Published online Feb 23, 2024.
https://doi.org/10.4111/icu.20230034
Histological and morphological development of the prepuce from birth to prepubertal age
,1
Mustafa Kaplan Caliskan,1
Meryem Ilkay Karagul,2
Erdem Akbay,1
Banu Coskun Yilmaz,3
and Yuksel Cem Aygun4
Abstract
Purpose
To study the histological changes of the preputial tissue from birth to prepubertal age in order to define unnoticed morphological changes.
Materials and Methods
Prepuce samples were obtained from 79 healthy boys who underwent routine ritual circumcision. Specimens were divided into six groups according to the boys’ age: newborn, 0–1 year of age, 2–3 years of age, 4–5 years of age, 6–7 years of age, and 8–9 years of age. Histologic analysis of the specimens was performed by H&E, Masson’s trichrome, Verhoeff–Von Gieson, immunohistochemical staining.
Results
Microscopic examinations showed that average epithelial thickness increased after the neonatal period (p=0.001). When collagen fiber density was evaluated, no significant differences between groups were found (p=0.083). When the elastic fibers in the dermis were evaluated, it was determined that the number and thickness of elastic fibers increased with age. Immunohistochemical examinations showed that the number of peripheral nerves marked with S100 was lower in the neonatal period than at other ages (p=0.048). When the vessels marked with CD105 antibody were counted, there was no significant difference between the groups (p=0.078).
Conclusions
This is the first study to examine the age-related structure of connective tissue elements in the foreskin. Our results showed that the prepuce’s prepubertal maturation process is continuous, and the first 2 years of life are appropriate not only in relation to the physiological effects of age but also the optimum structural changes for wound healing, such as vessel diameter, epithelium thickness, peripheral nerve count.
Graphical Abstract
INTRODUCTION
The prepuce is useful for medical and surgical purposes, such as hypospadias surgery. It can also be used as a graft or flap in reconstructive surgery to treat syndactyly, childhood burns, eyelid burns, and hand injuries [1, 2]. Recently, it has been considered as a donor site for stem cells. The prepuce’s anatomy, vessels, embryological, and postnatal development must be defined to improve clinical outcomes. Prepuce has features that are distinct from other parts of the body [3]. Minimal data are available about the postnatal differentiation of the prepuce. Accepting the prepuce as a tissue with stable histology and morphology may be one of the causes of the heterogeneous outcomes of procedures [4]. The epithelium of the prepuce consists of stratified squamous cells that are keratinized, and the dermis of the prepuce has connective tissue, blood vessels, nerve fibers, and Meissner corpuscles within the papillae, and occasionally scattered sebaceous and sweat glands. Although elastic fibers are abundant and dense, the density of the collagen fibers is less than that in most other body parts. There are scattered smooth muscle bundles located in the lower dermal zone. Canning stated that the penis anatomy, and possibly physiology, changes with age [5]. Determining the optimum time of surgery will be a critical factor for improving outcomes. Therefore, age-dependent changes in the donor site and the prepuce should be defined.
We hypothesized that the prepuce, a distinctive part of the penis, may be prone to histological changes from birth to prepubertal age.
MATERIALS AND METHODS
Preputial tissue was obtained from 79 Turkish boys undergoing routine ritual circumcision at the Mersin University, Türkiye, between 2011 and 2019. Boys who met the inclusion criteria had no extragenital pathology, no pathology of the male external genitalia, and parental or guardian approval to participate in the study. Clinical materials were divided into six groups according to the boys’ age: newborn (8 specimens), 0–1 year of age (15 specimens), 2–3 years of age (14 specimens), 4–5 years of age (14 specimens), 6–7 years of age (14 specimens), and 8–9 years of age (14 specimens). The study was approved by Mersin University Ethical Committee (approval number: 2009/178), and the written informed consent was obtained from all patients.
