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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Kellie W
KW
Edwards
Philip J
PJ
Chan
Albert K
AK
Wei
Johannah U
JU
Corselli
John D
JD
Jacobson
D000328:Adult; D001399:Azure Stains; D002843:Chromatin / Q000737:chemistry* / Q000145:classification / Q000473:pathology; D005260:Female; D005306:Fertilization; D006651:Histocytochemistry / Q000379:methods; D006801:Humans; D008297:Male; D008751:Methylene Blue; D011247:Pregnancy; D011897:Random Allocation; D012189:Retrospective Studies; D020554:Sperm Injections, Intracytoplasmic / Q000706:statistics & numerical data*; D013094:Spermatozoa / Q000737:chemistry* / Q000145:classification / Q000473:pathology; D014966:Xanthenes
D001399:Azure Stains; D002843:Chromatin; C067461:Diff Quik; D014966:Xanthenes; D008751:Methylene Blue