First unaffected pregnancy using preimplantation genetic diagnosis for sickle cell anemia.

CONTEXT: Sickle cell anemia is a common autosomal recessive disorder. However, preimplantation genetic diagnosis (PGD) for this severe genetic disorder previously has not been successful. OBJECTIVE: To achieve pregnancy with an unaffected embryo using in vitro fertilization (IVF) and PGD. DESIGN: Laboratory analysis of DNA from single cells obtained by biopsy from embryos in 2 IVF attempts, 1 in 1996 and 1 in 1997, to determine the genetic status of each embryo before intrauterine transfer. SETTING: University hospital in a large metropolitan area. PATIENTS: A couple, both carriers of the recessive mutation for sickle cell disease. INTERVENTIONS: Standard IVF treatment, intracytoplasmic sperm injection, embryo biopsy, single-cell polymerase chain reaction and DNA analyses, embryo transfer to uterus, pregnancy confirmation, and prenatal diagnosis by amniocentesis at 16.5 weeks' gestation. MAIN OUTCOME MEASURE: DNA analysis of single blastomeres indicating whether embryos carried the sickle cell mutation, allowing only unaffected or carrier embryos to be transferred. RESULTS: The first IVF attempt failed to produce a pregnancy. Of the 7 embryos analyzed in the second attempt, PGD indicated that 4 were normal and 2 were carriers; diagnosis was not possible in 1. Three embryos were transferred to the uterus on the fourth day after oocyte retrieval. A twin pregnancy was confirmed by ultrasonography, and subsequent amniocentesis revealed that both fetuses were unaffected and were not carriers of the sickle cell mutation. The patient delivered healthy twins at 39 weeks' gestation. CONCLUSION: This first unaffected pregnancy resulting from PGD for sickle cell anemia demonstrates that the technique can be a powerful diagnostic tool for carrier couples who desire a healthy child but wish to avoid the difficult decision of whether to abort an affected fetus.     

对β-地中海贫血携带者进行胚胎植入前遗传学诊断

目的 探讨对β-地中海贫血进行胚胎植入前遗传学诊断的方法。方法 对4例β-地中海贫血携带者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,巢式-PCR及反向点杂交进行胚胎植入前遗传学诊断,根据诊断结果选择健康的胚胎移植入子宫。结果 4例患者共获卵97个,受精率为83%,可供活检的胚胎47个,总扩增效率为84.8%。平均等位基因脱扣发生率为14.9%。共移植10个健康胚胎,获得1例三胎妊娠（其中1个为空囊）,现已分娩健康双胎。结论 对β-地中海贫血进行植入前遗传学诊断可使β-地中海贫血患者实现生育健康后代的愿望,达到优生的目的。     

Birth of healthy children after preimplantation diagnosis of β-thalassemia

Background Clinical programs for preventing β-thalassemia are presently based on prospective carrier screening and prenatal diagnosis. This paper report an achievement of a pregnancy with unaffected embryos using in vitro fertilization and embryo transfer (IVF-ET), in combination with preimplantation genetic diagnosis (PGD), for a couple at risk of having children with β-thalassemia.Methods A couple carrying different thalassemia mutations, both a codon 41-42 mutation and the IVS Ⅱ 654 mutation, received standard IVF treatment, with intracytoplasmic sperm injection, embryo biopsiy, single cell polymerase chain reaction (PCR) and DNA analysis. Only unaffected or carrier embryos were transferred to the uterine cavity. After confirmation of pregnancy, a prenatal diagnosis was performed.Results Of a total of 13 embryos analyzed for β-globin mutations, PGD indicated that 2 were normal,3 were affected, and 6 were carriers. Diagnosis could not be made in the other 2 embryos. Three embryos were transferred to the uterus on the third day after oocyte retrieval. Ultrasonography revealed a twin pregnancy with one blighted ovum. The prenatal genetic diagnosis revealed that both fetuses were unaffected, and two healthy boys were born, confirming the results of PGD.Conclusions We developed a single-cell based primer extension preamplification (PEP)-PCR assay for the detection of β-thalassemia mutations. The assays were efficient and accurate at all stages of the procedure, and resulted in the birth of PGD-confirmed β-thalassemia free children in China. PEP was used here in PGD for β-thalassemia.     

