Analysis of Spermatozoa from Seven ICSI Males with Constitutional Sex Chromosomal Abnormalities by Fluorescent In Situ Hybridization

Purpose: The objective was to estimate the risk for subfertilemales with a constitutional sex chromosomal abnormalityof transmitting such a chromosome abnormality to theirchildren, conceived by intracytoplasmic sperm injection(ICSI). Methods: Semen samples were obtained from seven severelyoligospermic ICSI candidates. Six of them had a numericalsex chromosomal abnormality, including mosaic 45,X/46,XY,mosaic 46,XY/47, XXY, 47,XXY (Klinefelter's syndrome), and47,XYY. One male had a structural abnormality, namely, aninversion of the Y chromosome. The semen was studied bythree-color fluorescent in situ hybridization (FISH) withprobes specific for chromosomes 18,X, and Y. Results: Chromosomal aneuploidy rates of any of the threechromosomes were significantly higher than the aneuploidyrates observed in three control samples but comparable tothe rates observed in 10 ICSI candidates witholigoasthenoteratozoospermia (OAT) and a normal constitutionalkaryotype. Conclusions: Our data indicate that males with (mosaic) sexchromosomal abnormalities have no higher risk of producingoffspring with a sex chromosomal abnormality by ICSI thanOAT males with a normal karyotype.     

畸形精子症患者精子染色体非整倍体的研究

目的:研究畸形精子症患者精子染色体的非整倍体率。方法:应用18号、X和Y染色体着丝粒探针,采用荧光原位杂交(FISH)技术比较畸形精子症患者(畸精组,n=18)和生育力正常且精子正常形态率、浓度、活力等均正常男性(对照组,n=5)精子中18号、X和Y染色体的非整倍体率。结果:畸精组共计数精子58 178条,对照组共计数精子16 369条。畸精组和对照组杂交效率分别为97.5%和98.3%;染色体非整倍体类型主要有二体(XX18、YY18、XY18、Y1818和X1818)和二倍体(1818XX、1818YY、1818XY)。畸精组和对照组的18号染色体二体率分别为0.29±0.16%和0.03±0.02%,性染色体二体率分别为0.65±0.24%和0.05±0.02%,二倍体率分别为0.14±0.12%和0.04±0.03%。18号、X和Y染色体非整倍体率组间均有统计学差异(P<0.05)。结论:与生育力和精液各参数均正常男性相比,畸形精子症患者18号、X和Y染色体非整倍体率明显升高。     

Fluorescence In Situ Hybridization with Chromosome Paint Probes: A Novel Approach To Assess Aneuploidy in Human Sperm Nuclei

Purpose: Fluorescence in situ hybridization (FISH) using whole-chromosome paint probes was performed to evaluate disomy and diploidy frequency for chromosomes 1, 18, 19, and 22 in human sperm nuclei. Methods: Ten subjects of proven fertility and normal spermatic parameters were included in the study. A dual-color FISH method was carried out. Results: A total of 157,896 spermatozoa was scored. The mean frequencies of disomic sperm for chromosomes 1, 18, 19, and 22 were 0.22% (range, 0.19 to 0.28%), 0.24% (range, 0.14 to 0.37%), 0.22% (range, 0.17 to 0.30%), and 0.25% (range, 0.21 to 0.29%), respectively. The mean frequency of diploidy was 0.14% (range, 0.09 to 0.18%). No interindividual and interchromosomal variations in the aneuploidy frequency were observed between the different subjects. Conclusions: FISH with whole-chromosome paint probes provides a novel and efficient approach for disomy assessment in human sperm nuclei.     

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.     

短时常规体外受精中废弃胚胎染色体非整倍体分析

目的:探讨短时常规体外受精周期中废弃胚胎中染色体非整倍体的情况。方法:收集常规体外受精周期及短时常规体外受精周期1原核(1PN)、2PN和≥3PN的废弃胚胎,运用2轮荧光原位杂交技术对胚胎染色体进行分析。结果:短时常规体外受精组与常规体外受精组废弃胚胎染色体异常率差异无统计学意义。结论:短时常规体外受精技术本身不会增加胚胎染色体异常的几率,是一种安全有效的受精方式。     

