Variability of Urinary Phthalate Metabolite and Bisphenol A Concentrations before and during Pregnancy

Background: Gestational phthalate and bisphenol A (BPA) exposure may increase the risk of adverse maternal/child health outcomes, but there are few data on the variability of urinary biomarkers before and during pregnancy. Objective: We characterized the variability of urinary phthalate metabolite and BPA concentrations before and during pregnancy and the ability of a single spot urine sample to classify average gestational exposure. Methods: We collected 1,001 urine samples before and during pregnancy from 137 women who were partners in couples attending a Boston fertility clinic and who had a live birth. Women provided spot urine samples before (n ≥ 2) and during (n ≥ 2) pregnancy. We measured urinary concentrations of monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-iso-butyl phthalate, monobenzyl phthalate (MBzP), four metabolites of di-(2-ethylhexyl) phthalate (DEHP), and BPA. After adjusting for specific gravity, we characterized biomarker variability using intraclass correlation coefficients (ICCs) and conducted several surrogate category analyses to determine whether a single spot urine sample could adequately classify average gestational exposure. Results: Absolute concentrations of phthalate metabolites and BPA were similar before and during pregnancy. Variability was higher during pregnancy than before pregnancy for BPA and MBzP, but similar during and before pregnancy for MBP, MEP, and ΣDEHP. During pregnancy, MEP (ICC = 0.50) and MBP (ICC = 0.45) were less variable than BPA (ICC = 0.12), MBzP (ICC = 0.25), and ΣDEHP metabolites (ICC = 0.08). Surrogate analyses suggested that a single spot urine sample may reasonably classify MEP and MBP concentrations during pregnancy, but more than one sample may be necessary for MBzP, DEHP, and BPA. Conclusions: Urinary phthalate metabolites and BPA concentrations were variable before and during pregnancy, but the magnitude of variability was biomarker specific. A single spot urine sample adequately classified MBP and MEP concentrations during pregnancy. The present results may be related to unique features of the women studied, and replication in other pregnancy cohorts is recommended.

Phthalates and bisphenol A (BPA) are multi functional compounds used in a variety of commercial and industrial products. Diethyl phthalate (DEP), dinbutyl phthalate (DBP), and benzylbutyl phthalate (BzBP) can be used in personal care and consumer products to hold scent and color [National Research Council (NRC) 2008]. DBP, BzBP, and di(2ethyl hexyl) phthalate (DEHP) can also be used in the manufacture of floor ings, carpet backings, adhesives, wallpaper, and polyvinyl chloride (PVC) plastics (NRC 2008). BPA can be used in food can linings, water supply pipes, medical tubing, thermal receipts, and cigarette filters (Biedermann et al. 2010;Chapin et al. 2008;Jackson and Darnell 1985). Exposure to BPA and phtha lates is nearly ubiquitous among persons in the United States Koch and Calafat 2009;Woodruff et al. 2011).
Animal studies demonstrate that gesta tional phthalate and BPA exposures are asso ciated with adverse health outcomes (Chapin et al. 2008;NRC 2008). Epidemiological studies suggest that exposure to some phtha lates and BPA may be associated with adverse neurodevelopmental outcomes (Braun et al. 2009;Miodovnik et al. 2011;Swan et al. 2010). However, the potentially epi sodic nature of exposure, combined with the short biological halflives of these compounds raises questions about the adequacy of a sin gle spot urine sample to classify gestational BPA and phthalate exposure. Variations in the patterns of food consumption may con tribute to the withinperson variability of phthalate and BPA exposure, while patterns of personal care product use and movement between environments with variable air and dust concentrations may create additional withinperson variability of phthalate expo sure (Adibi et al. 2008;Preau et al. 2010;Ye et al. 2011). In addition, pregnancy related changes in xenobiotic metabolism may contribute to the variability of urinary BPA and phthalate metabolite concentrations throughout gestation. As far as we are aware, no prior studies have examined the variability of urinary BPA and phthalate metabolite con centrations in the same woman before and during pregnancy.
