染色体易位的植入前遗传学诊断

目的 观察染色体平衡易位和罗伯逊(罗氏)易位基因携带者夫妇进行植入前遗传学诊断(PGD)后的胚胎染色体遗传特征和胚胎着床、妊娠情况,探讨PGD在染色体易位基因携带者夫妇实现正常生育中的意义.方法 用荧光原位杂交(FISH)技术对36对夫妇的胚胎进行PGD,其中14例为染色体平衡易位(平衡易位组),22例为染色体罗氏易位(罗氏易位组),并对诊断结果和胚胎着床、妊娠情况进行分析.结果 36例患者共活检胚胎253个,成功诊断胚胎225个,成功率为88.9%(225/253),获得可供移植的正常或平衡的胚胎共58个.平衡易位组和罗氏易位组PGD后胚胎着床率分别为36%(5/14)和14%(6/44),临床妊娠率分别为4/9和26%(5/19).结论 PGD可有效诊断胚胎染色体平衡易位和罗氏易位,避免反复流产和不必要的非意愿性终止妊娠,并获得理想的胚胎着床率和临床妊娠率.     

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。     

植入前遗传学诊断四例临床分析

目的 探讨对遗传病高危夫妇采用单细胞荧光原位杂交（FISH）进行胚胎植入前遗传学诊断（PGD）的临床价值。方法 对曾生育过遗传病患儿的4对夫妇通过超排卵获得卵子,体外受精,体外培养至6-8细胞胚胎,每个胚胎取1-2个细胞,采用FISH进行遗传学分析。筛选无遗传病发病风险的胚胎移植入子宫。结果 4例患者共进行4个治疗周期,获得可供活检的胚胎12个,活检细胞20个,固定后有核细胞17个,FISH后除2个细胞无杂交信号外,其余杂交信号清楚,结果明确,活检后的12个胚胎继续发育,结合遗传学诊断,8个胚胎可供移植,其中1例妊娠,于2001年9月14日足月剖宫产分娩一女婴,发育正常,体重4270g,出生后染色体检查为正常女性核型。结论 对遗传病高危夫妇采用FISH技术进行PGD具有临床应用价值。     

短串联重复序列在染色体罗氏易位植入前遗传学诊断的应用

目的:采用多重置换扩增(MDA)结合短串联重复序列(STR)建立一种基于PCR技术诊断染色体罗氏易位的植入前遗传学诊断(PGD)方法。方法:选择位于易位染色体上的STR位点,对家系采用荧光PCR进行分析,选择有多态性的位点,再采用MDA对单细胞进行全基因组扩增,根据家系分析的结果,对具有多态性的STR位点进行分析诊断。结果:对3个家系进行了4个取卵周期(3个PGD周期),每个家系分别采用7~15个具有多态性的STR位点进行分析,共对24个胚胎进行诊断。PGD的诊断效率为95.8%(23/24),平衡胚胎占52.2%(12/23),异常胚胎占47.8%(11/23),共移植了6个胚胎,获得2例临床妊娠,临床妊娠率为66.7%(2/3),出生了2个健康婴儿,染色体核型均正常。结论:采用依赖于STR的PCR分析法可以用于染色体罗氏易位的PGD。     

拷贝数变异测序在染色体易位夫妇植入前遗传学诊断中的临床应用

目的探讨拷贝数变异测序(copy number variation sequencing, CNV-seq)用于染色体易位夫妇胚胎植入前诊断的应用价值。 方法回顾性分析2017年1月至2018年12月,在广东省妇幼保健院生殖健康与不孕症科进行植入前遗传学诊断(preimplantation genetic diagnosis,PGD)的211对染色体易位夫妇患者的临床病例。使用CNV-seq对胚胎染色体进行检测,并对患者一般信息和PGD结果进行分析。 结果1210个胚胎中,被检出837个(79.2%)胚胎存在染色体异常,373个(30.8%)胚胎为整倍体。在241个PGD周期中,68个(27.6%)周期所有胚胎均存在染色体异常,178个(72.4%)周期至少含有一个整倍体胚胎。在176个移植周期中,130个(73.9%)确定临床妊娠,已出生46个健康婴儿,12例发生早期流产。 结论CNV-seq可准确地区分胚胎染色体是否存在异常,避免因胚胎含有染色体异常而被移植,是一种可靠而准确的PGD技术。     

