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Introduction: Hypospadias is an abnormal development of the urethral, ventral skin and corporeal bodies. Urethral meatus and ventral curvature have been historically the landmarks to define clinical severity. Genotyping has never been explored as a clinical predictor. Available reports have demonstrated a correlation between genetic mutations and syndromic hypospadias with poor surgical outcomes. We hypothesize that inclusion of genotyping can serve at classifying all types of hypospadias. We present the use of neural network algorithm to evaluate phenotype/genotype correlations and propose its potential clinical applicability., Methods: A systematic review was performed from January 1974 to June 2022. Literature was retrieved from Medline, Embase, Web of Science and Google Scholar. Included manuscripts were those that had an explicit anatomical description of hypospadias phenotype (urethral meatus location following an anatomical description) and a defined genotype (genetic mutation) description. Cases with more than one variant/mutation were excluded. A comprehensive phenotype-genotype statistical analysis using neural network non-linear data modeling SPSS™ was performed., Results: Genotype-Phenotype analysis was performed on 1731 subjects. Of those, 959 (55%) were distal and 772 (45%) proximal. 49 genes with mutations were identified. Neural network clustering predicted better for coronal (90%) and glanular (80%), and lowest for midshaft (22%) and perineal (45%). Using genes as predictor factor only, the model was able to highly and more accurately predict the phenotype for coronal and glanular hypospadias. The following genotypes showed association to a specific phenotype: AR gene n.2058G > A for glanular (p<0.0001), n.480C  >  T for coronal (p = 0.034), R840C for perineal (p = 0.002), MAMLD1 gene c.2960C > T for coronal (p< 0.0001), p. G289S for glanular (p<0.0001), gene SRD5A2 607G > A for scrotal (p<0.0001), c16C > T for penoscrotal (p<0.0001), c59 T  >  c for perineal (p = 0.042), V89L for midshaft and scrotal (p<0.0001, p = 0.041; respectively)., Discussion: Hypospadias phenotype has always been described from a purely anatomical perspective. Our results demonstrate that current phenotyping has poor correlation to the genotype. Higher genotype/phenotype correlation for distal hypospadias proves the clinical applicability of genotyping these cases. The concept and classification of differences in sexual development needs to be reconsidered given high positive yield reported for distal hypospadias. Given the better predictive value of genotyping in correlation to the phenotype, future efforts should be directed towards using the genotype., Conclusion: Hypospadias has poor phenotype/genotype correlation. Sequencing all hypospadias phenotypes may add clinical value if used in association to other predictive variables. Neural network analysis may have the ability to combine all these variables for clinical prediction., Competing Interests: Conflicts of interest None of the authors have any conflicts of interest to declare., (Copyright © 2023 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Objective: Hypospadias is a common congenital abnormality that has been increasing in prevalence over the last decades. Historically, 46, XY patients with severe hypospadias and descended scrotal testes at birth have frequently lacked a genetic diagnosis. Platforms for molecular genetic testing have become more readily available and can offer an insight into underlying genetic causes of severe hypospadias. The goal of this study was to define the anatomical characteristics of severe hypospadias that can accurately define patients with 46, XY severe hypospadias and determine the practical utility of performing molecular genetic testing in this group of patients., Methods: Patients who met the criteria for 46, XY severe hypospadias were offered a molecular genetic work-up in consultation with pediatric genetics. Patients were identified through chart review. Data extracted included karyotype, hypospadias phenotype including stretched penile length at diagnosis, age at genetic diagnosis, molecular genetic testing, pathogenic gene variant(s), gender identity, and clinical course. All patients underwent clinical genetic testing via 46, XY Disorders of Sexual Development (DSD) panels offered by Invitae®, GeneDx®, or Blueprint Genetics®., Results: Of the 14 patients that underwent genetic testing, there were 5 previously published and 3 novel pathogenic or likely pathogenic variants in genes associated with 46, XY severe hypospadias (Table). Pathogenic variants were identified in AR (3), SRD5A2 [1], NR5A1 [2], WT1 [1], and ARTX [1]. Two patients had a variant of unknown significance, one in FREM2 and another in CEP41. Four had negative gene panels. The patient with the WT1 pathogenic variant was subsequently found to have developed a Wilms tumor and the patients with NR5A1 pathogenic variants are now undergoing adrenal insufficiency surveillance., Discussion/conclusion: Patients with 46,XY severe hypospadias and descended testes in the scrotum at birth can benefit from molecular genetic testing as their underlying disorders may reveal pathogenic variants that could have potentially life-altering consequences and change surveillance and monitoring., Competing Interests: Declaration of competing interest The authors confirm that they have no conflicts of interest to declare for this publication., (Copyright © 2022 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Background: SRD5A2 deficiency leads to incomplete masculinization of individuals with a 46 XY karyotype. A definitive diagnosis in early infancy facilitates decisions concerning choice of sex of rearing and management., Aim: To review the clinical presentation, diagnosis, treatment and outcome of children with 46 XY DSD due to SRD5A2 deficiency at a Paediatric Gender Clinic., Study Design and Methods: Retrospective review of cases of SRD5A2 deficiency (2000-15) managed with a standard protocol at a multidisciplinary clinic. Demographic data, clinical presentation, physical findings, investigations (hormonal profile, imaging, genitoscopy), psychological evaluation (child, family), medical and surgical management, outcome and follow up were collated and analyzed., Results: There were 12 cases aged 3 days-14 years at presentation, 3 had parental consanguinity. Eight were reared as males and 4 as females. Specialist referral was sought for hypospadias (5), atypical genitalia (5) or incongruent pubertal masculinization (2). All had chordee, symmetrical inguinoscrotal gonads, rugose labioscrotum and proximal hypospadias (perineoscrotal -9, perineal -3). Both pubertal cases had significant masculinization and no gynecomastia. The median testosterone/dihydrotestosterone ratio was 22.1(IQR-8.6-55.7). Despite a classical phenotype, four (2 prepubertal, 2 pubertal) had a ratio <10. Genitoscopy showed urogenital sinus remnant (4) and hypoplastic verumontanum (5). Sex reassignment was done in 4. Surgical management was staged and completed by 4 years in those with infantile presentation. Besides correction of chordee and urethroplasty in 11, other procedures included orchidopexy (5), excision of a urogenital sinus remnant (4) and correction of penoscrotal transposition (4). The urethroplasty was single staged in 3. All operated cases were followed up (mean age at last follow up - 10.63 years, mean follow up period - 7.25 years). The overall cosmetic result was satisfactory, but the phallic structure remained relatively small across prepubertal period. Uroflowmetry curves were normal in 9. All showed penile tumescence/erection and two peripubertal cases had typical secondary sexual characters. All cases, including those with sex reassignment, have a well-adjusted male psyche., Discussion and Conclusion: The diagnosis, management and longitudinal follow up of cases of SRD5A2 deficiency at a multidisciplinary gender clinic is presented. Diagnostic dilemmas with low T/DHT ratios remained in a third of cases. Most were diagnosed in infancy and assigned a male sex of rearing, all underwent staged masculinizing genitoplasty. Those with sex reassignment also fared well with comprehensive management after family counseling., Competing Interests: Conflicts of interest There is no conflict of interest to declare., (Copyright © 2022 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Purpose: To test the hypothesis whether the number of the C(ytosine)-A(denine)-G(uanine) triplets in the androgen receptor (AR-) gene and further single-nucleotide polymorphisms in the androgen-responsive element of the promoter region of genes regulating the androgen pathway influence oncologic outcomes in patients with concomitant bladder (BC) and prostate cancer (PC) at a predisposing level., Material and Methods: A cohort of 36 patients was treated with radical cystectomy and histologically exhibited invasive BC and incidental PC. The number of cytosine-adenine-guanine (CAG)-triplets (rs4045402) in the AR gene was assessed in tumor-free lymph nodes as well as rs743572 in the CYP17A1 gene and rs676033, rs523349, rs9282858 in the SRD5A2. In addition, the clinical significance of incidental PC based on the Epstein-criteria was assessed with regard to BC-specific recurrence-free survival (RFS). The median follow-up was 26 months (range: 4-68)., Results: Patients with clinically significant PC had worse BC-specific RFS compared with patients with insignificant PC (P = 0.016). Patients with a PC volume of >0.2 cm 3 had shorter 3-year BC-specific RFS compared with patients with a PC volume ≤0.2 cm 3 (P = 0.025). The median number of CAG-triplets was 24 (mean ± SEM: 23 ± 2, interquartile range: 22-25, total range 18-29). Patients with a CAG-triplet number <23 exhibited significantly decreased 3-year BC specific RFS compared with patients with ≥23 repeats (27% vs. 65%; P = 0.026). No further significance were noted for the other tested SNPs and survival., Conclusions: A detailed description of incidental PC at radical cystoprostatectomy (RC) may be of greater prognostic importance than previously assumed in the literature. The CAG-repeat in the AR gene may predispose to worse oncologic outcomes after RC and should be further evaluated in larger studies., Competing Interests: Conflict of interest All authors declare that there are no competing financial interest in the relation to the work described., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Steroid 5α-reductase type 3 congenital disorder of glycosylation (SRD5A3-CDG) is a rare metabolic disease mainly characterized by psychomotor disability, visual impairment, and variable eye malformations caused by bi-allelic pathogenic variants in SRD5A3. So far, only 23 distinct mutations were described. Exome sequencing in 32-year old monozygotic male twins revealed only the heterozygous splice variant c.562+3delG in SRD5A3, but no second variant. The twins presented with psychomotor deficit and a complex eye disease including retinal dystrophy, pallor of the papilla, nystagmus, and strabismus suggestive of SRD5A3-CDG. Only when applying exome-based copy number analysis, we identified as a second compound heterozygous variant a previously not reported tandem duplication of exons 2-4 in SRD5A3. Next to the typical skeletal anomalies of SRD5A3-CDG such as kyphosis and scoliosis, extension deficits of the proximal interphalangeal (PIP) joints IV were observed. Since similar contractures were described once in a patient with SRD5A3-CDG, we suggest that this rare symptom is possibly associated with SRD5A3-CDG. Our findings further expand the mutational and clinical spectrum of SRD5A3-CDG and emphasize the importance of an intragenic copy number analysis in patients with strong clinical suspicion of SRD5A3-CDG and only one detectable sequence variant., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Introduction: Disorders of sex development (DSD) constitutes a group of congenital conditions that affect urogenital differentiation and are associated with chromosomal, gonadal and phenotypic sex abnormalities., Objective: To evaluate the clinical and genetic features of childhood DSD cases., Materials and Methods: DSD patients followed up between the years of 2002-2018 were evaluated in terms of their complaints, demographic, clinical features and genetic diagnoses., Results: Out of 289 patients, 143(49.5%) were classified as 46XY DSD, 62(21.5%) as 46XX DSD and 84(29%) as sex chromosomal DSD. Genetic diagnosis was achieved in 150 patients (51.9%). The distribution of the molecular diagnosis of the 46XY DSD patients were; 12 (26.6%) SRD5A2, 10 (22.2%) AR, 7 (15.5%) HSD17B3, 3 (6.6%) WT-1, 2 (4.4%) AMHR2, 2 (4.4%) AMH, 2 (4.4%) LHCGR, 2 (4.4%) HSD3B2, 1 (2.2%) NR5A1, 1 (2.2%) CYP17A1 and 1 (2.2%) SRY mutation. Fifty (80.6%) of the 46XX DSD patients received a diagnosis with clinical and laboratory findings. Twenty-four (38.7%) of them were 21-hydroxylase deficiency, 9(14.5%) Rokitansky-Küster-Hauser Syndrome, 4 (6.5%) 11-β hydroxylase deficiency, 3 (4.8%) gonadal dysgenesis and 2 (3.2%) aromatase deficiency. In 46XX group pathogenic mutations were detected in 21(33.8%) of the patients. Eighty-four (29%) patients were diagnosed as sex chromosomal disorder. Of these 66 (78.5%) were Turner Syndrome, 6 (7.2%) Klinefelter Syndrome and 10 (11.9%) mix gonadal dysgenesis. Gender re-assignment was decided in 11 patients. Malignant and pre-invasive lesions was diagnosed in 8 (2.7%) patients., Conclusion: Many of DSD's are clinically similar and etiology of numerous of them still cannot be established. A multi-disciplinary approach and new rapid genetic diagnostic methods are needed in the process from diagnosis to gender assignment and follow-up., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Background: The formation of the male urethra depends to enzyme-mediated testosterone (T) conversion into 5α-dihydrotestosterone (DHT). Two metabolic pathways could be operating in the fetal testis to synthesize androgens: 1) the "classic" route (T→DHT) mediated by SRD5A2 and 2) a "backdoor" pathway in which DHT is synthesized by aldo-keto reductase family 1, member C2 (AKR1C2), AKR1C3, and AKR1C4 enzymes without formation of a T intermediate., Objective: We studied four genes of the "backdoor" pathway in karyotypic males with hypospadias to ascertain whether gene defects in AKRs impair urethral DHT formation that result in hypospadias., Design and Patients: The coding regions of the AKR1C2-4 and HSD17B6 genes were analyzed by PCR-SSCP and sequencing in a cohort of 25 Mexican patients (0.3-9 year-old-children) with 46,XY-hypospadias. Chi-squared tests was performed to evaluate the distribution of genotypes, alleles, and the Hardy-Weinberg (H-W) equilibrium. The effect of the genetic variants was investigated by in silico studies., Results: Screening studies revealed distinct genotypic patterns at different exons of AKR1C2-4 whereas HSD17B6 presented a wild-type sequence. The DNA analyses detected two synonymous variants (c.327C>T, c.666T>C/unreported) in AKR1C2. The AKR1C3 had two variants (c.15C>G, c.230A>G), two unreported variants (c.538T>C, c.596G>A), and one silent variant (c.312G>A). Two variants (c.434C>G, c.931C>G) were identified in AKR1C4. All variants were in H-W equilibrium without structural changes., Discussion: Hypospadias have been associated with defects that alter androgen biosynthesis in the human fetal testis, specifically 5α-DHT. We selected four candidate genes involved in the "backdoor" pathway for the formation of 5α-DHT. Molecular assays of the AKR1C2, AKR1C3, and AKR1C4 genes revealed a total of nine genetic single nucleotide variants. Several variants in the AKR1C genes have been associated with a variety of human pathologies. However, our studies suggest that active steroid biosynthesis via AKR1C might not be involved in hypospadias. Additionally, genetic research suggests a low involvement in the "backdoor" 5α-DHT pathway during human sexual development, specifically, the differentiation of male external genitalia., Conclusion: These results indicate that substitutions in AKR1C2-4 are polymorphisms and all genetic variants lacks deleterious significant association with hypospadias. The data suggest that inactivating mutations in the AKR1C2-4 and HSD17B6 genes are an infrequent cause of hypospadias, which might weaken the contribution of the "backdoor" pathway to embryonic urethral masculinization., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Copyright © 2020 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Background and Aim: Multiple studies support a complex relationship between testosterone and type 2 diabetes mellitus (T2DM) and the transformation of testosterone is affected by several reductases. Thus, we aimed to explore the associations of steroid-5α-reductase type 1 (SRD5A1) gene polymorphism with impaired fasting glucose (IFG) and T2DM and the interactive effects of testosterone and genotypes on glycometabolism., Methods and Results: A case-control study including 2365 participants was performed. Genomic DNA was extracted from the whole blood and genotyped for the SRD5A1 single nucleotide polymorphisms (SNP) rs1691053. Multivariable logistic regression and linear regression were performed to estimate the associations of SRD5A1 rs1691053 alleles and genotypes with glycometabolism. Generalized linear models were used to investigate the modulatory effects of serum testosterone on glycometabolism indexes in males. After multivariable adjustment, the odds ratio (OR) of homozygous CC genotypes in male carriers was 2.62 (95%CI: 1.11-6.18) for IFG. Furthermore, significant associations of SRD5A1 rs1691053 polymorphisms with adverse indices of glycometabolism were observed in males. Interestingly, the opposite associations in females were observed. The interactive associations of SNP and testosterone were found and mutations were more likely to lead unfavorable metabolic phenotypes., Conclusion: These results showed that SRD5A1 rs1691053 gene polymorphism was independently associated with glycometabolism. The interaction between a genetic polymorphism from SRD5A1 and testosterone involved glycometabolism was identified in males. Although this preliminary data should be replicated with other rigorous researches, it highlighted the importance of the SNP-testosterone interaction over the present of glycometabolism., Competing Interests: Declaration of Competing Interest The authors have nothing to disclose., (Copyright © 2019 The Italian Society of Diabetology, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition, and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Background: Hypospadias is a common congenital malformation in males, in which the urethral orifice is found on the ventral side of the penis as a result of incomplete fusion of urethral folds. The etiology of hypospadias is poorly understood, and may be multifactorial, including genetic, endocrine and environmental factors. The steroid 5-alpha-reductase type 2 (SRD5A2) gene, which is mainly expressed in the ventral side of the urethra in the process of male genital development, plays an important role in urethral shaping., Objective: To investigate, with database searches of related published papers, whether SRD5A2 gene V89L polymorphism has an association with hypospadias risk., Methods: The following databases were searched for relevant papers, and all published case-control studies of hypospadias were used to perform a meta-analysis: PubMed, Embase, Springer Link, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang, and Weipu. A quality assessment was performed using the Newcastle-Ottawa scale of a case-control study. To assess the strength of the association under various genetic models, odds ratio (OR) and its 95% confidence interval (CI) were calculated using fixed-effect or random-effects model according to the heterogeneity. Overall and stratified subgroup analyses, including ethnicity, source of controls, sample for DNA extraction, and hypospadias classification, were performed. All data were analyzed using Review Manager 5.3., Results: This analysis included six eligible case-control studies with 1130 cases and 1279 controls. Overall, there was a statistically significant association between hypospadias risk and V89L polymorphism for allele contrast (C vs G: OR 1.91, 95% CI 1.13-3.23), P = 0.02), codominant model (CC vs GG: OR 2.97, 95% CI 1.25-7.04, P = 0.01; GC vs GG: OR 2.36, 95% CI 1.35-4.13, P = 0.003), dominant model (GC + CC vs GG: OR 2.46, 95% CI 1.28-4.72, P = 0.007), and recessive model (CC vs GC + GG: OR 1.91, 95% CI 1.00-3.66, P = 0.05). Moreover, there was also a statistically significant association in some subgroups. The positive results are shown in the Summary Table., Conclusion: This meta-analysis suggested that the V89L polymorphism definitely increases the risk of hypospadias, and the C allele is a genetic risk factor for hypospadias occurrence., (Copyright © 2017 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.)

