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Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005 ( C
2005)
DOI: 10.1007/s10815-005-6795-0
Male Factor Infertility
MTHFR C677T polymorphism associates
with unexplained infertile male factors
Jung Hoon Park,1Han Chul Lee,1Yu-Mi Jeong,1Tae-Gyu Chung,2
Hyun-Joo Kim,2Nam Keun Kim,3Sook-Hwan Lee,3and Suman Lee1,4
Submitted March 3, 2004; accepted May 10, 2005
Purpose:To determine whether 5,10-methylenetetrahydrofolate reductase (MTHFR C677T
and A1298C) genotype is associated with male infertility.
Methods:Analysis of cytogenetic, Y chromosomal microdeletion assay (Yq), and the C677T
and A1298C polymorphisms of the MTHFR gene by pyrosequencing and PCR-Restriction
Fragment Length Polymorphism (RFLP) method. SAS 8.1 assessed the statistical risk of
MTHFR genotype.
Results:The homozygous (T/T) C677T polymorphism of the MTHFR gene was present at a
statistically high significance in unexplained infertile men with normal karyotype, instead at
no significance in explained infertile men with chromosomal abnormality or Y chromosome
deletion. There was no statistically significance of A1298C variation in infertile males.
Conclusions:The MTHFR 677TT genotype may be a genetic risk factor for male infertility,
especially with severe OAT and non-obstructive azoospermia in unexplained infertile males.
KEY WORDS: Azoospermia; male infertility; MTHFR; pyrosequencing.
INTRODUCTION
5,10-Methylenetrahydrofolate reductase (MTHFR)
is the main regulatory enzyme of homocysteine
metabolism. There are two polymorphisms in the
MTHFR gene, C677T (A →V) (1) and A1298C
(E →A) mutation (2, 3), which can effect its bio-
chemical activity. The MTHFR C677T mutation de-
creases MTHFR activity and increases the homocys-
teine level. The other polymorphism, A1298C, also
1Functional Genomics Lab, Graduate School of Life Science and
Biotechnology, CHA Research Institute, Bundang Campus, Col-
lege of Medicine, Pochon CHA University, Seoul, Korea.
2Department of Urology, CHA General Hospital, College of
Medicine, Pochon CHA University, Seoul, Korea.
3CHA General Hospital, College of Medicine, Pochon CHA Uni-
versity, Seoul, Korea.
4To whom correspondence should be addressed at Suman
Lee Ph.D., Functional Genomics Lab, Bundang Campus, Col-
lege of Medicine, Pochon CHA University, 222 Yatap-Dong,
Bundang-Gu, Sungnam-Si, Kyunggi-Do, 463-836, Korea; E-mail:
suman@cha.ac.kr.
reduces MTHFR enzyme activity, but to a lesser ex-
tent than the C677T mutation (3).
Homocysteine metabolism is dependent upon vi-
tamins as cofactors or cosubstrates. In the trans-
sulfuration pathway, homocysteine is transformed
by cystathionin-β-synthase (CBS) into cystathion-
ine. Vitamin B6is also required for the transforma-
tion of cystathionine to cysteine and α-ketobutyric
acid (4). This reaction is important in one-carbon
metabolism because methionine is the precursor of S-
adenosylmethionine (SAM), the methyl group donor
for DNA, protein, and lipid methylation. If there
is a mutation in the MTHFR gene, the homocys-
teine level can be increased by decreased levels of
5-methyltetrahydrofolate and methionine.
Decreased MTHFR activity due to variations in
the MTHFR gene also results in DNA hypomethyla-
tion (5–7). We predicted that the C677T and A1298C
variations of the MTHFR gene may predispose to
aberrant DNA methylation and increase the risk of
meiotic nondisjunction.
361 1058-0468/05/1000-0361/0 C
2005 Springer Science+Business Media, Inc.
362 Park et al.
Abnormal folate level and methylation meta-
bolism can lead to abnormal chromosome segrega-
tion (8). Folate status in homocysteine metabolism
can play an important role. Folate deficiency occurs
frequently and the related hyperhomocysteinaemia is
considered a risk factor for various diseases includ-
ing infertility. According to a previous study (9), total
normal sperm count increase after combined zinc sul-
phate and folic acid treatment in both subfertile and
fertile men. James et al. (8) showed that the mutation
of MTHFR gene may be a direct genetic risk factor
for meiotic nondisjunction, such as Down syndrome.
