System and method for in vitro monitoring of embryos

Abstract

The system 100 and method according to the present invention are used for in vitro monitoring of embryos. The system 100 includes an incubator 110, oxygen tanks 162 and carbon dioxide tanks 164 connected to the incubator 110 through a gas control unit 160, a unit 170 for controlling the climate inside the incubator 110, and at least one dish 112 located at the bottom of the incubator 110 for storing embryos 140. The system 100 further includes a light-transmitting lens system located below the incubator 110, made of insulating material, connected from the outside to the bottom of at least one dish 112, and comprising an objective lens and a tube lens below the objective lens, a highly sensitive photon detector 120 located below the lens system and connected to the lens system, and a computer 130 connected to the photon detector 120.

Description

[Technical Field]

[0001] The present invention relates to a photon emission system and method for testing the development and viability of living embryos based on spontaneous photon emission. [Background technology]

[0002] In developed countries, the number of couples struggling with infertility has been steadily increasing over the past few decades, and the importance of assisted reproductive technologies (IVF) has also continued to grow. Among these procedures, in vitro fertilization (IVF) has become a standard practice in clinical medicine over the past 40 years and has experienced remarkable development since its introduction.

[0003] In most cases, efforts have been made to improve the efficiency of IVF techniques by increasing the number of embryos transferred. During the stimulation cycle, it is usually possible to fertilize multiple oocytes, which allows for the formation and transfer of multiple embryos, thereby increasing the chances of pregnancy. At the same time, multiple embryo transfer significantly increases the risk of multiple pregnancies. Multiple pregnancies are one of the most common causes of premature birth and are associated with an increased risk of premature birth. To avoid this risk, it would be important to select the most viable and transferable embryos.

[0004] At the same time, a crucial ethical and practical question arises: what method should be used to select the embryos to be transplanted? The most widely used selection method is based on examination of the embryo's morphology, ranging from simple use of a microscope to time-lapse photography. The latter technique provides important information not only about the embryo's morphology but also about the dynamics of embryonic development. Time-lapse photography has created the concept of stable culture conditions, along with the possibility of continuous observation and documentation of the embryo. However, a problem with this selection technique is that it involves exposing the embryos to visible light.

[0005] Early embryonic development is characterized by rapid cell division and activation of embryonic genes, which makes the embryo extremely vulnerable and sensitive to environmental influences. The ovaries produce oocytes, and the female body provides a safe environment for gametes and embryos. The human body protects itself from visible light through well-defined mechanisms. During IVF and especially intracytoplasmic sperm injection (ICSI) as fertilization methods, exposure can be harmful to cells during oocyte and sperm preparation, embryo culture and microscopy, and embryo transfer.

[0006] The harmful effects of ultraviolet (UV) light on living cells have long been known. However, the toxic effects of visible light (400-700 nm) are less well understood, despite several previous studies supporting the adverse effects of visible light on oocytes, sperm, and embryos. The harmful effects of light may be related to hydrogen peroxide formed in peroxisomes and mitochondria. Light can induce stress gene activation and DNA damage in embryos. The damaging effects of visible light depend on the wavelength of light. Blue light (400-500 nm) has been proven to be orders of magnitude more dangerous than visible light with longer wavelengths.

[0007] To protect spermatids, oocytes, and 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 microscopes and IVF workstations, are equipped with red filters.

[0008] It is well known from the literature that living cells (plant cells, animal cells, and human cells) spontaneously emit ultra-weak photon emission (UPE). UPE intensity can vary from a few photons per second to several hundred photons per square centimeter, mainly within the spectral range of 200–800 nm. The origin of UPE has been shown to be 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, and cancerous proliferation.

[0009] Patent EP3455340 discloses an apparatus for culturing living biological materials such as embryos, comprising a box with a closed but openable door, the box comprising two or more cell culture holding units, a video camera, and a fluorescence lifetime imaging microscope (FLIM) type microscope unit, wherein the camera and the FLIM unit, or parts thereof, can be moved relative to the cell culture holding units. The camera is used to determine several predetermined morphological features of the biological material. During the examination of the biological material, stimulation is used to induce photon emission by the cells.

[0010] The drawback of the above system is that neither light energy nor chemicals can be used during embryo examination to enhance embryo protection; in other words, the planned examination must be performed without external energy input. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] Patent EP3455340 [Non-patent literature]

[0012]

Linked Book1

Outdoor Tool2

Outdoor Tools3

Outdoor Tools 4

[0013] Therefore, an object of the present invention is to provide an embryo inspection system and a method that enable measurement of photon emission from an embryo during the culture stage without factors affecting embryo development, that is, without external energy.

