JP2010004789A - Embryo observation apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an apparatus for more simply and accurately deciding the quality of embryos. <P>SOLUTION: The embryo observation apparatus includes the following sections. An image-obtaining section 61 photographs an embryos set to a microscope as a sample with a camera in time series to obtain observation images, and supplies the obtained observation images to an observation section 62. The observation section 62 detects embryo cleavage on the basis of the obtained observation images and pursues each cell division in the embryo. A grading section 81 determines a time required for the division of each cell of the same generation in embryos at a prescribed timing on the basis of cell division information supplied from the observation section 62, and calculates the grading value of the embryo. A quality-deciding section 82 deciding the quality of the embryo on the basis of the grading value at a prescribed timing. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an embryo observation apparatus, and more particularly to an embryo observation apparatus for observing embryo growth.

  Conventionally, for the purpose of increasing the success rate of in vitro fertilization, etc., the state of the embryo is classified based on the morphology of the embryo in the image obtained by photographing a fertilized egg (hereinafter simply referred to as an egg), and the growth state of the embryo is evaluated. To be done. Known methods for evaluating the growth state of an embryo include, for example, a classification method by Veeck, a classification method by Gardner, and the like (for example, see Non-Patent Document 1).

Baczkowski, T., Kurzawa, R. and Glabowski, W., Methods of embryo scoring in in vitro fertilization, Reprod, Biol.v4.p.5-22

  However, the conventional classification method detects errors in DNA (Deoxyribo Nucleic Acid) in embryos that are judged to be good, or the embryos that are judged to be bad continue to crack normally. It is known that a determination occurs.

  The present invention has been made in view of such a situation, and makes it possible to determine the quality of an embryo more easily and accurately.

  An embryo observation apparatus according to one aspect of the present invention is an embryo observation apparatus that observes the growth of an embryo, detects cleavage of the embryo based on an image obtained by photographing the embryo in time series, Observation means for tracking the division of each cell, and evaluation means for evaluating the growth state of the embryo based on the time required for the division of each cell of the same generation in the embryo.

  In one aspect of the present invention, cleavage of the embryo is detected based on images taken in time series of the embryo, division of each cell in the embryo is tracked, and each cell of the same generation in the embryo Based on the time required for the division of the embryo, the growth state of the embryo is evaluated.

  According to the present invention, the quality of an embryo can be determined more easily and accurately.

  Embodiments to which the present invention is applied will be described below with reference to the drawings.

  FIG. 1 is a diagram showing a configuration example of an embodiment of an observation system to which the present invention is applied. This observation system includes a microscope 11, an operation unit 12, a controller 13, a camera 14, a personal computer 15, and an observation monitor 16.

  The microscope 11 is provided with an electric stage 21 driven by a stepping motor or the like (not shown). A container such as a glass bottom dish or a well plate in which a specimen such as a cell to be observed is placed is disposed on the electric stage 21, and the user can appropriately observe the specimen by operating the operation unit 12. Thus, the electric stage 21 is moved. Hereinafter, with respect to the moving direction of the electric stage 21, the left-right direction of the microscope 11 is defined as the x-axis direction, the front-rear direction is defined as the y-axis direction, and the up-down direction is defined as the z-axis direction.

  Further, the camera 14 images a sample in the field of view of the microscope 11 as a subject, and supplies an image obtained as a result (hereinafter referred to as an observation image) to the personal computer 15. Then, the personal computer 15 supplies the observation image from the camera 14 to the observation monitor 16 for display. Thereby, the user can observe the specimen in the visual field of the microscope 11 by viewing the observation image displayed on the observation monitor 16.

  The controller 13 records observation position information indicating the observation position of the specimen in time-lapse observation, which is registered by the user by operating the operation unit 12. The controller 13 supplies the recorded observation position information to the personal computer 15.

  The personal computer 15 records the observation position information supplied from the controller 13 and performs control for time-lapse observation based on the observation position information. That is, the personal computer 15 instructs the controller 13 to move the electric stage 21 to the registered observation position at regular time intervals based on the recorded observation position information. Then, the controller 13 moves the electric stage 21 to the registered observation position in accordance with an instruction from the personal computer 15, and the personal computer 15 acquires and records the sample image at the observation position from the camera 14.

  Hereinafter, the microscope 11 is an upright microscope, and the camera 14 photographs a sample from above the electric stage 21.

  FIG. 2 is a block diagram showing an example of a functional configuration of the embryo observation unit 51 realized by the personal computer 15 executing a predetermined control program.

  The embryo observation unit 51 is configured to include an image acquisition unit 61, an observation unit 62, and an evaluation unit 63, and realizes a function of observing the growth of the embryo and evaluating the growth state of the embryo.

  The image acquisition unit 61 controls the camera 14 based on a command from the observation unit 62 to photograph an egg (fertilized egg) installed as a specimen on the electric stage 21. The image acquisition unit 61 acquires an observation image of an egg photographed by the camera 14 and supplies it to the observation unit 62.

  The observation unit 62 is configured to include an egg detection unit 71, a vertical cleavage detection unit 72, a horizontal cleavage detection unit 73, and a cell recognition unit 74, and shoots eggs and embryos in the egg in time series. Based on the observed images, the cleavage of the embryo is detected and the division of each cell in the embryo is followed.

