应用荧光原位杂交技术进行人类早期胚胎性染色体嵌合型的初步研究

目的　应用双色荧光原位杂交技术对人类早期胚胎性染色体嵌合型进行初步分析,并进行单细胞种植前性别检测。方法　对体外受精与胚胎移植治疗周期中胚胎质量差,不适于胚胎移植和冷冻的１８ 个正常受精胚胎共７３ 个细胞和３ 个多精受精胚胎,进行双色荧光原位杂交。结果　有两个细胞以上的１４ 个正常受精胚胎中９ 个胚胎有不同程度的性染色体嵌合,其中男性胚胎４１ 个细胞中ＸＯ有５ 个,占１２ ％ ;女性胚胎２８ 个细胞中ＸＯ有４ 个,占１４％ 。结论　应用荧光原位杂交技术可准确地进行单细胞种植前性别诊断,性染色体嵌合型不会影响胚胎的性别诊断。     

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。     

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。     

应用荧光原位技术进行人类早期胚胎性染色体嵌合型的初步研究

应用双色荧光原位杂交技术对人类早期胚胎性染色体嵌合型进行初步分析,并进行单细胞种植前性别检测。方法对外受精与胚胎移植治疗周期中胚胎质量差,不适于胎胎移植植和冷冻的１８个正常受精胚胎共７３个细胞和３个多精受精胎胚胎,进行双色荧光原位杂交。     

应用荧光原位杂交技术进行植入前胚胎染色体诊断的价值

目的 初步探讨应用荧光原位杂交(FISH)技术进行植入前胚胎染色体诊断的价值。方法 对10对不孕夫妇进行植入前遗传学诊断(PGD)周期的超促排卵和卵母细胞浆内单精子注射,于受精后第3天进行胚胎活检及FISH分析,第4天选择染色体组成正常或平衡的胚胎进行移植。结果 10个PGD周期共获卵158个,对其中54个胚胎进行活检,51个胚胎获得明确诊断,诊断率为94％(51／54)。对染色体组成正常或平衡的24个胚胎进行官腔内移植,共4例获得妊娠,其中3例已足月分娩健康婴儿,1例为异位妊娠。结论 应用FISH技术进行植人前胚胎染色体诊断,是预防流产和染色体异常患儿出生的有效手段。     

植入前遗传学诊断中四种卵裂球固定方法的比较

目的:寻求卵裂球固定的最适方法。方法:IVF/ICSI受精治疗周期中不宜移植的24枚胚胎,采用OCTAX-LaserShot胚胎透明带激光打孔,用吸拉法共活检出154个完整卵裂球,分别用4种不同的固定方法(KCl组;Tween-20/HCl组;甲醇/冰醋酸组;Tween-20/HCl+甲醇/冰醋酸组)固定,固定后使用X、Ya-卫星DNA探针进行荧光原位杂交,比较其固定率和出信号率。结果:透明带激光打孔辅助下卵裂球吸拉法活检成功率为96.25%;4种固定方法固定率和出信号率分别为98.4%和95.2%,60.0%和72.2%、63%和73.7%、70%和76.2%。结论:透明带激光打孔辅助卵裂球吸拉法活检有效;第4种卵裂球固定方法能减少卵裂球丢失,简化操作程序,因此两者在植入前遗传学诊断中值得推广。     

植入前胚胎染色体嵌合型的初步分析

目的　应用荧光原位杂交 (FISH)技术对人类早期胚胎染色体的嵌合型进行初步分析 ,并探讨胚胎染色体嵌合型对常规体外受精 胚胎移植 (IVF ET)成功率以及植入前诊断的影响。方法　对IVF ET治疗周期中胚胎质量差、不适于胚胎移植和冷冻保存的正常受精胚胎以及在植入前胚胎性别诊断中诊断为男性胚胎、不能移植的 38个胚胎进行FISH。结果　 38个胚胎共有 2 93个核 ,杂交率为 96 .2 %。其中正常胚胎 12个 ,占 31.6 % ,二倍体胚胎 (正常胚胎 二倍体嵌合型胚胎 ) 2 8个 ,占73.7% ,嵌合型胚胎 (二倍体嵌合型胚胎 异常嵌合型胚胎 ) 19个 ,占 5 0 .0 %。在≤ 4细胞期胚胎中 ,嵌合型胚胎占 18.2 % ,但在 5～ 8细胞期和≥ 9细胞期胚胎中 ,嵌合型胚胎的比例分别增至 6 8.4%和5 0 .0 %。结论　随着卵裂次数的增加 ,嵌合型胚胎所占的比例也增加 ,但嵌合型胚胎中嵌合细胞的比例却随之而降低。胚胎染色体的嵌合型可能是影响常规IVF ET成功率的重要因素。应用FISH技术可准确地进行单细胞植入前性别诊断 ,但在进行植入前单基因诊断时 ,最好能同时活检 2个细胞 ,以增加准确度。     

