Steroid-converting enzymes in human adipose tissues and fat deposition with a focus on AKR1C enzymes.

Adipocytes express various enzymes, such as aldo-keto reductases (AKR1C), 11ß-hydroxysteroid dehydrogenase (11ß-HSD), aromatase, 5α-reductases, 3ß-HSD, and 17ß-HSDs involved in steroid hormone metabolism in adipose tissues. Increased activity of AKR1C enzymes and their expression in mature adipocytes might indicate the association of these enzymes with subcutaneous adipose tissue deposition. The inactivation of androgens by AKR1C enzymes increases adipogenesis and fat mass, particularly subcutaneous fat. AKR1C also causes reduction of estrone, a weak estrogen, to produce 17ß-estradiol, a potent estrogen and, in addition, it plays a role in progesterone metabolism. Functional impairments of adipose tissue and imbalance of steroid biosynthesis could lead to metabolic disturbances. In this review, we will focus on the enzymes involved in steroid metabolism and fat tissue deposition.

Comparison of Estradiol, Testostosterone, and CYP19 Methylation Levels between Full-Term and Preterm Newborns.

Estradiol (E), testosterone (T), and their ratio are crucial axis in life. Especially during intrauterine growth, they orchestrate the complex development of organs and their interaction, which have lifelong impact on health and an organism's capacity to respond to environmental stressors. The aim of this study was to compare for the first time E, T, and their ratio levels with aromatase (CYP19) gene methylation levels between preterm newborns (PN) and full-term newborns (FN) with respect to their mother's environmental exposure and diet. In this study, 56 FN of 37-42 weeks of gestation age (GA) and 46 PN at GA 27-36 weeks were analysed for E and T levels and CYP19A1 gene pI.3/II promoter region methylation. Results showed there was no difference in E levels between PN and FN, but there were significantly lower levels of T in PN than in FN (2.81 nmol vs. 3.76 nmol, respectively) and consequently a significantly higher E/T ratio in PN than in FN (5278.04 vs. 2891.23, respectively). CYP19A1 methylation was significantly lower in PN than in FN (86.04% vs. 90.04%, respectively). CYP19A1 methylation was significantly reduced in newborns whose mothers reported daily milk consumption. Our study is the first to provide referent values for CYP19A1 methylation levels in FN and PN and shows that PN and FN significantly differ in CYP19A1 methylation levels, T levels, and E/T ratio. Future research should further investigate the mechanisms involved in GA-dependent CYP19A1 methylation levels and mechanisms of sex hormone disturbances which may contribute to preterm birth.

Relationship of estrogen synthesis capacity in the brain with obesity and self-control in men and women.

Gonadal hormones are linked to mechanisms that govern appetitive behavior and its suppression. Estrogens are synthesized from androgens by the enzyme aromatase, highly expressed in the ovaries of reproductive-aged women and in the brains of men and women of all ages. We measured aromatase availability in the amygdala using positron emission tomography (PET) with the aromatase inhibitor [11C]vorozole in a sample of 43 adult, normal-weight, overweight, or obese men and women. A subsample of 27 also completed personality measures to examine the relationship between aromatase and personality traits related to self-regulation and inhibitory control. Results indicated that aromatase availability in the amygdala was negatively associated with body mass index (BMI) (in kilograms per square meter) and positively correlated with scores of the personality trait constraint independent of sex or age. Individual variations in the brain's capacity to synthesize estrogen may influence the risk of obesity and self-control in men and women.

Initial Studies with 11C-Vorozole PET Detect Overexpression of Intratumoral Aromatase in Breast Cancer.

