Hypertension is a common medical complication during pregnancy; pre-eclampsia is part of a group of hypertensive disorders in pregnancy that can be divided into gestational hypertension, chronic hypertension, pre-eclampsia, and pre-eclampsia superimposed on chronic hypertension 1. Pre-eclampsia occurs in 2–5% of pregnancies, but complicates up to 10% of pregnancies in developing countries, where emergency care is often inadequate or lacking 2. Severe pre-eclampsia is associated with significant maternal morbidity, including eclamptic seizures, intracerebral hemorrhage, pulmonary edema or heart failure, acute renal failure, liver dysfunction, and coagulation abnormalities. Fetal complications include abruptio placentae, intrauterine growth restriction, premature delivery, and intrauterine fetal death. Currently, there is no single reliable parameter for the prediction of pre-eclampsia, and considerable attention has focused on various blood-borne and urinary biomarkers.
Doppler ultrasound is a part of antenatal care that uses sound waves to detect the movement of blood in vessels. It studies blood circulation in the baby, the mother’s uterus, and the placenta. It has been used as a modality to evaluate placental circulation and fetal well-being for about three decades 3. Abnormal development of placental vasculature is considered as the pathophysiological basis for the development of pre-eclampsia 4 and this could be reflected in abnormal umbilical Doppler velocimetry. In normal pregnancies, the fetoplacental circulation acts as a low-resistance system unit. Thus, the blood velocity waveforms in the umbilical artery (UA) show continuous forward flow throughout the cardiac cycle 3.
High-resistance uterine artery Doppler in the third trimester of pregnancy can predict adverse postpartum outcome 5. Women with late-onset pre-eclampsia show a higher risk of perinatal complications if uterine resistance increases, although maternal outcome does not seem to be related to Doppler findings 6. Doppler waves of the middle cerebral artery (MCA) can detect most fetuses at a high risk in pregnancies complicated by pre-eclampsia 7. Several studies have shown the efficacy of the MCA Doppler assessment during pregnancy 8,9.
Several studies have reported that in patients with pre-eclampsia, the maternal plasma concentration of cell-free fetal DNA (cffDNA) using a Y-chromosomal DNA sequence is 2–15-fold higher than that in normotensive control individuals 2,10. It has been postulated that impaired trophoblastic invasion of the maternal spiral arteries leads to placental ischemia with the release of necrotic or apoptotic syncytiotrophoblast fragments that contain fetal DNA into the maternal circulation 11,12. In addition to evidence for increased entry of cffDNA into the maternal circulation, there is also evidence for reduced clearance of cffDNA from maternal plasma 13.
It is still controversial whether the amount of total circulating DNA can be used as a predictive marker for pre-eclampsia. Some studies found no changes, whereas others determined significant differences, between women with a normal outcome and those with this pathologic outcome. However, the quantification of total cell-free DNA levels in the plasma of women with established pre-eclampsia may serve to predict or diagnose further complications, such as hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome 14.
The aim of our study was to measure the plasma total cell-free DNA and cffDNA levels in pre-eclamptic pregnant women during the third trimester (gestational age>28 weeks) and whether it shows a correlation with uteroplacental blood flow.
Materials and methods
This study included 120 pregnant women during the third trimester (60 women diagnosed with pre-eclampsia, representing the study group, and another 60 normotensive pregnant women representing the controls). Cases were recruited among patients who attended the antenatal outpatient clinic – Kasr Al Aini Hospitals (Obstetrics and Gynecology Hospital) and the Medical Service Unit – National Research Center (Reproductive Health Department) in the period from January to December 2011. Informed consent was obtained from all the participants. The Ethical Committees of Cairo University and the National Research Centre provided approval for the study.
Patients enrolled in this study fulfilled the following criteria: singleton, viable pregnancy, gestational age more than 28 weeks, blood pressure equal or more than 140/90 with proteinuria, no history of hypertension before pregnancy, and no obstetric or medical complication of pregnancy apart from pre-eclampsia. Maternal complications other than pre-eclampsia, for example diabetes mellitus were excluded.
All pregnant women were subjected to full assessment of history and clinical examination (general and local). Ten milliliters of maternal venous blood was then collected. During the same visit, all of them were subjected to a fetal ultrasound scan to assess the fetal growth and exclude major fetal anomalies and was also a transabdominal color Doppler examination to measure umbilical resistance index (RI), uterine RI and pulsitility index (PI), and MCA/umbilical PI using a SONOACE X8 MEDISION U/S machine (Sonoace X8; Medison, Korea) equipped with pulsed and color Doppler options with a probe frequency of 3–5 MHz.
