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Keywords:

  • bcl-2;
  • CD 31;
  • cytokeratins;
  • human prenatal development;
  • utero-vaginal anlagen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Literature on the development of the human vagina is abundant; however, contributions concerning the prenatal development of the entire utero-vaginal anlagen (UVA) are rare or carried out in rodents. The primary epithelial characteristics in the adult vagina and uterus are determined during prenatal development and depend on epithelio-mesenchymal stroma interaction; thus an investigation summarizing the spatiotemporal distribution of relevant molecular markers in the entire human UVA will be of current interest. We phenotyped epithelial and mesenchymal characteristics in sagittal sections from 24 female fetuses of 14–34 weeks of gestation and two female newborns by immunostaining with cytokeratins 8, 13, 14 and 17, p63, bcl-2, bmp4, HOX A13, CD31, VEGF, SMA, Pax2 and vimentin. Epithelial differentiation followed a caudal-to-cranial direction in the UVA. Due to the cytokeratin profile of cytokeratins 8, 13 and 14, the characteristics of the different epithelial zones in the UVA could already be recognized in middle-age fetuses. Vaginal epithelium originated from the urogenital sinus in the lower portion and initiated the transformation of vimentin-positive Müllerian epithelium in the upper vaginal portion. During prenatal development the original squamo-columnar junction was clearly detectable from week 24 onwards and was always found in the cervical canal. Early blc-2 positivity within the surrounding mesenchyme of the entire vagina including the portio region pointed to an organ-specific mesenchymal influence. Prenatal findings in human specimens clearly show that fornix epithelium up to the squamo-columnar junction is of vaginal Müllerian origin, and the cervical epithelium cranial to the squamo-columnar junction is of uterine Müllerian origin and includes cells with enough plasticity to transform into squamous epithelium.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Anatomically, several internal female genital organs can be distinguished, the majority of which, i.e. vagina, uterus and uterine tube, develop from common embryonic structures, the fused Müllerian ducts. The morphology of the vagina and the uterine portions differs as to the epithelial lining and the muscular coats. The vagina is covered by a stratified squamous epithelium, the cervical canal is characterized by a columnar epithelium and cervical glands, and the uterine cavity is lined by a columnar epithelium and uterine glands that change in length during the uterine cycle (Marieb et al. 2008). There is a distinct border between the vaginal and cervical epithelium, which is called the original squamo-columnar junction (SCJ) and undergoes a positional change from the inside to the outside of the cervical canal during the different life (hormone) periods. According to their different functions, the walls of the vagina and uterus differ widely: whereas the smooth muscular coat of the vagina is thin, the uterine myometrium is bulky and consists of interlacing bundles.

Studies on the development of the entire human utero-vaginal anlagen (UVA) (Meyer, 1910; Hunter, 1930) or the human vaginal anlagen (VA) are rare (Mijsberg, 2007; Koff, 1933; Bulmer, 1957; Forsberg, 1973), mostly going back to the first decades of the last century; yet they are still the basis for embryological and clinical textbooks (Gray & Skandalakis, 1972; Ferris et al. 2004; Sadler,2004). With the exception of the early work of Meyer (1910) these studies do not lay special emphasis on the development of the transitory region between cervix and vagina. In contrast, Kurita's group has published several papers regarding the development of the female reproductive tract in mice including the vaginal/cervical transitory region (Kurita & Cunha, 2001; Kurita et al. 2005; Kurita, 2010). In summary, Kurita (2011) showed that during mice development the Müllerian ducts undergo a morphogenetic transformation from simple tubes into distinct organs and then differentiate to diverse epithelial cell types with a unique morphology in each organ. Within this process the transcription factor p63, induced by vaginal, cervical and uterine mesenchyme during development, was regarded to play a key role in the determination of epithelial cell fate (Kurita & Cunha, 2001). However, in adult mice, p63 is only expressed in the vaginal and cervical epithelium, but no longer in the uterine epithelium, when the developmental plasticity of epithelial cells is lost (Kurita et al. 2004). Kurita supposed rare epithelial plasticity to be restricted to small groups of so-called stem cells in cervix and uterus. In human specimens these cells are found to express combined CK17 and p63-positivity in the cervical epithelium (Martens et al. 2004, 2007), especially near the SCJ.

To provide up-to-date molecular correlates for the human utero-vaginal development that can be compared with the results of the above-mentioned cell biological studies in rodents we investigated the prenatal development of the entire UVA in complete series of human fetuses from week 14 to newborn and described our findings concerning the epithelial and mesenchymal differentiation. By application of antibodies raised against various cytokeratins, progenitor cells, transcription factors and smooth muscle actins as well as antibodies indicating cell survival, angiogenesis and mesenchymal origin (see Table 1), we were able not only to characterize the epithelial and mesenchymal structures at a defined stage but also to obtain detailed information about their spatiotemporal distribution. We presume that there is an interaction between the formation of mesenchyme and epithelial tissues, the knowledge of which is of high clinical relevance in regions of transformation (vagina/cervix).

