Inter- and trans-generational impacts of real-world PM2.5 exposure on male-specific primary hypogonadism.

Exposure to PM2.5, a harmful type of air pollution, has been associated with compromised male reproductive health; however, it remains unclear whether such exposure can elicit transgenerational effects on male fertility. Here, we aim to examine the effect of paternal exposure to real-world PM2.5 on the reproductive health of male offspring. We have observed that paternal exposure to real-world PM2.5 can lead to transgenerational primary hypogonadism in a sex-selective manner, and we have also confirmed this phenotype by using an external model. Mechanically, we have identified small RNAs (sRNAs) that play a critical role in mediating these transgenerational effects. Specifically, miR6240 and piR016061, which are present in F0 PM sperm, regulate intergenerational transmission by targeting Lhcgr and Nsd1, respectively. We have also uncovered that piR033435 and piR006695 indirectly regulate F1 PM sperm methylation by binding to the 3'-untranslated region of Tet1 mRNA. The reduced expression of Tet1 resulted in hypermethylation of several testosterone synthesis genes, including Lhcgr and Gnas, impaired Leydig cell function and ultimately led to transgenerational primary hypogonadism. Our findings provide insights into the mechanisms underlying the transgenerational effects of paternal PM2.5 exposure on reproductive health, highlighting the crucial role played by sRNAs in mediating these effects. The findings underscore the significance of paternal pre-conception interventions in alleviating the adverse effects of environmental pollutants on reproductive health.

PM2.5 caused ferroptosis in spermatocyte via overloading iron and disrupting redox homeostasis.

Fine particulate matter (PM2.5) has been reported to cause various types of damage to male reproductive system, but the research on the underlying mechanisms is still insufficient. This study attempted to explore the underlying mechanisms of this widely concerning environmental health problem through in vivo and in vitro exposure models. Significant pathological damage and abnormal mitochondria in spermatocytes were observed in the real-time PM2.5 exposure animal model. In addition, significant alterations in key biomarkers of iron metabolism and ferroptosis were found in testis tissues. Notably decreased cell viability was found in vitro. Moreover, the ferroptosis pathway was significantly enriched in the transcriptome enrichment analysis. Subsequent experiments showed that the two core events of ferroptosis, iron overload and lipid peroxidation, occurred in spermatocytes after PM2.5 treatment. Moreover, lipid metabolic genes (Acsl4 and Aloxe3) and the antioxidant gene Gpx4 were found to be key target genes of ferroptosis caused by PM2.5 in spermatocytes. Importantly, further studies showed that the damaging effect could be reversed by the iron chelator deferoxamine mesylate (DFOM) and the lipid peroxidation inhibitor ferrostatin-1 (Fer-1), which further confirmed the role of ferroptosis in PM2.5 toxicity. Our study revealed the vital role of ferroptosis in PM2.5-induced male reproductive damage, providing novel insights into the air pollution-induced decrease in male fertility.

Genome-wide alternation and effect of DNA methylation in the impairments of steroidogenesis and spermatogenesis after PM2.5 exposure.

The effects of ambient fine particles on male reproductive health have raised widespread concern. The particular underlying mechanisms of the damage remain largely unclear and demand more research in new directions. Previous research has revealed that DNA methylation plays an important role in male reproductive development and is also vulnerable to environmental influences. However, there hasn't been enough investigation into the involvement of DNA methylation in PM2.5-induced male reproductive toxicity. Here, we establish a real-time PM2.5 exposure model and revealed that PM2.5 exposure could lead to testicular dysfunction including spermatogenesis impairment and steroid hormone dysfunction. In particular, the decrease in the testicular global level of 5-methylcytosine (5mC) indicated a possible association of DNA methylation with testicular injury induced by PM2.5 exposure. Further genome-wide methylation analysis revealed genomic hypomethylation of testicular DNA and identified more than 1000 differentially methylated regions in both CAP and UA versus FA, indicating that PM2.5 exposure, even low-dose, could modulate the testicular methylome. Furthermore, integrated analysis of methylome and transcriptome identified some key methylated genes and networks, which may be involved in spermatogenesis and synthesis of steroid hormone. The testicular methylation levels of key genes especially Cyp11a1 and Pax8 raised, and their consequent reduced expression may impair the testosterone and sperm production process. Our research provides fundamental knowledge as well as novel insights into the possible involvement of DNA methylation in PM2.5-induced male reproductive harm.

Benzo[a]pyrene inhibits testosterone biosynthesis via NDUFA10-mediated mitochondrial compromise in mouse Leydig cells: Integrating experimental and in silico toxicological approaches.

