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Open Access

Peer-reviewed

Research Article

Maternal total cell-free DNA in preeclampsia and fetal growth restriction: Evidence of differences in maternal response to abnormal implantation

  • Tal Rafaeli-Yehudai ,

    Contributed equally to this work with: Tal Rafaeli-Yehudai, Majdi Imterat

    Roles Investigation, Methodology, Writing – original draft, Writing – review & editing

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Majdi Imterat ,

    Contributed equally to this work with: Tal Rafaeli-Yehudai, Majdi Imterat

    Roles Data curation, Methodology, Writing – original draft

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Amos Douvdevani,

    Roles Conceptualization, Investigation, Methodology, Project administration, Resources, Validation, Writing – review & editing

    Affiliation Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Dan Tirosh,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Neta Benshalom-Tirosh,

    Roles Data curation, Investigation, Methodology, Writing – review & editing

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Salvatore Andrea Mastrolia,

    Roles Investigation, Writing – original draft

    Affiliations Department of Obstetrics and Gynecology, Azienda Ospedaliera Universitaria Policlinico di Bari, School of Medicine, University of Bari "Aldo Moro", Bari, Italy, Department of Maternal Fetal Medicine, Fondazione MBBM, San Gerardo Hospital, School of Medicine, University of Milano Bicocca, Monza, Italy

    ⨯
  • Ruthy Beer-Weisel,

    Roles Investigation, Methodology, Writing – original draft

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Vered Klaitman,

    Roles Investigation

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Reut Riff,

    Roles Investigation, Resources

    Affiliation Department of Clinical Biochemistry and Pharmacology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Shirley Greenbaum,

    Roles Investigation

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Alex Alioshin,

    Roles Investigation

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Gal Rodavsky Hanegbi,

    Roles Investigation

    Affiliation Department of Obstetrics and Gynecology, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯
  • Giuseppe Loverro,

    Roles Investigation, Supervision

    Affiliation Department of Obstetrics and Gynecology, Azienda Ospedaliera Universitaria Policlinico di Bari, School of Medicine, University of Bari "Aldo Moro", Bari, Italy

    ⨯
  • Mariana Rita Catalano,

    Roles Investigation

    Affiliation Department of Obstetrics and Gynecology, Azienda Ospedaliera Universitaria Policlinico di Bari, School of Medicine, University of Bari "Aldo Moro", Bari, Italy

    ⨯
  • Offer Erez

    Roles Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Writing – review & editing

    erezof@bgu.ac.il

    Affiliation Maternity Department D, Obstetrical Day Care Unit, Soroka University Medical Center, School of Medicine, Ben Gurion University of the Negev, Beer Sheva, Israel

    ⨯

Abstract

Objectives

Preeclampsia and fetal growth restriction are obstetrical syndromes associated with abnormal placental implantation and changes in the activation status of maternal leukocytes. This study is aimed to determine by a simple, rapid fluorescent assay the changes in maternal serum total cell-free DNA (t-cfDNA) concentrations in women with preeclampsia and those with fetal growth restriction (FGR).

Study design

A cross-sectional study was conducted measuring maternal serum t-cfDNA concentrations. Women were classified into the following groups: 1) patients with preeclampsia (n = 21); 2) FGR-estimated fetal weight below the 10thpercentile (n = 28); and 3) normal pregnancy (n = 39). Serum samples were directly assayed for t-cfDNA using a rapid fluorescent SYBR Gold assay. Elevated maternal serum t-cfDNA concentrations were defined as a cutoff>850ng/ml. Nonparametric statistics were used for analysis.

Results

Women with preeclampsia had a higher median maternal serum concentration (802 ng/ml, 400–2272 ng/ml) than women with a normal pregnancy (499 ng/ml, 0–1892 ng/ml, p = 0.004) and those with FGR (484 ng/ml, 72–2187 ng/ml, p = 0.012). Moreover, even patients with FGR <5th percentile and abnormal Doppler had a lower median maternal serum t-cfDNA than those with preeclampsia (median 487 ng/ml, 144–1971 ng/ml, p = 0.022). The median concentration of t-cfDNA did not differ between women with a normal pregnancy and those with FGR (p = 0.54), as well as those with fetuses <5th percentile and abnormal Doppler (p = 0.7). Women with preeclampsia had a higher proportion of elevated t-cfDNA than those with a normal pregnancy (p = 0.015) and patients with FGR (p = 0.025).

Conclusions

Preeclampsia is associated with higher maternal serum t-cfDNA concentration than normal pregnancy or FGR. This observation may reflect an increased systemic activation of the maternal inflammation, rather than placental; this assumption is supported by the fact that we did not observe a significant change in the maternal serum t-cfDNA in patients with placental-mediated FGR.

Citation: Rafaeli-Yehudai T, Imterat M, Douvdevani A, Tirosh D, Benshalom-Tirosh N, Mastrolia SA, et al. (2018) Maternal total cell-free DNA in preeclampsia and fetal growth restriction: Evidence of differences in maternal response to abnormal implantation. PLoS ONE 13(7): e0200360. https://doi.org/10.1371/journal.pone.0200360

Editor: Víctor Sánchez-Margalet, Virgen Macarena University Hospital, School of Medicine, University of Seville, SPAIN

