Preliminary Comparison of a Modified cfDNA Extraction Protocol for Y-Chromosome Marker Detection in Maternal Plasma

Objectives: Noninvasive prenatal testing relies on the analysis of total cell-free DNA (cfDNA) in maternal plasma, where fetal-derived DNA constitutes only a minor fraction. This study aimed to preliminarily compare a modified TPY cfDNA extraction protocol with two commercial extraction kits for the downstream detection of Y-chromosome-specific markers in pregnancies carrying male fetuses. Methods: Plasma samples were obtained from 52 singleton pregnancies between 10 and 30 weeks of gestation with male fetal sex confirmed by ultrasonography. Total cfDNA was extracted from aliquots of the same maternal plasma samples using the modified TPY protocol, the QIAamp DSP Virus Kit, and the MagMAX™ Cell-Free DNA Isolation Kit. Quantitative real-time PCR was performed for the Y-chromosome-specific markers SRY and DYS14. At the same time, GLO was used as a reference marker to reflect the total cfDNA background. Extraction performance was assessed primarily using total cfDNA concentration and Ct values obtained from amplification of fetal-specific Y-chromosome markers. Results: Total cfDNA concentrations varied among the extraction methods, with the commercial kits yielding higher total cfDNA concentrations than the modified TPY protocol. In contrast, the TPY protocol yielded slightly lower mean Ct values for SRY and DYS14 than the commercial kits. SRY and DYS14 amplification was detected in 90.4% and 94.2% of samples, respectively. However, these Ct differences should be interpreted cautiously because fetal fraction, maternal DNA contamination, extraction recovery, and fragment size distribution were not directly measured. Conclusions: The modified TPY protocol showed preliminary technical feasibility for extracting total cfDNA from maternal plasma and enabling downstream amplification of Y-chromosome-specific markers in male pregnancies. Nevertheless, the observed lower Ct values do not establish selective fetal DNA enrichment, reduced maternal DNA contamination, or clinical superiority over commercial methods. Further analytical validation using standardized fetal fraction measurement, recovery efficiency testing, fragment size analysis, fetal-to-maternal DNA ratio assessment, and larger cohorts including both male and female pregnancies is required before broader clinical applicability can be determined.