Cell-free Fetal Deoxyribonucleic Acid Research Articles

Cell-Free Fetal Deoxyribonucleic Acid (cffDNA) Analysis as a Remarkable Method of Non-Invasive Prenatal Screening

The cell-free fetal DNA (cffDNA) analysis for screening fetal genetic anomalies has increased dramatically since its commercialization in 2011 worldwide. In the early weeks of pregnancy, it offers a hassle-free, non-invasive procedure of antenatal screening. It guides and protects mothers from undergoing unwanted risk-laden invasive prenatal testing. cffDNA testing is accurate at detecting the abnormal fetus chromosome among a large pool population. Patau syndrome, Edward syndrome, and Down syndrome are currently being accurately screened by this method. Due to their sensitivity and specificity, they now have become the screening method of choice, approaching almost 100% in various studies with a large sample pool. The latest procedures to analyze cffDNA, like the new digital droplet polymerase chain reaction (ddPCR) and sophisticated next-generation sequencing (NGS), haveincreased detection rates with decreased analyzing time. The latest techniques make it possible to screen large numbers of the population with faster report generation. Screening for Rh incompatibility and its timely prevention is now more accessible and more accurate with the help of cffDNA analysis. The problem arises when we deviate from the primary disease and start testing for anomalies not intended to be screened by cffDNA in the first place. Fetal sex chromosome aneuploidy screening by cffDNA is one area where the test gives mixed results either due to differences in machinery, laboratory parameters, or human error. Other rare occurrences like trisomes, such as trisomy 7, trisomy 16, trisomy 22, and a few microdeletion syndromes are also being screened but with less accuracy. Like every technology, cffDNA analysis is not entirely free of criticism. Its high testing cost, potential to accurately prognosticate the gender of the developing fetus and absence of standard testing practices will become an issue as the test becomes routine worldwide.

Cell-Free Fetal DNA: A Novel Biomarker for Early Prediction of Pre-eclampsia and Other Obstetric Complications

Hypertensive disorder of pregnancy, especially Pre-eclampsia is one of the major causes of increased maternal and perinatal morbidity and mortality all over the world. Early prediction of pre-eclampsia is the need of modern obstetrics, as this can timely prevent the progress of disease as well as related fetal and maternal morbidity and mortality. In addition to the screening of fetal aneuploidies, Rhesus-D status, fetal sex, single gene disorders, the cell-free fetal Deoxyribonucleic acid (DNA) quantification has emerged as a promising biomarker for the prediction of pre-eclampsia. Hence, its use can help in the early prediction of hypertensive disorders of pregnancy, especially pre-eclampsia even before the appearance of symptoms. Furthermore, in future, it can also help in the determination of the complete DNA sequence of every gene of the fetus. The present review focuses on recent literature concerning the use of cell-free fetal DNA in early prediction of preeclampsia as well as for non-invasive prenatal genetic screening of fetus for various disorders. Methods: The recent literature related to cell-free fetal DNA was searched from numerous English language journals and published peer-reviewed articles on Pubmed, Google Scholar, MEDLINE and various government agencies till 2016.

Cell-free Fetal Deoxyribonucleic Acid Results in Low-risk Pregnancy Screenings for Aneuploidies

Cell-free Fetal Deoxyribonucleic Acid Results in Low-risk Pregnancy Screenings for Aneuploidies

Alarmins at the maternal-fetal interface: involvement of inflammation in placental dysfunction and pregnancy complications 1.

Inflammation is known to be associated with placental dysfunction and pregnancy complications. Infections are well known to be a cause of inflammation but they are frequently undetectable in pregnancy complications. More recently, the focus has been extended to inflammation of noninfectious origin, namely caused by endogenous mediators known as "damage-associated molecular patterns (DAMPs)" or alarmins. In this manuscript, we review the mechanism by which inflammation, sterile or infectious, can alter the placenta and its function. We discuss some classical DAMPs, such as uric acid, high mobility group box 1 (HMGB1), cell-free fetal deoxyribonucleic acid (DNA) (cffDNA), S100 proteins, heat shock protein 70 (HSP70), and adenosine triphosphate (ATP) and their impact on the placenta. We focus on the main placental cells (i.e., trophoblast and Hofbauer cells) and describe the placental response to, and release of, DAMPs. We also covered the current state of knowledge about the role of DAMPs in pregnancy complications including preeclampsia, fetal growth restriction, preterm birth, and stillbirth and possible therapeutic strategies to preserve placental function.

