Normozoospermic Men Research Articles

L-Carnitine and Pentoxifylline Supplementation Improves Sperm Viability and Motility at Low Temperature.

In infertility clinics, preserving high-quality spermatozoa for a long time is a necessity. Pentoxifylline (PT) and L-carnitine (LC) are effective in improving sperm motility as well as protecting the sperm membrane. The present study aimed to investigate the protective impacts of PT and LC on the quality of the normal sperm motility, protamine content, and viability on prolonged storage for 12 days at 4-6°C. The present experimental work included 26 samples, which were first prepared based on the swim-up technique, of normozoospermic men. They were divided into three aliquots as untreated control, LC-treated, and PT-treated groups and incubated for up to 12 days at 4-6°C. Thereafter, chromatin maturity, sperm viability, and motility were assessed on 0, 1, 2, 5, 7, and 12 days. Data were analyzed using a one-way analysis of variance. The obtained data revealed that PT supplementation increased the percentage of motile spermatozoa in comparison with control and LC-treated specimens. On the other hand, LC supplementation increased the percentage of viable spermatozoa in comparison with the PT-treated and control samples. During the 12-day storage, the percentage of spermatozoa with a normal protamine content was nearly unchanged in the three groups (P>0.05). Although LC supplementation can be considered a better alternative than PT for preserving sperm viability, PT could better preserve sperm motility compared to LC during 12 days at 4-6°C.

Sexual dysfunction is more common among men who have high sperm DNA fragmentation or teratozoopermia

Men in couples that have experienced pregnancy loss have a higher risk of sexual dysfunction. Semen quality impairment is common in men of couples with pregnancy loss. The objective of this article is to evaluate the differences in the incidence of male sexual dysfunction in a cohort of pregnancy loss couples with different types of semen quality impairment. A cross-sectional analysis of 426 men who attended our outpatient clinic for couples’ pregnancy loss, those without genetic abnormalities were included in the final analysis covering June 2021 to October 2021. The patients were divided into 5 groups according to type of semen quality impairment: normozoospermia group (group normal; N = 161), high sperm DNA fragmentation group (group high-SDF; N = 87), isolated asthenozoospermia group (group iAstheno; N = 45), isolated teratozoopermia group (group iTerato; N = 44), and ≥ 2 abnormal sperm parameters group (group multiple; N = 89). All subjects underwent a complete physical inspection, including palpation of the male genitalia and semen analysis. Validated assessment tools for erectile dysfunction (the International Index of Erectile Function -IIEF-5) and anxiety (the seven-item Generalized Anxiety Disorder Scale- GAD-7) were also used. Men with high sperm DNA fragmentation and isolated teratozoopermia were associated with increased erectile dysfunction risk when compared with normozoospermic men, with an OR of 2.75 [1.49–5.09; p = 0.001] and 2.44 [1.22–5.31; p = 0.024], respectively. It is interesting to note that there was no difference in prevalence of erectile dysfunction between Group iAstheno and Group normal (20.0% vs. 18.0%; OR = 1.24 [0.52–2.97]; P = 0.625). More than half (50.6%) of the participants in Group high-SDF reported sexual intercourse less than once per week, much more than those in the normozoospermia group (23.2%, p < 0.05), followed by Group iTerato (44.4%) and Group multiple (46.1%). GAD-7 scores increased slightly but significantly among groups when compared with Group normal. Not surprisingly, GAD-7 scores remained higher in Group high-SDF. In males of pregnancy loss couples, men with high sperm DNA fragmentation and teratozoopermia suffer from a higher incidence of erectile dysfunction. This phenomenon is not significant in men with isolated asthenozoospermia. Proper counseling and treatment of impaired semen quality are warranted.

The Impact of a Very Short Abstinence Period on Conventional Sperm Parameters and Sperm DNA Fragmentation: A Systematic Review and Meta-Analysis.

