Plasticity in moth mating behavior and ejaculate proteomic diversity induced by male competition.

In male competition, large and costly ejaculates are advantageous. Prior research on male accessory gland secretions in Plutella xylostella left open questions about how males modulate their mating behaviors and ejaculate composition allocation in response to varying levels of competition. The current study aimed to delve deeper into these unexplored facets. A totally of 928 ejaculate proteins were identified across males exposed to different competition conditions. Notably, males courting under non-, low-, and high-competition scenarios exhibited 867, 635, and 858 ejaculate proteins, respectively. Approximately 10% of these ejaculate proteins displayed variations that aligned with changes in competition intensity. Subsequent analyses focused on the proteins transferred to females, revealing that 44% of ejaculate proteins were transferred, with 37 proteins exhibiting differential expression. Functional analyses uncovered their crucial roles in sperm maturation, motility, and capacitation. Our findings reveal adaptive adjustments in ejaculate protein abundance and transmission in P. xylostella as a response to varying competition levels. Moreover, fluorescent sperm labeling indicated higher sperm transfer during low competition correlated with shorter sperm length. Furthermore, evidence suggests that males shorten their courtship duration and extend their mating duration when faced with competition. These results illustrate how competition drives ejaculate investment and behavioral plasticity, offering valuable insights for advancements in assisted reproductive technologies and pest management strategies.

Small and Large Extracellular Vesicles of Porcine Seminal Plasma Differ in Lipid Profile.

Seminal plasma contains a heterogeneous population of extracellular vesicles (sEVs) that remains poorly characterized. This study aimed to characterize the lipidomic profile of two subsets of differently sized sEVs, small (S-) and large (L-), isolated from porcine seminal plasma by size-exclusion chromatography and characterized by an orthogonal approach. High-performance liquid chromatography-high-resolution mass spectrometry was used for lipidomic analysis. A total of 157 lipid species from 14 lipid classes of 4 major categories (sphingolipids, glycerophospholipids, glycerolipids, and sterols) were identified. Qualitative differences were limited to two cholesteryl ester species present only in S-sEVs. L-sEVs had higher levels of all quantified lipid classes due to their larger membrane surface area. The distribution pattern was different, especially for sphingomyelins (more in S-sEVs) and ceramides (more in L-sEVs). In conclusion, this study reveals differences in the lipidomic profile of two subsets of porcine sEVs, suggesting that they differ in biogenesis and functionality.

Looking into the Quantification of Forensic Samples with Real-Time PCR.

The quantification of human DNA extracts from forensic samples plays a key role in the forensic genetics process, ensuring maximum efficiency and avoiding repeated analyses, over-amplified samples, or unnecessary examinations. In our laboratory, we use the Quantifiler® Trio system to quantify DNA extracts from a wide range of samples extracted from traces (bloodstains, saliva, semen, tissues, etc.), including swabs from touched objects, which are very numerous in the forensic context. This method has been extensively used continuously for nine years, following an initial validation process, and is part of the ISO/IEC 17025 accredited method. In routine practice, based on the quantitative values determined from the extracts of each trace, we use a standard method or a low-copy-number method that involves repeating the amplification with the generation of a consensus genetic profile. Nowadays, when the quantification results are less than 0.003 ng/µL in the minimum extraction volume (40 µL), we do not proceed with the DNA extract analysis. By verifying the limits of the method, we make a conscious cost-benefit choice, in particular by using the least amount of DNA needed to obtain sufficiently robust genetic profiles appropriate for submission to the Italian DNA Forensic Database. In this work, we present a critical re-evaluation of this phase of the method, which is based on the use of standard curves obtained from the average values of the control DNA analysed in duplicate. Considering the various contributions to uncertainty that are difficult to measure, such as manual pipetting or analytical phases carried out by different operators, we have decided to thoroughly investigate the contribution of variability in the preparation of calibration curves to the final results. Thus, 757 samples from 20 independent experiments were re-evaluated using two different standards for the construction of curves, determining the quantitative differences between the two methods. The experiments also determined the parameters of the slope, Y-intercept, R2, and the values of the synthetic control probe to verify how these parameters can provide information on the final outcome of each analysis. The outcome of this revalidation demonstrated that it is preferable to use quantification ranges rather than exact quantitative limits before deciding how to analyse the extracts via PCR or forgoing the determination of profiles. Additionally, we present some preliminary data related to the analysis of samples that would not have been analysed based on the initial validation, from which genetic profiles were obtained after applying a concentration method to the extracts. Our goal is to improve the accredited analytical method, with a careful risk assessment as indicated by accreditation standards, ensuring that no source of evidence is lost in the reconstruction of a criminal event.

