Protein Fingerprinting of Seminal Plasma Reveals Dysregulation of Exosome-Associated Proteins in Infertile Men with Unilateral Varicocele

Panner Selvam Manesh Kumar,Agarwal Ashok,Sharma Rakesh,Samanta Luna,Gupta Sajal,Dias Tânia R.,Martins Ana Dias 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.2

Purpose: Aberrant expression of seminal plasma proteins are associated with altered homeostasis that may affect the fertilizing ability of spermatozoa. However, the precise roles of seminal exosomes on sperm function remain unclear. The objective of this study was to identify the differentially expressed proteins (DEPs) associated with varicocele-mediated infertility by comparing seminal plasma protein profile of unilateral varicocele patients with proven fertile donors. Materials and Methods: Semen samples were obtained from 10 proven fertile donors with normal semen parameters and 33 infertile patients with unilateral varicocele. For proteomic analysis, 5 samples from each group were pooled and run in triplicate. Key DEPs (ANXA2, TF, CD63, KIF5B, SEMG1) associated with the exosome function were selected by bioinformatic tools and validated using Western blotting. Results: A total of 47 seminal plasma proteins were differentially expressed in unilateral varicocele patients compared to fertile donors. Validation of exosome-associated DEPs in unilateral varicocele patients (n=7) and fertile donors (n=7) revealed significant upregulation of ANXA2 (p=0.0016) and downregulation of KIF5B (p=0.009). The main upstream regulators of the DEPs in seminal plasma of unilateral varicocele group were androgen receptor, YB1 and NRF2. Conclusions: This is the first report to identify DEPs in seminal plasma of unilateral varicocele patients compared to fertile donors. Based on the detection of DEPs associated with exosomal function, Western blotting was used to validate the presence of defective exosome machinery in seminal plasma of unilateral varicocele patients. KIF5B and ANXA2 can be utilized as potential biomarkers of infertility in unilateral varicocele patients.

Proteomic Profiling of Seminal Plasma Proteins in Varicocele Patients

Panner Selvam Manesh Kumar,Agarwal Ashok 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.1

Purpose: Seminal plasma provides a nutritive and protective milieu for spermatozoa. It contains factors/proteins required for sperm maturation, hyperactivation, capacitation and acrosome reaction. Alteration in the expression levels of seminal plasma proteins affect the fertilization process. The main objective of this study is to compare the seminal plasma proteome of healthy fertile men (control group) with varicocele patients in order to identify the differentially expressed seminal plasma proteins. Materials and Methods: Pooled seminal plasma samples from control (n=5) and varicocele (unilateral: n=5 and bilateral: n=5) subjects were used for proteomic profiling and functional bioinformatic analysis. Key differentially expressed proteins (DEPs) associated with binding of zona pellucida (acrosin; ACR), protein folding (heat shock related 70 kDa protein 2; HSPA2), oxidative stress (peroxiredoxin 2; PRDX2), lipid peroxidation and DNA fragmentation (apolipoprotein A2; APOA2) were validated by Western blot. Statistical analysis was conducted using Mann–Whitney test. Results: A total of 412 and 486 proteins were detected in seminal plasma of control group and varicocele patients respectively. Twenty-eight proteins were identified as DEPs between varicocele and control group. Validation of DEPs revealed downregulation of HSPA2 (p=0.0037) as well as APOA2 (p=0.0373), and upregulation of PRDX2 (p=0.0474). Conclusions: The seminal plasma protein profile of varicocele patients differ from healthy fertile men. Aberrant expression of seminal plasma proteins serve as an indicator of sperm pathology in varicocele patients.

Sperm and Seminal Plasma Proteomics: Molecular Changes Associated with Varicocele-Mediated Male Infertility

Manesh Kumar Panner Selvam,Ashok Agarwal 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.4

Male infertility is a rising problem and the etiology at the molecular level is unclear. Use of omics has provided an insight into the underlying cellular changes in the spermatozoa of infertile men. Proteomics is one the promising omics techniques for biomarker screening that can provide complete information on molecular processes associated with male infertility. Varicocele is a pressing issue in the field of male infertility and the search for an appropriate diagnostic and therapeutic biomarker is still ongoing. In this review, we discuss the reports on proteomic profiles of sperm and seminal plasma in male infertility and provide an in-depth insight into varicocele studies associated with male infertility.

