Oligoasthenozoospermic Men Research Articles

Telomeric RNAs, TERRA, as a Potential Biomarker for Spermatozoa Quality.

Characterization of long non-coding telomeric repeat-containing RNAs in sperm of normozoospermic and oligoasthenozoospermic men as new biomarker of idiopathic male infertility. We conducted an observational prospective study with two groups of men with normal or orligoasthenozoospermic spermiogram, aged 40 and above. Fertility parameters were analyzed in men undergoing intracytoplasmic sperm injection with donor oocytes, to avoid the female factor. Telomeric RNAs and telomere length were measured by quantitative fluorescent in situ hybridization. Data from seminal parameters and in-vitro fertilization were assessed according to IVIRMA protocols. Patients with oligoasthenozoospermia, who had worse seminal parameters, also obtained embryos with lower inner-cell-mass quality (p = 0.04), despite using donor oocytes. While mean levels of telomeric RNAs were similar for both groups, the percentage of spermatozoa with more than 3 foci was higher in oligoasthenozoospermic men (p = 0.02). Regarding telomere length, oligoasthenozoospermic men had shorter mean, a higher accumulation of short telomeres (15th percentile; p = 0.03) and a lower percentage of very-long telomeres (85th percentile; p = 0.01). Finally, a positive correlation was found between telomeric-RNAs intensity and total progressive motility in the spermatozoa of normozoospermic patients (r = 0.5; p = 0.03). Telomeric parameters were altered in the spermatozoa of the oligoasthenozoospermic group, which also showed lower quality embryos. Interestingly, in the normozoospermic group, a correlation was found between progressive motility and telomeric RNA levels, suggesting that they could be a good biomarker of sperm quality. Further studies are required to confirm these results and translate them into the clinical practice.Trial registration number: 1711-MAD-109-CB, 07/07/2021.

O-255 microRNA-23a/b-3p regulate expression levels of testis-specific transcripts in men with impaired fecundity

Abstract Study question To investigate whether microRNA-23a/b-3p targets spermatogenesis-related transcripts and whether this targeting impacts their expression contents in patients with subfertility. Summary answer The higher expression of microRNA-23a/b-3p and lower expression of 11 tested target genes are associated with men subfertility What is known already Spermatogenesis is a complex and highly regulated process, many genes are involved, the expression levels of which are strongly or partially coordinated by microRNAs (miRNAs). MiRNAs are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Transcriptome analysis shows that hundreds of genes are expressed exclusively or predominantly in male germ cells including CEP41, G2E3, GOLGA6B, GOLGA6C, LMLN, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 genes, which play a crucial role during spermatogenesis and/or sperm function. However, the expression regulation of these genes is still unclear. Study design, size, duration Reverse transcription-quantitative PCR (RT-qPCR), dual luciferase assay, Western blot, and bioinformatics analysis were used to validate the lower expression of 11 target genes as a result of the known higher expression of microRNA-23a/b-3p in men with subfertility. A total of 82 men were included for RT-qPCR, consisting of 41 oligoasthenozoospermic subfertile men who attended the IVF center for infertility treatment and 41 age-matched normozoospermic volunteers who served as controls. Participants/materials, setting, methods In silico prediction and dual-luciferase assays were performed to evaluate the potential links between the higher expression of microRNA-23a/b-3p and the lower expression of 11 genes. Total RNA, including miRNA, was isolated from the sperm of oligoasthenozoospermic (n = 41) and normozoospermic men (n = 41). RT-qPCR was used to detect the expression levels of 11 target genes. Correlation analyses between the mRNA expression levels and basic semen parameters were carried out. Main results and the role of chance The expression levels of microRNA-23a/b-3p were significantly up-regulated and 11 genes were significantly down-regulated in oligoasthenozoospermic men compared with age-matched normozoospermic men as determined by RT-qPCR. Using dual-luciferase assays, 9 genes including CEP41, G2E3, GOLGA6C, NOL4, PAPOLB, PCDHA9, RGPD1, SOX6, and ZNF695 were identified as direct targets of miR-23a-3p and 4 genes including GOLGA6C, PAPOLB, SOX6, and ZNF695 were identified as direct targets of miR-23b-3p. Mutations in the miR-23a/b-3p binding site within the 3ˊuntranslated regions (3ˊUTRs) of the 9 target genes, which target either miR-23a-3p and/or miR-23b-3p, resulted in abrogated responsiveness to microRNA-23a/b-3p and confirmed that CEP41, GOLGA6C, NOL4, PCDHA9, and SOX6 as direct targets for miR-23a-3p and NOL4, SOX6 and PCDHA9 as direct targets for miR-23b-3p. Correlation analysis highlighted sperm count, motility, and morphology were positively correlated with the lower expression level of these validated genes. Limitations, reasons for caution Despite the correlation between the higher expression of microRNA-23a/b-3p and the lower expression of the validated genes, further validation by Western blotting in human sperm and testicular tissues is needed. Wider implications of the findings Findings suggest that the higher expression of microRNA-23a/b-3p or the lower expression of validated target genes are associated with male subfertility, probably through influencing the basic semen parameters. This study lay the groundwork for future studies focused on investigating therapies for male infertility. Trial registration number Hedwig-Stalter foundation (2016)

