Fetal Fraction Research Articles

Non-invasive prenatal detection of dominant single-gene disorders in fetal structural abnormalities: a clinical feasibility study.

This study evaluated the accuracy of non-invasive prenatal testing (NIPT-SGDs) for dominant monogenic genetic diseases associated with fetal structural abnormalities and to assess the feasibility of clinical application. Pregnant women requiring prenatal diagnosis due to fetal structural abnormalities were enrolled. Maternal peripheral blood was analyzed for cell-free DNA (cfDNA) using coordinative allele-aware target enrichment sequencing (COATE-seq). This assessed fetal allele depth distribution, fraction and variation ratio. The variation's origin was then determined to obtain fetal variation information. Finally, NIPT-SGDs results were confirmed via invasive prenatal diagnosis (IPD). Upon examination of 113 samples using NIPT-SGDs, COATE-seq successfully analyzed 112 for fetal variation, excluding one due to hemolysis. The study detected six positive cases, yielding a 5.36% detection rate. These disorders included tuberous sclerosis complex (TSC1 and TSC2 being its causative genes), Noonan syndrome (PTPN11), polycystic kidney disease (PKD1), and Kabuki syndrome (KMT2D), occurring twice each, except for Noonan and polycystic kidney disease. Two false positives were due to the mother being a genetic mosaicism. Compared to invasive whole-exome sequencing (WES), NIPT-SGDs did not detect nine positive cases of IPD dominant monogenic diseases, accurately identifying 90.18% (101/112) of the actual positive and negative cases. Our findings demonstrate the clinical utility of NIPT-SGDs using COATE-seq in effectively identifying fetuses with dominant single-gene disorders. Furthermore, this method can be applied to all fetuses.

Evaluation of the clinical utility of NIPT-plus and analysis of adverse pregnancy outcomes.

To evaluate the performance of NIPT-plus in detecting fetal aneuploidies and CNVs and analyze the factors influencing adverse pregnancy outcomes. The retrospectively analyzed 8726 pregnant women who underwent NIPT-plus for fetal screening were classified into low- (who tested voluntarily) and high-risk (women with advanced age, abnormal ultrasound, abnormal serological screening, or a combination of indications) groups. Basic maternal information, prenatal findings, and pregnancy outcomes were recorded. NIPT-plus performance was assessed for various chromosomal abnormalities and the association between the fetal fraction and adverse pregnancy outcomes. Thirty-six (0.4%) patients had failed tests; 144 (1.65%) positive cases were detected, of which, 107 (74.31%) opted for invasive testing, and 51 were verified as true positives. The total positive predictive value was 45.45% and 48.65% in the low- and high-risk groups, respectively, and the difference was not significant. Among the subsequent cases with abnormal ultrasound monitoring, two false-negative cases were identified, and pathogenic CNV diagnosis was confirmed through amniocentesis, resulting in pregnancy termination. Fetal fraction was not associated with an increased adverse pregnancy outcome risk; however, ethnic differences may affect pregnancy outcomes. NIPT-plus technology use is no longer restricted to high-risk pregnant women, and it may produce false-positive results. The stakeholders should be aware of this limitation. The uncertainties and potential risks of the test results should be explained to the test takers to enable informed decision making and to minimize unnecessary anxiety and concerns. Ethnicity may influence adverse pregnancy outcomes in local multiracial settings.

Comprehensive evaluation of the impact of whole-genome bisulfite sequencing (WGBS) on the fragmentomic characteristics of plasma cell-free DNA

