Gene expression profiles separate endometriosis lesion subtypes and indicate a sensitivity of endometrioma to estrogen suppressive treatments through elevated ESR2 expression.

BACKGROUND: Endometriosis is a common, gynaecological disease characterised by the presence of endometrial-like cells growing outside the uterus. Lesions appear at multiple locations, present with variation in appearance, size and depth of invasion. Despite hormones being the recommended first-line treatment, their efficacy, success and side effects vary widely amongst study populations. Current, hormonal medication for endometriosis is designed to suppress systemic oestrogen. Whether these hormones can influence the lesions themselves is not yet clear. Evidence of hormone receptor expression in endometriotic lesions and their ability to respond is conflicting. A variation in their expression, activation of transcriptional co-regulators and the potential to respond may contribute to their variation in patient outcomes. Identifying patients who would benefit from hormonal treatments remain an important goal in endometriosis research. METHODS: Using gene expression data from endometriosis lesions including endometrioma (OMA, n = 28), superficial peritoneal lesions (SUP, n = 72) and deeply infiltrating lesions (DIE, n = 78), we performed principal component analysis, differential gene expression and gene correlation analyses to assess the impact of menstrual stage, lesion subtype and hormonal treatment on the gene expression. RESULTS: The gene expression profiles did not vary based on menstrual stage, but could distinguish lesion subtypes with OMA significantly differentiating from both SUP and DIE. Additionally, the effect of oestrogen suppression medication altered the gene expression profile in OMA, while such effect was not observed in SUP or DIE. Analysis of the target receptors for hormonal medication indicated ESR2 was differentially expressed in OMA and that genes that correlated with ESR2 varied significantly between medicated and non-medicated OMA samples. CONCLUSIONS: Our results demonstrate of the different lesion types OMA present with strongest response to hormonal treatment directly through ESR2. The data suggests that there may be the potential to target treatment options to individual patients based on pre-surgical diagnoses.

Histone H3K4me3 breadth in hypoxia reveals endometrial core functions and stress adaptation linked to endometriosis.

Trimethylation of histone H3 at lysine 4 (H3K4me3) is a marker of active promoters. Broad H3K4me3 promoter domains have been associated with cell type identity, but H3K4me3 dynamics upon cellular stress have not been well characterized. We assessed this by exposing endometrial stromal cells to hypoxia, which is a major cellular stress condition. We observed that hypoxia modifies the existing H3K4me3 marks and that promoter H3K4me3 breadth rather than height correlates with transcription. Broad H3K4me3 domains mark genes for endometrial core functions and are maintained or selectively extended upon hypoxia. Hypoxic extension of H3K4me3 breadth associates with stress adaptation genes relevant for the survival of endometrial cells including transcription factor KLF4, for which we found increased protein expression in the stroma of endometriosis lesions. These results substantiate the view on broad H3K4me3 as a marker of cell identity genes and reveal participation of H3K4me3 extension in cellular stress adaptation.

Overexpression of Human Estrogen Biosynthetic Enzyme Hydroxysteroid (17beta) Dehydrogenase Type 1 Induces Adenomyosis-like Phenotype in Transgenic Mice.

Hydroxysteroid (17beta) dehydrogenase type 1 (HSD17B1) is an enzyme that converts estrone to estradiol, while adenomyosis is an estrogen-dependent disease with poorly understood pathophysiology. In the present study, we show that mice universally over-expressing human estrogen biosynthetic enzyme HSD17B1 (HSD17B1TG mice) present with adenomyosis phenotype, characterized by histological and molecular evaluation. The first adenomyotic changes with endometrial glands partially or fully infiltrated into the myometrium appeared at the age of 5.5 months in HSD17B1TG females and became more prominent with increasing age. Preceding the phenotype, increased myometrial smooth muscle actin positivity and increased amount of glandular myofibroblast cells were observed in HSD17B1TG uteri. This was accompanied by transcriptomic upregulation of inflammatory and estrogen signaling pathways. Further, the genes upregulated in the HSD17B1TG uterus were enriched with genes previously observed to be induced in the human adenomyotic uterus, including several genes of the NFKB pathway. A 6-week-long HSD17B1 inhibitor treatment reduced the occurrence of the adenomyotic changes by 5-fold, whereas no effect was observed in the vehicle-treated HSD17B1TG mice, suggesting that estrogen is the main upstream regulator of adenomyosis-induced uterine signaling pathways. HSD17B1 is considered as a promising drug target to inhibit estrogen-dependent growth of endometrial disorders. The present data indicate that HSD17B1 over-expression in TG mice results in adenomyotic changes reversed by HSD17B1 inhibitor treatment and HSD17B1 is, thus, a potential novel drug target for adenomyosis.

