Embryo in-vitro culture medium and culture method in human periplanting period and later planting period

Abstract

The invention belongs to the field of human assisted reproduction, and particularly relates to a human embryo in-vitro culture medium in the periplanting period (6.5-8.5 days after fertilization) and the later planting period (8.5-13.5 days after fertilization) and a culture method for improving the development potential of in-vitro embryos. The culture medium comprises a culture medium used in the periplanting period and a culture medium used in the later planting period, multiple nutritional ingredients including folic acid, melatonin, L-alpha-lecithin, L-alpha-cephalin, ceramide, DHA, DPA, EPA and the like are added, and the invention further provides an embryo in-vitro culture method used in the periplanting period and the later planting period. The embryo in-vitro development success rate in the periplanting period and the later planting period is greatly improved, and necessary basic conditions are provided for exploring the embryo development mechanism in the periplanting period and the later planting period of human beings.

Description

Technical Field

[0001] The present invention belongs to the field of human assisted reproduction, and specifically relates to an in vitro culture medium for human peri-implantation and post-implantation embryos and a culture method for improving the developmental potential of in vitro embryos. Background Art

[0002] Embryo in vitro culture refers to placing embryos in an in vitro environment similar to the living environment in vivo, allowing them to grow and develop. The success or failure of early embryo growth and development is directly related to the conditions of its in vitro culture environment. The key link in embryo engineering technology is to culture embryos in vitro. With the research and discussion of in vitro culture, through the improvement of culture medium, improved culture system, physiologists' understanding of the maternal environment and the progress of mechanical engineering, the in vitro culture environment can be made closer and closer to the in vivo environment, solving the problems of poor quality of in vitro embryos, insufficient compactness of blastomeres, poor freezing resistance, low blastocyst rate, and low pregnancy rate.

[0003] Human embryos from day 1 to day 6 after fertilization are in the pre-implantation stage. The in vitro embryo culture technology for this stage is now mature and widely used in the field of human assisted reproductive treatment. From day 6 to day 9 after fertilization, the embryos are in the peri-implantation stage. The human embryos cultured in vitro show a blastocyst-like structure and grow in suspension in the culture medium; around day 8 to 9, the cystic cavity collapses and turns to adherent growth. Under physiological conditions, the embryos invade the endometrium during this period and complete embryo implantation; the embryos on day 9 after fertilization are in the late implantation stage. After the cystic cavity collapses, the embryos appear as a cell mass and gradually increase in size. Due to ethical principles, human embryos cultured in vitro cannot exceed 14 days after fertilization. The in vitro culture system for human embryos from day 1 to day 6 after fertilization is no longer suitable for embryos from day 7 to day 14 after fertilization, and the embryos will experience severe developmental stagnation. The traditional in vitro culture system for human embryos during the peri-implantation and late implantation stages has a low embryo development rate of only about 20%. The mechanism of human embryo development has always been a hot topic in the field of life sciences. The source of human embryos that can be used for scientific research is precious and scarce, and the low development rate has become a bottleneck problem restricting the study of the mechanism of human embryo development. The human peri-implantation period (for embryo culture from day 6.5 to 8.5) and post-implantation period (for embryo culture from day 8.5 to 13.5) in vitro embryo culture systems developed by the present invention have added polyunsaturated fatty acids and glycerophospholipid groups for the first time, further improving the embryo development rate, providing a more solid research foundation for exploring the mechanism of human embryo development. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of the present invention is to provide an in vitro culture medium and culture method for human peri-implantation and post-implantation embryos, which are used for in vitro culture of human embryos on days 6.5 to 8.5 and days 8.5 to 13.5 after fertilization, respectively. Specifically, it includes an embryo culture system and an operation method.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The first aspect of the present invention discloses an in vitro culture medium for peri-implantation embryos, characterized in that the culture medium comprises: DMEM / F12 culture medium, fetal bovine serum, L-glutamine, penicillin, 1× insulin-transferrin-seleno-ethanolamine, β-estradiol, progesterone, N-acetyl-cysteine, melatonin, Y-27632, L-α-phosphatidylcholine, L-α-cephalin, 18:2 cardiolipin-d5, ceramide, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA), and cis-5,8,11,14,17-eicosapentaenoic acid (EPA).

