Cell-free DNA In Maternal Blood Research Articles

Diagnostic accuracy of cell-free DNA in maternal blood in detecting chromosomal anomalies in twin pregnancies: systematic review and meta-analysis.

To report the diagnostic accuracy of cell-free DNA (cfDNA) in maternal blood in detecting chromosomal anomalies in twin pregnancies. Medline, Embase and Cochrane databases were searched. The inclusion criteria were twin pregnancies undergoing cfDNA screening for Trisomies 13, 18, 21, monosomy X0 and other sex chromosomal anomalies (SCA). The index test was represented by a positive results of cfDNA test. The reference standard was represented by the karyotype results (obtained either pre or postnatally) or, in case of negative cfDNA result, by a normal neonatal phenotype. The quality of the studies was assessed using the revised tool for the quality assessment of diagnostic accuracy studies (QUADAS-2). Summary estimates of sensitivity, specificity, positive and negative likelihood ratios (LR+ and LR-) and diagnostic odds ratio (DOR), with the corresponding 95% Confidence Intervals (95% CI), were computed using the bivariate random-effects model. Thirty-five studies were included. cfDNA had an overall high accuracy in detecting Trisomy 21 in twin pregnancies with a sensitivity of 98.8% (95% CI 96.5-100), a specificity of 100% (95% CI 99.9-100). Sensitivity and specificity were of 94.9% (95% CI 75.6-99.1) and 100 (95% CI 99.9-100) for Trisomy 18, and 84.6% (95% C% 54.6-98.1) and 100% (95% CI 99.9-100) for Trisomy 13 . We could not compute the diagnostic accuracy of cfDNA in detecting monosomy X0 in twins, while cfDNA had a sensitivity of 100% (95% CI 71.5-100) and a specificity of 99.8% (95% CI 99.7-99.9) in detecting other SCA (11 cases). The accuracy of cfDNA in detecting Trisomy 21, 18 and 13 was similar in dichorionic and monochorionic twin pregnancies. cfDNA has a high diagnostic accuracy in detecting Trisomy 18 and 21 in twin pregnancies, irrespective of chorionicity. Accuracy in the detection of Trisomy 13 and SCA was limited by the small number of affected cases and the difficulties in the confirmation of false negative cases in case of SCA and requires confirmation in larger studies. This article is protected by copyright. All rights reserved.

Bringing precision medicine to the management of pregnancy in women with glucokinase-MODY: a study of diagnostic accuracy and feasibility of non-invasive prenatal testing.

In pregnancies where the mother has glucokinase-MODY (GCK-MODY), fetal growth is determined by fetal genotype. When the fetus inherits a maternal pathogenic GCK variant, normal fetal growth is anticipated, and insulin treatment of maternal hyperglycaemia is not recommended. At present, fetal genotype is estimated from measurement of fetal abdominal circumference on ultrasound. Non-invasive prenatal testing of fetal GCK genotype (NIPT-GCK) using cell-free DNA in maternal blood has recently been developed. We aimed to compare the diagnostic accuracy of NIPT-GCK with that of ultrasound, and determine the feasibility of using NIPT-GCK to guide pregnancy management. We studied an international cohort of pregnant women with hyperglycaemia due to GCK-MODY. We compared the diagnostic accuracy of NIPT-GCK with that of measurement of fetal abdominal circumference at 28 weeks' gestation (n=38) using a directly genotyped offspring sample as the reference standard. In a feasibility study, we assessed the time to result given to clinicians in 43 consecutive pregnancies affected by GCK-MODY between July 2019 and September 2021. In terms of diagnostic accuracy, NIPT-GCK was more sensitive and specific than ultrasound in predicting fetal genotype (sensitivity 100% and specificity 96% for NIPT-GCK vs sensitivity 53% and specificity 61% for fetal abdominal circumference 75th percentile). In terms of feasibility, a valid NIPT-GCK fetal genotype (≥95% probability) was reported in all 38 pregnancies with an amenable variant and repeated samples when needed. The median time to report was 5 weeks (IQR 3-8 weeks). For the 25 samples received before 20 weeks' gestation, results were reported at a median gestational age of 20 weeks (IQR 18-24), with 23/25 (92%) reported before 28 weeks. Non-invasive prenatal testing of fetal genotype in GCK-MODY pregnancies is highly accurate and is capable of providing a result before the last trimester for most patients. This means that non-invasive prenatal testing of fetal genotype is the optimal approach to management of GCK-MODY pregnancies.

