PGT-SR with Catherine Welch Managing Director Ion ReproSeq PGS Kits Sequence46
Disclosures • Shareholder in Sequence46 • Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so • Speaker was provided travel and hotel support by Thermo Fisher Scientific for this presentation, but no remuneration 2
What is Preimplantation Genetic Testing? • PGT = testing performed on embryonic DNA in search of specific genetic abnormalities, with the goal of determining which embryos to prioritize for transfer in an IVF cycle • Previously referred to as PGD and/or PGS • Now categorized into PGT-A, PGT-SR, and PGT-M 3
Types of PGT PGT-A PGT-M PGT-SR Screens for chromosomal Screens for stru tructural Screens for mon onogen enic aneuploid idy, e.g. Trisomy 21 rearrangements, e.g. dis iseases, i.e. single gene unbalanced translocations disorders Previously referred to as PGS Previously referred to as PGD Previously referred to as PGD 4
Prevalence of Structural Rearrangements • General population: • Reciprocal translocations: 1 in 500 individuals • Robertsonian translocations: 1 in 1000 individuals • Inversions (including ‘benign’ variants): >1% • 4.7% of couples with recurrent pregnancy loss (2+ miscarriages) include one carrier partner • Of RPL couples with a structural rearrangement, a majority are reciprocal (62%) or Robertsonian (16%) translocations Gardner, R.J.M. and G.R. Sutherland. (1996) Gardner and Sutherland’s Chromosome Abnormalities and Genetic Counseling. 2 nd Edition. Oxford University Press, Oxford. De Braekeleer, M. and T.N. Dao. 1990. Cytogenic studies in couples experiencing repeated pregnancy losses. Human Reproduction 5(5):519-28. Franssen, M.T.M. et al. 2005. Selective chromosome analysis in couples with two or more miscarriages: case-control study. BMJ 331(7509): 137-41 5
PGT for Structural Rearrangements • Couples wherein at least one partner has a structural rearrangement have a substantially higher risk for miscarriage or having a child with a genetic disorder • PGT-SR can help these couples maximize their chances for a successful pregnancy and healthy live birth • PGT-SR utilizing next generation sequencing (NGS) can detect reciprocal or Robertsonian translocations as well as inversions and other complex rearrangements* 6 *Not all structural rearrangements can be detected using NGS. Case approval is required to ensure that the segments are large enough for accurate detection.
Our Experience with Ion ReproSeq PGS Kits • Platform • Reduced risk for sample mix-up due to sample barcoding process • Simplification of the lab workflow • Reduced noise/artifacts compared to other platforms • Software • Automatic detection of partial chromosomal abnormalities, with a resolution of 6Mb for standard PGT-A and an even higher resolution for PGT-SR • Customization of mosaicism thresholds for PGT-A • Ability to transfer data directly to a report template without human transcription errors • Service & Support • Rapid support with any hardware/equipment or software problems • Competitive and reasonable pricing • Research collaborations 7 For Research Use Only. Not of use in diagnostic procedures.
ReproSeq: Lab Workflow & Library prep Template prep Sequencing Analysis Combined DNA extraction, whole Automated clonal Simple, cartridge-loaded Automated Ion Reporter ™ software genome amplification (WGA), and amplification of libraries reagents and user interface sample barcoding • Flexible batch size (16-96 samples/run) • Rapid processing (<10-13 hours depending on batch size) • Standard sample processing regardless of test (PGT-A vs. PGT-SR) 8 For Research Use Only. Not of use in diagnostic procedures.
ReproSeq: Analysis w/ Ion Reporter Software • Preconfigured analysis workflows for aneuploidy detection • Can be adjusted based on lab preferences • Key Features • Improved plot smoothing • Updated mosaicism analysis to reduce risk of false positives • Smaller tile size baselines for small event calling – important for PGT-SR • Sequence46 settings: PGT-A = 2Mb vs. PGT-SR = 0.5Mb • NOTE: smaller tile size not recommended for standard PGT-A as results become too noisy and can lead to false positives 9 For Research Use Only. Not of use in diagnostic procedures.
Sample Embryo Profiles: Euploid 46, XY 46, XX Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 10 For Research Use Only. Not of use in diagnostic procedures.
