Preimplantation Genetic Diagnosis (PGD) Clinical Trial
Official title:
Establishment of Comprehensive Genetic Analysis From a Single Cell
Preimplantation genetic diagnosis (PGD) is the integration of both assisted reproductive technologies and molecular genetic technologies and was shown to improve implantation rate and reduce spontaneous abortions after implantation. The principal problems in single cell PCR include amplification failure, ADO and contamination. Multiple displacement amplification (MDA) is a technique used in the amplification of very low amounts of DNA and reported to yield large quantities of high-quality DNA. By this approach, the diagnosis of gene disorders form single cell will be more accurate and reliable.
Preimplantation genetic diagnosis (PGD) for couples at risk of conceptions with serious
genetic disorders is firmly established as a valid reproductive option for couples to
consider following appropriate genetic counseling. The procedure entails a balance of risks
between establishing a successful pregnancy and minimizing the risk of misdiagnosis. PGD of
single gene disorders relies on PCR-based tests performed on single cells (polar bodies or
blastomeres). Despite the use of increasingly robust protocols, allele drop-out (ADO; the
failure to amplify one of the two alleles in a heterozygous cell) remains a significant
problem for diagnosis using single cell PCR. In extreme cases ADO can affect >40% of
amplifications and has already caused several PGD misdiagnoses.
Fundamental to most genetic analysis is availability of genomic DNA of adequate quality and
quantity. Because DNA yield from human samples is frequently limiting, much effort has been
invested in developing methods for whole genome amplification (WGA) by random or degenerate
oligonucleotide-primed PCR. However, existing WGA methods like degenerate
oligonucleotideprimed PCR suffer from incomplete coverage and inadequate average DNA size.
Multiple displacement amplification (MDA) provides a highly uniform representation across
the genome. Amplification bias among eight chromosomal loci was less than 3-fold in contrast
to 4–6 orders of magnitude for PCR-based WGA methods. Average product length was >10 kb. MDA
is an isothermal, strand-displacing amplification yielding about 20–30 μg product from as
few as 1–10 copies of human genomic DNA. Amplification can be carried out directly from
biological samples including crude whole blood and tissue culture cells. MDA-amplified human
DNA is useful for several common methods of genetic analysis, including genotyping of single
nucleotide polymorphisms, chromosome painting, Southern blotting and restriction fragment
length polymorphism analysis, subcloning, and DNA sequencing. MDA-based WGA is a simple and
reliable method that could have significant implications for genetic studies, forensics,
diagnostics, and long-term sample storage.
In this study we will carefully vary reaction conditions in single cell amplifications from
isolated peripheral lymphocytes to minimize the rate of ADO. Consideration of the causal
factors identified during this study should permit the design of PGD protocols that
experience little ADO, thus improving the accuracy of PGD for single gene disorders.
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Observational Model: Case Control, Primary Purpose: Screening, Time Perspective: Longitudinal
| Status | Clinical Trial | Phase | |
|---|---|---|---|
| Terminated |
NCT01194531 -
Impact of Parental Support on Pregnancy Outcomes (IPSO) Trial- Day 3 Preimplantation Genetic Screening (PGS) With Day 5 Fresh Transfer
|
Phase 4 |