μChIP assay in combination with real time qPCR demonstrating loss of association of NSD1 promoter sequence with H4K12ac in subfertile patients with aberrant sperm chromatin condensation. (A) Representative qPCR demonstrating enrichment of μChIP experiments with anti-H4K12ac antibodies, with anti-protamine-1 antibodies from input material in individual fertile donors (fc = 5) and (B) subfertile patients (n = 8). Unmodified H3 was used as a positive and IgG as a negative, isotype control. (C) Enrichment of the immunoprecipitated sample from fertile and subfertile men compared to input materials was calculated as follows: ΔCt = Ct (input) - Ct (immunoprecipitated sample) and % total = 2ΔCt × 10 (according to 10% input chromatin of total immunoprecipitated chromatin). Depletion of binding and an increase of protamine occupancy to the NSD1 promoter were detected.