NOS3 gene polymorphism in male infertility [602717]

Role of endothelial nitric oxid e synthase (NOS3) gene polymo rphism in male infertility

Authors : Adel Abd El-kader Zalata , Mustafa Ahmed Neamatallah
, Noha Mohammed Hazem, Horya Moghazy Abdulaziz

Name and affiliation of the authors:
Adel Abd El-kader Zalata
Professor of Medical Biochemistry, Department of Medical Bio chemistry,
Faculty of Medicine, Manso ura University, Egypt.
Mustafa Ahmed Neamatallah
Assistant Professor of Medical Biochemistry, Departments of Med ical
Biochemistry, Faculty of Medici ne, Mansoura University, Egypt.
Noha Mohammed Hazem
Lecturer of Medical Biochemistry, Department of Medical Bioch emistry, Faculty
of Medicine, Mansoura University, Egypt.
Horya Moghazy Abdulaziz El-moghazy
Demonstrator in Medical Biochemistry Department, Faculty of Med icine,
Mansoura University, Egypt.

Corresponding Author: Dr. Adel Abd El-kader Zalata, Department of Medical
Biochemistry, Faculty of Medici ne, Mansoura University, Egypt.
Email: [anonimizat] .

Running title: NOS3 gene polymorphism in male infertility
Key words : eNOS gene, polymorphism, male infertility

Role of endothelial nitric oxid e synthase (NOS3) gene polymo rphism in male infertility
Adel Zalata , Mustafa Neamatallah,, Noha Mohammed Hazem, Horya Moghazy .
Department of Medical Biochemistry, Faculty of Medicine, Mansoura University, Egypt
ABSTRACT
Genetic abnormalities could be present in 15% of the cases of m ale infertility including
chromosomal aberrations and singl e gene mutations. Nitric oxide (NO) has been involved in
inducing poor sperm function. The main source of NO is the endo thelial isoform of NO
synthase (NOS3), encoded by the NOS3gene. Aim: The aim of the study is to investigate the
association of endothelial nitric oxide synthase (NOS3) gene po lymorphism and infertility in
males. Materials & methods: 100 blood and semen samples were obtained from men
attending the andrology outpatie nt clinic, Mansoura University Hospital. The semen samples
were grouped into Normozoospermia (n=25), Asthenozoospermia (A) (n=25), Astheno-
Teratozoospermia (AT) (n=25) and Oligo-Astheno-Teratozoospermia ( O A T ) ( n = 2 5 ) .
Computer assisted semen analysis, acrosin activity test and sem inal nitrite assay were
performed. Genotypes of the eNOS T786C and G894T SNPs were assessed by allelic
discrimination with real time PCR. Results: For NOS3 G894T polymorphism, the
homozygous mutant TT genotype and mutant T allele were higher i n infertile males (AT and
OAT groups) than controls with high odds ratio ( 2.9 , 4.5 res pectively). The NOS3 T786C
polymorphism might be related to increased incidence of male in fertility as the genotype CC
was up regulated in infertile males with higher odds ratio( 2.9 , 3.5, 5.8 for A, AT, and OAT
groups respectively) . The results showed significant relation between nitrite concentration
and both NOS3 T786C and G894T polymorphisms. Conclusions: The NOS3 G894T and
NOS3 786T/C polymorphisms seem to be linked to the risk of male infertility development in
Egyptian individuals. The association between both SNPs and th e overproduction of nitrite
have a potential pathogenetic role in affection of sperm parame ters.

INTRODUCTION
The most prevalent cause of male infertility is defective sperm function due to different
congenital or acquired factors. Genetic abnormalities could be present in about 15% of the cases
of male infertility including chromosomal aberrations and singl e gene mutations (Olayemi,
2010). Excess level of reactive oxygen species (ROS) is harmful to spe rmatozoa and leads to
DNA damage. ROS increase lipid peroxidation and so damage of sp ermatozoa plasma
membranes, which can impair sperm function via affecting metabo lism, acrosome reaction
reactivity, and ability of the sp ermatozoa to fuse with the ooc yte. This may result in loss of
fertility. Reactive nitrogen species (RNS) like peroxynitrite anion, nitro xyl ion, nitrosyl-containing
compounds, and nitric oxide has recently been involved in induc ing poor sperm function
(Choudhary et al., 2010). NO is known to be produced through enzymatic conversion of L-
arginine to L-citrulline by NO synthase (NOS),which has three i soforms namely, neuronal
(NOS1), endothelial (NOS3) and inducible (NOS2) synthases. The main source of NO is the
endothelial isoform of NOS, encoded by the NOS3gene (Jones and Hingorani, 2005) . Several
studies have been carried out on NOS3 gene polymorphisms in man y vascular and inflammatory
diseases (Srivastava et al., 2008). However, studies investigating the relationship between NOS3
gene polymorphisms and male infertility are very rare (Safarinejad et al., 2010). Bulderghini et
al., (2010) suggested that the T allele encoding for aspartic acid of the N OS3 (Glu298Asp) gene
may contribute to poor sperm motility, However Bianco et al.,(2013) demonstrated that in
Brazilian population the genetic variations, T786C, G894T, and 4a/4b of the NOS3 gene are not
associated with male infertility. Aim of work: The aim of the study is to investigate the
association of endothelial nitric oxide synthase (NOS3) gene po lymorphism and infertility in
Egyptien males.
MATERIALS AND METHODS
The present study included 100 subjects attending the Andrology Outpatient Clinic, Mansoura
University Hospital to obtain blood and semen samples. . All th e experiments were approved by