Table 1. Antibodies used in immunohistochemistry
Antibody (catalogue number)HostDilution in IHCHIERSupplierMarker for
  1. a

    Ready-to-use.

  2. HIER, heat-induced epitope retrieval; IHC, immunohistochemistry.

BCl-2 (760-4240)MouseRTUaCC1 standardVentana, Mannheim, GermanyCell survival (Hockenbery et al. 1990)
BMP4 (AP15370PU-N)Rabbit1 : 200CC1 mildAcris, Herford, GermanyBasal/progenitor cell compartments (Ter Harmsel et al. 1996)
CD 31 (SIG-3632-26)MouseRTUCC1 mildCovance, Dedham, MA, USAEndothelial cells and angiogenesis (Parums et al. 1990)
Cytokeratin 8 (760-2637)MouseRTUCC1 standardVentanaNon-squamous epithelium (Gown & Vogel, 1984)
Cytokeratin 13 (E018)MouseRTUCC1 shortLinaris, Wertheim-Bettingen, GermanyNon-cornified squamous epithelia, expressed in exocervix (Moll et al. 1982)
Cytokeratin 14 (RTU-LL002)MouseRTUCC1 mildNovocastra, Newcastle upon Tyne, UKStratified epithelial cell types (Purkis et al. 1990)
Cytokeratin 17 (790-4560)RabbitRTUCC1 standardVentanaBasal/stem cells in complex epithelia (Smedts et al. 1992)
HOX A13 (sc-133669)Rabbit1 : 400CC1 mildSanta Cruz Biotechnology, Santa Cruz, CA, USAHomeobox-gene, development of the upper vagina (Taylor et al. 1997)
p63 (MS-1084-P)Mouse1 : 400CC1 standardTermo Fisher ScientificBasal/progenitor cells of many epithelial tissues (Yang et al. 1999)
Pax2 (18-0483)Rabbit1 : 80CC1 standardInvitrogen Corporation, Camarillo, CA, USADuctal and mesenchyme of urogenital system including the MDs and Wolffian ducts (Shapiro et al. 2004)
SMA (E046)MouseRTU LinarisSmooth muscle actins, myofibroblasts and myoepithelial cells (Skalli et al. 1986)
VEGF (E2614)Rabbit1 : 160CC1 standardSpring Bioscience, Fremont, CA, USAInduction of angiogenesis (Ferrara et al. 2003)
Vimentin (E034)MouseRTUCC1 shortLinarisCells originating in the mesenchyme (Osborn et al. 1984)
Rabbit polyclonal lgG (ab27478)Rabbit1 : 200CC1 short, mild and standardAbcam, Cambridge, USAImmunoglobulin G rabbit isotype control

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Human fetal and newborn specimens

A total of 24 female fetuses of 14–34 weeks of gestation as well as two female newborns were used in this study. The specimens were obtained from the archival collection of the Division of Clinical and Functional Anatomy, Innsbruck Medical University (number of specimens/week: 3/14th, 4/15th, 3/16th, 3/17th, 1/19th, 2/20th, 1/21st, 2/24th, 2/25th, 2/29th, 1/30th and 2/newborn). The specimens, which had already been investigated in other studies (Fritsch et al. 2010), showed no macroscopic abnormalities and were categorized according to their postovulatory age (O'Rahilly & Müller, 2010), which was based upon the crown–rump length (CRL) and external and internal morphology, or upon their estimated gynaecological age.

Tissue preparation and conventional histology

The specimens were immediately fixed by immersion in cold 4% paraformaldehyde in phosphate-buffered saline (PBS), pH 7.4 for 24 h followed by rinsing in phosphate-buffered saline (PBS). After dehydration and embedding in paraffin wax (Paraplast regular; Sigma-Aldrich, St. Louis, MO, USA) using a routine histological infiltration processor (Miles Scientific Inc., Naperville, IL, USA) the specimens were cut into 4-μm-thick serial sagittal sections using a Microm ERGO Star Rotations microtome (Microm, Walldorf, Germany). The sections were mounted on glass slides (SuperFrost Plus, Menzel-Gläser, Braunschweig, Germany) and air-dried overnight, followed by an incubation for 2 h at 60°C to attach the sections firmly to the glass slides. Every 10th section of a series was dewaxed with xylene, rehydrated in a graded alcohol series and stained with haematoxylin and eosin (HE) for cytoarchitectural orientation.

