Proteinuria and hypertension are independent factors affecting fetal DNA values: a retrospective analysis of affected and unaffected patients.

Cell-free fetal DNA circulates in the plasma of pregnant women (1)(2)(3). Increased concentrations of fetal DNA have been reported in the plasma of pregnant women with various complications of pregnancy, including preeclampsia (PE) (4)(5)(6), preterm labor (7), invasive placenta(8), hyperemesis gravidarum (9), and aneuploidy(10)(11)(12). We recently reported that fetal DNA in maternal plasma is derived primarily from villous trophoblasts bordering the intervillous spaces, which are filled with maternal blood (13). Because trophoblast damage may be involved in the pathogenesis of PE, it may be possible to use fetal DNA as a marker to monitor the severity of PE. In this study, we assessed the relationship between fetal DNA concentrations in maternal plasma and clinical evidence of PE, such as proteinuria and/or hypertension. We conducted a retrospective study in which the control group included 116 women with uncomplicated male pregnancies between 28 and 40 weeks of gestation. Controls were matched with 45 consecutive pregnancies in which different grades of proteinuria and/or hypertension were observed at the time of blood sampling (28–40 weeks). PE was defined as gestational hypertension (systolic pressure >140 mmHg or diastolic blood pressure >90 mmHg on at least two occasions after 20 weeks of gestation) with proteinuria (>0.3 g/day). Severe PE (SPE) was defined as severe gestational hypertension (systolic pressure >160 mmHg or diastolic blood pressure >95 mmHg on at least two occasions after 20 weeks of gestation) with severe proteinuria (>3 g/day). Fetal growth restriction was defined as an estimated fetal weight 2.0 SD below the mean expected weight for gestational age (GA), as determined by ultrasonographic evaluation. None of the pregnant women had fetal aneuploidy, abnormalities of cord insertion, and/or maternal complications such as systemic lupus erythematosus, diabetes mellitus, or hyperthyroidism. All samples were obtained before the onset of labor. In the present study, 24 cases had PE, 8 had gestational hypertension, and 13 had proteinuria alone, the symptoms of which occurred after 20 weeks of gestation. All women presented at Showa University Hospital between August 2001 and December 2002. Fetal growth restriction was observed in 15 of 24 preeclamptic women. All study participants provided written informed consent for the use of their biological specimens for research purposes. The ethics committee of Showa University School of Medicine approved this protocol. Maternal blood samples (7 mL) were collected into tubes containing EDTA. After separation, the plasma samples were stored at -20 °C until use. DNA was extracted from 1.5-mL samples of plasma supernatant by use of a QIAamp Blood Mini Kit (Qiagen). The Y-chromosome-specific DYS14 sequence was subsequently quantified by PCR in a LightCycler (Roche Diagnostics), as described in previous reports (1)(6). Strict precautions were taken to avoid contamination, and water blanks were used as negative controls. A female staff member performed all procedures, including sample preparation, DNA extraction, and PCR amplification. Descriptive analysis of the available variables was performed by routine testing. Data were first stratified according to the presence of PE: 0 = absent; 1 = mild PE; and 2 = SPE. In addition, stratification based on the presence of proteinuria and/or hypertension was performed, including those cases affected by proteinuria alone or hypertension alone. Symptoms of proteinuria and hypertension were classified as follows: 0 = absent; 1 = mild; and 2 = severe. Two different log10 linear regressions were performed, the first to estimate the effect of mild and severe PE on fetal DNA concentrations, and the second to estimate the value of proteinuria and hypertension as independent predictors of increased fetal DNA. In the first regression analysis, we included only those cases with evidence of PE. All data (PE cases + hypertension...