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Journal of Gynecology
Volume 1| Issue 4
Review Article Open Access
Analysis of Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms (C677t & A1298c) in
Recurrent Pregnancy Loss (RPL)
Balasubramaniam, A. 1 *, Kotalawala, S. 2 and Amarasekara, R.3
1, 2, 3 BMS School of Science, Galle Road, Colombo 6, Sri Lanka
*Corresponding author: Balasubramaniam, A., BMS School of Science, Galle Road, Colombo 6, Sri Lanka;
Email: [email protected]
Citation: Balasubramaniam, A. (2017) Analysis of Methylenetetrahydrofolate Reductase (MTHFR) Polymorphisms
(C677t & A1298c) in Recurrent Pregnancy Loss (RPL): Nessa J Gynecology
Copyright: © 2017 Balasubramaniam, A., Kotalawala, S. and Amarasekara, R. et al. This is an open-access article
distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution,
and reproduction in any medium, provided the original author and source are credited.
Abstract
Introduction and Objectives
Recurrent Pregnancy Loss (RPL) is a multifactorial disorder responsible for 15 % of pregnancy failures. The
underlying etiology of RPL remains unknown and literature suggests that MTHFR polymorphisms to be the primary
genetic risk factors for RPL. Accordingly aim of this study was to find out the frequencies of MTHFR C677T and
A1298C MTHFR polymorphisms associated with RPL.
Method: From the eligible studies 17,780 cases and 22,346 controls were considered as study subjects.
Results: A positive MTHFR polymorphisms associated with RPL were seen in 75 % (26/35) of the study (p = 0.002).
Among the positive studies, 35 % had both C677T and A1298C polymorphisms. C677T polymorphism was the only
polymorphism associated with RPL in 50 % of cases, while 15 % found A1298C polymorphism associated with RPL.
Analysis also showed the prevalence of MTHFR-C677T polymorphism in the partners of women suffered from RPL.
Conclusion: The study confirms that the MTHFR C677T polymorphism is significantly associated with RPL (p =
0.043). Accordingly the C677T polymorphism could be used as a genetic marker for early diagnosis of RPL. Study
further suggests paternal screening is equally important as maternal screening in the early diagnosis of RPL.
Keywords: RPL, Recurrent Pregnancy Loss, Spontaneous Abortion, Methylenetetrahydrofolate reductase, MTHFR,
MTHFR Polymorphisms.
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Journal of Gynecology Volume 1| Issue 4
1. Introduction
1.1 Recurrent Pregnancy Loss (RPL), etiology, prevalence
Recurrent Pregnancy Loss (RPL) or spontaneous pregnancy loss is a multifactorial distressing disease that leads to 15 %
of pregnancy failures (Chen et al., 2015). “WHO defined miscarriage as loss of the fetus or embryo weighting less than
500 g which would normally be at 20-22 complete weeks of gestation” (Vanilla et al., 2015). Many scientific studies
reveal that 1 % to 2% of the female population suffer from RPL (Tiwari et al., 2015). The etiology underlying RPL is
still unknown and pathogenesis is complicated. Several reliable published data state that the risk of miscarriages could
be due to subsequent pregnancies and accounts up to 30 % following 2 losses and 33% following 3 losses without a
history of live birth (Zonouziet et al., 2012). RPL is thought to be a multifactorial complex disease with the involvement
of genetic, immunological, environmental, endocrine, and physio-pathological factors (Yousefian et al., 2014; Cao et al.,
2013).
Maintaining a normal pregnancy requires a series of differential gene expressions. Number of gene mutations are
suggested to be reasons for RPL, few of which are: the MTHFR gene, the CYP Family genes, and the Factor V Leiden
(FVL) mutation (Park and Chang, 2014; Nair et al., 2013). Finding abnormal expression of these genes may outline
general health during pregnancy (Chaithra et al., 2011).
Numerous investigations have revealed the possible association of MTHFR polymorphisms with recurrent spontaneous
miscarriages and reproductive failures (Nan and Li, 2015; Vanilla et al., 2015; Yalcintepe et al., 2014; Ocak and Ozyurt,
2013; Isotalo, et al., 2000).
In addition, in the year 2000, Isotalo and his colleagues stated the existence of human MTHFR gene mutation has its
potential role in compromised fetal viability. Almost 30 % of women with unexplained pregnancy loss showed MTHFR
polymorphisms (Cao et al., 2014).
1.2 MTHFR Gene polymorphisms in RPL
Folate pathway and progesterone pathway are the two most critical pathways contributing to a successful pregnancy
(Tiwari et al., 2015). Folate plays a key role in biosynthetic processes. Folate dependent mechanisms have shown to be
vital for the fetal growth and development, and also for the maternal well-being (D’Elia et al., 2014; Puri et al., 2013).
Therefore, any folate mechanism deficiency can influence various pregnancy complications.
Folate mechanism is regulated by the enzyme called 5-Methylenetetrahydrofolate reductase (MTHFR). The MTHFR
gene is located at position 36.3 on the short (p) arm of chromosome 1 (Figure 1) with 11 exons and the cDNA is 2220 in
length (Nan and Li, 2015). The best characterized single nucleotide polymorphisms (SNPs) are at the mRNA positions
677 and 1298 (Ren and Wang, 2006).
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Journal of Gynecology Volume 1| Issue 4
Figure 1: Molecular location of MTHFR Gene (Nan and Li, 2015)
MTHFR catalyzes the conversion of 5, 10-Methyleneterahydrofolate to 5-methylenetetrahydrofolate, the circulatory form
of folate. Mutations in the MTHFR gene lead to hyperhomocysteinemia and decreased activity of the enzyme (Aarabi et
al., 2015; Yousefian et al., 2014). Since several mutations were described in MTHFR gene, C677T and A1298C
polymorphisms are the two most common mutations involved in RPL (Cao et al., 2014). Table 1 describes the
characteristic features of these polymorphisms.
