Dish for embryo culture and method for collecting embryo culture solution for chromosome analysis by using said dish

Abstract

This dish for embryo culture comprises a plurality of wells each accommodating an embryo and a culture solution, wherein: the plurality of wells are arranged independently and adjacently at a bottom of the dish for embryo culture; a recess capable of holding the culture solution together with each of the wells is formed for each well at the bottom independently of another recess; a movement inhibiting part, which allows the passing of the culture solution and inhibits the movement of the embryo, is provided between the well and the recess; and the volume of the recess is set to be equal to or greater than the capacity of a culture solution suctioned for an aneuploidy test of chromosomes of an embryo cultured in the well. Consequently, a culture solution can be suctioned for an aneuploidy test of chromosomes of a cultured embryo, and a plurality of wells accommodating embryos can be arranged close to one another.

Description

Embryo culture dish and method for collecting embryo culture fluid for chromosome analysis using said dish

[0001] The present disclosure relates to a dish used for embryo culture and a method for collecting embryo culture medium for chromosome analysis using the dish.

[0002] With the increasing social demand for infertility treatment, various techniques for culturing fertilized treated eggs and embryos (hereinafter collectively referred to as embryos) and associated testing methods have been proposed. These methods are described in, for example, JP 2014-507664 A. In particular, in recent years, there has been an increasing need for chromosomal analysis of embryos during culture, and a method has been proposed for analyzing DNA released from the embryo in the culture medium in which the embryo is cultured, and analyzing chromosomal aneuploidy of the embryo. Such testing requires a certain amount of culture medium. Currently, several μl to several tens of μl of culture medium are aspirated and used for testing.

[0003] However, in recent years, the number of embryos cultured at one time in embryo culture devices has increased, and embryos are cultured in multiple microwells (e.g., 5 x 5 = 25) in a single dish. However, these microwells are unable to draw up the amount of culture medium necessary for testing. Human eggs have a diameter of approximately 150-200 μm, so a microwell with a diameter of, say, 300 μm can only draw up a small amount of culture medium. Drawing up the necessary amount of culture medium in this state could significantly alter the embryo culture environment, which is undesirable.

[0004] Furthermore, simply increasing the capacity of the wells that contain the embryos increases the distance between adjacent wells, which can lead to various problems. For example, in embryo culture devices equipped with a time-lapse function that captures embryos during cultivation, the number of wells that can be imaged at one time decreases, raising concerns that the structure of the imaging device may become more complex. Also, the distance required to transfer embryos between wells increases, increasing the possibility of errors during transfer.

[0005] The present disclosure can be realized in the following forms or application examples. One aspect of the present disclosure is an embryo culture dish including a plurality of wells for accommodating embryos together with a culture medium. The embryo culture dish may include: the plurality of wells arranged independently and adjacent to each other in a bottom portion of the embryo culture dish; a recess for each well that can hold the culture medium together with the well, formed in the bottom portion independently of the other recesses; a movement-inhibiting portion that allows the culture medium to flow between the wells but inhibits the movement of the embryo; and a volume of the recess that is equal to or greater than the volume of the culture medium to be drawn up for testing for aneuploidy of chromosomes of the embryos cultured in the wells.

[0006] Such an embryo culture dish has multiple adjacent wells. When used in an embryo culture device, the distance between the multiple wells is short, making it easy to handle. Furthermore, the process of aspirating culture medium for embryo chromosomal aneuploidy testing can be performed without adversely affecting the embryos being cultured. The short distance between the multiple wells facilitates the transfer of embryos within the wells and reduces transfer errors. Furthermore, when used in a time-lapse embryo culture device, multiple wells can be imaged simultaneously. This facilitates non-invasive chromosomal aneuploidy testing of embryos, with minimal adverse effect on the embryos being tested, and is expected to increase the success rate of embryo transfer (patient pregnancy rate) based on the results of aneuploidy testing.

[0007] 5 is a plan view of an embryo culture device according to a first embodiment; FIG. 6 is a plan view illustrating an example of an embryo culture dish arranged in a culture chamber; FIG. 7 is a view taken along the arrows III-III in FIG. 2; FIG. 8 is an explanatory diagram illustrating the electrical configuration of an embryo culture device; FIG. 9 is a plan view illustrating an example of the arrangement of a first container part, a first container part, and a second container part in an embryo culture dish; an explanatory diagram showing the state in which the embryo is cultured; a process diagram showing the contents of the collection and testing process of embryo culture medium for chromosome analysis; an explanatory diagram showing an example of the display screen of a chromosome aneuploidy detector; a plan view showing the shape of an embryo culture dish of the second embodiment; a plan view showing a modified example of an embryo culture dish of the second embodiment; an explanatory diagram showing another embodiment of the first storage unit and the second storage unit; a process diagram showing an overview of the embryo culture, storage, and transplantation process; and an explanatory diagram showing the state when the embryo culture dish is held in the hand.

[0008] A. First Embodiment: (A-1) Hardware Configuration: FIG. 1 is a plan view of an embryo culture device (also referred to as an incubator) 10, and FIG. 2 is a schematic explanatory diagram showing an embryo culture dish disposed in one culture chamber of the embryo culture device 10. This embryo culture device 10 is used to culture eggs that have undergone in vitro fertilization (hereinafter referred to as "treated eggs") for a certain period of time in a predetermined culture environment, i.e., at constant temperature and humidity. Treated eggs are not necessarily fertilized eggs, and therefore are cultured in the embryo culture device 10 for a predetermined period of time. Note that in vitro fertilization may be performed under a microscope using intracytoplasmic sperm injection (ICS), or it may be performed using conventional in vitro fertilization, in which eggs and sperm are placed together in a predetermined container. The fertilization method used to culture the treated eggs is not important.

