荧光原位杂交技术在自然流产绒毛染色体核型检测中的应用

目的 探讨荧光原位杂交(fluorescence in situ hybridization,FISH)技术在提高自然流产绒毛染色体核型分析准确性和异常核型检出率中的作用.方法 采用18、X、Y染色体着丝粒探针和13、21及16、22染色体单一序列探针,对100例自然流产绒毛标本同时进行FISH检测和常规染色体核型分析,比较并分析2种方法的一致性及差异.结果 (1)染色体核型分析:100例流产绒毛标本培养成功率为89.0％(89/100).检出异常核型51例,异常核型检出率为57.3％(51/89),其中常染色体非整倍体37例、性染色体非整倍体4例、三倍体2例、四倍体1例,还有1例核型为68,XX,结构异常6例.(2) FISH技术检测:100例流产绒毛标本均获得FISH结果,成功率为100.0％.共检出38例染色体异常,异常核型检出率为38.0％(38/100),其中常染色体非整倍体25例、性染色体非整倍体5例、三倍体3例,还有1例13、16、18、21、22号染色体均为三倍体,嵌合体4例.(3)核型分析与FISH结果的异同:在绒毛标本培养失败的11例中,FISH检测出染色体异常2例,占18.2％(2/11);核型为46,XY者中FISH检测出3例非整倍体嵌合体;核型为46,XX者中FISH检测出染色体异常2例.FISH能检测出的染色体异常占所有染色体异常核型的65.5％(38/58).结论 FISH技术能简便、快速的检测自然流产绒毛染色体非整倍体数目异常,联合常规染色体核型分析能提高染色体核型分析的准确性和异常核型的检出率.     

荧光原位杂交在未培养羊水细胞产前诊断中的临床应用研究

目的:建立稳定的荧光原位杂交(FISH)技术,探讨FISH技术在未培养羊水细胞产前诊断的-临床应用价值.方法:应用国产5条染色体探针(13、18、21、X、Y)对411例有产前诊断指征的孕妇进行未培养羊水细胞的FISH检测,同时进行细胞染色体核型分析,对两者检测结果进行比较.结果:411例中,正常核型398例,非整倍体核型5例(均为21-三体),结构异常4例(平衡易位2例、倒位2例),染色体多态4例.其中正常核型及5例非整倍体核型能被FISH检测出,而染色体结构异常和染色体多态未能检测出;FISH检测时间为48～72小时,较传统核型分析时间(14～21天)大大缩短;FISH检测一次成功率97.3%,并且不受孕周的限制;FISH检测与细胞培养核型分析相符;FISH检测没有受到羊水性状的影响;染色体非整倍体的诊断准确率达100%.结论:应用国产探针对未培养羊水细胞进行FISH分析,实验方法简便快速,可用于染色体非整倍体的产前诊断,有较大的临床应用价值.     

第一极体形态学与人类卵细胞非整倍体相关关系的研究

目的 :探讨第一极体的形态学和卵细胞染色体非整倍体异常间的相关性 ,为使用第一极体作为移植胚胎的筛选指标提供理论依据。方法 :采用多色荧光原位杂交法分析卵细胞中 1 3 ,1 6,1 8,2 1和 2 2号染色体的核型 ,并结合第一极体的形态学进行相关分析。结果 :72 .3 %的卵细胞核型正常 ,2 7.7%的卵细胞为非整倍体 ;在 4组具有不同形态第一极体的卵细胞中 ,非整倍体的发生率分别为 1 4.7% ,1 7.4% ,2 9.7%和 61 .1 %。第 1组卵细胞非整倍体的发生率与第 4组相比 ,差异有高度显著性 (P <0 .0 0 1 )。结论 :第一极体的形态学和卵细胞的染色体非整倍体异常之间存在一定的相关性。由于其非整倍体的发生率高 ,由具有异常大小第一极体的卵细胞发育而来的胚胎不适用于移植。     

