Clinical trial NCT00001322
Conditions: Healthy Volunteers
Keywords: Behavior Brain Function Progesterone Central Nervous System Function gonadotropin-releasing hormone (GnRH) Agonist
Sponsor: National Institute of Mental Health (NIMH)
Start date: 1994-06-09; Completion date: 2020-03-03
Enrollment: 100 participants; Allocation: NON_RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
This study evaluates the effects of estrogen and progesterone on mood, the stress response, and brain function in healthy women.

The purpose of this study is to evaluate how low levels of estrogen and progesterone (that occur during treatment with leuprolide acetate) compare to menstrual cycle levels of estrogen and progesterone (given during individual months of hormone add-back) on a variety of physiologic measures (brain imaging, stress testing, etc.) in healthy volunteer women without PMS.

This study will investigate effects of reproductive hormones by temporarily stopping the menstrual cycle with leuprolide acetate and then giving, in sequence, the menstrual cycle hormones progesterone and estrogen. Tests (such as brain imaging or stress testing, etc.) will be performed during the different hormonal conditions (low estrogen and progesterone, progesterone add-back, estrogen add-back). The results of these studies will be compared between women without PMS and women with PMS (see also protocol 90-M-0088).

At study entry, participants will undergo a physical examination. Blood, urine, and pregnancy tests will be performed. Cognitive functioning and stress response will be evaluated during the study along with brain imaging and genetic studies.

Detailed description:
Evidence suggests that the gonadal steroids may exert clinically significant effects on central nervous system function. For example, the menstrual cycle may influence the occurrence of seizures in some female epileptics and the performance on certain cognitive tests. Central nervous system effects of gonadal steroids have been inferred largely from changes in behavior occurring in association with presumed changes in gonadal steroids during the normal menstrual cycle, during the administration of ovarian hormones, or in a gender-specific context. These inferences are, by definition, indirect and associational in nature and further are incapable of disentangling the effects of hormones which are simultaneously present in women of reproductive age. This study is designed to address those problems by comparing measures during Lupron-induced hypogonadism with those during replacement with estrogen or progesterone. On the basis of prior findings from our group and from others, we will be asking the following questions: 1) Is the decreased r-CBF that we observed in the prefrontal cortex during the hypogonadal state confirmed in individual women using new imaging techniques; 2) Will variation in genotype (e.g., COMT val/met, BDNF val/met) confer differential sensitivity to ovarian steroids in brain circuitry and 3) Are the menstrual cycle phase-related changes in reward systems that we previously observed related to estradiol or progesterone actions within the brain (1). Additionally, this protocol will serve as a control study for protocol # 90-M-0088.

Arms: arm 1: Eight to 12 weeks of GnRH agonist treatment 3.75 mg given intramuscularly monthly. arm 2: 12 weeks of GnRH agonist treatment 3.75 mg given intramuscularly monthly. Additionally, 4 weeks of transdermal Estradiol (100mcg/day by skin patch) and placebo suppositories. Week 5 involves 100mcg/day transdermal Estradiol and active Progesterone suppositories (200mg vaginally twice/day). Followed by 1-2 weeks (weeks 6-7) washout period. Then crossover to 5 weeks (week 8-12) of Progesterone suppositories (200mg vaginally twice/day) and placebo patches. arm 3: 12 weeks of GnRH agonist treatment 3.75 mg given intramuscularly monthly. Additionally, 5 weeks of Progesterone suppositories (200mg vaginally twice/day) and placebo patches. Followed by 1-2 weeks (weeks 6-7) washout period. Then crossover to 4 weeks (weeks 8-11) of transdermal Estradiol (100mcg/day by skin patch) and placebo suppositories. Week 12 involves 100mcg/day transdermal Estradiol and active Progesterone suppositories (200mg vaginally twice/day).
Interventions: intervention 1: Leuprolide Acetate 3.75 intervention 2: Estradiol intervention 3: Progesterone intervention 4: Placebo suppository intervention 5: Placebo patch
Intervention descriptions: intervention 1: Eight to 12 weeks of GnRH agonist, Leuprolide Acetate 3.75 mg given intramuscularly monthly intervention 2: Transdermal Estradiol, 100mcg/day by skin patch intervention 3: Progesterone suppository, 200mg vaginally twice/day intervention 4: Placebo suppository twice daily intervention 5: Placebo by skin patch

Protocol documents:
Prot_SAP_000.pdf: 
Protocol Title: The central nervous system effects of pharmacologically induced 
hypogonadotropic hypogonadism with and without estrogen and progesterone replacement. 
 
Protocol Number: 92-M-0174 
 
Date of This Submission/Version: May 20, 2019 
Principal Investigator 
 
 
 
Research Contact 
Peter J. Schmidt, M.D. 
NIMH/ SBE 
E-mail peterschmidt@mail.nih.gov 
 
Medical Advisory Investigator 
 
Total requested accrual 
 0 Patients 
 150 Volunteers 
 
Human Research Protections Program Investigator and Staff Training: 
 
“Just in time” human subjects protection training courses are required for investigators and staff 
participating on this protocol:  None 
Peter J. Schmidt, M.D. 
NIMH/ SBE 
E-mail peterschmidt@mail.nih.gov 


2 
 
 
Project Uses Ionizing Radiation: 
 No 
Medically-indicated only 
Research-related only 
Both 
Yes (attach RSC/RDSC documentation) 
 
IND/IDE 
No 
 Yes (attach FDA documentation) 
Drug/Device/# depot Lupron 3.75 mg 
Sponsor PJ Schmidt 
 
Durable Power of Attorney 
 No 
Multi-institutional Project 
 No 
Yes  
Yes 
Institution 
FWA #  
 
Date of IRB approval 
(attach IRB documentation) 
 
Data and Safety Monitoring Board 
 No 
Yes 
 
Technology Transfer Agreement 
No 
 Yes 
Agreement type and number  
CTA 14-10 
Expiration 
Date   08/01/19  
 
 
Confidential Disclosure Agreement 
 No 
Yes 
 
Samples are being stored 
No 
 Yes 
 
Flesch-Kincaid reading level of consent forms: 
Standard 6 month: 9.3 
MDE: 
9.4 
Estradiol Gel : 9.4 
 
Standard 5 month: deactivated 
Stress Reactivity DEX/CRH: deactivated 
Estradiol suppository: deactivated 


3 
 
Precis: 
Evidence suggests that the gonadal steroids may exert clinically significant effects on 
central nervous system function. For example, the menstrual cycle may influence the occurrence 
of seizures in some female epileptics and the performance on certain cognitive tests. Central 
nervous system effects of gonadal steroids have been inferred largely from changes in behavior 
occurring in association with presumed changes in gonadal steroids during the normal menstrual 
cycle, during the administration of ovarian hormones, or in a gender-specific context. These 
inferences are, by definition, indirect and associational in nature and further are incapable of 
disentangling the effects of hormones which are simultaneously present in women of 
reproductive age. This study is designed to address those problems by comparing measures 
during Lupron-induced hypogonadism with those during replacement with estrogen or 
progesterone. On the basis of prior findings from our group and from others, we will be asking 
the following questions: 1) Is the decreased r-CBF that we observed in the prefrontal cortex 
during the hypogonadal state confirmed in individual women using new imaging techniques; 
2) Will variation in genotype (e.g., COMT val/met, BDNF val/met) confer differential sensitivity 
to ovarian steroids in brain circuitry and 3) Are the menstrual cycle phase-related changes in 
reward systems that we previously observed related to estradiol or progesterone actions within 
the brain (1). Additionally, this protocol will serve as a control study for protocol # 90-M-0088. 


4 
 
Table of Contents 
1) 
Introduction 
page 6 
 
A. Gonadal steroids, sexual dimorphisms, brain, and behavior 
page 6 
 
B. Mechanism by which gonadal steroids may alter mood and behavior 
page 9 
 
C. Observations since Protocol Commencement 
page 13 
 
D. Medication to be Employed in this Protocol 
page 14 
 
1. 
GnRH agonists 
page 14 
 
2. 
Estradiol 
page 15 
 
3. 
Progesterone page 17 
 
2) 
Study Objectives 
page 17 
 
3) 
Subjects 
page 18 
 
a) 
Description of study populations 
page 18 
 
b) 
Inclusion criteria 
page 18 
 
c) 
Exclusion criteria 
page 19 
 
4) 
Study Design and Methods 
page 20 
 
a) 
Study overview 
page 20 
 
b) 
Recruitment 
page 20 
 
c) 
Screening page 22 
 
d) 
Study Procedures 
page 23 
 
e) 
End of Participation 
page 31 
 
5) 
Management of data and samples 
page 32 
 
6) 
Additional Considerations 
page 32 
 
7) 
Risks and Discomforts 
page 32 
 
8) 
Subject Safety Monitoring 
page 38 


5 
 
9) 
Outcome Measures 
page 38 
 
10) Statistical Analysis 
page 38 
 
11) Human Subjects Protection 
page 39 
 
12) Anticipated Benefits 
page 42 
 
13) Classification of Risk 
page 42 
 
14) Consent Documentation and Process 
page 42 
 
15) Data Safety Monitoring 
page 43 
 
16) Quality Assurance 
page 44 
 
17) Reporting of Unanticipated Problems, Adverse Events and Protocol Deviations 
 
page 45 
 
18) Alternatives to Participation 
page 45 
 
19) Privacy 
page 45 
 
20) Confidentiality 
page 45 
 
21) Conflict of Interest 
page 46 
 
22) Technology Transfer 
page 46 
 
23) Research and Travel Compensation 
page 46 
 
24) References 
page 49 
 
25) Appendices 
 
Blood Drawing Schedules 
Flow Sheet 
Consent Documents 
 
Clinical Trials Database - Security Overview 


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1) 
Introduction 
 
A. 
Gonadal steroids, sexual dimorphisms, brain, and behavior 
 
For centuries medical observers have suggested that a special relationship exists between 
female reproductive function and/or dysfunction and disturbances in central nervous system 
activity. More recently a putative interaction between gonadal steroids, in particular estradiol 
and progesterone, and central nervous system function has been suggested to contribute to a 
number of observed epidemiologic, phenomenologic and treatment response characteristics in 
women compared to men. Studies have consistently identified a two-fold increased prevalence 
of depression in women compared with men (2-5). This increased prevalence has been observed 
in a variety of countries (4). A two- to three-fold increased prevalence of dysthymia and 
threefold increase in seasonal affective disorder (6) in women has also been noted (7), while 
bipolar illnesses is equi-prevalent in men and women (2, 8, 9) (and reviewed in (10)). Pre- 
pubertal depression prevalence rates are not higher in girls (11, 12), possibly reflecting 
ascertainment bias/reporting bias (depressed boys may be more likely to come to the attention of 
health care providers) or the possibility that pre-pubertal major depression is premonitory of 
bipolar illness (13). Women are more likely to present with anxiety, atypical symptoms, or 
somatic symptoms (6, 14-21); are more likely to report symptoms, particularly in self-ratings (6, 
14, 19); are more likely to report antecedent stressful events (22, 23); manifest a more robust 
effect of stress on the likelihood of developing depression during adolescence (24); and display 
increased comorbidity of anxiety and eating disorders (25-28), thyroid disease (29, 30), and 
migraine headaches (31), as well as lower lifetime prevalence of substance abuse and 
dependence (16, 28, 32). Reported differences in treatment response characteristics in women 
compared with men include poor response to tricyclics (33-36) particularly in younger women 