Aromatase inhibitors are the mainstay of hormonal therapy in estrogen receptor-positive breast cancer, although the response rate is just over 50% and in vitro studies suggest that only two thirds of postmenopausal breast tumors overexpress aromatase. The goal of the present study was to validate and optimize PET with 11C-vorozole for measuring aromatase expression in postmenopausal breast cancer in vivo. Methods: Ten newly diagnosed postmenopausal women with biopsy-confirmed breast cancer were administered 11C-vorozole intravenously, and PET emission data were collected between 40 and 90 min after injection. Tracer injection and scanning were repeated 2 h after ingestion of 2.5 mg of letrozole. Mean and maximal SUVs and ratios to nontumor tissue in the contralateral breast were determined at baseline and after letrozole. Biopsy specimens from the same tumors were stained for aromatase using immunohistochemistry and evaluated for stain intensity and the percentage of immune-positive cells. Results: Seven of the 10 women (70%) demonstrated increased mean focal uptake of tracer (SUV ratio > 1.1) coinciding with the mammographic location of the lesion, whereas the other 3 women (30%) did not (SUV ratio ≤ 1.0). All patients with an SUV ratio above 1.1 had mean SUVs above 2.4, and there was no overlap (SUV ratio ≤ 1; SUVmean, 0.8-1.8). The SUV ratio relative to breast around tumor was indistinguishable from the ratio to contralateral breast. Pretreatment with letrozole reduced tracer uptake in most subjects, although the percentage of blocking varied across and within tumors. Tumors with a high SUV in vivo also showed a high immunohistochemical staining intensity. Conclusion: PET with 11C-vorozole is a useful technique for measuring aromatase expression in individual breast lesions, enabling noninvasive quantitative measurement of baseline and posttreatment aromatase availability in primary tumors and metastatic lesions.

An assessment of the multifactorial profile of steroid-metabolizing enzymes and steroid receptors in the eutopic endometrium during moderate to severe ovarian endometriosis.

BACKGROUND: Previous studies of expression profiles of major endometrial effectors of steroid physiology in endometriosis have yielded markedly conflicting conclusions, presumably because the relative effects of type of endometriosis, fertility history and menstrual cycle phases on the measured variables were not considered. In the present study, endometrial mRNA and protein levels of several effectors of steroid biosynthesis and action in patients with stage III-IV ovarian endometriosis (OE) with known fertility and menstrual cycle histories were compared with the levels in control endometrium to test this concept. METHODS: Endometrial samples were collected from patients without endometriosis (n = 32) or OE stages III-IV (n = 52) with known fertility and cycle histories. qRT-PCR and immunoblotting experiments were performed to measure levels of NR5A1, STAR, CYP19A1, HSD17Bs, ESRs and PGR transcripts and proteins, respectively. Tissue concentrations of steroids (P4, T, E1 and E2) were measured using ELISAs. RESULTS: The levels of expression of aromatase and ERß were lower (P < 0.0001) and 17ß-HSD1 (P < 0.0001) and PRA (P < 0.01) were higher in OE endometrium. Lower aromatase levels and higher 17ß-HSD1 levels were detected in fertile (aromatase: P < 0.05; 17ß-HSD1: P < 0.0001) and infertile (aromatase: P < 0.0001; 17ß-HSD1: P < 0.0001) OE endometrium than in the matched control tissues. Both proliferative (PP) and secretory (SP) phase OE samples expressed aromatase (P < 0.0001) and ERß (PP: P < 0.001; SP: P < 0.01) at lower levels and 17ß-HSD1 (P < 0.0001) and PRA (PP: P < 0.01; SP: P < 0.0001) at higher levels than matched controls. Higher 17ß-HSD1 (P < 0.01) and E2 (P < 0.05) levels and a lower (P < 0.01) PRB/PRA ratio was observed in infertile secretory phase OE endometrium than in control. CONCLUSIONS: We report that dysregulated expression of 17ß-HSD1 and PGR resulting in hyperestrogenism and progesterone resistance during the secretory phase of the menstrual cycle, rather than an anomaly in aromatase expression, was the hallmark of eutopic endometrium from infertile OE patients. Furthermore, the results provide proof of concept that the fertility and menstrual cycle histories exerted relatively different effects on steroid physiology in the endometrium from OE patients compared with the control subjects.

Human chorionic gonadotropin-induced amphiregulin stimulates aromatase expression in human granulosa-lutein cells: a mechanism for estradiol production in the luteal phase.