1. Histologic measurements and assessments
For histologic analysis, preputial fragments were fixed in 10% neutral buffered formalin for 24 to 48 hours. After fixation, the routine light microscopic tissue processing protocol was performed, and tissues were embedded in paraffin. Section 5 µm thick were stained with H&E, Masson’s trichrome (for evaluation of collagen fibers), and Verhoeff–Van Gieson (for evaluation of elastic fibers) stains. The epithelial thickness was measured using LC micro image analysis software (Olympus BX50; Olympus Corporation) at 200× magnification in five different areas of each H&E-stained section. From each stained section with Masson’s trichrome stain to assess connective tissues (especially collagen fibers), color images of 640×480 pixel resolution were acquired with a light microscope (Olympus BX50; Olympus Corporation) and a digital camera (Olympus LC30; Olympus Corporation) using an imaging analysis program (ImageJ; National Institutes of Health). The collagen fiber density in the dermis was quantified at 200× magnification in five different areas of each section by RBG (red, blue, and green) color histogram. Elastic fibers were examined in each Verhoeff–Van Gieson-stained section.
2. Immunohistochemical staining protocol
Preputial fragments were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections 5 µm thick were deparaffinized, rehydrated, and treated with trypsin (pH 7.6, 37℃, 30 minutes) for antigen retrieval. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes. Then, the samples were incubated with blocking solution (ab93697, Mouse, and Rabbit Specific HRP Plus [ABC] Detection IHC Kit; Abcam) for 10 minutes. After blocking, sections were incubated overnight at 4℃ with a rabbit polyclonal primary antibody against CD105 (1:100, ab74462; Abcam) and rabbit polyclonal primary antibody against S100 (1:200, ab15520; Abcam). Negative control sections were incubated with a PBS-BSA (phosphate-buffered saline-bovine serum albumin) instead of the primary antibody. After that, they were incubated with a biotinylated goat anti-polyvalent secondary antibody (ab93697, Mouse, and Rabbit Specific HRP Plus [ABC] Detection IHC Kit; Abcam) for 10 minutes. They were incubated in streptavidin-peroxidase (ab93697, Mouse, and Rabbit Specific HRP Plus [ABC] Detection IHC Kit; Abcam) for 10 minutes. Diaminobenzidine (ACT500, DAB Chromogen/Substrate Kit; ScyTek Laboratories) was instilled. Counterstaining was carried out with hematoxylin.
The number of blood vessels and peripheral nerves was determined by labeling for CD105 and S100, respectively. Ten randomly chosen microscopic fields at 400× magnification were photographed. The number of blood vessels and peripheral nerves per field was counted.
3. Statistical analyses
Statistical analyses were performed using the IBM SPSS version 21.0 software package for Windows (IBM Corp.). Descriptive statistics for continuous variables were expressed and tabulated as mean±standard deviation. The one-way ANOVA test was used to compare parameters by age groups with the post hoc test. A p-value of <0.05 was considered statistically significant.
RESULTS
1. Histological findings
H&E and Masson’s trichrome staining of the prepuce tissue samples are shown in Figs. 1 and 2 (respectively).
Fig. 1
Epithelial skin thickness of the prepuce. Epithelium (E), dermis (DE), sebaceous gland (arrow). Newborn (A), 0–1 year old (B), 2–3 years old (C), 4–5 years old (D), 6–7 years old (E), 8–9 years old (F). H&E, ×200.
Fig. 2
Collagen fiber density in the dermis. Newborn (A), 0–1 year old (B), 2–3 years old (C), 4–5 years old (D), 6–7 years old (E), 8–9 years old (F). Masson’s trichrome, ×200. Collagen fibers are stained green.
Keratinized stratified squamous epithelium and underlying connective tissue had normal morphological features at the light microscope level. General histological structures of H&E-stained preputium tissues were similar among groups. We observed that the epithelial thickness in the newborn group was relatively thin compared with the other groups (Fig. 1). Mean epithelial thickness increased statistically significantly after the neonatal period (p=0.001, Tables 1, 2).
