Everything you need to know about Embryo Culture in an IVF Lab.

1. Decoding Embryo Cultur
Laboratory Incubation 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 Vitro culture 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
PRE COMPACTION (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
Gas Phase 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

6. 01
02
03
History
Expectations
Key Components
Embryo Culture Media

7. Expectations from Culture Media
Ensure Embryo
Growth
Decrease
Embryonic
Stress
Maintain
Gamete
Viability

8. History of Embryo Culture 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 Embryo Culture 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 Embryo Choose – Single Step Culture
Back To Nature – Sequential Culture
Comparison between the Protocols
Culture Media Type &
Protocols

11. Sequential Culture
• Mimic In 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
Company Medium 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
Fresh Stage 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
Characteristic Single 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 is Superior ?
STUDIES INCONCLUSIVE
If something is working for you, Good for You!

17. 01
02
03
Group Culture
Individual Culture
Advances in Culture Strategies
Embryo Culture Strategies

18. Single Embryo Culture
Group Culture
MicroDroplet Culture
Embryo Culture Strategies

19. Individual Culture
Advantages
• Individual evaluation 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 or Communal 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 Embryo Culture 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 & CO2 Concentration
Temperature
Oxygen Tension
Culture Condition

23. Temperature
FACT
Ovarian follicle and lumen 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

24. pH

25. Acceptable pH range for 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 in the 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 culture at 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

31. Thank You !

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 E Swain: 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