System and method for in vitro monitoring of embryos

Abstract

The system (100) and the method according to the invention are used for in vitro monitoring of embryos. The system (100) includes an incubator (110), an oxygen tank (162) and a carbon dioxide tank (164) connected to the incubator (110) through a gas control unit (160); a unit (170) for controlling the climate of the interior of the incubator (110); and at the bottom of the incubator (110) at least one dish (112) for storing embryos (140). The system (100) further comprises a light-transmitting lens system arranged under the incubator (110), made of heat- insulating material and coupled to the bottom of at least one dish (112) from outside, and consisting of an objective lens and a tube lens under the objective lens; a high-sensitivity photon detector (120) arranged under the lens system and coupled to the lens system; and a computer (130) connected to the photon detector (120).

Description

[0001] System and method for in vitro monitoring of embryos

[0002] The invention relates to a photon emission system and a method for testing the development and viability of living embryos on the basis of spontaneous photon emission.

[0003] In developed societies, the number of couples struggling with infertility problems has been continuously increasing in recent decades, while the importance of assisted reproductive technologies has also been continuously increasing. Among these procedures, the in vitro fertilization (IVF) has become a routine procedure in clinical practice over the past four decades, and has undergone significant development since its introduction.

[0004] In most cases, efforts were made to improve the efficiency of IVF technologies by increasing the number of implanted embryos. During stimulated cycles, it is usually possible to fertilize a plurality of oocytes, which enables the formation and implantation of a plurality of embryos, thereby increasing the chance of pregnancy. At the same time, multiple embryo transfer significantly increases the risk of multiple pregnancies. Multiple pregnancies are among the most common causes of preterm birth, along with an increased risk of prematurity. In order to avoid this risk, it would be important to select the most viable and implantable embryo.

[0005] At the same time, a very important ethical and even more practical question arises: what method should be used to select the embryo to be transferred? The most widely used selection method is based on the examination of the morphology of the embryo, the examination ranges from the simple use of a microscope to the time-lapse technique. The latter technique provides important information not only on the morphology of the embryo, but also on the dynamics of embryo development. The time-lapse technology created the concept of stable culture conditions with the possibility of continuous observation and documentation of the embryo. However, it is a problem that this selection technique involves exposing the embryos to visible light.

[0006] Early embryonic development is characterized by rapid cell division and the activation of embryonic genes, which makes the embryo extremely vulnerable and sensitive to environmental effects. The ovary produces oocytes and the female body provides a safe environment for gametes and embryos. The human body protects itself against visible light with well-defined mechanisms. During the use of IVF and especially Intracytoplasmic Sperm Injection (ICSI) as a fertilization method, light exposure can have a harmful effect on cells during the preparation of oocytes and sperm, embryo incubation and microscopic examination, as well as during embryo transfer.

[0007] The harmful effects of ultraviolet (UV) light on living cells have been known for a long time. However, the toxic effect of visible light (400-700 nm) is less well known, despite several previous studies confirming the negative effects of visible light on oocytes, sperm and embryos. The harmful effect of light can be related to hydrogen peroxide formed in peroxisomes and mitochondria. Light can induce stress gene activation and DNA damage in the embryos. The damaging effect of the visible light depends on the wavelength of the light. Blue light (400-500 nm) is proven to be orders of magnitude more dangerous than visible light with longer wavelengths.

[0008] In orderto protect the sperm cells, the oocytes and the embryos during IVF procedures, a dark environment is provided to eliminate the potentially harmful effects of light, and the laboratory lights, as well as the built-in light sources of the microscope and the IVF workstation, are equipped with a red color filter.

[0009] It is well known from the literature that living cells (plant, animal and human cells) emit spontaneous ultra-weak photon emission (UPE). The UPE intensity can vary from a few photons per second to hundreds of photons per square centimeter, mainly in the 200-800 nm spectral range. It has been shown that the origin of UPE is directly related to free radicals (reactive oxygen species, ROS). Changes in UPE intensity are associated with various physiological and pathological conditions. Such pathological conditions include thermal, chemical and mechanical stress, changes in the mitochondrial respiratory chain, changes in the cell cycle, cancerous growth, etc.

