Clinical trial NCT01811225
Conditions: Nicotine Dependence Contraception
Sponsor: University of Minnesota
Start date: 2012-12-01; Completion date: 2017-06-01
Enrollment: 95 participants; Allocation: RANDOMIZED; Intervention model: CROSSOVER

Brief summary:
The investigators aim to (1) examine the association between levels of progesterone (Prog), allopregnanolone (Allo), and the estradiol/progesterone (E2/P) ratios with smoking-related symptomatology during ad libitum smoking and (2) determine the association between Prog, Allo, and E2/P with the changes in smoking-related symptomatology and response to nicotine following overnight abstinence

Detailed description:
The sample population will consist of oral contraceptive users, both "low" and "high" dose levels of exogenous progesterone with a consistent dose of exogenous estrogen. Participants will complete data collection procedures including providing saliva (cortisol to measure stress), urine (cotinine and 3-Hydroxycotinine to measure nicotine exposure), and blood (progesterone, allopregnanolone, and estradiol) samples, as well as ecological momentary assessments (EMA) daily for seven-days. Participants will also complete a 3-hour smoking topography lab session after overnight abstinence and a 4.5-hour nicotine nasal spray lab session after a 14-hour abstinence. These sessions will contain a nicotine challenge via smoking a cigarette or nasal spray with timed-series physiological, subjective, and behavioral responses.

Arms: arm 1: Females, 18-35 years old, non-pregnant, using oral contraceptive for at least the last 3 months before enrollment will be given Tri-Sprintec and generic Prometrium.

Participants in this arm will begin with the "low" dose progesterone, which is seven days of placebo Tri-sprintec + placebo generic Prometrium twice daily (7AM and 7PM). Then participants in this arm will move to the "high" dose progesterone, which is seven days of placebo Tri-Sprintec + 200mg of generic Prometrium twice daily (7AM and 7PM). arm 2: Females, 18-35 years old, non-pregnant, using oral contraceptive for at least the last 3 months before enrollment will be given Tri-Sprintec and generic Prometrium.

Participants in this arm will begin with the "high" progesterone dose, which is seven days of placebo Tri-Sprintec + 200mg of generic Prometrium twice daily (7AM and 7PM). Then participants in this arm will move to the "low" dose progesterone, which is seven days of placebo Tri-sprintec + placebo generic Prometrium twice daily (7AM and 7PM).
Interventions: intervention 1: Low-dose Progesterone intervention 2: High-dose Progesterone
Intervention descriptions: intervention 1: The "low" progesterone dose is seven days of placebo Tri-sprintec + placebo generic Prometrium twice a day (7AM and 7PM). intervention 2: The "high" progesterone dose is seven days of placebo Tri-Sprintec + 200mg of generic Prometrium twice a day (7AM and 7PM).

Protocol documents:
Prot_SAP_000.pdf: 
Page 1 of 19 
 
 
 
 
 
 
 
 
 
 
 
Smoking, Sex Hormones and Pregnancy  
Study Protocol 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Version: 12.0 
Version Date: 6.2.2016 
 
 


Page 2 of 19 
SIGNIFICANCE 
Women and Smoking. Compared to men, women are at an increased risk for both smoking-related 
morbidity and mortality, as well as smoking relapse.1-6 Women respond less favorably to smoking cessation 
treatment and, on average, quit smoking at an older age than men.2-3 Women of childbearing age are of 
particular concern given the short-term and long-term risks associated with pregnancy such as placental 
previa, restricted fetal growth, sudden infant death syndrome, and, later in the child’s life, attention-deficit 
disorder and overweight/obesity. 7-9 More than 90% of women who quit smoking during pregnancy relapse 
shortly after delivery, resulting in the continuation of smoking-related risks well after delivery as mothers are 
the primary source of secondhand smoke (SHS) exposure for offspring. 10 SHS contains more than 50 
carcinogens, as well as other toxins. Children exposed to SHS have increased risk of respiratory and ear 
infections, and miss more days from school. 11 To date few, if any, interventions have addressed potential 
biological and/or neurobiological causal mechanisms associated with the sex differences in risk for smoking 
relapse. In sum, effective smoking cessation interventions for women of childbearing age are lacking, perhaps 
due to a limited understanding of the causal pathways involved in smoking relapse.  
Sex Hormones & Drug Abuse Behavior.  The evidence for the role of sex hormones on drug abuse 
behaviors is growing. The animal literature provides strong evidence indicating that progesterone is protective 
against drug abuse behaviors whereas estrogen promotes drug abuse behaviors.12-13 The clinical literature is 
less clear but recent work provides support for this theory. For example, one of our recent studies, utilizing a 
randomized prospective design, observed better smoking cessation outcomes among those women who quit 
smoking during the luteal phase (low estradiol/high progesterone) compared to those who quit in the follicular 
phase (high estradiol/low progesterone). 14 Further support comes from a second study that also observed 
improved smoking cessation outcomes among those with a quit date in the luteal phase compared to the 
follicular phase while using bupropion as a smoking cessation aid. 15 However, discordant results come from 
two additional studies. These studies observed improved smoking cessation outcomes in the follicular phase 
rather than the luteal phase.16, 17 In addition to differences in methodology, one of the primary differences 
between these four studies is that the first two 14, 15 did not use nicotine replacement therapy whereas the 
second two did. 16, 17  These data ultimately prompted a hypothesis suggesting that the timing of a quit attempt 
during the menstrual cycle and the pharmacotherapy used may be equally important in determining success of 
a quit attempt,18 indicating nicotine response may be a factor that needs to be addressed. Relatedly, there is a 
growing body of animal literature to support that a metabolite of progesterone – allopregnanolone – acts as a 
potential protective factor. 12 Allopregnanolone is a neuroactive steroid which increases the release of 
dopamine and has been shown to be associated with behavioral effects of alcohol in a non-dependent sample 
of women, as well as withdrawal symptoms in a sample of dependent women. 19, 20 One theory is that 
withdrawal symptom severity, and therefore risk for smoking relapse, may be associated with changes in 
dopamine release. This may be one of the mechanisms involved with these observed associations between 
sex hormones and smoking relapse. Increasing evidence exists indicating that the sex hormones influence 
addictive behaviors and sex hormones are known to change dramatically during pregnancy. However, to our 
knowledge, no study to date has investigated the association between sex hormones and smoking during 
pregnancy. 
Sex Hormones & Pregnancy.  Levels of progesterone, allopregnanolone, and estradiol all change 
dramatically during pregnancy (Figure 1). Progesterone is produced mainly from the corpus luteum during 
early pregnancy, then by the placenta in mid- to late-pregnancy. Levels of progesterone vary widely among 
women but levels generally increase from 
approximately 10 ng/ml in the first trimester up to 
200 ng/ml in the third trimester. 21 Similarly, 
allopregnanolone increases by ten-fold during 
pregnancy. 22 Estradiol, primarily from the ovaries, 
increases from 2 to 18 ng/ml during pregnancy. 21 
Given 
that 
the 
literature 
indicates 
that 
progesterone is protective against drug abuse 
behaviors whereas estradiol facilitates these 
behaviors, the ratio between these two hormones 
may be of critical importance. During pregnancy, 
the 
estradiol/progesterone 
(E2/P) 
ratio 
can 
decrease from 0.20 to 0.09. 21 In brief, while all 
three sex hormones increase during pregnancy, 


Page 3 of 19 
the sex hormones that may be protective against drug abuse behaviors (progesterone, allopregnanolone) 
increase more dramatically than the sex hormone that may facilitate drug abuse behaviors (estradiol); this 
results in the ratio to shift in favor of those hormones that are protective, possibly offering an overall protective 
effect against drug abuse behavior during pregnancy.  
Formulation of Central Hypothesis. Pregnancy provides an ideal clinical model for testing our central 
hypothesis given the literature to-date indicates the following: (1) while pregnancy is the most common time for 
women to quit smoking, the majority are unable to sustain smoking abstinence after delivery, 7 (2) sex 
hormones increase during pregnancy, with progesterone increasing the most dramatically, 21 and (3) 
progesterone and allopregnanolone appear to be protective against drug abuse behaviors. 12 Therefore, our 
central hypothesis is that higher levels of progesterone and allopregnanlone, along with lower 
estradiol/progesterone ratios, will be associated with a favorable change in smoking-related symptomatology 
and nicotine response. 
 
