Mouse Model Of Endometriosis Research Articles

Administration of red ginseng regulates microRNA expression in a mouse model of endometriosis.

ObjectiveRed ginseng (RG) exerts anti-inflammatory, anti-proliferative, and immunomodulatory effects on endometriosis through the regulation of microRNA (miRNA) expression. It may also ameliorate endometriosis by affecting the expression of multiple miRNAs simultaneously, rather than acting on a single miRNA at a given time. Since studies on the overall effects of RG on endometriosis via the regulation of miRNA expression are lacking, the current study aimed to explore the global effect of RG on miRNA expression in a mouse model of endometriosis.MethodsTo establish the mouse model, the uterine horn of donor mice was implanted into the lateral side of the recipients’ peritoneum, followed by vehicle or RG treatment for 8 weeks.ResultsTo confirm the effects of RG on the established mouse model, the size of the implanted uterus was measured; it was found to be lower in mice from the RG group than in mice from the control group. miRNA expression profiles in the implanted uterus of the mouse model of endometriosis after vehicle or RG administration were analyzed using microarray technology. Thereafter, seven candidate miRNAs and 125 candidate genes (miRNA targets) were identified through a bioinformatics analysis.ConclusionThe present findings suggest that RG regulates the expression of multiple miRNAs and mRNAs, thereby alleviating endometriosis in a mouse model of the disease.

Effects of dual inhibition of AKT and ERK1/2 pathways on endometrial pro-inflammatory, hormonal, and epigenetic microenvironment in endometriosis

Endometriosis is an estrogen-dependent and progesterone-resistant gynecological inflammatory disease of reproductive-age women. The prevalence of endometriosis is ~5–10% in reproductive-age women, increasing to 20–30% in women with subfertility. The current anti-estrogen therapies can be prescribed only for a short time because of the undesirable side effects on menstruation, pregnancy, bone health, and failure to prevent a recurrence. The causes of endometriosis-associated infertility are multifactorial and poorly understood. The objective of the present study was to determine the inhibitory effects of AKT and/or ERK1/2 pathways on the microenvironment of the endometrium in a xenograft mouse model of endometriosis of human origin. Results indicate that dual inhibition of AKT and ERK1/2 pathways, but not inhibition of either AKT or ERK1/2 pathway, suppresses the growth of the endometriotic lesions in vivo. Dual inhibition of AKT and ERK1/2 pathways suppresses the production of proinflammatory cytokines, decreases E2 biosynthesis and signaling, and restores progesterone receptor-B signaling components in the epithelial and stromal cells of the endometrium in a cell-specific manner. These results together suggest that dual inhibition of AKT and ERK1/2 pathways suppresses the estrogen-dominant state and concomitantly increases the progesterone-responsive state of the endometrium. Therefore, dual inhibition of AKT and ERK1/2 pathways could emerge as long-term nonsteroidal therapy for endometriosis.

Bioluminescent imaging in induced mouse models of endometriosis reveals differences in four model variations.

ABSTRACTOur understanding of the aetiology and pathophysiology of endometriosis remains limited. Disease modelling in the field is problematic as many versions of induced mouse models of endometriosis exist. We integrated bioluminescent imaging of ‘lesions’ generated using luciferase-expressing donor mice. We compared longitudinal bioluminescence and histology of lesions, sensory behaviour of mice with induced endometriosis and the impact of the gonadotropin-releasing hormone antagonist Cetrorelix on lesion regression and sensory behaviour. Four models of endometriosis were tested. We found that the nature of the donor uterine material was a key determinant of how chronic the lesions were, as well as their cellular composition. The severity of pain-like behaviour also varied across models. Although Cetrorelix significantly reduced lesion bioluminescence in all models, it had varying impacts on pain-like behaviour. Collectively, our results demonstrate key differences in the progression of the ‘disease’ across different mouse models of endometriosis. We propose that validation and testing in multiple models, each of which may be representative of the different subtypes/heterogeneity observed in women, should become a standard approach to discovery science in the field of endometriosis.

M1 Macrophage-Derived Nanovesicles Repolarize M2 Macrophages for Inhibiting the Development of Endometriosis.