[0010] The patent EP3455340 discloses an apparatus for incubating living biological material, such as an embryo, wherein the apparatus comprises a box with a closed but openable door, which comprises two or more cell culture holding units, a video camera and a FLIM (fluorescent lifetime imaging microscope) type microscope unit, wherein the camera and FLIM unit or a part of them can be moved relative to the cell culture holding unit. The camera is used to determine some predetermined morphological characteristics of the biological material. During the examination of the biological material, stimulation is used for the photon emission performed by the cells. The disadvantage of the above system is that neither light energy, nor chemical substances can be used when examining the embryos in order to increase the protection of the embryo, that is, the planned examinations must be carried out without the input of external energy.

[0011] It is therefore an object of the invention to provide an embryo examination system and a method that allows the photoemission measurement of embryos to be carried out during the incubation phase without factors affecting the development of the embryos, i.e. without external energies.

[0012] Another object of the invention is to provide a proper connection between the photon detector used for in vitro monitoring of embryos and the incubator storing the embryos, so that the embryos are always in ideal environmental conditions in the incubator during the test.

[0013] The invention is based on the recognition that if the photons spontaneously emitted by living cells, such as embryos, are detected with a high-sensitivity detector, qualitative information about the vital functions and viability of the examined cells can be examined based on the change in photon emission over time. It has been also realized that the detection of the photon emission of the embryos can be done by inserting a lens made of a photon-permeable material between the incubator and the detector.

[0014] The above objects are achieved by providing a system according to claim 1 and a method according to claim 5. Preferred embodiments of the system and the method according to the invention are defined by the dependent claims.

[0015] The invention will be described in detail below with reference to the drawing. In the drawings:

[0016] - Figure 1 is a functional block diagram of the system according to the invention;

[0017] - Figure 2 schematically illustrates the assembly of the incubator and photon detector forming parts of the system according to the invention;

[0018] - Figure 3 is a flow diagram illustrating the main steps of the method according to the invention;

[0019] - Figure 4 is a graph obtained as a result of the experiment with the system according to the invention, which illustrates the change in the photon emission of normal living embryos, living embryos produced from freezing and the background over time. The main functional units of the system 100 according to the invention are illustrated in the block diagram shown in Figure 1. The system 100 comprises an incubator 110, a high- sensitivity photon detector 120 and a computer 130 for image and data processing.

[0020] The incubator 110 comprises at least one light-transmitting dish 112, in each of which one embryo 140 is placed. The photon detector 120 detects the photon beams 150 originating from the ultra-weak spontaneous photon emission of the embryos 140. The detected signals of the photon emission are transmitted by the photon detector 120 to the computer 130, which produces information on the vital functions of the embryos and their viability based on the changes of the detected signals as a function of time.

[0021] An ideal environment for the embryos 140 must be provided inside the incubator 110. To this end, an oxygen tank 162 and a carbon dioxide tank 164 are connected to the incubator 110 via a gas control unit 160. The gas control unit 160 has the function to provide the desired oxygen and carbon dioxide concentration in the incubator 110.

[0022] In a preferred embodiment of the system 100 according to the invention, the operation of the gas control unit 160 is controlled by the computer 130, that is, the desired values of the gas concentrations, or even their change over time, can be set with the computer 130.

[0023] An air conditioning unit 170 is also connected to the incubator 110 for keeping the temperature and humidity of the incubator's interior space at a desired value. For example, a temperature of 37 °C and a humidity of 100% are to be maintained inside the incubator 110 during the measurements.

[0024] In a preferred embodiment of the system 100 according to the invention, the operation of the air conditioning unit 170 is controlled by the computer 130, that is, the desired values of the temperature and the humidity of the incubator 110, as well as their changes over time, can be set with the computer 130.

[0025] A recess at the bottom of the dish 112 arranged in the incubator 110 ensures that the embryos 140 placed therein are held without displacement since the possibility of the embryos 140 moving during the photon emission test must be minimized.

[0026] The incubator 110 may be, for example, the incubator unit forming a part of the bioluminescence imaging system „LV200 Luminoview" of Olympus Inc. (Japan).

[0027] In order to successfully detect the photon beams 150 from photon emission, the photon detector 120 is to be placed as physically close as possible to the examined and monitored embryos 140, therefore, as shown in Figure 2, on the lower side of the incubator 110, the detector 120 fits tightly through an interposed lens system 180 to the bottom of the one or more dishes 112. The lens system 180 consists of an objective lens 180a and a tube lens 180b arranged below it. Since the image recording unit of the photon detector 120 should be maintained at an extremely low temperature (about -20 °C), the lens system 180 fitted between the photon detector 120 and the at least one dish 112 is made of a special material. In a preferred embodiment of the system 100 according to the invention, the photon detector 120 has a high-resolution image sensor, for example a sensor of 9 MP (megapixel) resolution. The photon detector 120 may be, for example, the ORCA-Quest camera of CMOS type of Hamamatsu Photonics Inc. (Japan), which is capable of detecting even a single photon.

