Stesura Seveso Archivio Italiano di Urologia e Andrologia 2024; 96(4):12894 1 ORIGINAL PAPER ciated condition found in hypospadias, mostly the proxi- mal type, which is the proximal part of urethral folds that were fused incompletely and left an opening in the ven- tral penis in males (2, 3). Chordee may arise from the inadequacy or disorder of the dartos fascia, on the ventral side of the penis. The severity of the chordee may be asso- ciated with the degree of dartos fascia abnormality (4). In hypospadias patients, the composition of the extracellular matrix (ECM) was varied and different compared to the non-hypospadias patients, in which the main components are collagens, elastic fibers, and proteoglycans/glycosamino- glycans (GAGs). These composition changes might con- tribute to the thickening and stiffness of the tissue. A pre- vious study reported that the dartos fascia in hypospadias had thicker collagen fibers than a normal penis (5). To the best of our knowledge, there is no study reporting if there is any correlation between the degree of collagen thickness and the severity of the fibrotic condition of dar- tos fascia to the severity of ventral curvature. Therefore, this study aimed to compare the degree of collagen thick- ness and the severity of the fibrotic condition to the sever- ity of ventral curvature in hypospadias. MATERIALS AND METHODS A cross-sectional analytical study was performed on 100 hypospadias patients with various degrees of chordee who underwent urethroplasty between 2020 and 2022. Patients were differentiated into two groups based on the severity of penile curvature degree, namely mild to moderate (< 60 degrees) and severe (> 60 degrees). The penile curvature degree was measured, twice at least, using a goniometer intraoperatively before performing urethroplasty and a sin- gle surgeon (GWKD) performed urethroplasty for this study. Excision of ventral dartos fascia, including tunica albuginea, which manifests as chordee, was performed during surgery with a U-shape incision, which was a wide excision of urethra of about 8 mm in length, which started at 2 mm from proximal tip of urethral meatus to 2 mm from distal urethral meatus. These tissues were sent to the pathology laboratory to assess the fibrotic and collagen density conditions. Patients who underwent re-urethro- Purpose: Hypospadias, one of the congenital anomalies commonly associated with some degrees of ventral penile curvature that may arise from malfor- mation of dartos fascia, the chordee. Our study aims to deter- mine the correlation between the histopathology properties of dartos fascia and the severity of ventral penile curvature in hypospadias. Materials and methods: One hundred hypospadias patients with various degrees of ventral penile curvature were included in this cross-sectional analytical study from 2020 to 2022. During hypospadias repair, ventral dartos fascia was excised and ana- lyzed for the degree of collagen thickness and the severity of the fibrotic condition. Results: Out of 100 patients, the mean age was 6.58 + 4.28 years, who were classified as mild to moderate (66%) and severe (34%) ventral curvature cases. The analyses showed significant differences in the severity of fibrotic condition and collagen thickness of dartos fascia to the severity of penile ven- tral curvature with p-values of 0.002 and 0.017, respectively. Conclusions: The difference in histopathology properties of dartos fascia may affect the severity of penile curvature in hypospadias patients. KEY WORDS: Hypospadias; Chordee; Penile curvature; Collagen; Fibrotic. Submitted 2 August 2024; Accepted 12 October 2024 INTRODUCTION Hypospadias is a congenital anomaly found in children characterized by an abnormal location of urethral open- ing on the ventral side of the penis. The incidence of hypospadias varies around the world, with a prevalence of about 1 case in every 250 male newborn babies. This malformation is caused by abnormal development of the ventral side of the penis, hence an abnormal proximal position of the urethral opening (1). The abnormality has been classified into several cate- gories. Based on the position of the urethral opening, hypospadias can be differentiated into proximal and dis- tal types. Ventral curvature, the chordee, is another asso- Correlation between histopathology properties of dartos tissue and the severity of penile curvature in hypospadias Gede W.K. Duarsa 1, Pande M.W. Tirtayasa 2, Ni