Seminal plasma (SP) is the main vector of C. trachomatis (CT) during heterosexual transmission from male to female. It has immunomodulatory properties and impacts the susceptibility to HIV-1 infection, but its role has not been explored during CT infection. In the female reproductive tract (FRT), CT infection induces cytokine production and neutrophil recruitment. The role of neutrophils during CT infection is partially described, they could be at the origin of the pathology observed during CT infection. During this study, we developed an experimental in vitro model to characterize the impact of CT infection and SP on endocervical epithelial cell immune response in the FRT. We also studied the impact of the epithelial cell response on neutrophil phenotype and functions. We showed that the production by epithelial cells of pro-inflammatory cytokines increased during CT infection. Moreover, the pool of SP as well as individuals SP inhibited CT infection in a dose-dependent manner. The pool of SP inhibited cytokine production in a dose-dependent manner. The pool of SP altered gene expression profiles of infected cells. The culture supernatants of cells infected or not with CT, in presence or not of the pool of SP, had an impact on neutrophil phenotype and functions: they affected markers of neutrophil maturation, activation and adhesion capacity, as well as the survival, ROS production and phagocytosis ability. This study proposes a novel approach to study the impact of the environment on the phenotype and functions of neutrophils in the FRT. It highlights the impact of the factors of the FRT environment, in particular SP and CT infection, on the mucosal inflammation and the need to take into account the SP component while studying sexually transmitted infections during heterosexual transmission from male to female.

The establishment of pregnancy involves a fine-tuned balance between protection and tolerance within the maternal immune system, as the female needs to accept a foreign antigen (the semi-allogenic fetus) while still being able to combat pathogens from the uterus. In the horse, the first uterine exposure to paternal antigens is during mating when sperm is introduced to the tissue and draining lymphatics of the uterus. Additionally, it has been suggested that seminal plasma and its proteins within it play an essential role in preparing the female tract for a suitable immunologic environment but this has not been confirmed in the horse. Therefore, the objective of this study was to evaluate the endometrial transcriptome following insemination either with seminal plasma or with reduced seminal plasma. We hypothesised that reduced seminal plasma would alter the endometrial transcriptome and affect transcripts relating to immunotolerance, antigen presentation and embryo growth and development. To do so, six (n = 6) mares were inseminated in a randomised switch-back design over the course of four oestrous cycles. Mares were rectally palpated and scanned via ultrasonography for the detection of a pre-ovulatory follicle (>35 mm) alongside increasing uterine oedema and relaxed cervix, and then treated with one of four treatment groups including (1) 30 mL lactated Ringers solution (LRS; NegCon), (2) 500 × 106 spermatozoa in conjunction with 30 mL seminal plasma (SP+), (3) 30 mL lactated Ringers solution (LRS; wash out) and (4) 500 × 106 spermatozoa with seminal plasma reduced via gradient centrifugation and resuspended in 30 mL LRS (SP-). Human chorionic gonadotropin (hCG) was administered to standardise the time to ovulation and endometrial biopsies were collected 7 days after insemination. RNA was isolated utilising Trizol, and RNA-Seq was performed by Novogene, with 97.79% total mapping and 40 million read depth. p value was set to <0.05. When comparing SP+ to SP-, 158 differentially expressed genes (DEGs) were identified. Biological processes impacted included antigen processing and regulation, cholesterol synthesis, and immune/inflammatory response. Gene ontology (GO) enrichment analysis using DAVID v6.8 revealed that many of these DEGs were involved in biological process such as antigen presentation (HLA-DM beta chain, HLA-DRB, HLA-DQA and RASGRP1), immune cell signalling (CXCL9, CXCL1, DEFB1 and MIP-2B), embryo growth and development (INHA, KLF2, RDH10, LAMA3 and SLC34A2) and embryo metabolism (ABCA1, ABCA2, APOA1, LDL, INSR, IGFBP2 and IGFBP3). Overall, reduction of seminal plasma from the insemination dose impacted the endometrial transcriptome at the time of early embryonic exposure to the uterine environment. Further work is justified to evaluate these alterations impact on embryo maturation, placental development, pregnancy outcome and development of offspring.

