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
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Doppler ultrasound/fetal cells in maternal blood/fetal growth restriction
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Patients
The patients with fetal growth restriction were referred to our centre for further assessment at 2226 (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 crownrump 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 2226 (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 KleihauerBetke 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.
|
Statistical analysis
Comparison between the growth restricted group and controls was carried out using MannWhitney 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.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
The median percentage of fetal erythroblasts in the growth restricted group (8.5%, range 430%) was significantly higher than in the control group (1%, range 03%; 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 1736) was significantly higher than the control group (median 5, range 019; P < 0.001).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 2226 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 14 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 6070 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 2426 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.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bower, S., Bewley, S. and Campbell, S. (1993) Improved prediction of pre-eclampsia by two-stage screening of uterine arteries using the early diastolic notch and colour Doppler imaging. Obstet. Gynecol., 82, 7883.[Abstract]
Brosens, I.A., Robertson, W.B. and Dixon, H.G. (1972) The role of the spiral arteries in the pathogenesis of pre-eclampsia. Obstet. Gynaecol., 1, 177191.
Caballero, C., Vekemans, M., Lopez-del-Campo, J.G. and Robyn, C. (1997) Serum fetoprotein in adults, in women during pregnancy, in children at birth, and during the first week of life: a sex difference. Am. J. Obstet. Gynecol., 127, 384389.
Campbell, S., Diaz Recasens, J., Griffin, D. et al. (1983) New doppler technique for assessing uteroplacental blood flow. Lancet, i, 675677.
Crandall, B.F. (1981) -fetoprotein: a review. Critical Reviews in Clinical Laboratory Sciences, 15, 127185.[Medline]
Ganshirt-Ahlert, D., Borjesson-Stoll, Burschyk, M. et al. (1993) Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting. Am. J. Reprod. Immunol., 30, 194201.[ISI][Medline]
Hann, I.M., Gibson, B.E.S. and Letsky, E.A. (eds) (1991) The normal blood picture in neonates. In Fetal and Neonatal Haematology. Baillière Tindall, p. 40.
Holzgreve, W., Ghezzi, F., Di Naro, E. et al. (1998) Disturbed feto-maternal cell traffic in pre-eclampsia. Obstet. Gynaecol., 91, 669672.
Moore, T.R. and Cayle, J.E. (1990) The amniotic fluid index in normal human pregnancy. Am. J. Obstet. Gynaecol., 162, 11681173.[ISI][Medline]
Morssink, L.P., Kornman, H., Beekhuis, J.R. et al. (1995) Abnormal levels of maternal serum human chorionic gonadotropin and -fetoprotein in the second trimester: relation to fetal weight and preterm delivery. Prenat. Diagn., 15, 10411046.[ISI][Medline]
Naeye, R.L. (1974) Hypoxemia and the sudden infant death syndrome. Science, 186, 837838.[ISI][Medline]
Nicolaides, K.H., Clewell, W.H. and Rodeck, C.H. (1987) Measurement of human fetoplacental blood volume in erythroblastosis fetalis. Am. J. Obstet. Gynaecol., 157, 5053.[ISI][Medline]
Nicolaides, K.H., Thilaganathan, B. and Mibashan, R.S. (1989) Cordocentesis in the investigation of fetal erythroblastosis. Am. J. Obstet. Gynaecol., 161, 11971200.[ISI][Medline]
Sheppard, B.L. and Bonnar, J. (1976) The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation. Br. J. Obstet. Gynaecol., 83, 948959.[ISI][Medline]
Silber, R., LoBue, J. and Gordon, A.S. (1978) (eds) Factors thought to contribute to the regulation of egress of cells from marrow. In The Year of Hematology. Plenum, New York, pp. 243.
Snijders, R.J. and Nicolaides, K.H. (1994) Fetal biometry at 1440 weeks of gestation. Utrasound Obstet. Gynaecol., 4, 3448.
Snijders, R.J.M., Abbas, D.M., Melby, O. et al. (1993) Fetal plasma erythropoietin concentration in severe growth retardation. Am. J. Obstet. Gynaecol., 168, 615619.[ISI][Medline]
Soothill, P.W., Nicolaides, K.H. and Campbell, S. (1987) Prenatal asphyxia, hyperlacticaemia, hypoglycaemia, and erythroblastosis in growth retarded fetuses. Brit. Med. J., 294, 10511053.[ISI][Medline]
Walters, C. (1993) Poor pregnancy outcome associated with elevated maternal serum -fetoprotein in combination with increased risk for Down syndrome. Prenat. Diagn., 13, 221222.[ISI][Medline]
Yudkin, P.L., Aboualfa, M., Eyre, J.A. and Redman, C.W.G. (1987) New birthweight and head circumference centile for gestational ages 24 to 42 weeks. Early Human Development, 15, 4552.[ISI][Medline]
Submitted on May 26, 1999; accepted on September 28, 1999.