A Pilot Trial of 13-cis Retinoic Acid (Isotretinoin) for the Treatment of Men With Oligoasthenoteratozoospermia
Phase: PHASE2
Conditions: Male Infertility, Klinefelter's Syndrome, Y-chromosome Microdeletions
Sponsor: University of Washington

Protocol: 13-cis RA for OAT 
 
Title: A randomized, placebo-controlled trial of 13-cis retinoic acid for the 
treatment of men with oligoasthenoteratozoospermia  
 
 
 
IND #:  
 
application pending 
 
Clinical Phase: 
II 
 
 
Sponsor(s): University of Washington 
 
 
 
1959 NE Pacific St 
 
 
 
Seattle, WA 
 
 
 
 
National Institute of Child Health and Human Development 
 
 
 
6100 Executive Boulevard, Room 8B13G 
 
 
 
Bethesda, MD 20892-7510 
 
 
 
(301) 435-6988 
 
 
 
(301) 480-1972 fax 
 
 
Project Officer: 
Stuart Moss, PhD 
 
 
Medical Monitor: 
Bradley Anawalt, MD  
 
 
 
Date:  
 
October 29, 2013
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
2 
 
 
Principal Investigator 
 
John Amory MD, MPH 
Professor of Medicine  
University of Washington 
Box 356429 
1959 NE Pacific St 
Seattle, WA 98195 
 
  
 
 
Sub Investigators 
 
Thomas Walsh MD 
Associate Professor of Urology 
University of Washington 
 
Charles Muller PhD 
Head, Male Fertility Laboratory 
University of Washington 
 
Stephanie Page MD, PhD 
Associate Professor of Medicine 
University of Washington 
 
William Bremner MD, PhD 
Professor and Chairman 
Department of Medicine 
University of Washington 
 
Mara Roth MD 
Assistant Professor  
Department of Medicine 
University of Washington 
 
 
 
 
 
 
 

 
 
 
3 
 
TABLE OF CONTENTS 
 
 
List of abbreviations ......................................................................................................................5 
Synopsis ...........................................................................................................................................6 
1. Introduction and Background ..................................................................................................9 
2. Objectives .................................................................................................................................12 
2.1 
Primary Objective: ..................................... Error! Bookmark not defined. 
2.2 
Secondary Objectives: ............................... Error! Bookmark not defined. 
3. Rationale, Benefits and Risks .................................................................................................12 
4. Compliance ...............................................................................................................................12 
5. Study Design ............................................................................................................................13 
6. Duration of the Study ..............................................................................................................13 
7. Number of Subjects .................................................................................................................13 
8. Dosage and Administration ....................................................................................................13 
9. Selection of Subjects ................................................................................................................14 
9.1 
Inclusion Criteria .......................................................................................14 
9.2 
Exclusion Criteria ......................................................................................14 
10.............................................................................................................. Concomitant Treatment 
14 
11............................................................................................................................ Study Materials 
15 
11.1 
Study Medication .......................................................................................15 
11.2 
Storage ........................................................................................................... 
15 
11.3 
Disposal.......................................................................................................... 
15 
12......................................................................................................................... Study Procedures 
15 
12.1 
General ........................................................................................................... 
15 
12.2 
Laboratory Samples ...................................................................................16 
12.3 
Visits in Pre-treatment period ....................................................................16 
12.3.1 
Screening Visit .........................................................................................17 
12.4 
Visits in treatment period ...........................................................................18 
12.4.1 
Visit 1 (Treatment Day 1) ........................................................................18 
12.4.2 
Visit 2 (Treatment Day 21 + 2) ................................................................18 
12.4.3 
Visit 3 (End of Study Visit) .....................................................................19 
13............................................................................................................ Withdrawals From Study 
19 
14............................................................................................................ Statistical Considerations 
20 
14.1 
Sample size consideration ..........................................................................20 
Table 1: Estimated % and 95% CI of % gonadotropin suppression in 20 subjectsError! Bookmark not defined.
14.2 
Analysis Populations .................................. Error! Bookmark not defined. 
14.3 
Efficacy .......................................................................................................... 
20 
14.4 
Safety ............................................................................................................. 
21 

 
 
 
4 
 
15............................................................................................................................. Adverse Events 
21 
16.................................................................................................................Ethical Considerations 
22 
16.1 
Informed Consent.......................................................................................22 
16.2 
Conflicts of Interest....................................................................................22 
16.3 
Subject Recruitment ...................................................................................23 
17.............................................................................................................................. Confidentiality 
23 
18............................................................................................. Investigative Record Management 
23 
19....................................................................................................................... Data Transmission 
23 
20.........................................................................................................................Publication Policy 
23 
21........................................................................................................ Investigator Documentation 
23 
22..................................................................................................................................... References 
25 
23......................................................................................................................... List of appendices 
27 
Appendix 1:  Schedule of Events ................................................................................................28 
Appendix 2:  Informed Consent Document ................................... Error! Bookmark not defined. 
Appendix 3:  Informed Consent Document ................................... Error! Bookmark not defined. 
Appendix  4:  Questionnaire for the evaluation of Mood DisordersError! Bookmark not defined. 
Appendix 5:  Serious Adverse Event Report Form ..................................................................31 
 
 

 
 
 
5 
 
 
List of abbreviations 
 
BMI 
Body mass index 
BP 
Blood pressure 
CRF 
Case record form 
CBC 
Complete blood count 
CI  
Confidence interval 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonization 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
LC/MS 
Liquid chromatography/mass spectroscopy 
LH 
Luteinizing hormone 
MedDRA 
Medical Dictionary for Regulatory Activities 
NICHD 
National Institute of Child Health and Human 
Development 
OAT 
Oligoasthenoteratozoospermia 
SAE 
Serious adverse event 
T  
Testosterone 
 
