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Measured with Sperm Chromatin Dispersion (SCD) Technique: Will DNA

Integrity in Spermatozoa Increase After Micronutrient Supplementation?
Comparative Controlled Study

Article  in  Fertility & Reproduction · September 2019

DOI: 10.1142/S2661318219500142

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 ARTICLE
FERTILITY & REPRODUCTION

Measured with Sperm Chromatin Dispersion (SCD)

Technique: Will DNA Integrity in Spermatozoa
Increase After Micronutrient Supplementation?
Comparative Controlled Study
by 38.145.108.156 on 11/27/19. Re-use and distribution is strictly not permitted, except for Open Access articles.

Fiona Eidenberger1, Kim-Lien Huber², Patricia Fuchs2, Nikola Simkovicova2, Martin Imhof1,3,*
Karl Landsteiner Institute for Cell-Focused Therapy in Gynecology,
1

Wiener Ring 3–5, 2100 Korneuburg, Vienna, Austria

²Medical Students at the Medical University of Vienna, Spitalgasse 23, 1090 Vienna, Austria
3
Department of Obstetrics and Gynecology, General Public Hospital Korneuburg,
FandR 2019.01:136-140. Downloaded from www.worldscientific.com

Wiener Ring 3–5, 2100 Korneuburg, Vienna, Austria

ABSTRACT
Background: An elevated DNA Fragmentation Index (DFI) has been associated with male infertility; therefore measuring the
sperm nuclear DNA integrity appears to be useful in predicting the ability of spermatozoa to fertilize oocytes.
Aim: To evaluate the effect of micronutrient supplementation over DFI values in subfertile men.
Methods: This was a retrospective/comparative study that included a total of 306 subfertile males consulting the clinic from March
2011 to November 2017, who had performed the Sperm Chromatin Dispersion test (SCD) — a method used to detect DNA
fragmentation — along with the initial semen analysis. Of the included population, n = 146 had received two daily oral capsules of a
standardized combined micronutrient compound (Profertil®, Lenus Pharma, Vienna, Austria) for three months plus lifestyle change
(study group). Each capsule contains L-carnitine, L-arginine, coenzyme Q10, zinc, vitamin E, folic acid, glutathione and selenium.
Those who did not receive the active treatment (n = 160) but only engaged with lifestyle changes were considered controls. The SCD
test was performed in both groups at baseline and after 3 months.
Results: For the first statistical analysis, patients with an initial mean DFI of >15% were considered (n = 66 [37 were study and 29
were controls]). After 3 months, both groups displayed a significant decrease of mean DFI values. However, this decrease was more
evident in the study group as compared to controls (10.54% vs. 14.48%, α = 0.05; p = 0.013). For the second statistical approach the
entire population was considered (n = 306). After 3 months, only the study group displayed a significant decrease of the mean initial
DFI value (10.16% to 6.49%, p < 0.0001); decrease that was more evident as compared to controls (6.49% vs. 8.82%, α = 0.05; p =
0.000020).
Conclusions: Among subfertile men with a DFI >15%, both regimes significantly decreased sperm DNA fragmentation; however,
when any baseline DFI value was considered, only treatment with the active standardized micronutrient compound achieved a
significant better result.

Keywords: DNA Sperm Fragmentation; Male Infertility; Micronutrients; Supplementation.

INTRODUCTION Sub/infertility significantly increases the break-up rate in a

Couples who fail to conceive within a year of having regular and
unprotected intercourse are defined as subfertile (no pregnancy
within 6 cycles) or infertile (no pregnancy within 12 cycles).
“Primary subfertility/infertility” is the definition with no anamnesic
pregnancy while “secondary subfertility/infertility” defines
problems to conceive after a previous pregnancy. A 10–17% of all
couples wishing to conceive suffer primary or secondary subfertility.­
relationship (Templeton, 1995). A 25–30% of subfertility/infertility
cases is caused by male factors, which can be diagnosed by means
of a semen analysis. Semen analyses give an overview of the status
of semen quality: motility, morphology, vitality and concentration
of the sperm cells. A pathological semen analysis can be caused by
various diseases such as hypogonadism, varicoceles, infections of
the genital tract or autoantibodies against sperm cells. However, in

This is an Open Access article published by World Scientific Publishing Company. It is distributed under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivatives 4.0 (CC BY-NC-ND) License which permits use, distribution and reproduction, provided that the original work is properly
cited, the use is non-commercial and no modifications or adaptations are made.
Received 31 January 2019; Accepted 2 August 2019; Published 27 August 2019
*Correspondence should be addressed to: Dr. Martin Imhof, Karl Landsteiner Institute for Cell-Focused Therapy in Gynecology, Wiener Ring 3–5,
2100 Korneuburg, Austria. Email: martin.imhof@korneuburg.lknoe.at
VOLUME 1 • NUMBER 3 • SEPTEMBER 2019 • 136–140
DOI: 10.1142/S2661318219500142

