Reproductive Toxicology 59 (2016) 89–95

Contents lists available at ScienceDirect

Reproductive Toxicology
journal homepage: www.elsevier.com/locate/reprotox

Bisphenol A and other phenols in human placenta from children with

cryptorchidism or hypospadias
Mariana F. Fernández a,b,∗ , Juan P. Arrebola a,b , Inmaculada Jiménez-Díaz a,c ,
José María Sáenz a , José Manuel Molina-Molina a,b , Oscar Ballesteros c ,
Andreas Kortenkamp d , Nicolás Olea a,b
a
Instituto de Investigación Biosanitaria (ibs.GRANADA), University of Granada, San Cecilio University Hospital, 18071 Granada, Spain
b
CIBER en Epidemiología y Salud Pública (CIBERESP), Madrid, Spain
c
Research Group of Analytical Chemistry and Life Sciences, Department of Analytical Chemistry, University of Granada, Granada, Spain
d
Institute of the Environment, Health and Societies, Brunel University, Kingston Lane, Uxbridge, UB8 3PH, UK

a r t i c l e i n f o a b s t r a c t

Article history: Embryo-foetal exposure to low doses of endocrine disrupting chemicals (EDCs) has been related to repro-
Received 25 May 2015 ductive tract diseases in experimental animals but not convincingly in human populations. The aim of
Received in revised form 12 October 2015 this case—control study was to explore the relationship between exposure to non-persistent EDCs during
Accepted 2 November 2015
pregnancy and male genital development. Exposure to bisphenol-A (BPA), benzophenones (BPs) [BP-1,
Available online 19 November 2015
BP-2, BP-3, BP-6, BP-8 and 4-hydroxybenzophenone (4-OH-BP),] and parabens (PBs) [methyl-, ethyl-,
propyl- and butyl-PB] was analyzed by means of ultra-high performance liquid chromatography-tandem
Keywords:
mass spectrometry in placenta samples from a subsample of 28 cases and 51 healthy controls nested in a
Endocrine disrupting chemicals
Cryptorchidism
cohort of newborns recruited between 2000 and 2002. The multivariable regression analyses indicated a
Hypospadias statistically significant association between exposure to BPA and propyl-PB and the risk of malformations
Placenta [adjusted odd ratio (95% CIs) in the third tertile of exposure: 7.2 (1.5–35.5) and 6.4 (1.2–35.5) for BPA and
Foetal exposure propyl-PB, respectively].
BPA © 2015 Elsevier Inc. All rights reserved.
Parabens
Benzophenones

1. Introduction synthesis at background concentrations [3,6]. Some BPs, frequently

Biomonitoring studies have shown that most human pop-
ulations are continuously exposed to both persistent and
non-persistent environmental pollutants. For example, detectable
levels of Bisphenol A (BPA), parabens (PBs) and benzophenones
(BPs) have been found in different tissues of people from several
areas around the world [1,2]. BPA is a well-known endocrine-
disrupting chemical (EDC) [3,4]. In vitro studies have shown that
BPA exhibits estrogenic effects mediated by both genomic and
epigenomic estrogen-response mechanisms at very low doses [5].
Moreover, BPA could disrupt thyroid hormone action, cause pro-
liferation of human prostate cancer cells and block testosterone
Abbreviations: EDCs, endocrine disruptor chemicals; BPA, bisphenol A;
BP, benzophenone; BP-1, benzophenone-1; BP-2, benzophenone-2; BP-3,
benzophenone-3; BP-6, benzophenone-6; BP-8, benzophenone-8; 4-OH-BP, 4-
hydroxybenzophenone; PB, paraben; Methyl-PB, methyl paraben; Ethyl-PB, ethyl
paraben; Propyl-PB, propyl paraben; Butyl-PB, butyl paraben.
∗ Corresponding author at: University of Granada, Instituto de Investigación
Biosanitaria (ibs.GRANADA), Granada, Spain. Fax: +34 958 249953.
E-mail address: marieta@ugr.es (M.F. Fernández).
used as UV-filters in body-creams and in food packaging materials
[7], have been classified as potential EDCs. Thus, benzophenone-1
(BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3) and 4-
hydroxybenzophenone (4-OH-BP) have shown estrogenic and/or
anti-androgenic activity in various species [8,9]. Some parabens,
alkyl esters of p-hydroxybenzoic acid, which are widely used as
antimicrobial preservatives in all types of personal care product
[10] and in food and beverage processing [11], have demonstrated
estrogenic properties in in vitro and in vivo studies [12,13] as well
as antiandrogenic effects [14], with reports of decreased semen
quality and testosterone levels in parabens-exposed male rodents
[15].
Male sexual differentiation and reproductive functioning are
critically dependent on a balanced androgen:estrogen ratio
[16–18]. Thus, exposure of the developing male foetus to EDCs may
interfere with reproductive tract differentiation and development
and be responsible for sexual maturation and reproductive func-
tion anomalies in adult life [19,20]. In this regard, two common
male reproductive-tract malformations—cryptorchidism (failure of
one or both testicles to descend into scrotum) and hypospadias
(urethral opening on ventral side of penis)—are birth defects of

