Fetal DNA Levels Research Articles

Fetal trisomy 21 and maternal preeclampsia.

Placental trophoblast shedding into maternal circulation has been hypothesized as a potential cause of preeclampsia. Because pregnancies with a trisomy 21 fetus also have high levels of fetal cells and cell-free fetal DNA in maternal circulation, we examined whether trisomy 21 pregnancies have a higher risk of preeclampsia than euploid pregnancies. We used 2 population-based databases. We identified 7763 pregnancies with a singleton trisomy 21-affected fetus and 15,293 matched euploid gestations from the U.S. Natality files for the period 1995-1999. The second database consisted of 665 pregnancies with fetal trisomy 21 and 987 euploid controls in a population-based Down syndrome study in California. In the latter study, women were interviewed by telephone regarding characteristics and pregnancy complications. Gestational hypertension and preeclampsia are the outcomes of this study. The U.S. Natality files showed that in nulliparous women fetal trisomy 21 was associated with a reduced risk of pregnancy-induced hypertension (adjusted relative risk [aRR] = 0.67; 95% confidence interval [CI] = 0.53 to 0.85). Findings from the California study confirmed this association in nulliparous women, and further revealed that the decrease in overall risk of pregnancy-induced hypertension was mainly the result of a large reduction in the risk of preeclampsia (aRR = 0.19; CI = 0.04 to 0.88) rather than in gestational hypertension by itself (0.83; 0.37 to 1.84). Neither dataset showed these effects among multiparous pregnancies. Fetal trisomy 21 is associated with a reduced, rather than increased, risk of preeclampsia, specifically in nulliparous women.

Circulating Fetal DNA: Its Origin and Diagnostic Potential—A Review

Objective In contrast to the traditional teaching that the placenta forms an impermeable barrier, multiple studies show that both intact fetal cells and cell-free nucleic acids circulate freely in maternal blood. Complications of pregnancy, such as pre-eclampsia, or fetal cytogenetic abnormalities, such as trisomy 21, increase transfusion of both intact fetal cells and cell-free fetal nucleic acids into the maternal circulation. The objective of our research is to show that abnormal feto-maternal trafficking of nucleic acids is associated with fetal and placental pathology, and that these observations may lead to novel non-invasive diagnostic and screening tests. Methods Real-time quantitative PCR amplification of DYS1 is used to measure the levels of male fetal DNA in case-control sets of serum or plasma taken from pregnant women. In our laboratory, we use DYS1, a Y-chromosome specific gene, as a uniquely fetal DNA marker for the development of gestation-specific normal values and theoretical models. Results Women carrying fetuses with trisomies 21 or 13 (but not 18) have increased levels of fetal DNA in their fresh or archived serum and/or plasma samples. Women destined to develop pre-eclampsia have a characteristic bi-phasic elevation of cell-free fetal DNA that precedes clinical symptoms. Data obtained from a variety of clinical scenarios suggest that the placenta is the predominant source of the circulating fetal nucleic acids, although apoptotic haematopoietic cells may contribute to the pool as well. Conclusions Fetal cell-free DNA is elevated in a number of conditions associated with placental pathology. Widespread clinical implementation of fetal DNA as a screening tool awaits discovery of a reliable gender-independent marker, which may be DNA polymorphisms, epigenetic markers, or mRNA. Fetal cell-free nucleic acids have potential for non-invasive monitoring of placental pathology.

Fetal DNA in maternal plasma as a screening variable for preeclampsia. A preliminary nonparametric analysis of detection rate in low-risk nonsymptomatic patients.

Our objective was to examine whether plasma fetal DNA can be used as a screening variable in those women who developed preeclampsia but without any clinical symptom at the time of blood draw. Fetal DNA was extracted from 1.5-mL plasma samples, and the DYS14 gene was analyzed by real-time quantitative polymerase chain reaction. Plasma collected and frozen from six women were each paired with five matched control samples of identical specimen type from gravid women carrying a presumed normal male fetus. Matched rank-sum analysis and nonparametric receiver operating characteristic (ROC) curves analysis of estimated multiples of median (MoM) were used for calculating detection rate (DR) and false-positive rate (FPR). The mean observed rank of 5.08 in the cases was significantly higher than the expected 3.18 (p-value = 0.013). Pregnancies that will develop a preeclampsia exhibit 2.39-fold higher levels of maternal plasma cell-free fetal DNA compared to matched controls. DR was 33 and 50% at an FPR of 5 and 10% respectively. The estimated DR allows to consider fetal DNA as a potential variable to predict preeclampsia in a low-risk population. Further studies will be addressed to calculate a parametric statistical algorithm and to estimate a proper posterior risk of the disease by means of fetal DNA alone or combined with other markers.

