Are urinary bisphenol A levels in men

related to semen quality and embryo

development after medically
assisted reproduction?
Jure Knez, M.D.,a Roman Kranvogl, B.Sc.,b Barbara Pregl Breznik, B.Sc.,a Ernest Voncina, Ph.D.,b
and Veljko Vlaisavljevic, Ph.D., M.D.a
a
Department of Reproductive Medicine and Gynaecologic Endocrinology, University Medical Centre Maribor; and
b
Institute of Public Health, Environmental Protection Institute, Maribor, Slovenia

Objective: To evaluate whether urinary bisphenol A (BPA) levels in men adversely influence semen quality and embryo development
after medically assisted reproduction.
Design: Prospective, cohort study.
Setting: University-based tertiary care center.
Patient(s): A total of 149 couples undergoing their first or second IVF or intracytoplasmic sperm injection (ICSI) procedure.
Intervention(s): None.
Main Outcome Measure(s): Semen quality and embryo development parameters until the blastocyst stage after the IVF or ICSI
procedure.
Result(s): Bisphenol A was detected in 98% (n ¼ 146) of the samples with 0.1 ng/mL limit of detection. The geometric mean BPA con-
centration was 1.55 ng/mL. After the adjustment for potential confounders using linear regression models, an increase of natural log-
arithm transformed urinary BPA concentration was associated with lower natural logarithm transformed sperm count (b ¼ 0.241,
95% confidence interval [CI] 0.470 to 0.012), natural logarithm transformed sperm concentration (b ¼ 0.219, 95% CI 0.436
to 0.003), and sperm vitality (b ¼ 2.660, 95% CI 4.991 to 0.329). The embryo development parameters from oocyte fertilization
to the blastocyst formation stage were not affected by BPA exposure.
Conclusion(s): Urinary BPA concentrations in male partners of subfertile couples may influ-
ence semen quality parameters, but do not affect embryo development up to the blastocyst stage
after medically assisted reproduction. (Fertil SterilÒ 2014;101:215–21. Ó2014 by American So- Use your smartphone
ciety for Reproductive Medicine.) to scan this QR code
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B
isphenol A (BPA) is a monomer, most tested individuals (3, 4). It is A number of studies in laboratory
widely used in the production of considered as one of the potent rodents have demonstrated adverse
polycarbonate plastic and epoxy substances among the group of effects of BPA on male reproductive
resins. Hence, the exposure of the pop- endocrine-disrupting chemicals and function (7). However, the data are
ulation is ubiquitous (1, 2) and multiple its estrogenic effects in vitro are well less conclusive when exposed to low
studies have detected its presence in established (5, 6). concentrations of BPA (<50 mg/kg/d)
(8), as several studies failed to prove
Received August 5, 2013; revised September 15, 2013; accepted September 20, 2013; published online any effects on the male reproductive
October 29, 2013. system (9–12). Nevertheless, the
J.K. has nothing to disclose. R.K. has nothing to disclose. B.P.B. has nothing to disclose. E.V. has
nothing to disclose. V.V. has nothing to disclose.
studies investigating BPA's effect on
Supported by the Slovenian Research Foundation, grant P3-334-0327. human reproductive system are
Reprint requests: Jure Knez, M.D., Department of Reproductive Medicine and Gynaecologic Endocri- limited. The correlation between BPA
nology, University Medical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia (E-mail:
knez.jure@gmail.com). and free androgen index has been
demonstrated on the population of
Fertility and Sterility® Vol. 101, No. 1, January 2014 0015-0282/$36.00
Copyright ©2014 American Society for Reproductive Medicine, Published by Elsevier Inc.
fertile men; however, no connection
http://dx.doi.org/10.1016/j.fertnstert.2013.09.030 to sperm quality was evident (13). In

