Fetal Fraction Research Articles

Inconsistency between non-invasive prenatal testing (NIPT) and conventional prenatal diagnosis due to confined placental and fetal mosaicism: Two case reports.

We aimed to identify the causes of inconsistent results between non-invasive prenatal testing (NIPT) and invasive testing methods for trisomy 21. In the first case, NIPT was performed at 11 weeks of pregnancy, and the result showed a high risk of trisomy 21 [fetal fraction (FF) = 6.98%, 21 chromosome Z-score = 3.6]. The patient underwent quantitative fluorescent (QF)-PCR and karyotyping at 14 + 0 weeks of pregnancy through CVS showing mosaicism of 47, XX, + 21[11] and 46, XX [39] in karyotyping. The patient underwent amniocentesis at 15 + 6 weeks, showing a normal pattern in QF-PCR and 46, XX karyotyping in long term culture. The second case underwent NIPT at 16 + 5 weeks of pregnancy (FF = 7.52%, 21 chromosome Z-score = 2.503). She underwent an invasive test at 19 weeks through amniotic fluid sampling. As a result, trisomy 21 was detected by QF-PCR, and mosaicism of XX, +21[22]/46, XX [4] was identified by karyotyping. Despite significant advances in fetal chromosome analysis using NIPT, invasive testing is still needed as placenta-derived DNA does not reflect 100% fetal genetic information. Placental mosaicism can be detected by NIPT, but more research is needed to increase its sensitivity. Therefore, if the NIPT result is positive, an invasive test can confirm the result, and continuous monitoring is required even if the NIPT result is negative.

Comparison of The Foetal Fraction (ff) of Cell-Free DNA in In Vitro Fertilisation Versus Natural Conception. Evaluation of ff with IVF Parameters

Comparison of The Foetal Fraction (ff) of Cell-Free DNA in In Vitro Fertilisation Versus Natural Conception. Evaluation of ff with IVF Parameters

Factors affecting low fetal fraction in fetal screening with cell-free DNA in pregnant women: a systematic review and meta-analysis

Cell-Free DNA (cfDNA) is a non-invasive perinatal test (NIPT) used to assess fetal anomalies. The ability to detect fetal chromosomal aneuploidies is directly related to a sample's fetal to total DNA fraction, known as the fetal fraction (FF). The minimum FF is considered 4%, and the test result below 4% is uncertain due to low fetal fraction (LFF). This study aimed to conduct a systematic review and a meta-analysis to determine the possible factors affecting LFF in cfDNA testing for fetal screening. PubMed, Web of Science, Google Scholar, Since Direct, Scopus, CINHAL, Cochrane Library, and Persian databases, including Scientific Information Database, Irandoc, and Magiran were searched for studies investigating factors affecting LFF in cfDNA testing from 2000 until the end of 2021. Gathered data were analyzed using Comprehensive Meta-Analysis (CMA) software version 3.3.070. The quality of the included studies was assessed using the Joanna Briggs Institute Critical Appraisal of Cohort Studies tool. Thirteen articles related to the topic were included, and seven related articles were reviewed for meta-analysis. The other six were reviewed qualitatively. Four factors were identified that might have a potential effect on the LFF, of which only gestational age had a significant association with LFF (Pooled mean difference= -1.111, SE = 0.515, 95% CI= -2.121, -0.101, (P-value < 0.05)). Maternal age (P-value = 0.573), maternal weight (P-value = 0.113), and Body Mass Index (P-value = 0.104) had no statically significant effect. The effect size was pooled by mean difference and 95% confidence interval. Lower gestational age is significantly associated with LFF. Thus, this factor can be considered when interpreting prenatal cfDNA screening tests.

High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed—a 12-plex and a 57-plex—and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

Maternal Copy Number Imbalances in Non-Invasive Prenatal Testing: Do They Matter?

