Pre-eclampsia 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 part of the maternal phenotype; restricted intrauterinal growth and preterm delivery with increased mortality and postnatal morbidity is part of the the fetal syndrome of preeclampsia 2. The pathogenesis of preeclampsia is still unclear, although poor placentation and subsequent oxidative damage in the placenta are known to play a role 3. Epidemiological studies have shown that risk factors for preeclampsia include nulliparity, obesity, and extremes of maternal age 4. Preterm birth is more often and mortality is higher among male fetuses 5. Two large retrospective studies concluded that male sex of the fetus is an independent risk factor for adverse pregnancy outcome 6,7. This might be related to the sex-specific differences in microvascular functions 8. In addition, induced vasodilatation was found to be altered by preeclampsia and male fetus 9.
Circulating nucleic acids of fetal origin have been found in maternal plasma 10. The concentration of free fetal DNA is higher in pre-eclamptic pregnancies 11. There is a correlation between fetal DNA levels and severity of symptoms 12. The dynamics of fetal DNA concentrations during pregnancy is rather complex, suggested to be because of placental necrosis or apoptosis and also as a result of decreased DNA elimination 13. Cell-free fetal RNA has also been identified in maternal circulation 14. No differences were found between the analyzed mRNA from plasma of women with pre-eclamptic and healthy pregnancies 15.
Recently, the use of differential methylation patterns between the promoter of the maspin gene in maternal blood cells and the placenta as the first universal fetal DNA marker in maternal plasma was reported 16. This observation was based on the rationale that fetal DNA molecules in maternal plasma are mostly derived from the placenta 17 whereas background maternal DNA originate from maternal blood cells 18. After the discovery of maspin as a cell-free fetal DNA (cffDNA) marker, an opposite differential methylation pattern, namely, hypomethylation in blood cells and hypermethylation in the placenta, of the promoter of the RASSF1A gene was reported 19. These patterns allow the placental-derived hypermethylated RASSF1A in maternal plasma to serve as a universal fetal marker irrespective of fetal sex and genetic variations.
We aim to investigate whether the quantification of cffDNA using the RASSF1A system will be effective for detection of the severity of pre-eclampsia.
Materials and methods
The current study included 120 pregnant women [60 age-matched women with normal pregnancy representing the control group and 60 cases representing the pre-eclampsia group (30 mild and 30 severe)] among cases attending the antenatal care clinic – Kasr Al Aini hospitals (Obstetrics & Gynecology Hospital) and the Medical Service Unit – National Research Center (Reproductive Health Department) after approval from the local ethical committee. Informed consent was obtained from 120 pregnant women and they were informed about the nature of the test and any other test that may be necessary. All patients with any other comorbidities were not included in the study from the start. Preeclampsia cases were identified at the time of the disease at gestational age more than 28 weeks.
Severe pre-eclampsia was defined as blood pressure of at least 160 mmHg systolic or 110 mmHg diastolic with proteinuria more than 5 g in 24 h with or without other features such as oliguria (<500 ml of urine in 24 h), cerebral or visual disturbances, pulmonary edema or cyanosis, epigastric or right upper quadrant pain, impaired liver function, thrombocytopenia, hemolysis elevated liver enzymes low platelets syndrome, or intrauterine growth restriction.
Sample processing and DNA extraction
Ten to 15 ml 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. 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, and 35 µl of plasma DNA were digested with 100 U of BstUI, a methylation-sensitive restriction enzyme, at 60°C for 16 h 20.
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 primers, and 85 nmol/l probes. We used 7.15 µl of enzyme-digested plasma DNA mixture 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 Gmbh) was used as the standard 20.
Data analyses were carried out using real-time 7500 fast SDS software v. 2.05 (Applied Biosystems).
Statistical comparisons were performed using SigmaStat v.3.0.1a (SPSS, Systat Software, Inc., Chicago, Illinois). In general, a P-value less than 0.05 was considered statistically significant.
The current study was carried out on 120 pregnant women [60 women with normal pregnancy representing the control group and 60 cases representing the pre-eclampsia group (30 mild and 30 severe)].The age of the women ranged between 20 and 38 years and all of them at more than 28 weeks’ gestational age. Demographic data are summarized in Table 2.
There were no statistically significant differences between the two groups studied in the maternal age, parity, or gestational ages (P=0.073, 0.087, and 0.069, respectively).
