Genistein prevents the production of hypospadias induced by Di-(2-ethylhexyl) phthalate through androgen signaling and antioxidant response in rats.

Environmental estrogen exposure has increased dramatically over the past 50 years. In particular, prenatal exposure to estrogen causes many congenital diseases, among which reproductive system development disorders are extremely serious. In this study, the molecular mechanism of hypospadias and the therapeutic effect of genistein (GEN) were investigated through in vivo models prepared by Di-(2-ethylhexyl) phthalate (DEHP) exposure between 12 and 19 days of gestation. With increased DEHP concentrations, the incidence of hypospadias increased gradually. DEHP inhibited the key enzymes involved in steroid synthesis, resulting in decreasing testosterone synthesis. At the same time, DEHP increased reactive oxygen species (ROS) and produced inflammatory factors via NADPH oxidase-1 (NOX1) and NADPH oxidase-4 (NOX4) pathways. It also inhibited Steroid 5 α Reductase 2 (Srd5α2) and decreased dihydrotestosterone (DHT) synthesis. Additionally, DEHP inhibited the androgen receptor (AR), resulting in reduced DHT binding to the AR that ultimately retarded the development of the external reproductive system. GEN, a phytoestrogen, competes with DEHP for binding to estrogen receptor ß (ERß). This competition, along with GEN's antiestrogen and antioxidant properties, could potentially reverse impairments. The findings of this study provide valuable insights into the role of phytoestrogens in alleviating environmental estrogen-induced congenital diseases.

An experimental evaluation of the efficacy of perinatal sulforaphane supplementation to decrease the incidence and severity of vinclozolin-induced hypospadias in the mouse model.

Determining the mechanisms of toxicity induced by pollutants has long been a research priority in lieu of considering the mechanisms of resilience that prevent deleterious impacts. Protective mechanisms in many taxa can be therapeutically targeted to enhance resilience to synthetic toxicants. For example, the environmental sensor, Nuclear factor (erythroid-derived 2)-like 2 (Nfe2l2 or Nrf2), a transcription factor, facilitates transcription of many protective genes. Hypospadias is a common malformation of the penis. The risk of being born with hypospadias increases with pollutant exposure. We use vinclozolin-induced hypospadias in the mouse as a model to test the hypothesis that pollutant-induced birth defects can be prevented and reduced in severity by augmenting natural mechanisms of resilience. Pregnant mice were exposed to the demasculinizing toxicant, vinclozolin, in combination with increasing doses of the NRF2 activator, sulforaphane. The sulforaphane dose that most effectively increased masculinization (anogenital distance) was identified and used to test the hypothesis that sulforaphane reduces the hypospadias-inducing potency of vinclozolin. Finally, a Nrf2 knockout study was conducted to test whether NRF2 was required for the sulforaphane-induced rescue effects. Sulforaphane supplementation to vinclozolin exposed embryos increased anogenital distance in a nonlinear fashion typical of Nrf2 activators. The most effective dose of sulforaphane (45 mg/kg) reduced the occurrence and severity of vinclozolin-induced hypospadias and corrected penis morphogenesis. The sulforaphane-induced rescue effect was dependent on the presence of Nrf2. Nrf2 plays a critical role in protecting the fetus from vinclozolin and reduces the incidence and severity of hypospadias, the most common birth defect in boys in many countries. This work lays a foundation for developing prenatal supplements that will protect the fetus from pollutant-induced hypospadias. Studying the protective mechanisms that drive resilience to toxicants will facilitate innovation of protective therapies.

Downregulation of MicroRNA-494 inhibits the TGF-ß1/Smads signaling pathway and prevents the development of hypospadias through upregulating Nedd4L.

