Identification of potent pan-ephrin receptor kinase inhibitors using DNA-encoded chemistry technology.

EPH receptors (EPHs), the largest family of tyrosine kinases, phosphorylate downstream substrates upon binding of ephrin cell surface-associated ligands. In a large cohort of endometriotic lesions from individuals with endometriosis, we found that EPHA2 and EPHA4 expressions are increased in endometriotic lesions relative to normal eutopic endometrium. Because signaling through EPHs is associated with increased cell migration and invasion, we hypothesized that chemical inhibition of EPHA2/4 could have therapeutic value. We screened DNA-encoded chemical libraries (DECL) to rapidly identify EPHA2/4 kinase inhibitors. Hit compound, CDD-2693, exhibited picomolar/nanomolar kinase activity against EPHA2 (Ki: 4.0 nM) and EPHA4 (Ki: 0.81 nM). Kinome profiling revealed that CDD-2693 bound to most EPH family and SRC family kinases. Using NanoBRET target engagement assays, CDD-2693 had nanomolar activity versus EPHA2 (IC50: 461 nM) and EPHA4 (IC50: 40 nM) but was a micromolar inhibitor of SRC, YES, and FGR. Chemical optimization produced CDD-3167, having picomolar biochemical activity toward EPHA2 (Ki: 0.13 nM) and EPHA4 (Ki: 0.38 nM) with excellent cell-based potency EPHA2 (IC50: 8.0 nM) and EPHA4 (IC50: 2.3 nM). Moreover, CDD-3167 maintained superior off-target cellular selectivity. In 12Z endometriotic epithelial cells, CDD-2693 and CDD-3167 significantly decreased EFNA5 (ligand) induced phosphorylation of EPHA2/4, decreased 12Z cell viability, and decreased IL-1ß-mediated expression of prostaglandin synthase 2 (PTGS2). CDD-2693 and CDD-3167 decreased expansion of primary endometrial epithelial organoids from patients with endometriosis and decreased Ewing's sarcoma viability. Thus, using DECL, we identified potent pan-EPH inhibitors that show specificity and activity in cellular models of endometriosis and cancer.

CircFOXO3 mediates hypoxia-induced autophagy of endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disease that shares some common features of malignancy. Autophagy plays vital roles in endometriosis and influences endometrial cell metastasis, and hypoxia was identified as the initiator of this pathological process through hypoxia inducible factor 1 alpha (HIF-1α). A newly discovered circular RNA FOXO3 (circFOXO3) is critical in cell autophagy, migration, and invasion of various diseases and is reported to be related to hypoxia, although its role in endometriosis remains to be elucidated up to now. In this study, a lower circFOXO3 expression in ectopic endometrium was investigated. Furthermore, we verified that circFOXO3 could regulate autophagy by downregulating the level of p53 protein to mediate the migration and invasion of human endometrial stromal cells (T HESCs). Additionally, the effects of HIF-1α on circFOXO3 and autophagy were examined in T HESCs. Notably, overexpression of HIF-1α could induce autophagy and inhibit circFOXO3 expression, whereas overexpressing of circFOXO3 under hypoxia significantly inhibited hypoxia-induced autophagy. Mechanistically, the direct combination between HIF-1α and HIF-1α-binding site on adenosine deaminase 1 acting on RNA (ADAR1) promoter increased the level of ADAR1 protein, which bind directly with circFOXO3 pre-mRNA to block the cyclization of circFOXO3. All these results support that hypoxia-mediated ADAR1 elevation inhibited the expression of circFOXO3, and then autophagy was induced upon loss of circFOXO3 via inhibition of p53 degradation, participating in the development of endometriosis.

Long noncoding RNA BMPR1B-AS1 stability regulated by IGF2BP2 affects the decidualization in endometriosis patients through the SMAD1/5/9 pathway.

