Cell-free Fetal DNA Levels Research Articles

Comparative study between levels of maternal serum Cell-Free Fetal DNA and Uric acid in pregnant women with and without Preeclampsia

Comparative study between levels of maternal serum Cell-Free Fetal DNA and Uric acid in pregnant women with and without Preeclampsia

Association of Cell-Free Fetal DNA at 11-17 Weeks of Pregnancy and the Outcome of Pregnancy

Objectives: This study aimed to evaluate the association between cell-free fetal DNA (cffDNA) levels in the first and second trimesters and adverse pregnancy outcomes. Materials and Methods: This was a retrospective cohort study including all women who had cffDNA measurement as a primary or secondary screening test for chromosomal abnormalities in a screening laboratory in Iran. Results: Totally, 481 out of 634 pregnant women (75.9%) did not develop any pregnancy complications. On the other hand, 55 pregnancies (8.7%) led to preterm delivery. Based on the results, 33 pregnant women (5.2%) gave birth to intrauterine growth restriction (IUGR) babies and 16 pregnant women (2.52%) developed preeclampsia (PE). Moreover, 15 women developed gestational diabetes. Further, 11 pregnancies resulted in abortion and 6 pregnancies led to fetal death. There was a positive association between the duration of pregnancies and the weight of the babies at birth (r=0.362, P=0.000). There was a statistically significant association between the increase in cffDNA level and trisomy (P=0.000) and the decrease in cffDNA level and the incidence of PE (P=0.019). Conclusions: The reduction of plasma cffDNA level in pregnancies led to PE and the elevation of cffDNA results in fetal trisomy. However, further studies will be required to confirm these findings.

Circulating Maternal Serum Cell Free Fetal DNA Levels for prediction of Preeclampsia

Background: Pre-eclampsia is a pregnancy-specific disorder that has a worldwide prevalence of 5–15%. It is one of the main causes of maternal and perinatal morbidity and mortality globally and accounts for 50 000–60 000 deaths annually, with a predominance in the low- and middle-income countriesThere is a great need to have a test for predicition of women at high risk of developing preeclampsia. Aim of the Work: to assess the accuracy of maternal serum Cff-DNA concentration levels in primigravidas 10-20 weeks gestation as a predictive test for the development of preeclampsia. Patients and Methods: A nested case-control study was conducted on 26 patients with PE and 26 matched controls rimigravidas in 10-20 weeks gestation recruited from Ain-Shams University Maternity Hospital. Laboratory work was done in Ali Khalifa laboratory of the biochemistry department, Faculty of Medicine, Ain-Shams University. Results: The study showed a statistically significant increase median of severe preeclampsia compared to non severepreeclampsia and control group according to cff-DNA (GE/ml). Also the study results revealed a statistically significant increase median of preeclampsia group compared to control group according to BMI, GA (wks), SBP and DBP. Conclusion: Cff-DNA quantification can be considered as a promising marker for preeclampsia prediction, especially for the development of early-onset or severe preeclampsia.

Content of Free Fetal DNA in Maternal Blood and Expression of DNA Recognition Receptors ZBP-1 in Placental Tissue in Preeclampsia and Preterm Labor.

We evaluated the content of cell-free fetal DNA in maternal blood and expression of ZBP-1 receptors in the placental tissue of women with uncomplicated pregnancy, preeclampsia, and preterm labor. The study included 16 women with preeclampsia (early and late-onset preeclampsia, 8 cases each), 16 women with preterm labor, and 21 women with uncomplicated pregnancy. The concentration of cell-free fetal DNA was measured by PCR by detecting hypermethylated region of the RASSF1A gene. Immunohistochemistry was performed on paraffin-embedded sections of the placenta samples using primary polyclonal antibodies to ZBP-1. Significant increase in the level of cell-free fetal DNA was found in women with preeclampsia (both early and late-onset form) in comparison with uncomplicated pregnancy. The concentration of cell-free fetal DNA in preterm labor group did not differ from the control group; however, it was significantly lower than in early-onset preeclampsia, but not late preeclampsia. Immunohistochemical study showed higher expression of ZBP-1 in the villus syncytiotrophoblast in early-onset preeclampsia in comparison with that in preterm labor group (p=0.006). Fragments of damaged placental cells, predominantly trophoblast, enter maternal circulation and are the source of cell-free fetal DNA and a potential ligand for ZBP-1, which leads to further cell damage and the formation of a vicious circle. The increase in the content of cell-free fetal DNA in maternal blood and ZBP-1 expression in the syncytiotrophoblast in preeclampsia are interrelated processes reflecting impaired morphofunctional state of the placenta.