[0009] As shown in FIG. 1, this embryo culture device 10 has a total of nine culture units for embryo culture, with five culture units 11-15 arranged on the lower level and four culture units 21-24 arranged on the upper level. Hereinafter, when referring to components related to the culture units collectively, they will be referred to as culture units 11-24. Naturally, the number of culture units is not important. Each culture unit 11-24 includes a culture chamber R11-R24, a lid 17 that seals each of the culture chambers R11-R24, and switches SW11-SW24. By operating switches SW11-SW24, the lid 17 of the corresponding culture unit 11-24 is opened and closed by a drive mechanism (not shown). Naturally, the culture units 11-24 may be configured to be opened and closed manually. A silicone rubber seal is provided on the side of the cover 17 facing the culture chambers R11 to R24, and when the cover 17 is closed, the seal keeps the culture chambers R11 to R24 airtight.

[0010] The embryo culture device 10 is supplied with carbon dioxide gas (CO ) from an external gas cylinder. ２　 ) and nitrogen gas (N ２　 ) are supplied. Gas ports 18, 19 to which these gases are supplied are provided on the back of the embryo culture device 10. Also provided on the back of the embryo culture device 10 are filter ports 28, 29 to which a filter 27 is attached. This filter 27 is used to remove foreign matter such as dust from the outside air taken in by a built-in pump (not shown). Carbon dioxide gas CO 2 input from the gas ports 18, 19 is ２　 , nitrogen gas N ２　 The air-fuel mixture is generated by mixing the air-fuel mixture with the air that has been input through the filter 27 in a predetermined ratio. The ratio of each gas in the mixture is measured by a sensor (not shown) and is always kept constant.

[0011] The case body 20 has a display 70 on its surface. The surface of the display 70 is a touch panel, and various information can be displayed by tapping the displayed buttons. The case body 20 also has a general-purpose connector 26, such as a USB-C or Thunderbolt (registered trademark), to which an input device such as a keyboard or a pointing device such as a mouse can be connected to input various information. Examples of displays on the display 70 and connecting other devices to the general-purpose connector 26 will be described later.

[0012] FIG. 2 shows the culture unit 11 with the lid 17 open. Culture chambers R11 to R24 are provided in each of the culture units 11 to 24. Since the culture chambers R11 to R24 have the same structure, the following description will be given using the culture chamber R11 of the culture unit 11 as an example. The culture chamber R11 is provided with a holding frame 180 on which a dish 191 is placed. A supply port 43 and an exhaust port 44 are provided on the bottom of each of the culture chambers R11 to R24. The supply port 43 is an opening for supplying the gas mixture to the culture chamber R11. The exhaust port 44 is an opening for circulating the gas mixture. The exhaust port 44 is provided in a position covered by the holding frame 180. As mentioned above, the culture chamber R11 becomes almost airtight when the lid 17 is closed, so the internal gas environment is kept constant, but in order to make the temperature distribution within the culture chamber R11 uniform, the mixed gas is supplied from the supply port 43 and exhausted from the exhaust port 44.

[0013] The culture chamber R11 is provided with panel heaters (not shown) on its sidewalls and bottom. The panel heaters maintain a constant temperature within the culture chamber R11. Although not described here, the culture chambers R11 to R24 are equipped with not only heaters but also temperature sensors, gas concentration sensors, and humidity sensors (not shown), which can detect the temperature, gas concentration, and humidity within the culture chamber R11. By feeding back signals from these sensors, the culture chamber R11 is maintained at a constant temperature and humidity, and the gas concentration is also maintained constant. Instead of detecting the temperature, etc. within the culture chamber R11, the environment within the culture chamber R11 may be maintained constant by maintaining the temperature, humidity, gas concentration, etc. of the gas mixture supplied from the supply port 43 constant.

[0014] A dish 191 containing an embryo is placed in the holding frame 180 of the culture chamber R11. The dish 191 shown in FIG. 2 has a generally rectangular shape surrounded by an outer peripheral wall 194. The dish 191 is further surrounded by a wall 192 at the center of the bottom 196 and includes two rows of five storage sections 200 for storing embryos, for a total of ten storage sections 200. The detailed configuration of the storage sections 200 will be described later. A recess 197 is provided in one location on the outer peripheral wall 194 of the dish 191. The dish 191 can be positioned correctly by aligning this recess 197 with a protrusion 182 provided on the holding frame 180 and setting the dish 191 in the holding frame 180. Here, the correct position means an appropriate position for imaging a microwell 195 (described later) provided in the storage section 200.

[0015] The configuration for imaging the dish 191 from the bottom 196 side will be described with reference to FIG. 3 , a view taken along the line III-III in FIG. 2 . In the figure, reference symbol H11 indicates the continuous sidewall and bottom that form the culture chamber R11. As shown in the figure, an illumination unit 170 provided inside the lid 17 is disposed directly above the dish 191. An opening 198 is provided in the bottom H11 of the culture chamber R11 directly below a position corresponding to the center of the dish 191, and a camera module 51 and a lens module 52 attached to the case body 20 are disposed therein. The dish 191 is illuminated by the illumination unit 170 provided in the lid 17, and an image of the embryo 25 contained in the container 200 is captured by the camera module 51. As shown in the figure, the container 200 is filled with culture solution CS, and the embryo 25 is immersed in this culture solution CS. Furthermore, in this state, mineral oil OL is poured into the area surrounded by the wall 192 to cover the storage section 200 in order to prevent evaporation of the culture solution CS.

