miR-9-5p is Downregulated in Serum Extracellular Vesicles of Patients Treated with Biperiden After Traumatic Brain Injury.

Traumatic brain injury (TBI) is a prevalent and debilitating condition, which often leads to the development of post-traumatic epilepsy (PTE), a condition that yet lacks preventive strategies. Biperiden, an anticholinergic drug, is a promising candidate that has shown efficacy in murine models of PTE. MicroRNAs (miRNAs), small regulatory RNAs, can help in understanding the biological basis of PTE and act as TBI- and PTE-relevant biomarkers that can be detected peripherally, as they are present in extracellular vesicles (EVs) that cross the blood-brain barrier. This study aimed to investigate miRNAs in serum EVs from patients with TBI, and their association with biperiden treatment and PTE. Blood samples of 37 TBI patients were collected 10 days after trauma and treatment initiation in a double-blind clinical trial. A total of 18 patients received biperiden, with three subjects developing PTE, and 19 received placebo, with two developing PTE. Serum EVs were characterized by size distribution and protein profiling, followed by high-throughput sequencing of the EV miRNome. Differential expression analysis revealed no significant differences in miRNA expression between TBI patients with and without PTE. Interestingly, miR-9-5p displayed decreased expression in biperiden-treated patients compared to the placebo group. This miRNA regulates genes enriched in stress response pathways, including axonogenesis and neuronal death, relevant to both PTE and TBI. These findings indicate that biperiden may alter miR-9-5p expression in serum EVs, which may play a role in TBI resolution.

Mapping the human sperm proteome - novel insights into reproductive research.

INTRODUCTION: Spermatozoa are highly specialized cells with unique morphology. In addition, spermatozoa lose a considerable amount of cytoplasm during spermiogenesis, when they also compact their DNA, resulting in a transcriptionally quiescent cell. Throughout the male reproductive tract, sperm will acquire proteins that enable them to interact with the female reproductive tract. After ejaculation, proteins undergo post-translational modifications for sperm to capacitate, hyperactivate, and fertilize the oocyte. Many proteins have been identified as predictors of male infertility and also investigated in diseases that compromise reproductive potential. AREAS COVERED: In this review, we proposed to summarize the recent findings about the sperm proteome and how they affect sperm structure, function, and fertility. A literature search was performed using PubMed and Google Scholar databases within the past 5 years until August 2022. EXPERT OPINION: Sperm function depends on protein abundance, conformation, and PTMs; understanding the sperm proteome may help to identify pathways essential to fertility, even making it possible to unravel the mechanisms involved in idiopathic infertility. In addition, proteomics evaluation offers knowledge regarding alterations that compromise the male reproductive potential.

Influence of physical activity on male fertility.

Infertility is a worldwide issue impacting 15% of couples' population. Male-related infertility results in almost 50% of these cases. Considering lifestyle factors associated with infertility, here in this literature review article, we aimed to discuss training/sport effects on male-related infertility. Regarding this issue, human and animal model studies related to the subject were gathered and analysed. Exercise is well known as a general improving factor, however, excessive exercise can result in male infertility due to reduced hypothalamus-pituitary-gonadal axis (HPT) function, increased oxidative stress and chronic inflammation. Consequently, these underlying impacts result in a low testosterone production, and reduced semen quality, and can lead to infertility. In contrast, it has been revealed that exercise can improve male fertility status in lifestyle-induced infertility condition such as obesity and diabetes. Indeed, exercise, by increasing testicular antioxidant defence, reducing pro-inflammatory cytokines level and enhancing the steroidogenesis process, leads to improved spermatogenesis and semen quality in lifestyle-induced infertility. In fact, it seems that individual health status as well as exercise volume, intensity and duration are effective-involved co-factors that influence the impact that exercise will promote on male fertility. Regarding these findings, it is important to study exercise different impacts in further clinical trials in order to generate preservative guidelines for exercise and also considering exercise as a treatment option in lifestyle-induced disease management.

Seminal inflammasome activity in the adult varicocele.

Varicocele has been hypothesized to lead to seminal inflammation, which in turn interferes with sperm function. Thus, the aim of this study was to investigate the role of inflammatory cytokines in the pathogenesis of decreased semen quality observed in adult men with varicocele, and to determine if varicocelectomy corrects these potential alterations. A prospective study was carried out including fifteen control men without varicocele and with normal semen quality and 15 men with varicocele with surgical indication. Men with varicocele grades II or III underwent microsurgical subinguinal varicocelectomy. Controls collected one semen sample and men with varicocele collected one before and one 6 months after the surgery. Semen analysis, sperm function, and seminal lipid peroxidation levels were assessed. Seminal plasma inflammasome activity was evaluated by ELISA assays for IL-1ß, IL-18 and caspase-1 and by Western blotting for ASC (apoptosis-associated speck-like protein). Groups were compared by an unpaired Student's T test. Varicocelectomy samples were compared using a paired Student's T test (α = 5%). Men with varicocele had decreased semen quality, and increased seminal IL-1ß levels, when compared to control men. Varicocelectomy decreased levels of caspase-1, IL-18, and IL1ß. Thus, varicocelectomy improves sperm morphology and decreases seminal plasma inflammatory activity, after a six-month post-operative period.

