ANDROLOGY
Short Abstinence May Have Paradoxical Effects On Sperms With Different Level Of DNA Integrity:
A Prospective Study
Serajeddin Vahidi1, Nima Narimani2*, Taha Ghanizadeh1, Fatemeh Yazdinejad1, Maryam Emami2,
Kaveh Mehravaran2, Hossein Saffari2, Farhood khaleghiMehr2,Laleh Dehghan Marvast1
Purpose: To investigate the effect of short abstinence on sperm function tests and semen parameters.
Materials and methods: This prospective study included 65 male patients with increased DNA injury in their
ejaculated sperm and a history of recurrent pregnancy loss and/or assisted reproductive techniques failures. The
effects of antioxidants medical therapy and short abstinence on semen quality were assessed (TUNEL test and
CMA3 staining).
Results: Antioxidants have statistically significant effects on mean sperm concentration (untreated, 67.51 ± 44.40
million/ml, vs. treated, 56.09 ± 37.85 million/ml; P-value=0.005) and mean TUNEL score (untreated, 24.56% ±
9.49%, vs. treated, 20.64% ± 10.28%; P-value = 0.013).
Moreover, a short abstinence period might have positive effects as shown on the TUNEL assay (20.64% ± 10.28
vs. 17.38% ± 8.59 ; P-value = 0.028) and CMA3 staining (47.79% ± 20.78, vs. short 41.92% ± 18.49; P-value =
0.019), when considering all study subjects. However, different results were obtained using more precise analysis
based on a TUNEL cutoff score of 20%. The analysis showed that short abstinence might improve sperm DNA
integrity in patients with TUNEL score > 20% (mean TUNEL score from 27.85% ± 8.32% to 19.14% ± 8.90% ;
P-value =0.001%). However, it might have deleterious effects on sperm DNA integrity in patients with TUNEL
score < 20% (mean TUNEL score from 11.89% ± 3.21% to 15.17% ± 7.79%; P-value = 0.045%)
Conclusion: Our results showed that short abstinence may not be beneficial in all infertile males, and it should
only be used in selected patients with abnormal DNA integrity.
Keywords: CMA3; male infertility; short abstinence; sperm DNA integrity; TUNEL
1Department of Urology, Research & Clinical Center for Infertility, Shahid Sadoughi University of Medical Sciences, Yazd, Iran.
2Department of Urology, Hashemi Nejad Kidney Center (HKC), Iran University of Medical Sciences (IUMS), Tehran, Iran.
*Correspondence: Assistant Professor of Urology, Department of Urology, Hashemi Nejad Kidney Center (HKC), Iran University of
Medical Sciences (IUMS),Tehran, Iran.
Tel: (+98 21) 81161,Fax: (+98 21) 88644441, Email: Narimani.n@iums.ac.ir, nima_dr2001@yahoo.com.
Received October 2020 & Accepted July 2021
INTRODUCTION
Male fertility and sperm production ability have been traditionally assessed using conventional
semen analysis(1). The latest World Health Organisation
(WHO) guideline(2) recommends abstinence of 2 to 7
days prior to semen collection. On the other hand, some
reports recommend a single day of abstinence as opti-
mal for semen parameters(3).
In the era of assisted reproductive technology, special
consideration needs to be given to sperm DNA integ-
rity along with semen analysis. It has been estimated
that 15% of infertile men with normal semen analysis
(presumed to have idiopathic infertility) have increased
sperm DNA injury(4). A higher level of sperm DNA
fragmentation index (DFI) is associated with lower nat-
ural pregnancy rate and decreased assisted reproductive
technology (ART) outcome(5). In a state of oxidative
stress, reducing the epididymal transit effect may lead
to better semen quality. This goal may be achieved via
two means: short abstinence and testicular sperm re-
trieval(6).