[0016] Next, the processing performed in the embryo culture device 10 will be described. Figure 4 is an explanatory diagram showing the electrical configuration of the embryo culture device 10. As shown in the figure, the embryo culture device 10 is provided with a control unit 60. In addition to a well-known CPU 61, ROM 62, and RAM 63, the control unit 60 also includes a memory interface 64 for exchanging data with a memory card 65, a general-purpose I / O interface 66 for exchanging signals with external devices, an incubation chamber interface 67 for exchanging signals for controlling the environment within the incubation chambers R11 to R24, a camera interface 68 for issuing imaging instructions to the camera module 51 and acquiring captured images, and a video interface (abbreviated as video I / F) 69 for displaying images on a display 70.

[0017] The CPU 61 uses a high-speed processor that incorporates a DSP function for high-speed processing of fertilization determination based on images (described later) and a vector calculation function for performing determination processing using neural networks, etc. The ROM 62 stores programs that implement processing in the embryo culture device 10, including the time-lapse control processing (described later). The CPU 61 appropriately reads these programs from the ROM 62, loads them into the RAM 63, and executes them to implement the necessary processing and control. The memory card 65 stores images captured by the camera module 51 as well as information about the culture environment, such as the temperature of each culture chamber R11 to R24. Instead of the memory card 65, a magnetic storage medium such as a hard disk or a semiconductor storage medium such as an SSD may be used. Data may also be stored in a so-called cloud connected via a network.

[0018] The display 70 is provided with a touch panel on its surface, and buttons and the like displayed on the display 70 can be selected by touching the display 70. In this embodiment, the display 70 is provided integrally with the embryo culture device 10, but it may be provided separately from the embryo culture device 10 and connected by wire or wirelessly. Alternatively, the embryo culture device 10 may be connected to a computer via a network or the like, and the computer's display may be used as the display 70. Alternatively, a highly portable terminal such as a mobile phone or tablet may be used as the display 70.

[0019] The general-purpose I / O interface 66 is connected to switches SW11 to SW24 provided in the embryo culture device 10, a drive device 71 that opens and closes the lid 17, a warning device 72 that generates a warning sound, and a mixture adjustment device 73 that adjusts the mixture. The mixture adjustment device 73 includes a pressure adjustment valve that adjusts the pressure of carbon dioxide gas, nitrogen gas, etc. supplied to the gas ports 18 and 19, a pump that takes in air through a filter 27, and a pump that sends the mixture to the culture chambers R11 to R24. The culture chamber interface 67 is connected to panel heaters provided in each culture chamber R11 to R24 and control valves V11 to V24 that control the amount of mixture supplied. The control valves V11 to V24 are provided in the piping that extends from the mixture supply pipe 84 that supplies the mixture to the culture chambers R11 to R24.

[0020] A computer (hereinafter referred to as PC) 90 is connected to the general-purpose I / O interface 66 via a general-purpose connector 26. The PC 90 includes a rotary control device 95 in addition to a display 91 and a keyboard (not shown). The control device 95 includes a roughly circular base 97 with a small-diameter dial 96. By rotating the dial 96 left and right, multiple time-lapse images displayed on the display 91 can be displayed forward and backward in chronological order. This type of device is also called a rotary selector. The dial 96 can be rotated left and right, but can also be pressed down, allowing for a "confirm" operation similar to a mouse button when selecting one of the currently displayed images. Alternatively, a dedicated button may be provided on the base 97 to be assigned to the confirm or select operation. If time-lapse images are captured every 10 minutes for 24 hours, for example, 6 x 24 = 144 images will be captured for each embryo. This type of rotary pointing device is useful for quickly referencing multiple images to confirm fertilization.

[0021] (A-2) Details of the Dish: Fig. 5 is an enlarged view of a portion of the dish 191 surrounded by the wall 192, and Fig. 6 is a cross-sectional view taken along the line VI-VI in Fig. 5. As shown, five storage units 200 are arranged in two rows, symmetrically across the top and bottom of the figure, within the roughly rectangular area surrounded by the wall 192. Outside the ten storage units 200, near the wall 192, a plurality of drop placement units 186 are provided for placing the culture solution CS for cleaning. The drop placement units 186 may simply have drops of the culture solution CS placed on the bottom 196, but the bottom 196 may also be provided with a recess or a hydrophilic area to make it easier to place the culture solution CS.

[0022] As shown in the figure, each storage section 200 is formed by connecting a substantially circular first storage section 210 and a trapezoidal second storage section 220 that is elongated in the height direction. The first storage section 210 corresponds to a well, and the second storage section 220 corresponds to a recess. A microwell 195 with a diameter larger than that of an embryo 25 is further formed in the center of the first storage section 210. Therefore, the ten microwells 195 are arranged closely together and fit within one imaging area PA of the camera module 51 located below the dish 191. In other words, the areas where all the microwells 195 are present can be captured in a single imaging session. If an embryo 25 is contained in a microwell 195, the state of that microwell 195 can be captured all at once. The captured image is divided into images of each embryo 25 and saved on a memory card 65 or the like.

[0023] As shown in FIG. 6 , the first and second storage sections 210 and 220 are connected via a connecting portion. This portion is the passageway. A partition 230, slightly higher than the bottom surface of the second storage section 220, is formed in the passageway as a movement-inhibiting portion. The partitions 230 are provided so as to protrude toward the center from both sides of the width direction of the connection section. The partitions 230 protruding from both sides face each other, leaving a small gap at the center of the connection section, i.e., the position where the VI-VI section line passes. This gap is smaller than the expected minimum diameter of the embryo 25, approximately 50 μm in this embodiment. Therefore, even if each storage section 200 is filled with culture solution CS, the embryo 25 contained in the microwell 195 will not move toward the second storage section 220 due to the movement of the culture solution CS.