[0014] Another object of the present invention is to provide an appropriate connection between a photon detector used for in vitro monitoring of an embryo and an incubator storing the embryo so that the embryo is always in an ideal environmental condition in the incubator during the test. [Means for Solving the Problems]

[0015] The present invention is based on the recognition that when photons naturally emitted by living cells such as embryos are detected by a high-sensitivity detector, qualitative information regarding the life functions and viability of the test cells can be examined based on changes in photon emission over time. It is also recognized that detection of photon emission from an embryo can be achieved by inserting a lens made of a photon-transmissive material between the incubator and the detector.

[0016] The above objectives are achieved by providing the system described in claim 1 and the method described in claim 5. Preferred embodiments of the system and method according to the present invention are defined by the dependent claims.

[0017] The present invention will be described in detail below with reference to the drawings. [Brief explanation of the drawing]

[0018] [Figure 1] This is a functional block diagram of the system according to the present invention. [Figure 2] This figure schematically shows an assembly of the incubator and photon detector forming components of the system according to the present invention. [Figure 3] This is a flowchart showing the main steps of the method according to the present invention. [Figure 4] This graph shows the results of experiments using the system according to the present invention, illustrating the changes in photon emission over time in a standard live embryo, a live embryo created from frozen embryos, and the background. [Modes for carrying out the invention]

[0019] The main functional units of the system 100 according to the present invention are shown in the block diagram 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 is equipped with at least one light-transmitting dish 112, in which one embryo 140 is placed. A photon detector 120 detects photon beams 150 originating from the very weak spontaneous photon emission of the embryo 140. The photon emission detection signal is transmitted by the photon detector 120 to a computer 130, which generates information about the embryo's vital functions and viability based on the time-dependent changes in the detection signal.

[0021] An ideal environment for the embryo 140 must be provided within the incubator 110. For this purpose, 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 of providing the desired oxygen and carbon dioxide concentrations within the incubator 110.

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

[0023] An air conditioning unit 170 is also connected to the incubator 110 to maintain the temperature and humidity of the incubator's internal space at desired values. For example, during measurement, a temperature of 37°C and a humidity of 100% should be maintained inside the incubator 110.

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

[0025] Since the possibility of embryo 140 moving during the photon emission test must be minimized, a recess at the bottom of the dish 112 placed inside the incubator 110 ensures that the embryo 140 placed in the dish is held in place without displacement.

[0026] The incubator 110 may be, for example, an incubator unit that forms part of Olympus Corporation's bioluminescence imaging system "LV200 Luminoview".

[0027] To successfully detect the photon beam 150 from photon emission, the photon detector 120 should be positioned as physically as possible close to the embryo 140 being examined and monitored, and therefore, as shown in Figure 2, below the incubator 110, the detector 120 fits snugly to the bottom of one or more dishes 112 through an intervening lens system 180. The lens system 180 consists of an objective lens 180a and a tube lens 180b positioned below it. Since the image recording unit of the photon detector 120 should be maintained at an extremely low temperature (approximately -20°C), the lens system 180 fitted between the photon detector 120 and at least one dish 112 is made of a special material.

[0028] In a preferred embodiment of the system 100 according to the present invention, the photon detector 120 has a high-resolution image sensor, for example, a sensor with a resolution of 9 megapixels (MP). The photon detector 120 may be, for example, a CMOS-type ORCA-Quest camera from Hamamatsu Photonics K.K. that is capable of detecting even a single photon.

[0029] In a preferred embodiment of the system 100 according to the present invention, the system 100 also includes an illumination unit that directs three different colored lights, for example RGB colored lights, onto at least one dish 112. By using the illumination unit, one or more dishes 112 can be positioned without the use of external lighting (which typically includes UV light harmful to embryos) before the measurement begins.

[0030] The illumination unit is preferably a manually controlled unit that must be switched off before starting the photon emission measurement so that the embryo 140 is in complete darkness during the measurement.

[0031] The main steps of the in vitro embryo monitoring method performed using the system according to the present invention will be described below with reference to the flowchart shown in Figure 3.

