l323 semi closed incuchamber laura r. ksystem eshre 2009
DESCRIPTION
Dr. Laura Rienzi lecture on L323 semi-closed incubator and importance of Oxygen on cells.TRANSCRIPT
Why should we use the IVF chambers (L323IVF) during our procedures???
Laura Rienzi
Senior Embryologist, Laboratory Director
CLINICA VALLE GIULIA, Rome
www.generaroma.it
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.
Introduction
Whatever incubation system is chosen, a key to successful embryo culture is to minimize perturbations in the atmosphere around the embryo.
Temperature
Gas composition
Air quality
Media composition
Introduction
Preimplantation embryo is a free-living organism
Rizos et al., 2002
The early embryo represents a unique stage of development where environmental interactions must take place to set homeostatic mechanisms for the developmental program
Critical period determining oocyte competence
Critical period determining blastocyst quality
Stress tollerance
Oocyte preparation for “the great awakeness””
first cleavage division
embryonic genome activation morula
compaction blastocyst formation
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
Suboptimal in vitro conditions may lead to irreversibly long-term alterations in the characteristics of foetal and postnatal growth and development
metabolometranscriptome proteome
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).
Culture conditions
Gardner and Lane, 2004
5% CO5% CO22
in air in air
5% O5% O22
6% CO6% CO22
new medium new medium formulation formulation
Culture conditions
Stimulation protocol
PATIENT
Diet
Oocyte quality LABORATORY
Embryo transfer &
luteal supportOUTCOME
# of Incubators # of Embryologists & trainig level
Air quality
Gardner and Lane, 2007
Oocyte/embryo handling outside the incubator
CULTURE SYSTEM
Oil overlay
Gas phase
Tissue culture ware/ contact supplies
# of Embryo/dropCulture mediaQC & QA
Sperm quality
OXIDATIVE STRESS
Potential sources of oxidative stress:
• Exposure to light
• Presence of transitional metals in the culture media
• High oxygen tension (i.e., 20%)
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.
Culture system: Gas Phase
~20% 5%Oxygen Concentration
0
10
20
30
40
50
60
70
80
90
% B
last
ocys
t / C
ell n
umbe
r
***
Blastocyst (%)
Cell Number
*, P<0.05
**, P<0.01
Effect of Oxygen concentration on mammalian embryonic development
Gardner et al. personal comunication
Oxygen concentration
Prospective randomized trial: six hundred couples undergoing IVFMain 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
Oxygen concentration
Meintjes et al., Human Reproduction 2008
Oxygen concentration
Harvey et al., 2002; Thomas et al., 1997
• 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)
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.
REDOX state
Harvey et al., 2004
Oxygen is an essential key key energy substrateenergy substrate for oxidative phosphorylation Other cellular functions,
such as apoptosis and cell cycle control, are also significantly influenced by oxygen oxygen availability and REDOX availability and REDOX statestate, via transcription factors such as NFkB
REDOX state is considered to be an important mechanism that regulates several cell functions, particularly
in the preimplantation embryo.
a key modulator modulator of metabolic of metabolic
pathways pathways (OXPHOS and
glycolysis)
Hypoxia-inducible factors
Semenza et al., 2000
Under normoxic conditions, HIF-
1a protein is degraded rapidly by 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
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 hypoxia response element (HRE) regulatory region
[O2]Correct
gene expression
High O2
In-vivo Low O2
Effect of Oxygen concentration on gene and protein expression (mammalian embryos)
Katz-Jaffe et al., 2005
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
Oxygen is a physiological signal
Harvey et al., 2002; Thomas et al., 1997
Embryos encounter a decreasing O2 concentration gradient as they progress down the reproductive tract
post-compaction development is associated with a significant shift in the REDOX state to a more reduced state
O2 from a 5% atmosphere would be able to sustain OXPHOS throughout preimplantation development
glycolytic enzyme activity
glucose uptake
Oxigen gradient provide spatial information to cells within the
embryo
ICM TEC
Shift from glycolysisShift from glycolysis
[O2] ≈
7%
[O2] ≈
2%
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?
Effect of Oxygen concentration on human embryo development
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
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!!
Effect of Oxygen concentration on mammalian embryonic development
Gardner, personal comunication
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.
Buffer System and Hydrogen Ion Concentration (pH)Buffer System and Hydrogen Ion Concentration (pH)
HCO3-
+ H+
⇋ H2
CO3
⇋ CO2
+ H2
O
The pH
and hence the [H+] can be approximately estimated using the Henderson-Hasselbach equation: pH = 6.1 + log [[NaHCO3]/(PaCO2)]
pHi in somatic cells is implicated in:
Medicult7.2 7.4
7.1 7.3Quinn’s Advantage
Global7.2 7.35
G1.37.30 ±
0,10
HTF7.40 ±
0,10
COOK7.30 7.35
Recommended pH Ranges for Commercially Available IVF Media
pHo pHo ≠≠
pHipHi
DAY 5DAY 5--66
DAY 1DAY 1
DAY 2DAY 2
DAY 3DAY 3
7,17This elevation in pHi observed as development proceeds may reflect the very
different physiology of the early embryo in comparison to its later counterpart
7,217,19
7,24
Rather than pHo affecting pHi directly, it appears that the presence of weak acids or bases in the culture medium markedly affect pHi
60 minMorula / blastocystzigote zigote
Edwards et al., 1998
Embryos ability to recover from a pHi stressEmbryos ability to recover from a pHi stress
Altering pHi disrupts the organization of mitochondria
Bavister et al 2001, Edwards et al,1998
It has been established that even relatively small fluctuations in pHi can significantly retard subsequent developmental competence
Cellular buffer systems
• At cytoplasmatic level, locally and rapidly by intrinsic buffers such as the zwitterionic amino acids
• Ionic membrane transport mechanisms
Cellular buffer systems
Relieves alkalosis, with set point 7.3 pHi (AE gene family)
Na+/ Cl- HCO3- with set point pH<7
25mMCl-Na+
HCO3-
CO2
HEPES
5 mM [NaHCO3]
Relieves acidosis, with set-
point 6.8 pHi (NHE gene family)
Baltz et al., 2005
X
Cellular buffer system
Phillips and Baltz, 1999
Oviduct pH 7,7Oviduct pH 7,7
Uterin pH 7,1Uterin pH 7,1
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
Following the denudation procedure oocytes and early embryos cannot regulate their ionic homeostasis
Temperature: ~37°C
CO2
: 5-6%
O2 : ~5%
N2 : 89-90%
Standard micro-environment for embryo culture
Choosing the right incubator
• 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
Incubators
Out side the incubator?
Out side the incubator?
FOR HANDLING: chambers
CCOCs
screening, denudation, dishes
change, embryo
transfer
FOR INVERTED MICROSCOPE: stage top incubators
Oocyte evaluation, oocyte micromanipulation, embryo
evaluation
Quality
People forget how fast you did a job but they remember how well you did it
Howard W Newton
CLINICA VALLE GIULIA, Roma
www.generaroma.it
Ginecology:
Filippo Ubaldi
Elena Baroni
Antonio Ciconte
Silvia Colamaria
Antonio Lo Re
Fabio Sapienza
Embriology:
Laura Rienzi
Laura Albricci
Antonio Capalbo
Roberta Maggiulli
Stefania Romano
SALUS – ASI MEDICAL, Marostica