Identification of the body fluid donor in mixtures through target mRNA cSNP sequencing.

In forensic practice, mixture stains containing various body fluids are common, presenting challenges for interpretation, particularly in multi-contributor mixtures. Traditional STR profiles face difficulties in such scenarios. Over recent years, RNA has emerged as a promising biomarker for body fluid identification, and mRNA polymorphism has shown excellent performance in identifying body fluid donors in previous studies. In this study, a massively parallel sequencing assay was developed, encompassing 202 coding region SNPs (cSNPs) from 45 body fluid/tissue-specific genes to identify both body fluid/tissue origin and the respective donors, including blood, saliva, semen, vaginal secretion, menstrual blood, and skin. The specificity was evaluated by examining the single-source body fluids/tissue and revealed that the same body fluid exhibited similar expression profiles and the tissue origin could be identified. For laboratory-generated mixtures containing 2-6 different components and mock case mixtures, the donor of each component could be successfully identified, except for the skin donor. The discriminatory power for all body fluids ranged from 0.997176329 (menstrual blood) to 0.99999999827 (blood). The concordance of DNA typing and mRNA typing for the cSNPs in this system was also validated. This cSNP typing system exhibits excellent performance in mixture deconvolution.

Seminal plasma proteomics as putative biomarkers for male infertility diagnosis.

Male infertility represents a significant global public health issue that is currently emerging as a prominent research focus. Presently, laboratories adhere to the guidelines outlined by the World Health Organization (WHO) manuals for conducting routine semen analysis to diagnose male infertility. However, the accuracy of results in predicting sperm quality and fertility is limited because some individuals with a normal semen analysis report, an unremarkable medical history, and a physical examination may still experience infertility. As a result, the importance of employing more advanced techniques to investigate sperm function and male fertility in the treatment of male infertility and/or subfertility becomes apparent. The standard test for evaluating human semen has been improved by more complex tests that look at things like reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), sperm DNA fragmentation levels, DNA compaction, apoptosis, genetic testing, and the presence and location of anti-sperm antibodies. Recent discoveries of novel biomarkers have significantly enriched our understanding of male fertility. Moreover, the notable biological diversity among samples obtained from the same individual complicates the efficacy of routine semen analysis. Therefore, unraveling the molecular mechanisms involved in fertilization is pivotal in expanding our understanding of factors contributing to male infertility. By understanding how these proteins work and what role they play in sperm activity, we can look at the expression profile in men who can't have children to find diagnostic biomarkers. This review examines the various sperm and seminal plasma proteins associated with infertility, as well as proteins that are either deficient or exhibit aberrant expression, potentially contributing to male infertility causes.

Exploring correlations: Human seminal plasma and blood serum biochemistry in relation to semen quality.

Male infertility is a pressing global issue, prompting the need for biomarkers correlating with seminal parameters for diagnosis. Our study investigated 10 biochemical and energetic parameters in the seminal plasma and blood sera of fertile (25 subjects) and infertile (88 subjects) Polish men, correlations between their levels in seminal plasma and semen quality, and correlations between blood sera and seminal plasma levels of examined parameters. Infertile men displayed elevated seminal plasma glucose and fructose but reduced HDL levels compared to fertile men. We observed also weak negative correlations between seminal plasma triglycerides and sperm concentration in both groups. Moreover, infertile men exhibited positive correlations between seminal plasma HDL/LDL concentrations and sperm concentration. Fertile men showed moderate negative correlations between glucose/triglycerides concentrations and sperm count and between seminal plasma triglycerides levels and sperm vitality. Semen volume correlated with triglycerides (negative) and fructose (positive) concentrations in infertile men. Sperm motility correlated negatively with total cholesterol, LDL, and triglycerides concentrations in fertile men, and weakly with AMP-activated protein kinase in infertile men. Weak negative correlations between seminal plasma fructose/AMP-activated protein kinase concentrations and sperm progressive motility were observed in infertile men, whereas in fertile men seminal plasma AMP-activated protein kinase levels were positively correlated with progressive motility. Correlation analysis between blood serum and seminal plasma parameters revealed intriguing connections, notably regarding LDL, AMP-activated protein kinase, and carnitine, suggesting systemic influences on seminal plasma composition. These findings emphasize the complex interplay between metabolic factors and sperm parameters, offering promising directions for future research in male infertility diagnostics and therapeutics.

