Multi-Omics Analysis Reveals the Toxicity of Polyvinyl Chloride Microplastics toward BEAS-2B Cells.

Polyvinyl chloride microplastics (PVC-MPs) are microplastic pollutants widely present in the environment, but their potential risks to human lung health and underlying toxicity mechanisms remain unknown. In this study, we systematically analyzed the effects of PVC-MPs on the transcriptome and metabolome of BEAS-2B cells using high-throughput RNA sequencing and untargeted metabolomics technologies. The results showed that exposure to PVC-MPs significantly reduced the viability of BEAS-2B cells, leading to the differential expression of 530 genes and 3768 metabolites. Further bioinformatics analyses showed that PVC-MP exposure influenced the expression of genes associated with fluid shear stress, the MAPK and TGF-ß signaling pathways, and the levels of metabolites associated with amino acid metabolism. In particular, integrated pathway analysis showed that lipid metabolic pathways (including glycerophospholipid metabolism, glycerolipid metabolism, and sphingolipid metabolism) were significantly perturbed in BEAS-2B cells following PVC-MPs exposure. This study provides new insights and targets for a deeper understanding of the toxicity mechanism of PVC-MPs and for the prevention and treatment of PVC-MP-associated lung diseases.

High expression of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) associated with Diquat-induced damage.

Diquat (DQ) is a commonly used bipyridine herbicide known for its toxic properties and adverse effects on individuals. However, the mechanism underlying DQ-induced damage remain elusive. Our research aimed to uncover the regulatory network involved in DQ-induced damage. We analyzed publicly accessible gene expression patterns and performed research using a DQ-induced damage animal model. The GSE153959 dataset from the Gene Expression Omnibus collection and the animal model of DQ-induced kidney injury were used to identify differentially expressed genes (DEGs). Pathways including the regulation of DNA-templated transcription in response to stress, RNA polymerase II transcription regulator complex and transcription coregulatory activity were shown to be enriched in 21 DEGs. We used least absolute shrinkage and selection operator (LASSO) regression analysis to find possible diagnostic biomarkers for DQ-induced damage. Then, we used an HK-2 cell model to confirm these results. Additionally, we confirmed that 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) was the major gene associated with DQ-induced damage using multi-omics screening. The sample validation strongly suggested that HMGCS2 has promise as a diagnostic marker and may provide new targets for therapy in the context of DQ-induced damage.

Cytotoxicity prediction of nano metal oxides on different lung cells via Nano-QSAR.

In recent years, nanomaterials have found extensive applications across diverse domains owing to their distinctive physical and chemical characteristics. It is of great importance in theoretical and practical terms to carry out the relationship between structural characteristics of nanomaterials and different cytotoxicity and to achieve practical assessment and prediction of cytotoxicity. This study investigated the intrinsic quantitative constitutive relationships between the cytotoxicity of nano-metal oxides on human normal lung epithelial cells and human lung adenocarcinoma cells. We first employed quasi-SMILES-based nanostructural descriptors by selecting the five physicochemical properties that are most closely related to the cytotoxicity of nanometal oxides, then established SMILES-based descriptors that can effectively describe and characterize the molecular structure of nanometal oxides, and then built the corresponding Nano-Quantitative Structure-Activity Relationship (Nano-QSAR) prediction models, finally, combined with the theory of reactive oxygen species (ROS) biotoxicity, to reveal the mechanism of toxicity and differences between the two cell types. The established model can efficiently and accurately predict the properties of targets, reveal the corresponding toxicity mechanisms, and guide the safe design, synthesis, and application of nanometal oxides.

Modeling study for predicting altered cellular activity induced by nanomaterials based on Dlk1-Dio3 gene expression and structural relationships.

