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Endometriosis is a common, inflammatory pain disorder comprised of disease in the pelvis and abnormal uterine lining and ovarian function that affects ∼200 million women of reproductive age worldwide and up to 50% of those with pelvic pain and/or infertility. Existing medical treatments for endometriosis-related pain are often ineffective, with individuals experiencing minimal or transient pain relief or intolerable side effects limiting long-term use - thus underscoring the pressing need for new drug treatment strategies. In this study, we applied a computational drug repurposing pipeline to endometrial gene expression data in the setting of endometriosis and controls in an unstratified manner as well as stratified by disease stage and menstrual cycle phase in order to identify potential therapeutics from existing drugs, based on expression reversal. Out of the 3,131 unique genes differentially expressed by at least one of six endometriosis signatures, only 308, or 9.8%, were in common. Similarities were more pronounced when looking at therapeutic predictions: 221 out of 299 drugs identified across the six signatures, or 73.9%, were shared, and the majority of predicted compounds were concordant across disease stage-stratified and cycle phase-stratified signatures. Our pipeline returned many known treatments as well as novel candidates. We selected the NSAID fenoprofen, the top therapeutic candidate for the unstratified signature and among the top-ranked drugs for the stratified signatures, for further investigation. Our drug target network analysis shows that fenoprofen targets PPARG and PPARA which affect the growth of endometrial tissue, as well as PTGS2 (i.e., COX2), an enzyme induced by inflammation with significantly increased gene expression demonstrated in patients with endometriosis who experience severe dysmenorrhea. NSAIDs are widely prescribed for endometriosis-related dysmenorrhea and nonmenstrual pelvic pain. Our analysis of clinical records across University of California healthcare systems revealed that while NSAIDs have been commonly prescribed to the 61,306 patients identified with diagnoses of endometriosis, dysmenorrhea, or chronic pelvic pain (36,543, 59.61%), fenoprofen was infrequently prescribed to those with these conditions (5, 0.008%). We tested the effect of fenoprofen in an established rat model of endometriosis and determined that it successfully alleviated endometriosis-associated vaginal hyperalgesia, a surrogate marker for endometriosis-related pain. These findings validate fenoprofen as a potential endometriosis therapeutic and suggest the utility of future investigation into additional drug targets identified.

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Identifying biologic and genetic effects on DNA methylation (DNAm) in endometrium increases understanding of mechanisms that influence gene regulation predisposing to endometriosis and offers an opportunity for novel therapeutic target discovery. Herein, we characterize variation in endometrial DNAm and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genome-wide genotype data and methylation at 759,345 DNAm sites in endometrial samples from 984 deeply-phenotyped participants. We identify significant differences in DNAm profiles between menstrual cycle phases and at four DNAm sites between stage III/IV endometriosis and controls. We estimate that 15.4% of the variation in endometriosis is captured by DNAm, and identify DNAm networks associated with endometriosis. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independentcis-mQTL including some tissue-specific effects. We find significant differences in DNAm profiles between endometriosis sub- phenotypes and a significant association between genetic regulation of methylation in endometrium and disease risk, providing functional evidence for genomic targets contributing to endometriosis risk and pathogenesis.

Research questionAdenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis?DesignTranscriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples.ResultsTop biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis.ConclusionsOverall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

PURPOSE: To combine different independent endometrial markers to classify the presence of endometriosis. METHODS: Endometrial biopsies were obtained from 109 women with endometriosis as well as 110 control women. Nine candidate biomarkers independent of cycle phase were selected from the literature and NanoString was performed. We compared differentially expressed genes between groups and generated generalized linear models to find a classifier for the disease. RESULTS: Generalized linear models correctly detected 68% of women with endometriosis (combining deep infiltrating and ovarian endometriosis). However, we were not able to distinguish between individual types of endometriosis compared to controls. From the 9 tested genes, FOS, MMP7, and MMP11 seem to be important for disease classification, and FOS was the most over-expressed gene in endometriosis. CONCLUSION(S): Although generalized linear models may allow identification of endometriosis, we did not obtain perfect classification with the selected gene candidates. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01965-6) contains supplementary material, which is available to authorized users.

