Protective Effects of Ascorbate and Catalase on Human Spermatozoa During Cryopreservation

Human semen cryopreservation in the clinical management of male infertility is complicated by cryodamage to spermatozoa. We aimed to clarify the full pattern of cryodamage and evaluate the protective effects of ascorbate and catalase on cryopreserved spermatozoa. Semen samples were collected from 30 fertile males. Each sample was divided into 6 groups: fresh semen, cryopreserved semen without treatment, and samples cryopreserved with ascorbate (300 or 600 mu M) or catalase (200 or 400 IU/mL). Spermatozoa were examined for their viability, motility, reactive oxygen species (ROS) generation, mitochondrial membrane potential (MMP), apoptosis (positive for annexin V and negative for propidium iodide [ie, Ann(+)/ PI-]), and DNA damage (Olive tail moment [OTM]) in the presence or absence of ascorbate or catalase during cryopreservation. In comparison with the fresh spermatozoa, there was a significant decrease in the viability, motility, and MMP but increase in Ann(+)/PI- and OTM in the cryopreserved spermatozoa (P < .01 and P <.05, respectively). Concurrently, ROS levels in the postthaw spermatozoa also increased significantly, and this elevation was well correlated with the quality variations of postthaw spermatozoa (P < .01 for all). Ascorbate (300 mu M) and catalase (200 and 400 IU/mL) reduced the ROS levels in postthaw spermatozoa significantly, compared with those in the control (P < .05). Furthermore, these antioxidants also prevented those characteristics from being adversely affected (P < .05). This study demonstrated that cryopreservation results in cryodamage to human spermatozoa, possibly through the mechanism of ROS. Appropriate ascorbate or catalase supplementation of cryoprotective medium restrains ROS levels and the resultant cryodamage. Key words: Reactive oxygen species, mitochondrial membrane potential (MMP), DNA damage. J Androl 2010;31:437-444