WO2021121525A1

WO2021121525A1 - Rankl, opg and rank as seminal biomarkers for male infertility

Info

Publication number: WO2021121525A1
Application number: PCT/DK2020/050395
Authority: WO
Inventors: Martin Blomberg JENSEN
Original assignee: Fertilitypro Aps
Priority date: 2019-12-18
Filing date: 2020-12-18
Publication date: 2021-06-24
Also published as: EP4078185A1

Abstract

The present invention relates to the identification that RANKL, OPG and/or RANK in seminal fluid are strong predictor of the fertility potential of a male subject. The predictive value is almost as strong as the classically 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 e.g. similar to the established pregnancy test.

Description

RANKL, OPG AND RANK AS SEMINAL BIOMARKERS FOR MALE INFERTILITY

Technical field of the invention

The present invention relates to methods for testing male fertility potential. In particular, the present invention relates to methods for testing male fertility potential in a seminal fluid sample using soluble RANKL as biomarker.

Background of the invention

Decreased semen quality is a major factor of male infertility. 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.

In particular, it would be of great benefit to have a test, which could predict whether the person should continue at home the natural way or with the mild insemination or if the couple should proceed to the more invasive IVF or ICSI. Thus, developing a marker that can guide the doctor in this choice would be a clear advantage.

Hence, 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.

Summary of the invention

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.

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.

Thus, an object of the present invention relates to the provision of novel biomarkers for male fertility potential.

In particular, it is an object of the present invention to provide a novel method for predicting male fertility potential that solves the above mentioned problems of the prior art with complex and/or time consuming sample analysis. Thus, one aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising

• determining the level of RANKL and/or OPG and/or RANK in a semen sample from a male subject;

• comparing said level(s) to one or more corresponding reference levels; and

• predicting that said subject is unlikely to have normal fertility potential when: o said RANKL level is above said corresponding reference level; and/or o said OPG and/or RANK level(s) are below said corresponding reference level (s);

OR

• predicting that said subject is likely to have a normal fertility potential when: o said RANKL level are equal to or below said corresponding reference level; and/or o determining that said subject is likely to have a normal fertility potential, when said OPG and/or RANK level are equal to or above said corresponding reference level(s).

In a preferred embodiment, the level of RANKL is determined. Again, example 1 provides data for RANKL. For example, in Figure IB, 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

• determining a first level of RANKL and/or OPG and/or RANK in a semen sample from the male subject;

• determining a second level of RANKL and/or OPG and/or RANK in a blood sample, which has been obtained from the male subject;

• determining one or more ratios between the corresponding level(s) in the first sample and the level(s) in the second sample;

• comparing said determined ratio to one or more corresponding reference levels; and

• predicting that said subject is unlikely to have normal fertility potential when: o said RANKL ratio is above said corresponding reference level, and/or o said RANK and/or OPG ratio(s) are below said corresponding reference level(s),

OR · predicting that said subject is likely to have normal fertility potential when: o said RANKL ratio are equal to or below said corresponding reference level, and/or o said RANK and/or OPG ratio(s) are equal to or above said corresponding reference level(s).

As shown in in example 1 and specifically in Figure 1C, 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

• determining a first level of RANKL and/or OPG and/or RANK in a first semen sample from the male subject;

• determining a second level of RANKL and/or OPG and/or RANK in a second semen sample from the male subject, wherein the second sample has been obtained at a later time point than the first sample;

• comparing corresponding levels in the first and second sample;

• wherein 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; and o a lower RANKL level in the second sample compared to the first sample is indicative of an improved fertility potential; and/or 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. In yet a further aspect 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).

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.

Brief description of the figures

Figure 1 shows serum and seminal fluid concentrations of RANKL and semen quality. (A) Serum RANKL (pmol/L), (B) seminal RANKL (pmol/L) and (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.

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.

Figure 8 shows RANK concentrations (ng/ml) in (A) serum (p=0.12) and (B) seminal fluid (p=0.10), from subjects with normal fertility or infertile subjects.

Data presented as mean + sem.

The present invention will now be described in more detail in the following.