应用荧光聚合酶链反应对α地中海贫血进行植入前遗传学诊断

目的探讨应用跨越断裂点荧光聚合酶链反应（PCR）技术在α地中海贫血（简称α地贫）植入前遗传学诊断（PGD）中的应用。方法获取Ot地贫东南亚缺失型携带者单个淋巴细胞,建立了稳定的单细胞跨越断裂点荧光PCR检测技术,并对4对夫妇双方均为α地贫——SEA缺失型杂合子应用荧光PCR进行了α地贫的PGD。结果单个淋巴细胞平均扩增效率为90．0％（72／80）,平均等位基因脱扣（ADO）率为8．3％（6／72）。对4对夫妇进行4个周期PGO,共检测38个胚胎,获得38个卵裂球,其中34个卵裂球扩增成功,扩增效率为89．5％（34／38）,ADO率为5．9％（2／34）。经PCR分析,共获得11个正常胚胎,8个杂合子胚胎,15个重型地贫胚胎。移植了11个胚胎,获得2例临床妊娠。孕17周时经脐带血穿刺,分别证实为完全正常胚胎和杂合子胚胎,现已出生两名健康男婴。结论应用单细胞荧光PCR技术可对α地贫进行植入前遗传学诊断,达到优生的目的。     

Successful pregnancy outcome after in vitro fertilisation following Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening for single gene disorder (sickle cell anaemia) before embryo transfer: The clinical experience of an in vitro fertilisation clinic in Nigeria

A couple, both carriers of the sickle cell anaemia trait (Genotype HbAS) with an offspring already affected with the genetic disease underwent a Pre-implantation Genetic Diagnosis/Polymerase Chain Reaction screening of biopsied blastomeres. DNA analysis of single blastomeres was carried out to find out indicated a viable intra-uterine pregnancy with embryos which carried the sickle cell mutation, which resulted in a livebirth (HbAS). PGD/PCR in combination with IVF appears to be the most suitable treatment plan for patients who are at a higher risk of reproducing offspring affected with inheritable genetic diseases.     

对曾育21三体综合征患儿者行胚胎种植前遗传学诊断获妊娠1例

目的 在体外受精－胚胎移植过程中，对高危夫妇进行胚胎种植前遗传学诊断以避免21三体综合征患儿的出生。方法 常规激素替代治疗，对患者夫妇行胞母细胞质内精子注射（ICSI），正常受精培养到第3天，对6－10细胞期胚胎活检1个细胞，利用荧光原位杂交技术（FISH）进行胚胎种植遗传学诊断（PGD），挑选染色体数目正常的胚胎移植入患者子宫。结果 共对8个胚胎进行PGD，7个有诊断结果者中7个为正常胚胎、1个为21三体胚胎。挑选3个胚胎移植，获得妊娠并产下一健康男婴。结论 应用FISH对染色体数目的遗传疾病，如21三体综合征，进行胚胎种植前遗传学诊断是切实可行的。     

多重巢式聚合酶链反应在β地中海贫血植入前遗传学诊断中的应用

目的探讨应用多重巢式聚合酶链反应(PCR)技术在β地中海贫血(简称β地贫)植入前遗传学诊断(PGD)中的应用。方法获取β地贫基因携带者单个淋巴细胞,建立了稳定的单细胞多重巢式PCR检测技术,可同时检测β珠蛋白基因及与β珠蛋白基因紧密连锁的HumTH01基因,并对4例已出生的重型β地贫患儿及双方均为β地贫基因携带者的夫妇应用多重巢式PCR进行了β地贫的PGD。结果利用单细胞多重巢式PCR,可以同时检测中国人常见的16种β地贫突变类型,单个淋巴细胞平均扩增效率为91．3％,平均等位基因脱扣(ADO)率为17．0％。对4对夫妇进行4个周期PGD,共活检33个胚胎,获得33个卵裂球,其中30个卵裂球扩增成功,扩增效率为90．9％,ADO率为13．3％。26个胚胎经PCR分析后获得明确诊断,移植了8个胚胎,获得1例临床妊娠。孕17周时经脐带血穿刺,证实为完全正常胚胎,现已出生1名正常女婴。结论应用单细胞多重巢式PCR技术可对β地贫进行植入前遗传学诊断,达到优生目的。     

