Cyclical Neuroactive Steroid Changes, Arousal, and Proximal Suicide Risk: An Experimental Approach
Phase: PHASE4
Conditions: Suicidal Ideation
Sponsor: University of Illinois at Chicago

 
 
 
Cyclical Neuroactive Steroid Changes, Arousal, and Proximal Suicide 
Risk: An Experimental Approach 
Principal Investigator: 
Tory Eisenlohr-Moul, Ph.D.  
UIC Psychiatry 
Phone: 859-317-0503 
Email: temo@uic.edu 
Study Location(s): UIC Psychiatry Neuropsychiatric Institute 
 and Clinical Research Center 
Sponsor: NIMH 
Version: 14 
Date: April 19, 2024 
 
 

 
 
 
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LIST OF ABBREVIATIONS  
 
COI 
 
 
Conflict of Interest 
DHHS  
 
Department of Health and Human Services 
DMC  
 
Data Monitoring Committee 
DSMB  
 
Data and Safety Monitoring Board 
 
DSMP  
 
Data and Safety Monitoring Plan 
FERPA 
 
Family Educational Rights and Privacy Act 
FDA  
 
Food and Drug Administration 
GCP 
 
 
Good Clinical Practice 
HIPAA 
 
Health Insurance Portability and Accountability Act 
IBC 
 
 
Institutional Biosafety Committee 
ICD 
 
 
Informed Consent Document 
ICH 
 
 
International Conference of Harmonization 
IDE 
 
 
Investigational Device Exemption 
IDS 
 
 
Investigational Drug Service 
IND 
 
 
Investigational New Drug  
IRB 
 
 
Institutional Review Board 
LAR  
 
Legally Authorized Representative 
OHRP  
 
Office of Human Research Protections 
OPRS  
 
Office for the Protection of Research Subjects 
PHI 
 
 
Protected Health Information 
PI 
 
 
Principal Investigator 
PPRA  
 
Protection of Pupil Rights Amendment 
QA/QI  
 
Quality Assurance/Quality Improvement 
SAE 
 
 
Serious Adverse Event 
SOP 
 
 
Standard Operating Procedure 
NIMH  
 
National Institute of Mental Health 
 
E2 
17-beta-estradiol 
P4 
progesterone 
RCT 
Randomized controlled trial 
SITBI 
Self-Injurious Thoughts and Behaviors Interview 
UWRAP/LRAP 
University of Washington Risk Assessment and Management Protocol/Linehan Risk 
Assessment and Management Protocol 
EXP 
Experimental 
PL 
Placebo 
SCID 
Structured Clinical Interview for Diagnosis 
IDS 
Investigational Drug Services 
HC 
Healthy Control 
 

 
 
 
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1.0 
Project Summary/Abstract 
Suicide is the second leading cause of death among female of reproductive age, and female suicide attempts occur most 
frequently around menses (perimenstrually), when estradiol (E2) and progesterone (P4) fall rapidly. A recent prospective 
study demonstrated that suicidal ideation (SI) and attempts peak perimenstrually in natural cycles, and that this 
perimenstrual worsening of SI can be prevented by administering stabilizing doses of E2+P4 (relative to placebo). 
Therefore, while E2+P4 withdrawal is a viable model of proximal suicide risk in females with SI, mechanisms are unclear. 
GABAergic neuroactive steroid metabolites of ovarian hormones (e.g., allopregnanolone), exert potent sedative and 
antidepressant effects; we hypothesize that acute perimenstrual withdrawal from these hormone metabolites may increase 
suicide risk by increasing hyperarousal and hopelessness. The long-term objectives of this research are to (1) use the 
menstrual cycle as a model to probe the proximal mechanisms of suicide, and (2) develop long-term treatments that eliminate 
hormonal contributions to suicide. The objective of the current work is to use a crossover placebo-controlled trial of 
E2+P4 stabilization (vs. natural E2+P4 withdrawal under placebo) in the perimenstrual weeks to probe behavioral 
(hopelessness, hyperarousal) and molecular/genetic (neuroactive steroid levels, mRNA expression for neurosteroidogenic 
enzymes) mediators of perimenstrual suicide risk. Design: In this mechanistic trial, 90 female outpatients with past-month 
SI will complete two counterbalanced conditions: (1) two weeks of placebo during natural perimenstrual E2+P4 withdrawal, 
and (2) two weeks of perimenstrual E2+P4 stabilization (.1mg/day transdermal estradiol + 200mg/day oral micronized 
progesterone) to prevent withdrawal. Five labs per condition will capture changes in GC-MS quantified neuroactive steroids 
and mRNA expression for neurosteroidogenic enzymes, and a wearable device (Oura Ring) will index basal body 
temperature and physiological arousal (heart rate, heart rate variability). Our app (BiAffect) will passively track arousal via 
movement and typing speed instability. 40 healthy controls (HC) with no SI will be recruited for a two-month long 
observational substudy, in which five lab visits across one menstrual cycle (timed identically to mirror the observations 
across one medication phase of the experimental trial) will capture baseline molecular/genetic changes for between-group 
comparisons. Specific Aims. Aim 1 is to evaluate hyperarousal and hopelessness as interacting mechanisms by which 
perimenstrual E2+P4 withdrawal (vs. experimental E2+P4 stabilization) increases proximal suicide risk. Aim 2 is to 
evaluate neuroactive steroid withdrawal as a mechanism by which perimenstrual E2+P4 withdrawal (vs. stabilization) 
increases proximal suicide risk. If appropriate, a multilevel path model will test a path in which E2+P4 withdrawal (vs. 
stabilization)  causes neuroactive steroid withdrawal, which increases in hopelessness and hyperarousal, which in turn 
increases proximal suicide risk. Aim 3 (substudy) is to characterize GABA-A receptor gene expression plasticity across the 
cycle in individuals with and without perimenstrual changes in affect and suicide risk. Relevance. By conducting a 
mechanistic experiment to probe the mediators of a known cause of proximal suicide risk, the proposed research responds 
to public calls from the NIMH-sponsored Suicide Research Prioritization Agenda to identify modifiable causes of proximal 
suicide risk.  
  

 
 
 
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2.0 
Background/Scientific Rationale  
Among females of reproductive age, suicide is the second leading cause of death, accounting for 13% of all female deaths 
in America1.  Although males are more likely to die by suicide (due to higher-lethality methods), females are three times as 
likely to experience suicidal ideation (SI) or attempt suicide2.  This sex difference manifests between puberty3 and 
menopause4, implicating fluctuations in ovarian hormones. Despite this, no published longitudinal or experimental studies 
have directly examined ovarian hormone effects on suicide risk.  
 
In cross-sectional studies, suicide attempts occur most frequently peri-menstrually—that is, in the weeks before and 
during menses—when the ovarian hormones E2 P4 fall rapidly. The perimenstrual weeks have been repeatedly linked 
with greater risk of suicidal behaviors in cross-sectional hospital and postmortem studies5–8. While this further implicates 
hormone changes in proximal suicide risk, the impact of this work is limited by a lack of longitudinal or experimental data, 
and a lack of data regarding mechanisms.  
 
One possible mechanism of perimenstrual suicide risk is rapid perimenstrual withdrawal from E2 and P4 (Fig 2), as well as 
withdrawal from the antidepressant9 and sedative10,11 effects of their GABAergic neuroactive steroid metabolites. The 
PI’s recent longitudinal and experimental studies (K99-MH10966712), which form the premise for the proposed work, 
provide the first (1) longitudinal evidence that daily SI and behavior peak perimenstrually, and (2) experimental evidence 
that 
E2+P4 
withdrawal 
causes 
increased 
perimenstrual suicide risk that can be prevented by 
administering stabilizing doses of E2+P412. The 
proposed mechanistic trial responds to the RFA by 
integrating the PI’s experimental paradigm with 
molecular, genetic, and behavioral measurements to 
examine the mechanisms by which perimenstrual 
E2+P4 withdrawal (vs. experimental stabilization) 
increases proximal suicide risk. We examine 
mediation across levels of analysis, including changes 
in neuroactive steroid metabolites (serum neuroactive 
steroid levels, neuroactive steroid biosynthetic 
enzyme mRNA expression in peripheral monocytes) 
as well as hopelessness and hyperarousal.

 
 
 
Defining and Operationalizing Behavioral Constructs 
 
We define hyperarousal (RDoC Arousal and Regulatory Systems) as (1) self-reported agitation and overwhelm, (2) 
psychomotor agitation (smartphone accelerometer instability/movement13,14), (3) behavioral reactivity to external stimuli 
(smartphone typing speed instability13,14, attentional bias toward angry/fearful faces on task15), and (4) physiological 
hyperarousal (reduced resting parasympathetic autonomic function16, increased sympathetic and decreased parasympathetic 
autonomic function during an emotionally arousing task).  
 
We define hopelessness as a state of despair arising due to repeated experiences of loss or sustained threat (RDoC Negative 
Valence Systems). It will be measured primarily as self-reported hopelessness using the Beck Hopelessness Scale (BHS)17, 
and we will measure lifetime and daily exposure to loss and threat18,19.  
 
We define proximal suicide risk as the presence and severity of self-injurious thoughts and behaviors as assessed 
dimensionally using the Adult SI Questionnaire (ASIQ)20 and categorically using the Self-Injurious Thoughts and Behaviors 
Interview -Revised (SITBI-R).These instruments assess for passive and active SI; suicide plans; aborted, interrupted, and 
actual suicide attempts; and suicide death. Although non-suicidal self-injury (NSSI) is a distinct but related phenomenon, 
we also consider NSSI as an index of proximal risk given its predictive validity for suicide attempt21. 
 
 
 
In the first prospective study12 of daily 
suicide 
risk 
in 
females 
with 
SI 
(K99MH109667), the PI found that 
proximal 
suicide 
risk 
peaks 
perimenstrually, during periods of natural 
hormone (E2+P4) withdrawal (Fig. 3). 38 
females with past-month SI completed 
daily ratings on the ASIQ20 across a 
baseline menstrual cycle. ASIQ scores 
peaked during the perimenstrual weeks 
relative to late follicular week, γ=-.88, 
p<.001 and early luteal week, γ=-.56, 
p<.001. We also observed greater odds of 
endorsing suicidal planning items on 
perimenstrual vs. non-perimenstrual days 
(OR=4.29, p<.001). 28 of 30 participants 
reported 
the 
highest 
mean 
SI 
perimenstrually. These data support the 
premise 
that 
suicide 
risk 
peaks 
perimenstrually. 
 
Premise for Aim 1a 
Perimenstrual Hormone Withdrawal Causes Proximal Suicide Risk 

 
 
 
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Stabilization of E2+P4 Prevents Perimenstrual Worsening of Suicide Risk. In the PI’s recently-completed double-blind 
crossover trial12 (K99MH109667) in 30 females with past-month SI, SI severity once again peaked during perimenstrual 
weeks in the placebo condition (black solid line, Fig 4), and perimenstrual administration of E2 (.1mg/day) and P4 
(200mg/day) in the stabilization condition prevented this increase in SI (dashed red line, Fig 4; Cond×Phase γ=-.75, 
p<.0001). 
Further, 
as 
predicted, 
perimenstrual risk was then shifted in the 
active condition, appearing only later 
during 
withdrawal 
from 
exogenous 
hormones (γ=.59, p<.0001). This further 
demonstrates the causal effect of E2+P4 
withdrawal on proximal suicide risk. When 
results were person-standardized, the 
impact of stabilization was roughly a 1 
person-standard-deviation 
condition 
difference in severity of SI change. Further, 
more 
participants 
reported 
suicidal 
behaviors during placebo vs. active (NSSI: 
18 vs. 10; preparatory acts: 5 vs. 1; aborted 
attempts: 2 vs. 0). This experiment supports 
the premise that E2+P4 withdrawal causes 
proximal suicide risk. 
 
In order to develop safe long-term 
treatments that eliminate hormonal contributions to suicide, we need information about the mechanisms by which 
E2+P4 withdrawal cause proximal suicide risk. Unfortunately, recent meta-analytic work demonstrates that poor progress 
has been made toward predicting attempts among ideators21, and little is known about reliable time-varying prediction of 
proximal suicide risk22. Therefore, candidate mediators in the present proposal are informed primarily by our general 
understanding of the most robust mechanisms E2+P4 withdrawal, and preliminary findings from our prior trial of E2+P4 
stabilization in females with past-month SI. Below, we argue that neuroactive steroid withdrawal, hopelessness, and 
hyperarousal are strong candidate mediators of E2+P4 withdrawal effects on proximal suicide risk. 
 
 
 
Hopelessness features prominently in suicide theories. Although hopelessness has recently been framed as primarily a 
predictor of SI rather than suicide attempt or death, large prospective studies demonstrate the predictive validity of baseline 
hopelessness for suicide death in inpatients (n=102)23 and outpatients (n=1,958)24, and a meta-analysis of longitudinal 
studies reports a role for baseline hopelessness as a prospective predictor of attempt (OR=1.95), and death (OR=2.15)25. 
Although hopelessness is one of the best-studied constructs in suicide research, little evidence is available about dynamic 
changes in hopelessness and proximal suicide risk. One recent report described the use of daily surveys to evaluate links 
between risk factors and SI. They found that fluctuations in hopelessness successfully predicted SI severity at the next time 
point across two samples, suggesting that surges in hopelessness increase proximal suicide risk26.  
 
E2+P4 Withdrawal Causes Hopelessness. E2+P4 withdrawal paradigms increase behavioral despair in animals. While 
despair can be triggered by withdrawal from E227 or P428, combined E2+P4 withdrawal is most similar to the perimenstrual 
phase. Among other likely mechanisms of E2+P4 withdrawal on hopelessness (e.g., serotonergic), we focus on E2+P4 as 
regulators of GABAergic neuroactive steroids, since prevention of neuroactive steroid formation using finasteride 
administration (i.e., abrupt neuroactive steroid withdrawal) recapitulates E2+P4-withdrawal-induced despair in animals, 
even when E2 or P4 remain high29. In susceptible females, withdrawal from E2+P4 provokes sadness27,30,31; however, a key 
Premise for Aim 1b 
Hopelessness as a Mediator of E2+P4 Withdrawal Effects on Suicide Risk 

 
 
 
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limitation of this literature is that few experimental or longitudinal studies specifically examine the impact of hormone 
withdrawal on hopelessness. 
 