[0024] The filling and suction of the culture solution CS into the container 200 will now be described. FIG. 7 shows the process of filling the container 200 with the culture solution CS using a pipette 240. The culture solution CS is filled to nearly the upper limit of the container 200. The volume of the container 200 is predetermined. In this embodiment, the volume of the culture solution to be aspirated is set to 30 μl, as described later, so the volume of the container 200 is set to approximately 50 μl. Therefore, 40 μl of culture solution CS is aspirated into the pipette 240 and then transferred to the container 200. Of course, if the volume of culture solution to be aspirated is further reduced, the volume of the container 200 can be adjusted accordingly. The culture solution CS is then covered with mineral oil OL, and a glass capillary is placed on top of the microwell 195, and an embryo 25 is placed inside the microwell 195.

[0025] In this embodiment, after continuing to culture the embryo 25, the culture solution CS in the storage unit 200 is aspirated to detect chromosomal aneuploidy. In this case, as shown in FIG. 8 , the culture solution CS is sucked up from the storage unit 200 using a pipette 240. In FIG. 8 , the culture solution CS still remains at the bottom of the second storage unit 220. However, in reality, the first storage unit 210 and the second storage unit 220 are connected via the gap in the partition unit 230. Therefore, when all of the culture solution CS in the second storage unit 220 is sucked up, the level of the culture solution CS in the first storage unit 210 will be the same as the bottom of the second storage unit 220. Even in this state, the embryo 25 remains immersed in the culture solution CS. Furthermore, even if the culture solution CS in the first storage section 210 moves to the second storage section 220 as it is sucked up by the pipette 240, the gap between the partition sections 230 is narrow enough compared to the diameter of the embryo 25, so the flow of the culture solution CS will not cause the embryo 25 to flow out of the microwell 195 into the second storage section 220.

[0026] (A-3) Embryo Cultivation and Testing: Next, we will explain the testing of embryos cultured using this dish 191. Figure 9 is a flowchart showing the routine for collecting and testing embryo culture medium for chromosome analysis. In this embodiment, fertilized treated eggs are cultured in the embryo culture device 10, and chromosomal aneuploidy testing of the cultured embryos is performed during the culture process. This testing is called preimplantation chromosomal aneuploidy testing (PGT-A). Preimplantation chromosomal aneuploidy testing is often performed by embryo biopsy of a cultured blastocyst, i.e., by collecting approximately 6 to 10 cells from the trophectoderm of the blastocyst. However, in this embodiment, the culture medium in which the embryo was immersed is used. DNA released from the cultured embryo is present in the culture medium, and chromosomal aneuploidy testing can be performed by subjecting this DNA to a gene sequencer.

[0027] When this process begins, 40 μL of culture medium is filled into each of the ten storage sections 200 provided in the dish 191 (step S300). In this embodiment, a single medium (one-step culture medium) containing phenol red is used as the culture medium CS. Because the storage section 200 has a volume of 50 μL, even when filled with 40 μL of culture medium CS, the culture medium CS does not overflow from the storage section 200. After filling all ten storage sections 200 with the culture medium CS, mineral oil OL is filled inside the wall 192, and each storage section 200 filled with the culture medium CS is covered with mineral oil OL (step S310).

[0028] In this state, next, embryos 25 are placed in the microwells 195 of each storage section 200 (step S320). At this time, if necessary, a procedure is performed to remove any air bubbles present in the culture solution CS. One embryo 25 is placed in each microwell 195, and this process is repeated until all microwells 195 have been placed (step S325).

[0029] Once all the microwells 195 have been filled with embryos 25, the dish 191 is then set in one of the culture chambers R11 to R24 of the culture units 11 to 24 of the embryo culture device 10 (step S330). Specifically, the lid 17 of one of the culture chambers is opened, and the dish 191 is placed in alignment with the holding frame 180. The lid 17 is then closed, and culture begins.

[0030] As already explained, the embryo culture device 10 maintains the environment in the culture chamber R11 in a state suitable for culture, and performs a time-lapse culture process (step S340) to culture the embryos while capturing images of the embryos 25 contained in the microwells 195 of each container 200 of the dish 191 at predetermined intervals using the camera module 51 that photographs the dish 191 from below. This time-lapse culture process continues for 72 hours from the start of culture, which is set as the predetermined time (step S345).

[0031] After 72 hours have passed since the start of the culture, the lid 17 is opened, the dish 191 is removed from the culture chamber R11 (step S350), and the culture solution CS is sucked up from each of the containers 200 (step S360). Specifically, as shown in FIG. 8 , the tip of the pipette 240 is inserted into the second container 220 filled with each of the containers 200, and the culture solution CS is sucked up. At this time, all of the culture solution CS in the second container 220 is sucked up. When all of the culture solution CS in the second container 220 is sucked up, the culture solution CS in the first container 210 that is connected to it is also sucked up. However, because the microwell 195 at the bottom of the first container 210 is located deeper than the bottom of the second container 220, the culture solution CS in the microwell 195 is not sucked up. Therefore, even if the pipette 240 is inserted into the second storage section 220 and all of the culture solution CS in the second storage section 220 is aspirated, the embryo 25 remains immersed in the culture solution CS. When aspirating the culture solution CS with the pipette 240, the culture solution CS may be aspirated while measuring so that the amount of culture solution CS becomes 30 μl. The aspirated culture solution CS is individually placed in an analysis sample case for a gene sequencer, which will be described later.

