Original Article
The Intensity of Free Radical Processes and the Testosterone and Estradiol Levels in Seminal Fluid of Men with Different Types of Pathospermia - Personalized Approach
Authors
Tatiana Shkurat, Professor, Department of Genetics, Southern Federal University, Rostov-on-Don, Russia,
Ksenia Savikina, Embryologist, Department of Embryogenesis, Center for Human Reproduction and IVF, Rostov-on-Don, Russian Federation,
Svetlana Lomteva, Embryologist, Department of Embryogenesis, Center for Human Reproduction and IVF, Rostov-on-Don, Russian Federation,
Karina Sagamonova, Professor, Center for Human Reproduction and IVF, Rostov-on-Don, Russian Federation,
Alexander Shestel, Andrologist, Center for Human Reproduction and IVF, Rostov-on-Don, Russian Federation,
Viktor Prokofev, Researcher, Department of Biomedicine, Southern Federal University, Rostov-on-Don, Russia,
Tatiana Sherchkova, Senior Researcher, Department of Genetics, Southern Federal University, Rostov-on-Don, Russia,
Elena Shimanskaya, Senior researcher, Department of Genetics, Southern Federal University, Rostov-on-Don, Russia,
Ekaterina Derevyanchuk, Senior Researcher, Department of Genetics, Southern Federal University, Rostov-on-Don, Russia,
Anzhela Aleksandrova, Researcher, Department of Biomedicine, Southern Federal University, Rostov-on-Don, Russia.
Address for Correspondence
Tatiana Shkurat,
Professor, Department of Genetics,
Southern Federal University,
Rostov-on-Don, Russia,
194/1, Stachki avenue, Rostov-on-Don,
344090, Russian Federation.
E-mail:
tshkurat@yandex.ru
Citation
Shkurat T, Savikina K, Lomteva S, Sagamonova K, Shestel A, Prokofev V, Sherchkova T, Shimanskaya E, Derevyanchuk E, Aleksandrova A. The Intensity of Free Radical Processes and the Testosterone and Estradiol Levels in Seminal Fluid of Men with Different Types of Pathospermia - Personalized Approach. Online J Health Allied Scs.
2016;15(1):5. Available at URL:
http://www.ojhas.org/issue57/2016-1-5.html
Open Access Archives
http://cogprints.org/view/subjects/OJHAS.html
http://openmed.nic.in/view/subjects/ojhas.html
Submitted: Mar 28,
2016; Accepted: Apr 29, 2016; Published: May 30, 2016 |
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Introduction:
Recent years numerous studies were devoted to study oxidative stress, active forms oxygen
species (ROS) and antioxidants influence on male reproductive system.(1-3) Moderate amounts of ROS are necessary for the
physiological regulation of sperm function, hyperactivity and acrosomal reaction.(4,5) Under physiological conditions,
the sperm produces a small amount of ROS, which are necessary for fertilization, capacitation and acrosomal reaction.
Increased intensity of free radical processes are considered by some authors as a leading exogenous cause of spermatogenesis
processes violations.(6)
Molecular oxygen is the natural substrate and metabolite for aerobic life forms, being the
main source of energy, and therefore has no mutagenic activity in concentration not exceeding 20%, unlike its intermediators
- active oxygen species.(7-10) The increased concentration of oxygen and ROS are capable to induce mutations in the
spermatogonia and spermatozoa.(11,12)
It was shown that oxidative stress can reduce sperm motility, as well as it can prevent
the sperm penetration into the oocyte.(13) Oxidative stress, caused by an increase of ROS or reduction of antioxidants level
in the seminal plasma, plays an important role in the occurrence of the sperm parameters violations.(14) It is also known that
more than 25% of infertile men have an elevated level of ROS in seminal fluid.(15)
Standard methods of male fertility diagnosing, such as the study of the ejaculate parameters,
which includes macroscopic, microscopic, biochemical and functional indicators, not always let to judge about such causes of
male infertility, as increased production of ROS by spermatozoa.
The aim of this work is to conduct a comparative analysis of the oxidative processes intensity
and the hormones level produced in the seminal fluid in normal and in pathospermia.
Materials and Methods:
We studied 88 somatically healthy men aged from 20 to 50 years men who applied to the Center
for Human reproduction and IVF in 2014 with the problem of infertility in marriage and pathospermia in anamnesis. Of this
number patients were excluded with azoospermia, genetic diseases, inflammatory processes of various etiologies, as well as
patients from couples with unproven female infertility. The control group consisted of 22 healthy men with normozoospermia
and with proven fertility.
The patients were divided into five groups: 1) patients with normozoospermia (control group);
2) with asthenozoospermia (n = 28) oligozoospermia (n = 26), teratozoospermia (n = 20), oligoasthenozoospermia (n = 14).
