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Decoding Embryo Culture
This document summarizes a presentation on decoding embryo culture. It discusses how in vitro culture aims to mimic the in vivo environment by balancing nutrients, temperature, pH, oxygen tension, and preventing contaminants. It describes the in vivo conditions in the oviduct and uterus and how culture media and protocols try to replicate these. Key aspects of embryo culture systems like media, gas phase, incubators, and embryologists are outlined. Different culture media compositions and the expectations and history of development are summarized. Various culture protocols like single step, sequential, group and individual culture are compared. Factors like pH, temperature, oxygen tension and their optimal levels are highlighted.
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Decoding Embryo Culture
- 1. Decoding Embryo Cultur LaboratoryIncubation an Enigma Presented by: Ms. Doel Bose Pande B.E (Electronics), M.Sc Embryologist & Clinic Manager @ Indore Infertility Clinic Contact: 96444 44066
- 2. Decoding Embryo Culture •What are we trying to mimic – In Vivo? • Components of Embryo Culture System • Culture Medium • Culture Protocol & Culture Strategies • Culture Condition • Take Home Message
- 3. Embryos In-vivo In Vitroculture tries to mimic the in vivo environment that a fertilized oocyte is subjected to during the course of 3 to 5 days prior to it’s implantation within the uterine wall. What is the In Vitro Embryo Culture trying to achieve? Balanced Nutrients – Culture Media Formulation Optimum Temperature, Humidity, pH and Osmolality – Culture Media Buffering System and Incubator Optimum Oxygen Tension - Incubator Prevention from external contaminants & Oxidative stress – Incubator, Air Handling Units, Antibiotics & antioxidants in Culture Medium Removal of Metabolites from immediate vicinity & constant motion – Embryo Culture Protocol & Culture Platform
- 4. Embryos In Vivo PRECOMPACTION (D0 to D3/4) OVIDUCT • Control of Maternal Genome • Energy Source/Substrates Pyruvate & Lactate (Oxidative Metabolism) • Non Essential Amino Acids • Temperature ~ 35.5 to 36 °C • pH ~ 7.94 • Oxygen Tension ~ Appx 5% POST COMPACTION (D4 to D5/6) • Activation Of Embryonic Genome • Energy Substrates/Source Glucose (Glycolic Metabolism) • Non Essential Amino Acids, Essential Amino Acids • Temperature ~ 37.2 °C • pH ~ 7.2 to 7.35 • Oxygen Tension ~ Appx 2% UTERINE CAVITY
- 5. Embryo Culture System GasPhase for Equilibration Embryo Culture Dish Incubators Temperature and Humidity Embryo Culture Media Embryologists Ambient Air Quality Type of Oil Overlaid 01 02 03 04 05 06 07 08 Immediate environment of the developing Embryo
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- 7. Expectations from CultureMedia Ensure Embryo Growth Decrease Embryonic Stress Maintain Gamete Viability
- 8. History of EmbryoCulture Media Mouse Embryo in Blood Clot Kuhl and Chang Human embryos up to the blastocyst stage using Ham's F10 with human or fetal calf serum Steptoe et Al Simplex Optimized Medium, Co culture with Feeder cells Steptoe and Edward Rabbit Embryo cultured in Plasma Lewis and Gregory Mouse Embryo to Blastocyst stage cultured in Krebs-Ringers Bicarbonate that contained egg white Hammnod Earle's Simple Salt solution with pyruvate supplemented with the patient's serum First Successful IVF Baby Steptoe and Edward Sequential Media Back to Nature 1929 1949 1978 1941 - 1947 1971 1990 Onwards Single Step Media Let Embryo Choose 1984 Adding Serum Albumin ensures non adherence to glass surface Menezo
- 9. Composition of EmbryoCulture Media EDTA (Ethylenediaminetetraacetic Acid) ligand and chelating agent 06 Vitamins Ascorbic acid, cyanocobalamin, folic acid and tocopherol 07 Proteins Recombinant human albumin (AlbIX), human serum albumin (HSA and Buminate), and three complex protein supplements (SSS, SPS, LGPS) 08 Basic Salts Six inorganic ions: Na(+), K(+), Cl(-), Ca(2+), Mg(2+), and SO(4)(2-) 02 Carbohydrates Pyruvate, Lactate, Glucose 03 Amino Acids (AA) EAA, NEAA 04 05 Fatty Acids Oleic, linoleic and arachi- donic Anti Biotics & AntiOxidants Penicillin , Streptomycin and Gentamycin 09 Growth Factors IGF 1 & 2, LIF, GM-CSF, HB-EGF 10 01 Water Triple Glass Distilled Water The new generation Media contains more than 80+ components
