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 PDGFRß + 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.

Profiling the expression and function of oestrogen receptor isoform ER46 in human endometrial tissues and uterine natural killer cells.

STUDY QUESTION: Does the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function? SUMMARY ANSWER: ER46 is expressed in endometrial tissues, is the predominant ER isoform in first trimester decidua and is localised to the cell membrane of uterine natural killer (uNK) cells where activation of ER46 increases cell motility. WHAT IS KNOWN ALREADY: Oestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46-kDa truncated isoform of full length ERα (ER66, encoded by ESR1) that contains both ligand- and DNA-binding domains. Expression of ER46 in the human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. uNK cells are a phenotypically distinct (CD56brightCD16-) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative, but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor. STUDY DESIGN, SIZE, DURATION: This laboratory-based study used primary human endometrial (n = 24) and decidual tissue biopsies (n = 30) as well as uNK cells which were freshly isolated from first trimester human decidua (n = 18). PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of ERs (ER66, ER46 and ERß) was assessed by quantitative PCR, western blot and immunohistochemistry. uNK cells were isolated from first-trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with 17ß-oestradiol conjugated to bovine serum albumin (E2-BSA, 10 nM equivalent), the ERß-selective agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN; 10 nM) or dimethylsulphoxide vehicle control. MAIN RESULTS AND THE ROLE OF CHANCE: ER46 was detected in proliferative and secretory phase tissues by western blot and was the predominant ER isoform in first-trimester decidua samples. Immunohistochemistry revealed that ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression of ER46 by quantitative PCR and western blot and localised ER46 protein to the cell membrane by immunocytochemistry. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Expression pattern in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages, which may have precluded insights into gestation-specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ. WIDER IMPLICATIONS OF THE FINDINGS: E2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in the human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in the human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women. Given the importance of uNK cells to regulating vascular remodelling in early pregnancy and the potential for selective targeting of ER46, this may be an attractive future therapeutic target in the treatment of reproductive disorders. STUDY FUNDING/COMPETING INTEREST(S): These studies were supported by Medical Research Council (MRC) Programme Grants G1100356/1 and MR/N024524/1 to PTKS. H.O.D.C. was supported by MRC grant G1002033. The authors declare no competing interests related to the published work.

Endometrial Intracrinology: Oestrogens, Androgens and Endometrial Disorders.

Peripheral tissue metabolism of steroids (intracrinology) is now accepted as a key way in which tissues, such as the endometrium, can utilise inactive steroids present in the blood to respond to local physiological demands and 'fine-tune' the activation or inhibition of steroid hormone receptor-dependent processes. Expression of enzymes that play a critical role in the activation and inactivation of bioactive oestrogens (E1, E2) and androgens (A4, T, DHT), as well as expression of steroid hormone receptors, has been detected in endometrial tissues and cells recovered during the menstrual cycle. There is robust evidence that increased expression of aromatase is important for creating a local microenvironment that can support a pregnancy. Measurement of intra-tissue concentrations of steroids using liquid chromatography⁻tandem mass spectrometry has been important in advancing our understanding of a role for androgens in the endometrium, acting both as active ligands for the androgen receptor and as substrates for oestrogen biosynthesis. The emergence of intracrinology, associated with disordered expression of key enzymes such as aromatase, in the aetiology of common women's health disorders such as endometriosis and endometrial cancer has prompted renewed interest in the development of drugs targeting these pathways, opening up new opportunities for targeted therapies and precision medicine.

Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

The human endometrium undergoes regular cycles of synchronous tissue shedding (wounding) and repair that occur during menstruation before estrogen-dependent regeneration. Endometrial repair is normally both rapid and scarless. Androgens regulate cutaneous wound healing, but their role in endometrial repair is unknown. We used a murine model of simulated menses; mice were treated with a single dose of the nonaromatizable androgen dihydrotestosterone (DHT; 200 µg/mouse) to coincide with initiation of tissue breakdown. DHT altered the duration of vaginal bleeding and delayed restoration of the luminal epithelium. Analysis of uterine mRNAs 24 h after administration of DHT identified significant changes in metalloproteinases (Mmp3 and -9; P < 0.01), a snail family member (Snai3; P < 0.001), and osteopontin (Spp1; P < 0.001). Chromatin immunoprecipitation analysis identified putative androgen receptor (AR) binding sites in the proximal promoters of Mmp9, Snai3, and Spp1. Striking spatial and temporal changes in immunoexpression of matrix metalloproteinase (MMP) 3/9 and caspase 3 were detected after DHT treatment. These data represent a paradigm shift in our understanding of the role of androgens in endometrial repair and suggest that androgens may have direct impacts on endometrial tissue integrity. These studies provide evidence that the AR is a potential target for drug therapy to treat conditions associated with aberrant endometrial repair processes.-Cousins, F. L., Kirkwood, P. M., Murray, A. A., Collins, F., Gibson, D. A., Saunders, P. T. K. Androgens regulate scarless repair of the endometrial "wound" in a mouse model of menstruation.

Uterine NK cells regulate endometrial bleeding in women and are suppressed by the progesterone receptor modulator asoprisnil.

Uterine NK cells (uNK) play a role in the regulation of placentation, but their functions in nonpregnant endometrium are not understood. We have previously reported suppression of endometrial bleeding and alteration of spiral artery morphology in women exposed to asoprisnil, a progesterone receptor modulator. We now compare global endometrial gene expression in asoprisnil-treated versus control women, and we demonstrate a statistically significant reduction of genes in the IL-15 pathway, known to play a key role in uNK development and function. Suppression of IL-15 by asoprisnil was also observed at mRNA level (p < 0.05), and immunostaining for NK cell marker CD56 revealed a striking reduction of uNK in asoprisnil-treated endometrium (p < 0.001). IL-15 levels in normal endometrium are progesterone-responsive. Progesterone receptor (PR) positive stromal cells transcribe both IL-15 and IL-15RA. Thus, the response of stromal cells to progesterone will be to increase IL-15 trans-presentation to uNK, supporting their expansion and differentiation. In asoprisnil-treated endometrium, there is a marked downregulation of stromal PR expression and virtual absence of uNK. These novel findings indicate that the IL-15 pathway provides a missing link in the complex interplay among endometrial stromal cells, uNK, and spiral arteries affecting physiologic and pathologic endometrial bleeding.

RISING STARS: Androgens and immune cell function.

Androgens can modulate immune cell function and may contribute to differences in the prevalence and severity of common inflammatory conditions. Although most immune cells are androgen targets, our understanding of how changes in androgen bioavailability can affect immune responses is incomplete. Androgens alter immune cell composition, phenotype, and activation by modulating the expression and secretion of inflammatory mediators or by altering the development and maturation of immune cell precursors. Androgens are generally associated with having suppressive effects on the immune system, but their impacts are cell and tissue context-dependent and can be highly nuanced even within immune cell subsets. In response to androgens, innate immune cells such as neutrophils, monocytes, and macrophages increase the production of the anti-inflammatory cytokine IL-10 and decrease nitric oxide production. Androgens promote the differentiation of T cell subsets and reduce the production of inflammatory mediators, such as IFNG, IL-4 and IL-5. Additionally, androgens/androgen receptor can promote the maturation of B cells. Thus, androgens can be considered as immunomodulatory agents, but further work is required to understand the precise molecular pathways that are regulated at the intersection between endocrine and inflammatory signals. This narrative review focusses on summarising our current understanding of how androgens can alter immune cell function and how this might affect inflammatory responses in health and disease.

Endometrial macrophages in health and disease.

Macrophages are present in the endometrium throughout the menstrual cycle and are most abundant during menstruation. Endometrial macrophages contribute to tissue remodeling during establishment of pregnancy and are thought to play key roles in mediating tissue breakdown and repair during menstruation. Despite these important roles, the phenotype and function of endometrial macrophages remains poorly understood. In this review, we summarize approaches used to characterize endometrial macrophage phenotype, current understanding of the functional role of macrophages in normal endometrial physiology as well as the putative contribution of macrophage dysfunction to women's reproductive health disorders.

Single-cell RNA sequencing and lineage tracing confirm mesenchyme to epithelial transformation (MET) contributes to repair of the endometrium at menstruation.

