Reduced semen quality in patients with testicular cancer seminoma is associated with alterations in the expression of sperm proteins / 亚洲男科学杂志(英文版)

Testicular cancer seminoma is one of the most common types of cancer among men of reproductive age. Patients with this condition usually present reduced semen quality, even before initiating cancer therapy. However, the underlying mechanisms by which testicular cancer seminoma affects male fertility are largely unknown. The aim of this study was to investigate alterations in the sperm proteome of men with seminoma undergoing sperm banking before starting cancer therapy, in comparison to healthy proven fertile men (control group). A routine semen analysis was conducted before cryopreservation of the samples (n = 15 per group). Men with seminoma showed a decrease in sperm motility (P = 0.019), total motile count (P = 0.001), concentration (P = 0.003), and total sperm count (P = 0.001). Quantitative proteomic analysis identified 393 differentially expressed proteins between the study groups. Ten proteins involved in spermatogenesis, sperm function, binding of sperm to the oocyte, and fertilization were selected for validation by western blot. We confirmed the underexpression of heat shock-related 70 kDa protein 2 (P = 0.041), ubiquinol-cytochrome C reductase core protein 2 (P = 0.026), and testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (P = 0.016), as well as the overexpression of angiotensin I converting enzyme (P = 0.005) in the seminoma group. The altered expression levels of these proteins are associated with spermatogenesis dysfunction, reduced sperm kinematics and motility, failure in capacitation and fertilization. The findings of this study may explain the decrease in the fertilizing ability of men with seminoma before starting cancer therapy.

New Insights on the Mechanisms Affecting Fertility in Men with Non-Seminoma Testicular Cancer before Cancer Therapy

PURPOSE: Patients with non-seminoma testicular cancer (NSTC) cancer can be subfertile or infertile, and present reduced sperm quality, but the underlying mechanisms are unknown. The aim of this study was to compare the sperm proteome of patients with NSTC, who cryopreserved their sperm before starting cancer treatment, with that from healthy fertile men.MATERIALS AND METHODS: Semen volume, sperm motility and sperm concentration were evaluated before the cryopreservation of samples from patients with NSTC (n=15) and the control group (n=15). Sperm proteomic analysis was performed by liquid chromatography-tandem mass spectrometry and the differentially expressed proteins (DEPs) between the two groups were identified using bioinformatic tools.RESULTS: A total of 189 DEPs was identified in the dataset, from which five DEPs related to sperm function and fertilization were selected for validation by Western blot. We were able to validate the underexpression of the mitochondrial complex subunits NADH:Ubiquinone Oxidoreductase Core Subunit S1 (NDUFS1) and ubiquinol-cytochrome C reductase core protein 2 (UQCRC2), as well as the underexpression of the testis-specific sodium/potassium-transporting ATPase subunit alpha-4 (ATP1A4) in the NSTC group.CONCLUSIONS: Our results indicate that sperm mitochondrial dysfunction may explain the observed decrease in sperm concentration, total sperm count and total motile count in NSTC patients. The identified DEPs may serve as potential biomarkers for the pathophysiology of subfertility/infertility in patients with NSTC. Our study also associates the reduced fertilizing ability of NSTC patients with the dysregulation of important sperm molecular mechanisms.

Microtubular Dysfunction and Male Infertility

Ritalinic Acid Stimulates Human Sperm Motility and Maintains Vitality In Vitro

Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen / 亚洲男科学杂志(英文版)

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml-1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

Proteomic analysis of seminal plasma from bilateral varicocele patients indicates an oxidative state and increased inflammatory response / 亚洲男科学杂志(英文版)

