FETAL DEVELOPMENT OF THE FEMALE EXTERNAL URINARY SPHINCTER COMPLEX: AN ANATOMICAL AND HISTOLOGICAL STUDY

0022-5347/05/1735-1738/0 THE JOURNAL OF UROLOGY® Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 173, 1738 –1742, May 2005 Printed in U.S.A.

DOI: 10.1097/01.ju.0000154616.51979.da

FETAL DEVELOPMENT OF THE FEMALE EXTERNAL URINARY SPHINCTER COMPLEX: AN ANATOMICAL AND HISTOLOGICAL STUDY PHILIPPE SEBE, HELGA FRITSCH, JOSEF OSWALD, CHRISTIAN SCHWENTNER, ANDREAS LUNACEK, GEORG BARTSCH AND CHRISTIAN RADMAYR* From the Department of Pediatric Urology (JO, CS, AL, GB, CR) and Institute of Anatomy, Embryology and Histology (HF), Medical University, Innsbruck, Austria, and Department of Urology, Hoˆpital Tennon, Paris, France (PS)

ABSTRACT

Purpose: We investigated the fetal development of the smooth (lissosphincter) and striated (rhabdosphincter) female external urinary sphincter. Growth and organization of the muscle fibers around the urethra and morphological modifications due to the development of the vagina were analyzed in detail. Materials and Methods: A total of 28 human female fetal specimens were investigated in an anatomical and histological study. The sections were processed according to plastination technology. This technique allows examination of structures and organs of the small pelvis with minimal artifacts in all 3 planes. Results: At gestational week 9 the primordium of the external urethral sphincter complex was observed extending along the anterior aspect of the urogenital sinus, before the development of the primitive urethra and the vaginal primordium. From 15 weeks of gestation the lissosphincter and rhabdosphincter could be identified and clearly distinguished. After 20 weeks of gestation both elements acquired an omega-shaped configuration with a narrow posterior connective tissue raphe that was constantly present, fixing both components to the ventral vaginal wall. Both muscles were mainly located in the middle third of the urethra. In the proximal third of the urethra growth of the vagina led to disappearance of the striated muscle fibers of the rhabdosphincter, whereas the lissosphincter seemed to intermingle with the internal layer of the detrusor musculature of the bladder. Conclusions: The important morphological characteristics of the female adult rhabdosphincter and lissosphincter (omega-shaped configuration, presence of a narrow connective tissue raphe posteriorly and maximum thickness in the middle third of the urethra) are already evident early in fetal development and do not evolve during postnatal growth or by the influence of sex hormones. KEY WORDS: fetal development, urethra, vagina

Wilson was probably the first to describe an individual striated muscle surrounding the urethra in both sexes in 1809.1 Within the proceeding literature the existence of this individual muscle was denied by many authors, and it was supposed to be a part of the so-called “urogenital diaphragm.” The discovery that striated fibers of the sphincter musculature surrounding the urethra were significantly smaller than those of other pelvic muscles gave new support for the theory of an independent external urinary sphincter muscle.2, 3 From that point forward morphological descriptions were mainly dedicated to the whole muscle aspect, while the spatial extent and configuration remained subjects of discussion. Currently, it is widely accepted that the external urinary sphincter is built from a smooth (lissosphincter) and a striated (rhabdosphincter) component.4 In addition, several authors have suggested that the female rhabdosphincter and lissosphincter extend as a single unit along the entire urethra.5⫺7 More recent concepts of the external urethral sphincter system in the female were drawn in regard to urethra sparing cystectomy.8, 9 In contrast to previous studies, it was shown that the rhabdosphincter was predominantly located in the distal two thirds, whereas the lissosphincter layers were abundant only in the proximal two thirds of the ure-

thra.8, 9 Consequently, both sphincter components are overlapping only in the middle third of the urethra, corresponding to the point of maximum urethral closure pressure.10 Furthermore, as in the male, the rhabdosphincter in the female is supposed to have an omega-like shape in transverse sections due to a posterior connective tissue raphe. Nevertheless, some groups have described a nearly ring-shaped circular configuration with posteriorly crossing muscle fibers.7, 8 The extent and configuration of the female urethral sphincter have continued to be controversial issues. Embryological studies of the external sphincter apparatus of the female urethra have already been conducted to elucidate and understand its adult anatomy and function.9, 11–14 Nevertheless, it still remains a subject of confusion, since different configurations such as omega, ring and elliptical shapes are described. In addition, the extent of the sphincter components to either the proximal and/or the distal third of the urethra is characterized differently.9, 11–14 The aim of this study was to investigate further the fetal development of the urethral lissosphincter and rhabdosphincter. Growth, organization and spatial extent of the muscle fibers around the urethra and morphological modifications due to the development of the vagina were analyzed in detail.

