人精子间期核的双色荧光原位杂交

目的　建立人精子间期核双色荧光原位杂交 (dual colorfluorescenceinsituhybridization ,D FISH)的实验方法。方法　采用双色荧光原位杂交技术对 12例正常精子标本进行间期核的原位杂交 ,并统计精子间期核X、Y染色体的杂交信号颗粒数量。结果　在显微镜下可见精子头部有以Biotin标记的pBamX7探针显示 1个绿色杂交信号为X染色体精子 (X精子 ) ,以Digoxigenin标记的 pY3.4探针显示 1个红色杂交信号为Y染色体精子 (Y精子 ) ;精子头部有 2个荧光杂交信号的精子为染色体数目异常精子 (如XX、XY、YY精子 ) ;间期核背景经DAPI复染显示蓝色 ;统计 12例正常精子标本总共 4 80 0个精子间期核 ,X精子杂交信号阳性率为 4 8.5 6 % ,Y精子杂交信号阳性率为 4 9.73% ,总共统计 2 4 0 0 0个精子 ,3种双体总杂交率为 0 .197%。结论　本法具有荧光杂交信号直观易分辨、短时间内能大量分析精子数量和实验操作相对简便、结果准确可信等优点     

特异性DNA探针检测细胞因子诱导的杀伤细胞在患者体内的生存时间

目的 观察应用荧光原位杂交技术(FISH)研究细胞因子诱导的杀伤细胞(CIK)在性别错配的女性受者体内生存时间.方法 通过细胞采集仪采集男性供者的单个核细胞,经多种细胞因子体外诱导培养成CIK细胞,输注给女性受者.治疗数月后,用Y染色体特异性DNA探针测定女性受者体内淋巴细胞上Y染色体的表达.结果 流式细胞仪检测第14天时CIK细胞高表达CD3+ CD56+为55.4％.CSP X探针定位于Xp11.1-q11.1,杂交信号为绿色,CSP Y探针定位于Yp11.1-q11.1,杂交信号为红色,女性细胞染色体XX显示两个绿色荧光信号,男性细胞染色体XY 显示1个绿色信号,1个红色荧光信号.在女性受者体内淋巴细胞上有Y染色体的表达,表达比例为0.5％ ～3.0％.结论 Y染色体特异性DNA探针可以确定CIK细胞在患者体内的生存时间.     

200例稽留流产绒毛染色体的间期FISH分析

目的探讨荧光标记的原位杂交技术(FISH)检测稽留流产绒毛组织中染色体异常的临床价值。方法收集2016年1月至2018年9月在我院就诊/住院的稽留流产患者200例为研究对象,手术治疗后留取绒毛组织样本进行FISH检测,严格按照试剂盒说明书操作,应用13、16、18、21、22、X和Y染色体特异性DNA探针进行杂交处理,并对结果进行分析。结果 200例绒毛组织样本的间期FISH均获得杂交信号,其中112例(56.00%)杂交结果提示染色体异常。其中,常染色体单体2例,占1.79%(2/112);常染色体三体型72例,占64.29%(72/112);性染色体单体型19例,占16.96%(19/112);性染色体三体型2例,占1.79%(2/112);三倍体10例,占8.93%(10/112);四倍体、嵌合体各1例,分别占0.89%(1/112);其他5例,占4.46%(5/112)。结论染色体异常可能是稽留流产发生的主要因素之一;间期FISH的检测方法对于稽留流产的病因诊断有一定的预测价值,但是是否值得临床普及推广还需要进一步探讨验证。     

