Frequently Asked Questions EmbryoScope time-lapse system

Frequently Asked Questions EmbryoScope time-lapse system The Vision to Conceive www.fertilitech.com CE Class IIa Medical Device FDA 510(k) cleared for clinical use

Contents General 3 What is the EmbryoScope time-lapse system? 3 How does it work? 3 Safety and clinical results 3 How many clinics use the EmbryoScope time-lapse system? 3 Is the EmbryoScope time-lapse system approved (CE mark, FDA)? 3 How is the EmbryoScope tested prior to use? 3 Is the EmbryoScope time-lapse system safe for embryos? (CE mark, FDA)? 3 What are the experiences of using the EmbryoScope time-lapse system in a clinical setting? 4 How many patients can be treated in the EmbryoScope? 4 How are embryos transferred to the EmbryoSlide? 4 Can embryos be cultured to blastocyst in the EmbryoScope? 4 Which culture media can be used? 4 How is a media changed where necessary? 4 How are external validation of incubation conditions performed? 4 Is ph measurement possible? 5 Can patient data be viewed from a remote computer? 5 Research of improved embryo selection 5 Do you have a list of publications documenting the use of the EmbryoScope time-lapse system? 5 Have novel parameters which improve embryo selection been reported? 5 How do you facilitate sharing of knowledge between users? 5 Technical details 6 What type of incubation conditions are provided? 6 How many focal planes are acquired? 6 What is the frequency of image acquisition? 6 How is air quality and contamination controlled? 6 Why use a non-humid environment? 6 How was safety of light exposure to the embryo tested? 6 What imaging system is used in the EmbryoScope time-lapse system? 7 What kind of culture dish is used? 7 Support and service 7 What is the process for installation? 7 Can system alarms be connected to existing alarm systems? 7 Does the instrument require maintenance? 7 What precautions must we take in case of power failure? 7 What are the consequences of a power failure? 7 2

General What is the EmbryoScope time-lapse system? The EmbryoScope is a certified tri-gas IVF incubator with a built-in camera developed to improve IVF treatments. The instrument is the result of more than 9 years of research and uses several proprietary technologies; it was approved for clinical use in the EU in June 2009 and was cleared by FDA (510k) in January 2011. How does it work? Hoffman modulation images of individual embryos are automatically recorded at preset time intervals. These images are compiled using specialised software to create a time-lapse sequence of embryo development. Detailed information about timing of cell divisions, intervals between cell cycles and other important events including multinucleation, evenness of blastomeres and dynamic pronuclei patterns are annotated for analysis. This information is used by the embryologists to select the embryo that can be most likely to result in a pregnancy for transfer in an IVF treatment. Images are acquired without removing embryos from the incubator, and therefore manual handling of embryos outside the stable incubation conditions is minimised, leaving the embryos undisturbed at optimal growth conditions. Images and detailed information about incubation conditions are stored in patient data files for future reference. Safety and clinical results How many clinics use the EmbryoScope time-lapse system? The system has been widely adopted worldwide. More than 200 clinics are using this technology, many of which can be found on our website Find an EmbryoScope clinic feature to locate an EmbryoScope clinic near you (www.fertilitech.com). Please note that not all EmbryoScope clinics are listed on the website. Is the EmbryoScope time-lapse system approved (CE mark, FDA)? All systems are manufactured in accordance with ISO13485 and have been CE-marked as a Class IIa Medical Device since 29 June 2009. FDA 510(k) clearance of the EmbryoScope time-lapse system was obtained in January 2011. How is the EmbryoScope tested prior to use? Each individual EmbryoScope time-lapse system is subjected to a series of functional tests during assembly, recording >100 parameters that all have to perform within specification. The final test is a mouse embryo assay using 60 thawed mouse zygotes where development to fully expanded blastocysts is checked over a period of 96 hours with a minimum requirement of 80% expanded blastocysts. Currently, approximately 11,500 1-cell mouse embryos per year are used with an average expanded blastocyst rate of 93% in all our QC experiments. All materials in contact with the internal gas flow are tested in mouse embryo toxicity assays, assessing development from 1-cell to expanded blastocysts over 96 hrs. Is the EmbryoScope time-lapse system safe for embryos? (CE mark, FDA)? The clinical safety of the EmbryoScope time-lapse system was evaluated starting in June 2009 at IVI Valencia and IVI Alicante in Spain 1. The conclusions state: In the current study, our aim was to demonstrate that EmbryoScope incubation conditions are comparable to standard laboratory incubation circumstances by comparing embryo quality, development and ongoing pregnancy rates between the EmbryoScope (ES) and a standard incubator (SI)... Thus we concluded that time-lapse monitoring in the EmbryoScope does not impair embryo quality while allowing for morphological, spatial and temporal analysis of embryo development. These findings have been confirmed in a second prospective clinical trial conducted by Kirkegaard et al 2 comparing development in a standard incubator and in the EmbryoScope time-lapse system. Primary end-point was number of 4 cell embryos on day 2, and secondary endpoints was the proportion of 7-8 cell embryos on day 3 and blastocysts on day 5. 3

