Anorectal Malformations Group Research Articles

MiR-141-3p affects β-catenin signalingand apoptosis by targeting Ubtd2 in rats with anorectal malformations.

Anorectal malformations (ARMs) are the most common gastrointestinal malformations. miR-141-3p was obtained from whole-transcriptome sequencing, and Ub domain-containing protein 2 (Ubtd2) was predicted as the target gene. An ARM rat model was induced using ethylenethiourea. Fluorescence in situ hybridization and immunofluorescence were used to detect the spatiotemporal expression of miR-141-3p and Ubtd2, respectively. A dual-luciferase reporter assay confirmed their targeting relationship, and cell proliferation and apoptosis were investigated after transfection in the intestinal epithelium (IEC-6). Additionally, western blotting and co-immunoprecipitation were used to examine the protein levels and the endogenous binding relationship.miR-141-3p was downregulated in the ARM group, whereas Ubtd2 increased and colocalized with TUNEL-positive cells.After miR-141-3p inhibition, protein expression of USP5 and β-catenin was affected via Ubtd2, and USP5 could bind to both Ubtd2 and β-catenin. Flow cytometry analysisandcaspase 3/7 staining demonstrated that downregulatedmiR-141-3p promoted cell apoptosis through Ubtd2. In summary, targeting Ubtd2 decreased in miR-141-3p and promoted apoptosis of intestinal epithelium and regulated β-catenin expression. This may cause aberrant apoptosis during hindgut development and mediate the imbalance of β-catenin signaling in the cloaca, further affecting the occurrence of ARMs.

Sexual and Reproductive Health Outcomes in Females With Cloacal Malformations and Other Anorectal Malformations

To improve our understanding of reproductive health and sexual function in women with cloacal malformations and other anorectal malformations (ARMs) METHODS: An observational cross-sectional survey was administered to individuals assigned female at birth aged 12 to 55 with ARMs and cloacal malformations cared for at our institution. Data included age of thelarche/menarche and questions on body image, gynecologic anatomy, sexual function, and pregnancy. Twenty-one patients responded in the ARM group and 30 in the cloacal malformation group. There were no differences in median age of thelarche/menarche in patients with ARMs (11/12.5 years) compared with patients with cloacal malformation (11/12 years). Patients with ARMs were more likely to have native vaginal tissue than those with cloacal malformations (n=18, 82% vs n=12, 40%; P=.03). There were no differences between groups regarding concerns about dyspareunia and functionality of their vagina (P > .05). Forty-two percent of patients with cloacal malformations and 30% of patients with ARMs reported having been sexually active. Two patients with cloacal malformations and 2 with ARMs reported having been pregnant. Patients with cloacal malformations reported a lower quality of life score (80.4) compared with those with ARMs (87.0) (difference > 4.5). Patients with a cloacal malformation were less likely to have native vaginal tissue and reported a lower quality of life than those with ARMs. Despite this, patients with a cloacal malformation had similar reproductive health and sexual function compared with patients with ARMs. Our results reinforce the need for comprehensive sexual and reproductive health care for all women with ARMs.

Analysis of the efficacy of biofeedback for faecal incontinence after surgery for anorectal malformation

Objective To explore the therapeutic effects of biofeedback in the treatment of faecal incontinence (FI) after surgery for anorectal malformation (ARM). Methods Clinical data were collected from paediatric patients for postoperative biofeedback due to FI caused by ARM between May 2017 and November 2021. The data included the duration of symptoms, the integrity of the anal sphincter, anorectal manometry parameters, and FI scores. These patients were divided into the low ARM group (group A) and the high ARM group (group B). Results A total of 45 paediatric patients were enrolled in the study. There were 28 cases in group A and 17 cases in group B. The differences in age, gender, and body weight were not statistically significant between the two groups (p > 0.05). The differences in the clinical indicators were also not statistically significant between the two groups at the time of the initial evaluation. The duration of symptoms was 2.21 ± 0.71 years and 4.14 ± 1.89 years in groups A and B. There were 16 cases with an intact anal sphincter in group A and only two cases with an intact anal sphincter in group B. This difference was statistically significant between the two groups (p < .05). The anal resting pressure, initial sensitivity threshold, defaecation sensitivity threshold, defaecation urge threshold, and FI scores were significantly improved in both groups post-treatment compared to pre-treatment (p < .001). Strong impulses improved significantly in group A, while strong impulses did not improve significantly in group B. The multivariate logistic regression analysis with these variables further showed that symptom duration and anal sphincter integrity were the main factors influencing the therapeutic effects of biofeedback. Conclusion Biofeedback plays a positive role in the treatment of FI in paediatric patients following surgery for ARM. Symptom duration and anal sphincter integrity were found to be the main factors influencing the therapeutic effect of biofeedback.