双色间期荧光原位杂交技术在异性间造血干细胞移植中的应用

目的 探讨双色间期荧光原位杂交（FISH）法在检测异性异基因造血干细胞移植后植活标志及微量残留病灶（MRD）中的作用。方法 采用X、Y染色体探针和间期双色间期FISH法检测35例异性异基因造血干细胞移植受者移植后不同时期的性染色体荧光杂交信号。并设对照组，分析检测的有效率。结果 35例受者移植后的细胞遗传学及双色间期FISH性染色体分析均可见植入证据。当受者出现原病复发时，双色间期FISH性染色体可检测出受者源克隆的增加；其再次缓解后受者源克隆也相应减少。在移植后不同时间，当染色体分析为100％XX或100％XY结果时，双色间期FISH可显示不同比例的受者源染色体。结论 双色间期FISH应用于异性异基因造血干细胞移植后植活标志及MRD检测，操作简单快速，是细胞形态学及细胞遗传学检查很好的补充。     

精子荧光原位杂交分析男性臂间倒位携带者的减数分裂结果

目的:探讨用精子荧光原位杂交(fluorescence in situ hybridization,FISH)分析男性染色体臂间倒位携带者的减数分裂结果。方法:对4例男性染色体臂间倒位携带者的精子通过化学方法解聚,利用双色FISH,分析精子染色体组成并推断其分离类型。结果:4例染色体臂间倒位携带者中2例为46,XY,inv(9)(p11q12),1例为46,XY,inv(9)(p11q13),1例为46,XY,inv(6)(p22q24),其倒位片段长度分别占整条染色体的16.0%、16.0%、21.0%和76.0%,其重组精子分别为0.2%、0.4%、0.3%和43.9%(del(p)/dup(q)占22.4﹪,del(q)/dup(p)占21.5﹪)。结论:染色体臂间倒位携带者减数分裂的重组发生跟倒位片段占整条染色体长度比例有关。FISH分析可了解重组后染色体不平衡精子的比率,有助于提供更准确的遗传咨询。     

13号染色体重排所致严重少弱畸形精子症患者生精细胞减数分裂分析

目的:探讨13号染色体长臂相互缺失所致严重少弱畸形精子症患者生精阻滞的可能机制。方法:对1例13号染色体异常的严重少弱畸形精子症患者血液样本进行全基因组的寡核苷酸微阵列比较基因组杂交分析,以确定受累染色体的断裂点或基因缺失。对患者生殖细胞进行多色荧光原位杂交分析,以观察初级精母细胞13号染色体配对的情况。结果:寡核苷酸微阵列比较基因组杂交技术显示在13q12.3上有连续4个探针的缺失(A_16_P19757882,A_16_P02744617,A_14_P108858和A_16_P02744687),覆盖59.93 kb,位于基因FLT1和POMP之间,没有注释基因存在。初级精母细胞的减数分裂分析显示同源13号染色体配对错误,13q14和13qter信号彼此分离。结论:染色体重排导致的精子发生阻滞可能是由于生殖细胞第一次减数分裂同源染色体配对错误导致的。     

乙型肝炎病毒DNA序列整合入人类精子染色体的研究

目的 :研究乙肝病毒 (HBV)DNA整合到精子染色体的特点。方法 :选 14例精子染色体样本 ( 5例健康对照 ,9例乙肝病人 ,包括 1例急性乙型肝炎 ,2例慢性活动性乙肝 ,4例慢性迁延性乙肝 ,2例无临床症状的乙型肝炎表面抗原携带者 ) ,用于仓鼠无透明带卵母细胞和人类精子的种间体外受精。用生物素标记的全长HBVDNA探针进行荧光原位杂交至精子染色体 ,以检测人类精子染色体特异性HBVDNA序列。结果 :1例慢性迁延性乙肝病人精子染色体中检测到HBVDNA特异性荧光信号。 4 2对精子染色体补体中 ,9对含特异性荧光信号 ,其中 1对出现 5个明显的FISH点 ,其余有 2～ 4个信号点。荧光信号强度有显著差异 ,信号位点分布似乎是随机的。结论 :HBV可以整合入人类精子染色体中 ,结果提示HBV可能垂直传播到下一代     

