The Journal of Clinical Embryology™
Volume 13, Issue 4
Use of Temperature Regulated Centrifuge (Spermfuge)
Improves Sperm Quality
Ashok Agarwal, PhD, Reecha Sharma, MD, Rakesh K. Sharma, PhD, Vaishali Kale, MD, Aparna
Thiyagarajan, MSc, Sajal Gupta, MD,
Center for Reproductive Medicine, Cleveland Clinic, Cleveland, Ohio, USA
Corresponding author: Ashok Agarwal, Ph.D, HCLD
Professor and Director, Center for Reproductive Medicine , 9500 Euclid Avenue, Desk A19.1
Cleveland, Ohio 44195 • E-mail: [email protected]
Website: http://www.ClevelandClinic.Org/ReproductiveResearchCenter
Introduction
Materials and Methods
The goal of all sperm preparation techniques is
to improve sperm quality in ART. The ejaculated
spermatozoa have to be separated quickly and efficiently
from the seminal plasma so that they can be functional
[1]. The two routinely used preparation techniques are
swim-up and discontinuous density gradient techniques.
Since men with abnormal semen parameters do not yield
adequate motile sperm, it needs to be processed prior to its
use in ART [2]. Spermatozoa are sensitive to temperature
changes, dilution and centrifugation as it may adversely
affect sperm quality, especially in infertile patients, by
generating harmful reactive oxygen species (ROS) [2-4].
Sperm centrifugation is an essential prerequisite for
men with severe oligozoospermia and it is commonly
used for ART. Semen samples that appear azoospermic
upon initial wet mount microscopy should be
centrifuged at a minimum of 1,000 X g for 15 minutes
in order to identify presence of sperm [5]. Studies have
shown harmful effects of centrifugation on human
sperm at speeds greater than 500g and for longer
than 5-7 minutes continuously [6]. The process of
centrifugation leads to the generation of reactive oxygen
species [7]. Free oxygen radicals such as super oxide
radical (O2-), hydrogen peroxide (H2O2), and hydroxyl
radical (OH) cause damage to the spermatozoa.
Both time and speed of centrifugation affect sperm
quality [3]. The changes in the temperature during
centrifugation generate ROS from the spermatozoa
compromising the quality and quantity of normal
healthy spermatozoa [8]. The objective of our study
was to examine the affect of centrifugation with a
temperature regulated centrifuge (Spermfuge) at
34oC on sperm quality as compared to a standard
non-temperature regulated centrifuge.
The study was approved by the Institutional Review
Board of the Cleveland Clinic. Semen samples were
collected from 12 healthy men with unknown fertility.
Semen samples were obtained by masturbation
after at least 48 hours of abstinence. Samples were
collected into sterile containers. Semen analysis was
performed according to WHO (4th ed.) guidelines
[9] and included manual and computer assisted
sperm analysis (CASA). Sperm viability and seminal
levels of reactive oxygen species were assessed after
centrifugation.
Motility characteristics assessed by CASA
Manual and computer assisted semen analysis
(CASA) (IVOS 10.7s; Hamilton Thorne Research,
Beverly, MA) were performed. For each measurement,
a 5μL sample was loaded on a MicroCell slide
(Conception Technologies, San Diego, CA). Four to
eight representative fields containing 200 or more
spermatozoa were examined. Samples were analyzed
for concentration, percent motility, and complex motion
characteristics. Sperm motion kinetics measured by
CASA included the following: sperm concentration
(X106/mL), percentage motility, curvilinear velocity
(VCL, µm/sec), straight-line velocity (VSL, µm/sec),
average path velocity (VAP, µm/sec), linearity (%), and
amplitude of lateral head displacement (ALH, µm). In
addition to the computerized results, manual results
were calculated for sperm concentration and motility.
CASA results were used when the difference between
the manual and CASA values was less than 20%. In
cases where the difference was greater than 20%, the
manual results were reported.
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The Journal of Clinical Embryology™
Volume 13, Issue 4
Temperature controlled versus regular centrifuge
A temperature regulated bench top centrifuge
maintained at 34oC was used (Spermfuge, Fornax,
Shivani Scientific Industries Pvt. Ltd, Mumbai, India)
and compared with a non- temperature regulated bench
top centrifuge (IEC Centra GP8, Thermo Scientific).
