Official Title
Analysis of Human Genomic DNA in Embryo's Culture Media Targeting on a Single Gene Disease and Point Mutations
Brief Title
Analysis of Human Genomic DNA in Embryo's Culture Media
Protocol ID
NCT02359747
Lead Sponsor
Cervesi Hospital, Cattolica, Italy
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
Study Period
Enrollment Count
100 participants
Eligibility Criteria
Inclusion Criteria
- all the patients undergoing IVF treatment at Cervesi Hospital
Exclusion Criteria:
- patients with embryos that can't be cultured from day 0 (insemination day) to day
5/6 (blastocyst stage)
Filters
Single-Gene Disease
UNKNOWN
ADULT