应用荧光原位杂交技术(FISH)对罗伯逊易位携带者进行胚胎着床前遗传学诊断

目的用FISH对罗伯逊易住携带夫妇行胚胎着床前遗传学诊断，选择正常／平衡核型的胚胎移植。方法对14例罗伯逊易位携带者（男性9例，女性5例）行IVF／ET／PGD共15周期。9例男性和2例女性罗伯逊易位携带者体外受精后第3天卵裂球活检，3例女性携带者极体活检或极体活检加卵裂球活检；用特异位点探针或全染色体涂抹探针荧光原位杂交，选择信号正常或平衡的胚胎移植。结果15个PGD周期，共活检胚胎或MII卵母细胞120枚；取出卵裂球120个、第一极体18个，固定成功率92．8％（128／138）；杂交成功率为96．1％（123／128）。活栓后胚胎继续发育69．2％（83／120）；15例PGD周期中，移植13例；6例获得妊娠（6／13）；其中女性携带者妊娠4例（4／4），男性携带者妊娠2例（2／9），二者差异具有显著性（P〈0．05）。结论1、对存在不孕因素的罗伯逊易住携带者做胚胎着床前遗传学诊断，选出正常／平衡核型的胚胎移植可有较好的临床预后。2．对女性罗伯逊易住携带者哥首选极体活检，使患者有机会避免罗伯逊易位携带者出生。     

运用全染色体探针FISH技术进行罗伯逊易位携带者胚胎植入前诊断

目的运用全染色体探针荧光原位杂交技术(fluorescent in situ hybridization ,FISH)对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断,达到优生目的.方法应用FITC、Texas标记的21/14全染色体探针对14/21罗伯逊易位携带者的胚胎活检后的单个卵裂球进行遗传学检测,选择正常信号或携带者信号核型胚胎进行移植.结果常规单精子显微注射-胚胎移植(intracytoplasmic sperm injection,ICSI)后获取胚胎12个,活检优质胚胎11个,杂交后有信号胚胎7个,其中1个正常胚胎,1个携带者胚胎;1个14单体胚胎,1个21单体胚胎,1个21三体胚胎,另2个胚胎活检后均为21号二体,无14号信号.结论运用14/21全染色体探针荧光原位杂交技术对14/21罗伯逊易位携带者的胚胎进行植入前遗传学诊断是能够将正常核型胚胎挑选出来进行移植,从而达到优生目的.     

临沂地区58例罗伯逊易位携带者的遗传学分析北大核心CSCD

目的探讨临沂地区各种罗伯逊易位携带者异常妊娠的几率,计算各种核型的比例,统计罗伯逊易位携带者和罗伯逊易位型21三体患者的性别比,为遗传咨询提供依据。方法采用外周血淋巴细胞培养技术制备染色体,通过G显带进行核型分析,对异常核型者进行家系调查和遗传学分析。结果在10339例进行遗传咨询的人群中,共检出58例罗伯逊易位携带者和22例罗伯逊易位型21三体患者,罗伯逊易位携带者中der（13;14）和der（14;21）所占比例分别为50．00％和17．24％;易位型21三体患者只有der（14;21）和der（21;21）两种核型,分别占54．55％和45．45％。罗伯逊易位携带者男女之比为0．57：1（21／37）,与正常人群差异极为显著（P〈0．05）;易位型21三体中男女之比为1．75：1（14／8）,与正常人群无显著差异（P〉0．05）。53例非同源罗伯逊易位携带者中有7例男性因精子质量极差不育,4例未到生育年龄,其余42例平均流产率为92．86％（39／42）,平均流产（或其他异常妊娠）2．26次（95／42）,非同源罗伯逊异位携带者异常妊娠和正常妊娠的比例为7．4：1（96／13）。夫妇一方为同源罗伯逊易位携带者的异常妊娠率为100％,平均异常妊娠2．8次。结论罗伯逊易位是造成反复流产及生育易位型21三体患儿的主要遗传学原因之一,不同核型的个体在临床表现上有很大的差异。针对不同核型的罗伯逊易位携带者在遗传咨询时应有所侧重。对这些患者进行染色体检查、遗传咨询和生育指导非常必要。     

