Micro sperm cryopreservation method

Abstract

The invention relates to the technical field of sperm cryopreservation, in particular to a trace sperm cryopreservation method which comprises the following steps: S1, semen collection and pretreatment: collecting a semen sample, and liquefying in a thermostat at 37 DEG C for 30 minutes; detecting the sperm concentration (greater than or equal to 15 * 10 < 6 > / ml), the forward motion rate (PR is greater than or equal to 32%) and the normal form rate (greater than or equal to 4%) by adopting computer-aided semen analysis (CASA); s2, gradient addition of a protective agent: adding a precooled TEST-Yolk buffer solution (containing 10% of glycerol and 0.5 M of sucrose) in three times at the interval of 5 minutes until the final volume ratio is 1: 1; alpha-lipoic acid with the final concentration of 5 mu g / ml is added to inhibit oxidative damage; by avoiding direct contact for gradient cooling, accurate sample positioning and safety protection, the three major pain points of temperature fluctuation, operation risk and sample survival rate reduction in traditional freezing are solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sperm cryopreservation, and particularly relates to a method for cryopreservation of micro-amount of sperm. BACKGROUND

[0002] Environmental pollution is becoming more and more serious, and hormone abuse leads to continuous decline in the quality and quantity of male sperm, and the proportion of male infertility is increasing. About 40-50% of infertility worldwide is caused by male infertility, and about 10% of male infertility is azoospermia, which affects 1% of the male population worldwide. The incidence of azoospermia will gradually increase. Azoospermia patients cannot have their own genetically meaningful children in the past. With the development of assisted reproductive technology, azoospermia patients can obtain a small amount of sperm through percutaneous epididymal sperm aspiration (PESA), testicular sperm aspiration (TESA), micro-testicular sperm aspiration (MESE) and other techniques, and then inject the sperm into the oocyte through intracytoplasmic sperm injection (ICSI), and finally implant the formed embryo into the female uterine cavity to obtain clinical pregnancy. However, repeated sperm extraction may cause fibrosis of testicular and epididymal tissues, leading to testicular atrophy and degeneration of spermatogenic function. Therefore, cryopreservation of a small amount of motile sperm can not only reduce the pain and economic burden of patients due to repeated puncture, but also can greatly avoid the cancellation of fertilization due to the lack of available sperm on the day of ovum collection.

[0003] Compared with conventional semen freezing, micro-amount of sperm freezing has a low recovery rate and low survival rate of sperm after resuscitation due to the small amount of sperm, and the freezing difficulty is greater. Therefore, the appropriate sperm freezing carrier should be selected first. In order to effectively preserve these micro-amount of sperm, scholars at home and abroad have used various carriers to freeze micro-amount of sperm. These carriers include conventional freezing tubes, macaroni tubes, zonae pellucidae, freezing rings and ICSI injection needles. When freezing the carrier containing micro-amount of sperm, such as a freezing tube, the number of sperm in the freezing tube is small (usually only a few hundred to a few thousand), and the freezing process needs to be repeated to open and close the liquid nitrogen tank for gradient cooling, which causes temperature fluctuations and loss of cold air, seriously affecting the survival rate of sperm. At the same time, the operator needs to wear heavy protective equipment and manually adjust the position of the freezing tube several times, which not only increases the risk of liquid nitrogen frostbite, but also makes it difficult to ensure the continuity of the program cooling.

[0004] Therefore, it is particularly important to design a micro-amount of sperm cryopreservation method to solve the above problems. SUMMARY

[0005] In view of the shortcomings of the prior art, the present application designs a trace sperm freezing method, which aims to solve the technical problems that the traditional gradient cooling needs to open and close the liquid nitrogen tank multiple times, causing temperature fluctuations and cold air loss, which seriously affects the survival rate of sperm, and manual adjustment of the position of the freezing tube needs to frequently contact liquid nitrogen, which has the risk of frostbite.

[0006] To achieve the above object, the present application provides the following technical scheme:

[0007] A trace sperm freezing method, comprising the following steps:

[0008] S1, semen collection and pretreatment:

[0009] Collect the semen sample and place it in a 37℃ constant temperature box for 30 minutes;

[0010] Use computer-aided semen analysis (CASA) to detect sperm concentration (≥15×10 6 / ml), forward motility rate (PR≥32%) and normal morphology rate (≥4%);

[0011] S2, gradient addition of protective agent:

[0012] Add pre-cooled TEST-Yolk buffer solution (containing 10% glycerol and 0.5M sucrose) in three times, with an interval of 5 minutes each time, until the final volume ratio is 1:1;