A better understanding of the variability of urinary phthalate and BPA biomarkers can help investigators determine the adequacy of these markers to classify phthalate and BPA exposure during critical periods of develop ment such as pregnancy. The purpose of this study was to characterize the pattern, variabil ity, and reproducibility of DEHP metabo lites, mononbutyl (MBP), monoisobutyl phthalate (MiBP), monobenzyl phthalate (MBzP), monoethyl phthalate (MEP), and BPA concentrations in serial urine samples from 137 women before and during preg nancy. In addition, we determined whether a single spot urine sample during the first, second, or third trimester could accurately classify average gestational phthalate and BPA exposure. This information will help inform exposure assessment in epidemiological stud ies and aid in the evaluation of human studies that use a single spot urine sample to classify these exposures.

Methods
Women 18-45 years of age were recruited from partners seeking evaluation and treat ment for infertility at the Massachusetts General Hospital (MGH) Fertility Center in Boston between November 2004 and December 2009. The present analyses were from a larger prospective opencohort study, volume 120 | number 5 | May 2012 • Environmental Health Perspectives the Environment and Reproductive Health (EARTH) Study, which was designed to examine the relationship between environ mental chemical exposures and fertility/preg nancy outcomes. The study was approved by the human studies institutional review boards of the MGH, Harvard School of Public Health and the Centers for Disease Control and Prevention (CDC). Subjects signed an informed consent form after the study proce dures were explained by a research nurse and all questions were answered.
Women included in this analysis were recruited pre conception (herein referred to as pre or before pregnancy) and followed until delivery. Conception methods included natural conception, ovulation induction with timed intercourse, intrauterine insemination, or in vitro fertilization. Women provided spot urine samples in polypropylene containers at enrollment, on returning for subsequent clinic appointments before pregnancy, and during pregnancy (first, second, or third trimester). Enrollment urine samples were generally collected on entry into the study and before fertility treatment. Before stor ing samples at -80°C, urine was aliquoted and specific gravity (SG) was measured using a handheld refractometer calibrated with deionized water before each use (National Instrument Company Inc, Baltimore, MD). Samples were shipped on dry ice to the CDC for analysis.
An intrauterine pregnancy was confirmed by presence of a fetal heartbeat detected by transvaginal ultrasound. We used one of three methods to estimate the date of conception: oocyte retrieval date, which was abstracted from medical records; crown-rump length, which was measured during a fetal ultrasound between 6 and 8 weeks of gestation; or wom an's report of last menstrual period. When more than one dating method was available, priority was given to retrieval date > ultra sound > last menstrual period.
To examine variability of urinary phtha late metabolite concentration before and dur ing pregnancy, we restricted our analyses to women who delivered a liveborn infant and provided two or more pregnancy urine sam ples and two or more urine samples before that pregnancy. We excluded women with fewer than two urine samples during either or both time periods.
We measured the concentration of BPA and eight phthalate metabolites including MBP, MiBP, MBzP, MEP, and the following four DEHP metabolites: mono(2ethyl5 carboxypentyl) phthalate (MECPP), mono(2 ethyl5hydroxyhexyl) phthalate (MEHHP), mono(2ethyl5oxohexyl) phthalate (MEOHP), and mono(2ethylhexyl) phtha late (MEHP), using previously described ana lytical chemistry methods and quality control procedures. (Silva et al. 2007;Ye et al. 2008) We limited our statistical analyses to DEHP metabolites, MBP, MiBP, MBzP, and MEP because of their high frequency of detection in the U.S. population Woodruff et al. 2011) The limits of detection (LOD) for the target phthalate metabolites were in the low microgram per liter range (~ 0.1 to ~ 1 μg/L) and 0.4 μg/L for BPA. Values less than the LOD were given a value of the LOD/√ -2 (Hornung and Reed 1990). We applied correction factors of 0.66 and 0.72 to the MEP and MBzP concentrations, respectively, because the analytic standards used were of inadequate purity (Calafat A, personal communication).
