An integrative analysis of endometrial steroid metabolism and transcriptome in relation to endometrial receptivity in in vitro fertilization patients.

OBJECTIVE: To study the relationship between the steroid concentration in the endometrium, in serum, and the gene expression level of steroid-metabolizing enzymes in the context of endometrial receptivity in in vitro fertilization (IVF) patients. DESIGN: Case-control study of 40 IVF patients recruited in the SCRaTCH study (NTR5342), a randomized controlled trial investigating pregnancy outcome after "endometrial scratching." Endometrial biopsies and serum were obtained from patients with a first failed IVF cycle randomized to the endometrial scratch in the midluteal phase of the natural cycle before the next fresh embryo transfer during the second IVF cycle. SETTING: University hopsital. PATIENTS: Twenty women with clinical pregnancy were compared with 20 women who did not conceive after fresh embryo transfer. Cases and controls were matched for primary vs. secondary infertility, embryo quality, and age. INTERVENTION: None. MAIN OUTCOME MEASURE(S): Steroid concentrations in endometrial tissue homogenates and serum were measured with liquid chromatography-mass spectrometry. The endometrial transcriptome was profiled by RNA-sequencing, followed by principal component analysis and differential expression analysis. False discovery rate-adjusted and log-fold change >|0.5| were selected as the threshold for differentially expressed genes. RESULT(S): Estrogen levels were comparable in both serum (n = 16) and endometrium (n = 40). Androgens and 17-hydroxyprogesterone were higher in serum than that in endometrium. Although steroid levels did not vary between pregnant and nonpregnant groups, subgroup analysis of primary women with infertility showed a significantly lower estrone concentration and estrone:androstenedione ratio in serum of the pregnant group (n = 5) compared with the nonpregnant group (n = 2). Expression of 34 out of 46 genes encoding the enzymes controlling the local steroid metabolism was detected, and estrogen receptor ß gene was differentially expressed between pregnant and nonpregnant women. When only the primary infertile group was considered, 28 genes were differentially expressed between pregnant and nonpregnant women, including HSD11B2, that catalyzes the conversion of cortisol into cortisone. CONCLUSION(S): Steroidomic and transcriptomic analyses show that steroid concentrations are regulated by the local metabolism in the endometrium. Although no differences were found in endometrial steroid concentration in the pregnant and nonpregnant IVF patients, primary women with infertility showed deviations in steroid levels and gene expression, indicating that a more homogeneous patient group is required to uncover the exact role of steroid metabolism in endometrial receptivity. CLINICAL TRIAL REGISTRATION NUMBER: The study was registered in the Dutch trial registry (www.trialregister.nl), registration number NL5193/NTR5342, available at https://trialsearch.who.int/Trial2.aspx?TrialID=NTR6687. The date of registration is July 31, 2015. The first enrollment is on January 1, 2016.

Human monocytes differentiate into tumor-associated macrophages upon SKOV3 cells coculture and/or lysophosphatidic acid stimulation.

BACKGROUND: Serous ovarian carcinoma is the most common type of ovarian carcinoma. Tumor-associated macrophages (TAMs) promote ovarian cancer progression. Most macrophages are generated by monocyte differentiation. Lysophosphatidic acid (LPA) levels are high in blood, tissues and ascites of patients with ovarian cancer. This study investigated whether human monocytes can directly differentiate into TAMs in the serous ovarian carcinoma microenvironment. METHODS: Human monocytes were isolated and purified from umbilical cord blood. A serous ovarian carcinoma-like microenvironment was generated by coculturing monocytes and SKOV3 cells in 0.4-µm-pore-size Transwell chambers. Additionally, the effect of LPA was assessed. The two cultured cell types and supernatants were evaluated. RESULTS: The morphology and function of monocytes cocultured with SKOV3 cells and/or stimulated with LPA were significantly changed compared with those of non-stimulated monocytes. The CD14 + CD163 + and CD206 + phenotype indicated that stimulated cells were TAMs. The induced cells promoted SKOV3 cell proliferation and invasion, further proving that they were TAMs. The level of the cytokine interleukin-6R in the supernatant was significantly elevated in the treatment groups compared to the control monocyte group. Pathway enrichment analysis of ELISA results showed a strong influence of interleukin-6 family signaling, especially the JAK-STAT signaling pathway, further confirming the importance of IL-6R. CONCLUSION: Monocytes can differentiate into TAMs under coculture with SKOV3 cells and/or LPA stimulation. The induced TAMs promote SKOV3 cell proliferation and invasion. The cytokine receptor IL-6sR and the JAK-STAT signaling pathway play an important role in the differentiation of monocytes into TAMs.