BackgroundEndometriosis is a common nonmalignant gynecological disorder that affects 10–15% women of reproductive age and causes several symptoms that result in decreased quality of life and a huge social burden. In recent decades, extracellular vesicles (EVs) have gained attention as a potential therapeutic tool; however, the therapeutic effects of EVs against endometriosis have not been reported. Accordingly, in this study, we investigated the feasibility of nanovesicles (NVs) derived from M1 macrophages (M1NVs) in treating endometriosis.MethodsM1NVs were prepared by serial extrusion. Co-culture assays were performed to investigate changes in tube formation and migration/invasion of eutopic endometrial stroma cells (ESCs) obtained from patients with endometriosis (EM-ESCs). A mouse model of endometriosis was established, and mice were treated with phosphate-buffered saline, M0NVs, or M1NVs to evaluate the efficacy and safety of M1NV for treating endometriosis.ResultsM1NVs directly or indirectly inhibited the migration and invasion of EM-ESCs and reduced tube formation. In the mouse model, M1NVs suppressed the development of endometriosis through reprogramming of M2 macrophages, without causing damage to the organs.ConclusionsM1NVs inhibit the development of endometriosis directly, or through repolarizing macrophages from M2 to M1 phenotype. Hence, administration of M1NVs may represent a novel method for the treatment of endometriosis.

Metabolite profiles in the peritoneal cavity of endometriosis patients and mouse models

Research questionWhich metabolites are altered in the peritoneal cavity of women with endometriosis? Could the mouse endometriosis model simulate these alterations? DesignThirteen women with endometriosis and seven women with other benign gynaecological diseases, who underwent laparoscopic surgery, were included in this study. None had received hormonal therapy for 3 months before surgery. For the animal experiments, six and five mice were included in the endometriosis and control groups, respectively. Peritoneal fluid from the patients and peritoneal lavage fluid from the mice was collected and analysed. Non-targeted metabolomics via liquid chromatography with tandem mass spectrometry was used to identify the altered metabolites in the peritoneal fluid of endometriosis patients and mouse models. MetaboAnalyst 4.0 was used to visualize the data. ResultsSeveral metabolites in the peritoneal cavity were significantly altered in both humans and mice with endometriosis. Concentrations of lysophosphatidylcholine (LysopC) (P=0.017 in patients and P=0.041 in the mouse model) and derivatives of phosphoethanolamine (1-arachidonoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.027; 1-oleoyl-sn-glycero-3-phosphoethanolamine in patients, P=0.0086; and phosphorylethanolamine in the mouse model, P=0.0027) were significantly up-regulated in both, whereas concentrations of acylcarnitines (l-palmitoylcarnitine, P=0.047; and stearoylcarnitine, P=0.029) and kynurenine (P=0.045) were significantly increased only in humans. The human and mouse samples shared three altered enriched metabolite sets. ConclusionsWomen with endometriosis show an altered metabolic state in the abdominal cavity. The endometriosis mouse model shared half of the significantly altered metabolite sets found in the abdominal cavity of humans.

Effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha receptor 1 expression, granulosa cell apoptosis, and folliculogenesis repair in endometriosis mouse models.

Background and Aim:Endometriosis affects the ovaries and causes a decrease in the oocyte quality during endometrial receptivity. During the development of ovarian follicles, paracrine communication occurs between granulosa cells and oocytes. This study was conducted to determine the effects of bone marrow mesenchymal stem cell transplantation on tumor necrosis factor-alpha (TNF-α) receptor 1 (TNFR1) expression, granulosa cell apoptosis, and folliculogenesis in endometriosis mouse models.Materials and Methods:This study involved 42 female mice, which were divided into three groups: Healthy mice (T0), endometriosis mice without transplantation (T1), and endometriosis mice with bone marrow mesenchymal stem cell transplantation (T2). The mice were injected intraperitoneally with endometrial fragments (200 μL) to become endometriosis models. On day 15, the endometriosis models received mesenchymal stem cells. Sample collection was performed on day 29. Granulosa cell apoptosis and TNFR1 expression were examined using immunohistochemical staining, and folliculogenesis was assessed using hematoxylin and eosin staining of ovary samples. The data obtained from both examinations were statistically analyzed using Statistical Package for the Social Sciences.Results:The results showed that TNFR1 expression is significantly decreased in T2 (p<0.004). The apoptosis of granulosa cells was lower in T2 (p<0.000). The primary, secondary, and graafian follicle counts in T2 were significantly increased.Conclusion:Bone marrow mesenchymal stem cell transplantation in endometriosis mouse models can reduce TNFR1 expression and granulosa cell apoptosis and improve folliculogenesis.