http://dx.doi.org/10.1016/j.reprotox.2015.11.002
0890-6238/© 2015 Elsevier Inc. All rights reserved.
90 M.F. Fernández et al. / Reproductive Toxicology 59 (2016) 89–95
prenatal origin that may be related to in utero exposure to estro-
gens/androgens.
Because BPA, PBs and BPs are non-persistent chemicals with
short half-lives that are rapidly metabolized and excreted in urine
after exposure, urinary concentrations are commonly used in epi-
demiological studies to estimate exposure to these compounds
[2,4,10,21]. Alternative matrices, such as placenta tissue, might pro-
vide a more accurate measure of direct foetal exposure [22,23]. Our
research group recently developed a new multiclass ultra-high-
performance liquid chromatography-tandem mass spectrometry
(UHPLC–MS/MS) method to determine BPA, six BPs (BP-1, BP-
2, BP-3, BP-6, BP-8 and 4-OH-BP) and the most widespread PBs
(methyl-PB, ethyl-PB, propyl-PB and butyl-PB) in human placental
tissue samples [24].
The aim of this study was to estimate intrauterine exposure to
non-persistent EDCs (BPA, BPs and PBs) by measuring placental
exposure to these compounds in a case-control study nested in a
prospective birth cohort, and to evaluate the risk of cryptorchidism
and/or hypospadias (male urogenital malformations) related to this
exposure.
2. Material and methods
2.1. Study design and population
From October 2000 to July 2002, a prospective birth cohort with
668 mother–son pairs was established at the San Cecilio University
Hospital of Granada (one of the two reference public hospitals serv-
ing Granada province in Southern Spain), recruiting male newborns
registered at the Hospital. A case-control study was nested in the
cohort for investigation of the main risk factors for male urogenital
malformations.
Inclusion criteria for mothers were: single delivery, signing of
informed consent to participate and complete a questionnaire.
Exclusion criteria for mothers were: the presence of severe chronic
disease (e.g., diabetes, hypertension or thyroid pathology), the
development of any pregnancy complication that could affect foetal
growth or development and non-residency in the hospital refer-
ral area. All boys with urogenital malformations (cryptorchidism
and/or hypospadias) born in the study period were included [19].
Mothers were of Caucasian origin. All boys in the cohort
were examined at birth (within 2 days), and those diagnosed
with cryptorchidism and/or hypospadias were re-examined at 1
month of age. Only boys with congenital malformations at re-
examination were considered cases. The examination technique
and definition of cryptorchidism and hypospadias followed the
recommendations of a Danish–Finnish study [25], and all details
were previously reported [19]. Fifty boys were diagnosed with
cryptorchidism and/or hypospadias; two of these were excluded
from the investigation because their parents refused to partici-
pate. A maximum of three controls were selected for each case,
matched for gestational age (±1 week), date of birth (±7 days),
and parity (primiparous/multiparous). Although the case-control
ratio was 1:3, only 114 controls met the matching criteria. Cases
were: 27 boys born with undescended testes (19 unilaterally, 8
bilaterally) that persisted until 30 days of age, 19 boys born with
hypospadias, and 2 boys born with both malformations. Among
cryptorchidism cases, 17 were severe and 12 were mild cases, clas-
sifying non-palpable inguinal and/or suprascrotal cases as severe
and high scrotal cases as mild.
Out of the 50 cases of cryptorchidism and/or hypospadias and
114 matched controls, adequate biological samples were available
for the measurement of BPA and phenols from 28 cases and 51
control newborns that were selected for the final study. Missing
samples corresponded to samples with insufficient volume after
other assays had been performed. Cases were 15 boys born with
undescended testes (9 unilaterally, 6 bilaterally), 12 boys born with
hypospadias and 1 boy born with both malformations.
Information on potential confounding variables related to par-
ents, pregnancy/delivery and activities with potential for chemical
exposure were gathered from structured face-to-face interviews
with the mother within the first 48 h after delivery. The interviewer
was blinded to the case or control status of the child. The study
was approved by the Institutional Ethical Committee of the San
Cecilio University Hospital, and all participating mothers signed
informed consent. All subject information was coded to maintain
strict confidentiality.
Comparison of the cases and controls in the final sample with
those in the original cohort found no statistically significant dif-
ferences in maternal age, body mass index, smoking habit, weeks
of gestation, water consumption, residence, occupation or newborn
weight at delivery. However, there was a larger proportion of moth-
ers reporting historical (pre-pregnancy) use of oral contraceptives
in the selected versus non-selected cases (21% vs. 53%, p = 0.034)
although not in the selected versus non-selected controls (37% vs.
