Urorectal Septum Research Articles

Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development

Incomplete urethral tubularization (hypospadias) and anorectal abnormalities are two common and poorly understood birth defects that affect the extreme caudal midline of the human embryo. We now show that cell surface molecules essential for proper axon pathfinding in the developing nervous system, namely ephrin-B2 and the ephrin receptors EphB2 and EphB3, also play major roles in cell adhesion events that tubularize the urethra and partition the urinary and alimentary tracts. Mice carrying mutations which disrupt the bidirectional signals that these molecules transduce develop with variably penetrant severe hypospadias and incomplete midline fusion of the primitive cloaca. We further show that animals completely lacking ephrin-B2 reverse signaling present a fully penetrant failure in cloacal septation. This results in severe anorectal malformations characterized by an absence of the terminal-most hindgut (rectum) and formation of a fistula that aberrantly connects the intestines to the urethra at the base of the bladder. Consistent with an apparent requisite for both forward and reverse signaling in these caudal remodeling events, EphB2 and ephrin-B2 are coexpressed at the midline in the fusing urethral/cloacal endoderm and underlying lateral mesoderm of the urorectal septum that migrates toward the caudal midline as the cloaca septates. Our data thus indicate that B-subclass Eph and ephrin molecules play an important role in these clinically significant midline cell–cell adhesion and fusion events.

Normal and abnormal embryonic development of the anorectum in rats

Background The embryologic and pathologic aspect of anorectal malformation (ARM) remains poorly understood. There is no universally accepted theory to explain anorectal embryology and the abnormal development that produces ARM. The aim of this study was to observe the developmental processes of anorectum in rats and to explore the abnormal embryonic development that leads to ARM. Methods Rat embryos with ARM were obtained by treating pregnant rats with administration of ethylenethiourea (ETU). Normal rat embryos and embryos with ARM from gestational days 12.5 to 20 were sectioned serially and sagittally and stained with H & E. The relevant structure including cloaca and urorectal septum (URS) were examined in a temporospatial sequence. Results Characteristics of anorectum development in ARM rat embryos treated by ETU were as follows: (1) URS never fused with cloaca membrane. (2) Dorsal cloacal membrane was maldeveloped. (3) Cloacal configuration was abnormal. (4) Tail groove never appeared. All type of ARM was the rectourethal fistula and common cloaca in ETU-treated rat embryos and was discernible on gestation day 16. Conclusions Absence of the tail groove and maldevelopment of the dorsal cloacal membrane, cloacal configuration, and urorectal septum are likely to be responsible for the formation of ARM. Failure of fusion of the URS with cloacal membrane is the immediate reason for rectourethral fistula or common cloaca in ETU-treated rats.

Endocrine evaluation of hypospadias.

Hypospadias is a congenital defect of the penis resulting in an incomplete development of the anterior urethra (Duckett, 1998). The abnormal urethral opening may be any place along the shaft of the penis or open onto the scrotum or perineum. By the end of the first month of gestation, the hindgut reaches the surface of the embryo at the cloacal membrane on the ventral surface. The cloacal membrane is then divided by the urorectal septum into the anal and urogenital membrane. The genital tubercle and genital swellings appear around the urogenital membrane. A surge of luteinizing hormone from the pituitary gland leads to an increase of testosterone. Testosterone biosynthesis occurs at about 9 weeks gestation and hCG-LH receptors are present on fetal Leydig cells by at least 12 weeks of gestation. Androgens are critical for the induction, growth and differentiation of the genital tubercle into the male phenotype via the androgen receptor (AR), a nuclear transcription factor. Dihydrotestosterone, a metabolite of testosterone, mediates the action of androgens on the external genitalia and is known to bind to the AR with a higher affinity than testosterone. Masculinization of the external genitalia, namely elongation of the phallus, formation of the penile urethra from the urethral groove and development of the prepuce, depends on adequate testosterone synthesis, adequacy of the enzymatic pathway mediating the cascade and responsive end organs. During the third fetal month, the wolffian ducts complete their development and involution of the mullerian system occurs under the influence of mullerian inhibiting substance (MIS or AMH). A deficiency in the formation or delay in the maturation of the enzymes in the testosterone steroid cascade during these critical embryogenesis periods may be responsible for incomplete development.

