MOLECULAR MEDICINE REPORTS 14: 4659-4665, 2016
Abstract. Male infertility is a complex, multifactorial and
polygenic disease that contributes to ~50% cases of infertility.
Previous studies have demonstrated that excess weight and
obesity factors serve an important role in the development
of male infertility. An increasing number of studies have
reported that resveratrol may regulate the response of cells
to specic stimuli that induce cell injury, as well as decrease
germ cell apoptosis in mice or rats. In the present study, the
semen quality and serum sex hormone levels were evaluated in
324 men, which included 73 underweight, 82 normal weight,
95 overweight and 74 obese men. All patients were referred
to The Reproductive Medicine Center of Shanxi Women and
Infants Hospital (Taiyuan, China) between January 2013 and
January 2015. The aim of the present study was to investigate
the effects of resveratrol treatment on the motility, plasma
zinc concentration and acrosin activity of sperm from obese
males. The sperm concentration, normal sperm morphology,
semen volumes, DNA fragmentation rates and testosterone
levels in men from the overweight and obese groups were
markedly decreased when compared with men in the normal
weight group. In addition, the progressive motility, seminal
plasma zinc concentration and spermatozoa acrosin activity
were notably decreased in the obese group compared with the
normal weight group. However, estradiol levels were signi-
cantly increased in the overweight, obese and underweight
groups compared with the normal weight group. Notably,
semen samples from obese males with astenospermia treated
with 0-100 µmol/l resveratrol for 30 min demonstrated
varying degrees of improvement in sperm motility. When
these semen samples were treated with 30 µmol/l resveratrol,
sperm motility improved when compared to other doses of
resveratrol. Therefore, 30 µmol/l resveratrol was selected for
further experiments. Upon treatment of semen samples with
resveratrol (30 µmol/l) for 30 min, the seminal plasma zinc
concentration and spermatozoa acrosin activity increased
signicantly in the experimental group compared with the
control group. These data suggest that male obesity negatively
impacts on male reproductive potential, not only through
altering hormone levels, but also by directly altering sperm
function. In addition, resveratrol may have a therapeutic and
protective effect against obesity-induced abnormalities in
semen.
Introduction
Infertility is a devastating problem of human reproduction, and
male infertility contributes to 50% of all infertility cases (1,2).
In recent years, an increasing number of reports have demon-
strated a global trend indicating a signicant deterioration in
male reproductive function (3-5). In parallel, obesity rates are
increasing rapidly worldwide (6,7), which is not only associ-
ated with an increased risk of developing chronic diseases, but
has also been demonstrated to increase the risk of developing
reproductive disorders (8). Due to the reported association
between obesity and reproductive disorders, a greater clinical
awareness and understanding of the underlying mechanisms
of the effects of obesity on fertility are urgently required in
order to determine the appropriate interventions.
Studies have determined that the natural polyphenol, resve-
ratrol, has a similar molecular structure to diethylstilbestrol
and estradiol (E2), and is produced in several plants in response
to injury, stress, bacterial or fungal infection, ultraviolet (UV)
radiation or exposure to ozone (9-11). Resveratrol is known
for its anti‑inammatory, antioxidant, analgesic, cardiopro-
tective, anti-aging and neuroprotective roles (11-13). Studies
have demonstrated that resveratrol may inhibit cell apop-
tosis, thereby providing protection from numerous diseases,
including atherosclerosis, cerebral ischemia and myocardial
ischemic reperfusion injury (14,15). Juan et al (16) reported
that resveratrol is able to decrease germ cell apoptosis in mice
and rats, and serves a protective role in the male reproductive
tract, as well as enhancing blood testosterone levels, testicular
sperm count and epididymis sperm motility in rabbits.
The aim of the present study was to investigate the associa-
tion between obesity and a number of sperm parameters, and
Protective effect of resveratrol on spermatozoa function in
male infertility induced by excess weight and obesity
XIANGRONG CUI
1
, XUAN JING
2
, XUEQING WU
1
and MEIQIN YAN
1
1
Reproductive Medicine Center, Children's Hospital of Shanxi and Women Health Center of Shanxi, Taiyuan,
Shanxi 030000;
2
Clinical Laboratory, Shanxi Province People's Hospital, Taiyuan, Shanxi 030000, P.R. China
Received October 7, 2015; Accepted September 1, 2016
DOI: 10.3892/mmr.2016.5840
Correspondence to: Dr Xueqing Wu, Reproductive Medicine
Center, Children's Hospital of Shanxi and Women Health Center
of Shanxi, 13 Xinmin North Street, Xinghua Ling, Taiyuan,
Shanxi 030000, P.R. China
E-mail: wuxueqq@hotmail.com
Key words: overweight, obesity, sperm quality, resveratrol,
infertility
CUI et al: PROTECTIVE EFFECT OF RESVERATROL ON SPERMATOZOA FUNCTION
4660
to assess the protective effects of resveratrol on preventing the
harmful effects of obesity on spermatozoa. This may lead to
the development of strategies that facilitate the maintenance
of fer tilit y.
