Preimplantation diagnosis and HLA typing for haemoglobin disorders

Haemoglobin disorders are among the most frequent indications for preimplantation genetic diagnosis (PGD), introduced as an important option to couples at risk for producing offspring with thalassaemia and sickle cell disease. Previous experience mainly included PGD for beta-thalassaemia, while PGD for alpha-thalassaemia resulting in an unaffected pregnancy has not been reported. This study presents the results of the world's largest experience of 197 PGD cycles for haemoglobin disorders, which includes PGD for alpha-thalassaemia, resulting in 53 clinical pregnancies and birth of 45 healthy children, with five still ongoing. Fifty-four of these cycles were performed in combination with HLA typing, allowing the birth of thalassaemia-free children who were also HLA identical to the affected sibling, with successful stem cell transplantation in one case. As an increasing proportion of patients requesting PGD with HLA typing are of advanced reproductive age, aneuploidy testing was performed simultaneously with PGD. The results show that PGD has now become a practical approach for prevention of haemoglobin disorders, and is gradually being used also for improving access to HLA compatible stem cell transplantation for this group of diseases.     

Preimplantation diagnosis with HLA typing: birth of the first double hope child in France

Preimplantation genetic diagnosis (PGD) is authorized in France since 1999. After 10 years, technical results are encouraging. With the development of new technologies, our team is able to diagnosis the large majority of chromosome translocations and 75 monogenic diseases. However, PGD remains limited because of the growing augmentation of demands causing an increasing delay for the first procedure of more than 18 months. Since 2006, 19 couples asked for a PGD with HLA typing. In January 2011, 11 couples have already been included in our PGD program. The birth of the first child after PGD with HLA typing offers new perspectives of treatment for these couples.     

Preimplantation genetic diagnosis with HLA matching

Preimplantation genetic diagnosis (PGD) has recently been offered in combination with HLA typing, which allowed a successful haematopoietic reconstitution in affected siblings with Fanconi anaemia by transplantation of stem cells obtained from the HLA-matched offspring resulting from PGD. This study presents the results of the first PGD practical experience performed in a group of couples at risk for producing children with genetic disorders. These parents also requested preimplantation HLA typing for treating the affected children in the family, who required HLA-matched stem cell transplantation. Using a standard IVF procedure, oocytes or embryos were tested for causative gene mutations simultaneously with HLA alleles, selecting and transferring only those unaffected embryos, which were HLA matched to the affected siblings. The procedure was performed for patients with children affected by Fanconi anaemia (FANC) A and C, different thalassaemia mutations, Wiscott-Aldrich syndrome, X-linked adrenoleukodystrophy, X-linked hyperimmunoglobulin M syndrome and X-linked hypohidrotic ectodermal displasia with immune deficiency. Overall, 46 PGD cycles were performed for 26 couples, resulting in selection and transfer of 50 unaffected HLA-matched embryos in 33 cycles, yielding six HLA-matched clinical pregnancies and the birth of five unaffected HLA-matched children. Despite the controversy of PGD use for HLA typing, the data demonstrate the usefulness of this approach for at-risk couples, not only to avoid the birth of affected children with an inherited disease, but also for having unaffected children who may also be potential HLA-matched donors of stem cells for treatment of affected siblings.     

Preimplantation genetic testing for aneuploidy: A Canadian Fertility and Andrology Society Guideline

The objective of this guideline from the Canadian Fertility and Andrology Society is to synthesize the evidence on preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm biopsy and 24-chromosome analysis and to provide clinical recommendations using the Grading of Recommendations Assessment, Development and Evaluation (GRADE) framework. To date, randomized controlled trials have been limited to good-prognosis patients who were able to generate two or more blastocysts for biopsy. In this specific population the GRADE analysis of PGT-A shows an increase in the implantation rate and ongoing pregnancy or delivery rate per transfer. Clearly, it is difficult to generalize from this subgroup of patients to the infertility population at large. As a result, the application of PGT-A should be individualized, and patient factors such as age and ability to generate embryos will influence decision-making. Comprehensive patient counselling and informed consent are imperative before undertaking PGT-A. Potential benefits must be weighed against the costs and limitations of the technology, including the risk of embryo damage, false positives, false negatives and the detection of embryonic mosaicism. Future research is required, especially with regard to the use of PGT-A in poorer prognosis patients, and with respect to reporting outcomes per cycle start and cumulatively per retrieval.     

