Method for producing selected sperm population and method for assisting diagnosis of male infertility

Abstract

To provide a method for producing a selected sperm, capable of improving fertilization rate in in-vitro fertilization or artificial fertilization, and a method for assisting effective diagnosis of male infertility.SOLUTION: A method for producing a selected sperm population comprises a first step of preparing a first sperm population containing, as a subpopulation, specific sperms that improve the fertilization rate of the sperm population when their content in the sperm population is reduced, and a second step of removing the specific sperms from the first sperm population to obtain a second sperm population having a reduced content of the specific sperms compared with the first sperm population.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a selected sperm population and a method for assisting in the diagnosis of male infertility. [Background technology]

[0002] Patent Document 1 describes a method for determining the optimal time for utilizing a semen sample for insemination or storage as a method for improving fertility in artificial insemination. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2020-514772 Summary of the Invention [Problem to be solved by the invention]

[0004] However, because the causes of male infertility remain largely unknown, it has not been easy to improve fertilization rates in in vitro fertilization and artificial insemination. In addition, there have been cases where the cause of male infertility could not be identified during diagnosis.

[0005] The present invention has been made in consideration of the above-mentioned problems, and one of its objects is to provide a method for producing a selected sperm population that improves the fertilization rate in in vitro fertilization and artificial insemination, and an effective diagnostic aid method for male infertility. [Means for solving the problem]

[0006] [1] To solve the above problems, one embodiment of the present invention provides a method for producing a selected sperm population that improves fertilization rates in in vitro fertilization and artificial insemination, comprising: a first step of preparing a first sperm population containing, as a subpopulation, specific sperm whose content in the sperm population is reduced to improve the fertilization rate of the sperm population; and a second step of removing the specific sperm from the first sperm population to obtain a second sperm population having a reduced content of the specific sperm compared to the first sperm population.

[0007] [2] In the method of [1], the specified sperm may be a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface. [3] In addition, in the method of [1] or [2], the specified sperm may be a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface. [4] In the method of [1] or [2], the specified sperm may be a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface.

[0008] [4] To solve the above-mentioned problems, one embodiment of the present invention provides a diagnostic support method for male infertility, which uses the following indicators: (a) the content of specific sperm in a sperm population collected from a patient, which is a subpopulation whose content in the sperm population is reduced to improve the fertilization rate of the sperm population; and (b) the change in the content of the specific sperm in the sperm population before and after activation treatment or calcium treatment when the sperm population collected from a patient is subjected to the activation treatment or calcium treatment. According to the present invention, an effective diagnostic support method for male infertility is provided.

[0009] [5] In the method of [4], the specific sperm may be a subpopulation that expresses one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface. [Effects of the Invention]

[0010] According to the present invention, a method for producing a selected sperm population that improves the fertilization rate in in vitro fertilization and artificial insemination, and an effective diagnostic aid method for male infertility are provided. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is an explanatory diagram showing the results of evaluating the appearance of Lypd4-positive sperm in a mouse sperm population in Example 1. [Figure 2] FIG. 1 is an explanatory diagram showing the results of evaluating the expression of a marker antigen correlated with Lypd4 in a mouse sperm population in Example 1. [Figure 3] FIG. 1 is an explanatory diagram showing the results of examining the conditions for the appearance of Lypd4-positive sperm in a mouse sperm population in Example 1. [Figure 4] FIG. 1 is an explanatory diagram showing the results of evaluating the improvement in fertilization rate by removing Lypd4-positive sperm from a mouse sperm population in Example 1. [Figure 5A] FIG. 1 is an explanatory diagram showing the results (flow cytometry) of evaluating the appearance of CD55-positive sperm in monkey sperm populations in Example 2. [Figure 5B] FIG. 1 is an explanatory diagram showing the results (graph) of evaluating the appearance of CD55-positive sperm in monkey sperm populations in Example 2. [Figure 6] FIG. 1 is an explanatory diagram showing the results of evaluating the expression of a marker antigen correlated with CD55 in monkey sperm populations in Example 2. [Figure 7A] FIG. 1 is an explanatory diagram showing the results (flow cytometry) of evaluating the improvement in sperm penetration rate in a hamster test by removing CD55-positive sperm from a monkey sperm population in Example 2. [Figure 7B] FIG. 1 is an explanatory diagram showing the results (graph) of an evaluation of the improvement in sperm penetration rate in a hamster test by removing CD55-positive sperm from a monkey sperm population in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0012] An embodiment of the present invention will be described below, although the present invention is not limited to this embodiment.

[0013] In mammals, various regulatory mechanisms are at work to ensure proper fertilization. Sperm are incapable of fertilization immediately after they are produced in the testes. They must undergo a stepwise activation process, including capacitation, hyperactivation, and acrosome reaction, before meeting with an egg in the fallopian tube, where fertilization occurs. Each reaction in this activation process positively affects fertilization.

[0014] In response to this, the inventors of the present invention independently discovered that a specific subpopulation emerges during the activation of a sperm population, and that this specific subpopulation negatively regulates fertilization. Furthermore, the inventors of the present invention independently discovered that a selected sperm population obtained by removing the specific subpopulation that negatively regulates fertilization from an activated sperm population exhibits a higher fertilization rate than an activated sperm population from which the subpopulation has not been removed. The present invention is based on these independently discovered findings by the inventors.

[0015] A method for producing a selected sperm population according to one embodiment of the present invention (hereinafter referred to as "this method") includes a first step of preparing a first sperm population containing, as a subpopulation, specific sperm whose content in the sperm population is reduced to improve the fertilization rate of the sperm population, and a second step of removing the specific sperm from the first sperm population to obtain a second sperm population in which the content of the specific sperm is reduced compared to the first sperm population.

[0016] The first sperm population prepared in the first step of the present method includes specific sperm as a subpopulation within the first sperm population, and the specific sperm are a subpopulation whose concentration in the sperm population is reduced to improve the fertilization rate of the sperm population.

[0017] In other words, the specific sperm are identified as a subpopulation in which the fertilization rate of the sperm population in which the content of the specific sperm has been reduced by removing the specific sperm is higher than that of the sperm population before the removal. The fertilization rate of the sperm population can be evaluated by a known method such as the hamster test used in the Examples described below.

[0018] Alternatively, the specific sperm may be identified as a subpopulation whose content in the sperm population increases as a result of activating the sperm population, i.e., in this case, the specific sperm are identified as a subpopulation whose content in the activated sperm population increases compared to its content in the sperm population before activation.

