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Contents lists available at ScienceDirect

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

Semen quality and sperm DNA damage in relation to urinary bisphenol A among
men from an infertility clinic,
John D. Meeker a, , Shelley Ehrlich b , Thomas L. Toth c , Diane L. Wright c , Antonia M. Calafat d ,
Ana T. Trisini b , Xiaoyun Ye d , Russ Hauser b,c
a

Department of Environmental Health Sciences, University of Michigan, Ann Arbor, MI, United States
Department of Environmental Health, Harvard School of Public Health, Boston, MA, United States
Vincent Memorial Obstetrics and Gynecology Service, Massachusetts General Hospital, Boston, MA, United States
d
Centers for Disease and Control and Prevention, Atlanta, GA, United States
b
c

a r t i c l e

i n f o

Article history:
Received 28 January 2010
Received in revised form 16 April 2010
Accepted 12 July 2010
Available online xxx
Keywords:
Biomarkers
Endocrine disruption
Environment
Epidemiology
Estrogenic
Exposure
Fertility
Male
Reproduction

a b s t r a c t
Bisphenol A (BPA) impairs spermatogenesis in animals, but human studies are lacking. We measured
urinary BPA concentrations, semen quality, and sperm DNA damage (comet assay) in 190 men recruited
through an infertility clinic. BPA was detected in 89% of samples, with a median (interquartile range
[IQR]) concentration of 1.3 (0.82.5) ng/mL. Urinary BPA concentration was associated with slightly elevated, though not statistically signicant, odds for below reference sperm concentration, motility, and
morphology. When modeled as continuous dependent variables, an IQR increase in urinary BPA concentration was associated with declines in sperm concentration, motility, and morphology of 23% (95%CI
40%, 0.3%), 7.5% (17%, +1.5%), and 13% (26%, 0.1%), respectively, along with a 10% (0.03%, 19%)
increase in sperm DNA damage measured as the percentage of DNA in comet tail. In conclusion, urinary
BPA may be associated with declined semen quality and increased sperm DNA damage, but conrmatory
studies are needed.
2010 Elsevier Inc. All rights reserved.

1. Introduction
Bisphenol A (BPA) is a high production volume chemical used
in the manufacture of polycarbonate plastics, which can be used
in baby and water bottles, and epoxy resins used in food container linings and other applications; the use of these products can
lead to human exposure [1]. Over 6 billion pounds of BPA are produced annually, with a 610% growth in demand expected per year
[2]. Dietary ingestion is considered the primary source of general
population exposure to BPA. Other exposure sources may include

Work supported by grants ES009718 and ES00002 from the National Institute
of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH). The
authors gratefully acknowledge Amber Bishop, Tao Jia, and Jack Reidy (CDC, Atlanta,
GA) for measuring the urinary concentrations of BPA.
Disclaimer: The ndings and conclusions in this report are those of the author
and do not necessarily represent the ofcial position of the Centers for Disease
Control and Prevention.
Corresponding author at: Department of Environmental Health Sciences, University of Michigan School of Public Health, 6635 SPH Tower, 109 S. Observatory St.,
Ann Arbor, MI 48109, United States. Tel.: +1 734 764 7184; fax: +1 734 936 7283.
E-mail address: meekerj@umich.edu (J.D. Meeker).

water, air, and dust [1,3]. As a result, there is widespread general

population exposure to BPA [1,4]. BPA has been shown to alter
endocrine function through multiple pathways [5], and a number of
animal studies have reported adverse reproductive effects in males
exposed to low levels of BPA in early life or in adulthood [6]. To our
knowledge no human studies of BPA exposure and semen quality
or sperm DNA damage have been conducted to date. In the present
study we assessed the relationship between urinary BPA concentrations, which have been used as a biomarker of exposure to BPA
[4], and semen quality and sperm DNA damage in men recruited
through a United States (US) infertility clinic.
2. Methods
Subjects were recruited during 20002004 from an ongoing study on the relationship between environmental agents and reproductive health. Participating men
were partners in subfertile couples seeking treatment from the Vincent Andrology Lab at Massachusetts General Hospital. The study was approved by the Human
Studies Institutional Review Boards of the Massachusetts General Hospital, Harvard School of Public Health, the Centers for Disease Control and Prevention (CDC),
and the University of Michigan. After the study procedures were explained and all
questions answered, subjects signed an informed consent. Men between the ages of
1855 years without post-vasectomy status who presented to the Andrology Laboratory were eligible to participate. Of those approached, approximately 65% consented.

