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Evaluation of sperm DNA fragmentation using multiple methods: A comparison of their predictive power for male infertility

Authors:
  • IVF World
  • Vemsa Biotech Pvt Ltd

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Objective: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. Methods: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). Results: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001). Conclusion: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.
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ORIGINAL ARTICLE
https://doi.org/10.5653/cerm.2019.46.1.14
pISSN 2233-8233 · eISSN 2233-8241
Clin Exp Reprod Med 2019;46(1):14-21
Evaluation of sperm DNA fragmentation using
multiple methods: a comparison of their predictive
power for male infertility
Aamir Javed1, Muralidhar Srinivasaih Talkad2, Manjula Kannasandra Ramaiah1
1Department of Biotechnology, REVA University, Yelahanka; 2Department of Biotechnology, Dayananda Sagar University, Bangalore, India
Objective: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples.
However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of
male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict
male infertility and reactive oxygen species by Oxisperm kit assay.
Methods: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups
(50 patients in each group).
Results: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP
nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral
comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using
the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet
test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r=0.70 and r=0.68,
respectively; p< 0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r=0.77,
p< 0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r=0.59,
r= 0.57, and r=0.72, respectively; p<0.001).
Conclusion: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients,
and the alkaline comet test was the best predictor of male infertility.
Keywords: DNA fragmentation; Infertility; Sensitivity and specificity
Introduction
Sperm DNA fragmentation (SDF) has a major impact on fertility [1].
In the last decade, SDF has become a biomarker of male infertility,
since it was discovered that spermatozoa with poor-quality or frag-
mented genetic material may hinder embryonic growth and devel-
opment, increasing the risk of miscarriage in early pregnancy, issues
involving fetal development [2,3]. High levels of SDF have been asso-
ciated with repeated failure of assisted reproductive technology [4].
Various methods to gauge SDF have been assessed for clinical use in
studies that sought to identify threshold values for conception and
to investigate their significance, sensitivity, and specificity. The main
purpose of our case-control study was to compare the five most
widely used techniques of measuring DNA fragmentation index
(DFI), to identify correlations among them, and to determine their
Received: Oct 26, 2018 ∙ Revised: Jan 25, 2019 ∙ Accepted: Jan 29, 2019
Corresponding author: Manjula Kannasandra Ramaiah
Department of Biotechnology, REVA University, Rukmini Knowledge Park
Yelahanka, Kattigenahalli, Bengaluru, Karnataka 560064, India
Tel: +80-95716777 Fax: +80-66226622 E-mail: drkrmanjula@gmail.com
* This article has been corrected. A Corrigendum has been published: Clin Exp
Reprod Med 2019;46:211; https://doi.org/10.5653/cerm.2019.46.1.14.e1.
This is an Open Access article distributed under the terms of the Creative Commons Attribution
Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the
original work is properly cited.
www.eCERM.org
A Javed et al. Examination of DNA fragmentation and evaluation of index
15
See Corrigendum in Volume 46 on page 211.
sensitivity, specificity, and cut-off values for predicting male infertility
[5]. More specifically, the terminal deoxynucleotidyl transferase dUTP
nick end labelling (TUNEL) assay, the sperm chromatin dispersion
(SCD) test, the sperm chromatin structure assay (SCSA), and the com-
et assay were compared. Of these methods, the SCD test and SCSA
characterize SDF on basis of denatured chromatin in spermatozoa [6].
The SCSA utilizes acridine orange staining to label double- and sin-
gle-stranded DNA with green and red fluorescence, respectively, af-
ter treatment with an acidic denaturing agent; higher levels of dena-
turation are associated with lower levels of sperm DNA integrity, and
the clinical utility of this technique has been firmly established [7].
The SCD assay measures the extent to which chromatin in spermato-
zoa is dispersed based on the appearance of a radiant halo, and it en-
ables non-divided (with a halo) spermatozoa to be distinguished
from divided (without a halo) spermatozoa [8]. Although several clin-
ical investigations have investigated the predictive power of these
methods, few studies have explored correlations among the results
obtained using TUNEL, SCD, and SCSA and the studies that have
done so have not investigated the sensitivity and specificity of each
test [9]. However, studies have shown a relationship between em-
bryo quality and SDF based on SCSA, while other studies have not
found relationships between SDF and embryo growth or the out-
comes of assisted reproductive technology [10]. However, the design
of such studies does not allow the role of SCSA to be accurately de-
termined, since their results could have resulted from female factor
infertility, manifesting in the form of factors such as differences be-
tween oocytes in the function of the DNA repair system that ensures
successful fertilization [10]. We have discovered that extensive single-
strand SDF may prevent conception [10].
1. Oxidative stress
Reactive oxygen species (ROS) contribute to SDF and male infertility.
Oxidative stress occurs when ROS generation surpasses the body’s
own normal antioxidant defenses, bringing about cellular damage.
Seminal discharge consists of various types of cells, such as mature
and immature spermatozoa, round cells at various phases of sper-
matogenesis, pus cells, leukocytes, and epithelial cells [11]. The three
noteworthy sources of ROS in semen are pus cells, leukocytes, and
spermatozoa themselves. However, it has been suggested that pus
cells and leukocytes contribute the most to oxidative stress based on a
comparison to the effects of spermatozoa. The rate of ROS production
was observed to be almost 800 to 1,000 times higher in pus cells and
leukocytes than in spermatozoa. Furthermore, ROS generation is high-
er in patients who smoke and drink. However some medical condi-
tions also result in ROS generation, such as varicocele, genital tract in-
fections, and spinal cord injury. Age and infertility also play an impor-
tant role in ROS generation [12]. It has been observed that ROS gener-
ation was higher in semen samples of infertile patients than in those
of fertile patients, and that SDF increased as a result of the increased
concentration of ROS in semen samples. Nonetheless, ROS play a vital
role in sperm physiological and biochemical processes, such as activa-
tion, capacitation, acrosome reaction and signaling for fertilization. As
described above, oxidative stress has been linked to poor sperm motil-
ity and sperm function, leading to poor embryo formation, miscar-
riage, and infertility. Therefore, in this study, we examined ROS forma-
tion using the Oxisperm kit in the fertile and infertile samples [13].
