New insights into human female reproductive tract development

Abstract

We present a detailed review of the embryonic and fetal development of the human female reproductive tract utilizing specimens from the 5th through the 22nd gestational week. Hematoxylin and eosin (H&E) as well as immunohistochemical stains were used to study the development of the human uterine tube, endometrium, myometrium, uterine cervix and vagina. Our study revisits and updates the classical reports of Koff (1933) and Bulmer (1957) and presents new data on development of human vaginal epithelium. Koff proposed that the upper 4/5ths of the vagina is derived from Müllerian epithelium and the lower 1/5th derived from urogenital sinus epithelium, while Bulmer proposed that vaginal epithelium derives solely from urogenital sinus epithelium. These conclusions were based entirely upon H&E stained sections. A central player in human vaginal epithelial development is the solid vaginal plate, which arises from the uterovaginal canal (fused Müllerian ducts) cranially and squamous epithelium of urogenital sinus caudally. Since Müllerian and urogenital sinus epithelium cannot be unequivocally identified in H&E stained sections, we used immunostaining for PAX2 (reactive with Müllerian epithelium) and FOXA1 (reactive with urogenital sinus epithelium). By this technique, the PAX2/FOXA1 boundary was located at the extreme caudal aspect of the vaginal plate at 12 weeks. During the ensuing weeks, the PAX2/FOXA1 boundary progressively extended cranially such that by 21 weeks the entire vaginal epithelium was FOXA1-reactive and PAX2-negative. This observation supports Bulmer's proposal that human vaginal epithelium derives solely from urogenital sinus epithelium. Clearly, the development of the human vagina is far more complex than previously envisioned and appears to be distinctly different in many respects from mouse vaginal development.

Keywords: Cervix; Human Müllerian duct; Urogenital sinus; Uterovaginal canal; Uterus; Vagina.

Figure 1

Wholemount photos of developing human embryos from the Carnegie collection. Note increasing size and morphological complexity with developmental stage. Image from Dr. Brad Smith, University of Michigan (http://embryo.soad.umich.edu/carnStages/carnStages.html) NIH award N01-HD-6-3257 P/G F003637, with permission.

Figure 2

Wholemount photos of developing human fetal female internal genitalia staged by heel-toe measurements. Note (a) increase in size and morphological complexity with time, and (b) that landmarks distinguishing the uterine corpus, cervix and vagina are subtle/non-existent. Specimens photographed with transmitted light (9, 10, 11, 13 and 15 weeks) permit visualization of internal (epithelial) organization in regions not too thick. The 10-week specimen is shown at both low and high magnifications. Red arrowheads demarcate the epithelium defining the lumen of the uterine tube. Green arrowheads define the epithelium lining the uterus. Relative sizes of specimens are not exact, but increase with age.

Figure 3

Diagrammatic representation of the caudal growth of the Müllerian duct (MD) using the Wolffian duct (WD) as a “guide wire”. Note in (a) mesenchyme intervening between the Müllerian and Wolffian ducts, (b) contact of the basement membranes of Müllerian and Wolffian ducts, (c) direct contact of the epithelia of the Müllerian and Wolffian ducts.

Figure 4

Diagram of rudiments of human internal genitalia in the indifferent, bisexual stage (~54 days of gestation, Carnegie Stage 22). The Müllerian derivatives are red and Wolffian derivatives are purple. Note the changing anatomical relationships between the Müllerian and Wolffian ducts. Modified from Robboy et al with permission (Robboy et al., 2002).

