PREIMPLANTATION DIAGNOSIS OF THALASSEMIAS

Purpose: Preimplantation genetic diagnosis (PGD) is an important optio n for couples at risk of having children with beta-globin mutations to avoid selective abortions of affected fetuses following prenatal diag nosis. Methods: We performed PGD for thalassemia in 12 clinical cycles (IVSI-110, and IVS-745 mutations) using biopsy of the first and secon d polar bodies (PBs) extruded from oocytes during maturation and ferti lization, coupled with nested polymerase chain reaction analysis and r estriction digestion. Results: A total of 118 oocytes was obtained, of which 78 had results for both the first and the second PBs. This resu lted in the selection and transfer of 30 unaffected embryos (2.5 embry os per cycle). To avoid a possible misdiagnosis due to allele dropout (ADO), we have also introduced simultaneous detection of two highly po lymorphic linked markers, a short tandem repeat immediately at the 5' end of the globin gene and HUMTH01 which is a syntenic short tandem re peat. The application of multiplex polymerase chain reaction of the be ta-globin gene and linked polymorphic markers enabled detection of ADO in five first PBs, thus avoiding the transfer of potentially affected embryos resulting from their corresponding oocytes. Conclusions: Conf irmation studies of the embryos resulting from the oocytes predicted t o contain an affected gene confirmed the diagnosis in 98% of the cases , thus demonstrating the accuracy and reliability of PB PGD of thalass emia mutations. The application of PB analysis in six patients resulte d in two ongoing pregnancies with a thalassemia-free fetus already con firmed in both of them by prenatal diagnosis.