Clinical Preimplantation Genetic Diagnosis Cycles Research Articles

Preferential selection and transfer of euploid noncarrier embryos in preimplantation genetic diagnosis cycles for reciprocal translocations

To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. Retrospective and prospective study. Invitro fertilization (IVF) units. Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements.

Single-cell high resolution melting analysis: A novel, generic, pre-implantation genetic diagnosis (PGD) method applied to cystic fibrosis (HRMA CF-PGD)

BackgroundInstitutions offering CF-PGD face the challenge of developing and optimizing single cell genotyping protocols that should cover for the extremely heterogeneous CF mutation spectrum. Here we report the development and successful clinical application of a generic CF-PGD protocol to facilitate direct detection of any CFTR nucleotide variation(s) by HRMA and simultaneous confirmation of diagnosis through haplotype analysis. MethodsA multiplex PCR was optimized supporting co-amplification of any CFTR exon-region, along with 6 closely linked STRs. Single cell genotypes were established through HRM analysis following melting of the 2nd round PCR products and were confirmed by STR haplotype analysis of the 1st PCR products. The protocol was validated pre-clinically, by testing 208 single lymphocytes, isolated from whole blood samples from 4 validation family trios. Fifteen PGD cycles were performed and 103 embryos were biopsied. ResultsIn 15 clinical PGD cycles, genotypes were achieved in 88/93 (94.6%) embryo biopsy samples, of which 57/88 (64.8%) were deemed genetically suitable for embryo transfer. Amplification failed at all loci for 10/103 blastomeres biopsied from poor quality embryos. Six clinical pregnancies were achieved (2 twin, 4 singletons). PGD genotypes were confirmed following conventional amniocentesis or chorionic villus sampling in all achieved pregnancies. ConclusionsThe single cell HRMA CF-PGD protocol described herein is a flexible, generic, low cost and robust genotyping method, which facilitates the analysis of any CFTR genotype combination. Single-cell HRMA can be beneficial to other clinical settings, for example the detection of single nucleotide variants in single cells derived from clinical tumor samples.

The clinical utility of next-generation sequencing for identifying chromosome disease syndromes in human embryos

Next-generation sequencing is emerging as a reliable and accurate technology for pre-implantation genetic diagnosis (PGD) of aneuploidies and translocations. The aim of this study was to extend the clinical utility of copy number variation sequencing (CNV-Seq) to the detection of small pathogenic copy number variations (CNVs) associated with chromosome disease syndromes. In preliminary validation studies, CNV-Seq was highly sensitive and specific for detecting small CNV in whole-genome amplification products from three replicates of one and five cell samples, with a resolution in the order of 1–2 Mb. Importantly, the chromosome positions of all CNV were correctly mapped with copy numbers similar to those measured in matching genomic DNA samples. In seven clinical PGD cycles where results were obtained for 34 of 35 blastocysts, CNV-Seq identified 18 blastocysts with aneuploidies, one with an aneuploidy and a 4.98 Mb 5q35.2-qter deletion associated with Sotos syndrome, one with a 6.66 Mb 7p22.1-pter deletion associated with 7p terminal deletion syndrome and 14 with no detectable abnormalities that were suitable for transfer. On the basis of these findings, CNV-Seq displays the hallmarks of a comprehensive PGD technology for detection of aneuploidies and CNVs that are known to affect the development and health of patient's embryos.

Microsatellite Markers Within the α-Globin Gene Cluster for Robust Preimplantation Genetic Diagnosis of Severe α-Thalassemia Syndromes in Mediterranean Populations

In this study we report the development of a generic protocol for preimplantation genetic diagnosis (PGD) of severe α-thalassemia (α-thal) syndromes in α-thal carrier couples of Mediterranean origin. The in silico identification and design of primers for multiplex analysis of short tandem repeats (STRs), was followed by the optimization of polymerase chain reaction (PCR) conditions for multiplexed STR analysis within the α-globin gene cluster (16p3.3) and subsequent optimization and validation of a single-cell multiplex reaction including the selected STRs. Three simple dinucleotide repeats were selected based on their rate of heterozygosity, multiplex PCR efficiency and product size, and location within the α-globin gene cluster. The multiplex PCR was optimized in single lymphocytes with PCR efficiency ranging from 92.5 to 98% and an allele drop-out (ADO) rate of 0 to 9.0% for the three loci. The optimized method was applied in two clinical PGD cycles and genotypes were achieved in 17 out of 18 blastomeres (94%). Transfer of unaffected embryos led to a singleton pregnancy in one of the two couples. The triplex PCR validated for Greek and Cypriot populations is a robust generic method for α-thal PGD.

