M. Camargo

Differences in the seminal plasma proteome are associated with oxidative stress levels in men with normal semen parameters

OBJECTIVE To study the seminal plasma proteome in association with semen lipid peroxidation levels in men with normal semen parameters. DESIGN Cross-sectional study. SETTING University andrology and research laboratories. PATIENT(S) A total of 156 normozoospermic men. INTERVENTION(S) 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 Students 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. MAIN OUTCOME MEASURE(S) Seminal plasma protein profile and postgenomic pathways of seminal plasma are associated with seminal lipid peroxidation levels. RESULT(S) 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. CONCLUSION(S) The seminal plasma proteome does reflect semen lipid peroxidation status and, thus, oxidative stress.

Unbiased label-free quantitative proteomic profiling and enriched proteomic pathways in seminal plasma of adult men before and after varicocelectomy

STUDY QUESTION Does the seminal plasma proteomic profile and functional enrichment of gene ontology terms change after microsurgical varicocelectomy? Are there any potential targets for diagnosis or therapeutic intervention in varicocele? SUMMARY ANSWER A shift in state from a responsive-to-stress condition before varicocele correction to a responsive-to-environment condition after varicocelectomy was observed in enriched proteomic pathways. WHAT IS KNOWN ALREADY Varicocele may lead to many adverse effects, including failure of testicular growth and development, and is associated with decreased semen quality and increased semen oxidative stress. Varicocelectomy is the treatment of choice, and is associated with improved semen quality, but little is known regarding the underlying molecular mechanisms and post-genomic pathways following intervention. STUDY DESIGN, SIZE, DURATION A prospective study was carried out including 18 adult men with varicocele. These patients provided one semen sample before they were submitted for bilateral varicocele repair through microsurgical varicocelectomy, and one other semen sample 90 days after the surgery. PARTICIPANTS/MATERIALS, SETTING, METHODS An aliquot of each semen sample was used for unbiased proteomics analysis by a label-free quantitative approach (2D nanoUPLC-ESI-MS(E)). Samples were pooled according to group (normalized to protein content) and run in quadruplicate. These quadruplicate runs provided degrees of freedom in order to compare groups using a non-parametric Mann-Whitney test for quantified proteins. MAIN RESULTS AND THE ROLE OF CHANCE A total of 316 proteins were quantified or identified, of which 91 were exclusively identified or quantified in one of the groups (53 in the pre- and 38 in the post-varicocelectomy group), and 68 were quantified in both groups and submitted to statistical analysis, of which 5 were overrepresented in the pre-varicocelectomy group (P < 0.05). In enriched functional analysis, binding and response to stimulus functions were enriched in a common cluster (present in both groups), nitric oxide metabolism and tetratricopeptide repeat domain-binding functions were enriched in the pre-varicocelectomy group, and response to reactive oxygen species, gluconeogenesis, nicotinamide adenine dinucleotide-binding and protein stabilization were enriched in the post-varicocelectomy. LIMITATIONS, REASONS FOR CAUTION Because a shotgun proteomics analysis was chosen in order to generate a list of putative biomarkers, a targeted follow-up study should be performed to confirm these biomarkers. WIDER IMPLICATIONS OF THE FINDINGS The proteins found in both groups possess functions usually found in human semen. The enriched function analysis demonstrated a shift back to homeostasis after varicocelectomy, suggesting that varicocele correction promotes return of semen to a physiological state. STUDY FUNDING/COMPETING INTEREST(S) The funding for this project was received from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) as a scholarship for Ms Camargo. There was no conflict of interest.

Unraveling the sperm proteome and post-genomic pathways associated with sperm nuclear DNA fragmentation