Methods
1. Test collection
Raw semen samples from 50 fertile and 50 infertile subjects were
collected from patients receiving care at assisted reproductive center
facilities in Base Fertility Medical Science Pvt Ltd. Infertility was de-
fined as the inability of a sexually active couple not using contracep-
tion to achieve pregnancy in 1 year. Samples were gathered using
the antegrade technique with a period of sexual abstinence of 2–6
days and were analyzed using the World Health Organization criteria
(2010). A small amount of semen from each subject was frozen in liq-
uid nitrogen for SCSA, and the other five tests were carried out using
the fresh sample on the same day. Informed lawful consent (ICMR
BASED-NMAS-77-97) was procured from all participants. Applicable
approval was received from the ethical committee of the Base Fertili-
ty Medical Science, Department of Infertility and Reproductive Medi-
cine (approval No. BFM/ivf-80RI-78). The datasets used and/or ana-
lyzed during the current study are available from the corresponding
author upon a reasonable request.
2. TUNEL assay
The TUNEL evaluation was carried out using an in situ cell death de-
tection kit. For 30 seconds, the air-dried smeared sample was fixed in
3.9% paraformaldehyde at 28°C and further washed with phosphate-
buffered saline (PBS) at a pH of 7.4 and then permeabilized with 2%
Triton X-100. Under sterile conditions, the nucleotide mixtures la-
belled with TdT were layered onto individual slides and incubated in
a humidified chamber at 37°C for 58 minutes in the absence of light.
Subsequently, the humidified slides were washed three times and
stained with 8 mg/mL diamidino-2-phenylindole (DAPI), and nega-
tive controls without the TdT-tagged enzyme were run in each dupli-
cate for each sample. A total of 300 sperm per entity were examined
using fluorescence microscopy by the same surveyor. The spermato-
zoa stained with DAPI (blue) were counted first, followed by the sper-
matozoa dyed green (TUNEL-positive), and then the percentage of
these cells in the total sample was calculated [14].
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3. SCSA
Our lab modified protocol used for SCSA, and accordingly, each se-
men sample was diluted to a concentration of 2×106 spermatozoa/
mL in TE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.5) in
a total volume of 250 µL. The sample was treated with an acid solu-
tion for 35 seconds and restaining was completed with acridine or-
ange (5 µg/mL) for 150 seconds. A total of 1,000 spermatozoa were
analyzed in each examination, and the results are presented as the
DFI (%) calculated utilizing FAC scan program (BD Biosciences, Frank-
lin Lakes, NJ, USA). The proportion of spermatozoa with DNA frag-
mentation was shown by red fluorescence, in comparison to green
fluorescence in the non-fragmented sample [15].
4. SCD
The SCD test was carried out using the Halo sperm kit-Parque Tec-
nológico de Madrid Spain according to the manufacturer’s protocol.
The samples were smeared using a standard kit; 300 spermatozoa
were examined and identified as fragmented or non-fragmented
based on whether they formed a halo. More specifically, in this tech-
nique, whole spermatozoa (fresh or frozen) are immersed in an inert
agarose microgel on a pretreated slide. The initial treatment of sper-
matozoa with non-fragmented DNA with dilute acid denatures the
DNA, and the lysing buffer subsequently expels the vast majority of
the nuclear proteins. In the absence of colossal DNA breakage, nucle-
oids are produced with outsized coronas of spreading DNA coils, as-
cending from a focal center. Visualization can be performed under
bright field microscopy; however, if the staining is too concentrated,
the prestained slide can be gently washed with tap water [16].
5. Comet assays
Single- and double-stranded SDF can be measured using the alka-
line and neutral comet assays. The procedure was simultaneously
performed in semen samples on two distinct slides. Frozen semen
samples were liquefied and washed with HEPES/MOPS buffer, and
the sperm concentration was diluted to 10×106 spermatozoa/mL.
Then, 25-µL aliquots of spermatozoa were mixed with 50 µL of 1%
low-liquefying-point agarose in double-distilled water. Immediately,
10 µL of the combination was arranged on two pretreated slides of
agarose gel, encased with cover slips, and placed on a cold plate at
5°C for 4 minutes. Next, the cover slips were carefully isolated, and the
two slides were washed with an excess of lysing buffer for 30 minutes,
followed by a 10-minute wash in Tris-borate EDTA (TBE). In the neutral
comet assay, electrophoresis was performed with a TBE arrangement
of 20 V (1 V/cm) for 12 minutes and 30 seconds, with a subsequent
wash with 0.9% NaCl for 2 minutes. For the alkaline comet assay,
which measures the extent of denaturing, the slide was washed for 3
minutes at 5°C, and electrophoresis was then performed in 0.03 M
NaOH at 20 V (1 V/cm) for 4 minutes. Then, both slides were incubated
in a neutralizing solution for 5 minutes and with TBE for 2 minutes.
The slides were dried in a graded series of ethyl alcohol solutions
(75%, 85%, and 100%) for 2 minutes each. Finally, 500 spermatozoa
were assessed in terms of whether they showed divided or non-divid-
ed chromatin according to previously published criteria [17]
.
Table 1. Reactive oxygen species levels from the Oxisperm kit assay
for infertile and fertile patients
Color scheme-Oxisperm Kit Intensity (%)
Fertile (n =45) Infertile (n = 45)
L1 (Low) 39 16
L2 (Low–medium) 24 11
L3 (Medium) 11 31
L4 (High) 26 42
Figure 1. (A) Reactive oxygen species (ROS) intensity color scheme for semen samples using the Oxisperm kit assay. The figure was supplied by
halotech (Madrid, Spain). (B) ROS intensity plot for the infertile and fertile samples using the Oxisperm kit assay. L (level) 1, low; L2, low–medi-
um; L3, medium; L4, high.
A45
40
35
30
25
20
15
10
5
0
Oxisperm intensity (%)
Intensity parameter
L1 L2 L3 L4
ROS
Infertile (n = 45)
Fertile (n =45)
B
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A Javed et al. Examination of DNA fragmentation and evaluation of index
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6. Oxisperm kit
The human spermatozoon is highly vulnerable to oxidative stress.
