Quality control and long-term storage of semen samples in liquid nitrogen vapor

Abstract

OBJECTIVE: Cryopreservation of human spermatozoa is pivotal for fertility preservation in men at risk of fertility loss due to cancer or surgical intervention. Initial screening of semen prior to cryopreservation is often impractical. While screening should be offered prior to semen use, storage of samples in liquid nitrogen (LiN) may pose a theoretical risk of cross-contamination if storage vessel integrity is compromised. For this reason it has been suggested that storage in LiN vapor may be superior. Knowledge regarding long-term quality control and storage of semen samples in LiN vapor is wanting. Thus, objectives of this study were to provide information on quality control and storage success of semen samples over numerous years in LiN vapor. DESIGN: Observational IRB-approved study. MATERIALS AND METHODS: A semen sample was obtained from a single normozoospermic individual in 11/2003. After initial evaluation, it was aliquoted, cryopreserved, and stored in LiN vapor on the top rack, furthest from the LiN. One aliquot was thawed and assessed each month until 2/2008. Functional semen parameters assessed were percent motility, forward progression (FP), and speed. LiN level, usage, and intra-tank temperatures (top: TTT and middle: TTM) were assessed daily. Sperm cryosurvival was assessed for temporal change by mixed linear regression model, as well as compared to historical running average of sperm survival during storage in LiN using Student's t-test. Significance was considered at p≤0.05. RESULTS: Initial quality control sample evaluation showed 60% motility, 80% FP and speed of 3 (arbitrary units) prior to cryopreservation. First thawed evaluation occurred the next day noting 50% motility, 75% FP, speed 3 equating to an 83% cryosurvival which was not significantly different from the historical running average of 82% survival when stored in LiN. Fifty subsequent monthly quality control evaluations noted mean±SE motility, FP and speed of 49.8±0.14, 75.9±0.27, 3±0 respectively; with no significant change over time. Over the 51 month period, average LiN use: 12.8±0.4 mm/day, TTT: -168±0.9°C, and TTM: -189±0.5°C were not significantly different when compared over time. CONCLUSIONS: Liquid nitrogen vapor provides a stable environment for long-term storage of cryopreserved semen samples with similar cryosurvival rates as LiN storage. If properly maintained and quality controlled, LiN vapor provides a safe option for sample storage without potential interactions between intra-liquid nitrogen samples.