Effect of smoking on the functional aspects of sperm and seminal plasma protein profiles in patients with varicocele

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)What are the effects of smoking on the functional aspects of the sperm, the levels of lipid peroxidation and the protein profile of seminal plasma in patients with varicocele? In men with varicocele, smoking is associated with altered semen quality, decreased sperm functional integrity and seminal oxidative stress. Alterations in seminal plasma protein profiles are also present and may explain the altered semen phenotype. Varicocele is a major cause of male infertility. It reduces testicular blood renewal with a consequent accumulation of toxic substances. Thus, it can potentiate the toxic effects of environmental exposure to genotoxic substances such as those found in cigarette smoke. A cross-sectional study was performed in 110 patients presenting with variococele to the Human Reproduction Section of the Sao Paulo Federal University (20062010). The patients were divided into a control group of non-smokers, a moderate smokers group and a heavy smokers group. Semen parameters were analysed by standard methods. Sperm DNA integrity and mitochondrial activity were assessed by Comet assays and by 3,3-diaminobenzidine deposition, respectively. The level of lipid peroxidation in semen was determined by malondialdehyde quantification. Proteomic studies were performed by 2D-electrophoresis and mass spectrometry. Both groups of smokers showed reduced semen quality in comparison with the control group. In the groups of smokers, sperm DNA integrity and mitochondrial activity were also decreased and lipid peroxidation levels were increased. Proteomic analyses revealed 20 proteins differentially expressed between the study groups. A study including smokers without varicocele is still warranted as these results apply only to smokers who present varicocele. Patients with varicocele who are exposed to tobacco smoking present more important alterations to semen quality and sperm functional integrity and show changes in the seminal plasma proteome. This suggests testicular, and possibly systemic, adverse effects of smoking. Funding for the study was provided by Fundao de Amparo Pesquisa do Estado de So Paulo (Fapesp) (2007/59423-7) and by the Division of Urology, Human Reproduction Section at the So Paulo Federal University.271131403149Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Division of Urology, Human Reproduction Section at the Sao Paulo Federal UniversityFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2007/59423-7

Changes in the seminal plasma proteome of adolescents before and after varicocelectomy

Objective: To compare seminal plasma protein profiles before and after varicocele correction to assess if surgical intervention alters the protein profile. Design: Prospective study. Setting: Academic research environment. Patient(s): Nineteen adolescent boys with varicocele grades II or III. Intervention(s): Two semen samples were collected before bilateral subinguinal microsurgical varicocelectomy, and two semen samples were collected 3 months after surgery. Seminal plasma protein profiles were determined with the use of two-dimensional gel electrophoresis. Proteins were separated in 18-cm 3-10 pH strips and 10%-17.5% gradient gels. Gels were stained, scanned, and compared with the use of Imagemaster 2D platinum 7.0. Spots of interest were removed from gels, and protein digestion was performed with the use of trypsin. Digests were identified with the use of electrospray ionization-quadrupole/time-of-flight tandem mass spectrometry (ESI-QTOF MS/MS), and spectra were analyzed with the use of the Mascot software. Main Outcome Measure(s): Proteins uniquely or overexpressed in each period (before or after varicocelectomy). Result(s): Nineteen spots were differentially expressed between pre- and postsurgery samples. Identified proteins were albumin, proteasome subunit alpha type 6, alpha-1-antitrypsin, fibronectin, CD177, prostatic acid phosphatase, specific prostatic antigen, alpha-2antiplasmin, vitamin D-binding protein, gastricsin, clusterin, semenogelin-1, semenogelin-2, superoxide dismutase, protein-glutamine gamma glutamyltransferase-4, and prolactin-inducing protein. Conclusion(s): Varicocelectomy is associated with changes in the seminal plasma protein profile. Understanding specific pathways leading to male infertility may further assist physicians in demonstrating deviation from homeostasis in male infertility. In addition, it may be possible to observe if surgical intervention does indeed revert altered pathways toward a homeostatic state. ((C) 2013 by American Society for Reproductive Medicine.)100366767

Lipid profiling of follicular fluid from women undergoing IVF: Young poor ovarian responders versus normal responders

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This study identified possible lipid biomarkers in follicular fluid from women with poor ovarian response. These biomarkers indicate pathophysiological pathways and have potential diagnostic applications. An observational case-control study of young women undergoing ovarian stimulation for in-vitro fertilization was conducted. The participants were categorized into a poor ovarian response group and a normal ovarian response to stimulation group. All of the women underwent the same ovarian stimulation protocol, and follicular fluid was collected after ovarian aspiration. Analyses were performed using matrix-assisted laser desorption/ionization mass spectrometry. Principal component analysis and Volcano plots were used to describe follicular fluid classification models based on the lipid profiles. A total of 10 lipids were differentially expressed between the study and control groups. Of these lipid ions, three belonged to the phosphatidylcholine subclass and were present in higher concentrations in the control group. The other seven differential lipids were present in the study group and classified into four lipid subclasses: phosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, and diacylglycerols. These distinctive lipids may be involved in hormonal responses and oocyte development processes and may be useful as biomarkers for therapeutic intervention in women with poor ovarian response.164269277Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Proteomic analysis of follicular fluid from women with and without endometriosis: New therapeutic targets and biomarkers

