In-vitro culture method and device for improving early embryo development quality

Abstract

The invention relates to the technical field of embryo in-vitro culture, and discloses an in-vitro culture method and device for improving early embryo development quality, the method can accurately adjust the microenvironment pressure of a culture room and update culture environment gas in real time, and is especially suitable for embryos sensitive to environment pressure and gas components. The blastocyst rate of the embryo and the expression of densified related protein (CDH1) are obviously improved.

Description

Technical Field

[0001] This invention relates to the field of in vitro embryo culture technology, specifically to an in vitro culture method and apparatus for improving the quality of early embryo development. Background Technology

[0002] Under natural conditions, embryos complete fertilization and development within the mother's body, with the mother providing all the necessary nutrients, temperature, pH, gaseous environment, osmotic pressure, and microbiological environment. The advent of assisted reproductive technologies such as in-vitro fertilization (IVF) has offered hope for infertile couples. However, the differences between the in-vitro environment and the in-vitro microenvironment result in lower embryo development efficiency, quality, and pregnancy rates compared to embryos developing in vivo.

[0003] In-vivo fertilization occurs within the fallopian tubes. The early embryo develops in the physiological microenvironment of the fallopian tubes and uterus. The relatively sealed environment formed by the muscles in the fallopian tubes and uterus provides a micro-pressure mechanical environment for embryonic development, which promotes embryonic development.

[0004] Appropriate micropressure can activate relevant mechanomechanical signaling pathways, promote the expression of densification-related proteins, increase the densification degree of embryos, and ultimately increase the blastocyst rate. However, the in vitro environment created by existing technology is difficult to replicate the in vivo micropressure environment. In addition, in traditional box-type incubators, the gas composition inside the incubator is unstable due to changes in gas composition caused by cell metabolism, and fresh gas cannot be introduced continuously.

[0005] Therefore, in order to more realistically simulate the state of an embryo in a dynamically updated environment in vivo, there is an urgent need in the field of biological research for an embryo culture method that can dynamically update the gas phase environment while ensuring that the embryo is in an adjustable micro-pressure environment. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an in vitro culture method for improving the quality of early embryonic development, significantly increasing the blastocyst rate and the expression of compaction-associated protein (CDH1).

[0007] To achieve the above objectives, this invention provides an in vitro embryo culture method that improves blastocyst rate and the expression of densification-related proteins. This method promotes embryo densification by inducing increased expression levels of embryo densification-related genes through pressure, thereby increasing blastocyst development rate and the total number of blastocyst cells, thus improving embryo quality. The method specifically includes the following steps: S1. After 24 hours of in vitro maturation of bovine oocytes, COCs were separated from the oocytes by pipetting. Oocytes with uniform cytoplasm and extrusion of the first polar body were selected and transferred into microdroplets made from fertilization medium, which were then covered with paraffin oil. Next, the frozen bovine semen was thawed in a water bath. Semen that passed microscopic examination was transferred to the bottom of a round tube containing fertilization medium and placed in the upper part of an incubator to complete sperm capacitation. The supernatant was then aspirated, centrifuged, and discarded. Highly motile sperm were evenly added to microdroplets containing mature oocytes and then co-incubated in an incubator. 24 hours after in vitro fertilization, sperm adhering to the zygote surface were removed by pipetting. After washing with embryo culture medium, the sperm were transferred into microdroplets made from embryo culture medium and covered with paraffin oil. S2. Connect the gas cylinder to the inlet of the proportional valve, connect one end of the pneumatic switch valve to the outlet of the proportional valve, and connect the other end of the pneumatic switch valve to the inlet of the check valve. Adjust the pressure of the pressure reducing valve of the gas cylinder to supply pressure to the proportional valve. S3, connect a transformer and a proportional valve in series, and an analog signal generator and a proportional valve in parallel; adjust the voltage output value of the transformer to the normal operating voltage value of the proportional valve; S4. Connect the digital barometer to the outlet of the check valve, then adjust the pneumatic switch valve to the connected position, use the digital barometer to measure the air pressure, adjust the knob of the analog signal generator to adjust the air pressure until the air pressure value is the target pressure required, then adjust the pneumatic switch valve to the closed position so that the air path after the proportional valve is closed, and remove the digital barometer from the check valve. S5. Place the pressure incubator in a portable incubator to raise the internal temperature of the pressure incubator to the target temperature. Then, transfer the embryos from S1 to a culture dish, and place the culture dish containing the embryos into the container of the pressure incubator. After correctly positioning, close the lid, lock and seal the lid and container. Then, transfer the pressure incubator to a constant temperature water bath. During the transfer, use the portable incubator to keep the pressure incubator warm until it enters the constant temperature water bath. Then, fix the optical water level probe, start the electronic water replenisher, make the water level exactly at the target water level, and set the target water bath temperature. S6. Connect the outlet of the check valve to the inlet of the pressurized incubator. Connect the pneumatic speed control valve and the flow sensor to the outlet of the pressurized incubator in sequence. After connection, open the pneumatic switch valve between the proportional valve and the check valve so that the gas is pressurized by the proportional valve and enters the humidification bottle of the pressurized incubator through the check valve, and then overflows from the humidification bottle into the chamber of the pressurized incubator. Adjust the pneumatic speed control valve before the flow sensor to the target exhaust rate. S7. Replace half of the embryo culture medium every 48 hours during the culture process.