0890-6238/$ see front matter 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.reprotox.2010.07.005

Please cite this article in press as: Meeker JD, et al. Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from
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Most men that declined to participate in the study cited lack of time on the day of
their clinic visit as the reason for not participating.
2.1. Urinary BPA
A single spot urine sample was collected from each subject on the day of their
clinic visit in a sterile polypropylene cup. Because BPA is metabolized and excreted
from the body rapidly, and a single urinary measure likely reects an individuals
exposure in the hours to days leading up to urine sample collection [7], a second and
third urine sample were collected from a subset of men. These samples were generally collected between 1 week and 2 months following the rst sample at a follow-up
clinic visit. After measuring specic gravity (SG) using a handheld refractometer
(National Instrument Company, Inc., Baltimore, MD, USA), each urine sample was
divided in aliquots and frozen at 80 C. Samples were shipped on dry ice overnight
to the CDC, Atlanta, Georgia, USA, where the total urinary concentration of BPA (free
plus conjugated species) was measured using online solid-phase extraction (SPE)
coupled to isotope dilution high performance liquid chromatography (HPLC)
tandem mass spectrometry (MS/MS) on a system constructed from several HPLC
Agilent 1100 modules (Agilent Technologies, Wilmington, DE) coupled to a triple
quadrupole API 4000 mass spectrometer (Applied Biosystems, Foster City, CA) [8].
First, 100 L of urine was treated with -glucuronidase/sulfatase (Helix pomatia,
H1; Sigma Chemical Co., St. Louis, MO) to hydrolyze the BPA-conjugated species.
BPA was then retained and concentrated on a C18 reversed-phase size-exclusion
SPE column (Merck KGaA, Germany), separated from other urine matrix components using a pair of monolithic HPLC columns (Merck KGaA), and detected by
negative ion-atmospheric pressure chemical ionization-MS/MS. The limit of detection (LOD) for BPA in a 0.1-mL urine sample was 0.4 ng/mL. Low-concentration
(4 ng/mL) and high-concentration (20 ng/mL) quality control materials, prepared
with pooled human urine, were analyzed with analytical standards, reagent blanks,
and unknown samples [8]. Analysts at the CDC were blind to all information concerning subjects. BPA concentrations were corrected for urine dilution by specic
gravity (SG) using the following formula: Pc = P[(1.024 1)/SG 1)], where Pc is the
SG-adjusted urinary BPA concentration (ng/mL), P is the observed BPA concentration, and SG is the specic gravity of the urine sample. SG was measured using a
handheld refractometer (National Instrument Company, Inc., Baltimore, MD, USA).
2.2. Semen sample collection
Semen was collected on site at Massachusetts General Hospital in a sterile plastic
specimen cup after a recommended period of abstinence of 48 h. After liquefaction at 37 C for 30 min, semen quality parameters and motion characteristics were
measured at the clinic. The remaining unprocessed semen was frozen in 0.25 mL
cryogenic straws (CryoBiosystem, I.M.V. Division, San Diego, CA) by immersing
the straws directly into liquid nitrogen (196 C). Previous work in our laboratory
showed that this freezing method produced comet assay results that were highly
correlated with results from fresh, unfrozen samples [9]. Semen samples were later
analyzed in batches, where straws were thawed by gently shaking in a 37 C water
bath for 10 s and the semen was immediately processed for the comet assay.
2.3. Semen quality
In the MGH Andrology Laboratory, trained clinical staff analyzed semen samples
for sperm concentration, total sperm count, and motion parameters using computeraided semen analyzer (CASA, HTM-IVOS Version 10HTM-IVOS, Beverly, MA, USA).
Setting parameters and the denition of measured sperm motion parameters for
CASA were established by the Hamilton-Thorn Company. To measure both sperm
concentration and motility, 5 L of semen from each sample was placed into a prewarmed (37 C) Makler counting chamber (Se Medical instruments, Haifa, Israel).
A minimum of 200 sperm cells from at least four different elds was analyzed from
each specimen. Total sperm count (106 /ejaculate) was calculated by multiplying
sperm concentration (106 /mL) by semen sample volume (mL). Motile sperm was
dened as World Health Organization (WHO) grade a sperm (rapidly progressive
with a velocity 25 m/s at 37 C) plus b grade sperm (slow/sluggish progressive
with a velocity 5 m/s but <25 m/s) [10]. Measurement of CASA motion characteristics has been previously described [11,12]. Of seven CASA variables that were
measured, only three were chosen (straight-line velocity (VSL), curvilinear velocity
(VCL) and linearity (LIN = VSL/VCL 100)) for inclusion in the present analysis due
to a high degree of dependence between several of the measures.
At least two slides were made for each fresh semen sample. The resulting thin
smear was allowed to air dry for an hour before staining with a Diff-Quik staining kit (Dade Behring AG, Dudingen, Switzerland). Morphological assessment was
performed with a Nikon microscope using an oil immersion 100 objective (Nikon
Company, Tokyo, Japan). A minimum of 200 sperm cells was counted from two slides
for each specimen. Strict Kruger scoring criteria were used to classify men as having
normal or below normal morphology [13].
2.4. Neutral comet assay
The comet assay procedure used in the present study to assess sperm DNA damage has been previously described [14,15]. Briey, 50 L of a semen/agarose mixture