VOL. 101 NO. 1 / JANUARY 2014 215

ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY
contrast, a study by Li et al. (14) has shown that
environmental exposure to BPA adversely affects semen
quality parameters. Supporting these data, Meeker et al. (15)
have shown that exposure to BPA exerts a negative effect
on sperm quality parameters and sperm DNA damage in
men visiting infertility clinics.
At present only a few studies investigating the correlation
between environmental BPA exposure and embryo develop-
ment after medically assisted reproduction have been published.
Recently, it was discovered that female urinary BPA concentra-
tions are inversely correlated to E2 levels before follicular aspira-
tion, to the number of retrieved mature oocytes, and show a
negative trend toward lower quality embryo development
parameters (16, 17). The same research group also established
a correlation of the female environmental BPA exposure with
the implantation failure after IVF (18). In addition, an inverse,
although insignificant, association between male BPA
exposure and cleavage stage embryo quality has been
suggested in a study by Bloom et al. (19). The study was based
on a modestly sized sample of 27 couples. The goal of our
study was to investigate whether there exists a correlation
between male urinary BPA concentrations as a biomarker of
exposure to BPA (3), semen quality, and embryo development
parameters until the blastocyst stage after the IVF or
intracytoplasmic sperm injection (ICSI) procedure.
MATERIALS AND METHODS
Study Design and Eligibility Criteria
The prospective, observational cohort study included couples
seeking infertility treatment at the Department of Reproduc-
tive Medicine and Gynaecologic Endocrinology, University
Medical Centre, Maribor, Slovenia. Patients were recruited be-
tween February 2011 and June 2012. During the time of the
recruitment, the stimulation protocols and success rates in
terms of clinical pregnancies at the clinic were unchanged.
An informed consent was signed by both partners at the
time of recruitment. Patients also completed a questionnaire,
collecting the data on medical history, occupation, and life-
style. The study was approved by the National Medical Ethics
Committee of Slovenia.
During their first or second IVF or ICSI attempt, with the
female partner younger than 36 years of age, couples were
invited to participate in the study. Before the invitation, they
underwent a routine fertility evaluation according to our clin-
ical practice. Female partners diagnosed with polycystic
ovarian syndrome (PCOS) following the Rotterdam criteria
(20), the evidence of endometriosis, or FSH levels more than
10 mIU/mL in the early follicular phase of the menstrual cycle,
were not considered for the study. Only couples undergoing
oocyte retrieval were included. Low responding patients
with less than five retrieved oocytes were subsequently
excluded from the analysis of the BPA influence on embryo
development. There was a possibility of inferior embryo devel-
opment parameters in this group of patients that could bias the
results (21, 22). Male partners underwent clinical examination
and a routine semen analysis before the inclusion. Patients
with azoospermia, with the history of cryptorchidism or
varicocoele were not considered for recruitment.
216
Semen Analysis
Semen samples were collected on the day of follicle aspira-
tion. After liquefaction, the semen samples were analyzed
for volume, sperm concentration, motility, and vitality ac-
cording to the World Health Organization criteria (23). The
sperm concentration was assessed with an improved Neuba-
uer hemocytometer, according to the 2010 World Health Or-
ganization guidelines. Sperm motility was assessed with the
sperm class analyzer (Microoptics), using the SCA 5.0 soft-
ware. The analysis was performed in 20-mm-deep Leja two-
chamber slides, according to 2010 World Health Organization
guidelines (23). Each chamber was filled with 6.5 mL of the
semen sample. The tracks of at least 200 motile spermatozoa
were analyzed in six representative fields per chamber. Vital-
ity was assessed using the hypoosmotic swelling test. Seminal
smears were prepared for Papanicolau staining and were later
assessed for morphology, according to the strict Tygerberg
criteria (24). In addition to the standard measures of semen
quality, hyaluronan-binding assay was performed, following
the manufacturer's instructions (Origio). All measurements
were performed by a single, trained, clinical staff member
and every sample was processed within 1 hour of ejaculation
on the morning of oocyte retrieval.
Ovarian Stimulation and Embryo Development
Monitoring
Patients were treated with either long GnRH agonist (triptor-
elin; Diphereline) or GnRH antagonist (cetrorelix, Cetrotide;
Merck Serono) protocol, described in detail in our previous
publications (25). Briefly, in the case of the long agonist treat-
ment, the administration of 0.1 mg of triptorelin was started
on day 21 of the previous menstrual cycle, whereas in the
case of the antagonist protocol, 0.25 mg of cetrorelix was
started in a fixed protocol starting on day 7 of the menstrual
cycle. Ovarian stimulation was started on day 2 of the cycle by
administering a starting dose of 150 IU of recombinant FSH
(Gonal-F; Merck Serono). The dose could be adjusted on day
6 of the stimulation, according to the ovarian response, as
demonstrated by the ultrasound. Final oocyte maturation
was accomplished by administering 6,500 IU of hCG when
at least three follicles of 17 mm were observed by ultrasound.
Oocytes were retrieved 35 hours after the hCG administration.
An IVF or ICSI procedure was performed and a successful
fertilization of the oocytes was defined as the presence of
two pronuclei (2PN) 18–20 hours after the procedure. The em-
bryos were cultured in the BlastAssist System sequential em-
bryo culture media (Origio) for 5 days. Until the day of the ET,
the quality of embryos was assessed by an experienced
embryologist on a daily basis. A day-3 embryo was consid-
ered optimal if it consisted of at least eight cells without mul-
tinucleation and had less than 20% fragments. The
blastocysts were graded according to our established grading
system, as previously described (26). In brief, the blastocyst
was considered optimal if it was fully expanded and the blas-
tocoel completely filled the embryo. It contained a cohesive
trophectoderm and a compact inner cell mass (ICM) (26).
The ET was performed on day 5 after oocyte retrieval.
VOL. 101 NO. 1 / JANUARY 2014