the ethical committee (code M1701706 ). All cases were subjected to full history taking and
physical examination. Local exami nation of the testis, epididym is, vas deferens and inguinal
region was done for all patients. Samples were grouped into nor mozoospermia (n=25),
asthenozoospermia (n=25), astheno-Teratozoospermia (n=25) and o ligo-astheno-
teratozoospermia (n=25).
Blood samples: Five ml of venous blood were withdrawn from all patients and co ntrols by
venipuncture then it was divided into two aliquots. Two ml were collected into test tube
containing 200µl dipotassium ethylene diamine tetra-acetic acid (EDTA) solution. It was stored
as a whole anti-coagulated blood at -20 °C and subsequently use d for DNA extraction. The other
three ml were collected into dry test tube and left to stand at room temperature for 30 minutes,
then centrifuged at 5000 round per minutes (rpm) to obtain seru m. The sera were stored at -20 °C
and subsequently used for other biochemical investigations. Semen Samples: Fresh semen
samples were collected from cases by masturbation, after 2-7 da ys sexual abstinence and allowed
to liquefy for 15-30 minutes. After complete liquefaction, the liquefied semen was divided into 2
p a r t s . O n e p a r t w a s u s e d f o r p e r f o r m a n c e o f c o m p u t e r a s s i s t e d s emen analysis and acrosin
activity test according to the recommendation of the World Heal th Organization . The other part
was centrifuged at 1000 rpm for 15 minutes to separate seminal plasma which was divided into
two tubes one for α-glucosidase determination and the other one was stored at -20°c until nitrite
assay.
Computer-assisted method of semen analysis (AutoSperm, Fertipro , Belguim ) was used to
calculate sperm concentration, motility (%), velocity, average curve linea r velocity (expressed in
µm/sec), linear velocity and linearity index. Spermatozoa were classified according to their
motility characteristics as grade a, b, c, and d. Sperm morphol ogy was evaluated by phase
contrast microscope and Sperm Mac stain (Fertipro , Belguim ) . WBCs were determined by
peroxidase stain (Endtz, 1972) .
Assay of acrosin activity (by gelatin-covered micro slides and gelatinolysis): Semen samples

of 20 μL were diluted 1:10 in PBS containing 15.7mmol/L a-D-glu cose. Semen samples were
smeared on prepared gelatin covered slides and incubated in a m oist chamber at 37 C° for 2 h.
The halo diameter around any 10 spermatozoa shown to be represe ntative of sperm present in the
ejaculate was measured in phase contrast with an eyepiece micro meter. The halo formation rate
was calculated per slide as the percentage of spermatozoa showi ng a halo. One hundred
spermatozoa were evaluated. An acrosin activity index was calcu lated by multiplying the halo
diameter by the halo formation rate (Zalata et al., 2004). Collected sera were analyzed for
hormonal profile by enzyme linked immunosorbant assay (ELISA) t echnique which includes
follicle stimulating hormone (FSH), lutenizing hormone (LH), te stosterone and prolactin.
Molecular investigations: DNA extraction: The genomic DNA of blood samples was extracted
b y G – s p i n ™ T o t a l D N A E x t r a c t i o n K i t (iNtron Biotechnology Inc, South Korea). The DNA
extracted was kept at -20°C till use for real time PCR. Determination of DNA quantity and purity
was done.   Assessment of DNA quality by agarose gel electrophoresis (AGE).                            
Allelic discrimination (AD) with real time PCR : Two Single-nucleotide polymorphisms
(SNPs) of the NOS3 gene were investigated in this study. Allele typing of every DNA sample
was carried out with real-time PCR reaction on the apparatus (m odel 7500; Applied Biosystems)
consuming a readymade fluorescein-amidite–labeled SNP primers a nd probes (purchased from
Applied Biosystems). The allele specific probes were labeled wi th a fluorescent dye (VIC and
FAM). The oligonucleotide sequences flanking the two studied NO S3 SNPs were designed as
primers for TaqMan® allelic discrimination. the reaction was set according to the
recommendation of Applied Biosystems with an input DNA of 25 ng, 1X SNP Genotyping
Assay Mix, 1X TaqMan_ Universal PCR Master Mix (Applied Biosystems) and n ucleasefree
water to a total volume of 20 µl l. Probes and oligonucleotides sequences are available upon
request. Thermal cycling conditions were as following: 95˚C fo r 10 min, 40 cycles of: 95˚C for
15 sec and 60˚C for 1 min.
Determination of seminal pla sma nitrite (Nitric oxide): Seminal Plasma nitrite levels were