Materials and methods
Study population. The study population consisted of 324 men
(mean age, 37.2±0.89 years) referred to the Reproductive
Medicine Center of Shanxi Women and Infants Hospital
(Taiyuan, China) between January 2013 and January 2015. All
subjects were of Han ethnicity and from the Shanxi Province
in northern China. The present study was approved by the
Reproductive Medicine Ethics Committee of Shanxi Women
and Infants Hospital (Taiyuan, China) and written informed
consent was obtained from all subjects and their partners prior
to enrollment.
The inclusion criteria for the study were as follows:
i) Subjects had to be male and part of a couple that had been
unable to conceive for >1 year; ii) the couple had regular
intercourse, and iii) they were receiving infertility treatment
at the Reproductive Medicine Center, Shanxi Women and
Infants Hospital over the study period. Primary infertility
was diagnosed after the following medical assessments were
performed: The patient's medical history was examined;
a clinical examination; semen analysis; semen culture for
mycoplasma ureliticum and chlamydia detection; analysis of
follicle‑stimulating hormone (FSH), luteinizing hormone (LH),
testosterone and E2 levels; a prolactin assay; and sonography
of the genitalia. Each subject underwent a careful anamnesis
to exclude systemic diseases, alcohol consumption, smoking,
occupational chemical exposure, a history of (or presence of)
endocrine disorders, testicular diseases (for example, cryptor-
chidism, orchitis and varicocelle), infectious genital diseases,
leukocytospermia (seminal white blood cell count >1x10
6
/ml),
azoospermia and treatment with drugs or the use of antioxidant
supplements within 3 months prior to enrollment.
A brief medical background for each subject was collected
by conducting an informal interview, using clinical notes or
using a self‑report questionnaire. The male subjects were
divided into the following four groups according to the
International Association for the Study of Obesity criteria (17):
Underweight [body mass index, (BMI)<18.5 kg/m
2
], normal
weight (18.5≤BMI<23 kg/m
2
), overweight (23≤BMI<25 kg/m
2
)
and obese (BMI≥25 kg/m
2
) groups.
Serum collection and analysis. Blood samples were obtained
between 8:00 and 9:00 a.m. following a 12-h overnight fast, and
were immediately drawn and collected in a tube containing
ethylenediamine tetra-acetic acid. Samples were separated by
centrifugation at 18,00 x g for 15 min at room temperature
and the serum and buffy coat were separated. The serum was
stored at ‑80˚C until downstream analyses were performed.
The hormone levels, including FSH (reference value,
2.10-18.6 mIU/ml), LH (reference value, 1.7-11.2 mIU/ml), E2
(reference value, ≤77 pg/ml), progesterone (reference value,
≤0.46 ng/ml), testosterone (reference value, 262.0-870.0 ng/dl)
and prolactin (PRL; reference value: 2.10-18.60 mIU/ml) were
measured using an AIA-2000 ST Automated Immunoassay
Analyzer (Tosoh Corporation, Tokyo, Japan).
Semen collection and analysis. Semen samples were obtained
by masturbation following 2-7 days of abstinence from sexual
intercourse, in order to conduct a routine sperm count according
to the World Health Organization (WHO, 2010) criteria (18)
for sperm concentration, motility, morphology and viability.
Samples were collected in sterile containers and allowed
to liquefy at 37˚C for 20 min. Briefly, ejaculated volumes
were estimated by specimen weight, and a semen density of
1.0 g/ml was assumed. Sperm concentration, motility and
viability were assessed using a Sperm Class Analyzer CASA
System (Microoptic S.L, Barcelona, Spain). Sperm motility
was analyzed according to the WHO (2010) guidelines (18)
for determining progressive motility, non-progressive motility
and immotility. Sperm morphology was assessed using David's
classication system (19).
Sperm viability analysis. Sperm viability was assessed within
30 min of ejaculation using eosin Y staining. This was achieved
by dissolving 1 g eosin with 1 g fresh sperm. The percentage
of viable sperm (unstained sperm heads) and non-viable sperm
(stained sperm heads) was assessed by counting a minimum of
200 spermatozoa for each sample. Each sample was analyzed
in duplicate.
