Two Forms Of Urinary Phthalate Metabolites In Pregnant Women And Associations Between Maternal Phthalate Exposure And Birth Outcomes

Phthalates are synthetic compounds with endocine disrupting properties,and extensively used as plasticizers in various industrial and consumer products.Because phthalatea are not covalently bound to the matrix molecules in the product,it is easy to migrate from products to the environment over time,resulting in contamination of food,water,and air.There are multiple sources of exposure to phthalates,mainly including ingestion of food and water contaminated in the processing and packaging process,absorption from personal care products and air inhalation.Previous studies have found that detectable urinary concentrations of phthalate metabolite were found in more than 90%of the samples,suggesting that phthalates exposure is widespread in humans.Pregnant women and infants as a sensitive population of special concern,are more susceptible to the reproductive toxicity of phthalates.In addition,total urinary phthalate metabolites(the free plus conjugated forms)have been frequently measured in humans to be an exposure biomarker,while some evidence have shown that glucuronidation is a major route of detoxification for phthalates.Therefore,the free form may be more bioactive and might be used to reflect the bio?effective dose of human exposure.This is also confirmed in animal studies which found that free from of MBP was associated with more severe adverse effects on testicular damage than its total form.Furthermore,placental transfer of glucuronidated xenobiotics is typically inefficient,while the free form xenobiotics can freely pass through the placental barrier.Both animal and human studies have found that the free phthalate metabolites were predominant in amniotic fluid Thus,to some extent,the free form of phthalate metabolites can be used to reflect the biologically effective levels of human and intrauterine exposure.However,data are limited on the existence of free and conjugated forms of phthalate metabolites in urine of pregnant women,and further stdies are needed to evaluate the potential value of free phthalate metabolites as exposure biomarkers.Phthalate metabolites have been demonstrated to be able to transport from mother to embryo via placentas,and have been detected in umbilical cord blood,meconium and amniotic fluid in previous studies,indicating phthalate exposure to fetuses.Phthalates as a kind of environmental endocrine disruptors,can affect the homeostasis of mother and fetus which is vital for growth and metabolism of fetus,and thus lead to adverse birth outcomes.At present,it has been reported that prenatal phthalate exposure is associated with birth weight and birth length,but the evidence is limited and the conclusions are inconsistent so far.Further researches are needed to confirm these findings.In addition,toxicological evidence suggests that phthalate may interfere with the hypothalamic-pituitary-thyroid axis at multiple points and affect the thyroid hormone homeostasis.While maintaining thyroid homeostasis during pregnancy is essential for energy metabolism and normal fetal growth.Epidemiological studies have found that exposure to phthalate during pregnancy can affect thyroid hormone levels in mothers,but the evidence on impact of neonatal thyroid hormone homeostasis is limited.The aim in this study was to assess the levels and predictors of urinary phthalate metabolites among Chinese pregnant women,and investigate the main form of different phthalate metabolites in urine by measuring both free and total forms of phthalate metabolites.In addition,we aimed to evaluate the association of maternal phthalate exposure with birth outcomes based on nested case-control study and cohort study,and further investigate the relationship between maternal phthalate exposure and thyroid stimulating hormone levels in newborns,to explore its possible mechanism.Part I:Levels and Predictors of Urinary Phthalate Metabolites in Chinese Pregnant WomenObjective:We aimed to assess the phthalate exposure levels among pregnant women in Wuhan,and investigated the possible influence factors of phthalate exposure.Methods:The present study was a part of the Healthy Baby Cohort(HBC)study.We randomly selected 1002 pregnant women from participants in HBC who were recruitd in Wuhan City during 2014,and collected one spot urine of each pregnant woman before delivery.Concentrations of seven major phthalate metabolites(MMP,MEP,MBP,MECPP,MEHHP,MEOHP and MEHP)in urine were measured using ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS).The demographic information of pregnant woman was obtained from questionnaire and meicical records.One-way analysis of variance was used to evaluate the association between phthalate exposure levels and predictors.Results:Detectable MEHP was found in 80.0%of the urine samples,while other phthalate metabolites were found in 97.4-99.9%of the urine samples.Among the metabolites,MBP was present at the highest median concentrations(206 ?g/g Crea),followed by MMP(20.3 pg/g Crea),MECPP(17.6 ?g/g Crea),MEHHP(15.8 pg/g Crea),MEP(15.0 ?g/g Crea),MEOHP(13.8 ?g/g Crea)and MEHP(3.14 ?g/g Crea),respectively.Urinary levels of phthalate metabolites were influenced by maternal education,pre-pregnancy BMI,parity,passive smoking and season of sample collection.Conclusions:The results showed that the detection rate of PAEs metabolites in urine was high,suggesting the widespread exposure to phthalates among pregnant women in Wuhan.These exposures may be related to maternal characteristics,such as maternal education,pre-pregnancy