Infertility Clinical Trial
Official title:
Denosumab and Male Infertility: a Prospective Intervention Study
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.
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.
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