Optimize human sperm cryopreservation: influence of vial sealing methods and automated freezing protocols on preserved sperm quality

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Background: Cryopreservation of human sperm is an essential procedure for fertility preservation in oncological patients. Despite its clinical relevance apart from the recommendations of the World Health Organization (WHO) Laboratory Manual for the Examination and Processing of Human Semen, there is yet impaired guidance on practical implementation. The aim of this study was to optimize key steps during sperm cryopreservation, particularly the straw sealing method and the freezing protocol to ensure stable and consistently high outcomes with regard to sperm vitality and motility following thawing. Methods: Semen samples from 30 healthy donors were analyzed in this study conducted at the University of Tübingen, Germany. Validation of a heat-sealing closure method was performed by its comparison with the conventional method using metal ball seals, at sealing durations of 2.5 and 4.9 seconds (s), respectively. In addition, a novel, standardized and reproducible cryopreservation protocol was developed using the computer-assisted, fully automated IceCube XS-11 freezing system. This protocol was subsequently evaluated and validated by comparison with a standard, semi-automated freezing device (Nicool system) with regard to its potential application in routine clinical practice. Results: Heat sealing with the CryoSealer for 2.5 s proved to be fully airtight and produced equal or improved outcomes in vitality and motility compared to the conventional ball-capping method (medians 46.65% vs. 45.35% for vitality, 18.60% vs. 13.95% for total motility). After thawing, no significant differences were observed in total or progressive motility between the Nicool and IceCube systems. However, sperm viability was significantly higher in the IceCube system (56.0% vs. 48.5%; P=0.02). Conclusions: The developed IceCube protocol can be implemented as a new standard for cryopreservation. Further studies are warranted to evaluate remaining variables—such as cryoprotectant selection, thawing strategies, and sample-specific factors—to enable continued optimization of the cryopreservation process.

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