Free bisphenol A (BPA), BPA-Glucuronide (BPA-G), and total BPA concentrations in maternal serum and urine during pregnancy and umbilical cord blood at delivery

Emerging Contaminants 5 (2019) 279e287

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Research article

Free bisphenol A (BPA), BPA-Glucuronide (BPA-G), and total BPA concentrations in maternal serum and urine during pregnancy and umbilical cord blood at delivery Warren G. Foster a, *, Cariton Kubwabo b, Ivana Kosarac c, Sandra Gregorovich a, Garima Aryal a, Kaela Coleman b a b c

Department of Obstetrics & Gynaecology, McMaster University, Hamilton, Ontario, L8S 4K1, Canada Environmental Health Science and Research Bureau, Health Canada, Ottawa, Ontario, K1A 0K9, Canada Tobacco Control Directorate, Health Canada, Ottawa, Ontario, K1A 0K9, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 May 2019 Received in revised form 13 August 2019 Accepted 30 August 2019

While adult exposure to total bisphenol A (BPA) has been well documented, developmental exposure to BPA and fetal exposure to the bioactive form (free BPA) remains poorly defined. Therefore, pregnant women (n ¼ 199) between 28 and 35 weeks of pregnancy were invited to participate in this study. Maternal serum free of hemolysis (n ¼ 189) and urine (n ¼ 112) were collected at third trimester and delivery along with umbilical cord blood (UCB) at delivery. Free BPA, BPA mono-glucuronide (BPA-G) and total BPA concentrations were measured using gas chromatography coupled with tandem mass spectrometry. Circulating concentrations of free BPA were quantifiable above the method detection limit (MDL ¼ 0.026 ng/mL) in approximately 34% of serum samples in the third trimester and 21% of samples at delivery whereas BPA-G and total BPA were quantified in 43 and 70% of third trimester and delivery samples, respectively. The geometric mean of free BPA, BPA-G and total BPA concentrations in maternal serum during the third trimester and at delivery were 0.62 ng/L and
Keywords: Bisphenol A Free BPA Conjugated BPA Pregnancy Exposure Fetal development Cord blood

1. Introduction Bisphenol A (BPA) is a monomer used in the manufacture of polycarbonate plastics and some epoxy resins. First synthesized in 1891 and investigated as a potential estrogenic agent in the 1930s [1], BPA ultimately found commercial application as a plasticizer [2] in the 1950's. BPA has been used extensively in a broad range of commercial applications and products including reusable water bottles, sports equipment, compact discs, DVDs, paperboard packaging, adhesives, dental sealants, lenses for eye glasses, the lining of

* Corresponding author. Department of Obstetrics & Gynaecology, HSC-3N52D, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada. E-mail address: [email protected] (W.G. Foster). Peer review under responsibility of KeAi Communications Co., Ltd.

water pipes and sealants for tin cans, polyvinyl chloride stretch sealants, colour powder coatings, and thermal cash register receipts [3e5]. Incomplete polymerization and exposure to heat, acidic or alkaline conditions can cause unreacted BPA to leach into the surrounding medium including food products [5e9]. Widespread commercial use and the potential to leach from finished products including food-packaging results in environmental contamination and human exposure. BPA exposure has been linked with multiple adverse health outcomes including but not limited to: cardiovascular disease [10], cardiometabolic impairment [11,12], obesity in children [13e21] and adults [10,18,22e27], neurobehavioral effects in children [28e31], and reproductive dysfunction [32e34]. Although BPA has been linked with numerous adverse health outcomes, the mechanism(s) underlying these associations remains ill defined. BPA has

