FISH技术检测异常受精胚胎以及对胚胎单卵裂球核型分析技术的初步探索

发布时间：2018-08-07 06:31

【摘要】： 背景：人类植入前胚胎的染色体异常是很普遍的，常导致胚胎发育的停滞或引起自发流产。正常受精卵具有两个原核，而异常原核数目的受精卵包括单原核受精卵、多原核受精卵及无原核受精卵，这些受精卵一般都被丢弃，可一些研究显示这些原核形态异常的胚胎有一定比率的染色体正常率，有一定的移植价值。目前对人类植入前胚胎染色体异常的研究多采用免疫荧光原位杂交(FISH)的方法。然而，FISH只能检测有限数目的染色体，并且检测每一种染色体异常都需要相应的特定探针，局限了FISH技术的应用。目前，有研究者试图将胚胎单卵裂球的染色体诱导至中期相，可以在不需要特定探针的情况下，对卵裂球进行核型分析，弥补FISH的不足。目的： 1．检测原核数目异常胚胎中的染色体正常率，分析这些胚胎是否具有移植价值。 2．探索单卵裂球核型分析技术，以期为胚胎植入前遗传学诊断提供一种新的方法。方法： 1．将IVF及ICSI周期中原核评分为1PN的受精卵继续培养，第五天如胚胎发育为囊胚则分离出内细胞团后将余下滋养层细胞行双色FISH检测，如未发育至囊胚则将胚胎的全部卵裂球活检后分别行FISH； 2．对卵裂球进行体外操作以期得到染色体中期相，方法包括： 1)直接用秋水仙素阻断卵裂球至中期； 2)与体外成熟的人去核MⅡ卵母细胞融合，诱发PCC(染色体提前凝集)； 3)与小鼠MⅡ卵母细胞融合，诱发PCC； 4)与小鼠受精卵融合，诱导至中期。结果： 1．对138枚1PN胚胎行双色FISH检测，得到可分析的结果共87枚，其中来源于IVF的48枚，单体信号占18.8％、二体信号54.2％、嵌合体20.8％、多体信号6.2％；来源于ICSI的39枚，单体信号46.2％、二体信号25.6％、嵌合体23.1％、多体信号5.1％。87枚1PN胚胎中，有16枚发育到囊胚，其中12枚为二体信号(75.0％)，4枚为嵌合体(25.0％)； 2．对胚胎单卵裂球核型分析的结果为： 1)采用秋水仙素直接阻滞卵裂球57枚，得到中期染色体9枚，成功率15.8％； 2)采用体外成熟后人卵诱导卵裂球26枚，得到中期染色体4枚，成功率15.4％； 3)采用小鼠成熟卵母细胞诱导卵裂球13枚，电融合时小鼠卵母细胞皆被激活，成功率为0； 4)采用小鼠受精卵诱导卵裂球160枚，得到中期染色体25枚，成功率15.6％。结论： 1．ICSI来源的1PN多为单体信号，可能是由于精子未解聚而卵母细胞激活所致；IVF来源的1PN则多为正常二体信号，可能其主要的原因是原核形成不同步或原核融合。第五天发育到囊胚的胚胎则皆显示二体信号或嵌合体，可能是由于单倍体的胚胎缺乏发育至囊胚能力。 2．本实验采用四种方法所得到的中期染色体数都约为15.5％，成功率较低，，目前采用核转换技术来将卵裂球染色体诱导至中期相的效率还偏低，尚需要进一步改进。
[Abstract]:Background:
Chromosomal abnormalities in human preimplantation embryos are common, often leading to stagnation of embryonic development or spontaneous abortion. Normal fertilized eggs have two prokaryotes, while abnormal prokaryotic eggs include mono prokaryotic ovum, multiple prokaryotic and non prokaryotic fertilized eggs, and these fertilized eggs are generally discarded. Some studies show this Some embryos with abnormal nuclear morphology have a certain percentage of normal chromosomes and have certain transplantation value.
At present, the study of chromosomal abnormalities in human preimplantation embryos uses immunofluorescent in situ hybridization (FISH). However, FISH can only detect a limited number of chromosomes, and the detection of each chromosome abnormality requires a specific probe, limiting the use of FISH technology. At present, researchers are trying to dye the single blastomere of the embryo. Karyotype analysis of blastomeres can be made without the need for specific probes when the chromophore is induced to the metaphase phase, thus making up for the deficiency of FISH.
Objective:
1. detect the normal rate of chromosomes in the abnormal number of embryos and analyze whether these embryos have the value of transplantation.
2. explore the single blastomere karyotype analysis technology, in order to provide a new method for preimplantation genetic diagnosis.
Method:
1. the fertilized eggs of 1PN in IVF and ICSI cycle were continued to be cultured. On the fifth day, if the embryo developed into blastocyst, the remainder trophoblast cells were detected by double color FISH. If the blastocyst was not developed to the blastocyst, the whole blastomere of the embryo was performed FISH respectively.
2. in vitro operation of blastomere to obtain metaphase chromosomes.
1) direct colchicine to block the blastomere to the mid-term;
2) fusion with human mature M (II) oocytes and induce PCC (chromosome agglutination).
3) fusion with mouse M II oocytes and induced PCC;
4) fusion with mouse fertilized eggs, induced to mid-term.
Result:
1. pairs of 138 1PN embryos were detected by double color FISH. The results were 87, of which 48 were derived from IVF, 18.8% of the monomer, 54.2% for two body signals, 20.8% for chimerism and 6.2% in multibody signals, and from 39 of ICSI, two signal 25.6%, chimerism 23.1%, 5.1%.87 1PN embryos of multibody signals, 16 of them developed into blastocysts, of which 12 were two body signals (75%) and 4 were chimeras (25%).
2. the results of karyotype analysis of embryo single blastomere are as follows:
1) using colchicine to block 57 blastomere directly and get 9 metaphase chromosomes, the success rate was 15.8%.
2) 26 eggs from blastomere were induced by human egg maturation in vitro, and 4 metaphase chromosomes were obtained. The success rate was 15.4%.
3) 13 mature blastomere cells were induced by mouse oocytes, and the oocytes were activated by electrofusion, and the success rate was 0.
4) 160 fertilized eggs were used to induce blastomere, and 25 metaphase chromosomes were obtained, with a success rate of 15.6%.
Conclusion:
1.ICSI source 1PN is mostly mono signal, possibly due to sperm undisaggregation and oocyte activation; IVF source 1PN is mostly normal two body signal, which may be mainly due to the formation of prokaryotic asynchronous or prokaryotic fusion. The fifth day embryos developed to blastocyst were all two body signals or chimeras, probably due to unfolding. The body's embryo lacks the ability to develop to the blastocyst.
2. the number of metaphase chromosomes obtained by four methods is about 15.5%, and the success rate is low. The efficiency of using nuclear conversion technology to induce the blastomere chromosomes to medium-term phase is still low, and further improvement is needed.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R321-33

【共引文献】