The influence of semen quality on male pronucleus demethylation process during ICSI cycle.

There is growing evidence that the spermatozoon's epigenetic structure is of the utmost importance in the health of the future embryo. Following fertilization, sperm chromatin undergoes epigenetic reprogramming including DNA demethylation and remethylation, which resets gene expression. In some infertile patients, it is inevitable that sperm cells that are not within the range of normal human sperm parameters will be used for intracytoplasmic sperm injection. Understanding the relationship between the human sperm parameters and male pronucleus DNA demethylation seems necessary. We hypothesized that demethylation of the male pronucleus might be altered in zygotes conceived from a spermatozoa obtained from a sample exhibiting an abnormal semen analysis profile. To test the hypothesis, sperm cells from normal and abnormal human semen samples were injected into mouse oocytes. A group of cultured zygotes was fixed before the onset of DNA demethylation and the other group was fixed after DNA demethylation. Both groups were then labeled with a 5 methylcytosine antibody and the level of pronuclei methylation was detected as a function of fluorescent intensity. The level of demethylation was then determined as the difference between 5 methylcytosine fluorescent intensity before and after DNA demethylation. A negative correlation (p<0.05) was observed between sperm motility, morphology, percentage of head defects, protamine deficiency, and DNA demethylation level. However, no correlation was found between the demethylation level and sperm count. In conclusion, these observations suggest that demethylation is altered in the male pronucleus when low quality sperm samples are used.