Normozoospermic Subjects Research Articles

The association of sperm BAX and BCL-2 gene expression with reproductive outcome in Oligoasthenoteratozoospermia cases undergoing intracytoplasmic sperm injection: A case-control study.

The B-cell lymphoma 2 (BCL-2) protein is one of the members of the BCL-2 associated X (BAX) protein family that acts as an inducer of apoptosis. The present study aims to investigate the association between BAX and BCL-2 gene expression with reproductive outcome, in cases undergoing intracytoplasmic sperm injection. In this case-control study, 50 men were divided into healthy fertile and oligoasthenoteratozoospermic infertile men (n = 25/each). They were subjected to history taking, clinical examination, and semen analysis. Expression of BAX and BCL-2 genes were measured using real-time polymerase chain reaction. The DNA fragmentation index was measured using the sperm chromatin dispersion assay technique. Using World Health Organization criteria, sperm parameters were evaluated. Evaluation of apoptosis-related genes showed that oligoasthenoteratozoospermic significantly increased mRNA expression of BAX, and significantly decreased mRNA expression of BCL-2, when compared with control. Moreover, the BAX/BCL-2 ratio was significantly higher in oligoasthenoteratozoospermic compared to the normozoospermic group (p = 0.01). Also, this study showed that the BAX and BCL-2 genes expression had a significant correlation with sperm quality, and DNA fragmentation in the oligoasthenoteratozoospermic group (p = 0.01). The oligoasthenoteratozoospermic men, had a considerably lower proportion of fertilization rate and good-quality embryos at the cleavage stage than the normozoospermic subjects (p = 0.01). A significant correlation was observed between the expression of BAX and BCL-2 genes, fertilization, and embryo quality (p = 0.01). We concluded that the sperm BAX/BCL-2 ratio demonstrates a significant correlation with fertilization rate and embryo quality.

Sperm DNA damage in men with severe asthenozoospermia

Background. Sperm motility is a fundamental factor for sperm penetration into the oocyte and fertilization. According to the World Health Organization (WHO) 2021, less than 42% of motility is called asthenozoospermia, which is one of the most common causes of male infertility. This retrospective cohort study aims to investigate sperm DNA damage with TUNEL and SCSA methods and its relationship with sperm parameters, age, and body mass index (BMI) in severe asthenospermia (<2% sperm motility) and normozoospermia. Methods. The study parameters between 111 subjects with severe asthenozoospermia and 113 subjects with normozoospermia were investigated. The 2010 World Health Organization guidelines were used to evaluate sperm parameters, and TUNEL and SCSA methods were used to assess sperm DNA damage. The statistical analysis was done using the t-test of two independent samples. Pearson's correlation coefficient was used to investigate the relationship between sperm DNA damage and sperm parameters, age, and BMI P<0.05 was considered significant. Results. The mean seminal volume, sperm concentration and count, and the percentage of sperm total motility and progressive motility were significantly lower in severe asthenospermic subjects compared to normozoospermic subjects (P<0.001). In addition, the mean percentage of sperm DNA damage in severe asthenozoospermic individuals was significantly higher than in normozoospermic individuals (P<0.001). There was also a positive and significant relationship between sperm DNA damage and the age of severely asthenozoospermic men. Conclusion. Sperm motility, sperm DNA damage, and the father's age are essential in the successful conception, development, and health of the embryo. Therefore, the evaluation of sperm DNA damage is recommended in examining male fertility and selecting the appropriate treatment approach for men with severe asthenozoospermia. Practical Implications. Considering that the sperm genome constitutes 50% of the genetic material of the next generation, evaluating sperm DNA health to determine the appropriate treatment approach in infertile men can be influential in the success of assisted reproductive techniques and in maintaining the next generation’s health.

Evaluation of the serum zinc concentration in male infertility patients: an analysis of 2010 cases.