Submitted for publication September 5, 2004. * Correspondence: Department of Pediatric Urology, University of MATERIALS AND METHODS Innsbruck, Anichstr. 35, 6020 Innsbruck, Austria (telephone: 43–512A total of 28 human female fetuses that demonstrated no 504 – 4810; FAX: 43–512-504 – 8365; e-mail: christian.radmayr@ signs of maceration or macroscopic abnormalities after misuibk.ac.at). 1738

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Characteristics of 28 human female samples Fetal Specimen No.

Gestational Age (wks)

Crown-Rump Length (mm)

Section Thickness (␮m)

Plane

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

9 10–11 11–12 12 13–14 14 14–15 15 16 17 17 18 18 18 18 19–20 19–20 20 20 20 20 23 24 24 26–27 32 25 Newborn

41 58 71 80 100 111 118 125 135 138 145 151 153 154 159 165 185 174 184 185 185 218 225 229 245 305 341 360

600 300 300 300 500 500 500 500 400 600 500 500 300 500 400 600 600 400 500 500 500 400 500 500 500 500 600 500

Sagittal Transverse Transverse Coronal Transverse Transverse Transverse Coronal Transverse Transverse Transverse Transverse Transverse Transverse Sagittal Transverse Transverse Transverse Transverse Sagittal Transverse Sagittal Transverse Transverse Transverse Transverse Sagittal Transverse

carriage were included in the study (see table). Parental informed consent and ethical approval was given in all cases. Crown-rump length of the fetuses was measured as defined by Mall,15 and ranged from 41 to 341 mm. The age of the fetuses was estimated using the standard tables of Moore16 and Patten,17 and ranged from 9 to 35 weeks of gestation. Fetuses were further treated according to the epoxy resin based plastination technology protocol, allowing prolonged storage without deterioration of the slide quality. The specimens were prepared by immersion and stored in 4% formaldehyde solution for at least 3 months. Then the fetal pelves were dehydrated with acetone and impregnated with epoxy resin.18 After polymerization the epoxy blocks were sectioned serially with a diamond wire saw in either the transverse (21 fetuses), coronal (2) or sagittal (5) plane. Thickness of the sections ranged from 200 to 600 ␮m, being generally thinner in the smaller specimens. After mounting and polishing the sections were stained with azure II/methylene blue in alkaline solution and counterstained with basic fuchsin. Using this method, collagenous fibers were stained blue-violet and elastic fibers red. Muscle fibers could be distinguished from collagenous fibers by their blue-green color.18 This staining protocol provides the same morphological information as the Masson-Goldner technique. In the majority of cases evaluation of the fetal kidneys for embryological development of the morphology was possible, and no evidence of dysplasia in terms of reflux nephropathy or cystic dysplasia was found. All of the fetuses were clear of any nervous system and lower urinary tract anomalies. The stained sections were examined using light microscopy with computerized image analysis. Color photographs were taken using a Professional (Pixera, Los Gatos, California) closed circuit digital camera connected to a Pentium III/500 MHz 256 MB (Hewlett-Packard Company, Palo Alto, California) processor. Axio Vision version 3.1 (Carl Zeiss, Inc., Thornwood, New York) software was used for modular image acquisition and processing as well as interactive measurement of distances. The thickness of the urethral sphincter was evaluated in all transverse sections dorsally and medially at the same level. At each age the highest value was used to describe the growth of each component during develop-

FIG. 1. Sagittal (A) and transverse (B to D) sections with azure II/ methylene blue staining (counterstaining with basic fuchsin) of primitive urogenital sinus in 9-week (A), and urethra in 14-week (B and C) and 19-week (D) fetuses. ARC, anorectal canal. B, bladder. CTR, connective tissue raphe. GT, genital tubercle. L, lamina propria. LS, lissosphincter. PT, paramesonephric tubercle. R, rhabdosphincter. SUP, sphincter urethrae primordium. UC, uterine canal. UGS, urogenital sinus. URS, urorectal septum. V, vagina. VP, vaginal plate. Arrows indicate borderline between rhabdosphincter and lissosphincter.

