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ENDOCRINOLOGY & METABOLISM

ADVANCE ARTICLE: JCEM

Andrea Di Nisio, Iva Sabovic, Umberto Valente, Simone Tescari, Maria Santa Rocca,
Diego Guidolin, Stefano Dall’Acqua, Laura Acquasaliente, Nicola Pozzi, Mario Plebani,
Andrea Garolla, Carlo Foresta
The Journal of Clinical Endocrinology & Metabolism
Endocrine Society
Submitted: August 28, 2018
Accepted: November 01, 2018
First Online: November 06, 2018

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Endocrine disruption of androgenic activity by perfluoroalkyl substances:
clinical and experimental evidence

 The Journal of Clinical Endocrinology & Metabolism; Copyright 2018

DOI: 10.1210/jc.2018-01855

Endocrine disruption of androgenic activity by perfluoroalkyl substances:
clinical and experimental evidence
Andrea Di Nisio1, Iva Sabovic1, Umberto Valente1, Simone Tescari1, Maria Santa Rocca1 ,
Diego Guidolin2, Stefano Dall’Acqua3, Laura Acquasaliente4, Nicola Pozzi4, Mario Plebani5,
Andrea Garolla1, Carlo Foresta1*
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Department of Medicine, Operative Unit of Andrology and Medicine of Human Reproduction, University of
Padova, Via Giustiniani, 2, 35128 Padova, Italy.
Department of Neurosciences, University of Padova, via Gabelli 65, 35128 Padova, Italy.

Department of Pharmaceutical Science, University of Padova, via Marzolo 5, 35128 Padova, Italy

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Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine 1100 South
Grand Blvd. St. Louis, MO 63104, USA.

Department of Medicine, Laboratory Medicine, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy

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ORCiD numbers:

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0000-0002-6576-2183
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Carlo

Received 28 August 2018. Accepted 01 November 2018.

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Background: Considerable attention has been paid to perfluoroalkyl compounds (PFCs)
because of their worldwide presence in humans, wildlife, and environment. A wide variety of
toxicological effects is well supported in animals, including testicular toxicity and male
infertility. For these reasons, the understanding of epidemiological associations and of the
molecular mechanisms involved in the endocrine-disrupting properties of PFCs on human
reproductive health is a major concern.
Objective: To investigate the relationship between PFCs exposure and male reproductive
health.
Design: This study was performed within a screening protocol to evaluate male reproductive
health in high schools.
Patients: this is a cross-sectional study on 212 exposed males from the Veneto region, one of
the four areas worldwide heavily polluted with PFCs, and 171 non-exposed controls.
Main outcome measures: Anthropometrics, seminal parameters and sex hormones were
measured in young males from exposed areas, compared with age-matched controls. We also
performed biochemical studies in established experimental models.
Results: We found that increased levels of PFCs in plasma and seminal fluid positively
correlate with circulating T and with a reduction of semen quality, testicular volume, penile
length and AGD. Experimental evidence points towards an antagonistic action of PFOA on
the binding of T to AR in gene reporter assay, competition assay on AR-coated SPR chip and
AR nuclear translocation assay.
Discussion: This study documents that PFCs have a substantial impact on human health as
they interfere with hormonal pathways, potentially leading to male infertility.

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PFCs exposure leads to an impairment of male reproductive system, which is supported by
experimental evidence showing an interference of these chemicals on the binding of testosterone to
its receptor.

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Perfluoroalkyls disruption of androgenic activity

 The Journal of Clinical Endocrinology & Metabolism; Copyright 2018

DOI: 10.1210/jc.2018-01855

Introduction

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Methods

This study was performed within the annual screening protocol to evaluate male reproductive
health in the high schools of Padova and surroundings (Veneto Region, North-East of Italy).
The aim of this screening is to early diagnose possible risk factors and diseases of the male
reproductive system. Here, we report the findings of 383 subjects who voluntarily agreed to
complete the cross-sectional study between June 2017 and May 2018. Included subjects
underwent an accurate medical visit, measure of anthropometric parameters, ultrasound
examination of the testes and semen analysis at our medical center. Written informed consent
was obtained from all subjects, and the study was approved by the Research Ethics
Committee of the University Hospital of Padova (N. 2208P). The investigation was

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Perfluoroalkyl compounds (PFCs) are a class of organic molecules that are used in many
everyday products such as oil and water repellents, coatings for cookware, carpets, and
textiles. Their attractive physio-chemical characteristics (i.e., colourless, odourless, high
thermal stability, low chemical reactivity and durability), high availability and low cost
ensure widespread use in the industry but also drive persistent accumulation into the
environment, making them a potential biohazard for human health(1,2). Indeed, PFCs have
been found in human fluids and tissues including the brain, placenta, and testis, which are
protected by strong selective barriers(3–7). Interestingly, and for unknown reasons, there
seems to be a sex-dependent pharmacodynamic profile, with adult males having a much
higher tendency to PFCs accumulation and lower clearance(8–11).
Exposure pathways and toxicity mechanisms for PFCs are not well characterized, at least
in humans (reviewed in (12)). An attractive hypothesis emerges from recent
phenomenological studies correlating the dysfunction of the male reproductive system with
the environmental levels of PFCs(13). PFCs may be absorbed by the intestine or inhaled and,
once in the circulation, they may act as endocrine disruptors (ED) ultimately leading to
genital disorders, such as impaired spermatogenesis and reproductive defects, and
antiandrogenic-driven conditions, such as testicular dysgenesis syndrome (13), which is an
established risk factor for testis cancer(14,15). PFCs could exert their toxicity on the foetus,
new-born, as well as during development, especially in teenagers due to alterations in sex
hormones biosynthesis. Recent data suggest that in utero exposure to PFCs is associated to
lower sperm quality and higher levels of LH and FSH at adulthood(16). Furthermore, by
apparently acting as both anti-androgenic and anti-estrogenic molecules, PFCs might also
affect the downstream signalling pathways of sex hormones (17,18), down-regulate the
hypothalamic-pituitary axis activity and increase testicular toxicity during development(19–
21).
The crucial emerging role PFCs as pollutants of water, soil, and air, and their persistent
level in males warrant for more investigation on the mechanisms of PFCs toxicity in humans.
In this comprehensive study, we tested the hypothesis that human exposure to PFCs drives
androgenic dysfunction and deterioration of the male reproductive system by altering the
testosterone (T) interaction with its specific androgen receptor (AR). To investigate the
relationship between PFCs exposure and clinical alterations, we studied a cohort of 212
exposed young men from the Veneto region, in the North-East of Italy. With Mid-Ohio valley
in the USA, Dordrecht area in the Netherlands, and Shandong district in China, the Veneto
region is one of the four areas worldwide heavily polluted with PFCs. To fully characterize
the antiandrogenic action of PFCs, and the structural and functional interaction between
PFCs, AR, and T, we performed biochemical studies in established experimental models.