Endometriosis (EMs)-related infertility commonly has decreased endometrial receptivity and normal decidualization is the basis for establishing and maintaining endometrial receptivity. However, the potential molecular regulatory mechanisms of impaired endometrial decidualization in patients with EMs have not been fully clarified. We confirmed the existence of reduced endometrial receptivity in patients with EMs by scanning electron microscopy and quantitative real-time PCR. Here we identified an lncRNA, named BMPR1B-AS1, which is significantly downregulated in eutopic endometrium in EMs patients and plays an essential role in decidual formation. Furthermore, RNA pull-down, mass spectrometry, RNA immunoprecipitation, and rescue analyses revealed that BMPR1B-AS1 positively regulates decidual formation through interaction with the RNA-binding protein insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). Downregulation of IGF2BP2 led to a decreased stability of BMPR1B-AS1 and inhibition of activation of the SMAD1/5/9 pathway, an inhibitory effect which diminished decidualization in human endometrial stromal cells (hESCs) decidualization. In conclusion, our identified a novel regulatory mechanism in which the IGF2BP2-BMPR1B-AS1-SMAD1/5/9 axis plays a key role in the regulation of decidualization, providing insights into the potential link between abnormal decidualization and infertility in patients with EMs, which will be of clinical significance for the management and treatment of infertility in patients with EMs.

NEK2 promotes the development of ovarian endometriosis and impairs decidualization by phosphorylating FOXO1.

Ovarian endometriosis is a common gynecological disease, and one of its most significant symptoms is infertility. In patients with endometriosis, defects in endometrial decidualization lead to impaired endometrial receptivity and embryo implantation, thus affecting early pregnancy and women's desire to have children. However, the mechanisms underlying the development of endometriosis and its associated defective decidualization are unclear. We find that NEK2 expression is increased in the ectopic and eutopic endometrium of patients with endometriosis. Meanwhile, NEK2 interacts with FOXO1 and phosphorylates FOXO1 at Ser184, inhibiting the stability of the FOXO1 protein. Importantly, NEK2-mediated phosphorylation of FOXO1 at Ser184 promotes cell proliferation, migration, invasion and impairs decidualization. Furthermore, INH1, an inhibitor of NEK2, inhibits the growth of ectopic lesions in mouse models of endometriosis and promotes endometrial decidualization in mouse models of artificially induced decidualization. Taken together, these findings indicate that NEK2 regulates the development of endometriosis and associated disorders of decidualization through the phosphorylation of FOXO1, providing a new therapeutic target for its treatment.

Transcriptome-wide N6-methyladenosine (m6A) methylation profiling of long non-coding RNAs in ovarian endometriosis.

N6-methyladenosine (m6A) methylation is the most prevalent internal epigenetic posttranscriptional mechanism for regulating mammalian RNA. Despite recent advances in determining the biological functions of m6A methylation, its association with the pathology of ovarian endometriosis remains uncertain. Herein, we performed m6A transcriptome-wide profiling to identify key lncRNAs with m6A modification involved in ovarian endometriosis development by bioinformatics analysis. We found the total m6A level was lower in ovarian endometriosis than in normal endometrium samples, with 9663 m6A peaks associated with 8989 lncRNAs detected in ovarian endometriosis and 9902 m6A peaks associated with 9210 lncRNAs detected in normal endometrium samples. These m6A peaks were primarily enriched within AAACU motifs. Functional enrichment analysis indicated that pathways involving the regulation of adhesion and development were significantly enriched in these differentially methylated lncRNAs. The regulatory relationships among lncRNAs, microRNAs (miRNAs), and mRNAs were identified by competing endogenous RNA (ceRNA) analysis and determination of the network regulating lncRNA-mRNA expression. Several specific lncRNA, including LINC00665, LINC00937, FZD10-AS1, DIO3OS and GATA2-AS1 which were differently expressed and modified by m6A, were validated using qRT-PCR and its interaction with infiltrating immune cells was explored. Furthermore, we found LncRNA DIO3OS promotes the invasion and migration of Human endometrial stromal cells (THESCs) and ALKBH5 regulates the expression of the lncRNA DIO3OS through m6A modification in vitro. Our study firstly revealed the transcriptome-wide map of m6A modification in lncRNAs of ovarian endometriosis. These findings may enable the determination of the underlying mechanism governing the pathogenesis of ovarian endometriosis and provide theoretical basis for further deeper research on the role of m6A in the development of ovarian endometriosis.