INNOVATION  
 
This project incorporates at least three highly innovative aspects. First, this study will be among the first 
to systematically investigate the role of sex hormones on smoking-related symptomatology and nicotine 
response during pregnancy. This information will directly improve the state of the scientific knowledge by filling 
a gap in the literature and potentially offering a new direction in smoking cessation and relapse prevention 
interventions. For example, if progesterone proves to be associated with a reduction in smoking-related 
symptomatology during pregnancy, treatment of exogenous progesterone may facilitate cessation and help 
prevent relapse. Delivery of exogenous progesterone during the third trimester is an accepted treatment for 
pre-term delivery prevention;23 therefore investigation into the delivery of exogenous progesterone as a 
smoking cessation treatment during times of high risk for relapse (such as immediately prior to delivery and/or 
in the post-partum period) would be warranted. Further, this type of treatment may extend beyond the pregnant 
population, to non-pregnant women, who are at an increased risk for relapse compared to their male 
counterparts. 2 Second, to our knowledge no studies to date have investigated the role of allopregnanolone on 
drug abuse behavior during pregnancy in a clinical population. Adding this neurosteroid to our project 
represents a new focus in our research by investigating a possible neurobiological protective factor in smoking 
behavior. Considering the substantial role of allopregnanolone on drug abuse behavior in the animal literature, 
the knowledge gained from this project has 
the potential to directly influence and inform 
new postpartum smoking relapse prevention 
programs given that exogenous distribution of 
allopregnanolone has recently been proven to 
be safe in non-pregnant clinical populations. 
22 
Third, utilizing the two clinical models 
(pregnancy and oral contraceptives) will allow 
us to better assess the independent effects of 
sex 
hormones 
on 
smoking-related 
symptomatology 
and 
nicotine 
response. 
While motivations for smoking may change 
dramatically during pregnancy for a variety of 
reasons (i.e. infant health, social norms), 
utilizing these two clinical models will allow us 
to 
better focus on the relationship between sex 
hormones, smoking-related symptomatology and nicotine response by comparing the associations of sex 
hormones and study outcomes between clinical models.  
 
THE APPROACH  
PRELIMINARY STUDIES  
Study #1: Impulsivity Varies by Menstrual Phase after Exposure to Nicotine. Goal: This cross-over study 
aimed to measure differences in nicotine response between follicular (F) and mid-luteal (ML) menstrual 
phases in a sample of female smokers between the ages of 18 and 40 (n=20). Results: After exposure to 
nicotine nasal spray, participants in the F phase became significantly more impulsive on the Immediate 
Memory Task computer program 24 compared to participants in the ML phase (Figure 2). Relevance: Sex 
hormones appear to impact the level of impulsivity, such that higher levels of progesterone are associated 


Page 4 of 19 
with less impulsivity after exposure to nicotine.  
Study #2: Changes in Estradiol/Progesterone (E2/P) Ratio is Associated with Changes in Craving. 
Goal: This study aimed to assess the influence of the E2/P ratio on smoking-related symptoms of withdrawal 
(MNWS), 25 smoking urges (Brief-QSU), 26 and reinforcing effects of smoking (mCEQ) 27 using a within subject 
analysis in a sample of female smokers (n=50) with regular menstrual cycles between the ages of 18 and 40. 
Results: During ad libitum smoking, an increase in the E2/P ratio was associated with an increase in craving 
on two independent items of measurement: mCEQ (t=2.38, p=0.02) and MNWS (t=1.92, p=0.06). Relevance: 
Changes in sex hormone levels are associated with changes in craving for cigarettes.  
 Study #3: Smoking Satisfaction & Enjoyment Vary by Menstrual Phase during Ad Libitum Smoking. 
Goal: The purpose of this cross-sectional study was to investigate the differences in reinforcing effects 
(mCEQ) 27 of smoking by menstrual phase and 
depressive symptoms in a sample of female 
subjects (n=39) who were classified into four 
groups: no depressive symptoms follicular 
phase (NDS-F), no depressive symptoms luteal 
phase (NDS-L), depressive symptoms follicular 
phase (DS-F) and depressive symptoms luteal 
phase (DS-L). Results: Smoking Satisfaction 
varied by menstrual phase in the NDS group but 
not in the SDS group (F-NDS: 5.2±0.5; L-NDS: 
3.4±0.5; F-SDS: 4.5±0.4; L-SDS: 4.8±0.3; 
p=0.016; Figure 3) and a similar trend was 
observed within the Enjoyment subscale (F-
NDS: 4.4±0.6; L-NDS: 1.7±0.6; F-SDS: 3.6±0.5; 
L-SDS: 2.9±0.5; p=0.089). Relevance: Among those without depressive symptoms, sex hormones appear to 
impact the level of reinforcing effects of smoking, such that higher levels of progesterone are associated with 
less smoking satisfaction and less enjoyment.  
Study #4: Hormonal Contraceptive Use is Associated with Higher Levels of a Cigarette Biomarker. 
Goal: This cross-sectional study aimed to measure differences in NNAL, a biomarker of carcinogen exposure 
to cigarette smoking, between three groups of adolescent smokers (ages 13 to 19): females on hormonal 
contraceptives (F-HC; n=33), females not on hormonal contraceptives (F-no HC; n=27) and males (M; n=40). 
Results: After controlling for cigarettes/day, F-HC had the highest levels of NNAL values compared to F-no 
HC and M (1.00±0.89 vs. 0.46±0.31 vs. 0.71±0.59; f value=4.90, p=0.010). Relevance: Sex hormones 
appear to impact biomarker levels associated with the exposure to cigarette smoking.  
Study #5: Pharmacokinetics of Nicotine varies by Menstrual Phase in Women without Depressive 
Symptoms. Goal: This cross-over study aimed to measure 
menstrual phase differences in pharmacokinetics after 
exposure to nicotine nasal spray within a sample of 
abstinent female smokers with regular menstrual cycles 
who either had depressive symptoms (n=23) or did not 
(n=24). Results: There was a significant interaction 
between depressive symptoms status and menstrual 
phase such that those without depressive symptoms had a 
significant menstrual phase difference in their maximum 
nicotine 
concentration 
levels 
(p=0.020; 
Figure 
4). 
Relevance: In women without depressive symptoms higher 
progesterone levels are associated with lower nicotine 
response  
Study #6: Age of Menarche Predicts Onset of 
Smoking but Perception of Maturity does not. Goal: The goal of this retrospective study was to investigate the 
association of self-reported age of onset of daily smoking with self-reported age of menarche and perceived 
maturity level at age of menarche in a sample of female smokers (n=54) between the ages of 18 and 40. 
Results: Older age at menarche was associated with an older age at initiation of daily smoking (f-value=5.60, 
p-value=0.02). Perception of maturity was not significantly associated with smoking initiation (f-value=0.41, 
p=0.53). Relevance: Menarche represents marked sex hormone fluctuations and instability; it may be that the 
fluctuating sex hormones impact smoking behavior more than the perception of these life changes.  


Page 5 of 19 
 
PROGRESS REPORT  
For the past 20 years we have conducted research funded under NIDA grant R01-DA08075. This project’s 
overall goal is to investigate the influence of ovarian hormones, both with and without the use of nicotine 
replacement therapy, on multiple outcomes: women’s smoking behavior, relapse to smoking after a quit 
attempt, relapse-related factors including withdrawal symptoms, premenstrual symptoms, weight gain, caloric 
intake, negative mood, and nicotine response.  
 
