L323 Semi Closed Incuchamber Laura R. Ksystem Eshre 2009
1. www.generaroma.it
CLINICA VALLE GIULIA, Rome
Why should we use the IVF chambers
(L323IVF) during our procedures???
Laura Rienzi
Senior Embryologist, Laboratory Director
2. Introduction
Preimplantation embryos are known to be sensitive
to environmental conditions that can affect future
growth and developmental potential
Embryologists have the difficult task to select culture
media, supplies, instrumentations and methods
that better meet with the physiological
requirements of the developing embryo in the Lab.
3. Introduction
Whatever incubation system is chosen, a key to
successful embryo culture is to minimize
perturbations in the atmosphere around the embryo.
Temperature Air quality
Media composition Gas composition
4. Preimplantation embryo is a free-living organism
The early embryo represents a unique stage of development where environmental
interactions must take place to set homeostatic mechanisms for the developmental program
Stress tollerance
Critical period determining
oocyte competence
Oocyte preparation Critical period determining blastocyst quality
for “the great awakeness”
first cleavage embryonic
division genome activation morula
compaction blastocyst
formation
Rizos et al., 2002
5. Short-term effects of embryo environmetal
Working in vitro the embryos are exposed to several stress that may compromise
their physiology, gene expression and development
transcriptome metabolome proteome
Suboptimal in vitro conditions may lead to irreversibly long-term
alterations in the characteristics of foetal and postnatal growth and
development
6. Culture conditions
With the appropriate media and laboratory set-up, it is possible to obtain
blastocysts form on Day 4, such as occurs in vivo (in the mouse model)
and establish 80% pregnancy rates in humans (oocyte donor model).
5% CO2 5% O2 new medium
in air 6% CO2 formulation
Gardner and Lane, 2004
7. Culture conditions
Stimulation
protocol
Oocyte
Embryo
PATIENT quality LABORATORY OUTCOME
transfer &
Sperm luteal support
Diet quality
# of Incubators # of Embryologists Air quality
& trainig level
Oil overlay
CULTURE SYSTEM Tissue culture ware/
Gas phase contact supplies
QC & QA
Culture media # of Embryo/drop
Oocyte/embryo handling
outside the incubator
Gardner and Lane, 2007
8. OXIDATIVE STRESS
Potential sources of oxidative stress:
• Exposure to light
• Presence of transitional metals in the culture media
• High oxygen tension (i.e., 20%)
9. Culture system: Gas Phase
Human in vitro embryo culture is reported to be performed using a gas
phase containing either atmospheric (~20%) or reduced (~5%) oxygen
concentrations.
High oxygen concentration has been shown to adversely affect embryonic
development in several animal species. Quinn & Harlow 1978, Batt et
al., 1991, Fujitani et al., 1997; Gardner et al., 1996; Karagenic et al., 2004;
Booth et al., 2005
No beneficial (or too marginal) effect of low O2 concentrations has been
reported in IVF programs. Dumoulin et al., 1995, 1999
More recent data have, however, underlined the importance of the Gas
composition for human embryo culture.
10. Effect of Oxygen concentration on mammalian
embryonic development
90
**
% Blastocyst / Cell number
80
70
*
Blastocyst (%)
60
50 Cell Number
40
30 *, P<0.05
20 **, P<0.01
10
0
~20% 5%
Oxygen Concentration
Gardner et al. personal comunication
11. Oxygen concentration
Prospective randomized trial: six hundred couples undergoing IVF
Main Outcome Measure(s): live birth rate
Blastocyst culture with low-oxygen (5%) versus high-oxygen (~20%) concentration
yielded a better blastocyst outcome and a marked improvement in birth rate.
Waldenstròm et at., Fertility and Sterility 2008
13. Oxygen concentration
Under atmospheric oxygen conditions the main contributor to poor embryo
development is supposed to be ROS production. However a ‘cause and effect’
mechanism is yet to be demonstrated.
• There are some specific events in reproductive system that are positively associated
with ROS production
• Increase in antioxidant gene expression under 20% oxygen has not been observed
• Reducing O2 tension is more effective in promoting embryo development in vitro than
is treatment with detoxifying enzymes (superoxide dismutase and catalase)
Harvey et al., 2002; Thomas et al., 1997
14. REDOX state
REDOX state is considered to be an important mechanism that regulates
several cell functions, particularly in the preimplantation embryo.
Oxygen is an essential key
energy substrate for oxidative
Other cellular functions,
phosphorylation
such as apoptosis and
cell cycle control, are
also significantly
influenced by oxygen
availability and REDOX
state, via transcription
state
a key modulator factors such as NFkB
of metabolic
pathways
(OXPHOS and
glycolysis)
Harvey et al., 2004
15. Hypoxia-inducible factors
Members of the hypoxia inducible factor family of transcription factors are
influenced directly by the intracellular oxygen concentration, and are important
for embryonic development within the hypoxic reproductive tract
Core consensus sequence (A/G)CGTG in the
Under normoxic conditions, HIF- 1a protein is degraded rapidly by hypoxia response element (HRE) regulatory region
the ubiquitin–proteasome system
Under hypoxic conditions, the HIF-1a protein is not
targeted for degradation and can translocate to the
nucleus to form the active DNA-binding complex
Correct
[O2] gene
expression
Semenza et al., 2000
16. Effect of Oxygen concentration on gene and
protein expression (mammalian embryos)
A recent proteomic analysis of mammalian preimplantation embryonic
development revealed that specific pattern of 10 biomarkers effectively
discriminated in vitro embryos cultured at low or high oxygen concentration
High O2 In-vivo Low O2
Katz-Jaffe et al., 2005
17. Oxygen is a physiological signal
Embryos encounter a decreasing O2 concentration gradient as they
progress down the reproductive tract
O2 from a 5% atmosphere [O2] ≈ 7% post-compaction development
would be able to sustain is associated with a significant
OXPHOS throughout shift in the REDOX state to a
preimplantation more reduced state
development
glycolytic enzyme activity
glucose uptake
[O2] ≈ 2%
Shift from glycolysis
Oxigen gradient provide spatial
information to cells within the
embryo
ICM TEC
Harvey et al., 2002; Thomas et al., 1997
18. Effect of Oxygen concentration on human embryo
development
Oxygen concentration in the oviduct of hamster, rabbit and
rhesus monkey is similar to 8%. Oxygen concentration in the
uterus is significantly lower: 1.5% to 5%
In humans?
