荧光原位杂交技术在辅助生殖中的作用
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摘要
自1990年世界上诞生第一例胚胎植入前遗传学诊断试管婴儿(preimplantation genetic diagnosis,PGD)至今,已有近20年的历史。胚胎植入前遗传学诊断是对配子或胚胎进行遗传学分析和诊断,去除有遗传缺陷的配子或胚胎,选择正常的胚胎植入子宫的一种诊断方法。PGD将遗传学与辅助生殖技术相结合,克服了常规产前诊断技术因终止异常妊娠带给患者身心痛苦的缺陷,因此在辅助生殖技术中越来越受到重视。同时体外受精—胚胎移植(in vitrofertilization-embryo transfer,IVF-ET)技术的发展已日益成熟,也给PGD发展奠定了良好的基础。虽然新的植入前胚胎单细胞诊断方法不断出现如比较基因组杂交(comparative genomic hybridization,CGH)、array-CGH、Microarray等,但在现阶段仍然无法替代PGD中两个基本方法;单细胞聚合酶链式反应(polymerase chain reaction,PCR)和荧光原位杂交(fluorescence in situ hybridization,FISH)。由于单细胞PCR存在三个缺点:PCR扩增效率、污染及等位基因脱扣(Allele drop-out,ADO),而FISH不仅可以进行胚胎植入前性别诊断,更在研究胚胎嵌合现象、染色体数目及结构异常方面具有PCR所没有的优势。因此FISH是PGD中一种无法替代的、重要的检测方法。
在FISH-PGD中仍然存在难点:1、单细胞固定:在单细胞FISH诊断中,诊断的准确性与单个卵裂球的固定密切相关。理想的固定应该是彻底的去除胞浆蛋白,保留染色体DNA的完整性且简单易学。目前常用的单细胞固定方法有三种:甲醇/冰醋酸法、Tween-20/HCl法、Tween-20/HCl+甲醇/冰醋酸法。三种固定方法在各个生殖中心分别有使用。但三种方法在实际临床应用中仍然可能出现细胞核暴露不良、胞浆残留多、甚至细胞核丢失导致无法准确诊断等,因此认为固定方法仍需进一步改进和完善。2、植入前胚胎有高比例的非整倍体,这可能是FISH-PGD临床误诊、成功率低的重要原因之一。对于高生育风险的平衡易位携带者夫妇其胚胎非整倍体风险文献报道不一。同时在FISH诊断中信号在核中的位置的临床意义及其对诊断及胚胎发育的影响近年来成为研究热点,但仍然尚无明确定论。(3)临床上染色体异常PGD遗传咨询及预后估计如移植胚胎、妊娠结局等的研究逐渐成为热点,但目前进展缓慢。
本文就上述热点和难点问题从以下五个方面进行了研究:第一部分对Tween-20/HCl+甲醇/冰醋酸法进行了改良,并探讨了其对FISH信号及单细胞诊断的影响。第二部分探讨了平衡易位携带者胚胎中非整倍体的变化及卵裂球细胞核中染色体信号分布特点。第三部分探讨了精子FISH结果在男性染色体异常PGD及遗传咨询中的作用。第四部分将改良的卵裂球固定方法用于临床,进行了16个PGD周期的临床诊断,同时回顾性分析了染色体平衡易位对控制性超排卵过程中卵巢反应性的影响等。第五部分应用国产化探针对未培养的羊水细胞进行了FISH分析。
第一部分单细胞固定方法的改良及其对细胞核面积及荧光原位杂交信号的影响
研究对象:用于固定卵裂球细胞均来自不宜移植的人类2-10细胞阶段的胚胎,所有胚胎均经过病人知情同意后用于实验。
研究方法:获得的卵裂球使用3种不同的固定方法(甲醇/冰醋酸法;Tween-20/HCl+甲醇/冰醋酸法;改良的Tween-20/HCl+甲醇/冰醋酸法)固定。固定后进行FISH,同时利用荧光分析软件Imstar计算细胞核面积、比较各组的固定率、信号重叠率、信号分裂率。
结果:
1、细胞固定率上甲醇/冰醋酸法为86.73%,明显低于改良前后Tween-20/HCl+甲醇/冰醋酸法。
2、细胞面积上甲醇/冰醋酸法为55.3 um~3,明显高于改良前后Tween-20/HCl+甲醇/冰醋酸法。
3、三组间信号重叠率及信号分裂率无显著性差异。
结论:对Tween-20/HCl+甲醇/冰醋酸法进行了改良,同时对其及另外的卵裂球固定方法进行比较,改良的Tween-20/HCl+甲醇/冰醋酸法易于掌握、程序简单、固定细胞时间短、不增加误诊率等优点可在临床广泛推广使用。
第二部分平衡易位携带者胚胎非整倍体筛查及染色体位置与胚胎非整倍体相关性的研究
研究对象:2006.1～2009.3我中心针对平衡易位携带者进行11个PGD周期,包括罗伯逊易位9个周期,相互易位2个周期。
研究方法:
1、胚胎植入前遗传学诊断程序按我中心常规进行。
2、对固定好、信号明显的细胞核再次选择5种探针(LSI13/21及CEP18/X/Y),进行胚胎非整倍体检测。
3、采用Imstar荧光分析软件确定各个信号相对位置。信号相对位置(relativedistance)RD=SC/R。
结果:
1、11个针对平衡易位的PGD,选择细胞核固定良好、无多核、无核碎片的139个细胞核进行了PGS。选择信号完整的130个细胞核进行了分析。共分析了937个荧光信号,其中整倍体细胞核共38个,304个信号,余均为非整倍体细胞信号。
2、分析了来源于平衡胚胎的42个卵裂球和来源于非平衡胚胎的88个卵裂球的PGS结果。在平衡胚胎来源的细胞核中,整倍体率为71.4%(30/42),而非平衡胚胎来源的细胞核其整倍体率为9.1%(8/88),统计学上差别有显著性(x~2=53.4,P＜0.05)。其中非平衡胚胎来源的卵裂球主要表现为复杂的非整倍体(36.4%)。虽然染色体整倍体率在不同级别胚胎中分布无显著性差异,但优质胚胎(Ⅰ级+Ⅱ级)中非整倍体率仍占有60%(56/92)。
3、对38个整倍体卵裂球进行了分析,其中包括男性核型21个,女性核型17个。共分析了304个荧光原位杂交信号,在信号分布上未发现有显著差异。但信号13、21、X倾向于分布于细胞中央(RD＜0.5),而染色体18、Y则倾向分布于外周(RD＞0.5)。
4、对92个非整倍体卵裂球进行了分析,共分析了633个荧光原位杂交信号。非整倍体卵裂球中信号分布存在统计学差异。信号13、18、21、Y倾向分布于外周(RD＞0.5),尤其是染色体18号。但信号X的分布无统计学差异。
5、在整倍体细胞核中,染色体信号分布位置无统计学差异,但主要集中在核周1/4-3/4,而在非整倍体细胞核中,染色体信号分布主要在核周边。二者在信号分布上有统计学差异(p=0.00)。
结论:
1、平衡胚胎来源的卵裂球整倍体率为71.4%,明显高于非平衡胚胎来源的卵裂球整倍体率(9.1%)。
2、不同级别胚胎的卵裂球其整倍体率无显著差异,即使移植了优质胚胎仍然可能是非整倍体胚胎,最终导致妊娠失败。本研究显示优质胚胎非整倍体率仍占有60%,高于文献报道。因此对于针对平衡易位的胚胎植入前遗传学诊断建议同时进行胚胎的非整倍体筛查。
3、本研究显示整倍体卵裂球中染色体信号分布无显著差异。
4、非整倍体细胞核中,染色体信号分布主要在核周边。信号13、18、21、Y倾向分布于外周,尤其是染色体18号,信号X的分布无统计学差异。
第三部分精子荧光原位杂交在男性染色体异常PGD中的作用
研究对象:
2006.7～2008.8,9对不孕夫妇因男方染色体异常进行了精子荧光原位杂交分析和胚胎植入前遗传学诊断,包括罗氏易位7例,相互易位1例,克氏综合征1例。所有病例女方染色体均正常。患者禁欲3-5天手淫法取得的精液标本用于试验。所有患者均知情同意后行精子FISH和PGD。
研究方法:
1、胚胎植入前遗传学诊断程序按我中心常规进行。
2、精子预处理:精液预处理后用甲醇/冰醋酸(3:1)固定液调制精子浓度至20×10~6/ml,后滴片,使精子均匀分布于玻片并风干。后将风干玻片在1M NaOH溶液中浸泡5分钟,使精子核去凝集,后立即进行FISH分析。
3、FISH方法同第一部分。
结果:
1、9例染色体异常携带者男性进行了精子FISH分析,7例罗伯逊易位携带者中,正常或平衡精子比例为85.7%(6045/7053),1例相互易位携带者中正常或平衡精子比例为30.4%,克氏综合症正常精子比例为68.8%。
2、7例罗伯逊易位携带者中,正常或平衡胚胎比例为28.97%(31/107),1例相互易位携带者中正常或平衡胚胎比例为6.25%,1例克氏综合症正常胚胎比例为33.3%。
3、PGD中正常/平衡胚胎的比例与正常精子比例呈正相关(R=0.75,P=0.02＜0.05)。
结论:
1、罗伯逊易位携带者平衡精子平均比例为85.7%,克氏综合症患者的正常或平衡精子比例为68.8%,均高于相互易位携带者。
2、染色体正常精子比例与PGD中正常胚胎比例呈正相关,因此在PGD前生殖遗传咨询中,建议对携带者进行精子荧光原位杂交分析,了解平衡配子所占的比例,为PGD的应用提供客观依据。
第四部分胚胎植入前遗传学诊断16个周期的临床应用分析
研究对象:2006.1～2009.3年,我中心共进行胚胎植入前遗传学诊断16个周期,包括针对染色体罗伯逊易位9个周期,针对染色体相互易位2个周期,针对男性克氏综合症2个周期,针对男方AZFc缺失1个周期,针对21-三体高风险筛查2个周期。
研究方法:所有周期均经控制性超排卵、取卵、ICSI技术受精,受精后用Quinn's-1026培养液培养。受精后第三天观察胚胎发育情况,合适胚胎采用激光打孔法进行活检。可活检胚胎的标准为正常受精并发育到6-8细胞以上,碎片＜20%取1-2个卵裂球。活检出的卵裂球移入矿物油覆盖培养液Quinn's-1023微滴的培养皿(3001培养皿)准备行改良的Tween-20/HCl+甲醇/冰醋酸法固定。固定后选择相应探针杂交。相应的活检后的胚胎移入Quinn's-1029微滴中继续培养。D5根据FISH信号和胚胎质量等移植相应胚胎。