ment. Both sphincter components were distinguished by morphological and histological characteristics—the orientation of the layers, the presence of striations into the striated component (high magnification) and the intensity of the staining.1–3 RESULTS

Early fetal development. In the 9-week fetus a condensation of mononuclear cells could be identified in the anterior aspect of the urogenital sinus that corresponded to the sphincter urethrae primordium (SUP). The SUP extended without interruption from the bladder base to the genital tubercle. The SUP represented an independent dominant

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structure composed of myoblasts clearly distinct from the urogenital sinus (fig. 1, A). After week 12 of gestation 2 different configurations of the SUP could be distinguished. In the caudal aspect the SUP assumed a horseshoe-shaped configuration surrounding the urethra, which was fused with the vaginal plate. Those myoblasts were situated anteriorly and laterally around the urethra, adjoining the lateral aspects of the vaginal plate. No myoblasts were found posterior to the urethra (fig. 1, B). In the cranial aspect the SUP was thinner and displayed an incompletely ring-shaped configuration surrounding the urethra interrupted by a posterior connective tissue raphe that was linked to the connective tissue sheath of the vagina (figs. 1, C and 2, A). From week 15 of gestation the histological and macroscopic aspects of the striated and smooth components of the external sphincter were distinct, and a clear borderline could be drawn between them in all of the specimens. In transverse sections an outer layer of transverse bundles could be identified that were supposed to correspond to the rhabdosphincter. An inner layer of transverse muscle bundles that were half as thick surrounded the lamina propria of the urethra. These bundles presumably correspond to the lissosphincter (figs. 1, D and 2, B). Late fetal period. From gestational week 20 the vagina was distally separated from the urethra and had grown enormously. In median sagittal sections the extent of the smooth and striated components could be clearly identified. The rhabdosphincter was the main muscular structure in the middle and distal thirds of the urethra, whereas it was poorly represented cranially. In contrast, the lissosphincter was mainly found in the cranial aspect, where its muscle fibers intermingled with the musculature of the bladder neck on sagittal sections (fig. 3). At this time of development both sphincter components acquired an omega-shaped configuration in all investigated serial sections, representing almost the entire urethra due to a constantly present narrow connective tissue raphe posteriorly (fig. 2, C). In addition, the external sphincter complex was anchored to the anterior vaginal wall by the aforementioned raphe. From week 25 of gestation to term the extent of the rhabdosphincter was progressively limited to the middle and caudal thirds of the urethra (fig. 2, D). Caudally, the rhabdosphincter was the dominant muscular structure surrounding the female urethra. However, a transversely oriented horizontal muscle plate between the ischiopubic rami representing the urogenital diaphragm could not be identified. In the newborn the lissosphincter was mainly located in the middle and cranial thirds of the urethra. Its fibers were progressively arranged into an outer layer of transverse and an inner layer of longitudinal bundles intermingling with the smooth musculature of the bladder neck. As seen in figure 4, the thickness of the rhabdosphincter and lissosphincter grew proportionally during fetal development. The approximate rhabdosphincter-to-lissosphincter thickness ratio was 2:1. Growth of both components seems to be linear during fetal development. DISCUSSION

Early in the fetal period after the division of the cloaca the sphincter urethrae primordium emerges around the anterior circumference of the urogenital sinus. Consequently, the SUP appears before development of the vagina actually starts. The common primordium of the urethra and lower part of the vagina are outlined as the urogenital sinus, whereas the primordium of the upper portion of the vagina is associated with the uterine canal, which originates from the fused paramesonephric ducts.17 After the tip of the uterine canal reaches the urogenital sinus a solid vaginal plate grows out from the dorsal aspect of the urogenital sinus (fig. 5, A).

FIG. 2. Transverse sections with azure II/methylene blue staining (counterstaining with basic fuchsin) of urethra in 13-week (A), 20week (B), 27-week (C) and 38-week (D) fetuses. CTR, connective tissue raphe. R, rhabdosphincter. S, lissosphincter. SUP, sphincter urethrae primordium. V, vagina.