Hyperemesis Gravidarum, Diagnosis, and Pathogenesis

Nausea and vomiting are common in pregnancy and the condition may be mild or severe disabling disease. The severe form is known as Hyperemesis Gravidarum (HG) which is characterized by dehydration, electrolyte and metabolic imbalances, and nutritional deficiencies that may cause hospital admission. The severity of Nausea and Vomiting in Pregnancy (NVP) may be assessed by Pregnancy Unique Qualification of Emesis (PUQE) which showed a relatively accurate evaluation of the patient’s lifestyle including sleeping hours. Focused history and examination are needed to exclude differential diagnosis because the diagnosis of this condition is mainly determined clinically. Moreover, lab investigations are useful tools to assess complications as electrolyte or metabolic imbalances and renal affection. Besides, they help in determine etiologies in refractory cases. Many theories have been hypothesized to contribute to understanding the pathogenesis of HG. They have been postulated based on associations which are either causal, sequential or coincidental. Many efforts are needed to accurately establish these relationships in terms of well-designed studies of adequate sample size. The hormonal theory is one of the main theories involved in the pathogenesis of the disease specifically human Chorionic Gonadotropin (hCG), estrogen, progesterone, and serotonin. Psychological theory has conflicting evidencebased studies. Therefore, more studies are needed to decide whether psychological problems cause HG or this is a coincidental or sequential relationship. The immunological theory is newly defined and supported by high levels of cellfree fetal DNA and cytokines.

Extracellular DNA in the Dynamics of Uncomplicated Pregnancy.

We analyzed the concentration of extracellular DNA and its fractions in the dynamics of uncomplicated pregnancy Thirty women with singleton pregnancy were examined. The concentration of total, maternal, and fetal cell-free DNA in maternal blood was measured at gestation weeks 11-14, 24-26, and 30-32. The level of total cell-free DNA was evaluated by measuring the concentration of RASSF1A gene using quantitative PCR analysis, the level of cell-free fetal DNA was assessed by determining the hypermethylated part of RASSF1A gene. The concentration of total cell-free DNA and cell-free maternal DNA did not change during the first half of pregnancy, but increased after 24-26 weeks. The level of cell-free fetal DNA increased from the first to the second and third trimester: 14.15 (2.32-36.25), 24.87 (6.29-129.32), and 32.62 (8.97-133.52) GE/ml (p<0.05), respectively. Our results characterize the dynamics of the content of cell-free DNA and its fractions during pregnancy, which should be taken into account when using cell-free DNA for prediction of placenta-associated complications.

No increase in free fetal DNA level in ectopic pregnancy: A preliminary study

Objective:The aim of this study was to diagnose ectopic pregnancy in the early period by measuring cell-free fetal DNA (cffDNA) levels in maternal blood using spectrophotometry.Materials and Methods:Thirty patients with ectopic pregnancy and 30 patients with first trimester intrauterine pregnancy were enrolled in this prospective controlled study. cffDNA levels in maternal serum were measured using spectrophotometry.Results:There were no differences between the two groups in terms of cffDNA absorbance levels.Conclusion:Spectrophotometry is not suitable for measuring cffDNA levels to diagnose ectopic pregnancies in the early period. Practical and cost-effective methods should be found or larger patient series should be investigated.