Oxidative origin of sperm DNA fragmentation in the adult varicocele

ABSTRACT Purpose: Sperm DNA fragmentation is a major cellular mechanism underlying varicocele-related male infertility. However, the type of DNA fragmentation - whether oxidative or of another nature - remains unknown. Thus, the aim of this study was to evaluate single- and double-stranded sperm DNA fragmentation, and oxidative-induced sperm DNA damage in men with varicocele. Materials and Methods: A cross-sectional study was performed, including 94 normozoospermic adults, of which 39 men without varicocele (controls) and 55 men with varicocele grades II or III, uni- or bilaterally. All men collected semen by masturbation. After semen analysis, the remaining volume was used for evaluation of three types of sperm DNA damage: (i) total DNA fragmentation, using an alkaline comet assay, (ii) double-stranded DNA fragmentation, using a neutral comet assay, and (iii) oxidative DNA damage, using an alkaline comet assay associated with the DNA glycosylase formamidopyrimidine enzyme. In each assay, percentage of sperm with any degree of DNA fragmentation, and with high DNA fragmentation were compared between the groups using an unpaired Student's t test or a Mann-Whitney test. Results: The varicocele group presented a higher rate of sperm with fragmented DNA (both any and high DNA fragmentation), considering single-stranded DNA fragmentation, double-stranded DNA fragmentation, or a combination of both, as well as oxidative- induced DNA fragmentation. Conclusions: Patients with varicocele have an increase in sperm DNA fragmentation levels, particularly in oxidative stress-induced sperm DNA damage.

Evaluation of oxidative stress in seminal plasma of adolescents with varicocele.

Varicocele, defined by a dilation of efferent testicular veins, is the most commonly identifiable, surgically correctable lesion associated with male-factor infertility, starts at puberty and causes a progressive decline in fertility potential. The pathophysiology of infertility caused by this disease is still poorly understood, but it is suggested that the main mechanism is oxidative stress. Therefore, the aim of this study was to verify if the varicocele is associated with changes in enzymatic antioxidant mechanisms and seminal plasma lipid peroxidation levels in adolescents. We recruited 90 adolescents that were divided into control (C; n = 27); varicocele and normal semen (VNS; n =46); varicocele and altered semen (VAS; n =17). Seminal and serum levels of lipid peroxidation were quantified by thiobarbituric acid reactive substances (TBARS). Seminal plasma antioxidant profile was evaluated by the activities of catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD). The VAS group had increased lipid peroxidation levels when compared to the other groups. The levels of serum lipid peroxidation and activities of the enzymes SOD and GPx did not differ between groups. CAT was undetectable by the method used. In conclusion, in adolescents with varicocele and altered semen analysis, there is an increase in seminal lipid peroxidation levels compared to adolescents with varicocele and without seminal change and adolescents without evident varicocele. However, the observed oxidative stress is not caused by a decrease in superoxide dismutase and glutathione peroxidase activities, which did not differ between adolescents with and without evident varicocele. LAY SUMMARY: Varicocele, defined by a dilation of efferent testicular veins, is the most commonly identifiable, surgically correctable lesion associated with male-factor infertility, starts at puberty and causes a progressive decline in fertile potential. There is still much that is not understood regarding how exactly it affects semen quality, but most studies agree that oxidative stress, which is defined as excessive amounts of free radicals in relation to antioxidant defense, is an important mechanism. In this study, we aimed to verify if the varicocele is associated with changes in antioxidant defense and semen oxidation in 90 adolescents with and without varicocele. In adolescents with varicocele and abnormal semen, there is an increase in semen oxidation compared to controls or to the group with varicocele and normal semen quality. Our results can help to understand how varicocele leads to infertility in adolescents, identifying changes in oxidative activity in semen, since the onset of varicocele and before damage to sperm production can be detected.

Oxidative origin of sperm DNA fragmentation in the adult varicocele.