Therefore, the developing lower portion of the vagina seems to be fused with the distal urethra until week 20 of gestation. Our findings basically are in agreement with previous studies, in which the caudal part of the SUP was called the “urethrovaginal sphincter.”7, 11, 19 However, in early fetal development the SUP represents a continuous muscular unit, especially in its ventral aspect (fig. 5, A). Consequently, there seems to be no reason to divide the SUP into urethrovaginal and urethral parts. Furthermore, when the development proceeds the vagina grows super proportionately and its distal end is displaced in a caudal direction. Subsequently, the vagina disconnects from the SUP laterally and both sphincter components acquire an omega-shaped configuration. Other groups have indicated similar morphological characteristics of the female external sphincter apparatus (fig. 5, B).9, 11, 13, 20 The rhabdosphincter does not seem to start as a complete ring with circular and concentric fibers as described previously.12 The plastination technique offers the advantage of analyzing sections of tissue with much higher thickness (ratio 50:1) compared to conventional staining techniques. Therefore, the in situ morphology of both sphincter components could be magnified.18 Similar to the rhabdosphincter, the lissosphincter does not form a circular collar because of the persistent presence of a connective tissue raphe posterior to the urethra, which serves as an anchoring structure for the posterolateral fibers of each sphincter component. This fibrous connective tissue raphe adjoins the muscular wall of the urethra to the vagina, creating a so-called “urethrovaginal septum.”11

FETAL DEVELOPMENT OF FEMALE EXTERNAL URINARY SPHINCTER

FIG. 3. Sagittal section with azure II/methylene blue staining (counterstaining with basic fuchsin) of pelvis in 23-week fetus.

FIG. 4. Evolution of thickness of sphincter urethrae primordium (SUP), rhabdosphincter (RS) and lissosphincter (LS) from 13 weeks of gestation to term.

During fetal development the posteriorly oriented growth of the vagina leads to an additional effect. There is an increase in the length of the proximal portion of the urethra, partly caused by an elongation of the urethral plate as well as by an enlargement of the ventral wall of the vagina.11 This developmental change of the cranial urethra could give rise to a simple explanation for the progressive disappearance of the rhabdosphincter fibers in this region. At term we failed to find rhabdosphincter fibers in the cranial part of the urethra, which is in agreement with a previous study investigating fetal and adult specimens.9 Furthermore, the previous study revealed that the lissosphincter is predominantly developed in the 2 cranial thirds, as it is in adult females.9 The lissosphincter fibers seem to be intermingled with the internal layer of the detrusor muscle.17 Nevertheless, it is likely that detrusor and sphincteric smooth musculature has different embryonic origins, since the SUP might appear as a common primordium for the striated and smooth components. Both muscular parts of the external urinary sphincter are mainly represented in the middle third of the urethra, corresponding to the point of peak urethral closure pressure in urodynamics.10 In this region the boundary between smooth and striated fibers is well defined in transverse sections, and there seems to be no intermingling of fibers of the 2 components. The precise organization and direction of the different

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FIG. 5. Transverse sections with azure II/methylene blue staining (counterstaining with basic fuchsin) of urethra in 15-week (A) and 30-week (B) fetuses show evolution of configuration of urethral sphincter complex. CTR, connective tissue raphe. LS, lissosphincter. R, rhabdosphincter. SUP, sphincter urethrae primordium. V, vagina. VP, vaginal plate.

muscular layers are difficult to assess. Nevertheless, in transverse sections the striated fibers seem to be mainly arranged in transverse bundles, whereas the configuration of smooth bundles is transverse and longitudinal without a well-defined boundary. The functional role of the delineated muscle fiber architecture and its association with the posterior connective tissue raphe cannot be concluded from a morphological study, and, therefore, will be an issue of further investigations. At term the rhabdosphincter and the anterior part of the puborectalis muscle are the dominant striated muscular structures surrounding the urethra. The rhabdosphincter is an independent morphological unit during all fetal developmental stages, and our findings once again underline that the so-called “urogenital diaphragm” does not exist.7 An anatomical study does not provide data about the functional relevance of the 2 components of the urinary continence mechanism. Nevertheless, the results of our study, as well as previous approaches by other groups, indicate that the anatomical integrity of the urethral sphincter complex and the connective tissue raphe is likely to have a key role in maintaining urinary continence. CONCLUSIONS

The female rhabdosphincter and lissosphincter have a common sphincter urethrae primordium that consists of a myoblast plate located in the anterior aspect of the urogenital sinus. The SUP is already present before the beginning of the development of the vagina. The lissosphincter and rhabdosphincter acquire an omega-shaped configuration during fetal development due to a constant posterior connective tissue raphe. The maximum thickness of the external sphincter complex is achieved in the middle third of the urethra, corresponding to the point of peak urethral closure pressure in urodynamics.10 The development of the vagina leads to modifications of the configuration and spatial extent of the smooth muscular as well as the striated part of the external