An irreversible inhibitor of 17ß-hydroxysteroid dehydrogenase type 1 inhibits estradiol synthesis in human endometriosis lesions and induces regression of the non-human primate endometriosis.

Endometriosis is a gynecological disorder affecting about 10% of women and can lead to invalidating painful symptoms and infertility. Since there is no current definitive cure for this disease, new therapeutic options are necessary. 17ß-Hydroxysteroid dehydrogenase type 1 (17ß-HSD1) is involved in the production of estradiol (E2), the most potent estrogen in women, and of 5-androstene-3ß,17ß-diol (5-diol), a weaker estrogen than E2, but whose importance increases after menopause. 17ß-HSD1 is therefore a pharmacological target of choice for the treatment of estrogen-dependent diseases such as endometriosis. We developed a targeted-covalent (irreversible) and non-estrogenic inhibitor of 17ß-HSD1, a molecule named PBRM, and herein evaluated its efficiency for the treatment of endometriosis. In a cell-free assay containing estrone (E1), the natural substrate of 17ß-HSD1, PBRM was able to block the formation of E2 in a collection of 50 human endometriosis lesions from a different clinical feature type, location, and phase. When given orally by gavage at 15 mg/kg to baboons, the resulting plasmatic concentration of PBRM was found to be sufficiently high (up to 125 ng/mL) for an efficacy study in a non-human primate (baboon) endometriosis model. After 2 months of treatment, the number of lesions/adhesions decreased in 60% of animals (3/5) in the PBRM-treated group, compared to the placebo group which showed an increase in the number of lesion/adhesions in 60% (3/5) of animals. Indeed, the total number of lesions/adhesions decreased in treated group (-6.5 or -19% when excluding one animal) while it increased in the control group receiving a placebo (+11%). Analysis of specific endometriotic lesions revealed that PBRM decreased the number of red lesions (-67%; 8/12) and white lesions (-35%; 11/31), but not of blue-black lesions. Similarly, PBRM decreased the surface area of dense adhesions and filmy adhesions, as compared to placebo. Also, PBRM treatment did not significantly affect the number of menstrual days. Finally, this targeted covalent inhibitor showed no adverse effects and no apparent toxicity for the duration of the treatment. These data indicate that 17ß-HSD1 inhibitor PBRM is a promising candidate for therapy targeting endometriosis and supports the need of additional efforts toward clinical trials.

An Applicable Machine Learning Model Based on Preoperative Examinations Predicts Histology, Stage, and Grade for Endometrial Cancer.

Purpose: To build a machine learning model to predict histology (type I and type II), stage, and grade preoperatively for endometrial carcinoma to quickly give a diagnosis and assist in improving the accuracy of the diagnosis, which can help patients receive timely, appropriate, and effective treatment. Materials and Methods: This study used a retrospective database of preoperative examinations (tumor markers, imaging, diagnostic curettage, etc.) in patients with endometrial carcinoma. Three algorithms (random forest, logistic regression, and deep neural network) were used to build models. The AUC and accuracy were calculated. Furthermore, the performance of machine learning models, doctors' prediction, and doctors with the assistance of models were compared. Results: A total of 329 patients were included in this study with 16 features (age, BMI, stage, grade, histology, etc.). A random forest algorithm had the highest AUC and Accuracy. For histology prediction, AUC and accuracy was 0.69 (95% CI=0.67-0.70) and 0.81 (95%CI=0.79-0.82). For stage they were 0.66 (95% CI=0.64-0.69) and 0.63 (95% CI=0.61-0.65) and for differentiation grade 0.64 (95% CI=0.63-0.65) and 0.43 (95% CI=0.41-0.44). The average accuracy of doctors for histology, stage, and grade was 0.86 (with AI) and 0.79 (without AI), 0.64 and 0.53, 0.5 and 0.45, respectively. The accuracy of doctors' prediction with AI was higher than that of Random Forest alone and doctors' prediction without AI. Conclusion: A random forest model can predict histology, stage, and grade of endometrial cancer preoperatively and can help doctors in obtaining a better diagnosis and predictive results.

Clinical analysis and artificial intelligence survival prediction of serous ovarian cancer based on preoperative circulating leukocytes.

