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

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

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

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.

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

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

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

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 265-280 Vitamins – in blastocyst medium; role ill-defined

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

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

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

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

Lactate lactate Calcium-L-lactate = Ca L + isomer biologically active, ie metabolized lactate Calcium-L-lactate = Ca 2.04 mM = 4.08 mM L (+) lactate = 8.16 mM DL-lactate (HTF = 21.4 mM) Extra D (-) lactate  effects pHi 

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

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

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

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

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

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

Amino Acids Non-Essential Essential 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

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.

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

Tricarboxylic Acid (TCA) Cycle Krebs Cycle

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

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

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

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

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

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

Late Day 4 Blastocysts in medium containing Growth Factor

Day 5 Blastocysts in medium containing Growth Factor

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 #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

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 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

Life Global Comparisons

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

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.

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

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.

Comparison of Different Media Systems

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.

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.

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.

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

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.

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.