ORIGINAL ARTICLE

Effects of H2O2 exposure on human sperm motility

parameters, reactive oxygen species levels
and nitric oxide levels
S. S. du Plessis1, D. A. McAllister1, A. Luu2, J. Savia2, A. Agarwal2 & F. Lampiao1
1 Divison of Medical Physiology, University of Stellenbosch, Tygerberg, South Africa;
2 Center for Reproductive Medicine, Glickman Urological and Kidney Institute, Cleveland Clinic, Cleveland, OH, USA

Keywords Summary
Hydrogen peroxide—motility—nitric
oxide—oxidative stress—reactive oxygen Research has revealed that reactive oxygen species (ROS) negatively affect
species sperm function, both in vivo and in vitro. Sperm preparation techniques for
assisted reproductive technologies (ART) are potential causes for additional
Correspondence ROS production. This study aimed to correlate the concentration of exogenous
Dr SS du Plessis, PO Box 19063,
H2O2 with sperm motility parameters and intracellular ROS and nitric oxide
7505 Tygerberg, South Africa.
Tel.: +27 21 938 9388;
(NO) levels to reiterate the importance of minimising ROS levels in ART.
Fax: +27 21 938 9476; Human spermatozoa from 10 donors were incubated and exposed to different
E-mail: ssdp@sun.ac.za exogenous H2O2 concentrations (0, 2.5, 7.5 and 15 lm). Subsequently, motility
was determined using computer-aided semen analysis, while ROS (2,7-dichlo-
Accepted: June 30, 2009 rofluorescin diacetate) and NO (diaminofluorescein-2/diacetate) were analysed
using fluorescence-activated cell sorting. Results showed that H2O2 did affect
the sperm parameters. Exogenous H2O2 was detrimental to motility and
resulted in a significant increase in overall ROS and NO levels. A significant
increase in static cells was seen as well. It is important to elucidate the mecha-
nisms between intracellular ROS levels with sperm motility parameters. While
this experiment demonstrated a need to reduce exogenous ROS levels during
ART, it did not illustrate the cause and effect relationship of intracellular ROS
and NO levels with sperm motility. Further research needs to be conducted to
define a pathological level of ROS.

during the process of capacitation, possibly by inducing

Introduction
About 15–20% of couples deal with infertility, and within
these cases, around 50% of them are a result of male
factor infertility. Many turn to assisted reproductive
techniques (ART) to bypass their infertility woes.
Unfortunately, only 35% of ART procedures actually result
in live births (Wright et al., 2008). One of the causes for
the low live birth percentage is due to the susceptibility of
the spermatozoa to oxidative stress (OS) during sperm
preparation and selection (Du Plessis et al., 2008).
Oxidative stress is a result of increased levels of reactive
oxygen species (ROS) above physiological defence. ROS
may play a beneficial role in normal physiological func-
tion. Both Bize et al. (1991) and Griveau et al. (1994) sug-
gested that H2O2 plays a significant physiological role
membrane reorganisation to facilitate the fusion that takes
place during exocytosis of the acrosomal contents. But
once the presence of ROS is in excess and the antioxidant
defence system is overpowered, pathological conditions
are induced (Agarwal et al., 2006a,b). Specifically, ROS
have detrimental effects to sperm motility parameters
(Agarwal et al., 2003).
Sperm preparation and selection are based on the
motility and morphology of the spermatozoa (Allamaneni
et al., 2004). Increased levels of ROS have been correlated
to decreased motility parameters (i.e. total motility,
progressive motility and rapid motility), which were con-
cluded to have adverse effects on fertility (Padron et al.,
1997; Saleh & Agarwal, 2002; Saleh et al., 2003; Aziz
et al., 2004). Some ROS members include hydrogen

