Underrecognized triploidy and genome-wide uniparental disomy in human blastocysts revealed by a concurrent...

What is the incidence of triploidy and genome-wide uniparental disomy (gwUPD) in human embryos currently undetected by conventional preimplantation genetic testing (PGT)?

Triploidy and gwUPD were detected in 0.8% (8/1049) and 1.4% (15/1049) of blastocysts, respectively.

Concurrent PGT enhances embryo selection by simultaneously detecting monogenic disorders (PGT-M) or structural rearrangements (PGT-SR) and aneuploidy (PGT-A). However, triploidy and gwUPD, two genome-wide abnormalities causative of severe pregnancy outcomes, are not routinely detected by conventional PGT platforms and thus remain under-recognized at the preimplantation stage.

This study consisted of three phases: Phase I validated the detection capability of PGT-Plus on cell lines with previously ascertained genetic abnormalities; Phase II performed retrospective clinical validation on leftover trophectoderm biopsy whole-genome amplification (WGA) samples, including 65 for PGT-M, 67 for PGT-SR, and 130 for PGT-A received during January 2019 and July 2023; and Phase III was prospective implementation of PGT-Plus under a diagnostic setting on 529 blastocysts received from August 2023 to June 2024. Additionally, a cohort consisting of 258 embryo transfers, previously ascertained as euploid by conventional PGT-A methods but associated with unfavorable outcomes, was retested by PGT-Plus, which identified previously undetected cases of gwUPD or triploidy.

In Phase I, cell lines with ascertained genetic abnormalities were tested using at least one of the following methods, including G-banded Karyotyping, quantitative fluorescence PCR (QF-PCR), chromosomal microarray analysis (CMA), and mate-pair sequencing. The previous results were compared with those from PGT-Plus. In Phase II, results from conventional PGT platforms were compared in parallel with PGT-Plus to evaluate its accuracy. Cell lines or real trophoblast biopsy samples from blastocysts were amplified by the multiple displacement amplification (MDA) method. PGT-Plus data analysis was performed in various dimensions. For PGT-A analysis, except for canonical read-count analysis to assess the genome dosage, single nucleotide polymorphism (SNP) analysis was also performed to identify triploidy and gwUPD. Furthermore, for families requesting PGT-M and PGT-SR, linkage analysis was carried out for haplotype construction in addition to read-count analysis and SNP analysis. In addition to prospective cohort of Phase III, a unique cohort with adverse reproductive outcomes after transfer of a 'euploid' embryo, as determined by conventional PGT-A platforms, was retested to identify any triploidy and UPD among these copy-number neutral embryos. The unfavorable outcomes included no pregnancy, biochemical pregnancy, miscarriage, and termination of pregnancy due to fetal anomaly.

PGT-Plus detected all abnormalities ascertained by prior platforms in Phases I and II. In total, triploidy and gwUPD were detected in 0.8% (8/1049) and 1.4% (15/1049) of blastocysts, respectively. Notably, 3.5% (9/258) of the unfavorable transfers of embryos previously classified as 'euploid' would have been prevented if triploidy and gwUPD were detected. In addition, 45.7% (12/35) of embryos previously classified as balanced by PGT-SR were translocation carriers.

PGT-Plus, similar to other relative phasing methods, requires an additional family member for haplotype determination, although an embryo with a known genotype via prior independent testing is also acceptable. In addition, short-read next-generation sequencing (NGS) impedes the capacity of PGT-Plus to directly detect pathogenic SNVs located in highly repetitive regions of the genome, e.g. the HBA1 gene causative of thalassemia, which still require conventional methods, including short tandem repeats and PCR, to safeguard clinical diagnosis or can be addressed by long-read sequencing.

This newly established PGT-Plus approach additionally detected gwUPD and triploidy in preimplantation embryos. It explained another 3.5% of 'euploid' embryo transfer failures. Meanwhile, embryo selection was enhanced by integrating PGT-M, PGT-SR, or PGT-A with an analysis of parental origin and stage of non-disjunction within a single PGT-Plus workflow.

This work was funded by the Research Grant Council Collaborative Research Fund (grant no. C4062-21GF to K.W.C.), the National Natural Science Foundation of China (grant no. 82502033 to Y.L.), and the Research Grant Council General Research Fund (grant no. 14100822 to J.P.W.C.). All authors declare that they have no conflicts of interest.

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