estimated by colorimetric assay according to the method of Yousefniapasha et al. ( 2015 ), using
chemicals purchased from sigma laboratories (St-Louis, USA). Statistical Analysis: Statistical
analysis was done by using MedCalc® program version 12.2 2011 . (Schoonjans et al., 1995) .
RESULTS
Table (1 and 2) showed the distribution of NOS3 G894T and NOS3 T-786C genotypes that was
consistent with Hardy– Weinberg e quilibrium in cases and contro l. In table 3, nitrite level
correlations with sperm concentration, Grade A motility, grade (A+B) motility, normal
morphology, α- glucosidase, linearity index, linear velocity, v elocity , acrosin activity index and
WBCs were demonstrated. The relations between NOS3 T786C and G8 94T SNPs and semen
parameters are shown in table 4,5. The semen parameters include sperm concentration,
morphology, motility grade A, velocity and linearity index alph a glucosidase concentration,
acrosin activity index and seminal nitrite concentration.

Table (1): Genotype and allele f requencies of the NOS3 G894T po lymorphism in di fferent groups .
NOS3
polymorphis
m Populatio
n n Genotypes Alleles OR
(95%CI) n% n% n% n% n%
G894T GG% GT% TT% T% G%
Control 25 13(48%) 8(28%) 4(24%) 16(32%) 34(68%)
A 25 10(40%) 7(28%) 8(32%) 23(46%) 27(54%) 1.8
(0.8-4.08)
AT 25 7(28
%) 7(28%) 11(44%) 29(58%) 21(42%) 2.9**
(1.3-6.6)
OAT 25 5(20%) 6(28%) 14(52%) 34(68%) 16(32%) 4.5***
(1.9-10.5)
OR=odds ratio, . 95%CI=C onfidence interval. Normo. = Normozoospermia, A = Asthenozoospermia, AT = Astheno
teratozoospermia, OAT = Oligo-Astheno- teratozoospermia, n= number of cases. * S i g n i f i c a n t ( P ≤ 0 . 0 5 ) . * *
Moderately significant (P≤0.01). ***Highly significant (P≤ 0.001).

Table (2): Genotype and allele fre quencies of the NOS3 T786c polymorphism in different groups.
NOS3
polymorphism Population N Genotypes
n% Alleles
n% OR(95%CI)
T786C TT% TC% CC% C% T%
Control 25 11(44%) 9(36%) 5(20%) 19(38%) 31(62%)
A 25 5(20%) 8(32%) 12(48%) 32(64%) 18(36%) 2.9**
(1.3_6.5)
A T 25 4(16%) 8(32%) 13(52%) 34(68%) 16(32%) 3.5**
(1.5_7.9)
O A T 25 2(8%) 7(28%) 16(64%) 39(78%) 11(22%) 5.8***
(2.4_13.9)
O R = o d d s r a t i o . 9 5 % C I = C o n f i d e n c e i n t e r v a l N o r m o . = N o r m o z o o s p ermia, A = Asthenozoospermia, AT = Astheno
teratozoospermia, OAT = Oligo-Astheno- teratozoospermia, n= num ber of cases. * Significant (P≤0.05). ** Moderately
significant (P≤0.01). ***Highly significant (P≤0.001).

Table (3): Correlation of nitrite concentration with sperm para meters of all studied groups.
Variable Correlation
Coefficient 95% Confidence interval
Concentration
(million/ml) r = -0.5970*** -0.7101 to -0.4538
Motlity A % r = -0.6977*** -0.7864 to -0.5809
Motility A+B % r = -0.6709*** -0.7663 to -0.5465
Morphology %
r = -0.6609*** -0.7588 to -0.5338
WBCs
(million/ml) r = 0.2858** 0.09471 to 0.4566
Alpha Glucosidase
(mU/ml) r = -0.5613*** -0.6825 to -0.4101
Vel(μm / sec) r =-0.5722*** -0.6909 to -0.4233
Lin-index% r =-0.5910*** -0.7055 to -0.4464
Linear-vel
(μm / sec) r = -0.6045*** -0.7159 to -0.4630
Acrosin index r = -0.5843*** -0.7004 to -0.4382
* Significant (P≤0.05). ** Moderately significant (P≤0.01). * **Highly significant (P≤0.001). r=correlation coefficient .