Objective To phenotype transcriptomically M1 macrophages (Mϕ1) and M2 macrophages (Mϕ2) in the endometrium of women with endometriosis. Design Prospective experimental study. Setting University research laboratory. Patient(s) Six women with endometriosis and five controls without disease, in the secretory phase of the menstrual cycle. Intervention(s) Mϕ1, Mϕ2, uterine natural killer, and T regulatory cells were isolated from human endometrium using a uniquely designed cell-specific fluorescence activating cell sorting panel. Transcriptome profiles were assessed by RNA high sequencing, bioinformatics, and biological pathway analyses. Main Outcomes Measure(s) Differential gene expression between Mϕ1 and Mϕ2 in women with and without endometriosis and in Mϕ1 versus Mϕ2 in each group was determined and involved different biologic and signaling pathways. Result(s) Flow cytometry analysis showed no significant differences in total numbers of leukocytes between control and endometriosis groups, although Mϕ1 were higher in the endometriosis group versus controls. Statistical transcriptomic analysis was performed only in Mϕ1 and Mϕ2 populations due to larger sample sizes. Bioinformatic analyses revealed that in women with endometriosis, endometrial Mϕ1 are more proinflammatory than controls and that Mϕ2 paradoxically have a proinflammatory phenotype. Conclusion(s) As Mϕ are phenotypically plastic and their polarization state depends on their microenvironment, the altered endometrial environment in women with endometriosis may promote endometrial Mϕ2 polarization and an Mϕ1 proinflammatory phenotype. Moreover, aberrant phenotypes of Mϕ may contribute to abnormal gene expression of the eutopic endometrium and a proinflammatory environment in women with endometriosis relevant to the pathophysiology of the disease and compromised reproductive outcomes.

The uterine lining (endometrium) exhibits a pro-inflammatory phenotype in women with endometriosis, resulting in pain, infertility, and poor pregnancy outcomes. The full complement of cell types contributing to this phenotype has yet to be identified, as most studies have focused on bulk tissue or select cell populations. Herein, through integrating whole-tissue deconvolution and single cell RNAseq, we comprehensively characterized immune and nonimmune cell types in endometrium of women with or without disease and their dynamic changes across the menstrual cycle. We designed metrics to evaluate specificity of deconvolution signatures that resulted in single cell identification of 13 novel signatures for immune cell subtypes in healthy endometrium. Guided by statistical metrics, we identified contributions of endometrial epithelial, endothelial, plasmacytoid dendritic cells, classical dendritic cells, monocytes, macrophages, and granulocytes to the endometrial pro-inflammatory phenotype, underscoring roles for nonimmune as well as immune cells to the dysfunctionality of this tissue.Teaser SentenceCell type deconvolution and single cell RNAseq analysis identify altered endometrial cellular compositions in women with endometriosis

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

BACKGROUNDEndometriosis, a common oestrogen-dependent inflammatory disorder in women of reproductive age, is characterized by endometrial-like tissue outside its normal location in the uterus, which causes pelvic scarring, pain and infertility. While its pathogenesis is poorly understood, the immune system (systemically and locally in endometrium, pelvic endometriotic lesions and peritoneal fluid) is believed to play a central role in its aetiology, pathophysiology and associated morbidities of pain, infertility and poor pregnancy outcomes. However, immune cell populations within the endometrium of women with the disease have had incomplete phenotyping, thereby limiting insight into their roles in this disorder.OBJECTIVE AND RATIONALEThe objective herein was to determine reproducible and consistent findings regarding specific immune cell populations and their abundance, steroid hormone responsiveness, functionality, activation states, and markers, locally and systemically in women with and without endometriosis.SEARCH METHODSA comprehensive English language PubMed, Medline and Google Scholar search was conducted with key search terms that included endometriosis, inflammation, human eutopic/ectopic endometrium, immune cells, immune population, immune system, macrophages, dendritic cells (DC), natural killer cells, mast cells, eosinophils, neutrophils, B cells and T cells.OUTCOMESIn women with endometriosis compared to those without endometriosis, some endometrial immune cells display similar cycle-phase variation, whereas macrophages (Mø), immature DC and regulatory T cells behave differently. A pro-inflammatory Mø1 phenotype versus anti-inflammatory Mø2 phenotype predominates and natural killer cells display abnormal activity in endometrium of women with the disease. Conflicting data largely derive from small studies, variably defined hormonal milieu and different experimental approaches and technologies.WIDER IMPLICATIONSPhenotyping immune cell subtypes is essential to determine the role of the endometrial immune niche in pregnancy and endometrial homeostasis normally and in women with poor reproductive history and can facilitate development of innovative diagnostics and therapeutics for associated symptoms and compromised reproductive outcomes.