Table 1: Comparison of C677T and A1298C polymorphisms in MTHFR gene
C677T Polymorphism A1298C polymorphism
1. 70 % of the MTHFR mutation - 35 % of MTHFR mutation
2. Missense mutation - Point mutation
3. Occurs in the exon 4 of the gene - Occurs in the exon 7 of the gene
4. Converts an alanine to a valine codon - Glutamate to alanine substitution
5. Mutation at codon 222 in the N-terminal
catalytic domain of the protein
- At codon 429 within the C-terminal
regulatory domain of the protein
6. Mutation leads to thermolabile protein - No thermolability changes
7. Decreases the enzymatic activity – 50 to
70 % loss of activity in homozygotes
(677TT) and 30 % of loss in
heterozygotes (677CT).
- Decreases the enzymatic activity
C677T and A1298C polymorphisms decrease the conversion of homocysteine into methionine, hence homocysteine
accumulates in blood and results in hyperhomocysteinemia (HHcy) (Wiwanitkit, 2005). Hyperhomocysteinemia may
further influence common pregnancy outcomes, preterm delivery or intrauterine growth restriction, vasculopathy in the
placental vasculature which can lead to RPL (Hubacek et al., 2015; Li et al., 2015). Hyperhomocysteinemia may also
damage the coagulation system and influencing uteroplacental circulation selectively, which is closely related to the
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Journal of Gynecology Volume 1| Issue 4
occurrence of premature delivery (Parveen et al., 2013). MTHFR polymorphisms are also associated with fetal loss at
early stage of pregnancy (Varga et al., 2005).
However the role of MTHFR polymorphisms in RPL is still controversial based on the varying results that have been
obtained over a span of many years. Most of the studies are associated MTHFR with RPL while others have revealed no
association. Thus, this literature review is conducted to study the frequency of MTHFR polymorphisms in
hyperhomocysteinemia leading to RPL, and further examine respect to it.
2. Materials and Methodology
2.1 Eligible Studies
37 published reports were identified among which 2 that discussed about MTHFR mutation and non-pregnancy
complications were excluded. Hence, 35 reports were selected for this review. 29 case control studies, 4 meta-analysis
and one nested case control study and one case control study plus meta-analysis were taken into account for this study.
Further, 30 articles were on URPL, 3 on Hcy level and treatment, and one each on pre-matured delivery and pre-
eclampsia complications. All the URPL studies included women with cases of a minimum of two or more early fetal
losses. Studies were carried out with focus on worldwide, as elaborated in the table (Table 2) below. Table 2: Summarize
data of eligible studies
Country Number of Studies Year
1. China 07 2015, 2015, 2015, 2014, 2012,
2013, 2007
2. India 06 2015, 2015, 2013, 2013, 2013,
2012
3. Iran 04 2014, 2012, 2012, 2010
4. Turkey 04 2015, 2014, 2013, 2012,
5. Egypt 02 2012, 2011
6. Brazil 02 2015, 2014
7. Korea 02 2014, 2011
8. Italy 02 2008
9. Avicenna 01 2006
10. Slovenia 01 2015
11. Tunisia 01 2006
12. Mexico 01 2009
13. Canada 01 2000
14. USA 01 2007
15. Unknown 01 2005
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Journal of Gynecology Volume 1| Issue 4
Although the search was restricted to the last 10 years, one article from 2000 was used for the analysis as it was the first
ever evidence about ‘MTHFR polymorphisms and RPL’.
2.3 Data Extraction
From each study, year, authors, journal name, place of study, sample size and source, methods and detections, mutation
found, and mutation type and analysis information were extracted. Therefore from the eligible studies, 17,780 samples as
cases and 22,346 controls were taken into the analysis.
Year Aut
hor
Journ
al
Plac
e of
stud
y
Sample
Size
Type of
Sample
Sour
ce
Method
s &
Detectio
n
Mutatio
n
Found
Mutation Type and Analysis
2013
Cao
et
al.,
Gene
514
(2013)
105-11
China
46
reports
3559
URPL
cases,
5097
healthy
controls
Maternal
Meta-
analysis
MTHFR
C677T
# 68 % TT verus Total genotypes
# 34 % T verus total alleles
# East Asians people are higher
risk of URPL with 677TT
genotype.
# At a higher level TT and CT
genotypes influence the overall
association.
# 1298CC mutation was 9% in
both samples
2015
Tiwa
ri et
al.,
Meta
Gene
3
(2015)
31-42
Nort
h
East
Indi
a
209 PTD
cases
403
controls
Pregnant
women
18 - 45
years
whol
e
Bloo
d
PCR-
RFLP
analysis
of
MTHFR
polymor
phism
MTHFR
# MTHFR found in 198 bp band
# Preterm delivery was
significantly at a higher
distribution.
# significantly associated with
extremely
as well as moderately preterm
delivery
# For more than 3 folds, MTHFR
significantly correlates with
negative pregnancy outcome.
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2015
Vani
lla et
al.,
Bioche
mistry
section
9(2),
BC15-
BC18
Kola
r
Distr
ict -
Sout
h
Wes
t
Indi
a
15
couples
from each
control
and
sample
Blood
sample
from
male and
female
Whol
e
Bloo
d
PCR-
RFLP
Based
Screenin
g
MTHFR
C677T
# In RSA couple,
CC ( 80% M & 86.6 % F)
CT ( 13.3 M & 13.3 F)
TT ( 6.67 M & 0 Female)
# In healthy couples there is no
significant difference observed
with homozygous wild type CC,
CT, TT.
