KIinefeIter综合征的诊断：新型甲基化特异性实时定量PCR

本文研究目的为设计一种基于额外X染色体（X-ch）检测的分子检测方法用于Klinefelter综合征的诊断。从26名47，XXY男性，2名46，XY／47，XXY男性，22名46，XY男性和15名46，XX的女性的外周血标本中提取DNA，并进行脱氨基处理。甲基化特异性的定量多聚酶链反应（MS-qpPCR）以非甲基化和甲基化的X染色体非活化特异性转录基因（XIST-U和XIST-M）拷贝为模板。X染色体二倍体通过XIST基因甲基化状态来判定。46，XY／47，XXY男性的嵌合程度通过核型分析和原位荧光杂交（FISH）的结果比较来判定。数据分析应用Roche LightCycler software v．3．5.3，包括通过拟合点分析和溶解曲线分析决定交叉点（CPs）。所有对照组女性和Ks患者均检测到X染色体二倍体，而对照组男性只表达XIST-M。XIST-U和IXIST-M的交叉点范围分别是（29．5-32．5，SD0．8）和（29-31，SD0．6）。嵌合度的检测极限为1％。根据2名47，XXY/46，XY患者的XIST-U／）（IST-M比值分析，计算出的嵌合率（1．8％和17．8％）与FISH结果（2．3％和15％）相当。从DNA脱氨基到最终数据分析的间隔小于9小时。本文得出结论：MS-qpPCR是一种敏感、特异及快速的X染色体二体检测方法，可以用于KS的筛查和诊断，甚至在低嵌合水平的47，XXY／46，XY患者也适用。     

Routine screening for classical azoospermia factor deletions of the Y chromosome in azoospermic patients with Klinefelter syndrome

Aim： To evaluate the occurrence of classical azoospermia factor （AZF） deletions of the Y chromosome as a routine examination in azoospermic subjects with Klinefelter syndrome （KS）. Methods： Blood samples were collected from 95 azoospermic subjects with KS （91 subjects had a 47,XXY karyotype and four subjects had a mosaic 47,XXY/46, XY karyotype） and a control group of 93 fertile men. The values of testosterone, follicle stimulating hormone （FSH） and luteinizing hormone （LH） were measured. To determine the presence of Y chromosome microdeletions, polymerase chain reaction （PCR） of five sequence-tagged site primers （sY84, sY 129, sY 134, sY254, sY255） spanning the AZF region, was performed on isolated genomic DNA. Results： Y chromosome microdeletions were not found in any of the 95 azoosperrnic subjects with KS. In addition, using similar conditions of PCR, no microdeletions were observed in the 93 fertile men evaluated. The level of FSH in KS subjects was higher than that in fertile men （38.2 ± 10.3 mIU/mL vs. 5.4 ±2.9 mIU/mL, P 〈 0.001） and the testosterone level was lower than that in the control group （1.7 ±0.3 ng/mL vs. 4.3 ± 1.3 ng/mL, P 〈 0.001）. Conclusion： Our data and review of the published literature suggest that classical AZF deletions might not play a role in predisposing genetic background for the phenotype of azoospermic KS subjects with a 47,XXY karyotype. In addition, routine screening for the classical AZF deletions might not be required for these subjects. Further studies including partial AZFc deletions （e.g. gr/gr or b2/b3） are necessary to establish other mechanism underlying severe spermatogenesis impairment in KS.     