Circulating leukocytes are an important part of the immune system. The aim of this work is to explore the role of preoperative circulating leukocytes in serous ovarian carcinoma and investigate whether they can be used to predict survival prognosis. Routine blood test results and clinical information of patients with serous ovarian carcinoma were retrospectively collected. And to predict survival according to the blood routine test result the decision tree method was applied to build a machine learning model.The results showed that the number of preoperative white blood cells (p = 0.022), monocytes (p < 0.001), lymphocytes (p < 0.001), neutrophils (p < 0.001), and eosinophils (p < 0.001) and the monocyte to lymphocyte (MO/LY) ratio in the serous ovarian cancer group were significantly different from those in the control group. These factors also showed a correlation with other clinicopathological characteristics. The MO/LY was the root node of the decision tree, and the predictive AUC for survival was 0.69. The features involved in the decision tree were the MO/LY, differentiation status, CA125 level, neutrophils (NE,) ascites cytology, LY% and age.In conclusion, the number and percentage of preoperative leukocytes in patients with ovarian cancer is changed significantly compared to those in the normal control group, as well as the MO/LY. A decision tree was built to predict the survival of patients with serous ovarian cancer based on the CA125 level, white blood cell (WBC) count, presence of lymph node metastasis (LNM), MO count, the MO/LY ratio, differentiation status, stage, LY%, ascites cytology, and age.

Pharmacological inhibition of 17ß-hydroxysteroid dehydrogenase impairs human endometrial cancer growth in an orthotopic xenograft mouse model.

Endometrial cancer (EC) is the most common gynaecological tumor in developed countries and its incidence is increasing. Approximately 80% of newly diagnosed EC cases are estrogen-dependent. Type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD-1) is the enzyme that catalyzes the final step in estrogen biosynthesis by reducing the weak estrogen estrone (E1) to the potent estrogen 17ß-estradiol (E2), and previous studies showed that this enzyme is implicated in the intratumoral E2 generation in EC. In the present study we employed a recently developed orthotopic and estrogen-dependent xenograft mouse model of EC to show that pharmacological inhibition of the 17ß-HSD-1 enzyme inhibits disease development. Tumors were induced in one uterine horn of athymic nude mice by intrauterine injection of the well-differentiated human endometrial adenocarcinoma Ishikawa cell line, modified to express human 17ß-HSD-1 in levels comparable to EC, and the luciferase and green fluorescent protein reporter genes. Controlled estrogen exposure in ovariectomized mice was achieved using subcutaneous MedRod implants that released either the low active estrone (E1) precursor or vehicle. A subgroup of E1 supplemented mice received daily oral gavage of FP4643, a well-characterized 17ß-HSD-1 inhibitor. Bioluminescence imaging (BLI) was used to measure tumor growth non-invasively. At sacrifice, mice receiving E1 and treated with the FP4643 inhibitor showed a significant reduction in tumor growth by approximately 65% compared to mice receiving E1. Tumors exhibited metastatic spread to the peritoneum, to the lymphovascular space (LVI), and to the thoracic cavity. Metastatic spread and LVI invasion were both significantly reduced in the inhibitor-treated group. Transcriptional profiling of tumors indicated that FP4643 treatment reduced the oncogenic potential at the mRNA level. In conclusion, we show that 17ß-HSD-1 inhibition represents a promising novel endocrine treatment for EC.

Endometriotic cell culture contamination and authenticity: a source of bias in in vitro research?