Peroxidative injuries directly influence the lipid component of the
membrane and generate breaks in the strands of both nuclear and
mitochondrial DNA. Nitroblue tetrazolium (NBT; 0.1%) was combined
with PBS by adding 100 mg of NBT powder to 1,000 mL of PBS (pH
7.2) and mixed at 28°C for 60 minutes. The NBT solution was sieved
with a 0.2-µm filter channel. NBT (0.1%) was added to 0.5 mL of di-
lute semen and incubated for 45 minutes at 37°C. The sterile tubes
were centrifuged at 800 rpm for 5 minutes, and smears were set up
from the pellet and air-dried. The slide was recolored with Wright’s
stain, and an aggregate of 100 spermatozoa was scored under ×100
intensification. Two experienced examiners (AJ, STM) scored the NBT-
recolored slides in a blinded manner, using the following four pre-
defined levels of intensity: L1, low; L2, low-medium; L3, medium; L4,
high. The color of the sample was compared with the reclassified col-
or scheme (Table 1, Figure 1).
7. Statistical analysis
The statistical analysis was conducted utilizing the IBM SPSS ver.
20.0 (IBM SPSS Corp., Armonk, NY, USA). The Mann-Whitney U-test
was used to analyze the five different SDF measurement techniques.
The Spearman test was used to evaluate the correlations between
the techniques, and receiver operating characteristic (ROC) curve
analysis was performed to determine the sensitivity, specificity, and
cut-off values for each test.
Results
1. SDF and male infertility
Statistically significant differences were observed between the fer-
tile and infertile patients using TUNEL, SCD, SCSA, and the alkaline
comet test (p< 0.001), but not when the neutral comet test was used
(p= 0.865).
2. Oxidative stress (ROS) and male infertility
The presence of ROS was analyzed in 45 patients each from the fer-
tile and infertile groups. The color scheme pattern for the Oxisperm
kit assay represents the level of ROS in the semen sample. The distri-
bution of observed intensities in the fertile group was as follows: L1
(low), 39%; L2 (low–medium); 24%; L3 (medium), 11%; and L4 (high),
36%. In the infertile group, the distribution was as follows: L1 (low),
16%; L2 (low-medium), 11%; L3 (medium), 31%; and L4 (high), 42%.
Thus, ROS levels were higher in the samples from infertile men than
in those from fertile men (Table 1, Figure 1A).
3. Correlations between procedures
Spearman correlation analysis was used to assess the correlations
between all methods. Strong and significant correlations were ob-
served between the SCD test and SCSA (r= 0.70, p<0.001), between
the SCD test and the TUNEL assay (r=0.68, p<0.001), and between
SCSA and the TUNEL assay (r=0.77, p<0.001). Reasonably strong
and significant connections were detected between the alkaline
comet assay and the SCD test (r=0.59, p< 0.001), between the alka-
line comet test and SCSA (r=0.57, p< 0.001), and between the alka-
line comet test and the TUNEL assay (r=0.72, p< 0.001). No mean-
ingful correlation was found between the neutral comet assay and
the other four techniques.
Figure 2. Sensitivity and specificity under the receiver operating
characteristic curve for the six independent observations (95% confi-
dence interval). SCD, sperm chromatin dispersion; SCSA, sperm chro-
matin structure assay; TUNEL, terminal deoxynucleotidyl transferase
dUTP nick end labelling.
Alkaline comet
Neutral comet
0.918
0.84
0.319 0.996
0.322 0.991
0.92
0.734
0.872
0.594
0.942
0.754
Oxisperm
SCD
SCSA
TUNEL
0 0.2 0.4 0.6 0.8 1.0 1.2
Specificity
Sensitivity
Figure 3. Receiver operating characteristic curve for the sperm DNA
fragmentation assays. TUNEL, terminal deoxynucleotidyl transferase
dUTP nick end labelling; SCD, sperm chromatin dispersion; SCSA,
sperm chromatin structure assay.
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Sensitivity
0 0.2 0.4 0.6 0.8 1.0
1-Specificity
Neutral comet
TUNEL
SCD
Oxisperm
SCSA
Alkaline comet
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4. Sensitivity, specificity, cut-off values, and ROC analysis
The ROC curve analysis of the five different assays assessed their
sensitivity, specificity, and cut-off values for predicting male infertility.
The largest area under the curve 0.977 was observed for the alkaline
comet assay, with an SDF value of 48.74% yielding a sensitivity and
specificity of 0.840 and 0.918, respectively (Figure 2). Next, the TUNEL
assay resulted in an area under the curve of 0.901, with an SDF cut-off
value of 22.08% yielding a sensitivity and specificity of 0.754 and
0.942, respectively. For the SCD test, the area under the curve of was
0.871 with an SDF cut-off value of 24.74%, yielding a sensitivity and
specificity of 0.734 and 0.920, respectively. However, for SCSA, less
predictive power was found, with an area under the curve of 0.790 for
an SDF cut-off value of 19.90%, with a sensitivity of 0.594 and a speci-
ficity of 0.872, respectively (Figure 3). Moreover, the neutral comet as-
say and Oxisperm assay showed no correlations with male infertility,
with areas under the curve of 0.511 and 0.504, SDF cut-off values of
36.37% and 35.38%, and sensitivity and specificity values of 0.996,
0.991, 0.319, and 0.322, respectively (Table 2).
Discussion
Although the utilization of various approaches to survey sperm
DNA damage has been broadly examined, few reports have studied
the clinical utility and relationships between the most widely recog-
nized techniques in a comprehensive manner [5]. Therefore, we per-
formed this comparative investigation to evaluate correlations
among the most utilized procedures and to establish their clinical
cut-off values.
Significant differences in SDF were found between fertile and infer-
tile patients using the TUNEL test, SCSA, the SCD test, and the alka-
line comet test, as previous studies have reported [18]. However, no
significant distinctions were found between fertile and infertile pa-
tients using the neutral comet test. In previous studies, a bimodal
distribution was found in fertile donors, showing that fertile men are
a heterogeneous group in this regard [19]. The neutral comet assay
also demonstrated a normal distribution among the infertile sam-
ples, yielding generally high estimations of double-stranded SDF. A
difference was found in SDF in fertile patients depending on whether
the alkaline comet test or the SCD test, SCSA, or the TUNEL assay was
used, which may have been a direct result of the electrophoresis
step, since this step could amplify the sensitivity of the identification
of DNA, breaks [20].
