Sildenafil citrate improves sperm motility but causes

a premature acrosome reaction in vitro

David R. J. Glenn, M.R.C.O.G., Carmel M. McVicar, Ph.D., Neil McClure, F.R.C.O.G.,
and Sheena E. M. Lewis, Ph.D.
School of Medicine, Obstetrics and Gynaecology, Queen’s University Belfast, Institute of Clinical Science, Belfast,
United Kingdom

Objective: To determine whether sildenafil citrate, a cyclic monophosphate-specific type 5 phosphodiesterase

inhibitor, influences sperm motility or the acrosome reaction.
Design: Laboratory analysis of sperm motility after exposure to sildenafil citrate using computer-assisted semen
analysis and acrosome reaction by fluorescein isothiocyanate-labeled peanut agglutinin staining.
Setting: An assisted reproductive technology (ART) unit.
Patient(s): Fifty-seven male patients.
Intervention(s): Sperm were divided into 90% (those with the best fertilizing potential used in assisted
conception) and 45% (the poorer population) fractions by density centrifugation and incubated with sildenafil
citrate (0.67 ␮M) at 37°C for up to 180 minutes.
Main Outcome Measure(s): Both the number and velocity of progressively motile sperm were significantly
increased by sildenafil citrate between 15 and 135 minutes. Furthermore, samples revealed that these effects were
consistent in the 90% and 45% populations of sperm. In both populations, sildenafil also caused a significant
increase in the proportion of acrosome-reacted sperm—22.1% compared with 11.8% in the control group of the
good quality fraction and 16.6% compared with 9.4% in the control group of the poorer quality fraction.
Conclusion(s): The use of sildenafil citrate may adversely affect male fertility. (Fertil Steril威 2007;87:1064 –70.
©2007 by American Society for Reproductive Medicine.)
Key Words: Sildenafil citrate, sperm motility, acrosome reaction

Within 14 days of its introduction in 1998, doctors were
writing more than 110,000 sildenafil scripts a week. To date
it has been prescribed to more than 23 million men world-
wide and more than 1 billion doses of sildenafil have been
prescribed (1).
As a result of this huge success, marketing strategies have
extended to include younger men; therefore phosphodiester-
ase (PDE) inhibitors, like sildenafil, are used increasingly by
men of reproductive age (2, 3). These drugs are widely
available on the internet, which facilitates their use as rec-
reational drugs (2, 3) and there is now robust evidence (3)
that their use in recreation has gained credence in young,
healthy men as sexual enhancers as well for older men,
requiring them for erectile dysfunction problems. Further-
more, the reproductive age range for men has extended
dramatically during the past decades with older parenting
becoming more popular and second families with older fa-
thers constituting an increasing proportion of Western soci-
ety. Moreover, PDE inhibitors are reportedly sold illegally in
nightclubs to use along with illicit “club drugs” such as
ketamine and amyl nitrite. They are offered inappropriately
Received November 1, 2005; revised and accepted November 3, 2006.
Presented at Spring meeting, British Fertility Society, Cheltenham,
England, April 2004.
Reprint requests: Sheena E. M. Lewis, Ph.D., Obstetrics & Gynaecology,
Institute of Clinical Science, Grosvenor Road, Belfast BT12 6BJ, United
Kingdom (FAX: 44-2890328247; E-mail: s.e.lewis@qub.ac.uk).
through e-mail attachments, offering prospective purchasers
the opportunity to obtain them without visiting their doctors.
A further niche market for PDE inhibitors is to target the
rapidly increasing population of young men who suffer from
erectile dysfunction related to medical conditions such as
diabetes and spinal cord injuries to use them in the treatment
of their erectile dysfunction (4). Because the partners of
many of these men are hoping to conceive, it is vital to
determine the effects of sildenafil on sperm function.
Probably of more concern, however, is the extensive ac-
ceptance of this drug by assisted conception units in the U.K.
In a questionnaire audit of all the Human Fertilization and
Embryology Authority’s licensed assisted conception units
in the U.K., we demonstrated that 42% would prescribe
sildenafil to patients finding it difficult to produce semen
samples on demand and particularly in timed cycles of
treatment.
This widespread use of sildenafil is of concern as it is a
selective type 5 PDE inhibitor (PDE5), one of a large family
of different types of PDE inhibitors. There are 11 PDE
inhibitors and 21 subfamilies, most with high levels of
specificity. They act by modulating the activity of cyclic
nucleotides, the second messengers and controllers of nu-
merous cell functions, thus regulating their degradation. Sil-
denafil acts by increasing intracellular levels of cyclic
guanosine monophosphate (cGMP) and cyclic adenosine

