<clinical_study>
  <!-- This xml conforms to an XML Schema at:
    https://clinicaltrials.gov/ct2/html/images/info/public.xsd -->
  <required_header>
    <download_date>ClinicalTrials.gov processed this data on September 20, 2023</download_date>
    <link_text>Link to the current ClinicalTrials.gov record.</link_text>
    <url>https://clinicaltrials.gov/ct2/show/NCT03030196</url>
  </required_header>
  <id_info>
    <org_study_id>01112016</org_study_id>
    <secondary_id>2016-003546-84</secondary_id>
    <nct_id>NCT03030196</nct_id>
  </id_info>
  <brief_title>Denosumab and Male Infertility: a RCT</brief_title>
  <official_title>Denosumab and Male Infertility: a Randomized Controlled Double-blinded Intervention Study</official_title>
  <sponsors>
    <lead_sponsor>
      <agency>Martin Blomberg Jensen</agency>
      <agency_class>Other</agency_class>
    </lead_sponsor>
  </sponsors>
  <source>Rigshospitalet, Denmark</source>
  <oversight_info>
    <has_dmc>Yes</has_dmc>
  </oversight_info>
  <brief_summary>
    <textblock>
      To determine the significance of systemic RANKL inhibition for male reproduction, conducting&#xD;
      a clinical controlled randomized double blinded intervention study on infertile men, to&#xD;
      investigating whether Denosumab (Prolia) can increase semen quality and to investigate what&#xD;
      subgroup of infertile men that might benefit from treatment.&#xD;
    </textblock>
  </brief_summary>
  <detailed_description>
    <textblock>
      INTRODUCTION&#xD;
&#xD;
      Infertility is a serious problem estimated to affect 7-26% of all couples globally (1;2).&#xD;
      Approximately 9% of all newborns were conceived by assisted reproductive techniques in 2013&#xD;
      in Denmark (Danish Fertility Society). Impaired semen quality is the causal or contributing&#xD;
      factor in almost 50% of all cases of infertility (3;4). Today, there exist no treatments that&#xD;
      can improve semen quality of most infertile men. Instead, the vast majority of infertile&#xD;
      couples are treated with assisted reproductive techniques (ART) independently of the&#xD;
      aetiology (maternal/paternal) causing the infertility (3;5-8). The treatment choice ranges&#xD;
      from mild intrauterine inseminations (IUI) to more invasive in vitro fertilization (IVF) or&#xD;
      intracytoplasmic sperm injection (ICSI). ART is successful for more than 70% of all couples.&#xD;
      However, ART is very expensive and associated with maternal side effects due to the invasive&#xD;
      methodology and the need for hormonal treatment, often for several months (9-11). Infertility&#xD;
      is also a financial burden to society due to the costs of ART to maintain an acceptable&#xD;
      annual birth rate.&#xD;
&#xD;
      Fertility potential determined by semen quality is established already as a fetus (12). In&#xD;
      case the testes don't develop normally, reduced semen quality in adulthood will be the result&#xD;
      as a result of impaired Sertoli cell function. Up until today it has barely been investigated&#xD;
      whether intervention during adulthood can improve semen quality.&#xD;
&#xD;
      Our recent studies using both human testis and Vitamin D receptor knock-out mice revealed&#xD;
      that the vitamin D regulated bone factor RANKL is expressed in the testis (13;14). This is a&#xD;
      novel finding, because up until now RANKL has only been known to affect bone homeostasis and&#xD;
      to some extend influence the immune system, inflammation and lactation (15;16). In the&#xD;
      skeleton RANKL is expressed in osteoblasts and binds to its specific receptor RANK in&#xD;
      osteoclasts to induce osteoclastogenesis and bone resorption (17). This activation is&#xD;
      controlled by osteoprotegerin (OPG), which binds to the ligand domain of RANKL and thus&#xD;
      inhibits RANK activation and osteoclastogenesis, ultimately reducing bone resorption (18).&#xD;
      This knowledge has been utilized into development of an OPG-analogue drug, a recombinant&#xD;
      human monoclonal IGg2 antibody Denosumab (Prolia by Amgen), which inhibits RANKL and thereby&#xD;
      bone resorption and is indicated for use in both osteoporosis and bone metastases (19;20).&#xD;
      Prolia has in several clinical studies shown to be safe and is approved as treatment for&#xD;
      osteoporosis in both men and women (21-24). Newer data indicate that RANKL/OPG may be&#xD;
      involved in regulation of pancreatic function, insulin and glucagon production which support&#xD;
      a possible indirect effect on reproduction through bone-pancreatic-gonadal axis.&#xD;
&#xD;
      AIM OF STUDY&#xD;
&#xD;
      To determine the significance of systemic RANKL inhibition for male reproduction by&#xD;
      investigating whether Denosumab (Prolia) can increase semen quality and particularly to&#xD;
      identify the group of men that will benefit from the treatment.&#xD;
&#xD;
      DESIGN&#xD;
&#xD;
      Single center, prospective randomized double blinded* clinical controlled intervention.&#xD;
&#xD;
      Patients and methods:&#xD;
&#xD;
      95 infertile men, otherwise healthy, will be recruited among men referred to investigation&#xD;
      for infertility at the outpatient clinic at Dpt. of Growth and Reproduction, Rigshopitalet.&#xD;
      Neither choosing to participate, nor declining, will affect further outpatient clinic&#xD;
      treatment.&#xD;
&#xD;
      The 95 participants are randomly allocated to receiving either&#xD;
&#xD;
        -  Denosumab s.c. injection 60 mg x1, or&#xD;
&#xD;
        -  Placebo, Sodium Chloride s.c. injection x 1&#xD;
&#xD;
      All participants deliver semen samples, blood samples and have DXA-scan performed before and&#xD;
      after intervention. All receive supplements with Vitamin D and calcium to avoid&#xD;
      hypocalcaemia, a known, temporary side effect to Denosumab.&#xD;
&#xD;
      *double blinded studies require that placebo and active substance are identical. Placebo can&#xD;
      only be obtained from Amgen and we were not able to receive or purchase placebo. Instead the&#xD;
      study will be double blinded to patient, medical doctors and all who participates in&#xD;
      data-handling and analyses - only one nurse giving the medicine will be unblinded. She will&#xD;
      know whether she injects active substance or placebo.&#xD;
&#xD;
      Inclusion criteria&#xD;
&#xD;
        -  Men &gt; 18 years of age referred due to infertility in need for further investigation with&#xD;
&#xD;
        -  Sperm concentration ≥ 0,05 mio./ml.&#xD;
&#xD;
        -  Sperm concentration &lt;15 mio./ml or&#xD;
&#xD;
        -  &lt;40 % motile spermatozoer (ABC) or&#xD;
&#xD;
        -  &lt;4 % morphologically normal sperm (strict criteria)&#xD;
&#xD;
      Exclusion criteria&#xD;
&#xD;
        -  Men with chronic diseases (diabetes mellitus, thyroid disease, endocrine diseases&#xD;
           requiring treatment, malignant diseases or diseases known to be affected by- or&#xD;
           interfere with vitamin D supplements (granulomatous diseases such as sarcoidosis,&#xD;
           tuberculosis, wegeners, vasculitis as well as inflammatory bowel diseases e.g. chron's&#xD;
           disease or ulcerative colitis etc.).&#xD;
&#xD;
        -  Men with active or previous malignant disease&#xD;
&#xD;
        -  Any case with indication for testis biopsy,&#xD;
&#xD;
        -  Total calcium &lt; 2.14 mmol/l&#xD;
&#xD;
        -  25-OH Vitamin D &lt; 25 nmol/L&#xD;
&#xD;
        -  Poor dental status or dental implants&#xD;
&#xD;
        -  Men with obstructive oligospermia or who has been vasectomized&#xD;
&#xD;
        -  Serum Inhibin B &lt; 30 pg/ml&#xD;
&#xD;
        -  Abnormal karyotype&#xD;
&#xD;
        -  Patients practicing excessive exercise&#xD;
&#xD;
      SAMPLE SIZE CALCULATION AND STATISTICS&#xD;
&#xD;
      With a test level of 5% (level of significance), a power of 80% and 40 men in each group will&#xD;
      we be able to detect a change in total number of motile sperm of 75%, and a change in sperm&#xD;
      concentration (indexed to baseline value) of 25%.&#xD;
&#xD;
      We expect to include a total of 95 men, to take into account the possibility of dropouts.&#xD;
      This corresponds to a dropout rate of 10%, in case the rate is lower, chances of detecting a&#xD;
      significant difference will be above 80%.&#xD;
&#xD;
      Since we expect 50% of all infertile men in the outpatient clinic, to meet inclusion criteria&#xD;
      and 50% of these to consent to participation, we anticipate screening of 380 men in total.&#xD;
&#xD;
      BIOSTATISTICAL ANALYSIS&#xD;
&#xD;
      All the analyses will be performed according to Good Clinical Practice guidelines and the&#xD;
      primary analyses in the intention-to-treat population, which included all patients who&#xD;
      underwent randomization and received the first dose of medicine on day 1. We will analyze the&#xD;
      data in 2 ways. The primary analysis will proceed according to the randomized group&#xD;
      assignment. The secondary analysis will be based on stratifying the men according to subgroup&#xD;
      analyses in relation to the predefined primary and secondary endpoints.&#xD;
&#xD;
      The primary end points for this protocol will be changes in sperm production evaluated by:&#xD;
      total motile spermatozoa (106), total progressive motile spermatozoa (106), total spermatozoa&#xD;
      (106) or sperm concentration (106/ml). Due to the low sample size and large intraindividual&#xD;