Received: January 6, 2018; Accepted: June 24, 2018; Published: July 12, 2018

Copyright: © 2018 Rafaeli-Yehudai et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Circulating cell-free nucleic acids in plasma and serum are considered novel biomarkers with promising clinical applications in different medical conditions [1–9]. These biomarkers were reported in various aspects of obstetrics (especially in prenatal diagnosis) and in adverse pregnancy outcomes [10–13]. In serum samples, the vast amount of total cf-DNA is derived from the demise of maternal leucocytes [14]. This is why there is such a huge difference in the amount of this material with matching plasma samples. Placental or fetal cf-DNA (cfp-DNA) makes up for a small fraction (≈5%) of the total cf-DNA pool in maternal plasma, and is even lower in serum samples, as it is diluted by the significant increase in maternal leucocyte derived material [15]. The cfp-DNA can be detected in maternal circulation as early as the fifth or sixth weeks of gestation [16]. Its concentrations increase steadily with advancing gestational age. Maternal leucocyte, mainly neutrophils are the source of the elevated concentrations of cfDNA in the serum reflecting the enhanced capability of these cells to form Neutrophil Extracellular Traps (NETs) [14]. The small fragments of cfp-DNA predominantly originate from trophoblast cells [14, 17, 18]; nevertheless, the quantification of circulating cfp-DNA in maternal circulation requires the utilization of complex methods for DNA extraction, and real-time-polymerase chain reaction (rt-PCR). Our group previously reported [19–22] a rapid direct fluorescent assay for total cf-DNA concentration in biological fluids that uses the SYBR® Gold dye. Our test does not require a prior processing of the samples and therefore is a potential "point of care" assay.

Preeclampsia and fetal growth restriction (FGR) represent two of the “Great Obstetrical Syndromes”[23], constituting one of the leading causes of maternal and fetal/neonatal morbidity and mortality worldwide [24, 25]. These two conditions are the clinical end point of many underlying mechanisms (different as well as similar) and are frequently associated with one another [23]. The similar underlying mechanisms leading to FGR and preeclampsia include: 1) abnormal placentation, manifested as failure of transformation of the spiral arteries and shallow trophoblast invasion[26–28]; 2) an imbalance between angiogenic and anti-angiogenic factors in maternal blood[29–39]; 3) chronic utero-placental ischemia [40–42]; 4) increased trophoblast apoptosis/necrosis [43]; and 5) an enhanced maternal systemic inflammatory response [44–46]. However, In spite of these similarities in the underlying mechanisms, the two obstetrical syndromes differ in their clinical manifestation. [47].

Conditions that affect the placenta can directly impact its concentrations of cell-free fetal DNA in the maternal circulation, as its release is closely related to placental morphogenesis [48]. Preeclampsia is associated with elevated concentrations of cell-free fetal DNA in maternal circulation [49–55]. In pregnancies complicated with FGR, the cell-free fetal DNA concentrations were either normal or increased (yet to a lesser extent than that observed in early-onset preeclampsia) [56–59].

The aim of our study was to determine the changes in maternal serum total cell-free DNA (t-cfDNA) in women with preeclampsia and in women with FGR by using the fluorochrome SYBR® Gold assay.

Materials and methods

Study groups and inclusion criteria

A prospective cross-sectional study was conducted at the Soroka University Medical Center, a tertiary medical center in the southern region of Israel, which serves the entire obstetrical population of the region. Serum samples for total cf-DNA were collected from patients in the following groups: 1) women with normal pregnancies (n = 39); 2) patients with preeclampsia (n = 21); 3) patients with isolated FGR (without PE)(n = 28). Patients with multiple pregnancies or fetuses with congenital and/or chromosomal anomalies were excluded. All women provided a signed, informed consent prior to the collection of maternal blood. The Institutional Review Board of the Soroka University Medical Center approved the study.

Data collection.

Information regarding the pregnancy and delivery outcomes was collected from the birth files and medical records of the patients. Raw data is presented in S1 File.

Clinical definitions

Normal pregnancy was defined as women who delivered at term (37–42 weeks) a singleton neonate with a 5-minute Apgar score ≥7 and a birthweight between the 10th and 90th percentiles, and had no medical or obstetrical complications during pregnancy.

Preeclampsia was defined in the presence of new-onset hypertension in the second half of pregnancy (systolic blood pressure ≥140 mmHg or diastolic blood pressure ≥90 mmHg on at least two occasions, 4 hours to 1 week apart) and proteinuria (≥300 mg in a 24-hour urine collection or protein/creatinine ratio of at least 0.3 [each measured as mg/dl] or dipstick measurement ≥ 1) [60].

For the purpose of fetal growth evaluation, gestational age dating was primarily defined by last menstrual period (LMP) and correlated, when possible, with 1st trimester crown-rump length (CRL). If there was a difference of ≥7 days between gestational age by LMP and 1st trimester CRL gestational dating was set according to CRL measurements [61]. Fetal growth restriction (FGR) was defined as estimated fetal weight below the 10th percentile with or without abnormal umbilical, uterine, and middle cerebral artery Doppler velocimetry [62].

Sample collection and cell free DNA rapid direct fluorescent assay

Blood samples were collected using BD Vacutainer® gel tubes (Becton- Dickinson, Plymouth, UK) and samples were stored at 4°C for up to 24 hours before centrifugation. Serum was separated from the cellular fraction and frozen at -20°C until assayed. Total cf-DNA was detected directly in sera, according to our method [19]. Briefly, 20 μL of sera or DNA standard solutions were applied in duplicate to black 96-well plates (Greiner Bio-One; Frickenhausen, Germany). 80 μL of diluted SYBR® Gold was added to each well (final dilution 1:10.000), and fluorescence was measured with a 96-well fluorimeter at an emission wavelength of 535 nm and an excitation wavelength of 485 nm. Concentrations of unknown samples were calculated from a standard curve by extrapolation in a linear regression model. As described previously our assay correlates with the conventional quantitative PCR assay of ß-globin (R2 = 0.9987, p<0.001) [19].

