Piezo-ICSI device and piezo-ICSI kit

Abstract

The present invention relates to a working liquid for piezo-mediated microinjection. In particular, the present invention relates to the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working liquid for piezo-mediated intracytoplasmic sperm injection (piezo-ICSI). The present invention further relates to a method for in vitro fertilization of oocytes by piezo-ICSI using purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working liquid, and a method for assisted reproductive technology using the method of the present invention.

Description

[Technical Field]

[0001] The present invention relates to a working liquid for piezo-mediated microinjection. In particular, the present invention relates to the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working liquid for piezo-mediated intracytoplasmic sperm injection (piezo-ICSI). The present invention further relates to a method for in vitro fertilization of oocytes by piezo-ICSI using purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working liquid, and a method for assisted reproductive technology using the method of the present invention. [Background technology]

[0002] Male infertility is the cause of infertility in approximately 50% of couples. To address fertilization problems caused by male gamete dysfunction, many techniques were developed in the 1980s, including injecting a single sperm cell (sperm) into the subzonal cavity of the oocyte, but the success rate has been very limited. However, it was during the subzonal injection of oocytes, in which the egg membrane is accidentally ruptured and the sperm is delivered into the oocyte, that the development of intracytoplasmic sperm injection, which is still performed in humans today, was initiated.

[0003] Intracytoplasmic sperm injection (ICSI) is an in vitro fertilization technique in which sperm cells are directly injected into the cytoplasm of an oocyte. The first human pregnancy resulting from an ICSI-generated embryo was reported in 1992. ICSI is performed in a culture dish under a microscope by a human operator who manipulates the oocyte and sperm using a micromanipulator and micropipette. A fine glass micropipette (holding pipette) stabilizes the mature oocyte with gentle suction applied by a microinjector, while a thin, pointed glass micropipette (injection micropipette) collects single immobilized sperm by cutting their tails with the tip of the micropipette. The operator applies mechanical force through the micromanipulator to puncture the zona pellucida and plasma membrane (oolemma) of the oocyte with the injection micropipette. The sperm are then injected into the oocyte's cytoplasm.

[0004] To date, ICSI has been responsible for over 2 million live births worldwide. However, ICSI is a labor-intensive technique that requires highly skilled operators, and there is still room for improvement in terms of technical standardization and success rate. Because conventional ICSI did not solve the problems associated with fertilization in mouse IVF, an alternative oocyte microinjection method called "piezo-mediated" ICSI (piezo-ICSI) was developed for mouse oocytes in 1995.

[0005] Piezo-ICSI is similar to conventional ICSI (c-ICSI) in that a micropipette containing a single sperm punctures the oocyte, delivering the sperm into the oocyte cytoplasm. However, whereas in c-ICSI the zona pellucida and oolemma are punctured by mechanical force applied to the micropipette by the operator through a micromanipulator, in piezo-ICSI the oocyte is punctured using the piezoelectric effect. The piezoelectric effect is a phenomenon in which certain materials accumulate electrical charge in response to mechanical stress, and conversely, mechanical stress can be induced by the application of electricity. The piezoelectric effect has many technological applications. In piezo-ICSI, a short "piezo pulse" is applied to the sperm-containing micropipette, causing ultrafast, submicron forward momentum of the micropipette. This precise and rapid movement is used to puncture the zona pellucida and oocyte with less stress on the oocyte than the mechanical force used in c-ICSI.

[0006] The success rate of piezo-ICSI was initially lower than that of c-ICSI, but it was found that the success rate could be improved to higher than that of c-ICSI by including a small amount of mercury in the injection micropipette.

[0007] Thus, piezo-ICSI requires the presence of a "working fluid" in the injection micropipette. Currently, there is no complete scientific explanation for the physics of the piezo-ICSI penetration process. It has been suggested that piezoelectrically induced axial movement of the micropipette penetrates the zona pellucida. It has also been hypothesized that penetration occurs due to a pressure burst caused by the rapid forward movement of the working fluid. It has also been hypothesized that application of a piezo pulse moves the micropipette forward, but the working fluid remains stationary due to the law of inertia and the liquid's high specific gravity. The rapidly moving micropipette and stationary working fluid are thought to generate negative pressure at the micropipette tip, which then opens the zona pellucida. More recent studies have noted that significant lateral tip vibrations occur in the injection pipette when a piezoelectric pulse train is introduced. These studies argue that lateral dynamics play an important role in penetration, and that this process is also mediated by the working fluid. It is highly likely that all of the listed mechanisms contribute to some degree to the effective operation of piezo-mediated ICSI.

[0008] Mercury is toxic and its use is severely restricted, but piezo-ICSI is currently used for in vitro fertilization in animals. More recently, fluorinated compounds have been used as operating fluids.

[0009] Piezo-ICSI was first successfully used in mice in 1995, and was subsequently applied to microinjection in pigs and cattle. Piezo-ICSI achieved higher fertilization and survival rates than conventional ICSI (c-ICSI), and has since become the standard method in the art for microinjecting sperm into animal oocytes.

[0010] Successful human pregnancies achieved using piezo-ICSI technology were first reported in 1996, and improvements in fertility and survival rates were reported in 1999. However, piezo-ICSI has not been approved for clinical use in humans.

[0011] Further development of piezo-ICSI to allow its approval for clinical use has the potential to improve the success rate of assisted reproduction in humans. Summary of the Invention

[0012] The present invention relates to a working solution for piezo-mediated microinjection.

[0013] The present invention provides the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working fluid for piezo-mediated intracytoplasmic sperm injection (piezo-ICSI).

