Human Reproduction, Vol. 15, No. 7, 1624-1628,
July 2000
© 2000 European Society of Human Reproduction and Embryology
Increased fetal erythroblasts in women who subsequently develop pre-eclampsia
1 Harris Birthright Research Centre for Fetal Medicine and 2 Department of Haematological Medicine, King's College Hospital, London, UK
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Abstract |
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In pregnancies complicated by pre-eclampsia (PET) and/or intrauterine growth restriction (IUGR) there is an increased number of fetal cells in the maternal circulation. The aim of this study was to investigate whether this increase in fetal cells precedes the onset of these pregnancy complications. Doppler ultrasound studies at 24 weeks gestation have shown that increased impedance to flow in the uterine arteries identifies pregnancies with impaired placental perfusion that subsequently develop PET and/or IUGR. We obtained maternal blood from 18 pregnancies with abnormal Doppler results at 22–24 weeks gestation and from 10 normal controls. Fetal erythroblasts were enriched from maternal blood by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody, and the percentage of these erythroblasts was determined. The median proportion of fetal erythroblasts in the group with abnormal Doppler results was 4.5% (range 1–12%), which was significantly higher than in the control group [median 1% (range 0–3%; P < 0.001)]. Furthermore, within the group with abnormal Doppler the median proportion of fetal erythroblasts was higher in the 10 cases which subsequently developed PET and/or IUGR [median 5.5% (range 3–12%)], than in those with normal pregnancy outcome [median 2% (range 1–5%; P < 0.01)]. These findings suggest that impaired placental perfusion is associated with an increase in feto-maternal cell traffic, which precedes the onset of PET and/or IUGR by several weeks.
Key words: Doppler ultrasound/fetal cells in maternal blood/intrauterine growth restriction/pre-eclampsia/screening
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Introduction |
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In pregnancy, some fetal blood cells escape into the maternal circulation and about 1 in 103–107 of nucleated cells in maternal blood are fetal (Ganshirt-Ahlert et al., 1995
; Al-Mufti and Nicolaides, 1999a). Recent studies have reported that in pregnancies complicated by pre-eclampsia (PET) and/or intrauterine growth restriction (IUGR) the number of fetal erythroblasts in maternal blood is increased (Holzgreve et al., 1998; Lo et al., 1999; Al-Mufti et al., 2000). However, it is not certain if this increase in feto-maternal cell traffic precedes, or is the consequence of, placental damage associated with these pregnancy complications.
Pre-eclampsia is thought to be the consequence of abnormal trophoblast invasion of the maternal spiral arteries (Brosens et al., 1972; Sheppard and Bonnar, 1976), leading to impaired placental perfusion and placental insufficiency. Doppler ultrasound studies have shown that impaired placental perfusion at 22–26 weeks gestation is associated with high impedance to flow in the uterine arteries, which is characterized by the presence of an early diastolic notch in the waveform from these vessels. The presence of this abnormal waveform identifies a group of women at high risk of subsequent development of PET and/or IUGR (Steel et al., 1990; Bower et al., 1993; Harrington et al., 1996; Frusca et al., 1997).
The aim of this study was to determine if the number of fetal cells in maternal blood is increased before the onset of PET and/or IUGR by examining pregnancies with abnormal uterine artery Doppler results before the onset of these complications.
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Materials and methods |
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In our hospital all women attending for antenatal care are routinely offered a colour Doppler ultrasound examination at 23 weeks gestation for assessment of blood flow in the uterine arteries (Harrington et al., 1996
). Increased impedance to flow is considered to be present if there is an early diastolic notch in the waveforms obtained from both the left and right uterine arteries. Immediately after the Doppler examination, maternal blood (20 ml) was obtained from the antecubital vein from 18 women with abnormal flow velocity waveforms and from 10 women with normal waveforms (control group). The blood samples were used for measurement of 
-fetoprotein (AFP) and isolation of fetal erythroblasts. All pregnancies were singleton, and at the time of the ultrasound examinations (median 24; range 22–26 weeks) the women were healthy, normotensive and on no medication, the fetal growth was normal and there were no obvious fetal defects. Furthermore, in all cases the pulsatility index in the umbilical artery and fetal middle cerebral artery were normal. All but seven women were nulliparous, and in one of the multiparous women a previous pregnancy was complicated by PET. The women gave consent to participate in the study which was approved by the hospital Ethics Committee.
Details of pregnancy outcome were obtained from the patient notes. Pre-eclampsia was diagnosed as maternal blood pressure >140/90 mmHg on more than one occasion with `+' or more protein on reagent strip urinalysis or urine protein collection >300 mg/24 h. The diagnosis of IUGR was made if the birth weight was below the fifth centile of the normal range for gestation (Yudkin et al., 1987).