In infertile patients, the estimations of SDF made using the alkaline
comet test were statistically significantly higher than those obtained
with the SCD test, SCSA, and TUNEL techniques, demonstrating that
the comet assay appeared to have higher sensitivity for identifying
sperm DNA breaks, identifying that up to 100% of spermatozoa had
DNA fragmentation in some infertile patients. SCSA yielded statisti-
cally significantly lower SDF values than the SCD and TUNEL assays,
which did not have significant differences between their values.
These findings imply that different techniques may recognize differ-
ent aspects of SDF, as SCD and SCSA focus on chromatin fragmenta-
tion, while the comet test and the TUNEL assay directly identify DNA
breaks [21].
The closest correlation was found between the cytometric mea-
sures (TUNEL and SCSA), as previously reported [22]. This finding is
intriguing given that the two tests are believed to gauge distinct as-
pects of SDF. Additionally, it is important to standardize the TUNEL
technique, as minor technical variations in this method lead to varia-
tion in SDF. Nevertheless, despite the contrast between the TUNEL
assay and SCSA and the requirement for standardization of the for-
mer, both assays yielded fundamentally similar results for SDF. In ad-
dition, the Oxisperm kit showed higher levels of ROS in infertile pa-
tients than in fertile patients, which is directly related to sperm DNA
damage [22]. Additionally, the alkaline comet assay demonstrated a
moderate correlation with the SCD test, the TUNEL test, and SCSA, as
previously identified in various studies [22]. However, this correlation
was not as robust as the relationships found among the last three
techniques, which may have been a direct result of the higher sensi-
tivity of the alkaline comet test in comparison to alternate methods.
Interestingly, the neutral comet test did not demonstrate any correla-
tions with the other four techniques used to evaluate SDF. As pro-
Table 2. Cut-off values with sensitivity and specificity for each assay
Technique Number Areaa) Cut-off value (%) Sensitivity Specificity
TUNEL 95 0.901 22.08 0.754 0.942
SCSA 100 0.790 19.90 0.594 0.872
SCD 100 0.871 24.74 0.734 0.920
Oxisperm 90 0.504 35.38 0.991 0.322
Neutral Comet 100 0.511 36.37 0.996 0.319
Alkaline Comet 100 0.977 48.47 0.840 0.918
TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; SCSA, sperm chromatin structure assay; SCD, sperm chromatin dispersion.
a)Area under the receiver operating characteristic curve.
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A Javed et al. Examination of DNA fragmentation and evaluation of index
19
See Corrigendum in Volume 46 on page 211.
posed previously, the results of the neutral comet assay are associat-
ed with the risk of unsuccessful implantation, as the double-stranded
DNA breaks might reflect a non-extensive type of DNA damage
found only in a few loci along the genome, in the matrix connection
districts between toroids; such breaks may result from intense or
fractionated elucidation to radiation, as previously shown in tumor
cells [23]. Although the TUNEL test and SCSA identify both single-
and double-stranded DNA damage, our findings indicate that both
the TUNEL assay and SCSA showed correlations with the alkaline
comet test, which principally distinguishes single-stranded SDF.
However, these assays did not show a correlation with the neutral
comet assay, which has been demonstrated to primarily survey for
double-stranded DNA breaks [24]. Additionally, the neutral and alka-
line comet tests demonstrated a moderate correlation in infertile pa-
tients, which could have been associated with the likelihood that
many single-stranded DNA breaks in the same vicinity could prompt
double-stranded DNA breaks.
An ROC curve analysis was performed to assess the predictive pow-
er of these tests for male infertility. The alkaline comet test showed
the highest area under the curve, followed by the TUNEL test, the
SCD test, SCSA, and the neutral comet test (Table 3, Figure 3). The al-
kaline comet examination showed an area under the curve of 0.977
with an SDF threshold of 48.74%, demonstrating high sensitivity and
specificity. This finding is not directly comparable to those of past in-
vestigations, in which the percentage of damaged DNA was evaluat-
ed, not the percentage of fragmented sperm cells [25]. The TUNEL
test demonstrated a cut-off SDF value for male infertility of 22.08%,
with high values for the area under the curve and specificity (0.901
and 0.942, respectively); however, it was markedly less sensitive than
the alkaline comet test (0.754). These outcomes are practically identi-
cal to those obtained by Sharma et al. [26], who reported a cut-off
estimation of 19.25%, with an area under the curve, sensitivity, and
specificity of 0.890, 0.649, and 1.000, respectively. Our results show
an estimated limit of 19.9% for SDF, which is on the low end of the
distribution; however, this finding is consistent with those of other
studies that have reported values of approximately 20%. Additional-
ly, it is worth noting that SCSA is the most standardized procedure
across various research centers [26]. Furthermore, the neutral comet
assay demonstrated an exceptionally weak relationship with male
infertility, with fertile participants showing either low or high esti-
mated levels of double-stranded DNA fragmentation using this tech-
nique [27]. However, infertile patients dependably demonstrated a
high SDF. Hence, the cut-off SDF value of showed high sensitivity and
low specificity, due to the bimodal distribution in fertile participants,
as has been reported previously [28]. This finding is noteworthy be-
cause high values of SDF are related to miscarriage [29]. For further
evaluation, as various methods may gauge distinct aspects of chro-
matin integrity, a double investigation focusing on a single method
for measuring SDF would confirm these results.
This study presents clinical information from the five methods that
are most often used to assess SDF in the same set of patients. Based
on these results, it can be concluded that, aside from the neutral
comet test, the remaining four strategies are productive for distin-
guishing between fertile and infertile patients, with the alkaline
comet test being the best predictor of male infertility. In addition, the
Oxisperm kit assay showed that the semen samples from infertile
men had more ROS than those obtained from fertile men, which is
directly related to the greater SDF in the infertile samples.
Conflict of interest
No potential conflict of interest relevant to this article was reported.
Base Fertility Medical Science Pvt. Ltd. holds no conflict of interest.
