An Endocrine Model for Postpartum Mood Disorders
Phase: PHASE2
Conditions: Postpartum Depression
Sponsor: National Institute of Mental Health (NIMH)

 
Protocol Title: An Endocrine Model for Postpartum Mood Disorders 
Protocol Number: 95-M-0097 
Date of This Submission/Version:   12/21/2023 
 
Principal Investigator 
Peter Schmidt, M.D. 
NIMH/ 
BEB 
Bldg 
10CRC 
Rm 25330 
301-496-6120 
E-mail 
PeterSchmidt@mail.NIH.G
OV 
 
Total requested accrual: 100 
 (40) Patients 
 (40) Volunteers 
(20) Past Major Depression 
 
IND/IDE 
 
 
 No   
 Yes (attach FDA documentation) 
  
Drug/Device/#__163205 Lupron_____ 
  
Sponsor _NIMH (Maryland Pao, MD is the official representing the Institute) 
 
Durable Power of Attorney  
 No   
 Yes 
 
Multi-institutional Project 
 No   
 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 MTA 2017-0327________Expiration Date__None 
___________ 
 
Confidential Disclosure Agreement  
 No  
 Yes 
 
Samples are being stored  
 
 
 No   
 Yes 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
2
Table of Contents 
 
1. 
Introduction: ............................................................................................................. 4 
2. 
Study Objectives: ................................................................................................... 16 
3. 
Subjects: ................................................................................................................. 17 
4. 
Study Design and Methods .................................................................................... 19 
5. 
Management of Data and Samples: ....................................................................... 28 
6.  
Additional Considerations: .................................................................................... 30 
7. 
Risks and Discomforts: .......................................................................................... 30 
8.  
Subject Safety Monitoring: .................................................................................... 38 
9. Outcome Measures: ...................................................................................................... 40 
10. 
Statistical Analysis: ................................................................................................ 40 
12. 
Consent Documentation and Process: .................................................................... 43 
13. 
Data and Safety Monitoring: .................................................................................. 44 
14.  
Quality Assurance: ................................................................................................. 45 
15. 
Reporting for Unanticipated Problems, Adverse Events and Protocol Deviations:46 
16. 
Alternatives to Participation: ................................................................................. 46 
17. 
Privacy: .................................................................................................................. 46 
18. 
Confidentiality: ...................................................................................................... 46 
19. 
Conflict of Interest Statement: ............................................................................... 46 
20. 
  Technology Transfer: ........................................................................................... 47 
21. 
Research and Travel Compensation: ..................................................................... 47 
22. 
References: ............................................................................................................. 48 
Appendix I ........................................................................................................................ 60 

 
 
 
3
Precis: 
 
The appearance of mood and behavioral symptoms during pregnancy and the postpartum 
period has been extensively reported.  While there has been much speculation about possible 
biologically based etiologies for postpartum disorders (PPD), none has ever been confirmed.  
Preliminary results from two related studies (protocols 90-M-0088, 92-M-0174) provide 
evidence that women with menstrual cycle related mood disorder, but not controls, experience 
mood disturbances during exogenous replacement of physiologic levels of gonadal steroids.  The 
present protocol is designed to create a “scaled-down” hormonal milieu of pregnancy and the 
puerperium in order to determine whether women who have had a previous episode of 
postpartum major affective episode will experience differential mood and behavioral effects 
compared with controls and to determine whether it is the abrupt withdrawal of gonadal steroids 
or the prolonged exposure to gonadal steroids that is associated with mood symptoms.  
Supraphysiologic plasma levels of gonadal steroids will be established, maintained, and then 
rapidly reduced, simulating the hormonal events that occur during pregnancy and parturition.  
This will be accomplished by administering estradiol and progesterone to women who are 
pretreated with a gonadotropin releasing hormone (GnRH) agonist (Lupron).  After eight weeks, 
administration of gonadal steroids will be stopped in one group of patients and controls, and a 
sudden decline in the plasma hormone levels will be precipitated.  Another group will be 
maintained on supraphysiologic levels of estradiol and progesterone for an additional month. 
Outcome measures will include mood, behavioral, and hormonal parameters. 
 
 
 
 

 
 
 
4
1. 
Introduction: 
a. Background and Introduction 
 
Affective syndromes that occur during the postpartum period have traditionally been 
divided into three categories: 1) postpartum “blues”, 2) postpartum depression, 3) puerperal 
psychosis.  The present protocol will only deal with the last two categories, which are associated 
with more persistent symptoms and with a higher rate of morbidity. 
 
 
Puerperal psychosis is the most severe form of psychiatric disturbance in the puerperium.  
This condition is characterized by extreme agitation and confusion, lability of mood, severe sleep 
disturbances, possible paranoid or grandiose delusions, and in some cases prominent 
hallucinations.  Results of both retrospective case register studies and longitudinal prospective 
studies have observed that the majority of cases of puerperal psychosis begin during the first few 
weeks after parturition (1,2).  The prevalence of psychosis in the month after childbirth is 
approximately 22 times that of any month in the preceding two years (3).  Women with a history 
of bipolar or schizoaffective illness are particularly at risk, and about 50% of these women 
relapse in the first few months after delivery (4-6).  In fact most cases of postpartum psychosis 
are considered to be an extreme manifestation of a bipolar disorder (7), and the overall incidence 
of this condition is very low, occurring in 0.2% of all deliveries. 
 
 
Postpartum depression is clinically similar to the DSM-IV defined major depression, but 
its onset has a temporal relationship to parturition.  The main symptoms are appetite and sleep 
disturbance, excessive fatigue, sadness and anhedonia, excessive guilt, psychomotor and 
cognitive disturbances and suicidal ideation.  The two to three month prevalence rates of 
postpartum depression in studies using conventional diagnostic criteria (8) (e.g., RDC, DSM-III) 
have been reported to be in the range of 8.2% to 14.9% (9,10).  Some studies (1,10,11), but not 

 
 
 
5
all (12), have reported that the incidence of depression is increased significantly during the first 3 
months after birth as compared to during pre-pregnancy, pregnancy or the period after the first 
postpartum year.  Others have disputed this association, arguing that the prevalence of 
depression during the postpartum period is no greater than that for depression at other periods of 
a woman's life (12).  Factors associated with postpartum depression are a history of psychiatric 
illness, family history of psychiatric illness, marital disharmony and lack of confiding 
relationships, maternal age and the number of life events in the previous year (13-15).  
Additionally, several studies have suggested that a past history of postpartum disorders (PPD) 
may predispose women to develop an episode of PPD during subsequent pregnancies.  In a 
longitudinal prospective study (16) women with a past history of PPD had a significantly higher 
rate of PPD during a subsequent pregnancy than women without a history of PPD, specifically, 
28-40% compared with 10%.  These findings are corroborated by preliminary results from a 
longitudinal prospective study conducted at the Massachusetts General Hospital suggesting a 
higher relative risk of PPD during subsequent pregnancies in women with a prior episode of PPD 
(17,18). 
b. Biological Factors in Postpartum Disorders: 
 
Hormonal factors have been implicated as having an important etiological role in the 
development of PPD.  Pregnancy is accompanied by a slow but sustained rise in various steroid 
and peptide hormones, followed by a sudden drop over the first few days after delivery.  During 
the third trimester of pregnancy, plasma progesterone levels are approximately 150 ng/ml and 
estradiol reaches plasma levels of approximately 10-15 ng/ml.  These levels represent a 10 and 
50 fold increase, respectively, of maximum menstrual cycle levels (19).  After parturition 
progesterone and estradiol levels drop to early follicular phase levels within days (20). 

 
 
 
6
 
A role for progesterone and estradiol in postpartum mood disorders is suggested by their 
manifold neuroregulatory effects (21). 
 
1). Progesterone - In addition to its genomic effect, progesterone has rapid effects on 
neuronal membranes (22).  The rapid membrane action of progesterone metabolites is thought to 
be responsible for the anaesthetic action of progesterone (23) and occurs through positive 
modulation of the gamma-aminobutyric acid-A (GABA-A) receptor (24).  These progesterone 
metabolites (e.g., allopregnanolone) may be formed from circulating progesterone or may be 
synthesized de novo in the brain and are, therefore, known as neurosteroids.  Some researchers 
have postulated that the levels of neurosteroids reached in the brain are physiologically 
significant and compatible with their playing a neuromodulatory role during the estrous cycle 
and pregnancy, possibly associated with behavioral changes seen in these conditions (25,26).  
Studies, using different outcome measures, confirm that in female rats, GABA-A receptor 
sensitivity to specific neurosteroids such as allopregnanolone (26) is cycle-dependent.  
Pregnancy and the postpartum period have also been reported to both increase and decrease the 
affinity of GABA receptors in the brain for a variety of ligands (26,27).  These effects of 
progesterone could provide the theoretical ground for a “progesterone withdrawal” hypothesis 
for PPD. 
 