STUDY QUESTION: Are the primary cell cultures and cell lines used in endometriosis research of sufficient quality? SUMMARY ANSWER: Primary cells used in endometriosis research lack purity and phenotypic characterisation, and cell lines are not genotypically authenticated. WHAT IS KNOWN ALREADY: The poor reproducibility of in vitro research and the lack of authenticity of the cell lines used represent reasons of concern in the field of reproductive biology and endometriosis research. STUDY DESIGN, SIZE, DURATION: In the present study, past in vitro research in the field of endometriosis was systematically reviewed to determine whether the appropriate quality controls were considered. In addition, we explored the performance of Paired Box 2 (Pax2) as an endometrium specific marker in endometrial and endometriotic primary cell cultures; we also characterised the most diffused endometriosis cell lines with respect to important markers including the short tandem repeat (STR) profile. PARTICIPANTS/MATERIALS, SETTING, METHODS: Literature review part: almost 300 published protocols describing the isolation and creation of primary cell cultures from endometriosis were reviewed. Wet-lab part: primary cells isolated from 13 endometriosis patients were analysed by immunohistochemistry, immunofluorescence and FACS for the expression of Pax2. Cell lines Z11 and Z12, the most diffused endometriosis cell lines, were characterised with respect to the expression of Pax2, steroid hormone receptors and STR profile. MAIN RESULTS AND THE ROLE OF CHANCE: From the literature review work, we underscored the lack of sufficient cell purity and phenotypic characterisation of primary cell cultures, which present high risk of contaminations from surrounding non-endometriotic tissues. Past work based on the use of cell lines was reviewed as well, and it emerged that cell line authentication was never performed.In an effort to address these weaknesses for future research, we present data on the performance of Pax2, a suitable marker to exclude ovarian (and other non-endometrial) cell contaminations from primary cell cultures; STR profiles of cell lines Z11 and Z12 were analysed and indicated that the cells were authentic. These profiles are now available for authentication purposes to researchers wishing to perform experiments with these cells.A quality control pipeline to assure sufficient quality of in vitro research in the field of reproductive biology and endometriosis is proposed. We encourage scientists, research institutes, journal reviewers, editors and funding bodies to raise awareness of the problem and adopt appropriate policies to solve it in the future. LARGE-SCALE DATA: STR profiles of cell lines Z11 and Z12 are deposited at the Cellosaurus database-web.expasy.org. LIMITATIONS, REASONS FOR CAUTION: There may be additional markers suitable to assess cell quality. WIDER IMPLICATIONS OF THE FINDINGS: Future in vitro research in endometriosis and the reliability of outcomes can be improved by using the recommendations presented in this study. STUDY FUNDING/COMPETING INTEREST(S): The study was partly financed by the 'Stichting Fertility Foundation' (The Netherlands). The authors declare no existing conflict of interest. TRIAL REGISTRATION NUMBER: Non-applicable.

Blood Metabolites Associate with Prognosis in Endometrial Cancer.

Endometrial cancer has a high prevalence among post-menopausal women in developed countries. We aimed to explore whether certain metabolic patterns could be related to the characteristics of aggressive disease and poorer survival among endometrial cancer patients in Western Norway. Patients with endometrial cancer with short survival (n = 20) were matched according to FIGO (International Federation of Gynecology and Obstetrics, 2009 criteria) stage, histology, and grade, with patients with long survival (n = 20). Plasma metabolites were measured on a multiplex system including 183 metabolites, which were subsequently determined using liquid chromatography-mass spectrometry. Partial least square discriminant analysis, together with hierarchical clustering, was used to identify patterns which distinguished short from long survival. A proposed signature of metabolites related to survival was suggested, and a multivariate receiver operating characteristic (ROC) analysis yielded an area under the curve (AUC) of 0.820-0.965 (p ≤ 0.001). Methionine sulfoxide seems to be particularly strongly associated with poor survival rates in these patients. In a subgroup with preoperative contrast-enhanced computed tomography data, selected metabolites correlated with the estimated abdominal fat distribution parameters. Metabolic signatures may predict prognosis and be promising supplements when evaluating phenotypes and exploring metabolic pathways related to the progression of endometrial cancer. In the future, this may serve as a useful tool in cancer management.

Blood steroids are associated with prognosis and fat distribution in endometrial cancer.

BACKGROUND: Despite being a hormone dependent cancer, there is limited knowledge regarding the relation between level of steroids in blood and prognosis for endometrial cancer (EC) patients. METHODS: In this study we investigated plasma levels of 19 steroids using liquid-chromatography tandem mass-spectrometry in 38 postmenopausal EC patients, 19 with long, and 19 with short survival. We explored if estradiol levels were associated with specific abdominal fat distribution patterns and if transcriptional alterations related to estradiol levels could be observed in tumor samples. RESULTS: The plasma steroid levels for DHEA, DHEAS, progesterone, 21 OH progesterone and E1S were significantly increased (all p < 0.05) in patients with long survival compared to short. Estradiol levels were significantly positively correlated with visceral fat percentage (p = 0.035), and an increased expression of genes involved in estrogen related signaling was observed in tumors from patients with high estradiol levels in plasma. CONCLUSION: Several of the identified plasma steroids represent promising biomarkers in EC patients. The association between increased estradiol levels and a high percentage of visceral fat indicates that visceral fat is a larger contributor to estradiol production compared to subcutaneous fat in this population.

Intracrine Regulation of Estrogen and Other Sex Steroid Levels in Endometrium and Non-gynecological Tissues; Pathology, Physiology, and Drug Discovery.