Incidence of chromosome 8,10,X and Y aneuploidies in sperm nucleus of infertile men detected by FISH

We studied the frequency of aneuploidy in sperm nuclei of six infertile men with abnormal semen profile and normal karyotype, using fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 8, 10, X and Y. The control group consisted of four healthy fertile men with normal karyotype and semen profiles. The purpose of this study was to determine whether there are differences between infertile male donors and control donors for: (1) the incidence of sex chromosome aneuploidy, and (2) the number of disomies for chromosomes 8, and 10 cosegregating with chromosomes X and Y. FISH analysis showed no significant differences of sex ratios of the sperm nuclei in and between infertile and control groups. The most significant abnormalities in the infertile group were clusters of sperm nuclei bearing XY and XYY. In addition, the incidence of disomic sperm nuclei for chromosomes 8 and 10 consegregating with sex chromosomes was not significantly different between the patient and control groups, nor within them. However, the total frequency of aneuploid sperm nuclei was significantly different between the infertile group and the control group. We observed a significant excess of sperm nuclei bearing chromosome 10 along with disomy for chromosome Y (10YY). In conclusion, our results from FISH analysis demonstrate a significantly increased frequency of aneuploidy for the sex chromosomes in sperm nuclei from infertile men. Therefore it may be concluded that infertility is a risk factor for sex chromosome aneuploidy in sperm nuclei.     

A rare variant Klinefelter syndrome seen 40 years later: 47,X,del(Xq24),Y

Patients with Klinefelter syndrome (KS) show a typically 47,XXY karyotype; however, some variations have been observed, including 47,XX,der(Y), 46,XY/47,XXY, 48,XXXY, 48,XXYY, and mosaicism or structural sex chromosome abnormalities in some patients. In the literature, a rare KS variant, 47,X,del(Xq),Y karyotype, was reported in only a few cases prior to 1981. A 40-year-old man (IV-3) was referred to our department due to infertility. His phenotype did not differ from the classic KS phenotype. He had two siblings (1-male; 1-female). His brother (IV-5) had mental retardation and died one year earlier at age 32. Additionally, his sister (IV-2) also had a history of infertility due to her husband's azoospermia. His mother had a history of 12 miscarriages. Karyotype analysis revealed the 47,X,del(Xq24),Y karyotype, and no deletions were seen in the AZF and SRY regions. We thought this chromosomal abnormality in the patient might have resulted from X-autosome translocation in one of his parents since his mother had recurrent pregnancy loss and his sibling had mental retardation. However, we could not confirm it due to his parents were not alive. This study shows the first case of a long-arm X-chromosome deletion after a long period and reviews current knowledge concerning variant KS (deletion Xq).     

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。     

男性不育症中染色体特殊核型

在男性不育症的遗传咨询门诊中，发现的染色体异常绝大多数为典型的４７，ＸＸＹＫｌｉｎｅｆｅｌｔｅｒ综合征，但我们也发现一些较为特殊的核型，其中多Ｘ及多Ｘ嵌合体的３例，Ｙ染色体结构异常及Ｙ染色体与常染色体易位２例；常染色体之间的平衡易位６例。本文讨论了染色体的异常与男性不育症之间的可能关系。     

Detection of chromosomal abnormality and Y chromosome microdeletion in patients with azoospermia and oligozoospermia

Objective:To investigate the chromosomal abnormality and Y chromosome microdeletion in patients with azoospermia and oligozoospermia.Methods:Cytogenetic karyotype analysis and multiplex PCR were used to detect chromosomal abnormality and Y chromosome microdeletion in 99 azoospermic and 57 oligospermic patients(total 156).45 fertile men were includ-ed as controls.Results:31 patients were found with chromosomal abnormalities in 156 cases(31/156,19.9 %),20 cases showed 47,XXY,2 cases showed 46,XY/47,XXY,7 cases had Y chromosome structural abnormalities and 2 had autosomal chromosome abnormalities.There were significant differences between the frequency of AZF microde-letion in 125 cases with normal karyotype and 45 controls(P<0.01).The frequency of AZF microdeletion in 68 azoospermic and 57 oligospermic patients were 14.7%(10/68)and 15.8%(9/57)respectively,the difference was not significant(P>0.05).AZFa,AZFb,AZFa+b,AZFb+c,AZFa+b+d and AZFb+c+d mierodeletions were found in azoospermic patients.AZFb,AZFc,AZFd,AZFb+c+d and AZFc+d microdeletions were found in oligo-spermic patients.Conxlusion:The frequency of chromosomal abnormality was 19.9% and the frequency of Y chromosome mi-crodeletion was 15.2% in patient with azoospermia and oligozoospermia.We should pay close attention to this prob-lem.     

