This relatively low number of unaffected HLA-matched embryos detected in the present series may be influenced by advanced reproductive age of the women involved (approximately 35 years), known to affect significantly the number of available embryos for testing and also the success rate of assisted reproduction. The mean number of embryos available per cycle was under 10 from these women, which clearly limits the chances for finding a sufficient number ofunaffected embryos that are also HLA matched to a sibling. Assuming that one in four embryos is expected to be HLA matched and three of four unaffected, the overall probability of HLA-matched embryos to be also unaffected could not be expected to be higher than 1 in 5.3 embryos. So with the availability of only 8.5 embryos on the average with conclusive results in our material, only 1.6 HLA-matched unaffected embryos may have been detected. Assuming also that not all embryos develop to the status acceptable for transfer, approximately one embryo per cycle could have been available for transfer, which is of course below the optimal number of embryos to be replaced to ensure a clinical pregnancy and birth outcome. Overall, 53 unaffected HLA-identical embryos were transferred in 34 of 46 cycles performed (1.6 on the average), which resulted in 23.5% pregnancy rate, representing the range expected in women of advanced reproductive age.
The other limitation associated with advanced reproductive age is a higher prevalence of aneu-ploidies, which should be tested at least for those chromosomes in which the causative genes are located, and for chromosome 6, to which HLA genes are assigned, as this may affect the accuracy of PGD and HLA typing. Testing for chromosome 6 and for those chromosomes in which the causative genes for the above conditions are localized, revealed 39 (9.8%) embryos with aneuploidies, including a total of 14 trisomies, 24 monosomies, and 1 uniparental disomy, which may have affected the accuracy of diagnosis if not detected. This suggests that aneuploidies should be routinely detected by adding primers for microsatellite markers for chromosome 6 and other chromosomes in which causative gene is localized to the multiplex PCR system. This allows detecting the copy number of chromosomes as well as uniparental disomies of chromosome 6, which may result in misdiagnosis of the HLA matching.
Finally, one of the important phenomena affecting the accuracy of preimplantation HLA typing is a possible recombination in the HLA region, which may lead to misdiagnosis if not detected. As demonstrated in this study, the microsatellite markers linked to the HLA alleles may be tested simultaneously in the multiplex PCR system, allowing detection of recombination in the HLA region, observed in 4.3% of cases in our data (data not shown). Because preimplantation HLA typing may never be able to identify the HLA match for recombinant siblings, haplotype analysis prior to initiation of the actual cycle is required so that the couples maybe informed about their possible options. For example, in one of our cases performed for thalassemia, the fact that the child was recombinant became obvious only during PB1 analysis, without which maternal hyplotypes cannot be established. While paternal haplotypes maybe identified through sperm typing, the testing for maternal haplotypes requires the maternal somatic cell haploidization, which may be performed by somatic cell nuclei transfer and fusion with matured oocytes, as described in Chapter 2 .
Despite the need for further improvement of the technique as mentioned, the presented results shows that couples with affected children requiring HLA-compatible stem cell transplantation have a realistic option to undergo IVF and PGD with combined preimplantation HLA typing, so as to have an unaffected HLA-matched child as potential donor of compatible stem cells for sibling. Together with the previously reported application of PGD for FANCC, and the most recent report on preimplantation HLA typing not involving testing for causative gene, at least 10 HLA-matched children have already been born, confirmed to be HLA matched for affected siblings. Although not all these sibling have yet been treated, preliminary results provide encouraging results. At the present time, umbilical cord blood stem cells collected from these children have already been transplanted to the siblings with FANCC  thalassemia, HED-ID, and Diamond-Blackfan anaemia , resulting in a successful hematopoietic reconstitution in all of these conditions, which suggests that it is an acceptable approach for the couples at risk, despite being highly controversial. On the other hand, it also allows overcoming an important controversy of therapeutic cloning, as it avoids the use of surplus human embryos for obtaining embryonic stem cells (see Chapter 8). Also, no embryo is discarded based on the results of preimplantation HLA typing, as all unaffected embryos are frozen for future use by the couple.
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