In subjects in the initial study, 38 subjects failed

In our study, for the first time, the folicacid was administered with its epigenetic application regarding the expressionof methyltransferases, global DNA methylation and chromatin in human sperm.Of the 88 OAT subjects in the initial study, 38subjects failed to return in arranged time, and 20 others refused to continuethe treatment because of entering the IVF cycle. Finally, 30 infertile OATpatients were randomly divided into two treatment groups. No complications werereported at the time of taking the drugs. A study conducted by Wong et al.showed that the folate concentration in the blood and seminal fluid were withinthe normal range and similar in the fertile and infertile groups before theonset of drug intervention (22). For this reason, we did not examine folateconcentrations in this study.

 4.2. Treatment groups and sperm quality, spermchromatin and DNA integrity A significant improvement was observed in thethree-drug treatment group after treatment with respect to sperm parameters.Sperm parameters such as concentration, progressive motility, normal morphologyand sperm viability were significantly higher in the three-drug treatment. Inline with this finding, a study investigated the effect of 5 mg folic acid and66 mg zinc sulfate on semen variables in infertile men for 26 weeks, reportinga significant increase in the total number of sperms (22). The results of toluidine blue and TUNEL tests showed significant improvement in bothtreatment groups compared with pretreatment.

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In addition, the Aniline blue+results showed that there was a significant decrease in the amount of excess ofhistone in the three-drug treatment group compared with pretreatment. In thisregard, Amar et al. also found that treatment with antioxidants and B vitaminsincluding folic acid, needed for one-carbon cycles in metabolism, improvedsperm chromatin more than treatment with only antioxidants by decreasing spermDNA fragmentation level and Aniline blue+ results (23). Moreover, in the epigenetic role, folate canlead to fertility through its function in DNA synthesis because of itsimportant role in biosynthesis, of purine, pyrimidine, and certain amino acids (24).  4.3. Treatmentgroups, methyltransferases expressions and global DNA methylationExamining the methyltransferase transcriptsshowed that transcripts had an insignificant change after treatment.

On theother hand, global methylation of sperm DNA in a treatment group of three drugsincluding folic acid was significantly reduced compared with pretreatment. But,this did not occur for the two-drug treatment group.Since there is no study on the administrationof folic acid by examining epigenetic variables, it can be discussed in termsof folate poverty. Epigenetic changes in cancer caused by folate deficiency inrats significantly reduced S-adenosyl-L-methionine (SAM) concentrations in the liver tested in 9 weeks.

The level ofmRNA and protein of the Dnmts changed in response to the methyl deficiencyafter 9 months (25). Althoughmethyltransferase expression did not change, methylation increased in patientsand decreased after three- drug treatment, conditions can be like the cancer. Global hypomethylation is a common epigeneticincidence during the early stages of cancer, especially in repetitive elementsand oncogenes. The interesting observation is that in addition to thehypomethylation of DNA, folate and methyl deficiency can cause regionalhypermethylation. This finding manifested itself in the progressive increase intranscription and DNMT1 protein levels (26), very similar to disorders of methylation,hypermethylated and hypomethylated regions of the sperm genome in infertile men(27, 28). 4.4. Association between methyltransferaseexpression and sperm quality