Non-invasive prenatal determination of fetal gender using QF-PCR analysis of cell-free fetal DNA in maternal plasma

Abstract

Background

Detection of cell-free fetal DNA (cffDNA) in maternal plasma has given rise to the possibility of new non-invasive approaches for early prenatal diagnoses. We evaluated the feasibility and accuracy of non-invasive fetal gender determination using quantitative fluorescent-polymerase chain reaction (QF-PCR) analysis of circulating cffDNA in the first-trimester maternal plasma.

Methods

Plasma samples were prospectively collected from 202 singleton pregnancies at 4 to 13 weeks of gestation. Fetal gender was determined by QF-PCR with the sex-determining region Y (SRY) and amelogenin X/Y (AMELX/Y) genes. The result was confirmed by fetal karyotyping or phenotype at birth.

Results

Of the 202 pregnancies, 162 had pregnancy outcomes available and could be included in our evaluation. The accuracies of AMELX/Y, SRY, and combined AMELX/Y + SRY analysis for fetal gender determination were 83.3%, 82.1%, and 97.5%, respectively, compared with those of the invasive approach and the fetal gender outcome at birth (82 males and 80 females). Combined AMELX/Y + SRY analysis had the highest sensitivity (98.8%) for fetal gender determination with a specificity of 96.3%. Moreover, fetal gender detection by the combined AMELX/Y + SRY analysis at 11 to 13 weeks of gestation was 100% correct.

Conclusion

Fetal gender determination could be accurately determined from maternal cffDNA in the first-trimester using QF-PCR analysis of combined AMELX/Y + SRY.

Introduction

Early prenatal determination of fetal gender is required for fetuses at risk of X-linked genetic diseases, as identification of a male fetus indicates hemizygosity for the X chromosome and thus potential disease if the mother is a carrier. Traditionally, early fetal gender determination has relied on invasive procedures such as chorionic villus sampling (CVS) or amniocentesis. These procedures, however, still carry a 1% to 2% risk of miscarriage [1], [2] and cannot be performed until 11 weeks of gestation. Reliable determination of fetal gender by means of ultrasonography cannot be performed in the first-trimester, because development of the external genitalia is not complete [3].

The recent discovery of the presence of cell-free fetal DNA (cffDNA) in maternal plasma and serum has offered new approaches for non-invasive prenatal diagnoses. The concentration of cffDNA in maternal plasma was found to be much higher than that present in the cellular fraction [4]. Since cffDNA in maternal plasma circulates within the background of maternal DNA, it has been used mainly for non-invasive prenatal diagnosis of paternally-inherited disorders as well as fetal gender [5], [6], [7], [8], [9]. Many researchers have explored the possibility of circulating cffDNA on non-invasive prenatal diagnosis of fetal gender, RhD blood typing, aneuploidy, and paternally inherited single-gene disorders such as cystic fibrosis and Huntington's disease [10], [11]. Promising results have been reported from studies using the combination of markers and the application of various experimental methods [10], [11].

The specific sequences in Y chromosome such as DYS14, and DYZ3, the Y specific repeat sequences [12] and the sex-determining region Y (SRY) gene were usually amplified with polymerase chain reaction (PCR) technique [13], but the results were not completely reliable because of the lack of the amplification of internal control. The application of real-time quantitative PCR technique largely increases the sensitivity (close to 100%) and specificity (100%) of detecting the cffDNA in maternal plasma, which is a non-invasive fetal gender diagnosis by detecting the specific fragment in Y chromosome [14]. However, the expensive nature of real-time quantitative PCR machine limits its clinical applications, especially in developing countries. The common method of non-invasive early diagnosis of fetal gender still has important value.

The amelogenin gene is located in the sex chromosome. There is 6 bp difference in intron 3 of amelogenin gene between X-linked amelogenin (AMELX) and Y-linked amelogenin (AMELY). Detection of the 6 bp of difference may help us evaluate prenatal fetal gender [15]. In this prospective study, we determined fetal gender in the first-trimester pregnancies using circulating cffDNA in maternal plasma by quantitative fluorescent PCR (QF-PCR) analysis of AMELX/Y and SRY genes.