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Research article
The impact of COL1A1 and COL6A1 expressions on hypospadias and penile curvature severity
Prahara Yuri
Universitas Gadjah Mada Fakultas Kedokteran
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8403-5988
License:
This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License
Background: Hypospadias, the most frequent congenital male external genitalia abnormality, is usually associated with curvature of the ventral penile, i.e. chordee. Abnormality of dartos tissue has been suggested as the pathophysiology of the chordee. Collagen is one of the most abundant fibrous proteins within the extracellular matrix. In this study, we determined the expressions of collagen 1 (COL1A1) and COL6A1 in hypospadias patients and associated them with the severity of penile curvature.
Methods: We involved 60 children <18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 control in our institution from 2017 – 2020. The expressions of COL1A1 and COL6A1 from dartos tissue were determined by reverse-transcriptase polymerase chain reaction (qPCR). The penile curvature severity was classified as mild (<30 degree), moderate (30-60 degree), and severe (>60 degree).
Results: qPCR showed that the COL1A1 and COL6A1 expressions were significantly downregulated in distal (0.88 (0.38-2.53) and 0.54 (0.16-4.35), respectively) and proximal 0.76 (0.33-2.57) and 0.57 (0.18-1.38), respectively) hypospadias groups compared to controls (1.85 (0.24-4.61) and 0.93 (0.17-4.06), respectively) with p-value of 0.024 and 0.018, respectively. Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expressions (r=0.458, p<0.0001). Interestingly, the COL1A1 and COL6A1 was also significantly downregulated in the moderate and severe compared to the mild chordee groups with p-value of 0.003 and 0.037, respectively.
Conclusions: Aberrant COL1A1 and COL6A1 expressions might affect the abnormality of dartos tissue and penile curvature severity in hypospadias patients.
Keywords
chordee; COL1A1; COL6A1; expressions; hypospadias; penile curvature severity; qPCR
Figure 1
Hypospadias is the most frequent congenital penile anomaly which affects male external genitalia with the incidence of around 1 in 250 male newborns. It is caused by tissue underdevelopment on the ventral aspect of the penis resulting an abnormal location of the urethral opening on the penile underside (1). Elastosonography found deep alteration of hypospadias penile anatomy, in which the corpus spongiosum is stiffer and less elastic with less developed cavernous corpora (2).
Hypospadias is associated with curvature of the ventral penile, called chordee. It is caused by the insufficiency or disorganization of the complex growth (the vascular and fascial structures) of the ventral penile (3). Resection of dartos tissue usually straightens the penis in patients with chordee and buried penis, suggesting the pathophysiology of these anomalies is related to dartos tissue (4).
Extracellular matrix (ECM) is a non-cellular macromolecular network which supports cellular processes, such as proliferation, migration, differentiation, and homeostasis (5). One of the most abundant fibrous proteins within ECM is collagen. It has been considered the main component of the fibrous skeleton of the corpus cavernosum penis and is ubiquitous within the erectile tissues of the human penis (6, 7).
Fibrillar collagens are widely distributed in tissues providing the tensile strength. Most fibrillar collagen is type 1 collagen which can be found throughout the tissue beneath the urethral plate (6, 7). The type 6 collagen (COL6A1) binds to type 1 collagen (COL1A1) to form thicker collagen fibers. COL6A1 is a part of the beaded- filament-forming collagens and the most studied member of its group (8). In this study, we determined the expressions of collagen 1 (COL1A1) and COL6A1 in hypospadias patients and associated them with the severity of penile curvature.
We involved 60 children < 18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 control in our institution from 2017–2020. Detailed history and thorough examination of hypospadias patients were performed by one urologist.
Reverse-transcriptase polymerase chain reaction (qPCR) assays
Fragments of the foreskin were kept in RNAlater (Ambion, AM7021) solution before extraction. This tissue was extracted using Genezol RNA solution (GENEzol™, Cat. No. GZR100). cDNA was synthesized using Excel RT Reverse transcriptase kit (RP1300, SMOBIO, Hsinchu City, Taiwan) with PCR conditions of 25oC for 10 minutes (denaturation), 42oC for 50 minutes (annealing), and 85oC for 5 minutes (extension).
