Paraben exposure and semen quality of Japanese male partners of subfertile couples
Nishihama et al. Environmental Health and Preventive Medicine
Paraben exposure and semen quality of Japanese male partners of subfertile couples
Yukiko Nishihama 1
Hiroki Toshima 1
Jun Yoshinaga 0 1
0 Faculty of Life Science, Toyo University , Izumino 1-1-1, Itakura, Ora 374-0193, Gunma , Japan
1 Department of Environmental Studies, University of Tokyo , Kashiwanoha 5-1-5, Kashiwa, Chiba 277-8563 , Japan
Objectives: Possible relationship between semen quality (semen volume, sperm concentration and sperm motility) and parabens exposure was investigated in male partners of couples who visited a gynecology clinic in Tokyo for infertility consultation (n = 42, 36.8 ± 5.4 years). Methods: Semen parameters were measured according to WHO guideline at the clinic, and urinary methyl- (MP), ethyl- (EP), propyl- (PP) and butyl (BP) paraben concentrations were measured with liquid chromatography-tandem mass spectrometry. Results: Geometric mean urinary concentrations (geometric standard deviation) of the subjects were 48.2 (4.52), 1.88 (4.72), 1.13 (6.75) and 0.184 (11.1) ng/mL for MP, EP, PP and BP, respectively. No significant association was found between semen parameters and urinary paraben concentrations in multiple regression analyses and logistic regression analyses. Conclusions: Two reasons of the absence of adverse effects on semen quality might be suggested: lower paraben exposure level of the subjects and small sample size. Further investigation of effect of paraben exposure among general male population at environmental levels is warranted.
Paraben; Urine; Biomarker; Semen quality; Human
Alkyl esters of 4-hydroxy benzoic acid (parabens) are
used as a preservative in personal care products (PCPs)
such as cosmetics [1, 2]. Methyl paraben (MP), ethyl
paraben (EP), propyl paraben (PP) and butyl paraben
(BP) are commonly used parabens. They have been
thought to have low acute toxicity , but recently their
endocrine disrupting activity has been known . Recent
human biomonitoring programs found frequent
detection of parabens in urine samples from general
populations [5–7] indicating that human exposure to parabens
was widespread probably reflecting heavy usage of the
compounds in many PCPs.
Many in vitro and in vivo studies have reported that
parabens have estrogenic activity [8–10]. The feedback
systems of gonadotropin including FSH/LH are thought
to be regulated by exposure to xenoestrogen, especially
during development of Sertori cell, which results in
decrease of testes and sperm production . Oishi
[12–14] reported that exposure to parabens, especially
PP and BP, decreased serum testosterone
concentration by 20–70% in male rodents. His studies also
revealed that oral exposure to PP and BP decreased
dairy sperm production and epididymal sperm reserve
up to 50% [12–14]. Park et al.  showed that
sperm DNA hypermethylation could be caused by
oral exposure to BP in male rats. These studies
indicate that paraben exposure is one of the possible
factors that deteriorate male reproductive function.
On the other hand, as far as we know, there is only
one epidemiologic study that investigated the
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relationship between human male reproductive
function and paraben exposure: Meeker et al. 
examined the relationship between semen parameters and
sperm DNA damage measures and urinary paraben
excretion among male partners of couples attending
an infertility clinic in Boston, Massachusetts (n = 194).
They found no significant relationship between
urinary parabens excretion and semen parameters.
We have carried out a pilot study investigating
possible relationship between exposure to various endocrine
disrupting chemicals (pyrethroid insecticides, cadmium,
or phthalate esters) and semen parameters among male
partners of subfertile couples in Tokyo . In this
paper, we investigated possible association between
urinary paraben concentration and semen parameters of the
same study subjects.
Materials and methods
The subjects of the present study was the same as that
of our previous study . The subject was male partner
of couple who visited a gynecology clinic in Tokyo for
infertility consultation during January to June 2010.
Forty-two subjects who were randomly called to by a
gynecologist voluntarily agreed to participate in our
study after being explained the purpose and procedure
of the study from a gynecologist. [All of the subjects
who agreed to participate in this study were included
and selection of the subjects based on semen test result
was not done.] Methods of semen sampling and
analyses, urine sampling, and questionnaire were described
in Toshima et al. .
Urinary MP, EP, PP and BP analysis was carried out by
liquid chromatography-tandem mass spectrometry
according to our previous study . Limit of detections
(LODs) were 0.24, 0.021, 0.065 and 0.0090 ng/mL for
MP, EP, PP and BP, respectively, based on S/N = 3
An in-house quality control urine sample was included
in every batch of sample preparation and measurement
(typically 20 samples/batch) to monitor reproducibility.
Recoveries of the internal standards were monitored for
all of the samples and they were 34–44% for the 4
parabens (n = 42).
