Distinct Transcriptional Profiles of the Female, Male, and Finasteride-Induced Feminized Male Anogenital Region in Rat Fetuses.

A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that androgen receptor-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The antiandrogenic drug finasteride (10 mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days 7-21. The AGD was 37% shorter in exposed male fetuses compared with control males at gestation day 21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the 3 groups. The expression pattern of 4 genes of particular interest (Esr1, Padi2, Wnt2, and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment.

First trimester expression of anorectal malformation: Case report and review of the literature.

A 26-years-old woman, underwent an ultrasound examination at 13.4 weeks. A cystic structure was identified in the right lower abdomen. Gradually, the cystic mass was replaced by echogenic content and eventually attained the appearance of hyperechoic bowel. At 21.2 weeks, the anal sphincter could not be demonstrated which was consistent with the diagnosis of isolated anal agenesis. Amniocentesis revealed 46XY karyotype with normal comparative genomic hybridization. After termination of pregnancy at 23 weeks, an autopsy revealed an isolated high type anorectal malformation (ARM) without fistula. We reviewed all 14 cases reported in the literature of first trimester sonographic expression of ARM.

Tracheal Atresia with Segmental Esophageal Duplication: An Unusual Anatomic Arrangement.

An unusual anatomic configuration of segmental tracheal agenesis/atresia with esophageal duplication on autopsy in a fetus that demised in utero at 29 weeks is reported. The mother was scanned initially for a cardiac anomaly at 20 weeks and on follow-up scan at 27 weeks had polyhydramnios and underwent amnioreduction. The final autopsy diagnosis was vertebral, ano-rectal, cardiac, tracheoesophageal, renal, and limb malformations (VACTERL). We discuss the autopsy findings along with the embryological mechanisms and compare the configuration with Floyd's classification for tracheal agenesis. The difficulties in prenatal diagnosis are discussed.

Is it possible folic acid reduce anorectal malformations ethylenethiourea induced in rats?

PURPOSE: To investigate the effect of folic acid (FA) in an experimental model of anorectal malformations (ARMs) ethylenethiourea (ETU) induced.METHODS:Eight female Wistar rats were divided randomly in two groups. Group A - ETU; Group B - FA+ETU; Dams from group B received daily, since two weeks before pregnancy to the end of pregnancy, FA (50mg/kg) by gavage. Dams from groups A and B, received 1% ETU (125mk/kg) by gavage on gestational day (GD) 11. Their fetuses were harvested by cesarean section on GD21 and were examined looking for ARMs. The thickness of anal stratified squamous epithelium (ASSE) and intestinal epithelium (IE) were analyzed. p<0.05*.RESULTS:One hundred and one embryos were harvested. The number of embryos; number of ARMs; mean statistical % (± SD) were determined to be, respectively: ETU - 49 [30;65% (±24%)] versus FA+ETU - 52 [1;02% (±3%)] (p=0.025). AMRs were significantly lower in FA+ETU group than in ETU group (p=0.025). The thickness (µm) of ASSE (± SD) and IE (± SD) were measured, respectively: ETU - [27.75 (±0.56) and 18.88 (±0.93)] versus FA+ETU - [28.88 (±0.61) and 21.11 (±0.16)] (p=0.001). The thickness of IE was significantly enlarged when FA was given (p=0.001).CONCLUSION:Folic acid reduces the number and enlarged the IE of ARMs ETU-induced.

The great divide: septation and malformation of the cloaca, and its implications for surgeons.

The anorectal and urogenital systems arise from a common embryonic structure termed cloaca. Subsequent development leads to the division/septation of the cloaca into the urethra, urinary bladder, vagina, anal canal, and rectum. Defective cloacal development and the resulting anorectal and urogenital malformations are some of the most severe congenital anomalies encountered in children. In the most severe form in females, the rectum, vagina, and urethra fail to develop separately and drain via a single common channel known as a cloaca into the perineum. In this review, we summarize our current knowledge of embryonic cloaca development and malformation, and compare them to what has already been described in the literature. We describe the use of mouse models of cloaca malformation to understand which signaling pathways and cellular mechanisms are involved in the process of normal cloaca development. We also discuss the embryological correlation of the epithelial and stromal histology found in step sections of the common channel in 14 human cloaca malformations. Finally, we highlight the significance of these findings, compare them to prior studies, and discuss their implications for the pediatric surgeons. Understanding and identifying the molecular basis for cloaca malformation could provide foundation for tissue engineering efforts that in the future would reflect better surgical reconstruction and improved quality of life for patients.