qPCR was done by mixing the cDNA and Taq Master Mix (GoTaq®Green Master Mix, Cat.No. M7122) with these following primer genes: COL1A1 (F: 5’-TACAGCGTCACTGTCGATGGC-3’ and R: 5’-TCAATCACTGTCTTGCCCCAG-3’), COL6A1 (F: 5’-GACCTCGGACCTGTTGGGTAC-3’ and R: 5’-TACCCCATCTCCCCCTTCAC-3’) (9), and GAPDH (F: 5’-GCACCGTCAAGGCTGAGAAC-3’ and R: 5’-TGGTGAAGACGCCAGTGGA-3’) (10). qPCR conditions initiated with denaturation at 94 °C for 2 minutes, followed by up to 40 cycles consist of 94 °C for 10 seconds, annealing at 54 °C, 58 °C, 63 °C (COL6A1, COL1A1, and GAPDH, respectively) for 30 seconds, and 72 °C for 1 minute followed by final extension at 72 °C for 10 minutes. PCR products were then separated by 2% agarose gel along with a 100-bp DNA ladder (Bioron, Germany, Cat. No. 306009) (Fig. 1). Gene expressions was quantified using Image J software for densitometry analysis.
Statistical analysis
Data were analyzed for their normality distribution using Kolmogorov-Smirnov. Kruskal – Wallis and Mann–Whitney U tests were used for the data which were not normally distributed while the normally distributed data were analyzed with one-way ANOVA test. p < 0.05 was considered as statistically significant.
The baseline characteristics of our patients was described in Table 1. Most patients showed midshaft and penoscrotal hypospadias (67.5%) (Table 1). qPCR showed that the COL1A1 and COL6A1 expressions were significantly downregulated in distal (0.88 (0.38–2.53) and 0.54 (0.16–4.35), respectively) and proximal (0.76 (0.33–2.57) and 0.57 (0.18–1.38), respectively) hypospadias groups compared to controls (1.85 (0.24–4.61) and 0.93 (0.17–4.06), respectively) with p-value of 0.024 and 0.018, respectively (Table 2). Interestingly, the COL1A1 and COL6A1 was also significantly downregulated in the moderate and severe compared to the mild chordee groups with p-value of 0.003 and 0.037, respectively (Table 2).
| Characteristics | Mean ± SD; N (%) |
|---|---|
| Age (years) √ Hypospadias patients √ Controls | 7.85 ± 3.9 5.71 ± 3.92 |
| Hypospadias type (n = 40) √ Distal ♣ Glandular ♣ Subcoronal ♣ Midshaft √ Proximal ♣ Penoscrotal ♣ Scrotal ♣ Perineal | 1 (2.5) 5 (12.5) 14 (35) 13 (32.5) 6 (15) 1 (2.5) |
| Penile curvature (n = 40) √ Mild (< 30 degree) √ Moderate (30–60 degree) √ Severe (> 60 degree) | 14 (35) 12 (30) 14 (35) |
| Penoscrotal transposition √ Yes √ No | 2 (5) 38 (95) |
| Bifid scrotum √ Yes √ No | 6 (15) 34 (85) |
| Hypospadias type | ||||
|---|---|---|---|---|
| Gene | Distal Hypospadias | Proximal Hypospadias | Control | p-value |
| COL1A1 | 0.88 (0.38–2.53) | 0.76 (0.33–2.57) | 1.85 (0.24–4.61) | 0.024* |
| COL6A1 | 0.54 (0.16–4.35) | 0.57 (0.18–1.38) | 0.93 (0.17–4.06) | 0.018* |
| Penile curvature severity | ||||
| Gene | Mild (n = 14) | Moderate (n = 12) | Severe (n = 14) | p-value |
| COL1A1 | 1.56 (0.47–2.57) | 0.69 (0.33–1.59) | 0.73 (0.38–1.11) | 0.003* |
| COL6A1 | 0.90 (0.16–4.35) | 0.57 (0.23–1.04) | 0.44 (0.18–0.89) | 0.037* |
| *, p < 0.05 (Kruskal Wallis test) | ||||
Moreover, there was no significant difference of COL1A1 and COL6A1 expressions either between proximal and distal hypospadias or severe and moderate penile curvature (Table 3). Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expressions (r = 0.458, p = 0.000).
