To investigate the transgenerational effects of paternal PM_{2. 5} exposure on offspring growth and development, and to preliminarily elucidate the role of sperm DNA methylation modifications in mediating these effects.

Eight-week-old male C57BL/6 mice were randomly divided into filtered air (FA), unfiltered air (UA), and concentrated PM_{2. 5} (CAP) groups, with 10 animals in each group. The exposure was conducted from November 2019 to April 2020, and then, these male mice were mated with unexposed females to generate F1 offspring, which were bred successively to produce F2 and F3 generations. All the offspring were living in PM_{2. 5}-free environment. The birth body weight, birth number, and sex ratio of the offspring were recorded, body weight growth was monitored, and organ coefficients of the heart, liver, lung, and brain were calculated. Whole-genome methylation sequencing was performed on the sperm DNA of the CAP group, FA group, and their F1 generation offspring to screen for differentially methylated regions, and the genes and pathways associated with these regions were analyzed.

When compared with the F1~F3 offspring of the FA group, the CAP group had significantly reduced birth body weight in the F1 generation (P<0. 05), no statistical differences were observed in the birth body weight in the F2 and F3 generations (P>0. 05), or either in the sex ratio and birth number among the F1, F2 and F3 generations. Compared with the FA group offspring, the F1~F3 offspring of CAP group exhibited delayed body weight gain, especially in the males (P<0. 05), the CAP-F1 male generation had obviously elevated liver organ coefficient (P<0. 01), but no statistical changes were observed in the heart, lung, or brain coefficients among the F1~F3 generations. Between the FA group and the CAP group, 37997 differentially methylated regions were detected, with a reduction of approximately 50% in the number of differentially methylated regions in the F1 generation. Differentially methylated genes in F0 and F1 sperm were potentially related to developmental processes, including imprinting genes (Gnas, Igf2)and metabolic genes (Ppard, Rps6kb1).

Paternal exposure to PM_{2. 5} leads to reduced birth weight and intergenerational growth retardation in offspring. Its impact on phenotypic effects is gradually weakened during intergenerational transmission. Changes in the methylation of development-related genes in sperm may be one of the mechanisms mediating this intergenerational effect.

To analyze the changes in fertility trends of seasonally and calendar adjusted fertility rate (SAFR) in different countries before and after the COVID-19 pandemic outbreak and investigate the influence factors.

Monthly SAFR data of 28 countries during January 2012 and December 2022 were collected in the Human Fertility Database (HFD). The node of December 2020 (9-month delay in gestation from the start of the March 2020 outbreak) was used as the point of division for comparison between the pre- (2012.1-2020.11) and post-pandemic (2020.12-2022.12) periods. Interrupted time series was used to analyze whether fertility rate trends (short-term fluctuations and long-term trends) changed before and after the pandemic in each country. Wilcoxon test was employed to analyze whether pre-pandemic fertility rate, GDP per capita, public health and social measures (PHSM) and unemployment rate were associated with the changes in fertility trends.

Nineteen out of the 28 countries experienced a short-term decline in fertility rate after the pandemic, which subsequently rebounded. With respect to long-term trends, 2 countries shifted from a declining trend to an increasing trend, 8 countries shifted from an increasing trend to a declining trend, and 6 countries had their SAFR unchanged. The SAFR decline was mainly observed in some of the countries of Central Europe and the Western Mediterranean, while its increase was mainly in Northern and Western Europe. Pre-pandemic SAFR was lower in the countries with no short-term fluctuations in SAFR than those with short-term fluctuations (P=0.041). The countries with declining rate of SAFR had significantly lower pre-pandemic SAFR (P=0.005) and GDP per capita (P=0.027) than the countries with increasing rate of SAFR. No association was found between short-term fluctuations or long-term trends in fertility rate and the PHSM severity index or unemployment rate.

The COVID-19 pandemic has had varying short- and long-term impacts on SAFR in 28 countries, with the countries with relatively low economic levels and pre-pandemic SAFRs more vulnerable to further blows. The longer-term impacts of the COVID-19 pandemic on the populations of each country deserve further attention.