BACKGROUND: Hypospadias, as a congenital disorder of the urethra, is the second most common birth abnormality of the male reproductive system. This study primarily investigates the effects of microRNA-494 (miR-494) on the transforming growth factor-ß1 (TGF-ß1)/Smads signaling pathway and on the development of hypospadias by binding to neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4L). METHODS: We induced a mouse model of hypospadias through di-(2-ethylhexyl) phthalate treatment. The underlying regulatory mechanisms of miR-494 in this model were analyzed upon treatment of miR-494 mimic, miR-494 inhibitor, or small interfering RNA against Nedd4L in urethral epithelial cells isolated from mice with hypospadias. We then verified the binding site between miR-494 and Nedd4L and applied a gain- and loss-of-function approach to determine the effects of miR-494 on cell proliferation, cycle distribution, and apoptosis. RESULTS: Male mice with hypospadias exhibited significantly higher miR-494 expression and lower Nedd4L expression in urethral tissues than normal male mice. Nedd4L was verified as a target gene of miR-494. Treatment with miR-494 inhibitor suppressed the activation of the TGF-ß1/Smads signaling pathway, whereas down-regulation of miR-494 exerted protective effects on urethral epithelial cells by impeding cell proliferation and inducing cell apoptosis. CONCLUSIONS: The study indicates that downregulation of miR-494 inhibits the TGF-ß1/Smads signaling pathway and prevents the development of hypospadias through upregulating Nedd4L.

TGF-ß1 relieves epithelial-mesenchymal transition reduction in hypospadias induced by DEHP in rats.

BACKGROUNDS: To investigate the potential mechanism of hypospadias induced by DEHP in rats to reveal the preventative effect of TGF-ß1 in hypospadias induced by DEHP via the reduction of EMT. METHODS: Time-mated Sprague-Dawley rats underwent cesarean section, and the penises of male pups were collected after exposure to corn oil or DEHP to establish a rat model of hypospadias and to further study the molecular mechanisms of hypospadias in vivo. In addition, the penises were cultured and treated with MEHP or MEHP+TGF-ß1 in vitro. Subsequently, histomorphology and elements in TGF-ß/Smad signaling pathway changes were evaluated using scanning electron microscopy, immunofluorescence, polymerase chain reaction, and western blot. RESULTS: The development of rat penis and urethral seam fusion were delayed after the treatment with DEHP in vivo or MEHP in vitro compared with the Control group. Moreover, TGF-ß1, Smad2/Smad3, and the mesenchymal biomarkers, including α-SMA, N-cadherin, and Vimentin, were decreased. However, the epithelial biomarkers, including E-cadherin, ZO-1, ß-catenin, and occludin, were increased. In addition, TGF-ß1 could relieve all of the above changes. CONCLUSION: Gestational DEHP exposure could lead to hypospadias by reducing urethral EMT. Moreover, TGF-ß1 could prevent it by regenerating EMT through activating the TGF-ß/Smad signal pathway.

Hypospadias and maternal exposure to atrazine via drinking water in the National Birth Defects Prevention study.

BACKGROUND: Hypospadias is a relatively common birth defect affecting the male urinary tract. It has been suggested that exposure to endocrine disrupting chemicals might increase the risk of hypospadias by interrupting normal urethral development. METHODS: Using data from the National Birth Defects Prevention Study, a population-based case-control study, we considered the role of maternal exposure to atrazine, a widely used herbicide and potential endocrine disruptor, via drinking water in the etiology of 2nd and 3rd degree hypospadias. We used data on 343 hypospadias cases and 1,422 male controls in North Carolina, Arkansas, Iowa, and Texas from 1998-2005. Using catchment level stream and groundwater contaminant models from the US Geological Survey, we estimated atrazine concentrations in public water supplies and in private wells. We assigned case and control mothers to public water supplies based on geocoded maternal address during the critical window of exposure for hypospadias (i.e., gestational weeks 6-16). Using maternal questionnaire data about water consumption and drinking water, we estimated a surrogate for total maternal consumption of atrazine via drinking water. We then included additional maternal covariates, including age, race/ethnicity, parity, and plurality, in logistic regression analyses to consider an association between atrazine and hypospadias. RESULTS: When controlling for maternal characteristics, any association between hypospadias and daily maternal atrazine exposure during the critical window of genitourinary development was found to be weak or null (odds ratio for atrazine in drinking water = 1. 00, 95 % CI = 0.97 to 1.03 per 0.04 µg/day increase; odds ratio for maternal consumption = 1.02, 95 % CI = 0.99 to 1.05; per 0.05 µg/day increase). CONCLUSIONS: While the association that we observed was weak, our results suggest that additional research into a possible association between atrazine and hypospadias occurrence, using a more sensitive exposure metric, would be useful.

