C.G. Ekmekci

Seven years of experience of preimplantation HLA typing: a clinical overview of 327 cycles

Preimplantation human leukocyte antigen (HLA) typing allows the birth of healthy children who are potential donors of stem cells for their affected siblings. This technique can be used for acquired diseases such as leukaemia or can be used for single-gene disorders such as thalassaemia. This retrospective study presents clinical data obtained from 171 couples who had undergone 327 preimplantation HLA typing cycles: 262 cycles for HLA typing in combination with mutation analysis and 65 cycles for the sole purpose of HLA typing. Of the diagnosed embryos 17.6% were found to be HLA matched. Embryo transfer was performed in 212 cycles, 34.9% clinical pregnancy rate per transfer was achieved and 59 healthy and HLA-compatible children were born. Twenty-one sick children have been cured through haemopoietic stem cell transplantation. The effect of maternal age and ovarian reserve on reproductive outcome was assessed retrospectively. The data demonstrated that, once a mutation-free and HLA-compatible embryo was found, clinical pregnancy rate did not differ statistically significantly despite the presence of some cycle-related limitations such as advanced maternal age and/or diminished ovarian reserve. Preimplantation HLA typing is an effective therapeutic tool for curing an affected sibling even for poor-prognosis patients. Preimplantation human leukocyte antigent (HLA) typing allows the birth of healthy children who are potential donors of stem cells for their affected siblings. This technique can be used for acquired diseases such as leukaemia or can be used for single-gene disorders such as thalassaemia. This study presents clinical data obtained from 171 couples who underwent 327 preimplantation HLA-typing cycles. Of these, 262 cycles were performed for HLA typing in combination with mutation analysis and 65 cycles were performed for the sole purpose of HLA typing. A total of 17.6% of the diagnosed embryos were found to be HLA matched. Embryo transfer was performed in 212 cycles. The clinical pregnancy rate per transfer was 34.9% and 59 healthy and HLA compatible children were born. Twenty-one sick children have been cured through haemopoietic stem cell transplantation. The effect of maternal age and ovarian reserve on reproductive outcome was assessed retrospectively. The data demonstrated that, once a mutation-free and HLA-compatible embryo was found, clinical pregnancy rates did not differ statistically significantly by the presence of some cycle-related limitations such as advanced maternal age and/or diminished ovarian reserve. Preimplantation HLA typing is an effective therapeutic tool for curing an affected sibling even for poor-prognosis patients.

Birth of a healthy infant after preimplantation genetic diagnosis by sequential blastomere and trophectoderm biopsy for β-thalassemia and HLA genotyping

BACKGROUND Preimplantation genetic diagnosis (PGD) is a widely used technique for couples at genetic risk and involves the diagnosis and transfer of unaffected embryos generated through in vitro fertilization (IVF) techniques. STUDY DESIGN For those couples who are at risk of transmitting a genetic disease to their offspring, preimplantation embryos can be selected according to their genetic status as well as human leukocyte antigen (HLA) compatibility with the affected child. Stem cells from the resulting babys umbilical cord blood can be used for transplantation to the affected sibling without graft rejection. RESULTS Here we report successful hematopoietic stem cell transplantation (HSCT) after the birth of a healthy infant, who was born after successful PGD testing with both cleavage stage and blastocyst stage biopsy for the purpose of diagnosis of β-thalassemia and HLA compatibility. CONCLUSION The specific feature of this work is not only to have the first successful HSCT achieved in Bulgaria after using preimplantation HLA typing technique, it also demonstrates how to accomplish this success via cross-border collaboration of different units, which makes the application of these sophisticated methods possible in hospitals not having the necessary equipments and expertise.

Sperm fluorescence in situ hybridization analysis reveals normal sperm cells for 14;14 homologous male Robertsonian translocation carrier.