染色体平衡易位患者精子染色体荧光原位杂交分析

目的:探讨外周血染色体平衡易位患者在精子发生过程中染色体分离模式,了解各分离模式产生精子所占比例,预测染色体平衡易位携带者在胚胎移植前遗传学诊断(PGD)中得到正常表型胚胎的概率,评估其进行PGD的风险。方法:应用三色荧光原位杂交(FISH)对4例染色体平衡易位患者46,XY,t(9;11)(q22;q21)、46,XY,t(11;22)(q23;q11)、45,XY,t(13q;15q)、45,XY,t(13q;14q)进行精子相关染色体分析,计算出各染色体分离模式产生精子所占比例,同时以染色体正常男性的正常精液作为对照。结果:上述4例异常精子所占比例分别为50.86%、58.33%、13.00%和22.82%,均明显高于对照组(0.85%、1.63%、1.60%和1.37%)。结论:通过FISH检测染色体平衡易位患者精子染色体,有助于预测染色体平衡易位携带者在PGD过程中的风险,从而选择应用PGD。     

荧光原位杂交技术(FISH)在泌尿系统肿瘤领域的应用

一、荧光原位杂交（fluorescence in situ hybridization，FISH）技术概述 1969年，Gall和Pardue等首次将同位素探针用于原位杂交实验，获得成功。1987年，染色体原位抑制杂交法的创建，使FISH技术得以迅速发展。随后，Cremer等用生物素和汞或氨基乙酰荧光素等非放射性物质标记探针，创立了双色FISH技术。1990年，Nederlof等用3种荧光素成功探测出了3种以上的靶位DNA序列，从而宣告了多色FISH技术的问世。     

双色荧光原位杂交检测46,XY/47,XXY少精子症患者精子性染色体

目的 :观察 1例 4 6 ,XY/ 4 7,XXY少精子症患者精子性染色体分离的情况。　方法 :用双色荧光原位杂交技术对手淫取精液的精子进行X染色体和Y染色体数目检测。　结果 :受检的 10 0个精子中 ,X精子占 4 9% ,Y精子占 4 8% ,无杂交信号的精子占 3%。X精子与Y精子比例与预期值相同约为 1∶1。4 6 ,XY/ 4 7,XXY少精子症患者与正常对照男性所携带XX精子和XY精子频率比较无统计学差异。　结论 :可以用患者本人的精子进行卵细胞胞质内单精子注射以获得妊娠。     

Application of dual color fluorescence in situ hybridization （D—FISH） to the diagnosis of a 49,XXXXY chromosomal abnormality

To study the technique of D-FISH and its application in the diagnosis of a 49, XXXXY chromosomal abnormality. Methods: Biotin-labeled alpha satellite X chromosome DNA (pBamX7) probe and digoxi-genin-labeled Y chromosome long arm terminal repetitive sequence (pY3.4) probe in situ hybridized with pre-treated slides of peripheral blood chromosome and interphase nucleus. After washing, the slides were treated with avidin-FITC, rhodamine-FITC and anti-avidin, amplified with an additional layer and counter-stained with DAPI in an antifade solution. The hy bridization signals and chromosomal or interphase nucleus settings were observed respectively with WIB, WIG and WU filters under fluorescent microscope (Olympus AX-70) and the number of metaphase chromosome and interphase nucleus in the peripheral blood was counted. Results: The biotin-labeled pBamX7 probe showed 4 green hybridization signal and the digoxigenin-labeled pY3.4 probe showed 1 red hybridization signal. The chromosome or cytoplasm counter-stained     

Interchromosomal effect in sperm of males with translocations: report of 6 cases and review of the literature