[0013] Add alpha-lipoic acid with a final concentration of 5μg / ml to inhibit oxidative damage;

[0014] S3, programmed freezing:

[0015] Use a programmed cooling instrument for freezing, first reduce from room temperature to 4℃ at a rate of -1℃ / min, and then reduce to -80℃ at a rate of -5℃ / min;

[0016] The freezing tube uses CBS high-biocompatibility freezing macaroni, and each tube contains 0.25ml;

[0017] S4, liquid nitrogen gas phase preservation:

[0018] Transfer the freezing tube to the gas phase layer of the pre-cooled liquid nitrogen container at -180℃ for 24 hours, and then immerse the freezing tube in the liquid phase of the liquid nitrogen container at -196℃ for long-term preservation;

[0019] S5, thawing and evaluation:

[0020] Take the freezing tube out of the liquid nitrogen and place it in a 37℃ water bath for 60 seconds;

[0021] Use Percoll density gradient centrifugation method to remove the protective agent;

[0022] After thawing, evaluate sperm survival rate, require ≥ 50% compared with before freezing;

[0023] The liquid nitrogen container comprises a storage box, a storage cavity is formed in the left side of the inside of the storage box, a liquid nitrogen tank is placed at the right end of the inside of the storage box, the bottom end of the liquid nitrogen tank is connected with the inside of the storage cavity through a connecting pipe, a placing rack is movably installed in the inside of the storage cavity, a lifting mechanism is installed on the outside of the placing rack in the inside of the storage cavity, a plurality of groups of placing pipes are fixedly connected in the inside of the placing rack, a freezing tube is inserted into the inside of each of the plurality of groups of placing pipes, and a heat preservation plate is arranged on the top end of the storage cavity.

[0024] The lifting mechanism comprises moving grooves formed in the left and right sides of the inside of the storage cavity, an adjusting screw and a stabilizing slide rod are installed in the insides of the two groups of moving grooves respectively, the upper and lower ends of the adjusting screw are rotatably connected with the inside of the moving groove, the upper and lower ends of the stabilizing slide rod are fixedly connected with the inside of the moving groove, the left side of the placing rack is threadedly connected with the adjusting screw through a first connecting head, the right side of the placing rack is slidably connected with the stabilizing slide rod through a second connecting head, the top end of the adjusting screw is fixedly connected with an operation knob, and a locking mechanism is installed at the connection position of the operation knob and the adjusting screw.

[0025] As a preferred scheme of the present application, the TEST-Yolk buffer solution in step S2 needs to contain 20% egg yolk and 5mM HEPES (pH 7.4), and be sterilized by a 0.22μm filter membrane.

[0026] As a preferred scheme of the present application, the freezing tube in step S3 needs to be marked with patient ID, collection date and freezing batch two-dimensional code.

[0027] As a preferred scheme of the present application, the sample before freezing needs to be detected by sperm DNA fragmentation rate (SCSA method, DFI < 30%).

[0028] As a preferred scheme of the present application, the locking mechanism comprises a fixed gear fixedly connected at the connection position of the operation knob and the adjusting screw, a pull rod is slidably connected with the outside of the fixed gear at the top end of the storage cavity, one end of the pull rod close to the fixed gear is engaged with the fixed gear through a locking tooth block, and a first spring is sleeved on the outside of the pull rod.

[0029] As a preferred scheme of the present application, moving wheels are installed at the four corners of the bottom of the storage box, an operation handle is hinged to the front of the storage box, and a positioning sleeve is fixedly connected with the outside of the operation handle on the front of the storage box.

[0030] As a preferred scheme of the present application, a discharge pipe is fixedly connected with the outside of the storage box and at the bottom end of the storage cavity, and valves are installed on the discharge pipe and the connecting pipe.

[0031] As a preferred scheme of the present application, the transparent glass window is fixedly installed at the center of the heat preservation plate, the taking and placing opening is arranged at the inside of the storage cavity and above the plurality of placing tubes, and the cover is rotationally connected to the top of the heat preservation plate and outside the plurality of taking and placing openings.

[0032] As a preferred scheme of the present application, the upper and lower ends of the freezing tube are respectively threadedly connected with the tube cover and the discharge cover, the inside of the freezing tube is fixedly connected with the conical collecting sleeve, and the inside of the freezing tube and below the conical collecting sleeve is provided with the liquid nitrogen storage cavity.