The effect of short abstinence on sperm DNA have been
widely assessed by different groups, yet controversy
remains. Short abstinence is usually considered as ab-
stinence less than 24 hours (instead of recommended
abstinence of 2-7 days). Many reports(6,7) have shown
positive effects for short abstinence on sperm DNA
quality and ART outcomes, while others have report-
ed contrasting data(8,9). This discrepancy may be due to
selection bias, difference in sample size, or type of the
tests used.
In this context, the question to be addressed is wheth-
er the standard abstinence period(2) is only effective in
men with normal sperm DFI or if it is applicable in all
infertile males.
The present study assessed the effects of short absti-
nence ( ejaculation after 24 hours of abstinence) on se-
men and sperm DNA quality in a group of infertile men
with previous recurrent pregnancy loss and/or ART
failure and elevated sperm DFI.
MATERIALS AND METHODS
Following the approval by our institutional review
board (Ref Number: IR.SSU.RSI.REC.1398.004), 85
Urology Journal/Vol 18 No. 6/ November-December 2021/ pp. 682-687. [DOI: 10.22037/uj.v18i.6515]
Vol 18 No 6 November-December 2021 683
infertile men who provided written informed consent to
take part in the study were enrolled (referred to Yazd
center for infertility and research, Yazd, Iran, between
December 2018 and May 2019). The Yazd center of in-
fertility and research is a high-volume center with ap-
proximately 30 outpatient visits per day in the androl-
ogy clinic, most of which are referred from all over the
country due to recurrent pregnancy loss or ART failure.
The primary inclusion criteria were (i) couples with
recurrent early abortion (more than two abortions in
the first trimester) and (ii) recurrent failed ART (more
than three intrauterine inseminations (IUI) or more than
two in vitro fertilisation (IVF) sessions and more than
two intracytoplasmic sperm injection (ICSI) sessions).
Among them, men with disturbed DNA integrity, as as-
sessed using terminal deoxynucleotidyl transferase-me-
diated dUTP nick end labelling (TUNEL) test, and/or
increased level of chromomycin A3 (CMA3) staining
(TUNEL score > 20% and/or CMA3 score > 30) were
selected(10) and treated with antioxidant medical therapy
for 3 months (folic acid 1 mg/day, once daily seleni-
um plus-EuRho® Vital Selen Plus capsule, Euro OTC
Pharma GmbH, and once daily /250 mg L-carnitine ).
Because these tests have not been standardised yet, the
cutoff values were chosen based on previous studies
(10-14). At the end of the third month, the participants
provided us with two semen samples as described be-
low (in the semen collection section).
Exclusion criteria were (i) a history of smoking, (ii)
opium addiction, (iii) multiple sexual partners, (iv)
known hormonal abnormality, (v) clinically detected
varicoceles, or (vi) cryptorchidism. The same androlo-
gist performed a complete physical examination, eval-
uated the past medical history and recorded the results.
Finally, 21 patients were excluded, and 64 patients were
enrolled.
Semen collection
Each patient provided three semen samples: (i) the first
sample before antioxidant medical therapy (with absti-
nence of 2–7 days based on the WHO recommendation)
was considered as unntreated sample; (ii) the second
sample (treated sample ) after 3 months of antioxidant
medical therapy (with abstinence of 3 days) and (iii) the
third sample with 24 hrs of abstinence after the second
sample, was considered as the short abstinence (short
ejaculation) sample.
Semen analysis and sperm DNA integrity
Semen samples were obtained and analysed based on
WHO guidelines (2010)(2). Motility was classified as
(i) progressive, (ii) nonprogressive and (iii) immotile.
Sperm morphology was reported based on strict criteria.
Two different sperm DNA and chromatin tests were
performed on each semen sample: (i) a terminal deox-
ynucleotidyl transferase-mediated dUTP nick end la-
belling (TUNEL) test, which directly measures sperm
DFI, and (ii) chromomycin A3 staining (CMA3), which
evaluates sperm DNA protamination (or compaction).
TUNEL test
In brief, after fixation with paraformaldehyde, the sam-
ples were washed and treated with PBS and a mixture
of methanol and 3% H
2
O
2
.