(0.7% 3:1 high resolution agarose; Amresco, Solon, OH, USA) was embedded between
two additional layers of agarose on microgel electrophoresis glass slides (Erie Scientic, Portsmouth, NH, USA). Slides were then immersed in a cold lysing solution to
dissolve the cell membrane and make chromatin accessible for the enzyme digestion
steps. After 1 h cold lysis, slides were transferred to a solution for enzyme treatment with 10 g/mL of RNase (Amresco, Solon, OH, USA) and incubated at 37 C for
4 h. Slides were then transferred to a second enzyme treatment with 1 mg/mL proteinase K (Amresco, Solon, OH, USA) and incubated at 37 C for 18 h. The slides were
placed on a horizontal slab in an electrophoretic unit, equilibrated for 20 min, and
underwent electrophoresis for 1 h. DNA in the gel was then precipitated, xed in
ethanol, and dried. Slides were stained and observed with uorescence microscope.
Comet extent, tail distributed moment (TDM), and percent DNA located in the tail
(Tail%) were measured on 100 sperm in each semen sample using VisComet software
(Impuls Computergestutzte Bildanalyse GmbH, Gilching, Germany). Comet extent
is a measure of total comet length from the beginning of the head to the last visible
pixel in the tail. Tail% is a measurement of the proportion of total DNA that is present
in the tail. TDM is an integrated value that takes into account both the distance and
intensity of comet fragments:
TDM =


(I X)


I is the sum of all intensity values that belong to the head, body, or tail,
where
and X is the x-position of the intensity value.
2.5. Statistical analysis
Data analysis was performed using SAS version 9.1 (SAS Institute Inc., Cary, NC,
USA). Descriptive statistics on subject demographics were calculated, along with
the distributions of urinary BPA concentrations, semen quality, and sperm DNA
damage measures. To investigate differences between distributions or categories
and the potential for confounding, bivariate analysis was conducted between urinary BPA concentrations, and semen quality, sperm DNA damage, and demographic
variables. Differences were tested statistically using parametric or non-parametric
methods where appropriate. We also downloaded the most recent publicly available
urinary BPA data reported by the US National Health and Nutrition Examination Survey (NHANES 20052006) to compare BPA concentrations in our study with those
measured among men in the same age range in the general population [16].
The association between urinary BPA concentrations and sperm concentration,
motility and morphology were rst assessed by multiple logistic regression, where
subjects were dichotomized as either below or equal/above WHO reference levels for
total sperm count (40 million sperm), sperm concentration (20 million sperm/mL)
and sperm motility (50% motile sperm) [10]. The strict Kruger criteria (4% normal) was used as a cutoff for sperm morphology [13]. Comparison subjects were
those that were above the reference level for all three parameters. Urinary BPA
concentrations were lognormally distributed and transformed by the natural logarithm (ln) prior to statistical analysis. Samples with BPA concentrations below the
LOD were assigned a value equal to 1/2 the LOD in the statistical analyses. In all
models, specic gravity was included as a continuous variable to adjust for urinary dilution. Age, body mass index (BMI), race, abstinence period, smoking, and
time of day of the clinic visit (time urine/semen samples were collected: morning [9:00 a.m.12:59 p.m.] vs. afternoon [1:00 p.m.4:00 p.m.]) were considered as
covariates, and were included or excluded from models based on biologic and statistical considerations [17]. Covariates were included in the model if they are known
to be biologically important (i.e. abstinence period) or they acted as a confounder
(i.e. changed the BPA parameter estimate by >10%) in any of the statistical models; all models were adjusted for the same covariates for consistency. To improve
interpretability, all models were adjusted for the same covariates and effect estimates were expressed as odds ratios (OR) associated with an interquartile range
(IQR) increase in urinary BPA concentrations.
Multiple linear regression was also used to assess associations between urinary BPA concentrations and continuous measures of semen quality, sperm motion
parameters, and sperm DNA damage. Total sperm count and sperm concentration
were transformed using the natural logarithm, whereas all other semen quality,
sperm motion, and DNA damage measures were modeled untransformed. Urinary
BPA concentrations were also ln-transformed. The same covariates listed above
for the logistic regression models were considered, and specic gravity was again
included as a continuous variable in all models to adjust for urinary dilution. To
improve interpretability, the regression coefcients were back transformed and
expressed as a change in the dependent variable (i.e., semen quality or sperm DNA
damage measures) for an interquartile range (IQR) increase in urinary BPA concentrations.
Four sets of models were constructed: (1) using only urinary BPA concentrations
from a single urine sample collected on the same day as the semen sample; (2) using
the geometric mean urinary BPA concentration for each participant, where between
one and three values were used to calculate each individuals geometric mean (i.e.
the geometric mean for men with only one urine sample was equal to that single
value); (3) using the geometric mean urinary BPA concentration among only participants that contributed BPA data from at least two urine samples; and (4) using
only the single urinary BPA measure collected on the same day as the semen sample

Please cite this article in press as: Meeker JD, et al. Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from
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Table 1
Demographic categories by semen parameters (N = 190 men).
Comparison
subjects (N = 76)a , b