Urinary Bisphenol A Measurements
Spot urine samples of male partners were collected after the
sperm collection on the morning of oocyte retrieval. Sterile
polypropylene cups (Corning) were used for the urine collec-
tion and were previously tested to be BPA-free. All samples
were collected in the morning hours, between 7 and 9 AM.
An aliquot of sample was separated to determine urinary
creatinine concentration. Samples were immediately frozen
to -80 C and stayed frozen until the BPA testing. The testing
was conducted 2–6 months after the samples were obtained.
The frozen samples were shipped to the Institute of Public
Health, Maribor, Slovenia, for analysis.
The analysis was performed as a part of a larger study,
investigating the concentrations of several endocrine-
disrupting chemicals. The urinary concentrations of the sum
of free and conjugated BPA were measured using gas chroma-
tography coupled with the mass spectrometry method. The
urine sample (1 mL) was incubated with b-glucoronidase at
40 C for 90 minutes. The sample solution was then acidified
with 75 mL of HCl, which was previously extracted with di-
chloromethane and hexane. After adding sodium chloride
(150 mg) and acetonitrile, the sample solution was mixed
and then centrifuged. The next step was drying the sample
with azeotropic distillation because of the use of acetonitrile.
The dry residue with analytes was extracted using dichlorome-
thane and then the sample was loaded on a SiO2 column (with
10% HCl, sodium sulfate was added for water elimination),
which had been preconditioned with dichloromethane (5
mL). A fraction of endocrine compounds was eluted with
dichloromethane (3  10 mL). Dichloromethane was then
evaporated to dryness. A derivatization was performed with
N-methyl-N-trimethylsilyl trifluoroacetamide and penta-
fluoropyridine at 60 C for 30 minutes. An aliquot of sample
solution (1 mL) was injected into a gas chromatography
coupled with the mass spectrometry system. The extracts
were analyzed on a HP 6890 Gas Chromatograph with a
5,973 Mass Selective Detector (Agilent Technologies). The de-
tector was used in ion monitoring mode. The limit of detection
for BPA was 0.1 ng/mL urine. The matrix-matched calibration
curves were linear over the range from limit of quantification
to 200.0 ng/mL for the BPA. Linearity, expressed as the corre-
lation coefficient (R2), was 0.9996 for the linear range. To
determine the background levels of BPA originating from
the sample preparation, a blank test was carried out using
hexane-washed water instead of urine. To verify the absence
of analytes, representative specimen cups, tubes, pipette tips,
and autosampler vials were prescreened and found to be endo-
crine compound-free (%limit of detection). The quality con-
trol materials were prepared from pooled urine samples from
anonymous donors in the concentration level of 50 ng/mL.
A typical daily sample batch included two reagent blanks,
8–10 unknown samples, and one quality control sample.
Statistical Analysis
Basic demographic statistics were calculated, along with the
distributions of urinary BPA concentrations (adjusted and un-
adjusted for urinary creatinine concentration). A bivariate
analysis among urinary creatinine-adjusted BPA concentra-
VOL. 101 NO. 1 / JANUARY 2014
Fertility and Sterility®
tions, semen quality, and embryo development parameters
was conducted, to assess the potential correlation among
the parameters. Differences were tested using parametric
and nonparametric methods where appropriate.
Generalized linear modeling and multiple linear regres-
sion models were used to identify and calculate the coeffi-
cients for factors independently related to the urinary BPA
concentration. The BPA and urinary creatinine concentra-
tions showed skewed (non-normal) distributions and were
transformed using the natural logarithm before the analyses.
Samples with BPA concentration below the limit of detection
were assigned a value of limit of detection/O2 in the statisti-
cal analyses before transformation (27). In all models, BPA
was adjusted by urinary creatinine concentration (BPA
divided by urinary creatinine concentration and expressed
as nanograms per miligram creatinine). Considering sperm
quality parameters, total sperm count, and sperm concentra-
tion were also log-normally distributed and were trans-
formed by the natural logarithm before the statistical
analyses, whereas all the other variables were modeled
untransformed.
Age, body mass index (BMI), abstinence period, average
weekly alcohol consumption, and smoking were considered
as covariates and were included in the model if they were
known to be biologically important (i.e., abstinence period)
or if they acted as a confounder in any of the models (i.e.,
changed BPA parameter estimate by >10%). For consistency,
models assessing the BPA impact on sperm quality were
adjusted for the same covariates. The following covariates
were included in the final models: male age, male BMI, smok-
ing status (current smoker vs. noncurrent smoker), abstinence
period, and average alcohol consumption (units of alcohol/
week). All linear regression models were evaluated for quality.
Homoscedasticity was checked by plotting the standardized
residuals as the function of predicted values. Likewise, the
plots were constructed to check the normal distribution of
the residuals. Durbin-Watson statistic was checked to assess
for possible serial correlation.
Generalized linear models were used to assess the BPA
impact on embryo development and were adjusted for the
method of oocyte insemination (IVF or ICSI), male age, and
BMI, as well as for female age and BMI. In the sensitivity anal-
ysis, all models were rerun using the urinary creatinine con-
centration as a separate covariate, instead of using urinary
creatinine-adjusted BPA. In addition, nonlinear relationships
of urinary BPA concentrations with semen quality and em-
bryo development parameters were assessed using the gener-
alized linear model, categorizing urinary creatinine-adjusted
BPA concentrations into quartiles, and presenting the results
as the estimated mean values of the dependent variable asso-
ciated with the appropriate BPA exposure category adjusted
for confounders. Data were analyzed using the SPSS 20.0
(SPSS Inc.) statistical software package.
RESULTS
Characteristics of Patients
Of 195 invited couples, 32 declined participation. Most
of them expressed the lack of time as the reason for
217
ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY

nonparticipation. Seven couples were later excluded because

TABLE 1
of inadequate response to ovarian stimulation, one couple
conceived spontaneously awaiting treatment, and six cou- Patients' demographic characteristics and oocyte treatment
ples did not commence with the treatment plan and were outcome (N [ 149).
lost to follow-up for unknown reasons. In total, 149 couples
Characteristic Data
satisfying the inclusion criteria were included in the final
analysis. Of these, 12 couples were subsequently excluded Female age (y) 30.95 (2.96)
Female BMI (kg/m2) 21.93 (2.29)
from the embryo development analysis due to a low response Male age (y) 34.05 (4.76)
to ovarian stimulation (<5 retrieved oocytes). Furthermore, Male BMI (kg/m2) 27.45 (3.52)
20 couples opted for cleavage stage ET, leaving 117 couples Male smoking (%)
with all embryos cultured to the blastocyst stage Never/ever 76 (51.0) / 73 (49.0)
Current smoker (yes) 51 (34.2)
(Supplemental Fig. 1, available online). According to our Female current smoking (yes) 36 (24.2)
center policy, these were the couples with a low number of Male average alcohol consumption 2.9 (3.3)
optimal quality embryos (<3) available on day 3 after oocyte (units/wk)
IVF/ICSI treatment indication
fertilization. They decided not to proceed to blastocyst stage Tubal factor infertility 50 (33.6)
ET after a consultation with a doctor and an embryologist. Male factor 67 (45.0)
However, the BPA concentration in the 20 male partners opt- Unexplained 45 (30.2)
ing for cleavage stage ET was fairly evenly distributed; three Oocyte treatment (no. of patients)
IVF 13 (8.7)
of these men were in the lowest quartile; eight were in quar- ICSI 55 (36.9)
tile 2; one man was in quartile 3 and eight men were in quar- IVF/ICSI (on sibling oocytes) 81 (54.4)
tile 4. The mean urinary creatinine-adjusted BPA levels were Average no. of oocytes retrieved 12.26 (6.84)
comparable in both groups (2.29  2.22 ng/mg vs. 2.17  Fertilization rate (%) 76.7 (22.3)
Optimal day 3 embryos/2PN (%) 38.3 (31.4)
3.55 ng/mg for days 3 and 5 transfer, respectively; Blastocyst formation/2PN (%)a 52.0 (31.6)
P¼ .446). Therefore, it did not appear that these couples Optimal blastocysts/all blastocysts (%)a 28.2 (31.0)
differed significantly from the whole studied group in terms Note: Data are mean (SD) or number (%) unless otherwise noted. 2PN ¼ normally fertilized
oocytes; BMI ¼ body mass index; ICSI ¼ intracytoplasmic sperm injection.
of BPA exposure. a
n ¼ 117.
The average male age was 34.05  4.76 years, whereas Knez. Male BPA exposure and embryo development. Fertil Steril 2014.
the female partners were about 3 years younger in average
(30.95  2.96 years). In approximately one-third of the cou-
ples, tubal factor infertility was diagnosed; in another 45% the sperm by the Spearman rank correlation coefficient
male factor infertility was involved. The urinary BPA con- (P< .05). The sperm samples from men with higher average
centrations between groups were not statistically signifi- weekly alcohol consumption (Spearman rank correlation
cantly different regarding the treatment indications. coefficient) as well as from smokers compared with non-
However, although not of statistical significance, in the smokers (Mann-Whitney U test) exhibited lower ability to
group with male factor infertility, the mean creatinine- bind to hyaluronan (P< .05). Univariate representation of
adjusted BPA level was higher at 2.72  4.59 ng/mg BPA correlation to sperm quality parameters is provided
compared with couples with tubal factor infertility at 1.74 in Supplemental Table 1 and Supplemental Fig. 2, available
 1.81 ng/mg or when infertility was unexplained at 1.80 online.
 1.47 ng/mg (P¼ .229). The average BMI of male partners In multivariable regression models, BPA has shown an
was 27.45  3.52 kg/m2; 69% of all men were overweight inverse association to total sperm count, sperm concentra-
(BMI >25 kg/m2). One-third of male partners were identified tion, and sperm vitality (P< .05; Table 3). This means that
as current smokers. The mean urinary BPA concentrations for a natural logarithm unit increase in BPA, a decrease of
in smokers were not statistically significantly higher 0.241 units in natural logarithm total sperm count can be
compared with nonsmokers (2.76  5.21 ng/mg vs. 1.90  anticipated (b ¼ 0.241, 95% confidence interval, 0.470
1.58 ng/mg; P¼ .396). Patients' characteristics are presented to 0.012; P¼ .039). The removal of the confounding vari-
in Table 1. ables from the model increased the magnitude of BPA coef-
Bisphenol A was detected in 98% (n ¼ 146) of the tested ficient by 11.1%. In the sensitivity analysis, which included
samples. Geometric mean BPA concentration was 1.55 ng/mL urinary creatinine as a covariate rather than an adjusted uri-
for the whole tested population, which are the first data on nary BPA by creatinine, similar results were obtained (data
BPA exposure in the population of Slovenian men (Table 2). not shown).
The mean urinary creatinine concentration was 133.4  When urinary BPA concentrations were categorized
70.7 mg/dL (median, 124.4 mg/dL). into quartiles, a trend toward lower sperm concentration
and total sperm count with increasing BPA concentration
quartiles was still present, although not of statistical sig-
Bisphenol A Concentration Effect on Sperm nificance (Supplement Table 2, available online). The sig-
Quality nificant association between the increasing BPA exposure
Examining sperm quality parameters in a bivariate anal- and lower sperm vitality was retained also in this
ysis, the abstinence period was positively associated with analysis, but the magnitude of the effect was small
the volume of the semen sample and the concentration of (P¼ .024).

