Fetal DNA in maternal plasma in preeclamptic pregnancies

Cell-free fetal DNA present in maternal circulation has revolutionized non-invasive prenatal diagnosis of genetic diseases. In preeclampsia, the quantity of fetal DNA in maternal plasma has been studied and found to be higher in comparison to healthy pregnant women. Whether the quantity of fetal DNA can be used as a reliable predictive biomarker of preeclampsia is currently uncertain. This is a systematic review on studies quantifying fetal DNA in preeclamptic pregnancies. Using a PubMed search 22 studies were identified. In all of them, elevated levels of fetal DNA in maternal plasma in preeclampsia were found. In some of the studies, the higher concentration of fetal DNA was observed before the onset of clinical symptoms. This shows that fetal DNA levels might have a potential informative value as an early diagnostic biomarker of preeclampsia. However, in most of the studies important data are missing and there is an enormous variability in the reported results between the studies. From the available data it is currently not possible to perform a meta-analysis due to the variation between studies. If once fetal DNA should be used as a marker for determining preeclampsia at early stage, it is necessary to reduce these variations via standardized protocols for the quantification of cell-free fetal DNA as well as its reporting in the publications.     

Human chorionic gonadotropin levels in pregnancies with aneuploid fetuses

Maternal serum human chorionic gonadotropin (hCG) and the free alpha-hCG subunit were evaluated in 249 women from 9 to 11 weeks gestation who subsequently underwent chorionic villus sampling for determination of fetal karyotype and in 20 women of 18 or more weeks gestation who were ascertained to have an aneuploid fetus by genetic amniocentesis. Seven of the first-trimester pregnancies were determined to be aneuploid and six had hCG levels in the normal range (one triploid pregnancy had elevated hCG levels) whereas 12 of the 20 second-trimester cases had abnormal hCG levels and an additional three had elevated levels of alpha-hCG. This study confirms the previous report of abnormal maternal serum hCG levels in women with an aneuploid fetus at greater than or equal to 18 weeks gestation and demonstrates that hCG evaluation is not useful at 9-11 weeks gestation for selecting pregnancies at risk for fetal aneuploidy.     

First-trimester maternal serum progesterone in aneuploid pregnancies

BACKGROUND: First-trimester maternal serum screening for Down syndrome (DS) can be improved by the use of additional serum markers. We examined whether progesterone (P), synthesized by placenta, might be a first-trimester maternal serum marker for fetal DS. MATERIALS AND METHODS: P was quantified in first-trimester maternal serum from 42 DS, six trisomy 18 and two trisomy 13 pregnancies and 115 controls. Log-regression of P versus gestational age in days was used to convert P concentrations into multiples of the median (MoM). RESULTS: The P concentrations in controls increased with gestational age (p = 9.5 x 10(-7)). The log10MoM P distribution in DS pregnancies was not significantly different from that in controls. However, from day 58-67, the log10MoM P was elevated in DS pregnancies (n = 10) with a mean (SD) of 0.1040 (0.0956), compared to a mean (SD) of - 0.0109 (0.1661) in controls (n = 24) (p = 0.05). Five out of six trisomy 18 and both trisomy 13 pregnancies had a P MoM < 1. CONCLUSION: P is not a useful marker for DS in first trimester, except perhaps in a narrow gestational age window from day 58 to 67. P is a trisomy 18/13 marker.     

Fetal DNA in maternal plasma: emerging clinical applications

OBJECTIVE: To review the potential clinical diagnostic applications of fetal DNA analysis in maternal plasma or serum for noninvasive prenatal diagnosis and screening. DATA SOURCES: We conducted a MEDLINE search of articles published between January 1970 and March 2000 using the key terms "fetal DNA," "plasma," and "serum." METHODS OF STUDY SELECTION: All 369 articles describing the detection of fetal DNA in maternal plasma were reviewed. RESULTS: The diagnostic use of circulating fetal DNA in maternal plasma is currently limited to genes that are present in the fetus but not in the mother. From a clinical perspective, the most advanced application is for noninvasive detection of fetal rhesus D (Rh[D]) genotype. The results of studies performed by four different groups showed that prenatal diagnosis of fetal Rh(D) status by molecular analysis of maternal plasma or serum is routinely possible beginning in the second trimester. Noninvasive fetal genotyping should be useful for the treatment of sensitized Rh(D)-negative women whose partners are heterozygous for the Rh(D) gene because no further diagnostic or therapeutic procedures are necessary if the fetus is Rh(D) negative. Future clinical applications of fetal DNA may be in its use as a screening test for Down syndrome, preeclampsia, or preterm labor. However, these applications currently rely on the detection of Y chromosomal sequences and consequently are limited presently to male fetuses. CONCLUSION: The recent discovery of high concentrations of fetal DNA in maternal plasma represents a promising noninvasive approach to prenatal diagnosis. Compared with the analysis of the cellular fraction of maternal blood, the analysis of fetal DNA extracted from maternal plasma has the advantage of being rapid, robust, and easy to perform. The fetal DNA detected is limited to the current pregnancy. However, universal fetal gene sequences must be identified that allow analysis of genetic material from both male and female fetuses. Study of fetal DNA in maternal plasma can improve our understanding of fetomaternal biology and physiology. The long-term effects of maternal exposure to relatively high amounts of foreign DNA are unknown but represent an exciting area for future inquiry.     