206 ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 206–210

S. S. du Plessis et al.
peroxide (H2O2), nitric oxide (NO), superoxide (O2))
and hydroxyl radical (OH)). Exogenous addition of H2O2
has been shown to increase intracellular ROS levels in
spermatozoa (Mahfouz et al., 2008). Furthermore, NO,
while not as reactive as other ROS, can react with oxygen
to produce more potent oxidants, e.g. nitric dioxide and
peroxinitrite (ONOO)).
This experiment focused on H2O2, ROS and NO,
which are important intracellular signalling molecules that
are required at physiological levels to aid in sperm capaci-
tation, motility and fertilisation (de Lamirande &
Gagnon, 1993; Zini et al., 1995). First, it aimed to investi-
gate the detrimental effects of the in vitro addition
of increasing concentrations of exogenous H2O2 on sperm
motility. Secondly, it aimed to determine the effect of
exogenous H2O2 on intracellular NO and ROS produc-
tion. Additionally, this study aimed to relate ROS and
NO levels to sperm motility parameters. The significance
of this experiment is to reiterate the importance of mini-
mising OS introduced to the spermatozoa during sperm
preparation for ART.
Materials and methods
After Institutional Review Board approval from the Uni-
versity of Stellenbosch was received for this study, semen
samples were obtained from normozoospermic donors
(n = 10) according to World Health Organization guide-
lines. Motile sperm fractions were retrieved from the sam-
ples using the double wash in Hams F-10 medium
(400 g, 5 min) swim-up technique (3% human tubal
fluid-bovine serum albumin, 37 C, 5% CO2). After 1 h
of incubation, the supernatant containing motile sperma-
tozoa was collected and the concentration adjusted
accordingly (5 · 106 cells/ml), after which it was equally
divided into aliquots. The aliquots were subjected to
exogenous ROS stimulation with H2O2 (60 min, 37 C,
5% CO2) at different concentrations (2.5, 7.5 and 15 lm).
A control group was established with no exogenous H2O2
intervention. Motility, NO and ROS were subsequently
determined.
Motility was assessed by means of computer-aided
semen analysis on a Sperm Class Analyser (Microptics,
Barcelona, Spain), which evaluated the sample’s various
motility parameters including total, progressive and rapid
motility as well as the number of static cells.
NO and ROS analysis by means of flow cytometry was
performed after 60 min of H2O2 incubation. For detec-
tion purposes of NO and ROS, 4,5-diaminofluorescein-2/
diacetate (DAF-2/DA; Calbiochem, San Diego, CA, USA)
and 2,7-dichlorofluorescin diacetate (DCFH-DA; Sigma
Chemicals Co., St. Louis, MO, USA) were used respec-
tively. For NO measurements each sample was loaded
ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 206–210
H2O2 exposure affects sperm motility, ROS and NO
with DAF-2/DA (10 lm) and incubated in the dark
(120 min, 37 C) before analysis by fluorescence-activated
cell sorter (FACS) (Lampiao et al., 2006b). Analysis of
ROS required loading of samples with DCFH-DA (5 lm)
and incubation (15 min, 37 C) in the dark. The cells
were then washed twice and further incubated in the dark
in probe-free medium (30 min, 37 C) before analysis
with FACS (Lampiao et al., 2006a). Care was taken to
prevent exposure to light throughout the rest of the
experimentation as both probes are light-sensitive.
A Becton Dickinson FACSCaliburTM analyser was used
to quantify fluorescence (excitation wavelength 488 nm
and emission wavelength 530 nm for both DAF-2/DA
and DCFH-DA) at the single-cell level and data were
analysed using CellquestTM version 3.3 (Becton Dickin-
son, San Jose, CA, USA) software. The mean fluorescence
intensity of the analysed sperm cells was determined after
gating the cell population by forward and side scatter
light signals. In total, 100000 events were acquired, but
nonsperm particles and debris were excluded by prior
gating, thereby limiting undesired effects in overall fluo-
rescence. The final gated populations usually consisted of
8000–12000 sperm cells. Fluorescence in these cells was
recorded on a frequency histogram by logarithmic
amplifiers.
The sample size was 10 for all experiments. The
D’Agostino and Pearson omnibus normality test was used
to test for normal distribution. One-way analysis of vari-
ance (anova with Bonferroni post hoc test if P < 0.05)
and Pearson correlation tests were used to analyse the
results using the GraphPad PrismTM 4.0 statistical pro-
gram (GraphPad Software Inc., La Jolla, CA, USA). All
data were expressed as mean ± SEM. Fluorescence data
for DCFH-DA and DAF-2/DA were expressed as mean
fluorescence (percentage of control, control adjusted to
100%). Differences were regarded statistically significant if
P < 0.05.
Results
Significant differences with respect to the control group
were observed in motility parameters after exposure to
increased H2O2 concentrations (Fig. 1). With increasing
H2O2 concentrations, total motility (7.5 lm: 28.33 ± 4.014,
P < 0.001; 15 lm: 16.67 ± 3.333, P < 0.001; versus control:
60.33 ± 6.864; Fig. 1a), progressive motility (2.5 lm:
14.00 ± 1.653, P < 0.01; 7.5 lm: 10.00 ± 2.280, P < 0.001;
15 lm: 6.333 ± 1.687, P < 0.001 versus control: 29.33 ±