结果:
1、16个周期共取卵310个,正常受精253个,MⅡ卵共282个,2PN受精率89.7%(253/282),卵裂235个,卵裂率92.9%(235/253),优质胚胎165个,优质胚胎率73.0%(165/226)。共活检195个胚胎,活检成功180个,活检成功率为92.3%(180/195)。固定率为94.4%(170/180),经过FISH后诊断明确149个胚胎,诊断明确率为87.6%(149/170)。
2、16个周期妊娠成功4周期,成功率为25%。经检索为河南省首例针对罗伯逊易位、克氏综合症PGD妊娠成功。初步证实该诊断方法是准确、可行的
3、罗氏易位女性携带者组和男性携带者组平均年龄分别为30.75±2.87岁、28.67±0.58岁,P＞0.05差异无显著性。两组在体重指数(BMI)、基础FSH、LH、E_2、降调天数、Gn天数、2PN受精率、2PN卵裂率、移植胚胎数上比较,差异均无显著性(P＞0.05)。但女性携带者组的Gn用量为2493.75±555.79IU,明显高于男性携带者组1600.00±173.20 IU;HCG注射日E_2值、获卵数分别为4309.18±1687.26(pg/mL)、17.50±4.51个,明显低于男性携带者组,差别有统计学意义(P＜0.05)。
结论:
1、平衡易位携带者有较高比例的无规律分裂的胚胎,此类患者PGD在保障胚胎发育潜能的前提下倾向活检2个卵裂球以提高诊断的准确性。
2、染色体罗伯逊易位可能是卵巢反应不良的风险因素之一。
3、改良的Tween-20/HCl+甲醇/冰醋酸法应用于临床16个胚胎植入前遗传学诊断周期,4例获得妊娠,使其实现生育健康后代的愿望,达到优生的目的。
第五部分未培养羊水细胞FISH在产前遗传性疾病检测中的应用
研究对象:
2007～2008年来我院进行产前诊断的100例孕妇,年龄范围25-40岁,孕周范围17-25周。羊膜腔穿刺指征包括:1、孕妇年龄大于或等于35岁;2、孕妇曾经生育过染色体异常儿;3、夫妇一方有染色体结构异常;4、母血清生化筛查高风险;5、超声检查发现胎儿异常。并排除穿刺的禁忌症如具有先兆流产症状、体温高于37.5℃、有出血或盆腔宫腔感染征象者。
研究方法:
1、进行羊水核型分析及荧光原位杂交的产前诊断病例共100例。孕妇的年龄25-40岁,孕周范围17-25周。
2、羊水核型分析采用常规方法,由河南省人民医院遗传研究所完成。
3、未培养羊水细胞FISH检测方法按照探针说明进行。
结果:
1、研究对象的一般情况
进行羊水核型分析及荧光原位杂交的产前诊断病例共100例。孕妇的年龄25-40岁,孕周范围17-25周。男性胎儿55例,女性胎儿45例。
2、羊水细胞核型分析结果
100例羊水标本均同时行未培养羊水细胞荧光原位杂交和细胞核型分析,共发现1例胎儿染色体异常,为21-三体,9号染色体臂间倒位1例。此外染色体多态性变异2例,包括1例46 XX,1qh+,1例46 XY,Yqh+。
3、羊水细胞FISH结果
每一例羊水标本约5-6ml,一般均在24小时内出FISH结果,显示1例异常为47,XX,+21,其余FISH分析正常。
结论
1、未培养羊水细胞行FISH分析是可行的、可靠的。本研究中,针对特异探针而言,染色体非整倍体的诊断准确率达100%。该技术简便快速,能在24小时内出结果,有很大的应用前景。
2、本文利用国产探针进行未培养羊水细胞行FISH分析,结果可靠。同时可以降低FISH费用,易于临床推广应用。
全文小结
1、改良的Tween-20/HCl+甲醇/冰醋酸法易于掌握、程序简单、固定细胞所需时间短、不增加误诊率等优点可在临床使用。
2、平衡胚胎来源的卵裂球整倍体明显高于非平衡胚胎来源的卵裂球。
3、不同级别胚胎的卵裂球其整倍体率无显著差异。本研究显示优质胚胎非整倍体率仍占有60%,高于文献报道。因此对于平衡易位的PGD建议同时进行胚胎的非整倍体筛查。
4、本研究显示整倍体卵裂球中染色体信号分布无显著差异,而非整倍体细胞核中,染色体信号分布主要在核周边,尤其是18号染色体。
5、罗氏易位携带者及克氏综合症患者平衡精子比例高于相互易位携带者,其染色体正常精子比例与PGD中正常胚胎比例呈正相关。
6、平衡易位携带者有较高比例的无规律分裂的胚胎。
7、染色体罗伯逊易位是卵巢反应不良的风险因素之一。
8、改良的Tween-20/HCl+甲醇/冰醋酸法应用临床16个周期胚胎植入前遗传学诊断,4例获得妊娠,使其实现生育健康后代的愿望,达到优生的目的。同时PGD技术已得到卫生部批准,为进一步开展PGD的基础研究和临床应用打下基础。
9、未培养羊水细胞行FISH分析是可行的、可靠的。该技术简便快速,能在24小时内出结果,国产探针应用降低FISH费用,有很大的应用前景。
It has been 20 years since the first success of test tube baby of preimplantation genetic diagnosis(PGD) in 1990.Preimplantation genetic diagnosis(PGD) is introduced as a method to analyze embryo hereditary substance before implantation, and to identify which embryo is normal and suitable to transfer.Thus it prevents genetic disorders.Compared with prenatal diagnosis,PGD can select unaffected embryos to transfer,thus avoiding selective and repeated abortion and moral conflict resulted from abortion.As a result,PGD was received more and more emphasis.On the other hand,the rapid progress in the field of in vitro fertilization-embryo transfer lays solid basement for the development of PGD.Although many novel technologies have applied in this special field for single cell diagnosis such as comparative genetic hybridization(CGH),array-CGH and microarray,there was no other alternate methods which can replace two basic methods for PGD:Fluorescence in situ hybridization(FISH) and polymerase chain reaction(PCR),single-cell PCR has special limitations:amplification efficiency,contamination,and Allele drop-out.FISH has advantages over PCR especially in the study of mosaicism and chromosome number and structural abnormality.So at present FISH is an important and effective method in PGD.
Unfortunately,there are many difficulties encountered in clinical FISH-PGD. Firstly,the diagnosis accuracy of FISH depends on the fixation of blastomere in PGD. The ideal unicellular preparation should maintain the integrity of the chromosomal DNA,while removing the cytoplasm completely,and should be easy to learn and perform.At present,there are three different fixation methods for blastomere (Methanol/glacial acetic acid;Tween-20/HCl;Tween-20/HCl+ Methanol/glacial acetic acid),and each of them was adopted by many different PGD centers.However, the situation such as masking nucleus,residual cytoplasm,even cell loss often meet in the procedure of single cell fixation.Moreover,the results of fixation and signal