F2-isoprostane, a valid marker in the study of male infertility

The relevance of F2-isoprostanes (F2-IsoPs), produced by non-enzymatic oxidation of arachidonic acid has been explored in different applications of male reproductive field. In clinical studies, F2-IsoP levels were measured in semen of 192 subjects and correlated with sperm parameters; then they were analysed in the main infertility pathologies (varicocele, idiopathic infertility, urogenital infections). Semen analysis was performed following World Health Organization guidelines (2010) and F2-IsoPs quantified by gas chromatography/negative-ion chemical ionization tandem mass spectrometry, a highly sensitive and specific method. In the whole population, F2-IsoPs negatively correlated with sperm progressive and rapid motility, normal morphology, and vitality (p <0.01) and positively with slow motility (p <0.01). By grouping samples according to pathologies, increased F2- IsoP levels were detected in semen of men with varicocele and urogenital infection. The J index, summary measure of ROC curve, has revealed a F2-IsoP cut-off value of 29.96 ng/mL. Low quality semen showed F2-IsoPs higher than the cut-off value. Furthermore, the F2-IsoP levels were evaluated in semen of men from 49 infertile couples undergoing assisted reproductive technologies (ARTs) and correlated with reproductive outcome and embryo quality. Sperm chromatin maturity was assessed by aniline blue assay and sperm DNA integrity by acridine orange (AO) test. Then, the correlations among all variables and their impact on ART outcome and embryo quality were evaluated. F2-IsoPs were positively correlated with sperm DNA integrity and negatively with sperm mature chromatin (both p <0.001). Semen producing good- quality embryos showed increased percentage of sperm with double-strand DNA and levels of F2- IsoP (both p <0.01), still below the identified cut-off value. F2-IsoPs were used as in vitro marker to test the protective effect of chlorogenic acid (CGA), an antioxidant compound, against lipid peroxidation (LPO) induced by H2O2 in human spermatozoa and in cryopreservation protocols. Swim-up selected spermatozoa were treated with 100 μM CGA, 100 μM H2O2 and with both compounds and we evaluated the effects on mitochondrial membrane potential (MMP) by JC-1, DNA integrity by AO, and on sperm ultrastructure by transmission electron microscopy (TEM). CGA antioxidant activity was assessed by measuring the levels of malondialdehyde (MDA) and F2-IsoPs in media. CGA was not toxic for sperm motility, DNA integrity and MMP. The increased levels of F2-IsoPs and MDA (both p <0.01), after treatment with H2O2, were reduced in the presence of CGA. CGA protected also sperm DNA integrity and MMP (both p <0.01), as well as plasma membranes and acrosomes (both p <0.01). Frozen-thawed sperm treated with CGA showed increased percentage of progressive motility, DNA integrity, MMP and reduced levels of MDA (all p <0.01) and F2-IsoPs (p <0.05) compared to frozen untreated sperm. Finally, the formation of F2-IsoPs in the reproductive tissue of rabbits fed n-3 polyunsaturated fatty acid (PUFA) enriched diets was evaluated. Fifteen rabbit bucks were grouped in three experimental groups: FLAX group fed 10% extruded flaxseed, FISH group fed 3.5% fish oil and control group fed standard diet. Rabbit sperm traits were analysed by computer assisted sperm analysis (CASA). The amount of thiobarbituric reactive substances (TBARS) was assessed in blood and semen, finally the fatty acid profile was determined in sperm and testis. F2-IsoPs were measured in blood, semen, testicular and epididymal tissues. The immunolocalization of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in rabbit testes was also explored. FLAX and FISH diets were richer in n-3 PUFA content and lower in n-6 PUFAs than control diet. The dietary administration of n- 3 PUFA improved rabbit sperm track speed and motility percentage (both p ≤0.05). The TBARS concentration was higher in semen of FLAX and FISH (both p ≤0.05) groups than in controls, but similar in blood of the three groups. F2-IsoP levels decreased in blood, semen (both p ≤0.05), testes (p <0.001) and epididymides (p <0.001 and p <0.01, FLAX and FISH, respectively) of n-3 PUFAs dietary groups compared to controls. A wider distribution of DHA and EPA labelling in testes of rabbits fed n-3 PUFA enriched diets compared to those of controls was observed. In conclusion, F2-IsoPs are present in human semen, where they probably play a physiological role; their increased levels may be an effective index of oxidative damage, particularly evident in pathologies linked to an inflammatory status. The mechanisms underlying their biological function, associated with embryo quality, need to be better defined. F2-IsoPs are also a useful marker to evaluate in vitro LPO, resulting from gamete handling and in cryopreservation procedures, and the efficacy of in vitro antioxidant supplementation. Furthermore, their levels can be in vivo modulated, following the administration of definite diets rich in n-3 PUFAs.