[0007] The second aspect of the present invention discloses an in vitro culture medium for late implantation embryos, characterized in that it comprises: DMEM / F12 culture medium, KnockOut serum replacement, L-glutamine, penicillin, 1× insulin-transferrin-seleno-ethanolamine, β-estradiol, progesterone, N-acetyl-cysteine, folic acid, melatonin, Y-27632, L-α-phosphatidylcholine, L-α-cephalin, 18:2 cardiolipin-d5, ceramide, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA), and cis-5,8,11,14,17-eicosapentaenoic acid (EPA).

[0008] Furthermore, the culture medium also includes reinforced matrix gel.

[0009] Preferably, the reinforced matrix gel is prepared by mixing the matrix gel and the late planting culture medium in a ratio of 2:1.

[0010] The third aspect of the present invention discloses a method for culturing peri-implantation embryos in vitro, characterized in that the method comprises the following steps: Step 1: On the first day of culture (6.5 days after fertilization), prepare the above-mentioned peri-plantation medium, add 300 μL of the peri-plantation medium to a low-adsorption 96-well plate, and place it in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours; Step 2: Embryos before day 6.5 are cultured according to conventional methods. If the embryo has not yet separated from the zona pellucida on day 6.5, the zona pellucida is removed using laser or benchtop fluid; Step 3: Transfer the embryos to a low-adsorption 96-well plate and immediately place them in a 37°C, 21% O2, 5% CO2 incubator for 48 hours (8.5 days after fertilization).

[0011] A fourth aspect of the present invention discloses a method for culturing embryos in vitro at a late stage of implantation, characterized in that the method comprises the following steps: Step 1: On the 8.5th day after fertilization, prepare the above-mentioned late planting medium and enhanced matrix gel, use a new 96-well plate to prepare a late planting culture dish; place the late planting culture dish in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours; Step 2: Place the embryo in a new culture dish and place it in a 37°C, 21% O2, 5% CO2 incubator; Step 3: Thereafter, the late planting culture medium was pre-balanced every day. 150 μL of culture medium was aspirated from each well of the 96-well plate every day, and then 150 μL of the pre-balanced late planting culture medium was added, and the culture was terminated after 13.5 days.

[0012] The fifth aspect of the present invention discloses a method for culturing embryos in vitro, characterized in that the method comprises any of the methods described above.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0014] There are currently many culture media for human pre-implantation and peri-implantation embryos on the market. These culture media are only suitable for culturing embryos 1-5 days after fertilization. When the embryos are cultured to the 6th day after fertilization using existing culture media, slight cell degeneration can be observed. The embryo development rate of traditional peri-implantation and post-implantation human embryo in vitro culture systems is low, only about 20%. The human peri-implantation (for embryo culture from 6.5 to 8.5 days) and post-implantation (for embryo culture from 8.5 to 13.5 days) embryo in vitro culture systems developed by the present invention have added polyunsaturated fatty acids and glycerophospholipid groups for the first time, and the embryo development rate is further improved, providing the necessary basic conditions for exploring the peri-implantation and post-implantation embryo development mechanisms and embryo transplantation strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The embryos cultured using the culture medium and the culture method of the present invention developed normally to day 13.5.

[0016] Figure 2 Embryos cultured with traditional culture medium and method showed developmental arrest at day 11.5. DETAILED DESCRIPTION

[0017] The present invention is further described in detail below with specific embodiments, but it should not be understood that the scope of the above subject matter of the present invention is limited to the following embodiments, and all technologies realized based on the content of the present invention belong to the scope of the present invention.

[0018] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0019] Embodiment 1: An in vitro culture medium for peri-implantation embryos and a culture method thereof.

[0020] 1. In vitro culture medium for peri-implantation embryos: The remainder is basal culture medium.

[0021] 2. A method for culturing peri-implantation embryos in vitro, comprising the following steps: 1. Pre-balance the culture medium: balance the peri-implantation culture medium in a wet incubator at 37°C, 21% O2, 5% CO2 for at least 4 hours to obtain a balanced peri-implantation embryo in vitro culture medium; 2. In vitro culture of embryos during the peri-implantation period (6.5-8.5 days after fertilization): Transfer the blastocyst to the pre-balanced culture medium in step 1 above. If the blastocyst has not separated from the zona pellucida, use benchtop acid to remove the zona pellucida and culture in a wet incubator at 37°C, 21% O2, 5% CO2 for 48 hours until 8.5 days after fertilization.