| Gene | Hypospadias type | p | 95% CI | Penile Curvature | p* | 95% CI | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Lower | Upper | Lower | Upper | |||||||
| COL1A1 | Distal | Proximal | 0.957 | 0.963 | 0.972 | Moderate | Severe | 0.877 | 0.896 | 0.911 |
| COL6A1 | Distal | Proximal | 0.570 | 0.570 | 0.596 | Moderate | Severe | 0.123 | 0.125 | 0.142 |
| *Mann-Whitney test | ||||||||||
| Gen | Control | Hypospadia Type | Penile curvature | |||||||
| Distal Hypospadia | Proximal Hypospadia | Mild | Moderate | Severe | ||||||
| GAPDH | ||||||||||
| COL1A1 | ||||||||||
| COL6A1 | ||||||||||
Here, we are able to show the downregulated COL1A1 and COL6A1 expressions in hypospadias, moderate and severe penile curvature patients. These results are consistent with the previous research which found that the mean number of total collagen fibers of dartos tissue in hypospadias was lower but has thicker fibers compared to normal patients. The study also found the abnormality of hypospadias tissue which gave the thick and inelastic character although it was not followed by the increasing of collagen number (11). Another study showed no evidence of fibrous band or dysplastic tissue in subepithelial biopsies analyzed in 17 prepubertal boys undergoing hypospadias repair (12). Eros et al. proposed no difference in collagen intensity between normal areas and under urethral plate of hypospadias patients (13).
Type 1 Collagen is the most abundant collagen. It is the predominant component of interstitial membranes. It is also associated with fibrosis and fibrogenesis (14). Hayashi et al., (2011) explained the excess deposition of collagen type 1 occurs in the maturation phase of scar formation, making the fibrils become large and stiff bands. It may cause less tumescence in patients with scar formation or fibrosis of the corpus cavernosum penis (6).
This paper also investigated the gene expression of COL6A1 as the most studied type of collagen in the beaded- filament-forming collagens group. It plays a central role in cell attachments and connection between tissues and surrounding matrix (15). Type 6 collagen also regulates fibrogenesis by modulating the interactions among cells. It stimulates the activation of mesenchymal cells into myofibroblasts resulting the extracellular matrix deposition and tissue fibrosis (15). In lung fibrosis, type 6 collagen is expressed increasingly (15). However, this study found decreasing gene expression of COL6A1 in hypospadias groups compared to the control group. It might relate with the downregulation of COL1A1which was found in this paper, suggesting type 6 collagen is bound together to the sides of type 1 collagen to form thicker collagen fibers (7). We found significant positive correlation between COL1A1 and COL6A1.
The etiology of chordee in hypospadias remains unclear. Resection of dartos tissue usually can straighten the penis in patients with chordee and buried penis, suggesting the pathophysiology of these anomalies is related to dartos tissue (4). The composition of dartos fibromuscular tissue determines tissue elasticity and the skin mobility. (4, 12)
Collagen is a part of tissue backbone. During tissue turnover, it is formed and degraded to maintain tissue health and homeostasis. Imbalance of that process lead to fibrosis. Fibrosis is known as excessive formation of connective tissue which damage the structure and function of its tissue (16). Chordee occurs because of fibrosis in dartos tissue which disrupts the tissue elasticity and results in the curving of penile (17).
We also investigated the statistical difference of COL1A1 and COL6A1 expressions among groups based on penile curvature in hypospadias group. Mostly, severe forms of hypospadias are related to a significant chordee and a urethral meatus located proximal to the midshaft of the penis (18). Another study proposed that severity of chordee is generally proportional to the degree of hypospadias (19). Many factors, such as abnormal development of the urethral plate, fibrotic mesenchymal tissue at the urethral meatus, and ventral-dorsal corporal disproportion may be interconnected resulting different degree on final severity of curvature in each patient (20).
Aberrant COL1A1 and COL6A1 expressions might affect the abnormality of dartos tissue and penile curvature severity in hypospadias patients.
ECM : Extracellular matrix
COL1A1 : Collagen 1 subtype alpha 1
COL6A1 : Collagen 6 subtype alpha 1
GAPDH : Glyceraldehyde 3-phosphate dehydrogenase
qPCR : reverse-transcriptase polymerase chain reaction
Ethics approval and consent to participate
The Ethical Committee of Faculty of Medicine, Universitas Gadjah Mada/Dr. Sardjito Hospital gave approval for this study (KE/FK/0699/EC). Written informed consent was obtained from all parents for participating this study.
Consent to publish
Not applicable
Availability of data and materials
All data generated or analyzed during this study are included in the submission. The raw data can be requested to the corresponding author.
Competing interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article
Funding
This work financially supporting by Rekognisi Tugas Akhir No: 2640/UN1/DITLIT/DIT-LIT/PT/2020 from Universitas Gadjah Mada.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Authors’ Contribution
PY collected the patient data and PY, IS, NAD analyzed and interpreted data. PY, WA, NA RP, FP performed PCR examination of the dartos tissue. PY, IS, NAD was a major contributor in writing the manuscript. All authors’ read and agreed for the final manuscript.
Acknowledgement
We thank all staff and nurses who took care of patients.
- Marjan Waterloos PH, Spinoit A-F. Hypospadias Variants. In: Congenital Anomalies of the Penis. Springer - International Publishing Swizerland; 2017.
