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Technical Briefs |
Departments of
1
Chemical Pathology and
2 Obstetrics and Gynaecology, and
3
Center for Clinical Trials and Epidemiological Research, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR
aauthor for correspondence: fax 852-2194-6171, e-mail loym{at}cuhk.edu.hk
The discovery of fetal DNA in maternal plasma/serum has suggested new possibilities for noninvasive prenatal diagnosis (1) with applications in investigations of sex-linked diseases (2), rhesus D status (3), and ß-thalassemia (4). Biologically, fetal DNA concentrations have been found to increase with gestational age, with a sharp increase toward the end of pregnancy (5). This latter phenomenon has been postulated to be related to the imminence of delivery (6). Consistent with the latter hypothesis, women with preterm labor have been found to have a higher concentration of fetal DNA in their plasma (7)(8). These results suggest that circulating fetal DNA concentrations are undergoing a time of flux during the late third trimester, but detailed information on the dynamics of maternal plasma fetal DNA is not available for this stage of gestation. In this study, we performed a serial analysis of fetal DNA concentrations in maternal plasma during the late third trimester.
We recruited 33 healthy pregnant women from the Department of Obstetrics and Gynaecology, Prince of Wales Hospital, Hong Kong. All had ultrasonic evidence of male fetuses. Fetal sex was confirmed at delivery. Ethics approval was obtained from the Clinical Research Ethical Committee of the Chinese University of Hong Kong. Informed consent was obtained in all cases. Blood samples (in EDTA tubes) were taken from each of the women at 35 weeks of gestation. Serial samples (in EDTA tubes) were also collected from 26 of the women from 35 to 42 weeks or until delivery. All of these women were free of pregnancy complications in the index pregnancies.
The blood samples were centrifuged at 1600g for 10 min (Megafuge 1.0R; Heraeus Instruments), and plasma was carefully transferred into polypropylene tubes without disturbing the buffy coat layer. The plasma was recentrifuged at 16 000g for 10 min (Eppendorf Centrifuge 5415D), and the cell-free supernatant was collected into sterile microcentrifuge tubes. The plasma samples were stored at -20 °C until further analysis. DNA was extracted from 800 µL of plasma with the QIAamp Blood Kit (Qiagen) according to the "blood and body fluid spin protocol" as recommended by the manufacturer (5).
Real-time quantitative PCR was carried out for the SRY (as a fetus-specific marker) and ß-globin (as a marker for total DNA) genes as described previously (5). We used 5 µL of plasma DNA as a template for the amplification reaction, performed in an ABI Prism 7700 Sequence Detector (Applied Biosystems). Each sample was analyzed in duplicate, and the mean results were used for further calculations. A calibration curve was included in each assay. A conversion factor of 6.6 pg of DNA per cell was used for expressing the results as genome-equivalents (5). Plasma DNA concentrations were expressed as genome-equivalents/mL (5).
The median concentration of the SRY gene in maternal plasma was 74 genome-equivalents/mL (interquartile range, 53–141 genome-equivalents/mL). We observed a significant positive correlation between the concentration of the SRY gene and gestational age (Spearman rank-order correlation, r = 0.22; P = 0.04).
The concentration of ß-globin sequences in maternal plasma samples represented the total amount of extracted DNA, both maternal and fetal. The median concentration of the ß-globin gene was 668 genome-equivalents/mL (interquartile range, 491-1049 genome-equivalents/mL). There was no statistically significant correlation between the ß-globin concentrations and the gestational age (Spearman rank-order correlation, r = 0.175; P = 0.099). We found a positive correlation between the SRY and ß-globin DNA concentrations in maternal plasma (Spearman rank-order correlation, r = 0.608; P <0.0005).
To obtain an objective and quantitative assessment of the influence of gestational age on plasma DNA concentrations, we performed linear regression analysis on the fetal DNA concentrations and gestational age for all patients with more than two serial measurements of plasma fetal DNA. Because there is a wide interindividual variation in absolute fetal DNA concentrations, the change in the fetal DNA concentration was expressed relative to the first measured value from each individual before we performed linear regression analysis. This approach can avoid biasing the mean slopes toward those cases with high absolute concentrations. The mean slope of 0.293 was statistically different from 0 (Student t-test, P <0.001). We therefore conclude that the fetal DNA concentration in maternal plasma is positively correlated with gestational age during the late third trimester, with a mean increase of 29.3% each week during this gestational period. The slopes of the plasma fetal DNA concentrations of all individuals are presented in Table 1
, and the plots of the linear regression analyses are presented in Fig. 1, which is available in the Data Supplement accompanying the online version of this Technical Brief at http://www.clinchem.org/content/vol49/issue4/.
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This study provided detailed dynamic information on the variation of plasma fetal and total DNA concentrations during the late third trimester of pregnancy. The results provide normative values to which values in pathologic conditions, e.g., preterm labor and preeclampsia, can be compared. Previous results have indicated a quantitative aberration in circulating fetal DNA concentrations in these conditions (7)(9); it would thus be relevant to investigate whether there is also an abnormality in the rate of change of circulating DNA concentrations in these conditions.
The biological basis for the observed changes in maternal plasma fetal and total DNA concentrations also requires further investigation. The cellular origin of the increase in total plasma DNA, predominantly maternal in origin (5), is unclear at present. The positive correlation between the circulating fetal and total plasma DNA (predominantly maternally derived) concentrations suggests that the release of fetal and maternal DNA into the plasma and/or the clearance of these DNA species is/are somehow related. One possibility is that both of these DNA species may arise from cells at the fetomaternal interface. For example, fetal DNA may be released into the plasma by the trophoblasts, whereas maternal DNA may be released by the decidua into the circulation. Future research into these concepts will be expected to provide an improved understanding of the fetomaternal relationship.
Acknowledgments
Y.M.D.L. is supported by the Hong Kong Research Grants Council and the Innovation and Technology Fund (AF/90/99 and ITS/195/01). Supplementary graphs are available online (at http://www.clinchem.org/content/vol49/issue4/), showing the gestational variation of each of the cases.
References
The following articles in journals at HighWire Press have cited this article:
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  A. Yin, E.H.Y. Ng, X. Zhang, Y. He, J. Wu, and K.Y. Leung Correlation of maternal plasma total cell-free DNA and fetal DNA levels with short term outcome of first-trimester vaginal bleeding Hum. Reprod., June 1, 2007; 22(6): 1736 - 1743. [Abstract] [Full Text] [PDF]
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 M. L. Tjoa, T. Cindrova-Davies, O. Spasic-Boskovic, D. W. Bianchi, and G. J. Burton Trophoblastic Oxidative Stress and the Release of Cell-Free Feto-Placental DNA Am. J. Pathol., August 1, 2006; 169(2): 400 - 404. [Abstract] [Full Text] [PDF]
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 F. Z. Bischoff, D. E. Lewis, and J. L. Simpson Cell-free fetal DNA in maternal blood: kinetics, source and structure Hum. Reprod. Update, January 1, 2005; 11(1): 59 - 67. [Abstract] [Full Text] [PDF]
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 H. M. Muller, L. Ivarsson, H. Schrocksnadel, H. Fiegl, A. Widschwendter, G. Goebel, S. Kilga-Nogler, H. Philadelphy, W. Gutter, C. Marth, et al. DNA Methylation Changes in Sera of Women in Early Pregnancy Are Similar to Those in Advanced Breast Cancer Patients Clin. Chem., June 1, 2004; 50(6): 1065 - 1068. [Full Text] [PDF]
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