Maternal blood biomarkers of pregnancy and congenital heart disease

Introduction: Congenital Heart Disease (CHD) is the most common birth defect and the leading cause of infant mortality (Lopes et al., 2018). Early diagnosis improves outcomes, yet current testing is inadequate, with 50% of cases remaining undetected until birth (Walsh et al., 2014). At present, there are no clear biomarkers of CHD, however it has been shown that foetal-maternal communication occurs across the placenta, and that circulating micro-RNA (miRNA) and extracellular vesicles (EVs) can be detected in maternal circulation. This project aimed to identify potential biomarkers in hopes to one day develop a non-invasive antenatal screening test for CHD. Methods: Assay methods were developed during a rat pilot study; comparing pregnant rats (n=6) to non-pregnant controls (n=6) to identify plasma biomarkers of pregnancy. NanoString Technology was used to profile circulating miRNAs. As there is no clear workflow for miRNA analysis, the effect different variables have on the output were investigated. In silico recalculations and qPCR were then used to verify the results. Plasma EVs were purified using a precipitation/size exclusion chromatography kit and labelled with CFDA-SE. Flow cytometry was used to profile surface biomarkers (CD63, CD81, CD9) on CFSE+ EVs. The human CHD EV ‘ExoChase’ study recruited patients carrying CHD pregnancies (n=10), those carrying healthy pregnancies (n=20), and a group of non-pregnant women as controls (n=20). EVs were isolated from plasma and surface biomarkers (CD63, CD81, CD9, CD82) assayed. Results: Rat circulating miRNA: The pilot study showed that nCounter outputs are greatly influenced by different analysis variables and no background correction was suggested to be the most optimal. The study successfully identified and validated changes in 3 miRNAs with a known role in pregnancy (miR-196c, miR-183, miR-431). Rat EV biomarkers: Flow cytometry was used for the successful quantification of EVs expressing each surface biomarker. There were no changes in the EV concentration or number of CD9+ and CD81+ EVs between groups but a significant reduction in CD63+ EVs was recorded in pregnant rats. Human EV biomarkers: The ExoChase study was limited by a small disease group and even though all surface biomarkers were successfully quantified, no significant difference between groups was recorded. The concentration of EVs was reported as unchanged between non-pregnant and pregnant samples, but statistically increased in the CHD group compared to both non-pregnant and pregnant groups. Conclusion: Circulating miRNA and EV surface proteins can be effectively assayed in maternal circulation and show promise as biomarkers of disease. The findings demonstrate the importance of standardised analysis workflows in literature. In order to detect significant changes in the ExoChase study, future investigations will enlist a larger and more focused disease cohort.