An in-depth study of specific pathway linked to abnormal reproductive system: A meta-analysis in a specific Caucasian ethnicit

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Central Asian Journal of Medical and Pharmaceutical Sciences Innovation 1 (2021) 1–7
An in-depth study of specific pathway linked to
abnormal reproductive system: A meta-analysis in a
specific Caucasian ethnicit
Mohsen Mohammadi
Department of educational medicine, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Highlights Graphical Abstract
Article Info Abstract
* Corresponding author: mohsenmohammadi.tums@gmail.com (M. Mohammadi)
RESEARCH PAPER
• Methylene tetrahydrofolate
reductase gene has a chief role in
DNA truthfulness.
• The rs1801133 is a common
variation in MTHFR gene.
• The C677T gene variation is
associated with risk of male
infertility.
The human methylene tetrahydrofolate reductase (MTHFR) gene plays a vital role
in folate metabolism. This gene is located on chromosome 1 and has 12 exons.
Many single nucleotide polymorphisms are found in the MTHFR gene. One of the
key polymorphisms in this gene is C677T variation. In a literature review four
experimental works investigating the correlation of the mentioned polymorphism
with male infertility in Iranian population were enrolled (a specific Caucasian
ethnicity). To obtain more comprehensive outcomes, a meta-analysis was made by
pooling the data from four existed papers. Eligible studies were found the by a
search in appropriate electronic databases. The data from four studies were
extracted and so analyzed by Metagenyo online software. The results showed that
there are significant associations between C677T gene polymorphism and male
infertility in TT vs. CC, CT vs. CC, TT+TC vs. CC, and TT vs. TC+CC genetic
models within Iranian population without any publication bias. The results showed
that polymorphisms in the MTHFR gene could be associated with infertility in men.
Therefore, determining the genotype of a polymorphism in this gene will be very
useful for screening infertile people. © 2021 Published by CAS-Press.
Receive Date: 03 December 2020
Revise Date: 19 January 2021
Accept Date: 21 February 2021
Available online: 25 February 2021
Keywords:
Male reproductive system
MTHFR gene
Polymorphism
Meta-analysis
10.22034/CAJMPSI.2021.01.01

Cent Asian J Med Pharm Sci Innov 1 (2021) 1-7 Mohammadi
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Introduction
Infertility occurs in 15% of couples. Half of the causes of infertility are related to male factors Furthermore;
about 15-30% of these male factors are related to genetic factors (1). Defects in genes involved in
spermatogenesis can cause infertility (2). Polymorphisms in autosomal genes such as CFTR, BRCA2, etc., have
been shown to play an essential role in male infertility (3).
Another gene in which polymorphisms can increase the risk of infertility is the MTHFR gene. The product of
this gene is involved in the conversion of homocysteine to methionine (4). Deficiency in this gene causes
homocysteine accumulation and folate depletion, sleading to hyperhomocysteinemia and decreased serum
folate. Studies show that folate deficiency increases DNA failure (5). Hyperhomocysteinemia can be caused by
low MTHFR gene activity or a deficiency in vitamin B12 uptake (6). Low levels of vitamin B12 and folate in
serum are responsible for two-thirds homocysteinemia cases (7). The amount of vitamin B12 that a person
absorbs during the day determines a person's serum homocysteine level. High serum homocysteine levels can
be affected by environmental factors such as smoking, heavy coffee consumption, and low physical activity (8).
Single nucleotide polymorphisms (SNPs) are abundant in the MTHFR gene, but there are three common
SNPs C677T (rs1801133), A1298C (rs1801131), and G1793A (rs2274976) in this gene. C677T is more common
than the other two (9). Polymorphisms in this area are associated with many diseases such as cardiovascular
abnormalities, depression, and a wide range of cancers (10). The frequency of the 677T allele varies significantly
in different populations. Th allele frequency varies from 30% to 40% in Europe and the United States to 5% to
10% in Africa and Sri Lanka. Previous studies have shown that individuals with the 677TT genotype have more
deficits in intracellular methylation and higher serum homocysteine (6, 11).
There four studies regarding the correlation of C677T gene transition with male infertility in the Iranian men
population, but the outcomes are questionable. Thus, the relationship between this polymorphism and male
infertility in the Iranian population was investigated by meta-analysis with a creative approach.
Materials and Methods
Search procedure
Some suitable global databases were used such as Google Scholar, PubMed, Web of Science, and Persian
records such as Scientific Information (SID) and Magiran to discover eligible articles. Our electronic search
keywords were as follows: polymorphism, MTHFR gene, methylenetetrahydrofolate reductase, male infertility,
Iran, Iranian population, C677T, SNP, and mutation.
Study collection and information extraction
The numerous papers in the initial research were checked and moreover, the citations of all reached papers
were assessed. The following criteria were considered for inclusion of article in meta-analysis: (1) Papers on
human; (2) studies investigating the correlation of C677T variation with male infertility; (3) case-control studies;
(4) adequate data for computing the odds ratio (OR), and their 95% confidence interval (CI). However, the
exclusion criteria were as follows: (1) without sufficient data for the estimating of ORs and 95% CIs; (2) Studies
with other ethnicities except for the Iranian race.
Quantitative synthesis
A Chi-square test was employed to evaluate the Hardy-Weinberg equilibrium (HWE) in all included studies
in our meta-analysis. ORs and 95% CI measured the strength of correlation between MTHFR-C677T gene
variation and risk of male infertility.
The quantitative synthesis was performed for the four following models: Co-dominant homozygote (TT vs.
CC), Co-dominant heterozygote (CT vs. CC), dominant (TT+TC vs. CC), and recessive (TT vs. TC+CC) models.
The heterogeneity among studies was estimated by I2 score and Q test (12), and when the p-value of the
heterogeneity was >0.1, the fixed-effect model was employed (13). Otherwise, the random-effect model was