Benzo[a]pyrene (B[a]P), a representative of polycyclic aromatic hydrocarbons (PAHs), is ubiquitously spread in the environment and showing deleterious impacts on male steroidogenesis, including testosterone synthesis disorder. However, the precise mechanisms involved in B[a]P-induced steroidogenesis perturbation remains obscure. In the present study, we integrated in vivo tests, transcriptome profiling, in vitro assays, and conjoint in silico toxicological approaches to delineate the detailed mechanisms. In mouse models, we observed that B[a]P administration remarkably inhibited testosterone synthesis accompanied by ultrastructural impairments of mitochondria and mitophagosome formation in mouse Leydig cells. Transcriptome profiling showed that B[a]P down-regulated the expression of Ndufa9, Ndufa6, Ndufa10, and Ndufa5 in mouse testes, which are identified as critical genes involved in the assembly and functionality of mitochondrial complex I. In the in vitro tests, the bioactive B[a]P metabolite BPDE induced perturbation of testosterone synthesis by NDUFA10-mediated mitochondrial impairment, which was further exacerbated by mitophagy in TM3 Leydig cells. The findings of in silico toxicological analyses were highly consistent with the experimental observations and further unveiled that B[a]P/BPDE-involved PPARα activation could serve as a molecular initiating event to trigger the decline in Ndufa10 expression and testosterone synthesis. Overall, we have shown the first evidence that mitochondrial compromise in Leydig cells is the extremely crucial target in B[a]P-induced steroidogenesis perturbation.

Analysis by transcriptomics and metabolomics for the proliferation inhibition and dysfunction through redox imbalance-mediated DNA damage response and ferroptosis in male reproduction of mice and TM4 Sertoli cells exposed to PM2.5.

Sertoli cells play a pivotal role in the complex spermatogenesis process. This study aimed to investigate the effects of PM2.5 on Sertoli cells using the TM4 cell line and a real time whole-body PM2.5 exposure mouse model, and further explore the underlying mechanisms through the application of metabolomics and transcriptomics. The results in vivo and in vitro showed that PM2.5 reduced Sertoli cells number in seminiferous tubules and inhibited cell proliferation. PM2.5 exposure also induced Sertoli cell dysfunction by increasing androgen binding protein (ABP) concentration, reducing the blood-testis barrier (BTB)-related protein expression, and decreasing glycolysis capacity and lactate production. The results of transcriptomics, metabolomics, and integrative analysis of multi-omics in the TM4 Sertoli cells revealed the activation of xenobiotic metabolism, and the disturbance of glutathione and purine metabolism after PM2.5 exposure. Further tests verified the reduced GSH/GSSG ratio and the elevation of xanthine oxidase (XO) activity in the PM2.5-exposed TM4 cells, indicating that excessive reactive oxygen species (ROS) was generated via metabolic disorder caused by PM2.5. Moreover, the redox imbalance was proved by the increase in the mitochondrial ROS level, superoxide dismutase (SOD) and catalase (CAT) activity, as well as the activation of the Nrf2 antioxidative pathway. Further study found that the redox imbalance caused by PM2.5 induced DNA damage response and cell cycle arrest. Additionally, PM2.5 induced ferroptosis through iron overload and lipid peroxidation. Taken all together, our study provided new insights for understanding proliferation inhibition and dysfunction of TM4 Sertoli cells exposed to PM2.5 via metabolic disorder and redox imbalance-mediated DNA damage response and ferroptosis.

The prevalence of male rotating shift work correlates with reduced total fertility rate: an ecological study of 54,734 reproductive-aged males in 35 European countries between 2000 and 2015.

Europe has the lowest fertility rate in the world, and 19 European countries are already experiencing negative population growth. Previous studies have shown that light-dark cycle misalignment induces damages to semen quality in animals, but whether shift work impairs male human fertility remains controversial. The diverse types of shift work, which may have distinct health impacts, have not been differentiated in the few previous studies. The present study aims to assess the impact of different male shift work types, i.e., rotating shift work (RSW), permanent shift work (PSW), and daily split shifts (DSS), on male fertility in European countries, based on the European Working Conditions Survey of 54,734 men and 52,457 women of reproductive (15-49 years) age from 35 countries conducted between 2000 and 2015. Year-specific total fertility rate (TFR) for each country was obtained from the World Bank. Mixed linear model was used to analyze the association between the prevalence of shift work types and TFR among the countries during the 15 years span, with adjustment for latitude, per capita GDP, working duration, working frequency, median age of men or women engaged in shift work, education level of men or women, and female shift-work condition in reproductive aged participants. The results showed that the average prevalence of RSW declined from 15.6% in 2000 to 9.8% in 2010, and then rose to 11.1% by 2015, while TFR went up from 1.45 in 2000 to 1.60 in 2010, and then decreased to 1.56 in 2015. A 10% increase of RSW prevalence was associated with a 0.114 decrease of TFR (95% CI: -0.167, -0.061, P < .001), indicating 11.4 fewer births per 100 women throughout lifetime. The association remained significant (ß = -0.088, 95% CI: -0.160, -0.015, P = .020) after adjusting for confounding factors. However, no association was observed between TFR and the prevalence of male PSW (ß = -0.011, 95% CI: -0.094, 0.073, P = .801) or DSS (ß = 0.357, 95% CI: -0.171, 0.884, P = .189). In conclusion, male RSW, rather than PSW and DSS, may reduce TFR. Further studies are needed to validate our findings.