Development and evaluation of training resources to prepare health professionals for counselling pregnant women about non-invasive prenatal testing for Down syndrome: a mixed methods study

BackgroundThe availability of non-invasive prenatal testing (NIPT) for aneuploidies is expanding rapidly throughout the world. Training health professionals to offer NIPT in a way that supports informed choice is essential for implementation. The aim of this study was to develop and evaluate a training package for health professionals to support the introduction of NIPT into clinical practice.MethodsTraining on NIPT was offered to health professionals, primarily midwives, involved in Down syndrome screening and testing in eight hospitals located in England and Scotland as part of a research study evaluating the implementation of NIPT in the UK National Health Service. Training was evaluated using a mixed methods approach that included quantitative questionnaires at three time points and post-training qualitative interviews. The questionnaires measured confidence, self-perceived knowledge and actual knowledge about NIPT for Down syndrome. Interviews explored opinions about the training and experiences of offering NIPT.ResultsThe training provided to the health professionals was found to positively impact on their confidence in discussing NIPT with women in their clinic, and both their perceived and actual knowledge and understanding of NIPT was improved. Knowledge remained weak in four areas; cell-free fetal DNA levels increase with gestation; turnaround time for NIPT results; cell-free fetal DNA is placental in origin; and NIPT false positive rate.ConclusionsTraining materials, including a lesson plan, PowerPoint presentation and written factsheet on NIPT, have been developed and evaluated for use in educating midwives and supporting the introduction of NIPT. Implementation of training should include a greater focus on the areas where knowledge remained low. Some groups of midwives will need additional training or support to optimise their confidence in discussing NIPT with women.

Absolute first trimester cell-free DNA levels and their associations with adverse pregnancy outcomes

To study associations of first trimester cell-free fetal DNA levels (in this paper referred to as cell-free placental DNA (cfpDNA) levels) and preeclampsia (PE), pregnancy-induced hypertension (PIH), gestational diabetes (GDM) and spontaneous preterm birth (sPB). A nested case-control study was conducted in first trimester samples (gestational age 8+0 -13+6 weeks). A total of 226 cases and 301 controls were included. CfpDNA levels were quantified in male-bearing pregnancies using real-time DYS14-PCRs on DNA isolated from maternal serum. CfpDNA multiples of the median (MoMs) were calculated based on associations with patient characteristics (body mass index, parity, ethnicity and smoking). Associations between MoMs and adverse outcomes were studied. Cell-free placental DNA levels were negatively associated with body mass index (β = -0.297, p < 0.001) and smoking (β = -0.163, p = 0.006). MoMs were lower in women who later developed PIH (n = 84, p = 0.009) or GDM (n = 56, p = 0.037). There was no association between cfpDNA MoMs and PE (n = 37, p = 0.15) or sPB (n = 49, p = 0.19). CfpDNA was positively correlated with pregnancy-associated plasma protein A (r = 0.426, p < 0.001) but not with placental growth factor (r = 0.059, p = 0.179). Adjusted first trimester cfpDNA levels are associated with PIH and GDM but probably not with PE or sPB. © 2016 John Wiley & Sons, Ltd.

Circulating Maternal Total Cell-Free DNA, Cell-Free Fetal DNA and Soluble Endoglin Levels in Preeclampsia: Predictors of Adverse Fetal Outcome? A Cohort Study.

The diagnosis of preeclampsia (PE) is based on the measurement of maternal blood pressure and proteinuria; however, these parameters are not used in the prediction of adverse fetal outcomes that may occur due to fetal stress. The plasma concentrations of total cell-free DNA (cf-DNA), cell-free fetal DNA (cff-DNA) and soluble endoglin (sEng) are higher in women with established PE than in normotensive controls, and the increase is particularly marked in those with severe PE. We aimed to evaluate the levels of cf-DNA, cff-DNA and sEng in pregnant Egyptian women with PE in order to assess the severity of the disease and to detect their potential utility in the future prediction of time of delivery and adverse fetal outcome. The study included 107 pregnant females with established PE during their third trimester (51 with mild PE and 56 with severe PE), together with 93 normotensive pregnant women. Absolute quantitation of the hemoglobin subunit beta (HBB) and testis-specific protein, Y-linked 1 (TSPY1) genes for the measurement of cf-DNA and cff-DNA in maternal blood, respectively, was carried out using real-time polymerase chain reaction (PCR) together with the measurement of serum sEng using ELISA. An almost twofold increase in cf-DNA and cff-DNA was detected in the severe PE group over the mild group, and both were significantly different from the control group. Significantly higher levels of cf-DNA, cff-DNA and sEng, with variable magnitudes, were detected in the preterm labor and unfavorable fetal outcome groups compared with the term and favorable outcome groups, respectively. The three markers were almost equivalent with regard to the area under the curve for predicting adverse fetal outcome in the severe PE group. The same was also true for cf-DNA and cff-DNA within the mild PE group. Incorporation of cf-DNA, cff-DNA and sEng into the prenatal care service should be considered as a serious addition for the screening and detection of adverse pregnancy outcomes in view of their significant elevations in cases of preeclamptic women whose babies ultimately suffered a poor outcome.

