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Transfer of perfluoroalkyl substances from mother to fetus in a Spanish birth cohort
Cyntia B. Manzano-Salgado a,b,c*, Maribel Casas a,b,c, Maria-Jose Lopez-Espinosa c,d, Ferran
Ballester c,d, Mikel Basterrechea c,e,f, Joan Grimalt g,h, Ana-María Jiménez e,f, Thomas Kraus i,
Thomas Schettgen i, Jordi Sunyer a,b,c, Martine Vrijheid a,b,c
Affiliations
a Centre for Research in Environmental Epidemiology (CREAL), Barcelona, Spain b Universitat Pompeu Fabra (UPF), Barcelona, Spainc CIBER Epidemiología y Salud Pública (CIBERESP), Spain d FISABIO– Universitat Jaume I– Universitat de València Joint Research Unit of
Epidemiology and Environmental Health, Valencia, Spain e Public Health Department of Gipuzkoa, San Sebastian, Spain f Health Research Institute Biodonostia, San Sebastian, Gipuzkoa, Spain g Department of Environmental Chemistry, Institute of Environmental Assessment and
Water Research (IDAEA) , Barcelona, Spainh Spanish Council for Scientific Research (CSIC), Barcelona, Spain i Institute for Occupational Medicine, RWTH Aachen University, Aachen, Germany
Corresponding author:
Cyntia B. Manzano-Salgado, Centre for Research in Environmental Epidemiology (CREAL),
Doctor Aiguader, 88|08003 Barcelona, Catalonia, Spain. Tel.: + 34 932 147 314; Fax: + 34 932
045 904.
Email: [email protected]
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Abstract
Introduction: Prenatal exposure to perfluoroalkyl substances (PFAS) might affect child health;
thus estimating PFAS fetal burden is relevant. PFAS fetal burden is best estimated in cord
samples; previous studies have used either maternal plasma or serum during pregnancy as
proxy, but their validity is not clear. We aimed to evaluate PFAS transfer between mother and
fetus and determine its predictors in a Spanish birth cohort.
Methods: We measured perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate
(PFHxS), perfluorooctane sulfonate (PFOS), perfluorooctanoate (PFOA), and
perfluorononanoate (PFNA) in maternal blood and cord serum from 66 mother-child pairs. We
used Spearman’s rank coefficients to correlate PFAS concentrations in first trimester maternal
plasma and serum, with cord serum samples. We assessed PFAS placental transfer by
calculating maternal to cord ratios and examined their association with maternal socio-
demographic characteristics and child sex using linear regression models.
Results: Median concentrations of PFAS (ng/mL) of PFHxS, PFOS, PFOA, and PFNA in
maternal plasma (0.79, 6.18, 2.85 and 0.84, respectively) and serum (0.84, 6.99, 2.97 and 0.85)
were higher than in cord serum (0.40, 1.86, 1.90 and 0.32). PFBS was not detected. Positive
Spearman’s correlations (p-values<0.001) were found between maternal plasma and serum
(rho≥0.80), maternal plasma and cord (rho≥0.66), and maternal serum and cord samples
(rho≥0.67). Maternal plasma to cord ratios were above 1 (PFHxS: 2.35 [95%CI: 2.05, 2.70],
PFOS: 3.33 [3.05, 3.62], PFOA: 1.37 [1.27, 1.48], PFNA: 2.39 [2.18, 2.63]); maternal serum to
cord ratios were similar. Maternal to cord ratios decreased with maternal age, but not with other
socio-demographic factors.
Conclusions: Our results suggest that PFAS fetal body burden can be assessed using as proxy
maternal plasma or serum collected early in pregnancy. Maternal age might influence PFAS
placental transfer.
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Keywords: Perfluoroalkyl substances (PFAS); Pregnancy; Cord blood, Mother-child pairs;
INMA
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Funding Sources
This study was funded in part by grants from the European Union (FP7-ENV-2011 cod 282957
and HEALTH.2010.2.4.5-1), and from Spain: Instituto de Salud Carlos III and The Ministry of
Health (Red INMA G03/176, CB06/02/0041, FIS- PI12/01890, FIS-PI041436, FIS- PI081151,
FIS-PI06/0867, FIS-PS09/00090 FIS-FEDER 03/1615, 04/1509, 04/1112, 04/1931, 05/1079,
05/1052, 06/1213, 07/0314, 09/02647, 11/0178, 11/01007, 11/02591, 11/02038, 13/1944,
13/2032, 14/00891, 14/01687 and pre-doctoral grant PFIS 2014), the Conselleria de Sanitat,
Generalitat Valenciana, Department of Health of the Basque Government (2005111093 and
2009111069), the Provincial Government of Gipuzkoa (DFG06/004 and DFG08/001) and the
Generalitat de Catalunya-CIRIT (1999SGR 00241). This study has been reviewed and
approved by the accredited committees of the following institutions: The Municipal Institute of
Sanitary Assistance of Barcelona, La Fe University Hospital of Valencia and, The Donostia
Hospital.
