Important aspect of sperm function such as motility and capacitation appear to be mediated at least partially though hyaloronic acid (HA). Present study investigated effects of different doses of HA on sperm motility and vitality in human. Sperm was obtained from 20 male from IVF clinic in Imam Khomeini Hospital. Sperm motility and vitality in human semen was analyzed according to WHO criteria before and 4 hours after treatment with different doses of HA (0.750, 1000 and 1250 Î¼g/ml). The results showed that in 1000 Î¼g/ml the percent of stage 3 and 4 increased compare to control group. Percent of stage 1 and 2 decreased in group with 1000 Î¼g/ml HA, there was an increase in the percentage of stage 3 and 4 and decrease in percentage of stage 1 and 2 compare to control. In the group treated with 1250 Î¼g/ml stage 1 and 2 increased while stage 3 and 4 decreased. Vitality in all groups decreased except of the group treated with 1000 Î¼g/ml HA. The group with 1250 Î¼g/ml showed significantly decrease in vitality compare to fresh group (P < 0.05). The present study showed that the effects of HA on sperm motility and vitality is dose dependant and 1000 Î¼g/ml HA had the effective role on sperm parameters. Â© 2009 Tehran University of Medical Sciences. All rights reserved

Amanpour, S.

Nikzad, H.

Pasbakhsh, P.

Roushandeh, A.M.

Sobhani, A.

Sohrabvand, F.

kashan university of medical sciences

 ORIGINAL REPORT  
 
*Corresponding Author: Aligholi Sobhani 
Department of Anatomy and VALI-e-ASR Reproductive Health Research Center, School of Medicine, Tehran University of Medical Sciences, Te-
hran, Iran 
Tel: +98 21 64432348, Fax: +98 21 64432348, E-mail: sobhania@tums.ac.ir 
 
Effects of Different Doses of Hyaloronan on Human Sperm Motility,  
Vitality and Morphology 
Amaneh Mohammadi Roushandeh1, Farnaz Sohrabvand2, Saeid Amanpour2, Parichehr Pasbakhsh3,  
Abdollah Sobhani4*, Hossein Nikzad5, and Aligholi Sobhani6 
1
 Department of Anatomy, School of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran 
2
 VALI-e-ASR Reproductive Health Research Center, Tehran University of Medical Sciences, Tehran, Iran 
3
 Department of Anatomy, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran 
4
 Department of Animal Sciences, Faculty of Agriculture, University. of Mohghegh Ardabili, Ardabil, Iran 
5
 Department of Anatomy, Faculty of Medicine, Kashan University of Medical Sciences, Kashan, Iran 
6
 Department of Anatomy and VALI-e-ASR Reproductive Health Research Center, Faculty of Medicine, 
Tehran University of Medical Sciences, Tehran, Iran 
 
Received:  12 Mar. 2008; Received in revised form:  4 Apr. 2008; Accepted: 14 May 2008 
 
Abstract- Important aspect of sperm function such as motility and capacitation appear to be mediated at 
least partially though hyaloronic acid (HA). Present study investigated effects of different doses of HA on 
sperm motility and vitality in human. Sperm was obtained from 20 male from IVF clinic in Imam Khomeini 
Hospital. Sperm motility and vitality in human semen was analyzed according to WHO criteria before and 4 
hours after treatment with different doses of HA (0.750, 1000 and 1250 µg/ml). The results showed that in 
1000 µg/ml the percent of stage 3 and 4 increased compare to control group. Percent of stage 1 and 2 de-
creased in group with 1000 µg/ml HA, there was an increase in the percentage of stage 3 and 4 and decrease 
in percentage of stage 1 and 2 compare to control. In the group treated with 1250 µg/ml stage 1 and 2 in-
creased while stage 3 and 4 decreased. Vitality in all groups decreased except of the group treated with 1000 
µg/ml HA. The group with 1250 µg/ml showed significantly decrease in vitality compare to fresh group (P < 
0.05). The present study showed that the effects of HA on sperm motility and vitality is dose dependant and 
1000 µg/ml HA had the effective role on sperm parameters. 
© 2009 Tehran University of Medical Sciences. All rights reserved. 
Acta Medica Iranica 2009; 47(5): 369-372. 
 