Studies have been unable to identify a significant association between progesterone levels 
and either the “blues” (28,29) or postpartum depression (30).  However, one study did report a 
significant correlation between progesterone levels around parturition and the “blues.”  
Additionally, Dalton treated women at risk for the development of PPD with open label 
progesterone following parturition and observed no recurrence of PPD (31), suggesting a 
preventive effect of progesterone on the onset of PPD in this sample.  In contrast, there is at least 
one study that indicates that giving progestins during the postpartum period actually increases 

 
 
 
7
postpartum depressive symptoms (32).  It remains unclear whether there is a causal role for 
changing levels of progesterone in the onset of symptoms of PPD (30). 
 
2). Estradiol - Estradiol has been found to alter brain neurotransmitter activity in several 
ways: in the hypothalamus, estradiol (E2) is associated with decreased norepinephrine synthesis 
and increased choline acetyltransferase activity (33), decreased glutamic acid decarboxylase 
activity (34), and increased muscarinic receptors (35,36); in the cerebral cortex, E2 replacement 
in ovariectomized rats decreases beta-adrenergic and 5HT-1 receptors but increases 5HT-2 
receptors (37); and E2 facilitates excitability of hippocampal neurons in vitro (38,39) and in vivo 
(40), through both short term and long term mechanisms, and decreases the threshold to 
electrically-induced seizures in the medial amygdaloid nucleus (41,42).  Additionally, E2 
increases NMDA binding sites in the CA1 region of the hippocampus (43).  An anti-
dopaminergic effect of estradiol at both the pituitary and striatum has been described (44).  
Elevated levels of estradiol have been reported to decrease tyrosine hydroxylase activity and 
hence dopamine synthesis (45), while even low doses can modulate the striatal agonist affinity 
states of the D2 receptor (46).  Consistent with these putative antidopaminergic effects is the 
observation of improvement of L-DOPA-induced tardive dyskinesia with estradiol (47) and the 
amelioration of psychotic symptoms in schizophrenic women during the high estradiol phase of 
their menstrual cycle (48).  Conversely, it should be noted that enhanced dopamine receptor 
sensitivity following estradiol administration has been reported (49,50).  Regional brain MAO 
activity has been shown to be increased by progesterone (51) and decreased by estradiol (52). 
 
Most studies have failed to show an association between blues or depression and estradiol 
plasma levels around parturition (29,30).  However, there are preliminary data that indirectly 
suggest a role for estradiol in postpartum disorders.  Women who later have a recurrence of PPD, 
as opposed to women who remain well, show an increased D2 sensitivity postpartum as reflected 

 
 
 
8
by exaggerated growth hormone response to apomorphine (6,53).  This modification in D2 
sensitivity may result from altered effects of estradiol on D2 receptors in women with PPD, given 
the ability under normal conditions of E2 to inhibit D2 activity by uncoupling the D2 receptor 
from its second messengers and to decrease D2 receptor sensitivity (46,54).  Furthermore, 
estradiol replacement has been reported to be an effective treatment for postpartum depression 
under double-blind, placebo-controlled conditions (55). 
 
3) Other hormones - A significant association has been found between postpartum 
thyroid dysfunction and the development of postpartum depression (56,57).  It has also been 
shown that women with a positive thyroid antibody status, which occurs in approximately 10% 
of normal women, are prone to episodes of postpartum depression (58), and up to 1% of all 
postpartum women may develop a depression associated with autoimmune thyroid disease.  
More recently, Pedersen et al., in press, reported a negative correlation between antenatal 
thyroxine levels and self-reported depression scores in the postpartum period, suggesting that 
low antenatal thyroid status is a risk for developing PPD. 
 
Preliminary studies also indicate a possible association of HPA axis dysregulation and the 
development of postpartum depression. There is one study suggesting that symptoms of 
depression during pregnancy are associated with lower levels of CRH (59).  Our own 
preliminary data indicate a significant study phase effect on integrated cortisol secretion 
(increased during addback compared with the withdrawal phase) and a trend for a diagnosis by 
hormone condition interaction, reflecting increased cortisol in women with a history of PPD 
compared to controls during the addback phase (60). 
 
Other hormones such as prolactin, androgens, beta-endorphin, vasopressin, oxytocin, and 
growth hormone have marked fluctuations during pregnancy and parturition.  Association 
between these hormones and psychiatric symptoms have not been substantiated. 

 
 
 
9
 
Despite these observations, there are only a few systematic studies of the role of gonadal 
steroids in the etiology of PPD.  This probably reflects a number of factors, including the low 
prevalence of PPD in the community, the presence of numerous psychosocial and medical 
stressors during pregnancy and the puerperium, and the difficulties involved in recruiting and 
evaluating women who are either pregnant or lactating and nursing an offspring.  To avoid these 
problems, we propose to study women with a history of PPD compared with women without a 
history of PPD, in order to answer the questions, 1) “Are women who develop PPD specifically 
susceptible to the hormonal events of pregnancy and the puerperium?”; and 2) Is it the abrupt 
withdrawal of gonadal steroids or the prolonged exposure to gonadal steroids that is associated 
with mood symptoms in these women? 
 
Preliminary results of this study demonstrate that acute withdrawal of estradiol and 
progesterone is associated with a recurrence of mood and behavioral symptoms in women with a 
history of PPD but not in women lacking such history (61).  These findings document a 
differential sensitivity to changes in gonadal steroid levels in women with a history of PPD, 
similar to what is seen in women with premenstrual syndrome.  However the specific role of 
hormonal withdrawal is unclear as we observed that women with PPD were also significantly 
more symptomatic than controls during the addback phase of hormonal treatment (though less 
robustly than during the withdrawal phase).  It is possible, therefore, that the duration of 
exposure to gonadal steroids rather than their withdrawal was the critical determinant of 
symptom precipitation; i.e., continued exposure to estradiol and progesterone replacement in our 
study might have been associated with equal levels of mood symptoms.  Accordingly, we 
propose to mimic the hormonal events of pregnancy by randomly assigning patients and 
volunteers to either a withdrawal or continued replacement group (details in Clinical Assessment 
and Procedures).  Furthermore, in order to establish PPD as a distinct phenotype, we will study a 

 
 
 
10
group of women with histories of major depression occurring outside of the perinatal period to 
test whether the mood and HPA axis effect of this paradigm are specific to postpartum 
depression or a nonspecific response to any major depressive episode. 
 
  Certain forms of affective illness are distinguished by their appearance during periods of 
reproductive endocrine change, such as during pregnancy and the postpartum/puerperium. 
Unlike depression accompanying endocrinopathies (e.g., hypothyroidism), these peripartum 
mood disorders have been shown in our prior work to represent differential sensitivity to an 
otherwise normal reproductive endocrine stimulus (62). As such, these disorders offer a unique 
opportunity to identify underlying mechanisms of affective dysregulation: i.e., in this protocol 
we have identified the trigger, but the underlying susceptibility that transforms a normal 
endocrine signal into an abnormal response is undetermined. One approach we have undertaken 
is to study women with a history of peripartum mood disorders (PPMD) to determine whether 
blinded exposure to supraphysiologic levels of reproductive steroids will result in abnormal 
neural network activation in affective regulating and reward circuitries. These studies, will not, 
however, explain the underlying cellular/neural differences that transform a normal signal into an 
abnormal response. For that purpose, we propose to opportunistically collect blood samples from 
women already diagnosed with PPMD in order to generate human induced-pluripotent cells 
(iPSCs), neural progenitors, and possibly human induced serotonergic and dopaminergic neurons 
for transcriptomic analysis that can be compared with those from controls under baseline 
conditions and following stimulation with ovarian steroids (i.e., estradiol and progesterone). This 
strategy has been successfully employed by our group in a related disorder, premenstrual 
dysphoric disorder (PMDD), albeit with lymphoblastoid cell cultures (LCLs) rather than h-
iPSCs. h-iPSCs/neural progenitors offer the advantage of being a neural tissue and, therefore, 
physiologically more relevant to these conditions than the immune tissue represented by the 

 
 
 
11
LCLs. Given, however, observation of immune dysfunction in women with postpartum 
depression (women with PPD have a higher than expected rate of postpartum thyroiditis), we 
will also create LCLs to determine the following: 1) whether clear differences in genomic 
expression can be identified that may explain the observed relationship between immune 
dysregulation and PPD; and 2) whether the differential transcriptional responses to ovarian 
steroids  observed in women with a history of PMDD – a putative biomarker of hormone 
sensitivity – can be observed in women with a different disorder (PPD), albeit one characterized 
by susceptibility to reproductive hormone-induced affective dysregulation. 
   
