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

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

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)

all the mediun descarted after embryos culture in vitro