[0028] In a preferred embodiment of the system 100 according to the invention, the system 100 also comprises a lighting unit directing three different colored lights, for example RGB colored lights, to the at least one dish 112. By using the lighting unit, the positioning of the one or more dishes 112 can be performed without the use of external lighting (which usually includes UV rays harmful to the embryos) before starting the measurements.

[0029] Preferably, the lighting unit is a manually controlled unit, which has to be switched off before starting the photon emission measurement so that the embryos 140 are in complete darkness during the measurement.

[0030] In the following, the main steps of the in vitro embryo monitoring method carried out using the system according to the invention are described with reference to the flow diagram shown in Figure 3.

[0031] In a first step 300 of the method, preparation of the measurement takes place, during which, among other things, the embryo to be tested is put in a cup so that the embryo is placed in the recess of the cup, the temperature of the incubator is set to 37 °C, the desired humidity is set, and the volume ratio of the oxygen and the carbon dioxide is set. As a part of step 300, the duration and other parameters of the measurement, in particular the frequency of sampling, the duration of sampling, the signal amplification of the photon detector, the sensitivity of the image sensor of the photon detector, spectral characteristics of the photon detector, electronic control of the photon detector, the amount of data expected during the entire measurement etc., may be determined through preliminary measurements.

[0032] In the next step 310 of the method, during the natural development of the examined embryo(s), the spontaneous ultra-weak photon radiation emitted by the embryo(s) are detected according to a predetermined sampling procedure by means of the detector connected to the incubator, and the detected radiation signals are transmitted in digitized form to the image and data processing computer in step 315. The data transmitted by the photon detector carries information about the amount of photons detected by the detector, as well as the physical characteristics of the captured photons (e.g. position, temporal characteristics).

[0033] In step 320 of the method, the measurement data received by the computer are continuously stored in appropriate data structures on a data storage device or in a database. The measurement data are preferably represented using the Hyperstack Bio-Formats.

[0034] At the end of the measurement, the stored data are read into the computer in step 330 of the method, and multi-stage data processing is performed on the data. During the data processing, the change in the intensity of the photon emission is determined as a function of time. If during the measurement, the photon emission of several embryos is monitored simultaneously or subsequently, the time function of the intensity is determined for each embryo in the data processing step.

[0035] Finally, in step 340 of the method, information on the viability of each embryo is generated based on one or more photon emission intensity functions determined by the computer. If, in the case of a given embryo, the time change (slope) of the entropy-weighted spectral fractal dimension (EW-SFD)-based values of the recorded images is positive, the respective embryo is considered viable, otherwise the embryo is not considered viable.

[0036] The value of the entropy weighted spectral fractal dimension was calculated using the following scheme. The measurement data were recorded in the form of image data, so those parameters were selected for the evaluation of the data that corresponded both to the high- entropy energy output of the embryo and to the image data of the sensor. The choice of a digital characteristic suitable for the measurement of entropy-based quantities was obvious based on the previously expressed expectations of the energetic processes with respect to the living organisms, as well as the data of the imaging unit used for recording digital data. This is how the choice was made to measure the entropy of information theory, for the application of which to image data some practical results have already been published in many cases (see Berke, J. (2007): Measuring of Spectral Fractal Dimension, Journal of New Mathematics and Natural Computation, Print ISSN: 1793-0057, Online ISSN: 1793-7027, 3 / 3: 409-418, DOI: 10.1142 / S1793005707000872; Berke, J. - Biro, T. - Burai, P. - Kovats, L. D. - Kozma-Bognar, V. - Nagy, T. - Tomor, T. - Nemeth, T. (2013): Application of Remote Sensing in the Red Mud Environmental Disaster in Hungary, Carpathian Journal of Earth and Environmental Sciences , Vol . 8, No. 2, pp. 49-54, ISSN 1844-489X; Berke, J. - Polgar, Zs . - Horvath, Z. - Nagy, T. (2006): Developing you Exactly Quality and Classification System for Plant Improvement, Journal of Universal Computer Science, ISSN: 0948-695X, XII / 9, 1154-1164; and Kozma- Bognar, V., Berke J. 2015. Determination of Optimal Hyper- and Multispectral Image Channels by Spectral Fractal Structure. T. Sobh and K. Elleithy (eds.) Innovations and Advances in Computing, Informatics, Systems Sciences, Networking and Engineering. Lecture Notes in Electrical Engineering (LNEE). Vol 313. pp 255-262. DOI 10.1007 / 978-3-319-06773-5_l. Springer International Publishing, Switzerland 2015).

