Fetal DNA Research Articles

Fetal fraction of cell-free DNA in noninvasive prenatal testing and adverse pregnancy outcomes: a nationwide retrospective cohort study of 56,110 pregnant women

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.

Current controversies in prenatal diagnosis: Noninvasive prenatal testing should replace other screening strategies for fetal trisomies 13, 18, 21.

This is a written summary of the oral debate presented at the International Society for Prenatal Diagnosis annual conference in Edinburgh in 2023. The topic under debate is whether noninvasive prenatal testing (NIPT) using cell-free fetal DNA should replace other screening strategies for the detection of fetal trisomies 13, 18, 21. There is no disagreement that NIPT is far more sensitive and has better positive predictive values for identifying trisomies 13, 18, and 21 than traditional screening approaches using biochemical markers and measurement of nuchal translucency. The major issue lies in the potential adverse consequences associated with abandoning traditional screening methods. The source of disagreement stems primarily from whether you consider the role of ultrasound in the context of screening to be strictly for nuchal translucency measurement or whether it should be combined with a fetal anatomy scan. The debate featured two experts who presented evidence in favor of each argument.

Maternal and Fetal Factors Affecting Cell-Free Fetal DNA (cffDNA) Fraction: A Systematic Review.

Cell-free fetal DNA (cffDNA) is a novel screening method for fetal aneuploidy that facilitated non-invasive prenatal testing (NIPT) through analysis of cffDNA in maternal plasma. However, despite increased sensitivity, it has a number of limitations that may complicate of its results interpretation. Therefore, elucidating factors affecting fetal fraction, as a critical limitation, guides its clinical application. In this report, systematic search was carried out through PubMed, Web of Science, and Scopus databases until February 11, 2022 by using keywords consist of "noninvasive prenatal screening", "NIPT", "noninvasive prenatal", "cell free DNA" and "fetal fraction". The articles were screened for eligibility criteria before data extraction. A total of 39 eligible studies, most published between 2010 and 2020, were included. Based on the results of studies, a negative correlation between maternal age and BMI/body weight with fetal fraction was found. Furthermore, LDL, cholesterol, triglyceride level, metformin, heparin and enoxaparin therapy, hemoglobin-related hemoglobinopathies, and physical activity showed to have negative associations. Interestingly, it seems the ethnicity of patients from South and East Asia has a correlation with fetal fraction compared to Caucasians. Positive correlation was observed between gestational age, free β-hCG, PAPP-A, living in high altitude, and twin pregnancy. Considering each factor, there was significant inconsistency and controversy regarding their impact on outcomes. Indeed, multiple factors can influence the accuracy of NIPS results, and it is worth noting that the impact of these factors may vary depending on the individual's ethnic background. Therefore, it is important to recognize that NIPS remains a screening test, and comprehensive pre- and post-NIPS counseling should be conducted as part of standard clinical practice.

High sensitivity and specificity in fetal gender identification in the first trimester, using ultrasound and Noninvasive Prenatal Screening (NIPS) in twin pregnancies, a prospective study

IntroductionDetermination of the fetal gender in the first trimester is important in twin pregnancy cases of familial X-linked genetic syndromes and helps determine chorionicity. We assessed and compared the accuracy of first-trimester ultrasound scans, and cell-free fetal DNA (CfDNA) in determining fetal gender in the first trimester of twin pregnancies.MethodsWomen with twin pregnancies were recruited prospectively during the first trimester. Fetal gender was determined using both ultrasound scans and CfDNA screening. Both results were compared to the newborn gender after delivery.ResultsA total of 113 women with twin pregnancies were enrolled. There was 100% sensitivity and specificity in Y chromosome detection using CfDNA.Gender assignment using ultrasound in any first-trimester scans was 79.7%. Accuracy level increased from 54.2% in CRL 45-54 mm to 87.7% in CRL 55-67 mm and 91.5% in CRL 67-87 mm. Male fetuses had significantly higher chances of a gender assignment error compared to female fetuses, odds ratio = 23.574 (CI 7.346 - 75.656).ConclusionsCfDNA is highly sensitive and specific in determining the presence of the Y chromosome in twin pregnancies in the first trimester. Between CRL 55-87 mm, ultrasound scanning offers a highly accurate determination of fetal gender in twin pregnancies.

