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ORIGINAL RESEARCH |
From the Department of Obstetrics and Gynecology, University of Basel, Basel, Switzerland.
Address reprint requests to: Wolfgang Holzgreve, MD, University of Basel, Department of Obstetrics and Gynecology, Schanzenstrasse 46, Basel, CH 4031, Switzerland; E-mail: wolfgang.holzgreve{at}unibas.ch.
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ABSTRACT |
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METHODS: In a case-control study, we examined 47 pregnancies complicated by preterm contractions that occurred between 20 and 34 weeks’ gestation and an equal number of matched controls. Erythroblasts were enriched for by magnetic cell sorting and enumerated. These values were then correlated with subsequent pregnancy outcome.
RESULTS: In the study group 16 patients delivered prematurely (subgroup A). The other 31 (subgroup B) delivered at term, as did all those in the control group. No significant difference was noted in erythroblast numbers between either one of the subgroups and the controls.
CONCLUSION: Contrary to the reported increased levels of free fetal DNA in maternal serum, erythroblasts in maternal blood are not elevated significantly in pregnancies with threatened premature labor or in those that deliver preterm.
Research into the use of fetal cells (specifically, erythroblasts) enriched from the maternal circulation as a noninvasive method for prenatal diagnosis has yielded some interesting new insights into pathologic conditions of pregnancy.1,2 Our group made the novel observation that significant elevations in fetal cell traffic into the maternal periphery occur in pregnancies affected by preeclampsia.3 In a large-scale prospective study,4 we showed that this disturbance occurs as early as 20 weeks’ gestation in pregnancies at risk for preeclampsia. Similar observations have been made in independent studies.5 In addition, increases in fetal-maternal cell traffic have been noted in a pregnancy with polyhydramnios6 and in pregnancies with certain fetal aneuploidies. It is currently unclear if fetal cell traffic is elevated in pregnancies with growth-retarded fetuses, because of conflicting reports in the literature.4,7,8
A recent observation that has received much attention in this field is that of cell free fetal deoxyribonucleic acid (DNA) in maternal plasma or serum.9 As the analysis of this material is relatively facile by polymerase chain reaction (PCR), it has been shown that it can be readily used for the analysis of certain fetal genetic traits, such as fetal sex and rhesus D status in pregnancies with a rhesus constellation.1,10 The development of real-time PCR methods has permitted the accurate quantitation of this acellular fetal genetic material.1 By the use of this technology, we and others have shown that cell free fetal DNA levels are elevated in a manner analogous to fetal cells in pregnancies affected by preeclampsia11,12 and hydramnios6 and in pregnancy with trisomy 21 fetuses.13,14
Of particular interest is a recent report made by Leung et al,15 who found that pregnancies at risk for preterm labor were associated with elevated levels of cell free fetal DNA. In this report it was proposed that these elevations in cell free fetal DNA concentrations might be able to distinguish between true and false preterm labor. Because prematurity is one of the major unresolved problems in perinatal medicine,16 the ability to distinguish between true and false labor would be of considerable obstetric benefit. To evaluate this phenomenon more closely, we have investigated whether fetal cell traffic is altered in a manner similar to that of the reported release of cell free DNA in pregnancies with threatened prematurity.15 Previous studies from our group using both PCR and fluorescent in situ hybridization (FISH)3,6,17 have indicated that a significant proportion of the erythroblasts in maternal blood are of fetal origin. Therefore, in this study we made no attempt to distinguish between the two groups, but have solely used erythroblasts identified by morphology as a marker of fetal-maternal cell traffic.
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MATERIALS AND METHODS |
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For the enrichment of fetal erythroblasts, 20 mL of heparinized venous blood was collected. All samples were analyzed using our well-established protocol, whose performance we have previously validated.3,4,6,19 Analytic protocol included a single 1077 Ficol density gradient (Sigma, St. Louis, MO) and separation with magnetic cell sorting (Milteny Biotec, Bergisch Gladbach, Germany) using anti–CD 71 conjugated with magnetic microbeads (Milteny Biotec). The positively enriched cell fraction was transferred on to glass slides by cytocentrifugation (Shandon, Frankfurt, Germany), and the number of erythroblasts was enumerated after May Grü nwald staining (Sigma) using an Axioscope light microscope (Carl Zeiss, Jena, Germany). All blood samples were processed immediately or stored at room temperature up to a maximum delay of 24 hours. Analysis of erythroblast numbers was carried out without the knowledge of the outcome of the pregnancy.
