Purpose <p>To evaluate the impact of multiple vitrification-warming on clinical outcomes of embryos that have undergone preimplantation genetic testing (PGT).</p> Methods <p>This retrospective, single-center study involved 512 single frozen euploid blastocyst transfer cycles conducted between January 2018 and January 2024. Using propensity score matching, 105 patients who underwent double vitrification-warming with single biopsy (DVSB) were matched in a 1:4 ratio with 407 patients in the control group, who underwent single biopsy with single vitrification-warming (SVSB). Post-warming suitability for biopsy and clinical outcomes of subsequent frozen-thawed embryo transfer (FET) cycles were evaluated. Obstetrical and neonatal outcomes were also assessed.</p> Results <p>A total of 453 blastocysts were warmed, of which 417 (92.1%) exhibited sufficient quality to undergo trophectoderm biopsy. In FET cycles, a significant reduction in the live birth rate was observed with the additional vitrification-warming step (DVSB: 49.5% vs. SVSB: 61.2%). The pregnancy loss rate in the DVSB group was significantly higher compared to the SVSB group (20.6% vs. 12.2%). Neonatal outcomes, including sex ratio, preterm birth rate, and low birth weight rate, did not differ significantly between the two groups.</p> Conclusion <p>This study suggests that the inclusion of an additional vitrification-warming step in PGT may adversely affect pregnancy outcomes. Furthermore, only a portion of thawed embryos are able to re-expand and progress to the biopsy stage. PGT patients should be informed that undergoing a second vitrification-warming cycle may reduce the likelihood of a successful pregnancy.</p>