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。     

南宁地区864例男性不育患者的细胞遗传学分析

目的探讨男性不育与染色体异常的关系。方法通过对2009年1月-2012年12月来我院门诊检查的864例男性不育患者进行染色体核型分析。结果864例不育男性患者中,染色体异常发生率为12．0％（104／864）,其中性染色体异常占4．5％（39／864）,常染色体异常占7．5％（65／864）。结论染色体异常是造成男性不育的一个重要因素。因此．对少精子、弱畸精子、无精子等的不育患者,进行染色体核型分析是十分必要的。     

生精障碍患者的染色体核型分析

目的探讨染色体异常与男性生精障碍的关系。方法对429例生精障碍者进行外周血淋巴细胞培养,G显带染色体核型分析。结果检出染色体异常核型44例,检出率为10．3％。其中,无精子症、重度少精子症和少弱精子症的染色体异常检出率分别为15．8％、6．1％和4．7％,以无精子症最高。染色体异常包括性染色体数目、结构异常和常染色体异常,以性染色体数目异常为主。结论染色体异常是导致生精障碍的重要原因之一,生精障碍程度越高,染色体异常检出率越高。对生精功能障碍的男性患者有必要进行染色体核型分析。     

163例少、弱精子症和畸形精子症及无精子症患者的染色体分析

目的探讨染色体核型异常与男性精子质量异常的关系。方法对于临床诊断为少弱精子症,畸形精子症以及无精症患者进行外周血染色体核型分析。结果在163例患者中染色体核型中染色体异常11例,异常率6.7%,其中常染色体异常5例,性染色体异常6例;染色体多态21例,检出率12.8%。结论染色体异常是引起男性精子异常的重要原因,对于临床诊断为少弱精子症、畸形精子症及无精子症的患者进行染色体检查有非常重要的意义。     

男性不育患者的细胞遗传学分析

目的对男性不育患者进行染色体核型的检测,以指导其生育。方法1．应用规范的WHO精液检查方法检查男性不育患者精液标本。2．应用细胞遗传学检测男性不育患者外周血染色体核型分析。结果1．精液检查结果：男性不育组患者356例,包括无精子症82例、严重少精子症126例、少弱精子症148例,精液正常、核型正常男性组对照50例。2．细胞染色体检查：356例男性不育患者全部进行外周血淋巴细胞染色体核型分析,发现55例染色体核型异常,包括性染色体异常24例,常染色体异常21例和染色体多态性10例。其中,无精子症组的染色体异常率25．61％（21／81）,严重少精子症患者染色体核型异常率为11．90％（15／126）,少弱精子症患者染色体核型异常率为12．84％（19／148）。结论男性不育患者核型异常率为15．45％,远高于正常人群发病率。细胞遗传学染色体检查对男性不育的生育指导有重要意义。无精子症患者进行染色体检查尤其必要。     

联合多项技术筛查诊断出生缺陷和遗传病的研究

目的建立一套规范的产前筛查和诊断技术应用临床提高诊断率。方法对就诊遗传咨询的30866例。超声筛查诊断16933例；产前筛查337例；细胞遗传学检查10442例，其中外周血8987例，产前诊断1455例（包括绒毛、羊水、胎儿脐血细胞染色体和FISH）。基因诊断87例（产前基因诊断10例）。结果超声检查发现胎儿异常1829例。异常检出率10．80％。超声筛查诊断16933例中，随访结果与原报告不符的有3例，诊断率为99．98％，漏诊率0．02％。超声建立的软指标提示行羊水细胞染色体检查30例，发现3例18三体，可以看出超声在产前筛查中用于发现胎儿染色体异常，无疑是提供了一条新途径。细胞遗传学检查10442例，发现染色体异常1013例，其中常染色体异常489例（常染色体数目异常286例；常染色体结构异常199例），性染色体异常244例（性染色体数目异常111例；性染色体结构异常16例），脆性X染色体117例，76个家系）；染色体变异280例（常染色体变异109例；性染色体变异171例）；基因诊断87例。发现17例患者。结论联合应用多项诊断技术，提高了缺陷儿遗传病诊断率。     

609例无精子症和少精子症患者的细胞遗传学分析

目的探讨无精子症和少精子症患者染色体畸变的发病率和特点。方法常规G显带方法对609例无精子症和少精子症患者行细胞遗传学检测。结果总的染色体异常检出率为12.8%,其中包括46例性染色体非整倍体（59%）,7例性染色体结构异常（9%）,25例常染色体结构异常（32%）。其中克氏综合征达39例,占检出性染色体非整倍体83%。在206例无精子症患者中检出42例异常核型（20.4%）,在228例严重少精子症患者中检出24例异常核型（10.5%）,在175例少精子症患者中检出12例异常核型（6.9%）。结论染色体数目异常和结构异常在无精子症和少精子症患者中常见。我们需要更加有效的检测手段提高男性不育检出率。     

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.     

