JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):77
Poster Presentation
29th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263656
P-65. Does the cooling curve matter? Impact on sperm DNA fragmentation after cryopreservation.
Leticia Avi1, Ana Carolina Nogueira Martinhago1, Martina Cordini1, Vera Lucia Langaro Amaral2, Joao Paolo Bilibio1
1 FERTIBC Centro de Reprodução Humana - Balneário Camboriú - SC - Brasil
2 Universidade do Vale do Itajai – Itajai -SC - Brasil
Objective: To evaluate the impact of two different cooling curves during sperm cryopreservation on DNA fragmentation index after thawing.
Methods: This experimental study involved 21 semen samples from normozoospermic patients according to WHO (2021) criteria. Samples were divided into two groups according to the cooling protocol applied before immersion in liquid nitrogen (LN2). The Vapor Group was exposed directly to LN2 vapor using a floating support, with a cooling rate of -20.5°C per minute for 10 minutes. The Refrigerated Group underwent a pre-refrigeration step at 3°C (±2°C) for 20 minutes, with a slower cooling rate of -0.9°C per minute, followed by exposure to LN2 vapor. Subsequently, all samples were submerged in LN2 and stored in cryogenic tanks. Thawing was performed at 37°C for 10 minutes. DNA fragmentation was assessed before freezing and after thawing using the sperm chromatin dispersion (SCD) test. Sperm with large or medium halos were classified as non-fragmented, while sperm with small or absent halos were considered fragmented. The DNA Fragmentation Index (DFI) was calculated as the percentage of fragmented sperm among 100 counted cells. Statistical analysis was performed using paired t-tests and multivariate analysis, with a significance threshold of p<0.05.
Results: The mean DNA Fragmentation Index in fresh samples was 27.4%. A significant increase in DNA fragmentation was observed in both groups after cryopreservation compared to fresh samples (p≤0.001), in Refrigerated Group showed a mean DNA Fragmentation Index of 39.6%, while the Vapor Group exhibited a mean DFI of 35.9%. When comparing the two cooling protocols, the Refrigerated Group had significantly higher DNA Fragmentation Index than the Vapor Group (p=0.001), indicating that the additional refrigeration step before vapor exposure resulted in greater DNA damage. These findings suggest that the faster cooling protocol used in the Vapor Group is less harmful to sperm DNA integrity.
Conclusion: The protocol using direct exposure to LN2 vapor without prior refrigeration demonstrated lower DNA Fragmentation Index values compared to the Refrigerated Group, however both cooling curves resulted in a significant increase in sperm DNA fragmentation after cryopreservation. This indicates that avoiding the refrigeration step may help reduce DNA damage during sperm cryopreservation.