Search

Research Question: Does routine clinical practice require an increase in the resolution of preimplantation genetic testing for aneuploidies (PGT-A) to detect segmental aneuploidies ≤5 Mb?, Design: This retrospective study analysed 963 trophectoderm biopsies from 346 couples undergoing PGT between 2019 and 2023. Segmental aneuploidies ≥1 Mb were reported. The characteristics, clinical interpretation and concordance of segmental aneuploidies ≤5 Mb were analysed., Results: The incidence of segmental aneuploidies was 15.1% (145/963) in blastocysts, with segmental aneuploidies of ≤5 Mb accounting for 2.3% (22/963). The size of the segmental aneuploidies showed a skewed distribution. Segmental aneuploidies ≤5 Mb were found to occur more frequently on the q arm of the chromosome, compared with the p arm. Losses of ≤5 Mb segmental aneuploidies were more prevalent than gains, with 17 deletions compared with 5 duplications. Of the segmental aneuploidies, 63.6% (14/22) ≤5 Mb were de novo, and 50.0% (7/14) of de-novo segmental aneuploidies were pathogenic/likely pathogenic (P/LP) copy number variations, accounting for 0.7% of 963 blastocysts. For blastocysts carrying ≤5 Mb segmental aneuploidies, a re-analysis of back-up biopsy samples showed that 35.7% of de-novo segmental aneuploidies (5/14) were not detected in the back-up samples. Cases were reported in which prenatal diagnosis (amniocentesis) revealed the absence of embryonic ≤5 Mb segmental aneuploidies detected at the blastocyst stage., Conclusions: The incidence of P/LP de-novo ≤5 Mb segmental aneuploidies in human blastocysts is extremely low. There is no compelling need to increase the resolution of PGT-A to 5 Mb in routine clinical practice., (Copyright © 2024 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Preimplantation genetic testing (PGT) can minimize the risk of birth defects. However, the accuracy and applicability of routine PGT is confounded by uneven genome coverage and high allele drop-out rate from existing single-cell whole genome amplification methods. Here, a method to diagnose genetic mutations and concurrently evaluate embryo competence by leveraging the abundant mRNA transcript copies present in trophectoderm cells is developed. The feasibility of the method is confirmed with 19 donated blastocysts. Next, the method is applied to 82 embryos from 26 families with monogenic defects for simultaneous mutation detection and competence assessment. The accuracy rate of direct mutation detection is up to 95%, which is significantly higher than DNA-based method. Meanwhile, this approach correctly predicted seven out of eight (87.5%) embryos that failed to implant. Of six embryos that are predicted to implant successfully, four met such expectations (66.7%). Notably, this method is superior at conditions for mutation detection that are challenging when using DNA-based PGT, such as when detecting pathogenic genes with a high de novo rate, multiple pseudogenes, or an abnormal expansion of CAG trinucleotide repeats. Taken together, this study establishes the feasibility of an RNA-based PGT that is also informative for assessing implantation competence., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Purpose: To study the application of next-generation sequencing on preimplantation genetic testing for recurrent hydatidiform mole patients., Methods: A total of ten recurrent hydatidiform mole patients aged 27-34 years with a history of at least twice hydatidiform moles and no normal pregnancy were collected from 2019 to 2020. The diagnosis of hydatidiform mole type was clarified using short tandem repeat genotyping on products of conception, and whole-exome sequencing was applied for all patients and their partners. Seven recurrent hydatidiform mole patients with complete hydatidiform mole/partial hydatidiform mole type among previous hydatidiform mole tissues and no Pathogenetic/Likely pathogenetic/Uncertain significance variants in NLRP7/KHDC3L/MEI1/C11orf80 underwent a procedure of preimplantation genetic testing. Next-generation sequencing for analyzing the copy number variants and the numbers of heterozygous single nucleotide polymorphism was adopted to clarify the ploidy and parental origin of the embryo chromosomes in vitro. Embryos with biparental diploidy were selected for transfer., Results: Seven patients have undergone the procedure of preimplantation genetic testing, and twenty-three embryos were obtained, among which 82.6% (n = 19) were identified transferrable and 17.4% (n = 4) were identified aneuploid. Two patients have delivered healthy babies and another is currently in the second trimester after transfer., Conclusion: Analyzing the copy number variants and the numbers of heterozygous single nucleotide polymorphism on the basis of next-generation sequencing can be utilized in the procedure of preimplantation genetic testing among part of recurrent hydatidiform mole patients. The current study is effective to reduce the occurrence of hydatidiform mole with improved clinical strategy, the advanced testing technology and analysis methods, as three of seven patients have conceived or delivered successfully., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease characterized by the development of renal cysts and progression to renal failure. Preimplantation genetic testing-monogenic disease (PGT-M) is an alternative option to obtain healthy babies. However, de novo PKD1 mutation of one of the spouses or the absence of a positive family history poses a serious challenge to PGT-M. Here, we described a comprehensive strategy which includes preimplantation genetic testing for aneuploidies (PGT-A) study and monogenic diagnosis study for ADPKD patients bearing de novo mutations. The innovation of our strategy is to use the gamete (polar body or single sperm) as proband for single-nucleotide polymorphism (SNP) linkage analysis to detect an embryo's carrier status. Nine ADPKD couples with either de novo mutation or without a positive family history were recruited and a total of 34 embryos from 13 PGT-M cycles were examined. Within these nine couples, two successfully delivered healthy babies had their genetic status confirmed by amniocentesis. This study provides a creative approach for embryo diagnosis of patients with de novo mutations or patients who lack essential family members for linkage analysis., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Haplotyping reveals chromosome blocks inherited from parents to in vitro fertilized (IVF) embryos in preimplantation genetic diagnosis (PGD), enabling the observation of the transmission of disease alleles between generations. However, the methods of haplotyping that are suitable for single cells are limited because a whole genome amplification (WGA) process is performed before sequencing or genotyping in PGD, and true haplotype profiles of embryos need to be constructed based on genotypes that can contain many WGA artifacts., Results: Here, we offer scHaplotyper as a genetic diagnosis tool that reconstructs and visualizes the haplotype profiles of single cells based on the Hidden Markov Model (HMM). scHaplotyper can trace the origin of each haplotype block in the embryo, enabling the detection of carrier status of disease alleles in each embryo. We applied this method in PGD in two families affected with genetic disorders, and the result was the healthy live births of two children in the two families, demonstrating the clinical application of this method., Conclusion: Next generation sequencing (NGS) of preimplantation embryos enable genetic screening for families with genetic disorders, avoiding the birth of affected babies. With the validation and successful clinical application, we showed that scHaplotyper is a convenient and accurate method to screen out embryos. More patients with genetic disorder will benefit from the genetic diagnosis of embryos. The source code of scHaplotyper is available at GitHub repository: https://github.com/yzqheart/scHaplotyper.