- 10. 01 02 03 Let the EmbryoChoose – Single Step Culture Back To Nature – Sequential Culture Comparison between the Protocols Culture Media Type & Protocols
- 11. Sequential Culture • MimicIn vivo – Oviduct to Uterus • Pre to Post Compaction Embryos • Change from Maternal Genome to Embryonic Genome • Preference to Pyruvate Vs Glucose • Role of NEAA & EAA in Pre-implantation embryo’s • Inhibitory effect of EDTA on Blastulation. • The changing energy requirements of the preimplantation embryo and the inhibitory effect of glucose on early cleavage stage embryos, Glucose in all species inhibits the first cleavage division (2 Cell Block) – Change the quantity of Glucose in Cleavage & Blastocyst media • The inhibitory effect of (EDTA) on blastocyst development and the inner cell mass (ICM), EDTA removed from blastocyst media • The chemical breakdown of Lglutamine (Gln) in aqueous solution and the accumulation of ammonia. Media renewed to remove accumulated ammonia. • The role of amino acids on preimplantation embryo development: Cleavage Media contains only NEAA, whereas the Blastocyst Media, contains both NEAA and EAA. Back to Nature
- 12. Single Step /Monostep Culture Let the Embryo Choose As long as concentrations are within ‘tolerable ranges’, the embryo itself will adapt and utilize whatever it requires Pathophysiologic stress The switching of embryos may cause additional osmotic or other shock to the embryo and/or deprive it of any autocrine -paracrine factors it may have produced during the first culture period. • Based on the collected works of Biggers and coworkers • Inhibitory Effect of Glucose - Glucose is present in the natural environment of the Fallopian tubes, and there is evidence of culture media with glucose that do not inhibit embryo development • Role of EDTA - Negative effect of EDTA during blastulation refers to a higher concentration, presence of low quantity is not deleterious • Use of a mixture with all 21 AminoAcids. This way, the embryo would be able to choose which ones to use by itself. • The problem of accumulated ammonium from decomposing Gln is solved by using more stable dipeptides of Gln, such as glycol-L-glutamine (GlyGln) or L-alanyl-L-glutamine (AlaGln)
- 13. One media system CompanyMedium Culture period LifeGlobal global ® day-1 to day-5/6 Gynemed GM501 day-0 to day-5/6 IrvineScientific SSM™ day-0 to day-5/6 Sequential media Company Medium Culture period Cook Medical Cleavage K-SICM day-1 to day-3 Blastocyst K-SIBM day-3 to day-5/6 CooperSurgical Quinns Advantage ® Cleavage day-1 to day-3 Quinns Advantage ® Blastocyst day-3 to day-5/6 FertiPro FERTICULT™IVF Medium day-1 to day-2 FERTICULT™ G3 Medium day-3 to day-4 Sequential media Company Medium Culture period InVitroCare IVC-TWO™ day-0 to day-3 IVC-THREE™ day-3 to day-5 Irvine Scientific ECM ® day-0 to day-3 MultiBlast ® day-3 to day-5 Origio EmbryoAssist™ day-0 to day-3 BlastAssist™ day-3 to day-5 ISM1 day-0 to day-3 ISM2 day-3 to day-5 Vitrolife G-1™PLUS day-1 to day-3 G-2™PLUS day-3 to day-5 IVF™ day-0 to day-3 CCM™ day-3 to day-5 Comparison between Culture Period of Commercially available Media • D0 or D1 - Important to note that many media do not recommend culturing Oocytes in them • Sage IVM medium – Media specifically meant for IVM (However studies have shown that Blastulation media works same if not better. • Blastocyst medium may be a good choice in IVM/ICSI cycles as an alternative IVM medium.
- 14. Embryo Culture Protocols FreshStage Specific Medium Change every day Sequential Media Change of Media on Day 3 Sequential Media – Interrupted Culture Single Step Media renewed on Day 3 Single Step Media – Interrupted Culture Same media used throughout the 5/6 days. Single Step Media – Mono Culture
- 15. Comparison of Protocols CharacteristicSingle Step – MonoCulture Single Step – Interrupted/Renewed Sequential with Renewal on D3 Leave Embryo undisturbed Yes No No Replacement of Essential Nutrients No Yes Yes Accumulation of Toxins Yes Removed Removed Environmental Stress (change in pH/Temperature) No Yes Yes Accumulation of Endogenous Growth Factors Yes Lost Lost Labour Intensity of Embryologist Low Moderate High Cost & Time Low Moderate High Laboratory Management/Quality Control Easy (Single Media) Moderate Hard (Two Medias)
- 16. Which Protocol isSuperior ? STUDIES INCONCLUSIVE If something is working for you, Good for You!
