Method and device of sorting fertilized embryo

Abstract

To provide a method and a device of evaluating and sorting a nucleolus of an embryo based on morphology in a season specific manner.SOLUTION: An evaluating and sorting method of an embryo has: a step of determining whether or not at least 2 blastomeres of 4 cells (blastomeres) are blastomeres SN (single nucleolus) having a single large nucleolus or blastomeres MN (multi-nucleoli) having a plurality of small nucleoli, in an embryo of 4-cell stage of an animal other than human; and a step of sorting an embryo in which at least two or more blastomeres are SN out of the 4 blastomeres.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for selecting fertilized embryos according to their developmental potential. [Background technology]

[0002] On July 25, 1978, Dr. Robert Edwards and Dr. Patrick Steptoe gave birth to the world's first IVF baby, Louise Brown, in the UK. Since then, more than 8 million people have been born through in vitro fertilization (IVF) around the world. In Japan, more than 60,000 babies are born each year (as of 2020), and approximately one in 14 newborns is born through IVF. The history of IVF research in mammals can be traced back to the 19th century, but practical applications began in the 1950s with research using rabbits and mice. Dr. Edwards and his colleagues conducted IVF research using animals and humans for over 10 years, leading to success in 1978, after which they were awarded the Nobel Prize in Physiology or Medicine in 2010. It is known that IVF pregnancies have a lower birth rate, more placental abnormalities, and lower birth weight than those that occur in vivo (natural pregnancy). Additionally, experiments with mice have shown that offspring born through in vitro fertilization become obese and suffer from hyperglycemia.

[0003] In order to hope for even the slightest birth, it is necessary to return good embryos that are expected to have a high birth rate to the uterus. Currently, embryo evaluation methods generally used in humans (embryo evaluation methods that are also handled in textbooks and widely used) include, for example, the Gardner classification (Non-Patent Document 1) (Figure 1) for blastocysts, and the VeecK classification (Non-Patent Document 2) and the Istanbul Consensus (Non-Patent Document 3) for early cleavage stage embryos. Furthermore, there are recent evaluation methods using metabolomic profiling to look at the uptake of pyruvate and glucose, and oxygen consumption and amino acid metabolic turnover using spectrophotometric testing. However, these evaluation methods are influenced by the subjectivity of the doctor or embryologist who handles the embryo, and by evaluation systems such as embryo development evaluation, and have problems such as the need for expensive equipment. In fact, even when embryos are selected based on such morphological characteristics, the implantation rate does not exceed about 35% (Non-Patent Document 4).

[0004] In vitro fertilization is a basic technique used to obtain offspring in various industries, such as infertility treatment and livestock farming, for various animal species, including livestock, laboratory animals, and humans. However, in all cases, embryos fertilized in vitro have a limited rate of development into blastocysts and offspring, and it is particularly important to evaluate and select the developmental potential of the embryo when transplanting it into the fallopian tube or uterus of a surrogate mother, determining "which embryo is most likely to produce offspring." However, although various methods have been proposed and used, such as those shown in Patent Documents 1 to 5, the offspring rate is low and is still not sufficient. In embryo transfer, in the case of humans, who are singletons, the number of embryos that can be transferred back to the uterus is limited to one or two to avoid multiple births. On the other hand, in multifetal animals such as mice, the number of embryos that can be transferred back to the foster mother is large, such as about 20, so even if the development rate is somewhat low, offspring can be obtained from one embryo transfer. For this reason, embryo evaluation is more important in humans. Furthermore, an important point in this evaluation is that there is a restriction that the evaluation must be non-invasive because the embryos must not be damaged when evaluating individual embryos. For these reasons, morphological evaluation has remained the best and main method for selecting preimplantation embryos for the past 30 years (Non-Patent Document 5). [Prior art documents]

Chartered Documents

[0005] [Patent Document 1] Special form 2019-509057 [Patent Document 2] Special form 2021-519925 [Patent Document 3] Special opening 2023-7271 [Patent Document 4] Special form 2022-547900 [Patent Document 5] Special opening 2023-81880

Non-licensed documents

[0006]

