EP4078185A1 - Rankl, opg et rank utilisés en tant que biomarqueurs séminaux de l'infertilité masculine - Google Patents

Rankl, opg et rank utilisés en tant que biomarqueurs séminaux de l'infertilité masculine

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Publication number
EP4078185A1
EP4078185A1 EP20830065.7A EP20830065A EP4078185A1 EP 4078185 A1 EP4078185 A1 EP 4078185A1 EP 20830065 A EP20830065 A EP 20830065A EP 4078185 A1 EP4078185 A1 EP 4078185A1
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EP
European Patent Office
Prior art keywords
rankl
sample
level
rank
opg
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
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EP20830065.7A
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German (de)
English (en)
Inventor
Martin Blomberg JENSEN
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Xy Therapeutics Aps
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Fertilitypro Aps
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Publication date
Application filed by Fertilitypro Aps filed Critical Fertilitypro Aps
Publication of EP4078185A1 publication Critical patent/EP4078185A1/fr
Pending legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153 or CD154
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/342—Prostate diseases, e.g. BPH, prostatitis

Definitions

  • RANKL OPG AND RANK AS SEMINAL BIOMARKERS FOR MALE INFERTILITY
  • the present invention relates to methods for testing male fertility potential.
  • the present invention relates to methods for testing male fertility potential in a seminal fluid sample using soluble RANKL as biomarker.
  • Semen quality is a measure of the ability of the semen to accomplish fertilization. Evaluation of male fertility potential is today basically conducted through semen analysis. A semen analysis evaluates certain characteristics of a male's semen and the spermatozoa contained in the semen. The characteristics measured by the current tests for semen analysis are only some of the clinical important factors for semen quality. The most common variables measured to evaluate sperm quality are: sperm count, motility and morphology. Other variables are volume, fructose level and pH.
  • WO 2011/137906 discloses a method for predicting the fertility potential in a male mammal. In particular, it relates to a method for predicting the fertility potential in a male mammal by determining the expression of at least one protein of the vitamin D metabolising machinery in a semen sample.
  • EP3244911 B1 discloses a method for determining a likely effect of a treatment to improve male fertility, the method comprises determining the level of OPG in a blood serum sample.
  • WO 2015/018421 discloses RANKL targeting antibodies for use in the treatment of male infertility.
  • an improved method to test for male infertility would be advantageous, and in particular, a home kit to test for male infertility would be advantageous.
  • the present invention relates to the identification that RANKL, OPG and/or RANK in seminal fluid is a strong predictor of the fertility potential of a male subject.
  • the predictive value is almost as strong as the normally used predictors in the clinic such as sperm count, motility and morphology.
  • the method of the invention is suitable to be used in a home kit format similar to the established pregnancy test.
  • Example 1 provides data on seminal fluid RANKL and seminal fluid RANKL to serum RANKL ratio as biomarkers for the fertility potential of male subjects.
  • Example 2 shows that a known treatment of male infertility (Denosumab) lowers the RANKL levels in seminal fluid. In addition, the influence of Denosumab is different between RANKL in seminal fluid and blood.
  • Denosumab male infertility
  • Example 3 provides data on seminal fluid OPG as a biomarker for the fertility potential of male subjects.
  • Example 4 provides data on serum RANK and seminal fluid RANK as a biomarker for the fertility potential of male subjects.
  • an object of the present invention relates to the provision of novel biomarkers for male fertility potential.
  • one aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising
  • the level of RANKL is determined.
  • example 1 provides data for RANKL.
  • the seminal fluid RANKL levels are shown to be strong predictors of male fertility potential.
  • Another aspect of the present invention relates to a method for predicting the fertility potential of a male subject, the method comprising
  • semen RANKL to blood RANKL ratio is a strong predictor of male fertility potential.
  • Yet another aspect of the present invention is to provide a method for monitoring the development of the fertility potential for a male subject, the method comprising
  • o a higher RANKL level in the second sample compared the first sample is indicative of a worsened fertility potential
  • o a lower OPG and/or RANK level in the second sample compared the first sample is indicative of a worsened fertility potential
  • o equal RANKL and/or OPG and/or RANK levels in the first and second sample is indicative of an unchanged fertility potential
  • o a lower RANKL level in the second sample compared to the first sample is indicative of an improved fertility potential
  • o a higher OPG and/or RANK level in the second sample compared the first sample is indicative of an improved fertility potential.
  • Still another aspect of the present invention relates to the use of seminal RANKL levels, seminal OPG levels, and/or seminal RANK levels as a biomarker for male fertility potential.
  • the invention relates to the use of RANKL binding moieties, OPG binding moieties and/or RANK binding moieties to determine the fertility potential of a male subject in a semen sample from said subject.
  • An additional aspect of the invention relates to a detection device, such as a lateral flow device, comprising first binding moieties for RANKL, OPG and/or RANK; and second binding moieties functioning as positive controls, such as being specific for one or more protein components present in seminal fluid such as sperm specific (positive control).
