M. Vandervorst

Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis

Preimplantation genetic diagnosis (PGD) can be offered as an alternative to prenatal diagnosis (PND) to couples at risk of having a child with a genetic disease. The affected embryos are detected before implantation by fluorescent in situ hybridisation (FISH) for sexing (X‐linked diseases) and chromosomal disorders (numerical and structural) or by polymerase chain reaction (PCR) for monogenic disorders (including some X‐linked diseases). The accuracy and reliability of the diagnosis is increased by analysing two blastomeres of the embryo. However, the removal of two blastomeres might have an effect on the implantation capacity of the embryo. We have evaluated the implantation of embryos after the removal of one, two or three cells in 188 PGD cycles where a transfer was done. The patients were divided into five groups: a first group which received only embryos from which one cell had been removed, a second group which received only embryos from which two cells had been removed, a third group which received a mixture of embryos from which one and two cells had been taken, a fourth group where two and three cells had been removed, and a fifth group where three cells had been removed. The overall ongoing pregnancy rate per transfer was 26.1%, the overall implantation rate per transfer was 15.2% and the overall birth rate was 14.2%. Although pregnancy rates between the groups cannot be compared because the second group (two cells removed) contains more rapidly developing and therefore ‘better quality’ embryos, an ongoing pregnancy rate of 29.1% and an implantation rate of 18.6% per transferred embryo in this group is acceptable, and we therefore advise analysing two cells from a ≥7‐cell stage embryo in order to render the diagnosis more accurate and reliable. Copyright

Preimplantation diagnosis for Huntington's disease (HD): clinical application and analysis of the HD expansion in affected embryos

Huntingtons disease (HD) is an autosomal dominant disease characterized by motor disturbance, cognitive loss and psychiatric manifestations, starting between the fourth and the fifth decade, followed by death within 10–20 years of onset of the disease. The disease‐causing mutation is an expansion of a CAG triplet repeat at the 5′ coding end of the Huntington gene. We have developed a single‐cell PCR assay for the HD gene in order to propose preimplantation genetic diagnosis (PGD) for the couples at risk. We present here our first results with our first nine PGD cycles and also discuss the behaviour of the disease‐causing expansion in pre‐implantation embryos. Copyright

Preimplantation diagnosis for fragile X syndrome based on the detection of the non-expanded paternal and maternal CGG

Fragile X syndrome is the most common monogenic cause of mental retardation in boys. It is always characterized clinically by moderate mental retardation and often by a long face with large everted ears and macro‐orchidism. The causal mutation is an expansion of a CGG triplet repeat in a 5′ exon of the FMR‐1 gene in Xq27.3. We report here for the first time a method for preimplantation genetic diagnosis (PGD) for fragile X syndrome based on the amplification of the CGG triplet in the normal allele. Our candidate–patient population, as well as two clinical preimplantation genetic diagnosis (PGD) cycles which led to a pregnancy with an unaffected fetus, are presented in this paper. Copyright

Preimplantation genetic diagnosis for sickle‐cell anemia and for β‐thalassemia

We developed single‐cell polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples carrying mutations in the β‐globin gene. With PGD the genetic status of an embryo obtained after intracytoplasmic sperm injection (ICSI) is determined by PCR analysis in single blastomeres, allowing only healthy embryos to be transferred to the uterus. We carried out nine PGD cycles using fluorescent PCR for two couples in whom the partners carried sickle‐cell trait. Both couples achieved pregnancies, one of which was spontaneously aborted. We have developed two β‐thalassemia PGD protocols: one for the analysis of the 25‐26delAA and the IVS2+1G>A mutation, and the other for the simultaneous detection of the IVS1+6T>C and the IVS1+110G>A mutations. For the second protocol, both non‐labelled PCR and later fluorescent PCR were used. Both protocols were applied in clinical cycles (two non‐labelled PCR cycles and one fluorescent PCR cycle) for two couples. The patient with the fluorescent PCR‐PGD cycle became pregnant. Overall, the three fluorescent PCR assays were accurate and reliable with amplification efficiencies of minimum 93% and allele dropout (ADO) rates between 0 and 12%. Copyright