Towards a More Comprehensive Picture of the MicroRNA-23a/b-3p Impact on Impaired Male Fertility.

The expression levels of various genes involved in human spermatogenesis are influenced by microRNAs (miRNAs), specifically microRNA-23a/b-3p. While certain genes are essential for spermatogenesis and male germ cell function, the regulation of their expression remains unclear. This study aimed to investigate whether microRNA-23a/b-3p targets genes involved in spermatogenesis and the impact of this targeting on the expression levels of these genes in males with impaired fertility. In-silico prediction and dual-luciferase assays were used to determine the potential connections between microRNA-23a/b-3p overexpression and reduced expression levels of 16 target genes. Reverse transcription-quantitative PCR (RT-qPCR) was conducted on 41 oligoasthenozoospermic men receiving infertility treatment and 41 age-matched normozoospermic individuals to verify the lower expression level of target genes. By employing dual-luciferase assays, microRNA-23a-3p was found to directly target eight genes, namely NOL4, SOX6, GOLGA6C, PCDHA9, G2E3, ZNF695, CEP41, and RGPD1, while microRNA-23b-3p directly targeted three genes, namely SOX6, GOLGA6C, and ZNF695. The intentional alteration of the microRNA-23a/b binding site within the 3' untranslated regions (3'UTRs) of the eight genes resulted in the loss of responsiveness to microRNA-23a/b-3p. This confirmed that NOL4, SOX6, GOLGA6C, PCDHA9, and CEP41 are direct targets for microRNA-23a-3p, while NOL4, SOX6, and PCDHA9 are direct targets for microRNA-23b-3p. The sperm samples of oligoasthenozoospermic men had lower expression levels of target genes than age-matched normozoospermic men. Correlation analysis indicated a positive correlation between basic semen parameters and lower expression levels of target genes. The study suggests that microRNA-23a/b-3p plays a significant role in spermatogenesis by controlling the expression of target genes linked to males with impaired fertility and has an impact on basic semen parameters.

Sperm Motility Annotated Genes: Are They Associated with Impaired Fecundity?

Sperm motility is a prerequisite for achieving pregnancy, and alterations in sperm motility, along with sperm count and morphology, are commonly observed in subfertile men. The aim of the study was to determine whether the expression level of genes annotated with the Gene Ontology (GO) term 'sperm motility' differed in sperm collected from healthy men and men diagnosed with oligoasthenozoospermia. Reverse transcription quantitative real-time PCR (RT-qPCR), quantitative mass spectrometry (LC-MS/MS), and enrichment analyses were used to validate a set of 132 genes in 198 men present at an infertility clinic. Out of the 132 studied sperm-motility-associated genes, 114 showed differentially expressed levels in oligoasthenozoospermic men compared to those of normozoospermic controls using an RT-qPCR analysis. Of these, 94 genes showed a significantly lower expression level, and 20 genes showed a significantly higher expression level. An MS analysis of sperm from an independent cohort of healthy and subfertile men identified 692 differentially expressed proteins, of which 512 were significantly lower and 180 were significantly higher in oligoasthenozoospermic men compared to those of the normozoospermic controls. Of the 58 gene products quantified with both techniques, 48 (82.75%) showed concordant regulation. Besides the sperm-motility-associated proteins, the unbiased proteomics approach uncovered several novel proteins whose expression levels were specifically altered in abnormal sperm samples. Among these deregulated proteins, there was a clear overrepresentation of annotation terms related to sperm integrity, the cytoskeleton, and energy-related metabolism, as well as human phenotypes related to spermatogenesis and sperm-related abnormalities. These findings suggest that many of these proteins may serve as diagnostic markers of male infertility. Our study reveals an extended number of sperm-motility-associated genes with altered expression levels in the sperm of men with oligoasthenozoospermia. These genes and/or proteins can be used in the future for better assessments of male factor infertility.

