Endometriosis Model Research Articles

Modulation of the Proliferative Pathway, Neuroinflammation and Pain in Endometriosis.

Endometriosis is a chronic disease characterized by pelvic inflammation. This study aimed at investigating the molecular mechanisms underlying the pathology and how they can be modulated by the administration of a natural compound, Actaea racemosa (AR). We employed an in vivo model of endometriosis in which rats were intraperitoneally injected with uterine fragments from donor animals. During the experiment, rats were monitored by abdominal high-frequency ultrasound analysis. AR was able to reduce the lesion's size and histological morphology. From a molecular point of view, AR reduced hyperproliferation, as shown by Ki-67 and PCNA expression and MAPK phosphorylation. The impaired apoptosis pathway was also restored, as shown by the TUNEL assay and RT-PCR for Bax, Bcl-2, and Caspase levels. AR also has important antioxidant (reduced Nox expression, restored SOD activity and GSH levels, and reduced MPO activity and MDA levels) and anti-inflammatory (reduced cytokine levels) properties. Moreover, AR demonstrated its ability to reduce the pain-like behaviors associated with the pathology, the neuro-sensitizing mediators (c-FOS and NGF) expression, and the related central astrogliosis (GFAP expression in the spinal cord, brain cortex, and hippocampus). Overall, our data showed that AR was able to manage several pathways involved in endometriosis suppression.

Pingchong Jiangni recipe through nerve growth factor/transient receptor potential vanilloid 1 signaling pathway to relieve pain in endometriosis model rats

Ethnopharmacological relevancePingchong Jiangni recipe (PJR) is often used in the treatment of endometriosis (EM). This formula has been clinically validated by the State Administration of Traditional Chinese Medicine Key Specialties Collaborative Group for its therapeutic efficacy. Recently, our research team also confirmed that PJR has a shrinking effect on ovarian chocolate cysts. Additionally, PJR was also found to have a shrinking effect on EM lesions; however, the mechanism by which this effect occurs remains unclear. Aim of the studyTo explore the mechanisms by which PJR relieves pain in patients with EM. Materials and methodsA rat model of EM was established by autologous transplantation. PJR (3.78 g/kg, 7.56 g/kg, and 15.12 g/kg) was orally administered for 21 days. The rat grimace scale (RGS) score and paw withdrawal threshold (PWT) were measured at a fixed time during the experiment. Hematoxylin and eosin staining was performed to observe histopathological changes in EM rats after administration, enzyme-linked immunosorbent assay to evaluate the plasma expression of tumor necrosis factor-α (TNF-α) and nerve growth factor (NGF), and immunohistochemistry and western blotting to identify differences in the expression of pain-related factors in EM rats. ResultsThe medium-dose group of PJR (7.56 g/kg) had the best effect on relieving pain in EM rats by reducing RGS, increasing PWT, reducing the ectopic endometrium, improving the cellular structure of the lesion, and reducing TNF-α and NGF levels. However, PJR significantly decreased the expression of transient receptor potential vanilloid 1 (TRPV1), phosphorylated TRPV1 (p-TRPV1), protein kinase C (PKC), and NGF. ConclusionThe mechanism by which PJR relieves EM pain may be through the downregulation of NGF, PKC, and TRPV1 expression.

Detection of Endometriosis Lesions Using Gd-Based Collagen I Targeting Probe in Murine Models of Endometriosis

PurposeEndometriosis is a chronic condition characterized by high fibrotic content and affecting about 10% of women during their reproductive years. Yet, no clinically approved agents are available for non-invasive endometriosis detection. The purpose of this study was to investigate the utility of a gadolinium-based collagen type I targeting probe (EP-3533) to non-invasively detect endometriotic lesions using magnetic resonance imaging (MRI). Previously, this probe has been used for detection and staging of fibrotic lesions in the liver, lung, heart, and cancer. In this study we evaluate the potential of EP-3533 for detecting endometriosis in two murine models and compare it with a non-binding isomer (EP-3612).ProceduresFor imaging, we utilized two GFP-expressing murine models of endometriosis (suture model and injection model) injected intravenously with EP3533 or EP-33612. Mice were imaged before and after bolus injection of the probes. The dynamic signal enhancement of MR T1 FLASH images was analyzed, normalized, and quantified, and the relative location of lesions was validated through ex vivo fluorescence imaging. Subsequently, the harvested lesions were stained for collagen, and their gadolinium content was quantified by inductively coupled plasma optical emission spectrometry (ICP-OES).ResultsWe showed that EP-3533 probe increased the signal intensity in T1-weighted images of endometriotic lesions in both models of endometriosis. Such enhancement was not detected in the muscles of the same groups or in endometriotic lesions of mice injected with EP-3612 probe. Consequentially, control tissues had significantly lower gadolinium content, compared to the lesions in experimental groups. Probe accumulation was similar in endometriotic lesions of either model.ConclusionsThis study provides evidence for feasibility of targeting collagen type I in the endometriotic lesions using EP3533 probe. Our future work includes investigation of the utility of this probe for therapeutic delivery in endometriosis to inhibit signaling pathways that cause the disease.