218 VOL. 101 NO. 1 / JANUARY 2014

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TABLE 2

Distribution of unadjusted and urinary creatinine adjusted urinary bisphenol A (BPA) levels (n [149).
Geometric mean 5th centile 25th centile 50th centile 75th centile 95th centile
BPA (ng/mL) 1.55 0.30 0.81 1.64 3.27 6.68
BPA/urinary creatinine concentration (ng/mg) 1.33 0.24 0.69 1.40 2.63 5.66
Knez. Male BPA exposure and embryo development. Fertil Steril 2014.

Bisphenol A Concentration Effect on Embryo to investigate the association between male environmental
Development BPA exposure and embryo development until the blastocyst
stage after medically assisted reproduction.
Oocyte fertilization rates and embryo development parame-
Bisphenol A is an endocrine disruptor with a weak affin-
ters until the blastocyst stage are presented in Table 4. None
ity for estrogen (E) receptors (28), but it can also exert its
of the considered parameters showed any association to
effects through interference with the androgen production
increasing BPA concentrations. When oocytes were insemi-
(29–32) or directly with Sertoli cell function (33, 34). Many
nated by ICSI, compared with conventional IVF, a significant
animal studies are performed in vitro (35) or report of in
impact on a higher fertilization rate, a lower proportion of
utero or early postnatal BPA exposure (36) and are difficult
optimal day 3 embryos, and on the rate of blastocyst forma-
to correlate to adult exposure, but several animal studies
tion was observed. Hence, the fertilization rate and the em-
have also been performed after in vivo exposure in
bryo development parameters were modeled separately for
adulthood (37–39). Recently, also the evidence of human
IVF or ICSI, but in these analyses, BPA again showed no as-
epidemiologic studies is beginning to accumulate. A
sociation to any of the observed outcomes (data not shown).
possible negative influence of BPA on semen quality
Although all female partners were younger than 36 years, fe-
parameters was demonstrated, especially in occupationally
male age was negatively associated to the proportion of
exposed men (14) and in male partners of couples visiting
optimal day 3 embryos as well as to the proportion of optimal
infertility clinic (15). However, these correlations were not
blastocysts. Sensitivity analysis, including urinary creatinine
confirmed on the population of fertile men (partners of
as an independent covariate in the models, rather than adjust-
pregnant women) (13). Our study population was also
ing urinary BPA by creatinine concentrations, revealed
comprised of couples seeking infertility treatment and it is
similar results. Furthermore, BPA concentrations were cate-
possible that the partners of subfertile couples respond
gorized into quartiles, but also in these models, no association
differently to BPA exposure compared with men in couples
to any of the measured parameters was observed (Supplement
not seeking infertility treatment. Susceptible men, with
Table 3, available online).
possibly already impaired spermatogenesis, might be more
prone to harmful effects compared with men with intact
DISCUSSION spermatogenesis when exposed to environmental BPA. Still,
The results of our study demonstrate that environmental male the possible negative effect of BPA, observed in our
BPA exposure may influence semen quality parameters, but analysis, is likely to be small and when categorized to
does not affect embryo development parameters after an quartiles, not of statistical significance. When assessing
IVF or ICSI procedure. To our knowledge, this is the first study linear associations between continuous variables such as