Transcriptome analysis reveals the spinal expression profiles of non-coding RNAs involved in anorectal malformations in rat fetuses

BackgroundDespite improvements in anorectal malformation (ARM) therapy, patients might still experience post-operative problems such as fecal incontinence, constipation, and soiling. In particular, the dysplasia of the lumbosacral spinal cord in ARM patients is a major disorder that affects fecal function post-operation. However, the pathological mechanisms involved are still unclear. MethodsThe non-coding RNAs (ncRNAs) in the lumbosacral spinal cord of fetal rats with ethylenethiourea-induced ARM were identified using RNA sequencing (RNA-seq) and examined to determine their potential function. The lumbosacral spinal cord was isolated on embryonic day 17 for subsequent RNA extraction and RNA-seq. The transcriptome data was analyzed using bioinformatics analysis, followed by validation using quantitative reverse transcription PCR. ResultsCompared to the control group, 26 differentially expressed microRNAs (DEMs; 22 upregulated, 4 downregulated) and 112 differentially expressed long non-coding RNAs (63 upregulated, 49 downregulated) were identified in the ARM group. Several DEMs related to development, namely miR-200a-3p, miR-200b-3p, miR-200c-3p, miR-200a-5p, and miR-429, were selected for further analysis. Notably, compared to the control, the relative expression of miR-200 family members was highly upregulated in ARM fetal rats. Furthermore, GO and KEGG enrichment and miRNA-transcription factor-lncRNA/mRNA network analysis was explored to show molecular mechanism underlying DEMs. ConclusionsOur findings suggest the involvement of ncRNAs, especially the miR-200 family members, in the pathogenesis of lumbosacral spinal cord dysplasia in ARM fetal rats.

Prenatal detection of chromosomal abnormalities and copy number variants in fetuses with congenital gastrointestinal obstruction

BackgroundCongenital gastrointestinal obstruction (CGIO) mainly refers to the stenosis or atresia of any part from the esophagus to the anus and is one of the most common surgical causes in the neonatal period. The concept of genetic factors as an etiology of CGIO has been accepted, but investigations about CGIO have mainly focused on aneuploidy, and the focus has been on duodenal obstruction. The objective of this study was to evaluate the risk of chromosome aberrations (including numeric and structural aberrations) in different types of CGIO. A second objective was to assess the risk of abnormal CNVs detected by copy number variation sequencing (CNV-seq) in fetuses with different types of CGIO.MethodsData from pregnancies referred for invasive testing and CNV-seq due to sonographic diagnosis of fetal CGIO from 2015 to 2020 were obtained retrospectively from the computerized database. The rates of chromosome aberrations and abnormal CNV-seq findings for isolated CGIOs and complicated CGIOs and different types of CGIOs were calculated.ResultsOf the 240 fetuses with CGIO that underwent karyotyping, the detection rate of karyotype abnormalities in complicated CGIO was significantly higher than that of the isolated group (33.8% vs. 10.8%, p < 0.01). Ninety-three cases with normal karyotypes further underwent CNV-seq, and CNV-seq revealed an incremental diagnostic value of 9.7% over conventional karyotyping. In addition, the incremental diagnostic yield of CNV-seq analysis in complicated CGIOs (20%) was higher than that in isolated CGIOs (4.8%), and the highest prevalence of pathogenic CNVs/likely pathogenic CNVs was found in the duodenal stenosis/atresia group (17.5%), followed by the anorectal malformation group (15.4%). The 13q deletion, 10q26 deletion, 4q24 deletion, and 2p24 might be additional genetic etiologies of duodenal stenosis/atresia.ConclusionsThe risk of pathogenic chromosomal abnormalities and CNVs increased in the complicated CGIO group compared to that in the isolated CGIO group, especially when fetuses presented duodenal obstruction (DO) and anorectal malformation. CNV-seq was recommended to detect submicroscopic chromosomal aberrations for DO and anorectal malformation when the karyotype was normal. The relationship between genotypes and phenotypes needs to be explored in the future to facilitate prenatal diagnosis of fetal CGIO and yield new clues into their etiologies.

