Study population. The Study of Advanced Reproductive Age and Environmental Health (SARAEH) recruited 97 women who were referred for amniocentesis screening at the Mount Sinai Medical Center in New York, New York (USA). Recruitment occurred between 2005 and 2008. Study eligibility requirements were fluency in English or Spanish, maternal age between 18 and 40 years, singleton pregnancy, and intent to deliver at Mount Sinai Hospital. Women were approached for participation in the waiting area before their amniocentesis. Those who consented to participate in the study provided a spot urine sample immediately following their procedure. Two additional spot urine samples were collected from each participant later in their pregnancy, with collections planned at least 2 weeks apart, and the final sample collected after the 27th week of gestation. The present study comprises 71 participants who provided all three urine samples, including 69 for whom an amniotic fluid sample also was available. Following delivery, medical records were abstracted for information pertaining to fetal health.
This study was approved by the Mount Sinai Program for the Protection of Human Subjects, and all women gave informed consent to be part of the study. The involvement of the Centers for Disease Control and Prevention (CDC) laboratory was determined not to constitute engagement in human subjects research.
Exposure assessment. Following collection, amniotic fluid was delivered to the Mount Sinai Medical Center clinical cytogenetics laboratory for standard clinical care processing, which includes centrifugation and cell separation. Before being released for research purposes, amniotic fluid supernatant was stored in polypropylene containers at –20°C (time range, 0–38 weeks). Once the samples were released, SARAEH research personnel thawed the amniotic fluid overnight at 4°C, and then aliquoted the supernatant into 2-mL cryovials for storage at –80°C. Urine and amniotic fluid samples were shipped overnight on dry ice to the CDC for measurements of total (free plus conjugated) concentrations of 2,4- and 2,5-dichlorophenols, bisphenol A, benzophenone-3, triclosan, and methyl-, ethyl-, propyl-, and butylparabens by using online solid phase extraction–high performance liquid chromatography–isotope dilution tandem mass spectrometry (Ye et al. 2005 (link)). Specific gravity of urine was measured using a handheld refractometer in each thawed aliquot before shipment to the CDC, except for 11 urine samples that had specific gravity measured using an Atago PAL-10S refractometer (Atago, Bellevue, WA, USA) at the CDC. Urinary creatinine concentration was measured at CDC by an enzymatic reaction using a Roche Hitachi 912 chemistry analyzer (Hitachi, Pleasanton, CA, USA).
Statistical analyses. Analyses were conducted on natural-log (ln)–transformed concentrations. Amniotic fluid concentrations below the limit of detection (LOD) were replaced with the instrument reading values for phenols detected in at least 50% of the samples. Instrument reading values were not available for urine assays; therefore, urine phenol concentrations below the LOD were replaced with the LOD/√–2. The following formula was used to correct urinary concentrations for specific gravity: CSG = C × [(SGmean – 1)/(SG – 1)], where CSG is the specific gravity–corrected biomarker concentration, SGmean is the specific gravity arithmetic mean in our population, and C is the measured biomarker concentration. Creatinine-corrected concentrations (micrograms per gram) were calculated by dividing the phenol concentrations (micrograms per liter) by the creatinine concentration (milligrams per deciliter) and multiplying by 100.
Relation between maternal urine and amniotic fluid biomarker concentrations. We compared amniotic fluid and urinary biomarker concentrations collected on the same day, and computed ratios of uncorrected urine to amniotic fluid concentrations. For benzophenone-3 and propylparaben, the two phenols detected in > 50% of the amniotic fluid samples, we computed Spearman correlation coefficients comparing concentrations in amniotic fluid and urinary samples (uncorrected and specific gravity corrected) collected on the same day. To explore the possible predictors of benzophenone-3 and propylparaben concentrations in amniotic fluid, we performed Tobit regression models for a left-censored dependent variable (Lubin et al. 2004 (link)). We regressed amniotic fluid biomarker concentrations on maternal urinary concentrations in samples collected on the same day, fetal sex, gestational age at amniocentesis (< 17, 17–18.9, ≥ 19 gestational weeks), maternal age (< 31, 31–35.9, ≥ 36 years), maternal prepregnancy body mass index (BMI; < 25 or ≥ 25 kg/m2), race/ethnicity (white non-Hispanic or other), pregnancy complications related to placental function (including preeclampsia, placenta previa, small placenta, oligohydroamnios, and chorioamnionitis), and the time between amniotic fluid collection and processing (≤ 4, 4 to 16, 16–20, or > 20 weeks). Models were simultaneously adjusted for all of these factors. We also performed sensitivity analyses excluding four women with abnormal amniotic fluid conditions (oligohydramnios or polyhydramnios).
