Pathogenesis of Hypospadias
More Questions Than Answers
By Dietrich Kluth, Wolfgang Lambrecht, and Peter Reich Hamburg, Federal Republic of Germany 9 Most researchers believe that hypospadias arises. from malformation of the penile urethra. However, this~ concept has been recently rejected, and it has been suggested that the opening of the urethra is "pushed forward". by growth of the perineum. In order to obtain m o r e information on the development of the urethra, late stages of phallic development were studied in 220 rat embryos with scanning electron microscopy (SEM). In our study, signs of rupture of the urogenital membrane or fusion of. the urethral folds could not be found. Therefore, w e could not confirm the traditional concept for the development of the phallic urethra. A new concept of urethral developmentis suggested and the pathogenesis of hypospadias is discussed. 9 1988 by Grune & Stratton. Inc.
MATERIALS AND METHODS
In the 17-day-old rat embryo (Fig 1), sexual differentiation is not yet possible. The genital protuberance is very pronounced, and the cloacal membrane is to be recognized as a sagittal line. The integrity of the membrane is preserved. In a microdissected embryo of this age group (Fig 2), the anterior part of the cloaca extends up to the tip of the genital protuberance. The entoderm forms a tube-like structure that although delimited from the cloacal membrane is still closely apposed to this. A urethral plate in the actual sense is not to be found. On the contrary, the ventral cloaca has the structure of a hollow organ. In the 17.5-day-old embryo (Fig 3), folds arise from which the preputium is derived. At this stage, a partial disintegration of the cloacal membrane is to be discerned in its dorsal region in some embryos. Here, the urorectal fold reaches the cloacal membrane. It cannot be stated with certainty whether it penetrates the membrane or fuses with it. In the 18.5-day-old embryo (Fig 4), a certain sexual differentiation on the basis of external characteristics is not yet possible. The preputial folds are clearly limitable. At their upper edge, a furrow arises that might correspond to the coronary sulcus. In the region of the cloacal membrane, circumscribed irregularities are to be discerned. On the other hand, a disintegration of the cloacal membrane with formation of a urethral fold is not to be observed. The microdissection of the cloaca (Fig 5) shows that the ventral part of the cloaca still extends up to the tip of the phallus and forms a short cloacal membrane here with the ectoderm. In addition, a lumen runs dorsally to the ectoderm of the perineum. This lumen forms the "cloacal canal." The ventral part of the cloaca is completely enveloped by mesenchyme in the middle section, and thus no longer has any contact with the ectoderm of the genitals.
Twenty pregnant Sprague-Dawley rats were killed between the 16th and 20th day after copulation. Pregnancy was verified by means of the smear method, with the day of positive smear being rated as the first day of gestation (one day postconception). A total of 220 embryos were removed by laparotomy. Staging was performed according to Witsehi. 6 One hundred fifty-six embryos were fixed in glutaraldehyde for 24 hours. Sixty-four embryos were incubated for one hour in 0.75% trypsin solution at 370C. They were then fixed in Serra's solution for 24 hours. Mierodisseetion of all embryos was performed in 80% ethanol. After dehydration, the embryos were dried with the critical point method and then examined with a Novascan 30 scanning electron microscope (C. Zeiss, West Germany).
From the Department of Surgery, University Hospital, Hamburg, Germany. Supported by the Deutsche Forschungsgemeinschaft (DFG), Grant No. KI-596/I-1. Presented at the 19th Annual Meeting of the American Pediatric Surgical Association, Tucson, Arizona, May 11-14, 1988. Address reprint requests to Dietrich Kluth, MD, Chirurgische Universit~tskHnik, Martinistrafle 52, 2000 Hamburg 20, Federal Republic of Germany. 9 1988 by Grune & Stratton, Inc. 0022-3468/88/2312-0002503.00/0
INDEX WORDS: Urethral development; pathogenesis of hypospadias,
AULTY VENTRAL OPENINGS of the urethra
F constitute a broad spectrum of malformations which are subsumed under the term hypospadias. Many researchers TM believe that the urethra arises by fusion of the paired urethral folds after rupture of the urogenital membrane. Disturbance of this process is thought to result in the various forms of hypospadias? This concept was contradicted by van der Putte 5 in 1986. He assumes that the urethral opening initially situated dorsally is displaced to the tip of the genital anlage by the rapid ventral growth of the perineum. In order to obtain more detailed information on the development of the urethra in its late phase, the development of the urogenital region was investigated with scanning electron microscopy (SEM) in rat embryos. Answers to the following questions were to be established: When does the cloacal membrane rupture and does the urethra develop by fusion of the urethral folds?
