Human Reproduction, Vol. 15, No. 1, 218-221,
January 2000
© 2000 European Society of Human Reproduction and Embryology
Fetal cells in maternal blood of pregnancies with severe fetal growth restriction
1 Harris Birthright Research Centre for Fetal Medicine, King's College School of Medicine and Dentistry, London SE5 8RX and 2 Department of Haematological Medicine, King's College Hospital, London, UK
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Abstract |
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The aim of this study was to examine whether, in pregnancies with severe early onset fetal growth restriction, the number of fetal erythroblasts in maternal blood is increased. The percentage of fetal erythroblasts in maternal blood, enriched by triple density gradient centrifugation and anti-CD71 magnetic cell sorting, was determined in 10 singleton pregnancies with severe intrauterine growth restriction in which there was Doppler ultrasound evidence of impaired placental perfusion. The values were compared to those of 10 normal pregnancies at the same gestational range of 22–26 weeks. In the growth restricted pregnancies the median number of fetal erythroblasts per 100 nucleated cells in maternal blood enriched for fetal cells was significantly higher than the median value in the control pregnancies (8.5% compared with 1%; P < 0.001). These data suggest that impaired uteroplacental perfusion and severe fetal growth restriction may be associated with placental damage leading to increased feto-maternal cell traffic. Alternatively the rate of transfer of fetal cells into the maternal circulation is not altered but in growth restriction the proportion of fetal erythroblasts in fetal blood is increased.
Key words: Doppler ultrasound/fetal cells in maternal blood/fetal growth restriction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The main cause of fetal growth restriction is impaired placental perfusion leading to placental insufficiency. Other causes include fetal abnormalities and environmental problems such as maternal disease, congenital infection and maternal substrate abuse. Impaired placental perfusion is thought to be the consequence of defective trophoblast invasion of the maternal spiral arteries (Brosens et al., 1972
; Sheppard and Bonnar, 1976) and this condition is commonly associated with high impedance to flow in the uterine arteries assessed by Doppler ultrasound (Campbell et al., 1983). Screening studies have demonstrated that in pregnancies with abnormal flow in the uterine arteries there is increased risk for development of intrauterine growth restriction (IUGR) and/or pre-eclampsia (Bower et al., 1993).
Recent evidence suggests that in conditions of impaired placentation, such as pre-eclampsia, there is increased transfer of fetal cells into the maternal circulation (Holzgreve et al., 1998). The aim of this study is to examine if this is also true in pregnancies complicated by severe fetal growth restriction with Doppler evidence of impaired placentation.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The percentage of fetal erythroblasts in maternal blood, enriched by triple density gradient centrifugation and anti-CD71 magnetic cell sorting, was determined in 10 singleton pregnancies with severe IUGR and Doppler ultrasound evidence of impaired placental perfusion. The values were compared with those of 10 normal pregnancies at the same gestational range of 22–26 weeks. The study was approved by the hospital Research Ethics Committee. Recruitment was between May and October 1998.
Patients
The patients with fetal growth restriction were referred to our centre for further assessment at 22–26 (median 23.5) weeks. Ultrasound examination in our centre confirmed that the fetal abdominal circumference was below the 5th percentile of the normal range for gestation (Snijders and Nicolaides, 1994), which was calculated from the maternal last menstrual period and confirmed by sonographic measurement of the fetal crown–rump length in the first trimester. None of the fetuses had any obvious anatomical defects or sonographic markers of chromosomal abnormalities and in all cases the amniotic fluid volume was assessed by the four-quadrant technique and found to be below the 5th percentile of the normal range for gestation (Moore and Cayle, 1990). Additionally, in all cases colour Doppler ultrasound studies (Aspen®; Acuson Corp., Mountainview, CA, USA) demonstrated abnormal waveforms in the umbilical arteries (absent or reversed frequencies at the end of diastole) and uterine arteries (early diastolic notches and mean pulsatility index of more than 1.45).