https://doi.org/10.1016/j.emcon.2019.08.002 2405-6650/Copyright © 2019, KeAi Communications Co., Ltd. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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been shown to induce estrogenic effects in vivo using mouse and rat uterotrophic assays [35e37]. Although BPA is a more effective competitive agonist of estrogen receptor b (now called Esr2) than estrogen receptor-a (now referred to as Esr1), BPA activates both Esr1 and Esr2 with equal potency [38]. BPA concentrations reported in the contemporary literature are thought to be too low to bind with and activate estrogen receptors and cause adverse estrogenic effects in humans [39]. However, estrogenic effects can be induced thought BPA binding with and activation of alternative estrogen associated receptors including estrogen related receptor g (ERRg) and G protein-coupled receptor (GPR30) [40e43]. Recent data has emerged suggesting that BPA-G is not simply an inactive metabolite as previously thought but is able to induce adipogenesis at nM concentrations through non-estrogenic mechanisms [44]. Hence, measurement of BPA-G and total BPA are important for assessment of developmental exposure to BPA. Human exposure to BPA occurs primarily through ingestion of contaminated food and water [45] with minor contributions from dermal absorption and inhalation. BPA exposure is widespread having been quantified in human urine, blood, follicular fluid, amniotic fluid, placenta, meconium, and breast milk [32,46e59]. Since BPA does not bioaccumulate in human tissues but is quickly metabolized to what are thought to be biologically inactive metabolites [60], urine measures of BPA have been thought to be the most relevant measure of exposure total BPA and its metabolites have been reported for urine [61e64] and total BPA has been quantified in the blood of expectant mothers [49,65e68]. Total BPA concentrations in maternal and fetal serum were positively correlated and ranged between non-detectable to 4.46 and 4.60 ng/mL, respectively [65]. Placental perfusion studies have shown that BPA and its main metabolite BPA mono-glucuronide (BPA-G) can passively and bi-directionally cross the placental membranes suggesting that fetal exposure is equivalent to maternal serum concentrations [69]. However, the BPA levels measured in umbilical cord blood (UCB) collected at term were significantly higher than maternal plasma at 37 weeks of gestation [68], suggesting a potential increase in BPA exposure around the time of delivery, most likely the consequence of BPA leaching from medical devices (e.g. intravenous bags, lines and catheters). BPA conjugation and deconjugation reactions can produce sustained low free BPA exposure in fetal sheep [70] suggesting that measurement of free and BPA-G in maternal serum may also be important. Measurement of free and total BPA in fetal liver and placenta of archived human tissue samples [71] highlight concerns for developmental exposure. Pregnancy and foetal development are recognized as a critical period for exposure to hormonally active chemicals [72]. However, published reports of the concentrations of free BPA in maternal serum [54], urine [63], placenta [71], and UCB [73] are sparse. Therefore, the objective of this study was to quantify total BPA, free BPA, and BPA-G concentrations in third trimester and term blood and urine samples of expectant mothers as well as in UCB.

disorders, and pregnancy induced hypertension) that would preclude participation in this study. Participants who planned to participate in Stem Cell banking and those with prior participation in ongoing or competing studies were also excluded. All procedures were carried out in accordance with the study protocol approved by the McMaster University, Hamilton Integrated Research Ethics Board (HiREB# 09-172) and Health Canada Research Ethics Board (REB 2008-0049).

2. Materials and methods

2.4.1. Deconjugation of BPA mono-glucuronide Each 1 mL serum sample was fortified with 40 mL of a 500 ng/mL bisphenol A-d6 b-D-glucuronide spiking mix and vortexed. Following the mixing, 50 mL of b-glucuronidase from Escherichia coli source was added and the samples were incubated for 45 min at 37  C. For urine, each 1.5 mL sample and 30 mL of a 500-ng/mL bisphenol A-d6-b-D-glucuronide were used [74].

2.1. Study participants Pregnant women (n ¼ 199) were recruited through local midwifery clinics and obstetrician offices in Hamilton, Ontario over a two-year period from 2010 to 2012. Interested candidates were contacted by the research nurse and screened for eligibility. Participants were eligible provided that they were between 18 and 45 years of age, were in the third trimester of their pregnancy (28 weeks to birth) and able to understand and provide informed written consent to participate in this study. Exclusion criteria included known health problems (e.g. gestational diabetes, seizure

2.2. Specimen and infant data collection Study participants completed an obstetrical history questionnaire. Maternal pre-pregnancy body mass index (BMI), parity, and gestational age were recorded. In addition, a demographics questionnaire was used to collect data regarding study participant age, level of education, and household income. Participants provided two 10 mL tubes of blood collected during their third trimester clinic visit and again at delivery. In addition, a 50 mL spot mid-stream urine sample was collected during the third trimester clinic visit and then again immediately prior to delivery using a clean plastic urine collection hat. Urine was decanted into a sterile sample container and stored at 4  C for transport to the laboratory. Two 10 mL tubes of UCB were collected from the umbilical vein following delivery of the placenta at the time of delivery. Sample collection apparatus (blood and urine) and storage containers were tested for potential BPA contamination and shown to be BPA free. Briefly, sterile water, collected using the same collection apparatus for blood collection, was used as a field blank in this study. Specimens were stored and shipped frozen to the analytical laboratory (Health Canada, Ottawa, ON) for BPA analysis. 2.3. Serum cotinine assay For the purposes of the study, cigarette smoking in participating pregnant women was evaluated by both a self-report questionnaire and measurement of serum cotinine concentration in the third trimester serum samples using a commercial ELISA kit (Abnova, Walnut, CA, USA). The assay procedures and quality control were carried out according to the manufacturer's instructions. Absorbance was read at 450 nm on an ELISA plate reader (Biotek Synergy; Fisher Scientific, Ottawa, ON, Canada). Women with serum cotinine levels of 3 ng/mL were classified as smokers. 2.4. Chemical analysis We previously developed a novel analytical method for the quantification of free BPA in human serum [74] and urine [75]. A detailed description of the analytical methods employed have been reported elsewhere [74,75] and are summarized in the supplemental information included with this manuscript. Therefore, only a brief description is provided in the following sections.