Cellular models, involving neuronal cells, lymphoblastoid cell lines (LCLs), and more 
recently neural progenitor cells (63) have many advantages, but also distinct limitations for the 
study of brain diseases (64), (65), (66). Studies employing both LCLs and neural progenitor cells 
have been employed to investigate several forms of both inherited diseases (67), (68) and 
neuropsychiatric conditions (69), (64), (70), (71), (72). Despite limitations of peripheral blood 
cell-derived cell lines, these cellular models offer the opportunity to identify biochemical 
characteristics related to specific disease phenotypes. Additionally, recent evidence supports the 
use of LCLs for identifying both local and long-distance regulatory elements involved in 
immune-mediated disease states (73). 
 
2: Specific Aims 
 
Aim 1: Determine whether cellular substrates of differential hormone sensitivity can be 
identified through creating neuronal progenitors exposed to high dose estradiol (E) and 
progesterone (P)  (100nm each x 3 days) and withdrawal (3 days), with transcriptomic analyses 
conducted after each treatment. 

 
 
 
12
 
Aim 2: a) Determine whether transcriptional response in immune-relevant pathways to 
hormone response and withdrawal in LCLs will, in these long term cultured cells, distinguish 
women with PPD from controls and, therefore, further implicate differential immune responses 
in the pathophysiology of PPD; and b) replicate differences in the polycomb repressor complex 
under both basal and E- and P-stimulated conditions that we previously observed in premenstrual 
dysphoric disorder (PMDD).    
 
Hypotheses: H1) After exposure to combined E and P, neural progenitor cells from 
women with PPD will reveal a transcriptional pattern that differs from that in controls; H2) 
Similar to PMDD, LCLs from women with PPD will demonstrate a differential basal and 
hormone-stimulated transcriptional response in network and pathways analyses(e.g., Weighted 
Genome Correlation Network Analysis (WGCNA), Gene Set Enrichment Analysis (GSEA), and 
Database for Annotation, Visualization and Integrated Discovery (DAVID)). 
 
Experimental Plan: This component of our protocol will build on access to unique 
samples of women with peripartum mood disorders as follows: 1) to create human-induced 
pluripotent cells  
(h-iPSCs) and neuronal progenitor cells whose transcriptional responses to ovarian steroids in 
vitro will be assessed; and 2) to create lymphoblastoid cell lines (LCLs). 
  
Participants: PPD- Peripheral blood samples will be obtained from women with PPD and 
control women (matched for age, race and parity) who met the following criteria: 1) participated 
in the GnRH agonist and E/P replacement-then-withdrawal paradigms conducted at the NIMH 
IRP; and 2) clearly demonstrated recurrence of depression (in the women with past PPD but not 
controls) after E/P withdrawal. This group of women with past PPD and controls therefore will 
represent a highly selected phenotype in which we have demonstrated behavioral ovarian steroid 

 
 
 
13
sensitivity (in past PPD) or the absence of this phenotype (in controls). All women will be 
otherwise healthy, and medication free.  
 
h-iPSCs: h-iPSCs will be generated from Sendai virus-transformed T cells (74), a 
procedure already successfully accomplished in the LNG laboratory, and then differentiated to 
neuroprogenitor cells (75) (also successfully achieved by LNG), serotonergic (76) (experiments 
underway in LNG) and dopaminergic neurons (77), (69). Following differentiation, cells will be 
cultured in a sex steroid-free growth medium which is phenol red-free media to reduce possible 
exposure to steroid-like activity prior to experimental exposure. 
 
LCLs: EBV-transformed lymphoblastoid cell lines will be made from peripheral blood as 
described previously (78), and cells from women with PPD and matched controls will be 
cultured under identical conditions. As for IPSCs, 5 days before hormone exposure, LCLs will 
be cultured in a sex steroid-free growth medium of phenol red-free media supplemented with 
15% knock out serum (KOSR) (estrogen-depleted). 
 
PPD and control cells will be cultured and collected at baseline, after exposure to 
combined E/P at supraphysiologic doses of 100nm each (in DMSO) for 3 days, and after 
withdrawal to vehicle only (DMSO alone) or continued E/P treatment for 3 days (to control for 
latency following initial hormone exposure).  
 
RNA sequencing: Total RNA will be isolated from the cell pellets and then treated with 
DNase to remove any traces of genomic DNA contamination. Quality assessment of the RNA is 
done by an Agilent® 2100 Bioanalyzer. Whole-transcriptome RNA sequencing will be 
performed using an Ion Torrent NGS Sequencer, the Ion Proton. Total RNA will be polyAAA 
selected and used to prepare cDNA libraries using the Ion Total RNA-Seq Kit v2, barcoded with 
Ion Xpress™ RNA-Seq Barcodes.  

 
 
 
14
 
Analysis of RNA sequencing data: Reads will be mapped to reference genome Hg19 and 
normalized. Using edgeR analyses (or other similar platforms), performed in either CLC Bio 
Workbench 11 or “R” programming environment (or other appropriate environment), 
differentially expressed genes will be computed between conditions and multiple comparisons 
will be corrected using the False Discovery Rate (FDR, Benjamini Hochberg) method. For each 
gene of interest, normalized expression between untreated and hormone treated/withdrawn cell 
cultures will be further analyzed by 2-way ANOVAs, with hormone condition (i.e., untreated, 
hormone exposure, hormone withdrawal) as the within-groups factor and diagnostic group (i.e., 
PPP or PPD versus Controls) as the between-groups factor. The appropriate post-hoc tests will 
be performed should significant interactions be detected.  
 
Pathway/Network analysis of RNA sequencing data: Pathway/Network analyses (e.g., 
Gene Set Enrichment Analysis (GSEA)) will be performed on differentially expressed genes to 
identify significant biological processes, molecular functions and cellular components for 
samples in the baseline (untreated), E/P-treated condition, and after withdrawal. Additional 
analyses that could be performed include WGCNA to examine modules of differentially 
expressed genes between cells from women with PPD and controls under each hormone 
condition. Finally, we will subject all differentially expressed genes to other bioinformatics tools, 
such as the Database for Annotation, Visualization and Integrated Discovery (DAVID)to identify 
functionally related groups and networks, which can help to detect impacted biological, 
molecular and cellular functional domains. 
 
Validation of RNA-sequencing: mRNA expression analysis using qRT-PCR will be used 
to quantitate differentially expressed genes at baseline, after E/P exposure, and after E/P 
withdrawal. TaqMan qRT-PCR assays will be carried out according to the assay developer’s 

 
 
 
15
recommended conditions on a 7900HT Fast Real-Time PCR System. A two-tailed, unpaired 
Students t-test will be performed for each comparison.   
 
Additional Analyses: it is possible that whole exome sequencing will be performed in 
these samples, and evaluations of the methylome will be performed on repeated DNA samples 
across each of the hormonal conditions established in the clinical protocol (i.e., baseline, add-
back, withdrawal and follow-up). 
 
This study will be done in collaboration with Dr. David Goldman at the NIAAA. 
 
c. Medications Employed in this Study: 
 
In order to avoid fluctuating hormonal levels with subsequent menstruation, and to ensure 
reduction of gonadal steroids to hypogonadal levels during the withdrawal phase, suppression of 
ovarian function will be induced with the GnRH agonist leuprolide acetate (Lupron).  As 
described in detail in protocol # 90-M-0088, Lupron is a synthetic nonapeptide that functions as 
an agonist that is 80 to 100 times more potent than natural GnRH in inducing the release of LH 
(79).  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 estrone and 
estradiol in premenopausal females.  However, within two to four weeks after initiation of 
treatment, there is a subsequent decrease in the number of GnRH receptors and an inhibition of 
pituitary elaboration of gonadotropins, leading to levels of estradiol similar to those observed in 
post-menopausal women. 
 
Following suppression with Lupron, estradiol and progesterone will be administered in 
oral micronized form.  The administration of 17 beta estradiol via an oral route using micronized 
E2 was chosen for this study because it has several advantages.  First, it delivers the primary 
ovarian estradiol, into the circulation at a rate resulting in sustained levels of estradiol and 

 
 
 
16
constant rates of excretion of estradiol conjugates (80).  Second, at doses of 3-6 mg/day, it 
delivers sufficient estradiol into the circulation to raise estradiol plasma concentrations to levels 
similar to those of women around the ovulation phase of their menstrual cycle (80), levels that 
while far from reaching full pregnancy levels would give sustained supraphysiologic estradiol 
levels compatible with early pregnancy.  Third, micronized estradiol has a short half-life, thus 
facilitating rapid reduction in plasma levels.  Finally, the dose can be easily adjusted so that the 
presence of minor side effects such as nausea may not require complete cessation of the 
medication. 
 
