cfDNA In Maternal Plasma Research Articles

Non-invasive determination of gene expression in placental tissue using maternal plasma cell-free DNA fragmentation characters

BackgroundThe expression profiles of placental genes are crucial for understanding the pathogenesis of fetal development and placental-origin pregnancy syndromes. However, owing to ethical limitations and the risks of puncture sampling, it is difficult to obtain placental tissue samples repeatedly, continuously, multiple times, or in real time. Establishing a non-invasive method for predicting placental gene expression profiles through maternal plasma cell-free DNA (cfDNA) sequencing, which carries information about the source tissue and gene expression, can potentially solve this problem. MethodsPeripheral blood and placental samples were collected simultaneously from pregnant women who underwent cesarean section. Deep sequencing was performed on the separated plasma cfDNA and single-cell sequencing was performed on peripheral blood mononuclear cells (PBMC), chorioamniotic membranes (CAM), placental villi (PV), and decidua basalis (DB). The aggregation of corresponding information for each gene was combined with the transcriptome of PBMCs and a differential resolution transcriptome of the placenta. This combined information was then utilized for the construction of gene expression prediction models. After training, all models evaluated the correlation between the predicted and actual gene expression levels using external test set data. ResultsFrom five women, more than 20 million reads were obtained using deep sequencing for plasma cfDNA; PBMCs obtained 32,401 single-cell expression profiles; and placental tissue obtained 156,546 single-cell expression profiles (59,069, 44,921, and 52,556 for CAM, PV, and DB, respectively). The cells in the PBMC and placenta were clustered and annotated into five and eight cell types, respectively. A “DEPICT” gene expression prediction model was successfully constructed using deep neural networks. The predicted correlation coefficients were 0.75 in PBMCs, 0.84 in the placenta, and 0.78, 0.80, and 0.77 in CAM, BP, and PV respectively, and greater than 0.68 in different cell lines in the placenta. ConclusionThe DEPICT model, which can noninvasively predict placental gene expression profiles based on maternal plasma cfDNA fragmentation characteristics, was constructed to overcome the limitation of the inability to obtain real-time placental gene expression profiles and to improve research on noninvasive prediction of placental origin pregnancy syndrome.

Noninvasive prenatal screening in a pregnant woman with a history of stem cell transplant from a male donor: A case report and literature review.

As a screening method, inaccuracies in noninvasive prenatal screening (NIPS) exist, which are often attributable to biological factors. One such factor is the history of transplantation. However, there are still limited reports on such NIPS cases. We report an NIPS case of a pregnant woman who had received a stem cell transplant from a male donor. To determine the karyotype in the woman's original cell, we performed chromosome microarray analysis (CMA) on her postnatal blood and oral mucosa. To comprehensively estimate the cell-free DNA (cfDNA) composition, we further performed standard NIPS procedures on the postnatal plasma. Moreover, we reviewed all published relevant NIPS case reports about pregnant women with transplantation history. NIPS showed a low-risk result for common trisomies with a fetal fraction of 65.80%. CMA on maternal white blood cells showed a nonmosaic male karyotype, while the oral mucosa showed a nonmosaic female karyotype. The proportion of donor's cfDNA in postnatal plasma was 94.73% based on the Y-chromosome reads ratio. The composition of cfDNA in maternal plasma was estimated as follows: prenatally, 13.60% maternal, 65.80% donor, and 20.60% fetal/placental, whereas postnatally, 5.27% maternal and 94.73% donor. This study expanded our understanding of the influence of stem cell transplantation on NIPS, allowing us to optimize NIPS management for these women.