Improving the accuracy of noninvasive prenatal testing through size-selection between fetal and maternal cfDNA.

In general, fetal cfDNA is shorter than maternal cfDNA, and accuracy of noninvasive prenatal testing (NIPT) results can be improved by selecting shorter cfDNA fragments to enrich fetal-derived cfDNA. This study investigated potential improvements in the accuracy of NIPT by performing classification and analysis based on differences in cfDNA size. We performed paired-end sequencing to identify size ranges of fetal and maternal cfDNA from 62,374 pregnant women. We then developed a size-selection method to isolate and analyze both fetal and maternal cfDNA, defining fetal-derived cfDNA as less than 150 bp and maternal-derived cfDNA as greater than 180 bp. By implementing size-selection method, the accuracy of NIPT was improved, resulting in an increase in the overall positive predictive value for all aneuploidies from 89.57% to 97.1%. This was achieved by enriching both fetal and maternal-derived cfDNA, which increased fetal DNA fraction while the number of false positives for all aneuploidies was reduced by more than 70%. We identified the differences in read length between fetal and maternal-derived cfDNA, and selectively enriched both shorter and longer cfDNA fragments for subsequent analysis. Our approach can increase the detection accuracy of NIPT for detecting fetal aneuploidies and reduce the number of false positives caused by maternal chromosomal abnormalities.

Epigenetic mechanisms of intrauterine fetal senescence: little-known effects of hyperhomocysteinemia during pregnancy

Today, a whole body of evidence base has been formed showing the negative effect of hyperhomocysteinemia (HHC) during pregnancy on the intrauterine fetal development: risk factors associated with development of congenital neural tube defects, congenital heart defects and non-syndromic oral facial clefts. In addition, numerous studies have shown that HHC is a risk factor for serious pregnancy complications such as repeated pregnancy loss, preterm birth, preeclampsia, placental abruption, intrauterine growth restriction, gestational diabetes mellitus. At the same time, despite multiple clinical and experimental studies, the significance of HHC effects continues to support the interest to this issue. In recent years, new data were revealed about littleknown phenomena pregnancy-associated maternal HHC such as "epigenetic fetal aging" and "fetal atherogenesis". Epigenetic aging is an individual indicator of aging that captures interindividual differences at disease onset throughout life occurring during intrauterine development being reveaked as DNA hypomethylation. Moderate HHC during pregnancy causes fetal DNA hypomethylation, which, according to recent research, may represent the main mechanism underlying health in childhood and adulthood. "Fetal atherogenesis" is another understudied HHC effect emphasizing that the formation of atherosclerotic plaques begins not in adulthood, but in utero. A body of studies has shown that the formation of thickening and fatty streaks in vascular intima involved in the pathogenesis of atherosclerosis begins antenatally. The discovery of new aspects of negative intrauterine HHC impact on fetal development necessitates a detailed study of HHC causes as non-modifiable (genetic defects in enzyme systems), and more importantly, conditionally modifiable causes (lack of cofactors, excessive methionine consumption, medications) for their timely leveling. It is also important to study approaches to HHC correction at the preconception stage of a female reproductive function in order to correctly "program" fetus and newborn development.

