Three births after preimplantation genetic diagnosis for cystic fibrosis with sequential first and second polar body analysis

OBJECTIVE: The purpose of this study was to determine the accuracy and feasibility of sequential polar body removal and analysis for preimplantation genetic diagnosis of mendelian disorders. STUDY DESIGN: Three couples with risk factors for cystic fibrosis had preimplantation genetic diagnosis with the use of sequential polar body analysis. After stimulation, oocytes were harvested and the first polar bodies were removed and analyzed on the day of aspiration. The following day, after fertilization, the second polar bodies were aspirated. Only embryos known to have inherited the normal maternal allele were transferred. RESULTS: All three couples had successful pregnancies resulting in the births of unaffected infants. CONCLUSIONS: Preimplantation diagnosis with the use of sequential polar body removal is feasible and can prevent the establishment of genetically abnormal pregnancies for couples at risk. (Am J Obstet Gynecol 1998;178:1298-306.)     

单基因病植入前遗传学诊断难点及误诊原因分析

可供检测的遗传物质极少是单基因病植入前遗传学诊断（PGD）的瓶颈问题。单基因病PGD误诊的原因主要包括单细胞聚合酶链反应（PCR）固有问题、胚胎细胞固有问题以及与诊断技术不相关的人为错误等。文章首先分析单基因PGD的诊断难点,在此基础上介绍欧洲人类生殖与胚胎协会（ESHRE）PGD联盟报道的误诊案例及其对诊断技术的验证。     

植入前遗传学诊断技术风险环节

植入前遗传学诊断（PGD）相关的技术在近20年迅猛发展,随之而来的是在技术应用时可能存在的问题和风险。从遗传咨询到胚胎培养、活检,再到遗传学诊断的各个环节都有需要引起重视的问题。等位基因脱扣和早期胚胎的染色体嵌合现象是目前诊断中最主要的导致误诊的风险因素。文章就目前PGD中各个可能的风险环节以及相应的应对措施进行阐述。     

Polymerase chain reaction amplification specificity: Incidence of allele dropout using different DNA preparation methods for heterozygous single cells

Purpose: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. Methods: One- or two-cell lymphoblasts (n=100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the -hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN₂); (2) potassium hydroxide/dithiothreitol, heated to 65°C, followed by acid neutralization (KOH); (3) boiling only (BI); and (4) water only (H₂O). Cells were analyzed by polymerase chain reaction using nested primers. Results: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN₂ [38], 11, 16, 11; KOH [97], 91, 5, 1; BI [41], 17, 13, 11; and H₂O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN₂ [85], 53, 14, 18; and KOH [97], 96, 1, 0. Conclusions: KOH lysis was significantly greater than with all other methods (P<0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.Presented at the 42nd annual meeting of the Society for Gynecologic Investigation, Chicago, Illinois, March 15–18, 1995 and at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

植入前遗传学诊断及筛查咨询

随着分子生物学技术的飞速发展及其在生殖领域的应用,植入前遗传学诊断（PGD）、植入前遗传学筛查（PGS）的遗传咨询变得更加复杂。在PGD、PGS的遗传咨询中,医生应充分告知患者PGD、PGS的应用现状、利弊、可能的预后、技术缺陷与安全性问题。同时,经PGD、PGS成功妊娠的孕妇,仍需进行常规的产前诊断,这一点对于PGD、PGS的安全性至关重要。     

Assessing congenital anomalies after preimplantation genetic diagnosis

Background: Preimplantation genetic diagnosis is an exciting advance in prenatal diagnosis. However, the safety of embryo biopsy must be determined with respect to both pregnancy rate and cogenital anomalies. Analysis: Too few pregnancies have been reported to allow meaningful inferences to be drawn, for which reason data on pregnancy losses and anomalies after conventional IVF were first reviewed. Loss rates are approximately 25%, and anomaly rates are not increased over that observed in the general population. Unfortunately, considerable methodological problems exist in published surveys: lack of proper controls, failure to take into account potential confounding variables, anomaly surveillance that is inconsistent with respect to the vigor with which anomalies are sought, inclusion or exclusion of minor anomalies, inclusion or exclusion of anomalies evident only on ultrasound, and even inclusion or exclusion of anomalies present in terminated pregnancies. We recommend prospective surveillance for major anomalies, defined as those causing death, major handicap or requiring surgery. Prospective surveillance ideally dictates collection of intake information at the time pregnancy is diagnosed, surveillance during pregnancy to exclude teratogenic influences, and systematic neonatal anomaly surveillance.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

New perspectives on preimplantation genetic diagnosis and preimplantation genetic screening