Nanomaterials have been widely applied and developed due to its unique physicochemical characteristics, such as their small size. The environmental and biological effects caused by nanomaterials have raised concerns. In particular, some nanometal oxides have obvious biological toxicity and pose a major safety problem. The prediction model established by combining the expression levels of key genes with quantitative structure-activity relationship (QSAR) studies can predict the biotoxicity of nanomaterials by relying on both structural information and gene regulation information. This model can fill the gap of missing mechanisms in QSAR studies. In this study, we exposed A549 cells and BEAS-2B cells to 21 nanometal oxides for 24 h. Cell viability was assessed by measuring absorbance values using the CCK8 assay, and the expression levels of the Dlk1-Dio3 gene cluster were measured. By using the theoretical basis of the nano-QSAR model and the improved principles of the SMILES-based descriptors to combine specific gene expression and structural factors, new models were constructed using Monte Carlo partial least squares (MC-PLS) for the biotoxicity of the nanometal oxides on two different lung cells. The overall quality of the nano-QSAR models constructed by combining specific gene expression and structural parameters for A549 and BEAS-2B cells was better than that of the models constructed based on structural parameters only. The coefficient of determination (R2) of the A549 cell model increased from 0.9044 to 0.9969, and the Root Mean Square Error (RMSE) decreased from 0.1922 to 0.0348. The R2 of the BEAS-2B cell model increased from 0.9355 to 0.9705, and the RMSE decreased from 0.1206 to 0.0874. The model validation proved the proposed models have a good prediction, generalization ability and model stability. This study offers a new research perspective for the toxicity assessment of nanometal oxides, contributing to a more systematic safety evaluation of nanomaterials.

The Cytotoxicity of Tungsten Ions Derived from Nanoparticles Correlates with Pulmonary Toxicity.

Tungsten carbide nanoparticles (nano-WC) are prevalent in composite materials, and are attributed to their physical and chemical properties. Due to their small size, nano-WC particles can readily infiltrate biological organisms via the respiratory tract, thereby posing potential health hazards. Despite this, the studies addressing the cytotoxicity of nano-WC remain notably limited. To this purpose, the BEAS-2B and U937 cells were cultured in the presence of nano-WC. The significant cytotoxicity of nano-WC suspension was evaluated using a cellular LDH assay. To investigate the cytotoxic impact of tungsten ions (W6+) on cells, the ion chelator (EDTA-2Na) was used to adsorb W6+ from nano-WC suspension. Subsequent to this treatment, the modified nano-WC suspension was subjected to flow cytometry analysis to evaluate the rates of cellular apoptosis. According to the results, a decrease in W6+ could mitigate the cellular damage and enhance cell viability, which indicated that W6+ indeed exerted a significant cytotoxic influence on the cells. Overall, the present study provides valuable insight into the toxicological mechanisms underlying the exposure of lung cells to nano-WC, thereby reducing the environmental toxicant risk to human health.

Organ distribution of Nano-WC particles after repeated intratracheal instillation into the lungs of SD rats and subsequent organ injury.

Nano-tungsten carbide (nano-WC) is widely used in composite materials due to its special physical and chemical properties. Owing to their small size, nano-WC nanoparticles easily enter organisms through the respiratory tract, which may cause health hazards. However, only a few studies have reported the toxicity of nano-WC. In this study, a 10 mg/kg nano-WC suspension and 0.9% normal saline were quantitatively perfused into the lungs of two groups of healthy male SD rats by tracheal instillation, and the in vivo pulmonary toxic effects were systematically evaluated. Additionally, as multiple organs and tissues are involved, systemic effects were observed throughout the body and mainly manifested as inflammatory damage. The concentrations of tungsten ions in various organs and alveolar lavage fluid were measured by ICP-MS, and the results showed that the lung was the target organ, as it had the highest concentration of ions. In addition, the abnormal increases in the tungsten ion concentrations in the liver and kidney may be closely related to the immune damage we observed. This study provides a theoretical basis and data support for the systematic evaluation of the health hazards of nano-WC and a reference for the safe use of nanomaterials.

Multi-Omics Analyses Reveal the Mechanisms of Early Stage Kidney Toxicity by Diquat.

Diquat (DQ), a widely used bipyridyl herbicide, is associated with significantly higher rates of kidney injuries compared to other pesticides. However, the underlying molecular mechanisms are largely unknown. In this study, we identified the molecular changes in the early stage of DQ-induced kidney damage in a mouse model through transcriptomic, proteomic and metabolomic analyses. We identified 869 genes, 351 proteins and 96 metabolites that were differentially expressed in the DQ-treated mice relative to the control mice (p < 0.05), and showed significant enrichment in the PPAR signaling pathway and fatty acid metabolism. Hmgcs2, Cyp4a10, Cyp4a14 and Lpl were identified as the major proteins/genes associated with DQ-induced kidney damage. In addition, eicosapentaenoic acid, linoleic acid, palmitic acid and (R)-3-hydroxybutyric acid were the major metabolites related to DQ-induced kidney injury. Overall, the multi-omics analysis showed that DQ-induced kidney damage is associated with dysregulation of the PPAR signaling pathway, and an aberrant increase in Hmgcs2 expression and 3-hydroxybutyric acid levels. Our findings provide new insights into the molecular basis of DQ-induced early kidney damage.

