Does Prolonged Preservation of Blastocysts Affect the Implantation and Live Birth Rate? A Danish Nationwide Register-Based Study

<b>Background/Objectives:</b> Cryopreservation technology used in assisted reproductive technology (ART) has significantly improved live birth rates by enabling multiple embryo transfers with frozen embryos from a single ovarian stimulation cycle. However, there is conflicting data on the effect of prolonged cryopreservation of human blastocysts. <b>Methods:</b> This Danish nationwide cohort study includes all frozen embryo transfers (FETs) from 1 January 2012 to 31 March 2019. Biochemical pregnancy, clinical pregnancy, and live births were analyzed based on blastocyst storage time. Blastocyst storage time was stratified into five groups, ≤3 month, 4-6 months, 7-12 months, 13-24 months, and ≥25 months, with the shortest (≤3 months) as the reference. We also examined the risk of preterm birth, small and large for gestational age (SGA and LGA), and congenital malformations among live-born children. Multivariable analysis was used to estimate the odd ratios of the reproductive outcomes, accounting for potential confounders. <b>Results:</b> We identified 7042 women with 12,599 FETs. Characteristics of women at embryo transfer did not vary significantly by storage time, except for polycystic ovarian syndrome (PCOS), which increased from 2.6% in the reference group to 6.7% in the ≥25-month group. The clinical pregnancy rate was 35.7%. Blastocyst storage time did not significantly affect biochemical pregnancy rates, with adjusted odds ratios (aORs) of 0.94 (95% CI: 0.80-1.11) to 0.96 (95% CI: 0.82-1.12) for the 13-24-month and ≥25-month groups, respectively. Clinical pregnancy rates also did not decrease with storage time (aOR 0.96, 95% CI: 0.82-1.13) for ≥25 months. The live birth rate was 28.6%, with no significant decrease during storage (aOR 0.89, 95% CI: 0.75-1.06). However, the risk of LGA was slightly, but non-significantly, increased (aOR: 1.42, 95% CI: 0.84-2.42) in the ≥25-month group, whereas the aOR of SGA and congenital malformations was not increased. <b>Conclusions:</b> Our data indicates that storing blastocysts for a period of 25 months does not significantly affect pregnancy chances following assisted reproductive technology treatment.