title: Investigation on the Origin of Sperm DNA Fragmentation: Role of Apoptosis, Immaturity and Oxidative Stress.
abstract: Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of oxidative damage (8-hydroxy, 2'-deoxyguanosine [8-OHdG] and malondialdehyde [MDA]), apoptosis (caspase activity and cleaved poly[ADP-ribose] polymerase [cPARP]) and sperm immaturity (creatine phosphokinase [CK] and excess of residual histones). Active caspases and c-PARP were concomitant with sDF in a high percentage of spermatozoa (82.6% ± 9.1% and 53.5% ± 16.4%, respectively). Excess of residual histones was significantly higher in DNA-fragmented sperm versus sperm without DNA fragmentation (74.8% ± 17.5% and 37.3% ± 16.6%, respectively, p < 0.005), and largely concomitant with active caspases. Conversely, oxidative damage was scarcely concomitant with sDF in the total sperm population, at variance with live sperm, where 8-OHdG and MDA were clearly associated to sDF. In addition, most live cells with active caspase also showed 8-OHdG, suggesting activation of apoptotic pathways in oxidative-injured live cells. This is the first investigation on the origin of sDF directly evaluating the simultaneous presence of the signs of the hypothesized mechanisms with DNA breaks at the single cell level. The results indicate that the main pathway leading to sperm DNA breaks is a process of apoptosis, likely triggered by an impairment of chromatin maturation in the testis and by oxidative stress during the transit in the male genital tract. These findings are highly relevant for clinical studies on the effects of drugs on sDF and oxidative stress in infertile men and for the development of new therapeutic strategies.
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Muratori
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Lara
authors.initials: L
authors.lastname: Tamburrino
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Marchiani
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Cambi
authors.affiliation: Pediatric Section, Department of Health Sciences, University of Florence and Anna Meyer Children's University Hospital, Florence, Italy.
authors.forename: Biagio
authors.initials: B
authors.lastname: Olivito
authors.affiliation: Pediatric Section, Department of Health Sciences, University of Florence and Anna Meyer Children's University Hospital, Florence, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Azzari
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Gianni
authors.initials: G
authors.lastname: Forti
authors.affiliation: Sexual Medicine and Andrology Unit, Department of Experimental and Biomedical Sciences, Center of Excellence DeNothe, University of Florence, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Baldi
mesh_terms: D000080242:8-Hydroxy-2'-Deoxyguanosine; D017209:Apoptosis; D020169:Caspases / Q000378:metabolism; D003402:Creatine Kinase / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D053938:DNA Fragmentation; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D015870:Gene Expression; D006801:Humans; D008297:Male; D018384:Oxidative Stress; D013094:Spermatozoa / Q000378:metabolism*
chemical_list: D000080242:8-Hydroxy-2'-Deoxyguanosine; D003402:Creatine Kinase; D020169:Caspases; D003849:Deoxyguanosine