Department of Biomedical Sciences, School of Bio Sciences and Technology (SBST), VIT University, Vellore, Tamilnadu-
Department of Biomedical Sciences
School of Bio Sciences and Technology (SBST)
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Ramgir Shalaka S,
Association of CYP1A1*2A Polymorphism with Idiopathic
Non-Obstructive Azoospermia in A South Indian Cohort.
Int J Fertil Steril.
Infertility is the inability of a couple to conceive after one and a half years
of unprotected sex. Male infertility, which accounts for almost half of infertility cases, is
considered as a major problem all over the world. The aim of this study was to investigate
the association of CYP1A1 polymorphisms with idiopathic non-obstructive azoospermia
in a South Indian cohort.
Materials and Methods:
An experimental study was conducted with idiopathic nonobstructive
azoospermia. A total of 120 infertile and 80 fertile samples were collected,
and DNA was then extracted from all samples. The CYP1A1*2A polymorphism genotyping
was carried out by polymerase chain reaction (PCR) and restriction fragment length
The genotype distribution of CYP1A1*2A polymorphism showed significant
difference between patients and controls. Moreover, the CC genotype was associated
with decreased risk of idiopathic non-obstructive azoospermia in comparison with the
TT and TC genotypes.
The current experimental study identified that the CT genotype of
CYP1A1*2A polymorphism may contribute to the pathogenesis of male infertility in the
South Indian population.
Infertility is the inability of a couple to conceive
after one and a half years of unprotected
sex. It is one of the major medical problems
where about 10-15% of couples are affected
with infertility of which 50% of these cases are
male-related (1). The literature suggests that
about 15% of male infertility cases are due to
genetics factors (2). Besides genetic and environmental
factors, about 30% of cases of infertility
in men remain poorly understood in terms
of etiology and pathogenesis, and their condition
is thus considered idiopathic (3). A decrease in
sperm count and motility from 38.18 million/
ml and 61.16% in 1993-1994 to 26.61 million/
ml and 47.14% respectively by 2004-2005 was
recorded in a study on the Indian population.
Sperm morphology was 40.51% in 1993-1994
and was decreased to 19.75% by 2004-2005
(4). Ageing or environmental toxicants initiate
DNA strand break in the spermatozoa of affected
males, eventually leading to a mutation
in the embryo (5). Genetic factors can be identified
in male infertility with congenital hypogonadotropic
hypogonadism, congenital absence of
vas deferens and primitive testicular failure (6).
Epidemiological studies have been unequivocal
about the effects of lead (Pb2+) and cadmium
(Cd2+) on hormone concentrations, male fertility
and sperm parameters (7).
CYP1A1 (cytochrome P450, family 1, subfamily
A, polypeptide 1) (8) encodes the CYP1A1 enzyme
that catalyzes the bioactivation of polycyclic
aromatic hydrocarbons (PAHs). In the natural environment,
PAHs are capable of forming DNA adducts
after it has being activated to generate DNA
reactive metabolism. In sperm cells DNA adducts
may be considered as a sign of severe DNA damage
and infertility is thought to be associated with
such damage, which may affect meiotic division
during spermatogenesis (9). The four most important
enzyme families involved in the metabolism of
xenobiotics are the N-acetyltransferase (NAT), cytochrome
P450 (P450), glutathione-S-transferase
(GST) and microsomal epoxide hydrolase (mEH)
enzymes (10). A study on the Chinese population
suggested that a CYP1A1 polymorphism may
contribute to the pathogenesis of male infertility
(11). CYP1A1*2A (T→C substitution at nucleotide
3801 in the 3′-non-coding region; rs464693) is the
most prevalent in the Asian population (12). Increase
in smoking, alcohol consumption and high
exposure to chemicals may lead to infertility. The
study was therefore designed to investigate the association
of the CYP1A1*2A polymorphism with
idiopathic non-obstructive azoospermia and to assess
the impact of the status of life style factors
on the relationship between the polymorphism and
susceptibility to idiopathic non-obstructive azoospermia.
Materials and Methods
In this experimental study, 120 idiopathic azoospermic
men were included but excluding those
with known cases such as Y chromosome microdeletion,
obstructive azoospermia and Klinefelter
syndrome all of which were tested at the Andrology
Department, Stanley Medical College and
Hospitals, Chennai, India. The age of azoospermic
men ranged from 24-38 years and the 80 fertile
healthy subjects (control group) in the same age
range were included in the study. The criterion
for including healthy controls was to have at least
one child without assisted reproductive technologies.
Couples reported with female factors were
excluded from the study. With the help of an experienced
urologist at Stanley Hospital, for each
patient, a detailed case history was obtained and
a clinical examination was carried out. The lifestyle
habit and chemical exposure of the probands
were recorded including smoking habits, alcohol
drinking and exposure to toxic chemicals. Semen
was collected from both infertile and fertile males
after three days of abstinence from sex and semen
volume, sperm count, and motility were recorded.