[0019] Here, an activated sperm population is a sperm population that has acquired fertilization capacity, and an unactivated sperm population is a sperm population that has not yet acquired fertilization capacity (a sperm population that does not have fertilization capacity).

[0020] The method for activating the sperm population is not particularly limited as long as it allows the sperm population to acquire fertilization capacity, but for example, a method of subjecting the sperm population to an artificial activation treatment is preferably used. This activation treatment is not particularly limited as long as it is a treatment that artificially activates the sperm population, but for example, a treatment of contacting the sperm population with a sperm activation solution (specifically, a treatment of maintaining the sperm population in a sperm activation solution) is preferred.

[0021] The sperm activation solution is not particularly limited as long as it has a composition that activates a sperm population while maintaining the viability of the sperm population when brought into contact with the sperm population. For example, an aqueous solution containing calcium ions, bicarbonate ions, and albumin (e.g., bovine serum albumin) at concentrations that activate the sperm population is preferably used.

[0022] Specifically, preferred sperm activation solutions include HTF (Human Tubal Fluid), TYH medium (Toyoda Yokoyama Hoshi medium), which are artificially prepared solutions that mimic body fluids that activate sperm populations, such as oviductal fluid, or solutions having the same composition as HTF or TYH except that they contain methyl beta cyclodextrin instead of bovine serum albumin.

[0023] Furthermore, the inventors of the present invention independently discovered that by subjecting a sperm population to artificial calcium treatment, the content of specific sperm in the sperm population increases, even if the sperm population is not activated.

[0024] Therefore, the specific sperm may be identified as a subpopulation whose content in a sperm population increases when the sperm population is treated with calcium, i.e., in this case, the specific sperm are identified as a subpopulation whose content in a sperm population that has been treated with calcium increases compared to its content in the sperm population before the calcium treatment.

[0025] Here, calcium treatment is a process in which a sperm population that has not yet been activated and has not been subjected to the calcium treatment is contacted with calcium ions at a concentration that increases the content of specific sperm compared to before the calcium treatment.

[0026] That is, calcium treatment is, for example, a process in which a sperm population that has not yet been activated and has not been subjected to the calcium treatment is contacted with a solution containing calcium ions at a concentration that increases the content of specific sperm compared to before the calcium treatment (hereinafter referred to as a ``calcium treatment solution'') (specifically, a process in which the sperm population is maintained in the calcium treatment solution).

[0027] The calcium treatment solution is not particularly limited as long as it is a solution containing calcium ions at a concentration that, when brought into contact with a sperm population, maintains the viability of the sperm population while increasing the content of specific sperm in the sperm population. For example, the solution may be an aqueous solution containing calcium ions at a concentration that activates the sperm population but not containing bicarbonate ions and / or albumin (e.g., bovine serum albumin) at concentrations that activate the sperm population, or it may be the sperm activation solution described above.

[0028] The concentration of calcium ions to be brought into contact with the sperm population during calcium treatment (e.g., the calcium ion concentration in the calcium treatment solution) is not particularly limited as long as it is within a range in which the calcium treatment maintains the viability of the sperm population while increasing the content of specific sperm in the sperm population, but may be, for example, 30 μM or more, preferably 40 μM or more, more preferably 50 μM or more, even more preferably 100 μM or more, even more preferably 200 μM or more, even more preferably 500 μM or more, even more preferably 1 mM or more, even more preferably 3 mM or more, and particularly preferably 5 mM or more.

[0029] The calcium ion concentration contacted with the sperm population during calcium treatment may be, for example, 15 mM or less, 10 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, or 5 mM or less. The calcium ion concentration contacted with the sperm population during calcium treatment may be specified by combining any one of the above-mentioned lower limit values ​​with any one of the above-mentioned upper limit values.

[0030] In general, the sperm activation solution used in the above-mentioned activation treatment contains calcium ions at a concentration that increases the content of specific sperm in a sperm population, so as described above, the sperm activation solution can be used as a calcium treatment solution. Also, the activation treatment using the sperm activation solution can be said to be a calcium treatment.

[0031] However, calcium treatment may also be a treatment that increases the percentage of specific sperm in a sperm population without activating the sperm population, i.e., the sperm activation solution may contain bicarbonate ions and albumin at concentrations necessary for activation of the sperm population, but the calcium treatment solution may not contain either or both of bicarbonate ions and albumin at concentrations necessary for activation of the sperm population.

[0032] In this case, by contacting the sperm population with a calcium-treated solution that does not contain bicarbonate ions and / or albumin at a concentration required to activate the sperm population, the content of specific sperm in the sperm population can be increased without activating the sperm population.

[0033] Alternatively, the specific sperm may be identified as a subpopulation expressing a marker antigen specific to the specific sperm on the cell membrane surface. Specifically, the specific sperm may be identified as a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS on the cell membrane surface.

[0034] More specifically, the specific sperm are preferably identified as a subpopulation that expresses, for example, CD55 and / or CCR3 on the cell membrane surface, and particularly preferably as a subpopulation that expresses CD55 on the cell membrane surface.

[0035] That is, the specific sperm may be identified as a subpopulation expressing, on the cell membrane surface, one or more marker antigens selected from the group consisting of CCR3, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS in addition to CD55; or as a subpopulation expressing, on the cell membrane surface, one or more marker antigens selected from the group consisting of CD55, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS in addition to CCR3; or as a subpopulation expressing, on the cell membrane surface, one or more marker antigens selected from the group consisting of CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS in addition to CD55 and CCR3. The expression of marker antigens in the specific sperm is confirmed using a flow cytometer such as FACS (Fluorescence-Activated Cell Sorting).

[0036] The animal species from which the sperm population used in the present invention is derived is not particularly limited as long as the effects of the present invention can be obtained, but is preferably a mammal. The mammal is preferably a human, but may be a mammal other than a human. The non-human mammal is preferably a livestock, pet, or laboratory animal.

[0037] Specifically, the non-human mammal may be, for example, a primate (e.g., a monkey), a rodent (e.g., a mouse, a rat, a hamster, a guinea pig, or a rabbit), a carnivore (e.g., a dog, a cat), or an ungulate (e.g., a pig, a cow, a horse, a goat, or a sheep).

