Mouse Model Of Endometriosis Research Articles

Features of peritoneal dendritic cells in the development of endometriosis

BackgroundEmerging evidence of immunological dysfunction have been described in endometriosis. Dendritic cells (DCs), one of the main antigen-presenting cells, are specialized in the initiation and modulation of the adaptive immune response. Emerging studies demonstrated both endometrial and circulating differences in DCs populations in women with endometriosis. However, the role and mechanism of peritoneal DCs in endometriosis is still unclear. The present study was undertaken to explore the features of peritoneal DCs in the pathogenesis of endometriosis. This study is beneficial to further clarify the cause of endometriosis and provide a new insight into the medical treatment for endometriosis.MethodsThe study included 12 women with endometriosis and 11 women without endometriosis. The C57BL6 mouse model of endometriosis was established by intraperitoneal injection of endometrial segments. The peritoneal DCs of endometriosis patients and mouse models were analyzed by fluorescence associated cell sorting (FACS) examination.ResultsIncreased cell density of peritoneal DCs were observed in endometriosis patients. Moreover, the proportion of mature DCs (mDCs, CD80highCD1alow cells) in the peritoneal DCs was lower whereas the proportion of immature DCs (iDCs, CD80lowCD1ahigh cells) was increased in endometriosis patients. Similarly, the cell density of peritoneal DCs in murine models increased immediately after the injection of endometrial tissues and reached the highest level at 14 days. In addition, the proportion of mDCs (CD11chighCD80high cells) in the peritoneal DCs decreased immediately after the injection of endometrial tissues and then increased with the time until 42 days, but still lower than the control group. In contrast, the proportion of iDCs (CD11chighCD80low cells) in the peritoneal DCs showed the opposite dynamic changes. However, after treated with LPS, the mDCs proportion was significantly increased, leading to lower volume and weight of the endometriosis lesions.ConclusionsIncreased level of peritoneal DCs facilitated the pathogenesis of endometriosis lesions, especially in the early stage of the disease. Furthermore, peritoneal DCs maturation played an important role in the development of endometriosis.

Targeted Imaging of Endometriosis and Image-Guided Resection of Lesions Using Gonadotropin-Releasing Hormone Analogue-Modified Indocyanine Green

Objective. In this study, we utilized gonadotropin-releasing hormone analogue-modified indocyanine green (GnRHa-ICG) to improve the accuracy of intraoperative recognition and resection of endometriotic lesions. Methods. Gonadotropin-releasing hormone receptor (GnRHR) expression was detected in endometriosis tissues and cell lines via immunohistochemistry and western blotting. The in vitro binding capacities of GnRHa, GnRHa-ICG, and ICG were determined using fluorescence microscopy and flow cytometry. In vivo imaging was performed in mouse models of endometriosis using a near-infrared fluorescence (NIRF) imaging system and fluorescence navigation system. The ex vivo binding capacity was determined using confocal fluorescence microscopy. Results. GnRHa-ICG exhibited a significantly stronger binding capacity to endometriotic cells and tissues than ICG. In mice with endometriosis, GnRHa-ICG specifically imaged endometriotic tissues (EMTs) after intraperitoneal administration, whereas ICG exhibited signals in the intestine. GnRHa-ICG showed the highest fluorescence signals in the EMTs at 2 h and a good signal-to-noise ratio at 48 h postadministration. Compared with traditional surgery under white light, targeted NIRF imaging-guided surgery completely resected endometriotic lesions with a sensitivity of 97.3% and specificity of 77.8%. No obvious toxicity was observed in routine blood tests, serum biochemicals, or histopathology in mice. Conclusions. GnRHa-ICG specifically recognized and localized endometriotic lesions and guided complete resection of lesions with high accuracy.

Aberrant expression of histone deacetylase 8 in endometriosis and its potential as a therapeutic target.

To screen Zn2+-dependent histone deacetylase (HDAC) 1-11 in endometriotic cells and then evaluated the HDACs identified from the screening in ovarian endometrioma (OE) and deep endometriotic (DE) lesions, and to evaluate the therapeutic potential of HDAC8 inhibition in mice. Quantification of gene and protein expression levels of HDAC1-11 in endometriotic cells stimulated by TGF-β1, and immunohistochemistry analysis of Class I HDACs and HDAC6 in OE/DE lesion samples. The therapeutic potential of HDAC8 inhibition was evaluated by a mouse model of deep endometriosis. The screening identified Class I HDACs and HDAC6 as targets of interest. Immunohistochemistry analysis found a significant elevation in HDAC8 immunostaining in both OE and DE lesions, which was corroborated by gene and protein expression quantification. For other Class I HDACs and HDAC6, their lesional expression was more subtle and nuanced. HDAC1 and HDAC6 staining was significantly elevated in DE lesions while HDAC2 and HDAC3 staining was reduced in DE lesions. Treatment of mice with induced deep endometriosis with an HDAC8 inhibitor resulted in significantly longer hotplate latency, a reduction of lesion weight by nearly two-thirds, and significantly reduced lesional fibrosis. These findings highlight the progression-dependent nature of specific HDAC aberrations in endometriosis, and demonstrate, for the first titme, the therapeutic potential of suppressing HDAC8.

