Manusia Kesuburan, 2015; Awal Online: 1-5 © 2015 The British

Fertility Society ISSN 1464-7273 cetak / ISSN 1742-8149

secara online DOI: 10,3109 / 14647273.2015.1050462

ARTIKEL ASLI

Oral pengobatan antioksidan sebagian meningkatkan integritas DNA sperma manusia di kelas
subur I pasien varikokel

JOSEP Gual-FRAU 1. CARLOS ABAD 1. MAR Í A J. AMENGUAL 2. NAIM Hannaoui 1.

MIGUEL A. Checa 3,4 . Jordi RIBAS-MAYNOU 5,6 . IRIS LOZANO 6. ALEXANDROS Nikolaou 6.

Jordi Benet 5. Agust Í N GARC Í A-Peir Ó 5,6 * & JUAN PRATS 1*

1 Servei d 'Urologia, Corporaci ó Sanit à ria Parc Taul í, Institut Universitari Parc Taul í - UAB, Sabadell, Spanyol, 2 UDIAT, Pusat Diagn ò stic, Corporaci ó Sanit à ria Parc
Taul í, Institut Universitari Parc Taul í - UAB, Sabadell, Spanyol, 3 Departemen Obstetri dan Ginekologi, Parc de Salut Mar, Universitat Aut ò noma de Barcelona,
​Barcelona, ​Spanyol, 4 Andrologi Laboratorium, CIRH, Cl í nica Corachan, ANACER, Barcelona, ​Spanyol, 5 Departament de Biologia seluler saya Genètica Medica.
Universitat Autonoma de Barcelona, ​Bellaterra, Spanyol dan 6 Centro de Infertilidad Masculina y Sebuah á lisis de Barcelona (CIMAB), Edifi ci Eureka, PBM5, Parc de
Recerca de la UAB (PRUAB), Kampus de la UAB, Bellaterra, Spanyol
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Abstrak
laki-laki infertil dengan varikokel memiliki persentase tertinggi sel sperma dengan DNA yang rusak, dibandingkan dengan kelompok subur lainnya. pengobatan antioksidan
dikenal untuk meningkatkan integritas DNA sperma; Namun, tidak ada data tentang efek pada pasien varikokel. Dengan demikian kita menyelidiki ts benefi potensi
Untuk penggunaan pribadi saja.

pengobatan antioksidan Cally spesifik di kelas saya laki-laki varikokel. Dua puluh pasien rawat inap yang subur dengan grade I varikokel diberi multivitamin (1500 mg
L-Carnitine, 60 mg vitamin C, 20 mg koenzim Q10, 10 mg vitamin E, 200 μ g vitamin B9, 1 μ g vitamin B12, 10 mg seng, 50 μ g selenium) setiap hari selama tiga bulan.
parameter air mani termasuk Total jumlah sperma, konsentrasi, motilitas progresif, vitalitas, dan morfologi ditentukan sebelum dan sesudah ment memperlakukan. Selain
itu, fragmentasi DNA sperma dan jumlah sel sperma yang sangat terdegradasi dianalisis dengan Sperma Chromatin Dispersion. Setelah pengobatan, pasien menunjukkan
pengurangan relatif rata-rata 22,1% di fragmentasi DNA sperma ( p 0,02) dan memiliki 31,3% lebih sedikit sel sperma yang sangat terdegradasi ( p 0,07). Jumlah total sel
sperma meningkat ( p 0,04), tapi parameter air mani tidak terpengaruh. Data ini menunjukkan bahwa integritas DNA sperma di kelas I varikokel pasien dapat ditingkatkan
dengan pengobatan antioksidan oral.

Kata kunci: faktor infertilitas pria, DNA sperma, varikokel, antioksidan, L-karnitin, sperma, dispersi kromatin