[0022] Embodiment 2: An in vitro culture medium for late-implantation embryos and a culture method thereof.

[0023] 1. Embryo in vitro culture system.

[0024] 1. In vitro culture medium for late embryo implantation: The remainder is basal culture medium.

[0025] 2. Reinforced matrix glue.

[0026] Configuration method: (1) After freezing and thawing the matrix gel at 4°C, mix the matrix gel with a pre-cooled pipette tip; (2) Precool the late-stage planting culture medium in advance and mix it with the melted matrix gel on ice in a 2:1 ratio to obtain enhanced matrix gel.

[0027] 2. In vitro culture method of embryos in the late stage of implantation.

[0028] 1. Prepare the culture dishes.

[0029] (1) Place the 96-well plate on ice and add 100 μL of enhanced matrix gel to each well. Place the 96-well plate in a 37°C wet incubator for 30 minutes to allow the matrix gel to solidify.

[0030] (2) Add 300 μL of post-implantation culture medium to each well of a 96-well plate and cover it on the top layer of enhanced matrix gel.

[0031] (3) Equilibrate the 96-well plate in a 37°C, 21% O2, 5% CO2 wet incubator for at least 4 hours.

[0032] 2. In vitro culture of embryos in the late implantation stage (8.5-13.5 days after fertilization).

[0033] On day 8.5 after fertilization, embryos were transferred from the peri-implantation culture dish to the late-implantation culture dish. From day 9.5 onwards, the late-implantation culture medium was pre-balanced every day by aspirating 150 μL of culture medium from each well of a 96-well plate and adding 150 μL of late-implantation culture medium. The culture was terminated on day 13.5.

[0034] Example 3: Comparison of the culture effects of the culture system of the present invention and the traditional peri-implantation and post-implantation embryo culture medium (referred to as "traditional culture medium").

[0035] 1. Grouping.

[0036] 1 Control group: Human embryos were cultured from day 6.5 to day 13.5 using traditional culture medium.

[0037] 2 Invention Group: The present invention was used to culture human embryos from day 6.5 to day 13.5.

[0038] 2. Experimental methods.

[0039] 1. The culture system and culture method used in the control group and the present invention group on days 1 to 6.5 were operated according to conventional systems and methods.

[0040] 2. Embryo culture of the control group.

[0041] (1) Training system

[0042] Traditional medium 1 For human embryo culture from day 6.5 to day 8.5, its components include: L-glutamine (2 mM), penicillin (50 units / mL), 1× insulin-transferrin-seleno-ethanolamine, β-estradiol (8 nM), progesterone (0.64 μM), N-acetyl-cysteine ​​(25 μM), sodium lactate (0.22% by volume), sodium pyruvate (1 mM), and Y27632 (10 μM) were added to DMEM / F12 culture medium containing 20% ​​(volume ratio) heat-inactivated fetal bovine serum.

[0043] Traditional medium 2 For human embryo culture from day 8.5 to day 13.5, its components include: L-glutamine (2 mM), penicillin (25 units / mL), 1× insulin-transferrin-seleno-ethanolamine, β-estradiol (8 nM), progesterone (0.64 μM), N-acetyl-cysteine ​​(25 μM), sodium lactate (0.22% by volume), sodium pyruvate (1 mM), and Y27632 (10 μM) were added to DMEM / F12 medium containing 30% (volume ratio) KnockOut serum replacement.

[0044] (2) Training methods

[0045] ① On the first day of culture (embryos on day 6.5 after fertilization), prepare traditional culture medium 1, add 300 μL of traditional culture medium 1 to a low-adsorption 96-well plate, and place in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours.

[0046] ② If the blastocyst has not yet separated from the zona pellucida, use the laser method or desktop liquid method to remove the zona pellucida.

[0047] ③ The embryos were transferred to a pre-equilibrated low-adsorption 96-well plate and immediately placed in a 37°C, 21% O2, 5% CO2 incubator. The embryos were cultured for 48 hours, that is, until day 8.5 after fertilization.

