Association between maternal exposure to PM10 and risk of anorectal atresia/stenosis in offspring: a population-based case-control study in Liaoning Province, China.

The potential association between maternal exposure to PM10 ranging from 3 months prior to conception to the end of the early trimester and the risk of anorectal atresia/stenosis in offspring has not been established. Thus, we determined the association between maternal exposure to PM10 and risk of anorectal atresia/stenosis in offspring in this study. We recruited 713 patients including 480 male and 233 female with anorectal atresia/stenosis and 7950 randomly selected healthy offspring from the Maternal and Child Health Certificate Registry of Liaoning Province and delivered between 1 January 2010 and 31 December 2015. Monthly PM10 concentrations were retrieved from the Environment Protection Bureau of each city in Liaoning Province. We established a multivariable logistic regression model to calculate the adjusted odds ratios (ORs) and 95% confidence intervals (CIs). Maternal exposure to PM10 was associated with an increased risk for anorectal atresia/stenosis in offspring during the 3 months prior to conception (per 10 µg/m3 increment: OR = 1.15, 95% CI = 1.03-1.20; per SD [27 µg/m3] increment: OR = 1.33, 95% CI = 1.09-1.63) and the first trimester (per 10 µg/m3 increment: OR = 1.08, 95% CI = 1.00-1.17; per SD [28 µg/m3] increment: OR = 1.26, 95% CI = 1.01-1.57). Evaluation of the association with a shorter exposure window (1 month) revealed a positive association between anorectal atresia/stenosis and PM10 from the 3rd month prior to pregnancy to each month of the 1st trimester. Maternal exposure to PM10 3 months prior to conception and during the 1st trimester was associated with an increased risk of anorectal atresia/stenosis in the offspring. Future perspective cohort studies are needed to confirm our findings.

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

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.

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

BACKGROUND: To 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. METHODS: Anorectal 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. RESULTS: In 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). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

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

BACKGROUND: To 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. METHODS: Anorectal malformations was induced by ETU on the 10th gestational day (E10) in rat embryos. Spatiotemporal 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. RESULTS: In 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). CONCLUSIONS: The expression of TBX4 was downregulated in ETU-induced ARM embryos, which may play important roles in the pathogenesis of anorectal development.

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

BACKGROUND: This 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. METHODS: ARM 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). RESULTS: Immunohistochemical 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. CONCLUSION: These 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.

Microarray analysis of miRNAs during hindgut development in rat embryos with ethylenethiourea­induced anorectal malformations.

Anorectal malformations (ARMs) are one of the most common congenital malformations of the digestive tract; however, the pathogenesis of this disease remains to be fully elucidated. MicroRNAs (miRNAs) are important in gastrointestinal development and may be involved in the pathogenesis of ARMs. The present study aimed to profile miRNAs and examine their potential functions in rats with ethylenethiourea (ETU)­induced ARMs. Pregnant Wistar rats (n=36) were divided randomly into ETU­treated and control groups. The rats in the ETU­treated group were gavage­fed 1% ETU (125 mg/kg) on gestational day 10 (GD10), whereas the control group rats received a corresponding dose of saline. Embryos were harvested by cesarean section on GD14, GD15 and GD16. Hindgut tissue was isolated from the fetuses for RNA extraction and microarray analysis, followed by bioinformatics analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) validation. Overall, 38 miRNAs were differentially expressed (all upregulated) on GD14, 49 (32 upregulated and 17 downregulated) on GD15, and 42 (all upregulated) on GD16 in the ARM group compared with the normal group. The top 18 miRNAs with |log2(fold change)| >4.25 were selected for further bioinformatics analysis. Among these miRNAs, five were differentially expressed at two time-points and were involved in ARM­associated signaling pathways. The RT­qPCR analysis revealed that three miRNA (miR), miR­125b­2­3p, miR­92a­2­5p and miR­99a­5p, were significantly differentially expressed in rats with ARMs compared with the normal group. In conclusion, the results suggested that the differential expression of miR­125b­2­3p, miR­92a­2­5p and miR­99a­5p during key time-points of anorectal formation in rats may have functions in the pathogenesis of ARM.

High Prenatal Exposure to Bisphenol A Reduces Anogenital Distance in Healthy Male Newborns.

OBJECTIVE: To estimate the relationship between cord blood bisphenol A (BPA) levels and anogenital measurements in healthy newborns. METHODS: Pregnancy and birth history, together with body mass and length data, anogenital measurements, penile measurements and cord blood samples were obtained from healthy newborns. Cord blood concentration of BPA was analyzed by sandwich enzyme-linked immunosorbent assays kit. RESULTS: Among 130 healthy newborns (72 boys, 58 girls), mean anopenile distance was 45.2±6 mm and anoscrotal distance was 21.9±5.4 mm in boys; mean anoclitoral distance was 33.8±6.6 mm and mean anofourchette distance was 12.2±4.9 mm in girls. Mean cord blood BPA level was 4.75±2.18 ng/mL. 90th percentile value for cord blood BPA was 8.26 ng/mL and the analysis showed a statistically significant correlation between anoscrotal distance and cord blood BPA levels above the 90th percentile (p=0.047) in boys. The changes in anogenital distance in girls were not statistically significant. CONCLUSION: We showed a significant association between high cord blood BPA levels and shortened anoscrotal distance in male newborns. However, this result should be interpreted with caution since there were no significant external genital abnormalities in our study group.

Maternal exposure to di-n-butyl phthalate (DBP) induces combined anorectal and urogenital malformations in male rat offspring.

Anorectal malformations in combination with hypospadias (ARMs & hypospadias) are a type of complex congenital malformations. The underlying mechanisms of this deformity are largely unknown. In this study, we comprehensively characterized the dysplasia, histological malformations, and genetic changes of ARMs & hypospadias in male rats after maternal exposure to di-n-butyl phthalate (DBP) by gastric intubation at doses of 850mg/kg bw/day during GD11-15. On postnatal day 1, anatomical and histopathological analysis confirmed combined malformations of the genital tubercle (GT), terminal rectum (TR) and testes. DBP-induced dysplasia was also seen in the kidney, lung, spleen, heart and liver of ARMs & hypospadias male rats. Moreover, decreased levels of serum testosterone, as well as reduced expression of genes related to the androgen signaling pathway (Cyp11a1, Hsd3b, Scarb1, Star, AR, Srd5a2) were found in the testes of ARMs & hypospadias male rats after DBP exposure as compared to untreated controls. Further, decreased mRNA levels of Shh, Fgf10, Gli2, Gli3, Bmp4, Wnt5a, Hoxa13, Hoxd13, Fgfr2 and AR were observed in TR and GT in the ARMs & hypospadias group. These results provide evidence that prenatal exposure to DBP can lead to combined anorectal and urogenital malformations as well as dysplasia of the testes.