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Clinical Chemistry 50: 689-693, 2004; 10.1373/clinchem.2003.030064
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(Clinical Chemistry. 2004;50:689-693.)
© 2004 American Association for Clinical Chemistry, Inc.

Molecular Diagnostics and Genetics

Plasma {gamma}-Globin Gene Expression Suggests that Fetal Hematopoietic Cells Contribute to the Pool of Circulating Cell-Free Fetal Nucleic Acids during Pregnancy

Tuangsit Wataganara1, Erik S. LeShane1, Angela Y. Chen3,1, Lynn Borgatta3, Inga Peter2, Kirby L. Johnson1 and Diana W. Bianchi1,a

1 Division of Genetics, Departments of Pediatrics, Obstetrics and Gynecology, and2 Institute of Clinical Research and Health Policy Studies, Tufts-New England Medical Center, Boston, MA.
3 Department of Obstetrics and Gynecology, Boston Medical Center, Boston, MA.

aAddress correspondence to this author at: Division of Genetics, Departments of Pediatrics, Obstetrics and Gynecology, Tufts-New England Medical Center, 750 Washington St., Box 394 Tufts-NEMC, Boston, MA 02111. Fax 617-636-1469; e-mail dbianchi{at}tufts-nemc.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for {gamma}-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma.

Methods: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma {gamma}-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample.

Results: {gamma}-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P <0.01). The concentrations of {gamma}-globin mRNA were decreased in most women after TOP, but {gamma}-globin mRNA was increased in some patients when TOP was performed later than 9 weeks of gestation.

Conclusions: {gamma}-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma {gamma}-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination {gamma}-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of {gamma}-globin mRNA as a marker for fetomaternal hemorrhage.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The measurement of cell-free fetal DNA concentrations in maternal plasma or serum during pregnancy has several prospective clinical applications, including the determination of fetomaternal hemorrhage (FMH)2 (1). However, current methods of fetal DNA detection are based on Y sequences, thus limiting clinical application to pregnant women carrying male fetuses. The quantification of presumed fetal RhD sequences in the plasma of RhD-negative pregnant women has also been validated (2). Recent studies have demonstrated that it is also possible to detect placental mRNA sequences in the plasma of pregnant women (3)(4). These data suggest that the placenta is the predominant source of cell-free nucleic acids in the pregnant woman.

In the present study, we investigated {gamma}-globin gene expression in maternal plasma to determine whether hematopoietic cells also contribute to the pool of circulating fetal nucleic acids in the plasma of pregnant women. {gamma}-Globin is synthesized in fetal erythroid cells as a component of fetal hemoglobin (hemoglobin F) (5), and {gamma}-globin mRNA has also been used to identify fetal erythroblasts in blood samples of pregnant women (6). Although pregnancy can induce maternal {gamma}-globin synthesis (7), we wished to explore the clinical potential of this message as a gender-independent marker of FMH after termination of pregnancy (TOP).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
This study was approved by the Institutional Review Boards at Tufts-New England Medical Center and Boston University School of Medicine. Paired plasma samples obtained from 40 women before and within 8–20 min after elective first-trimester TOP by use of manual vacuum aspiration were analyzed. Samples were obtained between March and May 2003 and frozen at -80 °C for a maximum of 7 months. Gestational ages were established by ultrasonography and expressed as menstrual days. Fresh plasma samples were also obtained from five healthy, nonpregnant females and three healthy males as controls. Before enrollment, all patients received counseling and gave their written informed consent.

rna extraction and real-time quantitative reverse transcription-pcr
We centrifuged 1 mL of each plasma sample at 11 500g for 10 min to remove any residual cells. A 900-µL portion of the supernatant was used for RNA extraction via the Qiagen Viral RNA Kit (Qiagen Inc.). The manufacturer’s protocols were adjusted for the sample volume. The extracted RNA was eluted into a final volume of 50 µL.

The RNA concentration was determined by real-time quantitative reverse transcription-PCR with use of the EZ RNA PCR Kit (Applied Biosystems) and a Perkin-Elmer Applied Biosystems 7700 Sequence Detector (Applied Biosystems). The following {gamma}-globin primer sequences were used: forward primer, 5'-GGCAACCTGTCCTCTGCCTC-3'; reverse primer, 5'-GAAATGGATTGCCAAAAC-GG-3'. The dual-labeled fluorescent probe was 5'-FAM-CAAGCTCCTGGGAAATGTGCTGGTG-MGBNFQ-3', in which FAM is 6-carboxyfluorescein and MGBNFQ is a minor groove binder/nonfluorescent quencher (8). The probe is designed to prevent reporting of amplification of any possible contaminating genomic DNA. Calibration curves for {gamma}-globin mRNA quantification were prepared by assaying serial dilutions of HPLC-purified single-stranded synthetic DNA oligonucleotide specifying a {gamma}-globin amplicon (GenBank accession no. NM_000184), with a concentration ranging from 1 x 107 to 1 x 101 copies. Absolute concentrations of {gamma}-globin mRNA are expressed as copies/mL of plasma. The sequence of the synthetic DNA oligonucleotide for {gamma}-globin calibrations was 5'-GTGACAAGCTGCATGTGGATCCTGAGAACTTCAAGCTCCTGGGAAATGTGCTGGTGACCGTTTTGGCAATCCATTTCGG-3'.

