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- Title
Appropriate fixation technique of blastomere for spread of nucleus and removal of cytoplasm in preimplantation genetic diagnosis.
- Authors
Tarui, S.; Nakaoka, Y.; Ohgaki, A.; Fukuda, A.; Morimoto, Y.
- Abstract
Introduction: Preimplantation genetic diagnosis (PGD) has been widely conducted and the fluorescence in-situ hybridization (FISH) method is generally used. One of the important steps in the PGD procedure by FISH is the fixation process, which is required to obtain a good nuclear specimen from blastomeres. The first objective of the present study was to determine the appropriate duration for hypotonic treatment in gradual fixation method. The second objective was to compare three fixation methods in the appropriate hypotonic treatment depending on the first result to obtain good cell preparation with fully spread nuclei combined with sufficient cytoplasmic removal. Materials/Methods: Day-3 embryos to be discarded were applied for the present study under informed consent with patients after approval of IVF JAPAN IRB committee. Blastomeres of 50-60 µm in diameter derived from day 3 embryos were utilized. In study 1, blastomeres were treated with hypotonic solution (1% sodium citrate/0.5% BSA) for 5, 10, 15, 20 and 25 min and each blastomere was fixed by a gradual fixation method (method A). Blastomeres were treated by fixative I (methanol: acetic acid: water = 5:1:4) for 5 min and gently treated with a flow of fixative (methanol: acetic acid = 3:1) for 10 min on a slide glass. Those specimens were airdried and stained with Giemsa for analysis. In study 2, three different fixation methods (A, B and C) were compared after appropriate hypotonic treatment duration. Methods B and C were one-step fixation with an acetic acid/methanol method (3:1) and Tween-20 + acetic acid/methanol (0.1% Tween- 20/0.01N HCl) for 40 s, respectively. Those specimens were air-dried and stained with Giemsa for analysis. Results: In study 1, using hypotonic treatment for 5, 10, 15, 20 and 25 min, the rates of sufficient cytoplasmic removal were 56.7% (17/30), 86.7% (26/30), 90.0% (27/30), 86.7% (26/30) and 86.7% (26/30), respectively. Cytoplasmic removal rates were significantly higher in the group of 10 min or longer compared with the 5 min group. Diameters of the nuclei were 38.8 ± 11.3, 47.3 ± 13.6, 60.3 ± 11.7, 62.1 ± 11.6 and 59.8 ± 10.4 µm (mean ± SD), respectively. The nuclear diameter significantly increased with hypotonic treatment for 15 min or longer. In study 2 there was no significant difference in the rates of sufficient cytoplasmic removal among method A: 90.0% (3/30), B: 93.3% (2/30) and C: 93.3% (2/30). However, the diameters of nuclei by method A (60.3 ± 11.7) were significantly larger than those either by method B (48.8 ± 11.2) or C (43.0 ± 9.7) µm (mean ± SD). Conclusions: The present study indicates that preparation of blastomeres from day-3 embryos using a gradual fixation method after hypotonic treatment for 15 min or longer provides optimal specimens for FISH analysis in PGD.
- Subjects
IN situ hybridization; GENETICS
- Publication
Reproductive BioMedicine Online (Reproductive Healthcare Limited), 2008, Vol 16, Issue S2, pS-46
- ISSN
1472-6483
- Publication type
Abstract
- DOI
10.1016/S1472-6483(10)61581-7