Variability in the human-hamster in vitro assay for fertility evaluation

Sources of variability in the zona-free hamster egg penetration assay were evaluated. Internal consistency in the assay was examined in replicate experiments using sperm from 23 donors. The average difference in the percentage of fertilization between the replicates was 3.9%. Optimal preincubation conditions and insemination time were examined and shown to be 0.5 to 1.0 X 10(7) sperm/ml and 2 to 3 hours. Abstinence time was found to be a variable, and a critical abstinence of more than 12 hours was required. Prolonged exposure to seminal plasma (i.e., more than 30 minutes) produced a reduction in the fertilization test results. If the variables studied here are consistently controlled, then changes in the assay results greater than the experimental error should reflect true changes in the semen sample.