Antisera

Hosts, dilutions and sources of primary antibodies which were used in this study, as well as heat-induced epitope retrieval (HIER), where required, are listed in Table 1.

Immunohistochemistry

Immunohistochemistry was performed on paraffin sections in a Ventana Roche Discovery XT Immunostainer (Mannheim, Germany) according to the DAB-MAP discovery research standard procedure. If required, antigen retrieval was initiated by heat-induced unmasking of the epitopes while the slides were immersed, in accordance with the manufacturer's instructions (short, mild or standard for different incubation times) in EDTA buffer (Cell Conditioning Solution CC1, Ventana 950-124). After incubating the sections with primary antibodies at 37 °C, a biotinylated immunoglobulin cocktail of goat anti-mouse IgG, goat anti-mouse IgM, goat anti-rabbit IgG and protein block (Discovery Universal Secondary Antibody, Ventana 760-4205) was applied at room temperature. Detection was achieved using the DAB-MAP Detection Kit (Ventana 760-124) according to the diaminobenzidine development method with copper enhancement followed by light counterstaining with haematoxylin (Ventana 760-2021) for 4 min. The sections were then manually dehydrated, cleared in xylene, and cover-slipped. The immunohistochemical staining reaction was referred to positive controls (skin, tonsil, bone marrow, prostate) that were added to each experiment. In addition, representative sections were processed in the same way as previously described, omitting the primary antibodies or substituting them for isotype matching immunoglobulins for control purposes. These controls were consistently negative.

Double immunostaining of p63-vimentin experiments was done in compliance with the Ventana Roche Discovery XT double immunohistochemistry program. p63 immunostaining was detected with the DAB-MAP Detection Kit, and vimentin immunohistochemistry was determined using the RedMap Detection Kit (Ventana 760-123), a streptavidin-biotin alkaline phosphatase detection system with fast red detection. Denaturation between single immunostainings was achieved by heat treatment at 85 °C for 8 min.

Image analysis of immunohistochemistry

Digital images of immunostained slices were acquired in axiovision microscope software linked to an AxioCam HRc colour camera and an AxioPlan 2 microscope (Zeiss, Jena, Germany). Special attention concerning the immunohistochemical staining pattern was focused on the muscular coat, epithelial borders, folded portion, ribbon-like portion, cervical portion and the uterine portion of the utero-vaginal anlagen.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

As the conventional histological features of the UVA have been thoroughly described more than once in the literature (Meyer, 1910; Koff, 1933; Bulmer, 1957; Forsberg, 1973), we here refer to the prenatally occurring molecular characteristics of the epithelia and the surrounding mesenchyme.

Week 14/15

Muscular coat and mesenchymal differentiation

As indicated by SMA immunoreactivity, the UVA was covered by two different muscular coats (Fig. 1): a thin layer of longitudinal muscle fibres covered the VA (vaginal anlagen), starting above the UGS, ending and demarcating the future portio region where the longitudinal vaginal layer embraced a thicker uniform muscular coat of the uterus. Bcl-2 positivity was found in the mesenchyme adjacent to the vaginal epithelium (Fig. 1A) and in the muscular coat of the uterus (Fig. 1B). CD 31-positive vessels were sprouting towards the epithelium of the lower VA (Fig. 1C).

image

Figure 1. Neighbouring sagittal sections of the utero-vaginal anlagen (UVA) of a 13/14-week-old female fetus. The SMA-labelling (Fig. 1 overview) is a reconstruction of several pictures. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line. Scale bar: 500 μm. UGS, urogenital sinus; lVA, lower vaginal anlagen, uVA, upper vaginal anlagen, CS, cervical segment; US, uterine segment. (A,B) bcl-2. (C) CD 31. (D) Double labelling for p63 (brown) and vimentin (red). (E) bcl-2. (F) CK 14. (G) CK 13. (H) CD 31. (I) p63. (J) vimentin. Scale bars: (A,D,F-K) 20 μm, (B,C,E) 50 μm.

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Molecular characteristics of epithelia

As recently published (Fritsch et al. 2012), the VA consisted of a lower folded portion and an upper ribbon-like portion that were both mostly solid. The adjacent UA (uterine anlagen) was characterized by columnar epithelium and had a continuous lumen.

Lower VA

The epithelium was characterized by a lot of p63-positive basal and suprabasal cells (Fig. 1D), strong CK 17-positive cells (not shown), strong bcl-2-positive basal cells at the vaginal opening (Fig. 1E), scattered CK 14-positive basal cells (Fig. 1F), some suprabasal CK 13-positive cells (Fig. 1G), central vimentin-positive cells (Fig. 1D), some Pax2-positive cells (not shown) and some central CD 31-positive cells (not shown).

