Impact of repeated cryopreservation on embryo development and chromosomal ploidy

The potential adverse effects of repeated freezing and thawing processes on embryos remain controversial. This study aimed to investigate the impact of repeated embryo cryopreservation by comparing preimplantation genetic testing for aneuploidy (PGT-A) outcomes and clinical results of single vitrification and double-vitrification embryos.

This study analyzed the data of 2,335 embryos from 309 patients who underwent PGT-A. Embryos were divided into two groups based on the total number of vitrification procedures: (1) the CV - group (single vitrification, n = 1281), where fresh embryos were directly cultured to the blastocyst stage and vitrified once after trophectoderm biopsy, and (2) the CV + group (double vitrification, n = 1054), where cleavage-stage embryos underwent an initial vitrification, were subsequently warmed and cultured to the blastocyst stage for biopsy, and then vitrified again.Please check and confirm that the authors and their respective affiliations have been correctly identified and amend if necessary.We confirm that the author list and their respective affiliations have been carefully verified and are correct. We have made minor editorial amendments to the English wording of the institutional names to ensure accuracy and consistency with official nomenclature.

The CV + group exhibited significantly lower blastocyst formation rate (39.6% vs. 46.6%, adjusted difference: - 6.7%, P = 0.004), high-quality blastocyst formation rate (11.2% vs. 16.7%, adjusted difference: - 5.3%, P = 0.001), and euploid rate per cleavage-stage embryo (7.8% vs. 10.6%, adjusted difference: - 2.6%, P = 0.043) than the CV - group. In contrast, the euploid rate per biopsied blastocyst was not significantly different between groups (19.0% vs. 21.9%, P = 0.175). Complementary embryo-level mixed-effects logistic regression likewise showed no statistically significant association between double vitrification and embryo euploidy outcomes (adjusted OR 0.72, 95% CI 0.49-1.07; P = 0.101). Age-stratified analyses suggested that these detrimental laboratory effects appeared numerically greater in women aged ≥ 38 years, although formal interaction testing did not support statistically significant effect modification by age. No statistically significant differences were observed in live birth or neonatal outcomes following euploid blastocyst transfer; however, these exploratory comparisons were based on limited transfer numbers and, therefore, should be interpreted cautiously.

Repeated vitrification-warming exposure was associated with reduced embryo developmental efficiency and a numerically lower euploid embryo yield per cleavage-stage embryo. No statistically significant differences were observed in reproductive outcomes after euploid blastocyst transfer; however, these analyses were exploratory and substantially underpowered to exclude clinically meaningful differences. Larger adequately powered studies are required to further clarify the reproductive implications of repeated cryopreservation.