Table (4): Association of NOS3 p olymorphism (T786C) with semen parameters in infertile men.
T786C TT(n=22) TC(n=32) CC(n=45) P 1value P2 value P3 value
Concentration
(million/ml) 65.6
(7.5 – 96.0) 44.8
(4.5 – 91.2) 28.3
(3.1 – 96.0) 0.0141**
0.0004*** 0.0891
Normal
morphology % 46.5
(6.0 – 62.0) 36
(2.0 – 62.0) 16
(0.0 – 64.0) 0.0182* 0.0028** 0.3280
Grade
A motility(%) 40
(17.0 – 60.0) 28
(0.0 – 60.0) 20
(0.0 – 60.0) 0.0127** 0.0002*** 0.1690
Grade (A+B) motility (%) 51.5
(30.0 – 67.0) 37
(2.0 – 64.0) 31
(4.0 – 67.0) 0.0183* 0.0002*** 0.1068
Velocity (µm/sec) 69.9
(18.5 – 89.6) 52.9
(17.8 – 85.3) 40.2
(8.8 – 82.9) 0.0073** 0.0004*** 0.3135
Linearity index % 67.4
(30.3 – 86.3) 55.7
(31.4 – 85.9) 50.9
(14.8-86.3) 0.0099** 0.0004*** 0.0961
Linear velocity
(µm/sec) 58
(5.6 – 70.7) 28.6
(9.4 – 67.0) 19.4
(1.3 – 70.7) 0.0056** 0.0002*** 0.2491
WBCs (million/ml) 0.6
(0.4 – 3.6) 0.6
(0.4 – 4.8) 0.6
(0.4 – 4.8) 0.8578
0.6515 0.5365
α- glucosidase
(mU/ml) 59.9
(18.4 – 90.7) 42.4
(18.6 – 89.6) 36.1
(18.4-90.7) 0.0496* 0.0032** 0.1981
Acrosin index 11
(4.7 – 19) 8.9
(1.0 – 16.7) 6.2
(0.6 – 19.5) 0.0197* 0.0001*** 0.1223
Nitrite (µM\ml) 4.2
(3.1 – 6.7) 4.9
(3.1 – 7.2) 5.1
(3.2 – 6.8) 0.0362* 0.003**
0.4142
* Significant (P≤-0.05). ** Mod erately significant (P≤0.01). ***Highly significant (P≤0.001).
* P1 =TC versus TT *P2=CC v ersusTT * P3=CC versus T C
DISCUSSION
Male infertility is considered to be responsible for about 30-5 0% of all cases of infertility
(Abarikwu, 2013). Nitric oxide (NO) is a major component of ROS and plays various roles in the
physiology and pathology of spermatogenesis and sperm functions . At testicular level, NO
synthase utilizes L-arginine as a substrate for synthesis of en dogenous NO (Agarwal et al.,
2014) . NOS3 is expressed in testicula r endothelial cells, sertoli an d leydig cells throughout all
phases of spermatogenesis (Fujisawa et al., 2001) . In the present study, we evaluated the
association of T786C, G894T polymorphisms in NOS3 gene and thei r possible risk on male
infertility in Egypti an population. As re gard T786C polymorphis m, different genotypes and allele
frequencies were found among AT, A, OAT and control group. In G 894T significant difference was
found in the genotype TT and the allele T frequency of distribu tion between control and infertile
cases being more frequent in AT and OAT groups. These results, as observed by odds ratio, indicate

Table (5) : Association of NOS3 Polymorphism (G894T) With Semen Parameters in Infertile Men
G894T GG
(n=35) GT
(n=28) TT
(n=37) P1
Value P2
value P 3
Value
Concentration
(million/ml) 47.2
(3.1-91.2) 52.2
(4.5-96.0) 31.4
(3.3-96.0) 0.8034 0.0398*
0.0964
Normal
morphology % 44.0
(2.0-62.0) 39.0
(2.0-62.0) 16.0
(0.0-64.0) 0.8899 0.0604 0.1101
Grade A motility
(%) 33
(0.0-60.0) 23.3
(0.0-60.0) 22
(0.0-60.0) 0.3612
0.0836
0.5688
Grade (A+B) motility (%) 39
(2.0-67.0) 36.5
(4.0-66.0) 31
(4.0-67.0) 0.4425 0.0381*
0.1117
Velocity (µm/sec) 63.4
(17.8-86.6) 51.2
(8.8-85.3) 41.6
(9.2-82.5) 0.5566 0.0732
0.1852
Linearity index % 67.7
(31.4-86.3) 56
(14.8-86.3) 49.5
(29.3-86.1) 0.5659 0.0044** 0.0239*
Linear velocity
(µm/sec) 42.9
(9.4-69.8) 27.3
(1.3-70.7) 19.4
(2.7-70.7) 0.6731 0.0554 0.0991
WBCs (million/ml) 0.6
(0.4-4.4) 0.6
(0.4-4.8) 0.6
(0.4-4.8) 0.5698 0.7835 0.7474
α- glucosidase
(mU/ml) 46.2
(18.6-89.6) 42.2
(18.4-90.7) 38.7
(18.4-90.7) 0.7661 0.0741 0.2490
Acrosin index 10.7
(0.7-19) 6.7
(0.8-18.4) 7.5
(0.5-19.5) 0.1334 0.0197* 0.4462
Nitrite(µm/ml) 4.4
(3.1-7.3) 4.9
(3.2-7.3) 5.1
(4.1-6.8) 0.4849 0.0318* 0.1897