Platelets Sequester Extracellular DNA, Capturing Tumour-Derived and Free Fetal DNA

Introduction &amp; Hypothesis Platelets are small, multi-functional cells that lack a nucleus but contain RNA and translational machinery for protein synthesis. Platelet RNA mostly derives from parent megakaryocytes, but they also sense and sequester endogenous and pathogen-derived nucleic acids during circulation (D'Ambrosi, 2021; Koupenova, 2019). Nucleated cells release DNA after cell death or aberrant mitosis, resulting in ‘cell-free’ DNA in plasma (cfDNA). Excess cfDNA is deleterious. cfDNA isolated from platelet-poor plasma is emerging as a major liquid biopsy tool in cancer and antenatal screening, but a major limitation is its low abundance, especially in early-stage disease. Given their role in sensing pathogen-derived nucleic acids, we hypothesized that platelets may clear cfDNA from plasma, and that clinically-relevant insights may be derived from the analysis of DNA fragments contained in platelets. Methods Live/fixed imaging using specific DNA probes and fluorescence in-situ hybridization (FISH), droplet digital PCR (ddPCR), NGS and flow cytometry were applied to platelets isolated from Streck, EDTA tubes or apheresis bags from healthy donors, pregnant women, patients with cancer and murine platelets from cancer models. DNA was extracted from platelet pellets or platelet-depleted plasma (cfDNA) using the Qiagen QIAamp Circulating Nucleic Acid Kit. Results Using live cell imaging and flow cytometry, significant DNA content (Draq5+) was detected in ~8% of platelets from healthy donors, including in platelets with low RNA content (SYTO-13-negative). Overall Draq5+ platelets were larger and primed for agonist activation. FISH and ddPCR of platelets from pregnant women carrying male fetuses detected Y-chromosome fragments (n=10), confirming that platelet DNA is not solely derived from parent megakaryocytes but also sequestered during circulation (Fig. 1a). In contrast, Y-chromosome genes were not detected in DNA isolated from mononuclear cells or red blood cells pellets. Acute platelet depletion in an ITP mouse model (n=20) led to a &amp;gt;2-fold increase in cfDNA extracted from platelet-poor plasma, suggesting a role for platelets in plasma DNA clearance. Platelet uptake in vitro was rapid, visible using live cell microscopy within 2 minutes of co-culture with cancer cells labelled with a probe that irreversibly intercalates to nuclear DNA. ddPCR of platelet DNA following co-culture of healthy donor platelets with cancer cell lines detected a range of canonical cancer driver mutations, including in PI3K, BRAF and JAK2. DNase treatment of co-cultured platelets did not reduce mutant allele content, indicating that platelets encapsulate DNA and protect it from degradation. We explored the mechanism of platelet DNA uptake. Platelets internalized DNA contained in extracellular vesicles (EVs). Platelet uptake of “free” DNA was also confirmed, with uptake of synthetic DNA fragments of varying lengths (120 bp to 650 bp). Importantly, platelet DNA detection was enhanced following exocytosis inhibition, confirming that platelet DNA uptake and release is an active process. Whole genome sequencing revealed that platelets contain a repertoire of DNA fragments that map across the nuclear genome, similar to cfDNA, and that the majority of platelet DNA is of nuclear not mitochondrial origin. We also found tumor-derived DNA showing multiple copy number aberrations in a patient with pancreatic carcinoma. In a transgenic mouse model of colorectal cancer, mutant KRASG12D alleles were readily detectable in platelet DNA and, notably, in higher abundance in platelets than platelet-poor plasma (cfDNA) in 11/16 mice (Fig. 1b). Similarly, KRASG12D was detected in platelets of mice with orthotopic pancreatic adenocarcinoma (n=4). Finally, to explore utility in cancer screening, we analysed patients with high-risk, pre-malignant colonic lesions (serrated polyps). Remarkably, the driver mutation BRAFV600E was detected in platelets in 16% patients despite the small-size lesions (5/30, Fig.1b). Conclusions This study establishes a role for platelets in sequestration of cfDNA, an aspect of platelet biology that has not previously been highlighted and is of substantial clinical relevance. Their abundance, ease of isolation, and continuous tissue perfusion make platelets ideal ‘sentinels’ for genetic perturbations including in early stage/pre-malignant disease.