Statistical analysis

Statistical analysis was performed with the SPSS package, version 20 (SPSS, Inc.; Chicago, IL). Statistical significance of the categorical variables was tested using the x2 or Fisher’s exact test, as appropriate. Since data was not normally distributed, we used non-parametric statistics for the analysis of continuous variables, including Man–Whitney and Kruskal Wallis tests. Cox proportional hazard regression analysis was used to calculate the gestational age adjusted hazard ratio for the occurrence of preeclampsia according to the concentrations of total free cell DNA. A P value< 0.05 was considered statistically significant.

High total cell-free DNA concentrations were defined as a serum concentration above 850ng/mL, which represents a value of two standard deviations above the mean, previously reported in healthy adults using the same assay [22, 63, 64].

Results

Of the total 88 pregnancies that met the inclusion criteria, serum samples for total cf-DNA were collected from 21 patients with preeclampsia, 28 patients with FGR (14 of them ≤5th percentile), and 39 women with normal pregnancies.

Table 1 displays the demographic and clinical characteristics of the study groups. Patients with FGR had lower median gestational age at sample collection (p = 0.005), than women in the other study groups. Patients with preeclampsia had significantly higher rates of history of preeclampsia than women with either a normal pregnancy or FGR (p = <0.001). No significant differences were found among the study groups regarding maternal age, maternal BMI, ethnicity, gravidity, parity, smoking, diabetes, and history of SGA neonates, or the use of assisted reproductive technologies.

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Table 1. Demographic and clinical characteristics of the study population.

https://doi.org/10.1371/journal.pone.0200360.t001

Table 2 displays the perinatal outcome among the study population. Patients with FGR had lower median gestational age at delivery (p = <0.001) and lower median birthweight (p = <0.001) than women with normal pregnancies and women with preeclampsia. There was no significant difference in the low Apgar scores at 1 and 5 minutes among the groups.

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Table 2. Perinatal outcomes among the study groups.

https://doi.org/10.1371/journal.pone.0200360.t002

Changes in the median maternal serum concentrations of total cf-DNA in the different study groups

Maternal serum total cf-DNA concentrations differed significantly among the study groups (Kruskal-Wallis Test p = 0.006). The median maternal serum total cf-DNA concentrations were higher in patients with preeclampsia than in women with normal pregnancies (median:802 ng/ml; range 400–2272 vs.median:499 ng/ml;range 0–1892; p = 0.004, respectively) as well as from patients with FGR (median: 484 ng/ml; range 72–4219; p = 0.024) (Fig 1).

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Fig 1. Median maternal serum concentration of study groups.

https://doi.org/10.1371/journal.pone.0200360.g001

The rate of elevated total cf-DNA concentration was higher in women with preeclampsia than in those with both normal pregnancy (p = 0.015) and FGR (p = 0.02.5) (Fig 2).

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Fig 2. Elevated rate of maternal serum concentration of study groups.

https://doi.org/10.1371/journal.pone.0200360.g002

Moreover, the comparison between patients with preeclampsia and those with FGR ≤5th percentile, who had abnormal Doppler, yielded similar results (487 ng/ml, 144–1971 ng/ml, p = 0.022). The median concentration of total cf-DNA did not differ between women in the normal pregnancy group and those with FGR (p = 0.481), even when only those with fetuses ≤5th percentile and abnormal Doppler were included (p = 0.508) (Fig 3).

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Fig 3. Median maternal serum concentration of normal pregnancies and pregnancies with IUGR below 5th percentile.

https://doi.org/10.1371/journal.pone.0200360.g003

We used a Cox regression analysis to calculate the risk associated with total free cell DNA to developed preeclampsia according to gestational age after adjustment for confounding factors such maternal age and BMI. Increased maternal serum total cf-DNA concentrations (ng/ml) were independently associated with preeclampsia (Hazard ratio (HR) for preeclampsia of 1.002 (95% confidence interval 1.0003–1.003) while maternal BMI and age did not reach statistical significance (date not shown). In addition elevated cf-DNA was associated with an HR for preeclampsia of 12.36 (95%CI 2.53–60.41).

Discussion

Principal findings of the study

We found that women with preeclampsia have a higher median maternal serum total cf-DNA concentration than those of patients with normal pregnancy or those with pregnancies complicated with FGR, even if there were signs for placental origin of the FGR.

What are the changes in circulating cf-DNA in preeclampsia and FGR?

The observation that the total maternal serum cf-DNA is higher in women with preeclampsia than in women with a normal pregnancy or those who had an FGR neonate is novel. The elevation in maternal as well as fetal cf-DNA concentration in the maternal plasma and serum of patients with preeclampsia was previously reported [44, 49–52, 54, 55, 65–70].

In addition, several studies examined the relationship between FGR and cf-DNA levels. Sekizawa, et al [56] examined the concentration of total and cf-DNA in 9 pregnancies complicated by FGR and in 9 pregnancies complicated by preeclampsia; they found that the concentration of cf-DNA in FGR pregnancies were similar to those in women with normal pregnancies. In contrast to our study, however, the authors could not demonstrate a significant difference in total concentrations of cf-DNA between patients with preeclampsia and those with FGR. Since Sekizawa et al were using plasma and not serum, it is not surprising that their data do not match those of the current study, where serum was used.

Placental contribution to total cf-DNA

The human placenta is a dynamic organ with constant turnover of villous trophoblast. The turnover of villous trophoblast results in its final stage, in extrusion of apoptotic material (including cf-DNA) into the maternal circulation. In normal pregnancies, at term, grams of placental material are shed daily into the maternal circulation without causing inflammatory response [71]. As aforementioned, the release of cf-DNA is tied strongly to placenta morphogenesis. In cases of failure of the key mechanisms involved in placental development (proliferation, migration, invasion, and differentiation), such as in the case of preeclampsia, there are changes in placental cell composition, production of regulatory molecules, and cell turn-over, all of which directly impact the types and levels of placental inflammatory syncytiotrophoblast debris that enters maternal circulation [48, 71]. Furthermore, the report by Goswami et al. [72], increased concentration of syncytiotrophoblast microparticles was specific to women with preeclampsia, and not to those with normotensive FGR. This is in accord with our findings also suggesting difference in the pathophysiology of these two syndromes.