[0032] The embryo 25 remaining in the microwell 195 may be filled with new culture medium CS from the second container 220 and further cultured, or may be removed from the microwell 195 and frozen for transplantation. In the above example, the culture medium CS was aspirated 72 hours after the start of culture. However, taking advantage of the fact that the embryo culture device 10 is a time-lapse embryo culture device, the embryo may be continuously observed and the culture medium CS may be aspirated only after the completion of the blastocyst. In this case, this often occurs around 120 hours after the start of culture. The completion of the blastocyst may be determined by using AI that has previously performed supervised machine learning on the captured image.

[0033] This process is repeated for all the storage sections 200 of one dish 191, and once the culture solution CS has been aspirated from all the storage sections 200 and transferred to an analytical specimen case (step S365), the analytical specimen case is set in a genetic sequencer and aneuploidy testing is performed using the genetic sequencer (step S370). In this embodiment, the genetic sequencer used is "MiSeq" manufactured by Illumina, Inc. The process then exits to "END" to terminate this processing routine.

[0034] The culture medium CS placed in the analysis sample case contains DNA fragments released from the embryo during culture. The DNA fragments were originally isolated from 23 pairs of genes and contain the original genetic information. Therefore, when the culture medium contained in the analysis sample case is analyzed using a sequencer (a so-called next-generation sequencer) that utilizes the NGS method and has a semiconductor chip loaded with templates prepared using normal human genetic samples, it is easy to determine whether there are any excess or deficiency in the 23 pairs of genes. Examples of such next-generation sequencers include the NextSeq550 manufactured by Iiiumina, Inc. and the Ion GeneStudio GX5 manufactured by Ion Torrent.

[0035] An example of the analysis results is shown in Figure 10. As shown in the figure, the next-generation sequencer 300 serving as an analyzer is provided with a storage section 310 for storing analysis sample cases, an operation section 320 for issuing instructions for tasks such as template preparation and the start of analysis, and a display section 330 for displaying the analysis results. After preparations such as template preparation have been completed, the analysis sample cases containing the culture solution CS in which each embryo 25 has been cultured are placed in the storage section 310 of the next-generation sequencer 300, and an instruction to start the analysis is given. After several hours to several dozen hours, the number of genes contained in the culture solution CS is displayed, as shown in Figure 10.

[0036] In the illustrated example, three copies of the tenth gene pair were found instead of two, indicating an extra tenth chromosome, i.e., the embryo being analyzed has chromosomal aneuploidy. Using these analysis results, embryos cultured in the embryo culture device 10 that were not found to have aneuploidy can be transplanted into patients, thereby improving infertility treatment and, therefore, pregnancy rates. This is because embryos with chromosomal aneuploidy are known to either fail to implant after transplantation or, if implanted, have a high risk of miscarriage in the early stages of pregnancy. Furthermore, this method allows for non-invasive testing of the embryo 25. Compared to methods that extract several trophectoderm (TE) cells from a blastocyst and perform chromosomal aneuploidy testing, this method has less impact on the embryo. Furthermore, aneuploidy analysis can be performed while the embryo 25 remains immersed in the culture medium CS, minimizing the impact on the embryo.

[0037] As described above, with the dish 191 of this embodiment, when aneuploidy testing is performed using the culture solution CS in which the embryos 25 have been cultured, even if the culture solution CS is aspirated, the culture solution CS remains reliably in the microwells 195 containing the embryos 25, thereby minimizing the effects of aspirating the culture solution CS. Furthermore, the dish 191 of this embodiment has two rows of five microwells 195 closely spaced, facilitating the transfer of the embryos 25 within the microwells 195 and reducing transfer errors. When used in a time-lapse embryo culture device, the dish 191 of this embodiment can simultaneously capture images of the state of the embryos 25 within each microwell 195. Therefore, even if multiple microwells 195 are provided in one dish 191, the number of times images are taken during time-lapse embryo culture can be reduced without moving the camera module 51.

[0038] In this embodiment, the bottom of the second storage section 220 is rounded toward the center, similar to the first storage section 210, so that when the culture solution CS is sucked up, the culture solution CS in the second storage section 220 can be sucked up efficiently without any waste. Moreover, even if all of the culture solution CS in the second storage section 220 is sucked up with the pipette 240, the bottom is raised at the partition section 230, so the liquid level of the culture solution CS in the first storage section 210 does not drop below this bottom. Therefore, there is no risk of the culture solution CS in the microwell 195 containing the embryo 25 being accidentally sucked up.

[0039] B. Second Embodiment: (B-1) Dish Shape: Next, a second embodiment of the dish will be described. In the second embodiment, as shown in FIG. 11 , the shape of the dish 191A is different. However, other aspects, such as the collection and testing process routine for embryo culture medium for chromosome analysis, are the same as those in the first embodiment. The dish 191A of the second embodiment uses storage units 200 of the same shape as those in the first embodiment. The number of storage units 200 and the shapes of the first storage units 210 and second storage units 220 are also the same. However, in the second embodiment, the first storage units 210 of the ten storage units 200 are arranged closer together than in the first embodiment. To arrange the first storage units 210 closer together, the storage units 200 are arranged so that the outermost ones are more open outward. Therefore, the row-wise spacing L1 between the first storage units 210 in each row is narrower than the row-wise spacing L2 between the second storage units 220 corresponding to the first storage units 210. The interval L2 is the distance between the centers of the areas of the second receptacles 220. As a result, in the dish 191A of the second embodiment, the imaging range PA2 for simultaneously imaging ten first receptacles 210 can be made narrower than the imaging range PA1 of the first embodiment. This also makes it possible to increase the resolution of the embryos 25 in each microwell 195 imaged by the camera module 51. Note that the size of the imaging range PA2 may be the same as in the first embodiment, and the number of receptacles 200 may be increased to, for example, six receptacles x two rows.

