To improve outcomes of fetuses with spina bifida (SB), better knowledge is needed on the molecular drivers of SB and its comorbidities. We have recently shown in historical data that SB often associates with reduced fetal growth. We here use placental transcriptome sequencing and a novel nutrient-focused analysis pipeline to determine whether this association is due to placental dysfunction. We show that fetuses with SB have dysregulation in placental gene networks that play a role in nutrient transport, branching angiogenesis, and immune/inflammatory processes. Several of these networks are sensitive to multiple micronutrients, other than the well-known folic acid, and this deserves further investigation. An improved understanding of placental phenotype in fetuses with SB may help identify novel mechanisms associated with SB and its comorbidities, and reveal new targets to improve fetal outcomes in this population.

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Background: Neural tube defects (NTDs) remain among the most common congenital anomalies. Contributing risk factors include genetics and nutrient deficiencies, however, a comprehensive assessment of nutrient-gene interactions in NTDs is lacking. We hypothesised that multiple nutrient-gene interactions would be evident in NTD-associated gene signatures. Methods: We applied a novel, nutrient-focused gene expression analysis pipeline to identify nutrient-sensitive gene regulatory networks in amniocyte gene expression data (GSE4182) from fetuses with NTDs (cases; n=3) and fetuses with no congenital anomalies (controls; n=5). Differentially expressed genes (DEGs) were identified and screened for having nutrient cofactors. Transcription factors (TFs) with nutrient cofactors that regulated DEGs, and nutrient-sensitive miRNAs that had a previous link to NTDs, were identified and used to construct DEG regulatory networks. Results: Of the 880 DEGs in cases (vs. controls), 10% had at least one nutrient cofactor. DEG regulatory network analysis revealed that 39% and 52% of DEGs in cases were regulated by 22 nutrient-sensitive miRNAs and 10 nutrient-dependent TFs, respectively. Zinc- and B vitamin- dependent genes and gene regulatory networks (Zinc: 10 TFs targeting 50.6% of DEGs; B vitamins: 4 TFs [targeting 37.7% of DEGs], 9 miRNAs [targeting 17.6% of DEGs]) were dysregulated in cases. Two nutrient-dependent TFs predicted to target DEGs in cases (Tumor Protein 63 and Churchill Domain Containing 1) have not been previously linked to NTDs. Conclusions: We identified multiple novel nutrient-sensitive gene regulatory networks associated with NTDs, which may relate to NTD pathogenesis, and indicate new targets to explore for NTD prevention or to optimise fetal development.