[0032] In the first step 300 of the method, preparations for the measurement are made, during which, above all, the embryo to be tested is placed in a cup so that it is positioned in the recess of the cup, the incubator temperature is set to 37°C, the desired humidity is set, and the volume ratio of oxygen to carbon dioxide is set. As part of step 300, the duration of the measurement and other parameters, in particular the sampling frequency, sampling duration, signal amplification of the photon detector, sensitivity of the image sensor of the photon detector, spectral characteristics of the photon detector, electronic control of the photon detector, and the amount of data expected during the entire measurement may be determined through preliminary measurements.

[0033] In the next step 310 of the method, spontaneous ultra-weak photon emission emitted by the embryo during its natural development is detected by a detector connected to the incubator according to a predetermined sampling procedure, and the detected emission signal is transmitted in a digitized form to an image and data processing computer in step 315. The data transmitted by the photon detector conveys not only information about the amount of photons detected by the detector, but also the physical properties of the captured photons (e.g., position, time properties).

[0034] In step 320 of the method, the measurement data received by the computer is continuously stored in a data storage device or database in an appropriate data structure. The measurement data is preferably represented using Hyperstack Bio-Formats.

[0035] At the end of the measurement, the stored data is loaded into a computer in step 330 of the method, and multi-stage data processing is performed on the data. During data processing, changes in the intensity of photon emission are determined as a function of time. If photon emission from several embryos is monitored simultaneously or successively during the measurement, the time function of intensity is determined for each embryo in the data processing step.

[0036] Finally, in step 340 of the method, information regarding the viability of each embryo is generated based on the intensity function of one or more photon emissions determined by the computer. In the case of a given embryo, if the time change (slope) of the entropy-weighted spectral fractal dimension (EW-SFD)-based value of the entropy-weighted spectrum of the recorded image is positive, the embryo is considered viable; otherwise, the embryo is not considered viable.

[0037] The fractal dimension of the entropy-weighted spectrum was calculated using the following method. The measurement data was recorded in the form of image data, and therefore, its parameters were selected for the evaluation of data corresponding to both the high-entropy energy output of the embryo and the image data of the sensor. The selection of digital characteristics suitable for measuring entropy-based quantities was evident based on the data of the imaging unit used to record the digital data, as well as the predictions of energy processes for living tissue already mentioned. In this way, a selection for measuring entropy in information theory was made, and regarding its application to image data, several practical results have already been published in many cases (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, JPEG2026516357000002.jpg13165 , 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, and JPEG2026516357000003.jpg1190 , 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, edited by T. Sobh and K. Elleithy, Innovations and Advances in Computing, Informatics, Systems Sciences, Networking and Engineering, Lecture Notes in Electrical Engineering (LNEE), Vol313, pp. 255 - 262, DOI 10.1007 / 978 - 3 - 319 - 06773 - 5_1, Springer International Publishing, Switzerland 2015, see

[0038] The modern information theory concept of entropy was introduced by Claude E. Shannon in 1948 (see Shannon, CE, A Mathematical Theory of Communication, The Bell System Technical Journal, 27:379-423, 1948, and Shannon, CE, A Mathematical Theory of Communication, The Bell System Technical Journal, 28:623-656, 1948), and subsequently demonstrated through practical examples (see Shannon, CE, Prediction and entropy of printed English, The Bell System Technical Journal, 30:50-64, 1951). According to this theory, the average amount of information (entropy) of independent digital messages is:

number

[0039] The mathematical definition of entropy 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, pp. 547-561, 1960), and an independent measurement program was developed for measuring the entropy of 16-bit image data.

[0040] The entropy of a closed system in information theory is given by the following values: 0 ≤ H ≤ log2n (2) We can take the form of n, where n is the number of possible messages.

[0041] Entropy is minimized when a source always sends the same message, that is, when an image displays only one color or one intensity value. Entropy is maximized when all message possibilities are equal.

[0042] When the self-similar structure (fractal structure) of the data is also considered in addition to the amount of information (entropy), it is recommended to measure separate structural parameters. According to the inventors' assumptions, embryos should be treated as such objects, and therefore, fractal dimensions were chosen to measure the structural parameters. All of this was proven by the inventors' previous research results obtained during biological surgery. However, in this case, the pixel values ​​reaching the sensor from the embryo consist of three essential elements, namely, The intensity of photons emitted by the embryo, The structure of photons emitted at various moments within the integral time, and noise This also includes.