Effect of Endometriosis on Cumulus ATP, Number of Mitochondria and Oocyte Maturity in Cumulus Oocyte Complex in Mice.

Endometriosis causes a decrease in oocyte quality. However, this mechanism is not fully understood. The present study aimed to analyze the effect of endometriosis on cumulus cell adenosine triphosphate ATP level, the number of mitochondria, and the oocyte maturity level. A true experimental study with a post-test only control group design on experimental animals. Thirty-two mice were divided into control and endometriosis groups. Cumulus oocyte complex (COC) was obtained from all groups. Adenosine triphosphate level on cumulus cells was examined using the Elisa technique, the number of mitochondria was evaluated with a confocal laser scanning microscope and the oocyte maturity level was evaluated with an inverted microscope. The ATP level of cumulus cells and the number of mitochondria in the endometriosis group increased significantly (p < 0.05; p < 0.05) while the oocyte maturity level was significantly lower (p < 0.05). There was a significant relationship between ATP level of cumulus cells and the number of mitochondrial oocyte (p < 0.01). There was no significant relationship between cumulus cell ATP level and the number of mitochondrial oocytes with oocyte maturity level (p > 0.01; p > 0.01). The ROC curve showed that the number of mitochondrial oocytes (AUC = 0.672) tended to be more accurate than cumulus cell ATP level (AUC = 0.656) in determining the oocyte maturity level. In endometriosis model mice, the ATP level of cumulus cells and the number of mitochondrial oocytes increased while the oocyte maturity level decreased. There was a correlation between the increase in ATP level of cumulus cells and an increase in the number of mitochondrial oocytes.

Formononetin Inhibits Progression of Endometriosis via Regulation of p27, pSTAT3, and Progesterone Receptor: In Vitro and In Vivo Studies.

Formononetin is one of the phytoestrogens that functions like a selective estrogen receptor modulator (SERM). In this study, we evaluated the effects of formononetin on endometriosis progression in vitro and in vivo. After pathological confirmation, 10 eutopic and ectopic endometria were collected from patients with endometriosis. Ten eutopic endometria samples were collected from patients who did not have endometriosis. To determine the cytotoxic dose and therapeutic dose of formononetin, the concentration of 70% of the cells that survived after formononetin administration was estimated using a Cell counting kit-8 (CCK 8) assay. Western blot analysis was used to determine the relative expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 in the eutopic endometria without endometriosis, eutopic endometria with endometriosis, and ectopic endometria with endometriosis as the formononetin concentration was increased. We confirmed the effect of formononetin on apoptosis and migration in endometriosis using fluorescence-activated cell sorting (FACS) and wound healing assays, respectively. A mouse model of endometriosis was prepared using a non-surgical method, as previously described. The mice were intraperitoneally administered formononetin for four weeks after dividing them into control, low-dose formononetin (40 mg/kg/day) treatment, and high-dose (80 mg/kg/day) formononetin treatment groups. All the mice were euthanized after formononetin treatment. Endometriotic lesions were retrieved and confirmed using hematoxylin and eosin (H&E) staining. Immunohistochemical (IHC) staining of p27 was performed. We set the maximum concentration of formononetin administration to 80 μM through the CCK8 assay. Based on formononetin concentration, the expression levels of BAX, p53, pAKT, ERK, pERK, p27, and pSTAT3 proteins were measured using Western blot analysis (N = 4 per group). The expression level of pERK, p27, and pSTAT3 in eutopic endometrium with endometriosis tended to decrease with increasing formononetin concentration, and a significant decrease was noted at 80 μM. The expression of p27 in ectopic endometrium with endometriosis was also significantly decreased at 80 μM of formononetin. FACS analysis revealed that formononetin did not significantly affect apoptosis. In the wound healing assay, formononetin treatment revealed a more significant decrease in the proliferation of the eutopic endometrium in patients with endometriosis than in the eutopic endometrium without endometriosis. Relative expression of sex hormone receptors decreased with increasing formononetin doses. Although no significant differences were observed in the ER, PR-A, ERβ/ERα, and PR-B/PR-A, significant down-regulation of PR-B expression was noted after formononetin treatment at 80 μM. In the in vivo study, endometriotic lesions in the formononetin-treated group significantly decreased compared to those in the control group. The relative expression of p27 using IHC was highest in the control group and lowest in the high-dose formononetin treatment group. Formononetin treatment was shown to inhibit the proliferation of eutopic and ectopic endometria in patients with endometriosis through the regulation of p27, pSTAT3, and PR-B. In an endometriosis mouse model, formononetin treatment significantly reduced the number of endometriotic lesions with decreased p27 expression. The results of this study suggest that formononetin may be used as a non-hormonal treatment option for endometriosis.