  The egg detection unit 71 acquires an observation image from the camera 14 via the image acquisition unit 61. As will be described later with reference to FIG. 4 and the like, the egg detection unit 71 performs an egg detection process in the obtained observation image. The egg detection unit 71 supplies information indicating the position of the egg in the observation image and the observation image used for detecting the egg to the vertical cleavage detection unit 72.

  As described later with reference to FIG. 5 and the like, the vertical cleavage detection unit 72 performs cleavage detection processing when cells in the embryo divide in the vertical direction. The vertical cleavage detection unit 72 sets a cleavage boundary surface that divides the egg and the embryo in the vertical direction according to the detection result. The vertical cleavage detection unit 72 sets a unit region by dividing the egg by the set cleavage boundary surface. The vertical cleavage detection unit 72 supplies information indicating the set cleavage boundary surface and unit region to the horizontal cleavage detection unit 73.

  The horizontal cleavage detection unit 73 acquires a plurality of observation images having different focus positions from the camera 14 via the image acquisition unit 61. As will be described later with reference to FIG. 11 and the like, the horizontal cleavage detection unit 73 performs cleavage when cells in the embryo divide in the horizontal direction for each unit region based on the acquired observation images. The detection process is performed. The horizontal cleavage detection unit 73 sets a cleavage boundary surface that divides the eggs and embryos in the horizontal direction according to the detection result. The horizontal cleavage detection unit 73 stores information indicating the position of the egg and embryo in the observation image, and the position of the cleavage boundary set by the vertical cleavage detection unit 72 and the horizontal cleavage detection unit 73. This is supplied to the recognition unit 74. The horizontal cleavage detection unit 73 supplies the observation image used for detection of the cleavage to the cell recognition unit 74.

  The cell recognition unit 74 individually recognizes cells in the embryo in the observation image based on the set cleavage boundary surface. The cell recognition unit 74 assigns a label to each cell in order to uniquely identify each recognized cell. Moreover, the cell recognition part 74 detects the magnitude | size of each recognized cell based on the acquired observation image. The cell recognition unit 74 supplies the evaluation unit 63 with the label assigned to each cell and the cleavage information indicating the position and size of each cell.

  The evaluation unit 63 includes a grading unit 81 and a pass / fail determination unit 82, and evaluates the growth state of the embryo based on the cleavage information acquired from the cell recognition unit 74.

  The grading unit 81 records the cleavage information acquired from the cell recognition unit 74 in time series. The grading unit 81 performs a grading process for grading the growth state of the embryo based on the cleavage information recorded in time series. The grading unit 81 supplies the grading value calculated by the grading process to the pass / fail determination unit 82.

  The quality determination unit 82 determines the quality of the embryo based on the grading value calculated by the grading unit 81. The pass / fail determination unit 82 outputs information indicating the determination result to the outside.

  Next, an embryo observation process executed by the observation system of FIG. 1 will be described with reference to the flowchart of FIG. In this process, for example, an egg to be observed is placed on the electric stage 21 as a specimen, and when the user operates the operation unit 12, the execution of the embryo observation process is performed on the personal computer 15 via the controller 13. It starts when the command is input. Hereinafter, processing in the case where a human fertilized egg is an observation target will be described.

  In step S1, the embryo observation unit 51 acquires an observation image. Specifically, the egg detection unit 71 instructs the image acquisition unit 61 to acquire an observation image. The camera 14 captures an observation image under the control of the image acquisition unit 61 and supplies the captured observation image to the egg detection unit 71 via the image acquisition unit 61.

  In step S2, the egg detection unit 71 executes an egg detection process. Here, the details of the egg detection process will be described with reference to the flowchart of FIG.

  In step S21, the egg detection unit 71 applies a dispersion filter to the observation image. Here, the dispersion filter is a filter that calculates the dispersion of luminance within a filter window having a predetermined size. Therefore, the dispersion filter returns a larger value as some object exists in the filter window and the luminance variation of the object is larger, and returns 0 if nothing exists in the filter window. The egg detection unit 71 applies a dispersion filter to all regions of the observation image.

  In step S22, the egg detection unit 71 binarizes the observation image. That is, the egg detection unit 71 binarizes the pixel value of each pixel of the observation image after applying the dispersion filter using a predetermined threshold value. Thereby, the part in which an object exists in an observation image and the part which does not exist are distinguished and displayed.

  In step S23, the embryo observation unit 51 detects an object in the observation image based on the binarized image. The egg detection unit 71 assigns a different label to each object so that the detected objects can be individually distinguished.

  In step S24, the egg detection unit 71 calculates the area and moment (image moment) of each detected object.

  In step S25, the egg detection unit 71 selects an egg from the detected objects. Specifically, the egg detection unit 71 selects an object closest to the size and shape of a standard human fertilized egg based on the area and moment of each object. The egg detection unit 71 supplies information indicating the position of the egg in the observation image and the observation image before image processing to the vertical cleavage detection unit 72. Thereafter, the egg detection process ends.