Perineal ultrasound in infants with anteriorly displaced anus: A new decision-making tool for the surgeon?

BackgroundAnteriorly displaced anus (ADA) is defined as anterior displacement of the anus toward the perineum. Early radiologic characterization is a crucial step in guiding the first-line management. ObjectiveThe objective of this study was to assess the urethra-to-anus distance (UAD) on perineal ultrasound in female infants under the age of 3 months with anteriorly displaced anus and to retrospectively determine a cut-off to guide the indications for surgery. Materials and methodsPerineal ultrasound was performed prospectively in female infants under the age of 3 months with determination of the UAD, during screening for congenital hip dislocation. Determinations of the UAD on perineal ultrasound and pelvic MRI were performed for anteriorly displaced anus in girls between 2011 and 2018. Results230 patients were included, of whom 173 were in the control group, with 52 examinations performed for anteriorly displaced anus and 5 examinations performed for vestibular anus. The mean UAD for infants under the age of 3 months was: 22.9 mm (±1.7) in healthy infants, 21.4 mm (±2.4) in infants with non-operated ADA, 17.5 mm (±1.8) in infants with operated ADA, and 10.8 mm (±1.3) in infants with anorectal malformation (ARM). A statistically significant difference was observed between the control group and the ARM group (p = 0.0001) and between the control group and the operated ADA group (p = 0.0001). The mean UAD for infants over the age of 3 months was: 25.5 mm (±4.1) in infants with non-operated ADA and 26 mm (±3) in infants with operated ADA. ConclusionA urethra-to-anus distance of less than 17 mm appears to be a cut-off for anteriorly displaced anus in anorectal malformations.

RNA-Seq Profiling of Circular RNAs During Development of Hindgut in Rat Embryos With Ethylenethiourea-Induced Anorectal Malformations.

Anorectal malformations (ARMs) are among the most common congenital terminal digestive tract malformations. Circular RNAs (circRNAs), a novel type of endogenous non-coding RNAs, play roles in the development of the digestive system; however, their contributions to the pathogenesis of ARMs are not well-established. In this study, we explored the mechanism underlying ethylenethiourea (ETU)-induced ARMs by profiling circRNA expression via RNA-seq and constructing a regulatory circRNA-miRNA-mRNA network. Nine pregnant rats were gavage-fed a single dose of 125 mg/kg 1% ETU (ARM group) on gestational day 10 (GD10), and another 9 pregnant rats received a similar dose of saline (normal group) as a control. Embryos were obtained by cesarean section on the key time-points of anorectal development (GD14, GD15, and GD16). Hindgut samples isolated from the fetuses were evaluated by high-throughput sequencing and differentially expressed circRNAs were validated by reverse transcription-quantitative polymerase chain reaction, agarose gel electrophoresis, and Sanger cloning and sequencing. A total of 18295 circRNAs were identified in the normal and ARM groups. Based on the 425 differentially expressed circRNAs (|Fc| > 2, p < 0.05), circRNA-miRNA and miRNA-mRNA pairs were predicted using miREAP, miRanda, and TargetScan. A total of 55 circRNAs (14 up- and 41 downregulated in the ARM group compared to the normal group) were predicted to bind to 195 miRNAs and 947 mRNAs. Competing endogenous RNA networks and a Kyoto Encyclopedia of Genes and Genomes analysis revealed that novel_circ_001042 had the greatest connectivity and was closely related to ARM-associated signaling pathways, such as the Wingless Type MMTV integration site family, mitogen-activated protein kinase, and transforming growth factor-β pathways. These results provide original insight into the roles of circRNAs in ARMs and provide a valuable resource for further analyses of molecular mechanisms and signaling networks.

