The reactive oxygen species (ROS) test measures the oxidative stress the spermatozoa may be exposed to. High levels of oxidative stress result in damage to sperm DNA, RNA, and telomeres and, therefore, might provide a common underlying etiology of male infertility and recurrent pregnancy loss, in addition to congenital malformations.
ROS in semen are measured by chemiluminescence (luminol) according to WHO procedure.
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ROS includes many reactive molecules and free radicals derived from molecular oxygen. These molecules damage DNA and RNA and oxidize proteins and lipids (lipid peroxidation). The most common ROS include superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (HO-), and singlet oxygen (1O2), all of which are more reactive than oxygen (1O2) itself.
The physiological role of ROS in spermatozoa
Physiologically, ROS are considered regulators of several intracellular pathways, modulating the activation of different transcription factors. Several studies showed that higher ROS levels stimulate sperm capacitation and hyperactivation, acrosome reaction, motility and chemotaxis, and chromatin compaction in maturing spermatozoa. Furthermore, ROS can improve sperm capacity for binding to the zona pellucida, inducing sperm-oocyte fusion.
The pathological role of ROS in spermatozoa
In addition to their physiological role, excessive ROS generation and oxidative stress seem to be associated with harmful effects on spermatozoa. These effects result in alterations in morphological and dynamic cellular properties and, finally, in lower fertilization ability.
An altered redox balance in the seminal fluid may display deleterious effects on sperm homeostasis, leading to male infertility. ROS-mediated sperm oxidation may induce cellular dysfunctions, affecting spermatozoa concentration, total number, and motility.
Spermatozoa are particularly susceptible to ROS-induced oxidation due to their plasma membrane's elevated levels of polyunsaturated fatty acids.
Lipid peroxidation is strictly associated with fluidity and permeability membrane alterations, inhibition of membrane-bound enzymes and receptors, and activation of the apoptotic cascade, supporting oxidative stress involvement in motility and morphology sperms abnormalities.
It is accepted that nucleic acids represent another crucial target of oxidative stress. Both nuclear and mitochondrial DNA are vulnerable to hydroxyl radical (OH.) attack, forming several oxidative stress biomarkers. OH. can react with guanine to produce 8-hydroxy-2′-deoxyguanosine (8-OH-G), an essential marker of DNA oxidative damage, detectable in several biological samples.
The lack of adequate antioxidant systems makes spermatozoa highly susceptible to DNA oxidation. Sperm DNA oxidation is also due to the lack of complete DNA repair strategies in spermatozoa.
Several studies have indicated that ROS generation is associated with DNA fragmentation and poor chromatin packaging. This promotes apoptosis, which has relevant consequences for sperm count.
Main Sources of ROS
It is broadly accepted that several exogenous factors may contribute to inflammation and redox status alterations, promoting male infertility. Environmental pollution, lifestyle factors such as smoke, alcohol, obesity, varicocele, bacterial and viral infections, microorganism mutations, or sexually transmitted disorders are actively involved.
However, seminal fluid oxidative stress is primarily caused by leukocytes, which produce 1,000 times more ROS than normal and immature spermatozoa.
Leukocytospermia. Granulocytes and macrophages are the main cell types in the ejaculate and are responsible for generating ROS.
Immature spermatozoa. When spermatogenesis is defective, alterations in cytoplasmic extrusion mechanisms are observed, and spermatozoa are released with excess residual cytoplasm (cytoplasmic droplets). Immature spermatozoa are associated with higher ROS generation.
Mitochondria. Mitochondria represent another potential ROS source in spermatozoa. Different factors may alter the electron transport chain on the mitochondrial membrane, resulting in excessive ROS generation. Several reports indicate sperm mitochondrial dysfunction and oxidative stress as potential factors in asthenozoospermia.
