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Technical Briefs |
1 Department of Obstetrics and Gynecology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan
2 Institute of Embryology, Obstetrics and Gynecology, University of Bologna, Bologna, Italy
aauthor for correspondence: fax 81-33784-8355, e-mail sekizawa{at}med.showa-u.ac.jp
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-alone cases + proteinuria-alone cases) were included in the second regression analysis. The regressions were done with fetal DNA as the dependent variable and GA as a quantitative independent variable. Furthermore, data with regard to PE (for the first regression) and proteinuria and hypertension (for the second regression) were placed in the linear regression analyses as "dummy" variables; thus, they assumed values of 0 = absent, 1 = mild, or 2 = severe. The mean effect on fetal DNA was considered log-linear at any week and then estimated exactly halfway (mean) through the study interval (28–40 weeks of gestation).
The patient demographics for each group included in the regression analyses are shown in Table 1
. The GAs of the pregnancies in this study ranged from 28 to 40 weeks, with a mean (SD) GA of 34 (3.28) weeks. We thus used 34 weeks as a reference point by which to estimate the effect of various variables on fetal DNA values. GA was associated with an increase of 
12% in fetal DNA per week.
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The following equation was derived to estimate fetal DNA values according to severity of PE: log10DNA = 0.44 + 0.035 x GA + 0.355 x PE (see the Data Supplement that accompanies the online version of this Technical Brief athttp://www.clinchem.org/content/vol50/issue1/ for details). At 34 weeks, PE was associated with a 2.25-fold increase in the fetal DNA concentration, whereas SPE was associated with a 5.06-fold increase.
Both proteinuria and hypertension showed a strong independent association with fetal DNA values, including in the presence of GA, which represents a well-known source of variation in fetal DNA values. The following equation was derived to estimate fetal DNA values as a function of proteinuria (P) and hypertension (H): log10DNA = 0.62 + 0.045 x GA + 0.259P + 0.201H (see the online Data Supplement for details). Proteinuria was associated with greater increases in fetal DNA than hypertension (see the online Data Supplement for details). In fact, fetal DNA was 1.82 and 3.32 times greater in patients with mild and severe proteinuria, respectively, compared with controls at 34 weeks. The presence of hypertension was associated with mean increases of 1.58- and 2.52-fold in patients with mild and severe hypertension, respectively, compared with controls. Fig. 1
shows DNA estimations based on the second linear regression scale. It should be noted that similar fetal DNA values were calculated for the control group by the first and the second regression.
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The pathogenesis of PE is poorly understood, but it is likely associated with failure of the uterine vasculature to undergo adequate physiologic remodeling by extravillous trophoblasts in women with PE (14). Because there is insufficient invasion of extravillous trophoblasts into the uterine vasculature, placental vascular resistance is not reduced; there thus is inadequate oxygenation of blood within the placental intervillous spaces that border villous trophoblasts. This might damage the villous trophoblasts (15), leading to the release of DNA into the intervillous spaces as a result of cell damage or apoptosis (6)(13), after which it might enter the maternal circulation. Thus, both hypoxia and increased amounts of circulating fetal DNA might occur with insufficient trophoblast invasion. Lo et al. (15) have reported that the half-life of circulating fetal DNA (placental DNA) is 16.3 min. However, a fourfold increase in the clearance half-life of fetal DNA has been observed in patients with PE compared with controls (114 vs 28 min) (16). The rapid turnover of fetal DNA in women with PE might make fetal DNA a useful marker to monitor trophoblast damage over time.
In the present study, estimated fetal DNA concentrations in patients with mild and severe PE were 2.25 and 5.06 times greater than those in controls at 34 weeks (mean gestational age of the line of regression). We assumed a log-linear relationship between severity of PE and maternal fetal DNA concentrations. At the very least, our findings indicate that fetal DNA values increase with increasing severity of PE. This is likely attributable to more extensive villous trophoblast damage in patients with SPE than in those with milder PE. If there is indeed a correlation between fetal DNA concentrations and trophoblast damage, hypoxia within the intervillous spaces might be more pronounced in cases of SPE than cases of mild PE.
We also examined the relationship between proteinuria and hypertension and fetal DNA values. We found (a) that both proteinuria and hypertension were independently and strongly associated with increased concentrations of fetal DNA in maternal plasma, the extent to which depended on the severity of each symptom; and (b) that proteinuria was associated with greater increases in fetal DNA than hypertension. Again, we assumed a log-linear relationship between severity of proteinuria and hypertension and maternal fetal DNA values. We speculated that the pathogenesis of proteinuria might differ from that of hypertension and that proteinuria in pregnant woman might be more closely related to hypoxic damage to villous trophoblasts than hypertension. On the basis of our results, it appears that proteinuria is a better indicator of damage to villous trophoblasts or placental hypoxia than hypertension. We believe that further studies may be needed to help clarify these findings.
In summary, this is the first report to describe a relationship between fetal DNA in maternal plasma and severity of PE, as well as an association between fetal DNA and proteinuria and hypertension. Because fetal DNA is thought to come from villous trophoblasts and is associated with the severity of each of these findings, fetal DNA might represent a molecular marker that can be used to evaluate trophoblast damage and monitor the status of pregnancies affected by PE in clinical practice.
Acknowledgments
This work was supported in part by two Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sport and Culture of Japan (Grants 14770870 and 15591163), as well as the Takeda Science Foundation, Japan, and the Fondazione CARISBO Progetto Triennale–Molecular Genetics of Fetal DNA, Italy.
References
The following articles in journals at HighWire Press have cited this article:
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  A. F. Orozco, C. J. Jorgez, C. Horne, D. A. Marquez-Do, M. R. Chapman, J. R. Rodgers, F. Z. Bischoff, and D. E. Lewis Membrane Protected Apoptotic Trophoblast Microparticles Contain Nucleic Acids: Relevance to Preeclampsia Am. J. Pathol., December 1, 2008; 173(6): 1595 - 1608. [Abstract] [Full Text] [PDF]
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 Y. Purwosunu, A. Sekizawa, K. Koide, A. Farina, N. Wibowo, G. H. Wiknjosastro, S. Okazaki, H. Chiba, and T. Okai Cell-Free mRNA Concentrations of Plasminogen Activator Inhibitor-1 and Tissue-Type Plasminogen Activator Are Increased in the Plasma of Pregnant Women with Preeclampsia Clin. Chem., March 1, 2007; 53(3): 399 - 404. [Abstract] [Full Text] [PDF]
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A. Farina, C. W.M. Chan, R. W.K. Chiu, N. B.Y. Tsui, P. Carinci, M. Concu, I. Banzola, N. Rizzo, and Y.M. D. Lo Circulating Corticotropin-Releasing Hormone mRNA in Maternal Plasma: Relationship with Gestational Age and Severity of Preeclampsia Clin. Chem., October 1, 2004; 50(10): 1851 - 1854. [Full Text] [PDF]
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