3. Preimplantation genetic diagnosis

A. Successful hematopoietic stem cell transplantation for Fanconi anemia from an unaffected HLA-genotypically-identical sibling selected using preimplantation genetic diagnosis. Grewal SS, Kahn JP, MacMillan ML, Ramsay NKC, Wagner JE. Blood 2004;103:1147-1151.

The authors chose to treat a 6-year-old female with Fanconi anemia with a cord blood transplant. They used preimplantation genetics diagnosis to select an embryo produced by in vitro fertilization that was both unaffected by Fanconi anemia and HLA-identical with the proband. After 5 cycles of in vitro fertilization with intrauterine transfer of 7 embryos over a span of 4 years, successful pregnancy ensued. Twenty-eight days after delivery, the patient was transplanted with the donor’s HLA-identical cord blood hematopietic stem cells. The patient is well with normal hematopoiesis 2.5 years after the transplant. The authors discuss the medical, legal and ethical issues involved with this approach.

(Comment: For a brief summary and review of this article see: No donor? Then create one! Harris RE Blood 2004;103:757-758)

B. Novel universal approach for preimplantation genetic diagnosis of beta-thalassaemia in combination with HLA matching of embryos. Van de Velde H, Georgiou I, De Rycke M, et al. Hum Reprod. 2004;19:700-8.

The authors used an approach similar to that described above (in citation #2) in three families with a child with β-thalassemia. They performed in vitro fertilization (IVF) followed by preimplantation genetic diagnosis (PGD) to exclude affected embryos. Furthermore, they also selected for HLA-identical embryos in order to obtain an unaffected child that can be come an HLA-matched donor for its sibling. Six PGD cycles were performed in two families. Two transfers were done but only one pregnancy was obtained (defined by fetal heart beats on ultrasound) and that ended in an early miscarriage at 8 weeks. The authors reviewed the limitations of this procedure: (1) the method is labor-intensive and expensive, (2) only three of 16 embryos will be "unaffected" by the disease and HLA-matched, (3) the intrinsic implantation capacity of embryos is highly variable and the chance of a successful pregnancy may be not higher than 26%, and (4) embryo selection is likely to raise ethical controversy. (See next citation).

C. Preimplantation HLA testing. Verlinsky Y, Rechitsky S, Sharapova T, Morris R, Taranissi M, Kuliev A. JAMA. 2004;291:2079-85.

HLA matching procedures were conducted at a single site during 2002-2003 in an in vitro fertilization program for 9 couples with children affected by acute lymphoid leukemia, acute myeloid leukemia, or Diamond-Blackfan anemia requiring HLA-matched hematopoietic cell transplantation. In 13 clinical cycles, DNA in single blastomeres removed from 8-cell embryos following in vitro fertilization was analyzed for HLA genes simultaneously with analysis for short tandem repeats in the HLA region to select and transfer only those embryos that were HLA matched to affected siblings. As a result of testing a total of 199 embryos, 45 (23%) HLA-matched embryos were selected, of which 28 were transferred in 12 clinical cycles, resulting in 5 singleton pregnancies and birth of 5 HLA-matched healthy children. Thus, this report describes HLA typing performed without PGD for a causative gene as means of providing couples with a realistic option of having HLA-matched offspring to serve as potential donors of stem cells for their af-fected siblings.

(For further information see: Designer babies - are they a reality yet? Case report: simultaneous preimplantation genetic diagnosis for Fanconi anaemia and HLA typing for cord blood transplantation. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. Reprod Biomed Online. 2000;1:31.
Preimplantation diagnosis for Fanconi anemia combined with HLA matching. Verlinsky Y, Rechitsky S, Schoolcraft W, Strom C, Kuliev A. JAMA. 2001;285:3130-3133. )

D. Development and clinical application of a strategy for preimplantation genetic diagnosis of single gene disorders combined with HLA matching. Fiorentino F, Biricik A, Karadayi H, Berkil H, Karlikaya G, Sertyel S, Podini D, Baldi M, Magli MC, Gianaroli L, Kahraman S. Mol Hum Reprod. 2004;10:445-60.