Specific gravity (SG) of urine sample was measured
by a hand-held refractometer (Erma Inc., Tokyo,
Japan) by which urinary parabens concentration was
corrected for. Urinary creatinine concentration was
also measured with a commercial kit based on Jaffe
reaction (Wako Pure Chemicals Co. Ltd., Tokyo,
Japan) and used for correction.
The association between semen parameters and urinary
paraben concentrations were examined by using
multiple regression analysis and logistic regression analysis
by reference to Toshima et al. . In multiple
regression analyses, dependent variable was one of the semen
parameters and used as a continuous variable. Logistic
regression analysis were also performed. In this analysis,
subjects were classified into two by semen parameter
above or below WHO lower reference limit (LRL) of
2010 . Logistic regression analysis was carried out on
sperm motility only, because there were only one and
two subjects whose semen volume and sperm
concentration were below the LRL.
In these analyses, SG-corrected concentrations of
individual parabens were used as independent variables.
Urinary paraben concentrations skewed towards higher
value; therefore, the concentrations were transformed to
natural logarithm. When paraben concentration was
below LODs, 1/2 of LODs value was substituted in the
statistical analysis. In addition to individual paraben
concentrations, estrogen-equivalent total paraben (ETP) was
used in our statistical analysis by summing the individual
concentrations of the four parabens weighted by their
relative estrogenic activity according to Eq. 1 . The
weighing factor was derived from in vitro yeast estrogen
screen assays .
Other variables included as independent variable were
age, BMI, and abstinence period along with urinary
concentrations of 3-phenoxybenzoic acid (3-PBA), daidzein
and mono-n-butyl phthalate (MBP), current smoking,
consumption frequency of fruits and coffee, whether the
subject is equol producer and season of semen sampling
(February–March/ May–July) in multiple regression
analyses. Logistic regression analysis was also performed. In
this analysis, all of the continuous variables, including
urinary parabens, dichotomized by the median value
because distributions of urinary paraben concentration
were skewed and sample size was small. These statistical
analyses were performed by using SPSS ver 12.0 J.
Results and discussions
Urinary parabens level
Table 1 shows SG- and creatinine-adjusted geometric
mean and median urinary concentrations of MP, EP, PP
and BP of the present subjects. The relative contribution
of MP, EP, PP and BP to ETP was 12, 12, 38 and 38%,
Abbreviations: MP methyl paraben, EP ethyl paraben, PP propyl paraben, BP butyl paraben, ETP estrogen-equivalent total paraben
a Means and median were calculated by substituting 1/2 of LOD for subjects with urinary concentration below detection limit
b ETP was the sum of the urinary concentrations of 4 parabens weighted by relative estrogenic activity obtained in a yeast estrogen screen assay by
Routledge et al. 
c Not Applicable
Median urinary MP concentrations of the present
subjects was higher than that of healthy male subjects in
the USA  and China , whereas that of PP was
lower. Difference between the urinary MP and PP
concentrations in the present subjects and those in the US
and Chinese populations was most probably derived
from the differences in the usage of PCPs: the source of
parabens believed to be predominant for the general
The relationship between semen quality and urinary
Table 2 represents summary of semen parameters and
others of the present subjects, which were recapitulated
from Toshima et al. . One subject was excluded from
the statistical analysis since his abstinence period was
less than 2 days.
The present subjects were male partners of couples
who visited a gynecology clinic in Tokyo for infertility
consultation. Thus, both fertile and infertile male could
be included: actually, 19 out of the 42 subjects had all of
the semen parameters above the LRL of WHO of 2010
. Average semen volume, sperm concentration and
sperm motility of the present subjects were similar to
the levels of fertile Japanese men  and those of the
university male students . Therefore, in spite of the
fact that the present subjects had infertility test, their
semen quality was not significantly deviated from
Figure 1 shows the results of multiple regression
analyses with stepwise variable selection. Significantly
positive relationship between semen volume and urinary EP
and that between semen volume and ETP were found.
Other relationships between semen parameters and
urinary concentrations of parabens were not significant.
Logistic regression analysis also did not find significantly
negative relationship between paraben exposure and
Oishi [12–14] reported that parabens had adverse
effects on some of the male reproductive functions of
rodents, i.e., decreased sperm counts and dairy sperm
production, elongated spermatid counts and decreased
serum testosterone concentration, at the dose levels
(oral) of >125 mg/kg/day PP and >14.4 mg/kg/day BP.
