Systematic assessment of allele dropout in preimplantation genetic testing for monogenic disorders: incidence, detection, and clinical testing strategies.

OBJECTIVE

To systematically evaluate the allele dropout (ADO) rate in PGT-M and assess practical testing strategies to minimize its impact. Although preimplantation genetic testing for monogenic disorders (PGT-M) is widely applied in clinical practice, the incidence and clinical implications of ADO in direct variant detection remain inadequately characterized.

DESIGN

Retrospective study.

SUBJECTS: A total of 516 couples who underwent PGT-M treatment between December 2020 and September 2025 were included.

EXPOSURE: Trophectoderm biopsies were performed on day 5 or day 6 blastocysts, followed by whole-genome amplification using multiple displacement amplification (MDA). The PGT-M testing strategy comprised two components: a custom-designed next-generation sequencing (NGS) panel covering exon regions and adjacent single nucleotide polymorphisms (SNPs) of target genes, enabling family-based haplotype linkage analysis combined with direct variant detection in embryos; and Sanger sequencing for direct variant detection in MDA products.

MAIN OUTCOME MEASURES: ADO rate in PGT-M detected directly by NGS panel and Sanger sequencing.

RESULTS

A cohort of 2,949 embryos from 516 families was tested, encompassing 3,942 variant loci across 188 genes. Overall, ADO was observed at 425 loci (10.78%) distributed across 70 genes in 136 of 516 patients (26.36%). In the NGS panel, ADO occurred at 160 loci (4.06%) across 24 genes, while in the Sanger sequencing method, ADO occurred at 293 loci (7.43%) across 61 genes; this difference was statistically significant. Among all ADO events, cooccurrence of ADO in both methods was observed in only 6.59% (28/425) of loci, indicating minimal overlap. For all resulting pregnancies, prenatal diagnosis was recommended, and PGT-M results obtained by the NGS panel method were fully concordant with fetal amniotic fluid cell analysis.

CONCLUSION

The NGS-based PGT-M panel demonstrates a relatively low ADO rate while enabling concurrent haplotype analysis, supporting its use as the preferred first-line testing strategy. Routine Sanger sequencing may be omitted in clinical practice. In cases where NGS panel results show discordance between direct variant detection and haplotype linkage, targeted Sanger sequencing can be considered for confirmatory validation.