Because BstUI is a methylation-sensitive restriction enzyme, hypomethylated DNA sequences, such as the RASSF1A molecules derived from maternal blood cells, are digested and not detected after enzyme digestion. Our results showed that there was an increase in the level of cffDNA in mild and severe cases of pre-eclampsia (mean 166.1±130.38 and 283.62±222.64 copies/ml, respectively). In mild cases, the mean difference of cffDNA was 112.296 (with P<0.05). In severe cases, the mean difference of cffDNA was 227.402 (with P<0.05) (Table 3).
The comparison between all three categories of cases in terms of parity status showed that the cffDNA in primigravidas severe cases were significantly increased in comparison with the mild and control cases (P<0.05), whereas multipara cases did not show any significant difference (P>0.05) (Fig. 1).
Pre-eclampsia is one of the leading causes of maternal and fetal/neonatal mortality and morbidity worldwide 1. The disease occurs in 2–5% of pregnancies in the western countries, but it complicates up to 10% of pregnancies in the developing world, where emergency care is often inadequate or even lacking. Therefore, there is a need for widely applicable and affordable tests that can identify women at risk early in pregnancy and subsequently monitor them throughout pregnancy, and thus provide the best prenatal care for these patients and their children. Furthermore, if these tests could provide useful indications as to which women are likely to develop early-onset pre-eclampsia or a severe form of the disorder, this would allow an accurate risk categorization of women and would enable medical care providers to plan a more tailored course of action, for example a referral to a specialist center. This single action alone decreases neonatal mortality by ∼20% 21. Currently, there is no single reliable parameter for the prediction of pre-eclampsia, and considerable attention has shifted toward the development of noninvasive testing methods, including ultrasound examination and the quantification of various blood-borne and urinary biomarkers.
The value of cffDNA in maternal plasma as an indicator for preeclampsia was first reported by Lo et al.22, where cffDNA was increased approximately five-fold in women with preeclampsia in the third trimester compared with gestational age-matched controls. Also, the same effect was observed in the second trimester 23. Levine et al.13 studied 120 pre-eclamptic women and 120 controls: a two-fold to five-fold increase in cffDNA levels was observed starting from week 17 until 3 weeks before the onset of preeclampsia. As the amount of fetal DNA is routinely determined by quantifying Y-chromosome specific sequences, for example SRY (sex determining region Y) and DYS24, alternative approaches have been tested to overcome this limitation. Furthermore, approaches to analyze cffDNA independent of fetal sex, using epigenetic differences between maternal and fetal DNA, have been developed, for example the use of the maspin gene, which is hypomethylated in fetal tissue 25 or the hypermethylated fetal promoter sequence of RASSF1A26. Tsui et al.27 quantified cffDNA using the RASFF1A approach in 10 women with preeclampsia and 20 controls. Our results showed that there was an increase in the level of cffDNA in mild and severe cases of pre-eclampsia (mean 166.1±130.38 and 283.62±222.64 copies/ml, respectively). In mild cases, the mean difference in cffDNA was 112.296 (with P<0.05). In severe cases, the mean difference in cffDNA was 227.402 (with P<0.05).
In the study of Sifakis et al.28, the median cffDNA level was higher in those patients who developed early-onset pre-eclampsia compared with the controls, whereas in the case of late-onset pre-eclampsia, the levels were similar between cases and controls. Interestingly, cffDNA correlated significantly (P=0.038) with the pulsatility index of the uterine arteries, measured by transabdominal color Doppler in patients with subsequent pre-eclampsia, but not in control patients. Thus, the levels of cffDNA appeared to be already increased between 11 and 13 weeks of gestation in patients with subsequent early-onset pre-eclampsia 28,29.
Vlkova et al.30 hypothesize that fetal nucleic acids present in the maternal circulation can enter and be expressed by maternal cells. Especially, monocytes might phagocyte the subcellular particles that protect fetal RNA from degradation. This RNA can be used for translation of encoded proteins potentially breaking the immune tolerance against own maternal antigens (such as the angiotensin receptor 1). This may explain why the cffDNA level is significantly increased in primigravida on comparing multigravida cases in which the body tolerates the exogenous nucleic acids, which might lead to novel alternatives for prevention of pre-eclampsia including the use of RNases in vivo30.
