Enhancement of Frozen-Thawed Human Sperm Quality with Zinc as a Cryoprotective Additive.

BACKGROUND Cryopreservation preserves male fertility, crucial in oncology, advanced age, and infertility. However, it damages sperm motility, membrane, and DNA. Zinc (Zn), an antioxidant, shows promise in improving sperm quality after thawing, highlighting its potential as a cryoprotectant in reproductive medicine. MATERIAL AND METHODS Gradient concentration of ZnSO4 (0, 12.5, 25, 50, and 100 µM) was added in the Glycerol-egg yolk-citrate (GEYC) cryopreservative medium as an extender. Alterations in sperm viability and motility parameters after cryopreservation were detected in each group. Sperm plasma membrane integrity (PMI), acrosome integrity (ACR), DNA fragment index (DFI), and changes in sperm mitochondrial function were examined, including: mitochondrial potential (MMP), sperm reactive oxygen species (ROS), and sperm ATP. RESULTS We found that 50 µM ZnSO4 was the most effective for the curvilinear velocity (VCL) and the average path velocity (VAP) of sperm after cryo-resuscitation. Compared to the Zn-free group, sperm plasma membrane integrity (PMI) was increased, DNA fragmentation index (DFI) was decreased, reactive oxygen species (ROS) was reduced, and mitochondrial membrane potential (MMP) was increased after cryorevival in the presence of 50 µM ZnSO4. CONCLUSIONS Zn ion is one of the antioxidants in the cell. The results of our current clinical study are sufficient to demonstrate that Zn can improve preserves sperm quality during cryopreservation when added to GEYC. The addition of 50 µM ZnSO4 increased curve velocity, mean path velocity, sperm survival (or plasma membrane integrity), and mitochondrial membrane potential while reducing ROS production and DNA breaks compared to GEYC thawed without ZnSO4.

Exploring the mystery of colon cancer from the perspective of molecular subtypes and treatment.

The molecular categorization of colon cancer patients remains elusive. Gene set enrichment analysis (GSEA), which investigates the dysregulated genes among tumor and normal samples, has revealed the pivotal role of epithelial-to-mesenchymal transition (EMT) in colon cancer pathogenesis. In this study, we employed multi-clustering method for grouping data, resulting in the identification of two clusters characterized by varying prognostic outcomes. These two subgroups not only displayed disparities in overall survival (OS) but also manifested variations in clinical variables, genetic mutation, and gene expression profiles. Using the nearest template prediction (NTP) method, we were able to replicate the molecular classification effectively within the original dataset and validated it across multiple independent datasets, underscoring its robust repeatability. Furthermore, we constructed two prognostic signatures tailored to each of these subgroups. Our molecular classification, centered on EMT, hold promise in offering fresh insights into the therapy strategies and prognosis assessment for colon cancer.

The Essential Role of PGF2α/PTGFR in Molding Endometrial Breakdown and Vascular Dynamics, Regulated by HIF-1α in a Mouse Menstrual-like Model.

