The presence of cell-free foetal DNA (cffDNA) in maternal plasma was first discovered by Lo et al. in 1997, opening a new range of possibilities for non-invasive prenatal testing. cffDNA represents around 10% of total cell-free DNA in maternal circulation, and it is detectable in the blood from the fifth week of pregnancy. It allows us to obtain genetic information about the foetus without the necessity of using invasive procedures that imply a risk of miscarriage, such as amniocentesis and chorionic villous sampling. This makes the technique very useful in the identification of genetic pathologies.

Another common application of this technique is early sex determination, usually performed through PCR-based techniques, either end-point PCR or qPCR. While the most usual method for sex determination, sonography, is not reliable during the first trimester of pregnancy, the analysis of cffDNA allows us to perform gender determination as early as 9 weeks gestation. It can detect specific sequences of the Y-chromosome with great accuracy, which, if detected, would mean that the foetus is a male.

In the past, for the performance of this analysis, a blood sample needed to be taken from the patient’s arm. However, different tests have been developed recently to determine foetal sex with only a finger-prick blood sample, which is then sent to a laboratory for the cffDNA analysis. This makes it much more accessible and convenient.

For this study, a volume of 3-4 mL of maternal blood was collected from 241 participants between 9 and 35 weeks of pregnancy. Then the plasma was separated from the whole blood and the cffDNA was isolated from a micro-volume of it (100 µL). Finally, real-time quantitative PCR was performed using a multi-copy target sequence on the Y-chromosome to detect the presence of male cell-free DNA.

The accuracy of the test for foetal sex determination in this study was 99.6 %, identifying correctly 100% of the participants carrying a male foetus, and demonstrating its use as a simple and reliable method requiring only a small volume of maternal plasma.

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