[0037] The modern information theory concept of entropy was introduced in 1948 by Claude E. Shannon (see Shannon, C. E., A Mathematical Theory of Communication, The Bell System Technical Journal, 27:379-423, 1948; and Shannon, C. E., A Mathematical Theory of Communication, The Bell System Technical Journal, 28:623-656, 1948. ) , and then illustrated it through a practical example (see Shannon, C. E., Prediction and entropy of printed English, The Bell System Technical Journal, 30:50-64, 1951). According to this theory, the average information content (entropy) of independent digital messages can be determined as follows: where H is the information-theoretic entropy, and pi is the probability of occurrence of the i-th message (relative frequency in practice).

[0038] The definition of entropy in a mathematical sense was given by Alfred Renyi in 1961 (see Renyi, A., On measures of information and entropy, Proceedings of the 4th Berkeley Symposium Mathematics, Statistics and Probability , 547-561, 1960 .), for the measurement of which on 16-bit image data, an independent measuring program was developed.

[0039] The information-theoretic entropy of a closed system can take the following values:

[0040] 0 < H < log2n (2) where n is the number of possible messages. Entropy is the smallest when the source always sends the same message, i.e. the image shows only one color or one intensity value. Entropy takes on the highest value when the probability of all messages is equal.

[0041] In cases where, in addition to the information content (entropy), the self-similar structure of the data (fractal structure) is also of interest, it is recommended to also measure independent structural parameter(s). Since, according to the assumption of the inventors, the embryo should be treated as such an object, the fractal dimension was chosen to measure the structural parameter. All of this was justified by the inventors' previous research results obtained during the operation of living objects. However, in the present case, the values of the pixels reaching the sensor from the embryo also contain three essential elements: intensity of the photon emitted by the embryo, structure of photons emitted at different moments within the integration time, and noise.

[0042] To separate these data from a single region, a unique function (algorithm) called EW-SFD (Entropy-Weighted Spectral Fractal Dimension) has been developed.

[0043] The dimension of a fractal curve is a number that shows how the distance between two selected points of the curve increases when the resolution is increased. So, as long as the topological dimension of a line and a surface is always 1 and 2, respectively, the fractal dimension may be a value therebetween. The curves and the surfaces that exist in the real world are not fractals in fact, rather they were created by processes that can only create shapes in a specific size range. Thus, the value of the fractal dimension may change depending on the resolution. The change can help in understanding the processes characteristic to the living biological systems involved in their creation.

[0044] Several methods have been developed that are also suitable for calculating the fractal dimension (see Turner, M. T., - Blackledge, J. M. - Andrews, P. R., Fractal Geometry in Digital Imaging , Academic Press, 1998 and Rosenberg, E., Fractal Dimensions of Networks , Springer Nature Switzerland, 2020 ). These methods include the SFD method, which is a structure analysis method derived from the general fractal dimension (see Mandelbrot, B. B., The fractal geometry of nature, WH Freeman and Company, New York, 1983.), which represents a novel application of fractals. In addition to spatial structure, SFD is also suitable for measuring the color structure of spectral bands and provides sufficient information regarding the fractal properties of colors and shades. To calculate the SFD values (for two or more image bands with the same spectral resolution), the following definition of the spectral fractal dimension may be applied, calculated by simple mathematical averaging of the measured data, which are regarded as a function (number of valuable spectral boxes as a function of all spectral boxes), according to the following definition: wx

[0045] SFD^ =

[0046] 5-1 (3) where n is the number of image layers or image channels,

[0047] S is the spectral resolution in bits,

[0048] BMj is the number of spectral boxes containing a pixel of interest in case of j bits, and BTj is the total number of possible spectral boxes in case of j bits.

[0049] The number of possible spectral boxes for j bits can be calculated as follows:

[0050] BTj = (25)"

[0051] (4)

[0052] The SFDESR method defined above is a metric that satisfies the following conditions: it is nonnegative definite, symmetric and satisfies the triangle inequality. An additional condition for the metric to be satisfied is the fulfilment of the regularity condition. That is, the points of the discrete image plane should have a uniform density. In practice, before getting into the A / D converter, the image function is subject to a non-linear transformation, as a result of which the density function of the image function becomes constant. Thus, in the case of digital images, the condition of regularity is usually fulfilled or can be considered as such. Since SFDESR is a correlation metric, it can be used precisely to measure image data.

