Spatial transcriptomics reveals gene interactions and signaling pathway dynamics in rat embryos with anorectal malformation.

Anorectal malformation (ARM) is a prevalent early pregnancy digestive tract anomaly. The intricate anatomy of the embryonic cloaca region makes it challenging for traditional high-throughput sequencing methods to capture location-specific information. Spatial transcriptomics was used to sequence libraries of frozen sections from embryonic rats at gestational days (GD) 14 to 16, covering both normal and ARM cases. Bioinformatics analyses and predictions were performed using methods such as WGCNA, GSEA, and PROGENy. Immunofluorescence staining was used to verify gene expression levels. Gene expression data was obtained with anatomical annotations of clusters, focusing on the cloaca region's location-specific traits. WGCNA revealed gene modules linked to normal and ARM cloacal anatomy development, with cooperation between modules on GD14 and GD15. Differential gene expression profiles and functional enrichment were presented. Notably, protein levels of Pcsk9, Hmgb2, and Sod1 were found to be downregulated in the GD15 ARM hindgut. The PROGENy algorithm predicted the activity and interplay of common signaling pathways in embryonic sections, highlighting their synergistic and complementary effects. A competing endogenous RNA (ceRNA) regulatory network was constructed from whole transcriptome data. Spatial transcriptomics provided location-specific cloaca region gene expression. Diverse bioinformatics analyses deepened our understanding of ARM's molecular interactions, guiding future research and providing insights into gene regulation in ARM development.

Imaging of anorectal malformations in utero.

PURPOSE: To document the imaging findings suggestive of anorectal malformation (ARMs) on prenatal US and MRI. METHODS: Retrospective evaluation of the screening US and prenatal MRI exams of the rectum and ano-perineal region in normal fetuses and in patients with ARMs. RESULTS: Examples showing the normal rectal and anoperineal anatomy on prenatal US and MRI exams and the imaging findings observed in different types of confirmed ARMS. CONCLUSIONS: Prenatal diagnosis of ARMs requires both a systematic evaluation of the fetal pelvis and perineum and an appropriate knowledge of its suggestive imaging findings.

Prenatal imaging of anorectal malformations - 10-year experience at a tertiary center in Switzerland.

BACKGROUND: Anorectal malformation is a spectrum of congenital defects of the distal bowel, mostly diagnosed at birth. OBJECTIVE: To describe the prenatal imaging findings of anorectal malformations, explore the causes of the low rates of prenatal diagnosis, compare the accuracy of prenatal ultrasound (US) and magnetic resonnance imaging [MRI] and evaluate the relevance of information obtained at MRI. MATERIALS AND METHODS: Children treated for anorectal malformation at our hospital and with available prenatal studies were retrospectively identified and included in the study. We reviewed prenatal imaging exams, listed findings suggestive of the diagnosis, and compared results with the final classification. RESULTS: Fourteen fetuses and neonates - eight with intermediate-high type anorectal malformation and six with cloacae - fulfilled the inclusion criteria. All had associated congenital anomalies. Prenatal exams included 13 US and 8 MRI exams, with 7 children having both exams. Suggestive findings for anorectal malformation were detected in 50% of the cases prenatally and in 85% upon review. They were prospectively detected in 31% and 50% of the cases at US and MRI and retrospectively in 62% and 100% at US and MRI, respectively. MRI was superior to US because it improved the diagnosis, especially in cloacae, and provided relevant additional information that changed management in two cases. CONCLUSION: The most important signs suggesting anorectal malformation are an absent target sign and anomalous distal bowel wall and rectal fluid. Complementary prenatal MRI improves the diagnosis of anorectal malformation.

Involvement of proliferative and apoptotic factors in the development of hindgut in rat fetuses with ethylenethiourea-induced anorectal malformations.

