Fetal cell-free DNA circulates in the plasma of pregnant mice: relevance for animal models of fetomaternal trafficking

Kiarash Khosrotehrani, Tuangsit Wataganara, Diana W. Bianchi and Kirby L. Johnson1

Division of Genetics, Departments of Pediatrics and Obstetrics and Gynecology, Tufts–New England Medical Center, Boston, MA, USA

1 To whom correspondence should be addressed at: Division of Genetics, Department of Pediatrics, Tufts–New England Medical Center, Box 394, 750 Washington Street, Boston, MA 02111, USA. Email: kjohnson{at}tufts-nemc.org


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: Cell-free fetal DNA (fDNA) can be detected in maternal plasma throughout human pregnancy and is rapidly cleared after delivery. fDNA measurement has clinical application in many complications of pregnancy. Our aim was to determine if fDNA could be detected in maternal plasma during pregnancy in a mouse model system. We then compared the levels of fDNA during pregnancies in which the mother and fetus were either congenic or allogenic. METHODS: C57BL/6J (H-2b) or DBA/2J (H-2d) wild-type female mice were mated to C57BL/6J mice transgenic for the enhanced green fluorescent protein (gfp) and sacrificed while pregnant. C57BL/6J female mice that had previously given birth to three to six litters after mating with transgenic males were sacrificed after delivery. We used real-time quantitative PCR amplification to detect and measure gfp sequences in maternal plasma. RESULTS: fDNA was consistently detected in maternal plasma during pregnancy and was always absent after delivery [median 211 genome equivalents (GE)/ml vs 0 GE/ml, respectively, P=0.0001]. The level of fDNA was higher in allogenic matings compared to congenic matings (median 167 GE/ml/GFP + fetus vs 81 GE/ml/GFP + fetus, respectively). CONCLUSIONS: fDNA sequences can be reliably detected in maternal plasma during murine pregnancy. Our data lends further support to the use of nonhuman species to investigate the mechanisms involved in fetomaternal trafficking.

Key words: cell-free nucleic acids/fetomaternal trafficking/green fluorescent protein/mouse/pregnancy


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In humans, cell-free fetal DNA (fDNA) can be detected in maternal plasma as early as 32 days of gestation and throughout pregnancy (Ariga et al., 2001Go; Wataganara et al., 2004Go). After delivery, fDNA is rapidly cleared from the maternal circulation with an estimated half-life of 16–30 min (Lo et al., 1999Go; Lau et al., 2002Go). The abundance of fetal nucleic acids present in the maternal circulation makes this marker a valuable resource for the assessment of several fetal and maternal conditions. To date it has been used for the determination of fetal gender and rhesus status with high sensitivity and specificity (Costa et al., 2002Go; Turner et al., 2003Go; Rijnders et al., 2004Go). The potential clinical applications of cell-free fetal DNA measurement in the maternal circulation are expanding. Fetal cell-free DNA is increased in maternal plasma when the fetus is affected with trisomies 21 and 13 (Lee et al., 2002Go; Wataganara et al., 2003Go). In the former condition, fDNA has been proposed as an additional analyte to help identify women carrying fetuses affected with Down syndrome (Farina et al., 2003Go). In addition, fDNA levels are higher in women with preeclampsia even before clinical manifestations of the disease (Levine et al., 2004Go). However, the tissue origin, the mechanism of release and the fate of fetal cell-free DNA are still unknown (Bianchi, 2004Go). Experimental designs to answer these questions using human material are somewhat restricted by ethical issues and the difficulty in obtaining informative samples with appropriate accompanying clinical and reproductive histories.

It has recently been suggested that primates may be used as a model for the study of circulating fetal nucleic acids. Jimenez and Tarantal (2003a)Go showed that Y chromosome sequences could be detected and measured in the serum of pregnant rhesus monkeys, and could be reliably used for fetal gender determination and colony management (Jimenez and Tarantal, 2003bGo). The observed similarities of fetal and placental development in humans and rhesus monkeys strongly supports the idea that primates may be suitable experimental models for the study of nucleic acid trafficking during pregnancy. However, primates are expensive to maintain and require specialized facilities.

As experimental models, rodents possess several unique advantages over primates. Since they are smaller laboratory animals, they require lower maintenance costs and are easier to handle. A short reproductive cycle and duration of pregnancy (3 weeks) enable rapid experimental results. There is also a wide variety of genetically modified mouse models available, thus offering great opportunities to understand the physiology of nucleic acid trafficking. In addition, the use of the enhanced green fluorescent protein (GFP) transgenic animal eliminates the fetal gender limitation encountered with human and rhesus monkey fetal DNA measurement. These advantages allow the design of studies that would incur a large cost and ethical issues in humans and primates.

