control of variables; the key to success in the ivf laboratory a/prof cecilia sjoblom westmead...
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Control of Variables;Control of Variables;The key to success in the IVF laboratoryThe key to success in the IVF laboratory
A/Prof Cecilia SjoblomA/Prof Cecilia Sjoblom
Westmead Fertility Centre, University of SydneyWestmead Fertility Centre, University of Sydney
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BackgroundBackground
Success of assisted reproductive technology (ART) affected by:
Patient factorsQuality of the Laboratory Culture protocolsSupplies Methods
Equipment
(Alper et al., 2002; Higdon et al., 2007; Fujiwara et al., 2007)
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BackgroundBackground
• Embryos resembles primitive cells
• Pre-Compaction they can not regulate changes to– pH– Temperature– Osmolarity
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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TemperatureTemperature
• Temperature is a key determinant of gamete viability and embryonic growth
• The micro-environment for culture should be held a temperature of 37 ± 0.2 °C
• Various types of warming devices(Yeung et al., 2004; Hansen 2007)
Lane et al 2008
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TemperatureTemperature
• Meiotic spindles– Oocyte quality– Chromosome alignment and separation during MI and
MII
• Cooling and overheating produce disassembled meiotic spindles – Human (Wang et al., 2001)– Mouse (Sun et al., 2004)– Porcine (Suzuki et al., 2007)
– Cow (Pollard et al., 1996; Ju et al., 1999)
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TemperatureTemperature
Insemination of oocytes with disrupted meiotic spindles results in:
• Failed fertilization
• Abnormal fertilization– Aneuploidy
• Low embryo developmental competence
– Apoptosis/ fragmentation
– Gene expression?
(Wang et al., 2001; Sun et al., 2004; Massaro et al., 2007; Zeng et al., 2007; Lane et al., 2008)
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TemperatureTemperature
HyaluronidHyaluronidasease
ProbeProbe
WashWash
A
CD
B
HyaluronidaseHyaluronidase
ProbeProbe
WashWash
• Temperature measurements were taken every 20 seconds• Digital thermometer with ± 0.1°C accuracy
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TemperatureTemperature
36.6
29.75 32.02
0
5
10
15
20
25
30
35
40
45
UNDER OIL 4 WELL DISH 4 WELL DISH IN WARMING BLOCK
Tem
pera
ture
(°C
)
Denudation protocol
a
bc
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37°C
32 °C29.8°C
29.8 °C for 3 min
29.8 °C
32.3 °C
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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pHpH
7.6-7.9
7.2-7.3
• Mean pHi– Human 7.12 ± 0.01 (Phillips et al., 2000)
– Mouse 7.17-7.22 ± 0.01 (Edwards et al., 1998)
– Hamster 7.19-7.22 (Lane et al., 1998)
• During the in vitro manipulation of mammalian embryos the extra-cellular pH (pHo) should be maintained close to pHi in order to reduce stress(Edwards et al., 1998; Lane et al., 1998)
• Currently 7.35 ±0.05 in an environment of 5-6% CO2 (Sjöblom, 2004; Quinn, 2004)
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pHpH
A precise control over pHi is essential for numerous
cellular processes
– Enzyme activity (Lane et al., 1999a;1999b)
– Cell differentiation; growth and proliferation (Ozawa et al., 2006)
– Cell division; membrane transport; cell-cell communication
(Lane et al., 1998)
– Protein and DNA synthesis (Squirrell et al., 2001)
– Respiration (Lane, 2001)
– Metabolism, calcium level modulation and cytoskeletal
dynamics (Squirrell et al., 2001)
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Micro pH Probe in 50ul Drop under OilMicro pH Probe in 50ul Drop under Oil
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7.2
7.4
7.6
7.8
8
8.2
8.4
8.6
8.8
0 10 20 30 40 50 60 70
time (min)
pH
pH in a Drop under OilpH in a Drop under Oil
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7,3
7,4
7,5
7,6
7,7
7,8
7,9
0 5 10 15 20 25 30 35
time (min)
pH
3 minutes 5 minutes 10 minutes
pH in Drop under OilpH in Drop under Oil