BACKGROUND: Anorectal malformations (ARMs) are common congenital malformations of the terminal digestive tract, but little is known regarding their pathogenesis. Aberrant cell proliferation/apoptosis are believed to be involved in ARMs. However, there are no studies on proliferation/apoptosis-related genes. PURPOSE: We aimed to investigate the spatiotemporal expression patterns of two proliferation/apoptosis-related genes (MYC proto-oncogene and tumor protein p53) and explore their potential functions in the hindguts of ethylene thiourea-induced ARMs rat fetuses. METHODS: MYC and p53 expression was evaluated using immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction (RT-qPCR). Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and p53 costaining were performed to assay the colocalization of apoptotic and p53-expressing cells. RESULTS: Rat fetuses with ARMs displayed fusion failure of the urogenital septum and cloacal membrane. In the control group, MYC was persistently expressed from gestational day (GD)14 to GD16 and distributed throughout the hindgut, while p53 was weakly detected in the terminal segment of the urethra and hindgut; in the ARMs group, MYC expression was obviously reduced, while p53 was widely and highly expressed in the urethra and hindgut. Western blotting and RT-qPCR confirmed the decrease in MYC and increase in p53 expression in ARMs. TUNEL and p53 co-staining revealed considerable overlap between apoptotic and p53-expressing cells. CONCLUSION: The expression patterns of c-myc and p53 were disrupted in ARMs rat embryos, and the downregulation of c-myc and upregulation of p53 might be related to the development of ARMs at the key time points of ARMs morphogenesis.

Downregulation of P2Y2 and HuD during the development of the enteric nervous system in fetal rats with anorectal malformations.

Certain patients with anorectal malforma-tions (ARMs) continue to suffer from postoperative dysphoria. The enteric nervous system (ENS) is closely associated with defecation. The purinergic receptor P2Y2 (P2Y2) and Hu antigen D (HuD) proteins contain multiple motifs that enable their activation and direct coupling to integrin and growth factor receptor signaling pathways; thus, they may serve as key points in ENS development. The aim of the present study was to investigate the expression pattern of P2Y2 and HuD proteins during anorectal development in ARM embryos. The embryogenesis of ARM in rats was induced by ethylenethiourea (ETU) on the 10th gestational day. The expression patterns of P2Y2 and HuD proteins were evaluated by immunohistochemistry and western blot analysis in normal, ETU and ARM rat embryos on embryonic days E17, E19 and E21; their mRNA levels were assessed via reverse transcription­quantitative polymerase chain reaction (RT­qPCR) of the distal rectum of fetal rats. Immunohistochemistry of the distal rectum demonstrated that on E17, the expression levels of the two proteins were not different between the three groups. On E19, the expression of HuD was significantly decreased in the ARM group. On E21, the two proteins were significantly decreased in the ARM group. Additionally, the expression levels of the two proteins on E17 were significantly lower than on E21 in the ARM group. Western blotting and RT­qPCR also revealed that the P2Y2 and HuD proteins and mRNA expression levels were significantly decreased in the ARM groups when compared with the normal group on E17 and E21 (P<0.01). Thus, the present study demonstrated that downregulation of P2Y2 and HuD may partly be related to the development of the ENS in ARM embryos.

Prenatal Evaluation for Detection of Anorectal Atresia: Value of Ultrasound.

OBJECTIVES: To investigate the applicability and value of ultrasound (US) in the diagnosis of anorectal atresia. METHODS: Between January 2008 and January 2016, we prospectively evaluated 63,101 fetuses (gestational age, 20-38 weeks), including low- and high-risk populations using 2-dimensional US scans. An abnormal imaging finding was defined as an anal canal diameter of less than the 95% confidence interval (small anal canal) of the normal range or the absence of an anal canal and rectum. Imaging findings were considered normal on detection of an anal canal with a normal width and the absence of abnormalities. Prenatal imaging findings were confirmed by a postnatal or postmortem examination. RESULTS: Among the investigated fetuses, 28 showed evidence of anorectal atresia on US scans, and 22 of those with anorectal atresia had additional anomalies. Six cases of isolated anorectal atresia were successfully detected during the preclusive prenatal US scans. Four cases of a low imperforate anus (including 2 covered anuses) yielded false-negative results, indicating a diagnostic rate of 87.5% (28 of 32). The normal appearance of the fetal rectum and anal canal ruled out anorectal atresia in 30 fetuses with a dilated colon. Additionally, there were 3 false-positive cases, in which a narrow anal canal was observed. CONCLUSIONS: Identifying the abnormal appearance or absence of the fetal anal canal and rectum on preclusive US anomaly scans is useful for prenatal diagnosis or exclusion of anorectal atresia, which may help improve the detection of isolated anorectal atresia. Furthermore, a combined evaluation of the longitudinal and axial appearances of the fetal anal canal and rectum can improve diagnostic accuracy.

