Influence of Methylenetetrahydrofolate Reductase C677T Gene Polymorphisms in Algerian Infertile Men with Azoospermia or Severe Oligozoospermia

Abstract

Aims: The C677T allele of the methylenetetrahydrofolate reductase (MTHFR) gene has been suggested to represent a risk factor for male infertility. To confirm this association, the distribution of the single-nucleotide polymorphism C677T was investigated in idiopathic infertile Algerian patients with nonobstructive azoospermia (NOA) or severe oligoasthenoteratozoospermia (OAT). A case-control study was carried out, including 74 idiopathic infertile Algerian patients with NOA (n=46) or severe OAT (n=28) and 84 fertile men as controls. Polymorphism C677T was studied by polymerase chain reaction-restriction fragment length polymorphism, and the results were statistically analyzed. Results: The frequency of genotypes MTHFR 677CC, 677CT, and 677TT in idiopathic infertile men with NOA was 43.48%, 41.30%, and 15.22%; 39.29%, 50%, and 10.71% regarding the severe oligozoospermic men; and 42.86%, 45.24%, and 11.90% in the control group. Conclusions: The data suggest that the C677T MTHFR polymorphism is not a risk factor for idiopathic male subfertility in an Algerian population.

Introduction

Male infertility is a multifactorial disorder affecting 10%-30% of couples (Kara and Simoni, 2010). The underlying pathogenetic mechanisms can reflect the effect of environmental toxins, such as pesticides, glycol ethers, and heavy metals; systemic disorders, like hypothalamic pituitary disease, testicular cancers, and germ cell aplasia; and genetic factors, including aneuploidies and single-gene mutations (Skakkebaek et al., 1994). Spermatogenesis is governed by a tightly controlled series of gene expression events. The genes on the Y chromosome and autosomal genes are responsible for the regulation of spermatogenesis at different steps, and the aberrant expression of these genes can lead to infertility (Singh et al., 2010). One of the mechanisms regulating their expression is DNA methylation.

Folate is indispensable for DNA synthesis and methylation of DNA and histones. Methylenetetrahydrofolate reductase (MTHFR) is one of the key enzymes in folate metabolism that is essential for numerous cellular functions (Zhou-Cun et al., 2007). The enzyme catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for homocysteine in the synthesis of methionine. The homozygous C667T in the MTHFR gene is reported to be associated with the risk of certain human diseases, including some cardiovascular disorders, cancers, and neural tube defects (Frosst et al., 1995; Ma et al., 1997; Stern et al., 2000; Kirke et al., 2004). The activity of MTHFR is much higher in testes than in other major organs in the adult mouse, suggesting that it might play an important role in spermatogenesis (Chen et al., 2001).

A base change from C to T at the nucleotide position 677 of the MTHFR gene results in the substitution of valine for alanine (p.A222V). The single-nucleotide polymorphism (SNP) C677T in the gene that encodes MTHFR decreases the activity of the enzyme by 35% in persons who are heterozygous for the mutation and by 70% in those who are homozygous (Frosst et al., 1995). It is reported that the prevalence of the homozygous form is estimated to be between 5% and 10% (Guntram et al., 2001). Recently, several reports have suggested that the C677T (also known as C665T) polymorphism in the MTHFR gene may be associated with reduced sperm counts in humans, leading to male infertility in some populations (Tetik et al., 2008; Khazamipour et al., 2009; Ravel et al., 2009; Gava et al., 2010; Montjean et al., 2011; Safarinejad et al., 2011).

In the present study, which is the largest report in the medical literature and the first in Algeria, we investigated the frequency distribution of the common SNP C677T in the MTHFR gene in 74 infertile patients with idiopathic azoospermia or severe oligoasthenoteratozoospermia (OAT) and compared it with those in 84 fertile controls to explore the possible association between this gene variation and male infertility in an Algerian population. Our data suggest that genetic variants in MTHFR do not have a significant impact on sperm counts.

