Investigation on Parameters Influencing Fetal Fraction and Its Importance for Non-invasive Prenatal Diagnostic Application

Prenatal care has undergone a paradigm shift as a result of significant scientific efforts made in the field of circulating fetal nucleic acids. The rapid adoption of non-invasive prenatal testing (NIPT) for fetal aneuploidies in the high-risk pregnant population represents a remarkable landmark. More recently, a debate has emerged amongst medical professionals and in the scientific community about the implementation of NIPT in the general pregnant population. However, much remains to be elucidated about the observed variability of plasma fetal DNA fractions in normal and pathological pregnancies. Therefore, investigation into the factors affecting the levels of fetal DNA in maternal plasma may help shed light on the issues related to non-reportable results due to insufficient fetal DNA fractions.;The first part of the present thesis examines the relationships between first-trimester screening (FTS) parameters and fetal DNA fractions in maternal plasma. The lack of differences in fetal DNA fractions between different risk groups shown in this study offers solid evidence for the uniform performance of the NIPT-based aneuploidy screening across all pregnancies without regard to the a priori risk. The study demonstrates that the strongest predictor of fetal DNA fractions is maternal weight.;Given its importance in determining fetal DNA fractions, the next part of this thesis investigates the observed relationship with maternal weight and provides fundamental insights into the biology of plasma DNA molecules in pregnancies with a spectrum of maternal weights. I used an optimized protocol for the plasma DNA size-based approach I developed in Chapter 5 to measure plasma DNA size distributions in Chapter 6. The results of this study indicate that DNA molecules released by excessive maternal adipose tissue may not play a major role in the decrease in fetal DNA fractions among obese pregnant populations, as some investigators postulated. In fact, the altered plasma DNA size profile of the heaviest pregnant women points to the hematopoietic origin of these molecules. The plasma deconvolution analysis performed in this chapter elucidates the tissue origin of plasma DNA molecules through an examination of the methylation profile from plasma bisulfite sequencing analysis.;In the last section, I describe the development of non-invasive methods to detect a wide spectrum of mutations in the F8 and the F9 genes for NIPT of hemophilia using droplet digital PCR and targeted massively parallel sequencing. I focus on the detection of the F8 int22h inversion mutation, which is associated with the most severe clinical phenotype. Both technologies confirm the fetal hemophilia status in all tested pregnancies, although a small proportion of unclassified cases occur due to the low fetal DNA fractions in maternal plasma if one uses droplet digital PCR. The targeted sequencing approach appears to be the method of choice for the NIPT of hemophilia.;In summary, this thesis embraces a wide variety of methods and approaches to highlight the importance of fetal DNA fractions for applications in NIPT by studying characteristics of circulating DNA in maternal plasma. Progress in the NIPT of hemophilia represents a valuable development in the field.