Temporal Variability Of Urinary Bisphenol A And Its Substitutes And Their Associations With Male Semen Quality

Bisphenol A(BPA),as one of endocrine disrupting chemicals(EDCs),is widely present in various environmental media,resulting in multiple health hazards,including reproductive health damage.For the past few years,due to the highly adverse health effects,the use of BPA has been restricted or banned,which leads to the increasing usage of its substitutes such as bisphenol F(BPF)and bisphenol S(BPS).A large number of studies have shown that humans are widely exposed to BPA and its substitutes(BPF and BPS),and the exposure level of these substitutes is increasing.Accurate exposure assessment is a prerequisite to clarify the health hazards of exposure to environmental pollutants.Previous studies have reported that there is a high temporal variability in urinary BPA concentrations.Measurement of a single spot urine sample can lead to exposure misclassification.However,the temporal variability of BPF and BPS remains unclear.Toxicological studies have revealed that BPA can damage germ cells,disrupt spermatogenesis and reduce semen quality.To date,a large number of epidemiological studies have investigated the association between BPA exposure and male semen quality,but the results are inconsistent.Moreover,human studies on the associations between BPA substitutes and semen quality are very limited.Measurement of a single spot urine sample has been frequently used to evaluate the relationships between exposure to BPA and its substitutes and male semen quality in previous epidemiological studies.Such exposure assessment can easily lead to the false positive or false negative results.Furthermore,genetic factors and environment-gene interaction are also important factors affecting male semen quality.However,no studies to date have explored whether apoptosis-related gene polymorphisms modify the associations of exposure to BPA and its substitutes with male semen quality.Based on the above research background,we firstly investigated the temporal variability of urinary BPA and its substitutes(BPF and BPS)during male spermatogenesis,and also evaluated the accuracy of using one or more urine samples in predicting individual’s average 90-day exposure level.Secondly,we recruited reproductive-aged men who came to the reproductive center of Tongji Hospital in Wuhan for semen quality analysis,and collected two urine samples to improve the accuracy of individual exposure assessment.We further explored the associations between exposure to BPA and its substitutes and male semen quality.At last,the effect modifications by apoptosis-related gene polymorphisms on the associations of exposure to BPA and its substitutes with male semen quality were analyzed.The results will provide a scientific basis for the selection of accurate exposure assessment methods and human evidence for the reproductive health risk assessment for BPA and its substitutes.1 Temporal variability of urinary BPA and its substitute concentrations during male spermatogenesisObjective:To investigate the temporal variability of urinary BPA and its substitutes(BPF and BPS)concentrations during the spermatogenesis cycle(90 days)in reproductive-aged men.Methods:Eleven healthy and reproductive-aged men were recruited.All urine samples(N=520)were collected from 11 subjects at 8 days of one spermatogenesis cycle(90 days),including day 0,1,2,3,4,30,60 and 90.The concentrations of BPA,BPF and BPS in urine were detected by ultra-high performance liquid chromatography combined with mass spectrometer(UPLC-MS).Intraclass correlation coefficients(ICCs)were calculated to estimate the temporal variability of BPA and its substitute concentrations in spot(random urine samples),first morning voids(FMVs)and 24-hour urine.Mixed linear model was used to analyze the accuracy of using spot samples or FMVs in predicting 24-hour urinary concentrations of BPA and its substitutes.Specificity and sensitivity were used to evaluate the accuracy of using one or more urine samples in predicting the average concentrations of BPA and it substitutes during 90 days.Result:The detection rates of BPA and BPF in urine samples were≥85%,while the detection rate of BPS was only 13%(the follow-up analysis was not performed for BPS).The reproducibility of BPA and BPF in spot,FMVs and 24-hour urine was poor(ICCs=<0.01-0.28),among which the intra-individual variability was the biggest contributor,accounting for 72%-99%of the total variability.The accuracy of using spot urine sample to predict 24-hour concentrations of BPA and BPF in urine was higher than that of the FMVs.The specificity(0.66-0.85)was high but the sensitivity(0.37-0.65)was low when using one spot urine or one FMVs to predict subjects with urinary BPA and BPF concentrations in the highest exposure group(the top three or four individuals with average concentrations of all urine