Association of methyltetrahydrofolate reductase gene mutation, homocysteine level with semen quality of Iraqi infertile males

Infertility is very common condition and almost 50% of cases are due to male factors. Several genetic and environmental factors are responsible for the poor quality and reduced number of sperms in several cases of infertility. The present study was designed to investigate the association between semen parameters, homocysteine, and the risk of C677T polymorphism of MTHFR gene in infertile males of Iraqi population. This Case–control study has been conducted from February 2019 to July 2021 at a molecular laboratory in the Anatomy and Histology Department/college of Medicine/University of Kufa/Najaf/Iraq. It was composed of 353 infertile male patients. They were divided into five groups: 90 azoospermic, 84 oligospermia, 64 asthenospermic, 50 oligoasthenospermic, and 65 teratospermic with an age range 20–46 years compared with 100 fertile males as control with age range 21–49 years. In order to detect homocysteine levels, we used Hcy ELISA Kit. C677T mutation of MTHFR gene was employed by PCR–RFLP technique. Our data revealed three genotypes of MTHFR C677T, 167 (47.3%) subjects had CC genotype, 116 (32.9%) subjects had CT genotype and 70 (21.1%) subjects had TT genotype. Furthermore, T allele was associated with higher risk of infertility in all patients groups for any genetic model. In total infertile subjects (codominant model: CT vs. CC, OR = 2.0, 95% C.I = 1.2–3.3, P = 0.011; TT vs. CC, OR = 4.8, 95% C.I = 3.3–8.2, P = 0.0003; dominant model: CT + TT vs. CC, OR = 2.8, 95% C.I = 1.7–4.5, P = 0.0001). Oligoasthenospermic patients associated with higher risk in CT heterozygous genotype (OR = 2.8, 95% C.I = 1.0–4.9, P = 0.03) and TT homozygous of mutant allele (OR = 6.3, 95% C.I = 1.9–9.2, P = 0.002). Homocystein level was elevated in all infertile groups when compared with control group (P < 0.01), but the elevation was marked in oligoasthenospermia group. As well as, the level of Serum Hcy exhibited the highest value in TT mutant genotype (39.7 µmol/ml) followed by CT genotype (28.5 µmol/ml) while the lowest level of Hcy recorded in CC genotype (14.6 µmol/ml) for oligoasthenospermia group. By relating the MTHFR C677T gene mutation with a higher homocystein level, the results showed that Iraqi males with this mutation are more likely to suffer from infertility.


Background
Infertility is a common health problem, affecting approximately 15-20% of couples who approaching childbearing age. Almost 50% of infertility due to male factors [1]. About 12% of men suffer from male infertility and approximately 15-30% of male infertility is attributed to genetic abnormalities, which include Y chromosome microdeletions, chromosomal aberrations, translocation, and single gene mutation. Several factors may contribute to sperm quality and quantity reductions in some infertile men, including deleterious gene mutations in key genes which are involved in testicular function [2,3].
Mutations in genes involved in folate metabolism may influence spermatogenesis. The methylenetetrahydrofolate reductase gene (MTHFR), which is involved in folate metabolism, has been associated with abnormal spermatogenesis [4]. MTHFR is associated with an important pathway of folate mediated methyl group metabolism. It is also involved in metabolizing thymidine and purine, which are vital for dividing male germ cells to synthesize DNA. The 5,10-Methylenetetrahydrofolate is irreversibly reduced to 5-methyltetrahydrofolate by MTHFR, leading to the synthesis of methyl donor, which is required for methionine synthesis from homocysteine. Similarly, methionine forms S-adenosylmethionine, which is involved in a number of cellular reactions, including DNA, RNA, and histone methylation [5,6].
Homocysteine accumulation and inappropriate methylation lead to DNA damage in spermatozoa which may reduce semen quality, affecting concentration, motility, and morphology of sperm [7,8]. Therefore, deficiency in folate intake or mutation (s) in the folate pathway enzymes may result in aberrant DNA synthesis and methylation [9].
In adults testis of a mice, the activity of MTHFR is higher than other organs, representing its role in spermatogenesis [10,11]. Therefore, alterations in the MTHFR gene could change the process of spermatogenesis and predispose carriers to infertility [12,13].
MTHFR is a gene that regulates the synthesis of methylenetetrahydrofolate enzyme, is a key enzyme in homocysteine and folate metabolism, which acts as a carbon donor for remethylation of homocysteine to methionine. This enzyme reduces 5, 10-methylenetetrahydrofolate to 5, 10-methylenetetrahydrofolate, which is the predominant form of folate in the bloodstream. MTHFR gene is located on chromosome 1 p36.3, composed of 11 exons [14]. A total of 65 polymorphisms have been found in the MTHFR gene. The C677T (rs1801133) polymorphism results in a reduction of enzyme activity by 35%, which adversely affects nucleic acid metabolism [15].
Numerous studies have investigated the relationship between MTHFR gene mutation and male infertility, however, the conclusions are controversial [1, 4-6, 9, 16]. The cause of these conclusions can be attributed to small sample size, definition of infertility, and confounding factors such as ethnicity and race [17]. In the current study, we aim to identify a possible relationship between idiopathic sperm disorders, homocysteine concentration, and the MTHFR C677T mutation in the Iraqi population.