* Significant (P≤0.05). ** Moderately significant (P≤0.01). ***Highly significant (P≤0.001). *P1 =GT versus
GG. *P2=TT versus GG. *P3=TT versus GT.

a role of both G894T and T786C polymorphism in the pathogenesis of male infertility. Safarinejad
study on Iranian people are in harmony with these study results , a s e v i d e n t d i f f e r e n c e s w e r e
shown in the distribution of NOS3 variants in infertile patient s when compared with fertile men.
Until now the explanation for the relationship between the NOS3 polymorphism and male
infertility risk is not well known. Nevertheless, a defect in N O synthesis leads to low NO level
may participate in infertility (Safarinejad et al., 2010).
NO which is produced in majority by NOS3 gene is known as one o f the effective anti-
oxidants found in seminal plasma. The 786CC and 894TT genotypes studied previously might be
associated with lower NOS3 activ ity. However, mutation of NOS3, m a k e s t h e g e n e
dysfunctional and results in production of superoxide (Barbato and Tzeng, 2004) . It is well

recognized that impaired semen quality in majority results from imbalance in anti-oxidant status
in seminal plasma in addition to oxidative stress (Safarinejad et al., 2010).
The associations of NOS3 gene polymorphisms 786CC and 894TT wit h semen
parameters were also investigated in the present study. A signi ficant association was
demonstrated between NOS3 786CC genotype and concentration, gra de A motility, grade A+B
motility, normal morphology, velo city, linear velocity, lineari ty index, acrosin, nitrite, and α-
glucosidase. On the other hand, 894TT genotypes showed a signif icant association with sperm
concentration, grade A-B motility, linearity index, acrosin ind ex and nitrite. These results stands
with the hypothesis that NOS3 gene polymorphism may give an ide a about the severity of the
disease. Safarinejad et al. (2010) observed that there was a negative correlation between the
a b o v e – m e n t i o n e d N O S 3 g e n o t y p e s a n d t h r e e s e m e n p a r a m e t e r s n a m e l y (sperm concentration,
sperm morphology and sperm motility) . Yan and his companions carried out a study over
Chinese population and results revealed that NOS3 (G894T) was a ssociated with a borderline
significantly increased risk of male infertility (Yan et al., 2014) .
The NOS3 (G894T) is located in exon 7, when mutation occurs asp artic acid replaces
glutamic at codon 298. The explanation for the enhanced disease risk associated with G894T
polymorphism was related to the affected protein-protein intera ctions and localization of the
NOS3 protein which result from this mutation, and hence the pro tein function might be affected
(Joshi and Bauer, 2008) . Furthermore, T allele was revealed to generate protein produc ts with
different susceptibility to cleava ge, proposing that this polym orphism affects the NOS3 protein
function (Tesauro et al., 2000) . Additionally, the 894 T allele is associated with elevated pla sma
NO levels (Yoon et al., 2000) .
The SNP T786C is localized to the NOS3 gene promoter region. So me reports proposed
that the promoter activity was proposed to decrease to 50% in c ase of presence of the 786C
allele and decrease transcription efficiency , which results in decreased NOS3 expression in testis
tissue (Miyamoto et al., 2000) . Reduced expression of NOS3 in testis tissue leads to reductio n of

NO production and finally ends in idiopathic azoospermia (Fujisawa et al., 2001) . However,
over expression of NOS3 gene can speed up the rate of apoptosis of testicular germ cells (Erkan
et al., 2012) . Along with results of the current study Ying observed that the frequency of 786C
allele was significantly higher in total infertile patients and azoospermia group than in control
cases. This finding is suggestive of presence of association be tween that allele 786C and male
infertility (Ying et al., 2013) . Other study showed a significant relationship between eNOS
genotypes T786C, G894T polymorphisms with decreased sperm param eters and increased
seminal oxidative stress (Mostafa et al., 2015).
The present study demonstrated a significant increase in nitrit e concentration (the indicator of
endogenously produced NO) in cases with 786CC, 894TT than those having 786TT, 894GG
genotypes respectively. Studies supported our results proposed that NOS3 894TT genotype may
result in higher NOS3 enzyme levels and activity, and so a high concentration of NO; a high NO
concentration may lead to sperm DNA damage, thereby contributin g to male infertility (Yan et
al., 2014) . Reports have suggested that the NOS3 gene G894T polymorphism plays a role in
various conditions and diseases that are associated with abnorm al NO levels. These reports
support our hypothesis that NOS3 gene is associated with abnorm al levels of NO. Total NO
levels in the seminal plasma corresponded well with NOS activit y in sperm. In most of the
infertile subjects the semen presented with high NO and NOS act ivity compared with proven-
fertile controls (Ramya et al., 2011) . Thus, the relation between nitrite concentration in one
hand and NOS3 T786C and G894T polymorphisms on the other hand may suggest that
overproduction of this free radical and the consequent excessiv e exposure to oxidative conditions
in this genetic abnormality have a potential pathogenetic role in affection of sperm parameters.
The Current study demonstrated a negative correlation between n itrite concentrations and sperm
concentration, motility, morphology, velocity, wbc, alpha gluco sidase and acrosin activity index
indicating that subnormal sperm function as observed in current study might be due to increased

