Sperm enrichment apparatus and method for promoting fertilization

Abstract

The present invention relates to the technical field of biochips, and provides a sperm enrichment apparatus. The sperm enrichment apparatus comprises: an inverted microscope, comprising a stage; a high-speed CCD, disposed above the inverted microscope; a surface acoustic wave microfluidic chip, disposed on the stage and located below the high-speed CCD, and comprising a substrate and an interdigital electrode plated on the substrate; a signal generator, configured to generate ultrasonic waves; and an amplifier, configured to amplify the ultrasonic waves and then load the amplified ultrasonic waves to the surface acoustic wave microfluidic chip. The sperm enrichment apparatus, as a non-invasive method, has biocompatibility. The apparatus is mainly applied to raw sperm samples (including those with poor motility and no motility), and does not involve excessive manipulation of the sperm, thereby ensuring the naturalness of the fertilization process. The apparatus is reusable, simple to operate, and time-saving.

Description

Sperm enrichment device and method of promoting fertilization TECHNICAL FIELD

[0001] The present invention belongs to the field of biochip technology and provides a sperm enrichment device and a method of promoting fertilization. BACKGROUND

[0002] It is estimated that more than 48.5 million couples worldwide are unable to conceive within 5 years or more. About 30-50% of infertility cases are caused by male infertility. The most prominent abnormalities in semen are low sperm concentration, poor sperm motility and abnormal sperm morphology. Assisted reproductive technologies (ARTs) include in vitro fertilization (IVF), intrauterine insemination (IUI) and intracytoplasmic sperm injection (ICSI). To overcome the challenge of male infertility, IUI, IVF and ICSI have been implemented clinically. Artificial insemination is a method closest to natural fertilization, which directly introduces selected sperm populations into the female reproductive tract, shortening the fertilization process of sperm. For men with high sperm motility and women with normal healthy fallopian tubes, IUI is a suitable treatment method. To increase the chances of fertilization, IVF and ICSI, as more invasive methods, have also been introduced, in which fertilization (i.e. fusion of sperm and egg) occurs in vitro to produce embryos, which are then placed into the uterus after about 5 days of incubation to ensure blastocyst formation.

[0003] However, these technologies require healthy sperm cells to achieve successful IVF results. The success rate of test tube babies is only 25-30%. The main reason for the low success rate is not yet known, but sperm defects can be related to test tube baby results. Studies have shown that pregnancy rates are related to the movement and normal morphology of sperm cells. Defects in sperm cells reduce the binding affinity to the zona pellucida, which allows the egg cell to be fertilized. For fertilization in the ICSI procedure, motile and morphologically normal sperm cells are selected clinically and injected into the egg cell.

[0004] Traditional pre-fertilization sperm processing methods include swim-up, density gradient centrifugation, glass wool filtration, magnetic-activated cell sorting, microfluidic screening, and motility microscope morphological examination. These methods involve multiple centrifugation steps, which can damage sperm morphology, induce significant DNA fragmentation and ROS, have low yield, and are time-consuming. The selected sperm are different from the naturally selected sperm in FRT. The success rate of in vitro fertilization is only 25-30%, and the main reason for the low success rate is not yet known. In order to overcome the limitations of centrifuge-based methods, several advanced sperm selection methods have been developed, providing a wide range of possibilities for improving sperm fertilization potential. Microchannel screening selects sperm only according to swimming ability, which may not fully reflect the natural selection process of sperm under physiological conditions. Magnetic-activated cell sorting users tend to combine this procedure with DGC to eliminate contamination of white blood cells and immature germ cells, which makes the process both laborious and time-consuming. Today, acoustic methods have been proven to be very suitable for biological applications without causing damage to cell membranes or intercellular DNA structures. It has been proven that cells exposed to high-frequency ultrasound exhibit a higher metabolic rate, produce more energy needed for swimming, and are able to swim faster by jumping with higher frequency and larger amplitude. Compared with the traditional clinical methods currently used to improve sperm motility, this high-frequency acoustic-based method has biocompatibility.

[0005] In summary, the current assisted reproductive means has limitations, and further improvement of ART technology is needed, while ultrasound meets the necessary optimal conditions, including non-invasiveness, time efficiency, cost-effectiveness, and ease of operation. Therefore, it is crucial to carry out non-invasive ultrasound-assisted reproduction research, which will help to further develop and improve assisted reproductive technology and bring hope and gospel to more infertile families.

[0006] SUMMARY

[0007] The present invention provides an innovative non-invasive ultrasound pre-fertilization sperm processing technique, which aims to increase the probability of sperm-egg collision during fertilization, and thus is applied to moderate oligospermia and promotes fertilization in a more natural way.

[0008] The present invention provides a sperm enrichment device.