5% O2 10% O2 P
Patients (n) 27 27 NS
Oocytes inseminated (n) 371 422 NS
Fertilization rate 76% 74% NS
Clinical pregnancy rate 56% 70% NS
Implantation rate 27.2 39.5 0.09
Halicigil et al., Oral presentation ESHRE 2007
19. Effect of Oxygen concentration on mammalian embryonic
development
Even transient exposure to high O2 concentration reduce
embryo development in vitro
Culture dishes equilibrated at 20% oxygen concentration for
five hours prior culture in low O2 concentration decreases mouse
zygote development to the blastocyst stage and resultant
blastocyst cell number
IT TAKES MORE THAN 5 HOURS FOR THE OXYGEN
CONCENTRATION TO FALL TO EMBRYO SAFE LEVELS!!
Gardner, personal comunication
20. Culture system: pH and temperature
Most embryo culture media utilize a bicarbonate/CO2 buffer
system to maintain physiological pH of between 7.2 and 7.4 in
the medium. The inclusion of sodium bicarbonate in the media
requires the use of a CO2 incubator to maintain a 5-7% CO2
atmosphere.
Drawback - rapid pH increase when exposed to air
- impaired embryo development
To reduce gas exchange when culture dishes are taken out of
the incubator it is advisable to use oil overlay.
For prolonged manipulation the use of HEPES/MOPS is
required. Buffering capacity is temperature dependent.
21. Buffer System and Hydrogen Ion Concentration (pH)
pHi in somatic cells
is implicated in:
The pH and hence the [H+] can be approximately estimated using the
Henderson-Hasselbach equation: pH = 6.1 + log [[NaHCO3]/(PaCO2)]
HCO3- + H+ ⇋ H2CO3 ⇋ CO2 + H2O
22. Recommended pH Ranges for Commercially
Available IVF Media
7.1 7.3
Quinn’s Advantage
7.2 7.35
Global
7.30 ± 0,10
G1.3
7.40 ± 0,10
HTF
7.30 7.35
COOK
7.2 7.4
Medicult
23. pHo ≠ pHi
This elevation in pHi observed as development proceeds may reflect the very
7,17 different physiology of the early embryo in comparison to its later counterpart
7,19
7,21
7,24
DAY 1
DAY 2
DAY 3
DAY 5-6
5-
Rather than pHo affecting pHi directly, it appears that the presence of
weak acids or bases in the culture medium markedly affect pHi
zigote zigote Morula / blastocyst
60 min
Edwards et al., 1998
24. Embryos ability to recover from a pHi stress
It has been established that even relatively small fluctuations in pHi can
significantly retard subsequent developmental competence
Altering pHi disrupts the organization of mitochondria
Bavister et al 2001, Edwards et al,1998
25. Cellular buffer systems
• At cytoplasmatic level, locally and rapidly by intrinsic
buffers such as the zwitterionic amino acids
• Ionic membrane transport mechanisms
26. Cellular buffer systems
Relieves alkalosis, with set point
7.3 pHi (AE gene family)
Na+/ Cl- HCO3- with
set point pH<7
25mM
Cl-
Na+
HCO3-
CO2
X
Relieves acidosis, with set-
HEPES point 6.8 pHi (NHE gene family)
5 mM [NaHCO3]
Baltz et al., 2005
27. Cellular buffer system
It has been determined that the oocyte lacks any functional transport systems
to regulate pHi in either the acid or the alkaline ranges around 6 h following
fertilisation
Oviduct pH 7,7
Uterin pH 7,1
Following the denudation procedure oocytes and early embryos
cannot regulate their ionic homeostasis
Phillips and Baltz, 1999
29. Choosing the right incubator
Incubators
• Capacity
• Temperature distribution
• Heated door
• Fast recovery
• Gass tight split doors
• Ergonomic: all shelves easily accessible
• Reliable
• Failure Alarm
• Possibility to use independent probes
• Remote alarm system
• Easy to clean
• Easy to desinfect
• VOC filters
31. Out side the incubator?
FOR HANDLING: chambers FOR INVERTED MICROSCOPE:
stage top incubators
CCOCs screening, denudation, Oocyte evaluation, oocyte
dishes change, embryo transfer micromanipulation, embryo evaluation
32. Quality
People forget how fast you did a job
but they remember how well you did it
Howard W Newton
33. www.generaroma.it
CLINICA VALLE GIULIA, Roma
SALUS – ASI MEDICAL, Marostica
Ginecology: Embriology:
Filippo Ubaldi Laura Rienzi
Elena Baroni Laura Albricci
Antonio Ciconte Antonio Capalbo
Silvia Colamaria Roberta Maggiulli
Antonio Lo Re Stefania Romano
Fabio Sapienza