Clinical trial NCT03018366
Conditions: Estrogen Deficiency Cardiovascular Disease (CVD) Functional Hypothalamic Amenorrhea Endothelial Dysfunction
Keywords: estrogen premenopause menopause amenorrhea
Sponsor: Cedars-Sinai Medical Center
Start date: 2017-01-01; Completion date: 2023-02-01
Enrollment: 29 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
The purpose of this study is to determine whether young women with functional hypothalamic amenorrhea (premenopausal HypoE) is associated with risk factors for pre-clinical cardiovascular disease (CVD).

For this study, the investigators will measuring vascular function and inflammatory markers on:

* young women with functional hypothalamic amenorrhea (\>3 months of no menstrual cycle due to low estrogen)
* young women with regular menstrual cycles not on hormone therapy.
* recently menopausal women (\<3 years from final menstrual period) not on hormone therapy.

Premenopausal HypoE participants (women with functional hypothalamic amenorrhea) will be randomized to use either an estrogen patch or a placebo patch (no active medicine) for 12 weeks, followed by estrogen or placebo patch plus progesterone or placebo pills for 2 additional weeks. The investigators are looking to see if estrogen improves vascular and inflammation.

Detailed description:
Study Aims:

1. To test the hypothesis premenopausal HypoE (women with FHA) is associated with pre-clinical CVD as determined by reductions in vascular endothelial function.
2. To test the hypothesis premenopausal HypoE (women with FHA) is associated with increased immune-mediated inflammation.
3. To test the hypothesis whether estrogen replacement can reduce inflammation and improve vascular endothelial function in premenopausal HypoE women (women with FHA).

In a randomized, double-blind placebo-controlled trial in premenopausal HypoE women (women with FHA) the investigators will test 12 weeks of transdermal estradiol 0.1 mg/day patch or placebo followed by 2 weeks of estradiol plus progesterone 200mg (for endometrial safety) on vascular endothelial function and immune-mediated inflammation versus placebo. Patches will be applied by the participant to the lower abdomen twice weekly, alternating sides.

The investigators will be using non-invasive tests to measure vascular function to measures reactive hyperemic index (RHI) using peripheral arterial tonometry (PAT)

Arms: arm 1: 17Beta Estradiol (0.1mg/day) , Progesterone (100mg) or Medroxyprogesterone (10mg) for patient with a peanut allergy because progesterone 100mg is a peanut based product arm 2: Placebo Transdermal Patch, Placebo Pill
Interventions: intervention 1: 17beta Estradiol intervention 2: Transdermal placebo patch intervention 3: Progesterone intervention 4: Placebo Pill
Intervention descriptions: intervention 1: Participants will use a dose of transdermal estradiol 0.1 mg/day patch for 12 weeks +/- 1week. PAT index vascular measures and serum immune markers will be measured after 6 and 12 weeks +/- 1week on estrogen patches. Patches will be applied by the participant to the lower abdomen twice weekly, alternating sides. intervention 2: Participants will use a dose of placebo patches for 12 weeks +/- 1 week. Placebos will be applied by the participant to the lower abdomen twice weekly, alternating sides. intervention 3: After 12 weeks +/- 1week of transdermal estradiol patch, participants will use estrogen patch plus progesterone for 2 additional weeks +/- 3 days. Progesterone is a peanut based product and for patients with a peanut allergy we will replace this with a synthetic progestin at an equivalent dose, medroxyprogesterone 10mg. intervention 4: After 12 weeks +/- 1week of transdermal placebo patch, participants will use placebo patch plus placebo pill for 2 additional weeks +/- 3 days.

