Ion ReproSeq PGS Kits Sequence46 Disclosures Shareholder in - - PowerPoint PPT Presentation

PGT-SR with Ion ReproSeq PGS Kits

Catherine Welch Managing Director Sequence46

Disclosures

• Shareholder in Sequence46
• Thermo Fisher Scientific and its affiliates are not endorsing, recommending,
• r 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

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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

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Types of PGT

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PGT-A PGT-SR PGT-M

Screens for chromosomal aneuploid idy, e.g. Trisomy 21 Previously referred to as PGS Screens for stru tructural rearrangements, e.g. unbalanced translocations Previously referred to as PGD Screens for mon

• nogen

enic dis iseases, i.e. single gene disorders Previously referred to as PGD

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

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Gardner, R.J.M. and G.R. Sutherland. (1996) Gardner and Sutherland’s Chromosome Abnormalities and Genetic Counseling. 2nd 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

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

• ther complex rearrangements*

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*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

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For Research Use Only. Not of use in diagnostic procedures.

ReproSeq: Lab Workflow

• 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)

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Library prep

Combined DNA extraction, whole genome amplification (WGA), and sample barcoding

Template prep

Automated clonal amplification of libraries

Sequencing

Simple, cartridge-loaded reagents and user interface

Analysis

Automated Ion Reporter™ software

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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

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For Research Use Only. Not of use in diagnostic procedures.

Sample Embryo Profiles: Euploid

46, XY

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46, XX

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

Sample Embryo Profiles: Aneuploid

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46,X,+8 69,XXY

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

Sample Embryo Profile: Mosaic

46,XX / 47,XX,+15[45%]

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Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

Sample Embryo Profile: No Call

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Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

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

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Case Example #1: Known SR – t(5;7)

All segments are expected to be large (>50Mb), so the standard PGT-A workflow was utilized.

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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

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Case Example #1: Known SR – t(5;7)

Euploid/Balanced Embryo

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Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software.

Unbalanced Embryo

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
• r chromosome 7 segment
• 2 additional abnormal embryos involving other chromosomes

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Case Example #2: Suspected SR – t(2;7)

Embryo #7

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Embryo #11

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 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

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Case Example #2: Suspected SR – t(2;7)

Embryo #11 – PGT-A workflow

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Embryo #11 – PGT-SR workflow

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. 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)

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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

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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

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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 (1st and 2nd 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)

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Case Example #3: Suspected SR – Y chr.

Embryo #2 (1st cycle, ‘normal’ male) – PGT-A workflow

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Embryo #2 – PGT-SR workflow

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

Case Example #3: Suspected SR – Y chr.

Embryo #6 (2nd cycle, male with partial Y gain) – PGT-A workflow

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Embryo #6– PGT-SR workflow

Profiles generated by Thermo Fisher Scientific’s Ion Reporter™ Software. For Research Use Only. Not of use in diagnostic procedures.

Case Example #3: Known SR – Y chr.

Loss identified: Yq11.21/221q11.222/223 = ~2.9Mb Gain identified: Yq11.223q12 = ~4.9Mb

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No coverage via NGS

Case Example #3: Suspected SR – Y chr.

• New PGT-SR reports issued for both cycles indicating that all male

embryos appear to have the same Y chromosome related structural rearrangements

• Chromosomal microarray recommended for male partner (segments too small

to be reliably detected on standard or high resolution karyotype) – RESULTS PENDING

• Couple underwent genetic counseling a second time to re-review

results and discuss possible clinical implications

• Questio

ion: If If male ale par artner is is found to have a a Y c chromosome rear arrangement, ar are mal ale embry ryos sa safe to tran ansfer?

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PGT-SR Case Takeaways

• In our experience, ReproSeq’s standard PGT-A analysis workflow can assist

in the identification of previously-unknown parental chromosome abnormalities, especially those involving large chromosomal segments

• 6 patients/couples with suspected SRs over a ~1 year period
• The number of biopsy samples submitted greatly affects this, as data from a limited

number of samples may not reveal an obvious pattern – if a patient undergoes additional cycles, reviewing previous cycles’ results can be beneficial

• Karyotypes or microarrays are highly recommended for any suspected SR

cases to confirm the finding and ensure proper PGT-SR analysis for any subsequent cycles

• For atypical SR findings (ex: case #3 with the possible Y chromosome

abnormality) where the clinical effects are expected to be benign or are unknown, clinics will need to weigh ‘abnormal’ embryo transfers based on patient requests

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For Research Use Only. Not of use in diagnostic procedures.

Thank you!

catherine@sequence46.com

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