Machine Learning for Evaluating the Cytotoxicity of Mixtures of Nano-TiO2 and Heavy Metals: QSAR Model Apply Random Forest Algorithm after Clustering Analysis.

With the development and application of nanomaterials, their impact on the environment and organisms has attracted attention. As a common nanomaterial, nano-titanium dioxide (nano-TiO2) has adsorption properties to heavy metals in the environment. Quantitative structure-activity relationship (QSAR) is often used to predict the cytotoxicity of a single substance. However, there is little research on the toxicity of interaction between nanomaterials and other substances. In this study, we exposed human renal cortex proximal tubule epithelial (HK-2) cells to mixtures of eight heavy metals with nano-TiO2, measured absorbance values by CCK-8, and calculated cell viability. PLS and two ensemble learning algorithms are used to build multiple QSAR models for data sets, and the test set R2 is increased from 0.38 to 0.78 and 0.85, and RMSE is decreased from 0.18 to 0.12 and 0.10. After selecting the better random forest algorithm, the K-means clustering algorithm is used to continue to optimize the model, increasing the test set R2 to 0.95 and decreasing the RMSE to 0.08 and 0.06. As a reliable machine algorithm, random forest can be used to predict the toxicity of the mixture of nano-metal oxides and heavy metals. The cluster analysis can effectively improve the stability and predictability of the model, and provide a new idea for the prediction of cytotoxicity model in the future.

The regulation of LPCAT3 by miR-124-3p.1 in acute kidney injury suppresses cell proliferation by disrupting phospholipid metabolism.

Sepsis-associated acute renal injury (SA-AKI) is a common critical clinical disease. It is associated with increased mortality and increased risk of progression to chronic kidney disease. However, its pathogenesis is not fully known. We hypothesized that metabolic interactions mediate cell apoptosis and AKI. We found that phosphatidylcholine content in human renal tubular epithelial cells following lipopolysaccharide-induced injury was increased. The activity of lysophosphatidylcholine acyltransferase 3 (LPCAT3), a key enzyme in phospholipid metabolism, was increased, while the expression of miR-124-3p.1, which targets LPCAT3, was decreased. We also found that in the serum of SA-AKI patients, LPCAT3 activity was increased, and miR-124-3p.1 expression was decreased. Further experiments confirmed the specific binding of exocrine miR-124-3p.1 to LPCAT3. Our data reveal the molecular mechanisms of phospholipid metabolic disorder in early SA-AKI as well as the role of the miR-124-3p.1/LPCAT3 pathway in SA-AKI, which leads to ferroptosis. These results could provide the scientific basis for early diagnosis and renal replacement therapy in SA-AKI.

Nano-SAR Modeling for Predicting the Cytotoxicity of Metal Oxide Nanoparticles to PaCa2.

Nowadays, the impact of engineered nanoparticles (NPs) on human health and environment has aroused widespread attention. It is essential to assess and predict the biological activity, toxicity, and physicochemical properties of NPs. Computation-based methods have been developed to be efficient alternatives for understanding the negative effects of nanoparticles on the environment and human health. Here, a classification-based structure-activity relationship model for nanoparticles (nano-SAR) was developed to predict the cellular uptake of 109 functionalized magneto-fluorescent nanoparticles to pancreatic cancer cells (PaCa2). The norm index descriptors were employed for describing the structure characteristics of the involved nanoparticles. The Random forest algorithm (RF), combining with the Recursive Feature Elimination (RFE) was employed to develop the nano-SAR model. The resulted model showed satisfactory statistical performance, with the accuracy (ACC) of the test set and the training set of 0.950 and 0.966, respectively, demonstrating that the model had satisfactory classification effect. The model was rigorously verified and further extensively compared with models in the literature. The proposed model could be reasonably expected to predict the cellular uptakes of nanoparticles and provide some guidance for the design and manufacture of safer nanomaterials.

QNAR modeling of cytotoxicity of mixing nano-TiO2 and heavy metals.