Aims: Bladder urothelial carcinoma is increasing in incidence with age and its prognosis could become worse when accompanied with metastasis. Effective treatment of these advanced patients is required and it becomes important to understand its underlying biology of this neoplasm, especially with regard to its biological pathways. A potential proposed pathway is androgen receptor (AR)-mediated intracellular signaling but the details have remained relatively unexplored., Main Methods: The expression of AR, 5α-reductase type1 (5αR1) and 5α-reductase type2 (5αR2) were examined in the bladder cancer cell line T24 and surgical pathology specimens. We also evaluated the status of androgen related cell proliferation and migration using the potent, non-aromatizable androgen agonist 5α-dihydrotestosterone (DHT)., Key Findings: DHT treatment significantly increased AR mRNA expression level, but not those of 5αR1 and 5αR2 in T24 cells. DHT also suppressed cellular migration with weaker and opposite effects on cell proliferation. A significant inverse correlation was detected between pT stage and AR, 5αR1 and 5αR2 immunoreactivity., Significance: Inverse correlations detected between tumor grade and AR/androgen metabolizing enzyme also suggested that the loss of AR and androgen-producing enzymes could be associated with tumor progression. Effects of DHT on cells also suggest that androgens may regulate cellular behavior., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hypospadias, a common congenital malformation of male external genitalia, is characterized mainly by an aberrant opening of the urethra on the ventral side of the penis. Depending on the severity of the disease, it can be classified into three types: anterior, middle, and posterior. In our study, 33 patients with hypospadias were recruited, including eight with anterior hypospadias and 25 with posterior hypospadias. We performed mutation analysis of the SRD5A2, AR and HSD17B3 genes in these patients. Eight different SRD5A2 mutations were detected in 15 patients with posterior hypospadias (60%, 15/25), including six previously described mutations (p.Q6X, Q71X, p.L20P, p.G203S, p.R227Q, and p.R246Q) and two novel mutations (p.G196R and p.L73Pfs ∗ 17). One AR gene mutation (p.A597T) was found in a patient and no HSD17B3 mutations were detected. Additionally, we carried out routine semen analyses in all adult patients. Combing mutation analysis with semen examination results, showed that whole five adult patients who carried SRD5A2 mutations had abnormal semen quality. In summary, all the detected mutations were responsible for the clinical features observed in these 16 patients. Our data suggest that mutations of the SRD5A2 gene are the main causes of posterior hypospadias and seem to affect the semen quality of adult patients, whereas mutations in AR and HSD17B3 gene were rare in these hypospadias patients. Our study expanded the SRD5A2 mutation spectrum in the Han Chinese population and provided useful information for genetic and reproductive counselling for patients with hypospadias., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Perfluoroalkylated substances (PFASs), including perfluorooctyl sulphonate (PFOS) and perfluorooctane acid (PFOA), have been classified as persistent organic pollutants and are known to cause endocrine-disrupting effects in humans. The objective of the present study was to compare the potencies of four different PFASs, PFOS, PFOA, perfluorohexyl sulfonate (PFHxS), and perfluorobutyl sulfonate (PFBS), to inhibit neurosteroidogenic 5α-reductase 1 (SRD5A1), 3α-hydroxysteroid dehydrogenase (AKR1C9), and retinol dehydrogenase 2 (RoDH2) in rats. The potencies of PFASs to inhibit SRD5A1 are PFOS > PFOA > PFHxS = PFBS, with IC 50 values of PFOS and PFOA of 1.92 ± 0.07 and 14.24 ± 0.07 μM and no effects for PFHxS and PFBS at 100 μM, respectively. The potencies of PFASs to inhibit AKR1C9 and RoDH2 are PFOS > PFOA > PFHxS = PFBS, with IC 50 values of PFOS and PFOA on these two enzymes about 100 μM and no effects for PFHxS and PFBS at 100 μM, respectively. PFOS and PFOA competitively inhibited rat SRD5A1. In conclusion, PFOS and PFOA are potent inhibitors of rat SRD5A1, thereby controlling the biosynthesis of neurosteroids., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present study was undertaken to determine the role of different polymorphisms affecting the testosterone/oestrogen pathway in miscarriage. Alpha 5-reductase (SRD5A2) rs523349 and rs9282858, cytochrome P450 aromatase (CYP19A1) rs4646, rs10046 and rs2236722 and oestrogen receptor (ESR1) rs9340799, rs2234693 and rs6932902 polymorphisms were selected. The case group consisted of 94 samples of formalin-fixed and paraffin-embedded fetal tissue from a miscarriage at ≤24 weeks. The control group comprised a population of 331 young healthy subjects. Only those single nucleotide polymorphisms (SNPs) fitting the Hardy-Weinberg equilibrium (n = 4) and euploid miscarriage samples (n = 67) were included for downstream analysis. Interestingly, SRD5A2 rs523349 (Val89Leu) was significantly associated with the risk of undergoing miscarriage after Bonferroni correction (odds ratio = 11.245, P < 2.2 × 10 -9 ). Moreover, when Mantel-Cox regression analysis was performed, we observed that the effect was significantly constrained to the second trimester (P = 0.024, log rank). These results are compatible with an imbalance of testosterone/dihydrotestosterone, associated with a higher risk of miscarriage, especially in late pregnancy., (Copyright © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Di-(2-ethylhexyl) phthalate (DEHP) has reproductive toxicity and can affect male reproductive development. In order to clarify adverse effects of DEHP on testicular physiology and testosterone production, Sprague-Dawley (SD) rats were dosed daily with DEHP by gavage for 30days; TM3 cells (mouse Leydig cell line) were treated with DEHP for 24h after pretreatment with vitamin C or U0126. Results indicated that the hypothalamic-pituitary-testis (HPT) axis was disturbed and serum testosterone, LH and FSH levels were decreased following DEHP exposure. Histomorphological changes of rat testes were also observed, such as deformed seminiferous tubules, aggregated chromatin, multiple vacuoles, swollen mitochondria, apoptotic germ cells and Sertoli cells, as well as increased Leydig cell numbers. Moreover, DEHP caused oxidative stress in vivo and in vitro and then induced the ERK pathway, which was required to mediate 5α-Reductase 2 and scavenger receptor class B-1 (SRB1) levels. However, levels of steroidogenic acute regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), P450 17α-hydroxylase/17.20 lyase (P450c17), and P450 side-chain cleavage enzyme (P450scc) were not significantly altered after DEHP exposure. Taken together, DEHP-disturbed HPT axis and induced 5α-Reductase 2 contribute to the reduction of serum testosterone level. The activated ERK pathway is required to modulate expressions of 5α-Reductase 2 and SRB1., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Prostate cancer (PCa) is an androgen-dependent disease. Nonetheless, the role of single nucleotide polymorphisms (SNPs) in genes encoding androgen metabolism remains an unexplored area., Purpose: To investigate the role of germline variations in cytochrome P450 17A1 (CYP17A1) and steroid-5α-reductase, α-polypeptides 1 and 2 (SRD5A1 and SRD5A2) genes in PCa., Patients and Methods: In total, 494 consecutive Spanish patients diagnosed with nonmetastatic localized PCa were included in this multicenter study and were genotyped for 32 SNPs in SRD5A1, SRD5A2, and CYP17A1 genes using a Biotrove OpenArray NT Cycler. Clinical data were available. Genotypic and allelic frequencies, as well as haplotype analyses, were determined using the web-based environment SNPator. All additional statistical analyses comparing clinical data and SNPs were performed using PASW Statistics 15., Results: The call rate obtained (determined as the percentage of successful determinations) was 97.3% of detection. A total of 2 SNPs in SRD5A1-rs3822430 and rs1691053-were associated with prostate-specific antigen level at diagnosis. Moreover, G carriers for both SNPs were at higher risk of presenting initial prostate-specific antigen levels>20ng/ml (Exp(B) = 2.812, 95% CI: 1.397-5.657, P = 0.004) than those who are AA-AA carriers. Haplotype analyses showed that patients with PCa nonhomozygous for the haplotype GCTTGTAGTA were at an elevated risk of presenting bigger clinical tumor size (Exp(B) = 3.823, 95% CI: 1.280-11.416, P = 0.016), and higher Gleason score (Exp(B) = 2.808, 95% CI: 1.134-6.953, P = 0.026)., Conclusions: SNPs in SRD5A1 seem to affect the clinical characteristics of Spanish patients with PCa., (Copyright © 2015 Elsevier Inc. All rights reserved.)