7 
 
(34), superior response to SSRIs or MAOIs (37-39), and a greater likelihood of response to 
triiodothyronine (T3) augmentation (30, 40). The extent to which these differences reflect 
gender-related differences in pharmacokinetics (41-47) remains to be determined. Finally, while 
the prevalence of bipolar disorder is comparable in men and women, women are more likely to 
develop rapid cycling (10) and may be more susceptible to antidepressant-induced rapid cycling 
(48). 
The epidemiologic observations described above are increasingly complemented by 
demonstrations of sexual dimorphisms in brain structure and physiology in humans. Structural 
and functional brain imaging studies, for example, have shown the following: 1) differences in 
functional organization of the brain, with brain activation response to rhyming task lateralized in 
men but not women (49); 2) gender specific decreases in regional brain volume (caudate in 
males and globus pallidus, putamen in females) during development (50); 3) increased neuronal 
density in the temporal cortex in women (51); 4) greater interhemispheric coordinated activation 
of brain regions in women (52); 5) larger volume hypothalamic nucleus (INAH 3) in men (53); 
6) differences in both resting blood flow and the activation pattern accompanying self-induced 
mood change (54); 7) decreased 5-HT2 binding in the frontal, parietal, temporal, and singular 
cortices in women (55); 8) differences in whole brain serotonin synthesis (interpreted as 
decreased in women but possibly increased if corrected for plasma free tryptophan levels) (56); 
9) higher and more symmetric cerebral blood flow in women (57-61); 10) greater asymmetry in 
the planum temporale in men (62); and 11) greater brain glucose metabolism (19%) in women 
(63, 64). The potential relevance of gonadal steroids in some of these differences has also been 
demonstrated with the same technologies. Recent brain imaging studies in asymptomatic women 
(i.e., women without PMD) confirm for the first time in humans that physiologic levels of 


8 
 
ovarian steroids have the capacity to modulate the neurocircuitry thought to be involved in both 
normal and pathological affective states. First, Berman, et al. performed cognition-activated 
O15PET scans in women during conditions of GnRH agonist-induced hypogonadism and gonadal 
steroid replacement. They observed the elimination of Wisconsin Card Sort-activated regional 
cerebral blood flow (rCBF) in the dorsolateral prefrontal cortex as well as an attenuation of 
cortical activation in the inferior parietal lobule and posterior inferior temporal cortex 
(bilaterally) during GnRH agonist-induced hypogonadism (65). The characteristic pattern of 
cortical activation re-emerged during both estradiol and progesterone addback. Additionally, 
they observed a differential pattern of hippocampal activation with estradiol increasing and 
progesterone decreasing activation relative to hypogonadism. This was the first demonstration 
that ovarian steroids have activational effects on rCBF during cognitive stimulation in the brain 
regions 
(i.e., PFC) implicated in disorders of affect and cognition. Recent observations also suggest that 
estrogen’s modulatory effects on PFC function could be influenced by variation in catechol-O- 
methyltransferase gene (66). Second, Goldstein, et al. (67) observed an increase in amygdalar 
activity and arousal (as measured by fMRI and skin conductance, respectively) during the late 
follicular phase of the menstrual cycle (higher estradiol levels) compared to the early follicular 
phase (characterized by relatively low estradiol levels). Third, Protopopescu, et al. 
(68)employed an affective pictures task in an fMRI study and observed increased OFC activity (a 
region that in some studies exerts inhibitory control over amygdalar functioning) during the 
luteal compared with the follicular phase. Moreover, preliminary data from these same 
investigators in women with PMD (69) suggest a relative loss of OFC activity (decreased 
inhibition) in women with PMD during the luteal phase. Notwithstanding the caveat that 


9 
 
decreased cortical “activity” also could reflect more efficient or optimal function, these data 
suggest that a reduction in OFC inhibition of amygdalar function during the luteal phase is 
associated with PMD symptoms. Finally, Dreher, et al. (1), have initiated an event-related fMRI 
study of reward processing across the menstrual cycle in women with PMD and controls. The 
paradigm employed disentangles transient reward error prediction (PFC) from sustained response 
to reward uncertainty (ventral striatum). Preliminary data in the controls demonstrate, for the 
first time in humans, that ovarian steroids modulate reward system function, with increased 
follicular phase activation of the OFC and amygdala during reward anticipation and of the 
midbrain, striatum, and left ventrolateral PFC during reward delivery. New analytical 
approaches will allow for testing the hypothesis that the hormonally-induced alteration in 
function includes changes in interregional neural interactions. These findings then suggest that 
cognitive and affective information processes may serve as probes to identify candidate circuits 
for the mediation of gonadal steroid-dependent affective dysregulation. Additionally, 
neuroimaging studies in women suggest that ovarian steroids can influence many neural 
processes and systems relevant to PMD including arousal, stress-responsivity, and reward 
processing. The contribution of these and other effects of gonadal steroids to observed gender 
dimorphisms and reproductive-related mood disorders must, obviously, await further 
determination. 
B. 
Mechanism by which gonadal steroids may alter mood and behavior 
 
Animal studies have identified that gonadal steroids influence several of the 
neuroregulatory systems thought to be involved in both the development of affective disorders 
and the putative mechanisms underlying the efficacy of antidepressant or convulsive therapies. 


10 
 
The manifold interaction between gonadotropins/gonadal hormones and 
neurotransmitters/neuromodulators (70-74) suggest a neurobiological basis for the effect of 
changes in reproductive endocrine function, such as the menopause, on mood and behavior. For 
example, estrogen receptors (ER) (both α and β types of the ER) have been identified in several 
brain regions: in the preoptic area and the arcuate nucleus-median eminence region, where 
gonadotropin releasing hormone (GnRH), dopamine, and B-endorphin neurons are concentrated; 
and in extra-hypothalamic sites, including the interstitial nucleus of the striae terminalis, medial 
amygdaloid nucleus, forebrain, and brainstem (75, 76). Gonadal steroids have been shown to 
play a role in all stages of neural development, including neurogenesis, synaptogenesis, neural 
migration, growth, differentiation, survival, and death (77). These effects occur largely as a 
consequence of the ability of gonadal steroids to modulate genomic transcription. As 
transcriptional regulators, the receptors for gonadal steroids direct or modulate the synthesis of 
the synthetic and metabolic enzymes as well as receptor proteins for many neurotransmitters and 
neuropeptides (78). They additionally influence the levels of several critical enzymes involved 
in signal transduction. These actions permit gonadal steroids to influence all aspects of 
neurotransmitter activity. 
Estrogen’s role in mood regulation is not only suggested by its widespread actions on 
neurotransmitter system function but also by certain neuroregulatory actions shared by both 
estrogen and traditional therapies for depression (i.e., antidepressants, ECT). In some, but not all 
(79), experimental paradigms, estradiol has been observed to inhibit SERT mRNA (80) and 
decrease activity at 5HT1A receptors (81, 82), consistent with some reported actions of 
antidepressants on serotonergic system function. Moreover, in one study estradiol replacement 
facilitated imipramine-induced downregulation of 5HT2 receptors in the rat frontal cortex (83). 


11 
 
Additionally, several candidate neural signaling systems have also been identified as potential 
mediators of the therapeutic actions of antidepressants and ECT (e.g., guanine nucleotide- 
stimulated adenylyl cyclase and c-AMP dependent protein kinase (PKA) (84-87)), based on 
observations that these systems are modulated by a range of therapies effective in depression 
(e.g., serotonergic and noradrenergic agents and ECT) and exhibit a pattern of change consistent 
with the long term effects of antidepressants on mood (88). Similarly, estradiol has been 
reported to influence many of these same neuroregulatory processes. Specifically, ovariectomy 
has been reported to decrease, and estradiol increase, BDNF levels in the forebrain and 
hippocampus (89, 90). Estrogen has also been reported to increase CREB activity (91, 92) in rat 
brain and, additionally, to increase trkA (93). In contrast, an estradiol-induced decrease in 
BDNF has been reported to mediate estradiol’s regulation of dendritic spine formation in 
hippocampal neurons (94). It is also of interest that interactions between glucocorticoid and 
estrogen response elements and their receptors suggest additional ways by which gonadal 
steroids may modulate neural activity (95). For example, estrogen (ER) and glucocorticoid 
receptors (GR) compete for CREB binding protein (CBP) and glucocorticoid receptor interacting 
protein (GRIP), with the relative amounts of these receptors increasing (ER) or decreasing (GR) 
transcription at the AP-1 site (96, 97). 
Gonadal steroids may also exert important regulatory effects on the hypothalamic-pituitary- 
adrenal axis. Studies in animals indicate that estrogen administration decreases glucocorticoid 
receptor mRNA production in the thymus (98) and the pituitary (99-101), and enhances 
hypothalamic-pituitary adrenal (HPA) responses to stress (102-105). Moreover, evidence from 
animal studies further suggests that gonadal steroids may influence the serotonergic regulation of 
the HPA axis by altering the function of the 5-HT1a and 5-HT2 receptor systems in the cortex and 


12 
 
hippocampus (106-108). In humans, gender dimorphisms in the physiologic responses to stress 
(including HPA axis function) have been reported (109-115); however, the relationship between 
gonadal steroid hormones and HPA axis activity has not been systematically examined in 
humans. 
Our studies in women using an exercise stressor in the context of GnRH agonist-induced 
hypogonadism with and without estrogen and progesterone replacement, indicate that 
progesterone is a more important influence on the stress response than is estrogen (116). 
Further, in our menstrual cycle studies using the exercise stressor, we have found that 1) there is 
a decreased cortisol and ACTH response to stress during the follicular as opposed to the luteal 
phase of the cycle (117) and 2) that women with a history of PMD fail to demonstrate this 
follicular phase blunting of the axis (116). What accounts for the differential response to stress 
in women with PMD? Since we know that women with PMD respond differentially to gonadal 
steroids, such that they (but not controls) display mood dysregulation in this GnRH paradigm, 
one possibility is that the differences in HPA axis function that we observed in women with 
PMD may again represent a differential (abnormal) response to either estrogen or progesterone. 
To test this hypothesis, we will perform the exercise paradigm in patients (protocol # 90-M- 
0088) and control women under conditions of GnRH agonist-induced hypogonadism with and 
without estrogen and progesterone replacement. Protocol # 92-M-0174 provides a unique 
opportunity to investigate the effects of ovarian steroids on the regulation of HPA axis function 
in these women. 
Despite the possible effects of gonadal steroids on these neural systems, one cannot easily 
infer the mechanism underlying the observed psychotropic effects of these compounds. The 
extent to which gonadal hormones, gonadotropins, catecholamines, and neuropeptides may 