Validation of LensHooke® X1 PRO and Computer-Assisted Semen Analyzer Compared with Laboratory-Based Manual Semen Analysis

Agarwal Ashok,Panner Selvam Manesh Kumar,Ambar Rafael F. 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.3

Purpose: To compare two automated semen quality analysis systems (LensHooke® X1 PRO [X1 PRO]) and IVOS CASA) for accuracy, precision and agreement with laboratory-based manual semen analysis (MSA). Materials and Methods: Semen samples (n=31) were obtained from normozoospermic healthy male volunteers and infertile men with a minimum abstinence period between 2–3 days. After complete liquefaction, 101 seminal aliquots were prepared and tested according to WHO 5th Edition (2010) guidelines. The results obtained by X1 PRO and IVOS CASA were compared with that of MSA. Additionally, 10 samples were used to evaluate the intra- and inter-rater agreement for X1 PRO and MSA. Results: The semen parameters (sperm concentration, total, and progressive motility) showed strong correlation and agreement for both automated semen analyzers and MSA (Spearman’s rank correlation ≥0.92, p<0.0001). X1 PRO and IVOS CASA were able to differentiate samples with abnormal concentration with a positive predictive value (PPV) of 100%. Furthermore, the PPV for X1 PRO (86.5%) was higher than that for IVOS CASA (71.7%) in differentiating samples with abnormal motility. The X1 PRO device showed a high PPV (97.7%) in identifying normal sperm forms compared to MSA. Semen parameters evaluated showed a high inter-rater (kappa >0.91) and intra-rater (kappa >0.92) agreement for X1 PRO compared with MSA. Conclusions: Both automated semen analyzers demonstrated a high level of concordance and their performance was comparable with MSA analysis. Furthermore, high-levels of inter-and intra-rater reliability for semen analysis indicate that the new X1 PRO can be used in a clinical laboratory to offer accurate and quick test results.

Highly Cited Articles in the Field of Male Infertility and Antioxidants: A Scientometric Analysis

Agarwal Ashok,Selvam Manesh Kumar Panner,Baskaran Saradha,Finelli Renata,Leisegang Kristian,Barbăroșie Cătălina,Pushparaj Peter Natesan,Robert Kathy Amy,Ambar Rafael F.,Iovine Concetta,Durairajanayaga 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.4

Purpose: The objective of this scientometric analysis was to recognize the top 100 cited articles on ‘Male infertility and Antioxidants’ and analyze its publication characteristics. Materials and Methods: The Scopus database was used to retrieve related articles and the top 100 identified based on citation rate. Results: The articles were published in 56 journals between 1995 and 2019 with a median (interquartile range) citation score of 17 (5–62). Among the top 100 articles, 69 were clinical studies, which included controlled and blinded (33.33%), prospective (27.54%), randomized-controlled trials (26.09%), uncontrolled (11.59%), and retrospective (1.45%) studies. In addition to conventional semen parameters, advanced sperm function tests such as oxidative stress (51%) and sperm DNA damage (23%) were reported. Pregnancy rate (33%) was found to be the most reported reproductive outcome. Antioxidant therapy was mostly investigated in male cohorts with sperm abnormalities such as asthenozoospermia (28%) and clinical conditions such as idiopathic male infertility (20%), varicocele/varicocelectomy (17%) and general male infertility (16%). Conclusions: The most influential publications on antioxidants and male infertility were identified for the first time in the literature. This will serve as a reliable source of information for researchers and clinicians alike.