samples over a 90-day period).There were higher specificity(0.71-0.90)and sensitivity(0.43-0.75)in predicting urinary BPA and its substitutes by collecting two urine samples from different time points.However,the specificity(0.70-0.89)and sensitivity(0.43-0.75)did not improve when three random urine samples were collected.Conclusions:In this part of the study,the reproducibility of urinary BPA and its substitute concentrations was poor during the spermatogenesis.Using a single measurement of urinary BPA and its substitute concentrations to assess an individual’s average 90-day exposures can lead to exposure misclassification,whereas using two urine samples collected at different time points could improve the accuracy of the exposure assessment of BPA and its substitutes.2 Associations between urinary BPA and its substitute concentrations and male semen qualityObjective:Measurements of urinary BPA and its substitute concentrations at two different time points were used to improve the accuracy of exposure assessment.The associations between individual and mixture exposure to BPA and its substitutes and conventional semen quality and spermatozoa apoptosis parameters were analyzed.Methods:Two urine samples and one semen sample were collected from 984 reproductive-aged men who came to reproductive medicine center of Tongji Hospital in Wuhan from March to July 2013 for semen quality examination.The concentrations of BPA,BPF and BPS in all urine samples were determined using UPLC-MS,and the average concentrations were calculated as internal exposure biomarkers.Conventional semen quality parameters(i.e.,sperm concentration,total sperm count,sperm progressive motility and total motility)were measured according to the World Health Organization(WHO,fifth version)standards.Annexin V/PI apoptosis Kit coupled with the flow cytometry were used to measure spermatozoa apoptosis parameters(i.e.,dead spermatozoa rate,viable spermatozoa rate and apoptosis spermatozoa rate).Multivariate logistic regression models,multivariate linear regression models and Bayesian kernel machine regression(BKMR)models were used to assess the associations between individual or mixtures of BPA,BPF and BPS exposures and parameters of conventional semen quality and spermatozoa apoptosis.Stratification analyses were used to further investigate the modification effect of age,body mass index(BMI),alcohol consumption and smoking status on these above-mentioned associations.Result:The detection rates of BPA,BPF and BPS in both urine samples were more than83%.The averaged median concentrations of BPA,BPF and BPS were 2.24,0.90 and 0.38μg/L,respectively.Based on the WHO reference values of conventional semen quality parameters,91 men(9.2%)had sperm concentrations below 15×10~6/m L;121 men(12.3%)had total sperm counts below 39×10~6/one ejaculate;271(27.5%)men had sperm progressive motility lower than 32%;314(31.9%)men had sperm total motility lower than40%,and 621 men(63.1%)had all four semen quality parameters at or above the reference values.After adjusting for age,BMI,abstinence duration,alcohol use,smoking status,education level and ever having fathered a pregnancy,multivariate logistic regression models showed that higher BPA exposure was associated with increased odds ratios(ORs)of having below-reference sperm concentration,total sperm count,progressive motility and total motility(all P for trends<0.05).Compared with those in the first quartile of averaged urinary creatinine-adjusted BPA concentrations,men in the fourth quartiles had ORs of 2.16[95%confidence interval(CI):1.13,4.13],2.09(95%CI:1.20,3.64),2.09(95%CI:1.38,3.16)and 2.09(95%CI:1.41,3.11)in having below-reference sperm concentration,total sperm count,progressive motility and total motility,respectively.We also observed that higher BPS exposure was associated with increased ORs of having below-reference progressive motility and total motility(both P for trends=0.02).Compared with those in the first quartile of averaged urinary creatinine-adjusted BPS concentrations,men in the fourth quartile had ORs of 1.62(95%CI:1.07,2.43)and 1.57(95%CI:1.06,2.33)in having below-reference progressive motility and total motility,respectively.When conventional semen quality parameters were modeled as continuous variables,multivariate linear regression models indicated that increased urinary BPA and BPS concentrations were still negatively associated with conventional semen quality parameters(all P for trends≤0.06).However,the associations between increased urinary BPF concentration and decreased conventional semen quality parameters were not statistically significant.In addition,we found that increased urinary BPS concentration was associated with increased dead spermatozoa rate(P for trend=0.06).Compared to those in the first quartile of averaged urinary creatinine-adjusted BPS concentrations,men in the highest quartile had an increase in dead