ART reproductive outcome: Effects of sperm DNA fragmentation and methylation of H19 and IGF2 genes. A pilot study

Genomic imprinting is an epigenetic modification that results in monoallelic expression of specific genes depending on parental ori-gin. H19/IGF2 are among the most interesting imprinted genes inmale infertility. They are located on chromosome 11p15.5 and theirexpression is regulated by methylation. Alterations of methylation inH19/IGF2 genes have been reported in cases of impaired spermato-genesis and miscarriages. The purpose of this study was to evaluatesperm DNA fragmentation (SDF) and methylation profile of H19 andIGF2 genes in sperm DNA to investigate the possible relation withimpact on ICSI outcomes and embryo development. Men whose female partners underwent ICSI cycles were recruited.Following analyses were performed on semen: semen analysis accord-ing to WHO (2010); SDF evaluation by TUNEL assay; extraction ofsperm DNA performed using the Nucleospin kit that separate spermcells from epithelial and round cells; and sperm DNA methylation lev-els of H19/IGF2 genes by pyrosequencing. After sperm selection andbefore ICSI, an aliquote of semen was used for: semen analysis, SDF,and sperm borne miRNA analysis (miR-34c and miR-449b) throughddPCR.Twenty men were recruited and classified into three groups basedon ICSI outcomes: seven subjects who had achieved viable embryos(VE), seven subjects had achieved non-viable embryos (NVE), and sixsubjects who did not achieve fertilization (NF). The main causes ofinfertility were: 60% female, 25% male, and 15% couple factor. The mean age of men in three groups was: 37.7 ± 2.8 (VE), 39 ± 5.9 (NVE),and 47.7 ± 7.2 (NF). The mean age of female partners was 34.4 ± 3.5,36.6 ± 4.1, and 39.8 ± 2.2 years, respectively, in three groups (VE, NVE,and NF). The semen parameters were above the 5th percentile of theWHO 2010 reference values. No associations were found betweensemen characteristics and %SDF and H19 and IGF2 methylation. A negative correlation was observed between %SDF and viable embryos (r = −0.53; p = 0.041). Analysis of the four CpG islands present in theH19 gene promoter revealed a trend of difference in CpG1 methylation among the three groups (94.0 ± 2.0 in NF; 95.0 ± 4.0 in NVE;and 98.0 ± 2.0 in VE, p = 0.098). A significant positive correlation was also identified between CpG1 methylation and viable embryos(r = 0.53; p = 0.016). In the male/couple infertility subgroup, a negative correlation was found between H19 gene methylation and %SDF(r = −0.94; p = 0.05). Concerning the methylation of IGF2, the threeCpGs islands present on the gene were analyzed. No difference swere found between the methylation levels of IGF2 in the three CpGs islands among the three groups. A statistically significant positive correlation was found between the total methylation of the IGF2gene and levels of miRNA-34c (r = 0.474; p = 0.047) and miRNA-449b(r = 0.457; p = 0.05), that persist after adjustment for male factor (p adjusted 0.004 and 0.039, respectively). A negative trend was observed between total % methylation IGF2 and %SDF (r = −0.467;p = 0.086).This pilot study suggests that methylation profile of H19 gene promoter in spermatozoa may play an important role in embryo development. In addition, methylation of IGF2 and miRNA-34c/449b could be promising biomarkers of seminal quality.