Seminal plasma is a rich source of proteins and serves as an ideal sample for proteomic analysis of male infertility. In varicocele-associated infertility, the contributory role of seminal plasma proteins specific to unilateral and bilateral varicocele is not clear. Furthermore, there is a lack of specific protein biomarker to differentiate bilateral from unilateral varicocele. The main objective is to identify the differentially regulated molecular and cellular pathways in bilateral varicocele. Furthermore, we intend to identify seminal plasma biomarkers to differentiate bilateral and unilateral varicocele patients in comparison with fertile healthy men. Global proteomic analysis of seminal plasma proteins has identified the functionality of differentially expressed proteins (DEPs) in varicocele patients. Bioinformatic analysis has revealed response to reactive oxygen species and oxidative stress, and tissue homeostasis as top process pathways that are affected in bilateral varicocele patients compared to fertile healthy men. In comparison with unilateral varicocele patients, inflammatory response pathways were dysregulated, especially interleukin 6 (IL-6) signaling and Janus kinase-signal transducer and activator of transcription (Jak-STAT) pathways, in bilateral varicocele patients, owing to the involvement of underexpressed DEPs. Key DEPs associated with oxidative stress (peroxiredoxin 2; PRDX2), DNA fragmentation (fatty acid synthase; FASN), and inflammatory response (fibronectin 1; FN1) validated by western blot analysis revealed differential expression of these proteins in unilateral and bilateral varicocele groups. Altered expression of DEPs and its association with key processes show that the seminal plasma homeostasis is compromised in bilateral varicocele patients. Furthermore, we propose PRDX2, FASN, and FN1 as potential noninvasive seminal plasma markers for the differentiation of unilateral and bilateral varicocele patients.

Critical evaluation of two models of flow cytometers for the assessment of sperm DNA fragmentation: an appeal for performance verification / 亚洲男科学杂志(英文版)

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.

Proteomic analysis reveals dysregulated cell signaling in ejaculated spermatozoa from infertile men / 亚洲男科学杂志(英文版)

Dysfunctional sperm maturation is the primary reason for the poor sperm motility and morphology in infertile men. Spermatozoa from infertile men were fractioned on three-layer density gradient (80%, 60%, and 40%). Fraction 1 (F1) refers to the least mature stage having the lowest density, whereas the fraction 4 (F4) includes the most dense and morphologically mature motile spermatozoa. Fraction 2 (F2) and fraction 3 (F3) represent the intermediate stages. Proteins were extracted and separated by 1-dimensional gel. Bands were digested with trypsin and analyzed on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. A total of 1585 proteins were detected in the four fractions of spermatozoa. A dysregulated protein turnover and protein folding may lead to accumulation of defective proteins or proteins that otherwise would have been eliminated during the process of maturation, resulting in the impairment of sperm function. Aberrant chaperone expression may be a major contributing factor to the defective sperm function. Androgen receptor was predicted as a transcription regulator in one of the networks and the affected pathways were chaperone-mediated stress response, proteosomal pathway, and sperm function. The downregulation of key pathways and proteins which compromises the fertilizing potential of spermatozoa may provide insight into the mechanisms that lead to male infertility.

Multi-center evaluation of oxidation-reduction potential by the MiOXSYS in males with abnormal semen / 亚洲男科学杂志(英文版)

According to the World Health Organization (WHO), oxidative stress (OS) is a significant contributor to male infertility. Seminal OS can be measured by a number of assays, all of which are either costly or time sensitive and/or require large semen volume and complex instrumentation. One less expensive alternative is to quantify the oxidation-reduction potential (ORP) with the MiOXSYS. In this international multi-center study, we assessed whether ORP levels measured by the MiOXSYS could distinguish semen samples that fall within the 2010 WHO normal reference values from those that do not. Semen samples were collected from 2092 patients in 9 countries; ORP was normalized to sperm concentration (mV/106 sperm/ml). Only those samples with a concentration >1 × 106 sperm ml-1 were included. The results showed that 199 samples fell within the WHO normal reference range while the remaining 1893 samples did not meet one or more of the criteria. ORP was negatively correlated with all semen parameters (P < 0.01) except volume. The area under the curve for ORP was 0.765. The ORP cut-off value (1.34 mV/106 sperm/ml) was able to differentiate specimens with abnormal semen parameters with 98.1% sensitivity, 40.6% specificity, 94.7% positive predictive value (PPV) and 66.6% negative predictive value (NPV). When used as an adjunct to traditional semen analysis, ORP levels may help identify altered functional status of spermatozoa caused by OS in cases of idiopathic male infertility and in male partners of couples suffering recurrent pregnancy loss, and thereby directing these men to relevant medical therapies and lifestyle modifications.

