Hypertens Pregnancy, 2015; 34(1): 36–49 ! Informa Healthcare USA, Inc. ISSN: 1064-1955 print / 1525-6065 online DOI: 10.3109/10641955.2014.954722
ORIGINAL ARTICLE
Fetal DNA in maternal plasma in preeclamptic pregnancies Barbora Vlkova ´ ,1,2 Ja ´ n Turn ˇ a,3 and Peter Celec1,2,3,4 Hypertens Pregnancy Downloaded from informahealthcare.com by Selcuk Universitesi on 02/10/15 For personal use only.
1
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia, 2 Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia, 3 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 4 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Cell-free fetal DNA present in maternal circulation has revolutionized non-invasive prenatal diagnosis of genetic diseases. In preeclampsia, the quantity of fetal DNA in maternal plasma has been studied and found to be higher in comparison to healthy pregnant women. Whether the quantity of fetal DNA can be used as a reliable predictive biomarker of preeclampsia is currently uncertain. This is a systematic review on studies quantifying fetal DNA in preeclamptic pregnancies. Using a PubMed search 22 studies were identified. In all of them, elevated levels of fetal DNA in maternal plasma in preeclampsia were found. In some of the studies, the higher concentration of fetal DNA was observed before the onset of clinical symptoms. This shows that fetal DNA levels might have a potential informative value as an early diagnostic biomarker of preeclampsia. However, in most of the studies important data are missing and there is an enormous variability in the reported results between the studies. From the available data it is currently not possible to perform a meta-analysis due to the variation between studies. If once fetal DNA should be used as a marker for determining preeclampsia at early stage, it is necessary to reduce these variations via standardized protocols for the quantification of cell-free fetal DNA as well as its reporting in the publications. Keywords Fetal DNA, Preeclampsia, Quantification, Real-time PCR, Systematic review
INTRODUCTION Prenatal diagnosis undergoes revolutionary changes since the discovery of cell-free fetal (cff) DNA in maternal circulation (1). The source of extracellular fetal DNA fragments in plasma of pregnant women seems to be apoptotic fetal cells in placenta (2). This fact is supported by the properties of fetal DNA fragments in maternal circulation, which share several features of DNA fragments from apoptotic cells (3). The length of fetal DNA fragments present in maternal circulation is up to 313 base pairs (4). Surprisingly, the amount of cff nucleic acids in maternal plasma is relatively high and represents 5–10% of Correspondence: Barbora Vlkova´, MSc., PhD, Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. Tel: +421 2 59 357 296. E-mail: [email protected]
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Fetal DNA & preeclampsia
the total extracellular DNA present in maternal plasma (5,6). Even more surprising is the fact that the entire fetal genome is represented in maternal plasma (7). These characteristics of cff DNA enable the detection of fetal-specific sequences absent in the maternal genome usable in non-invasive prenatal diagnosis. Some of the tests such as the determination of fetal gender in sex-linked hereditary diseases and fetal RhD status in pregnancies at risk of incompatibility, already are becoming a part of the routine clinical practice (8). Preeclampsia is one of the most common and serious complications of pregnancy, affecting 3 to 5% of all pregnant women (9). This multisystem disease carries the risk of death for both, the mother and the child. So far, despite huge scientific effort the etiology of preeclampsia is unknown. Autoimmune inflammation and placental damage are part of the pathogenesis but the primary insult leading to these pathologies is still a matter of speculative hypotheses (10). Diagnostic criteria include hypertension, proteinuria and thrombotic complication. Although these criteria are clearly defined, they are often present only in the third trimester and clearly, there is a need for earlier diagnostics. A wide palette of biomarkers has been suggested for early detection of the disease and is currently under investigation. One of the potential biomarkers is the quantity of cff DNA present in the maternal circulation (11,12). In several studies focusing on pregnancies with preeclampsia, cff DNA concentrations in the maternal plasma were found to be significantly higher in comparison to healthy pregnant women (13). The cff DNA was elevated even before the first clinical symptoms (14). Thus, cff DNA in maternal plasma could be an early diagnostic marker of preeclampsia. The increase of cff DNA correlates with the degree of disease severity (15) and is higher in cases of severe preeclampsia compared with milder symptoms (16). In addition, a positive correlation was found between cff DNA concentrations and established clinical biomarkers of preeclampsia such as serum soluble fms-like tyrosine kinase-1 (17). This shows that cff DNA can be used not only as a qualitative, but also as a quantitative marker. Interestingly, higher levels of cff DNA in maternal plasma in pregnancies with symptomatic preeclampsia are accompanied by a similar increase in circulating maternal DNA (15). Before the onset of clinical symptoms cff DNA but not maternal DNA increases in the circulation (18). In another study higher total extracellular DNA was found in patients who subsequently developed preeclampsia (19). The increasing level of extracellular DNA in the circulation is associated with placenta damage, apoptosis and with the reduced ability to eliminate circulating DNA from the blood (12,20).
METHODS The PubMed database was systematically searched using keywords ‘‘preeclampsia and cell-free fetal DNA’’ to identify relevant studies. Data were extracted only from publications focusing on the association between preeclampsia and the quantity of cff DNA in the maternal circulation. Data from individual studies were summarized without any subsequent modifications.
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RESULTS The Pubmed search revealed 22 relevant studies. Table 1 contains information about the analyzed group of patients with preeclampsia and the control group. Data include number of patients in each group, gestation week at the time of blood sampling, type of preeclampsia, age of patients and BMI. The concentrations of cff DNA reported in the particular publications are shown for both, control and preeclampsia groups, as well as the ratio of these two values. Number of patients enrolled varied in the individual studies from 6 + 6 to 120 + 120 (control + preeclampsia). Gestation weeks at the time of blood sampling ranged from 11 to 41 gestation weeks and the age of patients was from 17 to 44 years. The gestation weeks were not reported in two studies. In several articles preeclampsia was subdivided into mild and severe and compared to the HELLP syndrome. This subdivision was missing in one study. BMI was reported only in three studies with values from 24.3 to 25.9 for the control group and from 26.6 to 30.0 for the preeclampsia group. The results from cff DNA quantification were reported in various ways. In three studies the results were not quantified at all. In 13 of 22 studies results are presented as median number of genome equivalents per milliliter of maternal plasma ranging from 22 to 527 for the control groups and from 41.9 to 3011 for the preeclampsia group. In two studies, results are reported as mean number of genome equivalent per milliliter of maternal plasma ranging from 16 to 333 for the control groups and from 36 to 3195 for the preeclampsia group. In other studies values of cff DNA concentrations are expressed as mean number of genome equivalents per PCR reaction, median number of copies per microliter of maternal blood or as median number of picogram per microliter of maternal plasma, respectively. The ratio of cff DNA concentration reported for the preeclampsia group and the control group ranged between 1.22 and 89.3. Table 2 provides details regarding sample processing, type of collection system and storage of samples. Details about the volume of the starting material and the purification method are presented. Except 7 studies without a description of sampling, in all other studies peripheral blood samples were collected in tubes containing EDTA. The blood/plasma/serum samples were frozen and stored at 20 C, 30 C, 70 C or 80 C, respectively. The sample volume for subsequent extraction of cell-free DNA was 200 ml of whole blood or 240–1500 ml of plasma/serum samples. In three studies the starting sample volume for the extraction was not reported. Commercially available kits were used to isolate cell-free nucleic acids. In most cases (14 studies) QIAamp Blood Kit (Qiagen, Hilden, Germany) was used. The following kits: QIAamp DSP Virus Kit (Qiagen, Hilden, Germany), High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany), Nuclisens easyMag Isolation Kit (bioMe´rieux, Boxtel, the Netherlands), Nucleospin Plasma XS (Macherey-Nagel, Du¨ren, Germany) were used in one or two studies each. In one study two kits were used – Gentra Puregene Blood Kit and QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The isolation procedure was not specified in two studies. In Table 3 details about the real-time PCR analysis are summarized: target and housekeeping gene, applied chemistry, size of the amplified PCR product and the number of replicates. In 14 out of 22 studies the PCR target was the
176 + 88 24 + 24 33 + 18 20 + 20 70 + 67 11 + 16 40 + 10 120 + 120
176 + 44 10 + 17
6+6 32 + 32
16 ± 4 11–14 11–22 27–41 20–41 20–28 19–25 17–28 29–41
32 (PE) 38 (C) 18–24 31.8 (±4.2) (PE) 32.5 (±4.8) (C) 11–13 26–34
22–40 33.7 ± 3.9 35–40 n.r.