The pro-inflammatory and anti-inflammatory role of hyaluronic acid in endometriosis

ObjectiveEndometriosis is a bothersome disease affected women worldwide, the mechanism of disease development is still under investigation. Several inflammatory responses after clinical hyaluronic acid (HA) use were reported. Cyclooxygenase (COX)-2 mediated inflammation pathway is involved in the pathogenesis of endometriosis. Thus, we tried to investigate the inflammatory role of hyaluronic acid in endometriosis. Materials and methodsPeritoneal fluid was collected in endometriosis and disease-free patients for the measurement of HA. Endometriotic stromal cells were treated with IL-1β and HA and expression of COX-2 was evaluated. Mice model of endometriosis was established and treated with fluid or gel form of HA. Endometriotic lesion size and weight were recorded and level of COX-2 was evaluated by immunohistochemistry staining. ResultsThe level of HA in the peritoneal fluid had no statistically significant difference between normal, early and advanced stage endometriosis patients. The overexpression of COX-2 protein was detected when treating endometriotic stromal cell with HA in the presence of IL-1β (P < 0.001). The endometriotic lesion size was reduced in mice model when treated with higher concentration gel form HA. It further showed less proportion of strong COX-2 expression compare of gel form HA to fluid form treatment in COX-2 expression score of endometriosis lesion. ConclusionBoth proinflammatory evidence, elevated COX-2 expression, and anti-inflammatory result, reduced endometriosis lesion size and COX-2 expression score, were noted in our study after treating HA in in vivo and in vitro models. We hypothesized it is possible that HA may induce an acute proinflammatory response followed by chronic anti-inflammatory reaction in the formation of endometriosis.

Differences in growth and vascularization of ectopic menstrual and non-menstrual endometrial tissue in mouse models of endometriosis.

Is there a difference in the growth and vascularization between murine endometriotic lesions originating from menstrual or non-menstrual endometrial fragments? Endometriotic lesions developing from menstrual and non-menstrual tissue fragments share many similarities, but also exhibit distinct differences in growth and vascularization, particularly under exogenous estrogen stimulation. Mouse models are increasingly used in endometriosis research. For this purpose, menstrual or non-menstrual endometrial fragments serve for the induction of endometriotic lesions. So far, these two fragment types have never been directly compared under identical experimental conditions. This was a prospective experimental study in a murine peritoneal and dorsal skinfold chamber model of endometriosis. Endometrial tissue fragments from menstruated (n = 15) and non-menstruated (n = 21) C57BL/6 mice were simultaneously transplanted into the peritoneal cavity or dorsal skinfold chamber of non-ovariectomized (non-ovx, n = 17), ovariectomized (ovx, n = 17) and ovariectomized, estrogen-substituted (ovx+E2, n = 17) recipient animals and analyzed throughout an observation period of 28 and 14 days, respectively. The engraftment, growth and vascularization of the newly developing endometriotic lesions were analyzed by means of high-resolution ultrasound imaging, intravital fluorescence microscopy, histology and immunohistochemistry. Menstrual and non-menstrual tissue fragments developed into peritoneal endometriotic lesions without differences in growth, microvessel density and cell proliferation in non-ovx mice. Lesion formation out of both fragment types was markedly suppressed in ovx mice. In case of non-menstrual tissue fragments, this effect could be reversed by estrogen supplementation. In contrast, endometriotic lesions originating from menstrual tissue fragments exhibited a significantly smaller volume in ovx+E2 mice, which may be due to a reduced hormone sensitivity. Moreover, menstrual tissue fragments showed a delayed vascularization and a reduced blood perfusion after transplantation into dorsal skinfold chambers when compared to non-menstrual tissue fragments, indicating different vascularization modes of the two fragment types. To limit the role of chance, the experiments were conducted under standardized laboratory conditions. Statistical significance was accepted for a value of P < 0.05. N/A. Endometriotic lesions were induced by syngeneic tissue transplantation into recipient mice without the use of pathological endometriotic tissue of human nature. Therefore, the results obtained in this study may not fully relate to human patients with endometriosis. The present study significantly contributes to the characterization of common murine endometriosis models. These models represent important tools for studies focusing on the basic mechanisms of endometriosis and the development of novel therapeutic strategies for the treatment of this frequent gynecological disease. The presented findings indicate that the combination of different experimental models and approaches may be the most appropriate strategy to study the pathophysiology and drug sensitivity of a complex disease such as endometriosis under preclinical conditions. There was no specific funding of this study. The authors have no conflicts of interest to declare.

