Document Type : Original Article
Authors
1 Department of Biology, Fars Science and Research Branch, Islamic Azad University, Fars, Iran;Department of Biology, Shiraz Branch, Islamic Azad University, Shiraz, Iran
2 Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;4Isfahan Fertility and Infertility Center, Isfahan, Iran
3 Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Abstract
Keywords
One of the main byproducts of sperm metabolism is
reactive oxygen species (ROS). Distinct roles have been
envisaged for ROS at physiological and pathological
levels. According to literature, a basal level of ROS are
needed for processes such as sperm capacitation, acrosome
reaction and sperm-oocyte fusion. But, uncontrolled or
excess production of ROS can have devastating effects on
sperm functions. Several studies have demonstrated that
induction of lipid peroxidation cascades and fragmentation
of DNA are two main pathological consequences of ROS
production in sperm (
Lipid peroxidation could lead to formation of
electrophilic lipid aldehydes such as malondialdehyde,
acrolein and 4-hydroxynonenal (4HNE). These aldehydes
further increase ROS level through binding to nucleophilic
centers of proteins, such as succinic acid dehydrogenase
in the mitochondrial electron transport chain (
This experimental study was performed from April 2016 to April 2018, and approved by the Ethics Committee of Royan Institute (IR.ACECR.ROYAN.REC.1396.270). Couples were informed of the study design and all the participants signed a written consent. Semen samples were obtained from 20 individuals referred to Isfahan Fertility and Infertility Center (IFIC) with male factor infertility.
Inclusion criteria: Couples with male factor infertility and at least one previous failed cycle after ICSI, without sign of varicocele and/reported genetic defect.
Exclusion criteria: Infertile couples with female factor infertility, individuals with leukospermia or varicocele, urinary infection, klinefelter syndrome, cancer and excessive alcohol or drug abuse.
Semen samples was collected from 20 infertile men
with previous failed cycles after ICSI by masturbation
after 3-7 days of abstinence. Part of the semen
sample was used for assessment of sperm parameters
(concentration, motility, morphology) with computerassisted sperm analysis (CASA) system (Video
Test, Version Sperm 2.1©, Russia) according to
World Health Organization (WHO) criteria (
The level of lipid peroxidation in sperm was evaluated
by BODIPY C11 loading BODIPY 581/591 C11 (D3861,
Molecular Probes) according to Aitken et al. (
Percentage of persistent histones in sperm samples was
assessed by aniline blue staining according to Nasr-Esfahani
et al. (
Percentage of protamine deficiency was assessed by
chromomycin A3 (CMA3) staining according to Razavi
et al. (
Sperm DNA fragmentation was assessed by two
procedures; sperm chromatin structure assay (SCSA)
and Terminal deoxynucleotidyl transferase dUTP nick
end labeling (TUNEL) according to Evenson (
For TUNEL assay: washed semen samples were fixed by 4% paraformaldehyde for 25 minutes and treated with 0.2% Triton X-100 for 5 minutes. Then, samples were washed with PBS and stained with a detection kit of DNA fragmentation (Apoptosis Detection System Fluorescein; Promega, Mannheim, Germany) according to the manufacturer’s instructions. For each sample, one positive control with an additional step [treatment of sperm with DNase I (1,000 U) after permeabilization with 0.2% Triton X-100] was considered for each sample. Finally, a minimum of 10,000 sperm were analyzed using BD Cell Quest Pro software, and the result was reported as TUNEL-positive spermatozoa for each sample.