Potent Dual Inhibitors of Steroid Sulfatase and 17β-Hydroxysteroid Dehydrogenase Type 1 with a Suitable Pharmacokinetic Profile for In Vivo Proof-of-Principle Studies in an Endometriosis Mouse Model.

Treating estrogen-dependent diseases like endometriosis with drugs suppressing local estrogen activation may be superior to existing endocrine therapies. Steroid sulfatase (STS) and 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1) are key enzymes of local estrogen activation. We describe the rational design, synthesis, and biological profilation of furan-based compounds as a novel class of dual STS/17β-HSD1 inhibitors (DSHIs). In T47D cells, compound 5 showed irreversible inhibition of STS and potent, reversible inhibition of 17β-HSD1. It was selective over 17β-HSD2 and displayed high metabolic stabilities in human and mouse liver S9 fractions. No effect on cell viability was detected up to 31 μM (HEK293) and 23 μM (HepG2), respectively, and there was no activation of the aryl hydrocarbon receptor (AhR) up to 3.16 μM. Single daily application to mice revealed steady-state plasma levels high enough to make this compound eligible for an in vivo proof-of-principle study in a mouse endometriosis model.

P-375 Effect of D-Chiro-Inositol in a mouse model of endometriosis

Abstract Study question Can D-Chiro Inositol administration mitigate the phenotype of endometriosis in a mouse model? Summary answer Based on an endometriosis mouse model, we demonstrated that administration of D-Chiro Inositol can reduce development of endometriotic lesions. What is known already Endometriosis, a disease affecting 5-10% of women of reproductive age, is characterized by the spread of endometrial-like tissue outside the uterine cavity that produces ectopic endometriotic lesions causing pain and infertility. The sensitivity of endometriosis to estrogens is a characteristic that can be used for therapeutic purposes. D-Chiro Inositol (DCI), one of the nine isomers of Inositol, is known to decrease the CYP19A1 aromatase gene expression in granulosa cells. Based on these premises, it was suggested that treatment with DCI may have clinical application in conditions where decreased estrogen levels is required. Study design, size, duration To address the study question, a mouse model of endometriosis was generated. Out of 20 CD1 mice, 4 mice were randomly selected as donors of uterine fragments and the remaining 16 were recipient mice. The first day after transplantation, mice were randomly assigned to four experimental group which received for 28 days 2ml of water containing: none (CTRL); DCI 0.4mg (DCI 0.4); DCI 0.2mg and Dienogest 0.33ng (DCI 0.2+DG 0.33); DG 0.67ng (DG 0.67). Participants/materials, setting, methods Uterine horns were removed from donor mice at the diestrous stage of the reproductive cycle. The tissue cut into fragments was inoculated in recipient mice by intraperitoneal injection. Four weeks after induction, all mice were sacrificed. Their endometriotic lesions were excised, measured by number and size, and examined for the presence of blood vessels vascularization under stereomicroscope. Then, lesions were processed for histology examination by hematoxilin-eosin (H&amp;E) and Azan Mallory staining. Main results and the role of chance Endometriotic lesions developed in recipient mice met all criteria for endometriosis, including the presence of endometrial epithelial and stromal cells, and encapsulation in neighboring tissues or organs. The lesions number was reduced in all the treatment groups when compared to control (p &amp;lt; 0.05, t-test), and no differences were observed among DCI 0.4, DCI 0.2+DG 0.33 and DG 0.67. Concomitantly the rate of vascularized lesions was lower in the treated groups, with more pronounced effect in the DCI 0.4 group where no vascularized lesions were observed (p &amp;lt; 0.05, t-test). The histological analysis revealed a marked reduction of endometriotic foci in all groups. These results provide evidence that DCI can reduce development and vascularization of endometriotic lesions in a mouse model, an effect that is not observed when it is employed at lower dose in association with DG. Although molecular mechanisms underlying DCI effects requires further investigation, present findings support the hypothesis that DCI could be more effective than DG in mitigating endometriosis phenotype. Limitations, reasons for caution Results from animal studies should be extrapolated to humans with caution. Wider implications of the findings Present findings may open new avenue in testing whether DCI may have clinical application in endometriosis therapy. Trial registration number Not Applicable