      variation in sperm production primary endpoints will also be evaluated after normalization to&#xD;
      the mean average level prior to the intervention. Based on the first intervention estimated&#xD;
      60% of the men will benefit from the treatment and this study is designed to determine&#xD;
      whether serum OPG, RANKL or phosphate can be used to identify men who will benefit from the&#xD;
      treatment. The use of these specified endpoints will be useful for determining efficacy.&#xD;
      Multiple secondary endpoints exist but for the initial investigation focus will be on: Sperm&#xD;
      DFI, morphology, motility, semen volume, pregnancy rate, Inhibin B, vitamin D and calcium&#xD;
      homeostasis. Subjects who terminate participation after visit at 80 days but before visit day&#xD;
      160 will be included for data analysis up to day 80. Men that only deliver one semen sample&#xD;
      or have missing data at any visit will still be included in the analysis. Men with fever up&#xD;
      to 3 months prior to semen analysis will be considered as a potential confounder. Those&#xD;
      values will then be carried forward for analyses. A significance level of 5% is used. For the&#xD;
      primary analyses Bonferroni-Holm p-value correction is calculated additionally. For the&#xD;
      secondary analysis no multiple test correction are used. Instead results are discussed in&#xD;
      view of the multiple testing situations.&#xD;
&#xD;
        1. Analyses between placebo versus active substance: The first step will be to compare the&#xD;
           changes in primary outcomes across the two groups. This analysis will show if there is a&#xD;
           significant difference between groups. For all endpoints measured at baseline and day 80&#xD;
           and 160, paired t-tests will be used to assess whether there is a significant difference&#xD;
           between the groups and determine whether the mean change within each group differs&#xD;
           significantly from zero. In both cases, data will be transformed as necessary to meet&#xD;
           model assumptions. Due to assumed differences in duration of abstinence the same&#xD;
           analyses will be conducted after exclusion of men with duration &lt;48 hours prior to or&#xD;
           after 80 days treatment. Afterwards, the initial analysis will be conducted by using&#xD;
           multiple regression with relevant confounders such as season, BMI, smoking, age,&#xD;
           duration of abstinence, time from ejaculation to motility assessment, fever etc. to see&#xD;
           if this changes the results. For outcomes measured that cannot be compared with t-test&#xD;
           or other parametric tests at day 1, day 80 and day 160, groups will be compared using&#xD;
           non-parametric tests such as Wilcoxon Mann-Whitney test. For Binary outcome the data&#xD;
           will be compared between the two groups by means of conditional logistic-regression&#xD;
           analysis with adjustment for relevant confounders (defined as being significantly p&lt;0.05&#xD;
           associated). For outcomes measured repeatedly, this will entail comparing the estimated&#xD;
           slopes, or rates of change, of each outcome between the groups. Mixed models allow for&#xD;
           the correlation between the repeated observations baseline-day 1-day 80- day 160 from&#xD;
           each man to be suitably incorporated into parameter estimation. Sperm production&#xD;
           variables will be normalized to baseline levels to avoid the huge interindividual&#xD;
           variation in sperm count and sperm concentration&#xD;
&#xD;
        2. Subgroup analysis will be essential in this study because we expect that around 60% of&#xD;
           the men will benefit from treatment with Denosumab. Analyses after stratification into&#xD;
           subgroups will therefore be used to identify a marker for selecting the men who will&#xD;
           benefit from the treatment Subjects will be grouped according to their serum OPG and/or&#xD;
           RANKL, RANKL/OPG ratio, their DNA-fragmentation-Index prior to the start of&#xD;
           intervention. Afterwards other potential predictors of treatment response will be tested&#xD;
           such as BMI, S-calcium, s-phosphate, BMD, semen quality, Inhibin b, FSH at day 80 and&#xD;
           day 160. For Serum OPG such stratification could be in two groups separated by for&#xD;
           instance 3 pg/ml or three groups, &lt;3, 3-4 and above 4pg/ml. Likewise for RANKL and&#xD;
           RANKL/OPG ratio and for DNA-fragmentation-Index stratification could also be in two&#xD;
           groups separated by e.g. DFI of 40%. The subgroup analyses will be in accordance with&#xD;
           normal clinical practice and stratification in appropriate groups according to the&#xD;
           clinical or highest/lowest groups.&#xD;
&#xD;
      SCREENING AND TIME COURSE&#xD;
&#xD;
      The study will be initiated January 2017 and ends when the last person has been included&#xD;
      latest January 2021.&#xD;
&#xD;
      OUTCOME MEASUREMENTS&#xD;
&#xD;
      Endpoints:&#xD;
&#xD;
      Primary endpoints&#xD;
&#xD;
      • Change in semen production (total motile spermatozoa, progressive motile spermatozoa, sperm&#xD;
      count, sperm concentration)&#xD;
&#xD;
      Secondary endpoints&#xD;
&#xD;
        -  Change in semen quality (-motility, -morphology, semen volume)&#xD;
&#xD;
        -  Change in DNA fragmentation (DFI)rate in sperm&#xD;
&#xD;
        -  Change in serum Inhibin-B&#xD;
&#xD;
        -  Change in serum levels of reproductive hormones (FSH, LH, AMH, testosterone, estradiol&#xD;
           and SHBG)&#xD;
&#xD;
        -  Change in serum prolactin level&#xD;
&#xD;
        -  Change in fasting glucose level&#xD;
&#xD;
        -  Change in fastin insulin level&#xD;
&#xD;
        -  Change in c-peptide&#xD;
&#xD;
        -  Change in HbA1c&#xD;
&#xD;
        -  Change in lipid profile&#xD;
&#xD;
        -  Change in bone mineral density evaluated by DXA.&#xD;
&#xD;
        -  Change in serum level of inactive vitamin D, 1,25(OH)2D3, 25-OHD3, 24,25(OH)2D3, PTH,&#xD;
           alkaline phosphatase, ionized calcium, phosphate, FGF23, Klotho, osteocalcin, matrix&#xD;
           gla, osteopontin, calcitonin, pnp, procollagen III, OPG, RANKL, Sclerostin as well as&#xD;
           other bone marker.&#xD;
&#xD;
        -  Change in choice of assisted reproductive assistance technique as well as conceived&#xD;
           pregnancies&#xD;
&#xD;
        -  Change in spontaneous conception rate&#xD;
&#xD;
        -  Change in live birth rate&#xD;
&#xD;
        -  Change in number of spermatozoa expressing RANKL&#xD;
&#xD;
        -  Change in semen pH, HCO3-, calcium, zink, phosphate, RANKL, RANK, OPG, FGF23, Klotho,&#xD;
           osteocalcin, osteopontin. VDB, 24,25OH2&#xD;
&#xD;
        -  Difference in infection rate in the two groups&#xD;
&#xD;
      SETTING, SCIENTIFIC PLAN AND RECRUITMENT&#xD;
&#xD;
      Participants will be included among men referred to the Department of Growth and Reproduction&#xD;
      (dept. of GR), Rigshospitalet (RH) for evaluation of male infertility&#xD;
&#xD;
      This is a prospective, double blinded, two-arm randomized controlled trial. Group of&#xD;
      intervention: Each man will receive Denosumab 60 mg x 1 sc at day 1 only Group receiving&#xD;
      placebo: Will follow the same chain of events, although Denosumab will be replaced with&#xD;
      saline&#xD;
&#xD;
      All men will be offered the same vitamin D and calcium supplementation starting prior to&#xD;
      initiation of the treatment start of Denosumab or placebo: vitamin D 15 microgram and calcium&#xD;
      400 mg once daily.&#xD;
&#xD;
      PARTICIPANTS&#xD;
&#xD;
      Screening of ~500 infertile men will be performed. The investigators assume that ~300 men&#xD;
      will be excluded and ~100 men do not wish to participate. Thus, 95 men will be included in&#xD;
      the study and half will be randomized to active treatment. The investigators expect a small&#xD;
      drop out (&lt;5) because of high motivation and no adverse effects.&#xD;
&#xD;
      ANALYSIS AND INTERVENTION&#xD;
&#xD;
      Reproductive hormones and semen will be analyzed at dept. of GR, Rigshospitalet.&#xD;
&#xD;
      ETHICS AND SIDE EFFECTS&#xD;
&#xD;
      All the patients will have full-filled their investigation, before they are invited to the&#xD;
      study. They will be informed of potential adverse effects, and they can leave the trial at&#xD;
      any point without any consequences.&#xD;
&#xD;
      Denosumab is proven to be safe in several randomized studies, and is already approved by both&#xD;
      FDA and EMA. All side effects will be closely monitored, registered and in case of either&#xD;
      unexpected frequency of adverse effects or unexpected clinical complications it will be&#xD;
      published.&#xD;
&#xD;
      Based on previous fertility studies at our department we expect a large proportion of the out&#xD;
      clinic-patients will wish to participate. In our opinion the outlined study is ethical sound,&#xD;
      and we believe that in order to generate future recommendations, randomized clinical trials&#xD;
      are necessary.&#xD;
&#xD;
      PUBLICATION OF RESULTS&#xD;
&#xD;
      All results, positive or negative will be submitted to peer reviewed scientific journals.&#xD;
      Data will successively be obtained and transferred to a statistical database.&#xD;