The current hypothesis to explain the increased levels of cffDNA long before the onset of the clinical symptoms of pre-eclampsia proposes that the failure of transformation of uterine spiral arteries during the early stages of placentation may induce aberrant placental perfusion 31,32. As a consequence, the placenta may become chronically hypoxic, or most likely, alternating periods of hypoxia/reoxygenation within the intervillous space may subsequently trigger tissue oxidative stress and may increase placental apoptosis and necrosis 33. What likely follows is an increased shedding of necrotic and/or apoptotic subcellular syncytiotrophoblast debris that contain fetal DNA into the maternal circulation 34. Further evidence that cffDNA may originate from the placenta by apoptosis comes from an in-vitro study examining the effects of oxidative stress induced by 0.5% oxygen for 1 h, followed by reoxygenation, on trophoblastic tissue. Also, the concentration of cell-free β-globin DNA in the tissue supernatant was significantly increased 20 h after hypoxia–reoxygenation 35. In addition to the evidence for an increased release of cffDNA into the maternal circulation in pre-eclampsia, there is also solid proof of reduced renal clearance of these molecules in this pregnancy condition. In normal pregnancy, cffDNA is detectable as soon as 5 weeks after coitus, its levels increase with gestational age, and it completely disappears after 2 h following delivery (the mean half-life for circulating fetal DNA is 16.3 min, range 4–30 min) 36. In contrast, Lau et al.37 reported a much slower clearance rate in pre-eclamptic patients, with a median half-life of cffDNA clearance that was four times longer than that in unaffected women (114 min in the pre-eclamptic group vs. 28 min in the control group). The fast disappearance rate may be because of an efficient renal clearance in normotensive women, which is impaired in pre-eclampsia. However, other organs, such as the liver, may also contribute toward the impaired clearance of circulating DNA in pre-eclamptic patients 38.
The current study shows that hypermethylated RASSF1A (fetal DNA) in maternal plasma of pre-eclampsia represents a useful noninvasive approach that is sex and polymorphism independent for assessment of pre-eclampsia.
This work was supported in part by the Science and technology development project, No. 549, 2010 to Dr Wael El-Garf and Dr Osama Azmy.
Conflicts of interest
There are no conflicts of interest.
1. Redman CW.Current topic: pre-eclampsia and the placenta.Placenta1991;12:301–308.
2. Roberts JM, Redman CWG.Preeclampsia – more than pregnancy-induced hypertension.Lancet1993;341:1447–1451.
3. Redman CW, Sargent IL.Latest advances in understanding preeclampsia.Science2005;308:1592–1594.
4. Sibai B, Dekker G, Kupferminc M.Pre-eclampsia.Lancet2005;365:785–799.
5. Ingemarsson I.Gender aspects of preterm birth. 1st International Preterm Labour Congress; 2002; Montreux, Switzerland. Amsterdam, the Netherlands I: Elsevier Science. pp. 34–38..
6. Sheiner E, Levy A, Katz M, Hershkovitz R, Leron E, Mazor M.Gender does matter in perinatal medicine.Fetal Diagn Ther2004;19:366–369.
7. Di Renzo GC, Rosati A, Sarti RD, Cruciani L, Cutuli AM.Does fetal sex affect pregnancy outcome?Gend Med2007;4:19–30.
8. Stark MJ, Clifton VL, Wright IMR.Neonates born to mothers with preeclampsia exhibit sex-specific alterations in microvascular function.Pediatr Res2009;65:291–295.
9. Stark MJ, Dierkx L, Clifton VL, Wright IMR.Alterations in the maternal peripheral microvascular response in pregnancies complicated by preeclampsia and the impact of fetal sex.J Soc Gynecol Investig2006;13:573–578.
10. Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CWG, et al..Presence of fetal DNA in maternal plasma and serum.Lancet1997;350:485–487.
11. Lo YMD, Leung TN, Tein MSC, Sargent IL, Zhang J, Lau TK, et al..Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia.Clin Chem1999;45:184–188.
12. Engel K, Plonka T, Bilar M, Orzinska A, Brojer E, Ronin-Walknowska E.The correlation between clinical characteristics of preeclampsia and the concentration of fetal DNA in maternal circulation.Eur J Obstet Gynecol Reprod Biol2008;139:256–257.
13. Levine RJ, Qian C, LeShane ES, Yu KF, England LJ, Schisterman EF, et al..Two-stage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia.Am J Obstet Gynecol2004;190:707–713.
14. Ng EKO, Tsui NBY, Lau TK, Leung TN, Chiu RWK, Panesar NS, et al..mRNA of placental origin is readily detectable in maternal plasma.Proc Natl Acad Sci USA2003;100:4748–4753.