Vaginal extracellular vesicles impair fertility in endometriosis by favoring Th17/Treg imbalance and inhibiting sperm activity.

Extracellular vesicles (EVs) play a key role in various diseases. However, their effect on endometriosis (EMs)-associated infertility is poorly understood. We co-cultured EVs from the female vaginal secretions with human sperm and also generated a mouse model of EMs by allogenic transplant to explore the effect of EVs on fertility. EVs from individuals with EMs-associated infertility (E-EVs) significantly inhibited the total motility (26.46% vs. 47.1%), progressive motility (18.78% vs. 41.06%), linear velocity (21.98 vs. 41.91 µm/s) and the acrosome reaction (AR) rate (5% vs. 22.3%) of human sperm in contrast to the control group (PBS). Furthermore, E-EVs dose-dependently decreased the intracellular Ca2+ ([Ca2+]i), a pivotal regulator of sperm function. Conversely, healthy women (H-EVs) increased human sperm motion parameters, the AR rate, and sperm [Ca2+]i. Importantly, the mouse model of EMs confirmed that E-EVs further decreased the conception rate and the mean number of embryo implantations (7.6 ± 3.06 vs. 4.5 ± 3.21) compared with the control mice by inducing the production of inflammatory cytokines leading to a Th17/Treg imbalance. H-EVs could restore impaired fertility by restoring the Th17/Treg balance. We determined the impact of EVs derived from the female genital tract on human sperm function and studied the possible mechanisms by which it affects fertility. Our findings provide a novel rationale to ameliorate EMs-associated infertility.

m6A methylation of RNF43 inhibits the progression of endometriosis through regulating oxidative phosphorylation via NDUFS1.

Oxidative phosphorylation is becoming increasingly important in the induction and development of endometriosis. Recently, it has been reported that ring finger protein 43 (RNF43) is involved in the process of oxidative phosphorylation, but the mechanism remains unclear. Our investigation is to delve into the roles of RNF43 in endometriosis and elucidate the related mechanisms. We found RNF43 was downregulated in ectopic endometrial tissue and primary ectopic endometrial stromal cells (ECESCs). Knockdown of RNF43 enhanced cell viability and migration by activating oxidative phosphorylation in eutopic endometrial stromal cells (EUESCs), while overexpression of RNF43 led to the opposite results. Moreover, RNF43 reinforced the ubiquitination and degradation of NADH dehydrogenase Fe-S protein 1 (NDUFS1) by interacting with it. Likewise to RNF43 overexpression, NDUFS1 silencing inhibited cell viability, migration, and oxidative phosphorylation in ECESCs. NDUFS1 was a downstream target of RNF43, mediating its biological role in endometriosis. Interestingly, the expression and stability of RNF43 mRNA were regulated by the Methyltransferase-like 3 (METTL3)/IGF2BP2 m6A modification axis. The results of rat experiments showed decreased RNF43 expression and increased NDUFS1 expression in endometriosis rats, which was enhanced by METTL3 inhibition. Those observations indicated that m6A methylation-mediated RNF43 negatively affects viability and migration of endometrial stromal cells through regulating oxidative phosphorylation via NDUFS1. The discovery of METTL3/RNF43/NDUFS1 axis suggested promising therapeutic targets for endometriosis.

The dysfunction of CD8+ T cells triggered by endometriotic stromal cells promotes the immune survival of endometriosis.