STUDY QUESTION: Does amphiregulin (AREG), the most abundant and important epidermal growth factor receptor (EGFR) ligand in the follicular fluid, regulate aromatase expression in human granulosa-lutein (hGL) cells? SUMMARY ANSWER: AREG mediates the hCG-induced up-regulation of aromatase expression and estradiol (E2) production in hGL cells. WHAT IS KNOWN ALREADY: AREG expression and secretion are rapidly induced by hCG in hGL cells and mediate physiological functions of LH/hCG in the ovary. EGFR protein is expressed in follicles not only in the pre-ovulatory phase but also throughout the luteal phase of the menstrual cycle. After the LH surge, the human corpus luteum secretes high levels of E2, which regulates various luteal cell functions. Aromatase is an enzyme responsible for a key step in the biosynthesis of E2. However, whether AREG regulates aromatase expression and E2 production in hGL cells remains unexplored. STUDY DESIGN, SIZE, DURATION: This study is an experimental study performed over a 1-year period. In vitro investigations examined the role of AREG in the regulation of aromatase expression and E2 production in primary hGL cells. PARTICIPANTS/MATERIALS, SETTING, METHODS: Primary hGL cells were obtained from women undergoing IVF treatment in an academic research center. Aromatase mRNA and protein levels were examined after exposure of hGL cells to recombinant human AREG, hCG or LH. The EGFR tyrosine kinase inhibitor AG1478, PI3K inhibitor LY294002 and siRNAs targeting EGFR, LH receptor, StAR and AREG were used to verify the specificity of the effects and to investigate the underlying molecular mechanisms. Reverse transcription quantitative real-time PCR (RT-qPCR) and western blot were used to measure the specific mRNA and protein levels, respectively. Follicular fluid and serum were collected from 65 infertile women during IVF treatment. Pearson's correlation analysis was performed to examine the correlation coefficient between two values. MAIN RESULTS AND THE ROLE OF CHANCE: Treatment of hGL cells with AREG-stimulated aromatase expression and E2 production. Using pharmacological inhibitors and specific siRNAs, we revealed that AREG-stimulated aromatase expression and E2 production via EGFR-mediated activation of the protein kinase B (AKT) signaling pathway. In addition, inhibition of EGFR activity and AREG knockdown attenuated hCG-induced up-regulation of aromatase expression and E2 production. Importantly, the protein levels of AREG in the follicular fluid were positively correlated with the E2 levels in serum after 2 days of oocyte pick-up and in the follicular fluid of IVF patients. LARGE-SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The in vitro setting of this study is a limitation that may not reflect the real intra-ovarian microenvironment. Clinical data were obtained from a small sample size. WIDER IMPLICATIONS OF THE FINDINGS: Our results provide the first evidence that hCG-induced AREG contributes to aromatase expression and E2 production in the luteal phase of the menstrual cycle. A better understanding of the hormonal regulation of female reproductive function may help to develop new strategies for the treatment of clinical infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China for Young Scientists (81601253), the specific fund of clinical medical research of Chinese Medical Association (16020160632) and the Foundation from the First Affiliated Hospital of Zhengzhou University for Young Scientists to Lanlan Fang. This work was also supported by an operating grant from the National Natural Science Foundation of China (81820108016) to Ying-Pu Sun. All authors declare no conflict of interest.

A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the standard radiometric assay.

Estrogens are key factors in the development of the estrogen receptor-positive (ER+) breast cancer. Estrogens, estrone (E1), and estradiol (E2) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER+ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E1 (ratio E1/AD) using isotopic analogues as internal standards. The method showed good linearity (r2 = 0.9908 for AD and 0.9944 for E1), high accuracy (more than 74% for AD and more than 66% for E1), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC50 values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC50 values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs. Graphical abstract.

High expression of adrenal P450 aromatase (CYP19A1) in association with ARMC5-primary bilateral macronodular adrenocortical hyperplasia.