BACKGROUND: Within-subject variability of semen parameters and molecular components of ejaculates in young men remains poorly understood.
OBJECTIVE: To investigate intraindividual variability (IIV) of semen parameters and molecular markers in repeated ejaculates from young men.
METHODS: Semen parameters were assessed in samples collected 6-8 days apart from 164 18-19-year old participants of the Russian Children\'s Study, a prospective cohort. Subsets of paired samples were used for label-free quantitation and targeted mass-spectrometry of proteins in seminal plasma (SP) and seminal extracellular vesicles (EVs), and for small non-coding RNA (sncRNA) profiling in EVs and spermatozoa using RNA-seq. The mean difference between two ejaculates, within-subject variation, intraclass correlation, and concordance correlation were used to assess IIV for all parameters. Low variability with high reproducibility and high reliability was considered if CVw < 15% and ICC > 0.90, respectively.
RESULTS: Analytical variability was low for all investigated parameters in technical replicates. IIV was assessed for basic semen parameters and proteins in SPs and EVs: 319 and 777 proteins, respectively, using untargeted analysis; 9 and 10 proteins using targeted quantification. We also described the IIV for sncRNA, including microRNA, piwi-interacting RNA, tRNA, and tRNA-derived small RNA (tsRNA) in EVs (409 sncRNA and 78 tsRNA) and in spermatozoa (265 sncRNA and 15 tsRNA). We identified 22 and 27 non-overlapping proteins in SP and EVs, respectively, and 46 and 9 sncRNA, including 5 and 0 tsRNA in seminal EVs and spermatozoa, respectively, with low variability. The fatty acid synthase (FAS) had the lowest IIV in both media in targeted protein quantification.
CONCLUSIONS: We identified a number of proteins and sncRNA with low variability among 111 proteins, 176 sncRNA, and 12 tsRNA which were previously suggested as biomarkers of male fertility and reproductive outcomes: lactotransferrin, cysteine-rich secretory protein 3, alpha-1-antichymotrypsin, epididymal sperm-binding protein 1, glutathione S-transferase Mu 3, alpha-1-acid glycoprotein 2, serum amyloid P-component, aminopeptidase N, neprilysin, FAS, and miR-10b-3p, miR-122-5p, miR-205-5p, miR-222-3p, miR-34c-5p, miR-509-3-5p, miR-888-5p, miR-892a, miR-363-3p, miR-941, miR-146a-5p, miR-744-5p.
CONCLUSIONS: These molecules have low IIV and may be promising candidate biomarkers of male fertility and reproductive health.

There is scarce information about the effect of sperm morphology and seminal plasma composition on cat semen freezability. Thus, this study aims to assess the effect of cat sperm morphology and seminal plasma cholesterol (CHOL) and triacylglyceride (TAG) concentrations on sperm post-thaw survival. Ejaculates (n = 49) were evaluated, and seminal plasma was separated and frozen until CHOL and TAG concentrations were measured. The sperm pellet was diluted in a tris-based egg yolk extender, frozen (n = 38), or processed for sperm ultrastructure study (n = 11). Abnormalities recorded were abnormal head shape and size, detached heads, knobbed or ruffled acrosomes, eccentric mid-piece insertion, proximal and distal cytoplasmic droplets, folded and coiled tails, and Dag defect. Ultramicroscopic evaluation detected several sperm abnormalities in fresh semen and some sperm damage in frozen semen. Seminal plasma lipids components were positively correlated with post-thaw motility and acrosome integrity. Higher freezability indices for motility and acrosome integrity were observed in frozen-thawed semen with high seminal plasma CHOL and TAG concentrations. No freezability differences were observed between teratozoospermic and normozoospermic ejaculates. Our results showed that even when seminal plasma was removed before cryopreservation, sperm survival after thawing was significantly higher in samples with high seminal plasma CHOL and TAG concentrations, indicating a rapid adherence to these compounds to the sperm plasma membrane, protecting sperm cells from temperature changes. Nevertheless, there were no differences in sperm freezability by sperm morphology.

UNASSIGNED: Testicular cancer (TC) is a relatively rare type of cancer in men. Early diagnosis of TC remains challenging. Metabolomics holds promise in offering valuable insights in this regard. In this study, a metabolic fingerprinting approach was employed to identify potential biomarkers in both serum and seminal plasma of TC patients.
UNASSIGNED: A total of 9 patients with testicular cancer and 10 controls were included in the study. The metabolic fingerprinting approach was utilized as a rapid diagnostic tool to analyze the metabolome in serum and seminal plasma of TC patients in comparison to fertile men. Raman spectroscopy was applied for the analysis of metabolites in these biological samples.
UNASSIGNED: Principal component analysis (PCA) and functional group analysis showed that the differentiation between serum samples from healthy men and TC patients was not possible. However, when analyzing seminal plasma, a significant difference was found between the two groups (p<0.05). Functional group analysis of serum only showed an increase in tryptophan concentration ratio in TC patients as compared to healthy men (p=0.03). In contrast, in seminal plasma of TC patients, this increase was observed in all analyzed compounds, including phenylalanine, tyrosine, lipids, proteins, phenols (p<0.001).
UNASSIGNED: Our study highlights the potential of metabolic fingerprinting as a fast diagnostic tool for screening TC patients, with seminal plasma serving as a valuable biological sample. Furthermore, several potential biomarkers, particularly phenylalanine, were identified in seminal plasma. This research contributes to our understanding of TC pathogenesis and has the potential to pave the way for early detection and personalized treatment approaches.

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.