BackgroundCell-free DNA (cfDNA) is non-randomly fragmented in human body fluids. Analyzing such fragmentation patterns of cfDNA holds great promise for liquid biopsy. Whole-genome bisulfite sequencing (WGBS) is widely used for cfDNA methylation profiling. However, its applicability for studying fragmentomic characteristics remains largely unexplored. MethodsWe performed paired WGBS and whole-genome sequencing (WGS) on 66 peripheral plasma samples from 58 pregnant women. Then, we systematically compared the fragmentation patterns of cell-free nuclear DNA and mitochondrial DNA (mtDNA) sequenced from these two approaches. Additionally, we evaluated the extent of the size shortening in fetal-derived cfDNA and estimated the fetal DNA fraction in maternal plasma using both sequencing methods. ResultsCompared to WGS samples, WGBS samples demonstrated a significantly lower genome coverage and higher GC content in cfDNA. They also showed a significant decrease in the size of cell-free nuclear DNA, along with alterations in the end motif pattern that were specifically associated with CpG and “CC” sites. While there was a slight shift in the inferred nucleosome footprint from cfDNA coverages in WGBS samples, the cfDNA coverage patterns in CTCF and TSS regions remained highly consistent between these two sequencing methods. Both methods accurately reflected gene expression levels through their TSS coverages. Additionally, WGBS samples exhibited an increased abundance and longer length of mtDNA in plasma. Furthermore, we observed the size shortening of fetal cfDNA in plasma consistently, with a highly correlated fetal DNA fraction inferred by cfDNA coverage between WGBS and WGS samples (r = 0.996). However, the estimated fetal cfDNA fraction in WGBS samples was approximately 7 % lower than in WGS samples. ConclusionsWe confirmed that WGBS can introduce artificial breakages to cfDNA, leading to altered fragmentomic patterns in both nuclear and mitochondrial DNA. However, WGBS cfDNA remains suitable for analyzing certain cfDNA fragmentomic characteristics, such as coverage in genome regulation regions and the essential characteristics of fetal DNA in maternal plasma.

Hierarchical Classification of Factors Associated With Noninvasive Prenatal Testing Failures and Its Impact on Pregnancy Outcomes

Abstract Objective To conduct a hierarchical classification analysis of the nonreportable results of noninvasive prenatal testing in an attempt to reduce failure rates and provide pregnant women with accurate information to alleviate their anxiety. Methods In this study, 30,039 singleton pregnancies who underwent noninvasive prenatal testing in a single center from May 2019 to April 2022 were collected, and 811 samples with initial noninvasive prenatal testing failure were retrospectively analyzed. Grouping was based on the reasons for initial test failure; tracking the noninvasive prenatal testing results and prenatal diagnosis results (if any) of the “z-scores in the gray area” group and analyzing the possible influencing factors of the “low fetal fraction” group in the pre-experimental and experimental period by using one-way analysis of variance, Mann-Whitney U test, and χ2 test; and tracking the pregnancy outcomes of the test failures samples to analyze the risk of perinatal complications and adverse pregnancy outcomes of the different types of test failures. Results None of the samples' initial nonresults because of z-scores in the gray area were ultimately found to have chromosomal aneuploidy. However, pregnancy complications (P = 0.018) and a high likelihood of adverse pregnancy outcomes (P = 0.048) may still occur. Maternal gestational age (P < 0.001), body mass index (P < 0.001), library concentration (P < 0.001), and fetal gender (P < 0.001) were considered to be the associated factors for the initial low fetal fraction results. This may be associated with pregnancy complications (P < 0.001) and a high likelihood of adverse pregnancy outcomes (P = 0.034). The body mass index (P = 0.015) and time between draws (P = 0.001) were associated with the second test’s success. The incidence of low fetal fraction samples was more frequent with blood collection tubes of the G type than with the K type (P < 0.001). Conclusion Initial nonresults because of z-scores in the gray area did not imply an increased risk of aneuploidy, but vigilance is needed for an increased risk of pregnancy complications and adverse pregnancy outcomes. Because of the low fetal fraction, the initial absence of results may be related to the assay method, as well as the effect of blood collection tubes and the need to be alert to the risk of pregnancy complications and adverse pregnancy outcomes.

Histone modifications of circulating nucleosomes are associated with changes in cell-free DNA fragmentation patterns