The normal control pregnancies were recruited from our routine antenatal clinic and they were also healthy singleton pregnancies at 22–26 (median 24) weeks gestation with sonographic evidence of normal fetal growth and amniotic fluid volume, no obvious anatomical defects or sonographic markers of chromosomal abnormalities and normal flow velocity waveforms from the uterine and umbilical arteries. All these control pregnancies were uneventful and resulted in healthy live births at term of babies that had appropriate weight for gestation and no obvious defects.
Enrichment of fetal cells
In all cases maternal blood was obtained for measurement of 
-fetoprotein, and measurement was made using Kryptor-AFP kit (CIS bio international, ORIS Group, Gif-Sur-Yvette Cedex, France). Maternal venous blood (20 ml) was also collected into Lithium heparinized bottles, 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 previously described and the middle layer containing the erythroblasts was separated and isolated (Ganshirt-Ahlert et al., 1993; Al-Mufti et al., 1999). 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 previously described (Ganshirt-Ahlert et al., 1993; Al-Mufti et al., 1999). 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: first, by morphology after staining by the Kleihauer–Betke method and counterstaining with methylene blue (Gurr-Giemsa, BDH Merck Ltd, Poole, Dorset, UK); and second, by cytoimmunochemistry using fluorescent antibody against 
-globin chain (Figure 1). At least 100 nucleated cells were counted on each slide, for each case.
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FISH analysis
The remaining cells in the positive fraction were centrifuged, treated with KCl and fixed with methanol/glacial acetic acid and frozen at –20°C. Fluorescent in-situ hybridization (FISH) was carried out with dual chromosome-specific DNA probes (Vysis Inc., Downers Grove, USA) to screen for X and Y chromosomes (Figure 1
). The slides were examined under fluorescence microscope (Nikon Corporation, Tokyo, Japan), using DAPI/FITC/TRITC triple band pass filter set. Image capture and processing was by a Microsoft computerized system – Winscan (Vysis). Only intact cells that were not overlapping were chosen for the analysis. Enrichment of fetal cells and analysis were carried out without knowledge of the clinical details of the patients.
Statistical analysis
Comparison between the growth restricted group and controls was carried out using Mann–Whitney test and two-sample t-test, for data that were not and those that were normally distributed respectively. The Spearman correlation coefficient was calculated for the different methods of fetal cell detection.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the growth restricted group one patient had pre-eclampsia at the time of sampling (23 weeks) and three others developed pre-eclampsia 1–8 weeks after sampling. There were six intrauterine deaths at 23–26 weeks and four livebirths after emergency Caesarean section at 27–32 weeks and in all cases the birth weight was below the 5th percentile (Table I
; Figure 2). None of the livebirths or stillbirths had any features suggestive of chromosomal abnormalities.
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) and livebirths (•) in the growth restricted fetuses, plotted on the normal range for gestation (mean, 5th and 95th percentile) (Yudkin et al., 1987Fetal erythroblasts, in maternal blood enriched for fetal cells, were detected in all cases of the growth restricted group and in nine of the 10 controls (Table I
). There were 13 pregnancies with male fetuses and seven with female fetuses. As expected for two techniques that stain for -globin, there was a significant association between the percentage of erythroblasts that stained positively with the Kleihauer–Giemsa stain and those that were positive for the -globin stain (r = 0.961, n = 20, P < 0.001) and the male pregnancies that stained positively with the Y-specific DNA probe (r = 0.755, n = 13, P < 0.01). None of the seven pregnancies with female fetuses had any cells that stained positively with the Y-specific DNA probe.
The median percentage of fetal erythroblasts in the growth restricted group (8.5%, range 4–30%) was significantly higher than in the control group (1%, range 0–3%; P < 0.001; Figure 3). The ratio of % fetal to % maternal erythroblasts, in the maternal blood enriched for fetal cells, in the growth restricted group (median 29, range 17–36) was significantly higher than the control group (median 5, range 0–19; P < 0.001).