[0038] The marker antigen for identifying the specific sperm may be appropriately selected depending on the animal species from which the specific sperm originate. Specifically, for example, when the first sperm population is a human or primate sperm population, the specific sperm contained in the first sperm population may be a subpopulation that expresses one or more selected from the group consisting of CD55, CCR3, CCR8, CD215 (IL15RA), and OPRD1 on the cell membrane surface.

[0039] In this case, the specified sperm are preferably identified as a subpopulation expressing CD55 and / or CCR3 on the cell membrane surface. That is, the specified sperm may be identified as a subpopulation expressing one or more marker antigens selected from the group consisting of CCR3, CCR8, CD215 (IL15RA), and OPRD1 in addition to CD55 on the cell membrane surface, or as a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR8, CD215 (IL15RA), and OPRD1 in addition to CCR3 on the cell membrane surface, or as a subpopulation expressing one or more marker antigens selected from the group consisting of CCR8, CD215 (IL15RA), and OPRD1 in addition to CD55 and CCR3 on the cell membrane surface.

[0040] Furthermore, for example, when the first sperm population is a rodent sperm population, the specific sperm contained in the first sperm population may be a subpopulation that expresses one or more selected from the group consisting of CD55, Lypd4, CCR3, and ICOS on the cell membrane surface.

[0041] In this case, the specific spermatozoa are preferably identified as a subpopulation expressing CD55 and / or Lypd4 on the cell membrane surface. That is, the specific spermatozoa may be identified as a subpopulation expressing one or more marker antigens selected from the group consisting of Lypd4, CCR3, and ICOS on the cell membrane surface in addition to CD55, or as a subpopulation expressing one or more marker antigens selected from the group consisting of CD55, CCR3, and ICOS on the cell membrane surface in addition to Lypd4, or as a subpopulation expressing one or more marker antigens selected from the group consisting of CCR3 and ICOS on the cell membrane surface in addition to CD55 and Lypd4.

[0042] The marker antigen for identifying a specific sperm may be an antigen other than the above-mentioned CD55, CCR3, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS, as long as it is an antigen that is specifically expressed on the cell membrane surface of the specific sperm. In other words, if a new antigen that is specifically expressed on the cell membrane surface of a specific sperm is found in the future, the new antigen can also be used as a marker antigen.

[0043] Furthermore, the inventors of the present invention analyzed the process of the appearance of specific sperm expressing CD55 on the surface of their cell membrane and found that the CD55 protein itself was also present on the back surface of the cell membrane of sperm that had not been activated or treated with calcium. Therefore, it is thought that when a sperm population is activated or treated with calcium, the CD55 protein on the back surface of the cell membrane of some sperm contained in the sperm population moves to the surface of the cell membrane, resulting in the appearance of specific sperm expressing CD55 on the surface of the cell membrane.

[0044] Therefore, the appearance of specific sperm in an activated or calcium-treated sperm population can also be considered to be a positive conversion of the marker antigen in some sperm contained in the sperm population (i.e., a change from marker antigen-negative sperm to marker antigen-positive sperm).

[0045] The first sperm population prepared in the first step of this production method is not particularly limited as long as it is a live sperm population that contains the above-mentioned specific sperm as a subpopulation, and may be, for example, a sperm population collected from a living organism, or a sperm population induced to differentiate from pluripotent stem cells (e.g., one or more selected from the group consisting of iPS (induced pluripotent stem) cells, ES (embryonic stem) cells, Muse (multilineage-differentiating stress-enduring) cells, and EG (embryonic germ) cells), but is preferably a sperm population collected from a living organism.

[0046] As a sperm population collected from a living organism, for example, a sperm population ejaculated from the living organism to outside the body (which may be a sperm population ejaculated by electrical stimulation or manually) or a sperm population surgically extracted from the epididymis or testis of the living organism is preferably used.

[0047] The first sperm population may be, for example, an activated or calcium-treated sperm population. In this case, the activated first sperm population may be a sperm population collected from a living organism and already activated at the time of collection, or a sperm population collected from a living organism and subjected to an activation treatment after collection. The calcium-treated first sperm population is a sperm population collected from a living organism and subjected to the calcium treatment after collection.

[0048] The first sperm population may also be, for example, a sperm population that has not yet been activated and has not been calcium-treated. In this case, the first sperm population may be a sperm population collected from a living organism, which includes specific sperm at the time of collection and has not yet been activated. The sperm population collected from a living organism may also be a sperm population that has been frozen after collection and then thawed.

[0049] The first sperm population contains specific sperm as a subpopulation, and also contains sperm other than the specific sperm (hereinafter referred to as "non-specific sperm"). The content of the specific sperm in the first sperm population (the value (%) calculated by dividing the number of specific sperm contained in the first sperm population by the total number of sperm contained in the first sperm population (the sum of the number of specific sperm and the number of non-specific sperm) and multiplying the result by 100) is not particularly limited as long as the effects of the present invention are obtained, but may be, for example, 1% or more, preferably 5% or more, more preferably 10% or more, even more preferably 15% or more, even more preferably 20% or more, even more preferably 25% or more, even more preferably 30% or more, even more preferably 35% or more, even more preferably 40% or more, even more preferably 45% or more, even more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 65% ​​or more, and particularly preferably 70% or more.

[0050] The content of the specific sperm in the first sperm population may be, for example, 95% or less, or 90% or less. The content of the specific sperm in the first sperm population may be determined by combining any one of the above-mentioned lower limit values ​​with any one of the above-mentioned upper limit values. The content of the specific sperm in the first sperm population is calculated based on the analysis results using a flow cytometer such as FACS.

[0051] The preparation of the first sperm population in the first step may be carried out by obtaining a first sperm population that has been activated or calcium-treated in advance, or by subjecting a sperm population that has not yet been activated or calcium-treated to an activation treatment or calcium treatment in the first step to prepare the first sperm population.

[0052] That is, in the first step, for example, a sperm population collected from a living body may be subjected to an activation treatment or calcium treatment to obtain a first sperm population having an increased content of specific sperm compared to before the treatment.

[0053] In the second step of this method, specific sperm are removed from the first sperm population prepared in the first step to obtain a second sperm population in which the content of the specific sperm is reduced compared to the first sperm population.

[0054] The method for removing specific sperm from the first sperm population is not particularly limited as long as it reduces the content of the specific sperm while obtaining a live second sperm population. For example, a method using a monoclonal antibody (hereinafter referred to as "anti-marker antibody") against a marker antigen specifically expressed on the cell membrane surface of the specific sperm is preferably used.