12 + 12 102 + 34 67 + 26 53 + 44
10 + 7
n.r.
Sampling time (gestation week)
35 + 24
Number of patients (C + PE)
Mild and severe Mild Mild Mild Mild Mild and severe Mild Mild and severe
Mild Mild and HELLP
Severe Mild
Mild and severe
Severe Mild and severe Mild Mild and severe
Mild and severe
Diagnosis of PE
21 ± 5
26 ± 5 29–31 n.r. n.r. 17–44 31 ± 6
25–38 24–38
28–35 25–29
n.r.
22–35 n.r. n.r. n.r.
n.r.
Age of patients
n.r. n.r. n.r. n.r. 25.9/30.0 n.r. n.r. 25.1 ± 6.1/27.3 ± 6.8
24.3/26.6 n.r.
n.r. n.r.
n.r.
n.r. n.r. n.r. n.r.
n.r.
BMI C/PE
28*y 1.61y 22*y 76*y 0.001* 42.5*y 128.5*y 16 y 75 y
51.5*y 92*y
n.r. 8.6*
227*y
51 z n.r. 65*y 333 y
320*y
Fetal DNA (C)
71.2*y 620 (mild)*y 3011 (HELLP)*y 280*y 4.34y 41.9*y 381*y 0.086* 326.2*y 422.9*y 36 y 176 y
n.r. 10.6*
843 (mild)*y 2606 (severe)*y 202 z n.r. 831*y 890 (mild) y 3195 (severe) y 521*y
Fetal DNA (PE)
(29) (54) (37) (55) (39) (56) (18) (57)
(52) (53)
(51) (28)
(50)
(47) (48) (49) (15)
(46)
Citation
(Continued )
1.37 6.74 (mild) 32.73 (HELLP) 25 1.88 1.9 5.01 89.3 7.58 3.28 2.25 2.35
2.39 1.22
3.96 2.62 12.78 2.67 (mild) 9.59 (severe) 2.30
2.63–8.14
PE: C ratio
Table 1. Data about the patients (age, BMI, type of diagnosis), blood sampling time and the results of quantitative fetal DNA analysis.
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Fetal DNA & preeclampsia
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I., II., III. trimester
29–36 24–37.6 25–38
60 + 20
20 + 9 77 + 49 89 + 28
n.r. Mild and severe Mild and severe
Mild and severe
Diagnosis of PE
n.r. 18–43 n.r.
n.r.
Age of patients
PE, preeclampsia; C, control; BMI, body mass index; n.r., not reported. *The values are expressed as median. The values are expressed as mean. yGenome equivalents per milliliter of maternal plasma. zGenome equivalents per PCR. Copies per microliter of maternal blood. Picogram per microliter of maternal plasma.
Sampling time (gestation week)
Number of patients (C + PE)
Table 1. Continued
n.r. n.r. n.r.
n.r.
BMI C/PE
191*y n.r. 58*y
46–527*y
Fetal DNA (C) 1672 (mild) *y 2346 (severe) *y 486*y n.r. 195.2*y
Fetal DNA (PE)
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3.17 (mild) 4.45 (severe) 2.12 1.22 3.6
PE: C ratio
(58) (16) (59)
(33)
Citation
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Fetal DNA & preeclampsia Table 2. Summary of sample processing, storage and isolation of the circulating extracellular DNA.
Blood sampling n.r. EDTA whole blood EDTA whole blood
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n.r. n.r. EDTA whole blood EDTA whole blood EDTA whole blood
EDTA whole blood
Sample type and storage
Starting volume (ml)
Plasma stored frozen Plasma stored at 80 C Plasma stored at 20 C Plasma stored frozen Plasma stored frozen Plasma stored at 20 C Plasma stored at 20 C Whole blood stored at 20 C Plasma stored at 80 C
400 n.r. 1500 400 400 200–800 1500 200
240
EDTA whole blood
Plasma stored at 30 C
500
EDTA whole blood
Whole blood stored at 20 C Plasma stored at 20 C Plasma stored at 70 C Serum stored at 70 C or 20 C Plasma stored at 80 C
200
n.r. EDTA whole blood n.r.
EDTA whole blood
EDTA whole blood n.r. n.r. EDTA whole blood
EDTA whole blood
Plasma stored at 80 C Plasma stored at 80 C Serum stored at 70 C Plasma stored at 80 C Plasma stored at 20 C
800 n.r.
Isolation kit
Citation
QIAamp Blood Kit (QIAGEN) Nuclisens easyMag (BioMerieux) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Kit (QIAGEN) QIAamp Blood Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN)
(46)
Nucleospin Plasma XS (MachereyNagel) PureGene & QIAquick (QIAGEN) QIAamp Blood Kit (QIAGEN)
(52)
QIAamp Blood Mini Kit (QIAGEN) n.r.
(47) (48) (49) (15) (50) (51) (28)
(53)
(29)
(54) (37)
400–800
QIAamp Blood Kit (QIAGEN)
(55)
400
High Pure PCR Template Preparation Kit (Roche Diagnostics) Nuclisens Isolation Kit (Boxtel) n.r.
(39)
1000 n.r. 400 1200 (control) 800 (preeclampsia) 1500
(56) (18)
QIAamp Blood Kit (QIAGEN) QIAamp Blood Kit (QIAGEN)
(57) (33)
QIAamp Blood Mini Kit (QIAGEN)
(58)
(Continued )
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B. Vlkova ´ et al. Table 2. Continued
Blood sampling EDTA whole blood
EDTA whole blood
Sample type and storage
Starting volume (ml)
Plasma stored at 20 C or 80 C n.r.
1100
QIAamp DSP Virus Kit (QIAGEN)
(16)
400
QIAamp Blood Mini Kit (QIAGEN)
(59)
Isolation kit
Citation
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EDTA, ethylene diamine tetraacetic acid; n.r., not reported.
Table 3. Description of details concerning the real-time PCR analysis.
Target gene SRY
Housekeeping gene n.r.
Paternal-specific n.r. polymorphisms SRY beta-Globin
SRY
GAPDH
SRY
GAPDH
SRY
beta-Globin
DYS14
n.r.
SRY
beta-Actin
DYS14
beta-Globin
SRY
Albumin
SRY
beta-Actin
DYS14
n.r.
Real-time chemistry Taqman PCR Core Reaction Kit (Pelkin Elmer) PCR buffer (Applied Biosystems) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) Taqman PCR Core Reagent Kit (Pelkin Elmer) Plexor qPCR System (Promega) TaqMan Universal Master Mix (Applied Biosystems) Taqman PCR Core Reagent Kit (Pelkin Elmer) Go Taq Flexi (Promega) SYBR Green (Molecular Probes)
PCR product Number of size (bp) replicates Citation n.r.
2
(46)
n.r.
4
(47)
136
3
(48)
n.r.
n.r.
(49)
n.r.
2
(15)
136
2
(50)
n.r.
n.r.
(51)
136
2
(28)
n.r.