Abnormal neural crest innervation in Sox10-Venus mice with all-trans retinoic acid-induced anorectal malformations.

BACKGROUND/PURPOSE: Despite technical advances in the surgical/medical care of anorectal malformation (ARM), persistent unsatisfactory postoperative bowel habit has been attributed to histopathologic abnormalities of the distal rectum/pouch (DRP) and hypoplasia of anal sphincter muscles (ASM). We used Sox10-Venus mice with ARM induced by all-trans retinoic acid (ATRA) to investigate neural crest cell (NCC) innervation in the DRP and ASM. METHOD: Pregnant Sox10-Venus mice were administered single doses of 50, 70, or 100 mg/kg of ATRA on embryonic day 8.5 (E8.5) then sacrificed on either E16.5 or E19.5. Bowel specimens comprising the anorectum were examined using fluorescence microscopy without immunohistochemical staining (FMIS). Anti-PGP9.5 was used to delineate ganglion cells and anti-SMA for smooth muscles. RESULTS: The appropriate dose of ATRA for inducing ARM was 50 mg/kg. Under FMIS, all ARM embryos (n = 5; all high type; 3 male:2 female) had less NCC innervation with thick Venus-positive nerve fibers in the DRP compared with normal embryos (n = 8); there was abnormal NCC innervation in the DRP and absent ASM in ARM mice. CONCLUSION: We are the first to delineate abnormal enteric nervous system innervation in the DRP of ARM mice without using immunohistochemical staining techniques thus allowing specimens to be examined without any distortion.

Long-term follow-up of functional outcome in patients with a cloacal malformation: a systematic review.

BACKGROUND: Reconstructive surgery is performed in patients with cloacal malformations to achieve anorectal, urological, and gynecological function. The aim of this study was to evaluate the functional outcome of cloacal malformation repair as reported in literature. METHODS: A systematic literature search was conducted according to PRISMA guidelines using PubMed, EMbase, and Web-of-Science. Records were assessed for the reporting of functional outcomes, which was divided into anorectal, urological, or gynecological function. Studies were used in qualitative (Rangel score) and quantitative syntheses. RESULTS: Twelve publications were eligible for inclusion. Voluntary bowel movements were reported in 108 of 188 (57%), soiling in 146 of 205 (71%), and constipation in 31 of 61 patients (51%). Spontaneous voiding was reported for 138 of 299 patients (46%). 141 of 332 patients (42%) used intermittent catheterization, and 53 of 237 patients (22%) had a urinary diversion. Normal menstruations were reported for 25 of 71 patients (35%). Centers with limited experience reported similar outcome compared to centers with more experience (≥1 patients/year). CONCLUSION: In this review we present functional outcome of the largest pooled cohort of patients with cloacal malformations as reported from 1993 to 2012. Functional disturbances are frequently encountered in anorectal, urological, as well as gynecological systems. Reporting of functional outcome in these patients should improve to increase knowledge about long-term results in patients with this rare malformation and to reach higher study quality. Especially, sacral and spinal anomalies should always be reported given their impact on functional outcome. Specialized care centers may be of great importance for patients with rare and complex conditions.

Effect of abnormal notochord delamination on hindgut development in the Adriamycin mouse model.

