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Background: Direct evidence for the relationship between a large prostate (≥80 ml) and androgen receptor/PSA signal remains lacking in benign prostatic hyperplasia (BPH). Our aim is to identify whether the cause of a large prostate is related to progesterone receptor (PGR) androgen receptor (AR), oestrogen receptor α, β (ERα,β) and prostate-specific antigen (PSA)., Materials and Methods: Surgical specimens of BPH in plasmakinetic resection of the prostate (PKRP) with three groups of different prostate-sizes with mean volumes of 25.97 ml, 63.80 ml, and 122.37 ml were collected for immunohistochemical analysis of the tissue microarray with PGR, AR, PSA and ERs. Rats were castrated and treated with testosterone replacement to explore androgen and PGR, AR and ERs expression levels in the prostate. Quantitative real-time reverse transcription polymerase chain reaction (Rt-PCR) for mRNA detection of above genes was conducted., Results: Immunoblotting, Rt-PCR and immunohistochemistry assays showed that PGR, PSA, AR, ERα expression levels were positively correlated with prostate size and that ERβ expression levels were negatively correlated with prostate volume. Animal experiments have shown that prostate volume is decreased in castrated rats with decreased PGR, AR, ERα and increased ERβ expression levels., Conclusion: PGR, AR, ERs signals can be regarded as important factors for large-sized prostates in BPH patients (≥100 ml).

Estradiol and progesterone have been recognized as important mediators of reproductive events in the female mainly via binding to their receptors. This study aimed to characterize the immunolocalization of the estrogen receptor alfa (ERα), estrogen receptor beta (ERβ) and progesterone receptor (PR) in the ovarian follicles of the lizard Sceloporus torquatus . The localization of steroid receptors has a spatio-temporal pattern that depends on the stage of follicular development. The immunostaining intensity of the three receptors was high in the pyriform cells and the cortex of the oocyte of previtellogenic follicles. During the vitellogenic phase, the granulosa and theca immunostaining was intense even with the modification of the follicular layer. In the preovulatory follicles, the receptors were found in yolk and additionally, ERα was also located in the theca. These observations suggest a role for sex steroids in regulating follicular development in lizards, like other vertebrates.

Aim: The aim of this study was to identify the biological characteristics and potential roles of endometrial progenitor cells in the pathogenesis of endometriosis., Background: It is generally believed that progenitor cells in human endometrium are responsible for rapid endometrial regeneration. However, the biological characteristics and potential roles of the paired eutopic and ectopic endometrial progenitor cells in endometriosis remain unclear., Objective: This study intends to isolate the epithelial progenitor (EP) cells and endometrial mesenchymal stem cells (eMSCs) from the eutopic and ectopic endometria from endometriosis patients, further to reveal their features and functions respectively., Methods: The distributions of EP cells and eMSCs and the expression of steroid hormone receptors in the endometrium and endometriotic tissues were assessed by immunohistochemistry. EP cells and eMSCs were sorted from paired eutopic and ectopic endometria with epithelial cell adhesion molecule (EpCAM) magnetic beads. The clonogenicity, cell viability after being treated with estradiol and progesterone, and cell markers expression were evaluated with colony forming on Matrigel, CCK-8 and immunofluorescence staining, respectively. The differentially expressed genes (DEGs) were further identified with RNA sequencing., Results: SSEA-1- and PDGFRβ-positive cells were distributed in the epithelial and stromal layers. The ERβ staining was much more intense in endometriotic tissues, but PR expression was almost absent. The ectopic EP cells exhibit strong clonogenicity and ERβ expression but weak PR expression, leading to progesterone resistance. There are 12604 and 13242 DEGs revealed by RNA sequencing between eutopic and ectopic EP cells or eMSCs. GO and KEGG analyses revealed that the functions and pathways of DEGs enriched in cellular energy metabolism and regulation of the immune response, respectively. Additionally, ERβ targets were mainly enriched in ectopic EP cells., Conclusion: Both EP cells and eMSCs may engage in ectopic lesion formation in endometriosis by modifying the metabolic mode and immune tolerance. These data not only help to understand the molecular mechanism of endometriosis but also could potentially contribute to the discovery of therapeutic targets for endometriosis., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

High-mobility group box 2 (HMGB2) is a chromatin-associated protein that is an important regulator of gene transcription, recombination, and repair processes. The functional importance of HMGB2 has been reported in various organs, including the testis, heart, and cartilage. However, its role in the ovary is largely unknown. In this study, ovary tissues from wild-type (WT) and HMGB2-knock-out (KO) mice were examined by histopathological staining and immunohistochemistry. The ovary size and weight were significantly lower in HMGB2-KO mice than in age-matched WT littermates. Histopathological analysis revealed ovarian atrophy and progressive fibrosis in 10-month-old HMGB2-KO mouse ovaries. Compared to age-matched WT mice, the numbers of oocytes and developing follicles were significantly decreased at 2 months of age and were completely depleted at 10 months of age in HMGB2-KO mice. Immunohistochemistry revealed the expression of HMGB2 in the granulosa cells of developing follicles, oocytes, some corpora lutea, and stromal cells. Importantly, HMGB2-positive cells were co-localized with estrogen receptor beta (ERβ), but not ERα. Estrogen response element-binding activity was demonstrated by southwestern histochemistry, and it was decreased in HMGB2-KO mouse ovaries. Cell proliferation activity was also decreased in HMGB2-KO mouse ovaries in parallel with the decreased folliculogenesis. These results indicated that the depletion of HMGB2 induced ovarian atrophy that was characterized by a decreased ovarian size and weight, progressive fibrosis, as well as decreased oocytes and folliculogenesis. In conclusion, we demonstrated the crucial role of HMGB2 in mouse ovarian folliculogenesis through ERβ expression., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Testing for antibodies against podocyte phospholipase A2 receptor-1 (PLA2R) allows clinicians to accurately identify primary membranous nephropathy (MN). Secondary MN is associated with a spectrum of pathology including solid organ malignancy. PLA2R positivity in these patients occurs, although no case of PLA2R-positive MN has been definitively linked to cancer., Case Presentation: We describe a case of biopsy-proven PLA2R-positive MN, in whom invasive ductal carcinoma of the breast was discovered. The patient underwent surgery and adjuvant chemotherapy (including cyclophosphamide) and went into a sustained complete remission of her nephrotic syndrome., Discussion and Conclusions: Case series have reported PLA2R positivity in patients with solid organ malignancy associated MN. Our case is unusual as it is a breast malignancy, and the patients nephrotic syndrome and anti-PLA2Rab titres improved with treatment of the cancer. Here we report, to the best of our knowledge, the first case of oestrogen receptor-2 positive breast cancer associated with PLA2R positive MN in a young lady that was treated successfully by treating the malignancy., (© 2021. The Author(s).)

The rapid decline of circulating 17β-estradiol (E2) at menopause leads to negative neurological consequences, although hormone therapy paradoxically has both harmful and positive effects depending on the age at which it is delivered. The inconsistent response to E2 suggests unappreciated regulatory mechanisms for estrogen receptors (ERs), and we predicted it could be due to age-related differences in ERβ phosphorylation. We assessed ERβ phosphorylation using a sensitive mass spectrometry approach that provides absolute quantification (AQUA-MS) of individually phosphorylated residues. Specifically, we quantified phosphorylated ERβ in the hippocampus of women (aged 21-83 years) and in a rat model of menopause at 4 residues with conserved sequence homology between the 2 species: S105, S176, S200, and Y488. Phosphorylation at these sites, which spanned all domains of ERβ, were remarkably consistent between the 2 species, showing high levels of S105 phosphorylation (80%-100%) and low levels of S200 (20%-40%). Further, S200 phosphorylation decreased with aging in humans and loss of E2 in rats. Surprisingly, Y488 phosphorylation, which has been linked to ERβ ligand-independent actions, exhibited approximately 70% phosphorylation, unaltered by species, age, or E2, suggesting ERβ's primary mode of action may not require E2 binding. We further show phosphorylation at 2 sites directly altered ERβ DNA-binding efficiency, and thus could affect its transcription factor activity. These findings provide the first absolute quantification of ERβ phosphorylation in the human and rat brain, novel insights into ERβ regulation, and a critical foundation for providing more targeted therapeutic options for menopause in the future., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Within the hypothalamic paraventricular nucleus (PVN), estrogen receptor (ER) β and other gonadal hormone receptors play a role in central cardiovascular processes. However, the influence of sex and age on the cellular and subcellular relationships of ERβ with ERα, G-protein ER (GPER1), as well as progestin and androgen receptors (PR and AR) in the PVN is uncertain. In young (2- to 3-month-old) females and males, ERβ-enhanced green fluorescent protein (EGFP) containing neurons were approximately four times greater than ERα-labeled and PR-labeled nuclei in the PVN. In subdivisions of the PVN, young females, compared to males, had: (1) more ERβ-EGFP neurons in neuroendocrine rostral regions; (2) fewer ERα-labeled nuclei in neuroendocrine and autonomic projecting medial subregions; and (3) more ERα-labeled nuclei in an autonomic projecting caudal region. In contrast, young males, compared to females, had approximately 20 times more AR-labeled nuclei, which often colocalized with ERβ-EGFP in neuroendocrine (approximately 70%) and autonomic (approximately 50%) projecting subregions. Ultrastructurally, in soma and dendrites, PVN ERβ-EGFP colocalized primarily with extranuclear AR (approximately 85% soma) and GPER1 (approximately 70% soma). Aged (12- to 24-month-old) males had more ERβ-EGFP neurons in a rostral neuroendocrine subregion compared to aged females and females with accelerated ovarian failure (AOF) and in a caudal autonomic subregion compared to post-AOF females. Late-aged (18- to 24-month-old) females compared to early-aged (12- to 14-month-old) females and AOF females had fewer AR-labeled nuclei in neuroendrocrine and autonomic projecting subregions. These findings indicate that gonadal steroids may directly and indirectly influence PVN neurons via nuclear and extranuclear gonadal hormone receptors in a sex-specific manner., (© 2020 Wiley Periodicals LLC.)

