Oocyte and Embryo Biopsy - Dr Paul Knaggs, Consultant Embryologist, Wales Fertility Institute

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Dr Paul Knaggs, a Consultant Embryologist at Wales Fertility Institute presented on Oocyte and Embryo Biopsy at the RI Istanbul Conference and Hands on Workshop on the 23rd and 24th January 2014.
He covered the following in his presentation:
- Why perform biopsy?
- The limitations of biopsy
- Regulations
- Considerations before biopdy
- Practical points
- Methods of analysis
- Methods of zona breaching
- Mechanical breaching
- Acid breaching
- Laser breaching
- Dish set up
- When to biopsy
- Polar body biopsy
- Day 3 biopsy
- Blastomere selection
- How many cells to biopsy
- Advantages of Day 3 Biopsy
- Blastocyst biopsy
- Biopsy documentation
- Things to remember when biopsying





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Oocyte and Embryo Biopsy - Dr Paul Knaggs, Consultant Embryologist, Wales Fertility Institute

  1. 1. Oocyte and Embryo Biopsy Dr Paul Knaggs Consultant Embryologist Wales Fertility Institute
  2. 2. Why perform biopsy? • Avoidance of a genetic condition • Avoidance of chromosomal abnormality • Earlier detection of abnormality to avoid need for termination • Improvement of ART outcome • Avoidance of miscarriage • Achievement of pregnancy and healthy live birth. • Family balancing • Saviour sibling
  3. 3. Limitations • • • • • Regulation Ethical considerations Biopsy practitioner Logistics Has the condition been diagnosed / mutation identified? • Analysis technique and its precision
  4. 4. Regulation • Some countries have none and others are highly regulated. • Canada-unregulated until 2004, now individual provincial jurisdiction. • Germany-Pre 2010, variable, 2011 onwards regulated and permitted for severe conditions. • UK-highly regulated by HFEA. Was licensed case by case but now has a registers of permitted conditions. NO SEX SELECTION! • USA-loose regulation with inter state variation.
  5. 5. Considerations before Biopsy • • • • • • • • • What is the indication? Male vs Female vs Post zygotic Embryo status and previous development. ‘Must test’ or ‘Selection’ Aneuploidy / Single Gene / TL Genetic test results turnaround time Freezing / Vitrification results at your clinic Is your Genetics Lab accredited (eg CPA) Have you performed a ‘DRY RUN’ ?
  6. 6. Practical points • Provide the optimum service for your patients • Flexibility is essential • Embryo development delay (D3 or D5/6) may require delay of biopsy • Temperature QC is essential • No result (re biopsy / rescue biopsy?) • Genetic counselling?/ Is the patient prepared? • Use of Ca / Mg free medium vs HEPES alone • Pre incubation in Ca/Mg free medium for compacted embryos? (>10mins detrimental!?)
  7. 7. Methods of Analysis 1st PGD births in the early 1990’s • PCR for single gene mutation • FISH for sex selection • Early diagnosis with FISH could be complete within 612 hours with ET possible on day of biopsy • Culture conditions were sub optimal/ blastocyst culture not widely used • PCR also possible with results available for same day ET • Array CGH now fast becoming the analysis method of choice
  8. 8. What and When to Biopsy? • • • • • PB 1 PB 1+PB2 Blastomere (D3) TE (D5 or D6) FET cycle (D3 or D5 / D6) • Some or all of the above!
  9. 9. Methods of Zona Breaching Mechanical • • • Cheap Slower More traumatic Acid • • • • Cheap Quicker than mechanical Detrimental to embryo Not very precise LASER • • • Hi-tech Rapid and very precise Expensive
  10. 10. Mechanical Breaching • Requires 3rd microtool (double holder) • Tearing action to breach zona • Traumatic+slower • Inexpensive
  11. 11. Acid Breaching • Requires 3rd microtool (double holder) • Poor control of hole size • Slower • Toxic environment to embryo
  12. 12. Laser Breaching • Now widely used • Compact diode 1.48nm laser • Creates groove in the zona • Benefits – Excellent control over hole position and size • Drawbacks – Expensive – Calibration/servicing – Potential heat damage
  13. 13. Dish Set Up • All dishes must be labelled • All movements of oocytes and embryos (culture dish – biopsy dish – post biopsy culture dish) should be witnessed • Individual dish or drop per embryo / oocyte (beware drop merge!) • Post biopsy culture in individual drops in 4 well dishes Acid Tyrodes HEPES Biopsy Drops HEPES Post-Biopsy Wash Drops
  14. 14. When to Biopsy TVOR (Day 0) PB1 Biopsy Results PB1± 2 Biopsy Results ICSI/ IVF Fert check (Day 1) Day 2 Cryo Day 3 Assisted Hatching Blastomere Biopsy Results ET ± Cryo Results ET ± Cryo Results ET ± Cryo Day 4 Cryo Day 5 Blastocyst Biopsy Cryo Day 6 Blastocyst Biopsy
  15. 15. Polar Body Biopsy • Established in Chicago • Originally 1st PB only – Preconception diagnosis • Require both PBs for accurate diagnosis • Potentially combined with cleavage stage biopsy • Used for single gene, maternal translocations, age related aneuploidy • Waste products or required for development??
  16. 16. Polar Body Biopsy • Benefits – Longer time period for Embryo integrity maintained – diagnosis • CGH – More ethically accepted in certain countries eg Germany, Italy – Can karyotye or paint chromsomes • Drawbacks – Not suitable for paternally derived defects – No detection of mitotic errors (mosaic/chaotic embryos – Fragility of polar bodies – Labour intensive