- 10.1016/j.jpurol.2016.02.007Camoglio FS, Zambaldo S, Bruno C, Zampieri N. Hypospadias anatomy: elastosonographic evaluation of the normal and hypospadic Penis. J Pediatr Urol [Internet]. 2016; Available from: http://dx.doi.org/10.1016/j.jpurol.2016.02.007.
- Putte SCJ, Van Der. Hypospadias and associated penile anomalies: a histopathological study and a reconstruction of the pathogenesis. J Plast Reconstr Aesthetic Surg. 2007;60:48–60.
- 10.1016/j.juro.2014.10.090Spinoit A, Praet C, Van, Groen L, Laecke E, Van, Praet M, Hoebeke P. Congenital penile pathology is associated with abnormal development of the dartos muscle: a prospective study of primary penie surgery at a tertiary referra center. J Urol [Internet]. 2015;193(5):1620–4. Available from: http://dx.doi.org/10.1016/j.juro.2014.10.090.
- Sampaio FJB. Collagen and elastic fibers in the penis of human fetuses at 28 weeks postconception. Eur Urol. 1999;970(36):158–63.
- Hayashi Y, Mizuno K, Kojima Y, Moritoki Y, Nishio H, Kato T, et al. Clinical investigation characterization of the urethral plate and the underlying tissue defined by expression of collagen subtypes and microarchitecture in hypospadias. 2011;18:317–22.
- Lodish H, Berk A, Zipursky S, Al E, Collagen: The Fibrous Proteins of the Matrix. In: Molecular Cell Biology [Internet]. 4th ed. New York: W.H.Freeman; 2000. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21582/.
- 10.1016/j.addr.2015.11.001Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev [Internet]. 2016;97:4–27. Available from: http://dx.doi.org/10.1016/j.addr.2015.11.001.
- Schnoor M, Cullen P, Lorkowski J, Stolle K, Robenek H, Troyer D, et al. Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity. J Immunol. 2008;180(8):5707–19.
- Liu ZH, Huo JL, Wu ZG, Sun Z, Bai F, Samartzis D, et al. RASSF7 expression and its regulatory roles on apoptosis in human intervertebral disc degeneration. Int J Clin Exp Pathol. 2015;8(12):16097–103.
- 10.1016/j.jpurol.2018.04.034Atmoko W, Shalmont G, Situmorang GR, Wahyudi I, Tanurahardja B, Rodjani A. Abnormal dartos fascia in buried penis and hypospadias: Evidence from histopathology. J Pediatr Urol [Internet]. 2018;14(6):536.e1-536.e7. Available from: https://doi.org/10.1016/j.jpurol.2018.04.034.
- Snodgrass W, JC KP. P, R G, ME M. Histology of the urethral plate: implications for hypospadias repair. J Urol. 2000;164:988–9.
- Erol A, Baskin L, Li Y, Liu W. Anatomical studies of the urethral plate: why preservation of the urethral plate is important in hypospadias repair. BJU Int. 2000;85(November 1999):728–34.
- Henriksen K, Karsdal MA. Type I, Collagen. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Sun S, Karsdal MA. Type VI Collagen. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Karsdal MA. Introduction. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Montag S, Palmer LS. Abnormalities of Penile Curvature: Chordee and Penile Torsion. Sci World J. 2011;11:1470–8.
- Abbas TO, Mahdi E, Hasan A, AlAnsari A, Pennisi CP. Current Status of Tissue Engineering in the Management of Severe Hypospadias. Front Pediatr. 2018;5(283).
- Azmy AF. Chordee (Penile Curvature). In: Hypospadias Surgery An Ilustrated Guide. Springer - Verlag Berlin Hiedelberg New York; 2004. p. 117.
- Moscardi PRM, Gosalbez R, Castellan MA. Management of High-Grade Penile Curvature Associated with Hypospadias in Children. Front Pediatr. 2017;5(189):1–8.