 

 
 
 
6 
 
 
Synopsis 
Study Title 
A randomized, placebo-controlled trial of 13-cis retinoic acid 
for the treatment of men with oligoasthenoteratozoospermia 
Study 
Phase 
II 
Sponsor 
National Institute of Child Health and Human Development 
Study Drug 
13-cis retinoic acid  
Objective 
Primary Objective:  To determine the effect of 13-cis 
retinoic acid administration on total motile sperm count in 
men with oligoasthenoteratozoospermia  
 
Approach: 
Sixty 
men 
with 
infertility 
due 
to 
oligoasthenoteratozoospermia (total, motile sperm count of 
<10 million), but not known genetic abnormalities, will be 
enrolled in a prospective six-month, randomized, double-
blinded, placebo-controlled 3-arm pilot trial of daily oral 
therapy with placebo, 20 or 40 mg daily of 13-cis retinoic 
acid. The impact of treatment on spermatogenesis will be 
determined by monthly semen analysis.  
 
Secondary Objectives: 
 To determine the chance of pregnancy during treatment 
with 13-cis retinoic acid 
 To determine the concentration of 13-cis retinoic acid in 
the seminal plasma of treated men 
 To determine the side effects associated with treatment 
with 13-cis retinoic acid 
 
Principal 
Investigator 
Dr. John K. Amory MD, MPH 
Rationale 
Men with infertility and normal gonadotropins have few 
options for treatment.  Recent research has demonstrated the 
lower intratesticular concentrations of 13-cis retinoic acid 
are associated with abnormal sperm parameters.  Older 
studies of 13-cis retinoic acid administration to normal men 
demonstrated increases in sperm concentrations, but the 
effect of 13-cis retinoic acid on sperm concentrations in men 
with infertility has never been studied.  If 13-cis retinoic 
acid increased sperm concentrations in infertile men, it 
could help infertile men father pregnancies without the need 
for IVF and/or ICSI. 

 
 
 
7 
 
 
Study 
Design 
This is a prospective, randomized, double-blinded, trial to 
evaluate 13-cis retinoic acid  for the treatment of 
oligoasthenoteratozoospermia  
 
We will conduct a prospective, three-arm, randomized, 
double-blinded, placebo-controlled interventional trial to 
determine the impact of therapy with 13-cis retinoic acid on 
sperm indices in infertile men. Sixty (n=20/group) infertile 
men with abnormal sperm analyses will be enrolled in a 24-
week study of 20mg or 40 mg of 13-cis retinoic acid or 
matching placebo daily. The subjects will be closely 
followed for side effects related to treatment. The impact of 
treatment on indices of spermatogenesis will be determined 
by monthly seminal fluid analyses. 
 
All aspects of this study will be performed in compliance 
with Good Clinical Practice (GCP) regulations, ICH 
guidelines, the Declaration of Helsinki, and under an FDA 
Investigational New Drug (IND) application. 
Number of 
Subjects 
Sixty men. 
Duration of 
Trial 
Enrolment should be completed in about 12 months, 
resulting in total study duration of about 24 months 
including screening, treatment phase, end of study visit 
procedures, close out and data analysis. 
Duration of 
Treatment 
The active treatment phase will be 24 weeks 
Dosage and 
Regimen 
13-cis retinoic acid at 20 or 40 mg daily or matching 
placebo 
Inclusion 
Criteria 
Subjects will be infertile men (no pregnancy with partner with 
normal cycles and normal hysterosalpingogram despite >1 year of 
unprotected intercourse) and abnormal sperm analyses with a 
total, motile sperm count of less than 10 million sperm as 
assessed by semen analysis on two occasions separated by one 
week 
Exclusion 
Criteria 
Exclusion criteria include: known genetic infertility (e.g. 
Klinefelter 
syndrome 
or 
Y-chromosome 
microdeletions), 
hypogonadotropic hypogonadism (that might respond to 
gonadotropin injections), the use of anabolic steroids, illicit 
drugs, or the consumption of more than 4 alcoholic beverages 
daily, severe mental health problems, or current therapy with 
retinoic acid (e.g. Accutane) or vitamin A. 

 
 
 
8 
 
Efficacy 
Parameters 
In this study, count of total, motile sperm after 24 weeks of 
treatment. 
Safety 
Parameters 
 CBC, clinical chemistry panel (glucose, liver and renal 
function tests including urea, creatinine, albumin, alanine 
aminotransferase, 
aspartate 
aminotransferase, 
alkaline 
phosphatase, bilirubin), full lipid panel including Total 
cholesterol, HDL, LDL, triglycerides at screening and at the 
end of treatment. Additional measures will be conducted if 
any abnormal values are found until the end of study visit.  
  Adverse event and concomitant medications throughout 
the study.  Other safety parameters include vital signs; and 
changes in pre- and post-treatment physical examination 
results and score on the PHQ9 depression questionnaire.  
Evaluations 
The following determinations will be made in all subjects: 
 Serum samples will be collected for the measurement of 
concentrations of vitamin A, all-trans and 13-cis retinoic 
acid, testosterone, FSH and LH will be collected before the 
beginning of the treatment, and monthly during treatment.   
Other 
Assessments 
Demographic characteristics, including age and race, will be 
collected 
Statistical 
Analysis 
The primary endpoint is the effect of treatment on the count 
of total, motile sperm between treatment groups after 24 
weeks.  This analysis will be conducted using a Kruskal-
Wallis ANOVA is the primary endpoint.  
 