Mohammadi Cent Asian J Med Pharm Sci Innov 1 (2021) 1-7
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used (14). The publication bias was evaluated by Egger's test (15) and Begg's funnel plots (16). The Metagenyo
online software was used for the statistical analysis.
Results
The search strategy and study selection procedure based on PRISMA criteria is shown in Figure 1. After the
screening possible suitable papers, it is concluded on four eligible articles (10, 17, 18). Some features of these
four eligible studies, including authors' names, publication date, genotyping method, genotype frequencies, are
detailed in Table 1. The frequencies of genotypes in control groups of all four studies met Hardy-Weinberg
criteria (19, 20).
Figure 1. Flowchart for search strategy.
Table 1. Features of included studies in the meta-analysis.
Study
P
HWE
Adjusted
HWE
Genotyping
method
TT
Cases
TC
Cases
CC
Cases
TT
Controls
TC
Controls
CC
Controls
Safarinejad et
al., (21)
0.83
0.826
PCR-RFLP
26
80
58
36
148
144
Nikzad et al.,
(19)
0.48
0.720
PCR-RFLP
24
109
109
13
98
144
Karimian and
Colagar, (13)
0.09
0.261
PCR-RFLP
8
59
51
3
52
77
Najafipour et
al., (18)
0.31
0.619
Sequencing
44
123
113
11
43
66
PCR: polymerase chain reaction; RFLP: restriction fragment length polymorphism.
PHWE: The p-value related to Hardy-Weinberg equilibrium in the control group.