Analysis by Metabolomics and Transcriptomics for the Energy Metabolism Disorder and the Aryl Hydrocarbon Receptor Activation in Male Reproduction of Mice and GC-2spd Cells Exposed to PM2.5.

Fine particulate matter (PM2.5)-induced male reproductive toxicity arouses global public health concerns. However, the mechanisms of toxicity remain unclear. This study aimed to further investigate toxicity pathways by exposure to PM2.5in vitro and in vivo through the application of metabolomics and transcriptomics. In vitro, spermatocyte-derived GC-2spd cells were treated with 0, 25, 50, 100 µg/mL PM2.5 for 48 h. In vivo, the real-world exposure of PM2.5 for mouse was established. Forty-five male C57BL/6 mice were exposed to filtered air, unfiltered air, and concentrated ambient PM2.5 in Tangshan of China for 8 weeks, respectively. The results in vitro and in vivo showed that PM2.5 exposure inhibited GC-2spd cell proliferation and reduced sperm motility. Mitochondrial damage was observed after PM2.5 treatment. Increased Humanin and MOTS-c levels and decreased mitochondrial respiratory indicated that mitochondrial function was disturbed. Furthermore, nontargeted metabolomics analysis revealed that PM2.5 exposure could disturb the citrate cycle (TCA cycle) and reduce amino acids and nucleotide synthesis. Mechanically, the aryl hydrocarbon receptor (AhR) pathway was activated after exposure to PM2.5, with a significant increase in CYP1A1 expression. Further studies showed that PM2.5 exposure significantly increased both intracellular and mitochondrial reactive oxygen species (ROS) and activated NRF2 antioxidative pathway. With the RNA-sequencing technique, the differentially expressed genes induced by PM2.5 exposure were mainly enriched in the metabolism of xenobiotics by the cytochrome P450 pathway, of which Cyp1a1 was the most significantly changed gene. Our findings demonstrated that PM2.5 exposure could induce spermatocyte damage and energy metabolism disorder. The activation of the aryl hydrocarbon receptor might be involved in the mechanism of male reproductive toxicity.

Female and male sleep duration in association with the probability of conception in two representative populations of reproductive age in US and China.

OBJECTIVE: Sleep duration has been found to affect some reproductive phenotypes but fecundability has been rarely researched. We aim to evaluate the association between female/male sleep duration and the probability of conception in two representative populations. METHODS: The present study uses two datasets, namely, a cross-sectional dataset of 9137 reproductive-age females in the US (National Health Interview Survey, NHIS) and a longitudinal dataset of 2687 reproductive-age females and their male mates in China (China Health and Nutrition Survey, CHNS). Logistic regression or mixed model was used to analyze the association between sleep duration and the probability of conception in the females of both populations and in CHNS males with adjustments for demographic, socioeconomic, behavioral, sleep health and reproductive factors. RESULTS: An inverse association was observed between male sleep duration (≥8 h/day) and their mates' conception probability in the CHNS population (P = 0.012). Sleep of 9 h/day and ≥10 h/day in men was associated with 0.65 (0.41-1.02) fold and 0.53 (0.31-0.90) fold of conception probability when compared to 8 h/day sleep. On the other hand, a U-shaped association between female sleep duration and conception probability was observed in both populations. Each hour/day departure (longer or shorter) from 7 h/day sleep was associated with 1.26 (1.12-1.42, P < 0.001) and 1.21 (1.03-1.41, P = 0.019) fold conception probability in the NHIS and CHNS populations, respectively. An adjustment for potential confounders, including spouse characteristics did not substantially attenuate these associations. CONCLUSIONS: Female and male sleep duration may be independent predictors of conception, suggesting there is an intervention target for reproductive health.

Adverse effects of circadian desynchrony on the male reproductive system: an epidemiological and experimental study.