2837例妊娠中期孕妇羊水间期细胞荧光原位杂交检测胎儿染色体异常的残余风险

目的 评估妊娠中期羊水间期细胞荧光原位杂交(fluorescence in situ hybridization,FISH)检测后漏诊非13、18、21、X或Y染色体非整倍体异常核型的残余风险. 方法 对中国医学科学院北京协和医学院北京协和医院2010年1月1日至2013年7月31日2 837例妊娠中期行羊膜腔穿刺术同时行间期细胞FISH检测和传统核型分析的病例及其结果进行回顾性分析.采用GLP 13/GLP21/CSP 18/CSPX/CSPY探针针对13、18、21、X或Y染色体进行间期细胞FISH检测.计算具有三大产前诊断指征(孕妇高龄、母体血清学筛查18或21-三体高风险、妊娠23周前超声发现胎儿有结构异常)的孕妇间期细胞FISH检测的检出率以及残余风险. 结果 2 837例标本中,共85例(3.0％)羊水细胞染色体核型分析异常.85例中的73例为13、18、21、X或Y染色体非整倍体的异常核型,间期细胞FISH结果均为阳性,其中21-三体共43例(包括2例嵌合体)、18-三体1 5例、13-三体l例、性染色体非整倍体14例(包括4例嵌合体).12例(14.1％)异常结果为非13、1 8、21、X或Y染色体的非整倍体,包括平衡性结构重排6例、非平衡性结构重排5例、其他染色体非整倍体1例.这12例中,间期细胞FISH结果阳性3例,均涉及21号染色体的不平衡性结构异常;阴性9例,其中4例遗传性平衡性重排和2例新生平衡性重排.2 837例病例间期细胞FISH分析对染色体异常的检出率为89.4％(76/85),异常核型漏诊率为14.1％(12/85),残余风险为0.43％(12/2 761). 结论 间期细胞FISH检测是核型分析的有效补充,但不能取代核型分析,单纯间期细胞FISH检测进行产前诊断可能漏诊部分染色体结构异常.在产前遗传咨询时,应向患者解释间期细胞FISH的检出率和残余风险,合理选择产前诊断方法.     

荧光原位杂交技术在未培养羊水细胞产前诊断中的应用研究

目的:探讨运用荧光原位杂交(FISH)技术对未培养羊水细胞的染色体非整倍体进行快速诊断的临床应用价值.方法:对符合产前诊断要求的孕妇于孕18～24周抽取羊水,用13、21、18及X、Y号染色体探针对其未培养羊水细胞进行诊断,并与核型分析结果比较.结果:125例标本的核型分析中,成功培养123例,其中117例为正常核型,6例为异常核型,异常核型为21-三体2例,9号染色体臂间倒位3例,6号和7号染色体平衡易位1例;与核型分析相比,FISH只检测出2例非整倍体核型,其余染色体结构异常未能检出,FISH检测时间为24～48小时,较传统核型分析时间2～3周大大缩短,且同核型结果一致,特异性100%.结论:荧光原位杂交技术具有简便、准确等优点,可快速地辅助核型分析,缓解患者及家属情绪,有临床推广价值.     

FISH技术在检测自然流产绒毛组织非整倍体异常中的应用研究

目的:探讨运用荧光原位杂交(FISH)检测自然流产绒毛组织的临床价值,评价它与传统经典的核型分析方法的关系。方法:对157例孕早期自然流产的绒毛组织进行FISH检测,均采用16、22、13、21、18、X、Y号染色体荧光探针检测,判断染色体非整倍体异常情况。同时进行绒毛细胞培养染色体核型分析,作为对照诊断标准。结果:核型分析成功率为48.4%,FISH检测成功率为100%。核型分析成功的76例样本中,64例结果与核型分析结果相一致,以细胞遗传学作为诊断标准,诊断的符合率为84.2%。结论:FISH技术与传统的绒毛细胞培养染色体核型分析相比,过程迅速,方法简单,提高了诊断的成功率,但无法完全取代传统的染色体核型分析,应两者结合应用于临床。     