Wayan Winarti 3, Andy Michael 4, Komang H.A. Duarsa 5 1 Department of Urology, Faculty of Medicine Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Denpasar, Indonesia; 2 Department of Urology, Faculty of Medicine Universitas Udayana/Universitas Udayana Teaching Hospital, Denpasar, Indonesia; 3 Department of Pathology Anatomy, Faculty of Medicine Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Denpasar, Indonesia; 4 Department of Surgery, Faculty of Medicine Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Denpasar, Indonesia; 5 Medical Doctor Study Program, Faculty of Medicine Universitas Udayana, Denpasar, Indonesia. DOI: 10.4081/aiua.2024.12894 Summary Archivio Italiano di Urologia e Andrologia 2024; 96(4):12894 G.W.K. Duarsa, P.M.W. Tirtayasa, N. Wayan Winarti, et al. 2 plasty due to a failed previous operation were excluded. The study has been approved by the research ethics committee of the Faculty of Medicine, Universitas Udayana, Prof. I.G.N.G. Ngoerah General Hospital, Denpasar with No. 2773/UN14.2.2VII.14/ LT/2022, and written informed consent has been acquired from the parents. The staining and evaluation of dartos specimens were conducted by a single pathologist (NWW) without prior infor- mation regarding the clinical condition of patients. The Masson’s trichrome (MST) and hematoxylin and eosin (H&E) stain- ing were performed to assess the colla- gen fibers thickness and the severity of the fibrosis, respectively. The images were then enlarged 200 times and five random points were marked to evaluate the specimen thoroughly. Regarding the thickness of collagen, patients were divided into two groups: thin to moderate and thick. Moreover, patients were also assigned into two groups in terms of the severity of fibrotic conditions, namely mild to moderate and severe. The cut-off points of thin to moderate and thick/severe are below or above 60% per high power field (HPF) for both variables. These percent- ages were obtained by dividing the area occupied by col- lagen by the area of the entire high power field. The Chi-square and the alternative Fisher’s exact tests were used to analyze the relationship between the severi- ty of chordee and collagen thickness and fibrotic severity on dartos tissue. P-value < 0.05 was considered statisti- cally significant. RESULTS The characteristics of all patients in our institution are shown in Table 1. The mean age of 100 patients was 6.58 + 4.28 years and they were classified into mild to moder- ate (66%) and severe (34%) ventral curvature cases. Moreover, in terms of collagen thickness of dartos fascia, there were 76% and 24% cases with thin to moderate and thick collagen density, respectively. In addition, 87% and 13% of patients suffered from mild to moderate and severe fibrotic conditions of their dartos fascia, respec- tively. Representation of thin to moderate and thick colla- gen density as well as mild to moderate and severe fibro- sis, shown in Figure 1. The analyses showed significant differences in the severi- ty of fibrotic and collagen thickness of dartos fascia to the severity of ventral curvature with p-values of 0.002 and 0.017 respectively, shown in Table 2. DISCUSSION A previous study revealed that the number collagen fibers of dartos fascia was significantly lower but the fibers were thicker in hypospadias compared to normal penis. Moreover, the reticulin to total collagen ratio was higher in hypospadias compared to normal penis. Reticulin, a type III collagen fibers, was found to increase during early stage of life and early healing condition (6). At the molec- ular level, Yuri et al. showed the downregulation of sever- al types of collagen gene expressions compared to normal patients. COL1A1 and COL6A1 were also significantly downregulated in the moderate and severe chordee Table 2. Analysis of collagen thickness and fibrotic severity on ventral curvature. Variable Ventral curvature degree P-value Mild-moderate Severe Fibrotic severity of dartos fascia Mild-moderate 61 26 0.002 * Severe 5 8 Collagen thickness of dartos fascia Thin-moderate 55 21 0.017 ** Thick 11 13 *: significant result (Fisher’s exact test); **: significant result (Chi-square test). Table 1. Characteristics of all patients. Variable Hypospadias patients (n = 100) Mean age (year + SD) 6.58 + 4.28 Ventral curvature, n (%) Mild-moderate 66 (66) Severe 34 (34) Fibrotic severity of dartos fascia, n (%) Mild-moderate 87 (87) Severe 13 (13) Collagen thickness of dartos