Preimplantation genetic diagnosis for HLA typing in a case of X-linked chronic granulomatous disease

Bone marrow transplantation may be life saving in cases of hematopoietic disease, severe congenital immunodeficiency or malignancy. An HLA-matching sibling often gives the best success, but this may not be an option, nor may an HLA-matching unrelated donor be found. Preimplantation genetic diagnosis with HLA-matching embryos may then be a solution. We report the first Danish child born after such diagnosis with identification of healthy embryos and HLA matching for a sibling with chronic granulomatous disease. Our patient had 13 in vitro fertilization (IVF) cycles; 286 oocytes were collected and 74 embryos analysed. Sixteen of these (22%) were either healthy or carriers. Five embryos were transferred in four stimulated fresh IVF cycles. Four embryos were frozen, and two were later transferred. Two successive clinical pregnancies ensued. In the first, prenatal diagnosis revealed trisomy 21, and the fetus was aborted. In the second pregnancy, chorionic villus sampling revealed a normal karyotype, the diagnosis was confirmed, and the pregnancy was normal. At delivery, 82 mL of cord blood was collected for later transplantation to the diseased sibling.     

Preimplantation testing for phenylketonuria

OBJECTIVE: To use preimplantation genetic diagnosis to achieve a phenylketonuria-free pregnancy in a couple at 50% risk for producing an affected child. DESIGN: DNA analysis of the first and second polar bodies (PB1 and PB2) obtained from oocytes of a heterozygous mother in IVF-ET, with the goal of identifying and transferring back to the patient the embryos resulting from mutation-free oocytes. SETTING: IVF program of Reproductive Genetics Institute, Chicago, Illinois. PATIENT(S): A mother carrying the R408W mutation and a father with compound heterozygosity for R408 and Y414C mutations in phenylalanine hydroxylase (PAH) gene. INTERVENTION(S): Removal and testing for maternal mutation in PB1 and PB2 from each oocyte after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of PB1 and PB2 indicating whether corresponding oocytes were mutation-free, for the purposes of transferring only unaffected embryos resulting from these oocytes. RESULT(S): Of 11 zygotes with both PB1 and PB2, 6 were predicted to be free of phenylketonuria. Of these, 4 were transferred, resulting in an unaffected twin pregnancy and birth of two healthy children. CONCLUSION(S): Preimplantation genetic diagnosis of phenylketonuria resulted in the birth of phenylketonuria-free children. Preimplantation genetic diagnosis by PB analysis in couples with a compound heterozygous male partner is clinically useful.     

Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages

OBJECTIVE: To determine the aneuploidy rate in embryos of women with idiopathic recurrent miscarriages and to evaluate whether preimplantation genetic diagnosis for aneuploidy screening could be a feasible approach to improve the possibility of successful pregnancy in these couples. DESIGN: Prospective cohort study. SETTING: Tertiary university referral center. PATIENT(S): Women (n = 49) with recurrent idiopathic miscarriages. INTERVENTION(S): In vitro fertilization with preimplantation genetic diagnosis for aneuploidy screening. MAIN OUTCOME MEASURE(S): Ongoing pregnancy rate (PR) and aneuploidy rate. RESULT(S): The aneuploidy rate was, respectively, 43.85% and 66.95% in the younger and older group. The ongoing PR per cycle was 25.71% in the younger and 2.94% in the older patients. CONCLUSION(S): There is no therapeutic evidence to prescribe IVF with or without preimplantation genetic diagnosis for aneuploidy screening for this heterogeneous group of patients.     

Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure

Chromosomal abnormalities are thought to be responsible for implantation failure, and among chromosomal abnormalities in normally developing embryos, aneuploidy is the most frequent. Genetic testing of preimplantation embryos for chromosomal aneuploidy allows selection of chromosomally normal embryos, and early detection of chromosomal aberration will increase the chance of conceiving. Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS), performed by polar body or blastomere analysis, is used in infertile patients treated with assisted reproduction technologies, especially in those with a poor prognosis, e.g. repeated IVF failure, advanced maternal age, or recurrent spontaneous abortion. The aim of this paper is to clarify the impact of PGD-AS in repeated implantation failure. In this review, the data collected so far regarding PGD-AS in this patient group will be discussed in depth.     

