First successful pregnancy after preimplantation genetic diagnosis for Hemophilia A in Asia
August 08, 2013First successful pregnancy after preimplantation genetic diagnosis for Haemophilia A in Asia
| Author | Hsing-Hua Lai, Huai-Ling Wang, Shih-Chieh Huang, Wen-Yi Chiang |
|---|---|
| Editor | Hui-Ling Kao |
| Seminar name | 2010 TSRM |
| Keyword | PGD, inherited disease, IVF |
| PDF download |
Haemophilia A, the most common X-linked disorder affecting males, results from deficient or defective coagulation factor VIII which impairs the coagulation cascade (Fig. 1) and causes bleeding symptoms. In Taiwan, there are about 1000 haemophilia patients, where the cost on medication is 2,300 million dollars per year. Different types of factor VIII gene mutation have been characterized and among which, intron 22 inversion is the most seen and responsible for 45% of severe HA cases.
The conventional way for HA patients to have an unaffected child is to play “reproductive roulette” and give up all male babies. Preimplantation genetic diagnosis (PGD) provides an alternative strategy for couples carrying HA to conceive a healthy child without sex selection hence to reduce the mental pressure and medical cost. This technique can be performed on blastomere or trophectoderm cells. The DNA in each cell is analyzed for a specific gene mutation, i.e. factor VIII gene in HA. In recent years, the need of PGD for HA is increasing.
Material and Method
Embryo culture, biopsy and cryopreservation
All embryos were cultured in single media (LifeGlobal). On day 3 post-oocyte retrieval, embryos that were in ≥6 cell stage and had ≤20% fragmentation were biopsied with non-contact laser. From each embryo, one blastomere was aspirated, collected separately into a PCR tube, stored at -20℃ and then sent to a standard PGD laboratory for factor VIII gene intron 22 inversion analysis.
Embryos were further cultured to day 5 and vitrified by Cryotop method. After the PGD results came out, frozen/thawed unaffected embryos with best quality were transferred. Thawing procedure was as described in Cryotop method.
Mutation analysis and PGD
Patient buccal cell DNA was obtained for construction of customized DNA probe. DNA from each blastomere was tested by a polymerase chain reaction (PCR) protocol using ABI 3130 Genetic Analyzer. Factor VIII gene amplification was followed by the second PCR round using the customized probes as primers. The final PCR products were subjected to capillary electrophoresis and analyzed by GeneMapper for allele identification.
Results
Patient
The patient is a 38-year-old HA carrier female whose first son died from HA in infant age. Family linkage analysis was performed earlier and the mutation type was determined as intron 22 inversion. 10 years after, she came to our clinic for the help of IVF because of diminished ovarian reserve. Pregnancy was achieved but ended in termination due to HA affected fetus. Later on, she approached PGD and went through IVF again.
IVF resultsIn total, ten mature oocytes were retrieved and nine zygotes were obtained. At day 3, all nine embryos were qualified to biopsy. Two embryos stopped growing at day 4. Till day 5, embryo vitrification was carried out on the other seven embryos.
PGD results
The factor VIII genotype was determined based on amplicon size compared with the maternal microsatellite markers. The data showed in Fig. 2 were two different markers and two representative embryo allele status (normal and affected). Nine specimens were successfully analyzed and the PGD results were: 1 carrier, 4 mutant and 4 normal embryos (Tab. 1). The genotyping rate was 100%.
Tab. 1 PGD results.
| No. | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| Embryo stage (D5) | Early blastocyst | Arrested | Early blastocyst | Early blastocyst | Early blastocyst |
| Genotype | carrier | mutant | normal | normal | mutant |
| No. | 6 | 7 | 8 | 9 |
|---|---|---|---|---|
| Embryo stage (D5) | Blastocyst | Blastocyst | Early blastocyst | Arrested |
| Genotype | mutant | normal | normal | mutant |
Clinical outcome
Single frozen/thawed blastocyst with normal factor VIII genotype (embryo 7 in Tab. 1) was transferred back on day 21 of the patient’s natural cycle. Pregnancy was achieved. Serum b-hCG was 2004.43 mIU/mL on day 16 after embryo transfer. Amniocentesis was concordant with the PGD result as a normal male baby.
Conclusion
We report a successful pregnancy of a HA carrier with the help of PGD, proving that PGD as an alternative diagnosis in HA is feasible.
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