    </textblock>
  </detailed_description>
  <overall_status>Completed</overall_status>
  <start_date type="Actual">January 2017</start_date>
  <completion_date type="Actual">May 2023</completion_date>
  <primary_completion_date type="Actual">January 2021</primary_completion_date>
  <phase>Phase 2</phase>
  <study_type>Interventional</study_type>
  <has_expanded_access>No</has_expanded_access>
  <study_design_info>
    <allocation>Randomized</allocation>
    <intervention_model>Parallel Assignment</intervention_model>
    <primary_purpose>Treatment</primary_purpose>
    <masking>Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)</masking>
  </study_design_info>
  <primary_outcome>
    <measure>Change in semen production (total motile spermatozoa, progressive motile spermatozoa, spermatozoa-count, spermatozoa-concentration)</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>semen analysis</description>
  </primary_outcome>
  <secondary_outcome>
    <measure>Change in semen quality (-motility, -morphology of semen volume)</measure>
    <time_frame>80 days,160 days after intervention</time_frame>
    <description>semen analysis</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in DNA fragmentation (DFI) in spermatozoa</measure>
    <time_frame>80 days after intervention</time_frame>
    <description>semen analysis</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in serum Inhibin-B concentration</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>blood sample</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in serum levels of reproductive hormonea (FSH, LH, AMH, testosterone, estradiol inhibin B/FSh ratio and SHBG)</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>blood sample</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in bone mineral density evaluated by DXA</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>DXA</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>• Change in serum level of inactive vitamin D, 1,25(OH)2D3, 25-OHD3, 24,25(OH)2D3, PTH, alkaline phosphatase, ionized calcium, phosphate, FGF23, Klotho, osteocalcin</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>blood sample</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in choice of assisted reproductive assistance technique as well as conceived pregnancies</measure>
    <time_frame>80 days,160 days and 365 days after intervention</time_frame>
    <description>patient file</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in spontaneous conception rate.</measure>
    <time_frame>80 days,160 days and 365 days after intervention</time_frame>
    <description>patient file, interview</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in number of spermatozoa expressing RANKL</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>cytospin</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in semen pH, HCO3-, calcium, zink, phosphate, RANKL, RANK, OPG, FGF23, Klotho, osteocalcin, osteopontin.</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>seminal anlysis</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Difference in infection rate in the two groups</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
    <description>patient history</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in serum level of osteopontin, calcitonin, pnp, procollagen III, OPG, RANKL, Sclerostin as well as other bone marker</measure>
    <time_frame>80 days and 160 days after intervention</time_frame>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in live birth rate</measure>
    <time_frame>80 days,160 days and 365 days after intervention</time_frame>
    <description>patient file telephone interview</description>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in serum prolactin level</measure>
    <time_frame>80 and 160 days after intervention</time_frame>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in fasting glucose level</measure>
    <time_frame>80 and 160 days after intervention</time_frame>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in lipid profile</measure>
    <time_frame>80 and 160 days after intervention</time_frame>
  </secondary_outcome>
  <secondary_outcome>
    <measure>Change in fasting insulin level Change in c-peptide Change in HbA1c</measure>
    <time_frame>80 and 160 days after intervention</time_frame>
  </secondary_outcome>
  <secondary_outcome>
    <measure>succesful fertilization (blastocyst) versus pregnancies versus live birth</measure>
    <time_frame>365 days after intervention</time_frame>
    <description>spontaneous, IVF, ICSI</description>
  </secondary_outcome>
  <number_of_arms>2</number_of_arms>
  <enrollment type="Anticipated">95</enrollment>
  <condition>Infertility, Male</condition>
  <arm_group>
    <arm_group_label>Denosumab</arm_group_label>
    <arm_group_type>Active Comparator</arm_group_type>
    <description>subcutaneous injection with 60 mg Denosumab once</description>
  </arm_group>
  <arm_group>
    <arm_group_label>Placebo</arm_group_label>
    <arm_group_type>Placebo Comparator</arm_group_type>
    <description>subcutaneous injection with NaCl once</description>
  </arm_group>
  <intervention>
    <intervention_type>Drug</intervention_type>
    <intervention_name>Denosumab</intervention_name>
    <description>NaCl 0.9% injection is used as placebo</description>
    <arm_group_label>Denosumab</arm_group_label>
    <arm_group_label>Placebo</arm_group_label>
    <other_name>Prolia</other_name>
  </intervention>
  <eligibility>
    <criteria>
      <textblock>
        Inclusion Criteria:&#xD;
&#xD;
          -  Men &gt; 18 years of age refered due to infertility in need for further investigation&#xD;
             with&#xD;
&#xD;
          -  Sperm count &gt; 0,05 mio./ml.&#xD;
&#xD;
          -  The men have either sperm count &lt;15 mio./ml or&#xD;
&#xD;
          -  &lt;40 % motile spermatozoer (A) or&#xD;
&#xD;
          -  &lt;4 % morphological normale spermatozoer (strict criteria)&#xD;
&#xD;
        Exclusion Criteria:&#xD;
&#xD;
          -  Men with chronic diseases (diabetes mellitus, thyroid disease, endocrine diseases&#xD;
             requiring treatment, malignant diseases or diseases known to be affected by- or&#xD;
             interfere with vitamin D supplements (granulomatous diseases such as sarcoidosis,&#xD;
             tuberculosis, wegeners, vasculitis as well as inflammatory bowel diseases e.g. chron's&#xD;
             disease or ulcerative colitis etc).&#xD;
&#xD;
          -  Men with active or previous malignant disease&#xD;
&#xD;
          -  Any case with indication for tesis biopsy,&#xD;
&#xD;
          -  Serum ionized calcium &lt; 1,15 mmol/l&#xD;
&#xD;
          -  Total calcium &lt; 2.14 mmol/l&#xD;
&#xD;
          -  Poor dental status og dental implants&#xD;
&#xD;
          -  Men with obstructive oligospermia or who has been vasectomized&#xD;
&#xD;
          -  Serum Inhibin B &lt; 30 pg/ml&#xD;
&#xD;
          -  Abnormal karyotype&#xD;
&#xD;
          -  Patients practising excessive exercise&#xD;
      </textblock>
    </criteria>
    <gender>Male</gender>
    <minimum_age>18 Years</minimum_age>
    <maximum_age>64 Years</maximum_age>
    <healthy_volunteers>No</healthy_volunteers>
  </eligibility>
  <overall_official>
    <last_name>Li J Mortensen, MD</last_name>
    <role>Principal Investigator</role>
    <affiliation>Rigshospitalet, Dpt. of Growth and Reproduction, section GR 5064, Blegdamsvej 9, 2100 Copenhagen, Denmark</affiliation>
  </overall_official>
  <overall_official>
    <last_name>martin Blomberg Jensen, MD</last_name>
    <role>Study Chair</role>
    <affiliation>Rigshospitalet, Dpt. of Growth and Reproduction, section GR 5064, Blegdamsvej 9, 2100 Copenhagen, Denmark</affiliation>
  </overall_official>
  <location>
    <facility>
      <name>Department of Growth and Reproduction, Rigshospitalet</name>
      <address>
        <city>Copenhagen</city>
        <zip>2100</zip>
        <country>Denmark</country>
      </address>
    </facility>
  </location>
  <location_countries>
    <country>Denmark</country>
  </location_countries>
  <reference>
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    <PMID>21411557</PMID>
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  <verification_date>June 2023</verification_date>
  <study_first_submitted>January 20, 2017</study_first_submitted>
  <study_first_submitted_qc>January 20, 2017</study_first_submitted_qc>
  <study_first_posted type="Estimate">January 24, 2017</study_first_posted>
  <last_update_submitted>June 11, 2023</last_update_submitted>
  <last_update_submitted_qc>June 11, 2023</last_update_submitted_qc>
  <last_update_posted type="Actual">June 13, 2023</last_update_posted>
  <responsible_party>
    <responsible_party_type>Sponsor-Investigator</responsible_party_type>
    <investigator_affiliation>Rigshospitalet, Denmark</investigator_affiliation>
    <investigator_full_name>Martin Blomberg Jensen</investigator_full_name>
    <investigator_title>MD</investigator_title>
  </responsible_party>
  <condition_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Infertility</mesh_term>
    <mesh_term>Infertility, Male</mesh_term>
  </condition_browse>
  <intervention_browse>
    <!-- CAUTION:  The following MeSH terms are assigned with an imperfect algorithm            -->
    <mesh_term>Denosumab</mesh_term>
  </intervention_browse>
  <patient_data>
    <sharing_ipd>No</sharing_ipd>
  </patient_data>
  <!-- Results have not yet been posted for this study                                          -->
</clinical_study>