Objective: To identify the risk factors associated with dysmenorrhea in adenomyosis and to discuss the potential hormone-based understanding of pain mechanisms., Study Design: Adenomyosis patients with mild or no dysmenorrhea (n = 40, Group 1) and moderate-to-severe dysmenorrhea (n = 80, Group 2) were recruited. Charts of all patients were recorded. An immunohistochemistry (IHC) analysis was performed to detect the cellular levels of estrogen receptor-α (ER-α), estrogen receptor-β (ER-β), gonadotropin-releasing hormone receptor (GnRH-R), and neurofilaments (NFs) in 60 cases., Results: A history of cesarean section (CS) was positively related to the degree of dysmenorrhea in adenomyosis (OR (95 % CI): 4.397 (1.371-14.104)). The ER-α levels in the eutopic endometrium (EUE) of Group 2 were higher than those in the ectopic endometrium (ECE) of Group 1. Group 2 had higher NF levels in the ECE than in the EUE., Conclusion: A history of CS is a risk factor for adenomyosis with moderate-to-severe dysmenorrhea. For patients with adenomyosis, high ER-α levels in the EUE and high NF levels in the ECE may be related to moderate-to-severe dysmenorrhea. These hormone-based mechanisms may contribute to our understanding of the pathogenesis of dysmenorrhea in adenomyosis., (Copyright © 2020. Published by Elsevier Masson SAS.)

Objectives: Prior studies have demonstrated that signaling via the estrogen and progesterone receptors (ER and PR) may affect prognosis in non-small cell lung cancer (NSCLC). The precise impact of hormone signaling on clinical outcomes in NSCLC, especially in the context of immune checkpoint blockade, remains unclear., Materials and Methods: We obtained RNA-Seq data from The Cancer Genome Atlas (TCGA) to determine mRNA expression levels of ESR1 (ER-α), ESR2 (ER-β), PGR (PR), CYP19A1 (aromatase), and immune-related genes. Tumor infiltration by activated T cells was predicted based on expression of immune metagenes., Results: High levels of both ESR1 and PGR were associated with significantly decreased tumor infiltration by CD4+ and CD8+ activated T cells. CYP19A1 expression was associated with decreased CD4+ but not CD8+ T cell infiltration. There were no significant differences based on ESR2. These findings persisted after stratifying patients based on sex and tumor histology. In addition, increased ESR1 was associated with high gene expression of immune checkpoint markers, while increased PGR was associated with high levels of TGF-β genes. In a multivariate logistic regression analysis, ESR1, PGR, TGFB1, and the total number of somatic variants were identified as independent factors predicting T cell infiltration., Conclusions: Increased gene expression of ER-α and PR was associated with decreased activated T cell infiltration in patients with NSCLC. The relevance of hormone receptor status should be validated clinically, including in the context of immune checkpoint inhibitors., (Copyright © 2021. Published by Elsevier Ltd.)

Given the high incidence and excellent prognosis of many papillary thyroid microcarcinomas, the Porto proposal uses the designation papillary microtumor (PMT) for papillary microcarcinomas (PMCs) without risk factors to minimize overtreatment and patients' stress. To validate Porto proposal criteria, we examined a series of 190 PMC series, also studying sex hormone receptors and BRAF mutation. Our updated Porto proposal (uPp) reclassifies as PMT incidental PMCs found at thyroidectomy lacking the following criteria: (a) detected under the age of 19 years; (b) with multiple tumors measuring >1 cm adding up all diameters; and (c) with aggressive morphologic features (extrathyroidal extension, angioinvasion, tall, and/or hobnail cells). PMCs not fulfilling uPp criteria were considered "true" PMCs. A total of 102 PMCs were subclassified as PMT, 88 as PMC, with no age or sex differences between subgroups. Total thyroidectomy and iodine-131 therapy were significantly more common in PMC. After a median follow-up of 9.6 years, lymph node metastases, distant metastases, and mortality were only found in the PMC subgroup. No subgroup differences were found in calcifications or desmoplasia. Expression of estrogen receptor-α and estrogen receptor-β, progesterone receptor, and androgen receptor was higher in PMC than in nontumorous thyroid tissue. BRAF mutations were detected in 44.7% of PMC, with no differences between subgroups. In surgical specimens, the uPp is a safe pathology tool to identify those PMC with extremely low malignant potential. This terminology could reduce psychological stress associated with cancer diagnosis, avoid overtreatment, and be incorporated into daily pathologic practice.

Aims: Characterising the factors responsible for metastatic triple-negative breast cancer (TNBC) is of significant importance, considering its high mortality rate and scant data. In this study, we evaluated the characteristics, clinical behaviour and role of biomarkers (androgen receptor (AR), oestrogen receptor beta (ERβ) and p53) in metastatic TNBC., Methods: Immunohistochemistry was performed for AR, ERβ and p53 on 125 primary TNBCs with known metastasis and correlated with clinicopathological parameters and outcome. AR and p53 mRNA profiling was also carried out on 34 tumours from the same series and correlated with outcomes., Results: In this cohort, grade 3 and pT2 tumours predominated. The most common site for metastasis was the lung and pleura (41, 32.8%), and 15 (12.0%) cases demonstrated metastasis in multiple sites. Among these, 92% of tumours metastasised without preceding local recurrences. Five- and ten-year overall survival (OS) rates were 27% and 7.2%, while 5- and 10- year survival rates after metastasis were 9.6% and 3.2% respectively. AR, ERβ and p53 protein expressions were observed in 16%, 96.8% and 58.1% of tumours, respectively. A combinational phenotype of AR-ERβ+p53+ tumours was associated with poorer OS (HR 1.543, 95%CI 1.030 to 2.310, p=0.035). Higher AR mRNA levels were significantly associated with favourable OS (p=0.015) and survival after metastasis (p=0.027)., Conclusions: Metastatic TNBC harboured aggressive behaviour and displayed predominantly visceral metastasis with most metastatic events occurring without intervening local recurrences. A combinational phenotype of AR-ERβ+p53+ was significantly associated with poorer OS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Supplementation with phytoestrogens and insoluble fibers has been reported to reduce duodenal polyps in colectomized familial adenomatous polyposis patients, with a mechanism involving, at least in part, upregulation of estrogen receptor-β subtype, whose expression is lowered during intestinal tumorigenesis. These data suggest a protective effect also in the colon, the main target organ for tumorigenesis in familial adenomatous polyposis and a major cancer type in non-familial (sporadic) cancers. Therefore, we tested whether a similar preparation might reduce tumorigenesis in the colon of Pirc rats (F344/NTac-Apc) mutated in the Apc gene and thus, like familial adenomatous polyposis patients, spontaneously developing multiple tumors in the colon. We first demonstrate that estrogen receptor-β expression in Pirc rat colon is significantly down-regulated compared to age-matched wt rats. Then, Pirc rats aged 1 month were treated for 3 months with Adipol (Adi), a patented preparation containing phytoestrogens and insoluble fibers. Colon tumorigenesis was significantly reduced by Adi treatment (colon tumors/rat were 5.3 ± 0.8 and 2.9 ± 0.3, Mucin Depleted Foci/rat 127 ± 6.6 and 97.1 ± 8.6 in Controls and Adi-treated rats, respectively, means ± SE, P < 0.01). The treatment also normalized colon proliferation pattern along the crypt and significantly increased apoptosis in colon tumors. Estrogen receptor-β expression was increased by Adi treatment, especially in the tumors. These positive effects suggest that Adipol may be exploited as a chemopreventive agent to reduce cancer risk in familial adenomatous polyposis patients and to postpone prophylactic colectomy. Moreover, given the similarities between familial adenomatous polyposis and sporadic colorectal cancer, it might also be used as chemopreventive agent in colorectal cancer patients at risk.