1950014.indd 136 10-09-2019 12:33:07


about 50% of all cases, the underlying cause for a pathological semen
analysis cannot be found (Templeton, 1995; Isidori et al., 2006; Park
et al., 2018). In addition, in about 15% of all couples with subfertility/
infertility caused by male factors, standard semen analysis does not
reveal abnormalities (Agarwal et al., 2005). One of the reasons may
be related to sperm DNA integrity. Recent studies have shown that
DNA integrity plays an important role during fertilization and early
pregnancy. Studies have shown a correlation between decreased
sperm DNA integrity and a decreased likelihood of pregnancy
(natural and artificial insemination), as well as an increased rate of
miscarriage (Zini et al., 2001a; 2001b; Carrel et al., 2003; Speyer et al.,
2010; Venkatesh et al., 2011; González-Marin et  al., 2012). DNA
damage can be caused by intrinsic (e.g. pathological maturation of
sperm cells, oxidative stress) or extrinsic factors (e.g. varicoceles,
infections or advanced age) (González-Marin et al., 2012). In 8%
of patients with decreased sperm DNA integrity, no abnormality
by 38.145.108.156 on 11/27/19. Re-use and distribution is strictly not permitted, except for Open Access articles.
can be found in a conventional sperm analysis (Zini et al., 2001a).
DNA integrity can be measured by various tests, one being the
Sperm-Hyaluronan Binding Assay (SHBA). SHBA is a method
that indirectly tests DNA integrity and is based on the premise that
only fully developed mature sperm cells with high DNA integrity
have the ability to bind to hyaluronic acid, which simulates the
FandR 2019.01:136-140. Downloaded from www.worldscientific.com
human pellucid zone (Lipovac et al., 2014). Although studies have
demonstrated that sperm cells bound to hyaluronic acid indeed
show high DNA integrity; a direct way to assess the level of DNA
integrity seems to be of a higher diagnostic value (Wong et al., 2000;
Fernández et al., 2003; Lipovac et al., 2014). In this sense, the Sperm
Chromatin Dispersion (SCD) test has been proven to be an effective
tool to directly assess DNA integrity (Gorczyca et al., 1993; Zhang
et al., 2009). This test detects the ability of the sperm chromatin
to disperse, following acid denaturation and removal of nuclear
proteins. Human sperm chromatin is organized in loops. In case of
a low rate of DNA breaks, the molecular torsion of chromatin will
open the chromatin into coils, however, DNA breaks will prevent
dispersion (Evenson, 2016). Therefore, fragmented DNA fails to
produce a characteristic halo surrounding the sperm head, whereas
non‐fragmented DNA does exactly that (Fernández et al., 2003).
The degree of DNA integrity is determined by the DNA
fragmentation index (DFI). The DFI is the percentage of sperm cells
with DNA damage. A DFI value above 15% is commonly understood
to be pathologic and is associated with subfertility/infertility.
However, so far 15% is only a threshold that still warrants more
research (Fernández et al., 2003; Lipovac et al., 2014).
For increased DFI, therapy options are limited and not final
proven (Baker, 2000). Studies among male subjects with elevated DFI
treated with standard fertility medications (e.g. gonadotropin) show
heterogeneous fertility outcomes (Templeton, 1995). On the other
hand, since trace elements, vitamins and amino acids are important
for DNA synthesis and spermatogenesis, antioxidant therapy
seems a promising option for the improvement of cell integrity.
Positive effects of micronutrients on fertility have already been
discussed in various studies (Wong et al., 2000; Ebisch et al., 2007;
Showell et al., 2011). Therefore, it is obvious that a combination of
micronutrients can have a significant influence on sperm quality and
pregnancy outcome (Wong et al., 2000; Ebisch et al., 2007; Lipovac
et al., 2014). A previous study demonstrated that DNA integrity in
sperm cells improved after three months of supplementing with a
standardized compound of micronutrients. However, this study
used the SHBA, an indirect technique to determine the level of DNA
integrity (Lipovac et al., 2014). Therefore, the present study aimed
at evaluating the effect of micronutrient supplementation over DFI
values, as measured with the SCD test — a method with higher
diagnostic value. Since therapeutic possibilities for increased DNA
1950014.indd 137
ARTICLE
fragmentation are currently limited, the findings of this study aim
at optimizing therapy regimens in cases with elevated DFI values.
MATERIALS AND METHODS
Study design and participants
This was a retrospective and comparative study, that included male
partners of couples who had consulted the clinic from March 2011 to
November 2017 due to problems incurred while trying to conceive.
All patient data were anonymized and the retrospective study was
