Insight toward inflammasome complex contribution to male infertility.

During the last decades, a wide range of factors involved in the physiopathology of male infertility disease have been discussed. The inflammation role in some of the main infertility-related diseases has been studied, such as varicocele, spinal cord injury and obesity. Inflammation is the main response of the immune system to infection or cell damage, leading to intense inflammatory cytokine release during the loss of homeostasis. One of the first steps toward pro-inflammatory cytokines release is the recognition of dangerous signals by the immune cells, including pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). These molecules can activate an important multiprotein complex, called inflammasome. Although these complexes have been studied during the last decades, their participation in male infertility has gained attention recently. Considering the inflammasome complex's high potential to be targeted for drug therapy, this review tries to shed light on current literature. Therefore, in the current review paper, we aimed to discuss the inflammasome complex activation, involvement in different male infertility conditions, and localization in the male reproductive tract.

Testicular torsion in vivo models: Mechanisms and treatments.

BACKGROUND: Testicular torsion is a condition in which a testis rotates around its longitudinal axis and twists the spermatic cord. This in turn results in a significant decrease in blood flow and perfusion of testicular tissue. During Testicular torsion, the testicular tissue is affected by ischemia, heat stress, hypoxia, and oxidative and nitrosative stress. The testicular torsion should be considered an emergency condition and surgical intervention (testicular detorsion ) as the sole treatment option in viable cases involves counter-rotation on twisted testes associated, when possible, to orchipexy, in order to avoid recurrence. Possible testicular detorsion side-effects occur due to reperfusion and endothelial cells injury, microcirculation disturbances, and intense germ cells loss. OBJECTIVES: To discuss testicular torsion surgery-based methods, different time frames for testicular torsion induction, and the associated pathophysiology by emphasizing cellular and molecular events as well as different therapeutic agent applications for testicular torsion. MATERIALS AND METHODS: We reviewed all original research and epidemiological papers related to testicular torsion condition. RESULTS: Testicular torsion causes germ cell necrosis, arrested spermatogenesis, and diminished testosterone levels, with consequent infertility. Among different involved pathophysiological impacts, testicular torsion/detorsion-induced ischemia seems to play the key role by leading the tissue toward other series of events in testis. Numerous studies have used adjuvant antioxidants, calcium channel blockers, anti-inflammatory agents, or vasodilating agents in order to decrease these effects. DISCUSSION AND CONCLUSION: To the best of our knowledge, no previously conducted study examined therapeutical agents' beneficial effects post clinical I/R condition in humans. Different agents targeting different pathophysiological conditions were used to ameliorate the ischemia/reperfusion-induced condition in animal models, however, none of the administrated agents were tested in human cases. Although considering testicular detorsion surgery is still the golden method to reverse the testicular torsion condition and the surgical approach is undeniable, the evaluated agents with beneficial effects, need to be investigated furthermore in clinical conditions. Thus, furthermore clinical studies and case reports are required to approve the animal models proposed agents' beneficial impacts.

Characterization of varicocele-induced animal models: Potential role of inflammasome complex in the varicocele pathophysiology.

Varicocele mechanisms and its impact in testicular dysfunction has been studied in order to understand the pathophysiology involved in this disease. However, study designs using testicular tissues from varicocele patients are restricted due to ethical limitations. Therefore, the use of animal models, mainly rats, that mimics varicocele and its effects is an option to develop new approaches. The surgical technique, that induces the varicocele in rats, is based on the partial obstruction of the left renal vein, leading to a dilation in the left spermatic vein and consequently to the pampiniform plexus, resulting in varicocele-induced condition. Thus, the study of the cellular and molecular mechanisms involved in varicocele development can be addressed in depth. Besides the animal model goal to uncover the exact varicocele pathophysiology, varicocele induced models are the best options to develop new non-surgical and less invasive therapies. Various animal model studies designed and investigated antioxidant and anti-inflammatory agents to face varicocele conditions. Minding this fact, we tried to discuss a newly uncovered complex in varicocele condition, known as inflammasome complex. Taking into consideration the possible inflammatory state present in varicocele, the inflammasome complex has been proposed to be involved in the pathophysiology of this disease.

Proteomic research and diagnosis in bladder cancer: state of the art review

ABSTRACT Purpose: Proteomic biomarkers have been emerging as alternative methods to the gold standard procedures of cystoscopy and urine cytology in the diagnosis and surveillance of bladder cancer (BC). This review aims to update the state of the art of proteomics research and diagnosis in BC. Materials and Methods: We reviewed the current literature related to BC research on urinary, tissue, blood and cell line proteomics, using the Pubmed database. Findings: Two urinary protein biomarkers are FDA-approved (NMP22® and BTA® tests), only if performed along with cystoscopy for surveillance after initial diagnosis, but not in the primary diagnostic setting due to high false-positive rates in case of infections, stones and hematuria. There are a great number of non-FDA approved proteins being studied, with good preliminary results; panels of proteins seem valuable tools to be refined in ongoing trials. Blood proteins are a bigger challenge, because of the complexity of the serum protein profile and the scarcity of blood proteomic studies in BC. Previous studies with the BC tissue proteome do not correlate well with the urinary proteome, likely due to the tumor heterogeneity. Cell line proteomic research helps in the understanding of basic mechanisms that drive BC development and progression; the main difficulty is culturing low-grade tumors in vitro, which represents the majority of BC tumors in clinical practice. Conclusion: Protein biomarkers have promising value in the diagnosis, surveillance and prognostic of BC. Urine is the most appropriate body fluid for biomarker research in BC due to its easiness of sampling, stability and enrichment of shed and secreted tumor-specific proteins. Panels of biomarkers may exhibit higher sensitivity than single proteins in the diagnosis of BC at larger populations due to clinical and tumor heterogeneity. Prospective clinical trials are warranted to validate the relevance of proteomic data in the clinical management of BC.