Our understanding of the intracrine (or local) regulation of estrogen and other steroid synthesis and degradation expanded in the last decades, also thanks to recent technological advances in chromatography mass-spectrometry. Estrogen responsive tissues and organs are not passive receivers of the pool of steroids present in the blood but they can actively modify the intra-tissue steroid concentrations. This allows fine-tuning the exposure of responsive tissues and organs to estrogens and other steroids in order to best respond to the physiological needs of each specific organ. Deviations in such intracrine control can lead to unbalanced steroid hormone exposure and disturbances. Through a systematic bibliographic search on the expression of the intracrine enzymes in various tissues, this review gives an up-to-date view of the intracrine estrogen metabolisms, and to a lesser extent that of progestogens and androgens, in the lower female genital tract, including the physiological control of endometrial functions, receptivity, menopausal status and related pathological conditions. An overview of the intracrine regulation in extra gynecological tissues such as the lungs, gastrointestinal tract, brain, colon and bone is given. Current therapeutic approaches aimed at interfering with these metabolisms and future perspectives are discussed.

Development of an Image-Guided Orthotopic Xenograft Mouse Model of Endometrial Cancer with Controllable Estrogen Exposure.

Endometrial cancer (EC) is the most common gynaecological malignancy in Western society and the majority of cases are estrogen dependent. While endocrine drugs proved to be of insufficient therapeutic value in the past, recent clinical research shows promising results by using combinational regimens and pre-clinical studies and identified potential novel endocrine targets. Relevant pre-clinical models can accelerate research in this area. In the present study we describe an orthotopic and estrogen dependent xenograft mouse model of EC. Tumours were induced in one uterine horn of female athymic nude mice using the well-differentiated human endometrial adenocarcinoma Ishikawa cell line-modified to express the luciferase gene for bioluminescence imaging (BLI). BLI and contrast-enhanced computed-tomograph (CE-CT) were used to measure non-invasive tumour growth. Controlled estrogen exposure was achieved by the use of MedRod implants releasing 1.5 µg/d of 17ß-estradiol (E2) in ovariectomized mice. Stable E2 serum concentration was demonstrated by LC-MS/MS. Induced tumours were E2 responsive as increased tumour growth was observed in the presence of E2 but not placebo, assessed by BLI, CE-CT, and tumour weight at sacrifice. Metastatic spread was assessed macroscopically by BLI and histology and was seen in the peritoneal cavity, in the lymphovascular space, and in the thoracic cavity. In conclusion, we developed an orthotopic xenograft mouse model of EC that exhibits the most relevant features of human disease, regarding metastatic spread and estrogen dependency. This model offers an easy to manipulate estrogen dosage (by simply adjusting the MedRod implant length), image-guided monitoring of tumour growth, and objectively measurable endpoints (including tumour weight). This is an excellent in vivo tool to further explore endocrine drug regimens and novel endocrine drug targets for EC.

Blocking 17ß-hydroxysteroid dehydrogenase type 1 in endometrial cancer: a potential novel endocrine therapeutic approach.

The enzyme type 1 17ß-hydroxysteroid dehydrogenase (17ß-HSD-1), responsible for generating active 17ß-estradiol (E2) from low-active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra-tissue generation of E2 in this estrogen-dependent condition. In this study, we explored the possibility of inhibiting 17ß-HSD-1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well-differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17ß-HSD-1 similar to human tissues. In these cells, HPLC analysis showed that 17ß-HSD-1 activity could be blocked by a specific 17ß-HSD-1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17ß-HSD-1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17ß-HSD-1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17ß-HSD-1-negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17ß-HSD-1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17ß-HSD-1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17ß-HSD-1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17ß-HSD-1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17ß-HSD-1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

High mRNA levels of 17ß-hydroxysteroid dehydrogenase type 1 correlate with poor prognosis in endometrial cancer.