Apoptosis appears to be the main mechanism controlling release of cf-DNA from the placenta. As a final event during the apoptosis cascade, old and late apoptotic nuclei are packed and released from syncytiotrophoblast in the form of exosomes to the maternal circulation [71, 73]. In addition to apoptotic mechanisms, accidental breakage or necrosis may also lead to the release of cf-DNA [65]; indeed, preeclampsia is associated with an increased placental apoptosis that leads to the apoptotic shedding and enhanced release of non-apoptotic molecules [71]. Our findings question the concept that the elevated total maternal cf-DNA observed in patients with preeclampsia is generated from the placenta. This doubt increased when we surveyed the results of the FGR groups: we could not demonstrate that patients, who had an FGR <5th percentile and abnormal Doppler (representative of placental origin), had an elevation in the median total circulating cf-DNA. This finding is in contrast to the report of Al Nakib et al [57], who demonstrated an increase in the total cf-DNA concentration in 19 pregnancies complicated by FGR due to placental origins. A possible explanation of the differences between the two studies is the method of measurement and sample size in each study group. Collectively we can summarize that even in cases of severe preeclampsia or placental FGR with evidence of substantial trophoblast apoptosis the overall contribution of fetal/placental DNA to the total circulating DNA in the maternal serum is minor.

Systemic maternal inflammation and changes in cf-DNA concentrations

In both obstetrical syndromes (preeclampsia and FGR), there is an enhanced maternal systemic inflammatory response; however, the magnitude and characteristics of this response differed between the two entities [44, 45, 74–76]. Oggé et al. [44] reported that patients with preeclampsia had a lower median mean channel brightness of CD62L on granulocytes as well as higher median basal radical oxidative species and oxidative bursts on monocytes than in patients with FGR. They concluded, in pregnancies complicated by FGR, there is enhanced activation of maternal peripheral leukocytes when compared to normal pregnancies but lower activation when compared to those complicated by preeclampsia [44]. These differences may explain the fact that patients with preeclampsia have a systemic maternal disease, while those with FGR have a mild activation of the inflammatory system but not a clinically evident disease.

What is the connection between systemic maternal inflammation and elevated cf-DNA?

The clinical syndrome of preeclampsia is, in part, a consequence of a wide systemic inflammatory response, including endothelium dysfunction [45, 71, 73, 74]. This exaggerated inflammatory response is characterized, among others, by increased expression of pro-inflammatory surface antigens (such as CD11b), increased basal iROS concentration, oxidative burst, and production and release of Neutrophil Extracellular Traps (NETs) [77, 78].

Brinkmann, et al. [79] reported that upon activation by bacterial endotoxin, inflammatory cytokines, or pharmacological agents, neutrophils release granule proteins and chromatin that, together, form extracellular fibers. These NETs degrade virulence factors and kill bacteria. A study in 2006 by Gupta, et al [80] demonstrated that NETs may also be elicited by placental syncytiotrophoblast micro-particles and the intervillous space in placentas of patients with preeclampsia was infiltrated by numerous NETs. Recently, a group from Basel, demonstrated that the increased total cf-DNA in the serum of women with preeclampsia results from increase NETs production by their neutrophils [14]. These findings support our hypothesis that the source of elevated concentrations of total cf-DNA in the serum of women with preeclampsia is mainly derived from the production of NETs resulting from systemic maternal inflammation. The contribution of the placental/fetal unit to the total cf-DNA is probably minor. Thus, we suggest that increased total cf-DNA observed in patients with preeclampsia results from neutrophil activation with the release of NETs full of DNA.

The strength of our study is that we measured the total free cell DNA by using our simple "mix and measure" fluorometric method. The estimated cost of reagents for our assay is 5 cents per sample, much lower than quantitative PCR (QPCR) based assays. In contrast to PCR-based assay that requires DNA extraction from large volume of blood, our assay is performed directly on small volume of serum (0.02 ml). Different from our assay, QPCR assays measure the number of gene copies under investigation and not DNA concentration. Thus, QPCR is affected by extraction losses, by DNA fragmentation, and PCR efficiency. In our view, our simple technique allows for rapid and effective evaluation of patients. We were able to demonstrate similar results as those previously published by more conservative and expensive methods in spite of the small sample size of patients. This assay can be simply applied directly to the relevant biological fluid serum.

Conclusions

In conclusion, preeclampsia is associated with an increase concentrations of total cf-DNA in the serum compared to normal pregnancies and pregnancies complicated with FGR. The lack of association between maternal serum total cf-DNA concentrations and FGR due to placental origin suggest that the main contribution to the elevated circulation total cf-DNA concentration observed in women with preeclampsia is derived from the maternal compartment and represents the systemic inflammation observed in this syndrome.