Non-patent document 1

Non-patent document 2

Non-patent document 3

[0007] Although morphological evaluation remains the primary method for selection of preimplantation embryos, it is still inadequate due to low birth rates. New embryo evaluation and selection methods based on stage-specific nucleolar morphology are needed. [Means for solving the problem]

[0008] According to the present invention, the following inventions are provided. [1] A method for selecting embryos, comprising the steps of: determining whether at least two of the four cells (blastomeres) in a four-cell stage embryo of an animal other than a human are SN (single nucleolus) blastomeres having one large nucleolus, or MN (multi-nucleoli) blastomeres having multiple small nucleoli; and selecting embryos in which at least two or more of the four blastomeres are SN and embryos in which fewer than two of the four blastomeres are SN. [2] The method for selecting embryos described in [1], wherein the 4-cell stage embryos are embryos obtained by in vitro fertilization (IVF) of ICR mouse strains. [3] A method for selecting embryos according to [1] or [2], characterized in that the method of determination is performed using a bright-field microscope. [4] An embryo sorting device comprising: an imaging unit that takes images of an embryo; a determination unit that determines whether a cell (blastomere) is an SN (single nucleolus) having a large nucleolus or an MN (multi-nucleoli) having multiple small nucleoli; a sorting unit that selects between embryos in which at least two of the four blastomeres at the four-cell stage are SN and embryos in which less than two of the four blastomeres are SN; and an output unit that outputs the results of the determination. Effect of the Invention

[0009] According to the present invention, the above-mentioned problems in the prior art can be solved and the above-mentioned object can be achieved. That is, evaluation of embryos based on the morphology of nucleoli at the 4-cell stage can be used as a simple and objective indicator of the embryos that will become offspring in the future. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows how blastocysts are graded according to the Gardner classification. [Diagram 2] Figure 2 shows comparative photographs of an in vitro fertilized embryo (top) and an in vivo fertilized embryo (bottom). [Diagram 3] FIG. 3 shows a flowchart of the screening and evaluation method of the present invention. [Figure 4]FIG. 4 shows the results of comparing the morphology of nucleoli in four-cell stage embryos. [Diagram 5] FIG. 5 shows the proportions of SN0 to SN4 in in vitro fertilized embryos and in vivo fertilized embryos. [Figure 6] FIG. 6 shows a photograph of a 4-cell stage embryo taken with a bright field microscope. [Figure 7] FIG. 7 shows photographs of offspring of ≧2SN and 1MN embryos. [Figure 8] FIG. 8 shows a comparison of birth rate (a), birth weight (b), and placental weight (c) of ≧2SN and 1MN embryos. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in detail below.

[0012] The present invention is based on the discovery that the morphology of nucleoli (one of the organelles in the nucleus of a cell, the site of ribosome synthesis) differs from the 4-cell stage onwards, as a result of comparing in vivo fertilization with in vitro fertilization. As shown in Figure 2, in IVF embryos at the 4-cell stage, the nucleoli of each cell (blastomere) are more common in IVF embryos with a single large nucleolus (C, red dotted arrow), whereas in IVF embryos, there are more cells with multiple nucleoli (C', white arrow). The arrows indicate nucleoli. As a result of comparing the changes in nucleoli during the developmental process of in vitro and in vivo fertilized embryos, it was found that the most significant difference was observed at the 4-cell stage, and that in IVF embryos, there were more cells with a single large nucleolus, and that these embryos had a higher probability of becoming liveborn (birth rate) than embryos consisting of blastomeres with multiple nucleoli. This led to the invention of a method for evaluating and selecting embryos based on the morphology of the nucleoli of the embryos at a stage specific to the stage.

