用全染色体涂抹探针进行卵母细胞第一极体荧光原位杂交

 【目的】建立用全染色体涂抹探针（WCP）对卵子第一极体进行荧光原位杂交（FISH）检测染色体的方法。【方法】收集单精子卵胞浆内注射（ICSI）中不成熟卵母细胞经体外培养成熟和常规试管婴儿（IVF）中未能成功受精的成熟卵母细胞，活检第一极体，固定后行13、14号染色体的全染色体涂抹探针荧光原位杂交。活检后，一部分卵母细胞固定行FISH以分析卵子自身的13、14号染色体，其余行ICSI受精，观察受精和卵裂情况。【结果】共获得成熟母细胞93个，成功活检85个，成功固定78个，共29个第一极体处于分裂中期，均有FISH结果。卵母细胞体外培养成熟后立即取极体进行固定，90．5％（19／21）的极体处于分裂中期，而取卵后30-48h和72h后活检的第一极体处于分裂中期的比例分别为27．3％（6／22）和11．4％（4／35），3组相比有统计学差异（P〈0．01）。11个卵母细胞同时获得了极体和相对应卵细胞的FISH结果，其中10个极体和相对应的卵细胞分别有1条13，14号染色体，剩余1个卵母细胞的极体有2条14号染色体和1条13号染色体，相对应的卵细胞仅有1条13号染色体，二者互补。活检后行ICSI受精的卵母细胞受精率为78．6％（11／14），优质胚胎率45．5％（5／11）。【结论】全染色体涂抹探针对卵子第一极体进行遗传分析可以有效、准确地推测相对应卵母细胞的染色体构成，从而应用于女性染色体易位患者的植入前遗传诊断。

Application of Whole Chromosome Painting Probes in Fluorescence In-situ Hybridization of First Polar Bodies

Objective] To establish the method of chromosome analysis of first polar bodies(1PBs)by using whole chromosome painting probes in fluorescence in-situ hybridization(FISH).Methods] Oocytes that failed to fertilize after in vitro fertilization(IVF)or oocytes matured in vitro from immature oocytes in intracytoplasmic sperm injection(ICSI)were used for this study.The first polar bodies were biopsied and fixed.Whole chromosome painting probes 13,14 were analyzed in FISH.After biopsy,parts of the oocytes were fixed for FISH,while the rest were fertilized by ICSI.Fertilization rate and cleavage rate were observed.Results] Of the 93 matured oocytes collected,85 were successfully biopsied and 78 were fixed.Altogether 29 1PBs were at metaphase stage and got FISH results.First polar body chromosomes fixed immediately after maturation in vitro were found to be at metaphase stage in 90.5 %(19/21)cases.However,the metaphase degenerated soon after oocyte retrieval.For the 1PBs biopsied 30~48 h and > 72 h after oocyte retrieval,only 27.3%(6/22)and 11.4%(4/35)were at metaphase stage respectively(P<0.01=.FISH analyses of the 1PBs and the corresponding MII oocytes(MII)showed concordance in the pair.Of the 11 oocytes with FISH results available for both the polar bodies and the complementary MII oocytes,numerically normal chromosome 13 and 14 were obtained in 10 1PB-MII doublets,the other one was chromosomally abnormal with two chromosome 14,one chromosome 13 in the polar body and only one chromosome 13 in the oocyte.The fertilization rate and good embryo rate of the oocytes fertilized by ICSI after biopsy were 78.6%(11/14)and 45.5%(5/11)respectively.Conclusions] The analysis of 1PBs by using whole chromosome painting probes is an accurate and effective way to diagnose the chromosome constitutes of the MII oocytes and can be applied to PGD for female carriers of translocation.