resulted in increased levels of phosphorylated AKT in endometriotic stromal cells compared with vehicle-treated cells (both P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Endometriosis involves a number of processes, such as invasion, metastasis, angiogenesis, and apoptosis resistance, and a variety of signaling pathways may be involved in promoting development and progression of the disease. In addition, further animal experiments are required to determine whether the AKT and ERK signaling pathways co-operate to support growth of endometriotic lesions in a fibrotic microenvironment in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Co-targeting the AKT and ERK pathways may be an effective therapeutic strategy for endometriosis treatment. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Antifibrotic properties of epigallocatechin-3-gallate in endometriosis.

STUDY QUESTION: Is epigallocatechin-3-gallate (EGCG) treatment effective in the treatment of fibrosis in endometriosis? SUMMARY ANSWER: EGCG appears to have antifibrotic properties in endometriosis. WHAT IS KNOWN ALREADY: Histologically, endometriosis is characterized by dense fibrous tissue surrounding the endometrial glands and stroma. However, only a few studies to date have evaluated candidate new therapies for endometriosis-associated fibrosis. STUDY DESIGN, SIZE, DURATION: For this laboratory study, samples from 55 patients (45 with and 10 without endometriosis) of reproductive age with normal menstrual cycles were analyzed. A total of 40 nude mice received single injection proliferative endometrial fragments from a total of 10 samples. PARTICIPANTS/MATERIALS, SETTING, METHODS: The in vitro effects of EGCG and N-acetyl-l-cysteine on fibrotic markers (alpha-smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin) with and without transforming growth factor (TGF)-ß1 stimulation, as well as on cell proliferation, migration and invasion and collagen gel contraction of endometrial and endometriotic stromal cells were evaluated by real-time PCR, immunocytochemistry, cell proliferation assays, in vitro migration and invasion assays and/or collagen gel contraction assays. The in vitro effects of EGCG on mitogen-activated protein kinase (MAPK) and Smad signaling pathways in endometrial and endometriotic stromal cells were evaluated by western blotting. Additionally, the effects of EGCG treatment on endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment with EGCG significantly inhibited cell proliferation, migration and invasion of endometrial and endometriotic stromal cells from patients with endometriosis. In addition, EGCG treatment significantly decreased the TGF-ß1-dependent increase in the mRNA expression of fibrotic markers in both endometriotic and endometrial stromal cells. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels were significantly attenuated at 8, 12 and 24 h after treatment with EGCG. Epigallocatechin-3-gallate also significantly inhibited TGF-ß1-stimulated activation of MAPK and Smad signaling pathways in endometrial and endometriotic stromal cells. Animal experiments showed that EGCG prevented the progression of fibrosis in endometriosis. LIMITATIONS, REASONS FOR CAUTION: The attractiveness of epigallocatechin-3-gallate as a drug candidate has been diminished by its relatively low bioavailability. However, numerous alterations to the EGCG molecule have been patented, either to improve the integrity of the native compound or to generate a more stable yet similarly efficacious molecule. Therefore, EGCG and its derivatives, analogs and prodrugs could potentially be developed into agents for the future treatment and/or prevention of endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Epigallocatechin-3-gallate is a potential drug candidate for the treatment and/or prevention of endometriosis. STUDY FUNDING/COMPETING INTERESTS: This study was supported in part by Karl Storz Endoscopy & GmbH (Tuttlingen, Germany). No competing interests are declared.

Targeting the Wnt/ß-catenin pathway in endometriosis: a potentially effective approach for treatment and prevention.

Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women. However, the underlying etiology, pathogenesis and pathophysiology remain to be fully elucidated. Knowledge of these factors is indispensable for the development of targeted therapies for prevention and treatment of endometriosis. Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/ß-catenin pathway may be involved in the pathophysiology of endometriosis. This is a review of the literature focused on the aberrant activation of the Wnt/ß-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis.

Involvement of the Wnt/ß-catenin signaling pathway in the cellular and molecular mechanisms of fibrosis in endometriosis.

BACKGROUND: During the development and progression of endometriotic lesions, excess fibrosis may lead to scarring, chronic pain, and altered tissue function. However, the cellular and molecular mechanisms of fibrosis in endometriosis remain to be clarified. OBJECTIVES: The objective of the present study was to investigate whether the Wnt/ß-catenin signaling pathway was involved in regulating the cellular and molecular mechanisms of fibrosis in endometriosis in vitro and to evaluate whether fibrosis could be prevented by targeting the Wnt/ß-catenin pathway in a xenograft model of endometriosis in immunodeficient nude mice. METHODS: Seventy patients (40 with and 30 without endometriosis) with normal menstrual cycles were recruited. In vitro effects of small-molecule antagonists of the Tcf/ß-catenin complex (PKF 115-584 and CGP049090) on fibrotic markers (alpha smooth muscle actin, type I collagen, connective tissue growth factor, fibronectin) and collagen gel contraction were evaluated in endometrial and endometriotic stromal cells from patients with endometriosis. In vitro effects of activation of the Wnt/ß-catenin signaling pathway by treatment with recombinant Wnt3a on profibrotic responses were evaluated in endometrial stromal cells of patients without endometriosis. The effects of CGP049090 treatment on the fibrosis of endometriotic implants were evaluated in a xenograft model of endometriosis in immunodeficient nude mice. RESULTS: Treatment with PKF 115-584 and CGP049090 significantly decreased the expression of alpha smooth muscle actin, type I collagen, connective tissue growth factor and fibronectin mRNAs in both endometriotic and endometrial stromal cells with or without transforming growth factor-ß1 stimulation. Both endometriotic and endometrial stromal cell-mediated contraction of collagen gels was significantly decreased by treatment with PKF 115-584 and CGP049090 as compared to that of untreated cells. The animal experiments showed that CGP049090 prevented the progression of fibrosis and reversed established fibrosis in endometriosis. CONCLUSION: Aberrant activation of the Wnt/ß-catenin pathway may be involved in mediating fibrogenesis in endometriosis.