Measurement of placental bed vascularization in the first trimester, using 3D-power-Doppler, for the detection of pregnancies at-risk for fetal and maternal complications.

OBJECTIVE: To evaluate the performance of placental bed vascularization in a low-risk population to predict severe pregnancy risks. Vascularization was measured in the first trimester, using 3D power-Doppler vascularization index. METHODS: All women who registered during a period of 3 years for delivery in our hospital were prospectively screened in the first trimester. Power Doppler vascularization index of the placental bed (PBVI) was measured in 4325 women and correlated to 7 outcome groups: 1) normal, 2) IUGR ≤ 3rd centile, 3) delivery ≤ 34 weeks, 4) pregnancy induced hypertension (PIH), 5) all pre-eclampsia (PE), 6) severe PE, 7) severe pregnancy problems (SPP i.e. PIH or PE plus IUGR ≤ 3rd centile and/or delivery ≤ 34 weeks). In addition, measurements of mean uterine artery Doppler at 12 and 22 weeks, placental volume and PAPP-A were also performed on all women and their predictive strength for pregnancy risks was compared with the PBVI. RESULTS: Severe PE and SPP occurred in 0.6 vs. 1.5% of all pregnancies. First trimester PBVI below the 10th centile detected 60% of severe PE and 66.2% of SPP, the odds ratio being 4.48 (95th CI 1.98-11.82) for severe PE and 9.92 (95th CI 5.55-17.71) for SPP. Second trimester uterine artery Doppler detected 72% of PE and 50.8% of SPP, the odds ratio being 14.58 (95th CI 5.78-36.79) and 5.46 (95th CI 3.18-9.36) respectively. All other measured parameters performed much worse compared to PBVI and 22 weeks uterine artery Doppler. CONCLUSION: Placental bed vascularization index could be used for a quick and reliable first trimester assessment of severe pregnancy risks.

First trimester placental and myometrial blood perfusion measured by 3D power Doppler in normal and unfavourable outcome pregnancies.

INTRODUCTION: To evaluate whether 3D placental and myometrial power Doppler blood perfusion in the first trimester can be used to detect risk pregnancies. METHODS: 3D power Doppler vascularization index (VI) and flow index (FI) of the entire placenta and the neighbouring myometrium were separately measured in the first trimester in all women with singleton pregnancies during a period of three months. In addition we measured placental volume, placental quotient, PAPP-A, as well as uterine artery at 12 and 22 weeks (mean PI and mean notch) and compared those data with the pregnancy outcome. RESULTS: Data from 383 women could be evaluated. 10 developed pre-eclampsia (PE). Both flow and vascularization were markedly lower in the placentas compared to the adjoining decidua and myometria. There was some correlation between placental vascularization Index (PVI) as well as deciduo-myometrial vascularization index (MVI) and placental volume, PAPP-A and number of pregnancies and a marked correlation between PVI and especially MVI to mean notch at 12 weeks and 22 weeks (PVI: -0.215, -0.274 MVI: -0.316,-0.322). PVI and MVI were significantly reduced in women with pregnancy problems and showed the greatest reduction in PE-pregnancies (p: 0.0018, 0.0004). Of all measured parameters MVI showed the best sensitivity for the detection of PE. CONCLUSION: The correlation between PVI and MVI in the first trimester and mean notch in the second shows that they provide valuable information at as early as 12 weeks which normally so far is only available at 22 weeks by uterine artery Doppler flow. As MVI measures the percentage of vessels in the deciduo-myometrial area it could also provide information on trophoblast invasion. This hypothesis is supported in particular by a marked decrease of the MVI in pregnancy problems especially in PE-pregnancies.

Comparison between three-dimensional placental volume at 12 weeks and uterine artery impedance/notching at 22 weeks in screening for pregnancy-induced hypertension, pre-eclampsia and fetal growth restriction in a low-risk population.

