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.

Correlation between seminal plasma biochemical markers and semen parameters in idiopathic oligoasthenoteratospermia: identification of biomarkers for L-carnitine therapy.

Background: L-carnitine therapy for idiopathic sperm abnormalities exhibits variable effectiveness, and currently, there are no established criteria to predict patient response. This study investigated correlations between seminal plasma markers and semen parameters to identify biomarkers that can guide indications for L-carnitine therapy indications in patients with idiopathic sperm abnormalities. Methods: A retrospective review was conducted on 223 male patients with idiopathic oligoasthenoteratospermia, who sought medical attention at our clinic between January 2020 and October 2022. These patients underwent a pretreatment seminal plasma biochemical analysis, followed by a three-month continuous L-carnitine treatment. The correlation between seminal plasma biochemical parameters and pretreatment semen parameters was analyzed. Semen quality was compared between cases with normal and abnormal seminal plasma biochemical parameters, both pretreatment and posttreatment. The correlation between the changes in semen parameters after treatment and seminal plasma biochemical parameters were investigated. Results: Correlation analyses revealed significant associations between all pretreatment semen parameters and seminal plasma biochemical markers, except for liquefying time and the ratio of normal morphology. Subgroup analysis, stratified by seminal fructose, zinc, citric acid, and neutral glycosidase levels, demonstrated that abnormal groups exhibited significantly different levels of semen parameters compared with the normal groups. The changing difference and changing ratio in the ratio of forward motile sperm showed a negative correlation with seminal fructose levels (r=-0.165 and -0.144). The changing difference in semen volume was negatively correlated with the level of seminal neutral glycosidase (r=-0.158). The changing ratio in semen volume, sperm concentration, total sperm count, and count of forward motile sperm all exhibited negative correlations with the levels of seminal neutral glycosidase (range from -0.178 to -0.224). Conclusion: Seminal plasma biochemical markers, particularly fructose and neutral glycosidase, may serve as valuable indicators for determining the eligibility of patients with idiopathic sperm abnormalities for L-carnitine therapy.

Mass spectrometry-based proteomic analysis of biological stains identifies body fluids specific markers.

The identification of biological stains and their tissue resource is an important part of forensic research. Current methods suffer from several limitations including poor sensitivity and specificity, trace samples, and sample destruction. In this study, we profiled the proteomes of menstrual blood, peripheral blood, saliva, semen, and vaginal fluid with mass spectrometry technology. Tissue-enhanced and tissue-specific proteins of each group have been proposed as potential biomarkers. These candidate proteins were further annotated and screened through the combination with the Human Protein Atlas database. Our data not only validates the protein biomarkers reported in previous studies but also identifies novel candidate biomarkers for human body fluids. These candidates lay the foundation for the development of rapid and specific forensic examination methods.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Quantification of mirtazapine in tablets via DNA binding mechanism; development of a new HPLC method.

Atypical antidepressant mirtazapine (MIR) is mostly prescribed for the management of major depressive disorder. The identification of MIR in pharmaceutical dosage forms was made possible by developing a novel, quick, sensitive high-performance liquid chromatography (HPLC) approach that was verified in accordance with ICH recommendations. In the first part of this study, HPLC investigations were optimized with regard to variables including pH, working column, mobile phase, temperature, and flow rate. The limit of detection (LOD) was 0.013 ppm, the limit of quantification (LOQ) was 0.044 ppm, and the linear range was computed as 0.5-15 ppm (R2 = 0.9998). The recovery investigation assessed the method's accuracy, which was shown to range between 98.82 and 100.97 %. In the second part, by using UV-vis spectroscopy, HPLC, thermal denaturation, and viscosity measurements, the mechanism of binding interaction of MIR with double-stranded fish sperm deoxyribonucleic acid (dsDNA) has been thoroughly studied. The DNA binding constants (Kb) were determined using UV-Vis absorption and HPLC methods. To investigate the interactions of MIR with dsDNA, molecular docking calculations and additionally, molecular dynamics simulations were performed. Results showed that MIR is located in the minor groove of dsDNA, and in addition to hydrogen bonding, electrostatic interaction is also formed between the aromatic ring of MIR and phosphate oxygen of dsDNA. Finally, a binding characterization study using MIR tablets was also conducted in order to assess the interaction mechanism of the DNA with the drug using the validated analytical procedure developed for the MIR molecule.