Orally administered micronized progesterone is biologically active and at doses of 600-
1600 mg/day will result in plasma levels of 30-40 ng/ml (81,82).  Progesterone in suppository 
form has been approved by the IRB for administration to patients with menstrual related mood 
disorders (MRMD) and controls under NIMH protocols # 92-M-0174, 90-M-0088. 
2. 
Study Objectives: 
 
There are several lines of evidence suggesting a potential role for the hormonal events 
that occur during pregnancy and parturition in precipitating mood and behavioral changes in a 
subgroup of women: 
 
1) Gonadal steroids modulate the activity of various neurotransmitter systems, 
 
2) Gonadal steroids may be efficacious in the treatment of PPD; 
 
3) Women with a history of PPD are vulnerable to the development of recurrent 
episodes of PPD during subsequent pregnancies. 
In our previous findings in this protocol, the specific role of hormonal withdrawal was unclear as 
we observed that women with PPD were also significantly more symptomatic than controls 
during the addback phase of hormonal treatment.  It is possible, therefore, that the duration of 
exposure to gonadal steroids rather than their withdrawal was the critical determinant of 

 
 
 
17
symptom precipitation. Thus, in this current protocol, we propose to mimic the hormonal events 
of pregnancy and randomly assign patients and volunteers to either a withdrawal or continued 
replacement group.  Furthermore, in order to establish PPD as a distinct phenotype, we will study 
a group of women with histories of major depression occurring outside of the perinatal period to 
test whether the mood and HPA axis effect of this paradigm are specific to postpartum 
depression or a nonspecific response to any major depressive episode. 
3. 
Subjects: 
a)   Description of Study Populations: 
This study will enroll -40 asymptomatic women with a past postpartum depression, 20 
asymptomatic women with a past major depression (not related to the postpartum), and 40 
asymptomatic women with no past Axis I psychiatric illness. 
 
b. Inclusion Criteria: 
 
A. Group 1: Women with a history of postpartum depression 
 
1) A history of DSM-IV major depression or hypomanic/manic episode that occurred 
within three months of childbirth (as determined by a SCID interview); 
 
2) has been well for a minimum of one year; 
 
3) a regular menstrual cycle for at least three months; 
 
4) age 18-50; 
 
5) not pregnant, not lactating and in good medical health; 
 
6) medication free (including birth control pills); 
 
7) no history of puerperal suicide attempts or psychotic episodes requiring 
hospitalization. 
 
B. Group 2: Women with a history of Major Depressive Disorder 

 
 
 
18
 
1) A history of DSM-IV major depression episode(s) occurring outside of pregnancy 
and not within three months postpartum; 
 
2) has been well for a minimum of one year; 
 
3) a regular menstrual cycle for at least three months; 
 
4) age 18-50; 
 
5) not pregnant, not lactating and in good medical health; 
 
6) medication free (including birth control pills); 
 
7) no history of suicide attempts or psychotic episodes requiring hospitalization. 
 
C. Group 3: Normal Controls 
 
1) Controls will meet all criteria specified except they must not have any past or present 
Axis I diagnosis or evidence of menstrually related mood disorders. 
 
 
c.) Exclusion Criteria for all Study Subjects: 
 
Patients will not be permitted to enter this protocol if they have important clinical or 
laboratory abnormalities including any history of the following: 
• endometriosis; 
• undiagnosed enlargement of the ovaries; 
• liver disease; 
• breast cancer; 
• a history of blood clots in the legs or lungs; 
• undiagnosed vaginal bleeding; 
• porphyria; 
• diabetes mellitus; 
• malignant melanoma; 

 
 
 
19
• gallbladder or pancreatic disease; 
• heart or kidney disease; 
• cerebrovascular disease (stroke); 
• cigarette smoking; 
• a history of suicide attempts or psychotic episodes requiring hospitalization; 
• recurrent migraine headaches; 
• pregnancy (patients will be warned not to become pregnant during the study and will be 
advised to employ barrier contraceptive methods; 
• pregnancy-related medical conditions such as hyperemesis gravidarum, pretoxemia and 
toxemia, deep vein thrombosis (DVT) and bleeding diathesis; 
• any woman with a first degree relative (immediate family) with premenopausal breast 
cancer or breast cancer presenting in both breasts or any woman who has multiple family 
members (greater than three relatives) with postmenopausal breast cancer will also be 
excluded from participating in this protocol; 
• any woman meeting the Stages of Reproductive Aging Workshop Criteria (STRAW) for 
the perimenopause (83) will be excluded from participation.  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. 
• subjects who are unable to provide informed consent. 
• NIMH employees and staff and their immediate family members will be excluded from 
the study per NIMH policy. 
4. 
Study Design and Methods 
a.) Study Overview: 

 
 
 
20
This is a mechanistic study designed to investigate the pathophysiology of postpartum 
depression.  The experimental model used in this protocol has been a benchmark in 
understanding the neurobiology of PPD, and a similar design is employed in our companion 
studies of severe premenstrual dysphoria to refine the hormone-related phenotype of these 
affective disorders.   Women undergo extensive assessments with standard measures to establish 
their individual patterns of mood symptoms in relation to the hormonal conditions induced in this 
study.  In addition to standard rating scales and interviews the study includes routine lab 
workThis study lasts five months plus two months of follow-up during which time the subject 
may have multiple out-patient visits in addition to completing daily mood assessments. 
 
 
 b.) 
Recruitment: 
 
 
Patients will be referred from community physicians and sought by advertising 
for women who suffered moderate postpartum-related, mood disturbances in the past.  NIH 
Employees and staff will not be directly recruited by or through their supervisors or co-workers 
to participate in this study. 
All recruitment materials will be approved by the IRB before use.   
Advertisements, both free and paid may be used to recruit subjects for this study.  The 
written advertisements will be used in color as submitted, or may be printed in black and white.  
The color of the ads may vary. Color changes will not be used to change the emphasis of an ad.  
The size of the ads may also vary, but all parts of the ads, including fonts and pictures, 
will be changed proportionately to the rest of that ad.  Disproportionate changes in size will not 
be used to change the emphasis of an ad. 
Flyers of various sizes, both with and without tear-off labels may be used in the following 
ways: 

 
 
 
21
• 
On bulletin boards on the NIH campus, 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. 
• 
Made available at outreach exhibits, speaking engagements, support group meetings, 
parent groups, professional meetings or conferences, association/trade meetings with 
approval of the venue or in accord with their policy. 
• 
May be given directly to those requesting study information. 
• 
May be sent using commercially-available mailing lists via direct mail.  If used for 
direct mail, the flyer will identify the source of the mailing list.  The flyer may be 
posted electronically on websites such as NIH or NIMH websites, advocacy/support 
group websites such as Postpartum Support groups, publications’ websites such as 
Washington Parent, or Gazette. 
Print ads will also be used as paid advertisements in local and national magazines with 
targeted advertising (e.g., Washington Parent, Bethesda Magazine, Washingtonian, More, 
Washington Parent, Washington Woman, Oprah, Women’s Health) as well as in local 
newspapers (e.g., The Washington Post, Express, Washington Examiner, Gazette, Washington Jewish 
Week, Military papers). 
IRB approved ads will be used on websites and e-newspapers.  All will link to the 
NIMH.NIH.gov landing page for this study.   Websites include advocacy websites such as Mental Health 
Association of America (MHA), MHA Montgomery County, NAMI, and DBSA.  They may also appear 
on the websites of vendors that run print or other paid ads, such as newspapers and magazines.  Twitter 
ads may be sent from Mental Health NIMH @NIMHgov, the NIMH extramural Office of 
Communications Twitter account. 

 
 
 
22
IRB-approved Listserv ads will be posted on listservs with the permission of the 
moderator and IRB-required statement on how the receiver was identified.  Listservs may 
include those of parenting organizations such as NIH Parenting Listservs and DC Urban Moms, 
women’s health organizations such as Postpartum Support Virginia and Postpartum support 
Maryland and professional groups, such as the Greater Washington Society of Social Workers 
and the Greater Washington Women’s Mental Health Consortium.  
 
c.)  
Screening: 
 
Subject eligibility for this protocol is determined in protocol 81-M-0126 at the NIH 
Clinical center.  
 
  
d.) 
 Study Procedures: 
Those selected will be personally interviewed to confirm the inclusion criteria by a psychiatrist, 
psychiatric nurse, or social worker to obtain a description of the following: 1) the nature of the 
relationships between mood disorders and the puerperium, and development and evolution of the 
disorder; 2) type, severity and duration of symptoms; 3) premorbid psychiatric history; 
4) medical and medication history, including medication taken for mood problems; 5) psychiatric 
history. 
 