原核形态、卵裂胚发育与染色体异常关系的研究

目的 探讨原核形态评分(原核评分)、卵裂胚发育与染色体异常的关系.方法 2005年9月至2006年3月在广州医学院第三附属医院对需要行胚胎植入前诊断(PGD)的7例患者,于受精后第3天,应用荧光原位杂交(FISH)方法 .选用13、18、21、X、Y 5种染色体探针筛查这些病人种植前胚胎的非整倍体,同时分析原核评分、胚胎发育与染色体异常的关系.结果 7例患者各行PGD 1个周期,共获得73个胚胎;根据原核评分将所有胚胎分成A、B两组.原核评分≥14分的胚胎为A组,共49个;＜14分者为B组,24个;在A组中,优质胚胎比例为46.9%(23/49).显著高于B组的20.8%(5/24),P＜0.05;对52个胚胎进行活检,51个胚胎有FISH诊断结果 ;在有FISH诊断结果 、原核评分≥14分的胚胎中,正常胚胎的比例为51.4%(19/37),显著高于评分＜14分胚胎的21.4%(3/14),P＜0.05;优质胚胎中,正常胚胎的比例为55.6%(15/27),显著高于非优质胚胎的29.2%(7/24),P＜0.05.原核评分≥14分的优质胚胎中,染色体正常胚胎的比例为70.0%(14/20),高于优质胚胎的55.6%,但差异无统计学意义(P＞0.05).结论 评分高的原核有更大的机会发育成优质胚胎和染色体正常的胚胎,优质胚胎中染色体正常胚胎的比例高于非优质胚胎.     

PGD周期中机械法和激光法活检对胚胎发育及妊娠结局的影响

目的:比较机械法和激光法进行卵裂球和极体活检对胚胎发育及着床前遗传学诊断(preimplantation genetic diagnosis,PGD)周期妊娠结局的影响。方法:本研究包括20对夫妇的21个PGD周期,其中18个周期分别用机械法或激光法于受精后第3天进行卵裂球活检,并用荧光原位杂交(fluorescence in situ hybridization,FISH)分析检出的卵裂球,于受精后第5或第6天移植信号正常的胚胎;2个周期分别用机械法和激光法在取卵后行极体活检,后行胞浆内单精子注射(introcytoplasmic sperm injection,ICSI)。同时将活检取出的极体进行FISH分析,于取卵后第3天移植经FISH检查正常的卵发育而来的胚胎。另外一个周期先用激光法实行了极体活检,由于FISH检查均无信号,后又用激光法对胚胎行卵裂球活检。结果:共活检胚胎145枚,其中109枚用机械法,36枚用激光法。活检后胚胎的继续发育率分别为72.48%和83.33%,囊胚形成率为33.94%和44.44%,临床妊娠率为38.46%和16.67%,着床率为21.43%和8.33%,两种方法无显著差异。对27枚卵行极体活检,其中12枚用机械法,15枚用激光法。活检后2PN受精率分别为58.33%和46.66%,继续发育率为66.67%和60.00%,亦无显著差异。对活检出的极体进行FISH分析,用机械法活检的极体信号阳性率为90.00%,显著高于激光法的28.57%。结论:用机械法和激光法行极体或卵裂球活检对胚胎发育的影响差异无统计学意义。但使用机械法活检卵裂球能获得较高的临床妊娠率和着床率。极体活检时能获得较高的受精率和继续发育率,故推荐使用机械法进行活检。     

荧光原位杂交技术用于1;12染色体平衡易位携带者胚胎植入前遗传诊断一例分析

染色体异常已成为不孕、不育的主要原因之一。染色体平衡易位患者因其具有完整的遗传物质，故往往表型正常，但在生殖细胞发生减数分裂时可产生多种不平衡配子而导致反复自然流产、死胎、死产、新生儿死亡、畸形或智力低下的后代。胚胎植入前遗传诊断(preimplantatation genetic diagnosis，PGD)通过选择正常的胚胎植入，从而降低流产风险，获得健康的后代。本例应用荧光原位杂交(fluorescence in situ hybridization，FISH)技术，成功对1例1；12染色体平衡易位的患者进行PGD。现报道如下。     