BMI,parity,passive smoking and season of sample collection.Part ?:Free and Conjugated Forms of Urinary Phthalate Metabolites in Pregnant WomenObjective:In this stusdy,we measured free and total forms(free form plus conjugated form)of urinary phthalate metabolites levels to study the glucuronidation capacity of different phthalate metabolites in pregnant women and explore the main forms of their metabolites in urine,and investigated their possibole influence factors.Methods:Pregnant women(n = 293)were randomly selected from the participants enrolled in HBC in Wuhan City during 2012-2014.The demographic information of pregnant woman was obtained from questionnaire and medical records.Concentrations of free and total forms of seven phthalate metabolites were measured using UPLC-MS/MS.We calculated the percentage of free metabolite to total metabolite concentration[free%],and investigated their possible influence factors using one-way analysis of variance.Results:Detectable urinary concentrations of free MMP,MEP,MECPP,MEHHP,MEOHP,and MBP were found in 81.2%,81.2%,100.0%,81.9%,92.8%and 78.2%of the samples,respectively.Among the free form phthalate metabolites,MBP was present at the highest median concentrations(6.18?g/g Crea),followed by MMP(6.18 ?g/g Crea),MECPP(8.19 ?g/g Crea),MEP(4.47 ?g/g Crea),MEHHP(1.26 ?g/g Crea)and MEOHP(1.02 ?g/g Crea),respectively.MECPP had the highest percentage of free concentration to the total form(Median:67.0%),whereas the medians free%of the other two metabolites of DEHP(MEHHP,MEOHP)were considerably lower(9.6%,11.0%).MEP was found to have the highest median free%values(52.4%)among low molecular weight phthalates(LMWPs),followed by MMP(40.7%)and MBP(9.0%).Urinary levels of free form phthalate metabolites were influenced by maternal education,pre-pregnancy BMI,parity and season of sample collection.Conclusions:This is the first study to report the free and total forms of phthalate metabolites among pregnant women.In this study,the main form of diferent phthalate metabolites in urine is different.MECPP,MEP and MMP presented in both free and conjugated form in urine.While MEHHP,MEOHP and MBP mainly existed in the free form.In addition,the levels of free form phthalate metabolites may be related to maternal characteristics,such as maternal education,pre-pregnancy BMI,parity and season of sample collection.Part ?:Associations of Maternal Phthalate Exposure with Birth Outcomes and Thyroid-stimulating Hormone Levels in Newborns Objective:To investigate the association between maternal phthalate exposure and low birth weight,and further evaluate associations of maternal phthalate exposure with birth weight,birth length and thyroid-stimulating hormone levels of newborns in a cohort study.Methods:Based on the birth cohort,we included 114 cases of low birth weight infants and 342 matched controls of normal birth weight infants.For every case selected,three controls were randomly selected and matched by infant sex and maternal age at conception.Concentrations of total forms of seven phthalate metabolites were measured using UPLC-MS/MS.A conditional logistic regression analysis was used to evaluate the assosiction between maternal phthalate exposure and low birth weight.Based on the birth cohort,we randomly selected 1002 mother-infant pairs,which is same to the Part ?.The information(e.g.birth weight,birth length and gestational age)of newborns was obtained from the medical records.Birth weight z-scores,a standardized measure of birth weight for gestational age,were calculated according to the INTERGROWTH-21st Newborn Birth Weight Standards and Z Scores.Multiple linear regression models were used to evaluate associations between maternal phthalate exposure and birth size(birth weight,birth weight z-scores,birth length,and ponderal index).973 mother-infant pairs with TSH measurement were selected from 1002 mother-infant pairs.Multiple linear regression models were used to evaluate associations between maternal phthalate exposure and TSH levels of newborns.Results:In crude models,both MEHP[OR= 1.34,95%confidence interval(CI):1.08,1.67]and EDEHP(OR=1.46,95%CI:1.03,2.08)were associated with low birth weight in boys.After covariates adjustment,phthalate metabolite concentration were no longer associated with low birth weight in boys.No significant association was observed in girls.In boys,the ln-transformed DEHP metabolite levels were significantly associated with increased birth weight and birth weight z-scores.Additionally,each ln-unit increase in MECPP was associated with a 0.25 kg/m3(95%CI:0.03,0.47)increase in ponderal index in boys.However,we observed no significant association of maternal phthalate metabolite levels with any of the outcomes in girls.Several phthalate metabolites were associated with birth weight and birth weight z-scores in patterns that varied by sex(p for sex interaction was 0.038 for MECPP and 0.037 for MEOHP).In boys,higher MEP levels were associated with increased TSH levels of newborns[MEP:Padjusted=0.40 for medium tertile,95%CI:0.11,0.69;padjusted=0.32 for highest tertile,95%CI:0.03,0.61].In girls,higher MMP level was associated with decreased TSH levels of newborns[Padjusted=-0.33 for highest tertile,95%CI:-0.63,-0.02].Conclusions:In this study,there is a sex difference in the associations of maternal phthalate exposure with increased birth weight and ponderal index,which were apparent only in boys.Additionally,we observed associations between maternal phthalate exposure and TSH levels in newborns,suggesting that phthalate exposure may affect the thyroid homeostasis in newborns.Further studies are needed to corroborate these findings.