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

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.

Protective effects of saffron against zearalenone-induced alterations in reproductive hormones in female mice (Mus musculus) / 대한생식의학회지

OBJECTIVE: Zearalenone (ZEA) is a mycotoxin with potent estrogenic effects. Saffron is an herbal product that has antioxidant activities. The objective of this study was to investigate the protective role of saffron against reproductive toxicity induced by ZEA in female mice. METHODS: Ninety 8-week-old female mice were randomly allocated into three treatment groups. The first group received an intraperitoneal injection of ZEA (2.5 mg/kg) on alternate days. The second group received ZEA (2.5 mg/kg) on alternate days plus oral saffron daily (50 mg/kg). The third group was treated with a vehicle of 1% dimethyl sulfoxide (DMSO) on alternate days, as a control. Ten mice were euthanized from each group at 30, 60, and 90 days of treatment. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and progesterone (P) were assessed. The uterus and ovaries were examined for changes in size or morphology. RESULTS: Serum levels of LH, FSH, E2, and P in the female mice treated with ZEA plus saffron were significantly higher than in those treated with ZEA alone, and were not significantly different from those treated with 1% DMSO. The female mice treated with ZEA alone showed a reduction in size of the uterus and abnormal architecture of the ovaries. CONCLUSION: The administration of saffron to female mice resulted in a significant reduction in ZEA-induced alterations in reproductive hormone levels, the size of the uterus, and the morphology of the ovaries.

Role of Antioxidants in Assisted Reproductive Techniques

Oxidative stress (OS) has been recognized as a significant cause of suboptimal assisted reproductive outcome. Many of the sperm preparation and manipulation procedures that are necessary in the in vitro environment can result in excessive production of reactive oxygen species (ROS) thereby exposing the gametes and growing embryos to significant oxidative damage. Antioxidants have long been utilized in the management of male subfertility as they can counterbalance the elevated levels of ROS inducing a high state of OS. Few studies have looked into the clinical effectiveness of antioxidants in patients undergoing assisted reproduction. While an overall favorable outcome has been perceived, the specific clinical indication and optimal antioxidant regimen remain unknown. The goal of our review is to explore the sources of ROS in the in vitro environment and provide a clinical scenario-based approach to identify the circumstances where antioxidant supplementation is most beneficial to enhance the outcome of assisted reproduction.

Spermatozoa protein alterations in infertile men with bilateral varicocele / 亚洲男科学杂志(英文版)

Among infertile men, a diagnosis of unilateral varicocele is made in 90% of varicocele cases and bilateral in the remaining varicocele cases. However, there are reports of under-diagnosis of bilateral varicocele among infertile men and that its prevalence is greater than 10%. In this prospective study, we aimed to examine the differentially expressed proteins (DEP) extracted from spermatozoa cells of patients with bilateral varicocele and fertile donors. Subjects consisted of 17 men diagnosed with bilateral varicocele and 10 proven fertile men as healthy controls. Using the LTQ-orbitrap elite hybrid mass spectrometry system, proteomic analysis was done on pooled samples from 3 patients with bilateral varicocele and 5 fertile men. From these samples, 73 DEP were identified of which 58 proteins were differentially expressed, with 7 proteins unique to the bilateral varicocele group and 8 proteins to the fertile control group. Majority of the DEPs were observed to be associated with metabolic processes, stress responses, oxidoreductase activity, enzyme regulation, and immune system processes. Seven DEP were involved in sperm function such as capacitation, motility, and sperm-zona binding. Proteins TEKT3 and TCP11 were validated by Western blot analysis and may serve as potential biomarkers for bilateral varicocele. In this study, we have demonstrated for the first time the presence of DEP and identified proteins with distinct reproductive functions which are altered in infertile men with bilateral varicocele. Functional proteomic profiling provides insight into the mechanistic implications of bilateral varicocele-associated male infertility.

