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.     

Preimplantation diagnosis for Fanconi anemia combined with HLA matching.

CONTEXT: The advent of single-cell polymerase chain reaction (PCR) has presented the opportunity for combined preimplantation genetic diagnosis (PGD) and HLA antigen testing. This is a novel and useful way to preselect a potential donor for an affected sibling requiring stem cell transplantation. OBJECTIVE: To perform in vitro fertilization (IVF) and preimplantation HLA matching combined with PGD for Fanconi anemia (FA). DESIGN: DNA analysis for the IVS 4 + 4 A-->T (adenine to thymine) mutation in the FA complement C (FANCC) gene in single blastomeres, obtained by biopsy of embryos, to identify genetic status and HLA markers of each embryo before intrauterine transfer. SETTING: In vitro fertilization programs at large medical centers in Chicago, Ill, and Denver, Colo. PARTICIPANTS: A couple, both carriers of the IVS 4 + 4 A-->T mutation in the FANCC gene with an affected child requiring an HLA-compatible donor for cord blood transplantation. MAIN OUTCOME MEASURES: DNA analysis of single blastomeres to preselect unaffected embryos representing an HLA match for the affected sibling. RESULTS: Of 30 embryos tested in 4 IVF attempts, 6 were homozygous affected and 24 were unaffected. Five of these embryos were also found to be HLA-compatible, of which 2 were transferred in the first and 1 in each of the other 3 cycles, resulting in a pregnancy and birth of an unaffected child in the last cycle. CONCLUSION: To our knowledge, this is the first PGD with HLA matching, demonstrating feasibility of preselecting unaffected embryos that can also be an HLA-compatible source for stem cell transplantation for a sibling.     

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.     

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.     

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 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.     

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。     

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.     

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。     

Prenatal diagnosis of hemoglobinopathies: evaluation of techniques for analysing globin-chain synthesis in blood samples obtained by fetoscopy.

Three techniques for analysing hemoglobin synthesis in blood samples obtained by fetoscopy were evaluated. Of the fetuses studied, 12 were not at risk of genetic disorders, 10 were at risk of beta-thalassemia, 2 were at risk of sickle cell anemia and 1 was at risk of both diseases. The conventional method of prenatal diagnosis of hemoglobinopathies, involving the separation of globin chains labelled with a radioactive isotope on carboxymethyl cellulose (CMC) columns, was compared with a method involving globin-chain separation by high-pressure liquid chromatography (HPLC) and with direct analysis of labelled hemoglobin tetramers obtained from cell lysates by chromatography on ion-exchange columns. The last method is technically the simplest and can be used for diagnosing beta-thalassemia and sickle cell anemia. However, it gives spuriously high levels of adult hemoglobin in samples containing nonlabelled adult hemoglobin. HPLC is the fastest method for prenatal diagnosis of beta-thalassemia and may prove as reliable as the CMC method. Of the 13 fetuses at risk for hemoglobinopathies, 1 was predicted to be affected, and the diagnosis was confirmed in the abortus. Of 12 predicted to be unaffected, 1 was aborted spontaneously and was unavailable for confirmatory studies, as were 3 of the infants; however, the diagnosis was confirmed in seven cases and is awaiting confirmation when the infant in 6 months old in one case. Couples at risk of bearing a child with a hemoglobinopathy should be referred for genetic counselling before pregnancy or, at the latest, by the 12th week of gestation so that prenatal diagnosis can be attempted by amniocentesis, safer procedure, with restriction endonuclease analysis of the amniotic fluid cells.     

罗式易位携带者胚胎植入前遗传学诊断的研究

【目的】应用荧光原位杂交(FISH)技术对罗式易位携带者进行胚胎植入前遗传学诊断。【方法】分别用化学打洞法及机械打洞法对3例罗式易位t（13q14q）患者的体外受精的胚胎活检,用VysisLSI13q14,TelVysion14q探针对卵裂球进行FISH分析,选取正常或平衡的胚胎移植入子宫腔。【结果】3个周期共获卵23个,受精率79%。活检14个胚胎,其中化学打洞法活检9个胚胎,活检后胚胎继续分裂率67%;机械打洞法活检5个胚胎,活检后胚胎继续分裂率为40%。2个周期分别有1个胚胎显示正常或平衡,进行移植,其中1例获得妊娠,妊娠20周时经羊水细胞核型分析证实为正常。【结论】对罗式易位携带者进行胚胎植入前遗传学诊断,可以解决患者的生育障碍。     

单角子宫患者50例辅助生殖助孕临床结局

目的 探讨单角子宫不孕患者行辅助生殖助孕情况。方法回顾分析行体外受精-胚胎移植/卵泡浆单精子注射-胚胎移植(IVF/ICSI-ET) 的50例单角子宫患者（观察组）的临床资料。利用SAS软件对妊娠年龄、不孕年限、孕前体质指数等影响助孕结局的因素按1∶3比例进行Case control matching匹配,选取150例同时期的不孕患者作为对照组。分别比较两组不孕患者的助孕情况及妊娠结局。结果(1) 两组患者基线资料包括年龄、不孕年限、体质指数、抗苗勒管激素（AMH）、基础卵泡刺激素（FSH）、不孕类型、不孕因素、平均获卵数、可利用胚胎数、优质胚胎数、移植胚胎数和HCG日子宫内膜厚度均差异无统计学意义(P>0.05)。(2)观察组50例患者行85个胚胎移植周期,临床妊娠23例共32个周期,其胚胎种植率为28.67%（41/143）,移植周期临床妊娠率37.65%（32/85）;对照组患者150例行288个胚胎移植周期,临床妊娠126例共156个周期,胚胎种植率为34.25%（186/543）,移植周期临床妊娠率54.17%（156/288）。观察组患者临床妊娠率明显低于对照组(P<0.05);(3)相对于对照组患者,观察组患者晚期流产率（28.13%,9/32）（P=0.027）和剖宫产率（70.59%,12/17）（P<0.001）显著升高,足月产率（37.50%,12/32）（P=0.032）显著降低,观察组患者平均孕周（36.01±2.23）周（P=0.039）更小,累积活产率更低（21.18%,18/85）（P=0.002）,余差异均无统计学意义(P>0.05)。结论单角子宫并不影响卵巢储备及促排卵巢反应性和胚胎培养结果,但降低IVF/ICSI助孕的临床妊娠率和累积活产率,增加妊娠流产及新生儿低出生体重风险。     

