Use of the comet assay™ to determine DNA integrity in single cells following laser assisted hatching and biopsy in a murine model

Abstract

Use of laser for assisted hatching is becoming popular with the recent FDA approval of two commercially available units. Cell integrity was evaluated following assisted hatching and biopsy using the Hamilton-Thorne Zilos-tk™ laser or acidified Tyrode's (AT) solution. The goal of this study was to compare DNA fragmentation from biopsied cells obtained by two methods of assisted hatching. Prospective Randomized Study. Frozen murine 2-cell embryos (N = 217) were thawed and cultured for 18 hours. Eight cell embryos were randomized between three treatment groups; Controls - non-biopsied, Laser hatching and biopsy (Laser) and Acidified Tyrode's hatching and biopsy (AT). The biopsied cells were exposed to the Comet Assay™ for detection of DNA damage. Positive and negative control cells, obtained from control embryos, were processed side-by-side with biopsied cells according to Comet Assay™ instructions. Endpoint outcomes include cell size and comet tail lenght as an indication of DNA fragmentation as analyzed using the Wilcoxon Rank Sums test. Cells obtained from Laser hatched embryos were larger than cells from AT hatched embryos (44.5 μm vs. 30.2 μm, respectively). However, there was no difference in comet tail length (36.4 μm vs. 41.5 μm, respectively). Comet tails were longer from cells obtained by both Laser and AT than those treated as negative controls (36.4 μm, 41.5 μm vs. 13.6 μm, respectively) and comet tails were shorter from cells obtained by both treatments than those treated as positive controls (36.4 μm, 41.5 μm vs. 52.4 μm, respectively). These data suggest that, althought there is a significant increase in size from cells biopsied from Laser hatched embryos compared to cells biopsied from AT, there is no indication of increased DNA fragmentation between the two treatments. However, because there was an increase comet tail length in cells from both Laser and AT hatched embryos compared to negative controls, some fragmentation is evident. Further investigation is indicated to determine clinical significance.