Hypospadias is a common congenital malformation of the urinary system in male children, with a prevalence of 18.6 per 10,000 births (1). It is defined as incomplete development of the male urethra leading to aberrant localization of the urinary meatus on the ventral side of the penis in a variable position from the glans to the perineum. The etiology of hypospadias remains unknown. Although some studies have implicated androgen receptors (ARs) in hypospadias development, recent studies report conflicting results regarding AR expression.

Vottero et al. demonstrated that AR expression in the preputial tissue of patients with hypospadias was lower than that of normal children (2). Chao et al. examined the AR in the prepuce of patients with hypospadias and controls using immunohistochemistry (IHC) and observed that AR expression in 68 hypospadias tissues was significantly lower than that in 68 normal Preputial tissues (3). Silva T et al. studied 17 hypospadias and 41 control patients and observed significantly less AR mRNA levels in the urethral mucosa of patients with hypospadias than in controls (4). Recent epigenetic studies further highlight transcriptional dysregulation of AR in preputial mucosa (5, 6). In contrast, Pichler et al. discovered that AR protein was higher in boys with hypospadias than in boys without hypospadias (133.25 ± 6.17 vs. 100 ± 4.45, P = 0.014) (7). Similarly, Kocaturk et al. documented higher AR expression in hypospadias-affected preputial tissue than in normal tissue (8). Hence, the expression of AR in patients with hypospadias remains controversial.

We conducted a large prospective study to clarify discrepancies in AR expression between hypospadias patients and normal children, and to assess the relationship between AR dysregulation and hypospadias development.

The hypospadias group included 40 patients; the mean age was 2.90 ± 0.79 (range, 1.6–5.2) years. Forty patients who underwent circumcision were in the control group, and the mean age was 3.2 ± 0.79 (range: 1.8–4.7) years. There was no difference in age between the hypospadias and control groups (Table 1). In addition, there was no difference in serum DHT concentration between the hypospadias and control groups (0.38 ± 0.11 vs. 0.39 ± 0.13 nmol/L, P = 0.88).

IHC of inner plate preputial tissues revealed that the AR protein was majorly expressed on the basal cell layer, while some were expressed in the fibroblasts (Figure 1). Furthermore, AR expression was lower in the hypospadias group than in the control group (AOD, 0.38 ± 0.09 vs. 0.56 ± 0.11, P = 0.01) (Table 1) (Figure 1). However, there was no difference in AR expression between the proximal and distal hypospadias (AOD, 0.40 ± 0.07 vs. 0.37 ± 0.10, P = 0.22) (Table 2).

Our study demonstrated significantly reduced AR expression in hypospadias patients compared to healthy controls, with no differential expression between proximal and distal subtypes.

Although extensive studies have characterized the histology of hypospadias, its etiology remains elusive. Multiple genes—including ZEB1, Hsa_circ_0000417, SRY, 5α-reductase, and AR—have been implicated as contributors, though none are established as causative (2, 3, 9).

The second trimester is a critical period for human fetal urethral development. During this period, androgens are critical for the differentiation and development of sexual genitalia in male fetuses (11). DHT-AR signaling is essential for urethral closure, with AR functioning as a ligand-dependent nuclear receptor transcription factor (12). AR deficiency disrupts androgen signaling, impairing male sex differentiation and potentially contributing to hypospadias pathogenesis.

The predominant localization of AR to the basal epithelial layer is biologically significant, as this compartment contains progenitor cells critical for urethral morphogenesis. Basal AR signaling likely orchestrates epithelial-mesenchymal interactions (EMT) during urethral closure by regulating downstream targets (e.g., ZEB1, FGF10). Reduced AR in basal cells may disrupt EMT, impairing urethral tubularization, resulting in failed urethral fusion and contributing to hypospadias pathogenesis (9, 10).

Numerous studies have investigated the AR-hypospadias relationship, yet yielded conflicting results. Vottero et al. reported reduced AR expression in preputial tissue from hypospadias patients vs. healthy controls (2). Conversely, Pichler et al. documented elevated AR protein in affected boys (133.25 ± 6.17 vs. 100 ± 4.45, P = 0.014) (7). These discrepancies likely reflect methodological variations and limited sample sizes in prior studies. To resolve this controversy, we employed a large cohort to definitively establish the AR-hypospadias association.

Our study demonstrated that AR expression is significantly decreased in patients with hypospadias than in healthy boys. This may be associated with the etiology of hypospadias. Lower AR expression causes abnormal combination and dysfunction of androgens; thus, urethral development is affected, and urethral closure cannot be achieved, contributing to various hypospadias. Vottero et al. documented reduced AR in preputial tissue from affected patients vs. controls (2), a result corroborated by Chao et al. using immunohistochemistry (3). Silva et al. further reported diminished AR mRNA in urethral mucosa (4), while An et al. noted AR reduction in penile skin, particularly within the urethral plate (10). Consistent with our protein-level findings, Yıldız et al. demonstrated downregulated AR expression and associated DNA methylation changes in foreskin tissue of hypospadias patients (6). Similarly, İnanç et al. reported dysregulation of AR and related genes (ESR1, FGFR2) in preputial tissues, reinforcing end-organ resistance in hypospadias pathogenesis (5). This convergence of protein and gene evidence reinforces AR deficiency as a hallmark of hypospadias. Conversely, several groups report contradictory data. Kocaturk H. described increased AR expression in affected preputial tissue (8). Balaji et al. similarly noted heightened AR levels, with proximal subtypes exhibiting greater expression than distal forms, potentially indicating end-organ overexpression (13).

We observed no difference in AR expression between the proximal and distal hypospadias. Although AR mediates external genitalia development, its levels did not correlate with phenotypic severity—a finding consistent with Celayir et al. who reported no AR-severity association (14). In contrast, Qiao et al. described elevated AR in severe vs. mild hypospadias (P < 0.05), though small sample sizes preclude definitive conclusions (15). Our null finding regarding AR expression across proximal/distal subtypes suggests that baseline AR deficiency may be a universal feature in hypospadias irrespective of severity. We speculate that compensatory mechanisms (e.g., local androgen synthesis) or alternative signaling pathways may modulate phenotypic expression beyond AR abundance alone.

Impaired AR transcriptional activity in hypospadias may be ameliorated by androgen supplementation. Kaya et al. demonstrated that preoperative DHT gel reduces postoperative complications (e.g., fistula rates) and improves cosmetic outcomes, potentially via enhanced angiogenesis and signaling rescue (16). Our data support DHT trials in AR-deficient hypospadias, reinforcing this therapeutic approach.

Our findings contrast with prior reports of AR overexpression in hypospadias (7, 8, 13). Specifically, we observed reduced AR expression in patients vs. healthy controls. These discrepancies likely reflect methodological differences. Pichler et al. quantified AR in whole-tissue lysates via Western blotting, whereas our IHC precisely localized AR to basal epithelial cells—the primary site for androgen signaling during urethrogenesis (7).

While our study identifies AR downregulation, future work should explore upstream mechanisms (e.g., AR promoter methylation, miRNA regulation) and functional consequences via in vitro models of urethral development.

In conclusion, the inner plate preputial tissues of patients with hypospadias showed decreased AR expression, suggesting that AR may be involved in hypospadias development and contributes to resolving a key controversy. However, further studies are necessary to assess whether epigenetic abnormalities or gene mutations are responsible for the decreased AR expression.