Patients and Methods

Patients and controls

This case-control study included a total of 74 infertile patients, including 46 with idiopathic azoospermia and 28 with severe OAT (semen count <20×10⁶/mL) aged from 24 to 48 years, who were recruited from the Ibn Sina Laboratory and Ibn Roch Clinic between May 2009 and May 2011. The control group consisted of 84 men who were proven fertile with normozoospermia. An informed consent was obtained from each subject to participate in this study.

These 74 nonobstructive infertile patients were classified by semen analysis. Semen analysis was strictly performed according to the World Health Organization guidelines (WHO, 1999). All patients and controls were of Algerian ethnic origin.

Polymerase chain reaction amplification

Genomic DNA was extracted from the peripheral blood leukocytes of patients and controls using a standard procedure. Primers 5′-TGAAGGAGAAGGTGTCTGCGGGA-3′ (forward) and 5′-AGGACGGTGCGGTGAGAGTG-3′ (reverse) were used to amplify the 198-bp fragment around the polymorphic site studied. Polymerase chain reaction (PCR) amplification was carried out in a total volume of 50 μL containing ∼100 ng of genomic DNA, 2 mM dNTPs, 8 pmol of each primer, 1.5 mM MgSO4 and 5 U Taq polymerase (Biomatik taq), and 2.5 μL of 10× PCR buffer.

The reaction profile was as follows: predenaturation at 94°C for 5 min followed by denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 40 s for 30 cycles, with a final extension at 72°C for 10 min.

Genotyping of SNP C677T in the MTHFR gene

PCR amplicons were digested with the restriction enzyme HinfI (Promega, Madison, WI). DNA products were separated on a 2% (or 2.5%) agarose gel, stained with ethidium bromide, and visualized under ultraviolet light.

Statistical analysis

All samples were genotyped, and the allele and genotype frequencies of the patients and controls were calculated by counting. The differences in allelic and genotypic frequencies of the C667T locus between groups were evaluated using the Chi-squared test with odds ratio (OR). p-values<0.05 were deemed as being statistically significant.

Results

We analyzed the polymorphism of the MTHFR gene in 74 infertile and 84 fertile men by PCR-restriction fragment length polymorphism. The representative results of genotyping for the C677T locus in the MTHFR gene by electrophoresis are shown in Figure 1.

FIG. 1.

The results of genotyping of C677T in the methylenetetrahydrofolate reductase (MTHFR) gene. M, DNA size marker; CC, wild-type homozygote; CT, heterozygote; TT, mutant homozygote.

The wild-type homozygote (CC), heterozygote (CT), and mutant homozygote (TT) showed one band (198 bp), two bands (198 and 175 bp), and one band (175 bp), respectively, because allele T produced a cut site for HinfI.

Also, the results of statistical analysis for the fertile and nonobstructive infertile men are summarized in Tables 1 and 2.

Table 1.

Distribution of the MTHFR C677T Genotype in Infertile (n=74) and Fertile Males (n=84)

	Fertiles males		Infertiles males
	%	n	%	n	OR	p
677 CC	42.86	36	41.89	31	—	—
677 CT	45.24	38	44.59	33	1.01 (0.49-2.08)	0.980
677 TT	11.90	10	13.51	10	1.16 (0.38-3.53)	0.769
CT+TT	57.14	48	58.11	43	1.04 (0.53-2.06)	0.902
C allele	65.48	110	64.19	95	—	—
T allele	34.52	58	35.81	53	1.06 (0.55-2.04)	0.848

MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio.

Table 2.

The Frequency of the MTHFR 677 Genotype According to the Two Subtypes of the Infertile Male Group

	677CC	677CT	677TT	CT+TT	T allele
Azoospermia	43.48%	41.30%	15.22%	56.52%	35.87%
n=46	n=20	n=19	n=7	n=26	n=33
OR (95% CI)	—	0.90 (0.39-2.10)	1.26 (0.36-4.36)	0.98 (0.44-2.15)	1.02 (0.48-2.17)
p	—	0.790	0.682	0.945	0.946
OAT	39.29%	50%	10.71%	60.71%	35.71%
n=28	n=11	n=14	n=3	n=17	n=20
OR (95% CI)	—	1.21 (0.44-3.31)	0.98 (0.18-4.97)	1.16 (0.45-3.04)	1.13 (0.45-2.86)
p	—	0.687	0.980	0.740	0.779
Fertile males	42.86%	45.24%	11.90%	57.14%	34.52%
n=84	n=36	n=38	n=10	n=48	n=58

CI, confidence intervals; OAT, oligoasthenoteratozoospermia.

