A trial for the clinical effectiveness of a Non-invasive Preimplantation Genetic Screening (PGS) method for determining the ploidy status of embryos of patients undergoing In Vitro Fertilisation (IVF) treatment that are not suitable for standard biopsy

The purpose of this study is to implement a clinic trial for a non-invasive Preimplantation Genetic Screening (PGS) method for human embryos that are not suitable for standard biopsy and PGS screening. Only embryos that are not suitable for biopsy and can not undergo standard PGS screening will be used for this trail, All embryos that are suitable for biopsy will undergo standard PGS screening. Embryos that are suitable for biopsy are Grade 1 and 2 expanded blastocysts. Embryos that are suitable for freezing but not for biopsy are Grade 3 non-expanded blastocysts ie. early blastocysts. Patients treatment will not be altered in any way and will proceed as per standard IVF cycle, including medications, oocyte retrieval, insemination and fertilization and embryo culture. Patient embryos will be cultured as per standard protocols; G1/G2 Plus media (Vitrolife) overlayed with oil (Ovoil, Vitrolife) in 6% CO2, 5% O2, 37 degrees. On day 6, embryos will be assessed for morphology and graded as per current clinical protocols by clinical staff not involved in the trial. All embryos suitable for biopsy (Grade 1 and 2 - expanded blastocysts) will undergo standard biopsy and PGS treatment. For all embryos that are suitable for freezing but not for biopsy (Grade 3 - non-expanded blastocysts ie. early blastocysts), spent culture medium will be sampled and assessed for aneuploidy as described below. A 4 micro litre drop of the spent culture medium will be sampled using PCR grade filtered tips, under laminar flow, and placed into PCR grade 0.6 ml tubes. DNA from the culture media sample will be amplified by WGA kit and Next Generation Sequencing using standard Illumina protocols. • Euploid embryos will be made available for transfer. If a pregnancy results from the transfer of the embryo, patients will be offered a NIPT (Non-Invasive Prenatal Testing) first trimester prenatal screening as per standard patient care. • Embryos assessed as aneuploid will have a confirmatory assessment using standard protocols. The main risk of this non-invasive technology is that a false positive will be obtained, i.e. indicating an embryo is aneuploid and the confirmatory biopsy screens the embryo as euploid. The screening is not 100% accurate and there remains a risk that the result could be incorrect. However, the thaw and confirmatory PGS biopsy assessment of embryos screened as aneuploid will minimise the risk of discarding a normal embryo. These embryos will be thawed, cultured on and biopsied as per standard protocols, It is described as a 'modified confirmatory biopsy' because embryos that are screened using biopsy and standard PGS are not ever thawed and biopsied a second time to confirm these initial screening results. Patients will have any embryo screened as euploid via standard PGS or via NEST4E non-invasive screening available for transfer. The decision on which embryo to transfer and how many to transfer is outside the scope of the trial. Standard patient care practices will be followed and this decision will be are made by the patients treating clinician in consultation with senior embryologists not involved in the trial. Patients typically have a single embryos transfer however double embryo transfers are requested by patients for various reasons but are only approved by their treating clinician upon informed consent once the of the risks involved have been given. For all stages/steps of the trial, patient treatment and embryology will be performed by those involved in standard patient care, ie. nurses, clincians, emrbyologyists. The only part of the trial that will involve someone outside of this scope is the informed consenting to be part of the trial will be performed by Dr Francesca Bell who is a research associate at Repromed. She will also be reposnible for the collection of culture media for embryos that will be screened non-invasively. The WGA DNA amplification, sequencing and screening of embryos will be formed by a qualified member of the genetics team at Repromed who is responsible for standard PGS patient embryo care.