2
(52)
86
2
(53)
136
2
(29)
n.r.
3
(54)
(Continued )
Fetal DNA & preeclampsia
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Table 3. Continued
Target gene
Housekeeping gene
SRY
beta-Globin
SRY
beta-Globin
SRY
beta-Globin
SRY SRY
n.r. GAPDH
DYS1
beta
RASSF1A
beta
DYS14
beta
DYS14
n.r.
SRY
n.r.
Real-time chemistry Taqman PCR Core Reagent Kit (Pelkin Elmer) Taqman PCR Core Reagent Kit (Pelkin Elmer) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) n.r. Taqman PCR Core Reaction Kit (Pelkin Elmer) TaqMan Universal MasterMix (Applied Biosystems) Premix Ex Taq (Applied TaKaRa) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) TaqMan Universal PCR Master Mix (Applied Biosystems) Taqman PCR Core Reagent Kit (Pelkin Elmer)
PCR product Number of size (bp) replicates Citation 136
2
(37)
136
2
(55)
n.r.
2
(39)
n.r. n.r.
2 2
(56) (18)
n.r.
3
(57)
n.r.
2
(33)
173
n.r.
(58)
n.r.
8
(16)
136
2
(59)
n.r., not reported.
SRY gene, in 6 studies DYS and in one study paternal specific polymorphisms or the RASSF1A gene. Housekeeping genes varied between the studies: beta globin (9 studies), GAPDH (3 studies), beta actin (2 studies), albumin (1 study) were used. In 7 studies the housekeeping gene was not reported. The most often used chemistry for real-time PCR amplification was Taqman Kit (Pelkin Elmer, Rodgau, Germany) in 14 studies. Kit LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics, Mannheim, Germany) was applied in 3 studies. The following kits were used in one study each: PCR buffer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), Plexor qPCR System (Promega, Madison, WI) and Premix Ex Taq (Applied TaKaRa, Kyoto, Japan). In one study was used combination of two sets of chemistry - Go Taq Flexi (Promega, Madison, WI) and SYBR Green (Molecular Probes, Thermo Fisher Scientific, Waltham, MA). Details regarding the PCR chemistry were not reported in one study. Size of the PCR product was not reported in 13 studies. In 7 studies the amplified product had a sequence length of 136 bp, in one study 86 bp and 173 bp. The PCR reaction was prepared in duplicates
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(14 studies), triplicates (3 studies), tetraplicates (1 study) or octaplicates (1 study). Number of replicates was not described in three studies.
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DISCUSSION Fetal DNA represents only a small fraction of total DNA present in maternal plasma with the high background of maternal DNA, is of relatively poor quality, consists of short DNA fragments and the fetal genome is to a half identical with the genetic information of the mother. Despite these apparent disadvantages, progress and the speed of the research in this field is enormous. The commercial interests and investments are adequate. The number of published reports describing diagnostic tests based on the analysis of cff DNA present in maternal plasma shows the importance of noninvasive prenatal diagnosis. The great effort so far resulted in the application of cff DNA analysis in the routine use for clinical testing, at least for early fetal gender determination in sex-linked hereditary diseases and the diagnosis of RhD status of the fetus in RhD negative pregnant women (21). The undoubted potential of cff DNA has exponentially risen with the emergence of new technologies of molecular biology. Massively parallel sequencing and digital PCR are approaches that allow complex applications such non-invasive prenatal diagnosis of fetal chromosomal aneuploidies (22). In the context of early diagnosis of preeclampsia – before the occurrence of clinical symptoms, there are no clear diagnostic criteria yet. Since this is relatively common disease complicating pregnancy, a suitable and reliable biomarker is needed. Several studies were conducted to cff DNA present in maternal plasma can fulfill these criteria. It was proved that in comparison to healthy pregnant women, in preeclampsia the concentrations of cff DNA was higher (23). However, the enormous variability of the reported results, especially between the studies prevents any general outcomes. It is even not possible to do a meta-analysis from the available data to see how well the quantification method works and to control the between-study variation (24). If standard procedures for such studies were used, it would be possible to avoid the variability associated with the study design and to overcome technical issues. So far, such a golden standard to quantify cff DNA has not been suggested. For fetal sex determination and analysis of fetal blood type in pregnancies at risk, sort of standard was discussed and proposed (21). The SAFE project was set up in order to implement routine non-invasive prenatal diagnosis and neonatal screening. This project played an important role in the standardization of noninvasive RhD genotyping. To ensure the accuracy, reliability, reproducibility and comparability of achieved results, standard protocols for cff DNA analysis in preeclampsia should be developed. Despite the relatively large variability, all studies were consistent in several parameters. In all studies (at least in those, where the collection procedure was specified) samples were collected as whole blood in EDTA. It was shown, that using cell stabilization tubes can preserve the proportion of cff DNA in the maternal plasma samples (25). The subsequent extraction of total nucleic acids was carried out using commercial column kits from different volumes of maternal plasma, serum or whole blood. The differences in sample size cannot
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Fetal DNA & preeclampsia
be the source of variability, since in most studies the results are converted to the same unit – genome equivalents per 1 ml of maternal plasma. However, sample size and type of kit used for isolation of extracellular DNA can have an impact on the reliability of the results. The extraction procedure with higher volume of plasma can reduce the number of inconclusive results and improve accuracy by avoiding false-negative results (25). The plasma sample of pregnant women is generally considered the most suitable material for the isolation of total non-cellular plasma DNA, which contains fetal DNA and maternal DNA. This is due to the fact, that unlike whole blood, plasma does not contain any cellular components of blood and after extraction the background of maternal DNA is lower in comparison to whole blood. Another reason for using plasma samples is that fetal cells remain in the maternal circulation for several years after delivery and therefore the results are susceptible for false positivity and the diagnostic error can occur when whole blood is used (26,27). Samples of whole blood were used for the isolation of maternal and fetal DNA only in two studies. Byrne et al. concluded that whole blood is not a suitable source of fetal DNA, which is then quantified in order to use it as a marker of preeclampsia (28). In comparison to other reports, Cotter et al. found very high levels of fetal DNA, which might be due to fetal cells present in a sample of whole blood (29). The quantitative approach using real-time PCR allows the identification and quantification of monitored DNA sequences. Commonly used genes (GAPDH, beta globin, beta actin or albumin) were used as housekeeping genes and positive controls, in order to confirm the presence of DNA in each sample (30). Segments specific for the Y chromosome, SRY or DYS gene were amplified as target genes to determine the presence of fetal DNA in the background of maternal nucleic acids (31). Of course, this is limited to the male gender of the fetus. In the future, if the concentration of cff DNA should be used as a biomarker for the prediction or diagnosis of preeclampsia, it would be appropriate to establish the amount of fetal DNA without the gender limitation by analysis of genes outside sex chromosomes (32). DNA of fetal origin can be detected and quantified using a gender – and genetic polymorphism – independent marker – the hypermethylated gene RASSF1A as the placental epigenetic signature (33). This approach has a potential to be a universal method for detecting fetal DNA in maternal plasma. However, standardization and wider use across laboratories is needed. Some of the variability may arise from the technical limitations of the realtime PCR. The efficiency of the reaction has not been indicated in the published reports, although this parameter is very important for the interpretation of the results (34). And alternative and more precise approach represents the digital PCR technology. This procedure allows counting the copy number of DNA present in a sample. By using of this method it is possible to avoid real-time PCR, normalization or taking into account the efficiency of the PCR reaction (4). Another possible and very promising technology is the massively parallel sequencing (35). Fetal aneuploidies (trisomy 13, 18, 21) can be reliably diagnosed by counting billions of DNA molecules extracted from maternal plasma. The massively parallel sequencing requires complex and expensive equipment as well as consumables. Even more important is the need for highly trained staff with a bioinformatic background. But improvements
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and simplifications will allow increasing throughput and decreasing costs in the future (36). Another source of variability in the results is probably the way how the values were calculated, which test was used for statistical data processing or which factors of correction were included in the calculation. The apparent enormous variability in the reported results between the studies is related to specific issues with the interpretation of results. In addition to the overlap observed in cff DNA concentrations between patients and controls in the particular studies (37), this is an important issue regarding potential clinical use in the diagnosis or prediction of preeclampsia. Low sensitivity and specificity might be improved either by combination with other monitored biochemical parameters or by decreasing the variability, both technical and biological, if possible. To do that, sources of variability have to be identified and eliminated. The important clinical parameter predisposing to preeclampsia – high body mass index (BMI) was not associated with cff DNA in some studies (38,39). In other studies there was a negative correlation – high BMI was associated with low cff DNA levels (40). This might be due to increased amounts of maternal cell-free DNA and, thus, lower cff DNA fractions (41). The reason for increased maternal circulating DNA is very likely the increased turnover of adipose cells in obese women (42). Importantly, higher concentrations of cff DNA were found in pregnancies with trisomy 21 (43). And, in addition, cff DNA is increased also in women with ectopic pregnancies in comparison to women with intrauterine pregnancies (44). These findings show that the association of preeclampsia and high cff DNA is not specific and can only be interpreted in combination with the clinical status and with other biomarkers.
