Oxidative Stress Induces Changes in Molecular Markers Associated with Ferroptosis in Human Spermatozoa

Ferroptosis is a type of iron-dependent regulated cell death characterized by increased bioavailability of redox-active iron, loss of GPX4 antioxidant capacity, and oxidation of polyunsaturated fatty acid-containing phospholipids mediated by reactive oxygen species (ROS). The aim of this study was to evaluate the effect of oxidative stress induced by arachidonic acid (AA) on ferroptotic cell death in human spermatozoa.

Spermatozoa from normozoospermic donors were exposed to AA (5, 25, and 50 µM) for 1 hour at 37 ℃, including an untreated control. Oxidative stress was confirmed by evaluation of cytosolic and mitochondrial ROS production, viability, mitochondrial membrane potential (ΔΨm) and motility. Subsequently, molecular markers of ferroptosis including iron content, levels of GPX4, SLC7A11, ACSL4, IREB2 and lipid peroxidation were evaluated. The analyses were carried out using either flow cytometry, a microplate reader or confocal laser microscopy.

AA-induced oxidative stress showed increased cytosolic and mitochondrial ROS production accompanied by impaired ΔΨm, viability and motility in human spermatozoa. These results were associated with biochemical and molecular markers related to ferroptotic cell death including an increase in iron content in the form of ferrous (Fe²⁺) ions, SLC7A11, ACSL4, IREB2, a decrease in the level of GPX4, and an increase in the level of lipid peroxidation compared to the untreated control.

This study revealed that AA-induced oxidative stress induces cell death with biochemical characteristics of ferroptosis in human spermatozoa, demonstrating another mechanism of alteration of sperm function induced by oxidative stress and could establish new therapeutic objectives to prevent the decrease in sperm quality mediated by oxidative stress.