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。     

荧光原位杂交技术及染色体核型分析在罗伯逊易位型21-三体检测中的应用

目的 通过对孕妇羊水进行染色体核型、荧光原位杂交(fluorescence in situ hybridization,FISH)分析,探讨两者联合检测在诊断罗伯逊易位型21-三体中的应用价值.方法 为2010年1月至2011年12月进行产前诊断的孕妇抽取羊水,经体外细胞培养后进行G显带染色体核型分析.对发现的5例罗伯逊易位采用FISH检测间期细胞13、18、21及X/Y的染色体数目,并分析孕妇及其丈夫外周血染色体核型.结果 两个胎儿父母外周血染色体核型正常,其中一个胎儿羊水染色体核型为46,XY,rob(21;21)(q10;q10),FISH检测提示其为21-二体,另一个胎儿核型为46,XY,rob(14;21)(q10;q10),FISH检测证实其为21-三体.另外3个胎儿母亲外周血染色体核型分别为45,XX,rob(14;21)(q10;q10)、45,XX,rob(15;21)(q10;q10)、45,XX,rob(21; 22) (q10; q10),其羊水染色体核型分别为46,XX,rob(14;21)(q10;q10)、46,XY,rob(15;21) (q10;q10)、46,XX,rob(21;22)(q10;q10).FISH检测证实其均为21-三体.结论 染色体核型分析结合FISH检测有助于明确罗伯逊易位型21-三体的诊断,但FISH检测同源罗伯逊易位型21-三体征有一定局限性.     

植入前诊断罗伯逊易位一例

目的　对 1例罗伯逊易位患者进行植入前胚胎遗传学诊断。方法　应用 Vysis L SI13q14 ,Tel Vysion14 q探针对卵裂球对进行荧光原位杂交分析。结果　荧光原位杂交结果显示 1个胚胎的染色体正常或为携带者 ;7个胚胎染色体异常 ,其中 1个 14三体 ,4个 14三体 13单体 ,2个 13三体 14单体 ;2个胚胎未见明确信号。移植 1个染色体正常或平衡易位的胚胎后 ,孕 10周复查 B超示胎儿发育良好。结论　罗伯逊易位的植入前胚胎遗传学诊断方法有效。     

一例罗伯逊易位携带者的胚胎植入前遗传学诊断

目的　探讨植入前遗传学诊断 (preimplantation genetic diagnosis,PGD)用于筛选罗伯逊易位携带者无遗传缺陷后代的可行性及风险。方法　 1对因男方携带易位 (13;14 )染色体并伴少、弱精的原发不孕夫妇 ,经激素超促排卵和单精子卵胞浆内注射 (intracytoplasmic sperm injection,ICSI)进行体外受精(in vitro fertilization,IVF) ,当胚胎发育到 6～ 8细胞阶段 (受精后第 3天 )时 ,用酸化法活检 ,从每个胚胎中取出单个分裂球 ,用 L SI 13q和 Tel 14 q探针进行荧光原位杂交 (fluorescence in situ hybridization,FISH)检测 ,继续培养活检后的胚胎到第 2天 ,并选择正常胚胎移植 ,获临床妊娠后 ,于妊娠中期行羊水细胞染色体检查。结果　活检 10个胚胎 ,获得 8个 FISH诊断结果 :5 0 % (4/8)正常或平衡的胚胎 ,37.5 % (3/8)不平衡的胚胎 ,12 .5 % (1/8)不确定。将诊断正常或平衡的胚胎 3枚于活检第 2天移植入母体宫腔 ,获临床单胎妊娠 ,产前诊断证实胎儿核型为 4 6 ,XY,完全正常 ,现分娩一正常男婴。结论　需行辅助生殖技术治疗的患者 ,当携带有罗伯逊易位时 ,PGD用于筛除异常胚胎 ,解决患者的生育障碍、预防严重遗传病胎儿的产生具有重要价值。     

植入前诊断t(5;14)(q11;q23)相互易位一例

目的对一例相互易位携带者进行胚胎植入前诊断，研究商业化探针组合在相互易位植入前诊断中的应用价值。方法受精后第3天对4个卵裂球进行活检，采用二步荧光原位杂交技术检测卵裂球中2对易位染色体状态，共选用5号，14号染色体断裂点远侧的ERG1和Tel 14q,以及5号染色体断裂点近侧的D5S23 3个探针。结果4个细胞裂解后均呈现单个完整细胞核，二步荧光原位杂交中各细胞核均有清晰明亮的杂交信号，各胚胎ERG 1/D5S23/Tel 14q信号数分别为：1号胚胎3/2/1；2号胚胎2/6/6；3号胚胎3/2/2；4号胚胎2/2/3。荧光原位杂交结果显示4个胚胎均为染色体不平衡胚胎，放弃移植。结论商业化探针的合理搭配使相互易位携带者胚胎植入前诊断大为简化，因此适合绝大多数相互易位携带者。     

