Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases.

STUDY QUESTION: What is the value of a new strategy for preimplantation genetic diagnosis (PGD) of monogenic diseases: blastocyst biopsy, cryopreservation and thawed embryo transfer? SUMMARY ANSWER: This strategy is highly effective for PGD of monogenic diseases and merits wide use. WHAT IS KNOWN ALREADY: PGD of monogenic diseases is conventionally performed on 6- to 8-cell embryos with fresh transfer. The diagnostic time is restricted and is subjected to amplification failure and allele drop-out (ADO). STUDY DESIGN, SIZE, DURATION: This is a prospective observational cohort study. A total of 33 couples were included from November 2008 to January 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 33 couples who were carriers of monogenic diseases underwent a total of 40 oocyte pick-up (OPU) cycles, with subsequent blastocyst biopsy, vitrification and thawed embryo transfer. DNA analysis was performed by whole genome amplification using multiple displacement amplification followed by real-time PCR and mini-sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: The diagnostic rate was 90% with 5% amplification failure and 5% ADO. The survival rate of vitrified blastocysts was 94%. Amongst 33 couples, 24 ongoing pregnancies were achieved (60% per OPU cycle) with an implantation rate of 50%. All of the genotyping results of prenatal diagnosis were consistent with those of PGD. There was no severe or late ovarian hyperstimulation syndrome (OHSS) and no hospitalization. LIMITATIONS, REASONS FOR CAUTION: The participants are limited to the carriers of monogenic diseases. WIDER IMPLICATIONS OF THE FINDINGS: This strategy achieves high rates of genotyping success, survival after warming and pregnancy. Cryopreservation of blastocysts after biopsy permits sufficient time for transportation of specimens and molecular diagnosis. In particular, cryopreservation of biopsied embryos without fresh transfer is an important strategy to prevent OHSS and circumvent a suboptimal endometrium in high responders. STUDY FUNDING/COMPETING INTEREST(S): This study is financially supported by the National Science Council of Taiwan (grants NSC 96-2628-B-002-063-MY3, NSC 98-2314-B-002-088-MY3 and 98-FTN13). No competing interests are declared.

A feasible strategy of preimplantation genetic diagnosis for carriers with chromosomal translocation: Using blastocyst biopsy and array comparative genomic hybridization.

BACKGROUND/PURPOSE: Patients with chromosomal translocation are highly vulnerable to produce unbalanced gametes that result in recurrent miscarriages, affected offspring, or infertility. Preimplantation genetic diagnosis (PGD) with blastomere biopsy and fluorescent in-situ hybridization (FISH) has been used to select normal/balanced embryos for transfer. However, FISH is inherent with some technical difficulties such as cell fixation and signal reading. Here we introduce a strategy of PGD using blastocyst biopsy and array comparative genomic hybridization (aCGH) for reproductive problems of patients with chromosomal translocation. METHODS: Twelve patients diagnosed as having chromosomal translocation who underwent PGD cycles were included in this single-center observational study. Blastocyst biopsy was performed and biopsied blastocysts were cryopreserved individually. Testing was performed with aCGH, and the euploid embryos were transferred in the following thawing cycles. RESULTS: The overall diagnostic efficiency was 90.2% (55/61) and the euploidy rate was 32.7% (18/55). Ten cycles of thawed embryo transfer (ET) were carried out, resulting in three live births and another three ongoing pregnancies with an ongoing pregnancy rate of 60%/transfer cycle. The prenatal diagnosis with chorionic villi sampling confirmed the results of PGD/aCGH in all six pregnant women. No miscarriage happened in our case series. CONCLUSION: Our study demonstrates an effective PGD strategy with promising outcomes. Blastocyst biopsy can retrieve more genetic material and may provide more reliable results, and aCGH offers not only detection of chromosomal translocation but also more comprehensive analysis of 24 chromosomes than traditional FISH. More cases are needed to verify our results and this strategy might be considered in general clinical practice.

Preimplantation genetic diagnosis of beta-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes.

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells ( approximately 5cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38weeks' gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.

Epigenetic profiling of the H19 differentially methylated region and comprehensive whole genome array-based analysis in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous congenital disorder characterized by severe growth retardation. Hypomethylation of the differentially methylated region (DMR) of the H19 gene and uniparental disomy of maternal chromosome 7 is present in ∼45% of the patients with SRS so more than half of these patients have no known genetic etiology. We combined several molecular technologies including multiplex methylation polymerase chain reaction, methylation-sensitive multiple ligation probe-dependent amplification, and methylation-sensitive high-resolution melting to assess the epigenetic status of 34 patients with SRS. Additionally, we applied a whole genome strategy to detect copy number changes and loss of heterozygosity. Thirteen patients (38.2%) had hypomethylation of the DMR of the H19 gene and none had uniparental disomy of maternal chromosome 7. The whole genome arrays identified five patients (14.7%) with microdeletions on chromosomes 1q23q24.3, 7p15.3, 13q31.3, 14q32.31, and 15q26.2qter, respectively. The overall mutation detection rate was 52.9% by the epigenetic study and the whole genome strategy. Although epimutation may be the major cause of SRS and can be identified by multiplex methylation polymerase chain reaction, the whole genome approach also provides information on the etiology of SRS. If no epimutation is identified in the patients with typical SRS, microdeletions should be suspected.

