Reliability of gender determination using the polymerase chain reaction (PCR) for single cells

Contamination with extraneous DNA sequences is a frequent problem when performing PCR analysis of single cells. This report describes our experience with eliminating contaminating DNA sequences from PCR reagents for the purposes of gender identification. We have used amplification of Y-specific sequences to identify the gender of single human amniocytes. Female cells consistently showed no Y-specific bands but only 80% of male cells showed the expected intense Y-specific band. This phenomenon could lead to incorrect gender identification of single cells. We developed a technique of simultaneous amplification of X- and Y-specific sequences to prevent misdiagnosis because of failed PCR, which allows accurate preimplantation gender determination for women at risk for conceiving children with X-linked genetic discoses. We analyzed the gender of 141 consecutive single cells in a blinded manner without a single incorrect gender assignment     

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.     

Allele dropout in sequential PCR and FISH analysis of single cells (cell recycling)

Purpose: Our purpose was to investigate the feasability of using sequential PCR and FISH analysis of single cells for preimplantation diagnosis. Methods: Protocols for sequential PCR and FISH analysis of a single fibroblast (cell recycling) were optimized for six loci and the rates of allele specific dropout (ADO) were determined. Results: Conditions that allow reliable genotyping of single cells in lysis buffer were not optimal for amplifying fibroblasts fixed to coverslips. After optimizing conditions, we observed a success rate of 85% for both analyses in sequential PCR-FISH experiments in single cells for the four loci studied. The individual success rates for each technique revealed a slightly higher rate for FISH (91–95%) than for PCR (85–87%) for single cells on coverslips. The presence of two hybridization signals in FISH experiments demonstrated that the failure to amplify both alleles from heterozygous cells on coverslips was due to true ADO, and not the loss of chromosomal material. The ADO rate observed on coverslips varied between 10 and 14%, which is significantly higher than that observed in solution, even after meticulous optimization. Conclusions: Sequential PCR and FISH analysis of single cells remains an attractive possibility. However, until the problem of the increased rate of ADO is resolved, cell recycling should not be applied to clinical preimplantation genetic analysis.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.     

Influence of mosaicism on sexing of human preembryos detected by the polymerase chain reaction

Purpose: Preimplantation sex determination using a single cell by the polymerase chain reaction (PCR) was investigated to elucidate the influence of mosaicism. Methods: The SRY and ZFX genes were coamplified as target sequences for the Y and X chromosomes, respectively. The sensitivity of the single and nested PCR method was examined initially followed by amplification of single amniocytes by the nested PCR. Then the sex of single blastomeres at the three- and nine-cell stages was determined by the nested PCR. Results: The nested PCR was 10⁴-fold more sensitive than the single PCR. Sex determination was possible in 97.5% (117/120) of the blastomeres tested. However, the correspondence rate for all blastomeres within a single embryo was only 60% (12/20 embryos). Among the remaining embryos for which sexing of all blastomeres was not consistent, only one blastomere showed findings indicating the presence of mosaicism (or pseudomosaicism). Conclusions: At least two blastomeres need to be assessed when determining the sex of an embryo in order to avoid misdiagnosis due to mosaicism (or pseudomosaicism).     

Rapid Trisomy Diagnosis (21, 18, and 13) Using Fluorescent PCR and Short Tandem Repeats: Applications for Prenatal Diagnosis and Preimplantation Genetic Diagnosis

Purpose and Methods: Prenatal diagnosis of fetal trisomies is usually performed by cytogenetic analysis from amniotic fluid. This requires lengthy laboratory procedures and high costs and is unsuitable for large-scale screening of pregnant women. An alternative method, which is rapid and inexpensive and may potentially be suitable for diagnosing trisomies even from single fetal cells, is the fluorescent polymerase chain reaction (F-PCR) using polymorphic small tandem repeats (STRs). Results: In this paper we present data demonstrating that fluorescent PCR amplification of STRs can be used for rapid diagnosis of trisomy 21, trisomy 18, and trisomy 13 and can be successfully applied to both prenatal diagnosis and diagnosis of single cells. This study also reports significant numbers of prenatal diagnoses using quantitative fluorescent PCR. Conclusions: We believe that further studies of greater numbers of samples will determine the absolute reliability of this technique. These results also provide a model for trisomy diagnosis from single cells using multiple STR markers for either preimplantation genetic diagnosis or, potentially, diagnosis from fetal cells isolated from maternal blood.     

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。     

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。     

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。     

Reduced allele dropout in single-cell analysis for preimplantation genetic diagnosis of cystic fibrosis

Background: For couples at risk of transmitting a known single-gene defect, preimplantation genetic diagnosis (PGD) allows the identification and transfer of only unaffected embryos followingin vitro fertilisation (IVF), single-cell biopsy at about the eight-cell stage, and genetic analysis by PCR. This technique therefore avoids the risk of terminating an affected pregnancy diagnosed later in gestation. Methods and Results: Using nested PCR, the F508 mutation causing cystic fibrosis can be detected in single cells and we previously reported successful PGD in a couple in whom both partners carry the F508 mutation. To date we have treated 12 couples in a total of 18 cycles. This resulted in five singleton births confirmed to be homozygous normal. Single blastomeres from disaggregated embryos which had not been transferred were analysed to confirm the original diagnosis and assess reliability in clinical practice. Amplification efficiency and accuracy were high, with blastomeres from embryos diagnosed as homozygous normal or affected. In a proportion of blastomeres from presumed carrier embryos, one of the parental alleles failed to amplify, apparently at random (allele dropout, ADO). A possible explanation is the relative inaccessibility of one of the target allele early in the PCR. To test this we have used single lymphocytes from F508 carriers and investigated the effects of various denaturation temperatures in the early cycles of amplification. Conclusion: Increasing the denaturation temperature reduced the rate of ADO without affecting amplification efficiency.Presented at the 5th Annual Meeting of the International Working Group on Preimplantation Genetics, Hamburg, Germany, June 28, 1995.