Cell-Free Fetal DNA Screening Analysis in Korean Pregnant Women: Six Years of Experience and a Retrospective Study of 9327 Patients Analyzed from 2017 to 2022.

Cell-free DNA (cfDNA) screening for normal fetal aneuploidy has been widely adopted worldwide due to its convenience, non-invasiveness, and high positive predictive rate. We retrospectively evaluated 9327 Korean women with single pregnancies who underwent a non-invasive prenatal test (NIPT) to investigate how various factors such as maternal weight, age, and the method of conception affect the fetal fraction (FF). The average FF was 9.15 ± 3.31%, which decreased significantly as the maternal body mass index (BMI) increased (p < 0.001). The highly obese group showed a 'no-call' rate of 8.01%, which is higher than that of the normal weight group (0.33%). The FF was 8.74 ± 3.20% when mothers were in their 40s, and lower than that when in their 30s (9.23 ± 3.34, p < 0.001) and in the natural pregnancy group (9.31% ± 3.33). The FF of male fetuses was observed to be approximately 2.76% higher on average than that of female fetuses. As the gestational age increased, there was no significant increase in the fraction of fetuses up to 21 weeks compared to that at 10-12 weeks, and a significant increase was observed in the case of 21 weeks or more. The FFs in the NIPT high-risk result group compared to that in the low-risk group were not significantly different (p = 0.62). In conclusion, BMI was the factor most associated with the fetal fraction. Although the NIPT is a highly prevalent method in prenatal analysis, factors affecting the fetal fraction should be thoroughly analyzed to obtain more accurate results.

EP15.04: The association of fetal growth and DNA methylation in placental serotonin system in gestational diabetes mellitus

EP15.04: The association of fetal growth and DNA methylation in placental serotonin system in gestational diabetes mellitus

Lung Hypoplasia in Fetuses with Skeletal Dysplasia Determined by Fetal Lung Weight: Which Ultrasound Measurement/Ratio Has the Highest Detection Rate

Introduction: To determine lung hypoplasia in cases with fetal skeletal dysplasia based on the total lung weight at autopsy as the most accountable surrogate marker for pulmonary hypoplasia. Methods: This retrospective cohort study included all pregnancies with antenatal diagnosis of skeletal dysplasia (2012–2018). We included only cases in which information on fetal biometry was available within 2 weeks before delivery and had autopsy and skeletal X-rays + molecular analysis using extracted fetal DNA. We compared the predictive accuracy of fetal sonographic body-proportional ratios (BPRs) including: (1) thoracic circumference-to-abdominal circumference ratio, (2) the femur length-to-abdominal circumference (FL/AC) ratio, (3) head circumference-to-abdominal circumference ratio, and (4) foot length-to-femur length ratio. Lung hypoplasia was defined as total lung weight below −2 SD from the expected mean for gestational age. Results: Fifty three pregnancies with antenatal diagnosis of skeletal dysplasia underwent autopsy included. Lung hypoplasia was determined in 34 (64.1%). Median of gestational age at last sonographic assessment was 21.3 (19.9–24.9) weeks. FL/AC ratio demonstrated the highest area under the curve of 0.817 (95% CI: 0.685–0.949; p < 0.0001). FL/AC ≤0.1550 demonstrated the highest detection rate of 88.2% along with the highest negative predictive value of 75%. Conclusion: Using a novel, more practical approach to predict lung hypoplasia in skeletal dysplasia, fetal sonographic BPRs and, specifically, FL/AC ratio demonstrate a high detection rate of lung hypoplasia.

