Once, twice, three times frozen: viability and post-transfer outcomes of fresh and vitrified-warmed mouse embryos.

study questionWhat is the impact of one, two, or three vitrification-warming cycles on embryo viability, implantation potential, and post-transfer development compared with fresh transfer? SUMMARY ANSWER: Repeated vitrification-warming cycles progressively impaired embryo cryosurvival, blastocyst quality, and foetal growth after two or more cycles. WHAT IS KNOWN ALREADY: Cryopreservation is central to embryo storage and preimplantation genetic testing, where embryos are biopsied and frozen while awaiting results. However, the impact of repeated vitrification-warming cycles remains unclear due to confounding factors including biopsy effects, prior implantation failure, the absence of a true non-cryopreserved control, and fresh transfer comparisons confounded by supraphysiological hormonal exposure. STUDY DESIGN, SIZE, DURATION: To overcome the constraints of clinical studies, this study used the mouse as a controlled preclinical model. This allowed precise experimental control over embryo stage at cryopreservation and vitrification protocol and enabled direct comparison with a non-vitrified (fresh) control group without the confounding effects of supraphysiologic hormone exposure or biopsy. While species differences are recognized, this model enables focused assessment of cryopreservation-specific effects. Outcomes included post-warming survival, cell lineage allocation, as well as post embryo transfer outcomes on Day 18.5 of development (pregnancy and implantation rates, foetal and placental weights). Post-warming survival was assessed after one, two, or three freeze-warm cycles (3035, 1465, and 447 blastocysts, respectively; 23 independent experimental replicates). Inner cell mass (ICM) and trophectoderm (TE) cell numbers were quantified following one, two, or three freeze-warm cycles and compared to a fresh (non-vitrified) control (35, 36, 33, and 28 blastocysts, respectively; four independent experimental replicates). Post-transfer outcomes were evaluated after one, two, or three freeze-warm cycles and compared to a fresh (non-vitrified) control (30, 33, 39, and 22 pseudo-pregnant recipients, respectively). Sample size calculations were performed to ensure the study was appropriately powered. PARTICIPANTS/MATERIALS, SETTING,

methodsMurine embryos were generated following IVF and cultured to the blastocyst stage, and underwent zero (fresh), one, two, or three repeated vitrification-warming cycles. Post-warming survival was assessed morphologically, and immunohistochemistry for OCT-3/4 (ICM) and CDX2 (TE) was used to assess cell lineage allocation. Embryo transfers were used to further evaluate developmental competence with pregnancy rate, implantation outcomes (implantation rate, viable implantation rate, resorption rate), foetal and placental outcomes (foetal weight, placental weight, and the foetal-to-placental weight ratio) measured on Day 18.5 of development. MAIN RESULTS AND THE ROLE OF CHANCE: Embryo cryosurvival declined significantly with increasing numbers of freeze-warm cycles, with marked reductions after two or three cycles compared with one cycle (P < 0.05) and a substantially higher proportion of non-viable embryos after three cycles (P < 0.001). ICM cell number was significantly reduced after two or three cycles compared with fresh embryos (P < 0.0001), while TE cell number increased after two cycles (P < 0.05). The ICM:TE ratio was significantly reduced after one, two, or three cycles compared with fresh (P < 0.01). Following embryo transfer, pregnancy rates were lower in all freeze-warm groups than in fresh controls, although these differences were not statistically significant. Relative risk of pregnancy compared with fresh transfer was 0.74 (95% CI 0.34-1.64) after one cycle, 0.75 (95% CI 0.35-1.62) after two cycles, and 0.59 (95% CI 0.27-1.32) after three cycles. Implantation and viable implantation rates showed similar nonsignificant reductions, while resorption rates were numerically higher after two or three freeze-warm cycles. Compared with the fresh group, foetal weights were significantly reduced after two and three cycles (P = 0.037 and P = 0.012), placental weight showed a nonsignificant trend towards increase, and the foetal:placental weight ratio was significantly decreased after two and three cycles (P = 0.009 and P = 0.002). While cryosurvival, ICM cell number, and foetal outcomes showed clear statistical effects, the nonsignificant pregnancy, implantation, and resorption measures indicate that chance may partly explain the post-transfer variability. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study used a murine model, and although procedures reflected clinical practice, species differences mean further mechanistic and translational studies are needed to evaluate relevance to human reproduction. Additionally, the study focused exclusively on blastocyst-stage embryos, limiting the applicability of the findings to other developmental stages. The effects of cryoprotectant exposure alone were not investigated. WIDER IMPLICATIONS OF THE

findingsThese findings provide quantitative evidence of the cumulative effects of repeated embryo vitrification-warming and may inform optimization of cryopreservation protocols, limit unnecessary re-freezing, and support refinement of clinical guidelines to improve embryo viability and foetal outcomes in IVF. STUDY FUNDING/COMPETING INTEREST(S): K.R.D. is supported by an Australian Research Council Future Fellowship (Adelaide University, FT240100291). The authors declare that there is no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.