Preliminary Data: Perimenstrual E2+P4 withdrawal (vs. stabilization) increases daily hopelessness. In our recent trial 
(K99MH109667), we found that ovarian steroid withdrawal (under placebo) was associated with increased daily reports of 
hopelessness in females with past-month SI, and that this was prevented by administering stabilizing doses of E2+P4 
(Condition X Phase γ=-.73, p=.023). Further, controlling for daily within-person changes in hopelessness partially mediated 
the effect of E2+P4 stabilization on SI severity (95% CI for Indirect Effect: -.21 to -.53). This preliminary work supports 
the premise that E2+P4 withdrawal causes perimenstrual hopelessness, effects which may explain proximal perimenstrual 
risk for suicide. 
 
 
 
Hyperarousal, defined as physiological hyperarousal, psychomotor agitation, and agitation or overwhelm32, is an acute 
precipitant of suicidal behavior32–35. In a meta-analysis, disturbances in arousal were among the strongest predictors of 
suicidal behavior25; however, this is an understudied area. In a meta-analysis focused specifically on agitation, agitation 
predicted suicide attempts35. However, little within-person work has been done to understand the time course of risk.  
Similarly, altered cardiovascular reactivity is linked to SI and attempts36–38, but no work examines acute changes in 
physiological arousal and proximal suicide risk.  
 
E2 and P4 withdrawal causes hyperarousal. In animals, E2+P4 withdrawal increases indices of hyperarousal, including 
autonomic function39 and heightened behavioral reactivity to stimuli10. Withdrawal from neuroactive steroids, which occurs 
to varying degrees during E2+P4 withdrawal in the perimenstrual phase, causes reactivity-promoting alterations in 
associated brain circuitry, including the amygdala40–42, the bed nucleus of the stria terminalis42,43, and the periaqueductal 
grey44,45. Further, variables linked to suicidal ideation and behavior, including social stress46 and isolation47, lifetime abuse48, 
impulsivity49, Borderline Personality Disorder50,51, and PTSD52,53, intensify effects of E2+P4 withdrawal on GABAergic 
neuroactive steroids or hyperarousal. 
 
Preliminary Data: Perimenstrual E2+P4 withdrawal (vs. stabilization) increases daily hyperarousal. In the previous 
hormone stabilization trial12, we found increased daily agitation54 and overwhelm55 during perimenstrual E2+P4 withdrawal 
under placebo, which was prevented by administering stabilizing doses of E2+P4 (Cond×Phase γ=-.87, p<.0001; 
Cond×Phase γ=-.92, p<.0001). Controlling for within-person levels of self-reported agitation on the Brief Agitation 
Measure partially mediated the effect of E2+P4 stabilization on SI severity (95% CI for Indirect Effect: -.30 to -.55) and 
probability of endorsing suicide planning20 (95% CI for Indirect Effect: -.18 to -.34). This work supports the premise that 
E2+P4 withdrawal causes hyperarousal in the form of self-reported agitation, which may explain proximal perimenstrual 
risk for suicide. 
 
Preliminary Data: Our recent meta-analysis demonstrates that the perimenstrual phase is robustly associated with 
the lowest parasympathetic nervous system activity, consistent with physiological hyperarousal during 
perimenstrual E2+P4 withdrawal. Our meta-analysis (under review) aimed to quantify within-person variations in 
measures of cardiac vagal activity (indices such as heart rate variability, that capture parasympathetic innervation of the 
heart) across different cycle phases. A literature search following the PRISMA-Statement yielded 38 empirical studies 
(N=1,018) reporting repeated measures of cardiac vagal activity in at least two cycle phases. We found a significant decrease 
in parasympathetic activity from the late follicular (stable P4 and neuroactive steroids) to the premenstrual phase (d=-1.32, 
95% CI [-2.35, -0.29]) during E2+P4 withdrawal. This meta-analysis supports the premise that autonomic hyperarousal is 
heightened during perimenstrual E2+P4 withdrawal. The proposed trial would experimentally test if physiological 
hyperarousal is caused by E2+P4 withdrawal, and whether these changes mediate effects on suicide risk. 
 
Premise for Aim 1c 
Hyperarousal as a Mediator of E2+P4 Withdrawal Effects on Suicide Risk 

 
 
 
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Hopelessness and agitation are core features of depression and hyperarousal, respectively. Notably, several studies 
document that the intersection of depression and hyperarousal signals increased risk of suicide attempt in inpatients and 
outpatients33. Although this has primarily been discussed in the context of “mixed” states related to bipolar disorder, the 
intersection of depression and hyperarousal is not unique to bipolar disorder, and may increase proximal suicide risk35. Thus, 
for the purposes of the proposed work we hypothesize that hopelessness translates into proximal suicide risk only during 
hyperaroused states such as psychomotor agitation, physiological hyperarousal, and subjective agitation or overwhelm. 
 
 
 
Because they rapidly change neuronal excitability, neuroactive steroids are excellent candidate mediators of 
proximal suicide risk. The neuroactive steroids allopregnanolone (ALLO; 3α,5α-THP) and pregnanolone (PA; 3α,5β-
THP) are cholesterol-derived molecules (see Fig. 5 for biosynthetic pathway) that act as potent positive allosteric modulators 
at synaptic and extrasynaptic GABAA receptors, amplifying Cl− ion flux 
during GABA binding at GABAA receptors56. Administration of ALLO 
induces rapid sedative, antidepressant, anxiolytic, anesthetic, analgesic, 
and anticonvulsant effects57. Therefore, ALLO withdrawal can cause mood 
disturbances (i.e., hopelessness) as well as psychomotor agitation and 
autonomic hyperarousal39. The biosynthetic pathways for these 
neuroactive steroids are illustrated in Figure 5. ALLO is produced via a 
two-step process in which P4 is first converted into 5α-DHP by the enzyme 
5α-reductase type 1 (5α-RI), which is converted to ALLO by the enzyme 
3α-hydroxysteroid dehydrogenase type 2 (3α-HSDII). PA is produced via 
a similar process substituting 5β-reductase. Our review will focus on 
ALLO, since it is the best studied. 
 
Neuroactive steroid withdrawal mediates many deleterious behavioral 
effects of E2+P4 withdrawal. Many deleterious behavioral effects of 
E2+P4 withdrawal are mediated by ALLO withdrawal, as evidenced by the 
fact that blockade of P4 metabolism to neuroactive steroids (i.e., acute ALLO withdrawal) recapitulates the depressogenic 
and anxiogenic effects of E2+P4 withdrawal even when E2+P4 remain stable9. Further, direct experimental manipulation 
of ALLO withdrawal also causes these behavioral phenotypes58.   
 
Sources of neuroactive steroid withdrawal: Changes in Precursor Levels. Changes in ALLO levels can be caused by 
both changes in the availability of steroid precursors (P4) and by changes in the expression of biosynthetic enzymes that 
control conversion of the precursor to ALLO. ALLO fluctuates across the menstrual cycle with changes in P4 and E2, 
showing lower and more stable levels in the follicular phase, increasing during the early luteal week, peaking in the midluteal 
phase, and falling during the perimenstrual weeks. Since P4 is a precursor, perimenstrual withdrawal from P4 generally 
causes corresponding withdrawal from ALLO around the onset of menses, although the trajectory and degree of this 
withdrawal may be influenced by many additional between- and within-person factors. For example, because E2 increases 
synthesis of ALLO by increasing expression of 3α-HSDII in the brain59, E2 changes also shape perimenstrual ALLO 
withdrawal.  
 
Sources of neuroactive steroid withdrawal: Changes in central biosynthetic enzyme activity. In addition to effects of 
neuroactive steroids that have been synthesized from precursors in the periphery, neuroactive steroids can be directly 
synthesized de novo in brain60, and ALLO’s enzymatic pathway (e.g. 5α-RI) is expressed particularly in brain corticolimbic 
Premise for Aim 1d 
Hyperarousal and Hopelessness as Interactive Mediators of Suicide Risk 
Premise for Aims 2a & 2b 
Neuroactive Steroids and Proximal Suicide Risk 
Fig. 5 

 
 
 
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regions relevant for emotion and stress responses61–64. Of note, individual differences and circumstances can affect the 
degree to which ALLO’s enzymatic pathway is expressed in the brain, and these changes in enzyme expression can be 
reliably measured via peripheral monocyte mRNA expression. Increases or decreases in expression of these biosynthetic 
enzymes (5α-RI, 3α-HSDII) have been shown to relate to risk for neuropsychiatric illness. Postmortem studies (from co-I 
Pinna) reported decreased 5α-RI mRNA expression in the PFC of depressed subjects compared with non-depressed 
controls65. Suppressed ALLO levels have been found in the CSF of females with post-traumatic stress disorder (PTSD)66 
and depression67, and elevated 5α-DHP/ALLO ratios have been observed in the serum of those with PTSD68, but not in 
those with a history of trauma who did not develop PTSD, suggesting a possible role of disturbed neurosteroidogenic enzyme 
activity in the etiology of both hyperarousal and hopelessness phenotypes. 
 
In sum, although changes in neuroactive steroids (both serum levels and biosynthetic enzyme mRNA expression) have been 
strongly implicated behavioral phenotypes associated with suicide, no studies have examined the roles of state changes 
in neuroactive steroids on proximal suicide risk. The proposed study would examine the roles of trait-like differences 
and day-to-day changes in neuroactive steroids in proximal suicide risk, during a high-risk period in which neuroactive 
steroids are rapidly changing. 
 
 
 
Exporatory substudy of healthy controls (HC): The menstrual cycle leads to predictable, cyclical changes in GABAergic 
neuroactive steroids (e.g., ALLO). The GABAAR has 19 known genes coding for subunits a1-6, b1-4, g1-3, d, e, q, π and 
r1-269, with the majority of receptors comprised of two a, two b, and one g or d subunit70 (e.g., abg or abd), although 
subunit expression is highly plastic. Preclinical work shows that the potency of ALLO’s positive allosteric modulatory 
binding to the GABAAR depends on the subunit composition; for example, g2-containing receptors are less sensitive to 
ALLO than a4-containing receptors, and d subunits augment steroid potency71. Further, preclinical models show that P4 
and ALLO levels alter gene expression (via mRNA) and/or protein levels (via Western blot) for the a4 subunit72–74, d 
subunit75–77, and g278–81 subunit, such that plasticity of the receptor is a function of reproductive cycles. However, no human 
studies have described how subunits change normatively across the cycle.  
 
There is further evidence that withdrawal from ALLO (i.e., perimenstrual phase) may also negatively impact affect and 
behavior82,83, suggesting that GABAAR plasticity to ALLO change is the trigger for perimenstrual exacerbation of 
psychiatric symptoms. In animal models, excessive subunit plasticity is associated with concurrent changes in negative 
affect73,74,84–88, such as an association between overexpression of a4 and d with anxiety (e.g., elevated plus maze)73 and 
depression (e.g., forced swim test)89. Notably, non-hormonal work in humans has also associated GABAAR subunits with 
suicidality90, further underlining the importance of subunit expression on negative affect. Together, research shows that 
GABAAR subunit expression is related to negative affect, which is a known predictor of suicidality91. The only translational 
study of hormone-regulated GABAAR subunit expression to date found that peripheral subunit expression differs by 
pregnancy status and depression92, suggesting peripheral GABAAR subunit expression is a viable biomarker of hormone-
related changes in affect. Given that these outcomes (GABAAR subunit expression and negative affect/suicidality) are 
both altered by ovarian steroids, there is an urgent need to translate GABAAR subunit expression to human females, 
as this could be a potential biomarker for cyclical affective change and related cyclical suicide risk. 
 
Significance of the Expected Contribution. Upon successful completion of the proposed trial, we expect to contribute a 
viable, malleable model of proximal suicide risk, complete with information about the molecular, genetic, and behavioral 
mechanisms that underlie its effects. This contribution is significant because it offers a novel solution to the field’s lack of 
proximal neurobiological treatment targets21(p50) for suicide.  These contributions are consistent with NIMH objectives to 
(1) “define the mechanisms of complex behaviors” (i.e., understand the multilevel causal mechanisms of hormone-related 
changes in suicide), to (2) “chart mental illness trajectories to determine when, where, and how to intervene” (see Figure 
3; i.e., during periods of hormone flux in females, during periods of neuroactive steroid flux in males and females), and (3) 
Premise for Aim 3 
GABA-A Receptor Subunit Composition  

 
 
 
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“to strive for prevention and cures” (i.e., by developing one of the first models of a naturally-occurring, biologically-driven 
cause of proximal suicide risk that could be prevented to improve outcomes). The public health relevance of this work is 
to immediately improve prediction of proximal suicide risk, depression, and arousal-related disorders in females (i.e., given 
the recurring and highly predictable nature of the cycle); to continue to develop an experimental model for understanding 
the role of ovarian hormone withdrawal in female-biased risk for psychopathology; and to pave the way for the long-term 
development of stabilization treatments to prevent suicide deaths. 
 
3.0 
Objectives/Aims 
 
Our aim is to test the central hypothesis is that perimenstrual E2+P4 withdrawal induces rapid withdrawal from calming 
GABAergic neuroactive steroids, which in 
turn increases hyperarousal and hopelessness, 
which then interact to cause proximal suicide 
risk (Figure 1).  P4 is metabolized to 
GABAergic neuroactive steroids, and E2 
promotes expression of enzymes that convert 
P4 to neuroactive steroids93. Perimenstrual 
E2+P4 withdrawal may increase suicide risk 
by causing rapid withdrawal from the 
antidepressant9 and sedative11,10 effects of 
neuroactive steroids94, as well as withdrawal 
from the antidepressant effects of E295. Our 
preliminary experimental work strongly 
suggests 
that 
perimenstrual 
E2+P4 
withdrawal 
(and 
neuroactive 
steroid 
withdrawal) heightens proximal suicide risk 
via hopelessness65  and hyperarousal12,66. 
 