OBJECTIVES: To compare the value of three-dimensional placental volume at 12 weeks and uterine artery Doppler at 22 weeks for predicting pregnancy-induced hypertension (PIH), pre-eclampsia and fetal growth restriction in a low-risk population. METHODS: Over a 20-month period we calculated the placental quotient (PQ = placental volume/crown-rump length) at 11-13 weeks' gestation in all women with singleton pregnancies who booked for delivery in our hospital. At 22 weeks, in the same population, we calculated the mean pulsatility index (PI) of both uterine arteries and the presence of an early diastolic notch was noted. Logistic regression models, the PQ and Doppler parameters were used to compare the two screening methods for subgroups of pregnancy outcome. RESULTS: Complete outcome data were obtained in 2489 consecutive singleton pregnancies. Logistic regression models for the detection of pre-eclampsia had a sensitivity of 38.5% (PQ) vs. 44.8% (Doppler); for the detection of small-for-gestational age (SGA) the sensitivity was 27.1% (PQ) vs. 28.1% (Doppler) at a specificity of 90%. Taking a PQ of or= 90th centile and a bilateral notch, the sensitivity for detection of SGA was 25.0%, 20.2% and 22.0%, respectively; for PIH it was 9.5%, 4.8% and 4.8%; for pre-eclampsia without SGA it was 20.0%, 28%, 12%; for PIH/pre-eclampsia with SGA it was 30.8%, 46.1% and 69.2%. In the group with the most severe complications, in which delivery took place before 34 weeks, the sensitivity was 50.0%, 50.0% and 38.9%, respectively. CONCLUSIONS: PQ at 12 weeks and uterine artery Doppler at 22 weeks have similar sensitivities for predicting pre-eclampsia and fetal growth restriction, although uterine artery Doppler is marginally more sensitive for the prediction of pre-eclampsia. While both methods are insufficient for screening in a low-risk population, the PQ method has the potential advantage of being performed in the first trimester.

The influence of leptin on placental and fetal volume measured by three-dimensional ultrasound in the second trimester.

For a couple of years mechanisms influencing placental and fetal growth and the functioning of leptin, the protein product of the ob/ob gene, have been subjects of intensive research. This study's aim was to investigate whether maternal serum leptin and amniotic fluid leptin have an influence on placental and fetal size measured by three-dimensional ultrasound in the second trimester. To determine this, 40 women with a singleton intrauterine pregnancy at the time of the amniocentesis were included in the study. Placental and fetal volume measurements were obtained and correlated to maternal serum leptin, amniotic fluid leptin, body mass index and gestational age. Multiple regression analysis identified amniotic fluid leptin as an independent negative predictor of placental and fetal volume (r = -2.29, p = 0.032 and r = -0.95, p = 0.011, respectively). In contrast, there was no correlation between maternal serum leptin and placental or fetal volume. The median leptin level in amniotic fluid (9.5 ng/ml) was significantly lower than in maternal blood (18.6 ng/ml). However, there was no significant correlation between maternal serum leptin and amniotic fluid leptin (r = 0.208, n.s.). Body mass index did not reveal any significant influences on placental or fetal volume. The relatively high level of amniotic fluid leptin and its inverse correlation on placental and fetal volume in the second trimester suggest that it possibly plays a role as an anti-placental growth hormone or feedback modulator of substrate supply to the fetus and placenta.

Increased nuchal translucency and congenital heart defects in a low-risk population.

