Background Cryopreservation is an essential tool for male fertility preservation, yet the freeze–thaw process is known to disrupt sperm function through oxidative stress and structural damage. This study aimed to compare the effects of slow freezing, rapid freezing, and vitrification on the sperm motion characteristics and oxidative profile in normozoospermic donors using a multi-parameter assessment approach. Materials and Methods Semen samples from normozoospermic donors (n = 50) were processed using conventional slow freezing with TEST-yolk buffer, rapid freezing with SpermFreeze™ medium, and a direct vitrification with sucrose and dextran supplementation. Post-thaw sperm quality outcomes included computer-assisted sperm analysis (CASA) kinematics; a multi-class reactive oxygen species (ROS) panel (global ROS, superoxide, hydrogen peroxide, hydroxyl radical); an antioxidant panel (total antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase, glutathione) and oxidative damage markers (malondialdehyde/MDA, protein carbonyls, 8-hydroxy-2'-deoxyguanosine/8-OHdG). Statistical analysis comprised one way ANOVA/Tukey, Spearman correlations, and delta comparisons. Results Vitrification consistently preserved the highest total motility (60.43 ± 4.10%) and progressive motility (43.86 ± 2.36%) among the cryopreserved groups (p < 0.001). Δ-from-fresh (Δ%) emphasized vitrification as most protective against the loss of post-thaw sperm motility (− 23.64%), slow freezing intermediate (− 39.17%), and rapid freezing least protective (− 50.18%) across endpoints. Vitrification resulted in the lowest hydroxyl radical levels (6.99 ± 0.24 RFU/106 sperm) and oxidative damage (0.15 ± 0.01 µmol MDA/106 sperm; 3.77 ± 0.48 ng 8-OHdG/µg DNA). Rapid freezing exhibited the most severe sperm impairment, including a reduced motility (39.43 ± 6.57%), elevated lipid peroxidation (0.27 ± 0.02 µmol MDA/106 sperm), and DNA fragmentation (5.89 ± 0.33 ng 8-OHdG/µg DNA). Strong inverse correlations were observed between total motility and ROS (r = − 0.893; p < 0.0001) and between progressive motility and 8-hydroxy-2'-deoxyguanosine (r = − 0.911; p < 0.0001). Conclusion Under standardized post-thaw handling, vitrification better maintained motility and redox homeostasis among the evaluated protocols. These findings support its potential integration into clinical sperm banking strategies; however, clinical generalizability should be cautious due to protocol-dependent effects.

Motility and Oxidative Status of Human Spermatozoa after Slow or Rapid Freezing, or Vitrification

期刊论文
发表日期：

2025年12月15日

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Eva Tvrdá,
Temidayo S. Omolaoye,
Fawzia AlObeidli,
Stefan S. Du Plessis

摘要

Background

Cryopreservation is an essential tool for male fertility preservation, yet the freeze–thaw process is known to disrupt sperm function through oxidative stress and structural damage. This study aimed to compare the effects of slow freezing, rapid freezing, and vitrification on the sperm motion characteristics and oxidative profile in normozoospermic donors using a multi-parameter assessment approach.

Materials and Methods

Semen samples from normozoospermic donors (n = 50) were processed using conventional slow freezing with TEST-yolk buffer, rapid freezing with SpermFreeze™ medium, and a direct vitrification with sucrose and dextran supplementation. Post-thaw sperm quality outcomes included computer-assisted sperm analysis (CASA) kinematics; a multi-class reactive oxygen species (ROS) panel (global ROS, superoxide, hydrogen peroxide, hydroxyl radical); an antioxidant panel (total antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase, glutathione) and oxidative damage markers (malondialdehyde/MDA, protein carbonyls, 8-hydroxy-2'-deoxyguanosine/8-OHdG). Statistical analysis comprised one way ANOVA/Tukey, Spearman correlations, and delta comparisons.

Results

Vitrification consistently preserved the highest total motility (60.43 ± 4.10%) and progressive motility (43.86 ± 2.36%) among the cryopreserved groups (p < 0.001). Δ-from-fresh (Δ%) emphasized vitrification as most protective against the loss of post-thaw sperm motility (− 23.64%), slow freezing intermediate (− 39.17%), and rapid freezing least protective (− 50.18%) across endpoints. Vitrification resulted in the lowest hydroxyl radical levels (6.99 ± 0.24 RFU/10⁶ sperm) and oxidative damage (0.15 ± 0.01 µmol MDA/10⁶ sperm; 3.77 ± 0.48 ng 8-OHdG/µg DNA). Rapid freezing exhibited the most severe sperm impairment, including a reduced motility (39.43 ± 6.57%), elevated lipid peroxidation (0.27 ± 0.02 µmol MDA/10⁶ sperm), and DNA fragmentation (5.89 ± 0.33 ng 8-OHdG/µg DNA). Strong inverse correlations were observed between total motility and ROS (r = − 0.893; p < 0.0001) and between progressive motility and 8-hydroxy-2'-deoxyguanosine (r = − 0.911; p < 0.0001).

Conclusion

Under standardized post-thaw handling, vitrification better maintained motility and redox homeostasis among the evaluated protocols. These findings support its potential integration into clinical sperm banking strategies; however, clinical generalizability should be cautious due to protocol-dependent effects.