|Experience On Preimplatation Genetic Diagnisis Combined With HLA Matching
ESHRE 2005 Copenhagen
19-22 June 2005
INTRODUCTION: Recently, Preimplantation Genetic Diagnosis (PGD) has been considered for several indications beyond its original purpose, not only to test embryos for a genetic disease, but also to select embryos for a non-disease trait, such as specific Human Leukocyte Antigen (HLA) haplotypes, related to immune compatibility with an existing affected child in need of an hematopoetic stem cell transplant.
We report our experience on preimplantation HLA matching, describing strategies and overall outcome data of 60 cycles (54 for -thalassemia, 1 for Wiscott-Aldrich syndrome, 2 for Diamond-Blackfan anemia and 3 for Acute Lymphoid Leukaemia) from 45 couples overall.
MATERIALS AND METHODS: An indirect single-cell HLA typing protocol based on a multiplex fluorescent polymerase chain reaction (PCR) of short tandem repeat (STR) markers scattered throughout the HLA complex was used. By selecting a consistent number of STR markers an accurate mapping of the whole region can be achieved. The HLA region can be indirectly typed by segregation analysis of the STR alleles and the HLA identity of the embryos with the affected sibling can be ascertained evaluating the inheritance of the matching haplotypes. A nested multiplex PCR assay was used to co-amplify all the selected loci. The first round PCR contained the external primers for the amplification of the informative HLA STR markers selected during the preclinical work-up of each PGD case, the gene regions involved by mutations, STR markers linked to these regions for ADO detection and STR markers used for detection of aneuploidies in patients of advanced reproductive age. The first round multiplex PCR was followed by separate second round PCR reactions for each locus. Mutation analysis was performed using the minisequencing method.
RESULTS: A total of 486 embryos were tested for HLA typing in combination with a genetic disease, and 44 embryos for HLA typing only. A total of 922 blastomeres was analysed, in 848 (92.0%) of which a successful amplification was obtained. A reliable HLA haplotype was obtained in 848/848 (100%) of the blastomeres with positive PCR results. Testing for chromosome 6 copy number revealed 47 (8.9%) embryos with aneuploidies, including a total of 4 (0.8%) trisomies, 43 (8.1%) monosomies, which affected the HLA matching diagnosis for these embryos, leading to a conclusive diagnosis in only 483/530 embryos (91.1%). Recombination was found in 23 (4.8%) embryos, 5 (1.0%) of which were still unaffected and HLA compatible, but were not considered for transfer. In total, 74 (15.3%) embryos revealed an HLA match with the affected siblings, 55 (11.4%) of which resulted unaffected and 46 (9.5%) have been transferred back to patients in 30 of the 60 cycles performed (1.5 on the average). Nine pregnancies were achieved (30.0% per transfer); two pregnancies resulted only biochemical, one spontaneously miscarried and one resulted ectopic, and was then terminated. Five healthy HLA matched children have been already delivered and cord blood stem cells were transplanted to 3 affected siblings, resulting in a successful hematopoietic reconstruction.
CONCLUSION: These results represent one of the most extensive experience in the field and, complemented by other similar experiences, demonstrate that preimplantation HLA matching is a reliable alternative for the achievement of a successful treatment in children affected by severe congenital or acquired bone marrow disorder, in the absence of a compatible related donor.