Reactive oxygen species are involved in several cellular signaling mechanisms and they can interact with lipids, proteins and DNA, leading to severe pathologic conditions. High ROS levels are detrimental to gametes, and compromise their function through lipid peroxidation, protein damage and DNA strand breakage . Although, spermatozoa physiologically produce ROS promoting sperm capacitation, in some pathological conditions the semen ROS levels exceeds the sperm antioxidant defenses and lead to a state of oxidative stress that could impair not only fertilization but also embryo development . In vivo studies suggested that oral administration of antioxidants improves semen quality and pregnancy rates in subfertile men [46, 47]. However, among several studies on the effect of antioxidants on semen quality, only a few were addressed to understand their direct action on sperm physiology in vitro. To this end, in the present study we evaluated the effects of zinc, D-Asp and CoQ10 on sperm physiology in vitro. Main results demonstrated that the in vitro treatment of human spermatozoa with zinc, D-Asp and CoQ10 1) preserves sperm motility and kinetics; 2) avoids sperm lipid peroxidation, and 3) DNA fragmentation.
To our knowledge, the present work represents the first addressing the effects of a combined in vitro treatment with zinc, D-Asp and CoQ10 on human spermatozoa. Several studies have been carried out on the role played by each of these molecules on spermatogenesis, sperm quality and fertility.
Zinc concentration in seminal plasma is generally higher than in serum and has been positively correlated with sperm count and motility [21, 48, 49]. On the other hand, a negative effect of high zinc levels in seminal plasma or in sperm tails on sperm motility has been reported by others [50, 51]. Despite these contradictory results, the role of zinc as an antioxidant is well established. Zinc in vitro is able to inhibit both superoxide anion generation and SOD-like activity in spermatozoa of infertile men .
Little is known about the role of D-Asp in human reproduction. The concentration of D-Asp in seminal plasma and in spermatozoa was significantly reduced in oligoasthenoteratospermic men . Moreover, DL-Aspartic acid administration has been reported to improve semen quality in rabbits .
CoQ10 has a bioenergetic and an antioxidant role and has been suggested to be involved in male infertility . High levels of oxidative stress and a low antioxidant capacity in varicocele patients has been correlated to an altered distribution of CoQ10 in spermatozoa and seminal plasma . CoQ10 administration has been reported to have a positive role in the treatment of asthenozoospermia .
Our results on the in vitro effects of zinc, D Asp and CoQ10 confirm the role played by the single molecules on human spermatozoa and demonstrate that they are able to protect spermatozoa from the oxidative stress during in vitro manipulation. Analysis of sperm motility and kinetics demonstrated that the supplementation of culture media with the three molecules prevents the drop of these values observed in medium alone. Such an effect was evident in 75% of samples in which a significant decrease of motility and kinetics was observed at 6 hours of incubation in medium alone. This indicate that a preliminary analysis of sperm motility dynamics on washed semen samples could help to identify responsive patients. The lack of response in 25% of samples analysed could be due to the presence of a correct balance between antioxidant defenses and ROS generation in those ejaculates.
Sperm plasma membrane plays fundamental roles during sperm transport within the female reproductive tract, in sperm capacitation, in sperm–egg interaction and, finally, in fertilization. For these reasons, the plasma membrane lipid composition of spermatozoa is different from somatic cells for the high content of highly polyunsaturated fatty acids. These unsaturated fatty acids confer to the sperm membrane a great fluidity needed to participate in the membrane fusion events associated with capacitation and fertilization. On the other hand this makes spermatozoa particularly vulnerable to the attack by ROS, and therefore more susceptible to undergo lipid peroxidation . Several studies examined the role of in vitro and in vivo antioxidant supplementation in protecting sperm from lipid peroxidation due to an imbalanced ROS production [55–57]. In the present paper, data demonstrated that treatment with zinc, D Asp and CoQ10 protects sperm plasma membrane from lipid peroxidation. This protection could be due to CoQ10 that has been previously reported to decrease lipid peroxidation when administered in a rat model of ischemia/reperfusion injury .
High levels of ROS can induce DNA damage in spermatozoa in every moment of their life. This damage can be produced during comigration of mature and immature spermatozoa from the seminiferous tubules to the caudal epididymis where sperm are highly packed and this would facilitate ROS-induced DNA damage . Moreover, the process of DNA fragmentation in spermatozoa progresses even after ejaculation. In vitro incubation of swim-up selected human spermatozoa results in a progressive increase in the percentage of DNA fragmented sperm .
In agreement with those studies we found that sperm culture for 6 hours induces an increase of sperm DNA fragmentation and, more interestingly, it is prevented by antioxidant treatment. According to Aitken et al.,  sperm oxidative stress not only could impair the sperm fertilizing ability but also its competence to sustain a correct embryo development. Moreover, DNA damage in human spermatozoa has been correlated with increased miscarriage rates and morbidity in the offspring [59, 60]. The possibility to answer these questions directly in the human is limited by a number of ethical and methodological reasons. Studies in animal models could provide insights into these fundamental questions.