The urorectal septum malformation sequence (URSMS) is characterized by the absence of urethral and vaginal perineal openings, ambiguous genitalia, internal genitourinary (GU) anomalies, and imperforate anus. Although URSMS was first described in 1542, its pathogenesis remains obscure. In this report, we characterize URSMS in discordant twin lambs and discuss its ontogeny with respect to genetic and teratogenic influences on blastogenesis.
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. Anat Rec 268:405–410, 2002. © 2002 Wiley-Liss, Inc.
MATERIALS AND METHODS
Delivery and Management of the Lambs
Twin lambs were delivered electively by Cesarean section at 126 days gestation (term = 147 ± 2 days) (Hasan and Rigaux, 1991). The ewe was mated on a specific day to allow precise knowledge of gestational age. The lambs were weighed (Table 1). Mechanical ventilation was begun with a high-frequency jet ventilator (420 cycles/min; Bunnell Life-Pulse, Salt Lake City, UT). Urine output was determined from diaper weights before and after each voiding. The lambs received sodium penicillin, 100 mg/kg, i.v. every 12 hr, and gentamicin, 2.5 mg/kg every 24 hr. The lambs received parenteral nutrition with solutions containing glucose, protein, electrolytes, trace metals, and vitamins. Enteral feeding was initiated on day 2 of life, using the ewe's colostrum and milk, which were administered by gravity feedings through an orogastric tube. The lambs were weighed daily to monitor fluid balance and nutritional status.
|Assessment||Twin A (normal)||Twin B (URSMS)|
|DOL 1||2,935 g||3,345 g|
|DOL 2||3,385 g||3,825 g|
|DOL 1||74 g/24 h||39 g/24 h|
|DOL 2||61 g/24 h||165 g/24 h|
|DOL 1||1 g/24 h||0|
|DOL 2||1 g/24 h||0|
The lambs were killed after 48 hr with .30 mL/kg Beuthanasia®-D Special (390 mg/mL pentobarbital sodium and 50 mg/mL phenytoin sodium; Schering-Plough Animal Health, Kenilworth, NJ). These studies were approved by the Institutional Animal Care and Use Committee at the University of Utah.
Normal placental size and absence of oligohydramnios were documented in both lambs at the time of delivery. Twin A had normal female internal and external anatomy and meconium staining. Blood gases obtained from the common carotid artery, with an intact umbilical cord, showed pH 7.29, pCO2 13 mm Hg, and pO2 56 mm Hg. Twin B, also a female, was not meconium stained. Blood gases showed pH 7.18, pCO2 45 mm Hg, pO2 64 mm Hg. Further examination of twin B revealed anal atresia and abnormal external genitalia (Fig. 1a). A phallus-like structure, likely derived from a genital tubercle, was present on the perineum. At its thin, elongated tip was an orifice, through which urine passed within 12 hr. The abdomen was not distended. Caudal to the phallus-like structure was a longitudinal furrow, where the anus should have been (imperforate anus, Fig. 1a). The remainder of the external physical exam was entirely within normal limits for gestational age. Specifically, the iris, hind limbs, and tail were normally formed. The clinical course of each lamb is summarized in Table 1.
Necropsy of twin B revealed severe GU anomalies. The cloaca was incompletely divided into bladder, vagina, and rectum (Fig. 1b–d). It drained via the phallus-like structure. The rectum was filled with urine and stool (Fig. 1c). The lamb had a normal bicornuate uterus, bilateral uterine tubes, and ovaries (Fig. 1b and c). There was no hydronephrosis or hydroureter, and the kidneys had grossly normal size and architecture. Further dissection revealed no anomalies in the sacrum, ribs, limbs, or heart, and no tracheo-esophageal fistula. Iris coloboma was not present. Twin A, also a female, had occupied the other uterine horn. Her exam was entirely normal for gestational age (not shown).