15. Schmidt M, Hoffmann B, Kimmig R, Kasimir-Bauer S.mRNA of placental origin in maternal serum of women with normal and preeclamptic pregnancies.Fetal Diagn Ther2009;25:269–276.
16. Chim SSC, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, et al..Detection of the placental epigenetic signature of the maspin gene in maternal plasma.Proc Natl Acad Sci U S A2005;102:14753–14758.
17. Lun FFM, Chiu RWK, Leung TY, Leung TN, Lau TK, Lo YMD.Epigenetic analysis of RASSF1A gene in cell-free DNA in amniotic fluid.Clin Chem2007;53:796–798.
18. Lui YY, Chik KW, Chiu RWK, Ho CY, Lam CW, Lo YMD.Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation.Clin Chem2002;48:421–427.
19. Chiu RWK, Chim SSC, Wong IH, Wong CS, Lee WS, To KF, et al..Hypermethylation of RASSF1A in human and rhesus placentas.Am J Pathol2007;170:941–950.
20. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al..Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.Clin Chem2006;52:2211–2218.
21. Sanderson M, Sappenfield WM, Jespersen KM, Liu Q, Baker SL.Association between level of delivery hospital and neonatal outcomes among South Carolina Medicaid recipients.Am J Obstet Gynecol2000;183:1504–1511.
22. Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al..Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia.Clin Chem1999;45:184–188.
23. Zhong XY, Holzgreve W, Hahn S.The levels of circulatory cell free fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia.Hypertens Pregnancy2002;21:77–83.
24. Zimmermann BG, Maddocks DG, Avent ND.Quantification of circulatory fetal DNA in the plasma of pregnant women.Methods Mol Biol2008;444:219–229.
25. Chim SS, Tong YK, Chiu RW, Lau TK, Leung TN, Chan LY, et al..Detection of the placental epigenetic signature of the maspin gene in maternal plasma.Proc Natl Acad Sci USA2005;102:14753–14758.
26. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, et al..Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.Clin Chem2006;52:2211–2218.
27. Tsui DW, Chan KC, Chim SS, Chan LW, Leung TY, Lau TK, et al..Quantitative aberrations of hypermethylated RASSF1A gene sequences in maternal plasma in preeclampsia.Prenat Diagn2007;27:1212–1218.
28. Sifakis S, Zaravinos A, Maiz N, Spandidos DA, Nicolaides KH.First-trimester maternal plasma cell-free fetal DNA and pre-eclampsia.Am J Obstet Gynecol2009;201:472–477.
29. El-Garf W, Salem M, Osman O, El Sirgany S, Bibers M, Salama S, Azmy O.Plasma circulating cell-free DNA and uteroplacental blood flow in pre-eclamptic patients.Med Res J2013;12:6–11.
30. Vlkova B, Szemes T, Minarik G, Turn J, Celec P.Circulating free fetal nucleic acids in maternal plasma and pre-eclampsia.Med Hypotheses2010;74:1030–1032.
31. Hung TH, Skepper JN, Charnock-Jones DS, Burton GJ.Hypoxia-reoxygenation: a potent inducer of apoptotic changes in the human placenta and possible etiological factor in pre-eclampsia.Circ Res2002;90:1274–1281.
32. Soleymanlou N, Jurisica I, Nevo O, Ietta F, Zhang X, Zamudio S, et al..Molecular evidence of placental hypoxia in pre-eclampsia.J Clin Endocrinol Metab2005;90:4299–4308.
33. Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, et al..Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation.Placenta2003;24:181–190.
34. Knight M, Redman CW, Linton EA, Sargent IL.Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies.Br J Obstet Gynaecol1998;105:632–640.
35. Tjoa ML, Cindrova-Davies T, Spasic-Boskovic O, Bianchi DW, Burton GJ.Trophoblastic oxidative stress and the release of cell-free feto-placental DNA.Am J Pathol2006;169:400–404.
36. Lo YMD, Poon L.The in and out of fetal DNA in maternal plasma.Lancet2003;361:193–194.
37. Lau TW, Leung TN, Chan LY, Lau TK, Chan KC, Tam WH, Lo YM.Fetal DNA clearance from maternal plasma is impaired in pre-eclampsia.Clin Chem2002;48:2141–2146.
38. Emlen W, Mannik M.Effect of DNA size and strandedness on the in vivo clearance and organ localization of DNA.Clin Exp Immunol1984;56:185–192.