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