[0014] The present invention further provides a method for in vitro fertilization of oocytes, comprising injecting sperm into oocytes by piezo-ICSI, wherein purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane is used as a working liquid for piezo-ICSI.

[0015] The present invention also provides a method for assisted reproduction, comprising: (a) in vitro fertilization of an oocyte using a method according to the present invention to form an embryo; (b) culturing the embryo; and (c) implanting the embryo into a subject; The present invention provides a method comprising:

[0016] Suitably, the use of purified perfluoro-n-octane as a working solution according to the present invention may provide advantages to assisted reproductive technology (ART) procedures. Such advantages may include improved fertilization success rates, improved embryo development rates (e.g., a higher proportion of embryos reaching the blastocyst stage), and improved implantation success rates. If the use of perfluoro-n-octane as a working solution for piezo-ICSI is approved for clinical use in humans, it may offer the potential to standardize ICSI procedures. The use of piezo-ICSI may be the first step toward the full automation of ART. Piezo-ICSI is also a technique that is easier to learn than c-ICSI, which requires a highly skilled operator. Therefore, the use of perfluoro-n-octane as a working solution for piezo-ICSI may simplify ART in humans and make it a more efficient method. [Brief explanation of the drawings]

[0017] [Figure 1] Fig. 1 shows the position of the operating liquid in an injection micropipette. Fig. 1 shows an injection micropipette 1 filled with an operating liquid 2. The operating liquid forms a liquid column approximately 10 mm to 15 mm long in the center of the micropipette. [Figure 2] 2A shows the insertion of an injection micropipette 1 into a micropipette holder 3. FIG. 2B shows the insertion of the micropipette holder 3 into an injector 4. [Figure 3] Figure 3A shows a piezo micromanipulator (PMM) / piezo driver. Figure 3A shows a piezo driver 5 attached to a micropipette holder 3. Figure 3B is a photograph of the piezo driver 5. [Figure 4] 4A shows an injection micropipette and culture medium. Fig. 4A shows that culture medium containing PVP and the like is aspirated by the injection micropipette. Fig. 4B is a photograph of PVP culture medium being aspirated using the injection micropipette. [Figure 5]Schematic diagram of piezo-ICSI. Figure 5 shows the steps in injecting sperm into an oocyte using piezo-ICSI. The tip of the injection micropipette is gently placed against the zona pellucida without deforming the oocyte (a). A piezo-electric pulse is applied to the micropipette (b), and it is advanced through the zona pellucida, perforating it (c). The injection micropipette is withdrawn from the zona pellucida, and the hollowed-out portion of the zona pellucida is expelled by draining the injection micropipette. This draining action is used to move the sperm to the tip of the injection micropipette (d). The injection micropipette is advanced through the zona pellucida (e) to approximately 80%-90% of the oocyte's diameter, where it presses against the oolemma, stretching it (f). A single piezo-electric pulse is applied to rupture the oolemma, surrounding the injection micropipette in the cytoplasm (g). The sperm are then injected (h). DETAILED DESCRIPTION OF THE INVENTION

[0018] The present invention provides the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working fluid for piezo-mediated intracytoplasmic sperm injection (piezo-ICSI).

[0019] The present invention also provides a method for in vitro fertilization of oocytes, comprising injecting sperm into oocytes by piezo-ICSI, wherein purified perfluoro-n-octane or a composition containing purified perfluoro-n-octane is used as a working liquid for piezo-ICSI.

[0020] [Perfluoro-n-octane] Perfluoro-n-octane consists of an unbranched chain of eight carbon atoms, with each carbon atom bonded to the maximum number of fluorine atoms (i.e., the terminal carbon has three fluorine atoms bonded, and each of the other carbons has two fluorine atoms bonded). The IUPAC name for perfluoro-n-octane is 1,1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-octadecafluorooctane. The CAS registry number for perfluoro-n-octane is 307-34-6. Perfluoro-n-octane has the chemical formula C8F 18 The structural formula of perfluoro-n-octane is as follows:

[0021] [ka]

[0022] At room temperature, perfluoro-n-octane is an odorless, colorless liquid. The physical properties of perfluoro-n-octane are shown in Table 1 below (temperature-dependent properties are estimated at 25°C).

[0023] [Table 1]

[0024] Perfluoro-n-octane is chemically inert. In other words, it is stable and does not readily react with other compounds. This stability is due to the numerous, strong carbon-fluorine bonds in its structure.

[0025] Perfluoro-n-octane is sometimes called a perfluorocarbon, a compound consisting only of carbon and fluorine atoms. Perfluoro-n-octane is sometimes called a perfluorocarbon liquid (PFCL). Perfluoro-n-octane is sometimes called a perfluoroalkane, a compound consisting only of carbon and fluorine atoms connected by single bonds.

[0026] Methods for synthesizing perfluoro-n-octane are known in the art.

[0027] For example, perfluoro-n-octane can be synthesized by electrofluorination, which uses electrolysis to replace hydrogen atoms in an organic compound with fluorine atoms from a donor compound. Two exemplary forms of electrofluorination that can be used are the Simons process and the Phillips-Petroleum process. In the Simons process (Simons and Harland 1949 Journal of the Electrochemical Society 95: 47-66), an organic compound is electrolyzed in a solution of hydrogen fluoride. The Phillips-Petroleum process is similar to the Simons process, but uses potassium fluoride in hydrogen fluoride as the fluorine source (Alsmeyer et al. 1994 Organofluorine Chemistry: Principles and Commercial Applications in Banks et al. (eds.) Organofluorine Chemistry (Boston, MA): Springer, pp. 121-143).