One of the twin lambs had a urorectal septum malformation sequence (URSMS). The afflicted lamb was likely a dizygous twin, since its sibling occupied a separate uterine horn and had a separate placenta and amniotic sac, ruling out mechanical errors in blastogenesis associated with monozygous twinning. The other lamb was normal, making teratogenic exposure unlikely. Moreover, variations in response to a given teratogen based on embryonic stage seem unlikely, because only one twin was affected. As discussed below, genetic defects can result in deficiencies in caudal mesoderm, interfere with normal anterior–posterior differentiation, and disrupt signaling between the endoderm and mesoderm, all hypothesized to contribute to the URSMS. The genes discussed all interact with Shh, and retinoic acid, a commonly used experimental teratogen, also regulates Shh gene expression (Chang et al., 1997).
First described in 1542 by Rocheus (cited by Padmanabhan et al., 1999), the URSMS is characterized by the absence of urethral and vaginal perineal openings, ambiguous genitalia, internal genitourinary (GU) anomalies, and imperforate anus. In humans, the reported prevalence of imperforate anus varies widely, ranging from 1/250,000 (Escobar et al., 1987) to 1/1,500 (Kimmel et al., 2000) live births. It is equally distributed between the sexes (Hall et al., 1970; Mesrobian et al., 1994; Stoll et al., 1997; Wheeler et al., 1997) and may be associated with severe congenital anomalies of other major organ systems (Hall et al., 1970; Carlton et al., 1973; Wiener and Kiesewetter, 1973; Escobar et al., 1987; Stoll et al., 1997; Wheeler et al., 1997; Endo et al., 1999). Fistulas between the rectum and GU system are frequent and contribute to the pathology of the condition, because they predispose to urinary tract infection with enteric flora. The location of these fistulous connections varies with gender; among 1,183 Japanese boys with imperforate anus, 333 (28%) had rectourethral connections and 42 (3.6%) had rectovesical fistulas, whereas 241 (30%) of 809 affected girls had anovesical fistulas and 93 (11%) had rectocloacal fistulas (Endo et al., 1999).
The natural incidence of URSMS in other species is less clear. There have been isolated reports of cloacal anomalies in foals (Richardson, 1985; Brown et al., 1988) and calves (Prieur and Dargatz, 1984; Dean et al., 1996), and a genetic porcine model (Van der Putte and Neeteson, 1984) has been described. This first report of ovine URSMS is noteworthy because it typifies the malformation in females, and provides insight into the pathogenesis of the disorder.
The cloaca is the most distal portion of the embryonic hindgut. It is lined with endoderm. At about the time of posterior neuropore closure (Kubota et al., 1998), the mesodermal urorectal septum (URS) proliferates caudally and fuses with the cloacal membrane. Simultaneously, lateral mesodermal urorectal folds grow inward from the sides (Johnson et al., 1972). The URS divides the cloaca into the urogenital (UG) sinus and rectum/cranial end of the anal canal (Fig. 1d). The cloacal membrane is thus divided into the UG diaphragm and the anal membrane. Subsequently, the cloacal membrane ruptures, creating anal and perineal openings (Escobar et al., 1987; Wheeler et al., 1997; Kimmel et al., 2000). In females, the Müllerian tubercle (the union of the Müllerian duct with posterior wall of the UG sinus) develops an opening into the UG sinus, now identified as the vagina and uterus (Escobar et al., 1987). Finally, inductive signals from the endoderm (Kimmel et al., 2000) and cloacal membrane appear to be required for normal differentiation of the external genitalia (Padmanabhan et al., 1999; Catala, 2002).
The URSMS occurs when this normal sequence of events is disrupted. Imperforate anus results from persistence of the dorsal cloacal membrane (Kimmel et al., 2000; Catala, 2002) and ventral persistence causes absent urethral and vaginal openings. Incomplete migration and/or fusion of the URS with the cloacal membrane leads to persistence of the cloaca and/or fistulae. In contrast, cloacal exstrophy ensues if the cloacal membrane is destroyed, confirming its requirement for normal formation of the perineum and adjacent structures (Escobar et al., 1987; Catala, 2002). Lack of inductive signals results in ambiguous genitalia and a phallus-like structure. Although the gonads are normal (Padmanabhan et al., 1999), Müllerian duct anomalies are common. In one study, 12 of 30 (40%) affected females had complete absence of the vagina, and the uterus was absent in 11 (37%) (Wheeler et al., 1997). Bicornuate uterus can occur, although this is the normal state in sheep, and thus does not constitute an anomaly in the case reported here. Renal anomalies are commonly reported (Mesrobian et al., 1994), but a patent urachus can protect against obstructive uropathy (Wheeler et al., 1997). In our case, the upper urinary tracts (defined as kidneys and ureters) were normal; therefore, we propose that the fistulous connection between the GU tract and the rectum, combined with the passage of some urine from the perineal orifice, decompressed the upper urinary tract.