OBJECTIVE: Increased first-trimester nuchal translucency (NT) is a possible marker for congenital heart defects in euploid fetuses. In this study, we wanted to determine the sensitivity for congenital heart defects using the 95th centile of the NT as a cut-off point. METHODS: All women who booked for delivery in our hospital in the first trimester underwent NT measurement at a crown-rump length (CRL) of between 35 and 75 mm. In all euploid fetuses and newborns with isolated or associated CHD, NT was examined retrospectively and classified as normal (<95th centile according to CRL-dependent centiles in our own data) or increased (> or =95th centile). RESULTS: From a total of 12,978 euploid fetuses screened, 27 had CHD (22 isolated and 5 cases associated with additional malformations). Moreover, 7 of the 27 fetuses also had increased NT (26%). Increased NT was significantly more frequent in fetuses with associated CHD (4/5) than in those with isolated CHD (3/22, Yates corrected chi2 p=0.012). In fact, the relative risk for CHD was 6.6 times higher in fetuses with increased NT compared to those with normal NT. CONCLUSION: Increased NT for the detection of CHD performed less well than in other studies. Nevertheless, it can be used as an indication for fetal echocardiography.

Placental growth from the first to the second trimester of pregnancy in SGA-foetuses and pre-eclamptic pregnancies compared to normal foetuses.

The aim of this study was to determine placental growth between 12-22 weeks in normal pregnancies compared to pregnancies complicated by foetal SGA and maternal pre-eclampsia (PE). The placentae of 1199 women were measured 3D sonographically at 12, 16 and 22 weeks of gestation. Placental volume growth was then calculated. Neonatal birthweight, birth centile and the occurrence of pre-eclampsia were recorded in every woman and correlated with placental growth (four groups: normals, SGA, PE, SGA+PE). SGA-placentae are already smaller at 12 weeks but then develop in a similar way to normal placentae. PE placentae are slightly, but significantly, larger at 12 weeks, grow rapidly until 16 weeks and then stop growing normally between 16 and 22 weeks. If SGA goes together with PE, both placental volume (PV) at 12 weeks as well as growth is reduced significantly. Nevertheless, placental growth between week 12 and 22 is too heterogeneous to justify using this method as a clinical tool, but it can provide new information on placental physiology underlying unfavourable obstetric outcomes.

Invasive intrauterine treatment of pulmonary atresia/intact ventricular septum with heart failure.

The mortality and morbidity of children with pulmonary atresia/intact ventricular septum (PA/IVS) are linked to the degree of right ventricular (RV) hypoplasia. Opening up the pulmonary valve (PV) in fetal life could result in improved growth of the RV making it amenable to biventricular repair postnatally. Successful valvulotomy of the PV was performed in a fetus with heart failure at 28 weeks. Following the procedure there was significant growth of the tricuspid valve and RV. The neonate was delivered at 38 weeks with a RV suitable for biventricular repair. In utero pulmonary valvulotomy is feasible and may change the natural history of the condition in affected fetuses with PA/IVS.

[Associations between birth weight and placental volume in the first trimester]. / Zusammenhänge zwischen Geburtsgewicht und Plazentavolumen im ersten Trimenon.

INTRODUCTION: Associations between the size of the placenta and birth weight have been described before. This connection has also been found in (sonographically estimated) second trimester placental size. The aim of this study was to find out if there are any differences in first trimester placental volume between various birth weight groups. METHODS: Placental volume was obtained from non-smoking women at the end of the first trimester during a period of eight months. After birth, the newborns were divided into four groups: below the 10th, 10th to 50th 50th to 90th and above the 90th centile. As is known from previous research, placenta size changes in proportion to crown-rump-length. Therefore, the medians of the "placenta quotients" (placental volume/CRL) of each group were compared in order to correct for differences in gestational age. RESULTS: Data from 1476 pregnancies could be evaluated. The overall median of the placenta quotient was 0.98. It was 0.85 in the group below the 10th, 0.92 between 10th and 50th, 1.02 between 50th and 90th and 1.10 above the 90th centile (p < 0.0001, median test). DISCUSSION: The finding of associations between early pregnancy placental size and birth weight at term gives hope for the development of new diagnostic methods for the recognition of placenta-associated problems. Further research is required to estimate the clinical possibilities for the detection of pregnancies at risk of severe growth retardation and other conditions.