Cell-Free DNA in Maternal Blood and Artificial Intelligence: Accurate Prenatal Detection of Fetal Congenital Heart Defects

(Abstracted from Am J Obstet Gynecol 2023;228(1):76.e1–76.e10) Congenital heart defects (CHDs) are one of the leading causes of child morbidity and mortality. Previous research has shown that prenatal detection of CHD can significantly reduce the risk of death for severe cases, yet the current techniques available for prenatal diagnosis detect approximately only half of cases.

Evaluation of The Relationship between Cell-Free DNA Fetal Fraction of The Circulatory System and Fetal and Maternal Pregnancy Prognosis: A Prospective Study.

Non-invasive prenatal testing (NIPT), sometimes called noninvasive prenatal screening (NIPS), is a non-invasive prenatal genetic test using cell-free DNA in maternal blood. This method is used to diagnose fetal aneuploidy disorders such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18) and Patau syndrome (trisomy 13), which causes disability disorders or significant postpartum defects. The aim of this study was to investigate the relationship between high and low fetal fraction (FF) and prognosis of maternal pregnancy. In this prospective study, after obtaining informed consent, 10 ml of blood was collected from 450 mothers with singleton pregnancies with gestational age above 11 weeks (11-16) at the request of NIPT for cell-free DNA BCT test. After obtaining the test results, maternal and embryonic results were evaluated based on the amount of non-cellular DNA FF. Data analysis was performed by using SPSS software version 21 and independent t test, chi-square statistical tests. Based on test results, 20.5% of women were nulli par. The mean FF index in the studied women was 8.3% with a standard deviation of 4.6. The minimum and maximum values were 0 and 27, respectively. The frequency of normal, low and high FFs was 73.2, 17.3 and 9.5%, respectively. High FF has fewer risks to the mother and fetus than low FF. The use of FF level (high or low) can help us determining the prognosis of pregnancy and using it to better manage the pregnancy.

Cell-free DNA in maternal blood and artificial intelligence: accurate prenatal detection of fetal congenital heart defects

DNA cytosine nucleotide methylation (epigenomics and epigenetics) is an important mechanism for controlling gene expression in cardiac development. Combined artificial intelligence and whole-genome epigenomic analysis of circulating cell-free DNA in maternal blood has the potential for the detection of fetal congenital heart defects. This study aimed to use genome-wide DNA cytosine methylation and artificial intelligence analyses of circulating cell-free DNA for the minimally invasive detection of fetal congenital heart defects. In this prospective study, whole-genome cytosine nucleotide methylation analysis was performed on circulating cell-free DNA using the Illumina Infinium MethylationEPIC BeadChip array. Multiple artificial intelligence approaches were evaluated for the detection of congenital hearts. The Ingenuity Pathway Analysis program was used to identify gene pathways that were epigenetically altered and important in congenital heart defect pathogenesis to further elucidate the pathogenesis of isolated congenital heart defects. There were 12 cases of isolated nonsyndromic congenital heart defects and 26 matched controls. A total of 5918 cytosine nucleotides involving 4976 genes had significantly altered methylation, that is, a P value of <.05 along with ≥5% whole-genome cytosine nucleotide methylation difference, in congenital heart defect cases vs controls. Artificial intelligence analysis of the methylation data achieved excellent congenital heart defect predictive accuracy (areas under the receiver operating characteristic curve, ≥0.92). For example, an artificial intelligence model using a combination of 5 whole-genome cytosine nucleotide markers achieved an area under the receiver operating characteristic curve of 0.97 (95% confidence interval, 0.87-1.0) with 98% sensitivity and 94% specificity. We found epigenetic changes in genes and gene pathways involved in the following important cardiac developmental processes: "cardiovascular system development and function," "cardiac hypertrophy," "congenital heart anomaly," and "cardiovascular disease." This lends biologic plausibility to our findings. This study reported the feasibility of minimally invasive detection of fetal congenital heart defect using artificial intelligence and DNA methylation analysis of circulating cell-free DNA for the prediction of fetal congenital heart defect. Furthermore, the findings supported an important role of epigenetic changes in congenital heart defect development.