spermatozoa rate of 18.36%(95%CI:-2.31%,43.41%).Stratification analyses showed that the inverse associations of BPA and BPS exposure with sperm concentration and total sperm count were only observed among older men(≥30 years old)(all P for interactions≤0.10),the inverse associations of BPF and BPS exposure with sperm concentration and total sperm count were only observed for smokers(all P for interactions<0.05),and the positive association between BPA exposure and apoptosis spermatozoa rate were only observed among non-drinkers(P for interaction=0.07).BKMR models showed that there were statistically significant increased risks of having below-reference sperm progressive motility and total motility when bisphenol mixtures were at≥55th percentiles compared to 50th percentiles.For conventional semen quality parameters which were modeled as continuous outcomes,statistically significant inverse associations with continuous sperm progressive motility and total motility were still observed when bisphenol mixtures were between 40th and 60th percentiles compared to50th percentiles.For spermatozoa apoptosis parameters,there were statistically significant increased dead spermatozoa rates when bisphenol mixtures were at≥55th percentiles compared to 50th percentiles,whereas there was a decreasing trend in the viable spermatozoa rate for bisphenol mixtures.The univariate exposure-response relationship results showed that there were nonlinear associations(e.g.,J-shaped,inverted J-shaped,U-shaped or inverted U-shaped curves)between individual bisphenol exposure and parameters of semen quality and spermatozoa apoptosis when setting the other bisphenols to their median levels.Conclusions:In this part of the study,exposure to individual BPA and BPS and bisphenol mixtures were associated with decreased conventional semen quality,and exposure to individual BPS and bisphenol mixtures were associated with spermatozoa apoptosis.3 Interactions between urinary bisphenol A and its substitute concentrations and apoptosis-related gene polymorphisms on male semen qualityObjective:To investigate the interactions between exposure to BPA and its substitutes and apoptosis-related gene polymorphisms on parameters of spermatozoa apoptosis and conventional semen quality.Methods:The subjects were from those reproductive-aged men who provided blood samples in the second part of the study(N=453).Single nucleotide polymorphisms(SNPs)of apoptosis-related genes(Fas,Fas L and caspase3)were detected by fluorescence quantitative polymerase chain reaction(PCR).The SNPs loci were Fas rs1800682 and Fas rs2234767,Fas L rs763110 and caspase3 rs12108497,respectively.The distribution of gene genotypes was analyzed for deviation from Hardy-Weinberg equilibrium by using Chi-square goodness of fit test.Multivariate linear regression models and multivariate logistic regression models were used to analyze the associations between polymorphisms of apoptosis-related genes and parameters of spermatozoa apoptosis and conventional semen quality,as well as the interactions between exposure to BPA and its substitutes and polymorphisms of apoptosis-related genes on parameters of spermatozoa apoptosis and conventional semen quality.Result:In addition to Fas rs1800682,Fas rs2234767,Fas L rs763110 and caspase3rs12108497 were in accord with Hardy-Weinberg equilibrium among the participants(all P>0.05).Multivariate linear regression models showed that Fas rs2234767,Fas L rs763110and caspase3 rs12108497 SNPs were not associated with conventional parameters of semen quality.But Fas L rs763110 polymorphisms was associated with increased dead spermatozoa rate.Men with Fas L rs763110 CT and CT/TT genotypes had 26.93%(95%CI:1.60%,58.57%)and 24.0%(95%CI:0.39%,53.21%)higher dead spermatozoa rate than those with Fas L rs763110 CC genotypes.Interaction analysis showed that there were statistical interactions between BPA and BPS exposures and Fas L rs763110 and caspase3rs12108497 gene polymorphisms on both dead spermatozoa rate and viable spermatozoa rate,respectively(all P for interaction≤0.10).BPA exposure in Fas L rs763110 CC carriers and BPS exposure in caspase3 rs12108497 TC/CC carriers were associated with increased both dead spermatozoa rate and viable spermatozoa rate.Moreover,there were also statistical interactions between BPF exposure and caspase3 rs12108497 gene polymorphisms on conventional semen quality parameters(all P for interaction≤0.10).BPF exposure in caspase3 rs12108497 TC/CC genotype carriers were associated with increased risk of having below-reference total sperm count,sperm progressive motility and sperm total motility,as well as with decreased sperm progressive motility and sperm total motility.Conclusions:Apoptosis-related gene polymorphisms(Fas L and caspase3)might modify the associations between exposure to BPA and its substitutes and spermatozoa apoptosis and conventional semen quality parameters.