rates of these methods have been inconsistent,and the fixation method is still in need of improvement.Secondly,there are high rate of aneuploidy in pre-implantation embryo which can lead clinical misdiagnosis and low clinical pregnancy rate.There are different standpoints about the embryo aneuploidy risk of chromosome balanced translocation carriers.And the effect of chromosome position on single diagnosis and embryo developmental potentiality remains unclear and become research hotspots. Thirdly,the prognostic factor and genetic counseling about chromosome aberration also become hot spots in the research of PGD.
According to the difficulties and hotspots in this special field,the study consists of five parts as follows:
PartⅠ:To simplify the procedure based on the Tween 20-HCL and acetic acid/methanol method,and compared it with the other standard methods to determine the suitability for FISH-PGD.
PartⅡ:To determine the aneuploidy in pre-implantation embryos of balanced chromosome translocation couples.And to analyze the distribution of chromosome position in nucleus.
PartⅢ:To determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorder carriers by FISH.
PartⅣ:To apply the modified unicellular fixation to clinical FISH-PGD.Till now,16 PGD cycles performed in our center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for 47,XXY,1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21.And to retrospectively analyze the effect of Robertsonian translocation on ovarian responsiveness in controlled ovarian hyperstimulation,et al.
PartⅤ:To perform FISH on uncultured amniocyte with domestic probes(Beijing Golden probe company).
PartⅠThe effect of modified unicellular spreading method on the nucleus area and FISH signal
Objective
To modify the unicellular fixation method based on Tween-20/HCl+ Methanol/glacial acetic acid and to analyzed the effect of modified unicellular spreading method on the nucleus area and FISH signal.
Methods
The embryos for this study were donated for research by patients treated with IVF/ICSI in our Reproductive Medical Unit.Written consent was obtained from all the patients.Only arrested cleavage-stage human embryos were used in this study.
Isolated blastomeres with visible nuclei were randomly divided into 3 groups and fixed by 3 different methods(Methanol/glacial acetic acid;Tween-20/HCl+ Methanol/glacial acetic acid,modified Tween-20/HCl+ Methanol/glacial acetic acid),then perform FISH on the spreading nuclei with 5 probes.To calculate the fixed nuclei area by the special FISH software Imstar2.1 and the overlap signal rate and splitted signal rate for each group.
Results
1.The Methanol/glacial acetic acid method resulted in 86.73%fixation,which was significantly low than other two methods.
2.The mean fixed nucleus area for Methanol/glacial acetic acid method was 55.3 um~3,which significantly larger than that of the other two methods.
3.As for the overlap signal rate and splitted signal rate,there was no statistical significance between the three groups.
Conclusion
The modified Tween-20/HCl+ Methanol/glacial acetic acid method do not increase the overlap signal rate and the splitted signal rate,but shorten the during of fixation. For the modified blastomere fixation method,it is easier to locate the nucleus of a blastomere and to learn which is critical for PGD.Considering all these points,the modified method of blastomere fixation for PGD is acceptable.
PartⅡThe study on the aneuploidy screening of 11 cycles of
PGD for balanced translocation carriers and the correlation of chromosome position and aneuploidy of preimplantation embryos
Objective
To perform aneuploidy screening of 11 cycles of PGD for balanced translocation carriers and analyze the correlation of chromosome position and aneuploidy of preimplantation embryos.
Methods
1.The procedure for PGD was the same as the SOP of our center.
2.The second round FISH was performed on the well-fixed nuclei for aneuploidy screening of chromosome 13,18,21,X and Y.
3.The relative distance(RD) of the signal was calculated with Imstar2.1 software. The RD=SC/R.