A relational database to identify differentially expressed genes in the endometrium and endometriosis lesions.

Endometriosis is a common inflammatory estrogen-dependent gynecological disorder, associated with pelvic pain and reduced fertility in women. Several aspects of this disorder and its cellular and molecular etiology remain unresolved. We have analyzed the global gene expression patterns in the endometrium, peritoneum and in endometriosis lesions of endometriosis patients and in the endometrium and peritoneum of healthy women. In this report, we present the EndometDB, an interactive web-based user interface for browsing the gene expression database of collected samples without the need for computational skills. The EndometDB incorporates the expression data from 115 patients and 53 controls, with over 24000 genes and clinical features, such as their age, disease stages, hormonal medication, menstrual cycle phase, and the different endometriosis lesion types. Using the web-tool, the end-user can easily generate various plot outputs and projections, including boxplots, and heatmaps and the generated outputs can be downloaded in pdf-format.Availability and implementationThe web-based user interface is implemented using HTML5, JavaScript, CSS, Plotly and R. It is freely available from https://endometdb.utu.fi/ .

Exploratory Analysis of CA125-MGL and -STn Glycoforms in the Differential Diagnostics of Pelvic Masses.

BACKGROUND: The cancer antigen 125 (CA125) immunoassay (IA) does not distinguish epithelial ovarian cancer (EOC) from benign disease with the sensitivity needed in clinical practice. In recent studies, glycoforms of CA125 have shown potential as biomarkers in EOC. Here, we assessed the diagnostic abilities of two recently developed CA125 glycoform assays for patients with a pelvic mass. Detailed analysis was further conducted for postmenopausal patients with marginally elevated conventionally measured CA125 levels, as this subgroup presents a diagnostic challenge in the clinical setting. METHODS: Our study population contained 549 patients diagnosed with EOC, benign ovarian tumors, and endometriosis. Of these, 288 patients were postmenopausal, and 98 of them presented with marginally elevated serum levels of conventionally measured CA125 at diagnosis. Preoperative serum levels of conventionally measured CA125 and its glycoforms (CA125-MGL and CA125-STn) were determined. RESULTS: The CA125-STn assay identified EOC significantly better than the conventional CA125-IA in postmenopausal patients (85% vs. 74% sensitivity at a fixed specificity of 90%, P = 0.0009). Further, both glycoform assays had superior AUCs compared to the conventional CA125-IA in postmenopausal patients with marginally elevated CA125. Importantly, the glycoform assays reduced the false positive rate of the conventional CA125-IA. CONCLUSIONS: The results indicate that the CA125 glycoform assays markedly improve the performance of the conventional CA125-IA in the differential diagnosis of pelvic masses. This result is especially valuable when CA125 is marginally elevated.

Transcriptomic responses to hypoxia in endometrial and decidual stromal cells.

Human reproductive success depends on a properly decidualized uterine endometrium that allows implantation and the formation of the placenta. At the core of the decidualization process are endometrial stromal fibroblasts (ESF) that differentiate to decidual stromal cells (DSC). As variations in oxygen levels are functionally relevant in endometrium both upon menstruation and during placentation, we assessed the transcriptomic responses to hypoxia in ESF and DSC. In both cell types, hypoxia-upregulated genes in classical hypoxia pathways such as glycolysis and the epithelial mesenchymal transition. In DSC, hypoxia restored an ESF-like transcriptional state for a subset of transcription factors that are known targets of the progesterone receptor, suggesting that hypoxia partially interferes with progesterone signaling. In both cell types, hypoxia modified transcription of several inflammatory transcription factors that are known regulators of decidualization, including decreased transcription of STATs and increased transcription of CEBPs. We observed that hypoxia-upregulated genes in ESF and DSC had a significant overlap with genes previously detected to be upregulated in endometriotic stromal cells. Promoter analysis of the genes in this overlap suggested the hypoxia-upregulated Jun/Fos and CEBP transcription factors as potential drivers of endometriosis-associated transcription. Using immunohistochemistry, we observed increased expression of JUND and CEBPD in endometriosis lesions compared to healthy endometria. Overall, the findings suggest that hypoxic stress establishes distinct transcriptional states in ESF and DSC and that hypoxia influences the expression of genes that contribute to the core gene regulation of endometriotic stromal cells.