Most endometrial cancers (ECs) are diagnosed at an early stage and have a good prognosis. However, 20-30% develop recurrence and have poor survival. Recurrence-risk prediction at diagnosis is hampered by the scarcity of prognostic markers. Most ECs are estrogen related, and recent studies show that estrogen exposure in EC is controlled intracrinally. We aim at assessing any association between patient prognosis and the pathways controlling the intracrine estrogen generation in EC: (a) the balance between 17ß-hydroxysteroid-dehydrogenase-type 1 (HSD17B1), that generates active estrogens, and HSD17B2, converting active into poorly active compounds; (b) the balance between steroid sulphatase (STS, that activates estrogens) and estrogen-sulphotransferase (SULT1E1, that deactivates estrogens); (c) the levels of aromatase (ARO), that converts androgen into estrogens. mRNA levels of HSD17B1, HSD17B2, STS, SULT1E1 and ARO were determined among 175 ECs using cDNA microarray. Proteins were explored by immunohistochemistry. Patients with high mRNA of HSD17B1 had a poorer prognosis compared with those with low levels. Combining the expression of HSD17B1 and HSD17B2, patients with high tumour expression of HSD17B1 and low levels of HSD17B2 had the poorest prognosis. Contrarily, women that had high tumour levels of HSD17B2 and low of HSD17B1 had the best outcome. No differences were seen between mRNA level of other the genes analysed and prognosis. At the protein level, HSD17B2, STS and SULT1E1 were highly expressed, whereas HSD17B1 was low and ARO was almost absent. In conclusion, HSD17B1 is a promising marker to predict EC prognosis. Immunohistochemical detection of this protein in ECs has low sensitivity and should be improved for future clinical applications.

Germ-line variants identified by next generation sequencing in a panel of estrogen and cancer associated genes correlate with poor clinical outcome in Lynch syndrome patients.

BACKGROUND: The risk to develop colorectal and endometrial cancers among subjects testing positive for a pathogenic Lynch syndrome mutation varies, making the risk prediction difficult. Genetic risk modifiers alter the risk conferred by inherited Lynch syndrome mutations, and their identification can improve genetic counseling. We aimed at identifying rare genetic modifiers of the risk of Lynch syndrome endometrial cancer. METHODS: A family based approach was used to assess the presence of genetic risk modifiers among 35 Lynch syndrome mutation carriers having either a poor clinical phenotype (early age of endometrial cancer diagnosis or multiple cancers) or a neutral clinical phenotype. Putative genetic risk modifiers were identified by Next Generation Sequencing among a panel of 154 genes involved in endometrial physiology and carcinogenesis. RESULTS: A simple pipeline, based on an allele frequency lower than 0.001 and on predicted non-conservative amino-acid substitutions returned 54 variants that were considered putative risk modifiers. The presence of two or more risk modifying variants in women carrying a pathogenic Lynch syndrome mutation was associated with a poor clinical phenotype. CONCLUSION: A gene-panel is proposed that comprehends genes that can carry variants with putative modifying effects on the risk of Lynch syndrome endometrial cancer. Validation in further studies is warranted before considering the possible use of this tool in genetic counseling.

Inhibition of type 1 17ß-hydroxysteroid dehydrogenase impairs the synthesis of 17ß-estradiol in endometriosis lesions.

CONTEXT: Endometriosis affects 10% of the women before menopause and has important personal, professional, and societal economic burdens. Because current medical treatments are aimed at reducing the symptoms only, novel therapeutic targets should be identified. Endometriosis is estrogen dependent and in some patients the endometriosis tissue is able to produce estrogens in an autocrine/paracrine manner. In a number of patients, this is the consequence of the high local activity of the 17ß-hydroxysteroid-dehydrogenases (17ß-HSDs), enzymes able to generate active estrogens from precursors with low activity. OBJECTIVE: The objective of the study was to identify the 17ß-HSD(s) responsible for the high local generation of estrogens in endometriosis and test the possibility to inhibit these enzymes for therapeutic purposes. DESIGN: The expression of different 17ß-HSDs involved in the estrogen metabolism was assessed by real-time PCR in eutopic and ectopic tissue from endometriosis patients (n=14). These biopsies had previously confirmed unbalanced local 17ß-HSD activity, which caused high estrogen generation. The possibility to block the synthesis of estrogens by one inhibitor specific for type 1 17ß-HSD was assessed by HPLC in tissue lysates from endometriosis tissues (n=27). RESULTS: In all but one of the patients, a high type 1 17ß-HSD level is associated with the unbalanced metabolism of estrogens, leading to higher estrogen synthesis in endometriosis than in the endometrium inside the uterus. Inhibition of type 1 17ß-HSD restores to various extents, depending on the patient, the correct metabolism. In 19 of 27 patients analyzed (70%), the 17ß-HSD type 1 inhibitor decreased the generation of 17ß-estradiol by greater than 85%. CONCLUSIONS: Inhibition of 17ß-HSD type 1 can be a potential future treatment option aimed at restoring the correct metabolic balance of estrogens in endometriosis patients with increased local 17ß-HSD type 1 enzyme activity.