[0008] As a further improvement of the present invention, in step S2, the output pressure of the gas cylinder pressure reducing valve is specifically adjusted to 0.04-0.06 MPa.

[0009] As a further improvement of the present invention, in step S4, the voltage of the analog signal generator is adjusted so that the pressure displayed on the digital barometer is 0.005-0.007 MPa.

[0010] As a further improvement of the present invention, in step S5, when the pressure incubator is placed in a portable constant temperature chamber, the internal temperature of the pressure incubator needs to be raised to 39 ℃; when the pressure incubator is heated in a constant temperature water bath, the specific target water bath temperature is 39 ℃; and the liquid level in the constant temperature water bath reaches half the overall height of the pressure incubator.

[0011] As a further improvement of the present invention, in step S6, the pneumatic speed control valve is adjusted so that the target exhaust rate is specifically 0.0065-0.0075 L / Min.

[0012] This invention also proposes an in vitro culture device for improving the quality of early embryonic development, applicable to the aforementioned in vitro culture method for improving the quality of early embryonic development, comprising: The system includes a pressure incubator, gas cylinder, proportional valve, pneumatic switch valve, check valve, transformer, analog signal generator, constant temperature water bath, pneumatic speed control valve, and flow sensor. The pressure reducing valve of the gas cylinder is connected to the inlet of the proportional valve. One end of the pneumatic switch valve is connected to the outlet of the proportional valve, and the other end is connected to the inlet of the check valve. The pressure incubator is placed in the constant temperature water bath, which is equipped with an electronic water replenisher and an optical water level probe to maintain a constant liquid level. The inlet of the pressure incubator is connected to the outlet of the check valve, and the outlet of the pressure incubator is connected in sequence to the pneumatic speed control valve and the flow sensor. The transformer is connected in series with the proportional valve to supply power. The analog signal generator is connected in parallel with the proportional valve to adjust the signal voltage input to the proportional valve, thereby adjusting the output gas pressure.

[0013] As a further improvement of the present invention, the pressurized incubator includes: a tank body and a cover body detachably connected to the tank body; the cover body is respectively provided with an air inlet, an air outlet, and a pressure measuring port penetrating through the inside and outside of the cover body; a humidification bottle is detachably connected to the air inlet body inside the cover body, and a humidification port is provided on the upper side of the humidification bottle; sterile water is placed inside the humidification bottle; the liquid level of the sterile water is lower than the setting height of the humidification port; an air inlet pipe is fixedly connected to the air inlet body inside the cover body, the air outlet end of the air inlet pipe is immersed in the sterile water and extends to the bottom of the inner side of the humidification bottle; a pressure gauge is provided at the pressure measuring port for monitoring the internal pressure value of the tank body.