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Abbreviations:
BMI Body mass index
CI Confidence interval
GM Geometric mean
HPLC High performance liquid chromatography
INMA Environment and Childhood Project
IQR Interquartile range
LC-MS-MS Liquid chromatography coupled with tandem mass spectrometry
LOQ Limit of quantification
LOD Limit of detection
LOG-KOW Logarithms of the octanol-water partition coefficients
MeOH Methanol
PFAS Perfluoroalkyl substances
PFBS Perfluorobutane sulfonate
PFHxS Perfluorohexane sulfonate
PFOS Perfluorooctane sulfonate
PFOA Perfluorooctanoate
PFNA Perfluorononanoate
RAM Restricted access material
SD Standard deviation
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Word count
Abstract: 281
Full text (i.e. Introduction, method, results, discussion and conclusions): 3, 547
Total (i.e. abstract + full text): 3,828
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1. Introduction
Perfluoroalkyl substances (PFAS) are synthetic chemicals widely used in many industrial and
commercial applications such as, the coating of paper and packaging, textiles and leather, fire-
fighting foam, photography industry, cleaning products and, pesticides (Casals-Casas and
Desvergne, 2011). The PFAS most studied are perfluorooctane sulfonate (PFOS) and
perfluorooctanoate (PFOA) because of their widespread use, environmental persistence and,
long biological half-lives (4-5 years) (Olsen et al., 2007). However, there are other PFAS (e.g.
perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS) and perfluorononanoate
(PFNA)) that are less frequently studied, but their production and use is also widespread
(Prevedouros et al., 2006). For example, PFBS is replacing PFOS (Oldham et al., 2012), and
PFNA blood concentrations are increasing in the US NHANES population (Kato et al., 2011).
PFAS can cross the placental barrier (Fei et al., 2007; Midasch et al., 2007; Monroy et al.,
2008). An increasing number of prospective studies have assessed the effects of prenatal PFAS
exposure on a range of child health outcomes, measuring PFAS concentrations either in
maternal or cord blood samples. For example, PFOA has been associated with a reduction in
birth weight in a recent meta-analysis of nine studies (Johnson et al., 2014; Lam et al., 2014).
Another review supports this conclusion (Bach et al., 2015), although more cautiously, because
confounding by glomerular filtration rate cannot be excluded (Verner et al., 2015). For PFOS
evidence is not consistent (Bach et al., 2015). Further, PFAS are suspected obesogens in
experimental studies (La Merrill and Birnbaum, 2012), but there are few human studies and
these show conflicting results with positive associations (Halldorsson et al., 2012; Høyer et al.,
2015; Maisonet et al., 2012) and null associations (Andersen et al., 2013; Barry et al., 2014) on
offspring body mass index (BMI) and weight at different ages. PFAS exposure may also be
associated with decreased antibody response to childhood vaccines (Grandjean et al., 2012;
Granum et al., 2013) and with childhood hypertension (Geiger et al., 2014). Studies evaluating
associations between fetal exposure to PFAS and neurodevelopmental outcomes are
inconsistent with several studies suggesting no association and only a few reporting adverse
effects (Roth and Wilks, 2014).
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PFAS fetal body burden may be best estimated in cord blood samples (Hanssen et al., 2010)
because PFAS bind to serum albumin (Salvalaglio et al., 2010). However, many studies have
used maternal blood during pregnancy as a surrogate; probably due to difficulties on cord blood
collection or low availability of samples. Furthermore, many studies alternately used either
maternal plasma or serum assuming that PFAS distribute evenly between both blood
compartments (Fei et al., 2007; Hanssen et al., 2010; Monroy et al., 2008; Porpora et al., 2013).
Only one study has studied the distribution of PFHxS, PFOS and PFOA between plasma and
serum samples from the same subject and concluded that they distributed evenly, but 78% of
their subjects were men (average age: 49) working in a fluorochemical factory (Ehresman et al.,
2007). Understanding if PFAS have a similar distribution between plasma and serum during
pregnancy could ease the comparison between studies.
Socio-demographic factors may determine PFAS concentrations in maternal and cord blood.
Some studies indicate that higher PFAS levels are associated with older maternal age (Kato et
al., 2014; Lien et al., 2013), lower parity and less previous breastfeeding (Ode et al., 2013),
higher maternal education (Lien et al., 2013) and Asian maternal race (Apelberg et al., 2007b)
but results are mainly for PFOS and PFOA and are still inconsistent (Ode et al., 2013).
Moreover, there is no information on the maternal determinants that influence the PFAS
transfer efficiency from the mother to the fetus.
In this study we aimed to evaluate PFAS transfer between mother and fetus and determine its
predictors in a Spanish birth cohort. Our objectives were (1) to determine the concentrations of
five PFAS (PFBS, PFHxS, PFOS, PFOA and PFNA) in maternal blood samples (plasma and
serum) and in cord serum samples; (2) to evaluate the correlations and transfer ratio between
PFAS concentrations matched maternal-cord samples and; (3) to evaluate which socio-
demographic factors predict PFAS transfer from mother to fetus.
