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Articles |
Departments of
1
Chemical Pathology and
2
Obstetrics and Gynecology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR.
a Author for correspondence. Fax 852-2194-6171; e-mail loym{at}cuhk.edu.hk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods: The fetus-to-mother transfer of nucleated cells and plasma DNA in pregnant women carrying male babies was studied using a real-time quantitative PCR assay for the SRY gene. For mother-to-fetus transfer, real-time quantitative PCR assays for the insertion/deletion polymorphisms involving the glutathione S-transferase M1 and angiotensin-converting enzyme genes were used.
Results: Of the 50 informative mother-baby pairs, maternal DNA was detected in the cellular fraction of umbilical cord blood in 24% of cases (12 of 50), at a median fractional concentration of 2.6 x 10-4 (interquartile range, 1.7 x 10-4 to 3.6 x 10-4). In the plasma fraction of cord blood, maternal DNA was detected in 30% (15 of 50) of cases at a median fractional concentration of 3 x 10-3 (interquartile range, 1 x 10-3 to 1.6 x 10-2). For the other direction of trafficking, fetus-to-mother transfer of nucleated cells was detected in 26% of cases (13 of 50) at a median fractional concentration of 3.2 x 10-4 (interquartile range, 0.6 x 10-4 to 7.6 x 10-4). In the plasma fraction, fetal DNA was detected in 100% of maternal plasma (50 of 50) at a median fractional concentration of 3 x 10-2 (interquartile range, 1.4 x 10-2 to 5.3 x 10-2).
Conclusions: This study indicated that significantly more fetal DNA is present in the plasma of pregnant women compared with DNA from the cellular fraction of maternal blood. In addition, maternal DNA was demonstrated in both the cellular and plasma fractions of cord blood after delivery. This study has therefore determined the fundamental quantitative values for the bidirectional fetomaternal cellular and plasma DNA traffic.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The reverse transfer of nucleated maternal cells into the fetal circulation has also been demonstrated (1)(14)(15)(16). This phenomenon has initially attracted attention because of the recent interest in the use of umbilical cord blood for bone marrow transplantation (17). The potential existence of maternal immunocompetent cells or their precursors in cord blood has been postulated to constitute a theoretical risk of graft-vs-host disease (18). Immunologically, the passage of maternal cells into the fetal circulation during pregnancy may also contribute to the observed partial immunological tolerance to the noninherited maternal HLA types (19)(20).
Recently, in addition to the fetomaternal trafficking of nucleated cells, plasma DNA transfer from the fetus to the mother has also been demonstrated (21). The concentration of fetal-derived plasma DNA has been shown to be several orders of magnitude higher than that of fetal nucleated cells per volume of maternal blood (22). In addition, the clearance kinetics of fetal cells and plasma DNA appear to be very different, with plasma fetal DNA being cleared much more rapidly (23) than fetal nucleated cells (24).
Several questions remain unanswered regarding the fetomaternal transfer of nucleated cells and plasma DNA. First, the possibility of plasma maternal DNA transfer into the fetal circulation in humans remains unexplored. Second, the relationship, if any, between the degree of fetomaternal transfer of nucleated cells and plasma DNA remains unknown. Third, most of the published data on the trafficking of maternal cells into the fetal circulation are qualitative in nature, with relatively little quantitative information. The recent development of real-time quantitative PCR (22)(25), with its high accuracy and wide dynamic range, has allowed us to undertake the present study to address these important questions. To maximize the informativeness of our study, we explored this bidirectional and bimodal (i.e., nucleated cells and plasma DNA) fetomaternal transfer in the same patient cohort.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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processing of blood samples
Blood samples were centrifuged at 1600g, and plasma was
carefully removed and transferred into plain polypropylene tubes. Great
care was taken to ensure that the buffy coat was undisturbed when
plasma samples were removed. The plasma was recentrifuged at
3000g, and the supernatants were collected into fresh
polypropylene tubes. The plasma and buffy coat samples were stored at
-20 °C until further processing.
