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Background: Gold nanoclusters (AuNCs) obtained by electroreduction have excellent electrochemiluminescence (ECL) properties, and its ECL intensity is regulated by the valence state. In addition, their ECL signals can be rapidly quenched by reactive oxygen species (ROS). Based on this observation, a sensitive ROS biosensor was designed based on valence regulation of AuNCs. Excessive ROS in seminal plasma can lead to male infertility, and the short half-life and instability of ROS pose a challenge for their detection. Since valence regulation can be done quickly and is very sensitive, this ECL biosensor holds promise to address this issue., Results: The ECL mechanism of AuNCs and the quenching mechanism of AuNCs by ROS were explored, mainly because ROS change the valence state of AuNCs. The ECL signals of the biosensor have a linear relationship with logarithm of the target concentration in the range of 1.0 × 10 -8 to 1.0 × 10 -3  M and 1.0 × 10 -3 to 1.0 × 10 -1  M, with a detection limit of 0.75 × 10 -10  M (S/N = 3). The biosensor enables rapid one-step detection of ROS and has the advantage of being stable and reusable. More notably, the results of 57 real samples showed that the biosensor can be used to accurately assess the concentration of seminal plasma ROS, guiding the monitoring of sperm quality and the diagnosis of male infertility., Significance: Compared with the traditional strategy of applying AuNCs only as a luminescent body, this strategy of regulating the valence state of AuNCs to achieve sensitive and rapid detection broadens the application of AuNCs in the field of analysis. Compared with other ROS detection strategies, the one-step immediate detection method effectively avoids the inaccuracy caused by the short half-life and natural dissipation of ROS, and is expected to improve the accuracy and efficiency of clinical diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Competing Interests: Declaration of competing interest The author declares that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objective: This randomized clinical trial study aims to investigate the effects of antioxidant food supplementation on the total antioxidant capacity of seminal plasma, sperm DNA fragmentation, sperm chromatin quality, and semen parameters., Methods: In this study, a total of 48 subfertile men with moderate physical activity were included. Group 1 was recommended to use the antioxidant supplements, while antioxidant food supplements were not given to Group 2. Total antioxidant capacity, sperm DNA fragmentation, sperm chromatin structure, hormone levels, physical activities, and semen parameters were evaluated before and after treatment. Total antioxidant capacity, sperm DNA fragmentation, and sperm chromatin structure were assessed using ELISA, transferase dUTP nick end labeling, and aniline blue staining, respectively., Results: Sperm DNA fragmentation (p=0.003) and histone/protamine ratio (p<0.001) were significantly decreased in the patients receiving antioxidant treatment. There was no statistical difference in the total antioxidant capacity values of the post-treatment groups., Conclusion: Antioxidant therapy seems to improve sperm DNA fragmentation and histone/protamine ratios in subfertile patients., Clinical Trial Registration Number: NCT06042738.

Objective: This study looked at how different concentrations of curcumin (Curc), zinc chloride (ZnCl 2 ), zinc oxide nanoparticles (ZnO-NPs) and Curc loaded on ZnO-NPs (Curc-co-ZnO-NPs) in cryopreservation dilution affected the quality of ram sperm after thawing., Methods: ZnO-NPs were synthesised using Berberis vulgaris leaf aqueous extract. Then, Curc was loaded on the ZnO-NPs that had been synthesised. We used analytical methods to look at the composition, morphology and size of green synthesised ZnO-NPs and Curc-co-ZnO-NPs, including UV-Vis, zeta potential, EDX, DLS, FE-SEM and FT-IR. Using a Tris-base extender containing various concentrations of Curc, ZnCl 2 , ZnO-NPs and Curc-co-ZnO-NPs (0, 1, 10 and 100 µg/mL), semen samples from four rams were combined. Sperm motility, viability, DNA and plasma membrane integrity, total abnormalities and malondialdehyde (MDA) generation were all evaluated in treatment groups after thawing., Results: The results showed that adding 1 µg/mL of ZnO-NPs and Curc-co-ZnO-NPs significantly reduced the level of MDA and total abnormalities (p < 0.05). Additionally, following the freeze-thawing procedure, the presence of 1 µg/mL of Curc-co-ZnO-NPs in the diluent of ram sperm significantly increased the percentage of sperm viability and motility in comparison to the control and other treatment groups (p < 0.05). Furthermore, as compared to the control group and other treatments, treatments containing 1 µg/mL of Curc-co-ZnO-NPs significantly improved membrane and DNA integrity (p < 0.05)., Conclusions: It appears that following freeze-thawing, the Curc-co-ZnO-NPs (1 µg/mL) enhanced sperm parameters., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

In cases of sexual assaults involving children, biological evidence such as semen is often found on clothes rather than on intimate swabs. Two cases involving young girls were submitted to one of the AFSP BFF forensic science laboratories instigating further research into the background levels of semen on children's underwear after being laundered, without sexual acts occurring. This study considered the potential for background levels of semen in the washing machine and the levels of semen transferred with varying laundry storage and washing arrangements. The results concur with previous studies that low levels of spermatozoa can transfer during washing; however, this study demonstrated that background levels of semen can be present on children's clothing during domestic laundering activities which include male underwear when there are sexually active males within the household. Further, that semen can persist in the washing machine even after three washes. This study has produced data which will assist forensic scientists to evaluate cases of child abuse in a domestic setting., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 Elsevier B.V. All rights reserved.)