The development of the male genitourinary system. I. The origin of the urorectal septum and the formation of the perineum

The embryological development of the male urinary system remains a subject of much controversy. As a result the pathogenesis of congenital anomalies such as hypospadias and epispadias, which are presented to the reconstructive surgeon remains poorly understood. A review of the literature identifies its three principal developmental stages: (1) division of the cloaca into the urogenital sinus and hindgut by the urorectal septum and the formation of the perineum; (2) the extension of the cloaca and its epithelium in the form of the urethral plate through the developing genital tubercle; (3) the separation of this extension from the surface during the formation of the urethra. This study, which uses a mouse model, examines these developmental stages in detail and together with a comprehensive review of the literature resolves many of the controversies relating to the development of the male urinary system. It reveals new insights into the origin of the associated congenital defects.

The absence of lateral fusion in cloacal partition

Background/purpose: The mechanism by which the cloaca becomes partitioned into a dorsal rectal part and a ventral genitourinary sinus has been the subject of speculation for more than a century. Despite repeated suggestions that partitioning of the cloaca by fusion of lateral folds does not occur, the concept continues to hold sway in many student and surgical texts. The authors reviewed the histologic and 3-dimensional appearance of the urorectal septum in human and rat embryos to see if there was any evidence of lateral fusion in its formation. Methods: Sprague-Dawley rat embryos (n = 143) were examined between 11 and 21 days’ gestation and compared with human embryo sections (57 embryos) held in historical collections in Europe. Rat embryos were examined by microscopy, dissection, and serial histologic section. In addition, some specimens were sectioned in wax until the lumen of the cloaca was reached, after which they were dewaxed and the internal cavities imaged with scanning electronmicroscopy (n = 18 of 143). Results: Cloacal “partitioning” resulted from a combination of growth of the mesenchyme of the hindgut and genitourinary sinus, an alteration in the position of the cloaca in relation to surrounding structures secondary to growth in the ventral, infraumbilical abdominal wall and changes in the curvature of the developing spine, and apoptosis in the dorsal wall of the cloaca with shortening of the dorsal cloacal wall. There was no septum, as it is usually defined, between the developing bladder and hindgut. There was no evidence on either histologic section or scanning electronmicroscopy of any process of fusion occurring between the 2 lateral folds within the lumen of the cloaca. Conclusions: Lateral fusion of the side walls of the cloaca does not play a role in cloacal “partition.” Development of the bladder and hindgut occurs by a process that involves growth, differentiation, and remodeling.

Fecal incontinence--anatomy/pathophysiology

The ano-rectal organ derives from two different structures, the proctodaeum and the hindgut. The uro-rectal septum splits off the hindgut from the anterior part of the cloaca. This means that the anorectum has the same origin of nerves and blood vessels as the anterior part of the cloaca, which derives from the uro-genital sinus. In order to understand continence and defaecation, exact knowledge of the anatomy and physiology is compulsory. Studies in embryology are essential to the understanding of the anatomic and topographic structures.

Urorectal septum malformation sequence: Insights into pathogenesis.

We characterize the urorectal septum malformation sequence (URSMS) in discordant fetal lambs and relate it to the human syndromes with which URSMS is associated. We found abnormal external genitalia, imperforate anus, and fistulous connections between the rectum, bladder, and vagina. Discordance among the dizygous twins eliminated teratogens as a likely etiologic factor. We summarize the relevant literature and propose a molecular model for the pathogenesis of the URSMS, in which alterations in sonic hedgehog and homeobox genes lead to caudal mesodermal deficiency during blastogenesis.

P63 Coordinates Anogenital Modeling and Epithelial Cell Differentiation in the Developing Female Urogenital Tract