dna extraction from plasma and buffy coat samples
DNA from plasma samples was extracted with a QIAamp Blood Kit
(Qiagen) using the "blood and body fluid protocol" (1997
version) as recommended by the manufacturer (22). Eight
hundred microliters of plasma sample was used for DNA extraction per
column. An elution volume of 50 µL was used. Buffy coat DNA was
extracted using a Nucleon DNA Extraction Kit from Scotlabs according to
manufacturer’s recommendations.
fetomaternal genotyping using conventional pcr
For detection of the transfer of nucleated cells and plasma DNA
from the fetus to the mother, the SRY gene that is present
on the Y-chromosome of a male fetus was used as a fetal marker
(22). For the detection of the transfer of nucleated cells
and plasma DNA from the mother to the fetus, two polymorphic markers
were used, namely, the insertion/deletion polymorphism involving the
glutathione S-transferase M1 (GSTM1) gene
(1)(26)(27) and the
insertion/deletion polymorphism involving intron 16 of the
angiotensin-converting enzyme (ACE) gene
(1)(28). Fetomaternal pairs informative for
these systems were those in whom the mother possessed an allele not
present in the fetus.
For the GSTM1 locus, 
50% of the population did not
possess the gene (27). Mother-baby pairs informative for
this locus would have the mother possessing the GSTM1 gene
and the baby possessing the null allele. GSTM1 genotyping of
fetomaternal pairs was carried out using a low-sensitivity system based
on conventional non-real-time PCR, using reagents from a GeneAmp DNA
Amplification Kit (PE Biosystems). The primer sequences are listed in
Table 1
. GSTM1 genotyping was carried out in 100-µL
reaction volumes using 100 ng of maternal or cord buffy coat DNA in a
mixture containing 1.5 mmol/L MgCl2, 12.5 pmol of
each of the primers GST-194F and GST-273R-2 (Table 1
), 0.6 U of
AmpliTaq Gold polymerase, and 200 µmol/L each of dATP, dGTP, dCTP,
and dUTP. The reaction mixture was initially denatured at 95 °C for
12 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for
30 s, and 72 °C for 1 min, and ended with a final extension at
72 °C for 5 min using a PE Biosystems model 480 thermocycler. On
agarose gel electrophoresis, DNA from individuals possessing a
GSTM1 gene would produce an 80-bp PCR product that was
absent in GSTM1-null homozygotes.
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For the insertion/deletion polymorphism involving a 287-bp fragment in
intron 16 of the ACE gene, mother-baby pairs would be
informative if the baby was homozygous for either the insertion (i.e.,
II) or deletion (i.e., DD) alleles, and
the mother was heterozygous (ID). Fetomaternal genotyping
was carried out using a low-sensitivity system involving primers ACE-P1
and ACE-P2 (Table 1
). PCR was carried out in 100-µL reaction volumes
using 100 ng of buffy coat DNA isolated from maternal and cord blood,
as for the GSTM1 system, except that
MgCl2 was kept at 3 mmol/L. Thermocycling was
initiated with a 12-min incubation at 95 °C, followed by 35 cycles
of 94 °C for 1 min, 58 °C for 1 min, and 72 °C for 1 min, and
ended with a final extension at 72 °C for 5 min. On agarose gel
electrophoresis, the insertion and deletion alleles were indicated by
479- and 191-bp PCR products, respectively.
real-time quantitative pcr
For the detection of the fetomaternal transfer of nucleated cells
and plasma DNA, real-time quantitative PCR was used. Real-time
quantitative PCR was based on the continuous optical monitoring of the
progress of an amplification reaction that was coupled to the
liberation of a fluorescent reporter (22)(25).
The fluorescent reporter was released when the 5'-to-3' exonuclease
activity of the Taq polymerase (29) cleaved it
from the oligonucleotide probe hybridizing to the PCR product
(30). Fluorescent signals were captured in real-time using a
PE Biosystems 7700 Sequence Detector (PE Biosystems).
