*1.3.3 Motility*

Decreased motility has been shown to be due to ROS-induced peroxidation of lipids in the spermatozoa membrane decreasing flexibility and by inhibition of motility mechanisms [24, 25]. The axosome and associated dense fibers of the middle pieces in spermatozoa are covered by mitochondria that generate energy from intracellular stores of ATP. It is well established that ROS can induce axonemal and mitochondrial damage, resulting in the immobilization of spermatozoa [26, 27]. In addition, ROS-induced damage of mitochondrial DNA leads to decreased ATP and energy availability and leads to activation of caspases and ultimately apoptosis, impeding spermatozoa motility [28, 29]. H2O2 can diffuse across the membranes of spermatozoa and inhibit the activity of some vital enzymes such as glucose-6-phosphate dehydrogenase (G6PD), which is an enzyme controlling the intracellular availability of NADPH. This is used as a source of electrons by spermatozoa to fuel the generation of ROS by an enzyme system known as NADPH oxidase [30]. Another hypothesis involves a series of interrelated events resulting in a ROS-reduced motility due to a decrease in axonemal protein phosphorylation and mitochondrial membrane damage and leakage of intracellular enzymes [31]. Meanwhile, cytochrome c release during the apoptotic pathway further increases levels of ROS, promoting DNA damage and fragmentation [32]. Especially after frozen–thawed cycles, spermatozoa with higher levels of oxidative stress have higher levels of caspase activation that can trigger apoptosis [33].

## *1.3.4 Apoptosis*

High levels of ROS disrupt the mitochondrial membranes, inducing the release of the cytochrome c protein and Ca2+ and activating the caspase-inducing apoptosis [34]. Apoptosis in spermatozoa also may be initiated by ROS-independent pathways involving the cell surface protein Fas, which is a type I membrane protein that belongs to the tumor necrosis factor-nerve growth factor receptor family and mediates apoptosis [35]. Mitochondrial exposure to ROS also results in the release of apoptosis-inducing factor (AIF), which directly interacts with the DNA to cause DNA fragmentation in spermatozoa [36, 37].

## *1.3.5 Fertilization, pregnancy and miscarriage*

Lipid peroxides and DNA damage are the most typical oxidative stress injury in sperm. Lipid peroxides are spontaneously generated in the sperm plasma

membrane, which induce decrease in fertility during storage of semen [38]. In addition, the importance of sperm DNA damage is brought to light when studies correlated the degree of DNA damage with various indices of fertility such as the fertilization rate, embryo cleavage rate, implantation rate, pregnancy rate and live birth rate of the offspring. If sperm DNA is unable to decondense after entering the ooplasma, fertilization may not take place or a postfertilization failure may occur when sperm DNA is defective by ROS. Higher miscarriage rate is observed with ROS-induced sperm DNA damage [39]. High-level sperm DNA fragmentation induced was related to lower pregnancy rates in in vitro fertilization (IVF) but not in intracytoplasmic sperm injection (ICSI) cycles, whereas it was associated with higher miscarriage rates in both IVF and ICSI cycles. In addition, ROS actively participate in metabolic pathways during sperm activation, which leads to cholesterol efflux, cyclic adenosine monophosphate (cAMP) production and tyrosine phosphorylation, important events that contribute to fertilization competence [40]. However, it has been also described that appropriate ROS (hydrogen peroxide stimulation) can promote the acrosome reaction and sperm hyperactivation with the mechanism of ROS-modulated tyrosine phosphorylation [41], thereby assisting the sperm's transit through the cumulus and zona pellucida [42].