Results
1.139 well-fixed nuclei were selected for re-analysis,and 130 nuclei got the integrated FISH signals.937 FISH signals were analysized,including 304 signals from euploid nuclei and the others from aneuploid nuclei.
2.The euploidy rate was 71.4%(30/42) from balanced embryos,while the euploidy rate was 9.1%(8/88) from unbalanced embryos,and there was significant difference between them(x~2 =53.4,P＜0.05)。Complex aneuploidy was the manifest phenomenon for nuclei from unbalanced embryos.The aneuploidy rate for good-quality embryo was 60%(56/92).
3.304 FISH signals from 38 euploid nuclei were calculated which include 21 male nuclei and 21 female nuclei.There was no significant difference between them, howener,signal 13,21,X were prone to locate in the center of nucleus(RD＜0.5).while signal 18,Y trend to distribute in the periphery of nucleus(RD＞0.5).
4.The distribution of FISH signal for aneupioid nuclei remains significant difference.
signal 13,18,21,Y were prone to locate in the center of nucleus(RD＞0.5),especial for chromosome 18.however it is different for chromosome X.
5.the signal distribution of euploidy was statistically significant different from that of aneploid nuclei.The signal location for euploid centralized the center of the fixed nucleus,however the signal distribution for aneuploidy centralized the periphery of the nucleus.
Conclusion
1.The euploidy rate for balance embryo was higher than that of the unbalance embryo.
2.There was no significant difference between the embryos form different grades. The embryos to transfer may be the aneuploid embryos which lead to pregnancy failure.The aneuploidy rate for good-quality was 60%,so it is suggestion that PGS was performed during the procedure of PGD for balanced embryos.
3.There is no significant difference for the distribution of chromosome position from euploid nuclei.
4.The signal distribution for aneuploidy centralized the periphery of the nucleus, especial for chromosome 18.however,it is different for chromosome X.
PartⅢThe Importance of Sperm Fluorescence in situ Hybridization in Preimplantation Genetic Diagnosis for Male Chromosome Abnormality
Objective
To determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorders.
Methods
1.Sperm fluorescence in situ hybridization(FISH) and preimplantation genetic diagnosis were done for 9 couples of male chromosomal abnormality including 9 cycles of Robertsonian translocation carriers,2 cycles for reciprocal translocation carriers,1 cycles for Klinefelter's syndrome(47,XXY).The partners have normal karyotype.Informative consent was obtained from all the patients.
2.The procedure for PGD was the same as the SOP of our center.
3.Sperm pre-treatment:After semen washing,the specimen was fixed with Methanol/glacial acetic acid(3:1),and to dilute the sperm concentration to 20×10~6/ml, then drop on the slide to air dry.The sperm was decondensized by rinsing in 1M NaOH for 5min.FISH was applied to the specimen immediately.
4.The program for FISH was the same as the PartⅠ.
Results
1.The rate of normal/balanced sperm of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 85.7%,30.4%and 68.8% respectively.
2.The rate of normal/balanced embryo of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 28.97%,6.25%and 33.3% respectively.
3.A correlation was found between the percentage of normal embryos and the percentage of normal sperms(R=0.75,P=0.02).
Conclusion
1.The rate of normal/balanced sperm of carriers for Robertsonian translocation and Klinefelter's syndrome are higher than that of reciprocal translocation carriers
2.Since the correlation was established between the percentage of normal embryos and the percentage of normal sperms,it is advisable to recommend for being routinely incorporated into the genetic screening offered prior to preimplantation genetic diagnosis.
PartⅣThe clinical analysis of 16 cycles of Preimplantation genetic diagnosis
Objective
To analyze 16 PGD cycles which were performed in our reproductive center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for Klinefelter's syndrome(47,XXY),1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21 from January,2006 to March,2009.
Methods
To perform FISH in clinical PGD.All treatment cycles consisted of controlled ovarian hyperstimulation(COH),oocyte retrieval,ICSI and culture in Quinn's-1026 media after fertilization.We observed the development of embryos on day3 after fertilization,then biopsied after laser-assisted ZP drilling.The standard for biopsy 1-2 blastomeres is the normally fertilized embryo which is above 6-8 cells,less than 20% fragment.The isolated blastomeres were fixed,then hybridized with corresponding probes;the embryos were cultured in Quinn's-1029 media.The normal embryos were transferred according to the diagnosis of FISH and the quality on D 4-5.
Results
1.We harvested 310 oocytes totally from 16 cycles.The number of MII oocyte was 282.The rate of normal fertilization,cleavage and good embryo were 89.7% (253/282),92.9%(235/253) and 73.0%(165/226) respectively.180 embryos were biopsied successfully in the 195 embryos which were suitable for biopsy,and the fixation rate was 94.4%(170/180).149 embryos were got the FISH result and the rate of FISH diagnosis rate was 87.6%(149/170).
2.4 couples had pregnancy in 16 PGD cycles.They were retrieved as the first success of pregnancy of PGD for Robertsonian translocation and Klinefelter's syndrome in Henan province.
3 In this study,we retrospectively reviewed 9 cycles controlled ovarian hyperstimulation in PGD for Robertsonian translocations including 4 cycles for female carriers(study group) and 5 cycles for male carriers(controlled group).In the two groups,we analysized the data including age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,total doses of gonadotropin,E2 level at hCG,number of oocytes retrieved, 2PN fertilization rate,2PN cleavage rate,number of ETs.There were no significant difference in age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,2PN fertilization rate,2PN cleavage rate,number of ETs between two groups.However,total doses of gonadotropin of study group was higher than that of controlled group(2493.75±555.79 vs 1600.00±173.20,P＜0.05).E2 level at hCG and number of retrieved oocytes of study group was lower than that of controlled group(4309.18±1687.26 vs 7711.00±380.76,17.50±4.51 vs 34.67±2.89,P＜0.05).
Conclusion
1.Modified modified Tween-20/HCl+ Methanol/glacial acetic acid method applied in clinical PGD.
2.Four couples had pregnancy in 16 PGD cycles.This enabled them realize their desire of having healthy offsprings,and laid solid foundation for further research and clinical application of PGD.
3.Robertsonian translocation carriers have high rate chaotic embryos.
4.Chromosome Robertsonian translocation maybe is one of the risk factors of poor ovarian responsiveness during controlled ovarian hyperstimulation.
PartⅤThe clinical application of Fluorescence in situ Hybridization for uncultured amniocyte in detection of prenatal genetic disorders
Objective
To detect the prenatal genetic abnormality with amniocyte FISH.
Methods
1.To performed FISH and karytyping of amniocyte for the cases needed to prenatal genetic diagnosis.
2.Standard amniocyte karytyping was adopted and performed in Genetic Research Institute of Henan renmin hospital.
3.The protocol FISH for uncultured amniocyte was strictly performed with the instruction of the probe manufacturer(Beijing Golden Probe company).
Results
1.Totally,100 cases were detected by amniocyte karytyping and FISH which consists of 55 male fetuses and 45 female fetuses.The age range was 25-40 years and the range of gestational week was 17-25 weeks.
2.The results of abnormal amniocyte karytypes consists of 1 case of 21-trisomy. The chromosomal variation includes 1 case of inversion(9),1 case of 46 XX,1qh+, 1 case of 46 XY,Yqh+.