2. Materials and methods
2.1 Study population
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The INMA (Environment and Childhood) Project is a network of prospective population-based
birth cohorts in Spain; aiming to understand the associations of pre and postnatal environmental
exposures and child health (www.proyectoinma.org). Details on the recruitment and follow-up
have been described elsewhere (Guxens et al., 2012). During 2003-2006 women from two
Spanish regions (Sabadell and Valencia) were recruited in their first trimester of pregnancy
(n=1,484) and were followed until delivery (n=1,409). From these two regions we randomly
selected a subsample of 66 mother-child pairs out of the 1,088 pairs that had available samples
of maternal blood and cord serum. Detailed information on the mother’s characteristics (e.g.
age, educational status, previous pregnancies, etc.) at 12 weeks of pregnancy and on the child at
birth was obtained from questionnaires and medical records. The regional hospital ethics
committees approved this study. We obtained written informed consent from the participants
prior to inclusion (Guxens et al., 2012).
2.2 Biological sample collection
Maternal blood was collected on gestational week 12 (mean: 13.5; standard deviation (SD:
1.7)) by trained personnel that followed the same protocol in the two birth cohorts (Guxens et
al., 2012). Maternal plasma and serum samples were aliquoted in 1.5mL criotubes and stored at
-80ºC. Venous cord blood was collected at birth. Cord serum was aliquoted in 1.5mL glass
criotubes and stored at -80ºC.
2.3 PFAS determination
PFAS analysis was carried out at the Institute for Occupational Medicine, RWTH Aachen
University (Aachen, Germany).
2.3.1 Chemicals
We purchased PFBS, PFHxS and linear PFOS (L-PFOS; 50 mg/L MeOH each) also the labeled
internal standards sodium perfluoro-1-hexane [18O2]-sulfonate and sodium perfluoro-1-[1, 2,
3, 4-13C4]-sulfonate (50 mg/L MeOH) from Wellington laboratories (Ontario, Canada). PFNA
plus the labeled internal standards [13C9]-perfluorononanoate and [13C8]-perfluorooctanoate
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(50mg/L MeOH each) were purchased from Cambridge Isotopes (Tewksbury, MA, USA).
PFOA, 96% was purchased from Sigma-Aldrich (Taufkirchen, Germany). Ammonium acetate,
acetic acid (100%, extra pure), acetonitrile and water (all of high performance liquid
chromatography (HPLC)-grade) were obtained from Merck, Darmstadt, Germany.
2.3.2 Sample preparation
We defrosted frozen samples at room temperature. We then mixed the samples and transferred
250µl aliquots to 1.8mL glass screw-cap vials. We added 10µl of the working solution of the
labeled internal standards ([13C4]-PFOS, [13C8]-PFOA: 250 ng/mL water; [18O2]-PFHxS,
[13C9]-PFNA: 50 ng/mL water) to the samples. Then, we added 500µl of HPLC solvent B
(2mM ammonium acetate buffer pH 4 in acetonitrile) to precipitate the proteins. The samples
were vortex mixed and centrifuged at 800g for 5min. We transferred 300µl of the supernatant
to a new 1.8mL glass screw-cap vial and, then added 700µl of HPLC solvent A (2mM
ammonium acetate buffer pH 4 in water). We then injected 100µl aliquot into the column-
switching liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS)
system for quantitative analysis.
2.3.3 Online column-switching LC-MS-MS analysis
We based our online column-switching method on the works of Mosch et al., 2010 and adapted
it to the available chromatographic system and columns. Liquid chromatography was carried
out on an Agilent 1100 Series HPLC apparatus (auto sampler G 1313A, binary gradient pump
G 1312A, vacuum degasser G 1379A) and an additional isocratic Agilent G 1310A pump. The
Agilent G 1310A was used to load the sample (100µl) on a restricted access material (RAM)
phase, a LiChrospher® RP-8 ADS (25µm) 24 x 4 mm RAM from Merck (Darmstadt,
Germany) using a solution of 2mM ammonium acetate buffer pH 4 in water (solvent A) and
2mM ammonium acetate buffer pH 4 in acetonitrile (solvent B) (80:20, v/v) as the mobile
phase and a flow rate of 0.3mL/min. After this clean-up and enrichment step, we transferred the
analytes after 2min to a reversed-phase HPLC column (Luna C 8 (2) 150 x 4.6 mm, 3µm
particle size from Phenomenex, Aschaffenburg, Germany) in back-flush mode through a six-
port valve (Valco Systems, Houston, Texas, USA) time-controlled by the autosampler and a
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pump gradient starting from 70% solvent B for 7min, then raising to 100% B until 13min,
staying there until 17min and returning to 70% B until 20min.
We performed our tandem mass spectrometric detection on a Sciex API 3000 LC-MS-MS
system in ESI-negative mode. We used two specific mass transitions to determine the analytes.
The limit of quantification (LOQ), determined as a signal-to-noise-ratio of 6 in the vicinity of
the analytes was 0.20 ng/mL for PFBS, PFHxS, PFOS and PFOA and, 0.10 ng/mL for PFNA.
The limit of detection (LOD) was 0.10 ng/mL for PFBS, PFHxS, PFOS and PFOA and, 0.05
ng/mL for PFNA.
2.3.4 Calibration and Quality control
We carried the calibration by spiking bovine serum with the analytes in the range concentration
of 1–100 ng/mL for PFOA and PFOS; as well as 0.1–10 ng/mL for PFBS, PFHxS and PFNA.