1064 Fertility and Sterility姞 Vol. 87, No. 5, May 2007 0015-0282/07/$32.00
Copyright ©2007 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:10.1016/j.fertnstert.2006.11.017
monophosphate (cAMP) (5). Previously, other phosphodies-
terase inhibitors, such as pentoxifylline, a type 4 PDE inhib-
itor, have been shown to affect sperm function (6). Sperm
exposed to pentoxifylline underwent motility alterations and
early acrosome reactions. If sperm are prematurely acrosome
reacted, they are rendered incapable of fertilization. These
were in vitro experiments and the effects persisted after the
drug was washed from the semen, suggesting that the effects
were irreversible. Another common property of pentoxifyl-
line and sildenafil is their ability to inhibit formation of
reactive oxygen species (ROS). Koupparis et al. (7) have
reported the inhibition of superoxide and expression of
gp47(phox) NAD [P]H oxidase by sildenafil in corpus cav-
ernosal smooth muscle cells. Similarly, our group has shown
the protective effects of pentoxifylline against ROS in hu-
man sperm (8). Since ROS (at low concentrations) are nec-
essary for normal sperm function, it is important that the
effects of sildenafil on human sperm are fully elucidated.
The aim of this study was to investigate the effects of
sildenafil on sperm motility using computer-assisted semen
analysis and acrosome reaction by fluorescein isothiocyanate-
labeled peanut agglutinin (FITC-PNA) staining with time.
MATERIALS AND METHODS
Subjects
Semen samples (n ⫽ 57) were obtained from unselected
men, aged 26 – 42 years, attending the Andrology Labo-
ratory, Royal Maternity Hospital, Belfast, for infertility
investigations. Their semen analyses indicated that they
had asthenozoospermic profiles (volume, 3.5 ⫾ 0.47 mL;
concentration, 82 ⫾ 11.3 ⫻ 106/mL; motility, 40.0% ⫾ 2.3%;
normal morphology 12.0% ⫾ 0.7%) as is the most common
profile of patients attending for infertility investigations in
Northern Ireland. Informed written consent for participation
was obtained from each recruit. The project was approved by
the Queen’s University Belfast Research and Ethics Com-
mittee and was in accordance with the Declaration of Hel-
sinki, as revised in 1983.
Samples were obtained by masturbation, into a sterile
container, after a minimum of 2 and a maximum of 5 days
abstinence. Subjects were only excluded if they were already
using sildenafil or other recreational drugs.
Preparation of Semen Using Density Centrifugation
Preparation of samples After liquefaction, routine semen
analyses were performed according to World Health Orga-
nization (WHO) recommendations (9) and then samples were
prepared using a two-step discontinuous Percoll gradient
(90%– 45%; Pharmacia Biotech AB, Uppsala, Sweden).
Each aliquot of liquefied semen was layered on top of the
gradient and centrifuged at 450 ⫻ g for 12 minutes. The
resulting sperm pellet was concentrated by centrifugation at
200 ⫻ g for 6 minutes and the low-density sperm removed
and concentrated by centrifugation at 200 ⫻ g for 6 minutes.
Fertility and Sterility姞
The final sperm preparations were suspended in a suitable
volume of Biggers, Whitten, and Whittingham (BWW) me-
dium (10) supplemented with 600 mg of albutein (Alpha
Therapeutic UK Ltd., Norfolk, England). Hence, two popu-
lations of sperm were prepared: that with the best fertilizing
potential as used in assisted conception treatments (90%
fraction), and that with the poorer population (45% fraction)
similar to the sperm profile of men with male infertility (11).
Soluble sildenafil citrate was prepared by dissolving 10
mg of pure sildenafil citrate (Pfizer, Sandwich, U.K.) in 100