Sample Embryo Profiles: Aneuploid 46,X,+8 69,XXY Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 11 For Research Use Only. Not of use in diagnostic procedures.
Sample Embryo Profile: Mosaic 46,XX / 47,XX,+15[45%] Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software . 12 For Research Use Only. Not of use in diagnostic procedures.
Sample Embryo Profile: No Call Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures. 13
Case Example #1: Known SR – t(5;7) • 31yo male patient with known reciprocal translocation • Karyotype: 46, XY, t(5;7) (q14.1;q21.1) • 31yo female partner • Clinical history: Two miscarriages 14
Case Example #1: Known SR – t(5;7) All segments are expected to be large (>50Mb), so the standard PGT-A workflow was utilized. 15
Case Example #1: Known SR – t(5;7) • 18 embryo biopsy samples received for PGT-SR • Results: • 4 euploid/balanced • 9 abnormal involving the translocation (+/- other abnormalities) • 5 additional abnormal/mosaic embryos not involving the translocation 16
Case Example #1: Known SR – t(5;7) Euploid/Balanced Embryo Unbalanced Embryo Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 17 For Research Use Only. Not of use in diagnostic procedures.
Case Example #2: Suspected SR - t(2;7) • 39yo female patient and 43yo male partner • Clinical History: AMA • 8 embryo biopsy samples received for PGT-A • Euploidy rate for 38-40yo female = 30-35% (i.e. 2-3 normal embryos expected) • Results: • No euploids • 4 abnormal embryos involving the same segments (+/- other abnormalities) • 2q35q37.3 - ~22Mb • 7q35q36.3 - ~15Mb • 2 abnormal embryos involving the same chromosome 2 or or chromosome 7 segment • 2 additional abnormal embryos involving other chromosomes 18
Case Example #2: Suspected SR – t(2;7) Embryo #7 Embryo #11 Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 19 For Research Use Only. Not of use in diagnostic procedures.
Case Example #2: Suspected SR - t(2;7) • Re-analyzed all samples on PGT-SR (high resolution) workflow • Results: • No euploids • 6 abnormal embryos involving the same segments (+/- other abnormalities) • 2q35q37.3 - ~22Mb • 7q35q36.3 - ~15Mb • 2 additional abnormal embryos involving other chromosomes 20
Case Example #2: Suspected SR – t(2;7) Embryo #11 – PGT-A workflow Embryo #11 – PGT-SR workflow Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 21 For Research Use Only. Not of use in diagnostic procedures.
Case Example #2: Suspected SR - t(2;7) • PGT-SR workflow identified the same abnormal segments in 6 of the 8 embryos • Results reported as suspected translocation with a recommendation for karyotypes in both partners • Upon follow up, male partner’s karyotype revealed a reciprocal translocation • 46XY, t(2;7)(q35;q35) 22
Case Example #3: Suspected SR – Y chr. • 36yo female patient and 38yo male partner • Clinical History: AMA • First cycle: 3 embryo biopsy samples received for PGT-A • Euploidy rate for 35-37yo female = 40-45% (i.e. 1-2 normal embryos expected) • First cycle results: • 2 euploids (1 male, 1 female) • 1 aneuploid (male): partial gain 10p15.3p15.1, partial gain Yq11.223q12 23
Case Example #3: Suspected SR – Y chr. • Second cycle: 5 embryo biopsy samples received for PGT-A • Euploidy rate for 35-37yo female = 40-45% (i.e. 2-3 normal embryos expected) • Second cycle results: • 1 euploid (female) • 4 aneuploid (all male): partial gain Yq11.223q12 • 1 also had partial loss Yq11.221q11.223 • Results reported as “suspected Y -chromosome structural rearrangement” with a recommendation for a karyotype in the male partner 24
Case Example #3: Suspected SR – Y chr. • Couple underwent genetic counseling the following day to review results and discuss possible outcomes of the karyotype and clinical interpretation • Further internal discussion regarding Y chromosome abnormality led to re-analysis of ALL 8 embryos (1 st and 2 nd cycle) with the PGT-SR workflow, with special attention to the Y chromosome • Results: ALL male embryos showed the same partial Y chromosome loss (q11.221q11.222/3) & partial Y chromosome gain (q11.223q12) 25
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