For SCSA assay: sperm concentration was adjusted to
2×106 in 1ml of TNE [Tris HCl (Merck, Germany)/NaCl
(Merck, Germany)/EDTA (Merck, Germany)] buffer. For
test group, 400 μl acid-detergent solution was added to 200
μl of diluted sample in TNE buffere and after 30 seconds,
1200 μl of acridine orange staining solution was mixed
with this suspension, while for control group, only 1200
μl of acridine orange (Sigma, St. Louis, USA) staining
solution was added to 200 μl of diluted sample. Finally, a
minimum of 10,000 sperm for each sample were counted
using a FACSCalibur flowcytometer, and the percentage
of DNA damage was reported as SCF (
For statistical analysis, correlation coefficients were carried out with the Statistical Package for the Social Sciences software (SPSS 16, SPSS, Chicago, IL, USA). The mean, standard error, and range of variables were presented according to descriptive analysis. P<0.05 was considered significant.
Description of sperm parameters, and couples age were presented in Table 1. Mean of female and male age were 32.5 ± 6.4 and 37 ± 6.2, respectively. Mean of sperm concentration, percentage of sperm total motility, and abnormal morphology were 46.1 ± 5.4, 34.5 ± 5.09, and 98.1 ± 0.4 respectively.
Description of sperm parameters, semen volume, and couples age (n=20)
Parameters | Minimum | Maximum | Mean ± SE |
---|---|---|---|
Female age (Y) | 20 | 49 | 32.5 ± 6.1 |
Male age (Y) | 29 | 51 | 37 ± 6.1 |
Sperm concentration (106/ml) | 8 | 80 | 46.1 ± 5.4 |
Sperm total motility (%) | 5.2 | 72 | 34.5 ± 5.09 |
Sperm abnormal morphology (%) | 95 | 100 | 98.1 ± 0.4 |
Semen volume (ml) | 2 | 5.5 | 4.1 ± 0.2 |
The correlations analysis between sperm parameters
with sperm functional tests such as DNA fragmentation,
protamine deficiency, persistent histones, and lipid
peroxidation (
In addition, correlations between sperm functional tests were analyzed together and results are presented in Table 3. There were significant correlations between percentage of persistent histones with percentage of CMA3- positive spermatozoa (r=o.6, P=0.008) and intensity of sperm lipid peroxidation (r=0.6, P=0.01). We also observed significant positive correlations between percentage of DNA fragmentation assessed by SCSA (DFI) with DNA fragmentation assessed by TUNEL (r=0.83, P<0.001), percentage (r=0.77, P<0.001) and intensity of lipid peroxidation (r=0.62, P=0.009). In regard to TUNEL test, we observed a positive significant correlation between DNA fragmentation assessed by TUNEL with percentage of lipid peroxidation (r=0.84, P<0.001). In addition, there was a positive significant correlation between percentage of CMA3- positive spermatozoa with intensity of lipid peroxidation (r=0.5, P=0.03).
Relationship between sperm parameters with sperm functional tests such as DFI and TUNEL+ and deficient protamine spermatozoa, persistent histones, lipid peroxidation (n=20)
Parameters | Concentration (106/ml) | Total motility (%) | Abnormal morphology (%) |
---|---|---|---|
Persistent histones (%) | -0.56* | -0.50 | 0.54* |
DFI (%) | -0.11 | -0.45 | 0.37 |
TUNLE+ (%) | -0.22 | -0.37 | 0.23 |
CMA3+ (%) | -0.43 | -0.28 | 0.47 |
Lipid peroxidation (%) | -0.19 | -0.43 | 0.32 |
Lipid peroxidation (intensity) | -0.43 | -0.45 | 0.62* |
The asterisks at the end of the correlation indicate that the correlation is significant at P<0.05. DFI; DNA fragmentation index, TUNEL; Terminal deoxynucleotidyl transferase dUTP nick end labeling, and CMA3; Chromomycin A3.