植入前遗传学诊断100个周期临床分析

目的 探讨染色体易位对早期胚胎发育的影响,以及植入前遗传学诊断(PGD)技术的诊断效率和可行性.方法 回顾性分析PGD中23个罗伯逊(罗氏)易位周期、19个平衡易位周期(染色体易位组),以及58个α地中海贫血周期(地贫组)共100个周期中的胚胎发育情况、PGD的诊断效率以及临床结局.结果 染色体易位组中有354个胚胎进行PGD,321(90.7%)个胚胎有荧光原位杂交(FISH)结果,其中罗氏易位者中正常和(或)平衡易位胚胎占38.3%(64/167),显著高于平衡易位者的20.8%(32/154).地贫组有537个胚胎进行PGD,单个卵裂球的扩增效率为82.5%(443/537),诊断出正常纯合子140个、杂合子112个、异常纯合子155个、另36个诊断结果不明确,总体诊断效率为75.8%(407/537).染色体易位组中,取卵后第3天卵裂球数≥7的胚胎中,正常和(或)平衡易位发生率(34.4%,77/224)显著高于卵裂球数＜7的胚胎(19.6%,19/97),在取卵后第4天,正常和(或)平衡易位胚胎的细胞融合率为59.4%(57/96),显著高于染色体不平衡胚胎的34.2%(77/225).染色体易位组共在37个周期移植了75个胚胎,获得10例临床妊娠,临床妊娠率27.0%(10/37).地贫组共在58个周期移植了170个胚胎,获得25例临床妊娠,临床妊娠率为43.1%(25/58).结论 PGD技术可有效为染色体易位和地中海贫血基因携带者提供优生选择.染色体易位可能对着床前胚胎的发育有一定的影响.

Abstract：

Objective To investigate influence of chromosomal translocations on early embryo development and to evaluate the efficacy and feasibility of preimplantation genetic diagnosis (PGD)techniques through clinical analysis on PGD cycles. Methods Embryo development, efficacy of PGD and clinical outcome of 100 cycles were studied retrospectively, including 23 cycles with Robertsonian translocations, 19 cycles with reciprocal translocations, and 58 cycles for α-Thalassaemia. Results Among 354 embryos biopsied by PGD for translocations, 321 (90. 7% ) presented fluorescence in situ hybridization (FISH) results. The rate of normal/balanced embryos in the Robertsonian translocation was 38. 3% (64/167),which was significantly higher than 20. 8% (32/154) in the reciprocal translocation group. Amplification was achieved in 443 blastomeres from 537 embryos in Thalassaemia group, which given to an amplification efficiency rate of 82. 5% ( 443/537 ). Totally, 140 normal homozygous, 112 heterozygotes and 155 affected homozygous embryos were identified, while 36 embryos had uncertain result. The successful diagnostic rate was 75.8% (407/537). After 3 days in the translocation groups, the rate of normal and/or balanced translocations in biopsed embryos with ≥7 cells was 34. 4% (77/224), which was significantly higher than 19. 6% ( 19/97 ) of biopsed embryos with ＜ 7 cells. After 4 days, the compaction rate in normal/balanced embryos was 59.4% ( 57/96 ), which was significantly higher than 34. 2% ( 77/225 ) in imbalanced embryos significantly. Seventy-five embryos transferred in 37 cycles with translocations group led to clinical pregnancy rate of 27.0% (10/37), and 170 embryos transferred in 58 cycles with Thalassaemia got a clinical pregnancy rate of 43. 1% ( 25/58 ) . Conclusions PGD can provide management efficiently for both chromosome translocations and Thalassaemia. Translocations might have slightly negative impact on embryo development before implantation.     

胚胎植入前遗传学诊断技术应用的安全性

<正>胚胎植入前遗传学诊断(preimplantation genetic diagnosis,PGD)技术是指在体外受精过程中,对具有遗传风险患者的胚胎进行植入前活检和遗传学分析,以选择无该检测遗传性疾病的胚胎植入宫腔,从而获得正常胎儿的诊断方法。这种方法是产前诊断的延伸,可有效地防止遗传性疾病患儿的出生及探讨出生缺陷发病机制。PGD技术的应用从单基因疾病、染色体结构和     

Multiple micromanipulations for preimplantation genetic diagnosis do not affect embryo development to the blastocyst stage

To determine the impact of multiple micromanipulation procedures for preimplantation genetic diagnosis (PGD) on embryo development, a retrospective analysis was performed of 9,925 embryos (862 PGD cycles), which were compared with 28,126 nonbiopsied embryos (2,751 consecutive intracytoplasmic sperm injection [ICSI] cycles) from the same time period. Because fertilization rates, the proportion of embryos with > or = 6 cells on day 3, and blastocyst rates were similar in the PGD and control groups, we conclude that multiple micromanipulations on oocytes and embryos can be performed safely for PGD.     