We classified non-obstructive infertile men into
two groups, explained and unexplained group by
karyotyping and Yq deletion analysis, and deter-
mined whether MTHFR (C677T and A1298C) geno-
type is associated with male infertility. In this study,
we used a pyrosequencing method to screen for the
two variations of MTHFR in infertile and fertile
men.
MATERIALS AND METHODS
Patients and Controls
Three hundred seventy-three infertile and 396
healthy fertile men were recruited from the CHA
General Hospital, College of Medicine, Pochon
CHA University (Seoul, Korea). From January 2000
to August 2002, three hundred seventy-three infertile
men were requested to Genetics Lab at CHA hos-
pital for cytogenetic and Y chromosomal microdele-
tion analysis, and then only non-obstructive infertile
men diagnosed by physical exam and hormone as-
say between 26 and 47 years of age (mean ±SD:
33.3±4.0 years) were included in case-control study.
Three hundred ninety-six fertile men between 26 and
71 years of age (mean ±SD: 42.7±8.4 years) who
have at least one child by direct survey and who
lacked any history of requiring assisted reproduction
technology were included as the control group. All
participants gave their written informed consent.
Semen analysis was performed strictly according to
World Health Organization (WHO, 1999) guidelines
and Kruger et al. (10) Non-obstructive infertile males
were classified into four subtypes, 286 azoosper-
mia, 26 oligoasthenoteratozoospermia (OAT), 59 se-
vere OAT, and 2 oligozoospermia. The diagnosis
of azoospermia (no spermatozoa in the ejaculate)
was made on the basis of two semen analyses per-
formed according to the WHO recommended proce-
dure. Non-obstructive azoospermia was determined
after historical and physical examination, sperm anal-
ysis (including assessment of sperm volume, pH
and evaluation of fructose concentration), endocrine
profile (FSH, luteinizing hormone, testosterone, an-
drostenedione), ultrasound testicular volume, and
seminal vesicle evaluation.
Cytogenetic and Y Chromosome
Microdeletion Assay
Cytogenetic analysis was performed on metaphase
spreads of cultured lymphocytes. For Yq deletion
studies, DNA was extracted from the peripheral
blood and amplified by a multi-PCR method with
primers to 13 loci on the Y chromosome, including
one SRY and 12 sequence-tagged sites (STSs) (AZF-
a region: sY84, sY86; AZF-b region: sY 134, sY 138,
MK5; AZF-c region: sY 152, sY 147, sY 254, sY 255,
SPGY1, sY 269, sY 158) (11, 12).
Of the 373 infertile men, 258 had a normal kary-
otype (46,XY) without any Yq deletion which was
defined as unexplained infertiles. One hundred and
fifteen men had abnormal chromosomes, which in-
cluded 28 men with 46,XY and Yq deletion; 4
with 46,XY and inversion; 1 with 46,XY, inver-
sion and Yq deletion; 10 with 46,XY and transloca-
tion; 1 with 46,XY, derivative (X) and translocation
(X:Y)(p22:q11); 1 with 46, XX male; 3 with 45,XY
and derivate (13:14)(q10:q10); 54 with 47,XXY; 1
with 47,XXY and Yq deletion; 1 with inversion (9);
1 with 47,XXY and translocation (9:12)(p13:q13); 1
with 47,XY and deletion(X)(q22); 2 with 47,XYY;
and 7 with chromosomal mosaicism which was de-
fined as explained infertiles.