fascia, n (%) Thin-moderate 76 (76) Thick 24 (24) Figure 1. The MST staining of collagen fibers (200x), the green arrow points to the thin to moderate collagen fibers in Figure (A) and thick collagen fibers in Figure (B). The H&E staining of fibrotic changes (200x), the green arrow points to the mild to moderate fibrosis in Figure (C) and severe fibrosis in Figure (D). Archivio Italiano di Urologia e Andrologia 2024; 96(4):12894 3 Histopathology related to penile curvature in hypospadias groups compared to the mild chordee groups, with p-val- ues of 0.003 and 0.037, respectively (7, 8). Therefore, we assessed the thickness of collagen fibers in hypospadias in several degrees of ventral curvature. Our study reported that collagen fibers of dartos fascia in hypospadias were significantly thicker in severe compared to mild to mod- erate ventral curvature. As far as we know, this is the first study comparing the histopathology aspect of dartos fas- cia in various degrees of ventral curvature of hypospadias. Fibrotic tissue leads to thickening and scarring of the tis- sue it affects (9, 10). Fibrotic tissue is a collection of extra- cellular matrix (ECM) and it may appear in various degree of elasticity and thickness that depends on its properties of collagen and elastin (11). Fibrosis is known as a dis- proportionate development of connective tissue that dis- rupts the structure and function of any tissue (12). Chordee exists due to the fibrosis of dartos fascia that results in elasticity disruption and leads to penile bending (13). Moreover, we reported the distinction of dartos fas- cia properties in various degrees of ventral curvature of hypospadias patients, especially in collagen thickness and fibrosis severity. The more severe ventral curvature degree patients suffered, the more severe and thicker the fibrotic condition and collagen thickness they would have. Hypospadias patients with severe ventral curvature had more abnormal and inelastic dartos fascia tissue that led to more severe ventral bending of the penile. Histopathology study on the hypospadias urethral plate revealed the characteristic of well-built connective tissue with adequate vascularization and no signs of fibrosis on the urethral plate (14). Moreover, there was no difference in collagen density in patients with hypospadias com- pared to the normal patient (15). However, the readouts of our study are different from those of previous studies. Previous studies evaluated the histopathology of the ure- thral plate, on the other hand, our study assessed the histopathology of the dartos fascia especially taken from the ventral side of the penile. In the current literature, there are no guidelines about the definition of collagen fiber thickness and fibrotic changes in micrometers, which might be a potential bias due to tissue sampling and handling for microscope analysis dif- ferences in size. Several pieces of literature have reported the abnormality of dartos fascia in hypospadias, buried penis and also in epispadias cases (3, 16). Atmoko et al. showed a reduction of total collagen to elastin and also an increased ratio of reticulin fibers in dartos fascia of hypospadias and buried penis (3). Moreover, there were thicker collagen and abnormal smooth muscle action distribution in hypospa- dias and buried penis compared to normal penile (3, 16). To the best of our knowledge, this is the first study to compare the histopathology of dartos fascia based on the severity of ventral curvature in hypospadias patients. This study showed that severe ventral curvature is correlated with thicker collagen fibers and more severe fibrosis con- dition of dartos fascia compared with mild to moderate ventral curvature in hypospadias patients. A more pro- gressive excision of the chordee is warranted in severe ventral curvature penile during hypospadias repair. In fact, total excision of the chordee is mandatory during urethroplasty regardless of the degree of penile curvature as this is an abnormal tissue. Leaving even a small rem- nant of chordee during reconstructive surgery may increase the risk of complications following surgery (17). We realized that there was a more objective measurement of fibrosis based upon immunohistochemical staining and/or quantification of relative protein expression such as collagen types and elastin. Therefore, we recommend- ed that further study is needed to evaluate the composi- tion of collagen in dartos fascia based