Proteomic Landscape of Human Sperm in Patients with Different Spermatogenic Impairments

Although the proteome of sperm has been characterized, there is still a lack of high-throughput studies on dysregulated proteins in sperm from subfertile men, with only a few studies on the sperm proteome in asthenozoospermic and oligoasthenozoospermic men. Using liquid chromatography-mass spectrometry (LC-MS/MS) along with bioinformatics analyses, we investigated the proteomic landscape of sperm collected from subfertile men (n = 22), i.e., asthenozoospermic men (n = 13), oligoasthenozoospermic men (n = 9) and normozoospermic controls (n = 31). We identified 4412 proteins in human sperm. Out of these, 1336 differentially abundant proteins were identified in 70% of the samples. In subfertile men, 32 proteins showed a lower abundance level and 34 showed a higher abundance level when compared with normozoospermic men. Compared to normozoospermic controls, 95 and 8 proteins showed a lower abundance level, and 86 and 1 proteins showed a higher abundance level in asthenozoospermic and oligoasthenozoospermic men, respectively. Sperm motility and count were negatively correlated with 13 and 35 and positively correlated with 37 and 20 differentially abundant proteins in asthenozoospermic and oligoasthenozoospermic men, respectively. The combination of the proteins APCS, APOE, and FLOT1 discriminates subfertile males from normozoospermic controls with an AUC value of 0.95. Combined APOE and FN1 proteins discriminate asthenozoospermic men form controls with an AUC of 1, and combined RUVBL1 and TFKC oligoasthenozoospermic men with an AUC of 0.93. Using a proteomic approach, we revealed the proteomic landscape of sperm collected from asthenozoospermic or oligoasthenozoospermic men. Identified abundance changes of several specific proteins are likely to impact sperm function leading to subfertility. The data also provide evidence for the usefulness of specific proteins or protein combinations to support future diagnosis of male subfertility.

Expression of SPAG7 and its regulatory microRNAs in seminal plasma and seminal plasma-derived extracellular vesicles of patients with subfertility

Seminal plasma contains a variety of extracellular vesicles (EVs) that deliver RNAs including microRNAs (miRNAs) molecules. However, the roles of these EVs along with their delivered RNAs and their interactions with male infertility are not clear. Sperm-associated antigen 7 (SPAG 7) is expressed in male germ cells and plays a crucial role in several biological functions associated with sperm production and maturation. In this study, we aimed to identify the post-transcriptional regulation of SPAG7 in seminal plasma (SF-Native) and seminal plasma-derived extracellular vesicles (SF-EVs) collected from 87 men undergoing infertility treatment. Among the multiple binding sites for miRNAs within its 3’UTR of SPAG7, we identified the binding of four miRNAs (miR-15b-5p, miR-195-5p, miR-424-5p, and miR-497-5p) to the 3’UTR of SPAG7 by the dual luciferase assays. Analyzing sperm, we found reduced mRNA expression levels of SPAG7 in SF-EVs and SF-Native samples from oligoasthenozoospermic men. By contrast, two miRNAs (miR-424-5p and miR-497-5p) form the SF-Native samples, and four miRNAs (miR-195-5p, miR-424-5p, miR-497-5p, and miR-6838-5p) from the SF-EVs samples showed significantly higher expression levels in oligoasthenozoospermic men. The expression levels of miRNAs and SPAG7 were significantly correlated with basic semen parameters. These findings contribute significantly to our understanding of regulatory pathways in male fertility by showing a direct link between upregulated miRNA, notably miR-424, and downregulated SPAG7 both in seminal plasma and in plasma-derived EVs likely contributing to oligoasthenozoospermia.

