Preconditioning of sperm with sublethal nitrosative stress: a novel approach to improve frozen-thawed sperm function.

RESEARCH QUESTION: Can sublethal stress induced by nitric oxide on fresh human spermatozoa protect the functional properties of post-thaw human spermatozoa?

DESIGN: Semen samples were obtained from 46 donors. Twenty semen samples were used to determine toxicity level of nitric oxide by incubation of semen with different concentrations of nitric oxide (0.01 to 400 μM). Then, 26 semen samples were cryopreserved with optimized ranges of nitric oxide: control (NO-0.00), 0.01 μM nitric oxide (NO-0.01), 0.1 μM nitric oxide (NO-0.1), 1 μM nitric oxide (NO-1), 10 μM nitric oxide (NO-10), 100 μM nitric oxide (NO-100). Frozen-thawed spermatozoa were assessed for motion characteristics, viability, morphology, apoptosis-like changes, caspase 3 activity, DNA fragmentation and intracellular reactive oxygen species levels. Fertilization potential was investigated by heterologous piezo-intracytoplasmic sperm injection (piezo-ICSI) of human spermatozoa into mouse oocytes.

RESULTS: In fresh spermatozoa, nitric oxide did not induce a negative effect, except a significant reduction in motility and viability at 200 µM and 400 µM (P < 0.05). Cryopreservation significantly reduced sperm motility and increased reactive oxygen species, apoptosis-like changes, caspase 3 activity, and DNA damage (P < 0.05). NO-0.01 significantly increased total and progressive motility versus the other groups (P < 0.05). The lowest percentage of caspase 3 activity was in the NO-0.01 and NO-0.1 compared with the other freezing groups. In the fertilization trial, the rate of two-cell embryo formation after heterologous piezo-ICSI was higher (P < 0.05) in NO-0.01 (69%) versus controls (42%).

CONCLUSIONS: Sublethal oxidative stress induced by nitric oxide might improve human sperm function after cryopreservation.