approved by the ethics committee of Lower Austria. All included
male patients must have had two sperm analyses as according to
WHO guidelines. Indeed, a sperm analysis has to be repeated in
order to validate the initial diagnosis. Also, a SCD test must have
been performed and patients who opted for active therapy regimen
must also have proven compliance for the intake of the standardized
micronutrient compound. Exclusion criteria was azoospermia,
aspermia, varicocele and recent urogenital infections.
Micronutrient supplementation and lifestyle modification
have proven to be effective in the treatment of male subfertility/
infertility and are therefore established as a standard procedure. The
applied standardized compound is a non-prescription micronutrient
compound that has proven to be safe and have a significant positive
effect on sperm quality. The males were advised to repeat the
sperm analysis and the SCD test after 3 months. All patients were
instructed to modify their lifestyles and had the option of taking
the standardized compound daily for the 3-month period between
the control sperm analyses and the SCD tests. Patients were then
retrospectively divided into 2 groups: the study group consisting of
those who opted for the therapy regimen including the standardized
micronutrient compound (plus lifestyle modification); and the
control group consisting of those who opted to improve their fertility
by lifestyle modification alone.
Active standardized micronutrient compound
Patients of the study group were instructed to take two daily oral
capsules of PROfertil® (Lenus Pharma GmbH, Vienna, Austria).
The  therapy contains: L-carnitine (440 mg), L-arginine (250 mg),
zinc (40 mg), vitamin E (120 mg), glutathione (80 mg), selenium
(60 μg), coenzyme Q10 (15 mg) and folic acid (800 μg).
The Sperm Chromatin Dispersion (SCD) test
Sperm DFI was assessed with Halosperm® (Halotech DNA SL,
Madrid, Spain) which is an in vitro diagnostic kit and is based on the
SCD technique. The sperm probe was fixed in a low-melting agarose
on a glass slide, covered by a coverslip. After the agarose hardened,
the coverslip was removed carefully and the slide was introduced to
an acid denaturation solution to denature chromatin and to generate
restricted single-stranded DNA from DNA breaks. After that, a
lysing solution was used, followed by staining of the coverslip. When
dried, the slide was put under an optical microscope, as the amount
of chromatin dispersion could then be easily assessed by simply
counting sperm cells with a halo surrounding the sperm head. Sperm
cells with fragmented DNA produce — if at all — very small halos,
but most commonly, they do not display any halos at all (Fernández
et al., 2003). If more than 15% of the counted sperm cells exhibited
signs of fragmented DNA, the test result was defined as pathological.
Statistical analysis
Statistical analysis was performed with the Statistical Package for
the Social Sciences version 21.0 (IBM SPSS, Armonk, NY, USA).
Data are presented as means, standard deviations and mean percent
differences. The Kolmogorow-Smirnov Test was used to determine
the normality of data distribution. For the comparison of normally
distributed data, the Student’s T-test was used; otherwise the
137
10-09-2019 12:33:07
 ARTICLE
Mann-Whitney U-test was used. The Wilcoxon test was used to
determine changes within each group. For all calculations, a p value
< 0.025 was considered as statistically significant.
RESULTS
A total of 306 males who had their DFI checked with the SCD test
were included in the retrospective, comparative study. Age of patients
ranged from 23 to 53 years (mean 35.7 ± 5.8 years). No differences
in age or body mass index were observed when comparing study or
controls in both statistical approaches to be described below.
We performed two statistical approaches. For the first, n = 66
patients were found to have a DFI above 15%, which is considered to
be pathological by study definition. Thirty-seven of these 66 males,
had opted to receive the standardized compound for 3 months
in conjunction with lifestyle modification (study group). The
29 males who did not opt to receive the standardized compound
by 38.145.108.156 on 11/27/19. Re-use and distribution is strictly not permitted, except for Open Access articles.
yet incorporated only a lifestyle modification were considered as
controls. Mean initial DFI values (DNA damage) did not differ
between studied groups (α = 0.05; p = 0.911). This confirmed
the comparability of the two assigned groups. After the 3-month
period, both groups displayed a significant decrease of mean DFI
values. However, this decrease was more evident in the study group
FandR 2019.01:136-140. Downloaded from www.worldscientific.com
as compared to controls (10.54% vs. 14.48%, α = 0.05; p = 0.013)
(Table 1).
Since the DFI value of 15% is not an established threshold, a
