Preterm birth is defined as childbirth occurring at less than 37 completed weeks; it is a major determinant of neonatal mortality and morbidity and has long-term adverse consequence for health 1. The pathological processes implicated in the preterm birth syndrome include intrauterine infection, uterine ischemia, uterine overdistension, abnormal allogenic recognition, allergic-like reaction, cervical disease, and endocrine disorders 2.
Preterm labor and birth continue to be a significant challenge to physicians in the obstetrics and neonatal fields. Until specific and effective therapeutic treatments are developed to prevent preterm labor, the best means of reducing preterm birth rate is early detection and diagnosis 3.
Both the mother and the fetus produce cell-free DNA. The primary source of cell-free fetal DNA (cffDNA) in the maternal circulation is considered to be apoptosis of placental cells (syncytiotrophoblast), whereas maternal hematopoietic cells are the source of most maternal cell-free DNA 4,5. Fetal DNA typically comprises 10–15% of the total DNA in maternal circulation during the late first and early second trimesters 6,7. The concentration increases with advancing gestational age and may be as high as 50% of the total DNA in the maternal circulation late in gestation 8. Clinical and in-vitro studies have found elevated concentrations of cffDNA in conditions related to significant placental ischemic disease 9. There is histopathological and clinical evidence to support the role of uteroplacental ischemia in the preterm labor etiology 2. In turn, this has led to the exploration of using cffDNA as an early marker of placental hypoxic dysfunction 10. cffDNA in maternal plasma can be first identified from the fourth week of gestation and increases throughout pregnancy. Temporarily, fetal DNA in maternal plasma and serum shows a progressive increase with gestational age, with a sharp increase during the last 8 weeks of gestation. It has been suggested that this sharp increase might be related to physiological processes that are associated with the imminent delivery 11. It is rapidly cleared from the maternal circulation after delivery and is undetectable within 2 hours after the birth 12. This suggests that maternal plasma DNA analysis would not be complicated by fetal DNA persistence from a previous gestation, making false-positive results unlikely 13. Elevation of cffDNA in maternal circulation has been found in pregnancies complicated by preterm labor 14.
Materials and methods
In this study, 60 women were enrolled, recruited from the antenatal care clinic and casualty unit of Obstetrics and Gynaecology Department, Kasr El Eini Hospital, Cairo University, and the antenatal clinic of Reproductive Health and Family Planning Department, Medical Research Unit, National Research Centre, Cairo, from February to August 2012. Informed consents were obtained from all participants. The Ethical Committee of the National Research Centre also approved the study. All patients were pregnant between 28 and 32 weeks’ gestation. Women were excluded from this study if they fulfilled any of the following criteria: multiple pregnancies, pre-eclampsia, gross fetal anomalies, intrauterine fetal death, intrauterine growth restriction, placenta previa, and placental abruption.
Women were divided into two groups. Group A included 30 women (study group) who presented with signs of either threatened or established preterm labor. Threatened preterm labor was diagnosed by uterine contractions occurring every 8 min or less with cervical dilatation of less than 3 cm and cervical effacement 50% or less with intact membranes, whereas established preterm labor was documented by uterine contractions occurring at a frequency of four in 20 min or eight in 60 min with a progressive change in the cervix, cervical dilatation greater than 1 cm, and cervical effacement of 80% or more. Group B included the controls: 30 women with no evidence of preterm labor. All cases were subjected to a full assessment of history. General, abdominal, and vaginal examinations were performed. Examination by a sterile speculum was performed before a digital vaginal examination to rule out rupture of membranes and high vaginal swabs were taken. Two-dimensional ultrasound was performed for fetal biometry, placental localization, and detection of possible gross fetal anomalies. Urine samples were taken for analysis. Venous blood samples were taken for complete blood count and quantification of cffDNA. All women were monitored using cardiotocography. Corticosteroids and tocolytics were administered according to the RCOG guidelines. All women were followed up until the end of pregnancy.
Sample processing and DNA extraction was performed in the National Research Centre laboratories.