# In RSA cases of male partners
are at a higher frequency of
getting the mutant allele.
2012
Zono
uzi
et
al.,
Obstet
rics
and
Gynec
ology
Nort
hwe
st of
Iran
139
women
( 89 RM,
50 C)
Maternal
Bood
leuko
cyte
PCR-
RFLP
Based
Screenin
g
ARMS-
PCR
Based
Screenin
g
MTHFR
C677T
MTHFR
A1298C
# 677 C/T - 33.7 % (P)
# 677 C/T - 44 % ©
# 677 T/T - 6.74 % (P)
# 677 T/T - 2% ©
# In both groups there is no
statistical significant difference in
the frequency of A1298CC
2013
Ocak
and
Ozy
urt
Africa
n
Health
Scienc
es
13 (2)
447-
452
Turk
ey
495
couples
Maternal
and
paternal
Bloo
d
(leuk
ocyte
)
Karyoty
ping
PCR-
RFLP
Screenin
g
MTHFR
C677T
# MTHFR polymorphism
(67.9%) is due to frequent
abnormality for heterozygote
mutation.
2015
Nan,
and
Li,
Int J
Clin
Exp
Pathol
8(6),
7397-
7402)
Chin
a
108
premature
delivery
pregnant
women
and
108
control
Maternal
Veno
us
blood
PCR-
RFLP
MTHFR
C677T
MTHFR
A1298C
#Than in controls TT allele
C677T is higher in cases.
# There is a lower distribution CC
allele A1298C in cases than
controls.
#Premature delivery risk could be
due to TT of MTHFR C677T.
# Premature delivery protection is
influenced by CC of A1298.
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2006
Coul
am
et
al.,
Am J
Repro
ud
immun
ol
55(5),
360-8
150 RPL
women
20 fertile
controls
Maternal
Bucc
al
swab
s
PCR-
RFLP
10 gene
mutatio
ns
were
found
# In the frequency of the specific
gene there is no difference.
#homozygous mutation with RM
was significantly higher than
normal
# 59 % found in RM and 10 % in
control
2015
Chen
et
al.,
Arch
Gynec
ol
Obstet
Chin
a
16 articles
1420 RPL
cases
1408
controls
Maternal
Meta-
analy
sis
Meta-
analysis
MTHFR
C677T
MTHFR
A1298C
# C677T was significantly
associated with RPL risk under
dominant
( TT + CC)
# Sensitivity analysis excluding
studied that deviated from HWE
did not change the direction of
effect
#A1298C - no significant
association was found
2005
Wiw
anitk
it, V
Clin
Appl
Throm
b
Hemos
t
11(3),
pp.343
-5
8 case
control
studies
752
patients
625
controls
Maternal
Meta-
analy
sis
Meta-
analysis
MTHFR
C677T
# 53% of subjects with T allele
have RPL while 55 % of subjects
without T allele have RPL
# Ethnicity of the study do not
correlate with pattern of MTHFR
C677T.
2015
Hub
acek,
J.A
et
al.,
Clin
Chim
Acta
440
p.104-
7.
Slav
onia
464
samples
486
controls
aborted
samples
Tissu
e
PCR-
RFLP
MTHFR
C677T
MTHFR
A1298C
# Carriers with dominant allele of
these
polymorphisms were associated
with
higher risk of abortion.
# Distinct combination of
MTHFR could be associated with
higher risk of abortions in
Caucasians.
2015 Li et
al.,
Gynec
ol
Obstet
Invest
79(2),
pp.107
-112
Chin
a
7,812
women Maternal
2,928
pregnant
women
were
tested
with
Taqman-
MGB
for
genotyp
e
4,884
women
MTHFR
C677T
MTHFR
A1298C
#MTHFR C677T were 49.1 %
and 50.9 %
#MTHFR A1298C were 80.2 %
and 19.8 %
#After folic acid supplement, the
complication rate in different age
groups were significantly reduced
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without
folic
acid
supplem
entation
(control)
2013
Parv
een
et
al.,
Arch
Gynec
ol
Obstet
,
288(5)
, 1171-
7
Nort
h
Indi
a
200
patients
300
controls
Maternal
Perip
heral
blood
sampl
es
PCR-
RFLP
MTHFR
MTHFD
PAI-1
# MTHFR C677T, A1298C,
MTHFD G1958A variant alleles
were found to be significantly
more prevalent than control
# MTHFR C677T is only twofold
and further decreased to only one
fold, and MTHFD-1958 lost its
significance upon meta-analysis
2012
Idali
et
al.,
Am J
Repro
ud
immun
ol
68(5),
400-7
Iran
177 RPL
women
100
control
Maternal
Perip
heral
blood
sampl
es
PCR-
RFLP
MTHFR
A1298C
PAI-1
4G/5G
#MTHFR A1298C and PAI-1
4G/5G
mutations in Iranian Women
suffering
from RPL with and without
PCOS
2012
Nair
et
al.,
Repro
d Sci,
19(2),
210-5
Nort
h
Indi
a
Case
control
Study
106 RPL,
140
control
Maternal Bloo
d
PCR-
RFLP
and
sequenci
ng
MTHFR
C677T
# statically significant association
was
found in RPL and the mutant T
allele
# homo and heterozygosity for
MTHFR
C677T polymorphism confer
increased risk of idiopathic REPL
2015
Boas
et
al.,
Revist
a
Brasile
ira de
Ginec
ologia
e
Obstet
rícia,
37(2),
Braz
il
Case
control
Study
89 RPL
women
150
healthy
women
Maternal Bloo
d
Serum
Folate
and B12
- ELISA
PCR-
RFLP
for DNA
analysis
MTHFR
C677T
MTHFR
A1298C
# No statistical difference
between in the frequency of the
genotypes between the studied
groups
#In study population
66.3% of presented with
C677T polymorphism
64 % presented with A1298C
polymorphism
# In control population
65% of presented with C677T
polymorphism
53.4 % f presented with
C677T polymorphism
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2007
Ren
and
Wan
g
Fertilit
y and
Sterilit
y
86(6)
Chin
a
Meta-
analysis
pregnant
women
18 - 45
years
2120
URPL
cases
2949
Healthy
controls
Meta-
analysis
MTHFR
C677T
#Certain scientific studies
revealed out a strong correlation
between C677T and URPL only
in Chinese studies.