588: Can high maternal level of cell-free fetal DNA be used as a predictor for spontaneous preterm birth?

588: Can high maternal level of cell-free fetal DNA be used as a predictor for spontaneous preterm birth?

773: Is high maternal level of cell-free fetal DNA associated with adverse pregnancy outcome?

773: Is high maternal level of cell-free fetal DNA associated with adverse pregnancy outcome?

The effect of exercise and metformin treatment on circulating free DNA in pregnancy

IntroductionSome pregnancy complications are characterized by increased levels of cell-free fetal (cffDNA) and maternal DNA (cfmDNA), the latter may also be elevated during physical strain. This study aims at assessing the impact of exercise and metformin intervention in pregnancy, and to compare the levels of cell free DNA in pregnant women with or without PCOS diagnosis. MethodsConsecutive women from two previous randomized controlled trials in pregnancy were included. Women came from a trial with organized exercise vs. standard antenatal care in pregnancy and a trial of metformin vs. placebo in PCOS women. Levels of cffDNA, cfmDNA and cell-free total DNA (cftDNA) were measured by qPCR. ResultsTraining in pregnancy did not affect the levels of cffDNA, cfmDNA or cftDNA.PCOS-women treated with metformin had lower levels of cfmDNA and cftDNA at week 32 (mean ± SD: 301 ± 162 versus 570 ± 337, p = 0.012, 345 ± 173 versus 635 ± 370, p = 0.019); otherwise the levels were comparable to PCOS-controls. Metformin-treated PCOS-women had higher cffDNA at inclusion, in the 1st trimester; later on in pregnancy the levels in the metformin and placebo groups were equal.A comparison of pregnant women in the exercise study (TRIP) to placebo-treated pregnant PCOS-women, showed the levels of cffDNA, cfmDNA or cftDNA during mid-pregnancy (weeks 18–36) to be equal. DiscussionTraining during pregnancy was not associated with altered levels of cffDNA cfmDNA or cftDNA, but metformin treatment may reduce cfmDNA and cftDNA in pregnant PCOS women.

Assessment of plasma cell-free fetal DNA using hypermethylated RASSF1A in maternal plasma in cases of spontaneous preterm labor

Objective The aim of this study was to determine the relation between the levels of cell-free fetal DNA (cffDNA) in maternal plasma and the occurrence of preterm labor. Materials and methods This was a prospective case–control study including 60 pregnant women between 28 and 32 weeks’ gestation, divided into a study group that included 30 patients with threatened or established preterm labor, and another 30 pregnant women with no symptoms or signs of preterm labor, representing the control group. cffDNA levels were quantified by real-time PCR using the RASSF1A gene before and after treatment by methylation-sensitive restriction enzyme, respectively, irrespective to fetal sex. Results The mean cffDNA levels in the study and control groups were 229.12±71.95 and 39.81±5.95 copies/ml, respectively. The cffDNA level was about six folds increased in group A (study group), with a statistical significance (P<0.05). Conclusion High levels of cffDNA between 28 and 32 weeks are associated with an increased risk of spontaneous preterm delivery.