[0048] ④ On day 8.5 after fertilization, prepare traditional medium 2 and place it in an incubator at 37°C, 21% O2, 5% CO2 for at least 4 hours.

[0049] ⑤ On day 8.5 after fertilization, carefully remove 150 μL of traditional culture medium 1 from the well using a sterile pipette tip, add 200 μL of pre-equilibrated traditional culture medium 2, and then immediately place in the incubator for continued culture.

[0050] ⑥ Culture the embryos in vitro from day 9.5 to day 13.5 after fertilization, and replace the culture medium every day according to method ⑤.

[0051] 3. Embryo culture of the present invention.

[0052] (1) Training system

[0053] Peri-implantation medium is used for culturing human embryos from day 6.5 to day 8.5 and includes: L-glutamine (2 mM), penicillin (50 units / mL), 1× insulin-transferrin-seleno-ethanolamine, β-estradiol (8 nM), progesterone (0.64 μM), N-acetyl-cysteine ​​(25 μM), melatonin (1 μM), Y-27632 (5 mM), L-α-phosphatidylcholine (0.2 μM), 18:2-aminobutyric acid (0.2 μM), and L-α-phosphatidylcholine (0.2 μM) were added to DMEM / F12 medium containing 20% ​​(volume ratio) heat-inactivated fetal bovine serum. Cardiolipin-d5 (50nM), ceramide (10nM), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) (50nM), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA) (50nM), cis-5,8,11,14,17-eicosapentaenoic acid (EPA) (50nM).

[0054] Post-implantation medium is used for culturing human embryos from day 8.5 to day 13.5, and its components include: The culture medium was supplemented with DMEM / F12 medium containing 30% (volume ratio) KnockOut serum replacement, L-glutamine (2 mM), penicillin (50 units / mL), 1× insulin-transferrin-seleno-ethanolamine, β-estradiol (8 nM), progesterone (0.64 μM), N-acetyl-cysteine ​​(25 μM), folic acid (2 μM), melatonin (1 μM), Y-27632 (5 mM), L-α-phosphatidylcholine (0.2 μM), L-α-cephalin (0.2 μM), 18:2 cardiolipin-d5 (50 nM), ceramide (10 nM), cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) (50 nM), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA) (50nM), cis-5,8,11,14,17-eicosapentaenoic acid (EPA) (50nM).

[0055] Enhanced Matrigel is used for culturing human embryos from day 8.5 to day 13.5 and is spread on the bottom of a 96-well plate. It is composed of Matrigel and late-stage culture medium mixed in a ratio of 2:1.

[0056] (2) Training methods

[0057] ① On the first day of culture (6.5 days after fertilization), prepare the peri-implantation medium, add 300 μL of pre-equilibrated peri-implantation medium to a low-adsorption 96-well plate, and place it in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours.

[0058] ② If the blastocyst has not yet separated from the zona pellucida, use the laser method or desktop liquid method to remove the zona pellucida.

[0059] ③ Transfer the embryos to a low-adsorption 96-well plate and immediately place them in a 37°C, 21% O2, 5% CO2 incubator for 48 hours (8.5 days after fertilization).

[0060] ④ On the 8th and 5th day after fertilization, prepare the late planting medium and enhanced matrix gel, and use a new 96-well plate to prepare the late planting culture dish. Place the late planting culture dish in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours.

[0061] ⑤ Place the embryo in a new culture dish and place it in an incubator at 37°C, 21% O2, 5% CO2.

[0062] ⑥Thereafter, fresh post-planting medium was balanced in advance every day, 150 μl of medium was aspirated from each well of the 96-well plate, and then 150 μl of pre-balanced post-planting medium was added, and the culture was terminated after 13.5 days.

[0063] 4. Measurement of embryonic growth and development.