pengantar
Varikokel merupakan penyebab yang diakui dari fungsi testis menurun
ditandai dengan ringan sampai dilatasi parah vena spermatika internal
dan pleksus vena pampiniformis. modifi kasi anatomi ini dianggap
sebagai salah satu penyebab paling umum dari produksi sperma yang
buruk, karena hal itu mempengaruhi kualitas semen dan mengganggu
jalur fisiologis ontogenesis sel sperma normal (Rodriguez-Rigau et al,
1978;. Hauser et al, 2001. ;. Perak et al, 2005). Varikokel
mempengaruhi 4,4-22,6% dari populasi umum. prevalensinya lebih
tinggi pada pria dengan infertilitas primer (21 - 41%) dan pada pria
dengan infertilitas sekunder (75-81%) (Saypol et al, 1981; Gorelick &
Goldstein, 1993).. Varicocele adalah klasifi ed sesuai dengan sistem
penilaian Dublin: kelas III: terlihat dan
teraba saat istirahat; kelas II: teraba saat istirahat; dan kelas I:
teraba selama manuver Valsalva. varicocele subklinis hanya
terdeteksi oleh USG Doppler (Miyaoka & Esteves, 2012).
Salah satu efek agunan dari kondisi ini adalah produksi dalam jumlah
tinggi spesies oksigen reaktif (ROS) yang mengakibatkan stres oksidatif,
yang pada gilirannya menyebabkan peroksidasi lipid dari kerusakan
membran plasma sperma dan DNA nuklir (Agarwal et al, 2008;. Makker et
al, 2009;. Aitken & De Iuliis, 2010). sel sperma sangat rentan terhadap
stres oksidatif, karena mereka mengandung jumlah hanya terbatas ROS-
melindungi enzim (Lewis & Simon, 2010). Selain itu, pasien dengan
varikokel memiliki signifi cantly lebih rendah total kapasitas antioksidan
dibandingkan laki-laki yang sehat (Ramadhan et al, 2002;.. Agarwal et al,
2006;. Smith et al, 2006;. Pasqualotto et al, 2008). Sebagai

* kepenulisan senior yang dibagikan.

Korespondensi: Dr. Agust í n Garc í a-Peir ó, Ph.D. Centro de Infertilidad Masculina y Sebuah á lisis de Barcelona (CIMAB). Parc de Recerca de la UAB, 08193 Bellaterra, Barcelona, ​Espa ñ a. Tel: 34
935868728. E-mail: agusti.garcia@uab.es

(Diterima 7 Januari 2014; diterima Desember 2014 8)

1
 2 J. Gual-Frau et al.

Hasilnya, tingkat tinggi sel sperma dengan DNA terfragmentasi
ditemukan di ejakulasi pria dengan varikokel (Saleh et al, 2003;. Chen
et al, 2004;. Fernandez et al, 2005;. Agarwal et al, 2014.).
Studi sebelumnya mencirikan fragmentasi DNA sperma (SDF)
menggunakan uji Dispersi Sperma kromatin (SCD) mengungkapkan
dua subpopulasi khas sperma spermatozoa terfragmentasi. standar
sperma terfragmentasi dan sangat DNA-terdegradasi sperma (DDS)
(Fern á ndez et al, 2005; Enciso et al, 2006;.. Garc í a-Peir ó et al,
2012). Sedangkan sel sperma yang sehat menampilkan halo besar
kromatin dalam tes dispersi kromatin sperma (SCD), halo kecil atau
hilang di sekitar inti sperma dikaitkan dengan DNA sperma
terfragmentasi (Gambar 1). DDS subpopulasi diakui oleh inti tidak
teratur dalam Selain halo hilang, yang refl Ects ​protein besar dan
degradasi DNA dalam sperma ini (Gos á lvez et al., 2014). sampel
sperma dari individu yang sehat biasanya berisi 1 - 4% dari DDS.
Pada pasien dengan varikokel, namun, tingkat DDS yang sampai
delapan kali lebih tinggi dari yang dilaporkan dalam sampel kontrol
(Enciso et al, 2006;. Garc í a-Peir ó et al, 2012;.. Rodr í guez et al,
2012) .
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administrasi antioksidan semakin digunakan sebagai obat potensi
kerusakan DNA sperma (Greco et al, 2005;. Tremellen, 2008; Tunc et
al, 2009.). Sebuah benefi efek resmi pada spermatogenesis setelah
pemberian pengobatan antioksidan lisan berdasarkan karnitin
sebelumnya telah dilaporkan oleh kelompok kami dalam kelompok
asthenoteratozoospermia (ATZ) laki-laki -infertile tanpa varikokel (Abad
et al., 2012). Selain itu, studi klinis lain menggunakan perbaikan laporan
karnitin kualitas air mani dari pasien infertil dengan faktor etiologi lainnya
(Vicari & Calogero, 2001; Lenzi et al, 2003;. Lenzi et al, 2004;.. Balercia
et al,
2005). The aim of this study was to investigate the potential benefi ts of
treatment with oral antioxidant containing carnitine on general semen
parameters and sperm DNA integrity, specifi cally in grade I varicocele
males.
Materials and methods
Study design and patient selection
Twenty infertile men known to have asthenoteratozoospermia and

clinical grade I varicocele (WHO, 1999)

Untuk penggunaan pribadi saja.