Pyrosequencing Assay for Screening of MTHFR
C677T and A1298C
The C677T mutation was analyzed by polymerase
chain reaction (PCR) of genomic DNA using the fol-
lowing primer pairs: 5–TAT TGG CAG GTT ACC
CCA AA–3(left primer) and 5–biotin–GAA AAG
CTG CGT GAT GAT GA–3(right primer). These
primers generate a 113 bp fragment following a stan-
dard three-step PCR at an annealing temperature
of 55◦C for 45 cycles. The second A1298C muta-
tion was also analyzed by PCR (MJ research thermal
cycle, Waltham, USA) using the following primer
pairs: 5–Biotin–TTT GGG GAG CTG AAG GAC
TA–3(left primer) and 5–TGG TTC TCC CGA
GAG GTA AA–3(right primer). These primers
Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005
MTHFR C677T polymorphism in unexplained infertile males 363
generate a 141 bp fragment following PCR at an an-
nealing temperature of 58◦C for 45 cycles. DNA frag-
ments were separated by electrophoresis on a 2%
agarose gel and visualized with ethidium bromide.
The biotinylated PCR product then underwent py-
rosequencing analysis for direct analysis of SNP on
PCR product according to the method published by
Ahmadian et al. (13).
Polymerase Chain Reaction–Restriction
Fragment Length Polymorphism for C677T
and A1298C of MTHFR
PCR–RFLP analysis was performed as described
by Friedman et al. (14) and Weisberg et al. (3).
Regions containing the C677T and A1298C poly-
morphisms in the MTHFR gene were amplified from
genomic DNA by PCR as 198 and 138 bp fragments,
respectively. The C677T and A1298C variation
created HinfI and Fnu4HI (New England Biolabs,
Beverly, MA, USA) restriction enzyme recognition
sequences, respectively, with the digest fragments
separable by 3% agarose gel electrophoresis and
visible under UV light.
Statistical Analysis
The statistical packages used to estimate the odds
ratio and 95% confidence intervals were SAS (r) Pro-
prietary Software Release 8.1 (SAS Institute, Cary,
NC.). Results for the enumeration of data (e.g.,
the number of individuals with various genotypes)
and comparison of percentages between groups were
evaluated with a chi-squared test and Fisher’s exact
test (two-sided). Allele frequencies were calculated
by allele counting. Tests for the Hardy–Weinberg
equilibrium and Odd ratios (OR) were carried out
by the chi-squared test. Pvalues <0.05 were taken as
statistically significant.
RESULTS
We analyzed the two variations of the MTHFR
gene in 373 infertile and 396 fertile men by py-
rosequencing. Figure 1A and B show the results
of the MTHFR C677T and A1298C analyses by
the pyrosequencing method. Pyrosequencing, a new
method for DNA sequencing, is gaining widespread
use for many different types of DNA analysis. Using
a four-enzyme mixture, this sequencing-by-synthesis
method relies on the luminometric detection of py-
rophosphate (PPi) released upon nucleotide incorpo-
ration (13).
To validate C677T and A1298C variation pyrose-
quencing data, we randomly selected 50 samples and
compared the pyrosequencing result with the PCR–
RFLP genotypes. Concordance was shown for all
genotypes by pyrosequencing and RFLP in fertile
and infertile men, shown in Fig. 1C and D.
The statistical analysis of the C677T and A1298C
results in fertile and infertile men are shown in
Table I. The frequencies of homozygous C677T (TT)
among the fertile and infertile men were 12.88 and
16.89% (p=0.0186), respectively. The allelic fre-
quency for fertile men (12.88%) with C677T vari-
ation in this study was similar to frequencies of
healthy Koreans in other study (13.4%, n=254)
(15). The MTHFR A1298C polymorphism data by
case-control analysis showed no significant differ-
ences in the individuals with infertility (p=0.4341;
data not shown). The frequencies of homozygous
A1298C (CC) among the fertile and infertile men
were 4.04 and 4.83%, respectively.
In Table II, the infertile men (n=373) were clas-
sified into azoospermia (n=286), severe OAT (n=
59), OAT (n=26) and oligozoospermia (n=2) sub-
types according to semen analysis. We analysed only
the C677T variation because no significant difference
in A1298C was observed in infertile men. Statisti-
cal differences were observed between the azoosper-
mia and severe OAT groups. The frequencies of the
heterozygous C677T (CT), homozygous C677T (TT)
and T allele in the group with azoospermia were
statistically significant (p<0.05). The frequencies
of heterozygous (CT) and 677 C →T substitution
in one or both alleles (CT +TT) of infertile men
with azoospermia were 57.34 and 73.78%, respec-
tively (p=0.0090, p=0.0042; Table II). Homozy-
gous (TT) and T allele frequencies in infertile male
patients with severe OAT had a high statistical sig-
nificance (p=0.0136 and p=0.0224, respectively)
when compared with 677 wild homozygous (CC) and
C allele frequencies.The odd ratio (OR) of 677 mu-
tant homozygous (TT) of severe OAT was 2.65.
