Hi I’m Stacy and I’m an Embryologist from the UK working at Cambridge IVF. You may have seen the Itegra 3 and Thermosafe on the RI stand – we have been fortunate to have the IT3 in our laboratory since September last year and today I would like to share with you a study that was carried out at our clinic which analysed temperature control and stability using the IT3.
Micromanipulation procedures such as ICSI, IMSI and embryo biopsy involve removing eggs or embryos from the incubator for extended periods of time. We know that when we remove eggs or embryos from the incubator for more than 3 minutes there are additional measures we need to take in order to maintain their environment, for example, we change the media from one that contains a bicarbonate based buffer to one that contains a HEPES or MOPS based buffer. Another important parameter we need to control is temperature CLICK We know that temperature stability is critical because numerous studies have demonstrated that destabilization of the meiotic spindle occurs when temperature is reduced. CLICK An interesting study published by a group from Brown University, Rhode Island demonstrated that we can stabilized and prevent this affect if temperature stability is controlled during micromanipulation procedures.
The study compared the affect of different heating systems during the ICSI procedure. The first heating system used a thermostated coverslip combined with an objective heater which maitained temperature stability within 0.1ْC and this was compared with a conventional stage warmer – in other words without an objective heater in which temperature fluctuations of up to 4ْC were recorded. Along the Y axis we have percentage and along the X axis you can see the different parameters that were assessed. Using the PolScope technique the group were able to visualise significantly more meiotic spindles when using the objective heater was used in comparison to the conventional method. A significant affect was also observed on both the fertilisation rate and the subsequent ongoing pregnancy rate – as you can see a significantly higher clinical pregnancy rate was achieved where temperature was controlled and stabilized using the objective warmer.
After reviewing this study it may seem strange that most ART laboratories perform micromanipulation procedures without an objective heater and therefore inadvertedly allowing the eggs or embryos to become vulnerable to high fluctuations in temperature. This is because objective heaters have a number of practical implications which makes them difficult to work with in clinical practice. They are size specific (small/medium and large) – which means you‘ll probably need a number of different sized objective heaters for each micromanipulation workstation. Depending on how many objective heaters you have in theory you may need to move the objective heater when changing to a different objective size which during an ICSI we all know we be impractical They need to be plugged into a power source when in use – which requires a reachable power socket in the laboratory – this also may become quite fiddly when changing between objectives as you will need to spend time plugging in the relavant objective warmer – by doing this you could risk knocking your dish as you would be spending a lot of time moving things underneath the heated stage and it also increases the amount of time the eggs or embryos are spending outside of the incubator – which is really something were trying to avoid. http://www.bioptechs.com/Objectives/Objective.html
At present there are 2 types of micromanipulation equipment that is mainly used in the ART laboratory. There is the heated stage with aperture – which is basically the heated workstation with a whole between the objective lens and the dish. There is also the heated stage with the ITO or glass platform.
In September 2013 Research Instruments brought out the Itegra 3 and theromosafe. The thermosafe is an air warming system which works by controlling the temperature of the air with is blown towards the objective lens underneath the heated stage.
In addition to the AWS the Integra 3 comes with a number of improved design innovations… The objective carousel lights up automatically when the operator changes the objective lens which makes it very easy to see what your doing. A green light indicates when the workstation has reached the temperature set-point and is therefore ready to use - there are also function buttons here if you would like to take a photo or a video you simply press these buttons. The heated platform is one large work area as opposed to 3 smaller work stations – this is more user friendly and safer as we do not need to pick-up and move our dishes as much. A counter allows operators to log how many eggs have been injected or embryos have been biopsied A stopwatch allows operators to time how long they are working for.
This study compared 3 micromanipulation workstations. WS1 was the heated stage with aperture WS2 was the heated stage with the glass platform And WS3 was the heated stage with the aperture and the AWS (the Integra3) Culture drop temperature was measured using a calibrated thermocouple every 5 minutes over the 1 hour test period. This was repeated 5 times for each workstation on each relevant objective which includes the x20, x40, x60 and the laser. In addition to this thermal images were also taken in order to identify potential “hot “or “cold” spots on each workstation using each objective.
Along the Y axis is the temperature and along the X axis is workstation 1, 2 and 3. Here you can see the mean temperature observed on the same workstation for each objective. You can see from this graph that no significant difference in mean temperature was observed on workstation 1 and workstation 3 between different objective lenses. However, a significant difference in mean temperature was observed on workstation 2 (remember that’s our glass platform!) Lets take a closer look – CLICK – here you can see the x40 read 1.3ْC lower than the x20, 0.8ْC lower than the x60 and 0.5ْC lower than the laser objective – further to this the x60 objective is 0.5 ْC lower than the x20 and the laser objective is also 0.8ْC lower than the x20.
Along the Y axis is temperature and along the X axis is time in minutes. Here you can see temperature stability over the 1 hour test period. You can see that on WS1 high temperature fluctuations were observed across the 1 hour test period for each objective lens. When we look at WS2 you can see that temperature stability has been improved, however, we are seeing a significant difference in mean temperature between different working objective lens. When we look at WS3 you see that the temperature stability has been maintained and the significant difference in mean temperature has been removed – you can see the positive affect the AWS is having by effectively giving us the best of both worlds.
