Neurovascular anatomy of the developing human fetal penis and clitoris

Abstract

The human penile and clitoral development begins from a morphologically indifferent genital tubercle. Under the influence of androgen, the genital tubercle forms the penis by forming a tubular urethra within the penile shaft. Without the effect of the androgen, the genital tubercle differentiates into the clitoris, and a lack of formation of the urethra within the clitoris is observed. Even though there are similarities during the development of the glans penis and glans clitoris, the complex canalization occurring along the penile shaft eventually leads to a morphological difference between the penis and clitoris. Based on the morphological differences, the main goal of this study was to define the vascular and neuronal anatomy of the developing penis and clitoris between 8 and 12 weeks of gestation using laser scanning confocal microscopy. Our results demonstrated there is a co-expression of CD31, which is an endothelial cell marker, and PGP9.5, which is a neuronal marker in the penis where the fusion is actively occurring at the ventral shaft. We also identified a unique anatomical structure for the first time, the clitoral ridge, which is a fetal structure running along the clitoral shaft in the vestibular groove. Contrary to previous anatomical findings which indicate that the neurovascular distribution in the developing penis and clitoris is similar, in this study, laser scanning confocal microscopy enabled us to demonstrate finer differences in the neurovascular anatomy between the penis and clitoris.

Keywords: anatomy; clitoris; fetal; neurovascular; penis.

FIGURE 1

Confocal fluorescent Imaging of the human fetal clitoris and penis from 8 to 12 weeks gestation immunostained with CD31 (green) a marker for blood vessels and PGP9.5 (magenta) and non‐specific marker for nerves. (a, b) 8 (c, d) 9 (e, f) 10 (g, h) 11, and (i, j) 12 weeks gestation, clitoris, and penis, respectively. Dorsal nerves are indicated are seen in c, e, g, f, and h and noted with magenta arrows in c, e, and h. Green arrowheads show vascular bundles (a, b). Asterisk shows the point of fusion of urethral folds (f). White arrowheads demonstrate the junction between the shaft and glans. (c–e) At 8 weeks gestation, the clitoris and penis neurovascular distribution look identical consistent with the indifferent stage. At 9 weeks gestation, the glans can be identified in both sexes (c, d). The open vestibular groove in the clitoris (c) and open urethral groove in the penis (d) is well defined. At 10 weeks gestation, the urethral folds are fusing in the proximal shaft of penis (f). Co‐expression of CD31 and PGP9.5 is noted at ventral part of the penile shaft at the site of the fusion process (asterisk, f). Oblique view of 11‐week clitoris reveals reticular pattern of circumflex vessels (g). At 11 weeks gestation, note the extensive vascular network of blood vessels based on CD31 staining in both sexes (g, h). In the penis, the dorsal nerve running along the shaft is well visualized (magenta arrow, h). Oblique view of the 12‐week clitoris and penis demonstrates a well‐vascularized prepuce that partially covers the glans (i, j). In lateral view of 12‐week penis, note the completely formed penile raphe (j).

FIGURE 2

Coronal section of 8‐week clitoris and penis at the indifferent stage immunostained with CD31(green). Green arrowheads indicate the vascular bundles running along the shaft and into the glans. Green arrows indicate numerous vascular branches that are radiating distally and laterally occupying the glans.

FIGURE 3

Confocal fluorescent immunostaining with CD31 (green) showing the vascular distribution in lateral, oblique, and ventral views of 9‐, 10‐, and 11‐week fetal human clitoris shown in a, aʹ, aʺ, b, b′, b″ and c, c′, c″, respectively. Note the complexity of circumferential vessels in the shaft from 9‐ to 11‐week clitoris (asterisks, a, b, b′ and c). Note the open vestibular groove seen at all time points illustrated (a″, b″, c′ dotted lines and c″). Note the dorsal clitoral prepuce starting to the cover the glans clitoris (c, c′, c″ white arrows).

