CatSper activity test for determining male sterility

Abstract

The present invention relates to an in vitro method for determining or diagnosing infertility in a male subject, the method comprising the steps of (a) contacting a sperm sample obtained from the subject with a calcium-free test solution, (b) detecting the motility of sperm in the sample under the test conditions of step (a), and (c) determining infertility in the male subject based on the motility detected in step (b).The present invention also relates to a kit for use in the method for determining or diagnosing infertility in a male subject, the kit comprising (a) a calcium-free test solution and (b) a calcium-containing control solution.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to an in vitro method for determining or diagnosing infertility in a male subject, the method comprising the steps of (a) contacting a sperm sample obtained from the subject with a calcium-free test liquid, (b) detecting the motility of sperm in the sample under the test conditions of step (a), and (c) determining infertility of the male subject based on the motility detected in step (b).The present invention further relates to a kit for use in the method for determining or diagnosing infertility in a male subject, the kit comprising (a) a calcium-free test liquid, and (b) a calcium-containing control liquid. [Background technology]

[0002] 2. Background of the Invention Unintentional childlessness is a serious problem that is caused by male infertility in about 40-50% of cases. However, both infertility itself and its underlying causes are often unreliable to diagnose. In about 30% of male patients, they remain unknown ("idiopathic infertility" or "unexplained sperm dysfunction"), due to the lack of adequate and timely diagnostic methods.

[0003] Human sperm have a complex repertoire of behaviors to access and fertilize eggs at the end of the fallopian tube. Oocytes and the cells around them secrete, for example, hormones to which sperm respond. Sperm can adjust their swimming direction along hormone gradients and pursue eggs by chemotaxis (Ralt et al., 1991, Proc. Natl. Acad. Sci. USA, 88; Giojalas et al., 1998, Int. J. Androl., 21; Eisenbach et al, 2006, Nat Rev Mol Cell Biol 7(4):276-285; Publicover et al. 2008, Front Biosci 13: 5623-5637). The egg cell is surrounded by a protective egg shell (zona pellucida) and a dense layer of cumulus cells - a mechanical barrier for sperm. Thus, to penetrate the cumulus cell layer, sperm change to a so-called "hyperactive" swimming behavior: their usual symmetric flagellar strike to cover long distances is replaced by an asymmetric, low-frequency pattern, which provides the mechanical force required to penetrate the cumulus cell layer (Suarez et al., 1991, Biol Reprod, vol. 44; Ren et al., 2001, Nature 413, 603-609; Quill et al., 2003, Proc Natl Acad Sci USA, vol. 100). The key to overcoming the zona pellucida is acrosomal exocytosis in addition to hyperactivation.That is, upon contact with the zona pellucida, hydrolases stored in the acrosome are released (Tulsiani et al., 1998, Experimental Cell Research 240 (Non-Patent Document 8); Gupta et al., 2011, American Journal of Reproductive Immunology 2011 (Non-Patent Document 9)), which digest the glycoproteins of the zona pellucida. Only after crossing the zona pellucida can the sperm fuse with and fertilize the egg (Wassarman et al., 2001, Nat. Cell Biol. 3 (Non-Patent Document 10)).

[0004] Hyperactive swimming, acrosomal exocytosis, and capacitation (the maturation process in the female reproductive tract that is essential for sperm to be able to fertilize) are driven by intracellular Ca 2+ (Publicover et al., 2007, Nat Cell Biol, vol. 9); successful navigation of sperm through the oviduct requires the incorporation of [Ca 2+ ] i Changes in Ca2+ levels are also thought to be involved (Breitbart, 2002, Mol. Cell Endocrinol. 187: 139-144 (Non-Patent Document 12); Publicover et al., 2008, Front Biosci 13 (Non-Patent Document 4)). 2+ The trigger for this change is the introduction of Ca from the outside into the sperm. 2+ In human sperm, Ca influx is crucial. 2+ The influx occurs via sperm-specific Ca2+ receptors present in the plasma membrane of the sperm flagella. 2+ Channel CatSper( cat ion channel of sperm). CatSper channels are essential for sperm function and fertilization. Mice lacking CatSper are infertile. CatSper-deficient sperm cannot hyperactivate and therefore cannot cross the eggshell or cumulus cell layer (Ren et al., 2001, Nature 413; Carlson et al., 2003, Proc Natl Acad Sci USA, 100) and reach the egg. However, CatSper is not significantly required for sperm production and their basic motility.

[0005] Thus, despite the essentiality of CatSper function for successful fertilization, there is currently no diagnostic method in the repertoire of andrology laboratories that can detect the absence or functional defects of CatSper channels. Sperm function is already measured based on easily detectable parameters under a microscope, such as sperm volume and sperm showing progressive motility or morphological changes, but this method can only diagnose easily detectable changes in sperm motility. Moreover, until now, sperm CatSper activity was only considered safe by laborious and expensive fluorescent optical or electrophysiological measurements, which did not allow these measurements to be integrated into the routine diagnostic process in andrology.

[0006] However, this contrasts with the high usefulness of diagnostic information on CatSper status for patients who want children. Indeed, in the absence or presence of severe functional defects of CatSper channels, three of the four standard techniques of reproductive medicine are known to fail from the outset: cycle optimization (CO; VZO in German), intrauterine insemination (IUI) and classical in vitro fertilization (IVF). Spermatozoa without active CatSper channels can only be successfully fertilized in the context of intracytoplasmic sperm injection (ICSI), which is the most expensive method and is therefore often only performed at the end of a fertility treatment history.

[0007] When the causes of male infertility remain unknown even after all currently available diagnostic methods, no evidence-based infertility treatment decisions can be made. If evidence-based decisions are replaced by the principle of "trial and error", the treatment becomes a game of chance for the couples involved.

[0008] Therefore, there is a need in the art to fill the diagnostic gap for idiopathic male infertility, thereby providing a safe, reliable and easy-to-handle method. In short, there is a need in the art to provide new means and methods that help diagnose or detect infertility in male subjects that are not characterized by reduced sperm count, altered morphology and / or significantly atypical swimming behavior, thus enabling evidence-based assisted reproductive therapy. Therefore, the technical problem underlying the present application is to meet these needs. This technical problem is solved by providing the embodiments reflected in the following claims, described in the detailed description and illustrated in the examples and drawings. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Ralt et al., 1991, Proc. Natl. Acad. Sci. USA, 88 [Non-Patent Document 2] Giojalas et al., 1998, Int. J. Androl., 21 [Non-Patent Document 3] Eisenbach et al, 2006, Nat Rev Mol Cell Biol 7(4):276-285 [Non-Patent Document 4] Publicover et al. 2008, Front Biosci 13: 5623-5637 [Non-Patent Document 5] Suarez et al., 1991, Biol Reprod, vol. 44 [Non-Patent Document 6] Ren et al., 2001, Nature 413, 603-609 [Non-Patent Document 7] Quill et al., 2003, Proc Natl Acad Sci USA, vol. 100 [Non-Patent Document 8] Tulsiani et al., 1998, Experimental Cell Research 240 [Non-Patent Document 9] Gupta et al., 2011, American Journal of Reproductive Immunology 2011 [Non-Patent Document 10] Wassarman et al., 2001, Nat. Cell Biol. 3 [Non-Patent Document 11] Publicover et al., 2007, Nat Cell Biol, vol. 9 [Non-Patent Document 12] Breitbart, 2002, Mol. Cell Endocrinol. 187: 139-144 [Non-Patent Document 13] Carlson et al., 2003, Proc Natl Acad Sci USA, 100 [Summary of the Invention]

[0010] The inventors have developed a novel, fundamentally simple assay, compared to fluorescent optical or electrophysiological methods, to screen sperm for functional CatSper channels; this assay can be used for reliable detection or diagnosis of infertility in male subjects. Measuring the CatSper channel activity in sperm obtained from male subjects thereby allows, for example, the attending physician of a couple hoping to have a child to make evidence-based treatment decisions. In fact, if a lack or severe impairment of CatSper channel activity is revealed despite an otherwise unremarkable spermiogram, this is an exclusion criterion for three of the four common reproductive medicine treatment strategies (CO, IUI, IVF).

[0011] In particular, the inventors have found a safe, reliable and easy-to-handle in vitro test assay for determining or diagnosing infertility in male subjects by detecting sperm motility in a calcium-free environment. The simplicity of the assay of the present invention is evident in that the assay can be performed directly using native ejaculate, without the need for laborious prior purification of sperm. Furthermore, no specific device is required to determine or diagnose infertility in male subjects according to the present invention. Finally, the amount of work required to analyze the motility of sperm itself is very limited. Therefore, by utilizing the assay of the present invention, it is possible to obtain the results or results very quickly, whether or not the male subject is actually determined or diagnosed as infertile.

[0012] In summary, the present invention is directed to detecting sperm motility in a calcium-free environment to determine CatSper channel activity and diagnose male infertility.

[0013] Thus, in a first aspect, the present invention relates to an in vitro method for determining or diagnosing infertility in a male subject, comprising the steps of: a) contacting a sperm sample obtained from said subject with a calcium-free test solution; b) detecting the motility of sperm in the sample under the test conditions of step (a); and c) determining the infertility of the male subject based on the motility of the sperm detected in step (b); The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0014] The invention may also include the methods described elsewhere herein, wherein the absence of a significant change in the amount of motile sperm under the test conditions of step (b) is indicative of infertility in the subject.

[0015] Further contemplated herein are the methods described elsewhere herein, wherein the calcium-free test solution contains less than about 20 nM free calcium.

[0016] The invention may also include the methods described elsewhere herein, wherein the calcium-free test solution comprises at least one agent that reduces free calcium concentration.

[0017] The present invention also relates to at least one agent that reduces free calcium concentration, said agent being Ca 2+ Preferably, the method includes at least one agent that reduces free calcium concentration, such as Ca 2+ The chemicals that bind to the are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca. 2+ Any one of the indicator dyes, more preferably EGTA.

[0018] Also included in the present invention are methods described elsewhere herein, wherein step (a) further comprises administering at least one CatSper channel activation stimulus.

[0019] Additionally, the invention may encompass the methods described elsewhere herein, wherein an activating stimulus is added before, simultaneously with, or after adding the calcium-free test fluid to the sperm sample.