Endometriosis is a gynecological disease that affects women of reproductive age. The protein profiles of women with endometriosis who were able or unable to achieve pregnancy and women without endometriosis who did achieve pregnancy were compared in this study. The follicular fluid was collected from 21 patients undergoing in vitro-fertilization treatment, according to the following groups: nine women in the control group (Group C), four women with endometriosis who achieved pregnancy (Group E.P), and eight women with endometriosis who did not achieve pregnancy (Group E.NP). Follicular fluid proteins were separated using 2D-electrophoresis,and their spots were compared, excised, and submitted to LC-ESI-MS/MS for proteins identification. The analysis showed 29 differentially expressed spots among the groups, and from these, 21 proteins were identified. Analysis showed some functional enrichment in the E.P group, including response to oxidative stress and apoptosis, while the E.NP group showed functions related to response to reactive oxygen species and positive regulation of apoptosis. These data suggest that endometriosis leads to differential protein expression in the follicular fluid, which can influences the outcome of pregnancy. These proteins may be potential targets for better diagnostics and new therapeutic intervention in affected women, as well as assisting in comprehending the physiopathologic mechanisms underlying endometriosis. Mol. Reprod. Dev. 80: 441-450, 2013. (c) 2013 Wiley Periodicals, Inc.806441450Center for Research in Urology, Human Reproduction Section, Sao Paulo Federal Universit

Testicular histopathological diagnosis as a predictive factor for retrieving spermatozoa for ICSI in non-obstructive azoospermic patients

OBJECTIVE: Histological testicular pattern has a predictive role in the possibility of finding spermatozoa for ICSI in cases of non-obstructive azoospermia because some individuals could show residual spermatogenic sites in the testis. The aim of this study was to evaluate the sperm retrieval rate in each of the histopathological groups (hypospermatogenesis-Hypo, spermatogenic maturation arrest-MA, Sertoli cell only-SCO and testicular hyalinization) in patients assisted in our clinic. MATERIALS AND METHODS: Retrospective study from March 1997 to October 2002. We analyzed 14 patients with mean age of 34.3 ± 0.7, with non-obstructive azoospermia. All patients were submitted to previous diagnostic biopsy (Bx) elsewhere and came to our institution for treatment. After an average of 12 months (8 - 20), they were submitted to a new Bx procedure to retrieve sperm. RESULTS: Previous diagnostic Bx showed the following histopathological results: 5 patients with MA, 4 with Hypo and 5 SCO. In the following Bx (for sperm retrieval) spermatozoa was found in 33% of the procedures in patients with MA, 50% in patients with Hypo and 40% of the procedures in patients with SCO. CONCLUSION: Previous diagnostic Bx can help in patient counseling concerning the result of sperm retrieval

Expression of the Fas-ligand gene in ejaculated sperm from adolescents with and without varicocele

To assess FasL mRNA levels in ejaculated sperm from adolescent patients with and without varicocele.Semen was obtained by masturbation following 2-4 days of ejaculatory abstinence, from 14 adolescents with varicocele grades II and III (study group), and 20 adolescents without varicocele (control group). Seminal analysis was done according to World Health Organization guidelines and morphology using Kruger's strict criteria. the Fas-ligand (FasL) gene expression was performed using reverse transcription and real-time quantitative polymerase chain reaction (RQ-PCR) analysis, according to the expression level of the housekeeping cyclophilin A gene. A Student's t-test was applied to compare the groups, and Spearman's rank test in order to verify possible correlations (p < 0.05).Quantitative RQ-PCR demonstrated that the expression of FasL mRNA in sperm from the varicocele group was higher than in the control group. Also, sperm concentration was higher in the controls, when compared to the varicocele group. When submitted to correlation analysis, adolescents with varicocele presented a correlation between sperm concentration and FasL gene expression levels (r = -0.470), not observed in controls.Our results allow us to conclude that, in adolescents with varicocele presenting lower sperm concentration, FasL mRNA levels are higher than in adolescents without varicocele.Universidade Federal de São Paulo, Dept Surg, Div Urol, BR-03320000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, BR-03320000 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04038032 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Surg, Div Urol, BR-03320000 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Med, Div Nephrol, BR-03320000 São Paulo, BrazilUniversidade Federal de São Paulo, BR-04038032 São Paulo, BrazilWeb of Scienc