应用荧光原位杂交产前诊断染色体异常的临床价值

目的:探讨应用荧光原位杂交(HSH)产前诊断染色体非整倍体的临床价值.方法:收集120例产前诊断孕妇的新鲜羊水进行FISH检测和染色体核型分析,并将结果与临床追踪确诊结果(随访的新生儿或引产的死胎脐血或外周血的染色体核型)作比较,同时根据FISH的检测效能和分析产前诊断方案,评价HSH的临床应用价值.结果:①HSH检测全部成功,其结果与临床追踪确诊的核型分析一致,并且染色体非整倍体检出率100%;1例孕晚期羊水细胞培养失败,2例羊水培养为四倍体镶嵌体胎儿经临床追踪确诊后为正常染色体.②产前诊断指征中,高龄、多项指征及其他因素的孕妇临床上对FISH及核型分析这两种方法的选择比较,差异无统计学意义(P>0.05);而血清唐氏筛查异常和超声筛查异常的孕妇分别倾向选择FISH(P=0.029)及核型分析(P=0.000).结论:HSH技术能快速准确检测染色体非整倍体的异常.母血清唐氏筛查异常孕妇产前诊断倾向选择FISH检测.FISH可作为孕晚期高危孕妇首选的产前诊断方法.     

国产探针荧光原位杂交技术用于产前诊断未培养羊水细胞染色体异常的研究

目的 探讨国产探针荧光原位杂交(FISH)技术用于产前诊断未培养羊水细胞染色体非整倍体异常的临床价值,评价国产探针的性能.方法 应用FISH技术埘全旧37家省级及地区级医院产前诊断中心就诊的孕16～24周的1369例孕妇的未培养羊水细胞进行快速产前诊断;应用多色FISH技术对5条染色体(21、13、18、X和Y)进行检测.同时将羊水细胞接种、培养,行常规细胞染色体核型分析,作为FISH检测结果的对照.结果 被检测的1369份样本中,1361例未培养羊水细胞获得诊断结果,检测成功率99.42%(1361/1369).共检出异常核型35例,异常核型枪出率为2.57%(35/1361),其中包括21三体22例;13三体4例;18三体6例;18二倍体、X0 1例;18二倍体、XXY 2例.FISH榆测结果与常规细胞染色体核型分析结果一致.结论 应用国产探针FISH技术检测未培养羊水细胞染色体数目异常具有快速、简便、所用样本量少的优势,结果准确可靠.     

FISH快速检测自然流产绒毛染色体非整倍体异常的临床价值

目的:探讨应用荧光原位杂交技术(FISH)对早期自然流产绒毛染色体非整倍体检测的临床价值。方法:对30例因自然流产行清宫术的绒毛组织行FISH分析,使用7种探针对13、16、18、21、22号和X、Y染色体进行了检测,并对这30例流产夫妇行外周血淋巴细胞染色体常规核型分析。结果:FISH分析的30例自然流产的绒毛组织中,有17例检测出了异常信号,检出率为57%,其中8例16-三体、2例22-三体、2例13-三体和5例三倍体。30例自然流产夫妇外周血淋巴细胞染色体核型未见异常。结论:FISH技术可以快速、简便地检测出流产物绒毛组织染色体非整倍体的异常,FISH技术的应用可以为自然流产夫妇遗传咨询提供重要的信息。     