STUDY QUESTION: Is circadian desynchrony a risk factor of male reproductive damage in semen parameters and/or reproductive hormones? SUMMARY ANSWER: Circadian desynchrony correlates with decrease of sperm count, which was improved when circadian desynchrony was attenuated. WHAT IS KNOWN ALREADY: Circadian desynchrony caused by work (shift work) and non-work-related reasons is prevalent worldwide and has been found to be associated with decreased female fertility, but whether it harms male reproductive health is unclear. STUDY DESIGN, SIZE, DURATION: A hybrid research was conducted. (i) A cross-sectional study of 1346 Chinese men in 2007 was used to analyze the association between semen/hormone biomarkers and work-related circadian desynchrony, which was divided into rotating shift work and permanent shift work against non-shift work. (ii) A cohort of 796 Chinese undergraduates from 2013 to 2014 was used to analyzed the association between semen/hormone biomarkers and non-work-related circadian desynchrony (between school days and days off). (iii) The biomarker identified simultaneously in both populations was further validated in male C57BL/6J mice housed under conditions simulating circadian desynchrony. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 17 semen/hormone biomarkers were compared among rotating shift workers and permanent shift workers against non-shift workers in the 1346 reproductive-age Chinese men. A total of 14 semen/hormone biomarker was analyzed in the undergraduate cohort for correlation with non-work-related circadian desynchrony (measured by Munich Chronotype Questionnaire) in 2013 and 2014 and compared between the 2 years. Photoperiod-shifting method was used to establish the mouse model, in which the biomarker was examined and molecular mechanism was explored by apoptosis analysis, DNA content analysis, transcriptome sequencing, real-time PCR and western blotting. MAIN RESULTS AND THE ROLE OF CHANCE: Among the semen/hormone biomarkers, sperm count was found to be lower in rotating shift workers, who had a higher risk of low sperm count defined by Chinese Ministry of Health (total sperm/ejaculate < 120 × 106) than non-shift workers (odds ratio = 1.26, 95% CI 1.05-1.52). This biomarker was replicated in the undergraduate cohort, where each hour of circadian desynchrony was associated with 1.16 (95% CI 1.02-1.31) fold odds of low sperm count, and sperm count increased during 2014 in men who reduced circadian desynchrony after 2013. A decrease of sperm count with circadian desynchrony and its recovery after removal of circadian desynchrony was also observed in the mouse model. During asynchrony, increased apoptosis was found in seminiferous tubules and the marker genes of post-spermatocyte stage cells were down-regulated. The most enriched functional pathway was homologous recombination, which happened during meiosis. LIMITATIONS, REASONS FOR CAUTION: The study of human beings was observational while the animal study has potential difference in circadian desynchrony exposure and species susceptibility. Further researches are needed to clarify the causal relationship in men. WIDER IMPLICATIONS OF THE FINDINGS: These findings provide novel insight to the effect of circadian desynchrony on male reproductive health and a potential strategy for prevention of reproductive damage. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Key R&D Program of China [2017YFC1002001] and National Natural Science Foundation of China [81871208]. There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: NA.

Role of Aliphatic Chain Characteristics on the Anti-Cracking Properties of Polymer-Modified Asphalt at Low Temperatures.

The anti-cracking properties of polymer-modified asphalt depend largely on the molecular structure of the polymer modifier. However, the mysterious structure-performance relationship is still elusive. In this paper, three kinds of polymers with different chain structures were selected to address this issue. The indices of styrene, trans-butadiene, aliphatic branched-chain, and aliphatic long-chain from the infrared spectrum were used to quantify the functional group compositions of polymer modifiers. Viscoelastic parameters, including relaxation time, dissipation energy ratios, and stiffness were assessed to illustrate the anti-cracking properties of polymer-modified asphalt. Results showed that relaxation time and dissipation energy ratios were mainly determined by the polymer network strength, molecular size, aliphatic chain feature, and the orientations speed of aliphatic chains. The short relaxation time and high dissipation ratio lead to the low stiffness and favorable low-temperature performance of asphalt. The improvement of these performances requires a polymer with high indices of an aliphatic long-chain, styrene, aliphatic branched-chain, and trans-butadiene, respectively. An aliphatic-long chain, aliphatic branched-chain, and trans-butadiene were soft segments in asphalt while styrene was the rigid segment. The soft segments affect the intramolecular friction, orientation, and thermal motion at low temperatures, whereas the rigid segment enhances the strength of polymer networks. Thus, the anti-cracking property of polymer-modified asphalt can be improved by adjusting the ratio of soft and rigid segments in the polymer modifier.