OBJECTIVE To report the presence of normal sperm cells for chromosome 14 in a homologous 14;14 Robertsonian translocation carrier. DESIGN Case report. SETTING In vitro fertilization clinic and genetics laboratory in a private hospital. PATIENT(S) Infertile couple referred for IVF. INTERVENTION(S) Conventional cytogenetic and fluorescence in situ hybridization (FISH) techniques were used in karyotype and sperm FISH analysis. Three IVF treatments were performed, two of which included preimplantation genetic diagnosis (PGD). MAIN OUTCOME MEASURE(S) Cytogenetic analysis revealed pure 45,XY,t(14;14)(q10;q10) karyotype. Sperm FISH analysis for chromosome 14 revealed 13% normal sperm cells in the sperm sample. RESULT(S) Sperm FISH analysis revealed 13% normal sperm cells for chromosome 14 in the homologous 14;14 Robertsonian translocation carrier. The couple underwent two IVF cycles together with PGD. In the first trial there was no suitable embryo for transfer. In the second trial one normal blastocyst was transferred on day 6. However, pregnancy was not established in this second PGD cycle. CONCLUSION(S) To our knowledge, this is the first sperm FISH study revealing the presence of normal sperm in the ejaculate of a pure homologous translocation carrier. The PGD study performed for this couple is also unique in the literature.

Preimplantation genetic diagnosis (PGD) for extremes—successful birth after PGD for a consanguineous couple carrying an identical balanced reciprocal translocation

OBJECTIVE To report a healthy birth after preimplantation genetic diagnosis (PGD) performed for a consanguineous couple carrying an identical familial reciprocal translocation in both partners. DESIGN Case report. SETTING In vitro fertilization (IVF) clinic and genetic laboratory in a private hospital. PATIENT(S) Consanguineous couple carrying the same balanced reciprocal translocation: 46,XX,t(1;16)(q12;q11.2) and 46,XY,t(1;16)(q12;q11.2). INTERVENTION(S) 25 oocyte-cumulus complexes were retrieved 36 hours after human chorionic gonadotropin injection; metaphase II oocytes were fertilized by intracytoplasmic sperm injection; single blastomere biopsy was performed on 15 embryos on day 3; one embryo was found to be normal or balanced according to fluorescent in situ hybridization studies, embryo transfer was performed on day 4. MAIN OUTCOME MEASURE(S) Healthy birth of homozygous double translocation carrier twins with 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat karyotype. RESULT(S) Healthy monozygotic male twins were born at 36 weeks of gestation. Karyotype studies of the babies revealed that they are double translocation homozygotes: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. They are healthy and more than 4 years old later show no physical or mental abnormalities. CONCLUSION(S) To our knowledge, this is the first PGD study performed for a couple who carry the same reciprocal translocation. The twins born after this study are rare examples in the literature of healthy balanced reciprocal translocation homozygotes.

O2 Outcomes of 303 cycles on preimplantation Human Leukocyte Antigen typing with or without mutation analysis

Aims: Single cell diagnosis for PGD requires simultaneous analysis of multiple linked polymorphic markers in addition to mutation analysis in order to reduce misdiagnosis. This type of analysis requires building family haplotypes spanning at least two generations. In cases of germ line mosaicism the wild type haplotype can be seen in both affected and unaffected children making the diagnosis more complex. We present a family with two children affected with Tuberous Sclerosis (TSC1 C1327T) and two healthy children in which both parents tested negative for the mutation. Material and Methods: Twelve informative markers flanking the TSC1 gene were used to construct haplotypes. Genomic DNA isolated from blood and buccal cells was used to analyze the 1327C>T mutation. Single sperm analysis was performed using a multiplex assay that included the 12 markers and the 1327C>T mutation. Results: The identified 1327C>T mutation was not detected in genomic DNA derived from blood or buccal cells from either parent. Both affected children shared the same paternal allele and different maternal alleles. However, one of the two healthy children also shared this same paternal allele, and in order to confirm paternal transmission we performed single sperm analysis for the mutation and 12 markers. Of 44 single sperm analyzed 4 bared the T allele allowing linkage between the mutation and markers. Conclusions: Germline mosaicism complicates allele assignment when constructing haplotypes for PGD. Sperm or polar body analysis are useful tools for verifying allelic linkage.