Impact of semen microbiota on the composition of seminal plasma.

Several studies have found associations between specific bacterial genera and semen parameters. Bacteria are known to influence the composition of their niche and, consequently, could affect the composition of the seminal plasma. This study integrated microbiota profiling and metabolomics to explore the influence of seminal bacteria on semen metabolite composition in infertile couples, revealing associations between specific bacterial genera and metabolite profiles. Amino acids and acylcarnitines were the predominant metabolite groups identified in seminal plasma. Different microbiota profiles did not result in globally diverse metabolite compositions in seminal plasma. Nevertheless, levels of specific metabolites increased in the presence of a dysbiotic microbiota. Urocanate was significantly increased in abnormal semen samples (adjusted P-value < 0.001) and enriched in samples dominated by Prevotella spp. (P-value < 0.05), which was previously linked to a negative impact on semen. Therefore, varying microbiota profiles can influence the abundance of certain metabolites, potentially having an immunomodulatory effect, as seen with urocanate.IMPORTANCEMale infertility is often considered idiopathic since the specific cause of infertility often remains unidentified. Recently, variations in the seminal microbiota composition have been associated with normal and abnormal semen parameters and may, therefore, influence male infertility. Bacteria are known to alter the metabolite composition of their ecological niches, and thus, seminal bacteria might affect the composition of the seminal fluid, crucial in the fertilization process. Our research indicates that distinct seminal microbiota profiles are not associated with widespread changes in the metabolite composition of the seminal fluid. Instead, the presence of particular metabolites with immunomodulatory functions, such as urocanate, could shed light on the interplay between seminal microbiota and variations in semen parameters.

Association of urinary and seminal plasma vanadium concentrations with semen quality: A repeated-measures study of 1135 healthy men.

Although animal studies have shown the reproductive toxicity of vanadium, less is known about its effects on semen quality in humans. Among 1135 healthy men who were screened as potential semen donors, we investigated the relationships of semen quality with urinary and seminal plasma vanadium levels via inductively coupled plasma-mass spectrometry (ICP-MS). Spearman rank correlation tests and linear regression models were used to assess the correlations between average urinary and within-individual pooled seminal plasma vanadium concentrations (n = 1135). We utilized linear mixed-effects models to evaluate the associations of urinary and seminal plasma vanadium levels (n = 1135) with repeated sperm quality parameters (n = 5576). Seminal plasma vanadium concentrations were not significantly correlated with urinary vanadium concentrations (r = 0.03). After adjusting for possible confounders, we observed inverse relationships of within-individual pooled seminal plasma vanadium levels with total count, semen volume, and sperm concentration (all P values for trend < 0.05). Specifically, subjects in the highest (vs. lowest) tertile of seminal plasma vanadium concentrations had - 11.3% (-16.4%, -5.9%), - 11.1% (-19.1%, -2.4%), and - 20.9% (-29.0%, -11.8%) lower sperm volume, concentration, and total count, respectively; moreover, urinary vanadium levels appeared to be negatively associated with sperm motility. These relationships showed monotonically decreasing dose-response patterns in the restricted cubic spline analyses. Our results demonstrated a poor correlation between urinary and seminal plasma levels of vanadium, and elevated vanadium concentrations in urine and seminal plasma may be adversely related to male semen quality.

Purification, identification and Cryo-EM structure of prostatic acid phosphatase in human semen.