UV spectrophotometric assay for spermatozoa acrosin
activity. Spermatozoa samples from each group were analyzed
for acrosin activity using the Human Spermatozoa Acrosin
Activity Quantitative assay kit (Huakang Biotechnology
Development Co., Ltd., Changsha, China) according to the
manufacturer's protocol. The required amount of semen
(7. 5x10
6
sperm) was transferred into a plastic tube and
centrifuged at 2,000 x g for 20 min at room temperature.
The supernatant was removed, and the tube was inverted on
absorbent paper to remove residual seminal plasma. A total of
100 µl inhibitor solution (0.3 g/ml HEPES) and 1 ml reactive
liquid (0.6 g/ml N-α-benzoyl-L-arginine-p-nitroanilide) were
added to the sample and control tubes, prior to the addition
of 100 µl stop solution (8.5 g/ml benzamidine) to the control
tube. Sample and control tubes were then thoroughly mixed
and incubated at 24˚C for 1 h. Stop solution (100 µl) was added
to the control tube and mixed thoroughly, and subsequently
both tubes were centrifuged at 2,000 x g for 10 min at 24˚C.
At this temperature, the amount of substrate that hydrolyzes
1.0 µmol N-benzoyl-DL-arginine-4-nitroanilide hydrochlo-
ride/min is dened as 1 IU acrosin activity. Acrosin activity
was determined using the following formula: Acrosin activity
(µIU/10
6
spermatozoa)=[sample optical density (OD)-control
OD] x [2/(495x7. 5 )] x10
6
.
Colorimetric assay for seminal plasma zinc. The zinc
concentrations in seminal uid samples for each group were
analyzed using the Seminal Plasma Zinc Quantitative assay kit
(Huakang Biotechnology Development Co., Ltd.) according to
the manufacturer's protocol. Seminal uid (1 ml) was centri-
fuged at 1,800 x g for 10 min at 24˚C. The supernatant was
subsequently transferred into a separate test tube for seminal
plasma analysis. Physiological saline solution (1 ml) was used
to wash the sediment, prior to the mixing of samples using a
vortex-type mixer for 30 sec, and centrifugation for at 1,800 x g
for 10 min at 24˚C. The supernatant was removed, and the
MOLECULAR MEDICINE REPORTS 14: 4659-4665, 2016
4661
sediment was used to determine the zinc concentration instead
of using 200 µl of liquid sample. The absorbance of the sample
solutions was read at 490 nm. Sample zinc concentrations were
calculated using the following formula: Seminal plasma zinc
(µmol) = zinc concentration (mmol/l) x semen volume (ml).
Detection of sperm DNA integrity. DNA integrity analysis
of sperm in fresh semen samples was performed using a the
Sperm DNA Fragments Staining kit (Huakang Biotechnology
Development Co., Ltd.), which is based on the emission of
fluorescence signals from individual sperm stained with
acridine orange (AO). The AO molecules intercalate into
double-stranded DNA, and green fluorescence is emitted
from the sperm nuclei. The DNA in sperm with immature
nuclei can be denatured into single strands, which leads to
the aggregation of AO molecules in the nuclei and emission
of an orange‑red uorescence signal. The cell suspension was
pipetted onto a glass slide and observed under a BX51 uores-
cence microscope (Olympus Corporation, Tokyo, Japan) with
a 480‑490 nm lter. The percentage of green (normal DNA
integrity) and orange-red (abnormal DNA integrity) sperma-
tozoa/200 spermatozoa in each sample was determined by a
single investigator. An abnormal sperm nuclear DNA integrity
was considered to be when >34% of sperm nuclei emitted
orange‑red uorescence signals following AO staining.
Resveratrol treatment and dose preparation. The sperm
suspensions from obese patients with astenospermia (60 cases)
were pooled and divided randomly into the following three
drug treatment groups: i) the control group, where sperma-
tozoa were treated with Quinn's Advantage™ Fertilization
(HTF) Medium (SAGE‑In vitro Fertilization, Inc., Trumbull,
CT, USA); ii) the negative control group, where spermatozoa
were treated with Quinn's Advantage™ Fertilization (HTF)
Medium plus 0.1% dimethyl sulfoxide (DMSO); and iii) the
experimental group, which was subdivided into six subgroups
based on the concentration of resveratrol (2.6, 6, 15, 30, 50,
100 µmol/l; Sigma-Aldrich; Merck Millipore, Darmstadt,
Germany) added to the medium, which was maintained at
37˚C, 5% CO
2
and 95% humidity. Samples were incubated
at 37˚C for 30 min with resveratrol before sperm motility,
seminal plasma zinc concentration and spermatozoa acrosin
activity were analyzed.