Key words: Human sperm, hyaloronan, sperm parameters 
 
Introduction 
  
Glycosaminoglycans (GAGs) are essential components 
of the extracellular matrix, contributing to cell recogni-
tion, cellular adhesion and growth regulation. The GAGs 
can be classified by their sugar residues and other char-
acteristics such as sulfation into four main groups of 
GAGs including the non-sulfated glycosaminoglycan, 
hyaluronic acid (HA), or hyaluronan (1). Important as-
pects of sperm function such as motility (2, 3) and ca-
pacitation (4, 5) appear to be mediated, at least partially, 
through HA. Addition of HA to fresh (6,7) and frozen–
thawed (8) sperm improves sperm motility in medium. 
Interestingly, HA not only improves sperm motility after 
freezing and thawing procedures, but it further appears 
to stabilize spermatozoa with already declining motility 
(7). The enhancement of sperm motility and velocity 
occurred as a direct response to HA. There is an instan-
taneous increase in sperm velocity and tail cross-beat 
frequency upon HA exposure and when, after density 
gradient centrifugation, the HA-exposed sperm is trans-
ferred to a standard medium, the motility and velocity 
properties return to those of the control sperm. It can be 
concluded that HA effects on sperm are likely to be re-
ceptor mediated, in line with the evidence established by 
various laboratories for the presence of the HA receptor 
in human sperm (8). Concurrently, with the sperm matu-
ration studies, in another line of experiments, scientists 
have investigated the effects of HA on human sperm 
function. Exogenous HA decreased polyspermy during 
Hyaloronan and sperm parameters 
370   Acta Medica Iranica, Vol. 47, No. 5 (2009) 
conventional porcine IVF (4), an effect probably related 
to the capacity of HA to delay sperm capacitation and/or 
the acrosome reaction (4, 5). It has been reported that 
effects of hyaluronan on sperm is dose dependant (9). 
There isn’t any report about the optimal dose of hyalu-
ronan on human sperm motility, viability and morphol-
ogy. The aim of the present study was to test the hy-
pothesis that supplementing with different dose of HA 
has an important role on the viability and motility of 
sperm.  
 
 
Patients and Methods 
 
This study was initially approved in the ethical and sci-
entific committee of the Vali-e-Asr Reproductive Health 
Research Center, Tehran, Iran. Semen samples were 
obtained from 20 couples referred to Vali-e-Asr Repro-
ductive Health Research Center for ICSI and IVF treat-
ment. The semen samples were collected by masturba-
tion after 3-4 days of abstinence on the day of oocyte 
recovery. Routine semen analysis was carried out by 
light microscopy according to World Health Organiza-
tion (WHO) criteria. After ART treatment, the remain-
ing samples were used for this research. Each sample 
was divided into five groups. The Group 1 (fresh con-
trol): These samples were analyzed freshly without any 
treatment. The Group 2 (control): They were maintained 
in HTF media (Sigma) without any hyaluronan (Sigma) 
supplementation for 2 h. In groups 3, 4 and 5, HA with 
the doses of 750, 1000 and 1250 µg/ml were added into 
HTF media, respectively. After 4 h incubation, the 
sperms were assessed for motility, vitality and morphol-
ogy. 
 
Sperm analysis 
Sperm motility was analyzed according to WHO 
manual. For evaluating sperm viability, we used the eo-
sin staining. Sperm and eosin (Sigma) were mixed (3:1) 
and number of sperm was counted by 400 magnifica-
tions by an invert microscope rapidly. Sperm smear was 
prepared on the slide for morphology analyzing. After 
fixing with 1:1 volume of alcohol (sigma) and ether 
(sigma), the slides were stained with papanicolaou me-
thods. Numbers of coiled sperm, stumped tail sperm, 
cytoplasmic droplet and remnant were analyzed by bin-
ocular microscope with 400 magnifications. 
 
Hyaluronan treatment 
After evaluation of fresh sperm, the samples were 
treated with different doses of Hyaluronan (0, 750, 1000 
and 1250µg/ml). The samples were put in incubator 
(37c° and 5% Co2 for 4 hours). After 4 hours the differ-
ent groups of sperm were analyzed again. 
 
Statistical analysis 
Data were analyzed by One-Way ANOVA followed 
up with post hoc test (Tukey). Statistical significance 
was set at P < 0.05. 
 
Results 
 
Sperm motility 
Table 1 indicates the various motility stages of sperm in 
different groups. As shown in this table, percentage of 
stage 1 in group 4 (1000 µg/ml) was decreased signifi-
cantly compared to control groups (P<0.05) but in group 
5 (1250 µg/ml) the stage 1 significantly increased. The 
percentage of stage 2 decreased in group 4 compared to 
other groups. In group 4, stage 4 motility significantly 
increased compared to all off other groups. In group 5 
there was significant decrease in stages 4 motility (P < 
0.05). 
 