␮L of 99% ethanol. The final concentration of ethanol to
which sperm were exposed was 0.0095%, which was shown
not to have any toxic effects on sperm function. This was
then diluted in BWW medium to give a final concentration
of 900 ng/mL Viagra. When mixed with an equal volume of
neat semen, the concentration was reduced to 450 ng/mL to
give a final concentration of 0.67 ␮M, equivalent to the
plasma concentration, 1 hour after oral ingestion, of 100 mg
of sildenafil—the maximum recommended dose.
Semen and prepared sperm (90% and 45%) were divided
into aliquots and incubated in the presence or absence (con-
trol; by adding the same volume of BWW medium) of
sildenafil.
Sperm motility assessment Quantitative sperm motility pa-
rameters were assessed using computer-assisted semen anal-
ysis (Hamilton Thorne Integrated Visual Optical System
sperm analyzer; version 10.7; Hamilton-Thorne Research,
Beverly, CA). The settings used for analysis were acquisi-
tion rate, 30 Hz; minimum contrast, 7; minimum size, 6;
low-size gate, 0.4; high-size gate, 1.6; low-intensity gate,
0.4; high intensity gate, 1.6; HTM magnification factor, 2.04.
Ten microliters from each sample were placed on preheated
slides (37°C) and inserted into the machine. Percentage
motility, percentage progressive motility, average path ve-
locity (VAP), progressive velocity, and curvilinear velocity
(VCL) were determined at intervals of 0, 15, 30, 45, 75, and
135 minutes. Cells were counted as progressively motile if
VAP was ⬎25 ␮msec⫺1.
Preparation of Sperm for Acrosome Reaction
Determination by FITC-PNA Staining
Two hours after ejaculation, prepared sperm was divided
into aliquots and incubated in the presence of an equal
volume of sildenafil in solution (to give a final concentration
of 0.67 ␮M) or absence (control) by adding the same volume
of BWW medium) of sildenafil. After incubation of the test
and control samples for 1 h at 37°C, sperm acrosome status
was assessed by FITC-PNA (Sigma, Dorset, UK) (12) stain-
ing and epifluorescence microscopy. Peanut agglutinin, from
Arachis hypogea, is specific for ␤-D-galactose residues and,
hence, binds to, and labels, the outer acrosomal membrane
(13).
Briefly, 20 ␮L of sperm suspension were spread over a
clean microscope slide and allowed to air-dry. The smear
1065
 FIGURE 1
The effects of sildenafil on progressive motility of
‘
human sperm control sperm ° sperm incubated
in the presence of 0.67 ␮M sildenafil citrate Values
are means for 22 samples; P⬍.001.
Glenn. Sildenafil citrate accelerates sperm function. Fertil Steril 2007.
was considered significant. Comparisons of the proportions
of sperm with intact, partially reacted, and fully reacted
acrosomes were made using ␹2 analysis.
RESULTS
Effects of Sildenafil Citrate on Total Sperm Motility
After incubation with the test substance for 15 minutes, the
percentage of progressively motile sperm in the sildenafil
aliquots had increased markedly compared with controls
(Fig. 1). This effect was observed at each time point mea-
sured. By 120 minutes there was a 52% increase (P⬍.001).
The drug also enhanced VSL of the sperm after 15 minutes
(⫹11%). Again, this effect was sustained through to 120
minutes (⫹14%; Fig. 2). Curvilinear velocity showed similar
increases at each time point (data not shown), therefore the
linearity of movement was unchanged. No significant differ-
ences were noted in amplitude of lateral head movements
and beat frequency between the groups (data not shown).
Within this patient group, 50% of the semen samples were
normozoospermic and the rest, asthenozoospermic. The per-
centage increase in progressive motility was similar in each
sample whether it was normal or asthenozoospermia.