  • a detection device such as a lateral flow device, comprising first binding moieties for RANKL, OPG and/or RANK; and second binding moieties functioning as positive controls, such as being specific for one or more protein components present in seminal fluid such as sperm specific (positive control).
  • An aspect of the invention relates to a kit comprising the detection device according to the invention; optionally a sampling container suitable for sampling a semen sample and/or for applying the sampled semen sample to the detection device; optionally instructions for using the kit in a method according to the invention; and optionally, washing and or dilution reagents for the semen sample optionally, devices and/or reagents to separate cells from fluid the component.
  • Figure 1 shows serum and seminal fluid concentrations of RANKL and semen quality.
  • A Serum RANKL (pmol/L)
  • B seminal RANKL (pmol/L)
  • C seminal/serum RANKL ratio in healthy and infertile men.
  • Figure 2 shows semen quality variables and seminal sRANKL levels in a pooled linear regression model of both healthy and infertile men.
  • A Total sperm
  • B Sperm motility
  • C Sperm concentration
  • D Progressive sperm motility
  • E Number of progressive motile Sperm
  • F Sperm morphology
  • Figure 3 shows seminal RANKL levels and semen quality stratified in groups according to WHO references for normal vs. low semen quality. Pooled analyses of all healthy and infertile men.
  • Figure 4 shows seminal/serum RANKL ratios and semen quality stratified in groups according to WHO classification of normal vs. low semen quality. Pooled analyses of all healthy and infertile men.
  • Figure 5 shows A) Receiver-operating characteristic (ROC) curve analysis and B) table showing sensitivity and 1-specificity for semen quality variables, seminal RANKL, and RANKL seminal/serum ratio as well as area under the curve (AUC) with 95 % confidence intervals (Cl). All beta and p-values are adjusted for duration of abstinence. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • ROC Receiver-operating characteristic
  • Figure 6 shows sRANKL levels before and after treatment with Denosumab (60 mg) in 10 infertile men.
  • A) Serum sRANKL (normalized, pmol/L) (n 10).
  • B) Seminal fluid levels of sRANKL (pmol/L) at day 80 to 120 compared with baseline (n 8). Dotted line indicates higher reference limit for the ELISA. Variables are presented after normalization to the two samples delivered prior to treatment start. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 7 shows an inverse correlation between seminal OPG and seminal sRANKL.
  • Receptor activator of nuclear factor kappa-B ligand also known as tumor necrosis factor ligand superfamily member 11 (TNFSF11), TNF-related activation- induced cytokine (TRANCE), osteoprotegerin ligand (OPGL), and osteoclast differentiation factor (ODF), is a protein that in humans is encoded by the TNFSF11 gene.
  • RNFSF11 tumor necrosis factor ligand superfamily member 11
  • TRANCE TNF-related activation- induced cytokine
  • OPGL osteoprotegerin ligand
  • ODF osteoclast differentiation factor
  • RANKL is known as a type II membrane protein and is a member of the tumor necrosis factor (TNF) superfamily. RANKL has been identified to affect the immune system and control bone regeneration and remodeling. RANKL is an apoptosis regulator gene, a binding partner of osteoprotegerin (OPG), a ligand for the receptor RANK and controls cell proliferation by modifying protein levels of Id4,
  • RANKL is expressed in several tissues and organs including: skeletal muscle, thymus, liver, colon, small intestine, adrenal gland, osteoblast, mammary gland epithelial cells, prostate and pancreas. Variation in concentration levels of RANKL throughout several organs reconfirms the importance of RANKL in tissue growth (particularly bone growth) and immune functions within the body.
  • RANKL NF-KB ligand
  • the RANKL system is a powerful regulator of bone resorption that comprises three components: RANKL, a transmembrane ligand that following binding to the receptor RANK on a neighbouring cell subsequently activates NF-KB and regulates cell cycle i.e. proliferation, differentiation and apoptosis.
  • the transmembrane RANKL protein resides in osteocytes and activates RANK in immature osteoclasts, which induces osteoclastogenesis and promotes bone resorption.
  • Osteoprotegerin (OPG) is an endogenous secreted decoy receptor that binds RANKL and blocks its signaling thereby preventing osteoclast differentiation and activation.
  • RANKL can also be found in circulation, suggesting a putative endocrine function of the protein. Indeed, recently novel extra-skeletal functions of RANKL have been proposed including regulation of glucose homeostasis. In the human seminiferous tubules, we found a marked transcriptional expression of RANKL, RANK and OPG was found (Fig. 3A).
  • RANKL All three isoforms of RANKL were expressed in the human testis and both the transmembrane and soluble forms of RANKL were expressed at moderate to high levels.
  • antibodies targeting transmembrane or extracellular domain respectively we found expression of RANKL in adult Sertoli cells and in male germ cells.
  • soluble RANKL or "sRANKL” refer to the free fraction of RANKL. sRANKL is not bound to OPG.
  • RANK nuclear factor k B
  • TRANCE receptor also known as TRANCE receptor or TNFRSF11A
  • TNFR tumor necrosis factor receptor
  • RANK is the receptor for RANK-Ligand (RANKL) and part of the RANK/RANKIVOPG signaling pathway that regulates osteoclast differentiation and activation. It is associated with bone remodeling and repair, immune cell function, lymph node development, thermal regulation, and mammary gland development.