Blood sample from each participant was collected
by a physician with written consent. The study was
approved by the University Human Ethical Committee
of the VIT University.
DNA was extracted from 2 ml of venous
blood according to lab procedure and stored at
+4°C and then subjected to agarose gel electrophoresis.
Oligonucleotide sequences of the
polymerase chain reaction (PCR) primers were
5ˊ-TAGGAGTCTTGTCTCATGCC-3ˊ, and the
product length was 340 bp. Three μl of DNA was
amplified with initial temperature of 95°C for 5
minutes, 30 cycles of denaturation at 94°C for 45
seconds, annealling at 60°C for 50 seconds and
extension at 72°C for 1 minute, followed by a final
extension at 72°C for 10 minutes in a thermal
cycling machine. The 20 μl PCR mixture contained
10 pmol of each forward and reverse primer,
6 μl of master mix, 9 μl of autoclaved miliq
water and 4 μl of DNA. The PCR products were
separated by gel electrophoresis on a 3% agarose
gel containing ethidium bromide (50 μg/μl) and
were visualized under UV illumination. The results
were analyzed with a gel analysis software
(MEDCARE). 2 μl of amplified PCR products
were then mixed with 7 μl of nuclease free water,
2 μl of 10X buffer and 2 units of Msp1 restriction
enzyme (Eurofins Genomic India pvt Ltd). The
digested fragments were visualized on an agarose
gel as above. When an Msp1 restriction site was
present, the fragment of 340 bp was digested into
two fragments of 140 and 200 bp. Homozygotes
for the ancestral allele lacked the 140 and 200 bp
fragments and had the PCR band of 340 bp while
heterozygous individuals had all the three bands
and homozygotes for the derived allele has the
two smaller bands (9).
Hardy-Weinberg equilibrium deviation was assessed
by using the Chi-Square goodness-of-fit
test. The difference in genotypic distribution was
analyzed using Fisher’s exact test (two-sided). The statistical package used to estimate the odds ratio
and 95% confidence intervals was Graphpad Prism
6.1. A P<0.05 was interpreted as statistically significant.
In total of 120 non-obstructive azoospermic
men, higher number of men with CT genotype
were observed in the 25-30 age group (>30%)
followed TT genotype with (>15%) than any
other groups with other genotypes (Fig .1,) and
this study revealed that 74% of TC, 70% of TT
and 40% of CC genotype men had reduced semen
volume (<1.5 ml) (Table 1,).
Bands corresponding to the 340 bp PCR fragment
were observed, confirming amplification
of this region of CYP1A1. The RFLP analysis of
CYP1A1*2A polymorphism results of the 120
patients (Fig .2,, RFLP results of a few samples),
showed that the genotype counts were 70 heterozygous,
40 homozygous for the ancestral allele
and 10 homozygous for the derived allele. In the
control group the counts were 27, 53 and 1 respectively.
The observed frequency of patients with
homozygous CC was 8.34% of which all were exposed
to smoking, the percentage of homozygous
TT was 33.34% of which 50% were exposed to
smoking and 50% were exposed to chemicals, and
the percentage of heterozygous CT was 58.34% of
which 57.14% were not exposed to any harmful
chemicals and 28.57% were exposed to smoking
and 14.2% were exposed to both alcohol as well
as smoking. In the control group homozygous
CC was 1.25% of which 100% were exposed to
smoking, the percentage of homozygous TT was
65.00% of which 61.53% were exposed to alcohol
and 38.46% were not exposed to any harmful
chemicals, and the percentage of heterozygous CT
was 33.75% of which 25.92% were not exposed
to alcohol and 74.07% were not exposed to any
harmful chemicals (Fig .3,).
The differences in allele frequencies of this
CYP1A1*2A polymorphism between fertile and
infertile men were found to be statistically significant
(P=0.0001). Differences in genotypic was
also observed between infertile and fertile men
(P=0.0001). The semen analysis report showed
the frequencies of TT, CT and CC genotypes in
azoospermic men were found to be 23.33, 43.33
and 3.33% in patients with less than 1.5 ml of reduced
semen volume and 10, 15, 5% in patients
with more than 1.5 ml of reduced semen volume
respectively. In fertile controls with normal semen
volume, we observed 65% TT, 33.75% CT and
1.25% CC genotypes (Table 2,).
Genotype frequencies of the CYP1A1*2A polymorphism among infertile and fertile men (controls) and their association with male infertility
Fertile men (Control) n=80
Infertile men (Patients) n=120
OR (95% CI)
OR; Odds ratio and CI; Confidence interval.
- Age-wise distribution of CYP1A1 polymorphism genotypes
in infertile and fertile men.
