Rapid sperm screening device and forming method

Abstract

The invention discloses a rapid sperm screening device and a forming method, the rapid sperm screening device comprises a working shell, a glass slide and at least one layer of screening assembly, and the working shell is provided with a first through groove and a second through groove; the working shell is arranged on the glass slide, so that a sample adding area is formed between the first through groove and the surface of the glass slide, a sample discharging area is formed between the second through groove and the surface of the glass slide, a containing concave position is arranged between the bottom of the working shell and the glass slide, the containing concave position is located between the first through groove and the second through groove, and the screening assembly is arranged in the containing concave position. Compared with the prior art, the sperm screening device has the beneficial effects that the sperm screening device is portable, can be operated in a common clinic or a home environment, completely depends on swimming of active sperms, the screened sperms have high DNA integrity, damage to the DNA integrity of the sperms is small, meanwhile, an integrated screening process is achieved, and the screening efficiency is improved. The seminal fluid is effectively prevented from being polluted in the whole assisted reproduction process.

Description

Technical Field

[0001] The invention relates to the technical field of sperm screening, and in particular to a sperm rapid screening device and a molding method. Background Art

[0002] About 15% of couples of childbearing age worldwide have infertility problems, and about half of them are caused by male factors. About 5% to 15% of male infertility is caused by immune factors, and about 10% of male infertility patients have anti-sperm antibodies (AsAb) in their bodies. AsAb is an antibody that can specifically bind to sperm surface antigens and is divided into circulating AsAb (serum) and local AsAb (genital tract). Serum AsAb is mainly IgG and IgM, while genital tract AsAb is mainly IgG and IgA. AsAb can directly act on sperm or indirectly affect sperm function by changing the local microenvironment, including causing sperm aggregation, complement-dependent sperm braking, inhibiting acrosome activity or inhibiting fertilized egg development, leading to infertility.

[0003] Sperm and its cell membrane are specific antigens with self- and foreign antigenicity. Both sexes can produce polyclonal anti-sperm antibodies through immune response, which leads to immune infertility. The causes of AsAb production are currently believed to be related to the following factors: 1) destruction of physiological barriers; 2) genital tract inflammation; 3) lack of seminal plasma immunosuppressants; 4) other reasons, such as abnormal local immune function of the genital tract. The presence of anti-sperm antibodies has the following effects on sperm: 1) agglutination and / or braking, interfering with sperm movement and the ability to penetrate cervical mucus; 2) direct or indirect killing of sperm; 3) hindering sperm-egg fusion and implantation. AsAb bound to the sperm head can interfere with the acrosome reaction and hinder sperm capacitation, affecting sperm-egg fusion and embryonic development. Therefore, anti-sperm antibodies have an effect on sperm capacitation, acrosome reaction and sperm-egg fusion.

[0004] The female reproductive tract is not a place of immune exemption, but a place where the immune system plays a local protective role against infectious agents and establishes and maintains pregnancy; among them, the production of AsAb has an adverse effect on fertility. AsAb may affect the parts of in vitro fertilization (IVF) including sperm binding to the zona pellucida, sperm penetration of the zona pellucida, zona pellucida reaction, gamete fusion, division and embryonic development. AsAb may also cause infertility because sperm are coated with anti-sperm antibodies and cannot pass through the cervical mucus normally to enter the uterus.

[0005] At present, with the development of clinical assisted reproductive technologies such as artificial insemination (IUI), in vitro fertilization and intracytoplasmic sperm injection (ICSI), there is a higher demand for in vitro sperm selection, especially for sperm with good vitality and high survival rate. Traditional sperm separation techniques include swim-up method, density gradient centrifugation method, etc. These methods all require in vitro centrifugation, which may cause mechanical and oxidative damage to sperm, thereby affecting sperm quality and sperm-egg fertilization. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a sperm rapid screening device and a forming method.

[0007] To achieve the above-mentioned purpose, the present invention discloses a sperm rapid screening device and a forming method, comprising a working shell, a glass slide and at least one layer of screening components, the working shell is provided with a first through groove and a second through groove, the working shell is arranged on the glass slide so that a sample loading area is formed between the first through groove and the surface of the glass slide, and a sample output area is formed between the second through groove and the surface of the glass slide, a receiving recess is provided between the bottom of the working shell and the glass slide, the receiving recess is located between the first through groove and the second through groove, the screening component is arranged in the receiving recess, the sample loading area is used to fill semen, and the sperm in the semen relies on its own swimming to be screened by the screening component and flow into the sample output area.

[0008] Furthermore, the screening component includes a support block and a plurality of micro-columns, wherein the plurality of micro-column arrays are arranged on the support block, and a micro-channel is formed between two adjacent micro-columns;

[0009] The end of the microcolumn is arranged in contact with the surface of the glass slide, and the top of the support block is arranged in contact with the top of the accommodating recess.

