Impact of repeated cryopreservation on embryo health and implantation potential

In IVF clinics, preimplantation genetic testing (PGT) is a common practice that involves a biopsy and cryopreservation of embryos for genetic evaluation. When testing fails or is required for already cryopreserved embryos, multiple freeze-thaw cycles occur. Though known to impact live birth rates, the exclusive influence of cryopreservation has not been elucidated. Here, we evaluate the effect of repeated cryopreservation on embryo health and implantation potential. Blastocyst-stage murine embryos were subjected to one, two or three freeze-thaw cycles with fresh embryos serving as a control. Outcomes assessed included post-thaw survival rate, allocation of cells to the inner cell mass (ICM) vs. trophectoderm cell lineages, implantation potential and offspring health. Post-thawing, embryos that were subjected to three freeze-thaw cycles had a significantly lower survival rates compared to embryos that had undergone one cycle (P<0.001). Additionally, the number of ICM cells was significantly reduced in embryos subjected to two or three freeze-thaw cycles compared to fresh or single-cycle embryos (P<0.001). No statistically significant differences were found for pregnancy rate, number of implantations, viable fetuses or resorption sites between treatment groups. We did however, find a non-significant yet interesting trend: three freeze-thaw cycles were associated with a 20% decrease in viable fetuses and a 20% increase in resorption sites compared to one freeze-thaw cycle group. These findings demonstrate that repeated cryopreservation adversely affects embryo health and may decrease implantation potential. Consequently, caution is advised regarding the repeated application of cryopreservation in IVF clinics, underscoring the need for further research to optimise cryopreservation protocols. ### Competing Interest Statement The authors have declared no competing interest.