Prostatic acid phosphatase (PAP) is a glycoprotein that plays a crucial role in the hydrolysis of phosphate ester present in prostatic exudates. It is a well-established indicator for prostate cancer due to its elevated serum levels in disease progression. Despite its abundance in semen, PAP's influence on male fertility has not been extensively studied. In our study, we report a significantly optimized method for purifying human endogenous PAP, achieving remarkably high efficiency and active protein recovery rate. This achievement allowed us to better analyze and understand the PAP protein. We determined the cryo-electron microscopic (Cryo-EM) structure of prostatic acid phosphatase in its physiological state for the first time. Our structural and gel filtration analysis confirmed the formation of a tight homodimer structure of human PAP. This functional homodimer displayed an elongated conformation in the cryo-EM structure compared to the previously reported crystal structure. Additionally, there was a notable 5-degree rotation in the angle between the α domain and α/ß domain of each monomer. Through structural analysis, we revealed three potential glycosylation sites: Asn94, Asn220, and Asn333. These sites contained varying numbers and forms of glycosyl units, suggesting sugar moieties influence PAP function. Furthermore, we found that the active sites of PAP, His44 and Asp290, are located between the two protein domains. Overall, our study not only provide an optimized approach for PAP purification, but also offer crucial insights into its structural characteristics. These findings lay the groundwork for further investigations into the physiological function and potential therapeutic applications of this important protein.

Exposure to particulate matter may affect semen quality via trace metals: Evidence from a retrospective cohort study on fertile males.

Particulate matter (PM) exposure may be associated with male semen quality. Besides, PM exposure induces up and down levels of trace metals in tissues or organs. The levels of trace metals in semen are critical for adverse male semen quality. This study aims to evaluate the concentrations of seminal-level trace metals in fertile men and assess its associations with PM exposure and to explore the mediation role of trace metals in seminal plasma plays in the relationship between PM exposure and semen quality. Total 1225 fertile men who participated in a cohort study from 2014 to 2016 were finally recruited. Multivariate linear regression was applied to explore associations between each two of PM exposure, trace metals and semen parameters. 1-year PM2.5 and PM10 exposure levels were positively associated with arsenic (As), mercury (Hg), lanthanum (La), praseodymium (Pr), neodymium (Nd) but negatively associated with vanadium (V), magnesium (Mg), strontium (Sr), barium (Ba) in semen. It was also found that most of the elements were associated with total sperm number, followed by sperm concentration. Redundancy analysis (RDA) also determined several strong positive correlations or negative correlations between 1-year PM exposure and trace metals. Mediation analysis found that trace metals had a potentially compensatory or synergetic indirect effect on the total effect of the association between 1-year PM exposure and semen quality. The retrospective cohort study provides long-term PM exposure that may cause abnormal semen quality by affecting seminal plasma element levels.

Using Bayesian and weighted regression to evaluate the association of idiopathic oligoastenoteratozoospermia with seminal plasma metal mixtures.