Non-invasive prenatal testing (NIPT) has become a routine practice in screening for common aneuploidies of chromosomes 21, 18, and 13 and gonosomes X and Y in fetuses worldwide since 2015 and has even expanded to include smaller subchromosomal events. In fact, the fetal fraction represents only a small proportion of cell-free DNA on a predominant background of maternal DNA. Unlike fetal findings that have to be confirmed using invasive testing, it has been well documented that NIPT provides information on maternal mosaicism, occult malignancies, and hidden health conditions due to copy number variations (CNVs) with diagnostic resolution. Although large duplications or deletions associated with certain medical conditions or syndromes are usually well recognized and easy to interpret, very little is known about small, relatively common copy number variations on the order of a few hundred kilobases and their potential impact on human health. We analyzed data from 6422 NIPT patient samples with a CNV detection resolution of 200 kb for the maternal genome and identified 942 distinct CNVs; 328 occurred repeatedly. We defined them as multiple occurring variants (MOVs). We scrutinized the most common ones, compared them with frequencies in the gnomAD SVs v2.1, dbVar, and DGV population databases, and analyzed them with an emphasis on genomic content and potential association with specific phenotypes.

Correlation of low fetal fraction of cell-free DNA at the early second-trimester and pregnancy complications related to placental dysfunction in twin pregnancy.

This study aimed to determine the correlation between fetal fraction (FF) of cell-free DNA (cf-DNA) and pregnancy complications related to placental dysfunction in Twin Pregnancy. This retrospective cohort study analyzed twin pregnant women who underwent non-invasive prenatal testing (NIPT) at 12+0-26+6 weeks of gestation from April 2017 to April 2021. Low fetal fraction (LFF) was defined individually as less than the 25th, 10th, 5th, and 2.5th percentile among all fetal fractions in the cohort. Primary outcomes included gestational hypertension (GH), preeclampsia (PE), gestational diabetes mellitus (GDM), and small for gestational age (SGA). Logistic regression analysis was used to assess the relationship between LFF and pregnancy complications. A total of 500 twin pregnancies (male-male twins, 245; female-female twins, 255) were included in this study. In LFF group (FF < 25th percentiles), maternal BMI was significantly higher than FF > 75th percentiles (23.6 kg/m2 vs. 21.3 kg/m2; P < 0.001). The risk of SGA increased gradually from FF < 25th percentiles [adjusted odds ratio (OR), 1.71; 95% confidence interval (CI), 1.07-2.99; P = 0.016] to FF < 2.5th percentiles (adjusted OR, 4.44; 95% CI,1.33-14.82; P < 0.015). In addition, the risks of SGA in both fetuses were higher than the risks of at least one fetus SGA in LFF group. LFF had no correlation with GH, PE, and GDM in twin pregnancy. LFF has a strong association with increased risk of SGA in twin pregnancy. Moreover, FF of cf-DNA may provide a new idea for the early screening of diseases related to placental dysfunction in twin pregnancy.

The effect of abnormal placentation on maternal serum fetal fraction of cell-free DNA.

Abnormal placentation may affect the maternal serum fraction of cell-free fetal DNA (fetal fraction) determined as part of non-invasive prenatal screening (NIPS). This study aimed to assess whether the fetal fraction can predict placenta accreta spectrum (PAS) with or without placenta previa (PP). We also investigated the impact of trophoblastic invasion depth on the fetal fraction. This is a retrospective case-control study of pregnant women with and without abnormal placentation carrying a singleton and having undergone NIPS prior to 20weeks of gestation. The eligible subjects were selected from a cohort managed at our institution for PAS suspected antenatally. We compared women with normal placentation (controls) to PAS, PP, or PAS+PP cases. Data were abstracted from electronic medical records, and PAS was confirmed histologically. Of the 146 patients in our cohort, 8 controls, 10 PP, 6 PAS, and 7 PAS+PP cases were eligible for the study. Among the groups, there were no significant differences in baseline demographic and clinical characteristics except the median number of prior uterine surgeries. Also, the groups did not significantly differ in their median fetal fraction. The fetal fraction did not discriminate any group when stratified according to the depth of placental invasion, i.e.,no PAS, abnormally adherent, and abnormally invasive placenta. The maternal serum fraction of cell-free fetal DNA measured before 20weeks of gestation is not predictive of PAS with or without concurrent PP or the depth of trophoblastic invasion.

Tricky TRIC: A replication study using trophoblast retrieval and isolation from the cervix to study genetic birth defects.

