JBRA Assisted Reproduction 2025;29(Suppl.2 SBRA 2025):142
Poster Presentation
29^th Annual Congress of the SBRA. São Paulo/SP - Brazil, 2025
doi: 10.5935/1518-0557.20263779

P-130. Impact of Different Cooling Curves on Sperm Motility and Vitality After Cryopreservation

Martina Cordini¹, Leticia Avi¹, Ana Carolina Nogueira Martinhago¹, Joao Paolo Bilibio¹, Vera Lucia Langaro Amaral¹

¹ FertiBC Centro de Reprodução Humana - Balneário Camboriú - SC – Brasil
² Universidade do Vale do Itajai-UNIVALI - Itajai - SC – Brasil

Objective: To evaluate the impact of different cooling curves during sperm cryopreservation on post-thaw sperm motility and vitality.
Methods: This experimental study included 21 semen samples from normozoospermic patients according to WHO (2021) criteria. Each sample was divided into two groups according to the cooling protocol applied prior to immersion in liquid nitrogen (LN2). The Vapor Group was exposed directly to LN2 vapor using a floating support, with a temperature decrease of -20.5°C per minute for 10 minutes. The Refrigerated Group underwent a pre-refrigeration phase at 3°C (±2°C) for 20 minutes, with a slower temperature decrease of -0.9°C per minute, followed by exposure to LN2 vapor. After cooling, all samples were submerged in LN2 and stored. Thawing was performed at 37°C for 10 minutes. Sperm motility (total and progressive) and vitality were evaluated before and after cryopreservation using standard optical microscopy and eosin staining for vitality assessment. Statistical analysis was performed using paired t-tests and multivariate analysis, with significance set at p<0.05.
Results: Both cooling protocols resulted in a significant reduction in total motility, progressive motility, and vitality when compared to fresh samples (p≤0.001). The total motility decreased from 76.3% in fresh samples to 58.6% in the Refrigerated Group and 53.3% in the Vapor Group. Progressive motility dropped from 47.9% to 29.7% in the Refrigerated Group and 31.4% in the Vapor Group. Vitality decreased from 85.1% in fresh samples to 64.7% in the Refrigerated Group and 63.7% in the Vapor Group. Despite these reductions compared to fresh samples, no statistically significant differences were observed between the two cooling protocols for total motility (p=0.361), progressive motility (p=0.341), or vitality (p=0.718). The recovery rates for motility and vitality were also similar between both protocols, indicating that the pre-refrigeration step did not provide additional benefits over direct vapor exposure.
Conclusion: Both cooling curves led to a significant reduction in sperm motility and vitality after cryopreservation compared to fresh samples. Furthermore, no significant differences were observed between the Vapor and Refrigerated protocols. These results suggest that adding a pre-cooling step before steam exposure does not improve sperm motility and vitality after thawing, and it also increases freezing time. Therefore, the direct LN2 vapor freezing technique, which takes less time, is more practical, and offers similar results in terms of motility and vitality, may be the technique of choice for sperm freezing.