Effect of varicocele on semen characteristics according to the new 2010 World Health Organization criteria: A systematic review and meta-analysis / 亚洲男科学杂志(英文版)

This study investigated the effects of varicocele on semen parameters in infertile men based on the new 2010 World Health Organization laboratory manual for the examination of human semen. Semen analysis results (volume, sperm count, motility, and morphology) were the primary outcomes. An electronic search to collect the data was conducted using the Medline/PubMed, SJU discover, and Google Scholar databases. We searched articles published from 2010 to August 2015, i.e., after the publication of the 2010 WHO manual. We included only those studies that reported the actual semen parameters of adult infertile men diagnosed with clinical varicocele and contained a control group of either fertile men or normozoospermic men who were not diagnosed with varicocele. Ten studies were included in the meta-analysis, involving 1232 men. Varicocele was associated with reduced sperm count (mean difference: -44.48 × 10 [6] ml-1 ; 95% CI: -61.45, -27.51 × 10 [6] ml-1 ; P < 0.001), motility (mean difference: -26.67%; 95% CI: -34.27, -19.08; P < 0.001), and morphology (mean difference: -19.68%; 95% CI: -29.28, -10.07; P < 0.001) but not semen volume (mean difference: -0.23 ml; 95% CI: -0.64, 0.17). Subgroup analyses indicated that the magnitude of effect was influenced by control subtype but not WHO laboratory manual edition used for semen assessment. We conclude that varicocele is a significant risk factor that negatively affects semen quality, but the observed pooled effect size on semen parameters does not seem to be affected by the WHO laboratory manual edition. Given most of the studies published after 2010 still utilized the 1999 manual for semen analysis, further research is required to fully understand the clinical implication of the 2010 WHO laboratory manual on the association between varicocele and semen parameters.

Bibliometrics: Tracking research impact by selecting the appropriate metrics / 亚洲男科学杂志(英文版)

Traditionally, the success of a researcher is assessed by the number of publications he or she publishes in peer-reviewed, indexed, high impact journals. This essential yardstick, often referred to as the impact of a specific researcher, is assessed through the use of various metrics. While researchers may be acquainted with such matrices, many do not know how to use them to enhance their careers. In addition to these metrics, a number of other factors should be taken into consideration to objectively evaluate a scientist′s profile as a researcher and academician. Moreover, each metric has its own limitations that need to be considered when selecting an appropriate metric for evaluation. This paper provides a broad overview of the wide array of metrics currently in use in academia and research. Popular metrics are discussed and defined, including traditional metrics and article-level metrics, some of which are applied to researchers for a greater understanding of a particular concept, including varicocele that is the thematic area of this Special Issue of Asian Journal of Andrology. We recommend the combined use of quantitative and qualitative evaluation using judiciously selected metrics for a more objective assessment of scholarly output and research impact.

Proteomic signatures of infertile men with clinical varicocele and their validation studies reveal mitochondrial dysfunction leading to infertility / 亚洲男科学杂志(英文版)

To study the major differences in the distribution of spermatozoa proteins in infertile men with varicocele by comparative proteomics and validation of their level of expression. The study-specific estimates for each varicocele outcome were combined to identify the proteins involved in varicocele-associated infertility in men irrespective of stage and laterality of their clinical varicocele. Expression levels of 5 key proteins (PKAR1A, AK7, CCT6B, HSPA2, and ODF2) involved in stress response and sperm function including molecular chaperones were validated by Western blotting. Ninety-nine proteins were differentially expressed in the varicocele group. Over 87% of the DEP involved in major energy metabolism and key sperm functions were underexpressed in the varicocele group. Key protein functions affected in the varicocele group were spermatogenesis, sperm motility, and mitochondrial dysfunction, which were further validated by Western blotting, corroborating the proteomics analysis. Varicocele is essentially a state of energy deprivation, hypoxia, and hyperthermia due to impaired blood supply, which is corroborated by down-regulation of lipid metabolism, mitochondrial electron transport chain, and Krebs cycle enzymes. To corroborate the proteomic analysis, expression of the 5 identified proteins of interest was validated by Western blotting.