The Effects of Age and Abnormal Sperm Count on the Nondisjunction of Spermatozoa

Purpose: The effect of paternal age on the nondisjunctionof sex chromosomes is controversial. Also, the prevalenceof chromosomal anomalies in infertile patients iscontroversial, it has been reported that the sex chromosomalaneuploidy rate following treatment with intracytoplasmic sperminjection (ICSI) is higher than in naturally conceivedpregnancies. We investigated the influence of paternal age andoligozoospermia on the nondisjunction of spermatozoa. Methods: We determined the rate of aneuploidy forgonosomes and autosomes, using two-color fluorescence in situhybridization (FISH) of the X and Y chromosomes andchromosomes 12 and 18 in 10 donors under 25 years of agewho had a normal sperm count (20 × 10⁶/ml), 10 donorsover the age of 39 years with idiopathic infertility andnormozoospermia (20 × 10⁶/ml), and 5 oligozoospermicdonors (<20 × 10⁶/ml). Results: There was no obvious relationship betweenincreasing age and autosomal disomy (disomy 12 and disomy 18).Neither autosomal disomy nor diploidy was increased inany group. The frequency of X-, Y-, XX-, and YY-bearingsperm did not differ significantly among groups, but thefrequency of XY-bearing sperm was significantly higher inthe older infertile group than in the control donors. Conclusions: The incidence of nondisjunction of paternalsex chromosome in meiosis I was higher in older men withidiopathic infertility. The present results suggest that therisk of producing XXY fetuses is higher among men >39years of age with idiopathic infertility.     

胚胎植入前遗传学诊断10个周期的临床分析

目的:初步探讨使用荧光原位杂交(FISH)方法对染色体异常患者进行胚胎植入前遗传学诊断(PGD)的临床意义。方法:7对不孕夫妇采用长方案控制性超排和卵胞浆内单精子注射,受精后d3胚胎活检、卵裂球固定和FISH,d4或d5择合适胚胎移植。结果:7对夫妇共进行10个PGD周期。获卵251个,可供活检胚胎133个,活检卵裂球207个,胚胎活检成功率为96.2%(128/133)。128个成功活检胚胎的197个卵裂球,其单细胞固定率为93.9%(185/197),FISH信号率为90.8%(168/185)。10个周期共移植22个胚胎,3例获得妊娠,并均足月分娩健康婴儿,其中1例孕妇平衡易位携带者于孕中期时,羊水核型分析为平衡易位携带者。结论:应用FISH方法进行PGD,是遗传病高危夫妇预防流产和染色体异常患儿出生的有效手段。     

Chromosome 21 Detection in Human Oocyte Fluorescence In Situ Hybridization: Possible Effect of Maternal Age

Purpose: The purpose of this study was to evaluate, among 100 uncleaved oocytes, the incidence of numerical and structural chromosome 21 and X abnormalities and to analyze the influence of various factors, such as in vitro (IVF) indications, follicle stimulation protocols, and women's age. Methods: We investigated 150 uncleaved oocytes from 128 patients after an IVF attempt. After cytogenetic analysis (Giemsa) 100 oocytes (66%) were selected for fluorescence in situ hybridization (FISH). Fluorescent probes for human chromosomes X and 21 were used simultaneously according to standard procedures for their hybridization and detection. Results and Conclusions: We analyzed by the FISH protocol 100 metaphase II oocytes with 22 to 25 chromosomes. Our results demonstrate a high rate of disomy for chromosome 21 in human oocytes. Among them, eight were disomic (8%) and three were nullosomic (3%) for chromosome 21. Only one disomy of chromosome X was noted. The various indications of IVF and the different folliculogenesis stimulating protocols did not seem to influence the results but suggested a correlation between the maternal age and the aneuploidy rate of chromosome 21.     

滋养层细胞活检法分析人废弃胚胎源性囊胚发育与染色体异常

目的:探讨年龄、卵裂期胚胎碎片、囊胚生长速率与形态对染色体异常的影响。方法:收集226例患者授精培养后第3日不适于移植和冷冻的正常受精来源的废弃胚胎988枚,激光辅助打孔后,依年龄和胚胎碎片比率分组序贯培养至囊胚期。待第5～7日囊胚形成且滋养外胚层细胞脱出2～9个时活检,同时评估囊胚生长速率与形态;机械法分离活检后存活囊胚的内细胞团细胞,应用13q14.2,21q22.13特异位点探针行FISH检测,分析染色体异常发生情况。结果:FISH检测99枚囊胚,其中58枚(58.6%)存在染色体异常;当患者年龄≥35岁时,其囊胚染色体异常风险增高;尤其胚胎碎片比率>25%者,形成的囊胚形态较差且染色体异常程度复杂(P<0.05)。随囊胚生长速率的延缓与形态分级的降低,染色体异常发生率增高。滋养层细胞活检后囊胚均存活,在分离内细胞团的36枚囊胚中,滋养层与内细胞团细胞的FISH荧光信号吻合率为91.7%。结论:体外受精治疗周期部分废弃胚胎可发育至囊胚,但由于受年龄、胚胎碎片等因素影响,囊胚染色体异常率较高,可能导致胚胎发育延迟且囊胚形态较差;FISH检测囊胚滋养外胚层细胞法可评估胚胎的染色体组成。     