Design: Using the PI’s hormone withdrawal 
paradigm (K99MH10966712), a new sample of 90 completers female outpatients with past-month SI will complete two 
counterbalanced conditions: (1) natural perimenstrual E2+P4 withdrawal (placebo), and (2) perimenstrual E2+P4 
stabilization. Five labs per condition will capture changes in neuroactive steroids, mRNA expression for neurosteroidogenic 
enzymes, physiological arousal, behavioral indices of arousal, and interview/self-report indices of proximal suicide risk, 
hopelessness, and hyperarousal. Our app (BiAffect) will passively track indices of arousal including psychomotor agitation 
(accelerometer) and reactivity (typing speed variability). Additionally, Metricwire will passively track movement using 
smartphone sensors. 40 healthy controls (HC) will complete five labs across one observational cycle to characterize 1) 
normative cycle-related variation in neuroactive steroids and mRNA expression, and 2) group-related differences (i.e., past-
month SI vs HC) in trajectories of GABAAR subunit change across the cycle.    
 
AIM 1: To evaluate hyperarousal and hopelessness as interacting mechanisms by which perimenstrual E2+P4 
withdrawal (vs. experimental stabilization) increases proximal suicide risk. We predict that (1a) individuals will 
demonstrate greater proximal suicide risk during perimenstrual E2+P4 withdrawal (placebo) than during perimenstrual 
E2+P4 stabilization, an effect mediated by (1b) indices of hyperarousal, (1c) indices of hopelessness, and (1d) their 
interaction (i.e., with hopelessness predicting suicide risk only during hyperarousal).  
 
AIM 2: To evaluate neuroactive steroid withdrawal as a mechanism by which perimenstrual E2+P4 withdrawal (vs. 
stabilization) increases proximal suicide risk. We expect that E2+P4 withdrawal (vs. stabilization) will induce 
neuroactive steroid withdrawal to some degree in all participants. However, we predict that the effect of hormone withdrawal 
(vs. stabilization) on proximal suicide risk at each lab will be mediated by (2a) degree of reduction in serum neuroactive 
steroid levels and (2b) degree of reduction in mRNA expression for neurosteroidogenic enzymes. We will also evaluate 

 
 
 
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within-person links between neuroactive steroids and all behavioral constructs; if appropriate, a multilevel mediation model 
will test the full model in Fig. 1. 
 
AIM 3: To characterize GABAAR gene expression plasticity across the cycle in individuals with and without 
perimenstrual changes in affective and suicide risk. In the HC group, we hypothesize that gene expression for a4 and d 
subunits (GABRA4 and GABRD genes, respectively) are upregulated and g2 (GABRG gene) is downregulated in the 
midluteal (high ALLO) and perimenstrual (ALLO decline) phases compared to the midfollicular phase (low ALLO). We 
also hypothesize that clinical group moderates the effect of cycle phase on GABAAR composition, such that the group with 
perimenstrual increases in affective symptoms and suicide risk shows higher slope of midluteal-to-perimenstrual change in 
a4 and d subunits, and lower g2 subunit replacement of d subunit expression than the HC group.   
 
4.0 
Eligibility 
 
Recruitment and Screening. We will recruit 170 females (90 completers) with past-month SI in outpatient treatment as 
usual with a mental health provider, and 60 healthy controls (HC) (40 completers) with no lifetime history of SI and no 
DSM-5 diagnoses in the past five years, for a total of 230 participants. Participants will be recruited via fliers, email listservs, 
online Research “match” websites (Research Match, Research & Me), and social media ads seeking volunteers to participate 
in a study “on the biology of depression, stress, and suicide” (with no mention of the cycle, hormones, female sex, or 
women). Participants will complete an online screener and will be scheduled for a phone screen if they meet basic inclusion 
criteria. Following phone screening, potential participants will be invited to an enrollment visit. 
 
4.1 Inclusion/Exclusion Criteria 
 
Participants will include 170 (90 completers) physically healthy premenopausal females reporting past-month suicidal 
ideation (but acceptably low imminent suicide risk for outpatient care) receiving treatment as usual at an outpatient mental 
health clinic in Chicago, and 60 (40 completers) physically healthy premenopausal females with no lifetime history of SI 
and no DSM-5 diagnoses in the past five years (total 230 participants). The HC group inclusion and exclusion criteria are 
identical except where indicated below. Although the HC group will not undergo exogenous hormone stabilization, they 
will be required to meet identical medical criteria in order to best match the experimental group. 
 
Of note, we will not exclude those without smartphones, and a smartphone will be provided to a subject if they do not have 
one.  
 
To participate, individuals must meet all of the following inclusion/exclusion criteria:  
 
(1) Must be biologically female assigned at birth and between the ages of 18-45 years.  
 
Justification: Females younger than 18 or older than 45 may have inconsistent menstrual cycles or may be 
postmenopausal and therefore no longer cycling. 
 
(2) Must self-report normal menstrual cycles between 21 and 35 days. 
 
Justification: Abnormal menstrual cycle lengths or variability in lengths could introduce error variance into the 
experiment and could reduce confidence regarding the timing of the experimental manipulation to cycle events. 
 
(3) Must be under the current care of an outpatient mental health provider with whom they are comfortable 
discussing history of suicidal ideation and are able to visit at least once every 3 months, or they must be 
willing to begin visiting an outpatient mental health provider at least once every 3 months. 

 
 
 
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Justification: Because we intend to recruit females with current suicidality, engagement with a mental health 
provider is required as a protective measure to ensure that participants have access to treatment if needed. 
 
HC group: criterion 3 not applicable; HC participants should not be under the care of an outpatient mental health 
provider for diagnosis or treatment of a psychiatric condition.  
 
(4) If the person has children, they must be at least 1 year postpartum. 
 
Justification: The postpartum period can be characterized by altered hormone levels and changes; therefore, 
postpartum females may introduce error variance into the experiment. 
 
(5) Must not be pregnant, breastfeeding, or trying to become pregnant. Pregnancy status will self-
reported at enrollment, confirmed using urine pregnancy test at the first in-person visit of the experimental 
condition, and confirmed again at the first visit of the second condition. Participants will be encouraged 
to use a barrier method of birth control during the study. 
 
Justification: Administration of ovarian hormones to a pregnant person could negatively influence the pregnancy 
in a variety of ways, including risks to the health of the fetus. 
 
(6) Must not be taking any form of exogenous hormones or intrauterine device (IUD) with hormones and 
must have ended previous use of hormonal preparations at least one month prior to the study. Those 
who were previously taking oral contraceptives or other hormonal medications must have one normal 
menstrual cycle (menstrual period) prior to enrollment in the study. Participants will be required to use 
licensed barrier methods of (non-hormonal) contraception during study participation; if they are 
unable to commit to this responsibility, they will not be eligible for participation. 
 
Justification: The use of additional exogenous hormones poses safety risks and would undermine experimental 
control of hormones.  
 
(7) Must be of normal weight (BMI between 18.000 and 34.999); measured via self-report at screening and 
confirmed via direct measurement in the laboratory at the Welcome Visit. 
 
Justification: While both obesity (BMI>=30) and use of ovarian steroid medications are each associated with 
slightly increased risk for thromboembolic events, the best evidence suggests that these risks are additive rather 
than interactive; further, use of transdermal estradiol (as in the present study) is associated with even smaller 
increases in thromboembolic risk relative to oral estradiol. Therefore, we have opted to include individuals with a 
BMI between 18 and 35.   
 
(8) Must report no personal history of any condition that could, in the opinion of the study psychologist or 
physician, significantly increase physical or behavioral risks to the participant over the course of the study, 
or which could interfere with the endocrine and immune endpoints of the study. This includes, but is not 
limited to, metabolic or autoimmune disease, epilepsy, endometriosis, fibroids, PCOS, cancer, diabetes, 
cardiovascular, gastrointestinal, hepatic, renal, or pulmonary disease, history of thromboembolic events, 
migraine with aura, or use of a pacemaker or other internal electrical device.  
 
Justification: Administration of exogenous hormones to females with these chronic medical conditions may 
increase the risk of an adverse health-related response to hormones or the Oura Ring. Further, the purpose of the 

 
 
 
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present study is to determine the impact of hormonal stabilization (vs. natural withdrawal) in otherwise physically 
healthy females.  
 
(9) Must not currently use nicotine regularly, and must not regularly smoke any combustible materials (i.e., 
marijuana cigarettes). 
 
Justification: Although more research is needed to clearly understand these risks, some evidence suggests that 
administration of ovarian hormones may interact with regular use of nicotine or regular smoking of combustible 
materials to synergistically increase risk of thromboembolic events. Therefore, any participant endorsing regular 
use of nicotine or smoking of combustible materials will be excluded.  
 
(10) Must not report substance use, mania, or psychosis symptoms that (in the opinion of the study 
psychologist or psychiatrist) impair the participant’s ability to follow study directions, safely take study 
medication, or communicate with the study team or their mental health provider.  
 
Justification: These factors may interfere with the individual’s ability to complete the study safely. They are also 
known sources of variability in suicidality and would therefore introduce error into our models as well as increasing 
the risk that a participant would experience a suicidal crisis during the study. Use of opioids, methamphetamine, 
and cocaine will be screened at the enrollment visit to validate interview measures of substance use. 
 
(11) 
Must report at least some recent suicidal ideation (in the past month) at enrollment. 
 
Justification: As reviewed in the research strategy, the purpose of the study is to examine the impact of natural 
perimenstrual hormone withdrawal (vs. prevention of either E2 or P4 withdrawal) on within-person changes in 
suicide risk. The present sampling strategy ensures generalizability of our findings to clinical populations of 
interest and will also prevent a floor effect (i.e., zero-inflation of daily outcome measures and associated limitations 
in power to detect effects) on primary daily outcome measures (e.g., mediating behavioral constructs, suicidality).  
 
HC group: must NOT report lifetime history of suicidal ideation in order to meet criteria as a control for the 
experimental study. 
 
(12) 
Must be categorized as having acceptably low imminent risk for suicidal crisis/attempt (such 
that they would be appropriate for outpatient—rather than inpatient—care) according to established 
clinical and research guidelines. Specific guidelines were developed during the PI’s K99/R00 studies 
(K99/R00109667) in collaboration with Dr. Mitch Prinstein, a suicide-focused co-mentor on the PI’s prior 
K99 training grant. As Dr. Prinstein notes in his 2014 methodological paper on research-based suicide 
risk assessment, evaluation of imminent risk of suicidal crisis/attempt is a complex clinical task, requiring 
integrative consideration of a variety of factors (including: general clinical presentation, ideation severity, 
frequency, recency, and associated consideration/intent; access to suicidal means; previous attempts 
and gestures; presence of suicidal planning, recent stressors, frequency of contact with mental health 
provider; and presence of social support). Therefore, our specific criteria for delineating whether a 
participant is eligible are based on an evidence-based determination, after reviewing all of the above 
information, that the participant is at acceptably low imminent risk of suicidal crisis/attempt such 
that they would be appropriate for ongoing care in an outpatient—rather than inpatient—setting. 
These decisions will be based on responses to a variety of questions from the Self-injurious Thoughts 
and Behaviors Interview- Revised (SITBI-R; clinical interviews at the beginning of an enrollment visit). 
We will exclude those judged to be at imminent risk of acute suicidal crisis/attempt. 
 

 
 
 
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HC group: N/A 
 
Justification: Individuals with current suicidality are not necessarily at imminent risk for suicidal 
crises/attempt (i.e., their risk is manageable on an outpatient basis through frequent clinical contact, such as the 
daily calls in the present study). Because the proposed hormone protocol simply prolongs the physiologic luteal 
hormone profile of E2 and P4, it is not expected to expose participants to additional risk of suicidal crisis beyond 
what they would naturally experience during their typical monthly menstrual cycles. However, out of an abundance 
of caution, we will exclude females with elevated imminent risk for suicidal crisis/attempt (determined by 
structured interview and established guidelines). Individuals with histories of suicidality often have ongoing, 
habitual thoughts about death or suicide that are highly distinguishable from imminent risk for suicide attempt. 
In further support of this notion and of the feasibility of recruiting this population, it should be mentioned that up 
to 1/3 of the American population experiences suicidal ideation at some point in their lives, yet less than .01% die 
by suicide each year.  Data from the preceding similar experiment (K99 experiment) provide clear evidence 
of the safety of this protocol; no participant was hospitalized for imminent risk or attempted suicide during 
the study.  
 
(13) 
Must not report symptoms consistent with primary dysmenorrhea that indicate immunological 
involvement (recurrent perimenstrual fever) or significant disruption in daily life (missing >= 2 days of 
work each month). 
 
Justification: The purpose of the present study is to determine the impact of hormonal stabilization (vs. natural 
withdrawal) in physically healthy females. Thus, females who present symptoms consistent with severe primary 
dysmenorrhea will be excluded.  
 
(14) 
Must not be (1) currently seeking treatment for premenstrual dysphoric disorder (as indicated by 
(a) treatment-seeking reported at screening AND (b) a PMDD diagnosis confirmed by the study team 
during baseline ratings using the daily record of severity of problems) and must not be (2) currently 
experiencing a mood episode that began during pregnancy or within four weeks of giving birth (Note: this 
criterion only applies if delivery occurred less than 2 years ago). 
 
Justification: Some evidence  suggests that the pattern of perimenstrual symptoms that have been linked with these 
reproductive mood disorders could differ meaningfully27 from that which we be expect in the more general clinical 
population of females with suicidality12. Because these distinctions are not yet clear, and because we do not wish 
to interfere with the evidence-based treatment of those who are currently diagnosed with these hormone-sensitive 
disorders, we are excluding these individuals.  
 
(15) 
Must NOT report family history of >=2 first degree relatives with hormone-related cancers 
(breast, ovarian, endometrial, uterine, prostate, colon), nor known genetic risk for hormone-related 
cancers (e.g., BRCA1, BRCA2, PTEN, or Tp53). 
 
Justification: Administration of exogenous hormones to females with increased genetic risk for hormone-related 
cancers may increase the risk of cancer. 
 
(16) 
Must NOT report increased risk for blood clots as described by personal history of (or at least 1 
first degree relative’s) diagnosis of blood clots (VTE, thromboembolism, etc.), nor evidence of genetic 
risk for clotting disorders (Factor V Leiden). 
 
Justification: Administration of exogenous hormones to females with increased genetic risk for these chronic 
thromboembolic medical conditions may increase the risk of a blood clot formation in response to hormones. 