Protocol documents:
Prot_SAP_000.pdf: 
Atherosclerosis, Immune Mediated 
Inflammation and Hypoestrogenemia in 
Young Women  
 
 
NCT# 03018366 
 
 
Date: 06/03/2022 
 
 
 


A. Introduction 
 
Premature CVD is the leading cause of death in young women, yet traditional risk factors 
underestimate CVD risk. While observational epidemiological study has identified ovulatory function 
and estrogen levels as potentially playing a role in CVD in women, specific study using well-
phenotyped subjects and accurate measures of atherosclerosis has not been performed. The loss 
of estrogen after menopause is associated with an unfavorable shift of CVD risk factors and is 
believed to contribute to increased rates of CVD. Young premenopausal women can have 
hypoestrogenemia due ovulatory cycle disruption. Functional hypothalamic amenorrhea (FHA) 
is the cessation of the menstrual cycle due to suppression of the hypothalamic gonadotropin-
releasing hormone (GnRH) resulting in severe hypoestrogenemia.  It is estimated that 1.62 
million women between the ages of 18 and 44 years in the US and 17.4 million women 
worldwide have functional hypothalamic amenorrhea (FHA) resulting in premenopausal 
hypoestrogenemia (HypoE). 
 
We hypothesize that a chronic state of premenopausal HypoE is associated with more adverse 
CVD risk factors and inflammatory biomarkers, and is associated with pre-clinical CVD.  Prior 
research has found an adverse association in young women with irregular menses and CVD 
events; young women with Turner’s syndrome, a genetic defect resulting in severe 
hypoestrogenemia from birth have premature deaths due to accelerated CVD; and 
hypoestrogenemia of hypothalamic origin in premenopausal women has been associated with 
premature obstructive coronary artery disease (CAD).   
 
Furthermore, the health impact of premenopausal HypoE in young amenorrheic woman has 
also been established on bone health resulting in early osteoporosis paralleling the changes 
seen after menopause estrogen loss, approximately 2.5% bone loss per year. Several 
epidemiological studies have found low bone mineral density has been associated with 
subclinical atherosclerosis and CVD mortality.1-3 While CVD and osteoporosis are both diseases 
of aging, more recent data suggests a common physiologic mechanism linking these two 
conditions beyond age, such as inflammatory markers and cytokines.4 To date, however, the 
impact of low estrogen in young premenopausal and the relation between bone mass and 
subclinical CVD unknown.  
 
Both the American Academy of Pediatrics and the American College of Obstetrics and 
Gynecology have advocated that the menstrual cycle should be considered a “vital sign” due to 
the importance of estrogen on tissues throughout the body. To date, however, there has been 
no study assessing the relationship between HypoE, CVD risk factors, bone mass and 
subclinical CVD. The overarching goals of this research proposal are to test whether 1) 
Premenopausal HypoE is associated with CVD risk factors and inflammatory biomarkers; 2) 
Premenopausal HypoE is a marker of subclinical CVD. In order to achieve our goals, we will 
compare Premenopausal HypoE women to regularly cycling women (normal controls) and 
women who are recently menopausal (<3 years from last menses) not on hormone replacement 
therapy ; 3) To determine that estrogen improves CVD inflammatory biomarkes and markers of 
subclinical CVD in premenopausal HypoE women. In a randomized, double-blind placebo-
controlled trial in premenopausal HypoE women we will test 12 weeks of transdermal estradiol 
followed by 2 weeks of estradiol plus progesterone on CVD biomarkers and subclinical CVD 
versus placebo. 4) to Results of the larger study could have readably translatable implications 
– use of hormonal contraceptive therapy provides sustained and safe levels of estrogen in 
premenopausal women and is a strategy that has been evaluated in a primate animal model. 
Hormonal contraceptive therapy could be tested as a next step on measures of atherosclerosis 
should the study results be as hypothesized.  
 
 


Specific Aim 1: Is premenopausal HypoE associated with CVD risk factors and 
biomarkers? 
To establish whether CVD risk factors and biomarkers are more adverse in premenopausal 
women with functional hypothalamic amenorrhea (FHA) resulting in premenopausal 
hypoestrogenemia (HypoE) compared to regularly cycling women and recently postmenopausal 
woman (<3 yrs) not on hormone replacement therapy. CVD risk factors will include systolic 
blood pressure (SBP), diastolic blood pressure (DBP), low density lipoprotein cholesterol (LDL) 
and high density lipoprotein cholesterol (HDL), triglycerides, body mass index (BMI), and 
inflammatory biomarkers such as high sensitivity C-reactive protein (hsCRP).  
 