download date: ClinicalTrials.gov processed this data on September 20, 2023
link text: Link to the current ClinicalTrials.gov record.
url: https://clinicaltrials.gov/ct2/show/NCT03030196
org study id: 01112016
secondary id: 2016-003546-84
nct id: NCT03030196
lead sponsor: 
has dmc: Yes
To determine the significance of systemic RANKL inhibition for male reproduction, conducting
      a clinical controlled randomized double blinded intervention study on infertile men, to
      investigating whether Denosumab (Prolia) can increase semen quality and to investigate what
      subgroup of infertile men that might benefit from treatment.
INTRODUCTION

      Infertility is a serious problem estimated to affect 7-26% of all couples globally (1;2).
      Approximately 9% of all newborns were conceived by assisted reproductive techniques in 2013
      in Denmark (Danish Fertility Society). Impaired semen quality is the causal or contributing
      factor in almost 50% of all cases of infertility (3;4). Today, there exist no treatments that
      can improve semen quality of most infertile men. Instead, the vast majority of infertile
      couples are treated with assisted reproductive techniques (ART) independently of the
      aetiology (maternal/paternal) causing the infertility (3;5-8). The treatment choice ranges
      from mild intrauterine inseminations (IUI) to more invasive in vitro fertilization (IVF) or
      intracytoplasmic sperm injection (ICSI). ART is successful for more than 70% of all couples.
      However, ART is very expensive and associated with maternal side effects due to the invasive
      methodology and the need for hormonal treatment, often for several months (9-11). Infertility
      is also a financial burden to society due to the costs of ART to maintain an acceptable
      annual birth rate.