512例无精子症患者染色体及性激素检查临床分析

目的；探讨无精子症患者染色体异常、性激素水平的变化及其与发病的关系。方法：对512例无精子症患者同时进行染色体G显带及性激素5项检测。结果：染色体异常169例，占总例数的33％。其中性染色体异常156例．占92．3％，其中又以Klinefelter’s综合征为多，137例，占性染色体异常的87．8％；常染色体异常13例，占2．5％。其中罗伯逊异位7例，占常染色体异常的53．8％；无精子症患者不论染色体核型异常与否，均有性激素水平的改变。睾酮（TT）明显降低，与正常均值对照有非常显著性差异（P＜0．01）：促卵泡素（FSH）、促黄体生成素（LH）、雌二醇（E2）、垂体泌乳素（PRL）均升高，与正常均值对比（P＜0．01）核型异常与核型正常者比较，TT降低更为显著，LH虽均升高，但前者低于后者，两组间比较（P＜0．01）。结论：512例无精子症患者中，染色体异常发生率为1／3，全部有性激素水平的改变。提示无精子症与性激素水平及染色体异常有密切关系。     

159例生精障碍患者的细胞遗传学分析

病例 159例息者主要来自本院不孕症门诊，年龄22-37岁，精液检查结果为无精子或少弱精子。采用外周血淋巴细胞培养，常规制片，G显带后进行染色体核型分析，镜下计数30个中期分裂相，分析4个核型，异常者加倍计数和分析。结果159例生精障碍患者中，正常核型102例，检出染色体异常57例。异常检出率为35．85％。其中性染色体数目异常34例（59．65％）；性染色体结构异常16例（28．07％），常染色体结构异常7例（12．28％），见表1。     

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.     

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。     

Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men

Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = -0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.     

Incidence of sperm chromosomal abnormalities in a risk population: relationship with sperm quality and ICSI outcome

BACKGROUND: An increased incidence of chromosome abnormalities has been reported in sperm samples of many infertile men by fluorescence in-situ hybridization (FISH). METHODS: Sperm aneuploidy and diploidy rates for chromosomes 13, 18, 21, X and Y were evaluated in 63 patients with normal karyotypes using dual and triple-colour FISH techniques. Indications for sperm FISH analysis were: recurrent miscarriages of unknown aetiology (RM, n = 40), repeated implantation failure after intracytoplasmic sperm injection (ICSI) (IF, n = 19), previous Down's syndrome pregnancies (n = 3), and meiotic abnormalities (MA, n = 1). Nine healthy normozoospermic donors were also evaluated as a control group. RESULTS: A significant increase in the incidence of sex chromosome disomies was found in the RM, IF and MA groups. Oligoasthenoteratozoospermic patients (n = 21) showed significantly higher rates of diploidy and disomies for sex chromosomes and chromosomes 18 and 21 than normozoospermic patients (n = 14). Thirty-one patients with normal and seven with abnormal FISH results had undergone several ICSI treatments (108 and 23 cycles respectively). Couples with abnormal sperm FISH results showed decreased pregnancy and implantation rates and increased miscarriage rates. CONCLUSIONS: Patients with a clinical background of recurrent miscarriages of unknown aetiology or implantation failure after ICSI are at risk of showing sperm chromosomal abnormalities, the incidence of which is higher in oligoasthenoteratozoospermic patients.     