[0028] Perfluoro-n-octane can also be synthesized by the Fowler method (Fowler et al. 1947 Ind. Eng. Chem. 39: 292-298), which uses cobalt(III) fluoride to fluorinate a hydrocarbon in the gas phase. Specifically, in the first step, cobalt(II) fluoride is fluorinated to cobalt(III) fluoride by exposing it to fluorine gas at high temperature. In the second step, cobalt(III) fluoride is exposed to a hydrocarbon (e.g., octane in the case of perfluoro-n-octane) at high temperature to fluorinate it. The hydrogen atoms of the hydrocarbon are replaced by fluorine atoms from cobalt(III) fluoride, and cobalt(III) fluoride is converted to cobalt(II) fluoride.

[0029] Perfluoro-n-octane is readily available from commercial sources.

[0030] Purified Perfluoro-n-octane The term "purified perfluoro-n-octane" refers to perfluoro-n-octane that contains low levels of low-fluorinated impurities.

[0031] Low-fluorinated impurities are by-products of the synthesis of perfluorocarbon compounds. Low-fluorinated impurities contain fewer fluorine atoms in their molecular structure than the desired perfluorocarbon synthesis products.

[0032] In particular, perfluoro-n-octane is fully fluorinated because each carbon atom in its molecular structure is bonded to the maximum number of fluorine atoms. However, during the synthesis of perfluoro-n-octane, other molecules may be additionally produced that are incompletely fluorinated, that is, still contain many hydrogen and / or carbon bonds in the molecule, and therefore contain fewer fluorine atoms than perfluoro-n-octane. These molecules are low-fluorinated impurities.

[0033] The low-fluorinated impurities of perfluoro-n-octane may contain a carbon-carbon double bond. The low-fluorinated impurities of perfluoro-n-octane may additionally or alternatively contain a carbon-carbon triple bond. The low-fluorinated impurities of perfluoro-n-octane may additionally or alternatively contain one or more hydrogen atoms in place of the fluorine atoms found in perfluoro-n-octane.

[0034] Low-fluorinated impurities may be referred to as low-fluorinated compounds, low-fluorinated by-products, under-fluorinated impurities, under-fluorinated compounds, or under-fluorinated by-products.

[0035] In contrast to perfluoro-n-octane, which is chemically inert, the low-fluorinated impurities are reactive, i.e., they may react with compounds that perfluoro-n-octane does not react with.

[0036] Purification can remove low-fluorinated impurities from the synthesized perfluoro-n-octane. In some embodiments of the present invention, purified perfluoro-n-octane includes perfluoro-n-octane that has been subjected to purification.

[0037] Purification methods for perfluorocarbon compounds that can be applied to perfluoro-n-octane are known in the art (see, for example, U.S. Pat. No. 3,696,156, U.S. Pat. No. 3,887,629, and U.S. Pat. No. 5,563,306, all of which are incorporated herein by reference). Such methods typically utilize the reactivity of low-fluorinated impurities to separate them from inert perfluoro-n-octane. In an exemplary method for purifying perfluoro-n-octane, low-fluorinated impurities are separated by the addition of Ca 2+ ions or Ba 2+ It is reacted with an amine or a strong base such as potassium hydroxide in the presence of ions.

[0038] The amount of low-fluorinated impurities in a preparation of perfluoro-n-octane can be expressed as the equivalent of the number of C-H bonds in these impurities. This value is called the H value and has units of parts per million (ppm). The amount of low-fluorinated impurities in a preparation of perfluoro-n-octane can be measured, for example, using fluoride selective ionometry.

[0039] The quantification of low-fluorinated impurities using fluoride ion selective ion measurement method is known in the art.The principle of this method is that perfluoro-n-octane is reacted with a strong base to cleave fluorine atoms from molecules in the preparation, producing fluoride ions.Then, fluoride ions are quantified, and the amount of low-fluorinated impurities can be calculated from the quantification.An exemplary methodology of fluoride ion selective ion measurement method is shown below.

[0040] Step 1: Chemical conversion of low-fluorinated impurities 10 mL of perfluorocarbon liquid (PFCL) is mixed with 3.4 g of 1,6-diaminohexane and 15 mL of nonane. This mixture is heated to 120°C for 8 hours with stirring in a 100 mL glass flask equipped with a reflux condenser. After cooling to room temperature, the solution is vigorously mixed with 30 mL of hydrochloric acid (1.3 molar), and the aqueous phase is separated.

[0041] Next, neutralize 15 mL of the aqueous phase with 1.3 molar aqueous hydrochloric acid using phenolphthalein as the indicator and dilute to 25 mL with deionized water. Transfer 10 mL of this neutralized, diluted solution to a 25 mL glass beaker and add 1 mL of TISAB-III while stirring.

[0042] Step 2: Fluoride ion-selective potentiometry Ion-selective potentiometry is used to quantify fluoride ions in the sample solution. Before the sample measurement, calibration must be performed using a sodium fluoride solution with a fluoride ion concentration between 0.005 mmol / L and 0.05 mmol / L as a reference standard. In addition, a blank value is recorded. The sample solution is then analyzed. The amount of reactive low-fluorinated impurities is expressed as the H value, which corresponds to the number of C—H bonds in these impurities. The H value (C F-C-H ) is calculated using the following formula:

number

[0043] During the ceremony, C F-C-H is the concentration of incompletely fluorinated contaminants in the sample (i.e., the H value), C F- is the measured concentration of fluoride ions in the sample, M￣ PFCL is the measured molecular weight of PFCL, ρ PFCL is the measured density of the PFCL, bν is the recorded blank value (reagent and boiling flask blank), 5 is a factor that compensates for the dilution process, and 1 / 3 is the stoichiometric coefficient (calculating the number of C-H bonds).