尿道下裂患儿细胞遗传学特点分析

目的:探讨尿道下裂患儿的细胞遗传学特点。方法:回顾性分析2008年6月至2018年5月于天津市儿童医院就诊的45例有细胞遗传学异常的尿道下裂患儿的临床资料。中位年龄为10个月(3 h～5岁)。45例中20例为近端型尿道下裂,1例为中段型尿道下裂;24例合并不同程度的泌尿生殖系统畸形,其中15例合并单侧或双侧隐睾,5例阴...     

Y chromosome microdeletions in azoospermic patients with Klinefelter＇s syndrome

Aim： To study the occurrence of Y chromosome microdeletions in azoospermic patients with Klinefelter＇s syndrome （KFS）. Methods： Blood and semen samples were collected from azoospermic patients with KFS （n = 14） and a control group of men of proven fertility （n = 13）. Semen analysis was done according to World Health Organization （WHO） guidelines. Blood samples were processed for karyotyping, fluorescent in situ hybridization （FISH） and measurement of plasma follicle stimulating hormone （FSH） by radioimmunoassay. To determine Y chromosome microdeletions, polymerase chain reaction （PCR） of 16 sequence tagged sites （STS） and three genes （DFFRY, XKRY and RBM1 Y） was performed on isolated genomic DNA. Testicular fine needle aspiration cytology （FNAC） was done in selected cases. Results： Y chromosome microdeletions spanning the azoospermia factor （AZF）a and AZFb loci were found in four of the 14 azoospermic patients with KFS. Karyotype and FISH analysis revealed that, of the four cases showing Y chromosome microdeletion, three cases had a 47,XXY/46,XY chromosomal pattern and one case had a 46,XY/47,XXY/48,XXXY/48,XXYY chromosomal pattern. The testicular FNAC of one sample with Y chromosome microdeletion revealed Sertoli cell-only type of morphology. However, no Y chromosome microdeletions were observed in any of the 13 fertile men. All patients with KFS had elevated plasma FSH levels. Conclusion： Patients with KFS may harbor Y chromosome microdeletions and screening for these should be a part of their diagnostic work-up, particularly in those considering assisted reproductive techniques. （Asian JAndrol 2006 Jan; 8： 81-88）     

Chromosomal abnormalities in 2 cases of testicular failure

This study investigated the underlying chromosomal abnormalities of testicular failure using molecular cytogenetic analysis. We report 2 cases of rare genetic anomalies that resulted in hypogonadism. The first patient presented with severe hypogonadism. Chromosome analysis revealed a mosaic 46,X,r(Y) (p11.3q11.23)/45,X karyotype, with a ring Y chromosome. A Y chromosome microdeletion assay showed a deletion in the azoospermia factor a region. The second patient presented with infertility and nonobstructive azoospermia. Cytogenetic and fluorescent in situ hybridization analysis revealed a 47,XY,+mar.ish i(15) (D15Z1++,SNRPN2,PML2) karyotype, with a small supernumerary chromosome derived from chromosome 15. These results emphasize the need for molecular cytogenetic evaluation in patients with testicular failure before using advanced reproductive techniques.     

Chromosomal investigation in infertile cases of azoospermia

Chromosomal examination in 85 patients with azoospermia revealed 62 patients (73%) with the karyotype 46, XY and 23 patients (27%) with other abnormal karyotypes. In this population, there were 17 patients with 47, XXY. Other anomalies were: 46, XYp+, 46, XXp+, 46, XYp-, 45, X, t (Ynf; 21), 45, XY, t (13q 15q), and 46, XY, r (18). Serum LH and FSH levels were high and testosterone level was low in the patients with 47, XXY compared with the other 6 patients (p less than 0.01). Also the mean value of testicular volume was low in the patients with 47, XXY (p less than 0.05).     

Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome

Aim:To study the occurrence of Y chromosome microdeletions in azoospermic patients with Klinefelter's syndrome(KFS).Methods:Blood and semen samples were collected from azoospermic patients with KFS(n=14)and acontrol group of men of proven fertility(n=13).Semen analysis was done according to World Health Organization(WHO)guidelines.Blood samples were processed for karyotyping,fluorescent in situ hybridization(FISH)andmeasurement of plasma follicle stimulating hormone(FSH)by radioimmunoassay.To determine Y chromosomemicrodeletions,polymerase chain reaction(PCR)of 16 sequence tagged sites(STS)and three genes(DFFRY,XKRYand RBM1 Y)was performed on isolated genomic DNA.Testicular fine needle aspiration cytology(FNAC)was donein selected cases.Results:Y chromosome microdeletions spanning the azoospermia factor(AZF)a and AZFb lociwere found in four of the 14 azoospermic patients with KFS.Karyotype and FISH analysis revealed that,of the fourcases showing Y chromosome microdeletion,three cases had a 47,XXY/46,XY chromosomal pattern and one casehad a 46,XY/47,XXY/48,XXXY/48,XXYY chromosomal pattern.The testicular FNAC of one sample with Ychromosome microdeletion revealed Sertoli cell-only type of morphology.However,no Y chromosome microdeletionswere observed in any of the 13 fertile men.All patients with KFS had elevated plasma FSH levels.Conclusion:Patients with KFS may harbor Y chromosome microdeletions and screening for these should be a part of their diagnosticwork-up,particularly in those considering assisted reproductive techniques.(Asian J Androl 2006 Jan;8:81-88)     

Sperm disomy in idiopathic severely oligoasthenoteratozoospermic males

This work aimed to determine the incidence of sperm disomy in infertile men with idiopathic severe oligoasthenoteratozoospermia (OAT). Fifty male subjects were included in this study: 30 infertile men with idiopathic severe OAT and 20 healthy fertile men as controls. Semen analysis, hormonal assay (follicle-stimulating hormone, luteinising hormone and testosterone), scrotal ultrasound examination and fluorescent in situ hybridisation of their semen samples were performed to determine the disomy levels of chromosomes X and Y. There was a significant higher frequency for XX disomy and XY disomy in spermatozoa from severe OAT patients than that in controls. There was nonsignificant difference in the percentage of YY disomy between OAT cases and controls. XX, YY and XY disomy showed nonsignificant correlation with the age. Sperm concentration and sperm motility demonstrated significant negative correlation with XX and XY disomy. Sperm abnormal forms had significant negative correlation with XX and XY disomy. Nonsignificant correlation was demonstrated between YY disomy and semen parameters. XX disomy showed significant positive correlation with XY disomy and nonsignificant correlation with YY disomy. YY disomy showed nonsignificant correlation with XY disomy. It is concluded that sperm disomy in severe OAT is increased, which should be taken into account when undergoing micromanipulation.     

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.     

Chromosomal abnormalities and polymorphisms in infertile men

The aim of this study was to determine the prevalence of alterations and normal variable chromosome features in males from infertile couples. Karyotyping was performed to 84 men attending the infertility clinic at the Hospital Clinic i Provincial of Barcelona (Spain). Sex chromosome abnormalities were detected in 19 patients (26.62%): 14 (16.67%) aneuploidies 47,XXY and 47,XYY, 3 (3.57%) Y-chromosome long arm deletions; 1 (1.19%) mosaic 45,x/46,XY and 1 (1.19%) Robertsonian translocation (45.X-15-Y+t(15p: Yq). Chromosomal polymorphisms were observed in 29 patients. Yqh+ was the most frequent variant in sex chromosomes and increased length in heterochromatin and satellites were present in autosomal chromosomes. The high prevalence of chromosomal abnormalities observed in infertile men justify the use of karyotyping to evaluate males enrolled in new assisted reproductive technologies programs.     

