Library Concentration After Whole Genome Amplification Improves Interpretation of Spent Culture Medium-Based Noninvasive PGT-A in Vitrified-Warmed Human Blastocysts

Purpose: To evaluate the feasibility and performance of spent culture medium (SCM)-based non-invasive pre-implantation genetic testing for aneuploidy (niPGT-A) after 8 h of post-warming in vitrified-warmed human blastocysts, and to determine whether post-amplification library concentration improves SCM-based result interpretation. Methods: This study included 26 discarded vitrified-warmed blastocysts from 17 patients. After 8 h of post-warming culture, SCM was collected and analyzed by next-generation sequencing together with trophectoderm (TE) biopsy samples and whole blastocysts (WB). Concordance at the chromosome and category levels was evaluated between SCM and WB and between TE biopsy and WB, and the association of library concentration with performance and euploid probability was assessed. Results: SCM analysis was informative in 92.3% (24/26) of embryos. Chromosome-level concordance with WB was lower for SCM than for TE (30.8% vs. 57.7%), whereas category-level concordance was 69.2% for SCM and 76.9% for TE. Combining SCM category with library concentration improved prediction of euploid embryos (AUC 0.9527 vs. 0.8077, p = 0.0158). The leave-one-out cross-validated AUC of the combined model was 0.896. Conclusions: SCM-based niPGT-A after 8 h of post-warming culture is feasible in vitrified-warmed human blastocysts. Library concentration improves interpretation of SCM-based results and may help identify embryos likely to be euploid.