Endometriosis is defined as an oestrogen-dependent and inflammatory gynaecological disease of which the pathogenesis remains unclear. This study aimed to investigate the cellular heterogeneity and reveal the effect of CD8+ T cells on the progress of endometriosis. Three ovarian endometriosis patients were collected, and single-cell RNA sequencing (scRNA-seq) progressed and delineated the cellular landscape of endometriosis containing five cell clusters. The endometrial cells (EMCs) were the major component, of which the mesenchymal cells were preponderant and characterized with increased inflammation and oestrogen synthesis in endometriosis. The proportion of T cells, mainly CD8+ T cells rather than CD4+, was reduced in endometriotic lesions, and the cytokines and cytotoxicity of ectopic T cells were depressed. CD8+ T cells depressed the proliferation of ESCs through inhibiting CDK1/CCNB1 pathway to arrest the cell cycle and triggered inflammation through activating STAT1 pathway. Correspondingly, the coculture with ESCs resulted in the dysfunction of CD8+ T cells through upregulating STAT1/PDCD1 pathway and glycolysis-promoted metabolism reprogramming. The endometriotic lesions were larger in nude mouse models with T-cell deficiency than the normal mouse models. The inhibition of T cells via CD90.2 or CD8A antibody increased the endometriotic lesions in mouse models, and the supplement of T cells to nude mouse models diminished the lesion sizes. In conclusion, this study revealed the global cellular variation of endometriosis among which the cellular count and physiology of EMCs and T cells were significantly changed. The depressed cytotoxicity and aberrant metabolism of CD8+ T cells were induced by ESCs with the activation of STAT1/PDCD1 pathway resulting in immune survival to promote endometriosis.

Causal effects of endometriosis on cancer risk: A Mendelian randomization study.

Endometriosis has been reported in epidemiological studies to be associated with certain types of cancer. However, the presence of reverse causality and residual confounding due to common risk factors introduces uncertainty regarding the extent to which endometriosis itself contributes to the development of cancer. We performed the Mendelian randomization (MR) to investigate the causal associations between endometriosis and 34 different types of cancers. The results of the inverse-variance-weighted (IVW) model suggested that genetic predisposition to endometriosis was causally associated with an increased risk for ovarian cancer (OR = 3.2913; p-value = .0320). The genetic liabilities to endometriosis had causal associations with the decreased risk for skin cancer (OR = 0.9973; p-value = .0219), hematological cancer (OR = 0.9953; p-value = .0175) and ER- breast cancer (OR = 0.6960; p-value = .0381). The causal association of the above combinations were robust by test of heterogeneity and pleiotropy. Together, our study suggests that endometriosis had causal effect on cancer risk.

Association between KRAS and PIK3CA Mutations and Progesterone Resistance in Endometriotic Epithelial Cell Line.

Although endometriosis is a benign disease, it is associated with cancer-related gene mutations, such as KRAS or PIK3CA. Endometriosis is associated with elevated levels of inflammatory factors that cause severe pain. In a previous study, we demonstrated that KRAS or PIK3CA mutations are associated with the activation of cell proliferation, migration, and invasion in a patient-derived immortalized endometriotic cell line, HMOsisEC10. In this study, we investigated the effects of these mutations on progesterone resistance. Since the HMOsisEC10 had suppressed progesterone receptor (PR) expression, we transduced PR-B to HMOsisEc10 cell lines including KRAS mutant and PIK3CA mutant cell lines. We conducted a migration assay, invasion assay, and MTT assay using dienogest and medroxyprogestrone acetate. All cell lines showed progesterone sensitivity with or without mutations. Regarding inflammatory factors, real-time quantitative RT-PCR revealed that the KRAS mutation cell line exhibited no suppression of Cox-2 and mPGES-1 on progesterone treatment, whereas IL-6, MCP-1, VEGF, and CYP19A1 were significantly suppressed by progesterone in both mutated cell lines. Our results suggest that KRAS mutation and PIK3CA mutation in endometriotic cells may not be associated with progesterone resistance in terms of aggressiveness. However, KRAS mutations may be associated with progesterone resistance in the context of pain.

The independent and joint associations of hysterectomy and uterine fibroids or endometriosis with ovarian cancer incidence: results from a US-based cohort.