2.4.2. Serum samples - extraction and sample cleanup Following incubation, each volume of 1 mL serum sample was fortified with 20 mL of 500 ng/mL 13C-BPA spiking mix. A volume of 1 mL of saturated aqueous ammonium sulphate, 2 mL of ethanol and 4 mL of hexane were added, and the samples were gently

W.G. Foster et al. / Emerging Contaminants 5 (2019) 279e287

inverted to mix and left to sit to allow for clear phase separation. The top organic layer was discarded, and an additional 4 mL of hexane was added to the bottom aqueous phase. Afterward, 3 mL of dichloromethane was added to the remaining phase and then samples were mixed and sonicated for 10 min and the bottom dichloromethane (DCM) phase was transferred into a 15 mL glass centrifuge tube. Extracts were evaporated to dryness under a gentle stream of nitrogen and the samples were reconstituted in 2 mL of HPLC-grade water. Sample clean up steps consisted of two solid phase extractions. In the first step 1g Envi-Florisil glass cartridges (Supelco, Bellefonte, PA, USA) were preconditioned using 5 mL DCM and 5 mL of hexane. Next, samples were loaded on to SPE cartridges and were percolated through and finally dried for 15 min. Cartridges were washed with 10 mL hexane and this fraction was discarded. Then, BPA was eluted with 25 mL DCM into a glass vial. Sample extracts were then evaporated to dryness and then reconstituted in 3 mL of HPLC-grade water. In the second step of the solid phase extraction clean up, 200 mg Oasis HLB glass cartridges (Waters, CA, USA) were first preconditioned using 9 mL of acetone, 9 mL of methanol and 5 mL of HPLC-grade water; then, the extracts were loaded and allowed to percolate through. The SPE cartridges were then dried for 30 min before BPA was eluted with 6 mL of methanol and 5 mL of acetone. The extracts were then concentrated to dryness and reconstituted with 150 mL of acetone and 50 mL of MSTFA was added to each sample for derivatization prior to GC-MS/MS analysis. 2.4.3. Urine samples - extraction and sample cleanup Following incubation, each 1.5 mL incubated urine sample as well as its corresponding nonincubated duplicate were fortified with 15 mL of 500 ng/mL 13C12-BPA spiking solution. Next, 6 mL of dichloromethane (DCM) were added to the sample. Samples were then mixed and sonicated. The bottom organic phase was collected and an additional 6 mL DCM were added to each sample, sonicated and an additional organic phase was collected. The combined DCM fraction was transferred into a 15 mL glass centrifuge tube. Sample extracts were evaporated to dryness under a gentle stream of nitrogen and then reconstituted in 2 mL of HPLC-grade water. For sample cleanup, extracts were loaded onto preconditioned (9 mL of acetone, 9 mL of methanol and 5 mL of HPCL-grade water) 200 mg Waters Oasis HLB glass cartridges (Milford, MA, USA) and then samples were loaded and allowed to percolate through. The SPE cartridges were then dried for 30 min and subsequently BPA was eluted using 6 mL of methanol and 5 mL of acetone. The extracts were concentrated to dryness and reconstituted with 100 mL of acetone and derivatized with 50 mL of MSTFA prior to GC-MS/MS analysis. 2.4.4. GC-MS/MS analysis The chromatographic separation was performed on a Zebron ZB-5HT capillary column (30 m  0.25 mm x 0.1 mm) from Phenomenex (CA, USA), using an Agilent 6980 gas chromatogram equipped with an Agilent 7683B Series Autosampler. Mass spectrometric experiments were performed using a Waters Quattro micro GC triple quadrupole mass spectrometer (Waters Corp., Milford, MA, USA). The following MRM transitions (m/z) were monitored: BPA (m/z 357 / 191), 13C12-BPA (m/z 369 / 197) and BPA-d6 (m/z 360 / 73). 2.5. Statistical analyses Results from field blanks revealed absence of BPA and thus correction for sample contamination from the collection apparatus was not required. Samples with results below the MDL were recorded as the MDL/√2. Data were tested for normality using the

