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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