Cent Asian J Med Pharm Sci Innov 1 (2021) 1-7 Mohammadi
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Table 2. Results of meta-analysis.
Genetic model
Analysis
Model
OR
(95%CI)
P-value
tau2
Q (df=3)
PH
I2
Codominant model
(TT vs. CC)
Random
effect
2.2136
(1.5262; 3.2106)
< 0.001
0.000
1.302
0.7266
0%
Fixed effect
2.2136
(1.5262; 3.2106)
< 0.001
-
1.302
0.7266
0%
Codominant model
(CT vs. CC)
Random
effect
1.5132
(1.2215; 1.8745)
< 0.001
0.000
0.7568
0.8598
0%
Fixed effect
1.5132
(1.2215; 1.8745)
< 0.001
-
0.7568
0.8598
0%
Dominant model
(TT+TC vs. CC)
Random
effect
1.6249
(1.3247; 1.9931)
< 0.001
0.000
0.9022
0.8249
0%
Fixed effect
1.6249
(1.3247; 1.9931)
< 0.001
-
0.9022
0.8249
0%
Recessive model
(TT vs. TC+CC)
Random
effect
1.8151
(1.2753; 2.5835)
< 0.001
0.000
1.126
0.7708
0%
Fixed effect
1.8151
(1.2753; 2.5835)
< 0.001
-
1.126
0.7708
0%
OR, odds ratio; CI, confidence interval; PH, P-values for heterogeneity from Q test.
The association results of meta-analysis are summarized in Table 2. As revealed in this table, a true
association between studied gene variation was observed with male infertility in Co-dominant model (TT vs.
CC), Co-dominant model (CT vs. CC), Dominant model (TT+TC vs. CC), Recessive model (TT vs. TC+CC)
genetic models. Also, there was not any significant heterogeneities among the included studies. Also,
publication bias evaluation revealed that there are no significant publication biases in our meta-analysis. The
data from publication bias is showed in Figure 2. The Egger's test was estimated for TT vs. CC as p-value=
0.0637, for CT vs. CC as p-value= 0.2444, for TT+TC vs. CC as 0.278, and for TT vs. TC+CC as 0.057.
Figure 2. Funnel plot. Results of meta-analysis in TT vs. CC (A), CT vs. CC (B), TT+TC vs. CC (C), and TT vs.
TC+CC (D) models.

Mohammadi Cent Asian J Med Pharm Sci Innov 1 (2021) 1-7
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Discussion
In the present study, the association of MTHFR-C677T gene mutation has been evaluated with male
infertility risk in the Iranian population by a meta-analysis. The obtained data revealed significant associations
between the mentioned polymorphism and male infertility in four heterozygotes, homozygote, dominant, and
recessive genetic models. These results could be strong evidence stating the biomarker feature of this variation.
On the other hand, there was no any significant publication bias in the outcomes. So this data could be a reason
why the obtained meta-analysis is reliable.
While the relation between polymorphisms in the MTHFR gene and infertility in men is unclear, number of
several mechanisms can explain the cause of infertility due to mutations in the MTHFR. First, spermatogenesis
is a complex process that involves several genes, and methylation of these genes has a significant effect on the
expression of these genes (20). DNA hypomethylation causes an error in the differentiation of germ cells into
spermatocytes (21). Induction of hypomethylation in mice by treatment with 5-aza-deoxycytidine prevents
spermatogonia from differentiating into spermatocytes (21). Second, mutations in the MTHFR gene cause auto-
oxidation and the production of reactive oxygen species (ROS) (22, 23). Increased ROS production increases
DNA damage mediated by increased homocysteine (24). Human sperm susceptible to oxidative stress and
antioxidants such as folate can relieve oxidative stress and protect sperm DNA against damage. In humans,
sperm produce ROS, leading to high spermatozoon activity, acrosome reaction, and sperm attachment to
oocytes (25, 26). Increased peroxidation of membrane lipids leads to changes in membrane fluidity, so sperm
function is impaired due to metabolic defects, defects in acrosome activation, and the inability of sperm to fuse
with oocytes. Increased membrane lipid peroxidation ultimately leads to a decrease in sperm count, sperm
motility, and loss of sperm morphology (27). Thus, increased DNA damage due to increased homocysteine by
oxidative stress can be infertility in the MTHFR gene (28). Genetic variations may change the RNA structure,
gene expression, and function of the protein. A previous study showed that MTHFR-C677T genetic
polymorphism could impact protein function and structure of RNA (18).
Conclusion
Based on results, the MTHFR-C677T mutation could be considered a genetic risk factor male infertility in
Iran's genetic risk factor for male infertility in Iran. However, there are some limitations in our study which
should be mentioned. For example, the number of eligible studies included in the meta-analysis was too low.
Therefore, more studies with larger sample sizes are essential to finding more precise outcomes. Moreover,
further studies regarding gene-gene, and gene-environmental factors such as folate intake could help reach
more accurate outcomes.
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How to cite this paper:
Mohammadi M. An in-depth study of specific pathway linked to abnormal reproductive system: A meta-
analysis in a specific Caucasian ethnicit. Cent Asian J Med Pharm Sci Innov 2021; 1(1): 1-7.