In women of childbearing age, extensive decidualization, shedding and remodeling of the endometrium during the menstrual cycle are fundamental for successful pregnancy. The role of prostaglandins (PGs) in menstruation has long been proposed in humans, and the rate-limiting enzyme cyclooxygenase was shown to play a key role in endometrial breakdown and shedding in a mouse menstrual-like model in our previous study. However, the specific types of PGs involved and their respective roles remain unclear. Therefore, our objective was to investigate the mechanism through which PGs regulate endometrial disintegration. In this study, the microscopy was observed by HE; the protein levels of prostaglandins E1 (PGE1), prostaglandins E2 (PGE2), prostaglandin F2α (PGF2α) and Prostaglandin I2 (PGI2) were detected by ELISA; the mRNA level of Pfgfr2, Vascular Endothelial Growth Factor(Vegf), Angiostatin and Hypoxia inducible factor-1α (Hif1α) were examined by real-time PCR; PTGFR Receptor (PTGFR), VEGF, Angiostatin and HIF-1α protein levels were investigated by western blotting; the locations of protein were observed by Immunohistochemistry; HIF-1α binding PTGFR promoter was detected by Chromatin Immunoprecipitation (ChIP) and real-time PCR. We found that the concentrations of PGE1, PGE2, and PGF2α all increased significantly during this process. Furthermore, Ptgfr mRNA increased soon after Progesterone (P4) withdrawal, and PTGFR protein levels increased significantly during abundant endometrial breakdown and shedding processes. PTGFR inhibitors AL8810 significantly suppressed endometrial breakdown and shedding, promoted Angiostatin expression, and reduced VEGF-A expressions and vascular permeability. And HIF-1α and PTGFR were mainly located in the luminal/gland epithelium, vascular endothelium, and pre-decidual zone. Interestingly, HIF-1α directly bound to Ptgfr promoter. Moreover, a HIF-1α inhibitor 2-methoxyestradiol (2ME) significantly reduced PTGFR expression and suppressed endometrial breakdown which was in accord with PTGFR inhibitor's effect. Similar changes occurred in human stromal cells relevant to menstruation in vitro. Our study provides evidence that PGF2α/PTGFR plays a vital role in endometrial breakdown via vascular changes that are regulated by HIF-1α during menstruation.

Combined Analysis of the Transcriptome, Proteome and Metabolome in Human Cryopreserved Sperm.

PURPOSE: This study aimed to identify the altered pathways and genes associated with freezing damage in human sperm during cryopreservation by multiomics analysis. MATERIALS AND METHODS: Fifteen fresh human semen samples were collected for transcriptomic analysis, and another 5 fresh human semen samples were obtained for metabolomic analysis. For each semen sample, 1 mL was cryopreserved, and another 1 mL was left untreated for paired design. The results were then combined with previously published proteomic results to identify key genes/pathways. RESULTS: Cryopreservation significantly reduced sperm motility and mitochondrial structure. Transcriptomic analysis revealed altered mitochondrial function, including changes in tRNA-methyltransferase activity and adenosine tri-phosphate/adenosine di-phosphate transmembrane transporter activity. Metabolomic analysis showed that the citrate cycle in mitochondria was significantly altered. Combining transcriptomic, proteomic, and metabolomic analyses revealed 346 genes that were altered in at least two omics analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that metabolic pathways were significantly altered and strongly associated with mitochondria. Five genes were altered in all three omics analyses: COL11A1, COL18A1, LPCAT3, NME1, and NNT. CONCLUSIONS: Five genes were identified by multiomics analysis in human cryopreserved sperm. These genes might have specific functions in cryopreservation. Explorations of the functions of these genes will be helpful for sperm cryopreservation and sperm motility improvement or even for reproduction in the future.

Novel biallelic variants in DNAH1 cause multiple morphological abnormalities of sperm flagella with favorable outcomes of fertility after ICSI in Han Chinese males.

BACKGROUND: Multiple morphological abnormalities of sperm flagella is an idiopathic asthenoteratozoospermia characterized by absent, short, coiled, angulation, and irregular-caliber flagella. Genetic variants of DNAH1 gene have been identified as a causative factor of multiple morphological abnormalities of sperm flagella and intracytoplasmic sperm injection is an available strategy for infertile males with dynein axonemal heavy chain 1 defects to conceive. OBJECTIVES: To identify novel variants and candidate mutant hotspots of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans. MATERIALS AND METHODS: The DNAH1 variants were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning and transmission electron microscopy, and immunostaining were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic DNAH1 variants. RESULTS: We identified 18 different DNAH1 variants in 11 unrelated families, including nine missense variants (p.A2564T, p.T3657R, p.G1862R, p.L2296P, p.T4041I, p.L611P, p.A913D, p.R1932Q, p.R2356W) and nine loss-of-function variants (c.2301-1G>T, p.Q1518*, p.R1702*, p.D2845Mfs*2, p.P3909Rfs*33, p.Q4040Dfs*33, p.Q4058*, p.E4060Pfs*61, p.V4071Cfs*54). A total of 66.7% (12/18) of the identified variants were novel. Morphological analysis based on Papanicolaou staining and scanning electron microscopy demonstrated the typical multiple morphological abnormalities of sperm flagella characteristics of dynein axonemal heavy chain 1-deficient spermatozoa. Immunostaining further revealed the absence of inner dynein arms but not outer dynein arms, which induced a general ultrastructural disorganization, such as the loss of central pair and mis-localization of the microtubule doublets and outer dense fibers. To date, seven affected couples have accepted the intracytoplasmic sperm injection treatment, and three of them have given birth to five healthy babies. DISCUSSION AND CONCLUSION: These findings further expand the variant spectrum of DNAH1 gene related to multiple morphological abnormalities of sperm flagella and male infertility in humans, thus providing new information for the molecular diagnosis of asthenoteratozoospermia. The favorable fertility outcomes of intracytoplasmic sperm injection will facilitate the genetic counseling and clinical treatment of infertile males with multiple morphological abnormalities of sperm flagella in the future.