Peripheral blood (10 ml) was drawn in an EDTA-containing tube and cell-free plasma samples were obtained by centrifugation of whole blood at 1600g for 10 min. Plasma was transferred to microcentrifuge tubes and centrifuged at 16000g for 10 min to remove residual cells. Cell-free plasma was stored at −80°C until further processing; thawing was carried out only once before DNA extraction. DNA extraction from 500 µl cell-free plasma samples was carried out using the QIAamp Mini Kit (Qiagen GmbH, Almeda, California) and eluted with 50 µl of H2O, and 35 µl of extracted DNA were digested with 100 U of BstUI, a methylation-sensitive restriction enzyme, at 60°C for 16 h 15.
Real-time detection of RASSF1A
PCR amplifications were performed using 7500 fast real-time PCR (Applied Biosystems, Foster City, California, USA). Each reaction contained 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 300 nmol/l of each primers, and 85 nmol/l probes. Enzyme-digested DNA (10 μl) or untreated DNA (5 μl) was used as a template for PCR. The thermal profile was 50°C for 2 min, 95°C for 10 min, 50 cycles of 95°C for 15 s, and 60°C for 1 min. All reactions were run in duplicate and the mean quantity was taken. A methylated DNA (Qiagen GmbH) was used as the standard 15.
Data were analyzed using IBM SPSS Advanced Statistics version 20.0 (IBM, Rochester, Minnesota). Numerical data were expressed as mean and SD or median and range as appropriate. Qualitative data were expressed as frequency and percentage. The χ2-test (Fisher’s exact test) was used to examine the relation between qualitative variables. For quantitative data, comparison between two groups was carried out using an independent-sample t-test or the Mann–Whitney U-test. Pearson’s product–moment was used to estimate the correlation between numerical variables. P-value less than 0.05 was considered significant.
In this study, the demographic criteria (maternal age, gestational age, gravidity, and parity) among the different groups of patients were compared and showed no statistically significant difference (P>0.05) (Table 1).
In our study, 90% of cases (27 women) in the study group were in established preterm labor, whereas only 10% of cases (three women) were in threatened preterm labor. The mean cervical dilatation was 5.2±2.3 cm and the mean cervical effacement was 71.3±12.2%.
The mean cffDNA levels in the study and control groups were 229.12±71.95 and 39.81±5.95 copies/ml, respectively. The calculated median was 234.89 and 39.92 copies/ml for the same groups, respectively. The cffDNA level was increased about six folds in group A (study group), with a statistical significance (P<0.05).
Our results showed no correlation between cffDNA levels and other parameters: maternal age, gestational age, cervical dilation, and cervical effacement (Table 2).
The presence of fetal DNA in maternal plasma may represent a source of genetic material that can be obtained noninvasively. We aimed to assess the relation between the level of cffDNA, detected by real-time PCR using the RASSF1A gene, in maternal plasma and the occurrence of preterm labor.
This association was first investigated by Leung et al.14 in 40 pregnant women between 26 and 34 weeks’ gestation. Twenty women had spontaneous preterm deliveries while the other 20 were taken as controls in the same age group. Measurement of cffDNA relies on the detection of Y chromosomal sequences in maternal serum as a fetal marker. They reported that cffDNA was present in higher concentrations in the plasma of pregnant women who were delivered preterm than in pregnant women who were delivered at term.
Our results showed a six-fold increase in the mean cffDNA levels in the study group compared with the control group, with a statistically significant difference (P<0.05). However, there was no correlation between cffDNA levels and other parameters: maternal age, gestational age, cervical dilation, and cervical effacement.
Farina et al.16 reported that high levels of cffDNA were related to the duration of pregnancy in women who are at high risk for preterm delivery following their study on 71 pregnant women with a male fetus between 20 and 42 weeks’ gestation.
Tanja et al.17 assessed whether spontaneous preterm delivery can be predicted from the amount of cffDNA through a cohort study including RhD-negative women participating in a routine RHD screening program at 25 weeks’ gestation. A highly significant association was found between preterm delivery and cffDNA levels.
Illanes et al.18 studied 56 women, with a male fetus, between 22 and 24 weeks’ gestation to evaluate the capability of cffDNA in increasing the accuracy of predicting preterm labor by cervical length. cffDNA was measured using Y chromosome-specific sequences. They reported that the median cffDNA level did not predict spontaneous preterm labor in women with a cervix less than 15 mm at 22–24 weeks.