was then fixed in 95% ethanol for 5 minutes and again

allowed to air-dry. Fixed slides were stained in FITC-PNA
(600-␮L aliquot of FITC-PNA in 15.4 mL of reagent water
in a foil-covered Coplin jar) for 15 minutes, at ambient
temperature. Slides were rinsed by dipping in phosphate-
buffered saline (PBS) twice before fixing for 15 minutes in
paraformaldehyde at ambient temperature. Slides were air-
dried, mounted, and stored in the dark until scoring. Between
100 and 250 sperm were counted per slide and scored into
three classes for PNA labeling (12).
Effects of Sildenafil Citrate on Motility in 90% and
45% Percoll Fractions
Sildenafil citrate had similar effects on the motility param-
eters of good (90% Percoll layer) and poor (45% Percoll
layer) quality populations of sperm. In the 90% layer, by 120
minutes, the percentage of highly progressive motile sperm
in the sildenafil group was substantially (⫹28%) increased.
Despite a very small proportion of sperm in the control
group of the poor quality (45%) fraction exhibiting progres-

I: acrosome intact—whole acrosome labeling, denotes an

intact outer acrosomal membrane;
II: partially acrosome reacted—patchy acrosome labeling, FIGURE 2
suggestive of a transition stage where the outer acroso-
The effect of sildenafil citrate on the velocity of
mal membrane is fenestrating;
III: acrosome reacted— equatorial segment only labeling, ‘
human sperm control sperm ° sperm incubated
in the presence of 0.67 ␮M sildenafil citrate Values
denoting a normally acrosome-reacted spermatozoa that
are means for 22 samples; P⬍.001.
has lost the outer acrosomal membrane over the anterior
cap of the acrosome but has retained the equatorial
segment of the acrosome intact.

Statistical Analysis
To determine changes in sperm motility parameters with
time after exposure to sildenafil, we calculated the time-
weighted values of percentage progressive motility and
straight-line velocity (VSL). This is equivalent to calculating
the area under the curve using the trapezium rule. These
time-weighted values were then compared using Student’s
paired t-test to avoid multiple comparisons and give a more
robust single statistic on which to determine the effects of the
drug. Student’s t-tests were also used to measure differences
Glenn. Sildenafil citrate accelerates sperm function. Fertil Steril 2007.
between prepared populations of sperm. A value of P⬍.05