[0020] Also contemplated herein is the method described elsewhere herein, wherein CatSper activation stimulus is any substance and / or condition that enhances CatSper channel activity.Preferably, the substance that enhances CatSper channel activity is a ligand.More preferably, the ligand is any one of steroids, prostaglandins, synthetic or natural chemicals that mimic the action of steroids or prostaglandins, serum albumin, zona pellucida protein, or combinations thereof.Most preferably, the steroid is progesterone.

[0021] Additionally, the invention may also include the methods described elsewhere herein, wherein the conditions that enhance CatSper channel activity include increasing intracellular pH and / or depolarizing the membrane potential.

[0022] The present invention may also envisage the method as defined elsewhere herein, wherein the sperm sample is contacted with the test fluid of step (a) for at least about 5 minutes.

[0023] The invention may also encompass the methods described elsewhere herein, where motile sperm are characterized by some movement compared to immotile sperm.

[0024] Also included herein may be the methods described elsewhere herein, in which sperm motility is measured using a light microscope.

[0025] Additionally, the invention may include the methods described elsewhere herein, wherein measuring the amount of motile sperm comprises counting at least about 10 sperm under the test conditions of step (b).

[0026] Furthermore, in preferred embodiments of the methods described elsewhere herein, the method may further comprise comparing the motility of spermatozoa detected in step (b) with the motility of spermatozoa from the subject detected under control conditions comprising a calcium-containing fluid. Preferably, a comparison of motility detected under test and control conditions is indicative of infertility in the subject.

[0027] Also included in the present invention, and as described elsewhere herein for certain embodiments of the invention, the comparison of sperm motility under test and control conditions includes determining a CatSper activity index, which comprises the following steps: a) obtaining percentage values ​​of motile sperm i) under test conditions and ii) under control conditions; b) subtracting the percentage value of motile sperm under the test conditions in i) from the percentage value of motile sperm under the control conditions in ii), thereby obtaining a subtraction score; c) determining the ratio of the subtraction score to the percentage value of motile sperm under the control condition of ii); d) multiplying the ratio of step c) by several hundred to obtain the CatSper activity index. The present invention relates to a method for producing a semiconductor device comprising the steps of:

[0028] The present invention may also envisage in this particular preferred embodiment further comprising comparing said index with a predetermined reference value.Preferably, if said index is below the reference value, it indicates infertility of said subject.The reference value obtained by this particular embodiment of the present invention may be about 20%.

[0029] The present invention may also include the methods described elsewhere herein, wherein the male subject is a vertebrate. Preferably, the vertebrate used as the male subject in the methods of the present invention is a mammal. More preferably, the mammal is any of a human, cow, buffalo, horse, donkey, camel, sheep, goat, pig, dog, cat, rat, or mouse, preferably a human.

[0030] Moreover, according to another aspect, the present invention relates to a kit for use in a method for determining or diagnosing infertility in a male subject, comprising (a) a calcium-free test solution, and (b) a calcium-containing control solution.

[0031] Furthermore, it may be envisaged that the kit for said use further comprises instructions for use and / or an imprint.

[0032] The present invention may also include a kit for the above-mentioned use, further comprising a validation solution containing a CatSper inhibitor.

[0033] The invention may also include kits for the above uses described elsewhere herein, in which the calcium-free test liquid comprises at least one agent that reduces free calcium concentration.

[0034] Preferably, the calcium-free test liquid contains at least one agent that reduces the free calcium concentration. 2+ More preferably, it is a chemical that binds to Ca. 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca. 2+ Any one of the indicator dyes, most preferably EGTA.

[0035] The present invention may also contemplate a kit for the above uses described elsewhere herein, wherein the calcium-free test solution and / or the calcium-containing control solution comprises at least one CatSper channel activating stimulus.

[0036] Preferably, the activation stimulus contained in calcium-free test solution and / or calcium-containing control solution is any one of ligand, weak base, buffer containing elevated potassium concentration, potassium or chloride ion channel inhibitor, or sodium ionophore.More preferably, said ligand is any one of steroid, prostaglandin, synthetic or natural chemical that mimics the action of steroid or prostaglandin, serum albumin, zona pellucida protein, or combinations thereof.Most preferably, said steroid is progesterone. [The present invention 1001] 1. An in vitro method for determining or diagnosing infertility in a male subject, comprising: a) contacting a sperm sample obtained from said subject with a calcium-free test solution; b) detecting the motility of sperm in the sample under the test conditions of step (a); and c) determining the infertility of said male subject based on said motility detected in step (b). A method comprising: [The present invention 1002] The method of claim 1001, wherein the absence of a significant change in the amount of motile sperm under the test conditions of step (b) is indicative of infertility in the subject. [The present invention 1003] The method of any one of claims 1001 to 1002, wherein the calcium-free test solution contains less than about 20 nM free calcium. [The present invention 1004] Any of the methods of the invention, wherein the calcium-free test solution comprises at least one agent that reduces free calcium concentration. [The present invention 1005] The at least one agent that reduces free calcium concentration is Ca 2+ The method of the present invention 1004, wherein the chemical compound binds to [The present invention 1006] Ca 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca 2+ The method of any one of claims 10 to 5, wherein the indicator dye is EGTA. [The present invention 1007] Any of the aforementioned methods of the invention, wherein step (a) further comprises administering at least one CatSper channel activating stimulus. [The present invention 1008] 1007. The method of claim 1007, wherein the activation stimulus is added before, simultaneously with, or after the calcium-free test fluid is added to the sperm sample. [The present invention 1009] The method of any one of claims 1007 and 1008, wherein the CatSper activating stimulus is any substance and / or condition that enhances the activity of the CatSper channel. [The present invention 1010] The method of claim 10, wherein the substance that enhances the activity of the CatSper channel is a ligand. [The present invention 1011] The method of any one of claims 10 to 12, wherein said ligand is any one of a steroid, a prostaglandin, a synthetic or natural chemical that mimics the action of a steroid or a prostaglandin, serum albumin, a zona pellucida protein, or a combination thereof. [The present invention 1012] The method of any one of claims 1 to 11, wherein said steroid is progesterone. [The present invention 1013] The method of claim 1009, wherein the conditions that enhance the activity of the CatSper channel comprise increasing intracellular pH and / or depolarizing the membrane potential. [The present invention 1014] The method of any of claims 1007 to 1013, wherein said sperm sample is contacted with said test solution of step (a) for at least about 5 minutes. [The present invention 1015] Any of the methods of the present invention 1002 to 1014, wherein the motile sperm are characterized by some movement compared to immotile sperm. [The present invention 1016] Any of the methods of the invention, wherein the motility of the sperm is measured using a light microscope. [The present invention 1017] Any of the methods of the present invention, wherein measuring the amount of motile sperm comprises counting at least about 10 sperm under the test conditions of step (b). [The present invention 1018] Any of the methods of the invention, further comprising the step of comparing the motility of sperm detected in step (b) with the motility of sperm from the subject detected under control conditions comprising a calcium-containing fluid. [The present invention 1019] The method of claim 1018, wherein a comparison of the motility detected under said test conditions and said control conditions is indicative of infertility in said subject. [The present invention 1020] Comparing the sperm motility under the test conditions and the control conditions comprises determining a CatSper activity index; a) obtaining a percentage value of motile sperm i) under said test conditions and ii) under said control conditions; b) subtracting the percentage value of motile sperm under said test conditions in i) from the percentage value of motile sperm under said control conditions in ii), thereby obtaining a subtraction score; c) determining the ratio of said subtraction score to the percentage value of motile sperm under said control conditions of ii); d) multiplying the ratio of step c) by a factor of hundreds to obtain the CatSper activity index. The method of the present invention 1018 or 1019, comprising: [The present invention 1021] The method of claim 1020, further comprising the step of comparing said index with a predetermined reference value. [The present invention 1022] The method of claim 1021, wherein the index being below the reference value indicates infertility of the subject. [The present invention 1023] The method of any one of claims 1021 to 1022, wherein said reference value is about 20%. [The present invention 1024] Any of the methods of the present invention, wherein said male subject is a vertebrate. [The present invention 1025] The method of claim 1024, wherein the vertebrate is a mammal. [The present invention 1026] The method of claim 1025, wherein said mammal is any of a human, cow, buffalo, horse, donkey, camel, sheep, goat, pig, dog, cat, rat, or mouse, preferably a human. [The present invention 1027] 1. A kit for use in a method for determining or diagnosing infertility in a male subject, comprising: (a) a calcium-free test solution, and (b) Calcium-containing control solution Including the kit. [The present invention 1028] A kit for use of the present invention 1027 further comprising instructions for use and / or an imprint. [The present invention 1029] A kit for use of the present invention 1027 or 1028, further comprising a verification solution containing a CatSper inhibitor. [The present invention 1030] A kit for use according to any one of claims 1027 to 1029, wherein the calcium-free test liquid comprises at least one agent that reduces free calcium concentration. [The present invention 1031] The at least one agent that reduces free calcium concentration is Ca 2+ A kit for use in the present invention 1030, which is a chemical compound that binds to. [The present invention 1032] Ca 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca 2+ A kit for the use of the present invention 1031, which is any one of the indicator dyes, preferably EGTA. [The present invention 1033] A kit for use according to any one of claims 1027 to 1032, wherein the calcium-free test solution and / or the calcium-containing control solution comprises at least one CatSper channel activating stimulus. [The present invention 1034] The kit for use of the present invention 1033, wherein the activating stimulus is any one of a ligand, a weak base, a buffer containing an elevated potassium concentration, a potassium or chloride ion channel inhibitor, or a sodium ionophore. [The present invention 1035] The kit for use of the present invention 1034, wherein said ligand is any one of a steroid, a prostaglandin, a synthetic or natural chemical that mimics the action of a steroid or a prostaglandin, serum albumin, a zona pellucida protein, or a combination thereof. [The present invention 1036] A kit for use of the present invention 1035, wherein the steroid is progesterone. [Brief description of the drawings]

