Non-invasive Preimplantation Genetic Testing for Aneuploidies Using Cell-free DNA in Spent Culture Media

Withdrawn

• Conditions: Secondary Infertility
• Interventions: Diagnostic Test: PGT-A / niPGT-A

• Registration Number: NCT04711239
• Lead Sponsor: ART Fertility Clinics LLC

Brief Summary

Analysis of embryonic cell-free DNA (cfDNA) present in the spent culture media (SCM) is a non-invasive alternative for preimplantation genetic testing for aneuploidies (PGT-A) that avoids the technical challenges and limitations of biopsy. Initial studies investigating this non-invasive PGT-A (niPGT-A) method reported variable concordance between cfDNA in SCM and the trophectoderm sample (\~ 30%-86%) and indicated a contribution from both the inner cell mass and trophectoderm to the cfDNA in SCM.

This study aims to evaluate the use of the embryo culture medium as a source of genetic material for PGT-A and validate a niPGT-A protocol using cfDNA in SCM.

Detailed Description

Multiple studies have demonstrated the ability to detect and amplify cfDNA from SCM, at different stages of embryonic development, with varying rates of amplification success. Differences in analytes, timing of SCM collection and the duration of embryo culture within the collected medium, performance of assisted hatching (AH), whole genome amplification methods, comprehensive chromosome screening methods and next generation sequencing (NGS) platforms, bioinformatic analyses and strategies for identifying maternal contamination all contribute to the ultimate performance of niPGT-A.

This study aims to validate a noninvasive PGT-A (niPGT-A) method utilizing cfDNA released from the human blastocyst into the SCM.

Patients undergoing a fertility treatment with PGT-A due to secondary infertility will be recruited. On day 6 post fertilization, SCM will be collected prior to blastocyst biopsy. The SCM is normally discarded at this stage. Trophectoderm biopsy and sample collection will follow the IVF laboratory's standard practices for clinical PGT-A.

Three aneuploidy screening kits, relying on different whole genome amplification methods followed by NGS on the Ion GeneStudio™ S5 Prime System (ThermoFisher Scientific), will be compared. The concordance between cfDNA and trophectoderm biopsies will be evaluated for approximately 150 blastocysts with the best performing niPGT-A protocol.

Selection of the embryo(s) for transfer will be based solely on the PGT-A result from the biopsied cells. The patient's IVF+PGT-A treatment plan and timeline will not be altered.

Study Timeline

• Study First Submitted: 2021-01-08
• Study First Submitted QC: 2021-01-12
• Study First Posted: 2021-01-15
• Study Start: 2021-10-15
• Primary Completion: 2021-10-15
• Study Completion: 2021-10-15
• Status Verified: 2023-03
• Last Update Submitted: 2023-03-17
• Last Update Posted: 2023-03-20

Recruitment & Eligibility

• Status: WITHDRAWN
• Sex: Female
• Target Recruitment: Not specified

Inclusion Criteria

• Patients undergoing fertility treatment with PGT-A (Recombinant FSH antagonist protocol with dual trigger)
• Secondary infertility
• BMI 18- 35 kg/m2
• Sperm: fresh ejaculated sperm (abstinence: 2-3 days)
• At least one blastocyst biopsied on day 6

Exclusion Criteria

• High progesterone on day of trigger (>1.5ng/ml)
• Vitrified oocytes
• Frozen sperm
• Indications for PGT-SR and PGT-M

Study & Design

• Study Type: OBSERVATIONAL
• Study Design: Not specified

Arm && Interventions

Group	Intervention	Description
Two types of samples (TE and SCM) will be collected for all blastocysts included in the study	PGT-A / niPGT-A	-

Primary Outcome Measures

Name	Time	Method
Discordance per chromosome between results for cfDNA in SCM and trophectoderm biopsies	4 months	Discordance per chromosome: number of misidentified chromosomal errors/24\*total number of embryos with cfDNA result
Concordance per chromosome between results for cfDNA in SCM and trophectoderm	4 months	Chromosome error concordance: number of correctly identified chromosomal errors/total number of chromosomal errors detected
Specificity of niPGT-A using cfDNA in SCM	4 months	False positive rate: 1- (true aneuploid result/total number of samples with a result)
General concordance between results for cfDNA in SCM and trophectoderm biopsies	4 months	General ploidy concordance rate: number of matched (euploid-euploid or aneuploid-aneuploid) result/total number cfDNA samples with a result
Sensitivity of niPGT-A using cfDNA in SCM	4 months	False negative rate: 1- (true euploid result/total number of samples with a result)

Secondary Outcome Measures

Name	Time	Method
Pregnancy outcome for patients having an embryo transfer - Implantation rate	12 months	Implantation rate = number of gestational sacs observed at echographic screening at 6 weeks of pregnancy / number of embryos transferred
Pregnancy outcome for patients having an embryo transfer - Implantation failure rate	12 months	Implantation Failure (%)
Pregnancy outcome for patients having an embryo transfer - Biochemical pregnancy rate	12 months	Biochemical pregnancy rate = positive βhCG test of \> 15IU but no foetal heartbeat / gestational sac on ultrasound examination
Pregnancy outcome for patients having an embryo transfer - Clinical pregnancy rate	12 months	Ongoing pregnancy rate (beyond 20 weeks)
Pregnancy outcome for patients having an embryo transfer - Miscarriage rate	12 months	Miscarriage rate (%) defined by ultrasonographic visualization of one or more gestational sacs, including ectopic pregnancies, with spontaneous pregnancy loss prior to 20 weeks