Preimplantation genetic diagnosis of Marfan syndrome with the use of fluorescent polymerase chain reaction and the Automated Laser Fluorescence DNA Sequencer

OBJECTIVE To develop and apply clinical preimplantation genetic diagnosis (PGD) for Marfan syndrome. DESIGN Case report. SETTING Centers for medical genetics and reproductive medicine in university hospitals. PATIENT(S) One couple in which the husband was affected with Marfan syndrome. INTERVENTION(S) The couple underwent three intracytoplasmic sperm injection cycles. MAIN OUTCOME MEASURE(S) The correct diagnosis was obtained for embryos in three PGD cycles. RESULT(S) Although all the PGD cycles were followed by ET, no pregnancy ensued. CONCLUSION(S) This assay can provide a reliable and accurate preimplantation diagnosis of Marfan syndrome.

Clinical application of preimplantation genetic diagnosis for cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease characterized by obstruction and chronic infections of the respiratory tract and pancreatic insufficiency. The gene was cloned in 1989 and the most frequent mutation was shown to be the ΔF508 mutation. During PGD, embryos obtained in vitro are checked for the presence or absence of the mutation, after which only embryos shown to be free of the mutation are returned to the mother. Up to 1999, 48 intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF) cycles had been carried out for PGD for CF in 24 couples, and different diagnostic tests had been used to select non‐affected embryos. Thirteen patients became pregnant and 12 healthy babies have been born. Copyright

Two pregnancies after preimplantation genetic diagnosis for osteogenesis imperfecta type I and type IV

Abstract. Osteogenesis imperfecta (OI) is an autosomal dominant genetic disorder characterized by the presence of brittle bones and decreased bone mass (osteopenia), as a result of mutations in the genes that encode the chains of type I collagen, the major protein of bone. The clinical features of the disease range from death in the perinatal period to normal life span with minimal increase in fractures. The present report describes two polymerase chain reaction (PCR)-based assays allowing preimplantation genetic diagnosis (PGD) on the one hand for OI type I, the mildest form, and on the other hand for OI type IV, which is intermediate in severity between OI type I and OI type III. In the couple referred for PGD for OI type I, the female partner carried a 1-bp deletion in exon 43 of the COL1A1 gene, resulting in a premature stop codon in exon 46. The synthesis of too little type I procollagen results from such a non-functional or COL1A1 null allele. In the other couple, referred for PGD for OI type IV, the male partner carried a G to A substitution in exon 19 of the COL1A2 gene, which results in an abnormal gene product due to an αGly247 (GGT) to Ser (AGT) substitution (G247S). Both mutations result in the loss of a specific restriction enzyme recognition site and can therefore be detected by PCR amplification followed by restriction fragment analysis. PCR amplification of genomic DNA of the parents-to-be with one of the two primers fluorescently labelled, followed by automated laser fluorescence (ALF) gel electrophoresis of the amplified and restricted fragments, allowed a distinction between the healthy and affected genotypes. PCR on single Epstein-Barr-virus (EBV)-transformed lymphoblasts resulted in acceptable amplification efficiencies (87% and 85% for OI type I and OI type IV respectively) and the allele drop-out (ADO) rate was assessed at 11.5% and 11.1% for OI type I and OI type IV respectively. With research blastomeres, 100% amplification rates were obtained and no contamination was observed in the blank controls, which validated the tests for clinical application. Embryos obtained after intracytoplasmic sperm injection (ICSI) were evaluated for the presence of the normal genotype of the non-affected parent. For OI type I, two frozen-thawed ICSI-PGD cycles and two fresh ICSI-PGD cycles were carried out for the same couple. The transfer of two unaffected embryos in the last cycle resulted in a twin pregnancy. A twin pregnancy was also achieved in one clinical ICSI-PGD cycle for OI type IV.