A Pilot Trial of 13-cis Retinoic Acid (Isotretinoin) for the Treatment of Men With Azoospermia
Phase: PHASE2
Conditions: Male Infertility, Azoospermia
Sponsor: University of Washington

 
 
 
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Title: A pilot trial of 13-cis retinoic acid (isotretinoin) for the treatment of men 
with azoospermia  
 
 
 
IND #:  
 
IND 120703 
Clinical Phase: 
II 
 
Version #1: 
1/12/17 
 
Sponsor(s): University of Washington 
 
 
 
1959 NE Pacific St 
 
 
 
Seattle, WA 
 
 
 
 
 
 
 
Funding from: 
 
 
 
“Retinoic Acid in Male Infertility”  
 
 
NIH/NICHD 
1K24HD082231 
Eunice Kennedy Shriver National Institute of Child Health and Human 
Development 
 
 
 
6100 Executive Boulevard, Room 8B13G 
 
 
 
Bethesda, MD 20892-7510 
 
 
 
(301) 435-6988 
 
 
 
(301) 480-1972 fax 
 
 
 
 
Medical Monitor: 
Bradley Anawalt, MD  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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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 
 
Kevin Ostrowski MD 
Acting Instructor of Urology 
University of Washington 
 
Charles Muller PhD 
Head, Male Fertility Laboratory 
University of Washington 
 
William Bremner MD, PhD 
Professor and Chairman 
Department of Medicine 
University of Washington 
 
 
 
 
 
 
 
 

 
 
 
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TABLE OF CONTENTS 
 
 
List of abbreviations ........................................................................................................................5 
Synopsis ...........................................................................................................................................6 
1. Introduction and Background ......................................................................................................9 
2. Objectives ..................................................................................................................................11 
3. Rationale, Benefits and Risks ....................................................................................................11 
4. Compliance ................................................................................................................................11 
5. Study Design .............................................................................................................................12 
6. Duration of the Study ................................................................................................................12 
7. Number of Subjects ...................................................................................................................12 
8. Dosage and Administration .......................................................................................................12 
9. Selection of Subjects .................................................................................................................13 
9.1 
Inclusion Criteria .......................................................................................13 
9.2 
Exclusion Criteria ......................................................................................13 
10.Concomitant Treatment ............................................................................................................14 
11.Study Materials .........................................................................................................................14 
11.1 
Study Medication .......................................................................................14 
11.2 
Storage .......................................................................................................14 
11.3 
Disposal......................................................................................................14 
12.Study Procedures ......................................................................................................................15 
12.1 
General .......................................................................................................15 
12.2 
Laboratory Samples ...................................................................................15 
12.3 
Visits in Pre-treatment period ....................................................................16 
12.3.1 
Screening Visit .........................................................................................16 
12.4 
Visits in treatment period ...........................................................................17 
12.4.1 
Visit 1 (Treatment Day 1) ........................................................................17 
12.4.2 
Visit 2 –Visit 9 .........................................................................................18 
12.4.3 
Visits 10 & 11 (Follow-up) ......................................................................19 
13.Withdrawals From Study ..........................................................................................................19 
14.Statistical Considerations ..........................................................................................................20 
14.2 
Analysis Populations ..................................................................................20 
14.3 
Efficacy ......................................................................................................20 
14.4 
Safety .........................................................................................................21 
15.Adverse Events .........................................................................................................................21 
16.Ethical Considerations ..............................................................................................................22 
16.1 
Informed Consent.......................................................................................22 

 
 
 
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16.2 
Conflicts of Interest....................................................................................22 
16.3 
Subject Recruitment ...................................................................................22 
17.Confidentiality ..........................................................................................................................23 
18.Investigative Record Management ...........................................................................................23 
19.Data Transmission ....................................................................................................................23 
20.Publication Policy .....................................................................................................................23 
21.Investigator Documentation ......................................................................................................23 
22.References .................................................................................................................................24 
23.List of appendices .....................................................................................................................26 
Appendix 1: Order of Study Procedures  .......................................................................................27 
Appendix 2:  PHQ9 Questionnaire for the evaluation of Mood Disorders....................................29 
Appendix 3:  Serious Adverse Event Report Form .......................................................................30 
 
 
 

 
 