[0037] [Figure 1A]Figure 1: Successive frames from a video recording of a sperm suspension after incubation (15 min). Control solution (A), Ca2+-free test solution in the presence of progesterone (10 µM) depicting non-motile sperm from a healthy donor with active CatSper (B), Ca2+-free test solution in the presence of progesterone (10 µM) and the CatSper blocker RU1968F1 (30 µM) depicting motile sperm from a healthy donor in which CatSper activity was pharmacologically blocked (C). Recording times are coded in greyscale: t = 0 s, light grey; 1 s, medium grey; 2 s, dark grey. Non-motile sperm appear white in the superposition of the three grey intensities (see (B); except for motile sperm, which are highlighted with circles). [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 2A] Figure 2: Consecutive frames from a video recording of sperm suspension after incubation (15 min). Control solution (A), Ca2+-free test solution in the presence of progesterone (10 μM) depicting non-motile sperm from a healthy donor with active CatSper (B), Ca2+-free test solution in the presence of progesterone (10 μM) and CatSper blocker RU1968F1 (30 μM) depicting motile sperm from a healthy donor in which CatSper activity was pharmacologically blocked (C). Recording times correspond to t = 0 s (left); 2 s (middle); 4 s (right). [Figure 2B] See legend to Figure 2A. [Figure 2C] See legend to Figure 2A. [Diagram 3]Figure 3: Motility of spermatozoa after incubation in the test solutions. Change in relative motility in a population of spermatozoa from healthy donors after incubation (15 min) in the different test solutions (calcium-containing control solution; calcium-free test solution + progesterone; calcium-free test solution + progesterone + CatSper inhibitor RU1968F1) compared to the motility of spermatozoa in calcium-containing control solution (set to 100%) approximately 30 s after the start of incubation. Mean values ​​and standard deviations are shown. Control solution = Ca2+, n = 8; Test solution = Ca2+-free + progesterone (10 μM), n = 8; Ca2+-free + progesterone (10 μM) + CatSper inhibitor RU1968F1 (30 μM), n = 6. [Figure 4] Figure 4: Characterization of CATSPER2-deficient human sperm by CatSper activity index. CatSper activity index of n=467 patients. Triangles in the frame represent n=5 patients with loss of CatSper function. [Diagram 5] Figure 5: Electrophysiological recordings of CATSPER2-deficient human sperm confirmed by the CatSper activity index. (A) Representative monovalent CatSper currents and corresponding current-voltage relationships (right) in CATSPER2- / - sperm (patients A-E) and sperm from healthy donors (controls). Membrane voltage was stepped from -100 mV to +100 mV in 10 mV increments from a holding potential of -80 mV. (B) Outward and inward current amplitudes (mean ± SD) at +100 mV and -100 mV, respectively, in CATSPER2- / - sperm (patients A-E) and sperm from healthy donors (controls). [Figure 6]Figure 6: Array comparative genomic hybridization analysis (array CGH) of CATSPER2-deficient patients confirmed by the CatSper Activity Index. Array CGH analysis of DNA copy number variants (CNVs) in a healthy donor (WT) and five patients (CATSPER2- / - 1-5) confirmed by the CatSper Activity Index. Black boxes represent the fluorescence intensity ratio (log2) upon co-hybridization of fluorescently labeled genomic DNA fragments of healthy donors or patients and controls (reference). Gene dosage variation is indicated by a fluorescence ratio ≠ 0. A ratio of <-1.5 indicates homozygous deletion. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention In order to overcome some of the shortcomings of the means previously described in the prior art of male infertility, the present inventors provide herein a promising new method and kit for determining or diagnosing the infertility of a male subject, based on detecting the motility of sperm obtained from said subject under calcium-free test conditions.In particular, the present inventors have surprisingly found that male infertility can be reliably detected or diagnosed by the method of the present invention.

[0039] In summary, the present invention opens up new avenues for determining or diagnosing infertility in male subjects based on detecting the motility of sperm obtained from said subjects under calcium-free test conditions.

[0040] As mentioned above, the present invention relates to an in vitro method for determining or diagnosing infertility in a male subject, the method comprising the steps of: (a) contacting a sperm sample obtained from the subject with a calcium-free test liquid; (b) detecting the motility of sperm in the sample under the test conditions of step (a); and (c) determining infertility of the male subject based on the motility of sperm detected in step (b).

[0041] The term "in vitro" refers to ex vivo.

[0042] The terms "determine" or "diagnose" used throughout the present invention include variations such as "detect", i.e., these terms can be used interchangeably. According to the present invention, the terms determine / detect / diagnose are generally understood to refer to infertility in male subjects. According to the methods and kits of the present invention, it may be possible to determine, diagnose or detect infertility in male subjects using the means described herein, thereby finding out whether the subject suffers from infertility.

[0043] The term "infertility" as used throughout the present invention refers to the inability of a vertebrate, preferably a mammal, to reproduce by natural means. In the present invention, the focus is on the infertility of male subjects. In other words, an infertile male subject is infertile and may not be able to father a child.

[0044] When applying the method of the present invention, first a sperm sample obtained from a male subject is contacted with a calcium-free test liquid, which corresponds to step (a) of the method of determining or diagnosing infertility in a male subject according to the present invention.

[0045] In this context, the term "contacting" as used throughout the present invention means adding a sperm sample obtained from said subject to a calcium-free test liquid. Preferably, about 50 μl of the sperm sample is added to the calcium-free test liquid. More preferably, about 50 μl of the sperm sample is added to 450 μl of the calcium-free test liquid to make a total volume of 500 μl. When referring to a preferred embodiment of the present invention, about 50 μl of the sperm sample is also added to a calcium-containing control liquid. Preferably, about 50 μl of the sperm sample is added to 450 μl of the calcium-containing control liquid to make a total volume of 500 μl. After adding the sperm sample to the tube (or vial) containing the calcium-free test liquid or the calcium-containing control liquid, the tube (or vial) containing the sperm sample and the calcium-free test liquid or the tube (or vial) containing the sperm sample and the calcium-containing control liquid may be gently mixed by inversion and / or low-speed vortexing.

[0046] Throughout the present invention, the plural term "sperm" or "spermatozoa" refers to more than one sperm cell. Therefore, more than one sperm cell refers to sperm of the present invention.

[0047] In this context, a "sperm sample" refers to a sample containing sperm. Such a sample can be obtained from said male subject by any method known to those skilled in the art, preferably by ejaculation. The so-called ejaculation refers to the orgasmic discharge of an adolescent or sexually mature male subject that contains sperm. The sperm sample of the present invention obtained by ejaculation from a male subject preferably does not contain about 90% or more, for example, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or even about 100% of motionless sperm. Therefore, sperm samples obtained by ejaculation from a male subject that contain a higher amount of immotile sperm, for example, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or even about 100% immotile sperm, are excluded from the method of the present invention. In other words, certain sperm samples that have a high amount of immotile sperm at the beginning, i.e., before the actual start of infertility testing, cannot be used and are not used in the method of the present invention. Therefore, the sperm sample that is contacted with the calcium-free test solution in step (a) of the method described elsewhere herein contains at least about 10% or more, for example, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more motile sperm. Initial sperm motility can be assessed prior to commencing the methods of the invention described elsewhere herein using a calcium-containing control fluid or native ejaculate and a means for detecting sperm motility in said control fluid.

[0048] Throughout the present invention, the term "motionless" used in conjunction with sperm refers to "immotile" sperm, and when a sperm cell is referred to as "immotile" or "non-motile", it means that it does not move at all. However, motile sperm cells (motile sperm) can be characterized by some movement compared to immotile sperm cells (sperm). Any movement as used herein means any detectable residual movement of the flagellum and / or head of the sperm cell. As known to those skilled in the art, the tail of a sperm cell, also called the flagellum, is the longest part and is capable of a wave-like movement that propels the sperm forward to swim and help it penetrate the egg. In general, sperm motility depends on four parts of the tail: the connecting, middle, main, and distal parts. The head of the sperm cell contains the nucleus and is surrounded by a thin flat sac called the acrosome, which contains the enzymes used to penetrate the female egg. In short, when sperm motility is detected in a sample used in the method of the present invention, any kind of independent / autonomous movement of the flagellum and / or head of the sperm cell may be a sufficient requirement to assign a sperm cell (sperm) to the category of motile sperm cells (sperm).

[0049] A "calcium-free test solution" according to the methods and kits of the present invention may contain less than about 20 nM, preferably less than about 15 nM, more preferably less than about 10 nM, more preferably less than about 5 nM, e.g., less than about 19 nM, less than about 18 nM, less than about 17 nM, less than about 16 nM, less than about 15 nM, less than about 14 nM, less than about 13 nM, less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, or less than about 1 nM of free calcium. Therefore, a solution containing less than about 20nM, less than about 15nM, less than about 10nM, or less than about 5nM, for example, about 19nM, about 18nM, about 17nM, about 16nM, about 15nM, about 14nM, about 13nM, about 12nM, about 11nM, about 10nM, about 9nM, about 8nM, about 7nM, about 6nM, about 5nM, about 4nM, about 3nM, about 2nM, or about 1nM of free calcium is qualified as a calcium-free test solution according to the present invention.The term "test solution" when used according to the method and kit of the present invention refers to a solution with a reduced calcium concentration, which is contacted with a sperm sample obtained from a male subject and used to test the motility of the sperm under calcium-free conditions.As mentioned elsewhere herein, the test solution according to the present invention preferably contains less than about 20nM of free calcium, and is therefore referred to as a "calcium-free test solution".

[0050] The term "free calcium" as used herein refers to calcium that is not bound to proteins such as albumin and / or globulins, or that is not bound (complexed) to, for example, bicarbonate, lactate, citrate and / or phosphate. In this context, the term "calcium" as used throughout the present invention refers to calcium that is free of Ca 2+ means...

[0051] The "calcium-free test solution" used in the present invention may contain at least one agent that reduces the free calcium concentration. The term "free calcium-reducing agent" can be used interchangeably with the term "agent that reduces free calcium concentration". Under physiological conditions, the calcium concentration in sperm cells is about 10,000 times lower than that in the extracellular fluid, which is on the order of about 200-500 nM versus about 2 mM. Along this concentration gradient, CatSper can mediate calcium influx into sperm. Even if the calcium pump or calcium exchanger in the sperm membrane transports calcium ions to the outside, it can compensate for the calcium influx to maintain calcium homeostasis. By using at least one agent that reduces the free calcium concentration to lower the extracellular free calcium concentration below the intracellular concentration, the calcium influx via CatSper can be suppressed, thereby increasing the intracellular calcium concentration to Ca 2+ The calcium concentration can be decreased because ions flow out of the cell through CatSper channel. This decrease in calcium concentration is intolerable to sperm, i.e. sperm cannot move. Thus, by using at least one agent capable of decreasing free calcium concentration, it is possible to decrease extracellular calcium concentration below the intracellular calcium concentration of sperm. Thus, decreasing free calcium concentration means that the free calcium concentration of the solution is decreased to less than about 20nM free calcium as defined above, thereby providing the "calcium-free test solution" described elsewhere herein.