Noninvasive Prenatal Diagnosis has recently been introduced for a limited number of monogenetic disorders. However, the majority of DNA diagnostics still require fetal material obtained using an invasive test. Recently, a novel technique, TRIC (Trophoblast Retrieval and Isolation from the Cervix), has been described, which collects fetal trophoblast cells by endocervical sampling. Since this technique has not been successfully replicated by other groups, we aimed to achieve this in the current study. Pregnant women referred for transvaginal chorionic villous sampling (CVS) were asked for an endocervical sample prior to CVS. The TRIC samples were processed to isolate trophoblast DNA. TRIC DNA was used in ForenSeq to determine the amount of maternal DNA contamination, and for Sanger sequencing in case of a monogenic disorder. 23%-44% of samples had a sufficiently high fetal DNA fraction to allow genetic testing, as calculated by Sanger sequencing and ForenSeq, respectively. We have been able to successfully replicate the TRIC protocol, although with a much lower success rate as described by the original study performing TRIC. As we obtained the samples in the actual clinical setting envisioned, the method in its current setup is not advisable for use in prenatal diagnostics.

Non-intuitive trends of fetal fraction development related to gestational age and fetal gender, and their practical implications for non-invasive prenatal testing

Discovery of fetal cell-free DNA fragments in maternal blood revolutionized prenatal diagnostics. Although non-invasive prenatal testing (NIPT) is already a matured screening test with high specificity and sensitivity, the accurate estimation of the proportion of fetal fragments, called fetal fraction, is crucial to avoid false-negative results. In this study, we collected 6999 samples from women undergoing NIPT testing with a single male fetus to demonstrate the influence of fetal fraction by the maternal and fetal characteristics. We show several fetal fraction discrepancies that contradict the generally presented conventional view. At first, the fetal fraction is not consistently rising with the maturity of the fetus due to a drop in 15 weeks of maturation. Secondly, the male samples have a lower fetal fraction than female fetuses, arguably due to the smaller gonosomal chromosomes. Finally, we discuss not only the possible reasons why this inconsistency exists but we also outline why these differences have not yet been identified and published. We demonstrate two non-intuitive trends to better comprehend the fetal fraction development and more precise selection of patients with sufficient fetal fraction for accurate testing.

Cell-free fetal DNA testing performance and fetal fraction estimation are not affected in ART-conceived pregnancies.

Does ART-based conception influence fetal fraction (FF) estimation and cell-free fetal DNA (cffDNA) testing performance? Mode of conception (ART versus natural) does not impact FF estimation or cffDNA test informativity rates. Pregnancies achieved via ART are increasing, and cffDNA testing is displacing traditional prenatal screening methods due to its high sensitivity and specificity and noninvasive nature. However, conflicting data exist on cffDNA testing performance and FF in ART pregnancies compared with natural pregnancies. We performed a case-control study that included 21 558 consecutive pregnancies (spontaneous, n = 15 707; ART, n = 5851). ART-conceived pregnancies were stratified into two groups according to oocyte origin. Samples were collected from April 2015 to September 2019. The study included women from different centers worldwide. Blood samples were drawn from the 10th week of gestation onward. Massive parallel whole-genome sequencing was used to analyze cffDNA content in blood plasma. Two different types of technologies (single-end and paired-end) were applied because of analysis technology changes made by the sequencing provider over time. FF was determined using different methods depending on the type of technology used. Cases with an FF <2% or with failure in any quality control metrics were classified as noninformative. An analysis of covariance model was selected to identify which qualitative (sequencing methodology, mode of conception, type (i.e. multiplicity) of gestation and age (women >35 or <35 years old)) and quantitative (gestational age, BMI) variables were predictors of FF value. Multinomial logistic regression was used to evaluate whether the mode of conception impacted cffDNA testing performance. A univariate t-test demonstrated no significant differences in FF values between ART (median FF = 9.2%) and spontaneous pregnancies (median FF = 9.2%). Also, a multivariate analysis showed that the mode of conception, did not strongly impact the percentage of FF. ART-treated women showed a lower incidence of high-risk cffDNA results compared to women who conceived naturally, specifically for trisomy (T)21 (0.7% versus 1.3%, P = 0.001) and T18 (0.1% versus 0.3%, P = 0.001). A multivariate model stratified by type of aneuploidy suggested that these differences were conditioned by oocyte origin, especially for the T21 risk classification (P < 0.0001). False-positive rates (FPRs) were significantly higher in the ART population, mainly for T13 (P = 0.001) and sexual chromosome aneuploidies (SCAs; P < 0.001). A multivariate model suggested that the differences observed in SCAs were caused by sequencing modality rather than by mode of conception. Likewise, ART-treated women who used their own oocytes had a higher probability of a false positive for T13 (P = 0.004). Our study lacks follow-up data for low- and high-risk cases of both ART-conceived and naturally conceived pregnancies. Therefore, the results comparing FPR in both populations should be interpreted carefully. Also, collecting information about different ART modalities and regarding preimplantation genetic testing for aneuploidy treatments would help draw definite explanations for the trends observed in this study. This is the first study that demonstrates, with a large sample size, that FF is not influenced by mode of conception, demystifying the notion that patients undergoing ART have a higher probability of noninformative cffDNA testing results. Multivariate models stratified by oocyte origin and type of aneuploidy demonstrated that ART-conceived pregnancies do not have a higher probability of classification as a high-risk pregnancy in prenatal testing. This information is especially valuable to clinicians and genetic counselors when informing patients about the risks and limitations of cffDNA testing in ART pregnancies. This study was financially supported by Igenomix Lab S.L.U. All authors declare no conflict of interest. N/A.