In this study, it was aimed to evaluate the effect of seminal plasma activity levels of enzymatic antioxidants such as catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), non-enzymatic antioxidant alpha-tocopherol (Vit-E), and also total antioxidant status (TAS), total oxidant status (TOS), and oxidative stress index (OSI) on post-thaw sperm quality. As well as it was aimed to investigate the possibility of the use of OSI as a marker for the estimation of bull semen freezability. For this study, 72 ejaculates were collected from 6 bulls and separated into two aliquots. The first one was centrifuged to separate seminal plasma. The latter one was cryopreserved and stored in liquid nitrogen until analysis. Post-thaw semen quality was examined in two groups (good-freezable semen (GFS) and poor-freezable semen (PFS)) through cluster analyses based on post-thaw total motility and plasma membrane and acrosome integrity. As a result of the analyses, seminal TAS, CAT, and Vit-E values were higher (P<0.05) in the GFS group, while TOS and OSI values were higher (P<0.01) in the PFS group. We also performed an ROC curve analysis to determine whether the seminal OSI value could be used to predict semen freezeability. The area under curve (AUC) value was found as 0,70 (P=0.006). In conclusion, it has been revealed that the seminal plasma antioxidant content is responsible for the freezability of semen, and the OSI value, which can be determined by performing TAS and TOS analyses instead of looking for separate antioxidant enzymes, can be used as a marker for the estimation of post-thaw semen quality at artificial insemination centers., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Operational forensic laboratories routinely perform immunological assays for detecting various body fluids. The ABAcard® p30 and HemaTrace® immunochromatographic tests from Abacus Diagnostics are used for detecting the p30 enzyme in human semen and human haemoglobin present in blood respectively. In early 2023, manufacturer modifications to the ABAcard® p30 and HemaTrace® tests resulted in a reduction in card size and volume of sample extract used in the recommended protocol. This change in card design and/or the reduced volume of sample extract may alter the sensitivity of the test. This study established and compared the limit of detection (LOD) for the old and newly modified ABAcard® p30 and HemaTrace® test cards. The LOD values showed that the new test cards were approximately 2.4-fold (HemaTrace® test) and 3.4-fold (p30 test) more sensitive than the old cards. Additionally, it was found that the new HemaTrace® test cards were more susceptible to the high dose hook effect. In response to the increased sensitivity, existing data pertaining to the reactivity of these test cards to non-target body fluid and substances warrants re-investigation to ensure positive results are interpreted correctly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Hyperspectral imaging (HSI) has become a crucial innovation in forensic science, particularly for analysing bodily fluids. This advanced technology captures both spectral and spatial data across a wide spectrum of wavelengths, offering comprehensive insights into the composition and distribution of bodily fluids found at crime scenes. In this review, we delve into the forensic applications of HSI, emphasizing its role in detecting, identifying, and distinguishing various bodily fluids such as blood, saliva, urine, vaginal fluid, semen, and menstrual blood. We examine the benefits of HSI compared to traditional methods, noting its non-destructive approach, high sensitivity, and capability to differentiate fluids even in complex mixtures. Additionally, we discuss recent advancements in HSI technology and their potential to enhance forensic investigations. This review highlights the importance of HSI as a valuable tool in forensic science, opening new pathways for improving the accuracy and efficiency of crime scene analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in the manuscript., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The inference of body fluids and tissues is critical in reconstructing crime scenes and inferring criminal behaviors. Nevertheless, present methods are incompatible with conventional DNA genotyping, and additional testing might result in excessive consumption of forensic scene materials. This study aims to investigate the feasibility of distinguishing common body fluids/tissues through the difference in mitochondrial DNA copy number (mtDNAcn). Four types of body fluids/tissues were analyzed in this study - hair, saliva, semen, and skeletal muscle. MtDNAcn was estimated by dividing the read counts of mitochondrial DNA to that of nuclear DNA (RR mt/nu ). Results indicated that there were significant differences in RR mt/nu between different body fluids/tissues. Specifically, hair samples exhibited the highest RR mt/nu (log 10 RR mt/nu : 4.3 ± 0.28), while semen samples showed the lowest RR mt/nu (log 10 RR mt/nu : -0.1 ± 0.28). RR mt/nu values for DNA samples without extraction were notably higher (approximately 2.9 times) than those obtained after extraction. However, no significant difference in RR mt/nu was observed between various age and gender groups. Hierarchical clustering and Kmeans clustering analyses showed that body fluids/tissues of the same type clustered closely to each other and could be inferred with high accuracy. In conclusion, this study demonstrated that the simultaneous detection of nuclear and mitochondrial DNA made it possible to perform conventional DNA analyses and body fluid/tissue inference at the same time, thus killing two birds with one stone. Furthermore, mtDNAcn has the potential to serve as a novel and promising biomarker for the identification of body fluids/tissues., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively., Competing Interests: Declaration of Competing Interest All authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (2 7 dilution) or 0.0218 μL (2 6 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called adverse analytical finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e., samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. SIGNIFICANCE STATEMENT: This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Biological fluid stains can be instrumental in solving crimes. Identification of semen can help reconstruct events in sexual assault cases and identify suspects via DNA profiling. Current methods for semen identification suffer from limitations, including destruction of the sample and potential false positives. One of the main unsolved issues is the elimination of underlying substrate interference. In this paper, chemometric approaches were developed to isolate and identify a biofluid stain on interfering substrates using Raman spectroscopy. The first approach, called Multivariate Curve Resolution with the Addition Method, combines the standard addition method with multivariate curve resolution. The second one uses a criterion based on reducing the spectrum complexity when a spectral component is removed from a Raman spectrum of a multi-component sample entirely. The results demonstrate the superiority of the first approach relative to the second for both small volume fraction of the fluid stain compared to the substrate and random noise., (© 2024. The Author(s).)

Abstract: Seminal fluid extracellular vesicles (SFEVs) have previously been shown to interact with spermatozoa and influence their fertilisation capacity. Here, we sought to extend these studies by exploring the functional consequences of SFEV interactions with human spermatozoa. SFEVs were isolated from the seminal fluid of normozoospermic donors prior to assessing the kinetics of sperm-SFEV binding in vitro, as well as the effects of these interactions on sperm capacitation, acrosomal exocytosis, and motility profile. Biotin-labelled SFEV proteins were transferred primarily to the flagellum of spermatozoa within minutes of co-incubation, although additional foci of SFEV biotinylated proteins also labelled the mid-piece and head domain. Functional analyses of high-quality spermatozoa collected following liquefaction revealed that SFEVs did not influence sperm motility during incubation at pH 5, yet SFEVs induced subtle increases in total and progressive motility in sperm incubated with SFEVs at pH 7. Additional investigation of sperm motility kinematic parameters revealed that SFEVs significantly decreased beat cross frequency and increased distance straight line, linearity, straightness, straight line velocity, and wobble. SFEVs did not influence sperm capacitation status or the ability of sperm to undergo acrosomal exocytosis. Functional assessment of both high- and low-quality spermatozoa collected prior to liquefaction showed limited SFEV influence, with these vesicles inducing only subtle decreases in beat cross frequency in spermatozoa of both groups. These findings raise the prospect that, aside from subtle effects on sperm motility, the encapsulated SFEV cargo may be destined for physiological targets other than the male germline, notably the female reproductive tract., Lay Summary: A male's influence over the biological processes of pregnancy extends beyond the provision of sperm. Molecular signals present in the ejaculate can influence the likelihood of pregnancy and healthy pregnancy progression, but the identity and function of these signals remain unclear. In this study, we wanted to understand if nano-sized particles present in the male ejaculate, called seminal fluid extracellular vesicles, can assist sperm in traversing the female reproductive tract to access the egg. To explore this, we isolated seminal fluid extracellular vesicles from human semen and incubated them with sperm. Our data showed that seminal fluid extracellular vesicles act to transfer molecular information to sperm, but this resulted in only subtle changes to the movement of sperm.