During the past funding period (May 2007 to October 2011), our findings were disseminated in eight peer-
reviewed journal articles and 20 poster/oral presentations at local, national, and international scientific 
meetings (see Section 2.5 for Progress Report Publication List). As of February 2011, we have begun 
analyzing data from our completed pharmacokinetics subgroup. Based on these results we have two 
manuscripts in various stages of publication. These manuscripts will describe the menstrual phase differences 
in the changes in serum cortisol and nicotine levels after a nicotine challenge via nicotine nasal spray in a 
sample of 52 female smokers who have been abstinent for four days. 
 
Menstrual Phase & Depressive Symptoms in Acute Smoking Abstinence (Continuation Grant R01-
DA008075)  Project Period reported on includes May 2007 to October 2011. This project’s specific aims are: 
Aim 1: Determine the effect of depressive symptoms, alone and in concert with ovarian hormones (i.e. 
menstrual phase: follicular versus luteal) on withdrawal symptoms, nicotine craving, smoking urges, 
premenstrual symptoms and cortisol levels (measuring stress response) during acute smoking abstinence; Aim 
2: Determine if depressive symptoms and menstrual phase moderate nicotine response following acute 
smoking abstinence. Study Design: This study utilized a randomized cross-over design to compare within 
subject effects of menstrual phase and between subject effects of depressive symptoms. A convenience 
sample is stratified by depressive symptoms, then randomized to testing order (follicular phase then luteal 
phase or vice versa). Each participant then completed two six-day testing sessions including two days of ad 
libitum smoking followed by four days of smoking abstinence. During these testing sessions, participants 
attended daily clinic visits to biochemically verify smoking status, collect blood samples of measurement of sex 
hormones and complete self-reported items assessing mood and withdrawal symptoms. Participants also 
completed two nicotine lab exposure sessions during alternate menstrual phases. Summary: As described in 
the Preliminary Results Section, our previous work demonstrates not only our expertise in studying the 
hormonal influence on smoking behavior in women, but also the importance of this work. As detailed above 
(Section D.1) our most recent work has provided additional evidence for a relationship between sex hormones 
and drug abuse behaviors. Specifically we have shown higher levels of progesterone appear to be associated 
with blunted levels of impulsivity, less favorable responses to nicotine, and changes in levels of craving. New 
Direction: We are proposing to continue our exploration of the possible causal mechanisms of sex hormones 
on smoking-related symptomatology and nicotine response in pregnant women. During pregnancy 
progesterone, allopregnanolone and estradiol increase dramatically. This will provide us with an ideal clinical 
model to further investigate the relationship between sex hormones and smoking behavior, as well as fill a gap 
in the current body of literature and potentially provide new information and direction to improve smoking 
cessation rates during pregnancy.  Our proposed study is further strengthened and innovative due to the 
inclusion of a more controlled study of sex hormone effects via the clinical model of oral contraceptives. 
 
RESEARCH PLAN  
Overview. Using two clinical models – pregnancy (Sample 1) and oral contraceptives (OC; Sample 2) – 
we aim to (1) examine the association between levels of progesterone (Prog), allopregnanolone (Allo) and the 
estradiol/progesterone (E2/P) ratios with smoking-related symptomatology during ad libitum smoking, and (2) 
determine the association between Prog, Allo, and E2/P with the changes in smoking-related symptomatology 
and response to nicotine following overnight abstinence. Sample 1 will enroll approximately 112 pregnant 
women to ensure a final sample size of 84 participants to participate in a cross-sectional study at two time 
points: 12 weeks to 22 weeks gestation (n=42) and 32 to 37 weeks gestation (n=42). Sample 2 will enroll 
approximately 78 OC users to ensure a final sample size of 52 participants in a randomized cross-over, 
double-blind study design to complete testing periods during “low” and ”high” dose levels of exogenous 
progesterone with a consistent dose of exogenous estrogen. All participants will complete identical data 
collection procedures including providing saliva (cortisol to measure stress), urine (cotinine and 3-
Hydroxycotinine to measure nicotine exposure, and medication compliance), and blood (progesterone, 
allopregnanolone, estradiol, and nicotine) samples, as well as ecological momentary assessments (EMA) daily 
for seven-days. Participants will also complete a three-hour smoking lab session (with the first morning 


Page 6 of 19 
cigarette) and a four and a half-hour nicotine exposure lab session (after at least 13 hours of overnight 
abstinence). These sessions will contain a nicotine challenge via nicotine nasal spray or smoking topography 
with timed-series of physiological, subjective and behavioral responses. 
Recruitment. Our primary recruitment strategy for both study samples is via two clinical sites: (1) 
Obstetrics & Gynecology Clinic at Hennepin County Medical Center (HCMC), the fourth largest hospital in 
Minnesota and is a safety net facility for low-income, uninsured and other vulnerable populations (see Section 
2.14 for letter of support) and (2) Fairview Riverside Women’s Clinic at Fairview University Medical Center 
(FUMC; see Section 2.14 for letter of support). Among these two clinics an estimated 4,900 infants are born 
each year. Given an estimated 23% of women of childbearing age currently smoke about 50% quit smoking 
during pregnancy, 7 about 15% of pregnant women experience a pregnancy-related complication 28 and an 
estimated 15% are using psychotropic medications, 29 we roughly estimate approximately 407 pregnant women 
will meet our inclusion/exclusion criteria (details described below) for Sample 1 each year (4,900 infant 
births/year x 23% smoking prevalence x 50% smoking cessation x 85% no pregnancy complications x 85% no 
psychotropic medications). We will also recruit non-pregnant women who are on oral contraceptives from these 
two clinical sites. Based on preliminary results from our current study, we estimate 40% of women who smoke 
are oral contraceptive users and 15% are currently being treated for psychiatric problems. At each clinical site  
we will post flyers and business cards in the waiting and exam rooms at the Hennepin County Medical Center 
Obstetrics & Gynecology Clinic, and the Fairview Riverside Women’s Clinic.  These flyers and business cards 
will instruct potential participants to call study staff for more information.  
In addition to recruiting at clinical sites we will also use two additional recruitment methods. The first is 
via the Minnesota Academy of Family Physicians Research Network. Approximately 80 MAFP members 
practice in the Minneapolis/St. Paul metro and greater surrounding area. The overall clinical populations within 
this network include 64% women who are White (82%), Asian (8%), Black (6%) and other/unknown (14%). 
Since MAFP physicians often have individual practices and are familiar with conducting clinical research, they 
will refer potentially eligible participants to our research study (see Section 2.14 for letter of support). The 
second recruitment method is advertising in the mainstream media. While this type of recruitment can be 
challenging, our team has been successful with this form of recruitment with both non-pregnant and pregnant 
women. For example, our current study recruiting non-pregnant women between the ages of 18-40 has had 
success with Facebook advertising. Specifically, over the past six months we have advertised on Facebook 
resulting in 4,121 clicks to our website, an estimated 130 phone calls to our clinic and an enrollment of 23 
participants (~4 participants/month). Similarly, Dr. Lewis’ (Co-I) ongoing study on pregnancy and depression 
recently sent out an email to the local newspapers listserv. This email was opened 21,700 times, resulting in 
338 clicks to the study website. A total of 109 participants were enrolled into the study, the majority of which 
were recruited directly from this single email. In addition to these two examples, we regularly use television, 
radio, mailings, and other internet sources to identify study participants with similar success.  
Study Samples. While all participants in Sample 1 (Pregnant Women) and Sample 2 (Oral 
Contraceptive Users) complete identical data collection procedures, there are some differences in 
inclusion/exclusion criteria, timing of the testing periods and follow-up procedures.  Those differences are 
described here and in Table 1.  
Sample 1 – Participants. To meet recruitment goals, we will aim to enroll 38 pregnant women per year 
(~3-4 women/month). Inclusion criteria: females 18-35 years old, in stable physical/mental health with 
confirmed pregnancy, self-reports smoking ≥ 5 cigarettes/day for the past year, has established prenatal care, 
single gestation, either 12-22 or 32-37 weeks gestation, English fluency, and able to provide informed consent. 
Exclusion criteria: use of psychotropic medications or illicit drugs with the exception of marijuana use < 2 times 
per month and a willingness to abstain from marijuana during the testing period, current use of other types of 
tobacco or nicotine, or pregnancy complications (i.e. gestational diabetes, known congenital anomaly, fetal 
growth restriction, pregnancy hypertension and history of >2 miscarriages). Finally, participants will not be 
eligible to participate in the study at more than one time point (i.e. 12-22 and 32-37 weeks gestation) due to the 
potential ethical issues associated with administering nicotine nasal spray multiple times during pregnancy 
and/or unintended encouragement to continue smoking to be eligible to participate again. 


