Cell-free DNA Screening Research Articles

Clinical Validation of a Prenatal Cell-Free DNA Screening Test for Fetal RHD in a Large U.S. Cohort.

To present a large U.S. clinical validation of a next-generation sequencing-based, noninvasive prenatal cell-free DNA test for fetal RHD. This clinical validation study assessed the performance of a commercially available, next-generation sequencing-based cell-free DNA test for fetal RHD status. Samples that passed quality metrics were included if the patient had a previously reported cell-free DNA result for fetal aneuploidy, maternal RhD-negative serology, newborn RhD serology, and maternal RHD deletion or RHD-CE-D hybrid(r's) genotype. Dizygotic twin pregnancies were excluded. Maternal and fetal RHD genotypes were evaluated with prospective cell-free DNA next-generation sequencing analysis. At the time of analysis, investigators were blinded to fetal RhD status. The cohort consisted of 655 pregnant patients with serologic results for RhD antigen. Patient demographics included a representative distribution of race and ethnicities in the RhD-negative U.S. population (74.0% White, 13.7% Hispanic, 7.0% Black, and 2.1% Asian). Cell-free DNA fetal RHD was not reported in two cases. There were zero false-negative cases; 356 of 356 fetuses were correctly identified as fetal RhD positive (sensitivity 100%, 95% CI, 98.9-100%). Of the 297 RhD-negative fetuses, 295 were correctly identified as RhD negative (specificity 99.3%, 95% CI, 97.6-99.8%). Of the fetuses with a negative RhD phenotype, the cell-free DNA test accurately identified three with the fetal RHD pseudogene (RHDΨ) genotype. Validation of this test in this large U.S. cohort of RhD-negative patients provides data on early and accurate noninvasive prenatal identification of fetal RHD genotype at 9 weeks of gestation or more. This test has the potential to assist patients and clinicians in the prevention and management of RhD alloimmunization.

Genomic Reporting Practices Across 5 Molecular Disciplines: A Study From the College of American Pathologists.

Genomic reports are primarily organized in a narrative and unstructured format with variations in content and format. Regulatory requirements and professional guidelines for genetic test reporting exist but provide little guidance for effective communication of information. To assess clinical genomic reporting practices across 5 disciplines within molecular diagnostics, including germline, somatic solid tumors, somatic hematologic malignancies, pharmacogenomics, and prenatal cell-free DNA screening. Reporting practices were assessed by using a structured review of clinical genomic reports from multiple laboratories in 5 molecular disciplines spanning different practice settings. Report content was reviewed by the presence/absence of from 27 to 44 elements, including 23 elements required by the College of American Pathologists and/or the Clinical Laboratory Improvement Amendments of 1988 (CLIA). If present, the element's location on the report was recorded. A total of 69 genomics reports from 31 laboratories were reviewed. Overall, the reports were compliant with regulatory requirements but showed variability in both format and content. Six of 7 required reporting elements (per CLIA, 42 CFR [Code of Federal Regulations] 493.1291) were included in 90% of the reports. However, these elements were often located in different report sections. Only patient demographics were always found in a specific report section (header). These results show that reports are overall compliant with regulatory requirements, despite some reporting elements being less consistently reported. The lack of consistent presentation of the data elements presents an opportunity to improve the communication of molecular testing results to clinicians and patients.

Fetal fraction amplification within prenatal cfDNA screening enables detection of genome-wide copy-number variants at enhanced resolution.