42%, p = 0.686).
2.2. Placenta sample collection and storage
Placentas were collected immediately after delivery at the
Maternity Unit. Each placenta was examined, weighed and frozen at
−86 ◦ C within 90 min of its collection. In order to ensure the homo-
geneity and representativeness of the whole placenta tissue, half of
the placenta (including maternal and foetal sides and central and
peripheral parts) was placed in the glass container of a mixer for its
homogenization (B-400 Buchi mixer). Once homogenized, aliquots
of 25 g together with the remaining half of the placenta were stored
and frozen at −86 ◦ C in the hospital bio-bank.
Placental tissue aliquots were additionally homogenized using
an ultrasonic spindle. The container was placed in a glass full of ice
in order to avoid sample heating and the spindle was in direct con-
tact with the placental tissue. Ultrasound setting consisted in pulses
of 30 s followed by 30 s without sonication, for a total of 5 min
of effective radiation. Once homogenized, samples were frozen at
−86 ◦ C and stored until their analysis in our laboratory.
2.3. Preparation/extraction protocol
All human placenta samples were simultaneously analyzed for
free BPA, BPs and PBs. Placenta tissue samples were not treated
with enzymes to deconjugate possible conjugated compounds. An
aliquot of placental tissue sample (1.5 g) was placed into an 8 mL
glass vial, fortified with 200 ␮L of methanol containing 100 ng/mL
of BPA-d16 and shaken for 10 min. Once fortified, the sample was
homogenized with 1.5 mL of deionized water and shaken for 1 min.
The homogenate was extracted by adding 3 mL of ethyl acetate and
shaken again for 10 min, and the mixture was then centrifuged for
10 min at 5000 rpm (4050 × g). The underlying organic layer was
transferred to a clean glass vial and evaporated to dryness at room
temperature under a nitrogen stream. The residue was dissolved in
a mixture of 100 ␮L of 0.1% (v/v) ammonia in methanol and 100 ␮L
of 0.1% (v/v) ammoniacal aqueous solution and shaken vigorously.
The extract was placed in a 1.5 mL Eppendorf tube and centrifuged
for 35 min at 16,500 rpm (24,960 × g) and finally, prior to its injec-
tion into the LC system, the extract was filtered through a 0.20 ␮m
(4-mm diameter) non-sterile regenerated cellulose filter.
2.4. UHPLC-tandem mass spectrometry conditions
Samples were analyzed in triplicate. Analyses were performed
using an Agilent 1200 series (Agilent Technologies Inc., Palo Alto,
 M.F. Fernández et al. / Reproductive Toxicology 59 (2016) 89–95
CA, USA) ultra-high-performance liquid chromatography system
equipped with a binary pump, a vacuum membrane degasser,
a thermostated column compartment, an automatic autosam-
pler and an automatic injector and connected on line to an API
2000 (Applied Biosystems, Foster City, CA, USA) triple quadrupole
mass spectrometer with atmospheric pressure chemical ionization
(APCI) interface.
Chromatographic separation of compounds was performed
using a Gemini C18 column (100 mm × 2 mm i.d., 3 ␮m particle
size) from Phenomenex (Torrance, CA, USA). The standards and
samples were separated using a gradient mobile phase consisting
of 0.1% (v/v) ammoniacal aqueous solution (solvent A) and 0.1%
(v/v) ammonia in methanol (solvent B). Gradient conditions were:
0–3.5 min, 60% B; 3.5–4.0 min, 60–100% B; 4.0–6.5 min, 100% B and
return to 60% in 0.5 min. Flow rate was 0.25 mL/min, injection vol-
ume 40 ␮L and the column temperature was maintained at 40 ◦ C.
Total run time was 8.0 min and the post-delay time for recondition-
ing the column with 60% B was 3.5 min.
APCI ionization was performed in the negative ion mode. The
tandem mass spectrometer was operated in the multiple reaction
monitoring (MRM) mode, and Q1 and Q3 quadrupoles were set
at unit mass resolution. The mass spectrometric conditions were
optimized for each compound by continuously infusing standard
solutions (10 mg/L). The ion source temperature was maintained
at 350 ◦ C. The IonSpray voltage was set at −3 kV. Nitrogen was
used as both curtain gas at 30 psi and ion source gas 1 and 2 at 50
and 30 psi, respectively; collision gas was helium at 10 psi. Other
adjustments and optimized parameters for each compound were
previously published [24,26,27].
2.5. Calibration and quality control
The calibration standards were prepared by adding 1.8 mL of
methanol containing the analytes to 6 g of the sample. The samples
were stirred and slightly heated to remove the added methanol
until they recovered their original weight. Then, 1.5 g of placen-
tal tissue was weighed in triplicate in 8 mL glass vials to apply
the above extraction procedure (Preparation/extraction protocol).
Matrix matched calibration curves were constructed, plotting the
analyte/surrogate peak area ratio against the analyte concentra-
tion. BPA-d16 (at 13.3 ng/g placenta) was used as surrogate. Limits
of detections (LODs) obtained were: 0.2 ng/g for BPA, BP-1 and
BP-2; 0.1 ng/g for BP-3, BP-6, BP-8 and 4-OH-BP; 0.06 ng/g for ethyl-
PB, propyl-PB and butyl-PB; and 0.03 ng/g for methyl-PB. Limits