PGD周期中机械法和激光法活检对胚胎发育及妊娠结局的影响

目的:比较机械法和激光法进行卵裂球和极体活检对胚胎发育及着床前遗传学诊断(preimplantation genetic diagnosis,PGD)周期妊娠结局的影响。方法:本研究包括20对夫妇的21个PGD周期,其中18个周期分别用机械法或激光法于受精后第3天进行卵裂球活检,并用荧光原位杂交(fluorescence in situ hybridization,FISH)分析检出的卵裂球,于受精后第5或第6天移植信号正常的胚胎;2个周期分别用机械法和激光法在取卵后行极体活检,后行胞浆内单精子注射(introcytoplasmic sperm injection,ICSI)。同时将活检取出的极体进行FISH分析,于取卵后第3天移植经FISH检查正常的卵发育而来的胚胎。另外一个周期先用激光法实行了极体活检,由于FISH检查均无信号,后又用激光法对胚胎行卵裂球活检。结果:共活检胚胎145枚,其中109枚用机械法,36枚用激光法。活检后胚胎的继续发育率分别为72.48%和83.33%,囊胚形成率为33.94%和44.44%,临床妊娠率为38.46%和16.67%,着床率为21.43%和8.33%,两种方法无显著差异。对27枚卵行极体活检,其中12枚用机械法,15枚用激光法。活检后2PN受精率分别为58.33%和46.66%,继续发育率为66.67%和60.00%,亦无显著差异。对活检出的极体进行FISH分析,用机械法活检的极体信号阳性率为90.00%,显著高于激光法的28.57%。结论:用机械法和激光法行极体或卵裂球活检对胚胎发育的影响差异无统计学意义。但使用机械法活检卵裂球能获得较高的临床妊娠率和着床率。极体活检时能获得较高的受精率和继续发育率,故推荐使用机械法进行活检。     

女性罗氏易位携带者辅助生育治疗5例临床分析

目的:探讨应用极体分析和分裂球分析法对反复流产的女性罗氏易位携带者进行着床前遗传学诊断(PGD)的临床策略。方法:采用荧光原位杂交(FISH)技术,对5例患者用全染色体涂抹探针检测第一极体和用特异位点探针检测分裂球中相应染色体的荧光信号。5例女性罗氏易位携带者的外周血淋巴细胞染色体核型分别为:45,XX,der (13;14)(q10;q10)3例,45,XX,der(14;21)(q10;q10)、45,XX,der(21;22)(q10;q10)各1例。结果:5例患者中4例获得妊娠并分娩,其中2例经分裂球分析后妊娠,出生3个婴儿:2个正常核型,1个罗氏易位携带者;1例经极体分析诊断后分娩一正常男婴;另1例极体分析未明确诊断又行分裂球分析选择胚胎移植后,出生1个罗氏易位携带者后代。结论:(1)女性罗氏易位携带者PGD应首选极体分析,争取避免携带者出生;(2)极体分析未能得到诊断的胚胎可在分裂球期再次PGD。     

Polar body diagnosis of common aneuploidies by FISH

Purpose: The purpose of this work was to investigate the reliability and accuracy of polar body analysis for preimplantation diagnosis of common aneuploidies in IVF patients of advanced maternal age. Design: We have previously introduced polar body analysis as an approach for nondestractive evaluation of the genotype of human oocytes. The method has recently been applied in a clinical trial involving 45 infertile patients, demonstrating the feasibility of preconception diagnosis of common aneuploidies by fluorescent in situ hybridization (FISH). The present paper describes the experience of polar body diagnosis in 135 IVF patients (161 cycles) of advanced maternal age. Results: FISH results of the first and/or second polar bodies were available in 648 (72.4%) of 895 biopsied oocytes subjected to FISH analysis. Of 648 oocytes with FISH results, 208 demonstrated chromosomal abnormalities. Of 440 oocytes predicted to be free from monosomy or trisomy of chromosomes X, 18, and/or 13/21, 314 were normally fertilized, cleaved, and transferred in 122 treatment cycles, resulting in 6 healthy deliveries and 12 ongoing pregnancies following confirmation of the polar body diagnosis by CVS or amniocentesis. Conclusion: The method may be useful for detection of oocytes with common chromosomal trisomies in IVF patients of advanced maternal age.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

A study of meiotic segregation in a fertile human population following ovarian stimulation with recombinant FSH-LH

Objective

The aim of the study is to investigate the meiotic segregation in fresh eggs from anonymous egg donors and to analyze the baseline levels of aneuploidy in this population.