Sample processing and DNA extraction
Ten milliliters of peripheral blood was drawn in an EDTA-containing tube and cell-free plasma samples were obtained by centrifugation of whole blood at 1600g for 10 min. Plasma was transferred to microcentrifuge tubes and centrifuged at 16 000g for 10 min to remove residual cells. The two centrifugation steps were performed within 24 h after blood collection. Cell-free plasma was stored at −80°C until further processing; thawing was carried out only once before DNA extraction. DNA extraction from 500 μl cell-free plasma samples was carried out using the QIAamp Mini Kit (Qiagen GmbH, Hilden, Germany) and eluted with 50 μl of H2O; 35 μl of plasma DNA were digested with 100 U of BstU I, a methylation-sensitive restriction enzyme, at 60°C for 16 h 15.
Real-time detection of RASSF1A
PCR amplifications were performed using 7500 fast real-time PCR (Applied biosystems, Foster City, California, USA). The sequences of the primers and probes are listed in Table 1. Each reaction contained 1× TaqMan Universal PCR Master Mix (Applied biosystems), 300 nmol/l of each primer, and 85 nmol/l probes. We used 10 μl of enzyme-digested plasma DNA or 5 μl of untreated DNA as a template for PCR. The thermal profile was 50°C for 2 min, 95°C for 10 min, 50 cycles of 95°C for 15 s, and 60°C for 1 min. All reactions were run in duplicate and the mean quantity was taken. A methylated DNA (Qiagen) was used as the standard 15.
Statistical comparisons were performed using SigmaStat v.3.0.1a (Systat Software, San Jose, California, USA). Data were statistically described in terms of range, mean±SD, frequencies (number of cases), and relative frequencies (percentages) when appropriate. The χ 2-test was used to compare qualitative variables between groups. An unpaired t-test was used to compare quantitative variables for parametric data (SD<50% mean). The Mann–Whitney test was used instead of an unpaired t-test for nonparametric data (SD>50% mean). Spearman correlation was used to rank different variables positively or inversely. An receiver operating characteristic curve was used to determine the best cut-off value in addition to validity. In general, a P-value less than 0.05 was considered statistically significant.
Our results showed that there was no statistically significant difference between normotensive pregnant women and pre-eclamptic patients according to the demographic data (maternal age, gestational age), P greater than 0.05, as shown in Table 2 using an unpaired t-test, but there was a statistically significant difference in hemodynamics parameters (P<0.05) as shown in Table 3 using an unpaired t-test.
Plasma cffDNA levels and total cell-free DNA levels were detected in both groups. There was a statistically significant difference (P<0.05) between plasma cffDNA concentration in pre-eclamptic and normotensive pregnant women groups using the Mann–Whitney test (Fig. 1). The median plasma cffDNA concentrations in pre-eclamptic and control pregnancies were 146 copies/ml (interquartile range, 219–471 copies/ml) and 33.63 copies/ml (interquartile range, 26–42 copies/ml), respectively. The mean was 199±160 copies/ml (mean±SD) in pre-eclamptic women and 38±25 copies/ml in the normotensive pregnant women.
Also, there was a statistically significant difference (P<0.05) between plasma total cell-free DNA concentration in pre-eclamptic than normotensive pregnant women using the Mann–Whitney test (Fig. 2). The median plasma total cell-free DNA concentration in pre-eclamptic and normotensive pregnant women was 322 copies/ml (interquartile range, 219–471 copies/ml) and 123 copies/ml (interquartile range, 69–137 copies/ml), respectively. The mean was 407±312 copies/ml (mean±SD) and 110±40 copies/ml in the same groups, respectively.
There was a significant association between plasma cffDNA and uteroplacental blood flow (UA RI, right and left uterine arteries’ resistance and pulsitility indices, and MCA/umbilical PI) in the pre-eclampsia group (P<0.05) but not in the control group (P>0.05) using the Spearman correlation test.
Also, a significant association was found between the plasma total cell-free DNA and uteroplacental blood flow (UA RI, right and left uterine arteries resistance and pulsitility indices, and MCA/umbilical PI) in the pre-eclampsia group (P<0.05).
However, in the control group, there was a significant association only between plasma total cell-free DNA and MCA/umbilical PI but no significant association with right and left uterine artery RI, right and left uterine artery PI, and UA RI (P>0.05) using the Spearman correlation test.
