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

Study Overview
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Brief Summary
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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
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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.

Official Title
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Non-invasive Preimplantation Genetic Testing for Aneuploidies (niPGT-A) - Evaluation of the Analysis of Cell-free DNA in Spent Culture Medium

Conditions
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Secondary Infertility

Intervention / Treatment
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* Diagnostic Test: PGT-A / niPGT-A


Participation Criteria
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Eligibility Criteria
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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

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 46 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Two types of samples (TE and SCM) will be collected for all blastocysts included in the study<br> | Diagnostic Test: PGT-A / niPGT-A<br>* PGT-A and niPGT-A will be performed using next generation sequencing (NGS) analysis for chromosome copy number variation (CNV). Embryo transfers will rely solely on the results of PGT-A for trophectoderm biopsies.<br>|

What is the study measuring?
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Primary Outcome Measures

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

Secondary Outcome Measures

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

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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cell-free DNA, PGT-A, spent culture media, NGS