The analysis of tissues of origin of cell-free DNA (cfDNA) is of research and diagnostic interest. Many studies focused on bisulfite treatment or immunoprecipitation protocols to assess the tissues of origin of cfDNA. DNA loss often occurs during such processes. Fragmentomics of cfDNA molecules has uncovered a wealth of information related to tissues of origin of cfDNA. There is still much room for the development of tools for assessing contributions from various tissues into plasma using fragmentomic features. Hence, we developed an approach to analyze the relative contributions of DNA from different tissues into plasma, by identifying characteristic fragmentation patterns associated with selected histone modifications. We named this technique as FRAGmentomics-based Histone modification Analysis (FRAGHA). Deduced placenta-specific histone H3 lysine 27 acetylation (H3K27ac)-associated signal correlated well with the fetal DNA fraction in maternal plasma (Pearson's r = 0.96). The deduced liver-specific H3K27ac-associated signal correlated with the donor-derived DNA fraction in liver transplantation recipients (Pearson's r = 0.92) and was significantly increased in patients with hepatocellular carcinoma (HCC) (P < 0.01, Wilcoxon rank-sum test). Significant elevations of erythroblasts-specific and colon-specific H3K27ac-associated signals were observed in patients with β-thalassemia major and colorectal cancer, respectively. Furthermore, using the fragmentation patterns from tissue-specific H3K27ac regions, a machine learning algorithm was developed to enhance HCC detection, with an area under the curve (AUC) of up to 0.97. Finally, genomic regions with H3K27ac or histone H3 lysine 4 trimethylation (H3K4me3) were found to exhibit different fragmentomic patterns of cfDNA. This study has shed light on the relationship between cfDNA fragmentomics and histone modifications, thus expanding the armamentarium of liquid biopsy.

Massive parallel sequencing-based non-invasive prenatal test (NIPT) identifies aberrations on chromosome 13

ObjectiveWe report data of non-invasive prenatal testing (NIPT) at Uppsala University Hospital between 2017–2022. Furthermore, we illustrate the potential capacity of massive parallel sequencing-based NIPT beyond identification of common trisomies. MethodsMaternal blood samples were analyzed using the Verifi NIPT or VeriSeq NIPT assays. Diagnostic testing, performed on amniotic fluid samples, included QF-PCR, microarray (SNP-array) and metaphase FISH. ResultsAmong 4532 NIPT tests performed between 2017–2022, 125 samples (2.76%) showed increased risk for trisomies 13, 18, 21 and sex chromosome aneuploidy. For three patients with normal NIPT result further microarray indicated other types of chromosomal rearrangement which were not analyzed by NIPT. For another patient (case 1) the Verifi NIPT indicated trisomy 13. Fetal fraction (FF) was estimated to be 10%. Confirmatory microarray detected a segmental duplication on chromosome 13, as well as a terminal duplication and a terminal deletion on chromosome 10. A complex karyotype was observed in the fetus with metaphase FISH. In the second case the VeriSeq NIPT indicated trisomy 13. FF was estimated to be 11%. Confirmatory microarray detected a mosaicism of trisomy 13 in 30 % of cells. ConclusionThis study illustrates detection of peculiar abnormalities of chromosome 13 and supports potential to screen copy number variations with genome-wide NIPT.

Fetal Fraction of Cell-Free DNA in the Prediction of Adverse Pregnancy Outcomes: A Nationwide Retrospective Cohort Study.

To assess the added value of fetal fraction of cell-free DNA in the maternal circulation in the prediction of adverse pregnancy outcomes. Retrospective cohort study. Nationwide implementation study on non-invasive prenatal testing (NIPT; TRIDENT-2 study). Pregnant women in the Netherlands opting for NIPT between June 2018 and June 2019. Two logistic regression prediction models were constructed for each adverse pregnancy outcome. The first model (base model) included prognostic clinical parameters that were selected from existing first-trimester prediction models for adverse pregnancy outcomes. The second model (fetal fraction model) included fetal fraction as a predictor on top of the prognostic clinical parameters included in the base model. The added prognostic value of fetal fraction was assessed by comparing the base and fetal fraction models in terms of goodness of fit and predictive performance. Likelihood ratio test (LRT), area under the curve (AUC) and Integrated Discrimination Improvement (IDI) index. The study cohort consisted of 56 110 pregnancies. The incidence of adverse pregnancy outcomes was 5.7% for hypertensive disorders of pregnancy (HDP; n = 3207), 10.2% for birthweight < p10 (n = 5726), 3.2% for birthweight < p2.3 (n = 1796), 3.4% for spontaneous preterm birth (sPTB; n = 1891), 3.4% for diabetes (n = 1902) and 1.3% for congenital anomalies (n = 741). Adding fetal fraction to the base model improved model fit for HDP, birthweight < p10, birthweight < p2.3, all sPTB, and diabetes, but not for congenital anomalies (LRT p < 0.05). For HDP, the AUC improved from 0.67 to 0.68 by adding fetal fraction to the base model (p = 0.14) with an IDI of 0.0018 (p < 0.0001). For birthweight < p10, the AUC improved from 0.65 to 0.66 (p < 0.0001) with an IDI of 0.0023 (p < 0.0001). For birthweight < p2.3, the AUC improved from 0.67 to 0.69 (p < 0.0001) with an IDI of 0.0011 (p < 0.0001). For all sPTB, the AUC was similar for both models (AUC 0.63, p = 0.021) with an IDI of 0.00028 (p = 0.0023). For diabetes, the AUC was similar (AUC 0.72, p = 0.35) with an IDI of 0.00055 (p = 0.00015). Fetal fraction has statistically significant but limited prognostic value in the prediction of adverse pregnancy outcomes in addition to known prognostic clinical parameters.