Uterine fibroids and endometriosis may be associated with an increased risk of ovarian cancer. Less is known about the role of hysterectomy in these associations. We estimated the independent and joint associations of hysterectomy, fibroids, and endometriosis with ovarian cancer incidence in the prospective Sister Study cohort (2003-2009). We used time-varying Cox proportional hazards models to estimate covariate-adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). By the end of follow-up, 34% of 40,928 eligible participants had fibroids, 13% had endometriosis, and 7% had both. A total of 274 women developed ovarian cancer during follow-up (median=12.3 years). In mutually adjusted models, fibroids (HR=1.65, 95% CI: 1.28, 2.12) and possibly endometriosis (HR=1.16, 95% CI: 0.82, 1.63), were positively associated with ovarian cancer. Hysterectomies (20% of participants) were also positively associated with ovarian cancer (HR=1.29, 95% CI: 0.95, 1.74). There was some evidence that hysterectomies may mitigate ovarian cancer risk among women with fibroids (HR=0.83, 95% CI: 0.56, 1.24), but not among women with endometriosis (HR=1.20, 95% CI: 0.65, 2.22). Identifying these joint associations adds to our understanding of ovarian cancer etiology and may help inform decisions about how women with fibroids, endometriosis, and hysterectomies are treated and surveilled for ovarian cancer.

Transcription factor EB-mediated autophagy affects cell migration and inhibits apoptosis to promote endometriosis.

Autophagy has emerged as an important process of cell metabolism. With continuous in-depth research on autophagy, TFEB has been a key transcription factor regulating autophagy levels in recent years. Studies have established that TFEB regulates autophagy and apoptosis in various diseases. However, the relationship between TFEB and the pathogenesis of endometriosis remains unclear. This study aimed to investigate the effect of TFEB on the mechanism of endometriosis progression. The results showed that TFEB and autophagy-related protein LC3 are highly expressed in ectopic endometrium of patients with endometriosis, overexpression of TFEB in cultured human endometrial stromal cells (HESCs) by lentivirus not only promoted autophagy but also inhibited apoptosis. In addition, the migration and invasion ability of HESCs were enhanced by TFEB overexpression. Furthermore, inhibiting autophagy with specific inhibitors can attenuate migration and invasion of HESCs induced by TFEB. The rat models of endometriosis show that TFEB knockdown can suppress lesion growth in vivo. Our results suggest that autophagy may be involved in the progression mechanism of endometriosis, and the mechanism of autophagy disorder in endometriosis is probably related to TFEB. TFEB may be a key molecule in promoting endometriosis.

IGF2BP3 promotes glutamine metabolism of endometriosis by interacting with UCA1 to enhances the mRNA stability of GLS1.

BACKGROUND: Insulin like growth factor II mRNA binding protein 3 (IGF2BP3) has been implicated in numerous inflammatory and cancerous conditions. However, its precise molecular mechanisms in endometriosis (EMs) remains unclear. The aim of this study is to examine the influence of IGF2BP3 on the occurrence and progression of EMs and to elucidate its underlying molecular mechanism. METHODS: Efects of IGF2BP3 on endometriosis were confrmed in vitro and in vivo. Based on bioinformatics analysis, RNA immunoprecipitation (RIP), RNA pull-down assays and Fluorescent in situ hybridization (FISH) were used to show the association between IGF2BP3 and UCA1. Single-cell spatial transcriptomics analysis shows the expression distribution of glutaminase 1 (GLS1) mRNA in EMs. Study the effect on glutamine metabolism after ectopic endometriotic stromal cells (eESCs) were transfected with Sh-IGF2BP3 and Sh-UCA1 lentivirus. RESULTS: Immunohistochemical staining have revealed that IGF2BP3 was upregulated in ectopic endometriotic lesions (EC) compared to normal endometrial tissues (EN). The proliferation and migration ability of eESCs were greatly reduced by downregulating IGF2BP3. Additionally, IGF2BP3 has been observed to interact with urothelial carcinoma associated 1 (UCA1), leading to increased stability of GLS1 mRNA and subsequently enhancing glutamine metabolism. Results also demonstrated that IGF2BP3 directly interacts with the 3' UTR region of GLS1 mRNA, influencing its expression and stability. Furthermore, UCA1 was able to bind with c-MYC protein, stabilizing c-MYC mRNA and consequently enhancing GLS1 expression through transcriptional promotion. CONCLUSION: These discoveries underscored the critical involvement of IGF2BP3 in the elevation and stability of GLS1 mRNA in the context of glutamine metabolism by interacting with UCA1 in EMs. The implications of our study extended to the identification of possible therapeutic targets for individuals with EMs.

