Article pubs.acs.org/est
Prenatal Exposure to Polybrominated Flame Retardants and Fetal Growth in the INMA Cohort (Spain) Maria-Jose Lopez-Espinosa,*,†,‡,⊥ Olga Costa,†,⊥ Esther Vizcaino,§,∥ Mario Murcia,‡,† Ana Fernandez-Somoano,‡,§ Carmen Iñiguez,†,‡ Sabrina Llop,†,‡ Joan O. Grimalt,∥ Ferran Ballester,†,‡ and Adonina Tardon§,‡ †
FISABIO−Universitat Jaume I−Universitat de València Joint Research Unit of Epidemiology and Environmental Health, Avenida de Catalunya 21, 46020 Valencia, Spain ‡ Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP), Calle Monforte de Lemos 3-5, 28029 Madrid, Spain § Department of Preventive Medicine and Public Health, University of Oviedo, Campus del Cristo s/n, 33006 Oviedo, Asturias, Spain ∥ Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona, 18, 08034 Barcelona, Spain S Supporting Information *
ABSTRACT: Our aim was to investigate the relation between PBDEs and fetal growth or newborn anthropometry in a Spanish cohort (2003−2008). PBDE congeners (BDE-47, -99, -153, -154, and -209) were determined in serum of 670 mothers at gestational week 12 and in 534 umbilical cord samples. Abdominal circumference (AC), estimated fetal weight (EFW), femur length (FL), and biparietal diameter (BPD) during gestation were measured by ultrasounds. At birth, weight (BW), head circumference (HC), and length (BL) were also measured. We assessed growth in the intervals between 12−20 and 20−34 weeks of gestation and size at birth by standard deviation (SD)scores adjusted for constitutional characteristics. We conducted multivariate linear regression analyses between PBDE congeners and their sum (ΣPBDEs) and outcomes. We found statistically significant inverse associations between ΣPBDEs and AC, EFW, and BPD at weeks 20−34 and HC at birth. Regarding congeners, the association was clearer with BDE-99, with inverse associations being found with AC, EFW, and BPD at weeks 20−34, and with BW and HC at delivery. These outcomes decreased between 1.3% and 3.5% for each 2-fold PBDE increase. Concerning matrices, we found statistically significant inverse associations with BPD, HC, and BW when using maternal serum, and for AC and EFW with cord serum. In conclusion, PBDEs may impair fetal growth in late pregnancy and reduce birth size.
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INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are flame retardants which were commonly used in the manufacture of many types of household and commercial products.1 The commercial Penta- and OctaBDE were added to the list of persistent organic pollutants under the Stockholm Convention in 2009.2 The commercial production and use of DecaBDE has been restricted in Europe and phased out in the United States. However, its production and use are still going on in other parts of the world.3,4 Humans are exposed to these pollutants via several sources, including diet and indoor environments.5,6 PBDEs can cross the placenta and have been found in cord blood of different birth cohorts worldwide.7,8 Even though PBDEs have been widely used for several decades and humans are exposed to them on a daily basis, little is known about their possible effects on development. Epidemiological reports on fetal growth are scarce, have chosen anthropometrical size at birth as a proxy measure of in utero © XXXX American Chemical Society
development, and have failed to yield conclusive results. Specifically, some of these PBDE studies have reported an inverse association with birth weight (BW),9−12 birth length (BL),10 or adverse birth outcomes (defined as low birth weight, prematurity, or still birth),13 whereas others found no association with BW,14,15 head circumference (HC),9,10 or BL.9,13 In addition, one study on birth size found associations in both directions depending on the PBDE congener studied.16 The possible biological mechanisms underlying the potential effects of PBDEs on fetal growth are not yet known, but could involve impairments in the endocrine system, since these pollutants may alter human levels of insulin-like growth factors Received: April 9, 2015 Revised: July 10, 2015 Accepted: July 16, 2015
A
DOI: 10.1021/acs.est.5b01793 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology and thyroid hormones during pregnancy,17−19 which play vital roles in fetal growth and development. Fetal growth is a good determinant of perinatal and postnatal health. In fact, the “fetal programming” hypothesis proposed by Barker20 suggests that impaired fetal growth leads to small but permanent deficits in childhood development and is also a risk factor of some chronic diseases in adulthood, such as diabetes, hypertension, and heart disease.21 Consequently, the possible impact of PBDEs on fetal growth is a matter of public concern and more studies are warranted. The aim of the present study was to investigate the relation between maternal and cord concentrations of PBDEs (BDE-47, -99, -153, -154, and -209, and their sum [ΣPBDEs]) and fetal growth (at 12−20 and 20−34 weeks of gestation) and birth size. This work was undertaken within the INMA (INfancia y Medio Ambiente − Environment and Childhood) Project in Spain.