TABLE 3

Adjusted linear regression coefficients (95% confidence intervals) for change in semen quality parameters associated with urinary BPA
concentration (n [ 149).
Variable Urinary BPA concentration b (95% CI) R2 P value
Sample volume (mL) a
0.065 (0.345 to 0.215) b
0.093 .647
Sperm concentration (106/mL)b 0.219 (0.436 to 0.003)a 0.071 .047
Total sperm count (106)b 0.241 (0.470 to 0.012)a 0.092 .039
Progressively motile sperm (%)a 1.870 (4.439 to 0.699)b 0.032 .152
Nonprogressively motile sperm (%)a 0.134 (1.237 to 0.968)b 0.033 .810
Total motile sperm (106)b 0.273 (0.537 to 0.009)a 0.075 .043
Morphologically normal sperm (%)a 0.293 (1.233 to 0.648)b 0.026 .539
Sperm vitality (%)a 2.660 (4.991 to 0.329)b 0.052 .026
HBA (%)a 2.354 (6.408 to 1.700)b 0.091 .253
Note: Adjusted for male age, BMI (kg/m2), current smoking status (yes/no), alcohol consumption (units/week), and abstinence period (days). BMI ¼ body mass index; BPA ¼ bisphenol A; CI ¼
confidence interval; HBA ¼ hyaluronan-binding assay; ln ¼ natural logarithm.
a
Urinary creatinine-adjusted BPA concentration ln-transformed.
b
Both variables ln-transformed.
Knez. Male BPA exposure and embryo development. Fertil Steril 2014.

VOL. 101 NO. 1 / JANUARY 2014 219

ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY
TABLE 4
Marginal effects (95% confidence intervals) for change in oocyte
fertilization and embryo development parameters associated with
urinary BPA concentration, adjusted for male and female age, BMI
(kg/m2), and oocyte insemination technique (IVF/ICSI), (n [ 137).
Urinary BPA concentration P
Variable marginal effect (95% CI) value
Fertilization rate (%) 0.014 (0.060 to 0.087) .716
Proportion of optimal day 3 0.018 (0.086 to 0.122) .732
embryos (%)
Blastocyst formation rate (%)a,b 0.053 (0.157 to 0.050) .312
Proportion of optimal 0.037 (0.167 to 0.093) .577
blastocysts (%)a,c
Note: BMI ¼ body mass index; BPA ¼ bisphenol A; CI ¼ confidence interval; ICSI ¼ intracy-
toplasmic sperm injection.
a
b
Data collected for 117 subjects.
Number of blastocysts per number of normally fertilized oocytes.
c
Number of optimal blastocysts per number of all formed blastocysts.
Knez. Male BPA exposure and embryo development. Fertil Steril 2014.
BPA and sperm quality, categorization of the variables is
suboptimal and compromises study power (40), but it was
used to enhance the data presentation and to evaluate
possible statistical nonlinear relationships.
Although our results acknowledge the possible negative
influence of BPA on sperm quality in the partners of subfer-
tile couples, we did not observe any disturbance in the devel-
opment of the embryos after IVF or ICSI to the blastocyst
stage at environmental levels of BPA exposure demon-
strated in our population. These findings are in contrast
with the previously suggested association of blood serum
unconjugated BPA concentrations to the quality of the em-
bryos at the cleavage stage (19). Considering the embryo
development physiology, the potential paternal negative ef-
fect on embryo development should be evident at the time of
embryonic genome activation, 3 days after oocyte fertiliza-
tion (41). We aimed to assess the possible BPA influence
through extended, blastocyst culturing of embryos. In fact,
it has been shown that possible damage to the sperm DNA
can be negatively correlated to blastocyst development
(41–43). However, if the damage to the sperm is low, the
oocyte can use its repair mechanisms and preserve the
development of the embryo (44, 45). It has to be
acknowledged that although we did not observe any trends
toward poorer embryo development with increasing BPA
exposure in our population, we cannot exclude the
possibility of subtle influences that could be elucidated
from a larger sample size analysis.
We used urine matrix as a method of BPA detection in our
study, which has been shown to be more accurate in detecting
the actual BPA exposure of an individual with a much lower
chance of potential errors or the contamination of samples
compared with the detection in the blood serum (46). There
are also differences in unconjugated BPA and total BPA
determination, which we measured. To assess BPA exposure
from all sources, total BPA measurement has been considered
to be more appropriate (4).
To reduce the possible confounding factors on embryo
development, our study design included only good prog-
nosis couples, with young female partner, undergoing their
220
first or second treatment attempt. Thus, the strong point of
our trial is that the possible negative female fertility factors
on embryo development were diminished. However, this
also means that a very robust population with good prog-
nosis was studied and the potential impact of male BPA
on the development of embryos among older women could
not be assessed in our study, which could present a potential
selection bias. In addition, when investigating the influence
of environmental contaminant, such as BPA, it should be
mentioned that both partners are exposed to the substance.
In fact, it has been shown that urinary BPA concentrations
are more similar among individuals who are partners than
those who are not partners (47). We could not account for
possible female BPA exposure influence on embryo devel-
opment parameters.
A potential limitation of our study was that we used a sin-
gle urine sample to assess BPA exposure. However, Mahalin-
gaiah et al. (47) observed that despite intraperson variability
in urinary BPA concentrations, a single urine sample is pre-
dictive of long-term exposure (weeks to months) and provides
good sensitivity to classify individuals in epidemiologic
studies (47). Potential selection bias and uncontrolled con-
founding are of concern in our trial as in other studies inves-
tigating environmental influences, semen quality, and
assisted reproduction outcome.
To conclude, our results suggest that low environmental
levels of BPA in male partners of subfertile couples may affect
semen quality. However, embryo development to the blasto-
cyst stage after medically assisted reproduction procedures
is unlikely to be influenced. Nevertheless, whereas environ-
mental BPA exposure could be of only minimal importance
for embryo development by itself, it still remains to be inves-
tigated whether it could contribute to the additive effects of
the mixture of endocrine disrupting compounds or even
lead to potentially greater-than-additive effects.
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ORIGINAL ARTICLE: ENVIRONMENT AND EPIDEMIOLOGY