Ejaculate analysis was performed according to WHO recommendations.(16) Spermoplasma were obtained by centrifugation of semen
at 2800 rpm for 10 min. The intensity of free radical processes and the testosterone and estradiol levels were determined in
spermoplasma.
For assessment of sperm plasma oxidative status, we used a method of induced chemiluminescence
(CL) in the H2O2-luminol system (5-amino-2,3-dihydro-1,4-phthalazinedione). It refers to chemical
activators of the CL and reacts with free radicals to form excited molecules. Luminol, without adverse cytotoxic effect and
photo effect, provides the high quantum yield during the oxidation process and forms a wide range of reactive oxygen species.(17)
Investigations were carried out on an automated chemiluminescence analyzer Berthold Autolumat Plus LB 953
(Berthold Technologies, GmbH).
The testosterone and estradiol levels were determine by enzyme immunoassay on automatic ELISA
analyzer «ALISEI QS» (Italy) in the Center for collective use «High Technology» (Southern Federal University).
Statistical analysis: Statistical analysis was performed using the software package
Excel and Statistica v.6.0. Comparison of different data was performed using Student's T test and Pearson (c2).
Differences were considered significant at 95% significance level (p < 0.05). To determine the phenomena
relationship Spearman correlation analysis was used by the correlation coefficient (r).calculation.
Results and Discussion:
According to the semen profile analysis and based on WHO guidelines,(16) we have formed the
following groups:
1) Normozoospermia (control) – the semen profile corresponds to normative values;
2) Oligoasthenozoospermia – sperm concentration was less than 15 million/ml, motility A+B was less than 40%;
3) Asthenozoospermia – sperm concentration was normal, sperm motility A+B was less than 40%, morphology on the strict Krueger's criteria was more than 4%;
4) Oligozoospermia - sperm concentration was less than 15 million/ml, motility A+B was more than 40%;
5) Teratozoospermia - sperm concentration was normal, sperm motility A+B was more than 40%, morphology by Kruger criteria was less than 4%.
Table 1 shows the results of the average concentration, motility and morphology of spermatozoa
in the studied groups. It was obtained that the groups significantly differed in the parameters of mobility and sperm count.
The increase in the number of immature germ cells in the group with oligozoospermia suggests that the maturation of the germ
cells in this group had unfinished character.
Table 1: Indicators of concentration, mobility and morphology of sperm in the studied groups |
Semen parameters Groups
|
Concentration, million/ml
|
Mobility (%) |
Morphology by Kruger (N) |
White blood cells |
The cells of spermatogenesis |
A |
B |
C |
D |
Normozoospermia (control) |
39.5 ± 6.8 |
35.1 ± 5.8 |
16 .1 ± 3 .1 |
7.6 ± 2.5 |
40.8 ± 8.7 |
6.0 ± 1.1 |
0.6 ± 0.3 |
0.6 ± 0.2 |
Oligoasthenozoospermia |
7.0 ± 4.1*** |
13,3 ± 3.9** |
13.2 ± 5.3 |
11.1 ± 4.6 |
62.3 ± 9.9* |
5.2 ± 1.2 |
0.6 ± 0.2 |
0.3 ± 0.1 |
Asthenozoospermia |
49.1 ± 8.8 |
15.1 ± 5.1** |
17.4 ± 7.4 |
10.0 ± 2.3 |
57.3 ± 7.8* |
4.8 ± 0.8 |
0.7 ± 0.3 |
0.7 ± 0.3 |
Oligozoospermia |
10.5 ± 2.1** |
29.8 ± 3.4 |
15.2 ± 4.5 |
9.0 ± 3.5 |
46.1 ± 5.5 |
4.2 ± 0.2 |
0.5 ± 0.2 |
1.2 ± 0.6* |
Teratozoospermia |
31.7 ± 7.5 |
31.6 ± 3.4 |
17.2 ± 4.4 |
8.7 ± 4.1 |
41.3 ± 7.1 |
2.6 ± 0.7** |
0.6 ± 0.1 |
0.7 ± 0.3 |
The study of the free radical processes intensity in the sperm of infertile men with
different pathospermia. Oxidative stress accompanies and/or is one of the key pathogenetic mechanisms in the development
of many types of reproductive pathology. It is known that free radical production and lipid peroxidation play an important
role in the regulation of physiological functions of the testis. Recent years numerous studies have shown that oxidative
stress of various etiologies is the main cause of testicular dysfunction.(4,18-20) Oxidative stress has a direct impact
on the processes of proliferation and differentiation of spermatogenic cells, induces apoptosis, disrupts steroidogenesis
in Leydig cells, causes the death of endocrinocytes by apoptosis.(21-23)
Dynamics of metabolic changes in sperm plasma was assessed according to the quick flashes
height and the light sum. The amplitude (height) of the flash CL (h) induced by H2O2, characterizes the
resistance of tissues to lipid oxidation. Its size is directly proportional to the oxidation of tissue lipids and the
concentration of variable valence metals and inversely proportional to the natural antioxidants content in the studied
biosubstrate. The light sum of CL (Sm) reflects the rate of consumption of lipid origin free radicals, due to their
interactions with antioxidants, and is caused, primarily, by the level of prooxidants in the system, and the influence of
antioxidant components has secondary character.