Quality control was prepared by spiking bovine serum at a 4 ng/mL concentration for PFOA
and for PFOS as well as 0.4 ng/mL for PFBS, PFHxS and PFNA. Moreover, we used the
aliquoted plasma of a 41-year-old German male for additional quality control. This was
included in every analytical series.
The between day imprecision for the spiked bovine samples (n=42) ranged from 6.4% for
PFOA (4 ng/mL) to 12.6% for PFHxS (0.4 ng/mL). In the human plasma sample, PFBS was
not detectable, the between day imprecision ranged from 8.7% for PFHxS (0.7 ng/mL) to
11.1% for PFNA (0.7 ng/mL). We participated biannually in successful round robins for the
determination of PFOS and PFOA in plasma organized in Germany (www.g-equas.de). During
the study period, certified material from this round robin was included in every analytical series
with all results in the acceptable range.
2.4 Statistical analysis
Our descriptive analyses included the median and the maternal to cord ratios (i.e. maternal
plasma-cord serum and, maternal serum-cord serum) for each PFAS. We substituted the values
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under the LOD with LOD/2. We used the non-parametric Mann–Whitney U test to compare
PFAS concentrations and ratios in maternal plasma and serum and, to compare PFAS
concentrations and ratios in maternal blood and cord serum (i.e. maternal plasma-cord serum,
and maternal serum-cord serum). Due to a skewed distribution in PFAS concentration, we
calculated the Spearman’s rank correlations coefficients (rho) to assess the correlations
between PFAS concentrations in maternal and cord samples (i.e. maternal plasma-maternal
serum, maternal plasma-cord serum, and maternal serum-cord serum). We used the maternal to
cord ratios to estimate the proportion of PFAS that crosses the placenta. We assessed if the
maternal to cord ratio varied by maternal characteristics (i.e. age, BMI, parity and, education)
and the sex of the child. For this we used linear regression models with the log10-transformed
PFAS maternal to cord ratio as the dependent variable. We interpreted the exponentiated beta
coefficients as the change in the geometric mean (GM) of the maternal to cord ratio (i.e. GM
ratio<1 meant higher PFAS transfer from the mother to fetus). P-values less than 0.05 (two-
sided) were considered statistically significant. For our statistical analysis we used STATA
version 12 (Stata Corporation, College Station, Texas).
3. Results
Women in our subsample were 32.1 (SD: 4.8) years of age on average, 42% were multiparous
and 29% had university studies (Table 1). We did not detect PFBS in any of the samples so it
was excluded from the analysis. PFHxS was detected in all but 2% and 4% of maternal plasma
and serum samples, respectively and, 12% of cord serum samples. Whereas PFOS, PFOA and
PFNA were detected in every maternal and cord sample. Table 2 describes PFAS
concentrations. PFAS concentrations were higher (p-values<0.001) in maternal plasma and
maternal serum than in cord serum samples (Table 2). PFOS was the most abundant PFAS in
maternal samples (maternal plasma p50= 6.18 ng/mL; interquartile range (IQR): 3.76; serum
p50= 6.99 ng/mL; IQR: 3.37) followed by PFOA (maternal plasma p50= 2.85 ng/mL; IQR:
1.71; serum p50= 2.97 ng/mL; IQR: 1.80). Meanwhile, PFOA was the most abundant in cord
serum samples (p50= 1.90 ng/mL; IQR: 1.26) followed by PFOS (p50= 1.86 ng/mL; IQR:
1.04). PFAS median concentrations in maternal plasma were slightly lower than in maternal
serum, although not statistically significant (data not shown).
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Spearman’s rank correlations between maternal plasma and serum were high (rho>0.80) for all
PFAS (Figure 1 and, Appendix Table A1). Positive Spearman’s rank correlations (p-
values<0.05) were also found between maternal samples and cord serum samples (rho>0.66),
but PFOA (rho=0.74) correlated the highest in comparison to rest of PFAS (see appendix Table
A1). Sensitivity analyses were done taking out extreme values for PFOS, PFOA (Figure 1) and
PFNA (see appendix Figure A1), and correlations remained positive and statistically significant
(data not shown). Moreover, all PFAS showed positive correlations between them; however the
highest correlation was seen between PFOA and PFNA (rho>0.70) in maternal plasma and
serum as well as in cord samples (see supplementary Table A2).
All maternal to cord ratios were above one (Table 2). Maternal to cord ratios were higher for
PFAS with longer carbon chain (i.e. PFOS and PFNA) than for PFAS with shorter carbon chain
(i.e. PFHxS and PFOA) (Figure 2). The maternal to cord ratios were lower for the sulfonates
(i.e. PFHxS and PFOS) than for carboxylates (i.e. PFOA and PFNA) (Figure 2). These ratios
were similar for maternal plasma-cord serum and, for maternal serum-cord serum (Table 2).
Maternal to cord ratios decreased with increasing age of the mother in the adjusted model,
especially for PFOS (GM ratio: 0.98; CI 95%: 0.96, 0.99) and PFNA (GM ratio: 0.97; CI 95%:
0.95, 0.98) (Table 3). These results were similar in nulliparous vs. multiparous women. We also
saw a decrease in PFNA maternal to cord ratio in girls compared to boys (GM ratio: 0.80; CI
95%: 0.69, 0.93) (Table 3). We saw no association with maternal BMI, parity or education
(Table 3).
