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EP-3942301-B1 - CATSPER-ACTIVITY-TEST FOR DETERMINING MALE INFERTILITY

EP3942301B1EP 3942301 B1EP3942301 B1EP 3942301B1EP-3942301-B1

Inventors

  • SCHIFFER, CHRISTIAN
  • Brenker, Christoph
  • STRÜNKER, Timo
  • YOUNG, Samuel Zachary

Dates

Publication Date
20260506
Application Date
20200323

Claims (15)

  1. An in vitro method for determining or diagnosing infertility of 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 the sperm in said sample under the test condition of step (a), and c) determining infertility of the male subject based on the motility detected in step (b).
  2. The method according to claim 1, wherein no significant change in the amount of motile sperm under the test condition of step (b) is indicative for infertility of said subject.
  3. The method according to claim 1 or 2, wherein a calcium-free test solution comprises less than about 20 nM free calcium, and/or wherein the calcium-free test solution comprises at least one agent reducing the free calcium concentration, wherein the at least one agent reducing the free calcium concentration is preferably a chemical binding Ca 2+ , wherein the chemical binding Ca 2+ is preferably any one of EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citric acid, phosphate, or a Ca 2+ -indicator dye, preferably EGTA.
  4. The method according to any one of the preceding claims, wherein step (a) further comprises applying at least one activation stimulus of the CatSper channel, wherein the activation stimulus is preferably applied prior to, simultaneously or after applying the calcium-free test solution to the sperm sample.
  5. The method according to claim 4, wherein the CatSper activation stimulus is any substance and/or condition increasing the CatSper channel activity, wherein (a) the substance increasing the CatSper channel activity is preferably a ligand, wherein the ligand is preferably any one of a steroid, prostaglandin, a synthetic or a natural chemical that mimics the action of a steroid or prostaglandin, a serum albumin, a zona pellucida protein, or a combination thereof, wherein the steroid is preferably progesterone, and wherein (b) the condition increasing the CatSper channel activity preferably comprises increasing the intracellular pH and/or depolarizing the membrane potential.
  6. The method according to claim 4 or 5, wherein the sperm sample is contacted for at least about 5 minutes with the test solution of step (a).
  7. The method according to any one of claims 2-6, wherein motile sperm is characterized by any movement in comparison to motionless sperm.
  8. The method according to any one of the preceding claims, wherein the motility of the sperm is determined using a light microscope, and/or wherein determining the amount of motile sperm comprises counting at least about 10 sperm under the test condition of step (b).
  9. The method according to any one of the preceding claims, further comprising comparing the motility of the sperm detected in step (b) to the motility of the sperm from said subject detected under a control condition comprising a calcium-containing solution, wherein the comparison of the motility detected under the test condition and the control condition is preferably indicative for infertility of said subject.
  10. The method according to claim 9, wherein the comparison of the motility of the sperm under the test condition and the control condition comprises determining a CatSper activity index, comprising the steps of a) obtaining a percentage value of motile sperm under i) the test condition and ii) the control condition; b) subtracting the percentage value of motile sperm under the test condition of i) from the percentage value of motile sperm under the control condition of 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); and d) multiplying the ratio of step c) with the number 100, thereby obtaining the CatSper-Activity-Index, further preferably comprising comparing the index with a predetermined reference value, wherein the index below the reference value is preferably indicative for infertility of said subject, wherein the reference value is preferably about 20%.
  11. The method according to any one of the preceding claims, wherein the male subject is a vertebrate, wherein the vertebrate is preferably a mammal, wherein the mammal is preferably any one of a human, a cattle, a buffalo, a horse, a donkey, a camel, a sheep, a goat, a pig, a dog, a cat, a rat, or a mouse, preferably a human.
  12. A kit for use in a method for determining or diagnosing infertility of a male subject comprising (a) a calcium-free test solution, and (b) a calcium-containing control solution, and further comprising a validation solution comprising a CatSper-inhibitor, and further preferably comprising instructions for use and/or an imprint.
  13. The kit for use according to claim 12, wherein the calcium-free test solution comprises at least one agent reducing the free calcium concentration, wherein the at least one agent reducing the free calcium concentration is preferably a chemical binding Ca 2+ , wherein the chemical binding Ca 2+ is preferably any one of EGTA, EDTA, BAPTA, HEDTA, NTA, DiBrBAPTA, citric acid, phosphate, or a Ca 2+ -indicator dye, preferably EGTA.
  14. The kit for use according to claims 12 or 13, wherein the calcium-free test solution and/or the calcium-containing control solution comprises at least one activation stimulus of the CatSper channel, wherein the activation stimulus is preferably any one of a ligand, a weak base, a buffer comprising increased potassium concentration, a potassium- or chloride ion channel inhibitor, or a sodium ionophore, wherein the ligand is preferably any one of a steroid, prostaglandin, a synthetic or a natural chemical that mimics the action of a steroid or prostaglandin, a serum albumin, a zona pellucida protein, or a combination thereof, wherein the steroid is preferably progesterone.
  15. Use of a kit comprising (a) a calcium-free test solution, and (b) a calcium-containing control solution in an in vitro method for determining or diagnosing infertility of a male subject.