Zinc is an important trace element, and its importance for male infertility has been reported. The aim of the study was to assess whether the serum zinc concentrations were related to semen quality in male infertility patients. In 2010 subjects who consulted at our male infertility clinic between November 2018 and May 2021, serum zinc concentrations were assessed along with age, sperm concentration, sperm motility, endocrine panel, and body mass index (BMI). A normal zinc concentration was observed in 1069 (53.2%), subclinical deficiency in 845 (42.0%), and deficiency in 79 subjects (3.9%). On the other hand, high a zinc level was observed in only 17 subjects (0.9%). The serum zinc concentration did not relate with age, sperm concentration, sperm motility, luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and body mass index (BMI). However, normozoospermic subjects showed significantly higher zinc concentrations than among azoospermic (included non-obstructive; NOA and obstructive; OA) and cryptozoospermic patients. Furthermore, the zinc concentration was lower in NOA subjects when comparing to oligozoospermia and/or asthenozoospermia. An association between zinc concentration and semen analysis remained unclear. This study was cross-sectional and retrospective, however, this is a largest investigation of the zinc concentration during reproductive life span in Japan. Further accumulation of cases are required to further examine the potential relationship between zinc concentration and semen quality.

Calprotectin as a novel diagnostic approach to screen male infertility risk: A pilot study

Research QuestionDiagnosis of male infertility is essentially based on the evaluation of semen quality (sperm concentration, motility, viability, and morphology). However, there is a lack of knowledge about possible molecules used as candidates for the early identification of male infertility risk. Calprotectin is a biological marker for inflammation, measured prevalently in stool specimens, widely used to discriminate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), and for the subsequent monitoring of gastrointestinal diseases’ development. Would it be possible to use calprotectin determination to identify also male infertility risk? DesignCross-sectional pilot study investigated calprotectin concentration in the seminal fluid of 45 men (range: 23–51 yrs) that were under evaluation for semen quality at our Center for Reproductive Medicine. Calprotectin concentration was determined with a commercially available immuno-chromatographic test and successfully detected in 37 of the 45 analyzed men (age: 37.38 ± 6.59). A correlation with semen quality (concentration, motility, morphology) was assessed. ResultsHigher calprotectin concentration seemed to indicate a better quality of the seminal fluid. Normozoospermic subjects (Group A) had on average a calprotectin value of 0.215 ± 0.162 µg/ml (mean ± SD), while subjects with at least one of the semen parameters below reference values (Group B) showed lower calprotectin concentration (mean ± SD: 0.126 ± 0.068, p-value < 0.05). A significant difference was clearly evident between calprotectin concentration measured in seminal fluids with physiological sperm morphology (≥4%) as compared with teratozoospermic samples (<4%) (p-value < 0.05). Indeed, the developed ROC curves showed a good diagnostic accuracy (around 67 %) using calprotectin concentration (threshold value: 0.121 μg/ml) as a preliminary test to discriminate subjects with and without abnormal semen parameters, especially morphology. ConclusionsCalprotectin determination in the seminal fluid may be proposed as a biological marker for preliminary screening in male subjects at risk of infertility due to one or more alterations of semen quality.

Tobacco and alcohol as factors for male infertility-a public health approach.

The study of reproductive characteristics of 430 male subjects of different age, fertility status and educational level who were involved in the program of extracorporeal fertilization at the Clinic of Gynecology and Obstetrics, Clinical Centre Niš, examined their knowledge, attitudes and behavior regarding tobacco and alcohol consumption as lifestyle risk predictors of their partial or full infertility. Consisted of the analyses of spermiograms to establish their fertility status and a survey of their attitudes towards smoking and alcohol use (behavior, knowledge of the general health and reproductive health consequences of such a lifestyle, and their determination to change it). The proportion with higher tobacco consumption and more severe forms of infertility increased significantly with ageing (P<0.001); the highest daily consumption of alcohol and the incidence of intoxication was seen among azoospermic patients; the level of awareness of the harmful effects of tobacco was highest among normozoospermic subjects and the highest level of determination to quit smoking was statistically significantly present among azoospermic subjects. Appropriate use of health promotion activities in relation to alcohol and tobacco use is through specially designed programs.

From subjective to objective: A pilot study on testicular radiomics analysis as a measure of gonadal function.