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Research article
The impact of COL1A1 and COL6A1 expressions on hypospadias and penile curvature severity
Prahara Yuri
Universitas Gadjah Mada Fakultas Kedokteran
Corresponding Author
ORCiD: https://orcid.org/0000-0001-8403-5988
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Complete author information
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CURRENT STATUS: Under Revision
Version 1
Posted 30 Jun, 2020
No community comments so far
Editorial decision: Major revision
On 31 Jul, 2020
Review #2 received
Received 28 Jul, 2020
Reviewer #4 agreed
On 26 Jul, 2020
Review #1 received
Received 24 Jul, 2020
Reviewer #3 agreed
On 23 Jul, 2020
Reviewer #2 agreed
On 29 Jun, 2020
Reviewer #1 agreed
On 29 Jun, 2020
Reviewers invited
Invitations sent on 28 Jun, 2020
Editor assigned
On 22 Jun, 2020
Submission checks complete
On 22 Jun, 2020
Editor invited
On 22 Jun, 2020
First submitted
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Background: Hypospadias, the most frequent congenital male external genitalia abnormality, is usually associated with curvature of the ventral penile, i.e. chordee. Abnormality of dartos tissue has been suggested as the pathophysiology of the chordee. Collagen is one of the most abundant fibrous proteins within the extracellular matrix. In this study, we determined the expressions of collagen 1 (COL1A1) and COL6A1 in hypospadias patients and associated them with the severity of penile curvature.
Methods: We involved 60 children <18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 control in our institution from 2017 – 2020. The expressions of COL1A1 and COL6A1 from dartos tissue were determined by reverse-transcriptase polymerase chain reaction (qPCR). The penile curvature severity was classified as mild (<30 degree), moderate (30-60 degree), and severe (>60 degree).
Results: qPCR showed that the COL1A1 and COL6A1 expressions were significantly downregulated in distal (0.88 (0.38-2.53) and 0.54 (0.16-4.35), respectively) and proximal 0.76 (0.33-2.57) and 0.57 (0.18-1.38), respectively) hypospadias groups compared to controls (1.85 (0.24-4.61) and 0.93 (0.17-4.06), respectively) with p-value of 0.024 and 0.018, respectively. Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expressions (r=0.458, p<0.0001). Interestingly, the COL1A1 and COL6A1 was also significantly downregulated in the moderate and severe compared to the mild chordee groups with p-value of 0.003 and 0.037, respectively.
Conclusions: Aberrant COL1A1 and COL6A1 expressions might affect the abnormality of dartos tissue and penile curvature severity in hypospadias patients.
Figure 1
Hypospadias is the most frequent congenital penile anomaly which affects male external genitalia with the incidence of around 1 in 250 male newborns. It is caused by tissue underdevelopment on the ventral aspect of the penis resulting an abnormal location of the urethral opening on the penile underside (1). Elastosonography found deep alteration of hypospadias penile anatomy, in which the corpus spongiosum is stiffer and less elastic with less developed cavernous corpora (2).
Hypospadias is associated with curvature of the ventral penile, called chordee. It is caused by the insufficiency or disorganization of the complex growth (the vascular and fascial structures) of the ventral penile (3). Resection of dartos tissue usually straightens the penis in patients with chordee and buried penis, suggesting the pathophysiology of these anomalies is related to dartos tissue (4).
Extracellular matrix (ECM) is a non-cellular macromolecular network which supports cellular processes, such as proliferation, migration, differentiation, and homeostasis (5). One of the most abundant fibrous proteins within ECM is collagen. It has been considered the main component of the fibrous skeleton of the corpus cavernosum penis and is ubiquitous within the erectile tissues of the human penis (6, 7).
Fibrillar collagens are widely distributed in tissues providing the tensile strength. Most fibrillar collagen is type 1 collagen which can be found throughout the tissue beneath the urethral plate (6, 7). The type 6 collagen (COL6A1) binds to type 1 collagen (COL1A1) to form thicker collagen fibers. COL6A1 is a part of the beaded- filament-forming collagens and the most studied member of its group (8). In this study, we determined the expressions of collagen 1 (COL1A1) and COL6A1 in hypospadias patients and associated them with the severity of penile curvature.
We involved 60 children < 18 years old, consisting of 20 distal hypospadias, 20 proximal hypospadias patients, and 20 control in our institution from 2017–2020. Detailed history and thorough examination of hypospadias patients were performed by one urologist.
Reverse-transcriptase polymerase chain reaction (qPCR) assays
Fragments of the foreskin were kept in RNAlater (Ambion, AM7021) solution before extraction. This tissue was extracted using Genezol RNA solution (GENEzol™, Cat. No. GZR100). cDNA was synthesized using Excel RT Reverse transcriptase kit (RP1300, SMOBIO, Hsinchu City, Taiwan) with PCR conditions of 25oC for 10 minutes (denaturation), 42oC for 50 minutes (annealing), and 85oC for 5 minutes (extension).
