title: Development of a specific method to evaluate 8-hydroxy, 2-deoxyguanosine in sperm nuclei: relationship with semen quality in a cohort of 94 subjects.
abstract: Oxidative stress (OS) is involved in many disoders including male infertility. Human spermatozoa are very sensitive targets of reactive oxygen species (ROS) and most sperm functions are impaired in the case of OS. In addition unbalanced production of ROS is considered one of the most important causes of sperm DNA fragmentation, a semen trait of infertile men. The relationship between oxidative damage and semen quality is partially controversial, probably due to the different methods and/or targets used to reveal the OS. In this study, by fluorescence microscopy and flow cytometry, we compared two methods to reveal 8-hydroxy,2-deoxyguanosine (8-OHdG), the hallmark of oxidative DNA damage: an immunofluorescence method and the commercial OxyDNA kit. We found that although both methods localized the labelling in sperm nuclei they yielded different measures, and only with the immunofluorescence method was the labelling specific for sperm 8-OHdG. The immunofluorescence method, coupled to flow cytometry, was thus selected to analyse the 8-OHdG content in semen samples from 94 subfertile patients and to investigate the relationship with semen quality. We found that the percentages of spermatozoa with 8-OHdG (mean±s.d., 11.4±6.9%) were related to sperm count (Pearson's correlation coefficient (r)=-0.27, P=0.04 (ANOVA and student's t-test)), motility (progressive: r=-0.22, P=0.04; non-progressive: r=0.25, P=0.01), and normal morphology (r=-0.27, P=0.01). In conclusion, we demonstrate that immunofluorescence/flow cytometry is a reliable and specific method to detect 8-OHdG at single-cell level and show that oxidative damage only partially overlaps poor semen quality, suggesting that it could provide additional information on male fertility with respect to routine semen analysis.
authors: Andrology Unit, Department of Clinical Physiopathology, University of Florence, Viale Pieraccini 6, I-50139 Florence, Italy.
authors: M
authors: M
authors: Cambi
authors: L
authors: L
authors: Tamburrino
authors: S
authors: S
authors: Marchiani
authors: B
authors: B
authors: Olivito
authors: C
authors: C
authors: Azzari
authors: G
authors: G
authors: Forti
authors: E
authors: E
authors: Baldi
authors: M
authors: M
authors: Muratori
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D000328:Adult; D000704:Analysis of Variance; D015415:Biomarkers / Q000032:analysis; D002467:Cell Nucleus / Q000737:chemistry* / Q000473:pathology; D015331:Cohort Studies; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D005434:Flow Cytometry; D006801:Humans; D007248:Infertility, Male / Q000175:diagnosis* / Q000378:metabolism / Q000473:pathology; D008297:Male; D008856:Microscopy, Fluorescence; D018384:Oxidative Stress; D011237:Predictive Value of Tests; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D055101:Semen Analysis / Q000379:methods*; D013076:Sperm Count; D013081:Sperm Motility; D013094:Spermatozoa / Q000737:chemistry* / Q000473:pathology
chemical_list: D015415:Biomarkers; D011933:Reagent Kits, Diagnostic; D000080242:8-Hydroxy-2'-Deoxyguanosine; D003849:Deoxyguanosine