Journal of PediatricSurgery,Vol 23, No 12 (December),1988: pp 1095-1101
KLUTH, LAMBRECHT, AND REICH
hypospadias is striking. Indications of fusion processes that might have been able to lead to formation of the raphe was not found. Findings in the female embryo (Fig 7) differ markedly from those in the male. A raphe does not exist. The perineum is shorter and flatter. At the root of the phallus, the ectoderm diverges, rendering the later urethral opening visible. DISCUSSION
Hypospadias are generally explained in terms of a malformation of the urethra. However, a review of the literature shows that observations on the formation of the urethra are contradictory and uncertain. Three development processes are controversial: the development of the cloacal membrane, of the urethral plate (precursor of the urethra?), and of the urethra.
Development of the Cloacal Membrane Fig 1. SEM picture of the developing genitalia in a 17-day-old rat embryo. A sex differentiation based on external signs is not possible. The cloacal membrane (CM) is seen as a segittal line without signs of its disintegration. Arrow indicates tip of the cloaca. GT, genital tubercle.
In 20-day-old embryos (Fig 6), the sex can now be clearly determined from outward appearance. In the male, the scrotum is to be discerned in the perineum. It is divided by a median fold, the raphe. The raphe reaches the base of the phallus, where it is concurrent with the preputial fold that covers the tip of the glans dorsally and laterally. The shaft of the phallus is distinctly curved. A urethral opening is not yet present. In terms of the outward appearence, the similarity to
According to Keibel,7 fusion of the urorectal septum with the cloacal membrane leads to its division into the ventral urogenital membrane and the dorsal anal membrane. A little later, the urogenital part of the membrane is thought to be destroyed; consequently, the urogenital sinus obtains a first opening. This view was shared by many investigators in the past, 1'2'4'8 Politzer and others 5'9"Hcontradicted this concept. They believe that the urorectal septum does not fuse with the cloacal membrane. On the contrary, they assume that the cloaeal membrane initially breaks down in the dorsal region, and that the arising opening is subdivided into an anal and urogenital orifice by the urorectal septum. In our investigation, we were unable to
Fig 2. A 17-day-old rat embryo. Microdissection of the entoderm of the cloaca, view from the left side, The anterior part of the cloaca (e) extends to the tip of the genital tubercle (GT). The ventral cloaca feetures a hollow organ rather than a plate. Arrow indicates urorectal fold. PU, proximal urethra; RE, rectum; BL, primitive bladder; CM, cloacal membrane.
PATHOGENESIS OF HYPOSPADIAS
Fig 3. SEM picture of a 17.5-day-old rat embryo. (A) View on the developing phallus from caudad. PF, folds that represent the anlage of the prepuce. (B) Higher magnification of the central part. Partial disintegration (') can be seen in the dorsal part of the cloacal membrane (CM).
observe the destruction of the entire cloacal membrane. The partial destruction in the dorsal region with formation of a urogenital meatus also could not be documented with certainty. Van der Putte 5'11'~2merely shows a schematic drawing of this phase of development in his studies. Unfortunately, as yet there is no histologic documentation. In our material, we merely found a phase of instability in the dorsal part of the cloaca, which is connected with the formation of the anal opening. On the other hand, indications for the existence of a urethral opening were not found in the male embryo. Neither the formation of the urethral
fossa nor fusion of lateral parts of the ectoderm could be observed in male embryos.
Development of the Urethral Plate The development of the urethra in the region of the phallus is also controversial. It is assumed that first of all a solid plate (the urethral plate) is formed. There are divergent views on its genesis. Kanagasuntheram ~3 regards it as a ventral outgrowth of the cloaca. Barnstein and Mossman ~4 believe that processes of invagination of the ectoderm lead to plate formation or contribute to plate formation ~4 Glenister 3 regards a
Fig 4. SEM picture of an 18.5-day-old rat embryo. (A) Same view as in Fig 3. Circumscribed irregularities (*) are seen, PF, Preputial folds; GL, glans; CS, coronal sulcus. (B) Higher magnification of the central part,
KLUTH, LAMBRECHT, AND REICH
Fig 5. (A) SEM picture of microdissscted 9 region of an 18.B-day-old rat embryo. (B) Diagram of the same embryo; view from the left, The cloaca is opened to show that it is a hollow organ that extends to the tip of the genital tubercle (GT). In the middle part, the cloaca has lost its contact to the phallic ectoderm (arrows). Therefore this part now Can be Called "'urethra'" (Ur). CC, cloacal canal (see t e x t in detail); CM, ventral part of the cloacai membrane; RE, rectum.
combination of the two processes as possible. In the penile region the plate arises by outgrowth of the cloaca, and in the glans region it arises ectodermally by invagination. Figure 2 shows that the ventral cloaca is not a plate but a hollow organ in a 17-day-old embryo. As such, it extends into the tip of the genital anlage. We did not see the formation of a plate by outgrowth of cloacal ectoderm or by ingrowth of ectoderm in the region of the developing phallus. We believe that the discussion about the urethral plate is the result of not entirely
sagittal seetions through the cloaeal region. The plate is to be regarded as an artifact resulting from the sagittal section.