[0043] To separate this data from a single region, a unique function (algorithm) called EW-SFD (Fractal Dimension of Entropy-Weighted Spectra) has been developed.

[0044] The dimension of a fractal curve is a number that indicates how much the distance between two selected points on the curve increases when the resolution is increased. Therefore, as long as the phase dimensions of lines and surfaces are always 1 and 2, respectively, the fractal dimension can be a value between them. Curves and surfaces that exist in the real world are not actually fractals, but rather created by processes that can only produce shapes within a specific size range. Therefore, the value of the fractal dimension can change depending on the resolution. This change can be helpful in understanding the processes characteristic of living biological systems involved in their generation.

[0045] Several methods have been developed that are equally suitable for calculating fractal dimensions (see Turner, MT, Blackledge, JM, Andrews, PR, 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 (see Mandelbrot, BB, The fractal geometry of nature, WH Freeman and Company, New York, 1983), a structural analysis method derived from general fractal dimensions, 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 about the fractal properties of color and hue. To calculate the SFD value (for two or more image bands with the same spectral resolution), the following definition of spectral fractal dimension may be applied, which is calculated by simple mathematical averaging of the measurement data,

number

[0046] The number of possible spectral boxes in the case of j bits is as follows: BT j =(2 S )n (4) can be calculated as follows.

[0047] The SFD defined above ESR method is a measurement criterion that satisfies the following conditions, namely, that it is non - negative definite, symmetric, and satisfies the triangle inequality. A further condition for the measurement criterion to be satisfied is the achievement of the regularity condition. That is, the points of the individual image planes should have a uniform density. In practice, the image function undergoes a non - linear transformation before entering the A / D converter, and as a result, the density function of the image function becomes constant. Therefore, in the case of digital images, the regularity condition is usually satisfied or can be considered to be so. The SFD ESR is a correlation measurement criterion and can thus be accurately used to measure image data.

[0048] To calculate the fractal dimension of the entropy - weighted spectrum (EW - SFD), the following measurable definition (5) of the fractal dimension of the spectrum, namely,

Number

[0049] The number of spectrum boxes is weighted by the weighting factor as follows,

Number

[0050] The number of possible spectral boxes in the case of j bits can be calculated according to equation (6).

[0051] Entropy weighting factor f j The following is

number

number

[0052] Based on the above, EW-SFD is as follows:

number

[0053] Since the signal-to-noise ratio for measurements of spontaneous photon emission is usually much lower than that for measurements of stimulated photon emission, in a preferred embodiment of the method, a preliminary, sample-free calibration measurement is performed by the photon detector in the preparation step 300 before the measurement is started, and the results are used when processing the results of a normal measurement, mainly for the purpose of removing measurement noise.

[0054] "Examples" Experimental photon emission measurements in animal embryos are presented below as an example.

[0055] Seven-week-old CD1 female mice and nine-week-old male mice were used to extract embryos. Two days after fertilization in female mice, two-cell or four-cell stage embryos were flushed out of the oviduct and cultured in groups in 50 μl droplets of KSOM medium (Millipore, England) supplemented with 0.4% bovine serum albumin (BSA) protein under mineral oil at a temperature of 37°C and an atmospheric CO2 content of 5%. The number of embryos in each droplet varied between 10 and 14.

[0056] During the procedure described above, unfertilized oocytes were cultured for 24 hours. If these oocytes had not divided after 24 hours, they were considered dead and used as degenerated oocytes for measurement. These embryos did not show significant differences compared to the background.

[0057] To produce live oocytes, females treated for superovulation with CD1 were mated with males that had undergone vasectomy (castration). Using a procedure similar to that described above, oocytes flushed from the fallopian tubes the following day were placed in KSOM medium and used directly as live oocytes for measurement.

[0058] Light treatment was applied to create stressed embryos, which were expected to have reduced transplantability. During light treatment, the embryos were transferred to M2 manipulation medium (Millipore, England). The light treatment of the embryos was performed in a dark room. Two small light bulbs were used as light sources. The intensity of the synchrotron radiation was 1130 lux. During spectral analysis of the synchrotron radiation, the highest peak appeared at 600-620 nm. Two-cell stage embryos were stressed for 50 minutes.

[0059] For frozen embryos, high-quality embryos with 6-8 cells were vitrified and heated. The vitrification and heating processes were performed using the Rapid-i vitrification kit and Rapid-i thawing kit (manufacturer: Vitrolife AG ​​Gothenburg, Sweden). The percentage of live embryos after thawing was higher than 90%. Measurements were taken for embryos derived from freezing after 1 or 2 hours of culture.

