Abstract Study question Does re-expansion grade at 1 hour post-biopsy correlate with post-thawing re-expansion rate and live birth rate? Summary answer Embryo re-expansion 1-hour post-biopsy is associated with post-warming re-expansion and increased live birth. What is known already Blastocyst cryopreservation is a key component of PGT-A procedures. In non-PGT-A cycles, collapsing blastocysts before vitrification is commonly recommended to improve blastocyst viability. However, the procedure becomes more complex in PGT-A cycles.The European Society of Human Reproduction and Embryology (ESHRE) recommends an immediate freeze post-biopsy, although a definitive timeframe remains elusive. To date, no conclusive timing has been agreed upon. Some studies support prolonged culture post-biopsy (> 3hrs), citing improved implantation and pregnancy rates. Conversely, others advocate for rapid cryopreservation, endorsing an immediate approach within an hour, while mostly blastocysts are still collapsed due to the biopsy procedure. Study design, size, duration Retrospective study of 1141 single euploid blastocyst transfers (seFET) conducted between April 2021 and February 2023. Analysed factors included patients' age, BMI, embryo quality at biopsy and 1-hour post-biopsy (at vitrification), and the biopsy day. Live birth and Ongoing pregnancy (OPR), defined as viable pregnancy beyond 24 weeks, were assessed. Blastocysts were scored at a standardized time (1hr post TE-biopsy): 0 for complete collapse, 1 for starting re-expansion, and 2 for clear re-expanded cavity. Participants/materials, setting, methods PGT-A patients undergoing a seFET. Only cycles with embryos being biopsied on Days 5,6 or 7 and with a known pregnancy outcome were included. Main results and the role of chance Out of 1141 transfers, 461 transfers resulted in live birth (40.4%) and 108 were still ongoing above 24 weeks’ gestation at the time of last contact (9.4%) for a total OPR/LB rate of of 49.8%. One-hour post-biopsy 64.0% of blastocysts re-expanded to Grade 2, 29.3% to Grade 1, and 6.7% showed no re-expansion (Grade 0). The OPR was significantly different between embryos that re-expanded to grade 2, 1 or 0 (54.2%, 44.3% and 32.5%, respectively, P < 0.001). Regression analysis showed re-expanding to Grade 2 at 1-hour post-biopsy was significantly and independently associated with OPR/LB compared to embryos that did not re-expand (i.e., Grade 0) (aOR: 1.77, 95% CI: 1.01–3.12, P = 0.047), after adjusting for embryo quality metrics (ICM, TE grade, day of biopsy), body-mass index and endometrial preparation type. . Embryos with greater re-expansion 1-hour post-biopsy had higher re-expansion slopes post-thawing, indicating faster and greater re-expansion to the point of transfer. Results were significant for all trophectoderm categories (Grade A, B, and C; P = 0.016, <0.001 and <0.001, respectively). Moreover, re-expansion rate 1-hour post-biopsy was significantly and independently associated with post-thawing re-expansion slope after adjusting for ICM, TE, expansion grade and biopsy day (P < 0.001). Limitations, reasons for caution The re-expansion grading remains an in-house system that involves subjective assessment with a possible inter-observer variation. Wider implications of the findings This study clearly demonstrates that post-biopsy re-expansion pattern, obtained in a standardized approach, can be helpful for predicting live birth. It is a novel independent marker that is not reflected by ICM, TE quality or day of biopsy for selecting embryos for thawing and transfer. Trial registration number not applicable