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.

Proteomic and genetic dissection of testis-specific histone 2B in infertile men reveals its contribution to meiosis and sperm motility

To investigate testis-specific histone 2B (TSH2B) and its gene anomalies in infertile men. Case-control study. Basic science laboratory. Fertile and infertile men. Not applicable. The histone and protamine status of sperm was studied by aniline blue and chromomycin A3 staining, respectively. Testis-specific histone 2B, total H2B, and phosphorylated TSH2B (pTSH2B) were estimated by Western blot analysis. The frequency of genetic polymorphisms and rare variants in H2BC1 was studied by Sanger sequencing. Phosphosites on TSH2B in sperm were identified by reverse-phase high-performance liquid chromatography purification of TSH2B followed by mass spectrometric analysis. Aniline blue and chromomycin A3 staining revealed significantly higher histone retention and low protamine in sperm of infertile men. Sperm TSH2B and total H2B levels were significantly lower in oligozoospermic and oligoasthenozoospermic men (in both groups). The TSH2B levels were comparable in asthenozoospermic men; however, the pTSH2B level was significantly low. The H2BC1 gene sequencing identified 6 variants, of which 2 are rare variants (rs368672899 and rs544942090) and 4 (rs4711096, rs4712959, rs4712960 and rs4712961) are single nucleotide polymorphisms. Minor allele frequency of 5'-untranslated region variant rs4711096 was significantly lower in infertile men (OR = 0.65). The rare nonsynonymous variant, rs368672899, p.Ser5Pro was seen in 1 oligoasthenoteratozoospermic individual. Interestingly, mass spectrometric analysis identified a site on TSH2B to bear a phosphate group in the sperm of fertile men. Our study reveals a defect in the replacement of somatic histones with testis-specific variants in infertile men. Chromatin compaction positively correlates with sperm motility, which is suggestive of its utility in diagnostic semen analysis of infertile individuals. Our observations with TSH2B and its cognate gene in sperm of infertile men indicate an essential role for TSH2B in meiosis and its phosphorylation in sperm motility, respectively.

Effects of paternal exposure to cigarette smoke on sperm DNA methylation and long-term metabolic syndrome in offspring

BackgroundAlthough paternal exposure to cigarette smoke may contribute to obesity and metabolic syndrome in offspring, the underlying mechanisms remain uncertain.MethodsIn the present study, we analyzed the sperm DNA-methylation profiles in tobacco-smoking normozoospermic (SN) men, non-tobacco-smoking normozoospermic (N) men, and non-smoking oligoasthenozoospermic (OA) men. Using a mouse model, we also analyzed global methylation and differentially methylated regions (DMRs) of the DLK1 gene in paternal spermatozoa and the livers of progeny. In addition, we quantified DLK1 expression, executed an intra-peritoneal glucose tolerance test (IPGTT), measured serum metabolites, and analyzed liver lipid accumulation in the F1 offspring.ResultsGlobal sperm DNA-methylation levels were significantly elevated (p < 0.05) in the SN group, and the methylation patterns were different among N, SN, and OA groups. Importantly, the methylation level of the DLK1 locus (cg11193865) was significantly elevated in the SN group compared to both N and OA groups (p < 0.001). In the mouse model, the group exposed to cigarette smoke extract (CSE) exhibited a significantly higher global methylation DNA level in spermatozoa (p < 0.001) and on the DMR sites of Dlk1 in 10-week-old male offspring (p < 0.05), with a significant increase in Dlk1 expression in their livers (p < 0.001). In addition, IPGTT and LDL levels were significantly altered (p < 0.001), with elevated liver fat accumulation (p < 0.05) in F1 offspring.ConclusionPaternal exposure to cigarette smoke led to increased global methylation of sperm DNA and alterations to the DMR of the DLK1 gene in the F1 generation, which may be inherited parentally and may perturb long-term metabolic function.

