Fetal fraction enrichment using in vitro high-resolution size-selection with NIPS: Advantages and considerations

The purpose of this noninvasive prenatal testing (NIPT) study was to compare the fetal fraction of singleton gestations by gestational age, maternal characteristics and chromosome-specific aneuploidies as indicated by z-scores. This study was a multicenter prospective cohort study. Test data were collected from women who underwent NIPT by the massively parallel sequencing method. We used sequencing-based fetal fraction calculations in which we estimated fetal DNA fraction by simply counting the number of reads aligned within specific autosomal regions and applying a weighting scheme derived from a multivariate model. Relationships between fetal fractions and gestational age, maternal weight and height, and z-scores for chromosomes 21, 18 and 13 were assessed. A total of 7740 pregnant women enrolled in the study, of which 6993 met the study criteria. As expected, fetal fraction was inversely correlated with maternal weight (P<0.001). The median fetal fraction of samples with euploid result (n=6850) and trisomy 21 (n=70) were 13.7% and 13.6%, respectively. In contrast, the median fetal fraction values for samples with trisomies 18 (n=35) and 13 (n=9) were 11.0% and 8.0%, respectively. The fetal fraction of samples with trisomy 21 NIPT result is comparable to that of samples with euploid result. However, the fetal fractions of samples with trisomies 13 and 18 are significantly lower compared with that of euploid result. We conclude that it may make detecting these two trisomies more challenging.

BackgroundThe diagnosis of aneuploidies via the detection of circulating fetal DNA from maternal circulation is impaired by low fetal fractions i.e. concentration of fetal DNA relatively to maternal DNA. Low...

ObjectiveDuring routine noninvasive prenatal testing (NIPT), cell‐free fetal DNA fraction is ideally derived from shallow‐depth whole‐genome sequencing data, preventing the need for additional experimental assays. The fraction of aligned reads to chromosome Y enables proper quantification for male fetuses, unlike for females, where advanced predictive procedures are required. This study introduces PREdict FetAl ComponEnt (PREFACE), a novel bioinformatics pipeline to establish fetal fraction in a gender‐independent manner.MethodsPREFACE combines the strengths of principal component analysis and neural networks to model copy number profiles.ResultsFor sets of roughly 1100 male NIPT samples, a cross‐validated Pearson correlation of 0.9 between predictions and fetal fractions according to Y chromosomal read counts was noted. PREFACE enables training with both male and unlabeled female fetuses. Using our complete cohort (nfemale = 2468, nmale = 2723), the correlation metric reached 0.94.ConclusionsAllowing individual institutions to generate optimized models sidelines between‐laboratory bias, as PREFACE enables user‐friendly training with a limited amount of retrospective data. In addition, our software provides the fetal fraction based on the copy number state of chromosome X. We show that these measures can predict mixed multiple pregnancies, sex chromosomal aneuploidies, and the source of observed aberrations.

The discovery of circulating fetal DNA in the plasma of pregnant women has greatly promoted advances in noninvasive prenatal testing. Screening performance is enhanced with higher fetal fraction and analysis of samples whose fetal DNA fraction is lower than 4% are unreliable. Although current approaches to fetal fraction measurement are accurate, most of them are expensive and time consuming. Here we present a simple and cost-effective solution that provides a quick and reasonably accurate fetal fraction by directly evaluating the size distribution of circulating DNA fragments in the extracted maternal cell-free DNA. The presented approach could be useful in the presequencing stage of noninvasive prenatal testing to evaluate whether the sample is suitable for the test or a repeat blood draw is recommended.

The relationship between fetal fraction and birth weight in twin gestations is poorly understood. This study aimed to investigate the relationship between first-trimester cell-free DNA (cfDNA) fetal fraction and birth weight <10th percentile in twin gestations.This is a planned secondary analysis of the Twin cfDNA Study, a 17-center retrospective cohort of twin pregnancies screened for aneuploidy using cfDNA in the first trimester from December 2011 to February 2022, excluding those with positive screen results for chromosomal aneuploidy. cfDNA testing was performed by a single laboratory using massively parallel sequencing. Baseline characteristics and birth weight of pregnancies with normal fetal fraction were compared with those with low (<5%) and high (>95%) fetal fraction using univariable analyses and multivariable regression.A total of 1,041 twin pregnancies were included. Chronic hypertension, elevated body mass index, and self-identified Black race were associated with fetal fraction <5th percentile. There was no difference in median fetal fraction between those with birth weight <10th percentile in at least one twin (median [interquartile range (IQR)] fetal fraction: 12.2% [9.8, 14.8] vs. those with normal birth weight (≥10th percentile) in both twins (median [IQR] fetal fraction: 12.3% [9.7, 15.2] for normal birth weight, p = 0.49). There was no association between high or low fetal fraction and birth weight <10th percentile for one (p = 0.45) or both (p = 0.81) twins, and there was no association between high or low fetal fraction and birth weight <5th percentile for one (p = 0.44) or both (p = 0.74) twins. The results were unchanged after adjustment for potential confounders.In this large cohort, there was no association between the extremes of cfDNA fetal fraction and birth weight <10th percentile, suggesting that first-trimester fetal fraction may not predict impaired fetal growth in twin gestations. · No association between fetal fraction and small for gestational age birth weight in twins.. · Results suggest that fetal fraction does not predict birth weight in twin gestations.. · These results differ from the relationship between fetal fraction and birth weight in singletons..

