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Sequential Culture

Sequential Culture. Patrick Quinn PhD, HCLD patrick.quinn@coopersurgical.com. Sage In-vitro Fertilization, Inc Redmond, Oregon, USA. Pre-compaction D1-D3, zygote  12/16-cell In oviduct maternal mRNA Undifferentiated Single cells Energy metabolism P  G Amino acids NEAA

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Sequential Culture

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  1. Sequential Culture Patrick Quinn PhD, HCLD patrick.quinn@coopersurgical.com Sage In-vitro Fertilization, Inc Redmond, Oregon, USA

  2. 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 Yes Preimplantation Development

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

  4. 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. Mimicing In vivo Conditions

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

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

  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 Amino Acids – Al-Gln, no alanine pH – defined with a specific % CO2 Osmolality – range between 265-280 Vitamins – in blastocyst medium; role ill-defined Components of ART Culture Media

  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 • QA Blastocyst Medium 0.1 mM

  13. Lactate • L + isomer biologically active, ie metabolized lactate • Calcium-L-lactate = Ca lactate • 2.04 mM = 4.08 mM L (+) lactate = 8.16 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 59:1000-1007, 1998 Rogers & Yanagimachi, Biol Reprod 15:614-619, 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. QA Fertilization Medium 0.2 mM QA Cleavage & Blastocyst Medium 2 mM Magnesium Concentrations in Media

  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-EssentialEssential Alanine omitted Arginine 0.1 mM Asparagine 0.1 mM Histidine 0.1 mM Aspartate 0.1 mM Leucine 0.2 mM Glutamate omitted Lysine 0.2 mM Glycine 0.1 mM Threonine 0.2 mM Proline 0.1 mM Valine 0.4 mM Serine 0.1 mM Taurine 0.1 mM Alanyl-glutamine 1.0 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 mediaLane & 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. Pyruvate Acting as a Sink for Ammonium Production During In Vitro Culture transaminase Alanine Pyruvate + NH3

  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; D3QA 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. 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 #353802.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 #353802.Overlay with 9 mL of oil. Prepare no more than two dishes at a time! 30 uL drops used for washing embryos 2 1 3 30 uL drop used for culture of grouped embryos A 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

  34. Life Global Comparisons

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

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

  37. 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 How different is the protocol of Single Step Culture from Sequential Culture?

  38. 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.

  39. Comparison of Different Media Systems

  40. Media Comparisons 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.

  41. 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.

  42. 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.

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

  44. 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.

  45. 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.

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