Seminal fluid is rich in sugars, but their role beyond supporting sperm motility is unknown. In this study, we found Drosophila melanogaster males transfer a substantial amount of a phospho-galactoside to females during mating, but only half as much when undernourished. This seminal substance, which we named venerose, induces an increase in germline stem cells (GSCs) and promotes sperm storage in females, especially undernourished ones. Venerose enters the hemolymph and directly activates nutrient-sensing Dh44 + neurons in the brain. Food deprivation directs the nutrient-sensing neurons to secrete more of the neuropeptide Dh44 in response to infused venerose. The secreted Dh44 then enhances the local niche signal, stimulating GSC proliferation. It also extends the retention of ejaculate by females, resulting in greater venerose absorption and increased sperm storage. In this study, we uncovered the role of a sugar-like seminal substance produced by males that coordinates reproductive responses to nutritional challenges in females., (© 2024. The Author(s).)

The identification of tissue-specific differentially methylated regions has significantly contributed to the field of forensic genetics, particularly in body fluid identification crucial for linking evidence to crimes. Among the various approaches to analyzing DNA methylation, the SNaPshot assay has been popularly studied in numerous researches. However, there is a growing interest in exploring alternative methods such as the use of massively parallel sequencing (MPS), which can process a large number of samples simultaneously. This study compares SNaPshot and MPS multiplex assays using nine cytosine-phosphate-guanine markers for body fluid identification. As a result of analyzing 112 samples, including blood, saliva, vaginal fluid, menstrual blood, and semen, both methods demonstrated high sensitivity and specificity, indicating their reliability in forensic investigations. A total of 92.0% samples were correctly identified by both methods. Although both methods accurately identified all blood, saliva, and semen samples, some vaginal fluid samples showed unexpected methylation signals at nontarget loci in addition to the target loci. In the case of menstrual blood samples, due to their complexity, independent typing criteria were applied, and successful menstrual blood typing was possible, whereas a few samples showed profiles similar to vaginal fluid. The MPS method worked better in vaginal fluid samples, and the SNaPshot method performed better in menstrual blood samples. This study offers valuable insights into body fluid identification based on the characteristics of the SNaPshot and MPS methods, which may help in more efficient forensic applications., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

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., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Alginate is the general term of a polysaccharide which is widely used in the area of pharmaceutics and the food industry and is known for its unique biological activities. However, due to the low water solubility and large viscosity of alginate, its development and utilization in the agricultural field are limited. Alginate oligosaccharide (AOS) is a degradable product derived from alginate and has attracted much attention in recent years because of its specific characteristics such as a low molecular weight, high water solubility, and non-toxicity. Boar semen quality, which is affected by various factors, is an important indicator for measuring reproductive performance of boars. With the development of artificial insemination technology, high quality semen has been more and more important. Therefore, increasing semen quality is an important means to improve the reproductive performance in swine industry. In this research review, we used the PubMed database and Google Scholar and web of science to search for relevant literature on the topic of AOS in relation to boar semen quality. Key words used were alginate oligosaccharide, boars, semen quality, microbiota and metabolites. The purpose of this review article was to describe the current knowledge on the relationship between AOS and boar semen quality, and provide an overview of solutions for the decline in the boar semen quality in specific conditions. Based on the existing literature, it is evident that AOS can be used as a new type of food additive. This review paper provides a theoretical basis for the production of high-quality boar sperm and, suggests that, in the future, AOS can even aid in treating human infertility., Competing Interests: Declaration of competing interest The remaining authors declare that the manuscript was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In forensic investigations, semen samples are a common form of biological evidence, especially in cases involving sexual assault. Therefore, accurately estimating the age of an individual is crucial in criminal cases. This study presents a novel age estimation model based on semen-specific CpG methylation patterns. A multiplex panel was developed, consisting of 12 CpG sites (PARP14, C5orf25, cg23488376, MXRA5, PFKFB3, DLL1, NOX4, cg12837463, TTC7B, KCNA7, NKX2-1, and SYNE4), which exhibit strong correlations with age. Additionally, this study investigates the resilience of these methylation markers under simulated environmental challenges. We collected ejaculate samples from a diverse cohort of 115 male individuals, aged 20-71 years, who underwent deoxyribonucleic acid extraction and bisulfite conversion. Methylation levels of the selected CpG sites were assessed using a SNaPshot assay, which revealed significant correlations with chronological age. We developed and validated two robust age estimation models through stepwise and enter regression analyses, achieving reliable accuracy with mean absolute errors ranging from 3.81 to 4.1 years. Additionally, the study also investigated the robustness of semen stains under diverse environmental conditions, including fabric type, washing, hematin exposure, and UV-C light. The selected methylation markers demonstrated remarkable resilience despite the challenges posed by washing procedures and environmental exposure, confirming their potential for age estimation in forensic genetics. This research presents successful age estimation models, emphasizing the strong correlations between methylation levels and chronological age. The proposed methodology's accuracy is affirmed through model validation on an independent test set, while also highlighting the resilience of semen stains on fabrics under varying storage and washing conditions., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

In forensic investigations, identifying the type of body fluid allows for the interpretation of biological evidence at the activity level. Over the past two decades, significant research efforts have focused on developing molecular methods for this purpose. MicroRNAs (miRNAs) hold great promise due to their tissue-specific expression, abundance, lack of splice variants, and relative stability. Although initial findings are promising, achieving consistent results across studies is still challenging, underscoring the necessity for both original and replication studies. To address this, we selected 18 miRNA candidates and tested them on 6 body fluids commonly encountered in forensic cases: peripheral blood, menstrual blood, saliva, semen, vaginal secretion, and skin. Using reverse transcription quantitative PCR analysis, we confirmed eight miRNA candidates (miR-144-3p, miR-451a, miR-205-5p, miR-214-3p, miR-888-5p, miR-891a-5p, miR-193b-3p, miR-1260b) with high tissue specificity and four (miR-203a-3p, miR-141-3p, miR-200b-3p, miR-4286) with lesser discrimination ability but still contributing to body fluid differentiation. Through principal component analysis and hierarchical clustering, the set of 12 miRNAs successfully distinguished all body fluids, including the challenging discrimination of blood from menstrual blood and saliva from vaginal secretion. In conclusion, our results provide additional data supporting the use of a small set of miRNAs for predicting common body fluids in forensic contexts. Large population data need to be gathered to develop a body fluid prediction model and assess its accuracy., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Semen traces are considered important pieces of evidence in forensic investigations, especially those involving sexsual offenses. Recently, our research group developed a fluorescence-based technique to accurately determine the age of semen traces. However, the specific compounds resonsible for the fluoresescent behaviour of ageing semens remain unknown. As such, in this exploratory study, the aim is to identify the components associated with the fluorescent behavior of ageing semen traces. In this investigation semen stains and various biofluorophores commonly found in body fluids were left to aged for 0, 2, 4, 7, 14 and 21 days. Subsequently, thin-layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) mass spectrometry were performed to identify the biofluorophores present in semen. Several contributors to the autofluorescence could be identified in semen stain, these include tryptophan, kynurenine, kynurenic acid, and norharman. The study sheds light on the., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