Variability in urinary concentrations. We evaluated variability in measures of dilution in spot urine samples across pregnancy by plotting urinary creatinine concentrations and urine specific gravity as a function of gestational age. To assess variability in phenol concentrations across pregnancy, we computed intraclass correlation coefficients (ICCs) between concentrations measured in the three spot urine samples using random intercept linear mixed models (Rabe-Hesketh 2008 ). The ICC is the ratio of the between-women variability to the total variability (between- plus within-woman variability). We also computed Spearman correlations between pairs of phenol concentrations measured in the three spot urine samples from each woman. We classified the comparability of samples based on ICCs and Spearman correlation coefficients according to the following general guidelines: < 0.4, weak; 0.4–0.6, moderate; > 0.6, good. We performed analyses of urinary concentration variability using uncorrected, specific gravity–corrected, and creatinine-corrected concentrations. Creatinine concentrations were missing for 11 of the first spot urine samples. Thus, analyses based on creatinine-corrected concentrations were restricted to 60 women with complete creatinine and environmental phenol biomarker data.
In a secondary analysis, we computed Spearman correlations among pairs of urine samples collected within specific time intervals: < 4 weeks apart, 4–6 weeks apart, 6–10 weeks apart, 10–12 weeks apart, 12–14 weeks apart, 14–16 weeks apart, and > 16 weeks apart. Samples were not independent within a given category because multiple samples from an individual woman could be included in a single category if they were collected at equally spaced intervals.
Finally, we estimated associations between urinary phenol biomarker concentrations and collection conditions (gestational age at collection; hour, day, and season of sampling; urine specific gravity) using a random intercept linear mixed model simultaneously adjusted for each collection condition along with BMI, maternal age, year of collection, maternal education, and maternal race/ethnicity (modeled as indicated previously) (Mortamais et al. 2012 (link)). We used the measured urinary biomarker concentrations and the estimated effects of collection conditions on the measured urine concentrations (for conditions that predicted urine concentrations with p < 0. 2) to derive standardized concentrations—concentrations that would have been observed if all samples had been collected under the same conditions (Mortamais et al. 2012 (link)). We estimated ICCs based on concentrations standardized for collection conditions to determine whether standardization improved reliability across repeated spot urine samples.
All analyses were performed using STATA/SE, version 12 (StataCorp, College Station, TX, USA).
This study was approved by the Mount Sinai Program for the Protection of Human Subjects, and all women gave informed consent to be part of the study. The involvement of the Centers for Disease Control and Prevention (CDC) laboratory was determined not to constitute engagement in human subjects research.
Exposure assessment. Following collection, amniotic fluid was delivered to the Mount Sinai Medical Center clinical cytogenetics laboratory for standard clinical care processing, which includes centrifugation and cell separation. Before being released for research purposes, amniotic fluid supernatant was stored in polypropylene containers at –20°C (time range, 0–38 weeks). Once the samples were released, SARAEH research personnel thawed the amniotic fluid overnight at 4°C, and then aliquoted the supernatant into 2-mL cryovials for storage at –80°C. Urine and amniotic fluid samples were shipped overnight on dry ice to the CDC for measurements of total (free plus conjugated) concentrations of 2,4- and 2,5-dichlorophenols, bisphenol A, benzophenone-3, triclosan, and methyl-, ethyl-, propyl-, and butylparabens by using online solid phase extraction–high performance liquid chromatography–isotope dilution tandem mass spectrometry (Ye et al. 2005 (link)). Specific gravity of urine was measured using a handheld refractometer in each thawed aliquot before shipment to the CDC, except for 11 urine samples that had specific gravity measured using an Atago PAL-10S refractometer (Atago, Bellevue, WA, USA) at the CDC. Urinary creatinine concentration was measured at CDC by an enzymatic reaction using a Roche Hitachi 912 chemistry analyzer (Hitachi, Pleasanton, CA, USA).