Salivary duct carcinoma (SDC) is an aggressive neoplasm that resembles high-grade invasive ductal carcinoma of the breast. It can develop de novo or from the malignant transformation of pleomorphic adenoma (PA). We performed immunohistochemical stains for phosphatase and tensin homologue [PTEN androgen receptor (AR)], HER2/neu, cytokeratin 5/6, estrogen receptor-beta, high-mobility group AT-hook 2 (HMGA2), and pleomorphic adenoma gene 1 (PLAG1) on tissue microarray samples of 75 SDCs and 31 adenocarcinomas, not otherwise specified (NOS). Our data showed the following in SDC samples: loss of PTEN was found in 17 of 60 (28.3%); AR was expressed in 43 of 62 (69.4%); HER2/neu was overexpressed in 25 of 58 (43.1%); cytokeratin 5/6 was expressed in 14 of 54 (25.9%); estrogen receptor-beta was expressed in 37 of 56 (66.1%); HMGA2 was expressed in 29 of 63 (46.0%); and PLAG1 was expressed in 0 of 62 (0%). In addition, there was no statistically significant difference in the age at onset between patients with HMGA2-positive SDCs (range 32-85 years; mean: 64.3 years; median: 64.5 years) and those with HMGA2-negative SDCs (range 41-79 years; mean: 62.5 years; median: 64.5 years). There was also no statistically significant difference in overall survival between patients with HMGA2-positive and HMGA2-negative SDCs (follow-up period range 3-201 months; mean: 49.8 months; median: 30 months). Among 10 patients with a definite PA component (SDC ex-PA), 6 were positive and 4 were negative for HMGA2. Our data were consistent with previous findings that AR and estrogen receptor-beta are expressed in most SDCs, whereas HER2/neu overexpression and loss of PTEN are expressed in a subset of SDCs. In our cohort of patients, HMGA2 was expressed in approximately half of SDCs. HMGA2 and PTEN are promising therapeutic targets for salivary gland tumors.

Many women experience some skin reaction or trouble in their monthly menstrual cycle, including the exacerbation of pre-existing diseases and skin eruptions directly associated with sex hormones. We herein report a Japanese woman who experienced repeated systemic urticaria in her premenstrual period, and was diagnosed as having estrogen dermatitis based on a positive result of intradermal estrogen skin test. Of note, the expression of estrogen receptor-β was increased in small dermal vessels of this case as well as in those of patients with other inflammatory skin diseases. These results suggest that inflammation may induce estrogen receptor-β expression in small dermal vessels, which potentially modifies the pathological skin inflammation during the menstrual period, leading to the development of estrogen dermatitis., (© 2019 Japanese Dermatological Association.)

Immunohistochemistry using formalin-fixed, paraffin-embedded tissue, chromogen label, and light microscopy has traditionally been used to semiquantify estrogen receptor (ER) to guide diagnosis and management of breast cancer. Quantitation of ER for this purpose currently only assesses levels of the ER-alpha subtype. Considerable variability in results reported has been due to protocol and fixation variability, intraobserver and interobserver variability, and different scoring systems and thresholds for scoring ER positivity. Results can also vary with low expression levels of ER. ER-beta expression is reduced in breast and ovarian cancers and requires quantitation.Herein we describe a novel approach to quantifying ERβ using older mouse ovarian surface epithelium, where ERβ is expressed at lower levels than ERα and is therefore harder to detect. We use an antibody highly specific to the ERβ1 isoform, together with immunofluorescence, confocal microscopy, and imaging and statistical software to achieve clear, reproducible, and unbiased quantitation of ERβ.

Renal cell carcinoma (RCC) has the third highest mortality rate among urological tumors, and 20-30% of RCC patients present with metastatic RCC at the time of diagnosis. Although recent studies have indicated that estrogen receptor β (ERβ) could play promoting roles in RCC progression, the detailed mechanisms remain to be clarified. In the present study, we found that expression of ERβ, but not ERα, increases with tumor stage and grade, and also observed that modification of ERβ signals using estrogens/anti-estrogens, shRNA knockdown of ERβ and overexpression of ERβ using ectopic cDNA affects RCC cell proliferation, migration and invasion. Mechanism analysis revealed that ERβ can promote RCC cell invasion via an increase in transforming growth factor β1 (TGF-β1)/SMAD3 signals, and interrupting TGF-β1/SMAD3 signals with a TGFβR1 inhibitor can reverse/block ERβ-increased RCC cell migration. Importantly, preclinical analyses using in vivo mouse models of RCC revealed that targeting of this newly identified ERβ/TGF-β1/SMAD3 pathway with either the FDA-approved anti-estrogen ICI182,780 (Faslodex) or a selective ERβ antagonist 4-[2-phenyl-5,7 bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl]phenol can significantly reduce RCC tumor growth and invasion, which may be suitable as the basis for novel therapies to more effectively suppress metastatic RCC., (© 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Becker's naevus is androgen-dependent. The aim of this study was to investigate whether oestrogen and progesterone receptors are involved in this disorder. Immunohistochemistry showed that epidermal expression of androgen receptors, oestrogen receptors (α, β) and progesterone receptors was higher in skin lesions of Becker's naevus than in perilesional and control skin. Androgen receptor overexpression was observed in pilosebaceous glands, while oestrogen and progesterone receptor overexpression was seen in hair follicles, but not in sebaceous glands in skin lesions compared with perilesional skin. Reverse tran-scription PCR and Western blot revealed that levels of androgen, oestrogen and progesterone receptors were generally upregulated in skin lesions compared with perilesional and control skin, and their expression was usually higher in perilesional than in control skin. These results suggest that simultaneous overexpression of androgen, oestrogen and progesterone receptors might be implicated in the pathogenesis of Becker's naevus.

Testosterone is often recommended in the treatment of several aging-related conditions. However, there are still questions about the consequences of this therapy in terms of hormonal and inflammatory parameters that are crucial for prostate homeostasis. Thus, we investigate if the testosterone therapy (TT) modulates the hormone receptors and inflammatory cytokines in the ventral prostate of adult rats. Wistar rats aging 150 days were divided into two experimental groups (n = 10/group): T: received subcutaneous injections of testosterone cypionate (5 mg/kg body weight) diluted in corn oil every other day for 4 weeks; and C: received corn oil as vehicle. Animals were euthanized at 180 days old by decapitation. Blood was collected to obtain hormone and cytokines concentrations. The ventral prostate was dissected and processed for light microscope and molecular analyses. Relative ventral prostate weight and epithelial compartment were increased after TT. The number of intact and degranulated mast cells was reduced in the T group. Plasma testosterone, DHT and intraprostatic testosterone concentrations were higher in the T group. TT leads to an increase in cell proliferation and up-regulation of AR, ERβ, PAR-4, and NRF2. Importantly, plasma concentration and tissue expression of IL-10 and TNF-α were higher after TT. In summary, these results indicate that TT can regulate inflammatory response, with impacts in cytokines and mast cell population, and modulates steroids receptors, important parameters for prostatic homeostasis., (© 2018 International Federation for Cell Biology.)

Purpose: Numbers of studies have demonstrated that estrogen receptor α and estrogen receptor β have been involved in development of gastric cancer. Hence, we analyzed studies and The Cancer Genome Atlas (TCGA) data of ERs expression and perform this meta-analysis to access the association between ERα or ER β and the clinicopathological characteristics, overall survival time, in GC., Method: A literature search was performed in PubMed, Cochrane Library, Web of Science, EMBASE database, and Chinese CNKI. Data on the relationship between ERα or ERβ expression and clinicopathological features were extracted. A TCGA dataset including information of the ERs expression and clinical data of GC patients was analyzed. Pooled odds ratios (ORs) and hazard ratios (HRs) with 95% confidence intervals (CIs) were calculated., Results: ERα was not associated with cancer risk, lymph node metastasis, infiltration degree, gender, and TNM stage. However, ERβ was negatively associated with lymph node metastasis. ERα expression may be associated with poor prognosis in GC patients., Conclusion: Estrogen receptors may be related to the progression and deterioration of gastric cancer. However, further high-quality studies are needed to provide more reliable evidence., (Copyright © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.)

Estrogen and progesterone are the main pregnancy hormones produced by the placenta. It is well understood that estrogen stimulates angiogenesis in the uterus during the reproductive cycle. Although the estrogen and progesterone signaling pathways are assumed to be associated with placental vascularization and preeclampsia, expression of estrogen receptors (ESRs) and progesterone receptor (PGR) in the placenta have not been well studied. The present study examined the expression patterns of steroid hormone receptors in placentas. Human placenta samples were collected and divided into normal and preeclampsia groups. Results revealed that expression levels of ESR1 were reduced, whereas ESR2 and PGR were elevated in preeclamptic placentas. To generate an in vitro preeclampsia environment, human placenta‑derived BeWo cells were incubated under hypoxic conditions, or treated with catechol‑O‑methyl transferase inhibitor (COMT‑in) or L‑NG‑nitroarginine methyl ester (L‑NAME). Expression levels of ESR1, ESR2 and PGR in hypoxic cells demonstrated similar regulation as those in placentas from women with preeclampsia. Although COMT‑in and L‑NAME did not significantly regulate the expression levels of the receptors, COMT‑in translocated ESR2 and PGR from the nucleus to the cytoplasm, indicating that these receptors were inactivated. These results suggested that ESRs and PGR are associated with symptoms of preeclampsia in the placenta. The expression of ESR1 was reduced in preeclamptic placenta and hypoxic BeWo cells. In addition, the activation of ESR2 and PGR was blocked in placenta cells subjected to COMT‑in treatment. The reduced ESR1 expression and inactivation of ESR2 and PGR proteins may affect the physiological complications of preeclampsia in the placenta.