Preimplantation genetic diagnosis is a procedure that involves the removal of one or more nuclei from oocytes (a polar body) or embryos (blastomeres or trophectoderm cells) in order to test for problems in genome sequence or chromosomes of the embryo prior to implantation. It provides new hope of having unaffected children, as well as avoiding the necessity of terminating an affected pregnancy for genetic parents who carry an affected gene or have balanced chromosomal status. Polymerase chain reaction-based molecular techniques are the methods used to detect gene defects with a known sequence and X-linked diseases. The indication for using this approach has expanded for couples who are prevented from having babies because they carry a serious genetic disorder to couples with conditions that are not immediately life threatening, such as cancer predisposition genes and Huntington disease. In addition, fluorescent in situ hybridization (FISH) has been widely applied for the detection of chromosome abnormalities. FISH allows the evaluation of many chromosomes at the same time, up to 15 chromosome pairs in a single cell. Preimplantation genetic screening, defined as a test that screens for aneuploidy, has been most commonly used in situations of advanced maternal age, a history of recurrent miscarriage, a history of repeated implantation failure, or a severe male factor. Unfortunately, randomized controlled trials have as yet shown no benefit with respect to preimplantation genetic screening using cleavage stage biopsy, which is probably attributable to the high levels of mosaicism at early cleavage stages and the limitations of FISH. Recently, two main types of array-based technology combined with whole genome amplification have been developed for use in preimplantation genetic diagnosis; these are comparative genomic hybridization and single nucleotide polymorphism-based arrays. Both allow the analysis of all chromosomes, and the latter also allows the haplotype of the sample to be determined. The promising results of these two approaches will inspire further validation of these array platforms, even at the single-cell level. It remains to be decided which embryo stage is the best for biopsy. Moreover, if randomized controlled trials are confirmed to play a role in increasing delivery rates, this will be a major step forward for assisted reproductive technology patients around the world.     

Simultaneous Single-Cell Detection of Two Mutations for Cystic Fibrosis

PURPOSE: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). METHODS: The reported test procedures made use of specific cell lines (lymphoblasts, fibroblasts) of known CF mutation status to determine the efficiency of signal generation and prevalence of allele dropout (ADO) during amplification. RESULTS: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. CONCLUSIONS: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.     

Reliability of polymerase chain reaction (PCR) analysis of single cells for preimplantation genetic diagnosis

Purpose We investigated the reliability of polymerase chain reaction (PCR) genotype analyses performed on single cells for the purposes of preimplantation genetic analysis.Methods We performed blind analysis of 130 single skin fibroblasts heterozygous for the -F508 mutation in the cystic fibrosis transmembrane regulator (CFTR) gene and 73 single skin fibroblasts from an individual heterozygous for the Xbapolymoporphic site of the Factor VIII gene.Results Amplification was successful for 116 cells and 52 cells respectively and in all but one case (a CFTR analysis) both alleles were amplified. The incidence of diagnostic error was 1 out of 203 analyses or 0.0043. We conclude that PCR is a reliable method for determining the genotype of single cells for the purposes of preimplantation genetic analysis.     

植入前遗传学诊断周期促排卵方案选择及特点分析

控制性促排卵是植入前遗传学诊断（PGD）中的关键步骤之一。不同遗传性疾病可供移植的胚胎比例不同,因此对获卵数的要求有所不同。某些遗传性疾病本身对卵巢反应性可能有一定影响。文章首先介绍遗传性疾病中遗传方式决定的可供移植胚胎比例,然后重点分析PGD中控制性促排卵的特点,包括遗传性疾病可能对卵巢反应性的影响以及PGD中促排卵方案的选择和目前文献报道PGD获卵数的截断值。     

Comparative perspectives: regulating preimplantation genetic diagnosis in Canada and the United Kingdom

Preimplantation genetic diagnosis (PGD) in the United Kingdom is governed by a centralized regulatory agency, while Canada's current approach to regulating PGD is neither integrated nor comprehensive. Though concerns have been raised about state regulation of assisted reproductive technologies (ARTs), Canada's move toward centralized oversight of these technologies will lead to improvements in uniformity and transparency of regulation, and will provide a central forum for policy debate and discussion.     

单核苷酸多态性微阵列在染色体易位植入前遗传学诊断中的应用

染色体易位是不孕不育的重要原因之一,也是植入前遗传学诊断 （PGD）的主要适应证之一。单核苷酸多态性微阵列（SNP array）是近年用来于PGD诊断的新技术。与传统的单细胞诊断方法相比较,SNP微阵列具有更多的优点。文章从SNP array原理、SNP array PGD国内外现状及适应证、优缺点及SNP array在PGD中的应用和展望等方面进行阐述。