3.FISH results were got less than 24hrs.Only one case was abnormal which indicate 47,XX,+21.
Conclusion
1.The result of uncultured amniocyte FISH is feasible and reliable.The diagnosis accuracy of FISH for amniocyte chromosome aneuploidy is 100%.
2.Considered the advantages of uncultured amniocyte FISH such as quickness and reliability,the technology has wonderful prospective application in the prenatal diagnosis,even with the low cost using domestic probes.
在FISH-PGD中仍然存在难点:1、单细胞固定:在单细胞FISH诊断中,诊断的准确性与单个卵裂球的固定密切相关。理想的固定应该是彻底的去除胞浆蛋白,保留染色体DNA的完整性且简单易学。目前常用的单细胞固定方法有三种:甲醇/冰醋酸法、Tween-20/HCl法、Tween-20/HCl+甲醇/冰醋酸法。三种固定方法在各个生殖中心分别有使用。但三种方法在实际临床应用中仍然可能出现细胞核暴露不良、胞浆残留多、甚至细胞核丢失导致无法准确诊断等,因此认为固定方法仍需进一步改进和完善。2、植入前胚胎有高比例的非整倍体,这可能是FISH-PGD临床误诊、成功率低的重要原因之一。对于高生育风险的平衡易位携带者夫妇其胚胎非整倍体风险文献报道不一。同时在FISH诊断中信号在核中的位置的临床意义及其对诊断及胚胎发育的影响近年来成为研究热点,但仍然尚无明确定论。(3)临床上染色体异常PGD遗传咨询及预后估计如移植胚胎、妊娠结局等的研究逐渐成为热点,但目前进展缓慢。
本文就上述热点和难点问题从以下五个方面进行了研究:第一部分对Tween-20/HCl+甲醇/冰醋酸法进行了改良,并探讨了其对FISH信号及单细胞诊断的影响。第二部分探讨了平衡易位携带者胚胎中非整倍体的变化及卵裂球细胞核中染色体信号分布特点。第三部分探讨了精子FISH结果在男性染色体异常PGD及遗传咨询中的作用。第四部分将改良的卵裂球固定方法用于临床,进行了16个PGD周期的临床诊断,同时回顾性分析了染色体平衡易位对控制性超排卵过程中卵巢反应性的影响等。第五部分应用国产化探针对未培养的羊水细胞进行了FISH分析。
第一部分单细胞固定方法的改良及其对细胞核面积及荧光原位杂交信号的影响
研究对象:用于固定卵裂球细胞均来自不宜移植的人类2-10细胞阶段的胚胎,所有胚胎均经过病人知情同意后用于实验。
研究方法:获得的卵裂球使用3种不同的固定方法(甲醇/冰醋酸法;Tween-20/HCl+甲醇/冰醋酸法;改良的Tween-20/HCl+甲醇/冰醋酸法)固定。固定后进行FISH,同时利用荧光分析软件Imstar计算细胞核面积、比较各组的固定率、信号重叠率、信号分裂率。
结果:
1、细胞固定率上甲醇/冰醋酸法为86.73%,明显低于改良前后Tween-20/HCl+甲醇/冰醋酸法。
2、细胞面积上甲醇/冰醋酸法为55.3 um~3,明显高于改良前后Tween-20/HCl+甲醇/冰醋酸法。
3、三组间信号重叠率及信号分裂率无显著性差异。
结论:对Tween-20/HCl+甲醇/冰醋酸法进行了改良,同时对其及另外的卵裂球固定方法进行比较,改良的Tween-20/HCl+甲醇/冰醋酸法易于掌握、程序简单、固定细胞时间短、不增加误诊率等优点可在临床广泛推广使用。
第二部分平衡易位携带者胚胎非整倍体筛查及染色体位置与胚胎非整倍体相关性的研究
研究对象:2006.1～2009.3我中心针对平衡易位携带者进行11个PGD周期,包括罗伯逊易位9个周期,相互易位2个周期。
研究方法:
1、胚胎植入前遗传学诊断程序按我中心常规进行。
2、对固定好、信号明显的细胞核再次选择5种探针(LSI13/21及CEP18/X/Y),进行胚胎非整倍体检测。
3、采用Imstar荧光分析软件确定各个信号相对位置。信号相对位置(relativedistance)RD=SC/R。
结果:
1、11个针对平衡易位的PGD,选择细胞核固定良好、无多核、无核碎片的139个细胞核进行了PGS。选择信号完整的130个细胞核进行了分析。共分析了937个荧光信号,其中整倍体细胞核共38个,304个信号,余均为非整倍体细胞信号。
2、分析了来源于平衡胚胎的42个卵裂球和来源于非平衡胚胎的88个卵裂球的PGS结果。在平衡胚胎来源的细胞核中,整倍体率为71.4%(30/42),而非平衡胚胎来源的细胞核其整倍体率为9.1%(8/88),统计学上差别有显著性(x~2=53.4,P＜0.05)。其中非平衡胚胎来源的卵裂球主要表现为复杂的非整倍体(36.4%)。虽然染色体整倍体率在不同级别胚胎中分布无显著性差异,但优质胚胎(Ⅰ级+Ⅱ级)中非整倍体率仍占有60%(56/92)。
3、对38个整倍体卵裂球进行了分析,其中包括男性核型21个,女性核型17个。共分析了304个荧光原位杂交信号,在信号分布上未发现有显著差异。但信号13、21、X倾向于分布于细胞中央(RD＜0.5),而染色体18、Y则倾向分布于外周(RD＞0.5)。
4、对92个非整倍体卵裂球进行了分析,共分析了633个荧光原位杂交信号。非整倍体卵裂球中信号分布存在统计学差异。信号13、18、21、Y倾向分布于外周(RD＞0.5),尤其是染色体18号。但信号X的分布无统计学差异。
5、在整倍体细胞核中,染色体信号分布位置无统计学差异,但主要集中在核周1/4-3/4,而在非整倍体细胞核中,染色体信号分布主要在核周边。二者在信号分布上有统计学差异(p=0.00)。
结论:
1、平衡胚胎来源的卵裂球整倍体率为71.4%,明显高于非平衡胚胎来源的卵裂球整倍体率(9.1%)。
2、不同级别胚胎的卵裂球其整倍体率无显著差异,即使移植了优质胚胎仍然可能是非整倍体胚胎,最终导致妊娠失败。本研究显示优质胚胎非整倍体率仍占有60%,高于文献报道。因此对于针对平衡易位的胚胎植入前遗传学诊断建议同时进行胚胎的非整倍体筛查。
3、本研究显示整倍体卵裂球中染色体信号分布无显著差异。
4、非整倍体细胞核中,染色体信号分布主要在核周边。信号13、18、21、Y倾向分布于外周,尤其是染色体18号,信号X的分布无统计学差异。
第三部分精子荧光原位杂交在男性染色体异常PGD中的作用
研究对象:
2006.7～2008.8,9对不孕夫妇因男方染色体异常进行了精子荧光原位杂交分析和胚胎植入前遗传学诊断,包括罗氏易位7例,相互易位1例,克氏综合征1例。所有病例女方染色体均正常。患者禁欲3-5天手淫法取得的精液标本用于试验。所有患者均知情同意后行精子FISH和PGD。
研究方法:
1、胚胎植入前遗传学诊断程序按我中心常规进行。
2、精子预处理:精液预处理后用甲醇/冰醋酸(3:1)固定液调制精子浓度至20×10~6/ml,后滴片,使精子均匀分布于玻片并风干。后将风干玻片在1M NaOH溶液中浸泡5分钟,使精子核去凝集,后立即进行FISH分析。
3、FISH方法同第一部分。
结果:
1、9例染色体异常携带者男性进行了精子FISH分析,7例罗伯逊易位携带者中,正常或平衡精子比例为85.7%(6045/7053),1例相互易位携带者中正常或平衡精子比例为30.4%,克氏综合症正常精子比例为68.8%。
2、7例罗伯逊易位携带者中,正常或平衡胚胎比例为28.97%(31/107),1例相互易位携带者中正常或平衡胚胎比例为6.25%,1例克氏综合症正常胚胎比例为33.3%。
3、PGD中正常/平衡胚胎的比例与正常精子比例呈正相关(R=0.75,P=0.02＜0.05)。
结论:
1、罗伯逊易位携带者平衡精子平均比例为85.7%,克氏综合症患者的正常或平衡精子比例为68.8%,均高于相互易位携带者。
2、染色体正常精子比例与PGD中正常胚胎比例呈正相关,因此在PGD前生殖遗传咨询中,建议对携带者进行精子荧光原位杂交分析,了解平衡配子所占的比例,为PGD的应用提供客观依据。
第四部分胚胎植入前遗传学诊断16个周期的临床应用分析
研究对象:2006.1～2009.3年,我中心共进行胚胎植入前遗传学诊断16个周期,包括针对染色体罗伯逊易位9个周期,针对染色体相互易位2个周期,针对男性克氏综合症2个周期,针对男方AZFc缺失1个周期,针对21-三体高风险筛查2个周期。