One-step multiplex polymerase chain reaction for preimplantation genetic diagnosis of Huntington disease

To develop a multiplex polymerase chain reaction (PCR) method for Huntington disease (HD) preimplantation genetic diagnosis (PGD) based on the coamplification of CAG repeats and three different polymorphic microsatellites in a single step of PCR. Techniques and instrumentation. Tertiary clinical and academic medical center. Thirty-six embryos from seven clinical PGD cycles. Patients underwent a PGD cycle with transfer of two unaffected embryos on day 5. PGD based on mutation identification or exclusion testing for at-risk HD carriers. Thirty-six embryos from seven clinical PGD cycles were analyzed with the new method here developed, and results were obtained for 34 of them. Two embryos were transferred on day 5, resulting in two singleton pregnancies. An interesting application of this approach can be considered for PGD cycles in which numerous markers must be used. We have also used this one-step multiplex method for PGD for other pathological conditions.

PGD for X-linked and gender-dependent disorders using a robust, flexible single-tube PCR protocol

X-linked genetic diseases include a wide range of disorders such as the dystrophinopathies. Additionally in some rare genetic diseases, severity of expression is gender dependent. Prevention of such disorders usually involves prenatal diagnosis and termination of affected pregnancies, while preimplantation genetic diagnosis (PGD) represents a specialized alternative that avoids pregnancy termination. To preclude the rejection of unaffected male embryos that cannot be differentiated from those affected when using fluorescence in-situ hybridization, a flexible protocol based on multiplex fluorescence polymerase chain reaction (PCR) was standardized and validated for gender determination in single cells, which can potentially incorporate any disease-specific locus. The final panel of nine loci included four loci on the Y chromosome, two on the X chromosome plus up to three microsatellite markers to either support the gender diagnosis or to further monitor extraneous contamination. The protocol, standardized on single lymphocytes, established a PCR efficiency of >93% for all loci with maximum allele dropout rates of 4%. Microsatellite analysis excluded external contamination and confirmed biallelic inheritance. Proof of principle for the simplicity and flexibility of the assay was demonstrated through its application to clinical PGD cycles for lipoid congenital adrenal hyperplasia, which presents a more severe clinical course in males, and Duchenne muscular dystrophy.

Successful preimplantation genetic diagnosis for alpha- and beta-thalassemia in China

In southern China, the average carrier rates of alpha-thalassemia and beta-thalassemia in the population are as high as 10.3% and 2.8%, respectively. Because of the high rates, they are known as 'social diseases' in some regions. In this study, the fluorescent gap PCR, which can detect the alpha-thalassemia Southeast Asia deletion (SEA deletion), was applied in four clinical applications of preimplantation genetic diagnosis (PGD) on four couples, among whom both partners were alpha-thalassemia carriers. Two patients became pregnant and two healthy babies were born, which confirmed the PGD results. The single cell multiplex nested PCR followed by reverse dot blot (RDB), which can simultaneously detect the 16 beta-thalassemia mutations in the Chinese population, was applied in four clinical PGD cycles on four couples among whom both partners were beta-thalassemia carriers. One pregnancy was achieved and it resulted in a live healthy birth, which confirmed the results of PGD. The amplification efficiencies of the two protocols described above were 89.5% and 93.9%, respectively. The allele drop-out (ADO) rates of these two protocols were 5.9% and 10.9%, respectively. These studies represent the successful applications of PGD protocols that can detect more than 95% of alpha- and beta -thalassemia mutations in the Chinese population.

