Novel Double Factor PGT strategy analyzing blastocyst stage embryos in a single NGS procedure

Abstract

In families at risk from monogenic diseases affected offspring, it is fundamental the development of a suitable Double Factor Preimplantation Genetic Testing (DF-PGT) method for both single-gene analysis and chromosome complement screening. Aneuploidy is not only a major issue in advanced-maternal-age patients and balanced translocation carriers, but also the aneuploidy rate is extremely high in patients undergoing in vitro fertilization (IVF), even in young donors. To adequate NGS technology to the DF-PGT strategy four different whole genome amplification systems (Sureplex, MALBAC, and two multiple displacement amplification systems-MDA) were tested using TruSight One panel on cell lines and blastocyst trophectoderm biopsies-TE. Embryo cytogenetic status was analyzed by Nexus software. Sureplex and MALBAC DNA products were considered not suitable for PGT diagnosis due to inconsistent and poor results on Trusight one (TSO) panel. Results obtained with both MDA based methods (GEH-MDA and RG-MDA) were appropriate for direct mutation detection by TSO NGS platform. Nevertheless, RG-MDA amplification products showed better coverage and lower ADO rates than GEH-MDA. The present work also demonstrates that the same TSO sequencing data is suitable not only for the direct mutation detection, but also for the indirect mutation detection by linkage analysis of informative SNPs. The present work also demonstrates that Nexus software is competent for the detection of CNV by using with TSO sequencing data from RG-MDA products, allowing for the whole cytogenetic characterization of the embryos. In conclusion, successfully development of an innovative and promising DF-PGT strategy using TSO-NGS technology in TE biopsies, performed in-house in a single laboratory experience, has been done in the present work. Additional studies should be performed before it could be used as a diagnostic alternative in order to validate this approach for the detection of chromosomal aneuploidies.

A systematic review on concurrent aneuploidy screening and preimplantation genetic testing for hereditary disorders: What is the prevalence of aneuploidy and is there a clinical effect from aneuploidy screening?

“…Title and abstract screening resulted in 73 papers. Full text screening resulted in 26 publications fulfilling the inclusion criteria . No randomized controlled trials were identified.…”

Preimplantation Genetic Testing for Chromosomal Abnormalities: Aneuploidy, Mosaicism, and Structural Rearrangements

“…Such a technique can be adapted to any mutation using appropriate primer spike-ins. Del Rey and colleagues have successfully combined NGS copy number analysis with large panels of common single gene conditions [ 194 ]. Alcaraz and colleagues reported a technique that combines chromosomal copy number calling with a SNP-based analysis of a mutation specific to a patient’s genetic profile, showing its feasibility in over 150 different mutations [ 195 ].…”

Preimplantation Genetic Testing for Monogenic Disorders

“…In another NGS approach, a target enrichment gene panel with nearly 5000 Mendelian disease-associated genes (TruSight One sequencing panel) was applied on MDA-based WGA products, thereby offering direct testing of family pathogenic variant(s) plus indirect pathogenic variant detection through haplotyping of SNPs together with chromosome copy number detection through the log ratio of signal intensities, i.e., PGT-M and PGT-A together in a single workflow [57].…”