Cotton rat ( Sigmodon hispidus) is a useful experimental rodent for the study of human infectious diseases. We previously clarified that cotton rats, particularly females, developed chronic kidney disease characterized by cystic lesions, inflammation, and fibrosis. The present study investigated female-associated factors for chronic kidney disease development in cotton rats. Notably, female cotton rats developed separation of the pelvic symphysis and hypertrophy in the vaginal parts of the cervix with age, which strongly associated with pyometra. The development of pyometra closely associated with the deterioration of renal dysfunction or immunological abnormalities was indicated by blood urea nitrogen and serum creatinine or spleen weight and serum albumin/globulin ratio, respectively. These parameters for renal dysfunction and immunological abnormalities were statistically correlated. These phenotypes found in the female reproductive organs were completely inhibited by ovariectomy. Further, the female cotton rats with pyometra tended to show more severe chronic kidney disease phenotypes and immunological abnormalities than those without pyometra; these changes were inhibited in ovariectomized cotton rats. With regard to renal histopathology, cystic lesions, inflammation, and fibrosis were ameliorated by ovariectomy. Notably, the immunostaining intensity of estrogen receptor α and estrogen receptor β were weak in the healthy kidneys, but both estrogen receptors were strongly induced in the renal tubules showing cystic changes. In conclusion, the close correlations among female reproductive organ-associated abnormalities, immunological abnormalities, and renal dysfunction characterize the chronic kidney disease features of female cotton rats. Thus, the cotton rat is a unique rodent model to elucidate the pathological crosstalk between chronic kidney disease and sex-related factors. Impact statement The increasing number of elderly individuals in the overall population has led to a concomitant age-related increase in chronic kidney disease. Moreover, the global prevalence of patients with chronic kidney disease is gradually increasing, which poses a serious public health problem. The limited number of spontaneous chronic kidney disease animal models, which resemble chronic kidney disease pathogenesis in elderly individuals, is a major limitation in the development of experimental and curative medicines for chronic kidney disease. This pathological study clarified that sex-related factors, including hormones, and abnormalities of the female reproductive system, such as pyometra, are closely associated with chronic kidney disease development by using cotton rats ( Sigmodon hispidus). Further, ovariectomy inhibited the phenotypes of the female reproductive system, immunological abnormalities, and chronic kidney disease. Thus, this laboratory rodent serves as a novel and useful spontaneous chronic kidney disease model to elucidate the candidate disease factors and the pathogenesis of chronic kidney disease both in human and experimental medicine.

Oestrogens play an important role in the development and progression of papillary thyroid carcinoma (PTC) through oestrogen receptor (ER)-α and -β, which may exert different or even opposing actions in PTC. The roles of ERβ in ERα-negative PTC are still not clear. This study investigated the expression dynamics of ERβ1 (wild-type ERβ) and its clinical significance in female ERα-negative PTC patients. ERβ1 expression was detected in thyroid tissues of 136 female patients diagnosed with PTC. The relationships between ERβ1 expression and clinicopathological/biological factors were also analysed in female ERα-negative PTC patients. The total score for ERβ1 was significantly lower in female ERα-negative PTC patients with LNM or ETE when compared to those without LNM or ETE (Z = -2.923, P = 0.003 and Z = -3.441, P = 0.001). Accordingly, the total score for ERβ1 was significantly higher in ERα-negative PTC patients expressing E-cadherin compared to patients negative for E-cadherin expression (Z = -2.636, P = 0.008). The total score was lower in ERα-negative PTC patients positive for VEGF expression compared to those negative for VEGF expression (Z = -1.914, P = 0.056). This preliminary study indicates that reduced expression of ERβ1 in female ERα-negative PTC patients is associated with greater progression of the disease. This may provide insights into the underlying molecular mechanisms of ERβ1 and could help design targeted approaches for treating or even preventing this disease., (© 2018 The Authors. International Journal of Experimental Pathology © 2018 International Journal of Experimental Pathology.)

Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9h, and 40nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E 2 ) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Follicle-stimulating hormone (FSH) promotes secretion of follicle fluid and follicle development. FSH acts via cognate FSH receptor (FSHR). It remains unknown whether the supplement of FSH-receptor binding inhibitor (FRBI) into the in vitro maturation (IVM)medium influence the estrogen receptor expression and signal pathway of oocytes in sheep. The present study aimed to investigate FRBI effects on inositol trisphosphate (IP3) of oocytes and protein kinase A (PKA) of sheep granulosa cells, further to elucidate the signal pathway of FRBI effects. Cumulus-oocyte complexes (COCs) were recovered from antral follicles. COCs were cultured for 24 h in the IVM medium supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40 μg/mL) and FSH (10IU/mL). ELISA was used to measure the concentrations of estradiol (E2) and IP3 in the IVM medium. Western blotting was utilized to detect protein expression of ERβ of COCs and protein kinase A (PKA) of granulosa cells. The results showed IP3 concentrations of FRBI-3 and FRBI-4 groups were less than that of CG and FSH groups at 22 h and 24 h (P < 0.05). PKA levels of FRBI-3 and FRBI-4 groups were significantly less than that of CG and FSH group (P < 0.05 or P < 0.01). Expression levels of ERβ mRNA and protein of FRBI-treated groups were gradually decreased in comparison to CG and FSH group. The minimum value was detected in the FRBI-4 group. ERβ protein level of the FRBI-4 group was significantly less than that of FSH group (P < 0.05). E2 concentrations of FRBI-treated groups were elevated as compared to CG, with the highest increment of FRBI-2 group (P < 0.05). Our results revealed a higher dose of FRBI reduced IP3 production. FRBI could suppress slightly expression levels of ERβ mRNA and protein of COCs and PKA of granulosa cells, additionally increased E2 production of sheep COCs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: Inflammatory bowel disease (IBD) increases the risk of developing colon cancer. This risk is higher in men compared to women, implicating a role for female hormones in the protection against this disease. Studies from our laboratory demonstrated that estradiol (E 2 ) protects against inflammation-associated colon tumor formation when administered following chemical carcinogen and induction of chronic colitis., Aim: This study seeks to better understand the effect of E 2 on acute colitis in the presence and absence of estrogen receptor β (ERβ)., Methods: Inflammation was induced by 2,4,6-trinitrobenzenesulfonic acid in wild-type (WT) and ERβ knockout (ERβKO) mice implanted with a control or E 2 -containing pellet and killed 5 days later. Inflammation and injury were scored by a pathologist. Apoptosis and proliferation were assessed by immunohistochemistry. Cytokines were measured by multiplex analysis., Results: E 2 treatment reduced inflammation in the middle colon in WT mice and the distal colon in ERβKO mice compared to control mice. WT mice had reduced IL-6, IL-12, IL-17, GM-CSF, IFN-γ, MCP-1, MIP-1α, and TNF-α, and ERβKO had reduced IL-6 and IFN-γ expression in response to E 2 . Injury scores were lower in E 2 -treated ERβKO mice compared to control ERβKO mice. ERβKO mice had increased proliferation in the basal third of crypts in the distal colon and decreased apoptosis in the proximal colon., Conclusions: These data suggest that E 2 has differential protective effects against acute colitis in the presence or absence of ERβ and provide insight into how E 2 may protect against IBD.

Estrogen exerts protective roles in amyotrophic lateral sclerosis (ALS). However, the expression of aromatase (ARO) and estrogen receptors (ERs) in the motoneurons of spinal cord, has not yet been elucidated. By immunohistochemistry, we found that ARO and ERs were present in the ventral horn of adult mice lumbar spinal cord, and colocalized with SMI-32, a motoneuron specific marker. Within motoneurons, we observed that ARO is detected primarily in the cytoplasm, with fewer ARO in the nucleus; ERα and ERβ mainly localized in the nucleus with less in the cytoplasm; while GPR30 is located in soma and processes. In conclusion, we found that ERs and ARO are expressed in the motoneurons of lumbar spinal cord in adult mice. These findings suggest that estrogen may be useful as a promising therapeutic agent for prevention of damage and improvement of locomotor function in ALS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The discovery of oestrogen receptor β (ERβ/ESR2) was a landmark discovery. Its reported expression and homology with breast cancer pharmacological target ERα (ESR1) raised hopes for improved endocrine therapies. After 20 years of intense research, this has not materialized. We here perform a rigorous validation of 13 anti-ERβ antibodies, using well-characterized controls and a panel of validation methods. We conclude that only one antibody, the rarely used monoclonal PPZ0506, specifically targets ERβ in immunohistochemistry. Applying this antibody for protein expression profiling in 44 normal and 21 malignant human tissues, we detect ERβ protein in testis, ovary, lymphoid cells, granulosa cell tumours, and a subset of malignant melanoma and thyroid cancers. We do not find evidence of expression in normal or cancerous human breast. This expression pattern aligns well with RNA-seq data, but contradicts a multitude of studies. Our study highlights how inadequately validated antibodies can lead an exciting field astray.