Supporting information

S1 File. Clinical information and cell-free DNA levels of the study participants.

https://doi.org/10.1371/journal.pone.0200360.s001

(XLS)

References

  1. 1. Kamat AA, Baldwin M, Urbauer D, Dang D, Han LY, Godwin A, et al. Plasma cell-free DNA in ovarian cancer: an independent prognostic biomarker. Cancer. 2010;116(8):1918–25. Epub 2010/02/19. pmid:20166213; PubMed Central PMCID: PMCPMC2854845.
  2. 2. Zhang R, Shao F, Wu X, Ying K. Value of quantitative analysis of circulating cell free DNA as a screening tool for lung cancer: a meta-analysis. Lung cancer (Amsterdam, Netherlands). 2010;69(2):225–31. Epub 2009/12/17. pmid:20004997.
  3. 3. Zancan M, Galdi F, Di Tonno F, Mazzariol C, Orlando C, Malentacchi F, et al. Evaluation of cell-free DNA in urine as a marker for bladder cancer diagnosis. The International journal of biological markers. 2009;24(3):147–55. Epub 2009/09/30. pmid:19787625.
  4. 4. Schwarzenbach H, Stoehlmacher J, Pantel K, Goekkurt E. Detection and monitoring of cell-free DNA in blood of patients with colorectal cancer. Annals of the New York Academy of Sciences. 2008;1137:190–6. Epub 2008/10/08. pmid:18837946.
  5. 5. Atamaniuk J, Hsiao YY, Mustak M, Bernhard D, Erlacher L, Fodinger M, et al. Analysing cell-free plasma DNA and SLE disease activity. European journal of clinical investigation. 2011;41(6):579–83. Epub 2010/12/07. pmid:21128939.
  6. 6. Zhong XY, von Muhlenen I, Li Y, Kang A, Gupta AK, Tyndall A, et al. Increased concentrations of antibody-bound circulatory cell-free DNA in rheumatoid arthritis. Clinical chemistry. 2007;53(9):1609–14. Epub 2007/08/23. pmid:17712000.
  7. 7. Saukkonen K, Lakkisto P, Pettila V, Varpula M, Karlsson S, Ruokonen E, et al. Cell-free plasma DNA as a predictor of outcome in severe sepsis and septic shock. Clinical chemistry. 2008;54(6):1000–7. Epub 2008/04/19. pmid:18420731.
  8. 8. El Tarhouny SA, Hadhoud KM, Ebrahem MM, Al Azizi NM. Assessment of cell-free DNA with microvascular complication of type II diabetes mellitus, using PCR and ELISA. Nucleosides, nucleotides & nucleic acids. 2010;29(3):228–36. Epub 2010/04/22. pmid:20408053.
  9. 9. Netz U, Perry Z, Mizrahi S, Kirshtein B, Czeiger D, Sebbag G, et al. Cell-free deoxyribonucleic acid as a prognostic marker of bowel ischemia in patients with small bowel obstruction. Surgery. 2017. Epub 2017/08/12. pmid:28797546.
  10. 10. Dugoff L, Barberio A, Whittaker PG, Schwartz N, Sehdev H, Bastek JA. Cell-free DNA fetal fraction and preterm birth. American journal of obstetrics and gynecology. 2016;215(2):231.e1-7. Epub 2016/02/16. pmid:26875947.
  11. 11. Thurik FF, Lamain-de Ruiter M, Javadi A, Kwee A, Woortmeijer H, Page-Christiaens GC, et al. Absolute first trimester cell-free DNA levels and their associations with adverse pregnancy outcomes. Prenatal diagnosis. 2016;36(12):1104–11. Epub 2016/10/14. pmid:27734510.
  12. 12. Stein W, Muller S, Gutensohn K, Emons G, Legler T. Cell-free fetal DNA and adverse outcome in low risk pregnancies. European journal of obstetrics, gynecology, and reproductive biology. 2013;166(1):10–3. Epub 2012/10/02. pmid:23021026.
  13. 13. Bauer M, Hutterer G, Eder M, Majer S, Leshane E, Johnson KL, et al. A prospective analysis of cell-free fetal DNA concentration in maternal plasma as an indicator for adverse pregnancy outcome. Prenatal diagnosis. 2006;26(9):831–6. Epub 2006/07/13. pmid:16832830.
  14. 14. Sur Chowdhury C, Hahn S, Hasler P, Hoesli I, Lapaire O, Giaglis S. Elevated Levels of Total Cell-Free DNA in Maternal Serum Samples Arise from the Generation of Neutrophil Extracellular Traps. Fetal diagnosis and therapy. 2016;40(4):263–7. Epub 2016/03/22. pmid:26998969.
  15. 15. Hahn S, Giaglis S, Buser A, Hoesli I, Lapaire O, Hasler P. Cell-free nucleic acids in (maternal) blood: any relevance to (reproductive) immunologists? Journal of reproductive immunology. 2014;104–105:26–31. Epub 2014/05/13. pmid:24815811.
  16. 16. Finning KM, Chitty LS. Non-invasive fetal sex determination: impact on clinical practice. Seminars in fetal & neonatal medicine. 2008;13(2):69–75. Epub 2008/02/05. pmid:18243829.
  17. 17. Ashoor G, Syngelaki A, Poon LC, Rezende JC, Nicolaides KH. Fetal fraction in maternal plasma cell-free DNA at 11–13 weeks' gestation: relation to maternal and fetal characteristics. Ultrasound in obstetrics & gynecology: the official journal of the International Society of Ultrasound in Obstetrics and Gynecology. 2013;41(1):26–32. Epub 2012/10/31. pmid:23108725.
  18. 18. Alberry M, Maddocks D, Jones M, Abdel Hadi M, Abdel-Fattah S, Avent N, et al. Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast. Prenatal diagnosis. 2007;27(5):415–8. Epub 2007/02/09. pmid:17286310.
  19. 19. Goldshtein H, Hausmann MJ, Douvdevani A. A rapid direct fluorescent assay for cell-free DNA quantification in biological fluids. Annals of clinical biochemistry. 2009;46(Pt 6):488–94. Epub 2009/09/05. pmid:19729503.