difficulties).24 The detection limits (LODs) for BDE-47, -99, -153, -154, and -209 were 2.59, 2.38, 1.24, 2.78, and 9.12 pg/ mL in Asturias, and 2.30, 2.10, 1.19, 0.43, and 5.94 pg/mL in Valencia. LODs were calculated as three times the standard deviation (SD) of the procedural blank levels.23 Samples were analyzed in two different batches, one per cohort, and therefore LODs were somewhat different for each cohort. The laboratory complies with the Arctic Monitoring and Assessment Program (AMAP) for persistent organic pollutants in human serum (Centre de Toxicologie, Institut National de Santé Publique du Québec). We used enzymatic techniques to determine total cholesterol and triglycerides, and total serum lipid concentrations were calculated as described by Phillips et al.25 Means ± SDs of total lipid contents in maternal (n = 473) and cord (n = 486) serum were 5.8 ± 1.3 and 2.6 ± 0.5 mg/mL, respectively. Fetal Outcomes. Specialized obstetricians performed ultrasound examinations in routinely scheduled antenatal care visits in gestational weeks 12, 20, and 34. We also had access to any other ultrasound scans performed during pregnancy at the same hospital. From two to eight valid ultrasound measurements were obtained per subject between the seventh and 41st weeks of gestation. Of a total of 1264 women from Asturias and Valencia INMA cohorts providing ultrasound data, 107 (9%) had two examinations, 901 (71%) had three, 214 (17%) had four, and 42 (3%) had five or more. Therefore, a total of 3991 ultrasound examinations were used to construct longitudinal growth curves for fetal parameters. The fetal parameters were abdominal circumference (AC), biparietal diameter (BPD: the transverse diameter of the head), and femur length (FL). Additionally, we calculated estimated fetal weight (EFW) using the Hadlock algorithm.26 An early crown−rump length (CRL) measurement was also obtained and used for pregnancy dating. Gestational age was based on the self-reported date of the last menstrual period, but an estimation based on the CRL measurement was considered if the self-reported and estimated dates differed by ≥7 days.27 Women with a difference of ≥3 weeks were excluded from the study (n = 8). Data of fetal parameters outside the range of the mean ± 4 SDs for each gestational age were also eliminated to avoid the influence of extreme values (n ≤ 5 for all parameters). Detailed information on ultrasound measures and gestational age is included in Table S2. We used linear mixed models28 separately in each cohort to obtain longitudinal growth curves for the four fetal parameters. Models were adjusted for those available covariates known to influence the growth potential of the fetus: parental anthropometric characteristics, sex of the fetus, parity, maternal age, and country of birth of the mother. These customized models provide individual rather than population-based fetal growth standards that are expected to reduce misclassification in the detection of “small for gestational age” by excluding constitutionally small fetuses and including those within normal population limits who should have reached a greater size.29 Fetal growth curves provided predictions for weeks 12, 20, and 34, and we used these predictions to calculate unconditional and conditional SD-scores. Unconditional SD-scores describe the size of a fetus at a given time point, while conditional SD-scores describe the fetal growth between two time points, i.e., they evaluate the size at the final time point according to the size at the starting time point by means of conditional probability.30,31 We calculated unconditional SDscores at 12, 20, and 34 weeks of gestation, and conditional SD-
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EXPERIMENTAL SECTION Study Design. The INMA Project is a mother-and-child cohort study established in different areas of Spain following a common protocol.22 This study included the INMA cohorts of Asturias and Valencia, since serum samples were only available for PBDE determinations in these cohorts. The Ethics Committees of the San Agustin Hospital in Aviles and La Fe Hospital in Valencia approved the research protocol, and all mothers gave their written informed consent prior to inclusion. We recruited a total of 1349 eligible women (≥16 years, singleton pregnancy, enrolment at 10−13 weeks of gestation, nonassisted conception, delivery scheduled at the reference hospital, and no communication impairment) in the first trimester of pregnancy. After excluding the women who withdrew from the study, were lost to follow-up, with induced or spontaneous abortions or fetal deaths, and with only one valid ultrasound, 1264 (94%) women were followed up to delivery (2003−2008). In the present study, the sample size was 686 mothers and their newborns with at least two valid ultrasounds and samples for PBDE determinations in maternal (n = 670) and/or umbilical cord (n = 534) serum. A flowchart