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sphincter apparatus. Alterations of the rhabdosphincter architecture are limited to the late fetal period. REFERENCES

1. Wilson, D.: A description of two muscles surrounding the membranous part of the urethra. Med Chirurg Soc Lond, 1: 175, 1809 2. Gosling, J. A., Dixon, J. S., Critchley, H. O. and Thompson, S. A.: A comparative study of the human external sphincter and periurethral levator ani muscles. Br J Urol, 53: 35, 1981 3. Von Hayek, H.: Das Faserkaliber in den MM. transversus perinei und sphincter urethrae. Z Anat Entwicklungsgesch, 121: 455, 1960 4. Dorschner, W. and Stolzenburg, J. U.: A new theory of micturition and urinary continence based on histomorphological studies. 3. The two parts of the musculus sphincter urethrae: physiological importance for continence in rest and stress. Urol Int, 52: 185, 1994 5. Elbadawi, A.: Functional anatomy of the organs of micturition. Urol Clin North Am, 23: 177, 1996 6. Gosling, J. A.: The structure of the female lower urinary tract and pelvic floor. Urol Clin North Am, 12: 207, 1985 7. Oelrich, T. M.: The striated urogenital sphincter muscle in the female. Anat Rec, 205: 223, 1983 8. Strasser, H., Ninkovic, M., Hess, M., Bartsch, G. and Stenzl, A.: Anatomic and functional studies of the male and female urethral sphincter. World J Urol, 18: 324, 2000 9. Colleselli, K., Stenzl, A., Eder, R., Strasser, H., Poisel, S. and Bartsch, G.: The female urethral sphincter: a morphological and topographical study. J Urol, 160: 49, 1998 10. Khullar, V. and Cardozo, L.: The urethra (UPP, MUPP, instability, LPP). Eur Urol, suppl., 34: 20, 1998 11. Droes, J.: Observations on the musculature of the urinary bladder and the urethra in the human foetus. Br J Urol, 46: 179, 1974 12. Kokoua, A., Homsy, Y., Lavigne, J. F., Williot, P., Corcos, J., Laberge, I. et al: Maturation of the external urinary sphincter: a comparative histotopographic study in humans. J Urol, 150: 617, 1993 13. Ludwikowski, B., Oesch Hayward, I., Brenner, E. and Fritsch, H.: The development of the external urethral sphincter in humans. BJU Int, 87: 565, 2001

14. Tanagho, E. A. and Smith, D. R.: Mechanism of urinary continence. 1. Embryologic, anatomic and pathologic considerations. J Urol, 100: 640, 1968 15. Mall, F. P.: On measuring human embryos. Anat Rec, 1: 129, 1907 16. Moore, K. L.: The Developing Human, 3rd ed. Philadelphia: W. B. Saunders Co., 1982 17. Patten, B. M.: Human Embryology, 3rd ed. New York: McGraw Hill Book Co., 1968 18. Fritsch, H.: Staining of different tissues in thick epoxy resinimpregnated sections of human fetuses. Stain Technol, 64: 75, 1989 19. Tichy, M.: The morphogenesis of human sphincter urethrae muscle. Anat Embryol, 180: 577, 1989 20. Yucel, S. and Baskin, L. S.: An anatomical description of the male and female urethral sphincter complex. J Urol, 171: 1890, 2004 EDITORIAL COMMENT The authors have performed an elegant anatomical study of the developing female urinary sphincter. They have reaffirmed the concept that the so-called urogenital diaphragm is a misnomer. The urinary sphincter develops as an integral part of the pelvic floor, enveloping the urethra as well as the vagina. Careful analysis of fetal specimens during development confirmed the omega-shaped configuration of the urinary sphincter. The authors noted a maximum thickness of the sphincter in the middle third of the urethra corresponding to the point of peak urethral closure pressures in adult urodynamic studies. These findings are in agreement with our recent studies and analogous to the development of the urinary sphincter in the male (reference 20 in article). Careful studies such as this will ultimately further the understanding of surgical reconstruction and the maintenance of urinary continence in the female as well as in the male. Further work analyzing age appropriate postnatal specimens is necessary to support the claim that the urinary sphincter in the female does not evolve during postnatal growth and/or is not influenced by sex hormones. Laurence Baskin Department of Urology UCSF Children’s Hospital San Francisco, California