双色FISH检测人精子染色体非整倍体方法的建立

目的　建立用双色FISH检测人精子染色体非整倍体的方法。方法　采用双色荧光原位杂交 (FISH)方法取适量精子标本用EDTA/PBS处理 ,然后用二硫苏糖醇 (DTT) ,使精子去凝集。固定后滴片 ,然后与双色荧光直接标记探针杂交。结果　在OLYMPUS荧光显微镜下可以清楚看到精子头部的蓝色杂交信号 ,头部有 1个绿色荧光杂交信号的精子为X染色体精子 (X精子 ) ,有 1个红色荧光杂交信号的精子为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。结论　双色荧光原位杂交 (FISH)方法可以用于测定人精子染色体非整倍体率的变化。     

外周血性染色体异常患者精子染色体分析及植入前遗传学诊断

目的探讨外周血性染色体异常患者的精子染色体组成，评估其胚胎性染色体异常的风险，为胚胎植入前遗传学诊断（preimplantation genetic diagnosis，PGD）的应用提供客观依据。方法应用三色荧光原位杂交技术fluorescence in situ hybridization，FISH）对3例性染色体异常的患者（例1为46，XY／47，XXY，例2为45，XO／46，X，Yqh-，例3为47，XYY）进行精子X、Y和18号染色体分析，并对例2进行PGD。结果例2的X18：Y18精子的比例为2．05：1，总异常精子比例达29．71％，其中XY18、O18和XO均明显高于其它组。例3总异常精子比例占4．91％，XY18占1．87％。对例2进行PGD，移植1个XX1818胚胎。结论通过FISH检测性染色体异常患者的精子，有助于评估其胚胎性染色体异常的风险，从而选择性应用胚胎植入前遗传学诊断。     

精子荧光原位杂交分析男性染色体相互易位携带者的减数分裂分离

目的探讨用精子荧光原位杂交（fluorescence in situ hybridization，HSH）分析男性染色体相互易位携带者减数分裂的分离。方法对4例男性染色体相互易位携带者的精子通过化学方法解聚，在2条易位染色体的断裂点两侧的4个染色体区域中分别选用位于其中3个区域的3个位点探针，进行多色FISH，分析精子染色体组成并推断其分离类型。结果4例染色体相互易位携带者的核型分别为46，XY，t（2；18）（p16；q23）、46，XY，t（4；6）（q34；q21）、46，XY，t（8；13）（q23；q21）和46，XY，t（4；5）（4q31；5q13），其分离结果是：对位分离精子占27．1％～49．4％，邻位-1分离精子占26．9％～37．6％，邻位-2精子占2．7％～15．7％，3：1分离精子占8．6％～33．7％，减数分裂Ⅱ不分离精子占0．2％～1．9％，4：0或二倍体精子占0．1％～0．4％。结论不同的男性染色体相互易位携带者减数分裂的分离结果可能不同，对其行精子FISH分析有助于提供更准确的遗传咨询和行胚胎植入前遗传学诊断的预后估计。     

FISH检测精子非整倍体率在Klinefelter综合征辅助生育治疗的应用首例分娩报告

目的分析Klinefelter综合征病人精子X,Y,18染色体的非整倍体率(aneuploid),指导其辅助生育治疗.方法应用荧光原位杂交(FISH)检测1例射出的少量精子X,Y和18号染色体,计算精子染色体非整倍体率,用卵胞浆内单精子注射(ICSI)治疗.结果ICSI前后拾取形态正常的精子,进行FISH后有荧光信号的精子数为156条,其中正常18/X精子有82条(52.6%),18/Y精子有74条(47.4%),未发现异常的精子信号.ICSI治疗后获得双胎妊娠,孕19w产前诊断,2个胎儿的染色体核型分别是46,XY和46,XX.孕36w分娩1男婴和1女婴.结论ISH分析证实Klinefelter综合征病人可以产生正常精子,有精子的克氏征病人可以通过ICSI获得正常婴儿.     