Figure 5

Fusion of the Müllerian ducts to form the uterovaginal canal. (A & B) are sections of the pelvis of a stage 23 Carnegie embryo (56 days). (A) shows the urogenital ridges containing Wolffian (WD) and Müllerian ducts (MD) prior to their fusion in the midline. In (B) after midline fusion of the urogenital ridges, the paired Wolffian (WD) and Müllerian ducts (MD) are present, and the two Müllerian ducts are separated by mesenchyme. (C) is a fortuitous pelvic section of a stage 22 Carnegie embryo (54 days) demonstrating midline fusion of the right and left Müllerian ducts (MD). The midline epithelial septum is present representing the fused medial walls of the Müllerian ducts. (D) is a transverse section through the Müllerian (MD) and Wolffian ducts (WD) near their junction with the urogenital sinus. The right and left Müllerian have fused and are in direct contact without intervening basement membranes (Reprinted with permission of Robboy Associates LLC). (E) A transverse section through the uterovaginal canal with flanking Wolffian ducts (WD) of a 9-week fetus. Images (A–C) are from the Virtual Human Embryo Project (http://virtualhumanembryo.lsuhsc.edu) with permission.

Figure 6

A pelvic section of a stage 23 Carnegie embryo (56 days) showing contact of the fusing Müllerian ducts (MD) with the urogenital sinus (UGS). The point of contact of the Müllerian ducts with the urogenital sinus is called the Müllerian tubercle. Wolffian ducts (WD) also join the urogenital sinus just lateral to the Müllerian tubercle. From the Virtual Human Embryo Project (http://virtualhumanembryo.lsuhsc.edu) with permission.

Figure 7

Diagrams of human (A) and mouse (B) urogenital tracts emphasizing the marked difference in the degree of Müllerian duct fusion.

Figure 8

High magnification images of the uterovaginal canal of an 8-week fetus stained with H&E. (A & B) are taken at cranial (A) and caudal (B) segments of the uterovaginal canal. (C) is also form a caudal domain and illustrates the pseudostratified nature of epithelium throughout the entire cranial-caudal extent of the uterovaginal canal at this stage.

Figure 9

Histologic sections of developing human uterine tubes. (A) Transverse section of that portion of an 8-week Müllerian duct destined to form the uterine tube. At this early stage the uterine tube consists of an undifferentiated simple columnar epithelium surrounded by loose mesenchyme. By 14 weeks, epithelial morphology of the isthmus (B), ampulla (C) and infundibulum (D) is more complex, and the stroma surrounding the epithelium is densely cellular (B–D). By the 18^th week (E & F) the ampullary mesenchyme has differentiated into a condensed stromal layer (doubled-headed black arrows) associated with the epithelium and a surrounding circularly oriented layer (green double-headed arrows) that has differentiated into α-actin-reactive smooth muscle (F). (A–E) = H&E, (F) = α-actin.

Figure 10

Early Müllerian duct growth and fusion to form the midline uterovaginal canal. Length of the uterovaginal canal increases with developmental age. Adapted from Koff.

Figure 11

Wholemount images of human fetal female reproductive tracts (A–F) photographed with transmitted light, and H&E stained transverse sections of the uterovaginal canal (G–H). Dotted lines indicate the contours of the uterotubal junction and the uterine cavity. Green arrowheads indicate the epithelium of the uterine tubes. Note the changing shape of the uterine lumen. The cranial portion is laterally expanded (G) and narrow caudally (H).

Figure 12

Uterine/cervical glands (red arrowheads). Glands are rudimentary at 14 weeks (A), but can be seen by weeks 15 and 16 week (B&C). (D) The glands appear early on as shallow outpouchings of simple columnar epithelium. (E) By the 18^th week, the uterine/cervical glands are elongated and branched. The fundus (C) is gland free (large arrow) at all ages examined, but appear later in development.

Figure 13

α-Actin reactivity in human female reproductive tracts at (A) 9, (B) 11, (C) 12, and (D) 18 weeks. Faint α-actin reactivity first appears focally in mesenchyme of the 9-week uterovaginal canal, increasing in intensity by the 11 weeks. By 12 weeks α-actin reactivity is strong in the middle 2/4^ths of the developing reproductive tract, and by 18 weeks, is strongly expressed through the developing female reproductive tract.