To explore the potential mechanism of Benzo (K) fluoranthene (BkF) on male reproductive injury in mice by proteomics and metabolomics.

Twenty healthy and clean male Kunming mice (6 weeks old, 18±2 g) were randomly divided into control group (corn oil group), low-, medium- and high-dose BkF groups (7.5, 15.0 and 30.0 mg/kg), with 5 mice in each group. The corresponding agents were gavaged at a dose of 10 mL/kg, 5 d per week, for 35 consecutive days. After modeling, the rats were fasted for 10 h, and then sperm samples and testicular tissues were harvested. Computer assisted sperm analysis (CASA) was used to detect and analyze semen parameters. HE staining was employed to observe the histopathological structure of the testicular tissue. Bioinformatics analysis was applied to analyze the differential protein pathways. Volcano plot were conducted to analyze the top 10 differentially expressed proteins (DEPs) in the control and high-dose BkF group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) untargeted metabolomics techniques were utilized to screen out differential metabolites. KEGG signaling pathway and KEGG annotation analyses and GO enrichment analysis were used to analyze the differential metabolites.

Compared with the control group, the sperm number and motility of BkF-treated mice showed a decreased trend, with statistical differences (P<0.05). Pathological observation showed that BkF treatment resulted in dilated seminal tubules and badly-arranged spermatogenic cells when compared with the control group. Proteomics analysis found that the protein levels of Spata46 and Rab5b were decreased, while those of Zscan21 and Aifm2 were increased (P<0.01). Proteomic KEGG enrichment analysis showed that it was mainly involved in phagosome, protein export, ribosome and other pathways. GO enrichment analysis indicated that it was mainly involved in male meiosis I, histone acetylation, regulation of p53 signaling pathway, positive regulation of cell cycle, positive regulation of cell death and other signaling pathways. Metabonomics KEGG displayed that amino sugar and nucleotide sugar metabolism were most closely related to other metabolic pathways.

Proteomics and metabolomics analyses show that BkF exposure is associated with spermatogenesis, apoptosis and cell cycle, DNA damage, amino sugar and nucleotide sugar metabolism.

To investigate the effect of polystyrene microplastic（PS-MPs）on the apoptosis of male germ cells.

Genes related to male urogenital disorders caused by polystyrene were searched in the Comparative Toxicogenomics Database（CTD）. Gene Ontology（GO）analysis and Kyoto Encyclopedia of Genes and Genomes（KEGG）analysis were used to analyze the involved functions and signaling pathways. A cell model was established based on mouse mesenchymal cells（TM3 cells）, and then 0, 10, 20 and 40 μg/mL PS-MPs with a particle size of 5 μm and 80 nm respectively were used to treat TM3 cells respectively for 48 h. Cell morphology was observed, cell viability was detected with CCK-8 assay, apoptosis by flow cytometry, and mRNA and protein expression levels of key molecules in the p53 signaling pathway were detected by RT-qPCR and Western blotting, respectively.

GO and KEGG enrichment analyses showed that the genes screened from CTD database were found to be significantly enriched in cell apoptosis and p53 signaling pathway（P<0.05）. PS-MPs of 5 μm and 80 nm in size treatment for 48 h resulted in significant inhibition of cell growth while enhancement in cell apoptosis（P<0.05）, up-regulated the mRNA and protein levels of p53 and Bax in the p53 signaling pathway（P<0.05）, enhanced downstream molecules Caspase3 and Caspase9（P<0.05）, and inhibited Bcl2 at mRNA and protein levels in TM3 cells（P<0.05）.

PS-MPs exposure may induce apoptosis of testicular mesenchymal TM3 cells via the p53 signaling pathway.

To investigate the association between exposure levels of air pollutants（PM₁₀, PM_2.5, SO₂, O₃, NO₂, and CO）and infertility risk in men of childbearing age.