  • Osteoprotegerin (OPG) is a decoy receptor for RANK, and regulates the stimulation of the RANK signaling pathway by competing for RANKL.
  • the cytoplasmic domain of RANK binds TRAFs 1, 2, 3, 5, and 6 which transmit signals to downstream targets such as NF-KB and JNK.
  • RANKL was expressed in the cytoplasm/membrane of mature Sertoli cells and in spermatocytes and spermatids (Fig. 1A). RANK was exclusively expressed in the cytoplasm of germ cells with the most prominent expression in spermatogonia.
  • OPG was expressed in the cytoplasm of peritubular cells, the junction between Sertoli and spermatogonia and in spermatids (see also example section) OPG
  • Osteoprotegerin also known as osteoclastogenesis inhibitory factor (OCIF) or tumour necrosis factor receptor superfamily member 11B (TNFRSF11B)
  • OPG osteoclastogenesis inhibitory factor
  • TNFRSF11B tumour necrosis factor receptor superfamily member 11B
  • OCIF osteoclastogenesis inhibitory factor
  • TNFRSF11B tumour necrosis factor receptor superfamily member 11B
  • RANK was exclusively expressed in the cytoplasm/membrane of the germ cells with the most prominent expression in spermatogonia, while OPG was expressed mainly in the cytoplasm of peritubular cells or on the border of the spermatogonia and in spermatids (Fig. 3B; Fig. S5B; Table SI).
  • the soluble isoform of RANKL (sRANKL, observed at 27-31 kDa) appeared to be expressed at a higher level than the transmembrane isoform (45 kDa) (Fig. S5C). Both forms of RANKL were together with RANK and OPG robustly expressed in samples with normal spermatogenesis. FertiHtv/fertiHtv potential
  • a person has a high sperm number (above 40 millions), above 50% motile sperm and more than 12 % morphological normal spermatozoa the person is regarded to have a normal fertility potential.
  • a person has a low sperm number (below 40 millions), below 50% motile sperm or less than 12 % morphological normal spermatozoa the person is regarded to unlikely to have a normal fertility potential.
  • Semen analysis is currently a cumbersome and lengthy process. It is performed by standard microscopy and is based on the subjective analysis of three key parameters: motility, morphology, and total sperm number.
  • the analysis has several drawbacks as it necessitates a trained medical staff, presence of the patient with a fresh semen sample (less than 1 hour old), laboratory facilities etc. making it a costly and time consuming procedure.
  • the analysis requires the patient to deliver a semen sample at the laboratory where the testing is performed, which for many patients is considered a major embarrassment.
  • semen analyses using the current methods is highly prone to intra- and inter observer variability due to the subjective assessment of the key parameters, which severely can affect the utility of the analysis, whereby the clinician is troubled in the guidance of the couple to the relevant assisted reproductive method.
  • references to a "male subject” or “subject” or an “individual” includes a human or non-human species of mamals including primates, livestock animals (e.g. sheep, cows, pigs, horses, donkey, goats), laboratory test animals (e.g. mice, rats, rabbits, guinea pigs, hamsters) and companion animals (e.g. dogs, cats).
  • livestock animals e.g. sheep, cows, pigs, horses, donkey, goats
  • laboratory test animals e.g. mice, rats, rabbits, guinea pigs, hamsters
  • companion animals e.g. dogs, cats.
  • the present invention has applicability, therefore, in human medicine as well as having livestock and veterinary and wild life applications.
  • the mammal is a human.
  • the mammal is a man. Semen sample
  • Semen is a mixture of an excreted fluid and cells.
  • the fluid is also known as seminal fluid that usually contains spermatozoa. It is secreted by the gonads (sexual glands) and other sexual organs of male or hermaphroditic animals and may be able to fertilize female ova.
  • seminal fluid may contain several components besides spermatozoa: proteolytic and other enzymes as well as fructose are elements of seminal fluid which promote the survival of spermatozoa and provide a medium through which they mature and get the ability to move or "swim". The process that results in the discharge of semen is called ejaculation.
  • the semen sample is to be understood as a sample comprising semen and/or components derived from semen.
  • the semen sample is a sample comprising spermatozoa.
  • the semen sample may be obtained after ejaculation, aspiration from the testis, epididymis or after testicular biopsy or microdissection of the testis.
  • the semen sample is obtained after ejaculation.
  • the semen sample is a sample comprising spermatozoa and/or components derived from an ejaculate. Procedures for collecting semen samples from human or animals such as farm animals is well described in the literature and well known for a person skilled in the art.
  • a minimum of handling steps of the sample is necessary before measuring the expression level(s).
  • the subject "handling steps” relates to any kind of pre treatment of the semen sample before determining the expression level(s) of the at least one protein of the vitamin D metabolizing machinery.
  • Pre-treatment procedures includes washing, lysis, immunocapture, cytospin, fixation, separation, spin down, filtration, dilution, distillation, concentration, inactivation of interfering compounds, centrifugation, heating, fixation, addition of reagents, or chemical treatment.