The first trimester 'combined test' for the detection of Down syndrome pregnancies in 4939 unselected pregnancies.

The high detection rate (DR) for Down syndrome (DS) pregnancies which can be achieved by measuring fetal nuchal translucency (NT) early in pregnancy can be improved by combining it with placental hormones [pregnancy-associated plasma protein A (PAPP-A) and free beta-human chorionic gonadotrophin (fbeta-hCG)] and maternal age ('combined test'). In this study we wanted to assess the DR using the 'combined test' in an unselected population of self-referred pregnant women at a false-positive rate (FPR) of about 5%. NT, PAPP-A, fbeta-hCG and maternal age were measured in all women with singleton pregnancies who booked for delivery in our hospital from 1 December 1997 to 31 April 2000 and who were between 10 and 13 completed weeks of gestation [crown-rump length (CRL) 35-70 mm]. The specific DS risk was calculated using the computer program Alpha Version 5aa (Logical Medical Systems, London, UK). A total of 4939 women were tested. Out of 14 DS pregnancies that occurred during this period of time, 12 were detected with the test. A total of 246 women had a false-positive test result in a non-DS pregnancy (FPR 5.0%). This makes the 'combined test' by far the best test for the detection of DS pregnancies in a low-risk population. The constant increase in maternal age at the time of delivery can also lead to an improved DR if a simple age-dependant protocol for DS detection is used, but only at the price of a much higher number of amniocenteses and subsequent abortions. The DR for DS can be increased much more markedly using the 'combined test' with a FPR that still remains at the level as it was in the early 1970s.

First-trimester placental volume as a marker for chromosomal anomalies: preliminary results from an unselected population.

OBJECTIVE: To compare first-trimester placental volume in chromosomally abnormal and normal pregnancies. METHODS: Placental volumes were routinely recorded at the time of nuchal translucency thickness measurement at 10-13 weeks of gestation. This was done using customized three-dimensional ultrasound equipment and measurements were then converted to the placental quotient (placental volume/fetal crown-rump length). The possible difference in placental quotient between chromosomally normal and abnormal pregnancies was examined. RESULTS: A total of 2863 pregnancies was evaluated, including 17 with major chromosomal defects (nine cases of trisomy 21, four of trisomy 18, two of trisomy 13, and one each of Turner syndrome and 48,XXY + 21). The median placental quotient in the chromosomally abnormal group (0.67) was significantly lower than that in the normal fetuses (0.98). In nine of the 17 affected pregnancies the quotient was below the 10th centile of the normal range. CONCLUSIONS: Assessment of placental volume may prove to be useful in first-trimester risk assessment for chromosomal anomalies.

Correlation of first trimester placental volume and second trimester uterine artery Doppler flow.

Uterine artery Doppler examination can identify impaired trophoblast invasion in the second trimester of pregnancy. High resistance and an early diastolic 'notch' show insufficient physiological conversion of the spiral arteries. Uterine artery Doppler is routinely performed between 22-24 weeks which is relatively late for treatment. In this study we wanted to find out whether women with increased uterine blood flow resistance at 22 weeks already have reduced placental volumes in the first trimester measured with 3D sonography.A total of 1060 women with singleton pregnancies had three dimensional (3D) volume measurements of their placentae between 11-13 weeks and uterine Doppler scans between 21-22 weeks. Stepwise logistic and linear regression analyses were used to show a correlation between placental volume (PV) and a CRL dependent placental quotient (PQ) with uterine perfusion parameters. Uterine perfusion at 21-22 weeks depends significantly on PV or PQ at 11-13 weeks (P< 0.0001 for both) and smoking behaviour (P=0.006). The occurrence of a notch also depends significantly on PV and PQ (P< 0.0001 for both) and also on gravidity (P< 0.0001) and age (P=0.0007) as well as on smoking behaviour (P=0.0094). PV and PQ did not show any dependency on age, gravidity, BMI or smoking habits. Placentae of women with high resistance uterine perfusion in the second trimester are already remarkably small in the first trimester. Placental volumetry is probably an efficient method for early and simple identification of impaired trophoblast invasion.