Insufficiency of Mrpl40 disrupts testicular structure and semen parameters in a murine model.

Approximately 31% of patients with 22q11.2 deletion syndrome (22q11.2DS) have genitourinary system disorders and 6% of them have undescended testes. Haploinsufficiency of genes on chromosome 22q11.2 might contribute to the risk of 22q11.2DS. In this study, we used mice with single-allele deletion in mitochondrial ribosomal protein L40 (Mrpl40 +/- ) as models to investigate the function of Mrpl40 in testes and spermatozoa development. The penetrance of cryptorchidism in Mrpl40 +/- mice was found to be higher than that in wild-type (WT) counterparts. Although the weight of testes was not significantly different between the WT and Mrpl40 +/- mice, the structure of seminiferous tubules and mitochondrial morphology was altered in the Mrpl40 +/- mice. Moreover, the concentration and motility of spermatozoa were significantly decreased in the Mrpl40 +/- mice. In addition, data-independent acquisition mass spectrometry indicated that the expression of genes associated with male infertility was altered in Mrpl40 +/- testes. Our study demonstrated the important role of Mrpl40 in testicular structure and spermatozoa motility and count. These findings suggest that Mrpl40 is potentially a novel therapeutic target for cryptorchidism and decreased motility and count of spermatozoa.

Determination of Parabens and Their Metabolites in Seminal Plasma from Chinese Men by Ultra High Performance Liquid Chromatography Tandem Mass Spectrometry (UPLC-MS/MS).

Parabens are endocrine-disrupting chemicals (EDCs) that have estrogen-like activities and may cause male reproductive disorders. Here, we developed a method for the simultaneous determination of four parabens (MeP, EtP, n-PrP, n-BuP) and two metabolites (4-HB and 3,4-DHB) in human seminal plasma by UPLC-MS/MS. The method was used to analyze 144 seminal plasma samples from Chinese males. MeP, EtP, n-PrP, and 4-HB were the dominant compounds. MeP, EtP, and n-PrP were significantly correlated to each other. In addition, 4-HB was significantly correlated to MeP, EtP, n-PrP, and 3,4-DHB, respectively. The results provide direct evidence that parabens and their metabolites are widely distributed in the male reproductive system. The study presents the paraben metabolites levels in human seminal plasma for the first time.

Effects of Qilin pills on spermatogenesis, reproductive hormones, oxidative stress, and the TSSK2 gene in a rat model of oligoasthenospermia.

BACKGROUND: Qilin pills (QLPs), a classic Traditional Chinese Medicine (TCM) formula for treating male infertility, effectively improve semen quality in clinical trials. This study was designed to evaluate the effects of QLPs on spermatogenesis, reproductive hormones, oxidative stress, and the testis-specific serinekinase-2 (TSSK2) gene in a rat model of oligoasthenospermia. METHODS: Forty adult male Sprague-Dawley (SD) rats were randomly divided into four groups. The rat model with oligoasthenospermia was generated by intragastric administration of tripterygium glycosides (TGs) once daily for 4 weeks. Then, two treatment groups were given different doses (1.62 g/kg and 3.24 g/kg) of QLPs once daily for 60 days. Sperm parameters, testicular histology and reproductive hormone measurements, oxidative stress tests, and TSSK2 expression tests were carried out. RESULTS: QLPs effectively improved semen parameters and testicular histology; restored the levels of FSH, LH, PRL, fT, and SHBG; reduced the levels of oxidative stress products (ROS and MDA); increased testicular SOD activity; and restored the expression of spermatogenesis-related gene TSSK2. CONCLUSION: QLPs have a therapeutic effect on a rat model of oligoasthenospermia, and this effect is manifested as improvement of semen quality and testis histology, gonadal axis stability, decreased oxidative stress, and the regulation of testis-specific spermatogenesis-related gene TSSK2.