CHIP induces ubiquitination and degradation of HMGB1 to regulate glycolysis in ovarian endometriosis.

Ovarian endometriosis is a common gynecological condition that can cause infertility in women of childbearing age. However, the pathogenesis is still unknown. We demonstrate that the carboxyl terminus of Hsc70-interacting protein (CHIP) is a negative regulator in the development of endometriosis and reduces HMGB1 expression in endometriotic cells. Meanwhile, CHIP interacts with HMGB1 and promotes its ubiquitinated degradation, thereby inhibiting aerobic glycolysis and the progression of endometriosis. Furthermore, the CHIP agonist YL-109 effectively suppresses the growth of ectopic endometrium in endometriosis mouse model, which could be a potential therapeutic approach for endometriosis. In conclusion, our data suggest that CHIP may inhibit the development of endometriosis by suppressing the HMGB1-related glycolysis.

Lipoxin A4 depresses inflammation and promotes autophagy via AhR/mTOR/AKT pathway to suppress endometriosis.

Endometriosis is a benign gynecological disease with the feature of estrogen dependence and inflammation. The function of autophagy and the correlation with inflammation were not yet revealed. Autophagosomes were detected by transmission electron microscopy. Gene Expression Omnibus (GEO) database was referred to analyze the expression of autophagy-related genes. Quantification of mRNA and protein expression was examined by qRT-PCR and Western Blot. Immunohistochemistry was performed to explore the expression of proteins in tissues. The mouse model of endometriosis was performed to analyze the autophagic activity and effect of LXA4. The expression of autophagy-related genes in endometriotic lesions were unusually changed. The number of autophagosomes and LC3B-II expression was diminished, and p62 was increased in ectopic lesions from both patients and mice. Interleukin 1β (IL1β) attenuated the expression of LC3B and promoted the level p62. The autophagy activator MG-132 upregulated the expression of LC3B and reduced IL1β, IL6, and p62. LXA4 reversed the inhibitory effect of IL1β on the expression of LC3B and p62, and blocking the receptor of LXA4 AhR (aryl hydrocarbon receptor) resulted in the incapacitation of LXA4 to influence the effect of IL1β. LXA4 depressed the phosphorylation of AKT and mTOR to against IL1β, and blocking AhR negatively regulated the effect of LXA4 on AKT/mTOR pathway. LXA4 reduced the ectopic lesions and the expression of IL1β and p62, but enhanced LC3B-II in endometriotic mouse models. In endometriosis, increased inflammation of ectopic lesions prominently depresses autophagy. LXA4 could regulate autophagy by suppressing inflammatory response through AhR/AKT/mTOR pathway.

MicroRNA-100 shuttled by human umbilical cord MSC-secreted extracellular vesicles induces endometriosis by inhibiting HS3ST2

In recent years, the function of human umbilical cord mesenchymal stem cell-originated extracellular vesicles (hUC-MSC-EVs) on endometriosis has been reported, while its specific mechanisms remain largely unknown. This study aimed at investigating the mechanisms underlying the modulation of EVs harboring miR-100 derived from hUC-MSCs in the growth dynamics of endometrial stromal cells in endometriosis. Endometriosis mouse models were established. miR-100 was upregulated and HS3ST2 was downregulated in endometriosis. Ectopic endometrial tissues and umbilical cord tissues were obtained to extract endometrial stromal cells and hUC-MSCs, from which EVs were isolated. Next, the endometrial stromal cells were co-cultured with hUC-MSC-EVs, during which gain- or loss-of-function approaches were employed for gene overexpression or silencing. The binding affinity among miR-100 and HS3ST2 was identified using multiple assays. It was unveiled that miR-100 could target and inhibit HS3ST2. miR-100 from hUC-MSCs could be transferred into the endometrial stromal cells via EVs. Moreover, miR-100 shuttled by hUC-MSC-EVs facilitated endometrial stromal cell proliferation, invasion, and migration, as well as EMT by inhibiting HS3ST2. In vivo experiments also confirmed that hUC-MSC-derived EVs carrying miR-100 induced the occurrence and development of endometriosis. Collectively, hUC-MSC-EV-loaded miR-100 downregulated HS3ST2 to facilitate the development of endometriosis, which highlights a promising therapeutic target for treating endometriosis.