 
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List of abbreviations 
 
BMI 
Body mass index 
BP 
Blood pressure 
CRF 
Case record form 
CBC 
Complete blood count 
CI  
Confidence interval 
CK 
Creatine kinase 
FDA 
Food and Drug Administration 
FSH 
Follicle stimulating hormone 
GCP 
Good Clinical Practice 
ICH 
International Conference on Harmonization 
ICSI 
Intracytoplasmic sperm injection 
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 
PSA 
Prostate Specific Antigen 
SAE 
Serious adverse event 
T  
Testosterone 
TESE 
Testicular sperm extraction  
 
 

 
 
 
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Synopsis 
Study 
Title 
A pilot trial of 13-cis retinoic acid for the treatment of men with 
azoospermia 
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 the appearance of sperm in the 
ejaculate among men presenting with azoospermia 
 
Approach: Twenty men with infertility, aged 21-60, due to 
azoospermia (no apparent sperm in the ejaculate on two 
separate occasions) will be enrolled in a single-arm pilot trial 
of daily oral therapy of 20 mg twice daily of 13-cis retinoic 
acid for 32 weeks. The impact of treatment on the appearance 
of sperm in the ejaculate will be determined by monthly 
semen analyses.  
 
Secondary Objectives: 
 To determine the side effects associated with treatment 
with 13-cis retinoic acid 
 
PI 
Dr. John K. Amory MD, MPH 
Rationale 
Men with infertility from azoospermia and normal 
gonadotropins have few options for treatment.  Recent 
research has demonstrated that lower intratesticular 
concentrations of 13-cis retinoic acid are associated with 
abnormal sperm production.  Older studies of 13-cis retinoic 
acid administration to normal men demonstrated increases in 
sperm concentrations. Our recent pilot study in men with 
oligoasthenozoospermia demonstrated increased sperm 
production in roughly ½ of men with treatment, including 
several who started with sperm counts very close to zero.  
However, the effect of 13-cis retinoic acid on sperm 
concentrations in men with azoospermia has never been 
studied.  If 13-cis retinoic acid allowed men with azoospermia 
to produce ejaculated sperm, it could help men with 
azoospermia father pregnancies using ICSI without the need 
for testicular biopsy or testicular sperm extraction (TESE). 

 
 
 
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Study 
Design 
 
We will conduct an unblinded, pilot study to determine the 
impact of therapy with 13-cis retinoic acid on sperm indices 
in azoospermic men. Twenty azoospermic men, ages 21-60 
will be enrolled in a 32-week study of 20 mg twice daily of 
13-cis retinoic acid.  The subjects will be closely followed 
for side effects related to treatment. The impact of treatment 
on the appearance of sperm in the ejaculate 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 
Twenty men. 
Duration of 
Trial 
Enrollment should be completed in about 18 months, 
resulting in total study duration of about 36 months 
including screening, treatment phase, end of study visit 
procedures, close out and data analysis. 
Duration of 
Treatment 
The active treatment phase will be 32 weeks. 
Dosage and 
Regimen 
13-cis retinoic acid at 20 mg twice daily with meals for 32 
weeks. 
Inclusion 
Criteria 
Subjects will be infertile men with azoospermia on at least 
two semen analyses separated by at least one week and no 
pregnancy with partner with normal cycles and normal 
hysterosalpingogram despite >1 year of unprotected 
intercourse. 
Exclusion 
Criteria 
Exclusion criteria include: 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. 
Efficacy 
Parameters 
The appearance of sperm in the ejaculate during treatment 
Safety 
Parameters 
 CBC, clinical chemistry panel (glucose, liver and renal 
function tests including urea, creatinine, albumin, calcium 
alanine 
aminotransferase, 
aspartate 
aminotransferase, 
alkaline phosphatase, bilirubin), CK, full lipid panel 

 
 
 
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including: Total cholesterol, HDL, LDL and fasting 
triglycerides at baseline, week 8, 16, 24 and 32 of treatment, 
and study exit. 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: 
 Semen analysis will be collected twice during screening 
and every four weeks during treatment and twice during 
recovery 
 Serum samples will be collected for the measurement of: 
vitamin A, all-trans and 13-cis retinoic acid, testosterone, 
FSH and LH.  Serum for these measures will be collected 
before the beginning of the treatment, and every two months 
during treatment and sy study exit.   
Other 
Assessments 
Demographic characteristics, including age and race, will be 
collected. 
Statistical 
Analysis 
The primary endpoint is the appearance of sperm in the 
ejaculate of treated men between baseline and 32 weeks.  
This analysis will be conducted using a Wilcoxon signed-
rank test.  
 