#Except in Chinese population,
MTHFR mutation is not genetic
risk factor.
2011
Park
et
al.,
Clinic
al
Experi
mental
Repro
ductiv
e
Medici
ne,
38(3)
Kore
a
39
patients
with
RM
50 control
women
Maternal
Veno
us
blood
#
Biochem
istry
measure
ment
#
MTHFR
genotypi
ng with
PCR
machine
# Flow
cytometr
ic
analysis
of pbNK
cells
MTHFR
C677T
# Homocysteine levels was
elevated in individual with TT
variant than those with CC and
CT variants in RM group.
# Between two groups' mean
plasma homocysteine level there
was no difference observed.
# Therefore it is very hard to use
plasma homocysteine as the
predictive marker of RM
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2000
Isota
lo et
al.,
The
Ameri
can
Journa
l of
Huma
n
Geneti
cs,
67(1),
Can
ada
119
samples
161
controls
119
neonatal
cord
blood
161 fetal
tissue
Neon
atal
PCR-
RFLP
#
MTHFR
677CC
677CT
677TT
#
MTHFR
1298AA
1298AC
1298CC
# First article described about
MTHFR mutation
# There is an evidence, fetal
viability compromization is due
to combination of MTHFR
C677T and A1298C mutation.
# In neonatal group, the combined
677CT / 1298CCand 677TT /
1298CC genotype with three or
four mutant alleles were not
observed
2008
D'Uv
a et
al.,
Biolog
ics:
Target
s &
Therap
y
Italy
115
UPRL
women
75
healthy
age-
matched
controls
Maternal
1st
and
2nd
blood
sampl
e
PCR
amplific
ation
with
specific
primer
and the
light
cycler
apparatu
s
MTHFR
C677T
FVL
PTHRA
20210G
among the study group 30% were
presented with MTHFR C677T
homozygosity (highest)
2014
D'Eli
a et
al.,
Repro
ductiv
e Bio
Medici
ne
Online
28(6):
733-8
Braz
il
82
women
1432 IVF
cycles
were
performe
d
maternal
and
paternal
Sper
m,
Bloo
d
Sperm
Count
Ovarian
Stimulat
ion
Genotyp
e
determin
ation by
Taqman
SNP
genotypi
MTHFR
C677T
MTHFR
A1298C
#MTHFRpolymorphism was
observed with a significant
decrease in oocytes maturity.
# Elevated homocysteine in
plasma which occurs individually
seems is not due to the presence
of MTHFR A1298C both in CC
and AC.
# When compared to homozygous
TT and CC genotypes, patients
with heterozygous CT genotypes
has high percentage of better
quality embryos and increase
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ng chance of pregnancy.
2014
Yous
efian
et
al.,
Iranian
Red
Cresce
nt
Medic
al
Journa
l,
16(7),
Iran
204 RPL
women
116
controls
Maternal Bloo
d
Reverse
hybridiz
ation
method
MTHFR
C677T
MTHFR
A1298C
# The prevalence of 677TT
patient - 8.8%
control- 8.6%
# The prevalence of 1298CC
Patient - 12.3%
control- 8%
#Between RPL patients and
healthy control subjects, MTHFR
C677T and A1298C did not
indicate a significant differences.
2014
Park
and
Chan
g
Vascul
ar
Specia
list
Interna
tional,
30(4),
Kore
a
146
samples
Arterial
and
venous
thrompisi
s
Real-
time
PCR for
screenin
g of
MTHFR
C677T
Fluoresc
ence
polarizat
ion
assay for
plasma
Hcy
level
measure
ment
MTHFR
C677T
# With high level of Hcy,
MTHFR C677 TT genotype gets
interacted.
2006
Mitr
aoui
et
al.,
Repro
ductio
n, 131,
Tuni
sia
200 RPL
patients
200 age
matched
controls
Maternal Bloo
d
PCR-
RFLP
Fasting
Hcy
level
measure
d by
ELISA
MTHFR
C677T
MTHFR
A1298C
# It was similar among patients
and controls higher Hcy levels
# In patients, there is a significant
higher frequency of MTHFR
677T/T and 1298C/C genotypes.
# In late and late-early RPL, there
is a higher prevalence of MTHFR
677T/T
# Higher prevalence of 1298C/C
was seen only in early-late RPL
# Between controls and patients
there is no statistical significant in
heterozygosity.
#Irrespectively for elevated total
Hcy level and risk factors of
RPLs is due to homozygosity of
MTHFR C677T/T and A1298C/C
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2007
Alto
mare
et
al.,
Throm
bosis
Journa
l,
5(17),
USA
3 of
whom
were
actively
pregnant
(were
referred
with
history of
RPL)
Bloo
d
MTHFR
C677T
#Relationship between recurrent
fetal loss and C677Tmutation an
evident support must be provided.