of quantification (LOQs) were: 0.5 ng/g for BPA, BP-1 and BP-2;
0.4 ng/g for BP-3, BP-6 and BP-8; 0.2 ng/g for 4-OH-BP, ethyl-PB,
propyl-PB and butyl-PB; and 0.1 ng/g for methyl-PB.
Validation in terms of the linearity, sensitivity, accuracy (true-
ness and precision), and selectivity was performed according to
the US Food and Drugs Administration guidelines for Bioanalytical
Method Validation [28]. Placenta samples (n = 79) were extracted
in batches of 10, with each batch containing approximately 4 cases,
4 controls and 2 quality-control samples. Laboratory analysts were
blinded to the case-control-blank status of samples. These quality-
control samples included 1 field blank (deionized water treated as
the biological sample) and 1 blank placenta sample spiked with
target compounds at a final concentration of 1.5 ng/mL, in order to
evaluate the trueness and reproducibility of the technique. The pre-
cision of the method was expressed as relative standard deviation
(RSD), and the trueness evaluated by a recovery assay. Recoveries
for all the target compounds in the quality control spiked sam-
ples ranged from 95% to 106%, with a mean RSD of 10%. Additional
control experiments were performed to ensure that BPA was not
leached from the UPLC–MS/MS itself or from the storage containers
or tubes and vessels used to collect tissue from the patients.
91
The analytical methodology is of major importance because the
selected EDCs are ubiquitous environmental contaminants, mak-
ing the assessment of its toxicity at low level challenging. In our
study, stringent measures were taken to avoid external contam-
ination, with the systematic use of glass materials and repeated
verification of the absence of contaminants released from labora-
tory materials. All study materials had chemical (BPA, BPs and PBs)
levels below LODs. The correct storage conditions are also essen-
tial, given the risk of secondary hydrolysis when samples are left too
long at room temperature [1]. This was not the case in the present
study, because placentas were frozen at −86 ◦ C within 90 min of
their collection, and homogenized aliquots were prepared while
the tissue remained frozen.
2.6. Statistical analysis
We performed statistical analyses to determine associations
between the exposure to environmental endocrine disrupting
chemicals (BPA, BPs and PBs) and the presence of cryptorchidism
and/or hypospadias. Descriptive statistics are reported as mean,
median, and range. Crude and adjusted odds ratios (ORs) with
their corresponding 95% confidence intervals (CIs) were calcu-
lated by conditional logistic regression to estimate the magnitude
of associations between EDCs and urogenital malformations. The
concentrations of EDCs were used as independent variables and
analyzed both as continuous variables and in tertiles, with the
first tertile as the reference group. EDC concentrations below the
LOQ were assigned a value of half of the LOQ. The tertiles were
determined using the distribution of concentrations among all par-
ticipants, even those below the LOQ.
Potential confounding variables were selected if they were
significantly associated with outcomes in bivariate analyses or
changed the beta coefficient by >20% in the multivariable analysis.
In the final adjusted model, only maternal age and newborn birth
weight had a substantial effect on results. In the bivariate analyses,
differences between groups were tested with Pearson’s chi-square
test or Fisher’s exact test, when appropriate. All statistical analy-
ses were performed using SPSS statistical software for Windows
version 20.0 (SPSS Inc., Chicago, IL, USA).
3. Results
The main clinical, socio-demographic and lifestyle character-
istics of cases and controls are summarized in Tables 1 and 2.
Cases and controls differed in the type of delivery, with a higher
prevalence of cesarean interventions in cases and of instrumental
deliveries in controls (p = 0.054).
All placentas studied were positive for at least one out of the
eleven endocrine disrupting chemicals studied (mean of 3.9 ± 1.3
residues). Table 2 exhibits the concentrations of free BPA, BPs [BP-
1, BP-2, BP-3, BP-6, BP-8 and 4-OH-BP] and PBs [methyl-, ethyl-,
propyl- and butyl-PB] in case and control samples, expressed as
ng/g of placenta. Methyl-PB was detected in 86% of analyzed pla-
centa samples (median: 0.82 ng/g placenta, range: LOQ-11.69 ng/g
placenta). Butyl-PB, propyl-PB and ethyl-PB were detected in 73%,
70% and 68% of samples, respectively, with median concentrations
(range) of 0.44 ng/g placenta (LOQ-1.60 ng/g placenta), 0.67 ng/g
placenta (LOQ-4.52 ng/g placenta) and 0.57 ng/g placenta (LOQ-
5.49 ng/g placenta), respectively. BPA was detectable in 58.2% of
placenta samples, with a median (range) concentration of 1.20 ng/g
placenta (LOQ-12.30 ng/g placenta). Detection frequencies of BPs
in placenta were much lower, ranging between 19% for 4-OH-
BP and 4% for BP-3 (only three placenta samples were positive
for BP-3), with mean (range) concentrations of 0.19 ng/g placenta
92 M.F. Fernández et al. / Reproductive Toxicology 59 (2016) 89–95