To determine the size of the study, a power analysis was performed based on the results published by Leung et al,15 which indicated that we needed to examine 34 patients and an equal number of controls for a power of 80% and a significance of .05. The variance of the number of fetal cells in maternal blood was taken from our previous findings.20 The data were analyzed using the SPSS statistics software package for Windows (SPSS Inc., Chicago, IL).
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RESULTS |
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. Of the study group, 16 pregnant women delivered prematurely (subgroup A), whereas 31 delivered at term (subgroup B). Table 2 summarizes the data of all 16 patients who delivered preterm, including the various and overlapping risks for prematurity and the number of erythroblasts. All the pregnant women in the control cohort delivered healthy, normal babies at term. In the study group we recorded six cases with vaginal bleeding, three instances of premature rupture of membranes, and four deliveries of growth-retarded fetuses. Twenty instances of infections (positive culture for bacterial vaginosis, Ureaplasma urealyticum, and/or group B streptococcus) occurred in the study group and four similar instances in the control group. No patients developed preeclampsia. Four patients in the study group had polyhydramnios, and two of them delivered preterm. Erythroblasts were elevated in maternal blood of nine patients who delivered preterm and in one patient who delivered at term. No patients in the control group developed polyhydramnios.
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and Figure 1).
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DISCUSSION |
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Because increments in cell free fetal DNA have recently been reported to precede the onset of true preterm labor,15 we have examined whether the traffic of fetal cells is affected in a similar manner. In our study we examined a cohort of 47 pregnant women hospitalized at our institution with premature contractions, of whom 16 delivered their newborns prematurely and 31 at term. A control cohort of 47 gestationally matched controls was examined at the same time. Our enumeration of enriched erythroblasts from these samples indicated that there was no significant difference between any of the groups. Because we have previously shown that almost half the erythroblasts in the maternal circulation are of fetal origin, both by FISH3 and single-cell PCR,17 the total number of enriched erythroblasts can be used as a reliable marker for fetal cell traffic.
From our results two conclusions can be drawn:
These data imply that the placenta provides a relatively impermeable barrier because gross physiologic pressures, such as those that occur during contractions, do not lead to an increased influx of fetal cells into the maternal periphery. They also indicate that the placental changes leading to preterm delivery are not such that they lead to an increase in fetal-maternal cell traffic.
Consequently, a notable feature of our study is that our data concerning fetal cell traffic do not parallel those of Leung and colleagues15 regarding the release of cell free fetal DNA in pregnancies with preterm labor. Currently the relationship between these two parameters is unclear because the main source of cell free fetal DNA appears to be the placenta, whereas fetal cell traffic occurs when a few rare fetal hemopoietic cells actually traverse the placenta. Indeed, studies from our laboratory have indicated that there is no significant correlation between the levels of cell free fetal DNA and fetal cell numbers in normal or preeclamptic pregnancies.22 This suggests that these two phenomena may occur independently of each other. Consequently, it is possible that certain conditions, such as preeclampsia, are associated with a placental lesion leading to both the increased release of cell free fetal DNA and an influx of fetal cells into the maternal periphery. On the other hand, in preterm labor it appears that only the release of cell free fetal DNA but not fetal cell traffic is affected. It will be of interest to examine cell free fetal DNA levels in the described study group and to compare these to fetal cell levels, as this will indicate the relationship between these two parameters. A further consequence of our findings is that alterations in fetal cell traffic do not have predictive value in determining pregnancies at risk for premature delivery in contrast to pregnancies at risk for preeclampsia.4
Increased levels of fetal DNA in the maternal periphery without increased fetal-maternal cell traffic, however, could mean that some degree of increased cellular apoptosis or necrosis in the placenta may precede premature labor.
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Footnotes |
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Received November 30, 2001. Received in revised form April 29, 2002. Accepted May 16, 2002.