Oleuropein suppresses endometriosis progression and improves the fertility of mice with endometriosis

BackgroundEndometriosis is an estrogen-dependent inflammatory reproductive disease. Therefore, systematic estrogen depletion and anti-inflammatory drugs are the current treatment for endometriosis. However, current endometriosis treatments have low efficacy and cause adverse effects in endometriosis patients. Consequently, alternative endometriosis treatments targeting endometriosis-specific factors are in demand. In this context, ERβ was selected as a druggable target for endometriosis due to its critical role in progression. Therefore, selective targeting of ERβ without inhibiting ERα activity would be a new paradigm for endometriosis treatment to overcome the low efficacy and adverse effects of hormonal endometriosis therapy.MethodsCell-based ERβ and ERα activity assay systems were employed to define a selective ERβ-inhibiting chemical product from a library of natural products. A surgically induced endometriosis mouse model was used to determine whether an ERβ inhibitory drug suppressed endometriosis progression. Mice with endometriosis were randomly separated and then orally treated with vehicle or 25 mg/kg oleuropein (once a day for 21 days), an ERβ inhibitory drug. The volume of endometriotic lesions or luciferase activity of endometriotic lesions was examined to define the growth of ectopic lesions in mice with endometriosis. The metabolite and levels of metabolic enzymes of the liver and kidney were determined in the serum of female mice treated with vehicle and oleuropein (25 mg/kg, once a day for 21 days) to define the toxicity of oleuropein. The in vitro decidualization assay was conducted with normal human endometrial stromal cells and endometriotic stromal cells to determine whether oleuropein overcomes decidualization in endometriosis patients. The pregnancy rate and pup numbers of C57BL/6 J female mice with endometriosis treated with vehicle or oleuropein (n = 10/group) were determined after mating with male mice. The cytokine profile in endometriotic lesions treated with vehicle and oleuropein (25 mg/kg) was determined with a Mouse Cytokine Array Kit.ResultsAmong natural products, oleuropein selectively inhibited ERβ but not ERα activity in vitro. Oleuropein treatment inhibited the nuclear localization of ERβ in human endometrial cells upon estradiol treatment. Oleuropein (25 mg/kg) treatment suppressed the growth of mouse (6.6-fold) and human (sixfold) ectopic lesions in mice with endometriosis compared to the vehicle by inhibiting proliferation and activating apoptosis in endometriotic lesions. Oleuropein treatment did not cause reproductive toxicity in female mice. Additionally, mice with endometriosis subjected to oleuropein treatment had a higher pregnancy rate (100%) than vehicle-treated mice (70%). Furthermore, oleuropein treatment partially recovered the decidualization impact of human endometriotic stromal cells from endometriotic lesions compared to the vehicle. Oleuropein-treated mice with endometriosis exhibited significantly lower levels of cytokines directly regulated by ERβ in ectopic lesions than vehicle-treated mice, illustrating the improvement in the hyperinflammatory state of mice with endometriosis.ConclusionsOleuropein is a promising and novel nutraceutical product for nonhormonal therapy of endometriosis because it selectively inhibits ERβ, but not ERα, to suppress endometriosis progression and improve the fertility of mice with endometriosis.

Re-evaluation of mouse models of endometriosis for pathological and immunological research.

Endometriosis is an estrogen-dependent gynecological disease with chronic pelvic inflammation. In order to study the pathophysiology of endometriosis and examine the therapeutic effects of new pharmaceuticals for endometriosis treatment, different animal models had been developed in the last two decades, especially mouse models. However, no study evaluated the effects of various modeling approaches on pathology and immunology in endometriosis. This study aimed to compare endometriotic lesion development and immune profiles under different methods of establishing endometriosis models in mice, including estrus synchronization (ovariectomy with estrogen supplement versus male urine-soaked transfer bedding), endometrium preparations (whole uterus including endometrium and myometrium fragments versus solely endometrium fragments), and surgical transplantation (subcutaneous transplantation versus intraperitoneal injection). Our results showed that lesion growth under estrus synchronization by ovariectomy with estrogen supplement had a higher success rate and more proliferative endometrium, apart from higher body weight gain. Immune responses in peripheral blood were similar in the whole uterus and solely endometrium fragments and in intraperitoneal injection and subcutaneous transplantation, but a more innate immune response in the peritoneal microenvironment was found in solely endometrium fragments and intraperitoneal injection than counterparts. In conclusion, different endometriosis modeling methods result in different pathological and immunological features. Ovariectomy with estrogen supplement, solely endometrium fragments, and intraperitoneal injection are more suitable for both pathological and immunological studies of endometriosis in mice, which are important for mechanistic studies and immunotherapy development.

