Global endometrial DNA methylation analysis reveals insights into mQTL regulation and associated endometriosis disease risk and endometrial function.

Endometriosis is a leading cause of pain and infertility affecting millions of women globally. Herein, we characterize variation in DNA methylation (DNAm) and its association with menstrual cycle phase, endometriosis, and genetic variants through analysis of genotype data and methylation in endometrial samples from 984 deeply-phenotyped participants. We estimate that 15.4% of the variation in endometriosis is captured by DNAm and identify significant differences in DNAm profiles associated with stage III/IV endometriosis, endometriosis sub-phenotypes and menstrual cycle phase, including opening of the window for embryo implantation. Menstrual cycle phase was a major source of DNAm variation suggesting cellular and hormonally-driven changes across the cycle can regulate genes and pathways responsible for endometrial physiology and function. DNAm quantitative trait locus (mQTL) analysis identified 118,185 independent cis-mQTLs including 51 associated with risk of endometriosis, highlighting candidate genes contributing to disease risk. Our work provides functional evidence for epigenetic targets contributing to endometriosis risk and pathogenesis. Data generated serve as a valuable resource for understanding tissue-specific effects of methylation on endometrial biology in health and disease.

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.

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.

Targeting colony stimulating factor-1 receptor signalling to treat ectopic pregnancy.

1-2% of pregnancies are ectopic, the majority implanting in the Fallopian tube. A single, systemic dose of methotrexate, a DNA-synthesis (S phase) inhibitor, has been used since 1991 for outpatient treatment of women with stable EP. However, methotrexate has limited clinical and cost effectiveness, restricting its use to 25-30% of these women. There is an unmet need for better medical treatment for EP. Colony stimulating factor-1 (CSF-1) promotes placentation and creates a pro-inflammatory environment that is fundamental for the maintenance of a normal pregnancy. We hypothesised that CSF-1 is also involved in the placentation and maintenance of an EP. Herein, we demonstrate the immunolocalisation of the CSF-1 receptor (CSF-1R) as well as its ligand (CSF-1) in immortalised first trimester trophoblast cells. We show that a specific CSF-1R kinase inhibitor, GW2580, abolishes CSF-1 induced trophoblast cell proliferation and migration and can be cytotoxic. We then demonstrate the expression of CSF-1R and CSF-1 in the cytotrophoblast and syncytiotrophoblast within ectopic implantation sites from women with EP. Our data suggests that CSF-1 is involved in the survival and proliferation of trophoblast cells in EP. This suggests that pharmacological disruption of CSF-1/CSF-1R signaling axis could be the basis of a new therapeutic for EP.

Author Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

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.

Publisher Correction: The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

The long non-coding RNA HOXB-AS3 regulates ribosomal RNA transcription in NPM1-mutated acute myeloid leukemia.

Long non-coding RNAs (lncRNAs) are important regulatory molecules that are implicated in cellular physiology and pathology. In this work, we dissect the functional role of the HOXB-AS3 lncRNA in patients with NPM1-mutated (NPM1mut) acute myeloid leukemia (AML). We show that HOXB-AS3 regulates the proliferative capacity of NPM1mut AML blasts in vitro and in vivo. HOXB-AS3 is shown to interact with the ErbB3-binding protein 1 (EBP1) and guide EBP1 to the ribosomal DNA locus. Via this mechanism, HOXB-AS3 regulates ribosomal RNA transcription and de novo protein synthesis. We propose that in the context of NPM1 mutations, HOXB-AS3 overexpression acts as a compensatory mechanism, which allows adequate protein production in leukemic blasts.

Effects of Aromatherapy on Pain and Anxiety Scores in Adult Patients Admitted to a Community Hospital on the Medical Unit or Telemetry Unit: A Pilot Study.

The purpose of the study was to determine the impact of aromatherapy intervention on pain and anxiety. The hypothesis was that the use of aromatherapy will improve pain and anxiety scores when assessed within 30 to 60 minutes of administration. The study design was a prospective comparison of aromatherapy using a pre-/postdesign study. A convenience sample of patients was recruited from both a medical unit and a telemetry unit with patients aged 18+ years from a 182-bed acute care Magnet community hospital. Pain and anxiety levels were assessed prior to administration of a medication, within 60 minutes of receiving pain medication, and within 60 minutes of receiving aromatherapy. Ninety-six percent of the participants would use aromatherapy if offered again, would use it in the future, and would recommend its use to family and friends. Both pain and anxiety improved after the aromatherapy with a P value of <.0001. This pilot study demonstrated that aromatherapy is safe and effective at reducing pain and anxiety and should be considered as a valuable adjunct to symptom management.

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.

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.

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.

Immunoprofiling of human uterine mast cells identifies three phenotypes and expression of ERß 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, ERß), 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 ERß+/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 ERß 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.

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.

Estrogen receptor (ER) agonists differentially regulate neuroangiogenesis in peritoneal endometriosis via the repellent factor SLIT3.

