Electromagnetic Field Stimulation and Sperm Parameters Evaluation

Electromagnetic Field Stimulation and Sperm Parameters Evaluation
D. Armanini1, C. Sabbadin1, P. Boccaccio2*, L. Bordin3, A. Andrisani4, A. Arcaro4, G. Clari3, G. Donà3, G. Moschini5
1
Dipartimento di Medicina dell'Università di Padova, Padova, Italy.
INFN, Laboratori Nazionali di Legnaro, Legnaro (Padova), Italy.
3 Dipartimento di Medicina Molecolare- sede di Chimica Biologica dell'Università di Padova, Padova, Italy.
4Dipartimento Di Salute Della Donna E Del Bambino dell’ Università di Padova.
5 Dipartimento di Fisica dell'Università di Padova, Padova, Italy, and INFN, Sezione di Padova, Padova, Italy.
*Member, IEEE.
2
INTRODUCTION
Spermatozoa, highly polarized and specialized cells with
a limited amount of cytosol and organelles, lost the
potential for gene expression during post-testicular
maturation. Sperm produced in the testis is immature, and
acquires maturational characteristics during its journey to
the female genital tract. The fluid environment surrounding
spermatozoa is involved in activities of sperm maturation,
survival and membrane modifications. Many proteins may
contribute to sperm composition or modifications and
alteration of fluid composition can lead to sperm
malfunction resulting in male infertility.
Recently, much attention has been paid to the influence
of electromagnetic fields on cells and biological molecules.
It is generally accepted that extremely low frequency
electromagnetic fields (ELF- EMFs) can exert influences
on biological functions of cells and tissues. These effects
include bone healing, nerve regeneration, influences on
cell calcium levels and oncogene activation. On the
contrary, the most part of studies concerning genotoxic
effects were negative.
At present, there is little information regarding possible
effects of ELF-EMFs on male reproductive system. In
mammals, the application of ELF-EMFs at 50 Hz with
intensities ranging from 1 mT up to 100 mT affected the
proliferative/differentiative
capacity
of
mouse
spermatogonia while the ELF-EMF treatments did not
induce clastogenic effects on human sperm chromosomes.
With regard to sperm motility, an increase in the
percentage of activated ejaculated sperm and a
prolongation of their viability were shown in fish after in
vitro sperm exposure to magnetic fields up to 100 mT [9].
Furthermore, it has been observed that mice exposure to a
constant intensity static magnetic field (0.7T) for different
periods of time did not produce any change in sperm
motility [1].
The aim of the present work was to investigate the
effects of ELF- EMFs on human sperm motility parameter
in fresh human semen.
MATERIALS AND METHODS
The motility of each spermatozoon was recorded by
computer assisted sperm analyzer (CASA) and was graded
as follows: Immotility (IM): no movement; Non-
progressive motility (NP): motility with an absence of
progression; Progressive motility (PR): spermatozoa
moving actively, either linearly or in a large circle,
regardless of speed.
Each measure consisted of VAP (average path velocity
in µm/s), VSL (progressive velocity in µm/s), LIN
(linearity as a %), ALH (amplitude of lateral head
displacement in µm) evaluations. The results are expressed
as means and standard errors for each time interval. The
mean values and the variances obtained for the control and
the treated samples at each time point were compared by
using the ANOVA method and the Student–Newman–
Keuls test. All tests were considered with a statistical
significance at p<0.05.
A picture of the cell exposure setup is shown in fig.1.
Cell exposure to pulsed electro-magnetic fields is achieved
by placing cell culture phials inside a Helmholtz Coil
Assembly. This consists of a pair of equal coils (22 cm
dia.) in a series electrical connection, fed by low-frequency
(f<100 Hz) current pulses. The cell culture temperature is
stabilized by a thermostatic bath. A commercial pulse
generator connected to a wide-band amplifier is employed
to energize the coils and the resulting magnetic field
intensity (B=100-800 T, typically) is periodically
measured by a Hall-effect magnetic field meter.
Fig. 1. Cell exposure setup, consisting of a Helmholtz Coil
Assembly and a thermostatic bath.
RESULTS
After initial spermiogram semen samples (n=30) were
divided in two groups depending on the percentage of
immotile cells (IM): Group 1 (Gr. 1), poorly motile group,
with more than 65% IM cells, and Group 2 (Gr. 2), motile
group, with percentages of IM cells under 65 (summarized
in Table 1).
Table 1. The motility of each spermatozoon is graded as follows:
Immotility (IM): no movement; Non-progressive motility (NP):
motility with an absence of progression; Progressive motility
(PR): spermatozoa moving actively, either linearly or in a large
circle, regardless of speed.
