The development of the external genitals in female human embryos and foetuses. Part 1: Perineal thick skin, clitoris and labia

2.1. Embryos and foetuses

This study used historical collections of anonymous human embryos and foetuses, which were compiled >50 years ago. These collections are exempt from ethical approval according to the Dutch regulations for the proper use of human tissue for (bio‐)medical research purposes. The study used digitized images of serial sections of specimens from the collections of human embryos and foetuses of the Departments of Anatomy and Embryology, Leiden University Medical Centre (LUMC), Leiden; Amsterdam University Medical Centers (AUMC), Amsterdam; Radboud Medical Center (RadboudMC), Nijmegen, The Netherlands; the Georg‐August‐Universität, Göttingen (Blechschmidt Collection); and the Ruhr Universität, Bochum, Germany (Hinrichsen collection) (Table 1). In addition, embryos of the Carnegie Collection in Washington DC, which are freely accessible on the internet, were included. The criteria of O'Rahilly and Muller (2010) were used to determine Carnegie Stages (CS) and ages of development of the embryos. The age of the foetuses (>8 weeks of development, Figure 1) was based on the comparison of their crown‐rump length (CRL) with that in reference studies (Table 2). The standard deviation for autopsies was ~1 week in the 2nd trimester and ~2 weeks in the last trimester (Bartosch et al., 2019; Hansen et al., 2003; Maroun & Graem, 2005; Szpinda et al., 2015), while that for ultrasound measurements was ~0.5 weeks in the 2nd half of 1st trimester and ~1 week in the 2nd trimester (Papageorghiou et al., 2014; Robinson & Fleming, 1975). Note that ultrasound measurements of CRLs were carried out only during the first 120 days of development.

We have prepared detailed reconstructions of the development of the external genitals and perineal thick skin in 13 embryos and foetuses between CS18 (~6 weeks of development) and ~ 20 weeks of development (Table 2). Specimens from the foetal period were relatively scarce. If appropriate, sections of male foetuses were also studied (and identified as such). Furthermore, we studied the serial sections of 13 female embryos between 8 and 13 weeks of development (35–105 mm CRL) of the Domènech‐Mateu collection at the Autonomous University of Barcelona (courtesy Prof. Rosa Mirapeix).

2.2. Image acquisition, 3D‐reconstruction and visualization

Serial sections of human embryos from the AMC, LUMC and RadboudMC were digitized with an Olympus BX51 or BX61 microscope and the DOTSLIDE program (Olympus, Zoeterwoude, The Netherlands). The Göttingen, Barcelona and Bochum embryos were scanned with a Zeiss Axioscan Z1 (Carl Zeiss Microscopy, Jena, Germany). Digital images of serial sections from the Carnegie collection were obtained via the Virtual Human Embryo Project (http://virtualhumanembryo.lsuhsc.edu). All images were converted into greyscale “JPEG” format and loaded into the Amira reconstruction program (version 2020.2; base package; FEI Visualization Sciences Group Europe, Merignac Cedex, France). The greyscale images were aligned automatically with the least‐squares alignment mode and then manually adjusted for the correct curvature of the embryonic body axis with the help of photographs and magnetic resonance images of human embryos of the same age (Pooh et al., 2011). Structures of interest were segmented manually and reconstructed three‐dimensionally with the Amira program. Small deformations of individual sections due to the histological processing and stepwise stacking of sections can confer a distracting noise on the 3D reconstructions. Therefore, polygon meshes from all reconstructed materials were exported via “vrml export” from Amira to Cinema4D (MAXON Computer GmbH, Friedrichsdorf, Germany) and remodelled using the Amira model as template (Figure S1). The accuracy of the remodelling process was safeguarded by the simultaneous visualization of the original output from Amira in Cinema4D and the remodelled Cinema model. The Cinema4D models were transferred via “wrl export” to Adobe Acrobat version 9 (https://www.adobe.com) to generate interactive 3D Portable Device Format (PDF) files, which are an easily accessible format for 3D visualization.