Primary bilateral macronodular adrenocortical hyperplasia (PBMAH) is a rare cause of ACTH-independent Cushing syndrome (CS), which has been associated with ectopic G-protein coupled receptors (GPCRs) in the adrenal cortex. We recently studied a 51-year-old male with PBMAH who presented with severe CS and hyperestronemia, manifesting clinically with a Cushingoid appearance, gynecomastia, and telangiectasias. Analysis of adrenal tissues following bilateral adrenalectomy showed high expression of P450 aromatase (CYP19A1). The patient carried a germline non-sense pathogenic variant in ARMC5 (p.R173*), with two independent somatic pathogenic variants identified in the right (p.S571*) and left (p.Q235*) adrenal tissues, respectively. The expression of ARMC5 was drastically decreased in the hyperplastic regions when compared to either the adjacent non-hyperplastic regions and samples from PBMAH without pathogenic variants in ARMC5. We found expression of CYP19A1 in other cases of PBMAH, although there were no differences in aromatase expression between ARMC5-mutant and ARMC5-non-mutant cases. We conclude that in select cases, PBMAH can be associated with aromatase expression resulting in elevated estrogens, irrespective of sex. Additionally, CYP19A1 expression does not appear to depend on the ARMC5 variant status.

Expression and characterization of the cyp19a gene and its responses to estradiol/letrozole exposure in Chinese soft-shelled turtle (Pelodiscus sinensis).

Cytochrome P450 aromatase (CYP19) catalyzes the conversion of androgens to estrogens and is critical in sex differentiation. CYP19 exists as the ovarian type and brain type. Herein, we cloned the full-length ovarian cyp19a gene from the Chinese soft-shelled turtle, Pelodiscus sinensis (pscyp19a). We determined the distribution of pscyp19a in adult tissue and evaluated its expression during embryonic development, following treatment with 17ß-estradiol (E2) or letrozole (LE). The pscyp19a complementary DNA is 2,285 bp in length and comprises a 1,512 bp open reading frame that encodes a protein of 503 AA. The nucleotide sequence and amino acid of pscyp19a shared significant identity with other vertebrate sequences. Expression of pscyp19a was high in the ovary (p < 0.01), and exhibited modest expression in the female brain and intestine. Expression of pscyp19a displayed significant differences between sexes during early embryo development stages; expression increased gradually during embryonic development in females, but the opposite trend was observed in males. Female embryos treated with different concentrations of E2 and LE displayed altered pscyp19a expression compared with untreated individuals, and E2 clearly induced pscyp19a expression. These results indicate that pscyp19a gene plays important roles in early developmental stages in Chinese soft-shelled turtle, and may assist future studies on sex differentiation and sex control in this and similar species.

The Mussel Mytilus galloprovincialis in the Bay of Naples: New Insights on Oogenic Cycle and Its Hormonal Control.

The aim of the present article was to investigate the oogenic cycle of Mytilus galloprovincialis sampled in the Bay of Naples, and to immunolocalize 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and P450 aromatase, enzymes involved in the synthesis of two sex hormones: testosterone and 17ß-estradiol. We demonstrate that the oogenic cycle starts in late summer-early fall and continues in early winter when the first event of spawning occurs; other spawning events take place until June, when the ovary is spent and contains a few empty ovarian follicles and numerous somatic cells, that is, adipogranular cells and vesicular connective tissue cells. During the oogenic cycle, apoptotic events occur at the level of oogonia, previtellogenic oocytes, as well as follicle cells; by contrast, necrosis events probably take place in vitellogenic oocytes, which, once degenerated, transfer their content to healthy oocytes. Finally, the present data demonstrate that 3ß-HSD, 17ß-HSD, and P450 aromatase are present in the ovary both during the reproductive and nonreproductive phases. The possible role of these enzymes during the Mytilus galloprovincialis reproductive cycle is discussed. Anat Rec, 302:1039-1049, 2019. © 2019 Wiley Periodicals, Inc.

Expression and localization of aromatase in human gastric mucosa : Immunohistochemical study using biopsy materials.

