TNFSF11 Inhibition and Fertility: a Prospective Study
Condition: Testis; Infertility; Sperm
MeSH terms: Infertility
Study type: Interventional
Phase: Phase 2

Summary: 
      The receptor activator of NF-kB ligand (RANKL) system is considered important for bone
      homeostasis and comprises three important factors. RANKL exist in three isoforms but the
      predominant function is mediated by the transmembrane ligand that binds to a specific
      receptor (receptor activator of NF-KB (RANK)) on a neighbour cell that subsequently activates
      NFKB and regulates cell cycle OPG is an endogenous secreted protein that binds RANKL and
      inhibits its signalling.

      Thus, the RANK/RANKL system is vital for activation of the bone resorbing cells
      (osteoclasts). In bone the bone synthesizing cells (osteoblasts) express RANKL that signals
      to RANK on the immature osteoclasts. This induces proliferation and activation of the cells
      they start to proliferate and resorp bone. OPG is produced by somatic cells in the bone and
      this production is regulated by sex hormones, TGF-B and various other substances. Today a
      human made recombinant antibody against RANKL, Denosumab is used to treat osteoporosis as it
      inhibits RANKL signalling and thus causes less bone resorption in humans.

      RANKL, RANK and OPG are expressed in the testis and this pathway appears to be a novel
      regulator of germ cell proliferation. Decreased semen quality is a major factor of male
      infertility. Semen quality is a measure of the ability of the sperm to accomplish
      fertilization. Evaluation of male fertility potential is today basically conducted through
      semen analysis. There is no treatment for men with no sperm in the ejaculate and there exist
      no drug that can increase sperm counts.Therefore, drugs that can lower RANKL
      expression/activity for instance an antibody against RANKL such as Denosumab may be used for
      this new indication: A new treatment option of infertile men with impaired semen quality.
    

Detailed description: 
      The receptor activator of NF-kB ligand (RANKL) system is considered important for bone
      homeostasis . RANKL exist in three isoforms and the effects of all isoforms are mediated
      through binding to a specific receptor (receptor activator of NF-KB (RANK)). RANKL is
      predominantly found as a transmembrane protein and signalling is therefore dependent on
      cell-cell interaction to a neighbouring cell expressing RANK that subsequently activates NFKB
      and regulates cellular activation through regulation of cell cycle i.e proliferation,
      differentiation and apoptosis. RANKL-RANK interaction is modified by osteoprotegerin (OPG),
      which is an endogenous secreted protein that binds RANKL and inhibits its signalling.

      RANK/RANKL triggers a network of TRAF-mediated kinase cascades that promote osteoclast
      differentiation. RANKL is expressed on osteoblast cells and its receptor, Rank, on pre-
      osteoclastic cells. RANKL expression is stimulated by a number of factors, such as IL-1 ,
      IL-6, IL-11 , IL-17, TNF- α, vitamin D, Ca2+, parathyroid, glucocorticoids, prostaglandin E2,
      and immunosuppressive drugs, and is down-regulated by TGF-α. The RANK/RANKL interaction
      induces differentiation and formation of multinucleated mature osteoclasts, causing bone
      resorption. The third protein agonist, osteoprotegerin (OPG), is also produced by osteoblasts
      and is known to exert an inhibitory effect on the pre-osteoclastic differentiation process.
      By binding to RANKL also known as osteoprotegerin binding protein (OPGbp), OPG inhibits the
      RANK/RANKL interaction and subsequent osteoclastogenesis. OPG is thus a very efficient
      anti-resorptive agent. It also serves as a decoy receptor for the tumour necrosis
      factor-related apoptosis-inducing ligand (TRAIL) and increases cell survival by blocking the
      apoptotic effects of this ligand. The fact that the overexpression of OPG in mice results in
      severe osteopetrosis and that OPG-null mice are osteoporotic is testimony to the
      physiological importance of OPG. The lack of RANK or RANKL induces osteopetrosis in mice.

      Thus, the RANK/RANKL system is vital for activation of the bone resorping cells
      (osteoclasts). In the skeleton the bone synthesizing cells (osteoblasts) express RANKL that
      signals to RANK on the immature osteoclasts. This induces proliferation and activation of the
      cells they start to proliferate and resorp bone. OPG is produced by somatic cells in the bone
      and this production is regulated by sex hormones, TGF-B and various other substances. Today a
      human made recombinant antibody against RANKL, Denosumab is used to treat osteoporosis as it
      inhibits RANKL signalling and causes less bone resorption in humans. RANKL signalling has
      only two other known additional functions in healthy humans where it is involved in lactation
      and the immune response.

      The investigators have data showing that RANKL, RANK and OPG are expressed at both RNA and
      protein level in the human testis. The Sertoli cells express RANKL, while the germ cells
      express RANK and the peritubular cells express OPG. Normally, RANKL activates NFKB and
      activation of this pathway in the male gonad appears to regulate whether the testicular cells
      proliferate or undergo apoptosis in the testis. The investigators' in vitro, ex vivo and in
      vivo data from functional models support this suggestion and this pathway appears therefore
      to be a novel regulator of germ cell proliferation.