In recent years, a growing number of studies have supported the beneficial effects of a very short abstinence period on sperm parameters, especially in patients with oligoasthenozoospermia. However, the results are controversial and no consensus exists regarding whether to request a second semen collection in clinical practice. Therefore, this systematic review and meta-analysis aimed to evaluate the influence of a very short abstinence period (within 4 h) on conventional sperm parameters and sperm DNA fragmentation (SDF) rate. The literature search was performed using Scopus and PubMed databases. The meta-analysis was conducted according to the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocol (PRISMA-P) guidelines. All eligible studies were selected according to the Population, Intervention, Comparison/Comparator, Outcomes, and Study design (PICOS) model. The quality of evidence of the included studies was analyzed through the Cambridge Quality Checklists. The standardized mean difference (SMD) was used to analyze the outcomes. Cochran-Q and I2 statistics were used to evaluate statistical heterogeneity. We assessed for eligibility 1334 abstracts, and 19 studies were finally included. All 19 articles evaluated the effects of a very short abstinence period on sperm parameters and, among these, 5 articles also evaluated the effects on SDF rate. The quantitative analysis showed a significant reduction in semen volume after a very short abstinence period in both normozoospermic men and patients with oligozoospermia, asthenozoospermia, and/or teratozoospermia (OAT) patients. We found a statistically significant increase in sperm concentration and total and progressive motility in the second ejaculation of patients with OAT. In contrast, the SDF rate decreased significantly in the second ejaculate of OAT patients. This is the first systematic review and meta-analysis investigating the impact of a very short abstinence period on sperm parameters and SDF rate. The results suggest that collecting a second consecutive ejaculation after a very short time from the first could represent a simple and useful strategy for obtaining better-quality spermatozoa, especially in patients with abnormal sperm parameters.

Bioinformatics analysis identifies potential hub genes and crucial pathways in the pathogenesis of asthenozoospermia

BackgroundAsthenozoospermia is a troublesome disease experienced by men in their reproductive years, but its exact etiology remains unclear. To address this problem, this study aims to identify the hub genes and crucial pathways in asthenozoospermia.MethodsWe screened two Gene Expression Omnibus (GEO) datasets (GSE92578 and GSE22331) to extract the differentially expressed genes (DEGs) between normozoospermic and asthenozoospermic men using the “Limma” package. Gene enrichment analyses of the DEGs were conducted using the “clusterProfiler” R package. The protein-protein interaction (PPI) network was then established using the STRING database. A miRNA-transcription factor-gene network was constructed based on the predicted results of hub genes using the RegNetwork database. The expression of four hub genes in asthenozoospermia and normal samples were verified using quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting.ResultsWe identified 271 DEGs, which included 218 upregulated and 53 downregulated in two asthenozoospermia datasets. These DEGs were observed to be markedly enriched in pathways with cell growth and embryonic organ development, phospholipase D signaling pathway, cGMP-PKG signaling pathway, and Wnt signaling pathway. The most significant genes were identified, including COPS7A, CUL3, KLHL7, NEDD4. We then constructed regulatory networks of these genes, miRNAs, and transcription factors. Finally, we found that the COPS7A was significantly upregulated in patients with asthenozoospermia, but CUL3, KLHL7 and NEDD4 were significantly downregulated compared with normal samples.ConclusionWe applied bioinformatics methods to analyze the DEGs of asthenozoospermia based on the GEO database and identified the novel crucial genes and pathways in this disease. Our findings may provide novel insights into asthenozoospermia and identify new clues for the potential treatment of this disease.

