Sperm immobilization test and quantitative sperm immobilization test using frozen-thawed sperm preparation applied with computer-aided sperm analysis

Purpose In a previous study, a new method was described using the sperm immobilization test (SIT) with computer-aided sperm analysis (CASA). However, obtaining high-quality sperm as needed was a known issue. Here, we compared the results of using frozen-thawed sperm and fresh sperm for the SIT using the CASA method. Methods For the frozen-thawed preparation, 500 mu L of condensed semen and 500 mu L of Sperm Freeze were mixed in a cryovial and cryopreserved in liquid nitrogen. Density gradient centrifugation was used for the collection of motile sperm in both the fresh and frozen-thawed sperm preparations. A total of 50 serum samples were prepared for both the fresh and frozen-thawed sperm with each sample tested containing 10 mu L of serum, 1 mu L of either fresh or frozen motile sperm suspension, and 2 mu L of complement. Sperm motilities were measured using CASA after a 1-hour incubation period for both fresh and frozen-thawed sperm. Results Both fresh and frozen-thawed sperm reacted similarly when exposed to serum containing sperm-immobilizing antibodies asserting the use of frozen-thawed sperm for the diagnosis of immunological infertility. Conclusion These results suggest the possibility of using cryopreserved sperm for the SIT when fresh sperm is unavailable.