Performance of whole-genome promoter nucleosome profiling of maternal plasma cell-free DNA for prenatal noninvasive prediction of fetal macrosomia: a retrospective nested case-control study in mainland China

BackgroundFetal macrosomia is common occurrence in pregnancy, which is associated with several adverse prognosis both of maternal and neonatal. While, the accuracy of prediction of fetal macrosomia is poor. The aim of this study was to develop a reliable noninvasive prediction classifier of fetal macrosomia.MethodsA total of 3600 samples of routine noninvasive prenatal testing (NIPT) data at 12+ 0–27+ 6 weeks of gestation, which were subjected to low-coverage whole-genome sequencing of maternal plasma cell-free DNA (cfDNA), were collected from three independent hospitals. We identified set of genes with significant differential coverages by comparing the promoter profiling between macrosomia cases and controls. We selected genes to develop classifier for noninvasive predicting, by using support vector machine (SVM) and logistic regression models, respectively. The performance of each classifier was evaluated by area under the curve (AUC) analysis.ResultsAccording to the available follow-up results, 162 fetal macrosomia pregnancies and 648 matched controls were included. A total of 1086 genes with significantly differential promoter profiling were found between pregnancies with macrosomia and controls (p < 0.05). With the AUC as a reference，the classifier based on SVM (CMA-A2) had the best performance, with an AUC of 0.8256 (95% CI: 0.7927–0.8586).ConclusionsOur study provides that assessing the risk of fetal macrosomia by whole-genome promoter nucleosome profiling of maternal plasma cfDNA based on low-coverage next-generation sequencing is feasible.

Plasma cell-free DNA analysis for COVID-19 and beyond.

In human, circulating cell-free DNA (cfDNA) in peripheral blood originates from various tissues/organs. In healthy subjects, cfDNA in plasma is mostly derived from the hematopoietic system; however, in various physiological and pathological conditions, the affected tissues would release DNA into circulation, thus allowing one to utilize cfDNA as a surrogate to monitor the dynamics of the inaccessible tissues in a non-invasive manner.1 Such approaches are commonly referred to as “liquid biopsies.” At present, three types of liquid biopsies are widely studied and adapted into clinical practices: non-invasive prenatal testing for genetic disorders of the fetus by sequencing and analyzing the cfDNA in maternal plasma; detecting circulating tumour-derived DNA (ctDNA) in the plasma of cancer patients makes it possible for early diagnosis, real-time tumour evolution monitoring and guidance in targeted therapy; donor-derived DNA in the plasma of transplantation recipients could be used for comprehensive monitoring of allograft rejection and injury. However, cfDNA applications in other clinical scenarios are much less explored. The COVID-19 pandemic lasted for more than 2 years. Due to the high variability in clinical symptoms of the COVID-19 patients, disease severity assessment is of clinical value in providing precision healthcare and better management for the large-number of patients. However, in most COVID-19 patients, a handful of organs is affected simultaneously, which makes the clinical assays incompetent for accurate and rapid severity assessment. To this end, in a recent work,2 Bai and colleagues developed a cfDNA-based approach for disease severity assessment of COVID-19 patients with high translational feasibility. Bai et al. performed whole-genome sequencing of plasma cfDNA and identified three types of features (including fragment length ratio, transcription start site coverage, and frequency of 4-nucleotide motifs at 5’ fragment ends3) in 399 consecutive hospitalized COVID-19 patients. They further combined these features with laboratory results and trained a model using machine learning approach, named M2Model, to distinguish critical from noncritical COVID-19 patients with a sensitivity of 85.19% at 93.33% specificity. In addition, for clinically diagnosed critical COVID-19 patients, Bai et al. successfully clustered them into three risk strata, and they showed that the M2Model was able to predict the risk of these patients towards deteriorating critical illness; moreover, survival analysis further showed that the high-risk critical COVID-19 patients predicted by the model required a significantly longer hospital stay than the other groups. Besides Bai et al. work, several studies from other groups had also explored the clinical applications of cfDNA as a biomarker for COVID-19 monitoring. For instance, Cheng et al.4 and other groups5 investigated the characteristics of cfDNA in COVID-19 patients and further developed various cfDNA-based approaches to appraise the internal organ injuries due to COVID-19. In particular, Cheng et al. reported that the concentration of cfDNA positively correlated with COVID-19 disease severity and the cfDNA profile at admission allowed one to identify patients who subsequently required intensive care or died during hospitalization. Together, these studies demonstrated the clinical potential of plasma cfDNA analysis in COVID-19, which is virtually an infectious disease. Apart from COVID-19, more and more studies have extended the significance of cfDNA in a broad spectrum of clinical scenarios in addition to its traditional applications (e.g., prenatal testing, cancer and organ transplantation). For examples, cfDNA analysis would provide precise subtyping of anaemia and effective monitoring of treatment responses6; Teo et al. showed that nucleosome signals inferred from cfDNA correlated with age-associated changes in the epigenome in vivo 7 Another recent study reported that cfDNA plays an important role in the pathogenesis of systemic lupus erythematosus and showed considerable diagnostic value.8 In the past several years, technical advances have also been developed, which allowed one to measure more types of cfDNA molecules, such as single-strand, ultralong and circular ones 9; moreover, besides concentration, genetic variation, DNA methylation profiles and copy number aberrations, the fragmentomic feature of plasma cfDNA is becoming an emerging direction in liquid biopsy, and it has been successfully implemented as novel diagnostic biomarkers for early cancer diagnosis, tumour-of-origin prediction, and tumour subtype.10 Hence, with the improvement of experimental and analytical methodologies, we believe that the next few years would witness the large expansion of the application range of plasma cfDNA in liquid biopsy, from traditional cancer fields to infectious diseases, and beyond. This work was supported by open project of BGI-Shenzhen, Shenzhen 518000, China (grant number: BGIRSZ20200007) and Shenzhen Bay Laboratory. The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Liquid Biopsy: A Family of Possible Diagnostic Tools.