p63 is a p53 homologue required for cutaneous development that is expressed in immature squamous epithelium and reserve cells of the cervix. Humans with p63 mutations exhibit defects in limb, accessory organ (skin appendage, breast, prostate), and genitourinary development. Because p63 expression patterns imply a strong role of the gene in the female genital tract development, newborn female p63-/-, +/-, and +/+ mice were examined in situ, dissected, and compared. Nuclear p63 protein was localized to the skin, vagina, bladder, urethra, and basal columnar cells of the caudal uterus in p63+/+ and +/- animals. p63-/- mice exhibited abnormal genital morphogenesis with hypoplastic genitalia, a single cloacal opening, and persistence of columnar epithelium at lower genital tract sites that normally undergo squamous and urothelial differentiation. The defects observed support p63-dependent pathways of genital tract development that permit externally, ectodermal basal cell replenishment integral to reciprocal epithelial stromal signaling, urorectal septation, and modeling of the external genitalia; and internally, the emergence of basal epithelial cell populations capable of divergent epithelial cell differentiation in the vagina, cervix, and urinary tract. Defects in the first pathway explain imperforate anus, vaginal septum, genital hypoplasia, and micropenis reported in humans with p63 mutations. The second is necessary for the generation of multipotential reserve cells in the cervix and may be operative in other epithelial stromal interactions integral to the emergence of uterine basal cells later in life.

Prenatal diagnosis of sex differentiation disorders: the role of fetal ultrasound.

We describe our experience with prenatal diagnosis of sex differentiation disorders, with focus on the role of ultrasound scans for coherent assessment of prenatal diagnosis. Over a 5-yr period all cases suspected of sexual ambiguity based on abnormal ultrasonographic scans (US) or US/genotype US discrepancy were evaluated prenatally by three modalities: 1) repeated fetal US; 2) genetic studies, primarily karyotype and fluorescence in situ hybridization analysis of sex-determining region on the Y gene (SRY); and 3) hormonal assays of amniotic fluid. Of approximately 10,000 gestations, 16 fetuses underwent prenatal evaluation. Twelve were referred because of an abnormal US and 4 because of genotype-phenotype discrepancy. Five fetuses were diagnosed with female pseudohermaphroditism (21-hydroxylase deficiency in 3 and urorectal septum malformation sequence in 2). Four fetuses were diagnosed with male pseudohermaphroditism (1 with steroid sulfatase deficiency, 1 with presumed camptomelic dysplasia, and 2 undetermined). Five cases had chromosomal abnormalities, and 2 had 46,XX+SRY sex reversal. In all genetic females the uterus was observed on US. In 11 cases initial US scan was performed at 13-15 wk; in 7 of 11, although the initial scan was normal, a repeated scan later in gestation revealed an abnormality. Repeated US scans performed at 13-15 and 22-24 wk gestation are a helpful tool in prenatal diagnosis of sex differentiation disorders. Our data suggest that both size and structure anomalies of the reproductive structures may evolve throughout pregnancy, and that they represent a developmental biological process rather than a single nonprogressive pathological event. US scan after approximately 19 wk enables detection of the uterus and provides pivotal information in cases of ambiguity. If the uterus appears normal, the most likely diagnosis is a virilized karyotypic female. Prenatal diagnosis allows for early parental counseling and anticipation of medical management postnatally.

Septation of the anorectal and genitourinary tracts in the human embryo: crucial role of the catenoidal shape of the urorectal sulcus.

Previous studies of the tracheoesophageal sulcus and the sulci of the developing heart have suggested that the catenoidal or saddle-shaped configuration of the sulcus had mechanical properties that were important to developmental processes by causing regional growth limitation. We examined the development of the human perineal region to determine if a similar configuration exists in relation to the urorectal septum. We wished to re-examine the controversial issue of the role of the urorectal sulcus in the partitioning of the cloaca. Digitally scanned photomicrographs of serial histologic sections of embryos from Carnegie stages 13, 15, 18, and 22, obtained from the Carnegie Embryological Collection were used. Each image was digitally stacked, aligned, and isolated using image-editing software. Images were compiled using 3-D image-visualization software (T-Vox), into full 3-D voxel-based volume renderings. Similarly, digital models were made of the urogenital sinus, anorectum, cloaca, allantois, mesonephric ducts, ureters, and kidneys by isolating their associated epithelium in each histologic section and compiling the data in T-Vox. Methods were developed to create registration models for determining the exact position and orientation of outlined structures within the embryos. Models were oriented and resectioned to determine the configuration of the urorectal sulcus. The results show that the urorectal sulcus maintains a catenoidal configuration during the developmental period studied and, thus, would be expected to limit caudal growth of the urorectal septum. The observations support the concept that the urorectal septum is a passive structure that does not actively divide the cloaca into urogenital and anorectal components.