Integrating lncRNAs and mRNAs expression profiles in terminal hindgut of fetal rats with anorectal malformations.

BACKGROUND: The detailed embryonic etiology and pathogenesis of anorectal malformations (ARMs) remains unclear. Recent studies have shown that gene expression abnormalities were the key factors that result in ARMs. Long non-coding RNAs (lncRNAs) were reported as the 'transcriptional noise' within the genome. The expression profiles of lncRNA and mRNA remain less characterized in the pathogenesis of ARMs. Furthermore, the function of lncRNAs in the regulation of this process has not been investigated so far. Therefore, this current study was aimed to integrate lncRNA and mRNA expression profiles in terminal hindgut of ethylenethiourea (ETU)-induced ARM rats using Agilents lncRNA and mRNA co-expression microarrays. METHODS: ARM model was induced with ethylenethiourea (ETU) on gestational day 10. Cesarean deliveries were conducted to collect the embryos on gestational day 20. For the extraction of total RNA, 1-cm terminal hindgut tissues were collected from three fetal rats with similair weights. The microarrays and quantitative RT-PCR analysis were conducted to evaluate the lncRNA and mRNA expression profiles in normal fetal rats and ARM fetal rats. RESULTS: Compared with control group, 164 lncRNAs were observed to be aberrantly expressed (FC ≥ 2; P < 0.05) in ARM group, including 36 upregulated and 128 downregulated, while 772 mRNAs were observed to be aberrantly expressed (FC ≥ 2; P < 0.05) in the terminal hindgut, including 350 up-regulated and 422 down-regulated. The differential expression profiles between the ARM and the control group were used for gene ontology (GO) and pathway analysis. A subset of those RNAs was identified to be closely related to the development process of ARMs. The four RNAs that were differentially expressed between the two groups were selected for qPCR validation, and the results were in line with the microarray data. In addition, the lncRNAs and mRNA co-expression network was established according to the correlation analysis. We predicted the functions of transregulatory lncRNAs by the TFs (transcription factors) which might modulate their expression. In the core network of lncRNA-TF pairs, the lncRNAs can be classified into 5 categories of pathways governed by Jun, c-Myc, Usf1, Alf2, and Stat3. CONCLUSION: From the above results, it can be suggested that these aberrant lncRNAs might participate in the pathogenesis of ARM, and our present work may provide new research directions for future studies of ARMs.

Bone morphogenetic protein 4 expression in the developing lumbosacral spinal cord of rat embryos with anorectal malformations.

Although there are improvements in treatment of anorectal malformations (ARMs), patients can still develop fecal incontinence, constipation, and soiling with loss in quality of life. Recent evidence suggests that malformations in the lumbosacral spinal cord are one of the factors that affect postoperative anorectal function. However, the underlying mechanism that produces these malformations has yet to be elucidated. The bone morphogenetic proteins (BMPs) comprise a large group of highly conserved molecules that are involved in multiple processes and play important roles in the formation, development, and differentiation of the spinal cord. This study was designed to investigate the levels of BMP4 expression in the lumbosacral spinal cord in ARMs rat embryos induced by ethylenethiourea (ETU). Specifically, we assessed the association of BMP4 levels with the maldevelopment of the lumbosacral spinal cord and whether BMP4 acted through the canonical intracellular pathway in embryonic rats with ARMs. BMP4 expression was confirmed with immunohistochemical staining, RT-qPCR and western blot analyses of embryonic day (E) 16, E17, E19 and E21 embryos, moreover Smad1/5 and pSmad1/5 expression were confirmed with western blot analyses at peak time point of BMP4 expression. Our results reveal that BMP4 expression in the lumbosacral spinal cord of ARMs rat embryos is decreased at both the mRNA and protein levels and could decrease the phosphorylation of smad1/5, when compared with their expression levels in normal tissue. These results also suggest that reductions in BMP4 expression were possibly responsible for dysfunction of the lumbosacral spinal cord during late developmental stages in ARMs fetal rats. Taken together, we conclude a role for BMP4 in the pathogenesis of lumbosacral spinal cord maldevelopment in developing ARMs rats.