Resveratrol protected against the development of endometriosis by promoting ferroptosis through miR-21-3p/p53/SLC7A11 signaling pathway.

Resveratrol is involved in regulating ferroptosis, but its role in Endometriosis (EMS) is not clear. In this study, we aim to investigate the effect of ferroptosis and resveratrol intervention in the pathogenesis of EMS cyst. Cell proliferation, migration, and oxidative stress level were analyzed. The interaction of miR-21-3p and p53 was analyzed by dual luciferase assay. The interaction between p53 and SLC7A11 were analyzed by chromatin immunoprecipitation (CHIP). The miR-21-3p, GPX4, ACSL4, FTH1, p53, SLC7A11, Ptgs2 and Chac1 expression were analyzed by RT-qPCR or Western blot. The Fe3+ deposition and miR-21-3p, GPX4, FTH1 and SLC7A11 expressions were increased, and ACSL4, p53, Ptgs2 and Chac1 expression were decreased in EMS patients. Resveratrol inhibited migration, induced Ptgs2 and Chac1 expression in EESCs. Overexpression of miR-21-3p inhibited p53, Ptgs2 and Chac1 expression, and promoted SLC7A11 expression, which was reversed by resveratrol. miR-21-3p bound to p53, which interacted with SLC7A11. Resveratrol promoted Ptgs2 and Chac1 expression in the sh-p53 EESCs. Resveratrol reduced miR-21-3p and SLC7A11 expressions, and increased p53, Ptgs2 and Chac1 expressions, and Fe3+ deposition in the lesion tissues of EMS mice, which were reversed by miR-21-3p mimics. Resveratrol activated p53/SLC7A11 pathway by down-regulating miR-21-3p to promote ferroptosis and prevent the development of EMS.

Is there an association between endometriosis and subsequent breast cancer? A retrospective cohort study from Germany.

PURPOSE: Given the relatively high incidence of both endometriosis and breast cancer, investigating the potential connection between these gynecological diseases is of substantial clinical significance. However, there is no clear consensus in the literature on the extent to which the risk of breast cancer is increased in patients with endometriosis. Therefore, we conducted a large-scale observational study investigating the association between endometriosis and breast cancer risk. METHODS: This study included women aged ≥ 18 years with an initial endometriosis diagnosis from one of 315 office-based gynecologists in Germany between January 2005 and December 2021. Non-endometriosis patients were matched 1:1 to patients with endometriosis based on age, index year, average yearly consultation frequency, and predefined co-diagnoses within 12 months before or on the index date, including obesity and benign breast disorders. The association between endometriosis and the 10-year incidence of breast cancer was studied using Kaplan-Meier curves and log-rank tests. Finally, a univariable Cox regression analysis was conducted to assess the association between endometriosis and breast cancer. RESULTS: Over a follow-up period of up to 10 years, no significant difference was observed between the endometriosis (2.4%) and the matched non-endometriosis group (2.5%) with regard to breast cancer diagnoses. Furthermore, the regression analysis revealed no significant association between endometriosis and subsequent breast cancer. CONCLUSION: In summary, our comprehensive 10-year study involving a substantial sample of women indicates that endometriosis is not significantly associated with an increased risk of subsequent breast cancer.

Predicting disease recurrence in patients with endometriosis: an observational study.