Real-time quantitative PCR systems for the SRY,
GSTM1, and ACE genes were used. The
SRY system has been described in detail previously
(22) and was used to detect the transfer of nucleated cells
and plasma DNA from a male fetus into the maternal blood. Real-time PCR
was set up with the primers SRY-109F and SRY-245R and the fluorogenic
probe SRY-142T. The primer and probe sequences are listed in Table 1
.
Amplification reagents were obtained from a TaqMan PCR Core Reagent Kit
(PE Biosystems). Detailed reaction setup conditions were as described
previously (22). dUTP and
uracil-N-glycosylase were included in the reaction
mixture as an anticontamination measure
(22)(31). The target consisted of either 500 ng
of buffy coat DNA or 5 µL of plasma DNA (out of a total elution
volume of 50 µL). Each assay was carried out in duplicate, and the
mean results were used for further analysis. For quantitative
measurement, a calibration curve was constructed using DNA from a
healthy male individual. A conversion factor of 6.6 pg of DNA per cell
was used for expressing the results as genome-equivalents
(32). One genome-equivalent was defined as the amount of
SRY sequence present in one diploid male cell. Serial
dilution experiments indicated that the SRY system was able
to detect a minimum of 1 genome-equivalent of target.
The GSTM1 and ACE systems were used to detect the
transfer of maternal nucleated cells and plasma DNA into the baby’s
circulation. The real-time GSTM1 system consisted of the
primers GST-194F and GST-273R-2 and the fluorogenic probe GST-215T
(Table 1
). Reaction setup conditions were as those for the
SRY system, with the target consisting of either 500 ng of
buffy coat DNA or 5 µL of plasma DNA. The amplification conditions
consisted of an initial incubation at 50 °C for 2 min (for the
uracil-N-glycosylase to act), followed by incubation at
95 °C for 10 min, and 40 cycles of 95 °C for 15 s and
56 °C for 1 min. Each assay was carried out in duplicate, and the
mean results were used for further analysis. For quantitative
measurement, a calibration curve was constructed using DNA from an
obligate GSTM1 heterozygote (i.e., a
GSTM1-positive mother of a GSTM1-null baby)
possessing one copy of the target GSTM1 gene. For the
purpose of conversion to genome-equivalent units, one cell from this
obligate GSTM1 heterozygote was defined as possessing 0.5
genome-equivalent of the GSTM1 sequence. Serial dilution
experiments indicated that the GSTM1 system was able to
detect a minimum of 1 genome-equivalent of target.
Two real-time PCR systems were constructed for the ACE gene:
one for the insertion allele and the other for the deletion allele. The
assay for the insertion allele involved the primers ACE-1721F and
ACE-1826R and the fluorogenic probe ACE-1745T (Table 1
). For the
deletion allele, the primers ACE-1428F and ACE-1826R and the
fluorogenic probe ACE-1745T (Table 1
) were used. In addition, further
allele specificity for the deletion allele was conferred by the
digestion of the insertion allele using the restriction enzyme
TaqI (10 U for 500 ng of buffy coat DNA or 5 µL of
extracted plasma DNA for 2 h at 65 °C; Life Technologies)
before DNA amplification (1). One unit of TaqI
was also included in each real-time PCR reaction to cleave any residual
DNA containing the insertion allele and any PCR product amplified from
the insertion allele. The thermal profile was same as that used for the
GSTM1 system, with 40 cycles being used for the
ACE insertion system and 50 cycles being used for the
deletion system. Each assay was carried out in duplicate, and the mean
results were used for further analysis.
For quantitative measurement, calibration curves were constructed using DNA from individuals homozygous for either the insertion or deletion alleles (i.e., possessing two copies of the respective alleles). For the purpose of conversion to genome-equivalent units, a single cell from each of these individuals was defined as possessing 1 genome-equivalent of the respective allele. Serial dilution experiments indicated that the ACE insertion and deletion systems were able to detect a minimum of 1 and 3 genome-equivalents of target, respectively.