Page 7 of 19 
 Sample 1 - Timing of Testing Period. The testing period will occur during either 12-22 or 32-37 weeks 
gestation (participants complete one testing period only). These gestational weeks were selected to capitalize 
on the expected variability in sex hormones (e.g. 12-22 weeks gestation expected progesterone values are 
19.5 to 89.4 ng/mL and 32-37 
weeks 
gestation 
expected 
progesterone values are 48.4 
to 200.0 ng/mL).20 We will not 
be assessing the first trimester 
(due to the high risk for 
miscarriage and delay of initial 
prenatal care) nor late third 
trimester (due to the possibility 
of early delivery).  
Sample 1 - Follow-up. 
After completing the testing 
period 
(described 
below) 
participants 
will 
receive 
smoking cessation behavioral 
counseling, self-help materials, 
advice 
for 
pharmacotherapy 
(and 
a 
prescription, 
if 
necessary) per the discretion of 
the study physician (Dr. Allen, 
PI) and referrals to smoking 
cessation programs. Additional 
weekly phone counseling will 
be offered.  
Sample 2 - Participants. 
To meet recruitment goals, we 
will aim to enroll 27 OC users 
per year (~3 women/month). Inclusion criteria: females 18-35 years old, in stable physical/mental health, self-
reports currently smoking ≥ 5 cigarettes/day for at least the past year, using OC for at least the past three 
months without complication, willing to switch to study supplied OC, English fluency, and able to provide 
informed consent. Exclusion criteria: use of psychotropic medications or illicit drugs with the exception of 
marijuana use < 2 times per month and a willingness to abstain from marijuana during the testing periods, 
current use of other types of tobacco or nicotine, current breast feeding, planning to become pregnant within 
the next three months, current use of finasteroid (propecia), efavirenz, red clover, ketoconazole and other 
drugs that are CYP3A4 inhibitors, and conditions contraindicated to progesterone treatment (including, but not 
limited to, thrombophlebitis, deep vein thrombosis, pulmonary embolus, clotting or bleeding disorders, heart 
disease, diabetes, history of stroke, allergy to peanuts, hypersensitive to progesterone and liver dysfunction). 
Sample 2 - Timing of Testing Period. As in Sample 1, the timing of the testing period has been selected 
to maximize the variability in sex hormones by OC use. Participants will be randomly assigned to order to 
complete two testing periods: “low” dose progesterone versus “high” dose progesterone. To obtain these 
values participants will be supplied with Tri-Sprintec (Barr Laboratories Inc.; Appendix A) and generic 
Prometrium 
(Teva 
Pharmaceuticals 
USA 
IncAppendix A). The “low” dose progesterone 
is seven days of placebo Tri-sprintec + 
placebo generic Prometrium twice a day (7AM 
and 7PM).  The “high” progesterone dose is 
seven days of placebo Tri-Sprintec + 200mg 
of generic Prometrium twice a day (7AM and 
7PM).  
The Wednesday after their Screening 
Visit (described below), participants will begin 
taking 
the 
Tri-Sprintec 
medication. 


Page 8 of 19 
Participants will be on Tri-Sprintec for three weeks prior to the first testing period. We have chosen this specific 
type of OC for four reasons. First, and foremost, it delivers a 
constant level of estrogen, allowing for an investigation of the varying levels of Prog, Allo and the E2/P ratio. 
Second, this type of progesterone (norgestimate, prometrium) has been shown to be converted into Allo (Dr. 
Richard Hauger, Personal Communication; 2/15/11). 30-31 Third, we have had success with reassigning women 
to this OC in our 
current 
study 
with 
little 
to 
no 
side 
effects. Finally, using 
an OC rather than 
attempting to track 
the natural menstrual 
cycle allows for ease 
in 
scheduling 
and 
related logistics.  
Given that the 
norgestimate dose in 
OC’s is relatively low 
during both the “low” 
and “high” dosage 
testing 
periods 
participants will also 
be given 200mg of 
generic 
Prometrium 
twice a day (7AM and 
7PM) 
during 
the 
“high” dose week and 
placebo pills during 
the “low” dose week. 
Participants 
will 
receive their first two 
doses of active or 
placebo 
Progesterone at the 
Baseline 
Visit 
(described 
below). 
They 
will 
be 
instructed 
to 
take 
their first dose at 
7PM.This technique 
has 
been 
successfully used in 
prior research 32 and 
is 
generally 
well 
tolerated. The most 
common 
adverse 
effect 
is 
sedation. 
Less common effects 
include breakthrough 
bleeding, nausea and breast tenderness. 33-34 While it is possible that progestins may contribute to risk for 
thromboembolism; it is rare and we will be using natural progesterone instead of synthetic progestins. Natural 
progesterone is not known to be associated with thromboembolic risk. 35 Using these techniques we will expect 
to see stable estradiol levels with varying levels of Prog and Allo (Figure 5). Participants will receive additional 
study medication as needed at the clinic visits. 
Sample 2 - Follow-up. Upon completion of the first testing period, participants in Sample 2 will be asked 
if they want to return to smoking. If they want to maintain their smoking abstinence, they will also receive 
Table 2.  Study Measures 
 
Screenin
g Visit 
Testing Period 
Baseline 
Visit 
(Day 0) 
 
(Day 
1-5) 
Follow-
up Visit 
(Day 6) 
Smoking 
Lab 
Session 
(Day 7) 
Nicotine 
Lab 
Session 
(Day 8) 
Independent Variables 
 
Serum Progesterone 
 
X 
 
X 
 
X 
 
Serum 
Allopregnanolone 
 
X 
 
X 
 
X 
 
Serum Estradiol/ 
Progesterone Ratio 
 
X 
 
X 
 
X 
Dependent Variables (Aims 1 & 2) 
 
Symptomatology 
Forms (MNWS, 1 Brief 
QSU, 2 mCEQ, 3 
PANAS, 4 PSS-10, 5 
CES-D, 6 LTEQ 7) 
X 
X 
X 
X 
X 
X 
 
Salivary Cortisol and 
mSSS 8 * 
 
 
X 
 
 
 
Dependent Variables (Aim 2) 
 
Physiological 
Response (Blood 
Pressure, Pulse) 
 
 
 
 
X 
X 
 
Subjective Response 
(SSS) 9 
 
 
 
 
X 
X 
 
Behavioral Response 
(FT, WR, 2B, IMT, 
DMT) 10 
 
 
 
 
X 
X 
 
Plasma Nicotine 
 
 
 
 
 
X 
Other Items 
 
Informed Consent 
X 
 
 
 
 
 
 
Liver Function Tests 
11 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
Smoking Behavior 12 
X 
 
 
 
 
 
 
Adverse Events 
(including C-SSRS) 
X 
X 
 
X 
X 
X 
 
Expired Carbon 
Monoxide 
X 
X 
 
X 
X 
X 
 
Urinary Cotinine and 
3-Hydroxycotinine 
 
X 
 
X 
 
X 
These items will be collected five times during the course of Day 5 only – at waking, 30 minutes later, two hours later, 8:00
and at bed time.   1 Minnesota Nicotine Withdrawal Scale, Hughes & Hatsukami, 1998; 2 Brief Questionnaire of Smoking U
Tiffany & Drobes, 1991; 3 Modified Cigarette Evaluation Questionnaire, Cappelleri et al, 2007; 4 Positive and Negative 
Scale, Watson et al, 1988; 5 Cohen Perceived Stress Scale, Chaaya et al, 2010; 6 Center for Epidemiologic Study – Depre
Knight et al 1997; 7 Modified Subjective State Scale, al'Absi, et al., 1998, al'Absi et al, 2003; 7 Lesiure Time Ex
Questionaire, Godin & Shephard, 1983;  8, 9 Subjective State Scale, al'Absi, et al., 1998 and al'Absi et al, 2003; 10 Beha
Tasks include Fingertapping, Hindmarch, 1980, Word Recall, Phillips & Fox, 1998, 2-Back, Meyers et al, 2008, Immediat
Delayed Memory Task, Dougherty et al, 2002; 11 Liver function tests of ALT and AST will be conducted in Sample 2 (OC u
only.  12 Includes Fagerstrom Test of Nicotine Dependence (FTND), age of smoking initiation, number of past quit atte
ongest past quit attempt, motivation to quit smoking and TimeLine FollowBack (TLFB). 