Clinically significant copy-number variants (CNVs) occur in 1% to 2% of pregnancies and are difficult to detect via prenatal cell-free DNA (cfDNA) screening because of the low fraction of fetal-derived cfDNA in maternal plasma. Here, we use fetal fraction amplification (FFA) and improved computational algorithms to enhance the resolution and sensitivity of CNV detection. We implemented and characterized the performance of a hidden Markov model that identifies fetal CNVs. This CNV caller was analytically validated on 117 FFA samples, including 57 fetal-CNV-containing samples, and applied retrospectively to a cohort of more than 300k patient samples. Our assay was concordant with orthogonal testing and detected fetal CNVs ≥5 Mb with estimated aggregate sensitivity and specificity of >95.1% and >99.7%, respectively. The resolution of CNV detection was fetal fraction dependent, but 97.2% of samples reached ≥5-Mb resolution. Overall, CNVs ≥5 Mb were found in 1 in 500 pregnancies. FFA improves the sensitivity and resolution of CNV detection in prenatal cfDNA screening, allowing accurate detection of fetal CNVs as small as 1 Mb. Using our approach, we found that clinically significant fetal CNVs were detected more frequently than the common trisomies 13 and 18 that are recommended as part of guideline-based screening.

Curation and reporting of pathogenic genome-wide copy-number variants in a prenatal cell-free DNA screen.

Advances in fetal fraction amplification in prenatal cell-free DNA screening now allow for high-resolution detection of copy-number variants (CNVs). However, approaches to interpreting CNVs as part of a primary screen are still evolving and require consensus. Here, we present a conservative, patient-centered framework for reporting fetal CNVs. Syndromes described in the literature were evaluated for inclusion based on a definable minimal critical region, disease severity, penetrance, and age of onset. The reporting framework required that a CNV overlap a defined minimal critical region and/or that it be ≥5 Mb and contain at least 1 OMIM disease-associated gene. This framework was then applied to CNVs identified from a cohort of 313,544 prenatal cfDNA screening patient samples. Patient-friendly terminology describing syndrome phenotypes was developed by scientists with training in genetic counseling. 65 syndromes met criteria for inclusion and represented the second most common class of CNVs in a retrospective cohort, more so than an established panel of microdeletions (1p36, 4p, 5p, 15q11.2-q13, and 22q11.2). Frequencies were concordant with reported syndrome incidence rates. The most common CNVs were those ≥5 Mb encompassing an OMIM disease gene(s). This framework for genome-wide fetal-CNV reporting carefully prioritizes findings with the potential to affect reproductive decision making.

'I Could Trust It': Experiences of Reciprocal Translocation Carriers and Their Partners With Prenatal Cell-Free DNA Screening for Unbalanced Translocations.

To explore the experiences of people having cfDNA screening to detect unbalanced translocations, and to understand motivations for choosing this option. We used a qualitative approach with in-depth semi-structured interviews with reciprocal translocation carriers and their partners. People who underwent cfDNA screening with translocation analysis through Victorian Clinical Genetics Services between 2015 and 2019 were invited to take part. Purposive sampling based on the participant's geographic location, requesting practitioner specialty and cfDNA screening result was used to capture a range of experiences. Interview transcripts were analysed using thematic analysis. Participants (n=13) had complex reproductive journeys associated with the translocation and opted for cfDNA screening rather than prenatal diagnosis to avoid risk to their pregnancy. Participants benefited from having a result early in pregnancy and had sufficient confidence in the result to decline a diagnostic testing procedure. Participants' experiences with cfDNA screening were intertwined with the experience of being a carrier of a reciprocal translocation. cfDNA screening with translocation analysis was perceived as an acceptable alternative to prenatal diagnosis and should be made more accessible to balanced translocation carriers. Access to specialist genetic counselling services is needed to ensure couples are provided with information about all prenatal testing options, including the benefits and limitations associated with cfDNA screening with translocation analysis.

The first 2-year prospective audit of prenatal cell-free deoxyribonucleic screening using single nucleotide polymorphisms approach in a single academic laboratory.