Insights into an Award-Winning Summer Internship Program: The First Six Years

Since its inception in 2008, the American Center for Reproductive Medicine's summer internship program in reproductive research and writing has trained 114 students from 23 states within the United States and 10 countries worldwide. Its fundamental goal is to inspire pre-medical and medical students to embrace a career as a physician-scientist. During this intensive course, established scientists and clinicians train interns in the essential principles and fundamental concepts of bench research and scientific writing. Over the first six years (2008~2013), interns have collectively published 98 research articles and performed 12 bench research projects on current and emerging topics in reproductive medicine. Interns have also developed and honed valuable soft skills including time management, communication and presentation skills, as well as life values, which all enhance personal and professional satisfaction. Program graduates are able to recognize the value of medical research and its potential to impact patient care and gain insight into their own career pathway. Between 2011 and 2014, the internship program was thrice awarded a Scholarship in Teaching Award by Case Western Reserve School of Medicine for its innovative teaching approach and positive impact on medical education and student careers. This report highlights the demographics, logistics, implementation, feedback, and results of the first six years of the American Center for Reproductive Medicine's summer internship program at Cleveland Clinic (Cleveland, OH, USA). This may be helpful to other research and academic institutions considering implementing a similar program. In addition, it creates awareness among potential physician-scientists of what the world of research has to offer in both scientific writing and bench research. Finally, it may stimulate further discussion regarding narrowing the gap between physicians and scientists and refinement of the current program.

Smoking and Male Infertility: An Evidence-Based Review

Many studies have reported that the contents of cigarette smoke negatively affect sperm parameters, seminal plasma, and various other fertility factors. Nevertheless, the actual effect of smoking on male fertility is not clear. The effect of smoking on semen parameters is based on the well-established biological finding that smoking increases the presence of reactive oxygen species, thereby resulting in oxidative stress (OS). OS has devastating effects on sperm parameters, such as viability and morphology, and impairs sperm function, hence reducing male fertility. However, not all studies have come to the same conclusions. This review sheds light upon the arguable association between smoking and male fertility and also assesses the impact of non-smoking routes of tobacco consumption on male infertility. It also highlights the evidence that links smoking with male infertility, including newly emerging genetic and epigenetic data, and discusses the clinical implications thereof.

Effect of Oxidative Stress on Male Reproduction

Infertility affects approximately 15% of couples trying to conceive, and a male factor contributes to roughly half of these cases. Oxidative stress (OS) has been identified as one of the many mediators of male infertility by causing sperm dysfunction. OS is a state related to increased cellular damage triggered by oxygen and oxygen-derived free radicals known as reactive oxygen species (ROS). During this process, augmented production of ROS overwhelms the body's antioxidant defenses. While small amounts of ROS are required for normal sperm functioning, disproportionate levels can negatively impact the quality of spermatozoa and impair their overall fertilizing capacity. OS has been identified as an area of great attention because ROS and their metabolites can attack DNA, lipids, and proteins; alter enzymatic systems; produce irreparable alterations; cause cell death; and ultimately, lead to a decline in the semen parameters associated with male infertility. This review highlights the mechanisms of ROS production, the physiological and pathophysiological roles of ROS in relation to the male reproductive system, and recent advances in diagnostic methods; it also explores the benefits of using antioxidants in a clinical setting.

Vitamin d deficiency among postmenopausal women with osteoporosis.

BACKGROUND: Hypovitaminosis D is widely prevalent in India and is a formidable issue especially in postmenopausal women. The study intends to estimate the prevalence of vitamin D deficiency among postmenopausal women with osteoporosis. METHODS: The study was performed at a referral teaching institute in north India between 2007 and 2009. One hundred and ninety postmenopausal osteoporotic women were enrolled and the clinical information was collected along with the assessment of biochemical parameters. RESULTS & CONCLUSION: Serum vitamin D was found to be de- ficient in two third of patients. A significant correlation was observed between body mass index and bone mineral density at lumbar spine. Prevention and early detection of hypovitaminosis D is the key to reduce the incidence of osteoporosis among postmenopausal women.