 
 
 

 
 
 
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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 testes 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. In theory, poor dietary intake of vitamin A could lead to 
infertility; however, a nutritional cause of infertility seems improbable in the U.S.  

 
 
 
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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.  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).  
We recently completed a pilot study in 20 men with infertility from 
oligoasthenoteratozoospermia.  In this study, roughly ½ of the men experienced 
significant improvements in their sperm production, suggesting this treatment is 
useful for men with oligoasthenoteratozoospermia.  In this proposal, we will 
perform a clinical trial of retinoic acid administration to infertility patients with 
azoospermia to determine whether retinoic acid therapy can initiate sperm 
production in men with azoospermia.    If the administration of retinoic acid were 
shown to improve spermatogenesis in some infertile men with azoospermia, it 
would have tremendous significance for our current approach to the treatment of 
men with infertility who could avoid the need for testicular biopsy and/or testicular 
sperm extraction.  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 allow a man to make sufficient sperm for ICSI, dramatically 
increasing his chances of fathering a pregnancy. 
 
 

 
 
  
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Objectives  
2.1 Primary Objective:   
To determine the effect of 13-cis retinoic acid administration on the appearance of sperm 
in the ejaculation in men with azoospermia. 
 
2.2 Secondary Objectives: 
 
o To determine the side effects and tolerability of  treatment with 13-cis retinoic acid 
in men with azoospermia 
 
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.  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, 
and have performed a pilot study in men with idiopathic oligoasthenozoospermia 
demonstrating that half of these men increase their sperm production on treatment.  In this 
study, we aim to determine if retinoic acid therapy can convert men with azoospermia into 
men with oligozoospermia.  The medication, 13-cis retinoic acid, is widely available and 
prescribed for the treatment of acne, suggesting that serious adverse events are unlikely, 
especially at the low doses to be tested.   
 
4. Compliance 
All aspects of this study will be performed according to Good Clinical Practice GCP 
regulations, the Declaration of Helsinki and ICH guidelines, under an active FDA 
Investigational New Drug (IND) (#120703). 
 

 
 
  
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5. Study Design 
 
6. Duration of the Study 
 
Subjects: Subjects will undergo a screening phase of up to 60 days. The first 20 subjects 
meeting the enrollment criteria will be enrolled. The treatment phase will last for 32 weeks 
and subjects will have two post-treatment visits, about 12 and 24 weeks after the end of 
treatment.  
 
Enrollment should be completed in approximately 18 months, resulting in total study 
duration of about 36 months including screening, treatment phase, end of study visit 
procedures, close out and data analysis. 
 
7. Number of Subjects 
20 men will participate in the study. We may need to screen up to 50 men to find 20 men 
eligible to participate.  
 
8. Dosage and Administration 
 
 
 
13-cis retinoic acid at 20 mg twice daily (purchased commercially) with meals 
 
 
 
We will conduct an open-label pilot trial to determine the impact of therapy with 13-cis retinoic 
acid on sperm indices in infertile men. Twenty infertile men with azoospermia will be enrolled in 
a 32-week study of 20 mg twice daily of 13-cis retinoic acid. 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.  
 
 

 
 
  
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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. Azoospermia (no sperm apparent in centrifuged semen) 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. 
Hypogonadotropic hypogonadism (that might respond to gonadotropin injections),  
5. 
The use of anabolic steroids, illicit drugs, or the consumption of more than 4 alcoholic 
beverages daily 
6. 
 Severe mental health problems requiring medications  
7. 
 Current therapy with retinoic acid (e.g. Accutane) or vitamin A 
8. 
Score of greater than 15 on the PHQ9 (mood) questionnaire 
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. 
Men with a personal history of serious psychiatric disorders 
11. 
Men currently receiving tetracycline containing medications 
12. 
Men currently receiving phenytoin 
13. 
Men with a history of inflammatory bowel disease 
14. 
Men with a history of bone disease 
15. 
Men who have used isotretinoin within eight weeks of the start of dosing 
16. 
Men with elevated serum triglycerides 

 
 
  
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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 
Concomitant medications are discouraged except as prescribed for the treatment of co-exising 
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 drug Isotretinoin 20 mg twice daily for 32 weeks. 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 until their next visit.  
 