  Returning to FIG. 3, in step S <b> 3, the vertical cleavage detection unit 72 performs a vertical cleavage detection process. Here, the details of the vertical cleavage detection process will be described with reference to the flowchart of FIG.

  In step S41, the vertical cleavage detection unit 72 removes noise from the observation image. In other words, the vertical cleavage detection unit 72 applies a low-pass filter to the observation image acquired from the egg detection unit 71 to remove noise of harmonic components.

  In step S42, the vertical cleavage detection unit 72 extracts an edge of the observation image. For example, the vertical cleavage detection unit 72 extracts the edge of the observation image by applying a secondary differential filter to the observation image.

  In step S43, the vertical cleavage detection unit 72 binarizes the observation image. That is, the vertical cleavage detection unit 72 binarizes the pixel value of each pixel of the observation image after performing edge extraction using a predetermined threshold value. Thereby, the edge part in an observation image and the part other than an edge are distinguished and displayed.

  In step S44, the vertical cleavage detection unit 72 detects the outer periphery of the embryo based on the binarized image. Specifically, based on the binarized image, the vertical cleavage detection unit 72 embeds an object surrounded by the extracted edges in the region within the egg of the observation image detected by the egg detection unit 71. And the outer frame part of the edge is recognized as the outer periphery (cell membrane) of the embryo.

  In step S45, the vertical cleavage detection unit 72 calculates polar coordinates of the outer periphery of the embryo. For example, as shown in FIG. 6, let us consider a case where an embryo 102 in the left egg 101 is divided into cells C1 and C2 as shown on the right side. FIG. 6 is a view of the egg 101 as viewed from above.

  The vertical cleavage detection unit 72 obtains the center O of the embryo 102 and expresses the coordinates of each point on the outer periphery of the embryo 102 by polar coordinates (θ, r) centered on the center O. FIG. 7 is a graph of polar coordinates on the outer periphery of the embryo 102 on the right side of FIG. 6, where the horizontal axis indicates the deflection angle θ and the vertical axis indicates the radius r.

  In step S46, the vertical cleavage detection unit 72 detects a dent on the outer periphery of the embryo based on the differential waveform of the polar coordinates on the outer periphery of the embryo. Specifically, the vertical cleavage detection unit 72 obtains a differential waveform (hereinafter referred to as a polar coordinate differential waveform) obtained by differentiating the polar coordinates on the outer periphery of the embryo with the deflection angle θ. For example, the polar coordinate waveform on the outer periphery of the embryo in FIG. 7 becomes minimal at the declination angles θ1 and θ2 at the vertices DP1 and vertices DP2 of the respective indentations on the outer periphery of the right embryo 102 in FIG. Therefore, for example, as shown in FIG. 8, the polar differential waveform in the vicinity of the deflection angle θ1 is minimized at the deflection angle θa slightly before the deflection angle θ1, and then becomes 0 at the deflection angle θ1. It becomes a maximum at a deviation angle θb slightly after θ1.

  The vertical cleavage detection unit 72 has a minimum to maximum value within a predetermined declination angle θ range (for example, within 5 degrees) in the polar differential waveform, and both the minimum value and the absolute value of the maximum value are both A section that is equal to or greater than a predetermined threshold is extracted. Then, in the extracted section, the vertical cleavage detection unit 72 detects a point on the outer periphery of the embryo corresponding to the deviation angle θ at which the polar coordinate differential waveform becomes 0 and its vicinity as a dent on the outer periphery of the embryo. In the extracted section, when there are a plurality of deflection angles θ at which the polar coordinate differential waveform becomes 0, for example, the point on the outer periphery of the embryo corresponding to the deviation angle θ closest to the center of the section and the vicinity thereof are It is detected as a dent on the outer periphery of the embryo. For example, in the case of the right embryo 102 in FIG. 6, as shown in FIG. 9, the vertex DP1 and its vicinity are detected as the dent D1, and the vertex DP2 and its vicinity are detected as the dent D2.

  In step S47, the vertical cleavage detection unit 72 determines whether or not the number of detected dents is greater than one. If it is determined that the number of detected dents is greater than 1, the process proceeds to step S48.

  In step S48, the vertical cleavage detection unit 72 sets a vertical cleavage boundary surface according to the position and number of dents on the outer periphery of the embryo. For example, in the case of the example shown in FIG. 9, the vertical cleavage detection unit 72 passes through the boundary line L1 connecting the vertex DP1 and the vertex DP2 and is parallel to the z axis, that is, the egg 101 and the embryo passing through the boundary line L1. The plane that divides 102 in the vertical direction is set as the cleavage boundary plane.

  Further, for example, as shown in FIG. 10, the cell C1 of the embryo 102 is divided into cells C11 and C12, the cell C2 is divided into cells C13 and C14, and a new dent D11 is formed in the outer periphery of the embryo 102. When D12 is detected, the vertical cleavage detection unit 72 passes through the boundary line L11 connecting the vertex DP11 of the dent D11 and the vertex DP12 of the dent D12, and passes through the plane parallel to the z axis, that is, the egg 101 passes through the boundary line L11. Further, a surface that divides the embryo 102 in the vertical direction is newly set as a cleavage boundary surface.

