Relationship between Fertilizing Ability of Ejaculated Human Spermatozoa and Its Chromatin Heterogeneity.

OBJECTIVE: The aim of this study was to examine the relationship between the fertilizing ability of ejaculated human sperm and its chromatin heterogeneity. METHOD: We used the D-AO staining method (acridine orange epifluorescence accompanied by diamide, a thiol oxidizing agent) to analyze the sperm chromatin structure of infertile patients with IVF-ET treatment. SDS-PAGE was performed to analyze the sperm nuclear proteins collected from patients with immature sperm (stained red with D-AO staining) and proven fertile men. RESULTS: 1. It was suggested that D-AO staining allowed the immature sperm to be divided into two groups. One was immature sperm, which had disturbance of S-S formation in the epididymides, and the other was sperm that which had an abnormal exchange process of nuclear proteins in the testes. 2. The stainability after D-AO staining showed no correlation with the findings of semen analysis. 3. The fertilization rate of IVF-ET was significantly correlated to the percentage of green sperm staining with D-AO staining. 4. In the pregnant group after IVF-ET, it was noticed that sperm of the green type with D-AO staining was increased in comparison with the non-pregnancy group. 5. The fertilization rate in the group of the sperm stained red with D-AO staining was increased to 73.5% by ICSI. 6. Definite differences were noticed between the protein components of the patients with immature sperm and those of the proven fertile men by analysing with SDS-PAGE. CONCLUSION: D-AO staining was an efficient method for evaluating the fertilizing ability of human ejaculated sperm, and to determine an appropriate ART tool such as ICSI.

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

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.