We compared the frequencies between the Yq
deletion and chromosome disorder (explained)
infertile group and the unexplained infertile group
(normal karyotype and no Yq deletions). Table III
shows that the mutant homozygous (TT) and T allele
in the unexplained infertile group was statistically
significant (p=0.0142 and p=0.0140, respectively).
However, the infertile group with Yq deletion
and chromosome disorder showed no significant
Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005
364 Park et al.
Fig. 1. The pyrosequencing and PCR–RFLP results from male control and infertile patients. The pyrosequencing analysis of the 677 C →T
(A) and 1298A →C (B) Polymorphism of MTHFR gene was carried out on all samples. The yaxis of the bar graph in the pyrosequencing
figures is scaled such that the value of one nucleotide was 0.5. PCR–RFLP analysis was carried out to confirm the pyrosequencing method.
For the MTHFR C677T genotype analysis, the 198-bp PCR amplicon of the MTHFR 677 CC remains after HinfII digestion, whereas the
TT genotype results in 23 and 175 bp fragments (C). (Lane M, 1 Kb PLUS DNA ladder; Lane 1, 198-bp PCR product as control; Lane
2, MTHFR 677 CC type; Lane 3, MTHFR 677 CT type; Lane 4, MTHFR 677 TT type). For the MTHFR A1298C genotype analysis, an
undigested 138 bp PCR amplicon was seen for the AA genotype (D), and the CC genotype define by the presence of both 19 and 119 bp
bands (Lane M, 1 Kb PLUS DNA ladder; Lane 1, 138-bp PCR product as control; Lane 2, MTHFR 677 AA type; Lane 3, MTHFR 677 AC
type; Lane 4, MTHFR 677 CC type).
Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005
MTHFR C677T polymorphism in unexplained infertile males 365
Table I. Distribution of MTHFR C677T and A1298C Genotypes in Infertile (n=373)
and Fertile Males (n=396)
Fertile males Infertile males
%n%nOR (95% CI) p
677CC 36.62 145 28.15 105 — —
677CT 50.50 200 54.96 205 1.42 (1.03–1.95) 0.0319a
677TT 12.88 51 16.89 63 1.70 (1.09–2.67) 0.0186a
CT +TT 63.38 251 71.85 268 1.48 (1.09–2.00) 0.0122a
T allele 38.13 302 44.37 331 1.29 (1.06–1.59) 0.0130a
1298AA 67.93 269 63.54 237 — —
1298AC 28.03 111 31.64 118 1.21 (0.88–1.65) NS
1298CC 4.04 16 4.83 18 1.27 (0.64–2.56) NS
AC +CC 32.07 127 36.46 136 1.22 (0.90–1.64) NS
C allele 18.06 143 20.64 154 1.18 (0.92–1.52) NS
Note. OR: odds ratio; CI: confidence interval. NS: not significant.
aStatistically significant pvalues compared to fertile male group (p<0.05) were noted.
difference in the frequencies of C677T CT, TT
and T allele (p>0.05, p=0.0821, 0.3089 and
0.1792).
In Table IV, we further classified the unexplained
infertile group into four subtypes: azoospermia, se-
vere OAT, OAT and oligozoospermia subtype. The
frequencies of 677 homozygous (TT) and T allele in
unexplained infertile males with severe OAT were
24.39% (p=0.0140) and 51.22% (p=0.0209). In-
terestingly, the odds ratio (OR) for the homozygous
(TT) in severe OAT of unexplained infertile group
was 3.2 (Table IV).
DISCUSSION
The MTHFR C677T polymorphism influences ho-
mocysteine metabolism, and thereby it may influ-
ence the process of DNA methylation, synthesis, and
damage.