1例嵌合型Klinefelter综合征患者细胞与分子遗传学研究

目的:观察嵌合型Klinefelter综合征的外周血染色体、Y染色体上SRY基因和AZF基因微缺失发生情况。方法:对1例嵌合型Klinefelter综合征患者及父母进行外周血染色体核型分析,确定9个实验用序列标签位点(STS),分别是:sY84、sY86、sY127、sY129、sY134、sY254、sY255、sY242、sY152,同时检测SRY基因,并以X/Y连锁锌指蛋白基因(ZFX/ZFY)为内对照进行多重PCR筛查AZF微缺失。结果:患者核型为46,XY/47,XXY/48,XXYY/49,XXXXY,其中48,XXYY占56%;47,XXY占30%;46,XY占12%;49,XXXXY占2%,患者父母核型正常;患者及父母SRY基因检测与染色体性别一致,患者检出Y染色体AZF微缺失,缺失位点为sY86和sY127,缺失类型为AZFa+AZFb缺失。结论:Klinefelter综合征患者存在Y染色体AZF微缺失,染色体核型分析和Y染色体AZF微缺失是Klinefelter综合征患者重要的遗传检测指标。     

Hypogonadotropic hypogonadism associated with another small supernumerary marker chromosome (sSMC) derived from chromosome 22, a case report

The idiopathic hypogonadotropic hypogonadism (IHH) is portrayed as missing or fragmented pubescence, cryptorchidism, small penis, and infertility. Clinically it is characterized by the low level of sex steroids and gonadotropins, normal radiographic findings of the hypothalamic-pituitary areas, and normal baseline and reserve testing of the rest of the hypothalamic-pituitary axes. Delay puberty and infertility result from an abnormal pattern of episodic GnRH secretion. Mutation in a wide range of genes can clarify ~40% of the reasons for IHH, with the majority remaining hereditarily uncharacterized. New and innovative molecular tools enhance our understanding of the molecular controls underlying pubertal development. In this report, we aim to present a 26-year-old male of IHH associated with a small supernumerary marker chromosome (sSMC) that originated from chromosome 22. The G-banding analysis revealed a karyotype of 47,XY,+mar. High-throughput DNA sequencing identified an 8.54 Mb duplication of 22q11.1-q11.23 encompassing all the region of 22q11 duplication syndrome. Pedigree analysis showed that his mother has carried a balanced reciprocal translocation between Chromosomes 22 and X[t(X;22)]. To the best of our knowledge, this is the second confirmed case of IHH with an sSMC deriving from chromosome 22. Based on our study, the duplicated chromosome fragment 22q11.1-q11.23 might be the reason for the phenotype of our case. Meanwhile, High-throughput DNA sequencing combined with cytogenetic analysis can provide a more accurate clinical diagnosis for patients carrying sSMCs.     

Meiotic segregation of sex chromosomes in mosaic and non-mosaic XYY males: case reports and review of the literature

The aim of this study was to determine the incidence of sex chromosome aneuploidy in spermatozoa of two males with a 47,XYY karyotype and one male with a 46,XY/47,XYY constitution. Spermatozoa obtained from two oligospermic patients and one volunteer semen donor were studied by multicolour fluorescence in situ hybridization. In the XY/XYY male, the frequencies of X-bearing to Y-bearing sperm were significantly different from the 1 : 1 expected ratio. Significantly increased frequencies were found in the mosaic and non-mosaic males for 24,XX and 24,YY sperm when compared with control donors. The number of 24,XY sperm was significantly different from the controls in the XYY males, but not in the mosaic male. The incidence of disomy 18 and the rate of diploidy also increased in the three patients. However, the mosaic male had the lowest cumulative rate of disomic and diploid spermatozoa when compared with the two XYY patients. Our data suggest that: (i) chromosome abnormalities observed in spermatozoa of the two XYY oligoasthenoteratospermic (OAT) males arise through segregation errors in XY germ cells rather than normal meiosis of XYY germ cells, (ii) mosaic XYY males with normal semen parameters have a lower risk of producing offspring with a sex chromosomal abnormality than OAT males with XYY karyotype.