- 17. 01 02 03 Group Culture Individual Culture Advancesin Culture Strategies Embryo Culture Strategies
- 18. Single Embryo Culture GroupCulture MicroDroplet Culture Embryo Culture Strategies
- 19. Individual Culture Advantages • Individualevaluation of embryo morphology and rate of development • Embryos cultured together may be exposed to secreted detrimental factors. • Depletion of necessary substrates by other embryo if cultured in a group. Disadvantages • Labour and Cost Intensive. • Loss of autocrine/paracrine growth factors affecting growth • Change in pH/temperature during manipulation - due to low volume of media
- 20. Group Culture orCommunal Culture Advantages • Easy Protocol • Uses accumulated autocrine/paracrine Growth Factors to it’s benefit • Less exposure to pH/temperature changes due to easy and quick manipulation Disadvantages • Does not permit continues evaluation of individual embryos • Embryos in groups may be exposed to negative effects of dying or delayed embryos cultured in the same drop Benefits of Group Culture more evident when you plan Blastocyst Transfer
- 21. Advances in EmbryoCulture Platforms Static Culture Platforms • Submicroliter platforms • Microwells • Microchannels Dynamic Culture Platforms • Shaking/rotation • Tilting • Vibration • Controlled fluid flow • Piezo-electric actuators The in vivo embryo is in constant motion, moved by gentle ciliated and muscular action of the female tract
- 22. 01 02 03 pH & CO2Concentration Temperature Oxygen Tension Culture Condition
- 23. Temperature FACT Ovarian follicle andlumen of oviduct (Fertilization Site) is 1-2ºC less then core body temp of 37 ºC Quiet Embryo Hypothesis Viable embryos have “Quiet” metabolism than their less viable counterparts, and that temperatures cooler could also lead to “quiet” metabolism. Results of some Randomized Control Trials are inconclusive CAUTION The minimum temperature may not be as critical as the maximum temperature Reduction in temperature will only slow down enzymatic functions Raising temperature even by a degree adversely affects oocyte maturation and fertilisation rates and retards further embryonic development
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- 25. Acceptable pH rangefor culture media may be set between 7.2 to 7.4 Factors affecting pH • Amount of bicarbonate in media • Concentration of Amino Acids added to the medium • Volume of media • Type of Culture Dish • Altitude of Lab • CO2 Concentration of Incubator • Temperature Inverse relationship between pH and CO2 / Temperature How to change pH without changing Temperature & CO2? By changing bicarbonate concentrations in Culture Media. pH in Culture Media Internal pH in Female Reproductive Track – not Constant Follicle 7.26-7.24 Oviduct 7.3 Uterus 7.12 Embryo 7.1 pH Temperature CO2 Higher pH may benefit fertilization, while a lower pH may benefit embryo development
- 26. Indicators of pH-Phenol Red ▪ Also known as Phenolsulfonphthalein • Exhibits a gradual transition from yellow to deep pink over a pH range of 6.2 to 8.2 ▪ Advantage It is for the most part an inert compound, acts as a visual pH indicator
- 27. Regulation of pH •Buffer is something that is added to keep the pH of a solution constant. • Buffering Capacity (pKa) - Resistance to changes in pH when strong acids or bases are added • Two most successful Buffers used in ART procedures safely are • HEPES-buffered medium • Zwitterionic organic buffer (Good Buffer) • Used as a buffered medium for human oocyte collection & embryo handling. • MOPS-buffered medium • Zwitterionic buffers (Good Buffer) • Slightly Superior compared to HEPES for Vitrification. • Mono Buffered Media & Dual/Combined Buffered Media • Combined Buffers • 1:1:1 HEPES:MOPS:DIPSO
- 28. Oxygen FACT: Oxygen concentration inthe Uterus is lower than in the oviduct • In Vivo, embryos pass through Sequential oxygen concentration varying from 5% in oviduct to around 2% in uterus. • Optimal oxygen tension in embryo culture may depend on the stage of development. • Post Compaction embryo’s benefit more from low Oxygen Concentration (2 to 5%) • In vitro embryos are subjected to monophasic oxygen concentrations
- 29. • Embryo cultureat 5% oxygen results in improved morphologic parameters, reduced embryo arrest, and higher live birth rates as compared to Atmospheric Oxygen concentration of 20% • There is an abundance of data on several mammalian species, including humans, showing that atmospheric oxygen negatively effects the preimplantation-stage embryo • Benchtop incubators typically use 5% physiological oxygen concentration More Cost due to Tri-gas. • Box incubators typically use atmospheric oxygen concentration of 20% • Ultra Low Concentration of oxygen ~2% to mimic physiological Oxygen concentration of Uterus is being studied. Evidence not conclusive Oxygen