4. Discussion
In this study we detected four different PFAS in maternal and fetal blood samples from a
Spanish birth cohort. Our median PFAS concentrations in both maternal and fetal matrices
tended to be higher than previous recent studies (Fromme et al., 2010; Hanssen et al., 2010;
Porpora et al., 2013), but lower than studies conducted before 2003 (Fei et al., 2007; Midasch
et al., 2007) (see Table 4). Differences in the year of sample collection should be considered
when interpreting our results. Our PFOS levels are lower than those of Fei et al., (2007)
probably because they collected their samples between the years 1996-2002. In INMA, we
collected samples during 2003-2006, at least one year after the PFOS voluntary phase-out by
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the major US worldwide producer in 2002 (EPA, 2000). Thus studies using samples collected
before or close to the year 2002 might show higher PFOS levels (Fei et al., 2007; Hanssen et
al., 2013; Midasch et al., 2007) than ours because less sources of exposure should be available
after the phase-out of PFOS. PFOS is being replace by PFBS because the latter exhibits a much
shorter half-life in humans (26 days vs. 5 years) (Olsen et al., 2009). PFBS was not detected in
our samples, probably because these were collected approximately 10 years ago when it was
not yet widely used (Wang et al., 2014). Further, the LOD for PFBS in our study may have
been too high. For example Glynn et al detected concentrations as low as 0.02 ng/mL, 5 times
less than our LOD (Glynn et al., 2012). Hence future studies with a more accurate LOD are
needed in order to understand PFBS distribution and evolution in recent years.
PFAS concentrations in our sample distributed evenly between maternal plasma and maternal
serum, showing a 1:1 concentration ratio. This supports the use of either maternal plasma or
maternal serum as a proxy to PFAS fetal exposure. Only one other study has assessed whether
PFAS concentrations were similar between both biological compartments from the same
individual and concluded that PFHxS, PFOS and PFOA distributed evenly in plasma and serum
from adults workers (Ehresman et al., 2007). Our results are in line with this previous study,
and we also added new information regarding PFNA. Thus results from studies measuring
PFAS in plasma and serum can be compared directly
PFAS concentrations in maternal plasma and serum at the first trimester of pregnancy
correlated well with PFAS concentrations in cord serum at birth, confirming that either
maternal blood compartments could be used as proxy for PFAS fetal exposure. This confirms
results from other studies comparing maternal and cord samples (see Table 4). However,
because PFAS concentrations decline during pregnancy (Fei et al., 2007; Fromme et al., 2010),
the time of sample collection during pregnancy could influence the comparison of
concentrations of PFAS in maternal blood between studies. Our maternal samples were
collected early in pregnancy (around gestational week 12) whereas most of the studies
comparing matched maternal-cord samples have collected them later in pregnancy (see Table
4). Glynn et al., (2012) suggested that prenatal exposure to PFOS, PFOA and PFNA is best
estimated using maternal blood samples collected prior to delivery or shortly after. If so, then it
is possible that our maternal to cord ratios underestimate PFAS transfer efficiency across the
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placenta since some hematological changes during pregnancy have not yet fully occurred at
gestational week 12, e.g. total volume expansion or differences in renal clearance during
pregnancy (Beesoon et al., 2011; Monroy et al., 2008). Nonetheless, we found good
correlations between our maternal and cord samples for PFHxS, PFOS, PFOA and PFNA,
indicating that maternal samples collected early in pregnancy can also be used to assess
prenatal exposure to PFAS in epidemiological studies. This can be an advantage over cord
blood given the logistic problems in its collection at the time of delivery (e.g. having onsite
personnel) or the lack of available archived samples, which is common in cohort studies.
Furthermore, since confounding by glomerular filtration rates may be an issue in
epidemiological studies and filtration rates increases during pregnancy (Morken et al., 2014;
Verner et al., 2015), samples collected earlier in pregnancy may indeed be preferable to those
collected later.
We used maternal to cord ratios to estimate the placental transfer efficiency of PFAS (Beesoon
et al., 2011). Maternal to cord ratios were lower for PFOA than for the other PFAS. Previous
studies have suggested that the carboxylic active group of PFOA might enable it to bind more
strongly to the protein fraction in the blood (albumin) than the other PFAS (Apelberg et al.,
2007a; Fromme et al., 2010). To this topic, Hanssen et al., (2013) reported that carboxylates
(i.e. PFOA and PFNA) transferred more efficiently across the placenta than sulfonates (i.e.
PFHxS and PFOS). Our findings also suggest that PFOA is more efficiently transferred across
the placenta than the other PFAS. PFAS transfer efficiency has further been related to the
carbon chain length. Shorter chained PFAS seem to transfer more easily across the placenta
than longer chained PFAS. For instance, Kato et al., (2014) recently reported a U-shape trend
for PFAS transfer across the placenta with increasing chain length. Although we cannot see an
exact U-shape trend because we did not measure as many PFAS as Kato et al. (2014) did, our
results are in line with this previous study especially when PFAS are divided into carboxylates
and sulfonates. The PFAS carbon chain length probably also plays a role in their hydrophobic
capacity so that the more hydrophobic PFAS (PFOS>PFHxS>PFNA>PFOA) (Arp et al., 2006)
were more retained in the maternal tissues and less transferred to the fetus; this is also
supported by our transfer ratios. More studies confirming these mechanisms are needed.