The authors describe a strategy optimized for preimplantation genetic diagnosis combined with HLA matching. The procedure involves a minisequencing-based genotyping of HLA regions A, B, C and DRB combined with mutation analysis of the gene regions involved by mutation. Analysis of at least eight polymorphic short tandem repeat (STR) markers scattered through the HLA complex has also been included to detect potential contamination and crossing-over occurrences between HLA genes. The strategy was clinically applied for HLA matching in 17 cycles (14 for beta-thalassemia, one for Wiscott-Aldrich syndrome and two for leukemia). A reliable HLA genotype was achieved in 255/266 (95.9%) of the blastomeres. In total, 22 (14.8%) embryos were obtained that were HLA-matched with the affected siblings, 14 (9.4%) of which were unaffected and transferred back to the patients. Four clinical pregnancies were obtained, three of which (one twin, two singletons) are ongoing and were confirmed as healthy and HLA-identical with the affected children. The major advantage of this approach is that the validation of a single assay can be done once and then used for the majority of the patients, reducing notably time needed for preclinical set-up of each case. The authors provide a detailed description of their methods including primers used for PCR amplification and minisequencing genotyping of the HLA regions investigated; and the description of genetic regions amplified, mutations investigated and primers used for β-thalassemia and Wiskott-Aldrich syndrome.

E. Ethical considerations on preimplantation genetic diagnosis for HLA typing to match a future child as a donor of haematopoietic stem cells to a sibling. Pennings G, Schots R, Liebaers I. Hum Reprod. 2002;17:534-8.

This article considers the ethical arguments for and against the application of preimplantation genetic diagnosis (PGD) to select embryos in the hope of conceiving an HLA identical donor sibling for couples with an affected child.They indicated that only embryos HLA matched with an existing sibling in need of a compatible donor of haematopoietic stem cells would be transferred. The main arguments are the instrumentalization of the child, the best-interests standard, the postnatal test for acceptability and the experience of the donor child. It is argued that conceiving a child to save a child is a morally defensible decision on the condition that the operation that will be performed on the future child is acceptable to perform on an existing child. The instrumentalization of the donor child does not demonstrate disrespect for its autonomy or its intrinsic worth.

F. Extending preimplantation genetic diagnosis: the ethical debate. Ethical issues in new uses of preimplantation genetic diagnosis. Robertson JA. Hum Reprod. 2003;18:465-71.

This article describes current and likely future uses of preimplantation genetic diagnosis, and then analyses the ethical issues posed by new uses to screen embryos for susceptibility and late-onset conditions, for HLA-matching for tissue donation to an existing child, and for gender selection. It also addresses ethical issues that would arise in more speculative scenarios of selecting embryos for hearing ability or sexual orientation. The article concludes that except for sex selection of the first child, most current extensions of PGD are ethically acceptable, and provides a framework for evaluating future extensions for nonmedical purposes that are still speculative.

G. Successful umbilical cord blood transplantation for Fanconi anemia using preimplantation genetic diagnosis for HLA-matched donor. Bielorai B, Hughes MR, Auerbach AD, Nagler A, Loewenthal R, Rechavi G, Toren A. Am J Hematol. 2004;77:397-9.

Stem cell transplantation is the only curative treatment for Fanconi anemia. In the absence of matched-sibling donor, an alternative mismatched family or matched unrelated donor can be used, but the results are inferior to the matched-sibling transplant. The use of preimplantation genetic diagnosis (PGD) for combined analysis of mutation and HLA-matching was reported for the first time in 2001. This enables the birth of an unaffected child who can serve as a donor for an affected sibling in need for stem cell transplantation. The authors report successful cord blood transplantation for a Fanconi anemia patient from his HLA-matched sibling, born after PGD that included mutation analysis for Fanconi anemia and HLA typing.