Antiphospholipid and Anti‐DNA Antibodies Are Not Associated with the Elevated Release of Circulatory Fetal DNA in Pregnancies Affected by Preeclampsia

Objectives: We have previously shown that the levels of circulatory fetal DNA are elevated in preeclampsia and that these increases correspond to disease severity. Several reports have indicated that increased levels of antiphospholipid (anti‐PL) and anti‐DNA antibodies may be associated with preeclampsia, in particular with the severe forms of the disorder. Since the release of cell‐free DNA by the placenta is attributed to some form of cell death or damage and as anti‐PL and anti‐double‐stranded DNA (dsDNA) antibodies have been proposed to lead to placental damage, we have studied the relationship between these parameters in preeclampsia. Methods: Circulating fetal DNA levels in samples taken from pregnant women with mild (n = 12) or severe (n = 12) preeclampsia and from normal pregnant controls (n = 35) were quantified using a Taqman real‐time Polymerase Chain Reaction (PCR) assay. The Anti‐PL antibodies (IgG and IgM) were assayed by anticardiolipin ELISA and by commercial anti‐β2‐Glycoprotein I (GPI) ELISA kits. Anti‐dsDNA antibodies (IgG and IgM) were analyzed by a commercially available anti‐dsDNA ELISA kit. Results: No correlation could be drawn with the quantity of circulatory fetal DNA in the samples analyzed and corresponding anti‐PL or anti‐dsDNA antibody levels. Furthermore, no significant difference existed between the levels of these antibodies in the two study groups and the control cohort. Conclusion: Our data suggest that the mechanism leading to the increased release of cell‐free circulatory DNA from the placenta does not involve trophoblast damage mediated by these agents. Our analysis also questions the reported involvement of anti‐PL and anti‐DNA antibodies in preeclampsia.

Inability to detect cell free fetal DNA in the urine of normal pregnant women nor in those affected by preeclampsia associated HELLP syndrome.

Recent reports have indicated that cell-free fetal DNA can be detected in the urine of pregnant women. We attempted to reproduce those data. Urine samples were collected from 18 normal pregnant women (11 with a male fetus). Urinary DNA was examined by Y-chromosome-specific nested polymerase chain reaction (PCR) or real-time PCR. Samples were also examined from two pregnancies complicated by HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome, which had very high levels of cell-free fetal DNA in the maternal plasma. To validate our data, a quantitative comparison of different DNA extraction procedures used in the previous reports was performed. In no instance were we able to detect any fetal DNA in maternal urine, although copious quantities of cell-free fetal DNA were present in the maternal plasma of those pregnancies affected by HELLP syndrome. Our quantitative analysis of the various extraction procedures used indicated that the commercial column elution method we used was comparable, if not superior, to the noncommercial methods used in previous reports. Our data strongly suggest that cell-free fetal DNA is not readily detectable in maternal urine, even under conditions known to increase kidney permeability.

Increased total cell-free DNA in the serum of pregnant women carrying a fetus affected by trisomy 21.

Analysis of the levels of cell-free fetal and total DNA in serum of women carrying a male fetus affected by trisomy 21, and comparison of these levels with those in women carrying a normal male fetus. DNA was extracted from archived second-trimester maternal serum samples collected as part of a prenatal screening program. A total of 10 cases with trisomy 21 male fetuses were compared with 10 controls (male fetuses) with samples matched for duration of storage and gestational age. Real-time quantitative PCR of the SRY and albumin genes was used to quantify fetal and total DNA respectively. The median fetal DNA level in the group of 10 pregnancies with trisomy 21 was 31.98 cell-equivalents per mL compared to 34.06 in the control group. The difference was not significant. The median total DNA level in women with a trisomy 21 fetus was significantly higher (P = 0.029) than that in controls (36 152.6 vs 5832.81 cell-equivalents per mL). Although we could not confirm previous studies of an increased amount of fetal DNA in pregnancies affected by trisomy 21, we did find increased levels of total DNA. The possible reasons for these observations are discussed with respect to previous findings. Larger studies are needed to elucidate the true value, if any, of measurement of fetal and total DNA in maternal serum in the context of prenatal screening for chromosomal abnormalities.

Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18.

Cell-free fetal DNA in the maternal circulation is a potential noninvasive marker for fetal aneuploidies. In previous studies with Y DNA as a fetal-specific marker, levels of circulating fetal DNA were shown to be elevated in women carrying trisomy 21 fetuses. The goal of this study was to determine whether cell-free fetal DNA levels in the serum of pregnant women carrying fetuses with trisomies 13 or 18 are also elevated. Archived maternal serum samples from five cases of male trisomy 13 and five cases of male trisomy 18 were studied. Each case was matched for fetal gender, gestational age, and duration of freezer storage to four or five control serum samples presumed to be euploid after newborn medical record review. Real-time quantitative polymerase chain reaction amplification of DYS1 was performed to measure the amount of male fetal DNA present. Unadjusted median serum fetal DNA concentrations were 97.5 GE/ml (genomic equivalents per milliliter; 29.2-187.0) for the trisomy 13 cases, 31.5 GE/ml (18.6-77.6) for the trisomy 18 cases, and 40.3 GE/ml (3.7-127.4) for the controls. Fetal DNA levels in trisomy 13 cases were significantly elevated ( P=0.016) by analysis of variance of the ranks of values within each matched set. In contrast, fetal DNA levels in trisomy 18 cases were no different from the controls ( P=0.244). Second trimester maternal serum analytes currently used in screening do not identify fetuses at high risk for trisomy 13. Fetal DNA may facilitate noninvasive screening for trisomy 13 provided that a gender-independent fetal DNA marker can be developed.

Down syndrome and cell-free fetal DNA in archived maternal serum

Objective: Increased levels of cell-free fetal DNA (f-DNA) in the maternal circulation are a potential noninvasive marker for fetal Down syndrome. Our objectives were to (1) determine whether f-DNA could be quantified by using archived serum and amniotic fluid, (2) examine whether serum f-DNA levels are elevated in Down syndrome pregnancies in a case-control series matched for gestational age and duration of sample storage, and (3) determine whether f-DNA levels are elevated in the amniotic fluid of Down syndrome fetuses. Study Design: Eleven serum and six amniotic fluid samples previously collected and stored at −20°C from gravid women carrying a 47,XY,+21 fetus were each paired with five matched control samples of identical specimen type from gravid women carrying a presumed euploid male fetus. f-DNA concentration was quantified blindly by real-time polymerase chain reaction amplification for a Y-chromosome sequence. Matched rank-sum analysis and analysis of variance were used for analysis. Results: The mean observed rank of 5.0 in the Down syndrome group was significantly higher than expected (P ≤.005). Adjusted mean serum f-DNA concentrations were 41.2 genomic equivalents (GE) per milliliter for the Down syndrome cases and 24.2 GE/mL for the euploid controls (P =.002). Differences among amniotic fluid samples were not statistically significant. There was a suggestion of a sample storage effect on f-DNA concentration on the order of −0.66 GE/mL per month (P =.071). Conclusion: Down syndrome pregnancies exhibit 1.7-fold higher levels of maternal serum cell-free f-DNA compared with matched controls. No such association is observed in amniotic fluid. Archived serum appears to be a useful source of clinical material for retrospective analyses but may require controlling for the duration of sample storage. (Am J Obstet Gynecol 2002;187:1217-21.)

Fetal cells and cell-free fetal DNA in maternal blood: new insights into pre-eclampsia.