levels of NO present in the semen. In support to this study res ults Amiri and his team showed
that a negative correlation was found between seminal plasma NO concentration and sperm
motility, concentration and viability (Amiri et al., 2007) . These findings support the conclusion
that a high concentration of NO plays a harmful effect on sperm atozoa viability (Huang et al.,
2006) . However , it has been reported that the concentration of NO in seminal p lasma does not
correlate with sperm concentration, sperm motility and leukocyt espermia (Revelli et al., 2001a) .
Investigations declared that NO production at supra-physiologic a l l e v e l c a n d i f f u s e a c r o s s
membranes and mediates it is action. NO in high concentration r eacts with superoxide anion
producing ONOO– and peroxy-nitrous acid (ONOOH). These molecules can cause mole cular
damages to various tissues (Stamler et al., 1992) . Additionally, cellular respiration by NO via
nitrosylation of heme in mitochondrial enzymes, aconitase, and glyceraldehyde phosphate
dehydro-genase, leading to a depletion of adenosine triphosphat e and a subsequent loss of
spermatozoal motility (Amiri et al 2006). Recent study showed that plasma membrane calcium
ATPase 4 (PMCA4) co-ordinates calcium and nitric oxide signalin g in regulating murine sperm
functional activity to maintain motility (Olli et al., 2018). The present study recognized a
negative correlation between nitrite and sperm motility but it is not considered the chief cause of
sperm immotility in infertile males. It is important to know th a t i n c a s e o f s e v e r e l y i m p a i r e d
sperm motility, factors other than the nitric oxide and other f ree radicals (e.g. the intrinsic
structure of sperm flagellum) might results in sperm damages (Amiri et al., 2007) .
Acrosin is a trypsin-like serine proteinase enzyme that can hyd r o l y z e t h e z o n a p e l l u c i d a i n
oocytes. It also plays a vital role in the process of fertiliza tion and is involved in acrosome
reaction (Nuzzo et al 1990). Thus, measuring acrosin activity is a suitable approach for
evaluating the fertilizing capacit y of human spermatozoa. Expos ing spermatozoa to oxidative
stress decreases the membrane fluidity and reduces the fusogeni c capacity leading to impaired
the acrosin activity (Zalata et al ., 2004 ). Ramaya and his group observed a negative
correlation between sperm functions in terms of hypo-osmotic sw elling, acrosomal status,