Upregulation of PPPDE1 contributes to anorectal malformations via the mitochondrial apoptosis pathway during hindgut development in rats

Congenital anorectal malformations (ARMs) are among the most prominent deformities of the gastrointestinal tract; however, their precise aetiology remains obscure. Immunohistochemistry demonstrated that, in the ARM group, the PPPDE1-positive cells were widely distributed in the hindgut epithelial tissue from GD13 to GD16. Immunofluorescence revealed that most TUNEL-, Bax-, and Cytochrome C (Cyt C)-positive cells overlapped with PPPDE1-positive cells in the urorectal septum (URS). Western blotting and quantitative real-time RT-PCR revealed that PPPDE1 levels were significantly higher in the ARM group from GD13 to GD14 (p < 0.05). IEC-6 cells were transfected with PPPDE1 overexpression plasmid/NC (negative control) or si-PPPDE1/si-NC. Flow cytometry analysis and CCK-8 assay (used to detect apoptosis and proliferation, respectively), as well as western blotting, showed that the levels of PPPDE1 were positively correlated with the pro-apoptotic molecules Bax and Cyt C. Accordingly, aberrantly high expression of PPPDE1 caused a spatiotemporal imbalance in foetal rats with ARMs during hindgut development. Therefore, the upregulation of PPPDE1 may promote epithelial apoptosis and reduce proliferation in the hindgut via the mitochondrial apoptotic pathway. This could affect the fusion of the URS and cloacal membrane, ultimately inhibiting the hindgut development and resulting in ARMs.

Rno_circ_0005139 regulates apoptosis by targeting Wnt5a in rat anorectal malformations

BACKGROUNDThe molecular mechanisms underlying anorectal malformations (ARM) are not fully established. Circular RNAs (circRNAs) are new born non-coding RNAs, and their role in ARM is unclear. We assumed that rno_circ_0005139 influences apoptosis and proliferation by acting as a miR-324-3p sponge, and downregulating Wnt5a in ARM.AIMTo identify the differential expression of circRNAs and mRNAs in a rat ARM model.METHODSSixty-six pregnant Wistar rats were randomly divided into two groups: ARM group (2-imidazolidinethione-induced) and control groups. Embryos were harvested by cesarean delivery, and anorectal tissue was taken on embryonic days 16 (E16), 17 (E17), 19 (E19), and 21 (E21). RNA sequencing and gene microarray analysis was used to identify differentially expressed circRNAs and mRNAs in the ARM in a rat model. We selected 6 circRNAs and 3 mRNAs in the Wnt signal pathway from the result of the RNA sequencing and gene microarray analysis, and quantitative reverse transcription polymerase chain reaction was performed to evaluate their tissue expression. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. We tested the function of rno_circ_0005139 and the binding sites between rno_circ_0005139 and miR-324-3p, miR-324-3p and Wnt5a by luciferase assays. Co-transfection of rno_circ_0005139 and miR-324-3p was to verify their functional consistency.RESULTSWe identified 38 upregulated and 42 downregulated circRNAs on E17 (P < 0.05), and 301 mRNAs were upregulated and 256 downregulated in the ARM on E17 (P < 0.05, fold-change > 2.0). We found that rno_circ_0006880 and rno_circ_0011386 were upregulated, whereas rno_circ_0000436, rno_circ_0005139, rno_circ_0009285, rno_circ_0014367, Wnt5a, Wnt10b, and Wnt2b were downregulated in ARM tissues. According to bioinformatics prediction, rno_circ_0005139 acted as a miR-324-3p sponge to regulate the expression of Wnt5a. We chose rno_circ_0005139 and Wnt5a as the final candidates. Because the role and molecular mechanism of rno_circ_0005139 are poorly understood, its effect on apoptosis and proliferation was investigated by in vitro plasmid transfection. A luciferase experiment showed that rno_circ_0005139 could bind with miR-324-3p, which negatively regulated Wnt5a expression. The expression of miR-324-3p was significantly higher in ARM anorectal tissues than that in control group on E17 and E19; Wnt5a expression showed the opposite trend. In addition, a miR-324-3p inhibitor attenuated the effects of rno_circ_0005139 knockdown on ARM development.CONCLUSIONRno_circ_0005139 influences cell proliferation and apoptosis by acting as a miR-324-3p sponge, thereby downregulating Wnt5a in ARM. Accordingly, rno_circ_0005139, miR-324-3p, and Wnt5a could be targeted therapeutic factors for ARM.