[0055] That is, for example, in the second step, a labeled anti-marker antibody may be mixed with the first sperm population, and the subpopulation to which the anti-marker antibody binds may be removed from the first sperm population as a specific sperm.

[0056] Methods for removing subpopulations to which anti-marker antibodies are bound include, for example, methods using magnetic beads and a magnetic column as employed in the Examples described below, or methods using a cell separation device such as a cell sorter.

[0057] The anti-marker antibody may be, for example, a monoclonal antibody against one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS (i.e., one or more anti-marker antibodies selected from the group consisting of an anti-CD55 antibody, an anti-CCR3 antibody, an anti-CCR8 antibody, an anti-CD215 (IL15RA) antibody, an anti-OPRD1 antibody, an anti-Lypd4 antibody, and an anti-ICOS antibody).

[0058] In this case, the anti-marker antibody to be used may be selected appropriately depending on the animal species from which the specific sperm originates, as in the case of selecting the marker antigen to be used to identify the specific sperm described above. For example, an anti-CD55 antibody and / or an anti-CCR3 antibody is preferably used, and an anti-CD55 antibody is particularly preferably used.

[0059] Furthermore, the anti-marker antibody used to remove specific sperm is not particularly limited as long as it is a monoclonal antibody against a marker antigen that is specifically expressed on the cell membrane surface of the specific sperm, and may be a monoclonal antibody against a marker antigen other than the above-mentioned CD55, CCR3, CCR8, CD215 (IL15RA), OPRD1, Lypd4, and ICOS.

[0060] In other words, if a new marker antigen that is specifically expressed on the cell membrane surface of a specific sperm is discovered in the future, the specific sperm may be removed from the first sperm population using a monoclonal antibody against the new marker antigen.

[0061] The content of specific sperm in the second sperm population obtained in the second step, which is lower than that in the first sperm population, is not particularly limited as long as the effects of the present invention are obtained, but the ratio of the content of specific sperm in the second sperm population to the content of specific sperm in the first sperm population (the value (%) calculated by dividing the content of specific sperm in the second sperm population by the content of specific sperm in the first sperm population and multiplying the result by 100) may, for example, be 80% or less, preferably 75% or less, more preferably 70% or less, even more preferably 65% ​​or less, even more preferably 60% or less, even more preferably 55% or less, even more preferably 50% or less, even more preferably 45% or less, even more preferably 40% or less, even more preferably 35% or less, even more preferably 30% or less, even more preferably 25% or less, even more preferably 20% or less, even more preferably 15% or less, and particularly preferably 10% or less.

[0062] According to this method, by carrying out the above-mentioned first and second steps, a selected sperm population that exhibits a higher fertilization rate than the first sperm population can be produced. That is, for example, the second sperm population obtained in the second step exhibits a higher fertilization rate than the first sperm population because the content of specific sperm is reduced compared to the first sperm population. Therefore, in this method, the second sperm population may be obtained as a selected sperm population as it is.

[0063] In this method, the second sperm population obtained in the second step may be further treated to obtain a third sperm population as the selected sperm population. The treatment of this second sperm population is not particularly limited, as long as the treatment results in a live third sperm population that exhibits a higher fertilization rate than the first sperm population.

[0064] The selected sperm population obtained by this method exhibits a high fertilization rate and can be used in a variety of applications. Specifically, the selected sperm population obtained by this method is preferably used for in vitro fertilization or artificial insemination.

[0065] That is, the present method is preferably a method for producing a selected sperm population for use in in vitro fertilization or artificial insemination. In this case, in the first step, a first sperm population collected from a living body is prepared for use in in vitro fertilization or artificial insemination. Then, in the second step, specific sperm are removed from the first sperm population to obtain a second sperm population in which the content of the specific sperm is reduced compared to the first sperm population. The selected sperm population thus obtained by the present method exhibits a higher fertilization rate in in vitro fertilization or artificial insemination compared to the first sperm population due to the reduced content of the specific sperm.

[0066] In this regard, in the past, when conventional in vitro fertilization or artificial insemination was unsuccessful, intracytoplasmic sperm injection (ICS) was attempted, but ICS had problems such as the need for skilled techniques, the risk of damaging the fertilized eggs, and the relatively high cost. In contrast, according to the present production method, a simple procedure of removing specific sperm can effectively produce a selected sperm population that shows an improved fertilization rate in in vitro fertilization or artificial insemination.

[0067] Furthermore, as mentioned above, by reducing the content of specific sperm in a sperm population, the fertilization rate of the sperm population is improved, and therefore it is believed that there is a cause of male infertility related to the specific sperm.

[0068] Therefore, a diagnostic support method for male infertility according to one embodiment of the present invention (hereinafter referred to as "this diagnostic support method") uses the following (a) and / or (b): (a) the content in a sperm population collected from a patient of specific sperm, which is a subpopulation whose content in the sperm population improves the fertilization rate of the sperm population when reduced; (b) when a sperm population collected from a patient is subjected to activation treatment or calcium treatment, the change in the content of the specific sperm in the sperm population before and after the activation treatment or calcium treatment as an indicator.

[0069] In this diagnostic support method, when the content of specific sperm in the sperm population collected from the patient (a) above is used as an index, specifically, (a1) the content of specific sperm in the sperm population collected from the patient that has been activated or calcium-treated may be used as the index, or (a2) the content of specific sperm in the sperm population collected from the patient that has not yet been activated or calcium-treated may be used as the index, but it is preferable to use (a1) as the index.

[0070] In this diagnostic assistance method, if the content of specific sperm in the sperm population collected from the patient is equal to or greater than a predetermined threshold, information is provided that indicates that male infertility may be caused in part by a high content of the specific sperm.

[0071] Furthermore, if the content of specific sperm in a sperm population collected from a patient for in vitro fertilization or artificial insemination is equal to or greater than a predetermined threshold, a proposal may be made to perform in vitro fertilization or artificial insemination using a selected sperm population obtained by removing the specific sperm from the sperm population (for example, a selected sperm population obtained by the above-mentioned manufacturing method).

[0072] In this regard, as mentioned above, in the past, when conventional in vitro fertilization or artificial insemination was unsuccessful, intracytoplasmic sperm injection (ICS) was sometimes attempted. However, ICS has problems such as the need for skilled techniques, the risk of damaging the fertilized egg, and the relatively high cost.