Male Oxidative Stress Infertility (MOSI): Proposed Terminology and Clinical Practice Guidelines for Management of Idiopathic Male Infertility

Ashok Agarwal,Neel Parekh,Manesh Kumar Panner Selvam,Ralf Henkel,Rupin Shah,Sheryl T. Homa,Ranjith Ramasamy,Edmund Ko,Kelton Tremellen,Sandro Esteves,Ahmad Majzoub,Juan G. Alvarez,David K. Gardner,Cha 대한남성과학회 2019 The World Journal of Men's Health Vol.37 No.3

Despite advances in the field of male reproductive health, idiopathic male infertility, in which a man has altered semen characteristics without an identifiable cause and there is no female factor infertility, remains a challenging condition to diagnose and manage. Increasing evidence suggests that oxidative stress (OS) plays an independent role in the etiology of male infertility, with 30% to 80% of infertile men having elevated seminal reactive oxygen species levels. OS can negatively affect fertility via a number of pathways, including interference with capacitation and possible damage to sperm membrane and DNA, which may impair the sperm’s potential to fertilize an egg and develop into a healthy embryo. Adequate evaluation of male reproductive potential should therefore include an assessment of sperm OS. We propose the term Male Oxidative Stress Infertility, or MOSI, as a novel descriptor for infertile men with abnormal semen characteristics and OS, including many patients who were previously classified as having idiopathic male infertility. Oxidation-reduction potential (ORP) can be a useful clinical biomarker for the classification of MOSI, as it takes into account the levels of both oxidants and reductants (antioxidants). Current treatment protocols for OS, including the use of antioxidants, are not evidence-based and have the potential for complications and increased healthcare-related expenditures. Utilizing an easy, reproducible, and cost-effective test to measure ORP may provide a more targeted, reliable approach for administering antioxidant therapy while minimizing the risk of antioxidant overdose. With the increasing awareness and understanding of MOSI as a distinct male infertility diagnosis, future research endeavors can facilitate the development of evidence-based treatments that target its underlying cause.

Sperm DNA Fragmentation: A New Guideline for Clinicians

Ashok Agarwal,Ahmad Majzoub,Saradha Baskaran,Manesh Kumar Panner Selvam,Chak Lam Cho,Ralf Henkel,Renata Finelli,Kristian Leisegang,Pallav Sengupta,Catalina Barbarosie,Neel Parekh,Marco G. Alves,Edmund 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.4

Sperm DNA integrity is crucial for fertilization and development of healthy offspring. The spermatozoon undergoes extensive molecular remodeling of its nucleus during later phases of spermatogenesis, which imparts compaction and protects the genetic content. Testicular (defective maturation and abortive apoptosis) and post-testicular (oxidative stress) mechanisms are implicated in the etiology of sperm DNA fragmentation (SDF), which affects both natural and assisted reproduction. Several clinical and environmental factors are known to negatively impact sperm DNA integrity. An increasing number of reports emphasizes the direct relationship between sperm DNA damage and male infertility. Currently, several assays are available to assess sperm DNA damage, however, routine assessment of SDF in clinical practice is not recommended by professional organizations. This article provides an overview of SDF types, origin and comparative analysis of various SDF assays while primarily focusing on the clinical indications of SDF testing. Importantly, we report four clinical cases where SDF testing had played a significant role in improving fertility outcome. In light of these clinical case reports and recent scientific evidence, this review provides expert recommendations on SDF testing and examines the advantages and drawbacks of the clinical utility of SDF testing using Strength-Weaknesses-Opportunities-Threats (SWOT) analysis.

An In-Depth Bibliometric Analysis and Current Perspective on Male infertility Research

Baskaran Saradha,Agarwal Ashok,Leisegang Kristian,Pushparaj Peter Natesan,Panner Selvam Manesh Kumar,Henkel Ralf 대한남성과학회 2021 The World Journal of Men's Health Vol.39 No.2

Purpose: Male infertility is emerging as a major, escalating global health problem that imposes the need to investigate research trends in male infertility. The purpose of this study is to analyze male infertility research trends in the past 20 years using the bibliometric database from Scopus. Materials and Methods: In order to perform an in-depth bibliometric analysis, we propose a ‘Funnel Model’, which includes several layers representing different sub-areas of male infertility research. Adopting this Funnel Model, using Scopus, we retrieved relevant bibliometric data (articles per year, authors, affiliations, journals, and countries) for various areas of male infertility research and performed descriptive statistics. Results: The bibliometric analysis showed an exponential increase in male infertility research in the last 20 years. USA dominated in research output, with Agarwal, A. as the most prolific researcher. Testicular cancer, obesity and metabolic syndrome, and azoospermia were found to dominate male infertility research, whereas erectile dysfunction and unexplained male infertility had lesser attention. Interestingly, prognostic/diagnostic and mechanistic studies have significantly increased in parallel over the last 20 years. Furthermore, our bibliometric analysis revealed fewer publications in proteomics, transcriptomics and metabolomics when compared to genomics. Also, an increasing trend in publication was seen in assisted reproductive technology (ART) research. Conclusions: An integrated and steep escalation in the field of omics and ART research appears to be a prerequisite for further development of future diagnostic and therapeutic strategies for male infertility.

