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Preimplantation genetic testing for aneuploidy (PGT-A) with trophectoderm (TE) biopsy is widely applied in in vitro fertilization (IVF) to identify aneuploid embryos. However, potential safety concerns regarding biopsy and restrictions to only those embryos suitable for biopsy pose limitations. In addition, embryo mosaicism gives rise to false positives and false negatives in PGT-A because the inner cell mass (ICM) cells, which give rise to the fetus, are not tested. Here, we report a critical examination of the efficacy of noninvasive preimplantation genetic testing for aneuploidy (niPGT-A) in the spent culture media of human blastocysts by analyzing the cell-free DNA, which reflects ploidy of both the TE and ICM. Fifty-two frozen donated blastocysts with TE biopsy results were thawed; each of their spent culture medium was collected after 24-h culture and analyzed by next-generation sequencing (NGS). niPGT-A and TE-biopsy PGT-A results were compared with the sequencing results of the corresponding embryos, which were taken as true results for aneuploidy reporting. With removal of all corona-cumulus cells, the false-negative rate (FNR) for niPGT-A was found to be zero. By applying an appropriate threshold for mosaicism, both the positive predictive value (PPV) and specificity for niPGT-A were much higher than TE-biopsy PGT-A. Furthermore, the concordance rates for both embryo ploidy and chromosome copy numbers were higher for niPGT-A than TE-biopsy PGT-A. These results suggest that niPGT-A is less prone to errors associated with embryo mosaicism and is more reliable than TE-biopsy PGT-A.

Dystrophinopathies are X-linked diseases, including Duchenne muscular dystrophy and Becker muscular dystrophy, due to DMD gene variants. In recent years, the application of new genetic technologies and the availability of new personalised drugs have influenced diagnostic genetic testing for dystrophinopathies. Therefore, these European best practice guidelines for genetic testing in dystrophinopathies have been produced to update previous guidelines published in 2010.These guidelines summarise current recommended technologies and methodologies for analysis of the DMD gene, including testing for deletions and duplications of one or more exons, small variant detection and RNA analysis. Genetic testing strategies for diagnosis, carrier testing and prenatal diagnosis (including non-invasive prenatal diagnosis) are then outlined. Guidelines for sequence variant annotation and interpretation are provided, followed by recommendations for reporting results of all categories of testing. Finally, atypical findings (such as non-contiguous deletions and dual DMD variants), implications for personalised medicine and clinical trials and incidental findings (identification of DMD gene variants in patients where a clinical diagnosis of dystrophinopathy has not been considered or suspected) are discussed.

Non-invasive prenatal testing (NIPT) has the potential to screen for a wider range of genetic conditions than is currently possible at an early stage of pregnancy and with minimal risks. As such, there have been calls to apply a ‘threshold of seriousness’ to limit the scope of conditions being tested. This approach is based on concerns about society at large and the potential impact on specific groups within it. In this paper, we argue that limiting the scope of NIPT using the criterion of ‘seriousness’ is arbitrary, potentially stigmatises certain disabilities over others and fails to respect reproductive autonomy. We contend that concerns about expanded NIPT are more appropriately addressed by the provision of adequate information, counselling and consent procedures. We recommend a decision-making process that helps healthcare providers support prospective parents to make informed decisions about the nature and scope of NIPT screening based on their own values and social context. In addition to addressing concerns about expanded NIPT screening, this process would help clinicians to obtain legally valid consent and discharge their duty of care (including the duty to inform) in the prenatal context.

We hypothesized that ethical criteria that guide the use of preimplantation genetic testing (PGT) could be used to inform policies about expanded use of non-invasive prenatal screening (NIPS). We used a systematic review of reasons approach to assess ethical criteria used to justify using (or not using) PGT for genetic conditions. Out of 1135 identified documents, we retained and analyzed 216 relevant documents. Results show a clear distinction in acceptability of PGT for medical vs. non-medical conditions. Criteria to decide on use of PGT for medical conditions are largely based on their severity, but there is no clear definition of “severity”. Instead, characteristics of the condition that relate to severity are used as sub-criteria to assess severity. We found that characteristics that are used as sub-criteria for assessing severity include monogenic etiology, high penetrance, absence of treatment, early age of onset, shortened lifespan, and reduced quality of life. Consensus about the use of PGT is highest for conditions that meet most of these criteria. There is no consensus around the acceptability of using PGT to detect non-medical conditions. We propose that the same severity criteria could be used by policymakers to assess the acceptability of using other genetic tests in screening and practice, including for the use of NIPS for additional conditions as indications broaden.

