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R E S E A R C H Open Access
COMT polymorphism influences decrease of
ovarian follicles and emerges as a predictive
factor for premature ovarian insufficiency
Emerson Barchi Cordts
1
, Monise Castro Santos
1
, Carla Peluso
1
, Erika Azuma Kayaki
1
, Bianca Bianco
1,2*
,
Caio Parente Barbosa
1
and Denise Maria Christofolini
1
Abstract
Background: Estrogens are important factors in the female reproductive functions and are processed by a number
of enzymes along their metabolic pathway. The COMT gene constitutes a crucial element in estrogen metabolism
and is assumed to be involved in the development of Premature Ovarian Insufficiency (POI). This study aimed to
determine whether the presence of the COMT Val/Met polymorphism (rs4680) is associated to the risk of
developing POI.
Findings: In this case–control study, we evaluated 96 infertile women with POI and 120 fertile women as controls,
after obtaining a detailed history of the disease and follicle-stimulating hormone measurements, besides karyotype
determination and fragile-X premutation syndrome investigation. COMT (Val/Met) genotypes were identified by real
time PCR (genotyping TaqMan assay), and the results were statistically analyzed. A statistically significant difference
was found in the distribution of COMT genotypes (p = 0.003) and alleles (p = 0.015) between the POI patients and
the control group.
Conclusion: We were able to demonstrate a strong association between the COMT Val/Met polymorphism and the
risk of premature ovarian insufficiency in the Brazilian women evaluated. However, further studies in larger
populations are necessary to confirm these findings.
Keywords: COMT, Estrogen metabolism, Infertility, POI, Polymorphism
Background
Premature ovarian failure (POF) is a disorder with a com-
plicated clinical presentation and course that is poorly de-
fined by its name. POF is classically defined as a process
in which the gradual decline of ovarian function results in
failure of folliculogenesis before the age of 40 years, ele-
vated FSH and low estradiol levels [1-3]. However, this
definition does not take into account the longitudinal pro-
gression towards the final menstrual cycle. A scientifically
more accurate term for the disorder is “primary ovarian
insufficiency”(POI), which can be appropriately modified
to describe the state of the ovarian function [4]. Indeed,
the process of ovarian senescence in this condition may
resemble that of natural menopause, which is preceded by
several years by elevated FSH levels and menstrual irregu-
larity [4].
The most common etiologies observed for this condi-
tion are chromosomal abnormalities, fragile X premuta-
tions and autoimmune causes. Once these were ruled out,
we can think that a non-obvious genetic pathway could be
implicated in the disease. Several genes have been identi-
fied as being expressed in the ovary and are postulated to
play a role in ovarian physiology and in maintaining nor-
mal homeostasis in the ovarian cycle. Alterations in these
genes can be associated with the development of POI [5].
A recently demonstrated example of that is that mutations
in estrogen receptors can affect regulatory pathways and
have been reported to be positively associated with the de-
velopment of POI [6-8].
* Correspondence: bianca.bianco@hotmail.com
1
Center of Human Reproduction and Genetics –Faculdade de Medicina do
ABC, Santo André/SP CEP 09060-650, Brazil
2
Faculdade de Medicina do ABC, Avenida Príncipe de Gales, 821, Santo
André/SP CEP: 09060-650, Brasil
© 2014 Cordts et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Cordts et al. Journal of Ovarian Research 2014, 7:47
http://www.ovarianresearch.com/content/7/1/47
It was demonstrated that the gene COMT (catechol-
O-methyltransferase - Gene ID: 1312) is expressed in gran-
ulosa cells [9], where it acts by processing the estrogen
metabolites. The increase of these metabolites can pro-
mote cellular atresia, with consequent anovulation. The
widely studied Val/Met polymorphism (rs4680) that oc-
curs in COMT is characterized by the substitution of val-
ine for methionine at codon 158, and this substitution
results in a less active enzyme form, which in turn pro-
motes an increase of metabolites in patients with the
mutated allele [9].
The main estrogens, estrone (E
1
) and estradiol (E
2
),
can suffer oxidative metabolism at different positions,
catalyzed by various cytochrome P450 isoforms. The oxi-
dation produces A-ring metabolites such as catecholes-
trogens (CEs) by 2-and 4-hydroxylation, and D-ring
metabolites by 16 α-hydroxylation [10]. The metabolism
of CEs and 16 α-OH-estrogens is catalyzed by phase II
metabolic enzymes such as COMT [11], which consti-
tutes a crucial element in estrogen metabolism by regu-
lating the inactivation and elimination of carcinogenic
metabolites by converting them into non-carcinogenic
methoxyestrogens such as 2-methoxyestradiol (2-ME
2
)
and 4-methoxyestradiol (4-ME
2
) [12,13]. 2-ME
2
is sug-
gested to have a potential physiologic role in follicle
homeostasis [5].