What events lead to the URSMS? The lateral compression theory originally proposed in 1891 by Dareste (cited by Padmanabhan et al., 1999), in which abnormal amniotic folds compress the caudal end of the embryo, suppressing development of pelvic structures and causing abnormal rotation of limbs, seems overly simplistic and does not explain other associated malformations (Table 2). In 1886, Weigert (cited by Padmanabhan et al., 1999) suggested that vascular steal might explain the URSMS, noting a single umbilical artery and hypoplastic aorta in an affected child. However, there was no evidence that the vascular anomaly preceded the caudal regression, and, again, this theory does not explain the associated anomalies. In our case, each lamb had two umbilical arteries and a normally sized aorta.
|Theory or event||Reference|
|URSMS first described||Rocheus (1542)a|
|Vascular steal theory proposed||Weigert (1886)a|
|Lateral compression theory proposed||Dareste (1891)a|
|Insulin causes rumplessness in chicks||Landauer (1945)|
|Deficiency of caudal mesoderm proposed||Kallen and Winberg (1974)|
|Currarino triad described, associated with HLXB9 mutations||Currarino and Weisbruch (1989)|
|Proposal: teratogens cause||Opitz (1993)|
|blastogenetic process of twinning and associated malformations||Kramer et al. (1997)|
|Retinoic acid acts on Hox genes, alters mesoderm||Mesrobian et al. (1994)|
|Hand-foot-genital syndrome associated with mutations in||Warot et al. (1997)|
|Polytopic developmental field defect proposed||Padmanabhan et al. (1999)|
|Martinez-Frias et al. (2000)|
|Shh acts via Gli3||Ruiz i Altaba (1999)|
|Kimmel et al. (2000)|
|Kim et al. (2001)|
|Mo et al. (2001)|
|Long range regulatory elements for SHH reside within the coding region of HLXB9||Mo et al. (2001)|
|Pallister-Hall syndrome associated with GLI 3||McKusick (2002)|
|mutations, characterized by imperforate anus|
|Townes-Brock syndrome caused by SALL-1 mutations, deficient mesoderm||McKusick (2002)|
Embryonic malformations can develop during blastogenesis, pattern formation or organogenesis. During blastogenesis, the embryo responds as a pluripotent single unit. Events in this primary field stage include formation of the midline axis, Hensen's node, and the primitive streak. Gastrulation produces the three germ layers (the ectoderm, mesoderm, and endoderm). Later events include segmentation of the paraxial mesoderm into somites, laterality determination, and the initial events of cardiogenesis (Padmanabhan et al., 1999). Pattern formation establishes upstream domains of specific transcription, signaling, and gene expression during the subdivision of the primary field into progenitor fields (Padmanabhan et al., 1999). Expression domains of key genes are present before there is any visible organogenesis, and two or more fields may share the same signaling molecules (Padmanabhan et al., 1999). The definitive structure is formed during organogenesis in secondary (epimorphic) fields.
The URSMS is commonly accompanied by vertebral defects, anal atresia, tracheo-esophageal fistula/esophageal atresia, radial and renal dysplasia, cardiovascular malformations, and limb anomalies (Chen et al., 1998). These associated abnormalities provide clues regarding the pathogenesis of URSMS. Collectively, they are considered to be primary polytopic developmental field defects, because they arise in response to a dysmorphogenetic stimulus, either during blastogenesis or when progenitor fields are being established (Padmanabhan et al., 1999; Martinez-Frias et al., 2000).
Kallen and Winberg (1974) suggested that a deficiency of caudal mesoderm and/or irregularities of the notochord and somites might lead to anorectal dysgenesis, as well as GU and vertebral malformations. Narrow defects would result in median anomalies such as sacral agenesis, whereas more extensive defects could lead to lateralized malformations such as limb defects.