[0009] The sperm enrichment device comprises:

[0010] An inverted microscope comprising a stage;

[0011] A high-speed CCD disposed above the inverted microscope;

[0012] The surface acoustic wave microfluidic chip is arranged on the object table and below the high-speed CCD, and comprises a substrate and an interdigital electrode plated on the substrate.

[0013] A signal generator is arranged for generating ultrasonic waves.

[0014] An amplifier is arranged for amplifying the ultrasonic waves and loading the amplified ultrasonic waves to the surface acoustic wave microfluidic chip.

[0015] Further, the substrate is made of lithium niobate single crystal, zinc oxide or aluminum nitride.

[0016] Further, the interdigital electrode is made of gold and / or chromium.

[0017] The droplet is asymmetrically dropped on the substrate outside the surface acoustic wave propagation axis of the surface acoustic wave microfluidic chip, and the ultrasonic waves are loaded to form a vortex in the droplet.

[0018] The application also provides a use of the sperm enrichment device in a diagnosis strategy and / or a treatment strategy for a human early embryo.

[0019] The application also provides a method for promoting fertilization.

[0020] The method for promoting fertilization comprises the following steps:

[0021] S1: Asymmetrical dropping of a sperm suspension on the substrate outside the surface acoustic wave propagation axis of the surface acoustic wave microfluidic chip as described above;

[0022] S2: Placing a single oocyte cumulus cell complex in the middle of the sperm droplet under the inverted microscope as described above;

[0023] S3: Loading of ultrasonic waves until the sperm are observed to gather on the surface of the oocyte.

[0024] Further, the volume of the sperm suspension in step S1 is 1 muL, and the sperm concentration is 8-9*10 6 per mL.

[0025] Further, the ultrasonic power in step S3 is 900 mW, and the duration of action is 5 min.

[0026] The application also provides a use of the method for promoting fertilization in a diagnosis strategy and / or a treatment strategy for a human early embryo. Advantages:

[0027] (1)Non-invasive, ultrasonic treatment of sperm is mainly through the enrichment of sperm on the surface of the egg cell by ultrasonic waves, without the need for contact with sperm and egg cells, and without the need for screening of sperm, which belongs to a non-invasive sperm treatment method.

[0028] (2)Applicability (small amount), the present application uses the surface acoustic wave chip microscope to analyze the effect of ultrasonic assisted reproduction from the quantitative point of view, and selects the unselected sperm and the concentration range belongs to the moderate oligospermia, which can realize that various sperm (without movement ability, progressive movement sperm) have the opportunity to contact the egg cell and be fertilized.

[0029] (3)Innovative, under the condition of limited number of sperm and single egg cell used in the experiment, the possibility of fertilization based on acoustic technology in extreme environment is studied, which is an innovative exploration. It challenges the traditional fertilization method and technology, and opens up a new path for scientific research and technology application.

[0030] (4)Repeatability, the preparation process of the surface acoustic wave microfluidic chip is the standard MEMS process, and the device performance has good consistency, which lays a foundation for the repeatability of the experiment. Moreover, all the cells used in the experimental operation are collected at the same time, and multiple groups of egg cells and sperm can be continuously experimented.

[0031] (5)Optical, PDMS compatibility, the movement trajectory of sperm needs to be recorded during the ultrasonic treatment of sperm in order to observe the state of sperm, and since the chip substrate has light transmission, it can be combined with traditional optical microscope and PDMS to record the fertilization process and differentiation process of fertilized eggs in real time. BRIEF DESCRIPTION OF DRAWINGS

[0032] Fig. 1 is a structure diagram of a sperm enrichment device.

[0033] Fig. 2 is a picture of a surface acoustic wave chip taken by a high-speed CCD.

[0034] Fig. 3 is a flow chart of the manufacturing method of the interdigital transducer of the surface acoustic wave microfluidic chip.

[0035] Fig. 4 is a sperm enrichment process.

[0036] Fig. 5 is different states of egg cells after enrichment of sperm and differentiation of culture. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the drawings, but it should not be understood as limiting the scope of the present application.

[0038] Referring to Figs. 1 and 2, the present application provides a sperm enrichment device, comprising:

[0039] an inverted microscope comprising a stage;

[0040] a high-speed CCD disposed above the inverted microscope;

[0041] an acoustic surface wave microfluidic chip disposed on the stage below the high-speed CCD, the acoustic surface wave microfluidic chip comprising a substrate and an interdigital electrode plated on the substrate;

[0042] a signal generator for generating ultrasonic waves;

[0043] an amplifier for amplifying the ultrasonic waves and loading the amplified ultrasonic waves to the acoustic surface wave microfluidic chip.