Europium Nanoparticle-Based Sialyl-Tn Monoclonal Antibody Discriminates Epithelial Ovarian Cancer-Associated CA125 from Benign Sources.

BACKGROUND: The Sialyl-Thomsen-nouveau antigen (STn) is abundantly produced on many types of human epithelial cancers including epithelial ovarian cancer (EOC). We previously developed an EOC-specific lectin sandwich immunoassay (CA125MGL) using a human macrophage galactose-binding lectin coated on fluorescent europium nanoparticles (Eu+3-NPs) as a tracer and an anti-CA125-specific mAb for capture. Here we have identified a novel STn-mAb that efficiently recognizes the EOC-associated STn antigen on CA125 when coated on Eu+3-NPs. METHOD: CA125 from the ovarian cancer cell line OVCAR-3, placental homogenate, and ascites fluid from patients with liver cirrhosis was captured by anti-CA125 antibody immobilized on microtitration wells and traced with anti-STn-mAb-Eu+3-NPs. Samples from EOC or patients with endometriosis with marginally increased conventional CA125 immunoassay (CA125IA; 35-200 U/mL) and healthy controls were analyzed. RESULTS: An analytically sensitive CA125STn assay that specifically recognized the CA125 isoform produced by OVCAR-3 was achieved. Serum CA125STn concentration was significantly higher in EOC patients than in those with endometriosis (P < 0.001). Furthermore, the sensitivity for detection of EOC with CA125STn assay was 73.3% when 95% of endometriosis cases were undetectable. CONCLUSION: Our findings suggest that Eu+3-NPs-based CA125STn assay could help reduce the false-positive rates of CA125IA to improve differential diagnosis. The results encourage studying further the potential use of CA125STn to detect EOC at earlier clinical stages. This approach warrants further investigation in other cancers as well.

Personalized Drug Sensitivity Screening for Bladder Cancer Using Conditionally Reprogrammed Patient-derived Cells.

Many patients with muscle-invasive bladder cancer (BC) are either ineligible for or do not benefit from cisplatin-based chemotherapy, and there is an unmet need to estimate individuals' drug sensitivities. We investigated the suitability of conditionally reprogrammed (CR) cells for the characterization of BC properties and their feasibility for personalized drug sensitivity screening. The CR cultures were established from six BC tumors with varying histology and stage. Four cultures were successfully propagated for genomic, transcriptomic, and protein expression profiling and compared to the parental tumors. Two out of four CR cultures (urothelial carcinoma and small cell neuroendocrine carcinoma [SmCC]) corresponded well to their parental tumors and underwent drug sensitivity screening to identify novel drugs for the respective tumors. Both cultures were sensitive to standard BC chemotherapy agents (eg cisplatin and gemcitabine) and to conventional drugs such as taxanes and inhibitors of topoisomerase and proteasome. The SmCC cells were also sensitive to statins (eg, atorvastatin and pitavastatin). In summary, after confirming their representativeness and origin, we conclude that CR cells are a feasible platform for personalized drug sensitivity testing and might thus add to the approaches used to personalize BC treatment strategies. PATIENT SUMMARY: We investigated the conditional reprogramming method for generating patient-derived bladder cancer cell cultures and studied their feasibility for planning personalized treatment strategies.

Role of hydroxysteroid (17beta) dehydrogenase type 1 in reproductive tissues and hormone-dependent diseases.