Non-informative results and monosomies in PGD: the importance of a third round of re-hybridization

The incidence of non-informative results after fluorescence in-situ hybridization (FISH) was analysed in preimplantation genetic diagnosis (PGD). FISH was performed on seven chromosomes (13, 16, 18, 21, 22, X, and Y) in two rounds of hybridization (one biopsied blastomere per day 3 embryo). A third round with telomeric probes was performed in order to analyse the chromosome(s) in question. A total of 702 embryos out of a total of 719 embryos from 95 cycles were analysed. The remaining 17 embryos were anucleated and/or had poor quality and could not be diagnosed. After FISH analysis, 52.7% of blastomeres were found to be abnormal, 27.1% euploid, and 20.2% had non-informative results. Abnormalities considered as non-informative included ‘monosomy in question’ (46.5%), ‘trisomy in question’ (40.2%), compound aneuploidy (8.5%), and ‘no result’ (4.9%) for a tested chromosome. Following re-hybridization with telomeric probes, euploidy was found in 42.4% of ‘monosomies in question,’ in 82.4% of ‘trisomies in question,’ in 16.7% of compound aneuploidies, and in 71.4% of ‘no results’ for a tested chromosome. Only 4.2% of non-informative results could not be rescued. This study clearly demonstrates the importance of re-hybridizing non-informative results and monosomies using a third round of hybridization with telomeric probes for chromosome(s) in question.     

Clinical pregnancy following blastomere biopsy and PGD for a reciprocal translocation carrier: analysis of meiotic outcomes and embryo quality in two IVF cycles

A couple were referred for preimplantation genetic diagnosis (PGD) following diagnosis of a reciprocal translocation in the female partner: 46,XX,t(14;22)(q11.2;q13.3). PGD was carried out using fluorescence in situ hybridization (FISH) with probes specific for the translocated and centric segments of chromosome 22. An initial cycle was unsuccessful, producing 11 embryos for biopsy, only one of which, when followed up on day 4, yielded more than 10 nuclei (median 7.5, n=10). In addition, five of the embryos showed mosaic or chaotic chromosome constitutions; some of these embryos had fragmented or multilobed abnormal nuclei, hindering interpretation of the FISH signals. The single embryo transferred did not result in a pregnancy. A second cycle, using a revised protocol, produced 10 embryos, three of which were transferred, resulting in an ongoing singleton pregnancy. All the remaining embryos yielded 12 to 23 nuclei by day 4 (median 17, n=7). Apart from some tetraploid nuclei, only one embryo showed mosaicism. The significance of the changes in protocol leading to the successful outcome is discussed, and the pattern of meiotic segregation products is analysed and compared with other previous reports of reciprocal translocations.     

Paternal gonadal mosaicism detected in a couple with recurrent abortions undergoing PGD: FISH analysis of sperm nuclei proves valuable

Many couples are now seeking preimplantation genetic diagnosis (PGD) and fluorescence in-situ hybridization (FISH) as an alternative approach to avoid spontaneous abortion by ensuring transfer of presumed chromosomally normal embryos. This case report describes unexpected findings in a couple having three spontaneous abortions and two failed IVF cycles. In two IVF PGD cycles, four of 13 (30.8%) embryos (blastomeres) demonstrated duplication involving the Down syndrome critical region, detectable by a locus specific chromosome 21 probe. The same duplication was subsequently detected by FISH in 66 of 1002 (6.6%) sperm nuclei, demonstrating paternal gonadal mosaicism. Cytogenetic studies of peripheral blood revealed normal karyotypes in both the male and female partners. This identification of paternal germ cell or gonadal mosaicism suggests that analysis of sperm nuclei prior to undergoing IVF with PGD may be of value in patients with recurrent spontaneous abortions or multiple failed IVF.     