[0013] The production of in vitro fertilized embryos is carried out according to a known standard method, in which sperm and eggs are collected from ICR mice, and in vitro fertilization (IVF) is carried out using HTF medium to produce in vitro fertilized embryos. Mouse embryos that have been fertilized in vitro and cultured in a standard culture medium CZB until they reach the four-cell stage are transferred to individual culture drops. For the embryos in each culture drop, carefully observe the nucleoli in at least two blastomeres of each embryo using a bright-field microscope with a 40x objective lens to determine their shape. A single large nucleolus (SN) was found in each blastomere, and embryos in which this was found in two or more blastomeres (embryos ≧2SN) were classified as "≧2SN embryos," while the remaining embryos were classified as "MN embryos." The "≧2SN embryos" judged to be ≧2SN are collected from each drop, and the remaining embryos are grouped as "MN embryos," and all embryos are divided into one of the two groups. Embryos from each group were transferred into the oviducts of surrogate mothers, and the presence or absence of offspring was examined on the 19th day. The group with "≧2SN embryos" showed a high offspring rate.

[0014] Therefore, by dividing in vitro fertilized embryos into "≧2SN embryos" and "MN embryos," it is possible to select good embryos with a high birth rate.

[0015] The method of the present invention can also be carried out using a device with an automatic control mechanism. The device can be configured as an embryo selection device, which includes a photographing unit equipped with a camera or the like to capture an image of an embryo, a determining unit that uses image processing or machine learning to determine whether a cell (blastomere) is a blastomere SN (single nucleolus) having a large nucleolus or a blastomere MN (multi-nucleoli) having multiple small nucleoli, a selecting unit that uses a sorting device such as a cell sorter equipped with a microchannel to select embryos in which at least two or more blastomeres are SN among four blastomeres at the four-cell stage, and an output unit that outputs the determination results to a printer, display, or connected equipment. In particular, in the production of a large number of experimental animals such as mice, a selection operation can be performed on a large number of embryos using an automated sorting device. EXAMPLES

[0016] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0017] This refers to materials used in in vitro fertilization, etc. (1) Experimental animals used (mice) ICR male and female mice aged 8 weeks or older (purchased from Japan SLC Co., Ltd. or bred in-house) were used. (2) Culture medium used The composition of the CZB stock solution is shown in Table 1.

[0018] [Table 1]

[0019] The composition of the Hepes-CZB stock solution is shown in Table 2.

[0020] [Table 2]

[0021] The composition of the HTF stock solution is shown in Table 3.

[0022] [Table 3]

[0023] Each stock solution was prepared in the same manner as above. 2+ -free stock solution, Hepes-CZB stock solution, and HTF stock solution were prepared. 100 ml of Milli-Q water was measured into a 200 ml medium preparation container using a graduated cylinder, a line was drawn on the liquid level of the 100 ml, and the Milli-Q water was discarded so that the container was about three-quarters full. Then, the reagents in Tables 1 to 3 were measured from top to bottom and dissolved in the container. Finally, the mixture was filled up to the marked line with Milli-Q water and refrigerated at 4°C overnight. The next day, the mixture was filter-sterilized using a 0.22 μm filter (NY-14831, CORNING) in a clean bench, and the stock solution was stored in a refrigerator at 4°C.

[0024] The composition of the CZB medium is shown in Table 4.

[0025] [Table 4]

[0026] The composition of the HTF medium is shown in Table 5.

[0027] [Table 5]

[0028] Each medium was prepared in the same manner: CZB medium, Ca 2+ -free CZB medium and HTF medium were prepared. Exactly 49.5 ml of filter-sterilized stock solution was dispensed into a 50 ml centrifuge tube (FALCON, 352098). Then, dissolve each of the reagents in order from the top of each table in Tables 4 and 5. Add 0.50 ml of CaCl2·2H2O to make a total of 50 ml. Finally, add BSA and leave it to dissolve naturally (without shaking) at 4°C overnight. The next day, filter-sterilize the solution in a clean bench using a 50 ml syringe (ss-50ESz, TERUMO) and a 0.22 μm filter (SLHU033RS, Durapove) and dispense into assist tubes (60.9221.530S, SARSTEDT).

