INTRODUCTION
Buried penis is a congenital anomaly, which is characterized by a phallus of normal size buried in suprapubic tissue.1 The incidence of buried penis is approximately 0.3% in Japanese infants at 4–5 years of age.2 It could lead to balanitis, poor hygiene, voiding difficulty, and psychological trauma.3,4
The etiology of buried penis is not clear. The dysgenetic dartos fascia plays an important role in the pathology,1 which needs to be treated during the surgery for correcting buried penis.3–6 Although several surgical methods could be applied, recurrence occurs.4,5,7,8 In the authors’ previous study, an investigation of the anatomy of penile fascia indicated the presence of sublayers in dartos fascia. The superficial layer was under the skin, which was thick and loose. The deep layer was under the superficial layer and on Buck’s fascia, which was thin and dense. No recurrence cases were observed after resecting the deep layer completely.9 Thus, the dysgenetic deep layer of dartos fascia was speculated to play an important role in the buried penis. The resection of the deep layer is helpful for avoiding recurrence.
In this study, patients with buried penis were subjected to anatomical resection of the deep layer of dartos fascia under a microscope. Penile length was measured before and after completely resecting the deep layer to investigate the role of this layer in penile retraction. Next, the histological and pathological characteristics of the superficial and deep layers of dartos fascia were investigated. The results showed that the abnormal deep layer of dartos fascia, characterized by disordered and fragmented elastic fibers, plays an important role in buried penis. Resecting the deep layer could help in avoiding recurrence.
PATIENTS AND METHODS
Patients
Forty-nine patients with buried penis were included in the study, which was conducted from June 2019 to August 2021 in The First Affiliated Hospital of Xi’an Medical University (Xi’an, China). Patients with acquired concealed penis were excluded. The median patient age was 14 (range: 5–25) years. The mean height was 162.9 (standard deviation [s.d.]: 13.8) cm. The mean weight was 63.0 (s.d.: 18.3) kg. The mean body mass index was 23.2 (s.d.: 3.9) kg m−2.
Penile length was measured before and after resecting deep layer of dartos fascia
The patients were subjected to anatomical resection of the deep layer of dartos fascia under a microscope (HIR1000/FS3-23, Muller, Hamburg, Germany). The surgery steps included degloving, separating the superficial and deep layers of dartos fascia, resecting the deep layer, and reconstructing the penoscrotal angle.9 Penile length was measured before and after completely resecting the deep layer. The surgery was performed under general anesthesia. The patients were in supine position. One examiner placed the penis at a 90° angle to the body. A flaccid, unstimulated penile length was measured. The penile length was defined as dorsal from the pubic bone to the tip of the glans penis. The room temperature was about 21°C–22°C.3 The Ethics Committee of The First Affiliated Hospital of Xi’an Medical University approved the study (Approval NO. XYYFY2018LSK-007). Every patient or one of their parents provided written informed consent before the surgery.
Samples
The superficial and deep layers of dartos fascia were collected from 49 patients with buried penis. The superficial layers of normal penis were collected from 25 children/adults who underwent circumcision for nonmedical reasons from June 2019 to April 2021. The deep layers of a normal penis were collected from 20 adult cadavers from the Department of Human Anatomy, Xi’an Jiaotong University Health Science Center (Xi’an, China). The Ethics Committee of Xi’an Jiaotong University approved all protocols (Approval No. 2018-02). The position of sampling was from 2:00 to 4:00 o’clock. The deep layers of normal penis were not collected from children/adults who underwent circumcision because of two reasons. First, when performing circumcision, only the foreskin and the superficial layer of dartos fascia need to be removed. Thus, collecting the deep layer of dartos fascia is in violation of ethical standards. Second, the deep layer ends at the coronal sulcus and it is fixed to the Buck’s fascia (Supplementary Figure 1 and 2). Distinguishing the superficial and deep layers of dartos fascia and collecting samples if not degloving are difficult.
Morphological examination
The collagen fibers were observed by Masson’s trichrome staining (G1006, Servicebio, Wuhan, China). Sirius red staining (G1018, Servicebio) was used to distinguish types I and III collagen. The contents of total collagen fibers and type I/III collagen were measured by ImageJ software (National Institutes of Health, Bethesda, MD, USA). The elastic fibers were stained by Verhoeff’s Van Gieson (EVG) method (G1042, Servicebio). The elastic fibers in the avascular zone were observed. The vessels, nerve fibers, and dartos muscle fibers were observed by hematoxylin–eosin staining.
