Noninvasive fetal sex determination in maternal plasma using Multiplex PCR

Background and Aim: Fetal gender determination at an early stage in pregnancy is clinically important in the management of X-linked genetic disorders. Prenatal screening and diagnosis offered in during pregnancy in order to monitor gestation outcome, that women to make aware choices about the resumption of pregnancies affected by genetic conditions. Since the discovery of cell-free fetal DNA (cffDNA) in maternal plasma, diagnostic non-invasive prenatal methods have been developed or optimized for fetal sex determination and identification of genetic diseases. In this study, non-invasive determination of fetal sex was performed by multiplex polymerase chain reaction (Multiplex PCR), and detection of Y-chromosome specific sequences (SRY gene and DYS۱۴) in maternal plasma. The absence of Y-chromosome sequences in maternal plasma implies that the fetus is female.Methods: The SRY and DYS۱۴ genes primers were designed according to specific sequence at the ۳' end of SRY and DYS۱۴ genes in chromosome Y, using Free-software Primer ۳. All oligonucleotide sequences were synthesized by Bioneer, Korea. Blood samples were collected from ۲۰ pregnant women using EDTA tubes. Approximately ۵ ml of blood was sampled at ۸ weeks of gestation with consent. Plasma was isolated within ۳ hours of blood collection and isolated plasma was used to extract free fetal DNA. Extraction of circulating cffDNA was extracted using the NucleoSpin® plasma XS Kit. To determine the sex of the embryo, we amplified a portion of the Y chromosome-specific SRY and DYS۱۴ genes using PCR. The PCR reaction was performed in ۲۵ µl volume containing ۵۰ ng cffDNA, and amplified in ۳۶ cycles for cffDNA extracted from plasma.Results: The specificity of the SRY and DYS۱۴ genes based PCR method was investigated to determine the sex of the fetus. The results of this study showed that the SRY and DYS۱۴ primers were specific for the extracted cffDNAs of known gender. Plasma samples were diagnosed correctly in ۱۶ cases and in ۲ cases the results did not match the sex of the born fetus. In that case it was false negative in ۲ cases.Conclusion: The results of this study showed that dual marker SRY and DYS۱۴ PCR analysis as a high-sensitivity method for early detection of fetal gender using cffDNA can be considered as a non-invasive pre-test.

کلیدواژه ها:

NIPT ، cffDNA ، chromosome Y ، PCR ، sex determination