Because DEHP is metabolized primar ily into MEHP, MEHHP, MECPP, and MEOHP, we used two summary measure ments: a) total molar sum of all four DEHP metabolites and b) molar sum of three oxida tive DEHP metabolites (MECPP, MEHHP, and MEOHP). We calculated the molar sum of DEHP metabolites by dividing each metabolite concentration by its molar mass and then summing the individual metabolite concentrations. We also present results sepa rately for MEHP concentrations to facilitate comparisons with prior studies.
We accounted for urine dilution by stan dardizing urinary phthalate metabolite and BPA concentrations using SG. Urine dilution was adjusted using a modified and previously described formula in all of our analyses of BPA and phthalate metabolites (Duty et al. 2005;Meeker et al. 2009). We excluded samples with SG values > 1.04 (Boeniger et al. 1993). All statistical analyses were conducted using SGstandardized biomarker concentrations unless otherwise noted.
Statistical analyses. Descriptive analyses. We first examined the sociodemographic characteristics of participating women (means and proportions). We computed the median and 25th and 75th percentiles of SGadjusted phthalate metabolites and BPA concentrations from the first (at enrollment) and last urine samples provided before women became preg nant and from samples provided during each trimester of pregnancy. We calculated univari ate characteristics of and correlation between the withinwoman geometric mean (GM) uri nary phthalate metabolite and BPA concentra tions for all pre pregnancy and pregnancy urine samples. We also compared the difference in phthalate metabolite and BPA concentrations before and during pregnancy using a linear mixed model with log 10 transformed phtha late metabolite or BPA concentrations as the outcome. We included an indicator variable to designate samples as being before or during pregnancy. We estimated the percent differ ence in pregnancy concentrations relative to prepregnancy concentrations.
Variability analyses. We conducted three analyses to characterize the variability and change in urinary phthalate metabolite and BPA concentrations before and during preg nancy. These analyses used log 10 transformed urinary SGadjusted phthalate metabolite and BPA concentrations because of their right skewed distribution. First, we estimated the variability of urinary phthalate metabolite and BPA concentrations before or during preg nancy by calculating the intraclass correlation coefficient (ICC) using a random intercept only linear mixed model. The ICC is a mea sure of reproducibility, calculated by dividing the betweensubject variability by the sum of the between and withinsubject variability. Values range from 0, indicating no reproduc ibility, to 1, indicating perfect reproducibility (Rosner 2000). Next we estimated the percent change in urinary phthalate metabolite and BPA concentrations over time during the pre pregnancy or pregnancy sampling frame using linear mixed models with subject specific inter cepts. For pre pregnancy samples we calculated the number of weeks since enrollment by sub tracting the date of enrollment from each sub sequent collection date. The enrollment urine sample was set to a time of 0. For pregnancy samples, we calculated the number of weeks since conception for each urine sample by subtracting the date of conception from each urine sample collection date. We estimated the percent change in phthalate metabolite and BPA concentration with each 4week change in time before and during pregnancy. Finally, using spaghetti plots, we graphed a random sample of urinary phthalate metabolite and BPA concentrations in 50 women before and during pregnancy as a function of time since enrollment or conception.
We evaluated the pattern and variabil ity of urine dilution before and during preg nancy by conducting the above analyses using untransformed SG values as the outcome, because changes in urine dilution (i.e., SG) during pregnancy may partially account for changes in urinary phthalate concentrations.