Proteomic research and diagnosis in bladder cancer: state of the art review.

PURPOSE: Proteomic biomarkers have been emerging as alternative methods to the gold standard procedures of cystoscopy and urine cytology in the diagnosis and surveillance of bladder cancer (BC). This review aims to update the state of the art of proteomics research and diagnosis in BC. MATERIALS AND METHODS: We reviewed the current literature related to BC research on urinary, tissue, blood and cell line proteomics, using the Pubmed database. FINDINGS: Two urinary protein biomarkers are FDA-approved (NMP22® and BTA® tests), only if performed along with cystoscopy for surveillance after initial diagnosis, but not in the primary diagnostic setting due to high false-positive rates in case of infections, stones and hematuria. There are a great number of non-FDA approved proteins being studied, with good preliminary results; panels of proteins seem valuable tools to be refined in ongoing trials. Blood proteins are a bigger challenge, because of the complexity of the serum protein profile and the scarcity of blood proteomic studies in BC. Previous studies with the BC tissue proteome do not correlate well with the urinary proteome, likely due to the tumor heterogeneity. Cell line proteomic research helps in the understanding of basic mechanisms that drive BC development and progression; the main difficulty is culturing low-grade tumors in vitro, which represents the majority of BC tumors in clinical practice. CONCLUSION: Protein biomarkers have promising value in the diagnosis, surveillance and prognostic of BC. Urine is the most appropriate body fluid for biomarker research in BC due to its easiness of sampling, stability and enrichment of shed and secreted tumor-specific proteins. Panels of biomarkers may exhibit higher sensitivity than single proteins in the diagnosis of BC at larger populations due to clinical and tumor heterogeneity. Prospective clinical trials are warranted to validate the relevance of proteomic data in the clinical management of BC.

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.

Willingness of Infertile Couples to Pay for In Vitro Fertilization Treatment in the Integrated Human Reproduction Section of the Escola Paulista de Medicina, São Paulo Federal University.

OBJECTIVE: To describe the willingness to pay (WTP) of infertile couples for in vitro fertilization (IVF) treatment. METHOD: This was a prospective study with an anonymous questionnaire for infertile couples in an academic setting. Clinical characteristics were analyzed by a Student's t test or Mann-Whitney test, categorical variables were compared by a chi-square or Fisher exact test, and correlations were assessed using a Spearman's test. An alpha of 5% was adopted. RESULTS: Mean female and male ages were 31.5 and 35.9 years, respectively; 80.2% were married; 19.8% were in consensual union; 48.1% of women had college degrees; and 49.4% of men had a high school education. Most women (77.8%) and men (75.3%) were white, with a household income of class C. Average duration of union was 8.5 years, and average infertility was 4.7 years. Using a willingness-to-pay (WTP) evaluation and the technique of "direct questioning," the average value was determined to be R$18 720.18 (by payment scale R$22 831.17). WTP positively correlated with household income and each woman's education level. Previous parenthood or use of public health system negatively correlated with WTP. CONCLUSION: We conclude that the higher the couple's monthly income and the woman's educational level, the higher the WTP for an IVF treatment; previous parenthood determined a lower WTP for an IVF treatment, and previous use of the Brazilian Unified Health System, determined a lower WTP for an IVF treatment.

Systemic arterial hypertension leads to decreased semen quality and alterations in the testicular microcirculation in rats.

Arterial hypertension is a cardiovascular disease that leads to important systemic alterations and drastically impairs normal organ function over time. Hypertension affects around 700 million men of reproductive age and hypertensive men present increased risk for reproductive disorders, such as erectile dysfunction. However, the link between arterial hypertension and male reproductive disorders is associative at best. Moreover, many studies have reported associations between decreased male fertility and/or semen quality and alterations to general male health. In this study we aim to investigate the effect of systemic high blood pressure in sperm quality, sperm functional characteristics and testicular physiology in a rat model. Hypertensive rats presented altered testicular morphology - mainly vascular alterations and impaired testicular vasomotion. Hypertensive rats also presented decrease in sperm concentration, DNA integrity and increased percentages of sperm with dysfunctional mitochondria, intracellular superoxide anion activity and abnormal morphology. This study provides mechanistic insights by which arterial hypertension affects the testes, evidencing the testes as another target organ for hypertension as well as its impact on sperm quality.

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.

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.

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.