BackgroundThe connection between testicular ultrasound (US) parameters and testicular function, including both spermato‐ and steroidogenesis has been largely suggested, but their predictive properties are not routinely applied. Radiomics, a new engineering approach to radiological imaging, could overcome the visual limit of the sonographer.ObjectivesThis study is aimed at extracting objective testicular US features, correlating with testicular function, including both spermato‐ and steroidogenesis, using an engineering approach, in order to overcome the operator‐dependent subjectivity.Materials and methodsProspective observational pilot study from December 2019 to December 2020 on normozoospermic subjects and patients with semen variables alterations, excluding azoospermia. All patients underwent conventional semen analysis, pituitary‐gonadal hormones assessment, and testicular US, performed by the same operator. US images were analyzed by Biolab (Turin) throughout image segmentation, image pre‐processing, and texture features extraction.ResultsOne hundred seventy US testicular images were collected from 85 patients (age 38.6 ± 9.1 years). A total of 44 first‐order and advanced features were extracted. US inhomogeneity defined by radiomics significantly correlates with the andrologist definition, showing for the first time a mathematical quantification of a subjective US evaluation. Thirteen US texture features correlated with semen parameters, predicting sperm concentration, total sperm number, progressive motility, total motility and morphology, and with gonadotropins serum levels, but not with total testosterone serum levels. Classification analyses confirmed that US textural features predicted patients’ classification according to semen parameters alterations.ConclusionsRadiomics texture features qualitatively describe the testicular parenchyma with objective and reliable quantitative parameters, reflecting both the testicular spermatogenic capability and the action of pituitary gonadotropins. This is an innovative model in which US texture features represent a mirror of the pituitary‐gonadal homeostasis in terms of reproductive function.

Kisspeptin and Spexin: lipid profile and sperm parameters in infertile and fertile male subjects

BACKGROUND &amp; OBJECTIVE: Kisspeptin (KP) and spexin (SPX) are neuropeptides that play a substantial role in metabolism and sexual function. Our objective is to explore the association of spexin and kisspeptin with sperm parameters and lipid profile in the infertile male population.&#x0D; METHODOLOGY: The cross-sectional study was conducted from March 2017 till July 2018 in Aga Khan University Hospital, Karachi, Pakistan, after acquiring ethical approval (4813-BBS-ERC-17). Convenient sampling was applied to recruit 44 normozoospermic subjects as controls and 44 patients with abnormal sperm parameters”. Serum levels of kisspeptin and spexin were measured by enzyme linked immunosorbent assay and Cobas c111 analyzer was used for estimation of high and low density-lipoprotein, triglyceride and serum cholesterol levels. Mann-Whitney U tests and spearman’s rank correlation test were used to analyze quantitative variables, and data was presented by means, standard error (SE) and correlations coefficient, with a statistical significance set at p≤0.05.&#x0D; RESULTS: The mean kisspeptin values of ‘altered sperm parameters’ males 13.23 ± 3.34 were lower than normal sperm parameters 31.89 ± 3.25 (p&lt;0.001). The mean spexin values of altered sperm parameters 383.57 ± 21.09 were less than compared to normal sperm parameters 502.76 ± 19.77 (p&lt;0.001). Correlation of KP and SPX levels was (r-value = 0.332, p= 0.002). There was no significant correlation was found between KP and SPX with lipid profile parameters.&#x0D; CONCLUSION: Spexin and kisspeptin levels observed were higher in male subjects with normal sperm parameters and had a significant positive correlation with each other. There was no impact of spexin on lipid profile and obesity.

MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY

You have accessJournal of UrologyInfertility: Basic Research & Pathophysiology (MP38)1 Apr 2020MP38-17 THE PROFILE OF HUMAN SPERM OXYSTEROLS IN NORMAL AND SUBFERTILE PATIENTS: RESULTS FROM A PROSPECTIVE MULTICENTRE STUDY Antonio Luigi Pastore*, Andrea Fuschi, Yazan Al Salhi, Gennaro Velotti, Lorenzo Capone, Alessia Martoccia, Pietro Paolo Suraci, Chiara Zerbinati, Luigi Iuliano, and Antonio Carbone Antonio Luigi Pastore*Antonio Luigi Pastore* More articles by this author , Andrea FuschiAndrea Fuschi More articles by this author , Yazan Al SalhiYazan Al Salhi More articles by this author , Gennaro VelottiGennaro Velotti More articles by this author , Lorenzo CaponeLorenzo Capone More articles by this author , Alessia MartocciaAlessia Martoccia More articles by this author , Pietro Paolo SuraciPietro Paolo Suraci More articles by this author , Chiara ZerbinatiChiara Zerbinati More articles by this author , Luigi IulianoLuigi Iuliano More articles by this author , and Antonio CarboneAntonio Carbone More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000887.017AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cholesterol is the main lipid component of sperm cell that is essential for sperm membrane fluidity, capacitation, and acrosomal reaction. Recent data obtained in bovine sperm showed that sperm capacitation is associated with the formation of oxysterols, oxidized products of cholesterol. The main objective of the present study was to identify and quantify, for the first time, the different species of oxysterols in human semen from normozoospermic, oligoasthenoteratozoospermic and asthenozoospermic patients. The secondary aim was to investigate the potential role of oxysterols in sperm pathophysiology. METHODS: To investigate the profile of human sperm oxysterols in subjects with normal and altered sperm characteristics, we recruited 150 consecutive subjects referring at the Centre of Andrology of Department of Urology for analysis of seminal fluid between March 2017 and June 2018. In order to investigate the possible correlation to oxidative stress and semen oxysterols profile, we included a group of patients with varicocele given the demonstrated relationship between varicocele and oxidative stress. Semen analysis was assessed by light microscope according to World Health Organization guidelines (WHO, 2010). Oxysterols were determined by GC–MS using deuterium-labelled internal standards as described by Dzeletovic et al. RESULTS: Complete oxysterol profile was obtained in 134 human semen of normozoospermic, oligoasthenoteratozoospermic, asthenozoospermic and varicocele patients. Oxysterols analyzed included seven autoxidation- and five enzymatically-generated oxysterols. Among the 12 oxysterols analyzed, 25-hydroxycholesterol (25-HC) resulted the most abundant oxysterol in normozoospermic subjects, and turned out to be the only one that differed significantly (p<0.0001) among the 4 groups. It was higher in the normozoospermic group (21.63±18.47 ng/mL, mean±SD) than oligoasthenoteratozoospermic (2.59±2.93, ng/mL, mean±SD), asthenozoospermic (5.59±3.17) and varicocele (13.48±11.81, ng/mL, mean±SD) groups. Furthermore, 25-HC positively correlated with the spermatozoa number (r=0.72, p<0.0001). CONCLUSIONS: In conclusion, to the best of our knowledge, this is the first study providing evidence for the feasibility of detection and quantification of oxysterols in human semen samples. We found in spermatozoa the presence of cholesterol 25-hydroxylase and its preferential accumulation in the neck and the post acrosomal area. 25-HC was able to induce calcium ions and cholesterol depletion in the acrosomal region, in an extent comparable to that of a capacitating medium. This finding offers the opportunity to identify a new molecular player involved in pre-fertilization processes. Source of Funding: None © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e575-e576 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Antonio Luigi Pastore* More articles by this author Andrea Fuschi More articles by this author Yazan Al Salhi More articles by this author Gennaro Velotti More articles by this author Lorenzo Capone More articles by this author Alessia Martoccia More articles by this author Pietro Paolo Suraci More articles by this author Chiara Zerbinati More articles by this author Luigi Iuliano More articles by this author Antonio Carbone More articles by this author Expand All Advertisement PDF downloadLoading ...

Alteration of neutral-alpha glucosidase in seminal plasma and correlation with sperm motility among men investigated for infertility Nigeria: a cross-sectional study

Introduction: Proteomic studies are becoming popular lately among reproductive biologists in the diagnosis and management of several diseases including male infertility. Objective: To evaluate the level of Neutral-alpha Glucosidase (NAG) in the seminal plasma of men being investigated for infertility in and to correlate its activity with sperm motility. Materials and Methods: Four hundred men age range 23–60 years were consecutively recruited in the study. After physical and clinical evaluation, the semen was collected by self or assisted masturbation and analysis was done according to the World Health Organization guidelines. The semen was then centrifuged and seminal plasma separated for the assay of NAG activity by ELISA technique using reagents supplied by Melson Medical Co, Shangai, China within one week of collection. The participants were categorized in to normozoospermia (191), oligozoospermia (98), severe oligozoospermia (70) and azoospermia (41) based on sperm count. Results: The mean levels of NAG activity decreased with decreasing levels of sperm count with values lowest among azoospermia and highest among normozoospermic subjects. The comparison of NAG activity between the various categories and control subjects was significantly different (P 0.05) when compared with controls. The mean NAG activity level was significantly lower among the asthenozoospermia (P

The higher risk for sperm DNA damage in infertile men.

