CHUN-I LEETSUNG-HSIEN LEE2026-10-162026-10-162025-12-01Pure ID: 15094870Pure UUID: 8262c8e5-9ce0-423e-9808-9255e7d5503e10.1038/s41598-025-18764-7https://www.scopus.com/pages/publications/105017950651https://scholars.csmu.edu.tw/handle/123456789/24291Non-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.enjournal article