Somatic chromosomal abnormalities are frequently found in infertile men, particularly in those with low sperm count and/or seeking intracytoplasmic sperm injection. These abnormalities mostly consist of numerical sex chromosome abnormalities and translocations (Robertsonian or reciprocal). In this study, we searched for the occurrence of non-disjunction of chromosomes not involved in translocations during meiosis, phenomenon called interchromosomal effect (ICE) and first described by Lejeune (1965). Ejaculate samples of two patients carrying a Robertsonian translocation and four a reciprocal translocation patients and four controls (men with a 46,XY karyotype and normal sperm parameters) were studied in dual FISH 7-9, dual FISH 13-21 and triple FISH X-Y-18. A statistically significant increase of disomy X, Y and XY (P = 0.009, P = 0.004, P < 0.001) was found in the Robertsonian der(13;14)(q10;q10) carrier but not in the der(14;21)(q10;q10) carrier compared with controls. Among reciprocal translocation carriers, a significant increase of disomy 21 (P = 0.033) was observed in a sole patient with a t(9;22)(q21;q11.2). The increase of meiotic non-disjunction for chromosome 21 and sex chromosomes is a recurrent event found in other studies. According to our results and published data, the ICE on some specific chromosomes is likely in men carrier of a translocation, although it cannot be excluded that the aneuploidy is related to the oligoasthenoteratozoospermia usually present in these men. Moreover, this phenomenon showed interindividual variations which cannot be predicted. The risk of aneuploidy in sperm of males used for ICSI need to be evaluated. It could be superadded to that of meiotic segregation of the translocation to give a more precise and personalized risk assessment of aneuploidy in the offspring of those men.     

Meiotic segregation analysis in male translocation carriers by using fluorescent in situ hybridization

Balanced reciprocal and Robertsonian translocations are the most common structural chromosome abnormalities in humans, with incidences of 0.7 and 1.23 per 1000. These translocations can affect fertility and/or pregnancy outcome because of possibly impaired production of gametes with an unbalanced zygote caused by the parental arrangement. Fertility problems in male translocation carriers are because of various degrees of sperm alterations that are directly related to the disturbance of the meiotic process. Investigation of human sperm chromosomes was performed by karyotyping spermatozoa after penetration of zona-free hamster oocytes, karyotype analysis now being possible to analyse the segregation patterns by using fluorescent in situ hybridization (FISH). Here, we document the results of meiotic segregation analysis for four Robertsonian and four reciprocal translocation carriers by FISH. In the sperm of Robertsonian translocation males, the majority of spermatozoa were normal/balanced. On the other hand, males with reciprocal translocations demonstrated a high rate of unbalanced spermatozoa of about 50% on meiotic segregation, with an unusually high rate (23.5%) of 3 : 1 segregation. This knowledge can be used for genetic counselling of families with these types of translocations.     

A study of meiotic segregation of chromosomes in spermatozoa of translocation carriers using fluorescent in situ hybridisation

In the infertile male population, there is a 2–20-time higher probability of having a structural chromosomal abnormality than in general population. Generally, these men have a normal phenotype but they can have sperm abnormalities. As they can produce a variable proportion of unbalanced gametes, it is important to evaluate the percentage of unbalanced chromosomal spermatozoa to assess the risk of injecting a chromosomally unbalanced gamete during ICSI procedure. We report here the meiotic segregation analysis of chromosomes in spermatozoa of 12 men with a balanced reciprocal translocation and 4 men with a Robertsonian translocation using a fluorescent in situ hybridisation analysis. The frequencies of normal or balanced spermatozoa ranged from 34.4% to 49.1% in balanced reciprocal translocation carriers. For Robertsonian translocation, the frequencies of normal or balanced spermatozoa ranged from 78.4% to 91.2%. These analyses allow us to define the orientation of genetic counselling according to the results of meiotic segregation obtained. As a last resort, it could then be discussed of the possibility of having recourse to donor spermatozoa or adoption.     