 
 

 
 
 
9 
 
 
1.0 Introduction and Background 
 
 
Infertility affects 10-15% of all couples, with 1.5 million couples seeking 
medical assistance for infertility yearly (1). Infertility attributable to the male 
partner accounts for 30-40% of all cases of infertility. The most common form of 
male infertility involves some type of impairment in spermatogenesis (2).  
Unfortunately, over 80% of men with infertility from impaired spermatogenesis 
do not have a medically treatable cause, such as a gonadotropin deficiency (3).  In 
men with idiopathic infertility, surgical sperm extraction from the testis coupled 
with in vitro fertilization or intra-cytoplasmic sperm injection offers some hope 
of fertility; however, these procedures are invasive, expensive, unsuccessful in 
30-40% of cases, and don’t address the underlying cause of infertility (4).  
Therefore, new approaches to the treatment of male infertility are sorely needed. 
 
 
The essential role of vitamin A (retinol) in spermatogenesis has been long 
appreciated.  Vitamin A deficient male mice are sterile due to impaired 
spermatogenesis and supplementation of deficient mice with vitamin A restores 
fertility (5, 6).  Vitamin A is required for spermatogenesis as it is converted to 
retinoic acid in the seminiferous tubules via the activity of testes-specific retinol 
and retinal dehydrogenases. Retinoic acid is known to mediate most of the effects 
of vitamin A in tissues.  In the testes, two retinoic acid receptors,  and , are 
present in both Sertoli cells and developing germ cells (7-10), and targeted 
deletion of these receptors in mice results in male infertility (11-13).  From this 
work, it is clear that retinoic acid plays several essential roles in spermatogenesis, 
including necessary functions in spermatogonial differentiation, spermatid 
adhesion to Sertoli cells and spermiation (14). Given the crucial role of retinoic 
acid in spermatogenesis, it seems quite possible that some men with “idiopathic” 
infertility have intratesticular concentrations of retinoic acid below those 
necessary to initiate and/or maintain optimal spermatogenesis, but whether 
relative deficiencies of intratesticular retinoic acid contribute to male infertility in 

 
 
 
10 
 
humans is unknown.  In theory, poor dietary intake of vitamin A could lead to 
infertility; however, a nutritional cause of infertility seems improbable in the US.  
More likely, low intratesticular concentrations of retinoic acid could occur in 
infertile men either due to impaired retinoic acid biosynthesis from vitamin A or 
increased metabolism of retinoic acid to inactive metabolites within the testes.   
 
 
Despite the known importance of retinoic acid for spermatogenesis in animals, there has 
been relatively little work examining the role of retinoic acid on spermatogenesis in 
humans.  In this proposal, we will perform a clinical trial of retinoic acid administration to 
infertility patients to determine whether retinoic acid therapy improves sperm quality in 
infertile men.  Intriguingly, there is a suggestion that the administration of retinoic acid to 
men may improve sperm production. During the development of 13-cis retinoic acid 
(Accutane) for acne, three human studies examining the effect of oral administration of 13-
cis-retinoic acid on sperm production in men were performed to determine if 13-cis 
retinoic acid was harmful to male fertility. Interestingly, men in these studies had increased 
sperm concentrations during treatment (see below), suggesting that 13-cis retinoic acid 
administration enhanced spermatogenesis in these normal men with acne (15-17).  
 
 
If the administration of retinoic acid were shown to improve spermatogenesis in some 
infertile men, it would have tremendous significance for our current approach to the 
treatment of male infertility.  Because of its widespread use in humans for over 25 years, 
13-cis retinoic acid is widely available in a generic formulation and could be quickly 
incorporated into male infertility treatment algorithms.  Ideally, retinoic acid therapy 
would increase an infertile man’s chances of successfully fathering a pregnancy without 
the need for assisted reproductive technologies such as intra-cytoplasmic sperm injection 
and/or surgical methods of sperm extraction that are invasive for both the man and the 
woman, expensive and fail in many of cases. 
 
 
Our group has completed the first ever study in humans to examine intratesticular 
retinoic acid concentrations.  With IRB approval, we enrolled 24 men having scrotal 
surgery in an observational study to examine the association between intratesticular 

 
 
 
11 
 
retinoids and spermatogenesis. Interestingly, we observed that intratesticular 13-cis 
retinoic acid is significantly lower in men with sub-normal sperm quality as compared to 
men with normal sperm quality as defined by the WHO criteria (18) (Figure 1).  
 
0
0.1
0.2
0.3
0.4
0.5
Intra-Testicular
13-cis-Retinoic
Acid (pmol/g
Tissue)
 
Sub-normal
      (n=7)
Normal 
(n=16)
B
p=0.04
Figure 1. Intratesticular 13-cis retinoic acid in 24 men expressed as median and interquartile ranges.  
13-cis-retinoic acid is significantly lower in men with sub-normal sperm quality as compared to men 
with normal sperm analyses (p=0.04) (41).
This data suggests that lower intratesticular retinoic acid is associated with impaired 
spermatogenesis; however, it is unclear from this cross-sectional data if the low retinoic acid is a 
result of the infertility or a cause of it.  Only an interventional trial of retinoic acid in infertile men 
will answer the question of the ability of retinoic acid to improve spermatogenesis in this 
population.  
 

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Objectives  
2.1 Primary Objective:   
To determine the effect of 13-cis retinoic acid administration on total motile sperm count 
in men with oligoasthenoteratozoospermia  
 
 
2.2 Secondary Objectives: 
 
o To determine the chance of pregnancy during treatment with 13-cis retinoic acid 
o To determine the concentration of 13-cis retinoic acid in the seminal plasma of 
treated men 
o To determine the side effects and tolerability of  treatment with 13-cis retinoic acid 
 
3. Rationale, Benefits and Risks  
Male infertility is common and difficult to treat unless men have an easily identified 
gonadotropin deficiency.  Many men have idiopathic infertility.  Retinoids are necessary 
for spermatogenesis, but no controlled studies of retinoic acid administration to men with 
infertility have been conducted.  We have shown that men with abnormal spermatogenesis 
have lower than normal levels of intratesticular retinoic acid, suggesting that intratesticular 
retinoic acid deficiency is associated with infertility.  In this study, we aim to determine if 
retinoic acid therapy can improve sperm counts in men with low sperm counts.  The 
medication, 13-cis retinoic acid is widely available and prescribed for the treatment of 
acne, suggesting that serious adverse events, especially at the low doses to be tested are 
unlikely. 
 