应用荧光原位杂交技术对精子染色体非整倍体的研究

目的 探讨人类精子染色体数目异常与自然流产的相关性。方法 采用多色荧光原位杂交（fluorescence in situ hybridization,FISH)方法，利用CEP（chromesome enumeration DNA probes)X、Y、18及LSI（locus specific identifier DNA probes)13、21两种探针混合液与二硫苏糖醇处理过的自然流产者丈夫的精子核杂交，记数杂交信号，与对照组进行比较。结果 经卡方检验，自然流产组丈夫精子染色体X、Y、18、13、21双体型及其它数目异常明显高于正常对照组（P＜0．005），说明流产者丈夫精子染色体异常是流产的重要原因之一。结论 FISH技术是一种快速、准确、简单、实用性强的检测精子染色体的好方法。     

Inv(Y)患者精子染色体荧光原位杂交分析

目的 探讨Y染色体臂间倒位患者精子减数分裂形成中性染色体的分离规律.方法 采用G带、C带及荧光原位杂交(fluorescence in situ hybridization,FISH)对中期分裂相进行分析.应用三色探针CEPX、Tel Xp/Yp、Tel Xq/Yq对5例inv(Y)(p11.1q11.2)患者精子进行FISH,同时以染色体正常男性的正常精液作为对照.结果 5例inv(Y)(p11.1q11.2)精于性染色体数目及重组Y染色体异常率与对照组比差异无统计学意义.结论 inv(Y)(p11.1q11.2)患者精子无明显性染色体数目与结构异常,精子FISH分析可为其提供更准确的遗传咨询及指导植入前遗传学诊断.     

应用荧光原位杂交技术对精子染色体非整倍性的研究

目的探讨人类精子染色体数目异常与自然流产的相关性. 方法采用多色荧光原位杂交(fluorescence in situ hybridization, FISH)方法,利用CEP(chromesome enumeration DNA probes)X、Y、18及LSI(locus specific identifier DNA probes)13、21两种探针混合液与二硫苏糖醇处理过的自然流产者丈夫的精子核杂交,记数杂交信号,与对照组进行比较.结果经卡方检验,自然流产组丈夫精子染色体X、Y、18、13、21双体型及其它数目异常明显高于正常对照组(P<0.005),说明流产者丈夫精子染色体异常是流产的重要原因之一.结论 FISH技术是一种快速、准确、简单、实用性强的检测精子染色体的好方法.     

精子细胞的荧光原位杂交

在新生儿中大约1／500出现染色体非整倍体（aneuploid）[1]，特别是在精子配子中其发生频率较高，是引起自然流产、婴儿死亡及神经系统发育迟缓的重要原因[2]。因而人类配子细胞已经成为筛查染色体非整倍体及估计染色体不分离发生的焦点之一。1970年Barlow等曾用阿的平对人类精子核Y染色体长臂染色，通过计数荧光信号判断Y染色体数目[3]，但由于非特异背景，两个Y的频率较高[4]。1978年Rudak首次描述了以“仓鼠穿卵法”制备精于染色体，并用来检测染色体数目及结构异常[5]。但方法复杂，技术难度大，并且只能对那些能穿透仓鼠卵细胞的精子进行分析，因而至今不能做一种常规的检测方法[6]。本文介绍一种简便而快速的检测精子染色体数目异常的方法，以D21Z1／D13Z1、TRX探针与精子问期核进行荧光原位杂交（fluorescenceinsituhybridization，FISH），能准确的计数精子核中染色体数目。     

多重定量荧光PCR技术在Y染色体AZF微缺失检测中的应用研究

目的建立检测Y染色体无精子症因子（AZF）微缺失的多重定量荧光PCR体系。方法以5’FAM、JOE和TAMRA荧光基团标记PCR引物,建立包含Y染色体AZF4个亚区（AZFa～d）15个序列标签位点（STS）的多重定量荧光PCR体系;并对无精子症组、严重少精子症组及精液正常组进行Y染色体AZF微缺失检测。结果成功建立了检测Y染色体AZF微缺失的多重定量荧光PCR体系;200例男性中检测到Y染色体AZF微缺失16例,其中72例无精子症组7例,缺失率为9．7％,78例严重少精子症组9例,缺失率为15．4％,50例精液正常组未检测到缺失。无精子症组、严重少精子症组缺失率与精液正常组比较差异均有统计学意义（P〈0．05）。结论多重定量荧光PCR技术是一种快速、简便检测Y染色体AZF微缺失的方法,具有重要临床应用价值。     