Parietal cells in the gastric mucosa are known not only as cells playing major roles in food digestion but also as cells bearing endocrine function. In addition to their production of gastrin and ghrelin, it has been recently revealed that these cells are also involved in the synthesis and secretion of estrogens with their expression of aromatase in experimental animals. Although aromatase activity has been detected in human gastric cancer cells and related cell lines, much less study has been done to ascertain the expression of the enzymatic activity in normal gastric mucosa. It has not been established which cell type is responsible for estrogen production in human gastric glands consisting of epithelial cells of several types. The aim of this study is to define the expression of aromatase by parietal cells in human gastric glands using immunohistochemical techniques. We retrieved formalin-fixed paraffin embedded materials of gastric biopsies from 16 patients (nine men, seven women). Colocalization of aromatase and H+/K+-ATPase ß-subunit indicated that positive cells are parietal cells, but not chief cells and mucous cells. Furthermore, immunoreactivity of aromatase was detected within gastric glands irrespective of age or sex. These results suggest that human parietal cells synthesize estrogens within gastric mucosa and subsequently secrete them to the portal vein via gastric vein, as they do in rats. These estrogens might influence liver functions in humans. The estrogenic effects related to liver dysfunction might also be attributed to them.

Differences in aromatase expression and steroid hormone concentrations in the reproductive tissues of male domestic turkeys (Meleagris gallopavo) with white and yellow semen.

1. To show hormonal differences between male turkeys with yellow semen syndrome (YSS) and white, normal semen (WNS), the expression of aromatase, oestrogen receptor α (ERα), and oestrogen receptor ß (ERß) as well as testosterone and oestradiol concentrations in YSS and WNS testes, epididymis, and ductus deferens were examined. 2. To measure gene expression levels of aromatase and oestrogen receptors (ERs), three complementary techniques (real-time PCR, Western blot, and immunohistochemistry) were used, whereas steroid hormone levels were determined radio-immunologically. 3. Upregulation of aromatase and ERα mRNAs in YSS testes (P < 0.05; P < 0.01), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.05; P < 0.01) compared to those of WNS tissues was detected. Significant increases in the levels of aromatase and ERα proteins were detected in YSS testes (P < 0.001; P < 0.05), epididymis (P < 0.001; P < 0.001), and ductus deferens (P < 0.001; P < 0.05). The expression of ERß mRNA and protein level was upregulated in the testes (P < 0.05; P < 0.01) and epididymis (P < 0.001; P < 0.01) but not in ductus deferens where it was downregulated (P < 0.01; P < 0.01). Increased intensity of immunoreactive proteins in YSS versus WNS reproductive tissues corroborated gene expression results. 4. Testosterone concentration diminished in YSS epididymis (P < 0.05) and ductus deferens (P < 0.05), but not in the testes, remaining at high level (P < 0.05) compared to WNS values. Concomitantly, increased oestradiol concentration was found in YSS testes (P < 0.05) and epididymis (P < 0.05) but decreased in the ductus deferens (P < 0.05). 5. From the published literature, this study is the first to demonstrate the ability for androgen aromatisation in the turkey reproductive tissues and to show the cellular targets for locally produced oestrogens. The data suggested that the androgen/oestrogen ratio is a mechanistic basis for amplification of differences between turkeys with white and yellow semen and that these results can have a relevance in applied sciences to widen the knowledge on domestic bird reproduction.

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques.

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large-scale production of functional food and nutraceutical ingredients.

Impacts of prenatal triclosan exposure on fetal reproductive hormones and its potential mechanism.

BACKGROUND: Triclosan (TCS) has been widely detected in pregnant women. The reproductive endocrine-disrupting effects of TCS have been observed in humans and animals. Little is known about the potential impact of prenatal TCS exposure on fetal reproductive development as well as its potential mechanism. OBJECTIVES: We investigated the potential effect of prenatal TCS exposure on fetal reproductive hormones in cord blood and its potential mechanism in relation to placental steroidogenic enzymes. METHODS: Urinary TCS was detected among 537 healthy pregnant women from a prospective cohort in China. Four reproductive hormones in cord blood, namely E2 (n=430), T (n=424), LH (n=428) and FSH (n=373), and three steroidogenic enzymes in placenta, namely P450arom (n=233), 3ß-HSD (n=227) and 17ß-HSD (n=222), were measured. RESULTS: Prenatal TCS exposure was associated with increased testosterone concentrations in cord blood in a dose-dependent manner. Infants with prenatal TCS levels >0.6µg/L had, on average, a 0.23ng/mL (95% CI: 0.05, 0.45, p=0.02) higher testosterone concentrations in cord blood compared to those with prenatal TCS levels <0.1µg/L. Of note, prenatal TCS exposure was associated with increased testosterone and decreased E2 concentrations in cord blood among male infants. Adverse associations were found between the prenatal TCS exposure and concentrations of three placental steroidogenic enzymes. 3ß-HSD and P450arom demonstrated mediating effects in the association between prenatal TCS exposure and testosterone concentrations in cord blood. CONCLUSIONS: Our findings suggested potential impacts of prenatal TCS exposure on reproductive hormones in cord blood mediated by steroidogenic enzymes, and male infants were more vulnerable.

