Severity-Dependent Profile of the Metabolome in Hypospadias

Abstract

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.

Keywords: etiology; hypospadias; metabolites; metabolome; metabolomics; severity.

Figure 1

Staging or classification systems that describe the anatomical position of the urethral meatus in hypospadias have been created over the years. From these, “Type I–Type III” terminology is used in this study. N values correspond to the number of hypospadias cases reported hereafter penile degloving and chordee release during each urethroplasty procedure.

Figure 2

Constituent ratio of the 35 metabolites identified by class.

Figure 3

Metabolome of Type I, II, and III hypospadias, and controls. Heatmap displays average metabolites concentrations for each group. Control: n = 8; Type I hypospadias: n = 7; Type II hypospadias: n = 7; Type III hypospadias: n = 6. Blue represents low concentration; red represents high concentration.

Figure 4

Type II and Type III hypospadias metabolome differs from Type I hypospadias and controls. (A) Principal component analysis (PCA) and (B) partial least squares-discriminant analysis (PLS-DA) analysis scores plots display variance and separation for Type II and Type III hypospadias between Type I hypospadias and control groups. The ellipses of the score plots illustrate 95% confidence region of the groups. Control: n = 8; Type I hypospadias: n = 7; Type II hypospadias: n = 7; Type III hypospadias: n = 6.

Figure 5

Significant downregulation of amino acids in Type II and Type III hypospadias. Heatmap represents metabolite concentrations (mM). Statistical analyses revealed that 14 amino acids had statistically significant lower concentrations in Type II and Type III hypospadias in comparison to Type I hypospadias and control groups. p < 0.05. Control: n = 8; Type I hypospadias: n = 7; Type II hypospadias: n = 7; Type III hypospadias: n = 6. Blue represents low concentration; red represents high concentration.