[0044] Suitably, purified perfluoro-n-octane may have an H value of about 1000 parts per million (ppm) or less. Purified perfluoro-n-octane may have an H value of about 500 ppm or less. Purified perfluoro-n-octane may have an H value of about 100 ppm or less. Purified perfluoro-n-octane may have an H value of about 50 ppm or less. Purified perfluoro-n-octane may have an H value of about 40 ppm or less. Purified perfluoro-n-octane may have an H value of about 30 ppm or less. Purified perfluoro-n-octane may have an H value of about 20 ppm or less. Purified perfluoro-n-octane may have an H value of about 10 ppm or less. Purified perfluoro-n-octane may have an H value of about 5 ppm or less. Purified perfluoro-n-octane may have an H value of about 1 ppm or less. In a preferred embodiment, the purified perfluoro-n-octane has an H value of about 10 ppm or less.

[0045] Suitably, the working fluid may have an H value of about 1000 parts per million (ppm) or less. The working fluid may have an H value of about 500 ppm or less. The working fluid may have an H value of about 100 ppm or less. The working fluid may have an H value of about 50 ppm or less. The working fluid may have an H value of about 40 ppm or less. The working fluid may have an H value of about 30 ppm or less. The working fluid may have an H value of about 20 ppm or less. The working fluid may have an H value of about 10 ppm or less. The working fluid may have an H value of about 5 ppm or less. The working fluid may have an H value of about 1 ppm or less. In a preferred embodiment, the working fluid has an H value of about 10 ppm or less.

[0046] Purified perfluoro-n-octane is available from commercial sources. In particular, purified perfluoro-n-octane is sold for use in vitreoretinal surgery. Examples of commercially available purified perfluoro-n-octane include PERFLUORON™ liquid sold by Alcon, BIO OCTANE™ PFS sold by Biotech, and OPTISOL sold by Moss Vision Inc.

[0047] Piezo-ICSI Piezo-mediated intracytoplasmic sperm injection (piezo-ICSI) is a technique known in the art for injecting sperm into the cytoplasm of oocytes in vitro. Piezo-ICSI is useful for in vitro fertilization in assisted reproductive technologies. Piezo-mediated ICSI is sometimes referred to in the art as "piezo-driven" or "piezo-actuated" ICSI. Examples of the use of piezo-ICSI are provided by Kimura and Yanagimachi (1995) Biol. Reprod. 52(4): 709-720 and Hiraoka et al. (2019) Piezo-ICSI in Chapter 39, pp. 481-489, of Nagy et al. (eds.) In Vitro Fertilization (Springer, Cham). Each of these documents is incorporated herein by reference.

[0048] Piezo-ICSI is performed on oocytes and sperm in a culture dish under a microscope. A human operator manipulates the oocytes and sperm using a micromanipulator and micropipette. A single oocyte is held in place by a holding micropipette through gentle suction, while a single sperm is captured in an opposing micropipette, called the "injection micropipette" to distinguish it from the micropipette that holds the oocyte in place. The injection micropipette is used to first puncture the zona pellucida of the oocyte and then the oolemma. The zona pellucida and oolemma are punctured using the piezoelectric effect. The piezoelectric effect is a phenomenon in which certain materials accumulate charge in response to mechanical stress, and conversely, mechanical stress can be induced by the application of electricity. In piezo-ICSI, short "piezo pulses" are applied electrically to the sperm-containing injection micropipette, causing ultrafast, submicron forward momentum of the injection micropipette. This precise and rapid movement is used to puncture the zona pellucida and oolemma. Next, the sperm are injected into the cytoplasm of the oocyte, and the injection micropipette is withdrawn from the oocyte.

[0049] The injection micropipette contains the operating solution necessary for safe penetration of the oocyte using the piezoelectric effect.

[0050] Thus, in some embodiments of the present invention, piezo-ICSI comprises the following steps: (a) preparing an injection micropipette containing sperm and an operating solution; (b) puncturing the oocyte with the injection micropipette using piezo pulses; (c) injecting sperm into the oocyte; Includes:

[0051] In some embodiments of the present invention, step (b) comprises: (i) puncturing the zona pellucida of the oocyte using a piezo pulse; (ii) moving the injection micropipette through the zona pellucida and pressing it against the oolemma of the oocyte; (iii) puncturing the oolemma using a piezo pulse, thereby moving the injection micropipette into the oocyte cytoplasm; Includes:

[0052] Thus, in some embodiments of the present invention, piezo-ICSI comprises the following steps: (a) preparing an injection micropipette containing sperm and an operating solution; (b) (i) puncturing the zona pellucida of the oocyte using a piezo pulse; (ii) moving the injection micropipette through the zona pellucida and pressing it against the oolemma of the oocyte; (iii) puncturing the oolemma using a piezo pulse, thereby moving the injection micropipette into the oocyte cytoplasm; puncturing the oocyte with the injection micropipette using piezo pulses according to (c) injecting sperm into the oocyte; Includes:

[0053] A "piezopulse" is an electrical current applied to an injection micropipette to induce ultrafast submicron forward momentum of the micropipette. This momentum results from the deformation of the crystal in response to an externally applied voltage. In addition to propelling the injection micropipette forward (axial momentum), lateral momentum is also generated. Both axial and lateral dynamics are thought to play important roles in oocyte puncture.