Kang et al.  also reported >50% decrease of sperm
Table 2 Semen parameters and other selected parameters of
the present subjects (n = 42) a
Urinary 3-PBA b c
Urinary daidzein b
Urinary MnBP b d
Season of semen sampling
(Winter and Summer)
February–March: 52.0 (22/42)
May–July: 48.0 (20/42)
a Recapitulated from Toshima et al. 
b SG-adjusted geometric mean concentrations (geometric standard
c 3-phenoxybenzoic acid (a metabolite of pyrethroid)
d Mono-n-butyl phthalate (a metabolite of di-n-butyl phthalate)
Fig. 1 Results of multi regression analyses a. a Stepwise method was used. b Non-smoker: 0, smoker: 1. c Urinary concentrations were
used as SG-adjusted and log-transformed values. d Values which were less than LOD value replaced the value of half of LOD. e Less
than 1 cup of coffee/ week: 0, more than 1 cup of coffee/ week: 1. f Less than once of fruit consumption/ week: 0, more than once of
fruit consumption: 1. g MnBP was an abbreviation for mono-n-butyl phthalate (a metabolite of di-n-butyl phthalate). h 3-PBA was an abbreviation
for 3-phenoxybenzoate acid (a main metabolite of pyrethroid). i Less than LOD of urinary equol concentrations: 0, more than LOD of urinary equol
concentrations: 1. * p < 0.05, ** p < 0.01 and *** p < 0.001
counts and >25% decrease of sperm motile activity at
oral BP intake levels of >100 mg/kg/day.
In contrast to the rodents studies, in which adverse
effects on semen quality were reported, we could not find
significantly negative relationships between semen
parameters and parabens exposure in the present human study.
Meeker et al.  also found no association between
paraben exposure and semen parameters (volume,
concentration and motility) of male partners attending an infertility
clinic in the USA. In the previous human studies, negative
relationships between semen parameters and exposure
levels of urinary chemicals with estrogenicity, e.g. DDT
 or phytoestrogen , have been found. The reason
why negative relationships between parabens exposure
and human semen parameters were not found in this
study may be due to low exposure levels of parabens of
the subjects. Much less paraben exposure levels of the
present subjects than the administrated doses in the
previous animal studies was noted: the exposure levels of PP
and BP in the present subjects roughly correspond to
intake of 50 and 5.5 ng/kg/day, respectively, when we
assume that 1) volume of dairy urine excretion is 2 L, 2)
the body weight is 60 kg  and 86% of parabens intake
is excreted from human body within 24 h . The
estimated exposure levels of the present subjects are lower
than the dose levels of PP and BP in rodents [13, 14] by
107. The exposure levels of parabens in the present study
may not be high enough to find negative association with
semen. Instead, we found a significantly positive
relationship between urinary EP and ETP concentrations and
semen volume (Fig. 1). These results were not consistent
with the results of animal study [12–14] in which
decreased sperm counts and sperm motility were
reported. The plausibility of the observed positive
correlation cannot be found with our current knowledge.
Limitation of the present study
A couple of limitations can be pointed out to this
study. Firstly, sample size was small (n = 42): this was
because the present study was designed as a pilot
study . Statistical power might be short to detect
a small effect. Secondly, the subjects of this study
included those who had normal semen quality and
those who did not: this might have obscured the
association between exposure and effect, if present.
Finally, the level of parabens exposure of our subjects
was assessed by the parabens concentrations in a
single spot urine. Since parabens are known to be
rapidly excreted after exposure , concentrations in a
spot urine may not represent long-term exposure
level. The intraclass correlation coefficient (ICC) of
urinary parabens concentration of male subjects was
reported as 0.2–0.5 [7, 16, 25] indicating that the
concentration in single spot urine only marginally
reflects long-term exposure levels of parabens of
We found no evidence of a negative association
between semen parameters and urinary paraben
concentrations at environmental levels in Japan. Even a
subtle negative effects, which were not detectable in
the present study that involved a small number of
subjects, can have a serious consequence because of
the widespread exposure to parabens among general
public in the world. It is particularly concerned
because of the accumulating evidence on adverse male
reproductive effects in rodents. It is warranted to
further investigate if adverse male reproductive effects
are seen among general male population at the
environmental levels of paraben exposure.
The authors thank all of the subjects who cooperated with this study.
This study was supported by Grant for Environmental Research from the
Sumitomo Foundation (2014).
YN statistically analyzed the data and prepared the manuscript. HT and YM
carried out urine sampling and measured specific gravity of urine samples.
JY designed the study and refined the manuscript. MY, DN, and HS analyzed
paraben concentrations in urine samples. ST analyzed semen parameters. All
authors read and approved the final manuscript.
The authors declare no conflict of interest for this study.
Ethics approval and consent to participate
All procedures performed in studies involving human participants were in
accordance with the ethical standards of the institutional and/or national
research committee and with the 1964 Helsinki declaration and its later
amendments or comparable ethical standards.
Informed consent was obtained from all individual participants included in
the study. The Ethical Committee of the University of Tokyo approved this
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