In both studies, time-lapse was not used for embryo assessment and embryos were evaluated by removal from the incubator to assess quality. These studies were conducted to validate that under similar evaluation conditions, incubation and image acquisition in the EmbryoScope did not present a risk to embryo quality. 1 Cruz, M., Gadea, B., Garrido, N., Pedersen, K.S., Martínez, M., Pérez-Cano, I., Muñoz, M. and Meseguer, M. (2011) Embryo quality, blastocyst and ongoing pregnancy rates in oocyte donation patients whose embryos were monitored by time-lapse imaging J Assist Reprod Genet 28(7): 569-573 2 Kirkegaard K., Hindkjaer J. J., Grøndahl, M. L., Kesmodel, U. S. and Ingerslev H.J. (2012) A randomized clinical trial comparing embryo culture in a conventional incubator with a time-lapse incubator J Assist Reprod Genet 29(6): 565-572 What are the experiences of using the EmbryoScope time-lapse system in a clinical setting? Users at current fertility clinics find that the technology ultimately provides valuable flexibility in the daily routines of the clinic, because embryologists are free to carry out evaluations of embryo development at times that suit them and can easily consult other specialists or colleagues if necessary. Embryologists are able to use more time evaluating embryos, but less time with physical processes such as removing and replacing from the incubator and taking manual notes. How many patients can be treated in the EmbryoScope? The EmbryoScope can hold six EmbryoSlides with embryos from up to six different patients. Each EmbryoSlide culture dish can accommodate up to 12 embryos; consequently the EmbryoScope can acquire time-lapse images of up to 72 embryos. The EmbryoScope time-lapse system has a capacity of 300 400 IVF treatment cycles per year per EmbryoScope instrument, depending on the duration of culture. How are embryos transferred to the EmbryoSlide? Embryos are handled with normal fine-tip pipettes (e.g. Stripper tips) under a normal dissection microscope. The time to load an EmbryoSlide is comparable to the time it takes to prepare a standard culture dish once you have passed the learning stage. Each well in the EmbryoSlide contains 25 µl medium and the 12 wells are covered with a confluent oil layer (1.2 ml). Two days on-site training is provided by a FertiliTech instructor as part of the installation procedure for new customers. Can embryos be cultured to blastocyst in the EmbryoScope? Yes, embryos can be cultured to day 5. Which culture media can be used? Culture in the EmbryoScope time-lapse system does not set specific requirements for media. Therefore, you may continue to use your preferred culture media. Media can be changed as often as you like. In general, most IVF clinics change the medium on day 3 for culturing to day 5. Always follow recommendations from the manufacturer of the specific medium. How is a media changed when necessary? A change of medium can be performed efficiently without moving embryos to a new EmbryoSlide. Each embryo resides in a central inner microwell (approximately 20nL) within an outer larger well of 25 µl. The medium can be changed by using a fine-tip pipette and removing the spent medium from the outer well, while the embryo remains safely in the inner well. The new medium can then be added to replace 80 90% of the original medium. Alternatively, the embryos can be transferred to a new EmbryoSlide culture tray. How are external validation of incubation conditions performed? Operators are required to run independent validations of incubation conditions. The responsibility of external validations rests upon the respective clinics. For external temperature validation, the EmbryoScope software is briefly interrupted to insert a temperature probe into the slide holder to measure the precise core temperature. For external validation of CO 2 and O 2, a gas valve allows sampling of the internal air from the embryo chamber. 4