Sperm viability 
As shown in figure 1, viability in all groups after 
treatment with HA was decreased except for group 4. In 
group 5 significant decrease (P < 0.05) in percentage of 
viability compared to fresh group was observed. 
 
 
 
Table 1. Various stages of motility in different groups 
1250 µg/ml HA 1000 µg/ml HA 750 µg/ml HA Control after 4 hr Fresh control    Groups       
Stages  
21% 12% 16% 15% 14% 1 
25% 10% 17% 17% 16% 2 
28% 33% 29% 28% 25% 3 
26% 45% 38% 40% 45%* 4 
 
 
 
A. Mohammadi Roushandeh, et al. 
    Acta Medica Iranica, Vol. 47, No. 5 (2009)   371 
 
65
70
75
80
85
90
95
fresh control 750 group 4
hr
1000 group 4
hr
1250 group 4
hr
Percent of viability in different groups
Figure 1. Percent of viability in different groups 
 
 
 
Discussion 
 
Hyaluronan (HA) is a linear polysaccharide which is a 
mediator of cell locomotion (9, 10). HA also occurs phy-
siologically in the reproductive system in cervical mu-
cus, the cumulus and the follicular and seminal fluids. 
The effects of HA on human spermatozoa have been 
explored in other clinical and research applications (12-
15). There are some reports regarding the effect of dif-
ferent doses of HA on sperm function(8,9). Sbracia et al. 
(8) showed that addition of 250µg/ml HA provided a 
significant motility increase for at least 4 h after the Per-
coll purification of spermatozoa. However, what re-
mains to be elucidated is determining the optimum doses 
of HA on sperm function. Therefore, we used different 
doses of HA were applied on sperm to evaluate their 
effect on motility, viability and morphology. The results 
of this study showed that effect of HA on sperm motility 
were dose dependant and 1000 µg/ml was optimal dose 
to improve the fast motility (stages 3 and 4). Further-
more, results showed that 1250µg/ml had a toxic effect 
on sperm and motility was decreased significantly. The 
mechanisms behind a HA-induced increase in sperm 
motility remains to be determined. A HA-receptor has 
been localized along the tail, mid piece, and head of 
human spermatozoa (16). Blocking this receptor with 
mono-specific antibodies resulted in inhibition of sperm 
motility. Moreover, enhanced phosphorylation of the 
34 kDa HA-binding protein (HABP) has been observed 
in response to HA supplementation, suggesting an im-
portant role of this glycosaminoglycan in initiating the 
transduction of signals controlling important processes 
of sperm physiology, such as motility (17, 18) that may 
result in an increase of the ATP levels and improved 
flagellar function(8). Bakhtiari et al. showed that, the 
hyaluronan may have an influence on motility, viability 
and fertilizing rate of mouse sperm and the dose of 750 µ 
g/ml had a significant effect on these factors. The results 
of this study, in addition, showed that viability in hyalu-
ronan group after four hours decreased except for 1000 
µg/ml. Sbracia et al. (8) showed that viability of incu-
bated with HA increased. These results are in agreement 
with those of other research (8, 9). This effect could be 
related to protective role of HA on sperm membrane. 
According to our study it seems that 1000 µ g/ml is the 
optimal dose that affects motility and vitality of human 
sperm. In this investigation different doses didn't have 
an effect on sperm morphology. Previous studies have 
shown that sperm morphology is controlled by spermio-
genic events such as acrosome formation and chromatin 
rearrangement. Protamine deficiency and aberrant P1/P2 
ratios are negatively related to abnormal morphology 
(19). After ejaculation sperm morphology isn’t affected 
by HA but it is possibly affected by osmolarity of sperm 
medium preparation. It seems that HA has a substantial 
advantage over other sperm motility enhancers, which 
have a temporary effect. Unlike other motility enhan-
cers, some of which cause short-term maintenance of 
improvement after removal of the agent, HA preserves 
motility in spermatozoa and therefore it can be useful in 
assisted reproduction technique (ART) in utilizing, es-
pecially when thawed semen samples are used. Also, 
when preparing sperm for IUI, IVF, application of HA 
which leads to extended motility  will increase fertiliz-
ing efficiency, while in intracytoplasmic sperm injection 
(ICSI) the improved sperm motility will enhance the 
selection of viable spermatozoa.  
 
Acknowledgements 
 
This project financially supported by research deputy of 
Tehran University of Medical Sciences. So, we would 
like to thank for their helps. 
 
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Effects of different doses of hyaloronan on human sperm motility, vitality and morphology

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Effects of different doses of hyaloronan on human sperm motility, vitality and morphology

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