Supplementary assays are needed for determination of relationships between sperm biomarkers and fertility potential. Therefore, our research was designed to determine the extent of sperm DNA fragmentation (SDF) and establish a discriminating threshold of SDF for fertility potential. Semen characteristics were evaluated according to World Health Organization recommendations, and SDF was assessed by sperm chromatin dispersion test on ejaculated spermatozoa from infertile and healthy normozoospermic men. A higher proportion of SDF was noted in infertile men (median 23.00%) than normozoospermic (median 14.00%). Significantly less subjects (17.03%) with low SDF level (≤ 15%) and more (35.17%) with high SDF level ( > 30%) were found for the infertile group vs the normooospermic (57.90% and 5.26%, respectively). Infertile group had significantly lower odds ratio (OR) for having a low SDF level (OR: 0.1493) and higher OR for having a high SDF level (OR: 9.7627). Receiver operating characteristic analysis [area under curve (AUC) = 0.785] revealed that 20% SDF is predictive value for discriminating between infertile and normozoospermic subjects. SDF was negatively correlated with the sperm number, morphology, progressive motility and vitality but positively with the teratozoospermia index. Our study demonstrates: (1) a significant difference in the extent of SDF and in the risk for having damaged sperm DNA between infertile and normozoospermic men, (2) > 20% SDF has negative predictive value for fertility potential, (3) coexistence of abnormal standard sperm parameters with sperm chromatin damages. Therefore, SDF should be considered as a highly valuable indicator of male fertility potential.

Alteration of Cholesterol Sulfate/Seminolipid Ratio in Semen Lipid Profile of Men With Oligoasthenozoospermia.

The reduction of sperm motility and count, or oligoasthenozoospermia, is one of the major causes of reduced fertility or infertility in men. Lipid composition of spermatozoa is important in determining their functional characteristics, in particular on motility, acrosomal exocytosis or fusogenic properties of the sperm. Here we investigated the levels of semen lipids in 11 infertile patients with severe oligoasthenozoospermia and 9 normozoospermic subjects with normal motility values. Sperm polar and neutral lipids were analyzed by thin-layer chromatography (TLC) and matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF/MS). Semen of patients with oligoasthenozoospermia showed a reduction of the degree of fatty acid unsaturation in the phospholipids chains that might affect the membrane fluidity. Furthermore, a significant higher cholesterol sulfate/seminolipid ratio was found in semen of oligoasthenozoospermic patients than in subjects with normal motility values, suggesting a critical role of sulfolipids in semen quality. The results may facilitate the understanding of the role of lipids on male fertility and offer interesting perspectives to find innovative treatments for oligoasthenozoospermia.

Oxidative stress-induced alterations in seminal plasma antioxidants: Is there any association with keap1 gene methylation in human spermatozoa?

Kelch-like ECH-associated protein 1 (keap1)-nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway is one of the master regulators of cellular defence against oxidative stress. Epigenetic alterations like hypermethylation of keap1 gene impair keap1-Nrf2 system in several oxidative stress-associated diseases. The objective of this study was to evaluate the epigenetic status of keap1 in sperm DNA of normozoospermic subjects, having different levels of reactive oxygen species (ROS) in seminal plasma. Semen samples were obtained from 151 apparently healthy male partners of couples who attended the Avicenna infertility clinic. Samples were categorised into four groups according to their ROS levels: group A (n=39, ROS<20RLU/s per 106 spermatozoa), group B (n=38, 20≤ROS<40RLU/s per 106 spermatozoa), group C (n=31, 40≤ROS<60RLU/s per 106 spermatozoa) and group D; (n=43, ROS≥60RLU/s per 106 spermatozoa). Keap1 methylation status was assessed using methylation-specific PCR along with seminal total antioxidant capacity. The results showed no significant alterations in keap1 methylation in any groups, whereas the total antioxidant capacity enhanced with increasing levels of ROS exposure. These results indicate that keap1 was not methylated during ROS elevation and oxidative stress, suggesting that the cells have adopted other mechanisms to elevate antioxidant level.