A calibration curve for quantification of the housekeeping sequence, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was prepared as described previously with results expressed in pg/mL (9).

Reverse transcription-PCR reactions were set up according to the manufacturer’s instructions (RZ rTth RNA PCR reagent set; Applied Biosystems) in a reaction volume of 50 µL. The fluorescent probe was used at concentration of 100 nM. PCR primers were used at a concentration of 200 nM for the {gamma}-globin system. We used 5 µL of extracted plasma RNA for amplification. Each sample was analyzed in triplicate, and the corresponding calibration curve was run in parallel with each analysis. Samples were also tested to ensure that they were negative for DNA by substituting the rTth polymerase with the AmpliTaq Gold enzyme (Applied Biosystems). Multiple negative water blanks were also included in every analysis.

The thermal cycle for the {gamma}-globin analysis was as follows: the reaction was initiated at 50 °C for 2 min for the uracil N-glycosylase to act, followed by reverse transcription at 56 °C for 30 min. After 5 min of denaturation at 95 °C, 40 cycles of PCR were carried out with denaturation at 94 °C for 20 s and 1 min of annealing/extension at 56 °C. The thermal cycle for GAPDH analysis was described previously (9).

statistical analysis
Descriptive statistics, including medians and 25th and 75th percentile ranges, were generated for all studied variables. The nonparametric (Wilcoxon) test was used to assess the difference in median plasma {gamma}-globin and GAPDH mRNA concentrations between cases and controls. Nonparametric paired t-tests were applied to detect the difference in median concentrations of plasma {gamma}-globin and GAPDH message between pre- and posttermination samples. The nonparametric unpaired t-test was used to detect the difference in the alteration of {gamma}-globin messages in the patients who underwent TOP before and after 9 weeks of gestation. This cutoff gestational age was chosen based on the reported embryologic development of functioning placental vascular structure (10). The effect of time lapse between the TOP procedure and the posttermination blood draw on the alteration of post-TOP plasma {gamma}-globin mRNA concentrations was also estimated by use of Pearson correlation. All statistical analyses were performed using SigmaStat 2.03 software (SPSS).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The median (25th, 75th percentiles) gestational age when the TOP was performed was 67 (62, 75) days. {gamma}-Globin and GAPDH mRNA sequences were detected in every plasma sample analyzed. The median (25th, 75th percentiles) concentrations of {gamma}-globin mRNA in the plasma of male and female controls were 640 (371, 855) and 534 (531, 591) copies/mL, respectively (P = 0.57). The median {gamma}-globin mRNA concentration in pretermination plasma samples was 8951 (1972, 26 892) copies/mL, which is significantly higher than in the controls (P <0.001).

The median (25th, 75th percentiles) {gamma}-globin mRNA concentration in posttermination plasma samples was 3267 (1259, 10 859) copies/mL, which is significantly lower than in the pretermination concentration (P <0.01), as demonstrated in Fig. 1A . Although most patients had decreased {gamma}-globin mRNA concentrations in the posttermination samples, some patients had increased concentrations, as shown in Fig. 2 . Increased posttermination {gamma}-globin mRNA concentrations were observed in 4 of 15 (27%) and 10 of 25 (40%) patients who had TOP before and after 9 weeks of gestation, respectively. The median (25th, 75th percentiles) percentage changes in plasma {gamma}-globin transcript among the patients who underwent TOP before and after 9 weeks of gestation were -40% (-83%, 8%) and -31% (-68%, 37%) respectively, which, however, did not reach statistical significance presumably because of the small sample size (P = 0.53).



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  		Figure 1. Boxplots of {gamma}-globin (A) and GAPDH (B) mRNA concentrations in plasma from nonpregnant controls and from pregnant women before and after TOP.

The box represents the first and third quartiles; the line within the box is the median. {gamma}-Globin and GAPDH gene expression is higher in the plasma of pregnant women than in the controls. This expression decreases after pregnancy termination.



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  		Figure 2. Alteration of {gamma}-globin mRNA concentrations in maternal plasma after elective TOP in the first trimester, expressed as a percentage change from baseline.