Dataset of seminal plasma proteome of Nellore bulls with high and low percentage of abnormalities sperm.

OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.

Evaluation of equine semen frozen in extenders free of egg yolk using two different freezing curves.

A chemically defined cryopreservation extender that maintains seminal parameters is relevant. Fifteen ejaculates from 5 stallions (n= 5; r=3) were diluted in 5 extenders: 1) EDTA-glucose based extender with egg-yolk and dimethylformamide (EY); 2) commercial equine extender (CE); 3) CE with dimethylformamide (CE-3); 4) bovine commercial extender with liposomes (OP); 5) bovine commercial extender with soybean lecithin (BIO), and frozen using a slow and a rapid temperature descent curve. Post-thaw evaluations were: sperm kinematic parameters, viability and acrosome status, membrane lipoperoxidation and DNA fragmentation. Sperm data were analysed using an ANOVA or Friedman test (results mean ± SD). Paired comparison between the two freezing curves was analysed using the Wilcoxon test. Total and progressive motility were significantly higher (P<0.05) in the EY and CE-3 samples using the slow curve, whereas for the fast curve, total and progressive motility were significantly higher (P<0.05) in the EY samples compared to all the extenders and the samples frozen in CE-3 were significantly higher than the remaining extenders (P<0.05). The percentages of live acrosome intact sperm and of live non-peroxidized sperm were significantly higher (P<0.05) in the EY extender when using either of the freezing curves and in turn, were significantly higher (P<0.05) in samples frozen in CE-3 compared to the remaining extenders. Intact DNA was significantly lower (P<0.05) in the BIO extender, using the rapid curve. To conclude, the commercial equine extender with 3% dimethylformamide, without egg-yolk, could be a suitable alternative for extenders with egg-yolk.

The importance of male underwear in cases of alleged sexual assault.

The potential evidential value of male underwear in cases of alleged sexual assault is often overlooked. Male underwear can be a critical item in the investigation of alleged sexual assaults. Body fluids/DNA, which may transfer to the penis during sexual contact, may in turn transfer to the inside front of the underwear, and persist for months or years, provided the underwear are not washed. Here, we demonstrate how the case circumstances drive the sampling strategy of male underwear, in order to maximize the effectiveness of the forensic analysis. Sampling considerations including recovery methods and sampling sequence are discussed, and a methodical examination strategy of male underwear is proposed. To highlight the pertinence of male underwear to the investigation of alleged sexual assaults, three real-life cases are discussed, in which male underwear were examined for multiple body fluids/DNA, and the findings obtained proved evidentially significant. The different cases demonstrate the versatility of male underwear examination in situations, where different body fluids and DNA may transfer based on the specific allegation, and emphasize how targeted sampling can allow the scientist to assess the probability of the findings based on two competing propositions. Accurate sampling strategies are imperative for robust probability assignment in evaluative reporting of scientific findings.

A green analytical method for the simultaneous determination of 17 perfluoroalkyl substances (PFAS) in human serum and semen by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS).

The ubiquity of perfluoroalkyl substances has raised concerns about the unintended consequences of PFAS exposure on human health. In the present study, an eco-friendly ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the simultaneous determination of 17 PFAS in human serum and semen samples. QuEChERS salts MgSO4:NaCl 4:1 (w/w) were used for the extraction. The separation of analytes was performed on an ACQUITY BEH C18 column (100 × 2.1 mm, 1.7 µm), using water:methanol 95:5 and methanol as mobile phases A and B, respectively, both containing 2 mM ammonium acetate. Multiple reaction monitoring (MRM) in negative ion mode was used, selecting two transitions for each analyte, except for perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA). The analytical method was validated according to the Organization of Scientific Area Committees (OSAC) for Forensic Sciences guidelines and AGREE approach software was used to evaluate the greenness of the method. The developed procedure was applied to the analysis of 10 paired human serum and semen samples, proving the suitability in high throughput laboratories due to the easy preparation and the reduced volume of toxic solvents. Moreover, it allows to perform further investigation on the correlation between serum and semen PFAS concentration, focusing on male reproductive system correlated pathologies, such as male infertility.