Detailed description of the invention

Definitions

Prior to discussing the present invention in further details, the following terms and conventions will first be defined: RANKL

Receptor activator of nuclear factor kappa-B ligand (RANKL), 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.

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,

Id2 and cyclin Dl. 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.

Studies in human testis and vitamin D receptor ( VDR ) knock-out mice have shown that several bone factors such as Runx2, osterix, and osteocalcin are expressed in the normal testis and testicular cancer. One of these factors is the receptor activator of NF-KB ligand (RANKL). 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). 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. By using antibodies targeting transmembrane or extracellular domain, respectively we found expression of RANKL in adult Sertoli cells and in male germ cells. The level of RANKL expression varied within samples but was detected in the majority of Sertoli cells and in spermatocytes and spermatids (see also example section).

In the present context, the terms "soluble RANKL" or "sRANKL" refer to the free fraction of RANKL. sRANKL is not bound to OPG.

RANK

Receptor activator of nuclear factor k B (RANK), also known as TRANCE receptor or TNFRSF11A, is a member of the tumor necrosis factor receptor (TNFR) molecular sub-family. 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.

In mice, RANKL, RANK and OPG were detected at transcriptional and protein level in the testis at 16-18 weeks of age. 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 (OPG), also known as osteoclastogenesis inhibitory factor (OCIF) or tumour necrosis factor receptor superfamily member 11B (TNFRSF11B), is a cytokine receptor of the tumour necrosis factor (TNF) receptor superfamily encoded by the TNFRSF11B gene.

In humans, 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

In the present context, if 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. In the present context, if 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.

The skilled person would know how to perform such analysis and set a different cut-off between when a male subject is considered to have normal fertility.

This is not an accurate estimate of male fertility potential and the result of the semen analysis does not always correlate with the ability to have children. This is also illustrated by the fact that WHO has recently changed the reference levels as a large proportion of the male population otherwise would be regarded as having a fertility potential below normal. No precise method for predicting the male fertility potential exists presently.

More than 9 % of all babies are born after assisted reproductive methods in Denmark, and the incidence of infertility is increasing worldwide. About 50% of cases are attributable to the male partner and the first step in the clinical evaluation involves semen analysis to evaluate male fertility potential. The clinicians use the analysis in deciding the appropriate reproductive method.

It is estimated that some 1.400.000 semen analyses are performed each year worldwide.

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. Logistically, 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. Furthermore, 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.

Due to above mentioned problems with the currently used methods, a large need exist for a new method for semen analysis, which accurately and objectively can assess male fertility potential in order to stratify patients into the correct reproductive method. The problems with the existing analysis are illustrated by the latest WHO recommendations from 2010, where the reference values for normal semen analysis were changed and caused a huge debate in fertility and andrology meetings worldwide.

Male subject

Reference 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). The present invention has applicability, therefore, in human medicine as well as having livestock and veterinary and wild life applications. In a preferred embodiment, the mammal is a human. In a particular preferred embodiment, 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. In humans, 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. In a preferred embodiment, the semen sample is to be understood as a sample comprising semen and/or components derived from semen.

In a preferred embodiment, the semen sample is a sample comprising spermatozoa. Rarely in the clinic subjects are seen with semen samples comprising very few or even no spermatozoa in their semen, samples from such a subjects is still of interest and thus included by the present definition of the sample base, as such samples would indeed be a sample which would be indicative of low fertility potential. The semen sample may be obtained after ejaculation, aspiration from the testis, epididymis or after testicular biopsy or microdissection of the testis.

In the most preferred embodiment, the semen sample is obtained after ejaculation.

In a particular preferred embodiment, 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.

In another embodiment of the present invention, a minimum of handling steps of the sample is necessary before measuring the expression level(s).

In the present context, 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.

In a preferred embodiment the semen sample is an ejaculate.

In one embodiment, the ejaculate may be lysed, fixed or otherwise modified upon direct ejaculation in to a container containing chemicals enabling this.

In an embodiment, the sample may be up-concentrated by centrifugation using a gradient such as Percoll gradient centrifugation. In another preferred embodiment, the sample may be up concentrated by centrifugation without a gradient.