Semen parameters

Total sperm count <40

million (N = 32)
Age, mean (SD)
BMI, mean (SD)

36.4 (4.1)
27.3 (5.2)

Sperm concentration
<20 million/mL (N = 37)

36.5 (5.8)
29.0 (5.5)

Sperm motility <50%

motile (N = 103)a

36.9 (6.0)
28.4 (4.4)

Sperm morphology
<4% normal (N = 49)a

36.9 (5.3)
27.4 (4.1)

36.4 (5.1)
28.0 (3.9)

Abstinence time, n (%)

3 days
40 (53)
4 days
14 (19)
5 days
8 (11)
6 or more days
13 (17)

22 (69)
4 (13)
4 (13)
2 (6)

24 (65)
4 (11)
5 (14)
4 (11)

54 (53)
29 (28)
9 (9)
10 (10)

22 (46)
15 (31)
3 (6)
8 (17)

Race, n (%)
White
Black/Afr-Amer
Hispanic
Other

68 (89)
3 (4)
1 (1)
4 (5)

24 (75)
1 (3)
1 (3)
6 (19)

27 (73)
1 (3)
2 (5)
7 (19)

84 (82)
3 (3)
4 (4)
8 (8)

40 (82)
2 (4)
1 (2)
6 (12)

Smoking status, n (%)

Never smoker
Ever smoker
Former
Current

54 (71)
22 (29)
16 (21)
6 (8)

18 (56)
14 (44)
9 (28)
5 (16)

18 (51)
18 (48)
12 (32)
6 (16)

63 (61)
40 (39)
30 (29)
10 (10)

29 (61)
19 (39)
13 (27)
6 (12)

a
b

Information on abstinence period missing for 1 man.

Comparison group consists of men with all three semen parameters above the WHO reference level.

among men with BPA data from at least two urine samples. The reason for including approach (4) was to assess the utility of collecting subsequent urine samples by
comparing effect estimates between approaches (3) and (4). In a sensitivity analysis,
the multivariable models were rerun after excluding men with highly concentrated
or highly dilute urine samples (SG above 1.03 or below 1.01) [18]. Models were
also rerun using specic gravity-corrected BPA concentrations rather than using
uncorrected urinary BPA concentrations but including specic gravity as a covariate. In addition, we assessed non-linear relationships of urinary BPA concentrations
with semen quality and sperm DNA damage parameters by categorizing urinary BPA
concentrations into quartiles in both linear and logistic regression models. Finally,
we modeled relationships between urinary BPA and semen quality parameters in a
stratied analysis where effect estimates from men with at least one semen parameter below WHO reference values were compared to those among men with all
semen parameters above WHO reference.

3. Results
Urinary BPA concentrations, specic gravity, and semen quality parameters from samples collected at the same clinic visit were
available for 190 men. Of these men, a second urine sample was
later collected from 78 of them, and a third urine sample was
collected from 4 men. As a result, the total number of urine samples collected and analyzed for BPA was 272. The amount of time
between consecutive urine samples ranged from 3 to 75 days,
with a median (25th, 75th percentile) of 29 (27, 34) days. Demographic variables stratied by semen quality reference values are
presented in Table 1. None of the demographic variables signicantly differed between semen quality groups (p-values > 0.05). The
distribution of semen quality, sperm motion, and sperm DNA damage measures are presented in Table 2. Because the comet assay
was introduced into the study at a later time than the assessment of

Table 2
Distribution of semen quality, sperm motion parameters, and comet assaymeasured DNA damage (N = 190).
Parameter

Mean

Selected percentiles
10th

Semen quality
Total sperm count (106 )
Concentration (106 /ml)
Motility (%)
Morphology (% normal)
Sperm motion
Straight-line velocity (m/s)
Curvilinear velocity (m/s)
Linearity (%)
Sperm DNA damagea
Comet extent (m)
Tail distributed moment (m)
Percent DNA in Tail (%)
a

25th

50th

75th

90th

407
145
66
10

643
225
77
14

279
99.6
45.8
7.2

22.0
8.8
13
1

68.7
23.8
26
3

186
64.1
49
7

42.4
73.0
55.2

26.2
46.7
46

35.7
62.8
53

43.6
73.7
57

51.8
87.7
63

59.8
100.6
68

149
62.7
40.0

94.0
41.4
17.4

111
50.2
23.6

143
60.6
40.2

179
74.6
50.4

215
87.4
66.0

N = 132 for DNA damage measures.

semen quality, DNA damage measures were only available for 132
of the men. The distribution of urinary BPA concentrations for the
194 urine samples collected on the same day as the semen sample
is presented in Table 3. BPA was detected in 89% of urine samples analyzed in the study. The geometric mean BPA concentration
in the present study (1.4 ng/mL) was signicantly lower than the
geometric mean concentration from adult men in the same age
range (1855 years) from the US general population reported in
NHANES 20052006 (2.3 ng/mL; p < 0.00001). Among the 78 men