Immunohistochemical examination
Sections with 4 µm thickness were deparaffinized, dehydrated, incubated in 3% hydrogen peroxide solution for 10 min, washed with phosphate-buffered saline (PBS), and incubated with normal goat serum for 15 min and anti-type III collagen antibody (22734-1-AP, Proteintech, Wuhan, China) or anti-elastin antibody (bs-1756R, Bioss, Beijing, China) overnight at 4°C. After the slides were washed with PBS, they were incubated with secondary antibody (SA1050, Boster, Wuhan, China) for 40 min and stained with diaminobenzidine (G1211, Servicebio). The optical density values were obtained using Image J software.
Scanning electron microscopy (SEM)
Collagen and elastic fibers were observed by SEM. The tissue masses of not more than 3 mm2 were washed with PBS, fixed in electron microscopic fixatives (G1102, Servicebio) for 2 h, and kept at 4°C. Then, the samples were washed thrice using 0.1 mmol l-1 phosphate buffer and post-fixed in 1% osmium tetroxide for 2 h at room temperature. Afterward, the samples were dehydrated in graded ethanol and isoamyl acetate for 15 min, critical-point dried, sputtered gold coated for 30 s, and observed using SEM.
Statistical analyses
Data were presented as mean ± s.d.. Categorical variables were analyzed using Chi-square test. Continuous variables with a normal distribution were analyzed using independent sample t-tests or paired t-test. All the data were analyzed using SPSS version 25.0 (SPSS, Inc., Chicago, IL, USA), and P < 0.05 was considered statistically significant difference.
RESULTS
Deep layer of dartos fascia plays an important role in retraction of buried penis
The penile lengths of 49 patients were measured during the operation. After the penis was degloved to the penopubic junction along Buck’s fascia, the corpus spongiosum and corpus cavernosum were protected using a wet gauze. The wet gauze was fixed to Buck’s fascia by suturing the side edges. The deep layer of dartos fascia is thin and tough. It was sutured to the wet gauze near the coronal sulcus to keep its tension, helping to distinguish the tissue boundary between the superficial and deep layer under the microscope (Supplementary Figure 3). The two layers were separated along the tissue boundary till the root of the penis. After the separation, only the deep layer pulled the penis toward the suprapubic fat and scrotum. Then, the deep layer was resected completely. The penile shaft (mean ± s.d.) was found to be significantly elongated after resecting the deep layer compared with that before resection (6.8 ± 1.9 cm vs 6.0 ± 1.6 cm, P < 0.001). These observations indicated the deep layer of dartos fascia plays an important role in the retraction of buried penis. After the penoscrotal angle was reconstructed and the superfluous mucosal prepuce and fibrotic ring were resected, the complete superficial layer of dartos fascia and penile skin was sutured at the coronal sulcus (Figure 1).
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Figure 1: The penile lengths were measured at different stages of the operation. (a) The appearance of buried penis of a 17-year-old patient. (b) The penile length was 4.0 cm before operation. (c) After the penis was degloved to the penopubic junction along Buck’s fascia, the penile length was 6.6 cm. (d) When the deep layer of dartos fascia was sutured to the wet gauze near the coronal sulcus, the penile length was 5.1 cm. (e) After the superficial and deep layers were separated completely, the penile length was 5.7 cm. (f) The deep layer was cut along the base of the penis. (g) When the deep layer was removed completely, the penile length was 6.6 cm. (h) After the skin and superficial layer were sutured at the coronal sulcus, the penile length was 6.7 cm. Purple area is the wet gauze. DD: the deep layer of dartos fascia; SD: the superficial layer of dartos fascia; BK: Buck’s fascia.
Histological characteristics of superficial and deep layer of dartos fascia were different in buried penis
Table 1 shows the differences in the histological characteristics of the superficial and deep layers in 49 patients with buried penis. The thickness of the superficial layer of dartos fascia was greater than that of the deep layer. The collagen fibers presented a transverse arrangement in the deep layer and nonpolar arrangement in the superficial layer. The collagen fiber content in the deep layer was higher than that in the superficial layer (Figure 2a). The type I/III collagen ratio in the deep layer decreased compared with that in the superficial layer (Figure 2b). The elastic fibers were few in the superficial layer. However, they exhibited continuous transverse arrangement in parallel with collagen fibers or disordered and fragmented in the deep layer (Figure 2c). The expression of type III collagen and elastin was higher in the deep layer than those in the superficial layer (Figure 2d and 2e). The quantities of nerves and vessels in the superficial layer were higher than those in the deep layer. Some vessels with diameters of >100 μm were observed in the superficial layer, but not in the deep layer. Many smooth muscle fibers of dartos, including thick and slender ones, were observed in the superficial layer, whereas few slender dartos muscle fibers were present in the deep layer (Figure 2f). The cross-sections of dartos muscles in the superficial layer were higher than those in the deep layer. The collagen and elastic fibers were further observed by SEM. Many elastic fibers in the deep layer formed an interwoven network and wrapped up the collagen fibers. Few elastic fibers were in the superficial layer (Figure 2g). These results indicated that the histological structures of the superficial and deep layers of dartos fascia obviously differ (Figure 2g and 2h).