We reported a performance during an implementation of prenatal cell-free (cf) DNA screening using single nucleotide polymorphism (SNP) approach in our accredited laboratory. Prospective audit with prompt intervention was set for the processing of 2,502 samples from singleton pregnancy from August 2017 to July 2019. Risks of trisomy 21 (T21), T18, T13, monosomy X (XO), and other sex chromosome aneuploidies (SCAs) were clarified by a proprietary bioinformatics algorithm. Laboratory failure occurred in 192 samples (7.7 %) as a result of inadequate sequencing (n=144), fundamental limitation of the testing (n=19), and obvious human error (n=29). Faulty setting of the calibration curve was the most common human error (n=22/29). After a redraw (n=110), 79 (71.8 %) were settled. Overall, 2,389/2,502 samples (95.5 %) were reportable. Thirty-five samples were high-risk for T21 (n=19), T18 (n=5), T13 (n=1), XO (n=3), and other SCAs (n=7), respectively. Positive predictive values calculated from either prenatal confirmatory tests or postnatal findings were 93.8 % (n=16), 100 % (n=4), 50 % (n=2), and 83.3 % (n=6) for T21, T18, XO, and other SCAs, respectively, with high sensitivity and specificity (>99.9 %). Vanishing twin was detected from 1 out of 4 samples with detected additional haplotypes. An overall performance of SNP-based prenatal cf-DNA screening during our initial implementation can be optimized with proactive approach. The technical know-how was a significant limiting factor for adopting the technology. The lessons learnt may be of interest to the academic laboratory considering adopting their own test instead of sending blood to a testing service of a supplier.

Recurrent Xp22.31-Yq11 Unbalanced Translocations: Molecular Diagnosis and Clinical Implications in Three Families.

Unbalanced translocation between chromosomes X and Y is a recurring chromosomal rearrangement. The presence of a derivative chromosome X (derX), where a Yq11-qter segment is attached to the short arm of chromosome X, replacing a terminal Xpter-p22.31, poses challenges for interpretation of findings by prenatal cell-free DNA (cfDNA) screening, establishing genotype-phenotype correlation in male and female individuals, and for genetic counseling. In this report, we provide clinical outcomes, inheritance, and clinical implications of derX in three families referred to diagnostic testing due to discrepant results for sex chromosomes reported by cfDNA, abnormal prenatal ultrasound findings, recurrent pregnancy losses, or affected family members with derX transmitted through multiple generations. Reports of discrepant sex and risk for sex chromosome aneuploidy such as 45,X, 47,XXY and 47,XYY are common false positive outcomes of a prenatal cfDNA screening if either a mother or a fetus has unbalanced Xp-Yq translocation. In addition, mothers who carry der(X) facing a recurrent risk of ambiguity in prenatal testing. Pregnancy loss and neonatal death/stillbirth of male offspring are common in affected families, but this risk does not directly correlate with the size of deleted Xp region. This study emphasizes the importance of CMA and familial testing for accurate diagnosis and genetic counseling.

Antenatal anti-D prophylaxis and D antigen negativity in pregnant women of the UAE: a cross-sectional analysis from the Mutaba’ah Study

ObjectiveThis study aimed to determine the prevalence of the negative D antigen phenotype, adherence to routine antenatal anti-D immunoglobulin prophylaxis (RAADP) administration and D antigen sensitisation among pregnant women in...

Unusual Maternal and Fetal Findings With Cell-Free DNA Screening.

With advances in prenatal cell-free DNA (cfDNA) technology, the information available with cfDNA continues to expand beyond the common fetal aneuploidies such as trisomies 21, 18, and 13. Due to the admixture of maternal and fetal/placental DNA, prenatal cfDNA remains a screening test with the possibility of false-positive and false-negative results. This review aims to summarize unusual incidental maternal and fetal genomic abnormalities detectable by cfDNA and to provide anticipatory guidance regarding management. Of 140 articles identified with keywords such as "incidental" and "discordant" cfDNA, 55 original research articles, review articles, case series, and societal guidelines were reviewed. Prenatal cfDNA may incidentally identify a spectrum of maternal genomic abnormalities such as malignancy, mosaicism, and copy number variants. When discordant with fetal diagnosis, these cases require additional investigation with maternal genetic testing and follow-up evaluation. Such incidental fetal/placental abnormalities may include rare autosomal trisomies, uniparental disomy, and triploidy. Further evaluation of fetal/placental abnormalities can be pursued with a combination of ultrasound and prenatal diagnosis with chorionic villous sampling and/or amniocentesis. Societal guidelines do not currently recommend cfDNA screening for rare autosomal trisomies, microdeletions, or copy number variants, and some experts suggest that sex chromosome screening should be opted in after counseling. Knowledge about possible incidental findings with prenatal cfDNA is needed to inform pretest and posttest counseling with appropriate follow-up evaluation. As cfDNA technology has advanced to include genome-wide findings, it is important for clinicians, genetic counselors, and societal guidelines to acknowledge the spectrum of possible results outside of the traditional and sex chromosome aneuploidies.