[0073] In contrast, this diagnostic assistance method can provide patients with useful suggestions for improving fertilization rates in in vitro fertilization or artificial insemination through the simple procedure of removing specific sperm, using the content of specific sperm in a sperm population collected from a patient as an indicator.

[0074] On the other hand, if the content of specific sperm in the sperm population collected from the patient is below a predetermined threshold, information may be provided that, for example, the presence of the specific sperm may not be one of the causes of male infertility.

[0075] Furthermore, if the content of specific sperm in the sperm population that has been activated or calcium-treated as described above in (a1) is below a predetermined threshold, it may be possible to provide information that the fact that the content of the specific sperm does not increase significantly even after the activation or calcium treatment (i.e., the specific sperm are not normally induced) may indicate some kind of abnormality (for example, information that the abnormality may be one of the causes of male infertility).

[0076] Furthermore, in this diagnostic support method, when the activation treatment or calcium treatment is applied to a sperm population collected from a patient (b) above, and the change in the content of the specific sperm in the sperm population before and after the activation treatment or calcium treatment is used as an index, specifically, for example, the difference and / or ratio between the content of the specific sperm in the sperm population before the treatment and the content of the specific sperm in the sperm population after the treatment is preferably used as an index.

[0077] More specifically, for example, the difference obtained by subtracting the content of a specific sperm in the sperm population before activation treatment or calcium treatment from the content of the specific sperm in the sperm population after the treatment may be used as an index, and / or the ratio of the content of a specific sperm in the sperm population before the treatment to the content of the specific sperm in the sperm population after the treatment to the other may be used as an index.

[0078] In this diagnostic assistance method, if the change in the content of specific sperm in the sperm population before and after the above treatment is equal to or greater than a predetermined threshold (for example, if the treatment results in a large increase in the content of specific sperm), information is provided that the large change in the content of the specific sperm may be one of the causes of male infertility.

[0079] Furthermore, if the change in the content of specific sperm in a sperm population collected from a patient for in vitro fertilization or artificial insemination is equal to or greater than a predetermined threshold, a proposal may be made to perform in vitro fertilization or artificial insemination using a selected sperm population obtained by removing the specific sperm from the sperm population (for example, a selected sperm population obtained by the above-mentioned manufacturing method).

[0080] In this case, this diagnostic assistance method can provide useful suggestions to patients, using the change in the content of specific sperm in a sperm population collected from a patient as an indicator, to improve the fertilization rate in in vitro fertilization or artificial insemination through the simple operation of removing the specific sperm.

[0081] On the other hand, if the change in the content of specific sperm in the sperm population before and after the above treatment is less than a predetermined threshold, information may be provided that the male infertility is not caused in part by the presence of the specific sperm, or information may be provided that the fact that the content of the specific sperm does not increase significantly due to the activation treatment or calcium treatment (i.e., the specific sperm are not normally induced) may indicate some kind of abnormality (for example, information that the abnormality may be one of the causes of male infertility).

[0082] In this diagnostic support method, the threshold value to be compared with the above-mentioned indicators (content rate of specific sperm and its change) may be determined statistically based on the evaluation results of sperm populations collected from an appropriate number of living organisms, evaluating the content rate of specific sperm in the sperm population, and, when activation treatment or calcium treatment is performed, evaluating the change in the content rate of specific sperm in the sperm population before and after the activation treatment or calcium treatment.

[0083] In this regard, the threshold value to be compared with the content rate of the specific sperm in (a) above may be, for example, within the range of 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more.

[0084] Furthermore, when the change in the content of specific sperm in (b) above is measured as the ratio of the content of specific sperm after activation treatment or calcium treatment to the content of specific sperm before the treatment, the threshold value for comparison with this ratio may be, for example, within the range of 1.5 or more, within the range of 2.0 or more, within the range of 2.5 or more, within the range of 3.0 or more, within the range of 3.5 or more, or within the range of 4.0 or more.

[0085] The sperm population collected from a patient for this diagnostic support method is preferably a sperm population ejaculated extracorporeally from the patient (which may be a sperm population ejaculated extracorporeally by electrical stimulation or manual means), or a sperm population surgically removed from the patient's epididymis or testis. The patient from whom the sperm population is collected for this diagnostic support method may be a patient who has already been diagnosed with male infertility, or a patient who has not yet been diagnosed with male infertility.

[0086] As described above, this diagnostic assistance method can provide useful information regarding the cause and / or treatment policy of male infertility by using the above (a) and / or (b) related to the content of specific sperm in a sperm population as indicators.

[0087] Next, a specific example according to this embodiment will be described. [Example]

[0088] [Identification of Lypd4] The present inventors have been conducting research focusing on the role of glycosylphosphatidylinositol-anchored proteins (GPI-APs) in fertilization (Reference 1: Kondoh, G., et al. FEBS let., 1999; Reference 2: Kondoh, G., Nat Med., 2005; Watanabe and Kondoh, J. Cell Sci., 2011). Based on this, the present inventors hypothesized that the sperm-specific and most abundantly expressed GPI-APs are important for sperm function, and identified the corresponding proteins using the following method.

[0089] First, a sperm population was collected from mouse epididymis and disrupted in an aqueous solution containing 50 mM Tris and 150 mM NaCl (pH 8.0). This was centrifuged at 100,000 × g, and the precipitate was collected. This precipitate was dissolved in an aqueous solution containing 50 mM Tris and 1% Triton-X114 (pH 8.0). The resulting solution was heated at 37 °C for 10 minutes to allow for phase separation. The phase-separated solution was centrifuged at 20,000 × g for 10 minutes at 25 °C, the upper water-soluble fraction was discarded, and the lower oil-soluble fraction was collected. An equal volume of an aqueous solution containing 50 mM Tris and 150 mM NaCl (pH 8.0) was added to this oil-soluble fraction, and the mixture was heated at 37 °C for 10 minutes to allow for phase separation. The phase-separated solution was then centrifuged at 20,000 × g for 10 minutes at 25 °C. This procedure was repeated three times, and then an equal volume of an aqueous solution (pH 8.0) containing 50 mM Tris and 150 mM NaCl was added. PI-PLC was then added and the mixture was incubated at 37°C for 16 hours. The incubation solution was centrifuged at 20,000 × g for 10 minutes at 25°C, and the upper water-soluble fraction was collected. This water-soluble fraction was separated and concentrated using an ultrafiltration membrane with a 10 kDa cutoff, applied to SDS-PAGE, stained with CBB, and the bands were excised and collected. As a result, two major bands (molecular weights of 30 kDa and 35 kDa) were observed (results not shown).