CONCLUSIONS The presence of certain levels of fetal DNA in maternal circulation is a physiological phenomenon. Increased level is associated with pregnancyrelated pathologies. Cell-free fetal DNA present in maternal plasma is a potential biomarker for early prediction of preeclampsia before the onset of clinical symptoms. The level of cff DNA correlates also with severity of the disease. These findings are promising, but will require further studies to confirm results and also to develop standardized protocols for its implementation in the routine non-invasive clinical diagnostics. A large prospective multi-center study proving a standardized consensus protocol with a very high diagnostic accuracy would then be needed. With such standards and improved efficiency pregnancies at increased risk could be identified at a very early stage of the disease. This will allow special monitoring and will help to improve the prenatal care of preeclamptic pregnancies. However, the reason for increased cff DNA in preeclampsia remains to be elucidated, but genetic factors will surely play a role (45).
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the Slovak research and development agency through contract APVV-0754-10.
Fetal DNA & preeclampsia
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REFERENCES 1. Lo YMD, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7. 2. Alberry M, Maddocks D, Jones M, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenatal Diagnosis 2007;27:415–18. 3. Giacona MB, Ruben GC, Iczkowski KA, et al. Cell-free DNA in human blood plasma: length measurements in patients with pancreatic cancer and healthy controls. Pancreas 1998;17:89–97. 4. Chan KCA, Zhang J, Hui ABY, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem 2004;50:88–92. 5. Lo YMD, Tein MSC, Lau TK, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75. 6. Lun FMF, Chiu RWK, Chan KCA, et al. Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma. Clin Chem 2008;54: 1664–72. 7. Lo YMD, Chan KCA, Sun H, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Translational Med 2010;2:61ra91. 8. Zhong XY, Holzgreve W, Hahn S. Detection of fetal Rhesus D and sex using fetal DNA from maternal plasma by multiplex polymerase chain reaction. Brit J Obstetr Gynaecol 2000;107:766–9. 9. Young BC, Levine RJ, Karumanchi SA. Pathogenesis of preeclampsia. Ann Rev Pathol 2010;5:173–92. 10. Harlow FH, Brown MA. The diversity of diagnoses of preeclampsia. Hypertens Pregnancy 2001;20:57–67. 11. Hahn S, Rusterholz C, Hosli I, Lapaire O. Cell-free nucleic acids as potential markers for preeclampsia. Placenta 2011;32:S17–20. 12. Bianchi DW. Circulating fetal DNA: its origin and diagnostic potential – a review. Placenta 2004;25:S93–101. 13. Lo YMD, Leung TN, Tein MSC, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45:184–8. 14. Leung TN, Zhang J, Lau TK, et al. Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia. Clin Chem 2001; 47:137–9. 15. Zhong XY, Laivuori H, Livingston JC, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstetr Gynaecol 2001;184: 414–19. 16. Alberry MS, Maddocks DG, Hadi MA, et al. Quantification of cell free fetal DNA in maternal plasma in normal pregnancies and in pregnancies with placental dysfunction. Am J Obstetr Gynaecol 2009;200:98.e1–e6. 17. Molvarec A, Szarka A, Walentin S, et al. Circulating angiogenic factors determined by electrochemiluminescence immunoassay in relation to the clinical features and laboratory parameters in women with pre-eclampsia. Hypertens Res 2010;33: 892–8. 18. 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 Pregnancy 2002;21:77–83. 19. Farina A, Sekizawa A, Iwasaki M, et al. Total cell-free DNA (beta-globin gene) distribution in maternal plasma at the second trimester: a new prospective for preeclampsia screening. Prenat Diagn 2004;24:722–6. 20. Hahn S, Holzgreve W. Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia. Hum Reprod Update 2002;8:501–8. 21. Maddocks DG, Alberry MS, Attilakos G, et al. The SAFE project: towards noninvasive prenatal diagnosis. Biochem Soc Trans 2009;37:460–5.
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22. Chiu RWK, Lo YMD. Noninvasive prenatal diagnosis empowered by highthroughput sequencing. Prenat Diagn 2012;32:401–6. 23. Levine RJ, Qian C, LeShane ES, et al. Two-stage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia. Am J Obstetr Gynaecol 2004;190: 707–13. 24. Kleinrouweler CE, Wiegerinck MMJ, Ris-Stalpers C, et al. Accuracy of circulating placental growth factor, vascular endothelial growth factor, soluble fms-like tyrosine kinase 1 and soluble endoglin in the prediction of pre-eclampsia: a systematic review and meta-analysis. BJOG 2012;119:778–87. 25. Barrett AN, Zimmermann BG, Wang D, et al. Implementing prenatal diagnosis based on cell-free fetal DNA: accurate identification of factors affecting fetal DNA yield. Plos One 2011;6:e25202. 26. Bianchi DW. Fetal cells in the maternal circulation: feasibility for prenatal diagnosis. Brit J Haematol 1999;105:574–83. 27. Bianchi DW, Zickwolf GK, Weil GJ, et al. Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum. Proc Natl Acad Sci USA 1996; 93:705–8. 28. Byrne BM, Crowley A, Taulo F, et al. Fetal DNA quantitation in peripheral blood is not useful as a marker of disease severity in women with preeclampsia. Hypertens Pregnancy 2003;22:157–64. 29. Cotter AM, Martin CM, O’Leary JJ, Daly SF. Increased fetal DNA in the maternal circulation in early pregnancy is associated with an increased risk of preeclampsia. Am J Obstet Gynecol 2004;191:515–20. 30. VanGuilder HD, Vrana KE, Freeman WM. Twenty-five years of quantitative PCR for gene expression analysis. Biotechniques 2008;44:619–26. 31. Zimmermann BG, Holzgreve W, Avent N, Hahn SH. Optimized real-time quantitative PCR measurement of male fetal DNA in maternal plasma. In: Swaminathan R, Butt A, Gahan P, eds. Circulating nucleic acids in plasma and serum IV. Vol 1075. Oxford: Blackwell Publishing; 2006:347–9. 32. Tang NLS, Leung TN, Zhang J, et al. Detection of fetal-derived paternally inherited X-chromosome polymorphisms in maternal plasma. Clin Chem 1999;45: 2033–5. 33. Zhao FX, Wang JA, Liu RH, et al. Quantification and application of the placental epigenetic signature of the RASSF1A gene in maternal plasma. Prenat Diagn 2010; 30:778–82. 34. Platts AE, Johnson GD, Linnemann AK, Krawetz SA. Real-time PCR quantification using a variable reaction efficiency model. Analytical Biochem 2008;380: 315–22. 35. Lo YMD. Non-invasive prenatal diagnosis by massively parallel sequencing of maternal plasma DNA. Open Biol 2012;2:120086. doi: 10.1098/rsob.120086. 36. Kinde I, Papadopoulos N, Kinzler KW, Vogelstein B. FAST-SeqS: a simple and efficient method for the detection of aneuploidy by massively parallel sequencing. Plos One 2012;7:e41162. 37. Leung TN, Zhang J, Lau TK, et al. Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia. Clin Chem 2001; 47:137–9. 38. Bodnar LM, Ness RB, Markovic N, Roberts JM. The risk of preeclampsia rises with increasing prepregnancy body mass index. Ann Epidemiol 2005;15:475–82. 39. Lazar L, Rigo Jr J, Nagy B, et al. Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia. BMC Med Genet 2009;10:120. 40. Gerovassili A, Garner C, Nicolaides KH, et al. Free fetal DNA in maternal circulation: a potential prognostic marker for chromosomal abnormalities? Prenat Diagn 2007;27:104–10. 41. Lapaire O, Volgmann T, Grill S, et al. Significant correlation between maternal body mass index at delivery and in the second trimester, and second trimester circulating total cell-free DNA levels. Reprod Sci 2009;16:274–9.
