Obstetrics & Gynecology:
Maternal Smoking: Effect on Circulating Cell-Free Fetal and Total DNA Levels in Maternal Plasma From the Second Trimester
Lapaire, Olav MD1; Volgmann, Thorsten MD2; Huang, Dorothy MD1; Hahn, Sinuhe PhD1; Holzgreve, Wolfgang MD1; Xiao Zhong, Yan MD1
From the 1University of Basel, Department of Obstetrics and Gynecology/Department of Research, Basel, Switzerland; and 2Medical Practice for Prenatal Medicine, Greifswald, Germany.
This study was funded in part by a grant from the Swiss National Science Foundation: 3200-066913.
The authors thank Andy Schötzau for statistical analysis.
Corresponding author: PD Dr. Xiao Yan Zhong, Laboratory for Prenatal Medicine/Gynecologic Oncology, Women’s Hospital/Dep. Research, Room 416, University of Basel, Hebelstrasse 20, CH-34031 Basel, Switzerland; e-mail: email@example.com.
Financial Disclosure The authors have no potential conflicts of interest to disclose.
OBJECTIVE: To estimate whether potential clinical applications of cell-free fetal and total DNA in the field of noninvasive prenatal diagnosis need to be adjusted for maternal smoking status.
METHODS: In this study, using 344 maternal blood samples from the second trimester of pregnancy, circulating cell-free DNA in maternal plasma samples, specific for the SRY and DYS14 loci (representing fetal DNA) and GAPDH sequence (representing total genomic DNA) were quantified by real-time polymerase chain reaction.
RESULTS: Fetal sex determination was 100% accurate using a combination of probes for SRY and DYS14. The levels of DYS14 and SRY detected were significantly correlated (r=0.884, P<.001). No significant difference was seen between the quantitative levels of cell-free male fetal DNA between the smoking groups and control group. Similarly, no significant difference was seen in the amount of total cell-free DNA in the study population.
CONCLUSION: In contrast to first- and second-trimester screening assays for Down syndrome, where smoking status significantly affect levels of maternal serum analytes, smoking status does not affect quantitative levels of cell-free fetal DNA or total cell-free DNA in maternal plasma.
LEVEL OF EVIDENCE: II
Circulating cell-free nucleic acids in plasma and serum are novel biomarkers with promising clinical applications in different medical fields, including prenatal diagnosis1,2 and oncology.3,4 The levels of cell-free DNA have been found to be elevated in acute medical emergencies, including trauma and stroke,5,6 and act as indicators of disease severity7
The discovery of cell-free fetal DNA in maternal blood created a new prospective in prenatal diagnosis. Trafficking of fetal cells and cell-free fetal DNA into the maternal circulation provides promising clues to the underlying physiology and to potential pregnancy-associated pathologies during all trimesters.8,9
Elevated fetal DNA concentrations have been found in several pregnancy-associated pathologies, including hyperemesis gravidarum,10 premature delivery,11 aneuploidies,12 and preeclampsia.13 Smoking is a highly important modifiable risk factor associated with adverse pregnancy outcomes.14 In populations with a high prevalence of smoking during pregnancy, it has been estimated that cessation during pregnancy could prevent 10% of perinatal deaths, 35% of low birth-weight births, and 15% of preterm deliveries.15 Smoking has been shown to influence the levels of maternal serum analytes currently used in prenatal screening for Down syndrome in the first and second trimester, lowering the levels of PAPP-A, unconjugated estriol, and β-hCG (free and total) and raising AFP and inhibin A levels, compared with nonsmokers.16–18 Maternal serum hCG may be reduced by up to 11–29% in euploid fetuses and up to 27–39% in affected pregnancies.19 In this large scale study to address the effect of smoking on cell-free fetal DNA, we sought to estimate whether potential clinical applications of cell-free fetal and total DNA in the field of noninvasive prenatal diagnosis need to be adjusted for maternal smoking status.
MATERIALS AND METHODS
Approval for this study was obtained from the institutional review boards of the University of Basel, Switzerland, and the University of Greifswald, Germany. Between 2004 and 2006, 344 plasma samples were collected from low-risk pregnant women with singleton fetuses in the second trimester between 20 and 21 completed weeks of gestation at the Department of Obstetrics and Gynecology, University of Greifswald, Germany, after written informed consent. Samples were stored at –80°C before shipment on dry ice to the University of Basel for further analysis. Gestational age was assessed by transabdominal ultrasonography, using embryonal crown-rump length measurements during the first trimester between 11+3 and 13+6 weeks of gestation.