13 
 
interact in the development of reproductive endocrine-related mood and behavioral disorders is 
purely a matter for speculation but may potentially elucidate relevant mechanisms in the 
pathophysiology of mood disorders. 
Previous studies infer the effects of gonadal steroids on CNS activity in humans from the 
occurrence of changes in CNS function in association with presumed changes in gonadal steroids 
during the menstrual cycle or during the administration of ovarian hormones (e.g., post 
menopausal estrogen replacement), or in the context of gender specific behaviors. As noted in 
the precis these methodologies suffer not only from their indirect and associational nature but as 
well from both their inability to disentangle the effects of hormones which are simultaneously 
present and the effects of aging and menopausal status on CNS function. In this study we 
propose to evaluate the selective effects of the gonadal steroids under three pharmacologically 
controlled conditions, specifically during GnRH agonist-induced hypogonadism, GnRH agonist- 
induced hypogonadism with estrogen replacement, and GnRH agonist-induced hypogonadism 
with progesterone replacement. Further, the mood and behavioral effects observed in subjects 
participating in this protocol will serve to control for our observations in protocol # 90-M-0088. 
C. 
Observations since Protocol Commencement: 
 
To date results of this protocol suggest the following: 1) the efficacy of GnRH agonist 
induced ovarian suppression compared to placebo in women with PMD; 2) the re-emergence of 
clinically significant dysphoria induced by both E2 and P4 (but not placebo) in women with 
PMD but not controls; 3) the absence of a significant effect of GnRH agonist induced 
hypogonadism, with or without E2 and P4 replacement, on the self-reports of cognitive abilities 
or neuropsychological test scores in both patients and controls (with the exception of 
significantly improved performance in motor dexterity tasks during progesterone compared to 


14 
 
other hormone conditions); 4) the elimination of Wisconsin Card Sort Test-activated regional 
cerebral blood flow (CBF) in the dorso-lateral prefrontal cortex during Lupron induced 
hypogonadism (p > 0.2) and the re-emergence of the characteristic pattern of activated CBF 
during E2 or P4 addback in both patients and controls; 5) both the increase (E2) and decrease 
(P4) in N-back-activated regional CBF in the hippocampus; 6) the altered pattern of correlative 
relationships between the prefrontal cortex and hippocampus during hypogonadism compared to 
estradiol replacement; 7) the significant increase in m-CPP stimulated plasma prolactin secretion 
during P4 hippocampus; 8) the significant increase in exercise-induced cortisol, ACTH, and 
AVP secretion during P4 addback compared to hypogonadism; and 9) the inability of estradiol 
suppositories at doses of 100 micrograms administered twice a day to maintain plasma estradiol 
levels between 80-120 pg/ml in women receiving Lupron during a 9 week pilot trial. 
D. Medication to be Employed in this Protocol: 
 
1. GnRH Agonists 
 
GnRH is produced by the hypothalamus and causes the anterior pituitary to release follicle 
stimulating hormone (FSH) and luteinizing hormone (LH). Leuprolide acetate (Lupron) is a 
synthetic nonapeptide that functions as an agonist that is 80 to 100 times more potent than 
synthetic native GnRH in inducing the release of LH (118, 119). Depot Lupron is a GnRH 
agonist that, administered monthly, results in a reversible cessation of pituitary ovarian axis 
function; i.e., both the gonadotropin and ovarian steroid secretion are effectively prevented. In 
the absence of ovarian activity, the effects of exogenously administered estrogen or progesterone 
on the CNS can be evaluated in isolation and free of the obfuscating effects of the usual changes 
in gonadal steroids. During the first week of Lupron therapy, there is an initial increase in the 
pituitary release of LH and FSH leading to a transient increase in levels of the gonadal steroids 


15 
 
estrone and estradiol in premenopausal females. However, with long-term administration of 
Lupron, there is a subsequent decrease in the number of GnRH receptors and an inhibition of 
pituitary elaboration of gonadotropins leading to levels of estrogens similar to those observed in 
post-menopausal women. These decreases in gonadal steroid levels occur within two to four 
weeks after initiation of treatment. Following a single Lupron depot injection (3.75 mg) to 
patients, mean peak leuprolide acetate plasma concentration was almost 20 ng/ml after four 
hours and .36 ng/ml at four weeks, levels associated with effective gonadotropin suppression. 
The plasma half-life of depot Lupron is not known; however, the half-life in the non-depot form 
is approximately three hours. 
2. 
Estradiol 
 
The administration of 17β-estradiol via a transdermal system was chosen for this study 
because it has several relevant advantages over oral preparations (120, 121). First, it delivers the 
primary ovarian estrogen, estradiol, into the circulation at a constant rate and results in sustained 
and easily measurable plasma levels of estradiol and in estrone/estradiol ratios less than one (as 
seen in the pre-climacteric period of life) (122). Second, it delivers sufficient estradiol into the 
circulation to raise estradiol plasma concentrations to levels similar to those of women in the 
early follicular to mid-follicular phases of the menstrual cycle (120, 123-125), levels reported by 
some investigators as the minimum necessary for the relief of menopausal symptoms, 
particularly hot flushes (123). In contrast to oral estrogen preparations, both transdermal and 
transvaginal systems avoid first-pass effects of hepatic metabolism, and, therefore, have less 
impact on hepatic protein synthesis including clotting factors, C-reactive protein and renin 
substrate. An increase in renin substrate has been suggested to accentuate or initiate the 
development of high blood pressure in susceptible women with other predisposing factors (126) 


16 
 
and has been implicated as a factor in the association of hypertension with the administration of 
oral conjugated estrogen (127). In our experience with the transdermal estradiol patch in over 
100 women in several protocols, it has been well tolerated and we have observed no unexpected 
adverse effects. With the application of the estrogen patch every three days, patient compliance 
has been reported to be excellent, and only occasional local irritation has been observed (123). 
Similarly, a transvaginal system has been employed to deliver progesterone in this protocol 
without expected adverse events (as detailed below). 
Formulation of Estradiol employed in this protocol: 
 
1. From 1972 until September 2013 – transdermal estradiol patches were employed at a dose 
of 100 micrograms per day. However, due to the unavailability of matched placebo 
patches and the prohibitive costs of manufacturing the small quantities of placebo patches 
needed for this protocol we ceased the employment of estradiol skin patches in this and 
related protocols (i.e., 90-M-0088, 05-M-0059). 
2. Between September 2013 and May 2014 – we performed a pilot study lasting 
approximately two months in which we evaluated estradiol vaginal suppositories (100 
micrograms once to twice daily) as a substitute for the transdermal estradiol patches. 
However, we could not achieve the desired plasma concentrations consistently in women 
and, therefore, we could not employ vaginal estradiol administration in this protocol. 
3. Between July 2014 and June 2016 – we performed a pilot study lasting approximately two 
months in which we evaluated estradiol transdermal skin gel (.06%) three to four 
applications per day (.75 to 3.0 mg estradiol per day) as a substitute for the transdermal 
estradiol patches. However, we could not achieve the desired plasma concentrations of 


17 
 
estradiol consistently in women and, therefore, we could not employ .06% estradiol gel 
administration in this protocol. 
4. Amendment: we intend to conduct a pilot trial of a higher dose of estradiol gel (.36% 
estradiol in hydro alcoholic base) at doses of 6 to 9 mg of estradiol per day (2-3 
applications per day) 
3. 
Progesterone 
 
In this protocol progesterone 200 mg will be administered twice daily. Progesterone is 
widely prescribed in gynecologic settings with proved indications including dysfunctional 
uterine bleeding, endometriosis, mastodynia, galactorrhea and precocious puberty (128). In our 
experience with the use of progesterone vaginal suppositories in over 80 women in several 
protocols, they have been well tolerated and we have observed no unexpected adverse events. 
Mild to moderate perineal irritation has been experienced rarely and when it occurs it is 
responsive to barrier creams such as vaseline. 
Estrogel .36% pilot study only: we will prescribe one of three forms of progestin to induce 
 
endometrial shedding and menstruation after women complete the four weeks of unopposed 
estradiol administration (Estrogel .36%). The decision to choose a specific form of progestin will 
be made by the PI and will depend on the availability of these progestins in the NIH CC 
Pharmacy. Thus, we will prescribe one of the following forms of progestin: progesterone by 
suppository at a dose of 200 mg twice a day for one week (as approved in the original protocol), 
or oral progesterone or oral medroxyprogesterone acetate (Provera) at standard clinical doses 
indicated for the induction of menses after unopposed estradiol exposure in healthy women. 
Finally, administration of depot Lupron up to a maximum of six monthly injections with 
and without the concurrent administration of the estraderm patch (alone for four weeks and in 


18 
 
combination with progesterone suppositories for one week) and progesterone suppositories (for 
five weeks) has been approved by the NIMH IRB for administration to patients with menstrual- 
related mood disorder under NIMH protocol # 90-M-0088. 
2) 
Study Objectives: 
 
The objectives of this study are to examine the effects of hypogonadism with and without 
physiologic replacement of estradiol and progesterone on measures of brain function and 
behavior. The use of GnRH agonists to suppress ovarian function (hypogonadism) permits us to 
examine the effects of estradiol and progesterone on brain function separately, and to standardize 
the exposure to these hormones across individual women and in our comparison in women with 
premenstrual dysphoria (PMD), who participate in an identical companion protocol (90-M- 
0088). The outcome measures currently employed in this study are as follows: 1) brain imaging 
with 015 PET and fMRI to examine brain activation and connectivity and the impact of genomic 
variation on these neuroimaging measures; 2) HPA testing with Dex/CRH stimulation; 
3) metabolomic profile of neurosteroid and neuroactive steroid metabolites, and 3) cell culture 
studies (lymphoid and IPCs) to examine cellular responses to exposures to ovarian steroids in 
controls and women with PMD (90-M-0088). 
3) 
Subjects: 
 
a) Description of study populations 
 
Healthy asymptomatic premenopausal women with no current Axis I diagnosis and no 
evidence of menstrual-related mood or behavioral disturbances. The targeted accrual of 
150 women reflects the use of this protocol to examine specific goals that have developed over 
the course of this protocol. 
b) Inclusion criteria 


19 
 
Volunteers participating in this study will be women meeting the following criteria: 
 
• between the ages of 18 and 50 years, 
 
• not pregnant, 
 
• in good medical health, 
 
• medication free, 
 
• no history of menstrual-related mood or behavioral disturbances. 
 