[0029] (3) Preparation of reagents PMSG (Pregnant Mare Serum Gonadotropin) Three ampoules of 1000 IU of serotropin (Asuka Animal Health Co., Ltd., E109A) were prepared and dissolved in two ampoules of 5 ml of the included physiological saline. A 50 ml syringe (TERUMO, SS-50ESG) and an 18G needle (TERUMO) were used. The dissolved hormone solution (3000 IU) was transferred to a 10 cm dish (BD FALCON, 8084478), and 50 ml of physiological saline (Otsuka Pharmaceutical, 5D77P) was added using an electric pipette. After pipetting, the solution was dispensed into 1.5 ml Eppendorf tubes (eppendolf, D156219J) and stored at -30°C until use.

[0030] hCG (human chorionic gonadotropin) One ampule of 3000 IU gonadotropin (Asuka Animal Health Co., Ltd., E764A) was prepared and dissolved in two ampoules of 5 ml of the included physiological saline. A 50 ml syringe (TERUMO, SS-50ESZ) and an 18G needle (TERUMO) were used. The dissolved 3000 IU hormone solution was transferred to a 10 cm dish (BD FALCON, 8084478), and 50 ml of physiological saline (Otsuka Pharmaceutical, 5D77P) was added using an electric pipette. After pipetting, the solution was dispensed into 1.5 ml Eppendorf tubes (eppendolf, D156219J) and stored at -30°C until use.

[0031] Shows the method of fertilization.

[0032] In vitro fertilization (IVF) (1) Superovulation treatment After 5 p.m. in the evening, 7.5 IU of PMSG was injected intraperitoneally into ICR female mice aged 8 weeks or older, and 48 hours later, 7.5 IU of hCG was injected intraperitoneally using a 1 ml syringe (TERUMO, SS-01T) and a 26G needle (TERUMO) to perform superovulation treatment. (2) Preparation of insemination dish for IVF On the day before the experiment, one 200 μl drop of HTF and several smaller drops of about 20 μl were prepared on a 6 cm dish, mineral oil (ORGANIC SPECIALTY CHEMICALS Mineral Oil Intestinal Lubricant Laxative NDC, 67618-108-16) was poured on top to cover the drops, and the mixture was equilibrated in an incubator (37°C, CO2:5%). (3) Preparation of capacitation dishes On the day before the experiment, a thin drop of 200 μl of HTF was prepared in a 6 cm dish, covered with mineral oil, and equilibrated in an incubator. (4) Semen collection ICR male mice aged 10 weeks or older were euthanized by cervical dislocation, and the tail of the epididymis was collected from the body of the male mouse. Fat and other substances were removed from the collected tail of the epididymis, and the tail of the epididymis was pierced two or three times with a 26G injection needle (TERUMO), and the semen was placed into a capacitation dish prepared the day before using tweezers. The semen was then cultured in an incubator for one hour to complete the capacitation. (5) Sperm count A Birker-Türk counting chamber was used to count sperm. 2 μl was taken from the capacitation drop, diluted 10-fold with 18 μl of ultrapure water, and placed in the counting chamber. Four sections were counted, with 16 squares in a 4 × 4 grid. The average of the four sections was used to determine the sperm concentration of 1.0 × 10 5 The amount was calculated and adjusted to sperm / ml. (6) Egg collection Superovulated ICR female mice were euthanized by cervical dislocation in the same manner as male mice, and the oviducts were collected from the body. The collected oviducts were placed in an Eppendorf tube containing mineral oil and brought to an insemination dish prepared the day before, and the oviduct was placed on the mineral oil in the dish. The ampulla of the oviduct was torn with tweezers, and the ovocumulus complexes were released and placed in a 200 μl HTF drop. (7) Insemination Count the sperm in the 200 μl drop of oocyte cumulus complexes in the insemination dish and find 1.0 x 10 5 Capacitation medium adjusted to sperm / ml was poured into the eggs using a pipette. After the injection, the eggs were inseminated in an incubator for 3 hours. (8) Preparation of culture dishes Several 20 μl drops of CZB were made on a 6 cm dish and covered with mineral oil. These were used as controls and equilibrated in an incubator. (9) Embryo washing, culture, and observation Five hours after insemination, the oocyte-cumulus complexes in the HTF drop had completed fertilization and the cumulus cells had been detached, so the oocytes were washed several times with a 20 μl drop using a glass capillary for manipulation.