Previous studies (Opitz, 1993; Kramer et al., 1997) indicated that teratogens cause both the blastogenic process of twinning and the associated malformations. Indeed, many animal models of the URSMS rely on teratogenic manipulation of the embryo. Padmanabhan et al. (1999) reported that cadmium, lead, trypan blue, retinoic acid, and ochratoxin A induce caudal dysgenesis in experimental animals. Another study (Liu and Hutson, 2000) found that rat fetuses exposed to Adriamycin on embryonic days 6–9 (E6–E9) had no bladders, and had severe hydroureter and hydronephrosis. Males had proximal blind-ending urethras communicating with dilated ureters. In the affected females, persistent urogenital sinuses communicated with the ureter, cervix, or uterus. Fifty-seven percent of the females had imperforate anus and some had rectal fistulae.
In a study by Mesrobian et al. (1994), a single dose of etretinate on E8 (equivalent to week 4 in human gestation) in the C57BL/6J mouse caused caudal regression (spina bifida, imperforate anus, GU anomalies, omphalocele, and limb anomalies). These defects appeared to result from a major insult to the primitive streak. On E9, the final day in which the primitive streak contributes to the embryonic trunk, etretinate caused anal and urethral atresia, bladder and ureteral dilation, and tail deficiencies. There was no evidence for spina bifida, limb anomalies, or renal agenesis. Narrowing of the genital tubercle was noted. Etretinate administration was associated with decreased caudal cell proliferation. There was no clear evidence of excess cell death in the primitive streak or hindgut; instead, apoptosis was deficient in etretinate-treated embryos. The authors hypothesized that deficient proliferation could result in diminished mesoderm available for completion of the cloacal region.
Some teratogens act by modulating gene activation. Exogenous retinoic acid alters homeobox (Hox) codes, resulting in homeotic transformations. Appropriately timed retinoic acid exposure may result in segmentation defects such as fusion or absence of vertebrae (Bohring et al., 1999; Padmanabhan et al., 1999). During normal embryogenesis, midline structures generate retinoic acid signals that sequentially activate Hox genes along the anterior-posterior axis (Padmanabhan et al., 1999). Hox genes act in part by controlling local rates of cell proliferation (Warot et al., 1997), and altered Hox gene expression could thus contribute to the diminished mesoderm available for completion of the cloacal region hypothesized by Mesrobian and colleagues (1994).
Hoxa-13 (human chromosome 7p15-p14.2, Genbank accession #142959) and Hoxd-13 (2q31-q32, Genbank #142989) are expressed in the mesenchyme of the genital tubercle, hindgut, cloaca, and limb in mouse, and their cognate genes are similarly expressed in chicken and zebrafish. In mice, Hoxa-13 null mutants had agenesis of the caudal ends of their Müllerian ducts, lacked bladder development, and displayed premature umbilical artery stenosis, resulting in midgestation lethality. Hoxa-13+/-/Hoxd-13-/- compound mutants reached adulthood. Females were sterile and had abnormal genitalia, with reduced spacing between the anus and genitalia. Either the caudal end of the urethra and vagina ended together, or the vagina and anus ended together. Malpositioning of the vaginal, urethral, and anal openings; improper separation of the vagina from the UG sinus; hydronephrosis; and anomalies of the muscular and epithelial layers of the rectum were noted. One of 14 fetuses showed almost complete absence of the terminal portion of the rectum. Hoxa-13-/-/Hoxd13-/- fetuses demonstrated no separation of the cloaca into the UG sinus and rectum, and no development of the genital tubercle. Hydronephrosis was noted, unaccompanied by urethral obstruction (Warot et al., 1997). In humans, the hand-foot-genital syndrome (OMIM 140000) is caused by a nonsense mutation in the third helix of the HOXA13 homeodomain (McKusick, 2002). Subtle digital anomalies and GU malformations characterize the syndrome. Females have “duplications” of the uterus, displaced urethral openings, and malpositioned ureteral orifices in the bladder wall. Males have hypospadias (Warot et al., 1997).