Table 1
Selected characteristics of parents, pregnancy, delivery, and infants in relation to urogenital malformations according to the case/control status of the newborn.

Variable Total (n = 79) Controls (n = 51) Cases (n = 28) p*

Percentiles Percentiles Percentiles

Median 25th 75th Median 25th 75th Median 25th 75th

Mother’s age (yrs) 30.0 25.0 32.0 30.0 25.0 32.0 29.0 25.0 31.0 0.241
Father’s age (yrs) 31.0 28.0 34.0 31.0 29.0 35.0 29.5 27.0 33.0 0.128
Child’s weight (g) 3250.0 2990.0 3570.0 3320.0 3070.0 3570.0 3080.0 2827.5 3597.5 0.215
Gestational age (weeks) 39.0 38.0 40.0 39.0 38.0 40.0 40.0 37.0 40.8 0.863
BMI before pregnancy (Kg/m2 ) 22.4 20.3 25.6 22.0 20.0 25.2 23.4 20.6 26.5 0.275
n % n % n % p**

Place of residence
Urban 20 25.3 14 27.5 6 21.4 0.38
Rural 59 74.7 37 72.5 22 78.6
Previous pregnancy
No 29 36.7 19 37.3 11 39.3 0.592
Yes 49 62.0 32 62.7 17 60.7
Oral contraceptive before pregnancy
No 55 69.6 33 64.7 22 78.6 0.306
Yes 24 30.4 18 35.3 6 21.4
Prenatal smoking
Yes 24 30.4 17 33.3 7 25.0 0.435
No 55 69.6 34 66.7 21 75.0

BMI: Body mass index; DS: Standard deviation; PB: Paraben; BPA: Bisphenol-A. BPA and all four PBs were detected in at least half of the samples.
*
Man–Whitney’s U-test.
**
Fischer’s exact test.

Table 2
Concentrations of several endocrine disrupter chemicals (EDCs) (ng/g) in placenta samples of cases and controls in relation to urogenital malformations.

