胚胎植入前诊断中多轮荧光原位杂交效果及影响因素分析

Efficiency of multi-round fluorescense in situ hybridization and its influencing factors in preimplantation genetic diagnosis

目的探讨胚胎植入前诊断（preimplantation genetic diagnosis，PGD）中多轮荧光原位杂交（fluorescense in situ hybridization，FISH）效果及其影响因素。方法48对夫妇因染色体异常（罗伯逊易位24对、相互易位16对、臂间倒位2对）或其他原因（女方高龄且生育过唐氏综合征患儿1对、男方或女方性染色体异常3对、反复自然流产史2对）行PGD，经72个采卵周期获得≥6细胞胚胎432个，受精后第3天行胚胎活检，对396枚完整核进行FISH，对信号不明确的核进行下一轮杂交，396枚核共进行了1～6轮870次杂交，分析比较各轮杂交信号明确率、不同探针对杂交效果的影响以及导致信号不明确的相关因素。结果870次杂交中535次信号明确，占比61．5％，第2轮和第3轮信号明确率分别为71．8％和77．4％，显著高于第1轮（52．8％，P〈0．01）、第4轮（55．8％，P〈0．05、P〈0．01）、第5轮（54．5％，P（O．05）和第6轮（27．3oA，P〈0．01）。着丝粒探针组（centromere specific probe，CEP）总体信号明确率80．3％，该组前3轮信号明确率最高，分别为85．7％、85．1％和88．0％，3轮间差异无统计学意义，第4轮起信号明确率明显下降，第4、第5轮与前3轮比有显著差异；其他探针组总体信号明确率55．2％（与CEP探针组比较，P〈0．01），该组第5轮信号明确率最高（72．7％），第2轮信号明确率（66．1％）显著高于第1轮和第6轮（P〈0．01），第3轮信号明确率（63．8％）与其他各轮相比差异无统计学意义。6轮FISH中335次信号不明确，原因依次为信号质量差（20．9％，182／870）、背景干扰（7．6％，66／870）、杂交失败（6．1％，53／870）、核破损（1．8％，16／870）、核丢失（1．1％，10／870）、信号分裂／重叠（0．9％，8／870）。结论多轮FISH能提高PGD中单个核的利用价值，以前3轮效果最好；CEP杂交效果优于其他探针；信号质量差是导致信号不明确的最常见原因。

Objective To investigate the efficiency of multi-round fluorescense in situ hybridization （FISH） and its influencing factors in preimplantation genetic diagnosis （PGD）. Methods A total of 48 couples accepted PGD because of various reasons： 24 with Robertsonian translocations, 16 with reciprocal translocations, 2 with pericentric inversions, one with advanced maternal age who had a previous liveborn of Down syndrome, 3 suffered from sex chromosome abnormalities and 2 repeated spontaneous miscarriages. After 72 retrieval cycles, 432 cleavage stage embryos with more than six cells were biopsied on day three. Only intact nuclei （396） were hybridized in order to verify the chromosomal status of the individual embryos. If previous FISHs failed to give conclusive results while the nuclei remained undamaged, the nuclei were hybridized once again. A total of 870 times of hybridization were conducted to 396 nuclei. Signal identification rates of each round as well as the influence of different probes to the hybridization efficiency were compared. Factors leading to unconclusive FISH results were analyzed as well. Results Five hundred and thirty-five out of 870 hybridizations gave identifiable signals （61. 5%）. The second and third round FISH showed the best signals with an identification rate of 71.8% and 77.4%, respectively, which were significantly higher than those of the first round （52.8%, P〈0.01）, the fourth round （55.8%, P〈0.05, P〈0.01）, the fifth round （54. 5%, P〈0. 05） and the the sixth round （27. 3%, P〈0. 01）. The identification rate of eentromere specific probe signals （CEP group） was 80.3 % and the former three rounds in this group got the best qulity of signals with an identification rate of 85. 7%, 85. 1% and 88. 0%, respectively, which was significantly higher than that of the latter three rounds. The identification rate of other probe was much lower than with the CEP probe （55.2% vs. 80.3%, P〈0.01） and the best quality of signal in this group was achieved in the fifth round （72.7 % ）, followed by the seeond round （66.1% ） and the third round （63.8%）. The identification rate of the first round （50.3%） and the sixth round （22.2%） were significantly lower compared with the second round （P（0.01）. During the 6 rounds of FISH, 335 hybridizations did not give conclusion results （38. 5%, 335/870）. The main cause of unidentification was weak signals （20.9%, 182/870）. Other common factors included background interference （7.6%, 66/870） and failed hybridization （6.1%, 53/870）. Rare causes included nucleus damage （1.8%, 16/870）, nueleus loss （1.1%, 10/870） and signal split/overlap （0.9%, 8/870）. Conclusion Multi-round FISH can improve the utility of single nucleus in PGD and the former three rounds have the highest efficiency. The hybridization effect of CEP is better than other probe. Poor signal quality is the common cause of unidentification results.