Formation of the Urethra Most researchers believe that the penile urethra results from the closure of the urethral fossa. The closure is believed to take place by fusion of the free edges of the urethral folds. This process is thought to begin at the base of the phallus and to end at its tip. ~'2'4']5 Glenister's explanation 3 is somewhat more
Fig 6. (A) SEM picture of the phallus of a 20-day-old male rat embryo. Note the similarity to the morphology of (B) the human penile hypospadia. SC, scrotum; RA, raphe; GL, glans; PF, preputial fold.
PATHOGENESIS OF HYPOSPADIAS
Fig 7. SEM picture of the phallus of a 20-day-old female rat embryo. A t the base of the phallus, the ectoderm diverges (arrow) to give rise to the future orifice of the urethra
complex, but nevertheless, the formation of the urethra finally takes place by processes of fusion. Van der Putte 11assumes that the phallic urethra is formed and that the urethral opening is displaced from dorsal to ventral by perineal growth processes. As already mentioned, we were unable to image either a urethral fossa or a urethral opening in the perineal region in our male embryos. Hence, it was also impossible to show the formation of the urethra by fusion of lateral folds or by displacement of the urethral opening. In our collection of normal rat embryos, we observed an intermediate stage in the development of the ventral
Fig 8. Schematic drawing of cloacal features in an 18.5day-old rat embryo (see Fig 5). This stage seems to be an intermediate one of urethral development. From this stage, the dorsal female urethra (short arrow) as well as the ventral male urethra (long arrow) is derived. (e), cloacal canal that establishes part of the femal urethra, C, rest of cloacal membrane which represents the future opening of the male urethra; Sy, symphysis; Ur, middle part of cloaca that shows "'urethral" features. Re, rectum.
cloaca in an 18.5-day-old embryo that displayed characteristics of both a male and female urethra (Figs 5 and 8). At this stage, the epithelium of the cloaca has contact with the ectoderm only in two places-ventrally in the region of the tip of the genitals and dorsally in the region of the perineum over the cloacal canal. We believe that the urethral opening of the female animal develops in the dorsal region in the further course, whereas the male urethra opens in the glans region of the genital anlage. Clinical experience shows that urethral duplications are possible in both sexes4 and these then display urethral openings precisely in these regions. In most cases of penis agenesis, a the urethra is also situated dorsally and opens perineally or even rectally. In our investigation of the development of external genitals in rat embryos, we were not able to support the traditional concept on the formation of the male urethra. This means that the traditional concept on the genesis of hypospadias also becomes doubtful. We found the following. (1) The urethra is already formed at an early stage from the ventral part of the cloaca. The ventral part of the cloaca always has contact with the tip of the genitals. (2) The urethra is initially formed as a double anlage. The differentiation into male and female urethra takes place in the 18.5day-old embryo. On the other hand, we found no indications for "rupture" of the urogenital membranes or fusion in lateral parts of the perineum. In our opinion, there is no uniform concept for the pathogenesis of hypospadias. The milder forms such as penile and glandular hypospadias constitute an inhibition malformation of the genitals. The point of departure of the malformation is a situation that corresponds
KLUTH, LAMBRECHT, AND REICH
to that in a 20-day-old embryo. In the further course, extension of the genitals is prevented, possibly by chorda tissue. The penis remains curved, and thus the urethra is too short in relative terms. For this reason, the penis and not the urethra is the primary organ of the malformation. Perineal and scrotal hypospadias
differ from those described previously. In these children, there are often marked signs of feminization. Hence, it is also logical to designate the urethra as a "female" urethra. The point of departure of these malformations is an indifferent stage of development, such as that seen in an 18.5-day-old rat embryo.