Purpose: While efforts have been made to establish blastocyst grading systems in the past decades, little research has examined the quality of biopsy specimens. This study is the first to correlate the morphology of biopsied trophectoderm (TE) cells to their quality and subsequent genetic testing results of preimplantation genetic testing (PGT), through an innovative Morphological Analysis and Genetic Integrality Criterion (MAGIC) system. Methods: Biopsied TE cells were first evaluated according to the MAGIC procedure, followed by whole-genome amplification (WGA) and library construction, and then sequenced using the Illumina X Ten Platform. Copy number variation (CNV) and allele drop-out (ADO) rates as well as test failure rates were compared and analyzed. Results: Our data explores the relationship between TE cell morphology and its quality and final genetic testing outcome, which is established based on the MAGIC system. MAGIC guarantees that only high- or good-quality TE cells are used for genetic testing to generate excellent data uniformity and lower ADO rates. Low-quality cells containing biopsied TE cell mass are responsible for the "background noise" of CNV analysis. Conclusion: The MAGIC application has effectively decreased the false-positive mosaicism, hence to ensure the stability and veracity of detection results, to avoid misdiagnoses, and to improve accuracy, as well as to avoid re-biopsy procedures. The study also contributes to understand how the IVF laboratory and the molecular biology laboratory depend on each other to achieve good-quality PGT results, which are clinically relevant for the patients. [ABSTRACT FROM AUTHOR]

Purpose: To determine whether next-generation sequencing (NGS) could be used to directly detect different mutations of Duchenne muscular dystrophy (DMD) during preimplantation genetic testing (PGT). Methods: From Sep. 2016 to Aug. 2018, a total of six couples participated in this study. Four cases carried DMD exon deletions and two carried exon duplications. Trophectoderm cells were biopsied at day 5 or 6 and NGS was used in the genetic testing of the biopsied cells after whole-genome amplification. We developed a new method—DIRected Embryonic Cell Testing of Exon Deletion/Duplication (DIRECTED) to directly detect the single-gene mutation by NGS. Linage analysis based on single-nucleotide polymorphism (SNP) was used to validate the results from DIRECTED. Results: In the four deletion cases, DIRECTED was used to detect DMD exon deletion in 16 biopsied embryos. All DIRECTED results were consistent with linkage analysis, indicating this method was reliable in detecting deletions around 1 Mb. In the two cases carrying exon duplications, no blastocyst was obtained for biopsy. Nonetheless, preliminary experiment results suggested that DIRECTED could also be used for direct detection of exon duplications in embryos. Conclusions: Exon deletions or duplications in DMD of preimplantation embryos could be detected directly by NGS-based methods during PGT. [ABSTRACT FROM AUTHOR]

Can preimplantation genetic testing for structural rearrangement (PGT-SR) based on low-coverage next-generation sequencing (NGS) accurately discriminate between normal and carrier embryos of reciprocal translocation (RecT) and Robertsonian translocation (RobT)? A total of 109 couples with RecT or RobT were included in this study. The ages, bad obsteric histories (BOH), blood karyotype and IVF cycle information, including the number of cumulus–oocyte complexes, metaphase II oocytes, two pronuclei oocytes and blastocysts were recorded. 0.1 × whole genome sequencing (WGS) of embryos followed by copy number variation (identifying unbalanced/balanced) and 2 × WGS of parents and embryos followed by haplotype analysis (discriminating between normal and carrier) were carried out in PGT-SR cycles. The embryos without translocation were transferred and clinical outcomes evaluated. Among all the couples in this study, 67 patients had RecT and 42 had RobT. After unbalanced and balanced detection, 103 balanced embryos underwent a further normal and carrier discrimination procedure, and 53 normal embryos were identified. Finally, 32 normal embryos were transferred, with an ongoing pregnancy rate of 46.88% (15/32). All ongoing pregnancies underwent amniocentesis, and the amninocentesis karyotyping results showed 100% concordance with PGT-SR diagnosis. Our low-coverage NGS-based PGT-SR method can accurately discriminate between normal and carrier status of balanced embryos. The method is cost-effective and has broad clinical applicability. [ABSTRACT FROM AUTHOR]