染色体核型分析及荧光原位杂交检测助孕夫妇自然流产绒毛组织染色体异常

发布时间：2018-03-01 21:24

本文关键词： G显带核型分析荧光原位杂交自然流产绒毛染色体染色体数目异常　出处：《山东大学》2014年硕士论文　论文类型：学位论文

【摘要】：研究背景自然流产一般是指孕不足22周、胚胎或者胎儿体重不足500g时,已死亡的组织或其附属物从母体中排出。造成自然流产的原因很多,主要包括遗传因素、解剖因素、免疫因素、感染因素、内分泌因素及环境因素等,其中遗传因素是导致其发生的主要原因。遗传因素中的胚胎染色体异常目前被认为是已知最为常见的自然流产诱发因素。绒毛膜的绒毛细胞具有和胚胎组织相同的遗传信息,因此,通过对绒毛细胞进行染色体检查,可以了解胚胎细胞的遗传性状,不仅能对自然流产的病因作出遗传学的判断,还可以为染色体病的早期产前诊断提供理论依据。目前检测染色体异常的主要手段是细胞培养后行染色体G显带核型分析,这种传统的方法存在较多的局限性,例如：细胞培养成功率较低,只能检测培养后的中期细胞,难以做出嵌合体的诊断,无法检测一些微小倒位、重排、缺失和标记染色体等,因而不能完全满足当前的要求。荧光原位杂交(fluorescent in situ hybridization,FISH)技术是在分子生物学、细胞遗传学和免疫学的基础上发展起来的一项新技术,其按照碱基互补配对原则,应用染色体特异性探针与待测DNA互补链杂交,通过观察荧光显微镜下信号的颜色和数目即可做出诊断,其检测快速、结果判读简单,对所检测的样本要求低,部分弥补了细胞遗传学方法的不足,显著提高了识别异常的能力,为检测染色体的数目及部分结构异常、确认标记染色体等研究提供了新的手段。胚胎染色体异常与孕早期胚胎停育关系密切,核型异常种类多样,如果能常规进行孕早期自然流产组织的染色体分析,将有助于提高异常核型胚胎的检出率。若有条件收集大样本、多区域的数据结果进行分析,将更有助于探讨自然流产的病因,为遗传咨询及优生优育提供指导。研究目的应用传统染色体核型分析及荧光原位杂交(FISH)技术对自然流产胎儿绒毛组织进行遗传学检测,分析导致自然流产发生的常见染色体异常的类型,探讨这；两种技术在检测自然流产绒毛组织染色体异常中的应用价值以及孕周、孕妇年龄、流产次数、助孕方式及其他相关因素与胚胎染色体异常的关系。研究方法收集2010年8月至2013年10月于山东大学附属生殖医院就诊的579例发生自然流产并以适当方式终止妊娠的孕妇的胎儿绒毛组织,其中449例绒毛组织进行细胞培养,培养成功的样本行G显带核型分析；228例绒毛组织应用FISH技术检测13、16、18、21、22、X、Y染色体数目异常。回顾性分析孕妇的临床资料,并对上述两种方法的检测结果进行总结、比较,采用统计软件SPSS19.0进行统计学分析。结果 1.在449例行传统染色体核型分析的标本中,因绒毛量少无法培养3例,细菌污染92例,不明原因细胞死亡101例,其他培养失败115例,核型报告率30.73%,失败率69.27%。共检出异常核型17例,异常率为12.32%(17/138)。有98例标本后又运用FISH方法检测； 2.在228例行FISH检测的自然流产标本中,共检测出异常标本86例,异常率为37.72%；有孕周记录的有效样本204例,在不同孕期的分布：早期妊娠(小于12周)、中期妊娠(12-23周)的样本数分别为196例、8例,各占比例96.08%、3.92%；前三位最常见的染色体数目异常为三体型46例(53.49%),三倍体12例(13.95%)和X单体5例(5.81%)； 3.有14例行传统核型分析有结果后又行FISH检测,两种结果相比较有4例不一致。其余10例结果一致,一致率为71.43%； 4.复发性自然流产患者绒毛染色体核型异常率为44.19%,高于初次自然流产的患者34.38%的异常率,既往流产次数多的患者绒毛染色体异常率有所升高,但差异不显著；采用IVF助孕的患者绒毛染色体核型异常率为31.36%,低于采用ICSI助孕患者44.44%的异常率,但二者差异不显著。结论 1.染色体数目异常是导致自然流产发生的重要因素,对自然流产孕妇行绒毛染色体检测及相关遗传咨询是有必要的； 2.自然流产组织中,排名前三位的染色体异常类型是常染色体三体型、三倍体和X单体； 3.传统核型分析是检测染色体异常的金标准,但由于这种方法有细胞培养时间长、影响因素多、分析过程复杂、失败率高、重复性差等局限性,因此不能完全胜任对自然流产组织的检查要求； 4. FISH技术在检测自然流产组织染色体异常时具有操作简单、快速及准确性、成功率高等优势,同时对所检测的样本要求低,是一种更适合临床要求的检测方法； 5.既往流产次数多的患者其流产组织染色体异常率有所升高,但差异不显著；采用不同方式助孕的患者,其自然流产组织染色体核型异常率没有显著性差异。
[Abstract]:Research background
Natural abortion generally refers to less than 22 weeks of gestation, embryonic or fetal weight is less than 500g, dead tissue or appendages discharged from the mother. Many causes of spontaneous abortion, including genetic factors, anatomical factors, immune factors, infection factors, endocrine factors and environmental factors, which is the result of genetic factors the main reasons for its occurrence. Genetic factors in embryonic chromosomal abnormalities is currently considered the most common predisposing factors of spontaneous abortion is known.
Chorionic villus cells have the same genetic information, and embryonic tissues therefore through chromosome examination of villus cells, can understand the genetic traits of embryonic cells can not only cause of spontaneous abortion to genetics judgment, can also provide a theoretical basis for chromosomal disease early prenatal diagnosis. The main means of chromosomal abnormalities cell culture after detection is chromosome G banding karyotype analysis, this traditional approach has limitations, for example: more cell culture success rate is low, can only detect metaphase cells after culture, it is difficult to make a diagnosis of mosaicism cannot detect small inversions, rearrangement, deletion and marker chromosomes, and therefore can not be completely meet the current requirements.
Fluorescence in situ hybridization (fluorescent in situ hybridization, FISH) is a technique in molecular biology, development of a new technology based on cell genetics and immunology, in accordance with the principle of complementary base, chromosome specific probe and detected DNA hybridization of complementary strands, make a diagnosis by fluorescence microscope and the number of color signal then, the detection is rapid, the interpretation of the results is simple, low requirement for the test part to compensate for the lack of cytogenetic methods, significantly improve the ability to identify abnormal, the number and structure detection of chromosome abnormalities, that provides a new means of marker chromosomes.