A-339 Utomated Flow of a Non-invasive Fetal Gender Determination Lab-developed Molecular Test

Abstract Background The discovery of cell-free fetal DNA in maternal circulation during pregnancy fostered the introduction of non-invasive tests. Fetal gender determination may be determined at early pregnancy by real-time PCR, identifying the presence or absence of Y chromosome markers in maternal plasma. Dasa has been offering this test for more than 10 years. In 2022, the assay was adapted to an automated flow. Methods The Flow Solution platform, powered by Roche®, was adapted to the in-house test, allowing the processing of 94 samples. Whole blood is obtained from pregnant women from the 8th week of pregnancy onwards. Each maternal plasma sample is extracted once and the DNA eluate submitted to real-time amplification targeting the DYS14 gene from the Y chromosome, in triplicate. The human RNAseP gene is co-amplified as an internal control. Full process takes 5 hours, with the possibility of overlapping a new routine every 2 hours, with a daily capacity of 12 routines (1128 samples/day). Results In 2022, 129 849 fetal gender determination results were released. Of these, 49% of the results indicated male and 51% female fetuses.A few samples displayed indeterminate results, showing amplification curves but with cycle threshold values that are above the cutoff. Other samples do not reach internal quality metrics and the analysis is repeated from the plasma or a new collection is requested. Results that, after the second collection, remain indeterminate, are forwarded with the patient's clinical history to the medical board. In contact with the patient, possible causes for inconclusive results are explained. In 2022, for every 10 000 tests there were 8 were medical contacts.The automated methodology developed and implemented, allowed a high processing volume, with 129 849 tests released in 2022, an increase of 33% compared to 2021 when the method had manual steps. The Total Service Time (TST) from collection to results release in 2022 was approximately 5 days, a 20% reduction compared to 2021, and the repetition rate of 2.43% in 2022 was 0.9% lower than 2021. Recollections also diminished from 1% in 2021 falling to 0.8% in 2022. Conclusion A new automated flow was successfully implemented in 2022 improving the productivity and quality of the service, achieving a high accuracy and leading to a significant reduction in errors and repetitions while reducing the turnaround time and recollections.

Clinical interpretation of cell-based non-invasive prenatal testing for monogenic disorders including repeat expansion disorders: potentials and pitfalls.

Introduction: Circulating fetal cells isolated from maternal blood can be used for prenatal testing, representing a safe alternative to invasive testing. The present study investigated the potential of cell-based noninvasive prenatal testing (NIPT) for diagnosing monogenic disorders dependent on the mode of inheritance. Methods: Maternal blood samples were collected from women opting for prenatal diagnostics for specific monogenic disorders (N = 7). Fetal trophoblasts were enriched and stained using magnetic activated cell sorting and isolated by fluorescens activated single-cell sorting. Individual cells were subject to whole genome amplification, and cells of fetal origin were identified by DNA-profiling using short tandem repeat markers. The amplified fetal DNA was input for genetic testing for autosomal dominant-, autosomal recessive-, X-linked and repeat expansion disorders by direct variant analysis and haplotyping. The cell-based NIPT results were compared with those of invasive testing. Results: In two cases at risk of skeletal dysplasia, caused by variants in the FGFR3 gene (autosomal dominant disorders), cell-based NIPT correctly stated an affected fetus, but allelic dropout of the normal alleles were observed in both cases. Cell-based NIPT gave an accurate result in two cases at risk of autosomal recessive disorders, where the parents carried either different diastrophic dysplasia causing variants in the SLC26A2 gene or the same cystic fibrosis disease-causing variant in the CFTR gene. Cell-based NIPT accurately identified an affected male fetus in a pregnancy at risk of Duchenne muscular dystrophy (DMD gene, X-linked recessive disorders). In two cases at risk of the myotonic dystrophy type 1 (DMPK gene, repeat expansion disorder), cell-based NIPT correctly detected an affected and an unaffected fetus, respectively. Discussion: Circulating fetal cells can be used to detect both maternally- and paternally inherited monogenic disorders irrespective of the type of variant, however, the risk of allelic dropout must be considered. We conclude that the clinical interpretation of the cell-based NIPT result thus varies depending on the disorders' mode of inheritance.