The Spermfuge temperature can be set between room
temperature (21o C) up to 42oC. The temperature
chosen for Spermfuge was 34o C based on the fact that
intrascrotal temperature is maintained around 32o
C to 34o C. This temperature is lower than the core
body temperature and is considered to be normal for
functionally normal sperm. An increase in intrascrotal
temperature of >34.1o C is associated with poor
spermatogenesis and epididymal maturation [10]. Each
specimen was divided into 2 aliquots and processed by
temperature regulated-Spermfuge (34oC) centrifugation
(group 1) and non-temperature regulated (group 2). We
used centrifugation speed of 500 g and time of 30 min
identical to actual sperm preparation protocol using
density gradient separation (DGS) used in most ART
labs. The standard DGS protocol uses centrifugation
at speeds producing <500g for 20 minutes followed
by another shorter centrifugation step that includes
removal of the seminal plasma, debris and the gradient
interphases. The sperm pellet is then resuspended in
sperm wash media and centrifuged at the same speed
for 7-10 min.
stain. Smears were made and allowed to air dry. Intact
spermatozoa are impermeable to the Eosin dye and
therefore do not stain and appear white, whereas
spermatozoa with damaged membrane allow the entry
of the stain and therefore appear pink. Sperm viability
was examined under X100 objective and percentage of
viable sperm was recorded. At least 200 sperms were
counted. An aliquot from each specimen was also tested
to determine the affect of centrifugation on sperm
longevity after 4 hours incubation at 37oC.
Statistical analysis
All parameters were examined for the mean, standard
deviation, and median. Student’s t-test was used for
comparison between the two groups. A P value < 0.05
was considered significant.
Results
Figures 1 – 3 demonstrate sperm quality results
following centrifugation of semen samples in group 1
compared to group 2.
Percent Motility
Spermfuge centrifuged samples in group 1 showed
significant increase in percent motility (66.4 ±
17.02) compared to group 2 (60.56 ± 16.59; p = 0.01)
illustrated in Figure 1. Group 1 specimens showed
lower amplitude of lateral head displacement (ALH)
obtained by CASA (5.98 ± 1.01) compared to samples
in group 2 (6.56 ± 1.03; p = 0.034) (Figure 2). This
increase in sperm motility (59.69 ± 18.9) in Group 1 was
maintained even after 4 hours of incubation (56.03 ±
15.5 (p = 0.016).
Measurement of reactive oxygen species (ROS)
Seminal ejaculates that had not undergone any
additional processing (neat samples) were used for ROS
measurement by chemiluminescence assay. Luminol
(5-amino-2, 3-dihydro-1, 4-phthalazinedione; Sigma
Chemical Co., St Louis, MO) was used as a probe. A
100 mmoL/ L stock solution of luminol was prepared
in dimethyl sulfoxide. For the analysis, 10 µL of the
working solution (5 mM) was added to 400 mL of a neat
sperm sample. Chemiluminescence was measured for
15 min using a Berthold luminometer (Autolumat LB
953; Berthold Technologies, 6LLC, Oak Ridge, TN).
Results were expressed6as X10 counted photons per
minute (cpm)/ 20 X 10 sperm [11, 12].
Figure 1
Sperm Viability and longevity measurement
Sperm viability was tested by Eosin-Nigrosin staining.
A 10 mL aliquot of well mixed semen sample was
mixed with 20 mL of Eosin stain and 20 mL of Nigrosin
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The Journal of Clinical Embryology™
Volume 13, Issue 4
Discussion
Figure 2
The aim of this study was to compare a temperature
regulated centrifuge to a traditional non-temperature
regulated centrifuge. Sperm motility is an important
parameter in predicting ART outcomes [13, 14]. The
most predictive parameter for estimating natural fertility
in both subfertile and fertile couples is the concentration
of progressively motile sperm [15, 16]. Reports show
that a significant positive correlation exists between
percent progressive motility, velocity, and morphology
parameters on both fertilization during IVF and
pregnancy rates [14]. Sperm motion variables, such
as motility, linearity, curvilinear velocity, and average
path velocity have been shown as reliable prognostic
indicators for fertilization potential of sperm [17].
Earlier studies have shown that the changes in
temperature affect the motility of the sperm [18-20].