Additional value of advanced ultrasonography in pregnancies with two inconclusive cell-free DNA draws.

We aimed to evaluate the additional value of advanced fetal anatomical assessment by ultrasound in pregnancies with twice inconclusive noninvasive testing (NIPT) due to low fetal fraction (FF). We performed a multicenter-retrospective study between 2017 and 2020 including 311 pregnancies with twice inconclusive NIPT due to low FF≤1%. Women were offered invasive testing and advanced fetal anatomical assessment at ≤18weeks' gestation. Ultrasound findings, genetic testing, and pregnancy/postnatal outcomes were evaluated. Ninety-two/311 (29.6%) women underwent invasive testing. Structural anomalies were diagnosed in 13/311 (4.2%) pregnancies (nine at the first scan and four at follow-up). In 6/13 (46.2%) cases, genetic aberrations were confirmed (one case of Trisomy 13 (detectable by NIPT), two of Triploidy, one of 16q12-deletion, HCN4-mutation and UPD(16) (nondetectable by NIPT). Genetic aberrations were found in 4/298 (1.3%) structurallynormal pregnancies (one 47XYY, two microscopic aberrations, one monogenic disorder found postpartum). Structural anomalies in genetically normal fetuses (2.0%) were not more prevalent compared to the general pregnant population (OR 1.0 [0.4-2.2]). In pregnancies with twice inconclusive NIPT due to low FF, fetal structural anomalies are not more prevalent than in the general obstetric population. The detailed anatomical assessment has the added value to detect phenotypical features suggestive of chromosomal/genetic aberrations and identify pregnancies where advanced genetic testing may be indicated.

Highlights from the International Twins Congress 2021

Highlights from the International Twins Congress 2021

232.11: Longitudinal Surveillance of a Kidney Transplant Recipient During Pregnancy Using Quantification of Fetal and Donorderived Cell-Free DNA

