PGT Applications and Biopsy procedures - COOK Media workshop- Dubai 2014
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This document discusses preimplantation genetic testing (PGT) techniques used to identify genetic defects in embryos prior to implantation, including preimplantation genetic screening (PGS) to detect chromosomal abnormalities and preimplantation genetic diagnosis (PGD) to detect single gene disorders. It provides details on the techniques of polymerase chain reaction, fluorescence in situ hybridization, microarrays, and biopsy methods at different embryonic stages. Key factors in selecting a PGT procedure include the accuracy, sensitivity, speed of the technique as well as the testing required, number of cells needed, and equipment and skills available.
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PGT Applications and Biopsy procedures - COOK Media workshop- Dubai 2014
- 1. Preimplantation Genetic Testing Ahmad Mustafa Metwalley, MSc, Molecular Pathology ART Lab. Director Al Baraka Fertility Hospital
- 2. PGT, is a technique used to identify genetic defects in embryos.
- 3. Chromosomes •PGS:Preimplantation Genetic Screening •CCS: Comprehensive Chromosomal Screening Genes •PGD: Preimplantation genetic Diagnosis •PGH: Preimplantation Genetic Haplotyping
- 4. 1968 1978 1990 1993 1996 2000 2004 2008 2009 2012 History of PGT IonTorrent/Next Generation
- 5. Genetic Study A- Preimplantation Genetic Diagnosis (PGD)
- 6. PGD is performed to differentiate affected, carrier or healthy embryo for transfer from abnormally genotyped parents. Step to avoid abnormality inheritance……….
- 7. • Single gene disorders – Recessive, dominant, X-linked: Sickle cell, cystic fibrosis.. – Triplet repeat disorders: fragile X syndrome – Late onset disorders: Cancers • Chromosome abnormalities – Translocations: Robertsonian Translocations – Inherited chromosome abnormalities: • X linked disease – Specific diagnosis: DMD, Hemophilia.. – Sex selection
- 8. Genetic Study B- Preimplantation Genetic Haplotyping
- 10. Chromosomal Study A- Preimplantation Genetic Screening (PGS)
- 11. •PGS are screening techniques for aneuploidy from normal genetic parents. •To improve IVF take home baby. 1. Advanced maternal age (>35/38) 2. Severe male factor 3. Recurrent IVF failure (2/3 or more) 4. Recurrent miscarriage (Normal karyotype)
- 12. Solution for a daily problem
- 13. Solution for a daily problem
- 15. IVF/PGT Setup
- 16. 1. IVF unit 2. Successful PGD Preparation 3. Basis of selection of procedures
- 18. Successful PGD Preperation Post-EC Post - ET • Documentation •Updating Genetic unite: Genetist/Embryologist •Patients Instructions: •Counter Check Pre-EC •Patient: proper counseling •Plan: type, •Proper coordination: with Genetic Unit •Biopsy Practitioner: Biopsy, Fixation, Tubing, •D5 facility: Incubator, Manipulations Practice, •Cryo service:
- 19. Basis of selection of procedures • Accurate • Sensitive • Fast
- 21. Diagnostics
- 23. Polymerase Chain Reaction (PCR) Multiple copies of specific DNA sequence ‘Molecular photocopying’
- 24. Fluorescence in situ hybridization (FISH) G G C T T C CG C A A G G C GC C T G GCG G C G T T C GC T G C GG GG T T A A A T A C C G A C C C C A Chromosome on slide / …Fixation Fluorescent Probes ….Coloring Chromosomes
- 25. Sex Selection Chromosome X Chromosome Y Chromosome 16 Normal Female Normal Male
- 26. Microarrays Array-Comparitive Genomic Hybridization (a-CGH) It is ultra sensitive and accurate technology to read 24 chromosomes together.