Have we done our last amniocentesis? Updates on cell-free DNA for Down syndrome screening

Prenatal aneuploidy screening changed significantly in 2012 when cell-free fetal deoxyribonucleic acid (DNA) was introduced as a noninvasive prenatal test. A noninvasive prenatal test detects cell free fragments of fetal DNA from the placenta circulating in maternal blood that coexist with cell-free DNA (cfDNA) of maternal origin. Using next-generation sequencing, the noninvasive prenatal test compares maternal and fetal cfDNA ratios for chromosomes of interest (i.e., 21, 18, 13, X, and Y) to assess chromosomal aneuploidy. Compared to traditional screening using ultrasound and serum markers, the noninvasive prenatal test has superior test characteristics, including a higher detection rate and positive predictive value, and a lower false-positive rate. The noninvasive prenatal test is already used for primary screening in high-risk women and is rapidly expanding to all women. Given its increasing use, understanding the noninvasive prenatal test's limitations is critical. Discordant results (i.e. noninvasive prenatal test is positive for aneuploidy with a normal fetal karyotype) can occur because of biological processes such as aneuploidy confined to the placenta, a vanished twin, maternal aneuploidy or maternal cancer. Use of the noninvasive prenatal test for screening beyond the most common aneuploidies is not recommended. The noninvasive prenatal test is a major advance in prenatal aneuploidy screening but it is not diagnostic and does not replace invasive testing (i.e. chorionic villous sampling or amniocentesis) for confirmation of fetal chromosomal disorders.

Detection of short tandem repeats at 5 loci and amelogenin with cell-free fetal DNA as a specimen in the development of prenatal paternity diagnostic tests

IntroductionA prenatal paternity test is one widely-used method of determining the paternity of an unborn child. Such tests using chorionic villus or amniocentesis may increase the risk of harm to both mother and foetus. In the present day, a prenatal paternity test using circulating cell-free fetal Deoxyribonucleic Acid (DNA) is one alternative method due to it being non-invasive and safe for both mother and foetus.AimThe aim of this study is to detect Short Tandem Repeat (STR) at 5 Loci (vWA, TH01, D13S317, D18S51, and D21S11) and amelogenin genes in circulating cell-free fetal DNA in paternity tests.Materials and methodsForty-one samples of maternal blood were obtained from pregnant woman. Circulating free fetal DNA was subsequently extracted. A paternity test was conducted using an STR test at loci vWA, TH01, D13S317, D18S51, D21S11 in circulating free fetal DNA. An analysis of the paternity test between loci > 200 bp and < 200 bp was also conducted to establish the sensitivity of the test.ResultsThere was a significant difference between maternal blood DNA and circulating cell-free fetal DNA (p = 0.000 D13S317; p = 0.000 D21S11; p = 0.000 D18S51; p = 0.000 vWA; p = 0.000 TH01; and p = 0.000 amelogenin genes). The locus < 200 bp also had a higher sensitivity than locus > 200 bp.ConclusionCirculating free fetal DNA can be used as an alternative sample for prenatal paternity tests because of its similarity with maternal DNA.

The Use of Ultrasound as a Potential Adjunct to Cell-Free Fetal DNA Screening for Aneuploidy at Weill Cornell Medical College, New York, USA.

