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DOI: 10.1369/jhc.4A6411.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (3): 315-317, 2005
Copyright ©The Histochemical Society, Inc.

First-trimester NRBC Count in Maternal Circulation : Correlation with Doppler Ultrasound Studies

Ariadni Mavrou, Aggeliki Kolialexi, Athena Souka, Athanasios Pilalis, Yannis Kavalakis, Panagiotis Antsaklis, Emmanuel Kanavakis and Aristidis Antsaklis

Medical Genetics (AM,AK,EK) and First Department of Obstetrics and Gynecology (AS,AP,YK,PA,AA), Athens University School of Medicine, Athens, Greece

Correspondence to: Dr. Ariadni Mavrou, Associate Professor of Genetics, Medical Genetics, Athens University School of Medicine, "Aghia Sofia" Children's Hospital, Thivon & Levadias, 11521 Athens, Greece. E-mail: ariamav{at}hol.gr


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This study aimed to determine whether the number of nucleated red blood cells (NRBCs) in maternal circulation during the first trimester of pregnancy could identify pregnancies that will have an anomalous Doppler in the second trimester. A total of 85 blood samples were obtained at 11–14 weeks of gestation with mean uterine arterial perfusion index >1.6, as noted by Doppler ultrasonography. NRBCs were enriched by magnetic automated cell sorting using anti-CD71 and were stained with May/Grunwald/Giemsa. A total of 4.8 NRBCs (range 1–75) were identified in 68 cases. Follow-up scans at 22–24 weeks were available in 46 cases. In 39 women, blood flow in the uterine arteries normalized, whereas in seven, high resistance was noted. One woman in the high-resistance group developed preeclampsia (PET; four NRBCs) and another delivered an intrauterine growth restriction (IUGR) baby (75 NRBCs). The number of NRBCs in women whose Doppler indices later normalized and in those who continued to have increased impedance was similar. The study indicates that NRBC number in maternal circulation during the first trimester cannot be used to screen pregnancies at high risk for developing preeclampsia (PET)/IUGR. High-impedance blood flow in the uterine arteries in the first trimester may be due to an unfinished process of trophoblastic invasion, most likely to be completed successfully by 22–24 weeks. (J Histochem Cytochem 53:315–317, 2005)

Key Words: intrauterine growth restriction • preeclampsia • nucleated red blood cells • screening Doppler ultrasound


   Introduction
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PREECLAMPSIA (PET) and intrauterine growth restriction (IUGR) are serious complications of pregnancy associated with high mortality and morbidity. Although the precise etiology of these conditions remains unclear, there is substantial evidence that they occur because the trophoblast is unable to effectively invade the decidua or to modify the spiral artery walls (Brosens et al. 1972Go; Sheppard and Bonnar 1976Go; Steel et al. 1990Go). At present, there is no reliable test to identify women at risk for developing these disorders early enough in pregnancy to permit preventive treatment and treatment is therefore usually symptomatic for hypertension.

Doppler ultrasound studies have shown that impaired placental perfusion at 22–24 weeks of 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 of these vessels (Harrington et al. 1996Go; Frusca et al. 1997Go). Therefore, Doppler ultrasound examination of the uterine arteries can be used to identify a group of pregnancies at high risk for subsequent development of PET/IUGR.

Recently, it has been demonstrated that fetal cells, as well as fetal DNA, in maternal circulation in weeks 22 and 23 of gestation precede the onset of PET, suggesting that impaired placental perfusion is associated with an increase in fetomaternal trafficking (Holzgreve et al. 1998Go,2000Go; Lo et al 1999Go; Al-Mufti et al. 2000aGo,bGo; Zhong et al. 2002Go).

Because PET is a major cause of fetal and maternal morbidity and mortality, it is important to develop a predictive screening test early in pregnancy so that we can anticipate pregnancies at high risk for this complication.

This is a preliminary study aimed at determining whether the number of nucleated red blood cells (NRBCs) in maternal circulation during the first trimester of pregnancy could identify pregnancies that will have an anomalous Doppler finding during the second trimester.


   Materials and Methods
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Materials
Twenty milliliters of peripheral blood was obtained from 85 pregnant women in the first trimester of pregnancy with increased impedance to both uterine arteries (mean uterine arterial perfusion index >1.6) as defined by color ultrasound examination. Ten women with normal flow velocity waveforms were used as controls. All pregnancies were singleton. Women were healthy, normotensive on no medication, and carried fetuses with no obvious defects and normal growth. All women gave consent to participate in the study.

Follow-up scans in weeks 22 and 23 of pregnancy were obtained for 46 of the 85 originally tested cases. Details of the pregnancy outcomes were available from the patients' files. PET was diagnosed as maternal blood pressure >140/90 mmHg with positive protein on reagent strip urinalysis of urine protein collection >300 mg/24 hr.

The diagnosis of IUGR was made if the birth weight was below the fifth percentile of the normal range of gestation.

Methods
Mononuclear cells were isolated by density gradient centrifugation with Histopaque 1083, magnetically labeled with CD71 antibody to the transferrin receptor, and positively selected using magnetic automated cell sorting. Isolated cells were next stained with May/Grunwald/Giemsa and NRBCs were identified and enumerated by morphology under a light microscope.


   Results
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NRBCs were identified in the blood samples of all cases tested (Figure 1). Of 85 women with abnormal first-trimester Doppler ultrasound, 39 were not available for reevaluation. In another 39, ultrasound findings normalized during the second trimester and seven women continued to have abnormal Doppler ultrasound findings during the second trimester. Of the latter, one woman later developed PET, one delivered an IUGR baby, and five had uncomplicated pregnancies and proceeded to deliver normal babies.



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  		Figure 1

NRBC isolated from maternal peripheral blood stained with May/Grunwald/Giemsa.