[0054] The equipment used to perform piezo-ICSI includes an inverted microscope (e.g., IX73) for visualizing the oocyte, sperm, and micropipette; a three-axis micromanipulator (e.g., XenoWorksPT) for manipulating the oocyte, sperm, and micropipette; an injector (e.g., a pneumatic microinjector such as the PNJ-T2) for controlling injection via the injection micropipette; and a piezo micromanipulator (PMM) (e.g., Piezo PMM4GD) for applying piezo pulses to the injection micropipette. Consumables required for piezo-ICSI include a micropipette for injecting sperm (e.g., ultra-fine PINU06-20FT), a micropipette for holding the oocyte, manipulation fluid, and a device for loading the manipulation fluid into the micropipette.

[0055] The present invention provides an apparatus for piezo-ICSI equipped with an injection micropipette containing purified perfluoro-n-octane as the working fluid.

[0056] In some embodiments, an apparatus for piezo-ICSI according to the present invention comprises an inverted microscope, a micromanipulator, an injector, an injection micropipette, and a piezo micromanipulator.

[0057] The present invention also provides a consumable kit for piezo-ICSI, comprising purified perfluoro-n-octane as a working fluid and one or more components selected from one or more injection micropipettes, one or more holding pipettes, and a device for loading the working fluid into the one or more injection micropipettes.

[0058] Suitably, the consumable kit according to the present invention comprises two or more components selected from one or more injection micropipettes, one or more holding pipettes, and a device for loading a working fluid into one or more injection micropipettes.

[0059] Suitably, the consumable kit according to the present invention comprises purified perfluoro-n-octane as a working fluid, one or more injection micropipettes, one or more holding pipettes, and a device for loading the working fluid into the one or more injection micropipettes.

[0060] Piezo-ICSI can be performed according to the steps shown below.

[0061] (a) Using a filling device, the working solution is filled into the injection micropipette to a depth of about 2 mm to 25 mm, for example, about 10 mm to 15 mm (see FIG. 1).

[0062] (b) Insert the injection micropipette into a micropipette holder (see Figure 2A), which is then attached to an injector (see Figure 2B) connected to a piezoelectric micromanipulator (PMM) (see Figure 3).

[0063] (c) Place the injection micropipette under the microscope.

[0064] (d) Using the injector, push the operating solution all the way to the tip of the injection micropipette, ensuring that no air remains.

[0065] (e) Move the tip of the micropipette into the drop of culture medium containing the sperm. The culture medium can be a nutrient broth or a culture medium containing polyvinylpyrrolidone (PVP), etc.

[0066] (f) A small amount of culture medium is aspirated and then expelled from the droplet into the injection micropipette, ensuring that the manipulation solution is not completely aspirated or expelled. This process is repeated until the interface between the culture medium and manipulation solution inside the injection micropipette moves smoothly (see Figure 4).

[0067] (g) Immobilize the sperm cells and aspirate them into the injection micropipette. For example, sperm can be immobilized by squeezing their tails with the tip of the micropipette. Sperm can be positioned within the injection micropipette approximately one oocyte diameter behind the tip of the injection micropipette.

[0068] (h) Position the oocyte using a holding micropipette. The oocyte should be positioned so that it can be punctured into a wide area of ​​the perivitelline space while avoiding the spindle.

[0069] (i) Gently place the tip of the injection micropipette against the zona pellucida without deforming the oocyte (see Figure 5a). Then, switch on the PMM and apply a piezo pulse to the micropipette (see Figure 5b). The ultrafast, submicron-level movement of the injection micropipette advances the injection micropipette through the zona pellucida, perforating it (see Figure 5c).

[0070] (j) The injection micropipette is withdrawn from the zona pellucida, and the hollowed-out portion of the zona pellucida remaining inside the injection micropipette is expelled by draining the injection micropipette. This draining operation of the injection micropipette is used to move the sperm to the tip of the injection micropipette (see Figure 5d).

[0071] (k) Set the PMM to single pulse setting. Only a single piezo pulse is required to rupture the egg membrane.

[0072] (l) Advance the injection micropipette through the zona pellucida (see Figure 5e) to approximately 80%–90% of the total diameter of the oocyte and press it against the oolemma, stretching it (see Figure 5f).

[0073] (m) The PMM is activated to rupture the oolemma with a single piezo pulse, and the injection micropipette is surrounded by the cytoplasm (see Figure 5g).

[0074] (n) Inject the sperm, taking care not to drain too much fluid into the cytoplasm (see Figure 5h).

[0075] (o) Withdrawing the injection micropipette from the oocyte.

[0076] In c-ICSI, a pointed or beveled injection micropipette is used to puncture the oocyte with mechanical force. In piezo-ICSI, mechanical force is not used, so a flat-tipped injection micropipette can be used to puncture the oocyte (see Hiraoka et al. 2019 Piezo-ICSI in Chapter 39, pp. 481-489, in In Vitro Fertilization, edited by Nagy et al. (Springer, Cham.)). Thus, in one embodiment, the injection micropipette has a flat tip.

[0077] In piezo-ICSI, injection micropipettes of various wall thicknesses have been used (Hiraoka and Kitamura 2015 J. Assist. Reprod. Genet. 32: 1827-1833). In the method of the present invention, injection micropipettes of any suitable wall thickness can be used. Suitably, the injection micropipette may have a wall thickness between 0.5 μm and 1 μm. The injection micropipette may have a wall thickness of 0.925 μm. The injection micropipette may have a wall thickness of 0.625 μm.

[0078] The injection micropipette that holds the sperm for piezo-ICSI is sometimes called a capillary or microcapillary.

[0079] In some embodiments of the present invention, piezo-ICSI involves the injection of mammalian sperm into mammalian oocytes. Accordingly, the present invention encompasses all piezo-ICSI applications performed on mammals in which the working fluid is purified perfluorooctane. In some embodiments, the sperm and oocytes are human. In some embodiments, the sperm and oocytes are murine. In some embodiments, the sperm and oocytes are bovine. In some embodiments, the sperm and oocytes are porcine. In some embodiments, the sperm and oocytes are equine.