PurposeSperm DNA fragmentation has been suggested as a marker for infertility diagnosis and prognosis. Hence, understanding its impact on male physiology and post-genomic pathways would be clinically important. We performed the proteomics and functional enrichment analyses of viable spermatozoa from ejaculates with low and high sperm DNA fragmentation to identify protein expression and pathways altered in association with sperm DNA fragmentation.MethodsSperm DNA fragmentation using the Comet assay and the Komet 6.0.1 software was assessed in raw samples from 89 subjects from a human reproduction service. The Low and High sperm DNA fragmentation groups were formed according to the Olive Tail Moment variable. Spermatozoa proteins from these groups were pooled and analyzed by a shotgun proteomic approach (2D nanoUPLC-ESI-MSE). Differentially expressed proteins were used for a functional enrichment study.ResultsTwo hundred and fifty-seven proteins were identified or quantified in sperm from the Low and High sperm DNA fragmentation groups. Of these, seventy-one proteins were exclusively or overexpressed in the Low group, whereas twenty-three proteins were exclusively or overexpressed in the High group. One hundred and sixty-three proteins were conserved between these groups. We also functionally related the differentially expressed proteins in viable spermatozoa from the groups. Processes such as triacylglycerol metabolism, energy production, protein folding, response to unfolded proteins, and cellular detoxification were found to be altered in these cells.ConclusionsSperm DNA fragmentation is associated with differential protein expression in viable spermatozoa. These proteins may potentially be used as biomarkers for sperm DNA integrity.

Analysis of the functional aspects and seminal plasma proteomic profile of sperm from smokers

To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile.

Sperm nuclear DNA fragmentation rate is associated with differential protein expression and enriched functions in human seminal plasma.

To analyse the proteomic profile of seminal plasma with the aim of identifying the proteins and post‐genomic pathways associated with sperm DNA fragmentation.

Association between the seminal plasma proteome and sperm functional traits

OBJECTIVE To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. DESIGN Cross-sectional study. SETTING University andrology and research laboratories. PATIENT(S) A total of 156 normozoospermic men. INTERVENTION(S) 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. MAIN OUTCOME MEASURE(S) Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. RESULT(S) 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. CONCLUSION(S) Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations.

Determination of testicular function in adolescents with varicocoele – a proteomics approach

The goal of this study was to determine seminal plasma biomarkers of testicular function in adolescents with varicocoele and to verify enriched gene ontology terms associated to these differential proteomes. An observational study was carried out in an academic research environment. A total of 77 adolescent patients were recruited from a local public school, of which 23 were without varicocoele and with normal semen analysis (control group), 37 were with varicocoele and normal semen (VNS) parameters, and 17 were with varicocoele and altered semen (VAS) parameters. Two semen collections were provided with a 1‐week interval, after 2–5 days of ejaculatory abstinence. Seminal plasma proteins were identified and quantified utilizing a label‐free shotgun proteomics approach, generating (i) proteins differentially expressed in each group (control, VNS, and VAS) and putative biomarkers using multivariate statistics followed by discriminant analysis. Confirmatory analysis was performed for two proteins by western blotting. Enriched biological processes and molecular functions were determined using gene ontology analysis. In total, 541 proteins were identified and quantified: 108 exclusive or overexpressed in controls, 26 in the VNS group, and 13 in the VAS group. The suggested biomarkers are Cab45/SDF4 (Q9BRK5), protein lefty‐1 (O75610), DNase I (P24855), PAP2‐alpha (O14494), IBP‐7 (Q16270), HDC (P01860), and CRISP‐3 (P54108). Western blotting results showed that Cab45 was significantly underexpressed in both varicocoele groups, and CRISP‐3 was significantly overexpressed in seminal plasma of adolescents with VAS. In conclusion, specific biomarkers of spermatogenesis and homeostasis are observed in adolescents without varicocoele, and the presence of a palpable varicocoele progressively shifts these adolescents toward initially an immune response, and finally toward a chronic inflammatory profile. This shift is accompanied by decreased semen quality.

Cysteine-rich secretory protein 3: inflammation role in adult varicocoele

Cysteine‐rich secretory protein (CRISP‐3), a protein involved in inflammatory response, is highly increased in seminal plasma of adolescents with varicocoele and altered semen analysis, but not in adolescents with varicocoele and normal semen. It is not known, however, whether this increased seminal concentration occurs as an acute marker during the initial stages of varicocoele or whether this persists as an altered protein pathway.

