Sperm mitochondrial dysfunction causes the generation of an insufficient amount of energy needed for sperm motility. This will affect sperm fertilization capacity, and thus, most asthenozoospermic men usually require assisted reproductive techniques. The etiology of asthenozoospermia remains largely unknown. The current study aimed to investigate the effect of mitochondrial genetic variants on sperm motility and intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study. One hundred five of the male partners were asthenozoospermic patients, and they were subdivided into three groups according to their percentage of sperm motility, while forty-five of the male partners were normozoospermic. Genetic variants were screened using direct Sanger’s sequencing in four mitochondrial genes (nicotinamide adenine dinucleotide hydrogen (NADH) dehydrogenase 1 (ND1), NADH dehydrogenase 2 (ND2), NADH dehydrogenase 5 (ND5), and NADH dehydrogenase 6 (ND6)). We identified three significant variants: 13708G>A (rs28359178) in ND5, 4216T>C (rs1599988) in ND1, and a novel 12506T>A in ND5 with P values 0.006, 0.036, and 0.013, respectively. The medians of sperm motility, fertilization rate, embryo cleavage score, and embryo quality score were significantly different between men showing 4216T>C, 12506T>A, 13708G>A and wild type, Mann-Whitney P values for the differences in the medians were < 0.05 in all of them. The results from this study suggest that 13708G>A, 12506T>A, and 4216 T>C variants in sperm mitochondrial DNA negatively affect sperm motility and ICSI outcomes.
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.
Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm-1, 731 cm-1, 785 cm-1, 858 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 were significantly correlated with at least one of the sperm staining methods. The median intensity of the Raman peaks at 670 cm-1, 731 cm-1, 785 cm-1, 1062 cm-1, 1098 cm-1, 1185 cm-1, 1372 cm-1, 1424 cm-1, 1450 cm-1, 1532 cm-1, 1618 cm-1 and 1673 cm-1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level.
Elevated concentrations of reactive oxygen species (ROS) in the semen can lead to oxidative protein damage as they react with the amino acids' side chains in the protein, leading to the generation of carbonyl groups. This study aimed to investigate the effect of protein carbonyl (PC) concentration on sperm motility and the laboratory intracytoplasmic sperm injection (ICSI) outcomes. A total of 150 couples from the ICSI cycle were enrolled in this study and were divided into three groups (G) according to the PC concentration as following, G1 included samples with PC concentrations <0.65 nmol/mg, G2 included samples with 0.65≤PC≤2.23 nmol/mg and G3 included samples with PC>2.23 (nmol/mg). PC concentrations were measured in all semen samples, and the laboratory ICSI outcomes were evaluated for all injected oocytes. The Kruskal–Wallis p‐values for the differences in the medians of sperm motility, fertilisation rate, embryo cleavage score and embryo quality score were <0.05. Furthermore, Dunn's post hoc test showed a significant difference between all groups, p‐values <0.05, except for the medians of embryo quality score between G2 and G3. In conclusion, our results showed that sperm motility and laboratory ICSI outcomes are affected negatively by higher concentrations of PC in the semen.
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.
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.
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)
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.
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