In a presently preferred embodiment said pre-treatment procedures comprises mixing the semen sample with a phosphate buffered saline solution and subsequently spinning down the sample.

In a particular preferred embodiment 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.

In another embodiment the sperm are separated from the seminal fluid before determining the expression level of the at least one protein of the vitamin metabolizing machinery.

In a particular preferred embodiment, no pre-treatment of the sample is necessary. In another particular preferred embodiment, the sample is a raw unmodified semen sample. The raw unmodified semen sample may be fresh. As outlined above, since the method of the invention is suited for home use, it is preferred that no pre-treatment is required.

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). In a preferred embodiment pre-treatment of the samples such as but not limited to cytospin of the sample prolongs the time the sample can be stored.

In a preferred embodiment the samples may be stored for at least one day, such as at least 7 days such as at least 30 days. In another preferred embodiment 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.

In one embodiment chemicals facilitating storage are added to the semen sample.

Thus, 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.

Expression level

The "expression level" or "level" as used herein refers to the absolute or relative amount of protein in a given sample. Thus, the expression level refers to the amount of protein in a sample. The expression level is usually detected using conventional detection methods. In another preferred embodiment, 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 (mAbs) 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.

The term "chimeric antibody", as used herein, 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. With this approach, 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. Alternatively, 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.

Reference level

In the context of the present invention, the term "reference level" relates to a standard in relation to a quantity, which other values or characteristics can be compared to.

In one embodiment of the present invention, 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.

Based on these results, 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. Risk Assessment

The present inventors have successfully developed a new method to predict the risk of a male subject to be infertile. To determine whether a patient has an increased risk of being 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.

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.

As obvious to one skilled in the art, in any of the embodiments discussed above, 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.

Preferably, 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.

Several terms are used along with the description of sensitivity and specificity; true positives (TP), true negatives (TN), false negatives (FN) and false positives (FP). If a disease is proven to be present in a sick patient, the result of the diagnostic test is considered to be TP. If a disease is not present in an individual (i.e. control, patient without disease), and the diagnostic test confirms the absence of disease, the test result is TN. If the diagnostic test indicates the presence of disease in an individual with no such disease, the test result is FP. Finally, if the diagnostic test indicates no presence of disease in a patient with disease, the test result is FN.

Sensitivity

As used herein 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.

Usually the 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).

Specificity

As used herein 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. For any test, there is usually a trade-off between each measure. For example in a manufacturing setting in which one is testing for faults, one may be willing to risk discarding functioning components (low specificity), in order to increase the chance of identifying nearly all faulty components (high sensitivity). This trade-off can be represented graphically using a ROC curve.

Selecting a sensitivity and specificity it is possible to obtain the optimal outcome in a detection method. In determining the discriminating value distinguishing mammals having a fertility potential below normal, the person skilled in the art has to predetermine the level of specificity. 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. However, 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%.

As will be generally understood by those skilled in the art, methods for screening for fertility potentials are processes of decision making and therefore the chosen specificity and sensitivity depends on what is considered to be the optimal outcome by a given institution/clinical personnel.

Method for predicting the fertility potential of a male subject As outlined above, 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. For example, 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.

Thus, an aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising

• comparing said level(s) to one or more corresponding reference levels; and

OR

In a preferred embodiment, the level of RANKL is determined, more preferably the level of sRANKL is determined. Again, example 1 provides data for RANKL. For example, in Figure IB, the seminal fluid RANKL levels are shown to be strong predictors of male fertility potential.

In another preferred embodiment, the level of RANK is determined. Again, example 4 provides data for RANK. For example, in Figure 8A, the serum RANK levels are shown to be strong (negative) predictor of male fertility potential. In Figure 8B, the seminal fluid RANK levels are shown to be strong (negative) predictor of male fertility potential.

In yet another preferred embodiment, the level of OPG is determined. Again, example 3 provides data for OPG. In another preferred embodiment, the semen sample comprises seminal fluid. In yet a preferred embodiment, the sample comprises semen.