REFERENCES 1. Felix W: Die Entwicklung der Harn- und Gesehleehtsorgane, in Keibel F, Mall FP (eds): Handbuch der Entwieklungsgesehichte des Menschen, vol 2. Leipzig, Hirzel, 1911, pp 923-955 2. Spaulding MH: The development of the external genitalia in the human embryo. Contrib Embryol Carneg Inst 13:67-88, 1921 3. Glenister TW: A correlation of the normal and abnormal development of the penile urethra and of the infraabdominal wall. Br J Uroi 30:117-126, 1958 4. Gray SW, Skandalakis JE: Embryology for Surgeons. Philadelphia, Saunders, 1972, pp 595-631 5. van der Putte SCJ: Normal and abnormal development of the anoreetum. J Pediatr Surg 21:434-440, 1986 6. Witsehi E: Development: Rat, in Altman PL, Dittmer DS (eds): Growth, Federation of American Society for Experimental Biology, 1962, pp. 304-314 7. Keibel F: Uber die Entwicklung yon Harnblase, Harnr6hre und Datum beim Mensehen, Verh Anat Ges 189-199, 1895 8. Patton BM, Barry A: The genesis of extrophy of the bladder and epispadias. Am J Anat 90:35-57, 1952
9. Politzer G: Ober die Entwicklung des Dammes beim Menschen. Z Anat Entw Geseh 102:690-709, 1934 10. Ludwig KS: Ober die Beziehung der Kloakenmembran zum Septum uroreetale bei menschlichen Embryonen von 9 bis 33 mm SSL. Z Anat Entw Gesch 124:401-413, 1965 11. van der Putte SCJ, Neeteson FA: The normal development of the anoreetum in the pig. Acta Morphol Neerl Scand 21:107-132, 1983 12. van der Putte SCJ, Neeteson FA: The pathogenesis of hereditary congenital malformations of the anoreetum in pigs, Aeta Morphol Neerl Scand 22:17-40, 1984 13. Kanagasuntheram R, Anandaraja S: Development of the terminal urethra and prepuce in the dog. J Anat 94:121-129, 1960 14. Barnstein NJ, Mossman HW: The origin of the penile urethra and bulbo-urethral glands with particular reference to the red squirrel (Tami-aseirius hudsonieus). Anat Rec 72:67-85, 1938 15. Hamilton W J, Boyd JD, Mossman HW: Human Embryology (ed 3). Baltimore, Williams & Wilkins, 1962, pp 294-299
Discussion H. Snyder (Philadelphia): It is interesting to postulate that the embryology that he has observed in the rat might also occur in the human, Certainly duplications of the urethra in humans which we have reviewed cannot be explained in any fashion. I would like to ask one question in relation to that, When urethral duplications occur, most commonly the orthotopic channel in the center of the penis is atretic and the ventral channel arrangements is the most workable channel. Do you have any feelings for why that might occur based on the embryologic assays that you have done? One other question that has always intrigued me. In the distal forms of hypospadias, it is very common to see an abortative of urethral duplication or pit extending in as much as 1/2 cm or more dorsal to the actual urethral channel that is tobe reconstructed. I could not quite follow your statement; do you see any difference in the embryology of the most distal portion in the rat as contrasted with the more proximal portion? Do you have an explanation for why there might be this dorsal duplication? D. Kluth (response): I am aware that our opinions about the development of the malformation are questionable. We do not have an animal model that can explain hypospadias as yet, and I think this would be
the main reason to find an animal with this malformation. Until then, I think this question cannot be answered sufficiently. I think the very distal part of the urethra is different because it has to open, and that is the main difference between the rest of the urethra that is already open through the entire time of development. I think that in the opening process, these kinds of malformations can occur. J. Noseworthy (Cincinnati): I have always been under the impression that we in hypospadias often dealt with two failures--that of tubularization which you seem to indicate doesn't occur, but also the more distal urethra develops by a different mechanism. Do you have any observations about the development of the glandular portion of the urethra in this rat model? Do you see in your model or in your observations any explanations for the etiology of chordee? D. Kluth (response): No, actually not. I have a slide in which you can see that the ectoderm in the region where the urethral opening is disintegrating. We have no signs that a urethral septum or something like that will come into existence in embryology. I have just checked the literature and I could not find any evidence of that theory.
PATHOGENESIS OF HYPOSPADIAS
I think that the chordee is the result of a very fundamental developmental step that can be seen very often in embryology. I think chordee is the result of stretching of mesenchyme or the rest of the urethra. The chordee in the ease of hypospadias occurs because the dorsal growth in those cases is more exuberant than
the ventral growth. The ventral part of the phallus is stretched passively. If some part of the embryo is stretched passively, it will become very stiff and very rigid, like a string. I think that the chordee occurs by this mechanism.