Factors affecting couples’ decision making about expanded prenatal cell-free DNA screening

PurposeTo assess intended parents’ preferences about expanded prenatal cell-free DNA screening. MethodsA survey was administered to couples who were pregnant or trying to conceive. Partners within couples were independently asked about willingness to seek prenatal cell-free DNA screening for diseases and traits that varied by severity, treatability, age of onset, and reliability. Additional questions explored couples’ decision-making processes and how various factors would affect those decisions. ResultsRespondents (n = 494) were most likely to seek a prenatal genetic test when the test is completely predictive (P value of predictability effect = .0116), for earlier onset (P value of onset effect = .0310), treatable diseases (P value of treatability effect = .0032), and of highest severity (P value of severity effect < .0001). Living in states where termination was not available was not associated with decreased interest in prenatal screening, with 45% reporting that it would make them more likely to seek testing. There was moderate concordance between couples about their testing decisions and the majority of pregnant persons (68.8%) and partners (66.7%) desired firm screening recommendations from their doctors. ConclusionThese data suggest that couples are highly information seeking when presented with all testing options neutrally, but most desire to be presented with firm testing recommendations from their clinician.

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.

CffDNA screening for Niemann-pick disease, type C1: a case series.

Cell-free fetal DNA (cffDNA) screening is a valuable tool in clinical practice for detecting chromosomal abnormalities and autosomal dominant (AD) conditions. This study introduces a novel proof-of-concept assay designed for autosomal recessive (AR) cffDNA screening, focusing on cases involving the NPC1 gene. We aim to illustrate the significant benefits of AR cffDNA screening in managing high-risk pregnancies, specifically where biallelic pathogenic variants in NPC1 cause Niemann-Pick disease, type C1 (NPC), a disorder marked by progressive neurodegeneration. Three participants for this study were recruited and gave consent to a hospital in Saudi Arabia. These participants were either carriers of NPC or had a first- or second-degree relative affected by the disorder. No specific criteria were set for the age of the participants. All were between 15 and 18 weeks of gestation. Using amplicon-based next-generation sequencing (NGS), we analyzed the zygosity and variants in cffDNA extracted from maternal peripheral blood. After amplicon NGS, analysis was completed by a custom data analysis pipeline that included in-house-built data processing scripts and commonly used software packages. Importantly, the results were not disclosed to the patients. Our findings showed that in all three cases, AR cffDNA screening results were consistent with standard invasive diagnostic testing. This screening method offers several advantages: it provides critical information to families earlier in the pregnancy compared to invasive diagnostic tests, and it helps to alleviate parental anxiety. Moreover, this non-invasive method can determine pregnancy status in the first trimester for known familial variants. Future research may extend this approach to screen for known disease-causing variants in common AR conditions.

Discordant Prenatal Cell-Free DNA Screening vs. Diagnostic Results of Sex Chromosome Aneuploidies: Implications for Newborn Screening and Genetic Counseling.