Maternal smoking status was assessed by self-reporting at counseling in the second trimester.
Pregnancy outcome and patient characteristics were subsequently taken from the patients’ clinical records. The degree of current maternal smoking was defined as smoking equal to or more than ten cigarettes a day, less than ten cigarettes per day, and nonsmoking.
The nonsmoking patients constituted the control group (n=260), whereas the smoking group included 58 patients who smoked less than ten cigarettes per day and 26 who smoked equal to or more than ten cigarettes per day.
Plasma DNA was extracted from 800 μL of plasma using the High Pure polymerase chain reaction (PCR) template kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. The DNA was eluted into 80 μL of elution buffer, of which 5 μL was used as template for the PCR reaction.
Cell-free fetal DNA and total DNA in the maternal plasma were analyzed by TaqMan real-time quantitative PCR using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Rotkreuz, Switzerland). The quantities of male fetal DNA were determined using the multiplex TaqMan assay for both the SRY and DYS14 sequences as described previously.20 The total levels of cell-free DNA, which represent the levels of cell-free maternal and fetal DNA, were determined by amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locus, which is present in all genomes, using primers and probes as described previously.21
The TaqMan assays were carried out in 25 μL of total reaction volume. Cycling conditions for all reactions consisted of a 2-minute incubation at 50°C to allow for UNGerase activity, an initial denaturation step of 95° for 10 minutes, and then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute.21 To determine the genome equivalents of male DNA and total maternal and fetal DNA present in the plasma, a standard dilution curve using known concentrations of male genomic DNA was used. For the conversion to genome equivalents, 6.6 pg was used, as described previously.22
To compare the parameters of SRY, DYS, and GAPDH levels among the study groups, a one-way analysis of variance with the three levels “control”, “less than 10”, and “10 or more” was performed. To achieve approximately normal distribution, SRY and DYS were square-root transformed, and GAPDH was log transformed. The distribution assumptions were checked using quantile plots.
The degree of correlation between the levels of fetal SRY and DYS14 detected were examined by using Pearson’s correlation coefficient. The medians in the two figures are indicated by a line inside each box, the 75th and 25th percentiles by the box limits, and the upper and lower error bars the 10th and 90th percentiles, respectively. All analyses were performed using SPSS 12.0 (SPSS Inc., Chicago, IL). A P<.05 was considered significant.
In this study, the amount of cell-free fetal DNA was measured in all of the 344 blood samples, including 132 patients with male fetuses in the control and 32 women with male fetuses in the smoking group. Patient characteristics are listed in Table 1. No significant difference in sex distribution was found among the groups. The real-time PCR experiments, using probes for SRY and DYS14, achieved a 100% accuracy for the identification of fetal sex. As expected, DYS14, being a multi-copy locus on the Y chromosome, was detected at 3- to 4.9-fold higher levels among all groups than SRY (Fig. 1).16 DYS14 and SRY were significantly correlated (r=0.884, P<.001).
No significant difference was seen between the quantitative levels of cell-free male DNA using SRY (P=.33) and DYS14 (P = .56) among the three different groups. Therefore, no subsequent comparisons among the three levels were performed (Fig. 1 and Table 1).
Comparing the levels of total maternal and cell-free fetal DNA, as measured by amplification of the GAPDH locus, no significant difference was found among the groups, P=.49. The control group, as well as the group in which the subjects smoked less than ten cigarettes a day, showed similar median values (1,742.0 genome equivalents [range 388–15,571] compared with 2,175.0 genome equivalents [range 433–16,730] (P=.24), whereas the group in which the women smoked 10 or more cigarettes a day showed a slightly lower median value (1,359.5 genome equivalents [range 447–48,903], P=.76, Fig. 2).
Fetal weight did not differ statistically between both smoking groups (3,320 g compared with 3,444 g in those smoking less than 10 cigarettes per day and those smoking 10 or more cigarettes per day, respectively (P=.34), and the control group (3,366 g P=.36).