[0044] Further, the substrate is made of lithium niobate single crystal, zinc oxide or aluminum nitride.

[0045] Further, the interdigital electrode is made of gold and / or chromium.

[0046] The droplet is asymmetrically dropped on the substrate outside the acoustic surface wave propagation axis of the acoustic surface wave microfluidic chip, and the ultrasonic waves are loaded to form a vortex in the droplet.

[0047] The application also provides a use of the sperm enrichment device as described in any one of the above in a diagnosis strategy and / or a treatment strategy of a human early embryo.

[0048] The application also provides a method for promoting fertilization by using the sperm enrichment device provided above.

[0049] The method for promoting fertilization comprises the following steps:

[0050] S1: asymmetrically dropping a sperm suspension on the substrate outside the acoustic surface wave propagation axis of the acoustic surface wave microfluidic chip as described above;

[0051] S2: placing a single oocyte cumulus cell complex in the middle of the sperm droplet under the inverted microscope as described above;

[0052] S3: loading ultrasonic waves until the sperm are observed to gather on the surface of the oocyte.

[0053] Further, the volume of the sperm suspension in step S1 is 1 muL, and the sperm concentration is 8-9 x 10 6 per mL.

[0054] Further, the ultrasonic power in step S3 is 900 mW, and the duration of action is 5 min.

[0055] The application also provides use of the method for promoting fertilization according to any one of the above in a diagnostic strategy and / or a therapeutic strategy for a human early embryo.

[0056] Example one

[0057] (I) Surface acoustic wave microfluidic chip

[0058] The surface acoustic wave chip is mainly made by plating gold and chromium interdigital electrodes on a piezoelectric substrate. In order to obtain a larger electromechanical coupling coefficient, 128°YX double-side polished lithium niobate is selected as the piezoelectric substrate. In the process of making the surface acoustic wave chip, the main processes include gluing, photoetching, film plating, and stripping.

[0059] Referring to FIG. 3, the method for preparing the interdigital transducer on the piezoelectric substrate is shown in FIG. 3(a-e), which includes the following steps:

[0060] (1) Gluing: On the surface of the completely cleaned piezoelectric substrate material, the positive photoresist AZ4620 is spin-coated at 5000 rpm for 30 s, and the chip is placed on a 120℃ hot plate for baking for 3 min. We use a step meter to test the thickness of the photoresist, and the thickness of the photoresist is about 5 μm, as shown in FIG. 3(a).

[0061] (2) Exposure and development: Then, the prepared film shown in FIG. 3(b) is covered on the above FIG. 3(a) for exposure, the patterned part is not transparent, the non-patterned part is transparent, the part with light transmission is solidified, and when the AZ400 is developed, the solidified part will not be dissolved, and the non-solidified part will be dissolved. After development, it is baked on a 150℃ hot plate for 10 min to form a pattern as shown in FIG. 3(c).

[0062] (3) Sputtering: The substrate with completed pattern transfer is subjected to magnetron sputtering to grow a metal layer with a thickness of about 200 nm, as shown in FIG. 3(d).

[0063] (4) Stripping: The substrate with the grown metal layer is placed in an acetone solution, and the photoresist is stripped by ultrasonic vibration of the ultrasonic cleaning machine, and the surface acoustic wave device is completed, as shown in FIG. 3(e).

[0064] (II) Preparation of sperm

[0065] The preparation of the 2-3 month old male C57BL / 6J mouse sperm sample is divided into 5 steps:

[0066] (1) Add TYH sperm capacitation solution (M2030, Aibei, China) to the culture dish, coat with mineral oil (M8410, Sigma-Aldrich, USA), and use it after balancing in a 37℃, 5% CO2 incubator for more than 4 hours.

[0067] (2) Anesthetize the mouse with sodium pentobarbital (100 mg / kg body weight).

[0068] (3) Disinfect the mouse's abdomen with a 70% alcohol cotton ball, cut the epididymis, and collect the epididymis.

[0069] (4) Place the epididymis in a culture dish filled with culture medium and cut it open to release the sperm.

[0070] (5) Place the culture dish in a 37°C incubator for 10 min, and collect the sperm after they have dispersed.

[0071] (Three) Preparation of oocytes.

[0072] Select 1.5-3-month-old female C57BL / 6J mice for superovulation, and collect the oocytes, including the following 3 steps:

[0073] (1) For superovulation treatment, the female is first injected with pregnant mare serum gonadotropin (PMSG, 5.0 IU, P9970, Solarbio, China). The purpose is to simulate the effect of endogenous follicle-stimulating hormone (FSH) to promote oocyte maturation. 48 h later, inject human chorionic gonadotropin (HCG, 5.0 IU per mouse, Sigma-Aldrich Corp) to simulate the luteinizing hormone (LH) to induce ovulation. Collect the oocytes 12-14 h after HCG injection.