  In step S49, the vertical cleavage detection unit 72 sets a unit region based on the cleavage boundary surface. That is, the vertical cleavage detection unit 72 divides the region in which the egg appears in the observation image by the set cleavage boundary surface, and sets each divided region as a unit region. For example, in the example of FIG. 9, the egg 101 is divided into a unit region R1 and a unit region R2 by an cleavage boundary surface passing through the boundary line L1. Further, for example, in the example of FIG. 10, the egg 101 is divided into unit regions R11 to R14 by an cleavage boundary surface passing through the boundary line L1 and a cleavage boundary surface passing through the boundary line L11. The vertical cleavage detection unit 72 supplies information indicating the set cleavage boundary surface and unit region to the horizontal cleavage detection unit 73. Thereafter, the vertical cleavage detection process ends.

  On the other hand, if it is determined in step S47 that the number of detected dents is 1 or less, the process proceeds to step S50.

  In step S50, the vertical cleavage detection unit 72 determines that no vertical cleavage has occurred. Then, the vertical cleavage detection unit 72 sets the entire region in which the egg in the observation image is shown as a unit region. The vertical cleavage detection unit 72 supplies information indicating the set unit area to the horizontal cleavage detection unit 73. Thereafter, the vertical cleavage detection process ends.

  Returning to FIG. 3, in step S <b> 4, the embryo observing section 51 performs horizontal cleavage detection processing. Here, the details of the horizontal cleavage detection process will be described with reference to the flowchart of FIG.

  In step S71, the embryo observation unit 51 acquires a plurality of observation images with different focus positions. Specifically, the horizontal cleavage detection unit 73 instructs the image acquisition unit 61 to acquire an observation image. The image acquisition unit 61 causes the camera 14 to capture an observation image at each position while moving the electric stage 21 at predetermined intervals in the z-axis direction. Thereby, a plurality of observation images having different focus positions in the depth direction (z-axis direction) of the egg (embryo) are photographed. The camera 14 supplies a plurality of taken observation images to the horizontal cleavage detection unit 73 via the image acquisition unit 61.

  In step S72, the horizontal cleavage detection unit 73 selects one unprocessed unit area and sets it as the attention area.

  In step S73, the horizontal cleavage detection unit 73 calculates a detection parameter in the region of interest. For example, the horizontal cleavage detection unit 73 calculates, for each observation image, a sum of contrast values in the attention area or a sum of differential values of luminance in the attention area as a detection parameter.

  In step S <b> 74, the horizontal cleavage detection unit 73 extracts a focus position where the detection parameter becomes maximum and is equal to or greater than a threshold value. Specifically, the horizontal cleavage detection unit 73 obtains a distribution of detection parameters in the attention area with respect to the focus position of the camera 14. The horizontal cleavage detection unit 73 extracts a focus position where the detection parameter is maximized and is equal to or greater than a predetermined threshold in the obtained distribution.

  Here, a specific example of the processing of steps S73 and S74 will be described with reference to FIG. In addition, FIG. 12 is the figure which looked at the egg 151 from the side, and a perpendicular direction is a z-axis direction. In addition, as seen from the direction of FIG. 12, the embryo 152 in the egg 151 is divided into the cell C51 and the cell C52, and the cleavage boundary surface in the vertical direction is not set. Note that a portion surrounded by a solid circle in FIG. 12 indicates a portion in which the horizontal extent of the outer periphery of the embryo 152 is maximized.

  As described above, a plurality of observation images having different focus positions in the depth direction of the embryo 152 can be obtained by capturing the observation image while moving the electric stage 21 in the z-axis direction. Since the embryo 152 has a translucent and rounded shape, when the embryo 152 is photographed from above the electric stage 21, the focus position of the camera 14 is a position F51 at which the horizontal width of the embryo 152 is maximized. Or when it corresponds with F52, the blur of the outer periphery of the embryo 152 in an observation image becomes the minimum, and the focus position of the camera 14 becomes suitable with respect to the embryo 152.

  The two types of detection parameters described above are parameters that indicate the degree of focus on the subject in the observation image. Therefore, in the z-axis direction (embryo depth direction) of the region of interest, the horizontal position of the embryo 152 is maximized by extracting the focus position of the camera 14 that has a maximum detection parameter and is equal to or greater than the threshold value. Positions F51 and F52 (hereinafter referred to as focus position F51 and focus position F52) can be detected.

  In step S <b> 75, the horizontal cleavage detection unit 73 determines whether or not the number of extracted focus positions exceeds 1. If it is determined that the number of extracted focus positions exceeds 1, the process proceeds to step S76.

  In step S76, the horizontal cleavage detection unit 73 sets the division position of the embryo in the region of interest based on the extracted focus position. For example, when the embryo 152 is vertically divided as in the example of FIG. 12, a dent is generated at the boundary between the cell C51 and the cell C52. The position of the dent exists between a focus position F51 at which the horizontal width of the cell C51 is maximized and a focus position F52 at which the horizontal width of the cell C52 is maximized. Therefore, the horizontal cleavage detection unit 73 determines that cleavage has occurred between the detected focus positions F51 and F52, and a position D51 at the center of the focus positions F51 and F52 in the z-axis direction is determined as an embryo. 152 division positions are set. Thereafter, the process proceeds to step S81.