Page 9 of 19 
support for cessation. Based on observations in our current study, it is expect that up to 20% of women will 
want to maintain their smoking abstinence. If the participants want to return to smoking, they will be instructed 
to smoke at their ‘regular rate’ for the next three weeks and then attend an identical second testing period 
during the opposite testing phase (i.e. “low” or “high” dose).  
Data Collection Procedures. All participants (Sample 1 and Sample 2) will complete identical testing 
procedures as described below and in Table 2. 
The first visit will be a screening visit. At this visit informed consent will be obtained, followed by 
collection of demographic and smoking behavior information. Medical histories will be collected to further 
evaluate eligibility.  The Columbia-Suicide Severity Rating Scale (C-SSRS) will be competed with each subject 
in order to assess suicidal ideation or behavior.  Results will be reviewed by study staff and participants will be 
referred to physicians (S. Allen, MD) or (D. Hatzukami, PhD) if necessary.  Measurements of height, weight 
(measurements taken with 
coats, shoes and other heavy items removed), blood pressure (measured 
after five minutes of rest), heart rate, and carbon monoxide will also be collected.  A blood sample will be 
collected for liver function tests (AST and ALT) for Sample 2 participants only.  At the end of this visit the all 
remaining clinic visits will be scheduled and participants will be compensated for their time. 
The baseline visit (Day 0) will be the first day of the testing period. At this visit participants will have 
their weight measured and then be shown into a private room to complete self-reported items of smoking-
related symptomatology and related factors (See Table 2 and Section E.5 for details). After the questionnaires 
are completed, blood pressure, and heart rate will be measured. Next, the participant will provide urine (for 
assessment of nicotine exposure) and blood (for assessment of sex hormones) samples. Remaining blood and 
urine samples will be saved in the biorepository. The participants will then be trained on how to use the study 
supplied Electronic Data Capture Device (EDCD).  Upon the completion of this visit, the upcoming visit 
dates/times will be confirmed and participants will be compensated for their time.  
During the testing period (Days 0-7) all participants will complete daily Ecological Momentary 
Assessments (EMA) using a study supplied Electronic Data Capture Device (EDCD). The EDCD will be 
programmed to “beep” to 
indicate an assessment is available. Given that recent research indicates that 
smoking-related symptomatology varies by time of day 36 participants will be prompted to complete 
assessments at random times (between the hours of 7AM and 11PM) up to four times per day, for an average 
of three times per day. Notification of an available assessment will happen via a “beep” sound. The EDCD will 
continue to “beep” once per every 15 minutes until the assessment is completed or after the two-hour window 
allotted for completion of assessment elapses. Each assessment will take an estimated 15 minutes to 
complete and will measure self-reported levels of smoking, withdrawal, craving, mood, stress, and physical 
activity (See Table 2 and Section E.5. for details). We have opted to use EMA due to several benefits versus 
standard paper-and-pencil format including improved quality of data, increased participant compliance, and 
participant preference. 37-38  
The interim clinic visit (Day 3 or Day 4), participants will return to the clinic. Upon arrival, participants 
will have their weight measured, followed by measurement of blood pressure, heart rate and carbon monoxide. 
Cigarettes smoked per day since Day 0 will be collected retrospectively from participants.  Participants will also 
be asked about any new medications or changes to current medications.  Participants will receive training on 
how to do the saliva samples on day 5 and receive the specific materials to do the saliva samples. Any 
difficulties the participant has had using the EDCD will be addressed before the participant leaves.  At the end 
of this visit the participants will be compensated for their time. 
On the day prior to the follow-up visit (Day 5) participants will collect salivary cortisol samples outside 
the clinic to measure physiological stress levels. Participants will be reminded to collect saliva samples via 
prompts from the EDCD. We are measuring saliva cortisol rather than blood serum levels, because samples 
are needed throughout the day and multiple blood draws are impractical, burdensome and would impose 
ethical issues in pregnancy. Saliva sample collection will occur at the following times to assess the diurnal 
pattern of cortisol: immediately after waking, 30 minutes later, two hours later, 8:00 pm and before going to 
sleep. We have successfully used this method in prior projects, 14, 39 as well as in our ongoing study. 
Participants are compliant with the testing procedures. Participants will be supplied with saliva sample swabs 
(Salivette tubes, Sarstedt, Rommelsdorf, Germany). Tubes will be labeled to indicate the date and time each 
sample should be completed. Previous research has shown that this ambulatory monitoring procedure can be 
adequately performed by participants, and that the integrity of the salivary cortisol is not compromised by 
storage at room temperature for 24 hours. 40 During each saliva sample collection, participants will complete 
the monitoring Subjective State Scale (mSSS) via the EDCD to monitor mood and potential confounders such 


Page 10 of 19 
as time of last cigarette, physical activity, and caffeine use (Appendix B).  
At the follow-up clinic visit (Day 6), participants will return to the clinic. Upon arrival, participants will 
have their weight measured and provide urine (nicotine exposure and medication compliance via visual 
inspection of urine color which will change due to riboflavin medication filler) and blood (sex hormones) 
samples. Remaining blood and urine will be saved in the biorepository. Next, participants will complete self-
reported items of smoking-related symptomatology and related factors (See Table 2 and Section E.5 for 
details) followed by measurement of blood pressure and heart rate. While participants are completing the 
questionnaires study staff may complete a NicAlert (Appendix C) to confirm self-reported smoking status 
(NicAlert level of ≥ 3 indicating a cotinine value of ≥ 100 ng/mL). Participants will then practice using the 
nicotine nasal spray but will be 
prompted to refrain from smoking 
for one hour prior to the visit to 
limit nicotine exposure. They will 
also practice smoking through the 
smoking 
topography 
machine 
and practice using the computer 
tasks and other measures used 
in the lab sessions (See Section 
E.5. 
for 
details). 
Finally, 
participants will be instructed to 
quit 
smoking 
by 
midnight 
(Appendix 
D). 
Upon 
the 
completion of this visit, the lab 
session will be confirmed and 
participants will be compensated 
for their time.  
The smoking lab session 
will occur on Day 7.  At this visit, participants will smoke their first morning cigarette (i.e. they will be instructed 
to not smoke on Day 7 until instructed to do so by study staff). Participants will attend a three hour visit 
beginning as close to their usual first morning cigarette as possible (starting at approximately 7am-10am, per 
participant preference).  Puff topography, a precise measure of smoking behavior,67,68,69 will be used to 
examine whether prolonged use of progesterone affects topography measures that may indicate smoking 
compensation.70 Puff topography will be assessed using a handheld topography device (type pending) that 
provides a valid measurement of puff number, puff volume, inter-puff interval and other indices.66 Carbon 
monoxide readings will be collected before and 2-5minutes after puff topography.  36 Prior to the initiation of the 
lab session, participants will have their weight, blood pressure, and heart rate measured, and they will 
complete subjective measures to assess their self-reported level of smoking-related symptomatology and 
related factors after overnight abstinence (See Table 2 and Section E.3.5 for details). Once the smoking lab 
session begins, participants will be instructed to turn off their cell phones and will not be allowed to consume 
caffeinated beverages or any food items. Measurements of smoking response will start 40 minutes prior to the 
participant smokes her cigarette and will be administered in a fixed order (Figure 6). Response to smoking will 
be quantified via seven performance domains known to be associated with nicotine response:41 (1) 
physiological (blood pressure, heart rate), (2) subjective (Subjective State Scale); (3) motor speed (Finger 
Tapping) (4) episodic memory (Word Recall); (5) working memory (2-Back); (6) attention/impulsivity 
(Immediate Memory Task), and (7) delayed memory (Delayed Memory Task). (See Section E.5. for more 
details.) In order to access plasma nicotine levels, a blood draw via an intravenous catheter will occur at 
baseline (-1 minute before smoking a cigarette) and +2 minutes after the last puff of the cigarette. A total of 
30cc of blood will be collected.  15 cc of blood will be analyzed for nicotine levels and hormone levels and 15 
cc of blood will be added to the biorepository.    
The nicotine exposure lab session will occur on Day 8. Participants will attend this four and a half-hour 
visit in the morning (beginning at 7am-10am depending on participant preference) to control for diurnal 
variation in outcome variables. While participants are completing the questionnaires study staff will download 
the information from the EDCDs.  Procedures will be identical to Day 7 except instead of subject smoking a 
cigarette, they will administer nicotine nasal spray and repeat the protocol twice.  Nicotine nasal spray 
(Nicotrol, Pharmacia & Upjohn; Appendix A) is an FDA-approved use of nicotine delivery. One spray delivers 
0.5 mg of nicotine; therefore, one dose (four sprays - two in each nostril) is approximately equivalent to the 