First-Trimester Cell-Free DNA Fetal Fraction and Birth Weight in Twin Pregnancies.

The relationship between fetal fraction and birth weight in twin gestations is poorly understood. To investigate the relationship between first trimester 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 12/2011 - 2/2022, excluding those with positive screen results for chromosomal aneuploidy. CfDNA testing was performed by a single lab using massively parallel sequencing (MPSS). Baseline characteristics and birth weight of pregnancies with normal fetal fraction were compared to those with low (<5%) and high (>95%) fetal fraction using univariable analyses and multivariable regression. A total of 1041 twin pregnancies were included. Chronic hypertension, elevated BMI, 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 [IQR] fetal fraction 12.2% [9.8, 14.8] versus 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 birthweight < 10th percentile, suggesting that first trimester fetal fraction may not predict impaired fetal growth in twin gestations.

Progress in fetal fraction prediction of placental diseases

Placental diseases may affect the outcome of pregnancy and long-term health of the mother and fetus. Fetal fraction is a key indicator for the success of non-invasive prenatal testing, and has been associated with gestational age, body mass index and fetal chromosomal aneuploidies. Many studies have found that fetal fraction is also related to placenta-derived diseases and may become a new predictor for such diseases. This article has summarized the association between the two, with an aim to provide new ideas for the prediction of placental diseases.

Performance evaluation of noninvasive prenatal testing on 24 chromosomes in a cohort of 118,969 pregnant women in Sichuan, China.

This study aimed to comprehensively analyze the detection capacity of non-invasive prenatal testing (NIPT) for chromosomal abnormalities of all 24 chromosomes, as well as high-risk indications for pregnancy and the fetal fraction, in a large cohort. We retrospectively enrolled 118,969 pregnant women who underwent NIPT at Sichuan Provincial Maternity and Child Health Care Hospital from March 2019 to June 2022. The sensitivity, specificity, positive predictive value, negative predictive value, and positive chromosomal abnormality rate were calculated. The fetal fraction based on gestational age, maternal body mass index, and number was examined. NIPT demonstrated > 99% sensitivity and specificity for almost all of the common trisomies (T21, T18, and T13), sex chromosomal aneuploidies, rare autosomal trisomies, and microdeletion/microduplication syndromes. Positive predictive values varied from 12.0% to 89.6%. Advanced maternal age was associated with an increased risk of three major aneuploidies. The fetal fraction was positively correlated with gestational age and negatively correlated with the maternal body mass index. NIPT can be used to effectively screen for chromosomal abnormalities across all 24 chromosomes. Advanced maternal age is a risk factor for high-risk pregnancy, and careful consideration of the fetal fraction is essential during NIPT.

The Effect of Self-Reported Race on Noninvasive Prenatal Screening Test Characteristics.

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..

Uterine fibroids and non-informative cell-free DNA screening results.