  20. 20. Shimony A, Zahger D, Gilutz H, Goldstein H, Orlov G, Merkin M, et al. Cell free DNA detected by a novel method in acute ST-elevation myocardial infarction patients. Acute cardiac care. 2010;12(3):109–11. Epub 2010/08/18. pmid:20712451.
  21. 21. Agassi R, Czeiger D, Shaked G, Avriel A, Sheynin J, Lavrenkov K, et al. Measurement of circulating cell-free DNA levels by a simple fluorescent test in patients with breast cancer. American journal of clinical pathology. 2015;143(1):18–24. Epub 2014/12/17. pmid:25511138.
  22. 22. Avriel A, Paryente Wiessman M, Almog Y, Perl Y, Novack V, Galante O, et al. Admission cell free DNA levels predict 28-day mortality in patients with severe sepsis in intensive care. PloS one. 2014;9(6):e100514. Epub 2014/06/24. pmid:24955978; PubMed Central PMCID: PMCPMC4067333.
  23. 23. Romero R. The child is the father of the man. Prenat Neonat Med. 1996;1:8–11.
  24. 24. Nardozza LM, Caetano AC, Zamarian AC, Mazzola JB, Silva CP, Marcal VM, et al. Fetal growth restriction: current knowledge. Archives of gynecology and obstetrics. 2017;295(5):1061–77. Epub 2017/03/13. pmid:28285426.
  25. 25. Bibbins-Domingo K, Grossman DC, Curry SJ, Barry MJ, Davidson KW, Doubeni CA, et al. Screening for Preeclampsia: US Preventive Services Task Force Recommendation Statement. Jama. 2017;317(16):1661–7. Epub 2017/04/27. pmid:28444286.
  26. 26. Lyall F, Simpson H, Bulmer JN, Barber A, Robson SC. Transforming growth factor-beta expression in human placenta and placental bed in third trimester normal pregnancy, preeclampsia, and fetal growth restriction. The American journal of pathology. 2001;159(5):1827–38. Epub 2001/11/07. pmid:11696443; PubMed Central PMCID: PMCPMC1867050.
  27. 27. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Pre-eclampsia and fetal growth restriction: how morphometrically different is the placenta? Placenta. 2006;27(6–7):727–34. Epub 2005/08/30. pmid:16125226.
  28. 28. Egbor M, Ansari T, Morris N, Green CJ, Sibbons PD. Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction. BJOG: an international journal of obstetrics and gynaecology. 2006;113(5):580–9. Epub 2006/04/04. pmid:16579806.
  29. 29. Levine RJ, Lam C, Qian C, Yu KF, Maynard SE, Sachs BP, et al. Soluble endoglin and other circulating antiangiogenic factors in preeclampsia. The New England journal of medicine. 2006;355(10):992–1005. Epub 2006/09/08. pmid:16957146.
  30. 30. Bujold E, Romero R, Chaiworapongsa T, Kim YM, Kim GJ, Kim MR, et al. Evidence supporting that the excess of the sVEGFR-1 concentration in maternal plasma in preeclampsia has a uterine origin. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2005;18(1):9–16. Epub 2005/08/18. pmid:16105786.
  31. 31. Erez O, Romero R, Espinoza J, Fu W, Todem D, Kusanovic JP, et al. The change in concentrations of angiogenic and anti-angiogenic factors in maternal plasma between the first and second trimesters in risk assessment for the subsequent development of preeclampsia and small-for-gestational age. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2008;21(5):279–87. Epub 2008/05/01. pmid:18446652; PubMed Central PMCID: PMCPMC2846114.
  32. 32. Korzeniewski SJ, Romero R, Chaiworapongsa T, Chaemsaithong P, Kim CJ, Kim YM, et al. Maternal plasma angiogenic index-1 (placental growth factor/soluble vascular endothelial growth factor receptor-1) is a biomarker for the burden of placental lesions consistent with uteroplacental underperfusion: a longitudinal case-cohort study. American journal of obstetrics and gynecology. 2016;214(5):629.e1–.e17. Epub 2015/12/22. pmid:26688491; PubMed Central PMCID: PMCPMC5769706.
  33. 33. Baltajian K, Bajracharya S, Salahuddin S, Berg AH, Geahchan C, Wenger JB, et al. Sequential plasma angiogenic factors levels in women with suspected preeclampsia. American journal of obstetrics and gynecology. 2016;215(1):89.e1–.e10. Epub 2016/02/02. pmid:26827880.
  34. 34. Kusanovic JP, Romero R, Chaiworapongsa T, Erez O, Mittal P, Vaisbuch E, et al. A prospective cohort study of the value of maternal plasma concentrations of angiogenic and anti-angiogenic factors in early pregnancy and midtrimester in the identification of patients destined to develop preeclampsia. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2009;22(11):1021–38. Epub 2009/11/11. pmid:19900040; PubMed Central PMCID: PMCPMC3427777.
  35. 35. Gotsch F, Romero R, Kusanovic JP, Chaiworapongsa T, Dombrowski M, Erez O, et al. Preeclampsia and small-for-gestational age are associated with decreased concentrations of a factor involved in angiogenesis: soluble Tie-2. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2008;21(6):389–402. Epub 2008/06/24. pmid:18570117; PubMed Central PMCID: PMCPMC2780324.
  36. 36. Stepan H, Kramer T, Faber R. Maternal plasma concentrations of soluble endoglin in pregnancies with intrauterine growth restriction. The Journal of clinical endocrinology and metabolism. 2007;92(7):2831–4. Epub 2007/04/12. pmid:17426082.