DNA methylation plays several functional roles,
including enhancement of genomic stability (16, 17)
and suppression of gene expression (18, 19). Subjects
with the homozygous (677TT) genotype have lower
levels of genomic DNA methylation than control
subjects (20). Genomic stability decreases through
Table II. The frequency of MTHFR 677 Genotype According to the Four Subtype of Infertile Male Group
677CC 677CT 677TT CT +TT T allele
Azoospermia 26.22% 57.34% 16.43% 73.78% 45.10%
(n=286) (n=75) (n=164) (n=47) (n=211) (n=258)
OR (95% CI) — 1.59 (1.12–2.24) 1.78 (1.10–2.89) 1.63 (1.16–2.27) 1.33 (1.07–1.66)
p— 0.0090d0.0189d0.0042d0.0098d
Severe OATa25.42% 50.85% 23.73% 74.58% 49.15%
(n=59) (n=15) (n=30) (n=14) (n=44) (n=58)
OR (95% CI) — 1.45 (0.75–2.79) 2.65 (1.20–5.88) 1.69 (0.91–3.15) 1.57 (1.06–2.31)
pNS 0.0136dNS 0.0224d
OATb50.00% 38.46% 11.54% 50.00% 30.77%
(n=26) (n=13) (n=10) (n=3) (n=13) (n=16)
OR (95% CI) — 0.56 (0.24–1.31) 0.66 (0.18–2.40) 0.58 (0.26–1.28) 0.72 (0.39–1.32)
p—NSNSNSNS
Oligozoospermiac100.0%
(n=2) (n=2) —
OR(95%CI)—————
p—— — — —
Fertile males 36.62% 50.50% 12.88% 63.38% 38.13%
(n=396) (n=145) (n=200) (n=51) (n=251) (n=302)
Note. OR: odds ratio; CI: confidence interval. NS: not significant.
aSevere oligoasthenoteratozoospermia (sperm count <5×106, progressive motility <10%, normal morphology <4%).
bOligoasthenoteratozoospermia (sperm count <20 ×106, progressive motility <50%, normal morphology <14%).
cOligozoospermia (sperm count <20 ×106/mL).
dStatistically significant pvalues compared to fertile male group (p<0.05) were noted.
Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005
366 Park et al.
Table III. The Frequency of MTHFR 677 Genotype in Infertile Malesab According to Cytogenetic and Y Chromosome Deletion Analysis
677CC 677CT 677TT CT +TT T allele
Unexplained infertile malesa28.29% 53.49% 18.22% 71.71% 45.00%
(n=258) (n=73) (n=138) (n=47) (n=185) (n=232)
OR (95% CI) 1.37 (0.96–1.96) 1.83 (1.13–2.98) 1.46 (1.04–2.06) 1.33 (1.06–1.66)
p— NS 0.0142c0.0274c0.0140c
Chromosome disorder and Yq deletionb27.83% 58.26% 13.91% 72.17% 43.04%
(n=115) (n=32) (n=67) (n=16) (n=83) (n=99)
OR (95% CI) — 1.52 (0.95–2.43) 1.42 (0.72–2.81) 1.50 (0.95–2.36) 1.23 (0.91–1.65)
p—NSNSNSNS
Fertile males 36.62% 50.50% 12.88% 63.38% 38.13%
(n=396) (n=145) (n=200) (n=51) (n=251) (n=302)
Note. OR: odds ratio; CI: confidence interval. NS: not significant.
abOnly infertile group (n=373) was classified into aunexplained group (n=258) and bexplained group (n=115) by cytogenetic and Yq
deletion analysis.
cStatistically significant compared to fertile male group: p<0.05.
DNA hypomethylation and, as a result of the process
of meiosis, methylation patterns are influenced and
epigenetic defects are created (21).
Other major possibilities reported include a high
level of homocysteine that can lead to the production
of auto-oxidation with the production of hydrogen
peroxide (H2O2), a harmful reactive oxygen metabo-
lite (22, 23). Moreover, the increased production of
reactive oxygen species may be associated with Hcy-
mediated DNA damage, and a recent study reported
that Hcy increases H2O2in intracellular generation
and this induces apoptosis because of DNA damage
(24). Oxidative stress induces peroxidative damage
in the sperm plasma membrane and DNA damage
in both the mitochondrial and nuclear genomes (25).
