Questions and Answers EmbryoScope
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1 Questions and Answers EmbryoScope The EmbryoScope is a time lapse system developed to improve IVF treatments. Recent clinical results revealed novel embryo selection criteria that will be evaluated in prospective trials. The instrument is based on more than 7 years of research and uses several proprietary technologies; it was approved for clinical use in EU in June 2009, and has recently been cleared by FDA (510k) for use by US clinics. 1
2 Contents General... 3 What is the EmbryoScope?... 3 What does it do?... 3 Who are using the EmbryoScope?... 3 Improved embryo selection... 3 What is the clinical improvement potential?... 3 Have you found novel selection parameters?... 3 How to proceed with the new selection parameters?... 3 How do you facilitate sharing of knowledge between users of time lapse systems?... 3 Safety and clinical results... 4 How many clinics use the EmbryoScope Embryo Monitoring System?... 4 Is the EmbryoScope Embryo Monitoring System approved (CE mark, FDA)?... 4 How is the EmbryoScope tested prior to use?... 4 Do you have clinical evaluation trials?... 4 Do you have published papers?... 5 Clinical use of the system... 5 How is it to use the EmbryoScope in a clinical setting?... 5 How many patients can an EmbryoScope handle?... 5 How to load embryos in the EmbryoSlide?... 5 Can you grow to blastocyst in the EmbryoScope?... 5 How often do you need to change media?... 5 How do you handle media change?... 6 How to perform independent validation and QC of incubation conditions?... 6 Is ph measurement possible?... 6 Technical details... 6 What about incubation conditions?... 6 How about air quality and contamination?... 6 Why is this a non humid environment?... 6 What about light exposure?... 6 Detailed description of the illumination system used in the EmbryoScope... 7 What about the culture dish (EmbryoSlide )?... 7 Support and service... 7 What about accidents and system failures?... 7 What about support and service?... 8 What happens in case of power failure?... 8 How fast does the temperature drop if you have a power failure?... 8 Embryo respiration... 8 Does the EmbryoScope measure respiration rate?... 8 Why did you not continue with respiration technology?... 8 Will you continue with respiration instruments?... 8 Unisense FertiliTech history... 8 How did it all start?
3 General What is the EmbryoScope? The EmbryoScope is an IVF incubator with a built in camera for automated time lapse imaging of fertilized oocytes in a safe incubation environment from conception until the time of transfer. What does it do? Images of each embryo are automatically recorded at preset time intervals. Detailed information about timing of cell divisions and other critical events provide extensive documentation that embryologists can use to select a viable embryo for transfer in an IVF treatment. All observations are performed inside the incubator so the embryo is never exposed to adverse growth conditions or temperatures. Images and detailed information about incubation conditions are stored in patient data files for future reference. Who are using the EmbryoScope? The EmbryoScope time lapse system is cleared for clinical use in Europe (CE) and in USA (FDA 510(k)). 58 EmbryoScope time lapse systems have been installed in IVF clinics in 18 countries (May 2011). They have been used in more than 3500 IVF treatments and the first healthy babies have been born after incubation and imaging in the EmbryoScope. Improved embryo selection What is the clinical improvement potential? We refer to the paper Morphokinetic Analysis of embryo development for a more detailed overview of relevant scientific literature. We can also provide a list of conference abstracts and published articles upon request. IVI believes that planned software upgrades and decision algorithms will improve embryo selection further. Unisense FertiliTech is actively working with IVI and several other clinics in Europe and US to develop new software to facilitate and improve embryo selection with the EmbryoScope time lapse system. Have you found novel selection parameters? Based on a very large database of embryo images (> 20 million images of >16,000 embryos) and a detailed analysis of the development pattern of embryos that successfully implanted and resulted in ongoing clinical pregnancies compared to embryos that failed to implant, we have found novel selection procedures to improve IVF treatments that will be used in a prospective trials. The novel procedures includes negative exclusion criteria based on detailed observations of the cell nuclei as well as dimensions of ensuing daughter cells. The procedure also includes positive selection criteria based on the duration, timing and synchrony of cell divisions as originally envisioned and tested in automated image analysis of bovine embryos by Unisense FertiliTech A/S in 2006 (patents pending), and abstracts will be presented at ASRM How to proceed with the new selection parameters? New selection/deselection criteria will be tested in a prospective trial, which was initiated by IVI clinics in November The clinical trial is expected to demonstrate an improvement in the clinical pregnancy rate by using the EmbryoScope. The next prospective trial will be initiated by Unisense FertiliTech A/S in This study will be a large multicenter study using elective single embryo transfer (eset) and is expected to demonstrate even larger improvement. How do you facilitate sharing of knowledge between users of time lapse systems? Twice a year we arrange a two day interactive workshop for experienced users of the EmbryoScope. We also arrange numerous smaller workshops and symposia in different countries as well as company symposia with late breaking news at the two large annual meetings, ESHRE in Europe and ASRM in the US.. 3