Statistical analyses. Analyses were conducted on natural-log (ln)–transformed concentrations. Amniotic fluid concentrations below the limit of detection (LOD) were replaced with the instrument reading values for phenols detected in at least 50% of the samples. Instrument reading values were not available for urine assays; therefore, urine phenol concentrations below the LOD were replaced with the LOD/√–2. The following formula was used to correct urinary concentrations for specific gravity: CSG = C × [(SGmean – 1)/(SG – 1)], where CSG is the specific gravity–corrected biomarker concentration, SGmean is the specific gravity arithmetic mean in our population, and C is the measured biomarker concentration. Creatinine-corrected concentrations (micrograms per gram) were calculated by dividing the phenol concentrations (micrograms per liter) by the creatinine concentration (milligrams per deciliter) and multiplying by 100.
Relation between maternal urine and amniotic fluid biomarker concentrations. We compared amniotic fluid and urinary biomarker concentrations collected on the same day, and computed ratios of uncorrected urine to amniotic fluid concentrations. For benzophenone-3 and propylparaben, the two phenols detected in > 50% of the amniotic fluid samples, we computed Spearman correlation coefficients comparing concentrations in amniotic fluid and urinary samples (uncorrected and specific gravity corrected) collected on the same day. To explore the possible predictors of benzophenone-3 and propylparaben concentrations in amniotic fluid, we performed Tobit regression models for a left-censored dependent variable (Lubin et al. 2004 (link)). We regressed amniotic fluid biomarker concentrations on maternal urinary concentrations in samples collected on the same day, fetal sex, gestational age at amniocentesis (< 17, 17–18.9, ≥ 19 gestational weeks), maternal age (< 31, 31–35.9, ≥ 36 years), maternal prepregnancy body mass index (BMI; < 25 or ≥ 25 kg/m2), race/ethnicity (white non-Hispanic or other), pregnancy complications related to placental function (including preeclampsia, placenta previa, small placenta, oligohydroamnios, and chorioamnionitis), and the time between amniotic fluid collection and processing (≤ 4, 4 to 16, 16–20, or > 20 weeks). Models were simultaneously adjusted for all of these factors. We also performed sensitivity analyses excluding four women with abnormal amniotic fluid conditions (oligohydramnios or polyhydramnios).
Variability in urinary concentrations. We evaluated variability in measures of dilution in spot urine samples across pregnancy by plotting urinary creatinine concentrations and urine specific gravity as a function of gestational age. To assess variability in phenol concentrations across pregnancy, we computed intraclass correlation coefficients (ICCs) between concentrations measured in the three spot urine samples using random intercept linear mixed models (Rabe-Hesketh 2008 ). The ICC is the ratio of the between-women variability to the total variability (between- plus within-woman variability). We also computed Spearman correlations between pairs of phenol concentrations measured in the three spot urine samples from each woman. We classified the comparability of samples based on ICCs and Spearman correlation coefficients according to the following general guidelines: < 0.4, weak; 0.4–0.6, moderate; > 0.6, good. We performed analyses of urinary concentration variability using uncorrected, specific gravity–corrected, and creatinine-corrected concentrations. Creatinine concentrations were missing for 11 of the first spot urine samples. Thus, analyses based on creatinine-corrected concentrations were restricted to 60 women with complete creatinine and environmental phenol biomarker data.
In a secondary analysis, we computed Spearman correlations among pairs of urine samples collected within specific time intervals: < 4 weeks apart, 4–6 weeks apart, 6–10 weeks apart, 10–12 weeks apart, 12–14 weeks apart, 14–16 weeks apart, and > 16 weeks apart. Samples were not independent within a given category because multiple samples from an individual woman could be included in a single category if they were collected at equally spaced intervals.
Finally, we estimated associations between urinary phenol biomarker concentrations and collection conditions (gestational age at collection; hour, day, and season of sampling; urine specific gravity) using a random intercept linear mixed model simultaneously adjusted for each collection condition along with BMI, maternal age, year of collection, maternal education, and maternal race/ethnicity (modeled as indicated previously) (Mortamais et al. 2012 (link)). We used the measured urinary biomarker concentrations and the estimated effects of collection conditions on the measured urine concentrations (for conditions that predicted urine concentrations with p < 0. 2) to derive standardized concentrations—concentrations that would have been observed if all samples had been collected under the same conditions (Mortamais et al. 2012 (link)). We estimated ICCs based on concentrations standardized for collection conditions to determine whether standardization improved reliability across repeated spot urine samples.
All analyses were performed using STATA/SE, version 12 (StataCorp, College Station, TX, USA).
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