Niclosamide targets the dynamic progression of macrophages for the resolution of endometriosis in a mouse model

Due to the vital roles of macrophages in the pathogenesis of endometriosis, targeting macrophages could be a promising therapeutic direction. Here, we investigated the efficacy of niclosamide for the resolution of a perturbed microenvironment caused by dysregulated macrophages in a mouse model of endometriosis. Single-cell transcriptomic analysis revealed the heterogeneity of macrophages including three intermediate subtypes with sharing characteristics of traditional “small” or “large” peritoneal macrophages (SPMs and LPMs) in the peritoneal cavity. Endometriosis-like lesions (ELL) enhanced the differentiation of recruited macrophages, promoted the replenishment of resident LPMs, and increased the ablation of embryo-derived LPMs, which were stepwise suppressed by niclosamide. In addition, niclosamide restored intercellular communications between macrophages and B cells. Therefore, niclosamide rescued the perturbed microenvironment in endometriosis through its fine regulations on the dynamic progression of macrophages. Validation of similar macrophage pathogenesis in patients will further promote the clinical usage of niclosamide for endometriosis treatment.

Impact of Chronic Exposure to Endometriosis on Perinatal Outcomes: Establishment of a Mouse Model

The purpose of this study was to establish a new mouse model of endometriosis that mimics real-world women’s health problems, in which women continue to be affected by endometriosis long before they wish to become pregnant, and to evaluate the impact of “chronic exposure to endometriosis” on perinatal outcome. Endometriosis was established by the intraperitoneal injection of homologous minced mouse uteri. Vehicle was injected for the control. Mating was initiated either 1 or 43 days after disease establishment (Young or Aged studies, respectively). Mice were sacrificed on 18 dpc. The number pups and resorptions were counted and pups’ body weights (BW) were measured, and the endometriosis lesion was identified and weighted. In the Young study, the number of resorptions and BW were comparable between the groups. In the Aged study, the number of resorptions was significantly higher and BW was significantly lower in endometriosis than that in control. The total weight of endometriosis lesion per dam was significantly lower in the Aged compared to the Young endometriosis group; however, not a single mouse was found to have any lesions at all. These results suggest that in addition to the presence of endometriosis per se, “chronic exposure to endometriosis” prior to pregnancy affect perinatal outcomes.

Uterine administration of C-X-C motif chemokine ligand 12 increases the pregnancy rates in mice with induced endometriosis

To study the effect of intrauterine injection of C-X-C motif chemokine ligand 12 (CXCL12), also known as a stem cell chemoattractant (stromal cell-derived factor 1), on fertility and endometrial receptivity in mice with endometriosis. Laboratory study. Academic Medical Center. Fifty-six mice underwent chemotherapy and bone marrow transplantation. Thirty-six of these mice underwent either surgery to induce endometriosis (n = 20) or sham surgery (n = 16). Injection of CXCL12 as a potential therapeutic agent to improve fertility in endometriosis. Pregnancy rate, bone marrow-derived cell (BMDC) recruitment and endometrial receptivity markers. The mice with or without endometriosis received a single uterine injection of either CXCL12 or placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. Mice were euthanized after delivery, and implantation markers homeobox A11, alpha-v beta-3 integrin, and progesterone receptor were analyzed by immunohistochemistry, whereas green fluorescent protein positive BMDC recruitment was quantified by immunohistochemistry and immunofluorescence. The sham surgery groups without endometriosis had the highest cumulative pregnancy rate (100%) regardless of CXCL12 treatment. The endometriosis group treated with placebo had the lowest pregnancy rate. An increased pregnancy rate was noted in the endometriosis group after treatment with CXCL12. There was also an increase in BMDC recruitment and endometrial expression of progesterone receptor and alpha-v beta-3 integrin in the endometriosis group that received CXCL12 compared with that in the endometriosis group that received placebo. Uterine injection of CXCL12 increased the pregnancy rates in a mouse model of endometriosis. These results suggest that CXCL12 has a potential role as a therapeutic agent in women with infertility related to endometriosis and potentially other endometrial receptivity defects.

Contribution of CD4+ cells in the emotional alterations induced by endometriosis in mice.