精子大头多尾畸形与性染色体异常

目的 探讨精子畸形与精子染色体异常的关系,了解畸形精子的病理学改变,方法 应用光学和电子显微镜,性染色体特异性探针荧光原位杂交（FISH）技术研究罕见的大头多尾畸形精子。结果 巴氏染色后观察的精子畸形率达98．75％（油镜下测量头部畸形达100％）,精子多尾率达60．25％（最多达8尾）,电镜观察证实,精子头部表面凹凸不平,核型极不规则,有大量细胞质结构,尾部除数量异常外,尚有中心粒,线粒体和鞭毛结构的异常,FISH结果证实,性染色体多体率为61．4％,与精子多尾的组成比有大致的平行关系。结论 尽管体细胞染色体正常,畸形精子人可伴有严重染色体异常。     

Albumin gradients do not enrich Y-bearing human spermatozoa

The aim of this study was to evaluate objectively whether or not discontinuous albumin gradients enrich the proportion of Y-bearing human sperm. A blinded, collaborative trial design was employed whereby a licensed centre prepared the sperm fractions using licensed procedures, coded the sperm slides and then sent them to an independent laboratory for determination of the X:Y ratio in each sperm fraction using X and Y chromosome-specific probes and double label fluorescence in-situ hybridization (FISH). The identification codes and FISH results were collated by an independent third observer. Two albumin gradient methods which are currently used by licensed centres for male sex pre- selection, protocol 3 and modified protocol 3, were tested. Essentially the same results were obtained for the two methods. Highly motile sperm fractions were recovered from the albumin gradients, and the recoveries of motile spermatozoa (1.3-8.5%) were within the optimal range reported to produce maximal enrichment of Y-bearing spermatozoa. FISH analysis, however, revealed no enrichment for Y-bearing spermatozoa with either method, and the overall X:Y ratios were not significantly different from 1.0. Some samples showed marginal enrichment of Y-bearing spermaotozoa, whereas others showed marginal enrichment of X-bearing spermaotozoa. In conclusion, this collaborative study has demonstrated that the protocol 3 and modified protocol 3 albumin gradient procedures do not enrich Y-bearing spermatozoa. The clinical use of albumin gradients for male sex preselection should be reconsidered in the light of this and other evidence.      

Application of modern molecular techniques to evaluate sperm sex selection methods

The aim of this paper is to review modern approaches which havebeen used to evaluate sex preselection procedures. Two approachescan be used, polymerase chain reaction (PCR) and fluorescencein-situ hybridization (FISH). FISH is currently the method ofchoice for evaluating sex selection procedures because: (i)FISH accurately identifies the sex chromosome of individualspermatozoa using specific probes for the X and Y chromosomesand a two-colour detection system; and (ii) large numbers ofspermatozoa can be screened in a short period of time. Of thepublished sex pre-selection methods tested using FISH, onlyflow cytometry has been shown to produce a clinically significantenrichment of X- and/or Y-bearing human spermatozoa. Studieshave shown that 12-step Percoll gradients produce a slight butclinically insignificant enrichment of X-bearing spermatozoa,swim-up techniques do not appgar to enrich either X- or Y-bearingspermatozoa, and discontinuous albumin gradients do not enrichY-bearing spermatozoa. Despite this evidence, some of thesemethods continue to be used clinically, so it is vital thatsex selection methods are properly evaluated using reliablemethods such as double-label FISH before they are introducedfor clinical use. FISH/PCR/sex selection/X chromosome/Y chromosome     

DNA and chromatin structure: Analysis of the sex chromosomal equipment in spermatozoa of a 47, XYY male using two-colour fluorescence in-situ hybridization