EDC LOD (ng/g) Mean Median Range Positives (%)

Methyl-PB 0.03 Cases 2.27 1.76 0.12–8.73 89.3

Controls 1.51 0.59 0.10–11.69 84.3
Total 1.78 0.80 0.10–11.69 86.1
Ethyl-PB 0.06 Cases 1.01 0.65 0.23–4.62 60.7
Controls 0.91 0.57 0.20–5.49 72.6
Total 0.95 0.57 0.20–5.49 68.4
Propyl-PB 0.06 Cases 1.40 1.13 0.25–4.04 75.0
Controls 0.74 0.47 0.20–4.52 66.7
Total 0.98 0.67 0.20–4.52 69.6
Butyl-PB 0.06 Cases 0.62 0.62 0.20–1.60 85.7
Controls 0.45 0.34 0.20–1.26 66.7
Total 0.51 0.44 0.20–1.60 73.4
BPA 0.2 Cases 4.01 3.40 0.60–12.30 67.9
Controls 1.82 0.89 0.50–10.20 52.9
Total 2.59 1.20 0.50–12.30 58.2
BP-3 0.1 Cases <LOD <LOD 1.28 3.6
Controls 0.12 <LOD 0.65–3.09 3.9
Total 0.11 <LOD 0.65–3.09 3.8
4OH-BP 0.1 Cases 0.28 <LOD 0.37–1.99 21.4
Controls 0.14 <LOD 0.20–2.72 17.6
Total 0.19 <LOD 0.20–2.72 18.9
BP-1 0.2 Cases 0.21 <LOD 3.32 3.6
Controls <LOD <LOD – 0.0
Total <LOD <LOD 3.32 1.3
BP-6 0.1 Cases <LOD <LOD – 0.0
Controls <LOD <LOD 0.33 2.0
Total <LOD <LOD 0.33 1.3
BP-2 0.2 Cases <LOD <LOD 0.38–1.92 10.7
Controls <LOD <LOD 0.52–1.47 3.9
Total <LOD <LOD 0.38–1.92 6.3
BP-8 0.2 Cases <LOD <LOD – 0.0
Controls <LOD <LOD – 0.0
Total <LOD <LOD – 0.0

PB: paraben; BP: benzophenone; BPA: Biphenol-A; LOD: limit of detection.

EDC exposure was analyzed by means of ultra-high performance liquid chromatography-tandem mass spectrometry in placenta samples. Mean and median EDC concentra-
tions were calculated assigning a value of half of the LOQ to those below the LOQ.

(LOQ-2.72 ng/g placenta) and 0.11 ng/g placenta, respectively. BP-8 following pairs: methyl-PB/propyl-PB, ethyl-PB/propyl-PB, ethyl-
was not detected in any of the placenta samples analyzed (Table 2). PB/BP-8, ethyl-PB/BPA and propyl-PB/BP-8 (Spearman coefficients
Positive linear correlations were found between concentrations ranging from 0.24 to 0.28). Spearman correlation coefficients
of specific phenols, which were statistically significant for the between the specific concentrations of chemicals in placenta sam-
 M.F. Fernández et al. / Reproductive Toxicology 59 (2016) 89–95 93

Table 3
Crude and adjusted ORs (95% CIs) for urogenital malformations among male offspring in relation to the presence in placenta samples of specific endocrine disruptors (ng/g
placenta), according to the case/control status of newborn.

EDCs Total Cases Controls OR 95.0% CI p-Value ORa 95.0% CI p-Value

Lower Upper Lower Upper

Methyl-PB
<0.40 26 19 7 1.00 1.00
0.44–1.91 26 18 8 1.00 0.32 3.09 0.997 1.04 0.33 3.26 0.949
1.96–11.69 26 13 13 3.18 0.88 11.48 0.078 3.24 0.83 12.69 0.092
Methyl-PB (continuous) 78 50 28 1.17 0.94 1.46 0.171 1.17 0.93 1.48 0.188

Ethyl-PB
<LOD 26 15 11 1.00 1.00
0.07–0.89 27 23 4 0.29 0.08 1.06 0.060 0.26 0.07 1.00 0.049
0.91–5.49 26 13 13 1.51 0.44 5.15 0.514 1.25 0.34 4.60 0.734
Ethyl-PB (continuous) 79 51 28 1.07 0.74 1.55 0.703 1.00 0.68 1.47 0.987

Propyl-PB
<LOD 26 19 7 1.00 1.00
0.06–1.15 27 20 7 1.23 0.30 5.04 0.769 1.39 0.33 5.91 0.654
1.16–4.52 26 12 14 4.72 1.08 20.65 0.039 6.42 1.16 35.47 0.033
Propyl-PB (continuous) 79 51 28 1.90 1.12 3.22 0.017 2.16 1.16 4.01 0.015

Butyl-PB
<0.08 26 20 6 1.00 1.00
0.16–0.74 27 16 11 2.29 0.65 8.05 0.195 2.26 0.62 8.21 0.216
0.79–1.60 26 15 11 2.31 0.72 7.46 0.162 2.11 0.62 7.16 0.232
Butyl-PB (continuous) 79 51 28 2.27 0.80 6.42 0.122 2.07 0.71 6.06 0.186