Sperm melatonin receptors, seminal plasma melatonin and semen freezability in goats.

Goat bucks are seasonal breeders that show variation in sperm quality, endogenous melatonin (MLT), and presumably in the expression of MLT receptors on the sperm throughout the year, which may modify sperm freezability. The aim of this study was to determine whether sperm freezability is associated with (i) endogenous melatonin levels in seminal plasma and (ii) the expression of sperm plasma membrane melatonin receptors (MT1, MT2). To evaluate this, spermatozoa from seven Saanen goat bucks were cryopreserved throughout the year in Mexico using a standard freezing protocol. Seminal plasma MLT concentrations were determined by ELISA and the expression and localization of MT1 and MT2 were detected by immunocytochemistry and confirmed by western blotting. The recovery rate of progressive motility after thawing was higher in spring than autumn and winter; in contrast, the F pattern (CTC assay) was higher in winter than in the other seasons. A proportional increase in the AR pattern (CTC assay) was smaller in winter than in the other seasons and the proportion of sperm showing high plasma membrane fluidity was higher in spring than in summer and autumn. The seminal plasma MLT concentrations showed no significant interseasonal differences. The MT1 receptor was immunolocalised at the apical region of the sperm head, while MT2 was mainly localised in the neck. The relative expression of MLT receptors showed significant differences between summer and winter for all bands, except at 75 kDa of MT2. In conclusion, there was an association between the relative expression of MT1 and MT2 receptors throughout the year and sperm freezability in goat bucks in México. Post-thaw sperm quality is enhanced in semen samples collected during breeding season.

Prevalence and implications of microplastic contaminants in general human seminal fluid: A Raman spectroscopic study.

Microplastics are ubiquitous environmental contaminants that have been detected in human semen from polluted areas, yet their prevalence and effects in the general population remain largely unexplored. To examine microplastic presence, abundance, polymer types, and associations with semen quality parameters in individuals without occupational exposures, this study was conducted by collecting semen samples from 40 participants undergoing premarital health assessments in Jinan, China. Raman microspectroscopy was employed to identify, quantify, and categorize microplastic polymers, sperm motility was assessed via computer-assisted analysis, and morphology was evaluated through Diff-Quik staining. Correlations between demographics, semen parameters, and microplastic content were examined by statistical analysis. We found that microplastics were detected in all semen samples, with 2 particles per sample (ranging from 0.72 to 7.02 µm). Eight distinct polymers were identified, with polystyrene (31 %) being most prevalent. Semen exposed to polystyrene demonstrated higher sperm progressive motility as compared to polyvinyl chloride exposure group (43.52 ± 14.21 % vs 19.04 ± 13.46 %). Sperm morphological abnormalities were observed but not significantly associated with specific plastic types. In conclusion, this study reveals microplastic contamination in semen from individuals without occupational exposure, with PS, PE, and PVC being the most prevalent and exhibiting differential correlations with sperm progressive motility, and highlight the need for further research into the potential reproductive impacts of microplastic exposure.

Identification and Quantification of Human Relaxin Proteins by Immunoaffinity-Mass Spectrometry.