Preimplantation HLA typing and stem cell transplantation: report of International Meeting, Cyprus, 27-8 March, 2004

There has been progress in the application of stem cell transplantation for the treatment of an increasing number of severe congenital and acquired bone marrow disorders that are currently restricted by the availability of human leukocyte antigen(HLA)-matched related donors. Preimplantation HLA typing has recently been introduced to improve the access to stem cell therapy for inherited bone marrow failures, and its possible use for the treatment of common sporadic malignant and non-malignant bone marrow disorders has also been explored. This paper describes the current experience of preimplantation HLA typing, reviewed by the International Meeting on the subject, which includes preimplantation HLA typing in 147 cycles, 109 of which were carried out as part of preimplantation genetic diagnosis (PGD) for Fanconi anaemia, thalassaemia, Wiscott-Aldrich syndrome, hyperimmunoglobulin M syndrome, hypohidrotic ectodermal dysplasia with immune deficiency, and X-linked adrenoleukodystrophy, and 38 for the sole purpose of HLA typing for leukaemias and aplastic and Diamond-Blackfan anaemias. The applied method resulted in the accurate pre-selection and transfer of HLA-matched embryos, yielding 25 clinical pregnancies and the birth of 14 HLA-matched children to the siblings who required stem cell transplantation.     

Preimplantation genetic diagnosis with HLA matching - a way to save a child

Preimplantation genetic diagnosis can be used to establish a pregnancy with an embryo that is human leukocyte antigen (HLA)-matched to a sibling having a hematological or immunological disease and needing a life-saving bone marrow transplantation. The ethical aspects of this procedure have been discussed intensively. The procedure applies where no unrelated HLA-matching donor is available or when transplantation from an HLA-matching sibling is considered a better solution. It is only offered in a limited number of centers in Europe as this is a challenging procedure. Where both HLA matching and diagnosis of a dominant disease are necessary, only a small proportion of the embryos can be used, and the procedure is not always technically feasible. The clinical pregnancy rate per cycle started is much lower than following normal in vitro fertilization (IVF) due to a high cycle cancellation rate, but the success rate is only somewhat lower when measured per transfer.     

Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review

Embryonic aneuploidies may be responsible for pregnancy failure in many IVF patients. In recent years, fluorescent in situ hybridisation (FISH) for multiple chromosomes has been used to document a high frequency of chromosomal errors and aneuploidy in human preimplantation embryos and, after embryo biopsy, to select embryos that are more likely to implant. Such studies suggest that women with recurrent miscarriage and advanced maternal age may benefit most from preimplantation genetic diagnosis with aneuploidy screening (PGD-AS). The success of PGD-AS is likely to be enhanced by new technologies, such as comparative genomic hybridisation, which enable full karyotyping of single cells.     

Preimplantation genetic diagnosis for Pelizaeus-Merzbacher disease with testing for age-related aneuploidies

Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive demyelinating disorder of the central nervous system, caused by mutations of the proteolipid protein 1 gene (PLP1 gene). As no specific therapy is available for PMD, preimplantation genetic diagnosis (PGD) may be a useful option for couples carrying this mutation. PGD was performed for a couple who had had one child with the L86P mutation in exon 3 of the PLP1 gene. Because of advanced maternal age, PGD for this single-gene disorder was performed together with testing for chromosomal abnormalities. Polar bodies and blastomeres were tested for the presence of maternal mutation and closely linked markers DXS8020 and PLP5' (CA)n. The same blastomeres were also tested for the copy number of chromosomes 13, 16, 18, 21, 22, X and Y, and five chromosomally abnormal embryos were identified. A total of three embryos predicted to be unaffected and free of chromosomal disorder were transferred back to the patient, resulting in a twin pregnancy and the birth of two healthy female infants confirmed to be free of PMD, representing the first PGD for PMD combined with aneuploidy testing.     

Preimplantation HLA haplotyping using tri-, tetra-, and pentanucleotide short tandem repeats for HLA matching

PURPOSE: To aid couples wishing to conceive children who are HLA matched to a sibling in need of a hematopoietic progenitor cell transplant, we developed a preimplantation HLA haplotype analysis of embryos that utilizes tri-, tetra-, and pentanucleotide STR markers. METHODS: For preimplantation HLA genotyping, we use polymorphic STR markers located across the HLA and flanking regions, selecting exclusively tri-, tetra-, and pentanucleotide repeats. These markers can be resolved using either capillary electrophoresis (CE) or polyacrylamide gels. RESULTS: We have developed 43 reliable STR markers for preimplantation HLA matching. Selected STR markers enabled unambiguous identification of embryos whose HLA haplotypes were matched with the affected patient using polyacrylamide gel or capillary electrophoresis. CONCLUSIONS: The use of tri-, tetra-, and pentanucleotide repeat markers and polyacrylamide gels for STR genotyping in HLA matching is a simple and cost effective approach to clinical testing.