Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERβ and glucocorticoid receptor

Background: Human mast cells (MCs) are long-lived tissue-resident immune cells characterised by granules containing the proteases chymase and/or tryptase. Their phenotype is modulated by their tissue microenvironment. The human uterus has an outer muscular layer (the myometrium) surrounding the endometrium, both of which play an important role in supporting a pregnancy. The endometrium is a sex steroid target tissue consisting of epithelial cells (luminal, glandular) surrounded by a multicellular stroma, with the latter containing an extensive vascular compartment as well as fluctuating populations of immune cells that play an important role in regulating tissue function. The role of MCs in the human uterus is poorly understood with little known about their regulation or the impact of steroids on their differentiation status. The current study had two aims: 1) To investigate the spatial and temporal location of uterine MCs and determine their phenotype; 2) To determine whether MCs express receptors for steroids implicated in uterine function, including oestrogen (ERα, ERβ), progesterone (PR) and glucocorticoids (GR). Methods: Tissue samples from women (n=46) were used for RNA extraction (n=26) or fixed (n=20) for immunohistochemistry. Results: Messenger RNAs encoded by TPSAB1 (tryptase) and CMA1 (chymase) were detected in endometrial tissue homogenates. Immunohistochemistry revealed the relative abundance of tryptase MCs was myometrium>basal endometrium>functional endometrium. We show for the first time that uterine MCs are predominantly of the classical MC subtypes: (positive, +; negative, -) tryptase+/chymase- and tryptase+/chymase+, but a third subtype was also identified (tryptase-/chymase+). Tryptase+ MCs were of an ERβ+/ERα-/PR-/GR+ phenotype mirroring other uterine immune cell populations, including natural killer cells. Conclusions: Endometrial tissue resident immune MCs have three protease-specific phenotypes. Expression of both ERβ and GR in MCs mirrors that of other immune cells in the endometrium and suggests that MC function may be altered by the local steroid microenvironment

Improving the diagnosis of endometrial hyperplasia using computerized analysis and immunohistochemical biomarkers

Endometrial hyperplasia (EH) is a precursor lesion to endometrial carcinoma (EC). Risks for EC include genetic, hormonal and metabolic factors most notably those associated with obesity: rates are rising and there is concern that cases in pre-menopausal women may remain undetected. Making an accurate distinction between benign and pre-malignant disease is both a challenge for the pathologist and important to the gynecologist who wants to deliver the most appropriate care to meet the needs of the patient. Premalignant change may be recognized by histological changes of endometrial hyperplasia (which may occur with or without atypia) and endometrial intraepithelial neoplasia (EIN). In this study we created a tissue resource of EH samples diagnosed between 2004 and 2009 (n = 125) and used this to address key questions: 1. Are the EIN/WHO2014 diagnostic criteria able to consistently identify premalignant endometrium? 2. Can computer aided image analysis inform identification of EIN? 3. Can we improve diagnosis by incorporating analysis of protein expression using immunohistochemistry. Our findings confirmed the inclusion of EIN in diagnostic criteria resulted in a better agreement between expert pathologists compared with the previous WHO94 criteria used for the original diagnosis of our sample set. A computer model based on assessment of stromal:epithelial ratio appeared most accurate in classification of areas of tissue without EIN. From an extensive panel of putative endometrial protein tissue biomarkers a score based on assessment of HAND2, PTEN, and PAX2 was able to identify four clusters one of which appeared to be more likely to be benign. In summary, our study has highlighted new opportunities to improve diagnosis of pre-malignant disease in endometrium and provide a platform for further research on this important topic

A Novel Mouse Model of Endometriosis Mimics Human Phenotype and Reveals Insights into the Inflammatory Contribution of Shed Endometrium

Endometriosis is an estrogen-dependent inflammatory disorder characterized by the presence of endometrial tissue outside the uterine cavity. Patients experience chronic pelvic pain and infertility, with the most likely origin of the tissue deposits (lesions) being endometrial fragments shed at menses. Menstruation is an inflammatory process associated with a dramatic increase in inflammatory mediators and tissue-resident immune cells. In the present study, we developed and validated a mouse model of endometriosis using syngeneic menstrual endometrial tissue introduced into the peritoneum of immunocompetent mice. We demonstrate the establishment of endometriotic lesions that exhibit similarities to those recovered from patients undergoing laparoscopy. Specifically, in both cases, lesions had epithelial (cytokeratin+) and stromal (vimentin/CD10+) cell compartments with a well-developed vasculature (CD31+ endothelial cells). Expression of estrogen receptor β was increased in lesions compared with the peritoneum or eutopic endometrium. By performing experiments using mice with green fluorescent protein–labeled macrophages (MacGreen) in reciprocal transfers with wild-type mice, we obtained evidence that macrophages present in the peritoneum and in menses endometrium can contribute to the inflammatory microenvironment of the lesions. In summary, we developed a mouse model of endometriosis that exhibits similarities to human peritoneal lesions with respect to estrogen receptor expression, inflammation, and macrophage infiltration, providing an opportunity for further studies and the possible identification of novel therapies for this perplexing disorder