罕见父源性染色体易位合并21三体综合征家系及文献综述

目的分析患儿及其父母的染色体核型,并对相关文献进行复习,探讨易位型21三体综合征的特点,为遗传咨询提供信息。方法取患儿及其父母外周血淋巴细胞进行培养,用常规G显带技术对中期细胞染色体进行核型分析并复习相关文献资料。结果患儿核型为46,XY,-14,+t(14q;21q),其父亲核型为45,XY,-14,-21,+t(14q;21q),其母亲核型为46,XX。文献复习表明易位型21三体综合征相对较为罕见,三体综合征的产生与生育年龄、有害物质接触史等多种因素有关。结论本家系患儿为易位型21三体携带者,其罗伯逊易位异常染色体可能来源于罗伯逊易位携带者父亲。21三体综合征的产生与多种因素有关,对易位型21三体综合征家系进行核型分析,有利于对患儿父母进行再生育指导和提高人口出生质量。     

二例罗伯逊易位的男性患者精液常规及精子形态学参数分析

罗伯逊易位在新生儿的出现率约为千分之二，也是最常见的人类染色体异常，这种核型只涉及人类核型中的D和G组染色体，在同源和／异源的端着丝粒染色体之问通过着丝粒融合或者着丝粒附近的断裂和重接丽形成的。大多数的罗伯逊易位包含两条非同源染色体，其中最常见的13q14q，其次是14q21q，他们的发生率分别占罗伯逊易位的73％和10％。     

Preimplantation genetic diagnosis of a reciprocal translocation t(3;11)(q27.3;q24.3) in siblings

Preimplantation genetic diagnosis (PGD) was performed in two couples to avoid chromosomally unbalanced progeny in a family in which a brother and a sister carry an identical maternally inherited balanced translocation t(3;11)(q27.3;q24.3). Embryos were biopsied 3 days after fertilization and blastomeres were analysed by fluorescent in-situ hybridization (FISH). Embryos were classified as unbalanced or normal/balanced. In the first case, the male carrier and his wife underwent one IVF/PGD treatment cycle. In all, 18 embryos were analysed. Of those, 15 revealed an unbalanced karyotype. For one embryo, results were not conclusive, from one embryo results were contradictory and one embryo was classified as normal/balanced and subsequently transferred. A singleton pregnancy was achieved. The PGD analysis was confirmed at 16 weeks gestation by amniocentesis. At term, a healthy girl with a balanced karyotype was born. Pregnancy and delivery were without complications. In the second case, the female carrier and her husband underwent two IVF/PGD treatment cycles. During the first cycle, three embryos were analysed. One embryo revealed an unbalanced karyotype and two embryos were designated a normal/balanced karyotype and transferred but no pregnancy was achieved. During the second PGD cycle three embryos were analysed. Of those, none appeared suitable for transfer. The couple decided not to undergo further treatment. Our results indicate that for individuals carrying a reciprocal translocation PGD is a feasible approach to obtain embryos with a normal chromosome balance and to avoid both spontaneous and induced abortion.     

Preimplantation genetic diagnosis and sperm analysis by fluorescence in-situ hybridization for the most common reciprocal translocation t(11;22).

In this study we describe the pre-clinical development and clinical application of preimplantation genetic diagnosis (PGD) by fluorescence in-situ hybridization (FISH) for two non-related carriers (one male and one female) of the most common balanced reciprocal translocation: t(11;22)(q25;q12). For the couple with the female carrier, enumeration of the sex chromosomes in the embryos was also indicated (husband: 47,XXY karyotype). Four-colour FISH analysis was performed on six blastomeres from three embryos. No embryo transfer was possible because all the embryos were unbalanced. Three PGD cycles, with two-colour FISH, were carried out for the couple with the male translocation carrier. A total of 35 embryos were biopsied and diagnosed by FISH; nine out of the 35 embryos (25. 7%) were normal and seven of them were transferred (two embryos from the first and four from the third cycle), six out of 35 embryos (17%) were unbalanced, three out of 35 embryos (5.7%) were triploid or polyploid, 10 out of 35 embryos (28.6%) were mosaic and seven out of 35 embryos (20%) were chaotic. Diagnosis failed in 2.9% of the embryos. The spermatozoa of the male carrier were also analysed using three-colour FISH. Only 29.1% of the sperm cells seemed to be balanced or normal. By choosing probes lying on both sides of the breakpoints and by using a combination of sub-telomeric or locus-specific probes and centromeric probes, the use of three-colour FISH enabled detection of all the imbalances in sperm and/or cleavage-stage embryos in the patients. This may improve risk assessment and genetic counselling in the future for translocation carriers.     