Endometriosis is a disease defined by the presence of endometrial tissue in extrauterine locations. This chronic condition is frequently associated with pain and emotional disorders and has been related with altered immune function. However, the specific involvement of immune cells in pain and behavioral symptoms of endometriosis has not been yet elucidated. Here, we implement a mouse model of non-surgical endometriosis in which immunocompetent mice develop abdomino-pelvic hypersensitivity, cognitive deficits, anxiety and depressive-like behaviors. This behavioral phenotype correlates with expression of inflammatory markers in the brain, including the immune cell marker CD4. Depletion of CD4 + cells decreases the anxiety-like behavior of mice subjected to the endometriosis model, whereas abdomino-pelvic hypersensitivity, depressive-like behavior and cognitive deficits remain unaltered. The present data reveal the involvement of the immune response characterized by CD4 + white blood cells in the anxiety-like behavior induced by endometriosis in mice. This model, which recapitulates the symptoms of human endometriosis, may be a useful tool to study the immune mechanisms involved in pain and behavioral alterations associated to endometriosis.

Leptin Stimulates Endometriosis Development in Mouse Models.

Endometriosis is a chronic inflammatory condition in women, and obesity leads to an inflammatory condition that is directly involved in the etiology of endometriosis. However, observational studies have shown an inverse correlation between endometriosis and a low body mass index (BMI). Obesity does not protect against endometriosis, and on the contrary, an increased BMI may lead to more severe forms of the disease. To determine the effect of obesity on endometriosis, diet-induced and genetically engineered obese mouse models were integrated with endometriosis mouse models with fluorescence-tagged ectopic lesions. High-fat diet-induced obese mice revealed a significant increase in endometriosis development compared with regular-diet control mice. However, obese recipient mice with leptin deficiency and leptin receptor deficiency showed suppressed endometriosis development compared with control mice. Furthermore, donor uterine tissues with leptin deficiency and leptin receptor deficiency suppressed endometriosis development compared with control donor in control recipient mice. Importantly, we revealed that aberrant high levels of leptin concentration significantly increased endometriosis development compared with vehicle treatment group in control mice with normal body weight. Our results suggest that leptin and its receptor are critical for endometriosis development.

Toll-like receptor signaling pathway triggered by inhibition of serpin A1 stimulates production of inflammatory cytokines by endometrial stromal cells.

Endometriosis is characterized by the presence of inflamed and fibrotic endometrial tissue outside the uterine cavity. Previously, we found decreased SERPINA1 (alpha-1 antitrypsin) expression in endometriosis-like lesions in a mouse model of endometriosis, suggesting that it exacerbated inflammation in these lesions. However, the molecular mechanism(s) by which SERPINA1 affects expression of inflammatory factors and development of endometriotic lesions have not been fully characterized. To investigate the role of intracellular SERPINA1 in endometrial stromal cells (ESCs), we performed RNA sequence analysis using RNA extracted from ESCs in which SERPINA1 was knocked down. The analysis identified several toll-like receptor (TLR)-related factors as being upregulated. Silencing of SERPINA1 increased expression of TLR3 and TLR4 in ESCs, as well as several TLR signaling pathway components, including MYD88, IRAK1/4, interleukin (IL)-1β, and interferon (IFN)-β. TLR3 or TLR4 agonists increased expression of inflammatory factors in SERPINA1-knockdown ESCs, whereas TLR3 or TLR4 inhibitors decreased expression. In addition, treatment with recombinant IL-1β or IFN-β increased expression of MYD88 and inflammatory factors in ESCs. Immunohistochemical analysis of endometriotic tissues showed that TLR3, TLR4, and MYD88 were localized in endometriosis lesions. Taken together, the data suggest that reduced expression of SERPINA1 induces expression of inflammatory factors by ESCs, which in turn are associated with TLR3/4, IL-1β, and IFN-β signaling. Regulation of intracellular SERPINA1 levels in ESCs may be a strategy to inhibit inflammatory responses in endometriotic lesions.

Anti-oxidant effect of metformin through AMPK/SIRT1/PGC-1α/SIRT3- independent GPx1 expression in the heart of mice with endometriosis.