Surrogate category analyses. We con ducted three additional analyses to examine the rankordering, predictive ability, and con sistency of a single urinary phthalate metabo lite and BPA concentration during pregnancy among women with all three urine samples (i.e., one sample for each trimester). First, we conducted a classification analysis (Hauser et al. 2004;Mahalingaiah et al. 2008). Using the GM of the three individual trimester phthalate metabolite or BPA concentrations, we calculated tertiles of average gestational exposure of the women. We then classified women into surrogate tertiles of phthalate metabolite/BPA concentrations using their trimesterspecific urine sample concentration. We categorized the women as being either in the top or bottom two tertiles of either of these measures. We then calculated the sensitivity, specificity, and positive predictive value (PPV) of the top tertile of the surrogate measure with the top tertile of the average gestational measure. The PPV is the probabil ity of being classified as having high average gestational concentrations given a high surro gate measure. We examined trimester specific surrogate categories to determine if the timing of sample collection influenced the predictive ability of a single spot urine sample.
Second, we examined whether surrogate categories of trimesterspecific urine samples were associated with average gestational urinary phthalate metabolite or BPA concentrations of women (Meeker et al. 2005;Teitelbaum et al. 2008). Similar to the first analysis, we calculated surrogate tertiles of trimester specific urinary phthalate metabolite/BPA concentra tions (i.e., surrogate categories of low, medium, and high). We then used box plots to exam ine the distribution of the average gestational exposure of women (e.g., GM of all three uri nary biomarker concentrations) within each of these surrogate categories. For example, we calculated the average gestational urinary BPA concentration of each woman using the values from all three trimesters. Based on the first trimester urinary BPA concentrations for all the women, we then categorized each woman's firsttrimester urinary BPA concentrations into surrogate tertiles. We then plotted the distribu tion of the average gestational urinary BPA concentration of women for the firsttrimester surrogate tertile variable. We then completed the same process for the second and third tri mesters. If the surrogate tertile variable pro vides reasonable rank ordering, then we would expect to see increasing average gestational BPA concentrations across the surrogate trimester categories (i.e., increasing average gestational BPA concentration as one goes from first to second to thirdtrimester surrogate categories).
Finally, we examined whether the women remained in the same tertile of exposure over the course of their pregnancy by counting the number of times (one, two, or three) her urine sample concentrations were in the same tertile for each phthalate metabolite or BPA. For instance, if all three urine samples for a woman were in the same tertile, she was assigned a count of three. We calculated sepa rate tertiles for each trimester. All analyses were conducted with SAS version 9.2 (SAS Institute Inc., Cary, NC).

Results
Descriptive analyses. A total of 221 women in our study had live births. For our BPA analy ses, 137 women provided two or more urine samples (n = 636 samples) before getting preg nant and two or more urine samples (n = 365 samples) during pregnancy. For phthalate metabolite analyses, 113 women provided 853 samples (544 before pregnancy and 309 during pregnancy). Women in our sample were predominately white, highly educated, and a mean (± SD) of 35 ± 4.1 years of age at enrollment (Table 1). The majority (59%) of women conceived using intrauterine insemi nation. Before pregnancy, women provided between 2 and 13 urine samples (median, 3 samples) during a period of < 1 to 110 weeks after enrollment (median, 12 weeks). During pregnancy, a median of three samples was col lected. On average, first, second, and third trimester urine samples were collected at 5 (range, 3-12 weeks), 20 (range, 12-29 weeks), and 33 (range, 26-38 weeks) weeks of gesta tion. A median of 5 weeks elapsed between the last pre pregnancy and first pregnancy urine sample collections (range, 1-40 weeks).