[0080] Preferably, the sperm and oocytes may be human.

[0081] Operating fluid The term "manipulating solution" refers to the liquid in the injection micropipette that assists in oocyte puncture during piezo-ICSI. If the micropipette does not contain a manipulating solution but only contains medium or oil, piezo-mediated oocyte puncture is less effective and results in a higher oocyte degeneration rate. Therefore, to ensure effective and safe oocyte puncture, it is recommended to use a manipulating solution in the injection micropipette.

[0082] Currently, there is no scientific consensus regarding the exact physical effect of the working fluid that improves the outcome of piezo-ICSI. Working fluids have a relatively high specific gravity. This high specific gravity is thought to result in the working fluid remaining in place due to inertia when the injection micropipette is moved. A static working fluid can enhance the axial (i.e., puncture) movement of the micropipette tip while stabilizing and dampening undesirable lateral vibrations. A static working fluid can also generate a slight vacuum when the micropipette is moved axially, creating a slight suction force that assists in opening the zona pellucida or oolemma.

[0083] The first operating liquid used was mercury, but its toxicity requires careful handling in the laboratory and it cannot be used in human piezo-ICSI.

[0084] Less toxic alternatives to mercury for use as operating fluids are fluorocarbons and fluoroethers.

[0085] In particular, fluorocarbon liquids such as perfluoro-n-alkylmorpholines (see Hiraoka et al. 2019 Piezo-ICSI in Chapter 39, pp. 481-489, in In Vitro Fertilization, edited by Nagy et al. (Springer, Cham.)) and perfluorohydrocarbon mixtures have been used as operating fluids in piezo-ICSI. Fluorocarbon liquids are clear, colorless, odorless, non-flammable fluids with high specific gravity but similar viscosity to water.

[0086] Commercially available hydrofluoroethers have also been used as working fluids in piezo-ICSI.

[0087] Although fluorocarbon liquids and hydrofluoroethers have been used in animal piezo-ICSI, these compounds have not been approved for use in human piezo-ICSI, and furthermore, these liquids are not as effective as mercury for piezo-ICSI.

[0088] The present invention relates to the use of purified perfluoro-n-octane or a composition comprising purified perfluoro-n-octane as a working fluid in piezo-ICSI.

[0089] As described herein, the term "purified perfluoro-n-octane" means that the perfluoro-n-octane contains a low level of reactive low-fluorinated impurities. Thus, the working liquid may contain other compounds as long as these compounds are not substantially low-fluorinated impurities (i.e., low-fluorinated compounds). In other words, the working liquid may contain other fully fluorinated compounds in addition to perfluoro-n-octane.

[0090] Therefore, while the working fluid used in the present invention is "purified perfluoro-n-octane," it may contain compounds other than perfluoro-n-octane. This is because "purified" refers to the proportion of low-fluorinated compounds in the working fluid. The proportion of low-fluorinated impurities is expressed by the H value (ppm) as described herein.

[0091] The proportion of purified perfluoro-n-octane in the working solution can be expressed as a percentage. Similarly, the proportion of any other compound in the working solution can also be expressed as a percentage. This percentage can refer to the percentage of the weight of the working solution that is occupied by purified perfluoro-n-octane or other compounds (wt %), or the percentage of the volume of the working solution that is occupied by purified perfluoro-n-octane or other compounds (vol %).

[0092] Suitably, the present use of purified perfluoro-n-octane as a working fluid for piezo-ICSI refers to a working fluid comprising about 90% or more purified perfluoro-n-octane, for example, about 95% or more purified perfluoro-n-octane, for example, about 96% or more purified perfluoro-n-octane, for example, about 97% or more purified perfluoro-n-octane, for example, about 98% or more purified perfluoro-n-octane, for example, about 99% or more purified perfluoro-n-octane, for example, 100% purified perfluoro-n-octane. In some embodiments, the working fluid comprises about 95% to about 100% purified perfluoro-n-octane.

[0093] Preferably, the working fluid contains about 95% to about 100% purified perfluoro-n-octane.

[0094] In some embodiments, the working fluid comprises about 95% or more perfluoro-n-octane and has an H value of about 100 ppm or less. In some embodiments, the working fluid comprises about 95% or more perfluoro-n-octane and has an H value of about 10 ppm or less.

[0095] The present invention also encompasses the use of a composition comprising purified perfluoro-n-octane as a working fluid for piezo-ICSI.

[0096] Suitably, the use of a composition comprising purified perfluoro-n-octane as a working fluid means that purified perfluoro-n-octane should be the major component of the working fluid (i.e., the compound that forms the majority of the working fluid). The composition used as a working fluid may contain other compounds in addition to purified perfluoro-n-octane.

[0097] For example, in some embodiments, the composition comprises purified perfluoro-n-octane as the predominant purified fully fluorinated compound, in other words, no other purified fully fluorinated compounds are present in the composition in amounts equal to or greater than the purified perfluoro-n-octane.

[0098] Suitably, the working liquid may be a composition comprising about 50% or more purified perfluoro-n-octane, such as about 60% or more purified perfluoro-n-octane, for example about 70% or more purified perfluoro-n-octane, for example about 80% or more purified perfluoro-n-octane, for example about 90% or more purified perfluoro-n-octane.

[0099] In some embodiments, the working fluid includes other compounds in addition to perfluoro-n-octane, where the other compounds are not substantially low-fluorinated compounds, or the other compounds are substantially fully fluorinated compounds.

