New frontiers on the molecular underpinnings of hypospadias according to severity.

Hypospadias, which is characterised by the displacement of the urethral meatus from its typical anatomical location in males, shows various degrees of severity. In this systematic review, we surveyed our current understanding of the genetics of isolated hypospadias in humans according to the severity of the condition. We found that sequencing and genotyping approaches were the preferred methods of study and that single nucleotide polymorphisms were the most common finding associated with hypospadias. Most genes fell into four gene-pathway categories related to androgens, oestrogens, growth factors, or transcription factors. Few hypospadias studies classify their findings by severity. Taken together, we argue that it is advantageous to take into consideration the severity of the condition in search of novel candidates in the aetiology of hypospadias. Abbreviations: AR: androgen receptor; ATF3: activating transcription factor 3; BMP4: bone morphogenetic protein 4; BMP7: bone morphogenetic protein 7; CYP17: steroid 17-alpha-hydroxylase/17,20 lyase; CYP1A1: cytochrome P450 1A1; CYP3A4: cytochrome P450 3A4; CNVs: copy number variants; DGKK: diacylglycerol kinase kappa; ESR1: oestrogen receptor 1; ESR2: oestrogen receptor 2; FGF8: fibroblast growth factor 8; FGF10: fibroblast growth factor 10; FGFR2: fibroblast growth factor receptor 2; HOXA4: homeobox protein Hox-A4; HOXB6: homeobox protein Hox-B6; HSD17B3: hydroxysteroid 17-beta dehydrogenase 3; MAMLD1: mastermind-like domain-containing protein 1; SF-1: splicing factor 1; SHH: sonic hedgehog; SNPs: single nucleotide polymorphisms; SOX9: SRY-box 9; SRD5A2: steroid 5 alpha-reductase 2; SRY: sex-determining region Y protein; STAR: steroidogenic acute regulatory protein; STARD3: StAR-related lipid transfer protein 3; STS: steryl-sulfatase; WT1: Wilms tumour protein; ZEB1: zinc finger oestrogen-box binding homeobox 1.

Severity-Dependent Profile of the Metabolome in Hypospadias.

Background & Objective: Hypospadias, characterized by the displacement of the opening of the urethra at any point in the medial-ventral side of the penis, is classified upon severity as mild (Type I) and severe (Type II and Type III) hypospadias. Hypospadias' etiology is idiopathic in the majority of cases, and underlying causes seem of multifactorial origin. Studies regarding genetic variants support this notion. It is unknown whether downstream gene products fit this profile. This study evaluated the metabolome of hypospadias by using the emerging technology of metabolomics in the search for distinct cellular processes associated with hypospadias' etiology according to the severity of this congenital urogenital condition. Methods: Foreskin samples were collected during urethroplasty from boys with Type I, II, and III hypospadias or undergoing elective circumcision (N = 28) between 5 and 28 months of age. Samples were processed and submitted to gas chromatography-mass spectrometry (GC/MS). MetaboloAnalyst (http://www.metaboanalyst.ca/) online platform was used for bioinformatic analyses. Results: Thirty-five metabolites across experimental groups were identified by GC/MS. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) showed that the metabolome of Type II and Type III hypospadias patients differs from the metabolome of Type I hypospadias and control patients. Of those 35, 10 amino acids were found in significantly low concentrations in severe hypospadias: aspartate, glutamate, glycine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, and tyrosine. A high concentration of the amino acid lysine was detected in mild hypospadias. Conclusions: The observed downregulation of specific amino acids in severe hypospadias provides alternative routes for future research aiming to identify disrupted networks and pathways while considering the severity of hypospadias.

A Proteomics Signature of Mild Hypospadias: A Pilot Study.

Background and Objective: Mild hypospadias is a birth congenital condition characterized by the relocation of the male urethral meatus from its typical anatomical position near the tip of the glans penis, to a lower ventral position up to the brim of the glans corona, which can also be accompanied by foreskin ventral deficiency. For the most part, a limited number of cases have known etiology. We have followed a high-throughput proteomics approach to study the proteome in mild hypospadias patients. Methods: Foreskin samples from patients with mild hypospadias were collected during urethroplasty, while control samples were collected during elective circumcision (n = 5/group). A high-throughput, quantitative proteomics approach based on multiplexed peptide stable isotope labeling (SIL) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was used to ascertain protein abundance changes in hypospadias patients when compared to control samples. Results: A total of 4,815 proteins were quantitated (2,522 with at least two unique peptides). One hundred and thirty-three proteins from patients with mild hypospadias showed significant abundance changes with respect to control samples, where 38 proteins were increased, and 95 proteins were decreased. Unbiased functional biological analysis revealed that both mitochondrial energy production and apoptotic signaling pathways were enriched in mild hypospadias. Conclusions: This first comprehensive proteomics characterization of mild hypospadias shows molecular changes associated with essential cellular processes related to energy production and apoptosis. Further evaluation of the proteome may expand the search of novel candidates in the etiology of mild hypospadias and could also lead to the identification of biomarkers for this congenital urogenital condition.

