Published on December 20, 2013
The finer points of trophectoderm biopsy Suzanne Cawood The Centre of Reproductive and Genetic Health, London. UK
Preparing for blastocyst biopsy Excellent blastocyst culture system - KPIs (Blastocyst formation rate, utilisation rate, %d5 vs. d6 formation rate Group/individual culture until d5. If group culture, separate blasts morning of d5/6 into individual culture Zona drilling d3 – ensure hole size optimum (too large, risk of premature cell loss; too small, risk of ‘pinching’ of trophectoderm cells) Biopsy in HEPES media. Make dishes 1 hour prior to procedure warmed at 37°C (non-gassed) Ensure microscope stage heated to temperature which ensures droplet temperature is at 37 °C Ensure optimum service is provided. Biopsy at d5AM/PM + d6 AM/PM is possible.
When and what to biopsy? WHEN? Take into account how long the diagnosis will take. Vitryfying blastocysts post biopsy? Or aiming for fresh embryo transfer? WHAT? Hatching or Hatched blastocysts. Hatching more ideal If herniating – trophectoderm cells will have to be ‘sucked’ from zona – more risk of lysis and damage to the embryo ICM position
The procedure Rapid technique Minimise trauma/damage to both embryo and cells biopsied Use blastocyst holding pipette Common difficulties: Positioning of embryo/pipettes (Hatched blastocysts, ICM hatching out) Do not take too many cells! Remember image is 3D Control at all times – both holding, suction and laser. Utilise foot pedal if possible. Minimise number of laser shots – maximise shot value. Laser shots between the junction of trophectoderm cells Post biopsy, cells to be washed (PBS-PBA) and tubed (hand-pipetted) into empty eppendorf tubes
Technique of excising cells Laser shot and tear cells Fewer laser shots and ‘brushing’ the cells off using the holding pipette. - Difficulty: Cells are sticky! - more likely to leave cells on holding/aspiration pipette (potential contamination so ensure pipettes changed if necessary) - this technique has to be used for completely hatched blastocysts
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