All epithelia were segmented following their basement membrane. Muscles become first detectable as dense mesenchymal masses and are identifiable initially only by their location. When muscle fibres develop, segmentation of different muscles can be challenging as long as perimysia are not yet well‐developed. The perineal skin of embryos and foetuses is referred to as the skin surrounding the pelvic outlets of the urogenital and intestinal tracts. The umbilical ring is located at the junction of the belly with the umbilical cord. Please note that the infraumbilical ventral abdominal wall of the embryo only begins to form in the 7‐week‐old embryo (Mekonen et al., 2015).

In addition to the Figures, the evidence for our descriptions can be inspected in the corresponding interactive 3D‐PDFs (Figures [Link], [Link]). The reader is, therefore, encouraged to read the text and inspect the corresponding interactive PDFs simultaneously. This is because their rotational options (“live” images) allow a much better understanding of the complex local topography than do “still” images and text. The 3D‐PDFs can also be used to identify or verify the identity of a structure in a Figure: after opening and positioning the reconstruction similar to the view in the Figure, marking or unmarking a structure in the model tree will link the image to a name.

2.3. Measurements

Volume measurements were performed with the statistical analysis mode in Amira. The length of structures was measured with the spline option in Cinema4D.

2.4. Orientation

The body axis is defined as the axis passing through the thoracal and lumbar vertebral column and is assumed to be perpendicular to the transverse plane. To describe (changes in) the position of a structure relative to another structure or an observer, we use the terms cranial or caudal (which are equivalent to superior or inferior) for differences in position along the body axis and ventral or dorsal (equivalent to anterior or posterior) and left or right to indicate (changes in) position perpendicular to the body axis. To obtain an inside view of the pelvic floor, oblique “inside‐out” views were often necessary.

3.1. Definition of the perineal region and its contents

In the present study, we investigated the developmental changes in the skin of the perineal region and the underlying external genitals of female human embryos and foetuses. The perineal region is delimited by the pelvic outlet and pelvic floor cranially, and the outlets of the urogenital and intestinal tracts and their surrounding skin caudally. The components in the perineal space of embryos and foetuses include the skin, the phallus (clitoris) with its glans and hood, the cavernous and spongy bodies, and the associated cutaneous muscles and nerves. These components all lie caudal to the pubococcygeal line (interrupted horizontal line, Figure 2), which reflects the position of the pelvic floor in adult women (Betschart et al., 2013). Striking features are that the dorsally situated structures (sacrum and rectum) maintain their orientation relative to the pubococcygeal line between 8 and 15 weeks, whereas the plane through the bladder and allantois relative to the pubococcygeal line gradually increases from −45° at 8 weeks to +45° at 11 weeks of development and later (Figure 2). The 15‐week foetus is the most advanced stage of which we could reconstruct the sacral and coccygeal vertebrae since the vertebral column is absent in the tissue block of the 20‐week foetus. The bladder, therefore, aligns its position with that of the rectum and sacrum between 8 and 11 weeks of development. This change in orientation occurs concurrently with the development of the infra‐umbilical ventral abdominal body wall starting at 7 weeks (Ji et al., 2020; Mekonen et al., 2015) and the return of the herniated midgut into the peritoneal cavity at 9.5 weeks (Kakeya et al., 2022; Soffers et al., 2015). Of note, the urethra and adjacent pubic bone did not change their position during this period. Another striking but temporary feature in the period shown in Figure 2 is the almost perpendicular position of the 2nd and following coccygeal vertebrae relative to the sacral vertebrae.

3.2. Appearance and fate of the “thick” perineal skin

At 6 weeks of development (CS18), the skin in the perineal region increases in thickness (Kruepunga et al., 2018). At this stage, the skin consists of basal, intermediate and periderm layers (Figure S2). The layer of thick skin in the perineal area differs from the skin outside this region in that its intermediate layer is at least 2‐fold thicker (Figure S2b,c). The thick perineal skin in a 6‐weeks‐old embryo extends between the two legs laterally and between the umbilical ring and the tip of the coccyx craniocaudally (orange‐coloured area in Figure 3a). Although the thick‐skin region continues to increase between 6 and 15 weeks of development (Figure 3g), the fraction of the perineal surface covered with thick skin declines due to a faster growth of surrounding “regular” skin (Figure 3a–f). From 9 weeks onward, areas of thick skin gradually transform to standard skin. This “thinning” of the thick epithelial layer of the skin is first seen at the cranial base of the clitoris and extends distally along the entire dorsal side of the clitoris and outer labial swellings (Figure 3c–f). At 11 weeks of development, the thick skin between the anus and urogenital outlet narrows, which results in separate peri‐urogenital and peri‐anal areas of thick skin at 15 weeks (Figure 3f). At 20 weeks of development, thick skin remains present in the vaginal vestibule and the anal canal only. The epithelial double layer between the prepuce and the glans cannot be distinguished from the thick skin at the tip of the clitoris.

