Isolation of Cell Free Fetal DNA From Spent Culture Medium and Its Potential Role for Preimplantation Genetic Diagnosis (PGD).

October 17, 2023 updated by: Damascus University

Isolation of Extracellular Embryonic DNA FromIn Vitro Fertilization (IVF) Embryonic Culture Medium and Its Potential Role for Preimplantation Genetic Screening (PGS).

Preimplantation genetic testing for chromosomal screening is, at present, the most reliable method to assess the genetic status of preimplantation embryos. DNA isolated and amplified from trophectoderm (TE) biopsiesis the method currently used for preimplantation genetic testing, but it has significant limitations. There is increasing evidence for a ''true'' noninvasive approach consisting of the analysis of cell-free DNA (cfDNA) released by the embryo into the spent blastocyst medium (SBM) during the late stages of preimplantation development.

This study is to assess whether Noninvasive prenatal genetic diagnosis using cell-free fetal DNA in spent culture medium will substitute for trophectoderm biopsy for heritable disorders screening and if it represents the total fetal genomic DNA.

A total of 27 spent blastocyst media (SBM) from women undergoing IVF will be enrolled with agesless than44 years, whose indications for preimplantation genetic diagnosis for Euploid/Aneuploid embryo determination, were advanced maternal age, recurrent miscarriage, or recurrent implantation failure.We examined the presence of the cell free DNA in spent culture media and whether this DNA is reliable and can be representative of the chromosomal constitution of a blastocyst. So we can affirm that Noninvasive prenatal determination of fetal sex using cell-free fetal DNA (used here as a marker) provides an alternative to invasive techniques for some heritable disorders.

Study Overview

Status

Completed

Conditions

Intervention / Treatment

Detailed Description

Noninvasiveness is a common theme across medical specialties in the 21st century. In obstetrics, noninvasive prenatal testing provoked a paradigm shift in prenatal clinical practice through the sequencing of fetal cell-free DNA (cfDNA) in maternal plasma. However, current practice for preimplantation genetic testing for aneuploidy (PGT-A) requires trophectoderm biopsy, which entails technical challenges and embryo viability concerns. The possibility to overcome these issues reached the horizon in recent years, with the discovery that cfDNA released by the embryo to the culture media during the latest stages of development offers a new strategy to assess the preimplantation embryo genetically.

Our study evaluates the possibility to isolate fetal cell-free DNA (cfDNA) from culture media and compare the results with TE biopsy samples from the same patients who underwent an IVF stimulation cycle with intracytoplasmic sperm injection at Al-Sharq Hospital between March 2022 and April 2023. During the study's time frame, all embryos were cultured until the blastocyst stage. All enrolled patients underwent niPGD for gender determination using spent culture media as well as standard PGD with TE biopsy using Polymerase chain reaction technique for SRY gene. The embryos were individually cultured in 30-mL media droplets and embryo development progressed using sequential culture media, consistent with routine laboratory practice. For niPGD analysis, the blastocyst-stage culture media on Day 5 was collected (10-20 µl) immediately before TE biopsy was performed. Only embryos achieving a grade 1 (fragmentation less than 15%), were considered eligible for biopsy and vitrification. The embryo culture media samples underwent whole-genome amplification (WGA) using the DOPlify® WGA kit (PerkinElmer) to generate representative amplification of total DNA from spent culture media. The DOP-PCR-based WGA reproducibly amplifies total DNA to produce microgram quantities of amplified and purified genomic DNA in less than 3 hours. We aim to evaluate the concordance rates between trophectoderm biopsy results and cfDNA collected from spent media with an optimized protocol to obtain an adequate quantity and quality of genomic DNA. This interesting work is an important effort to establish the feasibility of the new noninvasive approach that can be a reliable alternative for chromosomal abnormalities assessment of in vitro cultured embryos which will lead to the birth of a living and healthy baby.

Later on, we will perform the SRY gene detection for sex determination using the Polymerase Chain Reaction on the cell-free fetal DNA isolated from spent media samples and compare our results with the TB results.

Study Type

Observational

Enrollment (Actual)

27

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Locations

Participation Criteria

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Ages Eligible for Study

  • Adult

Accepts Healthy Volunteers

Yes

Sampling Method

Non-Probability Sample

Study Population

The study samples will be collected from couples attending Orient Hospital (assisted reproduction and genetics) to carry out the in vitro fertilization process and would like to perform PGD for gender selection. After obtaining the approval of the ethical committee of Damascus University, they will be informed of the research objectives and sign the approve consent.

Description

Inclusion Criteria:

  • At least two blastocysts suitable for PGD. Only embryos achieving a grade 1 (fragmentation less than 15%).

Exclusion Criteria:

  • All embryos do not achieve inclusion criteria as mentioned above.

Study Plan

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

What is the study measuring?

Primary Outcome Measures

Outcome Measure
Measure Description
Time Frame
Efficacy of cell free fetal DNA isolated from spent culture medium
Time Frame: At Day 5 of ICSI
Efficacy of cell free fetal DNA isolated from spent culture medium by Degenerate Oligonucleotide Primed PCR (DOP-PCR)-based Whole Genome Amplification (WGA) for preimplantation genetic analysis.
At Day 5 of ICSI

Secondary Outcome Measures

Outcome Measure
Time Frame
Increasing the live birth rate, increasing the chances of obtaining embryos free of genetic diseases, and the implantation rate.
Time Frame: 9 Months
9 Months

Collaborators and Investigators

This is where you will find people and organizations involved with this study.

Sponsor

Damascus University

Investigators

  • Study Chair: Marwan Alhalabi, PhD, Damascus University

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Helpful Links

Study record dates

These dates track the progress of study record and summary results submissions to ClinicalTrials.gov. Study records and reported results are reviewed by the National Library of Medicine (NLM) to make sure they meet specific quality control standards before being posted on the public website.

Study Major Dates

Study Start (Actual)

May 18, 2020

Primary Completion (Actual)

April 5, 2023

Study Completion (Actual)

September 23, 2023

Study Registration Dates

First Submitted

October 11, 2023

First Submitted That Met QC Criteria

October 11, 2023

First Posted (Actual)

October 16, 2023

Study Record Updates

Last Update Posted (Actual)

October 18, 2023

Last Update Submitted That Met QC Criteria

October 17, 2023

Last Verified

October 1, 2023

More Information

Terms related to this study

Keywords

Other Study ID Numbers

  • UDFP-Biochem-01-2023

Plan for Individual participant data (IPD)

Plan to Share Individual Participant Data (IPD)?

UNDECIDED

Drug and device information, study documents

Studies a U.S. FDA-regulated drug product

No

Studies a U.S. FDA-regulated device product

No

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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