Reporting experience on more than 100 blastomeres for diagnosing X-linked disorders

Background: Preimplantation genetic diagnosis (PGD) is a useful option for couples with a risk of transmitting a genetic disease to prevent birth of children affected with monogenic disorders. In this method, diagnoses performed on blastomeres biopsied from 8-cell stage embryos which are created by in vitro fertilization method (IVF). In this way, one could prevent medical abortion by selecting and transferring unaffected embryos. Other usages of PGD are sex selection and HLA typing. Also PGD allows infertile couples who are potentially candidate for IVF and just have one chance to get pregnant, assure their child’s sex or health before pregnancy.Objective: Here we present application of molecular PGD to select unaffected embryos for couples who are carrier of X-linked disorders such as hemophilia A, hemophilia B and Duchenne muscular dystrophy (DMD).Materials and Methods: 19 carrier couples who were candidate for PGD referred to our laboratory. Peripheral blood samples were collected and genomic DNA was extracted using salting out method. Mutation detection carried out using Sanger sequencing method. Fragment analysis and haplotype mapping performed to trace defective alleles in the family using multiplex short tandem repeats (STRs). On day 3 post fertilization a blastomere removed from each embryo. Selected mutation and informative STR markers were checked for each blastomere using nested PCR method. Linkage analysis performed and intended embryos were selected and implanted to mother’s uterus.Results: Since 2009, 140 blastomeres were checked for X-linked disorders. 89 of them were for diagnosing hemophilia A, 16 for hemophilia B and 35 for DMD. From this amount 60 embryos were checked for aneuploidy abnormalities (QF-PCR) in parallel. Totally 81 embryos were transferable which resulted in pregnancy and child birth in 8 cases.Conclusion: PGD is regarded as a powerful diagnostic tool for carrier couples who desire a healthy child and wish to avoid medical abortion. In case of having limitation for performing sequencing in some cases which a large deletion causes the disorder, which usually happens in diseases such as DMD, results obtained from linkage analysis and haplotype mapping are enough reliable to detect unaffected embryos.

Keywords:

PGD , IVF , STR , X-linked disorders