Endometriosis is a gynecological disease associated with an imbalance between oxidative species production and anti-oxidative defenses. In women, endometriosis has been reported to associate with increased incidence of cardiovascular events. As such, this study aimed to analyze the oxidation-responsive AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart of a mouse model of endometriosis. The effect of metformin, an insulin-sensitizing and anti-oxidative drug with already shown positive results in endometriotic tissue was studied. Thirty-six female B6CBA/F1 mice were divided into 4 groups (Control-C, Surgery-induced Endometriosis and Metformin-EM (50mg/kg/day orally administrated for 3months), Endometriosis-E and Metformin-M). Immunofluorescent labelling of SIRT1 and SIRT3 was performed in the heart tissue. Assessment of expression of AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPx1 was performed by Western Blotting. The quantification of microRNA(miR)-34a, miR-195, miR-217, miR-155 and miR-421, involved in the regulation of expression of SIRT1 and SIRT3, was performed by Real-Time PCR. Data showed an increase in phospho-AMPKα and in GPx1 expression in the EM group when compared to the C group, but not in the total AMPK, SIRT1, PGC-1α, SIRT3 and SOD2, suggesting a GPx1 expression increase independently of the AMPK/SIRT1/PGC-1α/SIRT3 pathway. MicroRNAs, excepting miR-217, showed a consistent trend of increase in the M group. Our study showed that endometriosis does not significantly affect the expression of the components of the AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart. However, it indicates that an oxidative condition underlying endometriosis is required for metformin to evidence an increment in the expression of the anti-oxidative enzyme GPx1.

Zearalenone affects the growth of endometriosis via estrogen signaling and inflammatory pathways

Endometriosis is a chronic, inflammatory, estrogen-dependent gynecological disease characterized by the growth of endometrial stromal cells and glands outside the uterine cavity in response to hormones, which commonly occurs in reproductive-age women. Zearalenone (ZEA) is a toxic metabolite produced by Fusarium, which acts as estrogen activity because of the similarity of its structure to estrogen. In this study, we used an endometriosis mouse model: 15 days after ovariectomy, endometrial fragments were sutured on the pelvic wall, and exogenous estrogen was supplied using an estrogen-releasing silicone tube embedded subcutaneously. Mice were treated with different doses of ZEA by gavage for 21 days. The results show that ZEA significantly inhibited the growth of ectopic endometrium in a dose-dependent manner. The proliferation of cells decreased while apoptosis increased in the ectopic tissues of ZEA-treated mice compared to the vehicle group. The expression of estrogen receptor-α and its downstream targets MUC1 and p-AKT decreased, indicating an impaired estrogen signaling activity by ZEA treatment. In addition, the decreased expression of pro-inflammatory cytokine Tnf-α, Il-1β, and Il-6, the lower number of macrophages and neutrophils cells, and the inhibited NF-κB signaling pathway suggest the inflammatory response in the ectopic endometrium was also suppressed by ZEA treatment. However, when the exogenous estrogen supply is removed, ZEA, in turn, plays an estrogen-like role that promotes cell proliferation in the ectopic endometrium. In summary, our data suggest ZEA acts as an antagonist in endometriotic tissue when estrogen is sufficient but turns to estrogenic activity in the absence of estrogen in the development of endometriosis. ZEA also inhibits ectopic tissue growth by inhibiting inflammatory response in the endometriosis model.

Sunitinib Reduced the Migration of Ectopic Endometrial Cells via p-VEGFR-PI3K-AKT-YBX1-Snail Signaling Pathway.

Endometriosis (EMs) is one of the most common gynecological diseases, lacking effective treatment. EMs are currently being treated with small molecule targeted therapy, which has resulted in a significant reduction in patient suffering. Our previous studies have shown that sunitinib plays an obvious role in migration. Consequently, the purpose of this study is to explore the molecular mechanism by which sunitinib suppressed the ectopic endometrial migration. The ectopic endometrial cells from patients were divided into two groups: the control group and the sunitinib group. Co-IP and protein spectrum assay were employed to filtrate differential proteins between two groups, and then, our study discovered a signaling pathway, p-VEGFR-PI3K-AKT-YBX1-Snail, in the cell of EMs. To confirm this signaling pathway, VEGF165 was added to the sunitinib group to upregulate the expression of VEGFR. Next, the expression of p-VEGFR, PI3K, AKT, YBX1, and snail was measured in the control group and sunitinib group (compared with the control group: p-VEGFR, PI3K, AKT, YBX1, and snail, ∗∗∗∗P < 0.0001) and the VEGFR+sunitinib group (compared with the sunitinib group: p-VEGFR, PI3K, AKT, and snail, ∗∗∗∗P < 0.0001; YBX1, ∗∗∗P < 0.001); finally, the outcome was as expected. In addition to in vitro experiments, we also conducted in vivo experiments in mice. In the EMs mouse model, we found sunitinib reduced the number of heterotopic foci (t = 11.16, ∗∗∗∗P < 0.0001) and inhibited the expression of p-VEGFR, YBX1, and snail by immunofluorescence. To sum up, sunitinib exactly reduced the migration of ectopic endometrial cells with the involvement of the p-VEGFR-PI3K-AKT-YBX1-Snail signaling pathway in both in vitro and in vivo experiments. This study suggests that sunitinib presents a potential targeted drug for EMs therapy.

