Among the sperm parameters, only sperm concentration was significantly associated with DNMT3B mRNA, and the sperm concentration decreased as a result of an increase in this transcript. Although no study has investigated methyltransferase in relation to sperm analysis, the presence of a polymorphism in DNMT1 may be associated with oligoasthenospermia (29).
A report on the mRNA content of 177 imprinted genes epigenetic regulation in male subfertility, DNMT3A was present in increased amounts in poor sperm motility, without any significant correlation with sperm concentration. These findings indicate the failure of abnormal sperm to obtain a proper methylation pattern (30).
4.6. Association between methyltransferase expression, sperm chromatin and DNA integrity
The results of TUNEL+ had a significant correlation with the DNMT3A mRNA and DNMT3 B mRNA level in such a way that an increase in these transcripts was followed by the increased fragmentation of sperm DNA. Although we found very little explanation in relation to these variables and mechanisms in the previous studies, it is possible to deal with the general association between chromatin integrity and the factors involved in epigenetic regulation. In line with these findings, in relation to apoptotic cells of sperm, some authors have pointed out that the initial response to oxidative stress may be due to an increase in DNA methyltransferase expression (31).
A report on the association between the expression of the two epigenetic genes, HDAC1 and DNMT3A, and chromatin integrity showed, if HDAC1 is expressed too much, it can be assumed that these histones are not replaced by protamine, which results in an immature chromatin structure sperm with a less compacted DNA. Therefore, incorrect and incomplete nuclear density due to the increased expression of HDAC1 and DNMT3A may affect the overall maturity of the sperm (30).
4.7. Association between global methylation and sperm quality
Global methylation has a significant correlation with concentration, progressive motility, non-motility and normal morphology. The increased global methylation in patients was associated with the decreased concentrations, progressive motility and normal morphology. However, Olszewska et al (2017) investigated the global methylation level in sperms containing chromosomal and normospermia disorders using chromatography and immunofluorescence techniques found no association between global methylation and sperm quality (31).
A report on oligoasthenospermic patients, about 45% with less than 10 million sperm per mL showed a defective methylation of H19 or MEST (27). The reason for this difference is probably due to the unknown environment, environmental interactions of genes, lifestyle, or other unmeasured factors that affect the expression and activity of DNA methyltransferases and the availability of methyl groups.
4.8. Association between global methylation, sperm chromatin and DNA integrity
Our study showed that global methylation is significantly correlated with all the results of chromatin integrity tests including TUNEL, Aniline blue, Toluidine blue, and an increase in the global methylation in patients leads to an increase in chromatin and sperm DNA disorders.
The results of the increased fragmentation of DNA in infertile men with the increased global methylation of sperm DNA showed that this status is followed by a decrease in the quality of semen, chromatin integrity and sperm DNA(32). Similarly, the analysis of the correlation between the 5mC global level and the chromatin-deprotaminization state using the Aniline Blue test indicated a positive association between these two variables in the patients group (31). On the other hand, there is a tendency to intrinsic apoptotic cascade in relation to the state of global methylation of sperm DNA (33). Some researchers have shown that low concentration of folate in seminal plasma is associated with further damage to sperm DNA in infertile men. Low concentration of folate and homocysteine disrupts the methylation cycle. When uracil misincorporation occurs with folate deficiency, instability will occur in the chromosome and the supply of methyl groups, which are important in protecting DNA, will decrease against exposure to free radicals (34).
Clinical application and conclusion
The most important modification occurs in the structure of sperm chromatin and at a higher level of nuclear organization shortly after fertilization, which is important for starting and regulating the activity of the paternal gene during the early stages of embryo development.This study has not dealt with the ART results to evaluate the effects of improving the epigenetic status, along with the expression of methyltransferase and global methylation on the success of IVF treatment.
Reducing global methylation of the sperm DNA after treatment including folic acid could affect the results of ART due to the population of sperm with higher epigenetic health. As the association between global methylation of sperm and fertilization or embryo quality has been observed (33). In conclusion, improvement by decreasing level in global methylation in three-drug treatment in order to improve the epigenetics, chromatin and sperm quality in patients may be associated with an increase in ART success due to the effects of folic acid.