Sex chromosome aneuploidies (SCAs) collectively occur in 1 in 500 livebirths, and diagnoses in the neonatal period are increasing with advancements in prenatal and early genetic testing. Inevitably, SCA will be identified on either routine prenatal or newborn screening in the near future. Tetrasomy SCAs are rare, manifesting more significant phenotypes compared to trisomies. Prenatal cell-free DNA (cfDNA) screening has been demonstrated to have relatively poor positive predictive values (PPV) in SCAs, directing genetic counseling discussions towards false-positive likelihood rather than thoroughly addressing all possible outcomes and phenotypes, respectively. The eXtraordinarY Babies study is a natural history study of children prenatally identified with SCAs, and it developed a longitudinal data resource and common data elements with the Newborn Screening Translational Research Network (NBSTRN). A review of cfDNA and diagnostic reports from participants identified a higher than anticipated rate of discordance. The aims of this project are to (1) compare our findings to outcomes from a regional clinical cytogenetic laboratory and (2) describe discordant outcomes from both samples. Twenty-one (10%), and seven (8.3%) cases were found to be discordant between cfDNA (result or indication reported to lab) and diagnosis for the Babies Study and regional laboratory, respectively. Discordant results represented six distinct discordance categories when comparing cfDNA to diagnostic results, with the largest groups being Trisomy cfDNA vs. Tetrasomy diagnosis (66.7% of discordance in eXtraordinarY Babies study) and Mosaicism (57.1% in regional laboratory). Traditional genetic counseling for SCA-related cfDNA results is inadequate given a high degree of discordance that jeopardizes the accuracy of the information discussed and informed decision making following prenatal genetic counseling.

Medical Findings in Infants Prenatally Identified with Sex Chromosome Trisomy in Year One of Life.

Sex chromosome trisomies (SCT), including XXY, XYY, and XXX syndromes, have been historically underdiagnosed. Noninvasive prenatal cell-free DNA screening has significantly increased identification of these conditions, leading to a need for pediatric care for a growing population of newborns with SCT. Our goal was to analyze and compare perinatal features, medical diagnoses, and physical features in infants with prenatal identification of SCT conditions through the first year of life. The eXtraordinarY Babies Study is an ongoing, prospective natural history study of prenatally identified children with SCT conducted by interdisciplinary teams in Colorado and Delaware. Participants were enrolled prior to 12 months of age and had pregnancy, birth, medical histories, and physical exams completed by board-certified pediatricians at 2, 6, and/or 12-month visits. Descriptive statistics were followed by comparisons between SCT groups using t-tests or ANOVA, Fisher exact, and correlations between medical features with alpha of 0.05. Relative risks were calculated compared to general population rates. 327 infants were included in the analysis (XXY=195, XXX=79, XYY=53). Major congenital anomalies were rare (1.7%). Relative risk compared to general population was elevated for breastfeeding difficulties (51.7%;RR 2.7), positional torticollis (28.2%;RR 7.2), eczema (48.0%;RR 3.5), food allergies (19.3%;RR 2.4), constipation requiring intervention (33.9%;RR 7.6), small cardiac septal defects (7.7%;RR 17.2), and structural renal abnormalities (4.4%;RR 9.7). Inpatient hospitalization was required for 12.4%, with 59.5% of hospitalizations attributable to respiratory infections. These findings of medical conditions with a higher prevalence can inform anticipatory guidance and medical management for pediatricians caring for infants with SCT. Medical findings in largest cohort of prenatally identified infants with XXY, Trisomy X, and XYY from birth to 12 months and implications for pediatric care.What's Known on This Subject: One in ∼500 individuals have an extra X or Y chromosome, or sex chromosome trisomy (SCT). Prenatal screening is now routinely identifying SCT, however there are few studies to guide perinatal and infant care for these individuals.What This Study Adds: This prospective observational study presents medical features for 327 infants with prenatally identified SCT from birth through the first year of life. Results identify where proactive screenings and/or interventions may be warranted for infants with SCT.