The study results of luminol-dependent chemiluminescence of seminal fluid in formed groups
patients are presented in Table 2. As it can be seen from the results in all men with pathospermia, regardless of its
manifestations (oligoasthenozoospermia, asthenozoospermia, oligozoospermia, teratozoospermia) a significant increase in the
free-radical processes intensity was observed. Thus in the seminal plasma of men with oligozoospermia and
oligoasthenozoospermia quick flash height was almost two times higher compared to normozoospermia and equal 19.82±2.86 and
24.44±3.06 mm, respectively. Quick flash height was significantly increased by 46% in asthenozoospermia, and by 42% in
teratozoospermia compared with the control (Figure 1).
Table 2: The intensity of free radical processes in the infertile men sperm with various pathospermia forms |
Groups |
Indicators of ejaculate chemiluminescence |
The height of the light sum quick flash H₂O₂-induced luminol dependent CL |
The validity to the control (P) |
Luminescence light sum for 10 seconds. H₂O₂-induced luminol dependent CL |
The validity to the control (P) |
Normozoospermia (control) |
11.78 ± 0.92 |
- |
30.05 ± 2.36 |
- |
Oligoasthenozoospermia |
19.82 ± 2.86 |
<0.02 |
45.6 ± 2.66 |
<0.02 |
Asthenozoospermia |
17.25 ± 1.89 |
<0.02 |
42.37 ± 2.68 |
<0.02 |
Oligozoospermia |
24.44 ± 3.06 |
<0.02 |
57.01 ± 2.29 |
<0.02 |
Teratozoospermia |
16.72 ± 2.43 |
> 0.05 |
38.89 ± 2.82 |
> 0.05 |
 |
Figure 1: The intensity of chemiluminescence changes in the seminal plasma in
different pathospermia types (% to normospermic): I - oligoasthenozoospermia; II – asthenozoospermia; III – oligozoospermia;
IV – teratozoospermia |
As it can be seen from the obtained data, normospermic individual differences in the magnitude
of quick flash sum ranged between 7.4 to 18.8 (relative chemiluminescence units), while in asthenozoospermia this indicator
value reached 36.9 (relative chemiluminescence units) in individual patients, in teratozoospermia - to 41.3, in
oligoasthenozoospermia - to 52.1, in oligozoospermia - to 56.4 (relative chemiluminescence units). Significant individual
scale was observed in the evaluation of the induced chemiluminescence light sum for 10 seconds (Ѕм10sec) in different
pathospermia types. So if normospermic Sm10sec maximum values did not exceed the level of 59.4 (relative chemiluminescence
units), then in individual patients with pathospermia this level was significantly increased: in teratospermia to 87.5; in
oligoasthenozoospermia to 102.9; in oligozoospermia to 118.2; in asthenozoospermia to 380.1 (relative chemiluminescence
units). Mechanism of tissue samples H2O2-induced chemiluminescence based on the interaction of
H2O2 with prooxidant components, primarily with iron-containing components, which leads to the
formation of reactive oxygen species such as hydroxyl radical, singlet oxygen and superoxide anion-radical. This may cause
activation of free radical processes leading to the formation of lipid radicals, the recombination of which is accompanied
by flashing of a light quantum. The CL flash amplitude (height) (H) induced by H2O2, characterizes the
sperm plasma resistance to peroxidation. Its size is directly proportional to the lipids oxidation and the variable valence
metals concentration and inversely proportional to the natural antioxidants content in the studied biosubstrate. Light sum
(Sm) of chemiluminescence for 10 seconds reflects the consumption rate of lipid nature free radicals, due to their
interaction with antioxidants, and is attributable primarily to the pro-oxidants level in the system; the antioxidant
components effect has secondary character.
The intensity of free radical processes and antioxidant system in the first place depend on
the nature of metabolic processes in different tissues. It is of great importance not only the absolute values of the activity
of pro- and antioxidant systems but also their ratio to each other, as well as the buffering capacity of antioxidant
protection. Aggressive oxidants damage not only DNA/RNA in somatic cells of the body, but also the genetic material of the
gametes. Chronic oxidative stress leads to Leydig cells hypoplasia,(24) which are the main source of testosterone production.