A Study drug log (diary) will be provided to the subjects for recording the times of taking study 
medication. These drug logs will be reviewed at each visit. The investigator will retain the copies 
of these logs as source documents.  
 
The prescriber Dr. Amory, is registered in iPLEDGE.  All subjects will be registered in iPLEDGE 
and undergo the extensive counselling recommended by this program. 
 
11.2 Storage 
Study drug will be stored at a temperature not to exceed 25oC in a locked storage area at the study 
site. The study drug will be accessible only to study personnel designated to handle study drug. 
 
11.3 Disposal 
Empty used pill bottles will be returned to the clinic and retained at the site in a locked storage 
area.   All unused study drug will be returned to the University of Washington investigational 
pharmacy for destruction once final drug accountability has been performed. 

 
 
  
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12. 
Study Procedures 
12.1 General 
 The study will consist of 3 periods: 
1. Pre-treatment period (including screening) for up to 60 days. 
2. Treatment period for 32 weeks 
3. Recovery period of 24 weeks 
 
The treatment period will begin with Day 1 of treatment, twice daily for 32 weeks. The recovery 
period will last for 24 weeks. 
 
12.2 Laboratory Samples 
Blood samples for laboratory analyses will be collected at the first screening visit, and at the 
treatment visits on weeks 8, 16, 24, 32 and 56 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 8 hours). A 
snack will be provided after the blood is drawn.  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. In terms of processing, the blood samples will be collected into vacutainers (containing 
serum separating tube gel and clot activator) wrapped aluminum foil to prevent light exposure that 
can degrade retinoic acid,, and gently mixed. The samples will be allowed to clot for 30 minutes at 
room temperature before centrifugation at 1000-2000g for 15 minutes. Following centrifugation, 
the serum samples will be frozen in (light protection?) tubes at –80 C.   
 
Semen Analysis: Semen will be collected after at least 48 hours of abstinence from ejaculation at 
the two screening visits, treatment visits 2-9, and all follow up visits. The semen will be assessed 
for the presence of sperm, and, if present, for count and motility.  In addition, a known volume of 
semen will be diluted and washed for making smears for morphology assessments.  These 

 
 
  
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procedures allow for accurate total sperm count and motility assessments following the WHO 
protocol (18). 
 
12.3 Visits in Pre-treatment period 
 
All visits will be scheduled between 7:00am and 3:00pm.   
 
12.3.1 Screening Visit 1 
The following screening procedures will be performed at the first 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 copy will be given to the subject. 
 Subjects will also complete an Isotretinoin-specific consent form as provided by the iPLEDGE 
program 
 A general medical history will be taken and will include information on current and previous 
diseases and treatment. 
 Vital signs (pulse, blood pressure) 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), creatine kinase (CK),  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 determine if sperm are present 
 Height and weight will be collected 
 Participants will be questioned regarding past? And concomitant medications and adverse 
events. 
 Once lab results are obtained and considered consistent with idiopathic infertility from 
azoospermia by the investigator as per the inclusion/ exclusion criteria for enrollment, the 

 
 
  
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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 one week later.   
  
12.3.2. Screening Visit 2 
Vital signs (pulse, blood pressure) will be taken and recorded. 
 Participants will be questioned regarding concomitant medications and adverse events. 
 
If this 2nd sperm collection also meets enrollment criteria, the subject is scheduled to begin 
on treatment. 
 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. 
 Subjects will be informed that they cannot donate blood during treatment and for 12 weeks 
after treatment is completed. 
 
12.4 Visits in treatment period 
Nine visits will occur in the treatment period: week 0, week 4, week 8, week 12, week 16, week 
20, week 24, week 28 and week 32.  All visits are ± 6 (does this include one week later?) eg 
following Tuesday?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) 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 5) 
 Subjects will be given enough supply of study medication for 30 days. This doesn’t allow 
enough medication, in case subject has to come in at week 4 + 6 days.  