The Quantitative Structure-Activity Relationship (QSAR) has been used to investigate organic mixtures but QSAR in the nanomaterial field (QNAR) is still new. Toxicity is a result of the interaction of many substances. QNAR research focuses on a single nanomaterial in the long-term. It is difficult to find an appropriate descriptor to build a model due to the complexity of the mixture. Here, we attempt to build a QNAR model to predict cell viability for HK-2 cells exposed to a mixture containing nano-TiO2 and heavy metals. HK-2 cells were exposed to four groups of mixtures containing heavy-metals and nanomaterials and CCK8 was added to obtain the number of living cells. At the same time, ROS was investigated to study this mechanism. Each descriptor of the components and mixtures were obtained using the formula Dmix= [Formula: see text] respectively. We used the Multiple Partial Least Squares Regression (PLS) and Random Forest Regression (RF) to build a QNAR model. Both models reliably predict and assess viability of HK-2 cells exposed to the mixture. The RF model showed greater stability and higher precision in toxicity predictability and can be applied to environmental nano-toxicology.

Highly Conductive P-Type MAPbI3 Films and Crystals via Sodium Doping.

To regulate the optical and electrical properties of the crystals and films of the intrinsic methylammonium lead iodide (CH3NH3PbI3), we dope them with sodium (Na) by selecting sodium iodide (NaI) as a dopant source. The highly conductive p-type sodium-doped CH3NH3PbI3 (MAPbI3: Na) perovskite single crystals and thin films are successfully grown using the inverse temperature crystallization (ITC) method and antisolvent spin-coating (ASC) method, respectively. With the increase of Na+ doping concentration, the grain size of the film increases, the surface becomes smoother, and the crystallinity improves. Hall effect results demonstrate that both the MAPbI3: Na thin films and single crystals change their quasi-insulating intrinsic conductivity to a highly conductive p-type conductivity. The room-temperature photoluminescence (PL) peaks of doped MAPbI3 films slightly blue shift, while the photocarriers' lifetime becomes longer. The optical fingerprints of the doped levels in MAPbI3: Na perovskites can be identified by temperature-dependent PL. Obvious fingerprints of Na-related acceptor (A0X) levels in the doped MAPbI3: Na were observed at 10 K. These results suggest that sodium doping is an effective way to grow highly conductive p-type MAPbI3 perovskites.

Adenomatous polyposis coli as a predictor of environmental chemical-induced transgenerational effects related to male infertility.

Exposure to toxic environmental chemicals during pregnancy is a ubiquitous threat to health with potentially transgenerational consequences. However, the underlying mechanism of how transgenerational effects occur as part of environmental chemical exposure are not well understood. We investigated the potential molecular changes associated with dibutyl phthalate exposure that induced transgenerational effects, using a rat model. Through the analysis of the Gene Expression Omnibus database, we found some similar studies of environmental exposure induced transgenerational effects. Then, we analyzed one of the studies and our results to identify the adenomatous polyposis coli (APC) gene. This gene participated the most of the pathways and was upregulated in both studies. We used the miRWALK data set to predict the microRNAs which targeted the APC gene. We confirmed the miR-30 family were significantly downregulated in F3 testis tissues and targeted the APC gene. In conclusion, the miR-30 family/APC interaction is a potential mechanism for the transgenerational effects induced by the environmental chemical.

Sensitive and multiplexed detection of antibiotics using a suspension array platform based on silica-agarose hybrid microbeads.

A multiplex suspension array detection platform of antibiotics has been developed based on silica-agarose hybrid microbeads (SAHMs). Chloramphenicol (CAP), sulfamethoxazole (SMX), metronidazole (MTZ) and amoxicillin (AMX) were employed as model analytes. The antigens (the antibiotics conjugated with BSA) were immobilized on the surface of four different types of SAHMs. Based on an indirect competition immunoassay, the selected antibiotics are detected through the competition of the specific monoclonal antibodies between the multiple antibiotics and the antigens. Due to high resistance to nonspecific protein absorption of SAHMs, the proposed method exhibited wide linear ranges (0.4˜72.9 ng/mL for CAP, 2.0˜108.5 ng/mL for SMX, 2.6˜142.2 ng/mL for MTZ, 1.0˜63.3 ng/mL for AMX) and low detection limits of 0.09˜0.8 ng/mL. Recoveries for spiked tap water samples were from 82% to 113%, with relative standard deviation lower than 14%, demonstrating the accuracy of the measurements performed with the developed method. This work offered a high-throughput, flexible and accurate tool, which provides a good platform for simultaneous detection of antibiotics.

