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.

The Level of Free Fetal DNA as Precise Noninvasive Marker for Chromosomal Aneuploidies: First Results from BALTIC Region.

Background and objectives: Noninvasive prenatal testing (NIPT), which has been introduced clinically since 2011, uses the circulating cell-free fetal DNA in the maternal blood to evaluate the risk of a chromosomal anomaly. The aim of this study was to examine the effectiveness of NIPT using a single nucleotide polymorphism method. Materials and Methods: A retrospective study was conducted between 2013 and 2019. The Natera Panorama test was used to analyze the risk of trisomies 21, 18, 13, X monosomy, trisomy, and other sex chromosome abnormalities. A positive result of NIPT for aneuploidy was confirmed by invasive testing. Results: 850 women with a singleton pregnancy participated in the study. The median fetal fraction was 9.0%. The fetal fraction was lower in the no-call group (3.1%) compared with the group that received a call (9.1%) (p < 0.001). A positive correlation was determined between the gestational age and the fetal fraction (r = 0.180, p < 0.001). The overall positive predictive value (PPV) of NIPT for trisomy 21 (n = 9), trisomy 18 (n = 3) and XYY syndrome (n = 1) was 100%. Conclusions: The results of present study showed 100% PPV effectiveness of NIPT Panorama test detecting trisomies of 21 and 18 chromosomes, as well as XYY syndrome in the studied cohort. Therefore, NIPT due to its high PPV, significantly reduces the need for invasive testing, thereby reducing the risk of miscarriage and stillbirth.

Elevated cell-free fetal DNA contributes to placental inflammation and antiangiogenesis via AIM2 and IFI16 during pre-eclampsia.

Accumulated evidence has shown that pre-eclampsia (PE) is related to both maternal and utero-placental antiangiogenesis and inflammation. Remarkably, an elevated cell-free fetal DNA (cffDNA) level has been found in maternal circulation; however, it remains unclear whether this DNA can induce activation of cytosolic DNA sensor signaling pathways and lead to the development of PE. In this study, we found that trophoblast cells constitutively expressed the cytosolic DNA sensors, absent in melanoma 2 (AIM2) and interferon-inducible protein 16 (IFI16). The cffDNA and pro-inflammatory and antiangiogenic factors were present at higher concentrations in PE compared with the control group and correlated with the severity of PE. DNA stimulation significantly increased the AIM2 and IFI16 levels, consistent with the elevated AIM2 and IFI16 expression in women with PE, and elicited increased production of AIM2-mediatedinterleukin IL-8 (IL-8), IL-6 and CC chemokine ligand 2 (CCL2) and IFI16-mediated sEndoglin, sFlt-1 and CXCL10. Furthermore, enhancement of the inflammatory response was found to be induced by DNA exposure, but DNA exposure did not induce PE-like symptoms in pregnant mice. It is possible that elevated cffDNA could reflect the degree of placental damage and trigger cytosolic DNA sensoractivation, which disrupts the immunity balance and, consequently, contributes to inflammatory and antiangiogenic responses. In conclusion, the results of this study suggest that circulating cffDNA levels are increased in preeclamptic women and act through AIM2 and IFI16 activation to promote the production of pro-inflammatory and antiangiogenic factors, which correlate with the severity of the disease, and may offer insights into the etiology and pathogenesis of PE.

Impact of long-term storage of plasma and cell-free DNA on measured DNA quantity and fetal fraction.

Optimal sample storage conditions are essential for non-invasive prenatal testing of cell-free fetal and total DNA. We investigated the effect of long-term storage of plasma samples and extracted cfDNA using qPCR. Fetal and total cfDNA yield and fetal fraction were calculated before and after storage of plasma for 0-6years at -25°C. Dilution experiments were performed to investigate PCR inhibition. Extraction with or without proteinase K was used to examine protein dissociation. Storage of extracted cfDNA was investigated by testing aliquots immediately, and after 18months and 3years of storage at -25°C. We observed a marked increase in the levels of amplifiable fetal and total DNA in plasma stored for 2-3years, and fetal fraction was slightly decreased after 3years of storage. cfDNA detection was independent of proteinase K during DNA extraction in plasma samples stored >2years, indicating a loss of proteins from DNA over time, which was likely to account for the observed increase in DNA yields. Measured fetal and total DNA quantities, as well as fetal fraction, increased in stored, extracted cfDNA. Fetal and total cell-free DNA is readily detectable in plasma after long-term storage at -25°C. However, substantial variation in measured DNA quantities and fetal fraction means caution may be required when using stored plasma and extracted cfDNA for test development or validation purposes.

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.

Диагностическая значимость определения уровня внеклеточной фетальной ДНК у беременных с преэклампсией и задержкой роста плода

Диагностическая значимость определения уровня внеклеточной фетальной ДНК у беременных с преэклампсией и задержкой роста плода

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.

Complex Analysis of Total and Fetal DNA and Cytokines in Blood Plasma of Pregnant Women with Preeclampsia.

We performed a complex analysis of total and fetal extracellular DNA, 8 cytokines (IL-2, IL-4, IL-6, IL-8, IL-10, granulocyte-macrophage CSF, IFNγ, and TNFα) in blood plasma obtained from women with preeclampsia prior to labor onset. Total (sensitivity 89.47%, specificity 93.75%) and fetal extracellular DNA (sensitivity 73.68%, specificity 87.5%) were the most accurate parameters determining preeclampsia. We revealed a high correlation (p=3×10-6) between total and fetal extracellular DNA levels in the group of preeclampsia. Preeclampsia significantly increased the levels of macrophage factors IL-10 and IL-6. These cytokines significantly correlated with the levels of total and fetal extracellular DNA in the preeclampsia group. In the control group, such correlations were not observed. These findings obtained suggest that preeclampsia develops upon increased macrophage activity, leading to destruction of the placenta trophoblast cells.