Introduction: Following kidney transplantation, women of childbearing age often experience rapid restoration of fertility as the feedback mechanisms of the hypothalamic-pituitary-gonadal axis normalize. Although rates of rejection during pregnancy are comparable to the non-pregnant transplant population, subtle indications of allograft injury such as increases in serum creatinine or proteinuria can be masked by pregnancy related changes. Here, we describe a novel approach of allograft surveillance using quantification of fetal and donor-derived cell-free DNA (dd-cfDNA) throughout gestation and postpartum. Methods: The patient was monitored longitudinally with dd-cfDNA (AlloSure, CareDx) pre-pregnancy, during gestation and postpartum. For this analysis, specific SNPs were selected that are heterozygous in kidney donor and homozygous (same allele) in recipient and fetal cells. An average minor allele frequency (MAF) was calculated from these selected SNPs. This average represents half of the expected donor cfDNA% due to Hardy-Weinberg principle based on Mendelian genetics. Hence, calculated average MAF was multiplied by factor of 2 to calculate the kidney donor percentage. Results: A 39 year old female with a living related kidney transplant (transplanted September 2018) conceived in February 2021. The patient had a baseline creatinine of 1.4 mg/dl and median dd-cfDNA level of 0.44%. dd-cfDNA levels remained stable throughout pregnancy (median 0.42%, IQR 0.31-0.44%) and were consistent with pre-pregnancy baseline, in keeping with absence of allograft injury. The percentage of fetal cfDNA saw an exponential rise in the first trimester, plateaued in the second trimester and showed a rapid decay post-delivery. Allograft function remained stable throughout pregnancy until development of pre-eclampsia with an acute rise in serum creatine to 2.1 mg/dl, prompting delivery at 31 weeks (Figure 1). Conclusions: The estimated percent of fetal fraction of cfDNA increased with gestational age and rapidly declined post-delivery. Throughout pregnancy, dd-cfDNA levels remained consistent with pre-pregnancy baseline in the absence of graft dysfunction, rejection, or clinical events. This case study suggests that longitudinal monitoring of fetal and dd-cfDNA contributions during pregnancy in kidney transplant recipients is a feasible, novel approach for allograft surveillance in this high-risk patient population.

Lower fetal fraction in clinical cell-free DNA screening results is associated with increased risk of hypertensive disorders of pregnancy.

To evaluate if fetal fraction (FF) reported on cell-free DNA (cfDNA) screening is a marker for adverse obstetric outcomes. We retrospectively reviewed medical records from a cohort of women with singleton pregnancies who had cfDNA screening. We evaluated if reported FF could predict the following pregnancy complications: hypertensive disorders of pregnancy (HDP), fetal growth restriction, preterm delivery, gestational diabetes mellitus, or a composite maternal morbidity, defined as the presence of at least one of these outcomes. Receiver operating curve analysis was performed on FF from 534 women to define the FF that differentiated a low FF group (<10%; N=259) and a high FF group (≥10%; N=275). Hypertensive disorders of pregnancy were more common for women in the low FF group (32.0% vs. 11.6% and p<0.001), who had a two-fold odds of developing HDP (p=0.006). Composite maternal morbidity was also more common for women in the low FF group (51.4% vs. 30.2% and p<0.001), who had a 1.7-fold odds of developing any of the adverse obstetrical outcomes (p=0.014). We found that low FF on cfDNA screening is associated with an increased risk of HDP. Fetal fraction reported that cfDNA screening reports have potential as a predictive marker for the development of HDP and adverse outcomes.

Experience in the Application of Non-invasive Prenatal Screening for the Detection of Down Syndrome in Russia: A Retrospective Cohort Study

Objectives: Assessment the effectiveness of non-invasive prenatal screening (NIPS) for identifying pregnant women at high risk of giving birth to a child with Down syndrome. Materials and Methods: The retrospective cohort study included 25798 pregnant women who underwent NIPS in Russia from January 2013 to December 2018. 21042 and 4756 participants underwent tests from the Natera laboratory, USA (non-invasive prenatal test 1, NIPT-1) and the Genomed laboratory, Russia (non-invasive prenatal test 2, NIPT-2), respectively. Results: A high risk of trisomy 21 (T21) was detected in 544 cases (2.59%). The mean age in women at high risk of T21 was 37.3 years. According to the NIPT results, in patients who revealed a low risk of T21, the mean age was 33.8 years (P&lt;0.001). In 535 cases, invasive prenatal diagnosis was performed. In 7 (1.3%) cases, the presence of T21 in the fetus was not confirmed. In 528 (97.3%) cases, T21 was confirmed by fetal karyotyping. Among women who revealed a low risk of T21 (N = 25086), in 4 cases (0.015%), fetal trisomy 21 was missed. Thus, the indicators of the effectiveness of NIPS in Russia in relation to T21 are as follows: sensitivity - 99.25%, specificity - 99.96%, PPV - 98.7%, NPV - 99.98%. In the first trimester, in the presence of fetal T21, the level of the fetal fraction of free-DNA is significantly lower. Conclusions: NIPS has good prospects for implementation in pregnancy management programs and increasing the effectiveness of prenatal detection of T21. The level of the fetal fraction is associated with the presence of fetal T21.