Liquid biopsies could be considered an excellent diagnostic tool, in different physiological or pathological conditions. The possibility of using liquid biopsies for non-invasive clinical purposes is quite an old idea: indeed many years ago it was already being used in the field of non-invasive prenatal tests (NIPT) for autosomal fetal aneuploidy evaluation. In 1997 Lo et al. had identified fetal DNA in maternal plasma and serum, showing that about 10–15% of cfDNA in maternal plasma is derived from the placenta, and biologic fluid represents an important and non-invasive technique to evaluate state diseases and possible therapies. Nowadays, several body fluids, such as blood, urine, saliva and other patient samples, could be used as liquid biopsy for clinical non-invasive evaluation. These fluids contain numerous and various biomarkers and could be used for the evaluation of pathological and non-pathological conditions. In this review we will analyze the different types of liquid biopsy, their potential role in clinical diagnosis and the functional involvement of extracellular vesicles in these fluids as carriers.

More attention should be paid to pregnant women who fail non-invasive prenatal screening

ObjectiveWe discuss how to handle failure of first-pass non-invasive prenatal screening (NIPS) and investigate the pregnancy outcomes after second-pass failure. MethodsA total of 35,187 pregnant women underwent NIPS in a single center. Those who failed first-pass NIPS were re-tested after a repeat blood draw. Those who failed again were offered genetic counseling. We recorded antenatal data and pregnancy outcomes. ResultsA total of 273 (0.78%) women failed the first test. On re-testing, 220 (80.59%) yielded reliable results and 53 failed the test again. Women with higher total cell-free DNA (cfDNA) levels evidenced a lower NIPS success rate (40%) and a higher incidence of adverse pregnancy outcomes. ConclusionsMost women who failed first-pass NIPS yielded reliable results on repeat testing, especially those with lower fetal fraction. Higher concentrations of cfDNA in maternal plasma were associated with poorer pregnancy outcomes. Such women require special attention, thus early medical intervention, to avoid an adverse prognosis.

Non-invasive prediction of fetal growth restriction by whole-genome promoter profiling of maternal plasma DNA: a nested case-control study.

