Effect of cryopreservation duration on pregnancy outcomes in embryos: a systematic review and meta-analysis

Is prolonged cryopreservation associated with adverse clinical pregnancy and live birth after frozen-thawed embryo transfer?

Prolonged embryo cryopreservation is associated with lower clinical pregnancy and live birth rates, particularly within the first 3-12 months of storage, but rates plateau after 2 years.

Evidence on the impact of cryopreservation duration on reproductive outcomes remains scarce and conflicting, with some studies reporting no adverse effects and others suggesting increased risks of pregnancy loss, preterm birth, and epigenetic alterations after prolonged storage.

Systematic review and meta-analysis of studies comparing pregnancy outcomes by cryopreservation duration. PubMed, Embase, and the Cochrane Library were searched from inception to 31 December 2025, for English-language articles. The search strategy combined terms for embryos (e.g. 'Embryo', 'Blastocyst'), cryopreservation techniques (e.g. 'vitrification', 'cryopreservation', 'freezing'), and pregnancy outcomes (e.g. 'pregnancy', 'live birth') using Boolean operators. A total of 5208 records were identified from PubMed, 9272 from Embase, and 331 from the Cochrane Library.

Two independent reviewers screened studies, extracted data, and assessed quality using the Newcastle-Ottawa Scale. Inclusion required reporting of clinical pregnancy rate (CPR) or live birth rate (LBR) stratified by cryopreservation duration. Studies on donated embryos, preimplantation genetic testing, or gamete cryopreservation were excluded. Pooled odds ratios (ORs) with 95% CIs were calculated using fixed- or random-effects models based on heterogeneity (I²). Subgroup analyses were performed for neonatal outcomes (singleton vs. multiple births). Publication bias was assessed via funnel plots.

A total of 14 811 records were screened (5208 from PubMed, 9272 from Embase, 331 from Cochrane). After removal of duplicates, 11 077 records were screened by title and abstract, and 785 full-text articles were assessed for eligibility. Twenty-three studies (254 017 transfer cycles/patients) were included. Embryos cryopreserved >5 years had lower survival rates (OR = 0.84, 95% CI: 0.72-0.98, P = 0.03; I ² = 0%, P = 0.99). Compared with ≤3 months storage, longer storage was associated with reduced CPR (>3 months: OR = 0.87, 95% CI: 0.78-0.96, P = 0.005; >6 months: OR = 0.70, 95% CI: 0.66-0.74, P < 0.00001; >12 months: OR = 0.71, 95% CI: 0.61-0.82, P < 0.00001; >2 years: OR = 0.77, 95% CI: 0.65-0.91, P = 0.003), reduced LBR (>3 months: OR = 0.90 95% CI: 0.84-0.95, P = 0.0008; >6 months: OR = 0.74, 95% CI: 0.69-0.80, P < 0.00001; >12 months: OR = 0.72, 95% CI: 0.63-0.84, P < 0.0001; >2 years: OR = 0.80, 95% CI: 0.72-0.89, P < 0.0001) and reduced biochemical pregnancy rate (>3 months: OR = 0.82, 95% CI: 0.73-0.92, P = 0.0007; >6 months: OR = 0.70, 95% CI: 0.63-0.77, P < 0.00001; >12 months: OR = 0.72, 95% CI: 0.65-0.80, P < 0.00001; >2 years: OR = 0.74, 95% CI: 0.61-0.90, P = 0.002). Miscarriage rate (OR = 1.16, 95% CI: 1.09-1.25, P < 0.0001) and preterm birth rate (OR = 1.10, 95% CI: 1.00-1.20, P = 0.04) were higher after >12 months storage. High birth weight and malformation rates were not significantly increased, but low birth weight was less frequent after >6 (OR = 0.83, 95% CI: 0.70-0.99, P = 0.04) or >12 months (OR = 0.75, 95% CI: 0.57-0.98, P = 0.03), and the proportion of male infants was lower after >3 months (OR = 0.92, 95% CI: 0.86-0.98, P = 0.02) and >2 years (OR = 0.79, 95% CI: 0.64-0.97, P = 0.02), compared with those cryopreserved for ≤3 months. No significant publication bias was detected.

Limitations include substantial heterogeneity across studies (design, cryopreservation method, embryonic stage, confounder adjustment), inability to evaluate frozen-thawed embryo transfer (FET) cycle type, maternal age, and embryo quality due to inconsistent reporting, retrospective observational design precluding causal inference, residual confounding, potential survivor bias, and combined singleton/multiple-birth data for neonatal outcomes. Despite a large sample size (254 017 cycles), these factors warrant cautious interpretation.

This comprehensive meta‑analysis reveals a non‑linear relationship between cryopreservation duration and outcomes, reconciling previous conflicting reports by demonstrating early declines (first 3-12 months) followed by a plateau beyond 2 years. The stability of outcomes in ultra‑long storage is reassuring for patients requiring delayed transfer. These findings support clinical counselling that encourages early transfer when feasible, but provides reassurance that ultra‑long storage does not lead to progressive deterioration in pregnancy or neonatal outcomes.

This work was funded by the First-class discipline innovation-driven talent program of Guangxi Medical University (to J.L.) and the College Students' Innovation Training Project of Guangxi Medical University (X202410598348).

The authors declare no conflicts of interest.

CRD42024555362.