[0040] The dish 191A of the second embodiment can narrow the imaging range of each of the multiple microwells 195, and can also ensure a sufficient size for the storage section 200 that stores the culture solution CS for chromosomal aneuploidy testing. Other effects are similar to those of the first embodiment.

[0041] (B-2) Modification 1: Dish 191B, a modification of dish 191A of the second embodiment, is shown in FIG. 12 . In this example, a replenishment fluid storage section is provided in the space on both sides of the first storage sections 210 in the direction of arrangement, resulting from the close placement of the first storage sections 210, for arranging equilibration drops 251, 252 for the additional addition of culture fluid CS. These drops are replenishment fluid and are prepared for injecting additional culture fluid CS into storage section 200 to continue culturing after the culture fluid CS has been sucked up from storage section 200. Because mineral oil OL is placed on dish 191B during culturing, it does not evaporate. Furthermore, because mineral oil OL is kept in the same environment (temperature, humidity, etc.) as the culture fluid CS in storage section 200, even when the additional culture fluid CS is added in place of the sucked-up culture fluid CS, the added culture fluid CS remains identical to the culture fluid that the embryos 25 had been in contact with up until that point, which is preferable.

[0042] In the first and second embodiments described above, the volume of the storage unit 200 is 50 μl, 40 μl of culture medium is filled, and 30 μl of culture medium is aspirated for testing. However, the volumes and capacities shown here are merely examples. If the volume of culture medium required for chromosomal aneuploidy testing becomes smaller and the volume of aspirated culture medium CS becomes, for example, approximately 10 μl, the volume of the storage unit 200 may be approximately 20 μl and the volume of filled culture medium CS may be approximately 15 μl. In general, if the volume of the storage unit 200 is V200, the volume of filled culture medium CS is Vcs, and the volume of aspirated culture medium CS for testing is Vout, the relationship Vout < Vcs ≦ V200 should be satisfied. Furthermore, if the volume of the microwell 195 is Vmw and Vout + Vmw ≤ Vcs is satisfied, when the culture solution CS is sucked out, the culture solution will remain in the microwell 195, which is preferable.

[0043] (B-3) Modification 2: Another example of the arrangement of the storage section 200 is shown in Figure 13. In this example, an even larger number of first storage sections 210 of the storage section 200 are arranged closely together. In this example, the storage section 200 is arranged circumferentially, with a total of 16 first storage sections 210 equipped with microwells 195, eight on the inner periphery and eight on the outer periphery. Even in this case, the same effects as in the second embodiment can be achieved, and the imaging range PA3 required to image the large number of microwells 195 can be made smaller.

[0044] C. Embryo Cultivation, Preservation, and Transfer Treatment: In the above-described embodiment, no particular explanation was given regarding the handling of the embryo after the culture medium CS has been drawn up. However, an example of the handling of the embryo for transfer will now be described. Figure 14 is a process diagram illustrating the embryo cultivation, preservation, and transfer treatment. After the processing routine shown in Figure 9 is completed, first, a process is performed in which the culture medium CS is added to each storage unit 200 (step S400). This process is continued until the addition to all storage units 200 is completed (step S405).

[0045] Once the addition of the fluid to all the containers 200 is complete, the dish 191 is returned to the incubation chamber R11 of the embryo incubation device 10 (step S410). Closing the lid 17 of the incubation chamber R11 resumes incubation, and the embryos continue to be cultured (step S420). After incubation resumes, the system waits for approximately 40 hours (step S425), and once 40 hours have elapsed, the results of the chromosomal aneuploidy testing are read from the results of the next-generation sequencer (step S430). Based on the results, normal embryos are selected from the multiple embryos that were tested for chromosomal aneuploidy (step S440).

[0046] If any of the tested embryos are found to have chromosomal aneuploidy, they are deemed to be abnormal, and the embryos other than those determined to be abnormal (hereinafter referred to as normal embryos) are cryopreserved. Once the patient is in a suitable condition for transplantation, the cryopreserved embryos are thawed and transplanted into the patient (step S450). This completes the embryo culture, storage, and transplantation procedures.

[0047] When removing the dish 191 from the culture chamber R11 ( FIG. 9 , steps S330 and S350) or returning it to the culture chamber R11 ( FIG. 14 , step S410), the user directly grasps and operates the dish 191 with their fingers HD. This is illustrated in FIG. 15 . The dish 191 used in each of the above-described embodiments is generally rectangular and is held between the thumb and opposing fingers, such as the middle finger. The outer peripheral wall 194 of the dish 191 is provided with a recess 400 as a gripping assistance structure. Therefore, when the dish 191 is grasped with the fingers HD, the fingers fit into the recess 400 in the outer peripheral wall 194, allowing the dish 191 to be firmly held. Note that the recess 400 is provided on at least one of the four sides of the generally rectangular dish 191. Recesses 400 may be provided at two locations where the thumb and opposing finger contact. Instead of the depressions 400, fine irregularities may be provided on the outer surface of the outer peripheral wall 194. In this case, the irregularities may be provided only at the locations of the depressions 400, or the outer peripheral wall 194 may have a widely irregular surface. The irregularities may be formed by making the surface of the molding die irregular when forming the dish 191, or may be formed later by roughening the surface using a method such as sandblasting. Note that the recesses 197 are not shown in the figures.