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Anthropometric and penile measurements included: height, weight, body mass index (BMI),
waist circumference, arm span, crown-to-pubis length, penile length and circumference.
These parameters are commonly used to suggest severe forms of congenital or pre-pubertal
hypogonadism, such as patients with Klinefelter and Kallmann syndrome(24). Every measure
was taken three times to the nearest millimeter. Height was accurately taken from the floor to
the crown of the head as described in previous studies(25,26). BMI was calculated using the
formula weight (kg)/height (m)2. Waist circumference was measured at the midpoint between
the superior border of the iliac crest and the lowest rib(27). The arm span was measured as
the distance between the tips of the middle fingers with the arms fully extended (28). The
pubis-to-floor distance was measured from the upper edge of pubic symphysis to the floor.
The crown-to-pubis length was consequently derived as the difference between height and
pubis-to-floor distance (29). The penile length was measured as the linear distance along the
dorsal side of the penis extending from the lower edge of the pubic bone to the tip of the
glans in the flaccid state. The penis circumference was measured at the middle of the shaft
(30). All subjects were evaluated by the same two clinicians. The intra-operator variations
were in all cases <5%. Testicular volumes were evaluated by ultrasound, using the standard
ellipsoid formula (width × height × length × π/6, CV<10%).

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The AGD was measured as previously described elsewhere(31), from the posterior base of
the scrotum to the center of the anus. The participant was placed in a supine, frog-legged
position with his thighs at a 45° angle to the examination table. In a subset of 50 randomly
chosen patients, AGD measures were repeated twice by the same technician, and then blindly
by the second examiner. Interclass correlation coefficients (ICC) were calculated for
repeatability estimation within and between examiners. Repeatability was very high within
individual (ICC= 0.979, 95% CI= 0.960 - 0.989) and slightly lower across examiners (ICC=
0.932, 95% CI= 0.873 – 0.964).

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Anthropometric measurements

Semen collection and analysis

Human semen samples were obtained by masturbation after 2–7 days of sexual abstinence
and stored in sterile containers. Samples were allowed to liquefy for 30 min at 37°C and were
examined for seminal parameters according to WHO criteria(32). Briefly: semen volume was
measured by weighing, assuming a semen density of 1.0 g/ml; sperm concentration was
evaluated by hemocytometer (Bürker-Türk; Paul Marienfeld GmbH&Co. KG, LaudaKönigshofen, Germany); sperm morphology was identified from semen smears prepared with
10  µl of well-mixed semen, stained with Papanicolaou and assessed using the Tygerberg

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performed according to the principles of the Declaration of Helsinki. Participants did not
receive any reimbursement. Based on geographical distribution of PFCs pollution (22)
subjects were then grouped on the basis of their residence. Regional authorities (23) have
defined two different zones within the exposed area, based on the degree of pollution: the red
area, which is the one with the highest PFCs levels, and the yellow zone, with slightly lower
levels, but at risk for the close proximity with the contamination plume and surroundings
(22). Among the 383 subjects included in the study, 83 were resident in the yellow zone, 129
in the red zone, and 171 outside the exposed area (green zone). Specific geographical origin
is reported in Supplementary figure 1(22). In order to increase the sample size for subsequent
statistical analyses, subjects from red and yellow zone were pooled together as a single
exposed group, since no difference has emerged between the two areas for the clinical
parameters considered (data not shown), except for non-progressive sperm motility and
immotile sperm that where mutually different between groups. Subjects from green zone
(non-exposed) were considered as control group.

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DOI: 10.1210/jc.2018-01855

Sex hormones quantification

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In a subset of patients (50 controls and 50 exposed subjects), PFCs were evaluated in serum
and seminal fluid. For serum analyses, cells are removed from plasma by centrifugation for
10 minutes at 2000 x g. Following centrifugation, the liquid component (plasma) was
transferred into a clean polypropylene tube. The quantification of PFOA and PFOS was
processed on reversed-phase (RP) liquid chromatography coupled with high-resolution mass
spectrometry (LC-MS) Agilent Varian 320 (Agilent Technologies, Santa Clara, CA, USA).
Briefly, each sample was dissolved in acetonitrile and fixed amounts of the stable isotopelabeled internal standard were added (MPFOA, MPFOS, Wellington Laboratories, Ontario,
Canada). To test the analytical response and to optimize calibration curve standard mixture
was used at increasing concentrations (PFAC-MXB, Wellington Laboratories) together with
isotope-labeled internal standards (MPFOA, MPFOS) at fixed concentrations. This solution
was analysed by LC-MS. The different perfluoroalkyl species were identified by comparing
the retention time and mass spectra (i.e. m/z value and isotopic pattern). Quantification of
each species was calculated using the corresponding calibration curve.
AR gene reporter assay

