Associations of environmental phthalate exposure with male steroid hormone synthesis and metabolism: An integrated epidemiology and toxicology study.

Humans are simultaneously and constantly exposed to various lipophilic chain phthalate acid esters. The association of urinary phthalate metabolites with altered male steroid hormone synthesis and metabolism was examined using epidemiology and toxicology studies. We measured 8 phthalate metabolites [monomethyl phthalate (MMP), monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), mono-n-octylphthalate (MOP), mono-(2-ethylhexyl) phthalate (MEHP), mono-(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) and mono (2-ethyl-5-oxohexyl) phthalate (MEOHP)] and two sex hormones [testosterone (T) and estradiol (E2)] in single serum and repeated spot urine samples among 451 reproductive-age males. Moreover, in vitro experiments with Leydig cell MLTC-1 steroidogenesis and liver cell HepG2 efflux in response to mixed and individual phthalates were designed to simulate real-world scenarios of human exposure. As a joint mixture, the phthalate metabolite was inversely associated with serum T and E2 concentrations but positively associated with urinary T and E2 concentrations. Combined with in vitro experiments, DEHP metabolites were identified as the predominant contributor to the decline in hormone synthesis, and ATP-binding cassette (ABC) gene activation might be involved in hormone excretion. Exposure to environmentally relevant phthalates was associated with both altered steroid synthesis and excretion, which provides additional insights into the endocrine-disrupting potential of phthalates.

SNP rs12794714 of CYP2R1 is associated with serum vitamin D levels and recurrent spontaneous abortion (RSA): a case-control study.

PURPOSE: Vitamin D (VD) deficiency seems to be associated with the risk of recurrent spontaneous abortion (RSA). Vitamin D receptor (VDR) and cytochrome P450 family 2 subfamily R member 1 (CYP2R1) are two genes which are vital for VD metabolism and actions. However, whether single-nucleotide polymorphisms (SNPs) in these genes are correlated with the risk of RSA are poorly understood. Therefore, we aimed to characterize the relationships among VDR SNPs, CYP2R1 SNPs and RSA. METHODS: This case-control study enrolled 75 RSA patients and 83 controls. Serum VD and some cytokines were detected with LC-MS/MS and flow cytometry, respectively. Genotyping for three SNPs of CYP2R1 (rs10741657, rs10766197 and rs12794714) and five SNPs of VDR (rs7975232, rs1544410, rs2189480, rs2228570 and rs2239179) was done with polymerase chain reaction (PCR) and high-throughput sequencing. All the data were analyzed with appropriate methods and in different models. RESULTS: The results revealed a significant correlation between the AG genotype of CYP2R1 rs12794714 and VD levels (OR 0.686; 95% CI 0.49-0.96; p = 0.028). Besides, the AG and GG genotypes of CYP2R1 rs12794714 were markedly related to the risk of RSA (OR 52.394, 59.497; 95% CI 2.683-1023.265, 3.110-1138.367; p = 0.009, 0.007, respectively). CONCLUSION: Our results indicate that CYP2R1 rs12794714 might be a risk factor for RSA. Hence, early screening of pregnant women for CYP2R1 rs12794714 is necessary to warrant proactive counseling and treatment against RSA.

[Impact of cigarette smoking on sperm quality and seminal plasma ROS in preconception males].