In addition to the SRY, GSTM1, and ACE systems,
all buffy coat and plasma DNA samples were also amplified using a
ß-globin real-time quantitative PCR system
using the primers beta-globin-354F and beta-globin-455R, and the
fluorogenic probe beta-globin-402T (Table 1
) (22). This
system was used to confirm the amplifiability of the extracted DNA, and
the quantitative results were used to calculate the fractional
concentration of the transferred cellular and plasma DNA involved in
fetomaternal trafficking. A normal diploid cell was defined as
possessing 1 genome-equivalent of ß-globin
sequence.
The data captured by the PE Biosystems 7700 Sequence Detector were
analyzed using the Sequence Detection System software, Ver. 1.6.3 (PE
Biosystems). The mean quantity of each duplicate was used for further
concentration calculation. For buffy coat DNA, the software would
calculate the amount of target sequences in the input DNA (500 ng). For
plasma DNA, the concentration expressed in genome-equivalents/mL was
calculated using the following equation:
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where, c is the target concentration in plasma (genome-equivalents/mL); Q is the target quantity (genome-equivalents) determined by sequence detector in a PCR; VDNA is the total volume of DNA obtained after extraction, typically 50 µL per Qiagen extraction; VPCR is the volume of DNA solution used for PCR, typically 5 µL; and Vext is the volume of plasma extracted, typically 0.8 mL.
statistical analysis
Statistical analysis was carried out using the Sigmastat 2.0
software.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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quantification of maternal nucleated cells and plasma dna in fetal
blood
In the cellular fraction, maternal DNA sequences were detected in
the cord blood buffy coat DNA in 12 of the 50 informative cases (24%;
Table 2
). Representative data illustrating the performance of the
GSTM1 system are shown in Fig. 1
. For the positive cases, the median fractional concentration of
maternal DNA (i.e., as a ratio to the ß-globin
real-time PCR results) in the buffy coat of cord blood was 2.6 x
10-4 (interquartile range, 1.7 x
10-4 to 3.6 x 10-4;
Table 2
). In the plasma fraction, maternal DNA sequences were detected
in cord plasma DNA in 15 of the 50 informative cases (30%; Table 2
).
For the positive cases, the median absolute concentration of maternal
plasma DNA in cord plasma was 56 genome-equivalents/mL (interquartile
range, 28–259 genome-equivalents/mL). When the results were expressed
as a ratio of maternal to total cord plasma DNA (i.e., maternal plus
fetal, using the ß-globin real-time PCR
results), the median fractional concentration was 3 x
10-3 (interquartile range, 1 x
10-3 to 1.6 x 10-2;
Table 2
). As negative controls, none of the maternal buffy coat and
plasma DNA samples from the 20 women carrying female babies were
positive for the SRY PCR.
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quantification of fetal nucleated cells and plasma dna in maternal
blood
In the cellular fraction, fetal-derived SRY sequences
were detected in the maternal buffy coat DNA in 13 of the 50
informative cases (26%; Table 2
). For the positive cases, the median
fractional concentration of fetal DNA in the total maternal buffy coat
DNA (i.e., as a ratio to the ß-globin real-time
PCR results) was 3.2 x 10-4 (interquartile
range, 0.6 x 10-4 to 7.6 x
10-4; Table 2
). In the plasma fraction,
fetal-derived SRY sequences were detected in maternal plasma
DNA in 50 of the 50 cases (100%; Table 2
). The median absolute
concentration of fetal DNA in maternal plasma was 443
genome-equivalents/mL (interquartile range, 271–925
genome-equivalents/mL). The median fractional concentration of fetal
DNA in maternal plasma was 3 x 10-2
(interquartile range, 1.4 x 10-2 to
5.3 x 10-2; Table 2
).