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All the transfections were performed in HeLa cells, obtained from the American Type
Culture Collection (ATCC, Manassas, VA, USA) as previously described(33). Briefly, cells
were cultivated in Dulbecco's modified Eagle medium (DMEM) (Thermofisher, Waltham,
MA, USA), supplemented with 5% fetal bovine serum (Sigma Aldrich, St. Louis, MO, USA),
antibiotics, and antimycotics in a humidified incubator at 37°C with 5% CO2. Transient gene
expression assay was performed in 96-well plates using Lipofectamine 2000 reagent
(Thermofisher, Waltham, MA, USA) and a Dual-Luciferase reporter assay system (Promega
Corp, Madison, WI, USA). HeLa cells were grown in 96-well plates and cotransfected at
70% confluence with 100 ng/well of the expression vector for the full-length human AR
(pSV-AR0), 100 ng/well of mouse mammary tumor virus (MMTV)-luciferase reporter
plasmid and 10 ng/well of pGL4.74 Renilla luciferase (Promega Corp.) (internal control for
transfection efficiency). pSV-AR0 and MMTV-Luc plasmids were a kind gift from Prof.
Claessens (University of Leuven, Belgium). Twenty-four hours after transfection, media was
replaced with fresh DMEM and test chemicals (PFOA and PFOS 1 µM, Wellington
Laboratories, Ontario, Canada) in the absence or presence of 10 nM T (positive control,
Sigma Aldrich) were added to each well. Flutamide (1 µM, Sigma Aldrich) served as
negative control. Treated cells were harvested 24 h later and lysed with lysis buffer of DualLuciferase reporter assay (Promega Corp.). Luciferase activity was measured with a
multilabel plate reader (Wallac Victor, Perkin-Elmer, Waltham, MA, USA) and all data were
standardized for luciferase activity. Results are shown as the mean ± SD of three independent
experiments, each performed in duplicate.

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PFCs quantification in serum and semen by mass-spectrometry

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Blood was collected in the fasting state between 08:00 and 10:00 AM. Serum total T, FSH
and LH were evaluated by commercial electrochemiluminescence immunoassay methods
(Elecsys 2010; Roche Diagnostics, Mannheim, Germany). For all parameters, the intra- and
interassay coefficient of variation were <8% and <10%, respectively. All determinations were
performed in duplicate.

Surface Plasmon resonance analyses
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strict criteria. Sperm motility was graded into total (progressive + non-progressive motility)
and progressive motility. Total sperm count (volume × sperm concentration) was also
calculated.
Seminal parameters were available for 211 exposed subjects and 170 controls, since one
subject in each group failed to collect semen.

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The clonal strain of mouse MA-10 Leydig cell line used for AR nuclear translocation assay
was purchased from ATCC® (CRL-3050™, ATCC, Manassas, Virginia, USA). Cells were
used at the second cell passage from original thawing, in order to maintain the phenotype as
close as possible to the one claimed by the manufacturer, and handled as previously
described(34). Briefly, cells were seeded on 0.1 % gelatine-coated plasticware and
maintained in DMEM/F12 medium, pH 7.7 (GIBCO-Invitrogen, Milano, Italy),
supplemented with 20 mM Hepes, 15% horse serum, and 50 µg/ml gentamicin. Starved MA10 cells seeded onto glass slides (BD Biosciences, Milano, Italy) and cultured at different
concentrations of T (1 to 100 nM, Sigma Aldrich) and PFOA (0.1 to 1 µM, Wellington
Laboratories), alone or in combination. After 24 hours, cells were fixed with 4 %
paraformaldehyde/PBS solution for 15 min at room temperature and were permeabilized with
1 % Triton X-100/PBS solution for 10 min at room temperature. Furthermore, samples were
saturated with 5 % BSA/5 % normal donkey serum in PBS for 30 min and then incubated
overnight at 4 °C with Rabbit polyclonal Anti-Androgen receptor antibody (ab74272, Abcam,
Cambridge, UK) for further assessment of AR nuclear translocation by the means of relative
quantification of fluorescence density. In the negative control, primary antibodies were
omitted. The following day, primary immunoreaction was detected by incubation with IgGFITC goat anti-Rabbit secondary antibody (K1715, Santa Cruz Biotechnology, Dallas, TX,
USA). Finally, cells were counterstained with DAPI, mounted with antifade buffer, and
analyzed with videoconfocal (VICO) fluorescence microscope (Nikon, Firenze, Italy). The
nuclear translocation of AR (the intensity of AR signal within the nucleus relative to the total
intensity) was quantified in 20-40 cells using the software ImageJ
(http://rsbweb.nih.gov/ij/download.html).
Statistical analyses

All statistics were calculated using SPSS (Version 23; SPSS Inc., Chicago, IL, USA). p
values <0.05 were considered as statistically significant. The results were expressed as means
± SD or as medians + interquartile ranges. The Shapiro-Wilk W test for normality was used
to check the distributions of the variables; as almost none of parameters was normally
distributed (except height, crown-to-pubis, pubis-to-floor and sperm progressive motility),
and almost all of log-transformed distributions did not satisfy normality, non-parametric
statistics was applied. Mann-Whitney test was used to assess differences between groups in
the anthropometric, seminal and hormonal parameters and in in the concentrations of serum e
seminal contaminants. Both raw and adjusted p values are reported; adjustment for multiple
comparisons was calculated with the Bonferroni-Holm method. Spearman's rank correlation

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AR nuclear translocation assay

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Surface Plasmon Resonance (SPR) experiments were performed on a BIAcore-S200
instrument (GE-Healthcare, Chicago IL USA) to monitor the interaction between T or PFOA
and the AR. The binding domain AF2 of the AR (650-920, Abcam, Cambridge, MA, USA)
was covalently immobilized on a CM5 sensor chip using an amine-coupling chemistry.
Binding experiments were carried out by injecting increasing concentrations of T (0-1mM;
Sigma Aldrich) and PFOA (0-4 M; Wellington Laboratories) at a flow rate of 30µl/min,
using 10mM Hepes pH7.4, 0.15M NaCl containing 3% MeOH (V/V) as running buffer. Each
cycle consists of 60-sec contact time, followed by 120-sec dissociation and 30-sec pulse with
100mM Hepes pH7.4, as regeneration step. The response units (RU) at the steady state were
plotted as a function of [analyte] and the dissociation constant (Kd) was obtained as a fitting
parameter of a binding isotherm. Competition experiments were performed in order to
investigate the effect of PFOA on T-AR interaction. Solutions of T (250 M) were incubated
with different concentrations of PFOA (0-4 µM) for 10 minutes, and then injected over the
AR-coated sensor chip. All experiments were performed in triplicate at 25ºC.