Figure 1. Characteristic types of sperm DNA damage as viewed by the SCD test. (a) Image of a semen sample of a grade I varicocele patient. (b) Sperm showing a large halo of
dispersed chromatin, indicative of intact DNA. (c) When sperm DNA presents fragmentation, a number of strands do not remain attached to the cell and will diffuse, which is refl ected
in the SCD test by a smaller halo from the remaining bound DNA. (d) Sperm with an aberrant core and a missing halo refl ect massive protein and DNA depletion, typically seen in
highly DNA-degraded sperms (DDS). Here, the DNA fragments diffuse to such an extent that no detectable signal above background can be observed by the SCD test, which is
observed as an irregularly stained core.

Kesuburan manusia
 Antioxidant treatment in infertile varicocele patients 3

were selected. Patients with genitourinary infl ammation, instructions. Slides were stained for optical microscopy using
leukocytospermia, history of autoimmune disease, or altered Diff-Quick (Panreac; Burlingame, CA), and 400 sperm were scored for
hormonal profi les were excluded from the study. None of the each experimental point. The proportion of fragmented sperm cells
selected patients had been pre-treated with antioxidants or other (SDF) and DNAdegraded sperm (DDS) was determined and
medication before the start of the study. Patients who required expressed as percentages vs the total number of sperm analyzed
treatment during the study for other reasons (mainly infl uenza) were (Enciso et al., 2006; Garc í a-Peir ó et al., 2011).
also excluded. In addition, the following exclusion criteria were
applied for women: the absence of hormonal imbalance,
chromosomal alterations and tubal obstruction, and the absence of Statistical analysis
bacterial or viral infection.
Data analysis was performed using the software Statistics Package for
the Social Sciences, version 15 (SPSS; Chicago, IL, USA). For normal
Semen parameters and sperm DNA fragmentation were
distribution of the variables, the Kolmogorov – Smirnov and Shapiro –
determined before and after three months of antioxidant treatment
Wilks tests were used. Depending on the presence of asymmetry,
with commercial multivitamins (Androferti, Q Pharma Laboratories;
values were compared using the Wilcoxon and Sign test for related
Alicante, Spain) containing L-Carnitine (1500 mg), vitamin C (60 mg),
samples, and the level of signifi cance was established at 95% of the
coenzyme Q10 (20 mg), vitamin E (10 mg), vitamin B9 (200 μ g),
confi dence interval (CI).
vitamin B12 (1 μ g), zinc (10 mg), and selenium (50 μ g) per day.
Successful pregnancies during treatment and three months
thereafter were followed-up. Written informed consent was provided
by all patients and the study was approved by the Institutional Ethics
Results
Committee.
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General semen parameters

Sperm parameters before and after oral antioxidant therapy with

multivitamins are given in Table I. Total sperm count was signifi
Sample collection and preparation
cantly increased by 63.3% relative to pre- treatment values ( p 0.04),
Patients provided a semen sample after three days of sexual while other semen parameters remained unaltered.
Untuk penggunaan pribadi saja.

abstinence, before and after three months of treatment with

multivitamins. Fresh ejaculates were allowed to liquefy, and a small
amount of sample reserved for the determination of sperm Sperm DNA integrity
parameters. The remaining sample was mixed at a ratio of 1/1 with
A typical SCD image is shown in Figure 1, and SDF and DDS values
test yolk buffer as a cryopreservation medium containing 14% (v/v)
are given in Table I. Sperm DNA fragmentation after antioxidant
glycerol, 30% (v/v) egg yolk, 1.98% (w/v) glucose and 1.72% (w/v)
treatment was signifi cantly reduced by 6.73% ( p 0.02), which
sodium citrate (Garc í a-Peir ó et al.,
represents a reduction of 22.1% SDF relative to the initial state of the
sample. The highly DNA-degraded sperm subpopulation was
2012), aliquoted and incubated overnight at 80 ° C in an isopropanol
reduced by 4.25% ( p 0.07) after treatment, which represents a
bath, and then cryopreserved in liquid nitrogen until the experiment
relative reduction of 31.3% compared to the initial DDS state.
was performed.

Determination of semen parameters

Semen parameters including total sperm count, concentration, motility
(progressive types a, b, and a b), vitality, and morphology were
determined according to the guidelines given by the World Health
Organization guidelines (WHO, 1999). Sperm Class Analyzer (SCA)
software v. 5.1 (Microptic; Barcelona, Spain) was used to determine
concentration and motility markers, vitality was assessed by
eosin/nigrosin staining, and morphology according to Menkveld et al.
(1990).
Assessment of sperm DNA fragmentation
Samples were washed three times in PBS, and sperm concentration
adjusted to 10 million per milliliter. Sperm DNA fragmentation (SDF),
defi ned as the percentage ratio of fragmented versus total
spermatozoa in the analyzed sample, was assessed using the Sperm
Chromatin Dispersion (SCD) test (Halosperm Kit, Halotech DNA,
S.L; Madrid, Spain) according to the manufacturer ’ s
© 2015 Th e British Fertility Society
Pregnancy rate following antioxidant treatment
Out of the twenty infertile couples, two couples participating in this
study achieved a pregnancy by natural conception by the end of the
treatment.
Discussion
In the present study, sperm quality based on seminal parameters
and two markers of sperm DNA integrity (SDF and DDS) were
determined in semen samples from a cohort of infertile
asthenoteratozoospermic males with clinical grade I varicocele.
Varicoceles are characterized by different factors such as reduced
testicular blood fl ow, increased testicular temperature, and a state of
chronic infl ammation (Hauser et al., 2001). In contrast to fertile and
some other infertile groups, considerable levels of DDS are often
observed in varicocele patients (Rodr í guez et al., 2012). This
intense degree
 4 J. Gual-Frau et al.

