UNASSIGNED: Infertility is a worldwide concern, and recent research indicates that vitamin B6 deficiency may play a role in male infertility, primarily by inducing hyperhomocysteinemia and oxidative stress. These processes can have a detrimental effect on semen quality, ultimately affecting male fertility. Here, we aim to evaluate the biochemical status of pyridoxine (vitamin B6) in relation to total glutathione and total antioxidant capacity.
UNASSIGNED: A case control study samples were collected of asthenozoospermic (n = 63) and normospermic (n = 43) cases, with average men age 30.35 ± 7.03 years old. Semen plasma specimens representing both fertile and sub-fertile men visiting two different secondary care health institute in Irbid province, Jordan. All samples were assessed according to WHO guidelines (2021) and by using spectrophotometry to evaluate the semen plasma levels of vitamin B6, glutathione (GSH) and total antioxidant capacity (TAC).
UNASSIGNED: Our main finding is there is significant positive correlations between the seminal plasma concentration of GSH (p < 0.0001) and TAC (p < 0.0073) are significantly correlated with vitamin B6 deficiency in asthenozoospermia group in comparison to normozoospermia cases. A significant decrease (p < 0.0001) the levels of vitamin B6 in men with asthenozoospermia compared to normozoospermic men (control) with an approximate 80 % percent reduction in the mean levels between groups.
UNASSIGNED: These findings suggest that pyridoxine deficiency may very well alter the GSH system, in so doing affecting the antioxidant defense mechanism against reactive oxygen species to sperm, impacting sperm development and maturation. leading to asthenozoospermia.

UNASSIGNED: Semen cryopreservation is the most popular practice for semen production for artificial insemination and in vitro fertilization in cattle. The Seminal plasma contains extracellular vesicles (spEVs) which modulate sperm viability and function during oocyte fecundation. The study of spEVs in frozen-thawed semen doses may yield novel indicators for predicting bull fertility, but the presence of the semen extender may hinder molecular profiling of spEVs. The aim of this study was to provide extensive characterization of EVs isolated from seminal plasma before and after the cryopreservation process and the addition of a commercial animal protein-free semen extender to understand the potential influence of EVs originating from the extender in hindering the use of spEVs derived biomarkers for assessment of bull fertility.
UNASSIGNED: EVs were isolated from the seminal plasma (with or without the extender), from the cryopreserved straw devoid of spermatozoa, and from the extender using two different methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), and characterized for their structure and composition.
UNASSIGNED: Physical characterization of EVs showed that size and particle numbers were related to the method of isolation. spEVs were larger but less abundant (UC: 168.9 nm, n = 2.68 × 109; SEC: 197.0 nm, n = 6.42 × 109) compared to extender EVs (UC: 129.0 nm, n = 2.68 × 1011; SEC: 161.8 nm, n = 6.47 × 1011). Western blotting analysis (WB) confirmed the presence of typical EV markers in spEVS: the membrane bound CD9 (25 kDa) and the luminal markers Alix (96 kDa) and TSG101 (48 KDa). Although Transmission Electron Microscopy confirmed the presence of a lipid bilayer structure in all preparations, no specific EV markers were detected in the vesicles isolated from extender when the Single Molecule Array (SiMoa) was used. A total of 724 Bos taurus miRNAs were identified in at least one preparation. The percentage of miRNAs identified in EVs from the extender (0.05%-0.49% of the total reads) was lower than in the preparation containing spEVs (10.56%-63.69% of the total reads). Edge-R identified a total of 111 DE-miRNAs between EVs isolated from the extender by two methods. Among them, 11 DE-miRNAs (bta-miR-11980, bta-miR-11987, bta-miR-12057, bta-miR-1246, bta-miR-125b, bta-miR-181b, bta-miR-2340, bta-miR-2358, bta-miR-2478, bta-miR-2898, and bta-miR-345-3p) were also abundant in EVs isolated from seminal plasma preparations with extender.
UNASSIGNED: This study clearly demonstrates that the presence of the extender does not prevent the characterization of spEVs in cryopreserved semen. However, the molecular profiling of spEVs can be influenced by the isolation method used and by the presence of some miRNAs from the extender. Therefore, in such studies, it is advisable to characterize both spEVs and the vesicles isolated from the extender.

Among the many factors with a proven relation to semen quality and male fertility, the determination of seminal plasma cytokines provides a promising direction for research into the identification of factors connected with male infertility. The interleukins: IL-1α, -1β, -2, -4, -6, -8, -10, -12p40, -12p70, -18, IFNγ, and GM-CSF, total oxidant (TOS) and antioxidant (TAS) status, were simultaneously examined in seminal plasmas and blood sera in terato- (n = 32), asthenoterato- (n = 33), and oligoasthenoteratozoospermic (n = 29) infertile men and in normozoospermic fertile men (n = 20). Our research shows different cytokine composition of the sera and seminal plasmas in all studied groups, along with much higher concentrations of seminal plasma GM-CSF, IFNγ, IL-1α, IL-4, IL-6, and IL-8 and lower IL-18 and TOS in the comparison to their sera levels. The seminal plasma concentrations of GM-CSF, IFNγ, IL-1α, -4, and -6 differ significantly between fertile and infertile as well as between teratozoospermic, asthenoteratozoospermic, and oligoasthenoteratozoospermic groups. The indication of the cause of different concentrations of cytokines in seminal plasmas of infertile men, and their associations with semen parameters and oxidative status, may be a promising direction for the search for new therapeutic targets that would directly affect the cells and tissues of male reproductive organs.