ObjectiveTo predict fetal growth restriction (FGR) by whole‐genome promoter profiling of maternal plasma.DesignNested case–control study.SettingHospital‐based.Population or Sample810 pregnancies: 162 FGR cases and 648 controls.MethodsWe identified gene promoters with a nucleosome footprint that differed between FGR cases and controls based on maternal plasma cell‐free DNA (cfDNA) nucleosome profiling. Optimal classifiers were developed using support vector machine (SVM) and logistic regression (LR) models.Main outcome measuresGenes with differential coverages in promoter regions through the low‐coverage whole‐genome sequencing data analysis among FGR cases and controls. Receiver operating characteristic (ROC) analysis (area under the curve [AUC], accuracy, sensitivity and specificity) was used to evaluate the performance of classifiers.ResultsThrough the low‐coverage whole‐genome sequencing data analysis of FGR cases and controls, genes with significantly differential DNA coverage at promoter regions (−1000 to +1000 bp of transcription start sites) were identified. The non‐invasive ‘FGR classifier 1’ (CFGR1) had the highest classification performance (AUC, 0.803; 95% CI 0.767–0.839; accuracy, 83.2%) was developed based on 14 genes with differential promoter coverage using a support vector machine.ConclusionsA promising FGR prediction method was successfully developed for assessing the risk of FGR at an early gestational age based on maternal plasma cfDNA nucleosome profiling.Tweetable abstractA promising FGR prediction method was successfully developed, based on maternal plasma cfDNA nucleosome profiling.

The fragmentation patterns of maternal plasma cell-free DNA and its applications in non-invasive prenatal testing.

The discovery of cell-free DNA (cfDNA) in maternal plasma has opened up new promises for the development of non-invasive prenatal testing (NIPT). Application of cfDNA in NIPT of fetus diseases and abnormalities is restricted by the low amount of fetal DNA molecules in maternal plasma. Fetus-derived cfDNA in maternal plasma are shorter than maternal DNA, thus leveraging the maternal and fetus-derived cfDNA molecules size difference has become a novel and more accurate method for NIPT. However, multiple biological properties such as size distribution of plasma DNA, proportion of fetal-derived DNA and methylation levels in maternal plasma across different gestational ages still remain largely unknown. Further insights into the size distribution and fragmentation pattern of circulating plasma cfDNA will shed light on the origin and fragmentation mechanisms of cfDNA during physiological and pathological processes in prenatal diseases and enhance our ability to take the advantage of plasma cfDNA as a molecular diagnostic tool. In the review, we start by summarizing the research techniques for the determination of the fragmentation profiles of cfDNA in maternal plasma. We then summarize the main progress and findings in size profiles of maternal plasma cfDNA and cffDNA. Finally, we discuss the potential diagnostic applications of plasma cfDNA size profiling.

Detection of microbial cell-free DNA in maternal and umbilical cord plasma in patients with chorioamnionitis using next generation sequencing.

