Effects of Prenatal Phthalate Exposure on Thyroid Hormone Concentrations Beginning at The Embryonic Stage

Limited studies have consistently shown an association of phthalates exposure with thyroid hormones (THs) in pregnant women. However, it remains unknown on which specific phthalates can affect THs and whether any effects could differ by gestational age. In the present study, we investigated associations between serum concentrations of phthalate monoesters [monoethyl phthalate (MEP), mono-(n + iso)-butyl phthalate (MBP) and mono(2-ethylhexyl) phthalate (MEHP)] and THs [thyroid-stimulating hormone (TSH), total thyroxine (TT4) and free thyroxine (FT4)] in Chinese pregnant women. 1,397 women were recruited from the China-Anhui Birth Cohort. Maternal serum samples were collected and used to measure phthalate metabolites and THs. Covariate-adjusted linear regression analyses showed that natural log (Ln)-transferred concentrations of MBP and LnMEHP were negatively associated with TT4 (β = −0.277 and –0.461, respectively; p < 0.001). Stratification analyses by gestational weeks showed significant associations of LnMBP and LnMEHP with TT4 in gestational weeks 5 to 8, 9 to 12, and 13 to 20. Our findings suggest an association of serum phthalates with lower TT4. The influence of MBP and MEHP on TT4 concentrations throughout the early pregnancy may begin from the embryonic stage (gestational weeks 5 to 8).

On the basis of the China-Anhui Birth Cohort (C-ABC), the objective of this pilot study was to investigate the cross-sectional association between prenatal serum concentrations of phthalate monoesters and maternal THs concentrations.

Results
Demographic characteristics. Among the 1,397 participants, the average age and gestational weeks of women were 26.6 ± 3.6 (range: 19-45) years and 12.0 ± 3.9 (range: 5−20) weeks, respectively. The pre-pregnancy body mass index (BMI) was 20.1 ± 2.3 kg/m 2 . Most women lived in urban areas (79.0%). Only 27.9% of women were none-passive smokers (Table 1). Pearson correlation and ANOVA tests showed that maternal age, pre-pregnancy BMI, gestational age, residence and passive smoking were associated with at least one type of THs.
Serum phthalate metabolites and THs concentrations. Phthalate metabolites and THs concentrations were measured in the serum samples ( Table 2). All the three metabolites (MEP, MBP, and MEHP) were detectable in over 86% samples. The concentration of MEHP (median = 6.14 ng/mL) was the highest, followed by that of MBP (median = 5.97 ng/mL). The concentration of MEP (median = 0.20 ng/mL) was the lowest. The median concentrations of FT4, TT4 and TSH were 0.96 ng/mL, 9.24 ng/mL, and 1.70 mIU/mL, respectively.
Stratification analyses based on gestational weeks. The addition of a product interaction term of gestational age with each phthalate metabolite to the model indicated that the associations of phthalate metabolites with TSH, FT4, or TT4 did not differ by time stages assessed in this study (data no shown). Stratification analyses

Discussion
In the present study, the detection rates of the three phthalate metabolites in serum were 86%−100%. The medians of serum MEP, MBP, and MEHP were 0.2, 5.97, and 6.14 ng/mL, respectively. The result suggested that Chinese women during gestational weeks 5 to 20 were widely exposed to low concentration of DEP, DBP, and DEHP. It was expected that concentrations of phthalate metabolites in urine were higher than those in serum. However, our reported concentrations of serum phthalates were different from those in other studies. The geometric means of serum MEP, MBP, and MEHP were 67.8, 14.7, and 1.39 ng/mL respectively, in females from the 2009-2010 National Health and Nutrition Examination Survey 29 . An infant congenital hypothyroidism study in Korea 30 observed that the mean plasma concentrations of MBP and MEHP were 19.87 and 9.82 ng/mL in mothers from control group, and 27.38 and 13.57 ng/mL in mothers from case group, respectively. The variation of phthalate exposure levels was partially attributed to the variations in sample collection time (e.g., gestational weeks) 31 and the differences in laws and regulations against application of phthalates in different countries. Our results showed negative associations of maternal serum LnMBP and LnMEHP with TT4 concentrations, which occurred as early as gestational weeks 5 to 8. Similarly, the association of "LnPAE" with TT4, which combined the three metabolites into a single class of chemicals, was also identified. Positive associations of serum LnMBP and LnMEHP with LnFT4 were also found. Data on the association of serum phthalates with thyroid function in pregnant women are limited. Previous animal studies showed that rats fed DEHP through the diet displayed histopathologic alterations in the thyroid indicative of hyperactivity, increased thyroglobulin turn over, and significantly decreased plasma T 4 concentrations 13,32-34 . A few epidemiological studies explored the associations of prenatal phthalates exposure with maternal thyroid function, which was assessed by metabolites concentrations in urine samples 13,16,[25][26][27][28] . Three studies reported that maternal phthalates exposures (e.g., MBP, MBzP, MEHP and MEHHP) were associated with reduced concentrations of TT4 13,25,28 , which were consistent with our results. However, Johns et al. 2016 27 reported that increased MEHP was associated with an increase of TT4. No significant associations of maternal TT4 concentrations were found by Kuo et al. 2015 16 and John et al. 2015 26 . Three previous studies revealed that some phthalates (e.g., MBP, MBzP and metabolites of DEHP) exposure were related to decreased FT4 concentrations 13,25,26 , whereas two investigations reported opposite results 16,27 . No significant relationships were observed for TSH in most previous studies. Our previous study 25 Table 2. Concentrations of phthalate metabolites and thyroid hormones in serum samples from the Chinese pregnant women (n = 1,397). Abbreviations: LOD = limit of detection. * p < 0.05; ** p < 0.001.
MiBP, and MCPP with TSH. Obviously diverse relationships between phthalates and THs were showed. We speculated that there were several potential reasons for these inconsistent findings. First, these studies were based on different sample sizes. Only our previous study recruited over 500 women (n = 2,521) 25 . Second, urine samples were collected in different time/gestational weeks. Two studies 16,25 obtained urine samples in the first trimester of pregnancy, and three studies 13,26,28 in the second trimester of pregnancy. Johns et al. 2016 27 averagely collected urine in 10th, 18th, 26th and 35th weeks' of gestation for a repeated measures analysis. The biological mechanisms of above abservations still remain unclear. Several studies have shown that thyroid hormone receptors (TRs) may be unintended targets of manufactured chemicals. Phthalates also have been reported to have TRs antagonist activity [11][12][13][14][15][16] and/or interfere triiodothyronine binding to transthyretin 35 . Some phthalates such as DBP and DEHP may induce thyroid hyperactivity and decrease thyroxin concentrations through modulating the transcriptional activity of NIS 36 .
There are some limitations in the present study. Firstly, we did not collect urine samples. Urine is the best matrix for measuring non-persistent chemicals including phthalates 37 . However, phthalate metabolites concentrations in serum significantly correlated to those in urine, suggesting that metabolites in serum could be used as biomarkers of human exposure 38 . Secondly, as the present study is a pilot research, the indicators of thyroid function and phthalates exposure were limited. We did not measure thyroid peroxydase antibody which was one of the most important determinants of thyroid function. In addition, only the primary metabolites of DEP, DBP and DEHP were analyzed since pregnant women were predominantly exposed to these three types of phthalates 13 . Finally, the cumulative exposure risks of multiple other phthalates could not be obtained in the present study.
Our findings indicate that serum LnMBP and LnMEHP during early pregnancy are associated with decreased maternal TT4 concentrations. Besides, it is suggested that the effects of MBP and MEHP exposure on TT4 could begin as early as at the embryonic stage.