on the severity of chordee using an electron microscope with specific immunohistochemical staining. Since we did not focus on and discuss the surgical outcomes in our study, we also recommended further study to assess the complication post-surgical to evaluate the outcome, which is not only studies at the protein level and molecular studies CONCLUSIONS There is a difference in the properties of dartos fascia in severe ventral curvature compared with mild to moderate ventral curvature of hypospadias patients, especially in the thickness of collagen fibers and severity of the fibrot- ic condition. The difference in histopathology properties of dartos fascia may affect the severity of penile curvature in hypospadias patients. REFERENCES 1. Waterloos M, Hoebeke P, Spinoit AF. Hypospadias variants. In: Congenital anomalies of the penis. Berlin: Springer; 2017. 2. Palmer LS, Palmer JS. Management of abnormalities of external genitalia in boys. In: Wein AJ, Kavoussi LR, Partin AW, Peters CA (Eds.) Campbell-Walsh urology. 11th ed. Philadelphia: Elsevier Ltd; 2016, pp. 3374-6. 3. Abbas M, Liard A, Elbaz F, Bachy B. Outcome of surgical man- agement of concealed penis. J Pediatr Urol. 2007; 3:490-4. 4. Van Der Putte SCJ. Hypospadias and associated penile anomalies: a histopathological study and a reconstruction of the pathogenesis. J Plast Reconstr Aesthet Surg. 2007; 60:48-60. 5. Atmoko W, Shalmont G, Situmorang GR, et al. Abnormal dartos fascia in buried penis and hypospadias: evidence from histopatholo- gy. J Pediatr Urol. 2018; 14:536.e1-536.e7. 6. Cheng W, Yan-hua R, Fang-gang N, Guo-an Z. The content and ratio of type I and III collagen in skin differ with age and injury. African J Biotechnol. 2011; 10:2524-9. 7. Yuri P, Gunadi G, Lestari RP, et al. The impact of COL1A1 and COL6A1 expression on hypospadias and penile curvature severity. BMC Urol. 2020; 20:189. 8. Srinivasan AK, Palmer LS, Palmer JS. Inconspicuous penis. Sci World J. 2011; 11:2559-64. 9. Almine JF, Bax DV, Mithieux SM, et al. Elastin based materials. Chem Soc Rev. 2010; 39:3371-9. 10. Daamen WF, Veerkamp JH, Van Hest JCM, Van Kuppevelt TH. Elastin as a biomaterial for tissue engineering. Biomaterials. 2007; 28:4378-98. 11. Neary R, Watson CJ, Baugh JA. Epigenetics and the overhealing wound: the role of DNA methylation in fibrosis. Fibrogenesis Tissue Repair. 2015; 8:1-13. Archivio Italiano di Urologia e Andrologia 2024; 96(4):12894 G.W.K. Duarsa, P.M.W. Tirtayasa, N. Wayan Winarti, et al. 4 12. Karsdal MA. Biochemistry of collagens, laminins and elastin structure, function and biomarkers. Berlin: Springer; 2016. 13. Montag S, Palmer LS. Abnormalities of penile curvature: chordee and penile torsion. Sci World J. 2011; 11:1470-8. 14. Snodgrass W, Patterson K, Plaire JC, et al. Histology of the ure- thral plate: implications for hypospadias repair. J Urol. 2000; 164:988-90. 15. Erol A, Baskin LS, Li YW, Liu WH. Anatomical studies of the ure- thral plate: why preservation of the urethral plate is important in hypospadias repair. BJU Int. 2000; 85:728-34. 16. Spinoit AF, Van Praet C, Groen LA, et al. Congenital penile pathology is associated with abnormal development of the dartos muscle: a prospective study of primary penile surgery at a tertiary referral center. J Urol. 2015; 193:1620-4. 17. Halaseh SA, Halaseh S, Ashour M. Hypospadias: A Comprehensive Review Including Its Embryology, Etiology and Surgical Techniques. Cureus. 2022; 14:e27544. Correspondence Gede Wirya Kusuma Duarsa (Corresponding Author) gwkurology@gmail.com Department of Urology, Faculty of Medicine, Universitas Udayana, Prof. Dr. I.G.N.G Ngoerah General Hospital, Denpasar, Bali, Indonesia Pande Made Wisnu Tirtayasa wisnu_tirtayasa@unud.ac.id Department of Urology, Faculty of Medicine Universitas Udayana/ Universitas Udayana Teaching Hospital, Denpasar, Indonesia Ni Wayan Winarti nw.winarti@unud.ac.id Department of Pathology Anatomy, Faculty of Medicine Universitas Udayana/Prof. dr. I.G.N.G. Ngoerah General Hospital, Denpasar, Indonesia Andy Michael andymichael91.am@gmail.com Department of Surgery, Faculty of Medicine Universitas Udayana/ Prof. dr. I.G.N.G. Ngoerah General Hospital, Denpasar, Indonesia Komang Harsa Abhinaya Duarsa abhinaya.duarsa@gmail.com Medical Doctor Study Program, Faculty of Medicine Universitas Udayana, Denpasar, Indonesia Conflict of interest: This study was supported by the PNBP Universitas Udayana funding grant 2022 No. B/78.174/UN14.4/PT/01/03/2022.