Acknowledgments
The authors are extremely grateful to the REVA University and Base
Fertility Medical Science Pvt. Ltd., Bangalore, India.
ORCID
Aamir Javed https://orcid.org/0000-0001-9552-5958
Table 3. SDF values for infertile and fertile patients with each assay
Technique Fertile Infertile
No. of patents Mean ±SD (range) No. of patents Mean ±SD (range)
TUNEL 50 12.65 ±5.65 (6.2–31.4) 45 27.55 ±11.65 (7.8–75.3)
SCSA 50 11.41 ±5.11 (4.9–27.5) 50 22.1 ±12.18 (7.8–75.6)
SCD 50 16.04 ±6.12 (4.2–31.2) 50 32.1 ±14.84 (6.1–79.1)
Neutral comet 50 62.23 ±30.24 (14.2–99.1) 50 67.21 ±17.02 (28.3–100.0)
Alkaline comet 50 26.32 ±10.25 (9.5–68.0) 50 61.25 ±16.45 (17.5–98.6)
SDF, sperm DNA fragmentation; SD, standard deviation; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling; SCSA, sperm chromatin struc-
ture assay; SCD, sperm chromatin dispersion.
https://doi.org/10.5653/cerm.2019.46.1.14
Clin Exp Reprod Med 2019;46(1):14-21
20
See Corrigendum in Volume 46 on page 211.
Muralidhar Srinivasaih Talkad
https://orcid.org/0000-0002-3306-9413
Manjula Kannasandra Ramaiah
https://orcid.org/0000-0002-2011-2662
Author contributions
Conceptualization: AJ. Data curation: AJ, MST. Formal analysis: MKR,
MST. Methodology: AJ, MKR. Project administration: MST, MKR. Visu-
alization: AJ. Writing - original draft: AJ, MKR, MST. Writing - review &
editing: MST, MKR.
References
1. Akashi T, Watanabe A, Komiya A, Fuse H. Evaluation of the sperm
motility analyzer system (SMAS) for the assessment of sperm
quality in infertile men. Syst Biol Reprod Med 2010;56:473-7.
2. Practice Committee of the American Society for Reproductive
Medicine. The clinical utility of sperm DNA integrity testing: a
guideline. Fertil Steril 2013;99:673-7.
3. Setti AS, Paes de Almeida Ferreira Braga D, Iaconelli A Jr, Aoki T,
Borges E Jr. Twelve years of MSOME and IMSI: a review. Reprod
Biomed Online 2013;27:338-52.
4. Perdrix A, Rives N. Motile sperm organelle morphology exami-
nation (MSOME) and sperm head vacuoles: state of the art in
2013. Hum Reprod Update 2013;19:527-41.
5. Evenson DP, Jost LK, Marshall D, Zinaman MJ, Clegg E, Purvis K,
et al. Utility of the sperm chromatin structure assay as a diagnos-
tic and prognostic tool in the human fertility clinic. Hum Reprod
1999;14:1039-49.
6. Sun JG, Jurisicova A, Casper RF. Detection of deoxyribonucleic
acid fragmentation in human sperm: correlation with fertiliza-
tion in vitro. Biol Reprod 1997;56:602-7.
7. Singh NP, McCoy MT, Tice RR, Schneider EL. A simple technique
for quantitation of low levels of DNA damage in individual cells.
Exp Cell Res 1988;175:184-91.
8. Fernandez JL, Muriel L, Rivero MT, Goyanes V, Vazquez R, Alvarez
JG. The sperm chromatin dispersion test: a simple method for
the determination of sperm DNA fragmentation. J Androl 2003;
24:59-66.
9. Collins JA, Barnhart KT, Schlegel PN. Do sperm DNA integrity
tests predict pregnancy with in vitro fertilization? Fertil Steril
2008;89:823-31.
10. Wang YJ, Zhang RQ, Lin YJ, Zhang RG, Zhang WL. Relationship
between varicocele and sperm DNA damage and the effect of
varicocele repair: a meta-analysis. Reprod Biomed Online 2012;
25:307-14.
11. Baehner RL, Boxer LA, Davis J. The biochemical basis of nitroblue
tetrazolium reduction in normal human and chronic granulo-
matous disease polymorphonuclear leukocytes. Blood 1976;48:
309-13.
12. Choi HS, Kim JW, Cha YN, Kim C. A quantitative nitroblue tetra-
zolium assay for determining intracellular superoxide anion pro-
duction in phagocytic cells. J Immunoassay Immunochem 2006;
27:31-44.
13. Esfandiari N, Sharma RK, Saleh RA, Thomas AJ Jr, Agarwal A. Util-
ity of the nitroblue tetrazolium reduction test for assessment of
reactive oxygen species production by seminal leukocytes and
spermatozoa. J Androl 2003;24:862-70.
14. Dominguez-Fandos D, Camejo MI, Ballesca JL, Oliva R. Human
sperm DNA fragmentation: correlation of TUNEL results as as-
sessed by flow cytometry and optical microscopy. Cytometry A
2007;71:1011-8.
15. Evenson D, Gharagozloo P, Aitken RJ. Sperm chromatin structure
assay (SCSA®): the clinical utility of measuring sperm DNA dam-
age and its potential improvement with supplemental antioxi-
dants. JSM Invitro Fertil 2017;2:1008.
16. Wang M, Sun J, Wang L, Gao X, Lu X, Wu Z, et al. Assessment of
density gradient centrifugation (DGC) and sperm chromatin dis-
persion (SCD) measurements in couples with male factor infer-
tility undergoing ICSI. J Assist Reprod Genet 2014;31:1655-63.
17. Van Kooij RJ, de Boer P, De Vreeden-Elbertse JM, Ganga NA,
Singh N, Te Velde ER. The neutral comet assay detects double
strand DNA damage in selected and unselected human sperma-
tozoa of normospermic donors. Int J Androl 2004;27:140-6.
18. Spano M, Bonde JP, Hjollund HI, Kolstad HA, Cordelli E, Leter G.
Sperm chromatin damage impairs human fertility. The Danish
First Pregnancy Planner Study Team. Fertil Steril 2000;73:43-50.
19.
Brandriff B, Pedersen RA. Repair of the ultraviolet-irradiated male
genome in fertilized mouse eggs. Science 1981;211:1431-3
.