Maternal-Fetal Medicine: The Journey Thus Far and Way Forward

Maternal-Fetal Medicine: The Journey Thus Far and Way Forward

GenomeMixer and TRUST: Novel bioinformatics tools to improve reliability of Non-Invasive Prenatal Testing (NIPT) for fetal aneuploidies

Non-invasive prenatal testing (NIPT) screens for common fetal chromosomal abnormalities through analysis of circulating cell-free DNA in maternal blood by massive parallel sequencing. NIPT reliability relies on both the estimation of the fetal fraction (ff) and on the sequencing depth (sd) but how these parameters are linked is unknown. Several bioinformatics tools have been developed to determine the ff but there is no universal ff threshold applicable across diagnostics laboratories. Thus, we developed two tools allowing the implementation of a strategy for NIPT results validation in clinical practice: GenomeMixer, a semi-supervised approach to create synthetic sequences and to estimate confidence intervals for NIPT validation and TRUST to estimate the reliability of NIPT results based on confidence intervals found in this study. We retrospectively validated these new tools on 2 cohorts for a total of 1439 samples with 31 confirmed aneuploidies. Through the analysis of the interrelationship between ff, sd and chromosomal aberration detection, we demonstrate that these parameters are profoundly connected and cannot be considered independently. Our tools take in account this critical relationship to improve NIPT reliability and facilitate cross laboratory standardization of this screening test.

Noninvasive prenatal screening in twin pregnancies with cell-free DNA using the IONA test: a prospective multicenter study

In singleton pregnancies, studies investigating cell-free DNA in maternal blood have consistently reported high detection rate and low false-positive rate for the 3 common fetal trisomies (trisomies 21, 18, and 13). The potential advantages of noninvasive prenatal testing in twin pregnancies are even greater than in singletons, in particular lower need for invasive testing and consequent fetal loss rate. However, several organizations do not recommend cell-free DNA in twin pregnancies and call for larger prospective studies. In response to this, we undertook a large prospective multicenter study to establish the screening performance of cell-free DNA for the 3 common trisomies in twin pregnancies. Moreover, we combined our data with that reported in published studies to obtain the best estimate of screening performance. This was a prospective multicenter blinded study evaluating the screening performance of cell-free DNA in maternal plasma for the detection of fetal trisomies in twin pregnancies. The study took place in 6 fetal medicine centers in England, United Kingdom. The primary outcome was the screening performance and test failure rate of cell-free DNA using next generation sequencing (the IONA test). Maternal blood was taken at the time of (or after) a conventional screening test. Data were collected at enrolment, at any relevant invasive testing throughout pregnancy, and after delivery until the time of hospital discharge. Prospective detailed outcome ascertainment was undertaken on all newborns. The study was undertaken and reported according to the Standards for Reporting of Diagnostic Accuracy Studies. A pooled analysis was also undertaken using our data and those in the studies identified by a literature search (MEDLINE, Embase, CENTRAL, Cochrane Library, and ClinicalTrials.gov) on June 6,2020. A total of 1003 women with twin pregnancies were recruited, and complete data with follow-up and reference data were available for 961 (95.8%); 276 were monochorionic and 685 were dichorionic. The failure rate was 0.31%. The mean fetal fraction was 12.2% (range, 3%-36%); all 9 samples with a 3% fetal fraction provided a valid result. There were no false-positive or false-negative results for trisomy 21 or trisomy 13, whereas there was 1 false-negative and 1 false-positive result for trisomy 18. The IONA test had a detection rate of 100% for trisomy 21 (n=13; 95% confidence interval, 75-100), 0% for trisomy 18 (n=1; 95% confidence interval, 0-98), and 100% for trisomy 13 (n=1; 95% confidence interval, 3-100). The corresponding false-positive rates were 0% (95% confidence interval, 0-0.39), 0.10% (95% confidence interval, 0-0.58), and 0% (95% confidence interval, 0-0.39), respectively. By combining data from our study with the 11 studies identified by literature search, the detection rate for trisomy 21 was 95% (n=74; 95% confidence interval, 90-99) and the false-positive rate was 0.09% (n=5598; 95% confidence interval, 0.03-0.19). The corresponding values for trisomy 18 were 82% (n=22; 95% confidence interval, 66-93) and 0.08% (n=4869; 95% confidence interval, 0.02-0.18), respectively. There were 5 cases of trisomy 13 and 3881 non-trisomy 13 pregnancies, resulting in a computed average detection rate of 80% and a false-positive rate of 0.13%. This large multicenter study confirms that cell-free DNA testing is the most accurate screening test for trisomy 21 in twin pregnancies, with screening performance similar to that in singletons and very low failure rates (0.31%). The predictive accuracy for trisomies 18 and 13 may be less. However, given the low false-positive rate, offering first-line screening with cell-free DNA to women with twin pregnancy is appropriate in our view and should be considered a primary screening test for trisomy 21 in twins.