BACKGROUND: Despite surgical and pharmacological interventions, endometriosis can recur. Reliable information regarding risk of recurrence following a first diagnosis is scant. The aim of this study was to examine clinical and survey data in the setting of disease recurrence to identify predictors of risk of endometriosis recurrence. METHODS: This observational study reviewed data from 794 patients having surgery for pelvic pain or endometriosis. Patients were stratified into two analytic groups based on self-reported or surgically confirmed recurrent endometriosis. Statistical analyses included univariate, followed by multivariate logistic regression to identify risk factors of recurrence, with least absolute shrinkage and selection operator (Lasso) regularisation. Risk-calibrated Supersparse Linear Integer Models (RiskSLIM) and survival analyses (with Lasso) were undertaken to identify predictive features of recurrence. RESULTS: Several significant features were repeatedly identified in association with recurrence, including adhesions, high rASRM score, deep disease, bowel lesions, adenomyosis, emergency room attendance for pelvic pain, younger age at menarche, higher gravidity, high blood pressure and older age. In the surgically confirmed group, with a score of 5, the RiskSLIM method was able to predict the risk of recurrence (compared to a single diagnosis) at 95.3% and included adenomyosis and adhesions in the model. Survival analysis further highlighted bowel lesions, adhesions and adenomyosis. CONCLUSIONS: Following an initial diagnosis of endometriosis, clinical decision-making regarding disease management should take into consideration the presence of bowel lesions, adhesions and adenomyosis, which increase the risk of endometriosis recurrence.

Gut microbiome in endometriosis: a cohort study on 1000 individuals.

BACKGROUND: Endometriosis, defined as the presence of endometrial-like tissue outside of the uterus, is one of the most prevalent gynecological disorders. Although different theories have been proposed, its pathogenesis is not clear. Novel studies indicate that the gut microbiome may be involved in the etiology of endometriosis; nevertheless, the connection between microbes, their dysbiosis, and the development of endometriosis is understudied. This case-control study analyzed the gut microbiome in women with and without endometriosis to identify microbial targets involved in the disease. METHODS: A subsample of 1000 women from the Estonian Microbiome cohort, including 136 women with endometriosis and 864 control women, was analyzed. Microbial composition was determined by shotgun metagenomics and microbial functional pathways were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Partitioning Around Medoids (PAM) algorithm was performed to cluster the microbial profile of the Estonian population. The alpha- and beta-diversity and differential abundance analyses were performed to assess the gut microbiome (species and KEGG orthologies (KO)) in both groups. Metagenomic reads were mapped to estrobolome-related enzymes' sequences to study potential microbiome-estrogen metabolism axis alterations in endometriosis. RESULTS: Diversity analyses did not detect significant differences between women with and without endometriosis (alpha-diversity: all p-values > 0.05; beta-diversity: PERMANOVA, both R 2 < 0.0007, p-values > 0.05). No differential species or pathways were detected after multiple testing adjustment (all FDR p-values > 0.05). Sensitivity analysis excluding women at menopause (> 50 years) confirmed our results. Estrobolome-associated enzymes' sequence reads were not significantly different between groups (all FDR p-values > 0.05). CONCLUSIONS: Our findings do not provide enough evidence to support the existence of a gut microbiome-dependent mechanism directly implicated in the pathogenesis of endometriosis. To the best of our knowledge, this is the largest metagenome study on endometriosis conducted to date.

Vaginal and rectal microbiome contribute to genital inflammation in chronic pelvic pain.