MicroRNA-targeting in spermatogenesis: Over-expressions of microRNA-23a/b-3p and its affected targeting of the genes ODF2 and UBQLN3 in spermatozoa of patients with oligoasthenozoospermia.

Male infertility is a multifactorial syndrome with diverse phenotypic representations. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Altered abundance levels of ODF2 and UBQLN3 have been reported in patients with different spermatogenic impairments. However, the transcriptional regulation of these two genes by miR-23a/b-3p is still unclear. To investigate experimentally whether miR-23a/b-3p targets the genes ODF2 and UBQLN3 and whether this targeting impacts abundance levels of ODF2 and UBQLN3 in patients with oligoasthenozoospermia. A total of 92 men attending a fertility clinic were included in the study, including 46 oligoasthenozoospermic men and 46 age-matched normozoospermic volunteers who served as controls. Reverse transcription-quantitative PCR (RT-qPCR), Western blot, and dual-luciferase (Firefly-Renilla) assays were used to validate the miRNAs and their target genes. RT-qPCR revealed that miR-23a/b-3p was more abundant and ODF2 and UBQLN3 targets were less abundant in men with impaired spermatogenesis. Besides, Western blot shows that ODF2 and UBQLN3 protein levels were reduced in men with impaired spermatogenesis. In silico prediction and dual-luciferase assays revealed that potential links exist between the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets. Mutations in the miR-23a/b-3p-binding site within the 3'UTRs (3'untranslated regions) of ODF2 and UBQLN3 genes resulted in abrogated responsiveness to miR-23a/b-3p. Correlation analysis showed that sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with the lower abundance level of UBQLN3, while ODF lower abundance level was positively correlated with sperm motility. Findings indicate that the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets are associated with oligoasthenozoospermia and male subfertility.

Double-blind, randomised, placebo-controlled trial on the effect of L-carnitine and L-acetylcarnitine on sperm parameters in men with idiopathic oligoasthenozoospermia.

Carnitine is essential for energy metabolism and spermatozoa maturation. Combining L‐carnitine and L‐acetylcarnitine with micronutrients has been investigated as a treatment for infertility in men. We evaluated the effects of a therapeutic formulation, Proxeed Plus, on sperm parameters in oligoasthenozoospermic men. This prospective, randomised, double‐blind, placebo‐controlled clinical trial involved 175 males (19–44 years) with idiopathic oligoasthenozoospermia who failed to impregnate their partners (12 months). Males received Proxeed Plus or placebo for 3 and 6 months. Sperm volume, progressive motility and vitality significantly (p < 0.001) improved after 6 months compared to baseline. Sperm DNA fragmentation index significantly decreased compared to baseline (p < 0.001) and the 3‐month therapy (p = 0.014) in treated men. Increased seminal carnitine and α‐glucosidase concentration also positively correlated with improved progressive motility. Decreased DNA fragmentation index was the good predictor of progressive sperm motility >10%, and simultaneous measurement of changes in sperm vitality and DNA fragmentation index gave the highest probability of sperm motility 10% (AUC = 0.924; 95% CI = 0.852–0.996; p < 0.001). Logistic regression analyses revealed DNA fragmentation index decrease as the only independent predictor of sperm motility 10% (OR = 1.106; p = 0.034). We have demonstrated the beneficial effects of carnitine derivatives on progressive motility, vitality and sperm DNA fragmentation. Combining metabolic and micronutritive factors is beneficial for male infertility.