[0033] As a preferred scheme of the present application, the inside of the liquid nitrogen storage cavity is fixedly connected with the two groups of connecting barrels, the opposite ends of the two groups of connecting barrels are slidably connected with the telescopic columns, the outside of the two groups of telescopic columns and inside the connecting barrels are sleeved with the second springs, the opposite ends of the two groups of telescopic columns are fixedly connected with the linkage blocks, the inside of the linkage blocks is slidably connected with the freezing tube, the inside of the linkage block and the placing tube are provided with the liquid inlet hole, and the inside of the freezing tube and the position corresponding to the liquid inlet hole are provided with the liquid guide groove.

[0034] Compared with the prior art, the present application has the following advantages:

[0035] 1、In the present application, the storage tank, the storage cavity, the liquid nitrogen tank, the connecting pipe, the placing rack, the lifting mechanism and the heat preservation plate are cooperatively designed, the liquid nitrogen in the liquid nitrogen tank is introduced into the storage cavity through the connecting pipe, the inside of the storage cavity is formed with the gas phase zone and the liquid phase zone, the lifting mechanism adopts the adjusting screw and the gear locking to prevent the false touch adjustment, the independent taking and placing opening and the transparent window on the heat preservation plate are combined to realize the cover-free operation, the direct contact for gradient cooling is avoided, and the risk of liquid nitrogen frostbite is avoided.

[0036] 2、In the application, through the cooperation design of the placing tube and the freezing tube, the upper end of the freezing tube is connected with the tube cover through threads, the closed protection of the sample access port is realized, the internal constant temperature environment of the freezing tube is ensured, the discharge cover connected with the lower end through threads is used for controlling the discharge of liquid nitrogen, and the discharge cover can be unscrewed to release the residual liquid nitrogen or clean the pipeline during operation, the conical collecting sleeve fixed in the freezing tube adopts an inclined wall surface to align the taking and placing port, the trace sperm sample is guided to accurately fall into the specified position, sample adhesion to the wall is avoided, the conical structure can be adapted to different specifications of the cryopreservation tube, the freezing sample recovery efficiency is improved, the liquid nitrogen storage cavity below the conical collecting sleeve is used for temporarily storing the evaporated liquid nitrogen, the deep low-temperature environment of the sample is maintained, the cavity is regularly cleaned of sediment impurities through the bottom discharge cover, the purity of the liquid nitrogen is ensured, the airtightness is ensured through the double-thread sealing, the sample positioning is optimized in combination with the conical collecting sleeve, and the liquid nitrogen storage cavity prolongs the cooling time, thereby forming an efficient and safe sperm freezing storage unit, when the freezing tube is not inserted into the inside of the placing tube, the second spring pushes the telescopic column to extend, the linkage block closes the liquid guide groove, and after the freezing tube is inserted into the inside of the placing tube, the linkage block is pressed to move to the inside of the freezing tube, the liquid inlet hole is communicated with the liquid guide groove, the liquid nitrogen is guided into the inside of the liquid nitrogen storage cavity, the accurate control of the liquid nitrogen passage is realized through mechanical linkage, and therefore the trace sperm recovery rate and the liquid nitrogen cooling time are improved, and the operation frostbite risk is reduced. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 It is a schematic view of the liquid nitrogen container structure of the application;

[0038] Figure 2 It is a schematic view of the internal structure of the storage cavity of the application;

[0039] Figure 3 It is Figure 2 the enlarged view of A in FIG. 1;

[0040] Figure 4 It is a schematic view of the placing rack structure of the application;

[0041] Figure 5 It is a schematic view of the placing tube structure of the application;

[0042] Figure 6 It is a state diagram of the freezing tube located in the inside of the placing tube of the application;

[0043] Figure 7 It is Figure 6 the enlarged view of B in FIG. 1;

[0044] Figure 8 It is a sectional view of the internal structure of the placing tube of the application;

[0045] Figure 9 It is a sectional view of the internal structure of the freezing tube of the application.

[0046] In the diagram: 1. Storage box; 101. Casters; 102. Operating handle; 103. Positioning sleeve; 2. Storage cavity; 201. Discharge pipe; 202. Valve; 3. Liquid nitrogen tank; 4. Connecting pipe; 5. Placement rack; 6. Lifting mechanism; 601. Moving slot; 602. Adjusting screw; 603. Stabilizing slide bar; 604. First connector; 605. Second connector; 606. Operating knob; 607. Locking mechanism; 608. Fixing gear; 609. Pull rod; 610. Locking tooth block; 611. First spring; 7. Placement tube; 8. Freezing tube; 801. Tube cap; 802. Discharge cap; 803. Conical collection sleeve; 804. Liquid nitrogen storage chamber; 805. Connecting cylinder; 806. Telescopic column; 807. Second spring; 808. Linkage block; 809. Liquid inlet; 810. Liquid guide groove; 9. Insulation plate; 901. Transparent glass window; 902. Removal port; 903. Sealing cap. Detailed Implementation