The examination of fetal cells, specifically erythroblasts, and cell-free fetal DNA from the blood of pregnant women is currently the subject of intense research with the aim of developing new risk-free methods for prenatal diagnosis. An unexpected finding made during these studies was that the traffic of fetal erythroblasts into the maternal peripheral circulation was enhanced in pre-eclampsia. Independent prospective studies examining samples collected in the second trimester indicated that this perturbation in fetal cell trafficking occurs early in pregnancy, well before the onset of pre-eclampsia symptoms. The quantitative analysis of cell-free fetal and maternal DNA levels indicated that these concentrations were elevated in a co-ordinate manner in manifest pre-eclampsia, and that these elevations corresponded to disease severity. On the other hand, analysis of prospectively collected samples indicated that only cell-free fetal but not maternal DNA levels were elevated before onset of symptoms in pregnancies which subsequently developed pre-eclampsia. These data support hypotheses suggesting that pre-eclampsia is a multi-step disorder, initiated by a placental lesion that occurs early in pregnancy and which subsequently leads to a systemic maternal inflammatory response and associated endothelial cell damage.

Quantitative analysis of DNA levels in maternal plasma in normal and Down syndrome pregnancies.

BackgroundWe investigated fetal and total DNA levels in maternal plasma in patients bearing fetuses affected with Down syndrome in comparison to controls carrying fetuses with normal karyotype.MethodsDNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and β-globin genes as markers. Twenty-one pregnant women with a singleton fetus at a gestational age ranging from 15 to 19 weeks recruited before amniocentesis (carried out for reasons including material serum screening and advanced material age), and 16 pregnant women bearing fetuses affected with Down syndrome between 17 to 22 weeks of gestation were involved in the study.ResultsThe specificity of the system reaches 100% (no Y signal was detected in 14 women pregnant with female fetuses) and the sensitivity 91.7% (SRY amplification in 22 of 24 examined samples). The median fetal DNA levels in women carrying Down syndrome (n=11) and the controls (n=13) were 23.3 (range 0–58.5) genome-equivalents/ml and 24.5 (range 0–47.5) genome-equivalents/ml of maternal plasma, respectively (P = 0.62). The total median DNA levels in pregnancies with Down syndrome and the controls were 10165 (range 615–65000) genome-equivalents/ml and 7330 (range 1300–36750) genome-equivalents/ml, respectively (P = 0.32). The fetal DNA proportion in maternal plasma was 0%-6 % (mean 0.8%) in women carrying Down syndrome and 0%-2.6 % (mean 0.7 %) in the controls, respectively (P=0.86).ConclusionsOur study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.

THE LEVELS OF CIRCULATORY FETAL DNA IN MATERNAL PLASMA ARE ELEVATED PRIOR TO THE ONSET OF PREECLAMPSIA

Objective: Elevations in cell free fetal DNA has previously been determined in pregnancies affected by preeclampsia. A recent report has indicated that cell free fetal DNA concentrations are elevated early in pregnancy before disease onset. As we have recently performed a prospective study to examine fetal cell traffic in pregnancies at risk for developing preeclampsia, we now quantify cell free fetal DNA concentrations in these samples. Methods: Blood samples were collected in the second trimester of pregnancy from pregnancies at risk for preeclampsia. Cell free fetal DNA amounts were quantified by real-time PCR. These results were then correlated with subsequent pregnancy outcome. Results: Free fetal DNA levels were significantly higher (median of 422.9 vs. 128.5 copies/mL maternal plasma; p=0.005) in plasma samples from women who developed preeclampsia (n=10) when compared to those who had unremarkable pregnancies (n=40). Conclusions: Our data independently confirm the finding that maternal plasma cell free fetal DNA levels are elevated early in pregnancies, which later develop preeclampsia.

Kinetics of fetal cellular and cell-free DNA in the maternal circulation during and after pregnancy: implications for noninvasive prenatal diagnosis.

Fetal genetic material is detectable in the maternal circulation and has been used for noninvasive prenatal diagnosis. However, few data are available concerning its quantity and natural history during gestation. This study prospectively characterized the kinetics of cellular and cell-free fetal DNA in the circulation of 25 healthy women during and after uncomplicated pregnancy. Real-time kinetic PCR was used to quantitate human Y-chromosome sequences, and liquid oligomer hybridization with (32)P-labeled probes was used to verify the identity of amplified products. In all male pregnancies, but no female pregnancies, low-level fetal Y-chromosome DNA was detected in both cellular and cell-free compartments beginning at 7 to 16 weeks but increasing steadily after 24 weeks and reaching a peak at parturition. The fetal DNA decreased rapidly after birth. Fetal genetic material can be detected throughout pregnancy, and its quantity is a function of gestational age and of whether the plasma or cellular compartment is examined. Both the absolute quantity of fetal DNA and its ratio to total DNA (maternal + fetal) are greater in the plasma than in the cellular compartment. Fetal DNA is cleared rapidly from both compartments after parturition, which suggests that turnover is dynamic. Because they provide prospective and quantitative data concerning fetal DNA levels, these observations and kinetic PCR methods may have implications for noninvasive prenatal diagnosis. Further studies will be needed to determine the immunologic implications of fetal-maternal DNA exchange and cellular microchimerism.