P-343 Metformin reduces endometrial implants and improves fertility in a mouse model of endometriosis

Abstract Study question Could endometriosis-associated infertility be mitigated by metformin? Summary answer Metformin treatment restored fertility to control rates in endometriosis-induced mice that present lower fertility. Endometrial implants presented a reduction in size with metformin treatment. What is known already Endometriosis is an inflammatory disease characterized by estrogen-dependent ectopic growth of endometrium causing pain and infertility. Hormone-based medication prescribed need to be interrupted when women intend to conceive. Corroborating observations in women, endometriosis decreases oocyte quality and pregnancy success in rodents. Thus, animal models of endometriosis constitute a valuable tool to elucidate the pathophysiology of the disease and putative therapies. Besides lowering glycaemia, metformin down-regulates estrogen secretion, inflammation and oxidation. Metformin can be safely used before and during pregnancy, it is currently used in gestational diabetes. Metformin has shown to induce regression of endometrial implants in an endometriosis rat model. Study design, size, duration B6CBA/F1 female mice were randomly divided in groups and subjected to treatment: 1-Endometriosis(E) (n = 20); 2-Sham-operated(S) (n = 12); 3-Endometriosis with metformin(EM) (n = 20); 4-Sham-operated with metformin(SM) (n = 20). Endometriosis was surgically induced by heterologous transplantation of endometrium from one donor in receptors from the same strain mice. Implants were confirmed and monitored by ultrasound. 50mg/kg/day of metformin was orally administrated during 3 months to Groups 3 and 4. Half of mice in each group were mated to fertility study. Participants/materials, setting, methods Endometrial implants were monitored at 3 timepoints during the experiments. Fertility rates were assessed by the average number of fetuses in each group. Histological characterization of eutopic and ectopic endometrium was performed by H&amp;E and Masson's trichrome staining. Immunofluorescence for PCNA and CYP17A1 proteins, markers of cell proliferation and estrogen secretion, respectively were performed. Western blotting quantification for these proteins is in course. Statistical analysis was carried out and significant differences were considered for p &amp;lt; 0.05. Main results and the role of chance A decrease of 30% of fertility rate was verified in mice with endometriosis (p = 0,01); treatment with metformin was able to revert this decrease (p = 0,04). Interestingly, no differences in fertility were found in sham-operated mice under metformin treatment relatively with those of group 2 (p = 0,16). No biometrical differences were found between mice with endometriosis receiving metformin and those that do not receive the drug. Implants size were reduced by metformin treatment. Histological similarities were observed between the ectopic tissue and uterus. It is possible to observe that the ectopic endometrium layer is thinner in the EM group compared with the E group. PCNA and CYP17a labeling was present in the same cells of eutopic and ectopic endometrium, with an apparent lower number in the ectopic tissue in the EM group. In addition, in untreated animals, the cavity of ectopic tissue has more flaking cells than in animals with metformin treatment. In sum, metformin seems to have a positive role in the control of cell proliferation and estrogen production of ectopic endometrium. We postulate that these findings owes to a metformin-mediated decrease of oxidative imbalance and inflammatory response, induction of regression of endometriomas and regulation of oestrogen secretion. Limitations, reasons for caution Extrapolation of data from animal models to human needs caution, considering that endometriosis pattern differs between species. Also, further investigation, focused in identification of molecular targets of metformin and molecular pathways activated in endometriosis, is needed and in course. Wider implications of the findings With these results, we suggest metformin as a novel and safe strategy to mitigate endometriosis-related oxidative stress and indeed could be used as a valid pharmacological approach to ameliorate endometriosis-associated infertility Trial registration number Not applicable

P-306 How ProEGCG achieves superior anti-angiogenic efficacy to prevent and treat endometriosis?

