Date post: | 07-May-2015 |
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Culture Media
Overview: What does media do?
• Keeps everything wet
• Feeds the cells
• Controls the environment
Media Components
Media is basically salt water with added vitamins and protein
• Salts• Carbohydrates• Protein• Metabolites
• Buffers• Antibiotics• Water
•LOTS OF CODES!!
•28 different codes
•Most are just volume changes
•Simplify by function
The four groups
–Media for gametes
–Media for fertilization
–Media for cleavage
–Media for blastocysts
• Gamete
• Fertilization
• Cleavage
• Blastocyst
GAMETE
• Media to prepare gametes (eggs and sperm) for IVF or ICSI– Creating zygotes from male and female
gametes
Sperm: Wash and Prepare
Large Volume
•Widely different sperm
Small Volume
•Highly concentrated & ?? high
quality sperm
Eggs: Retrieve and Wash
Ovum Retrieval:
flush buffer
Cumulus Oocyte
Complex wash
buffer
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
FERTILIZATION
• Media to create the zygote stage during embryo development
Media for the Fertilization Steps
IVF
ICSI
FERTILIZATION
• Products– Fertilization media– Culture Oil– Hyaluronidase – PVP
CLEAVAGE
• Media to culture early cleavage stage of embryos from Day 1 until Day 3 of development
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
CLEAVAGE
• Products– Cleavage Medium– Cryopreservation Kit– Thawing Kit– Embryo Biopsy Medium
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”
BLASTOCYST
• Media designed for the blastocyst stage of embryo development
Media for the blastocyst stepsDay 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
BLASTOCYST
• Products– Blastocyst Medium– Blastocyst Cryopreservation Kit– Blastocyst Thawing Kit– Blastocyst Vitrification Kit– Blastocyst Warming Kit
In Vivo
In Vitro
Fertilization Cleavage CompactionBlastulation
Day 1
Day 2
Day 3
Day 4
Day 5
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.
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)
Day (III)
• Post-ICSI Culture: Cleavage Medium.• Post-fertilization Culture: Cleavage
Medium.• Blastocyst Culture: Blastocyst Medium.• Biopsy: Biopsy Medium + Blastocyst
Medium for washing.
Others (IV)
• Embryo Transfer: Cleavage or Blastocyst Medium.
• Cryopreservation: Sperm Cryopreservation Buffer and Cryopreservation Kits.
• Thawing: Thawing Kits.• Vitrification: Blastocyst Vitrification and
Warming Kits
Results
• Nineteen studies involving 3008 patients were included.
CPR / woman
Embryo quality scoring
• Different media• Different diffinitions• Different parameters evaluated
D3 embryo transfer
Author Compared media Definition Parameter Embryo qualityBarrett 1997
HTF vs P1Morphological 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 IVFMorphological grade (4 to 1) x cell numbers
embryo score 31.9 ± 14 vs 33.4 ± 15.8
Bungum 2002 G1.2 vs r-S1Classification of Ziebe et al., 1997
No.good available embryos (mean/ET)
2.6 vs 2.5
Mayer 2003P1 vs G1.2P1 VS Sage
Morphological grade (1 to 5) Embryo grade (average)2.5 vs 2.62.7 vs 2.5
Zollner 2004 G2 vs BlastassistMorphological 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'sCumulative embryo classification scheme (Rienzi et al., 2002)
high quality embryos (%) 36.6 vs 49.6
Von During 2004 Sydney IVF vs Universal IVFEmbryos 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 2007Sydney 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 G5Morphological 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 BlastassistMorphological 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± 0.72.2± 0.7 vs 2.2 ± 0.8
Implantation rate
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
Conclusion• “Think like an embryo”
• Need constant temperature and pH, avoid environmental contaminants