[0090] Next, in-gel trypsin digestion was performed, and the resulting peptides were collected and applied to LC-MS / MS for protein identification. The 35 kDa band was identified as carbonic anhydrase 4 (CA4) (results not shown). Preliminary experiments showed that this GPI-AP is expressed systemically and delivered to sperm from the epididymal epithelium via exosomes. This molecule is also known to be involved in the production of bicarbonate ions, which are necessary for sperm activation.

[0091] On the other hand, the 30 kDa band was Ly6 / Plaur domain containing 4 (Lypd4) (results not shown). When the mRNA expression of this protein was examined in organs throughout the body, it was found to be testis-specific (results not shown).

[0092] [Identification of Lypd4-positive sperm] First, we generated fluorescently labeled monoclonal antibodies against Lypd4. Specifically, Ba / F3 cells expressing mouse and human Lypd4 were inoculated into C57BL / 6 mice multiple times, and serum was collected. This serum was used to immunostain sperm, Ba / F3 cells, and Ba / F3-Lypd4 cells to confirm the establishment of immunization. Spleen cells were collected from the immunized mice and fused with myeloma cells to generate hybridomas. The hybridomas were then subjected to limiting dilution to single cells, and after sufficient growth, the culture supernatants were collected and screened by immunostaining Ba / F3-Lypd4 cells. Ten anti-mouse antibody clones and four anti-human antibody clones were obtained. These clones were cultured in large quantities, purified on a protein G column, and a portion was fluorescently labeled with Alexa488. Sperm damaged by high-speed centrifugation were then immunostained with the purified antibodies. As a result, all 10 anti-mouse antibody clones were able to stain wild-type mouse sperm, but were unable to stain Lypd4 knockout mouse sperm.

[0093] Next, we collected live sperm populations from mouse epididymides and stained their membrane surfaces with fluorescently labeled antibodies. Immediately after collection, almost no staining was observed (results not shown). However, when the collected sperm populations were treated for 60 minutes with HTF, a sperm activation solution that can induce capacitation, a subpopulation of 10-30% of sperm expressed Lypd4 protein on the cell membrane surface.

[0094] Sperm from two epididymides were collected in 100 μL of HTF or calcium-free PBS and incubated at 37°C for 1 hour. Appropriate amounts of various antibodies and LiveDead reagent solution were then added and incubated for 30 minutes. 100 μL of sperm solution was then added to 10 mL of 0.2% BSA-supplemented PBS warmed to 37°C and incubated for 10 minutes. The mixture was then centrifuged at 800 rpm for 5 minutes at 10°C, and the supernatant was removed to an appropriate volume for FACS analysis using a flow cytometer (Cytoflex S, Beckman Coulter). Data analysis and FACS plots were generated using Flowjo (Tree Star Inc.).

[0095] As shown in Figure 1, when wild-type sperm (Lypd4+ / + in the figure) were incubated in sperm activation solution (HTF), 13.2% of the sperm were Lypd4-positive. When the sperm population was incubated in PBS, which does not activate the population, the percentage of Lypd4-positive sperm was only 1.79%, and in Lypd4-KO sperm (Lypd4- / - in the figure), it was less than 0.022%.

[0096] The percentage of Lypd4+ sperm in the wild-type sperm population (Lypd4+ / +) activated by incubation in sperm activation solution (HTF) was 13.2%, and the ratio of the percentage of Lypd4+ sperm in the activated sperm population to the percentage of Lypd4+ sperm in the unactivated sperm population was approximately 7.4 (=13.2 / 1.79).

[0097] [Motility and in vitro fertilization ability of Lypd4-positive sperm] The motility and in vitro fertilization capacity of Lypd4-positive sperm were examined. Specifically, sperm motility was assessed using a sperm motility analyzer (HTML-IVOS). Specifically, sperm populations were collected from mouse epididymides, cultured for 60 minutes, and then used for sperm motility analysis. Sperm motility was assessed based on motile, progressive motile, and sperm head amplitude (ALH), a marker of sperm capacitation. Here, motile refers to the percentage of sperm moving at 5 μm per second or more. Progressive motile refers to the percentage of sperm moving at 50 μm per second or more and with a straightness of 50% or more. ALH refers to the amplitude of sperm head swing when moving in a straight line.

[0098] Sperm populations were collected from the epididymis of male mice of each genotype and incubated in HTF for 90 minutes. Oocytes were collected from superovulated female mice and inseminated in HTF containing the activated sperm population. After 16 hours of incubation, the numbers of unfertilized and two-cell stage eggs were counted, and the fertilization rate was calculated. As a result, the motility and in vitro fertilization ability of Lypd4-positive sperm were both nearly normal (results not shown).

[0099] [Correlation of Lypd4 with CD55, ICOS, and CCR3] To characterize Lypd4-positive sperm, we performed FACS analysis using fluorescently labeled monoclonal antibodies (LEGENDScreen Mouse PE Kit, BioLegend) against 260 cell surface antigens. Sperm populations were collected from mouse epididymides and incubated in sperm activation solution (HTF) for 1 hour. Live, activated sperm populations were then stained for Lypd4 and then individually stained with 265 antibodies and subjected to FACS analysis.

[0100] As shown in Figure 2, the GPI-anchored protein CD55 and the membrane-bound proteins ICOS and CCR3 appeared on the sperm plasma membrane in correlation with the appearance of Lypd4-positive sperm. Among them, CD55 showed a particularly strong correlation with Lypd4. Furthermore, CD147 was expressed on the sperm plasma membrane upon sperm activation, regardless of the presence or absence of Lypd4 expression.

[0101] [Conditions for the appearance of marker antigen-positive sperm] We investigated the conditions under which Lypd4-positive sperm appear. First, we investigated the removal of essential components for activation, such as BSA and Ca, from the sperm activation solution (HTF). 2+ The sperm population collected from the epididymis of mice was treated with BSA, Ca, and bicarbonate ions. 2+ The sperm were incubated in a modified sperm activation solution (modified HTF) that had been deprived of either ions or bicarbonate ions, and the expression of Lypd4 on the cell membrane surface of the sperm was analyzed by FACS.