[0052] Preferably, the at least one agent that reduces the free calcium concentration is Ca 2+ In this context, the term "Ca" is used throughout the present invention. 2+ A "chemical that binds to calcium" refers to any substance or molecule with a defined molecular composition that is produced or used by a chemical process and is capable of binding to free calcium. More preferably, the Ca 2+ Chemicals that bind to Ca include chelators, citrate, phosphate, or Ca. 2+The present invention is preferably a Ca indicator dye, and more preferably a chelating agent. 2+ The chemicals that bind to are ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (abbreviated as EGTA), ethylenediaminetetraacetic acid (abbreviated as EDTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (abbreviated as BAPTA), N-(2-hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid (abbreviated as HEDTA), nitrilotriacetic acid (abbreviated as NTA), dibromo-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (abbreviated as DiBrBAPTA), citric acid, phosphate, or Ca. 2+ The indicator dye is one of the above, and most preferably EGTA. 2+ The chemical that binds to may be any derivative of EGTA, EDTA, BAPTA, HEDTA, NTA, or DiBrBAPTA.

[0053] As used herein, Ca 2+ EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA or their derivatives mentioned as agents that bind to Ca refer to chelating agents. The term "chelating agent" refers to an organic or inorganic compound that has more than one ion pair and is therefore capable of forming more than one coordinate bond with a central (metal) ion. In fact, said chelating agents coordinate with high affinity to divalent metal cations, making them unavailable to ion channels. Thus, such chelating agents may have the ability to fix divalent or polyvalent cations in stable complexes (so-called chelates). Any one of the chelating agents described elsewhere herein may be capable of binding Ca 2+When used as a chemical binding agent, at least about 2.5 mM, at least about 3 mM, at least about 3.5 mM, at least about 4 mM, at least about 4.5 mM, at least about 5 mM, at least about 5.5 mM, at least about 6 mM, at least about 6.5 mM, at least about 7 mM, at least about 7.5 mM, at least about 8 mM, at least about 8.5 mM, at least about 9 mM, at least about 9.5 mM, or at least about 10 mM, preferably about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, about 5 mM, about 5.5 mM, about 6 mM, about 6.5 mM, about 7 mM, about 7.5 mM, about 8 mM, about 8.5 mM, about 9 mM, about 9.5 mM, about 10 mM of said chelating agent can be used in accordance with the methods and kits of the invention. More preferably, the chelating agent can be used in the calcium-free test solution according to the method and kit of the present invention in the range of about 2.5 mM to about 10 mM, about 2.9 mM to about 8.8 mM, about 3.3 mM to about 7.5 mM, or about 4 mM to about 6 mM. Most preferably, the Ca concentration in the calcium-free test solution according to the present invention can be about 2.5 mM to about 10 mM, about 2.9 mM to about 8.8 mM, about 3.3 mM to about 7.5 mM, or about 4 mM to about 6 mM. 2+ Approximately 5 mM EGTA is used as the binding agent.

[0054] As used herein, "Ca 2+ "Indicator dye" refers to a reagent that indicates the presence or concentration of another substance, such as a metal ion, here calcium, by a characteristic color change. Thus, Ca 2+ The indicator dye forms a complex with calcium, which reduces the amount of free calcium in the test solution. 2+ Indicator dyes are also used in the present invention. 2+ It is possible to use the compound as an agent that binds to

[0055] According to the method for determining or diagnosing infertility in a male subject described herein, step (a) may further comprise administering at least one CatSper channel activating stimulus. The term "CatSper activating stimulus" can be used interchangeably with the term "CatSper channel activating stimulus".

[0056] The term "CatSper channel" as used throughout the present invention refers to the Ca channel of sperm (CatSper), which is specific to sperm and exists in the cell membrane of the sperm flagellum. 2+ The CatSper channel complex is composed of four homologous α subunits (CatSper 1-4) and at least five auxiliary subunits: CatSperβ, CatSperγ, CatSperδ, CatSperε, and CatSperζ.

[0057] The CatSper channel activation stimulus can be added before, simultaneously with, or after adding the calcium-free test solution to the sperm sample in step (a) of the method of the present invention. Thus, once a sperm sample is obtained from a male subject, the activation stimulus can be added to the sperm sample before adding the calcium-free test solution described in step (a), or both the activation stimulus and the calcium-free test solution can be added to the sperm sample simultaneously in step (a) of the method of the present invention. Also included herein is the addition of the activation stimulus to the sperm sample after adding the calcium-free test solution described in step (a) of the method of the present invention. The present invention also contemplates the use or addition of a combination of two or more CatSper channel activation stimuli described elsewhere herein, i.e., simultaneously.

[0058] In a preferred embodiment, the CatSper activation stimulus is any substance and / or condition that enhances CatSper channel activity. Thus, the present specification also includes that the CatSper activation stimulus is one or more substances that enhance CatSper channel activity. The present specification also includes that the CatSper activation stimulus is one or more conditions that enhance CatSper channel activity. The present specification also includes that the CatSper activation stimulus is a combination of any substance and any condition that enhances CatSper channel activity.

[0059] In this context, the term "enhance CatSper channel activity" used throughout the present invention means that a greater number of ions flow through the CatSper channel pore at a given membrane potential per unit time compared to the number of ions flowing through the CatSper channel pore at a given membrane potential per unit time without enhancement by any of the substances and / or conditions described herein. The term "enhance" CatSper channel activity can also refer to improving / enhancing CatSper channel activity, stimulating CatSper channel activity, or releasing the CatSper channel from inhibition. The term "enhance" CatSper channel activity can also refer to activating CatSper channel activity. If a substance or condition can stimulate CatSper channel activity, it means that the substance or condition can stimulate ion flow through the channel. If a substance or condition can release / unblock the CatSper channel from inhibition, it means that the substance or condition can help the CatSper channel to no longer be inhibited or blocked.

[0060] Preferably, the substance that enhances CatSper channel activity is a ligand. As used herein and throughout the present invention, "ligand" refers to a small organic molecule, protein, peptide, or chemical that directly binds to CatSper channel or binds to a receptor protein that affects the activity of CatSper channel. Thus, a "ligand" can enhance the activity of the channel by binding to the channel or receptor protein. More preferably, the ligand that enhances CatSper channel activity is any one of steroids, prostaglandins, synthetic or natural chemicals that mimic the action of steroids or prostaglandins, serum albumin, zona pellucida protein, or combinations thereof.

[0061] "Steroids" are generally biologically active organic compounds that contain a core structure of 17 carbon atoms in four rings arranged in a specific molecular form.The preferred steroid according to the present invention is progesterone as a ligand that enhances CatSper channel activity.In particular, in human sperm, progesterone and prostaglandins are two compounds in the oviduct that activate human CatSper channel.

[0062] As used herein, the term "synthetic chemicals that mimic the action of steroids or prostaglandins" refers to any synthetically produced or manufactured molecule / substance that can additionally mimic what a steroid or prostaglandin might do. "Natural chemicals that mimic the action of steroids or prostaglandins" refers to any molecule / substance formed in nature that can additionally mimic what a steroid or prostaglandin might do.

[0063] Also contemplated by the methods and kits of the present invention are combinations of two or more of the above-mentioned ligands, which are then capable of enhancing CatSper channel activity.

[0064] "Conditions that enhance CatSper channel activity" as described elsewhere herein may include an increase in intracellular pH and / or depolarization of the membrane potential that leads to activation of CatSper.

[0065] In this context, the term "increasing intracellular pH" refers to raising the pH inside (intracellularly) the sperm cell using a weak base. In other words, increasing the intracellular pH means making the pH alkaline. A weak base, as used throughout this invention, refers to a base that is only partially ionized in aqueous solution and has weak electrolytes. In addition, the pK, which indicates the degree to which a base is ionized in an equilibrium reaction with water, is also used. bBases with values ​​greater than 8 can be considered weak bases and are known to those skilled in the art. Weak bases of the present invention can include, but are not limited to, ammonium chloride, triethylamine (abbreviated as TEA), or imidazole. Any weak base known to those skilled in the art that can be used to increase intracellular pH can be present in the physiological aqueous solution. Increasing intracellular pH can be achieved by increasing extracellular pH (outside the sperm cell). When the extracellular pH is alkalized, the intracellular pH can also be alkalized (increased).

[0066] If the activation stimulus is in the form of an increase in intracellular pH and is applied before or after the addition of a calcium-free test solution to the sperm sample defined in step (a) of the method for assessing or diagnosing infertility in a male subject, the physiological aqueous solution comprising a weak base for increasing the intracellular pH of the sperm cells may not be the same as the calcium-free test solution. If the activation stimulus is in the form of an increase in intracellular pH and is applied simultaneously with a calcium-free test solution to the sperm sample defined in step (a) of the method for assessing or diagnosing infertility in a male subject, the calcium-free test solution may comprise a weak base for increasing the intracellular pH.

[0067] The term "depolarization" refers to an intracellular change in which a cell undergoes a shift in charge distribution, resulting in less negative charge within the cell. The difference in electrical potential between the inside and outside of the cell is called the membrane potential. Such membrane potential may be determined by applying the Goldman-Hodgkin-Katz voltage equation (GHK equation), more commonly known as the Goldman equation, as known to those skilled in the art. When the membrane potential decreases (i.e., becomes less negative), the membrane potential may be considered to be depolarized.

[0068] Thus, as used herein, "depolarize" refers to shifting the membrane potential to a less negative value or to a more positive value.

[0069] Depolarizing the membrane potential, i.e., depolarizing the membrane potential, can be achieved by increasing the potassium concentration in the solution used as the activating stimulus to a concentration higher than about 30 mM. In other words, the term "increasing the potassium concentration" refers to increasing the potassium concentration to a concentration greater than about 30 mM. The following definition of "increasing the potassium concentration" in the solution used as the activating stimulus can also be applied to the term "buffer containing an elevated potassium concentration" used in accordance with the present invention.

[0070] The cause of the membrane potential is the concentration gradient of ions across the membrane and their selective permeability. Inside the cell there is a high concentration of protein molecules (proteins), which exist as anions at a cellular pH of about 7.2; in addition, there are positively charged (cationic) K+ (potassium) ions in almost equimolar concentrations and Na+ and Cl- (sodium and chloride) in significantly lower concentrations. In contrast, completely different conditions prevail in the extracellular fluid: it contains much fewer protein molecules and has an inverse ratio with respect to other ions, i.e., a higher concentration of Na+ and Cl- ions and a significantly lower proportion of K+. The membrane potential is determined by the relative permeability of the membrane to the various ions (pK, pNa, pCl) and the concentration difference of the ions passing through the membrane. The relative permeability of the membrane is usually pK:pNa:pCl=1:0.05:0.45. The membrane potential can be calculated using the Goldman-Hodgkin-Katz formula (GHK formula) described elsewhere in this specification. Considering the relative ion permeabilities and GHK expression, it will be apparent to one skilled in the art that an increase in the extracellular K+ concentration depolarizes the membrane potential.