The human endometrium experiences repetitive cycles of tissue wounding characterised by piecemeal shedding of the surface epithelium and rapid restoration of tissue homeostasis. In this study, we used a mouse model of endometrial repair and three transgenic lines of mice to investigate whether epithelial cells that become incorporated into the newly formed luminal epithelium have their origins in one or more of the mesenchymal cell types present in the stromal compartment of the endometrium. Using scRNAseq, we identified a novel population of PDGFRb + mesenchymal stromal cells that developed a unique transcriptomic signature in response to endometrial breakdown/repair. These cells expressed genes usually considered specific to epithelial cells and in silico trajectory analysis suggested they were stromal fibroblasts in transition to becoming epithelial cells. To confirm our hypothesis we used a lineage tracing strategy to compare the fate of stromal fibroblasts (PDGFRa+) and stromal perivascular cells (NG2/CSPG4+). We demonstrated that stromal fibroblasts can undergo a mesenchyme to epithelial transformation and become incorporated into the re-epithelialised luminal surface of the repaired tissue. This study is the first to discover a novel population of wound-responsive, plastic endometrial stromal fibroblasts that contribute to the rapid restoration of an intact luminal epithelium during endometrial repair. These findings form a platform for comparisons both to endometrial pathologies which involve a fibrotic response (Asherman's syndrome, endometriosis) as well as other mucosal tissues which have a variable response to wounding.

The human uterus is a formidable organ. From puberty to menopause, it completely sheds off its internal lining every 28 days or so, creating what is in effect a large open wound. Unlike the skin or other parts of the body, however, this tissue can quickly repair itself without scarring. This fascinating process remains poorly understood, partly because human samples and animal models that mimic human menstruation are still lacking. This makes it difficult to grasp how various types of uterine cells get mobilised for healing. To fill this gap, Kirkwood et al. focused on fibroblasts, a heterogenous cell population which helps to support the epithelial cells lining the inside of the uterus. How these cells responded to the advent of menstruation was examined in female mice genetically manipulated to have human-like periods. A method known as single-cell RNAseq was used to track which genes were active in each of these cells before, one day and two days after period onset. This revealed the existence of a subpopulation of cells which only appeared when wound healing was most needed. These 'repair-specific' fibroblasts expressed a mixture of genes; those typical of fibroblasts but also some known to be active in the epithelial cells lining the uterus. This suggests that the cells were in the process of changing their identity so they could remake the uterine layer lost during a period. And indeed, labelling these fibroblasts with a fluorescent tag showed that, during healing, they had migrated from within the uterine tissue to become part of its newly restored internal surface. These results represent the first evidence that fibroblasts play a direct role in repairing the uterus during menstruation. From endometriosis to infertility, the lives of millions of people around the world are impacted by disorders which affect the uterine lining. A better understanding of how the uterus can fix itself month after month could help to find new treatments for these conditions. This knowledge could also be useful for to address abnormal wound healing in the skin and other tissues, as this process often involves fibroblasts.

A role for steroid 5 alpha-reductase 1 in vascular remodeling during endometrial decidualization.

Decidualization is the hormone-dependent process of endometrial remodeling that is essential for fertility and reproductive health. It is characterized by dynamic changes in the endometrial stromal compartment including differentiation of fibroblasts, immune cell trafficking and vascular remodeling. Deficits in decidualization are implicated in disorders of pregnancy such as implantation failure, intra-uterine growth restriction, and pre-eclampsia. Androgens are key regulators of decidualization that promote optimal differentiation of stromal fibroblasts and activation of downstream signaling pathways required for endometrial remodeling. We have shown that androgen biosynthesis, via 5α-reductase-dependent production of dihydrotestosterone, is required for optimal decidualization of human stromal fibroblasts in vitro, but whether this is required for decidualization in vivo has not been tested. In the current study we used steroid 5α-reductase type 1 (SRD5A1) deficient mice (Srd5a1-/- mice) and a validated model of induced decidualization to investigate the role of SRD5A1 and intracrine androgen signaling in endometrial decidualization. We measured decidualization response (weight/proportion), transcriptomic changes, and morphological and functional parameters of vascular development. These investigations revealed a striking effect of 5α-reductase deficiency on the decidualization response. Furthermore, vessel permeability and transcriptional regulation of angiogenesis signaling pathways, particularly those that involved vascular endothelial growth factor (VEGF), were disrupted in the absence of 5α-reductase. In Srd5a1-/- mice, injection of dihydrotestosterone co-incident with decidualization restored decidualization responses, vessel permeability, and expression of angiogenesis genes to wild type levels. Androgen availability declines with age which may contribute to age-related risk of pregnancy disorders. These findings show that intracrine androgen signaling is required for optimal decidualization in vivo and confirm a major role for androgens in the development of the vasculature during decidualization through regulation of the VEGF pathway. These findings highlight new opportunities for improving age-related deficits in fertility and pregnancy health by targeting androgen-dependent signaling in the endometrium.