[0102] As a result, as shown in Figure 3, in the sperm population incubated in modified HTF (in which either BSA or bicarbonate ions that induce capacitation have been removed) ("(-)BSA" and "(-)NaHCO3" in the figure), an increase in the content of Lypd4-positive sperm was observed, similar to that observed in the population incubated in unmodified HTF ("HTF" in the figure). In contrast, in the sperm population incubated in modified HTF (in which either BSA or bicarbonate ions that induce capacitation have been removed), an increase in the content of Lypd4-positive sperm was observed. 2+ In the sperm population incubated with ion-depleted modified HTF ("(-)CaCl2" in the figure), the content of Lypd4-positive sperm did not increase.

[0103] Specifically, the content of Lypd4-positive sperm in the unactivated sperm population maintained in PBS was 10.0%, whereas the content of Lypd4-positive sperm in the sperm population activated by maintenance in sperm activation solution (HTF) was 21.8%. Furthermore, the content of Lypd4-positive sperm in the sperm population maintained in modified HTF without BSA and in modified HTF without bicarbonate ions was 25.1% and 21.2%, respectively.

[0104] In contrast, Ca 2+ The content of Lypd4-positive sperm in the sperm population maintained in the ion-depleted modified HTF was 9.63%, which was not significantly different from that in the sperm population maintained in PBS.

[0105] Furthermore, the ratio of the content of Lypd4-positive sperm in the sperm population after activation with HTF to the content of Lypd4-positive sperm in the sperm population before activation was 2.18 (=21.8÷10.0).

[0106] A similar phenomenon was observed for CD55, whose expression correlates with Lypd4. Specifically, when we performed similar analyses on CD55 and CD147, the percentage of CD55-positive sperm showed almost the same behavior as that of Lypd4-positive sperm, but the percentage of CD147-positive sperm tended to decrease when BSA or bicarbonate ions were removed (results not shown).

[0107] Furthermore, detailed analysis by immunoelectron microscopy revealed that among the sperm populations maintained in HTF, there were indeed sperm in which Lypd4 and CD55 were localized on the cell membrane surface, and it was confirmed that such sperm had not undergone the acrosome reaction (results not shown).

[0108] These results suggest that the increase in the percentage of marker antigen-positive spermatozoa, in which Lypd4 and CD55 appeared on the cell membrane surface, is due to Ca 2+ Although it is ion-dependent, it is an event independent of capacitation and the acrosome reaction, which are important for sperm activation.

[0109] We further investigated whether marker antigen-positive sperm also appeared in the sperm population ejaculated into the female. Normal male and female mice were housed together, and sperm populations were collected from the uterus and oviduct after mating was confirmed and subjected to FACS analysis. Lypd4- and CD55-positive sperm began to appear approximately 90 minutes after mating, and approximately 20% of the sperm were Lypd4- and CD55-positive by 180 minutes (results not shown). Lypd4- and CD55-positive sperm were also detected in the oviduct around this time (results not shown). These findings suggest that marker antigen-positive sperm appear as a subpopulation in vivo and ultimately reach and function in the oviduct, where fertilization occurs.

[0110] [Calcium ion concentration in calcium treatment] We investigated the calcium concentration required to increase the percentage of Lypd4 / CD55-positive sperm (sperm expressing both Lypd4 and CD55 on their cell membrane surface). Sperm collected from mouse epididymides were incubated for 1 hour at 37°C in HTF containing calcium ions at concentrations of 0 μM (no added), 2 μM, 10 μM, 50 μM, 260 μM, 1.3 mM, or 6.5 mM.

[0111] Next, appropriate amounts of Alexa-488 fluorescently labeled anti-Lypd4 antibody and PE fluorescently labeled anti-CD55 antibody were added to each HTF containing the incubated sperm population and incubated for 30 minutes. Then, 100 μL of HTF containing the sperm population was added to 10 mL of PBS containing 0.2% BSA preheated at 37°C and incubated for 10 minutes. The incubated solution was then centrifuged at 800 rpm for 5 minutes at 10°C, and an appropriate amount of the supernatant was removed and subjected to FACS analysis using a flow cytometer (Cytoflex S, Beckman Coulter).

[0112] As a result, the content of Lypd4 / CD55-positive sperm in sperm populations incubated in HTF containing no calcium ions (calcium ion concentration 0 μM) was 10%, whereas the content of Lypd4 / CD55-positive sperm in sperm populations incubated in HTF containing calcium ions at concentrations of 50 μM or more (specifically, 50 μM to 6.5 mM) increased to more than 34%.

[0113] Specifically, the percentage of Lypd4 / CD55-positive sperm was 34% when the calcium ion concentration in HTF was 50 μM, 53% when it was 260 μM, 57% when it was 1.3 mM, and 64% when it was 6.5 mM. That is, the ratio of the percentage of Lypd4 / CD55-positive sperm in the calcium-treated sperm population to the percentage of Lypd4 / CD55-positive sperm in the untreated sperm population was 3.4 when the calcium ion concentration in HTF was 50 μM, 5.3 when it was 260 μM, 5.7 when it was 1.3 mM, and 6.4 when it was 6.5 mM.

[0114] [Improvement of fertilization rate in artificial insemination by removing marker antigen-positive sperm] Artificial insemination was performed to examine the in vivo fertilization capacity of the sperm population. First, Lypd4-positive sperm were removed from a mouse sperm population activated with HTF (32.5% Lypd4-positive sperm) using an anti-Lypd4 antibody, resulting in a sperm population with a Lypd4-positive sperm content of 3.3%. Specifically, biotin-labeled anti-Lypd4 antibody was first mixed with the activated sperm population, and then the resulting sperm population was mixed with magnetic beads coupled with anti-biotin antibody and applied to a magnetic column. As a result, Lypd4-positive sperm were removed with an efficiency of over 90% (96.7% to be exact) (results not shown).

[0115] The same number of sperm were then injected into the uterus of superovulated female mice with the same number of sperm from the activated sperm population that had been depleted of Lypd4-positive sperm and the sperm population that had not been depleted of Lypd4-positive sperm (i.e., the sperm population that had remained activated with HTF).The next day, all eggs were collected from the oviducts and the fertilization rate was calculated.

