Analysis of Human Genomic DNA in Embryo's Culture Media Targeting on a Single Gene Disease and Point Mutations
Overview
- Phase
- Not Applicable
- Intervention
- Not specified
- Conditions
- Single-Gene Disease
- Sponsor
- Cervesi Hospital, Cattolica, Italy
- Enrollment
- 100
- Locations
- 2
- Primary Endpoint
- genomic DNA in culture medium (pg)
- Last Updated
- 5 years ago
Overview
Brief Summary
try to find genomic DNA in culture medium after the embryos develop on Day 3 and Day 5 also in single step culture media. using direct PCR Polymerase Chain Reaction and also WGA Whole Genome Amplification before PCR and sequencing of the samples to find the point mutation
Detailed Description
Human fertilized oocytes were individually cultured from Day 1 to Day 3, and from day 3 to days 5/6, in 10 ul medium. A total of 100 samples (50 spent media from Day 3 and 50 from Day 5/6) will be use in the study. Double-stranded DNA (dsDNA) amplifiability will be evaluated in embryos culture media by quantitative PCR on a multicopy gene target (TBC1D3). Quantification will be made with a standard curve constructed with genomic DNA (with culture media -day 3 and day5-, to take into account media inhibitory effects) TSPY1 amplification will be use to assess the presence of Y chromosome WGA followed by PCR on a single copy gene (MTHFR gene) will be performed on a subset of some samples to monitor the C677T polymorphism (genotyping by sequencing) WGA followed by PCR on a single copy gene (MTHFR) will be also performed on blastocoele fluids
Investigators
Palini Simone
Senior Clinica Embryologist Lab Director
Cervesi Hospital, Cattolica, Italy
Eligibility Criteria
Inclusion Criteria
- Not provided
Exclusion Criteria
- •patients with embryos that can't be cultured from day 0 (insemination day) to day 5/6 (blastocyst stage)
Outcomes
Primary Outcomes
genomic DNA in culture medium (pg)
Time Frame: 1 year
Secondary Outcomes
- amplification of point mutation(1 year)