Noninvasive prenatal screening for common autosomal trisomies, sex chromosome aneuploidies and microdeletions vary by methodology and laboratory practice. The fetal portion of all cell-free DNA in the maternal circulation defines the fetal fraction (FF). The minimum specimen FF levels for reporting results vary between laboratories as well as the screening target (e.g., common trisomies vs select microdeletions). This variability can lead to confusion for both healthcare providers and patients. Participants in the College of American Pathologists Non-Invasive Prenatal Testing 2021-B Exercise provided FF estimates for 3 manufactured samples. Responses to supplemental questions were also collected and analyzed. Overall, 72 of 77 participants responded. FF was measured by 66 participants using sequence counts (40), single nucleotide polymorphisms (15), fragment length (24), and Y chromosome sequences (24). Nearly half (48%) used multiple methods. For common trisomies, minimum FFs were none or <1% (n = 7), 1.0% to 3.9% (n = 35), 4.0% to 6.9% (n = 23), and ≥7.0% (n = 1); 4 participants did not measure FF. Challenge-specific FFs were variable with CVs of 13%, 15%, and 36%; the latter rate appears due to that sample's fetal karyotype of 47,XYY. Comparing adjusted FF results for the 3 samples shows that 85% of participant results were within 20% of the consensus. Using multiple methods to estimate FF was common, and cutoff levels for sample suitability varied widely. Within-laboratory FFs were less variable than between laboratories. Current FF estimates from clinical laboratories are not standardized and should be considered laboratory-specific.

Direct haplotyping enables noninvasive prenatal testing (NIPT) without analyzing proband, which is a promising strategy for pregnancies at risk of an inherited single-gene disorder. Here, we aimed to expand the scope of single-gene disorders that NIPT using linked-read direct haplotyping would be applicable to. Three families at risk of myotonic dystrophy type 1, lipoid congenital adrenal hyperplasia, and Fukuyama congenital muscular dystrophy were recruited. All cases exhibited distinct characteristics that are often encountered as hurdles (i.e., repeat expansion, identical variants in both parents, and novel variants with retrotransposon insertion) in the universal clinical application of NIPT. Direct haplotyping of parental genomes was performed by linked-read sequencing, combined with allele-specific PCR, if necessary. Target DMPK, STAR, and FKTN genes in the maternal plasma DNA were sequenced. Posterior risk calculations and an Anderson–Darling test were performed to deduce the maternal and paternal inheritance, respectively. In all cases, we could predict the inheritance of maternal mutant allele with > 99.9% confidence, while paternal mutant alleles were not predicted to be inherited. Our study indicates that direct haplotyping and posterior risk calculation can be applied with subtle modifications to NIPT for the detection of an expanded range of diseases.

Background Uniparental disomy (UPD) is a specific type of chromosomal variation in which both chromosomes of a homologous pair are inherited from the same parent. It is responsible for a wide range of disorders. Monosomy rescue and trisomy rescue are the two main hypotheses of UPD generation. Methods An older parturient woman with a positive noninvasive prenatal screening (NIPS) test but a negative prenatal diagnosis was referred to the hospital. Trio whole exome sequencing (trio‐WES) and ddPCR were further performed. Results Utilizing Trio‐WES analysis, our research identified a maternal segmental UPD on chromosome 16, characterized by isodisomic genomic segments at the ends of the chromosome arms and heterodisomic genomic segments near the centromere. Moreover, several nuanced signs pointing to the paternal chromosome 16 were discovered, suggesting a low level of trisomy 16 mosaicism. A homozygous missense mutation (c.1499C>T; p.Ala500Val) was also detected in the fetal TBC1D24 gene, passed down from the heterozygous carrier mother. Furthermore, ddPCR analysis verified a 3% mosaic level of trisomy 16. Conclusion We have quantitatively verified for the first time a combination of trisomy 16 mosaicism and maternal segmental UPD 16 due to incomplete trisomy rescue, illuminating the cause of the mismatch between positive NIPS and negative prenatal diagnoses. We reported an elder parturient with a positive NIPS result but a negative prenatal diagnosis. Trio‐WES was performed to identify a homozygous missense mutation in TBC1D2. Trio‐WES data also revealed a maternal segmental UPD of chromosome 16 combined with a very low proportion of trisomy mosaicism (3%) in the fetus.

