l323 semi closed incuchamber laura r. ksystem eshre 2009

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

Presenter

Presentation Notes

To successfully fertilise oocytes and grow embryos outside the human body requires very specialised systems for oocyte collection and embryo culture. Human embryos are remarkably sensitive to environmental toxins that can come from plasticisers and other volatile chemicals present in the products they come in contact with and endotoxins from contaminating bacterial growth. As service technicians we need to be aware of this sensitivity and take appropriate actions not to harm any embryos. The use of chemicals/cleaning materials need to be examined prior to use in the presence of these embryos.

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

Presenter

Presentation Notes

In generale, possiamo considerare l’embrione preimpianto come un organismo free living, cioè un organismo che non è in dipendenza da un altro per la sopravivenza. Tuttavia questo unico e singolare stadio dello sviluppo, caratterizzato da una intrinseca plasticità evolutiva, gli impone il corretto espletamento temporale del potenziale genetico embrionale, rendendo estremamente critica e determinante la sua inteazione con l’ambiente. Analizzando globalmente il processo di sviluppo embrionale possiamo disinguerlo, sommariamente, in due momenti determinanti rispettivamente la capacità mitotica e quindi il numero di blastocisti che si formano, e la qualità relativa della blastocisti. Infatti è stato ampiamente riportato come Nonostante le condizioni di cultura possano influenzare la cinetica dello delle prime divisioni è ormai comprovato che la percentuale di blastocisti formata in vitro dipende principalmente dalla qualità maturativa dintrinseca dell’ovocita e non sia entro certi limiti strettamente dipendente dalle condizioni di coltura, che tuttavia risulta critica per fornire l’ambiente ideale peer lo svilupo la qualità e vitalità della blastocisti formata La fase di coltura ex vivo ha il compito di sostenere adeguatamente i maggiori eventi che accompagnano questo periodo e che risultano traduce queste potenzialità in relazione col fattore ambientale E’ possibile anche evidenziare come la tolleranza allo stress sia una funzione inversa della stadio maturativo. In particolare nelle primi divisione cellulare e quindi durante la routin dell’IVF l’embrione è trascrizionalemente silente e con molteplici apparati funzionali e strutture cellulari ancora indifferenziati(mitoconodri, sistemimi di trasporto omeostatici) . Queste crea una finestra di grande vulnerabilità in cui l’embrione nn è grado di rispondere alle variazioni ambientali. Con il procedere dello sviluppo l’embrione aquista sembiaza più propriamente somatiche fino alla generaione allo stadio di blastocisti di un epitelio di trasporto in grado di autoregolare l’omeostasi interna Le fasi di coltura sono critiche perché devono supportare eventi cruciali per la vitalità embrionale quali

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

Presenter

Presentation Notes

La realtà della coltura ex vivo nella pratica della medicina riprodittiva è quella di esporre gli embrioni a un ambiente fisico e biochimico differente da quello reale, cercando di approssimarlo al meglio Tuttavia anche se lo sviluppo mitotico procede, considerata l’lto rado di plasticità propro di un sistema altamente indifferenziato come quello embrionale, del periodo di coltura si traduce in uno stress capace di alterare globalmento il profilo fisiologico dell’embrione…………….. Inoltre essendo tra vivo e vitro e tra vitri diveri essendo alterazioni non siano specie-specifiche comuni in tutte gli embrioni di diverse specie questo evidezia il ruolo determinante dell’ambiente nella regolazione di queste funzioni. Tuttavia anche se grandi alterazioni di quste funzione portino nevitabilmente alla compromisione dela vitalità embrionale e quindi al fallimento dell’impianto e/o dello sviluppo fetale,

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

Presenter

Presentation Notes

Individual components are analysed

OXIDATIVE STRESS

Potential sources of oxidative stress:

• Exposure to light

• Presence of transitional metals in the culture media

• High oxygen tension (i.e., 20%)

Presenter

Presentation Notes

excessive generation of free O2 radicals (ROS: super oxide and hydrogen peroxide), apoptosis Burton et al., 2002, Wong et al., 1989

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

Presenter

Presentation Notes

Analizzando questo aspetto su grandi numeri possiamo concludere …Quanto poco realistico sia il nostro sistema prognostico


Meintjes et al., Human Reproduction 2008

Presenter

Presentation Notes

We also found no change in fertilization rate or cleavage rate when culturing embryos in a reduced O2 environment. Initial clinical outcomes (implantation rate and clinical pregnancy rate) were only affected by atmospheric O2 concentrations when considering Day 5 transfers When considering not only the initial clinical results in the present study but also the eventual live birth implantations and live births, it became apparent that even embryos transferred on Day 2–3 might have benefited fromculture in reduced O2 as more implantations were lost fromthe high-O2 treatment group.


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)

Presenter

Presentation Notes

Incubation in 5% O2 compared with 20% O2 increases oxygen uptake and pyruvate oxidation (Hooper et al.,2001) through its role as an electron acceptor in the electron transport chain.

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

Presenter

Presentation Notes

10 proteins down-regulated with 20% O2

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%

Presenter

Presentation Notes

Infatti nel sistema riproduttivo l’embrione è esposto a concentrazioni decrementai di ossigeno che determinano cambiando lo stato redox intracellulare verso una condizione più ridotta. A Questo consegue ‘aumento dell’attività degli enzimi glicolitici, aumenta l’uptake del glucosio che permettono lo shift metabolic profile of inner cell mass cells differs from that of trophectoderm

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

Presenter

Presentation Notes

Dire che

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

Presenter

Presentation Notes

Destabilizzazione la ben definita composizione perinucleare dei mitocondri

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

Presenter

Presentation Notes

Ionic pomp Hepes fototossic and low concentration of ions which do not allow the physiological antiport systems to function This has implications for the handling of human embryos in vitro, which are routinely manipulated in HEPES-buffered media with low HCO3– concentrations at atmospheric CO2

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

Presenter

Presentation Notes

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

l323 semi closed incuchamber laura r. ksystem eshre 2009

Education