4.566, Fig. 1c) and rapid motility (2.5 lm: 25.165 ± 1.887,
P < 0.01; 7.5 lm: 15.915 ± 0.822, P < 0.001; 15 lm:
10.50 ± 0.817, P < 0.001 versus control: 33.00 ± 1.500;
Fig. 1b) significantly decreased. Higher concentrations of
H2O2, namely, 7.5 lm (71.67 ± 2.197, P < 0.05) and 15 lm,
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H2O2 exposure affects sperm motility, ROS and NO
(83.33 ± 5.588, P < 0.01) led to a significant increase in the
percentage of static cells when compared with the control
(39.67 ± 4.094; Fig. 1d).
It was, furthermore, found that 15 lm H2O2 signifi-
cantly increased both the mean DAF-2/DA fluorescence
(172.40 ± 22.341% versus control; P < 0.05; Fig. 1e) and
the mean DCFH-DA fluorescence (130.40 ± 7.108%,
versus control; P < 0.05; Fig. 1f). A significant correlation
was also found between NO and ROS production
(r2 = 0.971, P = 0.0146). The percentage motile cells also
correlated negatively with an increase in both endogenous
NO (r2 = 0.991, P = 0.0041) and ROS (r2 = 0.965,
P = 0.0173) levels.
Discussion
The results support our assumption that exogenous H2O2
should adversely affect sperm motility parameters and
corresponds to the results of other studies (Ramos &
Wetzels, 2001; Misro et al., 2004; Mahfouz et al., 2008).
208
S. S. du Plessis et al.
Fig. 1 Effects of different concentrations of
H2O2 treatment on (a) motility; (b) rapid
motility; (c) progressive motility; (d) static cells;
(e) mean DAF-2/DA fluorescence and (f) mean
DCFH fluorescence (#P < 005; *P < 001;
**P < 0001). DCFH, 2,7-dichlorofluorescin
diacetate; DAF-2/DA, 4,5-diaminofluorescein-
2/diacetate; ROS, reactive oxygen species.
The results also suggest a correlation between an increase
in percentage of static cells and exogenous H2O2 concen-
tration. As both DAF-2/DA fluorescence and DCFH-DA
fluorescence increased after H2O2 exposure, we can
conclude that increasing H2O2 concentrations correspond
to increased intracellular NO and ROS production.
Despite the fact that both NO and ROS are important
signalling molecules, the increases detected in intracellular
NO and ROS levels present could be the reason for the
poor motility parameters. Excessive ROS levels have been
linked to lipid peroxidation of the sperm plasma mem-
brane, resulting in a loss of membrane fluidity, structure
and function (Allamaneni et al., 2005; Mahfouz et al.,
2009a). While the results in this experiment show a corre-
lation between increasing NO levels and decreasing sperm
motility function, further studies need to be performed to
elucidate whether or not this is just a correlation or a
cause and effect relationship.
On the other hand, the increased ROS and NO levels
present in our experiment could be a result of other
ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 206–210
S. S. du Plessis et al.
factors. It could be a direct consequence of the exogenous
H2O2, but could also result from the generation of ROS
and NO by damaged spermatozoa and mitochondria
(Koppers et al., 2008). Koppers et al. (2008) conclude
that mitochondria, the major site for sperm ROS produc-
tion, can contribute significantly to OS in defective sper-
matozoa. In our study, it is possible that the exogenous
H2O2 caused sperm cells to become damaged, in turn caus-
ing the mitochondria to leak high levels of free electrons. It
is therefore likely that these damaged mitochondria
contributed to the elevation in ROS and NO levels.
The previous studies have used chemiluminescence to
quantify ROS levels. A study by Mahfouz et al. (2009b)
demonstrated that both the DCFH% method, as used in
this study, and the chemiluminescence assay used in the
previous studies provide relatively similar results. There-
fore, our results are valid and signify a need to minimise
ROS levels during sperm preparation, because of the
harmful effects on sperm motility.
Overall, high ROS levels are not the only cause for
male factor infertility, but continue to play an essential
role in low live birth rates, because of the DNA damage
in spermatozoa caused by the induced OS. Further stud-
ies can be performed on measuring other types of ROS
and exogenous ROS to elucidate the relationship
between ROS production and exogenous ROS introduc-
tion. In addition, specific concentrations at which ROS
begin to compromise sperm function could be analysed.
Knowledge of the pathological levels allows clinicians to
treat infertility based on specific circumstances (i.e. high
ROS, but low physiological defence). It is also important
not to see these results in isolation, but to bear in mind
that the imbalance between ROS production and total
antioxidant capacity in seminal plasma will influence the
developing of OS and relate to sperm dysfunction
in vivo (Sharma et al., 1999).
The adverse effects of exogenous H2O2 on sperm
motility parameters further exemplify the importance of
minimising ROS introduction during sperm preparation
for various ART procedures, especially when spermatozoa
are removed from the seminal plasma during washing
procedures. Because sperm preparation is necessary to
ART procedures, maintaining sperm integrity is vital to
the success of the procedure.
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