Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review

Embryonic aneuploidies may be responsible for pregnancy failure in many IVF patients. In recent years, fluorescent in situ hybridisation (FISH) for multiple chromosomes has been used to document a high frequency of chromosomal errors and aneuploidy in human preimplantation embryos and, after embryo biopsy, to select embryos that are more likely to implant. Such studies suggest that women with recurrent miscarriage and advanced maternal age may benefit most from preimplantation genetic diagnosis with aneuploidy screening (PGD-AS). The success of PGD-AS is likely to be enhanced by new technologies, such as comparative genomic hybridisation, which enable full karyotyping of single cells. Copyright © 2002 John Wiley & Sons, Ltd.     

Normal pregnancy after preimplantation DNA diagnosis of a dystrophin gene deletion

To perform preimplantation DNA diagnosis for Duchenne muscular dystrophy (DMD) in a female carrier of a dystrophin gene deletion of exons 3–18, we developed a polymerase chain reaction (PCR)-based assay of exon 17 sequences. Exon 17 was efficiently amplified in all 50 single blastomeres of normal control embryos and in five blastomeres of one male embryo of the DMD carrier obtained after a first preimplantation diagnosis (PID) for gender determination. In ten blastomeres of another two male embryos of the DMD carrier, no PCR signals were observed, probably as a result of the deletion. After intracytoplasmic sperm injection, embryos were analysed for exon 17 and three of the four embryos showing normal PCR signals were replaced, resulting in a singleton pregnancy. Prenatal diagnosis showed a female karyotype and DNA analysis indicated that the fetus was not a DMD carrier.     

Aspects of biopsy procedures prior to preimplantation genetic diagnosis

Today, preimplantation genetic diagnosis (PGD) is offered in over 40 centres worldwide for an expanded range of genetic defects causing disease. This very early form of prenatal diagnosis involves the detection of affected embryos by fluorescent in situ hybridization (FISH) (sex determination or chromosomal defects) or by polymerase chain reaction (PCR) (monogenic diseases) prior to implantation. Genetic analysis of the embryos involves the removal of some cellular mass from the embryos (one or two blastomeres at cleavage-stage or some extra-embryonic trophectoderm cells at the blastocyst stage) by means of an embryo biopsy procedure. Genetic analysis can also be performed preconceptionally by removal of the first polar body. However, additional information is then often gained by removal of the second polar body and/or a blastomere from the embryo. Removal of polar bodies or cellular material from embryos requires an opening in the zona pellucida, which can be created in a mechanical way (partial zona dissection) or chemical way (acidic Tyrode's solution). However, the more recent introduction of laser technology has facilitated this step enormously. Different biopsy procedures at different preimplantation stages are reviewed here, including their pros and cons and their clinical applications. The following aspects will also be discussed: safety of zona drilling by laser, use of Ca²⁺/Mg²⁺-free medium for decompaction, and removal of one or two cells from cleavage-stage embryos. Copyright © 2001 John Wiley & Sons, Ltd.     

Mosaicism of autosomes and sex chromosomes in morphologically normal,monospermic preimplantation human embryos

We have previously detected chromosome abnormalities in human embryos whilst identifying the sex for preimplantation diagnosis of X-linked disease. In this study we assess the incidence of these abnormalities, both for sex chromosomes and autosomes 1 and 17, using dual fluorescent in situ hybridization (FISH). Sixty-nine normally fertilized embryos of good morphology at the 6–10 cell stage (day 3 post-insemination) were examined. The embryos were spread whole using HCl and Tween 20 to dissolve the cytoplasm. Thirty-four embryos were analysed for the sex chromosomes and 35 for autosomes 1 and 17. All probes were directly labelled with fluorochromes allowing analysis in 2 h. Control lymphocytes demonstrated that the probes were of high specificity. For the sex chromosomes, five embryos were mosaic (15 per cent) with the remaining 29 being uniformly XX or XY. In no case was an XX nucleus found in an otherwise XY embryo, indicating that even though mosaicism for the sex chromosomes is present, such abnormalities would not lead to a misdiagnosis of sex. For the autosomes, 16 embryos were abnormal (46 per cent); one embryo was triploid, one was monosomic for chromosome 1, and ten others were diploid mosaics (three diploid/aneuploid, three diploid/polyploid, and four diploid/haploid). A further four embryos had variable chromosome numbers in the majority of nuclei which appeared to be the result of uncontrolled mitotic division. The presence of haploidy or double monosomy, which occurred in 15 per cent of nuclei, has important implications for the diagnosis of trisomies and dominant disorders.     

Strategies for prenatal and preimplantation genetic diagnosis in Marfan syndrome (MFS)

Marfan syndrome (MFS) is an autosomal dominant disorder with a prevalence of 2–3 per 10 000 individuals. Symptoms range from skeletal overgrowth, cutaneous striae to ectopia lentis and aortic dilatation leading to dissection. Prenatal diagnosis was until recently mainly performed in familial cases by linkage analysis. However, mutation detection has become available with thorough screening methods. The phenotypic variability observed in MFS makes reproductive options difficult, as molecular diagnosis cannot predict clinical severity of the disease. Data are presented on 15 prenatal and/or preimplantation genetic diagnoses (PGD) in nine families, originating from Belgium, the Netherlands, Spain and France. In four families data from linkage analysis were used, whereas in five other families the causative FBN1 mutation was characterised. Four PGD cycles in two couples led to one ongoing pregnancy. In addition, two amniocenteses and nine chorionic villus (CV) samplings were performed. In five pregnancies an affected fetus was diagnosed. In one of them, the couple chose to continue the pregnancy and an affected child was born, whereas the other four couples decided to terminate the pregnancy. It is expected that the greater availability of mutation testing of the FBN1 gene will increase requests for prenatal diagnosis. PGD appears to be an acceptable alternative for couples facing ethical reproductive dilemmas. Copyright © 2002 John Wiley & Sons, Ltd.     

Personal desires of patients and social obligations of geneticists: applying preimplantation genetic diagnosis for non-medical sex selection

The arguments against the use of preimplantation genetic diagnosis (PGD) for non-medical sex selection are analysed. It is concluded that the distinction between medical and non-medical reasons is difficult to maintain, that the disproportionality of means and end is not a decisive counterargument and that the fear of damage to the reputation of PGD does not justify the refusal of controversial applications. Moreover, since non-medical sex selection does not belong to basic health care, it should not be equally accessible to all. The position defended in this article is founded on two basic principles: (1) medical reasons have priority on non-medical reasons, and (2) personal reasons do not qualify for public funding. In order to respect both principles, it is proposed that restrictions should be installed to control the number of requests for social sexing and that a tax should be imposed on these elective services. The tax should compensate the society for the investment it made in the training and education of the physician. Copyright © 2002 John Wiley & Sons, Ltd.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.