1. Culture Media

2. Overview: What does media do?
• Keeps everything wet
• Feeds the cells
• Controls the environment

3. Media Components
Media is basically salt water
with added vitamins and protein
• Salts
• Carbohydrates
• Protein
• Metabolites
• Buffers
• Antibiotics
• Water

4. • LOTS OF CODES!!
• 28 different codes
• Most are just volume
changes
• Simplify by function

5. The four groups
–Media for gametes
–Media for fertilization
–Media for cleavage
–Media for blastocysts

6. • Gamete
• Fertilization
• Cleavage
• Blastocyst

7. GAMETE
• Media to prepare gametes (eggs and sperm) for
IVF or ICSI
– Creating zygotes from male and female
gametes

8. Sperm: Wash and Prepare
Large Volume
• Widely different
sperm
Small Volume
• Highly concentrated
& ?? high quality
sperm

9. Eggs: Retrieve and Wash
Ovum
Retrieval:
flush
buffer
Cumulus
Oocyte
Complex
wash buffer

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. FERTILIZATION
• Media to create the zygote stage during
embryo development

12. Media for the Fertilization Steps
IVF
ICSI

13. FERTILIZATION
• Products
– Fertilization media
– Culture Oil
– Hyaluronidase
– PVP

14. CLEAVAGE
• Media to culture early cleavage stage of
embryos from Day 1 until Day 3 of
development

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. CLEAVAGE
• Products
– Cleavage Medium
– Cryopreservation Kit
– Thawing Kit
– Embryo Biopsy Medium

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. BLASTOCYST
• Media designed for the blastocyst stage of
embryo development

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. BLASTOCYST
• Products
– Blastocyst Medium
– Blastocyst Cryopreservation Kit
– Blastocyst Thawing Kit
– Blastocyst Vitrification Kit
– Blastocyst Warming Kit

21. In Vivo
In Vitro
Fertilization Cleavage Compaction
Blastulation

22. Day 1
Day 2
Day 3
Day 4
Day 5

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. 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. Day (III)
• Post-ICSI Culture: Cleavage Medium.
• Post-fertilization Culture: Cleavage
Medium.
• Blastocyst Culture: Blastocyst Medium.
• Biopsy: Biopsy Medium + Blastocyst
Medium for washing.

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. Results
• Nineteen studies involving 3008 patients
were included.

28. CPR / woman

29. Embryo quality scoring
• Different media
• Different diffinitions
• Different parameters evaluated

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. Implantation rate

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. Conclusion
• “Think like an embryo”
• Need constant temperature and pH, avoid
environmental contaminants