Multiple Aneuploidy Pattern Detected by Non-invasive Prenatal Screening in Successive Pregnancies [25K

INTRODUCTION: We present a case where non-invasive prenatal screening (NIPS) detected a similar pattern of multiple aneuploidies in sequential pregnancies. METHODS: Case Presentation (with patient consent): A 25 year-old G3P2 patient had NIPS, by massively parallel sequencing (MPS), in her second and third pregnancies. Her first NIPS result was positive for trisomies 18 and 21. Further bioinformatics review revealed possible partial trisomy 13 and complete trisomies 14 and 20, in addition to the previously reported trisomies. NIPS by MPS repeated at another lab was non-reportable. Amniocentesis revealed normal karyotype, microarray, FISH for the chromosomes of interest, and chromosome 14 uniparental disomy testing. Maternal karyotype, microarray, and chromosomal breakage studies were normal. Patient had a negative oncology evaluation. Patient had an insignificant medical and medication history. Patient delivered a healthy girl. Placenta was small with a normal karyotype. Her current pregnancy shows a similar NIPS pattern. Patient declined amniocentesis and further aneuploidy screening. Second trimester ultrasound detected fetal hyperechoic bowel. Repeat maternal chromosomal breakage studies were normal. CONCLUSION: NIPS analyzes cell-free DNA in maternal blood originating from maternal and cytotrophoblast cells. The similar NIPS pattern in both pregnancies indicates a likely maternal etiology. When multiple aneuploidies are observed by NIPS, possible etiologies include maternal benign tumor or malignancy and fetal, placental or maternal chromosome abnormalities. However, subsequent fetal and maternal evaluations were normal. This case highlights that NIPS may yield unexpected results that are not representative of the fetal or maternal karyotypes and an extensive multi-specialty work-up may be recommended in these scenarios.

541: Relationship between total cell-free DNA and hypertensive disorders of pregnancy using an epigenetic marker

To evaluate relation between total cell-free DNA in maternal blood and hypertensive disorders of pregnancy using an epigenetic marker. A nested case-control study was conducted. We collected 92 maternal blood samples with hypertensive disorders of pregnancy (63 with preeclampsia, and 29 with gestational hypertension), and 188 samples of normal controls between 6 and 41 gestational weeks. HYP2 was used as the total cell-free DNA marker, and we evaluated HYP2 levels in maternal plasma of normal and preeclampsia patients using real-time PCR. Measured HYP2 levels were converted to multiples of the median, and corrected for maternal age, body mass index, and gestational age at sampling. We compared the concentrations of HYP2 and multiple of the median values in normal controls and patient groups. In addition, we analyzed them with regard to onset-time of preeclampsia and accompanying small for gestational age neonates. HYP2 levels in patients with preeclampsia were significantly increased during the third trimester. However, these differences were not significant during the first and second trimester. When we divided the patients with regard to onset time of preeclampsia, significant difference in HYP2 levels was observed in patients who subsequently developed early-onset preeclampsia from the second trimester compare to normal controls. However, when we divided patients group with regard to birth weight, both median HYP2 concentrations and multiples of median values are significantly increased in patients who subsequently develop preeclampsia with small for gestational age during all trimesters in patients who subsequently developed preeclampsia and delivered small for gestational age neonates. The levels of cell-free total DNA in patients who later developed preeclampsia with intrauterine growth restriction were significantly increased from the first trimester.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies

Abstract Several externally blinded validation and implementation studies in the last 9 years have shown that it is now possible, through analysis of cell-free (cf) DNA in maternal blood, to effectively detect a high proportion of fetuses affected by trisomies 21, 18, and 13 at a much lower false-positive rate (FPR) than all other existing screening methods. This article is aimed at reviewing technical and clinical considerations for implementing cfDNA testing in routine practice, including methods of analysis, performance of the test, models for clinical implementation, and interpretation of results.