quantitative-directional correlation of fetomaternal cellular and
dna trafficking
The number of cases with or without transfer of nucleated cells
and plasma DNA from the mother to the baby are listed in Table 3
A. The proportions of cases with or without cellular transfer
were not affected by the plasma DNA transfer status (Fisher exact test,
P = 0.15). Fifteen of the 50 informative cases (30%)
had discordant results between the presence or absence of nucleated
cell and plasma DNA traffic. For the cases with detectable cellular
and/or plasma DNA transfer, there was no significant correlation in the
amounts of these transfers (Spearman rank-order correlation,
r = -0.20; P = 0.37). For the transfer
of nucleated cells and plasma DNA from the fetus to the mother, the
results are listed in Table 3
B. The plasma DNA transfer status did not
affect the proportions of cases with or without cellular transfer
(Fisher exact test, P = 1.0). Among the 50 cases, 37
(74%) had detectable fetal-derived plasma DNA without detectable
nucleated cell traffic. A comparison between the amounts of plasma
fetal DNA in cases with and without detectable amounts of fetal
nucleated cell traffic into the mother blood revealed no significant
difference (P = 0.20, Mann–Whitney rank-sum test). For
the cases with both detectable fetal cell and plasma DNA transfer into
the maternal circulation, there was no significant correlation between
the amounts of cellular and plasma DNA traffic (Spearman rank-order
correlation, r = -0.24; P = 0.41).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For nucleated maternal cell transfer into the fetal circulation, the proportion of cases with detectable maternal cell traffic into the fetal circulation was higher than those reported using less sensitive technologies, such as minisatellite analysis (14)(33). Our data were qualitatively consistent with those reported previously using methods of comparable sensitivity (1)(16)(34). However, our approach has the advantage that it is also quantitative in nature, thus allowing us to rapidly establish the amount of maternal cell transfer in each case. Our data indicated that maternal nucleated cells were present in umbilical cord blood at a median fractional concentration of 2.6 x 10-4 (interquartile range, 1.7 x 10-4 to 3.6 x 10-4). These results were consistent with those obtained by Hall et al. (15), who used fluorescence in situ hybridization techniques and determined that the fractional concentration ranged from 4 x 10-4 to 2 x 10-2. Our approach, however, was much more rapid and automated than that by Hall et al. (15) who had to manually score a minimum of 1000 nuclei for each case.
The current study was the first time maternal DNA was detected in human umbilical cord plasma. For the cases with detectable plasma DNA traffic, the median fractional concentration of maternal DNA in cord plasma was 3 x 10-3 (interquartile range, 1 x 10-3 to 1.6 x 10-2). It is interesting to note that this fractional concentration was >10-fold higher than the comparable values determined for nucleated cell traffic discussed above. One explanation for the existence of 15 cases with discordant mother-to-fetus cellular and plasma DNA transfer is that the cell types responsible for cellular transfer and plasma DNA release may be different. Future studies are necessary to test this hypothesis. It is currently unknown whether this transfer of plasma DNA from the mother to the fetus may have any biological consequence. An interesting analogy can be drawn between the present data and animal experiments documenting the transplacental transfer of exogenous DNA administered either intravenously or orally to pregnant animals (35)(36). In addition, recent data demonstrating the horizontal transfer of DNA (37)(38)(39) clearly suggest the interesting possibility that part of this plasma DNA may exert previously unknown biological functions. It is thus possible that some of the phenomena previously suggested to be mediated by cellular transfer, e.g., the induction of partial immunological tolerance to the noninherited maternal HLA antigens (19)(20), might actually be mediated by plasma DNA.