[0060] EmbryoSlide culture vessels (manufacturer: Vitrolife AG ​​Gothenburg, Sweden) were used to culture the embryos. Dishes were prepared according to the manufacturer's recommendations. The instrument's microwells allowed the embryos to remain in their original positions during measurement.

[0061] Photon emission measurements were performed using the ORCA-Quest CMOS photon detector (manufacturer: Hamamatsu Photonics), an extremely sensitive detector capable of detecting even a single photon. The change in photon emission intensity over time was monitored and recorded using the photon detector.

[0062] The photon detector was equipped with a CMOS image sensor having 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 × 2304 pixels, and the pixel size was 4.6 μm × 4.6 μm. The quantum efficiency was 90% at 475 nm and 33% at 900 nm.

[0063] The photon detector was used in conjunction with an incubator; in our tests, it was used with a microscope incubator from Olympus Corporation's LV200 Luminoview monitoring system, which provides ideal conditions for embryo development. Measurements were performed in a completely dark environment, without any visible light, to enable the detection of photons emitted by the embryo.

[0064] Figure 4 shows the events of a 50-hour continuous measurement using time-lapse photography with a dark incubator as a function of time, with the time axis compressed. During the measurement, a 1-minute exposure was performed. In the graph, the upper curve shows the photon emission intensity of a normal living embryo, the middle curve shows the photon emission intensity of a living embryo derived from freezing, and the lower curve shows the background photon emission intensity. In the graph, the vertical axis shows the EW-SFD (Fractal Dimension of Entropy-Weighted Spectrum) value. As can be clearly seen in the graph, the photon emission activity of a normal living embryo is significantly higher (average: approximately 0.35) than the activity of a frozen embryo (average: approximately 0.28) and the background activity (average: approximately 0.24). Photon emission activity of a degenerated embryo could not be detected. Therefore, using the method according to the present invention, viable and unviable embryos can be clearly distinguished based on the photon emission activity of the embryo.

Claims

1. A system (100) for monitoring an embryo outside the body, Incubator (110), An oxygen tank (162) and a carbon dioxide tank (164) connected to the incubator (110) via a gas control unit (160), A control unit (170) for controlling the climate inside the incubator (110), To store the embryo (140), at least one dish (112) located at the bottom of the incubator (110) and The system (100) is equipped with, A lens system (180) for transmitting light, positioned below the incubator (110), made of insulating material, externally connected to the bottom of at least one dish (112), and comprising an objective lens (180a) and a tube lens (180b) positioned below the objective lens, A highly sensitive photon detector (120) is positioned below the lens system (180) and connected to the lens system (180), A computer (130) connected to the photon detector (120) and A system (100) further characterized by comprising the following.

2. The system (100) according to claim 1, further comprising a lighting unit that directs three different colored lights onto 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 outside the body, Steps to prepare for measurement (300), during which at least one of the following operations is performed: placing the embryos to be tested into the microwells of the dish; setting the temperature of the incubator to a predetermined value; setting the humidity of the incubator to a predetermined value; setting the volume ratio of oxygen to carbon dioxide in the incubator to a predetermined value; determining the planned period of the measurement and other parameters, in particular the sampling frequency, sampling period, signal amplification of the photon detector, sensitivity of the image sensor of the photon detector, spectral characteristics of the photon detector, electronic control of the photon detector, and the amount of data expected to be collected during the entire measurement, based on preliminary measurements. The steps include: (300) detecting spontaneous ultra-weak photon emission emitted by the embryo during its natural development using the photon detector connected to the incubator according to a predetermined sampling procedure; and (315) transmitting the detected emission signal to a computer in a digitized form. The steps (320) include continuously storing the measurement data received by the computer in a data storage device or database, In order to determine the change in the photon emission intensity of the embryo over time, the stored data is read into the computer and the data is processed (330), Step (340) of generating a series of results based on a unique and multi-stage evaluation method for the viability of the embryo, based on the time function of the photon emission intensity of the embryo determined by the computer, A method characterized by including

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

7. The method according to claim 5, characterized in that the measurement preparation step (300) includes a preliminary, sample-free calibration measurement by the photon detector, and the results of the calibration measurement are used in the computer data processing step (330) in particular to remove measurement noise.

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