In yet an embodiment, the level is the protein level, such as protein level of RANKL, such as soluble RANKL.

Protein levels can be determined in different ways. Thus, in an embodiment, 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. In a preferred embodiment, said determination is performed by ELISA, immunocytochemistry or a lateral flow assay.

The semen sample may be provided in different forms for the method. Thus, in an embodiment, the semen sample is an unmodified, diluted, washed or purified semen sample, preferably an unmodified semen sample.

As also previously outlined, the reference level, or "cut-off" may be selected by the clinician and of course depends on a required sensitivity and specificity. In an embodiment, 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.

In an embodiment, 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.

In an embodiment, 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. It is noted that in an aspect of the invention the RANKL, RANK and/or OPG is determined in serum or in both seminal fluid and serum. Further in an embodiment, the ratio between serum and seminal fluid for one or more of RANKL, RANK and/or OPG is determined. In yet an embodiment seminal or serum RANKIVOPG ratio is determined.

Method for predicting the fertility potential of a male subject - ratio

The predictive value of the method of the invention may be further improved if a ratio between semen levels and blood levels are used. However, it is noted that a disadvantage of also using blood values is that the method becomes less suitable for home use, since a blood sample is also required. For e.g. hospital use, 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. Thus, another aspect of the invention relates to a method for predicting the fertility potential of a male subject, the method comprising

OR

• predicting that said subject is likely to have normal fertility potential when: o said RANKL ratio are equal to or below said corresponding reference level, and/or o said RANK and/or OPG ratio(s) are equal to or above said corresponding reference level(s). As shown in in example 1 and specifically in Figure 1C, semen RANKL to blood RANKL ratio is a strong predictor of male fertility potential. Thus, in an embodiment, 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. In a preferred embodiment, the ratio is semen (such as seminal fluid) RANKL to blood RANKL.

In an embodiment, the blood sample is a whole blood sample, a blood serum sample or a blood plasma sample.

Method for monitoring the development of the fertility potential

The methods of the invention may also be used for monitoring the development of the fertility potential for a male subject. Thus, 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

• comparing corresponding levels in the first and second sample;

• wherein 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; and o a lower RANKL level in the second sample compared to the first sample is indicative of an improved fertility potential; and/or o a higher OPG and/or RANK level in the second sample compared the first sample is indicative of an improved fertility potential.

In a preferred embodiment, 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). Thus, in an embodiment, a treatment against male infertility has taken place between the sampling of the first and second sample.

In an embodiment, the treatment is treatment with an RANKL inhibitor, such as an antibody binding to RANKL, such as Denosumab.

In another embodiment, the treatment is a change of diet, exercise, no smoking or reduction of smoking, Vitamin D supplement, and/or treatment with a RANKL inhibitor. Thus in the present context the treatment also includes a change in life style, such as change of diet, exercise, and reduction of smoking.

It is to be understood that the method of this aspect of the invention (monitoring the development of the fertility potential), 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. In such as case: 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; and 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. Uses

In a further aspect, 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.

In yet a further aspect 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. In a preferred use, the binding moiety is a RANKL binding moiety.

In an embodiment, 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. Preferably binds to the C- terminal of RANKL. Thus, preferably the epitope has to be in the extracellular region of the RANKL.

Examples of antibodies suitable for the present invention are listed in the table below.

Antibody Species Target Provider (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 C terminal ELISA, Biomedica antibodies

RANKL Mouse, Amino acids 74-308 of Novus biologicals extrac. monoclonal human RANKL (NBP2-61813, clone 8A7B9)

RANK C-term Rabbit Amino acids 317-616 of Santa Cruz Biotech human RANK (sc-9072)

RANK Rabbit Amino Acids 261-379 of Atlas Antibodies

Internal RANK (HPA027728)

In another embodiment, 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).

In yet a further embodiment, the use is performed in a dry-stick assay, a lateral flow assay, or a lateral flow immunochromatographic assay.

In another embodiment, the assay is a competitive assay or a sandwich assay.