Towards endometriosis diagnosis by gadofosveset-trisodium enhanced magnetic resonance imaging.

Endometriosis is defined as the presence of endometrial tissue outside the uterus. It affects 10-15% of women during reproductive age and has a big personal and social impact due to chronic pelvic pain, subfertility, loss of work-hours and medical costs. Such conditions are exacerbated by the fact that the correct diagnosis is made as late as 8-11 years after symptom presentation. This is due to the lack of a reliable non-invasive diagnostic test and the fact that the reference diagnostic standard is laparoscopy (invasive, expensive and not without risks). High-molecular weight gadofosveset-trisodium is used as contrast agent in Magnetic Resonance Imaging (MRI). Since it extravasates from hyperpermeable vessels more easily than from mature blood vessels, this contrast agent detects angiogenesis efficiently. Endometriosis has high angiogenic activity. Therefore, we have tested the possibility to detect endometriosis non-invasively using Dynamic Contrast-Enhanced MRI (DCE-MRI) and gadofosveset-trisodium as a contrast agent in a mouse model. Endometriotic lesions were surgically induced in nine mice by autologous transplantation. Three weeks after lesion induction, mice were scanned by DCE-MRI. Dynamic image analysis showed that the rates of uptake (inwash), persistence and outwash of the contrast agent were different between endometriosis and control tissues (large blood vessels and back muscle). Due to the extensive angiogenesis in induced lesions, the contrast agent persisted longer in endometriotic than control tissues, thus enhancing the MRI signal intensity. DCE-MRI was repeated five weeks after lesion induction, and contrast enhancement was similar to that observed three weeks after endometriosis induction. The endothelial-cell marker CD31 and the pericyte marker α-smooth-muscle-actin (mature vessels) were detected with immunohistochemistry and confirmed that endometriotic lesions had significantly higher prevalence of new vessels (CD31 only positive) than the uterus and control tissues. The diagnostic value of gadofosveset-trisodium to detect endometriosis should be tested in human settings.

Overexpression of 17ß-hydroxysteroid dehydrogenase type 1 increases the exposure of endometrial cancer to 17ß-estradiol.

CONTEXT: The local interconversions between estrone (low activity) and 17ß-estradiol (potent compound) by 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) can lead to high 17ß-estradiol generation in endometrial cancer (EC). OBJECTIVE: Examine the balance between the 17ß-HSDs reducing estrone to 17ß-estradiol (types 1, 5, 12, and 7) and those oxidizing 17ß-estradiol to estrone (2, 4, and 8), in EC. PATIENTS AND METHODS: Reducing and oxidizing 17ß-HSD activities (HPLC) and mRNA level (RT-PCR) were assessed in normal post-menopausal (n = 16), peritumoral endometrium (normal tissue beside cancer, n = 13), and 58 EC (29 grade 1, 18 grade 2, 11 grade 3). RESULTS: Grade 1 EC displayed a shifted estrone reduction/17ß-estradiol oxidation balance in favor of 17ß-estradiol compared with controls. This was more pronounced among estrogen receptor-α (ER-α)-positive biopsies. Type 1 17ß-HSD mRNA (HSD17B1 gene expression, real time PCR) and protein levels (immunohistochemistry) were higher in ER-α-positive grade 1 EC than controls. The mRNA coding for types 4, 5, 7, 8, and 12 17ß-HSD did not vary, whereas that coding for type 2 17ß-HSD was increased in high-grade lesions compared with controls. Three-dimensional ex vivo EC explant cultures demonstrated that 17ß-HSD type 1 generated 17ß-estradiol from estrone and increased tumor cell proliferation. Additional in vitro studies using EC cells confirmed that in the presence of 17ß-HSD type 1, estrone induced estrogen signaling activation similarly to 17ß-estradiol. Therefore, estrone was reduced to 17ß-estradiol. CONCLUSIONS: Type 1 17ß-HSD increases 17ß-estradiol exposure in grade 1 EC, thus supporting tumor growth. This enzyme represents a potential therapeutic target.

Deoxyribonucleic acid methyltransferases and methyl-CpG-binding domain proteins in human endometrium and endometriosis.