  17. 17. Polar Body Biopsy ICSI at 40 hours post hCG • Biopsy 1st PB • Or………. immediately after ICSI • Biopsy 1st and 2nd at • Biopsy 2nd PB 12 hours post ICSI 12 hours post ICSI • Improved integrity of PBs • Potentially more stressful • Increased risk of PB1 fragmentation • ?less stressful to oocyte
  18. 18. Polar Body Biopsy • 3 Methods • Bevelled pipette – pierce zona (no artifical zona breach) • Laser assisted – Channel formed to allow blunt pipette to pass through zona • Hybrid – bevelled pipette and laser assisted
  19. 19. Polar Body Biopsy
  20. 20. Day 3 Biopsy • Cleavage stage embryo can fragile • Increased risk of mosaicism • May need to delay biopsy if slow cleavage • Blastomeres are totipotent (or are they??)
  21. 21. Day 3 Biopsy Cell in focus but touching zona. Breach may not be clear. Cell not in focus. Breach will not be on same plane. Cell in focus with clear gap between zona. Access to 2nd cell.
  22. 22. Blastomere Selection • Single visible nucleus • Absence of multinucleation – Associated with chromosomal abnormalities • • • • Smaller cell (cytokinesis complete) Removal of vacuolated cell Minimal overlap of cells Easier with non-compacted embryos • Compacted embryos may require longer exposure to biopsy medium
  23. 23. Day 3 Blastomere Biopsy
  24. 24. How Many Cells to Biopsy? • Historically 2 cells were required for complete information • Does 2 cells increase confidence or introduce conflicting results? • Greater loss of embryo mass reduces blastulation rate. • Array aCGH allows immense data from 1 cell and can use linkage analysis (parental / family DNA) to make results more precise. • Reprogenetics has recently published the validation of array CGH (Gutierrez Mateo et al. 2011, Fertil Steril, 95:953–8) for aneuploidy diagnosis. • Error rates from D3 biopsy may be as low as 1.8% for false positive and 0.2% for false negative.
  25. 25. Advantages of D3 Biopsy • Advantages – Maternal and paternal genome analysable – Post zygotic errors identifiable – Multiple cells for accuracy – Relative resiliance of blastomere vs polarbody – Larger time window for biopsy • Disadvantages – Accuracy compromised by mosaicism – Removal of embryonic mass – Shorter time period for diagnosis – Ethically unaccepted in some countries – Normally interphase nuclei – no paint or karyotype
  26. 26. Procedure Biopsy Media – HEPES buffered, Ca2+ Mg2+ free – Loosen Gap junctions - Pre-incubation not essential Acid Tyrodes (not required for laser) HEPES Biopsy Medium 12 l culture drops under oil HEPES Post-Biopsy Wash Drops
  27. 27. Re-Biopsy of D3 Embryos • Same day – No nuclei seen on spreading blastomere – Use original hole – Relatively easy • Day 4 – All diagnosed embryos abnormal and others show: • No result • Inconclusive result – Use original hole – Increased compaction • Increased cell lysis • Preincubation in biopsy medium if required – ?Polarisation of blastomeres? • Both will add extra stress and remove mass from the embryo
  28. 28. Blastocyst Biopsy • • • • Zona breach Day 3/4/5 Preferably D3 Biopsy day 5 Differentiation of cell lines occurred – Inner Cell Mass (ICM) – Trophectoderm (TE) • Biopsy herniating TE • 5-8 cells excised – Glass needle – Laser
  29. 29. Blastocyst Biopsy • Advantages – Larger number of cells can be taken – Smaller proportion of embryonic mass taken – Not taking cells destined to contribute to the foetus • Disadvantages – Short time for diagnosis – <50% Blastocyst formation • Fewer embryos to test • Increased cycle cancellation – High levels of mosaicism • ?Preferential allocation of abnormal cells to TE?
  30. 30. Blastocyst Biopsy
  31. 31. Blastocyst Biopsy • Performed in HEPES medium (not biopsy medium!) • Preferable to hatch on D3 with laser (approx 10 m hole) • Biopsy performed with simultaneous suction, pulling of trophectoderm and laser shots • Hatched blastocysts can be biopsied with care • Avoid ICM • Carefully choose correct diameter of trophectoderm biopsy pipette
  32. 32. Biopsy Documentation
  33. 33. Things to remember • Protect your biopsies – thoroughly clean the work area to avoid contamination • Wear gloves, sterile gown or arm protectors • Use a separate pipette for each biopsy (avoids DNA) contamination if lysis occurs. • Label everything (Dishes, lids, tubes) with at least the patient surname and DOB • Witness all movements of embryos and biopsies to avoid mistakes
  34. 34. Things to remember Planning and preparation are key • Dish preparation in advance (biopsy and post biopsy) • Pipette choice: • PB (10µm) • Blastomere (30-35µm) • TE (20-30µm) – Bevelled or Non bevelled • • • • Wash dishes Slides / tubes for cell fixation 2nd embryologist for moving embryos / cell fix? Courier – if transport to external testing lab is required
  35. 35. Summary • Type and method of biopsy will depend on: – Testing required – maternal/paternal/postzygotic – Time required for diagnosis • Requires successful cryopreservation programme – Number of cells required • Efficiency of diagnosis – Equipment available • Low O2 incubation, laser – Skills of biopsy practitioner • Increasing evidence to suggest performing genetic test as late as possible during lab development (Capalbo et al 2012) • 47.6% of embryos had post zygotic errors detected • 20% of aneuploidies detected at PB and D3 corrected at the blastocyst stage
  36. 36. Review the literature for updates and results

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