BackgroundChorioamnionitis has been linked to spontaneous preterm labor and complications such as neonatal sepsis. We hypothesized that microbial cell-free (cf) DNA would be detectable in maternal plasma in patients with chorioamnionitis and could be the basis for a non-invasive method to detect fetal exposure to microorganisms.ObjectiveThe purpose of this study was to determine whether next generation sequencing could detect microbial cfDNA in maternal plasma in patients with chorioamnionitis.Study designMaternal plasma (n = 94) and umbilical cord plasma (n = 120) were collected during delivery at gestational age 28–41 weeks. cfDNA was extracted and sequenced. Umbilical cord plasma samples with evidence of contamination were excluded. The prevalence of microorganisms previously implicated in choriomanionitis, neonatal sepsis and intra-amniotic infections, as described in the literature, were examined to determine if there was enrichment of these microorganisms in this cohort. Specific microbial cfDNA associated with chorioamnionitis was first detected in umbilical cord plasma and confirmed in the matched maternal plasma samples (n = 77 matched pairs) among 14 cases of histologically confirmed chorioamnionitis and one case of clinical chorioamnionitis; 63 paired samples were used as controls. A correlation of rank of a given microorganism across maternal plasma and matched umbilical cord plasma was used to assess whether signals found in umbilical cord plasma were also present in maternal plasma.ResultsMicrobial DNA sequences associated with clinical and/or histological chorioamnionitis were enriched in maternal plasma in cases with suspected chorioamnionitis when compared to controls (12/14 microorganisms, p = 0.02). Analysis of the microbial cfDNA in umbilical cord plasma among the 1,251 microorganisms detectable with this assay identified Streptococcus mitis, Ureaplasma spp., and Mycoplasma spp. in cases of suspected chorioamnionitis. This assay also detected cfDNA from Lactobacillus spp. in controls. Comparison between maternal plasma and umbilical cord plasma confirmed these signatures were also present in maternal plasma. Unbiased analysis of microorganisms with significantly correlated signal between matched maternal plasma and umbilical cord plasma identified the above listed 3 microorganisms, all of which have previously been implicated in patients with chorioamnionitis (Mycoplasma hominis p = 0.0001; Ureaplasma parvum p = 0.002; Streptococcus mitis p = 0.007). These data show that the pathogen signal relevant for chorioamnionitis can be identified in both maternal and umbilical cord plasma.ConclusionThis is the first report showing the detection of relevant microbial cell-free cfDNA in maternal plasma and umbilical cord plasma in patients with clinical and/or histological chorioamnionitis. These results may lead to the development of a specific assay to detect perinatal infections for targeted therapy to reduce early neonatal sepsis complications.

Early second-trimester plasma cell free DNA levels with subsequent risk of pregnancy complications

ObjectiveTo investigate the association between cfDNA levels measured during non-invasive prenatal testing (NIPT) and the risk of pregnancy complications in a Chinese population. MethodsThis was a retrospective cohort study of 831 pregnant women who underwent NIPT at 12–22 weeks of gestation. Maternal plasma cfDNA levels and pregnancy outcomes were obtained from NIPT Screening System and hospitalization records, respectively. Logistic regression analysis was performed to investigate the relationship between cfDNA levels and pregnancy complications (after adjusting for confounding factors). ResultsMaternal cfDNA levels were significantly higher in women diagnosed with intrahepatic cholestasis of pregnancy (ICP) and preeclampsia (PE) compared to pregnant women with non-pregnancy complications (NPC) (median cfDNA 7.07, 6.42 vs. 5.99 ng/mL). Increase in cfDNA levels were associated with an increased risk for ICP (adjusted-OR = 1.20, 95% CI: 1.07–1.34) and PE (adjusted-OR = 1.14, 95% CI: 1.02–1.26). In addition, increase in cfDNA levels were associated with risk of GDM, and was dependent on maternal age (maternal age ≥ 35 years: adjusted-OR = 1.16, 95% CI: 1.04–1.29; maternal age < 35 years: adjusted-OR = 0.85, 95% CI: 0.73–0.99). ConclusionMaternal plasma cfDNA levels measured during NIPT are associated with pregnancy complications (ICP, PE and GDM). Maternal age may be an important effect modifier for the association between plasma cfDNA levels and GDM.

Particularities of pregnancy follow-up in obese patients

Since obesity has reached epidemic proportions even at very young ages, subsequently we notice an increased number of pregnancies in these categories of patients with higher rates of obstetrics complications such as: gestational diabetes, hypertensive disorders, and augmented rates of preterm birth and caesarean section. Maternal obesity has been proven to be associated with an increased risk of congenital malformations, including a significantly increased risk for neural tube defects, congenital heart defects, and orofacial clefts in the offspring, but despite their increased number, malformation detection rate is lower in obese patients. The screening procedures for detecting Down syndrome offered to pregnant women have changed over the last decades. Unfortunately, classical serum screening for Down syndrome testing performs worse in obese patients, the fetal fraction in maternal plasma cf-DNA being affected by maternal excessive weight. In this article, we aim to review the particularities of the available prenatal test and procedures in obese and overweight pregnant women.