Whereas most patients had decreased {gamma}-globin mRNA concentrations after pregnancy termination, the concentrations are increased in some patients, especially when the procedure was performed after 9 weeks of gestation.

The median (25th, 75th percentiles) time lapses between the TOP procedure and the posttermination blood draw in the patients who underwent TOP before and after 9 weeks of gestation were not different: 13 (9, 16) and 13 (12, 16) min, respectively. No correlation was found between this time lapse and the alteration of post-TOP plasma {gamma}-globin mRNA concentrations (r = -0.08; P = 0.61), as demonstrated in Fig. 3 .



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  		Figure 3. Alteration of {gamma}-globin mRNA concentrations in maternal plasma after elective TOP in the first trimester, expressed as a percentage change from baseline.

There is no significant correlation between the time lapse after the procedure and the change of posttermination {gamma}-globin mRNA concentrations (r = -0.08; P = 0.61).

The median (25th, 75th percentiles) concentrations of GAPDH mRNA in the plasma of male and female controls were 64 (48, 171) and 69 (36, 77) pg/mL, respectively, which were not significantly different (P = 0.77). The median GAPDH mRNA concentration in the pretermination samples was 387 (113, 1025) pg/mL, which was significantly higher than in the controls (P <0.01). The median GAPDH mRNA concentration in posttermination samples was 195 (85, 362) pg/mL, which was significantly lower than the pretermination concentration (P <0.001), as demonstrated in Fig. 1BUp .


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The quantitative analysis of fetal/placental specific mRNA sequences in the circulation of pregnant women provides an opportunity to study gene expression in various fetal tissues independent of fetal gender. In the present study, we demonstrated the feasibility of amplifying {gamma}-globin mRNA sequences in fresh and frozen plasma samples. We also showed that both plasma {gamma}-globin and GAPDH mRNA concentrations are increased in pregnancy.

{gamma}-Globin mRNA sequences are present in fetal nucleated erythrocytes isolated from peripheral blood of pregnant women (6), but we cannot completely exclude the possibility that the {gamma}-globin gene is expressed in the placenta. Fetal erythrocytes and trophoblasts derive from different cell types, and these cells have dissimilar functions. Therefore, our interpretation of the data is that transplacental trafficking of fetal hematopoietic cells, along with the physiologic increase in maternal {gamma}-globin synthesis during pregnancy, is likely to be responsible for the observed higher concentrations of {gamma}-globin mRNA in the plasma of pregnant women (7)(11).

In most patients, {gamma}-globin mRNA concentrations decreased after the TOP procedure. This is somewhat counterintuitive. We theorized that this decrease might be explained by the physiologic differences in how the fetal mRNA enters maternal circulation. It is generally believed that in pregnant women the source of cell-free fetal nucleic acids is placentally derived apoptotic cells (12), but it has recently been shown that some fetal DNA sequences are detectable in membrane-bound vesicles (13). This particle-associated form is believed to protect fetal nucleic acids from degradation by ribonuclease enzymes in maternal blood (14). After TOP, fetal mRNA is liberated directly by the sudden disruption of the fetomaternal interface; it thus may not be protected in apoptotic bodies. We therefore suggest that posttermination fetal mRNA sequences are vulnerable to destruction by maternal enzymes, leading to the rapid decrease in posttermination plasma {gamma}-globin mRNA concentrations in most patients.

Although many patients had decreased {gamma}-globin mRNA concentrations after TOP, the concentrations were increased in some patients, particularly when the procedure was performed after 9 weeks of gestation. This finding suggests an association between gestational age and the fetal contribution of {gamma}-globin mRNA. One possible reason could be that the switching of {epsilon}-globin to {gamma}-globin synthesis in fetal hematopoietic progenitors begins at 6–7 weeks of gestation (15). In addition, placental blood flow is not fully established until 8–9 weeks of gestation (10). For these reasons, it is conceivable that fetomaternal cellular and nucleic acid trafficking may not significantly occur until the gestational age is >9 menstrual weeks.

We chose GAPDH as a housekeeping sequence based on previous reports showing comparable concentrations of GAPDH mRNA during pregnancy and after delivery (3)(4). However, our findings suggest that GAPDH mRNA concentrations are affected by whether the individual is pregnant. In cancer patients, the origin of circulating GAPDH mRNA is thought to be from apoptosis occurring in the tumor (9). Similarly high apoptotic activity occurring in the placenta could also release this message into the circulation, causing the observed higher plasma GAPDH mRNA concentrations during pregnancy. Healthy, young, nonpregnant individuals without cancer, as in our control population, may simply not have significant amounts of cell death.

