Addition of eosin to the aniline blue assay to enhance detection of immature sperm histones.

OBJECTIVES

To compare the aniline blue assay with and without eosin, and to correlate the results with pregnancy outcome after intracytoplasmic sperm injection (ICSI) procedure.

DESIGN

A retrospective study.

SETTING

University-based fertility center.

PATIENT(S): One hundred thirty infertile patients.

INTERVENTION(S): Left-over washed sperm after each ICSI procedure were fixed on glass slides and stained with aniline blue with and without eosin.

MAIN OUTCOME MEASURE(S): Chromatin condensation, pregnancy, and age.

RESULT(S): Percentage chromatin condensation assessed by aniline blue-eosin was higher compared with standard aniline blue (72.4 +/- 2.4% vs. 64.0 +/- 2.4% [mean +/- SEM]). Chromatin condensation was higher in pregnant (86.6 +/- 0.9%) versus nonpregnant (80.9 +/- 2.1%) women age 35 years or more. In younger women, chromatin condensation was not correlated with pregnancy outcome. There was no correlation between chromatin condensation and ICSI fertilization or male age.

CONCLUSION(S): Adding eosin counterstain to aniline blue improved assessment of chromatin condensation, suggesting that the standard assay underestimated chromatin condensation. The association between chromatin condensation and pregnancy in older but not younger women suggests that oocytes of younger women had the capacity to compensate for the immature sperm shortcomings.