Background Noninvasive prenatal testing (NIPT) is commonly used to screen for fetal genetic abnormalities. However, the ability of NIPT to detect copy number variations (CNVs) has not been reported. Accordingly, in this study, we analyzed the efficiency of NIPT for the detection of fetal autosomal CNVs. Methods Patients who were positive for autosomal CNVs by NIPT and underwent diagnostic studies by karyotype analysis and chromosomal microarray (CMA) were evaluated. Samples were divided into groups according to age, in vitro fertilization, fetal‐free DNA concentration, uniquely mapped reads number, CNV size, and CNV type. Results Chromosomal microarray showed that the positive predictive value (PPV) of autosomal CNVs detected by NIPT was 14.89%. Increasing fetal DNA concentrations and uniquely mapped read numbers did not affect the PPV of CNVs detected by NIPT. There were no differences between microduplication and microdeletion PPVs detected by NIPT. The PPV of CNVs less than 10 Mb was significantly higher than that of CNVs greater than 10 Mb detected by NIPT. Conclusion The accuracy of NIPT for autosomal CNVs needs to be improved. The positive predictive value of copy number variations (CNVs) less than 10 Mb was significantly higher than that of CNVs greater than 10 Mb detected by Noninvasive prenatal testing (NIPT). NIPT was effective for detecting CNVs less than 10 Mb.

Background There is little evidence on the performance of non‐invasive prenatal testing (NIPT) for the detection of fetal sex chromosomal imbalances. In this review, we aimed to appraise and synthesize the literature on the performance of NIPT for the prenatal detection of fetal sex chromosome aneuploidies. Methods We performed our literature search in PubMed, Embase, Cochrane Library, Web of Science, and CADTH. Study selection and data extraction were performed by two reviewers independently. There were no restrictions on the study population. Meta‐analyses were performed with “R” software. Pooled sensitivities and specificities with their 95% CI were estimated using a random‐effects model. Heterogeneity between studies was assessed by a Q test. Results Based on 11 studies in high prior risk pregnancies, including 116 affected fetuses in aggregate, Massively Parallel Shotgun Sequencing (MPSS) had a sensitivity of 93.9% (95% CI 84.1%, 97.8%) and a specificity of 99.6% (95% CI 98.7%, 99.9%) for the detection of 45,X. Based on four studies in high‐risk pregnancies, with 83 affected fetuses in aggregate, Targeted Massively Parallel Sequencing (TMPS) had a sensitivity of 83.2% (95% CI 49.6%, 96.2%) and specificity was 99.8% (95% CI 98.3%, 100%) for the detection of 45,X. In mixed‐risk pregnancies, the sensitivity of TMPS for the detection of 45,X was 90.9% (2 studies; 95% CI 70%, 97.7%) and specificity 99.9% (2 studies; 95% CI 99.4%, 100%); MPSS data were not available in such pregnancies. Based on smaller numbers of studies, and small numbers of affected fetuses in either high‐risk or mixed‐risk pregnancies (using either MPSS or TMPS), the sensitivities and specificities were equal to or greater than 76.2% for 47,XXX, 47,XXY and 47, XYY. The test failures for SCAs were 0.2% (95% CI 0%, 13.6%) for MPSS and 5.6% (95% CI 3.7%, 8.4%) for TMPS. Conclusion High‐quality studies are still desirable in order to estimate the performance of NIPT for the detection of sex chromosome imbalances. MPSS based‐NIPT can be considered as performant for the detection of 45,X in high‐risk pregnancies, although this result is based on studies that might contain biases. For the other SCAs including 47,XXX, 47,XXY, and 47,XYY, more quality studies are still needed.

Background Expanding noninvasive prenatal testing (NIPT) to include the detection of fetal subchromosomal copy number variations (CNVs) significantly decreased the sensitivity and specificity. Developing analytic pipeline to achieve high performance in the noninvasive detection of CNVs will largely contribute to the application of CNVs screening in clinical practice. Methods We developed the Noninvasively Prenatal Subchromosomal Copy number variation Detection (NIPSCCD) method based on low‐pass whole‐genome sequencing, and evaluated its efficacy in detecting fetal CNVs and chromosomal aneuploidies with 20,003 pregnant women. Results Totally, NIPSCCD identified 36 CNVs, including 29 CNVs consistent and 7 CNVs inconsistent with amniocytes tests. Additionally, seven fetal CNVs identified by amniocytes testing were undetected by NIPSCCD. The sensitivities for detecting CNVs > 10 Mb, 5 Mb–10 Mb, and CNVs < 5 Mb were 91.67%, 100.00%, and 68.42%, respectively. Moreover, NIPSCCD identified 103/ true positive trisomy 21/18/13 cases and 21 false positives, producing an overall 100.00% sensitivity and 99.89% specificity. Conclusion NIPSCCD showed a good performance in detecting fetal subchromosomal CNVs, especially for CNVs >10 Mb, and can be incorporated into the routine NIPT chromosomal aneuploidies screening with high sensitivity and specificity. We developed the Noninvasively Prenatal Subchromosomal Copy number variation Detection (NIPSCCD) method based on low‐pass whole‐genome sequencing (WGS) in this study. Evaluation of NIPSCCD with clinical samples showed good performance on prenatal testing of fetal CNVs, and high sensitivity and specificity in the detection of trisomy 21/18/13.