Low first-trimester fetal fraction is associated with chronic inflammation in the placenta

The goal of this study is to evaluate the amount of free fetal DNA (ffDNA), total DNA, and 'fetal fraction' found in maternal plasma and whether these influence the enrichment ratios of differentially methylated regions (DMRs) and the correct classification of trisomy 21 using the methylated DNA immunoprecipitation-quantitative polymerase chain reaction (MeDIP-qPCR)-based noninvasive prenatal diagnostic methodology applied in peripheral blood. Absolute quantification of ffDNA using DYS14 and total DNA using β-globin was applied in 83 maternal plasma samples. The quantification values for all 83 samples were correlated with the enrichment ratios of all seven DMRs and D-values that were obtained from the diagnostic formula of MeDIP-qPCR method. Our analysis concluded that trisomy 21 samples had significantly higher ffDNA and total DNA levels compared with those of normal samples. Enrichment ratios of the majority of DMRs studied exhibited no association with ffDNA, total DNA, and 'fetal fraction', and only a small portion of DMRs exhibited moderate association. Correlation studies of ffDNA, total DNA, and fetal fraction with the diagnostic D-value showed weak to no association but without affecting the classification of trisomy 21. Overall, the variability of ffDNA and total DNA among maternal samples does not affect the correct trisomy 21 classification using MeDIP-qPCR methodology applied in peripheral blood.

BackgroundNon-invasive prenatal testing by cell-free DNA analysis is offered to pregnant women worldwide to screen for fetal aneuploidies. In non-invasive prenatal testing, the fetal fraction of cell-free DNA in the maternal circulation is measured as a quality control parameter. Since fetal cell-free DNA originates from the placenta, the fetal fraction might also reflect placental health and maternal pregnancy adaptation. ObjectiveTo assess the association between the fetal fraction and adverse pregnancy outcomes. Study DesignWe performed a retrospective cohort study of women with singleton pregnancies opting for non-invasive prenatal testing between June 2018 and June 2019 within the Dutch nationwide implementation study (TRIDENT-2). Multivariable logistic regression analysis was used to assess associations between fetal fraction and adverse pregnancy outcomes. Fetal fraction was assessed as a continuous variable and as <10th percentile, corresponding to a fetal fraction below 2.5%. ResultsThe cohort comprised 56,110 pregnancies. In the analysis of fetal fraction as a continuous variable, a decrease in fetal fraction was associated with an increased risk of hypertensive disorders of pregnancy (adjusted OR [aOR] 2.27 [95% CI 1.89 to 2.78]), small for gestational age neonates <p10 (aOR 1.37 [1.28 to 1.45]) and <p2.3 (aOR 2.63 [1.96 to 3.57]), and spontaneous preterm birth from 24 to 37 weeks of gestation (aOR 1.02 [1.01 to 1.03]). No association was found for fetal congenital anomalies (aOR 1.02 [1.00 to 1.04]), stillbirth (aOR 1.02 [0.96 to 1.08]), or neonatal death (aOR 1.02 [0.96 to 1.08]). Similar associations were found for adverse pregnancy outcomes when fetal fraction was <10th percentile. ConclusionsIn early pregnancy, a low fetal fraction is associated with an increased risk of adverse pregnancy outcomes. These findings can be utilized to expand the potential of non-invasive prenatal testing in the future, enabling the prediction of pregnancy complications and facilitating tailored pregnancy management through intensified monitoring or preventive measures.