FISH analysis of chromosome X,Y and 18 abnormalities in testicular sperm from azoospermic patients

BACKGROUND: Sperm extracted from testicular biopsies of azoospermic men can successfully be used for ICSI. The concern exists that testicular sperm from azoospermic men suffering from severe testicular failure may have a higher frequency of aneuploidy, which may lead to an increased risk for chromosomally abnormal offspring. METHODS: Testicular sperm from patients showing spermatogenic failure (n = 17) and from patients with normal spermatogenesis (n = 26) were analysed by fluorescence in-situ hybridization (FISH). Numerical chromosomal abnormalities for chromosomes X, Y and 18 were evaluated by FISH in a total of 1697 testicular sperm derived from 43 azoospermic patients. RESULTS: No difference was observed between the frequency of chromosomal abnormalities in testicular sperm from patients with normal spermatogenesis (5.6%) and from patients with spermatogenic failure (8.2%). However, the frequency of aneuploidy for chromosome 18 was higher in the group of azoospermic patients with spermatogenic failure than in the group with normal spermatogenesis (3.2 versus 1.3%). Within the obstructive group, sex chromosome aneuploidy (4.5%) occurred more frequently than chromosome 18 aneuploidy (1.3%; P < 0.001). Among testicular sperm derived from patients with spermatogenic failure, sex chromosomal aneuploidy (5.8%) was similar to that for chromosome 18 (3.2%). CONCLUSIONS: So far, no difference in the total frequency of chromosomal abnormalities has been observed between patients with normal spermatogenesis and patients with severe testicular failure. However, aneuploidy for chromosome 18 was higher in the group with spermatogenic failure.     

Frequency of aneuploidy in sperm from patients with extremely severe male factor infertility

BACKGROUND: A protocol for the chromosomal analysis of sperm samples with a severely reduced number of sperm cells was designed. METHODS: A severe male factor condition was the main cause of infertility for 38 couples: 27 were oligoasthenoteratospermic (OAT) and 11 with non-obstructive azoospermia underwent testicular sperm extraction (TESE). A two-round fluorescence in situ hybridization (FISH) protocol was performed with probes specific for the chromosomes X, Y, 13, 15, 16, 17, 18, 21 and 22. The recording of the position of each sperm cell at the microscope allowed diagnosis of each spermatozoon for the nine tested chromosomes. RESULTS: A mean number of 122+/-78.5 sperm were diagnosed per patient with an incidence of total abnormalities corresponding to 13.4%. chi2-tests for the observed frequencies and goodness-of-fit test were highly significant in all cases. A significantly higher proportion of total aneuploidy was detected in 79% of the tested samples compared to the normal population. Testicular sperm were significantly more prone to aneuploidy than ejaculated sperm. CONCLUSIONS: The designed FISH protocol for the analysis of severe OAT and TESE sperm samples is reliable, implying that the studied sample is representative of the original population. In view of the high incidence of aneuploidy in most severe OAT and TESE sperm, the FISH analysis of pathological sperm samples can be routinely performed in order to estimate the chances of the paternal contribution to aneuploidy in the resulting embryos.     

Andrology: Application of different in-situ hybridization detection methods for human sperm analysis

The detection of some types of aneuploidy in human spermatozoacan be based on the use of the fluorescence in-situ hybridizationtechnique (FISH). One of the crucial steps for FISH is to achievea proper decondensation and denaturation of the DNA in the specimen,so as to obtain efficient hybridization results. However, afterDNA decondensation the morphology of sperm heads is partly distortedand the majority of the tails is lost. This situation leadsto problems in the distinction between disomic and diploid spermatozoa,as well as between abnormal spermatozoa and somatic cells. Double-and triple-target FISH can partly solve this discriminationproblem. To improve these procedures we adapted the steps ofdecondensation and visualization of the single sperm cells.Firstly, DNA decondensation with 25 mM dithiothreitol in 1 MTris at pH 9.5 resulted in sperm cells with intact morphologyof both the head and the tail, and allowed efficient single-,double- and triple-target ISH to be performed. Secondly, weapplied a novel detection method, based on enzyme immunocyto-chemicalreactions, with coloured precipitation products. Thirdly, thisISH procedure was combined with Diff-Quik staining and bright-fieldmicroscopy. This absorption method has the advantage of a permanentsignal, and the adapted cytoplasmic staining of the sperm plasmamembrane allows the visualization of the outline of the singlespermatozoon. Using this approach, therefore, it is possibleto discriminate between disomic, diploid and abnormal spermatozoa,somatic cells and spermatozoa that overlap, because the morphologyof the cells is not distorted and the tails of the spermatozoaare intact and properly visualized.     