In pig production, the optimization of artificial insemination (AI) efficiency significantly relies on the accurate assessment of semen quality and fertility of boars. Traditional methods such as conventional seminogram techniques, although long-standing, exhibit limited sensitivity in predicting boar fertility, warranting the exploration of novel molecular markers. This review synthesizes the current knowledge on the utilization of molecular markers for semen quality evaluation and male fertility prediction in boars, providing an in-depth examination of molecular markers in this context. Specifically, the present work delves into the potential of OMICs technologies, encompassing genetic and genomic approaches, transcriptomics, proteomics, and metabolomics. A diverse array of molecular markers, including genomic regions associated with sperm quality and male fertility, chromatin integrity, mitochondrial DNA content, mRNA and non-coding RNA signatures, as well as proteins and metabolites in sperm and seminal plasma, are identified as promising molecular markers for fertility prediction in boars. Furthermore, the need of validating biomarkers and their practical implementation in AI centres is here emphasized. Addressing these considerations and integrating molecular markers within the swine breeding field holds the potential to enhance reproductive management practices and optimize productivity in boar breeding programs. This integration can significantly improve overall efficiency within the pig breeding industry., Competing Interests: Declaration of Competing Interest The author declares no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Male infertility along with altered semen parameters have been related to smoking. Smoking-related elevations in serum and seminal lead (Pb) and arsenic (As) may play a role in mediating the toxic effects of smoking on seminogram. This research aims to determine whether smoking has any significant impact on Pb and As levels in the seminal plasma and serum, as well as on the various semen parameters, when compared to nonsmokers. In total, 80 adult males were included: 60 smokers and 20 age-matched nonsmokers. Based on the number of cigarettes smoked/day (CPD), the smokers were categorized into mild (1-10), moderate (11-20), and severe (> 20). The analysis of semen was conducted in accordance with the 2010 WHO laboratory manual. Using an atomic absorption spectrophotometer, Pb and As concentrations in the serum and seminal plasma of all groups were determined. Compared to nonsmokers, smokers had a significantly reduced sperm count, motility, and viability, as well as a larger percentage of aberrant forms (P = 0.001, 0.025, 0.034, 0.002 respectively). Smokers had higher Pb concentrations in their serum and seminal fluid than nonsmokers (P = 0.002, 0.001 respectively). Seminal Pb had a significant negative correlation with sperm count (P = 0.004, r = -0.320). Serum Pb levels were found to positively correlate with seminal Pb levels (P 0.001, r = 0.648), and cigarette smokers had substantially greater seminal As levels than nonsmokers (P = 0.024). Sperm viability was strongly inversely related to seminal As (P = 0.042, r = -0.264). Seminal As levels and aberrant sperm shapes were found to be significantly correlated (P = 0.001, r = 0.414). In smokers, a significant positive relationship between seminal As and seminal Pb was observed. Therefore, semen parameters could be adversely affected by smoking through high levels of Pb and As (P = 0.012, r = 0.298)., (© 2024. The Author(s).)

Seminal extracellular vesicles (SemEVs) are repositories of biomolecules, including metabolites involved in the regulation of sperm function. The correlation between the metabolite profile of SemEVs and semen parameters, along with their role in regulating sperm function, is an unexplored area. This preliminary study evaluated the metabolomic content of SemEVs. Semen samples were obtained from 18 healthy men, and SemEVs were extracted from seminal plasma using the size exclusion chromatography qEV Gen 2-35 nm column coupled with an automatic fraction collector. The physical characterization of SemEVs was carried out with the ZetaView PMX-430-Z QUATT laser system. EV protein markers were detected using Western blot. In addition, these SemEVs were used for metabolomic profiling and functional bioinformatic analysis. The mean concentration of isolated SemEVs was 1.7 ± 1.1 × 10 11 /mL of seminal plasma, whereas SemEVs size and zeta potential were 129.5 ± 5.5 nm and -40.03 ± 3.99 mV, respectively. Western blot analysis confirmed the presence of EV specific markers such as CD81, ALIX, and TSG101. A total of 107 metabolites were identified using this untargeted metabolomic approach in SemEVs. Bioinformatics analysis further revealed that metabolites associated with tyrosine metabolism were highly enriched in these SemEVs. Ingenuity Pathway Analysis (IPA) also indicated that these metabolites present in SemEVs were involved in the regulation of the free radical scavenging pathway. Furthermore, our metabolomic results suggest that these SemEV-associated metabolites may play a pivotal role in the maintenance of seminal plasma redox homeostasis.