Could high levels of cell-free DNA in maternal blood be associated with maternal health and perinatal outcomes?

The aim of this study was to evaluate the maternal and foetal factors affect higher cell-free DNA (cfDNA) levels and to investigate a possible relationship between high cfDNA levels and adverse perinatal outcomes. From a total of 4594 women who underwent non-invasive prenatal testing from January 2016 to March 2018 in our hospital, 112 women had high levels of cfDNA, which was not appropriate for testing. Maternal characteristics and perinatal outcomes were compared between patients with high levels of cfDNA and normal levels of cfDNA. Patients with high levels of cfDNA had greater risks than patients with normal cfDNA levels of pregnancy complications but no statistically significant difference was found. Patients with high cfDNA levels had higher foetal death rates with a statistically significant difference (9.8% versus 1.8%, p = .024). An increase in foetal death could be expected in patients with increased cfDNA levels; therefore, these patients should be carefully followed up during pregnancy.IMPACT STATEMENTWhat's already known about this topic? Most studies about cfDNA levels are focussed on the foetal fraction. There are new arguments about maternal health and cfDNA. It is known that autoimmune diseases as systemic lupus erythematosus (SLE) and maternal obesity increase cell turnover. There are also clinical studies suggesting a relationship between low molecular weight heparin therapy and the amount of cfDNA.What do the results of this study add? This is the first study evaluating the maternal and foetal biological factors affecting cfDNA concentrations and investigating the possible relationship between high cfDNA levels and adverse perinatal outcomes in patients with high levels of cfDNA compared to patients with normal levels of cfDNA. In the present study, it was found that an increase in foetal death could be expected in patients with higher cfDNA levels.What are the implications of these findings for clinical practice and/or further research? If potential effects and underlying causes of increased cfDNA could be explained, cfDNA might be used as a biomarker for adverse perinatal outcomes.

The Urinary Peptidome as a Noninvasive Biomarker Development Strategy for Prenatal Screening of Down's Syndrome.

Prenatal screening for Down's syndrome based on maternal age, ultrasound measures, and maternal serum biomarkers is recommended worldwide, but the false-positive rate and poor diagnostic performance of these screening tests remain problematic. Genetic analysis of cell-free DNA in maternal blood has been developed as a new prenatal screening for Down's syndrome, but it has a number of limitations, including turnaround time and cost. Prenatal screening diagnostic innovation calls for new tests that are noninvasive, accurate, and affordable. We report original observations on potential peptide biomarkers in maternal urine for screening of fetal Down's syndrome. The peptidome of urine samples from 23 pregnant women carrying Down's syndrome fetuses and 30 pregnant women carrying fetuses with normal karyotype was fractionated by weak cation exchange magnetic beads and analyzed by MALDI-TOF mass spectrometry. Levels of six peptides (m/z 1022.1, 1032.1, 1099.5, 1155.9, 1306.6, and 2365.6) were significantly altered between the case and control groups after controlling for maternal and gestational age. A classification model was constructed based on these candidate peptides that could differentiate fetuses with Down's syndrome from controls with a sensitivity of 95.7%, a specificity of 70.0%, and an area under receiver operating characteristic curves of 0.909 (95% confidence interval, 0.835-0.984). Peptide peaks at m/z 1099.5 and 1155.9 were identified as the partial sequences of alpha-1-antitrypsin and heat shock protein beta-1, respectively. These new findings support the new idea that maternal urinary peptidome offers prospects for noninvasive biomarker discovery and development for the prenatal screening of fetal Down's syndrome.

Implementation of Maternal Blood Cell Free DNA Testing in Early Screening for Aneuploidies

Abstract In the past 9 years, several externally blinded validation and implementation studies have shown that it is now possible, through analysis of cell-free (cf) DNA in maternal blood, to effectively detect a high proportion of fetuses affected by trisomies 21, 18, and 13 at a much lower false positive rate than all other existing screening methods. This article aims to review the technical and clinical considerations for implementing cfDNA testing in routine practice, including methods of analysis, performance of the test, models for clinical implementation, and interpretation of results.