Single-cell RNA sequencing redefines the mesenchymal cell landscape of mouse endometrium

The endometrium is a dynamic tissue that exhibits remarkable resilience to repeated episodes of differentiation, breakdown, regeneration, and remodeling. Endometrial physiology relies on a complex interplay between the stromal and epithelial compartments with the former containing a mixture of fibroblasts, vascular, and immune cells. There is evidence for rare populations of putative mesenchymal progenitor cells located in the perivascular niche of human endometrium, but the existence of an equivalent cell population in mouse is unclear. We used the Pdgfrb‐BAC‐eGFP transgenic reporter mouse in combination with bulk and single‐cell RNA sequencing to redefine the endometrial mesenchyme. In contrast to previous reports we show that CD146 is expressed in both PDGFRβ + perivascular cells and CD31 + endothelial cells. Bulk RNAseq revealed cells in the perivascular niche which express the high levels of Pdgfrb as well as genes previously identified in pericytes and/or vascular smooth muscle cells (Acta2, Myh11, Olfr78, Cspg4, Rgs4, Rgs5, Kcnj8, and Abcc9). scRNA‐seq identified five subpopulations of cells including closely related pericytes/vascular smooth muscle cells and three subpopulations of fibroblasts. All three fibroblast populations were PDGFRα+/CD34 + but were distinct in their expression of Ngfr/Spon2/Angptl7 (F1), Cxcl14/Smoc2/Rgs2 (F2), and Clec3b/Col14a1/Mmp3 (F3), with potential functions in the regulation of immune responses, response to wounding, and organization of extracellular matrix, respectively. Immunohistochemistry was used to investigate the spatial distribution of these populations revealing F1/NGFR + cells in most abundance beside epithelial cells. We provide the first definitive analysis of mesenchymal cells in the adult mouse endometrium identifying five subpopulations providing a platform for comparisons between mesenchymal cells in endometrium and other adult tissues which are prone to fibrosis

Identifying the role of androgens in endometrial function

The endometrium is a complex multicellular tissue that undergoes dynamic alterations under the control of ovarian-derived sex steroid hormones. During the proliferative phase of the human menstrual cycle, oestrogen induces proliferation of the endometrial epithelium while during the progesterone-dominated secretory phase, the endometrial stromal compartment differentiates in preparation for pregnancy. This differentiation event is termed decidualisation and it is accompanied by immune cell infiltration, vascular remodelling and secretion of cytokines and growth factors, as well as a newfound capacity of active steroid synthesis in the endometrium. Defective decidualisation has been described in several endometrial-associated disorders such as endometriosis, a pathology of ectopic endometrial tissue in the peritoneal cavity, often associated with infertility. Rodent models have been used for the investigation of endometrial physiology and pathology due to the similarity in uterine tissue architecture, appropriate endometrial responses to steroid hormones and the opportunity to inform cellular mechanisms using genetic manipulation. While the impact of 17β-oestradiol and progesterone on endometrial function have been extensively studied, androgens have only recently emerged as potent potential regulators of the endometrium, however, their impact on cell function has not been fully elucidated. The aims of this study were to: Identify the impact of androgens on endometrial function using a mouse model of steroid depletion (ovariectomy) followed by administration of the potent androgen dihydrotestosterone (DHT). Investigate the capacity of endometrial stromal cells to synthesise androgens during decidualisation using human primary endometrial stromal cells (hESCs) decidualised in vitro. Elucidate the decidualisation response of hESCs from women with endometriosis after modulation of androgen receptor (AR) function during decidualisation. Novel results obtained provided evidence of a role for androgens in inducing a trophic effect in the mouse uterus characterised by: pronounced endometrial epithelial proliferation, altered expression pattern of AR, changes in the expression of genes involved in cell-cycle progression and stromal-epithelial cross-talk. In addition, androgen treatment resulted in a striking and unexpected increase in the number of endometrial glands. Decidualisation of hESCs resulted in time-dependent changes in expression of the androgen synthesising enzymes AKR1C3 and 5α-reductase (accompanied by biosynthesis of both testosterone and DHT in a dynamic time-dependent manner). Notably, blocking of AR action arising from local androgen signalling during decidualisation of hESCs culminates in sub-optimal decidualisation as detected by the expression of the classical decidualisation markers IGFBP1 and PRL. Women with endometriosis are reported to exhibit defective decidualisation, which may be accompanied with infertility. Treatment of hESCs from women with endometriosis with an AR agonist (DHT) or antagonist (flutamide) during decidualisation resulted in striking differences in decidualisation response as demonstrated in a case-study approach. Taken together, these findings highlight novel roles of androgens in regulating endometrial function by impacting on cell proliferation, gland formation and decidualisation. These striking new findings have implications for endometrial disorders such as endometriosis. Future studies will focus on the use of selective androgen receptor modulators, a novel class of compounds, with tissue-selective actions and without the undesired side-effects of potent androgens. The use of AR modulators would benefit from a personalised medicine approach, instructed by patient profiling to direct therapeutic targeting of endometrial disorders