OBJECTIVE: To investigate the influence of cigarette smoking on sperm quality and ROS in the seminal plasma of preconception males in Chongqing so as to provide some guidance for preconception couples. METHODS: Totally, 368 preconception males were enrolled in this study, including 196 smokers and 172 non-smokers, and the former divided into mild smokers (n = 88, ≤9 cigarettes per day for ≤5 years) and moderate to heavy smokers (n = 108, ≥10 cigarettes per day for ≥5 years or ≤9 cigarettes per day for ≥10 years). All the subjects underwent physical examination, medical history interview, health questionnaire investigation, and determination of semen parameters, ROS in the seminal plasma and sperm DNA fragmentation index (DFI). RESULTS: Among the 368 preconception males, 53.26% had a history of cigarette smoking, of whom 29.35% were moderate to heavy smokers. Only 55.4% of the subjects were found with normal sperm morphology and 52.6% with normal sperm progressive motility. Compared with the non-smokers, the moderate to heavy smokers showed significantly decreased semen volume (ï¼»3.33 ± 1.20ï¼½ vs ï¼»2.78 ± 1.08ï¼½ ml, P < 0.05), sperm concentration (ï¼»88.19 ± 70.33ï¼½ vs ï¼»75.16 ± 60.13ï¼½ × 106/ml, P < 0.05), and percentages of progressively motile sperm (PMS, ï¼»36.58 ± 13.90ï¼½ % vs ï¼»32.18 ± 15.24ï¼½ %, P < 0.05) and morphologically normal sperm (MNS, ï¼»3.85 ± 1.93ï¼½ % vs ï¼»3.52 ± 1.58ï¼½ %, P < 0.05), but increased sperm DFI (ï¼»10.45 ± 8.53ï¼½ % vs ï¼»14.53 ± 12.85ï¼½ %, P < 0.05) and ROS in the seminal plasma (ï¼»12.20 ± 8.10ï¼½ vs ï¼»24.10 ± 18.50ï¼½ nmol/mg prot, P < 0.05). Cigarette smoking was correlated positively with the ROS level in the seminal plasma (r = 0.235, P < 0.05), while the ROS level of the smokers negatively with the total sperm count (r = －136, P < 0.05), PMS (r = －0.381, P < 0.01) and MNS (r = －0.218, P < 0.01), but positively with sperm DFI in the preconception males (r = 0.387, P < 0.01). CONCLUSIONS: Cigarette smoking can increase the ROS level in the seminal plasma, decrease the total sperm count and sperm progressive motility, and induce sperm malformation and sperm DNA fragmentation in preconception males.

Phthalate exposure, even below US EPA reference doses, was associated with semen quality and reproductive hormones: Prospective MARHCS study in general population.

BACKGROUND: Environment-Protection-Agency Reference Doses (EPA RfDs) for phthalate intakes are based on limited evidence, especially regarding low-dose male-reproductive toxicity. This study investigates the association between phthalate exposure and semen parameters and reproductive hormones in a general population with low phthalate exposure compared to the EPA RfDs. METHODS: The MARHCS (Male-Reproductive-Health-in-Chongqing-College-Students) cohort recruited 796 male students, who experienced a relocation of campuses and shifting environmental exposure. Urine, semen and blood before and after the relocation was collected and investigated for: (1) the associations between 13 urinary phthalate metabolites and 11 semen/hormone outcomes (five semen parameters including semen volume, sperm concentration, total sperm number, progressive motility, normal morphology) and six serum reproductive hormones including estradiol, follicle-stimulating hormone, luteinizing hormone, prolactin, progesterone, testosterone; (2) re-analysis of the metabolite-outcome associations in the subjects with estimated phthalate intakes below the RfDs; (3) a change in phthalate metabolites and change in semen/hormone outcomes after the relocation; (4) the association between these changes. RESULTS: (1) All but two semen/hormone outcomes were associated with at least one phthalate metabolite, e.g., each quartile monoethyl phthalate was associated with a 5.3%, 5.7% and 2.6% decrease of sperm concentration, total sperm number and progressive motility respectively. (2) In the subjects with phthalate intakes below the RfDs, these metabolite-outcome associations remained significant. (3) All metabolites except mono(2-ethylhexyl) phthalate declined after relocation (P<0.001 respectively); at the same time, semen volume, normal morphology, estradiol and luteinizing hormone increased (by 5.9%, 25.0%, 34.2% and 10.0%) and testosterone decreased (by 7.0%). (4) The changes in semen volume, normal morphology, estradiol and testosterone, but not the change in luteinizing hormone after relocation, were associated with the changes in the phthalate metabolites. CONCLUSIONS: Phthalate exposure is associated with interrupted semen quality and reproductive hormones in the human population even with a dose given below the RfDs. These effects, however, may only partially revert back when exposure decreases, thus emphasizing the urgency of stricter phthalate administration.