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Fetal DNA & preeclampsia
42. Haghiac M, Vora NL, Basu S, et al. Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women. Obesity (Silver Spring) 2012;20:2213–19. 43. Lo YM, Lau TK, Zhang J, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45:1747–51. 44. Lazar L, Nagy B, Ban Z, et al. Presence of cell-free fetal DNA in plasma of women with ectopic pregnancies. Clin Chem 2006;52:1599–601. 45. Lasabova Z, Zigo I, Svecova I, et al. Association of specific diplotypes defined by common rs1800682 and rare rs34995925 single nucleotide polymorphisms within the STAT1 transcription binding site of the FAS gene promoter with preeclampsia. Gen Physiol Biophys 2014;33:199–204. 46. Hristoskova S, Holzgreve W, Hahn S. Anti-phospholipid and anti-DNA antibodies are not associated with the elevated release of circulatory fetal DNA in pregnancies affected by preeclampsia. Hypertens Pregnancy 2004;23:257–68. 47. Karina E, Tomasz P, Bilar M, et al. Assessment of the female fetal DNA concentration in the plasma of the pregnant women as preeclampsia indicator – preliminary report. Eur J Obstet Gynecol Reprod Biol 2009;146:165–8. 48. Farina A, Sekizawa A, Rizzo N, et al. Cell-free fetal DNA (SRY locus) concentration in maternal plasma is directly correlated to the time elapsed from the onset of preeclampsia to the collection of blood. Prenat Diagn 2004;24:293–7. 49. Zhong XY, Wang Y, Chen S, et al. Circulating fetal DNA in maternal plasma is increased in pregnancies at high altitude and is further enhanced by preeclampsia. Clin Chem 2004;50:2403–5. 50. Lau TW, Leung TN, Chan LY, et al. Fetal DNA clearance from maternal plasma is impaired in preeclampsia. Clin Chem 2002;48:2141–6. 51. Farina A, Sekizawa A, Sugito Y, et al. Fetal DNA in maternal plasma as a screening variable for preeclampsia. A preliminary nonparametric analysis of detection rate in low-risk nonsymptomatic patients. Prenat Diagn 2004;24:83–6. 52. Sifakis S, Zaravinos A, Maiz N, et al. First-trimester maternal plasma cell-free fetal DNA and preeclampsia. Am J Obstet Gynecol 2009;201:472.e1–e7. 53. Swinkels DW, de Kok JB, Hendriks JC, et al. Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome as a complication of preeclampsia in pregnant women increases the amount of cell-free fetal and maternal DNA in maternal plasma and serum. Clin Chem 2002;48:650–3. 54. Illanes S, Parra M, Serra R, et al. Increased free fetal DNA levels in early pregnancy plasma of women who subsequently develop preeclampsia and intrauterine growth restriction. Prenat Diagn 2009;29:1118–22. 55. Lo YM, Leung TN, Tein MS, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45:184–8. 56. Engel K, Plonka T, Bilar M, et al. The analysis of the correlation between extracellular fetal DNA concentration in maternal circulation and severity of preeclampsia. Ann Acad Med Stetin 2007;53:20–5. 57. Levine RJ, Qian C, Leshane ES, et al. Two-stage elevation of cell-free fetal DNA in maternal sera before onset of preeclampsia. Am J Obstet Gynecol 2004;190:707–13. 58. Sekizawa A, Jimbo M, Saito H, et al. Cell-free fetal DNA in the plasma of pregnant women with severe fetal growth restriction. Am J Obstetr Gynaecol 2003;188: 480–4. 59. Smid M, Galbiati S, Lojacono A, et al. Correlation of fetal DNA levels in maternal plasma with Doppler status in pathological pregnancies. Prenat Diagn 2006;26: 785–90.
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ORIGINAL ARTICLE
Fetal DNA in maternal plasma in preeclamptic pregnancies Barbora Vlkova ´ ,1,2 Ja ´ n Turn ˇ a,3 and Peter Celec1,2,3,4 Hypertens Pregnancy Downloaded from informahealthcare.com by Selcuk Universitesi on 02/10/15 For personal use only.