20. Evgeni E, Charalabopoulos K, Asimakopoulos B. Human sperm
DNA fragmentation and its correlation with conventional semen
parameters. J Reprod Infertil 2014;15:2-14.
21. Lewis SE, John Aitken R, Conner SJ, Iuliis GD, Evenson DP, Henkel
R, et al. The impact of sperm DNA damage in assisted concep-
tion and beyond: recent advances in diagnosis and treatment.
Reprod Biomed Online 2013;27:325-37.
22. Chohan KR, Griffin JT, Lafromboise M, De Jonge CJ, Carrell DT.
Comparison of chromatin assays for DNA fragmentation evalua-
tion in human sperm. J Androl 2006;27:53-9.
23. Speyer BE, Pizzey AR, Ranieri M, Joshi R, Delhanty JD, Serhal P.
Fall in implantation rates following ICSI with sperm with high
DNA fragmentation. Hum Reprod 2010;25:1609-18.
24. Brahem S, Mehdi M, Landolsi H, Mougou S, Elghezal H, Saad A.
www.eCERM.org
A Javed et al. Examination of DNA fragmentation and evaluation of index
21
See Corrigendum in Volume 46 on page 211.
Semen parameters and sperm DNA fragmentation as causes of
recurrent pregnancy loss. Urology 2011;78:792-6.
25. Robinson L, Gallos ID, Conner SJ, Rajkhowa M, Miller D, Lewis S,
et al. The effect of sperm DNA fragmentation on miscarriage
rates: a systematic review and meta-analysis. Hum Reprod 2012;
27:2908-17.
26. Sharma RK, Sabanegh E, Mahfouz R, Gupta S, Thiyagarajan A,
Agarwal A. TUNEL as a test for sperm DNA damage in the evalu-
ation of male infertility. Urology 2010;76:1380-6.
27. Osman A, Alsomait H, Seshadri S, El-Toukhy T, Khalaf Y. The effect
of sperm DNA fragmentation on live birth rate after IVF or ICSI: a
systematic review and meta-analysis. Reprod Biomed Online
2015;30:120-7.
28. Simon L, Zini A, Dyachenko A, Ciampi A, Carrell DT. A systematic
review and meta-analysis to determine the effect of sperm DNA
damage on in vitro fertilization and intracytoplasmic sperm in-
jection outcome. Asian J Androl 2017;19:80-90.
29. Agarwal A, Majzoub A, Esteves SC, Ko E, Ramasamy R, Zini A.
Clinical utility of sperm DNA fragmentation testing: practice rec-
ommendations based on clinical scenarios. Transl Androl Urol
2016;5:935-50.
... A módszer alkalmas egy-és kétszálú DNS-törések detektálására. A szakirodalomban nem lelhető fel egységes határérték spermium DF terén (25% és 50% között változik), amely annak köszönhető, hogy a mintakészítési protokoll számos olyan lépést tartalmaz, ami nagy tapasztalatot igényel -emiatt a klinikumban talán legkevésbé alkalmazott módszer -, emiatt rendkívül időigényes és nagy a laborok közötti szórás (38). n Sperm Chromatin Dispersion (SCD) assay: a DF vizsgálatára alkalmas technikák közül talán az egyik legkevesebb speciális berendezést igénylő technika, egyszerűen fénymikroszkópiával, brightfield optika segítségével végrehajtható. ...
... Egy-és kétszálú törések detektálására egyaránt alkalmas. A nemzetközileg megállapított határérték szerint 25%-os DF-indexnél már magas értékről beszélhetünk, azonban bizonyos SCD-kiteket forgalmazó márka ezt 30%-ban állapította meg, de ezektől teljesen eltérő határértékek is olvashatók a szakirodalomban (38). Nagy hátránya, hogy nem közvetlenül méri a DF-et, hanem közvetve, a denaturálásra való érzékenység alapján. ...
... Kiértékelése történhet fluoreszcens mikroszkópiával és áramlási citometriával is. A szakirodalomban fellelhető határérték 25% (38). Ez esetben is a módszer indirekt mivolta a legnagyobb hátrány, illetve leginkább az egyszálú DNS-törések kimutatására alkalmas. ...
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A férfi eredetű meddőség a meddőségi kezelésben résztvevő párok mintegy felét érintő probléma. A valóságban ez a szám sokkal nagyobb is lehet, mivel a legújabb kutatások egyre inkább alátámasztják, hogy az ismeretlen eredetű meddőség esetén (az esetek 15-30%-a), amikor a hagyományos andrológiai vizsgálatok során semmilyen problémát nem találunk, akkor sok esetben emelkedett, illetve magas spermium DNS fragmentációs érték detektálható. Azonban nem csak ebben az esetben figyelhető meg emelkedett DNS fragmentáció. Több kutatásban is a DNS fragmentációs index megemelkedését tapasztalták habituális vetélő hölgyek párjainál, többszöri sikertelen lombik beavatkozás esetén, leukocytospermia esetén, a varicocele-vel küzdőknél, magas spermium reaktív oxigén szabadgyök értéknél, stb. Kéziratunkban szeretnénk bemutatni a szakirodalomban felelhető indikációit a spermium DNS fragmentáció vizsgálatnak és a DNS fragmentáció vizsgálati módszereit. Male infertility is a problem that affects about half of couples in infertility treatment. In reality, this number may be much higher, as recent research confirms that infertility of idiopathic origin (15-30% of cases), when no problems are found in conventional andrological examinations, increased sperm DNA fragmentation value can be detected. However, it is not the only case when an increased DNA fragmentation can be observed. Several studies have found an increase in the DNA fragmentation index in couples with habituated abortions, multiple failed assisted reproduction cycles, leukocytospermia, varicocele, high sperm reactive oxygen radicals, and so on. In our manuscript we would like to present the indications in the literature for sperm DNA fragmentation testing and DNA fragmentation testing methods.
... Továbbá annak megállapítására, hogy a törések a DNS egyik vagy mindkét szálát érintik, a comet assay a legalkalmasabb módszer. A TUNEL assay és az SCSA assay leginkább az egyszálú DNS-törésekre érzékenyek, az SCD-teszt és a comet assay pedig az egy-és kétszálú törésekre [28]. Intézetünkben az SCD-technika alapján történik az SDF meghatározása. ...