BPA
<LOD 33 24 9 1.00 1.00
0.50–3.60 20 15 5 1.06 0.32 3.50 0.918 0.91 0.25 3.33 0.891
3.70–12.30 26 12 14 5.37 1.31 22.01 0.019 7.23 1.48 35.46 0.015
BPA (continuous) 79 51 28 1.35 1.10 1.67 0.005 1.39 1.10 1.76 0.006

Statistical analysis were performed using conditional logistic regression models for matched data sets (1:n matching, n = 1–3).
OR: odds ratio; CI: Confidence interval; PB: paraben; BPA: Biphenol-A; LOD: limit of detection.
Models were adjusted for mother’s age (years) and weight at birth (g)
BPA and all four PBs were detected in at least half of the samples

ples and maternal age, newborn birth weight and gestational
age were also analyzed, finding a borderline association between
methyl-PB placental exposure and gestational age alone (r = 0.198,
p = 0.085).
A larger number of phenols were detected in cases than in con-
trols (4.22 ± 1.4 vs. 3.71 ± 1.2, respectively; p = 0.130). There were
no statistically significant differences between cases and controls in
the median concentration of most of the selected chemicals except
for methyl-PB (median: 1.76 vs. 0.59 ng/g placenta, p = 0.072),
propyl-PB (median: 1.13 vs. 0.47 ng/g placenta, p = 0.024). The
median BPA concentration was 0.89 ng/g placenta (<LOQ-10.2 ng/g
placenta) in the control group vs. 3.4 ng/g placenta (<LOQ-12.3 ng/g
placenta) in the cases group (p = 0.024). Residues of butyl-PB were
also more frequently detected in cases than in controls (85.7% vs.
66.7%, respectively, p = 0.054) (Table 2).
In the unadjusted conditional regression model, urogenital mal-
formations showed a statistically significant association with the
placenta tissue concentrations of the phenols propyl-PB (OR = 4.72;
95% CI, 1.08–20.65) and BPA (OR = 5.37; 95% CI, 1.31–22.01) for
the 3rd tertiles of concentrations. When models were further
adjusted for maternal age and birth weight, both placenta tissue
BPA and propyl-PB remained significantly associated with cryp-
torchidism/hypospadias risk, with ORs (95% CI) for the 3rd tertiles
of BPA exposure (ORa = 7.2; 95% CI, 1.5–35.5) and propyl-PB expo-
sure (ORa = 6.4; (95% CI, 1.2–35.5). Table 3 lists these findings
and the results of conditional regression analysis with crude and
adjusted ORs for malformations in relation to the presence of BPA
and four PB residues.
Models in which the concentrations were entered as continu-
ous variables showed similar positive associations at all levels of
adjustment both for placenta tissue BPA and propyl-PB.
4. Discussion
We report here the simultaneous intrauterine exposure to dif-
ferent EDCs assessed in human placentas recruited at delivery from
an established birth cohort and the association of individual phe-
nol residues with the risk of male urogenital tract malformations.
All placentas had measurable concentrations of at least one of the
11 phenols quantified, with a mean value of four residues, reflect-
ing the widespread exposure of the population. Free BPA and all
four PBs were detected in at least half of the samples. The median
(range) BPA concentration was 0.89 ng/g placenta (LOQ-10.2 ng/g
placenta) in the controls versus 3.4 ng/g placenta (LOQ-12.3 ng/g
placenta) in the cases (p = 0.024), showing an excess risk of malfor-
mation associated with the concentration of BPA in placenta tissue
[ORa (95% CI) of 7.2 (1.5–35.5) for cryptorchidism/hypospadias in
the third tertile of BPA exposure].
Some studies suggest that BPA is transplacentally transferred to
the embryo/foetal compartment, supporting the proposition that
the foetus is continuously exposed to this compound [4,29–33],
and potentially could cause adverse birth outcomes in offspring.
Thus, some human studies have associated BPA exposure with
decreased gestational age [34], birth weight [34,35] and anogen-
ital distance [36]. BPA has also been related to the aetiology of
child hypospadias through inhibition of the expression of a spe-
cific matrix metallopeptidase (MMP11), a well-known effector of
development and normal physiology protein [37]. More recently,
Fenichel et al. [38] observed that BPA concentrations in cord blood
samples were not correlated with undescended testes in a nested
case-control study; nevertheless, mean levels of BPA were higher in
the cryptorchidism group and even higher in the non-palpable sub-
group [38,39]. Moreover, the foetus may be exposed to higher free
BPA levels because of the lower activity of hepatic enzymes in preg-
94 M.F. Fernández et al. / Reproductive Toxicology 59 (2016) 89–95
nant women [30,32] and the relative immaturity of the placenta’s
capability to metabolize BPA [40]. A recent report has evaluated
the importance of the conjugation–deconjugation cycle of the non-