Uterine fibroids are monoclonal tumors, which are often genetically abnormal and associated with false-positive genome-wide cell-free DNA (cfDNA) screening results, particularly when large. It is plausible that fibroids may also increase the risk of cfDNA failure by affecting fetal fraction or due to their genetic anomalies confounding cfDNA algorithms. We aimed to investigate a possible association between fibroids and cfDNA non-informative results. This was a retrospective cohort study of women undergoing cfDNA screening for fetal chromosomal abnormalities between 2013 and 2020, comparing pregnancies with vs without uterine fibroids recorded on any obstetric ultrasound before 24 weeks' gestation. Univariable and multivariable logistic regression models were used to investigate the association between fibroids and cfDNA failure, adjusting for gestational age, maternal age, weight and height at blood sampling, mode of conception, multiple gestation and test platform (chromosome-selective or genome-wide). Analyses were stratified according to the number of fibroids and total fibroid volume. The impact of fibroids on fetal fraction was assessed using linear regression, adjusting for the same covariates. Among 19 818 pregnancies undergoing cfDNA screening, fibroids were reported in 2038 (10.28%) and cfDNA failure at the first screening attempt occurred in 228 (1.15%) pregnancies. Non-informative results occurred in 1.96% of pregnancies with fibroids and 1.06% of pregnancies without fibroids (adjusted odds ratio (aOR), 2.40 (95% CI, 1.65-3.48)). The risk of failure in the first screening attempt increased progressively with the number of fibroids (aOR, 5.05 (95% CI, 2.29-11.13) in women with four or more fibroids) and total fibroid volume, with greater than a 5-fold and 14-fold increase in risk among women with fibroid volumes of 100.1-400 mL (aOR, 5.52 (95% CI, 2.30-13.25)) and > 400 mL (aOR, 14.80 (95% CI, 4.50-48.69)), respectively. Although test failure was more common with chromosome-selective than genome-wide screening, fibroids similarly increased the risk of failure of both screening platforms. Compared to pregnancies without fibroids, those with fibroids had a fetal fraction on average 0.61% lower (adjusted mean difference, -0.61% (95% CI, -0.77% to -0.45%)). Uterine fibroids are associated with lower fetal fraction and an increased risk of cfDNA screening failure. The strength of this association increases with increasing fibroid number and volume. © 2024 The Author(s). Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

High-Resolution Haplotyping of the PAH Gene Enables Early Gestation Noninvasive Prenatal Diagnosis of Phenylketonuria and Evolution Analysis of Recurrent Pathogenic Variations.

The clinical performance of RHDO-based NIPD for PKU during early gestation remains under-evaluated. Furthermore, studies focused on SNP loci obtained by next-generation sequencing to analyze the genetic evolution of pathogenic variations in PKU is limited. Maternal peripheral blood, along with proband and paternal samples, was collected between 7 and 12weeks of gestation. The PAH gene and surrounding high heterozygosity SNPs were targeted for enrichment and sequencing. Fetal genotypes were inferred using RHDO-based NIPD. High-resolution PAH haplotypes were used for the analysis of two common pathogenic variants in the Chinese population: c.728G>A and c.1238G>C. Sixty one PKU families participated with an average fetal fraction of 6.08%. The median gestational age was 8+6weeks. RHDO-based NIPD successfully identified fetal genotypes in 59 cases (96.72%, 59/62). Two cases failed because of insufficient informative SNPs. In addition, a recombination event was assessed in one fetus of 59 cases. Six, and three haplotypes were identified for c.728G>A(p.Arg243Gln) and c.1238G>C(p.Arg413Pro), respectively. Hap_3 and hap_8 were identified as the ancestral haplotypes for these pathogenic variants, with other haplotypes arising from mutations or recombination based on these ancestral haplotypes. This study validates the feasibility of an RHDO-based assay for NIPD of PKU in early pregnancy and introduces its application in the demonstration of founder effects in recurrent pathogenic variations, offering new insights into the evolutionary analysis of PAH variations.

Examining the predictive value of fetal fraction on hypertensive disorders of pregnancy