Complex chromosomal rearrangement and intracytoplasmic sperm injection: a case report

Complex chromosomal rearrangements (CCRs) are rare events in human pathology and are usually considered to induce severe reproductive impairment by disturbing the meiotic process and producing unbalanced gametes responsible for high reproductive risk. One-third of all CCRs are familial and tend to implicate fewer breakpoints and fewer chromosomes than de novo cases. CCRs are rarely transmitted through spermatogenesis and are primarily ascertained by male infertility. We report a familial balanced CCR, with seven breakpoints involving three chromosomes, which was detected prenatally in a female fetus conceived after intracytoplasmic sperm injection (ICSI) in a couple initially thought to be a carrier of a paternal reciprocal translocation involving two chromosomal breakpoints. Fluorescent in-situ hybridization (FISH) was used to elucidate the complexity of this CCR. The karyotype of the female CCR carrier was balanced and determined as 46,XX.ish t(1;4)(q42;q32)(WCP1+, D1Z5+, WCP4+, D1S3738-, D4S2930+; WCP4+, D4Z1+, WCP1+, D4S2930-, D1S3738+), ins(1;11)(q41;q23q24)(WCP1+,WCP11+, D11S2071-, MLL+; WCP11+, D11S2071+, WCP1-, MLL-), ins(4;11)(q23;q14q23)(WCP4+,WCP11+; WCP11+,WCP4-). The same balanced CCR was confirmed in her oligozoospermic father. We report, to our knowledge, the first case of ICSI performed in an infertile male with CCR, resulting in a balanced CCR carrier female with a normal clinical follow-up at 4 years of age. This particular case stresses the point of the relevance and feasibility of ICSI procedure in cases of balanced CCRs.     

Preimplantation genetic diagnosis for an insertional translocation carrier

BACKGROUND: While preimplantation genetic diagnosis (PGD) is well established for carriers of reciprocal terminal translocations, reports on PGD for insertional translocation carriers are lacking. Here, we report on the PGD of an insertional translocation carrier with karyotype 46,XX,ins(14;2)(q21;q31q35). Due to the possibility of crossovers within the inserted region, rather than a single probe, four probes are required for proper embryo selection. METHODS: Probes were generated for PGD using fluorescence in situ hybridization and two PGD cycles. RESULTS: Analysis of 10 embryos revealed four embryos to be normal diploid. Two embryos were consistent with 3:1 segregation of the theoretical quadrivalent and one was consistent with 2:2 or 1:1 segregation. Furthermore, one embryo was mosaic abnormal and one remained without diagnosis. CONCLUSIONS: With increased acceptance of PGD, it is likely that more carriers of complex translocations will enter PGD programmes. The present results suggest that a careful genetic work-up of complex translocations is essential for proper embryo selection. While theoretical modelling may predict that quadrivalents will form during the meiosis of insertional translocations, experimental proof for the occurrence of quadrivalents is still lacking and more research on the meiotic process of both female and male insertional translocation carriers is warranted.     

Robertsonian translocations--reproductive risks and indications for preimplantation genetic diagnosis.

BACKGROUND: Robertsonian translocations carry reproductive risks that are dependent on the chromosomes involved and the sex of the carrier. We describe five couples that presented for preimplantation genetic diagnosis (PGD). METHODS: PGD was carried out using cleavage-stage (day 3) embryo biopsy, fluorescence in-situ hybridization (FISH) with locus-specific probes, and day 4 embryo transfer. RESULTS: Couple A (45,XX,der(14;21)(q10;q10)) had two previous pregnancies, one with translocation trisomy 21. A successful singleton pregnancy followed two cycles of PGD. Couple B (45,XX,der(13;14)(q10;q10)) had four miscarriages, two with translocation trisomy 14. One cycle of PGD resulted in triplets. Couple C (45,XX,der(13;14)(q10;q10)) had four years of infertility; two cycles were unsuccessful. Couple D (45,XY,der(13;14)(q10;q10)) presented with oligozoospermia. A singleton pregnancy followed two cycles of PGD. Couple E (45,XY,der(13;14)(q10;q10)) had a sperm count within the normal range and low levels of aneuploid spermatozoa. PGD was therefore not recommended. No evidence for a high incidence of embryos with chaotic or mosaic chromosome complements was found. CONCLUSIONS: For fertile couples, careful risk assessment and genetic counselling should precede consideration for PGD. Where translocation couples need assisted conception for subfertility, PGD is a valuable screen for imbalance, even when the risk of viable chromosome abnormality is low.     

PGD in female carriers of balanced Robertsonian and reciprocal translocations by first polar body analysis.