1
Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Bratislava, Slovakia, 2 Center for Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia, 3 Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia, 4 Institute of Pathophysiology, Faculty of Medicine, Comenius University, Bratislava, Slovakia Cell-free fetal DNA present in maternal circulation has revolutionized non-invasive prenatal diagnosis of genetic diseases. In preeclampsia, the quantity of fetal DNA in maternal plasma has been studied and found to be higher in comparison to healthy pregnant women. Whether the quantity of fetal DNA can be used as a reliable predictive biomarker of preeclampsia is currently uncertain. This is a systematic review on studies quantifying fetal DNA in preeclamptic pregnancies. Using a PubMed search 22 studies were identified. In all of them, elevated levels of fetal DNA in maternal plasma in preeclampsia were found. In some of the studies, the higher concentration of fetal DNA was observed before the onset of clinical symptoms. This shows that fetal DNA levels might have a potential informative value as an early diagnostic biomarker of preeclampsia. However, in most of the studies important data are missing and there is an enormous variability in the reported results between the studies. From the available data it is currently not possible to perform a meta-analysis due to the variation between studies. If once fetal DNA should be used as a marker for determining preeclampsia at early stage, it is necessary to reduce these variations via standardized protocols for the quantification of cell-free fetal DNA as well as its reporting in the publications. Keywords Fetal DNA, Preeclampsia, Quantification, Real-time PCR, Systematic review
INTRODUCTION Prenatal diagnosis undergoes revolutionary changes since the discovery of cell-free fetal (cff) DNA in maternal circulation (1). The source of extracellular fetal DNA fragments in plasma of pregnant women seems to be apoptotic fetal cells in placenta (2). This fact is supported by the properties of fetal DNA fragments in maternal circulation, which share several features of DNA fragments from apoptotic cells (3). The length of fetal DNA fragments present in maternal circulation is up to 313 base pairs (4). Surprisingly, the amount of cff nucleic acids in maternal plasma is relatively high and represents 5–10% of Correspondence: Barbora Vlkova´, MSc., PhD, Institute of Molecular Biomedicine, Faculty of Medicine, Comenius University, Sasinkova 4, 811 08 Bratislava, Slovakia. Tel: +421 2 59 357 296. E-mail: [email protected]
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Fetal DNA & preeclampsia
the total extracellular DNA present in maternal plasma (5,6). Even more surprising is the fact that the entire fetal genome is represented in maternal plasma (7). These characteristics of cff DNA enable the detection of fetal-specific sequences absent in the maternal genome usable in non-invasive prenatal diagnosis. Some of the tests such as the determination of fetal gender in sex-linked hereditary diseases and fetal RhD status in pregnancies at risk of incompatibility, already are becoming a part of the routine clinical practice (8). Preeclampsia is one of the most common and serious complications of pregnancy, affecting 3 to 5% of all pregnant women (9). This multisystem disease carries the risk of death for both, the mother and the child. So far, despite huge scientific effort the etiology of preeclampsia is unknown. Autoimmune inflammation and placental damage are part of the pathogenesis but the primary insult leading to these pathologies is still a matter of speculative hypotheses (10). Diagnostic criteria include hypertension, proteinuria and thrombotic complication. Although these criteria are clearly defined, they are often present only in the third trimester and clearly, there is a need for earlier diagnostics. A wide palette of biomarkers has been suggested for early detection of the disease and is currently under investigation. One of the potential biomarkers is the quantity of cff DNA present in the maternal circulation (11,12). In several studies focusing on pregnancies with preeclampsia, cff DNA concentrations in the maternal plasma were found to be significantly higher in comparison to healthy pregnant women (13). The cff DNA was elevated even before the first clinical symptoms (14). Thus, cff DNA in maternal plasma could be an early diagnostic marker of preeclampsia. The increase of cff DNA correlates with the degree of disease severity (15) and is higher in cases of severe preeclampsia compared with milder symptoms (16). In addition, a positive correlation was found between cff DNA concentrations and established clinical biomarkers of preeclampsia such as serum soluble fms-like tyrosine kinase-1 (17). This shows that cff DNA can be used not only as a qualitative, but also as a quantitative marker. Interestingly, higher levels of cff DNA in maternal plasma in pregnancies with symptomatic preeclampsia are accompanied by a similar increase in circulating maternal DNA (15). Before the onset of clinical symptoms cff DNA but not maternal DNA increases in the circulation (18). In another study higher total extracellular DNA was found in patients who subsequently developed preeclampsia (19). The increasing level of extracellular DNA in the circulation is associated with placenta damage, apoptosis and with the reduced ability to eliminate circulating DNA from the blood (12,20).
METHODS The PubMed database was systematically searched using keywords ‘‘preeclampsia and cell-free fetal DNA’’ to identify relevant studies. Data were extracted only from publications focusing on the association between preeclampsia and the quantity of cff DNA in the maternal circulation. Data from individual studies were summarized without any subsequent modifications.
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RESULTS The Pubmed search revealed 22 relevant studies. Table 1 contains information about the analyzed group of patients with preeclampsia and the control group. Data include number of patients in each group, gestation week at the time of blood sampling, type of preeclampsia, age of patients and BMI. The concentrations of cff DNA reported in the particular publications are shown for both, control and preeclampsia groups, as well as the ratio of these two values. Number of patients enrolled varied in the individual studies from 6 + 6 to 120 + 120 (control + preeclampsia). Gestation weeks at the time of blood sampling ranged from 11 to 41 gestation weeks and the age of patients was from 17 to 44 years. The gestation weeks were not reported in two studies. In several articles preeclampsia was subdivided into mild and severe and compared to the HELLP syndrome. This subdivision was missing in one study. BMI was reported only in three studies with values from 24.3 to 25.9 for the control group and from 26.6 to 30.0 for the preeclampsia group. The results from cff DNA quantification were reported in various ways. In three studies the results were not quantified at all. In 13 of 22 studies results are presented as median number of genome equivalents per milliliter of maternal plasma ranging from 22 to 527 for the control groups and from 41.9 to 3011 for the preeclampsia group. In two studies, results are reported as mean number of genome equivalent per milliliter of maternal plasma ranging from 16 to 333 for the control groups and from 36 to 3195 for the preeclampsia group. In other studies values of cff DNA concentrations are expressed as mean number of genome equivalents per PCR reaction, median number of copies per microliter of maternal blood or as median number of picogram per microliter of maternal plasma, respectively. The ratio of cff DNA concentration reported for the preeclampsia group and the control group ranged between 1.22 and 89.3. Table 2 provides details regarding sample processing, type of collection system and storage of samples. Details about the volume of the starting material and the purification method are presented. Except 7 studies without a description of sampling, in all other studies peripheral blood samples were collected in tubes containing EDTA. The blood/plasma/serum samples were frozen and stored at 20 C, 30 C, 70 C or 80 C, respectively. The sample volume for subsequent extraction of cell-free DNA was 200 ml of whole blood or 240–1500 ml of plasma/serum samples. In three studies the starting sample volume for the extraction was not reported. Commercially available kits were used to isolate cell-free nucleic acids. In most cases (14 studies) QIAamp Blood Kit (Qiagen, Hilden, Germany) was used. The following kits: QIAamp DSP Virus Kit (Qiagen, Hilden, Germany), High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany), Nuclisens easyMag Isolation Kit (bioMe´rieux, Boxtel, the Netherlands), Nucleospin Plasma XS (Macherey-Nagel, Du¨ren, Germany) were used in one or two studies each. In one study two kits were used – Gentra Puregene Blood Kit and QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The isolation procedure was not specified in two studies. In Table 3 details about the real-time PCR analysis are summarized: target and housekeeping gene, applied chemistry, size of the amplified PCR product and the number of replicates. In 14 out of 22 studies the PCR target was the
176 + 88 24 + 24 33 + 18 20 + 20 70 + 67 11 + 16 40 + 10 120 + 120
176 + 44 10 + 17
6+6 32 + 32
16 ± 4 11–14 11–22 27–41 20–41 20–28 19–25 17–28 29–41
32 (PE) 38 (C) 18–24 31.8 (±4.2) (PE) 32.5 (±4.8) (C) 11–13 26–34
22–40 33.7 ± 3.9 35–40 n.r.
12 + 12 102 + 34 67 + 26 53 + 44
10 + 7
n.r.
Sampling time (gestation week)
35 + 24
Number of patients (C + PE)
Mild and severe Mild Mild Mild Mild Mild and severe Mild Mild and severe
Mild Mild and HELLP
Severe Mild
Mild and severe
Severe Mild and severe Mild Mild and severe
Mild and severe
Diagnosis of PE
21 ± 5
26 ± 5 29–31 n.r. n.r. 17–44 31 ± 6
25–38 24–38
28–35 25–29
n.r.
22–35 n.r. n.r. n.r.
n.r.
Age of patients
n.r. n.r. n.r. n.r. 25.9/30.0 n.r. n.r. 25.1 ± 6.1/27.3 ± 6.8
24.3/26.6 n.r.
n.r. n.r.
n.r.
n.r. n.r. n.r. n.r.
n.r.
BMI C/PE
28*y 1.61y 22*y 76*y 0.001* 42.5*y 128.5*y 16 y 75 y
51.5*y 92*y
n.r. 8.6*
227*y
51 z n.r. 65*y 333 y
320*y
Fetal DNA (C)
71.2*y 620 (mild)*y 3011 (HELLP)*y 280*y 4.34y 41.9*y 381*y 0.086* 326.2*y 422.9*y 36 y 176 y
n.r. 10.6*
843 (mild)*y 2606 (severe)*y 202 z n.r. 831*y 890 (mild) y 3195 (severe) y 521*y
Fetal DNA (PE)
(29) (54) (37) (55) (39) (56) (18) (57)
(52) (53)
(51) (28)
(50)
(47) (48) (49) (15)
(46)
Citation
(Continued )
1.37 6.74 (mild) 32.73 (HELLP) 25 1.88 1.9 5.01 89.3 7.58 3.28 2.25 2.35
2.39 1.22
3.96 2.62 12.78 2.67 (mild) 9.59 (severe) 2.30
2.63–8.14
PE: C ratio
Table 1. Data about the patients (age, BMI, type of diagnosis), blood sampling time and the results of quantitative fetal DNA analysis.