Concerning the transfer of nucleated cells and plasma DNA from the fetus to the mother, our data represent the first quantitative comparison of the degree of fetus-to-mother trafficking of cells and plasma DNA in the same subject cohort. The most striking feature of this quantitative analysis was the lack of correlation in the amount of cellular and plasma DNA transfer into the maternal circulation. One possible explanation of this observation is that the cell populations predominantly involved in cellular traffic and plasma DNA liberation are different, similar to the hypothesis suggested above for mother-to-fetus transfer. For example, whereas fetal nucleated red cells have been regarded by many investigators to be a predominant cell type involved in fetus-to-mother transfer (6)(40)(41), it is possible that other cell types, e.g., the trophoblasts, are more important for the liberation of plasma fetal DNA. Because of its ubiquitous presence in the studied samples, our data reemphasize our previous suggestion that plasma fetal DNA is a valuable and easily accessible source of fetal genetic material for prenatal diagnosis and monitoring (22). Conversely, the detection rate of fetal nucleated cells in maternal blood was just 26% in the present cohort. This latter much lower detection rate of fetal nucleated cells in maternal blood was less than in our previous study (1), possibly because of a reduction in the PCR reaction volume (from 100 µL to 50 µL), which thus concomitantly halved the amount of input DNA.
An impressive difference was observed between the detection rates of fetal DNA in maternal plasma (100%) and maternal DNA in cord plasma (30%). One possible explanation for this difference is the presence of a specialized "fetal-derived" structure, namely, the placenta, which separates the fetal and maternal circulation. If we postulate that the main constituent cell types of the placenta, i.e., the trophoblasts, are the predominant cell populations responsible for the liberation of fetal DNA into the maternal circulation, then the absence of an analogous maternal-derived cell type on the fetal side might contribute to the relatively low detection rate of maternal DNA in fetal plasma.
The present study establishes important baseline quantitative values for the two-way transfer of nucleated cells and plasma DNA between the mother and fetus in "normal" pregnancies. The biologic significance of the variations in the amount of trafficking in either direction is unclear at present. However, our methodology can be readily applied to answer the numerous unexplored areas in this field. First, relatively few data are available for the prenatal transfer of cells (42)(43) and plasma DNA from the mother to the fetus. It would therefore be very interesting to extend this type of quantitative bidirectional investigation to the prenatal scenario. Second, although it is established that the fetus-to-mother trafficking of nucleated cells and plasma DNA is perturbed in pathological conditions such as preeclampsia (7)(8)(44), it is currently unclear whether such abnormalities could also be detected in the mother-to-fetus arm of trafficking. Third, relatively little is known regarding the clearance of plasma DNA from the circulation. Recent data indicate that fetal DNA is cleared very rapidly from maternal plasma (23). It is currently unknown whether the same rapid clearance kinetics could also be observed for maternal DNA clearance from the fetal circulation. Because it is obviously difficult to carry out this type of analysis in humans, it would be highly informative to apply this bidirectional study approach to animal models of fetomaternal transfer. Fourth, van Wijk et al. (45) recently described the intriguing observation that apoptotic fetal and maternal nucleated cells are present in the plasma of pregnant women. The numbers of these "plasma-derived cells" (300–400 cells/mL of maternal blood, of which 1 in 500 to 1 in 2000 are of fetal origin, during gestational weeks 7–16) (45) appear to be much smaller than the total concentration of plasma DNA (mean of 3466 genome-equivalents/mL, of which an average of 3.4% is fetal-derived, during gestational weeks 11–17) (22). These preliminary comparisons therefore indicate that most of the DNA in the plasma fraction exists in a cell-free state, with the remaining minority being present in apoptotic cells. Further studies should attempt to confirm these deductions in the same subject cohort. In addition, it would be interesting to investigate whether apoptotic maternal and fetal cells are also present in fetal plasma. It is hoped that the answers to these questions would further improve our understanding of the fetomaternal relationship at a molecular level.
On a technological level, the real-time quantitative PCR systems that were developed as part of this study have numerous other applications. For example, these systems would be useful as sensitive and quantitative assays for the study of post-bone marrow transplantation chimerism. A second application would be for the study of autoimmune diseases in which recent data have suggested a possible role of fetal cell persistence in the mother’s body (11)(12)(13). The techniques described here might have application to study whether the persistence of maternal cells in the baby’s body (46) may also contribute to the development of certain autoimmune diseases.
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Acknowledgments |
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References |
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Rn, which is the
fluorescence intensity over the background (