# To achieve a full term
pregnancy and reduce risk of
recurrent abortion in future, the
hopes of therapeutic intervention
were suggested by lower dose of
lower molecular weight (LMW)
of heparin.
2013
Puri
et
al.,
Journa
l of
Prenat
al
Medici
ne,
1(41)
Nort
h-
Indi
a
107
URPL
women
343
controls
Maternal Bloo
d
PCR-
RFLP
MTHFR
C677T
# Within case and control, there is
no significant difference in
MTHFR genotypic distribution.
# In case group there is a C677T
found in increased Hcy
# T allele is detrimental with B12
deficiency
2013
Nair
et
al.,
Fertilit
y and
Sterilit
y,
99(5)
Indi
a
129 RPL
Patients
220
controls
+
For meta-
analysis
1080
cases and
709
controls
Maternal,
aborted
embryos
Bloo
d
PCR-
RFLP
MTHFR
A1298C
# Genetic risk factor for RPL is
MTHFR A1298C polymorphism.
# Increased risk of RPL could be
due to presence of rare allele "C"
and heterozygous and rare
homozygous genotypes.
# increased risk of carriers of AC
and CC genotypes showed due to
meta-analysis of both RPL and in
spontaneously aborted embryos.
2012
Wu
et
al.,
Geneti
c
Testin
g and
Molec
ular
Bioma
rkers,
16(7)
Chin
a
2427
cases and
3118
controls
Meta-
analysis
Meta-
analysis
MTHFR
C677T
# Except Caucasian, the increased
risk of RPL is associated with
idea of MTHFR C677T.
#Increase risk of RPL had
homozygous TT allele
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2012
Ozde
mir
et
al.,
Geneti
c
Testin
g and
Molec
ular
Bioma
rkers,
16(4)
Turk
ey
543 RPL
women
and 327
male
partners
(870
individual
s with
RPL)
and 106
fertile
couples as
control
group
Maternal
and
paternal
Bloo
d
Real-
Time
PCR
Reverse
Hybridiz
ation
method
#FVL
#Factor
V
H1299R
#Factor
II
prothro
mbin
G20210
A
#PAI-1
#GPIIIa
L33P
#MTHF
R
C677T
#MTHF
R
A1298C
#ACE
I/D
#Apo B
R3500Q
#Apo E
Genes
# RPL is associated with
heterozygous or homozygous
point mutation in FVL, FVR2,
ApoE2, PAI-1, MTHFR C677T,
MTHFR A1298C and ACE
genes.
# In women, homozygosity of
MTHFR C677T and 4G in PAI-1
gene play a critical role in RPL
also considered as a risk factor.
2012
Ibrah
im et
al.,
Middl
e East
Fertilit
y
Societ
y
Journa
l, 17
Egy
pt
44 pre-
eclamptic
women
44 normal
pregnanci
es women
Maternal Bloo
d
SSP-
PCR
MTHFR
C677T
#TT allele frequency is
significantly high in study group.
# In Egyptian pregnant women,
the development of pre-eclampsia
is due to TT genotypes risk
factor.
2010
Jeddi
-
Tehr
ani
et
al.,
Ameri
can
Journa
l of
Repro
ductio
n
Immu
nology
,
2011,
Iran
100 RPL
women
and
100
healthy
women
Maternal Bloo
d
PCR-
RFLP
PAI-I
BF
ITGB3
MTHFR
C677T
MTHFR
A1298C
# BF, MTHFR C677T, MTHFR
A1298C were found positive
# RPL risk increases due to
presence of both mutations of
MTHFR gene.
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2011
Setti
n et
al.,
Geneti
c
Testin
g and
Molec
ular
Bioma
rkers’,
15(12)
Egy
pt
70 RPL
cases
136
controls
Maternal Bloo
d
PCR-
RFLP
MTHFR
C677
TT
MTHFR
1298
CC
677T/12
98C
# Higher frequency of
homozygous mutant MTHFR
677TT, 1298CC genotypes are
showed in URPL.
# Haplotype 677T/1298C does
not reach statistical significance.
# For URPL mutation related to
MTHFR gene is not only the risk
factor but there are genetic
varient interactions might aid to
be as a risk factor.
2009
Rodr
igue
z-
Guill
en et
al.,
Salud
Public
a Mex,
51(1),
Mex
ico
Nested
case
control-
study
23 SA
women
and
74
controls
women
Maternal Bloo
d
# PCR-
RFLP
# Serum
homocys
teine by
HPLC
MTHFR
C677TT
MTHFR
A1298C
# Increased risk of SA is shown
by carriers of MTHFR C677TT
and A1298C genotypes
respectively.
# Risk of SA could be due to
smoking and paternal occupation
# Non-significant protection of
SA is shown by maternal of
young age and higher education.
# Than heterozygous,
homozygous alleles carried a high
risk factor of SA.
2014
Cao
et
al.,
Genes
Nutriti
on, 9,
Chin
a
82 RPL
women
166
Healthy
Women
Maternal
perip
heral
blood
#
Sequeno
m
MassArr
ay
# PCR
MTHFR
A1298C
MTHFR
677C
MTRR
SLC19
A1
# A significant association
between A1298C and URPL is
shown by the result
# RPL is associated with MTHFR
677C - MTHFR 1298C allele
combination.
# SLC19A1, 80GSLC had lower
association with RPL.