[0100] The term "other fully fluorinated compounds" means fully fluorinated compounds other than perfluoro-n-octane.

[0101] In some embodiments, the working fluid comprises no more than about 50% other fully fluorinated compounds, no more than about 40% other fully fluorinated compounds, such as no more than about 30% other fully fluorinated compounds, for example no more than about 20% other fully fluorinated compounds, for example no more than about 10% other fully fluorinated compounds, for example no more than about 5% other fully fluorinated compounds, for example no more than about 4% other fully fluorinated compounds, for example no more than about 3% other fully fluorinated compounds, for example no more than about 2% other fully fluorinated compounds, for example no more than about 1% other fully fluorinated compounds, e.g. no other fully fluorinated compounds.

[0102] Perfluoro-n-heptane and perfluoro-n-nonane are examples of other fully fluorinated compounds that may be included in the working liquid. In some embodiments, the working liquid includes perfluoro-n-heptane. In some embodiments, the working liquid includes perfluoro-n-nonane. In some embodiments, the working liquid includes perfluoro-n-heptane and perfluoro-n-nonane. In some embodiments, the working liquid includes perfluoro-n-heptane, perfluoro-n-nonane, and other compounds.

[0103] Perfluoro-n-heptane and perfluoro-n-nonane have the same structure as perfluoro-n-octane, except that they contain 7 and 9 carbon atoms, respectively, instead of 8. In other words, perfluoro-n-heptane consists of a chain of 7 carbon atoms, each carbon bonded to the maximum number of fluorine atoms, and perfluoro-n-heptane consists of a chain of 9 carbon atoms, each carbon bonded to the maximum number of fluorine atoms.

[0104] The structural formula of perfluoro-n-heptane is:

[0105] [ka]

[0106] The structural formula of perfluoro-n-nonane is:

[0107] [ka]

[0108] In some embodiments, the working liquid comprises about 5% or less perfluoro-n-heptane, such as about 4% or less perfluoro-n-heptane, for example about 3% or less perfluoro-n-heptane, for example about 2% or less perfluoro-n-heptane, for example about 1% or less perfluoro-n-heptane, for example no perfluoro-n-heptane.

[0109] In some embodiments, the working solution contains about 10% or less perfluoro-n-nonane, such as about 6% or less perfluoro-n-nonane, for example about 5% or less perfluoro-n-nonane, such as about 4% or less perfluoro-n-nonane, for example about 3% or less perfluoro-n-nonane, such as about 2% or less perfluoro-n-nonane, for example about 1% or less perfluoro-n-nonane, e.g., no perfluoro-n-nonane.

[0110] In some embodiments, the working liquid comprises about 5% or less perfluoro-n-heptane and about 5% or less perfluoro-n-nonane. In some embodiments, the working liquid comprises about 2% or less perfluoro-n-heptane and about 5% or less perfluoro-n-nonane.

[0111] In some embodiments, the working fluid comprises about 95% to about 100% perfluoro-n-octane, about 2% or less perfluoro-n-heptane, and about 5% or less perfluoro-n-nonane.

[0112] In some embodiments, the working fluid comprises about 95% to about 100% perfluoro-n-octane, about 2% or less perfluoro-n-heptane, and about 5% or less perfluoro-n-nonane, and has an H value of about 10 ppm or less.

[0113] Assisted reproductive technology The present invention provides a method for assisted reproductive technology, comprising: (a) in vitro fertilization of an oocyte using a method according to the present invention to form an embryo; (b) culturing the embryo; and (c) implanting the embryo into a subject; The present invention provides a method comprising:

[0114] In some embodiments of the assisted reproduction methods of the present invention, the subject is a human.

[0115] In some embodiments, methods of assisted reproduction according to the present invention include harvesting oocytes from a subject.

[0116] Technologies for assisted reproduction are known in the art. In particular, products and techniques for culturing embryos in vitro and then implanting them in a subject are known in the art. For example, Vitrolife produces needles for egg collection (e.g., Sense egg collection needles), gradients for optimal sperm preparation (e.g., SpermGrad™), media for culturing fertilized oocytes, and media for implanting viable and genetically healthy embryos into a subject (e.g., EmbryoGlue).

[0117] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range.

[0118] When a range of values ​​is expressed, each smaller range between any specified value or intervening value in the stated range and any other specified or intervening value in that stated range is included within the disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded within the range, and each range where either limit is included, neither limit is included, or both limits are included within the smaller range is also included within the disclosure, subject to any specifically excluded limits within the stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0119] Please note that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0120] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," and are non-exclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0121] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0122] The present invention will now be further illustrated by examples, which are intended to be used to assist those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example]

[0123] Example 1 - Suitability of Fluorinated Compounds as Working Fluids The suitability of three fluorinated test compounds for use as working fluids for piezo-ICSI was tested. The three test compounds were perfluorodecalin, perfluoro-n-octane, and perfluoroperhydrophenanthrene. The test compounds were compared with a commercially available hydrofluoroether previously used as a control working fluid. The findings of this comparison are shown in Table 2 below.

[0124] [Table 2]

[0125] In summary, perfluoro-n-octane was found to be as suitable for use as a working fluid for piezo-ICSI as standard hydrofluoroether working fluids, while other fluorinated compounds were less suitable or completely unsuitable.

[0126] Example 2 - Effects of Fluorinated Compounds on Embryonic Development The effect of incubation in various fluorinated compounds on embryonic development was examined.

[0127] 223 2PN (two pronuclear) mouse embryos were randomly divided into four groups: A) neat perfluoro-n-octane, B) a commercially available perfluorohydrocarbon mixture previously used as a working fluid for piezo-ICSI, C) purified perfluoro-n-octane, and D) untreated control.