Men with oligozoospermia had lower level of seminal plasma pyridoxine compared to normozoospermic men

The contribution of pyridoxine on various human disorders has been revealed in so many studies; however, this contribution on poor semen quality has yet to be investigated. Here, we intended to measure the level of seminal plasma pyridoxine (SPP) in men with oligozoospermia compared to normozoospermic men. Thirty-three men with oligozoospermia and forty-three normozoospermic men were randomly enrolled in this study. Collected semen were tested for sperm motility, sperm concentration, semen volume, and pyridoxine status. Liquid chromatography with tandem mass spectrometry was used to measure SPP in the collected samples. There was a significant reduction (p < 0.0001) in the concentrations of SPP in men with oligozoospermia (0.79 ± 0.41 μg L−1) compared to normozoospermic men (3.17 ± 0.96 μg L−1). Besides, SPP was not significantly correlated (p > 0.05) with sperm motility, sperm concentration, and semen volume in both tested groups, but, independently, it was found to be positively correlated (p = 0.0154) with male age in oligozoospermic group. Men with oligozoospermia had lower level of SPP compared to normozoospermic men. These results may open a new research gate for the use of pyridoxine in the management of male infertility.

Tissue specific age acceleration patterns in the sperm of oligozoospermic men.

To determine if disease can modify aging patterns in an affected tissue without altering the aging patterns of other tissues, blood and semen of individuals with oligozoospermia (n = 10) were compared to the blood and semen of individuals with normozoospermia (n = 24). DNA methylation data was obtained via Illumina's 850 K array. The Horvath and Jenkins age calculators were then utilized to predict the epigenetic age of blood and sperm. Epigenetic age of sperm was approximated using germ-line age differential (GLAD) values. Using nonpaired t-tests, it was found that sperm of oligozoospermic men (mean GLAD score of 0.078) were predicted to be significantly older than the sperm of normozoospermic men (mean GLAD score of -0.017), returning a p-value of 0.03. However, there was not a significant epigenetic age difference between the blood of those with oligozoospermia (mean GLAD equivalent score of -0.027) and normozoospermia (mean GLAD equivalent score of 0.048), producing a p-value of 0.20. These results lead to the conclusion that tissue specific aging is occurring in sperm of oligozoospermic individuals but not in unaffected somatic tissues (in this case, blood).

Effect of Radiofrequency Electromagnetic Radiation Emitted by Modern Cellphones on Sperm Motility and Viability: An In Vitro Study

BackgroundCellphones emit radiofrequency electromagnetic radiation (RF-EMR) for transmission of data for social media communication, web browsing, and music/podcast streaming. Use of Bluetooth ear buds has probably prolonged the time during which cellphones reside in the trouser pockets of men. It has been postulated that RF-EMR increases oxidative stress and induces free radical formation. ObjectiveTo investigate the effect of wireless-spectrum (4G, 5G, and WiFi) RF-EMR emitted by modern smartphones on sperm motility and viability and explore whether these effects can be mitigated using a physical barrier or distance. Design, setting, and participantsSemen samples were obtained from fertile normozoospermic men aged 25–35 yr. A current-generation smartphone in talk mode was used as the RF-EMR source. A WhatsApp voice call was made using either 4G, 5G, or WiFi wireless connectivity. We determined if exposure effects were mitigated by either a cellphone case or greater distance from the semen sample. Outcome measurements and statistical analysisThe semen samples were analyzed according to 2010 World Health Organization laboratory guidelines. Statistical analysis was performed using SPSS v.28. Results and limitationsWe observed decreases in sperm motility and viability with WiFi exposure but not with exposure to 4G or 5G RF-EMR. With large variability among smartphones, continued research on exposure effects is needed. ConclusionsOur exploratory study revealed that sperm motility and viability are negatively impacted by smartphones that use the WiFi spectrum for data transmission. Patient summaryWe looked at the effect of cellphone use on sperm motility and viability. We found that cellphones using WiFi connectivity for data usage have harmful effects on semen quality in men.

PROTECTIVE EFFECT OF L-PROLINE ON HUMAN SPERM CHARACTERISTICS DURING CRYOPRESERVATION

To explore the effectiveness of L-proline inclusion in freezing medium on sperm parameters, sperm DNA and chromatin quality, as well as the nitro-oxidative status of sperm culture medium in normozoospermic men who received antioxidants following the freezing-thawing process.

MicroRNA-targeting in male infertility: Sperm microRNA-19a/b-3p and its spermatogenesis related transcripts content in men with oligoasthenozoospermia.