  37. 37. Wathen KA, Tuutti E, Stenman UH, Alfthan H, Halmesmaki E, Finne P, et al. Maternal serum-soluble vascular endothelial growth factor receptor-1 in early pregnancy ending in preeclampsia or intrauterine growth retardation. The Journal of clinical endocrinology and metabolism. 2006;91(1):180–4. Epub 2005/11/03. pmid:16263826.
  38. 38. Malamitsi-Puchner A, Boutsikou T, Economou E, Makrakis E, Iliodromiti Z, Kouskouni E, et al. The role of the anti-angiogenic factor endostatin in intrauterine growth restriction. Journal of the Society for Gynecologic Investigation. 2005;12(3):195–7. Epub 2005/03/24. pmid:15784505.
  39. 39. Ahmed A, Perkins J. Angiogenesis and intrauterine growth restriction. Bailliere's best practice & research Clinical obstetrics & gynaecology. 2000;14(6):981–98. Epub 2001/01/06.
  40. 40. Granger JP, Alexander BT, Llinas MT, Bennett WA, Khalil RA. Pathophysiology of preeclampsia: linking placental ischemia/hypoxia with microvascular dysfunction. Microcirculation. 2002;9(3):147–60. Epub 2002/06/25. pmid:12080413.
  41. 41. Myatt L. Role of placenta in preeclampsia. Endocrine. 2002;19(1):103–11. Epub 2003/02/14. pmid:12583607.
  42. 42. Fisher SJ. The placental problem: linking abnormal cytotrophoblast differentiation to the maternal symptoms of preeclampsia. Reproductive biology and endocrinology: RB&E. 2004;2:53. Epub 2004/07/09. pmid:15236649; PubMed Central PMCID: PMCPMC493282.
  43. 43. Ishihara N, Matsuo H, Murakoshi H, Laoag-Fernandez JB, Samoto T, Maruo T. Increased apoptosis in the syncytiotrophoblast in human term placentas complicated by either preeclampsia or intrauterine growth retardation. American journal of obstetrics and gynecology. 2002;186(1):158–66. Epub 2002/01/26. pmid:11810103.
  44. 44. Ogge G, Romero R, Chaiworapongsa T, Gervasi MT, Pacora P, Erez O, et al. Leukocytes of pregnant women with small-for-gestational age neonates have a different phenotypic and metabolic activity from those of women with preeclampsia. The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstet. 2010;23(6):476–87. Epub 2009/11/18. pmid:19916874; PubMed Central PMCID: PMCPMC3426366.
  45. 45. Ness RB, Sibai BM. Shared and disparate components of the pathophysiologies of fetal growth restriction and preeclampsia. American journal of obstetrics and gynecology. 2006;195(1):40–9. Epub 2006/07/04. pmid:16813742.
  46. 46. von Dadelszen P, Watson RW, Noorwali F, Marshall JC, Parodo J, Farine D, et al. Maternal neutrophil apoptosis in normal pregnancy, preeclampsia, and normotensive intrauterine growth restriction. American journal of obstetrics and gynecology. 1999;181(2):408–14. Epub 1999/08/24. pmid:10454692.
  47. 47. Sibai B, Dekker G, Kupferminc M. Pre-eclampsia. Lancet (London, England). 2005;365(9461):785–99. Epub 2005/03/01. pmid:15733721.
  48. 48. Taglauer ES, Wilkins-Haug L, Bianchi DW. Review: cell-free fetal DNA in the maternal circulation as an indication of placental health and disease. Placenta. 2014;35 Suppl:S64–8. Epub 2014/01/07. pmid:24388429; PubMed Central PMCID: PMCPMC4886648.
  49. 49. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. American journal of human genetics. 1998;62(4):768–75. Epub 1998/06/13. pmid:9529358; PubMed Central PMCID: PMCPMC1377040.
  50. 50. Sekizawa A, Yokokawa K, Sugito Y, Iwasaki M, Yukimoto Y, Ichizuka K, et al. Evaluation of bidirectional transfer of plasma DNA through placenta. Human genetics. 2003;113(4):307–10. Epub 2003/07/29. pmid:12884005.
  51. 51. Farina A, Sekizawa A, Sugito Y, Iwasaki M, Jimbo M, Saito H, 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. Prenatal diagnosis. 2004;24(2):83–6. Epub 2004/02/20. pmid:14974111.
  52. 52. Leung TN, Zhang J, Lau TK, Chan LY, Lo YM. Increased maternal plasma fetal DNA concentrations in women who eventually develop preeclampsia. Clinical chemistry. 2001;47(1):137–9. Epub 2001/01/10. pmid:11148193.
  53. 53. Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clinical chemistry. 1999;45(2):184–8. Epub 1999/02/04. pmid:9931039.
  54. 54. Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. American journal of obstetrics and gynecology. 2001;184(3):414–9. Epub 2001/03/03. pmid:11228496.
  55. 55. 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. American journal of obstetrics and gynecology. 2004;190(3):707–13. Epub 2004/03/26. pmid:15042003.
  56. 56. Sekizawa A, Jimbo M, Saito H, Iwasaki M, Matsuoka R, Okai T, et al. Cell-free fetal DNA in the plasma of pregnant women with severe fetal growth restriction. American journal of obstetrics and gynecology. 2003;188(2):480–4. Epub 2003/02/20. pmid:12592259.
  57. 57. Al Nakib M, Desbriere R, Bonello N, Bretelle F, Boubli L, Gabert J, et al. Total and fetal cell-free DNA analysis in maternal blood as markers of placental insufficiency in intrauterine growth restriction. Fetal diagnosis and therapy. 2009;26(1):24–8. Epub 2009/10/10. pmid:19816026.
  58. 58. Crowley A, Martin C, Fitzpatrick P, Sheils O, O'Herlihy C, O'Leary JJ, et al. Free fetal DNA is not increased before 20 weeks in intrauterine growth restriction or pre-eclampsia. Prenatal diagnosis. 2007;27(2):174–9. Epub 2006/12/28. pmid:17191257.