22 Fetal down syndrome is associated with increased cell-free fetal DNA levels in archived maternal serum samples

22 Fetal down syndrome is associated with increased cell-free fetal DNA levels in archived maternal serum samples

Both maternal and fetal cell-free DNA in plasma fluctuate.

Elevations in the concentration of cell-free fetal DNA in maternal plasma have recently been determined in various pregnancy-related disorders, including preeclampsia, preterm labor, and polyhydramnios. In addition, almost 2-fold increments in cell-free fetal DNA levels have been recorded in pregnancies with certain aneuploid fetuses, in particular trisomy 21. These findings have led to the speculation that quantitative assessment of circulatory fetal DNA may be useful in the noninvasive prenatal diagnosis of certain fetal genetic constellations or pregnancy-related disorders. A premise for any quantitative analysis is that the quantity of the analyte being assayed does not vary greatly over time. As this aspect has not been examined for circulatory DNA levels, we examined these in normal healthy individuals as well as in pregnant women. Our data indicate that severalfold alterations in circulatory DNA amounts do occur over short periods of time. Of particular note is that we observed almost 2-fold variations in free fetal DNA levels over a period of 3 days, which are in a similar range to the elevations noted in aneuploid pregnancies. Our results, therefore, imply that caution should be used when using small increments in circulatory fetal DNA concentrations for potential diagnostic applications.

Circulatory fetal and maternal DNA in pregnancies at risk and those affected by preeclampsia.

Elevations in fetal cell traffic as well as increased release of cell-free fetal DNA into the maternal periphery have previously been shown to occur in pregnancies affected by preeclampsia. Our own investigations have shown that manifestation of preeclampsia is associated with an increased accumulation of circulatory fetal DNA as well as cell-free maternal DNA in maternal plasma. We further established that the increments in these two molecular genetic analytes corresponded to the severity of the disease and to each other. This latter phenomenon was evident in preeclamptic pregnancies, but not in normal ones. As we had recently performed a prospective study to investigate fetal cell traffic prior to onset of preeclamptic symptoms, we examined the levels of cell-free fetal and maternal DNA in these samples. This analysis indicated that circulatory fetal DNA concentrations were significantly elevated prior to onset of the disease symptoms. No similar feature was observed for cell-free maternal DNA levels.

Quantitative analysis of fetal DNA in maternal plasma in pathological conditions associated with placental abnormalities.

An increased fetal DNA concentration in maternal plasma has been observed in placental pathological conditions associated with hypertension and preeclampsia. To confirm these data, we performed real-time quantitative PCR on the SRY gene in a group of physiological and pathological male-bearing pregnancies. In 78 physiological pregnancies, fetal DNA concentration in maternal plasma was 20.7, 13.4, 23.6, and 74.8 genome-equivalents (g.e.)/mL during the first, second, and third trimesters and at term, respectively. In 10 preeclamptic women, fetal DNA concentration ranged from 59.3 to 615.2 g.e./mL (median: 332.9). In 7 women with preeclampsia and IUGR (intrauterine growth retardation), fetal DNA ranged from 96.5 to 859 g.e./mL (median: 146.8). In 4 women with IUGR and hypertension, fetal DNA ranged from 34 to 473.5 g.e./mL (median: 142.4). In 3 patients with IUGR, fetal DNA ranged from 168.6 to 519.7 g.e./mL (median: 308.1). In 2 patients with IUGR and HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, fetal DNA concentration ranged from 105 to 394.1 g.e./mL (median: 249.7). Four women who developed preeclampsia some weeks later showed fetal DNA levels within the physiological range. These data suggest that increased fetal DNA concentrations might represent a valuable marker of placental abnormalities and suggest that this rise may precede clinical manifestation of preeclampsia by only a few weeks.