- 30. Take Home Message •If in doubt, stay towards lower Temperature • Invest in Benchtop Incubators • Often simple things work best • If your cumulative results are good, then your culture condition must be good. • Do not be afraid of change, but change only ONE thing at a TIME
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- 32. References 1. Vajta a,*,1,Laura Rienzi c, Ana Cobo d, John Yovich a,b;Reproductive BioMedicine Online (2010) 20, 453– 469: Embryo culture: can we perform better than nature? 2. Ga´bor Elpiniki Chronopoulou, Joyce C. Harper:Human Reproduction Update, Volume 21, Issue 1, January/February 2015, Pages 39–55; IVF culture media: past, present and future 3. Ka Ying Bonnie Ng, Roel Mingels, Hywel Morgan, Nick Macklon, Ying Cheong:Reproduction Update, Volume 24, Issue 1, January-February 2018, Pages 15–34 : In vivo oxygen, temperature and pH dynamics in the female reproductive tract and their importance in human conception: a systematic review:Human 4. Irmhild Gruber, Matthias:(J Turkish-German Gynecol Assoc 2011; 12: 110-7):Embryo culture media for human IVF: which possibilities exist? 5. Petra L. Wale, David K. Gardner:Human Reproduction Update, Volume 22, Issue 1, January/February 2016, Pages 2–22: The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction: 6. Dean E. Morbeck, Ph.D., Ph.D. Dean E. Morbeck Ph.D.,Rebecca L. Krisher, Ph.D.c, Jason R. Herrick, Ph.D.c, Nikola A. Baumann, Ph.D.b, Dietrich,Matern, M.D., Ph.D.b, Thomas Moyer, Ph.D.b: Fertility & Sterility September 2014Volume 102, Issue 3, Pages 759–766.e9 ;Composition of commercial media used for human embryo culture 7. MARA SIMOPOULOU, KONSTANTINOS SFAKIANOUDIS and MICHAEL KOUTSILIERIS: From Media to Epigenetics: in vivo 32: 451-460 (2018): Considerations Regarding Embryo Culture Conditions 8. Arne Sunde, Daniel Brison, John Dumoulin, Joyce Harper, Kersti Lundin, M. Cristina Magli, Etienne Van den Abbeel, Anna Veiga:Volume 31, Issue 10, October 2016, Pages 2174–2182: Time to take human embryo culture seriously: Human Reproduction 9. John D. Biggers, D.Sc., Ph.D.,a and Michael C. Summers, MD., Ph.D: Fertility and Sterility Vol. 90, No. 3, September 2008; Choosing a culture medium: making informed choices 10. Gary D. Smith,2,3,4,5,6 Shuichi Takayama,6,7,8 and Jason E. Swain3,6:1:ISSN: 0006-3363: Rethinking In Vitro Embryo Culture: New Developments in Culture Platforms and Potential to Improve Assisted Reproductive Technologies 11. R. Michael Roberts:Embryo Culture Conditions: Endocrinology, Volume 146, Issue 5, 1 May 2005, Pages 2140–2141: What Embryos Like Best 12. Shuai Li 1 and Wipawee Winuthayanon 1:Journal of Endocrinology Volume 232: Issue 1: Oviduct: roles in fertilization and early embryo development 13. J.E. Swain, G.D. Smith: Human Reproduction Update, Volume 17, Issue 4, July-August 2011, Pages 541–557: Advances in embryo culture platforms: novel approaches to improve preimplantation embryo development through modifications of the microenvironment 14. Nora A Baak, Astrid EP Cantineau, Cindy Farquhar, Daniel R Brison:Cochrane Systematic Review - Intervention Version published: 17 September 2019: Temperature of embryo culture for assisted reproduction 15. Jason E Swain: Reproductive Healthcare Ltd. Published by Elsevier Ltd ,1472-6483/$ : Optimizing the culture environment in the IVF laboratory: impact of pH and buffer capacity on gamete and embryo quality
- 33. References 16. Jason ESwain: Journal of Assisted Reproduction and Genetics 28(8):711-24:Biological pH buffers in IVF: Help or hindrance to success 17. Maria Giulia Minasi & Gemma Fabozzi & Valentina Casciani & Anna Maria Lobascio & Alessandro Colasante & Filomena Scarselli & Ermanno Greco:1128: ESHRE 28th annual meeting, Istambul, Turkey, July 1 to 4, 2012. Improved blastocyst formation with reduced culture volume: comparison of three different culture conditions on sibling human zygotes 18. Petra L. Wale, David K. Gardner: Human Reproduction Update, Volume 22, Issue 1, January/February 2016, Pages 2–22: The effects of chemical and physical factors on mammalian embryo culture and their importance for the practice of assisted human reproduction 19. Scott J. Morin1,:Springer Science+Business Media New York 2017 Published online: 8 February 2017: Oxygen tension in embryo culture: does a shift to 2% O2 in extended culture represent the most physiologic system? 20. David K.Gardner: Reproductive BioMedicine Online Volume 32, Issue 2, February 2016, Pages 137-141:The impact of physiological oxygen during culture, and vitrification for cryopreservation, on the outcome of extended culture in human IVF