Specific Aim 2: Is premenopausal HypoE associated with relatively endothelial 
dysfunction? 
To evaluate whether endothelial dysfunction is greater in women with premenopausal HypoE 
compared to regularly cycling women and recently postmenopausal women using peripheral 
arterial tonometry (PAT). 
 
Specific Aim 3: To determine that estrogen improves CVD inflammatory biomarkers and 
markers of subclinical CVD in premenopausal HypoE women.  In a randomized, double-blind 
placebo-controlled trial in premenopausal HypoE women we will test 12 weeks of transdermal 
estradiol on inflammatory biomarkers and subclinical CVD versus placebo at baseline and 12 
weeks followed by 2 weeks of estradiol plus progesterone for endometrial safety. 
 
B. Background and Significance 
 
B.1. CVD is the leading killer of young women. Although significant advancements have 
been made in cardiovascular research, CVD remains the leading killer of women at all ages.5 
Traditional CVD risk factors are not adequately predictive of CVD mortality in young women and 
a significant amount remains unexplained. Notably, young, premenopausal women have a 2-4 
fold increased risk or mortality with CVD compared to age-matched males, despite adjustment 
for CVD risk factors and comorbidity.6 In the US each year: 1) approximately 480,000 women 
will die of CVD, 2) one in three women will die of heart disease regardless of race or ethnicity, 3) 
more women die of CVD than men, and 4) more women will die of CVD than the top 6 cancers 
combined.7 CVD remains a dominant contributor to the nation’s morbidity and health care 
expenditures in women. 
 
B.2. Pre-clinical atherosclerosis. The risk of CVD significantly increases in the 5th decade of a 
woman’s life; however the development of atherosclerosis represents a process that begins 
much earlier. In the Bologusa Heart Study, there is evidence that the pre-clinical fatty 
atherosclerotic streaks form in the teenage years, followed by fibrous atherosclerotic plaques in 
the 20’s and 30’s, and eventually clinical atherosclerosis resulting in CVD events.8  Autopsy 
data from this study showed that by age 30, the prevalence of a fibrous-plaque lesion is 60% in 
both the aorta and the coronary artery.8  In the Pathobiological Determinants of Atherosclerosis 
in Youth (PDAY) study, an autopsy study on men and women, age 15-34 years, approximately 
one-third of women had atherosclerosis by age 34.9  
 


B.3. CVD occurs later in women than in men. Women typically develop CVD 10 years later 
than men.10 (see figure 1) As shown in 
figure 1, the equivalent rate of myocardial 
infarction (MI) in women occur 10-years 
later than men, as illustrated by the 
dashed line.  After the age of 50, CVD risk 
factors shift unfavorably for women; which 
coincides temporally with estrogen loss 
and natural menopause. In women who 
undergo surgical removal of the ovaries, 
oophorectomy, the CVD risk increases.  
An increased risk of CVD is also observed 
among young women undergoing 
premature menopause, compared to age-
matched women.11    
Figure 1: Annual Rate of First Heart Attacks 
by Age and Sex  
 
B.4. Women with premature estrogen loss have early death and increased CVD mortality.  
Whether menopause is natural or surgical, common sequelae of the low estrogen environment 
are worsening CVD risk factors including dyslipidemia and hypertension.  Postmenopausal 
women in the Framingham study had significantly worse lipid profiles than same-aged 
premenopausal women. A shift to an increase in total cholesterol, LDL and triglycerides and 
decrease HDL has been shown to occur just 6 months after cessation of the last menses.12 
Compared to women who go through natural menopause, women who go through early 
menopause at or before age 46 have overall worse CVD risk factors, CVD mortality13-15and 
death16,17 Early menopause has also been associated with accelerated subclinical 
atherosclerosis by carotid IMT as compared to women who go through later menopause.18,19 In 
the Nurse’s Health Study, women who underwent surgical oophorectomy and were not treated 
with estrogen replacement therapy prior to the age of 50 were found to be at increased risk for 
coronary heart disease (HR 1.98, 95% CI 1.18-3.32), stroke (HR 2.19, 95% 1.16-4.14) and all-
cause death (HR 1.40, 95% 1.01-4.33).20 
 