Low fetal fraction (FF) on cell-free DNA (cfDNA)-based noninvasive prenatal screening (NIPS) is a common etiology for indeterminate results. As maternal Black race is implicated as a risk factor for low FF and more indeterminate results, we sought to evaluate this association. This was a single-institution, retrospective cohort study of cfDNA-based NIPS performed between May 2017 and May 2022 with complete clinical data abstraction. We compared FF, indeterminate rates, and total cfDNA concentration among self-reported Black, White, and Other groups from NIPS results from 2017 to 2022 with full clinical data abstraction. Using linear regression and interaction testing, we evaluated associations between self-reported race, FF, indeterminate rate, and total cfDNA concentration. In total, 1,591 participants met the inclusion criteria; 70.8% (n = 1,126) self-identified as White, 6.9% (n = 110) as Black, and 22.3% (n = 355) self-identified with another race. Mean FF was not different between the White, Black, or Other groups (11.8 vs. 11.2 vs. 11.7%, respectively, p = 0.52). This remained true after adjusting for body mass index (BMI), gestational age (GA) at draw, and fetal sex (all p > 0.17). Interaction testing for FF and total cfDNA by race with BMI, GA at draw, and fetal sex demonstrated no effect modification. In our population, maternal self-identified race, particularly Black race, does not affect FF. Biological plausibility for race-based differences on clinical tests requires ongoing thoughtful consideration. · NIPS is widely used to screen for fetal aneuploidy.. · FF is an important test metric, and low FF is associated with adverse outcomes, like aneuploidy.. · In existing studies, Black race is implicated as a risk factor for lower FF.. · Our study found no differences in FF between groups by self-reported race.. · Biological plausibility for race-based differences on clinical tests requires ongoing consideration..

BackgroundNon-invasive prenatal testing (NIPT)-derived total cell-free DNA (cfDNA) and fetal fractions (FFs) are potential indicators of placental dysfunction, a hallmark of preeclampsia (PE). However, their clinical utility in PE risk stratification, particularly in twin pregnancies, remains underexplored. This study evaluated the association between total cfDNA levels and the incidence of PE and assessed mediation by FFs.MethodsThis retrospective cohort study included 2,366 pregnant women who underwent NIPT at 12–22 weeks of gestation at a tertiary hospital. Total cfDNA levels and FFs were extracted from the NIPT data, and PE diagnoses were confirmed via medical records. Logistic regression and mediation analyses were used to assess associations and mediating effects.ResultsPE prevalence was 3.51% (83/2,366). Elevated total cfDNA tertiles (T2/T3 vs. T1) were associated with increased rates of PE (T2: OR = 2.36, 95% CI: 1.16–4.80; T3: OR = 3.00, 95% CI: 1.51–5.97). Each 1 µg/L increase in total cfDNA was associated with a 12% increase in PE odds (OR = 1.12, 95% CI: 1.05–1.20). Lower FFs were associated with PE risk (T1 vs. T3: OR = 2.70, 95% CI: 1.45–5.00; per 1% decrease: OR = 1.15, 95% CI: 1.07–1.23). In singleton pregnancies, total cfDNA levels ≤ 4.41 µg/L were significantly associated with increased PE risk (OR = 2.89, 95% CI: 1.32–6.29, P = 0.008) with a validated threshold effect [log-likelihood ratio test (LRT, P = 0.005)], while FF ≤ 8.86% showed a protective trend (OR = 0.81, 95% CI: 0.68–0.97, P = 0.021) despite a nonsignificant LRT (P = 0.192). In twin pregnancies, total cfDNA showed no significant threshold or linear association with PE risk (OR = 1.06, P = 0.382; LRT, P = 0.215), while FFs exhibited a stronger inverse linear association (OR = 0.77, 95% CI: 0.65–0.92, P = 0.003) with no significant threshold effect (LRT, P = 0.270). Total cfDNA was inversely correlated with FFs (T3 vs. T1: β=-2.96, 95% CI: -3.34 to -2.59). Mediation analyses showed FFs partially explained 24.4% and 32.5% of the total cfDNA-PE relationship in singleton pregnancies and all pregnancies, respectively.ConclusionThis study observed that elevated total cfDNA levels during NIPT were associated with an increased risk of PE, with FFs acting as partial mediators. These findings warrant further investigation into total cfDNA and FFs as potential biomarkers for early PE risk stratification, even in high-risk settings such as twin pregnancies, to determine whether their use in screening could contribute to the development of strategies aimed at improving maternal-fetal health.

Objective: To assess the association between lipid metabolism and fetal fraction, which is a critical factor in ensuring a highly accurate non-invasive prenatal testing (NIPT), and on the rate of screen failures or “no calls” in NIPT.Methods: A total of 4,514 pregnant women at 12–26 weeks of gestation underwent NIPT sequencing and serum lipid measurements. Univariate analysis and multivariate regression models were used to evaluate the associations of serum lipid concentrations with the fetal fraction and the rate of screen failures.Results: The fetal fraction decreased with increased low-density lipoprotein cholesterol and triglyceride (TG) levels, which were significant factors (standardized coefficient: −0.11). Conversely, high-density lipoprotein cholesterol and the interval between the two tests were positively correlated with the fetal fraction. The median fetal fraction was 10.88% (interquartile range, 8.28–13.89%) and this decreased with TG from 11.56% at ≤1.10 mmol/L to 9.51% at >2.30 mmol/L. Meanwhile, multivariate logistic regression analysis revealed that increased TG levels were independently associated with the risk of screen failures. The rate of screen failures showed an increase with TG levels from 1.20% at ≤1.70 mmol/L to 2.41% at >2.30 mmol/L.Conclusions: The fetal fraction and the rate of screen failures in NIPT are affected by TG levels. Meanwhile, in pregnant women with high TG levels, delaying the time between NIPT blood collections can significantly increase the fetal fraction.