4. Compliance 
All aspects of this study will be performed according to GCP and ICH guidelines, under an 
active IND. 
 

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5. Study Design 
 
 
 
6. Duration of the Study 
 
Subjects: Subjects will undergo a screening phase of up to 60 days. After fulfilling all 
entry criteria, the first 60 subjects will be randomly assigned to one of the three groups. 
The treatment phase will last for 24 weeks and subjects will have two post-treatment visits, 
about 4 and 12 weeks after the end of treatment.  
 
Enrolment should be completed in approximately 12 months, resulting in total study 
duration of about 24 months including screening, treatment phase, end of study visit 
procedures, close out and data analysis. 
 
7. Number of Subjects 
 
 
60 men will participate in the study (n=20/arm) 
 
8. Dosage and Administration 
 
 
 
13-cis retinoic acid at 20 or 40 mg daily (purchased commercially) or matching placebo 
 
We will conduct a prospective, three-arm, randomized, double-blinded, placebo-controlled 
interventional trial to determine the impact of therapy with 13-cis retinoic acid on sperm indices 
in infertile men. Sixty (n=20/group) infertile men with abnormal sperm analyses will be enrolled 
in a 24-week study of 20mg or 40 mg of 13-cis retinoic acid or matching placebo daily. The 
subjects will be closely followed for side effects related to treatment. The impact of treatment on 
indices of spermatogenesis will be determined by monthly seminal fluid analyses. All aspects of 
this study will be performed in compliance with Good Clinical Practice (GCP) regulations, ICH 
guidelines, the Declaration of Helsinki, and under an FDA Investigational New Drug (IND) 
application. 
 
 

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9. Selection of Subjects  
9.1.1 Inclusion Criteria 
1. Subjects will be infertile men (no pregnancy with partner with normal cycles and normal 
hysterosalpingogram despite >1 year of unprotected intercourse). 
2. Abnormal sperm analyses with a total, motile sperm count of less than 10 million sperm as assessed by 
semen analysis on two occasions separated by one week. 
3. In the opinion of the investigator, is able to comply with the protocol, understand and sign an           
informed consent and HIPAA form. 
 
9.2 Exclusion Criteria 
 
Men who meet any of the following criteria are NOT eligible for enrollment in the trial: 
1. 
Men participating in another clinical trial 
2. 
Men not living in the catchment area of the clinic 
3. 
Clinically significant abnormal findings at screening 
4. 
Known genetic infertility (e.g. Klinefelter syndrome or Y-chromosome microdeletions),  
5. 
Hypogonadotropic hypogonadism (that might respond to gonadotropin injections),  
6. 
The use of anabolic steroids, illicit drugs, or the consumption of more than 4 alcoholic 
beverages daily 
7. 
 Severe mental health problems requiring medications 
8. 
 Current therapy with retinoic acid (e.g. Accutane) or vitamin A. 
9.Abnormal serum chemistry values according to local laboratory normal values, which 
indicate liver or kidney dysfunction. Other abnormal lab values may also be exclusionary, 
at the discretion of the investigator 
 
10. 
Concomitant Treatment 
Concomitant medications that are exclusionary include: 
 Use of sex hormones for treatment 
 Use of androgens or other compounds for body building  
 Use of retin-A or Accutane for the treatment of acne 

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Concomitant medications are discouraged except as prescribed for the treatment of intercurrent 
medical conditions. All concomitant treatments will be recorded on the subject’s case record form 
(CRF), including the generic name of the drug, start and stop dates, and reason for use.   
 
11. 
Study Materials 
11.1 Study Medication 
Each subject will receive the study medication assigned per the randomization list. The first dose 
will be administered at the clinic under the observation of the study staff. The day and time of the 
start of treatment will be recorded on the CRF in the appropriate space provided. The subjects will 
be given enough supply of medication (for 28 days) until their next visit.  
 
Study medication log (diary) will be provided to the subjects for recording the time of application 
of gel. These medication logs will be reviewed at each visit. The investigator will retain the copies 
of these logs.  
 
11.2 Storage 
Study medications will be stored at a temperature not to exceed 25 C in a locked storage area at 
the study site. Study medication will be accessible only to study personnel designated to handle 
study medication. 
 
11.3 Disposal 
Empty used pill bottles will be returned to the clinic and retained at the site.   All unused 
medication will be returned to the University of Washington investigational pharmacy for 
destruction once final drug accountability has been performed. 
 
12. 
Study Procedures 
12.1 General 
 There will be a total of three treatment groups as described in Section 7. The study will consist of 
3 periods: 
1. Pre-treatment period (including screening) for up to 60 days. 
2. Treatment period for 24 weeks 

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3. Recovery period for about 12 weeks 
 
The treatment period will begin with Day 1 of treatment, daily for 24 weeks. The recovery period 
will last for about 12 weeks. 
 
12.2 Laboratory Samples 
Blood samples for laboratory analyses will be collected at the two screening visits, Day 1 and at 
the treatment visits every four weeks with the subject in a fasting state (no food or drink other than 
4 to 6 ounces of water or non-sweetened clear liquids for a minimum of 10 hours). If the subject is 
in a non-fasting state, the investigator will reschedule the visit within two days. The investigator 
will review all laboratory reports and file a copy with each subject’s chart and CRFs. The CBC 
and blood chemistry samples will be analysed at the local hospital laboratory at the University of 
Washington.    
 