Upper VA

We found the same distribution as in the folded portion with CK 17 scattered in basal cells (not shown) and with additional CD 31-positivity within the central epithelium (Fig. 1H) at the upper end of the VA.

Cervical segment

The columnar epithelium had scattered basal p63-positive cells (Fig. 1I) and was strongly positive for Pax2 (not shown) and vimentin, especially in small basal cells (Fig. 1J) and for bcl-2 (Fig. 1A), but no CK 8-, 13- or 14-positivity was found in the cervix epithelium.

The point at which the future squamous vaginal epithelium and the columnar cervico-uterine epithelium meet is generally known as SCJ. It could not exactly be demarcated in this age group.

Uterine segment

The columnar cells were only positive for bcl-2, Pax2 and vimentin (not shown).

Weeks 16/17

Muscular coat and mesenchymal differentiation

The form of the muscular coats was identical to that of the younger stage, but the thickness of both uterine and vaginal muscular coat had increased, as demonstrated by SMA staining (Fig. 2). The embracing muscular coats demarcated the future portio region. The caudal part of the vaginal muscular coat had become two-layered: the outer longitudinal layer was joined by a circumferential inner, subepithelial layer. The longitudinal muscular layer of the vagina was accompanied by a large vascular network (Fig. 2). The bcl-2-positivity of the surrounding vaginal mesenchyme was identical with that of the younger stage (not shown), there were also vessels sprouting beneath the epithelium. Two vascular layers were present in the uterine segment, an inner one beneath the epithelium and an outer one at the outer border of the muscular wall (Fig. 2A).

image

Figure 2. Neighbouring sagittal sections of the UVA of a 16-week-old female fetus (A-N). The SMA-labelling (Fig. 2 overview) is a reconstruction of several pictures. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line. Scale bar: 500 μm. UGS, urogenital sinus; lVA, lower vaginal anlagen, uVA, upper vaginal anlagen, CS, cervical segment; US, uterine segment. (A) CD 31. (B) Double labelling for p63 (brown) and vimentin (red). (C,D) CK 13. (E) p63. (F) Vimentin. (G) CK 8. (H) HOX A13. (I) CK 14. (J) Pax2. (K) Vimentin. (L) p63. (M) CK 8. (N) HOX A13. Scale bar: 50 μm. (O) Sagittal section of the UVA of a 17-week-old fetus. CK 14. Scale bar: 100 μm.

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Molecular characteristics of epithelia
Lower VA

At the entrance into the UGS a group of typical vaginal four-layered cells could be observed at the dorsal wall of the vagina. The basal and suprabasal cells were p63-positive (Fig. 2B), all cells were vimentin-negative (Fig. 2B) and Pax2-negative (not shown) but CK 13-positive (Fig. 2C). The characteristics of the cranially adjacent folded vaginal portion were identical to those of the younger stage, with the only difference that the cells had developed CK 13-positivity, especially in the suprabasal and superficial layers (Fig. 2D). CK 17-positive cells were found basally and centrally (not shown).

Upper VA

The same cellular characteristics were the same as in the younger stage, and CK 17-positivity was identical with that in the lower VA. In addition, a light positivity for CK 8 was found (not shown).

Cervical segment

As can be seen from the overview, crypt formation (formation of cervical glands) had already started at this stage. P63-positive cells were widely scattered (Fig. 2E), and vimentin- (Fig. 2F) and Pax2-positivity (Fig. 2J) was still found, especially in the basal cells. Bcl-2-positivity was the same as in the younger stage. CK 8- positivity (Fig. 2G) was observed as well as HOX A13-positivity (Fig. 2H).

Squamo-columnar junction

As in the younger stage, the SCJ could not be exactly defined, but the scattered vaginal CK 14-positive cells (Fig. 2I) nearly reached this border. Pax2-positive cells were found on both sides of the SCJ (Fig. 2J).

Uterine segment

Besides the still strong positivity for bcl-2, vimentin (Fig. 2K) and Pax2, p63-positive cells could now be detected in the cavum epithelium (Fig. 2L). The cells were CK 8- (Fig. 2M) and HOX A13-positive (Fig. 2N).

Weeks 19/20/21

Muscular coat and mesenchymal differentiation

The UVA had elongated, and the thickness of the UVA-muscular coat had increased (Fig. 3). The vaginal coat was two-layered throughout the entire organ and caudally reached the UGS level. Ventrally it was connected with the muscular wall of the urethra (Figs 2O and 3). The future portio region was demarcated by a slight flexion. Vessel sprouting could be detected in the subepithelial mesenchyme of this region (Fig. 3A). Bcl-2-positivity within the mesenchyme was comparable to the younger stages (not shown).

image

Figure 3. Neighbouring sagittal sections of the UVA of a 19-week-old female fetus. The SMA-labelling (Fig. 3 overview) is a reconstruction of several pictures and two different sections. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line, the future portio region is demarcated by an arrow. Scale bar: 500 μm. UGS, urogenital sinus; lVA, lower vaginal anlagen, uVA, upper vaginal anlagen, CS, cervical segment; US, uterine segment. (A) CD 31. (B) CK 13. (C) CK 14. (D) bmp4. (E) CD 31. (F) VEGF. (G) CK 17, (H) bmp4. (I) bmp4. Scale bar: 50 μm.