- CYP1A1 gene polymorphism was analyzed by polymerase
chain reaction (PCR). Description: Lanes L; Marker, Lanes 1-3;
Heterozygous genotype (CT), Lanes 4-6 and 8; Homozygous wild
(TT), and Lane 7; Homozygous mutant (CC).
- Association of smoke, alcohol and chemical exposure with
the CYP1A1 polymorphism in infertile and fertile men.
CYP1A1 is an important phase I enzyme and
plays a key role in the metabolism of lipophilic xenobiotics.
The enzyme is vitally expressed in male
reproductive organs and its polymorphisms may
be a determinant of individual susceptibility to infertility.
Metabolic activation or inactivation of xenobiotics
is catalyzed by hemethilate enzymes like
CYP1A1, which catalyzes PAHs in the first step
of metabolism. For instance, the process of converting
the carcinogen benzo[a]pyrene (B[a] P)
to its ultimate DNA-binding form is metabolized
by CYP1A1 (13). These metabolites have been
shown to cause small cell lung carcinoma (14),
recurrent pregnancy loss (15), coronary artery disease
and diabetes (16). It is thought that CYP1A1
also plays a vital role in metabolism of endogenous
substrates like steroid hormones through catalyzing
the hydroxylation of 17b-estradiol at the C-2
The genotypic distribution of CYP1A1*2A polymorphism
in the infertile male group deviated
from the Hardy-Weinberg equilibrium. There have
not been any reports describing such an incompatibility
for CYP1A1 polymorphisms in the South Indian
population. To remain in the Hardy-Weinberg
equilibrium, the population must be very large and
must follow random mating. Our study population
is relatively small and consanguineous marriages
are 5% common in this population. The observed
incompatibility may thus be inherent to the studied
population. In the overall analysis, we found that
individuals heterozygous for this polymorphism
had an increased risk. In the subsequent analysis,
we found that patients exposed to smoking, alcohol
or chemicals have an overrepresentation of the
homozygous ancestral genotype TT, leading to
male infertility. The patients with smoking, alcohol
consumption and high exposure to chemicals
may also have an increased risk in heterozygous
type polymorphism leading to male infertility.
It is suggested that in infertility, genetic polymorphisms
of xenobiotic metabolism may play
an important role (20). Based on an Indian study,
the pathogenesis of male infertility was associated
with the CC genotype of the CYP1A1*2A
polymorphism (9). Besides the study on the Indian
population, other studies have shown that being
homozygous for the CYP1A1*2A variant increased
susceptibility to estrogen-related breast cancer in
African-Americans (21). However, a case-control
study on Japanese women showed a decreased risk
with homozygous CYP1A1*2A among breast cancer
patients (22). A study of CYP1A1 in the Chinese
population showed that variants in this gene may contribute to the pathogenesis of male infertility
in the Han population (10). To completely understand
the etiology of idiopathic male infertility, an
understanding of the complex gene-environment
interactions is necessary. This is particularly relevant
for genes such as CYP1A1 which is in direct
contact with environmental toxins. Smoking,
which was reported at a moderately high percentage
in the infertile group of this study, could be an
additional contributory factor in the development
of male infertility by increasing levels of PAH in
the body (23). The study carried out by Abilash et
al. (24) estimated the frequency of Y chromosome
microdeletion in infertile men to explore the effect
of smoking, alcohol drinking, chemical exposure
and cellular chromosomal aberration among 34
azoospermia and 55 oligospermia patients. They
found that the chromosome aberrations per cell in
azoospermia and oligospermia were higher than
that of the control. The percentage of microdeletion
observed in unexposed azoospermia had 15%,
azoospermia smokers 22%, azoospermia smokers
and alcoholics 25%; whereas the unexposed
oligospermia had 7%, oligospermia smokers had
12%, and oligospermia smokers and alcoholics
had 37%. Based on these results, they concluded
that the etiology of male infertility may differ between
ethnicities and smoking, alcohol drinking
and chemical exposure may have deleterious effects
on human fertility (24).
Our study indicates that the CT genotype of
CYP1A1*2A may contribute to the pathogenesis
of idiopathic non obstructive azoospermia. This
result thus suggests that the relationship between
this genetic variation and the vulnerability to the
disease depends on personal habits such as smoking,
alcohol drinking and other environmental
factors such as exposure to chemicals and heavy
metals. Since this study is a preliminary step in
investigating this association, further studies are
needed to identiyfing the underlying mechanism
and to validate our results.
The authors wish to thank all the families who
participated in our study. The authors are grateful
to Dr R. Sudhakaran and his Research Scholar
of Aquatic Biotech Laboratory, Vellore Institute
of Technology University, for helping us to carry
out the molecular study. Finally the authors would
like to thank the VIT University management for
providing all the facilities needed for this project.
There is no conflict of interest in this study.
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