1. Sage In-vitro Fertilization, Inc Redmond, Oregon, USA
Sequential Culture
Patrick Quinn PhD, HCLD
Sage In-vitro Fertilization, Inc
Redmond, Oregon, USA

2. Preimplantation Development
Pre-compaction
D1-D3, zygote 12/16-cell
In oviduct
maternal mRNA
Undifferentiated
Single cells
Energy metabolism P G
Amino acids NEAA
Vitamins No
No growth
Growth factors?
Post- compaction
Morula  Blastocyst
In uterus
Embryonic mRNA
ICM, trophectoderm
Transport epithelium
P G  
NEAA + EAA
Yes
Growth, ie expansion

3. Sequential Culture Media
Imitative Principle
In vitro = In vivo
Location of embryo changes
Content of tract secretion changes

4. Mimicing In vivo Conditions
Fertilization.
Spermatozoa require glucose; need at least 2.8 mM. Quinn 1995 JARG 12:97-105
If using ICSI, transfer injected oocytes directly to Cleavage Medium that only has 0.1 mM glucose
Fertilization has to be separated from embryo culture in terms of culture conditions.

5. Quinn’s Advantage Sequential Media
QA Cleavage Medium
QA Fertilization Medium
QA Blastocyst Medium

6. Components of ART Culture Media
Ionic Composition
Energy Sources
Amino Acids pH
Osmolality
Vitamins
Growth Factors

7. Components of ART Culture Media Sage IVF
Inorganic Salts: NaCl, KCl, MgSO4, KH2PO4, NaHCO3, EDTA
Variation in Ca/Mg during fertilization and embryo development.
Energy Sources: Sodium pyruvate, calcium-L+-lactate, glucose, sodium citrate.
Only the bioactive L+ isomer of lactate present
Amino Acids: non-essential and essential, plus taurine, alanyl glutamine
pH: Specified under set CO2 level – 7.3 for Fertilization and Blastocyst medium, 7.2 for Cleavage medium
Osmolarity: 265 mosmoles/Kg
Vitamins: in Blastocyts medium
Other: Phenol red
Antibiotic: Gentamicin

8. Ionic Composition – Ca/Mg ratios Energy Sources – Ca lactate
Components of ART Culture Media
Ionic Composition – Ca/Mg ratios
Energy Sources – Ca lactate
Amino Acids – Al-Gln, no alanine
pH – defined with a specific % CO2
Osmolality – range between
Vitamins – in blastocyst medium; role ill-defined

9. Other Highlights Sodium citrate in Fertilization & Cleavage Medium
Lactate present as calcium lactate
EDTA present in precompaction but not post compaction media
Pantothenate, choline, inositol and other vitamins present in blastocyst medium
Osmolality 265 mOsm/Kg

10. Concentration of Pyruvate, Lactate & Glucose in Human Reproductive Tract Fluids
Gardner et al 1996

11. Pyruvate Included in nearly all ART media
Required for early development?
Amino acids can replace (Bavister)
This illustrates the plasticity in embryo metabolism

12. Pyruvate QA Fertilization & Cleavage Medium 0.33 mM 0.1 mM
QA Blastocyst Medium
0.1 mM

13. Lactate lactate Calcium-L-lactate = Ca
L + isomer biologically active, ie metabolized
lactate
Calcium-L-lactate = Ca
2.04 mM = mM L (+) lactate
= mM DL-lactate (HTF = 21.4 mM)
Extra D (-) lactate  effects pHi 

14. Glucose Required for sperm Precompaction
 glycolysis is best, low Glu, + EDTA
some required for pentose phosphate pathway
Postcompaction - need  glycolysis for growth & differentiation

15. Glucose QA Fertilization Medium
2.78 mM - sperm function, cumulus/corona cells
QA Cleavage Medium
0.1 mM - pentose phosphate pathway and other metabolism
QA Blastocyst Medium
2.78 mM - increased glycolysis