First-trimester maternal serum alpha-fetoprotein and chorionic gonadotropin in aneuploid pregnancies.

First-trimester maternal serum alpha-fetoprotein (AFP) and human chorionic gonadotropin (HCG) levels were measured in samples from 29 women with cytogenetically abnormal pregnancies and 145 women with cytogenetically normal pregnancies matched for gestational age, race, and sample storage time. All patients had a risk of fetal aneuploidy greater than or equal to that of a mother 35 years of age. AFP was significantly lower in samples from pregnancies affected with trisomy 21 (0.67 MoM; p less than 0.05), while HCG values were no different from those of matched controls. Trisomies 13 and 18 could not be distinguished from matched controls by AFP. However, levels of HCG were significantly lower in such pregnancy samples, with median values of 0.65 MoM in trisomy 13 and 0.32 MoM in trisomy 18 (p less than 0.05). Variations in AFP and HCG levels suggest that expressed differences between autosomal aneuploidies include differences in fetal and placental protein production in the first trimester.     

Correlation of fetal DNA levels in maternal plasma with Doppler status in pathological pregnancies

OBJECTIVES: To evaluate whether intrauterine growth restriction (IUGR) as seen in preeclampsia is associated with high levels of fetal DNA in maternal circulation, and whether fetal DNA is related to altered uterine and/or umbilical artery Doppler velocimetry. METHODS: Fetal DNA quantification was performed by real-time PCR on SRY sequences in 64 male-bearing pregnant women with IUGR and/or preeclampsia and 89 controls. RESULTS: Fetal DNA content was significantly elevated in IUGR pregnancies similar to preeclampsia and correlated with altered umbilical Doppler velocimetry, while no correlation was found with uterine Doppler status. CONCLUSION: Increased fetal DNA levels in maternal plasma may be a sign of placental or fetal pathology even in the presence of normal uterine Doppler velocimetry, allowing a more precise diagnostic evaluation. The finding that elevated fetal DNA in IUGR pregnancies correlates with abnormal umbilical Doppler velocimetry suggests that fetal DNA release is associated more with fetal chronic hypoxia than with fetal size.     

Fetal sex determination from maternal plasma in pregnancies at risk for congenital adrenal hyperplasia

OBJECTIVE: To determine first-trimester fetal sex by isolating free fetal DNA from maternal plasma. METHODS: The index case was a pregnant woman who previously delivered a girl with congenital adrenal hyperplasia. The SRY gene as a marker for the fetal Y chromosome was detected in maternal serum and plasma by quantitative polymerase chain reaction analysis. Simultaneously, we performed the same test in 25 and 19 women in the first and second trimester, respectively, and compared plasma results with fetal gender as assessed by prenatal karyotyping or as seen at ultrasound or birth. RESULTS: In 44 of 45 patients at gestational ages ranging from 8 3/7 to 17 3/7 weeks, we correctly predicted fetal sex using quantitative polymerase chain reaction analysis of the SRY gene in maternal plasma. In one case, the test result was inconclusive. Overall, fetal sex was correctly predicted in 97.8% of cases (95% confidence interval 88.2%, 99.9%). CONCLUSION: Amplification of free fetal DNA in maternal plasma is a valid technique for predicting fetal sex in early pregnancy. In case of pregnancies at risk for congenital adrenal hyperplasia, the technique allows restriction of dexamethasone treatment to female fetuses resulting in a substantial decrease of unnecessary treatment and invasive diagnostic tests.     