The results of our study were in agreement with those of Leung et al.14, Farina et al.16, and Tanja et al.17. In contrast, Illanes et al.18 reported that the median cffDNA level did not predict spontaneous preterm labor in women with a cervix less than 15 mm at 22–24 weeks’ gestation.
There was a difference between our study and the studies carried out by Leung et al.14, Farina et al.16, and Illanes et al.18 in the method of measurement of cffDNA. They depended on the detection of Y chromosomal sequences in maternal serum as a fetal marker and consequently limited to male fetuses. Tanja et al.17 depended on the detection of the RHD gene for measurement of cffDNA, which has been detected in the plasma of RhD-negative pregnant women carrying RhD-positive fetuses. However, in the present study, we used a sex-independent fetal epigenetic marker (DNA methylation). This test is based on the detection of a hypermethylated placental (fetal) DNA sequence in the maternal circulation. The methylation pattern of the RASSF1A promoter in the placenta and maternal blood cells allows the use of methylation-sensitive restriction enzyme digestion for specifically cutting the maternally derived background RASSF1A sequences while leaving placentally (fetal) derived RASSF1A sequences intact. It can be used as a marker irrespective of the fetal sex.
Detection of cffDNA using hypermethylated RASSF1A in maternal plasma could be used as a marker for prediction of preterm labor. Increased risk of spontaneous preterm labor is associated with high levels between 28 and 32 weeks’ gestation. Its value could be improved by combining it with other factors such as maternal obstetric history and cervical length.
Conflicts of interest
There are no conflicts of interest.
1. Wang ML, Dorer DJ, Fleming MP, Catlin EA.Clinical outcomes of near-term infants.Pediatrics2004;114:372–376.
2. Romero R, Espinoza J, Goncalves F, Kusanovic JP, Friel A, Nien JK.Inflammation in preterm and term labor and delivery.Semin Fetal Neonatal Med2006;11:317–326.
3. Hanna N, Kiefer D.A translational view of biomarkers in preterm labor.Am J Reprod Immunol2012;67:268–272.
4. Sekizawa A, Samura O, Zhen DK, Falco V, Farina A, Bianchi DW.Apoptosis in fetal nucleated erythrocytes circulating in maternal blood.Prenat Diagn2000;20:886–889.
5. Lui YY, Chik KW, Chiu RW, et al..Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation.Clin Chem2002;48:421–427.
6. Chiu RW, Akolekar R, Zheng YW, et al..Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study.BMJ2011;342:c7401.
7. Nygren AO, Dean J, Jensen TJ, et al..Quantification of fetal DNA by use of methylation-based DNA discrimination.Clin Chem2010;56:1627–1635.
8. Lo YM, Tein MS, Lau TK, et al..Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis.Am J Hum Genet1998;62:768–775.
9. Hahn S, Huppertz B, Holzgreve W.Fetal cells and cell free fetal nucleic acids in maternal blood: new tools to study abnormal placentation?Placenta2005;26:515–526.
10. 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 Diagn2009;29:1118–1122.
11. Bianchi DW.Fetal DNA in maternal plasma: the plot thickens and the placental barrier thins.Am J Hum Genet1998;62:763–764.
12. Homfray T.Progress in free fetal DNA (ffDNA)-based prenatal tests. Bionews, 2008.
13. Botezatu I, Serdyuk O, Potapova G, Shelepov V, Alechina R, Molyaka Y, et al..Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism.Clin Chern2000;46:1078–1084.
14. Leung TN, Zhang J, Lau TK, et al..Maternal plasma fetal DNA as a marker for preterm labour.Lancet1998;352:1904–1905.
15. Chan KC, Ding C, Gerovassili A, et al..Hypermethylated RASSF1A in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis.Clin Chem2006;52:2211–2218.
16. Farina A, LeShane ES, Romero R, Gomez R, Chaiworapongsa T, Rizzo N, et al..High levels of fetal cell-free DNA in maternal serum: a risk factor for spontaneous preterm delivery.Am J Obstet Gynecol2005;193:421–425.
17. Jakobsen TR, Clausen FB, Rode L, Dziegiel MH, Tabor A.High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery.Prenat Diagn2012;32:840–845.
18. Illanes S, Gomez R, Fornes R, Figueroa-Diesel H, Schepeler M, Searovic P, et al..Free fetal DNA levels in patients at risk of preterm labour.Prenat Diagn2011;31:1082–1085.