Pattern of aromatase mRNA expression in the brain of a weakly electric fish, Apteronotus leptorhynchus.

Aromatase is a steroidogenic enzyme involved in the conversion of testosterone into estradiol. Teleosts are unique among vertebrates in possessing two distinct aromatase genes that show different expression patterns within the body. Since the brain is the essential organ underlying the control of behavior, an understanding of the expression pattern of aromatase in the brain can help to identify neural circuits and behaviors that are most likely to be affected by aromatase activity. In addition, identifying species differences in aromatase expression in the brain can further our understanding of divergence in behaviors regulated by local estradiol production and estrogen signaling. Apteronotus leptorhynchus is a species of weakly electric fish in which little is known about sex steroid expression within the brain and its role in electric signaling behavior. The goal of this study was to identify the mRNA expression pattern of aromatase in the brain of A. leptorhynchus. Aromatase mRNA was detected in several parts of the forebrain and in the pituitary gland; however, no aromatase expression was detected in the midbrain or hindbrain. These findings in A. leptorhynchus support a role for aromatase activity in reproduction, but no direct role in electric signaling behavior in non-breeding fish. The findings of this study help to broaden the basis for making phylogenetic comparisons of aromatase expression across teleost lineages as well as different signaling systems, and provide information on behaviors and neural circuits that are potentially affected by local estradiol production in A. leptorhynchus.

Expression of aromatase and estrogen receptors in lumbar motoneurons of mice.

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 ERß 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.

Preparation of a novel antiserum to aromatase with high affinity and specificity: Its clinicopathological significance on breast cancer tissue.

Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between Aromatase inhibitors have been widely used for the endocrine treatment of estrogen-dependent breast cancer in postmenopausal patients. However, clinicopathological studies of aromatase have been limited due to unsatisfactory specificity and/or restricted availability of anti-aromatase antibodies. Here, we have generated a polyclonal antiserum with high affinity and specificity for human aromatase using a monoclonal antibody tagged immunoaffinity chromatography on an industrial production scale. Our preliminary immunohistochemical analysis of 221 invasive breast cancer cases indicated that 87.3% (193/221) had at least 5% aromatase positive cells. The histoscore for aromatase was inversely correlated with pT (p = 0.019), pN (p = 0.001), stage (p < 0.001), histologic grade (p = 0.003), lymphatic infiltration (p < 0.001), venous infiltration (p < 0.001), and Ki-67 index (p < 0.001). However, cancer aromatase expression was independent of estrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 statuses. This antiserum will be applicable to clinicopathological examination of aromatase in addition to ER and PgR for an appropriate use of aromatase inhibitor on the treatment of breast cancer. Further studies on the relationship between aromatase expression and aromatase inhibitors are warranted.

Abnormal lipid/lipoprotein metabolism and high plasma testosterone levels in male but not female aromatase-knockout mice.