The sex chromosomes in spermatozoa of a 47, XYY fertile malewere analysed simultaneously by dual fluorescence in-situ hybridization(FISH), with two probes (pHY2.1 and pXBR). Of the 100 000 cellsanalysed, 95 179 spermatozoa (95.18%) exhibited one or morehybridization signals. Of the hybridized nuclei, 85.37% showeda normal sex chromosome constitution (37.37% X-bearing cellsand 48.00% Y-bearing cells), with an X:Y ratio of 0.78:1. Atotal of 14.63% of the hybridized nuclei exhibited sex chromosomeaneuploidy with a majority of XY-and YY-bearing spermatozoa(9.37 and 4.65% respectively). Even if the majority of spermatozoahave chromosomal haploidy, a large proportion of them exhibitsnumerical errors for the sex chromosomes. These observationsraise questions about the commonly-admitted notions concerningthe absence of chromosomal risk for XYY male offspring. fluorescence interphase in-situ hybridization/sex chromosome/spermatozoa/XYY male     

A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47, XYY male

Two semen samples from a 47, XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95–98%. Ploidy levels were estimated by measurement against a microscope eyepiece graticule. The overall ratio of X-to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.     

Experiences in Hong Kong with the theory and practice of the albumin column method of sperm separation for sex selection [published erratum appears in Hum Reprod 1998 May;13(5):1414]

Controversy still surrounds the human serum albumin (HSA) method for separation of X- and Y-bearing human spermatozoa. There is doubt about whether the procedure does enrich sperm samples for the chosen sex chromosome. We have applied the HSA separation method in a clinic in Hong Kong, using the method as described by Ericsson et al. [Nature, 246, 421-424 (1973)] taking care to keep the sperm recovery to <5% of the initial number. Aliquots of separated spermatozoa were examined for X- and Y-bearing spermatozoa by fluorescent in-situ hybridization (FISH) using appropriate DNA probes. Of 18 couples wanting boys, 13 had single boys, one had twin boys, and one had twins comprising one boy and one girl. Only three single girls were born. This success rate of 83% is significantly different (P < 0.001) from the usual expected ratio. There were four miscarriages, one in the third and one in the fourth week of pregnancy. The times of the others are not definitely known, but are thought to have occurred early in pregnancy. We lack information on three couples. The FISH procedure showed no change in the normal and equal numbers of X- and Y-bearing spermatozoa after the HSA separation procedure. This study confirmed that the HSA sperm separation method can bias the number of babies in favour of males. However, the theory that it does so by enriching the sperm samples with Y-bearing spermatozoa appears to be incorrect and some other theory has to be postulated. It is tentatively proposed that passage through the HSA inactivates X-bearing spermatozoa more than Y-bearing spermatozoa, even though this is not apparent simply on inspection of sperm motility.      

Segregation of sex chromosomes in spermatozoa of 46,XY/47,XXY men by multicolour fluorescence in-situ hybridization

The sex chromosome disomy and diploidy rates on ejaculated spermatozoa from two patients with mosaic Klinefelter's syndrome were estimated, using X/Y/15 multicolour fluorescence in-situ hybridization (FISH). A 8/18 dual fluorescence in-situ hybridization analysis was also carried out. In triple FISH, a total of 1691 (patient 1) and 811 (patient 2) spermatozoa were analysed. Frequencies of cells with hyperhaploidies for sex chromosomes were 2. 01% and 3.45% for patients 1 and 2 respectively, with both patients showing a significantly increased incidence of 24,XY and 24,XX disomies and only patient 2 showing a significantly increased incidence of 24,YY disomy in comparison to the control (P < 0.001). The 46,XX diploidy rate in patient 1 was also significantly higher than the control (P < 0.01). The ratio of X-bearing to Y-bearing spermatozoa differed from the expected 1:1 ratio for only patient 1 (1.18:1). There was no significant difference for chromosomes 8, 15 or 18 disomy frequencies in comparison to those estimated in the control population. These results support the hypothesis that some 47,XXY cells are able to go through meiosis and form spermatozoa with an abnormal gonosomal complement. Thus, there is an increased risk, for these 46,XY/47,XXY men, of producing offspring with a gonosomal abnormality.     