 
 
  
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 A study drug log will be dispensed to record the date and time of administration of the study 
drug.  Subjects will be instructed to take the study drug twice a day, except on the day of the visit. 
Subjects will be scheduled to return to the clinic after 28 +/- 6 days.  
 
12.4.2 Visits 2-9 (Treatment Visits)(+/- 6 days)   
These visits are for weeks 4, 8, 12, 16, 20, 24, 28 and 32.  The following procedures will be done 
at these visits:  
 Vital signs (pulse, blood pressure) and weight will be taken and recorded. 
 The subject will complete the PHQ9 mood questionnaire 
 Complete physical exam including and testicular examination and volume measurement will be 
performed. 
 Subjects will be questioned regarding concomitant medications and adverse events. 
 Any unused study drug counted for drug accountability and returned to subject?  At treatment 
visit 32 or early exit vist, all unused study drug will be collected. 
 Study drug log will be reviewed to check the date and time of drug administration. These logs 
will be collected at each treatment visit. 
 A blood sample will be drawn at week 8, 16, 24 and 32. The time of day of the blood draws 
will be recorded in the source document.  
 A semen sample will be obtained for the presence of sperm, and, if present, 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, calcium, 
alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, alkaline 
phosphatase, bilirubin, and albumin), creatine kinase, full lipid panel including total cholesterol, 
HDL and LDL, triglycerides, hematology (hematocrit, hemoglobin), and testosterone, FSH and 
LH at weeks 8, 16, 24, and 32.  
 In addition, blood samples will be collected at each visit for two serum samples to be frozen for 
the future analysis of serum retinoid levels.  
 Subjects will be scheduled to return to the clinic for follow-up visits. 
 Subjects will be given the next month’s supply of study drug at visits 2-8 (weeks 4, 8, 12, 16, 
20, 24  and 28). 

 
 
  
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12.4.3 Visit 10 and 11 (Follow-up Visits) +/- 6 days 
These visits are for weeks 44 and 56. The following will be done at these visits: 
 Vital signs (pulse, blood pressure and respiratory rate) will be taken and recorded. 
 The subject will complete the mood (PHQ-9) questionnaire. 
 Subjects will be questioned regarding concomitant medications and adverse events. 
 Fasting blood samples (week 56 only) will be obtained for the following measurements: CBC, 
clinical chemistry including glucose, liver and renal function tests (urea, creatinine, albumin, 
calcium, alanine aminotransferase, aspartate aminotransferase, gamma glutamine transferase, 
alkaline phosphatase, bilirubin, and albumin), creatine kinase, 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.  
 A semen sample will be obtained to ascertain the presence of sperm, and, if present, the 
measurement of sperm count, concentration, motility and morphology 
 
 
13. 
Study Withdrawals  
Subjects withdrawn from the study can be replaced only if the withdrawal occurs before the start 
of the treatment. Subjects who have had study drug will not be replaced, regardless of the reason 
for discontinuation. All efforts will be made to contact any subject who decides to discontinue 
study participation before the end of study safety procedures (Visit 11). 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.   
 
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). 
 Non-compliance with study drug 

 
 
  
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 Subject lost to follow-up 
 A severe skin reaction judged by the investigator to potentially be related to the study drug 
 A persistent elevation of serum triglycerides 
 Onset of neutropenia 
 New onset of hearing disorder 
 New onset of visual changes 
 Onset of abdominal pain, rectal bleeding or severe diarrhea 
 Persistent, significant increase of liver enzymes or bilirubin 
 New onset of significant serum glucose elevation 
 New onset of headache associated with nausea and vomiting 
 Persistent elevation of serum calcium 
 Development of kidney stones 
 Increase in the PHQ9 score to >15 (suggestive of moderately severe depression) 
 
 
14. 
Statistical Considerations 
 
Data analysis and Interpretation of Results:  The primary endpoint is the presence of sperm in the 
ejaculate at week 32 of treatment compared with its absence at baseline.     The presence of sperm after 
32 weeks of treatment will be compared with baseline using a Wilcoxon sign-rank test.  Linear 
regression will be performed to determine if significant relationships between baseline (or delta) serum 
concentrations of 13-cis retinoic acid 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 will allow for an 80% power to detect a response rate of greater than 15% in terms 
of sperm production and allowfor a drop-out rate of 10% at an alpha of 0.05 (The primary outcome).  
 