Impact of Transanal Irrigation Device in the Management of Children With Fecal Incontinence and Constipation.

Children with fecal incontinence and constipation can be classified into 3 groups: neurogenic bowel dysfunction (NBD) related to spinal cord defects (NBD), refractory constipation (RC), or anorectal malformations (ARMs). The transanal irrigation (TAI) device (Peristeen) was approved in 2012 by the Food and Drug Administration. This system uses a pump rather than gravity to instill water as a colonic irrigant and uses balloon occlusion of the rectum. Our aim was to evaluate the effectiveness of TAI (Peristeen) in children who failed to respond to conservative measures for stool incontinence and constipation. Retrospective study of 147 patients prescribed TAI between January 2014 and January 2020. Data collected included demographics, prior bowel regimen, symptoms before and after, patient satisfaction scores, and NBD scores. Of the 147 patients initiated, 114 remain active users (13 lost to follow-up and 20 discontinued use). Multiple bowel regimens including laxatives (n = 139), cone enema (n = 40), and cecostomy (n = 7) were tried previously. The majority of our patients (n = 85) have NBD, primarily spina bifida, followed by RC (n = 43), and ARM (n = 19). For all patient groups, there was significant improvement in symptoms of fecal incontinence and constipation (P ≤ 0.001). Abdominal pain was improved in the NBD and RC group, but not significantly in the ARM group. We provide a single-center review of a large pediatric cohort using TAI (Peristeen) for management of fecal incontinence and constipation. Peristeen offered significant improvement in patients with NBD, RC, and ARM.

Upregulation of miR-92a-2-5p potentially contribute to anorectal malformations by inhibiting proliferation and enhancing apoptosis via PRKCA/β-catenin

Anorectal malformations (ARMs) is one of the most common gastrointestinal anomalies. Previous research revealed that miR-92a-2-5p was upregulated in ARMs. However, the underlying roles remains unknown. The current study was to further investigate the spatiotemporal expression patterns of miR-92a-2-5p and its target gene protein kinase C alpha (PRKCA) predicted by bioinformatic method, and to explore their potential functions in anorectal malformations (ARMs). Rat models with ethylenethiourea-induced ARMs were made for subsequent experiments. Direct target relationship between miR-92a-2-5p and PRKCA was validated using a luciferase reporter assay. The spatiotemporal expression pattern of miR-92a-2-5p was evaluated using fluorescence in situ hybridization (FISH), while the expression of PRKCA was revealed by immunohistochemical staining and western blotting. IEC-6 cells were transfected with mimics/mimics NC (Negative control)/inhibitor/inhibitor NC of miR-92a-2-5p or si-PRKCA/si-PRKCA NC, respectively. Then the downstream molecules of miR-92a-2-5p, PRKCA and β-catenin, were subsequently detected. Meanwhile, apoptosis and viability assays were measured. Dual luciferase assay confirmed the direct regulatory relationship between miR-92a-2-5p and PRKCA. FISH revealed that miR-92a-2-5p was expressed with a higher level in ARMs fetuses. Further analyses of PRKCA showed lower protein expression level in ARMs group, which was opposite to miR-92a-2-5p. In vitro experiments revealed that overexpression of miR-92a-2-5p or knockdown of PRKCA can down-regulate PRKCA, up-regulate and facilitate nuclear localization of β-catenin, increase apoptosis and decrease proliferation of IEC-6. Taken together, these findings suggest that aberrantly high expression of miR-92a-2-5p potentially contribute to ARMs by inhibiting proliferation and enhancing apoptosis of intestinal cells via negatively regulating PRKCA/β-catenin.

