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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
membrane reorganisation to facilitate the fusion that takes
About 15–20% of couples deal with infertility, and within place during exocytosis of the acrosomal contents. But
these cases, around 50% of them are a result of male once the presence of ROS is in excess and the antioxidant
factor infertility. Many turn to assisted reproductive defence system is overpowered, pathological conditions
techniques (ART) to bypass their infertility woes. are induced (Agarwal et al., 2006a,b). Specifically, ROS
Unfortunately, only 35% of ART procedures actually result have detrimental effects to sperm motility parameters
in live births (Wright et al., 2008). One of the causes for (Agarwal et al., 2003).
the low live birth percentage is due to the susceptibility of Sperm preparation and selection are based on the
the spermatozoa to oxidative stress (OS) during sperm motility and morphology of the spermatozoa (Allamaneni
preparation and selection (Du Plessis et al., 2008). et al., 2004). Increased levels of ROS have been correlated
Oxidative stress is a result of increased levels of reactive to decreased motility parameters (i.e. total motility,
oxygen species (ROS) above physiological defence. ROS progressive motility and rapid motility), which were con-
may play a beneficial role in normal physiological func- cluded to have adverse effects on fertility (Padron et al.,
tion. Both Bize et al. (1991) and Griveau et al. (1994) sug- 1997; Saleh & Agarwal, 2002; Saleh et al., 2003; Aziz
gested that H2O2 plays a significant physiological role 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. H2O2 exposure affects sperm motility, ROS and NO

peroxide (H2O2), nitric oxide (NO), superoxide (O2)) with DAF-2/DA (10 lm) and incubated in the dark
and hydroxyl radical (OH)). Exogenous addition of H2O2 (120 min, 37 C) before analysis by fluorescence-activated
has been shown to increase intracellular ROS levels in cell sorter (FACS) (Lampiao et al., 2006b). Analysis of
spermatozoa (Mahfouz et al., 2008). Furthermore, NO, ROS required loading of samples with DCFH-DA (5 lm)
while not as reactive as other ROS, can react with oxygen and incubation (15 min, 37 C) in the dark. The cells
to produce more potent oxidants, e.g. nitric dioxide and were then washed twice and further incubated in the dark
peroxinitrite (ONOO)). in probe-free medium (30 min, 37 C) before analysis
This experiment focused on H2O2, ROS and NO, with FACS (Lampiao et al., 2006a). Care was taken to
which are important intracellular signalling molecules that prevent exposure to light throughout the rest of the
are required at physiological levels to aid in sperm capaci- experimentation as both probes are light-sensitive.
tation, motility and fertilisation (de Lamirande & A Becton Dickinson FACSCaliburTM analyser was used
Gagnon, 1993; Zini et al., 1995). First, it aimed to investi- to quantify fluorescence (excitation wavelength 488 nm
gate the detrimental effects of the in vitro addition and emission wavelength 530 nm for both DAF-2/DA
of increasing concentrations of exogenous H2O2 on sperm and DCFH-DA) at the single-cell level and data were
motility. Secondly, it aimed to determine the effect of analysed using CellquestTM version 3.3 (Becton Dickin-
exogenous H2O2 on intracellular NO and ROS produc- son, San Jose, CA, USA) software. The mean fluorescence
tion. Additionally, this study aimed to relate ROS and intensity of the analysed sperm cells was determined after
NO levels to sperm motility parameters. The significance gating the cell population by forward and side scatter
of this experiment is to reiterate the importance of mini- light signals. In total, 100000 events were acquired, but
mising OS introduced to the spermatozoa during sperm nonsperm particles and debris were excluded by prior
preparation for ART. 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
Materials and methods
recorded on a frequency histogram by logarithmic
After Institutional Review Board approval from the Uni- amplifiers.
versity of Stellenbosch was received for this study, semen The sample size was 10 for all experiments. The
samples were obtained from normozoospermic donors D’Agostino and Pearson omnibus normality test was used
(n = 10) according to World Health Organization guide- to test for normal distribution. One-way analysis of vari-
lines. Motile sperm fractions were retrieved from the sam- ance (anova with Bonferroni post hoc test if P < 0.05)
ples using the double wash in Hams F-10 medium and Pearson correlation tests were used to analyse the
(400 g, 5 min) swim-up technique (3% human tubal results using the GraphPad PrismTM 4.0 statistical pro-
fluid-bovine serum albumin, 37 C, 5% CO2). After 1 h gram (GraphPad Software Inc., La Jolla, CA, USA). All
of incubation, the supernatant containing motile sperma- data were expressed as mean ± SEM. Fluorescence data
tozoa was collected and the concentration adjusted for DCFH-DA and DAF-2/DA were expressed as mean
accordingly (5 · 106 cells/ml), after which it was equally fluorescence (percentage of control, control adjusted to
divided into aliquots. The aliquots were subjected to 100%). Differences were regarded statistically significant if
exogenous ROS stimulation with H2O2 (60 min, 37 C, P < 0.05.
5% CO2) at different concentrations (2.5, 7.5 and 15 lm).
A control group was established with no exogenous H2O2
Results
intervention. Motility, NO and ROS were subsequently
determined. Significant differences with respect to the control group
Motility was assessed by means of computer-aided were observed in motility parameters after exposure to
semen analysis on a Sperm Class Analyser (Microptics, increased H2O2 concentrations (Fig. 1). With increasing
Barcelona, Spain), which evaluated the sample’s various H2O2 concentrations, total motility (7.5 lm: 28.33 ± 4.014,
motility parameters including total, progressive and rapid P < 0.001; 15 lm: 16.67 ± 3.333, P < 0.001; versus control:
motility as well as the number of static cells. 60.33 ± 6.864; Fig. 1a), progressive motility (2.5 lm:
NO and ROS analysis by means of flow cytometry was 14.00 ± 1.653, P < 0.01; 7.5 lm: 10.00 ± 2.280, P < 0.001;
performed after 60 min of H2O2 incubation. For detec- 15 lm: 6.333 ± 1.687, P < 0.001 versus control: 29.33 ±
tion purposes of NO and ROS, 4,5-diaminofluorescein-2/ 4.566, Fig. 1c) and rapid motility (2.5 lm: 25.165 ± 1.887,
diacetate (DAF-2/DA; Calbiochem, San Diego, CA, USA) P < 0.01; 7.5 lm: 15.915 ± 0.822, P < 0.001; 15 lm:
and 2,7-dichlorofluorescin diacetate (DCFH-DA; Sigma 10.50 ± 0.817, P < 0.001 versus control: 33.00 ± 1.500;
Chemicals Co., St. Louis, MO, USA) were used respec- Fig. 1b) significantly decreased. Higher concentrations of
tively. For NO measurements each sample was loaded H2O2, namely, 7.5 lm (71.67 ± 2.197, P < 0.05) and 15 lm,

ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 206–210 207


H2O2 exposure affects sperm motility, ROS and NO 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.

(83.33 ± 5.588, P < 0.01) led to a significant increase in the The results also suggest a correlation between an increase
percentage of static cells when compared with the control in percentage of static cells and exogenous H2O2 concen-
(39.67 ± 4.094; Fig. 1d). tration. As both DAF-2/DA fluorescence and DCFH-DA
It was, furthermore, found that 15 lm H2O2 signifi- fluorescence increased after H2O2 exposure, we can
cantly increased both the mean DAF-2/DA fluorescence conclude that increasing H2O2 concentrations correspond
(172.40 ± 22.341% versus control; P < 0.05; Fig. 1e) and to increased intracellular NO and ROS production.
the mean DCFH-DA fluorescence (130.40 ± 7.108%, Despite the fact that both NO and ROS are important
versus control; P < 0.05; Fig. 1f). A significant correlation signalling molecules, the increases detected in intracellular
was also found between NO and ROS production NO and ROS levels present could be the reason for the
(r2 = 0.971, P = 0.0146). The percentage motile cells also poor motility parameters. Excessive ROS levels have been
correlated negatively with an increase in both endogenous linked to lipid peroxidation of the sperm plasma mem-
NO (r2 = 0.991, P = 0.0041) and ROS (r2 = 0.965, brane, resulting in a loss of membrane fluidity, structure
P = 0.0173) levels. 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
Discussion
motility function, further studies need to be performed to
The results support our assumption that exogenous H2O2 elucidate whether or not this is just a correlation or a
should adversely affect sperm motility parameters and cause and effect relationship.
corresponds to the results of other studies (Ramos & On the other hand, the increased ROS and NO levels
Wetzels, 2001; Misro et al., 2004; Mahfouz et al., 2008). present in our experiment could be a result of other

