Associations between Sperm Epigenetic Age and Semen Parameters: An Evaluation of Clinical and Non-Clinical Cohorts.

The well-documented relationship between chronological age and the sperm methylome has allowed for the construction of epigenetic clocks that estimate the biological age of sperm based on DNA methylation, which we previously termed sperm epigenetic age (SEA). Our lab demonstrated that SEA is positively associated with the time taken to achieve pregnancy; however, its relationship with semen parameters is unknown. A total of 379 men from the Longitudinal Investigation of Fertility and Environment (LIFE) study, a non-clinical cohort, and 192 men seeking fertility treatment from the Sperm Environmental Epigenetics and Development Study (SEEDS) were included in the study. Semen analyses were conducted for both cohorts, and SEA was previously generated using a machine learning algorithm and DNA methylation array data. Association analyses were conducted via multivariable linear regression models adjusting for BMI and smoking status. We found that SEA was not associated with standard semen characteristics in SEEDS and LIFE cohorts. However, SEA was significantly associated with higher sperm head length and perimeter, the presence of pyriform and tapered sperm, and lower sperm elongation factor in the LIFE study (p < 0.05). Based on our results, SEA is mostly associated with defects in sperm head morphological factors that are less commonly evaluated during male infertility assessments. SEA shows promise to be an independent biomarker of sperm quality to assess male fecundity.

Moment of induction and duration of experimental varicocele in rats: effects on semen quality.

PURPOSE: Varicocele is a condition known to cause damage to seminal parameters and sperm function. Furthermore, it has been hypothesized that the varicocele effect on fertility is time-dependent; however, little is known about the consequences of its establishment time on reproductive organs and/or sperm function. This study aimed to evaluate the effect of the duration of experimental varicocele on reproductive organs, sperm parameters, and sperm function. MATERIALS AND METHODS: Varicocele induction surgeries were performed in Wistar rats aged 40 or 100 days old. At 160-day-old, analyses were performed, including biometry of reproductive organs (prostate, seminal vesicles, epididymis, and testis), sperm parameters (vitality, morphology, and motility), and sperm function tests (nuclear DNA integrity, acrosome integrity, and mitochondrial activity). RESULTS: The analysis of the biometry of reproductive organs showed no differences between distinct ages in which varicocele was induced. The total abnormal sperm morphology was bigger in animals with varicocele induced to 100 days old than in animals with varicocele induced to 40 days old. Regarding nuclear DNA integrity, animals of varicocele induced to 100 days old showed worse results compared to animals of varicocele induced to 40 days old. Other parameters analyzed showed no differences between varicocele groups. CONCLUSION: In this study conducted on rats, we conclude that varicocele adversely affects sperm, particularly its function. However, we did not observe a negative progressive effect on sperm.

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.

The '-ics' of male reproduction: genomics, epigenetics, proteomics, metabolomics, and microbiomics.

PURPOSE OF REVIEW: To review the most current findings, from the past 2 years, in various '-ics' fields in male infertility, with a specific focus on nonobstructive azoospermia, the most severe form, and varicocele, the most common correctable cause of male infertility. RECENT FINDINGS: Recent studies confirm previously identified causes and identify previously unknown genetic mutations as causes for nonobstructive azoospermia and varicocele. SUMMARY: Infertility is a common problem for couples with approximately half of cases attributable to male factor infertility. Although advances in assisted reproductive technology have permitted many more men with infertility to father biological children, the majority of infertile men continue to have unknown causes. The recent explosion of the '-ics' fields, including genomics, epigenetics, proteomics, metabolomics, and microbiomics, has shed light on previously unknown causes for various diseases. New information in these fields will not only shed light on the pathogenesis of these conditions but also may shift the paradigm in clinical testing that may allow clinicians to provide more precise counseling and prognostic information for men with infertility.

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.

Apoptotic balance during testicular detorsion after one hour induced torsion in rats.