Objective To evaluate the utility of ultrasound in identifying fetuses with uncommon chromosomal abnormalities that would be considered not detectable by cell-free fetal deoxyribonucleic acid (cfDNA). Study Design We performed a retrospective study of fetuses with chromosomal abnormalities that would be undetectable by cfDNA, who underwent an 11- to 14-week ultrasound from 2006 to 2016. Results There were 43 pregnancies included. First-trimester ultrasound revealed a fetal abnormality in 19 (44.2%) cases, of which 13 (30.2%) had a thickened nuchal translucency. There were an additional four fetuses with second-trimester sonographic abnormalities. Overall, 23 (53.5%) fetuses were found to have a major anomaly diagnosed by ultrasound. The rate of first-trimester sonographic abnormalities varied widely based on category of chromosomal abnormalities with high rates seen with triploidy (87.5%) and autosomal trisomy (80%) and lower rates seen with structurally abnormal chromosomes (33.3%), trisomy mosaicism (27.3%), other forms of mosaicism (11.1%), and deletions or duplications (25.0%), p < 0.001. Conclusion The majority of fetuses with uncommon chromosomal abnormalities in our cohort had major sonographic anomalies. The use of first-trimester ultrasound with nuchal translucency measurement may offer utility in identifying fetuses with risk of aneuploidy that would not be detectable with cfDNA.

Non-invasive prenatal testing - a new method in improving first trimester screening for chromosome-related abnormalities

In the last 15 years, major advances have been made in prenatal screening. Cohort studies have led to an understanding of the causes of many common diseases that are determined by the combined effects of genetic and phenotypic factors. Non-invasive prenatal testing (NIPT) is a technology used to isolate deoxyribonucleic acid (DNA) placental fragments from the mother’s blood at as early as 10 weeks of gestation, using cell free fetal DNA technology. NIPT screens for trisomy 13,18 and 21 for sex chromosome abnormalities with a high detection rate. Cohort studies have shown a high detection rate and a low false positive rate for NIPT, but it is still considered a screening test and not diagnostic. It is recommended confirmation in case of a positive test, with a diagnostic procedure as chorionic villus sampling or amniocentesis. It is essential that NIPT be used ethically and effectively. Because of its high sensitivity (true positive rate) and its specificity (true negative rate) many recommend that NIPT should be used as a diagnostic method. However, today NIPT is used as a screening method, an attractive alternative to the serum screens and invasive test currently in use. There is a continuing decline in sequencing costs and hopefully, soon, the cost will be reduced.

Cell-free DNA Testing: Where are We now?

ABSTRACTPrenatal screening for fetal aneuploidies has been focused on mainly Down syndrome in the last 40 years. The method of screening has changed from maternal age in the 1970s, with a detection rate of 30 and 5% false positive rate (FPR), to a combination of maternal age and second-trimester serum biochemical markers (triple test and quadruple test) in the 1980s and 1990s, with 60 to 75% detection rate and 5% false positive rate (FPR). Following this, the era of first trimester screening for Down syndrome has started with the clinical implementation of fetal nuchal translucency screening. The combination of maternal age, NT thickness and serum free beta-human chorionic gonadotropin (â-hCG) and pregnancy-associated plasma protein A (PAPP-A) in the first trimester has yielded a 90% detection rate with a 5% FPR. Starting from the year 2008, studies have shown that the performance of screening may be improved by analysis of cell-free deoxyribonucleic acid (DNA) (cfDNA) in maternal blood. Several studies in the last few years have reported the clinical validation of cell free fetal DNA test in the maternal serum in screening for trisomies 21, 18, and 13 and sex chromosome aneuploidies.Its widespread use is limited by the relatively high cost of the test and the lack of consensus about the optimal way for its clinical implementation. Until the optimal way of incorporating cfDNA into the clinical practice gets identified, it would be wise not to substitute cfDNA testing in place of first-trimester screening for fetal defects and other major complications of pregnancy. Furthermore, it would be preferable for clinicians managing individual patients not to counsel them for their result as positive or negative, rather the clinicians should use the risk estimate from the first-line method of screening as the prior risk and modify this by the appropriate positive or negative likelihood ratio from the cfDNA test.How to cite this articleSen C, Api O, Yayla M, Goynumer G. Cell-free DNA Testing: Where are We now? Donald School J Ultrasound Obstet Gynecol 2016;10(2):172-177.

Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation

ABSTRACT Objectives Early identification of fetal gender is important in management of X-linked and other metabolic disorders. Since ultrasound may not predict gender accurately during the first trimester, noninvasive fetal gender determination using deoxyribonucleic acid (DNA) amplification has been proposed. The aim of this study is to evaluate the feasibility of noninvasive prenatal gender determination by examining cell-free fetal DNA (cffDNA) from maternal plasma. Materials and methods Blood samples were collected from 49 pregnant women of gestational ages ranging from 12 to 41 weeks. Deoxyribonucleic acid was extracted from maternal plasma using a QIAamp DNA Blood Mini Kit. Real-time quantitative polymerase chain reaction (PCR) was performed to amplify the male specific DNA marker sex-determining region Y (SRY). Results From a total of 49 subjects, fetal gender was correctly determined in 13 out of 14 male fetuses and 32 out of 35 female fetuses, giving an overall accuracy of 92%. The sensitivity and specificity of the test to detect male fetuses was 93 and 91% respectively. There were three false-positive cases and one false-negative case. Conclusion Identification of fetal gender from maternal plasma using real-time PCR technique is feasible in a developing country, like Pakistan, and appears to be a promising tool for noninvasive prenatal diagnosis. How to cite this article Mohammed N, Nuruddin R, Hoodbhoy Z. Gender Determination by Isolation of Cell-free Fetal DNA from the Maternal Circulation. J South Asian Feder Obst Gynae 2016;8(1):29-32.

Has Noninvasive Prenatal Testing (NIPT) Come of Age?

Recent advances in genomic sequencing and bioinformatics have led to the development of noninvasive detection methods with detection rates approaching those obtained with amniocentesis and chorionic villus sampling (CVS) [1, 2]. Recently, a novel prenatal testing method has become available. This method, known as noninvasive prenatal testing (NIPT), is a molecular approach for assessing fetal aneuploidy using cell-free fetal deoxyribonucleic acid (cffDNA) from the plasma of pregnant women. NIPT has a false positive rate of about 0.2 % and detection rate of about 98 % for Down syndrome [1, 2]. NIPT has been used for assessing abnormalities such as trisomy 21, trisomy 18, and trisomy 13. Approximately 10–15 % of the cell-free deoxyribonucleic acid (cfDNA) in maternal blood comprises cffDNA [3, 4]. The half-life of cffDNA is short, and it clears from maternal circulation soon after delivery [3]. Hence, there is no risk of fetal DNA persisting from one pregnancy to the next and confounding test results. For women infected with hepatitis B, hepatitis C, and/or human immunodeficiency virus (HIV), the use of noninvasive methods of prenatal risk assessment is recommended, using tests with high sensitivity and low false-positive rates, such as serum screening combined (or not) with nuchal translucency, anatomic ultrasound, and noninvasive molecular prenatal testing [5]. Among other factors, cost implications for introducing this new technology in clinical practice will need to be considered.

The End of Amniocentesis? From TriTest to PrenaTestTM

ABSTRACT Purpose The rapid development in molecular biological technologies makes it possible to screen and to diagnosis thousands of genetic conditions, mutations and also predispositions to chronic diseases or traits, either prenatally or after birth. Clinical application of noninvasive prenatal diagnosis (NIPD) using fetal deoxyribonucleic acid (DNA) in maternal plasma has become a reality. We review the latest developments in screening and diagnosis of chromosomal diseases and a new noninvasive method of prenatal diagnosis. Materials and methods PrenaTest™ (LifeCodexx AG, Konstanz, Germany, patented and licensed by Sequenom Inc., San Diego, California, USA) is a molecular genetic test that can detect fetal trisomy 21 with a high precision level from maternal blood using new generation multiplex genome sequencing techniques. The test is based on the analysis of extracellular fetal DNA measured in the blood of pregnant women: Cell-free fetal DNA (cffDNA). Results In the case of trisomy 21, there were found 105 specific fragments of chromosome 21, 15 of fetal origin and 90 of maternal origin. The validity of PrenaTest™ has been demonstrated by many tests (427 cases), and the sensitivity was 95%, with a false negative rate of 5% and a specificity of 99.5%. Anyway, an additional ultrasound can always be performed to assess fetal morphology. Conclusion The arrival of the molecular genetic era also leads to many new ethical, social and medicolegal problems and dilemmas that obstetricians will have to face in the near future. There is an urgent need for the development of a new model for provision of genetic screening and diagnosis. How to cite this article Borruto F, Treisser A, Abdelkrim SB, Comparetto C. The End of Amniocentesis? From TriTest to PrenaTestTM. Donald School J Ultrasound Obstet Gynecol 2013;7(2):213-218.