Noninvasive prenatal diagnosis experience in the Çukurova Region of Southern Turkey: detecting paternal mutations of sickle cell anemia and β‐thalassemia in cell‐free fetal DNA using high‐resolution melting analysis

This study used a high-resolution melting (HRM) technique to detect paternal mutations for the noninvasive prenatal diagnosis (NIPD) of β-thalassemia and sickle cell anemia (HbS). We also determined the levels of cell-free fetal DNA and total cell-free DNA. We used the HRM technique for fetal genotyping of paternal mutations in maternal plasma from 32 pregnancies at risk of β-thalassemia and 57 pregnancies at risk of HbS. The DNA levels in maternal plasma were measured using real-time quantitative PCR. Multiples of the median (MoM) values were calculated in women at risk for β-thalassemia or HbS. Twenty-two paternal mutations were detected in 89 pregnant women. Although we were successfully able to detect the paternal β-thalassemia mutations, the mutant HbS fetuses could not be distinguished from maternal background in the early weeks of pregnancy. The detection of DYS14 in male fetuses was 100%. The MoM values of women at high risk of having HbS-affected fetuses were higher than those for the other groups. High-resolution melting is a useful method for NIPD of β-thalassemias by detecting paternal mutations in the maternal plasma. Cell-free fetal DNA quantification and MoM values were not informative for HbS or β-thalassemias in early pregnancy.

Plasma circulating cell-free DNA and uteroplacental blood flow in pre-eclamptic patients

Objective The aim of the present study is to evaluate the plasma total cell-free DNA and cell-free fetal DNA level in maternal venous samples obtained from pre-eclamptic pregnant women during the third trimester (gestational age>28 weeks) and whether it shows a correlation with uteroplacental blood flow. Materials and methods This was a case–control study in which plasma total cell-free DNA, cell-free fetal DNA levels, and uteroplacental blood flow including umbilical artery resistance index, uterine artery resistance index and pulsitility index, and middle cerebral artery/umbilical artery pulsitility index were measured in 60 women diagnosed with pre-eclampsia, representing the study group, and another 60 normotensives were taken as controls. Both total cell-free DNA and cell-free fetal DNA were quantified by real-time PCR using the RASSF1A gene before and after treatment by methylation-sensitive restriction enzyme, respectively, irrespective of the sex of the fetus. Results Our results showed that there was a positive correlation between plasma cell-free fetal DNA, total cell-free DNA, and uteroplacental blood flow in pre-eclamptic women (P<0.05). To detect pre-eclampsia, the best cut-off value for plasma cell-free fetal DNA level was 80 copies/ml and that for plasma total cell-free DNA level was 137 copies/ml. Conclusion Our results found that there was a positive correlation between plasma cell-free fetal DNA level and the severity of pre-eclampsia. In addition, we found a correlation between plasma cell-free fetal, total cell-free DNA level, and uteroplacental blood flow in pre-eclamptic women during the third trimester.

Cell-Free Fetal DNA and Cell-Free Total DNA Levels in Spontaneous Abortion with Fetal Chromosomal Aneuploidy

BackgroundCell-free fetal DNA and cell-free total DNA in maternal circulation have been proposed as potential markers for noninvasive monitoring of the placental condition during the pregnancy. However, the correlation of and change in cell-free fetal DNA and cell-free total DNA in spontaneous abortion (SA) with fetal chromosomal aneuploidy have not yet been reported. Therefore, we investigated cell-free fetal DNA and cell-free total DNA levels in SA women with fetal chromosomal aneuploidy.Methodology/Principal FindingsA nested case-control study was conducted with maternal plasma collected from 268 women in their first trimester of pregnancy. Subjects included 41 SA with normal fetal karyotype, 26 SA with fetal chromosomal aneuploidy, and 201 normal controls. The unmethylated PDE9A gene was used to measure the maternal plasma levels of cell-free fetal DNA. The GAPDH gene was used to measure the maternal plasma levels of cell-free total DNA. The diagnostic accuracy was measured using receiver-operating characteristic (ROC) curves. Levels of cell-free fetal DNA and cell-free total DNA were significantly higher in both SA women with normal fetal karyotype and SA women with fetal chromosomal aneuploidy in comparison with the normal controls (P<0.001 in both). The correlation between cell-free fetal DNA and cell-free total DNA levels was stronger in the normal controls (r = 0.843, P<0.001) than in SA women with normal karyotype (r = 0.465, P = 0.002) and SA women with fetal chromosomal aneuploidy (r = 0.412, P = 0.037). The area under the ROC curve for cell-free fetal DNA and cell-free total DNA was 0.898 (95% CI, 0.852–0.945) and 0.939 (95% CI, 0.903–0.975), respectively.ConclusionsSignificantly high levels of cell-free fetal DNA and cell-free total DNA were found in SA women with fetal chromosomal aneuploidy. Our findings suggest that cell-free fetal DNA and cell-free total DNA may be useful biomarkers for the prediction of SA with fetal chromosomal aneuploidy, regardless of fetal gender.