Cell-free fetal DNA (cffDNA) extraction from whole blood by using a fully automatic centrifugal microfluidic device based on displacement of magnetic silica beads

The purpose of this research was to design a fully automated centrifugal microfluidic system (Lab-on-a-Disk) for isolating cell free fetal DNAs (cffDNAs) from whole blood. To achieve this goal, magnetic silica beads were used, such that after attaching cffDNA to them, they were transferred between chambers by using external fixed magnets. All the standards and required steps for cffDNA extraction including plasma separation, adding proteinase K, lysis buffer, binding buffer, washing buffer, and elution buffer were considered in this designed disk. To evaluate the function of the disk, the collected samples were tested from several aspects. First, the purity of extracted plasma from whole blood was investigated which included hemoglobin test, hemocytometer, etc. This disk could extract 1.3 mL pure plasma from 3 mL blood with 45% hematocrit. The results of the extracted plasma showed 99% purity. Finally, the cffDNAs were examined by using a male fetal gender identification kit and real-time PCR machine. The results indicated the correct function of the disk in extracting cffDNAs in samples of 10 Landa from cycle 34 onwards. Compared to the clinical method, the disk not only was able to extract cffDNA in 20 min but also it led to less buffer consumption since the disk only required 1 mL plasma for extraction of cffDNAs.

Prenatal vs postnatal diagnosis of 22q11.2 deletion syndrome: cardiac and noncardiac outcomes through 1 year of age

The 22q11.2 deletion syndrome is the most common microdeletion syndrome and is frequently associated with congenital heart disease. Prenatal diagnosis of 22q11.2 deletion syndrome is increasingly offered. It is unknown whether there is a clinical benefit to prenatal detection as compared with postnatal diagnosis. This study aimed to determine differences in perinatal and infant outcomes between patients with prenatal and postnatal diagnosis of 22q11.2 deletion syndrome. This was a retrospective cohort study across multiple international centers (30 sites, 4 continents) from 2006 to 2019. Participants were fetuses, neonates, or infants with a genetic diagnosis of 22q11.2 deletion syndrome by 1 year of age with or without congenital heart disease; those with prenatal diagnosis or suspicion (suggestive ultrasound findings and/or high-risk cell-free fetal DNA screen for 22q11.2 deletion syndrome with postnatal confirmation) were compared with those with postnatal diagnosis. Perinatal management, cardiac and noncardiac morbidity, and mortality by 1 year were assessed. Outcomes were adjusted for presence of critical congenital heart disease, gestational age at birth, and site. A total of 625 fetuses, neonates, or infants with 22q11.2 deletion syndrome (53.4% male) were included: 259 fetuses were prenatally diagnosed (156 [60.2%] were live-born) and 122 neonates were prenatally suspected with postnatal confirmation, whereas 244 infants were postnatally diagnosed. In the live-born cohort (n=522), 1-year mortality was 5.9%, which did not differ between groups but differed by the presence of critical congenital heart disease (hazard ratio, 4.18; 95% confidence interval, 1.56-11.18; P<.001) and gestational age at birth (hazard ratio, 0.78 per week; 95% confidence interval, 0.69-0.89; P<.001). Adjusting for critical congenital heart disease and gestational age at birth, the prenatal cohort was less likely to deliver at a local community hospital (5.1% vs 38.2%; odds ratio, 0.11; 95% confidence interval, 0.06-0.23; P<.001), experience neonatal cardiac decompensation (1.3% vs 5.0%; odds ratio, 0.11; 95% confidence interval, 0.03-0.49; P=.004), or have failure to thrive by 1 year (43.4% vs 50.3%; odds ratio, 0.58; 95% confidence interval, 0.36-0.91; P=.019). Prenatal detection of 22q11.2 deletion syndrome was associated with improved delivery management and less cardiac and noncardiac morbidity, but not mortality, compared with postnatal detection.