Effect of Nano-SiO2 on Expression and Aberrant Methylation of Imprinted Genes in Lung and Testis.

Nanotechnology has been developing rapidly and is now used in many cutting-edge medical therapeutics. However, there is increasing concern that exposure to nanoparticles (NPs) may induce different systemic diseases as epigenetic mechanisms are associated with more and more disease. The role of NP epigenomic modification is important to disease etiology. Our study aimed to determine the epigenetic mechanisms of damage in lung and testis cells by exposing cells to SiO2 NPs. We used male C57BL/6 mice to characterize the damaging effect of SiO2 NPs on lung and testis cells as well as the resulting methylation state at the imprinted Dlk1/Dio3 domain region. The A549 cells exposed to SiO2 NPs had cell apoptosis, and male mice exposed to SiO2 NPs had altered lung and testis tissues. The genes in the imprinted domains Dlk1/Dio3 region changed in both tissues; Dlk1, Rtl1, and Dio3 are upregulated in testis while Dlk1 and Dio3 are also upregulated in lung tissues. Bisulfite sequencing PCR of male adult lung and testis were mostly hypomethylated, with a few hypermethylated CpGs. These findings indicate that nanoparticles play an important role in DNA methylation of imprinted genes.

From the Cover: Metabolomics Reveals a Role of Betaine in Prenatal DBP Exposure-Induced Epigenetic Transgenerational Failure of Spermatogenesis in Rats.

There is increasing concern that early-life exposure to endocrine disruptors affects male offspring reproduction. However, whether di-n-butyl phthalate (DBP), a widely used endocrine disruptor, has transgenerational effects and, if so, the exact underlying molecular mechanisms involved remain unknown. In our study, 5 of time-mated pregnant SD rats were exposed to 0 and 500 mg/kg DBP with corn oil as the vehicle via oral gavage from embryonic days (E8-E14). Epigenetic and metabolomic of testis were analyzed after post-natal 60 days. Sperm and testicular cell functions were examined to confirm the transgenerational effects. DBP exposure significantly decreased the sperm counts in F1 through F3 generation. We found distinct metabolic changes in the testis of both F1 and F3 generation offspring, specifically, a significantly increased level of betaine, which is an important methyl donor. In contrast, the expression of betaine homocysteine S-methyltransferase (BHMT), which catalyzes the transfer of methyl moiety from betaine to homocysteine, significantly decreased. There was accompanying global DNA hypomethylation, along with a reduction in follistatin-like 3 (Fstl3) promoter hypomethylation, which is a known modulator of Sertoli cell number and spermatogenesis. In summary, we conclude that metabolomic and epigenetic changes induced by the aberrant expression of BHMT represent a novel mechanism linking in utero DBP exposure to transgenerational spermatogenesis failure.

Genistein up-regulates miR-20a to disrupt spermatogenesis via targeting Limk1.

Genistein (GEN) is one of the isoflavones that has effect on male reproduction. However, the underlying mechanism remains unknown. miRNAs are a type of small non-coding RNAs that play important roles in spermatogenesis. We measured the GEN levels and miR-17-92 cluster expression in infertile subjects and found that miR-17-92 might be involved in GEN induced abnormal spermatogenesis. To clarify, we fed adult ICR mice with different doses of GEN (0, 0.5, 5, 50 and 250 mg/kg/day) for 35 days to study the underlying mechanism. We found that sperm average path velocity, straight-line velocity and eurvilinear velocity of the mice orally with GEN at 5mg/kg/day were significantly decreased, the expression levels of miR-17 and miR-20a in mice testis were higher in corresponding group. We also found miR-20a was the only miRNA that differentially expressed both in human and mice. By applying bioinformatics methods, Limk1 was predicted to be the target gene of miR-20a that is involved in spermatogenesis. Limk1 were significantly decreased in the corresponding group. Dual-luciferase report assay also proved that miR-20a could directly target Limk1. These results implied that Limk1 might be the target gene of miR-20a that is involved in GEN induced abnormal spermatogenesis.