281

Kolmogorov-Smirnov test. BPA concentrations were not normally distributed and therefore are expressed as the geometric mean together with the median, minimum, and maximum. Means were compared across groups using t-test and proportions were compared with Karl Pearson's chi-square test. Fisher's exact test of independence was run to compare proportions when the sample size was small. Correlations between circulating concentrations of BPA at different sampling time points were assessed using Pearson correlation. Potential relationships between circulating concentrations of BPA and maternal age, pre-pregnancy BMI, parity, and gestational age were analyzed using forward stepwise regression and Pearson correlation. All statistical analyses were performed using SigmaStat software (Sigma-Aldrich, Mississauga, ON). A p value of 0.05 was considered statistically significant. 3. Results 3.1. Participants characteristics The mean (±SD) age of the study participants was 32.0 ± 3.98 years with a pre-pregnancy BMI of 25.4 ± 6.13 (Table 1). The study participants were predominantly Caucasian and of the participants, 41.5% had completed university level education with a further 24.6% reporting postgraduate training and 57.2% of our sample reported annual incomes of greater than 80,000 dollars (CND). Consistent with questionnaire responses, five of the study participants were current smokers (2.5%) with serum cotinine levels of 3 ng/mL. 3.2. Serum BPA concentration Of the 199 women who agreed to participate in this study, a total of 189 serum samples collected during the first clinic visit were free of hemolysis and had adequate sample volume available for analysis. Free BPA and BPA-G was quantifiable at concentrations above the MDL (0.026 ng/mL) in 33.9% and 43.4% of samples, respectively (Table 2). In samples collected at the time of delivery, free BPA was quantifiable above the MDL in 17 of 83 samples (20.5%) whereas BPA-G and total BPA were measurable in 58 (69.9%) samples (Table 2). Since free BPA and BPA-G were quantified above the MDL in 33.9 and 43.4% of cases in samples collected during the first clinic visit, we explored potential differences in participant characteristics. Specifically, we compared maternal pre-pregnancy weight and BMI, age, education level, income, parity, and infant birth weight between women with detectable levels of free BPA in 3rd trimester serum vs. those in women with concentrations below the MDL (Table 1). No significant differences (p  0.05) between the groups were found. BPA concentrations were not normally distributed at any time point analyzed and all tended to cluster around the MDL (Table 2). The GM serum concentration of free-BPA during the third trimester was 0.62 ng/mL with a maximum level of 3.94 ng/mL, values that were decreased at delivery (
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Table 1 Study participant characteristics at and by whether free BPA was detected in maternal 3rd trimester serum samples. All (n ¼ 189)

Free BPA Detected (n ¼ 64)

Free BPA Undetected (n ¼ 125)

Mean ± SD

Mean ± SD

Mean ± SD

Age (yr) Pre-pregnancy wt. (lbs) Pre-pregnancy BMI Gestation (wks)

32.0 ± 3.98 154.5 ± 36.1 25.4 ± 6.13 39.4 ± 1.25

31.8 ± 3.33 152.3 ± 30.2 25.5 ± 5.28 39.5 ± 1.27

32.0 ± 4.20 154.7 ± 37.7 25.2 ± 5.78 39.3 ± 1.21

Education

N (%)

N (%)

N (%)

10 52 76 45

2 (3.45) 13 (22.41) 24 (41.38) 19 (32.76)

9 (8.00) 38 (32.0) 49 (41.0) 22 (19.0)

N (%)

N (%)

N (%)

29 (18.2) 39 (24.2) 91 (57.2)

4 (8.70) 9 (19.6) 33 (71.7)

23 (22.3) 25 (24.3) 55 (53.4)

N (%)

N (%)

N (%)

2 (1.50) 45 (34.1) 51 (38.6) 34 (25.8)

1 (2.50) 11 (27.5) 18 (45.0) 10 (25.0)

1 (1.20) 33 (39.3) 29 (34.5) 21 (25.0)

Mean ± SD

Mean ± SD

Mean ± SD

3551.1 ± 471.5 3345.3 ± 769.5

3456.9 ± 493.7 3396.5 ± 644.6

3657.6 ± 432.3 3290.2 ± 790.5

High School College Undergraduate Postgraduate Income (CND) 49,999 50,000e79,999 80,000 Parity 0 1 2 >3 Birth weight (g) Males Females

(5.50) (28.4) (41.5) (24.6)

p value

0.860 0.693 0.605 0.319

0.456 0.242 0.885 0.058

0.077 0.674 0.054

0.825 0.279 0.354 0.825

0.119 0.438

Table 2 Geometric mean of free BPA, BPA-G, and total BPA serum(MDL ¼ 0.026 ng/mL) and urine (MDL ¼ 0.027 ng/mL) concentrations presented along with 95% CI, median, and maximum values from samples collected during the 3rd trimester and at delivery Samples with results below the MDL were recorded as the MDL divided by the square root of 2. Visit Serum First visit