Inverse U-shaped Association between Sleep Duration and Semen Quality: Longitudinal Observational Study (MARHCS) in Chongqing, China.

STUDY OBJECTIVES: To investigate the association between sleep duration and semen parameters as well as reproductive hormone levels. METHODS: We designed a cohort of male college students in Chongqing, China. A total of 796 subjects were recruited in 2013 and 656 (82.4%) were followed up in 2014. Each time, semen and peripheral blood samples were collected for semen quality and reproductive hormone measurement. Sleep duration was estimated by revised Munich Chronotype Questionnaire. In 2014, sleep quality was also measured by Pittsburgh Sleep Quality Index (PSQI). RESULTS: There was a substantial inverse U-shaped association between sleep duration and two semen parameters (semen volume and total sperm number), with 7.0-7.5 h/day of sleep showing highest parameters. Either longer or shorter sleep was associated with decreased semen parameters in a dose-response manner (P = 0.002 and 0.001, respectively). Sleeping > 9.0 h was associated with a 21.5% (95% confidence interval 9.2, 32.2) reduction in semen volume and 39.4% (23.3, 52.1) reduction in total sperm number; sleeping ≤ 6.5 h was associated with 4.6% (-10.5, 22.3) and 25.7% (-1.2, 60.1) reduction. Increase of the two parameters was found in those who changed sleep duration toward 7.0-7.5 h/day from 2013 to 2014. The U-shaped association was independent from PSQI and was replicated in another dataset of 1,346 males. No association found between sleep duration and reproductive hormone. CONCLUSIONS: Either restricted or excessive sleep may impair semen quality. Further research is needed to validate this finding.

Lifestyles Associated With Human Semen Quality: Results From MARHCS Cohort Study in Chongqing, China.

Decline of semen quality in past decades is suggested to be potentially associated with environmental and sociopsychobehavioral factors, but data from population-based cohort studies is limited. The male reproductive health in Chongqing College students (MARHCS) study was established in June 2013 as a perspective cohort study that recruited voluntary male healthy college students from 3 universities in Chongqing. The primary objectives of the MARHCS study are to investigate the associations of male reproductive health in young adults with sociopsychobehavioral factors, as well as changes of environmental exposure due to the relocation from rural campus (in University Town) to metro-campus (in central downtown). A 93-item questionnaire was used to collect sociopsychobehavioral information in manner of interviewer-interviewing, and blood, urine and semen samples were collected at the same time. The study was initiated with 796 healthy young men screened from 872 participants, with a median age of 20. About 81.8% of this population met the WHO 2010 criteria on semen quality given to the 6 routine parameters. Decreases of 12.7%, 19.8%, and 17.0%, and decreases of 7.7%, 17.6%, and 14.7% in total sperm count and sperm concentration, respectively, were found to be associated with the tertiles of accumulated smoking amount. Fried food consumption (1-2  times/wk or ≥3  times/wk vs nonconsumers) was found to be associated with decreased total sperm count (10.2% or 24.5%) and sperm concentration (13.7% or 17.2%), respectively. Coffee consumption was found to be associated with increased progressive and nonprogressive motility of 8.9% or 15.4% for subjects consuming 1-2  cups/wk or ≥3  cups/wk of coffee, respectively. Cola consumption appeared an association with decreased semen volume at 4.1% or 12.5% for 1-2  bottles/wk or ≥3  bottles/wk. A cohort to investigate the effects of environmental/sociopsychobehavioral factors act on semen quality was successfully set up. We found smoking, coffee/cola/fried foods consumption to be significantly associated with semen quality from the baseline investigation.

Association of DNA methylation and mitochondrial DNA copy number with human semen quality.