BackgroundCell-free fetal DNA (cffDNA) screening is routinely performed in pregnancy. Abnormal fetal fraction has been associated with adverse pregnancy outcomes, including hypertensive disorders of pregnancy, which are associated with severe maternal and neonatal morbidity and mortality. ObjectiveThis study examined whether abnormal fetal fraction, defined in this study as fetal fraction either <6 or >15 on the basis of restricted-cubic-spline-plot within our study population, was associated with HDP in a retrospective sample, as well as whether fetal fraction improves the prediction of hypertensive disorders of pregnancy (HDP). We hypothesized that abnormal fetal fraction would be associated with HDP and that adding fetal fraction to a model would significantly improve its strength to predict HDP.Study Design: This was a retrospective cohort study of 729 patients delivering singleton, non-anomalous pregnancies with conclusive cffDNA screening. The primary outcome was HDP. Logistic regression models tested associations between fetal fraction and HDP. We evaluated the impact of including fetal fraction on the prediction of hypertensive disorders of pregnancy (HDP) by comparing the area under the receiver operating characteristic (ROC) curve (AUC) between predictive models with and without fetal fraction. ResultsAmong the study sample, there was an HDP rate of 11.5 %. Abnormal fetal fraction was defined as <6 % percentile and >15 %, HDP incidence was significantly higher in patients with fetal fraction <6 % compared to patients with fetal fraction in normal range (fetal fraction 6–15 %) (19.5 % vs 10.7 %, p = 0.006 on post hoc comparison). Model 1 had one predictor (fetal fraction) with an AUC of 0.59, Model 2 had three predictors (BMI, nulliparity, history of HDP) with an AUC of 0.71, and Model 3 had four predictors (BMI, nulliparity, history of HDP, and fetal fraction) with an AUC of 0.73. Models 2 and 3 were not significantly different (p = 0.18). ConclusionsMore patients who developed HDP had low fetal fraction and fewer patients who developed HDP had high fetal fraction compared to those patients who did not develop HDP. Based on results from multivariable regression models, we cannot conclude that fetal fraction improves HDP prediction. However, developing standardized values for abnormal fetal fraction may be clinically useful.

Cell-Free DNA Analysis for the Determination of Fetal Red Blood Cell Antigen Genotype in Individuals With Alloimmunized Pregnancies.

To evaluate the accuracy of next-generation sequencing-based quantitative cell-free DNA analysis for fetal antigen genotyping in individuals with alloimmunized pregnancies undergoing clinical testing in practices across the United States as early as 10 weeks of gestation, with the objective of identifying individuals with pregnancies at risk for hemolytic disease of the fetus and newborn and guiding management. This prospective cohort study included patients with alloimmunized pregnancies undergoing clinical fetal antigen cell-free DNA analysis between 10 0/7 and 37 0/7 weeks of gestation at 120 clinical sites. Both the pregnant person with the alloimmunized pregnancy and the neonates resulting from the pregnancies were included. The laboratory issued the cell-free DNA results prospectively as a part of clinical care. After delivery, neonatal buccal swabs collected between 0 and 270 days of life were sent to an outside independent laboratory for antigen genotyping. The outside laboratory was blinded to the fetal cell-free DNA results, and the results were compared. Concordance was reported for the fetal antigen cell-free DNA analysis for antigens to which the pregnant person was alloimmunized and for all antigens for which the pregnant person was genotype negative. A total of 156 pregnant people who received clinically ordered cell-free DNA fetal antigen testing provided neonatal buccal swabs for genotyping after delivery. Overall, 15.4% of participants were Hispanic, 9.0% were non-Hispanic Black, 65.4% were non-Hispanic White, 4.5% were Asian, 1.3% were more than one race or ethnicity, and 4.5% were unknown. The median gestational age at the time of testing was 16.4 weeks with a median fetal fraction of 11.1%. Concordance between cell-free DNA analysis results and neonatal genotype was determined for 465 antigen calls for the following antigens: K1 (n=143), E (124), C (60), Fya (50), c (47), and D(RhD) (41). These 465 calls included 145 in which the fetus was antigen positive and 320 in which the fetus was antigen negative. We observed complete concordance between prenatal fetal antigen cell-free DNA analysis results and neonatal genotypes for the 465 calls, resulting in 100% sensitivity, specificity, and accuracy. In a diverse multicenter cohort, cell-free DNA analysis was highly sensitive and specific for determining fetal antigen genotype as early as 10 weeks of gestation in individuals with alloimmunized pregnancies. Taken together with previously published evidence, this study supports the implementation of cell-free DNA testing to manage individuals with alloimmunized pregnancies in the United States.

A mathematical description of nonself for biallelic genetic systems in pregnancy, transfusion, and transplantation.