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In our study PFAS maternal to cord ratios seemed to slightly decrease with increasing age of
the mother, especially for PFOS and PFNA, suggesting a higher transfer efficiency with
increasing age. Moreover, PFNA placental transfer was higher in girls than in boys. Maternal
BMI, parity and, education did not influence the ratios. Changes in the physiology of the
placenta with increasing age are plausible (Reynolds et al., 2010) and may explain our findings.
However, due to our small sample size our results might not be generalizable to other
populations.
This study has some limitations. Firstly, we have a small sample size that might not be
generalizable to the general population. Secondly, we have only quantified the linear isomers,
which make difficult the comparison with studies that have assessed branched PFOS isomers.
This could be of special relevance if the linear isomers differ from the branched isomers in their
transfer across the placenta, thus we might have underdetected PFOS concentration in our
samples. Finally, our results indicated that sulfonates with longer carbon chains are less
transferred across the placenta than carboxylates with shorter carbon chains (i.e.
PFOS>PFHxS) and, PFNA>PFOA, respectively). However, PFAS carbon chain length and
active group might play a role in PFAS ability to bind to albumin in blood (Ng and
Hungerbühler, 2014) but we do not have information on albumin levels in our sample.
5. Conclusions
In this Spanish birth cohort we detected PFHxS, PFOS, PFOA and PFNA in maternal blood
(serum and plasma) and cord serum samples with good correlations between maternal-cord
matrices, confirming that PFAS transfer across the placenta. PFAS placental transfer was
higher with increasing maternal age. Our study suggests that maternal plasma or serum samples
collected early in pregnancy are good proxies to assess the fetal body burden of PFAS in
epidemiological studies.
6. Conflict of interest statement
There is no conflict of interest in our study.
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7. Acknowledgements
This study was funded in part by grants from the European Union (FP7-ENV-2011 cod 282957
and HEALTH.2010.2.4.5-1), and from Spain: Instituto de Salud Carlos III and The Ministry of
Health (Red INMA G03/176, CB06/02/0041, FIS- PI12/01890, FIS-PI041436, FIS- PI081151,
FIS-PI06/0867, FIS-PS09/00090 FIS-FEDER 03/1615, 04/1509, 04/1112, 04/1931, 05/1079,
05/1052, 06/1213, 07/0314, 09/02647, 11/0178, 11/01007, 11/02591, 11/02038, 13/1944,
13/2032, 14/00891, 14/01687 and pre-doctoral grant PFIS 2014), the Conselleria de Sanitat,
Generalitat Valenciana, Department of Health of the Basque Government (2005111093 and
2009111069), the Provincial Government of Gipuzkoa (DFG06/004 and DFG08/001) and the
Generalitat de Catalunya-CIRIT (1999SGR 00241). We would particularly like to thank all the
participants for their generous collaboration.
A full roster of the INMA Project Investigators can be found at:
http://www.proyectoinma.org/presentacion-inma/listado-investigadores/en_listado-
investigadores.html
8. Appendix
1) Summary of the characteristic of the participants not included in the present study; 2)
Spearman’s rank correlations for the detected PFAS concentration levels between maternal
matrices (plasma and serum) and cord serum; 3) Spearman’s rank correlation between PFAS in
maternal blood (plasma and serum) and cord serum samples; 4) a figure on PFHxS and PFNA
concentration in maternal blood (plasma and serum) and cord serum samples; 5) a figure of
PFAS maternal to cord ratios and; 6) certified material from PFAS round robins, are available
in the Appendix A.
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Tables and Figures
Table 1. Summary of the participants included and not included in the present study in INMA,
2003-2006.a
CharacteristicsSubjects
included (n=66)Subjects not
included (n=1,022)
Maternal age (years), mean (sd) 32.1 (4.8) 31.4 (4.5)
Maternal BMI (kg/m2), mean (sd) 23.7 (4.2) 23.8 (4.6)
Smoking during pregnancy (yes), n (%) 21 (32) 343 (34)
Alcohol consumption during pregnancy (yes), n (%)
17 (26) 237 (23)
Parity (multiparous), n (%) 28 (42) 455 (45)
Maternal education (university), n (%) 19 (29) 269 (26)
Birth weight (g), mean (sd) 3269 (516) 3259 (456)
Sex of the child (girl), n (%) 31 (47) 483 (47)a BMI: Body mass index; SD: standard deviation.