Preimplantation HLA typing: having children to save our loved ones

Preimplantation tissue typing has been proposed as a method for creating a tissue matched child that can serve as a haematopoietic stem cell donor to save its sick sibling in need of a stem cell transplant. Despite recent promising results, many people have expressed their disapproval of this method. This paper addresses the main concerns of these critics: the risk of preimplantation genetic diagnosis (PGD) for the child to be born; the intention to have a donor child; the limits that should be placed on what may be done to the donor child, and whether the intended recipient can be someone other than a sibling. The author will show that these concerns do not constitute a sufficient ground to forbid people to use this technique to save not only a sibling, but also any other loved one's life. Finally, the author briefly deals with two alternative scenarios: the creation of a human leukocyte antigen (HLA) matched child as an insurance policy, and the banking of HLA matched embryos.     

Preimplantation HLA testing

Context  Preimplantation genetic diagnosis (PGD) has become an option for couples for whom termination of an affected pregnancy identified by traditional prenatal diagnosis is unacceptable and is applicable to indications beyond those of prenatal diagnosis, such as HLA matching to affected siblings to provide stem cell transplantation. Objective  To describe preimplantation HLA typing, not involving identification of a causative gene, for couples who had children with bone marrow disorders at need for HLA-matched stem cell transplantation. Design, Setting, and Participants  HLA matching procedures conducted at a single site during 2002-2003 in an in vitro fertilization program for 9 couples with children affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched stem cell transplantation. In 13 clinical cycles, DNA in single blastomeres removed from 8-cell embryos following in vitro fertilization was analyzed for HLA genes simultaneously with analysis for short tandem repeats in the HLA region to select and transfer only those embryos that were HLA matched to affected siblings. Main Outcome Measures  Results of HLA matching and pregnancy outcome. Results  As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies and birth of 5 HLA-matched healthy children. Conclusion  This is the first known experience of preimplantation HLA typing performed without PGD for a causative gene, providing couples with a realistic option of having HLA-matched offspring to serve as potential donors of stem cells for their affected siblings.      

Is there an ethical difference between preimplantation genetic diagnosis and abortion?

When a person at risk of having a child with a genetic illness or disease wishes to have an unaffected child, this can involve difficult choices. If the pregnancy is established by sexual intercourse, the fetus can be tested early in pregnancy, and if affected a decision can be made to abort in the hope that a future pregnancy with an unaffected fetus ensures. Alternatively, preimplantation genetic diagnosis (PGD) can be used after in vitro fertilisation (IVF) to select and implant an unaffected embryo that hopefully will proceed to term and produce a healthy baby. We are aware that many individuals at risk regard the latter as ethically more acceptable than the former, and examine whether there is an ethical difference between these options. We conclude that PGD and implantation of an unaffected embryo is a more acceptable choice ethically than prenatal diagnosis (PND) followed by abortion for the following reasons: Choice after PGD is seen as ethically neutral because a positive result ("a healthy pregnancy") balances a negative result ("the destruction of the affected embryo") simultaneously (assuming the pregnancy proceeds to full term and a healthy baby is born). While there is usually the intention to establish a healthy pregnancy after an abortion, this is not simultaneous; A woman sees abortion as a personal physical violation of her integrity, and as the pregnancy proceeds she increasingly identifies with and gives ethical status to the embryo/fetus as it develops in utero and not in the laboratory; Many people see aborting a fetus as "killing", whereas in the case of PGD the spare embryos are "allowed to die". We argue that this difference of opinion gives further weight to our conclusion, but note that this has been addressed and debated at length by others.     

X、Y、18三色探针同时检测植入前人胚的实验研究

目的:探讨X、Y、18 三色探针同时检测植入前人胚,以鉴定其性别及倍体的可行性 .方法:对体外受精(in vitro fertilizatio n, IVF)所获18个剩余胚104个卵裂球,采用X、Y、18三色着丝粒探针进行FISH分析. 结果:FISH分析成功率97.5%(77/79), 9例(9/11,81.9%)单精受精胚胎为二倍体, 性染色体嵌合未影响胚胎性别的诊断, 7例双精受精胚胎呈多种倍体异常. 结论: 采用X 、Y、18三色探针FISH法同时检测植入前人胚准确、有效,每胚分析2 个卵裂球具有代表性.     