[0071] The potassium concentration in the solution used as the activating stimulus can be increased to enhance the CatSper channel activity described elsewhere herein.When the activating stimulus is in the condition of depolarizing the membrane potential and is added before or after adding calcium-free test solution to the sperm sample defined in step (a) of the method for determining or diagnosing infertility in male subjects, the solution with increased potassium concentration for depolarizing the membrane potential can be different from the calcium-free test solution.When the activating stimulus is in the condition of depolarizing the membrane potential and is added simultaneously with calcium-free test solution to the sperm sample defined in step (a) of the method for determining or diagnosing infertility in male subjects, the calcium-free test solution can have increased potassium concentration for depolarizing the membrane potential.

[0072] Furthermore, depolarizing the membrane potential, i.e. depolarizing the membrane potential, can also be achieved by using potassium and / or chloride ion channel inhibitors, or by using sodium ionophores. Potassium and chloride ion channel inhibitors lower pK and pCl, respectively, depolarizing the membrane potential according to the GHK formula. Sodium ionophores according to the present invention depolarize sperm by increasing pNa.

[0073] In this context, the term "inhibitor" as used throughout the present invention means a modulator capable of inhibiting the activity of something. In particular, "ion channel inhibitors" that depolarize the membrane potential may inhibit either potassium or chloride ion channels or both channels.

[0074] In this context, "ionophore" as used throughout the present invention refers to a chemical molecule that can reversibly bind to ions. Ionophores are lipid soluble and transport ions across cell membranes. Ionophores are sometimes called ion carriers. Ionophores may also be antibiotics. Ionophores may be selective for certain cations and anions, thereby inhibiting Na + or K+ Ionophores according to the present invention therefore are synthetic or natural compounds capable of increasing the permeability of membranes to (selected) ionic species such as sodium or potassium. Sodium ionophores according to the present invention may include, but are not limited to, gramicidin, fusafungin, monensin, salinomycin, narasin; gramicidin is preferred as it is selective for potassium and sodium but impermeable to anions. Ionophores according to the present invention artificially increase the relative permeability of membranes to Na+ (pNa), thereby depolarizing the membrane potential.

[0075] It is also envisioned herein that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH and depolarizing the membrane potential. Thus, it is also envisioned that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride and depolarizing the membrane potential by increasing the potassium concentration of a solution used as at least one activation stimulus. It is also envisioned that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as TEA and depolarizing the membrane potential by increasing the potassium concentration of a solution used as at least one activation stimulus. It is further envisioned that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as imidazole and depolarizing the membrane potential by increasing the potassium concentration of a solution used as at least one activation stimulus.

[0076] It is also contemplated that conditions that enhance CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride and depolarizing the membrane potential by utilizing a potassium and / or chloride ion channel inhibitor. It is also contemplated that conditions that enhance CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as TEA and depolarizing the membrane potential by utilizing a potassium and / or chloride ion channel inhibitor. It is also contemplated that conditions that enhance CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as imidazole and depolarizing the membrane potential by utilizing a potassium and / or chloride ion channel inhibitor.

[0077] It is also contemplated that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride and depolarizing the membrane potential by utilizing a sodium ionophore, preferably gramicidin. It is also contemplated that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as TEA and depolarizing the membrane potential by utilizing a sodium ionophore, preferably gramicidin. It is also contemplated that the conditions for enhancing CatSper channel activity include a combination of increasing intracellular pH by utilizing a weak base such as imidazole and depolarizing the membrane potential by utilizing a sodium ionophore, preferably gramicidin.

[0078] The disclosure of the present invention on the combination of increasing intracellular pH by using a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by increasing the potassium concentration of the solution used as at least one activation stimulus can also be combined with any substance that enhances CatSper channel activity. The disclosure of the present invention on the combination of increasing intracellular pH by using a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by using a potassium and / or chloride ion channel inhibitor can also be combined with any substance that enhances CatSper channel activity. The disclosure of the present invention on the combination of increasing intracellular pH by using a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by using a sodium ionophore, preferably gramicidin, can also be combined with any substance that enhances CatSper channel activity. In particular, the specific disclosures relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by increasing the potassium concentration of the solution used as at least one activation stimulus can be combined with any one of the ligands of the invention described elsewhere herein as a substance that enhances CatSper channel activity. In particular, the specific disclosures relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by utilizing a potassium and / or chloride ion channel inhibitor can be combined with any one of the ligands of the invention described elsewhere herein as a substance that enhances CatSper channel activity. In particular, the specific disclosures relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by utilizing a sodium ionophore, preferably gramicidin, can be combined with any one of the ligands of the invention described elsewhere herein as a substance that enhances CatSper channel activity.Preferably, the disclosure of the present invention relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by increasing the potassium concentration of the solution used as at least one activation stimulus is combined with progesterone as a substance that enhances CatSper channel activity. Preferably, the disclosure of the present invention relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by utilizing a potassium and / or chloride ion channel inhibitor is combined with progesterone as a substance that enhances CatSper channel activity. Preferably, the disclosure of the present invention relating to the combination of increasing intracellular pH by utilizing a weak base such as ammonium chloride, TEA or imidazole and depolarizing the membrane potential by utilizing a sodium ionophore, preferably gramicidin, is combined with progesterone as a substance that enhances CatSper channel activity.

[0079] Also in accordance with the present invention, according to the method for assessing or diagnosing infertility in a male subject described herein, which preferably comprises applying at least one CatSper channel activating stimulus in step (a) of said method, the sperm sample is exposed to the calcium-free test solution of step (a) for at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 21 minutes, at least about 22 minutes, at least about 23 minutes, at least about 24 minutes, at least about 25 minutes, at least about 26 minutes, at least about 27 minutes, at least about 28 minutes, at least about 29 minutes, at least about 30 minutes, at least about 31 minutes, at least about 32 minutes, at least about 33 minutes, at least about 34 minutes, at least about 35 minutes, at least about 36 minutes, at least about 37 minutes, at least about 38 minutes, at least about 39 minutes, at least about 40 minutes, at least about 41 minutes, at least about 42 minutes, at least about 43 minutes, at least about 44 minutes, at least about 45 minutes, at least about 46 minutes, at least about 47 minutes, at least about 48 minutes, at least about 49 minutes, at least about 50 minutes, at least about 51 minutes, at least about 52 minutes, at least about 53 minutes, at least about 54 minutes, at least about 55 minutes, at least about 56 minutes, at least about 57 minutes, at least about 58 minutes, at least about 59 minutes, at least about 6 It is envisaged that the sperm sample is contacted with the calcium-free test solution of step (a) for about 19 minutes, at least about 20 minutes, at least about 30 minutes, at least about 60 minutes, or preferably for about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, or for about 5 minutes to about 60 minutes, preferably for about 10 minutes to about 25 minutes, preferably for about 12 minutes to about 19 minutes, preferably for about 13 minutes to about 17 minutes, and then the infertility of the male subject is judged based on the motility detected in step (b) of the method. In a preferred embodiment of the present invention, the sperm sample is contacted with the calcium-free test solution of step (a) for about 15 minutes, and then the infertility of the male subject is judged based on the motility detected in step (b) of the method. In a further preferred embodiment, the sperm sample is contacted with the calcium-free test solution of step (a) for about 15 minutes at about 37° C. and then the infertility of the male subject is determined based on the motility detected in step (b) of the method.

[0080] In this particular context, the term "contacting" also means "incubating." Preferably, "incubating" means incubating by gentle mixing, such as by inversion and / or slow vortexing.

[0081] The method of the present invention for determining or diagnosing infertility in a male subject necessarily comprises, as a second step (b), detecting the motility of sperm in said sample under the test conditions of step (a), and, as a third step (c), determining the infertility of the male subject based on the motility detected in step (b).

[0082] In this context, the term "detecting motility" refers to determining whether the sperm in the sample are motile or non-motile as defined elsewhere herein using any technique to distinguish between motile and non-motile sperm. Thus, the term "determining motility" can be used interchangeably with the term detecting motility. Preferably, optical microscopy is used when detecting / determining whether the sperm in the sample are motile or non-motile as defined elsewhere herein.

[0083] Thus, sperm motility can be determined / detected in step (b) of the method of the present invention using a light microscope.

[0084] The term "light microscopy" may also include automated computer-assisted sperm motility analysis, known to those skilled in the art, called CASA. Thus, the present invention may also include a method of the present invention, in which sperm motility may be determined / detected in step (b) using CASA.

[0085] The motility of sperm in the sperm sample is detected under the test conditions of step (a), and if there is no significant change in the amount of motile sperm under the test conditions, this may indicate infertility of the subject. In other words, each subject is considered to be infertile.

[0086] As described elsewhere in this specification, calcium influx occurs through the CatSper channel along the gradient of intracellular and extracellular calcium concentrations. However, CatSper-deficient spermatozoa do not tolerate a decrease in extracellular calcium concentration as described elsewhere in this specification. 2+There is no exit for ions; that is, their motility is maintained for a long time even under calcium-free conditions. By comparing the ratio of motile sperm to non-motile sperm in the calcium-free test solution contacted with the sperm sample of the present invention, it can be determined whether the sperm have functional CatSper channels. If the sperm can maintain motility for a long time, there may be a CatSper defect or other serious defect in the calcium-dependent signaling process, leading to the conclusion that the male subject may be infertile. If the sperm cannot move, it is likely that the CatSper channel is functional.

[0087] Therefore, the term "no significant change in the amount of motile sperm" means that when the sperm sample obtained from a male subject is contacted with the calcium-free test solution, the amount of motile sperm is not significantly reduced or the amount of motile sperm remains approximately the same. In other words, when the term "no significant change in the amount of motile sperm" is used, it means that the amount of motile sperm is not reduced by more than about 20%. It means that less than about 20% of the motile sperm, for example, about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or even 0%, are immobile when the motility of the sperm in the sample as defined elsewhere herein is detected under the test conditions of step (b) of the method of the present invention.