      Fertility potential determined by semen quality is established already as a fetus (12). In
      case the testes don't develop normally, reduced semen quality in adulthood will be the result
      as a result of impaired Sertoli cell function. Up until today it has barely been investigated
      whether intervention during adulthood can improve semen quality.

      Our recent studies using both human testis and Vitamin D receptor knock-out mice revealed
      that the vitamin D regulated bone factor RANKL is expressed in the testis (13;14). This is a
      novel finding, because up until now RANKL has only been known to affect bone homeostasis and
      to some extend influence the immune system, inflammation and lactation (15;16). In the
      skeleton RANKL is expressed in osteoblasts and binds to its specific receptor RANK in
      osteoclasts to induce osteoclastogenesis and bone resorption (17). This activation is
      controlled by osteoprotegerin (OPG), which binds to the ligand domain of RANKL and thus
      inhibits RANK activation and osteoclastogenesis, ultimately reducing bone resorption (18).
      This knowledge has been utilized into development of an OPG-analogue drug, a recombinant
      human monoclonal IGg2 antibody Denosumab (Prolia by Amgen), which inhibits RANKL and thereby
      bone resorption and is indicated for use in both osteoporosis and bone metastases (19;20).
      Prolia has in several clinical studies shown to be safe and is approved as treatment for
      osteoporosis in both men and women (21-24). Newer data indicate that RANKL/OPG may be
      involved in regulation of pancreatic function, insulin and glucagon production which support
      a possible indirect effect on reproduction through bone-pancreatic-gonadal axis.

