Trophectoderm Biopsy: Methods and Tips

Dr Cristina Hickman, Head of EmbryologyCristina.Hickman@bostonplaceclinic.co.uk

Trophectoderm Biopsy:Methods and Tips

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Over 6000 patients per yearLargest Provider of IVF in the UK

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Why biopsy at the blastocyst stage?

Exclude embryos that are not capable of blastulating (25-50%)Many Cells for AnalysisBetter account of mosaicismLess No results (5% vs 10%)Easier to distinguish Cells from fragments (i.e. extruded cells)Extraembryonic tissue can be sampledSmaller proportion of embryonic mass takenLess likely to compromise embryo viabilitySurvival rates after vitrification >98%Knowledge of blastocyst qualityBetter clinical results

What are the barriers for some clinics?Requires a good blastocyst cryopreservation programmeShort time for diagnosis (if patient requests fresh ET)Requires good blastulation rateIncreased cycle cancellation rateHistoricalTechnically more difficult particularly if slow development

Blastocyst is better than cleaved biopsy

This lecture

PGS programme workflow

Workflow variationsSelecting Embryos for biopsyD3 Assisted HatchingDish PreparationFreeze EmbryoFETWorkflow variationsAssisted Hatching on day 3?Which embryos to biopsy (when)Freezeall vs fresh

ET (Day 6)Biopsy (By 10am)Freeze surplus (Day 6)Selecting Embryos for biopsyDish PreparationBiopsyFreeze EmbryoFETWarm embryosDish PreparationBiopsyWarm Blastocysts(Day 4)Dish PreparationBiopsyET (Day 5)Freeze surplus (Day 5)

Freeze all vs FreshTime cut-off for Fresh ET on Day 6 at 2pm, biopsy leave building by 9.30am day 5

Freeze all embryos can be biopsied and frozen at optimal stage Optimal time for endometrium?

Better chance of pregnancy

Warm Biopsy Refreeze

Fresh Warm Biopsy Post Biopsy ReVitrify Re-Warm Pregnancy

Wolff et al8

Assisted Hatching

Assisted Hatching

Assisted Hatching on day 3ProsConsTechnically easier if correct cells are herniatingICM may herniate

Additional task (weekend?)

Interruption of culture (risk?)

Alternative: AH during procedure, or on morning of procedure

Hatching at the time of biopsy

Capalbo techniqueCapalbo expel media through breach to unstick TE from zona

Hatching at the time of biopsy: Nudging

Tips of zona penetrationCollapse embryoHole just the right size

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Day of Procedure Setup

Day of ProcedureGrade embryos and select for biopsyAim for stage 4/5The more embryos biopsied, the higher the chance of a normalDo not exclude poor quality embryos

Gloves during tubing procedureDishes GMOPS warmed on day of procedureTest/Calibrate laserLine up micropipettes (Bevelled or Flat)Prime holder with HEPES/G-MOPS media and biopsy pipette with PVP

Dish PreparationPVP~15LBetter pipette control than mediaAvoid cells sticking to microtoolsMedia30-50uLMedia without debrisGMOPS or Ca/Mg free media if cleavage

Number of embryos per dishBuffer Drops between drops containing embryosDo not reuse a drop for another embryo!

Oil overlay

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PVP

Dish preparation Multiple embryos per dishUse of PVPSize of dropsOther tips on the day of procedureHolder sizeBevelled vs flat biopsy needle17

cell removal technique

Cell removal techniqueFocus on blastocyst, position holder in same focal plane

Position ICM on holder side

Aim for between 2 and 9 cells

Laser at junction of cells

Keep to 90%)implantation rate varies with embryo grade but not with patient age

Biopsy KPIsWhat to investigate if no result rate is too high or implantation of normal embryo is too low

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Competency assessment & Training

mouse blastocyst training (embryotools) 50 mouse embryos 25 human embryos (with consent)

Technical skills and Knowledge base

Confidence in all stages of blastulation

EQUIPMENTLaser (active)Inverted microscope: visualisation of cell junctionCamera ( monitoring) Pipettors LAF hoodTime-Lapse: Choice of embryos for transfer following biopsy

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Managing expectations

Chance of Normal Embryos

Managing expectations

Chance of No ET vs Implantation per normal ET

NOT ALL BLACK & WHITENo resultsPartialMosaics

Ingredients of a good biopsy programtraining progmonitoringblastFreeze allManage expecEquipmentHatch on D5Multiple biopsy practitionersGood cryo prog

Embryologists:Oriol OlianaSandy ChristiansenDanielle GwinnettThomas Wilkinson

Thank you

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LayoutWhy Blastocyst BiopsyWorkflow variationsAssisted Hatching on day 3?Which embryos to biopsy (when)Freezeall vs freshLaser Alignment Importance of testing and calibrating the laserSizeNumber of holesHole shapeImplications of too much laserDish preparation Multiple embryos per dishUse of PVPSize of dropsOther tips on the day of procedureHolder sizeBevelled vs flat biopsy needleTips of zona penetrationCollapse embryoHole just the right sizeTips of shooting cellsTypes of BiopsyTraditionalFlick12 o clockPossible causes of mosaicismCleaning pipetteSperm from ivfCumulus cell contaminationExtruded cellsRemoving extruded cells before biopsyReinserting embryo into shellBiopsied cellsAssessing number of cells biopsied how many should you biopsyLysed?Assessing survival post biopsyWarm biopsy refreezeBiopsy KPIsWhat to investigate if no result rate is too high or implantation of normal embryo is too low

Managing patients expectationsWhat you need for a good biopsy programImplications of no results

Blastocyst StageProsConsBoth parental genetic contributions can be analysed

Better account of mosaicism

Cell vs fragment

Extraembryonic tissue can be sampled

Many cells for analysis

Smaller proportion of embryonic mass taken

Survival rates after vitrification >90%

Low rate of Monozygotic twinningTechnically more difficult particularly if slow development

Mosaicism (although lower than earlier stage biopsies)

TE same as ICM in terms of ploidy?

ICM hatching unpredictable

Short time for diagnosis

Blastulation rate

Less time between biopsy and ET freeze all required? More cost to patient.

Increase of cycle cancellation rate

Ethical concerns