Clarification of the processes that lead to anorectal malformations in the ETU-induced rat model of imperforate anus

Background: Ethylenethiourea (ETU) administered to timed-pregnant rats can induce anorectal malformations (ARMs) in about 80% of rat fetuses, thus providing an ideal animal model to study the embryogenesis of ARMs. The current study was undertaken to investigate the embryogenetic events that may be responsible for the development of ARMs in rats. Methods: Time-mated pregnant rats were divided randomly into control and experimental groups. The experimental rats received 1% ETU (125 mg/kg) by gavage on gestational day 10, and control rats received only the vehicle. Their embryos were harvested by cesarean section on gestational days 13, 14, 15, and 16. They were fixed and embedded in paraffin and serially sectioned in either the sagittal or transverse plane. The sections were stained with H&E, examined, and photographed. The comparative morphogenesis of the hindgut, cloaca, and tailgut of age-matched embryos was studied. Results: The key abnormalities in the experimental embryos were: (1) maldevelopment of cloaca and urorectal septum with no sign of the fusion between the urorectal septum and the cloacal membrane, (2) delay of tailgut regression, (3) abnormal and massive apoptotic cell death involving the posterior cloacal wall, and (4) underdevelopment of the dorsal aspect of the cloaca and its membrane. The type of ARM that was developing was discernible by gestational day 15 and 16. Conclusions: ARMs induced by ETU in rat embryos seem to be caused by the cumulative effect of aberrations in the development of several components of the hindgut and cloaca. Variation in the extent of maldevelopment of these structures may result in a spectrum of ARMs. J Pediatr Surg 37:1305-1312. Copyright 2002, Elsevier Science (USA). All rights reserved.

Ontogeny of bladder agenesis in rats induced by adriamycin.

To determine the anatomical steps leading to bladder agenesis in rats prenatally exposed as fetuses on gestational days (GD) 6-9 to adriamycin. Timed-pregnant Sprague-Dawley rats were injected intraperitoneally with adriamycin at 2 mg/kg (n = 28) on GD 6-9 (vaginal plug = day 0). The control group (n = 21) received saline. Fetuses were harvested on GD 10, 11, 12, 13, 14, 15 and 16. Serial paraffin sections were prepared from a minimum of 10 experimental and five control fetuses at each gestational age, and stained with either trichrome or haematoxylin and eosin, and examined by light microscopy. In the control group the urorectal septum first became visible and the mesonephric ducts apparently abutting the anterior cloaca on GD 12. The presumptive urinary bladder was clearly defined on GD 14. On GD 15, the common excretory ducts became incorporated into the newly formed urogenital sinus and the ureters opened into the bladder. In the treated animals, beginning on GD 11, the undivided cloaca was noticeably smaller and by GD 13-14, the vesical extension of the urogenital sinus was conspicuously absent. Instead, opposite ureters joined to drain directly into the proximal blind-ending urethra or the persistent distal urogenital sinus. Prenatal exposure of rat fetuses to adriamycin resulted in primary agenesis rather than secondary resorption of the bladder. The ontogeny showed that the mechanism underpinning bladder development is unique and is under the influence of factors that can be targeted by adriamycin. Further work will elucidate the unique nature of bladder organogenesis and should have important applications in future research into artificial bladders.

OEIS complex (omphalocele-exstrophy-imperforate anus-spinal defects): a review of 14 cases.

OEIS complex refers to a combination of defects consisting of omphalocele, exstrophy of the cloaca, imperforate anus, and spinal defects. Possible embryologic mechanisms proposed for these findings have included: a single defect of early blastogenesis or a defect of mesodermal migration during the primitive streak period. Fourteen cases with OEIS complex and related malformations were reviewed for demographic features, prenatal and family histories, and clinical, radiological and pathological findings including the frequency and types of associated anomalies. The pathogenetic mechanisms causing OEIS complex and related malformations, such as anorectal and spinal defects, are discussed. The findings in these cases illustrate the spectrum of defects that can occur in the embryologic development of the cloaca and the urorectal septum. Differences in the timing and extent of mesenchymal ingrowth as well as cloacal membrane rupture may account for these variable findings. A developmental field defect involving the intraembryonic mesoderm suggests a possible etiologic role for homeobox genes, such as HLXB9 with mutations, resulting in anorectal and spine abnormalities, or retinoic acid receptors. OEIS complex with its mostly sporadic occurrence suggests etiologic heterogeneity with a possible role for environmental and genetic causes.

