Alternative Methods of Blastocyst Biopsy By Tracey Griffiths, Senior Clinical Scientist, Oxford Fertility Unit

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Alternative Methods of Blastocyst Biopsy By Tracey Griffiths, Senior Clinical Scientist, Oxford Fertility Unit

  1. 1. OXFORD FERTILITY UNITOXFORD FERTILITY UNIT Alternative Methods ofAlternative Methods of Blastocyst BiopsyBlastocyst Biopsy TRACEY GRIFFITHSTRACEY GRIFFITHS Senior Clinical Scientist
  2. 2. IntroductionIntroduction  Day 3 to drill or not to drill?  Dish & laser set up for day 5/6 biopsy  Blastocyst biopsy procedure  Licensing procedures  Factors Influencing outcome
  3. 3.  Pros – Technically easier day 5/6 biopsy for less experienced practitioners as cells already herniating through hole – Tool holder not required  Cons – Additional time out of incubator for embryo – No idea where ICM will form, may be in herniating portion – Day 3 may fall over the weekend, needing additional experienced staff Day 3 Zona Drilling for Blastocyst biopsy - Laser
  4. 4. Day 3 Zona Drilling for Blastocyst biopsy - Laser  Non-contact lasers operate in infrared spectrum - safer as their potential for damaging surrounding cells is far less  Laser can be fired at a single point to create a hole or at several points next to each other to create a slit  Size of hole dependent on power of laser and duration of firing: ie: 0.250ms pulse from Saturn Active laser gives a hole size of about 5µm  Drill successive small holes rather than attempt to breach zona with a single shot  Performed at 37O C  If using sequential media - drilling is performed prior to day 3 media change  Possible to drill in embryoscope dish
  5. 5. Biopsy Procedure Blastocyst stage biopsy  Grade all embryos, prepare paper work & biopsy dishes for all suitable blastocysts (see next slide for dish set up)  If hole drilled on Day3 would expect trophectoderm to be herniating through opening  Calibrate laser – follow step by step instructions on screen. Perform test fire on ink to confirm correct position  Set up micropipettes usually id 30 -> 35 µm used  Alternatively drill at time of biopsy on D5/6  Gloves are worn for all biopsy procedures
  6. 6. Biopsy Procedure Petri dish preparation for blastocyst stage biopsy using a laser 1 Blastocyst wash drop
  7. 7. Biopsy Procedure – Day 3 drilled  Balance pipettes  Position Blastocyst on holding pipette  Trophectoderm cells drawn into pipette.  Average number of 5 cells (range 2-9) reduces risk of misdiagnosis  Laser used to slice off cells – Aim at junction of the cells just behind trophectoderm cells inside lumen of biopsy pipette – pulses at between 0.400 – 0.500µs  Biopsy & holding pipettes simultaneously pulled apart to facilitate separation
  8. 8. Biopsy Procedure – Drilled at biopsy  Set up & balance pipettes as before  Position Blastocyst on holding pipette close to the ICM  On the opposite side to the ICM very carefully create an opening 10 - 20 µm in the zona. This is done on the lowest power level due to the thinness of the zona and it is important to start at the outside edge and work inwards.  Some of the cells should start to herniate as the hole is created. Gently aspirate between 2 -9 cells into the biopsy pipette and use the laser to slice off as before.
  9. 9. TROPHECTODERM BIOPSY WITHOUT ZONA OPENING AT THE CLEAVAGE STAGE ANTONIO CAPALBO¹ AND CHRISTIAN OTTOLINI² ¹ GENERA Reproductive Medicine Centers, Italy. ² The London Bridge Fertility, Gynaecology and Genetics Centre, London, UK  Position Blastocyst on holding pipette with ICM at 7 o’clock  Create an opening in the zona of about 10µm. Carefully press the biopsy pipette against the zona and gently expel media through the breach. This will release the TE cells from the internal surface of the zona (B and C, respectively). This step helps to avoid blastocyst collapse during the subsequent TE cell removal.
  10. 10. Another alternative  Drill on the morning ofDrill on the morning of biopsy & return to thebiopsy & return to the incubator for several hoursincubator for several hours  After cultureAfter culture  After biopsyAfter biopsy Post biopsy - 1hr in culture
  11. 11. Manual biopsy of the fully hatched blastocyst  Position the biopsy pipette with the blastocyst above the top of the holding pipette and get both pipettes in the same plane of focus. This is critical or the ‘rub off’ doesn’t work
  12. 12. Post Biopsy TLC  Wash the blastocyst through two wash droplets of blastocyst culture media before placing it in the correctly labelled blastocyst droplet.  If blastocyst’s have been cultured in an embryoscope dish up to the point of biopsy, it is important to transfer them to a culture dish after biopsy. Otherwise by the time of embryo transfer on day 6 they are fully hatched & stuck in the well! 1 2 Only 2 blastocysts /dish at opposite sides to prevent drops merging 50µl drops blastocsyt Culture media with oil overlay
  13. 13. Factors influencing outcomeFactors influencing outcome  Embryo development/qualityEmbryo development/quality  Media used- Major impact on reduction in preg in 2012/13Media used- Major impact on reduction in preg in 2012/13 – Sequential stage specific culture mediaSequential stage specific culture media – Advanced, ultra stable low oxygen culture systems (MINC/BT37Advanced, ultra stable low oxygen culture systems (MINC/BT37 /Panasonic incubators)/Panasonic incubators) – Adapted to changing metabolism of blastulating embryoAdapted to changing metabolism of blastulating embryo  Choice at transferChoice at transfer  Chromosomal constitutionChromosomal constitution  Cryopreservation?Cryopreservation?  Technical competence of biopsy practitonersTechnical competence of biopsy practitoners
  14. 14. Licensing processLicensing process  If ICSI qualified-25 embryosIf ICSI qualified-25 embryos  If not ICSI qualified-50 embryosIf not ICSI qualified-50 embryos  Detailed log book and videoDetailed log book and video  Application to HFEAApplication to HFEA  InspectionInspection  Impact on workloadImpact on workload  If 1 member of staff licensed as aboveIf 1 member of staff licensed as above they can assess others for competencethey can assess others for competence
  15. 15. Blastocyst BiopsyBlastocyst Biopsy • Technically demanding • Practice forbidden unless part of a research project • We have established consent for training • Multiple competent operators required

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