Isolation and detection of fetal cells
Maternal venous blood (20 ml) was collected into lithium heparinized vacutainers (Becton Dickinson, Franklin Lakes, NJ, USA) after the ultrasound examination, and stored at 4°C. The samples were processed within 24 h of collection. Triple density gradient centrifugation was carried out as described previously and the middle layer containing the erythroblasts was separated and isolated (Ganshirt-Ahlert et al., 1993; Al-Mufti et al., 1999b). Cells were incubated with magnetically labelled CD71 antibody to the transferrin receptor antigen (Miltenyi Biotech, Bergisch Gladbach, Germany) for 15 min at 4°C. Magnetic cell sorting was then performed to enrich these erythroblasts as described previously (Ganshirt-Ahlert et al., 1993; Al-Mufti et al., 1999b). Aliquots of the positively selected cells were cytocentrifuged at 14.3 g for 10 min (Shandon, Frankfurt, Germany), and the cells were cytospun onto slides. The fetal cells were detected and quantified using two methods: (i) by morphology after staining by the Kleihauer–Betke method (GTI, Brookfield, WI, USA) and counterstaining with methylene blue (Gurr–Giemsa; BDH Merck Ltd, Poole, UK); and (ii) by immunocytochemistry using monoclonal fluorescein isothiocyanate (FITC) conjugate fluorescent antibody against the 
haemoglobin chain (Caltac, Burlingame, CA, USA). Cells were fixed and permeabilized using commercial `Fix and Perm' reagents (Caltac); slides were incubated with the fluorescent antibody and mounted with 4,6-diamidino-2-phenylindole (DAPI), and then visualized under a fluorescence microscope (Zeiss Axioskop; Carl Zeiss, Gottingen, Germany). Nucleated cells that showed specific staining above the background stain were counted as positive. In each staining method at least 100 nucleated cells were counted on each slide for each case.
The remaining cells in the positive fraction were centrifuged, treated with KCl and fixed with methanol/glacial acetic acid, and frozen at –20°C. Cells were transferred to glass slides and fluorescent in-situ hybridization (FISH) was carried out as described previously (Al-Mufti et al., 1999b, 2000), using a dual chromosome-specific DNA probe (Vysis Inc., Downers Grove, IL, USA) to screen for X and Y chromosomes, as recommended by the manufacturers. At least 100 nucleated cells were examined on each slide, and the percentage of cells with one signal for the Y chromosome probe, and one, two and three signals for the X chromosome probe were calculated. Only intact cells that were not overlapping were chosen for the analysis. The slides were examined under a fluorescence microscope (Zeiss Axioskop), using a DAPI/FITC/TRITC triple band pass filter set. Image capture and processing was by a Microsoft computerized system (Vysis). Enrichment of fetal cells and analysis were carried out without knowledge of the clinical details of the patients.
Statistical analysis
Comparison between groups was carried out using Mann–Whitney U-Wilcoxon rank sum tests. Spearman correlation coefficient was carried out to examine the association between results obtained by the different methods of fetal cell detection.
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Results |
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A total of 28 women was studied. There was no significant difference between the two groups in maternal age, gravidity and parity. All 10 pregnancies with normal Doppler waveforms resulted in the delivery of healthy infants of appropriate birth weight at 37–42 weeks gestation; none of the women developed PET. In the group of 18 with abnormal waveforms, seven developed PET at 3–12 weeks (median 11 weeks) after the Doppler examination, five (including two from the PET group) delivered babies with birth weight below the fifth percentile of the normal range for gestation, and eight had uneventful pregnancies and delivered babies of appropriate birth weight (Figure 1
).
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, individual values for the control group; •, values for the abnormal Doppler group; 
, those patients that subsequently developed PET.Fetal erythroblasts in the maternal blood samples (Figure 2
) were identified in all cases from the abnormal Doppler group, and from nine of the 10 with normal Doppler waveforms.
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The median proportion of fetal erythroblasts (identified by Kleihauer–Giemsa staining) in the group with abnormal Doppler results was 4.5% (range 1–12%), which was significantly higher than in the control group [median 1% (range 0–3%), P < 0.001]. Furthermore, within the group with abnormal Doppler the median proportion of fetal erythroblasts was higher in the 10 patients who subsequently developed PET and/or IUGR [median 5.5% (range 3–12%)], than in those with normal pregnancy outcome [median 2% (range 1–5%), P < 0.01] (Figure 3
; Table I).
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There was a significant correlation between the percentage of erythroblasts that stained positively with the Kleihauer–Giemsa stain and those that were positively stained for the
-haemoglobin (r = 0.98, n = 28, P < 0.001). In 19 of the 28 pregnancies, the fetuses were male (Table II). There was a good correlation between the percentage of erythroblasts that stained positively with the Kleihauer–Giemsa stain and those that stained positively with the Y chromosome-specific DNA probe (r = 0.77, n = 19, P < 0.001). None of the 10 pregnancies with female fetuses had any cells that stained positively with the Y chromosome-specific DNA probe. Among the 19 male fetuses, six were in the PET outcome group, six in the normal outcome bilateral notches group, and seven in the control group. In the six cases with subsequent PET, the median proportion of erythroblasts positive for Kleihauer–Giemsa was 5.5% (range 3–12%) (Table I
) and for Y chromosome was 4% (range 0–9%). In the bilateral notches group with normal outcome, the median proportion of Kleihauer-positive erythroblasts was 2% (range 1–5%) and Y chromosome-positive erythroblasts was 0.5% (range 0–3%). In the control group, the median proportion of Kleihauer-positive erythroblasts was 1% (range 0–3%) and Y chromosome cells was 1% (range 0–1%).