persistent compounds, showing a dynamic balance between free
BPA and metabolites in the foeto-placental compartment [41].
Mean placenta levels of BPA and the other phenols are highly
consistent with the values described in previous studies, although
there is very little published research on the in utero exposure
of the human foetus to BPA and other phenols during pregnancy
[27,30,40,42,43].
The median (range) propyl-PB concentration in the control
group was 0.47 ng/g placenta (LOQ-4.52 ng/g placenta) versus
1.13 ng/g placenta (LOQ-4.04 ng/g placenta) (p = 0.024) in the cases
group, evidencing an excess risk of malformation associated with
the concentration of propyl-PB in placenta tissue [ORa (95% CI) of
6.4 (1.2–35.5) for cryptorchidism/hypospadias in the third tertile
of propyl-PB exposure]. The few previous studies on the possible
association between exposure to PB and human male reproductive
outcomes found no significant effects [10,44,45].
We could find no data on exposure to PBs or BPs in placenta
tissues except for studies by our research group [9,24,27]. In addi-
tion, limited information is available on the exposure of pregnant
women to BPs, and most studies have focussed on BP-3, the most
commonly used sunscreen agent [46].
Several studies confirmed that human foetuses are routinely
exposed to a complex mixture of EDCs [32,39,47], not only to
persistent and bioaccumulated substances stored in maternal adi-
pose tissue and mobilized during pregnancy [19], but also to EDCs
that are highly prevalent in the environment. Risk assessment in
relation to endocrine disruption is therefore highly complex, and
several authors have pointed out that even the most comprehen-
sive chemical analysis might explain only part of the biologically
effective impact of endocrine-disruption [48,49].
Our study has several limitations, including the relatively small
sample size, which prevented adjustment for some potential con-
founders, such as the type of delivery, foetal presentation, weeks
of gestation, child length, head size, presence of other malforma-
tion(s) and season of birth. Therefore, residual confounding cannot
be ruled out. Moreover, exposure assessment was made in term
placentas, which may have resulted in exposure misclassification.
These results should be interpreted with caution due to the vari-
ability observed in exposure patterns and because our series does
not fully represent the population of pregnant women in Spain.
Following proposals of separate genetic but common environmen-
tal components of risk for cryptorchidism and hypospadias, both
malformations were considered comparable entities in terms of
their aetiology, grouping together boys with one or both malfor-
mations in the statistical analysis. However, it should be taken into
account that hypospadias and cryptorchidism appear related to
inset mechanisms occurring at different critical stages in gestation
[50].
The use of placenta samples to investigate intrauterine expo-
sure to EDCs can be considered the main strength of this study,
largely because placental transfer represents the main access of
chemical agents to the foetus in a period of particular concern.
Moreover, this highly accessible organ can be used to assess chronic
exposure to persistent and non-persistent chemicals, avoiding
repeated maternal blood sampling and other invasive biomonitor-
ing [22,51].
Further studies are warranted to elucidate the implications of
exposure to mixtures of EDCs in sensitive human populations such
as newborn infants. Meanwhile, it is desirable to inform women as
early as possible during the preconception period about measures
to reduce or avoid their exposure to EDCs [52].
Conflict of interest
None declared.
Funding
This study was supported in part by research grants from the
EU Commission (QLK4-1999-01422, QLK4-2002-00603 and CON-
TAMED FP7-ENV-212502), the Spanish Ministry of Health (“INMA
Study” G03/176; FIS PI11/0610; and IJD-Sara Borrell Program, grant
number CD012/00462); CIBER de Epidemiología, Instituto de Salud
Carlos III, Government of Spain, and the Regional Government of
Andalucía-Spain (grant numbers SAS 202/04, P09-CTS-5488 Project
of Excellence, and SAS PI-0675-2010). The funding agencies had no
role in the analysis of data or preparation of the manuscript.
Transparency document
The Transparency document associated with this article can be
found in the online version.
Acknowledgements
We are indebted to all participants, without whom this work
would not have been possible. We are grateful to the nursing staff
for their cooperation and R. Davies for editorial assistance.
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