Results

The study includes the largest series of donor eggs so far studied: 203 eggs from donors aged between 20 and 31 years. No diagnosis was obtained in 10.8 % of cases (22/ 203). The biopsy of the first and second polar bodies was completed in a sequential manner on day 0 and day 1 of embryo development. Chromosomes 13, 16, 18, 21 and 22 are analyzed by means of the FISH test. The diagnosable fertilized eggs gave an aneuploidy rate of 19.1 % (31/162), with 83.8 % (26/31) of the errors produced during meiosis I, 12.9 % (4/31) produced during meiosis II, and 3.2 % (1/31) produced during both meiosis I and II. The premature division of sister chromatids is the main source of meiotic error during Meiosis I, resulting in the creation of oocyte aneuploidy.

Conclusions

FISH analysis of the first and second polar body in donor oocytes gave an aneuploidy rate of 19.1 %. This study shows the majority of errors occur during Meiosis I.     

Preimplantation Diagnosis of Common Aneuploidies by the First- and Second-Polar Body FISH Analysis

Purpose: A low pregnancy rate in in vitro fertilization (IVF) patients of advanced maternal age may be caused by aneuploidies originating from non disjunction in the first or second meiotic divisions. We introduced genetic testing of oocytes by sampling and fluorescent in situ hybridization (FISH) analysis of the first and second polar bodies, to avoid fertilization and transfer of aneuploid oocytes in IVF patients of advanced maternal age. Methods: Three hundred and sixty-three IVF patients 34 years and older participated in the study. Using micromanipulation procedures, the first and second polar bodies were removed following their extrusion from the oocytes and studied by FISH, using probes specific for chromosomes 13, 18, and 21 to detect oocytes with common aneuploidies. Results: Of a total of 538 IVF cycles, 3250 oocytes were available for FISH analysis, with conclusive FISH results in 2742 oocytes (84.3%). As many as 1102 (40%) of oocytes were predicted to be aneuploid and not transferred. Of 1640 embryos predicted to be normal, 1145 were transferred in 467 treatment cycles, resulting in 107 pregnancies (23%), from which 67 healthy children have been born, 32 pregnancies spontaneously aborted, and 15pregnancies are ongoing after being confirmed normal by prenatal diagnosis. Conclusions: Preimplantation diagnosis by first- and second-polar body FISH analysis allows us to avoid the age-related risk of common aneuploidies in IVF patients of advanced maternal age.     

Outcome of laser-assisted polar body biopsy and aneuploidy testing

Polar body biopsy and subsequent fluorescence in-situ hybridization (FISH) analysis allows detection of maternally derived chromosomal aneuploidies in human oocytes during IVF treatment. The development of a diode laser technique for the partial opening of the zona pellucida has stimulated the use of this technique to assist polar body biopsy. Laser-assisted polar body biopsy was performed in 140 IVF cycles from patients of advanced maternal age (> or =35 years). A total of 921 oocytes were treated by a laser for partial zona opening and polar body removal. FISH was performed for chromosomes 13, 16, 18, 21 and 22 and results were available for 903 oocytes (98%). In all, 443 oocytes (49.1%) were euploid and of these, 293 were fertilized. A total of 214 embryos were transferred in 120 embryo transfer cycles (1.78 per embryo transfer) resulting in 27 clinical pregnancies (22.5% per embryo transfer) with an implantation rate of 15.4%. Subsequently, five women aborted (18.5%) and 24 healthy children were born from the remaining 22 pregnancies, which gives a take home baby rate of 18.3% per transfer cycle. It is concluded that polar body biopsy using a diode laser system is as efficient as standard polar body biopsy using zona drilling.     