Materials and Methods
Study participants. The C-ABC study was conducted to examine the delayed, cumulative and interactive effects of maternal environmental exposures on birth outcomes and children's development. All details of in-and exclusion criteria were described in our previous studies 39,40 . Briefly, this cross-sectional survey, as a part of the C-ABC study, enrolled participants in the first 20 weeks' gestation in Ma'anshan city between November 2008 and September 2009. 1,599 women with singleton pregnancies were invited to participate at their first antenatal care visit. The response rate was 97.4% (n = 1,557). They were required to complete a self-administered questionnaire. Meanwhile, a single blood sample was collected from each woman for the laboratory examination. Finally, 1,397 pregnant women with complete data of both THs (measured in 2010) and phthalates (measured in 2012) were enrolled in the present study.  Table 4. Stratification analyses of associations between phthalate metabolites and THs on basis of gestational weeks. Adjustment for maternal age, pre-pregnancy BMI, residence and passive smoking. * p < 0.05, ** p < 0.001.
Informed consent was obtained from all participants in our study. The study (including the additional serum analysis of phthalates and thyroid hormones) was approved by the Biomedicine Ethical Committee of Anhui Medical University (approval NO. 2007002). All the study methods were performed in accordance with Helsinki declaration II.
Serum phthalate monoester analyses. At the prenatal visit, a single peripheral venous blood sample per woman was drawn from an antecubital vein into 10 mL glass vacuum vessels. After centrifugation, samples were aliquoted and stored at −80 °C until biochemical analyses. In the Anhui Provincial Key Laboratory of Population Health & Aristogenics (Anhui Medical University, Hefei City, China), a revised isotope dilution-solid phase extraction (SPE) coupled to high-performance liquid chromatograph-tandem mass spectrometry (HPLC-MS/ MS; HPLC, Agilent 1200; MS, Agilent 6410) method (Frederiksen et al., 2010) was used to analysis the serum concentration of phthalates. In brief, monoethyl phthalate (MEP) was measured through external standard method, while MBP (mono-n-butyl phthalate plus mono-iso-butyl phthalate) and MEHP were measured through isotope-labeled internal standard (MBP-d 4  The phthalate monoester concentrations below the LOD were replaced with a value of LOD/√2 for statistical analyses. To investigate the combined risks of multiple phthalate exposure, we calculated a single variable "PAE" through summing up the concentrations of all metabolites by weighting their molecular weight. Here is the equation. Firstly, we statistically described the concentrations of phthalate metabolites using several selected percentiles. Furthermore, as the distributions of phthalate metabolites and FT4 and TSH were right-skewed, their concentrations were natural logarithm (Ln)-transformed for statistical analyses. Multivariable linear regression analyses were used to explore the relationships between phthalate monoesters and THs. Finally, stratification analyses were applied by gestational weeks of serum sampling time (gestational weeks 5 to 8, 9 to 12 and 13 to 20). Potential confounders which were significantly correlated to THs according to Pearson correlation or ANOVA tests were included in the adjusted statistical models.