Successful preimplantation genetic diagnosis for alpha-thalassemia using fluorescent polymerase chain reaction

To develop single-cell fluorescent gap polymerase chain reaction (PCR) assay for preimplantation genetic diagnosis (PGD) in couples at risk of having child with alpha-thalassemia. Single cell fluorescent gap PCR which can detect the alpha-thalassemia Southeast Asia deletion (-SEA deletion), was applied to single lymphocytes and blastomeres which coming from four clinical PGD cycles. The Single cell fluorescent gap PCR can detect the alpha-thalassemia -SEA deletion, which account for 94% of hydrop fetalis in Chinese population with an amplification efficiency of 90.0% (72/80) and allele drop-out (ADO) rate of 8.3% (6/72) in lymphocytes. In four clinical PGD cycles, a total 38 embryos were detected and 38 blastomeres were obtained. Thirty-four blastomeres were amplified with the amplification efficiency of 89.5% (34/38) and ADO rate of 5.9% (2/34). Eleven embryos were shown to be normal homozygous, eight embryos were shown to be heterozygous and 15 embryos were shown to be affected homozygous. Eleven embryos were transferred back to the uterus of the patients. Two pregnancy achieved, resulting in two live healthy births, which confirmed the results of PGD. This first reported unaffected pregnancy resulting from PGD using fluorescent gap PCR for alpha-thalassemia demonstrates that this technique, as an alternative to prenatal diagnosis, is a reliable and effective way to help those carrier couples to get a healthy baby.

Successful preimplantation genetic diagnosis for beta-thalassemia using multiplex nested polymerase chain reaction

To develop single-cell multiplex nested polymerase chain reaction (PCR) assays for preimplantation genetic diagnosis (PGD) in couples at risk of having child with beta-thalassemia. Primers were designed and synthesized according to the documented mutation sites common among Chinese. Venous blood was collected from 4 pairs of husband and wife, all heterozygotes for beta-thalassemia, and underwent multiple nested PCR. Intraooplasmic sperm injection and mechanical bio psy was used to obtain single blastomere. Multiplex nested PCR was used to detect the CD41-42 mutation and the closely linked polymorphic marker, HumTHO1 gene or CD41-42, CD41-28, IVSII654 mutation and HumTHO1 gene in the single blastomeres from four clinical PGD cycles. The normal embryos with high scores capable of continuing to divide were transplanted into the uteri. The process of gestation was observed. 200 lymphocytes were amplified by nested PCR. The average amplification rate of the most common 16 beta-thalassemia mutations in Chinese population was 91.3% and the average rate of allele drop out for different sites was 17.0% without differences between any 2 sites. During the 4 PGD cycles 33 embryos underwent bioassay with a success rate of 100%. 33 blastomeres were obtained to undergo PCR, of which 30 were successfully amplified with an amplification rate of 90.9%. Explicit diagnosis was obtained in 26 of the 30 embryos: 7 normal homozygotes, 11 heterozygotes, and 8 abnormal or complex heterozygotes. One or more embryos were transferred back into the uteri of the 4 women and clinical pregnancy occurred in one woman. Five weeks after the implantation B-mode ultrasonography showed monocyesis, and in the 17th week of gestational period paracentesis of cord blood showed normal homozygote. At last a normal female infant confirming the PGD result had been born, which was the first reported unaffected pregnancy resulting from PGD using multiplex nested PCR for couples as beta-thalassemia gene carriers. The results of diagnosis for embryo all corresponded to those for blastomere. The average ADO rate of blastomere was 13.3% (4/30). PGD using multiplex nested PCR, as an alternative to prenatal diagnosis, is a reliable and effective way to help couples-carriers of pathogenetic genes to get a healthy baby.

Preimplantation genetic diagnosis for beta-thalassaemia using sequencing of single cell PCR products to detect mutations and polymorphic loci.

In order to carry out preimplantation genetic diagnosis (PGD) for beta-thalassaemia, we have applied direct sequencing of single cell PCR products to detect mutations and polymorphic loci within the beta-globin gene. Conventional duplex PCR was used to amplify two regions of the beta-globin gene with an amplification efficiency of 79% for blastomeres. Sequencing data were obtained for 100% of amplified products, with 12% having confirmed allele drop-out (ADO). A double ADO event was observed at least twice, confirming the real risk of such an event during PGD. In one couple, the presence of a polymorphism linked to the female partner's mutation enabled us to eliminate the risk of misdiagnosis due to double ADO without having to amplify both mutations within the same PCR product. We present here the data from eight clinical PGD cycles for three couples resulting in a singleton pregnancy and a twin pregnancy with all babies confirmed to be free from beta-thalassaemia (major).