Overview of Embryo Biopsy By Tracey Griffiths, Senior Clinical Scientist at Oxford Fertility Unit

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Overview of Embryo Biopsy

Overview of Embryo Biopsy

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  • 1. OXFORD FERTILITY UNITOXFORD FERTILITY UNIT Overview of Embryo BiopsyOverview of Embryo Biopsy TRACEY GRIFFITHSTRACEY GRIFFITHS Senior Clinical Scientist
  • 2. Embryo BiopsyEmbryo Biopsy  Biopsy procedure and pros and cons for eachBiopsy procedure and pros and cons for each different stage:different stage: – Polar body biopsyPolar body biopsy – Cleavage stage biopsyCleavage stage biopsy – Blastocyst biopsyBlastocyst biopsy
  • 3. Polar body biopsy-MII oocytePolar body biopsy-MII oocyte  Zona drilled byZona drilled by preferred methodpreferred method  Polar body removedPolar body removed  Can be performed onCan be performed on day of egg collection orday of egg collection or day of fertilisationday of fertilisation checkcheck
  • 4. Video- Laser polar bodyVideo- Laser polar body
  • 5. Polar body biopsyPolar body biopsy  ProsPros – Uses extra embryonic materialUses extra embryonic material – Useful in countries where screening ofUseful in countries where screening of embryos forbiddenembryos forbidden  ConsCons – Only maternal genetic contribution can beOnly maternal genetic contribution can be analysedanalysed  This restricts its application to prevention ofThis restricts its application to prevention of oocyte meiosis-based aneuploidies &oocyte meiosis-based aneuploidies & genetic disease in the maternal linegenetic disease in the maternal line – PB may be degenerate or fragmentedPB may be degenerate or fragmented
  • 6. Polar body biopsyPolar body biopsy  3 patients3 patients 33 polar bodies biopsied33 polar bodies biopsied 30 results30 results 91% diagnosis91% diagnosis 0 damaged0 damaged 5 normal5 normal 17%17% All negative pregnancy testsAll negative pregnancy tests
  • 7. Cleavage stage biopsyCleavage stage biopsy  To avoid misdiagnosis patients requiring PGD/STo avoid misdiagnosis patients requiring PGD/S need their oocytes totally stripping of cumulusneed their oocytes totally stripping of cumulus cells and ideally inseminated by ICSI. Althoughcells and ideally inseminated by ICSI. Although CIVF may be used if appropriate but only PGSCIVF may be used if appropriate but only PGS – Ensures no supernumerary cells present that mightEnsures no supernumerary cells present that might interfere with diagnosis.interfere with diagnosis.  Biopsy performed in calcium/magnesium freeBiopsy performed in calcium/magnesium free mediamedia – Maintenance of cell-cell connections depend on theMaintenance of cell-cell connections depend on the presence of these ions. Therefore blastomeres are heldpresence of these ions. Therefore blastomeres are held together less tightly in their absence, so facilitatingtogether less tightly in their absence, so facilitating biopsy procedurebiopsy procedure
  • 8. Biopsy ProcedureBiopsy Procedure Petri dish preparation for cleavage stage biopsyPetri dish preparation for cleavage stage biopsy using a laserusing a laser 15µl drop calcium/magnesium free media for balancing the pipettes 10µl drop PVP for balancing pipette & washing should cells lyse 1 embryo in bottom drop of ca +2 /mg +2 free media Drops covered with oil overlay Embryo number clearly marked on base & lid of each dish
  • 9. Cleavage stage biopsy
  • 10. Cleavage stage biopsyCleavage stage biopsy  ProsPros – Both parental contributions can be analysedBoth parental contributions can be analysed – Cells are still totipotent, not yet specialised (ieCells are still totipotent, not yet specialised (ie still have potential for developing in variousstill have potential for developing in various specialised ways in response tospecialised ways in response to external/internal stimuli). Biopsy doesn’t appearexternal/internal stimuli). Biopsy doesn’t appear to detrimentally affect developmentto detrimentally affect development – Early stage of biopsy allows sufficient time forEarly stage of biopsy allows sufficient time for diagnosis before embryo transfer without needdiagnosis before embryo transfer without need for cryopreservationfor cryopreservation
  • 11. Cleavage stage biopsyCleavage stage biopsy  ConsCons – Reducing cellular mass: Removal of 1 or 2 cells from 6-Reducing cellular mass: Removal of 1 or 2 cells from 6- 8 cell embryo depletes embryonic mass by 12.5%-25%.8 cell embryo depletes embryonic mass by 12.5%-25%. – Disproportionate segregation of mitochondrial DNA andDisproportionate segregation of mitochondrial DNA and active mitochondria among blastomeres.active mitochondria among blastomeres. – Risks inadvertent removal of critical cells or cells of highRisks inadvertent removal of critical cells or cells of high mitochondrial content which could be fatal to themitochondrial content which could be fatal to the embryo’s potential for energy production & adequateembryo’s potential for energy production & adequate nucleic acid synthesis required for survivalnucleic acid synthesis required for survival – Mosaicism:Mosaicism: – Aneuploidy:Aneuploidy: – Only 1-2 cells for analysisOnly 1-2 cells for analysis – Embryo may be compactEmbryo may be compact
  • 12. Cleavage Stage BiopsyCleavage Stage Biopsy  21 patients21 patients  11 Ets11 Ets 9 patients had no normal embryo’s &9 patients had no normal embryo’s & therefore no embryo transfer -> 43%therefore no embryo transfer -> 43% 1 patient had no development1 patient had no development  12 CGH – 3ETs 2/3 66.6% clin preg12 CGH – 3ETs 2/3 66.6% clin preg  9 PGD – 8ETs 3/8 37.5% clin preg9 PGD – 8ETs 3/8 37.5% clin preg
  • 13. Introduction to Blastocyst BiopsyIntroduction to Blastocyst Biopsy  Day 3 - to drill or not to drill?  Dish & laser set up for day 5/6 biopsy  Blastocyst biopsy procedure  Pros & Cons of Blastocyst biopsy  Licensing procedures  Factors Influencing outcome