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Anthropometrics and seminal measures of the two groups are reported on Table 1. In
particular, subjects from the exposed group showed significantly lower mean testicular
volume and shorter penile length and AGD, after adjustment for multiple comparisons. Prior
to adjustments, also crown-to-pubis and pubis-to-floor distances, and the respective ratio,
differed between groups. No significant difference was observed for age and other
anthropometric parameters.
For what concerns seminal parameters, exposed subjects showed significantly lower
sperm progressive motility and normal sperm morphology, together with higher semen pH
and immotile sperm (Table 1). In addition to the reduction in semen quality, also lower sperm
count was observed in exposed males, in terms of sperm concentration and total count,
although not statistically significant after correction for multiple comparisons (Table 1). The
overview of seminal and genital alterations is suggestive of an impairment of androgenic
signalling in these subjects.
To confirm this hypothesis, we evaluated gonadal-pituitary axis in a subset of 100
randomly-chosen subjects (50 from the control group and 50 from the exposed group) that
underwent also LC-MS quantification of PFOA and PFOS in serum and seminal plasma, in
order to confirm the geographic selection criteria based on exposure patterns. In the subgroup
from the exposed area, increased semen pH was confirmed, together with a lower percentage
of sperm with normal morphology, reduced penile length and circumference and smaller
testicular volume, but not AGD, although only after adjustment for multiple comparisons
(Table 2). No significant difference has emerged in terms of other seminal or anthropometric
parameters. PFOA was detected in serum from 98% subjects and in 96% of the respective
seminal plasma, whereas PFOS was detected in 90% of sera and 86% of seminal plasma.
PFCs quantification has confirmed higher serum levels of both PFOA and PFOS in exposed
subjects (Table 2), with the former being the prominent species in blood, with a mean of
14.99 ng/mL in the exposed group and 4.71 ng/mL in control subjects. In addition, the
concentration of PFOA, but not PFOS, was higher in the in seminal plasma from exposed
subjects, although lower than serum levels (Table 2). Hormonal analyses showed higher
levels of total T and LH in the exposed group, compared with control counterparts (Table 2).
In the correlation analyses, serum and seminal plasma levels of PFOS and PFOA were highly
correlated with each other (Spearman’s ρ =0.216, p =0.034 and ρ =0.294, p =0.003,
respectively), as were PFOA plasma and semen concentrations (ρ =0.449, p <0.001), but not
PFOS plasma and semen levels (ρ 0.163, p =0.111). Serum PFOA levels were positively
correlated with total T (ρ =0.305, p =0.002; Figure 1) and LH (ρ =0.224, p =0.046), as were
seminal PFOA (ρ =0.346, p <0.001 and ρ =0.259, p =0.021) and with the proportion of sperm

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Results

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coefficients were calculated to evaluate the correlations between the concentrations of each
contaminant and the variables of interest.
In the gene reporter assay, to examine differences between treatment groups and positive
control (T 10 nM), one-way analysis of variance (ANOVA) was performed with a Dunnett´s
post hoc test, given the normal distribution of data. Due to relatively few data points per
concentration and non-normality of the data, non-parametric statistics were used to analyse
AR nuclear translocation assay. The Kruskal-Wallis test was used to compare differences
between concentrations and the Jonckheere-Terpstra test (two-tailed) was used to analyze for
a linear trend between concentration and response. If one or both tests showed a significant
difference (p <0.05), the Mann–Whitney test with Bonferroni correction was used to compare
each concentration with the control. The same procedure was applied to comparisons
between different stimuli (PFOA 0.1, 1 and 10 µM and flutamide 1 µM) and T, within each T
concentration.

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with normal morphology (ρ =-0.303, p =0.002 and ρ =-0.225, p =0.025 respectively). Again,
seminal PFOA showed a positive correlation also with pH (ρ =0.203, p=0.042). Both serum
and semen PFOA, but not PFOS, were associated with reduced testicular volume (ρ =0.211,
p=0.037 and ρ =-0.277, p= 0.006, respectively).
Within this framework of clinical signs suggestive of an endocrine disruption of androgen
action by PFCs, we aimed to experimentally test the hypothesis of an interference of these
chemicals on the AR, the mediator of androgen signalling. To this end, an AR gene reporter
assay on HeLa cells transiently co-transfected with an MMTV-LUC reporter vector and an
AR expression plasmid pSV-AR0. PFOS and PFOA at a concentration of 1 µM acted as mild
agonists of AR (10.5% and 11.6%, Figure 2). Upon co-incubation with T 10 nM, both PFOS
and PFOA elicited a significant (p <0.001) antagonistic effect on T-induced activation of AR
at concentrations comparable with those reported in highly exposed populations. These
compounds antagonized the T-induced response (set to 100 %) down to 73.5 and 64.2 %,
respectively (Figure 2), with PFOA being the most potent AR inhibitor. The relative
potencies of the tested compounds were approximately twice lower than the inhibitor control
flutamide (Figure 2).
Given the highest occurrence of PFOA in the serum of exposed Italian populations and its
higher potency compared to PFOS in the gene reporter assay, we focused on PFOA to
elucidate the anti-androgenic mechanism of PFCs. SPR measurements were performed to
monitor the real-time interaction between T and PFOA with the AR. In this experiment, the
AF2 binding domain of AR was immobilized on a CM5 sensor chip and solutions of T (0 to 1
mM) and PFOA (0 to 4 µM) were injected separately at different concentrations. Despite the
low molecular weight of T (288 Da), SPR resolved the interaction and provided a
dissociation constant Kd =174±32 µM (Figure 3A-B). In contrast, no interaction between
PFOA (up to 4 µM) and AR was detected under the same experimental conditions (Figure
3C-D). Higher concentrations of PFOA were not tested because of the limited solubility in
the running buffer. Next, we performed competition experiment to assess whether the
presence of PFOA would reduce the binding of T to AR. We incubated a solution of T with
different concentrations of PFOA and the resulting mixture was flowed over the same ARcoated sensor chip. At the highest concentration tested, we observed a small but significant
(35%) decrease of T binding suggesting that the presence of PFOA reduces the binding of T
to its receptor (Figure 3E).
In a final set of experiments, we aimed to test in vitro the putative inhibitory effect of
PFOA on AR. To this end, nuclear translocation assay was performed on murine Leydig MA10 cells, cultured at different concentrations of T (1 to 100 nM) and PFOA (0.1 to 1 µM),
alone or in combination. Flutamide 1 µM served as negative control. In the positive control,
T elicited a significant (all p <0.001 vs unstimulated cells) and consistent AR nuclear
internalization, with approximately 90% of positive signal within the nucleus, even at the
lowest concentration (Figure 4, 5). By addition of androgen inhibitor flutamide, AR nuclear
signal decreased down to 17.9%, 20.9% and 36.4% at T concentrations of 1, 10 and 100 nM
respectively. A very low signal was detected in cells incubated with PFOA 0.1, 1 or 10 µM
(2.4, 3.4, 7.1%; Figure 4), which however was comparable with that of the negative
unstimulated control (4.1%). When Leydig cells were co-incubated with both PFOA and T, a
dose-dependent inhibition of AR nuclear translocation was observed for increasing
concentrations of PFOA, which was inversely correlated to T concentration (Figure 4, 5): at
the highest T concentration (100 nM), PFOA did not affect AR internalization at any
concentration, but at physiologically relevant T levels (10 nM), AR nuclear signal
significantly decreased at highest PFOA concentration (10 µM). On the other hand, at lower
levels of T (1 nM), PFOA induced a significant reduction of AR internalization at any tested
concentration (Figure 5). A Jonckheere-Terpstra test for ordered alternatives showed that