[0010] Furthermore, the dimensions of the slide glass are 75 mm×1.2 mm×25 mm in length, width and height.

[0011] Furthermore, the length, width and height dimensions of the first through groove and the second through groove are 45 mm×3 mm×6 mm.

[0012] Furthermore, the material of the working shell is polydimethylsiloxane.

[0013] Furthermore, the contour size of the accommodating recess is consistent with the contour size of the first through groove, and the periphery of the supporting block is flush with the periphery of the accommodating recess.

[0014] Furthermore, the array of the micropillars is any one of a rectangular array and a staggered array;

[0015] The spacing distance between the micro-pillars in two adjacent rows is 150 μm, and the spacing distance between the micro-pillars in two adjacent lines is 150 μm.

[0016] Furthermore, the diameter of a single microcolumn is 75 μm, and the height thereof is 75 μm.

[0017] Furthermore, the outer contour shape of the microcolumn is any one of a cylindrical shape, a semi-cylindrical shape and a C-shaped column.

[0018] Further, the following steps are included:

[0019] Step 1: Select microcolumns and array shapes of different shapes according to the use requirements, and make corresponding molds according to the microcolumn shapes and array shapes;

[0020] Step 2: Pour liquid polydimethylsiloxane into the mold. The liquid polydimethylsiloxane should cover all the molds and be at least 1mm higher than the mold height.

[0021] Step 3: Place the liquid polydimethylsiloxane together with the mold in a spin coater for spin coating. The spin coating should produce a layer of polydimethylsiloxane coating of about 1 mm on the mold, and the 1 mm coating serves as the support block;

[0022] Step 4: Place the mold together with the polydimethylsiloxane in a drying oven and dry at 70°C for about 40 minutes. At this time, the polydimethylsiloxane should be completely converted from liquid to solid;

[0023] Step 5: removing the solid polydimethylsiloxane from the mold to obtain a first layer screening component;

[0024] Step 6. Repeat steps 2-3;

[0025] Step 7: Place the mold and liquid polydimethylsiloxane in a drying oven and dry at 70°C for 20 minutes. At this time, the polydimethylsiloxane is not completely cured. The micro-column end of the first layer of the screening component is attached to the smooth surface of the second layer that is not cured at this time, and continue to place it in the drying oven and dry it at 70°C for about 40 minutes. At this time, layer 1 and layer 2 are bonded;

[0026] Step 8: Repeat steps 1 to 7 to produce a multi-layer screening component.

[0027] Compared with the prior art, the beneficial effects of the present invention are: it is portable and can be operated in ordinary clinics or home environments, it completely relies on the swimming of active sperms themselves, the sperm obtained by screening has high DNA integrity, and the damage to the DNA integrity of sperms is relatively small. At the same time, it has an integrated screening process, which effectively prevents semen from being contaminated during the entire assisted reproductive process. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the overall structure of this embodiment;

[0029] Figure 2 It is a cross-sectional schematic diagram of the overall structure of this embodiment;

[0030] Figure 3 (A) is a schematic diagram showing a rectangular array of several micropillars. Figure 3 (B) is a schematic diagram showing a staggered array of several micropillars;

[0031] Figure 4 (C) is a schematic diagram showing that the microcolumn is cylindrical. Figure 4 (D) is a schematic diagram showing that the microcolumn is semi-cylindrical. Figure 4 (E) is a schematic diagram showing that the microcolumns are C-shaped columns;

[0032] Figure 5 A flowchart of a multi-layer screening component was made for this embodiment; DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention clearer, the following will be combined with Figure 1 - Figure 5 The present invention is further described in detail with reference to the accompanying drawings.

[0034] Reference Figure 1 , Figure 2 As shown, a sperm rapid screening device and a forming method thereof include a working housing 1, a glass slide 2 and at least one layer of screening components 3. The working housing 1 is provided with a first through groove 11 and a second through groove 12. The first through groove 11 and the second through groove 12 are symmetrically arranged and are in conduction with the top and bottom of the working housing 1. When the working housing 1 is arranged on the glass slide 2, the bottom of the working housing 1 is arranged in contact with the surface of the glass slide 2. At this time, a sample adding area 4 is formed between the first through groove 11 and the surface of the glass slide 2, and a sample discharging area 5 is formed between the second through groove 12 and the surface of the glass slide 2.

[0035] The bottom of the working housing 1 is provided with a receiving recess 13, which is located between the first through groove 11 and the second through groove 12 and is in communication with the first through groove 11 and the second through groove 12. When the working housing 1 is disposed on the slide 2, the screening assembly 3 is disposed in the space between the receiving recess 13 and the surface of the slide 2.

