Expression data from ejaculated spermatozoa of normozoospermic and asthenozoospermic men
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ABSTRACT: Analysis of ejaculated spermatozoav from normozoospermic men and asthenozoospermic men. Some of genes were up-regulated or down-regulated in asthenozoospermia, and their abnormal expression were the causes of the impaired sperm motility. Results provide insight into the mechanisms by which asthenozoospermia is controlled. We used microarrays to detail the global programme of gene expression of ejaculated spermatozoa between normozoospermic and asthenozoospermic men and identified distinct classes of genes expressed differentially in two groups. The liquefied semen samples from normozoospermic and asthenozoospermic men were purified by Percoll on a discontinuous density gradients for RNA extraction and hybridization on Affymetrix microarrays. 30 sperm samples of each group were pooled to abtain enough total RNA.
Project description:Semen samples from men after a short ejaculatory abstinence show improved sperm quality and result in increased pregnancy rates, but the underlying mechanisms remain unclear. Herein, we report that ejaculates from short (1–3 hours) compared with long (3–7 days) periods of abstinence showed increases in motile sperm count, sperm vitality, normal sperm morphology, acrosome reaction capacity, total antioxidant capacity, sperm mitochondrial membrane potential, high DNA stainability, and a decrease in the sperm DNA fragmentation index (P < 0.05). Sperm proteomic analysis showed 322 differentially expressed proteins (minimal fold change of ±1.5 or greater and P < 0.05), with 224 up-regulated and 98 down-regulated. These differentially expressed proteins are profoundly involved in specific cellular processes, such as motility and capacitation, oxidative stress, and metabolism. Interestingly, protein trimethyllysine modification was increased, and butyryllysine, propionyllysine, and malonyllysine modifications were decreased in ejaculates from a short versus long abstinence (P < 0.05). Finally, the rates of implantation, clinical pregnancy, and live births from in vitro fertilization treatments were significantly increased in semen samples after a short abstinence. Our study provides preliminary mechanistic insights into improved sperm quality and pregnancy outcomes associated with spermatozoa retrieved after a short ejaculatory abstinence
Project description:The problem of the male reproductive system has both medical and social importance. It is highlighted that spermatozoa or seminal plasma are suitable samples for proteomic analysis of the male reproductive system. We suggested that the ejaculate could serve as one of the analytic specimens to determine fertility, which obtainable with relative ease and which contains many proteins. Moreover, the testis-specific proteins could form the basis of protein panel for detecting disorders associated with male infertility. The current study aimed to examine the ejaculate proteome compared to the spermatozoa and seminal plasma proteomes to determine a panel of proteins that can be used to assess sperm quality. Our results showed that the ejaculate represented a productive source for understanding the male reproductive physiology and could serve as a potential origin for the identification of novel protein biomarkers related to spermatozoa function in infertile. We believe that proteomic analysis based on testis-specific proteins of ejaculate along with spermatozoa (or even instead) can be useful for distinguishing the fertile, sub fertile, and infertile men.
Project description:The global trend on (male)infertility is concerning and the unidentifiable causes in half of the cases demands a better understanding of the molecular, biochemical and metabolic mechanisms as well as the identification of external and internal factors operating behind it, that might help to explain this apparently unjustified infertility. This can only be achieved through a comprehensive analysis of the infertile men, in which external (lifestyle, occupational and environmental factors) and internal factors (psychological distress) should also be evaluated and considered, but also assessing sperm function beyond the routine seminal analysis. In this prospective cohort study, 79 sperm samples, from men who were male partners in couples seeking for infertility treatment, were collected at the Reproductive Medicine Unit (CHUC) from July 2018 to July 2022 and analyzed by SWATH-MS. Based on couples’ clinical data, seminal/hormonal analysis, and strict exclusion criteria, samples were categorized in the different groups, namely control (CTRL; 50 individuals), idiopathic infertile (ID; 19 patients) and unexplained infertile (UMI; 10 patients) men. In general, ID patients presented the worst sperm functional profile, while the UMI patients were observed to be similar to controls. These differences were also observed at the proteomics levels, which revealed 145 differentially expressed proteins (DEP) between the three groups, with more than 120 proteins being altered between ID and the other groups, and only 14 proteins were considered altered between CTRL and UMI.
Project description:Analysis of ejaculated spermatozoav from normozoospermic men and asthenozoospermic men. Some of genes were up-regulated or down-regulated in asthenozoospermia, and their abnormal expression were the causes of the impaired sperm motility. Results provide insight into the mechanisms by which asthenozoospermia is controlled. We used microarrays to detail the global programme of gene expression of ejaculated spermatozoa between normozoospermic and asthenozoospermic men and identified distinct classes of genes expressed differentially in two groups.