OBJECTIVE: To investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters.
METHODS: The remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated.
RESULTS: The r2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1β and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05).
CONCLUSIONS: There are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches.

N-linked glycoproteins are rich in seminal plasma, playing essential roles in supporting sperm function and fertilization process. The alteration of seminal plasma glycans and its correspond glycoproteins may lead to sperm dysfunction and even infertility. In present study, an integrative analysis of glycoproteomic and proteomic was performed to investigate the changes of site-specific glycans and glycoptoteins in seminal plasma of asthenozoospermia. By large scale profiling and quantifying 5,018 intact N-glycopeptides in seminal plasma, we identified 92 intact N-glycopeptides from 34 glycoproteins changed in asthenozoospermia. Especially, fucosylated glycans containing lewis x, lewis y and core fucosylation were significantly up-regulated in asthenozoospermia compared to healthy donors. The up-regulation of fucosylated glycans in seminal plasma may interfere sperm surface compositions and regulation of immune response, which subsequently disrupts sperm function. Three differentiated expression of seminal vesicle-specific glycoproteins (fibronectin, seminogelin-2, and glycodelin) were also detected with fucosylation alteration in seminal plasma of asthenozoospermia. The interpretation of the altered site-specific glycan structures provides data for the diagnosis and etiology analysis of male infertility, as well as providing new insights into the potential therapeutic targets for male infertility.

Nerve growth factor (NGF) has long been known as the main ovulation-inducing factor in induced ovulation species, however, recent studies suggested the NGF role also in those with spontaneous ovulation. The first aim of this study was to evaluate the presence and gene expression of NGF and its cognate receptors, high-affinity neurotrophic tyrosine kinase 1 receptor (NTRK1) and low-affinity p75 nerve growth factor receptor (p75NTR), in the ram genital tract. Moreover, the annual trend of NGF seminal plasma values was investigated to evaluate the possible relationship between the NGF production variations and the ram reproductive seasonality. The presence and expression of the NGF/receptors system was evaluated in the testis, epididymis, vas deferens ampullae, seminal vesicles, prostate, and bulbourethral glands through immunohistochemistry and real-time PCR (qPCR), respectively. Genital tract samples were collected from 5 adult rams, regularly slaughtered at a local abattoir. Semen was collected during the whole year weekly, from 5 different adult rams, reared in a breeding facility, with an artificial vagina. NGF seminal plasma values were assessed through the ELISA method. NGF, NTRK1 and p75NTR immunoreactivity was detected in all male organs examined. NGF-positive immunostaining was observed in the spermatozoa of the germinal epithelium, in the epididymis and the cells of the secretory epithelium of annexed glands, NTRK1 receptor showed a localization pattern like that of NGF, whereas p75NTR immunopositivity was localized in the nerve fibers and ganglia. NGF gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis than in the other tissues. NTRK1 gene transcript was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.05) in all the other tissues examined. Gene expression of p75NTR was highest (p < 0.01) in the seminal vesicles and lowest (p < 0.01) in the testis and bulbourethral glands. NGF seminal plasma concentration was greater from January to May (p < 0.01) than in the other months. This study highlighted that the NGF system was expressed in the tissues of all the different genital tracts examined, confirming the role of NGF in ram reproduction. Sheep are short-day breeders, with an anestrus that corresponds to the highest seminal plasma NGF levels, thus suggesting the intriguing idea that this factor could participate in an inhibitory mechanism of male reproductive activity, activated during the female anestrus.

Indicators of male fertility are in decline globally, but the underlying causes, including the role of environmental exposures, are unclear. This study aimed to examine organic chemical pollutants in seminal plasma, including both known priority environmental chemicals and less studied chemicals, to identify uncharacterized male reproductive environmental toxicants. Semen samples were collected from 100 individuals and assessed for sperm concentration, percent motility, and total motile sperm. Targeted and nontargeted organic pollutant exposures were measured from seminal plasma using gas chromatography, which showed widespread detection of organic pollutants in seminal plasma across all exposure classes. We used principal component pursuit (PCP) on our targeted panel and derived one component (driven by etriadizole) associated with total motile sperm (p < 0.001) and concentration (p = 0.03). This was confirmed by the exposome-wide association models using individual chemicals, where etriadizole was negatively associated with total motile sperm (FDR q = 0.01) and concentration (q = 0.07). Using PCP on 814 nontargeted spectral peaks identified a component that was associated with total motile sperm (p = 0.001). Bayesian kernel machine regression identified one principal driver of this association, which was analytically confirmed to be N-nitrosodiethylamine. These findings are promising and consistent with experimental evidence showing that etridiazole and N-nitrosodiethylamine may be reproductive toxicants.