second statistical approach was performed to analyze the overall
improvement, including the entire population of 306 patients,
regardless of their initial DFI value. Hence, patients were divided
into two groups: those who opted for the therapy regimen of taking
the active standardized micronutrient compound (n = 146, study
group); and those who opted for lifestyle modification alone (n =
160, controls). Again, mean initial DFI values did not differ between
studied groups (α = 0.05; p = 0.819). After the 3-month period, only
the study group displayed a significant decrease of mean initial DFI
value (10.16% to 6.49%, p < 0.0001); decrease that was more evident
as compared to controls (6.49% vs. 8.82%, α = 0.05; p = 0.000020)
(Table 1).
DISCUSSION
The present study found that both micronutrient supplementation
with lifestyle changes and lifestyle changes alone decreased DNA
fragmentation in subfertile men with DFI baseline values above
15%; however, this decrease being more evident in the active group.
Independent of baselines DFI values, active group displayed a better
result as compared to controls. Micronutrients have proven being of
benefit in the treatment of male subfertility/infertility (Wong et al.,
2000; Ebisch et al., 2007; Showell et al., 2011; Lipovac et al., 2014).
Table 1.  The effect of each treatment over mean DFI values.
Group Study group
Parameter DFI > 15%
Total (n = 306) 37
Initial mean DFI [%] 20.84
Mean DFI after 3 months [%] 10.54
Mean percent difference [%] 10.30
Comparison of DFI before and after p < 0.0001
  of each group (α = 0.025)
138
1950014.indd 138
The investigated standardized compound consists of L-carnitine,
L-arginine, zinc, vitamin E, glutathione, selenium, coenzyme Q10
and folic acid all which are known to have positive effects on sperm,
e.g. by means of anti-oxidative protection. Therefore, it seems
obvious that a deficiency of these micronutrients could negatively
affect sperm cells. All these micronutrients have been examined for
decades, and have never shown any negative or adverse effects on
health.
L-carnitine helps promote sperm maturation, initiate sperm
motility and enhance the fertilizing potential of sperm cells. It also
protects sperm cells against oxidative damage and reduces apoptosis,
and improves sperm motility and chromatin quality (Aliabadi et al.,
2012). One study showed that a combination of L-carnitine and
coenzyme Q10 can improve semen parameters and the outcome of
clinical pregnancy in patients with oligoasthenozoospermia (Cheng
et al., 2018).
Coenzyme Q10 is an antioxidant and a component of the
respiratory chain (Lafuente et al., 2013). It plays an important role in
energy metabolism and is concentrated in the mitochondria of the
sperm cell’s midpiece (Lewin and Lavon, 1997; Lafuente et al., 2013).
Motility and all other energy-dependent processes in the sperm cell
therefore rely on the availability of coenzyme Q10 (Lewin and Lavon,
1997). Coenzyme Q10 supplementation improved fertilization rates
after intracytoplasmic sperm injection (Lewin and Lavon, 1997).
L-Arginine is a semi-essential amino acid and a precursor of
nitric oxide. Nitric oxide seems to be beneficial for sperm viability
and motility in both fertile and infertile individuals (Zhang et al.,
1996). Despite this, one study performed in healthy subjects failed
to evidence an improvement of nitric oxide production after acute
L-arginine supplementation (Alvares et al., 2012). Folic acid is
essential for DNA synthesis and can impact chromatin structure.
Studies have suggested that adequate folic acid intake in adulthood
is important for the prevention of chromatin damage, but they also
added that the dosage should probably be quite high in order to have
a notable effect (Swane et al., 2012).
One study found that vitamin E and selenium oral
supplementation improved semen quality and had a beneficial
effect on sperm motility (Keskes-Ammar et al., 2003). Selenium
supplementation can improve sperm motility in subfertile men with
low selenium status; however, only 56% of all patients responded
to the treatment (Scott et al., 1998). Another study suggested that
selenium may decrease sperm motility in healthy men by altering
thyroid hormone metabolism, but this study only included 11
patients who were given up to 297 μg of selenium per day, which is a
very high dosage (Hawkes et al., 2001).
Zinc has been suggested to act as an important anti-
inflammatory factor, also involved in sperm’s oxidative metabolism
Control Comparison of Study group Control Comparison of
both groups/ both groups/
DFI > 15% DFI 0–100% DFI 0–100%
p value p value
29 146 160
20.55 0.911 10.16 9.76 0.819
(α = 0.05) (α = 0.05)
14.48 0.013 6.49 8.82 0.000020
(α = 0.05) (α = 0.05)
6.07 3.67 0.94
p < 0.0001 p < 0.0001 p = 0.060
(α = 0.025) (α = 0.025) (α = 0.025)
10-09-2019 12:33:07