Polymorphism in the Alternative Donor Site of the Cryptic Exon of LHCGR: Functional Consequences and Associations with Testosterone Level

Selective splicing is a feature of luteinizing hormone receptor (LHCGR). A cryptic exon (LHCGR-exon 6A) was found to be derived from alternative splicing in intron 6 of the LHCGR gene, which including two transcripts LHCGR-exon 6A-long and LHCGR-exon 6A-short. We addressed the functional consequences of SNP rs68073206, located at the +5 position of an alternative 5′ splice donor site, and observed its association with male infertility in the subjects with azoospermia, oligoasthenozoospermia and normozoospermia. The translation product of splicing variant LHCGR-exon 6A was expressed in the cytoplasm and exhibited no affinity with [125I]-hCG. No dominant negative effect was observed in cells co-expressed with LHCGR-exon 6A and wild-type LHCGR. The long transcript (LHCGR-exon 6A-long) was significantly elevated in the granulosa cells with G/G genotypes, which could be reproduced in vitro by mini-gene construct transfection. Genotyping analysis showed no association between rs68073206 and male infertility. However, this polymorphism was significantly associated with testosterone levels in normozoospermic subjects (n = 210). In conclusion, SNP rs68073206 in the splicing site of the cryptic exon 6A of the LHCGR gene affect the splicing pattern in the gene, which may play a role in the modulation of the LHCGR sensitivity in the gonads.

Heat Sensing Receptor TRPV1 Is a Mediator of Thermotaxis in Human Spermatozoa.

The molecular bases of sperm thermotaxis, the temperature-oriented cell motility, are currently under investigation. Thermal perception relies on a subclass of the transient receptor potential [TRP] channels, whose member TRPV1 is acknowledged as the heat sensing receptor. Here we investigated the involvement of TRPV1 in human sperm thermotaxis. We obtained semen samples from 16 normozoospermic subjects attending an infertility survey programme, testis biopsies from 6 patients with testicular germ cell cancer and testis fine needle aspirates from 6 patients with obstructive azoospermia undergoing assisted reproductive technologies. Expression of TRPV1 mRNA was assessed by RT-PCR. Protein expression of TRPV1 was determined by western blot, flow cytometry and immunofluorescence. Sperm motility was assessed by Sperm Class Analyser. Acrosome reaction, apoptosis and intracellular-Ca2+ content were assessed by flow cytometry. We found that TRPV1 mRNA and protein were highly expressed in the testis, in both Sertoli cells and germ-line cells. Moreover, compared to no-gradient controls at 31°C or 37°C (Ctrl 31°C and Ctrl 37°C respectively), sperm migration towards a temperature gradient of 31–37°C (T gradient) in non-capacitated conditions selected a higher number of cells (14,9 ± 4,2×106 cells T gradient vs 5,1± 0,3×106 cells Ctrl 31°C and 5,71±0,74×106 cells Ctrl 37°C; P = 0,039). Capacitation amplified the migrating capability towards the T gradient. Sperms migrated towards the T gradient showed enriched levels of both TRPV1 protein and mRNA. In addition, sperm cells were able to migrate toward a gradient of capsaicin, a specific agonist of TRPV1, whilst capsazepine, a specific agonist of TRPV1, blocked this effect. Finally, capsazepine severely blunted migration towards T gradient without abolishing. These results suggest that TRPV1 may represent a facilitating mediator of sperm thermotaxis.

Fatty acids profiling reveals potential candidate markers of semen quality.

Previous reports showed altered fatty acid content in subjects with altered sperm parameters compared to normozoospermic individuals. However, these studies focused on a limited number of fatty acids, included a short number of subjects and results varied widely. We conducted a case-control study involving 155 patients allocated into four groups, including normozoospermia (n=33), oligoasthenoteratozoospermia (n=32), asthenozoospermia (n=25), and varicocoele (n=44). Fatty acid profiling, including 30 species, was analyzed by a validated gas chromatography (GC) method on the whole seminal fluid sample. Multinomial logistic regression modeling was used to identify the associations between fatty acids and the four groups. Specimens from 15 normozoospermic subjects were also analyzed for fatty acids content in the seminal plasma and spermatozoa to study the distribution in the two compartments. Fatty acids lipidome varied markedly between the four groups. Multinomial logistic regression modeling revealed that high levels of palmitic acid, behenic acid, oleic acid, and docosahexaenoic acid (DHA) confer a low risk to stay out of the normozoospermic group. In the whole population, seminal fluid stearic acid was negatively correlated (r=-0.53), and DHA was positively correlated (r=0.65) with sperm motility. Some fatty acids were preferentially accumulated in spermatozoa and the highest difference was observed for DHA, which was 6.2 times higher in spermatozoa than in seminal plasma. The results of this study highlight complete fatty acids profile in patients with different semen parameters. Given the easy-to-follow and rapid method of analysis, fatty acid profiling by GC method can be used for therapeutic purposes and to measure compliance in infertility trials using fatty acids supplements.