Idiopathic oligoastenoteratozoospermia (iOAT) affects 30% of infertile men of reproductive age. However, the associations between Cr, Fe, Cu, Se or Co levels and iOAT risk have not been determined. This research aimed to assess the associations between Cr, Fe, Cu, Se and Co levels as well as their mixtures in seminal plasma and the risk of iOAT and severe iOAT. Therefore, a case‒control study including 823 participants (416 iOAT patients and 407 controls) recruited from October 2021 to August 2022 at the reproductive medicine center of the First Affiliated Hospital of Anhui Medical University was conducted in Anhui, China. The concentrations of Cr, Fe, Cu, Se and Co in seminal plasma were detected via inductively coupled plasma‒mass spectrometry. Binary logistic regression models were used to assess the associations between the levels of Cr, Fe, Cu, Se and Co and the risk of iOAT and severe iOAT; additionally, Bayesian kernel machine regression (BKMR) and weighted quantile sum (WQS) regressions were performed to evaluate the joint effect of seminal plasma levels of Cr, Fe, Cu, Se and Co on the risk of iOAT and explore which elements contributed most to the relationship. We found significant associations between the concentrations of Fe, Cu and Se in seminal plasma and iOAT risk after adjusting for covariates (Fe, lowest tertile vs. second tertile: aOR = 1.86, 95% CI = 1.31, 2.64; Cu, lowest tertile vs. second tertile: aOR = 1.95, 95% CI = 1.37, 2.76; Se, lowest tertile vs. second tertile: aOR = 1.65, 95% CI = 1.17, 2.35). A lower Se concentration in seminal plasma (lowest tertile vs. second tertile: aOR = 1.84, 95% CI = 1.10, 3.10) was positively associated with the risk of severe iOAT. Additionally, we also observed an association between the concentration of Cr in seminal plasma and the risk of iOAT before adjusting for covariates (Cr, third tertile vs. lowest tertile: OR=1.44, 95% CI: 1.03, 2.02). According to the BKMR analyses, the risk of iOAT increased when the overall concentrations were less than the 25th percentile. The results from the WQS regression indicated that a negative WQS index was significantly associated with the iOAT risk, while a positive WQS index was not. Se and Fe had significant weights in the negative direction. In conclusion, lower Cu, Fe and Se levels in seminal plasma were positively associated with iOAT risk, while higher Cr levels in seminal plasma were positively associated with iOAT risk according to the single element model, and lower levels of Se were related to a greater risk of severe iOAT; when comprehensively considering all the results from BKMR and WQS regression, Fe, Se and Cr levels contributed most to this relationship.

New discovery of extremely high adsorption of environmental DNA on cuttlefish bone pyrolysis derivative via large pore structure and carbon film.

Environmental DNA (eDNA) carrying antibiotic resistance gene (ARG) has attracted a great deal of attention because of its threat to the ecology and human health. Traditional porous adsorbents, such as microporous biochar and natural mineral, are low-effective in removing eDNA from sewage. This study used cuttlefish-bone (CB), a fishery waste, as an anticipated material to adsorb a model compound of eDNA from herring sperm (hsDNA). An interesting result was firstly observed that extremely high DNA adsorption on cuttlefish-bone pyrolysis derivative (CCB) was up to 88.7 mg/g, 3-10 folds higher than that of most other adsorbents in the existing literatures, which was attributed to the carbon film and large pores. To achieve an adsorption rate of 75 %, hsDNA adsorption took 96 h on CB but only 24 h on CCB, which was attributed to the fluent channel of CCB. The ligand exchange, Ca2+ bridge and π-π interaction were identified as dominated adsorption mechanisms, based on FTIR and phosphate competition experiments. This study exploited a high-efficient, environmentally friendly, and low-cost adsorbent for treating ARG-contaminated soil and water.

Estimation of time since deposition of semen stain on different fabric types using ATR-FTIR spectroscopy and chemometrics.

Various body fluids such as blood, semen, vaginal secretions, and saliva are frequently encountered at crime scene. In cases of sexual assault, semen stains are one of the most reliable evidence of biological origin. In this study, our objective was to develop a method for estimating the time since deposition of semen stains on five different fabric types using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy, with a focus on a time frame of up to 8 weeks. Semen samples from six different volunteers were dripped onto five distinct fabric materials, and ATR-FTIR measurements were obtained at 17 different time points. Principal component analysis (PCA) and partial least squares (PLS) methods were employed to differentiate semen stains on various fabric samples and estimate the age of semen stains. Models constructed using PCA and PLSR achieved high R2 values and low root-mean-square error (RMSE). While the performance varies depending on fabric types, it was observed that age estimation of semen stains can be made within following intervals: 0.39-0.76 days for 0-7 day range, 2.59-3.38 days for the 1-8 week range, and 3.98-8.1 days for the 0-56 day range. This study demonstrates the effectiveness of using ATR-FTIR spectroscopy in combination with chemometrics to estimate the age of human semen stains on various fabric types based on time-dependent spectral changes.