[Impact of the freezing-thawing process on human sperm mitochondria].

OBJECTIVE: To study the structure and function of human sperm mitochondria before and after the freezing-thawing process. METHODS: Human sperm from healthy donors were subjected to the slow freezing-thawing process, and the sperm mitochondrion-related indexes compared before and after cryopreservation. The ultrastructural changes of the mitochondria were observed under the projection electron microscope, the mitochondrial membrane potential (MMP) and seminal adenosine triphosphate (ATP) content measured by immunofluorescence labeling and ELISA, respectively, and the sperm oxidative stress related indexes detected before and after sperm cryopreservation. RESULTS: Electron microscopy showed loose structures and widened crests of the sperm mitochondria, some with vacuole-like changes after the freezing-thawing process. The sperm after cryopreservation, compared with those before it, exhibited significantly increased contents of oxygen free radicals (ï¼»11.6 ± 3.8ï¼½% vs ï¼»9.6 ± 4.1ï¼½%, P < 0.05) and malondialdehyde (ï¼»3.2 ± 1.4ï¼½ vs ï¼»2.3 ± 1.2ï¼½ nmol/108, P < 0.05), but decreased antioxidant capacity (ï¼»0.6 ± 0.4ï¼½ vs ï¼»0.9 ± 0.4ï¼½ nmol/108, P < 0.05), superoxide dismutase activity (ï¼»0.9 ± 0.4ï¼½ vs ï¼»9.1 ± 3.9ï¼½ nmol/108, P < 0.05), MMP (ï¼»52.2 ± 6.2ï¼½% vs ï¼»55.7 ± 4.9ï¼½%, P = 0.026) and ATP production (ï¼»56.5 ± 9.0ï¼½ vs ï¼»61.3 ± 10.4ï¼½ pmol/106, P = 0.014). CONCLUSIONS: The freezing-thawing process can cause ultrastructural disorder of human sperm mitochondria, reduce their membrane potential and decrease their ATP production.

Quantitative proteomic characterization of human sperm cryopreservation: using data-independent acquisition mass spectrometry.

BACKGROUND: Human sperm cryopreservation is a simple and effective approach for male fertility preservation. METHODS: To identify potential proteomic changes in this process, data-independent acquisition (DIA), a technology with high quantitative accuracy and highly reproducible proteomics, was used to quantitatively characterize the proteomics of human sperm cryopreservation. RESULTS: A total of 174 significantly differential proteins were identified between fresh and cryoperservated sperm: 98 proteins decreased and 76 proteins increased in the cryopreservation group. Bioinformatic analysis revealed that metabolic pathways play an important role in cryopreservation, including: propanoate metabolism, glyoxylate and dicarboxylate metabolism, glycolysis/gluconeogenesis, and pyruvate metabolism. Four different proteins involved in glycolysis were identified by Western blotting: GPI, LDHB, ADH5, and PGAM1. CONCLUSIONS: Our work will provide valuable information for future investigations and pathological studies involving sperm cryopreservation.

Sperm Cryopreservation for Male Cancer Patients: More than 10 Years of Experience, in Beijing China.