There is evidence to suggest that H2O2treatment
causes more damage in asthenozoospermic infertile
men than in the normozoospermic infertile and fer-
tile men (26). The MTHFR C677T homozygous (TT)
is also associated with other human diseases, such
as, cardiovascular disease, congenital abnormalities,
pregnancy outcome and acute leukemia (27, 28).
In a previous study, Bezold et al. reported an asso-
ciation between C677T homozygosity (TT) and male
infertility (29). In this study, we designed to evaluate
the effect of the C677T and A1298C variations more
Table IV. Frequency of MTHFR 677 Genotype in Unexplained Infertile Male Group
Unexplained malesa(n=258) 677CC 677CT 677TT CT +TT T allele
Azoospermia 25.66% 56.54% 17.80% 74.35% 46.07%
(n=191) (n=49) (n=108) (n=34) (n=142) (n=176)
OR (95% CI) — 1.60 (1.07–2.38) 1.97 (1.15–3.39) 1.67 (1.14–2.46) 1.39 (1.08–1.77)
p— 0.0210b0.0132b0.0082b0.0095b
Severe OAT 21.95% 53.66% 24.39% 78.05% 51.22%
(n=41) (n=9) (n=22) (n=10) (n=32) (n=42)
OR (95% CI) — 1.77 (0.79–3.96) 3.16 (1.22–8.21) 2.05 (0.95–4.42) 1.70 (1.08–2.69)
p— NS 0.0140b0.0613c0.0209b
OAT 54.17% 33.33% 12.50% 45.83% 29.17%
(n=24) (n=13) (n=8) (n=3) (n=11) (n=14)
OR (95% CI) — 0.45 (0.18–1.10) 0.66 (0.18–2.40) 0.49 (0.21–1.12) 0.67 (0.35–1.27)
pNS NS NS NS
Oligozoospermia 100.0%
(n=2) (n=2)
OR(95%CI) —————
p—————
Fertile males 36.62% 50.50% 12.88% 63.38% 38.13%
(n=396) (n=145) (n=200) (n=51) (n=251) (n=302)
Note. OR: odds ratio; CI: confidence interval. NS: not significant.
aThe unexplained infertile group (n=258) without any chromosomal disorder and Yq deletion was classified into the four subtypes
by semen analysis.
bStatistically significant p values were noted.
cNot significant. (p>0.05), but showed the statistical tendency (p=0.0613).
Journal of Assisted Reproduction and Genetics, Vol. 22, Nos. 9/10, October 2005
MTHFR C677T polymorphism in unexplained infertile males 367
precisely in male infertility according to the semen
analysis, Yq deletion, and cytogenetic analysis. We
found that C677T heterozygous (CT) and homozy-
gous (TT) genotypes were specially associated with
azoospermia and severe OAT only in unexplained
infertility, not male infertility with other problems,
such as Yq deletion and cytogenetic defects.
Franco et al., have showed that the frequencies
of MTHFR C677T polymorphism among Whites
and Asians were similar to Caucasian populations
even though significantly heterogeneous distribution
among different ethnic or geographical populations
(30). The frequencies of the heterozygous (CT) and
homozygous (TT) genotypes in the Korean fertile
male group were a little higher than a German male
control group (29).
In the case of nonobsructive azoospermia, both the
C677T homozygous (TT) and the heterozygous (CT)
genotype showed statistical importance (Tables II
and IV). Specially, 677 CT heterozygotes were as-
sociated with azoospermia of unexplained infertile
male group (p=0.0090 and 0.0210, Tables II and
IV). Further investigation of larger groups of patients
with systematic research may resolve this role, if any,
of 677 CT heterozygotes in azoospermia.
We suggest that an extended study should be un-
dertaken that includes other possible genes that par-
ticipate in the homocysteine metabolism, such as me-
thionine synthase and cystathionine β-synthase gene
related with male infertility.
ACKNOWLEDGMENTS
This study was supported by a grant of the Korea
Health 21 R&D Project, Ministry of Health &
Welfare, Republic of Korea (01-PJ10-PG6-01GN13-
0002).
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