Additionally, we will recruit a subsample of 20 asymptomatic women who will meet all 
inclusion and exclusion criteria in this protocol except they will have a history of a past major 
depressive episode. 
Finally, a sample of 10 women who meet all the inclusion and exclusion criteria listed 
above for this protocol will be recruited to establish the dose range of transdermal .36% estrogen 
gel for this and related protocol (i.e., 90-M-0088). 
c) Exclusion criteria 
 
The following conditions will constitute contraindications to treatment with hormonal 
therapy and will preclude a subject's participation in this protocol: 
• Current Axis I psychiatric diagnosis (with the exception of those women with a past 
major depression who will be studied in this protocol); 
• History consistent with endometriosis; 
 
• Diagnosis of ill-defined, obscure pelvic lesions, particularly undiagnosed ovarian 
enlargement; 
• Hepatic disease as manifested by abnormal liver function tests; 
 
• History of mammary carcinoma; 
 
• History of pulmonary embolism or phlebothrombosis; 


20 
 
• Undiagnosed vaginal bleeding; 
 
• Porphyria; 
 
• Diabetes mellitus; 
 
• History of malignant melanoma; 
 
• Cholecystitis or pancreatitis; 
 
• Cardiovascular or renal disease; 
 
• Pregnancy. 
 
• Any woman meeting the Stages of Reproductive Aging Workshop Criteria (STRAW) 
for the perimenopause (129). Specifically, we will exclude any woman with an 
elevated plasma FSH level (> 14 IU/L) and with menstrual cycle variability of 
> 7 days different from their normal cycle length. 
 
NIMH employees/staff and their immediate family members will be excluded from the 
study per NIMH policy. 
Subjects taking birth control pills will be excluded from the study. Subjects taking 
diuretics, prostaglandin inhibitors, or pyridoxine (putative treatments for MRMD) will similarly 
be excluded from the study, as will patients taking psychotropic agents (e.g., lithium carbonate, 
tricyclic antidepressants). All subjects will be required to use non-hormonal forms of birth 
control (e.g., barrier methods) to avoid pregnancy during this study. Finally, participants who 
have an active condition that places them at an increased risk for osteoporosis (see Table I) will 
be excluded from this protocol. 
4) 
Study Design and Methods: 
 
a) 
Study overview 


21 
 
This is an out-patient study lasting approximately 5-7 months. Subjects will be asked to 
make visits to the clinic every one to two weeks. Each visit will last up to two hours. In 
addition, some subjects will complete additional procedures such as PET or fMRI scans for 
which there are separate consents. 
b) 
Recruitment 
 
Recruitment Screening Methods 
 
Participants will be sought by advertising for women who do not experience menstrually- 
related mood disturbances. 
• 
Flyers will be produced with tear-offs. Posters will not have tear-offs. 
 
• 
Flyers/poster will be used in color as submitted, or may be printed in black and 
white. Color changes to recruitment material will be made proportionately 
throughout and will not be used to change the emphasis of the material. 
• 
The size of the flyers/posters/ads may vary, but all parts, including fonts and 
pictures, will be changed proportionately. Disproportionate changes in size will 
not be used to change the emphasis of the material. 
• 
The size of the flyers/posters may vary, but all parts, including fonts and pictures, 
will be changed proportionately to the rest of that flyer. Disproportionate changes 
in size will not be used to change the emphasis of the flyer. 
• 
Flyers/posters may be posted on bulletin boards on the NIH campus, and at coffee 
shops, grocery stores, bookstores, libraries, fitness centers, community centers, or 
placed in venues, such as at advocacy group offices, in doctor’s office waiting 
rooms, and retail establishments with approval of the venue or in accord with their 
policy. 


22 
 
• 
Flyers/posters may be made available at outreach exhibits, speaking engagements, 
support group meetings, parenting groups, professional meetings, and 
association/trade meetings with approval of the venue or in accord with their 
policy. 
• 
Flyers/posters may be given directly to persons requesting study information. 
 
• 
Flyers/posters may be posted electronically on websites such as such as NIH or 
NIMH websites, advocacy/parenting group websites such as PTAs, athletic 
groups or neighborhood groups, publications’ websites such as Washington 
Parent, or Gazette. 
• 
Flyers/posters may be sent electronically to persons requesting study information 
 
• 
Flyers/posters may be sent electronically to listserv administrators. SBE will not 
post directly to listservs. Rather, an email with the material attached, including 
the following statement will be sent to the administrator of the listserv: 
You are receiving the email because your email address is included on this 
listserv. The purpose of this message is to inform you of an NIMH study that is 
recruiting subjects. The administrator of this listserv has permitted its use for this 
distribution. 
The administrator has the option of sharing the information with their list. 
Examples of types of listservs to be contacted include professional groups, 
parenting/ family, school, sport groups, women’s health and fitness. SBE will 
retain copies of all correspondence with the administrator of each listserv and 
submit as requested to the IRB. 


23 
 
• 
IRB-approved advertisements may be placed in local and national print 
publications of newspapers, magazines and support or health care organizations, 
such as The Washington Post, The Express, Washington Parent, Gazette, 
Washingtonian, Washington Jewish Week and others. 
• 
NIH Record announcements will appear as text in the NIH Record. 
 
c) 
Screening 
 
Subjects are initially screened for this protocol via participation in screening protocol 81- 
M-0126. All subjects will have a two month baseline screening period during which mood and 
behavioral ratings will be obtained. The absence of menstrual-related mood disorders will be 
prospectively confirmed during a two month period prior to the study entry when subjects will 
complete daily visual analogue rating scales monitoring both mood and behavior as outlined in 
NIMH protocol # 81-M-0126. 
The Structured Clinical Interview for DSM-IV (130) will be administered to controls 
prior to study entry. Any control with a current or past axis I psychiatric diagnosis will be 
excluded from participating in this protocol (except in the subgroup of asymptomatic women 
with a past major depressive episode who participate in this protocol). 
Prior to treatment, a complete physical and neurological examination will have been 
performed and the following routine laboratory data obtained: 
A. 
Blood 
 
Complete blood count; thyroid function tests; cortisol; renal function tests, such as BUN 
and creatinine; electrolytes; glucose; liver function tests, B-hCG for pregnancy test. 
B. 
Urine 
Routine urinalysis. 


24 
 
GnRH agonist will not be administered to any subject with significant clinical or 
laboratory abnormalities. 
Additional tests. 
 
Results of Pap smear performed not longer than 12 months prior to onset of treatment 
will be obtained. 
d) 
Study Procedures 
Medications: 
Following the baseline screening period, subjects will be administered 3.75 mg of GnRH 
agonist via intramuscular injection on a monthly basis in our clinic for a maximum of 24 weeks 
(see Figure I). 
After week eight of GnRH agonist treatment participants will be randomized to either 
Group # 1 or # 2 and receive, in a sequential fashion, estradiol and progesterone as follows. The 
subjects who elect to participate in the brain imaging studies will receive three months of Lupron 
alone (rather than two) to permit their data to be compared with those from protocol # 90-M- 
0088. Consequently, subjects in the brain imaging studies will be randomized to Groups 1 or 2 
after week 12 of Lupron alone. 
Group # 1: (Figure 1) While continuing on the GnRH agonist, subjects will be administered 
 
0.1 mg/day of 17ß-estradiol once or twice a day via vaginal suppository for a period of five 
weeks. After the fourth week, progesterone suppositories (200 mg BID) will be added to provide 
progesterone withdrawal induced shedding of the endometrium and menses in order to prevent 
any effects on the endometrium of prolonged exposure to unopposed estrogen. After the four 
weeks of estradiol administration and the one week of combined estradiol and progesterone, 
there will be a two week washout period when neither estradiol nor progesterone will be given. 


25 
 
Subjects will then be administered progesterone suppositories (200 mg) twice daily for a period 
of five weeks after which no progesterone will be taken. 
Group # 2: Group # 2 is identical to Group # 1 except that the order of estradiol and progesterone 
 
treatments will be reversed: progesterone suppositories (200 mg) will be administered twice 
daily for the first five weeks, and after a two week washout period estradiol suppositories will be 
administered for five weeks and in combination with progesterone during the fifth week. 
Symptom Ratings: 
 
Subjects will fill out the following symptom rating scales: (1) Self-ratings: daily ratings 
include a visual analogue scale (VAS) measuring the reported severity of 15 mood and 
behavioral symptoms, and the Daily Rating Form (131) (modified to include selected items from 
the DRSP (132)), which measures the severity of 15 mood and behavioral symptoms and 
functional impairment; biweekly clinic ratings include the Beck Depression Inventory (BDI) 
(133), the rating scale for Premenstrual Tension Syndrome (PMTS) – self (134), a self rating 
scale measuring the severity of mood and behavior symptoms, and a side effect checklist; the 
Derogatis Inventory of Sexual Function (135), which measures sexual interest and function, will 
be completed at baseline and during weeks 10-12, 14-16, and 21-23; (2) Observer ratings: the 
PMTS-rater form (134) will be completed during biweekly clinic visits. Symptom ratings, 
diagnostic interviews, and other questionnaires will be collected through an online system using 
a subject-specific log in and password to protect confidentiality (see Appendix I). Both 
participants and investigators will input data into CTDB/CTSS. Participant entered data will be 
reviewed during the clinic visit. 
Blood Samples - After the administration of the GnRH agonist, in addition to the 
completion of daily mood and behavioral rating scales, blood samples (50 ml) will be drawn at 


26 
 
each visit, if possible, for the duration of the study (20-24 weeks). Total blood volume will be 
550-660 ml. All blood sampling will be obtained in the following fashion. Subjects will arrive 
at the 4th or 9th floor outpatient clinic between 7:30 and 9:00 a.m.; they will be seated or placed 
at bed rest, a scalp vein needle will be placed in the forearm or hand via venipuncture, and 
approximately 50 ml of blood will be drawn. This blood will then be analyzed for several 
measures such as gonadotropins, estradiol, progesterone, (and its neurosteroid metabolites). 
Additionally, blood samples and skin biopsies (in combination with those obtained in 
 
protocol # 90-M-0088) will be analyzed for hormone and metabolic measures (as well as cellular 
function of the induced pluripotent cells from the skin fibroblasts taken by biopsy or peripheral 
blood T lymphocytes) that could distinguish either the symptomatic state in women with PMD or 
women with from those without PMD. In order to assess the effects of suppression of the 
hypothalamic pituitary gonadal axis and the re-introduction of (exogenous) estradiol and/or 
progesterone on cognitive function, subjects will be administered a series of cognitive test 
batteries at baseline, during the second month of depot Lupron treatment, and during the third 
weeks of both E2 and progesterone replacement. Further, subjects will be approached for their 
consent to participate in a number of related protocols attempting to assess and investigate the 
central nervous system effects of hypothalamic pituitary ovarian axis suppression as well as the 
effects of selective re-introduction of estradiol and/or progesterone. Related projects will include 
measures of regional cerebral blood flow, O 15 water PET, BOLD signal (protocol # 90-CC- 
0014), lymphoblastoid cells, and induced-pluripotent cells. 
Additional Procedures: 
 