In the next step, after being immersed in Triton X-100
and sodium citrate, the samples were washed with PBS
and then stained with a mixture of enzyme and fluo-
rescently labelled dUTP solution. Finally, they were
assessed using an In Situ Cell Death Detection Kit (Ro-
che Diagnostics GmbH, Mannheim, Germany) and flu-
orescence microscopy (BX51, Olympus, Tokyo, Japan)
(15). At least 200 sperms were counted and considered as
containing damaged DNA if they turned bright green.
Chromomycin A3
CMA3 is a guanine–cytosine binding fluorochrome
which competes with protamine for the same binding
locus. Therefore, CMA3 staining assesses chromatin
integrity (protamination). Briefly, air-dried samples
were fixed with Carnoy’s solution, stained with CMA3
for 20 min, rinsed and then mounted with glycerol buff-
er, and finally stored at a low temperature overnight.
The next morning, the samples were assessed using a
fluorescence microscope(16).
Sample size calculation
Considering the following formula, the estimated sam-
ple size for this before-after study, was 64 samples:
Where type I error rate (α), and power
(1-β)
were 0.05, 0.35 and 0.8, respectively(17)
Statistical methods
Continuous variables are reported as mean (standard de-
viation) and categorical data are presented as frequency
(percentage). In order to assess the difference between
two variables for the same subject, paired sample T test
was used for continuous variables. Also the association
between age, body mass index, smoking, alcohol con-
sumption, duration of infertility and the results of sperm
DFI were assesed using repeated measures ANOVA.
P-value < 0.05 was considered as statistically signif-
icant. Statistical analysis was performed using SPSS
version 25.0 (IBM, Chicago, Illinois, USA).
Variables Untreated Group (N= 64) Mean ± SD Treated Group (N=64) Mean ± SD P-value
Volume (ml) 3.40 ± 1.41 3.62 ± 1.44 0.161
Concentration (mil/ml) 67.51 ± 44.40 56.09 ± 37.85 0.005*
Progressive (%) 39.69 ± 12.50 38.76 ± 11.85 0.512
Nonprogressive (%) 10.55 ± 4.26 10.73 ± 3.34 0.787
Immotile (%) 50.02 ± 10.75 50.47 ± 11.59 0.746
Morphology (%) 3.54 ± 1.86 3.50 ± 1.66 0.837
TUNEL (%) 24.56 ± 9.49 20.64 ± 10.28 0.013*
CMA3 (%) 48.75 ± 15.96 47.79 ± 20.78 0.769
Table 1. Effect of antioxidants on semen parameters, sperm DNA integrity and chromatin compaction
*P-value < 0.05 considered as significant
Short abstinence and DNA integrity-Vahidi et al.
RESULTS
A total of 64 male patients were evaluated with a mean
age of 34.7 ± 4.66 yrs (range: 27–49 yrs).
The data indicate that antioxidants have statistically sig-
nificant effects on mean sperm concentration (untreat-
ed, 67.51 ± 44.40 million/ml, vs. treated, 56.09 ± 37.85
million/ml; P-value = 0.005) and the mean TUNEL
score (untreated, 24.56% ± 9.49%, vs. treated, 20.64%
± 10.28%; P-value = 0.013). However, there was no
significant relationship between antioxidant treatment
and other semen parameters, including CMA3 staining
(Table 1).
The effects of short abstinence on semen parameters
and sperm DNA integrity are demonstrated in Table 2.
Short abstinence had a statistically significant negative
effect on semen volume (recommended abstinence, 3.62
± 1.44 ml, vs. short abstinence, 2.92 ± 1.43 ml; P-value
< 0.001) but positive (decreasing) effects on TUNEL
score (recommended abstinence, 20.64% ± 10.28%,
vs. short abstinence, 17.38% ± 8.59%; P-value =0.028)
and CMA3 score (recommended abstinence, 47.79% ±
20.78%, vs. short abstinence, 41.92% ± 18.49%;
P-value = 0.019). Its effect on other semen parameters
was not statistically significant (P-value >0.05).