Immunohistochemistry of a transcription factor, GATA3, has been widely used as a promising urothelial marker in diagnostic surgical pathology practice. However, the expression status of GATA3 in upper urinary tract urothelial carcinomas (UUTUCs) and its prognostic significance have not been fully investigated. We immunohistochemically stained for GATA3 in 99 UUTUC samples and paired nonneoplastic urothelial tissues. GATA3 was positive in 51 (51.5%; 32 [32.3%] weak, 11 [11.1%] moderate, 8 [8.1%] strong) of 99 UUTUCs, which was significantly lower than in benign urothelium (79 [96.3%] of 82; 33 [40.2%] weak, 35 [42.7%] moderate, 11 [13.4%] strong; P<.001). However, there were no statistically significant associations between GATA3 expression and tumor grade, pT stage, lymph node involvement, or distant metastasis. Meanwhile, the rate of GATA3 positivity was significantly higher (P=.004) in ureteral tumors (66.0%) than in renal pelvic tumors (35.6%). Kaplan-Meier and log-rank tests revealed that GATA3 negativity was significantly associated with lower recurrence-free survival (P=.037 for all cases, P=.026 for muscle-invasive tumors) and cancer-specific survival (P=.007 for all cases, P=.012 for muscle-invasive tumors, P=.035 for cases with adjuvant chemotherapy) rates. Multivariate analysis further identified strong correlations of GATA3 expression with tumor progression (all cases: hazard ratio [HR], 0.479 [95% confidence interval {CI},0.229-1.003; P=.051]; muscle-invasive tumors: HR, 0.387 [95% CI, 0.166-0.903; P=.028) or cancer-specific mortality (all cases: HR, 0.354 [95% CI, 0.135-0.925; P=.034]; muscle-invasive tumors: HR, 0.402 [95% CI, 0.149-1.086; P=.072]). Thus, compared with nonneoplastic urothelium, a significant decrease in the expression of GATA3 in UUTUC was seen. Moreover, loss of GATA3 expression was found to be an independent predictor of poor patient outcomes. Of note was that only roughly half of high-grade and/or muscle-invasive UUTUCs were immunoreactive for GATA3., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: The aim of this study was to determine whether α-linolenic acid (ALA ω-3 fatty acid) enriched diet affects growth parameters when applied to a syngeneic model of mammary carcinoma., Materials and Methods: BALB/c mice were divided and fed with: 1) a chia oil diet, rich in ALA or 2) a corn oil diet, rich in linoleic acid (LA ω-6 fatty acid). Mice were subcutaneously inoculated with a tumor cell line LM3, derived from a murine mammary adenocarcinoma., Results: After 35 days, tumor incidence, weight, volume and metastasis number were lower in the ALA-fed mice, while tumor latency time was higher, and the release of pro-tumor metabolites derived from ω-6 fatty acids decreased in the tumor. Compared to the control group, a lower number of mitosis, a higher number of apoptotic bodies and higher T-lymphocyte infiltration were consistently observed in the ALA group. An ALA-rich diet decreased the estrogen receptor (ER) α expression, a recognized breast cancer promotor while showing an opposite effect on ERβ in tumor lysates., Conclusion: These data support the anticancer effect of an ALA-enriched diet, which might be used as a dietary strategy in breast cancer prevention.

Purpose: Isoform-specific tumor estrogen receptor β (ERβ) expression may hold prognostic information in breast cancer, especially among endocrine-treated breast cancer patients. The study's purpose was to evaluate ERβ isoform 1 (ERβ1) expression in relation to tumor characteristics, ESR2 genotypes, and prognosis in different treatment groups., Experimental Design: A population-based prospective cohort of 1,026 patients diagnosed with primary invasive breast cancer in Lund, Sweden, between October 2002 and June 2012 was followed until June 2014 (median 5 years). Associations between immunohistochemical ERβ1 expression, patient and tumor characteristics, as well as outcome within treatment groups were analyzed., Results: Tumor ERβ1 expression was available for 911 patients (89%) and was not associated with ESR2 genotypes. ERβ1 positivity, defined as >75% (ERβ1 75 + , 72.7%), was positively associated with established favorable tumor characteristics. Overall, ERβ1 75 + was associated with lower risk of breast cancer events [HR adj = 0.60; 95% confidence interval (CI), 0.41-0.89]. The magnitude of the association was larger in patients with ERα - tumors (HR adj = 0.30; 95% CI, 0.12-0.76), compared with ERα + tumors (HR adj = 0.66; 95% CI, 0.42-1.03). Among the 232 chemotherapy-treated patients, ERβ1 75 + tumors were associated with lower risk of breast cancer events compared with ERβ1 75 - tumors (HR adj = 0.31; 95% CI, 0.15-0.64). Among the 671 chemonaïve patients, ERβ1 75 status was not associated with the outcome., Conclusions: High ERβ1 expression was a favorable prognostic marker in this breast cancer cohort, especially in chemotherapy-treated patients, but not in endocrine therapy-treated patients. These results warrant confirmation, preferably via a biomarker study in a previously conducted randomized trial. Clin Cancer Res; 23(3); 766-77. ©2016 AACR., (©2016 American Association for Cancer Research.)

Objective: Male sex is a significant risk factor for abdominal aortic aneurysm (AAA). Female sex hormones have been reported to prevent aneurysm formation in animal models. The study aims to describe the expression profile of sex hormone receptors in the aneurysm wall of men and women with AAA and compare with unaffected controls., Methods: Aneurysm wall biopsies were obtained during elective open repair of AAA in men and women (n=16+16). Aortic vessel wall from controls were obtained at organ donation (n=6). Western blot-, mRNA expression- and immunohistochemical analyses were performed to assess the expression profile of the sex hormone receptors - androgen receptor (AR), progesterone receptor (PR), estrogen receptor α (ERα) and β (ERβ)., Results: The mRNA- and protein expression levels of AR were higher in AAA compared with control aorta (7.26 vs. 5.14, P=0.001). mRNA- and protein expression levels of ERβ were lower in AAA compared with control aorta (9.15 vs. 12.29, P<0.001). mRNA expression levels of PR were higher in AAA compared with control aorta (8.73 vs. 6.21, P=0.003), but could not be confirmed on protein level. The expression profile of sex hormone receptors in men and women with AAA was similar., Conclusion: Expression of sex hormone receptors differs in the aneurysmal aorta compared with unaffected aorta in men and women. A higher expression of AR and a lower expression of ERβ suggest that sex hormone activity could be associated with aneurysm development., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Sex steroids have been postulated to influence skin development and functions as well as its pathogenesis. MCC occurs in both sexes; however, the specific differences in pathogenesis among sexes have yet to be conclusively defined. The detailed status of sex steroid receptors (AR, PRA and PRB, and ERα, ERβ) are also unknown in MCC patients. We first immunolocalized sex steroid receptors and compared the results with immunolocalization of relevant transcription factors including SOX2, FOXA1, and Bcl-2 and Ki-67 in 18 cases of MCCs. AR, PRA, PRB, ERα, ERβ, Bcl-2, SOX2, and FOXA1 immunoreactivity was evaluated by using the modified H score method, and Ki-67 was quantified using labeling index. ERβ immunoreactivity was markedly present in all the cases of MCC examined, with relatively weak immunoreactivity of ERα, AR, PRA, and PRB. The status of ERβ immunoreactivity was also significantly correlated with Ki-67 labeling index and Bcl-2 score. These results demonstrated that ERβ could be associated with regulation of both cell proliferation and apoptosis in MCCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Basal-like breast cancer (BLBC) and triple-negative breast cancer (TNBC) are two entities of breast cancer that share similar poor prognosis. Even though both cancers have overlaps, there are still some differences between those two types. It has been reported that the c-Met high expression was associated with poor prognosis not only in breast cancer but also in many other cancers. The role of ERβ in pathogenesis and treatment of breast cancer has remained controversial. In this study, we firstly distinguished basal-like from nonbasal-like cancer patients in TNBC patients using CK5/6 and EGFR as markers and next determined the relationship of basal-like breast cancer with c-Met or ERβ expression levels and prognosis in TNBC patients. One hundred twenty-seven patients who had been diagnosed with TNBC were enrolled. The clinical and pathological characteristics of the patients were recorded. The expression of EGFR, CK5/6, ERβ, and c-Met were evaluated with immunohistochemical methods using paraffin blocks. The median age of patients was 50.7 years. CK5/6 immunopositivity was 31.5 % (40/127), and EGFR was 40.2 % (51/127). Of the TNBC cases, 55.1 % (71/127) were positive for either CK5/6 or EGFR and were thus classified as basal-like breast cancer. C-Met (P < 0.001) and ERβ (P = 0.002) overexpression, small tumor sizes, a ductal subtype, and high-grade tumor were significantly correlated with BLBC. High c-Met expression was detected in 43.3 % patients. Metastatic lymph nodes and tumor size (>5 cm), which were both important prognostic predictors, were significantly associated with recurrence and mortality. BLBC typically demonstrates a unique profile. CK5/6 and EGFR expression combination indicates a higher basal-like phenotype possibility. The expression of c-Met and ERβ were significantly related to the basal-like phenotype. The classical markers, lymph node metastasis, and tumor size were found to have important prognostic value. However, high c-Met expression and basal-like phenotypes did not show a direct correlation with poor prognosis.