Statistical analysis. Statistical analyses were performed
using the SPSS software program (version 17.0; SPSS, Inc.,
Chicago, IL, USA). Normally distributed data were expressed
as the mean ± standard deviation. To verify the normality of
the distribution, the Shapiro-Wilk test was performed, and
one-way analysis of variance was used to compare the mean
among the different groups. Variables with a non‑normal
distribution were analyzed using a Mann-Whitney U test or
Kruskal-Wallis variance analysis test. P<0.05 was considered
to indicate a statistically signicant difference.
Results
Distribution of male infertility frequency over the BMI groups.
A total of 324 men (mean age, 37.2 years) were recruited to the
study. Based on the analysis results of the semen parameters,
139 males (42.90%) were classied as fertile and 185 (57.10%)
were classied as infertile. The distribution of male fertility in
all BMI groups is shown in Table I. The general characteristics
of the participants were stratied according to the four BMI
groups, where 56/73 (76.71%) of underweight males were
infertile, 32/82 (39.02%) of males with a normal weight were
infertile, 47/95 (49.47%) of overweight males were infertile
and 50/74 (67.57%) of obese males were infertile.
Comparison of routine semen parameters and serum sex
hormone levels among BMI groups. As shown in Table II,
routine semen parameters and serum sex hormone levels
were assessed in abnormal weight groups, and the results
were compared with those of the normal weight group.
No signicant difference in FSH, LH and PRL levels were
observed among the abnormal weight groups and the normal
weight group (P>0.05). In addition, the underweight group
demonstrated no significant alterations in semen volume
when compared with the normal weight group (P>0.05),
whereas overweight and obese groups exhibited signicantly
lower semen volumes (P=0.0248 and P=0.0142, respec-
tively). The percentage of sperm with progressive motility
in the overweight group was not signicantly different when
compared with the normal weight group (P>0.05), whereas
the percentage of sperm with progressive motility in the
underweight and obese groups was signicantly decreased
(P=0.0009 and P=0.0419 respectively). When compared with
the normal weight group, the sperm concentration (under-
weight vs. normal weight, P<0.0001; overweight vs. normal
weight, P=0.0185; obese vs. normal weight, P=0.0034), the
percentage of sperm with a normal morphology (underweight
vs. normal weight, P<0.0001; overweight vs. normal weight,
P=0.0396; obese vs. normal weight, P=0.0004) and the
testosterone levels (underweight vs. normal weight, P=0.0011;
overweight vs. normal weight, P<0.0001; obese vs. normal
weight, P<0.0001) in abnormal weight groups were signi-
cantly decreased. By contrast, E2 levels were signicantly
increased in underweight, overweight and obese groups when
compared with the normal weight group (P=0.0003, P<0.0001
and P<0.0001, respectively).
Table I. Distribution of male infertility frequency across the
BMI groups.
BMI group
a
Fertile men (%) Infertile men (%) Total
BMI<18.5 17 (23.29) 56 (76.71) 73
18.8≤BMI<23 50 (60.98) 32 (39.02) 82
23≤BMI<25 48 (50.53) 47 (49.47) 95
BMI≥25 24 (32.43) 50 (67.57) 74
Total 139 (42.90) 185 (57.10) 324
a
Subjects were divided into BMI groups according to the International
Association for the Study of Obesity criteria (17). Male subjects
included in the current study were referred to the Reproductive
Medicine Center of Shanxi Women and Infants Hospital (Taiyuan,
China) between January 2013 and January 2015, and were all of Han
ethnicity. BMI, body mass index.
CUI et al: PROTECTIVE EFFECT OF RESVERATROL ON SPERMATOZOA FUNCTION
4662
Comparison of sperm viability among BMI groups. Sperm
viability was analyzed by eosin Y staining. The number of
viable sperm (Fig. 1A) and non-viable sperm (Fig. 1B) in each
group were examined using an Olympus CX31 microscope.
When compared with the normal weight group, the obese
group demonstrated a signicant reduction in sperm viability
(P= 0.0297; Fig. 1C).
Comparison of the plasma zinc concentration, sperma-
tozoa acrosin activity and DNA fragmentation rates among
BMI groups. As shown in Fig. 2A, the seminal plasma
zinc concentration was significantly reduced in the obese
group compared with the normal weight group (P=0.0233).