Endometriosis is an estrogen-dependent neurovascular disorder characterized by growth of endometrial tissue (lesions) outside the uterine cavity. Patients suffer chronic pelvic pain, and it has been proposed that co-recruitment of nerves/blood vessels (neuroangiogenesis) into the lesions is fundamental to the development of painful symptoms. We hypothesized that estrogen-dependent regulation of axonal guidance molecules of the SLIT/ROBO (Roundabout) family could play a role in neuroangiogenesis occurring in endometriosis lesions found on the peritoneal wall. In tissue samples from human patients and a mouse model of endometriosis, concentrations of mRNA encoded by SLIT3 were significantly higher in lesions than normal peritoneum. Estrogen regulation of SLIT3 was investigated using 17ß-estradiol and selective agonists for each subtype of estrogen receptor (ER) (ERα agonist, 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-tryl) trisphenol; ERß agonist, 2,3-bis(4-hydroxy-phenyl)-propionitrile [DPN]). In mice, DPN (EC50 0.85) increased Slit3 mRNA concentrations compared with hormone-depleted and 17ß-estradiol-treated (EC50 0.1) animals and decreased the density of nerves but not vessels in endometriosis lesions. SLIT3 mRNA concentrations were increased in DPN-treated human endometrial endothelial cells and in 4,4',4″-(4-propyl-(1H)-pyrazole-1,3,5-tryl) trisphenol-treated (EC50 200) rat dorsal root ganglia neurons. Functional assays (neurite outgrowth, network formation) revealed that SLIT3 promotes angiogenesis but decreases neurogenesis. In conclusion, these data suggest that estrogen-dependent expression of SLIT3 may play a key role in regulating nerve-vessel interactions within the complex microenvironment of endometriosis lesions.

Evidence of androgen action in endometrial and ovarian cancers.

Endometrial cancer (EC) and ovarian cancer are common gynaecological malignancies. The impact of androgen action in these cancers is poorly understood; however, there is emerging evidence to suggest that targeting androgen signalling may be of therapeutic benefit. Epidemiological evidence suggests that there is an increased risk of EC associated with exposure to elevated levels of androgens, and genetic variants in genes related to both androgen biosynthesis and action are associated with an increased risk of both EC and ovarian cancer. Androgen receptors (ARs) may be a potential therapeutic target in EC due to reported anti-proliferative activities of androgens. By contrast, androgens may promote growth of some ovarian cancers and anti-androgen therapy has been proposed. Introduction of new therapies targeting ARs expressed in EC or ovarian cancer will require a much greater understanding of the impacts of cell context-specific AR-dependent signalling and how ARs can crosstalk with other steroid receptors during progression of disease. This review considers the evidence that androgens may be important in the aetiology of EC and ovarian cancer with discussion of evidence for androgen action in normal and malignant endometrial and ovarian tissue.

ERß-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1.

STUDY QUESTION: What are the in vitro effects of estrogen receptor ß (ERß) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)? SUMMARY ANSWER: Studies conducted in vitro demonstrate that the ERß agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ERß-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus. WHAT IS KNOWN ALREADY: Estrogens, acting via ERs (ERα and ERß), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ERß. STUDY DESIGN, SIZE, DURATION: Cells were treated with a vehicle control or the ERß agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5). PARTICIPANT/MATERIALS, SETTING, METHODS: Analysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ERß-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ERß signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes. MAIN RESULTS AND THE ROLE OF CHANCE: HEECs and UtMVECs were ERß+/ERα-. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ≤ 0.001) but an increase in UtMVECs (P ≤ 0.05). Genomic analysis identified opposite changes in ERß target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ERß target genes via association with the transcription factor ('tethered' mechanism). LIMITATIONS, REASONS FOR CAUTION: The study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out. WIDER IMPLICATIONS OF THE FINDINGS: These results have implications for our understanding of the mechanisms responsible for ERß-dependent changes in EC gene expression in hormone-dependent disorders.

In silico analysis identifies a novel role for androgens in the regulation of human endometrial apoptosis.

CONTEXT: The endometrium is a multicellular, steroid-responsive tissue that undergoes dynamic remodeling every menstrual cycle in preparation for implantation and, in absence of pregnancy, menstruation. Androgen receptors are present in the endometrium. OBJECTIVE: The objective of the study was to investigate the impact of androgens on human endometrial stromal cells (hESC). DESIGN: Bioinformatics was used to identify an androgen-regulated gene set and processes associated with their function. Regulation of target genes and impact of androgens on cell function were validated using primary hESC. SETTING: The study was conducted at the University Research Institute. PATIENTS: Endometrium was collected from women with regular menses; tissues were used for recovery of cells, total mRNA, or protein and for immunohistochemistry. RESULTS: A new endometrial androgen target gene set (n = 15) was identified. Bioinformatics revealed 12 of these genes interacted in one pathway and identified an association with control of cell survival. Dynamic androgen-dependent changes in expression of the gene set were detected in hESC with nine significantly down-regulated at 2 and/or 8 h. Treatment of hESC with dihydrotestosterone reduced staurosporine-induced apoptosis and cell migration/proliferation. CONCLUSIONS: Rigorous in silico analysis resulted in identification of a group of androgen-regulated genes expressed in human endometrium. Pathway analysis and functional assays suggest androgen-dependent changes in gene expression may have a significant impact on stromal cell proliferation, migration, and survival. These data provide the platform for further studies on the role of circulatory or local androgens in the regulation of endometrial function and identify androgens as candidates in the pathogenesis of common endometrial disorders including polycystic ovarian syndrome, cancer, and endometriosis.