Preimplantation genetic diagnosis (PGD) using the first polar body (1PB) is a modality of PGD that can be used when the woman is the carrier of a genetic disease or of a balanced chromosomal reorganization. PGD using 1PB biopsy in carriers of balanced chromosome reorganizations has not become generalized. Here, we describe our experience based on the analysis of unfertilized or fresh, non-inseminated control oocytes, by fixing separately the 1PB and the corresponding oocyte, and on the study of six clinical cases of PGD using 1PB biopsy (four Robertsonian translocations and two reciprocal translocations). In fresh oocytes, the chromosome morphology of the 1PB was well preserved, and the results were always concordant for each oocyte-1PB pair. This indicates that the 1PB can be reliably used for the diagnosis of chromosome reorganizations. In these studies the technical problems encountered when performing PGD using 1PB biopsies for chromosome studies are also addressed. Three different strategies of 1PB biopsy (laser beam, partial zona dissection and acid Tyrode's) and two different protocols (intracytoplasmic sperm injection before or after 1PB biopsy) and their effect on the percentage of oocytes diagnosed and the fertilization rate, are discussed. In reciprocal translocation cases, published in the literature or studied by us, in which at least nine oocytes had been diagnosed, a correlation has been found between the frequency of nondisjunction observed and the theoretical recombination rate. To date, PGD by 1PB analysis alone or combined with blastomere biopsies in female carriers of chromosomal rearrangements has been used in 18 cases, with a further six cases reported here. A total of 325 cumulus-oocyte complexes have been obtained, of which 294 were biopsied and 224 were diagnosed. A total of 52 embryos was transferred, 19 of which implanted and 17 produced full-term pregnancies.     

Reproductive risk in mating between two translocation carriers: Case report and review of the literature

We conducted a study to determine the reproductive risk in a couple who were translocation carriers. This couple, who carried balanced reciprocal translocations, experienced habitual abortions. The wife had a karyotype of 46,XX,t(7;13)(p15.3;q12.3) and the husband of 46,XY,t(1;7)(p11.1;p11.1). Chromosome study of their fourth abortus demonstrated a chimera consisting of two cell lines with a 46,XY and a 46,XX,t(1;7)(p11.1;p11.1)pat, ?13, +der(7)t(7;13)(15.3;q12.3)mat, karyotype. A review of the literature indicates that the risk of having unbalanced live offspring or of spontaneous abortion/stillborns is similar in couples in whom both were translocation carriers and in couples in whom one individual was a translocation carrier. The apparent lack of increased reproductive failure may result from the selective disadvantage of aneusomic gametes at fertilization or very early spontaneous abortions of unbalanced conceptuses. © 1993 Wiley-Liss, Inc.     

Preimplantation genetic diagnosis for a known cryptic translocation: follow-up clinical report and implication of segregation products

This report describes preimplantation genetic diagnosis (PGD) of a couple with a known paternally-derived balanced cryptic translocation 46,XY.ish t(2q;17q)(210E14-,B37c1+;B37c1-,210E14+) in embryos from a couple who previously had a child with severe mental retardation and was previously described in this journal [Bacino et al., 2000]. This child inherited the unbalanced product of translocation from her father: 46,XX.ish der(2)t(2q;17q)pat(210E14-,B37c1+). The couple desired a normal offspring and sought PGD to avoid clinical pregnancy termination. They were treated three times with in vitro fertilization followed by PGD. Two sequential FISH hybridizations were performed. In the first hybridization, telomeric probes to 2q and 17q and a chromosome 17 centromere probe were employed. The second hybridization screened for maternal age-related aneuploidy (X,Y,13,18,21). Of the 18 informative embryos, only 4 (22%) were normal. The remaining 12 (67%) were abnormal; most with unbalanced products (10/12) from the paternally-derived rearrangement. The most frequent mode of segregation observed for this cryptic translocation was adjacent-1 (7/18, 39%). This suggests cryptic translocations are amenable to PGD and, as are traditional translocations, demonstrate higher frequencies of unbalanced segregants than the empiric risk of 10-15% observed at amniocentesis or chorionic villus sampling. Thus, cryptic translocations presumably behave like overt translocations, in that PGD must be performed on a relatively large number of embryos to assure even 2-3 transferable embryos.     

Mental retardation with 45 chromosomes 45,XX,--5,--14,+der(5) t(5,14)(p15;q13) mat due to familial balanced reciprocal translocation.

A girl with severe mental retardation and odd facies and some features of the cri-duchat syndrome was found to have only 45 chromosomes. Her karyotype was 45,XX, -5, -14,+der(5) t(5,14)(p15;q13) mat. Her mother and her two sisters were found to be balanced reciprocal translocation carriers having 46 chromosomes, one of which was a very small (14pter leads to 14q13::5p15leads to 5pter) that was missing in the proposita.