玻璃化冷冻法冻融经植入前遗传学诊断后囊胚的应用初探

目的:探讨玻璃化冷冻技术冻融经卵裂球活检后囊胚的可行性。方法:将活检后剩余的可移植囊胚用玻璃化冷冻保存,并在冷冻前人工皱缩囊胚腔,在需要移植时予以解冻囊胚进行移植。结果:24例共进行24个活检周期,活检了159个胚胎,活检后胚胎囊胚形成率60.38%(96/159)。有17个周期共移植26枚新鲜可用囊胚,成功种植13个(50.0%),11例获得临床妊娠(64.71%),7个周期因无可移植胚胎或卵巢过度刺激等因素而取消移植。10例患者(10个周期)有30个可移植囊胚进行了玻璃化冷冻保存,其中6例患者因未成功生育要求解冻其囊胚进行移植。共解冻8枚囊胚,全部存活并移植,5例获单胎妊娠;2例已分娩正常婴儿,3例继续妊娠中。结论:玻璃化冷冻技术结合人工皱缩囊胚腔能冷冻保存经卵裂球活检后的囊胚。     

小鼠及人胚胎分割技术的研究

植入前小鼠胚胎（ｐｒｅｉｍｐｌａｎｔａｔｉｏｎ ｅｍｂｒｙｏ）的透明带经软化后,用固定在显微操作臂上的微玻璃针行胚胎分割,２－、４－、８－细胞胚胎及桑椹胚分割成功率分别为９０．６％、８５．０％、７３．６％、７１．４％。所获半胚体外培养后,胚泡发育率分别为６２．１％、８０．１％、８３．０％、８９．２％。随胚胎细胞数目增多,分割成功率降低,而发育率则提高。将８－细胞胚组及桑椹胚组的半胚移植到假孕母鼠后,幼仔出生率分别为１４．６％、１５．１％。同法分割４－细胞人胚胎２个,获半胚４个,分割成功率为１００％。     

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.     

Clinical experience with preimplantation diagnosis of sex by dual fluorescent in situ hybridization

Purpose Our purpose was to assess the clinical application of dual fluorescent in situhybridization (FISH) for the diagnosis of sex in the human preimplantation embryo. Results Over a 2-year period, 18 couples at risk of transmitting X-linked recessive disorders underwent preimplantation diagnosis of embryo sex by dual FISH with X and Y chromosome-specific DNA probes. A total of 27 in vitro fertilization (IVF) treatment cycles led to nine pregnancies; 7 reached the stage of clinical recognition, of which 2 spontaneously aborted. There were five live births, three singleton and two twin: none in disagreement with the diagnosed sex. The diagnosis was corroborated in 51 of the 74 nontransferred embryos. The efficiency of the procedure improved throughout the four treatment cycles. This was reflected in the increased proportion of double embryo transfers (from 50% in series 1 and 2 to 100% in series 3 and 4), with a consequent improvement in pregnancy rate (from 28 to 71% per embryo transfer). The excess of male embryos (male∶female, 60∶40 overall) and the high proportion of biopsied embryos with abnormal numbers of X and Y chromosome signals (14.5%) effectively reduced the number of normal female embryos available for transfer. Conclusion Dual FISH is an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction, (PCR), makes FISH the preferred technique.     

引物延伸预扩增法应用于β-地中海贫血患者胚胎植入前遗传学诊断--附一例报告

目的 探讨对β-地中海贫血进行胚胎植入前遗传学诊断(PGD)的方法。方法 两对夫妇双方均分别为密码子41-42(-TCTT)及插入序列(IVS)-Ⅱ654(c→T)突变杂合子,在本中心进行超排卵和体外受精-胚胎移植治疗,胚胎活检后应用全基因组扩增技术及反向点杂交进行胚胎PGD,根据诊断结果选择健康的胚胎移植入子宫。结果 2例患者共获卵35个,受精率为87％,共活检胚胎16个,获卵裂球25个,单卵裂球总扩增效率为84％,等位基因脱扣率为15％。2例患者共移植5个胚胎,1例获得妊娠,已分娩健康双胎。结论 应用引物延伸预扩增技术可对β-地中海贫血进行PGD,达到优生的目的。     

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.     

Sex determination in single mouse blastomeres by polymerase chain reaction

In sex determination of mammalian preimplantation embryos, viability of biopsied embryos and accuracy of sexing are together important. In consideration of this point, single blastomeres were mechanically isolated from mouse embryos using a micromanipulator and then sexed by polymerase chain reaction (PCR) using mouse Y chromosome-specific primers. All of 260 embryos biopsied at the four-cell and morula stage survived. Developmental rate of the embryos to normal blastocysts was 93 and 94%, respectively. Sex determination of single blastomeres was performed by amplification of a mouse Y chromosome-specific DNA sequence using PCR technique. The ratio of male to female embryos was 53 and 47%, respectively. The sex-determined embryos were transferred to the uteri of pseudopregnant recipients to test the consistency of the assay system. The sex of 27 of 29 mice developed from male and female embryos agreed with the predicted sex. The method developed for embryo biopsy and sexing could be used for diagnosis of defective genes at the stage of the preimplantation embryos of human and other domestic animals.