Evaluation and Management of the Undescended Testis in Puerto Rico: A Single Surgeon's 10 Years of Experience.

OBJECTIVE: Cryptorchidism is an abnormality of the male genitourinary tract in which one or both testes fail to descend into the scrotum. The American Urological Association (AUA) clinical guidelines for the evaluation and treatment of cryptorchidism were recently published. We reviewed our experience with the evaluation and management of our patients and examined our findings with respect to the AUA and European Association of Urology (EAU) guidelines. METHODS: Data were obtained from pediatric patients who underwent a surgical intervention for an undescended testis from 2007 through 2017 at HIMA Hospital and the University Pediatric Hospital (both in Puerto Rico); all the surgeries were performed by the same surgeon. A total of 754 patients were identified; 142 patients were excluded due to lack of follow-up data (N = 612). The data obtained included age, testes locations, radiologic and surgical findings, and postoperative results. RESULTS: At their initial evaluations, a large proportion of the patients (46.4%) came accompanied with radiographic imaging. These findings were consistent with those of the physical examination in 58.5% of the patients and with the surgical findings in 63.1% (sensitivity 77.9%, specificity 45.8%). Our data showed that the median referral age was 24 months, which suggests that there was a significant delay in diagnosis. At the time of surgery, the average age of the patients who required an orchiectomy was 3.93 years, while those who underwent an orchiopexy had an average age of 3.28 years. CONCLUSION: Our data reveal that, despite its lack of usefulness, radiologic imaging continues to be included in the diagnostic workups of children newly identified with cryptorchidism in Puerto Rico. In addition, and contrary to the guidelines, there tends to be a significant delay in treatment with surgical intervention. It is important to continue to educate our referring physicians on the AUA and EUA guidelines in order to create awareness and encourage the proper diagnostic and treatment approach for cryptorchidism.

A General Method for Targeted Quantitative Cross-Linking Mass Spectrometry.

Chemical cross-linking mass spectrometry (XL-MS) provides protein structural information by identifying covalently linked proximal amino acid residues on protein surfaces. The information gained by this technique is complementary to other structural biology methods such as x-ray crystallography, NMR and cryo-electron microscopy[1]. The extension of traditional quantitative proteomics methods with chemical cross-linking can provide information on the structural dynamics of protein structures and protein complexes. The identification and quantitation of cross-linked peptides remains challenging for the general community, requiring specialized expertise ultimately limiting more widespread adoption of the technique. We describe a general method for targeted quantitative mass spectrometric analysis of cross-linked peptide pairs. We report the adaptation of the widely used, open source software package Skyline, for the analysis of quantitative XL-MS data as a means for data analysis and sharing of methods. We demonstrate the utility and robustness of the method with a cross-laboratory study and present data that is supported by and validates previously published data on quantified cross-linked peptide pairs. This advance provides an easy to use resource so that any lab with access to a LC-MS system capable of performing targeted quantitative analysis can quickly and accurately measure dynamic changes in protein structure and protein interactions.

Age of the mother as a risk factor and timing of hypospadias repair according to severity.

BACKGROUND & OBJECTIVES: Hypospadias is characterized by a displacement of the urethral opening in males that can change from the typical position within the glans penis to a subcoronal position (Type I), to anywhere along the ventral shaft (Type II), to penoscrotal, scrotal, or perineal positions (Type III). We and others have previously reported that age of the mother (≥ 40 years old) is a risk factor for having a child with hypospadias, but there is a scarcity of reports on whether such risk is higher for having a child with the mild (Type I) or the more severe forms (Types II and III). In addition, we aimed to assess the timing of hypospadias repair according to severity. METHODS: Parents of children with hypospadias were interviewed by using a series of questionnaires (n = 128 cases). Severity was confirmed in the clinic and age of the mother was self-reported. Number of surgeries, age of child by the first and the last intervention was also assessed. Ordered logistic regression and the Brant test were employed to calculate risk between mild (Type I) and severe cases (Types II and III), and the assumption of proportional odds, respectively. The Mann-Whitney U Test was used to compare number of surgeries and age by the last repair between mild and severe cases. One-way ANOVA was employed to compare age of the child at the time of first surgery across severities (Types I - III). RESULTS: Women ≥ 40 years of age are 3.89 times [95% CI: 1.20-12.64] at a higher risk for having a child with the more severe forms of the condition than younger women. Repair of Type I was accomplished with 1 intervention whereas more severe cases required 1 - 4 (2 ± 0.5) surgical interventions. The timing for hypospadias repair of Type I cases occurred at an average age of 16.2 ± 4.88 months, of Type II cases occurred at an average age of 20.3 ± 8.15 months whereas the average age of the first hypospadias repair among Type III cases was 12.68 ± 2.52 months. Number of surgeries according to severity (p ≤ 0.0018, z-ratio = 2.91) and age difference for the timing of last repair (p ≤ 0.045, z-ratio = 1.69) were statistically different, but not the age difference for the first repair. CONCLUSIONS: Increased maternal age is associated with the most severe forms of hypospadias. There is room for improvement for the timing of hypospadias repair according to severity.