Abnormal synthesis and metabolism of sex steroids is involved in the pathogenesis of various human diseases, such as endometriosis and cancers arising from the breast and uterus. Steroid biosynthesis is a multistep enzymatic process proceeding from cholesterol to highly active sex steroids via different intermediates. Human Hydroxysteroid (17beta) dehydrogenase 1 (HSD17B1) enzyme shows a high capacity to produce the highly active estrogen, estradiol, from a precursor hormone, estrone. However, the enzyme may also play a role in other steps of the steroid biosynthesis pathway. In this article, we have reviewed the literature on HSD17B1, and summarize the role of the enzyme in hormone-dependent diseases in women as evidenced by preclinical studies.

Analysis by LC-MS/MS of endogenous steroids from human serum, plasma, endometrium and endometriotic tissue.

An LC-MS/MS method was developed and validated to analyze simultaneously estrogens (estradiol, E2; estrone, E1), androgens (testosterone, T; androstenedione, A4; dehydroepiandrosterone, DHEA), progestagens (17a-hydroxypregnenolone, 17OHP5; 17a-hydroxyprogesterone, 17OHP4; progesterone, P4), glucocorticoids (cortisol, F; cortisone E; corticosterone, B; 11-deoxycortisol, S; 21-hydroxyprogesterone, 21OHP4), and mineralocorticoids (aldosterone, A) from 150 µl of human serum, plasma, or endometrium and endometriotic tissue homogenates. Samples spiked with isotope-labeled steroids as internal standards were extracted with toluene prior to LC-MS/MS analysis. The chromatographic separation of underivatized steroids was achieved on a biphenyl column with 0.2 mM NH4F as the eluent additive and a water-methanol gradient to improve E2 and E1 ionization. Method validation was performed with human plasma samples, and analysis of certified E2, T, F, and P4 reference serums (BCR-576, ERM-DA346, ERM-DA192, ERM-DA347), as well as homogenates of endometrium and endometriotic tissue. A total of 27 steroids were included in the method development to ensure the specificity of the method. After validation, the method was found suitable for quantitative analysis of 11 steroids: E2 (6.7 pM-13 nM), E1 (1.3 pM-6.6 nM), T (3.3 pM-13 nM), A4 (13 pM-33 nM), 17OHP5 (32 pM-65 nM), 17OHP4 (33 pM-13 nM), F (33 pM-133 nM), E (13 pM-130 nM), B (33 pM-134 nM), S (13 pM-129 nM), and A (32 pM-32 nM). In addition, DHEA (333 pM-32 nM), P4 (13 pM-13 nM) and 21OHP4 (13 pM-13 nM) can be analyzed semiquantitatively.

HSD17B1 expression induces inflammation-aided rupture of mammary gland myoepithelium.

Hydroxysteroid (17-beta) dehydrogenase type 1 (HSD17B1) converts low-active estrogen estrone to highly active estradiol. Estradiol is necessary for normal postpubertal mammary gland development; however, elevated estradiol levels increase mammary tumorigenesis. To investigate the significance of the human HSD17B1 enzyme in the mammary gland, transgenic mice universally overexpressing human HSD17B1 were used (HSD17B1TG mice). Mammary glands obtained from HSD17B1TG females at different ages were investigated for morphology and histology, and HSD17B1 activity and estrogen receptor activation in mammary gland tissue were assessed. To study the significance of HSD17B1 enzyme expression locally in mammary gland tissue, HSD17B1-expressing mammary epithelium was transplanted into cleared mammary fat pads of wild-type females, and the effects on mammary gland estradiol production, epithelial cells and the myoepithelium were investigated. HSD17B1TG females showed increased estrone to estradiol conversion and estrogen-response element-driven estrogen receptor signaling in mammary gland tissue, and they showed extensive lobuloalveolar development that was further enhanced by age along with an increase in serum prolactin concentrations. At old age, HSD17B1TG females developed mammary cancers. Mammary-restricted HSD17B1 expression induced lesions at the sites of ducts and alveoli, accompanied by peri- and intraductal inflammation and disruption of the myoepithelial cell layer. The lesions were shown to be estrogen dependent, as treatment with an antiestrogen, ICI 182,780, starting when lesions were already established reversed the phenotype. These data elucidate the ability of human HSD17B1 to enhance estrogen action in the mammary gland in vivo and indicate that HSD17B1 is a factor inducing phenotypic alterations associated with mammary tumorigenesis.