Early identification in maternal blood of fetal sex in the presence of fetal DNA from previous pregnancies

Abstract

The presence of cell-free fetal DNA in maternal plasma allows for the noninvasive identification of fetal sex. It was recently suggested, however, that fetal-cell DNA can persist in the maternal circulation from one pregnancy to the next [1], thereby severely hampering prenatal diagnostic analysis of fetal DNA from maternal plasma. A method for clearing fetal-cell DNA from a previous pregnancy in maternal plasma was developed to facilitate the identification of cell-free fetal DNA from the current pregnancy. Thirty-three of the 73 recruited women were pregnant, and the 40 who were not been pregnant with at least 1 male child. Characteristics of the entire sample are shown in Table 1. Blood samples were collected in ethylene diamine tetra-acetic acid (EDTA) tubes with 4% formaldehyde [2]. Samples from nonpregnant women underwent 3 different protocols. The first protocol consisted in centrifugation at 200 ×g for 10 min; the second protocol consisted of the first protocol, followed by centrifugation at 1600 ×g for 10 min, followed by centrifugation at 13,000 ×g for 10 min. The third protocol consisted in the second protocol plus a plasma filtration through 0.22 μm filters (Millex-GS, Millipore, Bedford, MA, USA). All samples from pregnant women were processed using the third protocol. DNA was extracted from 400 μL of plasma using aQIAampDNABloodMini Kit (Qiagen, Hilden, Germany). Real-time polymerase chain reaction with an ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA)was used to amplify SRY and GAPDH gene sequences. Of the extracted plasma DNA, 5 μL was amplified to a final volume of 25 μL under the following cycling conditions: 50 °C for 2min, 95 °C for 10min, 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The study findings are shown in Table 2. When the third protocol was used, fetal-cell DNA from previous pregnancies was entirely cleared out from the plasma of nonpregnant women. For this reason, the third protocol was also used in pregnant women. The GAPDH gene was detected in all samples and the SRY gene only in the samples of women carrying male fetuses. Sensitivity and specificity were 100% for the prediction of fetal sex. By the time recruitment for the present study was completed and DNA extracted using formaldehyde, finding that formaldehyde increased the amounts of cell-free fetal DNA in maternal plasma in [2] was no longer held accurate [3]. Nevertheless, this study's findings confirm and expand on recently published results regarding the diagnosis of fetuses at risk for X-linked diseases [4]. Clearing maternal plasma from fetal-cell DNA from previous pregnancies further enhances the potential of