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Table 2. Concentrations (ng/mL) of PFAS in maternal blood (serum and plasma) and in cord
serum samples, 2003-2006. a
N%<LO
DMin p25 p50 p75 p95 Max
M:C RatioGM (95% CI)
Maternal plasma
PFHxS 66 2
0.05
0.55
0.79
1.16 2.03 2.58 2.35 (2.05, 2.70)
PFOS 66 0
1.46
4.44
6.18
8.20
12.63 38.58 3.33 (3.05, 3.62)
PFOA 66 0
0.78
1.87
2.85
3.57 6.00 11.93 1.37 (1.27, 1.48)
PFNA 66 0
0.23
0.61
0.84
1.10 1.73 5.51 2.39 (2.18, 2.63)
Maternal serum
PFHxS 53
4 0.05
0.65
0.84
1.26 2.03 2.53 2.24 (1.94, 2.59)
PFOS 53
0 1.17
4.47
6.99
7.84
11.12 23.14 3.35 (3.04, 3.70)
PFOA 53
0 0.86
2.26
2.97
4.05 4.85 14.54 1.34 (1.23, 1.47)
PFNA 53
0 0.20
0.68
0.85
1.10 1.72 5.37 2.50 (2.26, 2.76)
Cord serum
PFHxS 66
12 0.05
0.27
0.40
0.52 0.90 1.93
PFOS 66
0 0.53
1.40
1.86
2.43 3.70 4.71
PFOA 66
0 0.60
1.45
1.90
2.70 4.70 10.56
PFNA 66
0 0.13
0.24
0.32
0.51 0.81 2.24
Abbreviations: LOD: limit of detection; M:C: maternal to cord; GM: geometric mean; CI:
confidence interval; p: percentile; Min: minimum; Max: maximum; PFHxS: perfluorohexane
sulfonate; PFOS: perfluorooctane sulfonate; PFOA: perfluorooctanoate; PFNA:
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perfluorononanoate. a Perfluorobutane sulfonate (PFBS) is not included because it was not
detected in any sample.
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Figure 1. PFOS and PFOA levels (ng/mL) in maternal blood (plasma and serum) and cord
serum samples, 2003-2006. a
0.00
1.00
2.00
3.00
4.00
5.00
0.00 5.00 10.00 15.00 20.00Maternal plasma
0.00
1.00
2.00
3.00
4.00
5.00
0.00 5.00 10.00 15.00 20.00Maternal serum
PFOS
1.00
2.00
3.00
4.00
5.00
0.00 2.00 4.00 6.00 8.00Maternal plasma
1.00
2.00
3.00
4.00
5.00
0.00 2.00 4.00 6.00 8.00Maternal serum
PFOA
Abbreviations: PFOS: perfluorooctane sulfonate; PFOA: perfluorooctanoate. a Two extreme
values are not included in the plots (1 for PFOS and 1 for PFOA). All correlations were
statistically significant (p-values<0.05).
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Figure 2. PFAS transfer eficciency from mother to fetus by carbon chain lenght in 66 matched
maternal-cord pairs, 2003-2006. a, b
Abbreviations: PFHxS: perfluorohexane sulfonate; PFOS: perfluorooctane sulfonate; PFOA:
perfluorooctanoate; PFNA: perfluorononanoate. a Higher maternal to cord ratios means lower
transfer efficiency. b Color legend: blue line are the PFAS sulfonates (i.e. PFHxS and PFOS)
and, red line are the PFAS carboxylates (i.e. PFOA and PFNA).
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Table 3. Association between PFAS maternal to cord ratios (transfer efficiency) and maternal characteristics and sex of the child, 2003-2006.
Covariates PFHxS PFOS PFOA PFNA GM ratio (CI95%)a GM ratio (CI95%)a GM ratio (CI95%)a GM ratio (CI95%)a
Maternal age 0.98 (0.95, 1.02) 0.98 (0.96, 0.99)c 0.98 (0.96, 1.00) 0.97 (0.95, 0.98)c
If Nulliparous b 0.99 (0.94, 1.04) 0.97 (0.94, 1.01) 0.99 (0.96, 1.02) 0.97 (0.94, 1.00)If Multiparous b 0.97 (0.92, 1.03) 0.97 (0.96, 0.99)c 0.97 (0.95, 0.99)c 0.97 (0.95, 0.99)c
Maternal BMI 1.00 (0.97, 1.04) 0.99 (0.97, 1.01) 1.00 (0.98, 1.02) 1.00 (0.98, 1.02)Parity
Nulliparous Reference Reference Reference ReferenceMultiparous 1.01 (0.74, 1.39) 1.02 (0.86, 1.22) 1.05 (0.89, 1.25) 1.03 (0.87, 1.22)
Maternal education None or primary Reference Reference Reference ReferenceSecondary 0.77 (0.53, 1.12) 0.95 (0.77, 1.17) 1.08 (0.89, 1.32) 0.97 (0.79, 1.18)University 0.76 (0.51, 1.14) 0.89 (0.71, 1.12) 0.97 (0.78, 1.21) 0.94 (0.76, 1.17)
Sex of the childBoy Reference Reference Reference ReferenceGirl 0.91 (0.68, 1.20) 0.86 (0.73, 1.01) 0.89 (0.77, 1.04) 0.80 (0.69, 0.93)c
Abbreviations: PFHxS: perfluorohexane sulfonate; PFOS: perfluorooctane sulfonate; PFOA: perfluorooctanoate; PFNA: perfluorononanoate;
GM: geometric mean; BMI: body mass index; CI: confidence interval; Ref.: reference group. a Interpretation of the GM ratio: GM ratio<1 means
higher transfer of PFAS from the mother to the fetus. The GM ratios shown are the exponentiated coefficients from a multivariate linear
regression model adjusted for: maternal age, BMI, parity, education and, sex of the child. The dependent variable was the log transformed PFAS
maternal to cord concentration ratios. b We stratified the analysis by parity (i.e. nulliparous vs. multiparous). c Statistically significant if p-
values<0.05.