- 27. & Amplify DNA WGA Biopsied cell(s) Control DNA Combine labelled DNA Blue Probes Biopsied cell DNA
- 28. Limitations and Misdiagnosis • Amplification failure – Degenerate nucleus – Un-Nucleated Blastomere – Multi- nucleated Blastomere – Fragments • Mosaicism – TE biopsy • Contamination – Cumulus cells: Eggs denudation – Sperm: ICSI – Practitioner
- 30. Biopsy
- 31. Techniques: Biopsy Polar Body Biopsy Cleavage D3- Biopsy Blastocyst D5-Biopsy Time Maternal Only Many Cells Freezing Single Cell Mosaicism
- 32. Days: 0/1,3 or 4 & 5 D3 Cleaving Biopsy D5 Blastocyst Biopsy
- 33. Drilling Acid Tyrodes solution, pH 2.2 Chemical Drilling Sharp NeedleMechanical Drilling Compact diode 1.48m laser Laser Drilling
- 34. Blastomere Extraction 2 holes, Injection media, control?!!Displacement 1 hole, Easy, fast, I like it??!!Extrusion 1 hole, blastomere lysisAspiration
- 35. Ca. Mg free medium Arrest EmbryosWashout Biopsy Media To avoid Embryo ArrestTemperature Proper Labeling Numerical Sequence Surface Oil Stripping CO2 StressTiming Post Biopsy
- 37. Considerations Ensure Biopsy medium will warmed Working with 3D Embryo stripper 300 LASER from OUT to IN Space between droplets Avoid CO2 with biopsy media Blastomere Stripper < 130 Drill wide hole Big droplets
- 38. 10 % HEPES NOT Ca Mg FreeMedia 1 hole, Easy, fast, I like it??!!LASER 1 hole, blastomer lysisMechanical Blastocyst Biopsy Trophoectoderm
- 39. Considerations If not expanded do not biopsy Differentiate ICM Hatching better than hatched Biopsy Media Vitrification Immediate Practice
- 40. Summary Type and method of biopsy will dependent on: •Testing required – maternal/paternal/ postzygotic •Time required for diagnosis •Successful cryopreservation programmed? •Number of cells required •Efficiency of diagnosis •Equipment available •Skills of biopsy practitioner
- 42. Contact us: https://www.facebook.com/EmbryologistsWW https://www.facebook.com/IVF Laboratory Techniques, From Basic To advanced
Editor's Notes
- Undiagnosed embryos never transferred Mostly fertile couple.
- by
- 62-64 hrs post insemination
- Original method •Acid Tyrodes solution, pH 2.2 –Applied locally using a 3rd microtool •Benefits: –Inexpensive •Drawbacks: –Poor control of hole size –Change in pH of biopsy drop – Slower – Resealing of hole (small degree) –Chemical contamination/toxicity Tearing zona using needle, 3rd microtool •Partial zona dissection •3D method reported •Benefits –Inexpensive –Quick –No heat damage/chemical toxicity •Drawbacks –Very poor control –Danger of piercing blastomeres –Irregularity in hole size Now widely used •Compact diode 1.48m laser •Creates groove in the zona •Benefits –Excellent control over hole position and size •Drawbacks –Expensive –Calibration/servicing –Potential heat damage
- Displacement •Reported clinically •2 holes made opposite each other •Medium is injected into one hole •Injection of media displaces blastomere out of the other hole (hopefully!) –No control –Detrimental to embryo –Difficult with compact embryos Extrusion •Only one report clinically •One hole is made •Embryo is pushed on opposite side to hole causing blastomere to pop out (hopefully!) –Poor control –Difficult with compact embryos –Tight junctions can pull cell back in again when pressure removed Aspiration •Most common method •One hole made •Embryo held in place with holding pipette •Aspiration pipette applies gentle suction through the hole to pull the cell out. •30-35μm internal diameter –Excellent control –BUT constriction of cell in pipette can cause lysis
- Displacement •Reported clinically •2 holes made opposite each other •Medium is injected into one hole •Injection of media displaces blastomere out of the other hole (hopefully!) –No control –Detrimental to embryo –Difficult with compact embryos Extrusion •Only one report clinically •One hole is made •Embryo is pushed on opposite side to hole causing blastomere to pop out (hopefully!) –Poor control –Difficult with compact embryos –Tight junctions can pull cell back in again when pressure removed Aspiration •Most common method •One hole made •Embryo held in place with holding pipette •Aspiration pipette applies gentle suction through the hole to pull the cell out. •30-35μm internal diameter –Excellent control –BUT constriction of cell in pipette can cause lysis
- Displacement •Reported clinically •2 holes made opposite each other •Medium is injected into one hole •Injection of media displaces blastomere out of the other hole (hopefully!) –No control –Detrimental to embryo –Difficult with compact embryos Extrusion •Only one report clinically •One hole is made •Embryo is pushed on opposite side to hole causing blastomere to pop out (hopefully!) –Poor control –Difficult with compact embryos –Tight junctions can pull cell back in again when pressure removed Aspiration •Most common method •One hole made •Embryo held in place with holding pipette •Aspiration pipette applies gentle suction through the hole to pull the cell out. •30-35μm internal diameter –Excellent control –BUT constriction of cell in pipette can cause lysis
- nselling
- Benefits –Maternal and paternal genome analysable –Post zygotic errors identifiable –Multiple cells for accuracy –Relative resiliance of blastomere vs polarbody –Larger time window for biopsy •Drawbacks –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
- Benefits –Maternal and paternal genome analysable –Post zygotic errors identifiable –Multiple cells for accuracy –Relative resiliance of blastomere vs polarbody –Larger time window for biopsy •Drawbacks –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