Preconception Reproductive Health and Birth Outcomes Cohort（PREBIC）was subjected by recruiting men of childbearing age who taking pre-pregnancy health examination in Chongqing Maternal and Child Health Hospital since November 2018. The baseline indicators were collected and established after their enrollment. Then they were followed up every 3 months, and failure to achieve a clinical pregnancy after 12 months of unprotected intercourse was defined as infertility. The individualized air pollution exposure levels of the subjects in 3 months prior to enrollment（spermatogenic cycle）were assessed by machine learning algorithm. Logistic regression model was established to determine the effect of air pollutant exposure on infertility and the potential susceptibility windows induced by single pollutant. Sensitivity analysis was carried out using two-pollutant model. The joint effects and interaction of air pollutants on infertility were analyzed by Bayesian kernel machine regression.

A total of 2 297 male subjects were included in the study. After multiple test adjustment, the relative risk of infertility was increased by 29.3%（OR=1.293, 95%CI: 1.022-1.636, P=0.008）in the highest and lowest quartiles of PM₁₀ exposure during the 90 d prior to enrollment. In the analysis of susceptibility windows, PM₁₀ was associated with infertility at all stages from 0 to 90 d, and no significant susceptibility window was observed. No significant results were observed in the analysis of the joint effects and interactions of air pollutants.

Male exposure to PM₁₀ may increase the risk of infertility.

To investigate the effect of 1-nitropyrene（1-NP）on the proliferation, cycle progression and sex hormone synthesis of human ovarian granulosa cell line KGN via inducing mitochondrial damage.

After KGN cells were treated with 1-NP at different concentrations（0.625, 1.25, 2.5, 5 and 10 μmol/L）for 48 h, cell proliferation, cell cycle, and contents of estradiol and pregnenolone in the supernatant were detected by EdU assay, flow cytometry and ELISA, respectively. And the levels of reactive oxygen species（ROS）, mitochondrial superoxide（MitoSOX）and mitochondrial membrane potential were examined with specific fluorescent probes including DCFH-DA probe, MitoSOX probe and JC-1 probe, respectively. In addition, ATP kit was adopted to measure the cellular ATP level, and Western blotting was also conducted to determine the expression of proteins related with the cell proliferation, cycle progression, DNA damage response and synthesis of sex hormones. Finally, the mitochondrial antioxidant idebenone（IDE, 1 μmol/L）and 1-NP（10 μmol/L）were combined to treat the cells for 48 h, and then the above indicators were determined correspondingly again.

As compared with the control cells, 1-NP（0.625~10 μmol/L）treatment significantly inhibited cell proliferation（P<0.05）. The expression of PCNA, a marker of cell proliferation, was reduced after treatment with 1-NP（2.5, 5 or 10 μmol/L）, and the percentage of cells at G₂/M phase was elevated at 5 and 10 μmol/L of 1-NP（P<0.05）. Western blotting showed that the expression of γ-H2AX began to rise after 1-NP treatment（10 μmol/L）for 3 h（P<0.01）, and the levels of DNA damage pathway proteins, p-ATM, p53 and p21^cip1 were also obviously increased（P<0.05）, while those of G₂/M phase block-related proteins, CDK-1 and CyclinB1, were notably decreased at different doses of 1-NP（P<0.05）. In addition, the contents of estradiol and pregnenolone in the supernatant of KGN cells were remarkably diminished after 1-NP treatment（P<0.05）, and the expression levels of sex hormone synthesis-related proteins CYP19 and CYP11A1 were inhibited as well（P<0.05）. The detection of mitochondrial and oxidative stress-related indicators indicated that the levels of ROS and MitoSOX in KGN cells were greatly up-regulated due to 1-NP treatment, while the mitochondrial membrane potential and ATP level were decreased in a dose-dependent manner. However, the combination of 1-NP and IDE significantly declined the percentage of G₂/M-phase cells, elevated the contents of estradiol and pregnenolone, and restored the expression levels of proteins related to DNA damage, cell cycle as well as sex hormone synthesis when compared with the cells treated with 10 μmol/L 1-NP. Moreover, the combination treatment also ameliorated the mitochondrial damage induced by 1-NP, including lowering ROS and MitoSOX levels and improving the mitochondrial membrane potential and ATP level.

1-NP can cause mitochondrial damage, which leads to proliferation inhibition, cycle arrest and disorder of sex hormone synthesis, improve mitochondrial function, and thus partially alleviate 1-NP-induced dysfunction in KGN cells.