Objective/background: Chronic venous disease (CVD) is a common and relevant problem affecting Western people. The role of estrogens and their receptors in the venous wall seems to support the major prevalence of CVD in women. The effects of the estrogens are mediated by three estrogen receptors (ERs): ERα, ERβ, and G protein-coupled ER (GPER). The expression of ERs in the vessel walls of varicose veins is evaluated., Methods: In this prospective study, patients of both sexes, with CVD and varicose veins undergoing open venous surgery procedures, were enrolled in order to obtain vein samples. To obtain control samples of healthy veins, patients of both sexes without CVD undergoing coronary artery bypass grafting with autologous saphenous vein were recruited (control group). Samples were processed in order to evaluate gene expression., Results: Forty patients with CVD (10 men [25%], 30 women [75%], mean age 54.3 years [median 52 years, range 33-74 years]) were enrolled. Five patients without CVD (three men, two women [aged 61-73 years]) were enrolled as the control group. A significant increase of tissue expression of ERα, ERβ and GPER in patients with CVD was recorded (p < .01), which was also related to the severity of venous disease., Conclusion: ERs seem to play a role in CVD; in this study, the expression of ERs correlated with the severity of the disease, and their expression was correlated with the clinical stage., (Copyright © 2016 European Society for Vascular Surgery. Published by Elsevier Ltd. All rights reserved.)

Purpose: Oncogenic driver mutations are critical for lung cancer development and serve as therapeutic targets. However, their associations with environmental factors are not fully understood. We aimed to elucidate the relationship between tumor developmental biology and exposure to environmental factors., Patients and Methods: This was a prospective, multicenter, molecular epidemiology study. Eligible patients were those with newly diagnosed stages I to IIIB non-small-cell lung cancer (NSCLC) who underwent surgery. The tumors were examined for somatic mutations in 72 cancer-associated genes by targeted deep sequencing, estrogen receptor β (ERβ) expression using immunohistochemical staining, and infection with any of 37 types of human papillomavirus (HPV) using a polymerase chain reaction-based microarray system. Detailed information on patient demographics and environmental factors was obtained from a comprehensive questionnaire., Results: From July 2012 to December 2013, 957 patients were enrolled, and molecular analyses were performed on 876 samples (from 441 ever- and 435 never-smokers). Oncogenic driver mutations in P53 and KRAS increased proportionally with smoking status, whereas mutations in EGFR and SMAD4 decreased. KRAS mutations in smokers and SMAD4 mutations were observed more frequently in proportion to body mass index. TP53 and NFE2L2 mutations were observed more frequently in advanced NSCLC stages. As for never-smokers, no environmental factors were significantly associated with mutational changes. EGFR mutations and TP53 mutations were observed more frequently in women and in men, respectively. Mutations in these two genes were also potentially associated with ERβ expression. Only three patients (0.3%) were HPV positive., Conclusion: The mutational spectrum is associated with smoking, body mass index, and other environmental factors, as well as with ERβ expression. Little association was observed between HPV and NSCLC., (© 2016 by American Society of Clinical Oncology.)

Aim: To investigate estrogen receptors expression in duodenal familial adenomatous polyposis (FAP) and any relationship with epithelial proliferation/apoptosis markers., Methods: Twenty-two patients affected by FAP undergoing duodenal resection for malignancies were recruited. Controls were 15 healthy subjects undergoing endoscopy for dyspeptic symptoms. ER-α, ER-α, Ki-67, TUNEL and caspase 3 expression (labeling index: percentage of positive cells) were evaluated by immunohistochemistry or immunofluorescence and examined by light or confocal microscopy. Samples were assigned to four groups: normal tissue, low (LGD) and high-grade dysplasia (HGD), adenocarcinoma (AC). One-way analysis of variance, corrected by Bonferroni's test, and Pearson's correlation test were applied for statistical analysis., Results: ER-beta showed a progressive decline: normal tissue (23.5 ± 4.9), LGD (21.1 ± 4.8), HGD (9.3 ± 3.5), AC (7.1 ± 3.1). The normal tissue of FAP subjects expressed ER-beta like the controls (23.9 ± 6.2). Conversely, ER-α showed a progressive increase from normal tissue (24.8 ± 5.6) to AC (52.0 ± 8.2); the expression in normal tissue was similar to controls (22.5 ± 5.3). Ki67 demonstrated a statistically significant progressive increase at each disease stage up to AC. TUNEL did not reveal differences between controls and normal tissue of FAP subjects, but progressive decreases were observed in LGD, through HGD to AC. Pearson's correlation test showed a direct relationship between ER-β and TUNEL LI (r = 0.8088, P < 0.0001). Conversely, ER-α was inversely correlated with TUNEL LI (r = - 0.7257, P < 0.0001). The co-expression of ER-β and caspase 3 declined progressively from normal to neoplastic tissue., Conclusion: This study confirmed that ER-β is strongly decreased in duodenal FAP carcinomas, declining in a multiple step fashion, thereby suggesting a putative anti-carcinogenic effect. ER-α showed the opposite trend. ER-β/caspase 3 co-expression suggests this hormone's possible involvement in apoptosis. Hormonal influences in FAP duodenal tumorigenesis, and modulation of these as a possible chemoprevention strategy, may be a promising approach.

Skin is a target organ of sex steroids which play important roles in skin health and disease. The aim of this study is to investigate the expression of estrogen receptor β (ERβ) and androgen receptor (AR) in human skin from different age groups for a better understanding of the hormonal regulation of skin aging. Using standard immunohistochemical techniques, biopsies of sun-unprotected and sun-protected skin were taken from 60 normal subjects. Sun-protected skin showed significantly higher immunoreactivity for ERβ and AR compared to sun-unprotected skin of all age groups. Significantly higher ERβ H score and percent of expression were associated with the 20-35 years age group compared to the groups that were 35-50 years and >50 years old (p < 0.02, p = 0.03, respectively) in sun-unprotected and sun-protected skin (p < 0.001, p = 0.01, respectively). AR H score showed a negative correlation with age (p = 0.04) with no significant difference in immunoreactivity in different age groups, either in sun-unprotected or sun-protected skin. There was also a significant correlation between ERβ H score and epidermal thickness in sun-unprotected (p = 0.04) and sun-protected skin (p = 0.04) in studied subjects regardless of age. The same relationships did not reach significance with AR expression. However, a significant positive correlation was detected between H scores and percent of expression of ERβ and AR in sun-unprotected (p = 0.01, p = 0.02, respectively) and sun-protected skin (p = 0.005, p = 0.02, respectively) regardless of age. In conclusion, both ERβ and AR decline gradually with intrinsic and extrinsic aging. This decline is more obvious with extrinsic aging. Further large-scaled studies are recommended to expand, validate and translate current findings to clinically significant, diagnostic and therapeutic applications. Molecular studies to investigate the probable ligand-independent action of both receptors are warranted. In addition, their gene expression patterns and associated signaling and metabolic pathways can also be tackled to provide a basis for further interventions in pathological processes that involve their dysregulation., (© 2016 S. Karger AG, Basel.)

