Reproducibility of "chaotic" embryo calls by non-invasive PGT-A: a prospective pilot study and implications for...

To evaluate the test-retest reproducibility of "chaotic" embryo calls (≥ 6 whole-chromosome abnormalities) by non-invasive preimplantation genetic testing for aneuploidy (niPGT-A) using cell-free DNA (cfDNA) from spent culture medium (SCM), by repeating niPGT-A after embryo warming and short-term reculture, and to discuss implications for diagnostic validity.

This prospective pilot study included seven vitrified blastocysts previously classified as "chaotic" by niPGT-A. After warming, embryos underwent 4 to 8 hours of individual reculture to enable additional cfDNA release. SCM was collected and processed for repeat niPGT-A using the same whole-genome amplification method (MALBAC), sequencing platform (Illumina MiSeq), and bioinformatic pipeline (ChromGo) employed in the initial test.Laboratory staff were blinded to the original results. An embryo-free negative media control was processed in parallel to monitor environmental contamination. Agreement between the initial and repeat classifications was assessed descriptively.

All cfDNA samples were successfully amplified and generated high-quality sequencing libraries. On repeat analysis, 3/7 embryos (43%) yielded a euploid call. The remaining 4/7 embryos (57%) showed abnormal findings; however, their chromosomal profiles differed from the initial reports, indicating limited agreement for the "chaotic" designation and for individual chromosomal calls.

In this pilot cohort, embryos initially classified as "chaotic" by SCM-based niPGT-A showed substantial discordance upon retesting after warming and short-term reculture. These findings raise concerns about end-to-end reproducibility and support a cautious interpretation of "chaotic" results until larger studies clarify technical from biological sources of discordance and validate SCM calls against embryo tissue and clinical outcomes.