A better understanding of how cell-free fetal DNA (cffDNA) is structured and transported in maternal blood could help overcome fundamental limitations of noninvasive prenatal testing and potentially expand its role beyond screening, according to a new review published in Science Advances.
Noninvasive prenatal testing has transformed prenatal care by allowing fetal genetic abnormalities to be screened from maternal blood from around 10 weeks’ gestation. However, positive results still require confirmation by invasive testing, such as chorionic villus sampling or amniocentesis.
Lead author Kieran Sparkes, from the Future Industries Institute, said, "For most parents, a negative test result provides reassurance about the health of their unborn baby. However, if NIPT does indicate a high risk for a chromosomal condition, parents have to wait over a month for confirmation and undergo an invasive diagnostic procedure. High-risk NIPT results are rare, but in 47 percent of cases, a high-risk result turns out to be a false alarm, creating unnecessary stress as well as an unnecessary surgical operation."
The review identifies three major biological barriers: cffDNA represents only a small proportion of circulating DNA, distinguishing fetal from maternal DNA remains difficult, and cffDNA does not always reflect the true fetal genotype.
One reason for the latter is that most DNA described as “fetal” actually originates from the placenta. Although placental and fetal genomes are generally considered identical, mosaicism can create differences. The authors therefore argue that “cell-free placental DNA” may sometimes be a more accurate description and that better differentiation between fetal and placental DNA could improve interpretation of noninvasive prenatal testing results.
The review also challenges the conventional picture of cffDNA as predominantly short, linear fragments generated through apoptosis. Most circulating cffDNA has a characteristic length of approximately 143 base pairs, but researchers have also identified circular DNA, ultrashort fragments of approximately 50 base pairs, and fragments exceeding 500 base pairs. Some have been measured at more than 23,000 base pairs, suggesting that several biological processes may contribute to circulating fetal DNA.
Exactly how this DNA circulates also remains unresolved. Although apoptotic extracellular vesicles have long been proposed as a mechanism for releasing placental DNA, the authors note that direct evidence is lacking. Fetal DNA could also circulate in other extracellular vesicles, nonvesicular particles, DNA-protein-lipid complexes, or as unbound DNA.
For diagnostics, resolving these questions could provide new ways to selectively isolate fetal DNA from the much larger background of maternal DNA. Differences in fragment size, density, protein or lipid associations, or placental surface markers could potentially be exploited to increase the fetal fraction before sequencing.
The authors suggest that research priorities should focus on determining the cellular origin of cffDNA and establishing how much circulating DNA is associated with extracellular particles. Approaches such as immunoaffinity enrichment and microfluidics could then be used to isolate particular DNA-containing particles or longer fetal DNA fragments.
Combined with advances in sequencing and bioinformatics, fetal fraction enrichment could improve the sensitivity and specificity of noninvasive prenatal testing and broaden the range of detectable genetic abnormalities. However, the review emphasizes that the underlying biology first needs to be better defined before noninvasive prenatal testing can progress reliably from screening to comprehensive genetic diagnosis.
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