FIGURE 4

Confocal fluorescent immunostaining with PGP9.5 (magenta) showing the vascular distribution in lateral, oblique and ventral views of 9‐,10‐, and 11‐week fetal human clitoris (a, a′, a″, b, b′, b″ and c, c′, c″, respectively). Note the dorsal nerves giving ventral branches (most notable to the glans) as they run through the clitoral shaft (a, a′, b, b′, c). Note the extensive neuronal distribution in the shaft at 11 weeks of fetal age which makes the vestibular groove evident.

FIGURE 5

Confocal fluorescent immunostaining with CD31(green) showing the vascular distribution of a 12‐week gestation human clitoris. Human fetal 12‐week clitoris fluorescent immunostained with CD 31. (a) Coronal view of 12‐week clitoris. (b–e) Transverse views indicated as horizontal lines in A. Note the clitoral ridge coursing through the clitoral shaft as a well‐vascularized structure in the midline of the vestibular groove (a). Note the clitoral ridge is evident at proximal shaft (e) and diminishes gradually (d) as it runs distally and completely disappears at the level of the glans (c).

FIGURE 6

Confocal fluorescent Imaging of the human fetal penis at 9 weeks gestation immunostained with CD31 (green) a marker for blood vessels and PGP9.5 (magenta) a non‐specific marker for nerves. Note the open urethral groove at this stage (a). Note the urethral plate at the junction of the penile shaft and glans (b). Note the urethral folds have not completely fused (c) at the proximal penile shaft.

FIGURE 7

Confocal fluorescent immunostaining with CD31 (green) showing the vascular distribution in lateral, oblique and ventral views of 9‐, 10‐, 11‐, and 12‐week human fetal penis is shown in a, a′, a″, b, b′, b″, c, c′, c″ and d, d′, d″, respectively. White arrowheads indicate the junction between the glans and shaft (a, b). Green arrows indicate ventrally radiating circumflex vessels (b, b′). Asterisks indicate the anastomosed vessels at the midline raphe (b′, b″). Green arrowheads indicate the penile shaft (b′, b″). White arrows indicate the finer vessels along the penile raphe radiating distally (c″). Note the extensive vascular network occupying the shaft and glans from 9 to 12 weeks fetal age. Note the prepuce starting to cover the glans at 12 weeks fetal age.

FIGURE 8

Confocal fluorescent immunostaining with PGP9.5 (magenta) showing the neuronal distribution in lateral, oblique, and ventral views of a 9‐ and 10‐week human fetal penis is shown in a, a′, a″ and b, b′, b″. Arrowheads indicate the branching dorsal nerves occupying the glans (b, b′). Note the dorsal nerves terminating in the glans via ventral (a, a′). Note the open urethral groove in the 9‐week fetal penis (dashed lines, a″) and 10‐week penis (b′, b″).

FIGURE 9

3D sagittal and oblique (a, b), coronal (c–f), and Transverse (g–j) view of a 10‐week fetal penis immunostained with CD31 and PGP9.5. Note the partially formed tubular urethra (e, g, and h). Note the circumferential vessels radiating ventrally anastomose at midline raphe (a, c, and h). Note the co‐expression of CD31 and PGP9.5 at the fusion point stained in yellow (b, c, and h). Note the co‐expression is most evident at the point of fusion (B & H).

FIGURE 10

Scanning Electron Microscopy (a) and confocal fluorescent immunostaining with CD31 (green) (b), PGP9.5 (magenta) (c), and merged channel (d) of a 10‐week human fetal penis. Asterisks indicate the fusion point. Note the similarity of vascular and neuronal distribution (b, c). Note the co‐expression of both markers where the urethral folds are actively fusing (d).

FIGURE 11

Confocal fluorescent immunostaining with CD31(green) showing the vascular distribution of a 11‐week gestation human penis (a, a′) and clitoris (b, b′). Note the difference in the pattern of distribution of circumflex veins between penile and clitoral shaft (a′, b′). Note the penile circumflex vessels running parallel to each other. In contrast, note clitoral circumflex vessels has more of a reticular pattern. Note the prepuce partially covering the dorsal part of the glans clitoris (b′) and penis (a′).