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Fetal DNA & preeclampsia
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I., II., III. trimester
29–36 24–37.6 25–38
60 + 20
20 + 9 77 + 49 89 + 28
n.r. Mild and severe Mild and severe
Mild and severe
Diagnosis of PE
n.r. 18–43 n.r.
n.r.
Age of patients
PE, preeclampsia; C, control; BMI, body mass index; n.r., not reported. *The values are expressed as median. The values are expressed as mean. yGenome equivalents per milliliter of maternal plasma. zGenome equivalents per PCR. Copies per microliter of maternal blood. Picogram per microliter of maternal plasma.
Sampling time (gestation week)
Number of patients (C + PE)
Table 1. Continued
n.r. n.r. n.r.
n.r.
BMI C/PE
191*y n.r. 58*y
46–527*y
Fetal DNA (C) 1672 (mild) *y 2346 (severe) *y 486*y n.r. 195.2*y
Fetal DNA (PE)
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3.17 (mild) 4.45 (severe) 2.12 1.22 3.6
PE: C ratio
(58) (16) (59)
(33)
Citation
40 B. Vlkova ´ et al.
Fetal DNA & preeclampsia Table 2. Summary of sample processing, storage and isolation of the circulating extracellular DNA.
Blood sampling n.r. EDTA whole blood EDTA whole blood
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n.r. n.r. EDTA whole blood EDTA whole blood EDTA whole blood
EDTA whole blood
Sample type and storage
Starting volume (ml)
Plasma stored frozen Plasma stored at 80 C Plasma stored at 20 C Plasma stored frozen Plasma stored frozen Plasma stored at 20 C Plasma stored at 20 C Whole blood stored at 20 C Plasma stored at 80 C
400 n.r. 1500 400 400 200–800 1500 200
240
EDTA whole blood
Plasma stored at 30 C
500
EDTA whole blood
Whole blood stored at 20 C Plasma stored at 20 C Plasma stored at 70 C Serum stored at 70 C or 20 C Plasma stored at 80 C
200
n.r. EDTA whole blood n.r.
EDTA whole blood
EDTA whole blood n.r. n.r. EDTA whole blood
EDTA whole blood
Plasma stored at 80 C Plasma stored at 80 C Serum stored at 70 C Plasma stored at 80 C Plasma stored at 20 C
800 n.r.
Isolation kit
Citation
QIAamp Blood Kit (QIAGEN) Nuclisens easyMag (BioMerieux) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Kit (QIAGEN) QIAamp Blood Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN) QIAamp Blood Mini Kit (QIAGEN)
(46)
Nucleospin Plasma XS (MachereyNagel) PureGene & QIAquick (QIAGEN) QIAamp Blood Kit (QIAGEN)
(52)
QIAamp Blood Mini Kit (QIAGEN) n.r.
(47) (48) (49) (15) (50) (51) (28)
(53)
(29)
(54) (37)
400–800
QIAamp Blood Kit (QIAGEN)
(55)
400
High Pure PCR Template Preparation Kit (Roche Diagnostics) Nuclisens Isolation Kit (Boxtel) n.r.
(39)
1000 n.r. 400 1200 (control) 800 (preeclampsia) 1500
(56) (18)
QIAamp Blood Kit (QIAGEN) QIAamp Blood Kit (QIAGEN)
(57) (33)
QIAamp Blood Mini Kit (QIAGEN)
(58)
(Continued )
41
42
B. Vlkova ´ et al. Table 2. Continued
Blood sampling EDTA whole blood
EDTA whole blood
Sample type and storage
Starting volume (ml)
Plasma stored at 20 C or 80 C n.r.
1100
QIAamp DSP Virus Kit (QIAGEN)
(16)
400
QIAamp Blood Mini Kit (QIAGEN)
(59)
Isolation kit
Citation
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EDTA, ethylene diamine tetraacetic acid; n.r., not reported.
Table 3. Description of details concerning the real-time PCR analysis.
Target gene SRY
Housekeeping gene n.r.
Paternal-specific n.r. polymorphisms SRY beta-Globin
SRY
GAPDH
SRY
GAPDH
SRY
beta-Globin
DYS14
n.r.
SRY
beta-Actin
DYS14
beta-Globin
SRY
Albumin
SRY
beta-Actin
DYS14
n.r.
Real-time chemistry Taqman PCR Core Reaction Kit (Pelkin Elmer) PCR buffer (Applied Biosystems) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) Taqman PCR Core Reaction Kit (Pelkin Elmer) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) Taqman PCR Core Reagent Kit (Pelkin Elmer) Plexor qPCR System (Promega) TaqMan Universal Master Mix (Applied Biosystems) Taqman PCR Core Reagent Kit (Pelkin Elmer) Go Taq Flexi (Promega) SYBR Green (Molecular Probes)
PCR product Number of size (bp) replicates Citation n.r.
2
(46)
n.r.
4
(47)
136
3
(48)
n.r.
n.r.
(49)
n.r.
2
(15)
136
2
(50)
n.r.
n.r.
(51)
136
2
(28)
n.r.
2
(52)
86
2
(53)
136
2
(29)
n.r.
3
(54)
(Continued )
Fetal DNA & preeclampsia
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Table 3. Continued
Target gene
Housekeeping gene
SRY
beta-Globin
SRY
beta-Globin
SRY
beta-Globin
SRY SRY
n.r. GAPDH
DYS1
beta
RASSF1A
beta
DYS14
beta
DYS14
n.r.
SRY
n.r.
Real-time chemistry Taqman PCR Core Reagent Kit (Pelkin Elmer) Taqman PCR Core Reagent Kit (Pelkin Elmer) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) n.r. Taqman PCR Core Reaction Kit (Pelkin Elmer) TaqMan Universal MasterMix (Applied Biosystems) Premix Ex Taq (Applied TaKaRa) LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics) TaqMan Universal PCR Master Mix (Applied Biosystems) Taqman PCR Core Reagent Kit (Pelkin Elmer)
PCR product Number of size (bp) replicates Citation 136
2
(37)
136
2
(55)
n.r.
2
(39)
n.r. n.r.
2 2
(56) (18)
n.r.
3
(57)
n.r.
2
(33)
173
n.r.
(58)
n.r.
8
(16)
136
2
(59)
n.r., not reported.
SRY gene, in 6 studies DYS and in one study paternal specific polymorphisms or the RASSF1A gene. Housekeeping genes varied between the studies: beta globin (9 studies), GAPDH (3 studies), beta actin (2 studies), albumin (1 study) were used. In 7 studies the housekeeping gene was not reported. The most often used chemistry for real-time PCR amplification was Taqman Kit (Pelkin Elmer, Rodgau, Germany) in 14 studies. Kit LightCycler-Fast Start DNA Master Hybridization Probes (Roche Diagnostics, Mannheim, Germany) was applied in 3 studies. The following kits were used in one study each: PCR buffer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA), Plexor qPCR System (Promega, Madison, WI) and Premix Ex Taq (Applied TaKaRa, Kyoto, Japan). In one study was used combination of two sets of chemistry - Go Taq Flexi (Promega, Madison, WI) and SYBR Green (Molecular Probes, Thermo Fisher Scientific, Waltham, MA). Details regarding the PCR chemistry were not reported in one study. Size of the PCR product was not reported in 13 studies. In 7 studies the amplified product had a sequence length of 136 bp, in one study 86 bp and 173 bp. The PCR reaction was prepared in duplicates
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(14 studies), triplicates (3 studies), tetraplicates (1 study) or octaplicates (1 study). Number of replicates was not described in three studies.