Preliminary study of noninvasive prenatal screening for 22q11.2 deletion/duplication syndrome using multiplex dPCR assay

ObjectiveThis study aimed to establish a cell-free fetal DNA (cffDNA) assay using multiplex digital PCR (dPCR) for identifying fetuses at increased risk of 22q11.2 deletion/duplication syndrome.MethodsSix detection sites and their corresponding probes were designed for the 22q11.2 recurrent region. A dPCR assay for the noninvasive screening of 22q11.2 deletion/duplication syndrome was established. A total of 130 plasma samples from pregnant women (including 15 samples with fetal 22q11.2 deletion/duplication syndrome) were blindly tested for evaluating the sensitivity and specificity of the established assay.ResultsDNA with different sizes of 22q11.2 deletion/duplication was detected via dPCR, indicating that the designed probes and detection sites were reasonable and effective. In the retrospective clinical samples, 11 out of 15 samples of pregnant women with 22q11.2 deletion/duplication were detected during the cffDNA assay, and accurate regional localization was achieved. Among the 115 normal samples, 111 were confirmed to be normal. Receiver operating characteristic curves were used for assessing the cut-off values and AUC for these samples. The sensitivity, specificity, and positive as well as negative predictive values were 73.3%, 96.5%, 73.3%, and 96.5%, respectively.ConclusionThe cffDNA assay based on dPCR technology for the noninvasive detection of 22q11.2 recurrent copy number variants in fetuses detected most affected cases, including smaller but relatively common nested deletions, with a low false-positive rate. It is a potential, efficient and simple method for the noninvasive screening of 22q11.2 deletion/duplication syndrome.

Invasive prenatal diagnosis in the era of cell-free fetal DNA: experience at a single center.

We aimed to assess procedure-related risk of fetal loss associated with amniocentesis and chorionic villus sampling and compare amniocentesis and chorionic villus sampling with cell-free fetal DNA in identifying chromosomal abnormalities. A retrospective observational study on 4712 women with singleton pregnancy who underwent invasive prenatal diagnosis, from January 2010 to December 2019. Postprocedural miscarriage rate (before 24+0 weeks gestation) was determined for the whole population and for the group of women aged ≥35 years who underwent the procedure for the sole maternal age. Miscarriage rate following amniocentesis and chorionic villus sampling were 0.50% and 1.25%, respectively. In our population of women undergoing invasive procedure for advanced maternal age cell-free fetal DNA would have identified only the 49 cases of trisomy 21, 13 and 18, whereas the other 21 more subtle chromosomal anomalies, diagnosed by amniocentesis and chorionic villus sampling, would have been missed. Patients who opt for cell-free fetal DNA test should be informed of the screening nature of the test and the possibility of false positive results. Invasive prenatal testing has probably lower risks than previously reported and has unquestionable advantages such as the certainty of diagnosis and the ability to detect a higher number of chromosomal abnormalities, when compared with cell-free fetal DNA.

The effectiveness of non-invasive prenatal test technology and the prenatal screening algorithm based on various methods for determining foetal aneuploidy.

The purpose was to evaluate the effectiveness of a non-invasive prenatal test (NIPT) using mass parallel sequencing (MPS) to detect trisomy 13, 18, 21 and fetal sex chromosome abnormalities in maternal blood samples by isolating freely circulating foetal extracellular DNA (eDNA), and to develop an algorithm for prenatal screening. The research methods used included blood sampling from patients, isolation of eDNA, determination of DNA concentration and quality, library preparation for sequencing, MPS using an Illumina HiSeq2000, positive and negative control samples, monitoring, and analysis of results using the distributed algorithms platform based on calculations of z-value and the average absolute deviation. Pregnant women were divided into two groups based on gestational age at sampling, group 1; 9-14 weeks and group 2; 15-27 weeks. A total of 377 pregnant women were included with a mean (range) age of 33 (23-44) years. The mean gestational age at the time of blood sampling in group 1 was 11 (9-14) weeks, and in group 2 was 21 (15-27) weeks. In the first group, three cases of trisomy 18 chromosomes were detected in patients aged 43 years old, and female children were subsequently born with Edwards syndrome. In the second group, one case of trisomy 21 was detected in a patient aged 36 years and the pregnancy was terminated at 25 weeks. The analysis of freely circulating foetal eDNA was a sensitive method for detecting chromosomal abnormalities. The study has a practical significance, since the NIPT for frequent aneuploidy considerably exceeds the effectiveness of traditional screening methods and allows identifying chromosomal disorders starting from the 9th week of the gestation period.