PURPOSE: Sperm DNA fragmentation is a major cellular mechanism underlying varicocele-related male infertility. However, the type of DNA fragmentation - whether oxidative or of another nature - remains unknown. Thus, the aim of this study was to evaluate single- and double-stranded sperm DNA fragmentation, and oxidative-induced sperm DNA damage in men with varicocele. MATERIALS AND METHODS: A cross-sectional study was performed, including 94 normozoospermic adults, of which 39 men without varicocele (controls) and 55 men with varicocele grades II or III, uni- or bilaterally. All men collected semen by masturbation. After semen analysis, the remaining volume was used for evaluation of three types of sperm DNA damage: (i) total DNA fragmentation, using an alkaline comet assay, (ii) double-stranded DNA fragmentation, using a neutral comet assay, and (iii) oxidative DNA damage, using an alkaline comet assay associated with the DNA glycosylase formamidopyrimidine enzyme. In each assay, percentage of sperm with any degree of DNA fragmentation, and with high DNA fragmentation were compared between the groups using an unpaired Student's t test or a Mann-Whitney test. RESULTS: The varicocele group presented a higher rate of sperm with fragmented DNA (both any and high DNA fragmentation), considering single-stranded DNA fragmentation, double-stranded DNA fragmentation, or a combination of both, as well as oxidative-induced DNA fragmentation. CONCLUSIONS: Patients with varicocele have an increase in sperm DNA fragmentation levels, particularly in oxidative stress-induced sperm DNA damage.

Restoration of the apoptosis pathways' proteins levels after orchiectomy in testicular tumour patients.

Seminal plasma proteins already demonstrated to reflect the testicular environment function and important regulatory mechanisms. However, it is crucial to understand which of these proteins participate in probable altered pathways in testicular germ cell tumours and after unilateral orchiectomy. In this study, we proposed to verify, by a multiplex approach, the levels of DNA damage and apoptosis pathways' proteins, in seminal plasma of men before and after unilateral orchiectomy, and also in control men. Comparing pre- and post-orchiectomy groups, just the apoptosis pathways' proteins presented different levels, in which Bad was lower and Bcl2, Akt, caspase-9, p53 and caspase-8 were higher after orchiectomy. When comparing pre- and post-orchiectomy groups with control, both presented lower levels of ChK1, Chk2, H2AX, p53 and p21, for DNA damage pathway. Regarding the apoptosis pathway, lower levels of JNK, Bcl2, Akt, caspase-9, p53 and caspase-8 and higher levels of Bad were observed before orchiectomy. The post-orchiectomy group did not differ from controls, demonstrating a probable restoration on its proteins levels. We can conclude that testicular tumours can alter both of the assessed pathways, and its removal is associated with a probable restoration of the apoptosis pathway.

Seminal plasma protein networks and enriched functions in varicocele: Effect of smoking.

To verify a possible synergistic effect of smoking and varicocele on the seminal plasma proteome and biological functions, a cross-sectional study was performed in 25 smokers and 24 nonsmokers. Samples were used for conventional semen analysis, functional analysis (DNA fragmentation, acrosome integrity and mitochondrial activity) and proteomics by a shotgun approach. Functional enrichment of biological pathways was performed in differentially expressed proteins. Smokers presented lower ejaculate volume (p = .027), percentage of progressively motile spermatozoa (p = .002), total sperm count (p = .039), morphology (p = .001) and higher percentage of immotile spermatozoa (p = .03), round cell (p = .045) and neutrophil count (p = .009). Smokers also presented lower mitochondrial activity and acrosome integrity and higher DNA fragmentation. We identified and quantified 421 proteins in seminal plasma, of which one was exclusive, 21 were overexpressed and 70 were underexpressed in the seminal plasma of smokers. The proteins neprilysin, beta-defensin 106A and histone H4A were capable of predicting the smoker group. Enriched functions were related to immune function and sperm machinery in testis/epididymis. Based on our findings, we can conclude that cigarette smoking leads to the establishment of inflammatory protein pathways in the testis/epididymis in the presence of varicocele that seems to act in synergy with the toxic components of the cigarette.

Author Correction: Semen levels of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) protein families members in men with high and low sperm DNA fragmentation.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Semen levels of matrix metalloproteinase (MMP) and tissue inhibitor of metallorproteinases (TIMP) protein families members in men with high and low sperm DNA fragmentation.