  On the other hand, if it is determined in step S75 that the number of extracted focus positions is 1 or less, the process proceeds to step S77.

  In step S77, the horizontal cleavage detection unit 73 detects the focus position where the detection parameter is maximized.

  In step S78, the horizontal cleavage detection unit 73 determines whether or not the detected focus position is closer to the camera 14 than the initial best focus position. The initial best focus position is a position where the horizontal width of the embryo is maximized and the focus of the camera 14 is assumed to be most appropriate when the embryo is not split horizontally. It is set at the center of the height direction (z-axis direction). If the horizontal cleavage detection unit 73 determines that the detected focus position is at a position closer to the camera 14 than the initial best focus position by a predetermined distance or more, the process proceeds to step S79.

  In step S79, the horizontal cleavage detection unit 73 sets the initial best focus position to the embryo division position in the region of interest. Thereafter, the process proceeds to step S81.

  Here, with reference to FIG. 13 and FIG. 14, a specific example of the processing of steps S77 to S79 will be described. FIG. 13 is a view of the egg 161 viewed from the side, and the vertical direction is the z-axis direction. As seen from the direction of FIG. 13, the embryo 162 in the egg 161 is divided into three cells C61 to C63. Further, in the egg 161, the cleavage boundary surface Bv61 is set by the vertical cleavage detection process, and the unit region R61 and the unit region R62 are set. Furthermore, the initial best focus position BF61 is set at the center of the embryo 162 in the height direction.

  When the egg 161 is photographed from above, the cell C63 is hidden by the cell C61 and the cell C62, so that it is difficult to detect the outer edge of the cell C63. As a result, the horizontal cleavage may be missed. In addition, in the drawing, the part surrounded by the solid line circle among the parts where the horizontal extent of the outer periphery of the embryo 162 is maximized indicates the part that can be observed from above, and the part surrounded by the dotted circle is This indicates a portion that is difficult to observe from above.

  On the other hand, in this case, in the unit region R61, the outer periphery in the horizontal direction of the embryo 162 is maximized, and the focus position F61 at which the detection parameter is maximized is closer to the camera 14 than the initial best focus position BF61. Therefore, the horizontal cleavage detection unit 73 has a horizontal cleavage in the unit region R61 because the focus position F61 is closer to the camera 14 than the initial best focus position BF61, and the cells are below. It is determined that it is hidden, and the division position in the unit region R61 is set to the initial best focus position BF61.

  Similarly, for the unit region R62, the horizontal cleavage detection unit 73 generates a horizontal cleavage in the unit region R62 because the focus position F62 is closer to the camera 14 than the initial best focus position BF61. Therefore, it is determined that the cell is hidden below, and the division position in the unit region R62 is set to the initial best focus position BF61.

  FIG. 14 is a view of the egg 171 viewed from the side, and the vertical direction is the z-axis direction. As seen from the direction of FIG. 14, the embryo 172 in the egg 171 is divided into four cells C71 to C74. Further, in the egg 171, the cleavage boundary surface Bv71 is set by the vertical cleavage detection process, and the unit region R71 and the unit region R72 are set. In addition, in the figure, among the portions where the horizontal extent of the outer periphery of the embryo 172 is maximized, the portion surrounded by a solid circle indicates a portion that can be observed from above, and the portion surrounded by a dotted circle is This indicates a portion that is difficult to observe from above.

  In this case, since the two focus positions of the focus positions F71 and F72 are extracted for the unit region R71, in step S76, the position D71 at the center of the focus position F71 and the focus position F72 in the z-axis direction is set as the division position. Is set.

  On the other hand, regarding the unit region R72, when the image is taken from above, the cell C74 is hidden by the cell C73, the outer edge of the cell C74 is difficult, and only the focus position F73 is extracted. However, since the focus position F73 is closer to the camera 14 than the initial best focus position BF71, the initial best focus position BF71 is set as a division position in the unit region R72 in the process of step S79.

  Returning to FIG. 10, if it is determined in step S78 that the detected focus position is not closer to the camera 14 than the initial best focus position, the process proceeds to step S80.

  In step S80, the horizontal cleavage detection unit 73 determines that no horizontal cleavage has occurred in the region of interest. Thereafter, the process proceeds to step S81.

  In step S81, the horizontal cleavage detection unit 73 determines whether all the unit areas have been processed. If it is determined that there is a unit area that has not been processed yet, the process returns to step S72, and the processes of steps S72 to S81 are repeatedly executed until it is determined in step S81 that all the unit areas have been processed. . Thereby, the process of setting the division position in the z-axis direction is executed in all unit areas.

  On the other hand, if it is determined in step S81 that all unit areas have been processed, the process proceeds to step S82.