Figure 14

Cervical development. (A–D) are sagittal sections of the female reproductive tract of a 16 week fetus (A=H&E stain, B=ISL1 immunostain, C–D=keratin 19 immunostain). Boundaries between the vagina, cervix and uterus are nebulous up to 18 weeks, when vaginal fornices become apparent (F). (B) ISL1 immunostaining is strong in vaginal stroma, absent in uterine stroma, with a sharp fall off in staining intensity at the mid-point of the uterovaginal canal (red arrow in B). Keratin 19 immunostaining (C–D) may also be indicative of vaginal-exocervical-endocervical boundaries. Cervical glands are prominent at 18 weeks of gestation (E).

Figure 15

Molecular mechanism of stratified squamous differentiation of vaginal epithelium. BMP4, activin A and FGF7/10 produced by vagina mesenchyme induce expression of p63 in Müllerian epithelium, which specifies and promotes vaginal epithelial differentiation.

Figure 16

PAX2 (A–E) and FOXA1 (F) immunostaining of human female fetal reproductive tracts at the ages indicated. PAX2 is expressed in the Müllerian duct and uterovaginal canal at 9 weeks (A–B), and in epithelium of the uterine tube, uterus and cervix (C–E). FOXA1 is expressed only in UGE derivatives (urethra and bladder) (F).

Figure 17

Sagittal sections of a 12-week human female fetal reproductive tract immunostained with PAX2 (A–B) and FOXA1 (C–D). PAX2-reactive epithelial cells extend to near the junction with the introitus/urethra (A–B). FOXA1-reactive epithelial cells extend only a short distance into the solid vaginal plate (C–D). Scale bar in A also refers to D. Scale bar in D also refers to C.

Figure 18

Sagittal sections of a 16-week human female fetal reproductive tract immunostained with PAX2 (A–B) and FOXA1 (C–D). FOXA1-reactive epithelial cells form all of the solid vaginal plate (C–D). PAX2-reactive epithelial cells constitute a stratified epithelium lining the lumen of the uterovaginal canal (A–B). Scale bar in A also refers to D. Scale bar in D also refers to C.

Figure 19

Sagittal sections of an 18-week human female fetal reproductive tract immunostained with PAX2 (A–B) and FOXA1 (C–D). PAX2-reactive epithelial cells line the lumen of the female reproductive tract from the uterine tube to the cranial aspect of the vagina (A–B). PAX2 staining is present, but weak in vaginal epithelium (B). FOXA1-reactive epithelial cells line the lower (caudal) vagina (C–D). Scale bar in A also refers to D. Scale bar in D also refers to C.

Figure 20

Sagittal sections of a 21-week female reproductive tract. Due to section orientation (A depicts the lower portion of the specimen (introitus to upper vagina), and (B) depicts the upper portion of the specimen (vagina, cervix and uterus) via H&E stain. The vaginal epithelium is many layers thick due to estrogenic stimulation (A–B). In (B) note the abrupt transition in epithelial differentiation at the vaginal/cervical border. PAX2 staining of the vaginal/cervical border (C) shows prominent PAX2 immunostaining of stratified but relatively thin cervical epithelium (indicative of Müllerian duct origin) and PAX2-negative vaginal epithelium (C). Epithelium of the uterine corpus (D) is strongly PAX2-reactive. FOXA1 immunostaining was seen uniformly throughout the entire vagina, and FOXA1 immunostaining abruptly stopped at the vaginal/cervical border (E–F) in mirror image to PAX2 immunostaining (C).

Figure 21

Transverse sections of the vaginal plate of 14- (A–F) and 16-week (G–H) female fetuses. Most of the vaginal plate (Vp) is PAX2-negative (A) and FOXA1-positive (B). In the cranial portion of the vaginal plate (C–D) a remnant of PAX2-positive Müllerian epithelium can be seen. Scattered at various cranial-caudal levels small islands of PAX2-positive islands are seen (E–H). Note “mirror images” of PAX2 and FOXA1 immunostaining. The urethra (Ur) is FOXA1-positive and PAX2-negative.