PGT-SR (reciprocal translocation) using trophectoderm sampling and next-generation sequencing: insights from a virtual trial.
Ontology highlight
ABSTRACT:
SUBMITTER: Scriven PN
PROVIDER: S-EPMC8417212 | biostudies-literature | 2021 Aug
REPOSITORIES: biostudies-literature
Publications
PGT-SR (reciprocal translocation) using trophectoderm sampling and next-generation sequencing: insights from a virtual trial.
Journal of assisted reproduction and genetics 20210327 8
<h4>Purpose</h4>The objective of this virtual study was to simulate a full cycle and assess the costs and benefits to a couple with a reciprocal translocation, using current techniques for preimplantation genetic testing and comparing reporting every chromosome with only reporting those involved in the rearrangement.<h4>Methods</h4>A simulation was constructed for women under the age of 35 years, where vitrified-warmed embryos were transferred one at a time in a first full cycle following preimp ...[more]
Similar Datasets
Project description:PurposePreimplantation genetic testing for monogenic disorders (PGT-M) allows early diagnosis in embryos conceived in vitro. PGT-M helps to prevent known genetic disorders in affected families and ensures that pathogenic variants in the male or female partner are not passed on to offspring. The trend in genetic testing of embryos is to provide a comprehensive platform that enables robust and reliable testing for the causal pathogenic variant(s), as well as chromosomal abnormalities that commonly occur in embryos. In this study, we describe PGT protocol that allows direct mutation testing, haplotyping, and aneuploidy screening.MethodsDescribed PGT protocol called OneGene PGT allows direct mutation testing, haplotyping, and aneuploidy screening using next-generation sequencing (NGS). Whole genome amplification product is combined with multiplex PCR used for SNP enrichment. Dedicated bioinformatic tool enables mapping, genotype calling, and haplotyping of informative SNP markers. A commercial software was used for aneuploidy calling.ResultsOneGenePGT has been implemented for seven of the most common monogenic disorders, representing approximately 30% of all PGT-M indications at our IVF centre. The technique has been thoroughly validated, focusing on direct pathogenic variant testing, haplotype identification, and chromosome abnormality detection. Validation results show full concordance with Sanger sequencing and karyomapping, which were used as reference methods.ConclusionOneGene PGT is a comprehensive, robust, and cost-effective method that can be established for any gene of interest. The technique is particularly suitable for common monogenic diseases, which can be performed based on a universal laboratory protocol without the need for set-up or pre-testing.
| S-EPMC10789686 | biostudies-literature
Project description:Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterization of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time-consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using "next-generation" (Illumina/Solexa) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows the determination of their exact nucleotide positions within a few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations.
| S-EPMC2493403 | biostudies-literature
Project description:Balanced chromosome rearrangements (BCRs) can cause genetic diseases by disrupting or inactivating specific genes, and the characterisation of breakpoints in disease-associated BCRs has been instrumental in the molecular elucidation of a wide variety of genetic disorders. However, mapping chromosome breakpoints using traditional methods, such as in situ hybridization with fluorescent dye-labeled bacterial artificial chromosome clones (BAC-FISH), is rather laborious and time consuming. In addition, the resolution of BAC-FISH is often insufficient to unequivocally identify the disrupted gene. To overcome these limitations, we have performed shotgun sequencing of flow-sorted derivative chromosomes using ânext generationâ (Solexa/Illumina) multiplex sequencing-by-synthesis technology. As shown here for three different disease-associated BCRs, the coverage attained by this platform is sufficient to bridge the breakpoints by PCR amplification, and this procedure allows to determine their exact nucleotide positions within few weeks. Its implementation will greatly facilitate large-scale breakpoint mapping and gene finding in patients with disease-associated balanced translocations. Array CGH was performed in three carriers of balanced translocations to exclude DNA copy number changes.