Time-dependent change in the microbiota structure of seminal stains exposed to indoor environmental.

Seminal stains acquired from fabric surfaces stand as pivotal biological evidence of utmost significance for elucidating sexual assault cases. The ability to determine the temporal aspect of a forensic incident via the analysis of a biological specimen found at the crime scene is crucial in resolving most cases. This study aimed to investigate the time-dependent change in the microbiota structure of human seminal stains exposed to indoor environmental conditions. Stains on polyester fabric generated using semen samples from five male volunteers were kept indoors for varying durations of up to 20 days, followed by sequencing of the V1-V9 regions of the 16S rRNA gene of the microbial DNA extracted from the stains. The acquired data provided the taxonomic composition, and microbial alterations across different days were examined. The most abundantly detected phyla in all samples were Firmicutes, Proteobacteria, and Bacteroidetes, and the relative abundances of bacteria were observed to change over time. Statistically significant changes at the species level were found for Treponema medium, Corynebacterium tuberculostearicum, Faecalibacterium prausnitzii, and Anaerostipes hadrus. Alterations observed in the samples between the analyzed time periods were investigated. The changes during the specified time periods were examined, identifying rare bacterial species that were initially present on certain days but later ceased to exist in the environment. Conversely, bacterial species that were absent before exposure but emerged at a later stage were also identified. The findings of this study demonstrate that species-level evaluations, in particular, can provide crucial insights into semen stain age.

A combined molecular approach utilizing microbial DNA and microRNAs in a qPCR multiplex for the classification of five forensically relevant body fluids.

Body fluid identification is an essential step in the forensic biology workflow that can assist DNA analysts in determining where to collect DNA evidence. Current presumptive tests lack the specificity that molecular techniques can achieve; therefore, molecular methods, including microRNA (miRNA) and microbial signature characterization, have been extensively researched in the forensic community. Limitations of each method suggest combining molecular markers to increase the discrimination efficiency of multiple body fluids from a single assay. While microbial signatures have been successful in identifying fluids with high bacterial abundances, microRNAs have shown promise in fluids with low microbial abundance (blood and semen). This project synergized the benefits of microRNAs and microbial DNA to identify multiple body fluids using DNA extracts. A reverse transcription (RT)-qPCR duplex targeting miR-891a and let-7g was validated, and miR-891a differential expression was significantly different between blood and semen. The miRNA duplex was incorporated into a previously reported qPCR multiplex targeting 16S rRNA genes of Lactobacillus crispatus, Bacteroides uniformis, and Streptococcus salivarius to presumptively identify vaginal/menstrual secretions, feces, and saliva, respectively. The combined classification regression tree model resulted in the presumptive classification of five body fluids with 94.6% overall accuracy, now including blood and semen identification. These results provide proof of concept that microRNAs and microbial DNA can classify multiple body fluids simultaneously at the quantification step of the current forensic DNA workflow.

Physiological evaluation of seasonal sperm quality in a biannual spawner, Pacific abalone: Effects on in-vitro fertilization and cryotolerance.