用双色荧光原位杂交检测人精子染色体非整倍体率

目的检测人精子染色体非整倍体率。方法采用双色荧光原位杂交（ＦＩＳＨ）方法,取少量精标本经洗后制片,用二硫苏糖醇（ＤＴＴ）和二碘水杨酸锂（ＬＩＳ）处理,使精子头部染色质去凝集。然后,与生物素标记的α卫星Ｘ染色体特异ＤＮＡ探针（ＤＸＺ１）和地高辛标记的α卫星Ｙ染色体特异ＤＮＡ探针（ＤＹＺ３）进行原位杂交。用ＣＹ３－链亲和素、山羊抗链亲和素检测Ｘ染色体探针杂交信号;用鼠抗地高辛抗体、与荧光素结合的兔抗鼠抗体检测Ｙ染色体探针杂交信号。结果在Ｎｉｋｏｎ荧光显微镜下可以清楚看到精子头部的杂交信号,头部有１个红色荧光杂交信号的精子为Ｘ染色体精子（Ｘ精子）,有１个绿色荧光杂交信号的精子为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。若用１条常染色体探针和１条性染色体探针进行ＦＩＳＨ,可以区别头部有２个相同颜色荧光杂交信号的精子属非整倍体精子或二倍体精子。结论双色荧光原位杂交（ＦＩＳＨ）方法,可以用于测定接触致突变剂和非整倍体诱导剂后,人精子染色体非整倍体率的变化。     

Size differences between human X and Y spermatozoa and prefertilization diagnosis

Normal human spermatozoa carry either the X or the Y chromosome. The differences between X and Y spermatozoa (X and Y haploid cells) may exist in two areas: the different chromosomes (i.e. different kinds and numbers of genes) and the different sperm structures and functions (i.e. different genetic expression). The aim of this study was to determine whether there are any size between X and Y spermatozoa and whether sperm size and shape varies between men. Identification of the Y (and X inferred) status of individual spermatozoa was carried out by polymerase chain reaction (PCR), amplifying the putative testis- determining gene (SRY) together with a control gene (ZP3). PCR amplification of 871 out of 895 (97.3%) single motile spermatozoa showed that 444 (51.0%) were Y and 427 (49.0%) were X-bearing spermatozoa. Of 233 normally-shaped but immobilized spermatozoa, 217 (93.1%) were photographed and measured. Statistically, the length, perimeter and area of the sperm heads, and the length of the sperm necks and tails of X-bearing spermatozoa were significantly larger and longer than those of Y-bearing spermatozoa. Some peculiarities (or variations) in the X and Y sperm shape and size in individual donors were found. The pre-screening by micro-measurement of these specific haploid characteristics of individual spermatozoa in different donors, which may be closely related to their different genetic conditions (or diseases), may be important in human medicine and animal husbandry, especially in sperm prefertilization diagnosis.      

Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods

Using three-colour fluorescence in-situ hybridization, sex chromosome ratios and frequencies of diploidy and disomy for chromosomes X, Y and 18 were compared in spermatozoa of good and poor motility after separation by swim-up, glass-wool and two-layer discontinuous Percoll methods. Semen samples were collected from seven normal males aged 26- 31 years. A minimum of 6000 sperm nuclei per sample were evaluated for each chromosome for a total of 308,432 sperm nuclei. Hybridization efficiency was 99.8%. A slight change in the ratio of X- to Y-bearing spermatozoa was noted after Percoll separation (from 49.3:49.5 to 50.0:48.9; P = 0.036 and P = 0.046), but not after separation by the other two methods. We did not observe significant differences in the disomy rates for sex chromosomes or chromosome 18 or in the diploidy rate between spermatozoa with good and poor motility after separation by any of the three methods. Our data indicate that separation of motile spermatozoa does not alter the ratio of X- to Y-bearing spermatozoa to a degree that represents sex chromosome selection.