[0036] Preferably, the length, width and height dimensions of the first through groove 11 and the second through groove 12 are all 45 mm×3 mm×6 mm.

[0037] Preferably, the length, width and height dimensions of the glass slide 2 are 75 mm×1.2 mm×25 mm.

[0038] Preferably, the material of the working housing 1 is polydimethylsiloxane (PDMS).

[0039] The screening component 3 includes a support block 31 and a plurality of microcolumns 32, and the plurality of microcolumns 32 are arranged in an array on the support block 31. A microchannel 33 is formed between two adjacent microcolumns 32 to simulate the environment in which sperms swim. When the working housing 1 is arranged on the glass slide 2, the ends of the plurality of microcolumns 32 are arranged in contact with the surface of the glass slide 2, and the top of the support block 31 is arranged in contact with the top of the accommodating recess 13, so that the sperm in the sample adding area 4 flows to the sample output area 5 through the microchannel 33, and the sperms with vitality are screened out through the microchannel 33 to the sample output area 5.

[0040] Furthermore, the outer contours of the first through groove 11 and the second through groove 12 of the present embodiment are rectangular and narrow, and their function is to ensure the opportunity for sperm to contact the microchannel. In actual applications, when sperm enters the microchannel, it will only enter from the side of the microchannel. If the contact surface is narrow, the contact area will be larger (compared to a groove with the same area but a square shape); another point is that the sperm is closer to the entrance of the microfluidic channel.

[0041] Furthermore, the outline size of the accommodating recess 13 is consistent with the outline size of the first through groove 11 .

[0042] Furthermore, the periphery of the support block 31 is flush with the periphery of the accommodating recess 13 , thereby ensuring that the sperms in the semen in the sample adding area 4 all flow to the sample discharging area 5 through the screening component 3 .

[0043] Reference Figure 3 As shown, the array of micropillars 32 in this embodiment is any one of a rectangular array and a staggered array.

[0044] Furthermore, when the micro-pillars 32 are arranged in a rectangular array or a staggered array, the spacing between two adjacent rows of micro-pillars 32 is 150 μm, and the spacing between two adjacent rows of micro-pillars 32 is 150 μm.

[0045] Furthermore, the diameter of a single micro-pillar 32 is 75-120 μm, and the height thereof is 75-120 μm.

[0046] Reference Figure 4 As shown, further, the outer contour shape of the micro-column 32 is any one of a cylindrical shape, a semi-cylindrical shape and a C-shaped column. This embodiment does not limit the outer contour shape of the micro-column 32.

[0047] When working, the device is assembled from bottom to top. Specifically, the glass slide 2 is placed on a plane, the screening component 3 is placed on the glass slide 2, and finally the working housing 1 is placed so that the receiving recess 13 covers the screening component 3;

[0048] Pre-treating the chip: Add a high-viscosity buffer to the sample outlet area 5 to simulate the cervical mucus in the sperm swimming environment, put the chip into a vacuum chamber for 30 minutes of vacuum treatment, so that the high-viscosity buffer fills the screening component 3, and ensure that there is no high-viscosity buffer in the sample addition area 4;

[0049] Add sperm sample to sample loading area 4 and wait for 30 to 60 minutes;

[0050] Recover the buffer in the sample area for observation.

[0051] Compared with the prior art, this device has the following advantages:

[0052] 1. The micro-column shape design is flexible and can meet different needs;

[0053] 2. This device is portable. Compared with the previous technology that relies on professional experimental equipment, this device can be operated in ordinary clinics or home environments;

[0054] 3. The screening method adopted by the device completely relies on the swimming of active sperms themselves, and the sperms screened have high DNA integrity. Compared with traditional technologies, the present invention has less damage to the DNA integrity of sperms.

[0055] Reference Figure 5 , this embodiment provides a molding method of a multi-layer screening component 3:

[0056] Step 1: Select microcolumns and array shapes of different shapes according to the use requirements, and make corresponding molds (masks) according to the microcolumn shapes and array shapes;

[0057] Step 2: Pour liquid PDMS into the mold. The liquid PDMS should cover all the molds and be more than 1mm higher than the mold height.

[0058] Step 3: Place the liquid PDMS together with the mold in a spin coater for spin coating. The spin coating speed and time vary depending on the PDMS manufacturer. The spin coating result should produce a PDMS coating layer of about 1 mm on the mold. The 1 mm coating serves as the support block 31.