Long-term impact of fistula status on growth and anemia in infants with congenital anorectal malformations.

To evaluate the impacts of fistula status over 12 months on growth and development and anemia in infants with anorectal malformation (ARM) and to analyze the effects of comorbidities. The ARM group included infants who suffered from ARM and underwent three operations including colostomy, anoplasty (group A) and fistula closure (group B). The normal group included infants aged approximately 6 months and 12 months who were classified as groups C and D. A 24-h dietary recall questionnaire was completed by the guardians of all the participants. Data on height, weight, hemoglobin (Hb) level and comorbidities were extracted from electronic medical records. Then, we compared the impacts of the 12-month fistula status on growth and development and anemia among the infants. In total, 47 patients in the ARM group and 100 infants in the normal group were included. The height-for-age z-score (HAZ) was lower in group A than in group B. Compared with those in group D, weight, height, the HAZ and the weight-forage z-score (WAZ) were lower in group B. The incidence of anemia was higher in the ARM group than in the normal group. Patients with ARM are at risk for growth problems and anemia. The long-term impacts of fistula status on infant length presented earlier than those associated with other physical indicators. Compared to infants with urogenital comorbidities, those with congenital heart disease (CHD) seemed to be more prone to growth disorders and anemia.

The effect of multidisciplinary colorectal center development on short-term hospital readmissions for patients with anorectal malformations or Hirschsprung disease

IntroductionHospital readmissions have become a quality metric for both hospital systems and individual surgeons. The medical literature is replete with studies describing readmission rates and factors contributing to readmissions following surgical procedures. Relatively little, however, has been done to define potential solutions to these problems. Over the past decade there has been a movement toward the development of multidisciplinary colorectal centers at high volume children's hospitals. We hypothesized that the development of a colorectal center at our children's hospital decreased readmissions in our colorectal surgery population. Materials and methodsA retrospective review was performed including all patients with the diagnosis of anorectal malformation (ARM) or Hirschsprung disease (HD) at our institution between the years of 2005–2017. Patient level outcomes were compared between the cohort treated prior to (2005–2010) and the cohort treated after the development of the colorectal center (2012–2017). ResultsA total of 354 patients were identified. One hundred seventy-eight patients (113 ARM, 65 HD) were treated prior to and 176 patients (110 ARM, 66 HD) were treated after the development of the colorectal center. Forty-five (25.3%) patients underwent neonatal repair prior to development of the center compared to 15 (8.5%) after. 111 (62.4%) patients underwent colostomy prior to the colorectal center comparted to 95 (54%) after. The rate of readmission within 120 days of discharge in the early group was 63% compared to 52% in those managed in the multidisciplinary colorectal center (p = 0.04). Conversely, the rate of emergency room visits increased from 8.4% to 27.3% (p = 0.01). The decrease in readmission rates was more pronounced in the ARM group, while the HD cohort had similar readmission rates before and after the establishment of the center. Multivariate logistic regression revealed an odds ratio of 0.59 (95% CI 0.37–0.92) for readmission following the development of the multidisciplinary colorectal center. DiscussionThe development of a multidisciplinary colorectal center at our institution was associated with decreased hospital readmissions, but an increase in emergency department resource utilization. These findings suggest improved efficiency in patient care with the implementation of a multispecialty, patient centered approach while also identifying areas of focus for future quality improvement initiatives. Level of EvidenceLevel III, retrospective comparative study.

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

Certain patients with anorectal malformations (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 transcription-quantitative polymerase chain reaction (RT-qPCR) 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 RT-qPCR 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.