[0088] That is, if a sperm sample is contacted with the calcium-free test solution defined in step (a) of the method of the present invention for determining or diagnosing infertility in a male subject, and the amount of motile sperm does not decrease or, conversely, the amount of motile sperm remains similar, the male subject can be considered to be infertile (Figures 1C and 2C).

[0089] The method of the present invention for determining or diagnosing infertility in a male subject also comprises measuring the amount of motile sperm, in other words determining / detecting the motility of sperm, under the test conditions of step (b), e.g. by using a light microscope, of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 110, at least about 120, at least about 130, at least about 140, at least about 150, at least about 160, at least about 170, at least about 180, at least about 190, at least about 200, at least about 210, at least about 220, at least about 230, at least about 240, at least about 250, at least about 260, at least about 270, at least about 280, at least about 290, at least about 300, at least about 350, at least about 400, at least about 450, or at least about 500, preferably about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 400, about 500, or at least about 500, preferably about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 250, about 300, about 400, about 500, or at least about 500, The method may comprise counting about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 350, about 400, about 450, about 500 or more spermatozoa. Preferably, measuring the amount of motile spermatozoa, in other words, determining the motility of the spermatozoa, comprises counting about 200 spermatozoa under the test conditions of step (b), e.g., using a light microscope.Thus, when the amount of motile sperm is measured to determine or diagnose infertility in a male subject of the present invention (which comprises counting at least about 10 sperm) under the test conditions of step (b) of the method of the present invention, and the motility of the sperm in the sample as defined elsewhere herein is detected under the test conditions of step (b) of the method of the present invention, if less than about 20% of the motile sperm, such as about 19% or less, about 18% or less, about 17% or less, about 16% or less, about 15% or less, about 14% or less, about 13% or less, about 12% or less, about 11% or less, about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less, or even 0% are immobile, it means that there is no significant change in the amount of motile sperm, and thus indicates infertility in the subject.

[0090] Alternatively, instead of assessing single sperm by counting at least 10 sperm under the test conditions in step (b), the entire sperm population can be assessed, preferably using image fluctuations. For this purpose, spatiotemporal fluctuations of intensity in time-lapse images from a sample are analyzed. These fluctuations are directly correlated with the average motility of the sample.

[0091] In a preferred embodiment, the method for determining or diagnosing infertility of a male subject of the present invention further comprises comparing the sperm motility detected in step (b) with the sperm motility of said subject detected under control conditions containing calcium-containing fluid.In this particular embodiment, a sperm sample obtained from a male subject as defined elsewhere herein can be contacted with a calcium-free test fluid as defined in step (a) of the method of the present invention (this is considered as test condition) and the sperm sample can be contacted separately with a calcium-containing fluid (this is considered as control condition).After this step, the sperm motility under test and control conditions can again be detected and compared with each other (compare Figure 1A / 2A with Figure 1B / 2B, or compare Figure 1A / 2A with Figure 1C / 2C). The relative motility change of sperm population under different test solutions compared to the motility of sperm under control solution is shown in Figure 3, which clearly shows that the sperm sample containing CatSper inhibitor (which corresponds to the sperm sample lacking CatSper) has no significant change in the amount of motile sperm under calcium-free test solution.Thus, the comparison of motility detected under test condition and control condition can indicate the infertility of the subject, in other words, the subject is infertile.As described herein, if there is no significant change in the amount of motile sperm under test condition compared to control condition, the male subject is considered to be infertile.

[0092] In this context, the term "calcium-containing solution" refers to a solution containing more than about 1,000nM, more than about 10,000nM, more than about 100,000nM, more than about 1mM of free calcium. Herein, calcium-containing solution may also be referred to as control solution. It may correspond to HTF buffer (human tubular fluid) as known to those skilled in the art. The calcium-containing solution of the present invention may contain an agent that reduces the free calcium concentration, in which case said solution contains more than about 1,000nM, more than about 10,000nM, more than about 100,000nM, more than about 1mM of free calcium. In a preferred embodiment of the method of the present invention, the calcium-containing control solution further comprises at least one CatSper channel activating stimulus as defined elsewhere herein.

[0093] Thus, according to a preferred embodiment, the present invention relates to an in vitro method for determining or diagnosing infertility in a male subject, comprising the steps of: a) contacting a sperm sample obtained from said subject with a calcium-free test liquid; b) contacting a sperm sample obtained from said subject with a calcium-containing control liquid; c) detecting sperm motility under conditions (a) and (b); and d) comparing the sperm motility detected under the test conditions of step a) with the sperm motility detected under the control conditions of step b), wherein a comparison of the motility detected under the test and control conditions is indicative of infertility in the subject.

[0094] In a further preferred embodiment of the method of assessing or diagnosing infertility in a male subject according to the invention, the comparison of sperm motility under test and control conditions comprises determining the CatSper Activity Index (CAI).

[0095] When determining a particular CatSper activity index, first, a percentage value of motile spermatozoa under test and control conditions can be obtained. In this context, the term "obtain a percentage value" means, in the examples, calculating the proportion / ratio of the amount of motile spermatozoa after contacting the spermatozoa sample with a test liquid described elsewhere herein and after contacting the spermatozoa sample with a control liquid described elsewhere herein to the total amount of spermatozoa counted in the sample under test and control conditions described elsewhere herein. The term "amount of motile spermatozoa" as used herein refers to the actual amount / number of spermatozoa with any detectable movement described elsewhere when counted using any available technique, such as optical microscopy. Thus, the term "percentage value of motile spermatozoa" can be expressed as a proportion / ratio of the amount of motile spermatozoa to the total amount of spermatozoa counted in the sample under test and control conditions described elsewhere herein. The amount of motile sperm after contacting the sperm sample with the test fluid (control fluid) relative to the total sperm counted in the sample under test (control) conditions can be expressed as a percentage, i.e., the amount of motile sperm after contacting the sperm sample with the test fluid (control fluid) divided by the total sperm counted in the sample under test (control) conditions.

[0096] Secondly, in the case of the comparison of sperm motility under test and control conditions, including the determination of CatSper activity index, in the embodiment, the percentage value of motile sperm under test conditions defined herein can be subtracted from the percentage value of motile sperm under control conditions described herein. In this context, the term "subtract" means to calculate the difference between two numerical values ​​expressed in %, and the percentage value defined above is determined by subtraction. The percentage value of motile sperm under test conditions can be subtracted from the percentage value of motile sperm under control conditions to obtain a subtraction score. In this context, "subtraction score" refers to the result of subtracting the percentage value of motile sperm under test conditions from the percentage value of motile sperm under control conditions. This subtraction score can also be expressed as a percentage (%).

[0097] Thirdly, in the comparison of sperm motility under test and control conditions, including determining the CatSper activity index, the ratio of the subtraction score to the percentage value of motile sperm under control conditions can be determined (see Examples). In particular, in this context, the term "determining" refers to calculating, for example, calculating the ratio of the subtraction score to the percentage value of motile sperm under control conditions.

[0098] Determining the ratio of the subtraction score to the percentage value of motile spermatozoa under control conditions defined above means comparing the subtraction score to the percentage value of motile spermatozoa under control conditions or correlating the subtraction score with the percentage value of motile spermatozoa under control conditions. Thus, determining the ratio of the subtraction score to the percentage value of motile spermatozoa under control conditions can also be included in the present specification. Therefore, the term "in relation to" or simply "to" used in this specification in this context, for example "level of A" to "level of B", generally means comparing "A" and "B". Preferably, this comparison is made by dividing A by B, thereby obtaining a ratio. Here, the relationship between "A" and "B" can be expressed as the quotient of "A" divided by "B", thereby also obtaining a ratio.

[0099] Fourth, in comparing sperm motility under test and control conditions, including determining the CatSper activity index, the ratio determined above can be multiplied by the number 100. By multiplying the ratio defined above by the number 100, the CatSper activity index (abbreviated CAI) can be obtained (see examples). In this context, the term "multiply" refers to "A" times "B", and therefore the "ratio" determined above times "the number 100".

[0100] Furthermore, a preferred embodiment of the method of the invention relating to the determination of the CatSper activity index may additionally comprise a further step of comparing said index determined as described above with a predetermined reference value.

[0101] The term "reference value" is generally understood to refer to a qualitative or quantitative value. Thus, when the reference value is understood to refer to a qualitative value, the index may be below the reference value. Also, when the reference value is understood to refer to a quantitative value, the index may deviate from the reference value. In another embodiment, when the reference value is understood to refer to a quantitative value, the index may deviate from the reference value by about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5 ...0%, about 5.0%, about 5.0%, about 5.0%, about 5.0%, about 5.0%, about 5.0%, about 5 The deviation may be about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. The greater the deviation of the index from the reference value, the greater the likelihood that the subject of the present invention is infertile.

[0102] The selection of the reference value includes, among others, the consideration of conditional probability, the distribution of true / false judgments at different test criteria, and the estimation of the outcome of the treatment (or failure of the treatment) based on the judgment. The appropriate criteria can be determined in various ways. Thus, a "predetermined reference value" can be obtained in advance, for example as described below, and can then be used as a criterion for determining whether a male subject is infertile / suffers from infertility.