BACKGROUND: Adriamycin mouse model (AMM) is a model of VACTERL anomalies. Sonic hedgehog (Shh) pathway, sourced by the notochord, is implicated of anorectal malformations. We hypothesized hindgut anomalies observed in the AMM are the result of abnormal effect of the notochord. METHODS: Time-mated CBA/Ca mice received two intraperitoneal injections of Adriamycin (6 mg/kg) or saline as control on embryonic day (E) 7 and 8. Fetuses were harvested from E9 to E11, stained following whole mount in situ hybridization with labeled RNA probes to detect Shh and Fork head box F1(Foxf1) transcripts. Immunolocalization with endoderm marker Hnf3ß was used to visualize morphology. Embryos were scanned by OPT to obtain 3D representations of expressions. RESULTS: In AMM, the notochord was abnormally displaced ventrally with attachment to the hindgut endoderm in 71 % of the specimens. In 32 % of the treated embryos abnormal hindgut ended blindly in a cystic structure, and both of types were remarked in 29 % of treated embryos. Endodermal Shh and mesenchymal Foxf1 genes expression were preserved around the hindgut cystic malformation. CONCLUSIONS: The delamination of the developing notochord in the AMM is disrupted, which may influence signaling mechanisms from the notochord to the hindgut resulting in abnormal patterning of the hindgut.

Anorectal malformation associated with a mutation in the P63 gene in a family with split hand-foot malformation.

PURPOSE: The aims of this study were to identify the mutation gene of a Chinese family with anorectal malformation (ARM) associated with split hand-foot malformation and to determine the spatiotemporal expression of the mutated gene during hindgut and anorectum development in human embryos. METHOD: A Chinese family with intrafamilial clinically variable manifestation was analyzed and primers were designed for exons 3-14 of P63, DLX5, DLX6, DAC, and HOXD13 as candidate genes and direct sequence analysis of the exons was performed. Immunohistochemical study of mutated gene in the hindgut and anorectum of human embryos of 4th-10th weeks was performed. RESULT: Affected individuals were found to have an Arg227Gln P63 gene mutation. From the 4th-10th weeks of gestation of the human embryo, the P63-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum ruptured during the 8th week, the P63 remained strongly immunoreactive on the epithelium of the anal canal and urethra, but the mucous membrane of the rectum exhibited no reaction. CONCLUSIONS: The mutation identified strongly suggests a causal relationship between the ARM phenotype and P63. The expression of P63 was persistently active during the dynamic and incessant septation of the cloaca and hindgut, suggesting that P63 may play a pivotal role in the morphogenesis of the hindgut and anorectum.

Pelvic exenteration for recurrent squamous cell carcinoma of the pelvic organs arising from the cloaca--a single institution's experience over 16 years.

AIM: Minimal data are available on the role of pelvic exenteration in patients with recurrent squamous cell carcinoma (SCC) of the pelvic organs. This study aimed to highlight our experience of pelvic exenteration in patients with recurrent and re-recurrent SCC of the pelvic organs. METHOD: A retrospective review of all patients who underwent pelvic exenteration for recurrent SCC of the pelvic organs arising from the embryological cloaca from 1994 to 2010 was performed. RESULTS: Twenty-four patients (median age 59, range, 27-79 years) underwent pelvic exenteration for recurrent SCC of the anus (18), cervix and upper vagina (2), lower vagina (1) and the vulva (3). Nine patients with anal SCC had undergone abdominoperineal excision prior to pelvic exenteration. Ten (41.7%) patients underwent a complete pelvic exenteration procedure, while sacrectomy was performed in 13 (54.2%) patients. There was no 30-day inpatient mortality. An R0 resection was achieved in 15 (62.5%) patients. Three (12.5%) had R1 resections while 6 (25%) had R2 resections. In the 15 patients with an R0 resection, 7 (46.7%) developed metastatic disease at a median of 18 (range 10-131) months. After a median follow-up of 26 (range 4-169) months, 1- and 2-year overall survival rates were 64% [95% confidence interval (CI), 44-84%] and 57% (95% CI 35-79%), respectively. CONCLUSION: Pelvic exenteration for recurrent SCC of the cloaca is safe and feasible even after previous salvage surgery. An R0 resection can be achieved in 62.5% of the patients with reasonable early survival though less than published recurrent rectal cancer studies.