Variability analysis. Before pregnancy, the concentrations of DEHP metabolites, MBzP, BPA, and SG exhibited substantial withinwoman variation, as evidenced by rela tively low ICCs (≤ 0.35) ( Table 3) (Table 3; see Supplemental Material, Figure 1). Most of the urinary phthalate metabolite concentra tions exhibited similar variability both before and during pregnancy (Table 3). MBzP and BPA concentrations were more variable dur ing pregnancy than before pregnancy. We also estimated the variability of MECPP before and during pregnancy to determine whether a DEHP metabolite with a longer halflife had less variability than other DEHP metabolites (Koch et al. 2006). Similar to the other two summary DEHP metabolite measures, the variability of urinary MECPP concentrations was similar both before (ICC = 0.19) and dur ing (ICC = 0.14) pregnancy. Overall, pre pregnancy urinary phthalate metabolite and BPA concentrations did not change over time, except for MEP and MBzP concentrations (Table 3). On average, uri nary MEP concentrations decreased 2% every 4 weeks (95% CI: -4, 0%) before conception. During pregnancy, concentrations of the sum of the four DEHP metabolites, the sum of the three oxidative DEHP metabolites, and the concentration of the hydrolytic monoester MEHP all decreased over time [ Table 3; see Supplemental Material, Figure 2 (http:// dx.doi.org/10.1289/ehp.1104139)]. The esti mated average decreases in MEHP concentra tions were larger than corresponding estimates for the sum of the three oxidative DEHP metabolites and the sum of all four DEHP metabolites. Decreases in urinary DEHP metabolite concentrations between the first and second trimester appeared to be respon sible for this trend (see Supplemental Material, Table 1). Because our spaghetti plots [see Supplemental Material, Figure 2) suggested that urinary DEHP metabolites decreased between the first and second trimester and then rose between the second and third trimes ter, we included a timesquared polynomial term in our model. Concentrations decreased 54% (95% CI: -67, -37%) between the 5th and 20th week of gestation and increased 18% (95% CI: -13, 62%) between the 20th and 33rd week of gestation. Urinary concentra tions of MBP, MBzP, and SG did not change over time during pregnancy, but MEP concen trations rose slightly during pregnancy (3%; 95% CI: -1, 7%) (Table 3).
Surrogate category analysis. In our classifi cation analysis using the GM concentration of urinary phthalate metabolites as the assumed gold standard of gestational exposure, the top tertile of trimesterspecific phthalate metabo lite and BPA concentrations accurately classi fied the highest tertile of gestational exposure in at least 54% of women (Table 4). The PPVs varied across trimesters for phthalate metabo lites and BPA. For instance, PPVs were highest for BPA concentrations in the first trimester (PPV = 0.70), but lowest in the second trimes ter (PPV = 0.60). Second and thirdtrimester concentrations of MBP and MEP accurately classified ≥ 69% of women. Classification probabilities for the sum of DEHP metabo lites and oxidative DEHP metabolites were similar to MEHP.
We observed that average gestational uri nary phthalate metabolite/BPA concentrations increased across tertiles of trimesterspecific concentrations (Figure 1). Average gestational phthalate metabolite/BPA concentrations were lowest among participants with trimester specific samples classified in the lowest tertile and highest among women in the top tertiles. However, the range of average gestational con centrations between adjacent surrogate cate gories overlapped in at least one trimester for each phthalate metabolite and BPA.
In our final surrogate analysis, at least 77% of women had two or more urine samples in the same tertile of urinary phthalate metabo lite or BPA concentrations during the course of pregnancy (Table 5). The proportion of women with all three urine samples in the same category during each trimester ranged from 16% (for DEHP) to 26% (for MBP).   a Assumes that the within-woman GM gestational urinary phthalate or BPA concentration is the gold standard. The surrogate measure of low, medium, or high is defined by the first, second, or third tertile of trimester-specific urine samples, respectively. Probabilities are calculated from the top versus bottom two tertiles of trimester-specific and average gestation urinary phthalate metabolite and BPA concentrations. Limited to women with all three urine samples. All concentrations are SG adjusted. b n = 77 for phthalates; n = 91 for BPA. c PPV is the probability of being classified in the top tertile of mean gestational phthalate metabolite or BPA concentration, given that the woman's trimester-specific urinary phthalate metabolite or BPA concentration is in the top tertile. d ΣDEHP metabolites: mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP), mono(2-ethyl-5-oxohexyl) phthalate (MEOHP), and mono(2-ethylhexyl) phthalate (MEHP).