Simultaneous Detection of Chromosomes X, Y, 13, 18, and 21 by Fluorescence In Situ Hybridization in Blastomeres Obtained from Preimplantation Embryos

Purpose. Simultaneous fluorescence in situ hybridization (FISH) was used in a preimplantation genetic diagnosis program to determine which embryos were normal for chromosomes X, Y, 13, 18, and 21. Methods: Single blastomeres were disrupted and attached to glass slides using acetic acid and ethanol. Using a ratio mixture of chromosome enumeration DNA probes in combination with locus-specific identifier DNA probes, FISH was performed for the identification of chromosomes X, Y, 13, 18, and 21. Results: Fourteen couples enrolled in IVF produced 134 embryos for biopsy. Blastomeres subjected to five-color FISH revealed that 22% of embryos were normal for chromosomes X, Y, 13, 18, and 21. In addition, 52% were abnormal and no results could be detected for 25%. Twelve couples underwent embryo transfer, two couples did not receive embryos due to lack of any normal embryos, and three couples became pregnant. Conclusions: The simultaneous detection of five-color FISH is a feasible method to detect aneuploidy in preimplantation embryos from women of advanced maternal age.     

用荧光原位杂交技术检测唐氏综合征

目的: 探讨用荧光原位杂交技术在检测唐氏综合征中的应用价值。方法: 以Biotin标记的DSCR21q22.3探针与经处理的20例唐氏综合征患者标本外周血中期染色体及其间期细胞核进行原位杂交,统计杂交信号数量。结果: 14例唐氏综合征患者出现3个杂交信号的细胞,染色体和间期细胞核杂交平均出现率分别为98.79％和98.46％,与染色体检测的结果一致;其余染色体核型检测为嵌合体的6例患者,染色体和间期细胞核中3个杂交信号细胞平均出现率分别为75.33％和7 3.50％, 2个杂交信号细胞平均出现率分别为22.67％和21.33％。结论: 荧光原位杂交技术检测唐氏综合征具有快速、敏感度高、信号强、背景低、直观安全等优点,故FISH技术在临床遗传病检测领域中具有重要的应用价值和发展前景。     

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。     

应用双色荧光原位杂交技术检测一例49,XXXXY性染色体异常

目的:探讨用双色荧光原位杂交技术(dual-color fluorescence in situ hybridization,D-FISH)检测性染色体数目异常的实验方法及其应用价值。方法:以Biotin标记的X染色体α-卫星DNA(pBamX7)探针和以Digoxigenin标记的Y染色体长臂末端重复顺序(pY3.4)探针与经处理的标本同时进行外周血染色体及间期细胞核的原位杂交,分别用Avidin-FITC和Rhodamine-FITC及其Anti-avidin进行信号的检测与放大,DAPI复染。于Olympus AX-70型荧光显微镜下,分别通过WIB、WIG及其WU滤光镜观察杂交信号及其染色体或间期核背景,并统计外周血中期染色体和间期细胞核的杂交信号颗粒数。结果:在显微镜下可见以Biotin标记的pBamX7探针显示4个绿色杂交信号,以Digoxigenin标记的pY3.4探针显示1个红色杂交信号,染色体或细胞质背景经DAPI复染显示兰色;统计350个中期染色体和间期细胞核,X染色体杂交信号阳性率分别为91.43％和92.57％;Y染色体杂交信号阳性率分别为99.5％和99.8％。结论:双色荧光原位杂交技术是检测49,XXXXY性染色体异常以及其他性染色体数目异常患者的一种十分有价值的技术,具有高效、灵敏、可靠等特点,可为临床提供良好的辅助诊断。     