Three-dimensional sonographic volumetry of the placenta and the fetus between weeks 15 and 17 of gestation.

OBJECTIVES: Three-dimensional sonographic volume measurement enables for the first time direct comparison of the increase in size of different but closely interacting structures like the placenta and fetus. Our aim was to calculate the fetal and placental volumes between weeks 15 and 17 of gestation, to monitor the difference in the increase of the fetal and placental sizes and to determine their mutual relationship. METHODS: Fetal and placental sonographic volume measurements were made in 356 singleton pregnancies. To measure the relationship between fetal and placental volumes, a quotient was calculated. Regression analyses were performed to analyze the dependence of the fetal and placental volumes and placental quotient on the week of gestation and other influencing variables. RESULTS: The mean of the fetal volume increased markedly from 67.8 to 76.6 mL (by 13%) within the 3 weeks of observation, whereas placental volume increased only slightly (111.1 to 114 mL (by 2.6%)). The random variation of placental volumes around the mean in all three gestational weeks was considerably higher than that of fetal volumes, indicating that in this early period of gestation there is little correlation between fetal and placental sizes. Fetal volume correlated better to gestational week than did placental volume. CONCLUSION: The quotient of fetal and placental volume might assist in the diagnosis of high-risk pregnancies and the assessment of a normal or large fetus with a small placenta.

Sequential screening for trisomy 21 by nuchal translucency measurement in the first trimester and maternal serum biochemistry in the second trimester in a low-risk population.

OBJECTIVE: To evaluate screening for trisomy 21 in a low-risk population utilizing a combination of nuchal translucency measurement in the first trimester and the triple test in the second trimester. METHODS: This was a retrospective study of 9342 women with singleton pregnancies who booked for delivery in our hospital over a period of 5 years. A nuchal translucency scan was carried out at 10-13 weeks' gestation and for those with a measurement of 3.5 mm or more chorionic villus sampling was performed. All other women were asked to return for the triple test at 16 weeks' gestation. Amniocentesis was offered to women in whom the nuchal translucency was 2.5-3.4 mm, the triple test showed a risk of > or = 1 : 250 and in women aged > or = 35 years. RESULTS: The detection rate using the combined screening method was 95% (18/19) with a screen-positive rate of 7.2%. In comparison, screening by maternal age alone would have identified nine (47%) trisomy 21 pregnancies with a screen-positive rate of 10.7%. CONCLUSION: Our data suggest that the combination of nuchal translucency measurement in the first trimester and the triple test in the second trimester is associated with a very high detection rate of trisomy 21 at a relatively low screen-positive rate.

Three-dimensional ultrasound measurement of the placental volume in early pregnancy: method and correlation with biochemical placenta parameters.

Placental size has been an interesting topic of research for many years. The main aim of this study was to investigate the feasibility of measuring the placental volume at the end of the first trimester using three-dimensional (3D) ultrasound and to correlate these volumes to known placental functional indices and to factors affecting the placenta. Women with singleton pregnancies at the end of the first trimester were included into this study. The volume data of the placentae were correlated to the crown-rump length (CRL), placenta-associated plasma protein A (PAPP-A), free beta-human chroangiogonadotropin (f-beta-hCG) and other factors that may affect the placental size or function. A total of 1462 pregnancies could be evaluated. Comparison between CRL and placental volume proved a significant correlation (r=0.43, P< 0.001). Due to the observed proportional growth of CRL and placental volume, a quotient (placental volume/CRL) was calculated for each case. There were no differences between placenta/CRL-quotients in relation to gravidity, parity or smoking. Correlations could be established between the placental volume and PAPP-A and f-beta-hCG (PAPP-A: r=0.28, P< 0.001, f-beta-hCG: r=0.10, P< 0.001). The measurement of the placenta in the first trimester can be performed in a high percentage of cases. The placenta/CRL quotient represents a simple method to compare placentae from different gestational days. The correlation between placental volume and maternal serum screening parameters might provide a chance to refine first trimester Down's syndrome serum screening. Future studies will be needed to evaluate the possible clinical use of first trimester placental volume measurements.

