Received April 16, 2026
Accepted May 06, 2026

Objective: To evaluate the analytical reproducibility of embryos classified as chaotic by non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) through repeat cell-free DNA (cfDNA) analysis and to explore implications for diagnostic validity. Methods: This prospective pilot study included seven vitrified blastocysts previously classified as chaotic (?6 chromosome abnormalities) by niPGT-A. Following warming and a short-term secondary culture to allow further cfDNA release, the spent culture medium (SCM) underwent repeat whole-genome amplification (MALBAC) and next-generation sequencing using the same laboratory platform and bioinformatic pipeline. All analyses were performed by experienced laboratory personnel blinded to initial results. Concordance between initial and repeat classifications was descriptively assessed. Results: All cfDNA samples were successfully amplified, providing high-quality libraries for sequencing. Upon repeat analysis, 3/7 embryos (43%) were reclassified as euploid (46,XX). The remaining four embryos retained abnormal findings; however, chromosomal profiles differed from initial reports. Overall, embryos previously classified as chaotic demonstrated limited analytical reproducibility under repeat cfDNA assessment. Conclusions: Embryos classified as chaotic by niPGT-A exhibited considerable analytical variability in this pilot cohort. These findings highlight the need for cautious interpretation of complex aneuploid profiles derived from low-input cfDNA and reinforce the importance of rigorous validation before such classifications guide clinical decision making in assisted reproduction.