[0103] For example, population studies can be used to select the reference value. Often, the receiver operating characteristic (ROC) is used to select the reference value that best distinguishes between good responding subpopulations (control condition) and bad responding subpopulations (test condition). A false positive in this case occurs when a person has a positive test result (a poor responder who is infertile) but is actually a good responder who may be fertile. A false negative occurs when a person has a negative test result (a good responder who may be fertile) but is actually infertile. To plot the ROC curve, the true positive rate (TPR) and false positive rate (FPR) are determined as the decision criterion is varied continuously. The ROC graph is sometimes called a sensitivity vs (1-specificity) plot because the TPR is equivalent to the sensitivity and the FPR is equal to 1-specificity. A perfect test would have an area under the ROC curve (AUC) of 1.0; a random test would have an area of ​​0.5. Preferably, the tests described herein provide an ROC curve area of ​​greater than 0.5, preferably at least 0.6, more preferably 0.7, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95. The reference value is selected to provide an acceptable level of specificity and sensitivity. The reference value is one that can provide an acceptable level of specificity and sensitivity in separating a population of subjects into "bins" such as a "first" subpopulation (e.g., infertile), a "second" subpopulation that may be fertile, etc. The reference value is selected to separate the first and second populations by one or more of the following measures of test accuracy: an odds ratio greater than 1, preferably at least about 2 or greater or about 0.5 or less, more preferably at least about 3 or greater or about 0.33 or less, even more preferably at least about 4 or greater or about 0.25 or less, even more preferably at least about 5 or greater or about 0.2 or less, and most preferably at least about 10 or greater or about 0.1 or less; a specificity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, even more preferably at least about 0.8, even more preferably at least about 0.9, and most preferably at least about 0.95, with a corresponding sensitivity of greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, even more preferably at least about 0.5, even more preferably about 0.6, even more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.9, and most preferably greater than about 0.95; a sensitivity of greater than 0.5, preferably at least about 0.6, more preferably at least about 0.7, even more preferably at least about 0.8, even more preferably at least about 0.9, and most preferably at least about 0.95, with a corresponding specificity of greater than 0.2, preferably greater than about 0.3, more preferably greater than about 0.4, even more preferably at least about 0.5, even more preferably about 0.6, even more preferably greater than about 0.7, even more preferably greater than about 0.8, even more preferably greater than about 0.9, and most preferably greater than about 0.95; A sensitivity of at least about 75% and a specificity of at least about 75%; a positive likelihood ratio (calculated as sensitivity / (1-specificity)) greater than 1, and more preferably at least about 2, more preferably at least about 3, even more preferably at least about 5, and most preferably at least about 10; or A negative likelihood ratio (calculated as (1-sensitivity) / specificity) that is less than 1, and is less than about 0.5, more preferably less than about 0.3, and most preferably less than about 0.1.

[0104] In addition to standard comparison, other methods for correlating assay results with patient selection or classification (e.g., likelihood of being a good or bad responder) include decision trees, rule sets, Bayesian methods, and neural network methods. These methods can provide a probability value that represents the degree to which a subject belongs to one classification among multiple classifications.

[0105] As mentioned above, a suitable test may exhibit one or more of the following results on these various scales: a specificity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding sensitivity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, even more preferably at least 0.5, even more preferably 0.6, even more preferably greater than 0.7, even more preferably greater than 0.8, even more preferably greater than 0.9, and most preferably at least 0.95; a sensitivity of greater than 0.5, preferably at least 0.6, more preferably at least 0.7, even more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95, with a corresponding specificity of greater than 0.2, preferably greater than 0.3, more preferably greater than 0.4, even more preferably at least 0.5, even more preferably 0.6, even more preferably greater than 0.7, more preferably greater than 0.8, more preferably greater than 0.9, and most preferably greater than 0.95; a sensitivity of at least 75% and a specificity of at least 75%; an ROC curve area of ​​at least 0.5, preferably at least 0.6, more preferably at least 0.7, more preferably at least 0.8, even more preferably at least 0.9, and most preferably at least 0.95; an odds ratio different from 1, preferably at least about 2 or greater or about 0.5 or less, more preferably at least about 3 or greater or about 0.33 or greater. a positive likelihood ratio (calculated as sensitivity / (1-specificity)) greater than 1 and at least 2, more preferably at least 3, even more preferably at least 5, and most preferably at least 10; and / or a negative likelihood ratio (calculated as (1-sensitivity) / specificity) less than 1 and less than 0.5, more preferably less than 0.3, and most preferably less than 0.1.

[0106] The comparison with the reference value can be performed manually, semi-automatically, or fully automatically.In some embodiments, the comparison can be computer-assisted.In computer-assisted comparison, the value stored in the database can be taken as the standard for comparing the obtained value or measured quantity, for example, through a computer-implemented algorithm.Similarly, the comparison with the reference measurement can be performed manually, semi-automatically, or fully automatically, including computer-assisted.

[0107] According to the present invention, if the index falls below the standard value, it may indicate infertility of the subject. Also, according to the present invention, if the index deviates from the standard value, it may indicate infertility of the subject. In a preferred embodiment, the index is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 1.5%, about 2.0%, about 2.5%, about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5 ... A deviation of 0.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, about 9.5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% may indicate infertility in the subject. The greater the deviation of the index from the reference value, the more likely the subject is infertile / suffers from infertility.

[0108] In a preferred embodiment, the reference value is about 20%. Therefore, if the CatSper activity index obtained from a male subject being evaluated for infertility is below the reference value of about 20%, it indicates that the subject is infertile (Figure 4). The infertility obtained by the CatSper activity index was verified by electrophysiological recording (Figure 5) and genetic analysis (Figure 6).

[0109] In this context, the term "below a reference value" means any numerical value that is less than (not equal to) the stated numerical value of the reference value, preferably rounded up to two decimal places.

[0110] The term "male subject", also referred to as a male individual, as used herein throughout the present invention refers to a vertebrate animal. Preferably, the vertebrate animal is a mammal. More preferably, the mammal is any of a human, cow, buffalo, horse, donkey, camel, sheep, goat, pig, dog, cat, rat, or mouse, and most preferably, a human.

[0111] In a further aspect, the present invention provides a kit comprising a calcium-free test solution as defined elsewhere herein and a calcium-containing control solution as defined elsewhere herein. Each kit is envisaged for use in a method for determining or diagnosing infertility in a male subject as described elsewhere herein. Thus, the present invention also relates to the use of the kits described herein in an in vitro method for determining or diagnosing infertility in a male subject as described elsewhere herein in detail.

[0112] According to the present invention, the calcium-containing control solution (abbreviated as control solution) and the calcium-free test solution (abbreviated as test solution) are preferably each provided in one container or vial as a kit (pharmaceutical pack). Thus, the present invention may include a kit consisting of one vial or container containing the control solution and one vial or container containing the test solution.

[0113] The kit of the present invention may further comprise a means of instructions / instructions (instruction sheet) and / or imprinting. The "instructions" may refer to a notice in a format prescribed by a governmental agency regulating the manufacture, use and / or sale of pharmaceutical or biological products (e.g., the control and test solutions of the kit of the present invention) and reflects the approval by the governmental agency of the manufacture, use and / or sale of the product for human administration or diagnosis. As used herein, an "imprint" may describe important information about the products / components of the kit and how one skilled in the art can use them to determine or diagnose infertility in male subjects. The description may be in a printout attached to the kit. Such information may include the composition of the components / products of the kit, storage conditions, shelf life and / or stability of the products / components of the kit.

[0114] The kit of the present invention may further comprise a verification solution containing a CatSper inhibitor. Any CatSper inhibitor may be used to inhibit / block the activity of the CatSper channel. Preferably, the kit of the present invention further comprises a verification solution containing a RU1968F1 CatSper inhibitor, which is known to those skilled in the art (Rennhack et al., 2018 British Journal of Pharmacology). This particular RU1968F1 inhibitor can pharmacologically mimic the phenotype of CatSper deficiency; that is, when such a verification solution containing a RU1968F1 CatSper inhibitor is utilized, this may correspond to the same effect as when an infertile male subject in which the CatSper channel is absent or functionally deficient is used in the method of the present invention. Such a CatSper inhibitor can be used for internal calibration to evaluate / define the reference value of the present invention. Thus, the present invention may include a kit comprised of one vial or container containing a control solution, one vial or container containing a test solution, and one vial or container containing a validation solution comprising a CatSper inhibitor, preferably comprising the RU1968F1 CatSper inhibitor.

[0115] Additionally, the kits of the invention may include a calcium-free test solution that includes at least one agent that reduces free calcium concentration, as described elsewhere herein. Thus, the invention may include a kit comprised of one vial or container containing a control solution and one vial or container containing a test solution that includes at least one agent that reduces free calcium concentration, as defined elsewhere herein.

[0116] Preferably, the at least one agent that reduces the free calcium concentration that can be included in the calcium-free test solution of the kit of the present invention is a Ca2+-containing agent as defined elsewhere herein. 2+ More preferably, the Ca-binding agent is at least one agent that reduces the free calcium concentration contained in the calcium-free test solution of the kit of the present invention. 2+ Chemicals that bind to Ca include chelators, citrate, phosphate, or Ca. 2+ The calcium indicator is preferably any one of the indicator dyes, more preferably a chelating agent. Most preferably, the calcium indicator is at least one agent that reduces the free calcium concentration contained in the calcium-free test solution of the kit of the present invention. 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca. 2+ The calcium-free test solution of the kit of the present invention is preferably any one of the indicator dyes, and most preferably EGTA. 2+ The chemical that binds to may be any derivative of EGTA, EDTA, BAPTA, HEDTA, NTA, or DiBrBAPTA.

[0117] The kit of the present invention may further comprise a calcium-free test solution comprising at least one CatSper channel activating stimulus as described elsewhere herein. It may also be included that the kit of the present invention may comprise a calcium-containing control solution comprising at least one CatSper channel activating stimulus. The present invention also contemplates kits in which the calcium-free test solution and the calcium-containing control solution comprise at least one CatSper channel activating stimulus. Thus, the present invention may also comprise a kit comprising one vial or container containing a control solution and one vial or container containing a test solution comprising at least one agent that reduces free calcium concentration as defined elsewhere herein and at least one CatSper channel activating stimulus as defined elsewhere herein. The present invention may further comprise a kit comprising one vial or container containing a control solution comprising at least one agent that reduces free calcium concentration as defined elsewhere herein and one vial or container containing a test solution comprising at least one agent that reduces free calcium concentration as defined elsewhere herein and at least one CatSper channel activating stimulus as defined elsewhere herein. Also included herein is a kit that includes one vial or container with a control solution, one vial or container with a test solution that includes at least one agent that reduces free calcium concentration, and further includes one vial or container with a solution that includes at least one CatSper channel activating stimulus. Thus, the present invention can also provide a kit that includes a calcium-free test solution and a calcium-containing control solution, and further includes a solution that includes at least one CatSper channel activating stimulus.

[0118] As defined elsewhere herein, the at least one activating stimulus contained in the calcium-free test solution and / or the calcium-containing control solution of the kit of the invention, or contained in another solution of the kit of the invention as defined above, may be any one of the ligands described elsewhere herein, a weak base, a buffer containing an elevated potassium concentration, a potassium or chloride ion channel inhibitor, or a sodium ionophore.

[0119] Preferably, the ligand used as at least one CatSper channel activation stimulus, which is included in the calcium-free test solution and / or calcium-containing control solution of the kit of the present invention or in another solution of the kit of the present invention, is any one of steroids, prostaglandins, synthetic or natural chemicals that mimic the action of steroids or prostaglandins, serum albumin, zona pellucida protein, or combinations thereof, more preferably the ligand is a steroid, and most preferably the steroid is progesterone.When at least one activation stimulus is used and is separately included in the solution of the kit of the present invention other than the calcium-free test solution or calcium-containing control solution, the kit can further include a carrier.In some embodiments, the carrier can also be included in one or more containers or vials that contain the solution that contains at least one activation stimulus as defined elsewhere herein.