Preliminary MALDI-TOF-MS analysis of seminal plasma N-glycome of infertile men

Glycosylation pattern within reproductive tract is now suggested to be involved in providing female immune tolerance for allograft sperm and developing embryo, but the information whether impaired glycosylation may influence male fertility potential is still limited. We have analyzed seminal plasma N-glycome in pooled samples derived from fertile and infertile men by means of MALDI-TOF/TOF tandem mass spectrometry. Among infertile subjects, normozoospermic, oligozoospermic, asthenozoospermic and oligoasthenozoospermic samples were obtained. Eighty-six oligosaccharides were identified in all the analyzed samples. Differences in the content of unique glycans: high mannose and hybrid type, lacking terminal sialic acid and highly fucosylated were found when samples derived from infertile subjects with different semen patterns were compared to the fertile control. The content of highly branched glycans was 3-fold elevated in normozoospermic infertile men, while the expression of highly fucosylated oligosaccharides was increased in asthenozoospermic, oligozoospermic and oligoasthenozoospermic samples. Sialylation of oligosaccharides was decreased in oligozoospermic, oligoasthenozoospermic and especially asthenozoospermic samples, but increased in infertile normozoospermic subjects. Altered glycosylation observed in seminal plasma may reflect similar changes in sperm surface glycoproteins, and may disturb sperm interaction with female immune system. We suggest that at least some cases of unexplained male infertility may be associated with impaired glycosylation.

Transient scrotal hyperthermia affects human sperm DNA integrity, sperm apoptosis, and sperm protein expression

This prospective randomized clinical study is aimed to evidence the reproductive impairment of frequent scrotal heat exposure. A total of 20 normozoospermic subjects were randomly divided into two groups to undergo testicular warming in a 43°C water bath 10 times, for 30min each time; the subjects in group 1 underwent testicular warming for 10 consecutive days and those in group 2 once every 3days. Sperm chromatin structure assay (SCSA), sperm mitochondrial membrane potential (MMP), apoptosis, and seminal plasma-soluble Fas (sFas) were analyzed before treatment and every 2weeks after, for a total of 10 times. In group 1, some critical proteins involved in heat stress, hypoxia, structure, and function of sperm mitochondria and flagella were evaluated before hyperthermia and 2, 6, 10, and 16weeks after hyperthermia. Both groups showed a reversible increase in the proportion of spermatozoa with a disrupted MMP (both p<0.05 when the minimums were compared with baseline levels, the same below), sperm apoptosis (both p<0.01) and high DNA stainability (both p<0.05). The sFas concentration in both groups showed no obvious changes except one: the value at week 2 was significantly increased over baseline in group 1 (p=0.036). The level of Bcl-2 decreased significantly at weeks 6 and 10 (p=0.017 and 0.05, respectively) and recovered to baseline at week 16. Proteins involved in heat stress and mitochondria functions were up-regulated, whereas in flagella structure and function was down-regulated (all p<0.05). This study demonstrated that transient and frequent scrotal hyperthermia severely and reversibly damaged spermatogenesis, consecutive heat exposure had more serious effects than intermittent exposure, whereas intermittent exposure led to a later recovery of sperm damage.

Alteration Expression of Bax, Bcl-2 and VDAC1 Genes in Oligozoospermic and Fertile Subjects.