... Ez utóbbi egyrészt azért jelent problémát, mert a módszer nem direkt méri az SDF-et, hanem a denaturációra való érzékenység alapján. Másrészről a denaturáció során alkalmazott oldatok közül több is potenciálisan toxikus, ami miatt fokozott odafigyelés és optimális esetben vegyi elszívófülke használata javasolt, bár hasonló "ellenérvek" a többi SDF-vizsgálati módszernél is elmondhatóak [28]. Utolsósorban pedig nagy lehet a vizsgálatokat végző technikusok közötti különbség. ...
... Az általunk használt SCD-módszer esetén a gyártó által közzétett határérték, amely felett magas SDF-indexről beszélhetünk, 30% [29]. Érdemes megemlíteni, hogy jelenleg nincs egységes konszenzus az egyes határértékek tekintetében, ami annak köszönhető, hogy nincs egységes protokoll a minta-előkészítésekhez, illetve több vizsgálati módszer is rendelkezésünkre áll, amelyek más-más elven mérnek (direkt vs. indirekt, egyszálú DNS-törések vs. kétszálú DNS-törések) [28]. Magas SDF esetén kétféle módon járhatunk el:  Andrológus kolléga irányításával terápiás javaslat kidolgozása, ha lehetséges. ...
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Számtalan olyan eset adódhat munkánk során, amikor egyszerűen nem sikerül megmagyaráznunk, hogy egy-egy meddőségi kezelésben résztvevő pár esetében miért nem sikerül a fogantatás, vagy ha meg is történik, akkor majdnem minden esetben vetéléssel végződik. A korábbiakban ilyenkor általában immunológiai és genetikai irányba tapogatóztunk, de a molekuláris biológiai módszerek fejlődésével új távlatok nyílnak előttünk. Ennek egyik alternatívájaként szolgálhatnak a különféle spermium DNS fragmentációs vizsgálati módszerek és berendezések, illetve a hozzájuk kapcsolódó spermium szeparálási eljárások. Az andrológiai oldalt kicsit mellőzve a spermium DNS fragmentáció potenciális jeleit a nőgyógyászati oldalról igyekeztük felvázolni, bemutatva az intézetünkben is alkalmazott indikációs protokollt, a vizsgálatainkhoz használt SCD assayt, valamint a magas fragmentációs indexnél használt spermium szeparálási technikákat. There are several cases during the infertility treatments of couples where we are unable to explain the recurrent implantation failure or if pregnancy occurs, it almost always results in a miscarriage. Earlier in such cases we usually looked for an immunological or a genetic discrepancy but with the development of molecular biological methods new perspectives have become an option. Alternatively, various sperm DNA fragmentation assays, equipments and sperm separation methods can be used. Slightly neglecting the andrological side we tried to outline the potential signs of sperm DNA fragmentation from the gynecological side presenting the indication protocol used in our institute: the SCD assay is used for our studies and sperm separation techniques are used at high fragmentation index.
... The reason of this controversy is related to the differences in both the methods for measuring DNA damage and the clinical outcomes used to compare these methods (26). Remarkably, while the techniques evaluating sperm DNA fragmentation in farm animals have not been compared, only four studies contrasted different DNA fragmentation methods in humans, focusing on the male infertility condition and IVF/ICSI outcomes (27)(28)(29)(30), and one analyzed the matter in mice (31). All these reports agreed in establishing correlations between TUNEL and alkaline Comet methods, and suggested that Comet would be one of the most sensitive methods to assess sperm DNA fragmentation (32). ...
... To the best of our knowledge, only five studies conducted in humans and mouse compared three or more methods of chromatin evaluation using the same sperm samples (27)(28)(29)(30)(31). In addition, this is the first study comparing eight methodological variants for sperm chromatin assessment in pigs. ...
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Although sperm chromatin damage, understood as damage to DNA or affectations in sperm protamination, has been proposed as a biomarker for sperm quality in both humans and livestock, the low incidence found in some animals raises concerns about its potential value. In this context, as separate methods measure different facets of chromatin damage, their comparison is of vital importance. This work aims at analyzing eight techniques assessing chromatin damage in pig sperm. With this purpose, cryopreserved sperm samples from 16 boars were evaluated through the following assays: TUNEL, TUNEL with decondensation, SCSA, alkaline and neutral sperm chromatin dispersion (SCD) tests, alkaline and neutral Comet assays, and chromomycin A3 test (CMA3). In all cases, the extent of chromatin damage and the percentage of sperm with fragmented DNA were determined. The degree of chromatin damage and the percentage of sperm with fragmented DNA were significantly correlated (p < 0.05) in direct methods (TUNEL, TUNEL with decondensation, and alkaline and neutral Comet) and CMA3, but not in the indirect ones (SCD and SCSA). Percentages of sperm with fragmented DNA determined by alkaline Comet were significantly (p < 0.05) correlated with TUNEL following decondensation and CMA3; those determined by neutral Comet were correlated with the percentage of High DNA Stainability (SCSA); those determined by SCSA were correlated with neutral and alkaline SCD; and those determined by neutral SCD were correlated with alkaline SCD. While, in pigs, percentages of sperm with fragmented DNA are directly related to the extent of chromatin damage when direct methods are used, this is not the case for indirect techniques. Thus, the results obtained herein differ from those reported for humans in which TUNEL, SCSA, alkaline SCD, and alkaline Comet were found to be correlated. These findings may shed some light on the interpretation of these tests and provide some clues for the standardization of chromatin damage methods.
... Indirect detection means reflecting the degree of fragmentation of sperm DNA by measuring its sensitivity to denaturation, with methods such as sperm chromatin diffusion test (17,18) and sperm chromatin structure analysis (SCSA) (19). These methods are limited in detecting fragmental types and are difficult to standardize (20). Therefore, current SDF detection methods are complex, expensive, and lack accuracy. ...