Comparison of Intracytoplasmic Sperm Injection Outcome with Sperm Selection Techniques in Oligoasthenozoospermic Males: A Randomized Controlled Trial

Background: Male infertility rate has increased over the recent years across the globe. Intracytoplasmic sperm injection (ICSI) is mainly used to treat male infertility; however, in this method, sperm selection plays a critical role in improving ICSI outcome. Objectives: The aim of this study was to evaluate the efficiency of three sperm selection techniques on ICSI outcomes in oligoasthenozoospermic men. Methods: This randomized clinical trial study was performed on 80 oligoasthenozoospermic (OAS) couples receiving ICSI in Milad Infertility Center, Mashhad, Iran from October 2016 to June 2017. The couples were randomly assigned to the three groups of control, hypo-osmotic swelling test (HOST) and hyaluronic acid (HA) according to the sperm selection technique for ICSI. Fertilization rate, embryo development (ED), embryo quality (EQ) and implantation rate (IR) were evaluated and compared among the groups. Results: Our results showed that there was no significant difference in fertilization rate between the studied groups. ED rate was increased in the HOST and HA groups compared to the control group, but this difference was not significant. EQ was increased in the HOST (P = 0.001) and HA groups (P = 0.001) compared to the control group. IR was enhanced in the HA group in comparison with the control group (P = 0.021), but there was no significant difference between the HOST and HA groups. Conclusions: The results of this study showed that for achieving successful clinical outcomes and improved embryo quality and implantation rate in oligoasthenozoospermic males undergoing Intracytoplasmic sperm injection, effective sperm selection through either the HOST or HA technique is necessary.

Supplementation of cryopreservation medium with TAT-Peroxiredoxin 2 fusion protein improves human sperm quality and function

To investigate the potential effects of TAT-PRDX2 protein supplementation to the cryopreservation medium on post-thaw sperm quality and function. Invitro prospective study. Medical university hospital. Fifty normozoospermic, 50 asthenozoospermic, and 50 oligoasthenozoospermic men undergoing semen analysis for couple infertility. Each semen sample was divided into three aliquots: fresh, cryopreserved control (without additive), and cryopreserved with TAT-PRDX2 protein. Sperm motility, viability, mitochondrial potential, and DNA damage as well as reactive oxygen species (ROS) levels and lipid peroxidation were analyzed. Acrosome reaction and zona-free hamster oocyte penetration tests were performed to assess the fertilization ability of cryopreserved spermatozoa. In normozoospermic and asthenozoospermic groups, the addition of 150μg/mL TAT-PRDX2 significantly reduced intracellular ROS and malondialdehyde levels and enhanced post-thaw sperm motility and viability when compared with the cryopreserved control of the respective groups but did not produce any significant protective effect in the oligoasthenozoospermic group. Mitochondrial potential was significantly increased, whereas DNA fragmentation was significantly decreased, after TAT-PRDX2 supplementation only in the asthenozoospermic group when compared with the cryopreserved control. Although the penetration rate and the penetration index were not markedly improved, TAT-PRDX2 supplementation obviously reduced spontaneous acrosome reaction and increased calcium ionophore-induced acrosome reaction in the normozoospermic and asthenozoospermic groups. TAT-PRDX2 protein effectively exerted cryoprotective effects on spermatozoa by reducing intracellular ROS level and thereby improved post-thaw sperm quality and function, especially for asthenozoospermic samples. TAT-PRDX2 protein is a promising additive for developing a new and highly efficient semen cryoprotectant.

Role of the Zeta Method in Intracytoplasmic Sperm Injection Outcomes in High Sperm DNA Fragmentation in Oligoasthenozoospermic Men

Background: New methods are needed to optimize intracytoplasmic sperm injection (ICSI) outcomes in oligoasthenozoospermic (OAS) men. We evaluated the level of DNA fragment index (DFI) in OAS men and its impact on ICSI outcomes. In addition, we used the zeta potential method for sperm selection to investigate the efficacy of this technique in improving ICSI outcomes. Materials and Methods: This cross-sectional study was performed on 95 couples. Sperm parameters and sperm DNA fragmentation (SDF) were measured. The couples were divided into the following 3 groups: group I (n=30) where SDF was between 15% and 30%, and routine sperm was selected on the basis of motility and morphology; group II (n=34) where SDF was more than 30%, and the routine sperm selection method was applied on the basis of motility and morphology; and group III (n=31) where SDF was more than 30%, and the sperm selection was performed on the basis of the zeta method. The fertilization rate, embryo development, embryo quality, and implantation rate were evaluated in these 3 groups. Results: The fertilization rate was significantly higher in group I compared with group II (P&lt;0.05). The embryo development rate in group I was significantly higher than that in group II (P&lt;0.001) and group III (P&lt;0.05), and it was significantly lower in group II compared with group III (P&lt;0.05). The embryo quality was higher in group III compared with group II (P&lt;0.01). The implantation rate in group I was significantly higher than that in group II (P&lt;0.05) and group III (P&lt;0.05). Conclusion: The present study indicated that a higher level of SDF has an adverse effect on the ICSI outcome. Furthermore, the zeta potential technique can be a useful method for sperm selection in OAS men. [GMJ.2018;7:e1107]