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Molecular characteristics of epithelia

This age group was characterized by the process of lumen formation in the vagina.

Lower VA

In the lower VA, typical multilayered vaginal squamous epithelium had overlain the subepithelial connective tissue papillae, and the organ had a lumen that started from the UGS (Fig. 3). The multilayered epithelium was strongly CK 13-positive (Fig. 3B). Bcl-2-positivity was comparable to the younger stages (not shown); p63-positive cells as well as CK 17- and CK 14-positive cells (Fig. 3C) were found basally and suprabasally. In the basal layers, slight positivity for bmp4 could be detected (Fig. 3D). Sprouting vessels beneath the epithelium (Fig. 3E) seemed to be attracted by basal VEGF-positive epithelial cells (Fig. 3F).

Upper VA

The number of epithelial layers had increased in comparison with the younger stages. Cellular positivity for p63, Pax2, vimentin, CK 13, CK 14, CK 17 (Fig. 3G) and CD 31 was identical with weeks 16/17 (not shown). At the cranial end of the upper VA where the fornix region was built up, a lumen had opened, running in caudal direction. The luminal cells were flat (Fig. 3A).

Cervical segment

Apart from some scattered CK 17-positive cells (Fig. 3G) p63-, Pax2-, vimentin- and CK 8-positivity was the same as in weeks 16/17 and there was no HOX A13-labelling, instead, slight bmp4-positivity was found (Fig. 3H).

Squamo-columnar junction

This border was situated between the Müllerian cuboidal vaginal epithelium and the Müllerian columnar cervical epithelium and could not be clearly distinguished.

Uterine segment

P63-, Pax2-, vimentin- and CK 8-positivity was the same as in weeks 16/17 and there was no HOX A13-labelling, instead, slight bmp4-positivity was found (Fig. 3I).

Weeks 24/25

Muscular coat and mesenchymal differentiation

The smooth muscle layers of the entire UVA were more differentiated: the complex, four-layered system of the uterus became visible, and the vaginal coat had an outer longitudinal layer that was intimately connected with the uterine coat at the portio level (Fig. 4). The muscular coat was joined ventrally by an extra layer beneath the portio, indicating the anteflexion of the uterus. This layer was connected to the bladder neck and the urethra. An inner more circumferential and loose vaginal muscle layer was situated between the epithelium and the longitudinal layer and filled the connective tissue papillae. The pattern of bcl-2-positivity within the mesenchyme of the UVA that was found in younger stages had gone (not shown).

image

Figure 4. Neighbouring sagittal sections of the UVA of a 25-week-old female fetus. The SMA-labelling (Fig. 4 overview) is a reconstruction of several pictures. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line. Scale bar: 1000 μm. lVA, lower vaginal anlagen, uVA, upper vaginal anlagen, P, portio; CS, cervical segment; US, uterine segment. (A) CD 31. (B) CK 17. (C) HE. (D) CK 8. (E) CK 17. (F) VEGF. (G) CD 31. Scale bar: 50 μm.

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Molecular characteristics of epithelia

This age group was characterized by a clearly demarcated epithelial border between the lower and upper VA.

Lower VA

The typical vaginal epithelium had ascended and was underlaid by vessel-filled connective tissue papillae (Fig. 4A). Positivity for CK13, 14 and p63 was the same as in the younger age groups, CK 17-positive cells were scattered basally (Fig. 4B) and positivity for bcl-2 and bmp4 was not observed (not shown).

Upper VA

The epithelial border between the lower and upper VA stood out clearly in conventional histology (Fig. 4C). The lumen of the upper VA was covered by a four- to six-layered cuboidal epithelium with scattered p63-positive as well as CK 14- and CK13-positive basal cells (not shown). The epithelium was slightly positive for CK 8 (Fig. 4D) and strongly positive for CK 17 (Fig. 4E). Luminar cells were flat and VEGF-positive (Fig. 4F). Vessels grew and spread into the epithelium (Fig. 4G). Thus, intraepithelial lacunae occurred between the basal and superficial cell layers.

Cervical segment

Basal p63-positive cells and some widely scattered CK 17-positive cells were found in a CK 8-positive epithelium. No HOX A13-positivity but slight bmp4-labelling could be observed (not shown).