  • the semen sample is an ejaculate.
  • the ejaculate may be lysed, fixed or otherwise modified upon direct ejaculation in to a container containing chemicals enabling this.
  • the sample may be up-concentrated by centrifugation using a gradient such as Percoll gradient centrifugation.
  • the sample may be up concentrated by centrifugation without a gradient.
  • said pre-treatment procedures comprises mixing the semen sample with a phosphate buffered saline solution and subsequently spinning down the sample.
  • the freshly delivered semen sample is centrifuged and cellular sediments collected before determining the expression level of the at least one protein of the vitamin D metabolizing machinery.
  • the cellular sediment may be fixed using an appropriate fixative such as but not limited to ethanol or formaldehyde.
  • Sperm may be "washed” by density gradient centrifugation or by a "direct swim- up” technique that doesn't involve centrifugation.
  • the sperm are separated from the seminal fluid before determining the expression level of the at least one protein of the vitamin metabolizing machinery.
  • the sample is a raw unmodified semen sample.
  • the raw unmodified semen sample may be fresh.
  • One aspect of the present invention relates to a method wherein the semen sample may be stored for several days before determination of the expression level(s).
  • pre-treatment of the samples such as but not limited to cytospin of the sample prolongs the time the sample can be stored.
  • the samples may be stored for at least one day, such as at least 7 days such as at least 30 days.
  • the samples may be stored for several years before detecting the expression level(s) such as at least one year, such as at least two years such as at least five years such as at least 10 years.
  • chemicals facilitating storage are added to the semen sample.
  • one embodiment of the present invention enables the male individual to make the semen sample in the privacy of his own home and subsequently sending the sample to laboratory for determining the fertility potential.
  • the expression level or “level” as used herein refers to the absolute or relative amount of protein in a given sample.
  • the expression level refers to the amount of protein in a sample.
  • the expression level is usually detected using conventional detection methods.
  • the expression level refers to the total protein level of the protein in question in a semen sample.
  • Antibodies of the invention include polyclonal, monospecific polyclonal, monoclonal, recombinant, chimeric, humanized, fully human, single chain and/or bispecific antibodies.
  • Antibody fragments include those portions of an anti-RANKL antibody which bind to an epitope on an RANKL polypeptide. Examples of such fragments include Fab F(ab'), F(ab)', Fv, and sFv fragments.
  • the antibodies may be generated by enzymatic cleavage of full-length antibodies or by recombinant DNA techniques, such as expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
  • Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen.
  • An antigen is a molecule or a portion of a molecule capable of being bound by an antibody, which is additionally capable of inducing an animal to produce antibody capable of binding to an epitope of that antigen.
  • An antigen can have one or more epitopes. The specific reaction referred to above is meant to indicate that the antigen will react, in a highly selective manner, with its corresponding antibody and not with the multitude of other antibodies which can be evoked by other antigens.
  • Polyclonal antibodies directed toward a RANKL polypeptide generally are raised in animals (e.g., rabbits or mice) by multiple subcutaneous or intraperitoneal injections of RANKL and an adjuvant.
  • Monoclonal antibodies contain a substantially homogeneous population of antibodies specific to antigens, which population contains substantially similar epitope binding sites. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
  • a hybridoma producing a monoclonal antibody of the present invention may be cultivated in vitro, in situ, or in vivo. Production of high titers in vivo or in situ is a preferred method of production.
  • Monoclonal antibodies directed toward OPGbp/RANKL are produced using any method, which provides for the production of antibody molecules by continuous cell lines in culture.
  • Examples of suitable methods for preparing monoclonal antibodies include hybridoma methods of Kohler et al., Nature 256, 495-497 (1975), and the human B-cell hybridoma method, Kozbor, J. Immunol. 133, 3001 (1984 ); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987 ); and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory (1988 ); the contents of which references are incorporated entirely herein by reference.
  • a particularly preferred method for producing monoclonal antibodies directed towards OPGbp/RANKL involves immunizing the XenoMouse as described in Green, LL, J. Immunol. Methods (1999), Vol. 231, 11-25, with a OPGbp/RANKL peptide, such as a full-length human RANKL protein.
  • Preferred anti-RANKL or anti-RANK antibodies include monoclonal antibodies which will inhibit partially or completely the binding of human RANKL to its cognate receptor, RANK, or an antibody having substantially the same specific binding characteristics, as well as fragments and regions thereof.
  • Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988 ), Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol., 92:589- 601 (1983).
  • Chimeric antibodies are molecules in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • chimeric antibody includes monovalent, divalent or polyvalent immunoglobulins.
  • a monovalent chimeric antibody is a dimer (HL) formed by a chimeric H chain associated through disulfide bridges with a chimeric L chain.
  • a divalent chimeric antibody is tetramer (H2L2) formed by two HL dimers associated through at least one disulfide bridge.
  • a polyvalent chimeric antibody can also be produced, for example, by employing a CH region that aggregates (e.g., from an IgM H chain, or [micro] chain).