Preimplantation genetic diagnosis (embryo screening) for enlarged vestibular aqueduct due to SLC26A4 mutation.

Preimplantation genetic diagnosis (PGD) is used to analyze embryos genetically before their transfer into the uterus. For families with genetic diseases, PGD offers a chance to have an unaffected child, without facing termination of pregnancy. Although PGD has been performed for many monogenic disorders, such as cystic fibrosis and beta-thalassemia, the application of PGD to hereditary hearing impairment has not been explored. In the present study, we reported the development and application of PGD protocols to address enlarged vestibular aqueduct (EVA), which is a common type of hereditary hearing impairment associated with mutations in the SLC26A4 gene. The family requesting PGD had a history of EVA, segregating the SLC26A4 c.919-2A-->G mutation. In short, the PGD process was composed of two steps: the development of a single-cell testing protocol and clinical PGD cycles (i.e., selection and implantation of unaffected embryos using the single-cell testing protocol). First, protocols for genetic testing in a single cell were established for the c.919-2A-->G mutation using GenomiPhi technology and primer extension mini-sequencing. These protocols were validated on single lymphocytes collected from both parents and their affected child. Two clinical PGD cycles were then performed for the parents, with the second cycle successfully leading to a singleton pregnancy. The baby was homozygous for the wild-type SLC26A4 allele and revealed a normal audiological phenotype after birth. To our knowledge, this is the first report in the literature describing successful PGD in families with genetic hearing impairment. In our opinion, the application of PGD in the field of hereditary hearing impairment involves fewer ethical controversies than other novel applications of PGD and traditional indications for PGD for other monogenic diseases. Therefore, the approach demonstrated in the present study can also be used in a large number of families with other types of hereditary hearing impairment.

PGD of beta-thalassaemia and HLA haplotypes using OmniPlex whole genome amplification.

A strategy was developed using the OmniPlex technology of whole genome amplification for preimplantation genetic diagnosis (PGD) of single gene diseases and human leukocyte antigen (HLA) haplotypes. The amplified genomic DNA library was subsequently examined separately for mutation analysis with mini-sequence and for short tandem repeat (STR) markers within the HLA loci. To evaluate the reliability of the protocol prior to PGD, tests of 50 single lymphocytes revealed an amplification efficiency of 92-96% and allele drop-out (ADO) rate of 6-16%. The strategy was validated in one beta-thalassaemia family having an affected boy. The couple underwent three cycles of ovarian stimulation and intracytoplasmic sperm injection for PGD. On 16 embryos tested, the amplification efficiency was 88-94% and ADO was 6-19%. Two cycles of embryo transfer were performed, and one pregnancy was achieved. The genotypes of the fetus were shown to be unaffected and HLA-identical, in agreement with PGD, by chorionic villus sampling. The cord blood stem cells from the newborn can be used to treat the affected sibling. This study demonstrates the first successful application of OmniPlex whole genome amplification in PGD of a single gene disorder for selecting unaffected and HLA-compatible embryos.

An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening.

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1.

OBJECTIVE: Preimplantation genetic diagnosis (PGD) offers an alternative for women to carry an unaffected fetus risk of hereditary diseases. Trophectoderm biopsy may provide more cells for accurate diagnosis. However, the time allowed for transportation of the specimens to the laboratory and performance of molecular diagnosis is limited. We designed a PGD program of trophectoderm biopsy, vitrification of blastocysts, whole genome amplification (WGA), double confirmatory genotypings, and thawed embryo transfer. CASE REPORT: We conducted this strategy for a woman of familial neurofibromatosis type I (NF-1). She had a genotype of heterozygous c.6709C>T mutation of NF1 gene. Trophectoderm biopsies were performed on 13 blastocysts. Then, individual blastocyst was vitrified. WGA was performed for the samples, followed by genotypings with both real-time polymerase chain reaction and sequencing. Eight embryos were diagnosed as unaffected, four were affected, and one was inconclusive because of allele drop-out. In the next cycle, two unaffected blastocysts were thawed and transferred, that resulted in a singleton pregnancy. The pregnancy was confirmed as unaffected by means of chorionic villi sampling. CONCLUSION: We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.