The objective of this study was to compare the plasma (PL) and seminal plasma (SP) pharmacokinetic profile of ceftiofur (CEFT) and desuroylceftiofur acetamide (DFCA) after administration of CEFT crystalline-free acid (CCFA) by SC route in two sites of the ear in beef bulls. Four clinically healthy Hereford bulls received a comprehensive physical exam and subsequently a breeding-soundness examination, CBC, and chemistry profile panel. All bulls were diagnosed healthy and satisfactory potential breeders. In one group (n = 2), a single dose of CCFA was administered SC route at the base of the ear (BOE) at a dose of 6.6 mg/kg of body weight. The second group (n = 2) was also administered by SC route in the middle third of the posterior aspect of the ear (MTE). The concentrations of CEFT and DFCA in PL and SP were determined by a high-performance liquid chromatography mass spectrometry (HPLC-MS). Blood and semen samples were collected before the administration of CCFA and at 12, 24, 36, 48, 72, 96, 120, 144, and 168 h after injection. No levels of CEFT were detected in PL and only in 20 of the 40 SP samples (P = 0.0001). The mean level of CEFT in SP was 0.11 % in comparison with the DFCA level. DFCA was found in all PL and SP samples. Therefore, DFCA was chosen to be utilized in the study of the pharmacokinetics parameters both in PL and SP. There were no differences in the mean PL levels of DFCA for the two sites of SC administration between the BOE (102.9 ± 78.9 ng/mL; X ± SD) and to MTE (116.1 ± 70.2 ng/mL; P = 0.58). The mean SP levels of DFCA after administration in the BOE was 857 ± 747 ng/mL, and for the MTE was 549 ± 488 ng/mL without differences between both sites (P = 0.15). The mean level of DFCA in PL was 109.5 ± 74.0 ng/mL, which was lower than the mean SP levels of 695 ± 103 ng/mL (P = 0.001). Moreover, the PL peak DFCA concentration (C max ) was 229 ± 46 ng/mL at 36.0 ± 29.4 h (T max ) post-administration. The SP C max was 1851 ± 533 ng/mL at 30.0 ± 28.6 h (T max ) post-administration. The C max between PL and SP were distinctive (P = 0.004) without any differences in T max between PL and SP (P = 0.60). The terminal half-life for PL DFCA (47.4 ± 29.3 h) was not different than in SP (53.1 ± 23.6 h; P = 0.77). The PL area under the curve concentration time from the first to the last sample (AUC 0-last ) was 18,984 ± 4841 ng/mL/h, which was significatively smaller compared with 125,677 ± 59,445 ng/mL/h for SP AUC 0-last (P = 0.04). The PL mean residence time from the first to the last sample (MRT 0-last ) was 69.7 ± 15.1 h, and it was similar than for SP of 66.5 ± 7.7 h (P = 0.69). From the present investigation, based in its pharmacokinetic features, it was concluded that CCFA should be an appropriate antibiotic that could be used for the treatment of bull genital infections when its indication is properly outlined. To study the pharmacokinetics of CCFA in SP, DFCA metabolite was appropriated., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Although seminal plasma extracellular vesicles (SPEVs) play important roles in sperm function, little is known about their metabolite compositions and roles in sperm motility. Here, we performed metabolomics and proteomics analysis of boar SPEVs with high or low sperm motility to investigate specific biomarkers affecting sperm motility. In total, 140 proteins and 32 metabolites were obtained through differentially expressed analysis and weighted gene coexpression network analysis (WGCNA). Seven differentially expressed proteins (DEPs) (ADIRF, EPS8L1, PRCP, CD81, PTPRD, CSK, LOC100736569) and six differentially expressed metabolites (DEMs) (adenosine, beclomethasone, 1,2-benzenedicarboxylic acid, urea, 1-methyl-l-histidine, and palmitic acid) were also identified in WGCNA significant modules. Joint pathway analysis revealed that three DEPs (GART, ADCY7, and NTPCR) and two DEMs (urea and adenosine) were involved in purine metabolism. Our results suggested that there was significant correlation between proteins and metabolites, such as IL4I1 and urea ( r = 0.86). Furthermore, we detected the expression level of GART, ADCY7, and CDC42 in sperm of two groups, which further verified the experimental results. This study revealed that several proteins and metabolites in SPEVs play important roles in sperm motility. Our results offered new insights into the complex mechanism of sperm motility and identified potential biomarkers for male reproductive diseases.

&lt;b&gt;Background and Objective:&lt;/b&gt; Biological exploration of male infertility is important for its treatment. Seminal plasma, by its composition, presents numerous molecules that can be exploited in the investigation of new sperm biomarkers. The evaluation of new biomarkers of azoosperm seminal plasma aims to identify vitamins A, D and E which can serve as discriminating biochemical markers in the exploration of azoospermia. &lt;b&gt;Materials and Methods:&lt;/b&gt; Thirty normozoospermic and 30 azoospermic sperm samples were collected by masturbation after three days of sexual abstinence from consulting patients at the Pasteur Institute of Côte d'Ivoire. After centrifugation of the sperm, the seminal plasma was collected and were analyzed for vitamin A, D and E. After extracting the vitamins from the seminal plasma, they were quantified using high-performance liquid chromatography. &lt;b&gt;Results:&lt;/b&gt; The concentration of vitamin A in seminal plasma from normal samples was 1.66±1.81 and 0.28±0.52 mg/L in pathological samples. The average vitamin D concentration in seminal plasma of normospermia was 0.27±0.40 and 0.08±0.12 mg/L in seminal plasma of azoospermia. For vitamin E, the results obtained show an average concentration of 2.56±3.58 mg/L in normal ejaculate and 0.33±0.51 mg/L in pathological ejaculate. Only vitamins A and E showed a significant difference in the two categories of sperms. &lt;b&gt;Conclusion:&lt;/b&gt; The determination of the concentration of vitamins A, D and E in seminal plasma showed that only vitamins A and E can serve as a biomarker for the differentiation of normozoospermic and azoospermic sperm.

Background: The unmet challenge in prostate cancer (PCa) management is to discriminate it from benign prostate hyperplasia (BPH) due to the lack of specific diagnostic biomarkers. Contemporary research on potential PCa biomarkers is directed toward methylated cell-free DNA (cfDNA) from liquid biopsies since epigenetic mechanisms are strongly involved in PCa development., Methods: In the present research, cfDNA methylation of the LGALS3 gene in blood and seminal plasma of PCa and BPH patients was assessed using pyrosequencing, as well as LGALS3 DNA methylation in tissue biopsies. Liquid biopsy samples were taken from patients with clinical suspicion of PCa, who were subsequently divided into two groups, that is, 42 with PCa and 55 with BPH, according to the histopathological analysis., Results: Statistically significant higher cfDNA methylation of LGALS3 in seminal plasma of BPH than in PCa patients was detected by pyrosequencing. ROC curve analysis showed that it could distinguish PCa and BPH patients with 56.4% sensitivity and 70.4% specificity, while PSA did not differ between the two patient groups. In contrast, there was no statistically significant difference in LGALS3 cfDNA methylation in blood plasma between the two patient groups. In prostate tumor tissue, there was a statistically significant DNA hypermethylation of LGALS3 compared to surrounding nontumor tissue and BPH tissue., Conclusions: The DNA hypermethylation of the LGALS3 gene represents an event specific to PCa development. In conclusion, LGALS3 cfDNA methylation in seminal fluid discriminates early PCa and BPH presenting itself as a powerful novel PCa biomarker highly outperforming PSA., (© 2024 The Author(s). The Prostate published by Wiley Periodicals LLC.)

