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Technical Briefs |
1
Department of Obstetrics and Gynecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, and
2
Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
a address correspondence to this author at: Department of Chemical Pathology, The Chinese University of Hong Kong, Prince of Wales Hospital, Room 38023, 1/F Clinical Sciences Bldg., 30-32 Ngan Shing St., Shatin, New Territories, Hong Kong SAR; fax 852-2194-6171,
loym{at}cuhk.edu.hk
Preeclampsia is a multisystem disorder specific to pregnant women. It remains one of the most important causes of maternal and fetal mortality and morbidity in developed countries (1). Although the pathogenesis of this condition is not fully understood, it is now widely accepted that vascular endothelial cell dysfunction is the final common pathway responsible for the maternal syndrome (2)(3). The underlying pathological changes that lead to the endothelial cell dysfunction remain incompletely understood, but poor placentation has been proposed as a major contributory factor (2)(4)(5). As a result of incomplete or failure of trophoblastic invasion of the spiral arteries, placental ischemia results, leading to the release of one or more factors that are responsible for the damage of the maternal vascular endothelium (5)(6). The normal process of trophoblastic invasion is complete by 20 weeks of gestation. Hence, the initiating placental pathology should exist prior to this stage of pregnancy, long before the onset of the clinical syndrome. Therefore, it might be possible to develop new plasma/serum biochemical markers for identifying subjects at increased risk of developing preeclampsia.
We previously have shown that women with established preeclampsia have a fivefold increase in circulating fetal DNA concentrations in their plasma compared with control pregnant subjects (7). However, it is unknown whether this is a late phenomenon or whether it precedes the onset of clinical symptoms and signs. In this study, we aimed to test whether the abnormal increase in circulating fetal DNA concentrations can be detected in susceptible subjects before onset of the clinical disease.
Pregnant women attending the Department of Obstetrics and Gynecology, Prince of Wales Hospital, Hong Kong were recruited with informed consent. The study was approved by the Clinical Research Ethics Committee of The Chinese University of Hong Kong. Only singleton pregnancies were included. The gestational ages of all studied subjects were confirmed by early ultrasound examination. Antecubital venous blood (10 mL) was collected from each subject between 11 and 22 weeks of gestation and placed immediately into EDTA tubes. After centrifugation at 3000g for 20 min, the plasma samples were collected into plain polypropylene tubes and stored at -70 °C until further processing. All subjects were followed until delivery, and all relevant clinical information was recorded.
Of those who carried male fetuses and had blood sampled, 18 subjects
subsequently developed preeclampsia as the only antenatal complication.
The gestational age at onset of clinical disease was 27.4–40.0 weeks.
Preeclampsia was defined essentially as described previously
(8), on the basis of a diastolic blood pressure >110 mmHg
on one occasion or >90 mmHg on two or more occasions at least 4 h
apart, with the presence of significant proteinuria in subjects with no
history of hypertension. Significant proteinuria was defined as
proteinuria >0.3 g/day or 
2+ on dipstick testing in two clean-catch
midstream urine specimens collected at least 4 h apart. Another 33
subjects who carried male fetuses and had no antenatal complication
were randomly selected as the control group. The mean gestational ages
of the preeclamptic and control subjects were 17.2 weeks (SD, 2.9
weeks) and 18.0 weeks (SD, 1.3 weeks), respectively. There was no
statistically significant difference in the gestational ages at blood
sampling between the preeclamptic and control groups (P
= 0.23).
Plasma samples from these two groups were assayed for circulating fetal DNA, using the SRY gene on the Y chromosome as a marker, as described previously (7). The sensitivity and precision of this assay have been reported previously (7). As a control for the amplifiability of plasma-extracted DNA, all samples were subjected to a TaqMan assay for the ß-globin gene on chromosome 11. Samples from 10 pregnant subjects carrying female fetuses during the second trimester were also assayed as negative controls. The laboratory staff responsible for the molecular analysis of the samples were unaware of the clinical status of the subjects from whom the samples were obtained.
Circulating fetal DNA was detected in all subjects carrying male
fetuses in both the preeclamptic and control groups. Fig. 1
shows the plasma fetal DNA concentrations of these two groups
of subjects. The median fetal DNA concentrations in preeclamptic and
control pregnancies were 41.9 genome-equivalents/mL (interquartile
range, 25.8–62.8; range, 36.3–2375) and 22.0 genome-equivalents/mL
(interquartile range, 15.3–31.5; range, 4.25–300), respectively.