Interaction between MTHFR 677C>T and periconceptional folic acid supplementation in the risk of Hypospadias.

BACKGROUND: Hypospadias is a congenital malformation with both environmental factors and genetic predisposition involved in the pathogenesis. The role of maternal periconceptional folic acid supplement use in the development of hypospadias is unclear. As folate levels may also be influenced by the C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene, we hypothesize that a gene-environment interaction between this polymorphism and folic acid use is involved in the etiology of hypospadias. METHODS: We conducted a case-control study among 855 hypospadias cases and 713 population-based controls from the AGORA data- and biobank. Folic acid supplement use was derived from maternal questionnaires and infant and maternal DNA was used to determine the MTHFR C677T polymorphism using Taqman assays. We performed separate analyses for different hypospadias phenotypes (anterior/middle/posterior). RESULTS: Hypospadias was neither associated with folic acid use or the MTHFR C677T polymorphism, nor with their interaction. However, we did find an association with middle hypospadias when no supplements were used (odds ratio = 1.6; 95% confidence interval, 1.1-2.4), especially in infants carrying the CT/TT genotype (odds ratio = 2.5; 95% confidence interval, 1.4-4.7). In addition, more infants with these genotypes seemed to have posterior hypospadias, regardless of folic acid use. CONCLUSION: Our study does not suggest a major role for folic acid supplements or the MTHFR C677T polymorphism in the etiology of hypospadias in general, but not using folic acid and/or carrying the MTHFR C677T polymorphism may be associated with middle and posterior hypospadias. Therefore, we stress the importance of studying gene-environment interactions preferably in stratified analyses for different hypospadias phenotypes.

Possible preventive effect of high doses of folic acid for isolated hypospadias: a national population-based case-control study.

Hypospadias is a common structural birth defect (congenital abnormality) of the male genital organ. The objective of this study was to test the hypothesis regarding the possible preventive effect of folic acid for isolated hypospadias (IH). Folic acid use was compared in 3,038 cases with IH and 24,814 male controls without any defects in the national population-based Hungarian Case-Control Surveillance of Congenital Abnormalities, 1980-1996. In Hungary only one kind of folic acid tablet, containing 3 mg/tablet was available during the study period. Hungarian obstetricians recommended daily use of 1-3 tablets, that is, 3-9 mg of folic acid during pregnancy; the estimated daily dose was 5.7 or 5.6 mg in the mothers of cases with IH and controls, respectively. Of 3,038 mothers of cases 1,474 (48.5%) were supplemented with high doses of folic acid during pregnancy and 13,509/24,814 mothers of controls (54.9%) as reference were supplemented at this level as well (OR 95% CI: 0.79, 0.73-0.85). If only medically recorded folic acid use in the critical period of IH was evaluated, the preventive effect was more apparent (OR 95% CI: 0.36, 0.32-0.41). The intake of folic acid among mothers of infants with severe IH was lower than among mothers of infants with mild IH, suggesting a dose-response relationship. In conclusion, this study suggests that high doses of folic acid are associated with a reduced risk of IH. However, this analysis was based on observational data; therefore, confirmation in a well-controlled study is needed.

Penile repair in patients with epispadias-exstrophy complex-can we prevent resultant hypospadias?

PURPOSE: Male genitalia reconstruction in patients with exstrophy-epispadias complex often leads to secondary hypospadias, thus requiring additional surgery and resulting in possible complications. We describe a technique of male epispadias repair to prevent resultant hypospadias. MATERIALS AND METHODS: The complete penile disassembly technique is modified by multiple Z-plasties, which enable creation of a funnel-like bladder neck and urethral plate lengthening. A total of 29 patients 2 months to 12 years old presenting with pure epispadias and bladder exstrophy were treated with this technique between 2004 and 2011. These patients were compared to 19 patients 1 day to 11 years old with exstrophy-epispadias treated with the standard penile disassembly technique between 2000 and 2004. RESULTS: Outcome measures were urethral orifice position, penile length and complications. There was no statistically significant difference between the 2 groups regarding penile length or complications. In the study group the urethra reached the tip of the glans in all patients, whereas the creation of intentional hypospadias was necessary in 6 patients (31.5%) in the control group (p <0.05). CONCLUSIONS: The modified technique was effective in obtaining appropriate meatal location without decreasing the penile length. The complication rate was not changed. Creation of resultant hypospadias at genitalia reconstruction in male epispadias should strongly be prevented.