[0053] For calculating the entropy-weighted spectral fractal dimension (EW-SFD), the following measurable definition (5) of the spectral fractal dimension is used:

[0054] The number of spectral boxes is weighted by a weighting factor as follows: iog(£?r)

[0055] EW-SFD measurement

[0056] 5-1 ( 6) where n is the number of image layers,

[0057] S is the spectral resolution in bits,

[0058] SBMj is the number of spectral boxes containing pixels of interest for j bits, SBTj is the total number of possible spectral boxes for j bits, fj is the weight factor based on entropy.

[0059] The number of possible spectral boxes for j bits can be calculated according to the formula (6).

[0060] The entropy weight factor fj is calculated as follows: where maxHj is the maximum entropy for pixels of j bits,

[0061] Pk is the relative frequency of the k-th pixel in the box SBMj, and

[0062] H(SBMj) can be calculated based on the following formula for independent pixels: where n is the number of image layers,

[0063] S is the spectral resolution of the entire image in bits, j represents the values of the pixels within the box SBMj, expressed in bits, SBMj is the number of spectral boxes containing pixels of interest for j bits, SBTj is the total number of possible spectral boxes for j bits, and Pk is the relative frequency of the k-th pixel in the box SBMj

[0064] Since the signal-to-noise ratio values for spontaneous photon emission measurements are usually much lower than for stimulated photon emission measurements, in a preferred embodiment of the method, before starting the measurement, in the preparatory step 300 of the measurement, a preliminary sample-free calibration measurement is performed with the photon detector and its result is used when processing the results of the normal measurement, primarily for the purpose of filtering out measurement noises.

[0065] Example

[0066] An experimental photon emission measurement on animal embryos is presented below as an example.

[0067] 7 -week old GDI female mice and 9-week old male mice were used to extract the embryos. On the second day after fertilization of the female mice, the two- or four-cell stage embryos were flushed from the oviduct and cultured in groups in 50 pl drops in KSOM medium (manufactured by Millipore, England), supplemented with 0.4% BSA (Bovine Serum Albumin) protein, under mineral oil, at a temperature of 37°C and 5% COz-content in the air. The number of embryos in the drops varied between 10 and 14.

[0068] During the procedure described above, unfertilized oocytes were cultured for 24 hours. If the oocytes did not divide even after 24 hours, they were considered dead and used as degenerated oocytes for the measurements. These embryos do not present significant difference with respect to the background.

[0069] To produce living oocytes, the GDI superovulated females were mated with vasectomized (sterilized) males. Using a procedure similar to the above mentioned one, the oocytes flushed from the oviduct on the next day were placed in KSOM culture medium and used directly as living oocytes for the measurements. For producing stressed embryos, which were expected to have a reduced ability to implant, light treatment was applied. The embryos were transferred to M2 manipulation medium (Millipore, England) during the light treatment. The light treatment of embryos was performed in a dark room. Two compact bulbs were used as light sources. The intensity of the emitted light was 1130 lux. During the spectral analysis of the emitted light, the highest peak appeared at 600-620 nm. Two-cell stage embryos were stressed for 50 minutes.

[0070] In the case of frozen embryos, high-quality embryos of 6 to 8 cells were vitrified and heated. The process of vitrification and heating was performed with the Rapid-i vitrification kit and the Rapid-i thawing kit (manufacturer: Vitrolife AG Gothenburg, Sweden). The proportion of live embryos after thawing was higher than 90%. The measurements were performed on the embryos originating from freezing, after one or two hours of culture.

[0071] EmbryoSlide culture vessels (manufacturer: Vitrolife AG Gothenburg, Sweden) were used for culturing the embryos. The dishes were prepared according to the manufacturer's recommendations. The microwell of the equipment allowed the embryos to be kept in their original position during the measurement.

[0072] The photon emission measurement was performed by an ORCA-Quest CMOS photon detector (manufacturer: Hamamatsu Photonics, Japan), which is an extremely sensitive detector capable of detecting even a single photon. The change in photon emission intensity over time was monitored and recorded with the photon detector.