Testicular torsion (TT) is an emergency complication that leads to oxidative stress and adversely affects spermatogenesis. Although immediate treatment consists of testicular detorsion (TD) to reverse TT-induced ischemia, mechanisms underlying recovery have yet to be fully understood. The current study aimed to investigate TD effects after a one-hour experimental TT by evaluating testicular antioxidant status and apoptosis-related proteins. Forty male Wistar rats were submitted to TT by testicular rotation, for one hour. Following TT, 32 rats were submitted to TD for 1, 2, 4, and 8 h (N = 8/group), the other 8 rats euthanized as TT-only. For controls, 8 rats were sham-operated. Testicular tissues were aseptically dissected for biochemical, histopathological, and immunohistochemistry analyses. The TD groups, especially after 4 h of TD, exhibited diminished MDA and increased TAC and GPX levels in testicular tissue. Levels of p53 and Caspase-3 were down-regulated in T1D4 and T1D8 groups versus torsion group. Bcl-2 was increased in T1D4 and T1D8 groups compared to the TT group. Moreover, spermatogenesis was recovered in T1D4 and T1D8 groups compared to the TT group. It can be concluded that after 1 h TT in rats, at least 4 h post-TD is needed for testicular tissue to initiate recovery.

Protein markers of spermatogenesis and their potential use in the management of azoospermia.

INTRODUCTION: Azoospermia, absence of sperm in the ejaculate is classified as obstructive (OA) and non-obstructive azoospermia (NOA). In OA, sperm are produced, but due to physical obstruction in the male reproductive tract, they are not released in the ejaculate. NOA, on the other hand, is defined as the absence of sperm in the ejaculate due to testicular dysfunction. In NOA, spermatogenesis is frequently preserved in specific sites, and proteomics studies have been employed in order to identify men with preserved spermatogenesis. AREAS COVERED: Differential protein expression in patients with male infertility is an indicator of impaired spermatogenesis. Here, we reviewed proteins with a potential role as biomarkers of spermatogenesis that could help in the management of non-obstructive and obstructive azoospermia. The following keywords were used for bibliographic research: seminal plasma, proteomics, male infertility, nonobstructive, obstructive, azoospermia, oligospermia. EXPERT OPINION: Biopsy is an invasive and potentially harmful technique for detecting spermatogenesis in men with OA and NOA. Seminal plasma proteins are highly promising as biomarkers for spermatogenesis. Current literature presents a number of potential candidate biomarkers for determining preserved spermatogenesis.

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.

Mechanisms of Sperm Oxidation: Breaking Down the Origins of Oxidative Activity.

Oxidative stress has been long considered an important cause of male infertility, and sperm-derived reactive oxygen species (ROS) have been considered one of the major sources. In article number 1900205, Issue 2, Netherton et al. have improved upon current technologies for detection of true ROS-derived oxidative events from those originated by mitochondrial compounds. Moreover, the authors have isolated proteins that bear lipid aldehyde adducts, in order to verify their origin. In their paper, Netherton et al. demonstrate that sperm-derived ROS do not contribute to sperm oxidative stress, which is an important finding, given that most studies consider altered sperm a major source of seminal ROS. Moreover, adducted proteins are mostly of prostatic origin, which leads to questions regarding how and where the oxidative events occur. Under the light of this study and the methodological improvements it has brought, it may be important to revisit some foundation studies in semen and sperm oxidative stress.

Carnosine treatment during human semen processing by discontinuous density gradient.

The aim of this article was to evaluate the effects of different concentrations of carnosine added during human semen processing. Semen samples from 34 patients were submitted to processing by discontinuous density gradient centrifugation without (control) or with different concentrations of carnosine supplementation as follows: (a) 20 mM of carnosine supplementation on the layers of Percoll; and (b) 50 mM carnosine supplementation. Sperm samples were then washed with human tubal fluid medium and evaluated according to sperm kinetics and functional assessment. For statistical analysis, data were evaluated by a general linear model or a Friedman test, whenever appropriate. The 50 mM carnosine supplementation led to improved sperm mitochondrial activity when compared to untreated samples. Motility variables, such as percentage of motile and progressively motile spermatozoa, average path velocity, straight line velocity, curvilinear velocity and linearity, showed an improvement after semen processing irrespective of carnosine supplementation. Both concentrations of carnosine increased the beat-cross frequency (BCF) when compared to samples before processing. We conclude that carnosine supplementation in semen samples benefits sperm mitochondrial activity and BCF.