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


S. S. du Plessis et al. H2O2 exposure affects sperm motility, ROS and NO

factors. It could be a direct consequence of the exogenous Agarwal A, Said TM, Bedaiwy MA, Banerjee J, Alvarez JG
H2O2, but could also result from the generation of ROS (2006b) Oxidative stress in an assisted reproductive
and NO by damaged spermatozoa and mitochondria techniques setting. Fertil Steril 86:503–512.
(Koppers et al., 2008). Koppers et al. (2008) conclude Allamaneni SS, Bandaranayake I, Agarwal A (2004) Use of
that mitochondria, the major site for sperm ROS produc- semen quality scores to predict pregnancy rates in couples
tion, can contribute significantly to OS in defective sper- undergoing intrauterine insemination with donor sperm.
matozoa. In our study, it is possible that the exogenous Fertil Steril 82:606–611.
H2O2 caused sperm cells to become damaged, in turn caus- Allamaneni SS, Agarwal A, Nallella KP, Sharma RK, Thomas
AJ Jr, Sikka SC (2005) Characterization of oxidative stress
ing the mitochondria to leak high levels of free electrons. It
status by evaluation of reactive oxygen species levels in
is therefore likely that these damaged mitochondria
whole semen and isolated spermatozoa. Fertil Steril 83:
contributed to the elevation in ROS and NO levels.
800–803.
The previous studies have used chemiluminescence to
Aziz N, Saleh RA, Sharma RK, Lewis-Jones I, Esfandiari N,
quantify ROS levels. A study by Mahfouz et al. (2009b)
Thomas AJ Jr, Agarwal A (2004) Novel association between
demonstrated that both the DCFH% method, as used in sperm reactive oxygen species production, sperm morpho-
this study, and the chemiluminescence assay used in the logical defects, and the sperm deformity index. Fertil Steril
previous studies provide relatively similar results. There- 81:349–354.
fore, our results are valid and signify a need to minimise Bize I, Santander G, Cabello P, Driscoll D, Sharpe C (1991)
ROS levels during sperm preparation, because of the Hydrogen peroxide is involved in hamster sperm
harmful effects on sperm motility. capacitation in vitro. Biol Reprod 44:398–403.
Overall, high ROS levels are not the only cause for Du Plessis SS, Makker K, Desai NR, Agarwal A (2008) The
male factor infertility, but continue to play an essential impact of oxidative stress on in vitro fertilization. Expert Rev
role in low live birth rates, because of the DNA damage Obstet Gynecol 3:539–554.
in spermatozoa caused by the induced OS. Further stud- Griveau JF, Renard P, Le Lannou D (1994) An in vitro
ies can be performed on measuring other types of ROS promoting role for hydrogen peroxide in human sperm
and exogenous ROS to elucidate the relationship capacitation. Int J Androl 17:300–307.
between ROS production and exogenous ROS introduc- Koppers AJ, De Iuliis GN, Finnie JM, McLaughlin EA, Aitken
tion. In addition, specific concentrations at which ROS RJ (2008) Significance of mitochondrial reactive oxygen
begin to compromise sperm function could be analysed. species in the generation of oxidative stress in spermatozoa.
Knowledge of the pathological levels allows clinicians to J Clin Endocrinol Metab 93:3199–3207.
treat infertility based on specific circumstances (i.e. high de Lamirande E, Gagnon C (1993) Human sperm
ROS, but low physiological defence). It is also important hyperactivation and capacitation as parts of an oxidative
process. Free Radic Biol Med 14:157–166.
not to see these results in isolation, but to bear in mind
Lampiao F, Strijdom H, Du Plessis S (2006a) Reactive
that the imbalance between ROS production and total
oxygen species measurement in human spermatozoa by
antioxidant capacity in seminal plasma will influence the
flow cytometry using the fluorescent probe, 2¢,7¢-dichloro-
developing of OS and relate to sperm dysfunction
fluorescein-diacetate (DCFH-DA). Med Technol SA
in vivo (Sharma et al., 1999).
20:7–8.
The adverse effects of exogenous H2O2 on sperm Lampiao F, Strijdom H, du Plessis SS (2006b) Direct nitric
motility parameters further exemplify the importance of oxide measurement in human spermatozoa: flow cytometric
minimising ROS introduction during sperm preparation analysis using the fluorescent probe, diaminofluorescein.
for various ART procedures, especially when spermatozoa Int J Androl 29:564–567.
are removed from the seminal plasma during washing Mahfouz R, Aziz N, Sharma R, Bykova M, Sabanegh E,
procedures. Because sperm preparation is necessary to Agarwal A (2008) Assessment of intracelular human sperm
ART procedures, maintaining sperm integrity is vital to reactive oxygen species after hydrogen peroxide exposure
the success of the procedure. using four different probes. Fertil Steril 90(Suppl 1):320–
321.
Mahfouz RZ, du Plessis SS, Aziz N, Sharma R, Sabanegh E,
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210 ª 2010 Blackwell Verlag GmbH Æ Andrologia 42, 206–210