Methods
All participants provided informed consent before specimens were taken. The medical ethics committee at the University of Kufa/Faculty of Medicine approved the consent protocol for this study. During the period from February 2019 to July 2021, total of 353 infertile male patients and 100 age-matched fertile males were examined for this study. All patients were tested for seminal fluid analysis. Culture media preparation, Giemsa stain, and chromosome cytology have been published elsewhere [18][19][20].
We selected the patients for the study based on andrology tests, including karyotyping and microdeletions of the Y chromosome. The study excluded individuals with cryptorchidism, atrophic azoospermia, numerical or structural chromosomal abnormalities, or Y-chromosome microdeletions.
A current case-control study was conducted in the Fertility center of Al-Sadder teaching Hospital and in the laboratories of the Faculty of Medicine/Kufa University in the Province of AL-Najaf /Iraq. The 353 infertile male patients were divided to five groups according to WHO 2020 criteria of semen parameters (colour, pH, semen volume, liquefaction time by macroscopic analysis while sperm count, sperm motility, and sperm morphology by using microscopic analysis). It is considered normal for men with ˃16 million sperms/milliliter [18]. There were 90 men with lack of sperm as azoospermic, 84 men with sperm count < 16 million/milliliter were oligospermic, 64 of men with total motility < 42% of sperm activity were asthenospermic, 65 of men with < 54% abnormal sperm morphology were teratospermic and 50 of men were oligoasthenospermic, there age range (20-46 years) compared with age matched 100 normal fertile males as control with age range (21-47 years).
Semen examinations were conducted twice a month for each participant after three days of abstinence, after centrifugation for 10 min at 1006 ×g, semen samples were analyzed, based on reference values published by the World Health Organization, the mean values of seminal fluid analyses were recorded and used as average results [18].
From all participants a total of five milliliter venous blood samples were collected. one ml of blood sample was drowning in EDTA tube for DNA extraction, another amount of blood samples were centrifuged for 10 min at 1006 ×g, and serum were separated and stored at − 17 °C until the assayed was performed. Total plasma homocysteine was analyzed according to the manufacturer procedure for determination by using microplate enzyme immunoassay ELISA kit method of Biorad laboratories. The amplification of DNA was done by polymerase chain reaction (PCR) using primers 5′-TGA AGG

Statistical analysis
An analysis of statistical data was performed using the SPSS software package (revision 20, Inc., Chicago, USA). Data are expressed as means ± SD. The Chi-square statistic was used to compare the genotype distributions between patients and controls. In order to estimate the risk of male infertility, odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated. P < 0.05 values were considered statistically significant.

Results
A total of 353 infertile men patients and 100 normal fertile men were enrolled in the current study. The patient's group was divided into five subgroups: 90 azoospermia, 84 oligospermia, 64 asthenospermia, 50 oligoasthenospermia, and 65 teratospermia. The test of chi-square (X 2 ) was done for analyzed the Hardy Weinberg equilibrium (HWE) among different patients groups and control groups, the results were indicated that an observed and expected frequencies were consistent among the three genotype groups (P > 0.05) indicating that the frequency of this sample was representative the Iraqi population.
As shown in Table 2, homocysteine level of each infertile group according to MTHFR gene polymorphism (rs:1801133) genotypes (co-dominant model), was compared with control group and the results showed significant increase in Hcy levels in infertile groups when compared with control group (P < 0.01). There were high levels of serum Hcy in homozygous genotype (TT) when compared with heterozygous genotype (CT) and normal allele genotype (CC) in all patient groups. But the highest levels of Serum Hcy was recorded in oligoasthenospermia group (31.5 µmol/ml) followed by the azoospermia group (28.7 µmol/ml), asthenospermia group (25.9 µmol/ml), teratospermia group (24.2 µmol/ml), and oligospermia group (21.3 µmol/ml), whereas the lowest level of Hcy appeared in the control group (12.1 µmol/ ml). As well as, the level of Serum Hcy exhibited the highest level in oligoasthenospermia group of TT mutant genotype (39.7 µmol/ml) followed by CT genotype (28.5 µmol/ ml) while the lowest level of Hcy was recorded in the CC genotype (14.6 µmol/ml).