To analyze the effect of subchronic exposure of polystyrene nanoplastics（PS-NPs）on reproductive function in adult male mice, and to explore the preliminary molecular mechanism of its impact on testosterone synthesis.

Sixty 8-week-old C57BL/6 male mice were fully randomly divided into control group（deionized water）and PS-NPs low（10 mg/kg, 10 PS）and high exposure group（40 mg/kg, 40 PS）, with 20 mice in each group. The mice were treated once per day for 60 d by gavage. The weights and organ coefficient of the testes, epididymis, brain, liver, spleen, lung and kidney of each group of mice were weighed and calculated at 24 h after the last exposure. HE staining was used to analyze the histopathological differences of testicular tissue, and transmission electron microscopy was used to observe the ultrastructural changes of testicular cells. The computer assisted sperm assay system was used to analyze semen parameters, the contents of serum testosterone（T）, luteinizing hormone（LH）and follicle-stimulating hormone（FSH）were detected by enzyme-linked immunosorbent assay（ELISA）, and the expression levels of key genes for T synthesis, which had been screened by transcriptome sequencing, were determined in testicular tissues by real-time PCR.

Compared with the control group, the weight of the epididymis in the 40 PS group was increased significantly（P<0.05）, the testicular organ coefficients of mice in the 10 PS group and the epididymis organs coefficients of mice in the 40 PS group were increased obviously（P<0.05）, and the weight and organ coefficients of the liver and spleen of the mice in the 10 PS and 40 PS groups were decreased remarkably（P<0.05）. The pathological results showed that compared with the testis of the control group, the lumen of the semininogenic tubule in the 10 PS and 40 PS groups were enlarged, the semimatogenic cells were loose, and the vacuoleization was increased. Transmission electron microscopy displayed that mitochondrial swelling and endoplasmic reticulum expansion were observed in the cytoplasm of Leydig cells in 10 PS and 40 PS groups. The sperm motility of mice in the 10 PS group was decreased significantly compared with the control group（P<0.05）. The serum T level of mice in the 10 PS and 40 PS groups was decreased, with that of the 40 PS group more significant（P<0.05）. The serum LH and FSH levels of mice in the 10 PS and 40 PS groups were increased notably. Compared with the control group, the mRNA levels of CYP11A1 in the testes of mice were significantly down-regulated in both 10 PS and 40 PS groups（P<0.05）.

PS-NPs can cause damage to the testes pathologically and functionally in mice, and may reduce T synthesis by inducing structural abnormalities of Leydig cells and down-regulating CYP11A1 expression.

To study the relationship of urinary total arsenic exposure level with sperm quality and sex hormone levels in young male population, and evaluate the potential impact of background arsenic exposure on parameters of sperm quality and sex hormone levels in Chongqing.

Generalized additive model and recursive algorithm were used to analyze the correlation of the baseline urine arsenic concentration with semen parameters and sex hormone levels in 703 male college students volunteers（18~22 years old）in the cohort of Chongqing college students constructed in 2013-2015. The inflection point of the nonlinear relationship was explored, and the threshold effect of arsenic exposure was analyzed. The median of urinary arsenic in this study was compared with the data from other reports at home and abroad.

There was a nonlinear relationship of urine arsenic level with sperm concentration, sperm total number, sperm DNA fragment index, and levels of prolactin, follicle-stimulating hormone and progesterone. Further threshold effect analysis showed that there were inflection points of urine arsenic concentration with the above indicators, and the key points were 15.17, 14.17, 50.97, 57.51, 11.36, and 70.44 μg/L, respectively（Log-likelihood ratio test P<0.05）. Compared with relevant literature, the urine arsenic level was at a relatively lower level in this cohort population.

Compared with some regions at home and abroad, the background urine arsenic level of this study population is quite lower, and significant correlation is found in the background level with the decline of sperm quality parameters and changes in sex hormone levels.

To establish a damage model of mouse spermatocyte GC-2spd cells induced by benzo[k] fluoranthene (BkF), a representative compound of high-molecular weight polycyclic aromatic hydrocarbons, so as to investigate the effect and mechanism of BkF on GC-2 cells.