Fusobacterium infection facilitates the development of endometriosis through the phenotypic transition of endometrial fibroblasts.

Retrograde menstruation is a widely accepted cause of endometriosis. However, not all women who experience retrograde menstruation develop endometriosis, and the mechanisms underlying these observations are not yet understood. Here, we demonstrated a pathogenic role of Fusobacterium in the formation of ovarian endometriosis. In a cohort of women, 64% of patients with endometriosis but <10% of controls were found to have Fusobacterium infiltration in the endometrium. Immunohistochemical and biochemical analyses revealed that activated transforming growth factor-β (TGF-β) signaling resulting from Fusobacterium infection of endometrial cells led to the transition from quiescent fibroblasts to transgelin (TAGLN)-positive myofibroblasts, which gained the ability to proliferate, adhere, and migrate in vitro. Fusobacterium inoculation in a syngeneic mouse model of endometriosis resulted in a marked increase in TAGLN-positive myofibroblasts and increased number and weight of endometriotic lesions. Furthermore, antibiotic treatment largely prevented establishment of endometriosis and reduced the number and weight of established endometriotic lesions in the mouse model. Our data support a mechanism for the pathogenesis of endometriosis via Fusobacterium infection and suggest that eradication of this bacterium could be an approach to treat endometriosis.

Host immunity and KLF 11 deficiency together promote fibrosis in a mouse model of endometriosis.

BackgroundEndometriosis is a debilitating disease typically characterized by prolific fibrotic scarring. Earlier we reported downregulation of two transcription factors belonging TGF-βR signaling pathway Sp/Krüppel-like factor 11 (KLF11) and 10 (KLF10) in human endometriosis lesions. Here we investigated the role of these nuclear factors and immunity in the scaring fibrosis associated with endometriosis. MethodsWe used a well characterized experimental mouse model of endometriosis. WT, KLF10 or KLF11 deficient mice were compared. The lesions were evaluated histologically, fibrosis was quantified with Masons' Trichome staining, immune-infiltrates were quantified by immunohistochemistry, peritoneal adhesions were score, gene expression was evaluated by bulk RNA sequencing. ResultsIntense fibrotic reactions and large changes in gene expression were detected in KLF11 deficient implants associated with squamous metaplasia of the ectopic endometrium, as compared to KLF10 deficient or WT implants. Fibrosis was mitigated with pharmacologic agents that blocked histone acetylation or TGF-βR signaling or with genetic deficiency for SMAD3. The lesions were richly infiltrated with T-cells, regulatory T-cells, and innate immune cells. Fibrosis was exacerbated when implants expressed ectopic genes implicating autoimmunity as a major factor contributing to the scaring fibrosis. ConclusionsOur findings identify KLF11 and TGF-βR signaling as cell intrinsic mechanisms and autoimmune responses as cell extrinsic mechanisms of scaring fibrosis in ectopic endometrium lesions. General significanceImmunological factors associated with inflammation and tissue repair drive scaring fibrosis in experimental endometriosis, providing the rationale for immune therapy of endometriosis.

CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis

Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.

Shaofu Zhuyu Decoction attenuates fibrosis in endometriosis through regulating PTEN/Akt/mTOR signaling pathway

