Sperm H3K9me3 levels are associated with embryo developmental dynamics and biochemical pregnancy in IVF patients with normozoospermia.

BACKGROUND: The posttranslational histone modification H3K9me3 is crucial for constitutive heterochromatin (cHC) and supports genome stability and gene regulation during development. This epigenetic mark persists in human sperm post histone-to-protamine transition and is transmitted to the embryo. Although H3K9me3 variability is linked to abnormal sperm parameters, its role in fertilization and embryo development remains unclear. Given its retention in sperm, aberrant H3K9me3 levels may underlie cases of unexplained male infertility. OBJECTIVE: Investigate the variability of H3K9me3 levels in sperm from normozoospermic men and assess its association with early embryo development and IVF outcomes. MATERIAL AND METHODS: H3K9me3 and histone H3 levels were quantified by Western blot in surplus sperm from 99 normozoospermic men undergoing IVF-treatment. Patients were stratified into quartiles based on the H3K9me3/H3 ratio. Pre-implantation embryo development was assessed by time-lapse imaging, focusing on nuclear precursor body (NPB) dynamics and morphokinetics. IVF outcomes were reported as cumulative biochemical and ongoing pregnancy rates per ovum pick-up and compared across H3K9me3/H3 quartiles. RESULTS: H3K9me3/H3 ratios exhibited substantial inter-individual variability among normozoospermic patients. Embryos from the third H3K9me3/H3 ratio quartile (Q3) demonstrated the highest proportion of zygotes with NPB clustering and faster, more consistent development through the first two cleavage divisions compared to other quartiles. A significant non-linear association was found between H3K9me3/H3 ratio and cumulative biochemical pregnancy rates: couples in the lowest quartile (Q1) had significantly reduced odds of biochemical pregnancy compared to Q3 (adjusted OR [95% CI]: 0.30 [0.09-0.97], p = 0.045). No significant association was found for ongoing pregnancy rates. DISCUSSION AND CONCLUSIONS: This study reveals that sperm H3K9me3 levels vary among normozoospermic men and correlate with early embryo development and biochemical pregnancy rates following IVF. However, no significant association was found with ongoing pregnancy, suggesting that additional mechanisms may determine long-term pregnancy viability. The non-linear relationship between H3K9me3/H3 ratio and embryo development suggests an optimal range for this epigenetic mark. These findings highlight the potential influence of paternal epigenetic variation, undetectable by standard semen analysis, on embryo quality and IVF outcomes. Further studies in larger cohorts are warranted to confirm these findings and clarify underlying mechanisms.