Table 1
Histological count results in prepuce samples collected at different ages
Table 2
p-values of the post hoc test for histological comparison among age groups
Masson’s trichrome staining showed collagen fiber density and distribution in tissues (Fig. 2). No statistically significant differences were found between the groups in light microscopic examinations (p=0.083, Tables 1, 2).
Verhoeff–Van Gieson dye was used to evaluate elastic fibers (Fig. 3). In this method, elastic fibers are dyed black (Verhoeff dye), whereas collagen fibers are dyed red (Van Gieson dye). In the light microscope examination, elastic fibers were observed to be few and thin in the newborn and 0- to 1-year-old age groups (Fig. 3A, B, respectively). The number and thickness of the elastic fibers increased in older age groups (2–3 years, 4–5 years, 6–7 years, and 8–9 years; Fig. 3C–F, respectively).
Fig. 3
Elastic fibers (arrows) in dermal tissues. Newborn (A), 0–1 year old (B), 2–3 years old (C), 4–5 years old (D), 6–7 years old (E), 8–9 years old (F). Verhoeff–Van Gieson, ×400. Elastic fibers are stained black.
2. Immunohistochemical evaluation
Peripheral nerves in preputial tissue were analyzed by labeling with S100 (Fig. 4). When we examined the tissues, we found that the number of peripheral nerves was lower in the neonatal period than in the other age groups. When we compared the number of peripheral nerves with the newborn group, we found that the number was statistically significantly higher in the other age groups (p=0.048, Fig. 4). There was no statistically significant difference between the age groups of 0–1, 2–3, 4–5, 6–7, and 8–9 years (Fig. 4, Tables 1, 2).
Fig. 4
Protein expression of S100 in the dermis. Newborn (A), 0–1 year old (B), 2–3 years old (C), 4–5 years old (D), 6–7 years old (E), 8–9 years old (F). Immunostaining for S100, ×400. Arrows, peripheral nerves; Asterisk, pacinian corpuscle.
The number of vessels in the preputial tissue was analyzed by labeling with CD105 (Fig. 5). There were no statistically significant differences in vessel count between the age groups (one-way ANOVA, p=0.078, Fig. 5, Tables 1, 2). However, when the data were analyzed according to vessel diameter, we found differences between the groups (Tables 1, 2). The median vessel diameter was 7.7989 µm. Vessel size data were divided into two subgroups of less than 7.80 µm and greater than or equal to 7.80 µm. In the subgroup with vessel diameter less than 7.80 µm, mean vessel diameter did not differ significantly by age (Table 3). In the subgroup with vessel diameter less than 7.80 µm, mean vessel diameter did not differ significantly by age (one-way ANOVA, p=0.779, Table 3). In the subgroup with vessel diameter greater than or equal to 7.80 µm, mean vessel diameter was statistically different between the age groups (one-way ANOVA, p=0.001, Table 3). Vessel diameter was higher in the newborn and 0- to 1-year-old age groups than in the other groups (post hoc, p=0.001 for all, Table 3).
Fig. 5
Protein expression of CD105 in the dermis. Newborn (A), 0–1 year old (B), 2–3 years old (C), 4–5 years old (D), 6–7 years old (E), 8–9 years old (F). Immunostaining for CD105, ×400. Arrows, blood vessels.
Table 3
Mean vessel diameter by age in subgroups with vessel diameter less than 7.80 µm or greater than or equal to 7.80 µm
DISCUSSION
The prepuce has distinctive features such as a papillary dermis rich in nerves, dense capillary networks, dense and abundant elastic fibers, and low counts of sweat glands, making it the main donor site for many medical and surgical procedures [3]. Its clinical use has expanded for procedures such as urethral augmentation, substation urethroplasty, skin resurfacing, ventral lengthening for correction of ventral curvature, and creation of a waterproofing layer [3]. The optimum time of surgery is a critical factor for improving outcomes. Therefore, age-dependent changes in the donor site and the prepuce should be defined.