2014
Ince
biyik
et
al.,
Obstet
rics
and
Gynec
ology
Scienc
e,
57(6),
Turk
ey
1507 RPL
women Maternal
Bloo
d
# FV-
PTH-
MTHFR
Strip A
kit
#
Hybridiz
ation of
PCR
amplifie
d DNA
products
FVL
Prothro
mbine
MTHFR
C677T
# FVL
Homozygous - 0.2%
Heterozygous - 5.51%
# Prothrombine Analysis
Homozygous - None
Heterozygous - 4.05%
# MTHFR
Homozygous - 8.29%
Heterozygous - 40.61%
# Screening seems to be
controversial
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Although the search was restricted to the last 10 years, one article from 2000 was used for the analysis as it was the first
ever evidence about ‘MTHFR polymorphisms and RPL’.
2.3 Data Extraction
From each study, year, authors, journal name, place of study, sample size and source, methods and detections, mutation
found, and mutation type and analysis information were extracted. Therefore from the eligible studies, 17,780 samples as
cases and 22,346 controls were taken into the analysis.
3 Results Analysis
From the analysis, 75 % (p = 0.002) of the studies (n=26) accepted that MTHFR polymorphisms effect recurrent
pregnancy loss. Whereas, 24 % of studies did not confirm this polymorphisms in RPL (Figure 2).
Figure 2: Ratio of the case studies
2015
Yalc
intep
e et
al.,
Interna
tional
Journa
l of
Molec
ular
and
Cellul
ar
Medici
ne,
4(2)
Turk
ey
23
abortion
materials
22
women
with
URPL
22 control
subjects
Maternal
and fetal
Spont
aneou
sly
abort
ed
fetal
mater
ials,
their
moth
ers
and
fertile
wom
en
Real
time-
PCR
FVL
Prothro
mbin-
G20210
A
MTHFR
C677T
PAI-1
4G/5G
ACE
I/D
eNOS
E298D
Apo E
E2/E3/E
4
# Compare to the mothers and
control group, PAI-1 4G/5G,
eNOS E2980, ApoE E2 / E3 / E4
genotypes and T' alleles were
spontaneous in aborted fetal
material
# Due to small sample size,
MTHFR C677T, ACE I / D were
not statistically significant.
75%
25%
Agree Disagree
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Journal of Gynecology Volume 1| Issue 4
Among the positive studies, 9 studies (35 %) discussed about both polymorphisms (C677T and A1298C) in RPL,
whereas 50 % of the cases specifically confirming the relationship between the C677T polymorphism in RPL and, 15 %
of studies discussed about the A1298C polymorphism’s specific role in RPL (Figure 3).
Figure 3: Percentage of frequency of both polymorphisms in RPL
4 Discussions
4.1 Hyperhomocysteinemia and risk for RPL
A successful pregnancy outcome is determined by several factors; maternal immunity, fetal vasculature and hemostatic
balance. Homocysteine metabolism cofactors; vitamin B12, B6, and folate are some of the cofactors that are essential for a
healthy pregnancy. Hyperhomocysteinemia (HHcy) is one of the leading factor, which has been stated as an independent
risk factor for several pregnancy-related complications, some are placental abruption, neural tube defects, pre-eclampsia,
and recurrent miscarriages. Hyperhomocysteinemia can occur as a result of folate deficiency when the dietary folate is
low (Nair et al., 2013). Now it was identified that in patients with chronic renal failure elevated level of creatinine can
cause an increase of plasma homocysteine levels (Park and Chang, 2014).
Low serum folate level in Rhesus monkeys has been related with granulosa cell impairment and with decreased estradiol
and progesterone level. Delayed ovulation and reduction of follicle growth are the indicators of embryonic growth and
malformation (Jangbloet et al., 2008). Thus folate metabolism is regulated by MTHFR gene, this makes the
polymorphisms in encoding MTHFR enzyme gene an important biomarker to assess the risk of RPL (Incebiyik et al.,
2014). Numerous latest studies are consistent with this fact and explains the significance of these polymorphisms with
RPL risk.
C677T MTHFR polymorphism
in RPL
50%
A1298C MTHFR polymorphism in RPL
15%
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4.2 MTHFR in RPL
In 2000, Isotalo and his colleagues provided the very first evidence to suggest “combined MTHFR 677T and 1298C
mutations may be responsible for compromised fetal viability”. Furthermore, they explained, the role of human MTHFR
677T/1298C and 677TT/ 1298CC genotypes in fetal viability.
Unfortunately, they couldn’t figure out how these polymorphisms present at 1298 and 677 positions interact with
pregnancy complications. Because, most of the recurrent miscarriages occur before the delivery, the real influence of
MTHFR polymorphisms to fetal viability is difficult to determine (Isotalo et al., 2000). However, they further suggested
that, combined MTHFR polymorphisms is believed to cause decreased fetal viability, especially when couple with folate
deficiency.
The hypothesis which was left by Isotalo and his colleagues was proved by Incebiyik and his mates in 2014. The cis
mutation identification is more significant, because it allows more than two mutant alleles to be present in the genome of
fetus. If c is MTHFR configuration happened, they would result in selection disadvantages because of the expression of
severe phenotypes include spontaneous abortion (Incebiyik et al., 2014). The probability for embryos with TT or CT
genotypes to terminate an early stage has been advanced as an explanation for the ‘unique distribution’ of 677CT and
677TT genotypes in spontaneously aborted embryos, irrespective of chromosomal integrity (Jangbloet et al., 2008).
Pregnancy complications were categorized into three stages: early (5-12 weeks), late (13-30 weeks), and combined early-
late (Gupta et al., 2014). Homozygous variant of both SNPs (677T/T and 1298C/C) plays a significant role in pregnancy
complications.
Homozygosity C677T is associated in combined early-late and late RPL, while A1298C homozygosity is related to
combine early-late idiopathic RPL (Mitraoui et al., 2006).