Pelvic pain correlates with peritoneal macrophage abundance not endometriosis.

Endometriosis is a chronic neuroinflammatory pain condition affecting ~180 million women worldwide. Surgical removal or hormonal suppression of endometriosis lesions only relieves pain symptoms in some women and symptomatic relapse following treatment is common. Identifying factors that contribute to pain is key to developing new therapies. We collected peritoneal fluid samples and clinical data from a cohort of women receiving diagnostic laparoscopy for suspected endometriosis (n = 52). Peritoneal fluid immune cells were analysed by flow cytometry and data compared with pain scores determined using the pain domain of the Endometriosis Health Profile Questionnaire (EHP-30) in order to investigate the association between peritoneal immune cells and pain symptoms. Pain scores were not different between women with or without endometriosis, nor did they differ according to disease stage; consistent with a poor association between disease presentation and pain symptoms. However, linear regression and correlation analysis demonstrated that peritoneal macrophage abundance correlated with the severity of pelvic pain. CD14high peritoneal macrophages negatively correlated with pain scores whereas CD14low peritoneal macrophages were positively correlated, independent of diagnostic outcome at laparoscopy. Stratification by pain subtype, rather than endometriosis diagnosis, resulted in the most robust correlation between pain and macrophage adundance. Pain score strongly correlated with CD14high (P = 0.007) and CD14low (P = 0.008) macrophages in patients with non-menstrual pain and also in patients who reported dysmennorhea (CD14high P = 0.021, CD14low P = 0.019) or dysparunia (CD14high P = 0.027, CD14low P = 0.031). These results provide new insight into the association between peritoneal macrophages and pelvic pain which may aid the identification of future therapeutic targets. LAY SUMMARY: Endometriosis is a common condition where cells similar to those that line the womb are found elsewhere in the body. It is associated with inflammation and pain in the pelvis and affects ~180 million women worldwide. Current treatments are not effective for all patients and we, therefore, need to understand what causes pain in order to develop new treatments. We investigated the types of immune cells present within the pelvis of women undergoing investigation for suspected endometriosis. Disease diagnosis and stage (I-IV) was recorded along with pain score determined by questionnaire. We characterised the immune cells present and compared them to disease stage and pain score. We found that pelvic pain was linked to the abundance of immune cells but, surprisingly, not to disease stage. These findings suggest that immune cells are closely associated with pain severity in endometriosis and may be good targets for future endometriosis treatments.

Menstruation Dysregulation and Endometriosis Development.

Endometriosis is a common gynecological condition characterized by the growth of endometrial-like tissue outside of the uterus which may cause symptoms such as chronic pelvic pain or subfertility. Several surgical and medical therapies are available to manage symptoms, but a cure has yet to be determined which can be attributed to the incomplete understanding of disease pathogenesis. Sampson's theory of retrograde menstruation is a widely accepted theory describing how shed endometrial tissue can enter the peritoneal cavity, but other factors are likely at play to facilitate the establishment of endometriosis lesions. This review summarizes literature that has explored how dysregulation of menstruation can contribute to the pathogenesis of endometriosis such as dysregulation of inflammatory mediators, aberrant endometrial matrix metalloproteinase expression, hypoxic stress, and reduced apoptosis. Overall, many of these factors have overlapping pathways which can prolong the survival of shed endometrial debris, increase tissue migration, and facilitate implantation of endometrial tissue at ectopic sites. Moreover, some of these changes are also implicated in abnormal uterine bleeding and endometrial diseases. More research is needed to better understand the underlying mechanisms driving dysregulation of menstruation in endometriosis specifically and identifying specific pathways could introduce new treatment targets. Analyzing menstrual fluid from women with endometriosis for inflammatory markers and other biomarkers may also be beneficial for earlier diagnosis and disease staging.