Turner’s syndrome (TS), 45 X, and primary ovarian insufficiency (POI), 46,XX are both 
conditions that result in severe estrogen deficiency in young women. TS is an X-chromosomal 
defect that results in ovarian dysgenesis and lifelong estrogen deficiency. These women have a 
reduced life expectancy with the main cause of death being cardiovascular complications 
including a seven-fold higher rate of CVD.21  Additionally, women with TS have higher rates of 
CVD risk factors including hypertension, hyperlipidemia, diabetes, and obesity.  Another model 
of severe estrogen deficiency in young women is 46, XX primary ovarian insufficiency (POI) 
resulting in primary amenorrhea.  In a cross-sectional study of women in their early 30’s with 
estrogen deficiency, either TS or POI, a statistically significantly thicker cIMT (mm) was found in 
TS and POI women compared to normal controls of similar ages. (0.61 + 0.07 mm; 0.60 + 0.05, 
and 0.55 + 0.06, p<0.001, respectively).22   
 
B.5. CVD risk in women with premature estrogen loss is attenuated by estrogen therapy. 
Estrogen therapy reduces oophorectomy-related and POI CVD risk,23-28,29 Estrogen therapy in 
young TS and POI results in improved high density lipoprotein cholesterol (HDL-C), serum 
glucose and cIMT. These important observational findings suggest that estrogen modulates 
CVD in young women with HypoE. 
 
B.6. Premenopausal HypoE due to Functional Hypothalamic Amenorrhea (FHA) may 
contribute to CVD risk. HypoE in premenopausal women is most often due to FHA; up to one 
third of adult-onset amenorrhea is due to estrogen disruptions.30 FHA is defined as no menses 
Per 1,000 Persons
Men
Women


due to low estrogen due to suppression of the hypothalamic-pituitary-ovarian axis and excludes 
pathologic diseases such as those related to a systemic disease or associated with a disorder of 
the reproductive tract. FHA is due to suppression of the hypothalamic gonadotropin-releasing 
hormone (GnRH) pulsatility resulting in lower levels of follicle stimulating hormone, luteinizing 
hormone, and a state of estrogen deficiency.  There are 3 types of FHA associated with stress, 
weight loss, or excessive exercise.   
 
Animal data suggest that premenopausal cardiovascular disease is strongly determined by 
estrogen status characterized by hypoestrogenemia of hypothalamic origin.31 Specifically, when 
young premenopausal monkeys are subjected to environmental stress (due to frequent cage 
rotation and constant re-establishment of social hierarchical position); they develop a stress 
reaction that is characterized by a reduction of the central brain hormones that stimulate the 
ovaries.32 Menstrual cycling becomes irregular or ceases, fertility is low or absent, and estrogen 
levels fall due to ovarian shut down. The monkeys have abnormally low follicle stimulating 
hormone (FSH) and luteinizing hormone (LH), as well as low estrogen (E2) and progesterone 
(PO) levels.32 These young female monkeys develop cardiovascular disease. Female monkeys 
that maintain normal ovarian function and estrogen levels are protected from cardiovascular 
disease. These results suggest strongly that environmental stress is a cause of infertility among 
monkeys, and that the resulting estrogen deficiency can cause cardiovascular disease. In 
addition, in premenopausal monkeys administering oral contraceptive therapy for 2-year period 
was found to be protective against the development of atherosclerosis in at-risk monkeys.33 The 
relevance of these findings to human females, however, is unknown.   
 
Menstrual cycle irregularities have been studied as a marker for cardiovascular disease.  In a 
cohort of 82,439 nurses aged 20-35yrs, compared to women with regular menses, women with 
usually irregular or very irregular menses have a significantly increased risk for CVD. (RR 1.22, 
95% CI 1.04-1.44, and RR 1.53, 95% CI 1.24-1.90, respectively).28 This 50% increased risk for 
CVD included nonfatal MI or fatal CHD adjusted for risk factors such as BMI, hormone use, 
smoking status and suggests that irregular menses may be a marker for increased risk of later 
CVD. Menstrual cycling irregularities have also been associated with uterine atherosclerosis.34 
This suggests that amenorrhea and cycling irregularity may be a metabolic marker useful to 
predict women at risk for developing CVD.  
 