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This study documents that PFCs have a substantial impact on human male health as they
directly interfere with hormonal pathways potentially leading to male infertility. We found
that increased levels of PFCs in plasma and seminal fluid positively correlate with circulating
T and with a reduction of semen quality, testicular volume, penile length and AGD.
Experimental evidence supports our observational results and points towards an antagonistic
action of PFOA on the binding of T to its natural AR.
The investigation covered an area of around 150 km2 in the provinces of Vicenza and
Padua and to some extent Verona, with 350,000-400,000 people potentially exposed(35,36).
These areas are heavily polluted with concentrations of PFCs up to 6872 ng/L for all PFCs
and up to 3733 ng/L for PFOA alone in surface waters and up to 3138 ng/l for all PFCs and
up to 1886 ng/l for PFOA in drinking water, that is >1000 fold higher than control values (0.5
to 8 ng/L)(37). Compared to median concentrations of PFOA and PFOS in blood serum of
the general population in Italy, we found levels of PFOA more than 5 times higher in plasma
and semen compared to control. Although slightly inferior to the PFCs levels calculated on
13856 subjects aged 14-40, during a surveillance programme promoted by the Veneto from
the most polluted “red zone”(38), our results are consistent with previous findings(35), and
our sensitive LC-MS method is able to differentiate between exposed population and
controls.
Interestingly, the majority of the exposed male population showed a reduction in
testicular volume, penile length, and AGD, but not anthropometrics in males aged 18-19.
These findings could be explained considering that AGD and anthropometric measures are
differentially determined during fetal and pre-pubertal development, respectively(24).
Accordingly, genital development is concomitant with AGD determination(39). Therefore we
could speculate an hypothetic involvement of PFC to in-utero rather than late ED exposure.
Prenatal exposure to androgens during the “masculinization programming window”, a critical
window during testicular development, is positively associated with AGD in mammals(39).
On these bases, AGD has been suggested as a putative marker of prenatal exposure to
chemicals with known anti-androgenic effect, or ED in general. For example, exposure to
phthalates(40), dioxins(41) and bisphenol A(42) has been associated with a reduction in
AGD. As the first report on water contamination of PFCs goes back to 1977(43), the
magnitude of the problem is alarming as it affects an entire generation of young individuals,
from 1978 onwards.
PFCs toxicity also concerns adult life independently from in-utero exposure. This implies
that healthy individuals living in territories contaminated with PFCs could present signs of
toxicity. In vitro and animal studies on PFCs toxicity have shown a detrimental effect of
PFCs on testicular function due to the alteration of steroidogenic machinery and subsequent
defect of spermatogenesis(44–48). Two cross-sectional studies reported negative associations
of PFOS, or high PFOA and PFOS combined, with the proportion of morphologically normal
spermatozoa in adult men(49,50). This is in agreement with our findings in which we observe
a significant reduction in progressive sperm motility in exposed subjects. The exact
mechanism, however, is not clear, and possibly involves an impairment of mitochondrial
activity, as observed in the endocrine disruptor bisphenol A(51).
Another important finding is the association between PFOA and seminal plasma pH,
indicative of an interference of PFCs at a prostatic level. The presence of PFCs in seminal
plasma reported by previous groups (52) and by us suggests either a prostatic or testicular
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there was a statistically significant trend of higher AR nuclear translocation scores with
increasing concentration of T, alone (z =2.416, p =0.016) or in combination with flutamide (z
=3.695, p<0.001) and PFOA, at any tested concentration (all p <0.001).