[Combined test in the first trimester of pregnancy in 3,316 unselected pregnant patients for diagnosing Down syndrome]. / Der combined test im ersten Schwangerschaftstrimenon bei 3316 nicht selektierten Schwangeren zur Diagnostik des M. Down.

UNLABELLED: The high detection rate (DR) for Down syndrome pregnancies which can be achieved by measuring fetal nuchal translucency (NT) early in pregnancy can be improved by combining it with placental hormones (PAPP-A, f beta hCG) and maternal age ("combined test"). In this study we wanted to assess the DR using the "combined test" in an unselected population of self-referred pregnant women at a false positive rate (FPR) of about 5%. MATERIALS AND METHODS: NT, PAPP-A, f beta hCG and maternal age were measured in all women with singleton pregnancies who booked for delivery in our hospital from 1.12.97 to 31.12.99 and who were between 10 and 13 completed weeks of gestation (crown-rump-length 35-70 mm). Calculation of the specific Down risk was done with the computer program Alpha, Version 5aa (Logical medical systems, London). RESULTS: A total of 3316 women were tested. Out of 10 Down pregnancies, which occurred in this period of time 9 could be detected with the test. 137 women had a positive test result but a non-Down pregnancy (FPR 4.1%). CONCLUSIONS: The combined test is an excellent test for the detection of Down syndrome pregnancies in a low-risk population. DISCUSSION: The constant increase in maternal age at the time of delivery can also lead to an improved DR if a simple age dependant protocol for Down-detection is used, but only at the price of a much higher number of amniocenteses and subsequent abortions. The DR for Down syndrome can be increased much more markedly using the "combined test" at a much lower FPR (approximately 5%).

Leiomyoma of the vesicovaginal septum.

A 69-year-old woman presented with a solid homogenous mass in the area of the posterior wall of the urinary bladder. A transvaginal approach was used to remove the mass located in the vesicovaginal septum. Histology revealed a benign smooth spindle cell tumor and immunohistochemistry confirmed leiomyoma.

Uterine artery Doppler and placental volume in the first trimester in the prediction of pregnancy complications.

OBJECTIVE: To evaluate placental volume and uterine artery Doppler in the first trimester in the prediction of pregnancies that subsequently develop pre-eclampsia, pregnancy-induced hypertension, preterm placental abruption or fetal growth restriction. METHODS: In 380 singleton pregnancies attending our center for nuchal translucency screening at 11-14 weeks of gestation, Doppler assessment of both uterine arteries was carried out for measurement of the pulsatility index and the mean pulsatility index of the two vessels was calculated. In addition, three-dimensional ultrasound was used to obtain images for subsequent measurement of placental volume. The 90th centile of the uterine artery mean pulsatility index and the 10th centile of the placental volume for crown-rump length (placental quotient) were calculated. These cut-offs were used for the prediction of pregnancy complications. RESULTS: Complications occurred in 36 (9.5%) of the 380 pregnancies, including 31 cases of fetal growth restriction, two of pregnancy-induced hypertension and abruption, two of pregnancy-induced hypertension, and one of abruption. The uterine artery mean pulsatility index was > or =90th centile in 38 (10%) pregnancies and this group contained nine (25%) of those that developed complications. The placental quotient was < or =10th centile in 39 (10%) pregnancies and this group contained eight (22%) of those that developed complications. In eight (2%) pregnancies the uterine artery mean pulsatility index was > or =90th centile and the placental quotient was < or =10th centile and this group contained six (17%) of those that developed complications. CONCLUSION: The combination of placental volume measurement and uterine artery Doppler in the first trimester may identify women at risk for subsequent development of pregnancy complications.