      AIM OF STUDY

      To determine the significance of systemic RANKL inhibition for male reproduction by
      investigating whether Denosumab (Prolia) can increase semen quality and particularly to
      identify the group of men that will benefit from the treatment.

      DESIGN

      Single center, prospective randomized double blinded* clinical controlled intervention.

      Patients and methods:

      95 infertile men, otherwise healthy, will be recruited among men referred to investigation
      for infertility at the outpatient clinic at Dpt. of Growth and Reproduction, Rigshopitalet.
      Neither choosing to participate, nor declining, will affect further outpatient clinic
      treatment.

      The 95 participants are randomly allocated to receiving either

        -  Denosumab s.c. injection 60 mg x1, or

        -  Placebo, Sodium Chloride s.c. injection x 1

      All participants deliver semen samples, blood samples and have DXA-scan performed before and
      after intervention. All receive supplements with Vitamin D and calcium to avoid
      hypocalcaemia, a known, temporary side effect to Denosumab.

      *double blinded studies require that placebo and active substance are identical. Placebo can
      only be obtained from Amgen and we were not able to receive or purchase placebo. Instead the
      study will be double blinded to patient, medical doctors and all who participates in
      data-handling and analyses - only one nurse giving the medicine will be unblinded. She will
      know whether she injects active substance or placebo.

      Inclusion criteria

        -  Men > 18 years of age referred due to infertility in need for further investigation with

        -  Sperm concentration ≥ 0,05 mio./ml.

        -  Sperm concentration <15 mio./ml or

        -  <40 % motile spermatozoer (ABC) or

        -  <4 % morphologically normal sperm (strict criteria)

      Exclusion criteria

        -  Men with chronic diseases (diabetes mellitus, thyroid disease, endocrine diseases
           requiring treatment, malignant diseases or diseases known to be affected by- or
           interfere with vitamin D supplements (granulomatous diseases such as sarcoidosis,
           tuberculosis, wegeners, vasculitis as well as inflammatory bowel diseases e.g. chron's
           disease or ulcerative colitis etc.).

        -  Men with active or previous malignant disease

        -  Any case with indication for testis biopsy,

        -  Total calcium < 2.14 mmol/l

        -  25-OH Vitamin D < 25 nmol/L

        -  Poor dental status or dental implants

        -  Men with obstructive oligospermia or who has been vasectomized

        -  Serum Inhibin B < 30 pg/ml

        -  Abnormal karyotype

        -  Patients practicing excessive exercise

      SAMPLE SIZE CALCULATION AND STATISTICS

      With a test level of 5% (level of significance), a power of 80% and 40 men in each group will
      we be able to detect a change in total number of motile sperm of 75%, and a change in sperm
      concentration (indexed to baseline value) of 25%.

