Sperm Imprinted Gene Methylation and DNA Fragmentation in ICSI Outcomes: A Pilot Study

Background/Objectives: Aberrant DNA methylation of imprinted genes and increased sperm DNA fragmentation (SDF) have been implicated in male infertility. However, their impact on assisted reproductive technology (ART) outcomes remains unclear. This pilot study aimed to investigate SDF and methylation status of H19, IGF2, and PEG1/MEST in relation to fertilisation and embryo development following intracytoplasmic sperm injection (ICSI). Methods: Twenty male partners of women undergoing ICSI were recruited and classified according to ART outcome into viable embryos (VEs, n = 7), non-viable embryos (NVEs, n = 7), and no fertilisation (NF, n = 6). Before sperm selection, an aliquot of each seminal sample was used for semen analysis according to WHO, 2021, SDF assessment (TUNEL assay), and sperm DNA methylation analysis of H19, IGF2, and PEG1/MEST (pyrosequencing). Results: Semen parameters were above the fifth percentile. SDF was significantly lower in the VE group compared with the other groups. H19 CpG1 methylation correlated positively with viable embryos (p = 0.016), while H19 CpG2 island showed a positive correlation with sperm concentration (p = 0.028). In male/couple infertility cases, total H19 methylation correlated negatively with SDF (p = 0.050). IGF2 CpG3 island methylation correlated positively with viable embryos (p = 0.027). Total PEG1/MEST methylation was positively correlated with fertilisation events (p = 0.002) and viable embryos (p = 0.011). PEG1/MEST CpG2 island also positively correlated with sperm motility (p = 0.034), while CpG3 and CpG4 showed significant correlations with fertilisation (p < 0.001; p = 0.004). Conclusions: This pilot study shows that SDF and sperm methylation levels of H19, IGF2, and PEG1/MEST are related to ICSI outcomes, supporting that sperm molecular and epigenetic features may influence fertilisation and embryo development.