  • Murine and chimeric antibodies, fragments and regions of the present invention may comprise individual heavy (H) and/or light (L) immunoglobulin chains.
  • a chimeric H chain comprises an antigen binding region derived from the H chain of a non-human antibody specific for RANKL, which is linked to at least a portion of a human H chain C region (CR), such as CHI or CH2.
  • a chimeric L chain according to the present invention comprises an antigen binding region derived from the L chain of a non-human antibody specific for RANKL, linked to at least a portion of a human L chain C region (CL).
  • Selective binding agents such as antibodies, fragments, or derivatives, having chimeric H chains and L chains of the same or different variable region binding specificity, can also be prepared by appropriate association of the individual polypeptide chains, according to known method steps, e.g., according to Ausubel et al., eds. Current Protocols in Molecular Biology, Wiley Interscience, N.Y. (1993 ), and Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988 ). The contents of these references are incorporated entirely herein by reference.
  • hosts expressing chimeric H chains (or their derivatives) are separately cultured from hosts expressing chimeric L chains (or their derivatives), and the immunoglobulin chains are separately recovered and then associated.
  • the hosts can be co-cultured and the chains allowed to associate spontaneously in the culture medium, followed by recovery of the assembled immunoglobulin, fragment or derivative.
  • the term "reference level” relates to a standard in relation to a quantity, which other values or characteristics can be compared to.
  • a reference level it is possible to determine a reference level by investigating the seminal fluid RANKL levels in samples from healthy subjects (in the present context fertile male subjects). By applying different statistical means, such as multivariate analysis, one or more reference levels can be calculated.
  • a cut-off may be obtained that shows the relationship between the level(s) detected and patients at risk.
  • the cut-off can thereby be used e.g. to determine the seminal fluid RANKL levels, which corresponds to for instance an increased risk of being infertile.
  • the present inventors have successfully developed a new method to predict the risk of a male subject to be infertile.
  • a cut-off (reference level) must be established. This cut-off may be established by the laboratory, the physician or on a case-by- case basis for each patient.
  • the cut-off level could be established using a number of methods, including: multivariate statistical tests (such as partial least squares discriminant analysis (PLS-DA), random forest, support vector machine, etc.), percentiles, mean plus or minus standard deviation(s); median value; fold changes.
  • multivariate statistical tests such as partial least squares discriminant analysis (PLS-DA), random forest, support vector machine, etc.
  • percentiles mean plus or minus standard deviation(s); median value; fold changes.
  • the multivariate discriminant analysis and other risk assessments can be performed on the free or commercially available computer statistical packages (SAS, SPSS, Matlab, R, etc.) or other statistical software packages or screening software known to those skilled in the art.
  • changing the risk cut-off level could change the results of the discriminant analysis for each subject.
  • Statistics enables evaluation of the significance of each level.
  • Commonly used statistical tests applied to a data set include t-test, f-test or even more advanced tests and methods of comparing data. Using such a test or method enables the determination of whether two or more samples are significantly different or not.
  • the significance may be determined by the standard statistical methodology known by the person skilled in the art.
  • the chosen reference level may be changed depending on the mammal/subject for which the test is applied.
  • the subject according to the invention is a human subject, such as a male subject considered at risk of being infertile.
  • the chosen reference level may be changed if desired to give a different specificity or sensitivity as known in the art.
  • Sensitivity and specificity are widely used statistics to describe and quantify how good and reliable a biomarker or a diagnostic test is. Sensitivity evaluates how good a biomarker or a diagnostic test is at detecting a disease, while specificity estimates how likely an individual (i.e. control, patient without disease) can be correctly identified as not at risk.
  • TP true positives
  • TN true negatives
  • FN false negatives
  • FP false positives
  • the sensitivity refers to the measures of the proportion of actual positives which are correctly identified as such - in analogy with a diagnostic test, i.e. the percentage of mammals or people having a fertility potential below normal who are identified as having a fertility potential below normal.
  • sensitivity of a test can be described as the proportion of true positives of the total number with the target disorder i.e. a fertility potential below normal. All patients with the target disorder are the sum of (detected) true positives (TP) and (undetected) false negatives (FN).
  • the specificity refers to measures of the proportion of negatives which are correctly identified - i.e. the percentage of mammal with a normal fertility potential that are identified as not having a fertility potential below normal.
  • the ideal diagnostic test is a test that has 100 % specificity, i.e. only detects mammal with a fertility potential below normal and therefore no false positive results, and 100 % sensitivity, and i.e. detects all mammals with a fertility potential below normal and therefore no false negative results.
  • the ideal diagnostic test is a test that has 100% specificity, i.e. only detects mammals with a fertility potential below normal and therefore no false positive results, and 100% sensitivity, and i.e. detects all mammals with a fertility potential below normal and therefore no false negative results.
  • 100% specificity i.e. only detects mammals with a fertility potential below normal and therefore no false positive results
  • 100% sensitivity i.e. detects all mammals with a fertility potential below normal and therefore no false negative results.
  • due to biological diversity no method can be expected to have 100% sensitive without including a substantial number of false negative results.