The serum samples for measurements of retinoic acid will be frozen for analysis at the end of 
study to prevent inadvertent breaking of the blind. Briefly, the blood samples will be collected into 
vacutainers (containing serum separating tube gel and clot activator) and gently mixed. After 
wrapping in aluminum foil to prevent light exposure that can degrade retinoic acid, samples will 
be allowed to clot for 1-2 hours at room temperature before centrifugation at 1000-2000g for 15 
minutes. Following centrifugation, the serum samples will be frozen at –20 C.   
 
 
12.3 Visits in Pre-treatment period 
 
All visits will be scheduled between 9:00am and 5:00pm.   
 

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12.3.1 Screening Visit 
The following screening procedures will be performed at the screening visit: 
 Each participant must sign an IRB-approved informed consent form before any of the study-
related procedures can be performed. The original signed informed consent form will be kept on 
file by the investigator with the subject’s record and a signed copy will be given to the subject. 
 A screening number will be assigned. 
 A general medical history will be taken and will include information on current and previous 
diseases and treatment. 
 Vital signs (pulse, blood pressure and respiratory rate) will be taken and recorded. 
 Complete physical exam including measurement of testicular volume will be performed. 
 Fasting blood samples will be obtained for the following measurements: Complete blood count 
(CBC), clinical chemistry including glucose, liver and renal function tests (urea, creatinine, 
albumin, alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, 
alkaline phosphatase, bilirubin, albumin, full lipid panel including total cholesterol, HDL, and 
LDL, triglycerides, hematology (hematocrit, hemoglobin), and hormones (LH, FSH, T) 
 Semen analysis will be done to measure sperm concentration, motility and morphology 
 Height and weight will be collected 
 Participants will be questioned regarding concomitant medications and adverse events. 
 Once lab results are obtained and considered consistent with idiopathic infertility by the 
investigator as per the inclusion/ exclusion criteria for enrollment, the participant is informed by 
telephone of his lab results. If conformance to the inclusion/exclusion is confirmed, he is 
scheduled to return to the clinic for the 2nd sperm collection.  If this 2nd sperm collection also 
meets enrollment criteria, the subject is schedule to begin on treatment 
 Successful volunteers will be assigned subject numbers in sequence as they meet the study 
requirements.    
 If any results are abnormal, according to local laboratory standards and in the clinical judgment 
of the investigator, the subject will be informed and excluded from the study and referred to his 
primary physician. 
 

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12.4 Visits in treatment period 
Seven visits will occur in the treatment period: week 0, week 4, week 8, week 12, week 16, week 
20 and week 24.  All visits are ± 4 days.  
 
12.4.1 Visit 1 (week 0) 
This is the day 1 of treatment. The following will be done at this visit: 
 Vital signs (pulse, blood pressure, respiratory rate) and weight will be taken and recorded. 
 Subjects will be questioned regarding concomitant medications and adverse events.  If the 
subject is taking a medication that is listed as disallowed the subject should not be enrolled in the 
study. 
 The subject will complete the PHQ9 questionnaire (see appendix xx) 
 A blood samples will be drawn, and the time of day of the blood draw will be noted in the CRF.  
 A semen sample will be obtained for the measurement of sperm count, concentration, motility 
and morphology. 
 Subjects will be given enough supply of study medication for 30 days (plus a supply for 2 
additional days in the event of scheduling issues) as per their assigned group.  
 A study medication log will be dispensed to record the date and time of administration of the 
study medication.  Subjects will be instructed to take the medication in the morning, except on the 
day of the visit. Subjects will be scheduled to return to the clinic after 28 + 4 days.  
 
12.4.2 Visits 2-7  
These visits are for weeks 4,8,16, 20 and 24.  The following procedures will be done at each of 
these visits:  
 Vital signs (pulse, blood pressure, respiratory rate) and weight will be taken and recorded. 
 The subject will complete the PHQ9 questionnaire 
 Complete physical exam including testicular examination and volume measurement will be 
performed. 
 Subjects will be questioned regarding concomitant medications and adverse events. 
 Any unused study medication will be collected at this visit.   
 Study medication log will be reviewed to check the date and time of drug administration. These 
logs will be collected at this visit. 

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 A blood sample will be drawn. The time of day of the blood draws will be recorded in the 
source.  
 A semen sample will be obtained for the measurement of sperm count, concentration, motility 
and morphology 
 Fasting blood samples will be obtained for the following measurements: CBC, clinical 
chemistry including glucose, liver and renal function tests (urea, creatinine, albumin, alanine 
aminotransferase, aspartate aminotransferase, gamma  
glutamine 
transferase, 
alkaline 
phosphatase, bilirubin, and albumin), full lipid panel including total cholesterol, HDL and LDL,  
triglycerides, hematology (hematocrit, hemoglobin), and testosterone, FSH and LH.  In addition, 
two serum samples will be frozen for the future analysis of serum retinoid levels.  
 Subjects will be given the next month’s supply of medication. 
  Subjects will be scheduled to return to the clinic after additional 4 weeks visit. 
 