3.3. Appearance and size of the labia majora

At 7 weeks of development (CS20), the swellings of the labia majora start to emerge on both sides of the clitoris (LM in Figure 4a,b). At 10 weeks, the labial swellings have produced a deep furrow between them and the clitoris. While “standard” skin covers the protruding parts of the labial swellings, the skin in the furrows remains “thick” (Figure 4c,e, and black arrowheads in panels (d) and (f)). At 20 weeks, the thick skin has also disappeared from this furrow, so that the labia majora are covered with “standard” skin only.

3.4. Development of the cavernous bodies, glans and prepuce of the clitoris

The mesenchymal condensation of the cavernous bodies and the glans of the clitoris could first be recognized at ~7 weeks of development (CS20; Figure 5a,e). Initially, they are part of a larger mesenchymal mass in the region of the pelvic floor, from which local bones and striated muscles also form. However, the contour of the cavernous bodies and glans can already be recognized in the upper part of this mesenchyme (Figure 5a). At 9 weeks, the glans has become a separate structure at the distal end of the clitoris (Figure 5b, b1). Although not far apart, the dense part of the glans and the cavernous bodies remain separate structures throughout subsequent development (Figure 5f–h). Between 15 and 20 weeks of development, the dense mesenchyme of the glans persists as an outer, cortical layer only (white arrowhead in Figure 5h1), while the inner, medullar part transforms into a looser mesenchyme (black star, Figure 5h1). The cavernous bodies retain a common part near the glans but separate more proximally in the shaft of the clitoris (the crura (C) in Figure S3a–c), with only loose connective tissue connecting them (arrowheads in Figure S3d–f).

3.5. Development of the labia minora

The labia minora (“lesser lips”) are skin folds between both labia majora that surround the urogenital exit. The labia minora become identifiable at 7 weeks of development as a skinfold covered with thick skin that surrounds the urogenital outlet (asterisk in Figure 4a). They are ventrolaterally continuous with the prepuce and ventromedially with the apex of the cavernous body. In the 8th week, the labia minora become ridges around the exit of the vaginal vestibule that remain covered with thick epithelium until at least 15 weeks of development (Figure 8) and are filled with dense mesenchyme (asterisks and dark green‐coded structure in Figure 9b, b1 and e). The transition of a dense to a loose mesenchymal organization demarcates the boundary between the labia minora and prepuce (Figure 9f; Figure S4). The urethral plate occupies the ventral midline of the vestibule and the caudal boundary of the glans (Figure S4). The labia minora (asterisks in Figure 7f) and the cavernous bodies (CB) are connected by the so‐called frenulum of the clitoris (“Fr” in Figure 7f), which becomes identifiable at ~12 weeks. After the disappearance of thick skin from the area between the urogenital sinus and anus (Figure 8a), the dorsoventral lengths of the labia majora and the vaginal vestibule continue to increase, but that of the labia minora does not (Figures 8, 10). Consequently, the labia minora border cover only the ventral ~50% of the vaginal vestibule at 14 weeks and the ventral ~30% at 20 weeks (Figures 8a, 10a). This reduction suggests that the craniocaudal growth of the vestibule and the labia majora exceeds that of the labia minora. Only the internal rim of the labia minora remains covered with thick stratified perineal skin (pink), whereas stratified squamous endoderm (brown) now covers the rest of the medial side (Figure 10a,c). At 20 weeks, both the standard and thick‐skin areas have developed a parakeratinized surface layer (Figure 10b,c).

