Diurnal Effects on the Fraction of Fetal Cell-Free DNA in Maternal Plasma

Abstract

Objective: The discovery of fetal cell-free DNA (cfDNA) has revolutionized prenatal diagnostics through non-invasive prenatal testing (NIPT), which depends on accurately measuring the fetal fraction (FF) in maternal plasma. While FF is known to be influenced by maternal and fetal factors, the impact of intraday rhythms remains unclear. This study investigated whether FF varies based on blood draw timing.

Methods: Data from 2519 euploid singleton pregnancies undergoing NIPT were analyzed. Key variables included maternal age, BMI, gestational age, fetal sex, and blood draw timing (06:50-21:00). FF was measured using the Harmony Prenatal IVD Test. Multiple linear regression identified independent predictors of FF, while intraday variation was assessed using Mann-Whitney U tests and boxplots.

Results: FF showed a significant positive relationship with blood draw timing (β = 0.00176 per hour, p < 0.005), with afternoon values approximately 10% higher than morning values (∼0.01 difference). Other predictors included BMI (negative), gestational age (positive), and fetal sex (higher in females). Blood draw timing appeared to be a stronger predictor of FF than gestational age or fetal sex, second only to BMI.

Conclusion: This novel finding demonstrates diurnal variation in FF, suggesting that optimizing blood draw timing could improve NIPT accuracy, particularly in borderline cases. Further research is needed to confirm the clinical implications.

FIGURE 1

Intraday variations of FF. (A) FF boxplot per hour of blood draw with n showing the number of samples, respectively. (B) Result matrix of the series of pairwise Mann‐Whitney U tests (FF). Black cells indicate significant different comparisons (p‐value below 0.05).

FIGURE 2

Relationship between FF and clock time of blood draw between three sets of morning (Population 1) and afternoon (Population 2) populations. (A) Set A: 06:50 and 12:00 & 12:01 and 21:00; (B) set B: 06:50–13:00 & 13:01–21:00; (C) set C: 06:50–14:00 & 14:01–21:00. Brackets with asterisks indicate a highly significant difference (p < 0.00001) in FF between morning and afternoon populations as found by the Mann‐Whitney U tests (see also Table 3). Corresponding density plots indicate, while largely overlapping, a greater relative concentration of lower FF values in the morning and a greater relative concentration of higher FF values in the afternoon populations, respectively. Samples belonging to the morning population are indicated in orange, while samples belonging to the afternoon population are blue.