Related genes of male reproductive diseases associated with BkF were screened in Comparative Toxicogenomics Database (CTD), and functional and pathway enrichment analysis is conducted subsequently to explore the possible pathway mechanism. Moreover, GC-2 cells were treated with BkF at a final concentration of 0, 10, 20, 40 or 80 μmol/L for 72 h, and then the cell cycle, oxidative stress and DNA damage effect were detected. Western blotting was used to determine the protein levels of the key genes as well as the core molecules of related pathways.

The key genes CYP1A1 and CYP17A1 associated with BkF-related reproductive system diseases were screened out from the CTD database. Gene Ontology (GO) enrichment analysis found that they were involved in male reproductive system and cell cycle arrest, obviously enriched in biological processes such as oxidative stress. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these genes were significantly enriched in cytochrome P450 pathway (P<0.01). After GC-2 cells were exposed to BkF for 72 h, the proliferation was significantly decreased in a dose-dependent manner, as compared with the control group. When the exposure dose was higher than 40 μmol/L, the cell cycle arrest was obvious (P<0.05); Both the intracellular reactive oxygen species (ROS) level (P<0.05) and the protein level of DNA damage marker γ-H2AX (P<0.01) were remarkably increased in a dose-dependent manner. In addition, the protein levels of CYP1A1 and CYP17A1 were greatly higher in the exposure group than the control group (P<0.05), while treatment of AhR inhibitor CH223191 resulted in notably down-regulated the expression of the 2 proteins (P<0.05).

BkF induces the activation of AhR receptors, then causes elevated expression of downstream genes CYP1A1 and CYP17A1 as well as increased intracellular ROS, and finally leads to oxidative stress and genetic damage in GC-2 cells.

To explore the role and mechanism of RNA demethylase FTO in the apoptosis of mouse spermatocyte GC-2 cells induced by bisphenol S (BPS).

GC-2 cells were treated with BPS at a final concentration of 0 (DMSO control group), 20, 40, 80, 160, and 320 μmol/L for 72 h. The cell morphology changes were observed with microscopy. CCK-8 assay was utilized to detect cell viability, and flow cytometry was used to detect cell apoptosis and cycle changes. Colorimetric assay was adopted to measure the global level of m6A RNA methylation. Western blotting was employed to detect the protein levels of RNA methyltransferase METTL3, RNA demethylase FTO, RNA binding protein, YTHDF1, apoptosis-related molecules P53, P21, BCL2, Caspase9, Caspase3, BAX and cell cycle-related molecules CyclinD1, CDK4, CDK2. After FTO gene interference cell model was established, CCK-8 assay was used to measure cell survival and flow cytometry to detect cell apoptosis. The protein levels of FTO, P53, BCL2 and BAX were detected by Western blotting.

Compared with the control group, the number of GC-2 cells was significantly reduced and more vacuole cells appeared in the BPS exposure group. The cell survival rate was significantly decreased and the apoptotic rate was markedly increased at 160 and 320 μmol/L of BPS (P<0.05). The protein levels of apoptosis-related molecules P53, P21, Caspase9 and Caspase3 were increased significantly, and that of BCL2 was decreased significantly (P<0.05). When the BPS concentration was 80, 160 and 320 μmol/L, cell cycle was arrested at G₁/S phase (P<0.05). The protein levels of G₁/S phase related molecules CyclinD1, CDK4, and CDK2 were significantly down-regulated (P<0.05). When the dose of BPS increased to 80~320 μmol/L, the global RNA methylation level was relatively increased (P<0.01). The protein levels of RNA methyltransferase METTL3 and RNA binding protein YTHDF1 did not change obviously, while that of RNA demethylase FTO decreased sharply (P<0.05). Compared with the control group, after interference with FTO expression, the number and viability of GC-2 cells were significantly reduced, and the apoptotic rate was notably increased (P<0.05). The protein level of P53 was remarkably up-regulated, and that of BCL2 down-regulated when FTO expression was knocked down (P<0.05).

RNA demethylase FTO may play an important role in BPS-induced apoptosis of GC-2 cells through regulating P53-P21 and BCL2 mitochondrial pathway.