Role of the Zeta Method in Intracytoplasmic Sperm Injection Outcomes in High Sperm DNA Fragmentation in Oligoasthenozoospermic Men

Background: New methods are needed to optimize intracytoplasmic sperm injection (ICSI) outcomes in oligoasthenozoospermic (OAS) men. We evaluated the level of DNA fragment index (DFI) in OAS men and its impact on ICSI outcomes. In addition, we used the zeta potential method for sperm selection to investigate the efficacy of this technique in improving ICSI outcomes. Materials and Methods: This cross-sectional study was performed on 95 couples. Sperm parameters and sperm DNA fragmentation (SDF) were measured. The couples were divided into the following 3 groups: group I (n=30) where SDF was between 15% and 30%, and routine sperm was selected on the basis of motility and morphology; group II (n=34) where SDF was more than 30%, and the routine sperm selection method was applied on the basis of motility and morphology; and group III (n=31) where SDF was more than 30%, and the sperm selection was performed on the basis of the zeta method. The fertilization rate, embryo development, embryo quality, and implantation rate were evaluated in these 3 groups. Results: The fertilization rate was significantly higher in group I compared with group II (P&lt;0.05). The embryo development rate in group I was significantly higher than that in group II (P&lt;0.001) and group III (P&lt;0.05), and it was significantly lower in group II compared with group III (P&lt;0.05). The embryo quality was higher in group III compared with group II (P&lt;0.01). The implantation rate in group I was significantly higher than that in group II (P&lt;0.05) and group III (P&lt;0.05). Conclusion: The present study indicated that a higher level of SDF has an adverse effect on the ICSI outcome. Furthermore, the zeta potential technique can be a useful method for sperm selection in OAS men. [GMJ.2018;7:e1107]&#x0D; Â

Role of the Zeta Method in Intracytoplasmic Sperm Injection Outcomes in High Sperm DNA Fragmentation in Oligoasthenozoospermic Men.

Background: New methods are needed to optimize intracytoplasmic sperm injection (ICSI) outcomes in oligoasthenozoospermic (OAS) men. We evaluated the level of DNA fragment index (DFI) in OAS men and its impact on ICSI outcomes. In addition, we used the zeta potential method for sperm selection to investigate the efficacy of this technique in improving ICSI outcomes. Materials and Methods: This cross-sectional study was performed on 95 couples. Sperm parameters and sperm DNA fragmentation (SDF) were measured. The couples were divided into the following 3 groups: group I (n=30) where SDF was between 15% and 30%, and routine sperm was selected on the basis of motility and morphology; group II (n=34) where SDF was more than 30%, and the routine sperm selection method was applied on the basis of motility and morphology; and group III (n=31) where SDF was more than 30%, and the sperm selection was performed on the basis of the zeta method. The fertilization rate, embryo development, embryo quality, and implantation rate were evaluated in these 3 groups. Results: The fertilization rate was significantly higher in group I compared with group II (P<0.05). The embryo development rate in group I was significantly higher than that in group II (P<0.001) and group III (P<0.05), and it was significantly lower in group II compared with group III (P<0.05). The embryo quality was higher in group III compared with group II (P<0.01). The implantation rate in group I was significantly higher than that in group II (P<0.05) and group III (P<0.05). Conclusions: The present study indicated that a higher level of SDF has an adverse effect on the ICSI outcome. Furthermore, the zeta potential technique can be a useful method for sperm selection in OAS men.

Progesterone requires heat shock protein 90 (HSP90) in human sperm to regulate motility and acrosome reaction.