Menstrual cycle irregularities are associated with hypoestrogenemia.  In the NHLBI-sponsored 
Women’s Ischemia Syndrome Evaluation (WISE) study, 95 premenopausal women with 
coronary risk factors underwent coronary angiography for evaluation of suspected 
cardiovascular disease.35 Thirteen premenopausal women with cardiovascular disease had 
significantly lower estradiol, bioavailable estradiol, and follicle-stimulating hormone (FSH) (all p 
< 0.05), compared to the 82 premenopausal women without angiographic cardiovascular 
disease, even after controlling for age. Estrogen deficiency of hypothalamic (central brain) 
origin, defined as estradiol <50 pg/ml, FSH <10 IU/L, and luteinizing hormone (LH) <10 IU/L, 
was significantly more prevalent among the women with cardiovascular disease compared to 
those without (69% vs 29%, respectively, p=0.01). This estrogen deficiency was the most 
powerful predictor of angiographic cardiovascular disease in statistical modeling.  
 
B.7. Meaures of pre-clinical CVD. Peripheral arterial tomometry (PAT) measures coronary and 
peripheral endothelial function and is a functional expression of atherosclerotic risk.36 PAT has 
been found to correlate in patients with coronary microvascular dysfunction,37 ischemic heart 
disease,38 and in menopausal women indicates an independent component of CV risk.39 WISE 
studies have demonstrated that the PAT index has a sensitivity of 85% and specificity of 80% 
for detection of endothelial dysfunction in CAD patients.36 
 
 
C. Research Design and Methods 


 
C.1. Specific Aim 1: Is premenopausal HypoE associated with CVD risk factors? To test 
the hypothesis that CVD risk factors are more adverse in premenopausal HypoE  compared to 
regularly cycling women and recently postmenopausal woman (<3 yrs) not on hormone 
replacement therapy.  
 
C.1.1. Recruitment: Women with HypoE, normal controls and recently postmenopausal 
woman  will be recruited from several different locations including the Women’s Hormone and 
Menopause Program at the Barbra Streisand Women’s Heart Center, Center for Fertility and 
Reproductive Medicine in the Department of Obstetrics and Gynecology at Cedars-Sinai 
Medical Center (CSMC) and CSMC OB/GYN staff physician’s offices by posting the study flyer 
in waiting rooms. We also plan to recruit women from the community by posting our study flyer 
on community bulletin boards at local college campuses, gyms and yoga studios as well as 
expand the social media posting to social media. I will also contact student health centers and 
obtain permission to post the study flyer at these locations. (see study flyers) To determine if a 
self-identified woman has HypoE, we will provide screening laboratories: FSH, LH, estradiol, 
progesterone, TSH, Free T4, prolactin, testosterone, free testosterone and androstenedione. 
Beta-HCG by urine or serum to ensure negative pregnancy prior to enrollment. These screening 
labs will be performed after a woman has signed the consent form. If a woman has already seen 
a physician and has had these laboratory results performed, we plan to use that report and not 
perform additional labs.  For women who qualify as HypoE, they will return within 7 days for a 
repeat estradiol blood draw to confirm HypoE. Regularly cycling women will be (age- and BMI-
match) defined as having self-reported menstrual cycle lengths of 24 to 38 days not on hormone 
therapy, and confirmation of one ovulation by either urinary LH surge or day 22-26 progesterone 
> 3 ng/ml. If ovulation is not confirmed that month, i.e. progesterone < 3 ng/ml then we would 
rescreen the subsequent month. Beta-HCG by urine or serum to ensure negative pregnancy 
prior to enrollment. For recently postmenopausal women will be determined by FSH>30 and 12 
months of amenorrhea,40 within 3 years of final menstrual period with natural menopausal not 
on hormone therapyFinally, we will offer a one-time $50 stipend to normal control and 
menopausal women, for the HypoE women up to $300 stipend will be offered for their research 
visit and time commitment. I have developed a pre-screening questionnaire which includes 
questions regarding the inclusion and exclusion criteria (see below).  For this project, I will 
recruit 30 women with  premenopausal HypoE, 30 regularly cycling women and 30 
postmenopausal women.  
 