[0047] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0048] Example: Please refer to Figures 1-9 The present invention provides a technical solution:

[0049] A method for cryopreservation of trace amounts of sperm includes the following steps:

[0050] S1. Semen Collection and Pretreatment:

[0051] Collect semen samples and liquefy them in a 37°C incubator for 30 minutes;

[0052] Sperm concentration (≥15×10⁻⁶) was detected using computer-assisted semen analysis (CASA). 6 / ml), forward motility rate (PR≥32%) and normal morphology rate (≥4%). Before freezing, the sample must be tested for sperm DNA fragmentation rate (SCSA method, DFI<30%).

[0053] Constant temperature liquefaction is used to maintain sperm physiological activity, triple quality control indicators ensure the quality of frozen samples, and DNA fragmentation detection excludes samples with genetic damage.

[0054] S2, Gradual addition of protective agents:

[0055] Pre-cooled TEST-Yolk buffer (containing 10% glycerol, 0.5M sucrose) was added in three times, the TEST-Yolk buffer needs to contain 20% egg yolk, 5mM HEPES (pH7.4), and sterilized by 0.22μm filter membrane, each addition interval 5 minutes, until the final volume ratio is 1:1;

[0056] Alpha-lipoic acid was added to a final concentration of 5μg / ml to inhibit oxidative damage;

[0057] The use of gradient addition reduces the osmotic shock to improve survival rate, egg yolk lipoprotein protects the integrity of cell membrane, and alpha-lipoic acid removes free radicals to reduce oxidative damage;

[0058] S3, programmed freezing:

[0059] The freezing was performed using a programmed temperature controller, first reducing from room temperature to 4℃ at a rate of -1℃ / min, and then reducing to -80℃ at a rate of -5℃ / min;

[0060] The freezing tube 8 uses CBS high biocompatibility freezing wheat tube, each tube contains 0.25ml, and the freezing tube needs to be marked with patient ID, collection date and freezing batch two-dimensional code;

[0061] The two-dimensional code traceability system is used to avoid sample confusion, two-stage cooling reduces ice crystal formation, and CBS wheat tube has high biocompatibility to reduce cold shock risk;

[0062] S4, liquid nitrogen gas phase storage:

[0063] The freezing tube 8 was transferred to the gas phase layer of the pre-cooled to -180℃ liquid nitrogen container for 24 hours, and then immersed in the liquid phase of the -196℃ liquid nitrogen container for long-term storage;

[0064] S5, thawing and evaluation:

[0065] The freezing tube 8 was taken out from the liquid nitrogen and placed in a 37℃ water bath for 60 seconds;

[0066] Percol l density gradient centrifugation method was used to remove the protective agent;

[0067] After thawing, the sperm survival rate was evaluated, which was required to be ≥50% compared with before freezing;

[0068] Through rapid rewarming to reduce recrystallization damage, three quality indicators ensure clinical usability.

[0069] First, in the present embodiment, the specific structure of the liquid nitrogen container is as follows:

[0070] The liquid nitrogen container comprises a storage box 1, a storage cavity 2 is formed in the left side of the inside of the storage box 1, a liquid nitrogen tank 3 is placed at the right end of the inside of the storage box 1, the bottom end of the liquid nitrogen tank 3 is connected with the inside of the storage cavity 2 through a connecting pipe 4, a placing rack 5 is movably installed in the inside of the storage cavity 2, a lifting mechanism 6 is installed in the inside of the storage cavity 2 and outside the placing rack 5, a plurality of placing pipes 7 are fixedly connected in the inside of the placing rack 5, a plurality of freezing pipes 8 are inserted into the inside of the plurality of placing pipes 7, and a heat preservation plate 9 is arranged at the top end of the storage cavity 2.

[0071] Then, in the embodiment, the specific structure of the lifting mechanism 6 is as follows:

[0072] The lifting mechanism 6 comprises two moving grooves 601 formed in the left and right sides of the inside of the storage cavity 2, two adjusting screws 602 and two stable slide rods 603 are installed in the inside of the two moving grooves 601 respectively, the upper and lower ends of the adjusting screw 602 are rotatably connected with the inside of the moving groove 601, the upper and lower ends of the stable slide rod 603 are fixedly connected with the inside of the moving groove 601, the left side of the placing rack 5 is threadedly connected with the adjusting screw 602 through a first connecting head 604, the right side of the placing rack 5 is slidably connected with the stable slide rod 603 through a second connecting head 605, the top end of the adjusting screw 602 is fixedly connected with an operation knob 606, a locking mechanism 607 is installed at the connection between the operation knob 606 and the adjusting screw 602, the liquid nitrogen tank 3 contains liquid nitrogen which is introduced into the inside of the storage cavity 2 through the connecting pipe 4, so that a gas phase zone and a liquid phase zone are formed in the inside of the storage cavity 2, the box cover at the top of the storage box 1 is opened, the adjusting screw 602 is rotated by rotating the operation knob 606, and the two ends of the adjusting screw 602 are rotatably connected with the moving groove 601; at the same time, the placing rack 5 is driven to move vertically under the thread engagement of the first connecting head 604, and is slidably connected with the stable slide rod 603 through the second connecting head 605, so as to ensure that the moving track is not deviated, until the placing rack 5 moves to the top end, then the prepared freezing pipe 8 is inserted into the placing pipe 7, the position of the placing rack 5 is adjusted up and down, so as to change the relative height of the freezing pipe 8 in the gas phase zone or the liquid phase zone, and at the same time, the risk of liquid nitrogen frostbite is avoided by avoiding direct contact.

[0073] Further, the locking mechanism 607 comprises a fixed gear 608 fixedly connected to the connecting position of the operating knob 606 and the adjusting screw 602, a pull rod 609 is slidably connected to the top end of the storage cavity 2 and located outside the fixed gear 608, one end of the pull rod 609 close to the fixed gear 608 is engaged with the fixed gear 608 through a locking tooth block 610, and the outside of the pull rod 609 is sleeved with a first spring 611, when it is necessary to adjust the height of the placing rack 5, the pull rod 609 is first pulled to disengage the locking tooth block 610 from the fixed gear 608, and then the adjusting screw 602 is unlocked and the operating knob 606 is rotated to rotate the placing rack 5, so as to adjust the height of the placing rack 5, and the locking mechanism 607 can effectively prevent the placing rack 5 from moving accidentally due to misoperation or external vibration during the operation of the liquid nitrogen container, thereby ensuring the safety and stability of the sperm cryopreservation.

[0074] Wherein, the four corners of the bottom of the storage box 1 are provided with moving wheels 101, the front of the storage box 1 is hingedly connected with an operating handle 102, and the front of the storage box 1 and outside the operating handle 102 is fixedly connected with a positioning sleeve 103, the four corners of the bottom of the storage box 1 are provided with moving wheels 101, which facilitates the overall movement and transportation, and the positioning sleeve 103 is used for fixing the operating handle in the inside when the operating handle is not used to avoid accidental opening, thereby improving the use convenience of the storage box 1.

[0075] Further, the outside of the storage box 1 and the bottom end of the storage cavity 2 are fixedly connected with a discharge pipe 201, and the discharge pipe 201 and the connecting pipe 4 are both provided with valves 202, liquid nitrogen in the liquid nitrogen tank 3 is introduced into the storage cavity 2 by opening the valve on the connecting pipe 4, and the discharge pipe 201 can discharge liquid nitrogen to control the storage amount in the storage cavity 2.

[0076] Furthermore, the center of the heat preservation plate 9 is fixedly provided with a transparent glass window 901, the inside of the storage cavity 2 and above the plurality of placing pipes 7 are provided with taking and placing openings 902, and the top of the heat preservation plate 9 and outside the plurality of taking and placing openings 902 are rotatably connected with covers 903, the transparent glass window 901 fixedly arranged at the center of the heat preservation plate 9 is made of high-transmittance low-temperature-resistant glass material, which can realize real-time observation of the sample state from the outside while maintaining the low-temperature environment in the storage cavity 2, avoids temperature fluctuation caused by frequent opening of the heat preservation plate, and guarantees the stability of the sperm cryopreservation environment, the inside of the storage cavity 2 is provided with independent taking and placing openings 902 above each group of placing pipes 7, forming a vertical channel directly reaching the placing pipe, facilitating accurate storage and taking operation of the cryopreservation tube, the cover 903 in the closed state is attached to the edge of the taking and placing opening 902, forming a sealed heat preservation barrier to reduce cold air leakage, and rotating the cover 903 completely exposes the taking and placing opening 902, so that the operator can store and take the cryopreservation tube through a special clamp, thereby improving the safety and operation efficiency of the frozen sample management.