 
 
 
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HC group: Criterion 16 removed for HC group. Increased clotting risk does not apply to the observational group 
not receiving exogenous hormones, and we have no expectation that menstrual cycle variables, neuroactive steroid, 
or gene expression outcomes would be altered by heightened risk for blood clot.   
 
(17) 
Must NOT be allergic to peanuts. 
 
Justification: The facility which processes the study pills indicates that the product is not suitable for those with a 
peanut allergy, so females with a peanut allergy will be excluded.  
 
HC group: Criterion 17 removed for HC group, who will not receive any study medication.   
 
 
SPECIFICS OF SUICIDE RISK-RELATED ELIGIBILITY CRITERIA  
 
As in the PI’s two prior trials, the proposed study will include only participants with current suicidal ideation (past-month 
endorsement of suicidal ideation) in order to maximize generalizability to our clinical population of interest. In order to 
minimize the risk of suicide attempts during the study, we will exclude females who appear to be at high imminent risk of 
acute suicidal crisis/attempt at the outset of the study. Although we acknowledge that the field’s ability to predict imminent 
risk of suicide attempts remains poor, these criteria are based on the available evidence about which factors sometimes 
predict imminent risk of suicide attempt or death.  
 
- 
A person will be excluded if they report more than 1 attempt (total of suicide attempts or interrupted 
suicide attempts) in the past year.  
 
- 
A person will be excluded if they report any suicide attempt or any interrupted suicide attempt in the past 
3 months, as the majority of repeated attempts occur within the first three months following an initial 
attempt. 
 
- 
A person will be excluded if they report any aborted suicide attempt in the past month.  
 
- 
A person will be excluded (and referred) if they report that they do not currently visit a mental health 
provider (psychiatrist, therapist) at least once every 3 months AND they are not willing to start visiting a 
mental health provider at least once every 3 months. If a participant opts to begin treatment prior to 
starting the study in order to become eligible, eligibility for starting clinical trial conditions will be based 
on self-report of having had at least one visit with the new provider.   
 
- 
A person’s participation will be delayed for 3 months if they report or expect any extreme external 
stressors (e.g., death of a loved one, loss of employment) in the past or coming month, as extreme 
stressors drastically increase the risk of suicidal crisis. 
 
- 
A person will be excluded if they report a history of manic, psychotic, or substance use disorder symptoms 
that may reduce the predictability and increase likelihood of suicidal crises/attempt.  
 
- 
Participants will be counseled in means restriction at the outset of the study; if a person reports access 
to a firearm or another highly lethal means of suicide and is not willing or able to restrict access to such 
means, they will not be eligible for the study.  
 

 
 
 
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5.0 
Subject Enrollment 
 
Recruitment and Screening. We will recruit 170 females (90 completers) with past-month SI in outpatient treatment as 
usual with a mental health provider, and 60 healthy controls (HC) (40 completers) with no lifetime history of SI and no 
DSM-5 diagnoses in the past five years, for a total of 230 participants. Participants will be recruited via fliers, email listservs, 
online research recruitment websites (Research Match, Research and Me) and social media ads seeking volunteers to 
participate in a study “on the biology of depression, stress, and suicide” (with no mention of the cycle, hormones, female 
sex, or women). The social media ad directs participants to complete an online screener. They will be scheduled for a phone 
screen (via HIPAA-compliant Webex teleconference software) if they meet basic inclusion criteria. Following phone 
screening, potential participants will be invited to an enrollment visit either in-person or virtually. Virtual enrollments 
(including digital signatures of the informed consent document hosted on UIC Qualtrics), will occur via UIC’s HIPAA-
compliant Webex teleconference software. For participants who completed virtual enrollment visits during the covid-19 
pandemic, their consent will be re-assessed during their first in-person Welcome Visit, where they will provide written 
consent and receive a copy of the consent form. 
 
 

 
 
 
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6.0 
Study Design and Procedures 
 
Overview of Research Design. The proposed primary experiment (identical to K99MH109667) tests the hypothesis that 
perimenstrual E2+P4 withdrawal causes varying degrees of neuroactive steroid withdrawal, which produces co-occurring 
hopelessness and hyperarousal, which interact to cause proximal suicide risk. In a crossover placebo-controlled experiment, 
90 completer female outpatients with recent SI (but acceptable suicide risk for outpatient treatment) will complete two 
counterbalanced perimenstrual conditions: (1) natural perimenstrual hormone withdrawal (placebo), and (2) prevention 
of perimenstrual hormone withdrawal (exogenous stabilization of E2+P4). Measures of neuroactive steroids, 
hopelessness, hyperarousal, and proximal suicide risk will be collected in the laboratory and via smartphone. The basic 
timeline for participation can be seen in Table 1a. We will utilize scientifically rigorous methods that promote transparency 
and reproducibility (NOT-OD-16-012): well-defined inclusion/exclusion criteria, validated measures, pre-registration of 
our protocol, and sharing of data. 
 
Table 1a. Primary study activities and timeline. 
 
SUB-STUDY TIMELINE 
START 
MONTH 1 
MONTH 2 
END 
Enrollment 
Visit 
Baseline 
Menstrual Cycle 
OBSERVATIONAL MENSTRUAL CYCLE 
  
Week 1 
Week 2 
Week 3 
Week 4   
Ovulation Testing 
  
  
  
X 
  
  
  
Laboratory Visits (60 mins) 
  
 
 
 
X X X X X 
  
Evening Surveys (3-5 mins) 
  
X 
X 
X 
X 
  
Using "BiAffect" App  
  
X 
X 
X 
X 
  
Using Metricwire App 
  
X 
X 
X 
X 
  
Table 1b. substudy activities and timeline. 
 
The proposed substudy characterizes 1) normative cycle-related variation in neuroactive steroids and 
mRNA expression, and 2) group-related differences (i.e., past-month SI vs HC) in trajectories of GABAAR 
subunit change across the cycle. 60 healthy controls (HC) with no SI will be recruited to gain a completer 
number of 40 for a two-month long observational substudy, in which five lab visits across one menstrual cycle 
(timed identically to mirror the observations across one medication phase of the experimental trial) will capture 
baseline molecular/genetic changes for between-group comparisons. The basic timeline for participation can 
be seen in table 1b. 
 
 

 
 
 
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The double-blind crossover experimental design is identical to that used during the PI’s recently-completed trial 
(K99MH109667; Fig. 6). The TOP (red) panel shows natural 
perimenstrual hormone withdrawal in the placebo condition; the 
BOTTOM (black) panel shows prevention of hormone withdrawal 
in the active E2+P4 stabilization condition. THIS IS NOT A 
CLINICAL EFFICACY TRIAL, but a RCT design will probe the 
effect of hormone withdrawal (vs. stabilization) on proximal suicide 
risk and mediators. Participants will be randomized to condition 
order A (see Table 1 above; Active E2+P4, washout cycle, then 
Placebo) or condition order B (Placebo, washout cycle, then Active 
E2+P4). Investigational Drug Services will manage the double-
blind. In each two-week condition, participants will wear a weekly 
patch (E2 or placebo) and take a daily pill (P4 or placebo). Because 
this manipulation simply delays natural E2+P4 changes96, and given 
our previous finding of benefit12, this manipulation is not expected 
to pose greater risk than a normal menstrual cycle.  
 
Timing and Duration. Participants will receive the first seven-day 
transdermal E2 patch (or placebo patch) and begin daily oral P4 (or 
oral placebo) at a lab visit scheduled seven days after a positive urine 
ovulation test (+7 in Fig 6). This coincides with a time in the natural 
cycle when E2+P4 are already elevated. The weekly patch will be 
changed at the lab visit on day +14.  The goal of this E2+P4 
manipulation is to extend and maintain the mid-luteal phase 
hormone profile beyond the point in the cycle (i.e., around day +10) 
that would normally be associated with natural hormone 
withdrawal. Therefore, the active condition effectively prevents 
perimenstrual E2 and P4 withdrawal.  
 
Formulation and Dose. Estradiol. Peak E2 with transdermal E2 
patches (~45 pg/mL) correspond roughly to normative luteal E2 
levels96. These E2 levels are expected to maintain stable mRNA expression of 3α-HSDII during the experimental period, 
stabilizing biosynthesis of neuroactive steroids. Transdermal E2 demonstrates reduced thromboembolic risks relative to oral 
E2. Progesterone. Although transdermal synthetic progestins exist, these molecules have confounding effects at androgen 
and mineralocorticoid receptors. Thus, oral micronized progesterone 200mg/day will maintain luteal ranges (~13 ng/dL)96 
as in the prior trial12; this dose of P4 is expected to buffer neuroactive steroid change by increasing precursors. Standardized 
times for evening dosing (7 pm) and lab visits (7-11am) will minimize variability in neuroactive steroid levels due to 
timing. Typical within-participant variation in emotional symptoms, physical premenstrual symptoms, and cycle length will 
maintain participant blind. Demand characteristics will be minimized by telling participants that responses to hormones 
differ, and they may experience positive or negative moods. Blinding was excellent in our completed trial, with medication-
day condition guesses only slightly more accurate than chance (53% accurate). Manipulation Checks. In the prior trial, we 
found that the hormone stabilization condition (E2+P4) was associated with elevated (maintained) levels of E2 and P4 on 
day +14 (see Figure 6) relative to a drop in E2 and P4 observed in the placebo condition. We will once again measure E2 
and P4 in this trial to confirm stabilization. 
 
 
EXPERIMENTAL 
Crossover RCT of perimenstrual E2+P4 stabilization 

 
 
 
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Overview of Assessment Protocols. Laboratory visits (90 minutes; 7-11 am). After a positive home urine ovulation test 
(day 0), five 90-minute lab visits will be scheduled on days +7, +10, +14, +18, and +22 across each condition to capture the 
dynamic changes in mediators and outcomes across the perimenstrual window (Figure 6). Consistent with standard practices 
in the field of reproductive mood disorders55, participants will complete brief (3 minute) daily evening surveys across the 
study. During the study participants will undergo passive tracking of physiological arousal, movement, and typing speed 
instability via the Oura Ring, Metricwire App, and BiAffect App, respectively. Participant Safety Monitoring. On 
medication days and 4 days following, daily phone calls (30-60 seconds) will evaluate physical adverse effects and suicide 
risk. 
 
Enrollment Laboratory Visit (5 hours; split into 2 visits) 
 
Participants will be asked to attend for a 5 hour enrollment session that is split into 2 visits either in-person or virtually in 
which participants will be learn more about the purpose of the study and will be assessed for eligibility.  
 
Baseline Interviews: The Structured Clinical Interview for DSM-5 (SCID-5)97 and Structured Clinical Interview for DSM-
5 Personality Disorders97, as well as a medical eligibility interview. Lifetime suicidal thoughts and behaviors will be assessed 
using the Self-Injurious Thoughts and Behaviors Interview -Revised (SITBI-R) and categorized with the C-CASA98. 
Participants will complete the Stress and Adversity Interview (STRAIN)18 interview, which captures lifetime exposure to 
adverse events (e.g., Negative Valence Systems of Loss, Threat).  
 
Baseline Self-Report Surveys: Participant complete the following measures with respect to the past two weeks: The Adult 
Suicidal Ideation Questionnaire (ASIQ)20, a 30-item questionnaire that provides a continuous measure of SI as well as items 
assessing the intensity of thoughts related to suicidal planning and intent; the Beck Hopelessness Scale (BHS)17, a widely-
used 20-item measure of hopelessness; Arousal will be measured using the Arousal items on the Profile of Mood States 
(POMS)99 as well as the Brief Agitation Measure (BAM)54. Other measures will be included to capture key transtheoretical 
constructs, including the Inventory of Depression and Anxiety Symptoms – ii (IDAS-ii)100, the UPPS-P Impulsivity Scale101, 
Interpersonal Needs Questionnaire (INQ; belongingness, burdensomeness)102, and the Fearlessness About Death Scale103.  
 
Enrollment Laboratory Visit for HC participants (Aim 3substudy) 
HC participants will complete a single enrollment session expected to last 3 hours, either in-person or virtually. Participants 
will complete all questionnaires and interviews listed above for primary study. Interviews are expected to take less time 
than the primary study because the HC substudy participants are recruited for a lack of current psychiatric symptoms. 
 
 
Welcome Visit (1 Hour) 
 
Participants will be asked to attend an in-person introductory session where they will receive ovulation test kits and Oura 
rings. Participants will also review the study consent form, fill out payment-related paperwork, and form a safety plan with 
the lab staff in case of elevated suicide risk.  
 
Welcome Visit for HC participants (Aim 3substudy) 
HC participants will not be asked to attend an in-person introductory session. Ovulation test kits will be mailed to HC 
participants after enrollment, paperwork will be signed at the first visit. 
 
 
 
ASSESSMENTS 
Laboratory and smartphone-based measures.  

 
 
 
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Repeated Laboratory Visits (90 minutes; 10 visits total; 5 visits across each condition)  
 
After a positive urine ovulation test (day 0), five 90 - minute lab visits will be scheduled on days +7, +10, +14, +18, and 
+22. Labs will be standardized to occur between 7am and 11am. Labs will begin with an assessment of side, after which 
participants will complete tasks, undergo a blood draw and urine pregnancy and standard drug screen (day +7), complete 
interviews and self-reports, and undergo patch applications (days +7, +14). Health behaviors and illness will be measured 
during labs and daily survey.  
During the study, each participant will also wear an Oura Ring, a wearable ring device (that includes a 
pulse plethysmograph (PPG) and temperature sensors) throughout the study; these rings, which have 
been validated to index heart rate and heart rate variability (measures of physiological arousal), as well 
as changes in basal body temperature, will serve the dual purpose of measuring both physiological 
arousal and progesterone-related temperature changes across the cycle.  
NOTE: A prior version of the protocols utilized lab-based measures of physiological arousal using a traditional 
EKG. However, given ongoing concerns regarding close contact during the COVID-19 pandemic, the NIMH 
program officer for the grant funding the present study has approved a change from lab-based cardiovascular 
physiological recording using EKG to ambulatory assessment of physiological arousal using the Oura Ring. 
Repeated Laboratory Visits for HC participants (Aim 3 substudy) 
HC participants will complete 5 60-minute lab visits across one menstrual cycle. Repeated lab visits will be timed 
identically in the HC substudy group as in the primary group (i.e., 5 lab visits scheduled on days +7, +10, +14, +18, and 
+22 relative to a positive urine ovulation test. Participants will undergo a blood draw, urine pregnancy and standard drug 
screen (day +7), and report health behaviors and illness. Participants will not receive any experimental patch 
application, will not complete cognitive tasks, and will not complete interviews. 
 