 
Prior to treatment, a complete physical and neurological examination will have been performed 
by a physician on the research team and the following routine laboratory data obtained: 
 
A. Blood 
 
Complete blood count; thyroid function tests; renal function tests, such as BUN and 
creatinine; electrolytes; glucose; liver function tests; and serum β-HCG (test for pregnancy). 

 
 
 
23
 
B. Urine 
 
Routine urinalysis. 
 
 
 
C. EKG 
D. Results of Pap smear performed not longer than one year prior to onset of treatment 
will be obtained.  Subjects who will not be able to perform a Pap smear privately or whose 
results seem to be a reason for concern will be sent for a gynecologic consultation.  All subjects 
will be required to use non-hormonal forms of birth control (e.g., barrier methods) to avoid 
pregnancy during this study.   
E. Subjects will be excluded if there is evidence of significant renal, cardiac, hepatic, or 
other serious medical disorder. 
Procedures: 
 
Initial screening lasting two to four months is completed under protocol 81-M-0126. 
Clinical Assessment: The amount of time assigned to evaluations may include a screening 
process time of up to two months.  All women will complete a four-item visual analogue self-
rating to confirm the absence of clinically significant mood and behavioral symptoms across the 
menstrual cycle.  This log also will serve to confirm the presence of regular menstrual cyclicity.  
After the first screening visit all participants will return to the clinic after approximately 4-
5 weeks to review the quality of their self-ratings and to address any problems about the ratings 
that they might have encountered.  After we receive the completed set of ratings (2 months), the 
ratings are reviewed by staff to determine the absence of clinically significant changes in mood 
symptoms throughout the menstrual cycle. 
 
Participants also will have a psychiatric history taken which includes diagnostic 
interviews such as the Structured Clinical Interview for Axis I DSM-IV Disorders (SCID) and 

 
 
 
24
the minor depression module of the SADS-L.  Urinary HCG will be determined to rule out 
pregnancy.  Subjects who meet the inclusion criteria and who are medication free may be asked 
to return for follow-up interviews.  During these interviews, phenomenological assessments may 
be performed by instruments such as the Premenstrual Tension Syndrome Scale (Steiner-Carroll 
Scale) (84), Beck Depression Inventory (85), Spielberger State-Trait Anxiety Inventory (86), 
Hamilton Rating Scales for Depression and Anxiety (87), Edinburgh Postnatal Depression Scale 
(88)) and related psychological measures.  Additionally, subjects will be asked to fill out 
symptom self-rating scales, a modified version of the Daily Symptom Rating Form (Halbreich 
and Endicott) (89) and visual analogue scales (90), on a daily basis.  During screening in 
protocol 81-M-0126 all subjects will receive a careful medical screening and will be excluded if 
there is evidence of significant renal, cardiac, hepatic, or other medical disorder.  All patients 
will receive routine physical and laboratory examinations such as urinalysis, hematocrit, 
hemoglobin, BUN, creatinine, liver function tests, electrolytes, HIV, and a serum pregnancy test. 
Additionally, all women will have a normal EKG, and a normal PAP and gynecologic exam 
within the past year.  
Subjects will be seen at the outpatient clinic on a regular biweekly basis.  (Total = three baseline 
and 15 visits during treatment and follow-up.)  During clinic visits, subjects will be monitored 
and managed for side effects, and blood samples will be drawn. 
 
Research Procedures: Following the baseline period patients will receive 3.75 mg of the 
gonadotropin releasing hormone (GnRH) agonist leuprolide acetate (Lupron) during the early 
follicular phase of the menstrual cycle via intramuscular injection on a monthly basis in our 
clinic for five months.  During the first two months of GnRH agonist administration, all subjects 

 
 
 
25
will receive placebo progesterone and placebo estradiol tablets and will be told that at some point 
the placebo pills will be switched to active medication. 
 
After the second month of Lupron-alone treatment, active estradiol and progesterone 
replacement will be initiated.  Subjects shall be randomly assigned to one of two groups, and 
treatment shall be administered as follows: 
 
Group 1 (“withdrawal” group) - while continuing to receive monthly Lupron injections, 
all women in this group will be started on 2 mg B.I.D. of 17 beta-estradiol and 200 mg B.I.D. of 
progesterone for a period of eight weeks.  After four months of GnRH agonist with placebo (two 
months) and estradiol/ progesterone replacement (two months), placebo will be substituted for 
the active medication while subjects continue on Lupron for another month.  Participants will 
then be followed for an additional two months while unmedicated.  Total duration of treatment 
with Lupron shall be five months.   
 
 
Group 2 (“continued replacement” group) - while continuing to receive monthly Lupron 
injections, all women in this group will be started on 2 mg B.I.D. of 17 beta-estradiol and 
200 mg B.I.D. of progesterone for a period of 12 weeks.  Subjects shall receive Lupron for a total 
of five months, with placebo (two months) and estradiol/ progesterone replacement (three 
months).  Participants will then be followed for an additional two months while unmedicated.  
Total duration of treatment with Lupron shall be five months.   
 
The difference between these groups is, therefore, the duration of treatment with estradiol 
and progesterone - eight weeks of hormonal addback in the withdrawal group and 12 weeks of 
hormonal addback in the continued addback group.  The fifth month of Lupron, then, provides 
the comparison between the effects of hormonal withdrawal and hormone continuation. 
 
During the active medication phase of the trial, blood levels of progesterone and estradiol 
will be determined and the doses will be titrated accordingly by an unblind co-investigator.  The 

 
 
 
26
blood levels that we expect to achieve and sustain in each woman will be approximately 
500 pg/ml of estradiol and 30-40 ng/ml of progesterone.  Doses will not exceed 10 mg of 
estradiol and 1600 mg of progesterone.  By maintaining a progesterone: estradiol plasma level of 
approximately 50:1, we expect to prevent the side effects associated with unopposed estradiol 
(Leon Speroff, personal communication).  As outlined in the “Hazards and Precautions” section 
of this amendment our collaborative gynecologist will be consulted should any such need arise.  
Serum pregnancy tests will be performed in all women prior to the administration of the GnRH 
agonist to exclude pregnancy.  No pregnant woman will be entered into the study.  Subjects will 
be seen at the outpatient clinic on a regular biweekly basis.  (Total = three baseline and 15 visits 
during treatment and follow-up.)  During clinic visits, subjects will be monitored and managed 
for side effects, and blood samples will be drawn. 
 
All blood samples (50 ml per visit, 50 ml for genetics on one occasion and 10 ml for the 
IPCs on only one occasion) will be drawn at the 4th floor outpatient clinic.  Fifty cc of blood will 
be drawn at each clinic visit (n = 15) in the 28 week study.  This blood will be analyzed for 
several hormonal (e.g., neurosteroid metabolites and metabolomics profile), biochemical, and 
metabolic measures that could distinguish either women with from those without PPD or the 
symptomatic state in women with PPD.   
 
Additionally, participants will be asked to continue daily self-ratings for mood and 
behavioral changes throughout the trial and will also have more extensive periodic assessments 
of mood, behavior and neuropsychological changes.  Mood and behavioral assessments include 
the following measures: repeat SCID interviews lasting approximately 30 minutes (psychological 
testing), Quality of Life Questionnaire (91) and Social Adjustment Scale (92)self- report scales 
requiring 15 minutes to complete  

 
 
 
27
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. Women will also be invited to participate in 
an additional optional research procedures as follows:  Peripheral Blood Samples for IPCs 
Women who participate in this protocol will be approached to obtain consent for a blood 
sample 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 obtain an additional 
blood sample.  Previous participants will be asked to provide consent using the revised genetics  
consent form.  All women will be asked to provide a 10 ml blood sample (93) to study functional 
genomics of neuronal and glial precursors.  If we are unable to produce cell lines from the initial 
blood sample obtained then participants will be re-contacted to allow us to take an additional 
10 ml blood sample.  Risks and potential adverse reactions from a second blood sample should 
be similar to those of the initial sample. 
e.)  
End of Participation 
 
After the completion of this trial, all participants will be evaluated for the presence of 
clinically significant mood symptoms.  Additionally, the results of symptom ratings and plasma 
hormone levels will be reviewed with each participant.  If negative mood symptoms are present, 
we will discuss therapeutic options prior to referral of each participant back to her community 
health care provider. The options for follow-up in those women who report persisting negative 
mood symptoms include no treatment with follow-up by community health care provider or 
initiation of standard antidepressant therapy such as selective serotonin uptake inhibitors with 
follow-up in community. Our experience in this protocol suggests that in the majority of women 
depressive symptoms that emerged during this study were of mild to moderate severity and time-

 
 
 
28
limited. In those women in whom depressive symptoms persisted and were accompanied by 
distress, standard antidepressant therapy led to a remission of symptoms. 
  