[0033] Collection of in vivo fertilized embryos (collected by irrigation from the oviduct) (1) Superovulation treatment After 5 p.m. in the evening, ICR female mice aged 8 weeks or older were intraperitoneally injected with 7.5 IU of PMSG, and 48 hours later, with 7.5 IU of hCG using a 1 ml syringe (TERUMO, SS-01T) and a 26G needle (TERUMO) to perform superovulation treatment. (2) Mating The female mice that had been superovulated (immediately after hCG injection) were placed in a cage with a male mouse that had been kept alone at around 5:00 pm on the evening of the same day to allow mating. The next morning at around 9:00 am, ICR female mice were judged to have mated if a vaginal plug (white plug or residue) was found in the vulva of the female mice by visual inspection using tweezers (NAPOX, A-7, A-5). The female mice with plugs were irrigated on the day the plugs appeared. (3) Preparation of perfusion dish A 200 μl drop of Hepes-CZB was prepared in a 6 cm dish for each oviduct to be collected, and several 20 μl drops were also prepared for washing. (4) Preparation of culture dishes It was prepared using the same procedure as for preparing IVF (8) culture dishes. (5) Oviduct On the day when the vaginal plug was confirmed in the ICR female mice, they were euthanized by cervical dislocation at around 12:00, an incision was made in the abdominal skin using scissors (NAPOX, B-3H), the skin was peeled off, the peritoneum was incised using scissors (NAPOX, B-13H), the oviduct was cut out as neatly as possible, and collected in a microtube containing CZB (approximately 100 μl). (6) Tubal irrigation The retrieved oviduct was placed on a 6 cm Petri dish (351007, FALCON) using tweezers, and an injection needle was inserted into the cut made during oviduct recovery, and CZB medium was poured in for perfusion. The fertilized embryos obtained by perfusion were retrieved with a mouth pipette, transferred to the previously prepared CZB for culture and CZB with each reagent added, and washed. After transferring to the last drop, the embryos were cultured in an incubator at 37℃ and 5% CO2. (7) Washing, culturing, and observation of embryos The collected fertilized embryos were washed several times with a 20 μl drop using a glass capillary, then distributed equally among the sections, transferred to a culture dish, washed several times, and cultured in the same manner.

[0034] 1 shows a method for fallopian tube transplantation. (1) Preparation of vasoligated mice ICR male mice aged 8 weeks or older were injected with 0.1 ml of a triple-anesthesia mixture per 10 g of body weight and put to sleep. An incision was then made on the ventral side, and fat and testes were removed from the body. Two points of the seminiferous tubules below the tail of the epididymis were tied with sutures, and the area between them was cut. After cutting, everything was returned to its normal place in the body, and the incision was finally secured with sutures and clips. Then, 0.1 ml of a medetomidine antagonist per 10 g of body weight was injected, and the mice were returned to their cages. (2) Preparation of pseudopregnant mice On the day before the transplantation surgery, estrus female mice were selected from ICR female mice aged 8 weeks or older and mated with vasectomized male mice. The following morning, female mice with white lumps or plugs in their vaginas were judged to be pseudopregnant. Female mice with plugs were transplanted into their oviducts on the 1.5th day, with the day of plug formation considered to be 0.5 days. (3) Fallopian tube transplant surgery On the day of fallopian tube transplantation, the female mice with plugs were injected with 0.1 ml of a triple-anesthesia mixture per 10 g of body weight and put to sleep. After that, the mice were placed in a prone position, and an incision was made at the midline and a little away from the center, and the ovaries and other organs were removed from the body. Under a stereomicroscope, the junction between the ovary and the fallopian tube was found, and an incision was made with microscissors near the fallopian tube just after the junction. The tip of the microscissors was used to check whether the incision had been made, and the embryos to be transplanted were retrieved with a glass capillary. After retrieval, a glass capillary was inserted into the incision in the fallopian tube, and the embryos were transplanted into the fallopian tube. After transplantation into the left and right fallopian tubes, the ovaries and other organs were returned to the body, and the incisions were closed with sutures and clips. After that, 0.1 ml of a medthemidine antagonist per 10 g of body weight was injected, and the mice were returned to their cages.