 
Table 1 shows the number of NRBCs isolated from maternal circulation during the first trimester of pregnancy. In the control group, an average of four cells (range 1–7) were identified, and in women with abnormal Doppler ultrasound waves, the mean NRBC number was 4.8 (range 1–75). Among women who continued to have an abnormal Doppler in the second trimester, the cell number was 4.9 (range 1–75). Four NRBCs were identified in the pregnant woman who subsequently developed PET, and 75 such cells were isolated from the blood of the woman with the IUGR baby.


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  		Table 1

Number of NRBCs isolated from maternal blood in the first trimester from pregnancies with abnormal Doppler and from controls

 

   Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
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Literature Cited
 
Early recognition of patients at risk for development of PET/IUGR might allow a reduction of the worldwide mortality and morbidity associated with this complication of pregnancy. The availability of screening tests for early detection of this problem will help to identify patients in need of more careful monitoring and preventive treatment, to evaluate the progress of the disorder, and to improve pregnancy outcome.

Previous studies demonstrated a correlation between the number of NRBCs and abnormal uterine artery Doppler ultrasound in women who develop PET/IUGR during the second trimester of pregnancy (Holzgreve et al. 1998Go,2000Go; Al-Mufti et al. 2000aGo,bGo). The present report, which must be considered preliminary because only seven of the 46 women tested continued to have high-impedance blood flow during the second trimester and only two of the seven developed PET or IUGR, indicates that the number of NRBCs in the maternal circulation during the first trimester cannot be used as a second-line screening to identify pregnancies at high risk for PET/IUGR. There was one case with high NRBC number and subsequent delivery of an IUGR baby, but this finding is not sufficient to establish a possible correlation and more pregnancies complicated by PET/IUGR must be tested to confirm this finding.

The NRBCs detected in our study must be both fetal and maternal, but we did not attempt to identify their origin because of the low cell numbers. However, it has been postulated that the increase in the number of fetal NRBCs during pregnancy precedes the increase in the number of maternal NRBCs (Al-Mufti et al. 2000aGo). In addition, female carriers of the ß-thalassemia trait were excluded from the study because we had previously demonstrated that under the stress of pregnancy, carriers of the ß-thalassemia trait produce and release into the circulation a significant number of NRBCs (Mavrou et al. 2003Go).

High-impedance blood flow in the uterine arteries in the first trimester may be due to an unfinished process of trophoblastic invasion, most likely to be completed successfully by 22–24 weeks. This may explain why the Doppler findings normalized during the second trimester.


   Footnotes
 
Presented in part at the 14th Workshop on Fetal Cells and Fetal DNA: Recent Progress in Molecular Genetic and Cytogenetic Investigations for Early Prenatal and Postnatal Diagnosis, Friedrich Schiller University, Jena, Germany, April 17–18, 2004.

Received for publication May 18, 2004; accepted September 15, 2004


   Literature Cited
Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

Al-Mufti R, Lees C, Albaiges G, Hambley H, Nicolaides KH (2000a) Fetal cells in maternal blood of pregnancies with severe fetal growth restriction. Hum Reprod 15:218–221[Abstract/Free Full Text]

Al-Mufti R, Hambley H, Albaiges G, Lees C, Nikolaides KH (2000b) Increased fetal erythroblasts in women who subsequently developed pre-eclampsia. Hum Reprod 15:1624–1628[Abstract/Free Full Text]

Brosens IA, Robertson WB, Dixon HG (1972) The role of the spiral arteries in the pathogenesis of pre-eclampsia. Obstet Gynaecol 1:177–191

Frusca T, Soregaroli M, Valcamonico A, Guandalini F, Danti L (1997) Doppler velocimetry of the uterine arteries in nulliparous women. Early Hum Dev 48:177–185[CrossRef][Medline]

Harrington K, Cooper D, Lees C, Hecher K, Campbell S (1996) Doppler ultrasound of the uterine arteries: the importance of bilateral notching in the prediction of pre-eclampsia, placental abruption or delivery of a small-for-gestational-age baby. Ultrasound Obstet Gynecol 7:182–188[CrossRef][Medline]

Holzgreve W, Ghezzi F, Di Naro E, Ganshirt D, Maymon E, Hahn S (1998) Disturbed feto-maternal cell traffic in pre-eclampsia. Obstet Gynaecol 91:669–672[CrossRef][Medline]

Holzgreve W, Li JC, Steinborn A, Kulz T, Sohn C, Hodel M, Hahn S (2000) Elevation in erythroblast count in maternal blood before the onset of preeclampsia. Am J Obstet Gynecol. 184:165–168

Lo YMD, Leung TN, Tein MSC, Sargent IL, Zhang J, Lau TK, Haines CJ, et al. (1999) Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 45:184–188[Abstract/Free Full Text]

Mavrou A, Kolialexi A, Antsaklis A, Korantzis A, Metaxotou C (2003) Identification of fetal nucleated red blood cells in the maternal circulation during pregnancy using anti-hemoglobin-epsilon antibody. Fetal Diagn Ther 18:309–313[CrossRef][Medline]

Sheppard BL, Bonnar J (1976) The ultrastructure of the arterial supply of the human placenta in pregnancy complicated by fetal growth retardation. Br J Obstet Gynaecol 83:948–959[Medline]

Steel SA, Pearce JM, McParland P, Chamberlain GV (1990) Early Doppler ultrasound screening in prediction of hypertensive disorders of pregnancy. Lancet 335:1548–1551[CrossRef][Medline]

Zhong XY, Holzgreve W, Hahn S (2002) The levels of circulatory fetal DNA in maternal plasma are elevated prior to the onset of preeclampsia. Hypertens Pregn 21:77–83[CrossRef][Medline]


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