  In step S82, the horizontal cleavage detection unit 73 sets a horizontal cleavage boundary surface according to the number and position of the set division positions. For example, in the case of the example of FIG. 12, the plane that passes through the dividing position D51 and divides the embryo 152 in the horizontal direction is set as the cleavage boundary surface. For example, in the case of the example of FIG. 13, a plane that divides the embryo 162 in the horizontal direction through the initial best focus position BF61 that is a division position is set as the cleavage boundary surface. Further, for example, in the case of the example of FIG. 14, for the unit region R71, the plane that divides the embryo 172 in the horizontal direction through the division position D71 is set as the cleavage boundary surface. The plane that passes through the initial best focus position BF71 and divides the embryo 172 in the horizontal direction is set as the cleavage boundary surface.

  The horizontal cleavage detection unit 73 stores information indicating the position of the egg and embryo in the observation image, and the position of the cleavage boundary set by the vertical cleavage detection unit 72 and the horizontal cleavage detection unit 73. This is supplied to the recognition unit 74. At this time, the horizontal cleavage detection unit 73 distinguishes between the cleavage boundary surface set based on the initial best focus position and other cleavage boundary surfaces, and notifies the cell recognition unit 74 of them. Further, the horizontal cleavage detection unit 73 supplies a plurality of observation images with different focus positions to the cell recognition unit 74 before image processing. Thereafter, the horizontal cleavage detection process ends.

  Returning to FIG. 3, in step S5, the cell recognition unit 74 executes a cell recognition process. Here, the details of the cell recognition process will be described with reference to the flowchart of FIG.

  In step S101, the cell recognition unit 74 recognizes each cell in the embryo based on the cleavage boundary surface. Specifically, the cell recognizing unit 74 recognizes the number and positions of cells in the embryo by dividing the embryo shown in the observation image based on the vertical and horizontal cleavage boundary surfaces.

  In addition, the cell recognition unit 74 assigns a label to uniquely identify each recognized cell. The cell recognition unit 74 recognizes each cell in the embryo at a predetermined interval, can track the division of each cell in the embryo, and from which cell each cell in the embryo You can see if it was split. Therefore, the cell recognition unit 74 assigns a label so that the mother cell can easily recognize the same cell.

  For example, in the example of FIG. 9 described above, the cell recognizing unit 74 assigns different binary labels 0000 and 1000 to the cells C1 and C2, respectively. When the embryo 102 changes from the state shown in FIG. 9 to the state shown in FIG. 10, the cell recognition unit 74 assigns label 0000 to the cell C11, assigns label 0100 to the cell C12, and cell C13. Is assigned the label 1000, and the cell C14 is assigned the label 1100. That is, the labels of the cells C11 and C12 are set to the same value 0 in the first digit and the different values 0 or 1 in the second digit so that it can be seen that the cells have divided from the same parent cell C1. In addition, the labels of the cells C13 and C14 are set to the same value 1 in the first digit so that it can be seen that the cells C11 and C12 have divided from the parent cell C2 different from the parent cell C1 of the cell C12. Different values are set to 0 or 1.

  The cell labeling method is not limited to this method, and a method that can uniquely identify each cell and easily identify the parent cell of each cell may be applied.

  In step S102, the cell recognizing unit 74 applies a secondary differential filter to the plurality of observation images acquired from the horizontal cleavage detection unit 73, and extracts the edge of each observation image.

  In step S103, the cell recognition unit 74 obtains the size of each cell. Specifically, the cell recognition unit 74 recognizes the shape of each cell recognized in step S101 in more detail based on each observation image from which the edge is extracted, and obtains the size of each cell. The cell recognizing unit 74 supplies the grading unit 81 with the label assigned to each cell and the cleavage information indicating the position and size of each cell. The grading unit 81 records the time information when the cleavage information is acquired. Thereafter, the cell recognition process ends.

  Returning to FIG. 3, in step S <b> 6, the grading unit 81 determines whether it is time to perform embryo grading. If it is determined that it is not time to perform embryo grading, the process proceeds to step S7. The timing of embryo grading is, for example, the timing when a predetermined time has elapsed since the start of the embryo observation process, the timing when a predetermined time has elapsed since the last grading, The timing at which a predetermined time has elapsed since the start of division, the timing at which division of all cells one generation before, and the like can be considered.

  In step S <b> 7, the egg detection unit 71 determines whether it is time to detect the cleavage. If it is determined that it is not the timing to detect the cleavage, the process returns to step 6, and it is determined in step S6 that it is the timing to perform embryo grading, or the timing to detect the cleavage in step S7. Steps S6 and S7 are repeatedly executed until it is determined that. Note that the timing for detecting the cleavage is set, for example, at predetermined time intervals.

  On the other hand, if it is determined in step S7 that it is time to detect cleavage, the process returns to step S1, and steps S1 to S7 are performed until it is determined in step S6 that it is time to perform embryo grading. This process is repeatedly executed.

  If it is determined in step S6 that it is time to perform embryo grading, the process proceeds to step S8.

  In step S8, the grading unit 81 performs a grading process. Here, the details of the grading process will be described with reference to the flowchart of FIG. 16 and FIG.

  First, the difference between a normal embryo and an abnormal embryo will be described with reference to FIG. FIG. 17 is a diagram schematically showing the state of cleavage of a human fertilized egg. Assuming that the embryo before cleavage is the 0th generation, the 0th generation embryo divides to generate two cells of the first generation. Then, each cell of the first generation divides into two, four cells of the second generation are generated, each cell of the second generation divides into two, and eight cells of the third generation are generated. Then, each cell of the third generation is divided into two, and 16 cells of the fourth generation are generated. That is, each cell in the embryo divides into two, and the number of cells in the embryo increases by a factor of two with each generation.