荧光原位杂交与染色体核型分析应用于自然流产病因学诊断的比较研究

目的:分析并比较荧光原位杂交技术(fluorescence in situ hybridization,FISH)及普通染色体核型分析技术在自然流产中的诊断意义。方法:以早孕自然流产的患者为研究对象,共201例。将同一孕周的患者随机分为A组和B组,A组(n=100)进行绒毛培养加染色体核型分析,B组(n=101)进行FISH分析,另在A、B组孕6~11周患者中每一孕周各随机选取1例,每组6例,共12例同时进行2种技术分析,并比较结果。结果:染色体核型分析成功率为66%,其中核型异常率为30.3%;FISH成功率为100%,其中核型异常率为46.5%;2种检测技术检测出的异常核型率比较有统计学差异(P=0.036)。结论:2种分析技术对异常核型的检出率有明显的差异,FISH更容易成功,更能反应胚胎的染色体数目;染色体核型分析结合FISH技术能有效诊断自然流产的染色体异常。     

不明原因的不育男性精子核碱性蛋白组型的电泳分析

本研究从15例正常生育力男性和37例不育男性的射出精子中提取核碱性蛋白,在酸性尿素系统聚丙酰胺凝胶中电泳,经微显像测密仪扫描获得各蛋白区带(组蛋白和HP1～3)的相对含量,并计算各蛋白条带的相对比值.实验结果表明:与正常生育力男性相比,不育组男性精子的TH(total histones)/HP1～3比值增高(P<0.01);而HP2+3/HP1比值降低(P<0.01).基于TH/HP1～3和HP2+3/HP1比值的分折,这些不育男性精子的核碱性蛋白组型可分为四种:1.TH/HP1～3比值较高,同时伴有较低的HP2十3/HP1比值;2.TH/HP1～3比值较高,而HP2+3/HP1比值正常;3,TH/HP1～3比值正常,而HP2十3/HP1比值较低;4.正常的TH/HP1～3和HP2十3/HP1比值.作者认为前三种异常的核蛋白组型可能与不育有关.     

Preimplantation Diagnosis by Fluorescence In Situ Hybridization Using 13-, 16-, 18-, 21-, 22-, X-, and Y-Chromosome Probes

Purpose: Our purpose was to select the proper chromosomes for preimplantation diagnosis based on aneuploidy distribution in abortuses and to carry out a feasibility study of preimplantation diagnosis for embryos using multiple-probe fluorescence in situ hybridization (FISH) on the selected chromosomes of biopsied blastomeres. Methods: After determining the frequency distribution of aneuploidy found in abortuses, seven chromosomes were selected for FISH probes. Blastomeres were obtained from 33 abnormal or excess embryos. The chromosome complements of both the biopsied blastomeres and the remaining sibling blastomeres in each embryo were determined by FISH and compared to evaluate their preimplantation diagnostic potential. Results: Chromosomes (16, 22, X, Y) and (13, 18, 21) were selected on the basis of the high aneuploid prevalence in abortuses for the former group and the presence of trisomy in the newborn for the latter. Thirty-six (72%) of 50 blastomeres gave signals to permit a diagnosis. Diagnoses made from biopsied blasotmeres were consistent with the diagnoses made from the remaining sibling blastomeres in 18 embryos. In only 2 of 20 cases did the biopsied blastomere diagnosis and the embryo diagnosis not match. Conclusions: If FISH of biopsied blastomere was successful, a preimplantation diagnosis could be made with 10% error. When a combination of chromosome-13, -16, -18, -21, -22, -X, and -Y probes was used, up to 65% of the embryos destined to be aborted could be detected.     

溶脲脲原体感染不育者精浆α－葡萄糖苷酶活性及果糖含量的分析

本文比较了溶脲脲原体感染的不育男性与正常生育男性精浆中α－１，４－葡萄糖苷酶活性及果糖含量。结果表明，溶脲脲原体感染的不育肾性精浆中α－１，４－葡萄糖苷酶活性明显低于正常生育男性（分别为：Ｘ±ＳＤ＝２７．９９±２０．３１ｍＩＵ／ｍｌ和Ｘ±ＳＤ＝４１．８６±２１．８６ｍＩＵ／ＭＬ，ｐ＜０．０５）；溶脲脲原体感染的不育男性精浆中果糖含量（Ｘ±ＳＤ＝２３３．９６±８９．２１ｍｇ／ｄｌ）稍高于正常生育男性（Ｘ±ＳＤ＝１７９．８１±１０６．４５ｍｇ／ｄｌ），但两者之间差异无显著性（Ｐ＞０．０５）。上述结果提示，生殖道溶脲脲原体感染对附睾功能具有干扰作用，而对精囊腺的功能影响尚缺乏依据，宜进一步探索。