[0035] How to select fertilized embryos The flow chart of the selection method is shown in Figure 3. Embryos of ICR mouse in vitro fertilization (IVF) were examined at the 4-cell stage under a bright-field microscope (Olympus IX71, Hoffman modulation contrast (relief contrast), objective lens 40x), and blastomeres with one large nucleolus were classified as SN (single nucleolus), and blastomeres with multiple small nucleoli were classified as MN (multi-nucleoli). However, it is not always possible to observe all four blastomeres in the bright field of all cells at the 4-cell stage. There are cases where the blastomeres overlap, or the nuclei are not visible during division. Therefore, it is confirmed whether at least two of the four blastomeres are SN or MN, and the embryos are classified as having at least two or more SN and the rest as MN, and those with two or more SN are evaluated as having high offspring. Comparing the morphology of nucleoli in 4-cell stage embryos, in vivo fertilized embryos and in vitro fertilized embryos were fixed and stained with an antibody to NOPP140, a nucleolus-specific marker, and it was found that each blastomere (cell) has one large nucleolus (SN) and multiple nucleoli (MN) (Figure 4). Using this classification, the number of cells with SN among the four cells in one embryo was counted, and each embryo was divided into SN0 to SN4. The classification is shown in Figure 5, and the numerical values ​​are shown in Table 6. In vivo fertilized embryos had more SN0 and SN1 types, accounting for more than half of the total embryos, whereas in vitro fertilized embryos, SN4 and SN3 types were more prevalent, accounting for more than half of the total embryos.

[0036] [Table 6]

[0037] Therefore, next, in order to avoid damaging the 4-cell stage embryos using a bright field microscope, we checked the IVF embryos for two or more cells (Figure 6), divided them into a group of embryos with two or more SNs (embryos ≧ 2SN) and another group, fixed the embryos, and verified whether the embryos could actually be divided. The verification results are shown in Table 7. As a result, in the "≧ 2SN embryos" group, more than half of the embryos were SN4 or SN3, while in the "MN embryos" group, conversely, more than half were SN1 or SN0, confirming that the grouping was successful.

[0038] [Table 7]

[0039] Next, embryos from each group were transferred into the oviducts of surrogate mothers, and the birth or non-birth of offspring was examined on the 19th day. Photographs of the offspring are shown in Figure 7. Table 8 shows the birth rate, Figure 8(a) birth rate, Figure 8(b) weight of offspring, and Figure 8(c) placental weight. Contrary to expectations, as shown in Figure 8(a), the "≧2SN embryo" group had a birth rate more than twice as high. There was also a tendency for placental weight to be lighter (Figure 8(c)).

[0040] [Table 8]

[0041] Table 9 shows the in vitro developmental rates after embryo selection. Although the development of MN embryos in vitro was slightly decreased, both embryos developed almost equally normally, indicating that they cannot be distinguished based on the in vitro developmental rates alone.

[0042] [Table 9]

[0043] These results indicate that evaluation of embryos based on nucleolar morphology at the 4-cell stage provides a new, simple and objective indicator of the embryos that will become future offspring.

Claims

1. In non-human animal 4-cell embryos, determining whether at least two of the four cells (blastomeres) are blastomeres SN (single nucleolus) having one large nucleolus or MN (multi-nucleoli) having multiple small nucleoli; selecting an embryo in which at least two of the four blastomeres are SN; A method for selecting embryos, comprising:

2. The 4-cell stage embryo is an in vitro fertilized (IVF) embryo of an ICR mouse. The method for selecting embryos according to claim 1,

3. The method of determination is performed using a bright-field microscope; The method for selecting embryos according to claim 1 or 2,

4. An imaging unit that captures images of the embryos. A determination unit for determining whether a cell (blastomere) is a blastomere SN (single nucleolus) having a large nucleolus or a blastomere MN (multi-nucleoli) having multiple small nucleoli; A selection section for selecting embryos in which at least two or more of the four blastomeres at the four-cell stage are SN; an output unit that outputs the result of the determination; An embryo selection device comprising:

Images