The human relaxins belong to the Insulin/IGF/Relaxin superfamily of peptide hormones, and their physiological function is primarily associated with reproduction. In this study, we focused on a prostate tissue-specific relaxin RLN1 (REL1_HUMAN protein) and a broader tissue specificity RLN2 (REL2_HUMAN protein). Due to their structural similarity, REL1 and REL2 proteins were collectively named a 'human relaxin protein' in previous studies and were exclusively measured by immunoassays. We hypothesized that the highly selective and sensitive immunoaffinity-selected reaction monitoring (IA-SRM) assays would reveal the identity and abundance of the endogenous REL1 and REL2 in biological samples and facilitate the evaluation of these proteins for diagnostic applications. High levels of RLN1 and RLN2 transcripts were found in prostate and breast cancer cell lines by RT-PCR. However, no endogenous prorelaxin-1 or mature REL1 were detected by IA-SRM in cell lines, seminal plasma, or blood serum. The IA-SRM assay of REL2 demonstrated its undetectable levels (<9.4 pg/mL) in healthy control female and male sera and relatively high levels of REL2 in maternal sera across different gestational weeks (median 331 pg/mL; N = 120). IA-SRM assays uncovered potential cross-reactivity and nonspecific binding for relaxin immunoassays. The developed IA-SRM assays will facilitate the investigation of the physiological and pathological roles of REL1 and REL2 proteins.

Associations between seminal plasma metals/metalloids and semen quality: A cross-sectional study.

Exposure to metals/metalloids is reported to potentially influence semen quality. While most studies have focused on single metal impacts, the link between exposure to multiple metals and semen quality has remained less explored. The study aimed to investigate the effects of both individual and mixed metal/metalloid exposure on semen quality. A total of 330 men were recruited from three reproductive centers in eastern China. Seminal plasma levels of 25 metals/metalloids and sperm parameters were determined. We used the Generalized Linear Model (GLM) and Restricted Cubic Spline (RCS) to assess the relationships between single metals/metalloids and semen quality. The weighted quantile sum (WQS) models were then applied to evaluate the combined effect of all these metals/metalloids. We observed positive associations of exposure to lithium (Li), zinc (Zn), and magnesium (Mg) with an increased risk of below reference values for progressive motility and total motility using a logistic regression model (P < 0.05). Additionally, our results also revealed a significant inverse relationship between aluminum (Al) and both sperm concentration and count, while cobalt (Co) demonstrated a positive association with sperm concentration (P < 0.05). Notably, the WQS model indicated a significant positive association between exposure to metal/metalloid mixtures and the risk of abnormal progressive motility (OR: 1.57; 95%CI: 1.10, 2.24) and abnormal total motility (OR: 1.53; 95%CI: 1.06, 2.19), with this association primarily driven by Li, Mg, and Zn. In summary, our findings indicate that exposure to metal/metalloid mixtures might have an adverse effect on semen quality.

Lipidomics random forest algorithm of seminal plasma is a promising method for enhancing the diagnosis of necrozoospermia.

BACKGROUND: Despite the clear clinical diagnostic criteria for necrozoospermia in andrology, the fundamental mechanisms underlying it remain elusive. This study aims to profile the lipid composition in seminal plasma systematically and to ascertain the potential of lipid biomarkers in the accurate diagnosis of necrozoospermia. It also evaluates the efficacy of a lipidomics-based random forest algorithm model in identifying necrozoospermia. METHODS: Seminal plasma samples were collected from patients diagnosed with necrozoospermia (n = 28) and normozoospermia (n = 28). Liquid chromatography-mass spectrometry (LC-MS) was used to perform lipidomic analysis and identify the underlying biomarkers. A lipid functional enrichment analysis was conducted using the LION lipid ontology database. The top 100 differentially significant lipids were subjected to lipid biomarker examination through random forest machine learning model. RESULTS: Lipidomic analysis identified 46 lipid classes comprising 1267 lipid metabolites in seminal plasma. The top five enriched lipid functions as follows: fatty acid (FA) with ≤ 18 carbons, FA with 16-18 carbons, monounsaturated FA, FA with 18 carbons, and FA with 16 carbons. The top 100 differentially significant lipids were subjected to machine learning analysis and identified 20 feature lipids. The random forest model identified lipids with an area under the curve > 0.8, including LPE(20:4) and TG(4:0_14:1_16:0). CONCLUSIONS: LPE(20:4) and TG(4:0_14:1_16:0), were identified as differential lipids for necrozoospermia. Seminal plasma lipidomic analysis could provide valuable biochemical information for the diagnosis of necrozoospermia, and its combination with conventional sperm analysis may improve the accuracy and reliability of the diagnosis.

Proteomic biomarkers in seminal plasma as predictors of reproductive potential in azoospermic men.