The frequencies of the heterozygote MTHFR 677CT and the homozygous MTHFR 677TT among the fertile and infertile men were 45.24% and 44.59% and 11.90% and 13.51%, respectively. For this polymorphism, we did not observe any statistically significant association with reduced sperm counts in the Algerian population (OR=1.01, p=0.980; OR=1.16; p=0.769, respectively).

In Table 2, the infertile men (n=74) were classified into azoospermia (n=46) and OAT (n=28) according to semen analysis. We analyzed the C677T variation between azoospermia and OAT groups. The frequencies of the homozygous TT were 15.22%, 10.71%, respectively, but no significant difference was observed (p=0.75).

Discussion

Data regarding the influence of MTHFR gene polymorphisms on male fertility status are scarce and conflicting. The 677C>T mutation of the MTHFR gene has been associated with a thermolabile enzyme with decreased activity that may cause an increase in plasma homocysteine concentrations when the folate status is poor (Mayor-Olea et al., 2008). This polymorphism is one of the most widely studied polymorphisms in humans, as it is related to various multifactorial disorders. There is considerable experimental evidence that key enzymes in the folate metabolism are necessary for male spermatogenesis. MTHFR plays an important role in folate metabolism, and tHcy levels could affect DNA synthesis and methylation. In adult male mice, severe MTHFR deficiency results in abnormal spermatogenesis and infertility (Tamara et al., 2005).

In humans, the allele T of SNP C677T can reduce MTHFR activity, and subjects with the genotype TT are associated with decreased globe genomic methylation compared those with the genotype CC (Zhou-Cun et al., 2007). In addition, individuals who are homozygotes for the MTHFR thermolabile mutation have elevated fasting homocysteine concentrations when the plasma folate concentration is in the lower range (Jacques et al., 1996), and a high level of homocysteine can induce the auto-oxidation that might cause DNA damage. Besides possible damage to the germ line DNA, oxidative stress could also damage the cell membranes (Sheweita et al., 2005). Also, an increase in sperm quality (such as sperm count and motility) after one cycle of spermatogenesis of treatment with folic acid has been reported (Bentivoglio et al., 1993). A follow-up study revealed that this effect occurred in men with a wild-type MTHFR 677CC, and there was no significant increase in the sperm concentration observed in either the MTHFR heterozygous 677C/T or MTHFR homozygous T/T cohorts (Ravel et al., 2009). These data indicate that folate metabolism plays a key role in the maintenance of spermatogenesis.

Recently, Safarinejad et al. (2011) have found a positive correlation between serum folate concentrations and sperm density (r=0.74, p=0.001), percentage of sperm with progressive motility (r=0.68, p=0.001), as well as percentage of sperm with normal morphology (r=0.72, p=0.001). Several studies reported the association of the SNP C677T polymorphism in the MTHFR gene with male infertility (Bezold et al., 2001) (Table 3). Han-Chul et al. (2006) have reported, by analysis of a large number of subjects and a more specific patient selection, the first genetic evidence that MTHFR C677T, methionine synthase (MS) A2756G, and methionine synthase reductase (MTRR) A66G genotypes were independently associated with male infertility in a Korean population. The MTHFR 677TT genotype has been suggested to be a genetic risk factor for male infertility, especially with severe OAT and nonobstructive azoospermia in unexplained infertile males (Park et al., 2005; Zhou-Cun et al., 2007; Tetik et al., 2008). Three studies on Brazilian, Portuguese, and Finnish males, respectively, all found an association between the SNP C677T in the MTHFR gene and infertility in men with azoospermia and severe oligozoospermia, and suggested that this mutation might be a genetic risk factor for infertility in these populations (Reyes-Engel et al., 2002; Castro et al., 2004; Gava et al., 2010).