Fetal DNA concentrations were significantly higher in preeclamptic than
control pregnancies (Mann–Whitney rank-sum test, P =
0.001; U-statistic = 340.5). None of the plasma samples
from the 10 women carrying female fetuses had any SRY
signal.
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Positive amplification signals from the ß-globin gene were detected in all tested samples, thus confirming the quality of the DNA samples. There was no significant difference in the plasma concentrations of the ß-globin gene between the preeclamptic and control groups (Mann–Whitney rank-sum test, P = 0.16).
Our data indicate that increased concentrations of maternal plasma fetal DNA could be detected in susceptible subjects before the onset of clinical presentation of preeclampsia. The mechanisms producing this increase require further investigation. Previously, we proposed that the possible pathways for maternal plasma fetal DNA increase after the onset of preeclampsia include increased liberation of fetal DNA into the maternal circulation and/or reduced clearance of circulating DNA from maternal blood (7). The latter mechanism was proposed because the kidney and liver have been suggested to be the main organs for the removal of circulating DNA (9)(10). Because pathologic changes involving the kidney and liver are well described in preeclampsia, it is likely that these processes might reduce the organs’ ability to remove DNA from the circulation. However, this mechanism is probably less likely to be important in the current study cohort, who had no renal or liver function abnormalities before the onset of preeclampsia.
It is therefore reasonable to suggest that increased liberation of fetal DNA into the maternal circulation is likely to be the main reason for the early increase in plasma fetal DNA concentrations in subjects who subsequently develop preeclampsia. Theoretically, increased fetal DNA liberation could be secondary to increased entry of fetal cells, such as trophoblasts (11)(12) and erythroblasts (8), into the maternal circulation. In support of this possibility is the recent demonstration of increased fetal erythroblast trafficking into the circulation of women who subsequently develop preeclampsia (13). Alternatively, fetal DNA could be liberated directly from dying cells in the placenta. In this regard, widespread apoptosis has been demonstrated in cytotrophoblasts obtained from the placental beds of preeclamptic pregnancies (14). Increased incidence of apoptosis involving syncytiotrophoblasts has also been reported in preeclampsia (15). Further research on the correlation between fetal DNA concentrations and incidence of placental apoptosis may help to confirm this link.
The potential clinical implication of our findings is that maternal
plasma fetal DNA might be used as marker for predicting preeclampsia.
However, our data showed that there was overlap in the fetal DNA
concentrations between the preeclamptic and control groups. This
implies that a relatively low sensitivity and specificity would result
if maternal plasma fetal DNA measurement is used as the sole predictor
for preeclampsia. Thus, ROC curve analysis (MedCalc 5.0)
revealed that the best discrimination between the preeclamptic and
control groups was obtained at a fetal DNA concentration of 33.5
genome-equivalents/mL. The sensitivity and specificity at this cutoff
concentration were 67% (95% confidence interval, 41–87%) and
82% (95% confidence interval, 65–93%), respectively. The area under
the ROC curve was 0.778 (SE = 0.073; 95% confidence interval,
0.639–0.882). Nonetheless, our data open up the possibility of
predicting preeclampsia using maternal plasma fetal DNA, especially
when used with other biochemical markers such as
corticotropin-releasing hormone, 
-fetoprotein, inhibin A, and
activin A (16)(17). In this regard, it is
also important to explore the use of fetal DNA markers outside the Y
chromosome so that this type of analysis can be extended to pregnant
women carrying female fetuses. Assays that are potentially applicable
in this capacity have recently been described (18)(19)(20).
Finally, our preliminary data would serve to stimulate further
large-scale studies to explore the possible correlation of this new
marker to the severity of the disease.
Acknowledgments
This work is supported by the Earmarked Research Grants Scheme from the Hong Kong Research Grants Council (CUHK 4255/99M).
References
-fetoprotein concentrations for predicting pre-eclampsia. Hum Reprod 2000;15:1813-1818.The following articles in journals at HighWire Press have cited this article:
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  H. S. Nielsen, R. Steffensen, K. Varming, A. G.S. Van Halteren, E. Spierings, L. P. Ryder, E. Goulmy, and O. B. Christiansen Association of HY-restricting HLA class II alleles with pregnancy outcome in patients with recurrent miscarriage subsequent to a firstborn boy Hum. Mol. Genet., May 1, 2009; 18(9): 1684 - 1691. [Abstract] [Full Text] [PDF]
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