Whether there is a relationship between quality, DNA methylation, and mitochondrial DNA (mtDNA) copy number in human-derived sperm specimens is unknown. A cohort (n = 118) of male partners of couples who were undergoing fertility assessment because of an idiopathic inability to conceive were recruited. Sperm motility parameters were determined by computer-aided sperm analysis (CASA), while sperm quality was assessed using World Health Organization criteria, mtDNA copy number was measured by real-time PCR, and DNA methylation patterns were analyzed employing high-melting resolution PCR and bisulfite sequencing PCR. The mtDNA copy number negatively correlated with semen parameters, including sperm motility, concentration, morphology, progression, and motion characteristics (r for -0.19 to -0.54; P < 0.05 for all). As a surrogate marker for global DNA methylation, LINE-1 negatively correlated with sperm motility (r = -0.25; P = 0.009). Meanwhile, after adjustment for age, length of abstinence, smoking, and alcohol intake, there was a suggested association for increased LINE-1 methylation and mtDNA copy number tertiles versus sperm motility (odd ratios were 1.0, 2.6, and 4.7, and 1.0, 2.5, and 4.9, respectively). Altered mtDNA copy number and DNA methylation may serve as genetic and epigenetic markers to assess human sperm quality together with CASA parameters.

Environmental exposure to arsenic may reduce human semen quality: associations derived from a Chinese cross-sectional study.

BACKGROUND: Recent observations in in vitro and in vivo models suggest that arsenic (As) is an endocrine disruptor at environmentally-relevant levels. When exposed to As, male rats and mice show steroidogenic dysfunction that can lead to infertility. However, the possible effects of As on human male semen quality remain obscure. METHODS: We monitored the profile of As species in the urine of a reproductive-age human cohort and assessed its association with semen quality. Men (n = 96) were recruited in an infertility clinic from July 2009 to August 2010 in the Affiliated Hospital of Chongqing Institute for Population and Family Planning. Five urinary As species were analyzed by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). Clinical information on the semen volume, sperm concentration and motility was employed to catalogue and evaluate semen quality according to WHO guidelines. As species concentrations in addition to other continuous variables were dichotomized by the medians and modelled as categorical variables in order to explore using the binary logistic regression possible associations between As exposure and semen quality. RESULTS: Urinary concentrations (geometric mean ± SD, µg g(-1) creatinine) of different As species were 7.49 (± 24.8) for AsB, 20.9 (± 13.7) for DMA, 2.77 (± 3.33) for MMA, and 4.03 (± 3.67) for Asi (Asi(III )and Asi(V)). DMA concentrations above the median were significantly associated with below-reference sperm concentrations (P = 0.02) after adjusting for age, body mass index (BMI), abstinence, smoking and drinking habits. In addition, smoking was positively associated with MMA. CONCLUSION: Reduced parameters in human semen quality are positively associated with As exposure in a reproductive-age Chinese cohort.

Phthalates exposure of Chinese reproductive age couples and its effect on male semen quality, a primary study.

Phthalates are suspected of having adverse effects on androgen-regulated reproductive development in animals and may be toxic for human sperm. The purposes of our study were to investigate the general exposure of a Chinese reproductive age cohort to these ubiquitous pollutants and to assess their potential effect on semen quality. Six phthalate metabolites, monomethyl phthalate (MMP), monoethyl phthalate (MEP), monobutyl phthalate (MBP), monobenzyl phthalate (MBzP), mono-2-ethylhexyl phthalate (MEHP), and mono-2-ethyl-5-oxohexyl phthalate (MEOHP) were measured in spot urines of 150 individuals recruited from a Chongqing, China, reproductive institute. The questionnaire and clinical data were evaluated, and the correlations of phthalate exposure and semen qualities like semen volume, sperm concentration, motility and sperm motion parameters, were determined by multiple logistic regression analysis. The creatinine adjusted average concentrations for MMP, MEP, MBP, MBzP, MEHP and MEOHP were 41.3, 300, 41.0, 0.78, 2.99 and 3.90 µg/g, respectively. After adjustment for age, body mass index (BMI), abstinence, smoking, drinking, and education, there was a borderline-significant dose-response relationship between MBP and sperm concentration, with odd ratios (ORs) 1.0, 6.8 and 12.0 for increasing exposure tertiles (p=0.05). Although the dose-response relationships for MMP and MEP versus sperm concentration were not significant, a significant positive correlation between MEP and straight-line velocity of sperm motion was observed. The present data may imply some effects of phthalate exposure on semen. However, due to the small sample size, our finding needs to be confirmed on a larger population.

[Semen quality of the men from six districts in Chongqing].