Pacific abalone, Haliotis discus hannai, is a highly valuable gastropod mollusk commonly found in Southeast Asia. The present study aims to analyze the seminal plasma quality, sperm quality, and cryotolerance of the Pacific abalone sperm during its reproductive season. The seminal plasma quality was evaluated by analyzing biochemical and metabolite composition, enzymatic activity (superoxide dismutase, catalase, and glutathione), and lipid peroxidation (LPO) activity. The sperm quality was evaluated by analyzing motility, concentration, volume, ATP content, acrosome integrity (AI), plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA integrity, and fertilization potential. The cryotolerance capacity was evaluated by analyzing post-thaw motility, AI, PMI, MMP, and DNA integrity. Seminal plasma osmolarity was significantly higher (1123.3 ± 1.5 mOsmL-1) in May compared to other reproductive periods, with Cl- (516.8 ± 0.5 mM) and Na+ (460.2 ± 0.4 mM) as the dominant ions. The seminal plasma pH remained constant at 6.8 throughout the reproductive season. Improved enzymatic activity and lower LPO were detected in May or June. Sperm quality indicators were similar in May and June, except for sperm production. The fertilization potential (May: 93.0 ± 4.4%, June: 86.0 ± 7.2%) and hatching rate (May: 86.6 ± 5.78%, June: 82.3 ± 3.2%) of Pacific abalone were significantly higher in May or June than they were in other reproductive seasons. The motility (May: 50.19 ± 2.35%, June: 49.96 ± 1.60%), AI (May: 44.02 ± 3.46%, June: 42.16 ± 3.61%), PMI (May: 54.12 ± 3.29%, June: 52.82 ± 2.58%), and MMP (May: 44.02 ± 3.46%, June: 42.16 ± 3.61%) of the cryopreserved sperm were similar in May and June compared with those preserved in other reproductive seasons. The DNA integrity of the cryopreserved sperm was similar in May (80.3 ± 6.7%) or June (78.9 ± 7.4%) and had a higher cryotolerance than in other reproductive seasons. Hence, it can be suggested that May and/or June are suitable periods for sperm physiology experiments, artificial reproduction, and sperm cryopreservation of Pacific abalone.

Effects of the adsorption behavior of polyamide microplastics on male reproductive health by reduction of testosterone bioavailability.

Microplastics (MPs) are global environmental pollutants with potential toxicity concerns, and their effects on the reproductive system have attracted increasing attention. This study investigated the interaction between MPs and mammalian biomolecules, focusing on the relationship between the testosterone adsorption behavior of MPs and male reproductive health. The adsorption capacity of different types of MPs for testosterone was evaluated in vitro experiments. Polyamide (PA)-MPs exhibited stronger adsorption, while polymethyl methacrylate (PMMA)-MPs displayed the weakest adsorption. Sorption equilibrium between PA-MPs and testosterone was achieved within 6 h, fitting the Pseudo-2nd-order model and Langmuir isotherm. The effects of MPs on male reproduction in mice was determined in vivo experiments. Male mice were treated with 0.1 and 0.5 mg/d PA-MPs/PMMA-MPs by gavage once per day for 28 days. The results showed that only 0.5 mg/d PA-MP exposure induced decreased serum testosterone levels, increased testicular testosterone levels compared to the control, and more severe damage to seminiferous tubule structure, sperm motility and sperm morphology compared to the PMMA-MPs group. Meanwhile, PA-MPs could reduce intracellular nuclear translocation of androgen receptor (AR) mediated by testosterone, while PMMA-MPs had no impact. The study revealed that PA-MP adsorption reduced testosterone bioavailability and caused sperm quality to decline, offering new insights into the combined toxicity mechanism of MPs in male mammals.

Comparative studies on the chemical composition and pharmacological effects of vinegar-processed antler glue modified from Lei Gong Pao Zhi Lun and traditional water-processed antler glue.