[0014] Compared with existing technologies, the present invention provides an in vitro culture method for improving the quality of early embryonic development, which has the following beneficial effects: (1) This invention solves the problem that the pressure cannot be stably controlled in the extremely low pressure environment during the existing pressurized culture process, such as the physiological gas pressure value in the fallopian tube is about 0.001 MPa; this invention can stably and controllably output the pressure within the extremely low pressure range of 0.0001 to 0.02 MPa, and compared with the problems of air leakage and pressure instability caused by traditional devices, our device has a function of automatically tracking the set target pressure in the output module, so that the target pressure is always maintained in the embryo culture device regardless of the change in the airtightness of the device; during the early development of the embryo, it will be affected by micromechanical forces such as the contraction of smooth muscle in the fallopian tube and uterus and the low pressure gas atmosphere. In the process of culturing embryos using this invention, it was found that a higher blastocyst rate and significantly improved expression of densification-related proteins can be obtained under the pressure of 0.006 MPa, which is better than the culture effect under the unstable pressure of traditional incubators.

[0015] (2) During embryo culture, whether the temperature is too high or too low, the blastocyst rate will be significantly reduced and the development of some embryos will be inhibited. The present invention adopts a semi-submersible water bath heating method, which optimizes the problem of excessive temperature fluctuations in the traditional electric heating wire heating process. At a water bath temperature of 39℃, a more stable and reliable culture effect is obtained, and the expression of heat shock protein and cold stress-related protein is downregulated.

[0016] (3) The present invention uses an adjustable flow sensor. Because there is positive pressure in the chamber of the culture device, the gas exchange rate in the chamber can be continuously maintained at a slow rate of 0.007 L / Min for 24 hours by adjusting the opening of the speed regulating valve. This ensures that the temperature, humidity, air pressure and other indicators in the culture chamber do not change drastically, and the gas composition and other indicators can be kept stable for 24 hours. Compared with the traditional micro air pump and the non-air exchange method (air outlet valve turned to 0- i.e. no gas is discharged), it can significantly stabilize the pH value of the culture medium and achieve higher cleavage rate and blastocyst rate. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the overall structure of an in vitro culture device for improving the quality of early embryonic development according to the present invention. Figure 2 This is a schematic diagram of the overall structure of a pressurized culture device for improving the quality of early embryonic development according to the present invention. Figure 3 This is a schematic diagram of the internal structure of a pressure culture device for an in vitro culture apparatus to improve the quality of early embryonic development according to the present invention; Figure 4 The effects of different pressure gradients on the in vitro development of early bovine embryos; Figure 5The effect of different pressure gradients on the total number of cells obtained in blastocysts; Figure 6 To compare the total number of blastocyst cells obtained under different culture pressures; Figure 7 To investigate the effect of different culture pressures on the compaction of blastocysts; Figure 8 To compare the degree of compaction of blastocysts obtained under different pressure cultures.