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.

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.

Current scenario of endourological treatment of kidney stones in brazil: results of a national survey.

OBJECTIVE: To elucidate the current scenario of endourology in Brazil for the treatment of urinary lithiasis, with an emphasis on regional differences and the reasons why certain techniques are still underutilized. MATERIALS AND METHODS: An electronic questionnaire was sent by email to the 4,745 members of the Brazilian Urological Society (BSU) in 2016 to collect information on the 3 main endourological procedures used in the treatment of nephrolithiasis: Semi-rigid ureteroscopy (URS), Flexible ureteroscopy (F-URS) and percutaneous nephrolithotripsy (PCNL). RESULTS: A total of 1,267 urologists answered the questionnaire. It was observed that the vast majority perform URS (95.6%), while 80.2% perform F-URS and only 72.1% perform PCNL. Regarding the surgical volume, most perform up to 10 procedures per month (73.4% to 88.2%) and the main impediment was the lack of patients with the pathology (42.1% to 67.7%). The lack of equipment or hospital infrastructure was one of the main limiting factors for rigid (23%) and flexible (38.1%) URS, mainly in the North and Northeast regions of the country. Regarding PCNL, most of them reported lack of practical experience in the method (29.9%). Finally, most urologists expressed interest in taking courses in endourology. CONCLUSION: Ureteroscopy, rigid or flexible, is already well established in the country, requiring the direction of more resources for its practice, especially in less developed regions. Regarding PCNL a significant part of Brazilian urologists still lack practical experience in this procedure, emphasizing the need for greater investment in teaching this technique.

Post-translational modifications of seminal proteins and their importance in male fertility potential.

Introduction: The seminal proteome has been shown to directly influence the male fertile potential. Post-translational modifications (PTMs) are significant changes that play a role in the biological regulation of proteins. Sperm cells are transcriptionally and translationally inactive and these modifications are essential to control protein function.Areas covered: Here we reviewed seven PTMs which importance for male reproductive function investigated in the past decade, namely S-nitrosylation and tyrosine nitration (both occurring by the action of NO), glycosylation, ubiquitination, acetylation, methylation, and SUMOylation. Since they were previously identified in human semen, we focus on their role in sperm function, as well as in physiological and pathophysiological processes which could contribute to the fertility potential. The following keywords were applied: 'post-translational modification', 'sperm', 'semen', 'seminal plasma', 'male infertility', 'nitrosylation', 'nitration', 'histone methylation', 'SUMOylation', 'ubiquitination', 'ubiquitilation', 'glycosylation', and 'acetylation'.Expert opinion: Most biological processes orchestrated by proteins require PTMs for their activation or inhibition. Most of them are dynamic and occur in mature sperm, modulating protein function, thus exerting a significant role in sperm function and fertility. Finally, the study of PTMs should be also addressed in pathophysiological processes, as different clinical conditions are known to alter the proteome.

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.

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.

Towards Understanding Male Infertility After Spinal Cord Injury Using Quantitative Proteomics.

The study of male infertility after spinal cord injury (SCI) has enhanced the understanding of seminal plasma (SP) as an important regulator of spermatozoa function. However, the most important factors leading to the diminished sperm motility and viability observed in semen of men with SCI remained unknown. Thus, to explore SP related molecular mechanisms underlying infertility after SCI, we used mass spectrometry-based quantitative proteomics to compare SP retrieved from SCI patients to normal controls. As a result, we present an in-depth characterization of the human SP proteome, identifying ∼2,800 individual proteins, and describe, in detail, the differential proteome observed in SCI. Our analysis demonstrates that a hyper-activation of the immune system may influence some seminal processes, which likely are not triggered by microbial infection. Moreover, we show evidence of an important prostate gland functional failure,i.e.diminished abundance of metabolic enzymes related to ATP turnover and those secreted via prostasomes. Further we identify the main outcome related to this fact and that it is intrinsically linked to the low sperm motility in SCI. Together, our data highlights the molecular pathways hindering fertility in SCI and shed new light on other causes of male infertility.