ETHNOPHARMACOLOGICAL RELEVANCE: Antler glue is a classic medicinal to enhance sexual function in traditional Chinese medicine (TCM), which was first recorded in Shen Nong Ben Cao Jing (Shennong's Classic of the Materia Medica). Vinegar-processing is a classic method of processing traditional Chinese medicine. The method of preparing antler glue by boiling antlers in vinegar and then concentrating them is recorded in Lei Gong Pao Zhi Lun (Master Lei's Discourse on Medicinal Processing). In modern times, the typical processing method of antler glue is water extraction and concentration. However, it is not clear whether there is a difference in the effect of these two processing methods on the chemical composition and pharmacological activity of antler glue. AIM OF THE STUDY: The Chinese Pharmacopoeia (2020) records that the processing method of antler glue is water extraction and concentration. But Lei Gong Pao Zhi Lun differs in Chinese Pharmacopoeia (2020), which records the processing method of vinegar extraction and concentration. The effect of the two processing methods on antler glue's chemical composition and pharmacological activity is unknown. So this study aimed to elucidate the difference between different processing methods on the chemical composition and the treatment effect on oligoasthenospermia of antler glue. MATERIALS AND METHODS: So the automatic amino acid analyzer is used to determine the amino acid content of two different processing methods of antler glue. Proteomics was performed to detect the protein components of two different processing methods of antler glue and analyze them. Cyclophosphamide-induced mice models of oligoasthenospermia were used to study the different pharmacological effects of antler glue in two different processing methods. An automatic sperm analyzer observed the quantity and quality of sperm in mice epididymis. Serum sex hormone testosterone (T), luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in mice were tested using the enzyme-linked immunosorbent assay (ELISA) kits. Hematoxylin-eosin (H&E) staining was used to analyze pathological alterations in mouse testicular tissue. The transcriptome has been used to reveal the potential mechanism of antler glue in treating oligoasthenospermia. Mitochondrial complex activity assay kits were used to assay the activity of mitochondrial respiratory chain complex I-V in mouse testicular tissue. Western blot was used to determine the expression of related proteins in mouse testicular tissue. RESULTS: Vinegar-processing can increase the alanine, proline, and glycine content in antler glue, reduce the length of protein peptides in antler glue, and produce a variety of unique proteins. Vinegar-processed antler glue (VAG) increased sperm density, sperm survival, sperm viability, and serum sex hormone levels in oligozoospermic mice. It reversed testicular damage caused by cyclophosphamide, and the effects were differently superior to those of water-processed antler glue (WAG). In addition, transcriptomics and related experiments have shown that VAG can increase the expression of Ndufa2, Uqcr11, Cox6b1, and Atp5i genes and proteins in mouse testis, thus promoting adenosine diphosphate (ATP) synthesis by increasing the activity of mitochondrial respiratory chain complexes I, III, IV and V. By promoting the oxidative phosphorylation process to produce more ATP, VAG can achieve the therapeutic effect of oligoasthenospermia. CONCLUSION: Vinegar-processing method can increase the content of active ingredients in antler glue. VAG increases ATP levels in the testes by promoting the process of oxidative phosphorylation to treat oligozoospermia.

In vivo exposure to electronic waste (e-waste) leachate and hydraulic fracturing fluid adversely impacts the male reproductive system.

Human health effects can arise from unregulated manual disassembly of electronic waste (e-waste) and/or hydraulic fracturing fluid spills. There is limited literature on the effects of e-waste and hydraulic fracturing wastewater exposure on the male reproductive system. Thus, this proof-of-concept study begins to address the question of how wastewater from two potentially hazardous environmental processes could affect sperm quality. Therefore, three groups of eight-week-old adult mice were exposed (5 d/wk for 6 wks) via a mealworm (Tenebrio molitor and Zophabas morio) feeding route to either: (1) e-waste leachate (50% dilution) from the Alaba Market (Lagos, Nigeria); (2) West Virginia hydraulic fracturing flowback (HFF) fluid (50% dilution); or, (3) deionized water (control). At 24-hours (hr), 3 weeks (wk), or 9-wk following the 6-wk exposure period, cohorts of mice were necropsied and adverse effects/persistence on the male reproductive system were examined. Ingestion of e-waste leachate or HFF fluid decreased number and concentration of sperm and increased both chromatin damage and numbers of morphological abnormalities in the sperm when compared to control mice. Levels of serum testosterone were reduced post-exposure (3- and 9-wk) in mice exposed to e-waste leachate and HFF when compared to time-matched controls, indicating the long-term persistence of adverse effects, well after the end of exposure. These data suggest that men living around or working in vicinity of either e-waste or hydraulic fracturing could face harmful effects to their reproductive health. From both a human health and economic standpoint, development of prevention and intervention strategies that are culturally relevant and economically sensitive are critically needed to reduce exposure to e-waste and HFF-associated toxic contaminants.