[0018] Explanation of reference numerals in the attached figures: 1. Pressure incubator; 2. Gas cylinder; 3. Proportional valve; 4. Pneumatic switch valve; 5. Check valve; 6. Transformer; 7. Analog signal generator; 8. Constant temperature water bath; 9. Pneumatic speed control valve; 10. Flow sensor; 11. Electronic water replenisher; 12. Optical water level probe; 13. Submersible pump; 14. Water pipe; 15. Water replenishment tank; 101. Tank body; 102. Cover; 103. Air inlet; 104. Air outlet; 105. Pressure measuring port; 106. Humidification bottle; 107. Humidification port; 108. Sterile water; 109. Air inlet pipe; 1010. Pressure gauge. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] Example 1 In this embodiment, see Figure 1 An in vitro culture method for improving the quality of early embryonic development includes the following steps: S1. After 24 hours of in vitro maturation of bovine oocytes, COCs were separated from the oocytes by pipetting. Oocytes with uniform cytoplasm and extrusion of the first polar body were selected and transferred into microdroplets made from fertilization medium, which were then covered with paraffin oil. Next, the frozen bovine semen was thawed in a water bath. Semen that passed microscopic examination was transferred to the bottom of a round tube containing fertilization medium and placed in the upper part of an incubator to complete sperm capacitation. The supernatant was then aspirated, centrifuged, and discarded. Highly motile sperm were evenly added to microdroplets containing mature oocytes, and the density was adjusted. The microdroplets were then co-incubated in an incubator. 24 hours after in vitro fertilization, sperm adhering to the zygote surface were removed by pipetting. After washing with embryo culture medium, the sperm were transferred into microdroplets made from embryo culture medium and covered with paraffin oil. S2. Connect gas cylinder 2 to the inlet of proportional valve 3, connect one end of pneumatic switch valve 4 to the outlet of proportional valve 3, and connect the other end of pneumatic switch valve 4 to the inlet of check valve 5. Adjust the pressure of pressure reducing valve of gas cylinder 2 to supply pressure to proportional valve 3. S3, series transformer 6 and proportional valve 3, parallel analog signal generator 7 and proportional valve 3; adjust the voltage output value of transformer 6 to the normal operating voltage value of proportional valve 3; S4. Connect the digital barometer to the outlet of the check valve 5, then adjust the pneumatic switch valve 4 to the connected position, use the digital barometer to measure the air pressure, adjust the knob of the analog signal generator 7 to adjust the air pressure until the air pressure value is the target pressure required, then adjust the pneumatic switch valve 4 to the closed position so that the air path after the proportional valve 3 is closed, and remove the digital barometer from the check valve 5. S5. Place the pressure incubator 1 in a portable constant temperature chamber to raise the internal temperature of the pressure incubator 1 to the target temperature; then transfer the embryos from S1 to a culture dish, and then place the culture dish containing the embryos into the tank 101 of the pressure incubator 1. After correctly positioning, close the lid 102, lock and seal the lid 102 and the tank 101; then transfer the pressure incubator 1 to the constant temperature water bath 8. During the transfer, use the portable constant temperature chamber to keep the pressure incubator 1 warm until the pressure incubator 1 enters the constant temperature water bath 8. Then fix the optical water level probe 12, start the electronic water replenisher 11 to make the water level exactly at the target water level, and set the target water bath temperature. S6. Connect the outlet of the check valve 5 to the inlet 103 of the pressurized incubator 1. Connect the pneumatic speed control valve 9 and the flow sensor 10 to the outlet 104 of the pressurized incubator 1 in sequence. After the connection is completed, open the pneumatic switch valve 4 between the proportional valve 3 and the check valve 5 so that the gas is pressurized by the proportional valve 3 and enters the humidification bottle 106 of the incubator through the check valve 5, and then overflows from the humidification bottle 106 into the chamber of the pressurized incubator 1. Adjust the pneumatic speed control valve 9 before the flow sensor 10 to the target exhaust rate. S7. Replace half of the embryo culture medium every 48 hours during the culture process.

[0021] Preferably, in S1, the volume of the droplets made from the fertilized fluid is 30 μL per drop, and each droplet contains 20 bovine oocytes.

[0022] Preferably, in step S1, the temperature during water bath thawing is specifically set to 37.5°C.

[0023] Preferably, in step S1, the semen spends 30 minutes in the incubator.

[0024] Preferably, in step S1, the specific centrifugation speed of the supernatant is 1500 r / min, and the specific centrifugation time is 5 min.

[0025] Preferably, in step S1, 4.5-5.5 μL of highly motile sperm is uniformly added to a microdroplet containing a mature oocyte, adjusting the density to 4.5 × 10⁻⁶. 6 -6.5×10 6 per mL.

