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Background: Placental development occurs concurrently with fetal cardiogenesis and share many developmental pathways. Recent data from animal models suggest a link between placental inflammation and abnormal heart development, but no studies have elucidated the molecular processes connecting specific placental lesions to fetal congenital heart disease (CHD) in humans. Objective: To determine differentially expressed genes and affected biological processes in placental tissue from pregnancies complicated by fetal left ventricular outflow tract obstruction (LVOTO) as well as chronic inflammatory and vascular placental pathologies. Methods: Total RNAs were extracted from archived Formalin-Fixed Paraffin-Embedded (FFPE) placental tissues from pregnancies complicated by LVOTO (n=24) and healthy control pregnancies (n=10) using AllPrep DNA/RNA FFPE Kit (Qiagen, Hilden, Germany) according to the kit protocol. The RNAs were subjected to strand-specific whole transcriptome library preparation, and sequenced using NextSeq 500/550 v2.5 High Output Kit (paired end reads - 75 cycles) (Illumina, San Diego, CA). Gene read counts were log-scaled and quantile normalized to generate expression levels. To determine the molecular factors that influences chronic placental pathologies, we performed unpaired t-test and expression fold change (FC) were used to compare groupwise differential gene expression: placentas with LVOTO and no placental pathology (Group 1) to those with LVOTO and specific placental lesions Group 2: fetal vascular malperfusion (FVM); Group 3: chronic villitis; Group 4: maternal vascular malperfusion (MVM). Gene set enrichment analyses (GSEA) were performed on ranked genes based on Log2FC to identify overlaps with common biological processes. Results: Group 1 showed 7143 genes that were differentially expressed (n=3859 genes upregulated; n=3284 genes downregulated) compared to healthy control P <0.05. Upregulated genes overlapped with circulatory and vascular development processes (False Discovery Rate FDR <0.0001). Downregulated genes overlapped with lipid biosynthesis and metabolic processes (FDR <0.002). There were 115 differentially expressed genes when compared between Group 2 (FVM) and Group 1 and GSEA analysis showed that downregulated genes overlapped with organ developmental processes (FDR<0.0005). Notably, GSEA analyses also showed that upregulated genes in chronic villitis (Group 3) overlapped with immune mediated processes, and downregulated genes in MVM overlapped with developmental and inflammatory response processes (FDR <0.05). Conclusion: Developmental and immune/inflammatory processes were most prominently represented in our transcriptomics data supporting a potential role for these biological processes in the chronic placental changes in pregnancies complicated by LVOTO. Validation of these results by expanding the cohort of patient FFPE tissues for additional gene expression analyses, and further establishing the mechanistic connections between these candidate genes, placental pathologies and specific forms of CHD are warranted. We acknowledge the following grants that funded this research: UT Southwestern Circle of Friends Synergy Grant (D.U and R.L.L), K23 HL161617-01A1 (R.L.L) and the AHA 23CDA1051500 (R.L.L).
Udayakumar et al. (Fri,) studied this question.