of the study population is available in the Supporting Information (Figure S1). Around 98% of the women from the Asturias cohort were included in the present study and population characteristics did not differ between included and excluded women (data not shown). A total of 28% of the women from the Valencia cohort were included and no differences in characteristics were found either, except in the case of a lower proportion of participants with a rural residence (3%) and not working during pregnancy (12%) compared to nonincluded women (7% and 19%, respectively) (Table S1). In Valencia, the lower proportion of available samples for PBDE determinations was due to logistic reasons. PBDE Concentrations. We analyzed concentrations of 14 PBDEs using gas chromatography coupled to a mass spectrometer, as described previously.23 In this study, we present the results of those PBDEs with maternal or/and cord concentrations with a detection frequency of >50% (BDE-47, -99, -153, and -154). In addition, we include analyses with BDE-209 because detection frequency was close to 50% in maternal serum, it is the only congener still in use in Europe4 (so concentrations in environmental samples are very likely to increase), and available data on prenatal BDE-209 exposure and their health effects are scarce (mainly because of analytical B
DOI: 10.1021/acs.est.5b01793 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
were measured in maternal (week 12 of pregnancy) and cord serum samples by gas chromatography with electron capture detection.32 Statistical Analysis. For descriptive purposes, we present numbers (percentages) for categorical variables, means ± SDs for continuous variables, and percentiles (P25, P50, and P75) for PBDEs. Pollutant concentrations were expressed in pg/mL and in ng/g lipid (using individual total lipid values to calculate PBDE concentrations on a lipid content basis). We used cohort-adjusted Pearson’s partial correlations to describe pairwise relationships between levels of log2(lipids) and log2(PBDEs) and between log2(PBDEs) measured in both matrices. PBDE values 10% after inclusion of this variable. Any possible confounder was previously selected if variables were associated with log2(PBDE) concentrations at the p < 0.2 level in Tobit regression analyses. Finally, we assessed the homoscedasticity and normality of regression residuals, and we excluded extreme outliers (Studentized residuals ≥4) or highly influential observations (Cook’s distance >0.5) from the final models. These models are the complete case analyses (i.e., models restricted to subjects with complete data in covariates and replacing PBDE values
Prenatal Exposure to Polybrominated Flame Retardants and Fetal Growth in the INMA Cohort (Spain) Maria-Jose Lopez-Espinosa,*,†,‡,⊥ Olga Costa,†,⊥ Esther Vizcaino,§,∥ Mario Murcia,‡,† Ana Fernandez-Somoano,‡,§ Carmen Iñiguez,†,‡ Sabrina Llop,†,‡ Joan O. Grimalt,∥ Ferran Ballester,†,‡ and Adonina Tardon§,‡ †
FISABIO−Universitat Jaume I−Universitat de València Joint Research Unit of Epidemiology and Environmental Health, Avenida de Catalunya 21, 46020 Valencia, Spain ‡ Spanish Consortium for Research on Epidemiology and Public Health (CIBERESP), Calle Monforte de Lemos 3-5, 28029 Madrid, Spain § Department of Preventive Medicine and Public Health, University of Oviedo, Campus del Cristo s/n, 33006 Oviedo, Asturias, Spain ∥ Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDÆA-CSIC), Jordi Girona, 18, 08034 Barcelona, Spain S Supporting Information *
ABSTRACT: Our aim was to investigate the relation between PBDEs and fetal growth or newborn anthropometry in a Spanish cohort (2003−2008). PBDE congeners (BDE-47, -99, -153, -154, and -209) were determined in serum of 670 mothers at gestational week 12 and in 534 umbilical cord samples. Abdominal circumference (AC), estimated fetal weight (EFW), femur length (FL), and biparietal diameter (BPD) during gestation were measured by ultrasounds. At birth, weight (BW), head circumference (HC), and length (BL) were also measured. We assessed growth in the intervals between 12−20 and 20−34 weeks of gestation and size at birth by standard deviation (SD)scores adjusted for constitutional characteristics. We conducted multivariate linear regression analyses between PBDE congeners and their sum (ΣPBDEs) and outcomes. We found statistically significant inverse associations between ΣPBDEs and AC, EFW, and BPD at weeks 20−34 and HC at birth. Regarding congeners, the association was clearer with BDE-99, with inverse associations being found with AC, EFW, and BPD at weeks 20−34, and with BW and HC at delivery. These outcomes decreased between 1.3% and 3.5% for each 2-fold PBDE increase. Concerning matrices, we found statistically significant inverse associations with BPD, HC, and BW when using maternal serum, and for AC and EFW with cord serum. In conclusion, PBDEs may impair fetal growth in late pregnancy and reduce birth size.