[0048] D. Other embodiments: (1) The embryo culture dish of the present disclosure can be embodied as an embryo culture dish including a plurality of wells for accommodating embryos together with a culture medium. In this embryo culture dish, the plurality of wells are arranged independently and adjacently in the bottom of the embryo culture dish, and a recess capable of holding the culture medium together with the well is formed in the bottom for each well, independently of the other recesses. A movement-inhibiting portion is provided between the wells to allow the culture medium to flow but inhibit the movement of the embryo, and the volume of the recess may be equal to or greater than the volume of the culture medium to be drawn up for testing for aneuploidy of chromosomes of embryos cultured in the wells.

[0049] Such an embryo culture dish has multiple adjacent wells. When used in an embryo culture device, the distance between the wells is short, making it easy to handle. Furthermore, the process of aspirating culture medium for chromosomal aneuploidy testing of embryos can be performed without affecting the embryos during culture. Furthermore, a movement-inhibiting section is provided between the well and the recess, allowing the culture medium surrounding the embryo to be aspirated while retaining the embryo in place. Furthermore, the short distance between the wells facilitates embryo transfer within the wells and reduces transfer errors. Furthermore, when used in a time-lapse embryo culture device, multiple wells can be imaged simultaneously. This facilitates non-invasive chromosomal aneuploidy testing of embryos, with minimal impact on the embryos being tested, and is expected to increase the success rate of embryo transfer (patient pregnancy rate) based on the results of aneuploidy testing.

[0050] The number of wells per embryo culture dish may be any number, with a minimum of two, as long as the wells are adjacent to each other and the storage section can be arranged. The wells may be hemispherical, elliptical in plan view, or parabolic in vertical cross section, depending on the shape of the embryos to be stored. Furthermore, the multiple wells do not necessarily have to be the same shape, and may each have a different shape. This allows visual recognition of which well's embryo is being manipulated in the microscope field of view.

[0051] (2) In such a configuration, at least some of the multiple wells may be arranged in two rows, and each of the recesses corresponding to wells in one row may be located on the opposite side of the well from the recesses corresponding to wells in the other row, and may have a shape extending a predetermined length outward from the corresponding well. This makes it easy to arrange multiple wells adjacent to each other and to arrange a storage section of sufficient size. The predetermined length may be adjusted according to the size of the storage section, particularly the amount of culture medium to be stored. The storage section may be rectangular or trapezoidal, with its horizontal length increasing as it moves away from the well. It may also be elliptical or oval in plan view. Furthermore, the depth of the storage section may be uniform throughout, or, like the wells, may have a curved shape that becomes deeper toward the center. In the latter case, when the culture medium is sucked up, the culture medium collects in the deepest part, allowing for efficient sucking of the culture medium in the storage section.

[0052] (3) In this configuration, the spacing between the wells in each row may be narrower than the spacing between the recesses corresponding to the wells. This allows for more efficient well placement and increases the number of wells that can be included in the embryo culture dish. Therefore, if this embryo culture dish is used in a time-lapse embryo culture device, the number of wells that can be imaged at one time can be increased.

[0053] (4) In this configuration, all of the wells in the embryo culture dish may be positioned within a range that allows a camera installed in a time-lapse embryo culture device that houses the embryo culture dish to capture images without moving relative to the embryo culture dish. This allows the camera installed in the time-lapse embryo culture device to capture images of all of the wells at once, eliminating the need to move the camera relative to the embryo culture dish. Alternatively, multiple sets of multiple wells and recesses may be provided in a single embryo culture dish, and images may be captured sequentially by moving the camera.

[0054] (5) In this configuration, the migration-inhibiting portion may be a passage connecting the well and the recess, and may include a passage shallower than the depth of the well. In this way, even if the culture medium in the recess is completely aspirated with a pipette or the like, the depth of the migration-inhibiting portion is shallower than the depth of the well, so the level of the culture medium in the well will not drop below this passage. Therefore, there is no risk of accidentally pumping out the culture medium in the well containing the embryo being cultured. Essentially, the migration-inhibiting portion only needs to be able to inhibit the embryo from moving out of the well, and its depth may be approximately the same as the depth of the well. In this case, it is possible to aspirate out all of the culture medium in the well, but if it is necessary to leave some of the culture medium around the embryo, the amount of aspirated can be adjusted. For example, an adapter may be provided on the tip of the pipette used to aspirate the culture medium, allowing it to be spaced a predetermined distance from the bottom of the recess. In this way, aspirating the culture medium with the pipette leaves a predetermined level of culture medium above the bottom of the recess.

[0055] (6) In this configuration, the movement-inhibiting portion may be provided at the connection between the well and the recess and include a passage portion having a gap through which the culture medium flows, the gap of the passage portion being narrower than the smallest diameter of the embryo contained in the well. This makes it easy to inhibit the movement of the embryo within the well. The passage may simply be formed as a gap narrower than the size of the embryo. In this case, the width of the passage itself may be narrowed, or a fence or mesh may be provided in a wide passage to inhibit the movement of the embryo.

[0056] (7) In this configuration, the well may have a microwell at the bottom thereof that is smaller than the outer shape of the well but larger than the outer shape of the embryo to be contained. This allows the embryo to be positioned within the microwell, thereby limiting the position of the embryo to be imaged. It also limits the movement of the embryo during culture, which is also desirable for embryo culture. Of course, the microwell need not be necessary. It is also possible to limit the movement of the embryo within the well by providing a structure, such as a ring-shaped protrusion with a diameter slightly smaller than the diameter of the embryo, at the bottom of the well.

[0057] (8) In this configuration, a replenisher container for accommodating a replenisher culture medium for replenishing the culture medium in the well may be provided on the bottom surface of the embryo culture dish, thereby allowing for easy replenishment of the equilibrated culture medium that has been acclimatized to the culture environment.