5-α Reductase type 2 (SRD5A2) is a critical enzyme for prostatic development and growth. Inhibition of SRD5A2 by finasteride is used commonly for the management of urinary obstruction caused by benign prostatic hyperplasia. Contrary to common belief, we have found that expression of SRD5A2 is variable and absent in one third of benign adult prostates. In human samples, absent SRD5A2 expression is associated with hypermethylation of the SRD5A2 promoter, and in vitro SRD5A2 promoter activity is suppressed by methylation. We show that methylation of SRD5A2 is regulated by DNA methyltransferase 1, and inflammatory mediators such as tumor necrosis factor α, NF-κB, and IL-6 regulate DNA methyltransferase 1 expression and thereby affect SRD5A2 promoter methylation and gene expression. Furthermore, we show that increasing age in mice and humans is associated with increased methylation of the SRD5A2 promoter and concomitantly decreased protein expression. Artificial induction of inflammation in prostate primary epithelial cells leads to hypermethylation of the SRD5A2 promoter and silencing of SRD5A2, whereas inhibition with tumor necrosis factor α inhibitor reactivates SRD5A2 expression. Therefore, expression of SRD5A2 is not static and ubiquitous in benign adult prostate tissues. Methylation and expression of SRD5A2 may be used as a gene signature to tailor therapies for more effective treatment of prostatic diseases., (Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Androgens in breast cancer have been studied alone and in correlation with estrogens as estrogen to testosterone ratio. 5-α-reductase is one of the important enzymes participating in androgen metabolism, which affects androgen activity by affecting conversion of testosterone to dihydrotestosterone. We hypothesized that polymorphisms in the SRD5A2 gene (encoding 5-α-reductase) may affect breast cancer risk by affecting total androgen activity. Complete coding region of the SRD5A2 gene was sequenced in a group of 628 patients and 244 control samples from three southern states (Tamil Nadu, Andhra Pradesh, and Karnataka) of India. We observed three common polymorphisms in this gene; namely, A49T, V89L, and (TA)n repeats. A49T locus was monomorphic in the study population, but V89L showed a strong correlation with breast cancer (P = 0.03, OR = 1.40, CI = 1.02-1.91). (TA)0/(TA)9 and (TA)9/(TA)9 genotypes were at a lower risk of breast cancer (P = 0.01, OR = 0.64, CI = 0.46-0.90). We conclude that SRD5A2 genotypes significantly affect breast cancer risk in the South Indian populations., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