Detection device

As outlined above, the method of the invention may be performed in a lateral flow device similar to the well-known pregnancy test format. Thus, 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. Thus, in an embodiment, 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. In yet an embodiment, 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.

In a preferred embodiment, the first binding moiety binds to RANKL.

For some devices, it may be advantageous if the binding moieties are tagged. Thus, in an embodiment, 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.

In an embodiment, the detection device according to the invention is in the form of a test strip, or a lateral flow device.

Kits

The methods and detection devices of the invention may also be incorporated in a kit format. Thus, in an aspect, 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, such as a filter.

There is relatively few cells and a lot of fluid in seminal fluid.

It should be noted that embodiments and features described in the context of one of the aspects of the present invention also apply to the other aspects of the invention.

All patent and non-patent references cited in the present application, are hereby incorporated by reference in their entirety.

The invention will now be described in further details in the following non-limiting examples.

Examples

Example 1 - RANKL in seminal fluid is a biomarker for male fertility potential

Aim of study

To determine if RANKL in seminal fluid is a biomarker for male fertility potential.

Materials and methods Semen analysis:

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.

Metab 103, 870 (2018) and Hum. Reprod. 26, 1307 (2011)). Briefly, semen volume was determined by weighing, sperm concentration was determined using a Burker-Turk hemocytometer, and total sperm count was calculated. Sperm morphology was assessed according to strict criteria on Papanicolaou-stained smears. Sperm motility classified as progressive motile (WHO class A+B), nonprogressive motile (class C), or immotile (class D) was determined in duplicate at two times and presented as AB or ABC motility. Spermatozoa DNA fragmentation was investigated at baseline and 20 and 120 days after intervention at SPZ Laboratory (Copenhagen, Denmark). In brief, 0.5 mL semen was diluted with TNE buffer, then mixed and frozen directly in liquid nitrogen until fluorescent staining according to the sperm chromatin structure assay protocol and then analyzed using a FACSCalibur (BD Biosciences, San Jose,CA) flow cytometer. All samples were run blinded in duplicate and recording stopped after 5000 events.

Statistics:

All values were expressed as mean ± SEM. p<0.05 was considered statistically significant. The following phenotypic variables: sperm count, organ weight, motility and gene-expression (some logarithmic transformed) were tested using Student's t-test or one-way analysis of variance (ANOVA) with Dunnets test to adjust for multiple comparions. No outliers were excluded from analyses. Cross- sectional data from Copenhagen bone gonadal study were expressed as mean ± SEM except sperm concentration and total sperm count (Median). Associations between serum or seminal fluid levels of RANKL and OPG with semen quality and reproductive hormones were conducted after natural logarithmic transformation and adjustment for BMI, age, and duration of abstinence for all semen variables. Subsequently, all men were stratified according to WHO criteria for semen quality variables. Evaluation of gaussian distribution was done by plotting residuals and as a result the following variables were transformed with natural logarithm: RANKL in seminal fluid, RANKL in serum, total number of sperm, semen concentration, concentration of progressive motile sperm, and 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.

Results

Interestingly, seminal fluid levels of sRANKL were 100-fold higher than corresponding serum levels (average serum RANKL = 0.27 pmol/L vs. seminal RANKL 26.19 pmol/L) in both infertile and normal men (Fig. 1A and IB), but there was no correlation between seminal and serum RANKL concentration (data not shown).

Infertile men had higher seminal RANKL concentration (p=7xl0 ⁹) and lower serum RANKL levels (p=7xl0^-28) compared with healthy men (Fig. 1A-C). Seminal sRANKL concentrations were positively associated with serum estradiol (b0.007, p=0.000002) and negatively with testosterone (b-0.30, p=0.004) after adjustment for BMI. sRANKL was not linked with LH, FSH, SHBG, inhibin B, or testicular size (data not shown).