Endometriosis is characterized by the abnormal presence of endometrium outside of the uterus, resulting in pelvic pain and infertility. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) has been postulated to be a marker of stem cells in the endometrium. However, LGR5+ cells have a macrophage-like phenotype in this tissue, so it is unclear what role LGR5+ cells actually play in the endometrium. Macrophages serve an important function in the endometrium to maintain fertility, while LGR5+ cells generally have a role in tumor progression and are involved in invasion in some cancers. We sought to determine whether LGR5+ cells vary across the menstrual cycle in women with endometriosis and whether there are implications for LGR5 in the aggressiveness of endometriosis and reproductive outcomes. We performed immunofluorescence, flow cytometry, and primary culture in vitro experiments on eutopic and ectopic endometrium from healthy and endometriosis patients and observed that neither LGR5+ cells nor LGR5 expression varied throughout the cycle. Interestingly, we observed that LGR5+ cell percentage overexpressing CD163 (anti-inflammatory marker) was higher in healthy endometrium, suggesting that in endometriosis, endometrium presents a more pro-inflammatory phenotype that likely leads to poor obstetric outcomes. We also observed higher levels of LGR5+ cells in ectopic lesions compared to eutopic endometrium and specifically in deep infiltrating endometriosis, indicating that LGR5 could be involved in progression and aggressiveness of the disease.

Objective To characterize leucine-rich repeat containing G protein-coupled receptor 5–positive (LGR5 + ) cells from the endometrium of women with endometriosis. Design Prospective experimental study. Setting University hospital/fertility clinic. Patient(s) Twenty-seven women with endometriosis who underwent surgery and 12 healthy egg donors, together comprising 39 endometrial samples. Intervention(s) Obtaining of uterine aspirates by using a Cornier Pipelle. Main Outcomes Measure(s) Immunofluorescence in formalin-fixed paraffin-embedded tissue from mice and healthy and pathologic human endometrium using antibodies against LGR5, E-cadherin, and cytokeratin, and epithelial and stromal LGR5 + cells isolated from healthy and pathologic human eutopic endometrium by fluorescence-activated cell sorting and transcriptomic characterization by RNA high sequencing. Result(s) Immunofluorescence showed that LGR5 + cells colocalized with epithelial markers in the stroma of the endometrium only in endometriotic patients. The results from RNA high sequencing of LGR5 + cells from epithelium and stroma did not show any statistically significant differences between them. The LGR5 + versus LGR5 − cells in pathologic endometrium showed 394 differentially expressed genes. The LGR5 + cells in deep-infiltrating endometriosis expressed inflammatory markers not present in the other types of the disease. Conclusion(s) Our results revealed the presence of aberrantly located LGR5 + cells coexpressing epithelial markers in the stromal compartment of women with endometriosis. These cells have a statistically significantly different expression profile in deep-infiltrating endometriosis in comparison with other types of endometriosis, independent of the menstrual cycle phase. Further studies are needed to elucidate their role and influence in reproductive outcomes.

Objective To study, isolate and characterize leucine-rich repeat–containing heterotrimeric guanine nucleotide–binding protein–coupled receptor 5 (LGR5)–positive cells from human endometrium to determine their functional relevance. Design Prospective experimental animal study. Setting University research laboratories. Animal(s) Nonobese diabetic mice (NOD-SCID) (strain code 394; NOD.CB17—Prkdc scid /NcrCrl). Intervention(s) Human LGR5 + cells were labeled with superparamagnetic iron oxide nanoparticles (SPIOs) and injected under the kidney capsule in immunocompromised mice. Main Outcome Measure(s) Epithelial and stromal LGR5 + cells were isolated from human endometrium by means of fluorescence-activated cell sorting, and phenotypic characterization was performed by means of flow cytometry with the use of hematopoietic and mesenchymal markers. Engrafted SPIO-labeled LGR5 + cells were localized with the use of Prussian blue staining and immunohistochemistry against CD9 and Vimentin. Deep transcriptomic profiling of LGR5 + cells was performed with the use of microarrays and RNA sequencing. Result(s) The percentage of LGR5 + cells in human endometrium represented 1.08 ± 0.73% and 0.82 ± 0.76% of total cells in the epithelial and stromal compartments, respectively. LGR5 + cells were phenotypically characterized by abundant expression of CD45 hematopoietic marker and no expression of surface markers CD31, CD34, CD133, CD73, and CD90. Coexpression with the macrophage marker CD163 was detected. Xenotransplantation of labeled LGR5 + cells into the kidney capsules of immunocompromised mice resulted in a weak endometrial reconstitution from this cell of origin. Transcriptomic profiling revealed new attributes for LGR5 + cells related to their putative hematopoietic origin. Conclusion(s) These data suggest that endometrial LGR5 is not an endogenous stem cell marker. Instead, LGR5 + cells appear to be recruited from blood to be part of the stem cell niche at the perivascular microenvironment to activate the endogenous niche.