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。     

植入前遗传学诊断"知情同意"的影响因素与对策

植入前遗传学诊断（Preimplantation Genetic Diagnosis PGD）是辅助生育技术与分子生物学技术相结合而发展的孕前遗传学诊断技术,在植入子宫前淘汰了遗传异常的胚胎,是产前诊断技术的重大进展.但是,由于技术本身存在着一定局限性和不确定性,同时,受到病人认知能力等因素的影响,由此引发了系列伦理学争议.在进行PGD前,一个明了、详尽的患者知情同意过程是必须的.包括通俗全面告知PGD有关信息、手术和检测的局限性和可能结果;充分告知通过PGD所获得的利益和风险.在此基础上针对不同的遗传病检测签署详细的书面知情同意书.     

体外受精-胚胎移植中胚胎碎片对临床妊娠率影响

目的 探讨体外受精-胚胎移植（IVF-ET）中胚胎碎片对临床妊娠率及种植率的影响。方法 观察246例行IVF-ET或卵母细胞浆内单精子注射病人胚胎碎片与临床妊娠率的关系。结果 与正常和轻度碎片的胚胎相比，严重碎片（〉50％）胚胎的临床妊娠率和种植率均明显降低（Х^2=10．112、9．510，P〈0．05），多胎妊娠率差异无显著性。结论 提高胚胎质量，减少移植胚胎数量，有助于提高IVF-ET的成功率。     

辅助生殖技术中选择性单胚胎移植的研究进展

辅助生殖技术（assisted reproductive technology, ART）已成为不孕症患者最有效的治疗方法,但多胎妊娠为其最突出的并发症之一。多胎妊娠常引起羊水过多、先兆子痫、低体质量儿、贫血等多项严重的母婴并发症。避免多胎妊娠的方法是单胚胎移植,选择性单胚胎移植已经成为生殖医学未来的发展方向,单胚胎的选择为临床难题。如何保证临床妊娠率又显著降低各类并发症？对于35岁以下的卵巢储备功能正常患者或有优质囊胚的高龄患者,可通过检测其胚胎发育过程中的代谢产物、胚胎持续监测或植入前遗传学诊断,筛选最具发育潜能的胚胎进行移植,改善临床结局。本文通过文献回顾,探讨选择性单胚胎移植的临床应用前景。     

胚胎早裂联合胚胎发育速度及形态评分在胚胎选择中的应用

目的 探讨胚胎早裂联合胚胎发育速度及形态评分对于体外授精/卵母细胞单精子显微注射(IVF/ICSI)周期中胚胎选择的意义.方法 IVF/ICSI 610周期,随机分为A、B两组.A组(269周期)单以授精后72 h(D3)胚胎发育速度及形态评分选择胚胎;B组(341周期)在此基础上联合胚胎早裂情况进行胚胎选择.比较两组的临床妊娠率和单胚着床率;比较B组移植胚胎中有早裂胚胎和无早裂胚胎者的妊娠结局.结果 B组临床妊娠率和单胚着床率均高于A组(P<0.05);B组移植胚胎中有早裂胚胎者的临床妊娠率和单胚着床率均高于无早裂胚胎者(P<0.01).结论 与单纯的胚胎发育速度及形态评分比较,胚胎早裂联合胚胎发育速度及形态评分进行胚胎评价可显著改善IVF/ICSI的临床结局.     

The ethics of using genetic engineering for sex selection

It is quite likely that parents will soon be able to use genetic engineering to select the sex of their child by directly manipulating the sex of an embryo. Some might think that this method would be a more ethical method of sex selection than present technologies such as preimplantation genetic diagnosis (PGD) because, unlike PGD, it does not need to create and destroy "wrong gendered" embryos. This paper argues that those who object to present technologies on the grounds that the embryo is a person are unlikely to be persuaded by this proposal, though for different reasons.     

男性染色体复杂平衡易位携带者助孕2例并文献复习

目的 探讨男性染色体复杂平衡易位(CCRs)携带者的适宜助孕手段。方法 2例男性CCRs携带者通过胚胎植入前染色体重排检测(PGT-SR)助孕,其中1例夫妇同时行携带者筛查。结果 2例CCRs携带者夫妇均获得可移植胚胎并生育健康子代。其中1例患者联合携带者筛查技术,移植1枚非携带型胚胎,确保生育携带正常核型的后代。2例携带者夫妇的胚胎整倍体率为8.82%(3/34)。结论 PGT-SR可有效帮助CCRs携带者筛选可移植胚胎。携带者筛查技术可进一步筛选非携带型整倍体胚胎,阻断亲代染色体异常向子代的传递。