909: Methylation profile of circulating cfDNA at term demonstrates relatively low placental contribution to hypomethylated cfDNA

The fetal fraction of circulating cell-free DNA (cfDNA) is placental in origin, and is a critical variable in noninvasive prenatal testing (NIPT). Although fetal cfDNA is known to be hypomethylated relative to maternal cfDNA, the methylation profile of circulating cfDNA in maternal plasma has not been well studied. We sought to determine whether hypomethylated cfDNA in maternal plasma: (i) could serve as a proxy for fetal fraction and (ii) was reduced in maternal obesity, as the fetal fraction of circulating cfDNA is known to be decreased in the setting of maternal obesity. In this prospective pilot study, maternal plasma was collected at admission and 24-48h postpartum (PP) from women admitted for delivery at term. Subjects were enrolled into 4 groups: lean (pre-pregnancy BMI <25 kg/m2) no labor (Lean-NL) (n=15); lean labor (Lean-L) (n=18); obese (pre-pregnancy BMI ≥30 kg/m2) no labor (OB- NL) (n=16); and obese labor (OB-L) (n=11). Total cfDNA was isolated and genome-wide methylation levels determined using 0.2x coverage whole-genome bisulfite sequencing (WGBS) in all 60 antepartum (AP) samples and in matching PP samples in ∼5 patients per study group. Calibration was performed using serial dilutions of fully methylated and unmethylated DNA. Hypomethylated cfDNA fraction was significantly lower in OB vs lean pregnant women (27.0 ±0.5% vs 28.5±0.3%, p=0.008). Hypomethylated fraction was significantly higher in AP vs PP samples overall, and when stratified by BMI (Fig 1A/B), demonstrating that the placenta contributes to hypomethylated cfDNA in maternal circulation. However, matched analyses of hypomethylation before and after delivery demonstrated a relatively small contribution of placental cfDNA to the overall hypomethylated fraction, ∼15%. The lower hypomethylated cfDNA fraction in OB patients is consistent with our prior observation of reduced placental cfDNA release in a mouse model of diet-induced obesity. Contrary to expectations, the placenta accounted for a relatively low proportion of all hypomethylated cfDNA in maternal plasma at term, suggesting that hypomethylation is not a good proxy for the fetal fraction. The finding of 20-25% hypomethylated cfDNA in non-pregnant healthy women is consistent with the oncology literature. Understanding the impact of pregnancy and maternal obesity on the methylation profile of cfDNA may have broader implications for NIPT, as well as for oncology researchers.

Factors affecting cell-free DNA fetal fraction and the consequences for test accuracy

Introduction: Biological factors are known to influence the fetal fraction (FF) of cell-free DNA and may also influence the accuracy of non-invasive prenatal testing.Material and methods: NIPT from 5267 mixed risk women across three specialist clinics in Australia were analyzed. Multivariable regression analysis was used to determine whether maternal characteristics, ultrasound, and placental biomarkers affect FF and test accuracy.Results: FF ranged from 4% to 37% (mean 11.6%). Body mass index (BMI), gestation, and placental biomarkers were found to be significant factors associated with FF. For each unit increase in BMI, the logarithmically transformed FF, (lnFF), mean value decreased by 0.027. Each week increases in gestation, lnFF increased by 0.023. Each unit increase in free BhCG, PAPPA, and PlGF, the lnFF increased by 0.065, 0.050, and 0.17, respectively. There was no significant association between FF with either maternal age or nuchal translucency. The false-positive cases and one false-negative case did not have lower FF than the true-positive cases.Discussion: The fetal fraction in maternal plasma cfDNA increased with gestational age, serum pregnancy-associated plasma protein A (PAPP-A), β-hCG, and PlGF and decreased with increasing maternal BMI. There was no significant correlation between low FF and test accuracy, when FF was above 4%.

