本文關(guān)鍵詞： G顯帶核型分析 熒光原位雜交 自然流產(chǎn) 絨毛染色體 染色體數(shù)目異常　出處：《山東大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景 自然流產(chǎn)一般是指孕不足22周、胚胎或者胎兒體重不足500g時(shí),已死亡的組織或其附屬物從母體中排出。造成自然流產(chǎn)的原因很多,主要包括遺傳因素、解剖因素、免疫因素、感染因素、內(nèi)分泌因素及環(huán)境因素等,其中遺傳因素是導(dǎo)致其發(fā)生的主要原因。遺傳因素中的胚胎染色體異常目前被認(rèn)為是已知最為常見的自然流產(chǎn)誘發(fā)因素。 絨毛膜的絨毛細(xì)胞具有和胚胎組織相同的遺傳信息,因此,通過對(duì)絨毛細(xì)胞進(jìn)行染色體檢查,可以了解胚胎細(xì)胞的遺傳性狀,不僅能對(duì)自然流產(chǎn)的病因作出遺傳學(xué)的判斷,還可以為染色體病的早期產(chǎn)前診斷提供理論依據(jù)。目前檢測(cè)染色體異常的主要手段是細(xì)胞培養(yǎng)后行染色體G顯帶核型分析,這種傳統(tǒng)的方法存在較多的局限性,例如：細(xì)胞培養(yǎng)成功率較低,只能檢測(cè)培養(yǎng)后的中期細(xì)胞,難以做出嵌合體的診斷,無法檢測(cè)一些微小倒位、重排、缺失和標(biāo)記染色體等,因而不能完全滿足當(dāng)前的要求。 熒光原位雜交(fluorescent in situ hybridization,FISH)技術(shù)是在分子生物學(xué)、細(xì)胞遺傳學(xué)和免疫學(xué)的基礎(chǔ)上發(fā)展起來的一項(xiàng)新技術(shù),其按照堿基互補(bǔ)配對(duì)原則,應(yīng)用染色體特異性探針與待測(cè)DNA互補(bǔ)鏈雜交,通過觀察熒光顯微鏡下信號(hào)的顏色和數(shù)目即可做出診斷,其檢測(cè)快速、結(jié)果判讀簡(jiǎn)單,對(duì)所檢測(cè)的樣本要求低,部分彌補(bǔ)了細(xì)胞遺傳學(xué)方法的不足,顯著提高了識(shí)別異常的能力,為檢測(cè)染色體的數(shù)目及部分結(jié)構(gòu)異常、確認(rèn)標(biāo)記染色體等研究提供了新的手段。 胚胎染色體異常與孕早期胚胎停育關(guān)系密切,核型異常種類多樣,如果能常規(guī)進(jìn)行孕早期自然流產(chǎn)組織的染色體分析,將有助于提高異常核型胚胎的檢出率。若有條件收集大樣本、多區(qū)域的數(shù)據(jù)結(jié)果進(jìn)行分析,將更有助于探討自然流產(chǎn)的病因,為遺傳咨詢及優(yōu)生優(yōu)育提供指導(dǎo)。 研究目的 應(yīng)用傳統(tǒng)染色體核型分析及熒光原位雜交(FISH)技術(shù)對(duì)自然流產(chǎn)胎兒絨毛組織進(jìn)行遺傳學(xué)檢測(cè),分析導(dǎo)致自然流產(chǎn)發(fā)生的常見染色體異常的類型,探討這；兩種技術(shù)在檢測(cè)自然流產(chǎn)絨毛組織染色體異常中的應(yīng)用價(jià)值以及孕周、孕婦年齡、流產(chǎn)次數(shù)、助孕方式及其他相關(guān)因素與胚胎染色體異常的關(guān)系。 研究方法 收集2010年8月至2013年10月于山東大學(xué)附屬生殖醫(yī)院就診的579例發(fā)生自然流產(chǎn)并以適當(dāng)方式終止妊娠的孕婦的胎兒絨毛組織,其中449例絨毛組織進(jìn)行細(xì)胞培養(yǎng),培養(yǎng)成功的樣本行G顯帶核型分析；228例絨毛組織應(yīng)用FISH技術(shù)檢測(cè)13、16、18、21、22、X、Y染色體數(shù)目異常。回顧性分析孕婦的臨床資料,并對(duì)上述兩種方法的檢測(cè)結(jié)果進(jìn)行總結(jié)、比較,采用統(tǒng)計(jì)軟件SPSS19.0進(jìn)行統(tǒng)計(jì)學(xué)分析。 結(jié)果 1.在449例行傳統(tǒng)染色體核型分析的標(biāo)本中,因絨毛量少無法培養(yǎng)3例,細(xì)菌污染92例,不明原因細(xì)胞死亡101例,其他培養(yǎng)失敗115例,核型報(bào)告率30.73%,失敗率69.27%。共檢出異常核型17例,異常率為12.32%(17/138)。有98例標(biāo)本后又運(yùn)用FISH方法檢測(cè)； 2.在228例行FISH檢測(cè)的自然流產(chǎn)標(biāo)本中,共檢測(cè)出異常標(biāo)本86例,異常率為37.72%；有孕周記錄的有效樣本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)的樣本數(shù)分別為196例、8例,各占比例96.08%、3.92%；前三位最常見的染色體數(shù)目異常為三體型46例(53.49%),三倍體12例(13.95%)和X單體5例(5.81%)； 3.有14例行傳統(tǒng)核型分析有結(jié)果后又行FISH檢測(cè),兩種結(jié)果相比較有4例不一致。其余10例結(jié)果一致,一致率為71.43%； 4.復(fù)發(fā)性自然流產(chǎn)患者絨毛染色體核型異常率為44.19%,高于初次自然流產(chǎn)的患者34.38%的異常率,既往流產(chǎn)次數(shù)多的患者絨毛染色體異常率有所升高,但差異不顯著；采用IVF助孕的患者絨毛染色體核型異常率為31.36%,低于采用ICSI助孕患者44.44%的異常率,但二者差異不顯著。 結(jié)論 1.染色體數(shù)目異常是導(dǎo)致自然流產(chǎn)發(fā)生的重要因素,對(duì)自然流產(chǎn)孕婦行絨毛染色體檢測(cè)及相關(guān)遺傳咨詢是有必要的； 2.自然流產(chǎn)組織中,排名前三位的染色體異常類型是常染色體三體型、三倍體和X單體； 3.傳統(tǒng)核型分析是檢測(cè)染色體異常的金標(biāo)準(zhǔn),但由于這種方法有細(xì)胞培養(yǎng)時(shí)間長、影響因素多、分析過程復(fù)雜、失敗率高、重復(fù)性差等局限性,因此不能完全勝任對(duì)自然流產(chǎn)組織的檢查要求； 4. FISH技術(shù)在檢測(cè)自然流產(chǎn)組織染色體異常時(shí)具有操作簡(jiǎn)單、快速及準(zhǔn)確性、成功率高等優(yōu)勢(shì),同時(shí)對(duì)所檢測(cè)的樣本要求低,是一種更適合臨床要求的檢測(cè)方法； 5.既往流產(chǎn)次數(shù)多的患者其流產(chǎn)組織染色體異常率有所升高,但差異不顯著；采用不同方式助孕的患者,其自然流產(chǎn)組織染色體核型異常率沒有顯著性差異。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R714.21

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