Objective: To elucidate and validate the potential regulatory function of miR-19a/b-3p and its spermatogenesis-related transcripts content in sperm samples collected from men with oligoasthenozoospermia. Methods: Men presenting at an infertility clinic were enrolled. MicroRNA (miRNA) and target genes evaluation were carried out using in silico prediction analysis, Reverse transcription-quantitative PCR (RT-qPCR) validation, and Western blot confirmation. Results: The expression levels of miRNA-19a/b-3p were significantly up-regulated and 51 target genes were significantly down-regulated in oligoasthenozoospermic men compared with age-matched normozoospermic men as determined by RT-qPCR. Correlation analysis highlighted that sperm count, motility, and morphology were negatively correlated with miRNA-19a/b-3p and positively correlated with the lower expression level of 51 significantly identified target genes. Furthermore, an inverse correlation between higher expression levels of miRNA-19a/b-3p and lower expression levels of 51 target genes was observed. Consistent with the results of the RT-qPCR, reduced expression levels of STK33 and DNAI1 protein levels were identified in an independent cohort of sperm samples collected from men with oligoasthenozoospermia. Conclusion: Findings suggest that the higher expression of miRNA-19a/b-3p or the lower expression of target genes are associated with oligoasthenozoospermia and male infertility, probably through influencing basic semen parameters. This study lay the groundwork for future studies focused on investigating therapies for male infertility.

Influence of ejaculatory abstinence period on semen quality of 5165 normozoospermic and oligozoospermic Nigerian men: A retrospective study.

Background and AimsSeveral studies have shown that the length of ejaculatory abstinence alters sperm quality. However, the available data are conflicting and none seems to exist in a Nigerian population. The present study aims to compare the semen quality in normozoospermic and oligozoospermic semen samples of a homogenous Nigerian population, following varying ejaculatory abstinence days (EAD); less than 2, 2–3, and 3–7 days.MethodsThe present retrospective study included 5165 semen samples collected over 5 years, from April 2015 to April 2020.ResultsIn normozoospermic samples, sperm count and total sperm count were significantly higher in prolonged EAD. In oligozoospermic patients, semen volume significantly increased with prolonged EAD, while sperm count, total sperm count, and progressive motility were significantly reduced with prolonged EAD. In addition, EAD and sperm volume positively correlated in oligozoospermic patients.ConclusionOur findings indicate that EAD affects sperm quality in both normozoospermic and oligozoospermic men with varying impacts. Prolonged EAD increased sperm count and total sperm count in normozoospermic patients, while EAD increased semen volume but reduced sperm count, total sperm count, and progressive motility in oligozoospermic patients.

Seminal plasma N-glycome as a new biomarker of environmental exposure associated with semen quality

Male infertility, a condition that has during the last decade raised significant concern, is a diagnostically demanding and socially sensitive topic. The number of unsolved issues on infertility etiology, especially potential environmental causes, in couples demonstrates the need for further investigations into infertility biomarkers. Semen parameters are often insufficient for reliable profiling of male infertility. Thus, this study aims to evaluate for the first time seminal plasma N-glycosylation as a biomarker of environmental exposure in semen samples from 82 normozoospermic men and 84 men with abnormal semen parameters and compare it with genome damage measured by DNA fragmentation. We obtained information about chronic exposure to environmental factors from the self-reported questionnaire, and determined sperm DNA fragmentation by sperm chromatin dispersion, while N-glycans were characterized with liquid chromatography-mass spectrometry (LC-MS). Based on previously published results, ten N-glycans were selected. Results show that the selected seminal plasma N-glycans were significantly associated with smoking, exposure to pesticides, air pollution, agents emitted during photocopying, alcohol consumption, and obesity. Some N-glycans showed a simultaneous association with DNA fragmentation, semen parameters, and environmental stressors. These subgroups of N-glycans are new potential candidates for biomonitoring of exposure to different environmental factors in men with semen abnormalities.