Alpha-linolenic acid regulates the gut microbiota and the inflammatory environment in a mouse model of endometriosis.

This study aims to investigate the effects of alpha-linolenic acid (ALA) on the gut microbiota (GM) and the abdominal environment in mice with endometriosis (EMS). The effects of faecal microbiota transplantation (FMT) from EMS mice on mice treated with antibiotic cocktail were conducted. The 16S rRNA sequencing and PICRUSt software were used to detect the structure and function of GM respectively. The protein levels of Claudin 4 and ZO-2 in the intestinal wall were detected using the western blotting. The level of LPS in the abdominal cavity was detected using enzyme-linked immunosorbent assay (ELISA). The content of macrophages in the abdominal cavity was detected using flow cytometry. The exogenous supplementation of ALA could restore the abundance of Firmicutes and Bacteroidota in EMS mice. After the ALA treatment, the abundance of 125 functional pathways and 50 abnormal enzymes related to GM in EMS mice was significantly improved (p<.05). The expression of the ZO-2 protein in the intestinal wall was decreased, and the level of LPS in the abdominal cavity was significantly increased after FMT from EMS mice (p<.05). ALA could increase the expression of the ZO-2 protein in the intestinal wall of EMS mice, reduce the level of LPS in the abdominal cavity (p<.05) and reduce the aggregation of peritoneal macrophages (p<.05). Alpha-linolenic acid can improve the GM, intestinal wall barrier and abdominal inflammatory environment and reduce the level of LPS in mice with EMS.

In vivo effects of AZD4547, a novel fibroblast growth factor receptor inhibitor, in a mouse model of endometriosis.

Endometriosis is a chronic disease, characterized by the growth of endometrial‐like cells outside the uterine cavity. Due to its complex pathophysiology, a totally resolving cure is yet to be found. The aim of this study was to compare the therapeutic efficacy of AZD4547, a novel fibroblast growth factor receptor inhibitor (FGFRI), with a well‐characterized progestin, etonogestrel (ENG) using a validated in vivo mouse model of endometriosis. Endometriosis was induced by transplanting uterine fragments from donor mice in proestrus into the peritoneal cavity of recipient mice, which then developed into cyst‐like lesions. AZD4547 and ENG were administered systemically either from the day of endometriosis induction or 2‐weeks post‐surgery. After 20 days of treatment, the lesions were harvested; their size and weight were measured and analyzed histologically or by qRT‐PCR. Stage of estrous cycle was monitored throughout. Compared to vehicle, AZD4547 (25 mg/kg) was most effective in counteracting lesion growth when treating from day of surgery and 2 weeks after; ENG (0.8 mg/kg) was similarly effective in reducing lesion growth but only when administered from day of surgery. Each downregulated FGFR gene expression (p < 0.05). AZD4547 at all doses and ENG (0.008 mg/kg) caused no disturbance to the estrous cycle. ENG at 0.08 and 0.8 mg/kg was associated with partial or complete estrous cycle disruption and hyperemia of the uteri. AZD4547 and ENG both attenuated endometriotic lesion size, but only AZD4547 did not disrupt the estrous cycle, suggesting that targeting of FGFR is worthy of further investigation as a novel treatment for endometriosis.

A mouse model of endometriosis that displays vaginal, colon, cutaneous, and bladder sensory comorbidities.

