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Letters to the Editor |
Departments of1
Obstetrics and Gynecology and2
Human Genetics Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
3 CREST, Japan Science and Technology Agency, Kawaguchi, Japan
aAddress correspondence to this author at: Department of Obstetrics and Gynecology, Graduate School of Biomedical Sciences, Nagasaki University, 1-7-1 Sakamoto, Nagasaki 852-8501, Japan. Fax 81-95-849-7365; e-mail kiyonori{at}net.nagasaki-u.ac.jp.
To the Editor:
Both maternal cells and maternal DNA are often present in the umbilical cord blood (UCB) (1). When UCB is used for bone marrow transplantation (2), the presence of maternal cells in UCB plasma is a theoretical risk factor for graft-vs-host disease and may lead to vertical transfer of infectious agents to a fetus. Previous studies showed that elective cesarean section (C/S) reduces these risks (3). Placental alkaline phosphatase is higher in cord blood of cases with labor than those without it (4), suggesting that labor increases transplacental maternal-fetal microtransfusion. As maternal DNA was found in only 75% of UCB plasma samples (5), we aimed to determine whether labor itself influences the frequency of maternal DNA contamination in UCB.
We studied 97 pregnant women with normal pregnancies. In group A (19 women), C/S was performed after the beginning of labor (labor with C/S); in group B (44 women), C/S was performed before labor (nonlabor with C/S); and group C (34 women) had normal vaginal delivery (labor with V/D). Group C did not have augmentation of labor, fundal pressure, or forceps/vacuum delivery. No statistical difference was observed among the three groups in clinical variables, such as maternal age, gestational age, gravidity, and birth weight of a newborn. None had a past history of placental abruption, preeclampsia, placenta previa, or infants with intrauterine growth retardation.
Soon after delivery, the umbilical cord was cleaned with saline, and UCB was collected from the umbilical vein between two clamped sites. Maternal blood was obtained at 1 month after delivery and stored at 4 °C. UCB was centrifuged once at 3000g, the plasma was stored at –20 °C, and the DNA was extracted. Whole-blood DNA was also extracted from mothers’ blood and UCB. The study protocol was approved by the Ethical Committee on Human Genome/Gene Analysis, Nagasaki University, and informed consent was obtained from all women.
We developed a set of nine tetra-repeat markers to detect maternal DNA in the Japanese. Forward primers for D21S11, D18S535, D7S1830, D4S1645, and D3S1359 were labeled with 6-carboxyfluorescein (green), and those for D11S2365, D8S1132, D3S2465, and FGA were labeled with hexachloro-6-carboxyfluorescein (yellow). Genotyping was performed by PCR in a 20-µL mixture containing 5 µL of DNA, 10 pM fluorescent primers, 250 mM deoxynucleotide triphosphates, 0.5 U of AmpliTaq Gold DNA polymerase [Applied Biosystems (ABI)], and 2 µL of 10x PCR buffer (ABI), using GeneAmp PCR System 9700 (ABI). PCR was cycled 40 times at 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s. PCR products were genotyped with an ABI 377 DNA sequencer and GeneScan 3.1.2 and Genotyper 2.5 software (ABI). Gradient dilution experiments (male DNA was gradually diluted in female DNA) showed that the assay could detect male-specific DNA in a female DNA background at 10–100 copy numbers of male DNA in a background of 100 000 copy numbers of female DNA (6). When maternal DNA had two alleles (heterozygous mother), and one of them was not shared by UCB (fetal) cellular DNA, we defined this allele as informative. Maternal DNA in UCB was judged to be present when an informative allele was found for at least two different loci and by at least triplicate experiments.
The detection rate (16 of 19; 84.2%) in group A was significantly higher than that (20 of 44; 43.2%) in group B (
2 = 9.05; P <0.005). Likewise, the rate (31 of 34; 91.2%) in group C was statistically higher than that in group B (2 = 19.2; P <0.0001). However, there was no significant difference in the rate between groups A and C (P >0.5).
The different rates of detection of maternal DNA in UCB plasma between the labor (with C/S or V/D) and nonlabor groups are most likely attributed to labor itself. Although the actual source(s) of maternal DNA in UCB plasma remains unknown, it is plausible that some maternal DNA in plasma passes through the placenta (4), and this may be increased by a mechanical force produced by uterine contraction, which could force placental blood into the umbilical cord. Our results suggest the possibility of trafficking of maternal cells into UCB during labor. Because such maternal DNA contamination may suggest the presence of microchimerism in UCB (7), the results are relevant to the use of cord blood for bone marrow transplantation and may give an insight into the mechanism of fetomaternal circulation and vertical transmission of infectious agents.
Acknowledgments
This work was supported in part by Grants-in-Aid for Scientific Research (15591761, 16591670, and 13854024) from the Ministry of Education, Sports, Culture, Science and Technology of Japan and CREST from the Japan Science and Technology Agency (JST).
Footnotes
1 These authors contributed equally to this work. 
References
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