Sex steroid hormones, such as estrogen and testosterone, are believed to play important roles in lipid metabolism. To elucidate the effects of estrogen depletion on lipid metabolism in male and female mice, we used aromatase-knockout (ArKO) mice, in which Cyp19 gene disruption prevented estrogen synthesis in vivo. These mice were divided into the following 4 groups: male and female ArKO mice and male and female wild-type (WT) mice. These mice were fed a normal-fat diet (13.6% fat) ad libitum. At 159 days after birth, the mice were tested for liver and plasma lipid content and hepatic hormone receptor- and lipid/lipoprotein metabolism-related gene expression. Interestingly, we found that hepatic steatosis was accompanied by markedly elevated plasma testosterone levels in male ArKO mice but not in female ArKO mice. Plasma lipoprotein profiles exhibited concurrent decreases in LDL- and small dense LDL-triglyceride (TG) levels in male ArKO mice. Moreover, male mice, but not female mice, exhibited marked elevations in androgen receptor (AR), sterol regulatory element-binding protein 1 (SREBP1), and CD36 expression. These results strongly suggest that Cyp19 gene disruption, which induces a sexually dimorphic response and high plasma testosterone levels in male mice, also induces hepatic steatosis.

4-(((4-Iodophenyl)methyl)-4H-1,2,4-triazol-4-ylamino)-benzonitrile: A Potential Imaging Agent for Aromatase.

Aromatase (CYP19) is a rate-limiting enzyme that catalyzes the biosynthesis of estrogens. Imaging agents based on aromatase inhibitors (AIs) have been developed for a PET/SPECT study. A series of compounds was synthesized based on YM511, which has previously been used for breast cancer treatment. Two examples of these derivatives, 4-(((4-iodophenyl)methyl)-4H-1,2,4-triazol-4-yl-amino)-benzonitrile (5) and 4-((1H-imidazol-1-yl)(4-iodobenzyl)amino)benzonitrile (11), displayed potent binding affinities to human aromatase (IC50 = 0.17 and 0.04 nM, respectively). Biodistribution and autoradiographic studies revealed that [125I]5 and [125I]11 were highly accumulated in the stomach (16.21 and 10.88% dose/g, respectively) and ovaries (8.56 and 3.32% dose/g, respectively) of female rats. Log P of [125I]5 was 2.49, meaning good brain penetration. Autoradiograms of brain sections showed a high uptake in the bed nucleus of the stria terminalis and amygdala. These results suggest that [125I]5 and [125I]11 are potent probes for aromatase imaging in both the brain and peripheral organs.

Combination of the non-invasive tests for the diagnosis of endometriosis.