Endometriosis is a painful inflammatory disorder affecting ~10% of women of reproductive age. Although chronic pelvic pain (CPP) remains the main symptom of endometriosis patients, adequate treatments for CPP are lacking. Animal models that recapitulate the features and symptoms experienced by women with endometriosis are essential for investigating the etiology of endometriosis, as well as developing new treatments. In this study, we used an autologous mouse model of endometriosis to examine a combination of disease features and symptoms including: a 10week time course of endometriotic lesion development; the chronic inflammatory environment and development of neuroangiogenesis within lesions; sensory hypersensitivity and altered pain responses to vaginal, colon, bladder, and skin stimulation in conscious animals; and spontaneous animal behavior. We found significant increases in lesion size from week 6 posttransplant. Lesions displayed endometrial glands, stroma, and underwent neuroangiogenesis. Additionally, peritoneal fluid of mice with endometriosis contained known inflammatory mediators and angiogenic factors. Compared to Sham, mice with endometriosis displayed: enhanced sensitivity to pain evoked by (i) vaginal and (ii) colorectal distension, (iii) altered bladder function and increased sensitivity to cutaneous (iv) thermal and (v) mechanical stimuli. The development of endometriosis had no effect on spontaneous behavior. This study describes a comprehensive characterization of a mouse model of endometriosis, recapitulating the clinical features and symptoms experienced by women with endometriosis. Moreover, it delivers the groundwork to investigate the etiology of endometriosis and provides a platform for the development of therapeutical interventions to manage endometriosis-associated CPP.

Kiwi Root Extract Inhibits the Development of Endometriosis in Mice by Downregulating Inflammatory Factors.

Purpose To determine whether the kiwi root extract inhibits the development of endometriosis in mice by suppressing inflammatory factors. Materials and Methods The mouse model of endometriosis was induced by surgery after which the mice were continuously injected with the drug for 14 days. On the 14th day, the mice were sacrificed, and the peritoneal fluid was obtained for enzyme-linked immunosorbent assay. Endometrial ectopic tissue was weighed and analyzed by tissue immunochemistry, RT-PCR, western blotting, and gelatin zymography experiment. Results Kiwi root extract significantly reduced endometriotic lesion volume and downregulated the proinflammatory cytokines IL-6, IL-8, IL-1β, and TNF-α, as well as the angiogenic factor VEGF-A. It also inhibited the mRNA and protein expression of COX-1 and COX-2, IL-6, TGF-β1, EP2 receptor, and ER-β in endometriotic lesions but did not affect the expression of MMP-9 and MMP-2. Conclusions Kiwi root extract could significantly inhibit the growth of surgery-induced endometriosis in mice. Our results suggest that the kiwi root extract may inhibit the development and progression of ectopic endometrium through disruption of neovascularization and reducing inflammation, which may be beneficial in treating this common gynecological disease.

CD206+\u2009macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model

In endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is unclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines and chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable to deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion of CD206+ MΦ decreased the total weight of endometriotic-like lesions significantly (p < 0.05). In the endometriotic-like lesions in the DT group, a lower proliferation of endometriotic cells and the decrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFβ1, angiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, respectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell marker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number of TGFβ1 expressing MΦ was significantly reduced compared to control. These data suggest that CD206+ MΦ promotes the formation of endometriotic-like lesions by inducing angiogenesis around the lesions.

Recepteur d'origine nantais contributes to the development of endometriosis via promoting epithelial-mesenchymal transition of a endometrial epithelial cells.

Endometriosis is a benign, chronic inflammatory disease that commonly occurs in reproductive‐aged women. Epithelial‐mesenchymal transition (EMT) of endometrial epithelial cells plays an important role in the development of endometriosis. Recepteur d'origine nantais (RON), a receptor tyrosine kinase, has been reported to promote EMT and progression in tumours. However, whether and how RON mediates the EMT and endometriosis development is not known. Here, we found that RON activation could improve the migratory and invasive capabilities, change cellular morphologies, and decrease expression of E‐cadherin and increase expression of N‐cadherin in endometrial epithelial cells. Inhibition or knockdown of RON expression suppressed the migration and invasion of endometrial epithelial cells. Our studies also indicated that RON played its part in endometrial epithelial cells through protein kinase B (Akt) and mitogen‐activated protein kinase (MAPK) pathways. Treatment with a RON inhibitor could decrease the number of ectopic lesions in a mouse model of endometriosis and mediate expression of EMT markers in endometriotic lesions. These data suggest that RON contributed to endometriosis development by promoting EMT of endometrial epithelial cells. Therefore, RON may be a new therapeutic target for endometriosis.

