Oxidative Stress and Human Semen: Clinical and in Vitro Studies

Male infertility is a complex condition caused by a wide range of genetic, environmental, and idiopathic factors, with oxidative stress (OS) playing a critical role in many pathologies such as varicocele and genitourinary infections. OS arises from an imbalance between reactive oxygen species and antioxidant defenses, leading to sperm dysfunction and DNA damage. There is an increasing need for stable and easily measurable biomarkers to detect OS in semen, particularly for idiopathic infertility cases. F2-isoprostanes (F2-IsoPs), due to their stability and physiological relevance, are considered a promising alternative to traditional markers like malondialdehyde (MDA). Moreover, human spermatozoa, thanks to their motility and structural characteristics, represent a reliable in vitro model to study the effects of OS and protective antioxidant compounds. This thesis aimed to explore the role of OS in male infertility through both clinical and in vitro studies. The research focused on the following issues: (1) identifying and evaluating biomarkers of OS, inflammation, and ferroptosis such as F2-IsoPs, MDA, apelin, interleukin (IL)-1β, acyl-CoA synthetase long chain family member 4 (ACSL4), and glutathione peroxidase 4 (GPX4) in semen samples from infertile men; (2) testing the efficacy of antioxidant compounds, including Olea europaea leaf extract (OE) and chlorogenic acid (CGA) used in different formulations, in mitigating OS during semen handling and cryopreservation protocols. Three clinical studies were conducted on semen samples from infertile men with varicocele, genitourinary infections, or idiopathic infertility. Biomarkers of inflammation (e.g., IL-1β, apelin), ferroptosis (e.g., ACSL4, GPX4), and OS (e.g., F2-IsoPs, MDA), were quantified using techniques such as ELISA and gas chromatography-mass spectrometry. In addition, the apelin-APJ receptor system was localized by immunofluorescence on human spermatozoa and testicular tissue and molecules involved in the lipid peroxidation (LPO) like 8-iso-PGF2α and arachidonate 15-lipoxygenase (ALOX15) were immunodetected in human spermatozoa. For in vitro studies, human spermatozoa were exposed to OS induced by H2O2 or freezing-thawing protocols. The protective effects of OE and CGA, tested in its free form and as liposome-encapsulated formulations, were assessed by evaluating sperm motility, DNA integrity with acridine orange test, acrosome status with TRITC-conjugated Pisum sativum agglutinin, mitochondrial membrane potential with JC-1 test, and LPO with F2-IsoP quantification. The Nrf2 expression, a key transcription factor involved in antioxidant responses, was analyzed through quantitative real-time PCR to investigate transcriptional activity in response to treatments. Clinical studies revealed significantly elevated levels of OS and inflammatory biomarkers in selected infertile patients compared to fertile controls. F2-IsoPs demonstrated a higher specificity as OS markers compared to MDA, supporting their potential for routine clinical evaluation. Moreover, the results suggested a mechanism of ferroptosis in spermatozoa, identified by increased ACSL4 levels and the occurrence of LPO; such events appear to be GPX4-independent in reproductive pathologies such as varicocele and genitourinary infections. The in vitro studies highlighted the protective effects of both OE and CGA on human spermatozoa. OE, rich in polyphenols, significantly improved sperm motility and DNA integrity while reduced LPO under H2O2-induced OS. Similarly, CGA enhanced motility and decreased the levels of OS biomarkers, with its effects mediated through the activation of the Nrf2 pathway. Particularly, CGA-loaded liposomes showed remarkable tolerability and efficacy in cryopreservation protocols, improving post-thaw sperm quality. These findings underscore the critical role of OS in male infertility and the need for clinically applicable OS markers like F2-IsoPs to ameliorate the routine clinical diagnostic accuracy. The use of human spermatozoa as a reliable in vitro model demonstrated their sensitivity to antioxidant compounds. Both OE and CGA exhibited significant protective effects against OS-induced damage, with CGA's performance enhanced through liposome encapsulation, especially in cryopreservation protocols. These studies pave the way for further research aimed at integrating OS assessment into routine semen analysis and developing antioxidant-based therapeutic strategies for male infertility.