Page 11 of 19 
nicotine delivery from two cigarettes.42-44 The nasal spray is the only available delivery method that closely 
mimics the kinetics of smoking, is easy to administer, and delivers a consistent dose.42-43 While the nicotine 
nasal spray results in an adverse burning sensation, this sensation resolves on its own within minutes. In our 
current study we have completed over 400 nicotine nasal spray lab sessions and have never had to postpone 
data collection due to problem with the adverse effects of the nasal spray. We also chose the nicotine nasal 
spray because: (a) there are ethical and practical difficulties of instructing people to inhale measurable 
amounts of tobacco smoke from cigarettes, (b) it is difficult to control nicotine intake via smoke inhalation, (c) 
the spray isolates the effects of nicotine from that of the many other compounds found in tobacco smoke, (d) it 
has the fastest delivery system and best approximates the rise in nicotine concentrations as seen with 
cigarette smoking, and (e) it has been given a Pregnancy Category Risk of D – indicating that although nicotine 
has known adverse effects, the benefits of use may outweigh the risks. While there may be some initial 
concerns with administering nicotine to a pregnant woman, nicotine replacement products have been 
recommended for smoking cessation during pregnancy.45 In a recent study by Oncken and colleagues, 46 11 
doses of nicotine nasal spray were administered over a six-hour period in a sample of pregnant smokers who 
abstained from smoking overnight. This study found that nicotine nasal spray reduced overall maternal nicotine 
exposure compared to active smoking. Compared to the 11 doses used in this study, our study will be using a 
single dose of nicotine nasal spray (less than 5% of the acceptable maximal dose and less than 10% of the 
dose used by Oncken and colleagues); therefore, there are limited ethical concerns with using nicotine nasal 
spray within our sample of pregnant smokers. Further this protocol was reviewed by a bioethicist at the 
University of Minnesota, Dr. Steven Miles, who felt there were no major ethical issues in administering this 
dose of nasal nicotine spray to pregnant women (see Section 2.14 for letter of support).  In order to access 
plasma nicotine levels, a blood draw via an intravenous catheter will occur at baseline (-1 minute before the 
nasal spray) and at the expected maximum concentration (+5 minutes after the nasal spray; Allen et al, 2012).  
Blood collected at baseline will also be used to analyze sex hormones, more specifically progesterone and 
allogregnanalone.  A total of 30cc of blood will be collected, 15 cc of blood will be analyzed for nicotine levels 
and hormone levels and 15 cc of blood will be added to the biorepository. Participants will also provide a urine 
sample to assessnicotine exposure and medication compliance via visual inspection of urine color which will 
change due to riboflavin medication filler. Remaining urine will be saved in the biorepository. Upon completion 
of the four and a half-hour lab session, any possible adverse events will be assessed for all participants. 
Finally, all participants will be compensated for their time and will be offered brief smoking cessation 
counseling and self help materials for smoking cessation.  
Study Participant Compensation. All participants will receive $10 at the screening visit, $20 at the 
baseline visit, $10 at the interim visit, $20 for Follow-up Visit on Day 6, $20 for Smoking Lab Session on Day 7 
and $50 for the Nicotine Exposure Lab Session on Day 8. They will also receive $5 for each EMA completed 
with a bonus of $20 for not missing any EMAs, and $1 for each cortisol salivary sample collected with a bonus 
of $20 for not missing any of the five samples. A bonus of $100 will be given for completing all study 
procedures and $120 for returning the study supplied EDCD. Thus, all participants in Sample 1 will be 
compensated a total of $510 for completing the study. The participants in Sample 2 will have the potential to 
earn $ 900 by completing the testing week twice. 
Study Measures. The independent measures will be the serum levels of progesterone (Prog), 
allopregnanolone (Allo), and the estradiol/progesterone (E2/P) ratio. Measurement of hormones will be done 
by collecting a blood sample on the baseline,follow-up, and nicotine nasal spray lab visits (Day 0, 6, and 8).  
We have opted to collect blood samples three times to allow for average hormone levels during the testing 
period to be obtained.   Blood (20 cc) will be drawn then centrifuged. The serum stored at -20°C in sealed 
storage tubes to prevent evaporation. Approximately two mL of serum will be analyzed by the Fairview 
University Medical Laboratories for progesterone and estradiol samples using chemiluminescence. The 
expected values for these tests are displayed in Figure 6. Approximately two mL of serum will be sent to Dr. 
Richard Hauger’s lab at the University of Califorina, San Diego (see Section 2.14 for letter of support) for Allo 
level measurement using radioimmunoassay techniques previously described. 47 The remaining serum 
(approximately two to four mL) will be stored as back-up. 
For Aim 1, the outcome is the level of smoking-related symptomatology during ad libitum smoking. Items 1-
7 below will be measured an average of three times a day for seven days via the EMAs. We selected the 
seven day testing period given that the hormones fluctuate fairly rapidly; thus, measuring symptomatology for 
longer than seven days would make the association between sex hormones and symptomatology more difficult 
to assess. For each item, participants will be instructed to rate their symptoms since the last assessment. The 
following nine dependent measures will be used (Appendix B):  