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Table 4. Previous studies that have assessed PFAS in paired maternal-cord blood samples.
Country NSampling
year Matrix (timing)
Concentration in ng/mL(M:C ratio; CI95%) a
PFHxS PFOS PFOA PFNA
Spain 53-66 2003-06 MP, MS (12w) Median: 0.79, 0.84 Median: 6.18, 6.99 Median: 2.85, 2.97 Median: 0.84, 0.85(present study) CS (birth) Median: 0.40 Median: 1.86 Median: 1.90 Median: 0.32
2.35 (2.05, 2.70) 3.33 (3.05, 3.62) 1.37 (1.27, 1.48) 2.39 (2.18, 2.63)
Canada 20 2007-08 MS (15w) Mean: 1.70 Mean: 5.50 Mean: 1.80 Mean: 0.90Beesoon et al., 2011 CS (birth) Mean: 0.70 Mean: 1.80 Mean: 1.10 Mean: 0.40
(3.0) b (1.66) b
Denmark 501996-2002 MB (1st & 2nd trimester) Mean: 35.30 Mean: 5.60
Fei et al., 2007 CB (birth) Mean: 11.00 Mean: 3.70
(Range: 2.96-3.40) (Range: 1.46-1.83)
Germany 33-44 2007-09 MB (34-37w; delivery) Median: 0.50, 0.50 Median: 3.20, 3.20 Median: 2.40, 1.90 Median: 0.60, 0.60Fromme et al., 2010 CB (birth) Median: 0.20 Median: 1.0 Median: 1.40 Median: <0.40
(3.0) b (1.4) b
South Africa 58-71 2005-06 MS (delivery) Median: 0.50 Median: 1.60 Median: 1.30 Median: 0.50Hanssen et al., 2010 CB (birth) Median: 0.30 Median: 0.70 Median: 1.30 Median: 0.20
(2.10; Range: 0.30, 9.90) (2.20; Range: 0.50, 7.90) (1.40; Range: 0.20,7.10)
Russia 7 2001-02 MP (delivery) Median: 0.26 Median: 11.0 Median: 1.61 Median: 0.6031
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Hanssen et al., 2013 CP (birth) Median: 0.07 Median: 4.11 Median: 1.00 Median: 0.29
Japan 15 2003 MB (38-41w) Range: 4.90-17.60 Range: <0.50-2.30Inoue et al., 2004 CB (birth) Range: 1.60-5.30 <LOD
USA 71 2003-06 MS (16w; delivery) Median: 1.20, 1.20 Median: 12.70, 8.50 Median: 4.80, 3.30 Median: 0.82, 0.66Kato et al., 2014 CS (birth) Median: 0.60 Median: 3.50 Median: 3.10 Median: 0.41
(2.15; 1.94, 2.38) (3.49; 3.28, 3.71) (1.72; 1.65, 1.80) (2.03; 1.92, 2.15)
(1.70; 1.54, 1.89) (2.47; 2.32, 2.63) (1.20; 1.15, 1.26) (1.56; 1.47, 1.65)
Korea 20 2007 MS (delivery) AM: 0.89 AM: 5.60 AM: 1.60 AM: 0.79Kim et al., 2011 CS (birth) AM: 0.58 AM: 2.00 AM: 1.10 AM: 0.37
(1.55) b (2.77) b (1.44) b (2.13) b
Germany 11 2003 MP (delivery) Median: 13.00 Median: 2.60Midasch et al., 2007 CP (birth) Median: 7.30 Median: 3.40
(1.74) b (0.82) b
Canada 101-105 2004-05 MS (24-28w; delivery) Median: 1.82, 1.62 Median: 16.6, 14.54 Median: 2.13, 1.81 Median: 0.73, 0.69Monroy et al., 2008 CB (birth) Median: 2.07 Median: 6.08 Median: 1.58 Median: 0.72Faroe Islands 12 2000 MB (32w) Median: 12.30 Median: 19.70 Median: 4.20 Median: 0.76Needham et al., 2011 CB (birth) Median: 9.10 Median: 6.60 Median: 3.10 Median: 0.37
(1.34) b (2.90) b (1.38) b (2.00) b
Sweden 237 1978- MS (delivery) <LOD Median: 15.0 Median: 2.10 Median: 0.24321
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2001Ode et al., 2013 CS (birth) <LOD Median: 6.50 Median: 1.70 Median: 0.20
(2.30) b (1.23) b (1.20) b
Italy 38 2008-09 MS (delivery) Median: 2.90 Median: 2.40Porpora et al., 2013 CS (birth) Median: 1.10 Median: 1.60
Abbreviations: PFHxS: perfluorohexane sulfonate; PFOS: perfluorooctane sulfonate; PFOA: perfluorooctanoate; PFNA: perfluorononanoate;
LOD: limit of detection; M:C: maternal to cord ratios; CI: confidence interval; AM: arithmetic mean; Range: minimum and maximum
concentrations a Only showing maternal to cord ratios (in brackets) if they were available in the original manuscript. b Approximated conversions
of maternal to cord ratios are showed.
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