motility, and concentration which support our finding (Ramya et al., 2011) . I t s e e m s t h a t t h e
controversial results achieved i n this and other studies regard ing NO concentration may be due to
different study populations and etiology of infertility in the subjects. A major limitation of our
study may be the small number of patients. Moreover, only two S NPs for NOS3 gene were
included in this study, and these markers may not be sufficient to provide full coverage for
genetic test (Buldreghini et al., 2010).
In conclusion, The NOS3 G894T and T786C polymorphisms seem to b e linked to the risk of
male infertility development in Egyptian individuals. The relat ion between nitrite concentration
in one hand and NOS3 T786C and G894T polymorphisms on the other hand may suggest that
overproduction of this free radical and the consequent excessiv e exposure to oxidative conditions
in this genetic abnormality hav e a potential pathogenetic role in affection of sperm parameters.
This work represents a small sized sample of Egyptian populatio n. So, additional studies with
larger number of patients would be useful to confirm the import ance of NOS3 gene
polymorphism in male infertility. Research on NOS3 and male inf ertility is still on few SNPs,
and more SNPs need to be studied to clarify whether there is a relation between their
polymorphisms and male infertility.
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ﺍﻟﻌﻘﻢ ﻋﻨﺪ ﺍﻟﺮﺟﺎﻝ ﻓﻲﺍﻹﻧﺰﻳﻢ ﺍﻟﺒﻄﺎﻧﻰ ﺍﻟﻤﻜﻮﻥ ﻷﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﺍﻟﺘﻌﺪﺩ ﺍﻟﺠﻴﻨﻲ ﻟﺠﻴﻦ ﺩﻭﺭ
ﺍﻟﻤﻠﺨﺺ ﺍﻟﻌﺮﺑﻲ
ﺍﻟﻤﻘﺪﻣﺔ:ﻌﻘﻢ ﻋﻨﺪ ﺍﻟﺬﻛﻮﺭ. ﻗﺪ ﺗﻮﺟﺪ ﺍﻟﺘﺸﻮﻫﺎﺕ ﺍﻟﺠﻴﻨﻴﺔ ﻳﻌﺪ ﺍﺧﺘﻼﻝ ﻭﻅﺎﺋﻒ ﺍﻟﺤﻴﻮﺍﻧﺎﺕ ﺍﻟﻤﻨﻮﻳﺔ ﻫﻮ ﺍﻟﺴﺒﺐ ﺍﻷﻛﺜﺮ ﺷﻴﻮﻋﺎً ﻟﻠ
.ﺃﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﻣﻦ ﺣــﺎﻻﺕ ﻋـﻘــﻢ ﺍﻟﺮﺟﺎﻝ ﺑﻤﺎ ﻓﻲ ﺫﻟﻚ ﺍﻟﺘﺸﻮﻫﺎﺕ ﺍﻟﻜﺮﻭﻣﻮﺳﻮﻣﻴﺔ ﻭﺍﻟﻄﻔﺮﺍﺕ ﺍﻟﺠﻴﻨﻴﺔ ﺍﻟﻮﺍﺣﺪﺓ %٥١ﺍﻟﻤﺨﺘﻠﻔﺔ ﻓﻲ
ﺎﺩﺗﻪ ﺗﺆﺩﻯ ﺇﻟﻰ ﺗﻠﻒ ﻟﻮﻅﺎﺋﻒ ﺍﻟﺤﻴﻮﺍﻧﺎﺕ ﺍﻟﻤﻨﻮﻳﺔ ﺍﻟﻤﺨﺘﻠﻔﺔ ﻛﺎﻟﻘﺪﺭﺓ ﺍﻟﺘﻠﻘﻴﺤﻴﺔ ﻭﺍﻟﺤﺮﻛﺔ ﻭﺍﻟﺸﻜﻞ ﻭﻗﺪﺭﺗﻬﺎ ﻋﻠﻰ ﺍﻟﺒﻘﺎء، ﻛﻤﺎ ﺃﻥ ﺯﻳ ﻣﻬﻢ
ﺍﻟﺤﻤﺾ ﺍﻟﻨﻮﻭﻱ ﻭﺍﻟﻐﺸﺎء ﺍﻟﺒﻼﺯﻣﻲ ﻭﺿﺮﺭ ﺍﻟﺤﻴﻮﺍﻧﺎﺕ ﺍﻟﻤﻨﻮﻳﺔ ﻭﻫﺬﺍ ﻳﺆﺩﻯ ﺍﻟﻰ ﻓﻘﺪﺍﻥ ﺍﻟﻘﺪﺭﺓ ﻋﻠﻰ ﺍﻟﺤﺮﻛﺔ ﻭﻓﻘﺪﺍﻥ ﺍﻟﺨﺼﻮﺑﺔ. ﺍﻟﻬﺪﻑ
ﻣﻦ ﺍﻟﺪﺭﺍﺳﺔ:ﺍﻹﻧﺰﻳﻢ ﺍﻟﺒﻄﺎﻧﻰ ﺍﻟﻤﻜﻮﻥ ﻷﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﻭﺍﻟﻌﻘﻢ ﺍﻟﻬﺪﻑ ﻣﻦ ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻫﻮ ﺍﻟﺒﺤﺚ ﻓﻲ ﻋﻼﻗﺔ ﺍﻟﺘﻌﺪﺩ ﺍﻟﺠﻴﻨﻲ ﻟﺠﻴﻦ
. ﻋﻨﺪ ﺍﻟﺮﺟﺎﻝ ﻁﺮﻳﻘﺔ ﺍﻟﺒﺤﺚ ﺍﻟﻤﻌﻤﻠﻴﺔ: ﺗﻢ ﺇﺟﺮﺍء ﺍﻟﺪﺭﺍﺳﺔ ﻋﻠﻰ ﻣﺠﻤﻮﻋﻪ ﻣﻦ ﺍﻟﺮﺟﺎﻝ ﺍﻟﺬﻳﻦ ﻳﺸﻜﻮﻥ ﻣﻦ ﺗﺄﺧﺮﺍﻻﻧﺠﺎﺏ ﻟﻤﺪﺓ ﻻ ﺗﻘﻞ
ﺷﻬﺮ ﻣﻘﺎﺭﻧﺔ ﺑﻤﺠﻤﻮﻋﻪ ﻣﻦ ﺍﻟﺬﻳﻦ ﻳﺘﻤﺘﻌﻮﻥ ﺑﺨﺼﻮﺑﺔ ﺛﺒﺘﺖ ﺑﻨﺠﺎﺡ ﺍﻟﺤﻤﻞ ﻓﻰ ﻏﻀﻮﻥ ﺍﻟﻌﺎﻣﻴﻦ ﺍﻟﻤﺎﺿﻴﻴﻦ ﻗﺒﻞ ﺑﺪء ﺍﻟﺪﺭﺍﺳﺔ. ٢١ﻋﻦ
ﻭﺳﺒﻌﻮﻥ ﺫﻛﺮﺍ ﻣﻤﻦ ﻳﻌﺎﻧﻮﻥ ﻣﻦ ﺗﺄﺧﺮ ﺍﻻﻧﺠﺎﺏ ﻭﻗﺪ ﺗﻢ ﺗﻘﺴﻴﻤﻬﺎ ﻋﻠﻰ ﺛﻼﺙ ﺗﻢ ﺍﺟﺮﺍء ﻫﺬﻩ ﺍﻟﺪﺭﺍﺳﺔ ﻋﻠﻰ ﻋﻴﻨﺎﺕ ﺩﻡ ﺗﻌﻮﺩ ﻟﺨﻤﺲ
ﻣﻦ ﺍﻟﺬﻛﻮﺭ ﺍﻻﺻﺤﺎء ﻣﻦ ﻧﻔﺲ ﺍﻟﻔﺌﺔ ﺍﻟﻌﻤﺮﻳﺔ ﻛﻤﺠﻤﻮﻋﻪ ﺿﺎﺑﻄﻪ. ﻭﺗﻢ ﺍﺟﺮﺍء ﺗﺤﻠﻴﻞ ﻟﻠﺴﺎﺋﻞ ﺍﻟﻤﻨﻮﻱ ٥٢ﻣﺠﻤﻮﻋﺎﺕ ﻭﻣﻘﺎﺭﻧﺘﻬﺎ ﻣﻊ
ﺍﻟﺠﻴﻨﻲ ﺍﻟﺘﺒﺎﻳﻦ ﺩﺍﺭﺳﺔﻭ ﺭﻳﺒﻮﺯﻯ ﺍﻟﺪﻳﺆﻛﺴﻰ ﺍﻟﻨﻮﻭﻱ ﺍﻟﺤﻤﺾ ﻓﺼﻞﺑﻮﺍﺳﻄﺔ ﺍﻟﺤﺎﺳﻮﺏ ﻭﻛﺬﻟﻚ ﺍﺧﺘﺒﺎﺭ ﻧﺸﺎﻁ ﺍﻻﻛﺮﻭﺯﻳﻦ. ﻛﻤﺎ ﺗﻢ
(ﺍﻟﻤﺘﺴﻠﺴﻞ ﺍﻟﺬﻱ ﻳﻌﻄﻰ ﻧﺘﺎﺋﺠﻪ ﻟﺤﻈﻴﺎ ﺃﺛﻨﺎء ﺍﻟﺘﻔﺎﻋﻞ ﻭﻟﻴﺲ ﺑﻌﺪﻩ ﺍﻟﺒﻠﻤﺮﺓ ﺗﻔﺎﻋﻞ ﺗﻘﻨﻴﺔ ﺑﺎﺳﺘﺨﺪﺍﻡ ﺍﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﺍﻟﺒﻄﺎﻧﻰ ﻟﺠﻴﻦ Real
time PCR) ﻭﺟﺪ ﻫﻨﺎﻙ ﺍﺭﺗﺒﺎﻁ ﺑﻴﻦ ﺍﻟﺘﻐﻴﺮ ﻓﻲ ﺍﻻﻧﻤﺎﻁ ﺍﻟﺠﻴﻨﻴﺔ ﻟﺠﻴﻦ :ﺍﻻﺳﺘﻨﺘﺎﺝ ﺍﻟﺒﻼﺯﻣﺎ ﺍﻟﻤﻨﻮﻳﺔ.ﻓﻲ ﻗﻴﺎﺱ ﺍﻟﻨﺘﺮﻳﺖ . ﻛﻤﺎ ﺗﻢ
ﻭﺟﻮﺩ ﻋﻼﻗﻪ ﺑﻴﻦ ﻣﺴﺘﻮﻯ ﺍﻟﻨﻴﺘﺮﻳﺖ ﻓﻲ ﻣﺠﻤﻮﻋﺎﺕ ﺍﻟﺬﻛﻮﺭ ﺍﻟﺘﻲ ﺗﺸﻜﻮ ﻣﻦ ﺗﺄﺧﺮ ﻣﻊ ﺍﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﺍﻟﺒﻄﺎﻧﻰ ﻭﺍﻟﻌﻘﻢ ﻋﻨﺪ ﺍﻟﺮﺟﺎﻝ
ﺍﻻﻧﺠﺎﺏ ﻭﺍﺭﺗﺒﺎﻁﻬﺎ ﺑﺎﻟﺘﻐﻴﺮ ﻓﻲ ﺍﻻﻧﻤﺎﻁ ﺍﻟﺠﻴﻨﻴﺔ ﻟﺠﻴﻦ ﺍﻛﺴﻴﺪ ﺍﻟﻨﻴﺘﺮﻳﻚ ﺍﻟﺒﻄﺎﻧﻰ ﻓﻰ ﺗﻠﻚ ﺍﻟﺤﺎﻻﺕ.