Maternal Plasma DNA and RNA Sequencing for Prenatal Testing.

Cell-free DNA (cfDNA) testing has recently become indispensable in diagnostic testing and screening. In the prenatal setting, this type of testing is often called noninvasive prenatal testing (NIPT). With a number of techniques, using either next-generation sequencing or single nucleotide polymorphism-based approaches, fetal cfDNA in maternal plasma can be analyzed to screen for rhesus D genotype, common chromosomal aneuploidies, and increasingly for testing other conditions, including monogenic disorders. With regard to screening for common aneuploidies, challenges arise when implementing NIPT in current prenatal settings. Depending on the method used (targeted or nontargeted), chromosomal anomalies other than trisomy 21, 18, or 13 can be detected, either of fetal or maternal origin, also referred to as unsolicited or incidental findings. For various biological reasons, there is a small chance of having either a false-positive or false-negative NIPT result, or no result, also referred to as a "no-call." Both pre- and posttest counseling for NIPT should include discussing potential discrepancies. Since NIPT remains a screening test, a positive NIPT result should be confirmed by invasive diagnostic testing (either by chorionic villus biopsy or by amniocentesis). As the scope of NIPT is widening, professional guidelines need to discuss the ethics of what to offer and how to offer. In this review, we discuss the current biochemical, clinical, and ethical challenges of cfDNA testing in the prenatal setting and its future perspectives including novel applications that target RNA instead of DNA.

Maternal cell-free DNA-based screening for fetal microdeletion and the importance of careful diagnostic follow-up.

BackgroundNoninvasive prenatal screening (NIPS) by next-generation sequencing of cell free DNA (cfDNA) in maternal plasma is used to screen for common aneuploidies such as trisomy 21, in high risk pregnancies. NIPS can identify fetal genomic microdeletions, however sensitivity and specificity have not been systematically evaluated. Commercial companies have begun to offer expanded panels including screening for common microdeletion syndromes such as 22q11.2 deletion (DiGeorge syndrome) without reporting the genomic coordinates or whether the deletion is maternal or fetal. Here we describe a phenotypically normal mother and fetus that tested positive for atypical 22q deletion via maternal plasma cell free DNA testing.MethodsWe performed cfDNA sequencing on saved maternal plasma obtained at 11 weeks of gestation from a phenotypically normal woman with a singleton pregnancy whose earlier screening at a commercial laboratory was reported to be positive for a 22q11.2 microdeletion. FISH and chromosomal microarray diagnostic genetic tests were done postnatally.ConclusionNIPS detected a 22q microdeletion that upon diagnostic work up, did not include the DiGeorge critical region. Diagnostic prenatal or postnatal testing with chromosomal microarray and appropriate parental studies to determine precise genomic coordinates and inheritance should follow a positive microdeletion NIPS result.

Quantification of cell-free DNA in normal and complicated pregnancies: overcoming biological and technical issues.

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal ‘fetal’ markers in maternal plasma, thus overcoming the limitations of gender- or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2–39.5 weeks). Two fetal- or placental- specific duplex assays (RPP30/SRY and RASSF1A/β-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p<0.0001; placental cfDNA (RASSF1A): R = 0.75, p<0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Maternal Plasma Cell-Free Fetal and Maternal DNA at 11-13 Weeks' Gestation: Relation to Fetal and Maternal Characteristics and Pregnancy Outcomes