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REFERENCES |
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2. Holzgreve W, Hahn S. Fetal cells in maternal circulation. What is the relationship to obstetric ultrasound? Ultrasound Obstet Gynecol 2001;17:1–3.[Medline]
3. Holzgreve W, Ghezzi F, Di Naro E, Gänshirt D, Maymon E, Hahn S. Disturbed feto-maternal cell traffic in preeclampsia. Obstet Gynecol 1998;91:669–72.[Abstract]
4. Holzgreve W, Li JC, Steinborn A, Kulz T, Sohn C, Hodel M, et al. Elevation in erythroblast count in maternal blood before the onset of preeclampsia. Am J Obstet Gynecol 2001;184:165–8.[Medline]
5. Al-Mufti R, Hambley H, Albaiges G, Lees C, Nicolaides KH. Increased fetal erythroblasts in women who subsequently develop preeclampsia. Hum Reprod 2000;15: 1624–8.
6. Zhong XY, Holzgreve W, Li JC, Aydinli K, Hahn S. High levels of fetal erythroblasts and fetal extracellular DNA in the peripheral blood of a pregnant woman with idiopathic polyhydramnios: Case report. Prenat Diagn 2000;20: 838–41.[Medline]
7. Simchen MJ, Barkai G, Lusky A, Guetta E. Fetal hemoglobin-expressing nucleated red blood cell frequencies in pregnancies with intrauterine growth restriction. Prenat Diagn 2001;21:31–5.[Medline]
8. Al-Mufti R, Lees C, Albaiges G, Hambley H, Nicolaides KH. Fetal cells in maternal blood of pregnancies with severe fetal growth restriction. Hum Reprod 2000;15: 218–21.
9. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485–7.[Medline]
10. Zhong XY, Hahn S, Holzgreve W. Prenatal identification of fetal genetic traits. Lancet 2001;357:310–1.[Medline]
11. Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45: 184–8.
12. Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 2001;184:414–9.[Medline]
13. Lo YM, Lau TK, Zhang J, Leung TN, Chang AM, Hjelm NM, et al. Increased fetal DNA concentrations in the plasma of pregnant women carrying fetuses with trisomy 21. Clin Chem 1999;45:1747–51.
14. Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000;20: 795–8.[Medline]
15. Leung TN, Zhang J, Lau TK, Hjelm NM, Lo YM. Maternal plasma fetal DNA as a marker for preterm labour. Lancet 1998;352:1904–5.[Medline]
16. Romero R, Gomez R, Chaiworapongsa T, Conoscenti G, Kim JC, Kim YM. The role of infection in preterm labour and delivery. Paediatr Perinat Epidemiol 2001;15 Suppl 2:41–56.
17. Troeger C, Zhong XY, Burgemeister R, Minderer S, Tercanli S, Holzgreve W, et al. Approximately half of the erythroblasts in maternal blood are of fetal origin. Mol Hum Reprod 1999;5:1162–5.
18. Goldenberg RL, Iams JD, Mercer BM, Meis PJ, Moawad A, Das A, et al. The Preterm Prediction Study: Toward a multiple-marker test for spontaneous preterm birth. Am J Obstet Gynecol 2001;185:643–51.[Medline]
19. Troeger C, Holzgreve W, Hahn S. A comparison of different density gradients and antibodies for enrichment of fetal erythroblasts by MACS. Prenat Diagn 1999;19: 521–6.[Medline]
20. Gänshirt D, Smeets FWM, Dohr A, Walde C, Steen I, Lapucci C, et al. Enrichment of fetal nucleated red blood cells from the maternal circulation for prenatal diagnosis: Experiences with triple density gradient and MACS based on more than 600 cases. Fetal Diagn Ther 1998;13: 276–86.[Medline]
21. Holzgreve W, Hahn S. Novel molecular biological approaches for the diagnosis of preeclampsia. Clin Chem 1999;45:451–2.
22. Zhong XY, Holzgreve W, Hahn S. Cell-free fetal DNA in the maternal circulation does not stem from the transplacental passage of fetal erythroblasts. Mol Hum Reprod 2002;8:864–70.
This article has been cited by other articles:
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  X. Y. Zhong, W. Holzgreve, I. Hoesli, and S. Hahn Circulatory Corticotropin-Releasing Hormone mRNA Concentrations Are Increased in Women with Preterm Delivery But Not in Those Who Respond to Tocolytic Treatment Clin. Chem., March 1, 2005; 51(3): 635 - 636. [Full Text] [PDF]
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