Debriefing Visit (1 hour)  
 
After completing both randomized conditions, participants will be asked to attend for a 1 hour debriefing session with Dr. 
Eisenlohr-Moul either in-person or virtually in which participants will be unblinded, learn more about the purpose of the 
study, have any concerns or questions participants may have about the study answered and addressed, and evaluate the 
participant’s current risk of suicide upon study completion. 
 
Debriefing Visit for HC participants (Aim 3substudy) 
HC participants will not be asked to attend a debriefing session, as they do not complete randomized double-blind 
experimental conditions, and they do not have suicide risk to assess. 
 
CLINICAL LABORATORY ASSESSMENTS AND SAMPLING: 
 
A point-of-care urine pregnancy test will be completed in our laboratory on site at the first visit of both conditions. A 
positive pregnancy test will result in termination of participation and will be managed by Dr. Wagner. 
 
We will also collect a urine sample and perform rapid urine toxicology screens for amphetamines, barbiturates, 
benzodiazepines, cocaine, opioids, cannabis, and methamphetamine on-site through the CRC nursing staff using Alere 
iCup 12-Panel Drug Test with temperature-sensitive strips. Positive tests will be used primarily for data quality assurance 
and covariate purposes. Individuals with positive test results will be allowed to continue the study provided that the 
participant demonstrates an ability to safely complete all study procedures. When there is any positive test that is not 

 
 
 
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accounted for by an existing prescription, the participant’s test results will be reviewed with the study physician and PI at 
routine team meetings. 
 
At all 10 laboratory assessments during active treatment with placebo or E2/P4, participants will provide blood (35ml at 
each visit) to be assayed for E2/P4, neuroactive steroids, and from which we will perform RNA gene expression assays, to 
evaluate the success of the hormone in achieving stable luteal phase levels of E2/P4/ALLO, as well as more stable gene 
expression markers of risk across various systems (neurosteroidogenic enzymes, GABA-A receptor subunits, and other 
mechanisms such as inflammatory cytokines). Blood will be collected as follows: (1) 30ml in red-top tubes (no additive), 
and (2) 5ml in PaxGene RNA tubes, at 2.5ml blood apiece. 30ml of blood (red-top tubes) will be centrifuged and plasma 
will be stored in cryovials, and the PaxGene tubes will be stored as well; when it is time for assay, samples will be 
transferred to Dr. Pinna’s laboratory. Standard freezer-proof labels will indicate participant ID and study session. 
Transport from the CRC to Dr. Pinna’s laboratory will be managed by Clinical Research Center staff. Samples will be 
analyzed in small batches with all of one participant’s samples being run in the same batch. Following assay, any 
additional serum will be stored by Dr. Pinna. Following assay, the PaxGene RNA blood samples will be destroyed. 
 
Clinical assessments and laboratory sampling for HC participants (Aim 3 substudy). 
Point-of-care pregnancy test, urine toxicology screen, and laboratory samples via blood draw are identical for HC 
participants as described above.  
 
Self-Reports. Self-report surveys from the enrollment visit will be repeated at each laboratory visit, with instructions to 
report symptoms in the past week or since the last visit.  The “since last visit” version of the C-SSRS104 interview will be 
used to precisely measure discrete self-injurious thoughts and behaviors at each laboratory visit; responses will be coded 
into suicide outcomes using the C-CASA98.  
 
We will operationalize behavioral hyperarousal (reactivity) as a longer response time when matching emotional faces 
relative to shapes. We will utilize these measures as independent predictors of proximal suicide risk, as well as variables 
with which to further validate BiAffect indices (below).  
 
Physiological Arousal. Following the laboratory visit, participants will be mailed an Oura Ring in the appropriate size, 
along with instructions for charging and use, and asked to wear the ring as much as possible while in the study. This 
commercially available product is intended for fitness tracking and recreational use.  It is worn on the middle finger and 
also uses PPG technology along with mechanical sensors to measure physical activity, sedentary behaviors, sleep, heart 
rate, oxygen saturation, and body temperature.   
 
Participants will also be asked to use the Oura app- this commercially available application is required to collect data from 
supported Oura products.  It is publically and free available for both Android and iOS devices in their respective app 
stores.  Data from the Oura Ring will be transferred to the Oura smartphone app, and later to the Oura cloud storage 
service (where it can be retrieved later by the study team) using an Oura Ring app account created by the study team using 
the participant's confidential study email address (e.g., clearlabparticipant+3999@gmail.com). Data collected and 
uploaded to the cloud will not include IP address or any other participant-identifying information.   
 
Self-reports and physiological arousal for HC participants (Aim 3 substudy). 
HC participants will not be asked to complete self-report surveys or cognitive tasks at laboratory visits. They will not 
wear an Oura Ring or use the Oura app. 
 
Neuroactive Steroids (Co-I Pinna). See sample collection, transportation, and preparation information above. In Dr. Pinna’s 
Laboratory, neuroactive steroid biosynthesis will be determined by levels in the plasma using gas chromatography-mass 
spectrometry (GC-MS of ALLO, PA, 5α-DHP, E2, and P4) and by real time PCR (qPCR) for measuring the peripheral 
blood mononuclear cells (PBMC) mRNA levels of key enzymes involved in the neuroactive steroid biosynthetic pathway, 

 
 
 
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3α-HSDII and 5α-RI (the most expressed isoforms in the brain105). The methodology to determine neuroactive steroid levels 
is a ‘state-of-the-art’ technique developed in co-PI Pinna's laboratory (GC-MS), which allows the simultaneous 
determination of neuroactive steroid from a single sample due to high sensitivity (fmolar) and unsurpassed structural 
specificity66,106–108. Comparative qPCR determination of 5α-RI and 3α-HSDII expression65 combined with complementary 
GC-MS measurement of neuroactive steroid has established a correlation among plasma and CSF neuroactive steroid 
biosynthesis in neurological disorders109. Highly significant correlations were also established among CSF and post-mortem 
brain110 and CSF and serum neuroactive steroid levels66,111,68 Importantly, Co-I Pinna’s study of ALLO levels and 5α-RI 
expression in human prefrontal cortex (BA9) is a further demonstration that down-regulation of neuroactive steroids in 
CSF67 and blood112 strongly reflects down-regulation of brain neuroactive steroid levels65. Neuroactive steroid levels will 
be corrected for procedural losses by adding to the samples 1 pmol of deuterium-labeled neuroactive steroid used as internal 
standards107,108. Measuring central mRNAs in peripheral tissues is easy to obtain, reliable, and known to closely reflect 
changes in neuroactive steroid biosynthesis occurring in the brain67,112. The best source of mRNA is represented by isolated 
PBMC, since hemoglobin from erythrocytes can inhibit PCR113. Several neurological/psychiatric clinical studies have 
correlated brain with lymphocytes findings114,115. Various groups confirmed the presence of 5α-DHP and ALLO (GC-MS), 
5α-RI and 3α-HSDII expression (PCR) in lymphocytes116,117. The qPCR methodology is a suitable method to quantify 
mRNAs encoding for neurosteroidogenic enzymes expressed in PBMC and is a powerful tool in combination with GC/MS 
to determine alterations in neuroactive steroid biosynthesis (e.g., 3α-HSDII and 5α-RI, to evaluate altered regulation of 
ALLO biosynthesis). 3α-HSDI and 5α-RII (peripheral isoform) mRNA will also be measured and used as an internal control.  
 
In addition to the specific gene expression assays for neurosteroidogenic enzymes performed by Dr. Pinna, We will also 
perform “whole transcriptome shotgun sequencing” in each sample collected across both conditions, which measures the 
presence and quantity of RNA (related to the entire genome) in a given sample at a given moment. This will be used to test 
hypotheses regarding the effects of the manipulation on expression of genes coding for all neurosteroidogenic enzymes (not 
just those measured by Dr. Pinna using qPCR), GABA-A receptor subunits, and inflammatory cytokines. These analyses 
will also be performed in-house at the University of Illinois.  
 
Neuroactive steroids for HC participants (Aim 3 substudy). 
Neuroactive steroids, mRNA, and genome sequence collection will be identical in the substudy as described above. 
 
Smartphone-Based Assessments 
 
Daily Symptoms Survey (3 minutes each evening throughout the 4-month Study). Each evening throughout the four 
months of the study, participants will complete the following self-report scales. First, the Daily Rating Form (DRF)55, 
capturing hormone-related symptoms listed in the DSM-5118(p5) (depression, hopelessness, worthlessness, anxiety, mood 
swings, rejection sensitivity, anger/irritability, interpersonal conflict, difficulty concentrating, lack of interest, insomnia, 
hypersomnia, overeating, lack of appetite, emotional overwhelm (hyperarousal), feeling out of control, fatigue, and physical 
discomfort). In addition to the DRF, participants will complete the Brief Agitation Measure (BAM)54, a 3-item measure that 
taps into feelings of agitation (e.g., “I felt so stirred up inside that I wanted to scream”; hyperarousal) as well as dimensional 
ratings of the three highest-loading items from the Beck Hopelessness Scale17 (e.g., “The future seems dark to me”). This 
daily survey will provide an integrated rating of the patient’s symptoms across the entire day, consistent with methods used 
in seminal experiments of hormones and mood30,119,120. Medication adherence, stressor exposure19, medication, drug, and 
alcohol use, sleep, pain, illness, and exercise will be recorded; any additional probing to clarify these online responses will 
be take place during the following day’s daily safety call.  
BiAffect App: Passive Tracking Measures of Hyperarousal (Co-I Leow). BiAffect App. Participants will be asked to type 
using a custom built virtual keyboard application on their smartphone that will replace the default keyboard. This keyboard 
will track keyboard dynamics "metadata" but not the actual words. Such metadata may include the start, finish and duration 
of each keyboard session, the time when a key is pressed and the category of the key. Such information can thus be used to 

 
 
 
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compute keystroke dynamics such as the mean typing rate. Metadata will be encrypted and transmitted across a secure 
network to Sage Bionetworks.  
The core technology of the BiAffect App is a custom-built keyboard that replaces the standard native smartphone keyboard 
in order to unobtrusively monitor keyboard dynamics metadata (such as typing speed and rhythm, mistakes in texts, and the 
use of backspace and auto-correct). The app also samples accelerometer displacement (both gross and fine movements of 
the phone). Together, these typing and movement data will measure:  
 
Psychomotor Changes. Higher accelerometer displacement values arise when participants hold their phones less stably, 
consistent with psychomotor agitation; therefore, accelerometer displacement will be used in the present study to measure 
psychomotor agitation. We will also capture and examine quadratic effects of overall physical movement as an index of 
both psychomotor retardation (infrequent, slow movement) and agitation (frequent, quick movement). Consistent with the 
intuitive face validity of directly measuring psychomotor activity using the smartphone accelerometer, two of our team’s 
prior studies using these BiAffect variables demonstrate within-person effects linking accelerometer displacement values 
to concurrent daily reports of disturbed mood121,122. These measures will be further validated through other measures of 
arousal in the study, including daily ratings (e.g., “slowed-down”, “sped up”) and autonomic measures. 
 
Behavioral Reactivity to External Stimuli: Typing speed instability will be calculated as a momentary and daily measure 
of reactivity to phone-based stimuli. Instability metrics will be calculated as the root mean square successive difference 
(rMSSD). This metric evaluates variability in consecutively recorded typing speeds and encapsulates both the size and the 
temporal order of within-subject fluctuations. In a prior study, our team has demonstrated that daily typing instability is able 
to prospectively predict disturbed mood122.  
 
A variety of other relevant indices will be available from the BiAffect Keyboard, including but not limited to the number of 
errors made while typing, diurnal activity patterns and inferred hours of sleep, and amount of phone usage123.  
 
Physiological Arousal: Oura Ring Devices will collect data on the subject’s body temperature, heart rate, interbeat 
interval) of sufficient quality for heart rate (HR) and heart rate variability (HRV) analysis.  
 
Metricwire is a multi-platform (iOS and Android) smartphone app (www.metricwire.com) that has been previously used 
in many health and behavioral studies. Participants who have a compatible mobile device will be emailed a link and given 
a lab-generated email and password to sign up for the study. The app is used for a variety of methods in the literature; here, 
it will be used as an adjunct application to capture accelerometer and movement data from the smartphone’s sensors. This 
is being collected in addition to the movement data from BiAffect since the sampling rate is higher and it can collect data 
even when the participant is not typing. Metricwire data will be collected up until July 31st, 2023. After this date, participants 
will no longer be enrolled into the Metricwire app and no data will be collected.  
 
Smartphone-based assessments for HC participants (Aim 3 substudy). 
HC participants will complete the following as described above: daily symptoms survey, BiAffect app (psychomotor 
changes, behavioral reactivity to external stimuli), Metricwire. HC participants will not receive Oura rings for physiological 
arousal. 
 
Participant Compensation. 
Participants will receive financial compensation (taxable) for participating in this research study based on the percentage 
of total possible study visits and surveys completed.  We collect social security number or Taxpayer Identification 
Number (TIN) to issue compensation and for tax reporting purposes to the United States Internal Revenue Service (IRS).  
 

 
 
 
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If participants complete all study activities and surveys, compensation is $2000 ($150 total for enrollment visits, and up to 
$925 for each of the two medication phases). The specific amount of study pay is based on how many of the study visits 
and surveys are completed as follows:  
• 
Enrollment visit payment is $150 total (split into 2 payments of $75) in cash after each in-person enrollment 
visit, or via digital cash transfer (using private modes in PayPal, Zelle, or Venmo smartphone applications) after 
each virtual enrollment visit. If participants complete any portion of the enrollment visit and are deemed 
ineligible, compensated is still provided for the visit(s) completed. 
• 
Pay for each the two study medication phases will be up to $925, with the two payments sent following each 
phase, mailed within approximately 30 days of completing that phase.  
o Within each medication phase, this $925 is broken down as follows:  
§ 
$250 for completing 100% of surveys (prorated if less than 100% complete) 
§ 
$75 for 100% of check-in calls (prorated if less than 100% complete) 
§ 
$120 for each laboratory visit completed  
• 
Participants will also receive the Oura Ring free of charge and will be allowed to keep the ring following the 
study.  
 