  59. 59. Smid M, Galbiati S, Lojacono A, Valsecchi L, Platto C, Cavoretto P, et al. Correlation of fetal DNA levels in maternal plasma with Doppler status in pathological pregnancies. Prenatal diagnosis. 2006;26(9):785–90. Epub 2006/07/13. pmid:16832835.
  60. 60. Hypertension in pregnancy. Report of the American College of Obstetricians and Gynecologists' Task Force on Hypertension in Pregnancy. Obstetrics and gynecology. 2013;122(5):1122–31. Epub 2013/10/24. pmid:24150027.
  61. 61. Committee Opinion No 700: Methods for Estimating the Due Date. Obstetrics and gynecology. 2017;129(5):e150–e4. Epub 2017/04/21. pmid:28426621.
  62. 62. Alexander GR, Himes JH, Kaufman RB, Mor J, Kogan M. A United States national reference for fetal growth. Obstetrics and gynecology. 1996;87(2):163–8. Epub 1996/02/01. pmid:8559516.
  63. 63. Czeiger D, Shaked G, Eini H, Vered I, Belochitski O, Avriel A, et al. Measurement of circulating cell-free DNA levels by a new simple fluorescent test in patients with primary colorectal cancer. American journal of clinical pathology. 2011;135(2):264–70. Epub 2011/01/14. pmid:21228367.
  64. 64. Tovbin D, Novack V, Wiessman MP, Abd Elkadir A, Zlotnik M, Douvdevani A. Circulating cell-free DNA in hemodialysis patients predicts mortality. Nephrology, dialysis, transplantation: official publication of the European Dialysis and Transplant Association—European Renal Association. 2012;27(10):3929–35. Epub 2012/07/27. pmid:22833622.
  65. 65. Sifakis S, Koukou Z, Spandidos DA. Cell-free fetal DNA and pregnancy-related complications (review). Molecular medicine reports. 2015;11(4):2367–72. Epub 2014/12/23. pmid:25530428; PubMed Central PMCID: PMCPMC4337476.
  66. 66. 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. Hypertension in pregnancy. 2002;21(1):77–83. Epub 2002/06/05. pmid:12044339.
  67. 67. Hahn S, Rusterholz C, Hosli I, Lapaire O. Cell-free nucleic acids as potential markers for preeclampsia. Placenta. 2011;32 Suppl:S17–20. Epub 2011/01/25. pmid:21257079.
  68. 68. Knight M, Redman CW, Linton EA, Sargent IL. Shedding of syncytiotrophoblast microvilli into the maternal circulation in pre-eclamptic pregnancies. British journal of obstetrics and gynaecology. 1998;105(6):632–40. Epub 1998/07/01. pmid:9647154.
  69. 69. Zeybek YG, Gunel T, Benian A, Aydinli K, Kaleli S. Clinical evaluations of cell-free fetal DNA quantities in pre-eclamptic pregnancies. The journal of obstetrics and gynaecology research. 2013;39(3):632–40. Epub 2012/10/31. pmid:23107423.
  70. 70. Lazar L, Rigo J Jr., Nagy B, Balogh K, Mako V, Cervenak L, et al. Relationship of circulating cell-free DNA levels to cell-free fetal DNA levels, clinical characteristics and laboratory parameters in preeclampsia. BMC medical genetics. 2009;10:120. Epub 2009/11/26. pmid:19930583; PubMed Central PMCID: PMCPMC2789064.
  71. 71. Huppertz B, Kingdom JC. Apoptosis in the trophoblast—role of apoptosis in placental morphogenesis. Journal of the Society for Gynecologic Investigation. 2004;11(6):353–62. Epub 2004/09/08. pmid:15350247.
  72. 72. Goswami D, Tannetta DS, Magee LA, Fuchisawa A, Redman CW, Sargent IL, et al. Excess syncytiotrophoblast microparticle shedding is a feature of early-onset pre-eclampsia, but not normotensive intrauterine growth restriction. Placenta. 2006;27(1):56–61. Epub 2005/11/29. pmid:16310038.
  73. 73. Bischoff FZ, Lewis DE, Simpson JL. Cell-free fetal DNA in maternal blood: kinetics, source and structure. Human reproduction update. 2005;11(1):59–67. Epub 2004/12/01. pmid:15569699.
  74. 74. Borzychowski AM, Sargent IL, Redman CW. Inflammation and pre-eclampsia. Seminars in fetal & neonatal medicine. 2006;11(5):309–16. Epub 2006/07/11. pmid:16828580.
  75. 75. Redman CW, Sargent IL. Pre-eclampsia, the placenta and the maternal systemic inflammatory response—a review. Placenta. 2003;24 Suppl A:S21–7. Epub 2003/07/05. pmid:12842410.
  76. 76. Redman CW, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. American journal of obstetrics and gynecology. 1999;180(2 Pt 1):499–506. Epub 1999/02/13. pmid:9988826.
  77. 77. Gervasi MT, Chaiworapongsa T, Naccasha N, Blackwell S, Yoon BH, Maymon E, et al. Phenotypic and metabolic characteristics of maternal monocytes and granulocytes in preterm labor with intact membranes. American journal of obstetrics and gynecology. 2001;185(5):1124–9. Epub 2001/11/22. pmid:11717645.
  78. 78. Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. American journal of obstetrics and gynecology. 1998;179(1):80–6. Epub 1998/08/15. pmid:9704769.
  79. 79. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science (New York, NY). 2004;303(5663):1532–5. Epub 2004/03/06. pmid:15001782.
  80. 80. Gupta A, Hasler P, Gebhardt S, Holzgreve W, Hahn S. Occurrence of neutrophil extracellular DNA traps (NETs) in pre-eclampsia: a link with elevated levels of cell-free DNA? Annals of the New York Academy of Sciences. 2006;1075:118–22. Epub 2006/11/17. pmid:17108200.