A. Skin Fibroblasts and Peripheral Blood Cells for Induced Pluripotent Cell Studies 
We wish to perform skin biopsies and collect an additional sample of venous blood in 


27 
 
women who have participated (or who currently are participating) in protocols 90-M-0088, titled 
“The treatment of menstrually-related mood disorders with the gonadotropin releasing hormone 
(GnRH) agonist, depot leuprolide acetate (Lupron),” and 92-M-0174, titled “The central nervous 
system effects of pharmacologically induced hypogonadotropic hypogonadism with and without 
estrogen and progesterone replacement.” Previous findings from these protocols have 
demonstrated a differential behavioral response (i.e., a recurrence of typical mood and behavioral 
symptoms experienced premenstrually) to physiologic levels of the ovarian steroids estradiol and 
progesterone in women with premenstrual dysphoria (PMD) compared with asymptomatic 
controls. The observed differential response appears to be tissue specific (i.e., neuronal) since 
the effects of ovarian steroids at several other target tissues does not differ in women with and 
without PMD (e.g., endometrium, pituitary, breast). The biological basis for this differential 
behavioral response to ostensibly the same physiologic stimulus is unclear; however, it is well 
established that tissue specificity in the response to sex steroids is mediated by factors at the 
level of cellular function. Receptors for both estradiol and progesterone are widely distributed 
throughout the brain, and recent neuroimaging studies have demonstrated that both estradiol and 
progesterone regulate the activation patterns in brain regions involved with the processes of 
affective adaptation and stress responsivity. 
We hypothesize that the capacity for phenotypic differences between women with and 
those without PMD will be preserved in the cellular function of induced pluripotential cells 
(IPCs). Although the phenotypic difference in response to sex steroids is tissue specific, the 
latent capacity for differential expression in response to steroids should be preserved in IPCs and 
expressed in the differentiation of the IPCs into neural elements. Thus, we plan to obtain 
fibroblasts from skin biopsies and T-lymphocytes from peripheral blood (136) in order to convert 


28 
 
these cells into IPCs to establish neuronal and glial cell lines in both women with PMD and 
controls. This study will be done in collaboration with Dr. David Goldman at the NIAAA. The 
goal of this project will be to develop neurons and glial cells from participants and to 
characterize their gene expression patterns, morphological properties, and cell signaling 
pathways in response to exposures to physiologic levels of estradiol and progesterone. These 
experiments, therefore, will allow us to evaluate the nature of the differential behavioral response 
by examining protein expression and changes in cellular behaviors associated with the exposure 
to physiologic levels of either estradiol or progesterone across the two different behavioral 
phenotypes. The two behavioral phenotypes will be constituted by those women with PMD 
whose symptoms remit during ovarian suppression and whose symptoms recur during 
physiologic addback of either estradiol or progesterone. This assures the phenotypic fidelity of 
the subjects and avoids the false positives that would otherwise be generated if diagnoses are 
established solely on the basis of baseline behavioral ratings. The selection of each woman will 
occur after completion of the six month study to ensure both the proper characterization of the 
behavioral phenotype and to include comparable numbers of women in whom symptoms recur 
after estradiol and progesterone exposure. To recruit sufficient numbers of women in each group 
we will contact women who have previously participated in these protocols, have met our 
baseline criteria for PMD or asymptomatic controls, and have further demonstrated either the 
elimination of symptoms during GnRH agonist and return of symptoms upon re-exposure to 
estradiol or progesterone (PMD) or the absence of negative behavioral symptoms during both 
GnRH agonist and ovarian steroid addback phases (controls). Each of these women who is 
interested in participating will be re-consented for this study. 
We hypothesize that the skin biopsy of the fibroblasts and the T-lymphocytes obtained 


29 
 
from women with PMD and those from asymptomatic controls can be converted into IPCs and 
differentiated into model neuronal and glial cell lines. The production of these pluripotential cell 
lines will retain genetic (or epigenetic) variations that will help us understand the differential 
behavioral response at a cellular level in this condition. 
Women who participate in these protocols will be approached to obtain consent for the skin 
biopsies as part of the core consent documents. Additionally, we wish to contact women who 
have previously participated in this study in order to obtain their consent to perform a skin 
biopsy. Previous participants will be asked to provide consent using the new consent form 
accompanying this amendment. All women will be asked to provide a skin sample to study 
functional genomics of neuronal and glial precursors. Skin biopsies will be performed using an 
instrument that cuts out one or two 4 mm cylindrical pieces of skin. These biopsies are 
performed under local anesthesia (2% Lidocaine with or without epinephrine). The level of 
discomfort associated with the anesthesia is analogous to a PPD skin test for tuberculosis (TB). 
The biopsy itself is painless. This type of biopsy site usually heals by secondary intention and is 
covered with a steri-strip type bandage. If necessary, it will be closed with 1 or 2 non-absorbable 
sutures. These sutures can be removed by the investigator or his/her assistant, in about 7 to 
10 days. No more than two biopsies will be taken at any one time. Samples derived from 
biopsies may be utilized for procedures such as special stains, immunohistochemistry, direct 
immunofluorescence or immunoelectron-microscopy for localization of immune deposits, PCR, 
or establishment of cell cultures. In our protocol, the samples will be used to isolate fibroblasts 
to grow a pluripotent cell lines. 
If we are unable to produce cell lines from the initial skin sample obtained then participants 
will be re-contacted to allow us to take additional samples from a different area of the skin. The 


30 
 
repeat sample will be obtained under the same conditions as those employed to obtain the first 
biopsy but from a different area of the body. Risks and potential adverse reactions from a second 
skin sample should be similar to those of the initial sample. 
B. 
Procedures for Sample to Determine Appropriate Dose of Transdermal Estradiol Gel: 
 
We wish to identify the lowest daily dose of transdermal estradiol gel to achieve plasma 
estradiol levels between 80 and 120 pg/ml; blood levels of estradiol comparable to those 
obtained with the previously employed transdermal system in this protocol. We do not expect 
sustained plasma estradiol levels above this range. Nonetheless, the normal follicular phase 
range of plasma estradiol is between 60 and 300 pg/ml and it is possible that a single high level 
of estradiol will be observed depending on how soon after gel application blood samples are 
obtained. Thus, we will recruit up to 10 women who meet the protocol’s selection criteria. Each 
woman will be administered Lupron for two months only. After the first month of Lupron 
treatment when ovarian suppression has been achieved, we will administer 2-3 applications 
(approximately 6.0– 9.0 mg estradiol gel (open label)) once a day for four weeks. Estradiol gel 
(EstroGel® 0.36% estradiol in a hydroalcoholic absorptive gel base; Ascend Therapeutics, 
Herndon, VA) is dispensed in a controlled release pump bottle calibrated to provide 1 ml of gel 
containing 3.0 mg of estradiol per pump. The colorless, odorless, and fast drying gel is applied to 
the skin over the arms, shoulders and outer thighs (as needed) once each day (usually in the 
morning). Plasma estradiol levels after use of 3.0 and 6.0 mg daily doses (i.e., 1 and 2 pumps 
respectively) for 7 days in men were approximately 86 and 125 pg/ml, respectively (137). Thus, 
we anticipate that plasma estradiol levels should reach approximately 100 pg/ml with the two to 
three applications proposed in this pilot study. Blood samples will be obtained at weekly clinic 
visits to evaluate whether this dose administered once a day is sufficient to achieve the plasma 


31 
 
estradiol levels targeted in this study (i.e., 80-120 pg/ml). If after one week of two applications 
of estradiol gel once a day, we observe that this dose schedule is insufficient to consistently 
obtain the desired blood levels, we will increase the dose to three applications (9.0 mg) each day 
for the next three weeks. Alternately, if adequate blood levels are observed after the two 
applications once a day regimen then women will continue at this dose for the remaining three 
weeks to evaluate the stability of plasma estradiol levels with the two applications each day 
regimen. We do not anticipate needing a dose greater than three applications each day. After the 
four weeks of estradiol treatment, all women will receive one week of progesterone suppositories 
(200 mg twice a day) to precipitate a progesterone withdrawal-induced menses. Upon 
completing this evaluation, we will provide the data to the FDA to modify the IND under which 
this protocol and its companion protocol (90-M-0088) are conducted. The duration of Lupron 
treatment is a third the length of time in which Lupron is administered in the regular six month 
protocol and the duration of estradiol treatment is the same as that approved in the original 
protocol. Women participating in this component of the protocol will not be asked to participate 
in any of the additional procedures employed in the six month protocol (i.e., brain imaging, IPCs, 
or metabolomics). 
Blood Samples for this component of the protocol (10 ml) will be drawn at each visit, if 
possible, for the duration of the study (8 weeks). Total blood volume will be 90 ml. All blood 
sampling will be obtained as described in the core protocol. This blood will then be analyzed for 
gonadotropins and estradiol. 
 
 
e) End of Participation 
 
When available, the results of this study will be discussed with each participant. Controls 


32 
 
will be discharged from this protocol with follow-up as needed by their private healthcare 
provider. 
5) 
Management of Data and Samples: 
 
a. Storage 
 
Samples and data will be stored using codes that we assign. Data will be kept in password- 
protected computers. Samples will be kept in locked storage. DNA samples and cell lines will 
be stored in locked freezers in David Goldman’s laboratory (NIAAA) on Fischer’s Lane in 
Rockville. Only study investigators will have access to the samples and data. Upon the 
completion of the study, blood samples, DNA and cell lines will be retained. Any loss or 
destruction of samples will be reported to the IRB. 
Data from structured diagnostic interviews and symptom ratings are kept in secure research 
files and electronically on the Branch server space or within the CTDB database. Access to 
these research files is only available to study investigators. Symptom ratings, diagnostic 
interviews, and other questionnaires will be collected through an online system using a subject- 
specific log in and password to protect confidentiality (see Appendix). 
b. 
Data and sample sharing plan 
 
This protocol is not subject to the Genomic Data Sharing (GDS) policy. The sharing of 
Genomic data according to the GDS policy is covered under the screening protocol 81-M-0126. 
Data and samples may be shared with dbGaP, collaborating laboratories at NIH or outside of 
NIH and/or submitted to NIH-designated repositories and databases if consent for sharing was 
obtained. Repositories receiving data and/or samples from this protocol may be open-access or 
restricted access. 
Samples and data will be stripped of identifiers and may be coded (“de-identified”) or 