BACKGROUND Fertility preservation is very important for male cancer patients, especially adolescents. Unfortunately, the use of fertility preservation is very low among Chinese male cancer patients. Additionally, the cumulative rate of frozen sperm use is also low. MATERIAL AND METHODS We performed a retrospective study by collecting available information at the Human Sperm Bank, National Research Institute for Family Planning from July 2006 to December 2017 to examine the data in China. RESULTS A total 145 male cancer patients underwent sperm cryopreservation. The patients were 29.3±6.9 years old, and 6.2% (9 out of 145) of the patients were adolescents under the age of 18 years old. As of June 2018, only 9.7% (14 out of 145) of patients returned to use their cryopreserved sperm for assisted reproduction technology (ART). Of the 33 ART cycles, conceptions were achieved in 51.5% (17 out of 33), and the rate of patients who had a baby was 71.4% (10 out of 14). The data indicate men with testicular cancer or leukemia had lower total sperm counts and recovery rate of progressive sperm than did men with other types of cancer, while men with sarcoma had the lowest progressive sperm. CONCLUSIONS The physician should make an effort to promote fertility preservation for male cancer patients in China. And patients with testicular cancer and leukemia require additional attention.

Glycolysis metabolic changes in sperm cryopreservation based on a targeted metabolomic strategy.

Our aim was to identify potential metabolomic pathway changes in the sperm cryopreservation process and to find new markers of human sperm freezability. Targeted metabolomic experiments were used to identify the quantitative metabolomic compound characterization of human sperm cryopreservation. A KEGG pathway analysis was used for these deregulated compounds. A total of 16 significantly deregulated compounds was identified between fresh and post-thawed sperm; of these, 7 were downregulated and 9 were upregulated in the frozen-thawed group. A bioinformatics analysis revealed that metabolic pathways play an important role in cryopreservation, including the citrate cycle (TCA cycle), glycolysis or gluconeogenesis, glyoxylate and dicarboxylate metabolism, pyruvate metabolism and galactose metabolism. We used immunoblotting and immunofluorescence to analyze the expression and localization of the three key enzymes in glycolysis. The glycolytic metabolic changes were noted in sperm cryopreservation. HK2 expression levels in fresh sperm were significantly higher than the levels in freeze-thawed sperm.

[Status quo of autologous sperm preservation in the human sperm bank in Beijing].

OBJECTIVE: To comprehensively analyze the status quo of autologous sperm preservation in the human sperm bank in Beijing and better utilize the existing resources for the preservation of male fertility. METHODS: We retrospectively analyzed the geographical data and semen quality of 251 males with autologous sperm preservation in the Human Sperm Bank, Science and Technology Research Institute, National Health and Family Planning Commission of China from July 2006 to December 2016. RESULTS: The rate of autologous sperm preservation in the Human Sperm Bank was as low as 8.76% between July 2006 and December 2010 but increased annually by 119% on average from 2011 to 2013. Of the 251 males involved, 204 (81.27%) were aged 20－39 years, 175 (69.72%) had bachelor's or master's degree, 223 (88.84%) had no child, 69 (27.49%) got less than 10 tubes of semen samples frozen, and 26 (10.36%) had their semen samples cryopreserved only once. The utilization rate of the cryopreserved sperm was only 5.58 % (n = 14). The main reason for autologous sperm preservation was carcinoma (55.78% ï¼»n = 140ï¼½), including blood cancer (22.31% ï¼»n = 66ï¼½), testicular cancer (13.15% ï¼»n = 33ï¼½) and other cancers (16.33% ï¼»n = 41ï¼½). Compared with the non-cancer males, the cancer patients had a significantly reduced mean sperm concentration (90.45 vs 60.53 ×106/ml, P < 0.05), total sperm count (311.3 vs 175.8 ×106, P < 0.05), percentage of progressively motile sperm (PMS) (49.21% vs 43.55%, P < 0.05) and recovery rate of PMS (68.13% vs 52.17%, P < 0.05). In the subgroups of testicular, blood and other cancers, the sperm concentration averaged 37.68, 57.98 and 90.69 ×106/ml, the semen volume 2.73, 2.82 and 3.41 ml, the total sperm count 93.29, 158.41 and 349.49 ×106, the percentage of PMS 45.32%, 43.47% and 44.49%, and the recovery rate of PMS 48.32%, 50.07% and 61.09%, respectively, the sperm concentration and total sperm count significantly lower in the testicular cancer patients than in the other two groups (P < 0.05). CONCLUSIONS: The number of the cases of autologous sperm preservation in Beijing is increasing year by year, and the majority of them are cancer patients. As most of the cancer patients have missed the best period for sperm preservation, sperm bank workers should endeavor to increase the public awareness of autologous sperm preservation.