Male Fertility and the COVID-19 Pandemic: Systematic Review of the Literature

Mohammad Ali Khalili,Kristian Leisegang,Ahmad Majzoub,Renata Finelli,Manesh Kumar Panner Selvam,Ralf Henkel,Moshrefi Mojgan,Ashok Agarwal 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.4

Purpose: Since its discovery in December 2019, the novel coronavirus SARS-CoV-2 has spread globally, causing the current COVID-19 (coronavirus disease-19) pandemic. As there is an increase of infections in the male population, concerns have emerged about the potential impact of COVID-19 on male reproductive organs and male fertility. Therefore, this study systematically investigates the current evidence of SARS-CoV-2 impact on male reproduction and pregnancy outcomes, discussing them in light of the evidence published on other coronaviruses. Materials and Methods: Literature search was carried out according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A total of 24 original articles were included for the analysis, investigating the effects of the infection on semen parameters, male reproductive hormones, and pregnancy outcomes. Further, a Strengths-Weaknesses- Opportunities-Threats (SWOT) analysis was conducted based on the available evidence linking the virus with male reproduction and conception. Results: Although there is limited data, viral mRNA has been identified in semen of infected men, with some evidence of altered seminal parameters. Low testosterone and dihydrotestosterone with raised luteinizing hormone has been reported as well as preterm delivery in pregnant women; however, data regarding vertical transmission remains contradictory and inconclusive. Conclusions: The recent literature provides evidence that male gonads may be potentially vulnerable to SARS-CoV-2 infection, recommending caution to pregnant women and couples planning natural pregnancy or assisted reproduction.

Alterations of Spermatozoa Proteomic Profile in Men with Hodgkin’s Disease Prior to Cancer Therapy

Ana D. Martins,Ashok Agarwal,Saradha Baskaran,Peter Natesan Pushparaj,Gulfam Ahmad,Manesh Kumar Panner Selvam 대한남성과학회 2020 The World Journal of Men's Health Vol.38 No.4

Purpose: Hodgkin’s disease (HD) is a type of cancer affecting men in the reproductive age with potential consequences on their fertility status. This study aims to analyze sperm parameters, alterations in proteomic profiles and validate selected protein biomarkers of spermatozoa in men with HD undergoing sperm banking before cancer therapy. Materials and Methods: Semen analysis was carried out in healthy fertile donors (control, n=42), and patients diagnosed with HD (patients, n=38) before cancer therapy. We compared proteomic profiles of spermatozoa from donors (n=3) and patients (n=3) using LTQ-Orbitrap Elite hybrid MS system. Results: A total of 1,169 proteins were identified by global proteomic in both groups. The ingenuity pathway analysis revealed that differentially expressed proteins involved in capacitation, acrosome reaction, binding of sperm to the zona pellucida, sperm motility, regulation of sperm DNA damage, and apoptosis were significantly downregulated in HD patients. Validation of proteins implicated in sperm fertility potential by Western Blot demonstrated that peroxiredoxin 2 (PRDX 2) was underexpressed (p=0.015), and transferrin (p=0.045) and SERPIN A5 (p=0.010) protein levels were overexpressed in spermatozoa of men with HD. Conclusions: Findings of this study indicates that the key proteins involved in sperm fertility potential are significantly altered in men with HD, which provides substantial explanation for the observed low sperm quality in HD subjects prior to cancer therapy. Furthermore, our results suggest PRDX 2, transferrin and SERPIN A5 as possible candidate proteins for assessing sperm quality in HD patients prior to cancer therapy.