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.

Follow-up of gestational trophoblastic disease/neoplasia via quantification of circulating nucleic acids of placental origin using C19MC microRNAs, hypermethylated RASSF1A, and SRY sequences.

The aim of the study was to evaluate the effectiveness of placental-specific markers, extracellular fetal DNA (sex-determining region Y and hypermethylated RASSF1A sequences) and circulating C19MC microRNAs (miR-516-5p, miR-517-5p, miR-518b, miR-520a-5p, miR-520h, miR-525, and miR-526a) for the diagnosis and consecutive follow-up of gestational trophoblastic disease/neoplasia. Increased levels of extracellular fetal DNA and C19MC microRNAs were detected in patients with active disease when compared with the period when the patients reached remission of the disease. The positive correlation between plasma levels of hypermethylated RASSF1A sequence, C19MC microRNAs, and human chorionic gonadotropin serum levels was found. MiR-520a-5p had the best performance to detect patients with active disease (a positive predictive value of 100% at a null false positive ratio (FPR)). MiR-516-5p and miR-525 were able to diagnose 100% of women with active disease at the FPR 3.9%/7.7%. The overall predictive capacity of single miR-526a (81.8% at null FPR), miR-517-5p (90.9% at 15.4% FPR), miR-518b (100% at 38.5% FPR), and miR-520h (90.9% at 26.9% FPR) biomarkers to detect active disease cases was slightly lower. Transient increase in C19MC microRNA plasma levels after the first cycle of chemotherapy indicated the decay of placental trophoblast residual tissue. The increased levels of extracellular fetal DNA and placental-specific C19MC microRNAs are associated with gestational trophoblastic disease/neoplasia. Screening of extracellular placental-specific biomarkers may represent an additional option to identify a significant proportion of women with active disease and to monitor the therapy response. Non-invasive follow-up of the decomposing residual tissue in the form of extracellular nucleic acids of placental origin packed into apoptotic bodies derived from placental trophoblasts is available.

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?

Cell free fetal DNA in the plasma of pregnant women with preeclampsia

Objective: Insufficient cytotrophoblast invasion to the myometrium is associated with preeclampsia, especially with the early-onset preeclampsia (before 34 gestational weeks). Several investigations have marked changes in the concentration of cell free fetal DNA in the maternal circulation of women with preeclampsia. However, these studies were not performed for early or late preeclampsia subgroups individually. The present authors planned to determine the levels of the cell free both fetal and maternal DNA in the maternal circulation in early preeclampsia subgroup and compare it with normotensive control cohort. Materials and Methods: A total of 16 women; eight of these with preeclampsia and eight normotensive control cohorts with singleton male pregnancy between 28 and 32 gestational weeks were included in the study. Real-time PCR analysis was performed for determining the circulating cell free DNA levels. Results: Cell free fetal DNA concentrations were higher in early preeclamptic women than control subjects. The authors found no statistically significant difference in each levels of maternal and total DNA between hypertensive and normotensive groups. Conclusions: The present findings suggest that the levels of cell free fetal DNA in maternal circulation were higher in pregnancies which are complicated with early preeclampsia than normotensive controls.

Genomic and proteomic investigation of preeclampsia.

The aim of this study was to use proteomic and transcriptomic approaches to examine differences in protein and gene expression in maternal plasma between normal and preeclamptic pregnancies. Preeclampsia and control groups were compared with respect to the expression of CD34 and CD133 genes by quantitative polymerase chain reaction (qPCR) and heat shock protein (Hsp)27 and 70 by western blotting in blood samples from the pregnant women. Blood samples were obtained at gestational week 12-14 from 65 healthy pregnant women. Fetal DNA was isolated from the maternal blood and CD34 and CD133 were amplified by qPCR. Western blot analysis was used to examine the expression levels of Hsp27 and Hsp70 proteins. The analysis of CD133 by qPCR was unsuccessful in 7 women as the levels of fetal DNA were in the collected maternal blood samples were insufficient. Measurements of CD34 and CD133 were performed in 57 and 50 women, respectively. Preeclampsia developed in 6 (10.5%) of 57 women. qPCR results of 8 healthy pregnant women were used for the calibration of CD34 and CD133 levels, and the results for the remaining women were compared with the calibration values. CD34 expression was decreased in 30 (52.6%) and increased in 27 (47.4%) of 57 women. CD133 expression was decreased in 14 (28%) and increased in 36 (72%) of 50 women. CD34 expression was increased in 2 (33%) and 25 (49%) and decreased in 4 (66%) and 25 (51%) women with and without preeclampsia, respectively (P=0.467). CD133 expression was increased in 4 (66%) and 32 (72%); and decreased in 2 (33%) and 12 (28%) women with and without preeclampsia, respectively (P=0.756). Western blotting showed that the expression of Hsp27 and Hsp70 in the maternal serum of the preeclampsia group was significantly higher than that in the normal pregnancy group. CD34 and CD133 were found to be inadequate for use in the prediction of preeclampsia. However, it is noteworthy that CD133 levels were increased in 66 and 72% of women with and without preeclampsia, respectively. Hsps are expressed under various pathological conditions. These results suggest that conditions of oxidative stress increased the Hsp27 and Hsp70 protein levels.