OBJECTIVE: To determine [1] expression levels of both DNA methyltransferases (DNMTs) and methyl-CpG-binding domain proteins (MBDs) in human endometrium throughout the menstrual cycle and in eutopic and ectopic endometrium of patients with endometriosis and [2] hormone responsiveness of DNMT and MBD expression in explant cultures of proliferative phase endometrium. DESIGN: In vitro study. SETTING: Academic medical center. PATIENT(S): Premenopausal women with and without endometriosis. INTERVENTION(S): Explant cultures of proliferative phase endometrium were treated with vehicle, 17ß-E(2), or a combination of E(2) and P (E(2) + P) for 24 hours. MAIN OUTCOME MEASURE(S): Expression levels of DNMT1, DNMT2, and DNMT3B and MBD1, MBD2, and MeCP2 with use of real-time quantitative polymerase chain reaction. RESULT(S): Expression levels of DNMT1 and MBD2 were significantly higher in secretory-phase endometrium compared with proliferative endometrium and menstrual endometrium. In explant cultures, treatment with E(2) + P resulted in significant up-regulation of DNMT1 and MBD2. Expression levels of several DNMTs and MBDs were significantly lower in endometriotic lesions compared with eutopic endometrium of women with endometriosis and disease-free controls. CONCLUSION(S): These findings suggest a role for DNMTs and MBDs in the growth and differentiation of the human endometrium and support the notion that endometriosis may be an epigenetic disease.

Olfactomedin-4 regulation by estrogen in the human endometrium requires epidermal growth factor signaling.

Olfactomedin-4 (OLFM-4) is an extracellular matrix protein that is highly expressed in human endometrium. We have examined the regulation and function of OLFM-4 in normal endometrium and in cases of endometriosis and endometrial cancer. OLFM-4 expression levels are highest in proliferative-phase endometrium, and 17ß-estradiol up-regulates OLFM-4 mRNA in endometrial explant cultures. Using the luciferase reporter under control of the OLFM-4 promoter, it was shown that both 17ß-estradiol and OH-tamoxifen induce luciferase activity, and epidermal growth factor receptor-1 is required for this estrogenic response. In turn, EGF activates the OLFM-4 promoter, and estrogen receptor-α is needed for the complete EGF response. The cellular functions of OLFM-4 were examined by its expression in OLFM-4-negative HEK-293 cells, which resulted in decreased vimentin expression and cell adherence as well as increased apoptosis resistance. In cases of endometriosis and endometrial cancer, OLFM-4 expression correlated with the presence of epidermal growth factor receptor-1 and estrogen receptor-α (or estrogen signaling). An increase of OLFM-4 mRNA was observed in the endometrium of endometriosis patients. No change in OLFM-4 expression levels were observed in patients with endometrial cancer relative with controls. In conclusion, cross-talk between estrogen and EGF signaling regulates OLFM-4 expression. The role of OLFM-4 in endometrial tissue remodeling before the secretory phase and during the predisposition and early events in endometriosis can be postulated but requires additional investigation.

Cyclin D1 regulates hepatic estrogen and androgen metabolism.

Cyclin D1 is a cell cycle control protein that plays an important role in regenerating liver and many types of cancer. Previous reports have shown that cyclin D1 can directly enhance estrogen receptor activity and inhibit androgen receptor activity in a ligand-independent manner and thus may play an important role in hormone-responsive malignancies. In this study, we examine a distinct mechanism by which cyclin D1 regulates sex steroid signaling, via altered metabolism of these hormones at the tissue and cellular level. In male mouse liver, ectopic expression of cyclin D1 regulated genes involved in the synthesis and degradation of sex steroid hormones in a pattern that would predict increased estrogen and decreased androgen levels. Indeed, hepatic expression of cyclin D1 led to increased serum estradiol levels, increased estrogen-responsive gene expression, and decreased androgen-responsive gene expression. Cyclin D1 also regulated the activity of several key enzymatic reactions in the liver, including increased oxidation of testosterone to androstenedione and decreased conversion of estradiol to estrone. Similar findings were seen in the setting of physiological cyclin D1 expression in regenerating liver. Knockdown of cyclin D1 in HuH7 cells produced reciprocal changes in steroid metabolism genes compared with cyclin D1 overexpression in mouse liver. In conclusion, these studies establish a novel link between the cell cycle machinery and sex steroid metabolism and provide a distinct mechanism by which cyclin D1 may regulate hormone signaling. Furthermore, these results suggest that increased cyclin D1 expression, which occurs in liver regeneration and liver diseases, may contribute to the feminization seen in these settings.