Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage.

Does cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing? Vitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing. A major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa. This was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol. All human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25-45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen. Among the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000). Extensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing. The presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.