Abnormal expression of TBX4 during anorectal development in rat embryos with ethylenethiourea-induced anorectal malformations

BackgroundTo assess the expression of T-box transcription factor 4 (TBX4) during the anorectal development in normal and ethylenethiourea (ETU)-induced anorectal malformations (ARM) rat embryos.MethodsAnorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatio-temporal expression of TBX4 was evaluated in normal (n = 490) and ETU-induced ARM rat embryos (n = 455) from E13 to E16 by immunohistochemical staining, Western blot analysis and real-time RT-PCR.ResultsIn the normal embryos, immunohistochemical staining revealed that TBX4 expression was detected in the epithelium of hindgut and urorectal septum (URS) on E13. TBX4-immunopositive cells were increased significantly in the epithelium of hindgut and URS, the future anal orifice part of cloacal membrane on E14. On E15, abundant stained cells were observed in the rectum, URS and dorsal cloacal membrane and the expression of positive cells reached its peak. On E16, only sporadic positive cells were distributed in the epithelium of the distal rectum. In the ARM embryos, the hindgut/rectum, URS and dorsal cloacal membrane were faint for TBX4 immunohistochemical staining. In the normal group, TBX4 protein and mRNA expression showed time-dependent changes in the hindgut/rectum from E13 to E16 on Western blot and real-time RT-PCR. On E13 and E15, the expression level of TBX4 mRNA in the ARM group was significantly lower than that in the normal group (P < 0.05). On E15, the expression level of TBX4 protein in the ARM group was significantly lower than that in the normal group (P < 0.05).ConclusionsThe expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

Histomorphological changes and expression of neuron-specific enolase, S-100 protein and c-kit in the terminal rectums in patients with high and intermediate anorectal malformation

Objective To investigate the morphological changes and development of enteric nerve in terminal rectums in patients with high and intermediate anorectal malformations (ARMs), and to explore the mechanism of bowel dysfunction postoperatively. Methods The hematoxylin and eosin (HE), Masson staining, IHC and Western blotting were used to evaluate the terminal rectums in patients with ARMs and healthy controls. Results Compared with the normal group, the most frequently observed histological finding in mucosa was inflammation and ulcer, submucosal congestion was the most common finding, disrupted muscularis propria and fibrosis was common. The number of submucosal and myenteric plexus also reduced significantly in ARMs group compared with controls (5.30±1.26 vs. 1.20±0.73, P=0.007). The expression levels of neuron-specific enolase (NSE), S-100, and c-kit in ARMs group were significant lower than that in normal group (NSE: 0.510±0.033 vs. 0.476±0.036; S-100: 0.445±0.009 vs. 0.420±0.012; c-kit: 0.982±0.043 vs. 0.756±0.017; P=0.023, 0.035, 0.028, respectively). Conclusion The terminal rectums in ARMs show various specific and non-specific histomorphological changes, along with ganglion cell dysplasia and aganglionosis and reduction of density and distribution of ICC. These findings maybe responsible for the defecation dysfunction after anorectoplasty. Key words: Anorectal malformation; Immunohistochemical staining; Western blotting

Integrative analysis of microRNA and mRNA expression profiles in fetal rat model with anorectal malformation.

BackgroundAnorectal malformations (ARMs) are the most common congenital malformation of the gut, and regulated by multiple signal transduction pathways. The microRNA (miRNA) expression profiles and their biologial functions in anorectal malformations (ARMs) remain unclear. The aim of our study was to evaluate miRNA and mRNA expression profiles in the ARM rats.Methods and MaterialsARM was induced with ethylenethiourea (ETU) on gestational day 10. Cesarean deliveries were performed to harvest the embryos on gestional day 20. For the extraction of total RNA, 1 cm terminal hindgut samples were obtained from three fetal rats that had similar weights. The microarrays and quantitative RT-PCR analysis were conducted to evaluate the miRNA and mRNA expression profiles in normal fetal rats (n = 3) and ARM fetal rats (n = 3).ResultsIn total, 33 miRNAs and 772 mRNAs were significantly and differentially expressed in terminal hindgut tissues of ARM group versus control group. Functional annotation was performed to understand the functions and pathways of differentially expressed mRNAs. Also, we constructed a miRNA-target gene regulatory network including 25 differentially expressed miRNAs and 76 mRNAs. Furthermore, the credibility of the microarray-based results were validated by using qRT-PCR.ConclusionThe miRNA and mRNA expression in terminal hindgut tissue of ARM fetal rats might provide a basis for further research on the pathogenesis of ARMs.