Gr.
1
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
IM
(%)
92
79
85
80
91
90
90
95
78
75
96
80
80
90
72
73
80
92
96
80
NP
(%)
2
2
1
3
2
0
1
0
3
2
2
2
2
4
1
5
0
1
1
0
PR
(%)
6
19
14
18
9
10
1
4
18
24
3
18
19
6
27
22
20
7
3
20
Gr.
2
1
2
3
4
5
6
7
8
9
10
IM
(%)
63
48
60
65
13
60
25
22
25
65
NP
(%)
4
3
4
0
16
6
25
6
12
10
PR
(%)
33
49
36
35
71
34
50
73
63
24
samples which were incubated at 37°C for 30 min in the
presence (irradiated, I) or absence (NI) of an EMF.
Samples were re-analysed and motility parameters
recorded (Table 2).
When incubated at 37°C without being purified from
semen envelopment, sperm are commonly affected by the
presence of capacitation inhibitors, which prevent sperm
from premature maturation leading to a consequent
increased fecundating inefficacy. Comparing values after
30’ with T0 (not incubated), sperm showed a slight
movement decrease involving almost all the evaluated
parameters. Cell exposure to EMF was achieved by setting
the current generator frequency at f=20 Hz and the
magnetic field intensity at B=760 T. Interestingly,
incubation with EMF emphasized this tendency, and in
both groups a significant decrease of the analysed motility
parameters, compared with the relative controls, was
evidenced.
Table 2. Each measure consisted of VAP (average path velocity
in µm/s), VSL (progressive velocity in µm/s), LIN (linearity as a
%), ALH (amplitude of lateral head displacement in µm).
Gr. 1
VAP (µm/s)
VSL (µm)
LIN
ALH (µm)
T0
48.7±9.8
42.8±9.8
69.5±8.2
2.3±0.6
30' NI
46.8±14.0
41.1±14.5
69.8±12.6
2.2±0.8
30' I
39.9±16.4 *
33.7±13.3 *
59.1±22.0 *
2.1±1.1
Gr. 2
VAP (µm/s)
VSL (µm/s)
LIN
ALH (µm)
T0
58.8±11.7
51.0±10.0
66.6±9.1
3.0±0.7
30' NI
47.2±12.0 *
41.0±10.0 *
62.8±5.2
2.5±1.0
30' I
51.0±11.0 *
42.0±9.3 *
59.0±8.3 *
2.9±0.7
DISCUSSION
The sperm motion pattern changes from progressive to
motility characterized by lateral head displacement, high
curvilinear velocity and large amplitudal flagellar waves.
This style of motility is termed ‘hyperactivation motility’
and essential in the fertilization process, as it allows the
sperm to travel through the cervical mucus and cumulus
oophorus and penetrate the dense zona-pellucida The exact
physiological mechanism for hyperactivation is thought to
be an increased intracellular calcium entry through sperm
calcium channels [1]. In addition, increased ATP content is
fundamental for sustaining energy-dependent increase
hyperactivation, and mitochondria are the principle
production site of chemical energy in the form of ATP
which can be used for supporting sperm motility. These
organelles, on the other hand, represent one of the major
determinants of male fertility. Accordingly, the presence of
structural and functional alterations in mitochondria from
asthenozoospermic subjects confirms the important role
played by these organelles in energy maintenance of sperm
motility, but also in cellular energy generation, apoptosis
regulation, and calcium homeostasis. Mitochondria are the
major source of ROS in the cell. Superoxide is continually
produced as a byproduct of normal cellular respiration. As
electrons are passed from complexes I to IV in the
mitochondrial ETC, continuous leakage of electrons
occurs, forming superoxide (1% of total rate of electron
transport). This superoxide is converted to hydrogen
peroxide by manganese superoxide dismutase in the
mitochondrial matrix under physiological conditions [2].
The formation of hydrogen peroxide from superoxide and
its transformation to hydroxyl radical is apparent,
especially when the mitochondrial ETC is abnormal or
compromised. In this line of evidence, the observation that
ELF-EMF can significantly reduce hyperactivation
parameters in semen may represent an intriguing aspect in
the sperm preparation. In the optic of assisted reproductive
techniques, semen manipulation may occur after a
significant period from production. In this time-lapse
mitochondria ROS production would result in sperm
denaturation leading to the worsening of the sperm sample
preparation. The ELF-EMF treatment, in contrast, would
result in a stand-by procedure thus allowing further
potential treatment.
[1] R. Iorio et al., Bioelectromagnetics 32 (2011) 15-27.
[2] N. Desai et al., Urology 75 (2010) 14-9.

Download Report