Telocytes Enhances M1 Differentiation and Phagocytosis While Inhibits Mitochondria-Mediated Apoptosis Via Activation of NF-κB in Macrophages

Telocytes (TCs), which are a recently discovered interstitial cell type present in various organs and tissues, perform multiple biological functions and participate in extensive crosstalk with neighboring cells. Endometriosis (EMs) is a gynecological disease characterized by the presence of viable endometrial debris and impaired macrophage phagocytosis in the peritoneal environment. Here, CD34/vimentin-positive TCs were co-cultured with RAW264.7 cells in vitro. M1/M2 differentiation-related markers were detected; phagocytosis, energy metabolism, proliferation, apoptosis, and pathway mechanisms were studied; and the mitochondrial membrane potential (ΔΨm) was measured. Furthermore, in an EMs mouse model, the differentiation of macrophages in response to treatment with TC-conditioned medium (TCM) in vivo was studied. The results showed that upon in vitro co-culture with TCM, RAW264.7 cells differentiated more toward the M1 phenotype with enhancement of phagocytosis, increase in energy metabolism and proliferation owing to reduced the loss of ΔΨm, and suppression of dexamethasone-induced apoptosis. Further, along with the activation of NF-κB, Bcl-2 and Bcl-xl, the expression of Bax, cleaved-caspase9, and cleaved-caspase3 reduced in RAW264.7 cells. In addition, the M1 subtype was found to be the dominant phenotype among tissue and peritoneal macrophages in the EMs model subjected to in vivo TCM treatment. In conclusion, TCs enhanced M1 differentiation and phagocytosis while inhibiting apoptosis via the activation of NF-κB in macrophages, which potentially inhibited the onset of EMs. Our findings provide a potential research target and the scope for developing a promising therapeutic strategy for EMs.

Knockdown of lncRNA H19 suppresses endometriosis in vivo

The long noncoding RNA (lncRNA) H19 is involved in the pathogenesis of endometriosis by modulating the proliferation and invasion of ectopic endometrial cells in vitro, but related in vivo studies are rare. This study aimed to investigate the role of lncRNA H19 in a nude mouse model of endometriosis. Ectopic endometrial stromal cells (ecESCs) were isolated from ectopic endometrium of patients with endometriosis and infected with lentiviruses expressing short hairpin RNA (shRNA) negative control (LV-NC-shRNA) or lncRNA-H19 shRNA (LV-H19-shRNA). The ecESCs infected with LV-NC-shRNA and LV-H19-shRNA were subcutaneously implanted into forty 6- to 8-week-old female nude mice. The size and weight of the endometriotic implants were measured at 1, 2, 3, and 4 weeks after implantation and compared, and lncRNA H19 levels in endometriotic implants were evaluated using real-time polymerase chain reaction (RT-PCR). All nude mice survived the experimental period, and no significant differences in body weight were observed between the experimental group and the control group. All nude mice developed histologically confirmed subcutaneous endometriotic lesions with glandular structures and stroma after 1 week of implantation. The subcutaneous lesions in the LV-NC-shRNA group after 1, 2, 3, and 4 weeks of implantation were larger than those in the LV-H19-shRNA group, and lncRNA H19 levels in subcutaneous lesions in the LV-NC-shRNA group were significantly higher than those in the LV-H19-shRNA group. Knockdown of lncRNA H19 suppresses endometriosis in vivo. Further study is required to explore the underlying mechanism in the future.

Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis.

Could erastin activate ferroptosis to regress endometriotic lesions? Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis. Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis. The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs. EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipid ROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration. N/A. This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated. Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis. This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.

