Updating quality control assays in the assisted reproductive technologies laboratory with a cryopreserved hamster oocyte DNA cytogenotoxic assay

Purpose: Despite advances in assisted reproduction, there is no progress in (quality control bioassays. The objectives were to develop a comet assay t o measure DNA fragmentation in thawed cryopreserved oocytes and compare thi s assay with one-cell mouse embryo bioassay. Methods: Thawed hamster oocytes from a commercial source were incubated in culture media with either 0-, 50-, or 100-muM hydrogen peroxide, or, in med ia exposed to different contact materials and unknown proficiency analytes. Incubation time was 1.5 h at 37 degreesC. The oocytes were dried filed, st ained with acridine orange, embedded in a mini-agarose layer and electropho resis was carried out. Fluorescent images were analyzed The results were co mpared with standard one-cell mouse assay data. Results: The 100-muM hydrogen peroxide treatment caused greatest DNA fragme ntation in the hamster oocytes at Hours 1 and 2. A dose response was observ ed. Intrassay coefficient of variation was 5.7%. Only one of the five mater ials tested passed both assays The data for the unknown proficiency analyte s were similar for both assays Conclusions: The oocyte comet assay demonstrated DNA fragmentation in the p resence of toxic substances. The detection of toxicity in two materials tha t passed the mouse bioassay suggested increased sensitivity in the new assa y The oocyte comet assay and the mouse bioassay results marched in the prof iciency test. However, more studies are still needed to determine optimal s ensitivity.