Sperm motion characteristics such as motility and
velocity are found to be higher in sperm incubated at 37o
C as compared to 20o C [21]. The changes in temperature
also affect the generation of ROS in the semen samples.
Agarwal et al. have also concluded that the levels of
ROS are significantly lower in samples incubated at 37o
C as compared to 25o C and 4o C [11]. We chose the
temperature controlled centrifugation at 34o C based
on the fact that intrascrotal temperature is maintained
around 32o C to 34o C. It is also seen that the increase in
intrascrotal temperature of > 34.1o C is seen in sub fertile
men [10]. Our results show improved sperm motility
in a temperature regulated centrifugation maintained
at 34o C as compared to non- temperature regulated
centrifugation. This may be beneficial in maintaining
sperm motility and sperm function for preparation of
semen specimen for ART purposes. Sperm motility
parameters such as ALH have been correlated with
fertilization rates [14, 22]. We also demonstrated a
significant correlation between temperature regulated
centrifugation and ALH levels in our study. Franken et al.
have demonstrated similar differences in sperm motion
characteristics by the use of Spermfuge as compared to
non-temperature regulated centrifugation [23]. Their
study did not compare sperm motility after incubation
of 4hours (longevity study) and the production of ROS
in semen samples. In our study we have compared
the production of ROS by using spermfuge and
non-temperature regulated centrifugation. Reactive oxygen species
ROS values log (ROS + 1) were lower in Group
1 (3.03 ± 1.86) compared to group 2 (3.15 ± 1.39)
However, these results were not significantly different.
Viability
No significant difference (p= 0.08) was found in
sperm viability between group 1 (Spermfuge) and group
2 (non-temperature regulated) (p= 0.48).
Longevity
Improvement in sperm motility was seen after
4 hours incubation at 37oC (longevity study) in the
Spermfuge group as compared to group 2. This is
illustrated in figure 3.
Figure 3
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The Journal of Clinical Embryology™
Volume 13, Issue 4
Higher sperm motility and lower ROS production is
reported at 37oC as compared with 4oC and 25oC [19]. The optimal temperature at which semen samples should
be collected, transported and processed has been shown
to be 37oC as this prevents the deleterious effects of ROS
[19]. Sperm preparation techniques involving repeated
washing, centrifugation times and subsequent total ‘g’
force have also been associated with increased ROS
production. An increase in intrascrotal temperature
has likewise been associated with an increase in ROS
and poor semen quality as the result of conditions such
as varicocele [24]. The regulation of a set temperature
(34oC) during processing and centrifugation may thus
prevent production of ROS by maintaining an even
physiological temperature. Reactive oxygen species are
known to cause DNA damage which may then adversely
affect in vitro embryo development [25]. Exogenous
ROS is mostly produced by spermatozoa that are used
to inseminate oocytes during IVF [26]. We used the
temperature regulated centrifuge so as to maintain an
optimal processing temperature and thereby reduce
the production of ROS. Even though we did not find a
significant decrease in seminal ROS production when
using the temperature regulated Spermfuge (group 1)
compared to a non- temperature related centrifuge
(group 2) in this study, further investigations using
specimens from subfertile or infertile male patients may
demonstrate the benefits of performing sperm workup
using strict temperature control.
In conclusion, an efficient sperm preparation
method is one that preserves sperm motility, viability
and fertilization capacity during assisted reproduction
procedures. Temperature regulated centrifugation
(Spermfuge) improves sperm motility and longevity
when compared to a non-temperature regulated
centrifuge. Since our sample size was small, our results
need to be validated using a larger sample size. n
5. Corea, M., J. Campagnone, and M. Sigman, The diagnosis of
azoospermia depends on the force of centrifugation. Fertil Steril, 2005. 83(4): p.
920-922.
6. Sharma, R.K., et al., Effect of centrifuge speed, refrigeration medium,
and sperm washing medium on cryopreserved sperm quality after thawing.
Arch Androl, 1997. 39(1): p. 33-8.
7. Alvarez, J.G., et al., Centrifugation of human spermatozoa induces
sublethal damage; separation of human spermatozoa from seminal plasma
by a dextran swim-up procedure without centrifugation extends their motile
lifetime. Hum Reprod, 1993. 8(7): p. 1087-92.