Free fetal DNA in maternal plasma in anembryonic pregnancies: confirmation that the origin is the trophoblast

OBJECTIVE: To test the hypothesis that free fetal DNA (ffDNA) circulating in maternal plasma originates mainly from the placenta we studied ffDNA levels in anembryonic pregnancies. METHODS: Maternal blood samples were collected from 15 normal first-trimester pregnancies in which fetal sex was subsequently determined and nine patients with a diagnosis of anembryonic gestation (AG). The Y chromosome DYS14 gene was quantified by real-time quantitative PCR (RT-PCR) for the determination of fetal sex in both plasma and chorionic tissue samples. Fetal sex in chorionic tissue samples was also determined using quantitative fluorescence PCR (QF-PCR). RESULTS: The correct sex result was obtained from maternal plasma in all. Four AG pregnancies were female (DYS14 negative) results. In five of the AG cases, the chorionic tissue was found to be male (by both QF-PCR and RT-PCR which agreed) and positive male signal was found in maternal plasma by RT-PCR. There was no statistical difference between median free fetal DNA concentration in plasma between the AG male cases (148.3 GE/mL) and controls (145.8 GE/mL). CONCLUSION: Since ffDNA levels are normal in pregnancies without a fetus, the data support the hypothesis that the trophoblastic cells are the major source ffDNA in maternal plasma.     

Significance of raised maternal serum alpha-fetoprotein in singleton pregnancies with normally formed fetuses

One hundred eighty-six pregnancies with elevated maternal serum alpha-fetoprotein (AFP) between 16 and 20 weeks' gestation, but with normally formed single fetuses, were analyzed retrospectively. In comparison with matched control subjects, there was an increased incidence of low birth weight, preterm delivery, intrauterine growth retardation, and other clinical complications, especially when maternal serum AFP was abnormally high on more than one occasion. These findings could not be explained by the occurrence of threatened abortion or the performance of amniocentesis. It is suggested that where maternal serum AFP screening for fetal neural tube defects is already established as a cost-effective routine procedure, the additional recognition of some pregnancies at very high risk of other, perinatal complications is of practical value. However, maternal serum AFP testing in the second trimester cannot be recommended as a cost-effective screening method for detecting low birth weight infants, having a sensitivity in this series of only 11%. Many (33%) of the low birth weight infants detected in this way were very small (birth weights less than 1.5 kg); 73% of the predicted preterm births were very premature (before 34 weeks of gestation), and 72% of the identified growth-retarded infants were severely effected (weighing less than the fifth percentile for gestational age).     

SRY-specific cell free fetal DNA in maternal plasma in twin pregnancies throughout gestation

Objectives

Levels of SRY-specific cell free fetal DNA (SRY-cffDNA) in maternal plasma were investigated in twin pregnancies with two male fetuses versus one male and one female fetus and singleton male pregnancies during second and third trimester. The aim was to evaluate at which gestational age the amount of SRY-cffDNA reflects the number of fetuses and placentas respectively.

Methods

251 venous blood samples were analyzed from a total of 178 women with male or mixed-gender twin pregnancies and male singleton pregnancies in the second and the third trimester. The concentration of SRY-cffDNA was determined by quantitative real time PCR using the Y-chromosome specific SRY assay. For statistical analysis these three groups were divided into four subgroups according to their gestational age.

Results

During second trimester levels of SRY-cffDNA showed no differences between twin and singleton pregnancies. After 28 weeks SRY-cffDNA of male twin pregnancies was significantly increased compared to singleton male pregnancies and mixed-gender twin pregnancies with no differences between the latter two.

Conclusion

The level of SRY-cffDNA in maternal serum of twin pregnancies reflects the number of fetuses only during the third trimester. Hence its use as a diagnostic tool for complications related to altered SRY-cffDNA levels in twin pregnancies should be evaluated at different weeks of gestation, especially during the second trimester.     

Non-invasive prenatal diagnosis using cell-free fetal DNA in maternal plasma from PGD pregnancies

Preimplantation genetic diagnosis (PGD) is usually used to establish a non-affected pregnancy for those couples facing a genetic risk of having an affected child. However, an invasive test is still recommended to all PGD patients due to the risk of misdiagnosis. The discovery of cell-free fetal DNA in maternal plasma provides the possibility for non-invasive prenatal diagnosis. Studies have shown that fetal single-gene disorders can be detected in cell-free fetal DNA by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single-allele base extension reaction (SABER) approach or by the size-fractionation approach, whereby cell-free fetal DNA is enriched on the basis of its smaller size compared with maternal DNA fragments. Recent studies have indicated that a combination of the two approaches increases the accuracy of detection. This study combined the two methods and examined fetal paternally inherited gene mutations in maternal plasma obtained from four PGD-conducted pregnancies. The presence or absence of mutations was correctly detected in all cases. This combined method could be used for risk-free prenatal diagnosis of diseases caused by single-gene mutations, and in particular for couples who undergo PGD who opt not to perform invasive prenatal confirmation due to the risk of abortion.     