Whole genome non-invasive prenatal testing in prenatal screening algorithm: clinical experience from 12,700 pregnancies

BackgroundA fast adoption of a non–invasive prenatal testing (NIPT) in clinical practice is a global tendency last years. Firstly, in Russia according a new regulation it was possible to perform a widescale testing of pregnant women in chromosomal abnormality risk. The aim of the study—to assess efficiency of using NIPT as a second-line first trimester screening test in Moscow.MethodsBased on the first trimester combined prenatal screening results 12,700 pregnant women were classified as a high-risk (cut-off ≥ 1:100) and an intermediate-risk (cut-off 1:101 – 1:2500) groups followed by whole genome NIPT. Women from high-risk group and those who had positive NIPT results from intermediate-risk group were considered for invasive prenatal diagnostic.Results258 (2.0%) samples with positive NIPT results were detected including 126 cases of trisomy 21 (T21), 40 cases of T18, 12 cases of T13, 41 cases of sex chromosome aneuploidies (SCAs) and 39 cases of rare autosomal aneuploidies (RAAs) and significant copy number variations (CNVs). Statistically significant associations (p < 0.05) were revealed for fetal fraction (FF) and both for some patient’s (body mass index and weight) and fetus’s (sex and high risk of aneuploidies) characteristics. NIPT showed as a high sensitivity as specificity for common trisomies and SCAs with an overall false positive rate 0.3%.ConclusionsNIPT demonstrated high sensitivity and specificity. As a second-line screening test it has shown a high efficiency in detecting fetus chromosomal anomalies as well as it could potentially lower the number of invasive procedures in pregnant women.

A fetal fraction enrichment method reduces false negatives and increases test success rate of fetal chromosome aneuploidy detection in early pregnancy loss

ObjectiveWe and others have previously demonstrated that the size-selection enrichment method could remarkably improve fetal fraction (FF) in the early gestational age (GA, 12–13 weeks), suggesting that 9 or 10 weeks should not be used as a threshold for GA in size-selection noninvasive prenatal screening (NIPS). Here, we assessed whether this method was reliable for detecting fetal chromosomal aneuploidy at the earliest GA (6–8 weeks).MethodsSize-selection NIPS for fetal chromosomal aneuploidy was applied to 208 pregnancy plasma samples (102 male and 106 female fetuses), while the 169 pregnancy samples with male fetuses also underwent standard NIPS. Multivariable linear regression models were used to evaluate the association between fold-change of FF and experimental factors.ResultsThe sensitivity of the cell-free DNA (cfDNA) test in detecting aneuploidy was 100% when screened with FF enrichment, whereas the sensitivity of the same patients was only 62.5% (5/8) without FF enrichment. In the 102 pregnancy samples with male fetuses, FF increased from 6.1% to 15.7%, and the median increase in FF was 2.8-fold with enrichment. Moreover, there was a trend toward an increasing success rate of the cfDNA test from 6 to 13 weeks of gestation, especially when the test success rate reached 100% after 7 weeks with FF enrichment. Multivariate linear regression analysis demonstrated that a lower initial FF, shorter cfDNA size, increased body mass index (BMI), and later GA were all independent predictors of a higher fold-change of FF. Compared with ≤ 120 bp cfDNA fragments, the mean fold-change of FF differences was 0.820 for 121–125 bp, 0.229 for 126–130 bp, − 0.154 for 131–135 bp, − 0.525 for 136–140 bp and − 0.934 for > 140 bp (Ptrend < 0.0001), suggesting that fold-change of FF significantly decreased with cfDNA fragments > 125 bp. These results were statistically significant after adjusting for confounding factors in the models for fold-change of FF.ConclusionsThe FF enrichment method is a reasonable strategy to detect fetal chromosomal aneuploidy in early pregnancy loss with reduced false negatives and increased test success rate after 7 weeks of GA and should be recommended for patients with early pregnancy loss.