With the presence of various genes in pregnancy regulations, MTHFR is believed to have a high tendency with the RPL.
Earlier it was suggested that FVL, prothrombin (G20210A) might cause RPL, however later it was described to be cause
by mutation in the MTHFR gene particularly C677T single nucleotide polymorphism (SNP) (Mitraoui et al., 2006).
Further, Jeddi-Tehrani and his colleagues' research in 2010 showed homozygous and heterozygous frequency of MTHFR
A1298 and C677T were significantly higher in cased compared to the control group and suggested an association of
these polymorphisms with RPL. This study also confirmed that combined heterozygosity for MTHFR mutations 677C/T
and 1298A/C may present as a genetic marker for placental abruption.
In 2012, Ozdemir et al., confirmed that, among the 12 Thromphilic mutations studied, MTHFR polymorphisms were the
second common mutation to be involved in RPL. In 2014, Incebiyik and his team conducted a research in Turkey to
identify the prevalence of thromogenic gene mutations in miscarried women. MTHFR polymorphisms were detected in
49 % cases, whereas other related mutations failed to produce a significant amount. The same result was in line with the
study conducted by Ocak et al., in 2013 in Turkey.
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Subsequently, several researches have stated the existence of the MTHFR polymorphisms to be associated with the
increased risk of RPL (Hubacek et al., 2015; Li et al., 2015; Tiwari et al., 2015; D’Elia et al., 2014; Ozdemir et al.,
2012; Zonouzi et al., 2012; Settin et al., 2011; Rodriguez-Gillen et al., 2009; Mitraoui et al., 2006; Wiwanitkit et al.,
2005; Isotalo et al., 2000).
On contrary, studies from different authors showed no significance difference in MTHFR polymorphisms in RPL (Boas
et al., 2015; Incebiyik et al., 2014; Yalcintepe et al., 2014; Yousefian et al., 2014; Puri et al., 2013; Coulam et al., 2006).
Variations of the frequency of MTHFR polymorphisms in different ethnic population were observed and this may be due
to the fact that some studies included women with 3 or more pregnancy losses and some studies included women with 2
or more pregnancy losses. Meanwhile MTHFR polymorphisms only result in the second and third trimester fetal loss
(Yousefian et al., 2014). Further, the gestational weeks used in defining pregnancy loss was different among the studies.
4.3 C677T in RPL
C677T polymorphism has been denoted as a risk factor for RPL because of expand tHcy. Homozygous TT genotype was
significantly higher in the RPL women in china (p = 0.003), and in East Asia (Chen et al., 2015; Nan and Li, 2015;
Vanilla et al., 2015; Park and Chang, 2014; Cao et al., 2013; Praveen et al., 2013; Ibrahim et al., 2012; Nair et al.,
2012; Wu et al., 2012; Park et al., 2011; Altomare et al., 2007; Ren and Wang, 2007).
C677T polymorphism is associated with hyperhomocysteinemia, and increased risk of arterial stiffness. Women with
hyperhomocysteinemia express an important association with defective chronic villous vascularization.
In embryogenesis, the survival and the growth of the embryo is stimulated by its own blood supply through angiogenesis.
A noble interchange between mother and fetus is necessary to ensure normal fetal growth; impaired chronic villous
vascularization can result in embryonic death and leading to miscarriages (Yalcintepe et al., 2014).
Yila et al., (2012) state, that MTHFR 677T allele has an association with low folate status. D’Elia et al., (2014) declared
that there is a statistically significant decrease in oocyte maturity when the MTHFR C677T was detected. Rodriguez-
Guillen et al., (2009) detected an increased risk of recurrent miscarriages among female carriers of MTHFR 677T.
Prevalence of the MTHFR genotype was higher among Mexicans (38-48 %), and a much lower frequency of 1298C
allele has been observed in Mexicans (0 - 2.4 %) than in Europeans (Rodriguez-Guillen et al., 2009). In 2010, Jeddi-
Tehrani and his team revealed MTHFR 677C/T was significantly more frequent in Iranian women with early pregnancy
loss. It was the highest MTHFR mutation rate found in patients with RPL.
Wu et al., (2012) state, MTHFR-C677 gene mutation was more prevalent in paternal side than maternal. MTHFR
paternal T-allele was connected with 2.37 fold risk, whereas maternal T-allele did not show any interference in risk of
RPL. Subsequently, high frequency of MTHFR T-allele was absorbed in recurrent spontaneous abortions (RSA)
couples’ paternal side in a study carried out in India (Vanilla et al., 2015). Accordingly, both studies suggest, paternal
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screening is equally important with maternal screening. When both parents are carrying mutant allele the chance of
inheriting the mutation in the offspring is much higher, which thereby influence the risk of miscarriages.
4.4 A1298C in RPL
Some of the individual studies have detected an increased risk of pregnancy loss in Indian, Turkish, and the Tunisian
population with 1298AC and 1298 CC polymorphisms (Li et al., 2015; Cao et al., 2014; Cao et al., 2013; Nair et al.,
2013; Idali et al., 2012; Zonouzi et al., 2012; Jeddi-Tehrani et al., 2010 ). Mean plasma homocysteine was elevated
among 1298AA homozygotes as compared with 1298C allele and was associated with RPL (Yila et al., 2012). MTHFR
1298 AC genotype allele has increased risk of spontaneous abortion along with MTHFR-C677T (Rodriguez-Guillen et
al., 2009).
MTHFR A1298C mutation was seen in Iranian women suffering from RPL (p< 0.001) (Idali et al., 2012). Meanwhile,
the frequency of both allele of A1298 were significantly higher in Iranian population and suggested an association with
RPL (p< 0.001) (Jeddi-Tehrani et al., 2010). D’Elia et al., (2014) found women with MTHFR 1298CC with In vitro
fertilization (IVF) were less probable to become pregnant than those with 1298AA genotype in Brazil.
