A Rapid Qc Testing Platform Using Frozen Semen

Prien, S. D.; Penrose, L. L.

Objective: The last twelve months have presented significant challenges for the ART laboratory. Mandatory shutdowns, lack of patient access, supply chain issues, and changing rules and recommendations brought on by COVID-19 have stretched laboratories to their limits in an attempt to maintain regular and required activities. One area of concern in the laboratory has been the lack of available fresh semen (FS) samples at the proper times for quality control (QC) and proficiency testing (PT). Cryopreserved semen (CS) would appear a reasonable alternative. However, the quality of CS is known to deteriorate much faster than FS, even in favorable culture conditions. The goal of the present study was to determine, given the limitations of CS samples, if a protocol could be developed for QC and PT testing using CS. Materials and Methods: Using materials of known quality from previous PT challenges, 7 commercial donor semen samples were thawed and prepared for quality control monitoring as follows. Samples were thawed using bank-specific protocols. Each thawed sample was split in half and prepared using an IUI wash protocol with the assigned PT challenge media, either tainted or un-tainted. Once prepared, samples were maintained at 37oC, room air, and 95% relative humidity. Starting at 0 hrs, the samples underwent a semen analysis hourly using an IVOS semen analyzer for a minimum of 6 hrs or until one sample in the pair reached 0% motility after the 6 hr time-point. The resulting data were compared using a paired student’s T-test. Further, results were compared with reports from laboratory PT to verify the efficacy of using frozen semen. Results: As expected semen parameters decreased over time regardless of treatment (P < 0.001). No pair of samples lasted more than seven hours of incubation. While sperm in the non-tainted media maintained at least 60% of its initial motility at 3 hrs (range 64-91%), none of the cells in the tainted media had more than 50% motility at that time point (range 12-43%;p < 0.001). Further by six hours, all but one of the seven samples in the tainted media had 0% motility (range 0-4%), while six of seven samples in the non-tainted media still maintained a minimum of 25% of their initial motility at thaw (22-37%: P < 0.001). Further, all samples correlated with previous PT results. Conclusions: The data suggest it is possible to perform a rapid sperm QC assay using CS. Having a secondary QC protocol would not only provide an alternative when fresh semen, mice embryos, or other methods are unavailable, it would also potentially allow for more standardized methods of QC and PT testing. Impact Statement: The past twelve months have taught that unexpected and uncontrollable events can disrupt routine procedures. Sperm QC assays, which are the mainstay for QC and PT in many andrology laboratories, are dependent on the availability of fresh semen. If a standardized CS method can be created, QC and PT could be done at the convenience of the lab without sacrificing quality or patient safety.