Developmental fluoxetine exposure and prenatal stress alter sexual differentiation of the brain and reproductive behavior in male rat offspring.

Depression during pregnancy and postpartum is a significant health problem and affects up to 20% of women. While selective serotonin reuptake inhibitor (SSRI) medications are the drug of choice for treatment of maternal depression, the combined effect of maternal depression and perinatal SSRI exposure on offspring development is poorly investigated. Our aim was to determine the role of exposure to fluoxetine during development on sexual behavior and sexually dimorphic brain structures in male offspring using a rodent model of maternal adversity. Sprague-Dawley rat dams were stressed during gestation and were chronically treated throughout lactation with either fluoxetine or vehicle beginning on postnatal day 1. Four groups of offspring were used: (1) Control+Vehicle, (2) Control+Fluoxetine, (3) Prenatal Stress+Vehicle, and (4) Prenatal Stress+Fluoxetine. We show here that developmental fluoxetine treatment decreases the anogenital distance in juvenile male offspring. In adult male offspring, maternal fluoxetine treatment results in a decrease in the number of intromissions, a longer latency to the first intromission, and a longer latency to the first ejaculation. Furthermore, developmental fluoxetine and/or prenatal stress decrease the area of the sexually dimorphic nucleus of the preoptic area (SDN-POA). Prenatal stress, but not exposure to developmental fluoxetine, decreases the number of tyrosine hydroxylase (TH)-positive cells in anteroventral periventricular nucleus (AVPv) and the volume of the posterior bed nucleus of the stria terminalis (pBST) in male offspring. These results provide important evidence for the long-term impact of maternal adversity and maternal fluoxetine use on the development of primary endocrinology systems in juvenile and adult male offspring.

Abnormal development of intrinsic innervation in murine embryos with anorectal malformations.

INTRODUCTION: Constipation, soiling, and incontinence are common problems after definitive repair of anorectal malformations (ARMs) in children. We studied the expression of substance P (SP), vasoactive intestinal peptide (VIP), and c-kit in the rectum of murine embryos with or without ARMs at later developmental stages. METHODS: On the 9th embryonic day (E9), pregnant Institute of Cancer Research mice were fed etretinate, a synthetic vitamin A analogue (60 mg/kg), whereas controls were fed only with sesame oil. Embryos were excised between E14 and E18, and prepared for histological examination. The SP, VIP, and c-kit expressions were examined by immunohistochemical staining for the SP, VIP, and c-kit antigens, respectively. RESULTS: On E14 and E15, the expression levels of the anti-SP and anti-VIP antibodies in the rectum did not differ between the control and etretinate-treated group. However, as compared to the controls, a decreased SP and VIP immunoreactivity was observed in the circular muscle layer of the rectum between E16 and E18. On the other hand, on E14 and E15, the expression of anti-c-kit antibody in the rectum did not differ between the etretinate-treated and control group. However, c-kit immunoreactivity was slightly higher in the circular muscle layer of the rectum in the controls on E16 and E17, and considerably higher on E18 than that of the muscle layer in the etretinate-treated group. CONCLUSION: At later developmental stages, the expression levels of SP, VIP, and c-kit reduced in the circular muscle layer of the rectum in mice with etretinate-induced ARMs. This result indicates that reduced SP, VIP, and c-kit expression levels in the circular muscle layer may cause severe constipation in children who develop severe ARMs after definitive surgery.

The effect of vitamin A deficiency during pregnancy on anorectal malformations.