Fluctuation of Maternal and Fetal Free Extracellular Circulatory DNA in Maternal Plasma

In Brief Objective To examine whether concentrations of free extra-cellular fetal circulatory DNA in maternal plasma are stable or fluctuate. Methods Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). Results In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. Conclusion The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes. Quantitative polymerase chain reaction analysis showed that levels of fetal and maternal circulating extracellular DNA in plasma vary over time.

Fluctuation of maternal and fetal free extracellular circulatory DNA in maternal plasma

Objective: To examine whether concentrations of free extracellular fetal circulatory DNA in maternal plasma are stable or fluctuate. Methods: Consecutive blood samples were drawn from 13 healthy nonpregnant volunteers and from 16 healthy pregnant women over 3 days. DNA was isolated from the plasma fraction and quantified by real-time polymerase chain reaction (PCR). Results: In nonpregnant controls the total amount of cell free DNA fluctuated by an average of 13.5-fold. In samples obtained from pregnant women the amount of maternal cell free DNA varied by an average of 21.5-fold. Because ten of those women were pregnant with male fetuses, the concentration of free fetal DNA in these cases was determined by a real-time PCR assay for the Y chromosome. The mean variation in free fetal DNA levels in male fetuses was 2.2-fold. Conclusion: The degree of variation in free fetal DNA concentrations observed in this study was similar to published values, so these results imply that care should be exercised when considering quantitation of this fetal material for potential diagnostic or screening purposes.

Uteroplacental insufficiency alters cerebral mitochondrial gene expression and DNA in fetal and juvenile rats.

Uteroplacental insufficiency increases the risk of perinatal and long-term neurologic morbidity by depriving the fetus of oxidative substrate and causing intrauterine growth retardation. Skeletal muscle and liver from growth retarded fetal and juvenile rats respond to this deprivation by altering mitochondrial gene expression and function. The objective of this study was to determine whether cerebral mitochondrial mRNA is similarly altered in fetal and juvenile growth retarded rats and to correlate these alterations with mitochondrial DNA and marker protein levels. To fulfill this objective, mRNA levels of four important mitochondrial proteins were quantified using RT-PCR in growth retarded and sham-operated control fetal and juvenile rat brains; these proteins were NADH-ubiquinone oxireductase subunit 4, subunit C of the F1F0-ATPase, and the adenine nucleotide transporters 1 and 2. Mitochondrial DNA/nuclear DNA ratios and mitochondrial 60 kD marker protein levels were also quantified in growth retarded and sham-operated control fetal and juvenile rat brains using PCR and Western Blotting, respectively. Cerebral mRNA levels of all four proteins were increased in the IUGR fetuses and decreased in the IUGR juvenile animals. Cerebral mitochondrial/nuclear DNA ratios and mitochondrial marker protein levels were not significantly altered in the IUGR fetuses; however, both were significantly diminished in IUGR juvenile pups. These studies suggest that the metabolic stresses associated with uteroplacental insufficiency in the rat cause altered fetal and postnatal cerebral mitochondrial mRNA and DNA levels.

Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses.

Current non-invasive screening methods for the prenatal diagnosis of fetal aneuploidies are hampered by low sensitivities and high false positive rates. Attempts to redress this situation include the enrichment of fetal cells from maternal blood, or the use of fetal DNA in the plasma of pregnant women. By the use of real-time quantitative polymerase chain reaction (PCR) it has recently been shown that circulatory male fetal DNA in maternal plasma is elevated in pregnancies with trisomy 21 fetuses. In this independent study we confirm and extend upon these results by showing that the levels of fetal DNA are also elevated in pregnancies with other chromosomal aneuploidies (mean=185.8 genome equivalents/ml; range=62.2-471.7) when compared to pregnancies with normal male fetuses (mean=81.9 genome equivalents/ml; range=28.8-328.9), p=0.005. This elevation was greatest for fetuses with trisomy 21, whereas it was not significant for fetuses with trisomy 18, p=0.356. These data suggest that a quantitative analysis of such fetal DNA levels may serve as an additional marker for certain fetal chromosomal abnormalities, in particular for trisomy 21.