1066 Glenn et al. Sildenafil citrate accelerates sperm function Vol. 87, No. 5, May 2007
 FIGURE 3
The effects of sildenafil citrate on sperm acrosome reactions  control sperm  sperm incubated in the
presence of 0.67 ␮M sildenafil citrate Values are means ⫾ SE for 33 samples.*P⬍.05, **P⬍.01, show
significant differences with Student’s t-test between control samples and those exposed to 0.67 ␮M of
sildenafil citrate.
Glenn. Sildenafil citrate accelerates sperm function. Fertil Steril 2007.
sive motility, significantly more sperm were progressively
motile under the influence of the drug. Similarly, increases
were seen in VSL and VCL, again leading to no alteration in
linearity (data not shown). No significant differences were
noted in amplitude of lateral head movements and beat
frequency between the groups in either layer (data not
shown).
Effects of Sildenafil Citrate on the Acrosome Status
Sildenafil citrate induced a significant increase in the per-
centage of acrosome-reacted sperm in both the 90% and 45%
populations (n ⫽ 33; Fig. 3). In the former, sildenafil in-
duced an 87% increase in the number of reacted sperm and
76% in the latter. A corresponding decrease in the number of
acrosome intact sperm was noted. Sildenafil induced a 13%
reduction in the number of acrosome intact sperm in the 90%
population and a 34% reduction in the 45% population. The
number of partially reacted sperm increased in the 90% layer
but decreased in the 45% layer.
DISCUSSION
Sildenafil is a potent and specific inhibitor of cGMP-specific
PDE5, which is the most common PDE in the corpus cav-
ernosum (14). This leads to its usefulness in preventing
cGMP breakdown, thereby increasing intracavernosal pres-
sure and penile erection (15, 16). Sperm function has also
been shown to be altered by PDE5 inhibitors (17) and by
other PDE inhibitors, namely PDE1, which degrades cAMP
and cGMP (18), and PDE4, which is cAMP-specific (19),
suggesting that this is not a response specific to only one of
the PDE family.
In this study we have demonstrated two effects of this
PDE5 inhibitor on sperm function, namely a sustained en-
Fertility and Sterility姞
hancement of motility, both in numbers of progressively
motile sperm and their velocity and also a premature activa-
tion of the acrosome reaction. Our findings on their effects
on motility confirm our previous studies with the more
general PDE inhibitor Pentoxifylline (20 –22).
Our findings also support those effects of PDE4 inhibitors
on sperm motility reported by Fisch et al. (23) and PDE5
inhibitors reported by Cuadra et al. (24) and Lefievre et al.
(25). Fisch’s group has shown type-specific PDE inhibition
on sperm motility and the acrosome reaction where PDE4
inhibitors enhance sperm motility over controls without
affecting the acrosome reaction, whereas PDE1 inhibitors
selectively stimulate the acrosome reaction. From these data,
they conclude that there are at least two distinct PDE types
in human spermatozoa. Our data suggest that there are also
common effects observed with more than one of the PDE
inhibitor family, as shown in this study, the specific PDE5
inhibitor sildenafil affects both sperm functions.
In contrast to these findings is a small (6 fertile and 6
infertile men) study by Burger et al. (26) reporting no effects
on motility, viability, or membrane integrity at a range of
concentrations (125–750 ng/mL) during a 3-hour period.
Also, Aversa et al. (27) found no changes in seminal param-
eters when a group of 20 men were given 100 mg of
sildenafil citrate orally and semen profiles determined 1 hour
after administration. This short time period between inges-
tion and semen production may have been insufficient for
any effects to become apparent, although another study (28),
using a second generation PDE inhibitor Tadalafil, reported
no detrimental effects on sperm concentration or motility
after oral administration for 6 months. In contrast, Andrade
et al. (29) showed no effect of sildenafil on sperm motility at
a concentration of 200 ␮g/mL, but reduced motilities at
higher concentrations (2,000 ␮g/mL). The reason of the
1067
reduction may be due to toxic effects at higher dosage, as
2,000 ␮g/mL is considerably higher than physiological se-
rum concentration. In contrast with this study, we chose a
concentration of sildenafil that is equivalent to the mean
maximum total plasma concentration (440 ng/mL) present
30 minutes after a single oral dose of 100 mg. Although the
manufacturers’ information sheet (30) shows negligible
amounts (0.0002% of 100-mg oral dose) of sildenafil in
semen, the concentration of sildenafil to which sperm are
exposed in the epididymis is unknown.
The rates at which drugs pass from blood (or plasma) and
concentrate in the biological fluids are regulated by their
physicochemical characteristics (31), with the concentration
and lipid solubility of the undissociated drug being the
physical properties most important in determining the disso-
ciation constant (32). These specific characteristics are not
published for sildenafil but given its hydrophobic nature, it is
probable that it crosses the blood–testis barrier without dif-
ficulty. We know that sperm are very vulnerable during the
prolonged period when they pass through the epididymis
(33). The manufacturer states that the final quantity of drug
in semen averages only 188 ng (30). This concentration is
substantially less than that to which sperm are exposed
before ejaculation. At that point semen is 5% sperm and 95%
seminal plasma, composed of contributions from the prostate
and seminal vesicles. Thus, it is difficult to determine the
sildenafil concentrations to which immature testicular and
epididymal sperm are exposed in the interval from ingestion
of the drug until the sperm are mixed with the ejaculatory
fluids as part of the ejaculatory process.
The effects of sildenafil on sperm function reported in this
study were all performed in vitro. Thus extrapolations to the