One of factors causing oligozoospermic circumstances is excessive apoptosis during spermatogenesis. Spermatogenesis known involves Bcl-2 family proteins in cytoplasm and Voltage Dependent Anion Channel 1 (VDAC1) in outer mitochondrial membrane to facilitate releasing of apoptosis factor such as cytochrome-c from inter-membrane space into cytoplasm. The study was aimed to analyze the mRNA expression of pro-apoptotic Bax, anti-apoptotic Bcl-2 and VDAC1 genes derived from 45 oligozoospermic subjects and 20 fertile subjects as control. Analysis of transcript expression was performed by two-steps real-time (PCR) and calculating by standard curve method. Stages of works were followed: Analysis of sperm basal characterization, isolation of spermatozoa to separate it from cement and resulted pellets. Pellets were saturated with PBS to obtain mRNA and reversed into cDNA. The cDNA were sequenced to investigate SNP of Bax, Bcl-2 and VDAC1 genes. Results showed that comparison of log mRNA copy number of Bax, Bcl-2 and VDAC1 genes for oligospemic and fertile subjects varied. The Bax, Bcl-2 and VDAC1 were significantly different between oligozoospermic and normozoospermic subjects (p = 0.000, p = 0.041, p = 0.000, respectively). It was suggested that oligozoospermia may be occurred by inducing the increase of Bax pro-apoptotic and VDAC1 genes expression and decreasing of Bcl-2 expression to lead the excessive of apoptosis.

Varicocele Negatively Affects Sperm Mitochondrial Respiration

To evaluate the effect of varicocele on oxidative stress, sperm mitochondrial respiratory efficiency, sperm morphology, and semen parameters. A total of 20 patients with varicocele and 20 normozoospermic subjects without varicocele (control group) were recruited from a medical center for reproductive biology. The levels of serum reactive oxygen metabolites and seminal lipid peroxides were assessed for both control and varicocele subjects. Sperm deoxyribonucleic acid fragmentation was measured by sperm chromatin dispersion test. Mitochondrial respiratory activity was evaluated with a polarographic assay of oxygen consumption carried out in hypotonically treated sperm cells. In this study, varicocele patients were compared with men without varicoceles. Oxidative stress was observed in the serum and seminal fluid of varicocele patients. These patients showed an increase of 59% (P <.05) in serum reactive oxygen metabolites and a 3-fold increase in the level of sperm lipid peroxides. A parallel and significant increase (a 2-fold increase; P <.05) in the degree of sperm deoxyribonucleic acid fragmentation was also observed. Varicocele patients showed a 27% decrease (P <.05) in mitochondrial respiratory activity in comparison to the control group. A 32% increase (P <.05) in sperm midpiece defects and a 41% decrease (P <.05) in sperm concentration and motility were also observed. Men with varicocele have increased markers of oxidative stress and decreased mitochondrial respiratory activity. These results correlated with abnormalities in semen parameters. For morphology, these correlated with midpiece defects.

A Comprehensive and Systematic Study of Semen Quality and Sperm Functional Status in Normozoospermic Controls and Infertile Males from South India

Objective: Despite, several global studies indicate the variations in semen characteristics that accounts for male infertility, the association of specific changes in semen quality and fertility status among different Indian communities are poorly investigated. With wide range of geographical locations, diverse lifestyle patterns, seasonal variations combined with heterogeneous population, India offers an excellent system to study genotype-to-phenotype correlation. Hence, the current study has been initiated in South Karnataka region of India, in order to examine the variations in semen quality and sperm functional status in infertile individuals compared with normozoospermic controls. Methods: WHO strict guidelines are followed for systematic semen analysis of 239 infertile and 244 normozoospermic control subjects. Results: Interestingly, compared to normozoospermic controls, higher percentage of physical abnormalities such as, low semen volume and reduced sperm count are observed in infertile men. Additionally, semen characteristics namely, vitality and motility values are significantly reduced in infertile than controls. Further, in sperm function test the lower scores are documented for hypo-osmotic swelling assay, but not for sperm chromatin decondensation and acrosome intactness examination, suggesting loss of sperm plasma membrane integrity in infertile men. Moreover, the observed changes in semen parameters and sperm function are also evident in different infertile sub-conditions with varied responses. Surprisingly, age wise analysis revealed reduction in sperm morphology scores, whereas, vitality, count, motility and volume remain unchanged with increasing age of infertile males. However, we recorded inverse relationship between age and sperm vitality as well as motility in normozoospermic control men. Together, though the scores for different semen parameters in normozoospermic control group are in accordance with WHO reference range, the infertile men displayed poor semen quality. Conclusion: Thus, our data establishes basic differences between infertile and normozoospermic control group in terms of semen characteristics and sperm functional status, but the cause may be attributable to genetic or environmental factors or interaction of the two, which necessitates further detailed examination in larger cohort among heterogeneous population.