Description

FIELD OF THE INVENTION The present invention relates to an in vitro method for determining or diagnosing infertility of 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 the sperm in said sample under the test condition of step (a), and (c) determining infertility of the male subject based on the motility detected in step (b). Further, the present invention relates to a kit for use in a method for determining or diagnosing infertility of a male subject comprising (a) a calcium-free test solution, and (b) a calcium-containing control solution, further comprising a validation solution comprising a CatSper-inhibitor, and further preferably comprising instructions for use and/or an imprint. Furthermore the present invention relates to a use of a kit comprising (a) a calcium-free test solution, and (b) a calcium-containing control solution in an in vitro method for determining or diagnosing infertility of a male subject. BACKGROUND OF THE INVENTION Unintentional childlessness is a serious problem that is caused by male infertility in approximately 40-50% of the cases. However, both the infertility per se and the underlying causes are often unreliable to diagnose. In about 30% of male patients they remain unclear ("idiopathic infertility" or "unexplained sperm dysfunction"). This is due to the lack of suitable and timely diagnostic methods. In order to access and fertilize the ovum at the end of the fallopian tube, human spermatozoa have a complex behavioral repertoire. The oocyte and surrounding cells secrete e.g. hormones to which the sperm react. It is believed that sperm may adjust their swim direction along the hormone gradient and track the ovum via 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 cloud of cumulus cells - mechanical barriers for the sperm. In order to penetrate the cumulus cell cloud, the sperm thus change to so-called "hyperactive" swimming behavior: The normal, symmetrical flagellar strike, with which they cover long distances, changes to an asymmetric, low-frequency pattern, which applies the necessary mechanical force to penetrate the cumulus cell cloud (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, in addition to hyperactivation, acrosomal exocytosis: hydrolytic enzymes stored in the acrosome are released by contact with the zona pellucida (Tulsiani et al., 1998, Experimental Cell Research 240; Gupta et al., 2011, American Journal of Reproductive Immunology 2011) and digest the zona pellucida glycoproteins. Only after the zona pellucida has been overcome, the sperm can fuse with the egg and fertilize it (Wassarman et al., 2001, Nat. Cell Biol. 3). Hyperactive swimming, acrosomal exocytosis, and capacitation, an indispensable process of maturation in the female genital tract, through which sperm become fertile, are controlled by intracellular Ca2+ (Publicover et al., 2007, Nat Cell Biol, vol. 9); it is also believed that the successful navigation of sperm through the fallopian tube also involves [Ca2+]i-changes (Breitbart, 2002, Mol. Cell Endocrinol. 187: 139-144; Publicover et al., 2008, Front Biosci 13). The Ca2 +-influx into the sperm from outside is crucial for triggering such changes in Ca2+. In human sperm, the Ca2+ influx is controlled by the sperm-specific CatSper (cation channel of sperm) Ca2+ channel that is located in the cell membrane of the sperm flagellum. The CatSper channel is essential for sperm function and fertilization. Mice lacking CatSper are infertile. CatSper-deficient sperm are unable to overcome the egg shell or the cumulus cell cloud because they can not hyperactivate (Ren et al., 2001, Nature 413; Carlson et al., 2003, Proc Natl Acad Sci USA, 100) and fail to reach the ovum. However, CatSper is expendable for sperm production and its basal motility. Hence, despite the indispensability of the CatSper function for fertilization success, no diagnostic procedure in the repertoire of andrology laboratories is currently able to detect the absence or a functional defect of the CatSper channel. Although sperm functionality is already determined based on microscopically easily detectable parameters like the amount of sperm and sperm showing progressive motility or morphological alterations, only easily detectable alterations of sperm motility can be diagnosed this way. Further, until now it has been considered safe to study the activity of CatSper in spermatozoa only with labor-intensive and cost-intensive fluorescence optical or electrophysiological measurements, which could