Antioxidant drugs may improve ( decreased TUNEL
test) or may have no effect on DNA integrity (TUNEL
test remains constant or even increase ). Therefore af-
ter three months of antioxidants medical therapy, the
samples may have TUNEL score below (responder) or
above (non-responder) normal cutoff ( 20%). In this
step, we tried to assess the effect of short abstinence on
sperm with normal TUNEL score (< 20%) and sperm
with abnormal DFI( TUNEL score > 20%)
For further robust evaluation of the effects of short
abstinence on sperms with normal or increased sperm
DNA integrity, table 2 were re-analysed based on a
TUNEL cutoff score of 20% as proposed by Sharma
and the colleagues.(10), and the results are presented in
Table 3.
At the TUNEL cutoff score of 20%, 28 samples had
low (TUNEL score < 20%) and 36 others had high
(TUNEL score > 20%) sperm DFI. Short abstinence
had a negative (increasing) effect on samples with low
sperm DFI (mean TUNEL score increased from 11.89%
± 3.21% to 15.17% ± 7.79%; P-value = 0.045) and a
positive (decreasing) effect on samples with abnormal
(high) sperm DFI (mean TUNEL score decreased from
27.85% ± 8.32% to 19.14% ± 8.90%; P-value < 0.001).
DISCUSSION
The present study showed that a short abstinence period
could improve sperm DNA integrity in patients with in-
creased sperm DNA damage. To the best of our knowl-
edge, the present study is the first to show that short
abstinence may have a paradoxical effect on sperm DFI
as evidenced by baseline DNA integrity status. In sub-
jects found to have low DNA damage using TUNEL
test (TUNEL score < 20%), short abstinence not only
failed to cause further improvement but also had cer-
tain deleterious effects. Therefore, short abstinence is
probably not applicable to all cases (as recommended
elsewhere)(3) and should be reserved for selected infer-
tile men with increased DFI.
In a functioning spermatogenesis system, epididymis
may act as a screening tool and may eliminate imma-
ture sperms due to its oxidative stress effects (induced
by epididymis epithelial cells, leukocytes, immature
sperms themselves, etc.). Therefore, epididymal pas-
sage may lead to subsequent improved semen quality
and seminal sperm DFI. In such systems, decreasing
epididymal passage time may result in increased sperm
DFI as explained in the current study (and in patients
with normal sperm DFI). On the other hand, in a mal-
functioning system ( e.g. due to incomplete protamina-
tion), epidiymal passage may lead to additional stress
on the vulnerable sperms and therefore may induced
sperm DNA injury apart from oxidative stress. In such
systems, short abstinence or testicular sperm extraction
may have great benefit in preserving sperm DNA in-
Table 2. Effect of short abstinence on semen parameters and sperm DNA integrity and chromatin compaction after previous medical
therapy in all the patients.
Variables Treated Group (N=64) Mean ± SD Short Abstinence (N=64) Mean ± SD P-value
Volume (ml) 3.62 ± 1.44 2.92 ± 1.43 < .001
Count (mil/ml) 56.09 ± 37.85 54.02 ± 37.51 0.699
Progressive (%) 38.76 ± 11.85 38.42 ± 11.32 0.887
Nonprogressive (%) 10.73 ± 3.34 11.32 ± 4.10 0.245
Immotile (%) 50.47 ± 11.59 50.08 ± 9.58 0.656
Morphology (%) 3.50 ± 1.66 3.38 ± 1.43 0.729
TUNEL (%) 20.64 ± 10.28 17.38 ± 8.59 0.028*
CMA3 (%) 47.79 ± 20.78 41.92 ± 18.49 0.019*
*P-value < 0.05 considered as significant.
Figure 1. TUNEL test, sperm with injured DNA depicted as bright
green.
Short abstinence and DNA integrity-Vahidi et al.
Andrology 684
Vol 18 No 6 November-December 2021 685
tegrity.