Table 3
Distribution of uncorrected and SG-corrected BPA in urine collected from men at initial clinic visit in the present study, and in men 1855 years of age from NHANES
20052006 (ng/mL).
N

Geometric mean

Selected percentiles
10th

Uncorrected BPA
SG-corrected BPA

194
190b

1.4
1.7

Uncorrected BPA, men aged 1855 from NHANES 20052006

540

2.3

a
b

NDa
NDa
0.7

25th

50th

75th

90th

0.8
1.0

1.3
1.6

2.5
2.9

5.7
5.8

95th
9.3
8.2

1.4

2.3

4.1

7.7

12.2

Max
36.4
29.1
150

ND = non-detectable, assigned a value of 1/2 the limit of detection for statistical analysis. Limit of detection for BPA was 0.4 ng/mL.
Specic gravity not measured in 4 samples.

Please cite this article in press as: Meeker JD, et al. Semen quality and sperm DNA damage in relation to urinary bisphenol A among men from
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Table 4
Adjusted odds ratios (95% condence intervals) for below reference semen quality parameter associated with an interquartile range increase in urinary BPA concentration.
Adjusted for specic gravity, age, BMI, abstinence period, current smoking, and time of urine sample.
Semen quality parameter

(1) BPA measure from

same day as semen sample
only (n = 190)a , d
Odds ratio (95%CI)

(2) Geometric mean BPA

from all participants
(n = 190)b , d
Odds ratio (95%CI)

(3) Geometric mean BPA

from men with 2 BPA
measures (n = 78)c , d
Odds ratio (95%CI)

(4) BPA from same day as

semen among men with 2
BPA measures (n = 78)a , d
Odds ratio (95%CI)

Total sperm count (<40 106 )

Sperm concentration (<20 million/mL)
Sperm motility (<50% motile sperm)
Sperm morphology (<4% normal)

1.20 (0.71, 2.03)

1.47 (0.85, 2.54)
1.23 (0.83, 1.80)
1.25 (0.77, 2.06)

0.98 (0.57, 1.68)

1.21 (0.69, 2.11)
0.96 (0.63, 1.46)
1.01 (0.60, 1.72)

0.30 (0.04, 2.69)

0.37 (0.05, 2.63)
0.74 (0.33, 1.64)
0.98 (0.35, 2.70)

1.50 (0.36, 6.22)

1.18 (0.35, 3.94)
1.54 (0.83, 2.84)
1.70 (0.73, 3.95)

a
b
c
d

Among men with urine sample from same visit as blood (hormone) sample only. N = 190.
Geometric mean of up to 3 repeated urine samples per subject, some of which were collected months after semen sample. N = 190.
Geometric mean value among men with at least 2 urine samples. N = 78.
Ln-transformations of bisphenol A concentration were used in the models.

from whom 2 urine samples were collected, BPA concentrations

in the two samples were weakly correlated (Spearman r = 0.18;
p-value = 0.10). Limiting this analysis to urine samples collected
within the median duration between repeated urine sample collection within the same individual (29 days) did not signicantly
strengthen this correlation.
When considering semen quality, sperm motion parameters,
and DNA damage measures as continuous variables, BMI was
inversely associated with Tail% and abstinence period was positively associated with total sperm count and sperm concentration
(p-values < 0.05). Geometric mean uncorrected urinary BPA concentration was also higher among men whose urine sample was
collected in the afternoon (1.6 ng/mL) compared to men providing
the urine sample in the morning (1.1 ng/mL; p = 0.02). The difference in BPA concentrations between urine samples collected in
the afternoon and morning was slightly lessened when comparing
SG-corrected concentrations (1.9 ng/mL vs. 1.5 ng/mL; p = 0.09).
Crude logistic and linear regression results (not shown) were
similar to the adjusted results presented in Tables 4 and 5. All
logistic/linear regression results in Tables 4 and 5 were adjusted
for urinary specic gravity, age, BMI, current smoking, abstinence
period, and time of day of clinic visit (time of urine/semen sample collection). As shown in Table 4, there were no consistent
relationships between urinary BPA concentrations and odds for
below reference semen quality parameters, though odds ratios
were somewhat elevated for below reference sperm concentration
in statistical approach 1 (rst column of odds ratios) and for below
reference sperm motility and morphology in statistical approach 4
(fourth column of odds ratios). Results from statistical approach 3
(third column of odds ratios) differed as reduced odds were found,
though the reported odds ratios were not statistically signicant
and considered unstable due to the reduced sample size.
In multivariable linear regression models using only urinary BPA
concentrations measured in the urine sample collected on the same
day as the semen sample (statistical approach 1, rst column of
regression coefcients in Table 5), we observed inverse associations
between urinary BPA concentrations and sperm concentration,
motility, morphology, VSL and VCL. For the median values of sperm
concentration (64 million/mL), motility (49% motile), and morphology (7% normal) for the study population, the coefcients represent
23% (95%CI 40% to 0.3%), 7.5% (95%CI 17% to +1.5%), and
13% (95%CI 26% to 0.1%) declines in these parameters, respectively, for an IQR increase in urinary BPA concentration (IQR 0.8
to 2.5 ng/mL). Urinary BPA concentration was also positively associated with Tail%, where an IQR increase in concentration was
associated with a 10% (95%CI 0.03% to 19%) increase in Tail% relative to the study population median. In sensitivity analyses, effect
estimates from the multivariable models were similar when including only men with SG 1.01 and 1.03 were included (n = 154
for semen quality parameters, n = 105 for DNA damage measures;
results not shown). Results were also similar when modeling

