title: Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers.
abstract: OBJECTIVE
  To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile.
  
  
  METHODS
  Sperm functional tests were performed in 20 non-smoking men with normal semen quality, according to the World Health Organization (2010) and in 20 smoking patients. These included: evaluation of DNA fragmentation by alkaline Comet assay; analysis of mitochondrial activity using DAB staining; and acrosomal integrity evaluation by PNA binding. The remaining semen was centrifuged and seminal plasma was used for proteomic analysis (liquid chromatography-tandem mass spectrometry). The quantified proteins were used for Venn diagram construction in Cytoscape 3.2.1 software, using the PINA4MS plug-in. Then, differentially expressed proteins were used for functional enrichment analysis of Gene Ontology categories, Kyoto Encyclopedia of Genes and Genomes and Reactome, using Cytoscape software and the ClueGO 2.2.0 plug-in.
  
  
  RESULTS
  Smokers had a higher percentage of sperm DNA damage (Comet classes III and IV; P < 0.01), partially and fully inactive mitochondria (DAB classes III and IV; P = 0.001 and P = 0.006, respectively) and non-intact acrosomes (P < 0.01) when compared with the control group. With respect to proteomic analysis, 422 proteins were identified and quantified, of which one protein was absent, 27 proteins were under-represented and six proteins were over-represented in smokers. Functional enrichment analysis showed the enrichment of antigen processing and presentation, positive regulation of prostaglandin secretion involved in immune response, protein kinase A signalling and arachidonic acid secretion, complement activation, regulation of the cytokine-mediated signalling pathway and regulation of acute inflammatory response in the study group (smokers).
  
  
  CONCLUSIONS
  In conclusion, cigarette smoking was associated with an inflammatory state in the accessory glands and in the testis, as shown by enriched proteomic pathways. This state causes an alteration in sperm functional quality, which is characterized by decreased acrosome integrity and mitochondrial activity, as well as by increased nuclear DNA fragmentation.
authors.affiliation: Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil.
authors.forename: Mariana Pereira
authors.initials: MP
authors.lastname: Antoniassi
authors.affiliation: Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil.
authors.forename: Paula
authors.initials: P
authors.lastname: Intasqui
authors.affiliation: Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil.
authors.forename: Mariana
authors.initials: M
authors.lastname: Camargo
authors.affiliation: Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil.
authors.forename: Daniel Suslik
authors.initials: DS
authors.lastname: Zylbersztejn
authors.affiliation: Fleury Group, Sao Paulo, Brazil.
authors.forename: Valdemir Melechco
authors.initials: VM
authors.lastname: Carvalho
authors.affiliation: Fleury Group, Sao Paulo, Brazil.
authors.forename: Karina H M
authors.initials: KH
authors.lastname: Cardozo
authors.affiliation: Department of Surgery, Division of Urology, Human Reproduction Section, Sao Paulo Federal University, Sao Paulo, Brazil. rbertolla@yahoo.com.|Hospital Sao Paulo, Sao Paulo, Brazil. rbertolla@yahoo.com.
authors.forename: Ricardo Pimenta
authors.initials: RP
authors.lastname: Bertolla
mesh_terms: D000177:Acrosome; D000328:Adult; D003430:Cross-Sectional Studies; D053938:DNA Fragmentation; D006801:Humans; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000502:physiology; D040901:Proteomics; D012661:Semen / Q000502:physiology*; D055101:Semen Analysis / Q000379:methods*; D029589:Seminal Plasma Proteins / Q000032:analysis*; D012907:Smoking / Q000503:physiopathology*; D013094:Spermatozoa / Q000502:physiology*; D055815:Young Adult
chemical_list: D029589:Seminal Plasma Proteins
keywords: DNA damage; acrosome; mitochondria; proteomics; seminal plasma