Spermatozoa acrosin activity was analyzed using a UV spec-
trophotometric assay. Compared with normal weight group,
the overweight and obese groups demonstrated a signicant
decrease in spermatozoa acrosin activity (P=0.0215 and
P=0.0193, respectively; Fig. 2B). DNA fragmentation rates
were analyzed by AO staining. As shown in Fig. 2C-E,
spermatozoa emitting green (normal DNA integrity) and
orange-red (abnormal DNA integrity) fluorescence signals
were visualized and counted using an Olympus BX51 uores-
Table II. Comparison of routine semen parameters and serum sex hormone levels among BMI groups.
Normal weight Underweight Overweight Obese
Parameter (18.8≤BMI<23) (BMI<18.5) (23≤BMI<25) (BMI≥25)
Semen volume (ml) 3.56±1.74 3.54±1.68 3.10±0.88
a
3.02±0.73
a
Sperm concentration (x10
6
/ml) 68.39±8.54 59.42±8.16
b
65.39±8.22
a
64.39±8.19
b
Progressive motility (%) 40.28±12.98 33.62±11.31
b
39.56±11.74 36.39±10.39
a
Morphology (% normal) 12.11±3.59 7.63±1.33
b
11.08±3.32
a
10.21±2.9
b
Follicle stimulating hormone (mIU/ml) 6.98±2.55 4.71±1.83 5.11±2.24 5.49±1.79
Luteinizing hormone (mIU/ml) 9.35±2.35 9.1±1.32 8.74±1.66 8.63±1.29
Estradiol (pg/ml) 29.32±7.90 34.11±8.27
b
36.63±7.53
b
37.21±8.94
b
Testosterone (ng/dl) 386.58±21.32 398.24±22.19
b
369.76±19.38
b
354.71±19.23
b
Prolactin (mIU/ml) 12.28±4.87 12.26±3.48 12.38±4.25 12.45±4.71
a
P<0.05 and
b
P<0.01 vs. the normal weight group. BMI, body mass index.
Figure 1. Comparison of sperm viability among BMI groups. Sperm viability was analyzed by eosin Y staining. Microscope images (magnication, x20)
of (A) viable sperm (unstained sperm head) and (B) non‑viable sperm (stained sperm head). (C) The obese group demonstrated a signicant decrease in the
number of viable sperm (P<0.05).
MOLECULAR MEDICINE REPORTS 14: 4659-4665, 2016
4663
cence microscope with a 480‑490 nm lter. When compared
with the normal weight group, the underweight, overweight
and obese groups demonstrated a significant increase in
DNA fragmentation rates (P=0.0347, P=0.0339 and P=0.0208
respectively; Fig. 2F).
Alterations in the progressive motility of sperm following
treatment with increasing concentrations of resveratrol. As
shown in Fig. 3, semen samples from obese patients with aste-
nospermia (60 cases) treated with 0-100 µmol/l resveratrol for
30 min, exhibited varying degrees of improvement in sperm
motility. When semen samples were exposed to 30 µmol/l
resveratrol, sperm motility was observed to increase compared
with the other doses of resveratrol. Therefore, 30 µmol/l resve-
ratrol was used for subsequent experiments.
Effect of resveratrol on plasma zinc concentration and sperma-
tozoa acrosin activity. Semen samples were treated with quinn's
Figure 3. Alterations in the progressive motility of sperm treated with
increasing concentrations of resveratrol. Semen samples from obese patients
with astenospermia (60 cases) were treated with 0-100 µmol/l resveratrol
for 30 min, and sperm motility was altered to varying degrees. Exposure to
30 µmol/l resveratrol demonstrated the most notable improvement in pro-
gressive motility compared with control and negative control samples. The
control and negative control groups were treated with Quinn's Advantage™
Fertilization (HTF) Medium and Quinn's Advantage™ Fertilization (HTF)
Medium plus 0.1% dimethyl sulfoxide, respectively.
Figure 2. Plasma zinc concentration, spermatozoa acrosin activity and DNA fragmentation rates among BMI groups. (A) The seminal plasma zinc concentra-
tion was signicantly reduced in the obese group compared with normal weight group (P=0.018). (B) The overweight and obese groups demonstrated a
signicant decrease in spermatozoa acrosin activity compared with the normal weight group (P=0.031 and P=0.021, respectively). Representative uorescence
images of spermatozoa with (C) normal DNA integrity, and (D and E) abnormal DNA integrity are shown. DNA fragmentation rates were analyzed using
acridine orange staining. (F) Spermatozoa from the underweight, overweight and obese groups demonstrated a signicant increase in DNA fragmentation rates
compared with those of the normal weight group (P=0.039, P=0.042, P=0.026, respectively).
*
P<0.05 vs. the normal weight group.