SG-corrected BPA concentrations as the independent variable as

compared to including SG as a covariate when modeling uncorrected BPA concentrations (not shown).
When urinary BPA concentrations were categorized into quartiles to assess potential non-linear relationships, there was no
evidence for elevated odds ratios for below reference semen
quality parameters in logistic regression models (not shown). Conversely, in multivariable linear regression models of semen quality
parameters as continuous dependent variables, there were suggestive trends for non-monotonic declines in sperm concentration,
motility and morphology, and a signicant increasing trend in
Tail%, when categorizing urinary BPA concentration into quartiles
(Fig. 1ad). BPA quartiles were not associated with total sperm
count (not shown; p-value for trend = 0.57). There were also statistically suggestive, though non-monotonic, relationships between
urinary BPA concentration quartiles and reduced VSL, VCL, and LIN
(results not shown; p-values for trend ranged from 0.05 to 0.13).
Results from the multivariable regression models using geometric mean BPA concentrations from multiple urine samples per
participant (statistical approaches 2 and 3, the second and third
columns of effect estimates in Tables 4 and 5), differed somewhat
from the models using only urine samples collected on the same day
as the semen samples. However, with the exception of the models
for sperm concentration and motility using statistical approach 3,
in which effect estimates changed in sign or direction, effect estimates were consistent though somewhat attenuated compared to
statistical approach 1. Effect estimates obtained from approach 4
(using only BPA concentration from the same day as the semen
sample and only from men with at least 2 urine samples) were also
consistent with approaches 1, 2, and 3, with the exception of sperm
concentration and motility effect estimates from approach 3. Wider
condence intervals were observed with approaches 3 and 4 as a
result of the smaller sample size, and these effect estimates should
be considered less stable.
Finally, we observed differences in effect estimates from multivariable linear regression models among (i.e., stratied by) men
with (n = 114) or without (n = 76) at least one semen parameter
below WHO reference values. When considering only BPA concentrations measured in urine samples collected on the same day
as the semen sample (statistical approach 1), an IQR increase urinary BPA was associated with a 27% decline in sperm concentration
(p = 0.048), a 6% decline in sperm motility (p = 0.2), and a 16% decline
in sperm morphology (p = 0.04) among men with at least one semen
parameter below WHO reference values compared to little change
[2% (p = 0.8), 2% (p = 0.6), and +1% (p = 0.9) for sperm concentration, motility and morphology, respectively], among men with all
semen parameters above WHO reference values. These results were
consistent when using geometric mean BPA concentrations (statistical approach 2), as the associations between urinary BPA and
sperm concentration (p = 0.06), motility (p = 0.10), and morphology
(p = 0.03) were stronger among men with at least one parameter

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(1) BPA measure from

same day as semen sample
only (n = 190)a , d

(2) Geometric Mean BPA

from all participants
(n = 190)b , d

(3) Geometric mean BPA

from men with 2 BPA
measures (n = 78)c , d

(95%CI)

p-Value

(95%CI)

p-Value

(95%CI)

p-Value

(95%CI)

p-Value

Semen quality
Sperm countd , e
Concentrationd , e
Motilityf
Morphologyf

0.85 (0.63, 1.14)

0.77 (0.60, 1.00)
3.70 (8.12, 0.74)
0.90 (1.79, 0.004)

0.27
0.047
0.10
0.049

0.92 (0.66, 1.28)

0.82 (0.62, 1.09)
2.43 (7.31, 2.45)
0.73 (1.70, 0.26)

0.61
0.17
0.33
0.15

1.38 (0.90, 2.12)

1.23 (0.80, 1.92)
0.74 (7.49, 8.97)
0.84 (2.55, 0.88)

0.14
0.33
0.86
0.33

0.93 (0.67, 1.30)

0.87 (0.64, 1.20)
3.92 (9.98, 2.13)
1.05 (2.33, 0.23)

0.66
0.41
0.20
0.11

Sperm motion
VSLf
VCLf
LINf

1.79 (4.00, 0.42)

3.97 (7.65, 0.27)
0.80 (0.85, 2.44)

0.11
0.04
0.34

2.69 (5.71, 0.35)

4.55 (9.62, 0.54)
1.17 (4.26, 1.92)

0.08
0.08
0.46

3.78 (8.75, 1.17)