The embryo chromosome abnormality and embryo growth arrest is closely related to abnormal karyotype analysis of species diversity, if can the routine of spontaneous abortion in early pregnancy tissue chromosomes, will help to improve the detection rate of abnormal karyotype embryos. If the conditions were collected from a large sample, multi regional data analysis results, will be helpful to the study of natural causes abortion, provide guidance for genetic counseling and prenatal and postnatal care.
research objective
The application of conventional karyotype analysis and fluorescence in situ hybridization (FISH) technique for genetic testing on spontaneous abortion fetal villi, analyze this leads to spontaneous abortion type, common chromosome abnormalities; application value of two kinds of technology in the detection of spontaneous abortion villus chromosomal abnormalities and the gestational age, maternal age, number of abortion the relationship between pregnancy, mode and other factors associated with chromosomal abnormalities.
research method
From August 2010 to October 2013 in Shandong University Hospital Affiliated reproductive 579 cases of spontaneous abortion and termination of pregnancy in a proper way of fetal villi, 449 cases of chorionic villus cell culture, cultured samples for G banding karyotype analysis; application of FISH technology in 228 cases of villi of detection of 13,16,18,21,22, X, Y chromosome abnormality the number of pregnant women. The clinical data were retrospectively analyzed, and the results of the above two methods are summarized, compared with SPSS19.0 statistical software for statistical analysis.
Result
1. in 449 patients were treated with conventional karyotype analysis of the specimens, because less villi cannot cultivate 3 cases, 92 cases of bacterial contamination, unexplained cell death in 101 cases, other training failed in 115 cases, karyotype report rate was 30.73%, the failure rate of 69.27%. were detected in 17 cases of abnormal karyotype, the abnormal rate was 12.32% (17/138) 98. After the cases were detected by FISH;
2. in 228 cases with FISH detection of spontaneous abortion specimens, were detected in 86 samples with abnormal, the abnormal rate was 37.72%; effective sample records of 204 cases of gestational age, distribution in different pregnancy: early pregnancy (less than 12 weeks), middle pregnancy (12-23 weeks) the number of samples were 196 cases, 8 the proportion of cases, 96.08%, 3.92%; the three most common chromosome abnormality in three cases of type 46 (53.49%), 12 cases of triploid (13.95%) 5 cases and X monomer (5.81%);
3. of the 14 cases of traditional karyotype analysis were detected by FISH and 4 cases were not consistent with the two results. The other 10 cases were consistent with the same rate of 71.43%.
The abnormal rate of 4. patients with recurrent spontaneous abortion villus karyotype was 44.19%, the abnormal rate was higher than the initial 34.38% patients with spontaneous abortion, abortion more patients with chromosomal abnormality rate was increased, but the difference was not significant; the villi of patients with chromosome karyotype IVF progesterone abnormal rate was 31.36%, lower than the rate of abnormal ICSI pregnancy 44.44% of the two patients, but the difference was not significant.
conclusion
Abnormal number of 1. chromosomes is an important factor leading to the occurrence of spontaneous abortion. It is necessary to detect chorionic chromosomes and related genetic counseling for pregnant women with spontaneous abortion.
In 2. spontaneous abortion groups, the top three chromosomal abnormalities were autosomal three, triploid and X monomers.
3., traditional karyotype analysis is the gold standard for detecting chromosomal abnormalities. However, because this method has many limitations, such as long cell culture time, many influencing factors, complicated analysis process, high failure rate and poor repeatability, it is not entirely competent for the examination requirements of spontaneous abortion.
4., FISH technology has advantages of simple operation, high speed and accuracy, and high success rate in detecting chromosomal abnormality in spontaneous abortion tissue. Meanwhile, it has low requirement for the tested samples, so it is a more suitable method for clinical requirements.
5. the rate of chromosomal abnormality in the abortion group increased but the difference was not significant. The abnormal rate of chromosome karyotype in spontaneous abortion group was not significantly different.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.21

【参考文献】