Finally, we used frozen-thawed sperm suspensions to validate
the effects of H2 treatment on the motility of damaged
sperm. Frozen sperm suspension is used routinely in
assisted reproduction treatment. However, freezing has
been reported to cause changes in sperm morphology, including
damage to mitochondria. Sperm motility is particularly
sensitive to freezing damage [34,35]. Effects were
evident in 13 out of 15 suspensions of low motile frozenthawed
sperm (86.7%) in which an increase of forward motility
was observed with sperm-wash medium containing
50% of H2 (Figure 6b), indicating that H2 treatment is clinically a potential approach to activate low motility
sperm. However, treatment with medium containing higher
concentration, 100%, of H2 was not effective, indicating the
possibility that very low concentration of O2 in the medium
might repress the sperm motility [36]. Further study is
needed to elucidate a dose-dependency of H2-treatment