OEIS complex (omphalocele-exstrophy-imperforate anus-spinal defects): A review of 14 cases

OEIS complex refers to a combination of defects consisting of omphalocele, exstrophy of the cloaca, imperforate anus, and spinal defects. Possible embryologic mechanisms proposed for these findings have included: a single defect of early blastogenesis or a defect of mesodermal migration during the primitive streak period. Fourteen cases with OEIS complex and related malformations were reviewed for demographic features, prenatal and family histories, and clinical, radiological and pathological findings including the frequency and types of associated anomalies. The pathogenetic mechanisms causing OEIS complex and related malformations, such as anorectal and spinal defects, are discussed. The findings in these cases illustrate the spectrum of defects that can occur in the embryologic development of the cloaca and the urorectal septum. Differences in the timing and extent of mesenchymal ingrowth as well as cloacal membrane rupture may account for these variable findings. A developmental field defect involving the intraembryonic mesoderm suggests a possible etiologic role for homeobox genes, such as HLXB9 with mutations, resulting in anorectal and spine abnormalities, or retinoic acid receptors. OEIS complex with its mostly sporadic occurrence suggests etiologic heterogeneity with a possible role for environmental and genetic causes.

Partial urorectal septum malformation sequence: a report of 25 cases.

We have identified 25 cases with what we are calling the partial urorectal septum malformation (URSM) sequence, which were seen in our institution over the past 27 years. The partial URSM sequence is defined as a single perineal/anal opening that drains a common cloaca in combination with an absent (imperforate) anus. In the 25 patients reported here, the partial URSM sequence was more common in females, with a female to male ratio of 18 to 7. Ambiguous genitalia were common in both sexes. Internal pelvic structures typically showed a cloaca with the bladder and rectum (and vagina in females) coalescing into a common canal that connected to the external surface in the perineal or anal area. Abnormalities of the internal genitalia were also common, with 12 females having a bifid or septate vagina and 11 having a bicornuate uterus. Renal anomalies were frequent in both sexes, with 10 of 25 patients having unilateral cystic renal dysplasia and 7 of 25 patients having unilateral renal agenesis. Twenty-one of 25 patients survived long term. By definition, the partial URSM sequence is a milder expression of the full URSM sequence, which is defined as having no perineal or anal openings and is typically associated with an internal cloaca. The URSM spectrum, which encompasses the partial and full URSM sequences, is believed to be caused by abnormalities of septation of the primitive cloaca. The URSM spectrum is distinct from the VATER association and conditions caused by sex hormone abnormalities, such as congenital adrenal hyperplasia.

Clarification of the process of separation of the cloaca into rectum and urogenital sinus in the rat embryo

Background/Purpose: The normal process of division of the cloaca into a rectum and urogenital tract is still not fully understood. The main controversies relate to how the urorectal septum (URS) divides the cloaca and whether the URS fuses with the cloacal membrane. This study used a 3-dimensional reconstruction technique, combined with histologic correlation, to observe the developmental and septational processes of the cloaca of the normal rat embryo from gestational days 11 to 16. Methods: Normal rat embryos from gestational days 11 to 16 were sectioned serially both transversely and sagittally and stained with H&E. 3-dimensional reconstructions were performed on embryos younger than day 13.5. The relevant structures were examined in a temporo-spatial sequence. Results: The tailgut started to regress by apoptosis on day 12 in a cranio-caudal direction. The URS, first evident in day-12.5 embryos, extended and fused with the cloacal membrane on day 15 of gestation, completing the separation of the cloaca into rectum and bladder. Regression of the tailgut and ventral protrusion of the urogenital sinus markedly changed the configuration of the cloaca. The cloacal membrane did not break down until after it had fused with the URS. Conclusions: These findings clarify the relative contributions made by active septation of the cloaca by the URS and configurational changes of the cloaca to produce a rectum and bladder. The URS fuses with the cloacal membrane before the anal and urogenital membranes break down. J Pediatr Surg 35:1810-1816. Copyright © 2000 by W.B. Saunders Company.

