To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profil... more To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. 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. 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). 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.
Journal of Assisted Reproduction and Genetics, 2016
To verify if the presence of varicocele (grades II and III) with and without seminal alterations,... more To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5(th) centile cutoff values in table A1.1 of the World Health Organization (WHO, 2010) manual, alters the seminal plasma levels of proteins DNASE1 (deoxyribonuclease-1) and IGFBP7 (Insulin-like growth factor-binding protein 7), which are related to apoptosis regulation and cell proliferation, respectively, demonstrating that these proteins are important for correct spermatogenesis. This cross sectional study was performed at the Sao Paulo Federal University Paulo between May 2014 and April 2016. A total of 61 male adolescents were included in this study, of which 20 controls without varicocele (C), 22 with varicocele and normal semen analysis (VNS) and 19 with varicocele and altered semen analysis (VAS). Seminal plasma from each patient was used for Western blotting analysis of individual protein levels. Values of each protein were normalized to a testicular housekeeping protein (PARK7-protein deglycase DJ-1). Levels of IGFBP7 protein are increased in varicocele. Levels of DNASE1 are progressively decreased in varicocele (lower in varicocele and normal semen analysis, lowest in varicocele and altered semen analysis) when compared to adolescents without varicocele. DNASE1 levels are positively correlated with sperm concentration and morphology (correlation values of 0.400 and 0.404, respectively; p values of 0.001 and 0.001, respectively). In conclusion, in adolescents, seminal plasma levels of IGFBP7, responsible for proliferative activity, are increased in varicocele grades II and III, and DNASE1, responsible for apoptosis regulation, are lower in varicocele, lowest in varicocele and low semen quality. These proteins demonstrate molecular alterations brought upon by varicocele. Moreover, DNASE1 is capable of discriminating a varicocele that causes alterations to semen quality from one that does not. We propose that the initial response of varicocele is to increase proliferative activity which, if followed by regulation of apoptosis, may lead to the ejaculation of a population of sperm that are in accordance with WHO cutoff values but, in the presence of dysregulated apoptosis, leads to lower sperm concentration and morphology.
To analyze the seminal plasma proteome and biological functions associated with sperm functional ... more To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.
To study the seminal plasma proteome in association with semen lipid peroxidation levels in men w... more To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Student's t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.
This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analy... more This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. For this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub-study 1.1), high and low sperm nuclear DNA fragmentation (sub-study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub-study 1.1 and 89 for sub-study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix-Assisted Laser Desorption Ionization Quadrupole-Time-of-Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters(®) MassLynx, and MetaboAnalyst online software was used for statistical analyses. For sub-studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS-DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5%. PLS-DA generated a list of 19, 24 and 23 VIP ions for sub-studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.
To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profil... more To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. 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. 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). 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.
Journal of Assisted Reproduction and Genetics, 2016
To verify if the presence of varicocele (grades II and III) with and without seminal alterations,... more To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5(th) centile cutoff values in table A1.1 of the World Health Organization (WHO, 2010) manual, alters the seminal plasma levels of proteins DNASE1 (deoxyribonuclease-1) and IGFBP7 (Insulin-like growth factor-binding protein 7), which are related to apoptosis regulation and cell proliferation, respectively, demonstrating that these proteins are important for correct spermatogenesis. This cross sectional study was performed at the Sao Paulo Federal University Paulo between May 2014 and April 2016. A total of 61 male adolescents were included in this study, of which 20 controls without varicocele (C), 22 with varicocele and normal semen analysis (VNS) and 19 with varicocele and altered semen analysis (VAS). Seminal plasma from each patient was used for Western blotting analysis of individual protein levels. Values of each protein were normalized to a testicular housekeeping protein (PARK7-protein deglycase DJ-1). Levels of IGFBP7 protein are increased in varicocele. Levels of DNASE1 are progressively decreased in varicocele (lower in varicocele and normal semen analysis, lowest in varicocele and altered semen analysis) when compared to adolescents without varicocele. DNASE1 levels are positively correlated with sperm concentration and morphology (correlation values of 0.400 and 0.404, respectively; p values of 0.001 and 0.001, respectively). In conclusion, in adolescents, seminal plasma levels of IGFBP7, responsible for proliferative activity, are increased in varicocele grades II and III, and DNASE1, responsible for apoptosis regulation, are lower in varicocele, lowest in varicocele and low semen quality. These proteins demonstrate molecular alterations brought upon by varicocele. Moreover, DNASE1 is capable of discriminating a varicocele that causes alterations to semen quality from one that does not. We propose that the initial response of varicocele is to increase proliferative activity which, if followed by regulation of apoptosis, may lead to the ejaculation of a population of sperm that are in accordance with WHO cutoff values but, in the presence of dysregulated apoptosis, leads to lower sperm concentration and morphology.
To analyze the seminal plasma proteome and biological functions associated with sperm functional ... more To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.
To study the seminal plasma proteome in association with semen lipid peroxidation levels in men w... more To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Seminal lipid peroxidation levels were assessed in individual samples through thiobarbituric acid reactive substances quantification. Subsequently, lipid peroxidation data were used to divide the samples into the experimental groups: low lipid peroxidation levels (control group, bottom 15%, n = 23) and high lipid peroxidation levels (study group, top 15%, n = 23). Seminal plasma proteins from these groups were pooled (four pools per group, with biological variation between the pools) and used for a shotgun proteomic analysis using a liquid chromatography-tandem mass spectrometry approach. Quantitative data were used for univariate (unpaired Student's t test) and multivariate (partial least-squares discriminant analysis, logistic regression, and discriminant analyses) statistical analyses. Significant proteins were also used for functional enrichment analysis. Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. In total, 629 proteins were quantified in seminal plasma. Of these, 23 proteins were absent or underexpressed and 71 were exclusive or overexpressed in the study group. The main enriched functions in association with seminal lipid peroxidation were unsaturated fatty acids biosynthesis, oxidants and antioxidants activity, cellular response to heat stress, and immune response. Moreover, we suggested mucin-5B as a potential biomarker of semen oxidative stress. The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.
This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analy... more This study proposed lipid fingerprinting of human seminal plasma by mass spectrometry as an analytical method to differentiate biological conditions. For this purpose, we chose infertile men as a model to study specific conditions, namely: high and low seminal plasma lipid peroxidation levels (sub-study 1.1), high and low sperm nuclear DNA fragmentation (sub-study 1.2), and intervention status: before and after subinguinal microsurgical varicocelectomy (study 2). Study 1 included 133 patients, of which 113 were utilized for sub-study 1.1 and 89 for sub-study 1.2. Study 2 included 17 adult men submitted to subinguinal varicocelectomy, before and 90 days after varicocelectomy. Lipids were extracted from seminal plasma and submitted to Matrix-Assisted Laser Desorption Ionization Quadrupole-Time-of-Flight Mass Spectrometry in the positive ionization mode. Spectra were processed using Waters(®) MassLynx, and MetaboAnalyst online software was used for statistical analyses. For sub-studies 1.1 and 1.2, and study 2, univariate analysis revealed 8, 87 and 34 significant ions, respectively. Multivariate analysis was performed through PCA and PLS-DA. PCA generated 56, 32 and 34 components respectively for each study and these were submitted to logistic regression. A ROC curve was plotted and the area under the curve was equal to 97.4, 92.5 and 96.5%. PLS-DA generated a list of 19, 24 and 23 VIP ions for sub-studies 1.1 and 1.2, and study 2, respectively. Therefore, this study established the lipid profile and comparison of patterns altered in response to specific biological conditions.
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Papers by Paula Intasqui