Squamo-columnar junction

This border could be recognized within the cervical canal.

Uterine segment

No changes in comparison with the younger age group were found. Pax2-positivity was now reduced to the epithelium of the cavum (not shown).

Weeks 29/30

Muscular coat and mesenchymal differentiation

The muscular coats of vagina and uterus had obtained their final structure (Fig. 5). The portio region and fornices were developed now.

image

Figure 5. Neighbouring sagittal sections of the UVA of a 29/30-week-old female fetus. The SMA-labelling (Fig. 5 overview) is a reconstruction of several pictures. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line. Scale bar: 1000 μm. uVA, upper vaginal anlagen; P, portio; CS, cervical segment. (A) bmp4. (B) CK 17. (C) CK 13. (D) CK 14. (E) CK8. Scale bar: 50 μm.

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Molecular characteristics of epithelia

This stage was characterized by the appearance of a uniform vaginal epithelium in both the lower and upper vagina as well as in the fornices. Furthermore, the SCJ was clearly demarcated now. None of the different epithelia labelled positive for vimentin any longer (not shown).

Lower and upper VA

The same epithelium was seen with basal p63-positive cells, strong CK 13- and 14-labelling, scattered CK 17-positive cells, slight positivity for HOX A13 in basal cells and in all epithelial layers for bmp4 (Fig. 5A). No vimentin-, bcl-2-, CK 8- or CD 31-positivity was found (not shown).

Cervical segment

The labellings were comparable to those at the younger stage. The number of basally situated subcolumnar CK 17-positive cells had increased (Fig. 5B). Apart from a slight bmp4-labelling, HOX A13-positivity was seen (not shown).

Squamo-columnar junction

This border was situated within the endocervical canal and clearly demarcated by CK 13- (Fig. 5C) and CK 14-labelling (Fig. 5D) in the squamous epithelium and by CK 8-labelling in the columnar epithelium (Fig. 5E).

Uterine segment

The uterine segment could not be described in this age group because this part of the organ was not included in the specimens.

Newborn

Muscular coat and mesenchymal differentiation

No obvious change in the development of the muscular coat (Fig. 6) could be detected in comparison with the younger stage.

image

Figure 6. Neighbouring sagittal sections of the UVA of a female newborn. The SMA-labelling (Fig. 6 overview) is a reconstruction of several pictures. The level of the squamo-columnar junction (SCJ) is indicated by a dotted line. Scale bar: 1000 μm. uVA, upper vaginal anlagen; P, portio; CS, cervical segment, US, uterine segment. (A) bcl-2. (B) CD 31. (C) Vimentin, arrowheads point to reserve cells. (D) CK 8. (E) HOX A13. Scale bar: (A) 100 μm, (B,D,E) 50 μm, (C) 20 μm.

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Molecular characteristics of epithelia

This stage was characterized by the appearance of cervical glands and the starting differentiation of the uterine glands.

Vaginal anlagen

The same distribution of p63-, CK 13-, CK 14-, HOX A13- and bmp4-positive cells could be detected as before. CK 17-positive cells were very rare (not shown). Basal and suprabasal cells showed slight positivity for bcl-2 (Fig. 6A).

Cervical segment

Cervical glands were spreading in a caudal and oblique direction. As indicated by CD-31-positivity (Fig. 6B), vessels sprouted beneath the cervical glands, and slight positivity for VEGF was found (not shown) within the columnar epithelium, which only contained some scattered vimentin-positive reserve cells (Fig. 6C). Some CK 17-positive cells but no more p63-, CK 8-, HOX A13- and bmp4-labelling was seen (not shown).

Squamo-columnar junction

This border was situated near the entrance of the endocervical canal and was as clearly demarcated as before.

Uterine segment

Apart from the surface epithelium, some developing uterine glands could be detected. No p63-positive cells were found, but the cells showed strong CK 8-positivity (Fig. 6D) and slight bcl-2- as well as Pax2-positivity (not shown). Some vimentin-positive cells were found in the glands (not shown), whereas HOX A13-positive cells were observed in the glands, the surface epithelium and the subepithelial mesenchymal cells (Fig. 6E).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The present study was undertaken to examine the development and differentiation of the human utero-vaginal epithelia and mesenchyme. We here report that:

  1. Epithelial differentiation runs in a caudal-to-cranial direction: vaginal epithelial differentiation is followed by cervico-uterine epithelial differentiation, and the characteristics of the different zones of the UVA can be recognized in middle-aged human fetuses (Table 2: marked in red).
  2. A dual mechanism for vaginal epithelialization is seen: the most caudal vaginal epithelium is exclusively UGS-epithelium-derived; all the upper vaginal epithelium is a converted or transformed Müllerian epithelium (Table 2: marked in lilac).
  3. The epithelium of the fornices, the portio (ectocervix) up to the SCJ at the beginning of the endocervical canal is of vaginal origin.
  4. Mesenchymal input seems to be important for the regionalization of Müllerian epithelium (vagina, cervix and uterus).
  5. The original SCJ is always situated in the cervical portion, i.e. within the cervical canal during prenatal life, and is clearly detectable from week 24 onwards (Table 2: asterisk).
Table 2. Summarized spatiotemporal distribution patterns of molecular markers in the entire UVA. Respective immunoreactivities were categorized in a semiquantitative manner: Ø, No immunoreactivity; + Ø, low; +, moderate; ++, high immunopositivity; [DOWNWARDS ARROW] bcl-2 pattern of mesenchyme is lost; × SCJ is clearly detectableThumbnail image of

Following the detailed investigations of Meyer (1910), Koff (1933) and Bulmer (1957), our study, based on nearly complete prenatal stages, is the first to contribute to the development of the human UVA. In the last decade, functional morphological studies concerning UVA development models were published by Kurita's group (Kurita & Cunha, 2001; Kurita et al. 2005; Kurita, 2010), who focused on mice and mice models and only in a few cases compared them with human probes. Although these publications present clear and strong results concerning the rodents, they suffer from the weakness that their human specimens were not complete, and their results cannot safely be connected and compared with the cell-biological findings in the laboratory animals or to clinical pathological aspects. A recent publication on human uterine development (Martens et al. 2007) lacks the younger human stages and does not consider the entire UVA. We are the first to analyse the immunohistochemistry of different cytokeratins, vimentin, Pax2, p63, bcl-2, bmp4 and HOX A13 in the entire human UVA in order to find new aspects as to the heterogeneity of the different epithelia in the UVA and their possible interaction with the underlying mesenchyme.

Our results clearly show that epithelial differentiation in the UVA does occur caudo-cranially, regardless of where the cells originate from. This is in accordance with the literature (Meyer, 1910; Forsberg, 1973; Orvis & Behringer, 2007; Kurita, 2010) and supports the results of our last study on early VA development (Fritsch et al. 2012) where we proposed that the caudo-cranial differentiation process is initialized by cells or a cellular stimulus from the UGS epithelium, itself is of endodermal origin. This is supported by the new results presented here – that in weeks 16/17 a group of UGS-derived squamous epithelial cells proliferates and extends into the lower vagina and initializes an epithelial differentiation first within the VA and then within the uterine portions.

We can be sure now that there is a dual mechanism of vaginal epithelialization, supporting the clinical compartment theory of the distal vagina (Höckel et al. 2011): the cells that extend into the caudal-most vaginal portion are CK 13-, 14- and p63- positive and vimentin-negative. However, those cells that are situated within the solid vaginal portion are vimentin-positive. Vimentin is a characteristic intermediate filament of mesoderm-derived cells, highly indicative of its derivation from the paramesonephric system (Van der Putte, 2005). The vimentin-positivity we noted therefore seems to be characteristic of the mesoderm-derived Müllerian epithelium and is observed neither in the UGS epithelium nor in the anorectal epithelia (not shown). The Müllerian-derived epithelium shows an ascending distribution of p63- (Table 2: marked in yellow) and CK 14-positive cells (Table 2: marked in red) that was shown by Kurita & Cunha (2001), which we assumed to be derived from the UGS in early vaginal development (Fritsch et al. 2012). In weeks 24/25 the solid Müllerian epithelium is transformed into a squamous epithelium by the ingrowth of blood vessels (Table 2: marked in pink). This transformed Müllerian squamous epithelium is found throughout the upper vagina, in the fornices, the ectocervix and in the entrance of the cervical canal. Our results revealed the Müllerian origin of this epithelium based on vimentin (Van der Putte, 2005) and Pax2 immunoreactivities that were found in the upper vagina, the cervix and the uterine segment at week 14. With increasing age the expression of Pax2 protein decreased in a caudo-cranial direction, and at week 24 it was restricted to the epithelium of the uterine segment.

In the adult, p63-positive cells are considered to be reserve cells in the vaginal and ectocervical epithelium as well as in subcolumnar cells at the SCJ (Martens et al. 2004). Furthermore, Martens et al. (2004) pointed out that in cervical intra-epithelial neoplasia, p63-immunostaining is strongly expressed irrespective of grade. Our results regarding human UVA development show an ascending p63-distribution from the vagina to the uterus in early fetal life (Table 2: marked in yellow). In later stages, p63-positive cells are regularly found in the basal cells of the vagina and the fornix (ectocervix), and they are scattered in the cervical and uterine epithelium (Table 2: marked in yellow). The latter cells are supposed to maintain the developmental plasticity, i.e. to be the targets of cervical or uterine squamous metaplasia (Kurita, 2011). As shown by Martens et al. (2004), HPV target cells are qualified not only by p63 but also by CK 17. We have shown that during prenatal life CK 17-positivity is first found in the lower vagina (Table 2: marked in violet), ascends towards the upper vagina, the fornix and portio, and is found in subcolumnar cells in the cervical epithelium in late fetal life. This finding is identical with the situation shown in the adult where CK 17 staining is only found in (endo)cervical reserve cells and reserve cell hyperplasia (Martens et al. 2004).