8. Mortimer, D., Sperm preparation techniques and iatrogenic failures of
in-vitro fertilization. Hum Reprod, 1991. 6(2): p. 173-6.
9. [Laboratory manual of the WHO for the examination of human semen
and sperm-cervical mucus interaction]. 4th ed. 1999, Cambridge Press.
10.Zorgniotti, A.W. and A.I. Sealfon, Measurement of intrascrotal
temperature in normal and subfertile men. J Reprod Fertil, 1988. 82(2): p.
563-6.
11.Agarwal, A., S.S.R. Allamaneni, and T.M. Said, Chemiluminescence
technique for measuring reactive oxygen species. Reprod Biomed Online, 2004.
9(4): p. 466-468.
12.Kobayashi, H., et al., Quality control of reactive oxygen species
measurement by luminol-dependent chemiluminescence assay. J Androl, 2001.
22(4): p. 568-74.
13.Berlinguer, F., et al., Semen molecular and cellular features: these
parameters can reliably predict subsequent ART outcome in a goat model.
Reprod Biol Endocrinol, 2009. 7: p. 125.
14.Donnelly, E.T., et al., In vitro fertilization and pregnancy rates: the
influence of sperm motility and morphology on IVF outcome. Fertil Steril, 1998.
70(2): p. 305-14.
15.Ayala, C., E. Steinberger, and D.P. Smith, The influence of semen
analysis parameters on the fertility potential of infertile couples. J Androl, 1996.
17(6): p. 718-25.
16.Larsen, L., et al., Computer-assisted semen analysis parameters as
predictors for fertility of men from the general population. The Danish First
Pregnancy Planner Study Team. Hum Reprod, 2000. 15(7): p. 1562-7.
17.Joshi, N., et al., The importance of computer-assisted semen analysis
and sperm function testing in an IVF program. Int J Fertil Menopausal Stud,
1996. 41(1): p. 46-52.
18.Milligan, M.P., S.J. Harris, and K.J. Dennis, The effect of temperature on
the velocity of human spermatozoa as measured by time-lapse photography.
Fertil Steril, 1978. 30(5): p. 592-4.
19.Esfandiari, N., et al., Effects of temperature on sperm motion
characteristics and reactive oxygen species. Int J Fertil Womens Med, 2002.
47(5): p. 227-33.
20.Appell, R.A. and P.R. Evans, The effect of temperature on sperm motility
and viability. Fertil Steril, 1977. 28(12): p. 1329-32.
21.Marin-Briggiler, C.I., et al., Effect of incubating human sperm at room
temperature on capacitation-related events. Fertil Steril, 2002. 77(2): p. 252-9.
22.Sukcharoen, N. and J. Keith, Evaluation of the percentage of sperm
motility at 24 h and sperm survival ratio for prediction of in vitro fertilization.
Andrologia, 1996. 28(4): p. 203-10.
23.Franken, D.A., K, Temperature Controlled Centrifugation: - The Effect
on Motion Characteristics of Human Sperm.
24.Hendin, B.N., et al., Varicocele is associated with elevated spermatozoal
reactive oxygen species production and diminished seminal plasma
antioxidant capacity. J Urol, 1999. 161(6): p. 1831-4.
25.Agarwal, A. and T.M. Said, Oxidative stress, DNA damage and
apoptosis in male infertility: a clinical approach. BJU Int, 2005. 95(4): p. 503-7.
26.Bedaiwy, M.A., et al., Relationship of reactive oxygen species levels in
day 3 culture media to the outcome of in vitro fertilization/intracytoplasmic
sperm injection cycles. Fertil Steril.
References
1. Mortimer, D., Sperm recovery techniques to maximize fertilizing
capacity. Reprod Fertil Dev, 1994. 6(1): p. 25-31.
2. Gardner, D., Weismann, A, ed. Textbook of Assisted Reproductive
Technologies: Laboratory and Clinical prespectives. 2004, Informa Health
Care.
3. Shekarriz, M., et al., A method of human semen centrifugation to
minimize the iatrogenic sperm injuries caused by reactive oxygen species. Eur
Urol, 1995. 28(1): p. 31-5.
4. Aitken, R.J. and J.S. Clarkson, Significance of reactive oxygen species
and antioxidants in defining the efficacy of sperm preparation techniques. J
Androl, 1988. 9(6): p. 367-76.
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