Non-invasive prenatal testing (NIPT) based on cell-free fetal DNA (cffDNA) is widely used for screening common fetal aneuploidies. Although fetal fraction (FF) increases with gestational age, the feasibility and performance of NIPT in the early first-trimester, particularly before 10weeks of gestation, remain incompletely defined. This retrospective cohort study included 9,708 singleton pregnancies undergoing first-trimester NIPT between 9weeks 0 days and 13weeks 6 days of gestation, which was determined by ultrasound using crown-rump length (CRL) measurement. Participants were stratified into two groups based on gestational age at testing (< 10weeks and ≥ 10weeks). Multivariable linear regression was used to assess factors associated with fetal fraction, and multivariable logistic regression was applied to evaluate predictors of no-call results (non-reportable NIPT outcome primarily attributed to fetal fraction <4%), adjusting for maternal age and body mass index (BMI). Fetal fraction increased significantly with advancing gestational age. After adjustment for maternal age and BMI, testing performed at ≥ 10weeks was associated with a higher fetal fraction compared with testing at < 10weeks (β = 1.58; 95% CI, 1.31 to 1.85; p < 0.001). However, gestational age was not independently associated with the risk of a no-call result (adjusted OR 0.88; 95% CI, 0.68 to 1.16; p = 0.40), and no significant difference in no-call rates was observed between the two gestational age groups (3.78% vs. 3.76%). Although fetal fraction increases after 10weeks of gestation, gestational age is not an independent determinant of no-call results once fetal fraction adequacy is achieved. NIPT performed in the early first-trimester (< 10weeks of gestation) demonstrates acceptable analytical feasibility and reliable performance, supporting its use for early prenatal screening under appropriate clinical conditions.

Improving Fetal Fraction of Noninvasive Prenatal Screening Samples Collected in EDTA-Gel Tubes Using Gel Size Selection: A Head-To-Head Comparison of Methods

Several non-invasive prenatal testing (NIPT) methods, which analyze circulating fetal cell-free DNA (cfDNA) in maternal plasma, suggest a fetal fraction (FF) ≥ 4% for a reportable result, with the assumption that fetal aneuploidies may not be detectable at lower FF. This study determined the actual limit of detection (LOD) of a massively parallel sequencing-based NIPT method and evaluated its performance in testing samples with low FF. An experimental model, involving the creation of artificial plasma mixtures with a final aneuploid FF ranging from 1% to 4%, simulated samples at different proportions of fetal cfDNA. We then analyzed 7103 blood samples, from pregnant women undergoing NIPT, to assess the impact of low FF on the performance of cfDNA testing. Detection of common aneuploidies in samples with an FF as low as 2% is well within the ability of this technology. Of 105 pregnancies confirmed chromosomally abnormal, 25 (23.8%) involving a 2% < FF < 4% were consistently detected. These high-risk pregnancies would have not been identified using the suggested 4% FF cut-off. This study underscores the importance of determining the actual LOD for each specific NIPT methodology. It may reduce the incidence of test cancelations and shorten the time required for the diagnosis of aneuploidy. © 2016 John Wiley & Sons, Ltd.

To determine the association between cell-free DNA fetal fraction (cffDNA) and various prenatal characters to better guide the clinical application of noninvasive prenatal screening (NIPS), a retrospective cohort study of 27,793 women with singleton pregnancies was conducted. Results indicated that no significant difference on cffDNA between trisomy/sex chromosome aneuploidy (SCA) and non-trisomy groups was found. However, the fetal fraction (FF) in the T18 and T13 subgroups were significantly lower than that in the non-trisomy group, while the FF in the T21 group was significantly higher than the non-trisomy group. Pearson’s correlation analysis revealed a positive correlation between √FF and gestational week in the T21, SCA, and non-trisomy groups. A negative correlation between maternal age and √FF in T21 and non-trisomy cases was found, but a positive correlation in SCA group. Compared to the decreasing trend in FF in the T21 group, no significant difference was observed in the SCA group. The √FF level was negatively correlated to maternal BMI in T21 and non-trisomy group, while a positive correlation in SCA group. FF was close related to the result of NIPS and related maternal factors. Though NIPS has increased accuracy, the complexity still should be recognized especially in clinical practice.