12.4.3 Visit 8 and 9 (Follow-up Visits) 
The following will be done at this visit: 
 Vital signs (pulse, blood pressure and respiratory rate) will be taken and recorded. 
 The subject will complete the behaviour questionnaire 
 Subjects will be questioned regarding concomitant medications and adverse events. 
 A blood sample will be drawn for the measurement of serum FSH, LH, T, and retinoids.  
 A semen sample will be obtained for the measurement of sperm count, concentration, motility 
and morphology 
 
 
13. 
Study Withdrawals  
Subjects withdrawing from the study can be replaced only if the withdrawal occurs before the start 
of the treatment. Subjects who have had medication will not be replaced, regardless of the reason 
for discontinuation. All efforts should be made to contact any subject who decides to discontinue 
study participation for the end of study safety procedures (Visit 9). The necessary clinical and 
laboratory examinations planned at the last visit should be performed at the end of the study 
whenever it occurs. The reasons for discontinuation will be documented.   
 

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Post-Admission Withdrawal Criteria 
Subjects may be discontinued prematurely for any of the following reasons: 
 Emergence of a severe condition(s) such that, in the judgment of the investigator, continuation 
in the trial would negatively impact the health of the subject. 
 Personal reasons (e.g. withdrawn consent). 
 Subject lost to follow-up 
 
All subjects in a treatment arm may be discontinued prematurely if greater than 25% of the 
subjects in an arm are discontinued for the emergence of the same severe condition that requires 
individual subjects to be discontinued.   
 
14. 
Statistical Considerations 
 
Data analysis and Interpretation of Results:  The primary endpoint is the count of total motile spermatozoa in 
the ejaculate at week 24 of treatment compared with baseline.  Secondary endpoints include other changes in 
sperm quality, and any pregnancies occurring during treatment as well as the incidence of side effects.   The 
number of total motile sperm after six months of treatment will be compared between the three groups using a 
Kruskal-Wallis ANOVA with a Wilcoxon rank-sum post-hoc test.  The change in the total motile sperm count 
from baseline (a secondary endpoint) will be performed with a Wilcoxon sign-rank test.  Linear regression will be 
performed to determine if significant relationships between baseline (or delta) retinoic concentrations and changes 
in sperm quality are present using STATA Version 10.0 (College Park, TX, USA).  For all comparisons, an alpha 
of 0.05 will be considered significant.  
 
14.1 Sample size consideration 
Inclusion of 20 men per group will allow for an 80% power to detect a greater than a 20% difference for total 
motile sperm count at week 24 compared with placebo with a variance of 25% and allowing for a drop-out rate of 
10% at an alpha of 0.05.  
 
14.2 Efficacy  
Primary:  The primary efficacy outcome is increase in the count of total, motile sperm after 
twenty-four weeks of study treatment.  

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Secondary: The magnitude and changes between baseline and week 24 in terms of total, motile 
sperm count will be described for subjects in each treatment arm.  
 
14.3 Safety 
Laboratory data, physical examination results, vital signs and chemistry levels will be assessed 
and compared for each subject from screening to end of study.  All adverse events will be 
recorded and coded using the body system coding of MedDRA. 
 
The incidence of adverse events will be examined by dose and where appropriate for trend by 
subject, clinic and time, using descriptive statistics.  Clinical chemistries and allied data will be 
examined for change over time.  
 
15. 
Adverse Events  
Adverse Events (AE) should be carefully monitored and an AE form is included in the CRF. 
Serious adverse events (SAE) as defined below will have to be reported on the SAE form included 
as an appendix. 
 
All adverse experiences must be recorded in the study event record of the subject’s CRF, and must 
include the following information (when applicable): 
 Specific condition or event 
 Indication of whether the condition was pre-existing or not and if yes, whether it has worsened 
in severity (including an increase in frequency) 
 Date of occurrence 
 Date of resolution 
 Relationship to study medication as evaluated by the investigator (causality assessment). The 
investigator must enter their opinion of causality on the AE forms.  
 Action taken (study medication continued or not) and outcome  
 Seriousness according to the approved regulatory classification {i.e. any event that is  fatal, 
life-threatening, disabling, incapacitating, results in or prolongs hospitalization, or is a medically 
significant event, e.g. an intervention to prevent one of the above outcomes, or any other serious 
criteria (cancer, congenital anomaly, overdose, other significant) is considered serious}. 
 

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When any serious adverse drug experience, regardless of causality, is encountered during 
this clinical trial at an investigator’s site, the investigator must notify the Study Monitor by 
facsimile using the form provided as an appendix. This report must be submitted within 2 
calendar days from the time the investigator’s staff is notified of the event.  
 
All serious adverse events that are unexpected will be reported to the FDA by the principal 
investigator or his designated surrogate. All serious adverse events will be followed until fully 
characterized.  The investigator will collect and forward to the FDA via fax, all available 
supporting documentation (with subject name redacted) for serious events, including at a 
minimum hospital discharge summaries and death certificates (where applicable). Additional 
supporting documentation that should be collected whenever possible, to verify the medical 
diagnosis, includes autopsy reports (where applicable), surgical procedure summaries, histology 
reports, and imaging reports. 
 
16. 
Ethical Considerations 
16.1 Informed Consent 
Principal investigators will provide the NICHD with a copy of the Informed Consent approved by 
their local Institutional Review Board (IRB). The appended Informed Consent (Appendix 2 and 
Appendix 3) will be translated and certified into the language of the respondent as needed. 
 
Under this consent, the subject shall understand that he is authorizing access to medical records as 
required for monitors, auditors, IRBs and regulatory authorities. To expedite collection of 
supporting documentation in the event of a serious adverse event, the subject should also be asked 
to provide a release of medical records authorization at the enrollment visit. 
 
16.2 Conflicts of Interest 
The investigators will not profit from results, either positive or negative, with regard to the 
product being evaluated.  
 

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16.3 Subject Recruitment 
The subjects will be recruited from our Infertility and Urology clinics as well as from greater 
Seattle Metropolitan infertility clinics, who see >300 such men annually.  The study coordinator 
will screen potential subjects over the phone by inquiring about their age and medical history. 
Potential subjects who qualify by phone screen will be scheduled for an appointment to consult 
with the investigator about further eligibility requirements for the study. 
 
