Purpose <p>This study evaluated the performance of preimplantation genetic testing for aneuploidies (PGT-A) using cell-free DNA (cfDNA) from spent culture media (SCM) of blastocyst embryos (non-invasive PGT-A; NiPGT-A) compared with conventional trophectoderm (TE) biopsy samples.</p> Methods <p>This prospective study was conducted at IVFMD, My Duc Hospital, Vietnam, from August to December 2020, and included patients with an indication for PGT-A. The culture medium was replaced on day 3, and SCM from day 3 to the day of TE biopsy (days 5 or 6) of all biopsied blastocysts was tested using next-generation sequencing. The total concordance rate, sensitivity, and specificity of NiPGT-A versus PGT-A for detecting aneuploid embryos were calculated. Outcomes after single blastocyst transfer are also reported.</p> Results <p>Forty-four couples participated; 100 paired TE PGT-A biopsies and SCM samples were evaluated. The whole-genome amplification success rate for SCM was 82%; 77 samples had clear NGS results and were further evaluated. The total concordance rate between NiPGT-A and PGT-A was 63.6%. For detecting aneuploidy, NiPGT-A had a sensitivity of 57.1%, specificity of 67.3%, positive predictive value of 50.0%, and negative predictive value of 73.3%. Of the 35 single euploidy embryo transfers, 8 had no NiPGT-A results, 21 were classified as NiPGT-A euploid, and 6 were classified as NiPGT-A aneuploid; the live birth rate was 51.4% (18/35). Four of the 6 NiPGT-A aneuploid blastocysts resulted in live births.</p> Conclusions <p>cfDNA in SCM has the potential for NiPGT-A. However, the NiPGT-A process is unreliable enough to replace traditional PGT-A using TE biopsy.</p>