Thermal images were taken in order to identify potential hot or colds spots on each workstation using different objectives. Figure 5 was taken underneath the objective turrent looking up towards the base of the dish on WS2 and WS3 – and I really think this image demonstrates on cold the objective actually is – we have this chunk of mental sitting directly underneath of dish. Figure was taken from WS2 and this is an thermal view taken above the glass and it is quite clear to see here the affect the x40 objective is having – you can visually see a reduction in temperature in the critical area of our workstation.
We began to think about what may be causing this ‘heat sink’ effect on WS2 and here we have plotted mean temperature for each objective against the objective mass – and you can see a clear negative correlation with the exception of the laser objective in that when the objective mass increases the mean dish temperature decreases… Here in figure 8 we plotted mean temperature against the working distance between the objective lens and the dish and here we see a positive correlation with the exception of the x60 objective in the when the working distance increases the mean temperature increases. This may serve as a starting point in why we are seeing a significant difference in mean temperature between different objectives using a workstation with a glass platform.
From this study we know that culture drops undergo changes in temperature when a WS with aperture is used – these fluctuations are concerning when procedures such as a difficult ICSI or IMSI can take hours, not minutes. We know that these subtle changes in temperature can impact on our clinical practice. The alternative approach of using a glass platform is susceptible to significant changes in temperature caused by different working objectives - this system is susceptible to intra-operator variation, for example, different embryologists may inject on a x20 or a x40 or switch between the two and in doing this they could be inadvertedly causing harm to the oocytes. The significant different in temperature may be due to the thermal mass and working distance between the objective and the dish causing a ‘heat sink’ effect – although this is the subject of ongoing investigation.
We can conclude that the AWS improves temperature control and stability during micromanipulation procedures when compared to alternatives currently available .
By controlling temperature during micromanipulations procedures we are able to obtain a number of clinical advantages. We can reduce the variation in temperature that may be caused by different operators using different objectives for micromanipulation procedures. We can use the x60 objective to select sperm during IMSI and inject on a x20 or a x40 with having to worry about temperature control Embryo biopsy again can be performed across a range of objectives and temperature can be controlled during the full time period of the biopsy – however long that my be! Procedures that involve the use of the laser such as assisted hatching and blastocyst collapse again can be performed at the optimum temperature outside of the incubator.
Temperature stability is improved by using a novel air warming system By Stacy Wheat, Cambridge IVF
Innovation and excellence in health and care Addenbrooke’s Hospital I Rosie Hospital
ESHRE 2014, Munich
Temperature stability is
improved by using a novel
air warming system
• ICSI, IMSI and embryo biopsy
• Removal from incubation for extended periods of time
• Control temperature stability
Is temperature stability important?
Figure 1: Meiotic spindle stability and subsequent fertilisation and pregnancy rates *p<0.05; from Wang et.al., 2002.
With objective heater
Without objective heater
Note, data in column 1 ‘without objective warmer’ was obtained using a thermostated coverslip as spindles could not be imaged using the conventional stage alone due to
Objective heaters: practical implications
• Size specific
• Changing between objectives
• Power source
Existing micromanipulation equipment
Heated stage with
Heated stage with
Integra Ti 2011 plus WIS metal stage,
Research Instruments, Falmouth.
Integra Ti 2011 plus ITO,
Research Instruments, Falmouth.
New innovation: Integra 3 and Thermosafe
Heated stage with aperture and air warming system (AWS)
Integra 3 2013 with ‘Thermosafe’, Research Instruments,
Improved functionality; design innovations
• Illuminated objective carousel
• Visual temperature indicator and function buttons
• Large flat heated stage area
• Novel counter function to track injections
• Stopwatch function to time procedures
• Workstation 1 (WS1): heated stage with aperture
• Workstation 2 (WS2): heated stage with glass platform
• Workstation 3 (WS3): heated stage with aperture + AWS
• x 20
• x 40
• x 60
• Temperature measured
every 5 minutes for 1 hour
• Thermal images taken to identify potential ‘hot-’ or ‘cold-
spots’ on each workstation using different objectives
Mean objective temperature on same workstation
Figure 2: Mean objective temperature over a 1 hour period for each workstation, *p<0.01Figure 3: Mean objective temperature over a 1 hour period for workstation 2, *p<0.01
Results: Temperature stability over time
readings over a 1 hour
period for WS1, WS2
Figure 5: View from the
objective turret to the
bottom of the dish on WS2
and WS3 (x20 objective).
Figure 6. Comparative
thermal images of x20
and x40 objective on
glass platform of WS2.
Are we seeing a ‘heat sink’ effect?
mass for WS2.
distance for WS2.
• Culture drops undergo temperature fluctuations when
a workstation with aperture is used.
• This is concerning as subtle changes in temperature
can impact on clinical outcome (Wang et al. 2002).
• The alternative approach of using a glass platform is
susceptible to significant changes in temperature
caused by different working objectives.
• Thermal mass of objectives and working distance
between objective and dish may cause a heat sink
effect – subject of ongoing investigation.
What can we conclude?
The AWS improves temperature control and
stability during micromanipulation procedures
when compared to currently available alternatives.
What are the potential clinical advantages?
No need to worry about
temperature control over time
during procedures –
IMSI and embryo
Laser – blastocyst
collapse & assisted