4 Safety and clinical results How many clinics use the EmbryoScope Embryo Monitoring System? Unisense FertiliTech have now (May 2011) 58 EmbryoScope time lapse systems, installed in 18 different countries (Spain, Sweden, Denmark, USA, Netherlands, Belgium, Germany, United Kingdom, Italy, Kuwait, France, Finland, Slovakia, Austria, Turkey, Czech Republic, Greece, Norway). Forty clinics (43 instruments) currently use the EmbryoScope for human clinical treatments, a total of approximately 3760 human clinical cycles have been performed using the EmbryoScope time lapse system, and the first babies after incubation in the EmbryoScope have been born. All customers and test sites have continued to use their EmbryoScope, and several have decided to purchase more instruments. Early 2011 the first clinic (Maigaard Fertility Clinic, DK) decided to switch completely to incubation in the EmbryoScope for all their fertility treatments. Is the EmbryoScope Embryo Monitoring System approved (CE mark, FDA)? We received our ISO13486 and CE marking as a Class IIa Medical Device on June 29, We have also received FDA 510(k) clearance for embryo cultivation in January How is the EmbryoScope tested prior to use? All individual instruments are subjected to a series of functional tests during assembly recording more >100 parameters that all have to perform within specifications. The final test is a mouse embryo assay using 60 thawed mouse zygotes in 6 EmbryoSlides where we check development to fully expanded blastocysts in 96 hours with a minimum requirement of 80% expanded blastocysts. The complete test procedure of each individual EmbryoScope is part of our CE mark approval. In 2010, we used cell mouse embryos with an average expanded blastocyst rate of 94% in all our QC experiments. We keep detailed records of the performance of every single EmbryoScope. All materials in contact with the internal gas flow are tested in mouse embryo toxicity assays, assessing development from 1 cell to expanded blastocysts in 96 hrs by incubating the embryos with large amounts of the material in a small enclosure throughout the development. We have chosen quality components for the instrument e.g. the use of German specialty motors developed for high energy physics experiments where it is essential to use non volatile vacuum lubricants. Do you have clinical evaluation trials? The EmbryoScope has been evaluated clinically since June 2009 at IVI Valencia and Alicante (IVI is Europe s largest chain of fertility clinics with more than 22,000 IVF treatments per year). Their initial outcome evaluation included 403 IVF treatments resulting in 203 clinical pregnancies (confirmed by presence of gestational sac in ultra sound scan on week 6). The resulting pregnancy rate of 50% was obtained without using any time lapse parameters for embryo selection but using only standard evaluation protocols based on images at four discrete time points). The purposes of this evaluation were; i) to evaluate the EmbryoScope as an embryo incubator and make sure that embryo development was similar to standard incubators, ii) to generate image data on embryo development to derive novel selection parameters for upcoming prospective studies. A clinical trial has been completed May 2011 (Clinical Trials gov. identifier: NCT ) comparing the embryo development in the EmbryoScope time lapse system with embryo development in a conventional IVF incubator. Two Danish clinics (Rigshospitalet and Skejby Sygehus) were enrolled in the study. Embryos were randomized by block randomization and evenly distributed in either the standard incubator or the EmbryoScope (split cycles). Embryo evaluation was based on discrete time points, thus time lapse information was not utilized for embryo selection. Embryo scoring, selection for transfer and freeze was blinded for the laboratory technician. Results from this study will be presented at the ESHRE Conference 2011, Stockholm. A retrospective study comparing embryo development in the EmbryoScope and in a standard incubator was presented at the annual meeting of the ASRM The clinical pregnancy rate after incubation in the EmbryoScope TM was 54% (66 out of 123) compared favorably to the matched control group where it was 47% (228 out of 481) but the difference was not significant. It should be noted that the time lapse information was NOT used to select embryos incubated in the EmbryoScope, as the purpose of the study was to evaluate embryo development. 4