[0058] (9) In such a configuration, an outer peripheral wall may be provided to define the outer periphery of the embryo culture dish, and the outer surface of the outer peripheral wall may be provided with a gripping assistance structure that assists the user in gripping the embryo culture dish with their fingers. This makes it easier to handle the embryo culture dish.

[0059] (10) In this configuration, the well and the recess of the embryo culture dish may be filled with the culture medium, an embryo to be cultured may be placed in the well filled with the culture medium, the well and the recess filled with the culture medium may be covered with oil to prevent evaporation of the culture medium, and a predetermined volume of the culture medium may be aspirated from the recess a predetermined time after the embryo is placed in the well, and the aspirated culture medium may be subjected to analysis by an analyzer capable of analyzing chromosomal aneuploidy using DNA contained in the culture medium. This allows chromosomal aneuploidy testing of embryos to be performed non-invasively, with little or no impact on the embryo being tested. Therefore, it is expected that the success rate of embryo transfer (patient pregnancy rate) based on the results of the aneuploidy testing may be increased.

[0060] The present disclosure is not limited to the above-described embodiments and can be realized in various configurations without departing from the spirit thereof. For example, the technical features in the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted.

[0061] The present disclosure is not limited to the above-described embodiments and can be realized in various configurations without departing from the spirit thereof. For example, the technical features of the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention section can be appropriately replaced or combined to solve some or all of the above-described problems or achieve some or all of the above-described effects. Furthermore, if a technical feature is not described as essential in this specification, it can be deleted as appropriate. For example, some of the configurations realized by hardware in the above-described embodiments can be realized by software.

[0062] 10...embryo culture device, 11 to 24...culture unit, 17...lid, 18...gas port, 20...case body, 25...embryo, 26...general-purpose connector, 27...filter, 28...filter port, 43...supply port, 44...exhaust port, 51...camera module, 52...lens module, 60...control unit, 61...CPU, 62...ROM, 63...RAM, 64...memory interface, 65...memory card, 66...general-purpose I / O interface, 67...culture chamber interface, 68...camera interface, 70...display, 71...driver, 72...warning device, 73...air-mixture adjusting device, 84...air-mixture adjusting device Gas supply piping, 91...display, 95...control device, 96...dial, 97...base portion, 170...illumination unit, 180...holding frame, 182...convex portion, 186...drop placement portion, 191, 191A, 191B...dish, 192...wall, 194...peripheral wall, 195...microwell, 196...bottom portion, 197...recess, 198...opening, 200...storage portion, 210...first storage portion, 220...second storage portion, 230...partition portion, 240...pipette, 251...balance drop for additional injection, 300...next-generation sequencer, 310...storage portion, 320...operation portion, 330...display portion, HD...fingers

Claims

1. An embryo culture dish having a plurality of wells for containing an embryo together with culture medium, the plurality of wells being arranged independently and adjacent to each other on the bottom of the embryo culture dish, a recess capable of holding the culture medium together with the well being formed on the bottom independent of the other recesses, a movement inhibition portion is provided between the well and the recess which allows the culture medium to flow but inhibits the movement of the embryo, and the volume of the recess is set to be equal to or greater than the volume of culture medium to be drawn up for aneuploidy testing of chromosomes of the embryos cultured in the wells.

2. An embryo culture dish as described in claim 1, wherein at least a portion of said plurality of wells are arranged in two rows, and each of said recesses corresponding to wells arranged in one row is located on the opposite side of the well from said recesses corresponding to wells in the other row, and has a shape extending a predetermined length outward from the corresponding well.

3. An embryo culture dish as claimed in claim 2, wherein the intervals between the wells in each row in the row direction are narrower than the intervals between the recesses provided corresponding to the wells in the row direction.

4. An embryo culture dish as described in any one of claims 1 to 3, wherein all of the wells in the embryo culture dish are positioned within a range that can be imaged by a camera provided in a time-lapse embryo culture device in which the embryo culture dish is housed, without moving relative to the embryo culture dish.

5. An embryo culture dish as described in any one of claims 1 to 4, wherein the movement-inhibiting portion is a passage portion connecting the well and the recess, and the passage portion is shallower than the depth of the well.

6. An embryo culture dish as claimed in any one of claims 1 to 5, wherein the movement-inhibiting portion is provided at the connection between the well and the recess and comprises a passage portion having a gap through which the culture medium flows, and the gap of the passage portion is narrower than the smallest diameter of the embryo to be contained in the well.

7. An embryo culture dish according to any one of claims 1 to 6, wherein the well is provided at the bottom with a microwell that is smaller than the outer shape of the well and larger than the outer shape of the embryo to be contained therein.

8. An embryo culture dish as described in any one of claims 1 to 7, comprising a supplementary culture medium storage section on the bottom surface of the embryo culture dish for storing supplementary culture medium for replenishing the culture medium in the recess.

9. An embryo culture dish as claimed in any one of claims 1 to 8, comprising an outer peripheral wall defining an outer periphery, the outer surface of the outer peripheral wall being provided with a gripping assistance structure that assists a user's fingers in gripping the embryo culture dish.

10. A method for collecting embryo culture fluid for chromosome analysis, comprising the steps of: filling the well and the recess of an embryo culture dish described in any one of claims 1 to 9 with the culture fluid; placing an embryo to be cultured in the well filled with the culture fluid; covering the well and the recess filled with the culture fluid with oil that suppresses evaporation of the culture fluid; sucking up a predetermined volume of the culture fluid from the recess a predetermined time after the embryo is placed in the well; and providing the sucked up culture fluid for analysis by an analyzer capable of chromosomal aneuploidy analysis using DNA contained in the culture fluid.

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