The present study aimed to investigate the protective role of Shaofu Zhuyu Decoction(SFZY) against endometriosis fibrosis in mice, and decipher the underlying mechanism through the phosphatase and tensin homolog deleted on chromosome ten(PTEN)/protein kinase B(Akt)/mammalian target of rapamycin(mTOR) pathway. Eighty-five BALB/c female mice were randomly assigned into a blank group, a model group, high-, medium, and low-dose SFZY(SFZY-H, SFZY-M, and SFZY-L, respectively) groups, and a gestrinone suspension(YT) group. The model of endometriosis was induced by intraperitoneal injection of uterine fragments. The mice in different groups were administrated with corresponding groups by gavage 14 days after modeling, and the blank group and model group with equal volume of distilled water by gavage. The treatment lasted for 14 days. The body weight, paw withdrawal latency caused by heat stimuli, and total weight of dissected ectopic focus were compared between different groups. The pathological changes of the ectopic tissue were observed via hematoxylin-eosin(HE) and Masson staining. Real-time PCR was employed to measure the mRNA levels of α-smooth muscle actin(α-SMA) and collagen type Ⅰ(collagen-Ⅰ) in the ectopic tissue. The protein levels of PTEN, Akt, mTOR, p-Akt, and p-mTOR in the ectopic tissue were determined by Western blot. Compared with the blank group, the modeling first decreased and then increased the body weight of mice, increased the total weight of ectopic focus, and shortened the paw withdrawal latency. Compared with the model group, SFZY and YT increased the body weight, prolonged the paw withdrawal latency, and decreased the weight of ectopic focus. Furthermore, the drug administration, especially SFZY-H and YT(P&lt;0.01), recovered the pathological and reduced the area of collagen deposition. Compared with the blank group, the modeling up-regulated the mRNA levels of α-SMA and collagen-Ⅰ in the ectopic focus, and such up-regulation was attenuated after drug intervention, especially in the SFZY-H and YT groups(P&lt;0.05,P&lt;0.01). Compared with the blank group, the modeling down-regulated the protein level of PTEN and up-regulated the protein levels of Akt, mTOR, p-Akt, and p-mTOR(P&lt;0.01, P&lt;0.001). Drug administration, especially SFZY-H and YT, restored such changes(P&lt;0.01). SFZY may significantly attenuate the focal fibrosis in the mouse model of endometriosis by regulating the PTEN/Akt/mTOR signaling pathway.

NLRP3 inflammasome-mediated Pyroptosis induce Notch signal activation in endometriosis angiogenesis

Endometriosis is characterized by the presence of endometrial tissue outside the uterus that not only causes severe pelvic pain and infertility but also increased risk for ovarian carcinogenesis in women of reproductive age. Here, we found that angiogenesis was increased and accompanied with up-regulation of Notch1 in human endometriotic tissue sample, which is associated with pyroptosis induced by activation of endothelial NLRP3 inflammasome. Further, in endometriosis model induced in wild type and NLRP3-deficient (NLRP3-KO) mice, we found that deficiency of NLRP3 suppressing the development of endometriosis. In vitro, inhibiting the activation of NLRP3 inflammasome prevents LPS/ATP-induced tube formation in endothelial cells. Meanwhile, knockdown NLRP3 expression by gRNA disrupt the interaction between Notch1 and HIF-1α under the inflammatory microenvironment. This study demonstrates that activation of NLRP3 inflammasome-mediated pyroptosis affects angiogenesis in endometriosis via Notch1-dependent manner.

Gut and reproductive tract microbiota: Insights into the pathogenesis of endometriosis (Review).

Endometriosis is characterized by the presence of endometrial-like tissue outside the uterus and is associated with an inflammatory immune response. The gut and reproductive tract microbiota constitute a protective barrier against infection by pathogens and regulate inflammatory and immune functions. This review summarizes microbiota imbalance (i.e., dysbiosis) in endometriosis and discusses how dysbiosis influences disease development. The literature was searched for studies published from inception to March 2022 in the PubMed and Google Scholar databases using a combination of specific terms. An altered gut and reproductive tract microbiome has been reported in numerous conditions, such as inflammatory bowel disease, allergies, autoimmunity, cancer and reproductive disorders (e.g., endometriosis). Furthermore, microbial dysbiosis is a hallmark of endometriosis and is characterized by a decrease in beneficial probiotics and an increase in pathogenic microbes, which leads to a series of estrobolomic and metabolomic changes. Gut or reproductive tract microbiome dysbiosis was reported in mice, nonhuman primates, and females with endometriosis. Animal models of endometriosis demonstrated the effects of the gut microbiome on lesion growth and vice versa. The immune system mediated by the microbiota-gut-reproductive tract axis triggers an inflammatory response that damages reproductive tract tissue, which possibly leads to endometriosis. However, whether the alteration of eubiosis (a balanced microbiota) to dysbiosis is a cause or a result of endometriosis is unclear. In conclusion, this review provides an overview of the relationship between the gut and reproductive tract microbiome and endometriosis, focusing on the mechanisms by which dysbiosis may increase the risk of disease.