Embryological and clinical implications of the association between anorectal malformations and spinal dysraphisms.

PURPOSE: To describe the association of anorectal malformation (ARM) and spinal dysraphism (SD) in terms of impact on the management of SD and embryogenetic implications. METHODS: Patients with SD associated with (A) or without (B) ARM were included. The two groups were further divided into operated on (A1/B1) or not (A2/B2) for SD. Groups A and B were compared for type of SD (embryogenetic classification) and prevalence of neurosurgery; Groups A1 vs. A2 for type of ARM (Wingspread classification); Groups A1 vs. B1 for age at neurosurgery, neurophysiology, and clinical symptoms. MAIN RESULTS: One hundred twenty-one patients with SD, 83 with and 38 without ARM were consecutively treated (1999-2015). Group A was associated only with SDs developing after primary neurulation, corresponding to the period of cloacal septation and organogenesis (p = 0.0007). Untethering surgery was significantly less frequent in Group A (p < 0.0001 and p = 0.04, respectively). Higher ARMs were not associated with increased risk for neurosurgery. No other significant differences were detected. CONCLUSIONS: In our series, ARMs were associated only with SD developing after primary neurulation, suggesting a single insult leading to both SD and the associated ARM. Neurosurgery is indicated less frequently in patients with ARM-associated SD, despite the similar preoperative clinical features.

Spatiotemporal expression of Wnt3a during striated muscle complex development in rat embryos with ethylenethiourea-induced anorectal malformations.

Numerous patients with anorectal malformations (ARMs) continue to experience fecal incontinence and constipation following surgical procedures. One of the most important factors that influences defecation is the striated muscle complex (SMC). Wnt signaling regulates the expression of myogenic regulatory factors, which serve an important role in muscle development. Therefore, the present study aimed to investigate the expression pattern of Wnt3a protein (by immunohistochemistry and western blot analysis) and mRNA [by reverse transcription­quantitative polymerase chain reaction (RT-qPCR)] in the SMC of ARM model rats that were exposed to ethylenethiourea. Immunostaining revealed that the expression of Wnt3a exhibits space­ and time­dependent changes in the developing SMC of ARM model rat embryos. Immunohistochemistry demonstrated that on embryonic day 17 (E17), Wnt3a­positive cells were observed in the SMC in normal embryos, and expression levels gradually increased as the rat embryos developed. Similar changes in Wnt3a protein expression were detected in ARM model rat embryos; however, the expression of Wnt3a was significantly reduced compared with the normal rat embryos. Western blotting and RT­qPCR also revealed lower expression levels of Wnt3a protein and mRNA, respectively, in the SMC of ARMs model rat embryos compared with normal rat embryos. These data revealed that the expression of Wnt3a in ARM embryos was notably reduced, indicating a potential role for Wnt3a in the maldevelopment of the SMC in patients with ARMs.

Prenatal Diagnosis of Vesicorectal Fistula.

Anorectal malformations are a rare condition difficult to diagnose in the prenatal period. It can be suspected if distal bowel appears dilated in the first-trimester ultrasound or if intraluminal echogenic foci are detected during the second-trimester scan. We report a case with these ultrasound signs (dilated sigmoid at the first trimester and intraluminal echogenic calcifications at the second trimester), in which a vesicorectal fistula image was obtained. This is the first published prenatal image of a vesicorectal fistula.