Thus, our current hypothesis is based on the fact that
COMT converts CEs to 2-ME
2
, and that polymorphisms
in the COMT gene could interfere in the concentration
of 2ME
2
, contributing to POI. So, the aim of this study
was to determine whether the Val/Met genetic variation
in the steroid hormone metabolism gene COMT is asso-
ciated with Premature Ovarian Insufficiency in Brazilian
women.
Material and methods
Patients
A total of 216 women were selected to participate in
the study. Ninety-six of these women, diagnosed with
POI, were recruited from the Human Reproduction
and Genetics Center of Faculdade de Medicina do ABC
(FMABC), Santo André, SP, Brazil. An important fact
about the clinical history of these women is that all of
them came to the Center with the wish of achieving preg-
nancy. All patients were diagnosed in FMABC hospitals,
based on two confirmed serum FSH level measurements
of > 25 IU/L before the age of 40 years. The serum FSH
levels were measured at two separate time-points within a
period of two months. The control group included 120
healthy women who had gone into physiological meno-
pause around 48 years of age (mean 46.3 ± 1.36 y), had
been fertile, with a normal menstrual history, regular
menses (every 25–35 days), no personal or family history
of premature or early menopause, and no consumption of
oral contraceptives or other hormonal medications at the
time of recruitment.
All women underwent a complete clinical examin-
ation, along with medical and gynecological history in-
cluding the reproductive health of the patient’s mother,
family history, consanguinity, and any other genetic condi-
tion in the family, age of menarche and age of menopause.
In the patient group, the mean age was 35.7 (±5.14) years,
and the mean age at menopause was 31.5 (±6.59) years.
All patients had normal karyotypes, determined by analyz-
ing 40 metaphases, and normal alleles for the FMR1 gene
(implicated in the Fragile-X syndrome). The FMR1 muta-
tion, the main cause of POI, has a high incidence in the
world population. The mean FSH serum value was 75.2
(±40.1) mUI/mL. Patients with a known cause of POI,
such as karyotype abnormalities, oophorectomy, chemo or
radiotherapy were excluded from the study.
Methods
DNA extraction
Peripheral blood was collected from each patient and
control in an EDTA-containing tube. Genomic DNA was
extracted from peripheral blood lymphocytes, using the
salting-out procedures described by Lahiri & Nurnberger
[14]. Clinical data and peripheral blood samples were only
collected after explaining the objectives of the study and
obtaining a signed informed consent form, as approved
by the Faculdade de Medicina do ABC ethics committee
(process no. 184/2007).
COMT genotyping
Detection of the COMT Val/Met (G/Val; A/Met) poly-
morphism was performed using TaqMan PCR. TaqMan
primers and probes are commercially available and were
purchased from Applied Biosystems® (Foster City, CA,
EUA) (rs4680, C__25746809_50). The assays were per-
formed with TaqMan Universal Master Mix, using 50 ng
of DNA per reaction. The PCR conditions were as recom-
mended by the manufacturer: 40 denaturation cycles at
95°C (15 sec), and annealing/extension at 60°C (1 min).
The reactions were performed on a Rotor gene 6000 real-
time PCR platform (Corbett, Mortlake, New South Wales,
Australia).
Statistical analysis
Thechi-squareandthelogisticbinaryregressiontests
were performed to compare the genotype frequencies
(rare homozygous, heterozygous and common homo-
zygous), odds ratios (OR), and confidence intervals (CI).
The analyses were made using the SPSS 18.0 (SPSS, Inc.,
Chicago, IL, USA) program. All p-values were two-tailed,
and 95% confidence intervals (CIs) were calculated. A
p-value < 0.05 was considered statistically significant.
Cordts et al. Journal of Ovarian Research 2014, 7:47 Page 2 of 4
http://www.ovarianresearch.com/content/7/1/47
Results
We found a differential distribution of genotypes and al-
leles between the studied groups. The GG (Val/Val), GA
(Val/Met) and AA (Met/Met) genotype frequencies of
the COMT polymorphism in the POI group were 9.4%
(9/96), 52.1% (50/960) and 38.5% (37/96), respectively, while
in the control group they were 27.5% (33/120), 39.1%
(47/120) and 33.3% (40/120) (p = 0.003), respectively.
According to the allelic distribution, allele G and A were
present, respectively, in 35.4% and 64.5% of the POI
group, and in 47.0% and 52.9%, respectively, of the
control group, showing a positive statistical correlation
between POI and allele A (p = 0.015, OR = 1.62, 95% CI =
1.10-2.39) (Table 1). Both the POI and the control group
were in Hardy-Weinberg equilibrium.
Discussion
The enzyme COMT is ubiquitously found in various
mammalian tissues, with high levels in the liver, kidney,
endometrium, breast and granulosa cells [15,16]. A com-
mon polymorphism, G/A in codon 158 of COMT, leads
to a substitution of a valine for a methionine in the gene
product, which is linked to low COMT activity, with an
up to 4-fold decrease in activity in red blood cells and
liver [16]. It has been hypothesized that reduced COMT
activity may increase the risk of hormone-dependent
diseases by enhancing the serum and tissue levels of es-
tradiol, as well as by the accumulation of catecholestro-
gens and the subsequent oxidative DNA damage [17].