Page 12 of 19 
(1) Minnesota Nicotine Withdrawal Scale (MNWS): Participants rate their nicotine withdrawal symptoms on 
eight-items on a scale of ‘0’ (not present) to ‘4’ (severe) resulting in summary scores overall withdrawal 
and craving. 25  
(2) Questionnaire on Smoking Urges-Brief (QSU-Brief): This ten-item version of the original form (QSU) 
was developed by Tiffany and Drobes 26 and has an excellent level of reliability (α = .97). Two factors 
are obtained: ‘Factor 1’ is a measure of primary intention and desire to smoke and ‘Factor 2’ is a desire 
to smoke in anticipation of relief from negative affect.  
(3) Modified Cigarette Evaluation Questionnaire (mCEQ): This 12-item questionnaire uses a seven-point 
Likert-type scale to assess the reinforcing effects of smoking resulting in five domains including 
smoking satisfaction, psychological reward, aversion, enjoyment of respiratory tract sensations and 
craving reduction. 27 
(4) Positive and Negative Affect Scale (PANAS): Participants rate 20 words associated with positive or 
negative affect using a five-point Likert-type scale. This scale has been shown to have high internal 
consistency. 48 
(5) Cohen Perceived Stress Scale (PSS): This ten-item questionnaire has been validated in a variety of 
populations including pregnant and postpartum women. It uses five-items to assess ten questions 
resulting in two factors negative feelings and inability to handle stress. It has high internal reliability 
(alpha = 0.75). 49  
(6) Center for Epidemiologic Studies – Depression (CES-D): This form contains ten-items to assess 
symptoms typically associated with depression. The items are ranked using a four-point Likert-type 
scale. It has high levels of sensitivity and specificity. 50-51 We have opted to use this item rather 
pregnancy-specific item to ensure that all participants had comparable data.  
(7) Leisure Time Exercise Questionnaire (LTEQ): This questionnaire has four-items to assess strenuous, 
moderate and mild exercise completed during leisure time and has high reliability (r=0.83, 0.85). 52 
(8) Monitoring Subjective State Scale (mSSS, Day 5 only): This form is identical to the Subjective State 
Scale (SSS) 53-54 except it also includes variables to assess possible confounders such as coffee 
consumption. Participants will be asked to mark each rating scale at the point that best describes how 
they felt over the past 30 minutes.  
(9) Salivary Cortisol (Day 5 only): Five saliva samples over the course of the day given that cortisol, a 
physiological measure of stress released by the hypothalamic-pituitary adrenal axis (HPAA), has a 
clear diurnal variation. Its peak activity occurs about 8:00 am in the normal sleeper, followed by steady 
decline until noon. The lowest activity occurs around 8:00 pm. The frequent sampling in the morning will 
be used to assess free cortisol responses to awakening. Awakening cortisol increase is considered a 
promising marker of adrenocortical functional status. 55-57 
For Aim 2, the outcomes will be assessed following overnight abstinence: (1) the change in smoking-
related symptomatology (items 1-6 above), and (2) response to nicotine (described below). We have a nearly 
identical timed series to assess nicotine response with success in our ongoing study. All items will be self-
administered per direction of study staff via a computer program (REDCap; http://project-redcap.org/) to 
improve the quality of data (by avoid missing data, illegible data, etc) and limit amount of time spent on data 
entry and cleaning. The following seven dependent measures were selected to assess seven domains of 
nicotine response: 41  
(1) Physiological Measures: Blood pressure (BP) and heart rate (HR) will be measured using a Dinamap S 
X/P. This device is an automated device that will read BP and HR simultaneously by placing a blood 
pressures cuff above the antecubital fossa region of the arm.  
(2) Subjective Response: The Subjective State Scale (SSS) questionnaire is comprised of 24-items to 
assess positive affect, distress and nicotine withdrawal symptoms. 53-54 Participants will rank each item 
based on how they felt over the past 5-30 minutes (depending on time of last completion) using a 
seven-point scale ranging from ‘not at all’ to ‘very strong.’ This form takes approximately two minutes. 
(3) Motor Speed: During the Finger Tapping (FT) task, 58 participants are instructed to use the index finger 
on the dominant hand to tap a key on a computer as quickly as possible for 30 seconds. This task takes 
about 1.5 minutes.  
(4) Episodic Memory: The Word Recall (WR) task presents 25 concrete nouns individually on a computer 
screen for three seconds each. Upon completion of the display participants will be given a short 
distractor of counting out loud from “20” to “0.” They then complete a written recall of the words within 
two minutes. The total number of words correct is each participant’s score. 59 This task is designed to 
assess short-term episodic memory and is estimated to take four minutes. 


Page 13 of 19 
(5) Working Memory: The 2-Back (2B) task presents stimuli (letters on a computer screen) for two seconds 
with a half-second between stimuli. Participants are instructed to press a key when a letter was 
repeated with one intervening letter (i.e. matched a letter two back in the series). The percent correct 
responding and mean response times are recorded.60 This task takes approximately six minutes. 
(6) Attention/Impulsivity: The Immediate Memory Task (IMT) is a continuous performance task that displays 
a series of randomly generated 5-digit numbers (e.g., 54983) on a computer monitor for ½ a second at 
a rate of one per second. The participant is instructed to click a button when the number displayed is 
identical to the preceding number. Responses to the target stimuli (i.e. correct identification of duplicate 
numbers) and filler stimuli (i.e. all other random 5-digit) are indicators of response inhibition and 
continuous attention. The higher the ‘catch stimuli’ (i.e. a number that differs from the preceding 
number by only one digit) indicates a more impulsive pattern. 24 It is estimated that this task will take 
approximately six minutes. 
(7) Delayed Memory: The Delayed Memory Task (DMT) is a continuous performance task functions nearly 
identical to the IMT except a series of distracter sequences (i.e. 12345) are presented between target 
stimuli. Participants are instructed to ignore these distracters and click the right mouse button in 
response to target stimuli. Therefore, this task measures delayed memory. 24 It is estimated that this 
task will take approximately six minutes 
(8) Plasma Nicotine (Day 8 only):  We will also collect two blood samples (30 cc) through an intravenous 
catheter one minute prior to nicotine nasal spray and five minutes after. The intravenous catheter will 
be placed by trained study staff approximately 45 minutes prior to the use of nicotine nasal spray to 
avoid influencing data collected during the baseline (Time -40 or -1 minutes) assessment.  These 
samples will be frozen and stored to be analyzed for plasma nicotine and sex hormones.  These time 
points were selected to allow for maximum concentrations of nicotine to be captured based on data 
from our prior work (Allen et al, 2012).  
Information on potential covariates is collected at the baseline visit. These items include number of 
cigarettes smoked per day during ad libitum smoking, Fagerstrom Test for Nicotine Dependence (FTND), 61 
self-reported number of past quit attempts, past longest quit attempt, motivation to quit smoking, social 
influences such as partner smoking and sociodemographic variables (Appendix B). Participants in both 
samples will also complete an adverse events form along with the Columbia-Suicide Severity Rating Scale at 
Screening, Day 0, and Day 8 to assess possible adverse events associated with the study medications.  The 
C-SSRS (Posner et al, 2009) assesses suicidal ideation, given this is listed as an SAE on the Prometrium 
label.  If any item of suicidality is endorsed referrals to the appropriate mental health care will be made.   
Sample Size & Power Analysis. In Sample 1, we expect an approximate drop-out rate of 25% based on 
our current, more burdensome protocol with a sample of 18-40 year-old non-pregnant females. Therefore, we 
will enroll a total of 112 participants (56 at 12-22-20 weeks and 56 at 32-37 weeks gestation) to ensure we 
have 84 participants to complete the nicotine exposure lab session. Based on a linear regression model, 
sample size of 84 will have 81% power to detect a correlation coefficient of 0.30 for a test of the association 
between Prog and self-reported craving with a two-sided 0.05 significance level. In Sample 2, we expect 
approximately 25% will drop out and an additional 20% will want to maintain smoking abstinence after 
overnight abstinence, therefore we will enroll a total of 78 participants to ensure a final sample size of 52. 
Based on a paired t-test (mean difference in craving outcome), this sample size will have 80% power to detect 
an effect size of 0.40 with a 0.05 two-sided significance level. These detectable effect sizes are realistic given 
observations in our current study (e.g. Study #1 in Section D.1, d=0.42) and other published literature (d=0.44 
to 0.77). 63-65 
Statistical Analysis. The analyses are intended to test each hypothesis from the specific aims (Table 3). 
Descriptive statistics will be used to summarize the data. For all analyses, assumptions regarding distributions, 
outliers and covariates (see details in Section E.5 under ‘potential covariates’) will be evaluated. We will use an 
average of self-reported levels of craving (primary), urges to smoke, stress and mood collected at Day 0, Day 
and during the seven-day EMA as the outcomes. For nicotine exposure outcomes, the changes in 
physiological measures (BP, HR [primary]), subjective measures (Subjective State Scale) and behavioral 
measures (Finger Tapping, Word Recall, 2 -Back, Immediate and Delayed Memory Task) will be calculated 
taking levels at specific time points after nicotine nasal spray administration minus the baseline measures at 
Time -1 (BP, HR) or Time -30 (all measures except BP, HR). The averages of the Day 0 and Day 7 Prog, Allo, 
and E2/P levels will be used as the independent variables. 