Extending preimplantation genetic diagnosis: medical and non-medical uses

New uses of preimplantation genetic diagnosis (PGD) to screen embryos prior to transfer raise ethical, legal, and policy issues that deserve close attention. Extensions for medical purposes, such as to identify susceptibility genes, late onset disease, and human leukocyte antigen (HLA) matching, are usually ethically acceptable. Whether embryo screening for gender, perfect pitch, or other non-medical characteristics are also acceptable depends upon the parental needs served and the harm posed to embryos, children, and society. Speculations about potential future uses of PGD should not prevent otherwise acceptable current uses of PGD.     

两种助孕方式下D5、D6囊胚行冻融移植后妊娠结局的比较

目的 比较体外受精-胚胎移植(IVF-ET)与胚胎植入前遗传学诊断或胚胎植入前遗传学筛查(PGD/PGS)周期中D5、D6囊胚形成情况及妊娠结局,同时探究D5、D6囊胚的发育潜能及两种助孕方式的优劣.方法 回顾性分析行IVF-ET助孕的138个周期及PGD/PGS筛查助孕的148个周期,按移植胚胎的发育天数分为IVF-D5组、IVF-D6组、PGD/PGS-D5组和PGD/PGS-D6组,分析比较各组的一般情况、囊胚形成情况及冻融移植后的种植率、临床妊娠率、流产率等.结果 IVF-D5组与IVF-D6组相比、PGD/PGS-D5组与PGD/PGS-D6组相比,D5组囊胚形成率、优质胚胎率、种植率、临床妊娠率均高于D6组(P＜0.05);PGD/PGS周期中D5组整倍体囊胚检出率高于D6组(p＜0.05);IVF-D5组与PGD/PGS-D5组相比、IVF-D6组与PGD/PGS-D6组相比,两组间的囊胚形成率、优质胚胎率、种植率及临床妊娠率均差异无统计学意义.结论 两种助孕方式下冻融移植D5囊胚比D6囊胚可获得较好的临床妊娠结局;与IVF相比,PGD/PGS并未明显改善患者的妊娠结局.     

常染色体显性多囊肾疾病行胚胎植入前遗传学诊断的实验研究

目的：建立由PKD1突变所致常染色体显性多囊肾疾病（autosomal dominant polycystic kidney disease,ADPKD）的胚胎植入前遗传学诊断（preimplantation genetic diagnosis,PGD）方法。方法：①通过微卫星连锁分析确定2个多囊肾家系的ADPKD致病基因。检测的微卫星包括为与PKDI连锁的KG8、SM6、CW4和CW2以及与PKD2连锁的D4S1534、D4S1563、D4S414和D4S423。②对18个淋巴细胞和1个PKD1突变所致ADPKD成员行常规体外受精胚胎移植后的5个废弃胚胎15个卵裂球行多重巢式PCR和毛细管电泳检测与PKD1连锁的微卫星分型。结果：①KG8、CW4和CW2可作为连锁微卫星分析外周血和单个细胞的PKD1突变;②2个家系的致病基因均为PKD1;③单个卵裂球扩增成功率为86.67％（13／15）,单个淋巴细胞扩增成功率为88.89％（16／18）,CW4等位基因脱扣率为25％（4／16）,CW2未发现等位基因脱扣,均未发现污染,2个胚胎携带致病基因。结论：PKD1连锁的微卫星分型可作为PKD1突变所致ADPKD的PGD诊断方法。     

用荧光原位杂交技术进行染色体异常者胚胎植入前遗传学诊断

目的：初步探索应用荧光原位杂交(FISH)技术为染色体异常患者进行胚胎植入前遗传诊断(PGD)。方法：针对染色体结构异常的种类．分别选择亚端粒探针及着丝粒探针，用荧光原位杂交技术为5例染色体异常患者进行植入前遗传学诊断。结果：5例患者共行7个PGD周期，共获卵134个，活检47个胚胎，FISH分析可供移植胚胎16个，6个移植周期移植其中15个胚胎，获得1例临床妊娠，并经产前诊断验证，已顺利诞生1健康男婴。结论：荧光原位杂交技术能有效地应用于染色体异常者胚胎植入前遗传学诊断。     