33 
 
unlinked from an identifying code (“anonymized”). When coded data is shared, the key to the 
code will not be provided to collaborators, but will remain at NIH. Data and samples may be 
shared with investigators and institutions with an FWA or operating under the Declaration of 
Helsinki (DoH) and reported at the time of continuing review. Sharing with investigators 
without an FWA or not operating under the DoH will be submitted for prospective IRB approval. 
Submissions to NIH-sponsored or supported databases and repositories will be reported at the 
time of Continuing Review. Submission to non-NIH sponsored or supported databases and 
repositories will be submitted for prospective IRB approval. 
Required approvals from the collaborating institution will be obtained and materials will be 
shipped in accordance with NIH and federal regulations. 
6) 
Additional Considerations: 
 
Estradiol gel (EstroGel® 0.36% estradiol in a hydroalcoholic absorptive gel base) is 
manufactured by Ascend Therapeutics, Herndon, VA. Progesterone suppositories will be 
manufactured and supplied by the NIH Pharmacy. 
7) 
Risks and Discomforts: 
 
A. Effects of Medications Administered in this Protocol 
 
Lupron 
 
The most frequent adverse effect of Lupron is hot flushes (flashes) reportedly occurring in 
4-89% of patients receiving the drug. Lupron-induced hot flushes have ranged in severity from 
occasional mild flushing to frequent sweating. In general, episodes of flushing persist with 
continued therapy in most patients receiving Lupron.  In a recently completed study of 
400 women of reproductive age with either uterine fibroids or endometriosis who each received 
 
3.75 mg depot Lupron every month for a period of six months, the most common side effects 


34 
 
reported to occur were as follows: 1) hot flashes of mild to moderate intensity (89%), 
 
2) headache (22%), 3) nervousness or irritability (11%), and 4) insomnia (10%). Local irritation 
at the injection site was complained of in less than 10% of the patients in this sample, and there 
was a mean decrease in bone density, as measured by bone densitometry, of 3.4 to 4.0%, which 
totally reversed after the medication had been discontinued for six months. Approximately ten 
patients of the original sample of 400 found the side effects to be severe enough to discontinue 
therapy. Regular menstrual cycle function returned within two months following the last 
injection of depot Lupron (Tapp Pharmaceuticals, personal communication). Blurred vision, 
lethargy, memory disorder, and numbness have been reported in less than 3% of patients 
receiving the drug. Thrombophlebitis, phlebitis and/or pulmonary embolism, and congestive 
heart failure have occurred rarely in patients receiving Lupron, but a causal relationship to the 
drug has not been established. Adverse GI effects occurring in 2% or more of patients receiving 
Lupron include nausea and/or vomiting, constipation, and anorexia. Diarrhea and a sour or 
unusual taste in the mouth have been reported less frequently. Other adverse effects of Lupron 
occurring in less than 3% of patients include decreased hematocrit and hemoglobin 
concentration, fatigue, fever, facial swelling, rash, hives, hair loss, and itching. Limited 
information is available on the acute toxicity of Lupron. Following subcutaneous administration 
of Lupron in rats at dosages 250-500 times the usual human dosage, dyspnea, decreased activity, 
and local irritation at the injection site were observed; however, there is no evidence to date that 
overdosage in humans produces similar adverse effects.  Lupron dosages up to 20 mg daily for 
up to two years have not produced unusual adverse effects in humans. There has been one report 
of an anaphylactic reaction in a patient following administration of a GnRH agonist. Recent 
longitudinal follow-up studies of girls and boys receiving GnRH agonists as a treatment for 


35 
 
precocious puberty report the development of normal reproductive function and fertility (138- 
140). The FDA recently published a caution about the effects of GnRH agonists in elderly 
males; however, the safe use of these agents in younger adults and children (141) was not 
questioned in the FDA statement. 
Estradiol 
 
Nausea is the most common side effect of estrogen administration. At conventional 
replacement doses, higher than those employed in this protocol, this complaint seldom interferes 
with eating, and no weight loss has been reported. Breast engorgement, endometrial hyperplasia 
and bleeding are also common side effects of estrogen administration. Pre-existing fibroid 
tumors of the uterus may enlarge under the effects of estrogen; however, at the dosage and for 
the duration of estrogen administration in this protocol this risk is small. 
The relationship between estrogen, both endogenous and exogenous, and the development 
of endometrial carcinoma has been suggested by several different lines of investigation (142). 
Numerous retrospective case control studies published since 1975 have indicated that post 
menopausal exposure to unopposed estrogens for more than one year results in a two to 12 fold 
increased relative risk for endometrial cancer. A relationship between the dose and duration of 
estrogen use and the risk for endometrial cancer has also been shown, the risk being increased 
after one to four years of estrogen use and rising also with the dosage employed. However, the 
addition of progesterone to estrogen therapy appears to decrease the risk of endometrial 
hyperplasia and endometrial cancer to equal or below that of women receiving no hormonal 
treatment. Recent studies suggest that the optimal regimen to prevent hyperplasia and thus, 
inferentially, the risk of carcinoma, consists of 12 to 13 days of progestin treatment each month 
when estrogens are administered (143). There is an increase in thromboembolism and stroke in 


36 
 
women receiving estrogen therapy (144-151) (and, possibly, ovarian cancer (152) and lung 
cancer (153, 154)); however, these complications are unlikely at the dose and duration of 
estrogen therapy employed in this protocol and in the younger age of the women participating in 
this trial. One study (123) reported no effect of the estrogen patch on the four clotting indices 
previously shown to be altered by oral contraceptive use (143, 155, 156). Additionally, recent 
studies (151, 157, 158) observed that an increased risk of venous thromboembolism was 
associated with oral but not transdermal estrogen compared with nonusers (in one study (157) 
odds ratios = .42 [95% CI, 1.5 to 11.6] and 0.9 [95% CI, 0.4 to 2.1] respectively). The 
mechanism underlying this observed difference in the risk of thromboembolism is not known; 
however, it has been suggested to reflect a lower activation of hepatic metabolism by the 
transdermal route of administration due to a smaller first past effect. Blood pressure, on 
average, appears to be unaffected by estrogen therapy, although both increases and decreases 
have been reported. Post menopausal estrogen therapy has been observed to increase the relative 
risk of cardiovascular disease in some (148, 159) but not all studies (160-162). Indeed recent 
analyses of the Women’s Health Initiative demonstrate that the adverse effects of estrogen 
therapy on cardiovascular outcomes were largely confined to older women compared with 
younger perimenopausal women (150, 163-171). High doses of oral estrogens have been 
reported to elevate hepatocellular enzyme levels and, less commonly, cause cholestatic jaundice. 
The risk for gall stones and hepatocellular adenomas has been reported to be increased in 
association with oral contraceptive use, and although uncommon these complications may also 
occur with the use of replacement doses of estrogen (172-174). Further, most studies have 
suggested an increased relative risk of breast cancer after four or five years’ use (150, 175-187), 


37 
 
similar to the risk expected if the onset of menopause was delayed for a comparable length of 
time. 
Due to the publicity surrounding the cancellation of the treatment arm of the Women’s 
Health Initiative study (188) that involved the administration of combined conjugated estrogens 
and medroxyprogesterone acetate (Prempro), we have included the following statement in the 
consent documents: 
Adverse Events Related to Combined Hormone Replacement and the Results of the Women’s 
 
Health Initiative (WHI): 
 
The WHI study demonstrated that continuous administration of one form of estrogen 
(conjugated estrogens) in combination with one form of progesterone (medroxyprogesterone 
acetate) is associated with an increased risk of dementia, heart attacks, stroke, blood clots, and 
breast cancer. Estradiol, the form of estrogen that we use in this study, is administered as a sole 
agent (with the exception of one week’s combination with progesterone) and, consequently, we 
do not expect that it will pose the increased risks observed with the chronic combination of the 
conjugated estrogens and medroxyprogesterone administered in the WHI study. Indeed, while 
the estrogen alone arm of the WHI trial was shown to be associated with an increased risk of 
stroke, no increased risk of either heart disease or breast cancer was observed (188, 189). 
Estrogens may precipitate migraine headaches, and depression has also been reported to 
occur with the use of estrogens. In general, considering the dose and duration of treatment that 
we propose to use in this protocol, the risk of developing such side effects is negligible. 
Progesterone 
 
Progesterone and the synthetic progestins are widely prescribed, with indications including 
dysfunctional uterine bleeding, endometriosis, mastodynia, galactorrhea, and precocious puberty 


38 
 
(128). Side effects reported in women taking progestins are uncommon but may include 
breakthrough bleeding, edema, change in weight (increase or decrease), cholestatic jaundice, 
rash (with or without pruritus), depression of mood, easy fatigue, lack of initiative, and 
chloasma. Since progestins are often used in women with antecedent menstrual irregularity, it is 
not clear whether the breakthrough bleeding represents an effect of the medication or 
refractoriness to treatment. In the large majority of patients, menstruation occurs predictably 
following withdrawal of progestins and is usually more regular than in spontaneous cycles. 
For the sake of completeness, it is also notable that side effects have been observed in 
women receiving combined oral contraceptives, including nausea, breast soreness, vaginal 
discharge, fluid retention, hypertension, and clotting abnormalities, which have been associated 
with the estrogen component of the oral contraceptive. Thromboembolic disorders including 
thrombophlebitis, pulmonary embolism, and cerebral and coronary thrombosis appear to occur 
with greater frequency in women taking oral contraceptives. While the increased incidence of 
these disorders has been associated with the estrogen component of the oral contraceptives, it is 
now believed that the progestogen component may, to a lesser extent, contribute to the increased 
risk. There are relatively few reports associating oral contraceptives with the development of 
carcinomas (vaginal, uterine, hepatic, and mammary) despite the vast use of these agents, 
although this may reflect the latent period needed for cellular transformation. Finally, several 
reports suggest an association between intrauterine exposure to female sex hormones and 
congenital anomalies. 
B. 
Effects of Additional Procedures 
 
Skin Biopsies 


39 
 
The risk of skin biopsy may be slight bleeding or infection. To avoid these complications a 
sterile dressing will be applied for 7-10 days until the scar is well formed. On occasion, the 
wound may be closed with 1 or 2 non-absorbable sutures. Acquiring skin fibroblasts from 
voluntary subjects is minimally invasive. Skin biopsy procedures can be considered as minimal 
risk since the risk is not greater than blood draws, a procedure already included in the protocol. 
The level of discomfort associated with the anesthesia is analogous to a PPD skin test for TB and 
the biopsy itself is painless. 
Blood Drawing 
 
One discomfort of this study may occur due to the venipuncture and multiple blood 
sampling. Total blood withdrawal (550-660 ml) falls within NIH guidelines (550 ml. per eight 
week period during this 20-24 month study). 
8) 
Subject Safety Monitoring: 
 
Subjects are evaluated by one of the associate investigators. Medical history and physical 
assessments occur at each clinic visit including interviews, symptom assessments, vital signs, 
and laboratory testing when clinically indicated. After completing the study, subjects will either 
be discharged from this protocol with a referral to the community, or will be referred to other 
NIMH studies. 
Patients will be warned not to become pregnant during the study and will be advised to 
employ barrier contraceptive methods. 
9) 
Outcome Measures: 
 
