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In our study, for the first time, the folic
acid was administered with its epigenetic application regarding the expression
of methyltransferases, global DNA methylation and chromatin in human sperm.

Of the 88 OAT subjects in the initial study, 38
subjects failed to return in arranged time, and 20 others refused to continue
the treatment because of entering the IVF cycle. Finally, 30 infertile OAT
patients were randomly divided into two treatment groups. No complications were
reported 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 within
the normal range and similar in the fertile and infertile groups before the
onset of drug intervention (22). For this reason, we did not examine folate
concentrations in this study.

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4.2. Treatment groups and sperm quality, sperm
chromatin and DNA integrity

A significant improvement was observed in the
three-drug treatment group after treatment with respect to sperm parameters.
Sperm parameters such as concentration, progressive motility, normal morphology
and sperm viability were significantly higher in the three-drug treatment. In
line with this finding, a study investigated the effect of 5 mg folic acid and
66 mg zinc sulfate on semen variables in infertile men for 26 weeks, reporting
a significant increase in the total number of sperms (22).

 

The results of toluidine blue and TUNEL tests showed significant improvement in both
treatment groups compared with pretreatment. In addition, the Aniline blue+
results showed that there was a significant decrease in the amount of excess of
histone in the three-drug treatment group compared with pretreatment. In this
regard, Amar et al. also found that treatment with antioxidants and B vitamins
including folic acid, needed for one-carbon cycles in metabolism, improved
sperm chromatin more than treatment with only antioxidants by decreasing sperm
DNA fragmentation level and Aniline blue+ results (23). Moreover, in the epigenetic role, folate can
lead to fertility through its function in DNA synthesis because of its
important role in biosynthesis, of purine, pyrimidine, and certain amino acids (24).

 

4.3. Treatment
groups, methyltransferases expressions and global DNA methylation

Examining the methyltransferase transcripts
showed that transcripts had an insignificant change after treatment. On the
other hand, global methylation of sperm DNA in a treatment group of three drugs
including 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 administration
of folic acid by examining epigenetic variables, it can be discussed in terms
of folate poverty. Epigenetic changes in cancer caused by folate deficiency in
rats significantly reduced S-adenosyl-L-methionine (SAM) concentrations in the liver tested in 9 weeks. The level of
mRNA and protein of the Dnmts changed in response to the methyl deficiency
after 9 months (25). Although
methyltransferase expression did not change, methylation increased in patients
and decreased after three- drug treatment, conditions can be like the cancer.

Global hypomethylation is a common epigenetic
incidence during the early stages of cancer, especially in repetitive elements
and oncogenes. The interesting observation is that in addition to the
hypomethylation of DNA, folate and methyl deficiency can cause regional
hypermethylation. This finding manifested itself in the progressive increase in
transcription 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 methyltransferase
expression and sperm quality

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