5 A validation study has been conducted at the Erasmus MC, University Medical Center, to validate the performance of the EmbryoScope, by comparing the development of surplus embryos in the EmbryoScope to development in a conventional incubator. Surplus embryos were collected from patients undergoing normal IVF treatments. Embryos not selected for transfer or freezing on day 3 and 4, respectively, were pooled and randomly divided into two groups at a low magnification to avoid a selection bias. It was estimated, based on previous data that approximately 5% of these poor quality embryos would develop to an expanding blastocyst. As a secondary parameter, survival rate of embryos were assessed. Survival rate was defined as a continued increase in cell number and limited fragmentation evaluated at defined time points, i.e. 120 h, 144 h and 168 h after insemination. Results from this study will be presented at the ESHRE Conference 2011, Stockholm. The abstract is pre selected for the ART Laboratory Award. Do you have published papers? The first peer reviewed paper on embryo quality, blastocyst and ongoing pregnancy rates from embryos incubated in the EmbryoScope are now available online in J Assist Reprod Genet (Cruz et al. 2011). A manuscript on the use of morphokinetics as a predictor of embryo implantation is recently accepted with minor changes in Human Reproduction. In 2010 there were 6 abstracts presented to ESHRE, Rome, and 8 presented to ASRM, Denver, showing results from research projects involving the EmbryoScope. This year to ESHRE, Stockholm, 11 abstracts based on data from the EmbryoScope will be presented. Furthermore, there are at least 10 abstracts submitted to ASRM 2011, Orlando, where 3 abstracts presents different aspects of automated image analysis. In addition, we can provide a list of peer reviewed publications describing correlations between morphokinetic parameters such as e.g. early cleavage, divisional timings, synchrony, fragmentation and ongoing pregnancy rate. All morphokinetic parameters can easily be followed using the time lapse image series acquired by the EmbryoScope, please contact us for our reference list and material about morphokinetic analysis of embryo development. Clinical use of the system How is it to use the EmbryoScope in a clinical setting? Users at IVI s fertility clinics find that the technology ultimately provides valuable flexibility in the daily work of the clinic because the embryologists freely can choose when to evaluate embryo development, and can easily consult other specialists if necessary. How many patients can an EmbryoScope handle? The EmbryoScope can hold up to 6 EmbryoSlides with embryos from six different patients. Each slide can hold up to 12 embryos; consequently the EmbryoScope can acquire time lapse images of up to 72 embryos sequentially within 20 min. We estimate that the EmbryoScope time lapse system can handle between 300 and 500 IVF treatment cycles per year per EmbryoScope instrument, depending how many days you culture. How to load embryos in the EmbryoSlide? Embryos are handled with normal fine tip pipette (e.g. Stripper tips) under a normal dissection microscope. You can use whatever medium you are using today and the time to load a slide is comparable to the time it takes to prepare a standard culture dish once you have passed the learning stage. However, as this is a new method, you have to allow for some time in the beginning to learnto use the system. Two day on site training is provided by Unisense FertiliTech as part of the installation procedure for new customers. Can you grow to blastocyst in the EmbryoScope? Yes, you can grow the embryos for as long as you would in a standard incubator. How often do you need to change media? You can change media as often as you like. In general, most IVF clinics change media at day 3 for culturing to day 5. However, always follow recommendations from the media manufacturer. 5
6 How do you handle media change? Media change can be performed efficiently without moving embryos to a new slide. Each embryo resides in a central inner microwell in an outer larger well. Media can be changed by using a fine tip pipette and removing spent media from the outer well, while the embryo remains safely in the inner well. New media can then be added to exchange 80 90% of the original media. How to perform independent validation and QC of incubation conditions? Operators are strongly encouraged to run independent validations of incubation conditions every week. The EmbryoScope software is briefly interrupted to insert a QC temperature probe into the slide holder to measure the core temperature. A gas valve allows sampling of the internal air from the embryo chamber. Is ph measurement possible? It is important to validate ph of the media at regular intervals. Precise calibration of CO 2 and temperature will result in a stable accurate media ph. Validation of media ph can be performed by placing a petri dish with a media sample and oil cover inside the embryo chamber of the EmbryoScope and leave it for equilibration for 24 hours (see explanation in our FertiliTechNote: Carbon dioxide and ph validation for IVF media in the EmbryoScope ). Technical details What about incubation conditions? The EmbryoScope is a state of the art tri gas incubator, with continuous recycling of the internal gas volume through filters to remove potentially harmful VOC and particles. Internal carbon dioxide