Delivery

Visit

Number of samples analyzed (N)

Number (%) samples with detectable BPA

Geometric Mean of BPA concentration 95% CI (ng/ Median [Maximum] (ng/ (ng/mL) mL) mL)

Free BPA BPA-G Total BPA Free BPA BPA-G Total BPA

189

64 (33.9)

0.06

0.55, 0.69


3.94

82 (43.4) 82 (43.4)

0.09 0.12


67.6 67.6

17 (20.5)


0.00, 1.51

0.27

0.98

58 (69.9) 58 (69.9)

0.28 0.31

0.25, 0.31 0.00, 0.77

0.29 0.71

10.9 11.6

Number of samples analyzed (N)

Number (%) samples with detectable BPA

Geometric Mean of BPA concentration 95% CI (ng/ Median [Maximum] (ng/ (ng/mL) mL) mL)

188

6 (3.2)

0.07

0.66, 0.71

0.06

1.67

60 (31.9) 60 (31.9)

0.36 0.36

0.00, 1.35 0.00, 1.36

0.20 0.20

54.7 54.7

4 (5.5)

0.06

0.00, 0.13

0.06

3.46

30 (41.1) 30 (41.1)

0.09 0.09

0.00, 0.42 0.00, 0.47

0.06 0.06

14.5 18.0

Visit Urine 3rd Trimester

Delivery

Free BPA BPA-G Total BPA Free BPA BPA-G Total BPA

83

73

95% CI ¼ 95% confidence interval.

was correlated with BPA-G and total in the UCB (r2 ¼ 0.587 and p < 0.0001, and r2 ¼ 0.596 and p < 0.0001). Circulating concentrations of BPA were not influenced by maternal age, pre-pregnancy BMI, parity, gestational age or sex of the foetus. 3.3. Urinary BPA concentration A small proportion (3.2%) of urinary samples analyzed from

third trimester had free BPA concentrations above the MDL (0.027 ng/mL), compared to a relatively higher proportion of samples with detectable urinary BPA-G and total BPA concentrations (31.9%). Similar patterns were observed for urine samples analyzed from participants at the time of delivery (Table 2) with the proportion of samples with quantifiable concentrations of BPA-G and total BPA in 41.1% of the samples. The GM concentrations of the specific gravity adjusted free BPA

W.G. Foster et al. / Emerging Contaminants 5 (2019) 279e287

in the urine of study participants at their first clinic visit (third trimester) were 0.07 ng/mL (maximum ¼ 1.67 ng/mL) and similar to the concentrations measured in the urine of women at delivery (0.06 ng/mL and maximum concentration ¼ 3.46 ng/mL) (Table 2). The GMs of the specific gravity-adjusted urinary BPA-G and total BPA concentration were identical at 0.36 ng/mL in third trimester samples. In the samples collected at the time of delivery the GM specific gravity adjusted BPA-G and total BPA concentrations were equivalent (0.09 ng/mL) and the maximum concentrations measured were similar (14.5 and 18.0 ng/mL, respectively). 3.4. UCB BPA concentration A total of 112 samples of UCB blood had adequate sample volume, were free of hemolysis and suitable for analysis. Summary data for free BPA, BPA-G, and total BPA is presented in Table 3. The GM of the sample free BPA concentration at the time of delivery was 0.05 ng/mL whereas the BPA-G concentration was higher (0.08 ng/ mL). The GM concentration of total BPA in the delivery samples was 0.10 ng/mL while the concentration in one study participant was profoundly greater (58.6 ng/mL). 4. Discussion Results of the present study describe the concentration of free BPA, BPA-G, and total BPA in the serum and urine of third trimester and term pregnant women as well as UCB concentrations. Results demonstrate that, in this Canadian cohort of pregnant women, free BPA was detected, at concentrations above the MDL, in the serum during the last trimester or at delivery in only a small fraction of cases and in less than a quarter of the UCB samples. The detection rates reported in the current study are lower than those reported in similar cohorts. Furthermore, the serum concentrations of free BPA, BPA-G and total BPA measured in the current study clustered in the low ng/mL range; concentrations that were only modestly above the MDL (0.026 ng/mL). Comparisons of study participant characteristics between women with quantifiable concentrations of free BPA with those in whom free BPA could not be quantified failed to reveal significant differences. While BPA concentrations in the serum were positively correlated within and across sampling time points the concentrations of free BPA in the serum at delivery were lower than during the third trimester. BPA concentrations were not associated with maternal age, pre-pregnancy BMI, parity, and gestational age. Taken together, our data suggest that, in our study cohort, human exposure to BPA is limited resulting in both low frequency of detection and low serum concentrations. Since BPA is quickly metabolized and does not bioaccumulate in human tissues [60], urine is the preferred biological matrix for estimates of exposure. In the current study, urine BPA sample concentrations were corrected for dilution using specific gravity. Correction for dilution in the urine has been shown previously to improve temporal reproducibility of results [76]. Both urinary creatinine concentrations and specific gravity follow similar patterns in the urine throughout pregnancy and thus either can be