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Primordial germ cell (PGC) preservation could allow the conservation of fish diploid genome of genetically valuable individuals, such as endangered species or species with reproductive dysfunctions, in contrast to the haploid genome of their gametes. Cryopreservation is the best technological tool for fish PGC storage. Moreover, after freezing/thawing, these cells can be used for surrogate production or other biotechnological purposes. However, cryopreservation can induce DNA damage. Considering that PGCs are the precursors of gametes, DNA integrity must be carefully preserved in order to avoid undesirable effects on the progeny. In this sense, several methods of global evaluation of DNA integrity have been described in different species such as comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), or sperm chromatin structure assay (SCSA). However, with all these techniques some specific DNA lesions cannot be revealed. For this reason, it is recommended to use such methods for global DNA integrity evaluation in combination with other analysis of higher sensitivity. In this chapter, we describe a detailed protocol for the cryopreservation of zebrafish PGCs and the subsequent molecular evaluation of DNA after a freezing/thawing cycle. Moreover, we discuss the advantages of this novel technique comparing to the traditional ones. Finally, we reflect about our contribution to improve DNA damage analyses in fish germ cells.
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The initial step in the diagnostic investigation of male infertility has been traditionally based on the conventional seminal profile. However, there are significant limitations regarding its ability to determine the underlying mechanisms that cause the disorder. Sperm DNA fragmentation has emerged as a potential causative factor of reproductive failure and its assessment has been suggested as a useful adjunct to the laboratory methodology of male infertility evaluation, especially before the application of assisted reproduction technology (ART). A review of recent bibliography was carried out in PubMed by the use of relevant keywords, in order to evaluate the possible correlation between the conventional seminal parameters and sperm DNA fragmentation assessment as diagnostic tools in male infertility evaluation. A comprehensive diagnostic approach of male infertility should be based on a combination of diagnostic attributes, derived from the conventional semen analysis, as well as the investigation of genomic integrity testing. Due to its strong correlation with several aspects of ART procedures and further consequences for the offspring, sperm DNA fragmentation is a parameter worth integrating in routine clinical practice. However, additional large scale studies focusing on specific subgroups of infertile men who may benefit from an efficient therapeutic management based on the optimization of sperm DNA integrity are needed.
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Sperm DNA damage is a useful biomarker for male infertility diagnosis and prediction of assisted reproduction outcomes. It is associated with reduced fertilization rates, embryo quality and pregnancy rates, and higher rates of spontaneous miscarriage and childhood diseases. This review provides a synopsis of the most recent studies from each of the authors, all of whom have major track records in the field of sperm DNA damage in the clinical setting. It explores current laboratory tests and the accumulating body of knowledge concerning the relationship between sperm DNA damage and clinical outcomes. The paper proceeds to discuss the strengths, weaknesses and clinical applicability of current sperm DNA tests. Next, the biological significance of DNA damage in the male germ line is considered. Finally, as sperm DNA damage is often the result of oxidative stress in the male reproductive tract, the potential contribution of antioxidant therapy in the clinical management of this condition is discussed. DNA damage in human spermatozoa is an important attribute of semen quality. It should be part of the clinical work up and properly controlled trials addressing the effectiveness of antioxidant therapy should be undertaken as a matter of urgency. © 2013, Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.
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A promising method for observing spermatozoa, motile sperm organelle morphology examination (MSOME) enables the evaluation of the nuclear morphology of motile spermatozoa in real time at high magnification and has allowed the introduction of a modified microinjection procedure, intracytoplasmic morphologically selected sperm injection (IMSI). Since its development, several studies have intensively investigated the efficacy of MSOME and IMSI. The objective of the present study is to review the current literature on the MSOME and IMSI techniques. A promising method for observing spermatozoa, motile sperm organelle morphology examination (MSOME), enables the evaluation of the nuclear morphology of motile spermatozoa in real time at high magnification and has allowed the introduction of a modified microinjection procedure, intracytoplasmic morphologically selected sperm injection (IMSI). Since its development, several studies have intensively investigated the efficacy of MSOME and IMSI. The objective of the present study is to review the current literature on the MSOME and IMSI techniques.
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A systematic review and meta-analysis was conducted to evaluate the relationship between the extent of sperm DNA damage and live birth rate (LBR) per couple and the influence of the method of fertilization on treatment outcome. Searches were conducted on MEDLINE, EMBASE and Cochrane Library. Six studies were eligible for inclusion in the meta-analysis. Overall, LBR increased signficantly in couples with low sperm DNA fragmentation compared with those with high sperm DNA fragmentation (RR 1.17, 95% CI 1.07 to 1.28; P = 0.0005). After IVF and intracytoplasmic sperm injection (ICSI), men with low sperm DNA fragmentation had significantly higher LBR (RR 1.27, 95% CI 1.05 to 1.52; P = 0.01) and (RR 1.11, 95% CI 1.00 to 1.23, P = 0.04), respectively. A sensitivity analysis showed no statistically significant difference in LBR between low and high sperm DNA fragmentation when ICSI treatment was used (RR 1.08, 95% CI 0.39 to 2.96; P = 0.88). High sperm DNA fragmentation in couples undergoing assisted reproduction techniques is associated with lower LBR. Well-designed randomized studies are required to assess the role of ICSI over IVF in the treatment of men with high sperm DNA fragmentation.
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BACKGROUND Approximately 10 years after the first publication introducing the motile sperm organelle morphology examination (MSOME), many questions remained about sperm vacuoles: frequency, size, localization, mode of occurrence, biological significance and impact on male fertility potential. Many studies have tried to characterize sperm vacuoles, to determine the sperm abnormalities possibly associated with vacuoles, to test the diagnostic value of MSOME for male infertility or to question the benefits of intracytoplasmic morphologically selected sperm injection (IMSI).METHODS We searched PubMed for articles in the English language published in 2001-2012 regarding human sperm head vacuoles, MSOME and IMSI.RESULTSA bibliographic analysis revealed consensus for the following findings: (i) sperm vacuoles appeared frequently, often multiple and preferentially anterior; (ii) sperm vacuoles and sperm chromatin immaturity have been associated, particularly in the case of large vacuoles; (iii) teratozoospermia was a preferred indication of MSOME and IMSI.CONCLUSION The high-magnification system appears to be a powerful method to improve our understanding of human spermatozoa. However, its clinical use remains unclear in the fields of male infertility diagnosis and assisted reproduction techniques (ARTs).