[0077] Secondly, the upper and lower ends of the freezing tube 8 are respectively threaded with a tube cover 801 and a discharge cover 802, the inside of the freezing tube 8 is fixedly connected with a conical collecting sleeve 803, the inside of the freezing tube 8 and below the conical collecting sleeve 803 is provided with a liquid nitrogen storage cavity 804, the upper end of the freezing tube 8 is threaded with the tube cover 801 to realize the airtight protection of the sample access port and ensure the constant temperature environment inside the freezing tube, the lower end is threaded with the discharge cover 802 for controlling the discharge of liquid nitrogen, which can be unscrewed to release the residual liquid nitrogen or clean the pipeline during operation, the conical collecting sleeve 803 fixedly arranged inside the freezing tube 8 is provided with an inclined wall surface aligned with the taking and placing port to guide the accurate falling of the trace sperm sample into the specified position, avoiding the adhesion of the sample to the wall, the conical structure can be adapted to different specifications of the cryopreservation tube to improve the recovery efficiency of the frozen sample, and the liquid nitrogen storage cavity 804 arranged below the conical collecting sleeve 803 is used for temporarily storing the evaporated and cooled liquid nitrogen to maintain the deep low temperature environment of the sample, the cavity is regularly cleaned of sediment impurities through the bottom discharge cover 802 to ensure the purity of the liquid nitrogen, the airtightness is ensured through double-thread sealing, the sample positioning is optimized in combination with the conical collecting sleeve, and the liquid nitrogen storage cavity prolongs the cooling time to form an efficient and safe sperm freezing storage unit.

[0078] Finally, the inside of the liquid nitrogen storage cavity 804 is fixedly connected with two groups of connecting barrels 805, the opposite ends of the two groups of connecting barrels 805 are slidably connected with telescopic columns 806, the outer sides of the two groups of telescopic columns 806 and inside the connecting barrels 805 are sleeved with second springs 807, the opposite ends of the two groups of telescopic columns 806 are fixedly connected with linkage blocks 808, and the two groups of linkage blocks 808 are slidably connected with the inside of the freezing tube 8, the inside of the linkage block 808 and the inside of the placing tube 7 are both provided with liquid inlet holes 809, the inside of the freezing tube 8 is provided with a liquid guide groove 810 at a position corresponding to the liquid inlet hole 809, the inside of the liquid nitrogen storage cavity 804 is fixedly connected with two groups of connecting barrels 805, the connecting barrel 805 is made of low-temperature-resistant aluminum alloy material to ensure stable work in-196℃ environment, the telescopic column 806 is slidably connected with the connecting barrel 805, the surface is chrome-plated to reduce the friction coefficient, and smooth extension in low-temperature environment is realized, the second spring 807 is made of stainless steel material, pre-compression design provides constant elastic force, and pushes the telescopic column 806 to reset, when the freezing tube 8 is not inserted into the inside of the placing tube 7, the second spring 807 pushes the telescopic column 806 to extend outward, the linkage block 808 closes the liquid guide groove 810, and after the freezing tube 8 is inserted into the inside of the placing tube 7, the linkage block 808 moves to the inside of the freezing tube 8 under pressure, so that the liquid inlet hole 809 and the liquid guide groove 810 are communicated, the liquid nitrogen is guided into the inside of the liquid nitrogen storage cavity 804, and the accurate control of the liquid nitrogen passage is realized through mechanical linkage.