OBJECTIVE: The aim of this study was to study the effect of vitamin A deficiency (VAD) on the embryological development of anorectal malformations (ARMs) and the enteric nervous system. MATERIALS AND METHODS: Female Sprague-Dawley rats were divided into 3 groups: VAD group, normal group (negative control), and ethylene thiourea (ETU) group (positive control) with a normal diet. On day 20 of pregnancy, cesarean section was performed on all rats. The incidence of ARMs in the fetal rats and Protein gene product 9.5 (PGP9.5) and S-100 protein expression by immunohistochemistry were determined. RESULTS: The incidence of ARMs in VAD and ETU groups was 64.8% (59/91) and 45.9% (61/133), respectively (P > .05). Anorectal malformations were not found in the normal group. Protein gene product 9.5 and S-100 protein expression in the non-ARM rectums of the VAD group was lower than the ETU (P = .0156 vs P = .0105) and normal groups (P = .0091 vs P = .0024). There was no significant difference in PGP9.5 and S-100 protein expression between ETU and normal groups. In the ARM rectums, PGP9.5 and S-100 protein expression in the VAD group was lower than the ETU group (P < .0001). Protein gene product 9.5 and S-100 protein expression was also lower in ARM than non-ARM rectums in the VAD and ETU groups (P < .0001, P = .0203, and P = .0122, respectively). CONCLUSION: Vitamin A deficiency during pregnancy may result in the embryological development of ARMs. Enteric nervous system development may be related to ARMs.

Clear cells of Toker in the developing anogenital region of male and female fetuses.

The clear cells of Toker are a mysterious population of intra-epidermal glandular cells. They were originally described in nipples, but were recently observed in the vulva as well. It was hypothesized that intra-epidermal embryonic remnants or underlying glands were a potential source. The embryological aspects were investigated by studying specimens of the anogenital region of 18 male and 15 female fetuses between 12 and 39 weeks gestation. The search for Toker cells was enhanced by cytokeratin (CK) 7 immunohistochemistry. The investigation showed that Toker cell elements are a normal, though highly variable constituent of the developing anogenital region. The study revealed the following: (1) single intra-epidermal glandular vesicles near follicular anlages in interlabial sulcuses of female fetuses of 15 and 16.5 weeks gestation; (2) CK7+ solitary cells, clusters, and vesicles which were related to developing intra-epidermal follicular canal tracks and tended to disperse inside the epidermis in fetuses of approximately 18 weeks gestation; (3) dispersed CK7+ cells in fetuses of 19-23 weeks gestation; (4) characteristic CK7+ Toker cell proliferations in fetuses more than 23 weeks gestation. These observations indicate that in the anogenital region, primordial follicular cells programmed to participate in the formation of apocrine and mammary-like glands, become displaced into the epidermis where they disperse, and proliferate into Toker cell populations. However, the proximity of Toker cells to CK7+ cells in excretory ducts of late fetal apocrine and mammary-like glands suggested a possible additional source. Consequences for Toker cells of the breast and primary Paget disease are discussed.

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus.

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.

Embryology of the hindgut.

Normal and abnormal development of the hindgut is still in debate. Normal development of the hindgut critically depends on the cloacal membrane. In this study, scanning electron microscopy of staged rat embryos between the gestational days 10-15 was performed to show the normal development of the hindgut and the abnormal development in Danforth's short tail (SD) mice. Our studies in normal and abnormal development indicate that the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in females. To explain the abnormal development in anorectal malformations, further studies are mandatory.

Spatiotemporal expression of Wnt5a during the development of the hindgut and anorectum in human embryos.

PURPOSE: The aim of the study was to determine the spatiotemporal expression of Wnt5a during hindgut and anorectum development in human embryos and to explore the possible role of Wnt5a during the morphogenesis of the human hindgut and anorectum. MATERIALS AND METHODS: The embryos (n = 107) were sectioned serially and sagittally, using Wnt5a immunohistochemical staining on the caudal midline from the 4th-9th weeks of gestation. RESULTS: From the 4th-7th week of gestation, the Wnt5a-positive cells were mainly located on the epithelium of the apical urorectal septum, hindgut, and cloacal membrane. After the anorectum and the urogenital sinus (UGS) opened to the amniotic cavity during the 7th week, the Wnt5a-positive cells disappeared and remained negative up to the 9th week on the epithelium of the anal canal. CONCLUSIONS: The expression of Wnt5a was constantly active during human hindgut and anorectum development and disappeared after the anus formed, suggesting that Wnt5a plays an important role in human hindgut and anorectal morphogenesis.

Abnormal innervation patterns in the anorectum of ETU-induced fetal rats with anorectal malformations.