The plasma cffDNA sensitivity was 99% and specificity was 97% in the detection of pre-eclampsia, but the plasma total cell-free DNA sensitivity was 95% and specificity was 99% in the detection of pre-eclampsia. The best cut-off value of the plasma cffDNA level was 80 copies/ml whereas for the plasma total cell-free DNA level, it was 137 copies/ml (Table 4 and Fig. 3).
Quantitative changes in cffDNA in maternal plasma have been reported as an indicator for impending pre-eclampsia in different studies using real-time quantitative PCR for the male-specific SRY or DYS14 loci. The increased levels of cffDNA before the onset of symptoms may be because of hypoxia within the intervillous space leading to tissue oxidative stress and increased placental apoptosis and necrosis. In addition to the evidence of increased cffDNA shedding into maternal circulation, there is also evidence of reduced renal clearance of cffDNA in pre-eclampsia. As the amount of fetal DNA is currently determined by quantifying Y-chromosome-specific sequences, alternative approaches such as the measurement of total cell-free DNA or the use of sex-independent fetal epigenetic markers, such as DNA methylation, offer a promising alternative 16.
A new test for the detection of fetal DNA in maternal plasma has been discovered. This test is based on the detection of a hypermethylated placental DNA (fetal) sequence in the maternal circulation. The methylation pattern of the RASSF1A promoter in the placenta and maternal blood cells allows the use of methylation-sensitive restriction enzyme digestion for specifically cutting the maternally derived background RASSF1A sequences while leaving the placentally (fetal) derived RASSF1A sequences intact. It can be used as a marker irrespective of the fetal sex. Also, this marker allows the detection of false-negative results caused by low fetal DNA concentrations in maternal plasma when applied to prenatal RhD genotyping 15.
There were no significant differences between maternal and gestational age among the study and control groups (Table 2), which was in agreement with the results of Lazar et al. 17, Sifakis et al. 18 and Shahnaz et al. 19.
Our study showed that the cffDNA level was significantly higher in pre-eclamptic women compared with normotensive pregnant women. The same results were reported by Lazar et al. 17, Lo et al. 20, Leung et al. 21, Zhong et al. 22 and Zhong et al. 23, but they measured fetal DNA using real-time quantitative PCR for the SRY gene on the Y chromosome.
The present study concluded that the plasma total cell-free DNA level was higher in the pre-eclamptic group compared with the normotensive group. Our data were in agreement with those of Lazar et al. 24, who confirmed in their study that circulating total free and fetal DNA levels are significantly elevated in pregnancies complicated by pre-eclampsia. Also, Al Nakib et al. 14 found that plasma total cell-free DNA level was higher in patients with placental insufficiency than normotensive pregnant women.
Zhao et al. 25 and Wang et al. 26 reported that there was a positive correlation between fetal-derived hypermethylated RASSF1A levels and the severity of pre-eclampsia. The same results were found in our study.
Our results showed a correlation between plasma cffDNA level and the uteroplacental blood flow in pre-eclamptic women during the third trimester, which was in agreement with Sifakis’s study. Sifakis et al. 18 examined 220 women at 11–13 weeks of gestation to determine whether, in pregnancies that experience pre-eclampsia, plasma cell-free fetal DNA increased and whether this increase was related to the uterine artery PI. All fetuses were males and cffDNA were assessed by amplification of the DYS14 gene. They found that the increase in plasma cffDNA in pregnancies complicated by pre-eclampsia is associated with the degree of impairment in placental perfusion. There was a significant association between cffDNA and uterine artery PI in the pre-eclampsia group but not in the control individuals. This may be attributed to the fact that there are Doppler changes associated with the development of pre-eclampsia because of placental pathology. The placental pathology and placental apoptosis are associated with an increased level of plasma cffDNA and the degree of placental apoptosis, which reflect the degree of pre-eclampsia. The difference between our study and that of Sifakis was that in our study, the women were examined during the third trimester whereas in Sifakis’s study, women were examined during first trimester. In addition, we detected cffDNA by detection of hypermethylated RASSF1A but in Sifakis’s study cffDNA was assessed by amplification of the DYS14 gene.
Both plasma cell-free fetal and total cell-free DNA level could be considered potential markers for pre-eclampsia. The best cut-off value of the plasma cffDNA level was 80 copies/ml whereas for the plasma total cell-free DNA level, it was 137 copies/ml.
Conflicts of interest
There are no conflicts of interest.
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© 2013 Medical Research Journal
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