[0079] In the embodiment, the specific implementation scene is that: the liquid nitrogen tank 3 stores liquid nitrogen, which is introduced into the storage cavity 2 through the connecting pipe 4, so that the inside of the storage cavity 2 forms a gas phase zone and a liquid phase zone; the box cover at the top of the storage box 1 is opened; the adjusting screw rod 602 is rotated by operating the knob 606; the two ends of the adjusting screw rod 602 are rotationally connected with the moving groove 601;Meanwhile, the placing rack 5 is driven to move vertically under the threaded engagement of the first connecting head 604, and is slidably connected with the stable slide rod 603 through the second connecting head 605, so that the moving track is ensured to be free of deviation, until the placing rack 5 moves to the top end, and then the prepared frozen tube 8 can be inserted into the placing tube 7, the position of the placing rack 5 is adjusted up and down, so that the relative height of the frozen tube 8 in the gas phase zone or the liquid phase zone can be changed, and meanwhile, the risk of frostbite caused by direct contact with liquid nitrogen is avoided, the locking mechanism 607 can be used to fix the adjusting rod 602, so as to effectively prevent the placing rack 5 from moving accidentally due to misoperation or external vibration during the operation of the liquid nitrogen container, the transparent glass window 901 fixedly installed at the center of the heat preservation plate 9 is made of high-transmittance low-temperature-resistant glass material, so that the sample state can be observed in real time from the outside while the low-temperature environment inside the storage cavity 2 is maintained, the temperature fluctuation caused by frequent opening of the heat preservation plate is avoided, and the stability of the sperm freezing and preservation environment is ensured, the independent taking and placing opening 902 is arranged above each group of placing tubes 7 in the storage cavity 2, a vertical channel directly reaching the placing tube is formed, and the accurate storage and taking operation of the cryopreservation tube is facilitated, the cover 903 is closed to fit the edge of the taking and placing opening 902, a sealed heat preservation barrier is formed, the cold gas leakage is reduced, the cover 903 is rotated to completely expose the taking and placing opening 902, and the operator can store and take the cryopreservation tube through a special clamp, the upper end of the frozen tube 8 is threadedly connected with the tube cover 801, the closed protection of the sample storage and taking opening is realized, the constant temperature environment inside the frozen tube is ensured, the lower end is threadedly connected with the discharge cover 802 for controlling the discharge of liquid nitrogen, the discharge cover 802 can be screwed to release the residual liquid nitrogen or clean the pipeline during operation, the conical collecting sleeve 803 fixedly installed inside the frozen tube 8 adopts an inclined wall surface aligned with the taking and placing opening, guides the accurate falling of a trace of sperm sample into a specified position, avoids the adhesion of the sample to the tube wall, the conical structure can be adapted to different specifications of cryopreservation tubes, and the recovery efficiency of the frozen sample is improved, the liquid nitrogen storage cavity 804 arranged below the conical collecting sleeve 803 is used for temporarily storing the evaporated liquid nitrogen, and maintains the deep low-temperature environment of the sample, the cavity is regularly cleaned of precipitated impurities through the bottom discharge cover 802, the purity of the liquid nitrogen is ensured, the airtightness is ensured through double-thread sealing, the sample positioning is optimized in combination with the conical collecting sleeve, the liquid nitrogen storage cavity prolongs the cold preservation time, and a high-efficiency and safe sperm freezing and storage unit is formed, when the frozen tube 8 is not inserted into the inside of the placing tube 7, the second spring 807 pushes the telescopic column 806 to extend, the linkage block 808 closes the liquid guide groove 810, and after the frozen tube 8 is inserted into the inside of the placing tube 7, the linkage block 808 is pressed to move to the inside of the frozen tube 8, so that the liquid inlet hole 809 is communicated with the liquid guide groove 810, and the liquid nitrogen is guided into the inside of the liquid nitrogen storage cavity 804, the accurate control of the liquid nitrogen passage is realized through mechanical linkage, the whole operation process is simple and convenient, the gradient cooling, accurate sample positioning and safety protection are realized by avoiding direct contact, and the three major pain points of temperature fluctuation, operation risk and reduced sample survival rate in the traditional freezing are solved.

[0080] While embodiments of the application have been shown and described, it is to be understood that the embodiments described are merely exemplary of the principles and application of the present application. Numerous modifications and adaptions can be effected without departing from the spirit and scope of the present application, which is not limited to the exact construction and arrangement described. It is intended, therefore, to cover all modifications and adaptions that fall within the scope of the claims and their equivalents.

Claims

1. A method for cryopreservation of microsperm, characterized by, Comprising the following steps: S1, semen collection and pretreatment: Collect semen samples and place them in a 37℃ incubator for 30 minutes; Computer assisted semen analysis (CASA) was used to determine sperm concentration (≥ 15 x 10 6 / ml), progressive motility (PR ≥ 32%) and normal morphology (≥ 4%). S2, protective agent gradient addition: Add pre-cooled TEST-Yolk buffer solution (containing 10% glycerol, 0.5M sucrose) in three times, each time with an interval of 5 minutes, until the final volume ratio is 1:1; Add α-lipoic acid with a final concentration of 5μg / ml to inhibit oxidative damage; S3, programmed freezing: Use a programmed cooling instrument to freeze, first reduce from room temperature to 4℃ at a rate of -1℃ / min, then reduce to -80℃ at a rate of -5℃ / min; The freezing tube (8) uses CBS high biocompatibility freezing wheat tube, each tube contains 0.25ml; S4, liquid nitrogen gas phase storage: Transfer the freezing tube (8) to the gas phase layer of the liquid nitrogen container pre-cooled to -180℃ for 24 hours, then immerse the freezing tube (8) in the liquid phase of the liquid nitrogen container at -196℃ for long-term storage; S5, thawing and evaluation: Take the freezing tube (8) out of the liquid nitrogen and place it in a 37℃ water bath for 60 seconds; Use Percol l density gradient centrifugation method to remove the protective agent; After thawing, evaluate the sperm survival rate, which should be ≥50% compared with before freezing; The liquid nitrogen container comprises a storage box (1), a storage cavity (2) is formed on the left side inside the storage box (1), a liquid nitrogen tank (3) is placed at the right end inside the storage box (1), the bottom end of the liquid nitrogen tank (3) is connected with the inside of the storage cavity (2) through a connecting pipe (4), a placing rack (5) is movably installed inside the storage cavity (2), a lifting mechanism (6) is installed on the outside of the placing rack (5) inside the storage cavity (2), a plurality of placing tubes (7) are fixedly connected inside the placing rack (5), a freezing tube (8) is inserted into each of the plurality of placing tubes (7), and a heat preservation plate (9) is arranged on the top end of the storage cavity (2); The lifting mechanism (6) comprises two moving grooves (601) formed on the left and right sides inside the storage cavity (2), an adjusting screw (602) and a stable sliding rod (603) are installed in the two moving grooves (601) respectively, the upper and lower ends of the adjusting screw (602) are rotatably connected with the inside of the moving groove (601), the upper and lower ends of the stable sliding rod (603) are fixedly connected with the moving groove (601), the left side of the placing rack (5) is threadedly connected with the adjusting screw (602) through a first connecting head (604), the right side of the placing rack (5) is slidably connected with the stable sliding rod (603) through a second connecting head (605), the top end of the adjusting screw (602) is fixedly connected with an operation knob (606), and a locking mechanism (607) is installed at the connection between the operation knob (606) and the adjusting screw (602).