Preeclampsia, according to the World Health Organization criteria (hypertension more than 140/90 mm Hg, increase of the systolic or diastolic blood pressure more than 30 mm Hg and 15 mm Hg, respectively, on two occasions at least 6 hours apart, in combination with proteinuria [more than 300 mg /24 hours]) was observed in 13 patients of the control group (5%) compared with three women in the smoking group (3.5%). This difference was not statistically significant (P=.18).
Trafficking of fetal cells and cell-free fetal DNA into the maternal circulation provides promising clues to the underlying physiology and to potential pregnancy associated pathologies during all trimesters.23–25 Most investigators are in agreement that the placenta is the predominant source of the circulating fetal DNA. This hypothesis is supported by the fact that fetal DNA and hCG concentrations are strongly correlated.26 Furthermore, direct evidence regarding the source of cell-free fetal DNA comes from the fact that placental-specific mRNA molecules are readily detectable in maternal plasma.27 Many studies have demonstrated that fetal cell-free DNA levels in maternal plasma are elevated in different clinical circumstances, such as fetal trisomy 21, hyperemesis gravidarum, or preeclampsia.28 Most of these studies have compared DNA values in affected pregnancies with so-called “normal controls.” However, other clinical variables may influence quantitative cell-free DNA levels in maternal plasma/serum, since maternal body mass index, ethnic background, and smoking status significantly affect levels of maternal serum analytes in maternal serum screening assays for the detection of aneuploidies in the first and second trimesters.29 Therefore, we hypothesized that cell-free DNA levels may be similarly affected by maternal smoking status. This large-scale analysis of circulating cell-free fetal and total DNA concentrations in pregnancies from smoking compared with nonsmoking women using two Y chromosome-specific real-time PCR assays and GAPDH locus, which is present in all genomes, showed no difference in the quantitative levels of cell-free fetal and total cell-free DNA in the maternal circulation, similar to the preliminary results of Wataganara et al30 which showed no correlation between maternal smoking status and cell-free DNA levels in the first and second trimesters.30 In contrast to this study,30 where the degree of smoking was not defined, we subdivided the smoking population into two subgroups, in which the women smoked 10 or more cigarettes per day or less than 10 cigarettes per day. Furthermore, our study included a large number of samples from the second trimester, whereas the previous study included 33 first-trimester and 10 second-trimester samples from smokers.30 In this study, maternal smoking status was assessed by self-reporting at counseling in the second trimester. It has been repeatedly questioned in the literature how reliably maternal smoking status can be assessed. The findings have been highly variable: some studies showed a considerable rate of nonreporting or underreporting of smoking by pregnant women,31,32 while other large-scale studies concluded that self-reporting by pregnant women was reliable when compared with biochemical or biophysical measures.18,33
Our findings that there is no significant difference between total cell-free and fetal cell-free DNA in the maternal circulation may be explained by the preexisting high amounts of placental material physiologically shed into the maternal circulation. A calculation of villous cytotrophoblast proliferation, syncytial fusion, and villous growth during the third trimester leads to the conclusion that several grams of trophoblast material are shed per day into the maternal circulation by term.34,35 It is important to note that the released material is physiologically packed into tightly sealed syncytial knots, thus preventing an inflammatory response by the mother’s blood vessels and organs. Although clinical experience suggests that the placentas of mothers who smoke are generally small in size, various epidemiological studies indicate that placental weight may be increased,36 decreased,37 or unchanged.38 In our study, the larger standard deviations of total cell-free DNA in the smoking groups compared with the nonsmoking group may suggest some evidence of possible tissue damage. Maternal smoking has minimal effects on fetal growth, with reductions in birth weight of only 90-200 g.39 This is in agreement with our data showing no statistical difference in birth weight among the groups. There is increasing evidence in the literature that smoking decreases the risk of preeclampsia.40 Different mechanisms have been implicated in smoking’s beneficial effect on the onset of preeclampsia, including the effects of nicotine and carbon monoxide.41,42 However, in our study, 5% of the control group compared with 3.5% in the smoking group developed preeclampsia, which was not a statistically significant finding.
In conclusion, maternal smoking status does not alter quantitative levels of cell-free fetal and total DNA. Therefore, this does not restrain their potential clinical applications in noninvasive prenatal diagnosis. This is in contrast to maternal serum screening for Down syndrome in the first and second trimesters, in which maternal smoking has a significant effect on the results, therefore making risk adjustments essential.
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