Reproductive oocyte/embryo genetic analysis: comparison between fluorescence in-situ hybridization and comparative genomic hybridization

Multiple displacement amplification (MDA) renders an increased quantity of genomic DNA available for comparative genomic hybridization (CGH), enabling it to be used to identify genomic imbalances in human blastomeres. The karyotypic lineage of 57 blastocysts derived from 11 ovum donors following ovarian stimulation was examined. CGH was performed on all first polar bodies, and linearly on corresponding second polar bodies and blastomeres. A diploid karyotype was propagated from the prefertilized oocyte to the embryo in 25 (44%) sets of analyses. In 32/57 sets (56%), aneuploidy was detected in the post-fertilized zygotes/embryos and in nine (28%) of such cases the aneuploidy was 'chaotic' (> or =3 chromosomes). In 4/57 cases (7%) mitotic aneuploidy was observed. CGH and fluorescence in-situ hybridization (FISH) were concurrently performed on two blastomeres removed from each of 44 embryos obtained from four patients. In 43 (98%) of these embryos there was a direct karyotypic correlation between nine-probe commercial FISH and CGH. CGH identified > or =15% more chromosomal abnormalities than through FISH alone. The linear propagation using MDA-CGH, of the same karyotypic abnormalities that affected the oocyte of origin, in the corresponding embryos, coupled with the fact that CGH confirmed the aneuploidies identified through FISH, validates the accuracy and reliability of CGH technology.     

How safe is germinal vesicle stage oocyte rescue? Aneuploidy analysis of in vitro matured oocytes

OBJECTIVE: To evaluate the rate and type of aneuploidies of chromosomes 13, 16, 18, 21 and 22, with respect to the length of in vitro maturation (IVM) period, and to compare the results to previously published studies on aneuploidy rates of unfertilized, uninseminated mature oocytes and first polar bodies. STUDY DESIGN: Two hundred and twelve immature germinal vesicle stage oocytes were assigned to two groups. After successful IVM, depending on their maturational period of 24h (Group A) or 36h (Group B), chromosomal analysis was performed by five color fluorescence in situ hybridization (FISH). In Groups A and B the rates of aneuploid oocytes were calculated and compared by chi-square test. Also the rates of hyperhaploidy, hypohaploidy, disomy and nullisomy were determined and compared by chi-square test. The difference was considered statistically significant at p-value of <0.05. RESULTS: The prolonged IVM did not significantly affect the aneuploidy rate compared to the shorter maturation period (48.1% and 45.0%, respectively). Regarding the unbalanced premature chromatid separation, no statistically significant difference was found between hyperhaploidy and hypohaploidy (14.8% versus 8.3%). For chromosome nondisjunction, higher frequency of disomy than nullisomy was observed (30.6% versus 14.8%; p<0.05). The estimated global aneuploidy rate was between 42% and 63%. CONCLUSIONS: The aneuploidy rate of IVM GV-oocytes is comparable to the aneuploidy rate of in vivo matured oocytes and first polar bodies, regardless of the length of maturation period. This suggests that the immature oocytes can be used in infertility treatment after they complete maturation.     

Demonstration of a mechanism of aneuploidy in human oocytes using Multifluor fluorescence in situ hybridization

OBJECTIVE: To evaluate the potential of Multifluor fluorescence in situ hybridization (M-FISH) for karyotyping the human oocyte and first polar body. DESIGN: Prospective case study. SETTING: Research laboratories, university hospital. PATIENT(S): A 33-year-old woman with polycystic ovary syndrome who was undergoing ovarian stimulation and ICSI. MAIN OUTCOME MEASURE(S): Karyotyping of all chromosomes within an oocyte and first polar body, using GV stage oocytes matured to metaphase II in vitro. RESULT(S): Oocyte hyperploidy was diagnosed by M-FISH to be 23, X +15 cht +19 cht +22 cht. The correspond- ing polar body was hypoploid, with a karyotype of 23, X -15 cht -19 cht -22 cht. This was due to unbalanced predivision at meiosis I. Reprobing confirmed karyotype assignments for chromosomes X, 13, 18, and 21. CONCLUSION(S): The mechanism involved in maternally derived aneuploidy can be defined by using M-FISH to simultaneously karyotype both oocyte and first polar body chromosomes at metaphase II. Multifluor FISH may be useful for investigative studies of maternally derived aneuploidy, which is a major cause of preimplantation waste in natural and assisted reproduction.