283

used to enhance estimates of urine contaminant concentrations [76]. In the current study, free BPA could only be quantified above the MDL (0.027 ng/mL) in 6 of 188 (3.2%) urine samples from the third trimester and 4 of 73 (5.5%) maternal urine samples at delivery whereas total BPA was quantified in 60 of 188 (31.9%) and 30 of 73 (41.1%) samples, respectively. In contrast to our results, free BPA was quantified in 43% of samples tested in a first trimester spot urine sample from another study of pregnant Canadian women [63]. Detection rates in maternal urine samples vary across studies with detection rates for total BPA in third trimester spot urine samples ranging from 80% of pregnant women in Mexico City [62] through to 100% of pregnant study participants in the Netherlands [77]. Reasons for divergent frequencies of free and total BPA detection between the current and previous studies are difficult to explain but are most likely the result of differences in study design and study population characteristics. The MDL used in the current study (0.027 ng/mL) was similar to that of other investigators although lower than that of another group [77] and thus cannot account for the observed lower frequency of detection in our study. In the present study, participants were recruited in midwifery and obstetrician offices with a large number of the participants opting for home deliveries. Consequently, fewer participants in the present study received i.v. fluids at the time of delivery compared to similar studies in which women delivered exclusively in hospital settings. It was postulated that an increase in BPA levels observed at delivery can be attributed to medical devices used in the delivery process such as catheters and tubing as they have been demonstrated to be a source of BPA exposure [78,79]. The present study population had lower BPA exposure compared to populations delivering in hospital settings and typically reported in the literature. It was further noted that higher BPA exposures have been associated with lower socioeconomic status [80]; however, the opposite relationship was observed in an European pooled study of mother-child cohorts [81]. In the present study, study participants were predominantly Caucasian with high socioeconomic status. Thus, taken together, study participant characteristics and home delivery could account for both lower rates of detection and lower urinary BPA concentrations compared to the concentrations generally reported in the published literature. Although urine is the most appropriate matrix for demonstrating human exposure to BPA, urine concentrations cannot provide insight into the concentration of free BPA in the circulation and available for distribution to target tissues. Methods for quantification of free BPA and conjugated BPA in serum have been developed and previously shown to yield reliable results from frozen samples [74,82,83]. Therefore, we quantified circulating concentrations of free BPA, the biologically active form of the chemical, which we suggest is the most biologically relevant to measure in the context of potential relationships with adverse health effects. Although serum BPA measures may provide the more direct evidence of BPA exposure to target tissues, at a given point in time, than can be achieved with urine samples, care must be taken to control for potential sample contamination from collection and processing devices and thus may be less feasible

Table 3 Geometric mean of free, BPA-G, and total BPA concentration (MDL ¼ 0.026 ng/mL), presented along with 95% CI, median, and maximum values as measured in the umbilical cord blood (UCB) at delivery. Samples with results below the MDL were recorded as the MDL divided by the square root of 2. UCB

Number of samples analyzed (N)

Free BPA 112 BPA-G Total BPA

Number (%) samples with detectable BPA

Geometric Mean of BPA concentration (ng/ 95% CI (ng/ mL) mL)

Median [Maximum] (ng/ mL)

25 (22.3) 26 (23.2) 26 (23.2)

0.05 0.08 0.10


0.00, 0.45 0.00, 0.16 0.00, 1.38

2.94 58.6 58.6

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than urine measures. The concentration of total BPA in maternal serum or plasma has been documented in several prior studies [49,65,66,68] (Table 4); however, circulating concentrations of freeBPA are less well defined. The frequency of serum samples with BPA

concentrations above the MDL in the present study were low with 33.9% for free BPA, and 43.4% for BPA-G and total BPA in the third trimester. However, in serum samples from delivery free BPA was detected in only 20.5% of samples vs. BPA-G and total BPA which

Table 4 Circulating and urine concentrations of free, BPA-G, and total BPA of pregnant women. Unless indicated otherwise the data cited are for total BPA. N

Sample

Samples > MDL

Mean (range) Concentration (ng/mL)