Understanding the seminal plasma proteome and its role in male fertility

Seminal plasma is a complex fluid comprised of secretions from the seminal vesicles, the prostate, bulbourethral glands and from the seminiferous tubule lumen / epididymides / vasa deferentia. While it has been established that seminal plasma serves not only as a medium to carry, protect, and nourish sperm after ejaculation up to fertilization, but also as a functional modulator of sperm function, there is still a need to properly characterize the molecular make-up of seminal plasma in fertile men, and to understand how this is altered in different causes of male infertility. The main purpose of this manuscript was to review articles that studied the human seminal plasma proteome, ranging from characterizing a fertile seminal plasma proteomic map to studies comparing seminal plasma from fertile and infertile men, and comparing seminal plasma of fertile or normozoospermic men to a diverse range of biological causes for male infertility. Finally, this review has focused on the association between semen and sperm functional quality and the seminal plasma proteome, in order to demonstrate cellular and molecular mechanisms of male infertility. Due to the untargeted nature of the majority of the studies presented in this review, and to the diverse range of techniques utilized to study the seminal plasma proteomic composition, many differentially expressed proteins were observed. However, in general, it seems that there is a seminal plasma proteome associated to male fertility, and that different biological conditions or cellular phenotypes shift its pathways away from its homeostatic condition to altered energy production pathways. Moreover, it seems there is an inflammatory component to the seminal plasma of infertile men. In conclusion, there are a number of studies focused on the proteomic composition of human seminal plasma; downstream confirmatory studies will help to understand specific pathways of infertility in different biological conditions.RésuméLe plasma séminal est un liquide complexe comprenant les sécrétions des vésicules séminales, de la prostate, des glandes bulbo-urétrales, et des sécrétions provenant de la lumière des tubes séminifères/épididymes/canaux déférents. Bien qu’il a été établi que le plasma séminal n’est pas seulement un milieu servant à transporter, protéger et nourrir les spermatozoïdes après l’éjaculation et jusqu’à la fécondation, mais qu’il constitue aussi un modulateur fonctionnel des fonctions spermatiques, il demeure nécessaire de caractiser de manière appropriée la constitution moléculaire du plasma séminal des hommes féconds, et de comprendre comment celle-ci est altérée dans les différentes causes d’infertilité masculine.Le principal objectif de cet article est de passer en revue les études du protéome du plasma séminal, en allant de celles ayant caractérisé une carte protéomique du plasma séminal fertile aux études ayant comparé le plasma séminal d’hommes féconds et inféconds et à celles ayant comparé le plasma séminal d’hommes féconds ou normozoospermiques à celui d’hommes présentant diverses causes d’infertilité. Pour finir, la présente revue est centrée sur l’association entre d’une part la qualité fonctionnelle du sperme et des spermatozoïdes et d’autre part le protéome du plasma séminal dans le but de démontrer les mécanismes cellulaires et moléculaires de l’infertilité masculine. En raison de la nature non ciblée de la majorité des études présentées dans cette revue, et de la grande diversité des techniques utilisées pour étudier la composition protéomique du plasma séminal, de nombreuses protéines différentiellement exprimées ont été observées.Cependant, d’une façon globale, il semblerait qu’il y ait un protéome séminal associé à la fertilité masculine et que des situations biologiques ou des phénotypes cellulaires particuliers l’éloignerait de son point d’équilibre vers des états associés à une production énergétique altérée. De plus, il semblerait exister une composante inflammatoire du plasma séminal chez les hommes infertiles. En conclusion, il existe de nombreuses études centrées sur la composition protéomique du plasma séminal humain; de futures études de confirmation seront utiles à la compréhension des voies spécifiques de l’infertilité dans ses différentes conditions biologiques.

Follicular fluid lipid peroxidation levels in women with endometriosis during controlled ovarian hyperstimulation

Abstract This observational study aimed to establishing a relationship between lipid peroxidation and endometriosis in women undergoing controlled ovarian hyperstimulation. A total of 79 women were divided into two groups: (i) controls (tubal or male factor); and (ii) endometriosis (stages III/IV). The endometriosis diagnosis was confirmed by videolaparoscopy and the controlled ovarian stimulation protocol was similar to all patients. Follicular fluid (FF) lipid peroxidation levels were determined through the quantification of malondialdehyde. Statistical analysis was performed using parametric and non-parametric tests, logistic regression was performed to estimate the chance of achieving a pregnancy in each group and a moving average was calculated for the endometriosis group. Peroxidation levels in the endometriosis group were significantly higher when compared to controls. The moving average showed a decrease of MDA levels in the endometriosis group with increasing female age. Moreover, women with endometriosis who were under 33 years of age were 4.3 times more likely to achieve a pregnancy than women above that age. In conclusion, endometriosis is associated with increased FF oxidative stress (OS) in patients undergoing in vitro fertilization (IVF). Also, increasing age is associated with a decrease in severity of the oxidative status, but a decreased chance of pregnancy.