16. Calcium/Magnesium Interactions in Early Embryonic Development
Early hamster embryo development is disrupted by increased free Ca-i
Increased Ca-i can be inhibited by > Mg2+ levels in the medium, the presence of the Ca-channel blocker, nifedipine and the intracellular Ca chelator, BAPTA
All of these treatments increased hamster embryo development
BUT, high Mg inhibits sperm capacitation
Lane & Bavister, Biol Reprod 59: , 1998
Rogers & Yanagimachi, Biol Reprod 15: , 1976

17. Why vary the Mg2+ concentration?
High Mg2+ decreases uptake of exogenous Ca2+. Therefore use with embryos to prevent damage to mitochondria and subsequent abnormal energy metabolism.
But keep Mg2+ low in Fertilization medium as sperm require a Ca2+ spike to undergo capacitation and acrosome reaction

18. Magnesium Concentrations in Media
QA Fertilization Medium
0.2 mM
QA Cleavage & Blastocyst Medium
2 mM

19. AMINO ACIDS IN ART MEDIA
Non-essential: 7 + Gln
- Used before and after compaction
-  cleavage rate precompaction
-  blastocoel,  trophectoderm,  hatching postcompaction
Essentials: 12
- Used after compaction
-  ICM

20. Amino Acids Non-Essential Essential Alanine omitted Arginine 0.1 mM
Asparagine mM Histidine mM
Aspartate mM Leucine mM
Glutamate omitted Lysine mM
Glycine mM Threonine mM
Proline mM Valine mM
Serine mM
Taurine mM
Alanyl-glutamine mM

21. Amino Acids
Cannot get maximum growth rates of 1-cell mouse zygotes to fully expanded blastocysts when medium contains essential amino acids, eg Blastocyst Medium.
Mouse embryos have to be cultured in Cleavage Medium (non-essential amino acids) and then placed in Blastocyst Medium to get good rates of blastocyst formation.
Highly likely that human embryos would react the same.

22. Role of Amino Acids in Preimplantation Development
Non Essentials
Role - Increase mitotic rate
Mechanisms
(i) Regulators of energy metabolism
(ii) Osmolytes; maintain intracellular physiology in high osmotic pressure of oviduct fluid
(iii) Buffer pHi
Essential
Role - Stimulate differentiation of ICM
Mechanism - unknown

23. Tricarboxylic Acid (TCA) Cycle Krebs Cycle

24. AMMONIUM PRODUCTION IN ART MEDIA
Dependent on levels of aa’s
Stabilized dipeptides, eg. Ala-Gln
Pyruvate acts as sink  Alanine

25. Ammonium production from different culture media Lane & Gardner, BOR 69:1109-17, 2003
No significant difference between QA and G.2 series

26. Appearance of Alanine During Incubation of Bovine Embryos
Partridge & Leese, Reprod Fertil Devel., 8:945-50, 1996

27. transaminase Alanine Pyruvate + NH3
Pyruvate Acting as a Sink for Ammonium Production During In Vitro Culture

28. Vitamins
Vitamins in Eagle’s MEM (+ AAs) maintain normal metabolic activity in mouse and rat blastocysts, and normal implantation rates and fetal weight
In hamsters, only pantothenate stimulates 1-cell  blastocyst. Several water soluble vitamins stimulate expansion and hatching
Included in postcompaction media, eg G2, Blastocyst Medium, QA Blastocyst Medium
Exact requirements and specificity for human embryos is unknown

29. 8-cell embryos; D3 QA Fertilization & Cleavage Media
Grade A( = 4); 0-5% fragmentation

