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Letters to the Editor |
Departments of1
Obstetrics, and Gynecology, and 2
Human Genetics, Nagasaki University Graduate, School of Biomedical Sciences, Nagasaki, Japan
3 Solution Oriented Research for Science and Technology (SORST), Japan Science and, Technology Agency (JST), Kawaguchi, Japan;
4 Sasebo Chuo Hospital, Sasebo, Japan
5 Department of Pediatrics, Clinical Genetics Program, Carolinas Medical Center, Charlotte, NC
aAddress correspondence to this author at: Department of Obstetrics and Gynecology, Nagasaki University Graduate School of Biomedical Sciences, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Fax 81-95-849-7365; e-mail kiyonori{at}nagasaki-u.ac.jp.
To the Editor:
Detection of circulating fetal nucleic acids in maternal plasma is a promising approach to noninvasive prenatal assessment and fetal monitoring. Cell-free fetal/placental (cff) DNA concentrations in maternal plasma undergo transient increases after amniocentesis, suggesting that cff DNA in maternal plasma is a sensitive marker for fetal-maternal hemorrhage (1). However, cff DNA measurement is a sex-dependent marker used only in pregnancies with male fetuses, whereas cell-free placental (cff) mRNA in maternal plasma, recently quantified with real-time reverse-transcription PCR, is considered a sex-independent marker for monitoring placental conditions (2). Evidence suggests that cff mRNA release into the maternal circulation is comparable to cff DNA release, but it is not known which molecular marker is more sensitive for detection of fetal-maternal hemorrhage. To investigate this question, we performed a quantitative analysis of cff DNA and cff mRNA in maternal plasma collected before and 10-min after amniocentesis and evaluated which molecular marker was more sensitive for detection of fetal-maternal hemorrhage.
Study participants were 32 pregnant women, at 15–17 weeks of gestation, who underwent amniocentesis at the Department of Obstetrics and Gynecology at Nagasaki University hospital. All participants gave written, informed consent, and the study was approved by the Research Ethics Committee of Nagasaki University. Blood samples were obtained from each woman before and 10 min after amniocentesis. We collected 8-mL samples into EDTA tubes and stored them at –20 °C until use. We performed karyotype analysis with G-banding on all samples, and on the basis of these results we selected 15 mothers with euploid male fetuses for this study.
We selected the hemoglobin ß gene (HBB) and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as targets to monitor changes in the total amount of maternal plasma cff DNA and cff mRNA before and after amniocentesis, respectively. To determine the amount of cff DNA and cff mRNA in maternal plasma, we used real-time PCR to measure the nucleotide sequence of deoxyphypusine synthase (DHPS) cff DNA, which is specific for the Y chromosome, and that of chorionic gonadotropin, ß polypeptide 7 (CGB7) mRNA. We then performed PCR analysis with an ABI 7900T Sequence Detector (Perkin-Elmer) to measure the cff DNA and cff mRNA concentrations in maternal plasma. The amount of cff DNA was measured with real-time PCR and of cff mRNA with 1-step real-time reverse-transcription PCR. Primer sequences and PCR conditions for the quantification analysis of plasma cff DNA and cff mRNA were as described in (3) and (2), respectively. We used Wilcoxon signed-rank tests to compare changes in the concentrations of cell-free nucleic acids before and after amniocentesis. Significant differences were defined as P <0.05.
Quantitative data for cell-free DNA in maternal plasma are shown in Fig. 1
, A and B. The mean concentrations of DHPS were 49.0 genome equivalents/mL before and 90.7 genome equivalents/mL after amniocentesis (P = 0.0106). Also, total plasma concentrations of cff DNA increased after amniocentesis (P = 0.0007). Our data regarding changes in cff DNA concentrations after amniocentesis were consistent with those reported previously (1). On the other hand, there was no substantial difference in the mean concentrations of cff CGB7 mRNA and cellfree GAPDH mRNA before and after amniocentesis (P = 0.0535 and P = 0.1118, respectively) (Fig. 1
, C and D).
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Recent evidence suggests that both cff DNA and cff mRNA in maternal plasma originate from the placenta (2)(4)(5). We found, however, that in maternal plasma cff DNA increased after amniocentesis but cff mRNA remained unchanged. The absence of change in cff mRNA concentrations before and after fetal and placental intervention was previously reported (6). Filtration of maternal plasma with a 0.22-µm filter led to a marked decrease of plasma cff GAPDH mRNA, but no change in plasma cff HBB-derived DNA (7), suggesting that cff DNA is smaller than cff mRNA. Furthermore, the 2 different nucleic acids are packaged and metabolized differently and may be released differently after tissue injury (7), possibly leading to the discrepancy between changes of cff DNA and cff mRNA concentrations after amniocentesis.
In conclusion, the concentration of cff DNA entering the maternal circulation was affected by amniocentesis, but that of cff mRNA was not changed. These data show that cff DNA rather than cff mRNA is a suitable molecular marker for assessment of fetal-maternal hemorrhage. Our results also suggest that plasma cff DNA and cff mRNA may be transferred into the maternal circulation by different mechanisms.
Acknowledgments
We thank Drs. Atsushi Yoshida, Takako Shimada, Terumi Tanigawa, Yuriko Kitajima, and Yoko Fujimoto for their help and valuable advice. H.M. and N.N. were supported in part by Grants-in-Aid for Scientific Research (Nos. 17591748 and 16390101, respectively) from the Ministry of Education, Sports, Culture, Scienceand Technology of Japan, and N.N. was supported by SORST from Japan Science and Technology Agency.
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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