Culture media for IVF: which to choose?

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Culture media for IVF: which to choose?

  1. 1. Culture Media
  2. 2. Overview: What does media do? • Keeps everything wet • Feeds the cells • Controls the environment
  3. 3. Media Components Media is basically salt water with added vitamins and protein • Salts • Carbohydrates • Protein • Metabolites • Buffers • Antibiotics • Water
  4. 4. • LOTS OF CODES!! • 28 different codes • Most are just volume changes • Simplify by function
  5. 5. The four groups –Media for gametes –Media for fertilization –Media for cleavage –Media for blastocysts
  6. 6. • Gamete • Fertilization • Cleavage • Blastocyst
  7. 7. GAMETE • Media to prepare gametes (eggs and sperm) for IVF or ICSI – Creating zygotes from male and female gametes
  8. 8. Sperm: Wash and Prepare Large Volume • Widely different sperm Small Volume • Highly concentrated & ?? high quality sperm
  9. 9. Eggs: Retrieve and Wash Ovum Retrieval: flush buffer Cumulus Oocyte Complex wash buffer
  10. 10. GAMETE • Products – Gamete Buffer – Sperm Medium – Sperm Gradient (40% & 80%) – Spermient (100% dilute with gamete buffer) – Sperm Cryopreservation Buffer – Follicle Flush Buffer – Oocyte Freeze – Oocyte Thaw
  11. 11. FERTILIZATION • Media to create the zygote stage during embryo development
  12. 12. Media for the Fertilization Steps IVF ICSI
  13. 13. FERTILIZATION • Products – Fertilization media – Culture Oil – Hyaluronidase – PVP
  14. 14. CLEAVAGE • Media to culture early cleavage stage of embryos from Day 1 until Day 3 of development
  15. 15. Media for the cell division/cleavage steps for fertilized oocytes (zygotes) Stripping cumulus cells post IVF The 2PN zygote on day 1 The 2 cell, early on day 2 The 8 cell on day 3
  16. 16. CLEAVAGE • Products – Cleavage Medium – Cryopreservation Kit – Thawing Kit – Embryo Biopsy Medium
  17. 17. Sequential (a Sequence of) Media • Provides a different formulation for each stage of embryo development • More viable blastocysts can be expected in culture with the use of sequential media: “extended culture”
  18. 18. BLASTOCYST • Media designed for the blastocyst stage of embryo development
  19. 19. Media for the blastocyst steps Day 3, 6-8 cell embryos are transferred to blastocyst media for further development. Compacting embryo day3/4 Blastocyst day 5 Hatched blastocyst day 5-6
  20. 20. BLASTOCYST • Products – Blastocyst Medium – Blastocyst Cryopreservation Kit – Blastocyst Thawing Kit – Blastocyst Vitrification Kit – Blastocyst Warming Kit
  21. 21. In Vivo In Vitro Fertilization Cleavage Compaction Blastulation
  22. 22. Day 1 Day 2 Day 3 Day 4 Day 5
  23. 23. Day 0 (I) • Follicular Flushing: Follicle Flush Buffer. • Oocyte Washing: Gamete Buffer. • Sperm Preparation: Gamete Buffer or Sperm Medium. • Sperm Culture: Sperm Medium. • Oocyte Culture: Fertilization Medium.
  24. 24. Day 0 (II) • Fertilization (IVF): Fertilization Medium. • Denudation: Hyaluronidase + Gamete Buffer or Cleavage Medium for washing. • Post-denudation culture: Cleavage Medium. • ICSI: Gamete Buffer or Cleavage Medium (+ PVP)
  25. 25. Day (III) • Post-ICSI Culture: Cleavage Medium. • Post-fertilization Culture: Cleavage Medium. • Blastocyst Culture: Blastocyst Medium. • Biopsy: Biopsy Medium + Blastocyst Medium for washing.
  26. 26. Others (IV) • Embryo Transfer: Cleavage or Blastocyst Medium. • Cryopreservation: Sperm Cryopreservation Buffer and Cryopreservation Kits. • Thawing: Thawing Kits. • Vitrification: Blastocyst Vitrification and Warming Kits
  27. 27. Results • Nineteen studies involving 3008 patients were included.
  28. 28. CPR / woman
  29. 29. Embryo quality scoring • Different media • Different diffinitions • Different parameters evaluated
  30. 30. D3 embryo transfer Author Compared media Definition Parameter Embryo quality Barrett 1997 HTF vs P1 Morphological grade (4 to 1) x cell numbers embryo quality 2.81 vs 2.94 Mayer 2001 P1/Blast vs G1.2 Morphological grade (1 to 5) Embryo grade (average) 2.5 ± 0.06 vs 2.5 ± 0.06 Cano 2001 Universal IVF vs IVF Morphological grade Embryo quality 4.0 ± 1.6 vs 4.0 ± 1.6 Mauri 2001 P1 vs IVF Morphological grade (4 to 1) x cell numbers embryo score 31.9 ± 14 vs 33.4 ± 15.8 Bungum 2002 G1.2 vs r-S1 Classification of Ziebe et al., 1997 No.good available embryos (mean/ET) 2.6 vs 2.5 Mayer 2003 P1 vs G1.2 P1 VS Sage Morphological grade (1 to 5) Embryo grade (average) 2.5 vs 2.6 2.7 vs 2.5 Zollner 2004 G2 vs Blastassist Morphological grade (4,3,2,1) x number of blastomeres Mean embryo score 23 vs 19.7 Baum 2004 Sydney IVF vs HTF NS No.of fair quality embryos 2.2 ±1.6 vs 2.0 ± 1.5 Fechtali 2004 Ferticult vs ISM1 Morphological grade (A to D) Good quality embryos (A+B)(%) 56.7 vs71.4 Rubino 2004 IVF vs Quinn's Cumulative embryo classification scheme (Rienzi et al., 2002) high quality embryos (%) 36.6 vs 49.6 Von During 2004 Sydney IVF vs Universal IVF Embryos available for replacement or cryopreservation % of cleaved embryos 66.9 vs 52.5 Yamamoto 2006 Multiblast vs Blastocyst Classification of Veeck % good grade embryos 81.2 vs 73.8 Arenas 2007 IVC vs G1.2 NS % good embryo quality 42.46 vs 76.55 Hoogendijk 2007 Sydney IVF medium vs Quinn's Advantage sequential culture media NS Day 3 good quality embryo (33/79 (42%) v. 40/67 (60%) Reed 2009 Global vs G5 Morphological score (Q 1-5) x cell numbers mean( SD) quality score for embryos replaced 2.4 (0.7) vs 2.5 ( 0.8) D5 embryo transfer Zollner 2004 G1.2/G2.2 vs Blastassist Morphological grade (4,3,2,1) x number of blastomeres Mean blastocyst grade 6.8 vs 6.7 Yamamoto 2006 Multiblast vs Blastocyst Classification of Gardner % good grade blastocyst 21 vs 36.7 Sepulveda 2009 Global vs ECM/Multiblast ICM : 3 is compact area, many cells present. TE: 3 many cells forming a tight epithelial network ICM grade (mean ± SD) TE grade (mean± SD) 2.3 ± 0.8 vs 2.4± 0.7 2.2± 0.7 vs 2.2 ± 0.8
  31. 31. Implantation rate
  32. 32. Conclusion • A clear treatment effect on either clinical outcomes as live birth rate, ongoing pregnancy rate, multiple pregnancy rate or laboratory outcomes as fertilization rate, embryo quality and cryopreservation rate could not be found
  33. 33. Conclusion • “Think like an embryo” • Need constant temperature and pH, avoid environmental contaminants

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