2. The method of claim 1, wherein the method is characterized by, The TEST-Yolk buffer solution in step S2 needs to contain 20% egg yolk, 5mM HEPES (pH7.4), and be sterilized by a 0.22μm filter membrane.

3. The method of claim 1, wherein the method is characterized by, The freezing tube in step S3 needs to be marked with patient ID, collection date and freezing batch two-dimensional code.

4. The method according to any one of claims 1 to 3, characterized in that: The sample before freezing needs to be detected by sperm DNA fragmentation rate (SCSA method, DFI <30%).

5. The method of claim 1, wherein the method is characterized by, The locking mechanism (607) includes a fixed gear (608) fixedly connected to the connection between the operation knob (606) and the adjusting screw (602), the top end of the storage cavity (2) is slidingly connected with a pull rod (609) outside the fixed gear (608), one end of the pull rod (609) close to the fixed gear (608) is engaged with the fixed gear (608) through a locking tooth block (610), and the outside of the pull rod (609) is sleeved with a first spring (611).

6. The method of claim 1, wherein the method is characterized by, The bottom of the storage box (1) is provided with four moving wheels (101) at the four corners, and the front of the storage box (1) is hingedly connected with an operation handle (102), and the front of the storage box (1) is fixedly connected with a positioning sleeve (103) outside the operation handle (102).

7. The method of claim 1, wherein the method is a method of cryopreservation of a small number of sperm. The outside of the storage box (1) is fixedly connected with a discharge pipe (201) at the bottom end of the storage cavity (2), and the discharge pipe (201) and the connecting pipe (4) are both provided with a valve (202).

8. The method of claim 1, wherein the method is a method of cryopreservation of a small number of sperm. The top end of the storage cavity (2) is provided with a heat preservation plate (203), the center of the heat preservation plate (203) is fixedly provided with a transparent glass window (204), the inside of the storage cavity (2) is provided with a taking and placing opening (205) directly above the plurality of placing pipes (7), and the top of the heat preservation plate (203) is rotatably connected with a cover (206) outside the plurality of taking and placing openings (205).

9. The method of claim 1, wherein the method is a method of cryopreservation of a small number of sperm. The upper and lower ends of the freezing tube (8) are respectively threadedly connected with a tube cover (801) and a discharge cover (802), the inside of the freezing tube (8) is fixedly connected with a conical collecting sleeve (803), and the inside of the freezing tube (8) is provided with a liquid nitrogen storage cavity (804) below the conical collecting sleeve (803).

10. A method for cryopreservation of trace sperm according to claim 9, characterized in that, The inside of the liquid nitrogen storage cavity (804) is fixedly connected with two groups of connecting barrels (805), the opposite ends of the two groups of connecting barrels (805) are slidingly connected with telescopic columns (806), the outside of the two groups of telescopic columns (806) and the inside of the connecting barrels (805) are sleeved with second springs (807), the opposite ends of the two groups of telescopic columns (806) are fixedly connected with linkage blocks (808), and the linkage blocks (808) are slidingly connected with the inside of the freezing tube (8), the linkage blocks (808) and the inside of the placing pipe (7) are both provided with liquid inlet holes (809), and the inside of the freezing tube (8) is provided with liquid guide grooves (810) corresponding to the liquid inlet holes (809).

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