Page 14 of 19 
For Sample 1, Hypothesis 1: We will investigate the association between Prog and self-reported craving 
with a linear regression model. A 
multiple linear regression model will be 
used to see if the relationship changes 
with consideration of other baseline 
measures 
(including 
cigarettes/day). 
Similar analyses will be performed 
comparing the change in Prog with the 
change in self-reported craving from 
Day 0 to Day 7. Hypothesis 2a: For this 
analysis, 
we 
will 
investigate 
the 
association between Prog and self-
reported 
craving 
after 
overnight 
abstinence with a linear regression 
model. A multiple linear regression model will be used to see if the relationship changes with consideration of 
other baseline measures, such as cigarettes/day and FTND score. Hypothesis 2b: We will investigate the 
association between Prog and heart rate outcome at five minutes post nicotine nasal spray using a linear 
regression model. A multiple linear regression model will be used to see if the relationship changes with 
consideration of other baseline measures (including cigarettes/day). We will also explore how Prog impacts the 
nicotine exposure outcomes over time using repeated measures models to account for within-subject 
correlation. For Hypothesis 1, 2a, and 2b, similar analyses will be performed for the secondary outcomes and 
the independent variables (Allo, E2/P). 
For Sample 2, Hypothesis 1: We will analyze self-reported craving (primary) using a repeated measures 
model. A random intercept will be included to model within subject correlation. OC level (”low” or “high” dose), 
testing period (first or second) and sequence will be included in the model as fixed effects. Hypothesis 2a: For 
this analysis, we will use self-reported craving, urges to smoke and stress collected after overnight abstinence. 
As in Hypothesis 1, repeated measures models will be used to compare the self-reported craving outcome 
between hormonal OC level groups. Hypothesis 2b: Again, repeated measures models will be used to 
compare the heart rate outcome between hormonal OC level groups at the first time point after baseline. We 
will also explore the heart rate outcome over time by using a covariance structure to model repeated measures 
on subjects at each visit. For Hypothesis 1, 2a, and 2b, similar analyses will be performed for the secondary 
outcomes and the independent variables (Allo, E2/P). 
To compare the two study samples we will use all data from Sample 1 and data from the first testing period 
only for Sample 2. To compare outcomes between Sample 1 and Sample 2, we will use a propensity score 65 
to balance the baseline covariates in the two groups. A linear regression model will be used to investigate the 
association between Prog and the self-reported craving outcome. An indicator variable for group, the 
propensity score quintile, Prog and a group by Prog level interaction will be included in the model as 
independent variables. Similar analyses will be conducted for the secondary outcomes and the independent 
variables (Allo, E2/P) 
Strengths & Limitations. This study has several strengths. First, and foremost, is the use of two unique 
clinical models to investigate the effects of sex hormones on smoking-related symptomatology. This study will 
be among the first to utilize this type of study design. Further, the cross-over study design used with Sample 2 
will limit confounding given that each participant is compared to herself. An additional strength is the detailed 
measurement of smoking status, sex hormones, smoking-related symptomatology, withdrawal symptoms and 
nicotine response. Finally, the study team has extensive experience and expertise, which provides unique 
qualifications to pursue this research project. Three limitations are worth noting. First, we do not have a sample 
of males for comparison. The relevance for needing this comparison group may be of lesser importance 
considering that female sex hormones are the main interest. Second, due to the experimental design and the 
use of a convenience sample, we will have limited generalizability. Finally, given that we will be completing the 
nicotine exposure session among pregnant smokers who report current daily smoking, it is possible that we will 
have a biased sample. However, the ethical concerns of administering nicotine to a pregnant woman who has 
quit smoking, makes this limitation unavoidable. Therefore, to investigate the possibility of a biased sample 
difference in associations between sex hormones (Prog, Allo, E2/P) with smoking-related symptomatology and 
nicotine response we will also look at a subgroup of women who reduced their smoking during pregnancy 
(>50% reduction) versus those who maintained their self-reported pre-pregnancy smoking rate. Specifically, 
we will include an indicator variable for smoking reduction and the interaction between this indicator and sex 


Page 15 of 19 
hormone in the models. Despite these limitations, this study provides a unique opportunity and excellent 
conditions for testing our overall study objective. 
Study Team. Our research team has unique breadth and depth of expertise in the following areas: 
effects of ovarian hormones (menstrual phase, hormone replacement therapy) on smoking cessation in women 
(Allen, 
PI); 
nicotine 
dependence, 
smoking cessation, and tobacco toxin 
exposure (Hatsukami, Co-I); cortisol 
measurement of stress (al’Absi, Co-I) 
and mental health and pregnancy 
(Lewis, Co-I). We will also be utilizing 
several experts (see Section 2.14 for letters of support): Dr. Neal Benowitz (safety of smoking cessation 
medications in pregnancy), Dr. Mehmet Sofoglu (sex hormones and addiction), Dr. Virginia Lupo (obstetrician), 
and Dr. Steve Heishman (neuropsychological nicotine response). 
Study Timeline. The timeline is displayed in Table 4.  
Future Directions. The expected outcomes of this project will be a better understanding of the relationship 
between sex hormones and smoking-related symptomatology. Upon completing this project, we intend to 
continue our exploration into the role of sex hormones on smoking behavior. Specifically, if our expectations 
are confirmed, we will conduct additional studies to investigate the possibility of exogenous progesterone as a 
treatment for smoking cessation and relapse prevention in several different populations including pregnant 
women, during the postpartum period and non-pregnant women.    
 
INCLUSION OF WOMEN AND MINORITIES 
This study will only be recruiting women because the research question addressed is only relevant to one sex.  
We are looking specifically at the effects of female sex hormones on smoking behavior and nicotine response 
in pregnant and oral contraceptive users.  The study will use participants recruited from the greater 
metropolitan area of Minneapolis and St. Paul, via two clinical sites and mass media advertisements.  The 
Twin Cities metropolitan area has a sufficiently large female smoking population to ensure an adequate 
sample.  We have been successful in recruiting similar numbers of women in similar periods of time in previous 
studies.  Estimates indicate that as of 2009 the Minneapolis and St. Paul, Minnesota Twin Cities metro area 
included approximately 50.5% female and 20.2% of the population in the racial minority groups (Black= 11.0%, 
Asian = 5.8%, American Indian = 1.2%, other = 4.0%) and the Hispanic population is 6.4%.   If minority women 
do not respond to the advertisements, we will make special efforts to solicit their participation by advertising in 
local neighborhood newspapers with high minority readership (such as the Southside Pride, 
Phillips/Powderhorn, and Riverside Editions); by posting flyers in free clinics in the metro area who service 
minorities (e.g., Community University Healthcare Center, Pilot City); and by identifying contacts in churches, 
health centers, and community centers with high minority participation and disseminating information regarding 
the study opportunity; and once we have garnered initial contact with participants we receive multiples word of 
mouth referrals.  Our current study has been successful in recruiting a diverse sample in that of the 212 
participants enrolled to date 48% are White, 29% are Black, 9% are more than one race and 14% are other.  
 
INCLUSION OF CHILDREN 
Children (under 18 years old) are not included since both menstrual cycle irregularities (e.g., anovulation) and 
pregnancy complications are more common in adolescents.  Further, since this study requires the continuous 
and regular use of cigarettes, it is unlikely adolescents will meet this criterion.  Young women (aged 18-20 
years) will be recruited for participation in our study.  The trial site is at the University of Minnesota, which has 
a large undergraduate population. Campus media will be also used to recruit young women.  We will also 
target venues and institutions frequented by young non-college women (bars, festivals, coffee shops, and 
community events) to ensure the sociodemographic diversity of the youngest component of our participant 
sample.  
 
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