BACKGROUND: Chronic pelvic pain (CPP) is a multifactorial syndrome that can substantially affect a patient's quality of life. Endometriosis is one cause of CPP, and alterations of the immune and microbiome profiles have been observed in patients with endometriosis. The objective of this pilot study was to investigate differences in the vaginal and gastrointestinal microbiomes and cervicovaginal immune microenvironment in patients with CPP and endometriosis diagnosis compared to those with CPP without endometriosis and no CPP. METHODS: Vaginal swabs, rectal swabs, and cervicovaginal lavages (CVL) were collected among individuals undergoing gynecologic laparoscopy. Participants were grouped based on patients seeking care for chronic pain and/or pathology results: CPP and endometriosis (CPP-Endo) (n = 35), CPP without endometriosis (n = 23), or patients without CPP or endometriosis (controls) (n = 15). Sensitivity analyses were performed on CPP with endometriosis location, stage, and co-occurring gynecologic conditions (abnormal uterine bleeding, fibroids). 16S rRNA sequencing was performed to profile the microbiome, and a panel of soluble immune mediators was quantified using a multiplex assay. Statistical analysis was conducted with SAS, R, MicrobiomeAnalyst, MetaboAnalyst, and QIIME 2. RESULTS: Significant differences were observed between participants with CPP alone, CPP-Endo, and surgical controls for body mass index, ethnicity, diagnosis of ovarian cysts, and diagnosis of fibroids. In rectal microbiome analysis, both CPP alone and CPP-Endo exhibited lower alpha diversity than controls, and both CPP groups revealed enrichment of irritable bowel syndrome-associated bacteria. CPP-Endo exhibited an increased abundance of vaginal Streptococcus anginosus and rectal Ruminococcus. Patients with CPP and endometrioma (s) demonstrated increased vaginal Streptococcus, Lactobacillus, and Prevotella compared to other endometriosis sites. Further, abnormal uterine bleeding was associated with an increased abundance of bacterial vaginosis-associated bacteria. Immunoproteomic profiles were distinctly clustered by CPP alone and CPP-Endo compared to controls. CPP-Endo was enriched in TNF⍺, MDC, and IL-1⍺. CONCLUSIONS: Vaginal and rectal microbiomes were observed to differ between patients with CPP alone and CPP with endometriosis, which may be useful in personalized treatment for individuals with CPP and endometriosis from those with other causes of CPP. Further investigation is warranted in patients with additional co-occurring conditions, such as AUB/fibroids, which add additional complexity to these conditions and reveal the enrichment of distinct pathogenic bacteria in both mucosal sites. This study provides foundational microbiome-immunoproteomic knowledge related to chronic pelvic pain, endometriosis, and co-occurring gynecologic conditions that can help improve the treatment of patients seeking care for pain.

Paradoxical role of phosphorylated STAT3 in normal fertility and the pathogenesis of adenomyosis and endometriosis†.

Signal transducer and activator of transcription 3 (STAT3), when phosphorylated at tyrosine 705, plays an important role in endometrial stromal cell decidualization and the receptivity of the endometrial epithelium during embryo implantation. However, the function of phosphorylated STAT3 (p-STAT3) in normal uterine receptivity is distinct from that in adenomyosis and endometriosis. In normal pregnancy, STAT3 phosphorylation in the endometrial epithelium determines the success of embryo implantation by regulating uterine receptivity. Additionally, p-STAT3 promotes cellular proliferation and differentiation during endometrial decidualization, which is crucial for embryonic development. In contrast, excessive STAT3 phosphorylation occurs in adenomyosis and endometriosis, which may lead to disease progression. Therefore, achieving a delicate balance in STAT3 activation is crucial. This review aimed to focus on the current understanding and knowledge gaps regarding the control of p-STAT3 activity in normal and pathological endometrial processes. This topic is important because precise control of p-STAT3 production could alleviate the symptoms of adenomyosis and endometriosis, improve endometrial receptivity, and potentially mitigate infertility without compromising normal fertility processes.

Targeted nanoparticles for imaging and therapy of endometriosis†.

In this brief review, we discuss our efforts to validate nanoplatforms for imaging and treatment of endometriosis. We specifically highlight our use of nonhuman primates and primate tissues in this effort. Endometriosis is a painful disorder of women and nonhuman primates where endometrium-like tissue exists outside of the uterus. There are no reliable, specific, and noninvasive diagnostic tests for endometriosis. Laparoscopic imaging remains the gold standard for identifying small endometriotic lesions in both women and monkeys. Visualizing and surgically removing microscopic lesions remains a clinical challenge. To address this challenge, we have created nanoparticle reagents that, when administered intravenously, enter endometriotic lesions both passively and by targeting endometriotic cells. The particles can carry payloads, including near-infrared fluorescent dyes and magnetic nanoparticles. These agents can be used for imaging and thermal ablation of diseased tissues. We evaluated this approach on macaque endometriotic cells, human and macaque endometrium engrafted into immunodeficient mice, in endometrium subcutaneously autografted in macaques, and in rhesus monkeys with spontaneous endometriosis. Employing these models, we report that nanoplatform-based reagents can improve imaging and provide thermal ablation of endometriotic tissues.