17. 
Confidentiality  
The information on individual subjects arising from this study is to be considered confidential and 
transmitted to the sponsor only in a form that will not permit identification of the individual.  
Regulatory and sponsoring agencies may request access to the study records and related medical 
records of each participating subject, and if requested, the subject’s identity will remain 
confidential to the extent permitted by the applicable laws and regulations.  All records will be 
kept in a secure storage area with limited access. 
 
18. 
Investigative Record Management 
All investigative site records will be kept in a secure storage area with limited access.  NICHD 
should be notified before destruction of any site records. 
 
19. 
Data Transmission 
Not applicable 
 
20. 
Publication Policy 
Data on the use of the study medication and results of all clinical and laboratory studies are 
considered private and confidential, and NICHD will encourage publication of the results of the 
study.   
 
21. 
Investigator Documentation 
Prior to beginning the study, the investigator will be asked to demonstrate compliance with ICH 
E6, 8.2 and 21 CFR 312 by providing the following essential documents, including but not limited 
to: 

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1. An original signed Investigator Agreement page of the protocol. 
2. An original signed acknowledgement of receipt of the Investigator’s Brochure. 
3. An Institutional Review Board (IRB) -approved Informed Consent (as described in section 
18.1) in the local language. 
4. Local IRB approval. 
5. Form FDA 1572, fully executed, and all updates on a new fully executed Form FDA 1572. 
6. Current curriculum vitae (CV) for each principal investigator and each sub investigator listed 
on Form FDA 1572.   
7. Financial disclosure information to attest to the absence of financial interests and arrangements, 
including any equity interests in the sponsor or proprietary interest in the tested product, which 
could introduce bias in the study conduct. 

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22. 
References  
 
1. Center for Disease Control.  Infertility Facts, acessed at 
http://www.cdc.gov/nchs/fastats/fertile.htm.   On June 3rd, 2013. 
2. Jequier AM, Holmes SC.  Primary testicular disease presenting as azoospermia or 
oligozoospermia in an infertility clinic.  Br J Urol 1993 71:731-735. 
3. deKretser DM. Male infertility.  Lancet 1997 349:787-790. 
4. Schlegel PN. Nonobstructive azoospermia: a revolutionary surgical approach and results. Semin 
Reprod Med 2009 27:165-170. 
5. Napoli J.  Retinoic acid: Its biosynthesis and metabolism.  In: “Progress in Nucleic Acid 
Research and Molecular Biology” (Ed. Moldave K.), Academic Press, San Diego, 2000 63:139-
188. 
6. Doyle TJ, Braun KW, McLean DJ, Wright RW, Griswold MD, Kim KH.  Potential functions of 
retinoic acid receptor A in Sertoli cells and germ cells during spermatogenesis.  Ann N Y Acad 
Sci 2007 1120:114-130. 
7. Koubova J, Menke D, Zhou Q, Capel B, Griswold MD, Page DC.  Retinoic acid regulates sex-
specific timing of meiotic initiation in mice.  Proc Natl Acad Sci USA 2006 103:2472-2479. 
8. Anderson EL, Baltus AE, Roepers-Gajadein HL, Hassold TJ, de Rooij DG, van Pelt AMM, Page 
DC.  Stra8 and its inducer, retinoic acid, regulate meoitic initation in both spermatogenesis and 
oogenesis in mice.  Proc Natl Acad Sci, USA 2008 105:14976-14980. 
9. Chung SS, Wang X, Wolgemuth DJ.  Expression of retinoic acid receptor alpha in the germline 
is essential for proper cellular association and spermiogenesis during spermatogenesis..  
Development 2009 136:2091-2100.  
10. Dufour JM, Kim KH.  Cellular and subcellular localization of six retinoid receptors in rat testis 
during postnatal development: identification of potential heterimeric receptors.  Biol Reprod 
1999 61:1300-1308. 
11. Lohnes D, Kastner P, Dierich A, Mark M, LeMeur M, Chambon P. Function of retinoic acid 
receptor gamma in the mouse.  Cell 1993 73:643-658. 
12. Lufkin T, Lohnes D, Mark M, Dierich A, Gorry P, Gaub MP, LeMeur M, Chambon P.  High 
postnatal lethality and testis degeneration in retinoic acid receptor alpha mutant mice.  Proc Natl 
Acad Sci USA 1993 90:7225-7229. 

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13. Kastner P, Mark M, Leid M, Gansmuller A, Chin W, Grondona JM, Décimo D, Krezel W, 
Dierich A, Chambon P. Abnormal spermatogenesis in RAR beta mutant mice.  Genes Dev 1996 
10:80-92. 
14. Ghyselinck NB, Vernet N, Dennefeld C, Giese N, Nau H, Chambon P, Viville S, Mark M.  
Retinoids and spermatogenesis: lessons from mutant mice lacking the plasma retinol binding 
protein.  Dev Dyn 2006 235:1608-1622. 
15. Hoting VE, Schutte B, Schirren C.  Isotretinoin and acne conglobata: andrological evaluations 
(German).  Fortschur Med 1992 23:427-430. 
16. Vogt HJ, Ewers R. 13-cis-retinoic acid and spermatogenesis (German). Der Hautarzt 1985 
36:281-286. 
17. Torok L, Kadar L, Kasa M. Spermatological investigations in patients treated with etretinate and 
isotretinoin.  Andrologia 1987 19:629-633. 
18. Nya-Ngatchou JJ, Arnold SLM, Walsh TJ, Muller CH, Page ST, Isoherranen N, Amory JK. 
Intratesticular 13-cis Retinoic Acid is Reduced in Men with Abnormal Semen Analyses. 
Andrology 2013 March; 1: 325-331 
 