Seminal fluid sRANKL concentrations were significantly negatively associated with all semen quality variables except semen volume (Figure 2). sRANKL remained inversely associated with total number of sperm (b-0.010, p=0.043) (Figure 2A), sperm concentration (b-0.012, p=0.015) (Figure 2C), progressive motility (b- 0.210, p=0.001) (Figure 2D), sperm motility (b-0.132, p=0.0120) (Figure 2B), sperm morphology (b-0.026, p=0.025) (Figure 2F), concentration of progressive motile sperm (b-0.018, p=0.001) (Figure 2E) , and concentration of morphologically normal sperm (b-0.019, p=0.002) after adjustment for duration of abstinence. All associations remained statistically significant after adjustment for both abstinence and BMI except total sperm count and morphology.

The seminal/serum sRANKL ratio was also negatively associated with all semen quality variables even after adjustment of relevant confounders (data not shown).

Seminal sRANKL concentration was significantly higher in poor versus good quality semen quality groups after stratification according to WHO criteria and adjusted for duration of abstinence; WHO threshold for total number of sperm 40 mill/sample (p=0.056) (Figure 3A), Sperm concentration = 15 mill/mL (NS) (Figure 3B), severe oligospermia sperm concentration <5 mill/mL (mean 3.14 vs. 2.90, p=0.035), low motility < 40% (mean 3.14 vs. 2.82, p=0.002) (Figure 3C), low sperm morphology <4% (mean 3.10 vs. 2.84, p=0.006) (Figure 3D). sRANKL in seminal fluid was determined in 31 infertile men delivering two semen samples on average 14 days apart, and there was a high correlation between the two samples (Spearman correlation r = 0.91) despite that the samples were diluted 1:60 prior to analyses.

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). Seminal fluid RANKL had an area under the curve of 67% [AUC=0.67] thus exceeding the predictive value for total sperm count [AUC=0.66] but being lower than sperm motility [AUC=0.85] and morphology [AUC=0.79]. Interestingly, seminal/serum RANKL ratio had an [AUC=0.83] and was a better predictor than all investigated variables except sperm motility.

Further, 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%.

Conclusion

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.

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

Aim of study

To determine if there is a difference on RANKL levels in seminal fluid and in blood after treatment with the RANKL inhibitor Denosumab. WO 2015/018421 discloses RANKL targeting antibodies for use in the treatment of male infertility.

Materials and methods

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. One man was lost to follow up and one man had high fever at the beginning of the trial and was therefore per protocol excluded from the analysis leaving 10 men in the study. Results sRANKL concentration in serum was completely repressed instantly in all men following injection of Denosumab and sRANKL was first detectable again (except in one man) at days 120-180 (Fig. 6A). Furthermore, Denosumab lowered sRANKL levels in the seminal fluid, but sRANKL was not completely repressed and remained detectable in most men at days 80 and 120 (Fig. 6B). Noteworthy, two of the 10 men enrolled in the study were responsible for a pregnancy during the study and their spouses conceived naturally with healthy live births.

Conclusion 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. In 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

Aim of study To determine if OPG in seminal fluid is a biomarker for male fertility potential.

Materials and methods

Semen analysis was conducted exactly as described in example 1. Results

Interestingly, seminal fluid levels of OPG were several fold higher than corresponding serum levels in both infertile and normal men (200 pm versus 4 pm). Serum OPG ranged from 1-6 pmol/l while seminal fluid OPG ranged from 90- 330 pmol/l.

Interestingly, there was an inverse correlation between serum OPG and sRANKL (r²=0.12) (data not shown). However, the inverse association was much stronger in the seminal fluid (r²=0.92) (figure 7).

This implies that 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).

Conclusion

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

Aim of study

To determine if RANK in serum and seminal fluid is a biomarker for male fertility potential.

Materials and methods

Semen analysis was conducted exactly as described in example 1. Results

Interestingly, serum RANK is several fold higher in infertile men compared with normal men (figure 8A). Also, seminal RANK is higher in infertile men compared with normal men (figure 8B). Serum and seminal fluid levels of RANK in normal men is at the same level while seminal RANK levels also are several fold higher than RANKL in seminal fluid of normal men. Again, considerable variability exist. This implies that RANK can be used as a negative predictive marker for male fertility potential.

Conclusion Serum and Seminal RANK levels are strong predictors for male fertility potential. It is therefore 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 or OPG as they may complement each other.