2.85 (11.5, 5.79)
0.38 (5.14, 4.37)

0.13
0.51
0.87

3.27 (6.93, 0.38)

5.04 (11.5, 1.43)
0.27 (3.19, 3.73)

0.08
0.12
0.88

0.19
0.58
0.048

4.02 (23.8, 15.8)

3.18 (9.17, 2.81)
3.67 (2.79, 10.1)

0.69
0.29
0.26

5.47 (20.0, 8.95)

0.80 (5.19, 3.60)
2.88 (1.73, 7.49)

0.45
0.72
0.22

DNA damageg
Comet extentf
TDMf
Tail%f
a

3.99 (6.43, 14.4)

1.12 (2.39, 4.63)
3.88 (0.01, 7.74)

0.45
0.53
0.048

8.11 (3.98, 20.2)

1.13 (2.93, 5.20)
3.65 (0.04, 8.83)

Among men with urine sample from same visit as semen sample only. N = 190.
Geometric mean of up to 3 repeated urine samples per subject, some of which were collected months after semen sample. N = 190.
c
Geometric mean value among men with at least 2 urine samples. N = 78.
d
Ln-transformations of bisphenol A, total sperm count, and sperm concentration were used. All other variables were modeled untransformed.
e
Coefcient represents a multiplicative change in sperm concentration for an IQR change in BPA concentration after back-transformation of both sperm concentration and BPA values. For an IQR change in BPA concentration,
a coefcient equal to 1.0 indicates no change in sperm concentration, a coefcient < 1.0 indicates a multiplicative decrease in sperm concentration, and a coefcient > 1.0 indicates a multiplicative increase in sperm concentration.
f
Coefcient represents the change in semen quality parameter or sperm DNA damage measure for an IQR change in urinary BPA concentration after back-transformation of urinary BPA concentration. For an IQR change in
BPA concentration, a coefcient equal to 0 indicates no change in semen/sperm measure, a coefcient < 0 indicates a decrease in semen/sperm measure, and a coefcient > 0 indicates an increase in semen/sperm measure.
g
N = 132 for comet assay measures in statistical approaches (1) and (2), N = 78 for comet assay measures in approaches (3) and (4).
b

J.D. Meeker et al. / Reproductive Toxicology xxx (2010) xxxxxx

(4) BPA from same day as

semen sample among men
with 2 BPA measures
(n = 78)a , d

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Table 5
Adjusted linear regression coefcients (95% condence interval) for change in semen quality or sperm DNA damage measure associated with an interquartile range increase in urinary BPA concentration. Adjusted for specic
gravity, age, BMI, abstinence period, current smoking, and time of urine sample.

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Fig. 1. Adjusted regression coefcients for a change in semen quality parameter or sperm DNA damage measure associated with increasing quartiles of urinary BPA concentration (n = 190): (a) ln-transformed sperm concentration (p-value for trend = 0.09); (b) sperm motility (p-value for trend = 0.04); (c) sperm morphology (p-value for
trend = 0.13); (d) Tail% (p-value for trend = 0.03). Adjusted for specic gravity, age, BMI, abstinence period, current smoking status, and time of day of urine sample.

below WHO reference compared to men with all parameters above

WHO reference (all p-values > 0.4).

4. Discussion
We found that urinary BPA concentrations measured in spot
urine samples collected on the same day as a semen sample were
associated with suggestive declines in semen quality parameters
and with increased sperm DNA damage (measured as Tail%). For the
semen quality parameters, these associations were only observed
when modeling the parameters as continuous variables in linear
regression models but not when dichotomizing them according
to widely accepted WHO reference values in logistic regression
models. Also, in linear regression models stratied by semen quality status, the relationships were only found among men with at
least one semen parameter below WHO reference values. When
one or two subsequent urine samples collected in the weeks and
months following the semen sample were taken into consideration
the associations were inconsistent and weakened, perhaps due to
reduced statistical power since repeated urines were only available
among a subset of the men, or because the repeated samples were
collected outside the most relevant exposure window of interest
for these outcome measures (i.e. weeks and months prior to the
semen sample).
Our ndings of suggestive relationships between urinary BPA
concentrations and semen quality parameters are consistent with
animal studies reporting adverse effects on Sertoli cell function and
sperm production in relation to BPA exposure [1929]. Though
some of these studies were conducted in vitro or report effects
stemming from in utero or early postnatal BPA exposure, and thus
limit our ability to directly compare with our ndings, several
studies have assessed effects following in vivo exposure in adulthood [2023]. Sakaue et al. [20] showed that oral administration
of BPA to adult SpragueDawley rats at concentrations as low as