Therefore, the next stage of our work was to study testosterone and estradiol levels in seminal fluid of men with different
pathospermia types. The study results of the testosterone (nm/l) and estradiol (PG/ml) levels in seminal fluid of infertile
men with different pathospermia types are presented in Table 3.
Table 3: Testosterone (nM/l) and estradiol (pg/ml) levels in the seminal fluid of infertile men with different types of pathospermia |
Groups |
The amount of testosterone and estradiol in the semen |
Testosterone (nM/l) |
The validity to the control (P) |
Estradiol (pg/ml) |
The validity to the control (P) |
Normozoospermia (control) |
13 .83 ± 2 .18 |
- |
51 .68 ± 4 .82 |
- |
Oligoasthenozoospermia |
15 .27 ± 3 .73 |
> 0.05 |
63 .13 ± 8 .20 |
<0.02 |
Asthenozoospermia |
20 .03 ± 1 93 |
<0.0 1 |
68 .40 ± 8 .41 |
<0.02 |
Oligozoospermia |
12 .90 ± 1 .95 |
> 0.05 |
53 .85 ± 4 .64 |
> 0.05 |
Teratozoospermia |
14 .82 ± 3 .26 |
> 0.05 |
50 .57 ± 5 .97 |
> 0.05 |
As it can be seen from the table a significant increase in the testosterone and estradiol
levels in the semen was observed in asthenozoospermia, estradiol levels also were tended to increase in oligoasthenozoospermia
associated with impaired sperm motility. The increase in the hormones level in seminal fluid in other pathospermia types was
not marked, but in some patients the individual level was significantly higher or below average normospermic values.
Estrogens are mostly considered as key hormones, which play a critical role in the female
body, but their role in the male body is no less significant, though remains poorly understood. Up to 80% of the estrogen in
the male body are synthesized as a result of aromatization of testosterone. Thus, the violation of the testosterone synthesis
and metabolism in men naturally lead to impaired estrogen synthesis and metabolism. Estrogen and testosterone usually work
together, and their function is sometimes reinforced by the effects of 5α-digidrotestosteron. Estrogens provide feedback of
the testicles with the pituitary gland and are able to decrease the testosterone level, but without estrogens testosterone
has only a limited influence on sexual behavior.(25)
To assess personalized conjugacy of free radical processes in the seminal fluid with the level
of sex hormones production the correlational analysis was conducted, the results of which are presented in Table 4.
Table 4: Correlation coefficients (r) between the studied parameters for different pathospermia types |
Compared options - Pathospermia types |
HSm / Sm |
HSm / Testosteron |
Sm / Testosterone |
HSm / Estradiol |
Sm / Estradiol |
Testosteron / Estradiol |
Normozoospermia (control) |
0.93 |
-0.02 |
-0.04 |
0.02 |
0.02 |
0.6 |
Oligoasthenozoospermia |
0.98 |
0.03 |
-0.06 |
0.26 |
0.2 |
0.57 |
Asthenozoospermia |
0.31 |
-0.3 |
-0.09 |
-0.14 |
0.24 |
0.35 |
Oligozoospermia |
0.96 |
0.7 |
0.62 |
0.5 |
0.4 |
0.8 |
Teratozoospermia |
0.91 |
-0.07 |
-0.2 |
-0.1 |
-0.1 |
0.48 |
For all studied pathospermia types except asthenozoospermia high correlation coefficient
(r >= 0.91) was obtained between indicators of induced chemiluminescence in semen, the total sum of illumination and quick
flash height (HSm / Sm), that shows a high antioxidante systems capacity in normospermia, oligoasthenozoospermia,
oligozoospermia and teratozoospermia. The lack of correlation parameters HSm / Sm in asthenozoospermia probably is due to
various reasons, resulting in violation of sperm motor activity. In some individuals it may be associated with increased
production of ROS, which is increased in a state of full rest, due to the uncoupling of the mitochondria respiratory chain,
(26) while in others decreased mobility may not be associated with violation of the ATP synthesis, and is caused by a
number of other reasons.
Conclusion:
Thus, the prooxidant and antioxidant processes conjugacy violation occurs in one of the
pathospermia types - asthenozoospermia, which is reflected in the correlation lack of all the studied parameters in the
seminal fluid.
Funding:
This research was supported by the internal grant of the Southern Federal University
№213.01-2015/003VG «The study of protein noncoding DNA elements in the structure of different genomes».
Conflict of interest:
None.
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