Seasonal variations of tris (2-chloroethyl) phosphate and cytotoxicity of organic extracts in water samples from Wuhan, China.

Tris (2-chloroethyl) phosphate (TCEP) is a typical phosphate flame retardant. Its potential adverse health effects have recently aroused concern. We investigated the seasonal variations of TCEP concentrations in the raw, finished and tap water samples from two drinking water treatment plants (DWTPs) in China, and evaluated the cytotoxicity and apoptosis/necrosis of organic extracts (OEs) in water samples. We enriched TCEP and OEs in water samples by solid-phase extraction method. The TCEP concentrations in water samples were determined by gas chromatography-mass spectrometry. Normal human liver cell line L02 was treated with OEs in the water samples, and then the cytotoxicity and apoptosis/necrosis were measured by 3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay and flow cytometry, respectively. The results showed that cytotoxicities of OEs in raw water samples from both DWTPs in summer and winter were stronger than those in spring and autumn, cytotoxicity of OEs in finished and tap water samples from both DWTPs in summer and autumn were stronger than those in spring and winter. In all seasons, the maximal concentrations (100 mL water/mL cell culture) of OEs in the raw water samples from both DWTPs induced late apoptosis/necrosis. The reasons for seasonal variations of TCEP in water samples and potential toxic effects of other pollutants in the water samples need to be further investigated.

Qilin pills alleviate oligoasthenospermia by inhibiting Bax-caspase-9 apoptosis pathway in the testes of model rats.

At present, the treatment of oligoasthenospermia with western medicine is ineffective. Qilin pill (QLP) is a Chinese traditional medicine for treating male infertility. Recent multicenter clinical studies in China reported that QLPs markedly improved sperm quality. However, the mechanism of action of QLPs on oligoasthenospermia remains unknown. In this study, we investigated the mechanistic basis for improvement of semen parameters and reversal of testis damage by QLPs in a rat model of oligoasthenospermia induced by treatment with tripterygium glycosides (TGs) (40 mg/kg) once daily for 4 weeks. Rats were administered QLPs (1.62 g/kg or 3.24 g/kg) each day for 60 days, with untreated animals serving as controls. The concentration and motility of sperm extracted from rat epididymis were determined, whereas histopathological examination and immunohistochemical apoptosis analysis of rat testes was performed. Expression profiles of apoptosis-related genes were determined by microarray analysis; the results were validated by quantitative real-time PCR, western blotting, and immunohistochemistry. Sperm concentration and motility in the QLP treatment group were increased relative to those in control rats. Testis tissue and DNA damage were reversed by QLP treatment. The improvement function of QLPs on sperm and testis works mainly by suppressing mitochondrial apoptosis in the testis via modulation of B cell lymphoma (Bcl)-2, Bcl-2-associated X protein (Bax), cytochrome C, caspase-9 and caspase-3 expression. QLPs could improve sperm quality and testis damage in a rat model of oligoasthenospermia by inhibiting the Bax-Caspase-9 apoptosis pathway and exerting therapeutic effects.

Obesity impairs male fertility through long-term effects on spermatogenesis.

OBJECTIVE: This study aimed to investigate the effect and possible underlying mechanisms of high-fat diet-induced obesity on spermatogenesis in male rats. METHODS: A total of 45 male rats were randomly divided into control (n = 15, normal diet) and obesity groups (n = 30, high-fat diet) and were fed for 16 weeks. Body weight and organ indexes were determined after sacrifice. Indicators of reproductive function, including sperm count, sperm motility, apoptosis of spermatogenic cells, and oxidative stress levels, were measured. Serum metabolic parameters and reproductive hormones were also assayed. RESULTS: Compared with the control group, epididymal sperm motility in the obese rats was significantly decreased (P < 0.01). Morphological analysis of the obesity group showed vacuolar changes in seminiferous tubules, spermatogenic cell dysfunction, and increased apoptosis of spermatogenic cells in testicular tissue (P < 0.05). The calculated free testosterone (cFT) concentration in serum was decreased (P < 0.05), whereas the serum sex hormone-binding globulin (SHBG) level was significantly increased (P < 0.01). The superoxide dismutase (SOD) concentration decreased and the malondialdehyde (MDA) concentration increased in testis tissues; however, neither changes were statistically significant (P > 0.05). RESULTS: Nutritional obesity can damage spermatogenesis in male rats due to long-term effects on spermatogenesis.