4.1. Programming window of the sexual phenotype

The external genitals and anus acquire their typically female appearance during the “masculinization programming period” between ~8 and ~14 weeks of development, although our measurements show that the phenotypic sex differences continue to become more pronounced until at least ~20 weeks. The masculinization programming period is associated with a rising production of androgenic steroid hormones in male foetuses (Connan‐Perrot et al., 2021; Siiteri & Wilson, 1974; Wilson et al., 1981). The anogenital distance is a lifelong readout of this sex‐dependent transient exposure of male and female foetuses to androgenic steroid hormones (Fowler et al., 2016; Sharpe, 2020). The increase in the anogenital distance is more rapid and more pronounced in male than in female foetuses (Politzer, 1932), but the perineal length also increases in female foetuses, growing ~2‐fold faster between 8 and 11 weeks than between 11 and 20 weeks (Figure 8 in (Hülsman et al., 2025)). Ultrasound measurements in live foetuses were only carried out in the latter period thus far (Fowler et al., 2016). The typically female structures in the perineal region (labia majora and minora, clitoris, urethral plate, vaginal vestibule and perineum) all start to differentiate and assume a female phenotype between 8 and ~15 weeks of development. The acquisition of the typically female phenotype, therefore, coincides with the masculinization programming period but does not seem to merely represent the default phenotype. Hence, it would be better to describe this period as the “sexual‐phenotype programming period”, since masculinization may only represent a quantitative aspect of this period.

4.2. Partial “descent” of pelvic organs into the perineal space

The developing vaginal vestibule and anal canal descend substantially below the pubococcygeal line, which identifies the level of the pelvic floor (Betschart et al., 2013). This change in position of pelvic organs starts at ~9 weeks of development and continues up to at least 15 weeks. The “descent” of the pelvic organs was previously reported to continue until birth (Tagaki, 1927) and probably is an important determinant of the typical funnel‐shaped appearance of the pelvic floor.

4.3. Thick perineal skin corresponds with the sex hormone‐sensitive area

A striking feature of the 6th week of development is the appearance of a pericloacal area with a selective thickening of the intermediate layer of the skin. In mammalian, including human embryos, a transient midline ridge of epithelial cells, the “Schwanzleiste”, arises caudally from the cloaca at 4 weeks of development, extends towards the tail tip and then regresses in reverse order at 4.5 weeks (Landolt, 1959). The same structure was identified as the “caudal ectodermal ridge” and shown to be a transient component of the ring of thick epithelium that encircles the lateral surface of the embryo (O'Rahilly & Müller, 1985). The early, midline appearance, the lack of cell layers in this thickened skin, and its brief presence seem to rule out a connection with the perineal thick skin described in the present study. As far as we are aware, therefore, the characteristic thickening of the intermediate layer of the ectodermal epithelium in the pericloacal skin was not reported earlier. We first noted this thick skin between both legs laterally, the umbilical cord cranially, and the base of the coccyx caudally at 6 weeks of development (Kruepunga et al., 2018). The thick skin area becomes restricted gradually to the urogenital and anal exits, where it persists in the superficial part of the vaginal vestibule and in the anal canal. The thick skin area around the urogenital exit roughly corresponds with the dihydrotestosterone‐sensitive pubic area (Imperato‐McGinley et al., 1974; Overland et al., 2016; Zouboulis et al., 2007), but its perianal counterpart has thus far not been associated with functional properties. Johnson (1914) mentioned the thick skin as the epithelium covering the anal canal but did not mention its extension over the perianal or pericloacal area. Although the function of thick skin in human embryos is unknown, in general, non‐cornified thick skin areas in foetal rodents seem to be more permeable to chemicals than thinner, cornified skin epithelia (Hardman et al., 1998).

It is tempting to compare the thick perineal skin with the anogenital swellings of female old‐world monkeys and some primates (Nunn, 1999). These swellings, which change in size during the ovarian cycle, have evolved independently in three separate primate lineages at the same anatomical location. Unfortunately, little is known about their development, except that they form at the location of the labia majora when Macaque foetuses pass through the sexual programming period (Dang, 1978). The labia majora are a genital skin region that is exquisitely sensitive to sex hormones (Imperato‐McGinley et al., 1974). This location and the appearance of the swellings in three independent primate lineages (Nunn, 1999) suggest, therefore, a strong predisposition. Unfortunately, this feature has not yet been studied developmentally.