qPCR was done by mixing the cDNA and Taq Master Mix (GoTaq®Green Master Mix, Cat.No. M7122) with these following primer genes: COL1A1 (F: 5’-TACAGCGTCACTGTCGATGGC-3’ and R: 5’-TCAATCACTGTCTTGCCCCAG-3’), COL6A1 (F: 5’-GACCTCGGACCTGTTGGGTAC-3’ and R: 5’-TACCCCATCTCCCCCTTCAC-3’) (9), and GAPDH (F: 5’-GCACCGTCAAGGCTGAGAAC-3’ and R: 5’-TGGTGAAGACGCCAGTGGA-3’) (10). qPCR conditions initiated with denaturation at 94 °C for 2 minutes, followed by up to 40 cycles consist of 94 °C for 10 seconds, annealing at 54 °C, 58 °C, 63 °C (COL6A1, COL1A1, and GAPDH, respectively) for 30 seconds, and 72 °C for 1 minute followed by final extension at 72 °C for 10 minutes. PCR products were then separated by 2% agarose gel along with a 100-bp DNA ladder (Bioron, Germany, Cat. No. 306009) (Fig. 1). Gene expressions was quantified using Image J software for densitometry analysis.
Statistical analysis
Data were analyzed for their normality distribution using Kolmogorov-Smirnov. Kruskal – Wallis and Mann–Whitney U tests were used for the data which were not normally distributed while the normally distributed data were analyzed with one-way ANOVA test. p < 0.05 was considered as statistically significant.
The baseline characteristics of our patients was described in Table 1. Most patients showed midshaft and penoscrotal hypospadias (67.5%) (Table 1). qPCR showed that the COL1A1 and COL6A1 expressions were significantly downregulated in distal (0.88 (0.38–2.53) and 0.54 (0.16–4.35), respectively) and proximal (0.76 (0.33–2.57) and 0.57 (0.18–1.38), respectively) hypospadias groups compared to controls (1.85 (0.24–4.61) and 0.93 (0.17–4.06), respectively) with p-value of 0.024 and 0.018, respectively (Table 2). Interestingly, the COL1A1 and COL6A1 was also significantly downregulated in the moderate and severe compared to the mild chordee groups with p-value of 0.003 and 0.037, respectively (Table 2).
| Characteristics | Mean ± SD; N (%) |
|---|---|
| Age (years) √ Hypospadias patients √ Controls | 7.85 ± 3.9 5.71 ± 3.92 |
| Hypospadias type (n = 40) √ Distal ♣ Glandular ♣ Subcoronal ♣ Midshaft √ Proximal ♣ Penoscrotal ♣ Scrotal ♣ Perineal | 1 (2.5) 5 (12.5) 14 (35) 13 (32.5) 6 (15) 1 (2.5) |
| Penile curvature (n = 40) √ Mild (< 30 degree) √ Moderate (30–60 degree) √ Severe (> 60 degree) | 14 (35) 12 (30) 14 (35) |
| Penoscrotal transposition √ Yes √ No | 2 (5) 38 (95) |
| Bifid scrotum √ Yes √ No | 6 (15) 34 (85) |
| Hypospadias type | ||||
|---|---|---|---|---|
| Gene | Distal Hypospadias | Proximal Hypospadias | Control | p-value |
| COL1A1 | 0.88 (0.38–2.53) | 0.76 (0.33–2.57) | 1.85 (0.24–4.61) | 0.024* |
| COL6A1 | 0.54 (0.16–4.35) | 0.57 (0.18–1.38) | 0.93 (0.17–4.06) | 0.018* |
| Penile curvature severity | ||||
| Gene | Mild (n = 14) | Moderate (n = 12) | Severe (n = 14) | p-value |
| COL1A1 | 1.56 (0.47–2.57) | 0.69 (0.33–1.59) | 0.73 (0.38–1.11) | 0.003* |
| COL6A1 | 0.90 (0.16–4.35) | 0.57 (0.23–1.04) | 0.44 (0.18–0.89) | 0.037* |
| *, p < 0.05 (Kruskal Wallis test) | ||||
Moreover, there was no significant difference of COL1A1 and COL6A1 expressions either between proximal and distal hypospadias or severe and moderate penile curvature (Table 3). Furthermore, there was a moderate correlation between COL1A1 and COL6A1 expressions (r = 0.458, p = 0.000).