(Fallah et al., 2018). Zinc levels in seminal plasma of infertile males
are significantly lower than those of fertile males; therefore zinc
supplementation could improve sperm quality (Zhao et al., 2016).
Glutathione is an antioxidant that reacts directly with reactive
oxygen species. Reduced levels have been found in patients with
oligozoospermia and azoospermia, suggesting that oxidative stress
plays an important role in male fertility (Atig et al., 2012).
Each micronutrient has shown positive effects on sperm
cells but only few studies have researched combined preparations
(Atig et al., 2012; Lipovac et al., 2014; Cheng et al., 2018; Alonge
et al., 2019). The present study found that combined micronutrient
supplementation plus lifestyle changes decreased sperm DNA
fragmentation more evidently than lifestyle modification alone
in those with pathological DFI (>15%); although, in fact both
decreased DFI values. When analysis was performed regardless of
baseline DFI values, micronutrient group displayed a better effect
by 38.145.108.156 on 11/27/19. Re-use and distribution is strictly not permitted, except for Open Access articles.
than controls. This finding suggests that all males benefit from the
intake of micronutrient supplementation, regardless of their baseline
DFI value. Besides the lack of studies related to the benefit of
combined micronutrient supplementation, effects of micronutrients
specifically on DNA fragmentation have been lacking until now.
The SCD test has been proven to be a reliable and easy to perform
FandR 2019.01:136-140. Downloaded from www.worldscientific.com
method to detect DNA fragmentation. However, some studies argue,
that it measures the absence of damage rather than the damaged
DNA in sperm (Evenson, 2016). Nevertheless, to our understanding
this fact does not make any difference to the test’s outcome though.
One study confirmed this assumption while describing that
sperms with a big halo (ergo undamaged DNA) showed a good
correlation with DFI values (Swane et al., 2012). It has been stated
that the SCD test allows the detection of an extreme degree of DNA
damage (possibly affecting the nuclear structure), which could not
be detected by means of other DNA damage assessing techniques
(Chen et al., 2004). The SCD test has also been described as a reliable
predictor of fertilization rate, pregnancy and embryo quality after
conventional IVF (Tandara et al., 2014). A higher rate of sperm cells
with increased nuclear dispersion was found in the patients with
recurrent pregnancy loss (Absalan et al., 2012). One study, however,
compared the sperm chromatin structure assay (SCSA) and the SCD
test by using them on thawed bull semen samples with and without
oxidative stress. The study found that SCD had a lower repeatability
and was not able to discriminate between samples with and without
oxidizing treatment, suggesting that SCSA might be the most useful
method to assess sperm chromatin in bulls (Martínez-Pastor et al.,
2009). Reports indicate that although the various methods used to
detect DNA damage generally correlate at least moderately with each
other, it might be possible that the tests are evaluating or expressing
different aspects of sperm DNA damage, leaving it up to further
discussion on which is the best method to use (Martínez-Pastor
et al., 2009; Ortiz et al., 2017).
As for the limitations of the present study, one can mention its
retrospective design. Lifestyle is not the only factor affecting sperm
DNA fragmentation, hence there is a need for a randomized trial
that can prospectively evaluate the combination of micronutrients,
also recording pregnancy rates and outcomes, and taking into
account all possible factors related to DNA fragmentation. Since to
date no method to test DNA fragmentation has been established as
the gold standard, further investigation has to be done to confirm
our findings. Setting a standard critical value for DNA fragmentation
should also be the subject of more studies, in order to provide
treatment for DNA fragmentation based on facts rather than on
assumptions. A DFI of >15% is only a threshold value so far and
therefore it is possible that the actual critical value could even be
1950014.indd 139
ARTICLE
lower. This needs to be the subject of further investigations in order to
offer subfertile/infertile patients the best treatment possible. Despite
the aforementioned limitations, data related to DNA fragmentation
and the use of combined micronutrients is scarce or null; hence our
data provides insights for future study designs.
In conclusion, among subfertile men with a DFI >15%, both
regimes significantly decreased sperm DNA fragmentation; however,
when any baseline DFI value was considered, only treatment with the
active standardized micronutrient compound achieved a significant
better result. In further studies, this effect should be analyzed in
relation to the pregnancy rate.
AUTHOR CONTRIBUTION
All authors have made substantial contributions in relation to the
research design, statistical analysis and interpretation. They all
approved the final version of this manuscript.
DECLARATION OF INTEREST
The authors report no conflicts of interest.
FUNDING
None.
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