P J Chan1, J H Calinisan, J U Corselli, W C Patton, A King. 1. Department of Gynecology and Obstetrics, Loma Linda University School of Medicine, Loma Linda, California 92350, USA. pchann@yahoo.com
Abstract
PURPOSE: Despite advances in assisted reproduction, there is no progress in quality control bioassays. The objectives were to develop a comet assay to measure DNA fragmentation in thawed cryopreserved oocytes and compare this 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-microM hydrogen peroxide, or, in media exposed to different contact materials and unknown proficiency analytes. Incubation time was 1.5 h at 37 degrees C. The oocytes were dried, fixed, stained with acridine orange, embedded in a mini-agarose layer and electrophoresis was carried out. Fluorescent images were analyzed. The results were compared with standard one-cell mouse assay data. RESULTS: The 100-microM hydrogen peroxide treatment caused greatest DNA fragmentation in the hamster oocytes at Hours 1 and 2. A dose response was observed. Intraassay coefficient of variation was 5.7%. Only one of the five materials tested passed both assays. The data for the unknown proficiency analytes were similar for both assays. CONCLUSIONS: The oocyte comet assay demonstrated DNA fragmentation in the presence of toxic substances. The detection of toxicity in two materials that passed the mouse bioassay suggested increased sensitivity in the new assay. The oocyte comet assay and the mouse bioassay results matched in the proficiency test. However, more studies are still needed to determine optimal sensitivity.
PURPOSE: Despite advances in assisted reproduction, there is no progress in quality control bioassays. The objectives were to develop a comet assay to measure DNA fragmentation in thawed cryopreserved oocytes and compare this 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-microM hydrogen peroxide, or, in media exposed to different contact materials and unknown proficiency analytes. Incubation time was 1.5 h at 37 degrees C. The oocytes were dried, fixed, stained with acridine orange, embedded in a mini-agarose layer and electrophoresis was carried out. Fluorescent images were analyzed. The results were compared with standard one-cell mouse assay data. RESULTS: The 100-microM hydrogen peroxide treatment caused greatest DNA fragmentation in the hamster oocytes at Hours 1 and 2. A dose response was observed. Intraassay coefficient of variation was 5.7%. Only one of the five materials tested passed both assays. The data for the unknown proficiency analytes were similar for both assays. CONCLUSIONS: The oocyte comet assay demonstrated DNA fragmentation in the presence of toxic substances. The detection of toxicity in two materials that passed the mouse bioassay suggested increased sensitivity in the new assay. The oocyte comet assay and the mouse bioassay results matched in the proficiency test. However, more studies are still needed to determine optimal sensitivity.