Detection of fetal mutations causing hemoglobinopathies by non-invasive prenatal diagnosis from maternal plasma

Prenatal diagnosis of hemoglobinopathies enables couples at risk to have a healthy child. Currently used fetal sampling procedures are invasive with some risk of miscarriage. A non-invasive approach to obtain fetal deoxyribonucleic acid (DNA) for diagnosis would eliminate this risk. To develop and evaluate a non-invasive prenatal diagnostic approach for hemoglobinopathies using cell-free fetal DNA circulating in the maternal plasma. Couples referred to us for prenatal diagnosis of hemoglobinopathies where the maternal and paternal mutations were different were included in the study. Maternal peripheral blood was collected at different periods of gestation before the invasive fetal sampling procedure was done. The blood was centrifuged to isolate the plasma and prepare DNA. A size separation approach was used to isolate fetal DNA. Nested polymerase chain reaction (PCR)-based protocols were developed for detection of the presence or absence of the paternal mutation. There were 30 couples where the parental mutations were different. Of these, in 14 cases the paternal mutation was absent and in 16 cases it was present in the fetus. Using cell-free fetal DNA from maternal plasma, the absence of the paternal mutation was accurately determined in 12 of the 14 cases and the presence of the paternal mutation was correctly identified in 12 of the 16 cases. Thus, this non-invasive approach gave comparable results to those obtained by the conventional invasive fetal sampling methods in 24 cases giving an accuracy of 80.0%. Although the nested PCR approach enabled amplification of small quantities of cell-free DNA from maternal plasma at different periods of gestation after size separation to eliminate the more abundant maternal DNA, an accurate diagnosis of the presence or absence of the paternal mutation in the fetus was not possible in all cases to make it clinically applicable.

New aids for the non‐invasive prenatal diagnosis of achondroplasia: dysmorphic features, charts of fetal size and molecular confirmation using cell‐free fetal DNA in maternal plasma

To improve the prenatal diagnosis of achondroplasia by constructing charts of fetal size, defining frequency of sonographic features and exploring the role of non-invasive molecular diagnosis based on cell-free fetal deoxyribonucleic acid (DNA) in maternal plasma. Data on fetuses with a confirmed diagnosis of achondroplasia were obtained from our databases, records reviewed, sonographic features and measurements determined and charts of fetal size constructed using the LMS (lambda-mu-sigma) method and compared with charts used in normal pregnancies. Cases referred to our regional genetics laboratory for molecular diagnosis using cell-free fetal DNA were identified and results reviewed. Twenty-six cases were scanned in our unit. Fetal size charts showed that femur length was usually on or below the 3(rd) centile by 25 weeks' gestation, and always below the 3(rd) by 30 weeks. Head circumference was above the 50(th) centile, increasing to above the 95(th) when compared with normal for the majority of fetuses. The abdominal circumference was also increased but to a lesser extent. Commonly reported sonographic features were bowing of the femora, frontal bossing, short fingers, a small chest and polyhydramnios. Analysis of cell-free fetal DNA in six pregnancies confirmed the presence of the c.1138G > A mutation in the FGRF3 gene in four cases with achondroplasia, but not the two subsequently found to be growth restricted. These data should improve the accuracy of diagnosis of achondroplasia based on sonographic findings, and have implications for targeted molecular confirmation that can reliably and safely be carried out using cell-free fetal DNA.