20 g/kg bw/day resulted in a reduction in daily sperm production of up to 40%. This was conrmed in a later study on adult
Swiss mice exposed to 25100 ng/kg bw/day for 1 month. Daily
sperm production, epididymal sperm concentration, and fertility
were signicantly decreased in the exposed groups compared to
controls [21]. Toyama et al. [22] also noted spermatid abnormalities
in adult Wistar rats and CD-1 mice injected with 20 g/kg bw/day
for 6 days. Finally, there was a dose-dependent reduction in epididymal sperm motility and sperm count in male rats following
ingestion of 0.2, 2, and 20 g/kg bw/day of BPA for a period of 45
days [23].
In the present study, we found evidence for an association
between urinary BPA concentrations and increased Tail%, but no
associations between urinary BPA concentrations and comet extent
or TDM. Inconsistent results between the various DNA damage
measures obtained by the neutral comet assay in relation to the
same independent variable have been observed in previous studies, and it has been hypothesized that the different comet assay
parameters may reect different types of DNA strand breaks [30].
Specically, a lack of correlation between TDM and Tail% has been
reported, and it was hypothesized that a high TDM may be more
likely to be associated with double-strand breaks, whereas a high
Tail% may reect single-strand breaks [30]. Thus, in the present
study, the positive association between urinary BPA concentration
and Tail% may reect a relationship between BPA exposure and
single-strand breaks. While BPA has expressed genotoxicity in a
number of in vitro and in vivo models [31,32], ndings have not
been fully consistent and details of the direct mechanisms involved
remain unclear. However, the adverse effects of BPA on adult male
reproduction may be through the induction of oxidative stress and
depletion of antioxidant defense mechanisms, as was reported in
epididymal sperm of rats orally dosed with BPA [23].
Urinary BPA concentrations in the present study were lower
than those reported for adult men in the same age range from the US
general population in NHANES 20052006 (Table 3). In our study

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the median and 95th percentile BPA concentrations (uncorrected

for specic gravity) were 1.3 and 9.3 ng/mL, respectively, compared
to 2.3 and 12.2 ng/mL among the 540 male participants aged 1855
years in NHANES 20052006. One potential reason for the discrepancy may involve the inclusion of urine samples collected in the
evening in NHANES, which had higher BPA concentrations than
samples collected in the afternoon in earlier NHANES datasets [4].
In addition, differences in the distribution of race/ethnicity and
income between the two study populations may also contribute
to the difference in urinary BPA concentrations. For example,
in NHANES 20032004 urinary BPA concentrations were higher
among non-hispanic blacks compared to non-hispanic whites, and
higher among participants with lower household incomes [4]. Differences in study years should also be considered. Urine samples
in the present study were collected in years 20002004 compared
with 20052006 in the most recent NHANES data. However, urinary
BPA levels reported in the previous NHANES investigation (NHANES
20032004) were even higher than in NHANES 20052006 [4].
Since the present study was conducted among men recruited
through an infertility clinic it may limit our ability to generalize the
results to the general population. However, the men were members
of couples seeking infertility diagnosis and treatment potentially
related to a male factor, a female factor, or both, resulting in a study
population that included both fertile men and men with a range of
fertility problems. In addition, in order for the generalizability of
our results to be limited, men recruited through an infertility clinic
would have to respond differently (i.e. be more or less susceptible)
to BPA exposure compared to men not in couples seeking infertility
diagnosis/treatment. Although we are not aware of evidence showing that men from an infertility clinic are more sensitive to BPA
exposure, we did nd stronger relationships between urinary BPA
concentrations and reduced semen quality parameters among men
with at least one parameter below WHO reference values. Thus,
our data suggest the possibility that men with subfertility are more
sensitive to BPA-related effects than men with normal fertility. As
more studies are published on BPA exposure and semen quality, it
is important to consider the subject populations when interpreting
the results.
We found that repeated urine samples collected from the same
man weeks to months apart from one another were weakly correlated (r = 0.18). Thus, another limitation in the present study is
the likelihood of exposure measurement error due to the high
within-individual temporal variability in BPA exposure and the
availability of multiple BPA measures from only a subset of participants. However, measurement error would be expected to be
non-differential, which would tend to reduce the ability to detect
associations between exposure and outcome. In addition, when
using broad exposure categories (e.g. quartiles in Fig. 1), a single
measure may adequately predict an individuals exposure category
over a longer period of time [7]. We also collected only a single
semen sample from each man, and the reliability of a single semen
sample to represent semen quality over a longer period of time is
not well characterized. However, two recent reports provide evidence that one sample may be representative of semen quality over
several weeks in large epidemiologic studies [33,34].
Another limitation of our study was its cross-sectional design
due to the availability of only a single semen sample from each
participant, as well as the availability of only a single urine sample from over half of the men. Thus, we cannot rule out reverse
causation in the explanation of our ndings in the event that an
underlying condition that causes poor semen quality may also lead
to altered BPA metabolism.
In conclusion, human exposure to BPA may be associated with
reduced semen quality and increased sperm DNA damage. However, due to some inconsistencies in our results between statistical
and exposure assessment approaches, these relationships need to

be assessed in other large and appropriately designed human epidemiologic studies that measure BPA in multiple urine samples
across the exposure window of interest.
Conict of interest statement
The authors declare that there are no conicts of interest.
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