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Acknowledgments:
We thank all the staff of the Unity of Andrology and Reproductive Medicine at the
University Hospital for the constant support.
Data availability
All data supporting the findings of this study are available within the article and
its Supplementary Information. All other data are available from the corresponding author
upon reasonable request.
Authors contribution:
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origin of PFCs, that could explain a weak association of plasma PFOS concentration with
incident prostate cancer (53). This aspect, however, requires further investigations.
Overall, the inefficient recognition between T and its receptor in the presence of PFCs
could explain the clinical symptoms in the exposed individuals. It would also explain why
higher levels of T are found in exposed subjects, which is a compensatory mechanism, as
supported by increased LH. Interestingly, in the only study that evaluated young males from
an exposed pregnancy cohort, prenatal exposure to PFOA was associated later in adult life
with lower sperm concentration and total sperm count(16). The same study also reported an
alteration of the hypothalamic-pituitary axis, with higher levels of LH and FSH, but not T.
Several experiments provide direct evidence that PFOA inhibits the binding of T to AR.
First, PFOS and PFOA elicit a significant antagonistic effect (~25%) on testosterone-induced
activation of AR in HeLa cells. This result is in agreement with the study by Kjeldsen and
colleagues(18), but in contrast with Behr(54) and Du(55), who have used different cell lines,
reporter plasmids and co-treatment conditions. Second, PFOA diminishes the binding of T to
the purified receptor. Third, PFOA significantly reduces the translocation of AR to the
nucleus in murine Leydig cells. Remarkably, co-incubation of physiological concentrations of
T in adults (10 nM) and PFOA led to a ∼20% reduction of AR nuclear signal, at
concentrations reported in regions with point source drinking water contamination (1
µM)(35,36,56) and in occupationally-exposed fluorochemical workers (10 µM)(57).
Despite the convincing biological effect, the mechanism of inhibition remains elusive and
requires more biochemical investigations to be unveiled. Moreover, quantification of
circulating androgens with more precise methods could unveil further associations with sex
steroids, and given the cross-sectional design of the study, further confounding factors could
be included, such as socioeconomic status. Because of the partial antagonist effect in our
assays, PFOA may act as an allosteric or non-competitive inhibitor thereby blocking
dimerization of the receptor and its translocation to the nucleus. This would explain as to why
SPR experiments failed to monitor the interaction between PFOA and covalently
immobilized monomeric AR. Alternatively, PFOA could interact with T thereby diminishing
the concentration of the bioactive hormone in the circulation. Furthermore, it remains to be
established how PFCs penetrate cells and barriers and what are the mechanisms of clearance.
In conclusion, we present both clinical and experimental evidence supporting the
endocrine disrupting activity of PFCs on androgenic function, which is mediated by the AR.
The interference of PFCs on the binding and activation of T on the AR, could explain the
resulting alterations of seminal parameters and the reduction in testicular volume and penile
length, together with shorter AGD, observed in young males from an exposure area. At the
hormonal level, the reduced activation of T results in increased serum T levels, possibly due
to the positive feedback on the hypothalamic-pituitary axis, as reflected by increased LH.
Importantly, the antagonistic activity on T by PFCs could also extend to other steroids, such
as DHT, progesterone or estradiol thereby affecting early and late development of male
genital tract to the different extents.

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*Corresponding author and person to whom reprint requests should be addressed:
Prof. Carlo Foresta, Department of Medicine, Unit of Andrology and Medicine of
Human Reproduction, University of Padova, Via Giustiniani, 2, 35128 Padova, Italy.
Phone: +39-049-8218517; Fax: +39-049-8218520; e-mail: carlo.foresta@unipd.it

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Disclosure statement:
The authors have declared that no conflict of interest exists.

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A.D.N., I.S. and C.F. designed the study; U.V. and A.G. conducted clinical evaluations;
A.D.N., I.S., M.S.R. performed molecular experiments, M.P., S.T., S.D.A. and D.G.
conducted analytical experiments; L.A. and N.P. conducted biochemical experiments; A.D.N.
, A.G. and C.F. participated to data analyses; A.D.N. and C.F. wrote the manuscript and all
authors approved the final version.

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Figure 1. Total testosterone levels are positively correlated with serum PFOA.
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Figure 2. PFOA and PFOS inhibit androgen receptor transactivation in HeLa
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activity is reported as relative (%) to positive control (T 10 nM, set to 100%). Data are

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Figure 4. Expression of androgen receptor in murine Leydig MA-10 cells under
different conditions. Immunofluorescence of AR (FITC, green) nuclear translocation in
Leydig Ma-10 cells stimulated with T and PFOA at different concentrations as reported in the
figure. Flutamide (1 uM) served as internal control. Nuclei are stained with DAPI (blue).
Cells were visualized by scanning confocal laser microscopy (60x magnifcation).

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Figure 5. Androgen receptor nuclear translocation induced by testosterone is reduced
by PFOA in murine MA-10 Leydig cells. Relative quantification (%) of nuclear
fluorescence intensity with respect to total fluoresce intensity in MA-10 Leydig cells
stimulated with T and PFOA at different concentrations. Results are the mean ± SE. The
Kruskal-Wallis test was used to compare differences between concentrations. The Mann–
Whitney test with Bonferroni correction was used to compare each concentration with the
control. The same procedure was applied to comparisons between different stimuli (PFOA
0.1, 1 and 10 µM and flutamide 1 µM) and T, within each T concentration. *= p<0.05 vs T;
**= p<0.001 vs T; †= p<0.05 vs T 0, 10 and 100 nM; ‡= p<0.05 vs T 0, 1 and 100 nM; §= p
<0.05 vs T 0, 1 and 10 nM; ¥= p<0.05 vs T 0 nM; ç= p<0.05 vs 0 and 1 nM

D

Table 1. Anthropometric and seminal parameters in 171 controls and 212 exposed subjects.
Parameters
Age (yrs.)

A

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LE

Figure 3. Binding of testosterone to immobilized androgen receptor is reduced by
PFOA. Solutions of testosterone (288.42 Da) (A) and PFOA (414.07 Da) (C) were injected
at a flow rate of 30 µl/min at 25°C, using 10mM Hepes pH7.4, 0.15M NaCl containing 3%
MeOH (V/V) as running buffer. Each SPR trace was subtracted for unspecific binding (<2%
of RUmax). The response units (RU) at the steady state were plotted as a function of [analyte]
and fitted to the Langmuir equation to yield the dissociation constant Kd (B, D). No
interaction between PFOA (up to 4mM) and AR was detected under the same experimental
conditions. (E) Next, we performed competition experiment to assess whether the presence of
PFOA would reduce the binding of testosterone to AR. A 250 mM solution of testosterone
was incubated with different concentrations of PFOA (0-4 µM) for at least 10 minutes before
injection over the same AR-coated sensor chip. We observed a ∼35% reduction of
testosterone binding to AR at 4 µM PFOA. Results are shown as the maximal association
response units (RU, expressed as the percentage relative to the response measured without
PFOA) achieved at increasing concentrations of PFOA. (F) Raw data showing the inhibitory
effect of PFOA at 1 µM (red) and the reproducibility of testosterone binding before and after
the competition experiment (black and blue).