Noninvasive fetal genotyping of single nucleotide variants and linkage analysis for prenatal diagnosis of monogenic disorders

BackgroundHigh-cost, time-consuming and complex processes of several current approaches limit the use of noninvasive prenatal diagnosis (NIPD) for monogenic disorders in clinical application. Thus, a more cost-effective and easily implementable approach is required.MethodsWe established a low-cost and convenient test to noninvasively deduce fetal genotypes of the mutation and single nucleotide polymorphisms (SNPs) loci by means of targeted amplification combined with deep sequencing of maternal genomic and plasma DNA. The sequential probability ratio test was performed to detect the allelic imbalance in maternal plasma. This method can be employed to directly examine familial pathogenic mutations in the fetal genome, as well as infer the inheritance of parental haplotypes through a group of selected SNPs linked to the pathogenic mutation.ResultsThe fetal mutations in 17 families with different types of monogenic disorders including hemophilia A, von Willebrand disease type 3, Duchenne muscular dystrophy, hyper-IgM type 1, glutaric acidemia type I, Nagashima-type palmoplantar keratosis, and familial exudative vitreoretinopathy were identified in the study. The mutations included various forms: point mutations, gene inversion, deletions/insertions and duplication. The results of 12 families were verified by sequencing of amniotic fluid samples, the accuracy of the approach in fetal genotyping at the mutation and SNPs loci was 98.85% (172/174 loci), and the no-call rate was 28.98% (71/245 loci). The overall accuracy was 12/12 (100%). Moreover, the approach was successfully applied in plasma samples with a fetal fraction as low as 2.3%.ConclusionsWe have shown in this study that the approach is a cost-effective, less time consuming and accurate method for NIPD of monogenic disorders.

Chromosomal phase improves aneuploidy detection in non-invasive prenatal testing at low fetal DNA fractions

Non-invasive prenatal testing (NIPT) to detect fetal aneuploidy by sequencing the cell-free DNA (cfDNA) in maternal plasma is being broadly adopted. To detect fetal aneuploidies from maternal plasma, where fetal DNA is mixed with far-larger amounts of maternal DNA, NIPT requires a minimum fraction of the circulating cfDNA to be of placental origin, a level which is usually attained beginning at 10 weeks gestational age. We present an approach that leverages the arrangement of alleles along homologous chromosomes—also known as chromosomal phase—to make NIPT analyses more conclusive. We validate our approach with in silico simulations, then re-analyze data from a pregnant mother who, due to a fetal DNA fraction of 3.4%, received an inconclusive aneuploidy determination through NIPT. We find that the presence of a trisomy 18 fetus can be conclusively inferred from the patient’s same molecular data when chromosomal phase is incorporated into the analysis. Key to the effectiveness of our approach is the ability of homologous chromosomes to act as natural controls for each other and the ability of chromosomal phase to integrate subtle quantitative signals across very many sequence variants. These results show that chromosomal phase increases the sensitivity of a common laboratory test, an idea that could also advance cfDNA analyses for cancer detection.

The association between fetal fraction and pregnancy-related complications among Chinese population.

To examine the association of fetal fraction with a wide spectrum of pregnancy-related complications among Chinese population, we carried out a single-institution retrospective cohort study of women with negative Noninvasive prenatal testing (NIPT) results and singleton pregnancies between May 2018 and May 2020. Indicators of pregnancy-related complications were examined individually, including preterm birth, low birth weight, hypertensive disorders of pregnancy, gestational diabetes, oligohydramnios and intrahepatic cholestasis. We evaluated disease odds ratios (ORs) and 95% confidence intervals (CIs), after controlling for potential confounders including body mass index (BMI), maternal age and gestational week at NIPT. A total of 3534 women were included in our analyses. Women with fetal fraction<15.15% had increased risk of gestational hypertension (OR 4.41, CI [1.65, 12.45]) and oligohydramnios (OR 2.26, CI [1.33, 3.80]) compared to women with fetal fraction≥15.15%. No significant associations with fetal fraction were found for preterm birth, low birth weight, gestational diabetes, and intrahepatic cholestasis. In Summary, fetal fraction is inversely associated with the risk of gestational hypertension and oligohydramnios.