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The median maternal serum AFP concentration in the group with abnormal Doppler waveform (22 ng/ml; range 5–52 ng/ml) was not significantly different to that in controls (18 ng/ml; range 3–111 ng/l). There was no significant correlation between the percentage of enriched fetal erythroblasts from maternal blood and maternal serum AFP concentration (r = 0.18, n = 28).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The findings of this study suggest that impaired placental perfusion is associated with an increase in feto-maternal cell traffic, which precedes the onset of PET and/or IUGR by several weeks. Thus, in pregnancies that subsequently developed PET and/or IUGR there was an increase in fetal erythroblasts in maternal blood. In contrast, the concentration of maternal serum AFP was not significantly different in cases that developed PET and/or IUGR from those with uncomplicated pregnancies.
Possible causes for the increase in fetal erythroblasts in maternal blood in pregnancies with abnormal uterine artery Doppler waveform that subsequently developed PET and/or IUGR include increased fetal production of erythroblasts or increased transfer across the placenta. Certainly, in severely growth-restricted hypoxaemic fetuses the fetal erythroblast count is increased (Nicolaides et al., 1987; Soothill et al., 1987; Snijders et al., 1993). However, in our pregnancies at the time of maternal blood sampling, fetal growth and fetal arterial Doppler results were normal and the mothers were normotensive. Increased transfer across the placenta may be due to placental damage secondary to impaired perfusion and placental oxygenation. However, the concentration of maternal serum AFP in the pregnancies that subsequently developed PET and/or IUGR was not significantly different from normal, and there was no significant association between the number of fetal erythroblasts and the concentrations of AFP. Since the half-life of AFP is only 5–7 days (Crandall, 1981; Caballero et al., 1997), whereas the life-span of erythroblasts is about 60–70 days (Hann et al., 1991), it is possible that the placental damage unmasked by the abnormal Doppler results at 22–26 weeks may have occurred several weeks previously. Indeed, there is evidence that elevated concentrations of AFP at around 15–20 weeks are associated with the subsequent development of pregnancy complications (Crandall, 1981; Walters et al., 1993; Morssink et al., 1995).
Previous reports have demonstrated that PET is associated with an increase in trophoblasts, free fetal DNA and fetal erythroblasts in the maternal circulation (Chua et al., 1991; Holzgreve et al., 1998; Lo et al., 1999). In the previous study examining fetal erythroblasts in maternal blood in pregnancies with PET (Holzgreve et al., 1998), it was suggested that the increased influx into the maternal circulation of either allogeneic immune effector cells or those that can develop into them could contribute to disease progression or at least exacerbate the condition. Although our findings that the increase in fetal erythroblasts in maternal blood precedes the onset of PET is compatible with the above hypothesis, our patients had Doppler evidence of impaired placental perfusion. To demonstrate a causative association between increased feto-maternal cell trafficking and PET, it would be necessary to show that the increase in fetal erythroblasts precedes the onset of abnormal Doppler results.
In a previous study (Holzgreve et al., 1998), despite there being a good correlation between the number of erythroblasts which stained positive with Giemsa and the number of cells positive for Y chromosome on FISH, the number of Y chromosome-positive cells was much lower than those cells stained by Giemsa. This difference was attributed to the presence of enriched maternal erythroblasts. Our data showed a similar pattern but not such a marked difference between the number and percentage of erythroblasts positive for Kleihauer–Giemsa staining and cells positive for Y chromosome on FISH.
Early diastolic notches in the Doppler waveform of the uterine arteries at about 24 weeks gestation identifies a group of pregnancies at high risk of subsequent development of PET and/or IUGR (Steel et al., 1990; Bower et al., 1993; Harrington et al., 1996; Frusca et al., 1997). The potential clinical application of our finding is in reducing the false-positive rate in patients with abnormal Doppler waveforms. However, the complexity and expense of investigating the presence of fetal erythroblasts in maternal blood at present are such to make it unlikely that this technique would have a useful role in screening for PET and/or IUGR. Nevertheless, the finding of increased feto-maternal cell traffic preceding the onset of the clinical manifestations of the disease provides further insight into the pathophysiology of impaired placentation.
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Acknowledgments |
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The study was funded by the fetal Medicine Foundation (Registered Charity No. 123456).
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Notes |
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3 To whom correspondence should be addressed at: Harris Birthright Research Centre for Fetal Medicine, King's College Hospital, Denmark Hill, London SE5 8RX, UK
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Submitted on November 10, 1999; accepted on April 13, 2000.
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