Lipoxin A4 inhibits the development of endometriosis in a mouse model by suppressing local estradiol synthesis

We investigated whether lipoxin A4 (LXA4) inhibits the development of endometriosis by suppressing local estradiol synthesis. An endometriosis mouse model was constructed by surgical transplantation to subcutanous tissue sites. The treatment group received daily injections of LXA4 (10 μg/Kg) for 21days after which lesions were recovered. We measured 17β-HSD1, 17β-HSD2, CYP11A1, CYP19A1, CYP17A1, and estrogen receptor mRNA expression levels using real-time RT-PCR. In addition, immunohistochemistry was performed to determine protein expression and localization. After LXA4 administration, the volume of endometrial lesions was significantly reduced. Administration of LXA4 resulted in a more rudimentary architecture with a reduced number of developed glands surrounded by a small amount of stroma. LXA4 downregulated the mRNA and protein expression levels of 17β-HSD1, CYP11A1, CYP19A1, CYP17A1, ERα, and ERβ. Furthermore, LXA4 downregulated the expression of ERβ, aromatase expression, and 17β-HSD1 enzyme activity, which affected local estradiol production, resulting in reduced endometriosis. Results from our endometriosis mouse model showed that treatment with LXA4 reduced expression of enzymes and receptors associated or implicated with estrogen-dependent regulation of extra-uterine tissue. We believe that LXA4 has a potential therapeutic value for the treatment of endometriosis.

Macrophage Immune Memory Controls Endometriosis in Mice and Humans

Endometriosis is a frequent, chronic, inflammatory gynecological disease characterized by the presence of ectopic endometrial tissue causing pain and infertility. Macrophages have a central role in lesion establishment and maintenance by driving chronic inflammation and tissue remodeling. Macrophages can be reprogrammed to acquire memory-like characteristics after antigenic challenge to reinforce or inhibit a subsequent immune response, a phenomenon termed "trained immunity." Here, whereas bacille Calmette-Guérin (BCG) training enhances the lesion growth in a mice model of endometriosis, tolerization with repeated low doses of lipopolysaccharide (LPSlow) or adoptive transfer of LPSlow-tolerized macrophages elicits a suppressor effect. LPSlow-tolerized human macrophages mitigate the fibro-inflammatory phenotype of endometriotic cells in an interleukin-10 (IL-10)-dependent manner. A history of severe Gram-negative infection is associated with reduced infertility duration and alleviated symptoms, in contrast to patients with Gram-positive infection history. Thus, the manipulation of innate immune memory may be effective in dampening hyper-inflammatory conditions, opening the way to promising therapeutic approaches.

Decreased mixed lineage leukemia 1 is involved in endometriosis-related infertility.

The aberrant histone methylation patterns contribute to the pathogenesis of endometriosis (EM). Mixed lineage leukemia 1 (MLL1), a histone methyltransferase, is crucial for gene expression by catalyzing the trimethylation of histone 3 lysine 4 (H3K4me3) in gene promoter. This study aimed to explore whether MLL1 is involved in EM-related infertility. The expressions of MLL1 and H3K4me3 were analyzed in the eutopic endometria from EM women with infertility (n = 22) and the normal endometria from EM-free women (n = 22). Mouse EM model was established. The MLL1 and H3K4me3 expression patterns in mice endometria of early pregnancy were also investigated. Immortalized human endometrial stromal cells (iESCs) were cultured and underwent in vitro decidualization. The chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) was performed to find the target gene of MLL1 during decidual process. Results showed that both MLL1 and H3K4me3 decreased in the eutopic endometrium from EM patients compared to that in the normal endometrium. During early pregnancy and the decidual process, MLL1 and H3K4me3 were significantly upregulated in stromal cells. ChIP-seq and ChIP-qPCR found that the cytochrome c oxidase subunit 4I 2 (COX4I2) was directly targeted by MLL1. The dominance of COX4I2-containing enzyme induced the expression of hypoxia-inducible factor-2α (HIF-2α), whose expression in the peri-implantation endometrium is essential for embryo implantation. Further results showed that MLL1 was directly regulated by progesterone (P4) - P4 receptors (PRs). Our study proved that MLL1 was involved in EM-related infertility, which may provide a novel approach to treat the nonreceptive endometrium in EM patients.