Peripheral and central neuroplasticity in a mouse model of endometriosis.

Chronic pelvic pain (CPP) is the most debilitating symptom of gynaecological disorders such as endometriosis. However, it remains unclear how sensory neurons from pelvic organs affected by endometriosis, such as the female reproductive tract, detect and transmit nociceptive events and how these signals are processed within the central nervous system (CNS) Using a previously characterised mouse model of endometriosis we investigated whether the increased pain sensitivity occurring in endometriosis could be attributed to i) changes in mechanosensory properties of sensory afferents innervating the reproductive tract, ii) alterations in sensory input from reproductive organs to the spinal cord, or iii) neuroinflammation and sensitisation of spinal neural circuits. Mechanosensitivity of vagina-innervating primary afferents was examined using an ex vivo single-unit extracellular recording preparation. Nociceptive signalling from the vagina to the spinal cord was quantified by phosphorylated MAP kinase ERK1/2 immunoreactivity. Immunohistochemistry was used to determine glial and neuronal circuit alterations within the spinal cord. We found that sensory afferents innervating the rostral, but not caudal portions of the mouse vagina, developed mechanical hypersensitivity in endometriosis. Nociceptive signalling from the vagina to the spinal cord was significantly enhanced in mice with endometriosis. Moreover, mice with endometriosis developed microgliosis, astrogliosis and enhanced substance P neurokinin-1 receptor immunoreactivity within the spinal cord, suggesting the development of neuroinflammation and sensitisation of spinal circuitry in endometriosis. These results demonstrate endometriosis-induced neuroplasticity occurring at both peripheral and central sites of sensory afferent pathways. These findings may help to explain the altered sensitivity to pain in endometriosis and provide a novel platform for targeted pain-relief treatments for this debilitating disorder.

Gut dysbiosis-derived β-glucuronidase promotes the development of endometriosis

To explore the role of gut dysbiosis-derived β-glucuronidase (GUSB) in the development of endometriosis (EMs). 16S rRNA sequencing of stool samples from women with (n = 35) or without (n = 30) endometriosis and from a mouse model was conducted to assess gut microbiome changes and identify molecular factors influencing the development of endometriosis. Experiments invivo in an endometriosis C57BL6 mouse model and invitro verified the level of GUSB and its role in the development of EMs. Department of Obstetrics and Gynecology, The First Affiliated Hospital of Sun Yat-sen University; Guangdong Provincial Clinical Research Center for Obstetrical and Gynecological Diseases. Women of reproductive age with a histological diagnosis of endometriosis were enrolled in the endometriosis group (n = 35) and infertile or healthy age-matched women who had undergone a gynecological or radiological examination in the control group (n = 30). Fecal and blood samples were taken the day before surgery. Paraffin-embedded sections from 50 bowel endometriotic lesions, 50 uterosacral lesions, 50 samples without lesions, and 50 normal endometria were collected. None. Changes in the gut microbiome of patients with EMs and mice and the effect of β-glucuronidase on the proliferation and invasion of endometrial stromal cells and the development of endometriotic lesions were assessed. No difference in α and β diversity was found between patients with EMs and controls. Immunohistochemistry analysis showed higher β-glucuronidase expression in bowel lesions and uterosacral ligament lesions than in the normal endometrium (p<0.01). β-Glucuronidase promoted the proliferation and migration of endometrial stromal cells during cell counting kit-8, Transwell, and wound-healing assays. Macrophage levels, especially M2, were higher in bowel lesions and uterosacral ligament lesions than in controls, and β-glucuronidase promoted the M0 to M2 transition. Medium conditioned by β-glucuronidase-treated macrophages promoted endometrial stromal cell proliferation and migration. β-Glucuronidase increased the number and volume of endometriotic lesions and number of macrophages present in lesions in the mouse EMs model. This β-Glucuronidase promoted EMs development directly or indirectly by causing macrophage dysfunction. The characterization of the pathogenic role of β-glucuronidase in EMs has potential therapeutic implications.