Project description:Infertility is a widespread problem, affecting around 15% of couples worldwide, and is defined as the inability to achieve pregnancy within one year despite unprotected intercourse 1. Infertility can be caused by either male or female reproductive issues. Various medical conditions including malignancies, infections, urogenital conditions, or genetic causes can contribute to male infertility. However, 30-40% of men in their reproductive age are affected by idiopathic infertility, according to the guidelines of European Association of Urology (EAU) 1. Towards a better understanding of male infertility, it is mandatory to achieve a comprehensive understanding of involved genes, their RNA transcripts, and regulatory factors, including miRNAs, which influence the expression level of proteins. Therefore, such proteins need to be identified to investigate their role in spermatogenesis and male infertility. Although there are numerous studies on RNAs, including miRNAs related to male infertility 2-9, there are few studies aiming to cover the whole proteome of human sperm 10. The sperm transcriptome comprises a total of 60,505 transcripts including 11,688 differentially expressed transcripts in infertile and fertile men, as reported by Joshi et al. (2022)11. The entire sperm proteome encompasses 6871 proteins, as summarized by Castillo et al. (2018)10. Nevertheless, there is still a lack of high-throughput studies aiming to identify dysregulated proteins in sperm from subfertile men. Only few studies focused on comparisons of the sperm proteome in men with asthenozoospermia and there is virtually no proteomic studies of oligoasthenozoospermic men 12. Some identified proteins in sperm have functions in maintaining sperm motility and enabling fertilization and are involved in structural composition and/or energy metabolism 12-14 and others are not yet functionally characterized. In this study, we employed Mass spectrometry (MS) technology that is still rarely used in the field of human reproductive research to investigate the proteomic landscape of human sperm and their differential expression patterns in men with subfertility.
Project description:The male gamete is not completely mature after ejaculation and requires further events in the female genital tract to acquire fertilizing ability, including the processes of capacitation and acrosome reaction. In order to shed light on dynamic protein changes experienced by the sperm cell in preparation for fertilization, a comprehensive quantitative proteomic profiling based on isotopic peptide labeling and liquid chromatography followed by tandem mass spectrometry was performed on spermatozoa from three donors of proven fertility under three sequential conditions: purification with density gradient centrifugation, incubation with capacitation medium, and induction of acrosome reaction by the exposure to the calcium ionophore A23187. After applying strict selection criteria for peptide quantification and for statistical analyses, 36 proteins with significant changes in their relative abundance within sperm protein extracts were detected. Moreover, the presence of peptide residues potentially harboring sites for post-translational modification was revealed, suggesting that protein modification may be an important mechanism in sperm maturation. In this regard, increased levels of proteins mainly involved in motility and signaling, both regulated by protein modifiers, were detected in sperm lysates following incubation with capacitation medium. In contrast, less abundant proteins in acrosome-reacted cell lysates did not contain potentially modifiable residues, suggesting the possibility that all those proteins might be relocated or released during the process. Protein-protein interaction analysis revealed a subset of proteins potentially involved in sperm maturation, including ERLIN2, GGH, TMED 10, and ATP synthases. These results contribute to the current knowledge of the molecular basis of human fertilization and propose new candidates to be validated as modulators of male fertility.
Project description:Artificial insemination in small ruminants is most commonly performed using fresh semen due to the low fertility rates typically achieved with frozen spermatozoa. Usually, when developing and applying assisted reproductive technologies, sheep and goats are often lumped together as one specie. In order to optimize sperm cryopreservation protocols in sheep and goat, differences in sperm proteomics between ram and buck are necessary to detect, which may contribute to differences in sperm function and fertility.
Project description:Alterations in the presence of sperm RNAs have been identified using microarrays in teratozoospermic (abnormal morphology) or other types of infertile patients. However, so far no studies had been reported on the sperm RNA content using microarrays in asthenozoospermic patients (low motility). We started the present project to with the goal to characterize the RNA expression in asthenozoospermic infertile patients as compared to normozoospermic fertile controls. We selected four normal fertile donors and four severe asthenozoospermic infertile patients. Equal amounts of RNA were extracted from the sperm samples, subjected to different quality controls and hybridized to the Affymetrix U133 Plus version 2 arrays.
Project description:Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research.
Project description:Data about the entire sperm DNA methylome are limited to two sperm donors whereas studies dealing with a greater number of subjects focused only on a few genes or were based on low resolution arrays. This implies that information about what we can consider as a normal sperm DNA methylome and whether it is stable among different normozoospermic individuals is still missing. The definition of the DNA methylation profile of normozoospermic men, the entity of inter-individual variability and the epigenetic characterization of quality-fractioned sperm subpopulations in the same subject (intra-individual variability) are relevant for a better understanding of pathological conditions. We addressed these questions by using the high resolution Infinium 450K methylation array and compared normal sperm DNA methylomes against somatic cells. Our study, based on the largest number of subjects (n = 8) ever considered for such a large number of CpGs (n = 487,517), provided clear evidence for i) a highly conserved DNA methylation profile among normozoospermic subjects; ii) a stable sperm DNA methylation pattern in different quality-fractioned sperm populations of the same individual. The latter finding is particularly relevant if we consider that different quality fractioned sperm subpopulations show differences in their structural features, metabolic and genomic profiles. We demonstrate, for the first time, that DNA methylation in normozoospermic men remains highly uniform regardless the quality of sperm subpopulations. In addition, our analysis provided both confirmatory and novel data concerning the sperm DNA methylome, including its peculiar features in respect to somatic and cancer cells. Our description about a highly polarized sperm DNA methylation profile, the clearly distinct genomic and functional organization of hypo- versus hypermethylated loci as well as the association of histone-enriched hypomethylated loci with embryonic development, which we now extended also to hypomethylated piRNAs-linked genes, provides solid basis for future basic and clinical research. Bisulphite converted DNA from the 26 normal sperm and 2 somatic cell samples were hybridised to the Illumina Infinium 450k Human Methylation Beadchip v1.2