研究方法:所有周期均经控制性超排卵、取卵、ICSI技术受精,受精后用Quinn's-1026培养液培养。受精后第三天观察胚胎发育情况,合适胚胎采用激光打孔法进行活检。可活检胚胎的标准为正常受精并发育到6-8细胞以上,碎片＜20%取1-2个卵裂球。活检出的卵裂球移入矿物油覆盖培养液Quinn's-1023微滴的培养皿(3001培养皿)准备行改良的Tween-20/HCl+甲醇/冰醋酸法固定。固定后选择相应探针杂交。相应的活检后的胚胎移入Quinn's-1029微滴中继续培养。D5根据FISH信号和胚胎质量等移植相应胚胎。
结果:
1、16个周期共取卵310个,正常受精253个,MⅡ卵共282个,2PN受精率89.7%(253/282),卵裂235个,卵裂率92.9%(235/253),优质胚胎165个,优质胚胎率73.0%(165/226)。共活检195个胚胎,活检成功180个,活检成功率为92.3%(180/195)。固定率为94.4%(170/180),经过FISH后诊断明确149个胚胎,诊断明确率为87.6%(149/170)。
2、16个周期妊娠成功4周期,成功率为25%。经检索为河南省首例针对罗伯逊易位、克氏综合症PGD妊娠成功。初步证实该诊断方法是准确、可行的
3、罗氏易位女性携带者组和男性携带者组平均年龄分别为30.75±2.87岁、28.67±0.58岁,P＞0.05差异无显著性。两组在体重指数(BMI)、基础FSH、LH、E_2、降调天数、Gn天数、2PN受精率、2PN卵裂率、移植胚胎数上比较,差异均无显著性(P＞0.05)。但女性携带者组的Gn用量为2493.75±555.79IU,明显高于男性携带者组1600.00±173.20 IU;HCG注射日E_2值、获卵数分别为4309.18±1687.26(pg/mL)、17.50±4.51个,明显低于男性携带者组,差别有统计学意义(P＜0.05)。
结论:
1、平衡易位携带者有较高比例的无规律分裂的胚胎,此类患者PGD在保障胚胎发育潜能的前提下倾向活检2个卵裂球以提高诊断的准确性。
2、染色体罗伯逊易位可能是卵巢反应不良的风险因素之一。
3、改良的Tween-20/HCl+甲醇/冰醋酸法应用于临床16个胚胎植入前遗传学诊断周期,4例获得妊娠,使其实现生育健康后代的愿望,达到优生的目的。
第五部分未培养羊水细胞FISH在产前遗传性疾病检测中的应用
研究对象:
2007～2008年来我院进行产前诊断的100例孕妇,年龄范围25-40岁,孕周范围17-25周。羊膜腔穿刺指征包括:1、孕妇年龄大于或等于35岁;2、孕妇曾经生育过染色体异常儿;3、夫妇一方有染色体结构异常;4、母血清生化筛查高风险;5、超声检查发现胎儿异常。并排除穿刺的禁忌症如具有先兆流产症状、体温高于37.5℃、有出血或盆腔宫腔感染征象者。
研究方法:
1、进行羊水核型分析及荧光原位杂交的产前诊断病例共100例。孕妇的年龄25-40岁,孕周范围17-25周。
2、羊水核型分析采用常规方法,由河南省人民医院遗传研究所完成。
3、未培养羊水细胞FISH检测方法按照探针说明进行。
结果:
1、研究对象的一般情况
进行羊水核型分析及荧光原位杂交的产前诊断病例共100例。孕妇的年龄25-40岁,孕周范围17-25周。男性胎儿55例,女性胎儿45例。
2、羊水细胞核型分析结果
100例羊水标本均同时行未培养羊水细胞荧光原位杂交和细胞核型分析,共发现1例胎儿染色体异常,为21-三体,9号染色体臂间倒位1例。此外染色体多态性变异2例,包括1例46 XX,1qh+,1例46 XY,Yqh+。
3、羊水细胞FISH结果
每一例羊水标本约5-6ml,一般均在24小时内出FISH结果,显示1例异常为47,XX,+21,其余FISH分析正常。
结论
1、未培养羊水细胞行FISH分析是可行的、可靠的。本研究中,针对特异探针而言,染色体非整倍体的诊断准确率达100%。该技术简便快速,能在24小时内出结果,有很大的应用前景。
2、本文利用国产探针进行未培养羊水细胞行FISH分析,结果可靠。同时可以降低FISH费用,易于临床推广应用。
全文小结
1、改良的Tween-20/HCl+甲醇/冰醋酸法易于掌握、程序简单、固定细胞所需时间短、不增加误诊率等优点可在临床使用。
2、平衡胚胎来源的卵裂球整倍体明显高于非平衡胚胎来源的卵裂球。
3、不同级别胚胎的卵裂球其整倍体率无显著差异。本研究显示优质胚胎非整倍体率仍占有60%,高于文献报道。因此对于平衡易位的PGD建议同时进行胚胎的非整倍体筛查。
4、本研究显示整倍体卵裂球中染色体信号分布无显著差异,而非整倍体细胞核中,染色体信号分布主要在核周边,尤其是18号染色体。
5、罗氏易位携带者及克氏综合症患者平衡精子比例高于相互易位携带者,其染色体正常精子比例与PGD中正常胚胎比例呈正相关。
6、平衡易位携带者有较高比例的无规律分裂的胚胎。
7、染色体罗伯逊易位是卵巢反应不良的风险因素之一。
8、改良的Tween-20/HCl+甲醇/冰醋酸法应用临床16个周期胚胎植入前遗传学诊断,4例获得妊娠,使其实现生育健康后代的愿望,达到优生的目的。同时PGD技术已得到卫生部批准,为进一步开展PGD的基础研究和临床应用打下基础。
9、未培养羊水细胞行FISH分析是可行的、可靠的。该技术简便快速,能在24小时内出结果,国产探针应用降低FISH费用,有很大的应用前景。
It has been 20 years since the first success of test tube baby of preimplantation genetic diagnosis(PGD) in 1990.Preimplantation genetic diagnosis(PGD) is introduced as a method to analyze embryo hereditary substance before implantation, and to identify which embryo is normal and suitable to transfer.Thus it prevents genetic disorders.Compared with prenatal diagnosis,PGD can select unaffected embryos to transfer,thus avoiding selective and repeated abortion and moral conflict resulted from abortion.As a result,PGD was received more and more emphasis.On the other hand,the rapid progress in the field of in vitro fertilization-embryo transfer lays solid basement for the development of PGD.Although many novel technologies have applied in this special field for single cell diagnosis such as comparative genetic hybridization(CGH),array-CGH and microarray,there was no other alternate methods which can replace two basic methods for PGD:Fluorescence in situ hybridization(FISH) and polymerase chain reaction(PCR),single-cell PCR has special limitations:amplification efficiency,contamination,and Allele drop-out.FISH has advantages over PCR especially in the study of mosaicism and chromosome number and structural abnormality.So at present FISH is an important and effective method in PGD.