Table 1: The histological characteristics of superficial layer and deep layers of dartos fascia in 49 patients with buried penis
Figure 2: Histological characteristics of superficial and deep layers of dartos fascia. (a) Masson’s trichrome staining showed the collagen fibers which were stained blue, presented a nonpolar arrangement in superficial layer but showed a transverse arrangement in deep layer. (b) Sirius red staining showed type I collagen in superficial layer presented a brilliant yellow, orange or red colour, whereas type III collagen in deep layer showed green colour. (c) EVG staining showed that the elastic fibers which were stained black band, were few in superficial layer but exhibited continuous transverse arrangement in parallel with collagen fibers (green arrow) or disordered and fragmented in deep layer (blue arrow). IHC showed that the expressions of (d) collagen III and (e) elastin were higher in deep layer than those in superficial layer. (f) HE staining showed some nerves (green triangle), vessels (red triangle), thick dartos muscle fibers (blue triangle) or slender dartos muscle fibers (yellow triangle) in superficial layer, whereas a few slender dartos muscle fibers (yellow triangle) were observed in deep layer. (g) SEM showed there were small quantities of elastic fibers in superficial layer, while (h) the collagen fibers were wrapped up in a large number of elastic fibers in deep layer. EVG: Verhoeff’s Van Gieson; IHC: immunohistochemical examination; SEM: scanning electron microscopy; CF: collagen fibers; EF: elastic fibers; HE: haematoxylin–eosin.
Elastic fibers were disordered and fragmented in the deep layer of dartos fascia of buried penis
The morphology of elastic fibers in the deep layer of buried penis was compared with that of normal penis by EVG staining. Two types were observed. Type I was characterized by a continuous band organized in a parallel configuration with collagen fibers, which represented a normal morphology of elastic fibers; this type was observed in 12.2% (6/49) of samples of buried penis and 100.0% (20/20) in the samples of normal penis from cadavers. Type II was characterized by disordered and fragmented elastic fibers, which represented elastic fiber defects.10 This type was observed in 87.8% (43/49) samples of buried penis but not in normal penis (Figure 3). The tissue thickness, collagen fiber content, the ratio of type I/III collagen, and the cross-section of dartos muscle in the deep layer showed no difference between the buried and normal penises (Table 2). No histological characteristic differences were found in the superficial layer between the buried and normal penises from children/adults (Supplementary Table 1). These observations indicated that the abnormal elastic fibers in the deep layer of dartos fascia are involved in the pathology of buried penis.
Figure 3: The morphology of elastic fibres in the deep layer of dartos fascia was observed by EVG staining. (a) The continuous elastic fibres were organized in a parallel configuration with collagen fibres (green arrow) in normal penis from cadavers. (b) The elastic fibres were disordered and fragmented (blue arrow) in congenital concealed penis. EVG: Verhoeff’s Van Gieson.
Table 2: The histological characteristics of deep layer of dartos fascia in buried penis and normal penis from cadavers
DISCUSSION
The main finding of this study is that the abnormal deep layer of dartos fascia, which is characterized by disordered and fragmented elastic fibers, plays an important role in buried penis. Thus, the resection of the deep layer is helpful for avoiding recurrence. In this study, few slender dartos muscle fibers were observed in the deep layer of dartos fascia. Therefore, “the deep layer of dartos fascia” is part of the deep fascia.
The inelasticity of dartos fascia with poor skin anchoring at the base of the penis was observed in 93% of all cases of the concealed penis.11 In the authors’ previous study, dartos fascia had sublayers. A new technique was also described, that is, anatomical resection of the deep layer of dartos fascia for correcting buried penis. No recurrence cases were observed after the resection of the deep layer. The present study aimed to determine whether the abnormal deep layer plays an important role in buried penis. First, the effect of the deep layer in the retraction of buried penis was investigated. The penile shaft was found to be elongated by approximately 13.3% after resecting the deep layer compared with that before resection. These observations indicated that the deep layer of dartos fascia plays an important role in the retraction of buried penis. Next, the histological and pathological characteristics of the superficial and deep layers in patients with buried penis were investigated. As the fusions of dartos fascia and Buck’s fascia at 2:00–4:00 o’clock positions were less, samples from these positions were collected under the microscope for accurate sampling. The thickness of the superficial layer is approximately three times greater than that of the deep layer. Many vessels could be observed in the superficial layer, which should be carefully protected during surgery to avoid severe prepuce edema after surgery. The deep layer is abundant in type III collagen and elastic fibers. Type III collagen provides tensile strength and integrity for organs,12 whereas elastic fibers maintain tissue elasticity, thereby allowing them to stretch when needed and then return to their original state.13 The abundant type III collagen and elastic fibers determine the tensile strength and elasticity of the deep layer. The abnormal type III collagen or elastic fibers in the deep layer were speculated to be involved in the pathology of buried penis.