Citation

189

Serum e maternal blood collected during 3rd trimester And at delivery

61 27

Serum e maternal blood collected at delivery Plasma e collected at 37 weeks gestation

0.62 (
This study

Canada Czech Republic Canada

Free BPA 33.9%) BPA-G (9.5%) Total BPA: 43.4% Free BPA 20.5%) BPA-G (49.4%) Total BPA (69.9%) 59/61; 97% 85.2%

58

Serum e maternal blood collected before delivery

58/58; 100%

United States

80

Plasma - maternal blood collected during 1st trimester NR And at delivery

30 189

Case: 2.80 (0.21e9.00)# Control: 3.00 (0.04e24.21)# Free BPA 4.8 (
Location Serum Canada

France

191

Serum e maternal blood collected before delivery 67% Maternal spot urine collected during 3rd trimester and Free BPA (3.2%) at delivery BPA-G (28.7%) Total BPA (31.9%) Free BPA: 5.5% BPA-G: 35.6% Total BPA (41.1%) Urine; 6-30 weeks gestation 98.5%

Mexico Netherlands United States Canada

60 10 181

Spot urine; Third trimester Spot urine sample in early pregnancy Spot urine gestation weeks 16 & 26

48/60; 80% NR NR

1,890

First trimester spot urine

Free BPA (43%) BPA-Glu (95%)

Canada Canada United States Denmark Denmark Korea United States United States Spain

80 2,000 506

Urine at multiple time points Spot urine; First trimester Spot urine; Third trimester

92% 88% 89%

565 200 757 1st visit: 407 2nd visit: 459 72

Spot urine Spot urine; 8-30 wks gestation Urine; Third trimester Spot urine; 1st and 2nd prenatal visit Urine

90% 179 (89.5%) e 79% of the samples at each visit NR

479

Spot urine; First and third trimester

100% e 1st Trimester

China Urine Canada

99.4% e 3rd Trimester China

452 (113 cases; 339 controls)

Puerto Rico 105 United 476 States United 112 States

China Mexico Unites States China

137 250 241 506

Netherlands 80

Urine collected at delivery

89.4%

Three spot urine samples Spot urine samples at first and second half of pregnancy Spot urine samples collected in the 3rd trimester

98% >82%

Free BPA: 0.01 (95% CI: 0.01, 0.01)* BPA-Glu: 0.91 (95% CI: 0.86, 0.95)* 1.2 (1.1e1.3)¶ 0.80 (0.76e0.85)* 1.4 (95% CI: 1.2, 1.5) 1.17 (95% CI: 1.05, 1.30) (
[65] [68] [49] [87]

[66] This study

[61] [62] [89] [90] [63]

[55] [85] [91] [92] [93] [86] [94] [95] [96]

[97]

[98] [99]

Free BPA 88% BPA-G 99% BPA-S 75% Total BPA 100% Spot urine samples collected in the 3rd trimester Total BPA 78.8% Spot urine samples collected during the 3rd trimester NR Spot urine samples collected during the 3rd trimester 97.5%

0.21 (
[80]

Spot urine sample collected upon hospital admission 86.6% for delivery Spot urine samples at 18, 18-25 and > 25 weeks of 80/80 (100%) pregnancy

1.33 (
[103]

1.6 (0.2e47.5)

[77]

*Creatinine-adjusted BPA Concentration in mg/g Cr; ¶specific gravity adjusted; NR ¼ not reported;

#

Median (range); ^Median (IQR); and $geometric mean.

[100] [101] [102]

W.G. Foster et al. / Emerging Contaminants 5 (2019) 279e287

were detected at concentrations above the MDL in 69.9% of samples. Concentrations of free BPA and BPA-G in the umbilical cord blood were only measured in 25 of 112 (22.3%) and 26 of 112 (23.2%) samples, respectively. The decrease in the frequency of detection and BPA concentrations in delivery samples vs. the third trimester may reflect differences in maternal habits (decreased activity and decline in food consumption) around the time of delivery. The detection frequency for total BPA was similar to that reported for pregnant women (67%) in China [66] but lower than reported in other Canadian cohorts [49,65]. Similarly, our detection frequencies for total BPA were also lower than those reported in umbilical cord blood in prior studies [49,65,66,73,84] (Table 5). The circulating concentrations of total BPA during the third trimester and at delivery, respectively were lower than the concentrations reported by others [65] for pregnant women in Canada (Table 4). Similarly, the GM concentrations of free BPA and BPA-G, although the latter was only reported in one other study [63], measured in the urine of expectant mothers in the third trimester and at delivery of the present study were similar to the values reported in another Canadian study [63] but lower than the concentrations reported by others [62,77,85,86]. Placental perfusion studies have shown that BPA and its main metabolite BPA-G can passively and bi-directionally cross the placental membranes suggesting that fetal exposure is equivalent to maternal serum concentrations [69]. However, recent data from a sheep study [70] suggests that BPA conjugation/deconjugation reactions can produce sustained low free BPA exposure in the fetus. Regardless, measurement of BPA concentrations in maternal and fetal serum ranged between non-detectable to 4.46 and 4.60 ng/mL, respectively [65] suggesting that conjugation/deconjugation may be less important in humans or exposures are too low to result in substantial fetal exposure. This proposal is supported by a prior study in which total BPA was only detected in 2 of 71 amniotic fluid samples studied [76] suggesting that fetal exposure is not common. Regardless, although sample contamination cannot be excluded in this prior report [76], measurement of free and total BPA in fetal liver and placenta of archived tissue samples [71] highlight