SUPPLEMENTAL FIGURE 1

Patient flow chart.

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SUPPLEMENTAL FIGURE 2

Correlation between urinary creatinine-adjusted BPA concentration and sperm quality parameters.
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SUPPLEMENTAL TABLE 1

Basic semen characteristics for the bisphenol A (BPA) exposure quartiles (N[149).
BPA quartile 25th 50th 75th 100th
Sample volume (mL) 3.81 (1.87) [1.29–9.78] 3.77 (1.79) [1.31–9.96] 3.84 (1.99) [1.04–9.23] 3.44 (1.49) [0.40–7.41]
Sperm concentration (106/mL) 55.8 (49.63) [2.2–223.0] 45.9 (45.7) [0.6–183.0] 36.6 (32.6) [0.7–138.0] 36.9 (40.3) [0.3–157.0]
Total sperm count (106) 203.5 (235.9) [8.8–1,232.3] 182.9 (224.2) [2.2–1,095.6] 134.8 (139.6) [6.5–705.2] 123.2 (129.1) [1.3–471.4]
Progressively motile 31.3 (14.3) [4.3–63.0] 31.0 (18.3) [3.1–68.5] 25.9 (14.8) [3.2–59.6] 26.4 (15.2) [2.8–74.1]
sperm (%)
Nonprogressively motile 21.4 (4.5) [13.0–30.7] 20.9 (7.5) [5.7–33.8] 20.7 (7.1) [5.7–37.2] 21.3 (7.6) [5.3–40.2]
sperm (%)
Morphologically normal 6.06 (4.13) [0–14.0] 7.00 (5.70) [0–21.0] 5.09 (4.36) [0–15.0] 5.90 (5.73) [0–21.0]
sperm (%)
Sperm vitality (%) 79.2 (9.2) [57.0–92.0] 73.5 (14.2) [41.0–92.0] 72.2 (11.1) [51.0–89.0] 71.7 (11.5) [49.0–89.0]
HBA (%) 69.7 (24.9) [2.0–96.0] 67.9 (27.0) [0–96.0] 70.4 (22.2) [3.5–97.0] 63.3 (27.6) [0–97.4]
Note: Data are means (SD), [range]. HBA ¼ hyaluronan-binding assay.
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SUPPLEMENTAL TABLE 2