4.5 MTHFR polymorphisms in other pregnancy complications
Maternal MTHFR 677T homozygosity was associated with lower birth weight among female infants in Japan (Yila et
al., 2012). Ibrahim and his colleagues study in 2012 showed, C677TT and the T allele polymorphisms were higher in
Egypt. TT allele of MTHFR gene was found to be associated with pre-eclampsia and other pregnancy complications.
MTHFR C677TT is considered as a good diagnostic test as it is highly sensitive and specific (Ibrahim et al., 2012).
Study of Nan and Li (2015) showed, C677TT is a candidate factor for premature delivery, and might increase the risk by
1.853 times than A1298C allele. Therefore A1298 polymorphisms act as a protective factor in premature delivery. A
study carried out in India also confirmed that, MTHFR polymorphism increased the preterm delivery by more than 2
folds. Further, MTHFR polymorphism was also focused to be significantly associated with fetal death and increased the
negative outcome by more than 3 folds (Tiwari et al., 2015).
4.6 Limitations
MTHFR mutation is a rare genetic mutation in leading to RPL, to obtain literature survey is difficult in its recent
publishing. Due to its pathetic conditions, therefore analysis was restricted for the past ten years from 2005 to 2015.
Since, MTHFR is related with other disease conditions the number of articles of MTHFR polymorphisms related to RPL
is limited.
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4.7 Suggestions, Treatments, Future aspects
Literatures suggest, carrying out a routine test for the defection of C677T MTHFR polymorphism gene using simple
technique such as Single Specific Primer-PCR (SSP-PCR) as a marker for pregnancy complication susceptibility
(Ibrahim et al., 2012). Zonouzi et al., (2012) study confirm that both Amplification Refractory Mutation System –PCR
(ARMS-PCR) and Restriction Fragment Length Polymorphism-PCR (RFLP-PCR) methods would detect the MTHFR
polymorphisms with the same specificity. Therefore, to detect large number of samples they proposed ARMS-PCR for
the detection of MTHFR polymorphisms as a diagnostic test since it is easy and cost effective.
Screening of pregnant women for MTHFR-C677T polymorphism during the first trimester can be used as a prognostic
marker in women who have a risk for miscarriages (Tiwari et al., 2015). Patients with 677CT genotype have a higher
proportion of better quality embryos and increased chances of a healthy pregnancy, when compared to the homozygous
allele. Therefore, it is recommended to detect for the MTHFR polymorphism prefer to perform an IVF (D’Elia et al.,
2014).
Women with folate deficiency have been identified to be at higher risk of early pregnancy loss compared to the women
having a higher or normal level of folate (D’Uva et al., 2008). RPL patients with MTHFR polymorphisms should be
advised to take a balanced diet with adequate intake of Vitamin B12, and folic acid to prevent RPL and poor pregnancy
outcomes (Nair et al., 2013; Ibrahim et al., 2012).
Several folate acid supplementation studies were concluded with positive outcomes in pregnant women with a risk for
RPL. A study carried out on 7,812 Chinese women confirmed that, periconceptional folic acid supplementation and
health care following gene polymorphism testing could be a powerful measure to decrease pregnancy complications (Li
et al., 2015).
Study carried out in Italy showed, in moderate hyperhomocysteinemia, Hcy concentration was reduced after folate-
enriched diet, and 5-MTHF or folic acid supplementation (p<0.001) (Zappacosta et al., 2013). Diverse supplementation
by enriched-diet (200 µg/ day), folic acid (200 µg/ day, 340 DFE) or 5-MTHF (200 µg/ day, 340 DFE) produced
significant decrease in Homocysteine concentration (Anderson et al., 2014).
Recent genetics studies predict that allosteric regulation of MTHFR gene structure plays a significant role in fetal
growth. In folate deficiency, cells get arrested in the S-phase of the cell cycle, which would increase the uracil
disincorporation and cause DNA damage. This would lead to complications in pregnancy and reduced fetal growth.
According to the 5, 10-MTHFR gene structure, the A1298C is located on the C-terminal regulatory domain, containing
protein retention signals that stop delivery of proteins to the secretory pathway. It has now been shown that allosteric
inhibitory interplay in the s-adenosyl methionine (SAM) and s-adenosyl-homocysteine (SAH) cycle is implicated in the
functional behavior of this SNP relative to folate level and mediation of fetal growth (Yila et al., 2012).
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5 Conclusions
This review analyzed the relationship of MTHFR polymorphisms with RPL. Based on the review the following
conclusions were drawn: Since 40 % of RPL cases are idiopathic, genetic factors tend to be highly associated with
miscarriages. Compared to other genetic mutations, MTHFR polymorphisms are the primary genetic risk factor for RPL
and MTHFR C677T and A1298C are the two most frequently occurring polymorphisms. Among both the
polymorphisms, C677T polymorphism is significantly associated with RPL (p = 0.043) compared to A1298C. MTHFR
polymorphisms could be easily and cost effectively detected using ARMS-PCR instead of RFLP-PCR. Screening of
pregnant women for MTHFR C677T polymorphism during the first trimester can be used as a prognostic marker in
women who have a risk for miscarriages. Hence, the C677T polymorphism can act as a genetic marker for early
diagnosis of RPL. Literature also strongly emphasizes that, MTHFR-C677 polymorphism is also prevalent in paternal
testing. Therefore, this review firmly concludes, paternal screening is also equally important along with maternal
screening.
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