Nevertheless, in the current study, short abstinence had
statistically significant effects on semen volume. It also
decreased sperm concentration, percentage of immotile
and progressive sperms, sperms with normal morphol-
ogy and the percentage of non-progressive motility, al-
though with no statistical significance (P-value > 0.05).
In the case of a longer abstinence period, three impor-
tant events occur. Sperms are stored in the epididymis
(with presumable stress effects )(18) , the seminal level of
reactive oxygen species (ROS) may increase(8) and the
antioxidant capacity of axillary sexual glands secretions
(prostate and seminal vesicle) may decrease significant-
ly(19). These changes may indicate better semen quality
in an efficient spermatogenesis process (act as a screen-
ing tool) but may lead to increased sperm DNA frag-
mentation and subsequent infertility in a non-efficient
system which is pathologically vulnerable to environ-
mental stressors (e.g. due to abnormal sperm chromatin
compaction) or in the oxidative stress status (excessive
production of ROS or decreased levels of seminal an-
tioxidants). Multiple ejaculations with short abstinence
periods may increase semen antioxidant capacity and
decrease seminal ROS, especially those originating
from the epididymis(6).
There are a few inconsistent reports regarding the ef-
fects of a short abstinence period on semen quality; the
discrepancies may be due to differences in the number
of recruited participants, fertility status and inclusion/
exclusion criteria.
Agrawal and his colleagues, in their study on seven
healthy men with unproven fertility, showed that sperm
DFI increased concomitantly with an increase in the
abstinence period. They reported that mean sperm DFI
increased from 9.9% in cases of short abstinence period
(less than 2 days) to more than 17% in cases with long
abstinence period (9–11 days). Their conclusion should
be cautiously interpreted as their cohort included only a
small number of normospermic men(6).
On the other hand, a study conducted by De Jong and
colleagues on 11 healthy volunteers and another study
by Mayorga-Torres and colleagues on six healthy vol-
unteers, using SCSA techniques, found that a shorter
abstinence period might not affect sperm DFI(9,20). Fur-
thermore, De Jong et al. reported that a shorter absti-
nence period might negatively affect sperm chromatin
compaction, making them susceptible to environmental
stressors (not observed in the present study). This dif-
ference may be due to differences in sample size, type
of sperm DFI test used, and fertility status of the re-
cruited men. Even if the small sample size in the above
study is not considered, results obtained from healthy
participants cannot be attributed to subfertile and infer-
tile men.
Our findings are in agreement with those of Pons and
co-workers and Sanchez and colleagues, who used the
sperm chromatin dispersion test and reported positive
effects of short abstinence on semen quality in 36 and
40 infertile men, respectively(21,22).
The result of previously published studies (in Pubmed)
on the effect of a short abstinence period on sperm DFI
are summaraized in Table 4.
Table 3. Effect of short abstinence on sperm DNA integrity based on baseline low or high level of sperm function tests (TUNEL test).
Variables Treated Group (N=64) Mean ± SD Short Abstinence Mean ± SD P-value
TUNEL < 20% (n = 28 samples) 11.89 ± 3.21 15.17 ± 7.79 0.045
TUNEL > 20% (n = 36 samples) 27.85 ± 8.32 19.14 ± 8.90 <.001
Study Number of Abstinence DFI Chromatin Semen Sperm Progressive morphology
participants time assessment compaction volume concentration motility
assessment
De jong et al. 2004(9) 11 24 hours SCSA: SCSA(HDS): decreased decreased No change No change
No change increased
Gosalvez et al, 2011 (29) 33 3 hours SCD: Not mentioned Not Not Not mentioned Not mentioned
and decreased mentioned mentioned
24 hours
Pons et al. 2013 (21) 34 24 hours SCD: Not mentioned decreased decreased decreased Not mentioned
decreased
Sanchez Martin et al. (22) 21 12 hours SCD: Not mentioned decreased No change No change Not mentioned
decreased
Mayorga Torres et al.(2015)(20) 6 24 hours SCSA: Not mentioned decreased No change No change Not mentioned
no change
Mayorga Torres et al.(2016)(8) 3 2 hours SCSA: Not mentioned decreased decreased decreased Not mentioned
decreased
Agrawal et al. 2016(6) 7 24 hours TUNEL: Not mentioned decreased decreased No change No change
decreased
Uppangala et al. 2016 (30) 16 24 hours SCD: Anilline blue: increased decreased decreased No change No change
decreased
Comar et al, 2017(31) 2458 Less than TUNEL: CMA3:increased decreased decreased increased No change
2 days decreased
Shen et al., 2018(32) 167 1-3 hours SCSA: SCSA:increased decreased increased increased Not mentioned
decreased
Borges et al. 2019 (7) 818 4.15± 2.72 SCD: Not mentioned decreased decreased No change No change
days decreased
Table 4. the results of the previous studies about the effects of short abstinence on Sperm DNA.