Financial Compensation for HC participants (Aim 3 substudy). 
Participants similarly will receive financial compensation (taxable) for participating in the research substudy based on the 
percentage of total possible study visits and surveys completed.  We collect social security number or Taxpayer 
Identification Number (TIN) to issue compensation and for tax reporting purposes to the United States Internal Revenue 
Service (IRS).  
 
If HC participants complete all study surveys and visits, compensation is $400.   
• 
Enrollment visit payment as $50 in cash after the in-person enrollment visit, or via digital cash transfer (using 
private modes in PayPal, Zelle, or Venmo smartphone applications). If a participant completes the enrollment 
visit and is deemed ineligible, compensation of $50 is still provided. 
• 
Baseline and observational phases: one payment of $350, mailed within approximately 30 days of completing 
the study. Study pay will be prorated based on completion of study surveys and activities.  
o Pay for each laboratory visit will be $50 (5 laboratory visits total)  
o Pay for completion of daily surveys will be $100 (prorated based on percent completed, where 100% 
survey completion = $100).  
 
 
7.0 
Expected Benefits/Risks 
EXPECTED RISKS 
1) Psychological Symptom Assessment. Clinical interviews and self-report assessments contain questions regarding 
sensitive personal information, including severe psychological symptoms such as suicidal and other impulsive behaviors 
(e.g., substance abuse). As a result, participants may become upset or embarrassed when discussing current or past 
distressing life events and behaviors. On the other hand, recent evidence suggests that research questions pertaining to 
potentially distressing topics such as psychiatric symptoms, suicidality, and sexual or physical abuse do not significantly 
increase distress or acute suicide risk in participants who report these issues124,125. The chance of increased distress is 
necessary in order to assess how symptoms change in response to perimenstrual hormone withdrawal (vs. stabilization). 
 
2) Effects of the Ovarian Hormone Stabilization Protocol on Mood/Anxiety and Behavior (for primary experimental 
group; N/A for HC group).  
 

 
 
 
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Mood/Anxiety. As the population being studied in this protocol will have elevated psychiatric symptoms, and as ovarian 
hormone changes are implicated in depression and anxiety symptoms in some females126, it is possible that adverse mood 
reactions may occur (1) during the administration of ovarian hormones in the stabilization condition or (2) naturally during 
the placebo (natural) perimenstrual withdrawal condition. However, because the proposed hormone manipulation mimics 
(i.e., stabilizes) the luteal hormone levels that cycling individuals naturally experience96, the proposed study is not expected 
to pose any greater risk to participants than what they experience in daily life. Furthermore, although the withdrawal from 
the experimental hormone condition could elicit negative mood changes, these are not expected to be any more severe than 
those that would otherwise have arisen from normal perimenstrual hormone withdrawal—that is, we will have simply 
delayed the normal withdrawal process. This argument is supported by the results of the prior K99 trial, where increases in 
symptoms observed during withdrawal from exogenous E2/P4 was less severe than that observed in the natural 
perimenstrual withdrawal condition under placebo.  
 
Suicidality. As detailed above, the population will be recruited to achieve a population of individuals that is simultaneously: 
(1) experiencing recent suicidal ideation, yet (2) at acceptably low risk for suicidal crisis/attempt (such that the 
participant is appropriate for outpatient rather than inpatient care) based on an assessment conducted during the enrollment 
visit. Because ovarian hormone changes are implicated in psychiatric symptoms in some females , and because such 
symptoms may increase suicide risk, it is possible that increases in suicidality may occur (1) during the administration of 
ovarian hormones in stabilization conditions or (2) naturally during the placebo (natural) perimenstrual withdrawal 
condition. However, because the proposed hormone manipulation mimics (i.e., stabilizes, then shifts) the normal 
physiologic range of luteal hormone levels and changes that cycling individuals naturally experience, the proposed study is 
not expected to expose participants to greater risk for suicidality than what they experience in daily life.  As above, this 
argument was supported in the prior K99 trial, where increases in proximal suicide risk observed during withdrawal from 
exogenous E2/P4 was less severe than that observed in the natural perimenstrual withdrawal condition under placebo.  
 
3) Hormone Side Effects (for primary experimental group; N/A for HC group). We do not expect any adverse side effects 
associated with the hormonal manipulations outlined in this protocol following reasons: First, we did not observe any side 
effects occurring significantly more frequently under active hormones than during placebo during the prior K99 trial using 
the same hormonal manipulation. Second, we will be administering the physiologically relevant steroid hormones (estradiol 
and/or progesterone) and not the substituted steroids (such as ethinyl estradiol or norethindrone) present in many oral 
contraceptives, the latter of which have been reported to have potentially more serious side effects127–129. Nevertheless, we 
describe the common side effects of estradiol and progesterone below.  
 
Estradiol. Nausea is the most common side effect of estrogen administration. At conventional hormone doses, this 
complaint seldom interferes with eating, and no weight loss has been reported as a result of nausea. Breast engorgement 
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 extremely small. 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 decreases the risk of endometrial hyperplasia and 
endometrial cancer to equal or below that of females receiving no hormonal treatment, and oral prometrium of 200 mg/day, 
as is present in the proposed work, is sufficient to prevent endometrial hyperplasia130. There is an increase in 
thromboembolism in females receiving non-contraceptive estrogen therapy131–133, but this risk is most evident in obese 
females and in those who smoke (exclusionary factors in the present research).  
 
It may be important to note that there were numerous concerns about increased risk for breast cancer and cardiovascular 
events related to the use of hormone replacement therapy (estrogen plus a progestin) that stemmed from the initial results 
of the Women’s Health Initiative (WHI), which randomized 27,000 postmenopausal females to HRT or placebo.  Critical 

 
 
 
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reviews subsequent to the initial WHI reports showing an increased incidence of non-fatal breast cancer and cardiovascular 
events have argued that the discordant findings between the WHI and the observational studies finding benefit of HRT for 
all cause and cardiovascular mortality reflected problems in the design of the WHI, the most important of which may have 
been the age and condition of the study subjects134.  As the risk of breast cancer, heart disease and metabolic dysregulation 
increase with age; the likelihood of adverse events upon exposure to E2 should also increase with age. Recent reports from 
the North American Menopause Society135 and the Endocrine Society136 confirm that the risk of CVD in the WHI was 
observed in older but not younger females. Additionally, data suggest that the formulation of E2 (conjugated equine 
estrogen) used in the WHI is associated with an increased profile of risk compared with transdermal E2, and the progestin 
used in the WHI (medroxy progesterone acetate) is particularly antagonistic (among progestins) to the beneficial 
cardiovascular effects of E2137.   
 
Given our plans to administer transdermal E2 to females: (1) for only 14 days, (2) no older than 45 years of age, (3) who 
are currently regularly menstruating and medically healthy, and (4) who are at no greater than average risk for CVD, breast 
cancer or VTE, the risk of serious adverse events is exceedingly low. 
 
Progesterone. Progesterone and the synthetic progestins are widely prescribed, with indications including dysfunctional 
uterine bleeding, endometriosis, mastodynia, galactorrhea, and precocious puberty130. In a recent study, a dose of 300 mg 
of oral micronized progesterone was given to a sample of females with premenstrual mood disturbance and was well 
tolerated by this sample138. Side effects reported included lightheadedness and fatigue; these were very mild and caused no 
dropouts.  
 
4) Confidentiality. A breach of confidentiality could indicate to others a participants’ history of mental and behavioral 
health concerns.  
 
5) Venipuncture. Venipuncture (the needle stick during blood draws) may result in syncope (lightheadedness), discomfort, 
nausea, and bruising. There is a very low risk of infection from venipuncture (less than 1 in 1000). 
 
6) Oura Ring. The small flashing light on the oura ring could theoretically increase risk of seizure among those with epilepsy 
(exclusionary in the present study), and could theoretically interfere with pacemakers and other internal electronic devices 
(also exclusionary in the present study).  
 
PROTECTION AGAINST RISKS 
 
Protections Against Risks Associated with Psychological Assessment. The PI (Dr. Eisenlohr-Moul) is a licensed clinical 
psychologist with ten years of experience in risk assessment and empirically-supported treatment of chronic suicidality (e.g., 
Dialectical Behavior Therapy). The PI has personally conducted roughly ten studies assessing daily emotional symptoms, 
three of which included measures of daily suicidal ideation and depressive symptoms in females with borderline personality 
disorder or chronic suicidality. Based on these experiences and the available literature, participants are unlikely to become 
acutely upset in response to the psychological assessments conducted in the present study. If participants do become upset 
during an assessment, they will be reminded of their right to discontinue participation, suicidal ideation will be assessed and 
managed using the University of Washington Risk Assessment and Management Procedure (UWRAP) as recommended by 
NIMH, and appropriate referrals will be made. Participants will be reminded each day that they do not have to answer any 
question that they do not want to answer.  
 
1) Protections Against Risks Associated with E2 and P4 Administration (for primary experimental group; N/A for HC 
group). Hormone-related risks are minimized in our protocol by the following: 1) including only young, medically healthy 
premenopausal females; 2) the use of a very short-term administration of estradiol in combination with oral micronized 
progesterone will virtually prevent the chance of endometrial hyperplasia; 3) exposure to E2 for less than 3-5 years has not 
been shown to be associated with an increased risk of breast cancer133,139;  4) the exclusion of individuals with a family 

 
 
 
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history indicative of increased breast cancer risk or thromboembolic disorders; 5) the frequent assessment of side effects at 
every phone call; 6) provision of a drug information sheet with the instruction to call the study doctors immediately if any 
of the following symptoms are experienced: abnormal or prolonged vaginal bleeding, severe headaches, changes in vision 
or speech, dizziness or faintness, pains in calves or chest, or sudden shortness of breath; 7) banking a prescription for a 
back-up, additional 3 pills with the investigational drug service for each participant in an active condition will prevent 
participants from missing study medication doses on days they are in the lab should they forget to bring their study 
medication with them to the lab visit; 8) the development of a safety monitoring plan (see below); and 9) monitoring for 
adverse events by a Data and Safety Monitoring Committee.   
 
2) Effects of the Ovarian Hormone Stabilization Protocol on Mood and Suicidality (for primary experimental group; 
N/A for HC group).  
 
The following measures will be taken to mitigate any negative impact of the experiment on mood or suicidality: 
 
Depressive symptoms and suicidality will be monitored during a daily evening telephone call during each condition and 
will also be measured and monitored via smartphone surveys. These daily calls will be carried out by trained research 
assistants, and the PI will be on-call during these calls. In the case of (A) severe mood changes (defined below) or (B) 
imminent risk for suicide attempt (requiring inpatient hospitalization), participation will be discontinued and the 
individual will be referred for an appropriate level of psychiatric care. Protocols for assessing severity of mood symptom 
changes, evaluating suicide risk, and discontinuation and referral are described below.  
 
Protocol for Assessing Emergence of Severe Mood Changes. Because they will be recruited for recent suicidality, 
participants are expected to show elevated baseline levels of mood symptoms. Therefore, the emergence of severe mood 
symptoms will be defined as three days of daily depression scores (defined as depression, hopelessness, and 
worthlessness/guilt on the daily symptom record as defined in the research strategy) that are 1) severe or extreme in nature 
(i.e., above “moderate” on the response scale), 2) at least 50% increased over their self-reported trait levels of depression 
(as defined by mean scores on these items across the baseline month), 3) maintained for three consecutive days, and 4) a 
clinical interview must reveal that this worsening of mood is significantly impacting functioning in relationships or ability 
to engage with work or school. Should such an acute, impairment-related worsening of depression persist for three days, 
the Protocol for Discontinuation and Referral described below will be followed to remove the participant from the study 
and ensure proper engagement with their mental health provider at the appropriate level of psychiatric care.  
 
Protocol for Assessing the Emergence of Imminent Risk of Suicide Attempt. Participants will be recruited to be at 
acceptably low current risk for suicide attempt given historical factors as described above.  Daily telephone assessments 
will end with a structured screening for current suicide risk, including suicidal ideation, planning, and intent using the 
University of Washington Risk Assessment and Management Protocol (or UWRAP; also known as the Linehan Risk 
Assessment Procedure or LRAP)125. The UWRAP is the suicide risk assessment and protocol recommended by the NIMH 
for use in RCTs when participants are not in an ongoing treatment relationship with the assessor. The purpose of this daily 
UWRAP screening is to determine (1) whether the participant’s current risk of a suicide attempt has elevated to 
imminent risk for suicide attempt according to established guidelines125 and to (2) intervene as appropriate to reduce 
any emergent imminent suicide risk. The UWRAP allows the screener to determine the participant’s current risk status 
for a suicide attempt, and provides concrete, structured guidelines for appropriate actions at each level of risk specifically 
in the context of a research study. UWRAP protocols will be followed closely. The UWRAP also includes a template for 
documenting specific risk classifications based on the participant’s responses and the screener’s actions. A UWRAP note 
will be written for each daily contact where nonzero suicidality is reported; notes in which any suicidal ideation is reported 
will be immediately reviewed with the PI, and will be discussed at weekly and monthly team meetings. Should an 
individual’s responses to the UWRAP indicate imminent current risk for suicide attempt according to established standards, 
the Protocol for Discontinuation and Referral described below will be followed to remove the participant from the study 
and to ensure the appropriate level of psychiatric care. 