[0120] The term "carrier" refers to a diluent, adjuvant, or vehicle, and at least one activation stimulus is formulated / mixed with the carrier in the solution of the kit that contains at least one activation stimulus.Such carrier can be sterile liquid, for example, water, buffer such as PBS, HTF, and oil, including petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc.Carrier can also be any organic solvent, such as DMSO or alcohol, preferably ethanol, methanol, etc., as known to those skilled in the art.Water or HTF is the preferred carrier.

[0121] All of the above relating to the methods of the invention is applicable to the kit aspects of the invention and the use of such kits.

[0122] It should be noted that the singular forms "a," "an," and "the" used herein include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the method described herein.

[0123] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to every element in the series. The term "at least one" refers to one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0124] As used herein, the term "and / or" includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0125] The terms "less than" or "more than" do not include specific numbers.

[0126] For example, less than 20 means less than the indicated number. Similarly, more than or greater than means more than or greater than the indicated number, for example, more than 80% means more than or greater than 80% of the indicated number.

[0127] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises," "comprising," and the like, are understood to mean the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of other integers or steps or group of integers or steps. As used herein, the term "comprising" can be replaced with the terms "containing" or "including," and, as used herein, can also be replaced with the term "having." As used herein, "consisting of" excludes elements, steps, or ingredients not specified.

[0128] The term "including" means "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0129] The term "about" means plus or minus 10%, preferably plus or minus 5%, more preferably plus or minus 2%, and most preferably plus or minus 1%.

[0130] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as unity, unless the context otherwise requires.

[0131] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, substances, etc. described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0132] All publications cited throughout the text of this specification (including all patents, patent applications, scientific publications, manuals, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. In the event that material incorporated by reference conflicts or is inconsistent with the present specification, the present specification shall control over such material.

[0133] The contents of all publications and patent documents cited herein are incorporated herein by reference in their entirety.

[0134] A better understanding of the present invention and its advantages will be had from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLES

[0135] Materials used in the present invention The control solution used in the method and kit of the present invention corresponds to the HTF buffer (human tubular fluid) that has been used for many years in sperm research (Struenker et al. 2011, Nature 471, 382-386). The calcium-free test solution is also based on this buffer. The test can likely be performed with buffers of different composition, as long as they are kept within the physiological range.

[0136] Table 1. Composition of control and test solutions used in the method and kit of the present invention TIFF0007629408000001.tif93158

[0137] Flowchart for conducting CatSper activity test The flow chart shown below is to be considered as a guide for carrying out the method of the present invention. It represents a preferred embodiment of the method of the present invention. Number code: 1 = calcium-containing control solution; 2 = calcium-free test solution in the presence of progesterone (10 μM).

[0138] To perform the CatSper activity test, 450 μl each of calcium-containing control solution (labeled "1") and calcium-free test solution (labeled "2") are used (see specific compositions above in Table 1). Both solutions are stored in the kit at 4°C and warmed to 37°C before use.

[0139] Sperm (semen) samples are then collected from the male subjects. Initial motility is measured in native ejaculates using established methods for standard sperm analysis. Samples with less than 10% motile sperm are excluded from the study. 50 μl of native ejaculate is added to the buffer (1 and 2) in each microreaction tube. A homogenous sperm suspension is obtained by slow vortex mixing or repeated inversion of the microreaction tubes. Both microreaction tubes are then incubated at 37° C. for 15 min. After incubation, the mixing step is repeated and sperm motility in both conditions is measured by counting a total of 200 sperm. Condition 1 (= calcium-containing control solution, labeled "1") is counted prior to condition 2 (= calcium-free test solution, labeled "2"). Finally, the CatSper Activity Index (CAI) is calculated as described elsewhere. The results of the characterization of CATSPER2-deficient human sperm by this CatSper Activity Index are shown in FIG. 4. - Samples with less than 10% motility should be excluded from this test! - Warm the buffer to 37°C (Tube 1, Tube 2; 37°C) - Add 50 μl of native ejaculate to each tube (50 μl to tube 1, 50 μl to tube 2), use a new pipette tip for each step! - Mix gently (inversion, slow vortex) - Incubate the tubes at 37°C for 15 minutes (Tube 1, Tube 2; 37°C, 15 minutes) - Mix gently (inversion, slow vortex) - Measure the motile sperm in each tube by counting 200 sperm (counting order: tube 1, tube 2) - Calculate the "CatSper Activity Index" (CAI) TIFF0007629408000002.tif17128

[0140] Electrophysiological recordings and genetic analysis confirm CatSper deficiency To verify CatSper deficiency, five patients (patients A–E) who were confirmed as "CatSper deficient" when using the CatSper activity test (open triangles in Figure 4) were further analyzed by electrophysiological recordings (Figure 5). For comparison, sperm recordings from a control subject with intact CatSper channels are shown (Control). Panel A (left) shows monovalent CatSper currents measured in whole-cell derivatives. The current-voltage curve derived from this is shown on the right. Panel B shows the CatSper deficient patients (CATSPER - / - ) are shown. As a control, the current amplitudes of sperm from seven subjects with intact CatSper channels are shown (control). In CatSper-deficient sperm, no CatSper currents could be measured. Electrophysiological recordings were performed as described in Struenker et al., 2011.

[0141] Furthermore, genetic analysis was performed on these five patients (Figure 6). From so-called array CGH analysis (for example, by Tuettelmann et al. 2011), it was found that all five patients have chromosomal deletions including the CATSPER2 gene (CatSper 2 protein is an essential subunit of the CatSper channel complex). In summary, the CatSper deficiency determined according to the means and methods of the present invention can be verified by electrophysiological recording and genetic analysis.

Claims

1. 1. An in vitro method for determining infertility in a male subject, comprising: The method comprises: (a) contacting a sperm sample obtained from the subject with a calcium-free test solution; (b) detecting the motility of sperm in the sample under the test conditions of step (a); (c) contacting a sperm sample obtained from the subject with a calcium-containing control solution; (d) detecting the motility of sperm in the sample under the control conditions of step (c); and (e) determining infertility of the male subject based on the motility detected in steps (b) and (d), wherein the absence of a significant change in the amount of motile sperm under the test conditions of step (b) compared to the amount of motile sperm under the control conditions of step (d) indicates infertility of the subject. Including; The method comprises: comparing the motility of spermatozoa detected in step (b) with the motility of spermatozoa from said subject detected under control conditions comprising a calcium-containing fluid. Further comprising: A comparison of the motility detected under test conditions and under control conditions indicates infertility in the subject, and the comparison of sperm motility under test conditions and under control conditions comprises determining a CatSper activity index; a) obtaining a percentage value of motile sperm i) under said test conditions and ii) under said control conditions; b) subtracting the percentage value of motile sperm under said test conditions in i) from the percentage value of motile sperm under said control conditions in ii), thereby obtaining a subtraction score; c) determining the ratio of said subtraction score to the percentage value of motile sperm under said control conditions of ii); d) multiplying said ratio of step c) by the number 100 to obtain said CatSper activity index. The method comprising:

2. 2. The method of claim 1, further comprising the step of comparing the index to a predetermined reference value, wherein the index being below the reference value indicates infertility in the subject.

3. 3. The method of claim 1 or 2, wherein the calcium-free test solution contains less than about 20 nM free calcium.

4. The method of any one of claims 1 to 3, wherein the calcium-free test liquid comprises at least one agent that reduces free calcium concentration.

5. The at least one agent that reduces free calcium concentration is Ca 2+ It is a chemical that binds to Ca 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca 2+ 5. The method of claim 4, wherein the indicator dye is any one of the indicator dyes.

6. The method of any one of claims 1 to 5, wherein step (a) further comprises administering at least one CatSper channel activating stimulus.

7. 7. The method of claim 6, wherein the at least one CatSper channel activating stimulus is any substance and / or condition that enhances activity of a CatSper channel.

8. The method of claim 7, wherein the substance that enhances the activity of the CatSper channel is a ligand.

9. 9. The method of claim 8, wherein the ligand is any one of: a steroid; a prostaglandin; a synthetic or natural chemical that mimics the action of a steroid or a prostaglandin; serum albumin; a zona pellucida protein; or a combination thereof.

10. The method of claim 9, wherein the steroid is progesterone.

11. The method of claim 7, wherein the conditions that enhance the activity of the CatSper channel include an increase in intracellular pH and / or a depolarization of the membrane potential.

12. 12. The method of any one of claims 1 to 11, wherein measuring the amount of motile sperm comprises counting at least about 10 sperm under the test conditions of step (b).

13. 1. A kit for use in a method for determining or diagnosing infertility in a male subject, comprising: (a) a calcium-free test solution, and (b) Calcium-containing control solution Including, The kit for use in the method for determining the male subject according to any one of claims 1 to 12.

14. The kit for use according to claim 13, further comprising a validation solution comprising a CatSper inhibitor.

15. 15. The kit for use according to claim 13 or 14, wherein the calcium-free test liquid comprises at least one agent that reduces free calcium concentration.

16. The at least one agent that reduces free calcium concentration is Ca 2+ It is a chemical that binds to Ca 2+ The chemicals that bind to are EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citrate, phosphate, or Ca 2+ 16. The kit for use according to claim 15, wherein the indicator dye is any one of the indicator dyes.

17. The kit for use according to any one of claims 13 to 16, wherein the calcium-free test solution and / or the calcium-containing control solution comprises at least one CatSper channel activating stimulus.

18. 18. The kit for use of claim 17, wherein the activating stimulus is any one of a ligand, a weak base, a buffer containing an elevated potassium concentration, a potassium or chloride ion channel inhibitor, or a sodium ionophore.

19. 19. The kit for use according to claim 18, wherein the ligand is any one of: a steroid; a prostaglandin; a synthetic or natural chemical that mimics the action of a steroid or a prostaglandin; serum albumin; a zona pellucida protein; or a combination thereof.

20. The kit for use according to claim 19, wherein the steroid is progesterone.

21. In vitro method for determining infertility of a male subject according to any one of claims 1 to 12, (a) a calcium-free test solution, and (b) Calcium-containing control solution Use of the kit, including:

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