T is converted to a more potent androgen, DHT by the action of microsomal membrane enzyme 5α reductase 2. Defects in 5α reductase 2 isozyme results in incomplete virilisation of external male genitalia. Mutations in SRD5A2 gene leads to diminished enzyme activity, thus hampering DHT synthesis from T. We describe two unrelated patients from India with 5αRD2 due to novel insertion of nucleotides in the exon 1 of SRD5A2 gene that lead to premature termination of protein. Master S (case 1; III.8) was 3 years old at initial evaluation, had perineoscrotal hypospadias, microphallus and both testes were palpable in the inguinal region. Master P (case 2; III.9) was born as normal full term baby. He had primary complaint of microphallus, penoscrotal hypospadias and gonads in the inguinal region. Diagnosis of 5αRD2 was made, as T/DHT ratio in the two cases was 41 and 131.2 respectively. Sequence analysis of SRD5A2 gene showed an insertion of nucleotides TA in exon 1 (c.188&#95;189). This resulted in premature termination of the protein due to stop codon at amino acid position 7. The protein formed is drastically truncated and inadequate protein synthesized explains the phenotypic characteristics of our patients., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Endometriosis is defined as the presence of endometrial glands and stroma outside the uterine cavity. This disease is associated with diminished protective effects of progesterone, which are usually explained by inadequate activation of progesterone receptors and also enhanced pre-receptor metabolism of progesterone. Endometriosis is often treated with progestins, which act as progesterone receptor agonists, although their exact mechanisms of action are not completely understood. The aim of the present study was to investigate how the progestins medroxyprogesterone acetate, dydrogesterone and dienogest, as well as progesterone, impact on the expression of genes of pre-receptor progesterone metabolism in Z-12 epithelial cell line, a model system of peritoneal endometriosis. Our data demonstrate that these progestins affect local pre-receptor metabolism to a different extent. The most favorable effects were seen for dydrogesterone and dienogest, where the first, dydrogesterone, significantly reduced SRD5A1, AKR1C2 and AKR1C3 expression, and additionally had a nonsignificant impact on progesterone receptor B (PR-B) protein levels. This might slow down the first step of pre-receptor metabolism, the conversion of progesterone to 5α-dihydroprogestrone by SRD5A1, and it might also affect further reduction of 3-keto and 20-keto groups catalyzed by AKR1C2 and AKR1C3. Similarly favorable effects were seen for dienogest, which promoted significant reduction of AKR1C1 and AKR1C2 expression and also showed no effect on PR-B protein levels. Different effects were seen for progesterone, which significantly decreased SRD5A1, PR-B and HSD17B2 protein levels. In contrast, treatment with medroxyprogesterone acetate resulted in increased AKR1C1 expression and decreased levels of PR-B, which might lead to enhanced progesterone metabolism and reduced signaling through progesterone receptors. Altogether, our data in this Z-12 cell model suggest that the beneficial effects of treatment with progestin observed in endometriosis patients might arise from decreased pre-receptor metabolism of the protective progesterone by the SRD5A1 and AKR1C enzymes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Endometrial cancer (EC) is one of the most common gynecological malignancies worldwide. It is associated with prolonged exposure to estrogens that is unopposed by the protective effects of progesterone, which suggests that altered progesterone biosynthesis, metabolism and actions might be implicated in the development of EC. Our aim was to evaluate these processes through quantitative real-time PCR expression analysis in up to 47 pairs of EC tissue and adjacent control endometrium. First, we examined the expression of genes encoding proteins associated with progesterone biosynthesis: steroidogenic acute regulatory protein (STAR); a side chain cleavage enzyme (CYP11A1); and 3β-hydroxysteroid dehydrogenase/ketosteroid isomerase (HSD3B). There were 1.9- and 10.0-fold decreased expression of STAR and CYP11A1, respectively, in EC versus adjacent control endometrium, with no significant differences in the expression of HSD3B1 and HSD3B2. Next, we examined expression of genes encoding five progesterone metabolizing enzymes: the 3-keto and 20-ketosteroid reductases (AKR1C1-AKR1C3) and 5α-reductases (SRD5A1 and SRD5A2); and the opposing 20α-hydroxysteroid dehydrogenase (HSD17B2). These genes are expressed in EC and adjacent control endometrium. No statistically significant differences were seen in mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1. Expression of HSD17B2 was 3.0-fold increased, and expression of SRD5A2 was 3.7-fold decreased, in EC versus adjacent control endometrium. We also examined mRNA levels of progesterone receptors A and B (PGR), and separately the expression of progesterone receptor B (PR-B). Here we saw 1.8- and 2.0-fold lower mRNA levels of PGR and PR-B, respectively, in EC versus adjacent control endometrium. This down-regulation of STAR, CYP11A1 and PGR in endometrial cancer may lead to decreased progesterone biosynthesis and actions although the effects on progesterone levels should be further studied., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Aims: The aim of this study was to investigate the effect of testosterone treatment on the proliferation index and the mRNA expression levels of 5α-reductase, CYP7B1, androgen receptor (AR), and estrogen receptor β (ΕRβ) in the canine prostate., Main Methods: Immature dogs were treated with testosterone for one month, after which prostate gland growth was assessed by comparing the proliferation index in prostates from testosterone-treated dogs with that of untreated control dogs. The relative mRNA expression levels of the aforementioned genes in the prostate glands of testosterone-treated and untreated dogs were determined by real time PCR., Key Findings: Testosterone treatment induced a highly significant reduction in proliferation index in prostate gland. This inhibition of prostate gland growth was associated with differential mRNA expression of 5α-reductase, CYP7B1, AR, and ΕRβ by the prostate gland of testosterone-treated dogs, as compared to that of untreated dogs. While the expression levels of 5α-reductase and CYP7B1 mRNA were significantly down-regulated by testosterone treatment, the expression level of ER-β mRNA was highly up-regulated. In contrast, AR mRNA expression was not significantly altered., Significance: Prostate gland proliferation appeared to be associated with the expression levels of genes that encode proteins that control intra-prostatic levels of testosterone metabolites and their respective receptors. Testosterone treatment may regulate gene expression in the prostate to generate a phenotype that suppresses growth-promoting signaling through AR and enhances anti-proliferative signaling through ERβ. Therefore, targeting disturbances of this genetic machinery in benign prostate hyperplasia and prostate cancer is of a therapeutic potential., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Paranoia is commonly a mood-incongruent psychotic symptom of mania which may be related to dopamine dysregulation. Progesterone and its metabolite allopregnanolone (ALLO) have been found in animals to antagonize the effects of dopamine. We therefore examined serum progesterone, its endogenous antagonist DHEAS and polymorphisms of the genes coding for certain steroidogenetic enzymes (AKR1C4, HSD3B2, and SRD5A1) in 64 males and 96 females with bipolar 1 or 2 disorder with or without paranoid ideation during mood elevation. Euthymic morning serum progesterone, DHEAS and cortisol concentrations were measured in males and in premenopausal women who were in follicular phase and not taking oral contraceptives. In women only, SNPs in AKR1C4 reduced the likelihood of having exhibited paranoid ideation by circa 60%. The haplotype of all 4 SNPs in the AKR1C4 gene reduced the risk of exhibiting paranoia by 80% (OR 0.19, 95% CI 0.06-0.61, p=0.05). A history of paranoid ideation was not, however, related to progesterone or DHEAS concentration. Serum DHEAS and progesterone concentrations were lower in men who had shown paranoid ideation during mania/hypomania compared with those who had not (F=7.30, p=0.006) however this was not coupled to polymorphisms in the selected genes. The ancestral G in rs4659174 in HSD3B2 was in men associated with a lower risk of paranoid ideation (likelihood ratio χ(2) 3.97, p=0.046, OR 0.31 (95% CI 0.10-0.96)) but did not correlate with hormone concentrations. Hence, gene variants in the steroidogenetic pathway and steroids concentration differences may be involved in the susceptibility to paranoia during mood elevation., (Copyright © 2012 Elsevier B.V. and ECNP. All rights reserved.)