Recently, we have used genetically modified rodents to study fetomaternal trafficking. By mating a transgenic male mouse to a wild-type female, the transgene can be tracked as a fetal marker in the maternal circulation. We used mice with the enhanced green protein fluorescent (GFP+) transgene under the control of a chicken beta-actin promoter and a cytomegalovirus (CMV) enhancer. Our aim in this study was to determine whether it was possible to track the inherited transgene in maternal plasma during and after pregnancy using real-time quantitative PCR in order to understand the natural history of fetal cell-free nucleic acid trafficking in mice. We also used this model to compare the level of fetal cell-free DNA during pregnancies in which the mother and the fetus were either congenic or allogenic.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Mice
The Institutional Animal Care and Use Committee (IACUC) of the Tufts University Division of Laboratory Animal Medicine approved the present protocol. The enhanced green fluorescent protein transgenic mouse (GFP+) (Jackson Laboratories stock #03291, Bar Harbor, ME) has a C57BL/6J genetic background (H-2b) with the enhanced GFP transgene under the control of a chicken beta-actin promoter and a CMV enhancer (Okabe et al., 1997Go).

We used four groups of mice to study the levels of fDNA during and after pregnancy. The first group (A to K) consisted of 8-week-old C57BL/6J virgin female mice that were bred to congenic GFP+ males and sacrificed during pregnancy. The second group (D1 to D5) consisted of 8-week-old DBA/2J virgin female mice (H-2d) that were bred to allogenic GFP+ males and were also sacrificed during pregnancy. In this group the fetuses (H-2b/d) will be recognized as allogenic tissue by the mother. We recorded the total number of fetuses and the number of GFP+ fetuses (based on observed green fluorescence after UV excitation after dissecting the mice) for most of the mice. The third group consisted of C57BL/6J female retired breeders that were previously bred to congenic male GFP+ mice, and gave birth to an average of three to four litters (RB1 to RB7). These mice were directly purchased at Jackson Laboratories and no pregnancy histories were available. They were not pregnant at the time of sacrifice. Finally, C57BL/6J female mice that had been bred three times to congenic GFP+ males and that had delivered three litters were also sacrificed at the end of the lactation period (3 weeks after delivery) of their last pregnancy (LW1 to LW6). The number of GFP+ pups and the total number of pups resulting from each of their pregnancies were recorded.

During pregnancy, or after delivery, animals were sacrificed using carbon dioxide inhalation. Gestational age was based on weeks post-coitum, as the precise date of conception could not be reliably determined in all animals. Approximately 0.5–1 ml of blood was drawn by cardiac puncture and plasma was prepared after centrifugation of whole blood as previously described (Wataganara et al., 2003Go). The presence or absence of pregnancy was confirmed during the dissection of the animals.

DNA extraction and real-time PCR
DNA extraction was performed on all samples using the QIAamp DNA Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. We used 200–400 µl of the plasma to perform DNA extraction. We eluted the extracted DNA into a 60 µl volume and used 10 µl of this per reaction well. A 75 base pair region of the gfp transgene was detected with primers and probe (see below) as described previously (Pan et al., 2002Go): forward primer: 5'-ACTACAACAGCCACAACGTCTATATCA-3', reverse primer: 5'-GGCGGATCTTGAAGTTCACC-3', and Taqman probe: 5'-FAM-CCGACAAGCAGAAGAACGGCATCA-TAMRA-3', where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine. As an internal control for the presence of total DNA, we used probes and primers to amplify a genomic sequence of the mouse apolipoprotein b (apob) gene: forward primer: 5'-CGTGGGCTCCAGCATTCTA-3', reverse primer: 5'-TCACCAGTCATTTCTGCCTTTG-3', and Taqman probe: 5'-FAM-CCTTGAGCAGTGCCCGACCATTC-TAMRA-3'. Real-time PCR amplifications were performed using an ABI 7700 Sequence Detection System with the SDS v1.9 software (Applied Biosystems, Foster City, CA). All experiments were performed in triplicate and a sample was considered to be positive if the amount of gfp transgene detected was equivalent to at least one cell. The quantity of DNA, one genome equivalent (GE), present in a C57BL/6J and a DBA/2J female mouse cell is 6.25 and 5.99 pg, respectively (Capparelli et al., 1997Go). Results were presented as the amount of DNA in GE per ml of plasma, based on apob being present in two copies per cell and gfp, which maps to chromosome 15, being present in one copy per transgenic cell.

Statistical analysis
Descriptive statistics, including median and ranges, were generated for all studied variables. The nonparametric unpaired t-test was used to assess the difference in median plasma gfp and apob sequences levels between different groups of mice. Results were considered to be significant when the P-value was <0.05.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We first developed a PCR assay to detect small quantities of gfp sequences. We were able to amplify both the gfp transgene and the apob genes in as low as 6.4 pg of DNA from GFP+ mice, which is the equivalent of one cell. Our observation that apob always amplified approximately one cycle before gfp in standard curve reactions (data not shown) supported verbal information provided by Jackson Laboratories that gfp is present at one copy per transgenic cell. We never amplified the gfp sequence in genomic DNA from wild-type C57BL/6J at any DNA concentration. We were also able to amplify the gfp transgene at comparable levels (the same cycle threshold, Ct) with background amounts of mock maternal DNA <5 µg. Altogether, our assay enabled us to detect as low as one GFP+ GE in a background of 100 000 wild-type GE.