 
 
 
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More about the use of the UWRAP to manage suicide risk both virtually and in-person. The UWRAP is included as 
an Appendix to this Document. The UWRAP protocol has been utilized to assess and manage suicide risk in a variety of 
clinical trials sponsored by NIMH, and was developed by Marsha Linehan, the creator of Dialectical Behavior Therapy 
(the only evidence-based treatment for chronic self-injury). It is designed to be carried out either in person or remotely 
(on a phone or video call). The protocol includes assessment of risk, and a variety of individualized options for facilitating 
the participant’s use of coping skills to help the participant avoid having to be hospitalized (either voluntarily or via an 
emergency services call). Ultimately, if the participant’s suicide risk cannot be managed appropriately with the brief mood 
improvement and other risk management protocols included in the UWRAP, they are referred to the emergency 
department. If the participant is being evaluated in-person for a visit and needs immediate referral to the emergency 
department, we follow existing clinical protocols for hospitalization in the UIC Department of Psychiatry, which includes 
calling a security official at UIC who is trained to facilitate a safe walking of the participant from the psychiatry 
department offices to the emergency department. If the participant is being evaluated over the phone or videoconference 
(as in the daily phone calls during active conditions, the virtual enrollment option, or the virtual debriefing option), this 
will occur by either recruiting the help of a family member (often the emergency contact provided at enrollment) or by 
contacting 911 to facilitate an emergency safety check at the participant’s location.  
 
If a participant must be referred for emergency treatment (via emergency services/911 safety check, or walking the patient 
to the emergency room) at any point in the study, the blind will be broken for the participant and their study participation 
will be discontinued. If it is revealed that the participant was currently in the placebo condition, they will simply be 
referred to their mental health provider (or, as necessary, to the emergency department) for treatment. However, if it is 
revealed that the participant was currently in an active hormone stabilization condition, the supervising physician (Dr. 
Wagner) will oversee a gradual tapering of the hormonal intervention and the patient will be referred to their mental 
health provider (or, as necessary, the emergency department) for treatment.  
 
Protocol for Discontinuation and Referral. The protocol for discontinuation and referral is as follows. First, if the patient 
needs immediate inpatient evaluation or care, we will follow the protocol outlined in the UWRAP (see Appendix) to 
facilitate referral to emergency department (either via coordination with the emergency contact, by involving emergency 
services to perform a safety check at the participant’s home, or by recruiting the help of UIC security to escort the patient 
from the outpatient psychiatry offices to the UIC emergency department. While the patient’s immediate need for care is 
being attended to, the study staff will contact the investigational drug pharmacy and the blind will be broken for the study 
staff, the PI, and the participant. The participant will be notified that their study participation is discontinued and provided 
with instructions for either tapering medication (if active medication was being taken) or to follow up as needed with 
outpatient providers. If it is revealed that the participant was currently in an active hormone stabilization condition, Dr. 
Wagner-Schuman will oversee (or communicate with the participant’s physician to recommend) a gradual tapering of the 
hormonal intervention.  
 
3) Hormone Side Effects (for primary experimental group; N/A for HC group).  As discussed above, we do not expect 
any adverse side effects associated with the hormonal manipulations outlined in this protocol. However, any patient 
experiencing clinically significant side effects (e.g., nausea, hypertension, vomiting) deemed by Dr. Wagner-Schuman to 
be due to the hormonal manipulation will be discontinued from the study. The blind will be broken for that participant and 
their study participation will be discontinued. If it is revealed that the participant was currently completing the placebo 
condition, they will be informed of this and referred for outpatient medical care. However, if it is revealed that the participant 
was currently completing the active hormone stabilization condition, Dr. Wagner-Schuman will oversee a gradual tapering 
of the hormonal intervention and the patient will be referred for outpatient medical care.  
 
4) Confidentiality.  
 
For each of the above measurements, participants will be identified by study ID number on all research documents, 

 
 
 
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electronic data files, and collected specimens. All paper data will be stored in locked cabinets inside locked offices, and 
electronic data will be stored only on password-protected file servers only accessible from computers in the Psychiatry 
Department. The file linking the study ID number to identifying information will be kept separate from any study data on a 
UIC Psychiatry password protected drive. Only study personnel will have access to these data. 
Biaffect Data Confidentiality. Sage Bionetworks, the company who stores the behavioral and EMA response 
data collected by Biaffect and other Apple HealthKit apps, will provide a business associate agreement to ensure 
continued HIPAA-compliant handling of data. They will not collect information about location or any other 
identifying information. Nonetheless, to maintain strict confidentiality of the BiAffect Data linked to this study, the 
research staff will create an anonymous email address for each study participant and create a linked BiAffect 
account for the study; this email address (which defines the account) will be linked to the participant’s study ID 
for the trial only on a password-protected spreadsheet in the UIC server. Therefore, Sage Bionetworks will 
process only deidentified data and will not be able to link study data to any individual.   
MetricWire Data Confidentiality. Metricwire is the company that will collect the phone sensor data (movement, 
acceleration) from participant smartphones as an augment to the use of the Biaffect app. A study-generated 
email address and password will be used to register each participant’s phone to the Metricwire App, and 
collection of IP address will be turned off; these measures eliminate any identifiers from being coded by the 
Metricwire app. However, Metricwire is HIPAA-compliant and a Business Associate’s Agreement is on file with 
UIC through our laboratory for data collection and storage through this company. 
Oura Ring Data Confidentiality. No identifiable data will be collected from the Oura Ring. The serial number of 
rings given to participants will not be recorded. A premade study email will be used to create an Oura Ring App 
account. Therefore, no identifiable information will be collected related to the Oura App or Ring. 
5) Venipuncture. The use of a trained phlebotomist who is fully trained in safety protocols will minimize any risk associated 
with venipuncture. 
 
6) Oura Ring. We will exclude participants with epilepsy, pacemakers, and other internal electronic devices to reduce any 
possible/theoretical risks associated with use of the Oura Ring. 
 
 
POTENTIAL BENEFITS OF THE PROPOSED RESEARCH TO HUMAN PARTICIPANTS AND OTHERS 
 
This is a mechanistic study to determine the role of ovarian hormone changes in constructs that are relevant to suicidality. 
There is no direct benefit to the participants other than the benefit of knowing that they are contributing to research on the 
causes of suicidality.   
 
8.0 
Quality Control and Quality Assurance 
 
The project manager Sabina Raja will check over all databases in Qualtrics and generated from tasks for quality 
assurance monthly. Any missing data or data anomalies will be communicated to the PI for clarification/resolution.  
The investigational site will provide direct access to all trial related source data/documents and reports for the purpose of 
monitoring and auditing by NIMH, and inspection by local and regulatory authorities. 

 
 
 
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9.0 
Data and Safety Monitoring 
 
Digital Data Safety. All surveys and tasks, including those that request information about demographics which could 
theoretically be used to identify an individual, will be completed digitally and immediately stored on UIC’s secure, 
HIPAA compliant server in a password-protected server that can be accessed only by the PI and her designees (i.e., 
postdoctoral fellows or project manager affiliated with the study). All additional biological assay data will similarly be 
stored on UIC’s secure server.  
 
With respect to data collected using the BiAffect App, all data will be captured digitally and stored on the HIPAA-
compliant server at Sage Bionetworks, where all data from apps in the Apple HealthKit (and associated Android app 
counterparts) are hosted. As noted in the Protection of Human Subjects section, we will generate an anonymous email 
address and password to correspond to each study identification number, and this email will serve as the link to match 
deidentified participant BiAffect data on the Sage Bionetworks server with their local study data. Given that this unique 
email “ID” will be used to identify participants, no PHI will be collected using the BiAffect App, since there are no survey 
responses that contain PHI.  
 
Similarly, with respect to data collected using the Metricwire App, all data will be captured digitally and stored on the 
HIPAA-compliant server at Metricwire. As noted in the Protection of Human Subjects section, we will generate an 
anonymous email address and password to correspond to each study identification number, and this email will serve as the 
link to match deidentified participant Metricwire data on the Metricwire server with their local study data. Given that this 
unique email “ID” will be used to identify participants, no PHI will be collected using the Metricwire App. Metricwire 
data will be collected up until July 31st, 2023. After this date, participants will no longer be enrolled into the Metricwire 
app and no data will be collected. 
 
As noted above, the Oura Ring app will not record any identifying information. 
 
Paper Document Safety. Paper documents containing PHI include those used to register patients in the UIC vendor 
system to be paid for their participation, which include SSN, address, and name, as well as the SCID-5, which includes 
date of birth and other demographic information. These documents will be digitized (see notes regarding digital data 
protections above) and shredded.  Any paper documents that have not yet been digitized (e.g., SCID-5 interviews awaiting 
data entry) will be stored in a locked file drawer in the PI’s locked office until they are digitized (see above). Only the PI 
and the project manager will have keys to this file drawer. Once digitized, all paper documents (except consent forms) 
will be immediately shredded per UIC protocol for protected health information. Consent forms will be kept in a locked 
file drawer in the PI’s office as per UIC ethics protocol.  
 
Data Monitoring and Report Generation. Each month, the project manager will generate a report from the database, 
including information about participant flow, demographics, and data completeness. This report will be sent to the PI and 
all Co-Is for review. Reports will also include detailed (but deidentified information about any Adverse Events (AEs) 
(e.g., suicide risk elevation) and follow-up procedures.  
 
Monitoring and Reporting of Mood Changes, Suicide Risk, and Adverse Events. The PI will supervise the clinical 
postdoctoral fellow as well as the project manager in monitoring changes in suicidal risk and mood symptoms obtained 
from laboratory visits, daily online surveys, and daily telephone risk screenings. Research staff will be specifically trained 
to identify significant changes in mood or suicidality in various assessments (see protocol in Protection of Human 
Subjects section), which will be reported immediately to the PI and further monitored for the duration and impairment 
criteria described in the Protection of Human Subjects Section (i.e., symptoms must interfere with either relationships or 
occupational health, and must remain elevated for three days). As described above, participants who experience 
significant deterioration in mood or who are judged to be at imminent suicide attempt risk will be discontinued from the 

 
 
 
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protocol and referred for an appropriate level of psychiatric care.  
 
In addition, specific events to be reported to the data safety and monitoring board (DSMB) will be defined by the DSMB 
at their first meeting prior to the start of the research. 
  
DATA SAFETY AND MONITORING BOARD  
 
Similar to the PI’s ongoing clinical trials, the safety of the study will be monitored by a specially-convened Data and 
Safety Monitoring Board at UIC. This will ensure the independent oversight of issues related to safety/adverse events. The 
DSMB will monitor the study for AEs, including a significant worsening of symptom severity, imminent suicide risk, 
substantial functional impairment, and physical effects (e.g., migraine, thromboembolic events). The DSMB will specify 
the tables and data it wishes to have presented, including moderate to severe side effects and all serious adverse events. 
DSMB review of this study will occur every six months. At these meetings the DSMB will also review data from 
participants who have been discontinued or who have dropped out of the study, and reasons for discontinuation. The 
DSMB will identify if any study procedures should be altered or stopped in the event of an indication of harm to 
participants attributable to the study interventions. The DSMB will evaluate issues of participant safety as well as the 
adequacy and integrity of accumulating data, review of enrollment data, and making recommendations regarding safety. 
 
Members of the DSMB:  
 
Karen Bernstein, MD, MPH (CHAIR), Associate Professor and Director of Division of Adolescent Medicine, expertise 
with hormonal manipulation in vulnerable populations. 
 
Jamie Haley Kienast, LCSW, Social worker at UIC with professional competence in female mental health and suicide 
risk assessment among diverse populations such as those seen at UIC. 
 
Deepika Laddu, PhD, Assistant Professor in Department of Physical Therapy, expertise in clinical research with female 
populations.  
 
 
Definitions of Adverse Events 
 
Adverse event means any untoward medical occurrence associated with the use of an intervention in humans, whether or 
not considered intervention-related. 
 
AE SEVERITY 
 
• 
Mild – Events require minimal or no treatment and do not interfere with the participant’s daily 
activities.  
• 
Moderate – Events result in a low level of inconvenience or concern with the therapeutic measures. 
Moderate events may cause some interference with functioning.  
• 
Severe – Events interrupt a participant’s usual daily activity and may require systemic drug therapy 
or other treatment. Severe events are usually potentially life-threatening or incapacitating. 
• 
Serious adverse event or serious suspected adverse reaction. An AE or suspected adverse reaction is 
considered "serious" if, in the view of either the investigator or sponsor, it results in any of the following 
outcomes: death, a life-threatening adverse event, inpatient hospitalization or prolongation of existing 
hospitalization, or a persistent or significant incapacity. Important medical events that may not result in death, 
be life-threatening, or require hospitalization may be considered serious when, based upon appropriate 

 
 
 
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medical judgment, they may jeopardize the patient or subject and may require medical intervention to prevent 
one of the outcomes listed in this definition.  
 
o *Notable examples of serious adverse events in the present study include development of a venous 
thromboembolism or suicide attempt.  
 
AE STUDY-RELATEDNESS 
For all AEs, Dr. Eisenlohr-Moul will urgently collaborate with Dr. Wagner to determine the AE’s causality based on 
temporal relationship and clinical judgment. The degree of certainty about causality will be graded using the categories 
below.  
Definitely Related – There is clear evidence to suggest a causal relationship, and other possible contributing factors can 
be ruled out. The clinical event occurs in a plausible time relationship to drug administration and cannot be explained by 
concurrent disease or other drugs or chemicals. The response to withdrawal of the drug (dechallenge) should be clinically 
plausible. The event must be pharmacologically or phenomenologically definitive, with use of a satisfactory rechallenge 
procedure if necessary.  
Probably Related – There is evidence to suggest a causal relationship, and the influence of other factors is 
unlikely. The clinical event occurs within a reasonable time after administration of the drug, is unlikely to be 
attributed to concurrent disease or other drugs or chemicals, and follows a clinically reasonable response on 
withdrawal (dechallenge). Rechallenge information is not required to fulfill this definition.  
 
Possibly Related – There is some evidence to suggest a causal relationship (e.g., the event occurred within a reasonable 
time after administration of the trial medication). However, other factors may have contributed to the event (e.g., the 
participant’s clinical condition, other concomitant events). Although an AE may rate only as “possibly related” soon after 
discovery, it can be flagged as requiring more information and later be upgraded to “probably related” or “definitely 
related,” as appropriate. 
 
Unlikely to be related – A clinical event whose temporal relationship to drug administration makes a causal relationship 
improbable (e.g., the event did not occur within a reasonable time after administration of the trial medication) and in 
which other drugs or chemicals or underlying disease provides plausible explanations (e.g., the participant’s clinical 
condition, other concomitant treatments). 
 
