Improved fixation technique reduces time consuming and increases fish efficiency

Abstract

To modify current blastomere fixation technique and then evaluate the time consuming and FISH efficiency for PGD. Laboratory study. Day 3 human embryos were biopsied by mechanical aspiration after laser zona opening. After biopsy, two different methods were used for fixation. In Method I (traditional method), each blastomere was placed in the hypotonic solution for 5 min and then fixed on slide. The slides were kept at room temperature until procedures for FISH. In Method II (modified method), all blastomeres were individually placed in the hypotonic solution drops covered by oil at least 5 min and then fixed on slide with 0.1% Tween. The slides were placed in a freezer for at least 30 min and then kept cold until the procedures for FISH. Time consuming and the percentages of blastomeres with FISH signals were compared between two methods. As shown in Table 1, a total of 329 blastomeres from 30 cases were fixed by Method I and the average time for fixation is 8-10 min for each blastomere. While with the Method II, a total of 362 blastomeres from 32 cases were fixed and the average time for fixation is 3-4 min for each blastomere. After FISH, 86.9% of the nuclei had positive signals with the traditional fixation method while 97.2% of nuclei had signals with the modified method.Table 1Comparison of traditional method and modified method for fixation of biopsied blastomeres for FISH diagnosis in term of time consuming and PGD efficiency.No. of casesNo. of blastomeresFixation methodsAverage time (min) consumingNo. (%) of blastomeres with signals30329Method I8-10286 (86.9)aSignificantly different, P<0.01.32362Method II3-4352 (97.2)bSignificantly different, P<0.01.a,b Significantly different, P<0.01. Open table in a new tab With these modifications, laboratory technician can use less time to fix the blastomeres. Most importantly, the modifications increase the numbers of embryos with FISH signals, hence increase the efficiency of PGD.