Women’s Attitudes Toward Invasive and Noninvasive Testing When Facing a High Risk of Fetal Down Syndrome

Noninvasive prenatal testing (NIPT) using cell-free DNA in maternal blood is increasingly common compared with invasive testing (IT) in routine antenatal detection of Down syndrome (DS). To assess attitudes and decision making in pregnant women facing a risk of fetal DS greater than 1 in 250 as established by combined first trimester screening at 11 to 14 weeks of gestation. Survey study in which data were collected from pregnant women at high risk of fetal DS participating in a randomized clinical trial. Data were collected from April 8, 2014, to April 7, 2016, in 57 prenatal diagnosis centers in France. Data were analyzed in 2018. Data on attitudes were collected prior to offering randomization between NIPT and IT, whereas data on decision making and test results were collected as part of the clinical trial. The primary outcome related to attitudes. A hierarchical cluster analysis was conducted to identify clusters with contrasting attitudes. Logistic regression analyses were used to identify factors associated with attitudes. All 2436 consecutive women to whom the study was proposed (mean [SD] age, 36.3 [5.0] years) answered the questionnaire: 515 (21.1%) expressed preference toward IT with complete karyotyping, whereas 1843 (75.7%) favored NIPT with almost certain but limited information. Hierarchical cluster analysis yielded 4 different clusters that mainly differed in attitudes toward risk taking and extent of information seeking. Factors likely associated with attitudes driven by risk aversion were mostly age and religious beliefs (adjusted odds ratio [aOR], 1.03; 95% CI, 1.00-1.05; P = .03 and aOR, 1.62; 95% CI, 1.29-2.04; P < .001, respectively), whereas higher nuchal translucency measurements by ultrasonography were associated with attitudes driven by ambiguity aversion (aOR, 1.67; 95% CI, 1.27-2.20; P < .001). For attitudes involving both risk and ambiguity aversion at different extents, lower education was associated with highly valuing all possibilities of getting information on pregnancy, whereas higher education was associated with highly valuing information on fetal DS as a primary concern (aOR, 0.54; 95% CI, 0.44-0.67; P < .001 and aOR, 1.44; 95% CI, 1.20-1.74; P < .001, respectively). In all, decision making was in line with attitudes. Aversion to risk of fetal loss related to IT and aversion to ambiguity generated by incomplete information from NIPT played a major role in shaping attitudes and decision making. Informed decision making should require pregnant women at high risk of DS to receive extensive information on targeted abnormalities by both tests.

Cell-free RNA as a Fetal Timekeeper.

Cancer and fetal development share a lot. This is nothing new. The 19th century pathologist John Beard noted the similarities between a tumor and a trophoblast. Building on that, Lloyd Old, a pioneer in cancer immunology said, “If you've ever seen the trophoblast invading the uterus, it invades, spreads, creates a blood supply. It also suppresses the maternal immune system. All of those are characteristics of cancer” (1). Today, tumors are being diagnosed noninvasively through the ability to find traces of circulating malignant material in blood. The same holds true for fetal development. The ability to reliably detect fetal cell-free DNA in maternal blood is changing clinical practice. I spoke with Howard Hughes Investigator and bioengineering, physics, and applied physics professor at Stanford University, Stephen Quake, who has played a central role in these innovations. Motivated to replace invasive biopsies with blood tests, Quake developed a polymorphism-independent shotgun DNA sequencing test that could detect fetal aneuploidy from maternal plasma (2). “After 10 years, this has had an enormous impact,” he says, “three million women are tested each year, and these methods have been widely adopted in the liquid biopsy space.” Quake has his sights on another challenge around childbirth—estimating gestational age and the risk for preterm delivery. “It started with the idea of following human development, and using RNA to do that,” Quake explains. In a manuscript in which he and colleagues combined high-throughput microarrays for RNA analysis with next-generation sequencing …

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.