In the present study, we defined age-related histological and morphological changes of the foreskin. In this aspect, we agree with Canning [5], who when discussing Dossanova’s study [4], which reported histological and morphological changes of the prepuce in different age groups, stated that “studies like this remind us that penile anatomy and likely physiology changes with age” and we need a greater understanding of “wound healing and age as they apply to hypospadias.” For this reason, unlike in previous studies, we carried out our examination of histochemical staining of prepuce connective tissue elements (collagen and elastic fiber density) from newborn to prepubertal age by using image analysis software. In addition, blood vessels in the foreskin were immunohistochemically labeled with CD105, and vascularization was evaluated by counting the newly formed blood vessels. The sensitivity of the prepuce was evaluated by immunohistochemically labeling the peripheral nerves with S100.
Wound healing is a complex process in which different components take a role. Without re-epithelialization, wound healing is not possible [5, 6, 7, 8, 9]. The extracellular matrix was reported as a vital factor in wound healing [3]. The major components of the supportive tissue are type I and type III collagens. While type I collagen count, which is necessary for the wound healing process, decreases with aging, type III collagen increases. The increase in type III collagen has a negative impact on wound healing. The other component of the extracellular matrix is fibroelastic fibers, which help wound healing by contracting wound tissue. In this study, we discussed the components of the extracellular matrix including collagen and fibroelastic fibers. Although we observed no increase in the number of collagen fibers, we did observe an increase in the number and thickness of fibroelastic fibers, except in the newborn and 0- to 1-year-old age groups. This finding suggests that fibroelastic fibers may contribute to wound healing after the newborn and 0- to 1-year-old periods.
The flap survival rate mainly depends on the blood supply [2, 3, 10, 11, 12, 13, 14], and this explanation applies to all flaps. Not only is a well-vascularized flap needed for tube reconstruction, but a second layer of well-vascularized tissue is also required for wound healing after hypospadias surgery. Dossanova et al. [4] showed that arterioles with lumen diameters of 0.15 mm or more were more common in the under 3 age group than in the groups aged 3–5 years and 5–7 years. In addition, it has been reported that the number of vessels increases with age [4]. In the current study, when vessels in specimens were analyzed, we found that vessel counts increased slightly after the newborn period and then remained stable. However, the more critical factor, the number of large vessels (≥7.80 µm), was higher in the age groups of newborn, 0–1 year, and 2–3 years. As defined by Tonnesen et al. [15], larger vessels naturally have more endothelial cells. During wound healing, larger vessels and more endothelial cells may also lead to an acceleration of the healing process. Additionally, the presence of larger diameter blood vessels may be an indication of activated extracellular matrix elements that are also vital for wound healing [15].
Nerve fibers generate electrical impulses, which are needed in the complex processes of the inflammatory response, contraction, angiogenesis, and preventing inflammation during the wound healing process. Barker et al. [11] reviewed the impact of denervation and reported that it prevents wound flap healing [11, 16, 17]. In our study groups, peripheral nerve count was increased after the newborn period and remained stable afterward. Ages of 0–1 year and 2–3 years may be more appropriate for using the prepuce as a graft compared to other age periods because of higher nerve fiber density and higher large vessel counts. In the newborn period, although a higher large vessel count was observed, epithelium thickness and nerve fiber density were low compared with the other age groups.
Our goal was to define the age-related changes of the prepuce. In our country, religious circumcision is routinely performed, and this is an advantage for studying the morphology of the prepuce compared with other countries in which circumcision is rare. The maturation process of the prepuce is important to understand not only to help with appropriate timing of penile surgeries but also to better understand a neglected tissue [4, 5]. In their study, Dossanova et al. [4] also aimed to determine the role of age in the prepuce’s histological and morphological characteristics, but the age group of the patients they studied and the comparative indexes differ from ours, making the results hard to compare. Further studies may reveal the histological changes of the preputium with age.