Fatty-acid biosynthesis in a branched-chain alpha-keto acid dehydrogenase mutant of Streptomyces avermitilis.

Fatty-acid biosynthesis by a branched-chain alpha-keto acid dehydrogenase (bkd) mutant of Streptomyces avermitilis was analyzed. This mutant is unable to produce the appropriate precursors of branched-chain fatty acid (BCFA) biosynthesis, but unlike the comparable Bacillus subtilis mutant, was shown not to have an obligate growth requirement for these precursors. The bkd mutant produced only straight-chain fatty acids (SCFAs) with membrane fluidity provided entirely by unsaturated fatty acids (UFAs), the levels of which increased dramatically compared to the wild-type strain. The levels of UFAs increased in both the wild-type and bkd mutant strains as the growth temperature was lowered from 37 degrees C to 24 degrees C, suggesting that a regulatory mechanism exists to alter the proportion of UFAs in response either to a loss of BCFA biosynthesis, or a decreased growth temperature. No evidence of a regulatory mechanism for BCFAs was observed, as the types of these fatty acids, which contribute significantly to membrane fluidity, did not alter when the wild-type S. avermitilis was grown at different temperatures. The principal UFA produced by S. avermitilis was shown to be delta 9-hexadecenoate, the same fatty acid produced by Escherichia coli. This observation, and the inability of S. avermitilis to convert exogenous labeled palmitate to the corresponding UFA, was shown to be consistent with an anaerobic pathway for UFA biosynthesis. Incorporation studies with the S. avermitilis bkd mutant demonstrated that the fatty acid synthase has a remarkably broad substrate specificity and is able to process a wide range of exogenous branched chain carboxylic acids into unusual BCFAs.

Quantitative FISH analysis and in vitro suspension cultures of erythroid cells from maternal peripheral blood for the isolation of fetal cells.

Several techniques for the enrichment of nucleated fetal red blood cells present in maternal blood have been reported. Here we describe the use of a quantitative fluorescence in situ hybridization (FISH) method and in vitro suspension cultures of erythroid cells from newborn cord blood and maternal peripheral blood. Together with a rapid high performance liquid chromatography (HPLC) method, that allows us to determine as few as 100 cells containing haemoglobin F (HbF), we have scrutinized the reported enrichment methods for fetal nucleated cells in peripheral maternal blood. One hundred FISH analyses on maternal peripheral blood were performed. The method comprises a cell lysis method for depletion of red cells with minimal losses of nucleated cells, uniform numbers of cells (750 000 cells each) on microscopic slides, and inclusion of internal controls to monitor the efficacy of hybridization. Twenty-six cultures of pure erythroid progenitor cells from maternal peripheral blood were analysed for the expansion of fetal cells. To generate these in vitro cultures, nucleated cells from 10-20 ml of peripheral blood from 26 pregnant women were grown in media containing growth factors and hormones to yield over 10(7) of immature erythroid cells within two weeks. Of those, 13 cultures were from pregnancies with confirmed male fetuses. A total of approximately 8x10(8) maternal cells were added into tissue culture medium for these 13 cultures, resulting in about 2x10(8) nearly pure erythroid cells after two weeks. Whereas fetal cells, alone or added into cultures of peripheral blood, grow rapidly and can be detected quantitatively, we could not find any fetal cells in cultures from maternal blood. Likewise, in 7.5x10(7) peripheral blood cells probed by FISH analysis (half of which were from pregnancies with male fetuses) no single Y chromosome was detected. In summary, suspension cultures of erythroid cells can be established routinely and easily. With the quantitative FISH technique used, 750 000 cells per slide can be screened reliably for cells with Y chromosomes. However, the stringent quality-criteria and most elaborate methods indicate that fetal cells in maternal peripheral blood can not be found using the current technology.