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

NCT ID: NCT06084377

Last Updated: 2023-10-18

Study Results

Results pending

The study team has not published outcome measurements, participant flow, or safety data for this trial yet. Check back later for updates.

Basic Information

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Recruitment Status

COMPLETED

Total Enrollment

27 participants

Study Classification

OBSERVATIONAL

Study Start Date

2020-05-18

Study Completion Date

2023-09-23

Brief Summary

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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.

Detailed Description

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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.

Conditions

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Females Undergoing PGD Technology for Medical Conditions

Study Design

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Observational Model Type

OTHER

Study Time Perspective

CROSS_SECTIONAL

Interventions

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Preimplantation Genetic Screening

Non-invasive technique to investigate the presence of genetic disorders.

Intervention Type GENETIC

Other Intervention Names

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PGS

Eligibility Criteria

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Inclusion Criteria

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

22 Years

Maximum Eligible Age

44 Years

Eligible Sex

FEMALE

Accepts Healthy Volunteers

Yes

Sponsors

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Damascus University

OTHER

Sponsor Role lead

Responsible Party

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Responsibility Role SPONSOR

Principal Investigators

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Marwan Alhalabi, PhD

Role: STUDY_CHAIR

Damascus University

Locations

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Damascus University

Damascus, , Syria

Site Status

Countries

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Syria

References

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Huang J, Rong L, Zeng L, Hu L, Shi J, Cai L, Yao B, Wang XX, Xu Y, Yao Y, Wang Y, Zhao J, Guan Y, Qian W, Hao G, Lu S, Liu P, Qiao J. Embryo selection through non-invasive preimplantation genetic testing with cell-free DNA in spent culture media: a protocol for a multicentre, double-blind, randomised controlled trial. BMJ Open. 2022 Jul 27;12(7):e057254. doi: 10.1136/bmjopen-2021-057254.

Reference Type BACKGROUND
PMID: 35896299 (View on PubMed)

Sousa LN, Monteiro PB. Non-invasive preimplantation genetic testing: a literature review. JBRA Assist Reprod. 2022 Aug 4;26(3):554-558. doi: 10.5935/1518-0557.20210102.

Reference Type RESULT
PMID: 35238503 (View on PubMed)

Rubio C, Rienzi L, Navarro-Sanchez L, Cimadomo D, Garcia-Pascual CM, Albricci L, Soscia D, Valbuena D, Capalbo A, Ubaldi F, Simon C. Embryonic cell-free DNA versus trophectoderm biopsy for aneuploidy testing: concordance rate and clinical implications. Fertil Steril. 2019 Sep;112(3):510-519. doi: 10.1016/j.fertnstert.2019.04.038. Epub 2019 Jun 11.

Reference Type RESULT
PMID: 31200971 (View on PubMed)

Navarro-Sanchez L, Garcia-Pascual C, Rubio C, Simon C. Non-invasive preimplantation genetic testing for aneuploidies: an update. Reprod Biomed Online. 2022 May;44(5):817-828. doi: 10.1016/j.rbmo.2022.01.012. Epub 2022 Jan 31.

Reference Type RESULT
PMID: 35307298 (View on PubMed)

Yang L, Lv Q, Chen W, Sun J, Wu Y, Wang Y, Chen X, Chen X, Zhang Z. Presence of embryonic DNA in culture medium. Oncotarget. 2017 Jun 29;8(40):67805-67809. doi: 10.18632/oncotarget.18852. eCollection 2017 Sep 15.

Reference Type RESULT
PMID: 28978073 (View on PubMed)

Cornelisse S, Zagers M, Kostova E, Fleischer K, van Wely M, Mastenbroek S. Preimplantation genetic testing for aneuploidies (abnormal number of chromosomes) in in vitro fertilisation. Cochrane Database Syst Rev. 2020 Sep 8;9(9):CD005291. doi: 10.1002/14651858.CD005291.pub3.

Reference Type RESULT
PMID: 32898291 (View on PubMed)

Other Identifiers

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UDFP-Biochem-01-2023

Identifier Type: -

Identifier Source: org_study_id

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