[0026] Preferably, in step S1, the temperature inside the incubator is 38.5°C, the carbon dioxide concentration is 5%, and the humidity is saturated humidity.

[0027] Preferably, in S1, the volume of the microdroplets made from the embryo culture medium is 30 μL per drop, and each microdroplet made from the embryo culture medium contains 10 early embryos.

[0028] Preferably, in S1, the pH value of the embryo culture medium is 7.2-7.4.

[0029] Furthermore, in S2, the output pressure of the pressure reducing valve of gas cylinder 2 is specifically adjusted to 0.04-0.06 MPa.

[0030] Furthermore, in step S4, the voltage of the analog signal generator 7 is adjusted so that the pressure displayed on the digital barometer is 0.005-0.007 MPa.

[0031] Furthermore, in step S5, when the pressure incubator 1 is placed in a portable constant temperature chamber, the internal temperature of the pressure incubator 1 needs to be raised to 39 ℃; when the pressure incubator 1 is heated in the constant temperature water bath 8, the specific target water bath temperature is 39 ℃; the liquid level of the constant temperature water bath 8 reaches half the overall height of the pressure incubator 1.

[0032] Furthermore, in S6, the pneumatic speed control valve 9 is adjusted so that the target exhaust rate is specifically 0.0065-0.0075 L / Min.

[0033] Example 2 This embodiment provides an in vitro culture device for improving the quality of early embryonic development, applicable to the aforementioned in vitro culture method for improving the quality of early embryonic development. The device includes: a pressurized culture unit 1, a gas cylinder 2, a proportional valve 3, a pneumatic switching valve 4, a check valve 5, a transformer 6, an analog signal generator 7, a constant temperature water bath 8, a pneumatic speed control valve 9, and a flow sensor 10. The pressure reducing valve of the gas cylinder 2 is connected to the inlet of the proportional valve 3. One end of the pneumatic switching valve 4 is connected to the outlet of the proportional valve 3, and the other end of the pneumatic switching valve 4 is connected to the inlet of the check valve 5. The check valve 5 is used to prevent the proportional valve from stopping pressure supply (0) after a power outage. The pressure of MPa caused the residual pressure in the pressurized incubator 1 to force the water in the humidification bottle 106 into the proportional valve 3, burning out the proportional valve 3. The pressurized incubator 1 is set in the constant temperature water bath 8, which is equipped with an electronic water replenisher 11 and an optical water level probe 12 to achieve a constant liquid level. The air inlet 103 of the pressurized incubator 1 is connected to the air outlet of the check bottle 5. The air outlet 104 of the pressurized incubator 1 is connected in sequence to the pneumatic speed control valve 9 and the flow sensor 10. The transformer 6 is connected in series with the proportional valve 3 to supply power to the proportional valve 3. The analog signal generator 7 is connected in parallel with the proportional valve 3 to adjust the signal voltage input to the proportional valve 3, thereby adjusting the output air pressure of the proportional valve 3.

[0034] Preferably, the proportional valve 3 is a German FESTO proportional pressure regulating valve, which converts the input electrical signal into a supply pressure value. The principle of stable pressure control is as follows: when the analog signal generator 7 outputs a voltage signal to the proportional valve 3, it controls the pilot chamber inlet solenoid valve to operate, so that the pilot chamber obtains a gas pressure value (target gas pressure value) that matches the input voltage signal. At the same time, there is a pressure difference between the upper and lower parts of the pilot chamber (the lower part is interconnected with the inner cavity of the pressurized incubator 1, and the pressure values ​​of the two are consistent), which causes the exhaust valve core to move up and down. When the pressure of the upper and lower parts of the pilot chamber is consistent, the pressure sensor pressure difference value is 0, that is, the exhaust valve core stops operating, ensuring that no matter how the pressure inside the pressurized incubator 1 changes, the exhaust valve core is always fine-tuning, so that the pressure value inside the pressurized incubator 1 always approaches the target pressure.