Estimated marginal mean values (with corresponding 95% confidence intervals) of sperm quality variables for the bisphenol A (BPA) exposure
quartiles (n [ 149).
P
BPA quartile 25th 50th 75th 100th value
Sample volume (mL) 3.81 (3.24–4.38) 3.77 (3.20–4.34) 3.84 (3.27–4.41) 3.44 (2.88–4.00) .747
3.80 (3.25–4.35) 3.80 (3.25–4.36) 3.70 (3.12–4.28) 3.42 (2.88–3.97) .748a
Sperm concentration (106/mL) 55.8 (42.2–69.3) 45.9 (32.4–59.4) 36.6 (23.1–50.1) 36.9 (23.6–50.3) .158
54.6 (41.2–68.0) 45.1 (31.5–58.7) 33.7 (19.6–47.7) 36.7 (23.3–50.0) .144a
Total sperm count (106) 203.5 (143.7–263.3) 182.9 (123.1–242.7) 134.8 (75.0–194.6) 123.2 (64.2–182.9) .190
198.3 (140.3–256.4) 184.3 (125.4–243.1) 122.2 (61.4–183.0) 124.4 (66.6–182.3) .156a
Progressively motile sperm (%) 31.3 (26.3–36.4) 31.0 (26.0–36.0) 25.9 (20.9–30.9) 26.4 (21.5–31.3) .271
32.1 (27.0–37.1) 31.0 (26.0–36.1) 25.5 (20.2–30.7) 25.7 (20.7–30.7) .144a
Nonprogressively motile sperm (%) 21.4 (19.2–23.6) 20.9 (18.7–23.0) 20.7 (18.5–22.8) 21.3 (19.2–23.5) .958
21.5 (19.3–23.7) 20.9 (18.7–23.1) 20.4 (18.1–22.6) 21.3 (19.2–23.5) .899a
Total motile sperm (106) 102.9 (65.5–140.3) 112.7 (75.7–149.6) 73.2 (36.3–110.2) 69.4 (33.0–105.9) .271
101.4 (65.1–137.7) 113.7 (77.4–150.1) 64.5 (26.9–102.0) 69.1 (33.4–104.8) .159a
Morphologically normal sperm (%) 6.06 (4.34– 7.78) 7.00 (5.33– 8.67) 5.09 (3.42– 6.76) 5.90 (4.21– 7.60) .468
6.26 (4.54–7.97) 6.90 (5.20–8.61) 4.86 (3.11–6.60) 5.71 (4.00–7.42) .376a
Sperm vitality (%) 79.2 (75.0–83.3) 73.5 (69.4–77.7) 72.2 (68.1–76.2) 71.7 (67.4–76.0) .048
79.3 (75.1–83.4) 73.0 (68.8–77.2) 71.2 (66.9–75.4) 71.2 (66.9–75.6) .024a
HBA (%) 69.6 (61.3–78.0) 67.9 (59.6–76.1) 70.4 (62.2–78.6) 63.3 (55.3–71.3) .616
70.4 (62.3–78.4) 65.5 (57.5–73.6) 66.8 (58.4–75.1) 62.0 (54.2–69.8) .544a
Note: HBA ¼ hyaluronan-binding assay.
a
Adjusted for male age, body mass index (kg/m2), current smoking status (yes/no), alcohol consumption (units/week), and abstinence period (days).
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SUPPLEMENTAL TABLE 3

Estimated marginal mean values, unadjusted and adjusted for covariates (with corresponding 95% confidence intervals) of embryo development
variables for the bisphenol A (BPA) exposure quartiles (N [ 137).
P
BPA quartile 25th 50th 75th 100th value
Fertilization rate (%) 79.7 (74.1–85.4) 71.4 (65.5–77.3) 77.6 (71.9–83.4) 76.9 (71.0–82.8) .237
79.2 (73.6–84.8) 70.1 (64.9–76.6) 76.4 (70.6–82.1) 75.2 (69.2–81.2) .211a
Proportion of optimal day 3 embryos (%) 41.5 (33.5–49.4) 31.8 (23.3–40.3) 42.4 (34.2–50.6) 39.1 (30.8–47.3) .288
42.9 (35.1–50.7) 33.3 (25.1–41.6) 43.7 (35.7–51.6) 40.2 (32.0–48.4) .275a
Blastocyst formation rate (%)b,c 50.3 (41.5–59.1) 52.7 (43.3–62.1) 51.6 (42.5–60.7) 53.6 (44.5–62.7) .962
52.6 (44.3–60.9) 56.0 (47.1–65.0) 52.7 (44.0–61.3) 55.3 (46.7–64.0) .922a
Proportion of optimal blastocysts (%)b,d 20.0 (11.2–28.8) 35.3 (25.3–45.4) 32.8 (22.6–42.2) 27.5 (18.2–36.8) .112
21.6 (13.0–30.3) 35.1 (25.2–45.0) 32.7 (23.1–42.3) 26.1 (16.9–35.3) .155a
Note: BMI ¼ body mass index; ICSI ¼ intracytoplasmic sperm injection.
a
Adjusted for male and female age, BMI (kg/m2), oocyte insemination technique (IVF/ICSI).
b
Data collected for 117 cases.
c
Number of blastocysts per number of normally fertilized oocytes.
d
Number of optimal blastocysts per number of all formed blastocysts.
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