Unfortunately,there are many difficulties encountered in clinical FISH-PGD. Firstly,the diagnosis accuracy of FISH depends on the fixation of blastomere in PGD. The ideal unicellular preparation should maintain the integrity of the chromosomal DNA,while removing the cytoplasm completely,and should be easy to learn and perform.At present,there are three different fixation methods for blastomere (Methanol/glacial acetic acid;Tween-20/HCl;Tween-20/HCl+ Methanol/glacial acetic acid),and each of them was adopted by many different PGD centers.However, the situation such as masking nucleus,residual cytoplasm,even cell loss often meet in the procedure of single cell fixation.Moreover,the results of fixation and signal rates of these methods have been inconsistent,and the fixation method is still in need of improvement.Secondly,there are high rate of aneuploidy in pre-implantation embryo which can lead clinical misdiagnosis and low clinical pregnancy rate.There are different standpoints about the embryo aneuploidy risk of chromosome balanced translocation carriers.And the effect of chromosome position on single diagnosis and embryo developmental potentiality remains unclear and become research hotspots. Thirdly,the prognostic factor and genetic counseling about chromosome aberration also become hot spots in the research of PGD.
According to the difficulties and hotspots in this special field,the study consists of five parts as follows:
PartⅠ:To simplify the procedure based on the Tween 20-HCL and acetic acid/methanol method,and compared it with the other standard methods to determine the suitability for FISH-PGD.
PartⅡ:To determine the aneuploidy in pre-implantation embryos of balanced chromosome translocation couples.And to analyze the distribution of chromosome position in nucleus.
PartⅢ:To determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorder carriers by FISH.
PartⅣ:To apply the modified unicellular fixation to clinical FISH-PGD.Till now,16 PGD cycles performed in our center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for 47,XXY,1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21.And to retrospectively analyze the effect of Robertsonian translocation on ovarian responsiveness in controlled ovarian hyperstimulation,et al.
PartⅤ:To perform FISH on uncultured amniocyte with domestic probes(Beijing Golden probe company).
PartⅠThe effect of modified unicellular spreading method on the nucleus area and FISH signal
Objective
To modify the unicellular fixation method based on Tween-20/HCl+ Methanol/glacial acetic acid and to analyzed the effect of modified unicellular spreading method on the nucleus area and FISH signal.
Methods
The embryos for this study were donated for research by patients treated with IVF/ICSI in our Reproductive Medical Unit.Written consent was obtained from all the patients.Only arrested cleavage-stage human embryos were used in this study.
Isolated blastomeres with visible nuclei were randomly divided into 3 groups and fixed by 3 different methods(Methanol/glacial acetic acid;Tween-20/HCl+ Methanol/glacial acetic acid,modified Tween-20/HCl+ Methanol/glacial acetic acid),then perform FISH on the spreading nuclei with 5 probes.To calculate the fixed nuclei area by the special FISH software Imstar2.1 and the overlap signal rate and splitted signal rate for each group.
Results
1.The Methanol/glacial acetic acid method resulted in 86.73%fixation,which was significantly low than other two methods.
2.The mean fixed nucleus area for Methanol/glacial acetic acid method was 55.3 um~3,which significantly larger than that of the other two methods.
3.As for the overlap signal rate and splitted signal rate,there was no statistical significance between the three groups.
Conclusion
The modified Tween-20/HCl+ Methanol/glacial acetic acid method do not increase the overlap signal rate and the splitted signal rate,but shorten the during of fixation. For the modified blastomere fixation method,it is easier to locate the nucleus of a blastomere and to learn which is critical for PGD.Considering all these points,the modified method of blastomere fixation for PGD is acceptable.
PartⅡThe study on the aneuploidy screening of 11 cycles of
PGD for balanced translocation carriers and the correlation of chromosome position and aneuploidy of preimplantation embryos
Objective
To perform aneuploidy screening of 11 cycles of PGD for balanced translocation carriers and analyze the correlation of chromosome position and aneuploidy of preimplantation embryos.
Methods
1.The procedure for PGD was the same as the SOP of our center.
2.The second round FISH was performed on the well-fixed nuclei for aneuploidy screening of chromosome 13,18,21,X and Y.
3.The relative distance(RD) of the signal was calculated with Imstar2.1 software. The RD=SC/R.
Results
1.139 well-fixed nuclei were selected for re-analysis,and 130 nuclei got the integrated FISH signals.937 FISH signals were analysized,including 304 signals from euploid nuclei and the others from aneuploid nuclei.
2.The euploidy rate was 71.4%(30/42) from balanced embryos,while the euploidy rate was 9.1%(8/88) from unbalanced embryos,and there was significant difference between them(x~2 =53.4,P＜0.05)。Complex aneuploidy was the manifest phenomenon for nuclei from unbalanced embryos.The aneuploidy rate for good-quality embryo was 60%(56/92).
3.304 FISH signals from 38 euploid nuclei were calculated which include 21 male nuclei and 21 female nuclei.There was no significant difference between them, howener,signal 13,21,X were prone to locate in the center of nucleus(RD＜0.5).while signal 18,Y trend to distribute in the periphery of nucleus(RD＞0.5).
4.The distribution of FISH signal for aneupioid nuclei remains significant difference.
signal 13,18,21,Y were prone to locate in the center of nucleus(RD＞0.5),especial for chromosome 18.however it is different for chromosome X.