Hoxd13 expression is expanded in Gli3 mutant mice (Moribe et al., 2000) and a human GLI3 (7p13, Genbank #165240) mutation causes Pallister-Hall syndrome (OMIM #146510), which is characterized by anal stenosis or imperforate anus (McKusick, 2002). Gli proteins are sonic hedgehog (Shh, 7q36, Genbank #600725) responsive transcription factors (McKusick, 2002). Whether they act as positive or negative mediators of Shh signaling depends upon their C-terminal truncation. Their N-termini mediate nuclear translocation and act as dominant negatives, whereas their C-termini are important in maintaining a cytoplasmic location and in positively mediating the inducing activity of Shh (Ruiz i Altaba, 1999). Gli2-/- mutant mice have imperforate anus and rectourethral fistula, whereas Gli3-/- mutant mice have anal stenosis and ectopic anal openings in which the hindgut opens into the urogenital sinus. Gli2-/-/Gli3+/- compound mutants have persistent cloacal abnormalities, with thinner, multilayered cloacal membranes; in these mice, the hindgut joins the distal end of the ureter (Kimmel et al., 2000; Kim et al., 2001; Mo et al., 2001).
Features of the URSMS characterize several other human syndromes. The homeobox gene HLXB9 (Genbank #142994) is mutated in the Currarino triad (OMIM #176450), which is composed of dominantly inherited anorectal and GU malformations, a presacral mass, and partial sacral agenesis (Currarino and Weisbruch, 1989; Currarino, 1996; Belloni et al., 2000; McKusick, 2002). The observed pattern of anomalies can be explained by a malformation in the caudal portion of the notochord, resulting in aberrant secondary neurulation. Interestingly, HLXB9 mutant mice lack a urogenital phenotype; however, human HLXB9 is located in close proximity to SHH at 7q36, with a LOD score of 4.82 and a theta of 0.05 (OMIM), and SHH long-range regulatory elements reside within the coding region of HLXB9 (Mo et al., 2001). Thus, the features of the Currarino triad may be caused by altered SHH signaling. HLXB9 is expressed in the developing pancreas, and 25% of infants with the Currarino triad are infants of diabetic mothers (Ross et al., 1998; Hagan et al., 2000; Lynch et al., 2000). Maternal diabetes is associated with developmental field defects (Chen et al., 1998; Padmanabhan et al., 1999), but animal studies show that hyperglycemia is not the only causal factor because insulin therapy failed to abolish teratogenic potential of serum from diabetic rats (Wentzel and Eriksson, 1996). Moreover, insulin may cause rumplessness in chicks (Landauer, 1945). Thus, the association of maternal diabetes with HLXB9 expression in the fetal pancreas, and mutations in the gene in the Currarino triad is intriguing.
In addition to mutations in HLXB9 and SHH at chromosome 7q36, two families have been reported with caudal deficiency sequence, sacral agenesis, and deletion of chromosome 7q69-72 (Wang et al., 1999). Cat eye syndrome (inv dup (22)(q11), OMIM #115470), named for its iris coloboma, is due to partial tetrasomy of chromosome 22 (McKusick, 2002). It has substantial clinical variability, but can include anal atresia with a rectal fistula into the bladder, vagina, urethra, vulva, or perineum. Mutations in the SALL 1 (Genbank #602218) putative transcription factor gene on human chromosome 16q12.1 have been associated with rectovaginal fistula and imperforate anus in Townes-Brocks syndrome (OMIM #107480 (McKusick, 2002)). Chen et al. (1998) reported a girl with isochromosome 18q (monosomy 18p, trisomy 18q) who had congenital megacystis, cloacal dysgenesis, oligohydramnios, pseudohermaphroditism, imperforate anus, and a phallus-like structure.
Our lamb was likely a dizygous twin, unaffected by teratogens, since the co-twin was normal. Many of the gene defects summarized above result in deficiencies in caudal mesoderm via interaction with Shh, and retinoic acid, a commonly used experimental teratogen, also regulates Shh gene expression (Fig. 2) (Chang et al., 1997). Thus, it is plausible that a genetic mutation caused the malformations reported in the affected lamb.
The authors are supported by the National Institutes of Health, grants HL62875 (to K.H.A.) and DK02490 (to T.J.M.), and the March of Dimes Birth Defects Foundation, grant 6-FY00-75 (to T.J.M.).