| Gene | Hypospadias type | p | 95% CI | Penile Curvature | p* | 95% CI | ||||
|---|---|---|---|---|---|---|---|---|---|---|
| Lower | Upper | Lower | Upper | |||||||
| COL1A1 | Distal | Proximal | 0.957 | 0.963 | 0.972 | Moderate | Severe | 0.877 | 0.896 | 0.911 |
| COL6A1 | Distal | Proximal | 0.570 | 0.570 | 0.596 | Moderate | Severe | 0.123 | 0.125 | 0.142 |
| *Mann-Whitney test | ||||||||||
| Gen | Control | Hypospadia Type | Penile curvature | |||||||
| Distal Hypospadia | Proximal Hypospadia | Mild | Moderate | Severe | ||||||
| GAPDH | ||||||||||
| COL1A1 | ||||||||||
| COL6A1 | ||||||||||
Here, we are able to show the downregulated COL1A1 and COL6A1 expressions in hypospadias, moderate and severe penile curvature patients. These results are consistent with the previous research which found that the mean number of total collagen fibers of dartos tissue in hypospadias was lower but has thicker fibers compared to normal patients. The study also found the abnormality of hypospadias tissue which gave the thick and inelastic character although it was not followed by the increasing of collagen number (11). Another study showed no evidence of fibrous band or dysplastic tissue in subepithelial biopsies analyzed in 17 prepubertal boys undergoing hypospadias repair (12). Eros et al. proposed no difference in collagen intensity between normal areas and under urethral plate of hypospadias patients (13).
Type 1 Collagen is the most abundant collagen. It is the predominant component of interstitial membranes. It is also associated with fibrosis and fibrogenesis (14). Hayashi et al., (2011) explained the excess deposition of collagen type 1 occurs in the maturation phase of scar formation, making the fibrils become large and stiff bands. It may cause less tumescence in patients with scar formation or fibrosis of the corpus cavernosum penis (6).
This paper also investigated the gene expression of COL6A1 as the most studied type of collagen in the beaded- filament-forming collagens group. It plays a central role in cell attachments and connection between tissues and surrounding matrix (15). Type 6 collagen also regulates fibrogenesis by modulating the interactions among cells. It stimulates the activation of mesenchymal cells into myofibroblasts resulting the extracellular matrix deposition and tissue fibrosis (15). In lung fibrosis, type 6 collagen is expressed increasingly (15). However, this study found decreasing gene expression of COL6A1 in hypospadias groups compared to the control group. It might relate with the downregulation of COL1A1which was found in this paper, suggesting type 6 collagen is bound together to the sides of type 1 collagen to form thicker collagen fibers (7). We found significant positive correlation between COL1A1 and COL6A1.
The etiology of chordee in hypospadias remains unclear. Resection of dartos tissue usually can straighten the penis in patients with chordee and buried penis, suggesting the pathophysiology of these anomalies is related to dartos tissue (4). The composition of dartos fibromuscular tissue determines tissue elasticity and the skin mobility. (4, 12)
Collagen is a part of tissue backbone. During tissue turnover, it is formed and degraded to maintain tissue health and homeostasis. Imbalance of that process lead to fibrosis. Fibrosis is known as excessive formation of connective tissue which damage the structure and function of its tissue (16). Chordee occurs because of fibrosis in dartos tissue which disrupts the tissue elasticity and results in the curving of penile (17).
We also investigated the statistical difference of COL1A1 and COL6A1 expressions among groups based on penile curvature in hypospadias group. Mostly, severe forms of hypospadias are related to a significant chordee and a urethral meatus located proximal to the midshaft of the penis (18). Another study proposed that severity of chordee is generally proportional to the degree of hypospadias (19). Many factors, such as abnormal development of the urethral plate, fibrotic mesenchymal tissue at the urethral meatus, and ventral-dorsal corporal disproportion may be interconnected resulting different degree on final severity of curvature in each patient (20).
Aberrant COL1A1 and COL6A1 expressions might affect the abnormality of dartos tissue and penile curvature severity in hypospadias patients.
ECM : Extracellular matrix
COL1A1 : Collagen 1 subtype alpha 1
COL6A1 : Collagen 6 subtype alpha 1
GAPDH : Glyceraldehyde 3-phosphate dehydrogenase
qPCR : reverse-transcriptase polymerase chain reaction
Ethics approval and consent to participate
The Ethical Committee of Faculty of Medicine, Universitas Gadjah Mada/Dr. Sardjito Hospital gave approval for this study (KE/FK/0699/EC). Written informed consent was obtained from all parents for participating this study.
Consent to publish
Not applicable
Availability of data and materials
All data generated or analyzed during this study are included in the submission. The raw data can be requested to the corresponding author.
Competing interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article
Funding
This work financially supporting by Rekognisi Tugas Akhir No: 2640/UN1/DITLIT/DIT-LIT/PT/2020 from Universitas Gadjah Mada.
The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Authors’ Contribution
PY collected the patient data and PY, IS, NAD analyzed and interpreted data. PY, WA, NA RP, FP performed PCR examination of the dartos tissue. PY, IS, NAD was a major contributor in writing the manuscript. All authors’ read and agreed for the final manuscript.