To investigate whether anorectal malformations (ARMs) were associated with a global neuromuscular maldevelopment of the lower gastrointestinal (GI) tract and anorectum, the distribution of neuronal markers protein gene product (PGP9.5), nitric oxide synthases (NOs), neuromuscular junction markers (synaptophysin, SYP), interstitial cells of Cajal (ICC) marker (c-kit) within the terminal rectum were analyzed by immunohistochemistry and Western blot in rat embryos with ethylenethiourea (ETU) induced ARMs. From Gestational day16 (Gd16) to Gd21, neural crest-derived cells (NCC) migrated from the proximal gut into the terminal colon, colonising it along its entire length, gradually proliferated and differentiated to innervate the distal gut. From Gd19 to Gd21, significant gross-morphological differences of the anorectum of normal (n=90) and ARMs (n=90) embryos were found. Different myenteric plexus (MPs) development of the anorectum suggested that ARMs were associated with a global abnormal innervation patterns in the anorectum in gestational course and might have some postoperative effect.

Embryonic development of the internal anal sphincter in rats with anorectal malformations.

BACKGROUND/PURPOSE: The embryogenesis of the internal anal sphincter (IAS) in anorectal malformations (ARMs) remains unclear. This study aimed to investigate the development of the smooth muscle in the terminus of the digestive tract in normal and abnormal rats. METHODS: Rat embryos with ARMs were generated by administration of ethylenethiourea to pregnant rats. The normal rat embryos and embryos with ARMs from E13.5 to E21 were serially sectioned in the sagittal plane and stained immunohistochemically using specific antibody to α-smooth muscle actin (SMA). Temporospatial study was carried out on circular muscle of the distal portion of the hindgut. RESULTS: α-Smooth muscle actin immunolabeling cells could not be observed in the hindgut on E13.5, E14, and E14.5. On E15, there were α-SMA immunolabeling circular muscle cells in the hindgut; and the distal portion of the circular muscle was not thickened in the normal and ARMs rats. From E16 onward, the smooth muscle with slight dilated terminus, which was characterized by the features of IAS, could be noted in the primitive anorectum. In the normal group, the circular muscle in the distal portion of the hindgut thickened slightly and became the musculature with shutter-like bundles. In the ARMs group, the α-SMA immunolabeling myogenic precursors of the smooth muscle could be observed in the primitive anorectum as well. The musculature was similar to that in the normal group. On E15 and E16, there was no significant difference in the development of the circular muscle in the 2 groups. Moreover, the terminus of the circular muscle in the hindgut did not reach the orificium fistulae in ARMs rats. From E17 onward, in ARMs rats, the funnel-shaped distal hindgut communicated the genitourinary tract with a narrow fistula; the dilated musculature at this portion thinned gradually and formed an acute angled extremity in the ARMs group rather than formed blunt extremity in the normal group; the terminus circular muscle in the dorsal hindgut reached the orificium fistulae. During the following gestational days, the circular muscle of the hindgut in both normal and ARMs rats continued its own tendency. CONCLUSION: The IAS primordium started to appear at the terminus of the hindgut on E15 in the 2 groups. The IAS in the ARMs group failed to develop as well as that in the normal group. The IAS dysplasia occurred in the late embryonic development (E17-E21).

Embryology of anorectal malformations.

Today, the normal and abnormal development of the hindgut is still a matter of speculation. However, as the result of recent studies in appropriate animal models, most embryologic events that finally lead to abnormal hindgut development are better known than in the past: (1) the process of maldevelopment starts in early embryonic stages; (2) the cloacal membrane is always too short in its dorsal part, thus, the dorsal cloaca is missing; and (3) as a result, the hindgut remains attached to the sinus urogenitalis, forming the recto-urethral fistula. In the past, an impaired process of septation was believed to be the main cause of abnormal hindgut development. In contrast to this, our results indicate that the development of the septum is more passive than active. Furthermore, the results of our studies in normal and abnormal development indicate that (1) the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in female embryos, and (2) to explain abnormal development, studies in abnormal embryos are mandatory.