Discussion
Spermatogenesis is a complex development process that involves mitosis, meiosis, and spermiogenesis. Methyl group formation mediated by folate, is one of many pathways regulating this complex process. Besides providing the bases for DNA synthesis in rapidly dividing male germ cells, this pathway is also critical for the removal of homocysteine and several methylation reactions such as methylation of DNA, RNA, histones, and other molecules [5,21]. Folate deficiency is associated with various illnesses including male infertility [22]. As a result of changes in folate status, spermatogenesis can be affected by DNA hypomethylation and uracil misincorporation during DNA synthesis, resulting in errors in DNA repair and chromosomal anomalies [23]. There is clear evidence that hyperhomocysteinemia increases the risk of cardiovascular disease [24] and neural tube defects [25,26], but its effects on spermatogenesis remain unclear. The epigenetic architecture of sperm DNA has been studied and it has been shown that sperm DNA, histones methylation patterns are vital for normal sperm function, as well as there is significant association between methylation of certain genes and sperm concentration, motility, and morphology [27][28][29][30][31][32][33]. Numerous studies have suggested that mutations in the MTHFR C677T gene may be associated with decreased sperm counts in the human that lead to male infertility in a number of populations [34][35][36][37]. Infertility in men has been studied in different populations to estimate the association between homocysteine levels, MTHFR gene and infertility. In fact, the results were conflicted due to differences in sample size,  ethnicity, race, geographic variations, genetic variation, and exposure to environmental risk factors [38][39][40]. Up to our knowledge, there is no data regarding the relationship between the MTHFR C677T polymorphism, homocysteine and semen parameters in Iraqi infertile patients, so this study is designed to assess the relationship of MTHFR gene polymorphism, homocysteine levels, and semen parameters quality in Iraqi population.
The results revealed that MTHFR C677T gene mutation is a risk factor for male infertility in our population, also, it appeared high homocysteine levels in azoospermic, oligospermic, asthenospermic, teratospermic, and oligoasthenospermic patients groups. Highest level was in oligoasthenospermic group. TT mutant genotype was more than CT genotype while the lowest level of homocysteine recorded in CC genotype.
Our results were consistent with the Egyptian study which one [41] out of four studies in Africa showed a high association of 677 T with men's infertility [41][42][43][44]. A two Iranian studies were found that carriers of the 677 T allele (CT and TT genotypes) at a higher risk of infertility than individuals with other genotypes [45,46], In Chinese men with azoospermia and severe oligozoospermia, an SNP in the MTHFR gene appeared to be associated with infertility [47], an Italian study of Lombardo et al. [48] who recorded that a function of C677T mutation in MTHFR leads to hyperhomocysteinemia, is associated with erectile dysfunction mainly in homozygous 677 TT patients. Also in France Montjean et al. [49] and Ménézo et al. [50] was recorded the MTHFR 677TT genotype is combined with significantly increased plasma homocysteine levels, in Netherlands Boxmeer et al. [51] was recorded increased sperm damage by increased homocysteine and decreased folate in seminal plasma. A number of Asian studies [52][53][54][55] expressed this association, Mutant genotypes are significantly associated with fertility by Gupta et al. [56] and Kumari et al. [57] studies in India, another study in Italy that conducted by Paracchini et al. [58] was found the risk of infertility increased with TT genotype in the C677T locus of MTHFR gene.
In contrast, the MTHFR 677 T mutation was found to be protective for Moroccan infertile men [59]. Other studies in the Indian population [60,61] were deserved no significant differences of C677T variants between fertile and infertile males. Celia et al. [62] found no evidence for an association between C677T polymorphism in enzymes involved in folate metabolism and reduced sperm counts in the French population.

Conclusion
By relating the MTHFR C677T gene mutation with a higher homocysteine level, the results showed that Iraqi males with this mutation are more likely to suffer from infertility. Additional data are needed to confirm our conclusion and provide an important relationship between genes and the environment that affects male infertility risk.