Is ph measurement possible? It is important to validate ph of the medium at regular intervals. Precise calibration of CO 2 and temperature will result in a stable accurate ph of the medium. Validation of ph can be performed by placing a Petri dish with a medium sample and oil cover inside the embryo chamber of the EmbryoScope and leave to equilibrate for 24 hours (see explanation in our FertiliTechNote: Carbon dioxide and ph validation for IVF media in the EmbryoScope ). We are currently in the process of developing improved procedures for ph measurements. Can patient data be viewed from a remote computer? We are currently working on a software solution to provide EmbryoScope users with easy and safe remote access to their time-lapse embryo images and running conditions. This will facilitate off-site access to embryo development and incubation conditions. Research of improved embryo selection Do you have a list of publications documenting the use of the EmbryoScope time-lapse system? There are many publications and abstracts available documenting the use of this system. Please refer to our Science page at www.fertilitech.com where you will find a list of references, publications and other scientific material. Have novel parameters which improve embryo selection been reported? Evidence from the literature indicates that certain morphokinetic parameters may be strong indicators for embryo deselection. One of these which appear to be particularly clinically relevant is deselection of embryos which develop directly to 3 cells or proceed from first to second division in less than 5 hours 3. More recently, Campbell et al 4 reported that annotating the time from insemination to start of blastulation and time from insemination to blastocyst formation may be used to categorize aneuploidy risk. However, it is becoming apparent that embryo development kinetics may vary between clinics, and that using cell division timings must be independently validated at each clinic to be implemented successfully. Please refer to our list of Relevant Abstracts and Publications on our website at www. fertilitech.com. 3 Rubio, I., Kuhlmann, R., Agerholm, I., Kirk, J., Herrero, J., Escriba, M.J., Bellver, J. and Meseguer, M. (2012) Limited implantation success of direct-cleaved human zygotes: A time-lapse study Fert Steril doi.org/10.1016/j.fertnstert.2012.07.1135 4 Campbell, A., Fishel, S., Bowman, N., Duffy,S., Sedler, M., and Hickman, C.F.L. (2013) Modelling a risk classification of aneuploidy in human embryos using non-invasive morphokinetics Reprod Biomed Online 26: 477-485 How do you facilitate sharing of knowledge between users? We arrange interactive workshop for users of the EmbryoScope time-lapse system. Our EmbryoScope User Workshops include presentations of results and ongoing clinical studies (presented by EmbryoScope users) and hands-on exercises on embryo selection using time-lapse information. The primary aim of our User Workshop is to facilitate discussion, collaboration and exchange of ideas and results between EmbryoScope users. In addition, we arrange courses on statistical methods suitable for the analysis of time-lapse data for our EmbryoScope users. We also arrange numerous smaller workshops and symposia in different countries. 5