Androgens, oestrogens and endometrium: a fine balance between perfection and pathology.

The endometrium is a complex multicellular tissue that is exquisitely sensitive to the actions of sex steroids synthesised in the ovary (endocrine system). Recent studies have highlighted a previously under-appreciated role for local (intracrine) metabolism in fine-tuning tissue function in both health and disease. In this review we have focused on the impact of oestrogens and androgens on endometrial function summarising data from studies on normal endometrial physiology and disorders including infertility, endometriosis and cancer. We consider the evidence that expression of enzymes including aromatase, sulphatase and AKR1C3 by endometrial cells plays an important role in tissue function and malfunction and discuss results from studies using drugs targeting intracrine pathways to treat endometrial disorders. We summarise studies exploring the spatial and temporal expression of oestrogen receptors (ERalpha/ESR1, ERbeta/ESR2 and GPER) and their role in mediating the impact of endogenous and synthetic ligands on cross-talk between vascular, immune, epithelial and stromal cells. There is a single androgen receptor gene and androgens play a key role in stromal-epithelial cross-talk, scar-free healing of endometrium during menstruation and regulation of cell proliferation. The development of new receptor-selective drugs (SERMs, SARMs, SARDs) has reinvigorated interest in targeting receptor subtypes in treatment of disorders including endometriosis and endometrial cancer and some show promise as novel therapies. In summary, understanding the mechanisms regulated by sex steroids provides the platform for improved personalised treatment of endometrial disorders as well as novel insights into the impact of steroids on processes such as tissue repair and regeneration.

The ERß5 splice variant increases oestrogen responsiveness of ERαpos Ishikawa cells.

Endometrial cancer is a common gynaeological malignancy: life time exposure to oestrogen is a key risk factor. Oestrogen action is mediated by receptors encoded by ESR1 (ERα) and ESR2 (ERß): ERα plays a key role in regulating endometrial cell proliferation. A truncated splice variant isoform (ERß5) encoded by ESR2 is highly expressed in cancers. This study explored whether ERß5 alters oestrogen responsiveness of endometrial epithelial cells. Immunhistochemistry profiling of human endometrial cancer tissue biopsies identified epithelial cells co-expressing ERß5 and ERα in stage I endometrial adenocarcinomas and post menopausal endometrium. Induced co-expression of ERß5 in ERαpos endometrial cancer cells (Ishikawa) significantly increased ligand-dependent activation of an ERE-luciferase reporter stimulated by either E2 or the ERα-selective agonist 1,3,5-(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT) compared to untransfected cells. Fluorescence recovery after photobleaching (FRAP) analysis of tagged yellow fluorescent protein (YFP)-ERß5 transfected into Ishikawa cells revealed that incubation with E2 induced a transient reduction in intra-nuclear mobility characterised by punctate protein redistribution which phenocopied the behaviour of ERα following ligand activation with E2. In ERαneg MDA-MD-231 breast cancer cells, there was no E2-dependent change in mobility of YFP-ERß5 and no activation of the ERE reporter in cells expressing ERß5. In conclusion, we demonstrate that ERß5 can act as heterodimeric partner to ERα in Ishikawa cells and increases their sensitivity to E2. We speculate that expression of ERß5 in endometrial epithelial cells may increase the risk of malignant transformation and suggest that immunostaining for ERß5 should be included in diagnostic assessment of women with early grade cancers.

Selective androgen receptor modulators (SARMs) have specific impacts on the mouse uterus.