Non-surgical Syngeneic Model of Endometriosis in Ovary-intact Outbred Mice

Intraperitoneal injection of endometrial tissues into inbred mice such as C57BL/6J is widely used as a model to study endometriosis, a disease characterized by the abnormal proliferation of endometrial cells which invade various tissues within the peritoneal cavity. However, most of these inbred mouse strains have a weak immune system and are often ovariectomized, which is not reflective of the human population in general. Hence, this study used the ovary intact ICR mouse strain as a model to study the immune response during endometriosis development using a non-surgical syngeneic model with no estrogen supplementation. We showed that ICR mice developed ectopic endometrial tissues after 8 wk, but these were mostly necrotic. Reducing the induction period to 4 wk increased the number of ectopic tissues, and endometriotic lesions were also formed in 30% of the induced recipient mice, albeit with a relatively low incidence rate. Endometriotic lesions in ICR mice were also associated with fewer lesion-resident macrophages and lesser vascularization than in C57BL/6J mice. This is further supported by a significantly downregulated expression of genes involved in angiogenesis and M2 macrophage activity in ICR versus C57BL/6J donor endometrium. Conversely, inflammatory response genes were significantly upregulated in the endometrium of ICR versus C57BL/6J mice. Overall, these data implicate the role of inflammation in inhibiting the establishment of endometrial lesions in ICR mice and the involvement of macrophage in promoting endometriosis in C57BL/6J mice. The present work reports the establishment of endometriotic lesions in outbred ICR mice by a less invasive syngeneic intraperitoneal injection procedure.

Loss-of-STING function by nitro-fatty acids reduces endometriotic lesion formation in a syngeneic model of endometriosis

Endometriosis is a painful, chronic inflammatory disease that impacts fertility and the overall well-being in 10% of reproductive age women. Current therapies include hormonal treatment to induce a hypoestrogenic state, causing serious complications from side effects. STING (Stimulator of Interferon Genes) plays an essential role in innate immunity but also exacerbates inflammation in chronic inflammatory diseases. Recently, STING was found aberrantly upregulated in endometriosis patients. Nitro-fatty acids (NO2-FAs) are bioactive lipids that exert anti-inflammatory and protective roles in preclinical models of inflammation and fibrosis, in part via inhibition of STING. We hypothesized that NO2-FAs protect against STING-dependent endometriosis lesion formation and tested its therapeutic potential using a syngeneic transplant model of endometriosis. Mice expressing green fluorescent protein (EGFP) were treated with Pregnant Mare Serum Gonadotrophin (PMSG) to induce decidualization. Decidualized uterine fragments were transplanted into wild-type (WT) female recipients randomized to receive NO2-FA or vehicle. Endometriosis was allowed to develop for 28 days. In vivo imaging of EGFP donor-derived endometrial lesions demonstrates that NO2-FA administration significantly reduces peritoneal endometriotic lesion formation in female recipients. Furthermore, loss-of-STING function, using STING KO female mice as recipients, demonstrates that STING plays a systemic functional role on endometriosis development. Finally, our studies demonstrate that NO2-FAs inhibit cyclic GMP-AMP Synthase (cGAS)-induced type I interferon signaling in bone-marrow derived macrophages (BMDM) and in the peritoneum of WT female mice but not STING KO mice. In summary, we show that STING is a novel, essential factor contributing to the pathogenesis of endometriosis and that NO2-FA administration may serve as a novel non-hormonal therapy against endometriosis by targeting STING. This study is funded by the American Physiological Society William Townshend Predoctoral Fellowship (MP), NIMHD U54-MD007602 (WT), R01-HL162294 (MGB) and R01-HL123333 (LV). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The effect of Chromolaena odorata on endometriosis implants in mice model of endometriosis