In conclusion, quantification of {gamma}-globin mRNA in plasma suggests that hematopoietic cells contribute to the pool of circulating cell-free nucleic acids. Fetal nucleic acids transferred during a physiologically "unexpected" removal of the fetus and placenta as a result of elective TOP may not be particle associated and may therefore be more vulnerable to destruction. Further evaluation of plasma {gamma}-globin mRNA concentrations after the events that potentially cause FMH at gestational ages later than 9 weeks is necessary to validate its clinical value as a gender-independent marker for FMH. Future studies should also include the comparison of this potential novel marker to the standard Kleihauer–Betke test.


   Acknowledgments
 
This study was supported in part by institutional funding, an anonymous foundation, and Grants N01-HD42053 and R01-43204. Dr. Wataganara’s maternal-fetal medicine fellowship was supported in part by the Anandamahidol Foundation, Thailand. We thank Olivera Vragovic, who helped organize the study and enroll the patients. Special thanks go to Helene Stroh, Dongkai Zhen, Betty Ann McIsaac, Jill Maron, Reeval Segel, Kiarash Khosrotehrani, and Donghyun Cha for technical advice with regard to the study.


   Footnotes
 
1 Current affiliation: Department of Obstetrics and Gynecology, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA.

2 Nonstandard abbreviations: FMH, fetomaternal hemorrhage; TOP, termination of pregnancy; and GAPDH, glyceraldehyde-3-phosphate dehydrogenase.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Wataganara T, Chen AY, LeShane ES, Sullivan LM, Borgatta L, Bianchi DW, et al. Cell-free fetal DNA levels in maternal plasma after elective first trimester termination of pregnancy. Fertil Steril 2004: in press..
  2. Rijnders RJ, Christiaens GC, Bossers B, van der Smagt JJ, van der Schoot CE, de Hass M. Clinical applications of cell-free fetal DNA from maternal plasma. Obstet Gynecol 2004;103:157-164.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  3. Ng EK, Tsui NB, Lau TK, Leung TN, Chiu RW, Panesar NS, et al. mRNA of placental origin is readily detectable in maternal plasma. Proc Natl Acad Sci U S A 2003;100:4748-4753.[Abstract/Free Full Text]
  4. Ng EK, Leung TN, Tsui NB, Lau TK, Panesar NS, Chiu RW, et al. The concentration of circulating corticotropin-releasing hormone mRNA in maternal plasma is increased in preeclampsia. Clin Chem 2003;49:727-731.[Abstract/Free Full Text]
  5. Peschle C, Migliaccio G, Lazzaro D, Petti S, Mancini G, Care A, et al. Hemopoietic development in human embryo. Blood Cells 1984;10:427-441.[Web of Science][Medline] [Order article via Infotrieve]
  6. Larsen RD, Schønau A, Thisted M, Petersen KH, Lohse J, Christensen B, et al. Detection of {gamma}-globin mRNA in fetal nucleated red blood cells by PNA fluorescence in situ hybridization. Prenat Diagn 2003;23:52-59.[CrossRef][Web of Science][Medline] [Order article via Infotrieve]
  7. Lee JC, Hayashi RH, Shepard MK. Fetal hemoglobin in women with normal and with hydatidiform molar pregnancy. Am J Hematol 1982;13:131-139.[Web of Science][Medline] [Order article via Infotrieve]
  8. Smith RD, Li J, Noguchi CT, Schechter AN. Quantitative PCR analysis of HbF inducers in primary human adult erythroid cells. Blood 2000;95:863-869.[Abstract/Free Full Text]
  9. Ng EK, Tsui NB, Lam NY, Chiu RW, Yu SC, Wong SC, et al. Presence of filterable and nonfilterable mRNA in the plasma of cancer patients and healthy individuals. Clin Chem 2002;48:1212-1217.[Abstract/Free Full Text]
  10. Jauniaux E, Hempstock J, Greenwold N, Burton G. Trophoblastic oxidative stress in relation to temporal and regional differences in maternal placental blood flow in normal and abnormal early pregnancies. Am J Pathol 2003;162:115-125.[Abstract/Free Full Text]
  11. Lui YY, Chik KW, Chiu RW, Ho CY, Lam CW, Lo YMD. Predominant hematopoietic origin of cell-free DNA in plasma and serum after sex-mismatched bone marrow transplantation. Clin Chem 2002;48:421-427.[Abstract/Free Full Text]
  12. Lo YMD. Fetal DNA in maternal plasma: biology and diagnostic applications. Clin Chem 2000;46:1903-1906.[Abstract/Free Full Text]
  13. Bischoff FZ, Dang D, Horne C, Marquez-Do D, Brinkley WR, Lewis DE, et al. Fetal DNA in maternal plasma circulates as apoptotic bodies: elucidation of the structural nature of fetal DNA for non-invasive prenatal genetic diagnosis [Abstract]. Am J Hum Genet 2003;73(Suppl):189.
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