concentration is measured with a high quality IR sensor, and the Oxygen with a state of the art amperometric sensor. Oxygen and carbon dioxide concentration is regulated by mixing CO 2 and N 2 into the internal airstream. The tri gas mixer ensures a stable gas composition and a low consumption. Proportional gas regulation ensures a fast response to openings of the load door so incubation conditions are rapidly restored (< 5 min for CO 2 and <15 min for O 2 regulation). Direct thermal contact between the EmbryoSlide and the slideholder that is made of a high heat conductance aluminum alloy ensures that the temperature is stable and virtually unperturbed in case of load door openings. All together this gives exceptionally stable incubation conditions with clean controlled gas and constant temperature for the incubation. How about air quality and contamination? Internal circulation of the air inside the EmbryoScope through a volatile organic compound (VOC) filter, a high efficiency particulate air (HEPA) filter and an ozone free 254 nm UV light illumination box (EU only) ensures a highly controlled, clean and stable air supply. The internal air volume is approximately 20 L and the flow rate is >120 L/hr, so all air passes through the cleaning filters several times per hour. The UV illumination decontaminates any microorganisms attached to airborne particles in the gas stream and the HEPA VOC filter retains them. The growth environment is non humidified which eliminates the problems with fungal growth and contamination that can arise in humidified environments. Why is this a non humid environment? The risk of fungal and bacterial contamination is greatly reduced in a non humid environment. The EmbryoSlides are not permeable to water vapor. When working with oil cover it is not necessary to incubate in a humid environment as evaporation is barely detectable (< 1% of volume per day). There is no need to replenish water reservoirs and accidental condensation of water vapor within the instrument is eliminated. What about light exposure? Low intensity light from a single LED is used to acquire images with very short exposure time (typically sec. per image) and only using low energy red light (635 nm) to eliminate any possible damage to the embryo due to image acquisition. The total light dose to which the embryo is exposed has been measured with a light microsensor and was found to be substantially lower than the dose used in conventional IVF procedures with 6
7 normal microscopes (see FertiliTechNote; Exposure to light during image acquisition in the EmbryoScope time lapse system). Intensity measurements with a normal light sensor are inadequate because the sensor area is too large and thus not able to discern between light systems whose lenses focus all light on a smaller or on a larger area. The relevant intensity measurement (W/m 2 ) must be made with a light sensor with the adequate spatial resolution. When evaluating embryonic light exposure in IVF it is essential to use a scalar irradiance microsensor with the same dimensions as an embryo. A scalar irradiance microsensor is a tapered optical fiber with a light diffusion sphere at the tip, so that incident light from all angles are measured including reflected light etc. Only by using a microsensor with the same dimensions as an embryo placed in the same spot as the embryo, is it possible to obtain a true recording of the incident light hitting the embryo in different experimental setups. Detailed description of the illumination system used in the EmbryoScope The imaging system in the EmbryoScope uses low intensity red light (635 nm) from a single LED with short illumination times of 40 ms per image or less (typically 15 ms) to minimize embryo exposure to light and to avoid damaging short wavelength light (Takenaka et al. 2007; Oh et al. 2007; Ottosen et al. 2007). The optics use modified Hoffmann modulation contrast with a 20x speciality Leica objective to provide optimal light sensitivity and image resolution using the red wavelength. Embryo exposure to light during incubation was measured with a scalar irradiance microsensor with a tip diameter of 100 µm placed within the EmbryoScope at the position of the embryo in the EmbryoSlide. Similar measurements were made on standard microscopes used in fertility clinics. The total exposure time in the time lapse system during 3 day culture and acquisition of 1420 images were 57 seconds, which compares favourably with the 167s microscope light exposure time reported for a standard IVF treatment (Ottosen et al. 2007). As the light intensity measured within the EmbryoScope with the scalar irradiance microsensor was much lower than the light intensity in microscopes used in IVF clinics, we found the total light dose during 3 day incubation in the time lapse system to be 20 J/m 2 (i.e µj/embryo) as opposed to an exposure of 394 J/m 2 during microscopy in normal IVF treatments (i.e. 4.8 µj/embryo) based on average illumination times from (Ottosen et al. 2007) and measured average intensities with the scalar irradiance microsensor. Furthermore, the spectral composition of the light in the EmbryoScope was confined to a narrow range centered around 635 nm, and thus devoid of low wavelength light below 550 nm, which