Second trimester cell-free placental DNA methylation profiles in preterm birth subtypes

Second trimester cell-free placental DNA methylation profiles in preterm birth subtypes

Non-Invasive Biomarker Detection for Pregnancy Complications Using Cell-Free RNA

Pregnancy complications pose a significant threat to maternal health as they may result in a higher risk for issues during pregnancy or labor relative to the risk for these issues in a typical pregnancy. Many cases of maternal deaths and complicated pregnancies can be avoided with a richer understanding of maternal health early on in pregnancy. Genetic analysis of fetal DNA in maternal blood is becoming increasingly common1, and while genetic screening efforts have progressed substantially in recent years, they have focused on fetal health rather than the health of the mother2. This work focuses on the detection of common complications of pregnancy including preeclampsia, gestational diabetes, and chronic hypertension using non-invasive circulating cell-free RNA data. We developed interpretable supervised machine learning methods that had high performance in identifying pregnancy complications from healthy pregnancies (AUC = 0.86). Using our models, we found various relevant transcripts, related to pregnancy biology. These included S100A9, which encodes for a protein involved in inflammation and was elevated in complicated pregnancies, as well as two small RNAs involved in cell proliferation and body mass, RNY4 and RNY3, which were reduced in preeclampsia and GDM and have previous roles in pregnancy. Our findings highlight several promising non-invasive biomarkers for the early diagnosis of complications of pregnancy that have the potential to be easily integrated into existing clinical workflows.

Associations between nutrients in one-carbon metabolism and fetal DNA methylation in pregnancies with or without gestational diabetes mellitus

BackgroundGestational diabetes mellitus (GDM), characterized by hyperglycemia that develops during pregnancy, increases the risk of fetal macrosomia, childhood obesity and cardiometabolic disorders later in life. This process has been attributed partly to DNA methylation modifications in growth and stress-related pathways. Nutrients involved with one-carbon metabolism (OCM), such as folate, choline, betaine, and vitamin B12, provide methyl groups for DNA methylation of these pathways. Therefore, this study aimed to determine whether maternal OCM nutrient intakes and levels modified fetal DNA methylation and in turn altered fetal growth patterns in pregnancies with and without GDM.ResultsIn this prospective study at a single academic institution from September 2016 to June 2019, we recruited 76 pregnant women with and without GDM at 25–33 weeks gestational age and assessed their OCM nutrient intake by diet recalls and measured maternal blood OCM nutrient levels. We also collected placenta and cord blood samples at delivery to examine fetal tissue DNA methylation of the genes that modify fetal growth and stress response such as insulin-like growth factor 2 (IGF2) and corticotropin-releasing hormone (CRH). We analyzed the association between maternal OCM nutrients and fetal DNA methylation using a generalized linear mixed model. Our results demonstrated that maternal choline intake was positively correlated with cord blood CRH methylation levels in both GDM and non-GDM pregnancies (r = 0.13, p = 0.007). Further, the downstream stress hormone cortisol regulated by CRH was inversely associated with maternal choline intake (r = − 0.36, p = 0.021). Higher maternal betaine intake and serum folate levels were associated with lower cord blood and placental IGF2 DNA methylation (r = − 0.13, p = 0.049 and r = − 0.065, p = 0.034, respectively) in both GDM and non-GDM pregnancies. Further, there was an inverse association between maternal betaine intake and birthweight of infants (r = − 0.28, p = 0.015).ConclusionsIn conclusion, we observed a complex interrelationship between maternal OCM nutrients and fetal DNA methylation levels regardless of GDM status, which may, epigenetically, program molecular pathways related to fetal growth and stress response.