4.4. Glans and cavernous bodies become separate structures

The shape of the connective tissue core of the emerging clitoris resembles that of a paper airplane (see Figure 5a,e). The initially single mesenchymal mass soon divides into a smaller ventral part, the clitoral glans, and a larger dorsal part, the cavernous bodies. The wings will transform, starting at 8 weeks of development, into the crura of the cavernous bodies, while the frenulum of the clitoris will form the connection between the apical, non‐divided part of the cavernous bodies and the base of the prepuce of the glans. The clitoral frenulum becomes identifiable at ~12 weeks. The structure was already described almost 90 years ago as bilateral (Popper, 1937) but is nowadays usually described as a midline structure (Clerico et al., 2017; Pauls, 2015). Although the glans of the clitoris and the cavernous bodies have a common mesenchymal origin, they become separate structures early in their development. Our findings in this respect differ from the schematic drawings presented by Puppo (e.g. Figure 9 in (Puppo & Puppo, 2015)).

4.5. Angular growth of the clitoris

Both the clitoris and the labia majora initially grow in ventral direction, but especially the declining growth of the clitoris slows down its ventral extension to reach an apparent dorsoventral length of ~2.5 mm at ~13 weeks (Figure 6) or 3.1 mm at 14 weeks (Ozguner et al., 2017), compared to 6.5–6.8 mm at birth (Adekoya et al., 2021; Ozguner et al., 2017). The decline in the ventral extension of the clitoris results from the formation of a caudal bend (“angle”) in its shaft (compare Figure 6a–c). This bend does not develop in the foetal penis, so that its presence or absence, known as the “sagittal sign”, can be used to distinguish males and females externally after ~10 weeks (Emerson et al., 1989; Mazza et al., 2001). The apparent decline in dorsoventral length of the clitoris between 15 and 20 weeks of development reveals that this angle continues to become more acute. In rare cases, the bending and possibly also the slowdown in clitoral growth is delayed by 8–12 weeks, so that the clitoris continues to protrude ventrally from between the labia majora until 22–26 weeks of development (“clitoromegaly”; Zimmer et al., 2012). In the spotted hyena, the entire ensemble of external genitals differentiates permanently in a near masculine way with a straight clitoris, whereas the internal genitals remain female in phenotype (Cunha et al., 2005). The mechanism inducing the caudal bending of the clitoris remains unknown but most likely resides in the bend between the shaft and the crura of the cavernous bodies. Irrespective of the mechanism, the outcome is that the labia majora largely covers the clitoris.

4.6. Clitoral hood is a skin fold that edges over the glans

The development of the prepuce or clitoral hood was described accurately already in 1937 (Popper, 1937). The prepuce begins to form at ~10 weeks of development and extends the next 4 weeks as a fold of tissue over the glans, starting dorsally. The inner surfaces of the prepuce and glans adhere mutually at first, the adhesion resembling a thick epithelial lamella. Mechanistically, the forming of the female and the male prepuce (Hunter, 1935; Liu et al., 2018) appear to be very similar.

4.7. Labia minora are initially short

The labia minora become identifiable at 7 weeks of development as bilateral ridges covered with the thick ectodermal epithelium that border the urogenital exit ventrolaterally. This thick epithelium is clearly visible in, e.g. Plate 2 of (Glenister, 1954) and Figure 3 of (Bulmer, 1959), but was not commented upon. The labia minora are continuous ventrolaterally with the clitoral hood and ventromedially with the frenulum of the clitoris and the apex of the cavernous body. The size of the labia minora and their ventral connections (frenulum and clitoral hood) varies substantially (Clerico et al., 2017). The labia minora cover only the ventral ~50% of the vaginal vestibule at 14 weeks and the ventral ~30% at 20 weeks (Figure 10), which suggests that the dorsoventral growth of the vestibule exceeds that of the labia minora. This observation seems in line with old illustrations (e.g. 11–18 week‐old foetuses in (Mijsberg, 1924)), and a recent conclusion that the labia minora are short in young girls (Wroblewska‐Seniuk et al., 2021).