They reported increases in arteriole diameters, cellular components (fibroblasts, fibrocytes, histiocytes, and lymphocytes) of nerve fibers, and bundles of collagen fibers at 5–7 years of age [4]. Their study’s limitation was dividing the samples into age groups of less than 3 years, 3–5 years, and 5–7 years. In our study, we grouped patients as newborns, who were under the mother’s hormonal influence; 0–1 year of age, in which mini-puberty occurs; 2–3 years of age; 4–5 years of age; 6–7 years of age; and 8–9 years of age, which might be under early pubertal hormonal changes. We also used reported serum androgen levels and penile growth charts to group specimens. In our opinion, evaluating newborns, 0 to 1 year old, and 2 to 3 years old as a single group may lead to confusing results.
The penis is a target organ for androgens, and thus the skin of the penis may also be a target for androgens. In 1987, the presence of androgenic receptors in the prepuce was also reported by Roehrborn et al. [6]. A peak in the serum testosterone level during the first year of life is followed by a return to basal levels and another significant rise at puberty, followed by adult hormone levels. This peak at first age and its phenotypic result is called “mini-puberty.” The structural changes of the prepuce were concordant with changes in serum androgen levels reported in previous studies [18, 19, 20]. Our results provide significant clues about the concordance of androgen levels and structural changes of the prepuce. However, we did not analyze the serum androgen levels of the patients. One of the limitations of the study is the absence of a pubertal age group, which could provide important results about the impact of androgenic activity on preputial tissue morphology. However, early ages are being accepted as the optimum age for penile surgery. Therefore, in our study, we focused on prepubertal ages. In our opinion, the other limitation of our study is the lack of experimental or clinical results on whether these age-related changes affect wound healing because of the complex interplay during wound healing of the studied indexes.
CONCLUSIONS
We observed changes in the morphology and histology of the prepuce after birth. The data provided in this study may be helpful for determining optimal surgical times.
CONFLICTS OF INTEREST:The authors have nothing to disclose.
FUNDING:This work was supported by Mersin University Department of Scientific Research Projects (project number: BAP-TF CTB (MKÇ) 2013-3).
AUTHORS’ CONTRIBUTIONS:
Research conception and design: Erim Erdem, Mustafa Kaplan Caliskan, and Erdem Akbay.
Data acquisition: Erim Erdem, Mustafa Kaplan Caliskan, Meryem Ilkay Karagul, and Banu Coskun Yilmaz.
Statistical analysis: Erim Erdem, Mustafa Kaplan Caliskan, Erdem Akbay, and Yuksel Cem Aygun.
Data analysis and interpretation: Erim Erdem, Meryem Ilkay Karagul, Banu Coskun Yilmaz, and Yuksel Cem Aygun.
Drafting of the manuscript: Erim Erdem, Meryem Ilkay Karagul, and Erdem Akbay.
Critical revision of the manuscript: Erim Erdem, Meryem Ilkay Karagul, Banu Coskun Yilmaz, and Yuksel Cem Aygun.
Obtaining funding: Erim Erdem and Mustafa Kaplan Caliskan.
Supervision: Erim Erdem, Meryem Ilkay Karagul, and Banu Coskun Yilmaz.
Approval of the final manuscript: all authors.
References
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Canning DA. Re: histological and morphological characteristics of the prepuce of penis skin structure in different age groups. J Urol 2019;202:1086
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Zirkin BR, Papadopoulos V, Hardy MP. Leydig cell development and function. In: Lipshultz LI, Howards SS, Niederberger CS, editors. Infertility in the male. 4th ed. Cambridge University Press; 2009. pp. 29-47.
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Lam YT, Lecce L, Bursill CA, Ng MKC. The role of sex steroids in angiogenesis. In: Mehta J, Mathur P, Dhalla N, editors. Biochemical basis and therapeutic implications of angiogenesis. 2nd ed. Springer; 2017. pp. 445-471.
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Lecce L, Lam YT, Ng MKC. Role of sex steroids in angiogenesis. In: Mehta J, Dhalla N, editors. Biochemical basis and therapeutic implications of angiogenesis. Springer; 2013. pp. 159-180.
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Funding Information
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Mersin Üniversitesi
BAP-TF CTB (MKÇ) 2013-3