Molecular cytogenetic detection of meiotic segregation patterns in sperm nuclei of carriers of 46,XY,t(15;17)(q21; q25)

Structural chromosomal abnormalities in gonadal tissue represent an important category of parentally transmittable unbalanced chromosomal abnormalities to the offspring. A child with multiple anomalies was sent for cytogenetic analysis, and his karyotype was 46,XY,der(17)t(15;17)(q21; q25). This abnormality was transferred from his grandfather to his father and to the proband. In this family, 5 persons (1 female and 4 male) are the carriers of this abnormality. In this study, fluorescence in situ hybridization (FISH) on sperm nuclei of 4 male carriers was studied to determine the distribution of segregation patterns of the balanced translocation 15q;17q. The segregation results showed that the segregation products in the third carrier (the grandfather) were different, but they were not statistically significant. The segregation patterns in the other carriers were similar. Overall, 50.3% of the sperm nuclei (mean value for 4 carriers) analyzed were the result of alternate segregation; 36.9%, of adjacent I segregation; 9.0%, of adjacent II segregation; and 2.4%, of 3:1 segregation; the remaining 1.3% could be diploid sperm nuclei or of 4:0 segregation. Multicolor FISH analysis appears to be a rapid and reliable method for the direct analysis of segregation patterns in sperm nuclei of carriers of balanced reciprocal translocation, and it also provides interesting information for determining the possible risks for the offspring.     

Studies on the integration of hepatitis B virus DNA sequence in human sperm chromosomes

Aim: To study the integration of hepatitis B virus (HBV) DNA into sperm chromosomes in hepatitis B patients and the features of its integration. Methods: Sperm chromosomes of 14 subjects (5 healthy controls and 9 HB patients, including 1 acute hepatitis B, 2 chronic active hepatitis B, 4 chronic persistent hepatitis B, 2 HBsAg chronic carriers with no clinical symptoms) were prepared using interspecific in vitro fertilization between zona-free hamster oocytes and human spermatozoa. Fluorescence in situ hybridization (FISH) to sperm chromosome spreads was carried out with biotin-labeled full length HBV DNA probe to detect the specific HBV DNA sequences in the sperm chromosomes. Results: Specific fluorescent signal spots for HBV DNA were seen in sperm chromosomes of one patient with chronic persistent hepatitis B. In 9(9/42) sperm chromosome complements containing fluorescent signal spots, one presented 5 obvious FISH spots and the others 2 to 4 signals. The fluorescence intensity showed significant diffe     

Severe hypospadias associated with Robertsonian translocation

In this study, we report a 3-year-old boy with severe scrotal hypospadias with Robertsonian translocation [45,XY,t(13q;14q)]. The patient was born at term with a low birth weight and hypospadias. There was no endocrinological abnormality. His father also has a balanced 13-14 Robertsonian translocation. Two-stage hypospadias repair was carried out. The presence of this chromosomal anomaly and hypospadias are unique to our patient, compared to others with the 45,XY,t(13q;14q) translocation. Although no such association has been reported so far, we thought that severe hypospadias in this case might be associated with this translocation.     

Cytogenetic alterations in renal tumors. Applications for comparative genomic hybridization and fluorescence in situ hybridization

The WHO classification of renal cell carcinomas (RCC) takes into account chromosomal alterations. New cytogenetic techniques such as comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH) offer alternative methods to the classic cytogenetic banding technique. Clear cell (classic) RCC frequently show the loss of 3p. Papillary RCC are characterized by trisomies and tetrasomies as well as loss of the Y chromosome. CGH analysis demonstrates that DNA copy increase is more common in type I papillary RCC compared to type II. Chromophobe RCC are characterized by losses in chromosomes 1, 2, 6, 10, 13, 17, and 21. Oncocytomas can be divided into cases with rearrangements in the 11q13 region and those with loss of chromosome 1 and the sex chromosomes. Translocations involving chromosome 3, such as t(3;8)(p14;q24.13) and t(2;3)(q35;q21) have been described in familial clear cell RCC. The most recent class of RCC, seen only in men, is referred to as translocation tumors. These tumors demonstrate a tubulopapillary growth pattern and have a t(X;1)(p11.2;q21.2) translocation. Although not required for most clinical diagnoses, CGH and FISH complement the standard histologic diagnosis of RCC and may provide a definitive diagnosis in a small number of challenging cases.