Abstract Study question What are the therapeutic targets and molecular mechanisms that bring superior pharmacological effects of ProEGCG against angiogenesis in endometriosis? Summary answer ProEGCG binds to PXK and downregulates PXK-mediated EGF expression in inhibiting angiogenesis via HIF-1α pathway during the growth and development of endometriosis. What is known already Current endometriosis treatment shows great effects in managing the symptoms, but associated with significant recurrence risk and hormonal side effects. Previously, we demonstrated the greater efficacies and potency of prodrug of green tea epigallocatechin gallate (ProEGCG) over EGCG to inhibit angiogenesis and oxidative stress in experimental endometriosis mouse models ( Xu, et al., 2011; Wang, et. al., 2013; Hung, et. al. 2021). However, the therapeutic targets and molecular mechanism to bring the superior anti-angiogenic effects of ProEGCG are still not known. Study design, size, duration Efficiency of ProEGCG as preventive or therapeutic medication were compared with that of EGCG in endometriosis mice model Therapeutic targets were studied by Proteome Integral Stability Alteration and proteomic analysis (Gaetani et al.,2019) using endometriotic cells, and further validated by computational simulation, target kinetic analysis and immunostaining. Anti-angiogenic mechanism were confirmed in endometriotic cell lines and mouse models. Participants/materials, setting, methods Endometriotic (Hs832(C)T) cell line was treated with EGCG or ProEGCG to identify the binding target with chemical proteomics analysis. The stability of ProEGCG-protein complexes was assessed by molecular docking analysis and bio-layer interferometry. Gene and protein expressions of the binding targets and downstream angiogenesis pathway were assessed by qRT-PCR, ELISA and immunostaining to characterise the molecular pathways in vitro and in vivo. Main results and the role of chance ProEGCG significantly reduced the endometriotic lesions in both preventive and treatment regimens with no recurrence risk after discontinuation in mice. MTDH and PXK were identified as differential protein targets of EGCG and ProEGCG respectively in Hs832(C)T cells via proteomics analysis. Molecular docking and kinetic studies validated stabilities of the drug-target complexes. Protein expressions of MTDH and PXK were significantly higher in various kinds of human endometriotic tissues compared with normal endometrium, which were found significantly downregulated in mouse endometriotic tissues after treatment of EGCG and ProEGCG, respectively. Angiogenesis was significantly inhibited by ProEGCG and EGCG in endometriosis in mice and downstream angiogenic factors HIF-1α and VEGF were downregulated in both mRNA and protein levels. However EGCG bound to MTDH and inhibited AKT-mediated angiogenesis via AKT/PI3K signalling pathway, while ProEGCG bound to PXK and inhibited EGF-mediated angiogenesis via more profound EGF/AKT1/HIF-1α/VEGF signalling pathways. It indicated distinct and specific potency of ProEGCG as an anti-proliferation and anti-angiogenesis agent in inhibiting the endometriosis progression. Limitations, reasons for caution The crosstalk of binding targets-induced therapeutic effects in endometriosis and their corresponding effects on angiogenesis are still unclear. Clinical studies are also needed to confirm the efficacy and mechanisms in humans. Wider implications of the findings Novel drug discovery identifies an ideal pharmaceutical for clinical use in endometriosis, which alleviates the symptoms, cures the disease and prevents the recurrence. Molecular targets and mechanisms as preclinical research priorities will optimize pharmacological and physiochemical properties of new drugs, as well as maximize specificity and efficacy for endometriosis treatment. Trial registration number NA

The effect of hyperbaric oxygen therapy in the inflammatory response in a mouse model of endometriosis: An experimental study.

BackgroundEndometriosis pathogenesis is related to the inflammation shown by the secretion of pro-inflammatory mediators. This hypoxia condition can stimulate this condition.ObjectiveTo investigate the effect of hyperbaric oxygen therapy (HBOT) on the inflammation reaction of endometriosis-induced mice.Materials and MethodsThe animals were designated into 3 groups: I) the pre-test group, II) the post-test group receiving the HBOT, and III) the post-test group without HBOT. All groups were subjected to induction of endometriosis by xenotransplantation for 15 days. HBOT was given 30 min 3 times a day for 10 days. The evaluation of the HBOT effect was conducted by examining the endometrial tissue. The inflammation level was evaluated using the Klopfleisch semiquantitative scoring system (index remmele scale), whilst the expression of nuclear factor kappa (NFB) beta was measured by immunohistochemical staining.ResultsThe results showed that group I demonstrated the highest level of inflammation degree (9.41 1.99) compared to the post-test groups (group II: 1.60 0.53; group III: 2.42 0.53). The HBOT-groups was found to have the lowest inflammation level compared to the non-HBOT group (p = 0.020). The results demonstrated that HBOT lowered the peritoneal inflammation degree caused by the endometrial lesion in mice. NFB expression on the post-test groups was significantly decreased, compared to the pre-test group (p 0.001), with a strong correlation between the NFB expression and the peritoneal inflammation level (p 0.001, r = 0.670).Conclusion HBOT significantly reduced the inflammation level on the endometrial lesion in mice, involving the NFB pathway.