      Decreased semen quality is a major factor of male infertility. Semen quality is an indirect
      measure of the ability of the sperm to accomplish fertilization. Evaluation of male fertility
      potential is evaluated by semen analysis. Semen analysis evaluates certain characteristics
      and the most common variables measured to evaluate sperm quality are: sperm count, motility
      and morphology.

      There is no treatment for men with no spermatozoa in the ejaculate or even a drug that can
      increase sperm number in infertile men.Therefore, we suggest that antibodies against RANKL
      such as Denosumab, may be used as a novel treatment option for male infertility , which
      highlights a new indication for Denosumab treatment.

      The investigators will therefore test whether inhibition of RANKL by Denosumab in humans
      increases sperm production and semen quality in this small prospective intervention study.

      We will invite 15 infertile men for detailed screening to secure inclusion and study
      completion of anticipated 12 infertile men

      BIOSTATISTICAL ANALYSIS

      All the analyses will be performed according to Good Clinical Practice guidelines and the
      primary analyses in the intention-to-treat population, which included all patients who
      received the first dose of medicine on day 1. We will analyze the data in two ways. The
      primary analysis will proceed according to baseline values compared with outcomes variables
      after intervention. The secondary analysis will be based on stratifying the men according to
      subgroup analyses in relation to the predefined primary and secondary endpoints.

      Data analysis and quality The primary end points for this protocol will be changes in sperm
      production evaluated by total sperm number, sperm concentration followed by number of
      progressive and motile sperm, morphologically normal sperm, sperm motility, progressive
      motility and morfology which will be compared by paired t-test. Multiple secondary endpoints
      exist but for the initial investigation focus will be on changes of the following secondary
      endpoints: Sperm DFI, FSH, Inhibin B, serum OPG, RANKL, OPG, vitamin D and calcium
      homeostasis. Subjects who terminate participation after visit day 1 but before visit day 180
      will be included for data analysis up to the last day they provided semen and bloodsample.
      Men that only deliver semen samples occasionally or have missing data at any visit will still
      be included in the analysis. Men that do not meet the criteria in the protocol will be
      excluded from the analysis. Men with fever above 38.5 degrees Celsius will not be included in
      the analyses up to 3 months after the fever episode. Semen samples obtained with an
      abstinence period less than 24 hours or with a semen volume < 1.5 ml will not be included in
      the analyses. Those values will then be carried forward for analyses. A significance level of
      5% is used. For the primary analyses Bonferronu-Holm p-value correction is calculated
      additionally. For the secondary analysis no multiple test correction are used. Instead
      results are discussed in view of the multiple testing situations.

        1. Analyses between baseline versus different timepoints The first step will be to compare
           the changes in primary outcomes between baseline versus the different timepoints.
           Spermatogenesis normally takes up to 70 days in men and we will therefore determine the
           difference to all the individual timepoints and calculate average for days 80, 120 and
           180 and compare with baseline values to determine the effect of RANKL inhibition on the
           whole length of spermatogenesis. This analysis will show if there is a significant
           difference between groups. For outcomes measured repeatedly, this will entail comparing
           the estimated slopes, or rates of change, of each outcome between the groups. Mixed
           models allow for the correlation between the repeated observations baseline-day 1-day
           80- day 180 from each man to be suitably incorporated into parameter estimation. For all
           endpoints measured at baseline and day 180, paired t-tests will be used to assess there
           is a significant difference between the groups and determine whether the mean change
           within each group differs significantly from zero. In both cases, data will be
           transformed as necessary to meet model assumptions. Afterwards, the same analysis will
           be conducted by using multiple regression with relevant confounders such as season, BMI,
           smoking, duration of abstinence, time from ejaculation to motility assessment, fever
           etc. to see if this changes the results For outcomes measured that cannot be compared
           with t-test or other parametric tests at day 1, day 80 and day 180, groups will be
           compared using non-parametric tests such as Wilcoxon Mann-Whitney test. For Binary
           outcome the data will be compared between the two groups by means of conditional
           logistic-regression analysis with adjustment for relevant confounders (defined as being
           significantly p<0.05 associated).

        2. Analyses after stratification into subgroups Subjects will be grouped according to their
           BMI, semen quality, serum RANKL, OPG, calcium, PTH, osteocalcin or other bonefactors
           evaluated at the day of screening. The subgroup analyses will in accordance with normal
           clinical practice and stratification in appropriate groups according to the clinical
           (BMI <25, 25-30, >30 etc.), tertiles or highest/lowest versus remaining at baseline.
           Analyses will be performed on each timpeoint compared with baseline in addition to
           mean/median of visit day 80, 120, 180 with baseline and mean/median of all visits after
           intervention.
    

Intervention name(s): Denosumab
Intervention type(s): Drug
Other intervention name(s): Prolia
Intervention MeSH terms: Denosumab
Intervention model: Single Group Assignment
Masking: None (Open Label)
Primary purpose: Treatment
Keywords: RANKL