Calibration of sperm concentration for in vitro fertilization in a mouse reprotoxicity model

Xenobiotics, such as chemicals and pesticides, may result in adverse effects on reproduction in human and animals. Using in-vitro embryo production as a testing system reveals details of fertilization (IVF) and early embryonic development (IVC). The aim of our study was to perform a systematical calibration of sperm concentration in an IVF/IVC system, using an outbred mouse strain, and further determine the sperm concentration that furnishes a sensitive assessment of sperm fertilizing capacity in relation to reprotoxic evaluations. By performing breakpoint analysis, the results revealed a maximum two-cell percentage (51%, 95% CI: 38 to 69%) at 3.6 × 10⁴ sperm/ml (95% CI: 2.1 × 10⁴ to 6.1 × 10⁴). For future application of the IVF/IVC system, a sperm concentration lower than this breakpoint concentration is required to be within the responsive range for determining sperm fertilizing capacity. We conclude that a relatively low sperm concentration (2.5 × 10⁴ sperm/ml) is a precondition in a mouse IVF/IVC system in order to detect potential reprotoxic effects on sperm fertilizing capacity. Our study illustrates that a systematic approach is necessary for validation and appropriate use of such in-vitro system used for reproductive toxicity testing.