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Mouse EmbryosMouse Embryos
The effect of exposure time on cell number in blastocysts cultured in vitro
(11)
(12)(18)
(10) (10)
0
10
20
30
40
50
60
70
In vivo Control 3 min 5 min 8min
Time spend outside the incubator (min)
Nu
mb
er o
f ce
lls
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pHpH
• The pH becomes sub optimal after just 3 min outside
the incubator
• No activity should take longer than 3 min
• A stop watch is an embryologists best friend
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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OsmolarityOsmolarity
• Zygotes are more sensitive than 2-cell embryos• In response to changes in osmolarity• Embryos will act by changing their volume to
regulate osmotic pressure across their membrane • Irreversible damage to cyto-skeleton• Changes to gene expression and possibly
imprinted genes
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OsmolarityOsmolarity
• The osmolarity of the reproductive tract is high for most species– Mouse 310- 360 mOs/L (Borland et al., 1977; Van Winkle et al., 1990;
Dawson et al., 1998) – Human 320-360 mOs/L (Collins and Baltz, 1999; Li et al., 2007)
– Bovine 330-370 mOs/L (Baltz 2001; Hwang et al., 2008)
– Rat 290 mOs/L (Baltz 2001; Hwang et al., 2008)
• Attempts to culture embryos in vitro at these high osmolarities have failed
• The optimal osmolarity for pre-implantation embryos is 260 mOS/L
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Osmolarity
240
250
260
270
280
290
300
0 24 hrs 48 hrs 72 hrs
4 Well Dish60 mm dishCentre well dish
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OsmolarityOsmolarity
Osmolarity Total cell count
(Mean±SD)
Control (260) mOsM 82.25±4.0311
280 mOsM 59.75±4.113
300 mOsM 43.5±4.1231
310 mOsM 26.5±3.7
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OsmolarityOsmolarity
• Making up dishes in advance is common practice in the IVF lab– To allow for pre-equilibration– For smooth running of the day
• Making up dishes too early can effect the osmolarity of the culture medium
• This can in turn have detrimental effects on embryo development
• Cover micro-drops IMMEDIATELY
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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Gas CompositionGas Composition
• CO2 is used in cell culture as a part of the CO2 - HCO3-
buffer system
• CO2 is NOT a metabolite for cells or embryos
• Concentration of CO2 is depending on
• What pH you aim for
• The concentration of HCO3-
• O2 is a metabolite and crucial in the embryos
utilisation of Pyruvate as an energy substrate
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Gas CompositionGas Composition
• A total of 573 patients, 7312 oocytes• 689 consecutive IVF and ICSI cycles at
NURTURE • Prospectively randomised to culture in
– 7% oxygen (275 patients, 325 cycles)– Ambient conditions at approximately 20% oxygen (298
patients, 364 cycles).
• No difference between the two groups in;– IVF:ICSI ratio (56:44)– Patient age (34±0.3 years)– Infertility background
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Gas CompositionGas Composition
Lowering oxygen to more physiological levels
is associated with; • Significant improvement in embryo quality
• Significantly higher pregnancy rate per oocyte retrieval
(47% versus 39% p< 0.05)
• There was no difference in the pregnancy rate per ET
(48% and 42% respectively, p=0.11)
• Significant difference in live birth rate
(45% and 39% respectively, p< 0.05)
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Control of Physical ParametersControl of Physical Parameters
• Temperature
• pH
• Osmolarity
• Gas Composition
• Full Control
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Full ControlFull Control
• You can improve your results by having full
control over variables
• It is crucial that the laboratory has full
control over physical parameters and that
embryologists are fully aware of the
implications of their actions
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High IVF Live Birth Rates is achieved High IVF Live Birth Rates is achieved through Full Controlthrough Full Control