Significant Correlation Between Maternal Body Mass Index at Delivery and in the Second Trimester, and Second Trimester Circulating Total Cell-free DNA Levels

The use of cell-free fetal deoxyribonucleic acid (DNA) requires the knowledge of variables that may influence the levels of cell-free DNA, such as maternal body mass index (BMI). In this study, using 406 maternal blood samples from the second trimester of pregnancy, cell-free fetal DNA specific for the SRY and DYS14 loci and glyceraldehyde-3-phosphate dehydrogenase sequence were quantified by real-time polymerase chain reaction. No significant correlation was seen between the levels of cell-free fetal DNA and maternal BMI, whereas total cell-free DNA was significantly associated with maternal BMI at 20 to 21 weeks of gestation (P = .034) and at the end of pregnancy (R2 regression: 0.016, P = .014). Quantitative levels of cell-free fetal DNA are not affected by maternal BMI, whereas total DNA levels in the second trimester significantly correlate with maternal BMI at the moment of blood drawing and at delivery.

Fetal sex determination from maternal blood at 6 weeks of gestation when at risk for 21-hydroxylase deficiency

Background 21-hydroxylase deficiency can lead to masculinization of female fetuses. Corticosteroid therapy may reduce these effects. When the fetus is male, this approach means that unnecessary treatment, with theoretic side effects, is given until the result of chorionic villus sampling (CVS), a procedure with known risks, is available. Case A woman was referred for prenatal assessment at 6 weeks’ gestation because her first daughter had been born virilized from 21-hydroxylase deficiency. A real-time polymerase chain reaction assay was performed on maternal blood to detect the fetal Y chromosome–associated SRY gene. A positive signal for the SRY gene was observed. The assay was repeated a few days later, and the result was again consistent with a male fetus. Conclusion Analysis of cell-free fetal deoxyribonucleic acid in maternal plasma for fetal sex determination might reduce the need for corticosteroid administration and CVS in women with fetuses at risk for 21-hydroxylase deficiency.

Circulating erythroblasts in maternal blood are not elevated before onset of preterm labor

OBJECTIVE: Preterm labor has recently been reported to be associated with an increased release of cell free fetal deoxyribonucleic acid (DNA) into the maternal circulation. We have previously observed increases in both fetal cell traffic and cell free fetal DNA in preeclamptic pregnancies. In this study, we investigated whether fetal cell traffic is also disturbed in pregnancies with preterm labor. METHODS: In a case-control study, we examined 47 pregnancies complicated by preterm contractions that occurred between 20 and 34 weeks’ gestation and an equal number of matched controls. Erythroblasts were enriched for by magnetic cell sorting and enumerated. These values were then correlated with subsequent pregnancy outcome. RESULTS: In the study group 16 patients delivered prematurely (subgroup A). The other 31 (subgroup B) delivered at term, as did all those in the control group. No significant difference was noted in erythroblast numbers between either one of the subgroups and the controls. CONCLUSION: Contrary to the reported increased levels of free fetal DNA in maternal serum, erythroblasts in maternal blood are not elevated significantly in pregnancies with threatened premature labor or in those that deliver preterm.

Cell-free fetal deoxyribonucleic acid in maternal circulation as a marker of fetal-maternal hemorrhage in patients undergoing external cephalic version near term

Objective: Our aim was to investigate whether external cephalic version performed near term increases the concentration of cell-free fetal deoxyribonucleic acid in maternal plasma. Study Design: Forty-five patients who had singleton male fetuses and were undergoing external cephalic version at or beyond 36 weeks of gestation were recruited during a 20-month period. Maternal venous blood samples were taken before and within 10 minutes after external cephalic version. Deoxyribonucleic acid was extracted from the plasma samples. The amount of fetal deoxyribonucleic acid was quantified by means of the SRY gene on the Y chromosome as a fetal marker. The change in SRY gene concentration before and after external cephalic version was compared by paired sample t test. Results: There was a significant increase in the concentration of fetal deoxyribonucleic acid in maternal serum after external cephalic version (before, 296 ± 209 copies per milliliter; after, 369 ± 228 copies per milliliter; P =.014). This increase in the concentration of deoxyribonucleic acid was most profound among the nulliparous patients after a successful version and in the presence of a posterior placenta. The location of the placenta was found to be the most significant factor accounting for the change in the deoxyribonucleic acid concentration. Conclusions: External cephalic version near term imposed a significant disturbance to the maternalplacental interface. Fetal deoxyribonucleic acid is a sensitive marker that is useful in the assessment of subclinical fetal-maternal hemorrhage. (Am J Obstet Gynecol 2000;183:712-6.)