Efficacy and clinical outcome of preimplantation genetic diagnosis using FISH for couples of reciprocal and Robertsonian translocations: the Korean experience

OBJECTIVE: To evaluate the efficacy and clinical outcome of preimplantation genetic diagnosis (PGD) using fluorescence in situ hybridization (FISH) for couples with chromosomal translocations. METHODS: PGD using FISH was performed in 59 cycles of 43 couples with reciprocal translocations, and 11 cycles of 6 couples with Robertsonian translocations. The diagnostic and clinical data were reviewed in a series of 70 treatment cycles of 49 couples from January 2001 to June 2002 at Samsung Cheil Hospital, Korea. RESULTS: A total of 1408 oocytes were retrieved, and 938 (81.7%) out of 1148 matured oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Single blastomere biopsy and FISH analysis were successfully carried out in 99.3% (890/896) and 94.4% (840/890), respectively. Among 193 normal or balanced embryos, 169 embryos were transferred in 64 cycles (91.4% per started cycle). Twenty clinical pregnancies including two ectopic pregnancies and three spontaneous miscarriages (28.6% per started cycle, 31.3% per transfer cycle, 40.8% per couple) were established. Of the three spontaneous miscarriages, one was karyotyped as normal, one had an unbalanced arrangement and one was tetraploid. One case of preterm twin delivery occurred and 16 healthy babies were delivered in 12 single and 2 twin pregnancies. CONCLUSION: The clinical outcome was successful in 28.6% (14/49) of the treated couples with translocations after PGD. The spontaneous abortion rate was significantly reduced from 95.8% (69/72) to 16.7% (3/18) in these couples.     

荧光原位杂交技术在胚胎植入前性别诊断中的应用

目的 探讨荧光原位杂交技术在人光胚胎植入前性别诊断中的应用价值。方法对２例因友病基因携带者和２例Ｙ染色体异常的患者进行了５个周期的超排卵治疗,胚活检后取单个细胞进行固定,然后用荧光原位杂交技术检测胚胎的性别,最后选择女性胚胎移植入子宫腔。结果 ４例患者５个治疗周期共取卵１１０个,受精率为６８．２％,可供活检的胚胎５５个,活检成功率为８５．５％,活检后继续分裂率为６１．７％,活检细胞固定率为９７．     

Normal birth following PGD for reciprocal translocation after serial vitrification of oocytes from a poor responder: a case report

This case study reports the first successful birth outcome following preimplantation genetic diagnosis (PGD) for a chromosome translocation in embryos generated by serial vitrification of oocytes. A couple presented to the fertility clinic with 2 years of primary infertility. The woman was diagnosed with poor ovarian reserve and her partner was diagnosed with severe oligoteratozoospermia and the reciprocal translocation 46,XY,t(1;7)(p36.1;q11.23). Following counselling, the couple opted for serial vitrification of oocytes followed by PGD. A total of 31 oocytes were obtained in five egg collection cycles over a period of 12 months and 27 metaphase-II oocytes were vitrified. Nineteen of the 27 vitrified oocytes survived warming: 14 oocytes from the vitrified group and three oocytes from the fresh cycle were fertilized by intracytoplasmic sperm injection. Eleven embryos, including three from the fresh cycle, were biopsied on day 3 post insemination. Fluorescence in-situ hybridization was performed for the specific chromosomes involved in translocation. Only two embryos from the cryopreservation cycles were diagnosed as normal/balanced, one of which was transferred on day 5 post insemination. A normal healthy female infant was born at week 42 of gestation.     

Conversion and non-conversion approach to preimplantation diagnosis for chromosomal rearrangements in 475 cycles

Due to the limitations of preimplantation genetic diagnosis (PGD) for chromosomal rearrangements by interphase fluorescent in-situ hybridization (FISH) analysis, a method for obtaining chromosomes from single blastomeres was introduced by their fusion with enucleated or intact mouse zygotes, followed by FISH analysis of the resulting heterokaryons. Although this allowed a significant improvement in the accuracy of testing of both maternally and paternally derived translocations, it is still labour intensive and requires the availability of fertilized mouse oocytes, also creating ethical issues related to the formation of interspecies heterokaryons. This method was modified with a chemical conversion procedure that has now been clinically applied for the first time on 877 embryos from PGD cycles for chromosomal rearrangements and has become the method of choice for performing PGD for structural rearrangements. This is presented within the context of overall experience of 475 PGD cycles for translocations with pre-selection and transfer of balanced or normal embryos in 342 (72%) of these cycles, which resulted in 131 clinical pregnancies (38%), with healthy deliveries of 113 unaffected children. The spontaneous abortion rate in these cycles was as low as 17%, which confirms an almost five-fold reduction of spontaneous abortion rate following PGD for chromosomal rearrangements.