Short abstinence and DNA integrity-Vahidi et al.
The present study also showed that antioxidants, de-
spite showing statistically significant improvement in
TUNEL tests, could not remarkably improve chromatin
compaction. It may be due to the fact that ROS, although
are harmful in high amounts, considered as physiologic
in small quantity, and are essential for sperm matura-
tion (chromatin compaction)(23). Antioxidant overuse,
especially in cases with normal seminal ROS levels,
may eliminate such physiologic amount and may lead
to decreased sperm maturation ( disturbed chromatin
compaction). This is so-called reductive stress phenom-
enon(24,25). This may be the reason for decreased semen
quality after antioxidants therapy in some patients.
Measurement of the ROS level has not been performed
in our center, possibly leading to antioxidant overpre-
scription and related side effects.
Menezo and co-workers previously reported a similar
finding. In their cohort of 54 patients using the SCSA
technique, they reported a significant decrease in sperm
DFI (32.4% to 26.2%; P-value < 0.05) and increase in
sperm decondensation rate (17.5% to 21.5%; P-value <
0.05) after antioxidant therapy(26).
Because increased sperm DFI may lead to a lower ART
outcome(27), methods that improve sperm DNA integrity
appear necessary. Nonresponders to antioxidant thera-
py (those with disturbed sperm DNA integrity) could
be treated with a simple, cost-effective, noninvasive ap-
proach (short abstinence) instead of invasive/expensive
alternatives (such as testicular sperm extraction, mag-
netic activated cell sorting and physiologic intracyto-
plasmic sperm injection).
There are some limitations in the current study. First,
the present study showed that short abstinence might
improve both sperm DFI and chromatin compaction
status, but its probable positive effect on ART outcome
remains to be elucidated.
Second, in the current study, the sperm DNA integrity
was assessed by TUNEL test and CMA3 staining, using
a fluorescence microscope. Since this method may be
operator dependent, our results need to be further as-
sessed with flow cytometric techniques, in future stud-
ies.
Third, it has been reported that testicular sperms have
a higher rate of chromosomal aneuploidy in compari-
son with that in ejaculated samples(28); the question of
whether this is true in cases of short abstinence will be
addressed in future studies.
Finally, the present study mainly included men with
subfertility (increased DFI with approximately normal
semen analysis) and having no control group. Future
studies are anticipated assessing the effects of short ab-
stinence on males with abnormal semen parameters and
sperm DNA integrity.
CONCLUSIONS
The present study showed that short abstinence ( absti-
nence time less than 24 hours) would improve sperm
DNA integrity in patients with high sperm DFI and
previous attempts of failed medical therapy. However,
because the effects of such a strategy on ART outcome
and chromosomal aneuploidy are unknown, it must be
applied cautiously until further investigations verify its
safety.
ACKNOWLEDGEMENT
We would like to thank Prof Allan Pacey at the Uni-
versity of Sheffield for his excellent scientific advice
during the writing of this manuscript.
DECLARATION OF INTEREST
No potential conflict of interest was reported by the au-
thors.
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