Use of diamide–acridine orange fluorescence staining to detect aberrant protamination of human-ejaculated sperm nuclei

Abstract

Objective To investigate the influence of human sperm nuclear chromatin on fertilization. Design Prospective study. Setting Assisted reproductive technology unit at a university teaching hospital. Patient(s) Fifty men starting an IVF-ET program. Intervention(s) Epifluorescent microscopic observation of human-ejaculated sperm nuclei stained with diamide–acridine orange. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of extracted sperm nucleoproteins. Main outcome measure(s) Usefulness of diamide–acridine orange in analysis of human sperm nuclear chromatin and fertilization ability. Result(s) There was no correlation between the semen parameters and the diamide–acridine orange observation. A positive correlation was observed between the fertilization rate after conventional IVF and the green-type increase ratio (percentage of green-pattern sperm after diamide–acridine orange staining/percentage of green-pattern sperm after acridine orange staining). Furthermore, it was suggested by SDS-PAGE that structural differences were noticed between the fertile men and the men with sperm immaturity diagnosed after diamide–acridine orange staining. Conclusion(s) Diamide–acridine orange staining was a more precise method for detecting chromatin abnormalities in human-ejaculated sperm and evaluating fertilization ability than acridine orange staining alone. This method can be used as a diagnostic tool to assess the fertilization ability of human-ejaculated spermatozoa before IVF procedures.