Discussion
The absolute differences in urinary concentra tions of DEHP metabolites, MBP, MiBP, MBzP, and BPA were relatively small before and during pregnancy, considering the vari ability of our analytic chemistry methods. Urinary concentrations of MBP, MBzP, and MEP were modestly correlated before and during pregnancy, whereas DEHP metabolites and BPA were less correlated during the two time periods. Serial urine concentrations of BPA and some phthalate metabolites were highly variable before and during pregnancy. The variability of most urinary phthalate metabolites was similar before and during pregnancy, whereas the variability of BPA and MBzP increased during pregnancy. During pregnancy, urinary concentrations of DEHP metabolites decreased and were the most vari able. MBP and MEP concentrations were less variable, and MEP and MiBP concentrations increased during pregnancy. The variability of urinary phthalate metabo lite and BPA concentrations in our study par ticipants was remarkably similar to that of most previous studies that collected multiple urine samples over weeks to months from   (Adibi et al. 2008;Baird et al. 2010;Braun et al. 2011;Hauser et al. 2004;Irvin et al. 2010;Peck et al. 2010). Previous studies of phthalate metabolites have all shown a similar pattern where the con centration of MEHP is the most variable and concentrations of MBP, MEP, and MBzP are less variable. MEP concentrations (ICC = 0.21) among pregnant women from New York City were more variable than MEP con centrations in our study participants (Adibi et al. 2008). Source population characteristics and variations in exposure sources could influ ence withinwoman variability across cohorts. To our knowledge, no studies have exam ined whether phthalate metabolite and BPA concentrations or their withinperson variabil ity are different before or during pregnancy in the same woman. Differences would suggest that physiological changes during pregnancy may influence the absorption, distribution, metabolism, or excretion of phthalate metabo lites and BPA. The absolute differences in urinary concentrations of phthalate metabo lites and BPA were relatively small, suggesting that women did not change their behaviors to reduce these exposures. We did not observe a consistent increase or decrease in most phthalate metabolites or BPA over the course of pregnancy, which suggests that urinary concentrations of these compounds are not impacted by pregnancyinduced changes in pharmacokinetics. However, urinary concen trations of DEHP metabolites decreased dur ing pregnancy. Behavioral changes may be responsible for the decrease in urinary DEHP metabolite concentrations between the first and second trimesters of pregnancy, because women may have begun eating more meals consisting of fresh foods with less packag ing after learning of their pregnancy (Rudel et al. 2011).
The different variability among urinary phthalate metabolite and BPA concentra tions may be attributable to their exposure sources. Diet is believed to be the main source of DEHP and BPA exposure, primarily from PVC materials used in the processing/ storage of food and polycarbonate food/ beverage containers, respectively (Chapin et al. 2008;LopezCervantes and PaseiroLosada 2003;Petersen and Jensen 2010;von Goetz et al. 2010). Other sources of BPA exposure may include contact with thermal receipts (Braun et al. 2011;Liao and Kannan 2011). Higher daytoday and withinday variability in dietary sources is likely responsible for the higher variability of urinary DEHP metabo lites and BPA concentrations relative to MEP and MBP concentrations, whose primary sources include personal care and beauty products (Preau et al. 2010;Ye et al. 2011). Although the halflives of the DEHP metabo lites vary, we observed similar variability of both MEHP and MECPP, suggesting that the sources of exposure may be more impor tant than halflife of the metabolite.
MEP and MBP concentrations were rela tively stable and reproducible in repeated sam ples collected before and during pregnancy. Preau et al. (2010) reported that urinary MEP concentrations among eight adult volunteers were variable during the day but exhibited similar patterns across days within the same individual. One possible explanation may be that people use the same DBP and DEP containing personal care or cosmetic products from day to day and at similar times during the day, which may be responsible for the reduced variability in DBP and DEP urinary metabolites relative to other phthalate metab olites (Duty et al. 2005;RomeroFranco et al. 2011). Furthermore, phthalates found in personal care products will be absorbed across the skin over a longer period of time and bypass firstpass metabolism in the liver, which might increase their apparent halflife and reduce the variability of their metabolite concentrations in urine within a given day.