2010-05-22 | E-GEOD-10115 | biostudies-arrayexpress
Project description:PurposeTo assess the costs and benefits of carrier screening and preimplantation genetic testing (PGT) for recessive autosomal monogenic disorders for couples attempting assisted conception.MethodsA simulated first full cycle for women less than 35 years transferring embryos one at a time. The effect of testing on pregnancy outcomes was evaluated for different reporting scenarios. A Monte Carlo method utilising 1000 trials for 10,000 couples, testing 4, 16 and 38 genes, was used to assess the numbers likely to be at high risk and to estimate the incremental cost of screening and PGT to avoid an affected child.ResultsPGT for high-risk couples: testing embryos only for the monogenic condition avoided 1 affected pregnancy for 4 cycles started. Combined with testing for chromosomal aneuploidy: ranking test results avoided 1 adverse pregnancy (affected, biochemical, clinical miscarriage) from 3 cycles started; 1 in 2 when excluding from transfer all embryos with an abnormal test result, within 1 in 25 fewer women achieving an unaffected live birth. Carrier screening for 4, 16 and 38 gene scenarios, where 1:250, 1:196 and 1:29 couples were at high risk: the incremental cost to prevent 1 affected live birth was estimated to be less than GBP 1,150,000 (US $1,587,000), < 836,642 (1,154,566) and < 137,794 (190,156), respectively, in 95% of trials.ConclusionsCarrier screening combined with PGT, with and without testing for unrelated chromosomal abnormalities, for couples attempting assisted conception is complex but likely to be effective and also expensive.
| S-EPMC8956760 | biostudies-literature
Project description:The detection of chromosomal aneuploidies and mosaicism degree in preimplantation embryos may be essential for achieving pregnancy. The aim of this study was to determine the robustness of diagnosing homogenous and mosaic aneuploidies using a validated algorithm and the minimal resolution for de novo and inherited deletions and duplications (Del/Dup). Two workflows were developed and validated: (a,b) preimplantation genetic testing for uniform whole and segmental aneuploidies, plus mixtures of euploid/aneuploid genomic DNA to develop an algorithm for detecting mosaicism; and (c) preimplantation genetic testing for structural rearrangements for detecting Del/Dup ≥ 6 Mb. Next-generation sequencing (NGS) was performed with automatic library preparation and multiplexing up to 24-96 samples. Specificity and sensitivity for PGT-A were both 100% for whole chromosomes and segmentals. The thresholds stablished for mosaicism were: euploid embryos (<30% aneuploidy), low mosaic (from 30% to <50%), high mosaic (50-70%) or aneuploid (>70%). In the PGT-SR protocol, changes were made to increase the detection level to ≥6 Mb. This is the first study reporting an accurate assessment of semiautomated-NGS protocols using Reproseq on pools of cells. Both protocols allow for the analysis of homogeneous and segmental aneuploidies, different degrees of mosaicism, and small Del/Dup with high sensitivity and specificity.
| S-EPMC7397276 | biostudies-literature
Project description:We synthesized reversible terminators with tethered inhibitors for next-generation sequencing. These were efficiently incorporated with high fidelity while preventing incorporation of additional nucleotides, and we used them to sequence canine bacterial artificial chromosomes in a single-molecule system that provided even coverage for over 99% of the region sequenced. This single-molecule approach generated high-quality sequence data without the need for target amplification and thus avoided concomitant biases.
| S-EPMC2719685 | biostudies-literature
Project description:Next-generation sequencing is a cutting edge technology, but to quantify a dynamic range of abundances for different RNA or DNA species requires increasing sampling depth to levels that can be prohibitively expensive due to physical limits on molecular throughput of sequencers. To overcome this problem, we introduce a new general sampling theory which uses biophysical principles to functionally encode the abundance of a species before sampling, SeQUential depletIon and enriCHment (SQUICH). In theory and simulation, SQUICH enables sampling at a logarithmic rate to achieve the same precision as attained with conventional sequencing. A simple proof of principle experimental implementation of SQUICH in a controlled complex system of ~262,000 oligonucleotides already reduces sequencing depth by a factor of 10. SQUICH lays the groundwork for a general solution to a fundamental problem in molecular sampling and enables a new generation of efficient, precise molecular measurement at logarithmic or better sampling depth.