[0128] Embryos in groups A, B, and C were incubated in each compound for 3 minutes before being transferred to culture medium. Embryos in all groups were then cultured for 5 days in an incubator (37.5°C, 100% relative humidity, 5% CO2 in air).

[0129] The number of embryos in each group showing signs of degeneration was counted.

[0130] The number of embryos in each group that reached the 2-cell, 3-cell, expanded blastocyst, and hatching / hatched blastocyst development stages within 5 days of incubation was counted.

[0131] The average time taken for the embryos to reach the 2-cell stage (T2) and the average time taken for the embryos to develop from the 2-cell stage to the 3-cell stage (T3) was measured.

[0132] The results are shown in Table 3 below.

[0133] [Table 3]

[0134] A small proportion of embryos incubated in a commercially available perfluorohydrocarbon mixture operating solution showed signs of degeneration, whereas none of the embryos incubated in perfluoro-n-octane showed signs of degeneration.

[0135] The working fluid was a commercially available mixture of perfluorohydrocarbons (Group B) or neat perfluoro-n-octane (Group C). A ) embryos incubated in Untreated Control embryos (group D ) developed more slowly than the expanded blastocyst and hatching / post-hatching blastocyst stages. This slowdown is indicated by a lower percentage of embryos reaching the expanded blastocyst and hatching / post-hatching blastocyst stages and an increased time required to develop from the 2-cell to 3-cell stage (average T2 to T3).

[0136] In contrast, purified perfluoro-n-octane (group C ) were incubated in the untreated control group. (Group D) They developed at a pace comparable to that of embryos.

[0137] These data indicate that purified perfluoro-n-octane, in contrast to commercially available perfluorohydrocarbon mixtures used in the operating fluid, does not adversely affect embryonic development.

[0138] Example 3 - Assisted Reproduction Using Piezo-ICSI with Perfluoro-n-octane Working Fluid The efficacy of c-ICSI in assisted reproductive technology was compared with that using piezo-ICSI. Purified perfluoro-n-octane was used as the operating fluid for piezo-ICSI.

[0139] Specifically, 69 patients with at least six mature oocytes were randomly divided into two groups: one group underwent assisted reproduction using c-ICSI to transfer sperm into oocytes, and the second group underwent assisted reproduction using piezo-ICSI to transfer sperm into oocytes.

[0140] c-ICSI was performed using an ICSI micropipette (TPC, Cooper Surgical) with an Eppendorf 2K control. Piezo-ICSI was performed using a PMM and a Piezo micropipette (Primetech). Purified perfluoro-n-octane was used as the operating fluid in piezo-ICSI.

[0141] Patients undergoing ART using piezo-ICSI had a significantly increased fertilization rate compared with patients undergoing ART using c-ICSI (65.9% of oocytes successfully fertilized using c-ICSI vs. 80.6% of oocytes successfully fertilized using piezo-ICSI, p<0.05). The use of piezo-ICSI also resulted in a decreased rate of oocyte degeneration compared with c-ICSI (10.2% with c-ICSI vs. 3.8% with piezo-ICSI). Utilization rates were similar for c-ICSI and piezo-ICSI (47.3% with piezo-ICSI vs. 45.6% with c-ICSI). Clinical pregnancy rates were also comparable for c-ICSI and piezo-ICSI (55% with piezo-ICSI vs. 50% with c-ICSI).

[0142] The use of piezo-ICSI produced an average of one additional embryo for vitrification compared with c-ICSI (average of 2.7 embryos for vitrification from c-ICSI vs. average of 3.8 embryos for vitrification from piezo-ICSI).

[0143] These data indicate that piezo-ICSI using purified perfluoro-n-octane as the working fluid in assisted reproduction is more effective than c-ICSI in generating embryos for assisted reproduction.

[0144] All publications cited in the above specification are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications and variations can be made in the described methods and systems of the invention without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in obstetrics and reproductive technology or assisted reproductive technology or molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. Apparatus for piezo-ICSI comprising an injection micropipette containing purified perfluoro-n-octane as the working fluid.

2. 2. The apparatus for piezo-ICSI according to claim 1, comprising an inverted microscope, a micromanipulator, an injector, the injection micropipette, and a piezo micromanipulator.

3. a purified perfluoro-n-octane working fluid and one or more injection micropipettes; The kit for piezo-ICSI further comprises one or more components selected from one or more holding pipettes and a device for filling a working fluid into the one or more injection micropipettes.

4. 3. The device for piezo-ICSI according to claim 1 or claim 2, wherein the purified perfluoro-n-octane has an H value of 1000 parts per million (ppm) or less.

5. 3. The piezo-ICSI device according to claim 1, wherein the purified perfluoro-n-octane has an H value of 10 ppm or less.

6. 3. The piezo-ICSI device according to claim 1, wherein the operating liquid contains 90% or more purified perfluoro-n-octane.

7. 3. The piezo-ICSI device according to claim 1, wherein the operating liquid contains 95% or more purified perfluoro-n-octane.

8. 4. The kit for piezo-ICSI according to claim 3, wherein the purified perfluoro-n-octane has an H value of 1000 parts per million (ppm) or less.

9. 4. The piezo-ICSI kit according to claim 3, wherein the purified perfluoro-n-octane has an H value of 10 ppm or less.

10. 4. The piezo-ICSI kit according to claim 3, wherein the operating solution contains 90% or more purified perfluoro-n-octane.

11. 4. The piezo-ICSI kit according to claim 3, wherein the operating solution contains 95% or more purified perfluoro-n-octane.

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