Uterine leiomyomas are benign myometrial neoplasms that function as one of the common indications for hysterectomy. Clinical and biological evidences indicate that uterine leiomyomas are estrogen-dependent. Estrogen stimulates cell proliferation through binding to the estrogen receptor (ER), of which both subtypes α and β are present in leiomyomas. Clinically, leiomyomas may be singular or multiple, where the first one is rarely recurring if removed and the latter associated to a relatively young age or genetic predisposition. These markedly different clinical phenotypes indicate that there may different mechanism causing a similar smooth muscle response. To investigate the relative expression of ERα and ERβ in multiple and solitary uterine leiomyomas, we collected samples from 35 Chinese women (multiple leiomyomas n = 20, solitary leiomyoma n = 15) undergoing surgery to remove uterine leiomyomas. ELISA assay was performed to detect estrogen(E2) concentration. Quantitative real-time PCR analysis was performed to detect ERα and ERβ mRNA expression. Western blot and immunohistochemical analysis were performed to detect ERα and ERβ protein expression. We found that ERα mRNA and protein levels of in multiple leiomyomas were significantly lower than those of solitary leiomyomas, whereas ERβ mRNA and protein levels in multiple leiomyomas were significantly higher than those in solitary leiomyomas, irrespectively of the menstrual cycle stage. In both multiple and solitary leiomyomas, ERα expression was higher than that of ERβ. E2 concentration in multiple and solitary leiomyomas correlated with that of ERα expression. ERα was present in nuclus and cytoplasma while estrogen receptor β localized only in nuclei in both multiple and solitary leiomyomas. Our findings suggest that the difference of ERα and ERβ expression between multiple and solitary leiomyomas may be responsible for the course of the disease subtypes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background/aim: Benign acini in benign prostatic hyperplasia (BPH) are lined with pseudostratified cylindrical epithelium with a continuous basal cell layer. Adenocarcinoma of the prostate is the most common cancer in men. High gradus-prostatic intraepithelial neoplasia (HGPIN) lesions precede invasive cancer. Prostate adenocarcinoma (PCa) implies a complete absence of basal cells and stromal invasion by malignant acini. Estrogen receptor (ER) is located in nuclei of acinar basal and secretory cells and partially in stromal cells. The aim of this research was to demonstrate and localize ER in BPH and in PCa of different Gleason scores. Considering literature data for ER-beta. expression in different morphologic prostate lesions, it is assumed that there is expression of ER-beta in most moderately differentiated PCa, and that the observed receptor expression is lost with increasing of the Gleason score., Methods: Four groups of patients were formed: the control with BPH and three experimental groups with PCa of different grades and scores, according to the Gleason grading system. The patients were male of various ages suspected of PCa, based on clinical and laboratory parameters. The study was conducted in a period 2010-2012. None of the patients received prior hormonal therapy. Sextant byopsies with BPH and PCa were treated for ER-beta (Novacastra). Localization and intensity of ER-beta expression is reported through the score: 0 = zero; 1 = < 1%; 2 = 1-10%; 3=11-33%; 4= 34-66%; 5- > 66%. Positive fibroblasts and endothelial cells are used for comparison., Results: ER-beta expression in acinar epithelial cells was the weakest in well-differentiated adenocarcinoma. A decline of ER-beta expression was noticed in malignant lesions of the prostate vs. benign ones. Less differentiated adenocarcinomas showed a decrease of ER-beta expression in basal and in the secretory cells. ER-beta expression in basal cells was stronger than in secretory ones in BPH and well-differentiated adenocarcinoma., Conclusion: ER-beta expression was most pronounced in BHP samples and declined in malignant prostate lesions. This finding supports statement on anticiproliferative role of ER-beta in prostatic tissue.

We report embryo-induced alterations occurring in endometrial stromal cells (ESCs) during the embryo-attachment stage in bonnet monkeys (Macaca radiata). Laser micro-dissected ESCs obtained from pregnant and non-pregnant animals were compared for levels of selected proliferation and decidualization-associated factors by analysis with quantitative real-time polymerase chain reaction or immunohistochemistry. Stromal cells exhibited extensive cellular proliferation, as indicated by cellular compaction and significantly higher (P < 0.05) levels of proliferating cell nuclear antigen and of estrogen receptor 1, c-Myc, and Cyclin D1 transcripts in pregnant animals as compared with non-pregnant animals. A significant decrease (P < 0.05) was observed in the transcript levels of stromal interleukin-6 (IL-6) in pregnant animals. Cell proliferation was accompanied by a significant increase (P < 0.001) in the levels of decidualization-associated molecules such as IL-1β in the luminal and glandular epithelium and of stromal insulin-like growth-factor-binding protein-1 (IGFBP-1) and prostaglandin-endoperoxide synthase-2 (PTGS-2) proteins. In pregnant animals, proliferation was evident throughout the gestational stroma, whereas decidualization was more pronounced in the embryo-attachment zone than in the non-attachment zone. To our knowledge, this is the first report of alterations in the endometrial stroma during the embryo-attachment stage in a non-human primate model.

To investigate whether Period 1 (PER1) and Estrogen receptor-beta (ER2) are associated with occurrence and development of Chinese colorectal cancers. By using RT-quantitative PCR, tissue microarray (TMA) and immunohistochemistry, we detected mRNA levels and protein levels of PER1 and ER2 in the cancerous tissues and paired normal adjacent tissues in patients with colorectal cancer. Survival analyses were performed by the Kaplan-Meier method utilizing log-rank test and univariate and multivariate Cox proportional modeling to measure 5-year disease-free survival (DFS) and overall survival (OS). Real-time PCR showed that, the delta Ct value (tumor tissue vs. normal mucosa) of PER1 or ER2 is 8.51 ± 2.81 vs. 7.34 ± 2.08 or 12.39 ± 2.43 vs. 9.76 ± 1.75, expression of PER1 and ER2 decreased significantly in tumor tissues compared with noncancerous mucosas of patients with or without metastasis (both of P values <0.001). Spearman test revealed that PER1 and ER2 were significantly down-regulated in cancerous tissues (r=0.283; P<0.001) which was also confirmed by immunohistochemistry of specimens from 203 colon cancer patients in a TMA format. The reduction of PER1 was associated with gender and distant metastasis (P=0.037 and P<0.001, respectively) whereas the decline of ER2 was associated with age (P=0.043) by analyzing the clinical data. However, we were not capable of detecting any association between PER1 level or ER2 level and overall survival (OS) or disease free survival (DFS). It is the first observation of correlated reduction of PER1 and ER2 in Chinese colon cancers, and they do play a certain role in colorectal cancer.

Estrogen and estrogen receptor (ER)-α and -β play a role in the development and progression of thyroid cancer. ERβ2 is one major splicing variant of ERβ. In this study, we investigated the clinical significance of ERβ2 protein expression in the papillary thyroid carcinoma (PTC) lesion. ERβ2 expression was immunohisto-chemically examined in formalin-fixed, paraffin-embedded thyroid tissues from 106 patients with PTC by Elivision™ plus two-step system as previously described. The relationships between ERβ2 expression and clinicopathological/biological factors were then analyzed. ERβ2 protein was expressed in all the PTC patients studied. It was positively associated with Ki-67 expression in female PTC patients with advanced reproductive age (>45 years, in low-estrogen status) and with VEGF expression in male PTC patients with reproductive age (18~45 years, in low-estrogen status) (P=0.005 and P=0.044, respectively). There was no association between ERβ2 expression and tumor size, extrathyroidal extension and tumor-node-metastasis stage in PTC patients. In addition, ERβ2 expression was lower in female patients of reproductive age (18~45 years, in relatively high-estrogen status) with lymph node metastasis than that in those patients without lymph node metastasis (P=0.035). The present results suggest that the expression of ERβ2 in PTC is associated with the progression of the disease. Its potential effect may vary with different estrogen status. Further study will assess the underlying molecular mechanisms of ERβ2 in PTC.