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); the open circles (
) are the pregnancies that resulted in intrauterine death.The median maternal serum
-fetoprotein was not significantly different between the growth restricted group (19 ng/ml, range 7–195) and the normal controls (18 ng/ml, range 3–111). Furthermore there was no significant association between the percentage of fetal erythroblasts, in enriched blood, and maternal serum -fetoprotein concentration (r = 0.254, n = 20). |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The findings of this study suggest that in pregnancies with severe IUGR due to impaired placental perfusion there is an increase in number of fetal erythroblasts that can be recovered from the maternal circulation. The fetal origin of these cells was confirmed by three methods and there was a significant association between the findings of these techniques.
Four of our growth restricted pregnancies were complicated by pre-eclampsia and in this group there was no obvious difference in erythroblast count or -fetoprotein from those that did not develop pre-eclampsia. A previous study of women with pre-eclampsia also reported the presence of increased numbers of erythroblasts in maternal blood (Holzgreve et al., 1998). In that study it was found that the number of maternal erythroblasts also increased and that actually the ratio of fetal to maternal erythroblasts was not significantly different between the pre-eclamptic group and the controls. It was postulated that an erythropoietic cytokine of fetal origin could be driving the production of erythroblasts in both the fetus and the mother. In our study of severe growth restriction there was a major increase in fetal erythroblasts, but also in the ratio of fetal to maternal erythroblasts in the enriched maternal samples.
One possible explanation for increased number of fetal erythroblasts in maternal blood in growth restricted pregnancies is increased transfer across the placenta. Several studies have reported that increased maternal serum -fetoprotein, during routine screening at 16 weeks for neural tube defects or trisomy 21, is associated with subsequent intrauterine death or growth restriction (Crandall, 1981; Walters, 1993; Morssink et al., 1995). In our study (at 22–26 weeks), the median maternal serum -fetoprotein was not significantly different between the growth restricted group and the normal controls and there was no significant association between the percentage of fetal erythroblasts and serum -fetoprotein. Additionally, in six of our cases there was intrauterine death within 1–4 weeks after maternal blood sampling and in this group there was no obvious difference in -fetoprotein compared to the group resulting in live births. Since the half-life of -fetoprotein in maternal serum is less than 1 week (Crandall, 1981; Caballero et al., 1997) whereas the life-span of fetal erythroblasts is 60–70 days (Hann et al., 1991), the observed increase in fetal erythroblasts may be the consequence of either chronic leakage through the placenta or a fetomaternal haemorrhage that predated by several weeks the time of our assessment of growth restriction.
An alternative explanation for our findings in growth restricted pregnancies is that placental transfer is not altered but the concentration of erythroblasts in fetal blood is increased. We have previously shown that in severely growth restricted fetuses there is increased production of erythropoietin and erythroblastosis (Snijders et al., 1993). In normal human fetal life, the number of circulating erythroblasts decreases exponentially with gestation, reaching a plateau at 24–26 weeks gestation (Nicolaides et al., 1989). This decrease coincides with the switch from hepatic to medullary erythropoiesis. With liver erythropoiesis, erythroblasts enter the peripheral circulation freely whereas with marrow erythropoiesis, the nucleated erythroid precursors are confined to the parenchyma in which haematopoiesis takes place (Silber et al., 1978). Erythroblastosis in growth restricted fetuses may be due to a delay in the switch from hepatic to marrow erythropoiesis or erythropoietin-mediated premature release of erythroblasts from the bone marrow, or recruitment of extramedullary erythropoiesis (Naeye, 1974; Nicolaides et al., 1987; Soothill et al., 1987).
Our data suggest that impaired uteroplacental perfusion and severe fetal growth restriction is associated with placental damage, which leads to increased fetomaternal cell traffic. The other possibility is that the rate of transfer of fetal cells into the maternal circulation is not altered but the proportion of fetal erythroblasts in the blood of those growth restricted fetuses is increased. The extent to which in pregnancies with impaired placentation the increase in fetal erythroblasts in maternal blood precedes the development of growth restriction and pre-eclampsia remains to be determined.
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Acknowledgments |
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The study was funded by the Fetal Medicine Foundation, registered charity number 123456.
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Notes |
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3 To whom correspondence should be addressed
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Submitted on May 26, 1999; accepted on September 28, 1999.
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