  In normal embryos, the speed at which cells of the same generation divide is approximately the same, and divide at approximately the same timing. Therefore, when the phylogenetic diagram of the cells as shown in FIG. 17 is created along the time series, the phylogenetic diagram extends almost symmetrically, and there is no timing within the embryo except for the timing at which cleavage is performed. Only cells of the same generation exist. In normal embryos, the size of cells of the same generation is almost the same. Therefore, except for the timing when cleavage is performed, the sizes of cells in the embryo are almost equal.

  On the other hand, in an abnormal embryo, the speed at which cells of the same generation divide varies, or the division of some cells stops. Therefore, when a phylogenetic diagram of cells as shown in FIG. 17 is created in chronological order, the phylogenetic diagram does not extend symmetrically, and cells of different generations are mixed in the embryo. In an abnormal embryo, there is a difference in the size of cells of the same generation. Therefore, the size of the cells in the embryo is not uniform and varies.

  In the grading process of FIG. 16, the grading of the embryo is performed based on the difference in characteristics between the normal embryo and the abnormal embryo.

  In step S121, the grading unit 81 performs grading based on the time taken for the cell of the previous generation to divide. Specifically, the grading unit 81 takes the time required for each cell of the previous generation to divide into cells of the current generation based on the recorded cleavage information (hereinafter referred to as division time). Is calculated. Then, the grading unit 81 obtains the grading value A of the current embryo based on the division time of each cell one generation before, in other words, based on the speed at which each cell one generation before divided. The grading value A is the smallest when, for example, the division time of each cell one generation before is within the normal range, that is, when the division speed of each cell one generation before is all normal, The larger the number of cells whose division time is not within the normal range, and the larger the difference from the normal division time, the larger the cell.

  In step S122, the grading unit 81 performs grading based on the cell size. Specifically, the grading unit 81 calculates the ratio of cells having a size that is recognized as fragmentation in the embryo based on the cleavage information. The grading unit 81 obtains the current grading value B of the embryo based on the calculated ratio. For example, the grading value B is smaller as the proportion of cells having a size recognized as fragmentation is smaller, and is larger as the proportion is larger. Note that the size of a cell refers to an area based or an area based shape.

  In step S123, the grading unit 81 performs grading based on a statistic such as a dispersion value or an average value of the time taken for the cell of the previous generation to divide. Specifically, the grading unit 81 calculates a statistic such as a dispersion value or an average value of the division time of each cell one generation before based on the cleavage information. Then, the grading unit 81 obtains the current grading value C of the embryo based on the dispersion value of the division time of each cell one generation before. For example, the grading value C becomes smaller as the dispersion value of the division time of each cell one generation before becomes smaller, and becomes larger as the dispersion value becomes larger.

  In step S124, the grading unit 81 performs grading based on a statistic such as a variance value or an average value of the size of each cell in the current generation. Specifically, the grading unit 81 calculates a statistic such as a variance value or an average value of the size of each cell in the current generation based on the cleavage information. Then, the grading unit 81 obtains the current grading value D of the embryo based on the variance value of the size of each cell in the current generation. For example, the grading value D becomes smaller as the variance value of the size of each cell in the current generation is smaller, and becomes larger as the variance value is larger.

  In step S125, the grading unit 81 integrates the grading values. Specifically, the grading unit 81 adds the grading values A to D calculated by the current grading process to the integrated value of the grading values of the embryo so far. The grading unit 81 supplies the integrated grading value to the pass / fail determination unit 82. Thereafter, the grading process ends.

  In addition, the grading unit 81 may select and integrate two or more grading values as appropriate from among the grading values A to D according to a user setting. Furthermore, the grading unit 81 may select and output one of the grading values A to D.

  Returning to FIG. 3, in step S <b> 9, the quality determination unit 82 determines whether it is time to determine the quality of the embryo. If it is determined that it is not the timing for determining the quality of the embryo, the process returns to step S6, and the processing of steps S1 to S9 is repeatedly executed until it is determined in step S9 that the timing for determining the quality of the embryo is determined. The The timing for determining the quality of the embryo is, for example, the timing when a predetermined time has elapsed since the start of the embryo observation process, the timing when the embryo grading is performed a predetermined number of times, and the cells in the embryo being a predetermined generation ( For example, the timing at which the third generation or the fourth generation) is split, the timing at which the integrated value of the grading values exceeds a predetermined threshold, and the like can be considered.

  On the other hand, if it is determined in step S9 that it is time to determine whether the embryo is good or bad, the process proceeds to step S10.

  In step S10, the pass / fail determination unit 82 determines pass / fail of the embryo. For example, when the integrated value of the grading value is less than a predetermined threshold, the pass / fail determination unit 82 determines that the embryo being observed is normal, and when the integrated value of the grading value is greater than or equal to the predetermined threshold, Is determined to be abnormal. The pass / fail determination unit 82 outputs information indicating the determination result to the outside. Thereafter, the embryo observation process ends.