Introduction: Azoospermia, characterized by an absence of sperm in the ejaculate, represents the most severe form of male infertility. While surgical sperm retrieval in obstructive azoospermia (OA) is successful in the majority of cases, patients with non-obstructive azoospermia (NOA) show retrieval rates of only about 50% and thus frequently have unnecessary surgery. Surgical intervention could be avoided if patients without preserved spermatogenesis are identified preoperatively. This prospective study aimed to discover biomarkers in seminal plasma that could be employed for a non-invasive differential diagnosis of OA/NOA in order to rationalize surgery recommendations and improve success rates. Methods: All patients signed written informed consent, underwent comprehensive andrological evaluation, received human genetics to exclude relevant pathologies, and patients with azoospermia underwent surgical sperm retrieval. Using label-free LC-MS/MS, we compared the proteomes of seminal plasma samples from fertile men (healthy controls (HC), n=8) and infertile men diagnosed with 1) OA (n=7), 2) NOA with successful sperm retrieval (mixed testicular atrophy (MTA), n=8), and 3) NOA without sperm retrieval (Sertoli cell-only phenotype (SCO), n=7). Relative abundance changes of two candidate markers of sperm retrieval, HSPA2 and LDHC, were confirmed by Western Blot. Results: We found the protein expression levels of 42 proteins to be significantly down-regulated (p ≤ 0.05) in seminal plasma from SCO NOA patients relative to HC whereas only one protein was down-regulated in seminal plasma from MTA patients. Analysis of tissue and cell expression suggested that the testis-specific proteins LDHC, PGK2, DPEP3, and germ-cell enriched heat-shock proteins HSPA2 and HSPA4L are promising biomarkers of spermatogenic function. Western blotting revealed a significantly lower abundance of LDHC and HSPA2 in the seminal plasma of men with NOA (SCO and MTA) compared to controls. Discussion: The results indicate that certain testis-specific proteins when measured in seminal plasma, could serve as indicators of the presence of sperm in the testis and predict the success of sperm retrieval. Used in conjunction with conventional clinical assessments, these proteomic biomarkers may assist in the non-invasive diagnosis of idiopathic male infertility.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

[Tumor necrosis factor in the ejaculate as an indicator of reduced fertility].

INTRODUCTION: Pro-inflammatory cytokine - tumor necrosis factor-alpha (TNF) is one of the components of the seminal plasma proteome; its meaning has not been definitively revealed. A comparative analysis of the concentration of this protein in the blood serum and in the ejaculate and changes in its level in the semen of men with infertility is f scientific interest. THE PURPOSE OF THE STUDY: determination of TNF- level in the blood serum and seminal plasma of healthy men and patients with reduced fertility. MATERIALS AND METHODS: 70 men of reproductive age with azoospermia (main group, n=18), with oligoastenozoospermia (comparison group, n=18) and with normal spermogram parameters (control group, n=34) were examined. The ejaculate was examined using an SQA-V semen analyzer (MES, Israel). In seminal plasma samples, the concentration of TNF was determined using the alpha-TNF-ELISA-BEST test system (A-8756, Vector-Best LL, Russia). RESULTS: The concentration of TNF- in blood serum had a significant variation (CV=85.31%) and amounted to 2.75+/-2.18 pg/ml, which is 2.55 times lower than the same indicator in seminal plasma (7.01+/-5.98 pg/ml, CV=126.15%, p<0.00001). When comparing the content of TNF- in seminal plasma, significant differences were found in the examined patients (Kruskal-Wallis test H=24.75991; p<0.00001). Pairwise comparison revealed a statistically significant difference in the level of TNF- in seminal plasma between the comparison and control groups (p2-3=0.000023), as well as between the main group and the comparison group (p1-2=0.000043); there were no significant differences between the main and control groups (p>0.05). When determining the content of TNF- in the blood serum, there was no statistically significant difference between the groups (p>0.05). There were no correlations between the concentration of TNF- in blood serum and in seminal plasma (R=0.295374), and the total number of spermatozoa in the ejaculate (R=-0.027945); and the concentration of spermatozoa in the ejaculate (R=-0.042902). DISCUSSION: It is unlikely that TNF crosses into seminal plasma from serum against a concentration gradient. It is most likely that TNF is produced locally in the organs of the reproductive system by resident immune cells or cells involved in spermatogenesis. An increased content of TNF- in seminal plasma in patients of the comparison group may indicate the presence of an inflammatory process in the reproductive system and a reduced fertility of the ejaculate. CONCLUSION: The physiological role of TNF in sperm, its sources in the organs of the male reproductive system, and the pathogenetic mechanisms of the participation of the TNF in pathological processes in male reproductive system still remain unclear. All this justifies the need for further study of the TNF level in seminal plasma in normal conditions and in diseases of the urogenital tract in men.