The outcome measures are as follows: 1) brain imaging measures including O15 PET and 
fMRI (performed as part of NIMH protocol 90-M-0014); 2) Dex/CRH stimulation tests; 
3) metabolomic profile; 4) DNA for genotyping (performed under protocol 81-M-0126; and 


40 
 
5) cell culture studies (lymphoid and IPCs). Additionally, we will employ the symptom ratings 
(described above) to evaluate the effects of GnRH-agonist-induced hypogonadism and estradiol 
and progesterone replacement on measures of mood and behavior. 
10) Statistical Analysis: 
 
In this protocol we originally proposed using 30 subjects. Calculations of the minimum 
sample size that will provide statistical significance between the three different hormonal states 
(hypogonadal, hypogonadal plus estrogen, and hypogonadal plus progesterone) with a power of 
80% were performed using the results obtained in the Hampson and Kimura study (190). 
Additionally, we supplemented this analysis with our own preliminary neuropsychological 
testing results from protocol # 90-M-0088. In the Hampson and Kimura article standardized 
differences of 0.6, 0.7, and 0.5, were calculated for the finger tapping, Purdue Pegboard, and the 
manual sequence box, respectively. The standardized difference obtained from five women who 
have been tested during the hypogonadal and during the hypogonadal plus estrogen phase of 
protocol # 90-M-0088 results in a standardized difference of .6. Thus, the originally calculated 
sample size required to provide 80% power at an alpha of 0.05 with the standardized difference 
obtained from these two investigations was approximately 30 subjects. 
Amendments to Sample Size 
Brain Imaging 
On the basis of our prior work (65), and as described in protocol # 90-M-0014 we 
anticipate that the 20 subjects recruited for the HPA axis studies will be ample for showing 
differences in rCBF. 
Skin Biopsies 
 
To recruit sufficient numbers of women in each group we will contact women who have 


41 
 
previously participated in these protocols, have met our baseline criteria for PMD or 
asymptomatic controls, and have further demonstrated either the elimination of symptoms during 
GnRH agonist and return of symptoms upon re-exposure to estradiol or progesterone (PMD) or 
the absence of negative behavioral symptoms during both GnRH agonist and ovarian steroid 
addback phases (controls). Each of these women who is interested in participating will be re- 
consented for this study. 
11) Human Subjects Protection: 
 
a. Subject selection 
 
All subjects must meet the inclusion and exclusion criteria listed in Section 6. We will 
select physically healthy adult female individuals. The proportion of ethnic minorities 
(vs. Caucasians) in the total sample, will be approximately consistent with the overall U.S. 
population proportions. 
We are limiting the upper age range of the women in this study to 50 years of age to match 
the age ranges with those in the existing companion protocols, 90-M-0088 and 05-M-0059. 
Women over the age of 50 may have age-related changes in brain function, HPA axis function, 
or reproductive hormone secretion, which could have confounding effects on the outcome 
measures in this and the companion protocols. 
b. Justification for exclusion of children 
 
We will exclude children or minors because the study population is menstruating 
premenopausal women. 
c. Justification for exclusion of other vulnerable subjects 
 
We will exclude subjects that are unable to provide their own consent given the length of 
the protocol and associated risks. 


42 
 
d. Justification of sensitive procedures N/A 
 
e. Safeguards for vulnerable populations 
 
Protections for NIH employees and staff participating in this study include 1) assuring 
that the participation or refusal to participate will have no effect, either beneficial or 
adverse, on the subject’s employment or position at the NIH, 2) giving employees and 
staff who are interested in participating the “NIH Information Sheet on Employee 
Research Participation” prior to obtaining consent, and 3) assuring that there will be no 
direct solicitation of employees or staff. 
f. Qualifications of investigators 
 
Peter J. Schmidt, M.D., is an investigator with the Section on Behavioral Endocrinology, 
NIMH. He has over 20 years experience performing studies that examine the effects of 
reproductive hormones on mood and behavior in women with reproductive endocrine-related 
mood disorders.  He will be allowed to obtain consent. 
David R. Rubinow, M.D., is a special volunteer and collaborator within the Section on 
Behavioral Endocrinology, NIMH. He has over 30 years experience in reproductive 
endocrinology and psychiatry. He will not be involved in obtaining consent nor will he have 
access to PII. 
Lynnette K. Nieman, M.D., is a senior investigator in the Reproductive Biology and 
Medicine Branch, NICHD and has extensive experience with clinical research studies in 
endocrinology and reproductive biology. She will not be involved in obtaining consent. 
Karen F. Berman, M.D., is a senior investigator within the Section on Integrative 
Neuroimaging, CDBD, NIMH. Dr. Berman has 25 years of experience in neuroimaging. She 
will not be involved in obtaining consent. 


43 
 
Pedro E. Martinez, M.D., is a staff clinician within the Section on Behavioral 
Endocrinology, NIMH and has performed endocrine studies examining the effects of aging and 
reproductive hormones on mood and behavior in both adults and children. He will be allowed to 
obtain consent. 
Shau-Ming Wei, PhD has an extensive experience in experimental design, and she has 
designed and implemented both behavioral and neuroimaging experiments over the past several 
years. Her graduate training at the NIH/Brown Program included collaborative neuroimaging 
studies on the effects of genotype and reproductive hormones on brain function. She has worked 
with our program for more than ten years (as part of our ongoing collaborations with Dr. Karen 
Berman’s group), first as a doctoral candidate, then as a postdoctoral fellow. Currently, she is a 
research fellow within our Branch and in the future will be offered a position as a staff scientist. 
Over the last decade Dr. Wei has worked closely with our clinical program and this protocol 
involving the administration of a GnRH agonist with and without ovarian steroid hormone 
replacement. She will be involved in imaging data collection and analysis, and manuscript 
preparation. She will not obtain informed consent. 
Qualifications of telephone interviewers: 
 
Screenings are performed by Annie Shellswick, who is a licensed social worker in our 
group, and who is in charge of our recruitment and outreach activities. Personally identifiable 
information is not obtained in callers who are determined to be ineligible, and the screening 
forms are not retained in the research records. 
12) Anticipated Benefits: 
 
There are no direct benefits to participants in this protocol. This protocol allows us to 
selectively examine the central nervous system effects of the gonadal steroids and, thus, provides 


44 
 
the opportunity to explore the linkages between changes in mood and behavior and gonadal 
steroid activity. Further, as a control for protocol # 90-M-0088 this study will extend our 
understanding of potential biological mechanisms underlying menstrual-related mood disorders 
and offers the possibility of uncovering some etiopathogenic mechanisms involved in these and 
related mood disorders. 
13) Classification of Risk: 
 
The overall risks for this study are more than minimal. 
 
There are low risks to individual subjects in the use of medication and procedures under the 
conditions stated in this protocol. 
14) Consent Documentation and Process: 
 
a. Designation of those obtaining consent 
 
Study investigators designated as able to obtain consent in section 12 above, will obtain 
informed consent. 
b. Consent procedures 
 
Each patient will receive a verbal and written explanation of the purpose, procedures, and 
potential hazards of this project. A record of the communication of this information and of the 
consent to participate in this study will be placed in the medical record. The right of the subjects 
to withdraw from the study or to refuse any procedure will be made clear. Any patient whose 
side effects become excessive during either the GnRH alone or estrogen/progesterone 
replacement phases will be offered the option to withdraw without completing the five or six 
month trial. Confidentiality of patients will be assured according to the laws of the State of 
Maryland. In case of published data resulting from the study, care will be taken to protect the 
anonymity of patients. 


45 
 
c. Consent documents 
 
Each consent contains all required elements. There are 3 consent forms used for this 
protocol: Standard (6 months), Estradiol gel (pilot study), and MDE (major depressive episode). 
15) Data and Safety Monitoring 
 
a) Data and safety monitor 
 
The PI will serve as the data and safety monitoring official. 
 
b) Data and safety monitoring plan 
 
As we are administering doses of estradiol and progesterone designed to produce 
physiologic levels, we expect - and have seen - no unexpected adverse events. Similarly, we do 
not anticipate unexpected serious adverse events with the dose of Lupron that we propose to use. 
Nonetheless, we see subjects every one to two weeks during their clinic visit and advise them in 
the consent form that if they experience side effects, they should notify the investigator 
immediately. Any adverse events will be reported as per NIH policy. The PI will review data 
and safety parameters at least annually. The PI will document the data and safety review in the 
research records and at the time of continuing review. 
c) Criteria for stopping the study or suspending enrollment or procedures 
 
The study will be stopped if the estradiol suppositories become unavailable or if new 
findings emerge that indicate it would not be safe to continue the study. The study will be 
stopped if there is any Serious Adverse Event related to the research. 
The PI and IRB will determine if changes are needed for the research to continue or if it will be 
closed. 
16) Quality Assurance: 
 
A. Quality assurance monitor 


46 
 
Quality assurance will be monitored by the PI, the research team and the NIMH Office of 
Regulatory Oversight (ORO). 
B. Quality assurance plan 
 
ORO monitors intramural research studies to ensure compliance with GCP, organizational 
policies and regulations. Audit frequency is determined by the ORO SOP based on the study 
level of risk. Results of ORO audits are provided to the PI, The Clinical Director and the CNS 
IRB. As an IND study, this protocol will be subject to GCP audits at study initiation and after the 
first enrolled subject. Timing of subsequent review will be established by ORO but no less 
frequent than every other year. 
17) Reporting of Unanticipated problems, adverse events and protocol deviations: 
 
The Principal Investigator is responsible for detecting, documenting, and reporting unanticipated 
problems, adverse events (AEs), including serious adverse events (SAEs), and deviations in 
accordance with NIH policy, IRB requirements, and federal regulations. Relatedness to the 
research of all serious adverse events will be determined by the PI in consultation with the CD. 
 
 
Serious unanticipated problems, serious adverse events (including deaths) that are not 
unanticipated problems, and serious protocol deviations will be reported to the IRB and CD as 
soon as possible and in writing not more than 7 days after the PI first learns of the event, unless 
immediate reporting is waived for specific serious adverse events as noted below. Not serious 
unanticipated problems and not serious deviations will be reported to the IRB and CD as soon as 
possible and in writing not more than 14 days after the PI first learns of the event. Written 
reports will be submitted in PTMS. 


47 
 
All adverse events, deviations, and unanticipated problems will be summarized and reported at 
the time of Continuing Review. 
18) Alternatives to Participation: 
 
Subjects do not receive any treatment in this study or forego any treatment in order to 
participate in this study.  The alternative, therefore, is not to participate. 
19) Privacy 
 
All research activities will be conducted in as private a setting as possible. 
 