[0074] (2) When collecting the ovary, take the oocyte culture droplet, and use a 200 μL pipette to drop the HTF culture medium (M1130, Aibei, China) into a 60 mm culture dish in small droplets, cover the HTF droplet with mineral oil (M8410, Sigma-Aldrich, USA), and place it in a 37°C, 5% CO2 incubator for more than 4 hours. HTF creates a glucose-rich environment to promote the effective metabolism of oocyte cumulus complexes and sperm cells.

[0075] (3) Anesthetize the mouse with sodium pentobarbital (100 mg / kg body weight), and disinfect the abdominal cavity with 75% alcohol. Open the abdominal cavity, and place the oviduct in a culture dish containing HTF culture medium. Under a stereomicroscope, the upper part of the oviduct (ampulla) is visibly swollen. Use forceps to fix the oviduct, and then use a 1-milliliter syringe needle to open the ampulla longitudinally. This method will open the entire ampulla, thereby releasing the oocytes into the liquid. Use an oocyte transfer tube to transfer the oocytes to another oocyte droplet, and then use the remaining 2 droplets for washing to remove contaminants such as blood cells. After completion, transfer the oocytes to the prepared HTF droplet, and return it to the incubator for 1 hour before further use.

[0076] (Four) Sperm enrichment experiment.

[0077] (1) About 1 μL of 10-fold diluted sperm suspension was directly and asymmetrically dropped on the LiNbO3 substrate outside the propagation axis of surface acoustic wave using a micropipette. At this time, the sperm was not selected, and the concentration of sperm in 1 μL suspension was 8-9 x 10 6

[0078] (2) In each experiment, a single oocyte-cumulus cell complex was aspirated into a micropipette and placed into the sperm droplet on the substrate. Under the observation of a microscope, the oocyte-cumulus cell complex was placed in the middle of the droplet.

[0079] (3) After the sperm and oocyte were placed, the input ultrasonic power was 900 mW, and the duration of action was 5 min. The process of sperm gathering on the surface of the oocyte under the continuous ultrasonic excitation was observed by a microscope.

[0080] The state of sperm and oocyte without ultrasonic stimulation and the process of sperm gathering on the surface of the oocyte after ultrasonic action are shown in Fig. 4.

[0081] (5) Subsequent culture of oocyte. After ultrasonic treatment, the sperm and oocyte were transferred into HTF culture solution by a micropipette under the assistance of a microscope, and then transferred into an incubator (37°C, 5% CO2) for further culture. The fertilization result of oocyte was observed by a microscope after 36 hours, and whether the second polar body and the multi-cell state appeared was judged.

[0082] Referring to Fig. 5, Figs. 5(a-c) show the fertilized oocyte and its differentiated two-cell, four-cell, eight-cell states produced after 36 h of ultrasonic treatment, which indicates the success of fertilization.​

Claims

1. A sperm enrichment device, characterized in that, include: Inverted microscope, including stage; A high-speed CCD is positioned above the inverted microscope; A surface acoustic wave (SAW) microfluidic chip is disposed on the stage below the high-speed CCD. The SAW microfluidic chip includes a substrate and interdigitated electrodes deposited on the substrate. A signal generator used to produce ultrasonic waves; An amplifier amplifies the ultrasonic waves and then loads them onto the surface acoustic wave microfluidic chip.

2. The sperm enrichment device as described in claim 1, characterized in that, The substrate is made of lithium niobate single crystal, zinc oxide, or aluminum nitride.

3. The sperm enrichment device as described in claim 1, characterized in that, The interdigitated electrodes are made of gold and / or chromium.

4. The use of the sperm enrichment device as described in any one of claims 1-3 in diagnostic and / or therapeutic strategies for early human embryos.

5. A method for promoting fertilization, characterized in that, Includes the following steps: S1: Asymmetrically drop a sperm suspension onto a substrate outside the surface acoustic wave propagation axis of the surface acoustic wave microfluidic chip as described in claim 1; S2: Under the inverted microscope as described in claim 1, a single oocyte-cumulus cell complex is placed in the middle of a sperm droplet; S3: Apply ultrasound until sperm are observed to converge on the surface of the egg cell.

6. The method for promoting fertilization as described in claim 3, characterized in that, The volume of the sperm suspension in step S1 is 1 μL, and the sperm concentration is 8–9 × 10⁻⁶. 6 per mL.

7. The method for promoting fertilization as described in claim 3, characterized in that, In step S3, the ultrasonic power is 900mW and the duration of action is 5min.

8. The application of the fertilization-promoting methods as described in claims 5-7 in diagnostic and / or therapeutic strategies for early human embryos.

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