Table I. Summary of key seminal parameters of grade I varicocele patients before and after oral antioxidant treatment.

Before treatment After 3 months of treatment

mean SD; rank mean SD; rank P value

SDF (%) 30.38 13.32 (14.5 – 42.9) 23.65 7.5 (14.5 – 38.5) 0.02 *
DDS (%) 13.55 6.21 (7.5 – 27.5) 9.3 2.78 (5.5 – 13) 0.07
Sperm ml 1 71.14 51.2 (12 – 160) 109.65 108.77 (22 – 346) 0.21
Total number of sperm 183.64 153.54 (28 – 480) 299.85 275.45 (90 – 750) 0.04 *
Progressive motility (%) 39.86 23.37 (9 – 89) 43.14 15.59 (18.3 – 66) 0.55
Viability (%) 53 21.13 (23 – 90) 48.19 13.01 (40 – 78) 0.48
Normal morphology (%) 2.6 2.01 (0 – 6) 3.8 3.2 (0 – 10) 0.10

* 0.05 Statistical signifi cance threshold.

of DNA damage and nuclear protein depletion is indicative of a
defective process of spermiogenesis (Enciso et al., 2006; Garc í
a-Peir ó et al., 2012).
Our group and others have shown that oral antioxidant treatment
based on carnitine improves semen quality in different groups of
infertile males (Vicari & Calogero, 2001; Lenzi et al., 2003; Lenzi
et al., 2004; Balercia et al., 2005; Abad et al., 2012). In addition,
low levels of vitamin C are associated with poorer semen quality and
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In summary, we showed that oral antioxidant treatment
augmented the integrity of sperm DNA in patients with grade I
varicocele. A modest increase in pregnancy rate by natural
conception was also observed. Higher rates of pregnancy loss and
childhood diseases are seen after assisted reproductive technology
(ART) such as in vitro fertilization or intracytoplasmic sperm injection,
possibly linked to sperm DNA damage (Basatemur & Sutcliffe, 2008;
Zini et al., 2008; Katari et al., 2009). The next step would therefore

more sperm DNA fragmentation (Song et al., 2006). A critical be to investigate the potential benefi ts of antioxidant treatment on
review of 20 clinical studies of antioxidant treatment showed that 19 sperm DNA quality in improving ART success rates.
of these studies report a reduction in oxidative stress (Gharagozloo
& Aitken, 2011). We therefore hypothesized that the intake of
antioxidants produces a more appropriate testicular environment for
Untuk penggunaan pribadi saja.

sperm production by partial attenuation of ROS. Here, we confi rm

that antioxidant treatment signifi cantly improved sperm DNA integrity
and increased total sperm counts, specifi cally in infertile
asthenoteratozoospermic males with clinical grade I varicocele.
Other semen parameters were unaffected and no adverse effects on
sperm quality were observed in these patients. Of note, DDS
reduction did not reach signifi cance ( p 0.07); however, we speculate
that more conclusive results could be achieved with a larger patient
sample size.
Acknowledgements
Supported by ISCIII, Ministry of Health, Spain (FIS, PI080623 and
PI11/00630), Generalitat de Catalunya (2009 SGR 1107), the
Ministry of Education and Science, Spain (Grant
BFU2010-16738/BFI) and Centro de Infertilidad Masculina y An á
lisis de Barcelona (CIMAB).

Declaration of interest : The authors report no confl icts of interest.

Two couples with a history of repeated failed pregnancies
achieved a pregnancy, suggesting that DNA integrity may be an
important factor for conception. Numerous studies show an
association between sperm DNA damage and unusually long
conception times, implantation failures, and an increased risk of
miscarriages (Evenson & Wixon, 2006; Zini et al., 2008;
RibasMaynou et al., 2012). Of signifi cance, poor sperm DNA quality
is even linked to severe childhood diseases such as cancer or
neurological disorders (Aitken et al., 2009). Since varicoceles have a
The authors alone are responsible for the content and writing of the
paper.
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