BACKGROUND: About 10% of women of reproductive age suffer from endometriosis, a costly chronic disease causing pelvic pain and subfertility. Laparoscopy is the gold standard diagnostic test for endometriosis, but is expensive and carries surgical risks. Currently, there are no non-invasive tests available in clinical practice to accurately diagnose endometriosis. This review assessed the diagnostic accuracy of combinations of different non-invasive testing modalities for endometriosis and provided a summary of all the reviews in the non-invasive tests for endometriosis series. OBJECTIVES: To estimate the diagnostic accuracy of any combination of non-invasive tests for the diagnosis of pelvic endometriosis (peritoneal and/or ovarian or deep infiltrating) compared to surgical diagnosis as a reference standard. The combined tests were evaluated as replacement tests for diagnostic surgery and triage tests to assist decision-making to undertake diagnostic surgery for endometriosis. SEARCH METHODS: We did not restrict the searches to particular study designs, language or publication dates. We searched CENTRAL to July 2015, MEDLINE and EMBASE to May 2015, as well as the following databases to April 2015: CINAHL, PsycINFO, Web of Science, LILACS, OAIster, TRIP, ClinicalTrials.gov, DARE and PubMed. SELECTION CRITERIA: We considered published, peer-reviewed, randomised controlled or cross-sectional studies of any size, including prospectively collected samples from any population of women of reproductive age suspected of having one or more of the following target conditions: ovarian, peritoneal or deep infiltrating endometriosis (DIE). We included studies comparing the diagnostic test accuracy of a combination of several testing modalities with the findings of surgical visualisation of endometriotic lesions. DATA COLLECTION AND ANALYSIS: Three review authors independently collected and performed a quality assessment of the data from each study by using the QUADAS-2 tool. For each test, the data were classified as positive or negative for the surgical detection of endometriosis and sensitivity and specificity estimates were calculated. The bivariate model was planned to obtain pooled estimates of sensitivity and specificity whenever sufficient data were available. The predetermined criteria for a clinically useful test to replace diagnostic surgery were a sensitivity of 0.94 and a specificity of 0.79 to detect endometriosis. We set the criteria for triage tests at a sensitivity of 0.95 and above and a specificity of 0.50 and above, which 'rules out' the diagnosis with high accuracy if there is a negative test result (SnOUT test), or a sensitivity of 0.50 and above and a specificity of 0.95 and above, which 'rules in' the diagnosis with high accuracy if there is a positive result (SpIN test). MAIN RESULTS: Eleven eligible studies included 1339 participants. All the studies were of poor methodological quality. Seven studies evaluated pelvic endometriosis, one study considered DIE and/or ovarian endometrioma, two studies differentiated endometrioma from other ovarian cysts and one study addressed mapping DIE at specific anatomical sites. Fifteen different diagnostic combinations were assessed, including blood, urinary or endometrial biomarkers, transvaginal ultrasound (TVUS) and clinical history or examination. We did not pool estimates of sensitivity and specificity, as each study analysed independent combinations of the non-invasive tests.Tests that met the criteria for a replacement test were: a combination of serum IL-6 (cut-off >15.4 pg/ml) and endometrial PGP 9.5 for pelvic endometriosis (sensitivity 1.00 (95% confidence interval (CI) 0.91 to 1.00), specificity 0.93 (95% CI, 0.80, 0.98) and the combination of vaginal examination and transvaginal ultrasound (TVUS) for rectal endometriosis (sensitivity 0.96 (95% CI 0.86 to 0.99), specificity 0.98 (95% CI 0.94 to 1.00)). Tests that met the criteria for SpIN triage tests for pelvic endometriosis were: 1. a multiplication of urine vitamin-D-binding protein (VDBP) and serum CA-125 (cut-off >2755) (sensitivity 0.74 (95% CI 0.60 to 0.84), specificity 0.97 (95% CI 0.86 to 1.00)) and 2. a combination of history (length of menses), serum CA-125 (cut-off >35 U/ml) and endometrial leukocytes (sensitivity 0.61 (95% CI 0.54 to 0.69), specificity 0.95 (95% CI 0.91 to 0.98)). For endometrioma, the following combinations qualified as SpIN test: 1. TVUS and either serum CA-125 (cut-off ≥25 U/ml) or CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.79 (95% CI 0.64 to 0.91), specificity 0.97 (95% CI 0.91 to 1.00)); 2. TVUS and serum CA 19.9 (cut-off ≥12 U/ml) (sensitivity 0.54 (95% CI 0.37 to 0.70), specificity 0.97 (95% CI 0.91 to 1.0)); 3-4. TVUS and serum CA-125 (cut-off ≥20 U/ml or cut-off ≥25 U/ml) (sensitivity 0.69 (95% CI 0.49 to 0.85), specificity 0.96 (95% CI 0.88 to 0.99)); 5. TVUS and serum CA-125 (cut-off ≥35 U/ml) (sensitivity 0.52 (95% CI 0.33 to 0.71), specificity 0.97 (95% CI 0.90 to 1.00)). A combination of vaginal examination and TVUS reached the threshold for a SpIN test for obliterated pouch of Douglas (sensitivity 0.87 (95% CI 0.69 to 0.96), specificity 0.98 (95% CI 0.95 to 1.00)), vaginal wall endometriosis (sensitivity 0.82 (95% CI 0.60 to 0.95), specificity 0.99 (95% CI 0.97 to 1.0)) and rectovaginal septum endometriosis (sensitivity 0.88 (95% CI 0.47 to 1.00), specificity 0.99 (95% CI 0.96 to 1.00)).All the tests were evaluated in individual studies and displayed wide CIs. Due to the heterogeneity and high risk of bias of the included studies, the clinical utility of the studied combination diagnostic tests for endometriosis remains unclear. AUTHORS' CONCLUSIONS: None of the biomarkers evaluated in this review could be evaluated in a meaningful way and there was insufficient or poor-quality evidence. Laparoscopy remains the gold standard for the diagnosis of endometriosis and using any non-invasive tests should only be undertaken in a research setting.