A central issue in immunology is the immunological response against nonself. The prerequisite for a specific immunological response is the exposure to the immune system of a nonself antigen. Mathematical equations are presented, which define the fraction of all outcomes with a nonself allele in biallelic systems at the population level in pregnancy and transfusion/transplantation medicine. When designing assays, the mathematical descriptions can be used for estimating the number of genetic markers necessary to obtain a predetermined probability level in detecting nonself alleles of a given frequency. For instance, the equations can be helpful in the design of assays, where the nonself allele can be detected by analysis of cfDNA in plasma from pregnant women, to estimate fetal fraction or to monitor changes in cfDNA in plasma of transplantation patients. The equations give exact, quantitative descriptions of all nonself situations in pregnancy and transfusion/transplantation.

Single-cell transcriptomics reveal differences between chorionic and basal plate cytotrophoblasts and trophoblast stem cells.

Cytotrophoblast (CTB) of the early gestation human placenta are bipotent progenitor epithelial cells, which can differentiate into invasive extravillous trophoblast (EVT) and multinucleated syncytiotrophoblast (STB). Trophoblast stem cells (TSC), derived from early first trimester placentae, have also been shown to be bipotential. In this study, we set out to probe the transcriptional diversity of first trimester CTB and compare TSC to various subgroups of CTB. We performed single-cell RNA sequencing on six normal placentae, four from early (6-8 weeks) and two from late (12-14 weeks) first trimester, of which two of the early first trimester cases were separated into basal (maternal) and chorionic (fetal) fractions prior to sequencing. We also sequenced three TSC lines, derived from 6-8 week placentae, to evaluate similarities and differences between primary CTB and TSC. CTB clusters displayed notable distinctions based on gestational age, with early first trimester placentae showing enrichment for specific CTB subtypes, further influenced by origin from the basal or chorionic plate. Differential expression analysis of CTB from basal versus chorionic plate highlighted pathways associated with proliferation, unfolded protein response, and oxidative phosphorylation. We identified trophoblast states representing initial progenitor CTB, precursor STB, precursor and mature EVT, and multiple CTB subtypes. CTB progenitors were enriched in early first trimester placentae, with basal plate cells biased toward EVT, and chorionic plate cells toward STB, precursors. Clustering and trajectory inference analysis indicated that TSC were most like EVT precursor cells, with only a small percentage of TSC on the pre-STB differentiation trajectory. This was confirmed by flow cytometric analysis of 6 different TSC lines, which showed uniform expression of proximal column markers ITGA2 and ITGA5. Additionally, we found that ITGA5+ CTB could be plated in 2D, forming only EVT upon spontaneous differentiation, but failed to form self-renewing organoids; conversely, ITGA5-CTB could not be plated in 2D, but readily formed organoids. Our findings suggest that distinct CTB states exist in different regions of the placenta as early as six weeks gestation and that current TSC lines most closely resemble ITGA5+ CTB, biased toward the EVT lineage.

Accuracy of fetal fraction measurements in a single-nucleotide polymorphism-based noninvasive prenatal test.

Noninvasive prenatal testing (NIPT) for fetal aneuploidy relies on the analysis of fetoplacental cell-free DNA (cfDNA) found in maternal plasma. A minimum cfDNA fetal fraction (FF) is required for reliable test performance, but some methods may have suboptimal accuracy for FF measurement. This study investigated the accuracy of a single-nucleotide polymorphism- (SNP-) based NIPT method to assess FF. FF measurements using SNP-based NIPT in consecutive samples from singleton male pregnancies were compared with FF measured using a "gold standard" Y-chromosome method. In a cohort of 106,846 samples, the SNP-based FF method showed a standard deviation (SD) of 0.42%. Compared to the Y chromosome FF method, a correlation coefficient, r, of 0.995, and bias of 0.17% were observed. The SD was not substantially different across specific FF ranges or for samples with high-risk NIPT results. The SNP-based NIPT method estimates FF with good accuracy, with a SD three to eight times better than other NIPT methods (0.42% vs. 1.3%-3.4%). FF is an important quality control parameter and should be routinely reported as part of NIPT.

Early Non-Invasive Prenatal Testing at 6-9 Weeks of Gestation.