Clinical evaluations of cell‐free fetal DNA quantities in pre‐eclamptic pregnancies

Quantitative changes of cell-free fetal DNA in maternal plasma as an indicator for impending pre-eclampsia was reported in different studies. Cell-free fetal nucleic acids can be detected in maternal circulation during pregnancy. Our aim was to determine the higher rate of fetal DNA levels in maternal blood in pre-eclampsia compared to normal pregnancies and the clinical use of real-time polymerase chain reaction (PCR) in the Turkish population as a marker. According to their gestational ages, the plasma levels of 30 pre-eclamptic women at 26-40 weeks of pregnancy were matched with 18 healthy pregnant women. Cell-free fetal DNA levels in maternal plasma were compared using real-time PCR technology. For the quantitative measurement of fetal DNA from maternal blood, the relative quantification PCR process was applied to all samples, using SRY and GAPDH genes. These patients were classified as pre-eclamptic and control groups and were matched according to weeks of pregnancy. Free fetal DNA levels of 30 pre-eclamptic patients were compared to healthy pregnant women and an average 3.06-fold increase was observed. During the second trimester, free fetal DNA levels were 1.5 times higher in pre-eclamptic patients. This increase was 3.5-fold during the third trimester. The DNA increase of pre-eclamptic patients was 4.1-fold and 3.4-fold during 29th-33rd and 34th-40th weeks, respectively. Cell-free fetal DNA in maternal blood could be used as a marker for identifying subjects at increased risk of developing pre-eclampsia.

Relative quantitation of cell-free fetal DNA in maternal plasma using autosomal DNA markers

BackgroundCell-free fetal DNA in maternal plasma presents interest as it can possibly serve as a pregnancy associated biomarker. For its quantitation, we investigate the use of fluorescent polymerase chain reaction (PCR) amplification of short tandem repeats (STRs) in pregnancies irrespective of sex. MethodsArtificially chimeric DNA samples were prepared so as to simulate cell-free fetal DNA in maternal plasma at different proportions. The samples were tested with fluorescent PCR amplification of informative STRs and the percentage of the population simulating fetal DNA was plotted against a relative ratio of the alleles detected in each sample. The graphs were used for the quantitation of cell-free fetal DNA in 50 maternal plasma samples. ResultsDetection of at least one paternally inherited fetal STR was possible in 46/50 pregnancies leading to the estimation of the relative percentage of cell-free fetal DNA. Four pregnancies failed to reveal any fetal allele which may reflect undetectable levels (<1%) of fetal DNA. Among pregnancies fetal DNA ranged from 0 to 20% (mean value of 7%). ConclusionOur system can readily estimate the relative levels of cell-free fetal DNA in maternal circulation, normal gestations of either sex and can be exploited for estimating possible variations of this analyte between normal and pathological pregnancies.

Quantitative variation analysis of fetal DNA in maternal plasma samples collected before and after amniocentesis

The aim of this study was to evaluate possible procedure-related variations in the levels of cell-free fetal DNA (fDNA) in maternal plasma of women undergoing genetic amniocentesis. Blood samples were collected at 16-18 weeks' gestation from 33 pregnant women attending the Fetal Medicine Unit for genetic amniocentesis. For each woman, two blood samples were obtained: the first immediately before amniocentesis and the second one 15 min after the procedure. A real-time quantitative polymerase chain reaction assay, using primers for SRY and beta-globin genes, was used to assess fDNA concentrations in maternal plasma. A Wilcoxon signed-rank test was used for statistical analysis. The karyotype on cultured amniocytes showed that 15 out of 33 women had a male fetus. Real-time quantitative polymerase chain reaction results, on maternal plasma sample pairs from known male pregnancies, showed no significant variations of fDNA correlated to amniocentesis (P=0.394). Our preliminary study suggests that amniocentesis, although invasive, could be associated with minimal, if any, disruption at the fetal-maternal interface, as revealed by the lack of substantial modifications of fDNA levels in maternal circulation.