This article has been removed: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been removed at the request of the Editor-in-chief and the corresponding author for legal reasons regarding the patient consent., (Copyright © 2011. Published by Elsevier Masson SAS.)

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objectives: The incidence of endometrial cancer has recently increased substantially and studies have shown that altered levels of exogenous and endogenous hormones are associated with individual variation in endometrial cancer risk. The environmental and reproductive risk factors that influence these hormones are well known, however, genetic variants involved in hormone biosynthesis and estrogen metabolism have not been well established in endometrial cancer., Methods: To determine whether polymorphisms in genes of the steroid hormone biosynthesis and metabolism pathways are associated with endometrial cancer risk, 28 polymorphisms in 18 genes were genotyped in 191 endometrial cancer cases and 291 healthy controls., Results: The GSTM1 deletion and the variant (GG) genotype of the CYP1B1 rs1800440 polymorphism were associated with a decreased risk of developing endometrial cancer. Furthermore, combinations of haplotypes in CYP1A1, CYP1B1 and GSTs were associated with a decreased risk. The analysis of the repeat polymorphisms revealed that women with the long repeat allele length of the ESR1 (GT)n repeat polymorphism were at an increased risk of developing endometrial cancer. Conversely, women with two long repeat length alleles of the (CAG)n repeat polymorphism in the AR correlated with a decrease in endometrial cancer risk compared to women with one or two alleles with the short repeat length., Conclusions: The findings are consistent with our hypothesis that variability in genes involved in steroidogenesis and estrogen metabolism may alter the risk of developing endometrial cancer, suggesting that they may be useful as biomarkers for genetic susceptibility to endometrial cancer., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Androgens, such as testosterone, play important roles in regulation of diverse physiological process of target tissues. Recently, we reported that steroidogenic enzymes exist in skeletal muscle and regulate local production of testosterone in response to exercise. Testosterone is transformed into a bioactive androgen metabolite, dihydrotestosterone (DHT) by 5alpha-reductase. However, it is unclear whether exercise stimulates local bioactive androgen metabolism in the skeletal muscle in both sexes. In the present study, we examined sex differences in the levels of dehydroepiandrosterone (DHEA), free testosterone, DHT, and steroidogenesis-related enzymes 5alpha-reductase and androgen receptor (AR) in rat's skeletal muscle before and after a single bout of exercise. Basal muscular free testosterone and DHT levels were higher in males than females, whereas the levels of DHEA did not differ between the sexes. Muscular DHEA, free testosterone, and DHT levels were increased in both sexes after the exercise. There were no differences of 5alpha-reductase and AR transcripts and proteins between the sexes, and the expression of 5alpha-reductase was significantly increased in both sexes after the exercise. Finally, the expression of AR was significantly higher in female rats, but not in males after the exercise. These data suggest that acute exercise enhances the local bioactive androgen metabolism in the skeletal muscle of both sexes., (Copyright 2009 Elsevier Inc. All rights reserved.)