Evaluation of PGK2 and ACR proteins in seminal plasma: suggestion of potential new biomarkers for prediction of sperm retrieval in non-obstructive azoospermia patients

This study aimed to assess the role of testis-specific proteins, PGK2 and ACR, in the prediction of sperm retrieval results by microdissection testicular sperm extraction (micro-TESE) in men with non-obstructive azoospermia (NOA). This was a case-control study including 48 semen samples of NOA patients undergoing the micro-TESE procedure, 15 semen samples from normozoospermic men as the positive control, and 12 semen samples from obstructive azoospermia/post-vasectomy (OA/PV) as negative controls. We investigated the levels of PGK2 and ACR proteins by ELISA tests in seminal plasma samples. The ELISA results revealed a significantly higher concentration of PGK2 and ACR in the NOA patients with successful sperm retrieval (NOA+) in comparison to NOA patients with failed sperm retrieval (NOA−) group (p = 0.0001 in both cases). For the first time, the data from this study suggests that a seminal PGK2 concentration of 136.3 pg/ml and ACR concentration of 21.75 mIU/ml can be used as cut-off values for the prediction of micro-TESE outcomes in NOA patients. These findings may be useful to avoid unnecessary micro-TESE operations. Overall, the seminal levels of the PGK2 and ACR proteins may be useful in predicting sperm retrieval success by micro-TESE in NOA patients.

Possible Implications of Bacteriospermia on the Sperm Quality, Oxidative Characteristics, and Seminal Cytokine Network in Normozoospermic Men.

This study focused on the identification of bacterial profiles of semen in normozoospermic men and their possible involvement in changes to the sperm structural integrity and functional activity. Furthermore, we studied possible fluctuations of selected cytokines, oxidative markers, and antibacterial proteins as a result of bacterial presence in the ejaculate. Sperm motility was assessed with computer-assisted sperm analysis, while sperm apoptosis, necrosis and acrosome integrity were examined with fluorescent methods. Reactive oxygen species (ROS) generation was quantified via luminometry, sperm DNA fragmentation was evaluated using the TUNEL protocol and chromatin-dispersion test, while the JC-1 assay was applied to evaluate the mitochondrial membrane potential. Cytokine levels were quantified with the biochip assay, whilst selected antibacterial proteins were quantified using the ELISA method. The predominant species identified by the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry were Staphylococcus hominis, Staphylococcus capitis and Micrococcus luteus. The results revealed that the sperm quality decreased proportionally to the increasing bacterial load and occurrence of conditionally pathogenic bacteria, including Enterococcus faecalis, Staphylococcus aureus and Escherichia coli. Antimicrobial susceptibility tests revealed a substantial resistance of randomly selected bacterial strains to ampicillin, vancomycin, tobramycin, and tetracycline. Furthermore, an increased bacterial quantity in semen was accompanied by elevated levels of pro-inflammatory cytokines, including interleukin-1, interleukin-2, interleukin-6, tumor necrosis factor alpha as well as ROS overproduction and lipid peroxidation of the sperm membranes. Our results suggest that semen quality may be notably affected by the bacterial quantity as well as quality. It seems that bacteriospermia may be associated with inflammatory processes, oxidative stress, sperm structural deterioration, and a subsequent risk for the development of subfertility, even in normozoospermic males.

Evaluation of sperm integrin α5β1 as a potential marker of fertility in humans.