  • the chosen specificity determines the percentage of false positive cases that can be accepted in a given study/population and by a given institution. By decreasing specificity an increase in sensitivity is achieved.
  • One example is a specificity of 95% that will result in a 5% rate of false positive cases. With a given prevalence of 1% of e.g. a fertility potential below normal in a screening population, a 95% specificity means that 5 individuals will undergo further physical examination in order to detect one (1) fertility potential below normal if the sensitivity of the test is 100%.
  • the present invention relates to the identification that RANKL, OPG and RANK in seminal fluid are strong predictors of the fertility potential of a male subject.
  • the predictive value of RANKL is almost as strong as the normally used predictors in the clinic such as sperm count, motility and morphology.
  • the method of the invention is suitable to be used in a home kit format similar to the established pregnancy test.
  • Example 1 provides data on seminal fluid RANKL as biomarkers for the fertility potential of male subjects.
  • an aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising
  • the level of RANKL is determined, more preferably the level of sRANKL is determined.
  • example 1 provides data for RANKL.
  • the seminal fluid RANKL levels are shown to be strong predictors of male fertility potential.
  • the level of RANK is determined.
  • example 4 provides data for RANK.
  • the serum RANK levels are shown to be strong (negative) predictor of male fertility potential.
  • the seminal fluid RANK levels are shown to be strong (negative) predictor of male fertility potential.
  • the level of OPG is determined. Again, example 3 provides data for OPG.
  • the semen sample comprises seminal fluid. In yet a preferred embodiment, the sample comprises semen.
  • the level is the protein level, such as protein level of RANKL, such as soluble RANKL.
  • Protein levels can be determined in different ways.
  • said determination of the at least one protein level is performed using a method selected from the group consisting of immunohistochemistry, immunocytochemistry, FACS, ImageStream, Western Blotting, ELISA, Luminex, Multiplex, Immunoblotting, TRF-assays, immunochromatographic lateral flow assays, Enzyme Multiplied Immunoassay Techniques, RAST test, Radioimmunoassays, immunofluorescence and immunological dry stick assays, such as a lateral flow assay.
  • said determination is performed by ELISA, immunocytochemistry or a lateral flow assay.
  • the semen sample may be provided in different forms for the method.
  • the semen sample is an unmodified, diluted, washed or purified semen sample, preferably an unmodified semen sample.
  • the reference level may be selected by the clinician and of course depends on a required sensitivity and specificity.
  • the reference level is in the range 1-76 pmol/l seminal sRANKL, such as 20-76 pmol/l seminal sRANKL, 40-76 pmol/l seminal sRANKL, 1-50 pmol/l seminal sRANKL, 1-30 pmol/l seminal sRANKL, or 1-20 pmol/l seminal sRANKL.
  • the reference level for RANK is in the range 0-500 ng/ml seminal RANK, such as 0-300 ng/ml seminal RANK, 0-200 ng/ml seminal RANK, 0-150 ng/ml I seminal RANK, 1-120 ng/ml seminal RANK, or 0-100 ng/ml seminal RANK.
  • the reference level is in the range 0-1000 pmol/l seminal OPG, such as 0-800 pmol/l seminal OPG, 0-500 pmol/l seminal OPG, 0-400pmol/l seminal OPG, 5-400 pmol/l seminal OPG, or 5-350 pmol/l seminal OPG.
  • the RANKL, RANK and/or OPG is determined in serum or in both seminal fluid and serum.
  • the ratio between serum and seminal fluid for one or more of RANKL, RANK and/or OPG is determined.
  • seminal or serum RANKIVOPG ratio is determined.
  • the predictive value of the method of the invention may be further improved if a ratio between semen levels and blood levels are used.
  • a disadvantage of also using blood values is that the method becomes less suitable for home use, since a blood sample is also required.
  • such method using multiple sample types may still be relevant if it provides a stronger indication of the fertility potential for a male subject considered at risk of having a low fertility potential.
  • another aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising
  • semen RANKL to blood RANKL ratio is a strong predictor of male fertility potential.
  • the ratio is selected from the group consisting of semen (such as seminal fluid) RANKL to blood RANKL, semen (such as seminal fluid) OPG to blood OPG and semen (such as seminal fluid) RANK to blood RANK.
  • the ratio is semen (such as seminal fluid) RANKL to blood RANKL.
  • the blood sample is a whole blood sample, a blood serum sample or a blood plasma sample.
  • the methods of the invention may also be used for monitoring the development of the fertility potential for a male subject.
  • a further aspect of the invention relates to a method for monitoring the development of the fertility potential for a male subject, the method comprising
  • o a higher RANKL level in the second sample compared the first sample is indicative of a worsened fertility potential
  • o a lower OPG and/or RANK level in the second sample compared the first sample is indicative of a worsened fertility potential
  • o equal RANKL and/or OPG and/or RANK levels in the first and second sample is indicative of an unchanged fertility potential
  • o a lower RANKL level in the second sample compared to the first sample is indicative of an improved fertility potential
  • o a higher OPG and/or RANK level in the second sample compared the first sample is indicative of an improved fertility potential.
  • At least the level of RANKL is measured in the first and second sample.
