Optimizing human semen cryopreservation by reducing test vial volume and repetitive test vial sampling.

OBJECTIVE: To investigate optimal test vial (TV) volume, utility and reliability of TVs, intermediate temperature exposure (-88°C to -93°C) before cryostorage, cryostorage in nitrogen vapor (VN2) and liquid nitrogen (LN2), and long-term stability of VN2 cryostorage of human semen. DESIGN: Prospective clinical laboratory study. SETTING: University assisted reproductive technology (ART) laboratory. PATIENT(S): A total of 594 patients undergoing semen analysis and cryopreservation. INTERVENTION(S): Semen analysis, cryopreservation with different intermediate steps and in different volumes (50-1,000 µL), and long-term storage in LN2 or VN2. MAIN OUTCOME MEASURE(S): Optimal TV volume, prediction of cryosurvival (CS) in ART procedure vials (ARTVs) with pre-freeze semen parameters and TV CS, post-thaw motility after two- or three-step semen cryopreservation and cryostorage in VN2 and LN2. RESULT(S): Test vial volume of 50 µL yielded lower CS than other volumes tested. Cryosurvival of 100 µL was similar to that of larger volumes tested. An intermediate temperature exposure (-88°C to -93°C for 20 minutes) during cryopreservation did not affect post-thaw motility. Cryosurvival of TVs and ARTVs from the same ejaculate were similar. Cryosurvival of the first TV in a series of cryopreserved ejaculates was similar to and correlated with that of TVs from different ejaculates within the same patient. Cryosurvival of the first TV was correlated with subsequent ARTVs. Long-term cryostorage in VN2 did not affect CS. CONCLUSION(S): This study provides experimental evidence for use of a single 100 µL TV per patient to predict CS when freezing multiple ejaculates over a short period of time (<10 days). Additionally, semen cryostorage in VN2 provides a stable and safe environment over time.