We next analyzed plasma samples from the four groups of mice. In groups 1 and 2, fDNA was consistently detected (median 211 GE/ml, range 59–1000) in maternal plasma obtained during pregnancies in which GFP+ fetuses were present (Table I). The median fDNA value in congenic pregnancies was 81 GE/ml/GFP+ fetus. In groups 3 and 4, fDNA was always absent in post-partum mice, including during lactation and after completion of weaning (Table II). The differences in circulating fDNA levels during and after pregnancy were highly significant (211 GE/ml compared to 0 GE/ml, P=0.0001). The average estimated ratio between fetal and maternal DNA in the plasma of pregnant mice was 1.2% (range: 0.3 to 6.2%). Fetal cell-free DNA in maternal plasma increased with gestational age (Figure 1). In a univariate analysis, the level of fDNA was also higher in allogenic matings compared to congenic matings (167 vs 81 GE/ml/GFP+ fetus, respectively). However, this was not statistically significantly different, due to the small sample size. The amount of apob sequence was significantly higher in the plasma of pregnant mice (P=0.004) and lactating mothers (P=0.03), indicating more circulating maternal cell-free DNA during pregnancy and lactation.


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Table I. Fetal cell-free DNA detection and quantitation in maternal plasma during congenic and allogenic pregnancies. Mice A through K had a single congenic pregnancy. Mice D1 through D5 were mated to allogenic males

 

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Table II. Fetal cell-free DNA detection and quantitation in maternal plasma after congenic pregnancies

 


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Figure 1. Relationships of fetal cell-free DNA to gestational age and fetomaternal histocompatibility. Closed diamonds represent the level of fDNA in congenic matings of a C57BL/6J female to a C57BL/6J male transgenic for GFP. Open squares represent the level of fDNA in allogenic matings of a DBA/2J female to a C57BL/6J male transgenic for GFP. GE/ml: genome equivalents per milliliter.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Analysis of fDNA in maternal plasma has broad potential for translation to prenatal diagnosis and screening. Current applications, however, are limited by a lack of understanding of the production and clearance of the DNA, as well as its restriction to the analysis of male fetuses. In this study, we developed a real-time quantitative PCR assay to measure fetal DNA resulting from a transgenic mouse mating to study nucleic acid trafficking during pregnancy. We were able to quantify gfp sequences in every plasma sample obtained from the wild-type pregnant mouse confirmed to be carrying GFP+ fetuses. The gfp sequences were never found in the plasma of female mice that had previously given birth to GFP+ pups, suggesting that the fDNA was efficiently cleared from the circulation after delivery. This indicates that this sequence is a marker specific for the current pregnancy, and is similar to the data from human pregnancies (Lo et al., 1998Go).

Although in our model there is no gender limitation, only 50% of the fetuses carry the gfp gene. The small circulating blood volume in the mouse made it necessary for us to sacrifice the animal for fDNA analysis. This precludes a longitudinal study in the same subject, unless the sensitivity of the technique can be improved. Using this model, we compared the level of fDNA in pregnancies in which the mother and the fetus were congenic to pregnancies in which they were allogenic. This comparison was performed in a univariate analysis with a small sample size. Median fDNA levels were found to be higher in allogenic pregnancies, although this did not achieve statistical significance. The results suggest that an increase in placental apoptosis occurs as a result of a more robust maternal immune response to the fetuses' allo-antigens (Shiraishi et al., 1996Go), although further study with a larger number of animals would be useful to determine if this is significant.

In many aspects, our results validate previous findings in human pregnancies. Fetal cell-free DNA is detected in all pregnancies and only during pregnancy. The ratio between fetal and background plasma DNA is comparable to those reported in pregnant women (Lo et al., 1998Go). This was somewhat surprising, as the mouse has a labyrinthine placenta and the human has a hemochorial placenta (Georgiades et al., 2002Go).

The total amount of cell-free DNA in the plasma is higher during pregnancy and lactation (Tsui et al., 2002Go); it is possible that this increase during lactation is due to increased mammary gland apoptosis during remodeling of the breasts that occurs at the end of lactation and the beginning of the weaning period.

With the model described here, other studies could be performed that would be difficult or impossible in humans and primates. Mouse models of preeclampsia have already been developed and could be used to assess the origin of fDNA (Davisson et al., 2002Go). Genetically modified mice lacking essential genes in the apoptosis pathway (Watanabe-Fukunaga et al., 1992Go) could also be used to conclusively demonstrate whether cell-free DNA is released by apoptotic bodies.

In conclusion, we demonstrate here for the first time that fetal cell-free DNA can be reliably detected in maternal plasma during murine pregnancy. Our data lends further support to the use of nonhuman species to investigate the mechanisms involved in fetomaternal trafficking.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Dr Wataganara's maternal–fetal medicine fellowship was supported in part by the Anandamahidol Foundation, Thailand. This study was supported by NIH HD42053 to Dr Bianchi, by a Tufts–New England Medical Center internal grant, and by the GRASP Center NIH/NIDDK, P30 DK34928.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on April 7, 2004; accepted on July 12, 2004.