[0116] As a result, as shown in Figure 4, the fertilization rate when a sperm population from which Lypd4-positive sperm had been removed ("(+)" in the figure) was administered was approximately twice as high as when a sperm population from which Lypd4-positive sperm had not been removed ("(-)" in the figure).

[0117] Based on the results of this artificial insemination test and the above-mentioned motility and in vitro fertilization tests, Lypd4-positive sperm were thought to potentially have sufficient fertilization ability, but to negatively regulate fertilization in vivo. [Example]

[0118] [Appearance of CD55-positive sperm] To investigate whether the emergence of the above-mentioned subpopulations upon sperm activation is universal across mammals, we performed a surface antigen screening of monkey sperm. Here, we used sperm from Japanese macaques, a species of macaque monkey more closely related to humans. Because macaque proteins generally share high homology with human proteins, anti-human antibodies can be used. Therefore, we first generated an anti-human Lypd4 monoclonal antibody that reacts with monkey Lypd4. However, unlike mouse sperm, we found that activated monkey sperm did not express Lypd4 at all (results not shown).

[0119] Therefore, we analyzed CD55, which has a strong correlation with Lypd4 expression in mouse sperm. Sperm populations were collected from the epididymis of anesthetized Japanese macaques and incubated for 2 hours in either HTF, which activates sperm populations, or PBS, which does not activate sperm populations, and then CD55 expression was examined.

[0120] As shown in Figures 5A and 5B, the percentage of CD55-positive sperm increased significantly in the sperm population activated by incubation with HTF. That is, the percentage of CD55-positive sperm in the non-activated sperm population ("PBS" in the figure) was 18.8%, while the percentage of CD55-positive sperm in the activated sperm population ("HTF" in the figure) was 78.8%.

[0121] The ratio of CD55-positive sperm in the activated sperm population to the unactivated sperm population was approximately 4.2 (=78.8 ÷ 18.8). This trend was observed in all four individuals examined, and a subpopulation of activated spermatozoa expressed CD55 protein on the cell membrane surface at a rate of 60-80%.

[0122] To clarify the differences between CD55-positive and CD55-negative sperm, we performed FACS analysis using fluorescently labeled monoclonal antibodies (LEGENDScreen Human PE Kit, BioLegend) against 370 cell surface antigens, similar to the mouse model. Sperm populations were collected from monkey epididymides and incubated in HTF or PBS for 2 hours. Live sperm populations were then stained with anti-CD55 antibodies, then individually stained with 370 antibodies, and subjected to FACS analysis (results not shown).

[0123] As a result, as shown in Figure 6, the expression of CCR3, CCR8, CD215 (IL15RA), and OPRD1 on the sperm cell membrane surface correlated with the expression of CD55.

[0124] These results revealed that monkey CD55-positive spermatozoa show differential expression of multiple cell membrane surface proteins, forming a subpopulation distinct from CD55-negative spermatozoa.

[0125] [Removal of CD55-positive sperm improves sperm penetration rate in hamster tests] To perform functional analysis of monkey CD55-positive sperm, a hamster test was performed using a sperm population with a CD55-positive sperm content of 1.66%, which was obtained by removing CD55-positive sperm from a sperm population activated with HTF (CD55-positive sperm content was 56.1%).

[0126] First, we reacted monkey sperm populations with five antibodies: PE-Cy7-labeled anti-CD55 antibody, PE-labeled anti-CCR3 antibody, anti-CCR8 antibody, anti-CD215 (IL15RA) antibody, and anti-OPRD1 antibody. As mentioned above, CCR3, CCR8, CD215 (IL15RA), and OPRD1 are molecules whose expression on the sperm plasma membrane correlates with that of CD55. In preliminary studies, we found that the anti-CD55 antibody alone was insufficient to remove CD55-positive sperm. Therefore, in this experiment, we used a total of five antibodies to remove CD55-positive sperm.

[0127] Next, the sperm population was reacted with anti-PE antibodies coupled to magnetic beads and then applied to a magnetic column. As shown in Figure 7A, CD55 expression correlated with the expression of PE-tagged cell membrane proteins, and CD55-positive sperm were removed with an efficiency of over 95%. In Figure 7A, "(-)" indicates the results of analyzing the sperm population before removal of CD55-positive sperm, while "(+)" indicates the results of analyzing the sperm population after removal of CD55-positive sperm.

[0128] Next, a sperm population from which CD55-positive sperm had been removed and a control sperm population from which CD55-positive sperm had not been removed were added to unfertilized hamster eggs from which the zona pellucida had been removed. Because sperm that penetrated the egg membrane have enlarged nuclei, the percentage of hamster eggs penetrated by sperm (sperm penetration rate) was calculated by microscopic observation. As shown in Figure 7B, the sperm penetration rate when using the sperm population from which CD55-positive sperm had been removed ("+" in the figure) was significantly higher than when using the sperm population from which CD55-positive sperm had not been removed ("-" in the figure).

[0129] These results confirmed that CD55-positive sperm negatively regulate fertilization efficiency in primates, and that fertilization rates can be improved by removing CD55-positive sperm and using a sperm population with a reduced content of CD55-positive sperm.

Claims

1. a first step of preparing a first sperm population that includes a subpopulation of specific sperm whose concentration in the sperm population is reduced to improve the fertilization rate of the sperm population; a second step of removing the specific sperm from the first sperm population to obtain a second sperm population having a reduced content of the specific sperm compared to the first sperm population; Including, Method for producing selected sperm populations.

2. The specific sperm are a subpopulation that express one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface. The method for producing a selected sperm population according to claim 1.

3. 1. A method for producing a population of selected sperm for use in in vitro fertilization or artificial insemination, A method for producing a selected sperm population according to claim 1 or 2.

4. The following (a) and / or (b): (a) the prevalence in a sperm population collected from a patient of a particular sperm subpopulation whose prevalence in the sperm population is reduced to improve the fertilization rate of the sperm population; (b) when a sperm population collected from a patient is subjected to activation treatment or calcium treatment, the change in the content of the specific sperm in the sperm population before and after the activation treatment or calcium treatment; Using this as an indicator, A diagnostic aid for male infertility.

5. The specific sperm are a subpopulation that express one or more marker antigens selected from the group consisting of CD55, CCR3, CCR8, CD215, OPRD1, Lypd4, and ICOS on the cell membrane surface. The method for assisting in the diagnosis of male infertility according to claim 4.

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