Oct4基因在猪孤雌激活和体外受精早期胚胎中的表达特征

摘 要：[目的]研究植入前胚胎发育重要基因Oct4在猪孤雌和体外受精胚胎中的表达特征。[方法]收集成熟卵母细胞、孤雌和体外受精2细胞、4细胞、8细胞和囊胚，做荧光定量PCR，以成熟卵母细胞做对照分析相对表达量。[结果]孤雌组和体外受精组在8细胞期Oct4表达量均最高（p<0.05），在孤雌和体外受精组囊胚相对于其他时期Oct4表达量最低（p<0.05）。在同一时期孤雌和体外受精Oct4表达并没有差异。[结论]多能性基因Oct4在卵裂发育时期表达量动态变化，孤雌胚胎在一定程度上可作为体外胚胎基因表达的模型，且不同的胚胎培养条件可能导致基因表达的不同。     

TaqMan-MGB探针实时荧光PCR在β地中海贫血植入前诊断中的应用研究

目的建立单细胞MGB探针荧光PCR检测β地贫的方法以及在β地贫PGD中的应用研究。方法获取β地贫基因携带者单个淋巴细胞,建立稳定的单细胞MGB探针荧光PCR检测技术,并对2例双方均为β地贫基因携带者的夫妇应用MGB探针荧光PCR进行了β地贫的PGD。结果利用单细胞MGB探针荧光PCR检测了β地贫基因携带者的200个单个淋巴细胞的基因型,平均扩增效率为94%,平均等位基因脱扣(ADO)率为2.5%。对两对夫妇进行4个周期PGD,共活检16个胚胎,获得16个卵裂球,其中15个卵裂球扩增成功,扩增效率为93.8%,ADO率为6.2%。14个胚胎经PCR分析后获得明确诊断,移植了4个胚胎,获得1例临床妊娠。孕11周时经腹吸取绒毛,证实为完全正常胚胎,最终分娩了一正常女婴。结论应用单细胞MGB探针荧光PCR技术可对β地贫以及其它单基因遗传病进行植入前遗传学诊断,达到优生目的。     

Trophinin在人卵母细胞及着床前胚胎细胞上的表达与意义

目的 研究trophinin在人卵母细胞、着床前胚胎细胞上的表达与意义.方法 采用间接免疫荧光技术、应用激光扫描共聚焦显微镜对9个卵母细胞、16个分裂胚胎及12个囊胚进行了荧光检测.结果 trophinin蛋白在人卵母细胞、分裂胚胎以及囊胚上均有表达,且表达逐渐显著增强(P＜0.05).结论 人囊胚可能通过trophinin与表达有相同粘附分子的着床窗期子宫内膜发生同种粘附,从而在人胚胎着床过程中起重要作用.     

种植前人胚胎细胞荧光原位杂交检测染色体非整倍体

建立应用13/21染色体特异性探针荧光原位杂交技术(FISH)检测种植前人类胚胎13/21染色体非整倍体的方法。方法:应用FITC标记的13/21染色体着丝粒部位杂交的α卫星重复序列探针对27个未受精卵母细胞和36个多精受精胚胎的卵裂球进行原位杂交。结果:单倍体卵母细胞核呈现2个杂交信号,固定率80%,杂交效率67%;多精受精胚胎的卵裂球核呈现6个杂交信号,固定率72%,杂交效率65%。结论:应用荧光原位杂交技术在卵母细胞和胚胎细胞可检测13/21染色体非整倍体。     

Preimplantation genetic diagnosis of chromosomal abnormalities by multicolour fluorescence in situ hybridisation

Preimplantation genetic diagnosis (PGD) is an early diagnosis of genetic disorders, prior to the onset of pregnancy. PGD incorporates the latest techniques in assisted reproduction and molecular genetics. Embryos or oocytes are biopsied during culture in vitro and genetic analysis is carried out on the blastomeres or polar bodies. Embryos shown to be free of the genetic disease under investigation are transferred to the uterus. Multicolour fluorescence in situ hybridisation (FISH) is used to diagnose numerical and certain structural abnormalities of chromosomes in the embryo. The common probes used are for chromosomes 13, 18, 21, X and Y. FISH can also be used for PGD of translocations, when one of the parents is a carrier. PGD was carried out recently in 4 cases using multicolour FISH. In one of the embryos, trisomy 18 was detected. Tetraploidy was seen in another embryo. Only chromosomally normal embryos were transferred back to the uterus. Care has to be taken while interpreting FISH signals as the signal may be split, diffused, superimposed or in a different focus.