[0035] Preferably, the proportional valve 3 of the German Festo model requires an operating voltage of 24 V, and in S3, the output value of the transformer 6 is specifically adjusted to 24 V.

[0036] Preferably, the analog signal generator 7 needs to be able to output any signal voltage required by the proportional valve 3, ensuring that the proportional valve 3 can output any air pressure value within the range of this model. Therefore, the analog signal generator 7 is a handheld voltage and current signal generator with the optional model QH-VISG2-ED (with battery), and its accuracy is adjusted to two decimal places; rotating the adjustment knob outputs the corresponding voltage signal so that the proportional valve 3 outputs the corresponding air pressure value.

[0037] Preferably, in order to achieve the target pressure of 0.006 MPa, the voltage of the analog signal generator 7 is specifically adjusted to 3.07V; the analog voltage signals corresponding to other corresponding air pressures output by the proportional valve 3 are shown in Table 1.

[0038] Table 1: Analog Voltage Signal - Corresponding Air Pressure Value Output by Proportional Valve Preferably, the pneumatic speed control valve 9 is an SMC speed control valve, model AS2002FS-04; a 4 mm rigid pipe is used to connect the air outlet 104 of the pressurized incubator 1 and the air inlet 103 of the pneumatic speed control valve 9.

[0039] Preferably, the flow sensor 10 is a Festo flow sensor from Germany, and the unit of the flow sensor 10 is adjusted to L / Min; a 4 mm rigid pipe is used to connect the air outlet of the pneumatic speed control valve 9 and the air inlet of the flow sensor 10.

[0040] Preferably, the constant temperature water bath 8 is a super low temperature bath of model THD-0510 from Shaoxing Dongpu Instrument Co., Ltd.; the electronic water replenisher 11 is electrically connected to a submersible pump 13, which is installed in the water replenishment tank 15. The submersible pump 13 draws water from the water replenishment tank 15 to the constant temperature water bath 8 through the water pipe 14; the electronic water replenisher 11 is specifically a Camel model ATO ONE 2 submersible pump version intelligent electronic water replenisher, which is equipped with a power adapter and can be plugged into a power source independently.

[0041] Furthermore, the pressurized incubator 1 includes: a tank body 101 and a cover 102 detachably connected to the tank body 101; the cover 102 is provided with an air inlet 103, an air outlet 104, and a pressure measuring port 105 penetrating inside and outside the cover 102; a humidification bottle 106 is detachably connected to the air inlet 103 on the inner side of the cover 102, and a humidification port 107 is provided on the upper side of the humidification bottle 106; sterile water 108 is placed inside the humidification bottle 106; the liquid level of the sterile water 108 is lower than the setting height of the humidification port 107; an air inlet pipe 109 is fixedly connected to the air inlet 103 on the inner side of the cover 102, and the air outlet of the air inlet pipe 109 is immersed in the sterile water 108 and extends to the bottom of the inner side of the humidification bottle 106; a pressure gauge 1010 is provided at the pressure measuring port 105 to monitor the internal pressure value of the tank body 101.

[0042] Preferably, the container 101 is a semi-cylindrical structure with a sealed bottom, a bottom diameter of 13 cm, a height of 12 cm, and an opening diameter of 14 cm. Two 35 mm culture dishes can be placed at the bottom at the same time. The lid 102 has a sealing ring with a diameter of 14 cm inside, and locking and fastening switches are provided on both sides to seal the container 101 and the lid 102.

[0043] Preferably, the barometer 1010 can be selected as a mechanical barometer or a digital barometer, depending on the requirements.

[0044] Example 3 In this embodiment, 0, 0.002, 0.004, 0.006, and 0.008 MPa were selected as the culture pressure for in vitro culture, and the following developmental indicators were statistically analyzed (see details). Figure 4 (and Table 2), the results are as follows: 1. There was no significant difference in the cleavage rate of the embryos among the various pressure groups (0, 0.002, 0.004, 0.006, 0.008 MPa).