In several publications (Cunha, 1976; Kurita & Cunha, 2001; Kurita, 2011) it has been shown that in rodents the functional differentiation of the vaginal and uterine epithelia requires organ-specific factors. For the first time now, our results present evidence of this phenomenon in the human UVA. Bcl-2-positivity is found in the surrounding mesenchyme of all the VA as well as in the portio region until weeks 19/20, whereas there is no bcl-2-positivity in the neighbouring mesenchyme of the cervical and uterine portion of the UVA (Table 2: arrow). Vaginal mesenchyme bcl-2-positivity is lost in weeks 24/25 when the differentiation process into squamous epithelium is in progress. As bcl-2 is an apoptosis regulator and not tissue-specific, our results can only be considered an indirect hint, further underlined by the position of the border of the vaginal muscular coat illustrated by the SMA staining pattern in weeks 13/14 and 16/17. Up to week 24 the vaginal epithelium is also bcl-2-positive (Table 2: marked in blue), the following differentiation and transformation process in the human vagina seems to be supported by HOX A13 and bmp4 (Table 2: marked in green) which, according to Cai (2009), play a decisive role in the conversion of the intermediate mesoderm nature of the Müllerian duct in mice. According to our results, HOX A13-positivity in human specimens is not restricted to the vagina but is also found in the cervical and uterine portion (Table 2: marked in black) just at the time when the glandular structures develop and migrate from the epithelial surface into the underlying mesenchyme. In contrast to the literature (Jaubert et al. 2009), smooth muscle appearance in the UVA was detected in early fetal life, whereas the completion of the uterine muscular coat about week 24 was found to be in accordance with the literature. Our results clearly show that the mesenchymal surroundings of vagina and uterus are different. Therefore we suppose that the different mesenchymal environment may drive the epithelial cells to different cell fates within the vaginal anlagen and the uterine segment of the UVA. To examine the epithelial–mesenchymal interactions further investigations using other methods (e.g. in situ hybridization) will be necessary before using this study as the basis for revealing the epithelial differentiation influenced by the adjacent mesenchyme.

The original SCJ is situated within the cervical canal during all stages of fetal life. In the newborn this border descends towards the vagina. Thus our results are in complete agreement with those of Meyer (1910), gained from the observation of more specimens than we had at our disposal. Ferris et al. (2004), however, proposed a variable position of the SCJ in late fetal life and were not able to explain why squamous epithelial cells partially replace the ‘Müllerian’ columnar epithelium in the fetal cervix. We think that the SCJ may have been confused with the border of the two squamous vaginal epithelia, and that this may have led to a misleading interpretation. We have shown that the cervical glands appear in the newborn, and that they grow caudally towards the cervical orifice; consequently the SCJ descends towards the fornices. This process cannot be considered to represent a replacement of epithelia (Ferris et al. 2004) but must be seen as a displacement or dislocation of the squamous cervical epithelium. Malpica & Robboy (2009) pointed out that during adolescence cervical growth leads to a descending original SCJ and an exposure of cervical tissue outside the cervical os, i.e. to a repositioning of cervical epithelium to a vaginal environment. In accordance to Martens et al. (2004) we have shown that the cervical epithelium includes cells with the plasticity to transform into squamous epithelium.

In the course of our investigations we found that there is a probable dual mechanism causing vaginal epithelialization, but we also considered the possibility of a second dual mechanism in which the human cervix develops into three compartments: (i) the Müllerian columnar epithelium of the uterus and cervix, (ii) the Müllerian squamous epithelium of the cervix and the upper vagina, and (iii) the vaginal squamous epithelium of the lower vagina. This approach is an interesting one and might offer explanations concerning the genesis/development of lesions and carcinomata in this region. However, as pursuing this was far outside the scope of this study, we intend to follow up our present investigations with another study considering not only the theory of this approach but also its clinical consequences, ranging from human papillomavirus to carcinomata of the cervix and vagina, thus our findings concern data which may become of lifelong clinical relevance for affected persons.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We would like to thank Prof. Dr. Höckel for reading and amending our study and Mrs. Claudia Siemon for revising the English.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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