Not Related – The AE is completely independent of study drug administration, and/or evidence exists that the event is 
definitely related to another etiology. There must be an alternative, definitive etiology documented by Dr. Wagner or Dr. 
Eisenlohr-Moul. 
 
AE EXPECTEDNESS 
Dr. Eisenlohr-Moul will collaborate with Dr. Wagner to make determinations about whether an AE is expected or 
unexpected. An AE will be considered unexpected if the nature, severity, or frequency of the event is not consistent with 
the risk information previously described for the study agent. 
 
10.0 Data Analysis and Statistical Considerations 
 
ANALYTIC STEPS 
 

 
 
 
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For each outcome, we will use model fit indices to select the best-fitting hierarchical model before proceeding 
with analyses. Based on our prior study (K99MH109667) with a similar experimental design and similar data 
collection structure for daily surveys and lab visits, we expect that the data structure for primary analyses will 
be observations nested within participants (two-level regression model). In our prior models, inclusion of a third 
level of nesting (i.e., observations nested within condition nested within individuals) did not improve model fit 
and did not substantively alter hypothesis tests.  Based on the prior study, we also expect that a combination of a 
random intercept for participant and an autoregressive (day-1) within-person error structure will be included 
based on model fit.  
 
Testing Experimental Effects: Aim 1 and 2 require that we first evaluate the impact of the experiment on each 
of the 3 primary suicide outcomes and 9 (RDoC, neurosteroid) mediators described above. Therefore, we will 
first examine effects of the within-person hormone stabilization experiment on repeated measures outcomes 
across the menstrual cycle.   
1. For daily outcomes, we will utilize multilevel models (with 52 days—26 per condition--nested 
within each individual; two-level models) to predict each repeated measures outcome from 
covariates (daily pain, illness, PRN medication use) and the interaction between (1) experimental 
condition (a dichotomous variable coded as 0 for natural hormone withdrawal condition and 1 for 
hormone stabilization condition) and (2) condition phase, a categorical (contrast) variable coded 
as follows: midluteal baseline phase, coded as days +1 to +7 after the positive ovulation test, 
perimenstrual experimental phase, coded as days +11 to +17 after the positive ovulation test, and 
the medication withdrawal phase, coded as days +22 to +26 after positive ovulation test (see Figure 
6 in grant for study design and timeline). Number of daily observations per person. For the 
primary contrast between the midluteal and perimenstrual experimental phases, there are 28 
observations per person—14 per condition (7 midluteal, 7 perimenstrual). Based on our preliminary 
data from the K99 trial (identical experimental manipulation), we anticipate receiving an average of 
24 observations per participant. 
 
2. For laboratory visit outcomes, we will again utilize multilevel models (with 10 labs per person—5 
per condition—nested within individuals; two-level models). A two-level model will predict each 
repeated measures lab outcome from covariates (daily pain, illness, PRN medication use) and the 
interaction between (1) experimental condition (a dichotomous variable coded as 0 for natural 
hormone withdrawal condition and 1 for hormone stabilization condition) and (2) condition phase, 
a categorical (contrast) variable coded as follows: midluteal baseline visit, perimenstrual 
experimental visit, and the medication withdrawal visit. Number of lab observations per person. 
For the primary contrast between the midluteal and perimenstrual experimental phases, there are 4 
observations per person—2 per condition. Based on our preliminary data from the K99 trial 
(identical experimental manipulation), we anticipate very minimal missing laboratory data (see grant 
for details) but have estimated that on average each completer participant will miss .25 visits. 
 
3. Hypothesized Pattern of Effects for Daily and Lab outcomes: A significant interaction between 
experimental condition and condition phase is hypothesized for all twelve specified outcomes such 
that, in the natural withdrawal condition we expect deleterious changes from the midluteal phase to 
the premenstrual and menstrual phases (due to corresponding hormone withdrawal), but in the 
hormone stabilization condition we expect no deleterious changes (or less severe changes) from 
the midluteal to the premenstrual and menstrual phases (since hormone withdrawal is prevented).  

 
 
 
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Testing Associations Between Mediators and Suicide Outcomes: Aim 1 and 2 next require that we 
evaluate the day-to-day or week-to-week associations between single mediators (listed above) and suicide 
outcome variables on the same day or visit. This will be carried out in similar multilevel models as above, 
predicting each of the three primary suicide outcomes from person-centered values for each of the 9 mediators 
(listed above).  
 
Testing Interactive Effects of Hopelessness and Arousal on Suicide Outcomes: Interactions among 
hopelessness and arousal will be examined as predictors of suicide outcomes and as mediators of the 
experimental effect. In order to test this, we examine day-to-day interactive effects of daily person-centered 
hopelessness (noted above) and daily person-centered arousal indices (noted above) predicting same-daily 
suicide outcomes (noted above). We predict that higher-than-usual hopelessness on a given day will predict 
greater same-day risk on suicide outcomes, but only when arousal is also higher than usual for that individual.  
 
4. Evaluating Mediation Where Appropriate. Finally, each aim indicates that mediation will be explored as 
appropriate. Only when analyses indicate significant effects of the experiment on both the suicide outcome and 
any mediator, as well as significant associations between the person-centered variable (or interaction variable) 
and the suicide outcome, we will utilize methods provided by Bauer, Preacher, and Gil (2006) to construct a 
confidence interval for the level 1 indirect effect of experimental condition (condition X phase) on the suicide 
outcome via the mediator. 
 
Testing for normative cyclical change in the HC group. Aim 3 seeks to characterize how gene expression 
for GABAAR subunits varies across pre-specified menstrual cycle phase in response to hormonal change. In 
N=40 HC participants, we will run multilevel growth models to predict subunit gene expression (one model per 
gene) from specific cycle phase. We will test all pairwise comparisons between midluteal, perimenstrual, and 
midfollicular phases, controlling for ALLO level, to capture high P4 and ALLO states (midluteal), P4 and ALLO 
withdrawal (perimenstrual), and low P4 and ALLO (midfollicular). 
Testing for group differences between participants with past-month SI and HC. We hypothesize that clinical 
group moderates the effect of cycle phase on GABAAR composition, such that the primary experimental group shows higher 
slope of midluteal-to-perimenstrual change in a4 and d subunits, and lower g2 subunit replacement of d subunit expression 
than the HC group. In one model of N=80 total participants, we will test the interaction of group and cycle phase on gene 
expression outcomes. We also hypothesize a correlation with symptoms, such that increases in a4 and d subunit expression 
will be associated with increases in individuals with perimenstrual increases in SI. We also predict that even in the HC 
group, higher-than-average a4 or d subunit expression will be associated with higher-than-average negative affect (e.g., 
depressed mood, anxiety, irritability, or perceived stress). We predict that higher g2 expression will be associated with lower 
negative affect and perceived stress. We will calculate a person-mean for each subunit’s expression (mean level across 5 
visits) and a deviation score at each visit (visit level minus person mean). We will test overall correlations (e.g., do 
participants with higher mean a4 expression have higher negative affect?) and interaction with cycle phase (e.g., does a 
perimenstrual increase in a4 expression compared to person-mean correlate with an increase in negative affect?). To further 
refine hypotheses, we will generate credible intervals using Bayesian methods in the context of multilevel modeling.  
 
 
POWER ANALYSIS 

 
 
 
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80 completers achieves 80% power to detect conventionally small-to-medium sized effects that, in our 
estimation, are clinically meaningful (i.e., f2 = .10, which would mean that the interaction of condition and phase 
had uniquely accounted for 10% of the variance in the outcome in the perimenstrual window; Selya et al., 
2012, “A Practical Guide to Calculating Cohen’s f2, a Measure of Local Effect Size”). We posit that if hormone 
withdrawal (vs. experimental stabilization) does not account for at least 10% of the variance in the outcome 
during this critical risky window of the menstrual cycle, then this line of research, which involves significant 
participant burden, is not worth pursuing further. 
 
11.0 Regulatory Requirements 
11.1 Informed Consent  
 
During the SCREENING PROCESS, the screening website and the research staff (or PI) will obtain digital or verbal 
consent from each participant (see online screener and phone screener, attached). Potential participants will be 
contacted by phone to assess their interest in participating; although we will note in the phone script that we are 
affiliated with UIC, we will also explain that they are under no obligation to participate and that their decision to 
participate has no influence on their clinical care at UIC (if applicable).  
 
In addition, at the ENROLLMENT VISIT, trained members of the research team will obtain full written or digitally-
signed informed consent from the participant using the following documented process: 
 
 
Interested participants will visit the laboratory in-person or have virtual online sessions for their enrollment session 
where they will each read the consent form. 
 
 
Research personnel will then ask if the participant has any questions they would like answered. 
 
The participant will sign the consent form, witnessed by research personnel. The original consent for will be 
maintained in the participant's file, along with documentation that the participant's questions were answered by the 
informational sheet or that research personnel gave them an opportunity to ask questions. These documents will be 
secured in Dr. Eisenlohr-Moul's locked laboratory. A copy of the consent form will be provided to the participant. 
For participants who completed virtual enrollment visits during the covid-19 pandemic, their consent will first be 
obtained through a secure online survey and then be re-assessed during their welcome visit, where they will provide 
written consent and given a copy of the consent form. 
11.2 Subject Confidentiality  
 
Participant confidentiality is strictly held in trust by the participating investigators, their staff, and the sponsor(s) and their 
agents. This confidentiality is extended to cover testing of biological samples in addition to the clinical information 
relating to participants. Therefore, the study protocol, documentation, data, and all other information generated will be 
held in strict confidence. No information concerning the study or the data will be released to any unauthorized third party. 
 
Study participant research data, which is for purposes of statistical analysis and scientific reporting, will be transmitted to 
and stored on protected servers at UIC. Identifying information will be linked to ID number only in one separate 
password-protected file on the UIC server. These ID numbers will not contain any identifying information (e.g., initials, 

 
 
 
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birthdates, SSN) and the file linking the study ID number to identifying information will be kept in a password-protected 
file, separate from any study data on a UIC Psychiatry password protected drive.  De-identified, coded data may be 
transmitted by hand or scanned and transmitted via email among the research team.  
 
Post-Study Disposition of identifiable data or human biological materials. Once data collection is completed and data are 
cleaned for analysis (within 1 year after the completion of data collection), data will be de-identified and the dataset 
including any identifiers will be destroyed. Recordings of study interviews will be deleted immediately following weekly 
review by the PI.  Biological samples will be destroyed according to OSHA procedures once assay is completed. 
 
Certificate of Confidentiality 
To further protect the privacy of study participants, a Certificate of Confidentiality will be obtained from the NIH. This 
certificate protects identifiable research information from forced disclosure. It allows the investigator and others who have 
access to research records to refuse to disclose identifying information on research participation in any civil, criminal, 
administrative, legislative, or other proceeding, whether at the federal, state, or local level. By protecting researchers and 
institutions from being compelled to disclose information that would identify research participants, Certificates of 
Confidentiality help achieve the research objectives and promote participation in studies by helping assure confidentiality 
and privacy to participants. 
11.3 Unanticipated Problems 
 
Definition of Unanticipated Problems (UPs) 
For the purposes of this study, we will consider unanticipated problems involving risks to participants or others to 
include, in general, any incident, experience, or outcome that meets all of the following criteria:  
• 
Unexpected in terms of nature, severity, or frequency given (a) the research procedures that are 
described in the protocol-related documents, such as the IRB-approved research protocol and informed 
consent document; and (b) the characteristics of the participant population being studied;  
• 
Related or possibly related to participation in the research (“possibly related” means there is a 
reasonable possibility that the incident, experience, or outcome may have been caused by the 
procedures involved in the research); and  
• 
Suggests that the research places participants or others at a greater risk of harm (including physical, 
psychological, economic, or social harm) than was previously known or recognized.  
 
Reporting of Unanticipated Problems 
 
Incidents or events that meet criteria for UPs require the creation and completion of an UP report form. The PI will 
report UPs to the IRB and to the study sponsor. The UP report will include the following information:  
• 
Protocol identifying information: protocol title and number, PI’s name, and the IRB project number; 
• 
A detailed description of the event, incident, experience, or outcome; 
• 
An explanation of the basis for determining that the event, incident, experience, or outcome represents 
an UP; 
• 
A description of any changes to the protocol or other corrective actions that have been taken or are 
proposed in response to the UP. 
 

 
 
 
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To satisfy the requirement for prompt reporting, UPs will be reported using the following timeline:  
• 
All UPs will be reported to the IRB, the DSMB, and to the study sponsor within 1 week of the 
investigator becoming aware of the event. 
 

 
 
 
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Canetto SS, Sakinofsky I. The gender paradox in suicide. Suicide {&. 
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Kann L. Youth Risk Behavior Surveillance — United States, 2015. MMWR Surveill Summ. 
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Usall J, Pinto-Meza A, Fernández A, et al. Suicide ideation across reproductive life cycle of 
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Baca-Garc ’ıa E, Diaz-Sastre C, Ceverino A, Saiz-Ruiz J, Diaz FJ, de Leon J. Association 
between the menses and suicide attempts: a replication study. Psychosom Med. 
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Baca-Garc ’ıa E, D ıaz-Sastre C, de Leon J, Saiz-Ruiz J. The relationship between menstrual 
cycle phases and suicide attempts. Psychosom Med. 2015;62(1):50-60. 
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Baca-Garcia E, Diaz-Sastre C, Ceverino A, et al. Suicide attempts among women during low 
estradiol/low progesterone states. J Psychiatr Res. 2010;44(4):209-214. 
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SAUNDERS KEA, HAWTON K. Suicidal behaviour and the menstrual cycle. Psychol Med. 
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Beckley EH, Finn DA. Inhibition of progesterone metabolism mimics the effect of progesterone 
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10.  Gallo MA, Smith SS. Progesterone withdrawal decreases latency to and increases duration of 
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12.  Eisenlohr-Moul T, Prinstein M, Rubinow D, et al. S104. Ovarian Steroid Withdrawal Underlies 
Perimenstrual Worsening of Suicidality: Evidence From a Crossover Steroid Stabilization Trial. 
Biol Psychiatry. 2018;83(9, Supplement):S387. doi:10.1016/j.biopsych.2018.02.995 

 
 
 
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