      We expect to include a total of 95 men, to take into account the possibility of dropouts.
      This corresponds to a dropout rate of 10%, in case the rate is lower, chances of detecting a
      significant difference will be above 80%.

      Since we expect 50% of all infertile men in the outpatient clinic, to meet inclusion criteria
      and 50% of these to consent to participation, we anticipate screening of 380 men in total.

      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
      underwent randomization and received the first dose of medicine on day 1. We will analyze the
      data in 2 ways. The primary analysis will proceed according to the randomized group
      assignment. The secondary analysis will be based on stratifying the men according to subgroup
      analyses in relation to the predefined primary and secondary endpoints.

      The primary end points for this protocol will be changes in sperm production evaluated by:
      total motile spermatozoa (106), total progressive motile spermatozoa (106), total spermatozoa
      (106) or sperm concentration (106/ml). Due to the low sample size and large intraindividual
      variation in sperm production primary endpoints will also be evaluated after normalization to
      the mean average level prior to the intervention. Based on the first intervention estimated
      60% of the men will benefit from the treatment and this study is designed to determine
      whether serum OPG, RANKL or phosphate can be used to identify men who will benefit from the
      treatment. The use of these specified endpoints will be useful for determining efficacy.
      Multiple secondary endpoints exist but for the initial investigation focus will be on: Sperm
      DFI, morphology, motility, semen volume, pregnancy rate, Inhibin B, vitamin D and calcium
      homeostasis. Subjects who terminate participation after visit at 80 days but before visit day
      160 will be included for data analysis up to day 80. Men that only deliver one semen sample
      or have missing data at any visit will still be included in the analysis. Men with fever up
      to 3 months prior to semen analysis will be considered as a potential confounder. Those
      values will then be carried forward for analyses. A significance level of 5% is used. For the
      primary analyses Bonferroni-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 placebo versus active substance: The first step will be to compare the
           changes in primary outcomes across the two groups. This analysis will show if there is a
           significant difference between groups. For all endpoints measured at baseline and day 80
           and 160, paired t-tests will be used to assess whether 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. Due to assumed differences in duration of abstinence the same
           analyses will be conducted after exclusion of men with duration <48 hours prior to or
           after 80 days treatment. Afterwards, the initial analysis will be conducted by using
           multiple regression with relevant confounders such as season, BMI, smoking, age,
           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 160, 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). 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 160 from
           each man to be suitably incorporated into parameter estimation. Sperm production
           variables will be normalized to baseline levels to avoid the huge interindividual
           variation in sperm count and sperm concentration

        2. Subgroup analysis will be essential in this study because we expect that around 60% of
           the men will benefit from treatment with Denosumab. Analyses after stratification into
           subgroups will therefore be used to identify a marker for selecting the men who will
           benefit from the treatment Subjects will be grouped according to their serum OPG and/or
           RANKL, RANKL/OPG ratio, their DNA-fragmentation-Index prior to the start of
           intervention. Afterwards other potential predictors of treatment response will be tested
           such as BMI, S-calcium, s-phosphate, BMD, semen quality, Inhibin b, FSH at day 80 and
           day 160. For Serum OPG such stratification could be in two groups separated by for
           instance 3 pg/ml or three groups, <3, 3-4 and above 4pg/ml. Likewise for RANKL and
           RANKL/OPG ratio and for DNA-fragmentation-Index stratification could also be in two
           groups separated by e.g. DFI of 40%. The subgroup analyses will be in accordance with
           normal clinical practice and stratification in appropriate groups according to the
           clinical or highest/lowest groups.

      SCREENING AND TIME COURSE

      The study will be initiated January 2017 and ends when the last person has been included
      latest January 2021.

      OUTCOME MEASUREMENTS

      Endpoints:

      Primary endpoints

      • Change in semen production (total motile spermatozoa, progressive motile spermatozoa, sperm
      count, sperm concentration)

      Secondary endpoints

        -  Change in semen quality (-motility, -morphology, semen volume)

        -  Change in DNA fragmentation (DFI)rate in sperm

        -  Change in serum Inhibin-B

        -  Change in serum levels of reproductive hormones (FSH, LH, AMH, testosterone, estradiol
           and SHBG)

        -  Change in serum prolactin level

        -  Change in fasting glucose level

        -  Change in fastin insulin level

        -  Change in c-peptide

        -  Change in HbA1c

        -  Change in lipid profile

        -  Change in bone mineral density evaluated by DXA.

        -  Change in serum level of inactive vitamin D, 1,25(OH)2D3, 25-OHD3, 24,25(OH)2D3, PTH,
           alkaline phosphatase, ionized calcium, phosphate, FGF23, Klotho, osteocalcin, matrix
           gla, osteopontin, calcitonin, pnp, procollagen III, OPG, RANKL, Sclerostin as well as
           other bone marker.

        -  Change in choice of assisted reproductive assistance technique as well as conceived
           pregnancies

        -  Change in spontaneous conception rate

        -  Change in live birth rate

        -  Change in number of spermatozoa expressing RANKL

        -  Change in semen pH, HCO3-, calcium, zink, phosphate, RANKL, RANK, OPG, FGF23, Klotho,
           osteocalcin, osteopontin. VDB, 24,25OH2

        -  Difference in infection rate in the two groups

      SETTING, SCIENTIFIC PLAN AND RECRUITMENT

      Participants will be included among men referred to the Department of Growth and Reproduction
      (dept. of GR), Rigshospitalet (RH) for evaluation of male infertility

      This is a prospective, double blinded, two-arm randomized controlled trial. Group of
      intervention: Each man will receive Denosumab 60 mg x 1 sc at day 1 only Group receiving
      placebo: Will follow the same chain of events, although Denosumab will be replaced with
      saline

      All men will be offered the same vitamin D and calcium supplementation starting prior to
      initiation of the treatment start of Denosumab or placebo: vitamin D 15 microgram and calcium
      400 mg once daily.