DOES THE URORECTAL SEPTUM FUSE WITH THE CLOACAL MEMBRANE?

Traditional theories of cloacal embryogenesis assume that the urorectal septum fuses with the cloacal membrane before the anal membrane disintegrates. However, recent observations in humans and other species raise doubt about this assumption. We determined whether urorectal septum fusion occurs in rats. Rat embryos were harvested at specific times between days 11 and 16 of gestation. We evaluated the morphology, growth and relationship of the urorectal septum to the cloacal membrane on serial histological sections. The urorectal septum consistently fused with the cloacal membrane on day 15 of gestation before the cloacal membrane began to disintegrate. In rats the urorectal septum fuses with the cloacal membrane, after which the urogenital membrane and anal membrane disintegrate by a process of apoptosis.

Apoptosis during regression of the tailgut and septation of the cloaca

Purpose: Apoptosis is involved in the embryonic morphogenesis of many organs. The current study was undertaken to ascertain the role of apoptosis during cloacal development in the rat. Methods: One hundred five rat embryos, ranging from gestational days 11 to 16, were sectioned serially in the transverse or sagittal planes and stained with H&E. The cloaca, urorectal septum, rectum, urogenital sinus, Wolffian ducts, and tailgut (TG) were examined consecutively in temporospatial sequence. Results: The tailgut immediately distal to the hindgut starts to regress by apoptosis on day 12 of gestation in a craniocaudal direction and has regressed completely by day 13.5. A large number of apoptotic cells and debris can be identified in the urorectal septum during cloacal septation. Vacuoles are formed by coalescence of apoptotic cells at the tip of urogenital sinus from day 15 to 16, and, at the same time, sporadic apoptotic bodies in the anal membrane contribute to its thinning. Conclusion: Results of the current study confirm that apoptosis occurs in a specific temporo-spatial sequence in the hindgut and cloaca and appears to be an important mechanism in TG regression, uro-rectal separation, urethral opening, and rupture of the anal membrane. J Pediatr Surg 35:1556-1561. Copyright © 2000 by W.B. Saunders Company.

Urorectal septum malformation sequence: prenatal sonographic diagnosis in two sets of discordant twins.

Urorectal septum malformation sequence (URSMS) is a rare congenital malformation, which includes ambiguous genitalia, a phallus-like structure, imperforate anus, bladder, vaginal and rectal fistulas and Müllerian duct defects. We report two cases of prenatally diagnosed URSMS, both occurring in two sets of discordant twins. To the best of our knowledge, this is the first antenatal description of such an anomaly. The first fetus, one of a set of monochorionic, monoamniotic twins was detected sonographically at 21 weeks of gestation due to an enlarged phallus-like formation. The second fetus, one of dichorionic, diamniotic twins, was suspected of having an abnormally enlarged rectum at 13 weeks of gestation. The diagnosis of URSMS was established at 29 weeks of gestation by showing abnormal female external genitalia, with a dilated bowel that contained echogenic foci due to enterolithiasis. The diagnosis of both cases was confirmed postnatally. Sonographic findings and differential diagnosis are presented.

P49Prenatal diagnosis of myelocystocele‐cloacal exstrophy in a male neonate with a abnormal penis and urethra

IntroductionCloacal exstrophy results from a migration failure of the lateral mesodermal folds of the infraumbilical anterior abdominal wall, and rupture of the resulting enlarged, persistent cloacal membrane before the eighth week of gestation. Myelocystocele is a defect of the medular channel characterizased by a cystic that contain dural and arachnoid layer and the ependymal lining layer, respectively. The association of persistent cloaca and caudal spinal anomalies is thought to be related to cell loss in the caudal cell mass, which affects caudal spinal column formation and cleavage of the cloaca by the urorectal septum. Cloacal malformation occurs in approximately 1 in 65 000 live male births. Prenatal ultrasound may lead to the diagnosis in selected cases.Case reportWe report an unusual case of prenatally detected with a myelocystocele and cloacal malformation. Additional anomalies included an omphalocele and malformations of the upper urogenital tract, skeleton, and vertebral column. Labor was induced at 34 weeks' estimated gestational age. It finish by eutocical delivery. A 2154‐g male was born, Apgar 6/8, pH 7.32. The newborn was taken to NCIU and the neonatal outcome was favourable.