Sperm aneuploidy and meiotic sex chromosome configurations in an infertile XYY male

BACKGROUND: There is little information regarding the behaviour of the extra Y chromosome during meiosis I in men with 47,XYY karyotypes and the segregation of the sex chromosomes in sperm. We applied immunofluorescent and FISH techniques to study the relationship between the sex chromosome configuration in meiotic germ cells and the segregation pattern in sperm, both isolated from semen samples of a 47,XYY infertile man. METHODS: The sex chromosome configuration of pachytene germ cells was determined by immunostaining pachytene nuclei for synaptonemal complex protein 3 (SCP3) and SCP1. FISH was subsequently performed to identify the sex chromosomes and chromosome 18 in pachytene cells. Dual- and triple-color FISH was performed on sperm to analyse aneuploidy for chromosomes 13, 18, 21, X, and Y. RESULTS: 46,XY/47,XYY mosaic pachytene cells were observed (22.2% vs. 77.8%, respectively). The XYY trivalent, and X+YY configurations were most common. While the majority of sperm were of normal chromosomal constitution, an increase in sex and autosome disomy was observed. CONCLUSIONS: The level of germ cell moscaicism and their meiotic sex chromosome configurations may determine sperm aneuploidy rate and fertility status in 47,XYY men. Our approach of immunostaining meiotic cells in the ejaculate is a novel method for investigating spermatogenesis in infertile men.     

Molecular cytogenetic evaluation of the aneugenic effects of teniposide in somatic and germinal cells of male mice

The ability of topoisomerase II inhibitor, teniposide, to induce aneuploidy and meiotic delay in somatic and germinal cells of male mice was investigated by fluorescence in situ hybridisation (FISH) assay using labelled DNA probes and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, respectively. Colchicine and mitomycin C were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. Using FISH assay with centromeric and telomeric DNA probes for erythrocyte, micronuclei (MN) showed that teniposide is not only clastogenic but also aneugenic in somatic cells in vivo. The assay also showed that chromosomes can be enclosed in the MN before and after centromere separation. By using the BrdU incorporation assay, it could be shown that the meiotic delay caused by teniposide in germ cells was ～48 h. Disomic and diploid sperms were shown in epididymal sperm hybridised with DNA probes specific for chromosomes 8, X and Y after teniposide treatment. The prevalence of autodiploid (XX88 and YY88) sperm and disomic XX8 or YY8 sperm indicates that the second meiotic division was more sensitive to teniposide than the first meiotic division. The results also suggest that earlier prophase stages contribute relatively less to teniposide-induced aneuploidy. Both the clastogenic and the aneugenic potential of teniposide can give rise to the development of secondary tumours and abnormal reproductive outcomes in cured cancer patients and medical personnel exposing to drug regimens that include teniposide. Thus, genetic counselling of these patients should take place before the start of chemotherapy and should take the present results into consideration.     

Aneuploidy rate in spermatozoa of selected men with abnormal semen parameters

A large proportion of patients with oligoasthenoteratozoospermia (OAT) have an abnormal karyotype and hence they produce aneuploid gametes. However, a normal karyotype does not exclude the chance of having germ cell aneuploidy, since an altered intra-testicular environment not only damages spermatogenesis, but may also disrupt the mechanisms controlling chromosomal segregation during meiosis. Therefore, this study was undertaken to evaluate the rate of aneuploidy in the spermatozoa of selected patients with abnormal sperm parameters. For this purpose, sperm aneuploidy rate for chromosomes 8, 12, 18, X and Y was evaluated by multicolour fluorescence in-situ hybridization (FISH) in nine patients with teratozoospermia alone and 19 OAT patients of presumably testicular origin. Thirteen normozoospermic healthy men served as controls. Patients with teratozoospermia or OAT had significantly greater disomy and diploidy rates compared with controls, whereas the rate of nullisomy was similar. XY disomy was very low in all groups, suggesting that chromosomal non-disjunction occurs mainly during the second meiotic division. Autosome 12 disomy rate was low in both patients and controls. There was a marked variability of total sperm aneuploidy rate in both groups of patients. Sperm aneuploidy rate was negatively correlated with sperm concentration and particularly with the percentage of normal forms. In conclusion, patients with teratozoospermia or OAT have an increased rate of sperm aneuploidy. This increase is similar in both groups, suggesting that teratozoospermia may be the critical sperm parameter associated with aneuploidy.