Sperm selection for assisted reproduction techniques is generally based on basic parameters, while key aspects of sperm competence and its journey from the deposition site to the fertilization site are overlooked. Consequently, identifying molecular markers in spermatozoa that can efficiently predict the fertility of a semen sample could be of great interest, particularly in cases of idiopathic male infertility. When spermatozoa reach the female reproductive tract, it provides to them the cellular and molecular microenvironment needed to acquire fertilizing ability. In this sense, considering the role that integrin α5β1 of spermatozoa plays in reproduction-related events, we investigated the correlation between the subcellular localization of sperm integrin α5β1 and early embryo development outcome after in vitro fertilization (IVF) procedures in human. Twenty-four semen samples from normozoospermic men and metaphase II (MII) oocytes from healthy women aged under 38 years, from couples who underwent IVF cycles, were used in this work. Sperm α5β1 localization was evaluated by immunofluorescence assay using an antibody against integrin α5 subunit. Integrin α5β1 was mainly localized in the sperm acrosomal region (45.33±7.89%) or the equatorial segment (30.12±7.43%). The early embryo development rate (data obtained from the Fertility Center) correlated positively with the localization of α5β1 in the acrosomal region (number of usable embryos / inseminated oocytes: ρ = 0.75; p<0.01 and number of usable embryos/total number of two pronuclear zygotes: ρ = 0.80; p<0.01). However, this correlation was not significant when the equatorial segment mark was evaluated. In addition, human sperm released from co-culture with bovine oviductal epithelial cells (BOEC) showed a significant enrichment in the acrosomal localization pattern of α5β1 compared to those sperm that were not co-cultured with BOEC (85.20±5.35% vs 35.00±17.09%, respectively, p<0.05). In conclusion, the evaluation of sperm integrin α5β1 immunolocalization could be a useful tool to select sperm with fertilizing ability from human semen samples before IVF procedures.

Evaluation the level of antisperm antibodies, some cytokines and zinc for oligozoospermia, asthenozoospermia patients compared with normozoospermia

Evaluation of the level of anti-sperm antibodies, some cytokines and zinc for oligozoospermia patients, asthenozoospermia patients and normozoospermia patients. Semen samples were collected from asthenozoospermic, oligozoospermic, and normozoospermic men who attended the fertility center. The collected samples of normozoospermia patients were (15), Asthenozoospermia patients were (15), and Oligozoospermia patients were (13). Seminal plasma for all study groups was evaluated for interleukin-1, interleukin-6, and zinc levels. The result showed there were statistically significant differences (p &lt; 0.05) in sperm concentration, progressive motility, and liquefaction time in the asthenospermia and oligospermia groups compared with the normospermia group. This study found that antisperm antibody, interleukin 1, and interleukin 6 were higher in the oligozoospermia and asthenozoospermia groups than in the normozoospermia group. The levels of antisperm antibodies and interleukins increase, while zinc levels decrease in the seminal plasma of patients with oligospermia, as well as those with asthenospermia, compared to patients with normospermia.

Altered methyltransferase gene expression, mitochondrial copy number and 4977-bp common deletion in subfertile men with variable sperm parameters.

Semen parameters have been found to predict reproductive success poorly and are the most prevalent diagnostic tool for male infertility. There are few conflicting reports regarding the correlation of DNMT genes expression, mitochondrial DNA copy number (mtDNAcn) and deletion (mtDNAdel) with different sperm parameters. To investigate DNMT mRNA level, mtDNAcn and deletion in infertile men, with different sperm parameters, compared with fertile men, semen samples from 30 men with unknown male infertility and normal sperm parameters (experimental group I), 30 infertile patients with at least two abnormal sperm parameters (experimental group II) and 30 fertile normozoospermic men (control group) were collected. After semen analysis, total RNA and DNA were extracted. The isolated DNA was used for assessing the respective mtDNAcn and the presence of common 4977 bp deletion in mtDNA by applying real-time quantitative PCR and multiplex PCR, respectively. Synthesized cDNA from total RNAs was used to quantify DNMT1, DNMT3A and DNMT3B transcripts in study groups by using real-time quantitative reverse-transcription PCR. Significantly higher proportions of mtDNAcn were found in experimental group II. DNMT1 was significantly downregulated in both experimental groups and 4977 bp deletion was not detected. Progressive motility and normal morphology were significantly and negatively correlated with mtDNAcn. A significant positive correlation was detected between sperm parameters and DNMT1 mRNA levels. In conclusion, infertile men with different sperm parameter qualities showed elevated mtDNA content. Abnormal sperm parameters associated with DNMT1 gene expression indicate the possibility of changes in some epigenetic aspects of spermatogenesis in subfertile men with different sperm parameters.