Effects of the Neuropeptides Pituitary Adenylate Cyclase Activating Polypeptide and Vasoactive Intestinal Peptide in Male Fertility.

Background and Objectives: The neuroendocrine system plays a crucial role in regulating various bodily functions, including reproduction, with evidence suggesting its significant involvement in male fertility and sperm development. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) are expressed in both male and female reproductive tissues, influencing penile erection and regulating steroidogenesis in males. Therefore, our study aimed to compare the protein levels of VIP and PACAP in seminal plasma between healthy controls and sub-fertile patients. Additionally, we sought to correlate the levels of these biomarkers with clinical, functional, and laboratory findings in the participants. Materials and Methods: The study included a total of 163 male participants for analysis. The participants were further stratified into subgroups of fertile and sub-fertile men of four subgroups according to the 2021 WHO guidelines. Seminal plasma concentrations of the neuropeptides VIP and PACAP were measured using human enzyme-linked immunosorbent assay technique. Results: The findings showed statistically significant differences in total sperm count, sperm concentration, total motility, and vitality (p < 0.001) between the fertile group and the sub-fertile group. Specifically, significant differences found between healthy males and oligoasthenospermic patients (p = 0.002), and between asthenospermic and oligoasthenospermic patients (p = 0.039). An ROC analysis showed associated sensitivity and specificity values of 62.2% and 55.6%, respectively, to PACAP seminal levels differentiated between sub-fertile patients from fertile males (p = 0.028). No significant difference in seminal levels of VIP was found between the sub-fertile and fertile groups. Conclusions: Previous research leads to the point of PACAP active involvement in spermatogenesis. In accordance to our study, in human semen samples, we have seen a significance change in PACAP levels amongst patients with low sperm count or with both low sperm count and low motility, hinting at its contribution and acting as a possible factor in this complex process. Thus, alterations in the levels or actions of these neuropeptides have been associated with certain reproductive disorders in males.

Developing an interpretation model for body fluid identification.

Criminal investigations, particularly sexual assaults, frequently require the identification of body fluid type in addition to body fluid donor to provide context. In most cases this can be achieved by conventional methods, however, in certain scenarios, alternative molecular methods are required. An example of this is the detection of menstrual fluid and vaginal material, which are not able to be identified using conventional techniques. Endpoint reverse-transcription PCR (RT-PCR) is currently used for this purpose to amplify body fluid specific messenger RNA (mRNA) transcripts in forensic casework. Real-time quantitative reverse-transcription PCR (RT-qPCR) is a similar method but utilises fluorescent markers to generate quantitative results in the form of threshold cycle (Cq) values. Despite the uncertainty surrounding body fluid identification, most interpretation guidelines utilise categorical statements. Probabilistic modelling is more realistic as it reflects biological variation as well as the known performance of the method. This research describes the application of various machine learning models to single-source mRNA profiles obtained by RT-qPCR and assesses their performance. Multinomial logistic regression (MLR), Naïve Bayes (NB), and linear discriminant analysis (LDA) were used to discriminate between the following body fluid categories: saliva, circulatory blood, menstrual fluid, vaginal material, and semen. We identified that the performance of MLR was somewhat improved when the quantitative dataset of the original Cq values was used (overall accuracy of approximately 0.95) rather than presence/absence coded data (overall accuracy of approximately 0.94). This indicates that the quantitative information obtained by RT-qPCR amplification is useful in assigning body fluid class. Of the three classification methods, MLR performed the best. When we utilised receiver operating characteristic curves to observe performance by body fluid class, it was clear that all methods found difficulty in classifying menstrual blood samples. Future work will involve the modelling of body fluid mixtures, which are common in samples analysed as part of sexual assault investigations.