Acknowledgement
We thank all staff and nurses who took care of patients.
- Marjan Waterloos PH, Spinoit A-F. Hypospadias Variants. In: Congenital Anomalies of the Penis. Springer - International Publishing Swizerland; 2017.
- 10.1016/j.jpurol.2016.02.007Camoglio FS, Zambaldo S, Bruno C, Zampieri N. Hypospadias anatomy: elastosonographic evaluation of the normal and hypospadic Penis. J Pediatr Urol [Internet]. 2016; Available from: http://dx.doi.org/10.1016/j.jpurol.2016.02.007.
- Putte SCJ, Van Der. Hypospadias and associated penile anomalies: a histopathological study and a reconstruction of the pathogenesis. J Plast Reconstr Aesthetic Surg. 2007;60:48–60.
- 10.1016/j.juro.2014.10.090Spinoit A, Praet C, Van, Groen L, Laecke E, Van, Praet M, Hoebeke P. Congenital penile pathology is associated with abnormal development of the dartos muscle: a prospective study of primary penie surgery at a tertiary referra center. J Urol [Internet]. 2015;193(5):1620–4. Available from: http://dx.doi.org/10.1016/j.juro.2014.10.090.
- Sampaio FJB. Collagen and elastic fibers in the penis of human fetuses at 28 weeks postconception. Eur Urol. 1999;970(36):158–63.
- Hayashi Y, Mizuno K, Kojima Y, Moritoki Y, Nishio H, Kato T, et al. Clinical investigation characterization of the urethral plate and the underlying tissue defined by expression of collagen subtypes and microarchitecture in hypospadias. 2011;18:317–22.
- Lodish H, Berk A, Zipursky S, Al E, Collagen: The Fibrous Proteins of the Matrix. In: Molecular Cell Biology [Internet]. 4th ed. New York: W.H.Freeman; 2000. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21582/.
- 10.1016/j.addr.2015.11.001Theocharis AD, Skandalis SS, Gialeli C, Karamanos NK. Extracellular matrix structure. Adv Drug Deliv Rev [Internet]. 2016;97:4–27. Available from: http://dx.doi.org/10.1016/j.addr.2015.11.001.
- Schnoor M, Cullen P, Lorkowski J, Stolle K, Robenek H, Troyer D, et al. Production of type VI collagen by human macrophages: a new dimension in macrophage functional heterogeneity. J Immunol. 2008;180(8):5707–19.
- Liu ZH, Huo JL, Wu ZG, Sun Z, Bai F, Samartzis D, et al. RASSF7 expression and its regulatory roles on apoptosis in human intervertebral disc degeneration. Int J Clin Exp Pathol. 2015;8(12):16097–103.
- 10.1016/j.jpurol.2018.04.034Atmoko W, Shalmont G, Situmorang GR, Wahyudi I, Tanurahardja B, Rodjani A. Abnormal dartos fascia in buried penis and hypospadias: Evidence from histopathology. J Pediatr Urol [Internet]. 2018;14(6):536.e1-536.e7. Available from: https://doi.org/10.1016/j.jpurol.2018.04.034.
- Snodgrass W, JC KP. P, R G, ME M. Histology of the urethral plate: implications for hypospadias repair. J Urol. 2000;164:988–9.
- Erol A, Baskin L, Li Y, Liu W. Anatomical studies of the urethral plate: why preservation of the urethral plate is important in hypospadias repair. BJU Int. 2000;85(November 1999):728–34.
- Henriksen K, Karsdal MA. Type I, Collagen. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Sun S, Karsdal MA. Type VI Collagen. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Karsdal MA. Introduction. In: Biochemistry of Collagens, Laminins and Elastin Structure, Function and Biomarkers. Springer - International Publishing Swizerland; 2016.
- Montag S, Palmer LS. Abnormalities of Penile Curvature: Chordee and Penile Torsion. Sci World J. 2011;11:1470–8.
- Abbas TO, Mahdi E, Hasan A, AlAnsari A, Pennisi CP. Current Status of Tissue Engineering in the Management of Severe Hypospadias. Front Pediatr. 2018;5(283).
- Azmy AF. Chordee (Penile Curvature). In: Hypospadias Surgery An Ilustrated Guide. Springer - Verlag Berlin Hiedelberg New York; 2004. p. 117.
- Moscardi PRM, Gosalbez R, Castellan MA. Management of High-Grade Penile Curvature Associated with Hypospadias in Children. Front Pediatr. 2017;5(189):1–8.