MSX2 and BCL2 expressions in the development of anorectal malformations in ethylenethiourea-induced rat embryos

BackgroundThis study aimed to determine Msh homeobox 2 (MSX2) and B cell lymphoma-2 (BCL2) expression patterns during anorectal development in anorectal malformations (ARM) and normal rat embryos, with the goals of determining the role of MSX2 and BCL2 in ARM pathogenesis. MethodsARM was induced in rat embryos with ethylenethiourea administered to dams on gestational day 10 (GD10). Embryos were harvested by cesarean deliveries from GD14 to GD16. MSX2 and BCL2 expression was evaluated via immunohistochemical staining, immunofluorescence, western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). ResultsImmunohistochemical staining of ARM embryos revealed that MSX2 was mainly expressed in the epithelium of the hindgut and urorectal septum (URS) on GD14. On GD15 and GD16, MSX2-immunolabeled cells were noted in the epithelium of the rectum, fistula and URS. However, in normal embryos, faint immunopositivity for MSX2 was demonstrated in the epithelium of the rectum and URS from GD14 to GD16. As for BCL2, in normal embryos, BCL2-immunopositive cells were extensively expressed in the epithelium of the hindgut and URS on GD14 and GD15. In ARM embryos, weak immunopositivity for BCL2 was detected in the epithelium of hindgut and URS on GD14 and GD15. Immunofluorescence revealed that MSX2 and BCL2 colocalized in the hindgut. In ARM embryos, we observed more MSX2-positive than BCL2-positive cells on GD14; the normal embryos had the opposite pattern. Analyses by western blot and qRT-PCR showed that MSX2 protein and mRNA expression was significantly increased in ARM embryos compared with the normal embryos on GD15 and GD16 (p < 0.05). However, BCL2 protein and mRNA expression was significantly decreased in ARM embryos compared with the normal embryos on GD14 (p < 0.05). The MSX2/BCL2 ratio of protein and mRNA expression level in the ARM group was the highest on GD15. ConclusionThese results indicate that upregulation of MSX2 and downregulation of BCL2 during cloacal development into the rectum and urethra might be related to the ARM development, and MSX2 promoted apoptosis through reduction of BCL2 expression during the development of anorectal development in ARM.

Clinical characteristics, prognosis, and its risk factors of anorectal malformations: a retrospective study of 332 cases in Anhui Province of China

Objective: To analyze the clinical characteristics and prognosis of anorectal malformations (ARMs) and explore the influencing factors of postoperative anal function in Anhui Province of China.Methods: We performed a retrospective study of ARM patients diagnosed from 2013 to 2016 at Anhui Provincial Children’s Hospital. A total of 332 infants with ARM were enrolled in the survey. Demographic characteristic and clinical data were collected. Follow-up study was required to evaluate anal function after the operation and Logistic regression analysis was used for analyzing the influencing factors of prognosis.Results: A total of 253 males and 79 females were studied, with a ratio of 3.2:1. Abdominal distention was the most common presenting symptom, followed by vomiting. Of the cases, 53.0% (176/332) combined with other congenital malformations. The incidence of other malformations in intermediate and high ARM group was significantly higher than that in the low ARM group. Of the cases, 280 underwent anoplasty. 188, 73, 19 cases were treated with one-stage perineal anoplasty, posterior sagittal anorectoplasty, laparoscopically assisted anorectal pull-through, respectively. The result of follow-up study showed that the excellent and good rate of postoperative anal function was up to 83.3%. Type of ARM, combined with other malformations and the times of anoplasty were related to the anal function postoperatively.Conclusions: ARM occurs mainly in male infants. The incidence of associated abnormalities in ARM patients was high. Intermediate- and high-type, combined with other malformations and more times of anoplasty increased the risk of anal dysfunction postoperatively. Multicenter, prospective randomized-controlled studies were needed to clarify the curative effect of different surgical approaches.

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

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. 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. 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. 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.