Olea europaea Leaf Extract: Antioxidant Properties and Supplement in Human Sperm Cryopreservation

Abstract

Background: Semen handling and cryopreservation increase reactive oxygen species (ROS) production, exposing spermatozoa to oxidative stress (OS) that can be minimised by antioxidant supplementation. We studied if Olea europaea leaf extract (OE) could have antioxidant and protective activity during sperm manipulation. Methods: The extract was characterized by high-performance liquid chromatography with diode array detection (HPLC-DAD) and antioxidant activity was determined by 2,2-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays. Then, spermatozoa of 35 normozoospermic donors were treated with OE. First, swim-up selected spermatozoa were incubated with OE (1:100–1:400) and sperm motility and DNA integrity (acridine orange test) were assessed. Then, swim-up selected sperm were treated with 100 μM H₂O₂ to induce OS with and without OE; motility, DNA integrity and F₂-Isoprostanes (F₂-IsoPs quantified by gas chromatography/negative ion chemical ionization tandem mass spectrometry analysis), an OS marker, were assessed. Finally, sperm were frozen with and without OE. Beside the previous endpoints, acrosome shape was evaluated by Tetramethylrhodamine (TRITC)-conjugated Pisum sativum agglutinin (PSA). Results: OE was enriched in polyphenols (0.34%) and triterpenes (0.72%) and the synergistic action of phytocomplex components was responsible for antioxidant activity. Since OE was not toxic for spermatozoa, 1:100 dilution was used for the other experiments. OE protected motility, DNA integrity and reduced F₂-IsoPs (p < 0.001) in in vitro experiment with OS induction. OE treated frozen-thawed spermatozoa showed increased motility, DNA integrity, reduced F₂-IsoP concentration (p < 0.001) and normal acrosomes versus non-supplemented samples. Conclusions: OE is characterized by high concentration of polyphenols and exhibits protective properties against oxidative damage induced by H₂O₂ in human ejaculated sperm. After cryopreservation, the samples supplemented with the extract showed increased sperm quality. OE could represent a supplement of culture media during semen handling where OS is exacerbated.