Relationship between indicators of oxidative stress and idiopathic infertility in men

Male infertility is a major health problem worldwide. According to a study of the WHO and the European Association of Urologists, the incidence of infertility is increasing worldwide. The male factor causes up to 50% of all cases of infertility in married couples and approximately 7% of men worldwide suffer from infertility. No cause can be found in approximately 30% of infertile men, and these cases are called idiopathic infertility. In many such cases, oxidative stress is believed to be the main causative factor. Oxidative stress, which is characterized by an imbalance between the production of reactive oxygen species and the antioxidant defense system, which is responsible for their neutralization, leads to damage to many cellular structures, especially phospholipids of cell membranes. The study is aimed at studying the adverse effect of lipid peroxidation on spermatogenesis in idiopathic male infertility. The study was conducted on the sperm of 56 men with normozoospermia and 30 men with oligoasthenozoospermia. The levels of malondialdehyde, zinc, fructose, NO-synthase activity and glutathione peroxidase (GP) in seminal plasma were correlated with sperm count and motility. Ejaculate malondialdehyde and NO-synthase activity were significantly higher, while zinc, fructose and GP were significantly lower in oligoasthenospermic than in normospermic men (p&lt;0.001). A 1.8-fold decrease in the level of fructose in the spermogram of patients of group oligoasthenospermia, compared to the data of normozoospermic men correlated with processes of impaired motility and viability of spermatozoa. This was confirmed by the data obtained in the scientific work, in the form of a statistically significant difference in the average indicator of the quantitative content of fructose in the studied groups of patients. In particular, we found a positive correlation between the fructose content in the ejaculate and sperm motility (r=0.69), as well as a positive correlation between fructose content and the number of spermatozoa in the ejaculate (r=0.53). There was a significant negative correlation between malondialdehyde and NO-synthase with sperm count and motility in oligoasthenospermia. Elevated levels of malondialdehyde, NO-synthase in sperm, and decreased levels of zinc and GP in oligoasthenospermia may be responsible for impaired sperm membrane integrity and play a role in sperm DNA damage. The positive correlation of zinc with sperm count and sperm motility indicates an important role of zinc in spermatogenesis. Thus, these parameters may be useful in determining the fertilization potential of sperm in the diagnosis, prognosis and treatment of male infertility, especially idiopathic infertility. Keywords: sperm plasma, malondialdehyde, glutathione peroxidase, NO-synthase, zinc, fructose.

Polymorphisms in the HSF2, LRRC6, MEIG1 and PTIP genes correlate with sperm motility in idiopathic infertility.

The aim of this study was to investigate the association of 24 functionally important single nucleotide polymorphisms (SNPs) with male infertility. In this cross-sectional study, we genotyped 24 functionally important single nucleotide polymorphisms in 24 infertility candidate genes in 500 oligo-/astheno-/oligoastheno-/normo-zoospermic infertile men with idiopathic infertility. Sequenom iPlex gold assay was used for genotyping. Sperm count and motility were compared between prevalent genotypes at each test locus. We did not observe any significant difference in the average sperm count between the alternate genotypes for the loci in the KLK3, LRRC6, MEIG1, HSF2, ESR2 and PTIP genes. However, we observed a significant difference in sperm motility between the alternate genotypes for the loci in the LRRC6, MEIG1, HSF2 and PTIP genes. Polymorphisms in the LRRC6 (rs200321595), MEIG1 (rs150031795), HSF2 (rs143986686) and PTIP (rs61752013) genes show association with sperm motility.