It is now well documented that brain tissue is capable of synthesizing de novo bioactive steroids, named neurosteroids, which are involved in the regulation of various functions in the brain, including behavioral, neuroendocrine and metabolic processes. In this chapter, we have summarized the current knowledge about the expression of enzymes involved in the biosynthesis and metabolism of steroids in the brain with special emphasis on the morphological localization of those enzymes. The results obtained following use of immunocytochemistry and/or in situ hybridization indicate that the enzymes are expressed in both neurons and glial cells distributed throughout the brain with some species-related variations. As observed at the electron microscopic level, the enzymes localized in the neurons or glial cells are not associated with any specific organelles, being distributed throughout the cytoplasm. Since usually one nerve cell expresses only one enzyme, it might be suggested that the neuroactive steroid synthesis that requires the action of several enzymes involves the intervention of several cells (neurons and/or glial cells). More work is required to fully establish the migration of precursors and active steroids in the brain.

There is an increasing number of differences reported between the steroidogenesis pathways described in the traditional literature related to gonadal steroidogenesis and the more recent observations achieved using new technologies, especially molecular cloning, pangenomic expression studies, precise quantification of mRNA expression using real-time PCR, use of steroidogenic enzymes stably transfected in cells, detailed enzymatic activity analysis in cultured cell lines and mass spectrometry analysis of steroids. The objective of this chapter is to present steroidogenesis in the light of new findings that demonstrate pathways of biosynthesis of estradiol (E(2)) and dihydrotestosterone (DHT) from adrenal dehydroepiandrosterone (DHEA) in peripheral intracrine tissues which do not involve testosterone as intermediate as classically found in the testis and ovary. Steroidogenic enzymes different from those of the ovary and testis act in a tissue-specific manner to catalyze the transformation of DHEA into active sex steroids. These new pathways are especially important in post-menopausal women where all estrogens and practically all androgens are made at their site of action in peripheral tissues from DHEA, the precursor of adrenal origin. In men, on the other hand, from 40 to 50% of androgens are made in peripheral tissues from adrenal DHEA, thus indicating the major importance of the intracrine pathways in both men and women. We also examine the molecular evolution of steroidogenic enzymes which explains the major differences in steroid metabolism observed between laboratory animals and humans.

Oleoyl-estrone (OE) induces a marked loss of body fat in rats by maintaining energy expenditure, body protein and blood glucose despite decreasing food intake. OE increases glucocorticoids, but they arrest OE lipid-mobilization. We studied here whether OE induces a direct effect on adrenal glands function as part of this feedback regulation. Dietary overweight male rats were given oral 10nmol/g OE gavages for ten days. A group (PF) of pair-fed to OE rats, and controls received vehicle-only gavages. OE rats lost slightly more body than PF, but had larger adrenal glands. Tissue corticosterone levels, and gene expressions for glucocorticoid-synthesizing enzymes were increased in OE versus controls and PF; thus, we assumed that adrenal growth affected essentially its cortex since OE also lowered the expression of the medullar catecholamine synthesis enzyme genes. Serum corticosterone was higher in PF than in OE and controls, but liver expression of corticosteroid-disposing steroid 5alpha-reductase was 3x larger in OE than PF and controls. Circulating glucocorticoids changed little under OE, in spite of higher adrenal gland and liver content, hinting at modulation of glucocorticoid turnover as instrumental in their purported increased activity. In conclusion, we have observed that OE considerable enhanced the expression of the genes controlling the synthesis of glucocorticoids from cholesterol in the rat and increasing the adrenal glands' corticosterone, size and cellularity, but also the liver disposal of corticosteroids, suggesting that OE increases corticosterone synthesis and degradation (i.e. serum turnover), a process not driven by limited energy availability but directly related to the administration of OE.

The prostate gland is an androgen-dependent, and polymorphisms in androgen synthesis gene steroid 5-alpha reductase type II (SRD5A2) may be associated with benign prostatic hyperplasia (BPH) and prostate cancer. We evaluated the association between 3 polymorphisms in the SRD5A2 gene (2 single nucleotide polymorphism: alanine-49 to threonine [A49T] and valine-89 to leucine [V89L], and a (TA)n dinucleotide repeat in the 3' untranslated region), and BPH and prostate cancer within a multiethnic population. Men between 60 and 86 years of age were recruited from annual prostate cancer screening programs and from a large urology clinic. Unconditional logistic regression was used to compute odds ratios (OR) and 95% confidence intervals (95% CI). We genotyped 606 men (412 Hispanic, 98 Caucasian, 73 African-American, and 23 Asian), of whom 100 had prostate cancer, 393 had BPH (280 symptomatic and 113 asymptomatic), and 113 had normal prostates. Overall, the V89L variant was associated with prostate cancer; the OR for men with the leucine-leucine (LL) genotype compared to men with the valine-valine (VV) genotype was 4.47 (95% CI, 1.24-16.18). This association was stronger in Hispanics (OR=7.26; 95% CI: 1.49-35.47). Although V89L was nonsignificantly associated with BPH in overall population, BPH risk increased significantly with the number of L alleles in Hispanics (P for trend=0.03). Prostate cancer and BPH were not associated with the alanine-49 to threonine single nucleotide polymorphism and the (TA)n repeat. These results suggest that the SRD5A2 gene may play an important role in both BPH and prostate cancer.

Background/aims: Genetic polymorphisms of enzymes involved in hormone metabolism can influence hormonal activities and risk of hormone-dependent cancers. As progression of chronic hepatitis C and risk of liver cancer is higher in males than in females, we evaluated whether the polymorphisms of three enzymes participating in the pathway of estrogen and androgen biosynthesis and inactivation, 5alpha-reductase type II (SRD5A2), cytochrome P450c17alpha (CYP17) and catechol-O-methyltransferase (COMT), might affect the expression of hepatitis C virus (HCV)-related liver disease., Methods: The study included 78 healthy subjects and 387 HCV patients: 100 asymptomatic carriers, 105 hepatitis, 90 cirrhosis and 92 hepatocellular carcinomas (HCC). Variant positions SRD5A2 V89L and A49T, CYP17 (-34)T/C and COMT V108M were analysed by polymerase chain reaction and restriction fragment length polymorphism. A cross-sectional study of association was performed, considering carriers as reference category., Results: The CYP17 (-34)C/C genotype was over-represented in HCC patients as compared to carriers (22.5 vs. 11.2%, odds ratio (OR): 2.29, P: 0.05). Females mostly contributed to this association (OR: 4.95, P: 0.01) and OR values increased in post-menopausal women (OR: 6.00, P: 0.03). No differences were observed for SRD5A2 and COMT gene polymorphisms., Conclusions: CYP17 high-activity alleles associated with increased circulating levels of estrogens and androgens may affect liver cancer risk in HCV-infected women.