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DISCUSSION Fetal DNA represents only a small fraction of total DNA present in maternal plasma with the high background of maternal DNA, is of relatively poor quality, consists of short DNA fragments and the fetal genome is to a half identical with the genetic information of the mother. Despite these apparent disadvantages, progress and the speed of the research in this field is enormous. The commercial interests and investments are adequate. The number of published reports describing diagnostic tests based on the analysis of cff DNA present in maternal plasma shows the importance of noninvasive prenatal diagnosis. The great effort so far resulted in the application of cff DNA analysis in the routine use for clinical testing, at least for early fetal gender determination in sex-linked hereditary diseases and the diagnosis of RhD status of the fetus in RhD negative pregnant women (21). The undoubted potential of cff DNA has exponentially risen with the emergence of new technologies of molecular biology. Massively parallel sequencing and digital PCR are approaches that allow complex applications such non-invasive prenatal diagnosis of fetal chromosomal aneuploidies (22). In the context of early diagnosis of preeclampsia – before the occurrence of clinical symptoms, there are no clear diagnostic criteria yet. Since this is relatively common disease complicating pregnancy, a suitable and reliable biomarker is needed. Several studies were conducted to cff DNA present in maternal plasma can fulfill these criteria. It was proved that in comparison to healthy pregnant women, in preeclampsia the concentrations of cff DNA was higher (23). However, the enormous variability of the reported results, especially between the studies prevents any general outcomes. It is even not possible to do a meta-analysis from the available data to see how well the quantification method works and to control the between-study variation (24). If standard procedures for such studies were used, it would be possible to avoid the variability associated with the study design and to overcome technical issues. So far, such a golden standard to quantify cff DNA has not been suggested. For fetal sex determination and analysis of fetal blood type in pregnancies at risk, sort of standard was discussed and proposed (21). The SAFE project was set up in order to implement routine non-invasive prenatal diagnosis and neonatal screening. This project played an important role in the standardization of noninvasive RhD genotyping. To ensure the accuracy, reliability, reproducibility and comparability of achieved results, standard protocols for cff DNA analysis in preeclampsia should be developed. Despite the relatively large variability, all studies were consistent in several parameters. In all studies (at least in those, where the collection procedure was specified) samples were collected as whole blood in EDTA. It was shown, that using cell stabilization tubes can preserve the proportion of cff DNA in the maternal plasma samples (25). The subsequent extraction of total nucleic acids was carried out using commercial column kits from different volumes of maternal plasma, serum or whole blood. The differences in sample size cannot
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be the source of variability, since in most studies the results are converted to the same unit – genome equivalents per 1 ml of maternal plasma. However, sample size and type of kit used for isolation of extracellular DNA can have an impact on the reliability of the results. The extraction procedure with higher volume of plasma can reduce the number of inconclusive results and improve accuracy by avoiding false-negative results (25). The plasma sample of pregnant women is generally considered the most suitable material for the isolation of total non-cellular plasma DNA, which contains fetal DNA and maternal DNA. This is due to the fact, that unlike whole blood, plasma does not contain any cellular components of blood and after extraction the background of maternal DNA is lower in comparison to whole blood. Another reason for using plasma samples is that fetal cells remain in the maternal circulation for several years after delivery and therefore the results are susceptible for false positivity and the diagnostic error can occur when whole blood is used (26,27). Samples of whole blood were used for the isolation of maternal and fetal DNA only in two studies. Byrne et al. concluded that whole blood is not a suitable source of fetal DNA, which is then quantified in order to use it as a marker of preeclampsia (28). In comparison to other reports, Cotter et al. found very high levels of fetal DNA, which might be due to fetal cells present in a sample of whole blood (29). The quantitative approach using real-time PCR allows the identification and quantification of monitored DNA sequences. Commonly used genes (GAPDH, beta globin, beta actin or albumin) were used as housekeeping genes and positive controls, in order to confirm the presence of DNA in each sample (30). Segments specific for the Y chromosome, SRY or DYS gene were amplified as target genes to determine the presence of fetal DNA in the background of maternal nucleic acids (31). Of course, this is limited to the male gender of the fetus. In the future, if the concentration of cff DNA should be used as a biomarker for the prediction or diagnosis of preeclampsia, it would be appropriate to establish the amount of fetal DNA without the gender limitation by analysis of genes outside sex chromosomes (32). DNA of fetal origin can be detected and quantified using a gender – and genetic polymorphism – independent marker – the hypermethylated gene RASSF1A as the placental epigenetic signature (33). This approach has a potential to be a universal method for detecting fetal DNA in maternal plasma. However, standardization and wider use across laboratories is needed. Some of the variability may arise from the technical limitations of the realtime PCR. The efficiency of the reaction has not been indicated in the published reports, although this parameter is very important for the interpretation of the results (34). And alternative and more precise approach represents the digital PCR technology. This procedure allows counting the copy number of DNA present in a sample. By using of this method it is possible to avoid real-time PCR, normalization or taking into account the efficiency of the PCR reaction (4). Another possible and very promising technology is the massively parallel sequencing (35). Fetal aneuploidies (trisomy 13, 18, 21) can be reliably diagnosed by counting billions of DNA molecules extracted from maternal plasma. The massively parallel sequencing requires complex and expensive equipment as well as consumables. Even more important is the need for highly trained staff with a bioinformatic background. But improvements
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and simplifications will allow increasing throughput and decreasing costs in the future (36). Another source of variability in the results is probably the way how the values were calculated, which test was used for statistical data processing or which factors of correction were included in the calculation. The apparent enormous variability in the reported results between the studies is related to specific issues with the interpretation of results. In addition to the overlap observed in cff DNA concentrations between patients and controls in the particular studies (37), this is an important issue regarding potential clinical use in the diagnosis or prediction of preeclampsia. Low sensitivity and specificity might be improved either by combination with other monitored biochemical parameters or by decreasing the variability, both technical and biological, if possible. To do that, sources of variability have to be identified and eliminated. The important clinical parameter predisposing to preeclampsia – high body mass index (BMI) was not associated with cff DNA in some studies (38,39). In other studies there was a negative correlation – high BMI was associated with low cff DNA levels (40). This might be due to increased amounts of maternal cell-free DNA and, thus, lower cff DNA fractions (41). The reason for increased maternal circulating DNA is very likely the increased turnover of adipose cells in obese women (42). Importantly, higher concentrations of cff DNA were found in pregnancies with trisomy 21 (43). And, in addition, cff DNA is increased also in women with ectopic pregnancies in comparison to women with intrauterine pregnancies (44). These findings show that the association of preeclampsia and high cff DNA is not specific and can only be interpreted in combination with the clinical status and with other biomarkers.
CONCLUSIONS The presence of certain levels of fetal DNA in maternal circulation is a physiological phenomenon. Increased level is associated with pregnancyrelated pathologies. Cell-free fetal DNA present in maternal plasma is a potential biomarker for early prediction of preeclampsia before the onset of clinical symptoms. The level of cff DNA correlates also with severity of the disease. These findings are promising, but will require further studies to confirm results and also to develop standardized protocols for its implementation in the routine non-invasive clinical diagnostics. A large prospective multi-center study proving a standardized consensus protocol with a very high diagnostic accuracy would then be needed. With such standards and improved efficiency pregnancies at increased risk could be identified at a very early stage of the disease. This will allow special monitoring and will help to improve the prenatal care of preeclamptic pregnancies. However, the reason for increased cff DNA in preeclampsia remains to be elucidated, but genetic factors will surely play a role (45).
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the Slovak research and development agency through contract APVV-0754-10.
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