Selective androgen receptor modulators (SARMs) have been proposed as therapeutics for women suffering from breast cancer, muscle wasting or urinary incontinence. The androgen receptor (AR) is expressed in the uterus but the impact of SARMs on the function of this organ is unknown. We used a mouse model to compare the impact of SARMs (GTx-007/Andarine®, GTx-024/Enobosarm®), Danazol (a synthetic androstane steroid) and dihydrotestosterone (DHT) on tissue architecture, cell proliferation and gene expression. Ovariectomised mice were treated daily for 7 days with compound or vehicle control (VC). Uterine morphometric characteristics were quantified using high-throughput image analysis (StrataQuest; TissueGnostics), protein and gene expression were evaluated by immunohistochemistry and RT-qPCR, respectively. Treatment with GTx-024, Danazol or DHT induced significant increases in body weight, uterine weight and the surface area of the endometrial stromal and epithelial compartments compared to VC. Treatment with GTx-007 had no impact on these parameters. GTx-024, Danazol and DHT all significantly increased the percentage of Ki67-positive cells in the stroma, but only GTx-024 had an impact on epithelial cell proliferation. GTx-007 significantly increased uterine expression of Wnt4 and Wnt7a, whereas GTx-024 and Danazol decreased their expression. In summary, the impact of GTx-024 and Danazol on uterine cells mirrored that of DHT, whereas GTx-007 had minimal impact on the tested parameters. This study has identified endpoints that have revealed differences in the effects of SARMs on uterine tissue and provides a template for preclinical studies comparing the impact of compounds targeting the AR on endometrial function.

Androgens and endometrium: New insights and new targets.

Androgens are synthesised in both the ovary and adrenals in women and play an important role in the regulation of female fertility, as well as in the aetiology of disorders such as polycystic ovarian syndrome, endometriosis and endometrial cancer. The endometrium is an androgen target tissue and the impact of AR-mediated effects has been studied using human endometrial tissue samples and rodent models. In this review we highlight recent evidence that endometrial androgen biosynthesis and intracrine action is important in preparation of a tissue microenvironment that can support implantation and establishment of pregnancy. The impact of androgens on endometrial cell proliferation, in repair of the endometrial wound at the time of menstruation and in endometrial disorders is discussed. Future directions for research focused on AR function as a therapeutic target are considered.

Animal models of endometriosis: Replicating the aetiology and symptoms of the human disorder.

Endometriosis is a chronic incurable disorder that affects 1 in 10 women of reproductive age: associated symptoms include chronic pain and infertility. The aetiology of endometriosis remains poorly understood but patients, clinicians and researchers are all in agreement that new non-surgical therapies are urgently needed to reduce the severity of symptoms. Preclinical testing of drugs requires the development and validation of models that recapitulate the key features of the disorder. In this review we describe the best-validated animal models (primate, rodent, xenograft) and their contributions to our understanding of the factors underpinning the development of symptoms. We consider the evidence that these models have provided the platform for identification of new therapeutic interventions and reflect on future directions for research and drug validation.

SULFATION PATHWAYS: Contribution of intracrine oestrogens to the aetiology of endometriosis.

Endometriosis is an incurable hormone-dependent inflammatory disease that causes chronic pelvic pain and infertility characterized by implantation and growth of endometrial tissue outside the uterine cavity. Symptoms have a major impact on the quality of life of patients resulting in socioeconomic, physical and psychological burdens. Although the immune system and environmental factors may play a role in the aetiology of endometriosis, oestrogen dependency is still considered a hallmark of the disorder. The impact of oestrogens such as oestrone and particularly, oestradiol, on the endometrium or endometriotic lesions may be mediated by steroids originating from ovarian steroidogenesis or local intra-tissue production (intracrinology) dependent upon the expression and activity of enzymes that regulate oestrogen biosynthesis and metabolism. Two key pathways have been implicated: while there is contradictory data on the participation of the aromatase enzyme (encoded by CYP19A1), there is increasing evidence that the steroid sulphatase pathway plays a role in both the aetiology and pathology of endometriosis. In this review, we consider the evidence related to the pathways leading to oestrogen accumulation in endometriotic lesions and how this might inform the development of new therapeutic strategies to treat endometriosis without causing the undesirable side effects of current regimes that suppress ovarian hormone production.

The impact of 27-hydroxycholesterol on endometrial cancer proliferation.

