Analysis of Human Genomic DNA in Embryo's Culture Media

• Conditions: Single-Gene Disease
• Interventions: Genetic: Polymerase Chain Reaction and Whole Genome Amplification

• Registration Number: 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 Timeline

• Study First Submitted: 2015-02-03
• Study First Submitted QC: 2015-02-09
• Study First Posted: 2015-02-10
• Study Start: 2020-01-12
• Status Verified: 2021-02
• Last Update Submitted: 2021-02-08
• Last Update Posted: 2021-02-10
• Primary Completion: 2021-11
• Study Completion: 2022-12

Recruitment & Eligibility

• Status: UNKNOWN
• Sex: All
• Target Recruitment: 100

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)

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Arm && Interventions

Group	Intervention	Description
In Vitro Fertilization treatment	Polymerase Chain Reaction and Whole Genome Amplification	culture medium discarded after IVF treatment

Primary Outcome Measures

Name	Time	Method
genomic DNA in culture medium (pg)	1 year

Secondary Outcome Measures

Name	Time	Method
amplification of point mutation	1 year	amplification of point mutation with PCR (polymerase chain reaction)

Trial Locations

Locations (2)

Cervesi Hospital; 🇮🇹 Cattolica, Italy

cervesi Hospital; 🇮🇹 Cattolica, Italy