Document Type : Original Article
Authors
1 ACECR Institute of Higher Education (Isfahan Branch), Isfahan, Iran;Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
2 Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
3 Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
4 4Department of Reproduction and Development, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
5 Department of Reproductive Biotechnology, Reproductive Biomedicine Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran;5Isfahan Fertility and Infertility Center, Isfahan, Iran
Abstract
Keywords
Lack of pregnancy following one year of unprotected
sexual intercourse is termed” infertility”, and its
frequency is around 15%, that 40% of which is related
to male infertility factors. Male infertility can be cured
by intracytoplasmic sperm injection (ICSI), which almost
bypasses all-natural selection barriers that sperm faces
during natural fertility (
Quality of oocyte and sperm are two critical parameters
determining ICSI outcomes. Quality of sperm is
commonly defined based on the assessment of routine
seminal indices, such as sperm concentration, motility,
and morphology which reflect the efficiency of the male
reproductive system (
One of the approaches for the separation of functional
spermatozoa according to cellular and molecular
characteristics is the selection of sperm cells based on
surface negative electric charge or “zeta potential”,
which is induced by sialic acid added to sperm surface
during maturation or the passage through the epididymis
(
Despite novel approaches for sperm selection/
preparation, routine sperm processing has a historical
background and lies in the way of assisted reproductive
techniques (ARTs), especially intrauterine insemination
(IUI). Previous studies indicate that several approaches
have been taken to process spermatozoa for insemination,
including swim-up, swim-down, DGC, albumin
gradient, glass wool filtration, and Sephadex beads (
One of the critical aspects of sperm selection/preparation
procedures and sperm process techniques such as DGC
is the genomic integrity of sperm cells. Spermatozoa
have very highly condensed nucleus protected against
any chemical and physical insults during
Telomeres are guanine-rich sequences that are more
prone to undergo DNA break than non-telomeric DNA
regions. They are considered important targets for free
oxygen radicals. In this line, several studies showed
significant negative correlations between sperm telomere
length and sperm parameters, such as DNA fragmentation,
protamine deficiency, and oxidative stress (
Considering the fundamental roles of the DGC method in andrology or ARTs, as well as Zeta potential for sperm preparation as a novel approach to select the most fecund sperm, we aimed, for the first time, to evaluate and compare the sperm telomere length as a parameter of sperm quality between DGC and Zeta potential methods used for sperm preparation.
In this experimental study, was approved by the
Research Ethics Committee of the Royan Institute (IR.
ACECR.ROYAN.REC.1397.89). Between September
2017 and December 2017, semen samples were obtained
from 15 infertile men with normozoospermia who
referred to the Andrology Unit of the Isfahan Fertility and
Infertility Center for semen analysis. Total sperm count,
sperm concentration, sperm motility, and morphology
of spermatozoa were equal to or above the lower
reference limit according to the criteria for the selection
of normozoospermia established by World Health
Organization (WHO) (
Semen samples were collected after 2-7 days of sexual
abstinence and standard semen analysis was performed
according to WHO (
Semen samples were washed with sperm washing media
(VitaSperm, Inoclon, Iran) supplemented with 10% human
serum albumin. Then, the DGC procedure was performed
with PureSperm (Nidacon International, Sweden). In this
method, 1.5 ml of 45% PureSperm was layered over 1.5
ml of 90% PureSperm, and then, 1.5 ml washed samples
were mounted on the 45% PureSpermand layer and
centrifuged for 15 minutes (300 g). Subsequently, sperm
pellet was regarded as processed spermatozoa and used
for the assessment of sperm telomere length and DNA
fragmentation (
The zeta potential method was carried out based on
a study conducted by Chan et al. (
Subsequently, the surface of the tube was washed thoroughly with VitaSperm plus 10% human serum albumin to detach adhering spermatozoa from the tube wall. Subsequently, the selected spermatozoa were centrifuged and used for further assessments.
