Further evidence supporting the Diff-Quik stain for sperm chromatin integrity testing

To correlate intracytoplasmic sperm injection (ICSI) fertilization with chromatin status assessed by the Diff-Quik procedure modified with a one-minute soak step, and to determine the association of chromatin status with in vitro fertilization (IVF) pregnancy.

This was a retrospective study of 81 IVF patients. Gradient-centrifuge washed sperm remaining after ICSI were fixed, stained by Diff-Quik, immersed in water for 1 minute, and analyzed under oil immersion light microscopy. Sperm nuclear coloration (types A-D), strict morphology, fertilization, and pregnancy status were determined.

Sperm with light purple staining (type A) were correlated (R = 0.48, p < 0.05) with ICSI fertilization. The intraassay and interassay coefficients of variation were 5.9% and 4.1%, respectively. Sperm strict normal morphology was correlated neither with ICSI fertilization (R = 0.24, p > 0.05) nor with type A sperm (R = 0.35, p > 0.05). Sperm incubated in Fenton reagent that damaged DNA showed a time-dependent decrease in percent type A sperm. However, there was no correlation with IVF pregnancy status.

This retrospective study showed that the inclusion of a one-minute soak step post-Diff-Quik staining enhanced the detection of sperm chromatin abnormalities related to ICSI fertilization. Fenton reagent-treated sperm suggested that the staining patterns correlated with DNA damage. A large prospective trial should be undertaken to confirm these findings.