Our results suggest that MEP and MBP concentrations measured in a single spot urine sample in the second or third trimester of pregnancy might reasonably classify gesta tional exposure to DEP and DBP, respectively. However, reproducibility was substantially lower in samples collected in the first trimes ter. Given that DEHP metabolites, MBzP, and BPA were more variable, more than one sample may be necessary to adequately clas sify gestational exposure to these compounds. Most sampling strategies will not completely eliminate all variability and will still result in some exposure misclassification.
Our surrogate category analysis has at least two limitations. First, we assumed that GM phthalate metabolite and BPA concentrations from three urine samples during pregnancy were indicative of exposure during the entire gestation. However, it is likely that there were additional exposures and sources of exposure variability that we were unable to capture. Serial urine collections will not be possible in every epidemiological study, and researchers are encouraged to conduct their own expo sure validation when possible. Second, our surrogate analyses may overestimate the accu racy of a single spot urine sample because of the non independence of the surrogate and average measures. Other studies with more than three urine samples during pregnancy are needed to determine whether a single spot urine sample, not included in the calculation of average exposure, can accurately classify gestational exposure.
The primary strength of this study was the availability of multiple urine samples obtained both before and during pregnancy from the same woman. Furthermore, we col lected urine samples very early in pregnancy, allowing us to examine exposures and expo sure variability across the entire pregnancy. However, we were not able to examine BPA and phthalate metabolite variability within specific trimesters, which may be important in identifying critical windows of exposure dur ing pregnancy. Depending on the end point of interest, it may be more relevant to classify exposure during a narrow window of gesta tion early in pregnancy instead of during the entire period of gestation (Ge et al. 2007). Although we were unable to estimate variabil ity within narrower windows of gestation, our findings may be used to guide and evaluate studies examining gestational phthalate/BPA exposures and maternal/child health outcomes. Given the consistent magnitude of phthalate metabolite/BPA variability observed in previ ous studies conducted over time spans ranging from days to months, we believe the variability within trimesters or narrower windows would be similar to the variability over the course of pregnancy, especially considering the short halflife and non persistent nature of phthalates and BPA (Adibi et al. 2008;Baird et al. 2010;Braun et al. 2011;Hauser et al. 2004;Irvin et al. 2010;Peck et al. 2010;Preau et al. 2010;Ye et al. 2011).
Source population characteristics could influence phthalate/BPA exposures and their absorption, distribution, metabolism, and excretion. Therefore, these results may not be generalizable to other populations. Women in this study were from higher socioeconomic position and were aware that this study was examining the health effects of phthalates and other environmental chemicals. These factors might increase the likelihood of behavioral changes before and during pregnancy that would not be observed in other source popu lations. In addition, these women were older than women from studies among couples con ceiving naturally and were partners in couples seeking treatment for infertility. Physiological changes associated with advanced age or sub fertility may be responsible for some of the observed results in this cohort.
In conclusion, urinary phthalate metabo lite and BPA concentrations were variable before and during pregnancy in this cohort. MEP and MBP concentrations were less vari able and more correlated before and during pregnancy than DEHP metabolites, MBzP, and BPA. Our findings suggest that a single spot urine sample may permit relatively accu rate classification of DBP and DEP exposure during pregnancy, but accurate classification of DEHP, BzBP, and BPA exposure may require multiple urine samples. Our variabil ity and surrogate category estimates can be used to assess the extent to which phthalate and BPA exposure misclassification may bias results in epidemiological studies. Future studies should investigate the relative contri bution of pharmacokinetic and behavioral factors to urinary phthalate metabolite and BPA concentration variability.