| S-EPMC6932819 | biostudies-literature
Project description:Introduction Uniform chromosome abnormalities are commonly seen in early pregnancy loss, with analyses of the product of conception suggesting the presence of mosaic autosomal trisomy in ∼10% of cases. Although chromosomal mosaicism occurs in a minority of embryos, their relative commonality and uncertainty regarding associated transfer outcomes have created discussion at both the clinical and research levels, highlighting the need to understand the clinical conditions associated with the incidence of embryo mosaicism. Methods We took advantage of a preimplantation genetic testing for aneuploidy (PGT-A) database created from 2019 to 2022 in more than 160 in vitro fertilization (IVF) clinics in Brazil, the second-largest world market for IVF. We carried out descriptive statistical and associative analyses to assess the proportions of mosaicism associated with clinical conditions and reported incidence by chromosome, clinic origin, and biopsy operator. Results Chromosomal analysis revealed that most mosaic aneuploidies occurred in the last three chromosomes, with 78.06% of cases having only one chromosome affected. Low mosaicism in trisomy represented the most ordinary form, followed by low mosaicism in monosomy. We identified associations between low (negatively-associated) and high mosaicism (positively-associated) and maternal age, indication (male factor and uterus/ovarian factor negatively associated with low and high mosaic, respectively), day of blastocyst development (day five has an overall better outcome), morphology grade (lower quality increased the chances of low and high mosaicism), origin (vitrified oocyte and embryo increased the rates of low and high mosaicism, respectively), and embryo sex (male embryos negatively associated with low mosaic). Discussion With these results, we hope to foster an improved understanding of the chromosomal mosaicism linked with distinct clinical conditions and their associations in Brazil.
| S-EPMC10083245 | biostudies-literature
Project description:BackgroundDespite the gradual application of third-generation long read sequencing (LRS) or reference embryo establishment to preimplantation genetic testing for structural rearrangement (PGT-SR) without familial involvement, there are still limitations to their extensive clinical application yet. This study developed a novel virtual NGS-based long read method (ViLR) and preliminarily evaluated its clinical feasibility of breakpoint characterization and direct SNP haplotyping for de novo chromosomal structural rearrangements (CSR).MethodsA total of 10 families with de novo CSR risk were enrolled in this study for ViLR analysis. In contrast to LRS, ViLR is a virtual long read solution that used the same barcoded labeling and assembly of different long gDNAs differently barcoded. Notably, ViLR could generate an average fragment length of over 30 Kb, with an N50 block size of up to 16 Mb in a single assay, allowing to achieve accurate breakpoint mapping and direct carrier's haplotyping. An approximately 2 Mbp region flanking upstream and downstream of each breakpoint was selected for informative SNP collection. Embryo haplotype determination was based on the established carriers' haplotypes after whole genome amplification and sequencing. To confirm PGT-SR results, we performed prenatal genetic diagnosis.ResultsThis study achieved an average mapping rate of 99.5%, > 90% coverage depth (> 10X), an average number of effective barcode (> 5 kb length) counts of 11,000,000 and an average fragment length of 40 kb, which generated sufficient informative SNPs for breakpoint characterization and haplotype phasing. ViLR analysis of 10 de novo PGT-SR carriers precisely identified breakpoints and haplotypes. Seven families obtained 18 euploid embryos, in which 10 were euploid/normal embryos, 7 were euploid/balanced carrier embryos, and the remaining one unknown was due to homologous recombination of the breakpoint region. Prenatal genetic diagnosis was performed for four women, and the outcomes coincided with the results from embryo PGT-SR. At the time of writing this paper, four healthy babies had been delivered uneventfully.ConclusionHere, we demonstrated the clinical potential of ViLR as a novel solution for breakpoint identification and direct SNP haplotyping in de novo PGT-SR families without proband involvement.Clinical trial numberNot applicable.
| S-EPMC11881346 | biostudies-literature
Project description:Improved embryo prioritization is crucial in optimizing the results in assisted reproduction, especially in light of increasing utilization of elective single embryo transfers. Embryo prioritization is currently based on morphological criteria and in some cases incorporates preimplantation genetic testing for aneuploidy (PGT-A). Recent technological advances have enabled parallel genomic and transcriptomic assessment of a single cell. Adding transcriptomic analysis to PGT-A holds promise for better understanding early embryonic development and implantation, and for enhancing available embryo prioritization tools. Our aim was to develop a platform for parallel genomic and transcriptomic sequencing of a single trophectoderm (TE) biopsy, that could later be correlated with clinical outcomes. Twenty-five embryos donated for research were utilized; eight for initial development and optimization of our method, and seventeen to demonstrate clinical safety and reproducibility of this method. Our method achieved 100% concordance for ploidy status with that achieved by the classic PGT-A. All sequencing data exceeded quality control metrics. Transcriptomic sequencing data was sufficient for performing differential expression (DE) analysis. All biopsies expressed specific TE markers, further validating the accuracy of our method. Using PCA, samples clustered in euploid and aneuploid aggregates, highlighting the importance of controlling for ploidy in every transcriptomic assessment.
| S-EPMC6393576 | biostudies-literature