The histological and pathological characteristics of the deep layer between the buried and normal penises were compared. Disordered and fragmented elastic fibers defects were observed in 87.8% of buried penises, whereas no abnormal elastic fibers were observed in normal penises. No difference in type III collagen in deep layer was observed. Then, the superficial layers were investigated, and no difference was found in the histological characteristics between the buried and normal penises. The lack of elastic fibers leads to decreased tissue compliance and increased stiffness.14,15 The highly fragmented elastic fibers in the aortic wall decrease the resistance to stretch.10 The lack of elastin, fibulin-4, or lysyl oxidase causes absent or highly fragmented elastic fibers in the aortic wall.16 Thus, during the development of penis, the deep layer with decreased tissue compliance may pull the penile shaft toward the suprapubic fat and scrotum, causing the hidden state of penis. The mechanisms of elastic fiber defects in buried penis are worth further investigating.
Thus, the decreased elasticity of the deep layer of dartos fascia, which is characterized by disordered and fragmented elastic fibers, plays an important role in buried penis. The resection of the deep layer is helpful for avoiding recurrence. The superficial layer should be kept completely to prevent severe prepuce edema after surgery because it is abundant in vessels. In this study, the superficial and deep layers were separated under the microscope. The method is helpful to precisely identify and resect the deep layer and keep the superficial layer, especially in children with undeveloped penis. Mild prepuce edema was observed, which then disappeared within 2 weeks, similar to circumcision.
This study has some limitations. First, comparing histological data to cadavers is not an ideal comparison and it may not be directly representative to other live controls. Second, the samples were derived from a large Chinese university hospital. The characteristics of penile fascia may differ amongst different races. The difference of elastic fibers in the deep layer between the prepuberal and postpubertal patients was not studied because of small sample size. Multicenter randomized controlled studies are necessary to demonstrate the importance of resection of deep layer.
CONCLUSIONS
The abnormal deep layer of dartos fascia plays an important role in buried penis. The resection of the deep layer is helpful for avoiding recurrence.
AUTHOR CONTRIBUTIONS
JC carried out the study design, acquisition of data, analysis and interpretation of data, statistical analysis, and supervision. HYZ carried out the study design, acquisition of data, and drafting of the manuscript. GGZ carried out the acquisition of data, and analysis and interpretation of data. YTS carried out the acquisition of data, and critical revision of the manuscript. KBX carried out the analysis and interpretation of data. HFL carried out the analysis and interpretation of data, and critical revision of the manuscript. All authors read and approved the final manuscript.
COMPETING INTERESTS
All authors declare no competing interests.
Supplementary Information is linked to the online version of the paper on the Asian Journal of Andrology website.
Supplementary Figure 1
Diagram of penile fascia.
Supplementary Figure 2
When the penis is flaccid, the part of the superficial layer of dartos fascia folds with the foreskin, while the deep layer of dartos fascia is still tightly attached to Buck’s fascia; When the penis is erect, the superficial layer of dartos fascia is stretched and clings to the penis shaft. At this time, the two layers have the same dimensions.
Supplementary Figure 3
The tissue boundary between the superficial and deep layer under microscope. DD, the deep layer of dartos fascia; SD, the superficial layer of dartos fascia; Dotted line represent the tissue boundary.
Supplementary Table 1
The histological characteristics of superficial layer of dartos fascia in buried penis and normal penis from children/adults
The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (https://links.lww.com/AJOA/B132).
The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (https://links.lww.com/AJOA/B133).
The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (https://links.lww.com/AJOA/B134).
The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (https://links.lww.com/AJOA/B135).
ACKNOWLEDGMENTS
We would like to thank the Department of Pathology, The First Affiliated Hospital of Xi’an Medical University (Xi’an, China) for the help in histological analysis, and Department of Human Anatomy, Xi’an Jiaotong University Health Science Center (Xi’an, China) for the help in sample collection.
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