has been shown to be inhibitory to embryo development (Takenaka et al. 2007, Oh et al. 2007) and comprise about 15% of the light encountered in a normal IVF microscope. The internal optics are very light sensitive and designed to work with illumination from a single red LED providing light at 635nm. The discrete wavelength provided by the LED means that the wide spectral composition of light used in a standard microscope, including damaging light shorter than 550nm is totally eliminated. What about the culture dish (EmbryoSlide )? The EmbryoSlide is CE approved (Type IIa) and FDA cleared 510k for human clinical use. It is composed of medical grade polystyrene and tested by exactly the same tests as other IVF approved culture dishes like the Nunc 4 well dish for IVF. All production lots are QC tested by external companies according to approved practices for: i) Sterility (ISO11137 with SAL 10 6 ), ii) Cytotoxicity (USP method <87> and ISO ); iii) Non pyrogenic (< 20 Endotoxin units/device) and iii) Embryo toxicity (Tested with mouse zygote development to fully expanded blastocysts in 96 hrs. The acceptance criterion is 80% expanded blastocysts). A certificate to this effect is included with all shipments of the EmbryoSlide. Support and service What about accidents and system failures? The EmbryoScope is equipped with two audible alarm systems and a CTS system that can convey the emergency signal to any standard pre existing external alarm system. External alarm systems that can be configured to notify personnel by phone, SMS or E mail according to the needs of the clinic must be purchased and installed separately. The error conditions that activate the alarm system comprise deviations in: temperature, CO 2 concentration, CO 2 pressure, O 2 concentration, O 2 pressure, external power, prolonged opening of the load door, software errors, etc. 7
8 What about support and service? Under our service agreement, we provide free service telephone hotline 24/7 and software updates on a regular basis. Each user can choose to upload anonymous data to our servers for free quality checks to detect deviations in running parameters (e.g. readings from eight internal temperature sensors, current consumptions of subcomponents, fan operations). At installation we provide 2 days of extensive training on site. Service visits, scheduled every 6 months, are performed by qualified technicians for validation and recalibration. What happens in case of power failure? The EmbryoScope must ALWAYS be connected to a battery operated uninterrupted power supply (UPS) to ensure uninterrupted image acquisition and to allow IVF personnel time to re establish power if possible. In the very rare case of both power failure and failure of the UPS the EmbryoScope will stop taking images and temperature and gas will equilibrate with the environment. In such a situation, the EmbryoScope can be opened manually for retrieval of the EmbryoSlides. The EmbryoScope is normally automatically locked to avoid accidental opening of the load door during motor movement. How fast does the temperature drop if you have a power failure? The temperature drops by 2 degrees in 10 minutes and we therefore require that the EmbryoScope is connected to an online battery operated by an uninterrupted power supply (UPS). Embryo respiration Does the EmbryoScope measure respiration rate? Measurement of embryo respiration rate is not a feature of this instrument. In a previous research only version (The EmbryoScope C ) did measure respiration rates, and there is several abstracts presenting results from research on embryo respiration rate. Why did you not continue with respiration technology? We recognized the untapped assessment potential that can be realized with repiration rate observations. The developmental pattern of cell divisions appears to relate to embryo viability and we designed the current instrument acknowledging that this was an easier instrument to certify and to use in a clinical setting. Will you continue with respiration instruments? We will likely resume development of a clinical respiration rate measurement instrument in the future. Unisense FertiliTech history How did it all start? Unisense FertiliTech A/S is a small University spin off company that made advanced research systems based on electrochemical microsensors. We initially made systems to quantify oxygen consumption of oocytes and preimplantation embryos. For these instruments we developed time lapse image analysis systems to observe and quantify cellular divisions in animal model systems. We developed the first fully automated time lapse systems capable of predicting bovine blastocyst development based on time lapse images of early development. We presented results that demonstrated that the accuracy of the developed algorithms were better that the predictions made by trained embryologists with access to the same images. These results were presented at ASRM in 2006 and The proprietary algorithms and original observation that novel parameters such as duration of cell division and interval between cell divisions are indicative of embryo viability form the basis of the selection criteria we will employ in the future prospective studies. 8
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