Chromomycin A3-staining as an indicator of protamine deficiency and fertilization

Mature mammalian spermatozoa have a compact and stable nuclear structure conferred by protamines instead of histones, which are present in all other cellular types. Chromomycin A3 (CMA3) is a useful tool for the detection of protamine deficiency in sperm chromatin. The purpose of this study was to correlate the percentage of spermatozoa staining positively for CMA3 with sperm parameters and in-vitro fertilization of human oocytes. Spermatozoa were collected from 56 fertile and 18 infertile men, and washed twice in PBS, fixed in two changes of methanol : acetic acid (3 : 1 v : v) spread on rinsed slides treated with APES and dried. Twenty-four of the semen samples were subjected to both Percoll and swim-up, and were stained subsequently with CMA3. CMA3-stained spermatozoa were expressed as a percentage in a count of 200 spermatozoa. A substantial variation in the percentage of CMA3-stained cells was observed in ejaculated human spermatozoa, varying between 8% and 77%. A strong negative correlation (r = -0.64, p < 0.001) was found between sperm count and the percentage of CMA3-stained spermatozoa. No correlation was found between CMA3-stained spermatozoa and their motility, while excessive sperm morphological abnormalities were related positively to CMA3-staining. Spermatozoa in samples exhibiting low (8-62%) CMA3-staining had significantly higher fertilizing rates in vitro than did samples exhibiting high (49-77%) CMA3-staining. The mean percentage of CMA3-stained spermatozoa after swim-up or Percoll preparation (26% vs 31%) did not differ significantly. These results demonstrate a close relationship between CMA3-staining, fertilization and sperm count, and suggest potential application of this marker for the prediction of sperm quality and fertilizing capacity.