Technical details What type of incubation conditions are provided? The EmbryoScope is a benchtop style tri-gas incubator. Continuous recycling of the internal gas volume through filters removes potentially harmful VOC and particles. Internal carbon dioxide concentration is measured with a high-quality IR sensor and the oxygen with an amperometric sensor. Oxygen and carbon dioxide concentrations are regulated by mixing CO 2 and N 2 into the internal airstream (it is not possible to use pre-mixed gas). 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 slide holder ensures that the temperature is stable and virtually unperturbed in situations of load door openings. All told, this results in stable incubation conditions with clean controlled gas and a constant temperature for the entire incubation period. How many focal planes are acquired? Images of each embryo are acquired in 1, 3, 5, 7 or 9 equidistant focal planes. In general, most clinics acquire time-lapse videos of embryo development in 7 focal planes. The distance between the focal planes can be changed as required by the user in the software. What is the frequency of image acquisition? Acquisition of images in 7 focal planes of 72 embryos is acquired approximately every 10 minutes. How is air quality and contamination controlled? Internal circulation of the air inside the EmbryoScope passed through a volatile organic compound (VOC) filter, a high-efficiency particulate air (HEPA) filter. An ozone-free 254 nm UV-light illumination lamp (EU only) further ensures a purified and clean air supply. The total air volume is approximately 20 L (the incubation area 3 L) and the flow rate is >120 L/hr, so all air passes through the cleaning filters several times an hour. The UV illumination kills any microorganisms attached to airborne particles in the gas stream and the HEPA VOC filter retains them. Why use 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 vapour. Covering culture medium droplets with an oil film a humid environment unnecessary as evaporation is barely detectable (< 1% of volume per day). There is no need to replenish water reservoirs and accidental condensation of water vapour within the instrument is eliminated (see abstract from ASRM 2011, 96(3) Suppl, Okimura et al Efficiency of dry culture system in human blastocyst culture ). How was safety of light exposure to the embryo tested? The total embryo light exposure has been measured with a light microsensor and was found to be substantially lower than in conventional IVF procedures with normal microscopes. Total exposure time in the EmbryoScope time-lapse system over three days of culture and acquisition of 3024 images is 136s which compares favorably with the 167s microscope light exposure reported for a standard IVF treatment (Ottosen et al 2007). (see FertiliTechNote; Exposure to light during image acquisition in the EmbryoScope time-lapse system). 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 fibre with a light-diffusion sphere at the tip, so that incident light from all angles is measured - including reflected light. Only by using a microsensor with the same dimensions and shape as an embryo and placed in the embryo s position, is it possible to obtain a true recording of the incident light hitting the embryo in different experimental setups. Furthermore, the spectral composition of the light in the EmbryoScope was confined to a narrow range and thus devoid of low wavelength light below 550 nm, which has been shown to be inhibitory for embryo development (Takenaka et al 2007; Oh et al 2007) and makes up about 15% of the light encountered in a normal IVF microscope. 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. 6

What imaging system is used in the EmbryoScope? The imaging system in the EmbryoScope uses internal optics which are very light-sensitive and designed to work with illumination from a single red LED providing light at 635nm. 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. What kind of culture dish is used? The EmbryoSlide is a CE-marked Type IIa Medical Device 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. 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 10993-5); iii) Nonpyrogenic (< 20 Endotoxin units/device) and iv) Embryo toxicity (tested with mouse zygote development to fully expanded blastocysts over 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 is the process for installation? As soon as a clinic orders an EmbryoScope, FertiliTech takes over the management of all logistics and planning related to the installation. The physical and technical installation and training of the laboratory staff is undertaken by a skilled FertiliTech instructor in an intensive two-days training session. Can system alarms be connected to existing alarm systems? The EmbryoScope system 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 staff 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, N 2 pressure, external power, prolonged opening of the load door, software errors. Does the instrument require maintenance? Under our service agreement we provide a service telephone hotline 24/7 and software updates on a regular basis. Service visits, scheduled every 6 months for validation and recalibration, are performed by qualified technicians. What precautions must we take in case of power failure? The EmbryoScope must be connected to a battery-operated uninterrupted online power supply (UPS) to ensure uninterrupted image acquisition and to allow IVF personnel time to re-establish power. If needed, the EmbryoScope can always be opened manually for retrieval of embryos. What are the consequences of a power failure? The temperature drops by 2 degrees every 10 minutes and we therefore insist that the EmbryoScope system should be connected to an online, battery-operated uninterrupted power supply (UPS). The CO 2 and O 2 concentrations remain stable for a long period, similar to a conventional incubator, if the EmbryoScope system remains closed. 7

Unisense FertiliTech A/S, Denmark www.fertilitech.com info@fertilitech.com F7.797.5 (Ver2) (13.06.2013) EU - FertiliTech A/S