Prenatal cell-free DNA screening for chromosomal aneuploidies after euploid embryo transfer shows high concordance with preimplantation genetic testing for aneuploidy results and low positive predictive values

To evaluate the positive predictive value (PPV) of prenatal cell-free DNA screening for chromosomal aneuploidies in pregnancies achieved either after single euploid transfer in IVF/PGT cycles or transfer of single untested embryo, and to assess the concordance of prenatal-cfDNA-screening and PGT-A results. Single centre retrospective cohort study SUBJECTS: 2973 prenatal-cfDNA-screening results for the most common trisomies(T)(T13,T18,T21,X,Y) and microdeletions(1p36;4p16.3;5p15.2;15q11.2;22q11.2) from singleton pregnancies allocated into 2 groups: PGT-A group (n=1204) pregnancy after single euploid transfer and non-PGT-A group (n=1769) pregnancy after transfer of single untested embryo, between 2016 and 2023. Primary outcome measure was accuracy of prenatal-cell-free-DNA-screening. Positive and negative prenatal-cell-free-DNA-screening results, and subsequent prenatal or postnatal diagnostic testing were used to classify each positive prenatal-cell-free-DNA-screening result as a true or a false positive. Secondary endpoints were to evaluate the concordance of PGT-A and prenatal-cell-free-DNA-screening results and to assess the differences of the fetal fraction of cell-free-DNA used for prenatal-cell-free-DNA-screening report between the study groups. Prenatal-cell-free-DNA-screening was performed at mean 11.3±1.8weeks gestational age (GA) and yielded results in 99.9% of the patients (0.1% cancellation rate). There was no difference in the fetal fraction between PGT-A tested and not tested pregnancies (9.5%±4% vs 10.3%±4%). 13 positive prenatal-cell-free-DNA-screening results (2-T21,2-X0,4-XXX,1-XYY, 1-indeterminate sex, 2-22q11 del/dup, 1-15q11.2) were received for PGT-A group. Only one (22q11 dup) was confirmed with amniocentesis and fetal autopsy, giving a PPV for an abnormal prenatal-cfDNA-screening of 7.7%, the rest had results concordant with PGT-A. Sex chromosomes were 100% concordant between prenatal-cell-free-DNA-screening and PGT-A results, giving a 100% PPV for PGT-A for sex chromosomes and 100% NPV for aneuploidies. Positive prenatal-cell-free-DNA-screening results were received for 27 pregnancies from untested embryos (1.5%), follow up testing was electively performed for 21, and 8 had confirmed the prenatal-cell-free-DNA-screening result, giving a PPV for the non-PGT-A group of 38%. This study demonstrates that patients undergoing IVF/PGT and single euploid embryo transfer can reliably do prenatal-cell-free-DNA-screening during their first trimester. Fetal fraction in singleton pregnancies after PGT-A tested embryos is not different from pregnancies with untested embryos. PPV for an abnormal prenatal-cell-free-DNA-screening result after euploid embryo transfer was reassuringly low (7.7%). PGT-A reliably selects against aneuploidy with 100% concordance with fetal sex.

Amniocentesis in pregnancies at or beyond 24 weeks: an international multicenter study

BackgroundAmniocentesis for genetic diagnosis is most commonly done between 15 and 22 weeks of gestation, but can be performed at later gestational ages. The safety and genetic diagnostic accuracy of amniocentesis have been well-established through numerous large-scale, multicenter studies for procedures before 24 weeks, but comprehensive data on late amniocentesis remain sparse. ObjectivesTo evaluate the indications, diagnostic yield, safety, and maternal and fetal outcomes associated with amniocentesis performed at or beyond 24 weeks of gestation. Study DesignWe conducted an international, multicenter retrospective cohort study examining pregnant individuals who underwent amniocentesis for prenatal diagnostic testing at gestational ages between 24w0d and 36w6d. The study, spanning from 2011 to 2022, involved nine referral centers. We included singleton or twin pregnancies with documented outcomes, excluding cases where other invasive procedures were performed during pregnancy or if amniocentesis was conducted for obstetric indications. We analyzed indications for late amniocentesis, types of genetic tests performed, their results, and the diagnostic yield, along with pregnancy outcomes and post-procedure complications. ResultsOf the 752 pregnant individuals included in our study, late amniocentesis was primarily performed for the prenatal diagnosis of structural anomalies (91.6%), followed by suspected fetal infection (2.3%) and high-risk findings from cell-free DNA screening (1.9%). The median gestational age at the time of the procedure was 28w5d, and 98.3% of pregnant individuals received results of genetic testing before birth or pregnancy termination. The diagnostic yield was 22.9%, and a diagnosis was made 2.4 times more often for fetuses with anomalies in multiple organ systems (36.4%) compared to those with anomalies in a single organ system (15.3%). Additionally, the diagnostic yield varied depending on the specific organ system involved, with the highest yield for musculoskeletal anomalies (36.7%) and hydrops fetalis (36.4%) when a single organ system or entity was affected. The most prevalent genetic diagnoses were aneuploidies (46.8%), followed by copy number variants (26.3%) and monogenic disorders (22.2%). The median gestational age at delivery was 38w3d, with an average of 59 days between the procedure and delivery date. The overall complication rate within two weeks post-procedure was 1.2%. We found no significant difference in the rate of preterm delivery between pregnant individuals undergoing amniocentesis between 24-28 weeks and those between 28-32 weeks, reinforcing the procedure's safety across these gestational periods. ConclusionsLate amniocentesis, at or after 24 weeks gestation, especially for pregnancies complicated by multiple congenital anomalies, has a high diagnostic yield and a low complication rate, underscoring its clinical utility. It provides pregnant individuals and their providers with a comprehensive diagnostic evaluation and results before delivery, enabling informed counseling and optimized perinatal and neonatal care planning.