Semen quality is an important indicator that can directly affect fertility. In mammals, miRNAs in seminal plasma extracellular vesicles (SPEVs) and sperms can regulate semen quality. However, relevant regulatory mechanism in duck sperms remains largely unclear. In this study, duck SPEVs were isolated and characterized by transmission electron microscopy (TEM), western blot (WB), and nanoparticle tracking analysis (NTA). To identify the important molecules affecting semen quality, we analysed the miRNA expression in sperms and SPEVs of male ducks in high semen quality group ((DHS, DHSE) and low semen quality group (DLS, DLSE). We identified 94 differentially expressed (DE) miRNAs in the comparison of DHS vs. DLS, and 21 DE miRNAs in DHSE vs. DLSE. Target genes of SPEVs DE miRNAs were enriched in ErbB signaling pathway, glycometabolism, and ECM-receptor interaction pathways (P < 0.05), while the target genes of sperm DE miRNAs were enriched in ribosome (P < 0.05). The miRNA-target-pathway interaction network analyses indicated that 5 DE miRNAs (miR-34c-5p, miR-34b-3p, miR-449a, miR-31-5p, and miR-128-1-5p) targeted the largest number of target genes enriched in MAPK, Wnt and calcium signaling pathways, of which FZD9 and ANAPC11 were involved in multiple biological processes related to sperm functions, indicating their regulatory effects on sperm quality. The comparison of DE miRNAs of SPEVs and sperms found that mir-31-5p and novel-273 could potentially serve as biomarkers for semen quality detection. Our findings enhance the insight into the crucial role of SPEV and sperm miRNAs in regulating semen quality and provide a new perspective for subsequent studies., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: This study focuses on the association between seminal concentration of prosaposin and ambient air pollutants and whether the association affects the normal fertilization rate in vitro fertilization (IVF) treatment., Methods: The cohort of 323 couple participants aged 22-46 was recruited from Jan. 2013 to Jun. 2018. At enrollment, resident address information was obtained and semen parameters of male counterparts were evaluated according to WHO criteria. We used inverse distance weighting interpolation to estimate the levels of ambient pollutants (SO 2 , O 3 , CO, NO 2 , PM 2.5, and PM 10 ) in the surrounding area. The exposure of each participant was estimated based on the data gathered from air quality monitoring stations and their home address over various periods (0-9, 10-14, and 0-90 days) before semen sampling. The generalized linear regression model (GLM) and the Bayesian kernel machine regression (BKMR) were used to analyze the associations between pollutants, semen parameters, prosaposin, and normal fertilization. Additionally, the mediating effect of prosaposin and semen parameters on the link between pollutants and normal fertilization was investigated., Results: GLM and BKMR showed exposure to ambient air pollutants was all associated with the concentration of seminal prosaposin, among them, O 3 and CO were also associated with normal fertilization (-0.10, 95 %CI: -0.13, -0.06; -26.43, 95 %CI: -33.79, -19.07). Among the semen parameters, only the concentration of prosaposin and total motile sperm count (TMC) was associated with normal fertilization (0.059, 95 %CI: 0.047, 0.071; 0.016, 95 %CI: 0.012, 0.020). Mediation analysis showed that prosaposin played a stronger mediating role than TMC in the relationship between short-term exposure to O 3 and fertilization (66.83 %, P<0.001 versus 3.05 %, P>0.05)., Conclusion: Seminal plasma prosaposin showed a stronger meditating effect reflect the correlation between ambient air pollutants and normal fertilization rate than conventional semen parameters, which may be used as one of the indicators between pollution and fertilization in IVF., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of competing interest none., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Bisphenol A (BPA) is a widespread industrial chemical, used as the key monomer of polycarbonate plastics and epoxy resins. BPA has been detected in human seminal fluid and has been correlated with changes in sperm parameters, crucial in determining male fertility. In this study, semen samples were collected from 100 patients aged 29-47 years undergoing fertility assessment between 2021 and 2023 and analyzed according to WHO guidelines. BPA levels in the seminal plasma were then measured through an enzyme-linked immunosorbent assay (ELISA) and compared to sperm quality metrics. The relative mRNA/miRNA expression of key genes associated to male reproduction, including androgen receptor, miR-34c, miR-21, miR-130a, was then quantified and compared between groups with high or low BPA content. Our results revealed that BPA levels were increased with age and were negatively correlated with sperm counts (p<0.05). The negative correlation remained significant when patients were age-matched. No other relationships between seminal BPA and motility, morphology or DNA fragmentation levels were observed. qPCR analysis showed that androgen receptor mRNA expression was significantly greater in sperm with high seminal BPA (p<0.05). Moreover, we found that the expression of miR-21 and miR-130a was also upregulated in the higher BPA group (p<0.05). These results display a relationship between BPA content in the semen and male fertility parameters, and provide insights into the molecular mechanisms through which BPA may be affecting male reproductive capability. Ultimately, this research can potentially drive changes to guidelines and exposure limits for BPA exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Excessive reactive oxygen species (ROS) in seminal plasma can trigger male infertility. Therefore, the development of simple and rapid ROS detection methods is urgently needed, particularly for the early self-screening of preconception couples. Herein, a gold nanobipyramid (Au NBP)-based colorimetric hydrogel for convenient and fast ROS detection is described. In the hydrogel, Au NBP is etched efficiently by ROS under the synergistic effect of Fe 2+ and I - , which finally causes color variations. Besides, agarose gel with the function of molecular sieve enables the separation of biomacromolecules, improving the interference resistance of the system and the stability of Au NBP. This chemical sensor can complete all the tests within 20 min, covering two detection range of 10-125 μM at relative low H 2 O 2 concentration and 125-1000 μM at relative high H 2 O 2 concentration, with the detection limits of 1.76 μM and 12.10 μM (S/N = 3) respectively. Furthermore, via visual observation of the color variations, it allows the initial interpretation of ROS concentration without any additional equipment. We applied this device to the detection of ROS in clinical seminal plasma samples and obtained promising results, demonstrating its potential for rapid and convenient detection in clinical applications., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Confirmatory identification of dyes in the physical pieces of evidence, such as hair and fabric, is critically important in forensics. This information can be used to demonstrate the link between a person of interest and a crime scene. High performance liquid chromatography is broadly used for dye analysis. However, this technique is destructive and laborious. This problem can be overcome by near-Infrared excitation Raman spectroscopy (NIeRS), non-invasive and non-destructive technique that can be used to determine chemical structure of highly fluorescent dyes. Analyzed fabric materials often possess body fluid stains, which may obscure the accuracy of NIeRS-based identification of dyes. In this study, we investigate the extent to which fabric contamination with body fluids can alter the accuracy of NIeRS. Our results showed that NIeRS coupled with partial-least squared discriminant analysis (PLS-DA) enabled on average 97.6% accurate identification of dyes on fabric contaminated with dry blood, urine and semen. We also found that NIeRS could be used to identify blood, urine and semen on such fabric with 99.4% accuracy. Furthermore, NIeRS could be used to differentiate between wet and dry blood, as well as reveal the presence of blood on washed fabric. These results indicate that NIeRS coupled with PLS-DA could be used as a robust and reliable analytical approach in forensic analysis of fabric., (© 2024. The Author(s).)