[0045] 2. When the pressure culture was set at 0.006 MPa, the blastocyst rate of the embryos was significantly improved.

[0046] Table 2: Effects of different pressure gradients on in vitro development of early bovine embryos Example 4 Embryo compaction and total cell count are key indicators for assessing embryo quality. In this example, blastocysts were obtained by culturing under different pressures (0, 0.002, 0.004, 0.006, and 0.008 MPa). Immunofluorescence staining was used to detect the expression level of CDH1, a key protein for embryo compaction, in each group of blastocysts. DNA binding agent staining was used to determine the number of embryonic cells. (See details...) Figure 5 - Figure 8 The results show: A culture pressure of 1.0006 MPa can significantly increase the number of cells in the blastocyst.

[0047] A culture pressure of 2.006 MPa can significantly increase the expression of densification-associated protein (CDH1) (the stronger the fluorescence intensity, the higher the expression level).

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. An in vitro culture method for improving the quality of early embryonic development, characterized in that, Specifically, the following steps are included: S1. After 24 hours of in vitro maturation of bovine oocytes, COCs were separated from the oocytes by pipetting. Oocytes with uniform cytoplasm and extrusion of the first polar body were selected and transferred into microdroplets made from fertilization medium, which were then covered with paraffin oil. Next, the frozen bovine semen was thawed in a water bath. Semen that passed microscopic examination was transferred to the bottom of a round tube containing fertilization medium and placed in the upper part of an incubator to complete sperm capacitation. The supernatant was then aspirated, centrifuged, and discarded. Highly motile sperm were evenly added to microdroplets containing mature oocytes and then co-incubated in an incubator. 24 hours after in vitro fertilization, sperm adhering to the zygote surface were removed by pipetting. After washing with embryo culture medium, the sperm were transferred into microdroplets made from embryo culture medium and covered with paraffin oil. S2. Connect the gas cylinder (2) to the inlet of the proportional valve (3), connect one end of the pneumatic switch valve (4) to the outlet of the proportional valve (3), and connect the other end of the pneumatic switch valve (4) to the inlet of the check valve (5). Adjust the pressure of the pressure reducing valve of the gas cylinder (2) to supply pressure to the proportional valve (3). S3, series transformer (6) and proportional valve (3), parallel analog signal generator (7) and proportional valve (3); adjust the voltage output value of transformer (6) to the normal operating voltage value of proportional valve (3); S4. Connect the digital barometer to the outlet of the check bottle (5), then adjust the pneumatic switch valve (4) to the connected position, measure the air pressure with the digital barometer, adjust the knob of the analog signal generator (7) to adjust the air pressure until the air pressure value is the target pressure required, then adjust the pneumatic switch valve (4) to the closed position so that the air path after the proportional valve (3) is closed, and then remove the digital barometer from the check bottle (5). S5. Place the pressure incubator (1) in a portable constant temperature chamber to raise the temperature of the inner cavity of the pressure incubator (1) to the target temperature. Then transfer the embryo in S1 to a culture dish. Next, place the culture dish containing the embryo in the tank (101) of the pressure incubator (1). After correctly positioning, close the lid (102) and lock and seal the lid (102) and the tank (101). Then transfer the pressure incubator (1) to the constant temperature water bath (8). During the transfer, use the portable constant temperature chamber to keep the pressure incubator (1) warm until the pressure incubator (1) enters the constant temperature water bath (8). Then fix the optical water level probe (12), start the electronic water replenisher (11) to make the water level just at the target water level, and set the target water bath temperature. S6. Connect the outlet of the check valve (5) to the inlet of the pressurized incubator (1), and connect the pneumatic speed control valve (9) and the flow sensor (10) in sequence to the outlet of the pressurized incubator (1); after the connection is completed, open the pneumatic switch valve (4) between the proportional valve (3) and the check valve (5), so that the gas is pressurized by the proportional valve (3) and enters the humidification bottle (106) of the incubator through the check valve (5), and then overflows from the humidification bottle (106) into the chamber of the pressurized incubator (1); adjust the pneumatic speed control valve (9) before the flow sensor (10) to the target exhaust rate; S7. Replace half of the embryo culture medium every 48 hours during the culture process.

