Three-hour analysis of non-invasive foetal sex determination: application of Plexor chemistry

Adalgisa Pietropolli, Maria Vittoria Capogna, Raffaella Cascella, Chiara Germani, Valentina Bruno, Claudia Strafella, Simona Sarta, Carlo Ticconi, Giusy Marmo, Sara Gallaro, Giuliana Longo, Luigi Tonino Marsella, Antonio Novelli, Giuseppe Novelli, Emilio Piccione, Emiliano Giardina

The knowledge of the individual genetic "status" in the prenatal era is particularly relevant in the case of positive family history for genetic diseases, in advanced maternal age and in the general screening for foetal abnormalities. In this context, here, we report an innovative molecular assay which utilizes the cell-free foetal DNA (cffDNA) as a source for the early and fast detection of the foetal sex. The study involved 132 pregnant women in their first 3 months of pregnancy, who agreed to give a blood sample. All the collected samples were immediately subjected to the separation of the plasma, which was utilized for the extraction of the cffDNA. Successively, the extracted cffDNA was analysed by a quantitative PCR (qPCR) method based on Plexor-HY chemistry, which is able to simultaneously identify, quantify and discriminate the autosomal DNA from the sex-linked DNA. Overall, the Plexor-HY assay demonstrated to be sensitive and specific for the determination of low-template DNA, such as the cffDNA. In fact, the Plexor-HY assay has been successfully performed in all the samples, identifying 70 males and 62 females. As the foetal sex can be provided in 120 min just by utilizing a maternal blood sample as cffDNA source, the assay represents a very fast, safe and non-invasive prenatal method. The possibility of determining the foetal sex in the early prenatal life consents the application of our assay as a helpful screening test for subjects and families at risk of sex-linked disorders. Moreover, the early knowledge of the foetal sex may be of great help even for the specialist, who might promptly advise the patients concerning the foetal risk of inheriting sex-linked disorders and the clinical utility of performing an invasive prenatal diagnosis.