Non-invasive prenatal testing (NIPT) is usually performed beyond 10 weeks of gestation, because earlier in pregnancy, the fetal fraction is low, resulting in failure to obtain reliable results. This study aimed to evaluate the clinical performance of NIPT earlier in pregnancy using a method for cell-free DNA (cfDNA) analysis that eliminates the need for polymerase chain reaction (PCR), DNA sequencing, or microarrays (Vanadis® system, PerkinElmer, Waltham, MA, USA). Cell-free DNA was extracted from the maternal plasma of 30 singleton pregnancies at 6-9 weeks of gestation (group 1) and at 11-14 weeks of gestation of the same patients (group 2). The mean crown-rump length (CRL) and gestational age in group A was 16.12 mm and that in group B was 61.45 mm. In group A, results were obtained in all, but one, cases (97%). From the remaining pregnancies, one miscarried at 8 weeks and, therefore, the follow-up NIPT at 12 weeks could not be performed. The fetal sex was diagnosed correctly in the 28 cases that had a successful early test, and the results were in accordance with the examination at 12 weeks. There were no cases of aneuploidies and disomy was diagnosed correctly in all. The "Vanadis" prenatal NIPT assay can successfully be used early during the first trimester at 6-9 weeks of gestation (early NIPT) to identify the fetal sex. Further studies are needed to explore the diagnostic potential for aneuploidies.

P-538 Liquid biopsy (niPOC) as a new approach to genetic studies in early pregnancy loss. First clinical experience in Spain

Abstract Study question How accurate is the use of liquid biopsy as a new tool for aneuploidy testing in cases of pregnancy losses? Summary answer This study demonstrates that genome-wide cfDNA testing in the maternal bloodstream (niPOC) constitutes a simple, tissue-independent, straightforward and reliable complement/alternative to traditional POC analysis. What is known already Around 50-70% of clinical miscarriages are caused by aneuploidies, mostly trisomies. To date, conventional cytogenetic and advanced molecular techniques are used for the analysis of POC to identify the genetic cause of miscarriage, providing valuable information for genetic counseling. However, both approaches are based in the direct analysis of the abortive tissue, which entails several limitations due to a risk of culture failure and/or maternal cell contamination. To solve these drawbacks, maternal cell-free DNA (cfDNA) testing emerges as a promising alternative due to the accumulated evidence. Study design, size, duration Retrospective study and first clinical experience, both conducted in our facilities from January 2021 to December 2023. Before carrying out the fetal tissues collection for the POC analysis, a blood sample was drawn to evaluate possible aneuploidies by cfDNA testing. Using NGS+STR POC results as the gold standard, results derived from both studies were compared to assess the percentage of concordance and the cases of non-informativeness (fetal fraction (FF) &amp;lt;2%), false positives, and false negatives. Participants/materials, setting, methods Herein we present a clinical validation (n = 152) and the first clinical experience (n = 103). Maternal peripheral blood samples were extracted for aneuploidy evaluation using Illumina’s sequencing platform. Genetic testing on POCs was performed using NGS (Thermo Fisher Scientific) to detect aneuploidies, and short tandem repeats to detect cases with MCC and/or polyploidy. In both groups (niPOC and POC), the percentage of concordance and cases of non-informativeness (by fetal fraction &amp;lt;2%, or CCM) were evaluated. Main results and the role of chance In the validation set, no significant differences in non-informativity were observed between POC and niPOC [9.2% (14/152) vs 13.8% (21/152)]. Excluding triploids, the sensitivity of niPOC compared with the pregnancy tissue sequencing result was 90.1%, specificity of 97.1% and an accuracy of 88%. In the first clinical experience, non-informativity was similar to that found in the validation (17/103, 16.5%). The percentage of abnormal cases was 43%, very close to the 50% expected according to the scientific literature. Regarding turnaround time, reports in niPOC were released in an average of 3.8 working days. Limitations, reasons for caution Current cfDNA testing technology fails in polyploidy, uniparental disomy identification, which is a potential cause of pregnancy loss. Aneuploidy detection is only feasible if the blood collection was performed before the fetal remains evacuation. Wider implications of the findings CfDNA testing before curettage or pharmacological treatment could be an alternative to POC analysis. If optimized, cfDNA testing could be used contingently with the molecular POC analysis in cases where maternal cell contamination is present. As a result, the overall success rate in the POC program could be substantially improved. Trial registration number Not applicable