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 MgSO 4 :NaCl 4:1 (w/w) were used for the extraction. The separation of analytes was performed on an ACQUITY BEH C 18 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

Sexual assault cases typically involve intense physical contact between victim and perpetrator, resulting in a high chance of trace transfer. In addition the victim's, and sometimes the suspect's, declaration is available, and can help direct the forensic investigation. However, many cases are still closed because of insufficient proof or an unknown perpetrator.Previously in Belgium, the forensic analysis strategy was often limited to the detection of human sperm fluid and if not found, the forensic part of the case was closed.Therefore, the Forensic Advice service from the Belgian National Institute for Criminalistics and Criminology (NICC) together with the public prosecutor's office of the Judicial district Antwerp, department Antwerp started "Project code 37". In Belgium every type of crime receives a code and sexual assault cases are categorized under code 37. Because a forensic advisor focuses on the potential of forensic examination on physical objects, the project focused on cases with samples taken on the victim or clothing available.The goal of the project was to perform a contextual analysis in order to go beyond the standard sperm fluid-orientated approach and evaluate if this had a positive impact on the conviction rate. The project also aimed to evaluate the impact of the consistent use of toxicological analyses on conviction rates. Through this systematic contextual analysis, it became clear that four clear categories could be defined: (1) cases with an unknown perpetrator, (2) cases where the suspect denies all contact, (3) cases where the suspect denies (part of) the sexual contact, and (4) cases where only the consent of the victim is disputed. These categories all assumed one female victim and one male perpetrator. Together with the NICC's DNA, trace evidence and toxicology lab, an analysis strategy was drafted for the four categories. All cases that do not fit this description were gathered in a fifth category (custom-made cases), as the victim's/perpetrator's sex has a significant effect on the forensic analysis strategy. At the end of the project, a comparison was made with the same set of cases from another judicial year where the standard analysis strategy was used. For all the categories for which data was available, there was a rise of the conviction rate and a decrease in the number of dismissed cases., Competing Interests: Declaration of Competing Interest I hereby declare that the disclosed information is correct and that no other situation of real, potential or apparent conflict of interest is known to me., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Some research has identified the prevalence and motivation of using condoms by assailants during sexual assault cases proving the necessity of analyzing condom trace evidence. The majority of the papers published have discussed forensic analysis of lubricants from condoms retrieved at sexual assault scenes but those discussing the identification of semen from condoms are rare. Therefore, the present study aims to provide insight into the detectability of the semen that remained in a condom, to examine the effect of exposure time, environmental conditions, and condom type, and ultimately to determine the capability of the AP test and Microscopic examination for identification of this sample type. In the study, samples were collected from three male donors after being instructed on the proper way of collecting the semen sample. The received samples from the donors were checked first by microscopic examination to observe the sperm to confirm that the sample being handled was semen. After confirmation, samples were transferred to 4 prepared condoms (brands: dkt xxx and Manforce) and kept in conditions i.e. two condoms in a refrigerator maintained from 2 to 10°C and other ones at ambient temperature (weather status: summer season of average 39°C). The samples were analyzed into two batches, the first analysis batch was conducted after the samples were exposed to the conditions within 11-60 days. After analysis from the first batch, the samples were continuously kept in the same condition for the consecutive second batch conducted when the samples reached 40-90 days. This study has determined that semen remaining in a condom can be detected and each test studied is appropriate according to the exposure stage, i.e., time and conditions of exposure. It has been found that nonmotile spermatozoa can be observed when semen remains in the condom for a few days. However, if the sample reaches approximately 25 days at room temperature above 25°C or 54 days below 10°C, the semen may dry out limiting the effectiveness of microscopic examination. Despite this, even semen that remained in a condom for up to 90 days can be identified by Acid Phosphatase. Results on condom type used reveal that condom constituents can crossreact with semen but none of them can limit the semen identification with Acid Phosphatase., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Determining the biological source of a stain can be important information for both investigators and the judiciary in criminal cases. Immunochromatographic assays are commonly used in forensic science for the identification of human biological material. It has previously been demonstrated that various environmental, thermal and chemical insults can affect the efficacy of ABAcard® HemaTrace® in the detection of human blood. In this study, the efficacy of three tests - ABAcard® HemaTrace®, ABAcard® p30, and RSID™-Saliva - was determined for the detection of blood, semen, and saliva respectively, after the fluids had been exposed to adverse environmental conditions. Each biological fluid was deposited on cotton swatches and exposed to infrared (IR) light using a 100 W heat lamp emitting IR light between 620 and 750 nm and heat of 32° for 24, 36 and 48 h. Cotton swatches bearing biological fluids were also buried in outdoor soil for 3, 4 and 5 weeks. To test common forensic scenarios where biological material may be exposed to solar light, samples were placed on a car bonnet and left for 24, 36 and 48 h. ABAcard® HemaTrace® was able to detect haemoglobin in blood that had been exposed to IR and solar light up to 48 h. False negative ABAcard® HemaTrace® results were obtained from 60 % of blood samples buried for 3 and 4 weeks, and 80 % of blood samples buried for 5 weeks. ABAcard® p30 was able to detect p30 in semen that had been exposed to IR and solar light up to 48 h, except for one false negative after 48 h of IR exposure. False negative ABAcard® p30 results were obtained from all semen samples buried for 3, 4 and 5 weeks. RSID™-Saliva was able to detect α-amylase in saliva in all instances, with no false negative results observed. The findings from this study highlight the need to consider the context in which human blood, semen and saliva are found when reporting on negative immunoassay results., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to identify if biological material could be detected on the opposite side to deposition on fabric by commonly used presumptive and/or secondary tests. Additionally, this study aimed to ascertain if there is a difference in the DNA quantity and quality from samples obtained from both sides of the same substrate: cotton, polyester, denim, or combined viscose and polyester swatches. Blood, semen, or saliva (25 μL) was deposited on one side of 5 replicates of each fabric type and left for 24 h. Blood swatches were tested using Hemastix® and the ABACard® HemaTrace® immunoassay, semen swatches were tested using acid phosphatase (AP) reagent, the ABACard® p30® immunoassay and hematoxylin and eosin staining, and saliva swatches were tested using Phadebas® paper and the RSID-Saliva™ immunoassay. Both sides of each swatch were separately wet/dry swabbed and subjected to DNA analysis. Blood was able to be detected on the underside of all fabrics using both tests. Semen was able to be detected on the underside of swatches using the presumptive AP test but not p30®, and sperm was rarely observed. Saliva was able to be detected by RSID-Saliva™ but not Phadebas® paper when the underside of swatches were tested. Across all biological materials, DNA was able to be recovered from the top side of all 60 swatches. For the underside, DNA was able to be recovered from 54 swatches. Of the 6 swatches that DNA was unable to be recovered from, one sample was from semen and the rest were from saliva. This study has demonstrated that DNA and components of interest in forensically relevant biological material can be recovered from the opposite side to where it was originally deposited, and that observing biological material and/or DNA on one side of fabric does not definitively indicate direct deposition on that side., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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.