Several association studies in POI candidate genes
have been widely used in the search for susceptibility al-
leles, but only few definitive associations have been
established. These inconsistencies in results probably re-
flect an actual variation in the underlying association be-
tween populations studied and the low penetrance of
mutations in these multigenic pathways [18]. In the present
study, the patient group was homogeneous and especially
selected by the presence of infertility, defined according to
the minimum propedeutics of the infertile couple. In
addition, all patients were investigated as to other causes
of POI, such as chromosome aberrations, and positive
findings were used as exclusion criteria.
To our best knowledge, this is the first study that cor-
relates COMT polymorphism and premature ovarian in-
sufficiency. Here, we found a positive association of allele
A of the studied polymorphism with POI, reflecting on
the genotype, as we observed a high frequency of hetero-
zygous and mutated homozygous (p= 0.003) and allelic
distribution (p = 0.015). The allele acts in a dominant
mode. We suppose that it may produce increased damage
to ovarian cells, leading to POI.
The estrogen oxidative DNA damage effect has pre-
viously been shown to affect both female and male
reproduction [19,20]. The risk of oxidative damage and
lipid peroxidation was found to be especially high in
steroid-synthesizing tissues, because, in addition to oxida-
tive phosphorylation, they use molecular oxygen. Indeed,
it has been shown that free radicals inhibit steroidogenesis
by interfering with cholesterol transport to the mitochon-
dria and/or the catalytic function of P450 enzymes, which
leads to an increase of lipid peroxidation and to the
decline of the antioxidant barrier. Considering males,
all these changes can alter the testicular cells, including
spermatozoa and, therefore, the sperm production, leading
to an alteration in male fertility [21]. In females, it can de-
termine cellular apoptosis, corpus luteum regression and
follicular atresia [19,22].
Salih et al. [5] proposed that the metabolite 2-ME
2
has
a potential physiological role in follicle homeostasis:
under normal conditions, its level is low in early follicu-
logenesis, rising (probably to the inhibitory range) with
the augmented E production in the fully developed dom-
inant follicle. Modulation of COMT activity and, there-
fore, the concentration of 2-ME
2
might be part of the
ovarian physiological apparatus. Thus, a pathologic alter-
ation of COMT could lead to a major perturbation of
folliculogenesis.
In PCOS (Polycystic Ovary Syndrome), catechol O-
methyltransferase overexpression and increased levels of
2-ME
2
in ovarian granulosa cells represent a mechanism
leading to abnormalities of steroidogenesis, follicular ar-
rest, and anovulation [5]. We propose that the opposite
occurs in POI. Here, we found that most of the POI pa-
tients presented an increased incidence of the low-
activity COMT allele, promoting decreased formation of
2-ME
2.
As suggested by Salih et al. [5], we believe that dis-
turbed levels of 2-ME
2
could be associated with increased
follicular depletion, eventually leading to follicular ar-
rest. This disturbed pathway seems to be an important
Table 1 Genotype and allele frequencies of the COMT Val/Met polymorphism in POI patients and a control group
Population
studied
nCOMT G/A genotypes COMT Alleles Dominant model p
GG (%) GA (%) AA (%) p G (%) A (%) p OR (95% CI) GG GA + AA
POF patients* 96 9 (9.4) 50 (52.1) 37 (38.5) 0.003 68 (35.4) 113 (58.8) 0.015 1.62 (1.10 –2.39) 9 87 0.0008
Control group* 120 33 (27.5) 47 (39.2) 40 (33.3) 113 (47.1) 127 (52.9) 33 87
*Sample groups are in Hardy Weinberg equilibrium.
Cordts et al. Journal of Ovarian Research 2014, 7:47 Page 3 of 4
http://www.ovarianresearch.com/content/7/1/47
mechanism associated with Premature Ovarian Insuffi-
ciency in Brazilian women. However, further studies in lar-
ger sample sets are needed to confirm these findings.
Competing interests
None of the authors has any conflict of interest to disclose.
Authors’contribution
EBC participated in patients’evaluation and selection and study design; MS,
CP and EAK participated in samples preparation, molecular genetics studies
and statistical analysis; BB and CPB participated in manuscript design and
DMC participated in the conception of the idea, supervision of the research
and manuscript design. All authors read and approved the final manuscript.
Acknowledgments
The authors would like to thank CNPq for Research grant provided to the
study ( 470333/2013-8), for student scholarship granted to Erika Azuma
Kaiaky (PIBIC), and productivity grant provided to Denise Maria Christofolini
(301242/2013-5), Bianca Bianco (300825/2013-7) and Caio Parente Barbosa
(300816/2012-0).
Received: 11 February 2014 Accepted: 21 April 2014
Published: 2 May 2014
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Cite this article as: Cordts et al.:COMT polymorphism influences
decrease of ovarian follicles and emerges as a predictive factor for
premature ovarian insufficiency. Journal of Ovarian Research 2014 7:47.
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