Extended blastocyst culture improves DNA yield in non-invasive preimplantation genetic testing for aneuploidy but diagnostic specificity remains limited

Non-invasive preimplantation genetic testing for aneuploidy (niPGT-A), which analyzes cell-free DNA (cfDNA) from spent culture media (SCM), has emerged as a proposed alternative to trophectoderm (TE) biopsy in in vitro fertilization (IVF). However, its clinical reliability remains unproven. This prospective paired-sample study compared niPGT-A and TE biopsy in 212 blastocysts from 98 couples undergoing IVF, evaluating diagnostic performance and clinical outcome correlation. All embryos underwent simultaneous niPGT-A and TE biopsy, analyzed using next-generation sequencing, with embryo transfers based solely on TE biopsy results. Extended culture to Day 6 significantly improved the informative rate of niPGT-A (from 69.4 to 97.9%). The overall ploidy concordance between niPGT-A and TE biopsy was 75.9%. niPGT-A classified more embryos as aneuploid (75.3%) than TE biopsy (58.5%), including 33 false positives. While niPGT-A showed high sensitivity (91.6%) for aneuploid detection, its specificity was low (50.7%), and discordant embryos (euploid by TE, aneuploid by niPGT-A) achieved unexpectedly high pregnancy (94%) and live birth (88%) rates. These findings highlight that false-positive classification by niPGT-A may result in the unnecessary exclusion of transferable embryos. Despite its non-invasive appeal, niPGT-A lacks sufficient diagnostic accuracy for clinical use. Further refinement of cfDNA analysis, contamination control, and standardization are needed before niPGT-A can be considered for routine clinical implementation.