The aims of this paper were to study whether heat shock protein 90 (HSP90) is a regulator of sperm functions and to determine its association with oligoasthenozoospermia. The levels of HSP90 in sperm lysates were measured by ELISA. Localization of HSP90 and its isoforms was evaluated by immunofluorescence. Sperm motility and kinetics were assessed by computer-assisted sperm analysis. Acrosome reaction was determined by lectin staining. The levels of HSP90 were lower in oligoasthenozoospermic men and correlated positively with the number of motile spermatozoa. In capacitated human spermatozoa, HSP90α was mostly found in residual nuclear envelope, and the HSP90β isoform was higher in the flagella. Inhibition of HSP90 by geldanamycin or 17-AAG did not affect basal motility, but suppressed progesterone-mediated forward progressive motility, hyperactivation and acrosome reaction. Progesterone treatment dephosphorylated both HSP90α and HSP90β at Ser/Thr-Pro residues, but not Tyr residues. HSP90 levels are downregulated in oligoasthenozoospermia, and its functional inhibition attenuates progesterone-mediated sperm motility and acrosome reaction.

Altered micro-ribonucleic acid expression profiles of extracellular microvesicles in the seminal plasma of patients with oligoasthenozoospermia

To determine whether microRNA (miRNA) expression profile is different in extracellular microvesicles collected from seminalplasma of men with oligoasthenozoospermia, to gain further insight into molecular mechanisms underlying male infertility. Microarray with quantitative real-time polymerase chain reaction validation and Western blot analysis confirmation. University research and clinical institutes. A total of 24 men, including 12 oligoasthenozoospermic subfertile men and 12 normozoospermic men. None. Statistically significant altered miRNA expression profiles in oligoasthenozoospermic subfertile men compared with normozoospermic fertile men. Extracellular microvesicles including exosomes were isolated from seminal plasma by ultracentrifugation. Presence of exosome-specific proteins was confirmed by Western blotting. In the extracellular microvesicles, we analyzed 1,205 miRNAs by microarray and identified 36 miRNAs with altered expression levels in oligoasthenozoospermic compared with normozoospermic fertile men. Seven miRNAs were overexpressed and 29 miRNAs were underexpressed in oligoasthenozoospermic men. Using quantitative real-time polymerase chain reaction as an independent method, we confirmed the significantly higher expression levels of miR-765 and miR-1275 and the significantly lower expression level of miR-15a in oligoasthenozoospermic subfertile men as compared with the normozoospermic men. We identified altered expression levels of miRNAs in extracellular microvesicles from seminal plasma as part of the molecular events in the male genital tract. These miRNAs may help to understand the molecular mechanisms underlying male infertility.

Epigenetic germline mosaicism in infertile men.

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.

Levels of Tektin 2 and CatSper 2 in normozoospermic and oligoasthenozoospermic men and its association with motility, fertilization rate, embryo quality and pregnancy rate

To compare the expression profiles of Tektin 2 and CatSper 2 motility proteins in the spermatozoa of normozoospermic and oligoasthenozoospermic men and determine its correlation with sperm motility, fertilization rate, embryo quality and pregnancy rate. Tektin 2 and CatSper 2 protein expression was studied using Western Blotting and immunofluorescence. Tektin 2 and CatSper 2 protein levels were quantified by ELISA. Oligoasthenozoospermic men were found to have lower fertilization rates, poor embryo quality and lower pregnancy rates as compared to normozoospermic men. The levels of Tektin 2 and CatSper 2 are significantly lower in spermatozoa of oligoasthenozoospermic men as compared to normozoospermic controls; the levels were also lower in immotile fraction as compared to motile fraction of spermatozoa obtained from normozoospermic individuals. The levels of Tektin 2 and CatSper 2 were higher in individuals demonstrating sperm motility >60 % as compared to sperm motility <30 %. Tektin 2 but not CatSper 2 levels were positively associated with fertilization rate, embryo quality and pregnancy rate. Levels of Tektin 2 and CatSper 2 proteins are positively associated with sperm motility parameters. Measurements of Tektin 2 levels can be correlated with the clinical outcome of ICSI.