Fetal and maternal levels of lipid peroxides in term pregnancies

OBJECTIVE: To examine the relationships between maternal and fetal concentrations of lipid peroxides in term pregnancies before the onset of labor. METHODS: Umbilical cord arterial and venous blood samples were collected from 114 singleton term pregnancies delivered by elective cesarean section. Base excess, oxygen, carbon dioxide and pH were measured in both samples and compared to identify double venous samples. Maternal venous and umbilical cord arterial and venous concentrations of organic hydroperoxides and malondialdehyde were assayed. RESULTS: Maternal plasma malondialdehyde was, on average, double that of cord blood, whereas maternal organic hydroperoxide was only 18% higher. Maternal organic hydroperoxide was correlated with cord arterial and venous levels of organic hydroperoxide but not with pH, carbon dioxide, oxygen or base excess. Maternal malondialdehyde concentration was significantly correlated with both umbilical arterial and venous values of malondialdehyde and with arterial oxygen. Multiple regression shows that 70% of the variation in maternal malondialdehyde can be accounted for by variation in arterial and venous malondialdehyde, and arterial oxygen and base excess. A similar regression analysis with maternal organic hydroperoxide as dependant variable incorporated only umbilical arterial organic hydroperoxide concentration. CONCLUSION: These findings suggest that there is significant trans-placental transport of malondialdehyde from the fetal circulation.     

Fetal heart rate patterns in pregnancies complicated by maternal diabetes

Objective: To compare the fetal heart rate (FHR) pattern between fetuses of well controlled diabetic and non diabetic mothers using a computerized analysis of FHR. Study design: Weekly fetal surveillance was performed in 99 fetuses of mothers with diabetes class A, 21 fetuses of mothers with diabetes class B-R, and 55 fetuses of non-diabetic women, starting at 30 weeks' gestation. All diabetic patients were well controlled. Fetal surveillance included a computerized analysis of the FHR, umbilical and uterine Doppler velocimetry, and a biophysical profile. Changes of FHR variation, frequency of FHR accelerations, and umbilical and uterine Doppler velocimetry were calculated using a regression analysis for each patient. The average slopes and the intercept at 30, 34, and 38 weeks' gestation of these variables were compared among the three groups. Results: The slope of FHR variation and the frequency of accelerations had a lower rate of increase during the third trimester in fetuses of mothers with diabetes class A (0.84 ± 0.25 ms/week and 0.06 ± 0.02/20 min/week, respectively) compared with fetuses of non-diabetic mothers (1.34 ± 0.55 ms/week and 0.5 ± 0.1/20 min/week, respectively). In fetuses of mothers with diabetes class B-R, FHR variation did not change with gestation (−0.011 ± 0.2 ms/week) with a small increase in the frequency of accelerations (0.02 ± 0.004/20 min/week. While no differences were observed at 30 weeks' gestation, FHR variation and the frequency of accelerations were significantly reduced at 34 weeks' gestation in fetuses of mothers with diabetes class B-R compared with fetuses of non-diabetic mothers (P < 0.01). At 38 weeks' gestation, fetuses of mothers with diabetes class B-R and diabetes class A had both significantly reduced FHR variation as well as frequency of accelerations compared with fetuses of non-diabetic mothers (P < 0.01). The rate of decrease of the umbilical and uterine artery S/D ratios were similar among the three groups. Conclusions: The FHR pattern appears to be different in fetuses of well controlled diabetic mothers when related to fetuses of non-diabetic mothers. Disease specific standards should be considered for interpretation of FHR patterns in diabetic pregnancies.     

Fetal and maternal Doppler blood flow parameters in postterm pregnancies

In this preliminary investigation, we sought evidence of increasing impedance to placental blood flow from both sides of the placenta and evidence of compromised fetal aortic blood flow in 35 human pregnancies exceeding 42 weeks' gestation. Fetal age was confirmed by biparietal diameter (BPD) measurements obtained before 21 weeks. Pourcelot's Index, calculated from Doppler sonograms recorded with a noninvasive technique from small arteries in the myometrium and from an umbilical cord artery, did not correlate with the duration of the pregnancy beyond term. However, this "resistance index" of Pourcelot was higher in the umbilical cord arteries of fetuses with a worse clinical outcome. Doppler blood cell velocity in the fetal descending aorta correlated significantly and negatively with the prolongation of gestation. Fetal aortic velocities appeared to be lower in fetuses who passed meconium before delivery. Our findings suggest that fetal compromise in prolonged pregnancy is more a fetal-placental problem than a uteroplacental problem.     

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

BACKGROUND: We 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. METHODS: DNA levels in maternal plasma were measured using real-time quantitative PCR using SRY and beta-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. RESULTS: The 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). CONCLUSIONS: Our study revealed no difference in fetal DNA levels and fetal DNA: maternal DNA ratio between the patients carrying Down syndrome fetuses and the controls.