Claims

1. A method for predicting the fertility potential of a male subject, the method comprising

• comparing said level(s) to one or more corresponding reference levels; and

OR

2. The method according to claim 1, wherein the level of RANKL is determined, preferably the level of sRANKL.

3. The method according to any of the preceding claims, wherein the semen sample comprises seminal fluid.

4. The method according to any of the preceding claims, wherein the level is the protein level, such as protein level of RANKL, such as soluble RANKL.

5. The method according to claim 4, wherein 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.

6. The method according to claim 5, wherein said determination is performed by ELISA, immunocytochemistry or a lateral flow assay.

7. The method according to any of the preceding claims, wherein the semen sample is an unmodified, diluted, washed or purified semen sample, preferably an unmodified semen sample.

8. The method according to any of the preceding claims, wherein 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.

9. A method for predicting the fertility potential of a male subject, the method comprising

• determining a first level of RANKL and/or OPG and/or RANK in a semen sample from the male subject; · determining a second level of RANKL and/or OPG and/or RANK in a blood sample, which has been obtained from the male subject;

• predicting that said subject is unlikely to have normal fertility potential when: o said RANKL ratio are above said corresponding reference level, and/or o said RANK and/or OPG ratio(s) are below said corresponding reference level (s).

OR

• predicting that said subject is likely to have normal fertility potential when: o said RANKL ratio are equal to or below said corresponding reference level, and/or o said RANK and/or OPG ratio(s) are equal to or above said corresponding reference level(s),

10. The method according to claim 9, wherein the ratio is selected from the group consisting of seminal fluid RANKL to blood RANKL, seminal fluid OPG to blood OPG and seminal fluid RANK to blood RANK.

11. The method according to claim 9 or 10, wherein the ratio is semen RANKL to blood RANKL.

12. The method according to any of claims 9-11, wherein the blood sample is a whole blood sample, a blood serum sample or a blood plasma sample, preferably a blood serum sample.

13. A method for monitoring the development of the fertility potential for a male subject, the method comprising

• comparing corresponding levels in the first and second sample;

• wherein o a higher RANKL level in the second sample compared the first sample is indicative of a worsened fertility potential; and/or 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; and o a lower RANKL level in the second sample compared to the first sample is indicative of an improved fertility potential; and/or o a higher OPG and/or RANK level in the second sample compared the first sample is indicative of an improved fertility potential.

14. The method according to claim 13, wherein at least the level of RANKL is measured in the first and second sample.

15. The method according to claim 13 or 14, wherein a treatment against male infertility has taken place between the sampling of the first and second sample.

16. The method according to claim 15, wherein the treatment is treatment with an RANKL inhibitor, such as an antibody binding to RANKL, such as Denusomab.

17. The method according to claim 16, wherein the treatment is a change of diet, exercise, no smoking or reduction of smoking, Vitamin D supplement, and/or treatment with a RANKL inhibitor.

18. Use of seminal RANKL levels, seminal OPG levels, and/or seminal RANK levels as a biomarker for male fertility potential.

19. 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.

20. The use according to claim 19, wherein the binding moiety is a RANKL binding moiety.

21. The use according to claim 20, wherein 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, preferably binds to the C-terminal of RANKL.

22. The use according to any of claims 19-21, wherein 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).

23. The use according to any of claims 19-22, wherein the use is performed in a dry-stick assay, a lateral flow assay, or a lateral flow immunochromatographic assay.

24. The use according to claim 23, wherein the assay is a competitive or a sandwich assays.

25. 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).

26. The detection device according to claim 25, wherein the second binding moieties have affinity for protamins, Catsper, solube adenylyl cyclase or septin4.

27. The detection device according to claim 25 or 26, wherein the first binding moiety binds to RANKL.

28. The detection device according to any of claim 25-27, wherein 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.

29. The detection device according to any of claims 25-28, being in the form of a test strip, and/or a lateral flow device.

30. A kit comprising

• the detection device according to any of claims 25-29;

• optionally instructions for using the kit in a method according to any of claims 1-17; and

• optionally, washing and or dilution reagents for the semen sample