Cell-free DNA screening for common autosomal trisomies using rolling-circle replication in twin pregnancies.

To evaluate the performance of prenatal screening for common autosomal trisomies in twin pregnancies through the use of rolling-circle replication (RCR)-cfDNA as a first-tier test. Prospective multicenter study. Women who underwent prenatal screening for trisomy (T) 21, 18 and 13 between January 2019 and March 2022 in twin pregnancies were included. Patients were included in two centers. The primary endpoint was the rate of no-call results in women who received prenatal screening for common autosomal trisomies by RCR-cfDNA at the first attempt, compared to that in prospectively collected samples from 16,382 singleton pregnancies. The secondary endpoints were the performance indices of the RCR-cfDNA. 862 twin pregnancies underwent screening for T21, T18 and T13 by RCR-cfDNA testing at 10-33weeks' gestation. The RCR-cfDNA tests provided a no-call result from the first sample obtained from the patients in 107 (0.7%) singleton and 17 (2.0%) twin pregnancies. Multivariable regression analysis demonstrated that significant independent predictors of test failure were twin pregnancy and in vitro fertilization conception. All cases of T21 (n=20/862; 2.3%), T18 (n=4/862; 0.5%) and T13 (n=1/862; 0.1%) were correctly detected by RCR-cfDNA (respectively, 20, 4 and 1 cases). Sensitivity was 100% (95% CI, 83.1%-100%), 100% (95% CI 39.8%-100%) and 100% (95% CI 2.5%-100%) for T21, T18 and T13, respectively, in twin pregnancies. The RCR-cfDNA test appears to have good accuracy with a low rate of no-call results in a cohort of twin pregnancies for the detection of the most frequent autosomal trisomies.

Non-invasive prenatal screening: Testing motivations and decision making in the low-risk population.

Non-invasive prenatal screening provides a risk assessment for aneuploidies by utilizing cell-free DNA (cfDNA). It is recommended that cell-free DNA screening (cfDNA screening) be offered to all pregnant people regardless of a priori risk for aneuploidy. In the absence of an increased risk, alternative motives for electing cfDNA screening and different levels of informed decision making may arise. Therefore, our study aimed to characterize low-risk patients' motivations for cfDNA screening election, determine how often informed decisions are being made, and compare motivations between informed and uninformed decision makers. A survey that included a modified, validated measure of informed choice (MMIC) and questions to assess patients' motivations for cfDNA screening was offered at four MFM clinics following genetic counseling. It was found that 44% of participants (n = 100) made an uninformed decision about testing. Participants with private insurers were 4.25 times more likely to make an informed decision (95% CI = 1.10-16.37). Informed decision makers scored avoiding invasive procedures higher (p = 0.007) and ranked doing what family/friends desire lower (p = 0.005) than uninformed decision makers. While most participants scored receiving information about genetic conditions highest, 12% of participants reported fetal sex disclosure as a priority. However, this was not found to be associated with uninformed decision making. This study ultimately established that following genetic counseling, a low-risk population shared motivations with high-risk populations which highlights the importance of complete pre-test counseling for all. Future research should investigate the effect of modifying variables, such as socioeconomic status, on the performance of informed choice measures and critically evaluate the parameters that determine informed choice.