Metatranscriptomic characterization of six types of forensic samples and its potential application to body fluid/tissue identification: A pilot study.

Microorganisms are potential markers for identifying body fluids (venous and menstrual blood, semen, saliva, and vaginal secretion) and skin tissue in forensic genetics. Existing published studies have mainly focused on investigating microbial DNA by 16 S rRNA gene sequencing or metagenome shotgun sequencing. We rarely find microbial RNA level investigations on common forensic body fluid/tissue. Therefore, the use of metatranscriptomics to characterize common forensic body fluids/tissue has not been explored in detail, and the potential application of metatranscriptomics in forensic science remains unknown. Here, we performed 30 metatranscriptome analyses on six types of common forensic sample from healthy volunteers by massively parallel sequencing. After quality control and host RNA filtering, a total of 345,300 unigenes were assembled from clean reads. Four kingdoms, 137 phyla, 267 classes, 488 orders, 985 families, 2052 genera, and 4690 species were annotated across all samples. Alpha- and beta-diversity and differential analysis were also performed. As a result, the saliva and skin groups demonstrated high alpha diversity (Simpson index), while the venous blood group exhibited the lowest diversity despite a high Chao1 index. Specifically, we discussed potential microorganism contamination and the "core microbiome," which may be of special interest to forensic researchers. In addition, we implemented and evaluated artificial neural network (ANN), random forest (RF), and support vector machine (SVM) models for forensic body fluid/tissue identification (BFID) using genus- and species-level metatranscriptome profiles. The ANN and RF prediction models discriminated six forensic body fluids/tissue, demonstrating that the microbial RNA-based method could be applied to BFID. Unlike metagenomic research, metatranscriptomic analysis can provide information about active microbial communities; thus, it may have greater potential to become a powerful tool in forensic science for microbial-based individual identification. This study represents the first attempt to explore the application potential of metatranscriptome profiles in forensic science. Our findings help deepen our understanding of the microorganism community structure at the RNA level and are beneficial for other forensic applications of metatranscriptomics.

Alterations in sperm DNA methylation may as a mediator of paternal air pollution exposure and offspring birth outcomes: Insight from a birth cohort study.

Paternal exposure to environmental risk factors influences the offspring health. This study aimed to evaluate the association between paternal air pollution exposure mediated by sperm DNA methylation and adverse birth outcomes in offspring. We recruited 1607 fertile men and their partners from 2014 to 2016 and collected semen samples to detect sperm DNA methylation. Multivariate linear regression and weighted quantile sum regression models were used to assess the associations between paternal air pollution exposure and offspring birth outcomes. A critical exposure window was identified. Reduced representation bisulfite sequencing was used to detect sperm DNA methylation. The results demonstrated that high paternal exposure to PM2.5 (ß = -211.31, 95% CI: (-386.37, -36.24)), PM10 (ß = -178.20, 95% CI: (-277.13, -79.27)), and NO2 (ß = -84.22, 95% CI: (-165.86, -2.57)) was negatively associated with offspring's birthweight, especially in boys. Additionally, an early exposure window of 15-69 days before fertilization was recognized to be the key exposure window, which increased the risk of low birth weight and small for gestational age. Furthermore, paternal co-exposure to six air pollutants contributed to lower birthweight (ß = -51.91, 95% CI: (-92.72, -11.10)) and shorter gestational age (ß = -1.72, 95% CI: (-3.26, -0.17)) and PM2.5 was the most weighted pollutant. Paternal air pollution exposure resulted in 10,328 differentially methylated regions and the IGF2R gene was the key gene involved in the epigenetic process. These differentially methylated genes were predominantly associated with protein binding, transcriptional regulation, and DNA templating. These findings indicate that spermatogenesis is a susceptible window during which paternal exposure to air pollution affects sperm DNA methylation and the birth outcomes of offspring.