  • Such monitoring may also be used to monitor the effect of a treatment against male infertility (low fertility potential).
  • a treatment against male infertility has taken place between the sampling of the first and second sample.
  • the treatment is treatment with an RANKL inhibitor, such as an antibody binding to RANKL, such as Denosumab.
  • an RANKL inhibitor such as an antibody binding to RANKL, such as Denosumab.
  • the treatment is a change of diet, exercise, no smoking or reduction of smoking, Vitamin D supplement, and/or treatment with a RANKL inhibitor.
  • the treatment also includes a change in life style, such as change of diet, exercise, and reduction of smoking.
  • the method of this aspect of the invention may also include determining the corresponding levels in blood plasma and using ratio between the levels in the seminal sample and the level in the blood sample to monitor the development of the fertility potential of a male subject.
  • o a higher seminal RANKL level to blood RANKL level ratio in the later obtained samples is indicative of a worsened fertility potential;
  • o a lower seminal OPG level to blood OPG level ratio in the later obtained samples is indicative of a worsened fertility potential;
  • o a lower seminal RANK level to blood RANK level ratio in the later obtained samples is indicative of a worsened fertility potential;
  • o an equal RANKL ratio and/or OPG ratio and/or RANK ratio in the samples obtained at different time points is indicative of an unchanged fertility potential;
  • o a lower seminal RANKL level to blood RANKL level ratio in the later obtained samples is indicative of an improved fertility potential;
  • o a higher seminal OPG level to blood OPG level ratio in the later obtained samples is indicative of an improved fertility potential;
  • o a higher seminal RANK level to blood RANK level ratio in the later obtained samples is indicative of an improved fertility potential;
  • the invention relates to the use of seminal RANKL levels, seminal OPG levels, and/or seminal RANK levels as a biomarker for male fertility potential.
  • the invention relates to the use of RANKL binding moieties, OPG binding moieties and/or RANK binding moieties to determine the fertility potential of a male subject in a semen sample from said subject.
  • the binding moiety is a RANKL binding moiety.
  • the RANKL binding moiety binds to soluble RANKL, such as binds to full length RANKL, or truncated versions of RANKL, such as binds to C- terminal or N-terminal truncated versions of RANKL.
  • soluble RANKL such as binds to full length RANKL
  • truncated versions of RANKL such as binds to C- terminal or N-terminal truncated versions of RANKL.
  • the epitope has to be in the extracellular region of the RANKL.
  • Antibody Species Target Provider catalog (cat. no.) designation RANKL C- Rabbit Human soluble receptor Abeam (Ab-9957) term activator of NF-Kappa B Ligand
  • RANKL TM Rabbit Amino acids 46-317 of full Santa Cruz Biotech length RANKL of human (sc-9073) origin
  • RANKL Mouse Amino acids 74-308 of Novus biologicals extrac. monoclonal human RANKL (NBP2-61813, clone 8A7B9)
  • the binding moiety is selected from the group consisting of polyclonal antibody, a monoclonal antibody, an antibody wherein the heavy chain and the light chain are connected by a flexible linker, an Fv molecule, an antigen binding fragment, a Fab fragment, a Fab' fragment, a F(ab')2 molecule, a fully human antibody, a humanized antibody, a chimeric antibody and a single domain antibody (sdAb) (nanobody).
  • sdAb single domain antibody
  • the use is performed in a dry-stick assay, a lateral flow assay, or a lateral flow immunochromatographic assay.
  • the assay is a competitive assay or a sandwich assay.
  • an aspect of the invention relates to a detection device, such as a lateral flow device, comprising • first binding moieties for RANKL, OPG and/or RANK; and • second binding moieties functioning as positive controls, such as being specific for one or more protein components present in seminal fluid such as sperm specific (positive control).
  • the second binding moieties may be specific for semen and/or seminal fluid.
  • the second binding moieties have affinity for protamins, Catsper, soluble adenylyl cyclase or septins such as septin4. These biomarkers are specific for that there is sperm in the sample, thus being sperm specific.
  • the second binding moieties have affinity for sperm specific proteins.
  • a positive control may also simply be an antibody only having affinity for a binding moiety in the detection device, thereby only being a positive control for the colouring reaction. Thus not having affinity for an analyte in the (semen) sample.
  • the first binding moiety binds to RANKL.
  • the binding moieties are tagged.
  • at least some of the first and/or second binding moieties are coupled/conjugated to tags, such as gold, latex, fluorophores; and/or wherein at least some of the first and/or second binding moieties a coupled to the surface on the detection device, such as on a test line of a lateral flow device or the control line of a lateral flow device.
  • the detection device according to the invention is in the form of a test strip, or a lateral flow device.
  • kits comprising
  • sampling container suitable for sampling a semen sample and/or for applying the sampled semen sample to the detection device
  • Example 1 - RANKL in seminal fluid is a biomarker for male fertility potential
  • Semen samples were delivered in an adjacent room in the out-patient clinic and information on duration of abstinence, fever, and spillage was obtained. The two semen samples were delivered 10-16 days apart prior to treatment start and analysis was conducted as described in detail previously (J. Clin. Endocrinol.