Matrix Metalloproteinases (MMPs) and their regulators - Tissue Inhibitors of Matrix Metalloproteinases (TIMPs) - participate in extracellular matrix remodeling, fibrosis, and semen liquefaction, as well as to inflammatory activity. Seminal plasma has been shown to contain MMPs (MMP-2 and MMP-9) and TIMPs (TIMP-1 and TIMP-2). Also, a link between MMPs gene expression and excessive reactive oxygen species (ROS) has been established. In semen, ROS are associated with altered sperm function and increased DNA fragmentation. In this study, it is hypothesized that seminal MMPs and TIMPs levels are associated with sperm DNA fragmentation due to the fact that MMPs have been associated with semen quality. We also hypothesized that these proteins could predict DNA fragmentation status in sperm. Therefore, this study set out to verify if sperm DNA fragmentation levels relate to seminal levels of members of the MMP and TIMP protein families. The High sperm DNA fragmentation group presented lower seminal plasma levels of MMP-2, MMP-7, TIMP-1, TIMP-2 and TIMP-4 when compared to Low sperm DNA fragmentation group. Also, samples in the high sperm DNA fragmentation group presented higher acrosome integrity and lower mitochondrial activity levels when compared to low sperm DNA fragmentation samples. In the logistic regression analysis, MMP-2, MMP-7, and TIMP-4 classified samples as low and high sperm DNA fragmentation, with an overall model fit of 74.5%. Results from this study may demonstrate a specific inflammatory mechanism in samples with high sperm DNA fragmentation. This, in turn, can lead to the development of new studies regarding this mechanism and, in the future, create an opportunity to treat these patients for sperm DNA fragmentation by treating inflammatory seminal activity.

Effect of orchiectomy on sperm functional aspects and semen oxidative stress in men with testicular tumours.

To verify if quality of spermatozoa from men with testicular germ cell tumours is better before or after orchiectomy. This prospective study was carried out including 24 patients with testicular germ cell tumours, who provide one semen sample before they were submitted to unilateral orchiectomy and one other semen sample 30 days after the surgery. After collection by masturbation and liquefaction, an aliquot of the semen sample was used for semen analysis and another was used to evaluate sperm mitochondrial activity, DNA fragmentation and acrosome integrity. Seminal plasma was used to evaluate lipid peroxidation levels. Pre-orchiectomy sample and post-orchiectomy sample were compared using a paired Student's t test (normal distribution) or a paired Wilcoxon test, when appropriate (p Ë‚ 0.05). No significant difference was observed in semen analysis. A significant decrease in DNA fragmentation and lipid peroxidation and an increase in mitochondrial activity were observed after orchiectomy. Based on our findings, the semen quality from men with testicular germ cell tumours is better after orchiectomy.

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.

Le 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.

Prostate cancer proteomics: clinically useful protein biomarkers and future perspectives.

INTRODUCTION: Although prostate cancer constitutes one of the most important, death-related diseases in the male population, there is still a need for identification of sensitive biomarkers that could precociously detect the disease and differentiate aggressive from indolent cancers, in order to decrease overtreatment. Proteomics research has improved understanding on mechanisms underlying tumorigenesis, cancer cells migration and invasion potential, and castration resistance. This review has focused on proteomic studies of prostate cancer published in the recent years, with a special emphasis on determination of biomarkers for cancer progression and diagnosis. Areas covered: Shotgun and targeted-proteomic studies of prostate cancer in different matrices are reviewed, i.e., prostate tissue, prostate cell lines, blood (serum and plasma), urine, seminal plasma, and exosomes. The most important biomarkers for cancer diagnosis and aggressiveness characterization are highlighted. Expert commentary: In general, results demonstrate alteration in cell cycle control, DNA repair, proteasomal degradation, and metabolic activity. However, these studies suffer from low reproducibility due to heterogeneity of the cancer itself, as well as to techniques utilized for protein identification/quantification. Downstream confirmatory studies in separate cohorts are warranted in order to demonstrate accuracy of these results.

Alterations in the proliferative/apoptotic equilibrium in semen of adolescents with varicocele.

PURPOSE: To verify if the presence of varicocele (grades II and III) with and without seminal alterations, using the 5th 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. METHODS: 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). RESULTS: 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). CONCLUSION: 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.

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

OBJECTIVE: To evaluate the effect of smoking on sperm functional quality and seminal plasma proteomic profile. PATIENTS AND 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). CONCLUSION: 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.

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.

Proteomic profile of seminal plasma in adolescents and adults with treated and untreated varicocele.

Varicocele, the most important treatable cause of male infertility, is present in 15% of adult males, 35% of men with primary infertility, and 80% of men with secondary infertility. On the other hand, 80% of these men will not present infertility. Therefore, there is a need to differentiate a varicocele that is exerting a deleterious effect that is treatable from a "silent" varicocele. Despite the growing evidence of the cellular effects of varicocele, its underlying molecular mechanisms are still eluding. Proteomics has become a promising area to determine the reproductive biology of semen as well as to improve diagnosis of male infertility. This review aims to discuss the state-of-art in seminal plasma proteomics in patients with varicocele to discuss the challenges in undertaking these studies, as well as the future outlook derived from the growing body of evidence on the seminal proteome.

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 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. 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.

Maldi-tof fingerprinting of seminal plasma lipids in the study of human male infertility.

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.