Intrauterine Growth Restriction Programs the Hypothalamus of Adult Male Rats: Integrated Analysis of Proteomic and Metabolomic Data.

Programming of hypothalamic functions regulating energy homeostasis may play a role in intrauterine growth restriction (IUGR)-induced adulthood obesity. The present study investigated the effects of IUGR on the hypothalamus proteome and metabolome of adult rats submitted to 50% protein-energy restriction throughout pregnancy. Proteomic and metabolomic analyzes were performed by data independent acquisition mass spectrometry and multiple reaction monitoring, respectively. At age 4 months, the restricted rats showed elevated adiposity, increased leptin and signs of insulin resistance. 1356 proteins were identified and 348 quantified while 127 metabolites were quantified. The restricted hypothalamus showed down-regulation of 36 proteins and 5 metabolites and up-regulation of 21 proteins and 9 metabolites. Integrated pathway analysis of the proteomics and metabolomics data indicated impairment of hypothalamic glucose metabolism, increased flux through the hexosamine pathway, deregulation of TCA cycle and the respiratory chain, and alterations in glutathione metabolism. The data suggest IUGR modulation of energy metabolism and redox homeostasis in the hypothalamus of male adult rats. The present results indicated deleterious consequences of IUGR on hypothalamic pathways involved in pivotal physiological functions. These results provide guidance for future mechanistic studies assessing the role of intrauterine malnutrition in the development of metabolic diseases later in life.

Antioxidant enzyme profile and lipid peroxidation products in semen samples of testicular germ cell tumor patients submitted to orchiectomy.

PURPOSE: To determine enzymatic antioxidant and lipid peroxidation levels in seminal plasma of patients orchiectomized for testicular tumors. MATERIALS AND METHODS: The study included 52 patients: 26 control men and 26 orchiectomized patients for testicular tumor, of which 12 men had seminoma tumor and 14 men non-seminoma tumor. After semen analysis performed according to the WHO guidelines, an aliquot of semen was centrifuged and the seminal plasma was collected. Lipid peroxidation was performed by thiobarbituric acid reactive substances(TBARS) assay and antioxidant profile was assessed by analyzing catalase, glutathione per-oxidase (GPx) and superoxide anion (SOD) activities using colorimetric assays with a standard spectrophotometer. Data were tested for normality and compared using one-way ANOVA (p<0.05). RESULTS: Seminoma and non-seminoma groups presented lower sperm concentration and morphology when compared to control group (p=0.0001). Both study groups (seminoma and non-seminoma) presented higher TBARS levels when compared to control group (p=0.0000013). No differences were observed for SOD (p=0.646) and GPx (p=0.328). It was not possible to access the enzymatic activity of catalase in any group. CONCLUSION: Patients with testicular tumor present increased semen oxidative stress, but no differences were observed in antioxidant levels, even after orchiectomy. This indicates that most likely an increased generation of oxidative products takes place in these patients.

Sperm proteomics: potential impact on male infertility treatment.

Spermatozoa are unique cells that have highly compact DNA, motility (and hypermotility) patterns, a specific morphology, localized mitochondria and an apical acrosome. They are the end product of a dynamic process termed spermatogenesis. Sperm are therefore produced with specific proteins in order to effect different traits, such as the presence of cysteine-rich protamines in DNA, which effectively compacts DNA. Moreover, specific proteins are transferred during epididymal maturation and after ejaculation in order to render sperm capable of undergoing post-ejaculatory alterations, generally termed capacitation, which confers capacity to fertilize a mature oocyte. In addition, sperm exhibit several post-translational modifications, which are fundamental to their function, such as SUMOylation and ubiquitination. Discussed in this review is the current knowledge of the sperm proteome in terms of its composition and the function that these proteins determine, as well as their post-translational modifications and how these alter sperm functional integrity. Studies are emphasized that focus on shotgun proteomics--untargeted determination of the protein constituent of a cell in a given biological condition--and technologies currently applied toward that end are reviewed.