First-Trimester Contingent Screening for Trisomy 21 by Fetal Nuchal Translucency and Maternal Serum Biomarkers and Maternal Blood Cell-Free DNA Testing

AbstractAnalysis of cell-free DNA in maternal blood has been proposed as a novel screening method for evaluation of aneuploidies. The higher performance of this technique in screening of trisomies compared to all currently available methods would lead to widespread use of this technique in clinical settings. In total, 1,066,829 singleton pregnancies referred to Nilou Clinical Laboratory were screened for chromosomal trisomies during a period of 12 years. First-trimester screening (FTS), Triple and Quad markers of second-trimester screening (STS) as well as integrated results have been obtained from 444,515, 34,984, 560,857 and 26,473 singleton pregnancies respectively. Non-invasive prenatal test (NIPT) using cfDNA was applied in 3500 pregnant women. Risk cutoffs, detection rates (DRs) and false positive rates (FPRs) were assessed for combinations of screening strategies to identify the most efficient strategy for contingent cfDNA testing. Contingent screening including FTS and NIPT offer to 20% of cases would lead to detection of 98% of fetuses with trisomy 21 at a total invasive testing rate of 1.1%. Contingent screening including STS and NIPT offer to 9.0% of cases would lead to detection of 95.5% of fetuses with trisomy 21 at a total invasive testing rate of 4.5%. Contingent screening including FTS or STS and cfDNA testing are efficient strategies for screening of trisomy 21.

A cost-effectiveness analysis comparing two different strategies in advanced maternal age: Combined first-trimester screening and maternal blood cell-free DNA testing

ObjectiveTo estimate cost efficacy of first-trimester screening strategies based on nuchal translucency (NT) and maternal blood cell-free DNA (cfDNA) testing in women with advanced maternal age (AMA). Materials and methodsThis was a retrospective population-based analysis of all pregnant women with AMA booked for combined first-trimester screening (cFTS) in China over a 3-year period. The assumed screening strategies were the following: cFTS (Strategy 1), cfDNA testing as a first-tier investigation replacing biomarkers after NT measurement (Strategy 2), and cfDNA testing combined with dating ultrasound for all women (Strategy 3). The direct costs were compared between strategies. ResultsStrategy 1 was completed in 6443 women with AMA. The respective detection rates were 94.5% and 90.9% for trisomies 21 and 18, with a total screen-positive rate of 13.5%. Such a policy resulted in 871 invasive tests and a total cost of $747,870 or a cost of $116 per person tested. Strategy 2 would result in a total cost of $1,812,570, or a cost of $281 per person tested, with increased detection rates for trisomies 21 and 18, and a decreased number of invasive tests compared with strategy 1. The total cost of Strategy 3 would be $1,675,430, or a cost of $260 per person tested with the least number of invasive tests. ConclusionThe cfDNA modalities have the advantages of higher detection rate for common trisomies and lower screening-positive rate. However, the cost of cfDNA testing needs to decrease significantly if it is to replace the current cFTS practice in a population of AMA on a purely cost effectiveness basis.

Non-Invasive Prenatal Testing (NIPT) in pregnancies with trisomy 21, 18 and 13 performed in a public setting – factors of importance for correct interpretation of results

ObjectivesWe have established an open source platform for non-invasive prenatal testing (NIPT) based on massively parallel whole-genome sequencing in a public setting. The objective of this study was to investigate factors of importance for correct interpretation of NIPT results to ensure a high sensitivity and specificity. Study designThis investigation is a retrospective case-control study performed in a public NIPT center. The study included 108 aneuploid cases and 165 euploid controls. MPS was performed on circulating cell-free DNA in maternal blood. The pipeline included automated library preparation and sequencing on a HiSeq1500 (Illumina). The software programmes WISECONDOR and SeqFF were used for data analysis of aneuploidy status and fetal fraction of cell-free DNA, respectively. Lower limit of fetal fraction for aneuploidy testing was 0.02. ResultsWe identified four false negative aneuploidy cases of which two were explained by a vanishing twin. The number of no-call cases due to low fetal fraction was 8 out of 273 (2.9%). The sensitivity and specificity, when no-calls and vanished twins were excluded, were 100% and 99.5% for T21, 91% and 99.2% for T18, and 100% and 99.6% for T13. By multiple regression analysis we found a significant association between fetal fraction and gestational age, maternal BMI and ART treatment. ConclusionWith a non-commercial open source NIPT set-up having the same high test-performance as reported by large private laboratories, we show that fetal fraction, a vanishing twin, BMI, gestational age and ART treatment are important factors in the interpretation of NIPT results.