5. 
Management of Data and Samples: 
 
a. Storage 
Samples of plasma are being stored for future analysis.  These samples are stored, in 
coded vials in secured freezers located in the Fourth Floor Clinic in Building 10 of the NIH 
campus.  Samples are inventoried by codes we assign.  Blood samples will be stored in locked 
freezers on the NIH campus.  The key to the code will be kept in a separate, secure area and will 
only be accessible to the PI.  These samples will be used for the study described in this consent 
form. 
 
The inventory of samples and mood ratings are maintained on the Branch server and 
database. Upon the completion of the study, plasma samples 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 I). 
b. Data and sample sharing plan 
 
This protocol is subject to the Genomic Data Sharing (GDS) policy. Data and samples will 
be shared with dbGaP, and The Perinatal Psychiatry Genetic Consortium - Postpartum 
depression: Action towards Causes and Treatment (PACT), which is headquartered at The 
University of North Carolina at Chapel Hill.  

 
 
 
29
 
Data and samples may also be shared with 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 
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.    
 
The proposed research with our collaborators will be in accord with the purpose of the 
study stated in the consent that participants signed from the start of the protocol in 1995 to 
present. Starting in the 2008 consent forms, subjects were given the option whether they wanted 
to share their samples or not.  
 
The ability to perform a meta-analysis of data will move the field forward.  The plan is to 
focus on investigating the heterogeneity of Perinatal Depression (PND) in order to identify 
clinical subtypes. 
Human tissue samples are being shared with collaborators of the PACT Consortium with a 
pending Material Transfer Agreement from the Office of Technology Transfer. 

 
 
 
30
All personally identifiable information will be removed from the data and data will be coded 
with a research identifier that is not derived from or related to information about the subjects.  
The codes will be stored in a secure database in the Section database and server. 
6.  
Additional Considerations: 
Depot leuprolide acetate (TAP, pharmaceuticals) is administered in this protocol under the IND # 
163205 sponsored by NIMH (Maryland Pao, MD is the official representing the Institute). 
Estradiol, progesterone, and placebo capsules will be manufactured by either NIH Pharmacy, 
Knowles Pharmacy or Pine Pharmaceuticals. Knowles Pharmacy, a PCAB-accredited 
compounding pharmacy located at 10400 Connecticut Avenue #100, Kensington, MD, will 
prepare the estradiol, progesterone, and placebo capsules on a per-prescription basis under 
FDA 503a and USP <795> regulations. Pine Pharmaceuticals, Inc. is located at 100 Colvin 
Woods Parkway, Tonawanda, NY. Pine Pharmaceuticals, Knowles Pharmacy and the NIH 
Pharmacy will produce these capsules under current Good Manufacturing Practice (cGMP) 
regulations. The medications administered in this protocol are dispensed, stored, and monitored 
by the NIH Pharmacy Investigational Drug Management and Research Section. 
7. 
Risks and Discomforts: 
Potential discomforts in this study could arise from the medications administered in this study, 
the repeated venipuncture (i.e., bruising or pain at site of needle entry), the completion of rating 
scales, and the behavioral and endocrine challenge studies.  
Medications: We do not expect any adverse side effects associated with the hormonal 
manipulations outlined in this protocol for the following reasons: First, we will be administering 
the physiologically relevant steroid hormones (estradiol and progesterone) and not the 
substituted steroids (such as ethinyl estradiol or norethindrone) present in many oral 
contraceptives and which have been reported to have a potentially more serious profile of side 

 
 
 
31
effects.  Second, the doses of Lupron, estradiol and progesterone, and the duration for which they 
will be administered in this protocol, will result in plasma hormone levels comparable to those 
commonly used for in vitro fertilization (IVF) protocols lasting 1-3 months.  Therefore, based on 
current IVF procedures (80 ,94-96) (Leon Speroff, personal communication) we do not anticipate 
any adverse incidents arising from the proposed doses of estradiol and progesterone.  Third, no 
adverse reactions or events were encountered in the 25 women who completed the study to date.  
Finally, comparable extended, uninterrupted gonadal steroid treatment (such as oral 
contraceptives) for 6 to 12 weeks has been shown to be well-tolerated (97). 
 
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.  Episodes of flushing appear to 
decrease with continued therapy in most patients receiving Lupron; however, in at least one 
study, the incidence of hot flushes did not appear to decrease with continued therapy.  In a 
recently completed study of 400 women of reproductive age with either uterine fibroids or 
endometriosis who each received 3.75 mgs depot Lupron every month for a period of six 
months, the most common side effects 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.  In the majority of women regular menstrual cycle 
function returned within two months following the last injection of depot Lupron (Tapp 
Pharmaceuticals, personal communication).  Blurred vision, myalgias, lethargy, memory 

 
 
 
32
disorder, and numbness have been reported in less than 3% of patients receiving the drug.  
Thrombophlebitis, pulmonary embolus, 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.  From our experiences with 20 PMS patients and 
20 controls, Lupron is well tolerated (no dropouts) with the most common side effects being hot 
flushes and a decrease in libido.  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 precocious puberty report the 
development of normal reproductive function, skeletal growth, and fertility (98,99). 
 
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. 

 
 
 
33
 
The relationship between estrogen, both endogenous and exogenous, and the 
development of endometrial carcinoma has been suggested by several different lines of 
investigation (100).  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 replacement 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 during long term ERT and thus, inferentially, the risk of carcinoma, consists 
of 12 to 13 days of progesterone treatment each month when estrogens are administered (101).  
There is an increase in thromboembolism in women receiving non-contraceptive estrogen 
therapy (102-106).  Additionally, some but not all studies report an increase in risk of stroke 
(107,108) in older women taking estrogen therapy.  However, these complications are unlikely at 
the dose and duration of estrogen replacement employed in this protocol, and in the younger age 
group of women who participate in this study.  One study (94) reported no effect of the estrogen 
patch on the four clotting indices previously shown to be altered by oral contraceptive use 
(101,109,110).  Blood pressure, on average, appears to be unaffected by estrogen therapy, 
although both increases and decreases have been reported.  In observational studies, post-
menopausal estrogen therapy has been observed to lower the relative risk of cardiovascular 
disease in some but not all studies (107,111).  In contrast, recent randomized controlled trials in 
older postmenopausal women (e.g., Women’s Health Initiative [WHI]) report an increased risk 
of cardiovascular disease (112).  Emerging data suggest that these disparities in findings may be 

 
 
 
34
related to the timing of initiation of estrogen therapy in relation to the proximity of menopause.  
Subgroup analyses of the combined estrogen and progestin (EPT) arm of the WHI demonstrated 
a significant interaction between coronary heart disease (CHD) risk and time since initiation of 
EPT, with an increased risk in the early years following initiation and a decreased risk in later 
years.  Additionally, the increased risk of CHD was observed in older but not younger 
perimenopausal women (113-118).  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 (119,120).  Estrogen therapy also may increase the risk of urinary 
incontinence in older postmenopausal women (121-123).  Further, most studies have suggested 
an increased relative risk of breast cancer after four or five years' use (124-136), 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 (137) 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 

 
 
 
35
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 (137,138). 
 
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 (139).  Side effects reported in women taking progestins may include 
breakthrough bleeding, edema, change in weight (increase or decrease), cholestatic jaundice, 
rash (with or without pruritus), depression, easy fatigue and sedation, 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.  In a 
recent study, an average dose of 1750 mg of oral micronized progesterone was given to 
59 women with PMS for a period of three months and was well tolerated by this sample.  The 
side effects reported on progesterone were lightheadedness, fatigue, forgetfulness, and 
headaches.  These were very mild and caused no dropouts (Ellen Freeman, personal 
communication). 
 
For the sake of completeness we will also describe the side effects reported when 
estradiol and progesterone are combined in the form of oral contraceptives.  Side effects 
observed in patients receiving combined oral contraceptives include nausea, breast soreness, 

 
 
 
36
vaginal discharge, fluid retention, hypertension, and clotting abnormalities that have been 
associated with the estradiol 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 estradiol 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. 
 
Any patient experiencing clinically significant side effects such as nausea, hypertension, 
vomiting or extreme fluid retention from the medication will have the dose titrated to achieve 
relief of the symptoms.  If adequate relief cannot be achieved in this manner, drug treatment will 
be discontinued.  Similarly if menopausal-like symptoms occurring secondary to GnRH agonist 
treatment are intolerable, drug treatment will be discontinued. 
 
