The pathophysiology of hypospadias is not fully understood [10]. While the tissue proximal to the meatus displays normal morphology, hypospadias specimens distal from the meatus exhibit abnormal characteristics, including granular areas and less organized smooth muscles around large blood vessel channels, in contrast to the structure seen in healthy tissue [13].

Under the urethral plate, Snodgrass et al. [14] discovered well-vascularized connective tissue composed of collagen, smooth muscle, blood vessels, and nerves with no signs of fibrous bands or dysplastic tissue. Similarly, Erol et al. [13] identified large blood vessels, glands, and muscles, suggesting a connection to the corpus spongiosum. Additionally, Hayashi et al. [15] utilized transmission electron microscopy (TEM) to observe nerves, smooth muscle cells, and capillaries, further supporting its similarity to the corpus spongiosum. It was discovered that the glans tissue beneath the urethral plate displayed the same cytokeratin staining as the normal urethra, suggesting that in hypospadias, the glans under the urethral plate represent an incomplete attempt at urethral formation [13]. Some studies support the findings of a well-vascularized tissue area beneath the urethral plate [13,14,15].

Camoglio et al. [16] observed that the penis in hypospadias was stiffer than that of normal penis; although not explicitly stated in the publication, elastography of the tissue surrounding the tunica spongiosum indicated significantly increased stiffness in hypospadias compared to the normal penis. Similarly, Spinoit et al. [17] reported that 70% of the patients with hypospadias exhibited structural abnormalities in the dartos fascia. Resection of dartos tissue often helps straighten the penis in patients with chordee, indicating that the pathophysiology of this condition is associated with dartos tissue [17]. Structural abnormalities in the dartos tissue are closely related to the clinical severity of congenital penile malformations. The composition of the fibromuscular dartos tissue along the penile shaft determines the elasticity of the subcutaneous tissue and skin mobility [17, 18]. Based on the nature of each subtype of collagen and its distribution, the level of expression and type of collagen will affect the consistency of the tissue [19].

Atmoko et al. [20] reported that the dartos tissue in hypospadias had a thicker, less elastic consistency but with lower levels of total collagen and elastin, and a higher reticulin-to-collagen ratio. Our previous study in hypospadias compared to normal penis showed that expression of COL1A1 and COL6A1 might affect dartos tissue elasticity [10], but no difference in vascularity [21]. Our results support the idea that total collagen expression was reduced in patients with hypospadias compared to those with a normal penis, as we observed lower levels of COL1A1 and COL2A1 mRNA in the hypospadias group.

Type I collagen is notably soft and fragile when unbound, but can form a denser matrix network by crosslinking with other collagen types [22]. Our previous study indicated that mRNA levels of COL1A1 and COL6A1 were downregulated in the dartos tissue of patients with moderate to severe hypospadias, and there was also a moderate correlation observed between COL1A1 and COL6A1 [11]. Hypospadias arise from abnormal growth of the urethral fold and ventral foreskin of the penis, reflecting failure in the canalization of the urethral fold [23]. Furthermore, cell differentiation can be assessed by measuring the ratio of COL2A1 to COL1A1 [24].

Type II collagen is primarily expressed in cartilage tissue, but proteins derived from the translation of COL2A1 have also been detected in malignant melanoma and breast cancer [26]. This indicates that type II collagen can be found in non-cartilage tissues, which aligns with our findings. Additionally, proteins from COL2A1 mRNA have been linked to chemokines such as transforming growth factor-β (TGF-β), which plays a role in tissue fibrosis and possesses anti-inflammatory properties [25]. This indirectly supports the notion that collagen expression may be correlated with the expression of other collagen subtypes.

Collagen type II has a structure similar to collagen I. Collagen type II has a homotrimeric molecular structure with a chain composition [α1(II)]3 [26]. Several studies have suggested COL2A1:COL1A1 ratio usage in the cell differentiation index [24], anabolic chondrocyte marker [27] and osteoarthritis severity [28]. In addition to our study, no study has discussed the COL2A1:COL1A1 ratio in dartos tissue of hypospadias patients. We found no differences between hypospadias and phimosis in COL2A1:COL1A1 ratio.

Lastly, the thick and inelastic dartos tissue seen in patients with hypospadias does not necessarily indicate higher collagen levels. In our study, we discovered that this tissue showed lower expression levels of COL1A1 and COL2A1, although this was not reflected in the COL2A1: COL1A1 ratio. Furthermore, mRNA levels of COL2A1 do not always correlate with protein expression, as fluctuations in COL2A1 and COL1A1 protein levels can arise from transcriptional and post-transcriptional modifications [29]. This research represents the first assessment of COL2A1 and COL1A1 gene expression and its ratio in the dartos tissue of hypospadias patients. However, our study has limitations, including the small sample size and the absence of immunohistochemical examinations to validate this finding.