Objective: To examine the possible relationship between maternal and fetal characteristics and pregnancy outcomes on fetal and maternal cell-free (cf) DNA in maternal plasma at 11-13 weeks' gestation. Methods: cfDNA was extracted from maternal plasma of 1,949 singleton pregnancies and chromosome-selective sequencing was used to determine the proportion of cfDNA and total cfDNA counts which was of fetal and maternal origin. Multivariate regression analysis was used to determine whether specific maternal and fetal characteristics and pregnancy complications, such as preeclampsia (PE), early spontaneous preterm birth (SPB) and delivery of small for gestational age (SGA) neonates, were significant predictors of fetal and maternal cfDNA in maternal plasma. Results: The fetal and maternal cfDNA plasma concentration increased with serum pregnancy-associated plasma protein-A and free β-human chorionic gonadotropin level, was higher in women of Afro-Caribbean and East-Asian racial origin than in Caucasians, and lower in smokers, but it was not significantly altered in pregnancies complicated by PE, SPB or SGA. The fetal cfDNA level was inversely related to maternal weight and uterine artery pulsatility index, and maternal cfDNA increased with maternal weight. Conclusions: The fetal and maternal cfDNA level in maternal plasma is affected by maternal and fetal characteristics, but it is not altered in pregnancy complications.

Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10(9) tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.

Fetal fraction in maternal plasma cell‐free DNA at 11–13 weeks' gestation: relation to maternal and fetal characteristics

To examine the possible effects of maternal and fetal characteristics on the fetal fraction in maternal plasma cell-free (cf) DNA at 11-13 weeks' gestation and estimate the proportion of pregnancies at high risk of non-invasive prenatal testing (NIPT) failure because the fetal fraction is less than 4%. In 1949 singleton pregnancies at 11-13 weeks' gestation cf-DNA was extracted from maternal plasma. Chromosome-selective sequencing of non-polymorphic and polymorphic loci, where fetal alleles differ from maternal alleles, was used to determine the proportion of cf-DNA that was of fetal origin. Multivariable regression analysis was used to determine significant predictors of the fetal fraction among maternal and fetal characteristics. The fetal fraction decreased with increased maternal weight, it was lower in women of Afro-Caribbean origin than in Caucasians and increased with fetal crown-rump length, serum pregnancy-associated plasma protein-A, serum free β-human chorionic gonadotropin, smoking and trisomy 21 karyotype. The median fetal fraction was 10.0% (interquartile range, 7.8-13.0%) and this decreased with maternal weight from 11.7% at 60 kg to 3.9% at 160 kg. The estimated proportion with fetal fraction below 4% increased with maternal weight from 0.7% at 60 kg to 7.1% at 100 kg and 51.1% at 160 kg. Fetal fraction in maternal plasma cf-DNA is affected by maternal and fetal characteristics.

Fetal Fraction in Maternal Plasma Cell-Free DNA at 11–13 Weeks’ Gestation: Effect of Maternal and Fetal Factors

Objective: It was the aim of this study to examine the possible effects of maternal and fetal characteristics on the fetal fraction in maternal plasma cell-free DNA (cfDNA) at 11–13 weeks’ gestation. Methods: In a nested case-control study, cfDNA was extracted from maternal plasma obtained before chorionic villous sampling from 300 euploid, 50 trisomy 21 and 50 trisomy 18 pregnancies at 11–13 weeks’ gestation. Chromosome-selective sequencing of maternal cfDNA non-polymorphic and polymorphic loci, where fetal alleles differ from maternal alleles, was used to determine the proportion of DNA which is of fetal origin. Multivariate regression analysis was used to determine which of the factors amongst maternal weight, racial origin, smoking status, plasma storage time, serum pregnancy-associated plasma protein (PAPP)-A and free β-subunit of human chorionic gonadotropin (β-hCG), fetal crown-rump length, nuchal translucency thickness, gender and karyotype were significant predictors of the fetal fraction. Results: Significant independent prediction of fetal fraction was provided by maternal weight, serum PAPP-A and serum free β-hCG multiples of the median, but not by other maternal characteristics, fetal karyotype, crown-rump length or nuchal translucency thickness. Fetal fraction increased with serum metabolite levels and decreased with maternal weight. Conclusions: The fetal fraction in maternal plasma cfDNA increases with serum PAPP-A and free β-hCG and decreases with maternal weight.