[0073] The photon detector comprised a CMOS image sensor with a maximum spectral sensitivity between 300 nm and 1000 nm. The CMOS image sensor was cooled to -20 °C. The image resolution of the image sensor was 4096 x 2304 pixels, and the pixel size was 4.6 pm x 4.6 pm. The quantum efficiency was 90% at 475 nm and 33% at 900 nm.

[0074] The photon detector was used together with an incubator, in our test with a microscope incubator of the monitoring system LV200 Luminoview of Olympus Inc., which provided ideal conditions for breeding embryos. The measurement was performed in a fully dark space, excluding any visible light, which allowed the detection of photons emitted by the embryos.

[0075] Figure 4 shows the events of a 50-hour continuous measurement of time-lapse type using a dark incubator, as a function of time, where the time axis is collapsed. During the measurement, exposures of 1 minute were made. In the graph, the upper curve shows the photoemission intensity of normal live embryos, the middle curve shows the photoemission intensity of live embryos originating from freezing, and the lower curve shows the photoemission intensity of the background. In the graph, the vertical axis shows the EW-SFD (Entropy-Weighted Spectral Fractal Dimension) values. As can be clearly seen in the graph, the photoemission activity of normal living embryos is significantly higher (mean value: approx. 0.35) than the activity of the embryos originating from freezing (mean value: approx. 0.28) and also the activity of the background (mean value: approx. 0,24). The photoemission activity of the degenerated embryos could not be detected. Consequently, viable and non-viable embryos can be clearly distinguished based on the photoemission activity of the embryos using the method according to the invention.

Claims

Claims1. A system (100) for monitoring embryos in vitro, comprising- an incubator (110),- an oxygen tank (162) and a carbon dioxide tank (164) connected to the incubator (110) through a gas control unit (160),- a control unit (170) for controlling the climate of the interior of the incubator (110),- at the bottom of the incubator (110), at least one dish (112) for storing the embryos (140), characterized in that the system (100) further comprises- a light-transmitting lens system (180) arranged under the incubator (110), made of a heatinsulating material and coupled to the bottom of at least one dish (112) from outside, said lens system consisting of an objective lens (180a) and a tubular lens (180b) arranged under said objective lens,- a high-sensitivity photon detector (120) arranged under the lens system (180) and coupled to the lens system (180), and- a computer (130) connected to the photon detector (120).

2. The system (100) according to claim 1, characterized in that it further comprises a lighting unit that directs light of three different colors to the dish (112).

3. The system (100) according to claim 1 or 2, characterized in that the climate control unit (170) is configured to control at least the temperature and humidity of the air space of the incubator (110).

4. The system (100) according to any one of claims 1 to 3, characterized in that the computer (130) is connected to the gas control unit (160) and / or the climate control unit (170).

5. A method for monitoring embryos in vitro, characterized in that the method comprises the following steps:- preparing (300) the measurement, during which at least one of the following operations is performed: placing an embryo to be tested in a dish by placing the embryo in a microwell of the dish; setting the temperature of the incubator to a predetermined value; setting thehumidity of the incubator to a predetermined value; setting the volume ratio of oxygen and carbon dioxide in the incubator to a predetermined value; on the basis of preliminary measurements, determining the planned duration of the measurement and other parameters, in particular the frequency of sampling, the duration of sampling, the signal amplification of the photon detector, the sensitivity of the image sensor of the photon detector, the spectral characteristics of the photon detector, the electronic control of the photon detector, the amount of data expected during the entire measurement,- during the natural development of the examined embryo, detecting (300) the spontaneous ultra-weak photon radiation emitted by the embryo according to a predetermined sampling procedure by means of the photon detector connected to the incubator, and transmitting (315) the detected radiation signals to a computer in digitized form,- continuously storing (320) the measurement data received by the computer on a data storage device or in a database,- reading the stored data into the computer and processing (330) the data for determining the change in the photon emission intensity of the embryo over time, and- based on time function of the embryo's photon emission intensity determined by the computer, producing a series of results on the basis of a unique and multi-step assessment methodology for the viability of the embryo (340).

6. The method according to claim 5, characterized in that in the information generation step (340), if the entropy-weighted spectral fractal dimension (EW-SFD) based value of the intensity function for a given embryo exceeds a predetermined reference value, then the embryo is considered viable, otherwise the embryo is not considered viable.

7. The method according to claim 5, characterized in that the measurement preparatory step (300) comprises a preliminary, sample-free calibration measurement by means of the photon detector, and using the calibration measurement result in the computer data processing step (330), in particular for filtering out measurement noises.

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