Seasonal fluctuations in the activity of bird testes are regulated by a complex mechanism where androgens play a key role. Until recently, the role played by estrogens in males has been significantly underestimated. However, there is growing evidence that the proper functioning of the testes is associated with optimal estradiol (E2) concentration in both the plasma and testes of many mammalian species. Estrogens are gradually emerging as very important players in hormonal regulation of reproductive processes in male mammals. Despite the previously mentioned, it should be noted that estrogenic action is limited by the availability of specific receptors--estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Interestingly, there is a general scarcity of information concerning the estrogen responsive system in the testes of male birds, which is of particular interest in exploring the phenomenon of seasonality of reproduction. To address this question, we have investigated for the first time the simultaneous expression of testicular ERα and ERβ genes and proteins with the accompanying plasma and testicular E2 concentrations during the annual reproductive cycle of male bird. The research model was the domestic goose (Anser anser f. domestica), a species whose annual reproductive cycle can be divided into 3 distinct phases characterized by changes in testicular activity. It has been revealed that the stable plasma E2 profile did not correspond to changing intratesticular E2 profile throughout the experiment. The expression of ERα and ERβ genes and proteins was detected in gander testes and it fluctuated on a seasonal basis with lower level in breeding and sexual reactivation stages and higher level during the nonbreeding stage. Our results demonstrated changes in testicular sensitivity to estrogens in male domestic goose during the annual reproductive cycle. The seasonal pattern of estrogen receptors (ERs) expression was analyzed against the hormonal background and a potential mechanism of ERs regulation in bird testes was proposed. The present study revealed seasonal variations in the estrogen responsive system, but further research is needed to fully explore the role of estrogens in the reproductive tract of male birds., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Little is known about the reaction of the posterior vaginal wall influenced by steroid receptor expression. The aim of our study was to evaluate change of estrogen and progesterone receptor expression in the posterior vaginal wall after local estrogen therapy for vaginal prolapse surgery and to compare this expression with a group of untreated women of same age. Furthermore we examined the steroid receptor expression among untreated women in each period of life. In a prospective clinical study we examined staining intensity and ratio of positive cytoblasts in specimens of vaginal tissue sampled during posterior colporrhaphy and as control group specimens from autopsied corpses to proof the change in steroid receptor expression in treated women after local estrogen therapy compared to treatment-naive women of all ages. We compared 60 premenopausal female corpses to 43 postmenopausal female corpses and 80 postmenopausal women undergoing posterior colporrhaphy to 43 postmenopasusal female corpses. Estrogen-receptor alpha score was significantly higher in the intervention group in basal epithelium (p=0.004), stroma (p≤0.001) and connective tissue (p=0.005). Estrogen-receptor beta score was significantly higher in basal epithelium (p=0.048), progesterone-receptor score was significantly higher in the intervention group in stroma and connective tissue (p≤0.001) and in superficial epithelium (p=0.017). Local vaginal estrogen therapy leads to increase in estrogen-receptor alpha and progesterone-receptor expression of the posterior vaginal wall in postmenopausal women, while estrogen-receptor-beta-expression keeps nearly unchanged. This explains the fact that proliferation of the vaginal tissue is mediated by estrogen-receptor alpha and improves the condition for our prolapse surgery., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Purpose: The natural history of human malignant melanoma suggests that steroid hormones may affect the biological behavior of this tumor. The purpose of the current study was to investigate the specific immunostaining patterns of estrogen receptors in malignant melanomas and their sentinel lymph nodes (SLNs), as well as to examine any possible association with patients' prognosis and overall survival., Materials and Methods: A retrospective analysis of prospectively collected data was conducted during a 12-year period (2001-2012). Sixty patients with mean age of 54.4 ± 14.5 years diagnosed with melanomas of varying depth (Clark) and thickness (Breslow) after excision biopsy of pre-existing melanocytic lesions, were included in the study. All patients underwent wide excision of the primary tumor and SLN identification. Determination of estrogen receptor alpha (ERa) and beta (ERb) status by immunohistochemistry on tumor and nodal paraffin blocks was performed in all feasible cases., Results: ERb but not ERa was the predominant estrogen receptor found in all primary tumors and SLNs examined. The most intense ERb immunostaining was seen in negative SLNs associated with thinner, less invading melanomas. ERb expression in the primary tumor seems to correlate with the cellular microenvironment, possibly altering the process of SLN invasion., Conclusions: ERb expression is down-regulated in aggressive melanomas with sentinel nodal metastatic disease, suggesting its possible usefulness as a surrogate marker for metastatic potential and prognosis in malignant melanoma.

Background: The clinicopathological importance of Ki-67 in breast cancers has been intensely studied; however, there have been few systematic large studies of patients treated with predefined adjuvant therapy. Further, Ki-67 evaluation methodology differed among studies, which prevents Ki-67 from being used for clinical practice. We performed a large systematic study using routinely processed tissues and compared various scoring methods., Methods: Representative slides of archival tissue blocks of 442 consecutive invasive breast cancers from women treated with adjuvant tamoxifen monotherapy and having a long follow-up period were subjected to immunohistochemistry using anti-Ki-67 monoclonal antibody, Mib-1. Both the average score across the section and the score at the hottest spot were assessed., Results: Ki-67 evaluated at the hottest spot, not the average score across the section, independently predicted poor clinical outcomes of patients with hormone receptor-positive/HER2-negative cancer. Ki-67 was not a predictor of clinical outcome in patients with triple-negative breast cancer. Overall, high Ki-67 level significantly correlated with classic unfavorable clinicopathological factors, correlating negatively with the status of estrogen receptor (ER)-α and progesterone receptor (PR), and positively with HER2 status and grade. ER-β status positively correlated with the Ki-67 level., Conclusions: Ki-67 evaluation at the hottest spot was superior to that determined by average score across the section as a predictor of outcome in patients with hormone receptor-positive/HER2-negative breast cancers treated with endocrine monotherapy. The different result obtained in patients with triple-negative carcinomas needs to be further investigated.

Estrogen signaling is considered to play an important role in spermatogenesis, spermiogenesis and male fertility. Estrogens can act via the two nuclear estrogen receptors ESR1 (ERα) and ESR2 (ERβ) or via the intracellular G-protein-coupled estrogen receptor 1 (GPER, formerly GPR30). Several reports on the localization and expression of all three receptors in the human testis have been published but are controversial particularly in case of ERα. Contrary to previous studies, we decided therefore to evaluate expression of all three receptors in the testis by a number of different methods and in comparison with MCF-7 cells. Using qPCR, we could show that mRNA expression of ERα is considerably lower and expression of ERβ and GPER much higher in the testis than in MCF-7 cells. RT-PCR after laser-assisted microdissection of tubular and interstitial compartments from normal and Sertoli cell only syndrome testes plus in situ hybridization and immunohistochemical analyses of the same samples demonstrated that there is very low expression of ERα in germ cells and in single interstitial cells, very high expression of ERβ in germ cells and Sertoli cells and high expression of GPER in interstitial cells and less in Sertoli cells.

The world-wide occurrence of zearalenone (ZEN) as a contaminant in feed for farm animals and fish requires the evaluation of toxicity mechanisms of action of ZEN. The present study investigates possible metabolization of ZEN in fish cell lines suggesting that mainly glucuronidation takes place. It demonstrates that concentrations up to 20,000 ng ml(-1) ZEN are capable of influencing cell viability in permanent fish cell cultures in a dose-response manner with different response patterns between the five tested cell lines, whereby lysosomes appeared to be the main target of ZEN. ZEN toxicity is often discussed in the context of oxidative stress. Our study shows a biphasic response of the cell lines when reactive oxygen species (ROS) production is monitored. Damage in cells was observed by measuring lipid peroxidation, DNA strand breaks, and alterations of intracellular glutathione levels. Metabolization of ZEN, especially at concentrations above 7500 ng ml(-1) ZEN, does not prevent cytotoxicity. ZEN as an estrogenic compound may involve processes mediated by binding to estrogen receptors (ER). Since one cell line showed no detectable expression of ER, an ER-mediated pathway seems to be unlikely in these cells. This confirms a lysosomal pathway as a main target of ZEN in fish cells., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Methylations in estrogen receptor (ER) α and ERβ are known to be involved in the pathogenesis of breast cancer. Here, we explore the role of promoter methylation of estrogen receptors, ERα and ERβ, in sporadic breast cancer cases from a North Indian population. To this end, association between ERα and ERβ methylation status along with different clinicopathological parameters and its correlation with protein expression was examined. Four hundred eighty paired breast cancer tissue samples and adjacent normal controls from 240 sporadic breast cancer patients were included, and their clinical and demographic profiles were recorded. ERα and ERβ methylation was determined by methylation-specific polymerase (MSP) chain reaction. Our findings demonstrate that methylation of ERα and ERβ occurs in high frequency and appears to be a mechanism of gene silencing in our population. Furthermore, on performing stratified analysis, we observed strong associations between ERα/ERβ methylation and ER, PR, and HER2 status, tumor size, clinical stage, and triple negative tumors. Thus, our study not only highlights the role of ERα/ERβ methylation in breast cancer but also suggests the ERα/ERβ methylation pattern as a biomarker for assessing breast cancer risk.

Objectives: Estrogen is suggested to participate in pathogenesis of irritable bowel syndrome (IBS), but expression of G protein-coupled estrogen receptor (GPER) in the colon of IBS patients has never been investigated. The aim of this study was to investigate the expression of GPER and classical estrogen receptors in the colon of IBS patients and healthy controls., Methods: Colonic biopsies were obtained by endoscopy from patients with IBS (n=46) and healthy subjects (n=13). Expression of GPER, estrogen receptor α (ERα) and estrogen receptor β (ERβ) in mast cells were measured by double-labelling immunofluorescence. Quantification of mRNA expression was performed for GPER, ERα and ERβ by real-time polymerase chain reaction., Results: Differential distribution of GPER, ERα and ERβ were detected in human colonic mucosa. The expression of GPER in the cytoplasm of mast cells and GPER-positive cells was significantly higher in diarrhea-predominant IBS (D-IBS) patients than that in constipation-predominant IBS (C-IBS, P<0.001) patients and healthy subjects (P=0.005). ERα and ERβ were not detected in majority of mast cells in colonic mucosa and no difference of immunostaining results for ERα and ERβ was found among these three groups. A positive correlation (r=0.451, P=0.011) between GPER-positive cell counts and abdominal pain severity was observed in D-IBS group. Relative mRNA expression of GPER in D-IBS was also higher than that in C-IBS (P=0.018) and healthy subjects (P=0.011)., Conclusions: The present study, for the first time, demonstrated the expression of GPER in human colonic mucosa and its correlation with abdominal pain severity.