  • 14.  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 – Even blastocysts that are at a very early cavitating stage may herniate, when their cells are still very large Day 3 Zona Drilling for Blastocyst biopsy - Laser
  • 15. 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
  • 16. 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
  • 17. Biopsy Procedure Petri dish preparation for blastocyst stage biopsy using a laser  The day before biopsy place enough Falcon 1006 dishes (1 dish per embryo), 1ml hepes buffered media & a new bottle of oil into a non CO2 incubator.  On the morning of biopsy – on the underside of each dish, draw a line from top to bottom to mark the centre + round RI tag.  On the left side of the line pipette 2 x 15ul drops of hepes buffered media and on the right side of the line opposite the bottom drop pipette 1 x 5ul drop of pvp (mark with a circle on the base of the dish)  Pipette 1 x 15ul drop of hepes buffer above the pvp. This is used for rinsing the pipettes if necessary during the procedure.  Cover quickly with enough pre-warmed oil to cover the drops and replace the lid. Place in a non CO2 incubator.  Repeat for each dish (1 embryo per dish).
  • 18. Biopsy Procedure Petri dish preparation for blastocyst stage biopsy using a laser Blastocyst wash drop
  • 19. Blastocyst BiopsyBlastocyst Biopsy
  • 20. 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 – < 9 pulses at between 0.400 – 0.500µs  Biopsy & holding pipettes simultaneously pulled apart to facilitate separation
  • 21. 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.
  • 22. 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.
  • 23. Other alternatives  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
  • 24. Post Biopsy TLC  Immediately after biopsy return to the flow hood - It is more important to deal with the blastocyst than the biopsy at this stage since its time out of the incubator needs to be kept to a minimum.  Visualize the blastocyst and biopsied trophectoderm, carefully pick up the blastocyst (taking care not to pick up the biopsied trophectoderm cells) and transfer the blastocyst to its new culture dish.  A witness is required to verify that the patient and blastocyst number are correct.  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
  • 25. After Lunch time cut off or Day 6  If biopsy can be performed before 12 o’clock on day 5 aIf biopsy can be performed before 12 o’clock on day 5 a fresh day 6 embryo transfer is possiblefresh day 6 embryo transfer is possible  Any blastocyst’s biopsied after this time must be vitrifiedAny blastocyst’s biopsied after this time must be vitrified immediately after biopsy, before re-expansion startsimmediately after biopsy, before re-expansion starts  It is therefore essential to have everything needed for vitrification ready before starting the biopsy  NB: We label the straws with the EMBRYO NUMBER and NOT the straw number as in our normal vitrification sop – For example EMB 1
  • 26. Blastocyst stage biopsyBlastocyst stage biopsy  ProsPros – Both parental contributions can be analysedBoth parental contributions can be analysed – Extraembryonic tissue can be sampledExtraembryonic tissue can be sampled – MANY cells for analysisMANY cells for analysis – Blastocysts have lower prevalence of lethalBlastocysts have lower prevalence of lethal chromosomal monopolies & chaotic abnormalitieschromosomal monopolies & chaotic abnormalities than cleavage stage embryosthan cleavage stage embryos – Survival rates high, even after cryopreservationSurvival rates high, even after cryopreservation (close to 90%)(close to 90%) – Rate of Monozygotic twinning lowRate of Monozygotic twinning low – Level of mosaicism in blastocyst lower than levelLevel of mosaicism in blastocyst lower than level in cleavage stage embryosin cleavage stage embryos
  • 27. Blastocyst stage biopsyBlastocyst stage biopsy  ConsCons – Extraembryonic tissue-same as embryo?Extraembryonic tissue-same as embryo? – Vitrification required for some blastocysts, reducing survival?Vitrification required for some blastocysts, reducing survival? – Reduced number of embryos for biopsy?Reduced number of embryos for biopsy? Reported figures suggest only 50% of all embryos areReported figures suggest only 50% of all embryos are capable of reaching blastocystcapable of reaching blastocyst What we are seeing in our own patients - analysis of overWhat we are seeing in our own patients - analysis of over 10,000 embryo’s shows >66% blastocyst development rate10,000 embryo’s shows >66% blastocyst development rate across all ages & media types usedacross all ages & media types used – Effects of extended culture and biopsy?Effects of extended culture and biopsy? – Biopsy technique can be difficult if embryo development slowBiopsy technique can be difficult if embryo development slow or quality reducedor quality reduced
  • 28. Blastocyst Stage BiopsyBlastocyst Stage Biopsy 2012 - 132012 - 13  26 patients26 patients 3 patients had no normal embryo’s & therefore3 patients had no normal embryo’s & therefore no embryo transfer -> 12% (compared to 43%no embryo transfer -> 12% (compared to 43% for day 3 biopsy)for day 3 biopsy) 6 had embryo’s frozen6 had embryo’s frozen 1 FET1 FET 7/16 = 43.8% clin preg7/16 = 43.8% clin preg  23 CGH 7/13 = 53.8% clin preg23 CGH 7/13 = 53.8% clin preg  3 PGD 0 pregnancy (prior to Karyomapping)3 PGD 0 pregnancy (prior to Karyomapping)
  • 29. 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
  • 30. 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
  • 31. Blastocyst BiopsyBlastocyst Biopsy • Technically demanding • Practice forbidden unless part of a research project • We have established consent for training • Multiple competent operators required