The liver is a multifunctional organ responsible for steroid hormones catabolism. Thus, the enzymes responsible for steroid catabolism are located in the liver, including the steroid 5alpha-Reductase (5alpha-R) (EC 1.3.99.5) which catalyzes the conversion of compounds with Delta(4,5) double bonds such as testosterone (T) into their respective reduced derivatives such as dihydrotestosterone (DHT), which are more hydrosoluble, therefore facilitating their excretion. We present precise measurements of mRNA levels of steroid 5alpha-Reductase type 1 isozyme (5alpha-R1) in the liver of male rats with different androgen status, using a quantitative RT-PCR coupled to laser-induced fluorescence capillary electrophoresis (LIF-CE). By means of this technique, we demonstrate a high level of expression of the gene that encodes 5alpha-R1 isozyme in male rat liver, and both T and DHT exert a positive control on the genetic expression of liver 5alpha-R1 isozyme. Since DHT does not contain a Delta(4,5) double bond, our results raise the possibility that hepatic 5alpha-R type 1 not only participates in the catabolism of steroids with Delta(4,5) double bonds, but also in other physiological functions, perhaps in the masculinization of the external genitalia in males with 5alpha-R type 2 gene deficiency.

Background: The conversion of testosterone to dihydrotestosterone; 5alpha-androstan-17beta-ol-3-one by 5alpha-reductase plays a crucial role in hair baldness and prostatomegaly. Recent approach showed specific inhibitors for 5alpha-reductase type 2 such as finasteride promoted hair growth in male pattern alopecia., Objective: In order to search for effective medicinal plant extracts applied topically for androgenetic alopecia, we screened natural plant extracts having inhibitory activities of 5alpha-reductase type 2 and demonstrated its biological function in androgen-related animal models., Methods: We evaluated the inhibition activities of numerous plant extracts by contact cell based metabolic method using a stable HEK 293 cell line expressing human 5alpha-reductase (type 2). To elucidate the biological activity in vivo, the Thujae occidentalis semen (TOS) extract was topically applied to fuzzy rat and androchronogenetic alopecia (AGA) mouse, respectively. The secreted sebum and the size of sebaceous glands of fuzzy rat were measured after 6 weeks. Also, after the topical treatment with TOS extract and androgen receptor antagonist (cyproterone acetate) simultaneously with subcutaneous injection of testosterone (1 mg/mice/day), hair loss patterns of female B6CBAF1/j hybrid mouse were observed., Results: TOS extract showed higher inhibition activity of 5alpha-reductase type 2(IC(50) value=2.6 microg/ml) than that of gamma-linolenic acid, but lower than that of finasteride. When applied to fuzzy rat, the amount of sebum and sebaceous gland size decreased remarkably. In AGA model, alopecia degrees of two groups, treated with TOS extract (P<0.015) or cyproterone acetate (P<0.01), were lower than that of vehicle (propylene glycol:ethanol=7:3) and there was no difference between above two groups., Conclusion: We have demonstrated the inhibitory activity of TOS extract for 5alpha-reductase type 2 and its biological action in two animal models, suggesting that TOS extract would be used as an effective agent for male pattern baldness by modifying androgen conversion.

Background: Genetic polymorphisms of steroid 5alpha-reductase have been studied in androgenetic alopecia in Caucasians, but the genes encoding the two isoenzymes were not associated with male pattern baldness. Genetic polymorphisms and ethnic variations have not been studied for Asians, although it is suggested that racial difference could exist and influence clinical phenotypes., Objective: The purpose of our study is to investigate the genetic polymorphisms of steroid 5alpha-reductase type 1 and 2 (SRD5A1 and SRD5A2) genes in Korean population, and to study the association of these polymorphisms with the development, clinical types (female or male pattern) and therapeutic response of androgenetic alopecia., Methods: Sixty-six patients with androgenetic alopecia and controls consisted of 92 healthy men were included. Twenty-four patients were treated with finasteride for at least 6 months, and clinical responses were assessed by a simple classification. For type 1 isoenzyme, HinfI and NspI restriction fragment length polymorphisms (RFLPs) were detected using polymerase chain reaction method. For type 2 isoenzyme, RsaI RFLPs detected valine/leucine polymorphisms at codon 89, and MowI RFLPs detected alanine/threonine polymorphisms at codon 49., Results: We could not find any significant associations of the genetic polymorphisms of these two isoenzyme genes with androgenetic alopecia in Koreans (P>0.05). These polymorphisms were not associated with the clinical types of baldness or the response to finasteride (P>0.05)., Conclusion: These results suggest that polymorphisms of SRD5A1 and SRD5A2 genes may not be directly associated with the development of baldness or generation of different clinical phenotypes.

Selective REM sleep deprivation was carried out under the conditions designed to minimize the adverse influence of environmental conditions and restricted movement, and the influence of REM sleep deprivation on adrenocortical steroid metabolism was investigated by measuring the steady-state levels of mRNAs encoding steroid metabolism-related genes, steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc) and steroid 5alpha-reductase (5alpha-R), in rat adrenal glands. Selective REM sleep deprivation caused a significant decrease in StAR mRNA and an increase in 5alpha-R mRNA levels without any notable change in P450scc mRNA levels in the adrenal gland. In contrast, non-selective sleep disturbance, resulting in the partial reductions of non-REM and REM sleep, tended to increase both StAR and P450scc mRNA levels without any statistical significance. These results indicate that REM sleep deprivation by itself may affect the expression of steroid metabolism-related genes in the adrenal gland, suggesting a possible relation between REM sleep and adrenocortical steroid metabolism.

Induction of apoptosis and androgen ablation are two major approaches for treating human prostate carcinoma. In a study of the bioactive components of the soft coral Nephthea chabroli, we found that lemnabourside is a 5alpha-reductase inhibitor, as shown by its ability to inhibit the conversion of testosterone into the more potent dihydrotestosterone in rat prostate homogenate. The compound also inhibited the incorporation of tritiated thymidine into human prostate androgen-dependent carcinoma LNCaP cells, and thus blocking the cell proliferation (IC50 = 37.5 microM). The expression of prostate marker genes, including 5alpha-reductase, prostate-specific antigen, prostatic acid phosphatase and androgen receptor, and the anti-apoptotic bcl-2 gene were markedly reduced, but the transcription of apoptosis-related caspase 3 gene showed a dose-dependent increase in lemnabourside-treated LNCaP cells. Immunofluorescent microscopy and flow cytometric analysis further demonstrated apoptotic changes in these cells. Taken all results together, a relatively weak 5alpha-reductase inhibitory activity on LNCaP cells (EC50 > 250 microM), and a similar growth inhibitory activity on both androgen dependent- and independent-prostate cells (IC50 approximately 37.5 microM) indicated that caspase-3 apoptosis pathway is one of the possible antiproliferative activities mediated by lemnabourside.

We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.

Background: Prostate cancer is a very common disease in more-developed countries, but its cause is largely unknown. It is an androgen-dependent cancer, and androgens have been proposed as having a substantial role in predisposition to the disease. Thus, variations in androgen metabolism genes may affect risk of this disease., Methods: We screened 216 African-American and 172 Hispanic men with prostate cancer, and 261 African-American and 200 Hispanic healthy men (controls), from a large prospective cohort study (the Hawaii-Los Angeles Multiethnic Cohort Study) for a mis-sense substitution in the human prostatic (or type II) steroid 5alpha-reductase (SRD5A2) gene, the product of which controls metabolic activation of testosterone to dihydrotestosterone. This mis-sense substitution results in an alanine residue at codon 49 being replaced with threonine (A49T). We also reconstructed this mutation in the SRD5A2 cDNA, and overexpressed the enzyme in mammalian tissue culture cells., Findings: The A49T aminoacid substitution in the SRD5A2 gene increased the risk of clinically significant disease 7.2-fold in African-American men (95% CI=2.17-27.91; p=0.001) and 3.6-fold in Hispanic men (1.09-12.27; p=0.04). The mutant enzyme had a higher in-vitro Vmax than the normal enzyme (9.9 vs 1.9 nmol min(-1) mg(-1))., Interpretation: The A49T variant of the SRD5A2 gene may be a significant contributor to the incidence of prostate cancer in African-American and Hispanic men in Los Angeles. We estimate that the population attributable risk due to this aminoacid substitution for clinically significant disease is about 8% in both populations. Increased conversion of testosterone to dihydrotestosterone catalysed by this variant steroid 5alpha-reductase enzyme may be the cause of the increased risk.