Correlation between sperm lipid peroxidation, DNA fragmentation and chromatin status
Parameters | Blue-stained (%) | DFI (%) | TUNEL+ (%) | CMA3+ (%) | Lipid peroxidation (%) |
---|---|---|---|---|---|
Persistent histones (%) | 1 | 0.45 | 0.44 | 0.60** | 0.30 |
DFI (%) | 0.45 | 1 | 0.83** | 0.30 | 0.77** |
TUNLE+ (%) | 0.44 | 0.83** | 1 | 0.33 | 0.84** |
CMA3+ (%) | 0.60** | 0.30 | 0.33 | 1 | 0.06 |
Lipid peroxidation (%) | 0.30 | 0.77** | 0.84** | 0.6 | 1 |
Lipid peroxidation (Intensity) | 0.60** | 0.62** | 0.34 | 0.50* | 0.23 |
The asterisks at the end of the correlation indicate that the correlation is significant at * P<0.05 and **P<0.01. DFI; DNA fragmentation index, TUNEL; Terminal deoxynucleotidyl transferase dUTP nick end labeling, CMA3; Chromomycin A3, and LO; Lipid peroxidation.
Oxidative stress has been reported in 30-80% of
infertile men and has toxic effects on sperm functions.
Oxidative stress is mainly mediated through endogenous
generation of hydrogen peroxide. Medium or moderate
concentrations of hydrogen peroxide could result in sperm
immobilization due to depletion of ATP and reduction
of the phosphorylation in axonemal proteins while high
concentrations of hydrogen peroxide can induce apoptosis
in sperm (
In the normal condition, numerous antioxidants
present in seminal plasma and sperm, support male
gametes against oxidative stress. However, reduced
antioxidant capacity and excessive generation of ROS,
prone sperm to damage in infertility condition. One
of the main reasons of sperm susceptibility to damage
is the low volume of cytoplasm and high content of
unsaturated fatty acids (
Our results show a negative significant correlation
between percentage of sperm persistent histones with
sperm concentration while similar correlation was
not observed between percentage of CMA3 positive
spermatozoa with sperm concentration. According to
literature background, aniline blue dye discriminates
lysine-rich histones from arginine-and cysteine-rich
protamine, while CMA3 dye compete with the protamines
for binding to the minor groove of DNA in sperm (
In addition, we also observed significant positive
correlations between percentage of sperm abnormal
morphology with percentage of sperm persistent histones
and intensity of lipid peroxidation. These results suggest
that abnormal sperm contain high level of excessive
histones with more relaxed chromatin configuration
compared to sperm chromatin that was packed with
protamines, producing higher amount of hydrogen
peroxide which prone sperm to lipid peroxidation. Based
on previous study by professor Aitken group, lipid
peroxidation by product not only exposed DNA to damage
but also induces mitochondrial to produce higher amount
of H2O2
, the consequence of which DNA fragmentation
and apoptosis. Therefore, antioxidant therapy to minimize
the level of oxidative stress has been suggested for these
type of patients. In this regard, we recently demonstrated
that supplementation of One-Carbone Cycle, which
improves chromatin remodeling and allowse proper
exchange of histone to protamine to take place resulting
in the reduction of sperm lipid peroxidation and DNA
damage in varicocelized rat model (
According to the literature background, the final
consequence of the increased level of oxidative stress and
protamine deficiency is fragmentation of DNA in sperm.
Therefore, we assessed SDF by two methods; TUNEL,
and SCSA and observed there was a strong significant
correlation between these methods. In addition, there
were significant correlations between percentage of DNA
fragmentation assessed by two methods with percentage
and intensity of sperm lipid peroxidation. This result
shows that the intensity of lipid peroxidation in sperm
is in line with the fragmentation of DNA. Based on
previous proposed theory by professor Aitken group, the
lipid peroxidation is induced mainly by H2O2
derived
from mitochondrial and leucocytes (
The result of these studies clearly showed that there are strong significant correlations between oxidative stress, chromatin packaging and DNA fragmentation in sperm sample of infertile men with at least one failed cycle after ICSI. It seems a reduction of oxidative stress through clinical approach like varicocelectomy and therapeutic approaches like antioxidant therapy and subsequently improvement of sperm function can be expected to provide satisfactory results in the next assisted reproduction cycle.