OBJECTIVE: To investigate the quality and spatial distribution features of semen and to evaluate the reproductive health of the males in the Chongqing section of the Three-Gorge Reservoir area. METHODS: We collected semen samples by masturbation after 2 -7 days of abstinence from the men in Nan'an, Shapingba, Zhongxian, Wanzhou, Yunyang and Wushan of Chongqing, which are geographically and demographically representative of the Three-Gorge Reservoir area. We analyzed the semen quality of all the samples and evaluated the reproductive health of the men. RESULTS: The mean value of the five semen parameters of the male subjects from the six districts was within the normal range, including semen volume, sperm concentration, total sperm count, rapid progressive motile sperm, and total motile sperm. Those from Shapingba, Yunyang and Zhongxian exhibited abnormal sperm motility. According to the WHO criteria, normal value of all the semen parameters was found in less than 50% of the semen samples from the six districts, in 47% of those from Yunyang, and only 16% of those from Wanzhou. Spatial distribution maps of the semen parameters revealed significant spatial differences in seminal quality among the six districts, the highest in Yunyang, and the lowest in Wanzhou and Wushan that are located in the middle and lower reaches of the Three-Gorge Reservoir area. CONCLUSION: The mean value of semen parameters was low in a large proportion of men in the Chongqing section of the Three-Gorge Reservoir area, with spatial differences along the Changjiang river.

[Effects of smoking on sperm apoptosis and semen quality of adult males in the main urban area of Chongqing].

OBJECTIVE: To investigate the effects of smoking on sperm apoptosis and semen quality of healthy adult males in the main urban area of Chongqing. METHODS: According to the smoking habit, we divided 235 healthy adult males into a non-smoking group (n = 89) and a smoking group (n = 146). Then we detected the routine semen parameters by the computer-assisted semen analysis system and obtained the parameters of sperm apoptosis (the ratios of AN-/PI-, AN+/PI-, AN+/PI+ and AN-/PI+ sperm) by flow cytometry combined with Annexin V-FITC/PI fluorescence staining. RESULTS: The rate of early apoptotic sperm (AN+/PI-) was higher in the smoking than in the non-smoking group ([8.1 +/- 5.1]% vs [6.8 +/- 3.8]%; P = 0.039), but there were no significant differences between the two groups in the rate of late apoptotic sperm (AN+/PI+) ([5.6 +/- 5.2]% vs [5.5 +/- 5.1]%; P = 0.87), as well as in such routine semen indexes as semen volume, sperm density, sperm motility, sperm vitality and normal sperm morphology (P = 0.30, 0.82, 0.37, 0.81 and 0.84, respectively). CONCLUSION: The rate of early apoptotic sperm is higher in smokers than in non-smokers, suggesting that smoking may induce early damage to sperm cells. Compared with routine semen parameters, sperm apoptosis is a more sensitive biomarker to reflect smoking-induced damage to sperm.

Semen quality of 1346 healthy men, results from the Chongqing area of southwest China.

BACKGROUND: Large studies on semen quality of the general healthy population from China are rare. METHODS: A cross-sectional study was performed to evaluate the semen quality of 1346 healthy men residing in Chongqing area of southwest China in 2007. The semen parameters were measured and compared with the World Health Organization (WHO) criteria. A linear regression model was used to examine the determinants of semen quality. RESULTS: The medians were 2.3 ml for semen volume, 77.8 x 10(6) per ml for semen concentration, 167.7 x 10(6) for total sperm count, 33% for sperm rapid progressive motility, 52.6% for sperm progressive motility and 70.9% for total motility. According to WHO criteria, 61.1% of healthy males had at least one semen parameter below normal threshold values. Season and abstinence duration were found to be significantly associated with semen quality (P < 0.001). Age, smoking, alcohol use and BMI had little or no effect on semen parameters. CONCLUSIONS: A high proportion of healthy males in Chongqing area of southwest China had abnormal semen parameters values according to WHO criteria. The semen parameters in the study population were markedly different from those reported for the other Chinese, USA and European populations. The differences remain unexplained and may be due to demographic characteristics, lifestyle, environmental factors or genetic variation.