2. The in vitro culture method for improving the quality of early embryonic development according to claim 1, characterized in that: In S2, the output pressure of the pressure reducing valve of the gas cylinder (2) is specifically adjusted to 0.04-0.06 MPa.

3. The in vitro culture method for improving the quality of early embryonic development according to claim 1, characterized in that: In step S4, the signal voltage of the analog signal generator (7) is adjusted so that the pressure displayed on the digital barometer is 0.005-0.007 MPa.

4. The in vitro culture method for improving the quality of early embryonic development according to claim 1, characterized in that: In S5, when the pressure incubator (1) is placed in a portable constant temperature box, the internal temperature of the pressure incubator (1) needs to be raised to 39 ℃; when the pressure incubator (1) is heated in a constant temperature water bath (8), the specific target water bath temperature is 39 ℃; the liquid level of the constant temperature water bath (8) is half the overall height of the pressure incubator (1).

5. The in vitro culture method for improving the quality of early embryonic development according to claim 1, characterized in that: In S6, the pneumatic speed control valve (9) is adjusted so that the target exhaust rate is 0.0065-0.0075 L / Min.

6. The in vitro culture method for improving the quality of early embryonic development according to claim 1, characterized in that: In S6, the internal pressure of the pressure incubator (1) is stably controlled at 0.006 MPa.

7. An in vitro culture device for improving the quality of early embryonic development, applicable to any one of the in vitro culture methods for improving the quality of early embryonic development according to claims 1-6, characterized in that, include: The components include a pressurized incubator (1), a gas cylinder (2), a proportional valve (3), a pneumatic switch valve (4), a check valve (5), a transformer (6), an analog signal generator (7), a constant temperature water bath (8), a pneumatic speed control valve (9), and a flow sensor (10). The pressure reducing valve of the gas cylinder (2) is connected to the inlet of the proportional valve (3), one end of the pneumatic switch valve (4) is connected to the outlet of the proportional valve (3), and the other end of the pneumatic switch valve (4) is connected to the inlet of the check valve (5). The pressurized incubator (1) is placed in the constant temperature water bath (8). An electronic water replenisher (11) and an optical water level probe (12) are provided to achieve a constant liquid level. The air inlet (103) of the pressurized incubator (1) is connected to the air outlet of the check bottle (5). The air outlet (104) of the pressurized incubator (1) is connected in sequence to the pneumatic speed control valve (9) and the flow sensor (10). A transformer (6) is connected in series with the proportional valve (3) to supply power to the proportional valve (3). An analog signal generator (7) is connected in parallel with the proportional valve (3) to adjust the signal voltage input to the proportional valve (3) and thus adjust the output air pressure of the proportional valve (3).

8. The in vitro culture device for improving the quality of early embryonic development according to claim 7, characterized in that: The pressurized incubator (1) includes: a tank (101) and a cover (102) detachably connected to the tank (101); the cover (102) is provided with an air inlet (103), an air outlet (104), and a pressure measuring port (105) penetrating inside and outside the cover (102); a humidification bottle (106) is detachably connected to the air inlet (103) on the inner side of the cover (102), and a humidification port (107) is provided on the upper side of the humidification bottle (106); the humidification bottle (106) The container (101) contains sterile water (108); the water level of the sterile water (108) is lower than the height of the humidification port (107); an air inlet pipe (109) is fixedly connected to the air inlet (103) on the inside of the cover (102), and the air outlet of the air inlet pipe (109) is immersed in the sterile water (108) and extends to the bottom of the inside of the humidification bottle (106); a pressure gauge (1010) is installed at the pressure measuring port (105) to monitor the internal pressure value of the tank (101).

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