Table 3.

Genotype Distribution of MTHFR C677T in Infertile Patients and Controls from Different Studies

			Frequency of genotypes
Population	Group	Number of cases	CC	CT	TT	References
German	Controls	200	46.0	44.5	9.5	Bezold et al. (2001)
	Patients	225	44.7	36.5	18.8^a
Netherlandic	Controls	112	44.2	42.5	13.3	Ebisch et al. (2003)
	Patients	77	54.5	36.4	9.1
Italian	Controls	105	31.4	41.0	27.6	Stuppia et al. (2003)
	Patients	93	39.8	38.9	20.4
Korean	Controls	396	36.6	50.5	12.9	Park et al. (2005)
	Patients	373	28.15	55.0	16.9^a
Indian	Controls	200	81.5	18.5	0	Singh et al. (2005)
	Patients	151	69.5	26.5^a	4.0^a
Chinese	Controls	252	50.8	37.7	11.5	Zhou-Cun et al. (2007)
	Patients	355	36.6	45.1	18.3^a
Turkish	Controls	50	60	40	0	Tetik et al. (2008)
	Patients	100	44.0	44.0	12.0^a
French	Controls	114	42.98	45.62	11.4	Ravel et al. (2009)
	Patients	252	47.18	41.59	11.23
Brazilian	Controls	233	71.7	53	5.6	Gava et al. (2010)
	Patients	156	52.85	36.3	10.9^a
Iranian	Controls	328	ND	ND	ND	Safarinejad et al. (2011)
	Patients	164	ND	ND	ND^a

Significant difference between the patient and control group was observed.

CC, wild-type homozygote; CT, heterozygote; TT, mutant homozygote; ND, not done.

In contrast, Ebisch et al. (2003) concluded that the C677T polymorphism is not a risk factor for male infertility and also indicated the importance of folates in sperm production. Stuppia et al. (2003) reported that there was no significant difference in the frequency of the CT heterozygote and T homozygotes and also suggested that some genes other than the MTHFR gene in the DNA methylation pathway could affect male infertility. Additionally, two French studies have examined the incidence of polymorphisms in the genes MTHFR (R68Q, A222V, and E429A), MTRR (I22M and S175L), and cystathionine beta-synthase (CBS; G307S). Ravel et al. (2009) and Montjean et al. (2011) have reported that there was no significance between reduced sperm counts and polymorphisms in enzymes involved in folate metabolism in the French population.

Our results also showed that the MTHFR C 677T is not a risk factor for male factor subfertility. The explanation of our data may lie in the effect that environmental factors and other genes have also a role on the semen quality.

These contradictory results from studies on different populations suggest that the role of C677T in susceptibility to male infertility might depend on ethnic or geographic factors (Zhou-Cun et al., 2007). Moreover, gene-nutrient/environmental and gene-racial/ethnic interactions have been shown to affect the impact of these MTHFR genetic variants (Toffoli and De Mattia, 2008). Also, it is reported that association studies with a larger sample size and hence power of study need to be performed to identify a modest risk associated with these polymorphisms (Ravel et al., 2009). In whole-genome scans, standard power calculations show that up to 1000 participants are required to detect major gene main effects, and at least 4000 samples are required to detect small effects (Wang et al., 2005). However, some studies have suggested that strong associations can be detected even in modestly sized samples (Purcell et al., 2003). It is reported that the studies with larger cohorts are those who report modest associations with MTHFR polymorphisms (Ravel et al., 2009).

Conclusion

We suggest that an extended study should be undertaken that includes other possible polymorphisms and genes that participate in homocysteine metabolism, such as MTHFR A1298C and G1793C for polymorphisms of MTHFR, MS, and MTRR gene related with male infertility.

Footnotes

Acknowledgments

The authors appreciate the kind participation of the patients and controls. We are grateful to all members of the Biology and Genetics Laboratory, the PMA Ibn Rochd Centre, and private laboratories of Algeria for their contributions to this work.

Author Disclosure Statement

No competing financial interests exist.

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