Introduction: Endometriosis is a benign gynecological disease whose pathogenesis mechanism is not fully understood with treatment modality with hormonal and surgical methods aiming to relieve symptoms, suppress progression, and increase fertility. This study aims to observe the differences in the administration of Chromolaena odorata leaf extract on the area of endometriosis implants in the mice model of endometriosis Materials and Methods: This study was an experimental study with a Randomized Separated Pre and Post Test Design research conducted with the control group. Thirty mice (Mus musculus) were injected with 0.2 ml/mice of cyclosporine A. Then endometrial tissue was injected into the peritoneal cavity, followed by 0.1 ml Ethinyl estradiol/mice injections on the 1st and 5th days. Mice were reared for 14 days until the endometriosis model was formed. Mice models of endometriosis were randomly divided into five groups: endometriosis mice that were terminated on day 14 (pretest), then the other groups are endometriosis mice given Aquadest as placebo (P0), endometriosis mice treated with Chromolaena odorata leaf extract at a dose of 400 mg per kilograms of body weigh (mg/kgBW) (P1), 800 mg/kgBW (P2) and 1200 mg/kgBW (P3) for 14 days. On the twenty-eighth day, mice were dissected to examine and assess the extent of endometriosis implantation macroscopically in hyperemic areas by taking samples in the most hyperemic areas of the peritoneal wall. Research data were analyzed using SPSS Version 22. Results: From this study, the average value of endometriosis implant area in the group given Chromolaena odorata leaf extract was 400 mg/kgBW (P1 = 15.60 mm2), 800 mg/kgBW (P2=14.23 mm2) and 1200 mg/kgBW (P3=0.00 mm2) was lower than the pretest group (P=104.72 mm2) and the placebo group (P0 = 153.89 mm2), showing a significant difference (p&lt;0.001). There was a tendency that the area of endometriosis implants was also lower in the group receiving a higher dose of Chromolaena odorata leaf extract 1200 mg/kgBW (P3=0.00 mm2) than the group receiving a dose of 400 mg/kgBW (P1=15.60 mm2) and 800 mg/kgBW (P2=14.23 mm2). Conclusions: It can be concluded that the administration of Chromolaena odorata leaf extract can reduce the area of endometriosis implants in mice models of endometriosis. Higher doses result in the lower endometrial implant area.

Genistein Effect on Estrogen Receptor especially Interleukin-6 and Tumor Necrosis Factor-α in Mice Model of Endometriosis: a Systematic Review and Meta-analysis

Introduction: Endometriosis is the endometrial tissue proliferation outside the uterine space. Anti-angiogenic agents from different groups of substances are currently being discussed as promising therapies for the treatment of endometriosis in the future, one of which is therapy using the phytoestrogen genistein. At the molecular level, genistein's effects on cells include inhibition of proliferation, induction of differentiation, apoptosis, and cell arrest in the cell cycle, which are related to the transforming growth factor. Genistein inhibits cell growth by modulating the signal-transforming growth factor pathway. Methods: We searched PubMed, Scopus, Cochrane Library and Proquest from its inception to December 2022 to collect observational and interventional studies of Genistein consumption in endometriosis mice. Two authors independently screened the literature according to the inclusion and exclusion criteria, extracted data, and assessed the risk of bias. Safety analysis was assessed through TNF-α and IL-6 values of estrogen expression. Results: No significant difference in the values of IL-6 and TNF-α in rats with endometriosis that were given genistein at these three doses. However, if analyzed further, the IL-6 outcome compared to the 0.78mg/day dose most affected the IL-6 value between the two groups, and the TNF-α output compared to the 0.78 mg/day dose most affected the TNF-α value between the two groups. So, a genistein dose of 0.78 mg/day can affect IL-6 and TNF-α in mice with endometriosis. Conclusion: Genistein modulated estrogen expression of TNF-α and IL-6 at all doses, but the best dose for its effect was 0.78 mg/day.

Suppressive effects of linzagolix, a novel non-peptide antagonist of gonadotropin-releasing hormone receptors, in experimental endometriosis model rats.

Endometriosis is an oestrogen-dependent disease in which endometrial-like tissue grows outside the uterus in women of reproductive age. Accordingly, control of oestradiol (E2) levels is an effective treatment for endometriosis. Because gonadotropin-releasing hormone (GnRH) is the main controller of E2 secretion, control of GnRH signalling by GnRH antagonism is an effective strategy for the treatment of sex hormone-dependent diseases such as endometriosis. The purpose of the present study was to evaluate the effects of the potent, orally available and selective GnRH antagonist linzagolix on experimental endometriosis in rats and compare them with those of dienogest, which is used clinically to treat endometriosis. Experimental endometriosis was induced in female rats at the proestrus stage of the oestrous cycle via autotransplantation of endometrial tissue into the renal subcapsular space. Linzagolix significantly decreased cyst volumes compared with the control group at doses of 50 mg/kg or more. Indeed, a suppressive effect of dienogest on cyst volume was observed only at the highest dose evaluated (1 mg/kg). The effective concentration of linzagolix, calculated as the free form of the last-observed drug concentration, was ~1 μmol/L in endometriosis model rats. The present study also reveals that linzagolix exerts a sustained inhibitory effect on E2 secretion, indicating that the suppressive effect on endometriosis cyst volumes could be attributed to its pharmacological suppression of GnRH signalling and serum E2 concentrations. Altogether, our findings indicate that linzagolix may be a useful therapeutic intervention for hormone-dependent diseases including endometriosis.