30. Late Day 4 Blastocysts in medium containing Growth Factor

31. Day 5 Blastocysts in medium containing Growth Factor

32. oocytes /embryos in a dish!!
Microdrop culture to enhance effects of autocrine/paracrine growth factors produced by embryo itself
4-well Nunc Multi dish
Microdrops in 60 mm diameter dishes, Falcon # Overlay with 9 mL of oil
50 uL to 1.0 mL
Place NO MORE than 6
oocytes /embryos in a dish!!
30 uL drops used for washing 2PN embryos
Oil ART-4008
Cleavage Medium ART-1026 or Protein Plus Cleavage Medium ART-1526
10 uL drops used for culture of individual embryos

33. Group Culture for same effect
IF YOU WANT TO DO GROUP CULTURE OF EMBRYOS RATHER THAN SINGLE EMBRYO CULTURE, PREPARE THE FOLLOWING TYPES OF DISHES FOR D1-3 AND D3-5/6 WITH THE APPROPRIATE MEDIUM.
HOWEVER, it is important to stress that for best pH and temperature control, Place NO MORE than 6 embryos in a dish!!
Place the following microdrops in 60 mm diameter dishes, Falcon # Overlay with 9 mL of oil. Prepare no more than two dishes at a time!
30 uL drops used for washing embryos 1 2 3 A
30 uL drop used for culture of grouped embryos B
Wash the embryos through the two 30 uL drops 1 and 2 in each row and then place in the third drop 3 for culture. Up to 6 embryos can be cultured in one 30 uL drop. C

37. Life Global Comparisons

38. Life Global Comparisons
Original version 2006
Version 3, 2006
Be careful about what some ART media companies claim and/or tell you

39. It is the composition of the media that is different.
How different is the protocol of Single Step Culture from Sequential Culture?
One still has to change embryos on Day 3 to fresh medium.
It is the composition of the media that is different.

40. How different is the protocol of Single Step Culture from Sequential Culture?
Commercially, Sage can provide both options of culture.
Several labs have used the Sage Cleavage Medium for culture from ICSI to D3, then change to fresh Cleavage Medium for D3 to D5/6, and have obtained high quality blastocysts.
Lynette Scott, Boston
Serdar Coskun, Saudi Arabia

41. The unanswered question of this debate is that there has been no real trial of the two culture systems. And, there may be subtle differences between different programs, eg patients, stimulation protocols, laboratory procedures, so each laboratory should do its own trial to compare the different culture systems.

42. Comparison of Different Media Systems

43. Media Comparisons Phase I:
Two Phase Trials
Phase I:
Within patient, sibling oocyte split between two media sources in vitro data parameters, eg fertilization, cleavage rate, morphology.
Phase II:
Between patient, randomized allocation to media in vivo data, eg PR & IR.

44. Media Comparisons
Two Phase Trials
In both Phase I and II, try to make the comparisons as balanced as possible, eg:
Same protein supplement.
Same pH, O2 and, if possible, same incubator.
Prospective randomization of everything.
Same embryologist, physician etc working on a within patient comparison.

45. Design of Study
Have a Balanced Comparison in time and patients. For example, one week (or month etc) with Sage media, one week with current medium. Make patients similar in age, diagnosis, etc.
Compare for 3-4 time periods with a minimum of 20 patients in each group.
Repeat comparison if problems arise, eg fertilization, development and pregnancy rates decline (<10%) for no apparent reason.

46. Laboratory and Clinical End Points
Lab End Points
Fertilization Rate
Development Rate
Degree of Fragmentation
Lab specific endpoints
Multinucleate
PN score
blastocyst development
Clinical End Points
Implantation Rate

47. Comparison of media with protein supplement as SPS or HSA
Suggested design:
Patients ≤ 38 years old
1st or 2nd cycle
No severe male or female pathology
Minimum of 10 embryos to do a within patient 50:50 split
Attempt to do ETs with embryos from a single medium group.

48. The unanswered question of this debate is that there has been no real trial of the two culture systems. And, there may be subtle differences between different programs, eg patients, stimulation protocols, laboratory procedures, so each laboratory should do its own trial to compare the different culture systems. Only in this way can a lab get appropriate evidence as to whether Single Step Culture versus Sequential Culture system is best for them.