      PARTICIPANTS

      Screening of ~500 infertile men will be performed. The investigators assume that ~300 men
      will be excluded and ~100 men do not wish to participate. Thus, 95 men will be included in
      the study and half will be randomized to active treatment. The investigators expect a small
      drop out (<5) because of high motivation and no adverse effects.

      ANALYSIS AND INTERVENTION

      Reproductive hormones and semen will be analyzed at dept. of GR, Rigshospitalet.

      ETHICS AND SIDE EFFECTS

      All the patients will have full-filled their investigation, before they are invited to the
      study. They will be informed of potential adverse effects, and they can leave the trial at
      any point without any consequences.

      Denosumab is proven to be safe in several randomized studies, and is already approved by both
      FDA and EMA. All side effects will be closely monitored, registered and in case of either
      unexpected frequency of adverse effects or unexpected clinical complications it will be
      published.

      Based on previous fertility studies at our department we expect a large proportion of the out
      clinic-patients will wish to participate. In our opinion the outlined study is ethical sound,
      and we believe that in order to generate future recommendations, randomized clinical trials
      are necessary.

      PUBLICATION OF RESULTS

      All results, positive or negative will be submitted to peer reviewed scientific journals.
      Data will successively be obtained and transferred to a statistical database.
allocation: Randomized
intervention model: Parallel Assignment
primary purpose: Treatment
masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
measure: Change in semen production (total motile spermatozoa, progressive motile spermatozoa, spermatozoa-count, spermatozoa-concentration)
time frame: 80 days and 160 days after intervention
description: semen analysis
measure: Change in semen quality (-motility, -morphology of semen volume)
time frame: 80 days,160 days after intervention
description: semen analysis
measure: Change in DNA fragmentation (DFI) in spermatozoa
time frame: 80 days after intervention
description: semen analysis
measure: Change in serum Inhibin-B concentration
time frame: 80 days and 160 days after intervention
description: blood sample
measure: Change in serum levels of reproductive hormonea (FSH, LH, AMH, testosterone, estradiol inhibin B/FSh ratio and SHBG)
time frame: 80 days and 160 days after intervention
description: blood sample
measure: Change in bone mineral density evaluated by DXA
time frame: 80 days and 160 days after intervention
description: DXA
measure: • Change in serum level of inactive vitamin D, 1,25(OH)2D3, 25-OHD3, 24,25(OH)2D3, PTH, alkaline phosphatase, ionized calcium, phosphate, FGF23, Klotho, osteocalcin
time frame: 80 days and 160 days after intervention
description: blood sample
measure: Change in choice of assisted reproductive assistance technique as well as conceived pregnancies
time frame: 80 days,160 days and 365 days after intervention
description: patient file
measure: Change in spontaneous conception rate.
time frame: 80 days,160 days and 365 days after intervention
description: patient file, interview
measure: Change in number of spermatozoa expressing RANKL
time frame: 80 days and 160 days after intervention
description: cytospin
measure: Change in semen pH, HCO3-, calcium, zink, phosphate, RANKL, RANK, OPG, FGF23, Klotho, osteocalcin, osteopontin.
time frame: 80 days and 160 days after intervention
description: seminal anlysis
measure: Difference in infection rate in the two groups
time frame: 80 days and 160 days after intervention
description: patient history
measure: Change in serum level of osteopontin, calcitonin, pnp, procollagen III, OPG, RANKL, Sclerostin as well as other bone marker
time frame: 80 days and 160 days after intervention
measure: Change in live birth rate
time frame: 80 days,160 days and 365 days after intervention
description: patient file telephone interview
measure: Change in serum prolactin level
time frame: 80 and 160 days after intervention
measure: Change in fasting glucose level
time frame: 80 and 160 days after intervention
measure: Change in lipid profile
time frame: 80 and 160 days after intervention
measure: Change in fasting insulin level Change in c-peptide Change in HbA1c
time frame: 80 and 160 days after intervention
measure: succesful fertilization (blastocyst) versus pregnancies versus live birth
time frame: 365 days after intervention
description: spontaneous, IVF, ICSI
arm group label: Denosumab
arm group type: Active Comparator
description: subcutaneous injection with 60 mg Denosumab once
arm group label: Placebo
arm group type: Placebo Comparator
description: subcutaneous injection with NaCl once
intervention type: Drug
intervention name: Denosumab
description: NaCl 0.9% injection is used as placebo
arm group label: Denosumab
arm group label: Placebo
other name: Prolia
criteria: 
gender: Male
minimum age: 18 Years
maximum age: 64 Years
healthy volunteers: No
last name: Li J Mortensen, MD
role: Principal Investigator
affiliation: Rigshospitalet, Dpt. of Growth and Reproduction, section GR 5064, Blegdamsvej 9, 2100 Copenhagen, Denmark
last name: martin Blomberg Jensen, MD
role: Study Chair
affiliation: Rigshospitalet, Dpt. of Growth and Reproduction, section GR 5064, Blegdamsvej 9, 2100 Copenhagen, Denmark
facility: 
country: Denmark
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responsible party type: Sponsor-Investigator
investigator affiliation: Rigshospitalet, Denmark
investigator full name: Martin Blomberg Jensen
investigator title: MD
mesh term: Infertility
mesh term: Infertility, Male
mesh term: Denosumab
sharing ipd: No