Height (cm)

Mea
± SD
n
18.7 ± 1.0

179.2 ± 6.2

Weight (kg)
73.2 ± 8.5
BMI (kg/m²) 22.8 ± 2.3
WC (cm)
81.8 ± 7.1
Arm span
182.1 ± 10.2
(cm)
Crown-topubis
81.8 ± 4.9
distance
(cm)
Pubis-to97.4 ± 5.3
floor

Controls (N=171)*
Media
min-max
n
18.0-24.0
18.0
162.0180.0
192.0
54.0-96.0
73.0
18.2-31.0
22.5
64.0-103.0
81.0
87.0-200.0

(IQR)

(18.0-19.0)
(175.0184.0)
(67.0-78.0)
(21.1-24.0)
(77.0-85.6)
(178.0182.0
187.6)

Exposed (N=212)*
Media
min-max
n
18.0-22.0
18.0
160.0178.8 ± 6.9
179.0
203.0
73.9 ± 11.9 47.0-120.0
73.0
23.1 ± 3.1
16.6-35.8
22.5
84.0 ± 10.5 63.5-140.0
82.0
160.0182.0 ± 8.2
181.5
204.0
Mea
± SD
n
18.5 ± 0.8

(IQR)
(18.0-19.0)
(175.0183.0)
(65.0-80.0)
(21.0-24.5)
(77.0-88.0)
(176.5187.0)

Raw
Adj.
pa
pb
0.081 0.567
0.575

1.0

0.897
0.492
0.174

1.0
1.0
0.87

0.276

1.0

70.0-94.0

82.0 (78.0-85.2)

82.9 ± 5.5

68.0-98.0

83.0 (79.0-86.0)

0.041

0.328

84.5-110.0

97.8 (93.0-101.2)

95.9 ± 5.7

79.0-117.0

96.0 (93.0-100.0)

0.009

0.09

14

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reported as mean±SD of three independent experiments. *p <0.001 calculated with one-way
ANOVA with a Dunnett´s post hoc test with T 10 nM as reference category.

 0.68-1.01

0.8 (0.8-0.9)

0.9 ± 0.1

0.6-1.2

0.9 (0.8-0.9)

7.6-26.5

16.0 (14.1-18.0)

14.7 ± 3.2

6.8-24.5

14.5 (12.5-16.5)

<0.001 <0.001

6.0-13.5

10.0 (8.5-11.0)

8.6 ± 1.7

2.0-13.5

9.0 (8.0-10.0)

<0.001 <0.001

5.0-13.0

10.0 (9.5-10.5)

9.9 ± 1.1

7.0-13.0

10.0 (9.0-10.5)

2.5-7.2

4.5 (4.0-5.0)

4.1 ± 0.9

2.0-7.0

4.0 (3.5-4.5)

0.3-7.5

2.5 (1.5-3.5)

2.6 ± 1.3

0.2-7.0

2.5 (1.5-3.3)

7.0-8.0

7.5 (7.4-7.6)

7.6 ± 0.2

7.0-8.5

7.7 (7.5-7.7)

66.2 ± 53.2

0-327.0

57.0 (25.3-99.0)

0.045

0.180

154.
5

0-817.5

123.0 (43.4-258.0)

0.032

0.160

135.0 (66.0-281.1) 166.8 ±
53.0 (42.0-62.0)

44.1 ± 17.1

0-85.0

44.0 (32.0-57.0)

0-32.0

6.0 (3.0-10.0)

8.2 ± 7.6

0-63.0

6.0 (4.0-10.0)

0-91.0

38.0 (30.0-50.3)

46.8 ± 17.0

0-30.0

6.0 (4.0-12.0)

0-98.0

85.0 (78.0-90.0)

T

0-91.0

R

0-90.0

0.134

0.126

0.804

<0.001 <0.001
0.512

0.568

LE

0-2240

65.0 (33.2-115.6)

IC

0-800.0

0.014

47.0 (35.0-57.0)

<0.001 <0.001

<0.001 <0.001
0.284

0.568

<0.001 <0.001

6.1 ± 4.3

0-20.0

6.0 (2.0-8.0)

0.006

0.036

81.1 ± 9.7

0-95.0

83.0 (78.0-88.0)

0.057

0.180

E

A

IQR: interquartile range 25th – 75th percentiles. Significant p values are in bold. *In the seminal parameters
analyses, 1 subject within each group failed to collect semen and was therefore omitted. aMann-Whitney test
was used to assess differences between groups. bAdjustment for multiple comparisons was calculated with the
Bonferroni-Holm method.

C

Table 2. Sex hormones, PFOA and PFOS levels in serum and semen from 50 controls and 50
exposed subjects, with respective anthropometrics and seminal parameters.
Parameter

4.71 ± 2.08

1.2-8.0

4.70 (3.5-6.6)

0.89 ± 0.7

0.6-1.8

± 4.06

6.8-29.4

± 1.26
± 1.63

1.2-6.6
1.4-8.3
18.233.6
64.0114.0
165.0200.0
71.088.5
87.0106.0

0.82 (0.4-1.3)
(0.080.1
0.11)
(0.08-00.11
13)
(12.918.98
17.9)
2.89 (2.0-3.8)
4.18 (2.9-6.8)
(21.122.19
24.7)
(77.080.75
86.0)
182.7 (178.05 185.8)
(79.082.0
85.0)
(93.097.0
101.1)

0.7-1.0

0.85 (0.8-0.9)

A

Serum PFOA (ng/ml)
Serum PFOS (ng/ml)

0.1 ± 0.01

0.0-0.1

0.11 ± 0.03

0.1-0.2

V

Semen PFOA (ng/ml)
Semen PFOS (ng/ml)