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., Competing Interests: Declaration of Competing Interest The author(s) declare(s) that there is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Purpose: Inadvertent and/or unknowing exposure to drugs and drug residues has been frequently debated in situations of so-called adverse analytical finding (AAF) in the context of sports drug testing programs. Transfer of drug residues via unprotected intercourse is a conceivable scenario but scientific data and authentic case reports are scarce. Herein, investigations into two AAFs with the peroxisome proliferator-activated receptor delta (PPARδ) agonist GW1516 are reported and discussed., Methods: To probe for a contamination scenario involving sexual intercourse, two assays were used to determine semenogelin in human urine, with one employing an immunochromatographic lateral flow approach and another based on liquid chromatography-tandem mass spectrometry. Further, drug-residue testing using patients' ejaculate was conducted by utilizing liquid chromatography in conjunction with a triple quadrupole mass spectrometer, followed by re-analysis of suspect samples (i.e., samples indicating the presence of relevant compounds) using high resolution/high mass accuracy mass spectrometry., Results: In one case, but not the other, the possibility of intimate contact as the source of the AAF was confirmed after a thorough investigation of potential contamination scenarios. Subsequent research revealed analytical evidence for the presence of seminal fluid in one of the female athlete's doping control urine samples, and the analysis of clinical ejaculate specimens provided first data on an authentic concentration level of GW1516 and its metabolites in human seminal fluid., Conclusions: The combined facts substantiate the possibility of an AAF caused by unprotected sexual intercourse and the plausibility of the case-related arguments., (© 2024. The Author(s).)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

DNA methylation plays essential roles in regulating physiological processes, from tissue and organ development to gene expression and aging processes and has emerged as a widely used biomarker for the identification of body fluids and age prediction. Currently, methylation markers are targeted independently at specific CpG sites as part of a multiplexed assay rather than through a unified assay. Methylation detection is also dependent on divergent methodologies, ranging from enzyme digestion and affinity enrichment to bisulfite treatment, alongside various technologies for high-throughput profiling, including microarray and sequencing. In this pilot study, we test the simultaneous identification of age-associated and body fluid-specific methylation markers using a single technology, nanopore adaptive sampling. This innovative approach enables the profiling of multiple CpG marker sites across entire gene regions from a single sample without the need for specialized DNA preparation or additional biochemical treatments. Our study demonstrates that adaptive sampling achieves sufficient coverage in regions of interest to accurately determine the methylation status, shows a robust consistency with whole-genome bisulfite sequencing data, and corroborates known CpG markers of age and body fluids. Our work also resulted in the identification of new sites strongly correlated with age, suggesting new possible age methylation markers. This study lays the groundwork for the systematic development of nanopore-based methodologies in both age prediction and body fluid identification, highlighting the feasibility and potential of nanopore adaptive sampling while acknowledging the need for further validation and expansion in future research., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Epidemiological studies on the associations between the levels of oxidative stress (OS) indicators (MDA, SOD, and GSH) in seminal plasma and the risk of idiopathic oligo-asthenotera-tozoospermia (OAT) are still inconsistent. Additionally, whether the associations can be altered by the status of essential trace elements is still unknown. To investigate the relationship between MDA, SOD, and GSH levels in seminal plasma and the risk of idiopathic OAT, and further to examine whether levels of iron (Fe), copper (Cu), and selenium (Se) in seminal plasma can alter the associations. A total of 148 subjects (75 idiopathic OAT cases and 73 controls) were included in this study. Seminal plasma samples from all the participants were measured for levels of MDA, SOD, GSH, Fe, Cu, and Se. Unconditional logistic regression models were used to examine the associations between three oxidative stress indicators and the risk of idiopathic OAT. Bayesian kernel machine regression was performed to determine the joint effects of levels of three OS indicators on the risk of idiopathic OAT. Subgroup analyses were performed to explore whether the above associations can be different when Fe, Cu, and Se were in different levels. The level of MDA in seminal plasma was positively associated with the risk of idiopathic OAT, with adjusted odds ratio (OR) and 95% confidence interval (CI) of 2.38 (1.17, 4.83), and SOD and GSH levels were not associated with the risk of idiopathic OAT. In BKMR analyses, we found a significant positive association between the mixture of MDA, SOD, and GSH levels and the risk of idiopathic OAT at concentrations below the 65th percentile, while a negative association at concentrations above it. In subgroup analysis, a positive association was observed between MDA levels in seminal plasma and the risk of idiopathic OAT in the high-Cu group (adjusted OR = 3.66, 95%CI = 1.16, 11.57), while no significant association was found in the low-Cu group (adjusted OR = 1.43, 95%CI = 0.44, 4.58). Additionally, a negative association was found between GSH levels in seminal plasma and the risk of idiopathic OAT in the high-Se group (adjusted OR = 0.34, 95%CI = 0.11, 0.99), while no significant association was observed in the low-Se group (adjusted OR = 1.96, 95%CI = 0.46, 8.27). The levels of MDA, SOD, and GSH in seminal plasma were associated with the risk of idiopathic OAT, and the levels of Cu and Se in seminal plasma may alter the associations., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)