Prenatal Screening and Diagnosis: Time for a Paradigm Shift.

Recent advances in genetics and imaging have ushered substantial breakthroughs in screening and diagnosis for chromosomal and structural abnormalities. Thus, it is imperative that health care providers caring for pregnant individuals should reexamine established practices in prenatal screening and diagnosis. In the past, screening for chromosomal abnormalities was based almost entirely on Down syndrome. Pregnant individuals aged > 35 years were considered at "high risk" or of "advanced maternal age" based on age alone; however, the advent of tests with high sensitivity for prenatal detection of chromosomal abnormalities should lead to abandoning that concept, at least from the perspective of chromosomal abnormalities. Given that first-trimester and second-trimester screenings will fail to detect between 5 and 20% of Down syndrome, in most situations, noninvasive testing with cell-free DNA should be the first-line screen for Down syndrome. The fact that over 99% of fetuses with Down syndrome will be detected prenatally with cell-free DNA gives other fetal chromosomal and structural abnormalities increasing prominence. Chromosomal microarray analysis (CMA) permits prenatal detection of several clinically important chromosomal aberrations that cannot be detected by karyotype and may exist in structurally normal fetuses with low-risk cell-free DNA screening. As such, CMA should be more readily conducted when invasive testing is performed, regardless of the presence of a structural abnormality. Isolated sonographic "soft markers" have no clinical significance in patients who have normal cell-free DNA screening, can cause unwarranted anxiety and a negative impact on pregnancy, and perhaps it is time to stop discussing them. Detailed first-trimester ultrasound allows early detection of several severe fetal anomalies and, therefore, in settings with adequately trained personnel and resources, should be used more frequently. This opinion traces the evolution of prenatal screening and diagnosis and advocates for a paradigm shift that aligns with recent developments in prenatal screening and diagnostic capabilities. KEY POINTS: · Noninvasive prenatal testing with cell-free DNA should be available to all pregnant individuals.. · Chromosomal microarray should be available to all pregnant individuals undergoing amniocentesis.. · Patients >35 years with low-risk screening are not at "high risk" for chromosomal abnormalities..

The impact of confined placental mosaicism on prenatal cell-free DNA screening: Insights from a monocentric study of 99 cases

IntroductionConfined placental mosaicism (CPM) is thought to be one of the main sources of false-positive prenatal cell-free DNA (cfDNA) screening results, but extensive and systematic studies to prove this statement are limited. We evaluate the contribution of CPM to false-positive prenatal cfDNA screening results in the largest cohort published to date. MethodWe systematically offered postnatal analysis on placenta and umbilical cord to women who had a negative amniocentesis following a positive prenatal cfDNA screening result. A standardized protocol was used in which (when available) biopsies were taken at five locations in the placenta and umbilical cord. ResultsWe analyzed a series of 99 placentas. CPM could be confirmed in 32.3 % of cases (32/99). CPM was detected across all subtypes of chromosomal aberrations (common and rare autosomal trisomies, sex chromosome abnormalities, copy number variations and autosomal monosomies). A lower detection rate was present in umbilical cord biopsies in comparison with placental biopsies. When comparing different sections of the placenta, no clear difference could be observed with regard to the probability of CPM being present nor to the grade of mosaicism. DiscussionWe confirm an important role for CPM in explaining false-positive prenatal cfDNA screening results. Placental regional differences are common. Given its limited clinical relevance, we do however not advocate placental studies in a diagnostic setting.