MicroRNAs (miRNAs) are bio-active elements cargoed by seminal plasma extracellular vesicles extracellular vesicles (SPEVs) which are crucial for sperm function and fertility modulation. This study aimed to isolate, characterize, and identify the miRNA expression profiles in the SPEVs from high (HSM) and low sperm motility (LSM) groups that could serve as fertility biomarkers and explain the underlying mechanisms. The isolated SPEVs were round spherical structures of approximately 50-200 nm in diameter expressing molecular markers. A total of 1006 and 1084 miRNAs were detected in HSM and LSM, respectively, with 34 being differentially expressed. Their targeted genes involved in SNARE interactions in vesicular transport, Metabolic pathways, and Apelin signaling pathway, etc. The joint analysis with mRNAs of sperm and sperm storage tubules cells highlighted the cellular communication mediated by SPEVs miRNAs, where they may rule fertility by affecting sperm maturation and amino acid metabolism. SPEVs as additives could improve fertility of fresh and frozen sperm, while the knockdown of one of the differentially expressed miRNAs, miR-24-3p, diminished this effect, indicating its crucial roles. This study expands our understanding of SPEVs miRNAs mediated sperm maturation and fertility modulation, and may help to develop new therapeutic strategies for infertility and sperm storage.

Seminal plasma induces immune tolerance towards paternal allogenic antigens within the female reproductive tract and during foetal development. Recent evidence suggests a role for extracellular vesicles in seminal plasma (spEVs). We isolated spEVs from seminal plasma that was donated by vasectomized men, thereby excluding any contributions from the testis or epididymis. Previous analysis demonstrated that such isolated spEVs originate mainly from the prostate. Here we observed that when isolated fluorescently labelled spEVs were mixed with peripheral blood mononuclear cells, they were endocytosed predominantly by monocytes, and to a lesser extent also by T-cells. In a mixed lymphocyte reaction, T-cell proliferation was inhibited by spEVs. A direct effect of spEVs on T-cells was demonstrated when isolated T cells were activated by anti-CD3/CD28 coated beads. Again, spEVs interfered with T cell proliferation, as well as with the expression of CD25 and the release of IFN-γ, TNF, and IL-2. Moreover, spEVs stimulated the expression of Foxp3 and IL-10 by CD4+CD25+CD127- T cells, indicating differentiation into regulatory T-cells (Tregs). Prior treatment of spEVs with proteinase K revoked their effects on T-cells, indicating a requirement for surface-exposed spEV proteins. The adenosine A2A receptor-specific antagonist CPI-444 also reduced effects of spEVs on T-cells, consistent with the notion that the development of Tregs and their immune suppressive functions are under the influence of adenosine-A2A receptor signalling. We found that adenosine is highly enriched in spEVs and propose that spEVs are targeted to and endocytosed by T-cells, after which they may release their adenosine content into the lumen of endosomes, thus allowing endosome-localized A2A receptor signalling in spEVs targeted T-cells. Collectively, these data support the idea that spEVs can prime T cells directly for differentiation into Tregs.

Seminal plasma (SP) is rich in extracellular vesicles (EVs), which are still poorly studied, especially in livestock species. To better understand their functional role in both spermatozoa and endometrial epithelial cells, proper characterization of EVs is an essential step. The objective was to phenotypically characterize porcine seminal EVs (sEVs) using cryogenic electron microscopy (cryo-EM), which allows visualization of EVs in their native state. Porcine ejaculates are released in fractions, each containing SP from different source. This allows characterization sEVs released from various male reproductive tissues. Two experiments were performed, the first with SP from the entire ejaculate (n:6) and the second with SP from three ejaculate fractions (n:15): the first 10 mL of the sperm-rich ejaculate fraction (SRF-P1) with SP mainly from the epididymis, the remainder of the SRF (SRF-P2) with SP mainly from the prostate, and the post-SRF with SP mainly from the seminal vesicles. The sEVs were isolated by size exclusion chromatography and 1840 cryo-EM sEV images were acquired using a Jeol-JEM-2200FS/CR-EM. The size, electron density, complexity, and peripheral corona layer were measured in each sEV using the ImageJ software. The first experiment showed that sEVs were structurally and morphologically heterogeneous, although most (83.1%) were small (less than 200 nm), rounded, and poorly electrodense, and some have a peripheral coronal layer. There were also larger sEVs (16.9%) that were irregularly shaped, more electrodense, and few with a peripheral coronal layer. The second experiment showed that small sEVs were more common in SRF-P1 and SRF-P2, indicating that they originated mainly from the epididymis and prostate. Large sEVs were more abundant in post-SRF, indicating that they originated mainly from seminal vesicles. Porcine sEVs are structurally and morphologically heterogeneous. This would be explained by the diversity of reproductive organs of origin.