20) Confidentiality 
 
a. For research data and investigator medical records 
 
Samples and data will be stored using codes that we assign. Data will be kept in password- 
protected computers. 
No sensitive information is collected, so employee information will be treated the same as all 
other participants. 
b. For stored samples 
 
Samples will be kept in locked storage. Only study investigators will have access to the 
samples and data. Samples and data will be stored using codes that we assign. DNA samples and 
cell lines will be stored in locked freezers in David Goldman’s laboratory (NIAAA) on Fischer’s 
Lane in Rockville. Only study investigators will have access to the samples and data. 
c. Special precautions: N/A 
 
21) Conflict of Interest 
 
a. Distribution of NIH guidelines 
 
NIH guidelines on conflict of interest have been distributed to all investigators. 
 
b. Conflict of interest 
 
There are no conflicts-of-interest to report. 


48 
 
22) Technology Transfer 
 
There is an active CTA with Besins Healthcare which was executed on 08/01/14 and 
expires on 08/01/19. Samples and data will be stored using codes that we assign. Data will be 
kept in password-protected computers. Samples will be kept in locked storage. DNA samples 
and cell lines will be stored in locked freezers in David Goldman’s laboratory (NIAAA) on 
Fischer’s Lane in Rockville. Only study investigators will have access to the samples and data. 
23) Research and Travel Compensation: 
 
Each volunteer will be compensated according to the following schedule: 
 
Core Protocol 
Initial evaluation, physical exam (2 hours) 
100.00 
Screening phase 
100.00 
Clinic visits (weekly) 2 hour x 13 
390.00 
Multiple venipuncture x 13 
260.00 
Psychological testing x 13 (at each clinic visit) 
100.00 
Symptom rating scales x 6 months 
150.00 
Investigational drugs 
a) Depot Lupron injections 100.00 x 6 
600.00 
b) Vaginal suppositories/transdermal patch daily x 3 months 
800.00 
Total 
2500.00 
Dose-finding protocol for estradiol gel: 
Initial evaluation, physical exam (2 hours) 
100.00 
Screening phase 
100.00 
Clinic visits (weekly) 2 hour x 7 
210.00 


49 
 
Multiple venipuncture x 9 
180.00 
 
Symptom rating scales x 2 months 
50.00 
 
Investigational drugs : 
 
a) Depot Lupron injections 100.00 x 2 
200.00 
 
b) Estradiol gel application daily x 1 month 
60.00 
 
Total 
900.00 
 
Reimbursement of travel and subsistence will be offered consistent with NIH guidelines. 
Compensation will be prorated for parts completed if subjects do not complete the study. No 
escort fee will be provided. 
Employees and staff who participate during work hours must have permission from their 
supervisor. NIH employees must either participate outside of work hours or take leave in order to 
receive compensation. 
 
 
Additional Procedures 
 
Skin biopsies 
$100 per biopsy 
 
Blood Drawing Schedules: 
 
(A) Core Protocol (24 weeks) –660 ml (six months) 


50 
 
24) 
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61 
 
Flow Sheet: 
 
 
 
 
Central Nervous System Effects of GnRH Agonist & 
Gonadal Steroid Replacement in Asymptomatic Controls— 
Schematic Baseline(2cycles)
 
 MCDay2-5
 Procedures
Estradiol 0.1 mg/d 
Progesterone 200 mg BID 
OR Time
(weeks) 0
 4
 8
 12
 16
 20
 24
 
Leuprolide Acetate (3.75 mg IM QM) 


62 
 
Table I 
 
Conditions Associated with Increased Risk for Osteoporosis 
 
AIDS/HIV 
Hyperparathyroidism 
Multiple Sclerosis 
Amyloidosis 
Hypogonadism (primary or 
Multiple Myeloma 
Ankylosing Spondylitis 
secondary) 
Pernicious Anemia 
COPD 
Hypophosphatasia 
Rheumatoid Arthritis 
Congenital Porphyria 
Hypophophosphatemia 
Severe Liver Disease, esp 
Cushings Syndrome 
Idiopathic Scoliosis 
PBC 
Eating Disorders 
Inadequate Diet 
Spinal Cord Injury 
Female Athlete Triad 
Inflammatory Bowel 
Sprue 
Gaucher’s Disease 
Disease 
Stroke/CVA 
Hemochromotosis 
Insulin-Dependent Diabetes 
Thallasemia 
Hemophilia 
Lymphoma/Leukemia 
Thyrotoxicosis 
Malabsorption Syndromes 
Tumor 
Mastocytosis 
 
 
Adapted from National Osteoporosis Foundation Physician’s Guide 


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Clinical Trials Database - Security Overview 
 
There are multiple aspects to the security framework for the Clinical Trials Database (CTDB) and 
Clinical Trials Survey System. The following features allow for the safe and secure collection of 
research variables: 
 
• Application Firewall- The NICHD has recently upgraded their application firewall which 
protects both the front end web server and back end database server for the CTDB. Strict 
policies are in place which control exactly who has pre-defined, limited access to the 
application. This firewall is a state-of-the-art hardware solution which blocks access to 
everyone but authorized users for the CTDB system. 
• Data Encryption and SSL Certificates - The CTDB system makes use of military grade 
encryption both for the session and the data storage. The CTSS collects de-identified self- 
reported data. Both systems protect information from interception by encrypting the data 
flow using SSL with a 1024 bit signed certificate. The entire communication session from 
the time a client requests a connection to the system to the time a user logs out is encrypted 
using a certificate from an industry recognized vendor. The result is a secure 
communications channel for our partners, providing data confidentiality and integrity. In 
further, the CTDB program encrypts data stored within the system, thereby providing an 
additional layer or security for the sensitive CTDB clinical data. 
• CTSS HIPAA Requirements - Below is the list of identifiers to be removed for the de- 
identification of health information under HIPAA. This is found in 45 C.F.R. 
164.514(a),(b)&(c) 
 
(A) Names; 
(B) All geographic subdivisions smaller than a State, including street address, city, 
county, precinct, zip code, and their equivalent geocodes, except for the initial three 
digits of a zip code if, according to the current publicly available data from the Bureau of 
the Census: 
(1) The geographic unit formed by combining all zip codes with the same three initial 
digits contains more than 20,000 people; and 
(2) The initial three digits of a zip code for all such geographic units containing 20,000 or 
fewer people is changed to 000. 
(C) All elements of dates (except year) for dates directly related to an individual, 
including birth date, admission date, discharge date, date of death; and all ages over 89 


64 
 
and all elements of dates (including year) indicative of such age, except that such ages 
and elements may be aggregated into a single category of age 90 or older; 
(D) Telephone numbers; 
(E) Fax numbers; 
(F) Electronic mail addresses; 
(G) Social security numbers; 
(H) Medical record numbers; 
(I) Health plan beneficiary numbers; 
(J) Account numbers; 
(K) Certificate/license numbers; 
(L) Vehicle identifiers and serial numbers, including license plate numbers; 
(M) Device identifiers and serial numbers; 
(N) Web Universal Resource Locators (URLs); 
(O) Internet Protocol (IP) address numbers; 
(P) Biometric identifiers, including finger and voice prints; 
(Q) Full face photographic images and any comparable images; and 
(R) Any other unique identifying number, characteristic, or code, except as permitted by 
paragraph (c) of this section 
 
• Logical Access Controls (Role/Privileges) – Logical access controls are in placed using role 
based security for database access and application account access. Security controls are in 
place to detect unauthorized access attempts. The application is further protected by the 
NICHD firewall and NIH firewall. Inactive user accounts are monitored and removed when 
not needed, and users are disconnected after a specific period of inactivity. Encryption is 
used (1024bit SSL key) and data is HIPPA compliant. Access is monitored and apparent 
security violations are investigated when identified. Insecure protocols are disabled on all 
application servers. Guest and anonymous accounts/access is disabled. 
• Audit Trails – Activity involving access to and modification of sensitive or critical files is 
logged and monitored for possible security violations. Access to these audit trails is strictly 
controlled and can be used to support after-the-fact investigations of how, when and why 
normal operations ceased should this occur. Off-line storage of audit logs is retained for a 
period of at least 1 year. Suspicious activity is investigated and appropriate action is taken 
when warranted. 
• Physical and Environmental Protection – The servers are physically located in a secured 
NIH data center with controlled limited access. All work products from the system including 
Data backup tapes are rotated to off-site storage with must be authorize and are recorded. All 
visitors to sensitive areas are escorted with entry codes changed periodically. Fire prevention 


65 
 
and suppression devices are installed and in working condition. All heating and air-cooling 
systems are periodically checked to ensure proper working condition. 
• Production Input and Output Control – Audit trails are in place to record data changes. 
Only authorized system administrators are allowed access to this data, as well as any data 
backup tapes. Damaged media is sanitized or destroyed, and any hardcopy media is shredded 
when no longer needed. 
• Contingency Planning – All critical data files, database files and web server files have been 
identified. A regularly scheduled data backup solution is in place with identified resources 
supporting critical operations. A comprehensive contingency plan has been developed and 
documented. This plan has been approved by key affected parties. The Contingency 
plan/Disaster recovery plan is regularly tested and adjusted as appropriate. 
• Hardware and Software System Maintenance – Access is limited to the hardware and 
software infrastructure. Restrictions are in place as to who performs maintenance activities. 
Procedures are in place to monitor the use of system resources. All new and revised hardware 
and software are tested and approved before implementation. All system and application 
components are tested, documented and approved prior to promotion to production 
environment. Detailed system specification as prepared and reviewed by management. A 
version control system is in place for all key application and operating system files. The 
systems are actively managed to monitor and reduce vulnerabilities with unnecessary 
services eliminated. 
 
Strategic framework – The tactical security framework provides a mechanism whereby the 
Clinical Trials support personnel provide day-to-day operational support activities for the regular 
maintenance of the CTDB system. These initiatives incorporate application and software 
security. The CTDB application was designed using the latest Java technology. This allows the 
adaptation of the application to ever-changing business rules within the application. An Oracle 
10g relational database provides the repository for the clinical data. The use of a robust, industry 
standard relational database provides a modular architecture design of CTDB which allows for 
the CTDB program to assign role based security to the participants in the system. This allows 
roles to be defined and implemented for different users- such as investigators, study participants, 
report writes, etc- in order to secure database access and the application data stored within the 
system. This implies that the NICHD CTDB partners can implement custom roles and maintain 
their own clinical data with a high level of confidence that the data will not be compromised nor 
shared with non-participants. As technology evolves, this attention to the strategic framework 
allows us to address individual software components and target them for enhancements or 
upgrades all while maintaining the integrity and confidentiality of the CTDB system. Another 


66 
 
example of this strategic framework is the reporting interface. The modular design of CTDB 
allows provides the ability to upgrade to Cognos reporting with the result being more detailed 
comprehensive reporting abilities. The net result is a feature-enhanced system while maintain the 
strict security framework of the system.