14.2 Efficacy  
The primary efficacy outcome is the presence of sperm in the ejaculate of formerly azoospermic 
men. 

 
 
  
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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 using descriptive statistics.  Clinical 
chemistries and allied data will be examined for change over time.  
 
15. 
Adverse Events  
Adverse Events (AE) will be carefully monitored and an AE form is included in the CRF. Serious 
adverse events (SAE) as defined below will be reported on the SAE form included as appendix #3. 
 
All adverse experiences must be recorded in the study event record of the subject’s CRF, and will 
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 drug as evaluated by the investigator (causality assessment). The 
investigator must enter their opinion of causality on the AE forms.  
 Action taken (study drug 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}. 
 
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.  

 
 
  
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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 Informed Consent 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.  
 
16.2 Conflicts of Interest 
The investigators will not profit from results, either positive or negative, with regard to the 
product being evaluated.  
 
16.3 Subject Recruitment 
The subjects will be recruited from UW and Community Infertility and Urology clinics who see 
>200 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. 
 

 
 
  
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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 drug and results of all clinical and laboratory studies are considered 
private and confidential.  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: 
1. An Institutional Review Board (IRB) -approved Informed Consent (as described in section 
18.1) in the local language. 
2. Local IRB approval. 
3. Form FDA 1572, fully executed, and all updates on a new fully executed Form FDA 1572. 
4. Current curriculum vitae (CV) for each principal investigator and each sub investigator listed 
on Form FDA 1572.   

 
 
  
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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. 

 
 
  
13-cis RA for Azoospermia, v.1, 1/12/17 
 
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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. 
19. WHO. WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical 
Mucus Interaction. Cambridge, United Kingdom: Cambridge University Press; 1999. 
 
 

 
 
  
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23. 
List of appendices 
 
 
1. Schedule of Events 
2. PHQ-9 Questionnaire  
3. Serious Adverse Event Report Form 
 
 
 

 
 
  
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Appendix 1: Schedule of Events 
 
 
Screening 
Treatment 
Follow-up 
Visit # 
S1 
S2 
T1 
T2-T9 
F1 
F2/exit 
Study Day 
-60 to 
-1 
-53 to 
1 
1 
Wks: 4, 
8,12,16, 20, 
24, 28, 32 
Wk 
44 
Wk 56 
Administrative 
Informed 
consent/HIPAA 
X 
 
 
 
 
 
Vital Signs 
X 
X 
X 
X 
X 
X 
Medical 
Medical History 
X 
 
 
 
 
 
Physical Exam 
X 
 
X 
X 
X 
X 
AE & Con Med 
X 
X 
X 
X 
X 
X 
Semen Analysis 
X 
X 
X 
X 
X 
X 
PHQ9 Questionnaire 
X 
 
X 
X 
X 
X 
Blood 
Sampling 
CBC,Chemistry/Lipids, 
CK 
X 
 
X 
X** 
X 
X 
FSH, LH, T & 
Retinoids 
X 
 
X 
X** 
X 
X 
Drug 
Administration 
Dispense Study Meds 
 
 
X 
X* 
 
 
Dispense study med 
log 
 
 
X 
X 
 
 
Collect and review 
med log 
 
 
 
X 
 
 
Collect unused study 
meds 
 
 
 
X 
 
 
Reimbursement 
($) 
 
30 
20 
40 
40 
30 
30 
 Visit T9, week 32: No drug dispense **Blood draws only week 8, 16, 24 and 32

 
 
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 Abbreviations: 
 HIPAA: Health insurance portability and accountability act 
 PHQ9: Patient health questionnaire, #9 
 FSH: follicle-stimulating hormone 
 LH: luteinizing hormone 
 T: testosterone 
 AE: adverse event 
 Con Med: concomitant medications 
 S1/S2: screening visit 1/2 
 T1-T7: treatment visits 1-7 
 F1/F2: follow-up visit 1/2 
 Wk: week 
 
 
 

 
 
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Appendix 2: PHQ9 Questionnaire 
 

 
 
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Appendix 3:  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 
 
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