■
INTRODUCTION Polybrominated diphenyl ethers (PBDEs) are flame retardants which were commonly used in the manufacture of many types of household and commercial products.1 The commercial Penta- and OctaBDE were added to the list of persistent organic pollutants under the Stockholm Convention in 2009.2 The commercial production and use of DecaBDE has been restricted in Europe and phased out in the United States. However, its production and use are still going on in other parts of the world.3,4 Humans are exposed to these pollutants via several sources, including diet and indoor environments.5,6 PBDEs can cross the placenta and have been found in cord blood of different birth cohorts worldwide.7,8 Even though PBDEs have been widely used for several decades and humans are exposed to them on a daily basis, little is known about their possible effects on development. Epidemiological reports on fetal growth are scarce, have chosen anthropometrical size at birth as a proxy measure of in utero © XXXX American Chemical Society
development, and have failed to yield conclusive results. Specifically, some of these PBDE studies have reported an inverse association with birth weight (BW),9−12 birth length (BL),10 or adverse birth outcomes (defined as low birth weight, prematurity, or still birth),13 whereas others found no association with BW,14,15 head circumference (HC),9,10 or BL.9,13 In addition, one study on birth size found associations in both directions depending on the PBDE congener studied.16 The possible biological mechanisms underlying the potential effects of PBDEs on fetal growth are not yet known, but could involve impairments in the endocrine system, since these pollutants may alter human levels of insulin-like growth factors Received: April 9, 2015 Revised: July 10, 2015 Accepted: July 16, 2015
A
DOI: 10.1021/acs.est.5b01793 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology and thyroid hormones during pregnancy,17−19 which play vital roles in fetal growth and development. Fetal growth is a good determinant of perinatal and postnatal health. In fact, the “fetal programming” hypothesis proposed by Barker20 suggests that impaired fetal growth leads to small but permanent deficits in childhood development and is also a risk factor of some chronic diseases in adulthood, such as diabetes, hypertension, and heart disease.21 Consequently, the possible impact of PBDEs on fetal growth is a matter of public concern and more studies are warranted. The aim of the present study was to investigate the relation between maternal and cord concentrations of PBDEs (BDE-47, -99, -153, -154, and -209, and their sum [ΣPBDEs]) and fetal growth (at 12−20 and 20−34 weeks of gestation) and birth size. This work was undertaken within the INMA (INfancia y Medio Ambiente − Environment and Childhood) Project in Spain.
difficulties).24 The detection limits (LODs) for BDE-47, -99, -153, -154, and -209 were 2.59, 2.38, 1.24, 2.78, and 9.12 pg/ mL in Asturias, and 2.30, 2.10, 1.19, 0.43, and 5.94 pg/mL in Valencia. LODs were calculated as three times the standard deviation (SD) of the procedural blank levels.23 Samples were analyzed in two different batches, one per cohort, and therefore LODs were somewhat different for each cohort. The laboratory complies with the Arctic Monitoring and Assessment Program (AMAP) for persistent organic pollutants in human serum (Centre de Toxicologie, Institut National de Santé Publique du Québec). We used enzymatic techniques to determine total cholesterol and triglycerides, and total serum lipid concentrations were calculated as described by Phillips et al.25 Means ± SDs of total lipid contents in maternal (n = 473) and cord (n = 486) serum were 5.8 ± 1.3 and 2.6 ± 0.5 mg/mL, respectively. Fetal Outcomes. Specialized obstetricians performed ultrasound examinations in routinely scheduled antenatal care visits in gestational weeks 12, 20, and 34. We also had access to any other ultrasound scans performed during pregnancy at the same hospital. From two to eight valid ultrasound measurements were obtained per subject between the seventh and 41st weeks of gestation. Of a total of 1264 women from Asturias and Valencia INMA cohorts providing ultrasound data, 107 (9%) had two examinations, 901 (71%) had three, 214 (17%) had four, and 42 (3%) had five or more. Therefore, a total of 3991 ultrasound examinations were used to construct longitudinal growth curves for fetal parameters. The fetal parameters were abdominal circumference (AC), biparietal diameter (BPD: the transverse diameter of the head), and femur length (FL). Additionally, we calculated estimated fetal weight (EFW) using the Hadlock algorithm.26 An early crown−rump length (CRL) measurement was also obtained and used for pregnancy dating. Gestational age was based on the self-reported date of the last menstrual period, but an estimation based on the CRL measurement was considered if the self-reported and estimated dates differed by ≥7 days.27 Women with a difference of ≥3 weeks were excluded from the study (n = 8). Data of fetal parameters outside the range of the mean ± 4 SDs for each gestational age were also eliminated to avoid the influence of extreme values (n ≤ 5 for all