in vivo situation must be considered with care. Although
these concentrations are similar to physiological concentra-
tions in the male reproductive tract, the presence of poten-
tially modulating factors in the body may alter its effects.
Nonetheless, an additional characteristic of PDE inhibitors
that causes concern is the persistence of their effects. These
effects have been observed for at least 3 hours after at-
tempted removal by repeated washings (6). In a case report
of Viagra use for human-assisted reproduction the investi-
gators noted that there was no fertilization of the oocytes
despite the intracytoplasmic injection of the sperm (34).
They attributed the failed fertilization to the delay in obtain-
ing the semen sample and the resultant advanced age of the
oocytes. However, it is possible that Viagra had a deleterious
effect on sperm function and it was the reason for the
fertilization failure. When Viagra is given to patients by oral
administration there is no opportunity to remove either the
sperm or the embryos from its potentially persistent and
damaging effects.
In this study we have also shown an early activation of the
acrosome reaction with this PDE5 inhibitor. This has impor-
tant clinical implications because sperm that acrosome- react
before contact with the oocyte are incapable of fertilization
1068 Glenn et al. Sildenafil citrate accelerates sperm function
(35, 36). Given that the majority of sperm acrosome react on
exposure to sildenafil, the drug may cause significant im-
pairment to their fertilizing potential.
Again the literature regarding the effects of PDE inhibi-
tors on sperm acrosome reactions is conflicting. Fisch et al.
(23) have shown that PDE1 inhibitors selectively stimulate
the acrosome reaction, whereas, in their hands, PDE4 inhib-
itors have no effect. Furthermore, Lefievre et al. (37) and du
Plessis et al. (38) found no effect with PDE5, whereas
Cuadra et al. (24) found a marked early acrosome reaction.
The latter are in agreement with the well-documented acro-
some activation effects of general PDE inhibitors such as
pentoxifylline (39, 40) and caffeine (41). Indeed, Tasdemir
et al. (42) showed the direct benefits of PDE in an IVF
program where Pentoxifylline-enhanced acrosome reaction
led to an improved fertilization rate and because of that fact
Yovich (43) recommended the addition of pentoxifylline in
cases of assisted reproduction where the acrosome reaction
was suboptimal.
Most recently, an in vivo study (44) has reported that
another PDE, PDE11, the most recently discovered family of
human PDEs, is highly expressed in testis, prostate gland,
and developing sperm. This suggests that PDEs have a
physiological role in the male reproductive tract, although it
is as yet unproven. In support of this, Wayman et al. (45)
found that ejaculated sperm from PDE11 (⫺/⫺) mice dis-
played reduced sperm concentration, rate of forward pro-
gression, and percentage sperm viability. This group also
observed a marked premature spontaneous capacitance of
sperm from PDE11 knockout mice. This adds to concerns
that drugs that inhibit PDEs may disrupt the delicate phys-
iological balance and “lead to reduced fertilizing ability.”
These researchers suggest that the role of PDE inhibitors on
sperm fertilizing capacity requires clarification. The role of
PDE11 in human testicular function and its interaction with
PDE inhibitors remains controversial (44).
The mechanism by which PDE inhibitors exert their phar-
macological effects is by inhibiting phosphodiesterase, an
enzyme responsible for the degradation of cGMP to GMP
(46). This creates an environment with increased energy
substrates. These raised levels of cGMP affect many intra-
cellular functions including calcium transport (47). Altered
levels of calcium are known to affect sperm motion and an
energy-dependent influx of calcium into the sperm cell is
believed to be responsible for initiation of the acrosome
reaction (48).
An additional function of cGMP is as a second messenger
for nitric oxide (NO) and PDE inhibitors, which are also
known to augment NO production (49). We have shown
(50, 51) that NO assumes a key role in the maintenance of
sperm motility, but is only beneficial within a very carefully
regulated concentration range. Nitric oxide is a potent reac-
tive oxygen species that will interact with superoxide anions
to form hydroxyperoxyl radicals or peroxynitrite, which will
form the hydroxyl radical; all of which are detrimental to
Vol. 87, No. 5, May 2007
sperm (51). Thus, sildenafil has the potential to alter sperm
function by in vivo effects on the NO concentration range, as
the beneficial effects of NO at nanomolar concentrations
become highly detrimental at millimolar concentrations.
The PDE inhibitors are known to impair both preimplan-
tation (52, 53) and postimplantation (44) embryo develop-
ment. Cell numbers of blastocysts and development of the
early embryo are both significantly reduced after exposure to
pentoxifylline (54). Lacham-Kaplan and Trounson (52) also
observed embryonic arrest and retarded development after
exposure to pentoxifylline. Elevated levels of cAMP have
also been shown to inhibit germinal vesicle breakdown and
oocyte maturation (55). Morales et al. (56) have reported that
although sildenafil is a selective inhibitor of type 5, it has
inhibitory effects similar to types 1 and 6 PDE. In agree-
ment, we have shown no differences between general PDE
and specific PDE5 inhibitors in sperm function. If they act
similarly in embryo development, we have further cause for
concern. Studies on their effects on embryo development are
urgently needed before their use in the clinic is advocated.
There are also considerable implications in terms of public
health education and the need to inform recreational users
of its potential side effects. These concerns make it all the
more crucial to elucidate fully any effects sildenafil has on
human reproduction.
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