285

concerns for developmental exposure and potential adverse health effects of BPA. While we did not quantify BPA in the placenta in the current study, we did quantify free and total BPA in the umbilical cord blood. The GM concentrations of free and total BPA measured in the present study were lower than the concentrations reported previously [65,73,84]. Divergent results may reflect differences in study population and methodological procedures. Prior studies have shown that BPA exposure is associated with lower socioeconomic status [80,87] and affected by race and parity [87]. Participants in the present study were primarily Caucasian and from high socio-economic groups. Racial disparities in blood BPA concentrations have been documented previously with higher BPA concentrations found in the serum of African Americans vs. Caucasians [88] whereas higher free BPA levels were found in the serum of women with multiracial backgrounds or reported belonging to Asian, native Hawaiian, Pacific Islander or Hispanic background [87]. We cannot exclude that women in this study were potentially more attuned to possible associations between exposure to environmental chemicals thought to affect pregnancy and thus avoided products containing BPA. Finally, while sources of BPA were not explored in the present study, a prior report found that pregnant women with frequent thermal cash register receipt contact had higher BPA exposures whilst consumption of foods thought to contain BPA was not associated with exposure [80]. The present study has many strengths including quantification of serum and urinary free BPA and total BPA, measures that provide a more complete picture of both exposure and the internal dose available for distribution to target tissues. The prospective nature of the study allowed for sample collection, control measures to minimize potential sources of contamination, and assessment of relationship with pregnancy outcomes. In addition to these strengths, our study also has several limitations as well including the relatively small sample size. Although reliable methods for the quantification of free and conjugated BPA in serum and urine samples have been described [74,75,82,83], the stability of BPA in frozen samples over time remains unknown. The use of spot samples also precludes ability to determine exposure to BPA over the

Table 5 Concentration of free, BPA-G, and total BPA in the placenta and umbilical cord blood. Unless indicated otherwise the data cited are for total BPA. Location

N

Placenta United States 12

UCB

Samples > MDL

Mean BPA Concentration (range) in ng/mL

Citation

Placenta - elective 2nd trimester pregnancy terminations

Free BPA: 10/12 Conjugated BPA: 5/12 200/200 (100%) 58/58 (100%)

Free BPA: 0.54# (<0.05e25.4) Conjugated BPA: 0.07# (<0.05e9.1) 103.4 ± 61.8 (4.4e273.9)* Case: 9.40# (0.40e101) Control: 3.00# (0.30e36.1) Free BPA: 12.6 (0.55e165)* BPA-G: 17.2 (0.10e178)* Total BPA: 30.2 (1.43e280)* Free BPA: 0.05 (
[104]

[66] [65] [84]

Case: 1.26 ± 1.13 (0.14e4.75) 0.132 (0.049e0.304)^

[68]

United States 200 Canada 58

Placenta at delivery Placenta at delivery

Canada

128

Early to mid-gestation placenta

Canada

112

UCB at delivery

China Canada France

30 61 Control: 106 Case: 46 27

UCB at delivery Fetal cord blood UCB

Czech Republic United States 85

#

Sample

Term UCB

27% 58/61 (95%) Control: 106/106 (100%) Case: 46/46 (100%) 75.1%

United States 80

UCB at delivery

BPA: 47% BPA-G: 76% BPA-Sul: 96% NR

Canada

Fetal cord blood

58/58 (100%)

58

Umbilical Cord Serum- elective 2nd trimester pregnancy terminations

Free BPA: 113/128 Total BPA: 51/54 BPA-G: 50 samples 22.3%

Median in g/g; * BPA concentration in ng/g; $geometric mean and range; ^Median (IQR).

BPA 0.16 (
[48] [49] [71]

This stud

[73]

[87] [49]

286

W.G. Foster et al. / Emerging Contaminants 5 (2019) 279e287

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