parameters). Detailed information on ultrasound measures and gestational age is included in Table S2. We used linear mixed models28 separately in each cohort to obtain longitudinal growth curves for the four fetal parameters. Models were adjusted for those available covariates known to influence the growth potential of the fetus: parental anthropometric characteristics, sex of the fetus, parity, maternal age, and country of birth of the mother. These customized models provide individual rather than population-based fetal growth standards that are expected to reduce misclassification in the detection of “small for gestational age” by excluding constitutionally small fetuses and including those within normal population limits who should have reached a greater size.29 Fetal growth curves provided predictions for weeks 12, 20, and 34, and we used these predictions to calculate unconditional and conditional SD-scores. Unconditional SD-scores describe the size of a fetus at a given time point, while conditional SD-scores describe the fetal growth between two time points, i.e., they evaluate the size at the final time point according to the size at the starting time point by means of conditional probability.30,31 We calculated unconditional SDscores at 12, 20, and 34 weeks of gestation, and conditional SD-
■
EXPERIMENTAL SECTION Study Design. The INMA Project is a mother-and-child cohort study established in different areas of Spain following a common protocol.22 This study included the INMA cohorts of Asturias and Valencia, since serum samples were only available for PBDE determinations in these cohorts. The Ethics Committees of the San Agustin Hospital in Aviles and La Fe Hospital in Valencia approved the research protocol, and all mothers gave their written informed consent prior to inclusion. We recruited a total of 1349 eligible women (≥16 years, singleton pregnancy, enrolment at 10−13 weeks of gestation, nonassisted conception, delivery scheduled at the reference hospital, and no communication impairment) in the first trimester of pregnancy. After excluding the women who withdrew from the study, were lost to follow-up, with induced or spontaneous abortions or fetal deaths, and with only one valid ultrasound, 1264 (94%) women were followed up to delivery (2003−2008). In the present study, the sample size was 686 mothers and their newborns with at least two valid ultrasounds and samples for PBDE determinations in maternal (n = 670) and/or umbilical cord (n = 534) serum. A flowchart of the study population is available in the Supporting Information (Figure S1). Around 98% of the women from the Asturias cohort were included in the present study and population characteristics did not differ between included and excluded women (data not shown). A total of 28% of the women from the Valencia cohort were included and no differences in characteristics were found either, except in the case of a lower proportion of participants with a rural residence (3%) and not working during pregnancy (12%) compared to nonincluded women (7% and 19%, respectively) (Table S1). In Valencia, the lower proportion of available samples for PBDE determinations was due to logistic reasons. PBDE Concentrations. We analyzed concentrations of 14 PBDEs using gas chromatography coupled to a mass spectrometer, as described previously.23 In this study, we present the results of those PBDEs with maternal or/and cord concentrations with a detection frequency of >50% (BDE-47, -99, -153, and -154). In addition, we include analyses with BDE-209 because detection frequency was close to 50% in maternal serum, it is the only congener still in use in Europe4 (so concentrations in environmental samples are very likely to increase), and available data on prenatal BDE-209 exposure and their health effects are scarce (mainly because of analytical B
DOI: 10.1021/acs.est.5b01793 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
Article
Environmental Science & Technology
were measured in maternal (week 12 of pregnancy) and cord serum samples by gas chromatography with electron capture detection.32 Statistical Analysis. For descriptive purposes, we present numbers (percentages) for categorical variables, means ± SDs for continuous variables, and percentiles (P25, P50, and P75) for PBDEs. Pollutant concentrations were expressed in pg/mL and in ng/g lipid (using individual total lipid values to calculate PBDE concentrations on a lipid content basis). We used cohort-adjusted Pearson’s partial correlations to describe pairwise relationships between levels of log2(lipids) and log2(PBDEs) and between log2(PBDEs) measured in both matrices. PBDE values 10% after inclusion of this variable. Any possible confounder was previously selected if variables were associated with log2(PBDE) concentrations at the p < 0.2 level in Tobit regression analyses. Finally, we assessed the homoscedasticity and normality of regression residuals, and we excluded extreme outliers (Studentized residuals ≥4) or highly influential observations (Cook’s distance >0.5) from the final models. These models are the complete case analyses (i.e., models restricted to subjects with complete data in covariates and replacing PBDE values