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23. 
List of appendices 
 
 
1. Schedule of Events 
2. Informed Consent Document  
3. HIPPA Informed Consent Document  
4. PHQ-9 Questionnaire  
5. Serious Adverse Event Report Form 
 
 
 

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Appendix 1:  Schedule of Events 
 
Screening 
Treatment 
Follow-up 
 
Visit Number 
1 
2 
1 
2-7 
3 
 
Study Day 
6
0 
-30 
0 
Wk 4, 
8, 12, 
16, 20 
and 24 
Wk 28 
and 36 
Administ
rative 
Inform consent/HIPAA signed 
X 
 
 
 
 
Clinical 
Vital signs 
X 
 
X 
X 
X 
Medical history  
X 
 
 
 
 
Physical exam  
X 
 
X 
X 
X 
AE & ConMed 
X 
 
X 
X 
X 
Semen analysis  
X 
X 
X 
X 
X 
PHQ9 questionnaire 
X 
X 
X 
X 
X 
Blood 
Sampling  
CBC and Clinical Chemistry  
X
 a 
 
X 
X a 
X 
 
FSH, LH, T, retinoids 
X 
 
X b 
X b 
X 
Drug 
Administrat
ion 
Dispense study medication 
 
 
X 
 
 
Dispense study medication log 
 
 
X 
 
 
Collect and review study 
medication log 
 
 
 
X 
 
Collect unused study medication 
 
 
 
X 
 
 
 
a: 
This blood collection requires fasting. 
 
 
b: 
Sampling of blood will be collected every 15 minutes for one hour during which a total of five 
samplings (0, 15,      30, 45 and 60 minutes) will be collected for this visit. 
 
  
 
 

 
 
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Appendix 4: PHQ9 Questionnaire 
 

 
 
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Appendix 5:  Serious Adverse Event Report Form 
SERIOUS ADVERSE EVENT REPORT 
CBR SERIAL NO:  
I. EVENT INFORMATION 
1. 
PATIENT 
INITIALS 
 
 
1.a 
COUNTR
Y 
 
 
2. 
DATE 
OF 
BIRTH 
Day      Month     
Year 
 
 
 
2.a. 
AG
E 
 
      
year
s 
3. 
SE
X 
 
 
3.a. HEIGHT 
 
           cm 
3.b. 
WEIG
HT 
 
           
kg 
7.- 13. DESCRIBE EVENT. 
 
 
     EXPECTED 
 
     
UNEXPECTED 
 
DIAGNOSIS: 
…………………………………………………………………………………………………………………. 
 
Description: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
4.-6. EVENT ONSET 
Day Month Year  
 
 
 
_______________________
_______ 
8.-12. 
SERIOUSNESS 
CRITERIA 
CHECK 
ALL 
APPROPRIATE 
TO EVENT 
 
     DEATH (DATE) 
     Day 
Month
 
Year 
 
 
 
 
 
     HOSPITALIZATION 
 
     DISABILITY 
OR 
INCAPACITY 
 
     LIFE-THREATENING 
 
     OTHER 
SERIOUS 
CRITERIA 
     (Cancer, 
congenital 
anomaly, 
       overdose, significant) 
 
 
     NOT APPLICABLE 
II. SUSPECT DRUG INFORMATION 
14. SUSPECTED DRUG(S) (include generic name(s)) 
 
 
20. DID EVENT ABATE 
      AFTER 
STOPPING 
DRUG? 
     YES 
15. DAILY DOSE (include schedule) 
 
 
16. ROUTE OF ADMINISTRATION 
 
 
     NO 
     NOT APPLICABLE 
 

 
 
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17. INDICATION(S) FOR USE 
 
 
21. 
DID 
EVENT 
RE-
APPEAR 
AFTER 
REINTRODUCTION? 
 
18. THERAPY DATES (from/to) 
Day       Month        Year 
Day        Month         Year 
 
 
 
 
 
 
19. THERAPY DURATION UNTIL REACTION 
ONSET 
 
 
 
     YES 
     NO 
     NOT APPLICABLE 
 
III. CONCOMITANT DRUGS AND HISTORY 
22. CONCOMITANT DRUGS AND DATES OF ADMINISTRATION (exclude those used to treat reaction) 
 
 
 
 
 
23. OTHER RELEVANT HISTORY (e.g. diseases, allergies, pregnancy etc.) 
 
 
 
 
 
IV.IND HOLDER INFORMATION 
V. INVESTIGATOR’S ASSESSMENT 
24.a. NAME AND ADDRESS OF IND HOLDER 
Population Council  
Center for Biomedical Research 
1230 York Avenue 
New York, NY 10021 USA 
Fax:+1(212)327 86 73 
23.d. OUTCOME 
 
     Recovered 
     Recovering 
     Not Recovered 
     Sequelae 
     Fatal 
     Unknown 
 
23.e. CAUSALITY 
 
     Not Related 
     Unlikely 
     Possible 
     Probable 
     Highly Probable 
     Insufficient                
Data 
 
BATCH 
NO. 
(when 
relevant) 
 
 
24.b. 
PATIENT 
IDENTIFIER NO 
 
 
24.c. DATE RECEIVED 
Day Month Year 
 
 
 
24.d. TRIAL NUMBER 
 
 
26. NAME AND ADDRESS OF REPORTING PHYSICIAN 
 
 
 
 
 
 
 
24.e. 
DATE 
OF 
THIS  
REPORT 
Day Month Year 
 
 
 
25.a. REPORT TYPE 
 
     INITIAL 
     FOLLOW-UP 
 
Add another page if not sufficient space. If this case is from literature, please attach an original article