[0059] Step 4: Place the mold and PDMS in a drying oven and dry at 70°C for about 40 minutes. At this point, the PDMS should be completely transformed from liquid to solid;

[0060] Step 5: removing the solid PDMS from the mold to obtain the first layer of screening components;

[0061] Step 6. Repeat steps 2-3;

[0062] Step 7: Place the mold and liquid PDMS in a drying oven and dry at 70°C for 20 minutes. At this time, the PDMS is not completely cured. Attach the end of the micro-column of the first layer of the screening component to the smooth surface of the second layer that is not cured at this time, and continue to place it in the drying oven and dry it at 70°C for about 40 minutes. At this time, layer 1 and layer 2 are bonded;

[0063] Step 8: Repeat steps 1 to 7 to make a multi-layer screening component to form a high-throughput screening area.

[0064] Of course, the above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with this technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any modifications made according to the spirit of the main technical solution of the present invention should be included in the protection scope of the present invention.

Claims

1. A sperm rapid screening device, characterized in that: The invention comprises a working shell (1), a glass slide (2) and at least one layer of screening components (3); the working shell (1) is provided with a first through groove (11) and a second through groove (12); the working shell (1) is arranged on the glass slide (2) so that a sample loading area (4) is formed between the first through groove (11) and the surface of the glass slide (2); a sample output area (5) is formed between the second through groove (12) and the surface of the glass slide (2); a receiving recess (13) is arranged between the bottom of the working shell (1) and the glass slide (2); the receiving recess (13) is located between the first through groove (11) and the second through groove (12); the screening component (3) is arranged in the receiving recess (13); the sample loading area (4) is used for filling semen; sperm in the semen flows to the sample output area (5) through screening by the screening component (3) by their own swimming.

2. The sperm rapid screening device according to claim 1, characterized in that: The screening component (3) comprises a support block (31) and a plurality of micro-pillars (32), wherein an array of the plurality of micro-pillars is arranged on the support block (31), and a micro-channel (33) is formed between two adjacent micro-pillars (32); The end of the microcolumn (32) is arranged in contact with the surface of the glass slide (2), and the top of the support block (31) is arranged in contact with the top of the accommodating recess (13).

3. The sperm rapid screening device according to claim 1, characterized in that: The dimensions of the slide glass (2) are 75 mm x 1.2 mm x 25 mm in length, width and height.

4. The sperm rapid screening device according to claim 1, characterized in that: The length, width and height dimensions of the first through groove (11) and the second through groove (12) are both 45 mm×3 mm×6 mm.

5. The sperm rapid screening device according to claim 1, characterized in that: The material of the working housing (1) is polydimethylsiloxane.

6. The sperm rapid screening device according to claim 2, characterized in that: The outline size of the accommodating recess (13) is consistent with the outline size of the first through groove (11), and the surroundings of the supporting block (31) are flush with the surroundings of the accommodating recess (13).

7. The sperm rapid screening device according to claim 2, characterized in that: The array of the micropillars (32) is any one of a rectangular array and a staggered array; The spacing distance between two adjacent rows of micro-pillars (32) is 150 μm, and the spacing distance between two adjacent lines of micro-pillars (32) is 150 μm.

8. The sperm rapid screening device according to claim 2, characterized in that: The diameter of a single micro-pillar (32) is 75 μm, and its height is 75 μm.

9. The sperm rapid screening device according to claim 2, characterized in that: The outer contour shape of the microcolumn (32) is any one of a cylindrical shape, a semi-cylindrical shape and a C-shaped column.

10. A method for forming a screening component according to claim 2, characterized in that: The steps include: Step 1: Select microcolumns and array shapes of different shapes according to the use requirements, and make corresponding molds according to the microcolumn shapes and array shapes; Step 2: Pour liquid polydimethylsiloxane into the mold. The liquid polydimethylsiloxane should cover all the molds and be at least 1mm higher than the mold height. Step 3: Place the liquid polydimethylsiloxane together with the mold in a spin coater for spin coating. The spin coating should produce a layer of polydimethylsiloxane coating of about 1 mm on the mold. The 1 mm coating serves as the support block (31). Step 4: Place the mold together with the polydimethylsiloxane in a drying oven and dry at 70°C for about 40 minutes. At this time, the polydimethylsiloxane should be completely converted from liquid to solid; Step 5: removing the solid polydimethylsiloxane from the mold to obtain a first layer screening component; Step 6. Repeat steps 2-3; Step 7: Place the mold and liquid polydimethylsiloxane in a drying oven and dry at 70°C for 20 minutes. At this time, the polydimethylsiloxane is not completely cured. The micro-column end of the first layer of the screening component is attached to the smooth surface of the second layer that is not cured at this time, and continue to place it in the drying oven and dry it at 70°C for about 40 minutes. At this time, layer 1 and layer 2 are bonded; Step 8: Repeat steps 1 to 7 to produce a multi-layer screening component (3).

Images