[Levels of serum reproductive hormones and metabolic syndrome in adult males].

OBJECTIVE: To investigate the prevalence of metabolic syndrome (MS) and its relationship with reproductive hormones in adult males in China. METHODS: Using the cluster and stratified sampling methods, we conducted an investigation among 3 600 adult males aged over 20 years in Hebei, Shaanxi and Jiangsu provinces. We obtained their reproductive hormone levels, biochemical indicators and basic body indexes and compared them between the two groups of subjects. RESULTS: A total of 3 332 valid serum samples were collected, which revealed a prevalence rate of MS of 38.5% in the adult males. Compared with the non-MS subjects, the MS males showed a significantly higher free testosterone index (FTI, 0.39 ± 0.15 vs 0.45 ± 0.19, P < 0.01) but lower levels of total testosterone (TT, ï¼»16.35 ± 4.78ï¼½ vs ï¼»13.37 ± 4.23ï¼½ nmo/L, P < 0.01) and sex hormone-binding globulin (SHBG, ï¼»47.13 ± 20.50ï¼½ vs ï¼»33.32 ± 14.91ï¼½ nmol/L, P < 0.01) and testosterone secretion index (TSI, 3.64 ± 1.92 vs 3.14 ± 1.80, P < 0.01). There were no statistically significant differences between the two groups in the levels of calculated free testosterone (cFT) and LH (P > 0.05). Spearman rank correlation analysis showed that the levels of serum TT and SHBG were correlated with all the indicators of MS (P < 0.01) except systolic blood pressure (SBP), while that of cFT only with the levels of high-density lipoprotein (HDL), fasting blood glucose (FBG) and SBP (P < 0.01). After adjusted for age, smoking, drinking, and body mass index (BMI), all the MS indicators were significantly associated with the SHBG level (P < 0.01), but not high blood pressure with the serum TT level (P > 0.05). After adjusted for the age, smoking, drinking, BMI and TT, the serum SHBG level was the main independent risk factor for MS (OR: 0.965, 95% CI: 0.958－0.973, P < 0.01). CONCLUSIONS: The level of serum cFT is not correlated with while that of SHBG is the main independent risk factor for metabolic syndrome in adult males in China.

Tris (2-chloroethyl) phosphate induces senescence-like phenotype of hepatocytes via the p21Waf1/Cip1-Rb pathway in a p53-independent manner.

Tris (2-chloroethyl) phosphate (TCEP) has been widely used as a plasticizer and flame retardant. TCEP as a potential carcinogen is often detected in the occupational and nature environments. To investigate effects of TCEP on human hepatocytes, we assessed cell growth rate, cellular membrane integrity, senescence-associated ß-galactosidase (SA-ß-Gal) activity and analyzed expression of regulators involved in the p53-p21Waf1/Cip1-Rb pathway in TCEP-treated L02 cells. The results showed TCEP increased the percentage of SA-ß-Gal positive cells, decreased IL-6 levels, down-regulated the regulators of p38MAPK-NF-κB pathways, but up-regulated the regulators of p21Waf1/Cip1-Rb pathway in L02 cells. Furthermore, we measured the SA-ß-Gal activity and expression of regulators involved in the p53-p21Waf1/Cip1-Rb pathway in L02-p53 cells and p53-null Hep3B cells. Similar results were found in L02-p53 cells and Hep3B cells. The findings demonstrated that TCEP induced senescence-like growth arrest via the p21Waf1/Cip1-Rb pathway in a p53-independent manner, without activation of the IL-6/IL6R, p38MAPK-NF-κB pathways in hepatocytes.