 
Venipuncture: 
One discomfort of this study may occur due to the venipuncture and multiple blood 
sampling.  The total blood withdrawal is estimated to be 50 ml per clinic visit for a total of 
750 ml for the 28 week study (i.e., 15 visits).  An additional 60 ml sample of blood will be 
obtained for genetics (50ml) and IPCs (10ml) that will be timed to coincide with one of the 
regular visits in those women currently participating in this protocol. Total blood 
withdrawal over 28 weeks of study duration - 1,080 ml) and falls within NIH guidelines 

 
 
 
37
(550 ml per eight week period).  The total blood volume collected will be 200 ml every 
eight weeks (4 visits= 200 ml = 200 ml) plus one 10 mL additional sample for IPCs and 
one 50 ml sample for genetics. 
Psychological and Cognitive tests: 
The risks and discomforts of the symptom evaluations and baseline exams are minimal.  
No discomfort is expected to be associated with the physical examination or the clinical 
interview other than potential stress of answering personal questions.  All clinical assessors have 
extensive experience in clinical psychiatric assessment and will make every effort to implement 
protocol procedures in a sensitive and supportive manner.  Research interviews will be 
interrupted if subjects become distressed or object to answering questions.  Other measures to 
minimize risks include the careful assessment of each subject before the study, and close clinical 
scrutiny during all aspects of the study. 
 
Intravenous catheter insertion may be momentarily painful and there is a slight risk of 
infection or inflammation at the site of the catheter insertion.  Some people faint when needles 
are inserted, so this procedure will be performed with the subject seated. 
 
History and Physical Exam and Laboratory Tests:  It is possible that during the evaluation 
portion of the study a subject may be found to have a medical problem of which they were 
previously unaware.  In that event, the participant will be given the results and an appropriate 
referral made as indicated by the condition identified. 
Donation of blood for genetic material: 
Under some circumstances, it can be a risk for genetic information about an individual to 
be known.  Variation in some genes is known to be directly related to risk for certain illnesses.  
Other genes may be shown at some point in the future to be related to illness.  Since the results 
of these genetic tests may allow prediction of risk of illness in some cases, it is possible this 

 
 
 
38
information could be used against a subject participating in the study.  Although there are no 
physical risks associated with participation in genetic studies, apart from those routinely 
associated with phlebotomy, psychological and social/economic risks associated with genetics 
studies of the kind described here are difficult to define, and remain the subject of heated 
controversy in the ethics community.  The major risk, to the degree that any exists, is that a 
breach of confidentiality regarding genetic studies that resulted in third parties finding out 
genetic information about a person could theoretically place a person at risk for loss of insurance, 
loss of employment, etc. because of genotype-based discrimination.  To our knowledge, no 
person has ever suffered harm for the reasons just described as a result of participating in a 
genetics research study.  Regardless, our written informed consent process will go over these 
risks carefully.  In addition, subjects will have the right to “withdraw” from the research by 
having their DNA sample destroyed.  Finally, all research records containing any subject-
identifying information will be stored under lock and key, or in secured computing environments 
by the CTDB.  Personal identifiers are never associated directly with genotypes in the same data 
file - all genotype information is indexed only to de-identified subject codes.  With these 
safeguards in place, we are confident the research is virtually without psycho-social-economic 
risk to subjects. 
8.  
Subject Safety Monitoring: 
Subjects, both patients and healthy controls, are monitored by one of the associate 
investigators during all study procedures.  Medical history and physical assessments occur at 
each clinic visit including interviews, symptom assessments, vital signs, and laboratory testing 
when clinically indicated. 
 
As the population being studied in this protocol may be at high risk to develop 
subsequent depressions and as gonadal steroids have been implicated in the etiology of 

 
 
 
39
postpartum depression, some depressive reactions may occur during this protocol.  However, 
based on experience with Lupron and estradiol/progesterone addback in women with 
menstrually-related mood disorders and preliminary observations of 16 women participating in 
this protocol, we would expect mood symptoms to be of mild severity.  Adverse mood symptoms 
will be monitored by administering the Beck Depression Inventory (BDI); if BDI scores of > 20 
are observed at any period in the protocol then the Hamilton Rating Scale for Depression will be 
administered on consecutive days.  Anyone scoring > 20 on the Hamilton for three or more 
consecutive days, or any subject with suicidal ideation, or anyone with concerns about their 
ability to continue in the study, will be considered to have severe mood symptoms and be 
discontinued from the protocol.  In the event of the occurrence of severe mood symptoms, either 
the protocol will be terminated, or if symptoms occur during the withdrawal phase, hormone 
replacement will be re-instituted.  Should this step prove to be unsuccessful, conventional 
medication will be prescribed as needed.  Further, although we do not anticipate severe adverse 
reactions, we have arranged for inpatient hospital admission at the Clinical Center if symptoms 
are otherwise unmanageable. 
Criteria for individual subject withdrawal: 
 
Any subject requiring immediate treatment will be referred for private treatment.  In the 
event that private treatment cannot be arranged promptly, the subject will be treated openly with 
an antidepressant until successful referral can be achieved. 
        Subjects presenting as “healthy” but found to have a psychiatric diagnosis will be 
discharged from this protocol and either be referred to the community for further evaluation and 
treatment or consented to another NIMH protocol.  In the case a psychiatric diagnosis is 
unveiled; the assessment and disposition plan will be reviewed with a senior clinician who 
reviews the case with the Dr. Schmidt. 

 
 
 
40
9. Outcome Measures: 
 Outcome measures will include the scores on several standardized cross-sectional rating scales 
(Beck Depression Inventory, Hamilton Depression and Anxiety Scales, and Edinburgh 
Postpartum Depression Scale), symptom rating scales completed daily, plasma hormone 
measures (including neurosteroid levels and metabolomic profile), induced pluripotent cells and 
immortalized lymphoid cells mRNA expression after exposure to estrogen and progesterone 
withdrawal, and the response to the o-CRH stimulation tests.  
10. 
Statistical Analysis: 
 
Analysis of Data: A repeated measures design has four groups of ten subjects each for a 
total of forty subjects.  Each subject is measured during the hypogonadal baseline, at the end of 
the two months of addback, and during the following month (i.e., continued addback in group 2 
or withdrawal in group 1).  The effect of hormonal addback and withdrawal in subjects and 
controls on mood and biological parameters will be calculated using ANOVA-R with post-hoc 
Bonferroni t-tests. 
 
Sample Size Justification: Based on previous data (61), the between-subject standard 
deviation is 0.69 and the within-subject standard deviation is 0.28.  This design achieves 67% 
power when an F test is used to test the Groups factor at a 5% significance level and the actual 
standard deviation among the appropriate means is 0.30 (an effect size of 0.43), achieves 81% 
power when an F test is used to test the Times factor at a 5% significance level and the actual 
standard deviation among the appropriate means is 0.16 (an effect size of 0.57), and 
achieves 60% power when an F test is used to test the group by time interaction at a 5% 
significance level and the actual standard deviation among the appropriate means is 0.13 (an 
effect size of 0.46). 
 
11. 
Human Subjects Protection: 

 
 
 
41
 Subject selection: 
 
All subjects must meet the inclusion and exclusion criteria listed in Section 3.  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. 
Justification for inclusion/exclusion of children: 
We will exclude children or minors because the study population is women with 
postpartum depression. 
Justification for symptom challenge procedure: 
The justification for employing a symptom challenge is three-fold as follows:  
 
a. The study of PPD is very difficult because of pharmacologic limitations and child 
care constraints.  A hormonal model of pregnancy and parturition may help overcome some of 
these difficulties. 
 
b. The development of mood and behavior symptoms in this study may predict 
psychiatric problems in future pregnancies and may suggest a subgroup of women with an 
affective disorder who are differentially susceptible to the hormonal changes of pregnancy and 
parturition. 
 
c. This model may be helpful in understanding underlying biological mechanisms in 
depression and affective disorders in general. 
 
Safeguards: As described above in the Subject Safety Monitoring: Adverse mood symptoms will 
be monitored by administering the Beck Depression Inventory (BDI); if BDI scores of > 20 are 
observed at any period in the protocol then the Hamilton Rating Scale for Depression will be 
administered on consecutive days.  Anyone scoring > 20 on the Hamilton for three or more 

 
 
 
42
consecutive days, or any subject with suicidal ideation, or anyone with concerns about their 
ability to continue in the study, will be considered to have severe mood symptoms and be 
discontinued from the protocol.  In the event of the occurrence of severe mood symptoms, either 
the protocol will be terminated, or if symptoms occur during the withdrawal phase, hormone 
replacement will be re-instituted.   
Participation of NIH Staff or family members of study team members 
 
NIH staff and family members of study team members may be enrolled in this study as 
this population meets the study entry criteria.  Neither participation nor refusal to participate as a 
subject in the research will have an effect, either beneficial or adverse, on the participant’s 
employment or position at NIH. 
 