Inguinal hernias and genital abnormalities in young Jordanian males.

This study was carried out to document the prevalence of inguinal hernia, hypospadias, undescended testis and varicocele in 3057 male applicants to the military wing of Mu'ta University in the south of Jordan. Age range was 17-20 years. A total of 250 men had one the 4 conditions: 93 (3.0%) had inguinal hernia; 15 (0.5%) had undescended testis (26.7% bilateral); 59 (1.9%) had hypospadias; 83 (2.7%) had varicocele (98.79% on the left side). Prevalence of inguinal hernia and undescended testis were comparable with international prevalence rates, while the rate for hypospadias was higher and that for varicocele lower. A birth defects registration system would help in planning preventive and treatment strategies.

Complete repair of bladder exstrophy in boys: can hypospadias be avoided?

INTRODUCTION: Complete repair of classic bladder exstrophy in male newborns has been successful with minimal morbidity. However, the technique may create hypospadias in some cases. We have recently adopted a modification to obtain an orthotopic meatus in bladder exstrophy boys. MATERIALS AND METHODS: Between November 1998 and December 2002 complete repair of classic bladder exstrophy was carried out in 27 boys. Complete penile disassembly was performed in 22 boys including 4 newborns and 18 older children; mean age 3+/-2 years old. Modified disassembly was used in the last 5 boys including 4 newborns and a 9-month-old boy. During repair of epispadias, the dissection starts on the ventral aspect of the penis as usual. The urethral plate is separated from both corpora cavernosa to allow ventral transposition of the plate. In the complete disassembly technique, the urethral plate is completely separated from both hemiglans. In this modification, while the urethral plate is completely separated from both corpora cavernosa, the extreme distal end of the urethral plate remains attached to the distal ends of both hemiglans. Thus, when the plate is tubularized with fine interrupted sutures the meatus ends up at the tip of the glans penis. The symphysis pubis is re-approximated as usual and corpora cavernosa are approximated dorsally in the midline. RESULTS: There was no major complication. Mean follow-up was 43+/-7 and 7+/-4 months for complete and modified disassembly groups respectively. Of the 22 boys, who underwent complete disassembly, 15 (68%) ended up with hypospadias and 7 (32%) had an orthotopic meatus. Modified disassembly has resulted in an excellent cosmetic appearance and orthotopic meatus in the 5 boys. Penile length was not shorter than boys who underwent complete disassembly. Parents of the 5 boys noticed normal straight morning erection. CONCLUSION: Although complete penile disassembly allows ventral placement of the urethra, hypospadias is created in approximately two thirds of the cases. When the extreme distal end of the urethral plate remains attached to the distal ends of both hemiglans (modified disassembly), orthotopic meatus can be obtained in all boys. With modified disassembly, posterior mobilization of the bladder and urethra does not result in corporal angulations or shortening because of the proportionate inward movement of the corpora that accompanies symphyseal approximation. The modification is feasible in newborns and infants. These short-term results may obviate the need for later penile reconstructive procedure.

Iatrogenic hypospadias--a preventable injury?

Iatrogenic hypospadias is a preventable injury to the ventral male urethra produced by the downward pressure of an indwelling urethral catheter. In spite of avoiding long-term urethral catheterisation, we have acquired 16 patients with this injury in the last 9 years either in our unit or discovered in patients referred to our unit. Although not a life endangering complication, the majority of such patients find the penile appearance so produced unacceptable. We have reconstructed any penis that has more than meatal cleavage in a total of six patients, with good results. The majority of patients have been fully investigated so that an alternative therapy to catheterisation may be provided. Where possible a catheter free state has been achieved. If not, a suprapubic catheter has been inserted. Those not reconstructed were due to reasons of ill health, and in two instances, due to patient choice.