Effects of Gold Nanorods on Imprinted Genes Expression in TM-4 Sertoli Cells.

Gold nanorods (GNRs) are among the most commonly used nanomaterials. However, thus far, little is known about their harmful effects on male reproduction. Studies from our laboratory have demonstrated that GNRs could decrease glycine synthesis, membrane permeability, mitochondrial membrane potential and disrupt blood-testis barrier factors in TM-4 Sertoli cells. Imprinted genes play important roles in male reproduction and have been identified as susceptible loci to environmental insults by chemicals because they are functionally haploid. In this original study, we investigated the extent to which imprinted genes become deregulated in TM-4 Sertoli cells when treated with low dose of GNRs. The expression levels of 44 imprinted genes were analyzed by quantitative real-time PCR in TM-4 Sertoli cells after a low dose of (10 nM) GNRs treatment for 24 h. We found significantly diminished expression of Kcnq1, Ntm, Peg10, Slc22a2, Pwcr1, Gtl2, Nap1l5, Peg3 and Slc22a2, while Plagl1 was significantly overexpressed. Additionally, four (Kcnq1, Slc22a18, Pwcr1 and Peg3) of 10 abnormally expressed imprinted genes were found to be located on chromosome 7. However, no significant difference of imprinted miRNA genes was observed between the GNRs treated group and controls. Our study suggested that aberrant expression of imprinted genes might be an underlying mechanism for the GNRs-induced reproductive toxicity in TM-4 Sertoli cells.

Common SNP in hsa-miR-196a-2 increases hsa-miR-196a-5p expression and predisposes to idiopathic male infertility in Chinese Han population.

MicroRNA plays an important role in spermatogenesis. Whether pre-miRNAs polymorphisms are associated with idiopathic male infertility remains obscure. In this study, 1378 idiopathic infertile males and 486 fertile controls were included between 2006 and 2014. Genotype of three polymorphisms (hsa-mir-146a rs2910164, hsa-mir-196a-2 rs11614913, and hsa-mir-499 rs3746444) and expression of miRNA in seminal plasma were examined by TaqMan method. The role of hsa-miR-196a-5p in cell proliferation, apoptosis and cell cycle were also examined in GC-2 cells. Our results demonstrated that rs11614913 of hsa-miR-196a-2 was significantly associated with idiopathic infertility (TT vs. CT: P = 0.014; TT vs. CC: P = 0.005; TT vs. CT + CC: P = 0.003). In following stratified analysis, we found that rs11614913 exhibited a significantly higher risk of asthenospermia, oligozoospermia and azoospermia. However, no significant association was observed between the other two polymorphisms and idiopathic male infertility risk. In a genotype-expression correlation analysis, rs11614913 CC was significantly associated with elevated expression of hsa-miR-196a-5p (P < 0.05). Additionally, apoptosis levels were significantly increased in hsa-miR-196a-5p mimic treated GC-2 cells, while decreased in hsa-miR-196a-5p inhibitor treated GC-2 cells. Our data revealed a significant relationship between hsa-miR-196a-2 polymorphism and idiopathic male infertility.

Association between DAZL polymorphisms and susceptibility to male infertility: systematic review with meta-analysis and trial sequential analysis.

Several studies have investigated the association between polymorphisms in the Deleted in AZoospermia-Like (DAZL) gene and male infertility risk, but with inconsistent results. We aimed to derive a more precise estimation of the relationship, therefore a meta-analysis was performed. A total of 13 case-control studies, including 2556 cases and 1997 controls, were selected. Two polymorphisms in DAZL were investigated, namely T12A (Thr12 → Ala) and T54A (Thr54 → Ala). Our meta-analysis showed that A > G is a risk factor for male infertility (P = 0.047, OR = 1.262, 95%CI = 1.003-1.587). However, when using trial sequential analysis (TSA) to confirm, we found that A > G risk effect turned out to be false positive. In addition, significant association was found between the T54A polymorphism and male infertility under co-dominant model (AG vs. AA: OR = 4.364, 95%CI = 2.207-8.630, P < 0.001) and dominant model (OR = 4.584, 95%CI = 2.320-9.058, P < 0.001). Stratified analysis showed that significantly strong association between T54A polymorphism and male infertility was present only in Asians, but not in Caucasians. Further studies of T12A and T54A with their biological functions are needed to understand the role of these polymorphisms in the development of male infertility.