Effects of Caffeic Acid Supplementation on Human Sperm Against In Vitro-Induced Oxidative Stress: Nrf2 Molecular Pathway

Oxidative stress (OS) is a major cause of defective sperm function. During laboratory handling, gametes are exposed to OS, potentially mitigated by in vitro antioxidant supplementation. This study evaluates the protective role of caffeic acid (CAF) on basal human semen and under induced OS. First, six semen samples from normozoospermic donors were incubated with CAF concentrations ranging from 50 to 500 µM at 37 °C for 2 h. Sperm motility and DNA integrity (acridine orange) were evaluated. Then, ten semen samples were divided into four aliquots and incubated, respectively, with CAF at 100 µM, H₂O₂ at 2 mM, or H₂O₂ at 2 mM + CAF at 100 µM, or untreated. Motility, DNA integrity, acrosome status (<i>Pisum sativum</i> agglutinin), OS quantified by F₂-isoprostanes (ELISA), and expression of Nrf2, Keap1, and HO-1 (qRT-PCR) were assessed. CAF at 100 µM improved progressive motility without damaging DNA and was selected for subsequent experiments. CAF showed protective effects on sperm damage induced by H₂O₂ treatment, restoring motility, DNA integrity, and acrosome status and reducing F₂-isoprostane levels. Nrf2 and HO-1 expression were upregulated by CAF, downregulated by H₂O₂, and restored by the co-treatment. CAF supplementation may protect human spermatozoa during in vitro handling by reducing OS, improving several sperm parameters, with a possible mechanism of action involving the Nrf2 pathway.