  • RANKL in seminal fluid RANKL in serum
  • total number of sperm RANKL
  • semen concentration concentration of progressive motile sperm
  • concentration of morphological normal sperm concentration of morphological normal sperm.
  • Seminal RANKL levels were set as dependent in linear regression analyses with hormones, whereas it was set independent in linear regression with seminal parameters.
  • RANKL levels, hormonal levels, and seminal parameters were compared in infertile men and healthy men, followed by a pooled analyses of all the men. All hormonal analyses were adjusted for BMI and analyses on semen quality were also adjusted for duration of abstinence.
  • sRANKL was not linked with LH, FSH, SHBG, inhibin B, or testicular size (data not shown).
  • ROC analysis was used to determine whether seminal and seminal/serum sRANKL had any predictive value for distinguishing infertile from normal men compared with semen quality variables (Fig. 5).
  • a threshold of 20 pmol/l gives a specificity 68% and a sensitivity 63% and a threshold of 30 pmol/l gives a specificity 79% and a sensitivity 42%.
  • Seminal RANKL level is a strong predictor for male fertility potential seminal/serum sRANKL ratio is an even stronger predictor, but of course requires a serum sample.
  • kit for home use By using only seminal RANKL as predictor for male fertility potential, it is possible to develop a kit for home use to test fertility. Such as kit could be in a lateral flow format, similar to the well-known pregnancy test.
  • Example 2 - RANKL inhibitor Denosumab can block RANKL in serum but not to same extent in seminal fluid
  • RANKL targeting antibodies for use in the treatment of male infertility.
  • Denosumab To test the influence of Denosumab on RANKL levels in seminal fluid and blood in infertile men, a single dose of Denosumab (Prolia®, 60 mg) was injected subcutaneously into 12 infertile men. sRANKL, and OPG were evaluated at day 5, 20, 40, 80, 120, and 180 after injection to avoid missing an early antiapoptotic effect, but most importantly to assess semen quality after a full cycle of spermatogenesis. Therefore, the prespecified statistical analysis stated that direct comparisons between timepoints starting at day 80 with baseline to be conducted initially to avoid adjustment for multiple adjustments. RANKL levels were normalized to the average of the two samples delivered prior to treatment start.
  • Denosumab clearly has a different influence on RANKL levels in seminal fluid and blood, clearly indicating a difference.
  • One injection of Denosumab 60 mg completely suppresses sRANKL in serum already 5 days after injection to zero.
  • sRANKL is undetectable in serum for 120 days then it starts to recover and is back to baseline levels.
  • the seminal fluid there is a lower sRANKL after denosumab treatment but very few are fully repressed and most are detectable. This implies that RANKL system here is regulated very differently.
  • Example 3 - OPG in seminal fluid is a biomarker for male fertility potential
  • OPG is a positive predictive marker for semen quality variables except semen volume since seminal fluid sRANKL concentrations were significantly negatively associated with all semen quality variables except semen volume ( Figure 2 and example 1).
  • Seminal RANKL level is a strong predictor for male fertility potential seminal/serum sRANKL ratio is an even stronger predictor, but of course requires a serum sample.
  • OPG is also present in much higher abundance in seminal fluid compared with serum and is strongly inversely associated with seminal RANKL fluid levels. This implies that OPG in seminal fluid is a positive predictor of male fertility potential and it is possible to develop a kit for home use to test fertility. Such as kit could be in a lateral flow format, similar to the well-known pregnancy test either alone or in combination with RANKL as they may complement each other.
  • Example 4 - RANK in serum and seminal fluid is a biomarker for male fertility potential
  • kits for home use could be in a lateral flow format, similar to the well-known pregnancy test either alone or in combination with RANKL or OPG as they may complement each other.

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Abstract

Selon la présente invention, RANKL, OPG et/ou RANK contenus dans le liquide séminal sont de forts prédicteurs du potentiel de fertilité d'un individu de sexe masculin. Leur valeur prédictive est presque aussi forte que celle des prédicteurs couramment utilisés dans la clinique, tels que la numération, la motilité et la morphologie des spermatozoïdes. La méthode selon l'invention est appropriée pour être utilisée sous un format de kit domestique, similaire à un test de grossesse par exemple.
EP20830065.7A 2019-12-18 2020-12-18 Rankl, opg et rank utilisés en tant que biomarqueurs séminaux de l'infertilité masculine Pending EP4078185A1 (fr)

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US20130045890A1 (en) 2010-05-03 2013-02-21 Rigshospitalet Copenhagen University Hospital Test for male fertility
JP6599861B2 (ja) 2013-08-07 2019-10-30 リグスホスピタレット コペンハーゲン ユニバーシティ ホスピタル 男性不妊治療用の抗体、化合物、およびその誘導体
DK3244911T3 (da) * 2015-01-12 2019-12-16 Rigshospitalet Copenhagen Univ Hospital Fremgangsmåde til bestemmelse af en sandsynlig virkning af en behandling til forbedring af mandlig ufrugtbarhed

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