  As described above, by tracking the division of each cell in the embryo at a predetermined time interval, the growth state of the embryo can be grasped more accurately, and the quality of the embryo can be accurately determined. Further, it is possible to easily determine the quality of an embryo without performing complicated image processing and computation.

  In the present invention, it is assumed that the pass / fail judgment is made when the embryo is divided into 8 or 16 parts.

  Further, the method for detecting the cleavage of an embryo is not limited to the method described above, and other detection methods may be used. Here, with reference to FIG. 18, another example of a method for detecting the cleavage of an embryo will be described. FIG. 18 shows the same egg 101 as in FIG.

  For example, first, a circle 201 that approximates the outer periphery of the embryo 102 is obtained. Then, in the portion where the outer periphery of the embryo 102 enters the inside of the circle 201, a point (maximum DP1 and vertex DP2 in this case) where the distance from the circle 201 is maximal and is equal to or greater than a predetermined threshold is detected. Then, the vicinity of the detected point may be detected as a dent.

  Furthermore, the type of grading value used to determine the quality of an embryo is not limited to the above-described example. For example, the type of grading value may be increased or decreased, You may make it use the grading value based on other judgment elements, such as the deviation of the length of the boundary line which shows a division position.

  The series of processing of the embryo observing unit 51 described above can be executed by hardware or can be executed by software. When a series of processing is executed by software, a program constituting the software executes various functions by installing a computer incorporated in dedicated hardware or various programs. For example, it is installed from a program recording medium in a general-purpose computer (for example, personal computer 15).

  The program executed by the computer may be a program that is processed in time series in the order described in this specification, or in parallel or at a necessary timing such as when a call is made. It may be a program for processing.

  Further, in the present specification, the term “system” means an overall apparatus composed of a plurality of apparatuses and means.

  Furthermore, the embodiments of the present invention are not limited to the above-described embodiments, and various modifications can be made without departing from the gist of the present invention.

It is a figure which shows one Embodiment of the observation system to which this invention is applied. It is a block diagram which shows the example of a functional structure of an embryo observation part. It is a flowchart for demonstrating an embryo observation process. It is a flowchart for demonstrating an egg detection process. It is a flowchart for demonstrating a vertical direction cleavage detection process. It is a figure for demonstrating the example of the detection method of the dent of the outer periphery of an embryo. It is a graph which shows the example of the polar coordinate of the outer periphery of an embryo. It is a graph which shows the example of the differential waveform of the polar coordinate of the outer periphery of an embryo. It is a figure for demonstrating the example of the detection method of the cleavage of a vertical direction. It is a figure for demonstrating the other example of the detection method of the cleavage of a vertical direction. It is a flowchart for demonstrating a horizontal direction cleavage detection process. It is a figure for demonstrating the example of the detection method of the cleavage in a horizontal direction. It is a figure for demonstrating the other example of the detection method of the cleavage of a horizontal direction. It is a figure for demonstrating the further another example of the detection method of the cleavage in a horizontal direction. It is a flowchart for demonstrating a cell recognition process. It is a flowchart for demonstrating a grading process. It is a figure which shows the example of the systematic diagram of a human embryo. It is a figure for demonstrating the other example of the detection method of the dent of the outer periphery of an embryo.

Explanation of symbols

  11 Microscope, 13 Controller, 14 Camera, 15 Personal computer, 21 Motorized stage, 51 Embryo observation unit, 61 Image acquisition unit, 62 Observation unit, 71 Egg detection unit, 72 Vertical cleavage detection unit, 73 Horizontal cleavage detection Part, 74 cell recognition part, 81 grading part, 82 pass / fail judgment part

Claims (7)

In an embryo observation device for observing embryo growth,
Observation means for detecting cleavage of the embryo based on images taken in time series of the embryo and tracking division of each cell in the embryo;
And an evaluation means for evaluating the growth state of the embryo based on the time required for dividing each cell of the same generation in the embryo. The embryo observation apparatus according to claim 1, wherein the evaluation unit evaluates the growth state of the embryo based on a dispersion value of time required for division of each cell of the same generation in the embryo. The embryo observation apparatus according to claim 1, wherein the evaluation unit evaluates a growth state of the embryo based on a speed at which each cell in the embryo divides. The observation means further detects the size of each cell in the embryo,
The embryo observation apparatus according to claim 1, wherein the evaluation unit further evaluates the growth state of the embryo based on the size of cells of the same generation in the embryo. The embryo observation apparatus according to claim 4, wherein the evaluation means evaluates the growth state of the embryo based on a variance value of the size of cells of the same generation in the embryo. The embryo observation apparatus according to claim 1, wherein the observation unit detects cleavage of the embryo by detecting a dent on the outer periphery of the embryo. The observation means is based on a plurality of images obtained by photographing the embryo while changing the focus position in the depth direction of the embryo, and the parameter indicating the degree of focus in the embryo depth direction is maximized, and The focus position that is equal to or greater than a predetermined threshold value is detected, and when there are a plurality of detected focus positions, it is determined that cleavage occurs between the detected focus positions. Embryo observation equipment.

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