For each sample, washed semen that obtained
spermatozoa after DGC and zeta potential methods were
used for assessment of DNA fragmentation according
to the terminal deoxynucleotidyl transferase dUTP
nick end labeling (TUNEL) assay (
The extraction of DNA sperm and peripheral blood
leukocytes were carried out by the QIAamp DNA Mini
Kit (Qiagen, Italy) according to the manufacturer’s
recommendations. Real-time PCR was performed
according to the study by Cawthon (
Statistical analyses were performed by the Statistical Program for Social Sciences (SPSS Inc., Version 11.0, Chicago, IL, USA). Data are expressed as the means and standard error of the mean (means ± SEM), except for the age reported as the standard deviation of the means (means ± SD). One-way ANOVA was used, followed by LSD t tests to analyze the differences of parameters before and after semen preparation. Pearson’s correlation coefficient was applied to calculate the association between different parameters. The P<0.05 was considered statistically significant.
For this study, the sample size was determined according to the sample size formula mentioned below:
In this formula, σ1=2.5; σ2=3.8; µ1=6.51; µ2=9.73, Z1- β=0.8, and ɑ=0.05. Accordingly, the minimum number of cases in each group was 15.
Table 1 shows the semen characteristics of 15 infertile
men with normozoospermia that participated in this
study. Sperm parameters, such as sperm concentration,
motility, morphology, and semen volume, were higher
than the defined threshold levels in accordance with
the criteria established by the WHO (
The comparison of the mean percentage of DNA fragmentation among washed samples, density gradient centrifugation (DGC), and zeta potential-processed samples. Common letter indicate significant differences between groups.
Fresh semen characteristics of men with normozoospermia (n=15)
Parameters | Mean ± SE | Min | Max |
---|---|---|---|
Male age (Y)* | 32 ± 5.02 | 25.00 | 45.00 |
Sperm concentration(106/ml) | 91.40 ± 4.1 | 70.00 | 125.00 |
Sperm count (106/ejaculate) | 339.34 ± 34.14 | 121.00 | 621.6 |
Sperm motility (%) | 63.66 ± 1.5 | 55.00 | 70.00 |
Abnormal sperm morphology (%) | 95.93 ± 0.43 | 92.00 | 97.00 |
Semen volume (ml) | 3.78 ± 0.38 | 1.1 | 7.4 |
*; Mean ± SD.
The results of absolute and relative sperm telomere
length among washed samples, DGC, and zeta potentialprocessed samples were compared (
The comparison of sperm telomere length (STL) between experimental groups. A. Comparison of absolute and B. Relative of STL among washed semen samples, density gradient centrifugation (DGC), and zeta-processed samples (n=15).
The correlative analysis between absolute sperm telomere length and sperm parameters revealed a significant correlation between this parameter with sperm abnormal morphology (r=-0.561, P=0.03). The results of the correlation analysis of absolute and relative telomere length, sperm parameters, and sperm DNA fragmentation with the male age are presented in Table 2. The results indicated significant correlations of the male age with sperm abnormal morphology (r=-0.75, P=0.001), absolute (r=+0.64, P=0.009) and relative telomere length (r=+0.64, P=0.01).
The correlation of male age with semen parameters, absolute, and relative sperm telomere length, as wel as sperm DNA fragmentation (n=15)
Parameters | r (P value) |
---|---|
Semen volume (ml) | 0.17 (0.54) |
Sperm concentration (×106/ml) | 0.31 (0.26) |
Total sperm count (×106) | 0.29 (0.28) |
Sperm motility (%) | -0.11 (0.69) |
Abnormal sperm morphology (%) | -0.75 (0.001) |
Sperm DNA fragmentation (%) | -0.008 (0.97) |
Absolute sperm telomere length | 0.64 (0.009) |
Relative sperm telomere length | 0.64 (0.01) |
Numerous studies in the field of andrology emphasize
on sperm telomere length as a sperm marker which has
the ability to distinguish fecund sperm from non-fecund
ones (
As mentioned above, sperm selection/preparation
procedures play a pivotal role in the management of
ARTs and have profound effects on ICSI outcomes (
Comparison of absolute and relative sperm telomere
length among the three groups demonstrated the lack
of a significant difference among experimental groups.
In contrary to our results, Yang et al. (
In accordance with the literature, in this study, we
detected a significant positive correlation between sperm
telomere length and male age, indicating spermatozoa
derived from old age men present higher telomeres
length. It is also important to note that numerous factors,
including oxidative stress, aging, psychological stress,
obesity, infection, smoking, lifestyle, diet, etc., can affect
telomere length (
The results of this study show that both DGC and zeta potential procedures can select sperm population with higher DNA integrity, but no difference was observed between the sperm selected samples in terms of telomeres length.