5.the signal distribution of euploidy was statistically significant different from that of aneploid nuclei.The signal location for euploid centralized the center of the fixed nucleus,however the signal distribution for aneuploidy centralized the periphery of the nucleus.
Conclusion
1.The euploidy rate for balance embryo was higher than that of the unbalance embryo.
2.There was no significant difference between the embryos form different grades. The embryos to transfer may be the aneuploid embryos which lead to pregnancy failure.The aneuploidy rate for good-quality was 60%,so it is suggestion that PGS was performed during the procedure of PGD for balanced embryos.
3.There is no significant difference for the distribution of chromosome position from euploid nuclei.
4.The signal distribution for aneuploidy centralized the periphery of the nucleus, especial for chromosome 18.however,it is different for chromosome X.
PartⅢThe Importance of Sperm Fluorescence in situ Hybridization in Preimplantation Genetic Diagnosis for Male Chromosome Abnormality
Objective
To determine whether the proportion of normal sperm is predictive of the proportion of normal embryos from couples in which the males are chromosomal disorders.
Methods
1.Sperm fluorescence in situ hybridization(FISH) and preimplantation genetic diagnosis were done for 9 couples of male chromosomal abnormality including 9 cycles of Robertsonian translocation carriers,2 cycles for reciprocal translocation carriers,1 cycles for Klinefelter's syndrome(47,XXY).The partners have normal karyotype.Informative consent was obtained from all the patients.
2.The procedure for PGD was the same as the SOP of our center.
3.Sperm pre-treatment:After semen washing,the specimen was fixed with Methanol/glacial acetic acid(3:1),and to dilute the sperm concentration to 20×10~6/ml, then drop on the slide to air dry.The sperm was decondensized by rinsing in 1M NaOH for 5min.FISH was applied to the specimen immediately.
4.The program for FISH was the same as the PartⅠ.
Results
1.The rate of normal/balanced sperm of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 85.7%,30.4%and 68.8% respectively.
2.The rate of normal/balanced embryo of carriers for Robertsonian translocation, reciprocal translocation and Klinefelter's syndrome was 28.97%,6.25%and 33.3% respectively.
3.A correlation was found between the percentage of normal embryos and the percentage of normal sperms(R=0.75,P=0.02).
Conclusion
1.The rate of normal/balanced sperm of carriers for Robertsonian translocation and Klinefelter's syndrome are higher than that of reciprocal translocation carriers
2.Since the correlation was established between the percentage of normal embryos and the percentage of normal sperms,it is advisable to recommend for being routinely incorporated into the genetic screening offered prior to preimplantation genetic diagnosis.
PartⅣThe clinical analysis of 16 cycles of Preimplantation genetic diagnosis
Objective
To analyze 16 PGD cycles which were performed in our reproductive center including 9 cycles of Robertsonian translocation carrers,2 cycles for reciprocal translocation carriers,2 cycles for Klinefelter's syndrome(47,XXY),1 cycles for AZFc deletion,and 2 cycles for aneuploidy screening for chromosome 21 from January,2006 to March,2009.
Methods
To perform FISH in clinical PGD.All treatment cycles consisted of controlled ovarian hyperstimulation(COH),oocyte retrieval,ICSI and culture in Quinn's-1026 media after fertilization.We observed the development of embryos on day3 after fertilization,then biopsied after laser-assisted ZP drilling.The standard for biopsy 1-2 blastomeres is the normally fertilized embryo which is above 6-8 cells,less than 20% fragment.The isolated blastomeres were fixed,then hybridized with corresponding probes;the embryos were cultured in Quinn's-1029 media.The normal embryos were transferred according to the diagnosis of FISH and the quality on D 4-5.
Results
1.We harvested 310 oocytes totally from 16 cycles.The number of MII oocyte was 282.The rate of normal fertilization,cleavage and good embryo were 89.7% (253/282),92.9%(235/253) and 73.0%(165/226) respectively.180 embryos were biopsied successfully in the 195 embryos which were suitable for biopsy,and the fixation rate was 94.4%(170/180).149 embryos were got the FISH result and the rate of FISH diagnosis rate was 87.6%(149/170).
2.4 couples had pregnancy in 16 PGD cycles.They were retrieved as the first success of pregnancy of PGD for Robertsonian translocation and Klinefelter's syndrome in Henan province.
3 In this study,we retrospectively reviewed 9 cycles controlled ovarian hyperstimulation in PGD for Robertsonian translocations including 4 cycles for female carriers(study group) and 5 cycles for male carriers(controlled group).In the two groups,we analysized the data including age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,total doses of gonadotropin,E2 level at hCG,number of oocytes retrieved, 2PN fertilization rate,2PN cleavage rate,number of ETs.There were no significant difference in age,body mass index(BMI),basal FSH/LH/E2 level,number of down-regulation days,number of days of Gn stimulation,2PN fertilization rate,2PN cleavage rate,number of ETs between two groups.However,total doses of gonadotropin of study group was higher than that of controlled group(2493.75±555.79 vs 1600.00±173.20,P＜0.05).E2 level at hCG and number of retrieved oocytes of study group was lower than that of controlled group(4309.18±1687.26 vs 7711.00±380.76,17.50±4.51 vs 34.67±2.89,P＜0.05).
Conclusion
1.Modified modified Tween-20/HCl+ Methanol/glacial acetic acid method applied in clinical PGD.
2.Four couples had pregnancy in 16 PGD cycles.This enabled them realize their desire of having healthy offsprings,and laid solid foundation for further research and clinical application of PGD.
3.Robertsonian translocation carriers have high rate chaotic embryos.
4.Chromosome Robertsonian translocation maybe is one of the risk factors of poor ovarian responsiveness during controlled ovarian hyperstimulation.
PartⅤThe clinical application of Fluorescence in situ Hybridization for uncultured amniocyte in detection of prenatal genetic disorders
Objective
To detect the prenatal genetic abnormality with amniocyte FISH.
Methods
1.To performed FISH and karytyping of amniocyte for the cases needed to prenatal genetic diagnosis.
2.Standard amniocyte karytyping was adopted and performed in Genetic Research Institute of Henan renmin hospital.
3.The protocol FISH for uncultured amniocyte was strictly performed with the instruction of the probe manufacturer(Beijing Golden Probe company).
Results
1.Totally,100 cases were detected by amniocyte karytyping and FISH which consists of 55 male fetuses and 45 female fetuses.The age range was 25-40 years and the range of gestational week was 17-25 weeks.
2.The results of abnormal amniocyte karytypes consists of 1 case of 21-trisomy. The chromosomal variation includes 1 case of inversion(9),1 case of 46 XX,1qh+, 1 case of 46 XY,Yqh+.
3.FISH results were got less than 24hrs.Only one case was abnormal which indicate 47,XX,+21.
Conclusion
1.The result of uncultured amniocyte FISH is feasible and reliable.The diagnosis accuracy of FISH for amniocyte chromosome aneuploidy is 100%.
2.Considered the advantages of uncultured amniocyte FISH such as quickness and reliability,the technology has wonderful prospective application in the prenatal diagnosis,even with the low cost using domestic probes.
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