Objective: This study was designed to test the hypothesis that mutations in the gene for type 2 steroid 5alpha-reductase (SRD5A2) may be the cause of a phenotype characterized primarily by oligospermia or azoospermia., Study Design: Deoxyribonucleic acid from control subjects and subjects with oligospermia (n = 12) and azoospermia (n = 6) were evaluated for mutations in SRD5A2. Methods used for mutation analysis included polymerase chain reaction, Southern blotting, and denaturing gradient gel electrophoresis., Results: Denaturing gradient gel electrophoresis of all 5 amplified exons resulted in similar migration patterns in samples from both control and study subjects. Genomic deoxyribonucleic acid subjected to denaturing gradient gel electrophoresis after restriction digest revealed melting polymorphisms. Direct sequencing of the gene in a single patient with a unique melting polymorphism yielded a normal sequence., Conclusions: Melting polymorphisms for SRD5A2 were detected in a group of patients with oligospermia or azoospermia. Sequence analysis did not demonstrate functional mutations in the coding sequence of this gene.

Previous studies have shown that the reduced nicotinamide adenine dinucleotide phosphate (NADPH)- binding domain of rat liver microsomal steroid 5alpha-reductase isozyme-1 (r5alphaR-1) is in a highly conserved region of the polypeptide sequence (residues 160-190). In this study, we investigated, by site-directed mutagenesis, the role of hydroxylated and aromatic amino acids within the NADPH-binding domain. The r5alphaR-1 cDNA was cloned into a pCMV vector, and the double strand site-directed mutagenesis method was used to create mutants Y179F, Y179S, Y189F, Y189S, S164A, S164T, and Y187F, which were subsequently expressed in COS-1 cells. Kinetic studies of the expressed enzymes showed that the mutation Y179F resulted in an approximately 40-fold increase in the Km for NADPH versus wild-type, with only a 2-fold increase in the Km for testosterone. The mutants Y189F and S164A showed smaller increases (4 and 6-fold) in Kms for NADPH and no significant change in the Km for testosterone, whereas Y189S had kinetic properties similar to the wild-type r5alphaR-1. Mutants Y179S and S164T both resulted in inactive enzymes, whereas F187Y showed an approximately 5-fold decrease in Km for NADPH and a significant increase (approximately 18-fold) in the Km for testosterone. The results suggest that the -OH functionality of Y179 is involved in cofactor binding, but is not essential for the activity of the enzyme, whereas the -OH functionalities of Y189 and S164 play lesser roles in cofactor binding to r5alphaR-1 and may not be required for enzyme activity. On the other hand, the residue F187 may be important for the binding of both NADPH and testosterone.

We have previously shown that the photoactive 4-azasteroid, [1,2 3H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5alpha-androst an-17beta-carboxamide is an effective probe of rat steroid 5alpha-reductase (isozyme-1) (5alphaR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5alphaR-1 activity were ultraviolet (UV)-photolyzed with [3H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5alphaR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55-56 min. Rechromatography of this fraction using a modified gradient (elution 54-55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15-18 of the 5alphaR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an approximately 12-fold increase in the Km for testosterone, whereas the Km for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.

Genetic predisposition and androgen dependence are important characteristics of the common patterned loss of scalp hair known as male pattern baldness. The involvement of the 5alpha-reductase enzyme in male pattern baldness has been postulated due to its role in the metabolism of testosterone to dihydrotestosterone. There are two known isozymes of 5alpha-reductase. Type I has been predominantly localized to the skin and scalp. Type II, also present on the scalp, is the target of finasteride, a promising treatment for male pattern baldness. We conducted genetic association studies of the 5alpha-reductase enzyme genes (SRD5A1 on chromosome 5 and SRD5A2 on chromosome 2) using dimorphic intragenic restriction fragment length polymorphisms. From a population survey of 828 healthy families comprising 3000 individuals, we identified 58 young bald men (aged 18-30 y) and 114 older nonbald men (aged 50-70 y) for a case control comparison. No significant differences were found between cases and controls in allele, genotype, or haplotype frequencies for restriction fragment length polymorphisms of either gene. These findings suggest that the genes encoding the two 5alpha-reductase isoenzymes are not associated with male pattern baldness. Finally, no clear inheritance pattern of male pattern baldness was observed. The relatively strong concordance for baldness between fathers and sons in this study was not consistent with a simple Mendelian autosomal dominant inheritance. A polygenic etiology should be considered.

The enzyme steroid 5 alpha-reductase (5 alpha R) catalyzes the reduction of testosterone (T) to 5 alpha-dihydrotestosterone (DHT). In this study, the baculovirus expression system was used to overexpress rat 5 alpha R type I isozyme (r5 alpha R 1). The full length of r5 alpha R1 cDNA was inserted into the Autographa californica nuclear polyhedrosis virus (Ac-MNPV) genome and expressed in Spodoptera frugiperda, Sf 21, insect cells. The expressed recombinant r5 alpha-R1 showed maximal enzymatic activity when the infected cells were harvested on day 3 of post-transfection. The K(m) values for NADPH and T were 17 microM and 2.7 microM, respectively. Inhibition of the recombinant r5 alpha R1 by N,N diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide (4MA) was competitive with respect to the substrate (T), and a Ki of 3 nM was obtained. The enzyme was located primarily in the nuclear fraction, and the maximum velocity for the recombinant r5 alpha R1 in this fraction was 60 nmoles DHT/min/mg. Immunoblot analysis indicated a single immunoreactive band at 26 kDa, which corresponds to the molecular weight of r5 alpha R1. Photoaffinity labeling by [2'-32P]-2-azido-NAD P+ ([2'-32P]2N3-NAD P+) and [1,2(3)H] N-(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha androstane-17 beta-carboxamide ([3H]-4MABP) also showed a labeled protein band at 26 kDa.

The human prostate is a complex organ composed of four glandular zones that differ in their histology and biology. In addition to this anatomical organization, the prostate is mainly composed of epithelial and mesenchymal tissues. Two pathologies affect the growth of the prostate: cancer and benign prostatic hyperplasia. Anatomical and pathological studies have shown that stromal enlargement is the first step in the process of BPH and that both transition and periurethral regions are the exclusive sites of origin. Because the first step of androgen action is the reduction of testosterone into dihydrotestosterone by the 5 alpha-reductase, it have been postulated that modification of this enzymatic activity may play an important role in BPH development. However DHT production alone cannot explain the hyperplastic development of the gland. Thus it has also been postulated that androgen-growth factor interaction may be an important feature of this growth: these growth factors include the IGF axis. An interaction between androgen pathway and sympathethic system, via alpha-adrenoreceptor may also be suspected.

Hormone-sensitive cancers, namely those of the prostate, breast and uterus, constitute one third of all cancers. In addition to the classical steroidogenic tissues, namely the ovaries, testes, adrenals and placenta, a large series of peripheral tissues possess all the enzymatic systems required for the formation of active androgens and estrogens from a relatively large supply of precursor steroids namely, dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S) provided by the adrenals. This report describes the structure, function and tissue-specific expression and regulation of the 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase, 17 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase gene families as well as some information about the aromatase gene. While, so far, most therapeutical approaches have been aimed and limited at controlling steroid formation by the classical steroidogenic tissues, it is clear that major efforts should now be turned towards intracrinology in order to better understand the physiological mechanisms controlling local steroid formation in peripheral target tissues and to develop improved therapy for hormono-sensitive diseases, especially breast, prostate, and uterine cancers.