P-104 production and glucose-dependent secretion of insulin by human spermatozoa

Abstract Study question Do human spermatozoa express insulin and the enzymes responsible for the cleavage of proinsulin? Is the secretion of insulin by spermatozoa responsive to glucose stimuli? Summary answer Human spermatozoa express insulin and PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. Spermatozoa produce and release insulin in a glucose concentration-dependent manner. What is known already Spermatogenesis is dependent on a finely regulated hormonal and metabolic control. Insulin is considered one of the most important regulators in the metabolic regulation of spermatogenesis, acting directly on the differentiation of spermatogonia into primary spermatocytes or indirectly through the metabolic modulation of both Sertoli and Leydig cells. In addition, insulin is hypothesized to play a major role in human spermatozoa capacitation, although the mechanisms that regulate its production and secretion remain to be disclosed. Study design, size, duration The main objective of this study was to estimate the capacity of insulin production by human spermatozoa and if they express PC1/3 and PC2, which are enzymes responsible for the cleavage of proinsulin. In addition, we hypothesized that insulin production could respond to glucose stimuli. Seminal samples of normozoospermic men (n = 15) seeking fertility treatment were collected for these experiments. Participants/materials, setting, methods Spermatozoa were submitted to a density gradient centrifugation and two fractions were collected according to their motility (high vs low motility). The expression of insulin, PC1/3 and PC2 mRNA was evaluated by RT-qPCR. The protein expression of insulin, PC1/3 and PC2 was evaluated by immunofluorescence. High motility spermatozoa were incubated in BWW medium with increasing glucose concentrations (0, 5.5, 11, and 22 mM). After 6 h, the concentration of secreted insulin was quantified by ELISA. Main results and the role of chance We were able to identify the expression of insulin, PC1/3, and PC2 mRNA, as well as the respective proteins, in human spermatozoa. Through immunofluorescence, we observed that insulin and both enzymes are mainly expressed in the midpiece. Human spermatozoa also express insulin receptor, mainly in the midpiece and tail. The mRNA expression of insulin and PC1/3 was found to be higher in the highly motile spermatozoa. Human spermatozoa produce very small quantities of insulin (pg/mL range), which suggests an autocrine or paracrine action. The release of insulin to the medium was found to occur in a glucose concentration-dependent manner. Limitations, reasons for caution This is an in vitro study with relatively small sample size. Future studies are required to further evaluate the role of insulin and its signalling pathway on human spermatozoa capacitation and fertilization capacity. Wider implications of the findings This study shows that human spermatozoa express PC1/3 and PC2, enzymes responsible for the cleavage of proinsulin. In addition, human spermatozoa release insulin in a glucose concentration-dependent manner. The study of insulin signalling opens the path for a deeper understanding of human spermatozoa bioenergetics and the development of novel therapies. Trial registration number Not applicable

Sperm DNA damage in men with spinal cord injury: the relative incidence of single- and double-strand DNA breaks.

Men with spinal cord injury (SCI) show a high proportion of sperm DNA damage in their ejaculate but the underlying pathology remains elusive. To investigate the relative incidence of single (SSBs) and double-strand DNA breaks (DSBs) and DNase activity in men with SCI. This study included ejaculates of 20 men with SCI and 27 normozoospermic (sperm donors). A TwoTails comet assay (TTComet) allowed visualization of three categories of sperm DNA damage corresponding to SSBs, DSBs and those with a combination of SSBs and DSBs, facilitating accurate calculation of the total proportion of SSBs and DSBs. A subset of 15 individuals (sperm donors and SCI patients) was used to test for DNase activity in the seminal plasma. While the proportion of DSBs in men with SCI (median-57.5%) was higher (P=0.000) than normozoospermic samples (median-4.6%), the proportion of SSBs was higher (P=0.022) in the normozoospermic ejaculates (median-6.0%) compared to men with SCI (median-2.5%). The relative proportion of the total DSBs with respect to the total SSBs was 3.3× in men with SCI but 0.9× in normozoospermic samples. We further confirmed the high DNase activity in the seminal plasma of men with SCI. The TTComet assay provided new insights to the pathology of sperm DNA in men with SCI and may have diagnostic value in developing sperm selection methodologies to reduce DSBs prior to ART. Men with SCI are characterized by a high proportion of sperm with DSBs and high levels of DNase activity in the seminal plasma compared to normozoospermic men.