[0064] Observe the embryos daily and measure the diameter of the embryos. The measurement method is the average value of the long axis and the short axis (unit: μm). If the embryo diameter grows longer than 10 μm, it is judged as normal growth; if the embryo diameter grows longer than 10 μm or the diameter shrinks, it is judged as developmental arrest. The embryo is in the blastocyst stage from day 6.5 to day 8.5. Its morphological feature is a hollow cystic cavity. Around day 8-9, the cystic cavity collapses and its volume rapidly decreases. The embryo morphology becomes a cell mass, and the embryo volume gradually increases thereafter. In addition, the optical morphology of embryonic cells can be used to judge whether it is developing normally. If the embryo grows normally, the cytoplasm is translucent and the cell membrane boundaries are clear. If the embryo develops stagnates, the cytoplasm becomes darker, the cell membrane boundaries are blurred, and vacuoles appear, marking cell degeneration.

[0065] 3. Results

[0066] 1. Embryo development rate: A total of 62 embryos were cultured in the control group, and 64 embryos were cultured in the present invention. Compared with the control group, the embryo development rates of the present invention on days 11.5, 12.5, and 13.5 were all higher than those of the control group (Table 1, Figure 1 , Figure 2 ).

[0067] Table 1 Embryo development rate of the patent group and the control group .

[0068] 2. Embryo development diameter: Compared with the control group, the embryonic development diameters of the present invention group on days 11.5, 12.5, and 13.5 were all higher than those of the control group (Table 2).

[0069] Table 2 Mean values ​​of embryo diameters in the present invention group and the control group .

[0070] The above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An in vitro culture medium for peri-implantation embryos, characterized in that: include: DMEM / F12 medium, fetal bovine serum, L-glutamine, penicillin-streptomycin, 1× insulin-transferrin-selenoethanolamine, β-estradiol, progesterone, N-acetyl-cysteine, melatonin, Y-27632, L-α-phosphatidylcholine, L-α-cephalin, 18:2 cardiolipin-d5, ceramide, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA).

2. An in vitro culture medium for late embryo implantation, characterized in that: include: DMEM / F12 medium, KnockOut serum replacement, L-glutamine, penicillin-streptomycin, 1× insulin-transferrin-selenoethanolamine, β-estradiol, progesterone, N-acetyl-cysteine, folic acid, melatonin, Y-27632, L-α-phosphatidylcholine, L-α-cephalin, 18:2 cardiolipin-d5, ceramide, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), all-cis-7,10,13,16,19-docosapentaenoic acid (DPA), cis-5,8,11,14,17-eicosapentaenoic acid (EPA).

3. The culture medium according to claim 2, characterized in that The culture medium also includes reinforced Matrigel.

4. The culture medium according to claim 3, characterized in that The preparation method of the reinforced matrix glue is to mix the matrix glue and the late planting culture medium in a ratio of 2:

1.

5. A method for culturing peri-implantation embryos in vitro, characterized in that: The method comprises the following steps: Step 1: On the first day of peri-implantation embryo culture (equivalent to the 6.5th day after fertilization), prepare the peri-implantation medium according to claim 1, add 300 μL of the pre-equilibrated peri-implantation medium to a low-adsorption 96-well plate, and place in a 37°C, 21% O2, 5% CO2 incubator for at least 4 hours; Step 2: If the blastocyst is at stage 4 or stage 5, that is, the blastocyst has not yet separated from the zona pellucida, use the laser method or benchtop liquid method to remove the zona pellucida to reach the stage 6 blastocyst; Step 3: Transfer the stage 6 blastocysts to a low-adsorption 96-well plate and immediately place them in a 37°C, 21% O2, 5% CO2 incubator for 48 hours (8.5 days after fertilization).

6. A method for culturing embryos in vitro at the late stage of implantation, characterized in that: The method comprises the following steps: Step 1: On the 8.5th day after fertilization, prepare the late planting culture medium and the reinforced matrix gel as described in claim 2, use a new 96-well plate to prepare a late planting culture dish, and place the late planting culture dish in a 37° C., 21% O2, 5% CO2 incubator for at least 4 hours; Step 2: Place the embryo in a new culture dish and place it in a 37°C, 21% O2, 5% CO2 incubator; Step 3: Thereafter, 150 μL of culture medium was removed from each well of the 96-well plate every day, and 150 μL of pre-balanced late planting culture medium was added, and the culture was terminated after 13.5 days.

7. A method for culturing embryos in vitro, characterized in that: The method comprises the method according to any one of claims 5 to 6.

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