Endometrial cancer (EC) is the most common gynaecological malignancy. Obesity is a major risk factor for EC and is associated with elevated cholesterol. 27-hydroxycholesterol (27HC) is a cholesterol metabolite that functions as an endogenous agonist for Liver X receptor (LXR) and a selective oestrogen receptor modulator (SERM). Exposure to oestrogenic ligands increases risk of developing EC; however, the impact of 27HC on EC is unknown. Samples of stage 1 EC (n = 126) were collected from postmenopausal women undergoing hysterectomy. Expression of LXRs (NR1H3, LXRα; NR1H2, LXRß) and enzymes required for the synthesis (CYP27A1) or breakdown (CYP7B1) of 27HC were detected in all grades of EC. Cell lines originating from well-, moderate- and poorly-differentiated ECs (Ishikawa, RL95, MFE 280 respectively) were used to assess the impact of 27HC or the LXR agonist GW3965 on proliferation or expression of a luciferase reporter gene under the control of LXR- or ER-dependent promoters (LXRE, ERE). Incubation with 27HC or GW3965 increased transcription via LXRE in Ishikawa, RL95 and MFE 280 cells (P < 0.01). 27HC selectively activated ER-dependent transcription (P < 0.001) in Ishikawa cells and promoted proliferation of both Ishikawa and RL95 cells (P < 0.001). In MFE 280 cells, 27HC did not alter proliferation but selective targeting of LXR with GW3965 significantly reduced cell proliferation (P < 0.0001). These novel results suggest that 27HC can contribute to risk of EC by promoting proliferation of endometrial cancer epithelial cells and highlight LXR as a potential therapeutic target in the treatment of advanced disease.

Dehydroepiandrosterone enhances decidualization in women of advanced reproductive age.

OBJECTIVE: To investigate the impact of the androgen precursor dehydroepiandrosterone (DHEA) on the decidualization of human endometrial stromal cells isolated from women of advanced reproductive age. DESIGN: In vitro study. SETTING: University research institute. PATIENT(S): Proliferative phase primary human endometrial stromal fibroblasts (hESFs) were isolated from women of advanced reproductive age (n = 16; mean age, 44.7 ± 2.3). None of the women were receiving hormone therapy or had endometriosis. INTERVENTION(S): Isolated hESFs were decidualized in vitro by incubation with P (1 µM) and cAMP (0.1 mg/mL) in the presence, or absence, of DHEA (10 nM, 100 nM). MAIN OUTCOME MEASURE(S): Secretion of androgens was assessed by ELISA. Expression of decidualization markers and endometrial receptivity markers was assessed by quantitative polymerase chain reaction and ELISA. RESULT(S): Decidualization responses were retained in hESF isolated from women of advanced reproductive age. Supplementation with DHEA increased androgen biosynthesis and concentrations of T and dihydrotestosterone were ∼3× greater after coincubation with DHEA compared with hESF stimulated with decidualization alone. Addition of DHEA to decidualized hESF increased expression of the decidualization markers IGFBP1 and PRL and the endometrial receptivity marker SPP1. DHEA enhanced secretion of IGFBP1, PRL, and SPP1 proteins maximally by day 8 of the decidualization time course concomitant with peak androgen concentrations. CONCLUSION(S): These novel results demonstrate DHEA can enhance in vitro decidualization responses of hESF from women of advanced reproductive age. Supplementation with DHEA during the receptive phase may augment endometrial function and improve pregnancy rates in natural or assisted reproductive cycles.

SULFATION PATHWAYS: A role for steroid sulphatase in intracrine regulation of endometrial decidualisation.

In women, establishment of pregnancy is dependent upon 'fine-tuning' of the endometrial microenvironment, which is mediated by terminal differentiation (decidualisation) of endometrial stromal fibroblasts (ESFs). We have demonstrated that intracrine steroid metabolism plays a key role in regulating decidualisation and is essential for time-dependent expression of key factors required for endometrial receptivity. The primary aim of the current study was to determine whether sulphated steroids can act as precursors to bioactive sex steroids during decidualisation. We used primary human ESF and a robust in vitro model of decidualisation to assess the expression of genes associated with sulphation, desulphation and transport of sulphated steroids in human ESF as well as the impact of the steroid sulphatase (STS) inhibitor STX64 (Irosustat). We found evidence for an increase in both expression and activity of STS in response to a decidualisation stimulus with abrogation of oestrone biosynthesis and decreased secretion of the decidualisation marker IGFBP1 in the presence of STX64. These results provide novel insight into the contribution of STS to the intracrine regulation of decidualisation.