Every effort will be made to protect participant information, but such information may be 
available in medical records and may be available to authorized users outside of the study team 
in both an identifiable an unidentifiable manner. 
The NIH Frequently Asked Questions (FAQs) for Staff Who are Considering Participation in 
NIH Research will be made available.  Please see section 12 for consent of NIH Staff. 
 
 This study collects sensitive information on medical and psychiatric diagnoses and drug 
and alcohol use. The PI will train study staff regarding obtaining and handling potentially 
sensitive and private information about co-workers through staff discussions and written 
branch/section procedures.  Prior to enrollment, potential participants will be informed that this 
sensitive information will be in their NIH medical record. 
 
Incidental and Secondary Findings 
In this project, we are analyzing ribonucleic acid (RNA) information which could inform us 
whether particular genes or gene groups are being expressed in women with PPD.  We will not 
be looking for incidental findings associated with each woman’s specific DNA. We may have 

 
 
 
43
incidental findings on a group basis that may be relevant to diseases other than PPD. These 
findings will not be reported on an individual basis but may be included in future publications. 
12. 
Consent Documentation and Process: 
 
Prospective participants will receive an explanation of the objectives, procedures, and 
hazards of this protocol that is appropriate to their level of understanding.  The attached consent 
forms will be reviewed and written or electronic informed consent obtained. When a document 
that is in electronic format is used for the documentation of consent, this study will use the iMed 
platform, which is 21 CFR, Part 11 compliant, to obtain the required signatures. The participant 
will be selected in CRIS, and their identification will be confirmed prior to selecting the study 
consent in iMed. During the consent process, participants and investigators will view the same 
approved consent document simultaneously in the same location at the Clinical Center. After all 
questions have been answered, and if the subject would like to participate, they will both sign the 
electronic consent using a finger, stylus or mouse, and a timestamp will be generated and 
recorded on the consent form.  This informed consent documentation will be included as part of 
the medical record maintained for each subject.  The right of the subject to decline to participate 
or to withdraw from the study at any time will be made clear.  Subject confidentiality will be 
assured in accordance with the Federal Privacy Act.  Subject anonymity will be protected in any 
published form of the collected data.  Separate consent forms are provided for genetic tests 
involved in the study.  The consent forms contain all required elements.  
Considerations for Consent of NIH staff, or family members of study team members: 
Consent for NIH staff will be obtained as detailed above with following additional protections: 
Consent from staff members will be obtained by an individual independent of the staff member’s 
team whenever possible.  Otherwise, the consent procedure will be independently monitored by 

 
 
 
44
the CC Department of Bioethics Consultation Service in order to minimize the risk of undue 
pressure on the staff member. 
13. 
Data and Safety Monitoring: 
a. Data and safety monitor 
 
We will have an independent safety monitor (ISM) for the study.  The Independent 
Safety Monitor (ISM) will be a sole physician with relevant expertise. William Regenold, 
M.D.C.M. will serve as the independent safety monitor (ISM) for this protocol for the Behavioral 
Endocrinology Branch. He is a psychiatrist, board certified in Adult and also Geriatric Psychiatry 
who is Senior Research Physician and Medical Director for the Noninvasive Neuromodulation 
Unit in the Experimental Therapeutics and Pathophysiology Branch of the Intramural Research 
Program of NIMH, working primarily with mood disordered patients. He provides clinical care, 
assists in consenting subjects for research protocols, conducts research procedures, assists in data 
analysis and administers and monitors noninvasive neuromodulation therapies including 
electroconvulsive therapy (ECT) and repetitive transcranial magnetic stimulation (rTMS), for 
treatment resistant depression.  In addition, he was he was at the University of Maryland 
Department of Psychiatry for 24 years where he was Director of the Division of Geriatric 
Psychiatry, the Adult Inpatient Service and the Electroconvulsive Therapy Service.  His research 
at the University of Maryland focused on mood disorder pathophysiology and electroconvulsive 
therapy. Dr. Regenold’s primary responsibility will be to provide independent safety monitoring 
in a timely fashion. This will be accomplished by review of SAEs or unexpected adverse events, 
immediately after they occur, with follow-up through resolution.  The ISM will evaluate 
individual and cumulative participant data when making recommendations regarding the safe 
continuation of the study. The ISM focus will be on SAEs or unexpected adverse events rather 
than all adverse events. The ISM will be provided with quarterly reports on safety and 
enrollment as well as all protocol revision and other pertinent documents relating to the study.  

 
 
 
45
Such reports will include information on laboratory tests and SAEs or unexpected adverse 
events.  The ISM will also be notified of SAEs within 24 hours.  The ISM will not have direct 
involvement in the conduct of the study. 
 
b. Data and safety monitoring plan 
 
The PI will prepare a quarterly report on data and safety parameters for the Independent 
Monitor.  The Independent monitor will provide a written monitoring report to be submitted to 
the IRB at the time of continuing review. 
 
c. Criteria for stopping the study or suspending enrollment or procedures 
The study will be stopped if there is any Serious Adverse Event related to the research.   
The PI/Independent Monitor and IRB will determine if changes are needed for the research to 
continue or if it will be closed. Any changes required as conditions for resuming the research 
must be submitted as an amendment and IRB-approved before the changes can be implemented. 
14.  
Quality Assurance: 
 
A. Quality assurance monitor 
 
Quality assurance will be monitored by the PI, the research team and the Office of 
Regulatory Oversight (ORO) serving NIAAA, NIDA and NIMH. 
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 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.  

 
 
 
46
15. 
Reporting for Unanticipated Problems, Adverse Events and Protocol Deviations: 
Reporting will be in compliance with policy 801.  
 
 
16. 
Alternatives to Participation: 
Patients may elect not to participate in this study without jeopardizing their participation in other 
studies at NIH. 
17. 
Privacy: 
All research activities will be conducted in as private a setting as possible. 
18. 
Confidentiality: 
Data and samples will have all personally identifying information removed and will be assigned 
a code.  The key to the code will be kept separately and securely. The inventory of samples and 
mood ratings are maintained on the Branch server and database.  Hard copies are stored in 
locked cabinets within a locked room, human tissues are stored in locked freezers within a 
locked room with pass key access, electronic data (including the sample inventory) is stored on 
the NIMH server and CTDB database in which access is restricted to members of the Behavioral 
Endocrinology Branch and CTDB staff. 
This study collects sensitive medical information. The PI will train study staff regarding 
obtaining and handling potentially sensitive and private information about co-workers through 
staff discussions and written branch/section procedures. 
19. 
Conflict of Interest Statement: 
The NIH policy on conflict-of-interest has been distributed to all investigators. None of the 
investigators have a conflict of interest to report regarding this protocol. No NIH investigator 
involved in this study receives payments or other benefits from any company whose drug, product 
or device is being tested. 

 
 
 
47
20. 
Technology Transfer: 
Human tissue samples are being shared with collaborators of the PACT Consortium pending a 
Material Transfer Agreement obtained from the Office of Technology Transfer. 
21. 
Research and Travel Compensation: 
For this study, the subject may be compensated for research-related discomfort and 
inconveniences. Compensation will be in the form of a check payment. This will be given at the 
end of each study phase. 
Volunteers will be paid in accordance with NIMH guidelines as follows: 
Study 95-M-0097 
Clinic visits (15-18) 
 
 540.00 
Repeated blood draws (16) 
 
 350.00 
Mood and behavioral ratings 
 
 150.00 
Investigational drugs 
a) Depot $100.00 x 5 
 
 500.00 
b) Estrogen tablets 
 
 100.00 
c) Progesterone tablets 
 
 100.00 
 
Total Compensation for 95-M-0097 is up to: 
$1740.00 
 
Additional tests (separate consent) 
 
 
 
Blood Sample for IPCs 
3 x $20.00 per 10 ml sample  
  60.00 
 
 
 
 
 
This study does not offer reimbursement for, or payment of, travel or meals. If an overnight stay 
is necessary, lodging may be provided. 
Employees and staff who participate during work hours must have permission from their 
supervisor. NIH employees and staff must either participate outside of work hours or take leave 
in order to receive compensation. 
 

 
 
 
48
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Appendix I 
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 

 
 
 
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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 
and suppression devices are installed and in working condition. All heating and air-cooling 
systems are periodically checked to ensure proper working condition.  

 
 
 
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• 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 

 
 
 
63
individual software components and target them for enhancements or upgrades all while 
maintaining the integrity and confidentiality of the CTDB system.  Another 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.