D

Testosterone (nmol/L)
FSH (U/L)
LH(U/L)
BMI (kg/m²)
WC (cm)

Arm span (cm)
Crown-to-pubis distance
(cm)
Pubis-to-floor distance
(cm)
Crown-to-pubis/pubis-tofloor ratio

Controls (N =50)
minMedi
max
an (IQR)

N

Mea
n ± SD

A

ADVANCE ARTICLE: JCEM

distance
(cm)
Crown-topubis/pubis0.8 ± 0.1
to-floor ratio
Testicular
16.1 ± 3.2
Volume (ml)
Penis length
9.7 ± 1.6
(cm)
Penis
circumferenc 10.0 ± 1.0
e (cm)
AGD (cm)
4.5 ± 0.8
Semen
2.7 ± 1.4
volume (mL)
pH
7.5 ± 0.2
Sperm
concentratio
89.2 ± 97.9
n (10⁶/mL)
Total sperm
292.
230.5 ±
6
count (10⁶)
Progressive
51.8 ± 15.5
motility (%)
Non
progressive
7.5 ± 6.5
motility (%)
Immotile
40.2 ± 14.2
sperm (%)
Normal
morphology
7.9 ± 5.8
(%)
Viability (%) 82.4 ± 10.0

DOI: 10.1210/jc.2018-01855

15.4
2
3.03
4.24
23.0
2
82.4
8
182.
19
81.5
3
96.8
0

± 2.86
± 8.42
± 6.61
± 4.17
± 5.20

0.84 ± 0.06

Testicular Volume (ml)

16.8
± 3.61
6

9.7-26.5

Penis length (cm)

10.0 ± 1.87

6.0-13.0

(14.816.13
19.0)
10.0 (9.0-11.0)

15

Exposed (N =50)
minMedi
max
an (IQR)
2.37.35 (4.7-14.9)
156.7
0.0-4.0
1.11 (0.8-1.3)
(0.110.0-5.3
0.24
0.99)
(0.010.06
0.0-1.1
0.11
0.14)
(16.35.27 9.3-35.0 18.98
21.8)
1.53
1.5-7.3
2.99 (2.2-7.0)
1.79
2.0-8.4
5.37 (4.3-7.0)
16.6(20.43.24
22.40
30.5
25.5)
11.5
65.0(76.581.50
2
140.0
85.4)
160.0179.0 (174.27.47
198.0
0 187.0)
74.0(79.04.92
82.0
96.0
84.0)
84.0(90.35.27
95.0
102.0
99.0)
0.730.08
0.86 (0.8-0.9)
1.14
(12.63.32 9.5-24.5 14.00
16.0)

Mea
n ± SD
14.9
25.0
±
9
8
1.11 ± 0.3
0.90
0.67 ±
8

Raw Adj.
pa
pb
<0.00 <0.00
1
1
0.012 0.048
<0.00 <0.00
1
1

0.12 ±

0.916
0.916
<0.00 <0.00
1
1
0.228 0.576
0.003 0.015

19.3
4
3.48
5.47
22.9
0
83.1
3
180.
45
82.1
4
94.5
9

±
±
±
±
±
±
±
±

0.87 ±
14.6
±
7

8.75 ± 1.82

4.0-12.0

9.00 (8.0-10.0)

0.847 1.000
0.757 1.000
0.246 0.738
0.592 1.000
0.064 0.320
0.110 0.440
<0.00 <0.00
1
1
<0.00 <0.00
1
1

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 Penis circumference (cm)

Total sperm count (10⁶)
Progressive motility (%)

± 0.90

9.0-13.0

10.10 (9.9-11.0)

9.65 ± 0.90

7.8-12.0

± 0.9
± 1.91
± 0.21
133.
±
87
347.
±
40
13.3
±
9

2.7-7.2
0.5-9.0
7.0-7.9
6.0800.0

4.2
2.76
7.62
89.7
6
226.
93
55.3
1

2.5-5.7
0.3-6.0
7.0-8.0
6.0264.0

30.085.0

4.50 (4.0-5.2)
2.55 (1.5-3.9)
7.60 (7.5-7.7)
(27.449.50
94.3)
146.2 (70.05 270.3)
(45.054.50
64.8)
4.50 (3.0-9.8)

Non progressive motility
(%)

6.88 ± 6.38

0.0-27.0

Immotile sperm (%)

39.0
12.3
±
8
4

15.068.0

Normal morphology (%)

8.72 ± 5.51

2.0-20.0

Viability (%)

82.4
± 7.81
4

53.092.0

38.0

(30.246.5)

15.8-680
15.088.0

5.06 ± 3.35

1.0-21.0

39.6
16.4
±
3
9

7.0-79.0

7.0 (4.0-12.0)

4.55 ± 2.31

2.0-10.0

(80.089.5)

79.6
± 7.67
9

60.091.0

82.50

th

a

4.00 (3.5-5.0)
3.00 (1.6-3.4)
7.70 (7.6-7.7)
(34.454.50
96.5)
171.5 (77.25 301.7)
(43.057.0
68.0)
4.0 (3.0-6.0)
40.50

(25.553.5)

<0.00
1
0.019
0.373
0.005

<0.00
1
0.114
1.000
0.042

0.771 1.000
0.596 1.000
0.992 1.000
0.106 0.636
0.624 1.000

4.0 (2.0-6.0)

82.0

(75.085.0)

<0.00 <0.00
1
1
0.048 0.336

D

V

A

N

C

E

A

R

T

IC

IQR: interquartile range 25 – 75 percentiles. Significant p values are in bold. Mann-Whitney test was used to
assess differences between groups. bAdjustment for multiple comparisons was calculated with the BonferroniHolm method.

A

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6.1-2240

± 0.8
± 1.37
± 0.23
59.3
±
5
264.
±
38
16.4
±
1

9.50 (9.0-10.0)

16

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AGD (cm)
Semen volume (mL)
pH
Sperm concentration
(10⁶/mL)

10.3
1
4.5
3.09
7.55
92.4
0
241.
78
54.7
3

DOI: 10.1210/jc.2018-01855

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