C.1.2. Eligibility/Inclusion Criterion: Entry criteria for HypoE and normal controls will 
include premenopausal by WISE criteria currently not on hormone therapy, English speaking 
(for the purposes of complete psychosocial assessment), able to give informed consent, a 
gynecological age (age since menarche) > 10 and < 25 years, and chronological age > 18 
years. Within 90-110% of ideal body weight as determined by the 1983 Metropolitan height and 
weight table for women. All patients with hypothalamic amenorrhea will be diagnosed based on 
exclusion of other etiologies for their amenorrhea, including pregnancy, thyroid dysfunction, 
hyperprolactinemia, POI, and polycystic ovary disease.  For recently postmenopausal women 
will be determined by FSH>30 and 12 months of amenorrhea,40 within 3 years of final menstrual 
period with natural menopausal not on hormone therapy, English speaking, able to give 
informed consent.  
 
Exclusion criteria for normal control and postmenopausal groups are current use of hormone 
contraceptive or any estrogen or progestin therapy, smoking, hypertension, hyperlipidemia, 
diabetes, medical, neurological, or ophthalmologic disease except acuity problems, a major Axis 
I disorder other than depression, parturition in the last 12 months and/or lactating in the last 6 


months or the patient has any concurrent disease or condition that in the opinion of the 
investigator would make the patient unsuitable for participation in the study. 
For HypoE women, exclusion criteria includes current use of hormone contraceptive or any 
estrogen or progestin therapy, allergy to adhesive or tape allergy, or the patient has any 
concurrent disease or condition that in the opinion of the investigator would make the patient 
unsuitable for participation in the study.  
For postmenopausal women exclusions also include surgical or chemotherapy induced 
menopause or premature ovarian failure. 
 
C.1.3. Data Collection: All laboratory data will be collected and analyzed according to our 
published WISE Core Lab methods.41 Data collection will occur at the Cedars-Sinai Women’s 
Heart Center. Collection will include: reproductive hormone assays (estradiol, progesterone, 
FSH, LH, bioavailable testosterone, fasting lipid panel (LDL, HDL, Trigylcerides) and metabolic 
and inflammatory markers (glucose, cortisol, hsCRP). Blood pressure, pulse and body mass 
index computed as weight in kg/height in m2 will be collected. 
 
C.2. Specific Aim 2: Is premenopausal HypoE associated with relatively endothelial 
dysfunction? To establish whether subclinical atherosclerosis is greater in premenopausal 
HypoE compared to regularly cycling women and recently postmenopausal women using 
peripheral arterial tonometry (PAT).  
 
C.2.1. Data Collection: Measure of Vascular Function:  PAT is a proven, non-invasive 
measure of vascular function is method of detecting endothelial function by Endo PAT 2000 
(Itamar® Medical Ltd), which we and others including the Framingham Study have successfully 
used.42 The device consists of a finger probe to assess digital volume changes accompanying 
pulse-waves. A pressure of 40-70mmHg is applied by the probe to the index fingers of both 
hands to eliminate venous stasis and arterial pulse amplitude is recorded. Device calculates a 
reactive hyperemia peripheral arterial tonometry (RH-PAT) index, which reflects peripheral 
vascular function.  
 
C.3. Specific Aim 3: To determine that estrogen improves CVD inflammatory biomarkers 
and markers of subclinical CVD in premenopausal HypoE women.  In a randomized, double-
blind placebo-controlled trial in premenopausal HypoE women we will test 12 weeks of 
transdermal estradiol on inflammatory biomarkers and subclinical CVD versus placebo at baseline 
and 12 weeks followed by 2 weeks of estradiol plus progesterone for endometrial safety. 
 
30 HypoE women will be randomized to transdermal estrogen for 12 wks followed by transdermal 
estrogen plus oral progesterone for 2 weeks. We will compare markers of endothelial function 
and inflammatory response at baseline and 12wks in women randomized to hormone therapy vs 
placebo. 
 
C.3.1. Rationale. Estrogen treatment has been shown to improve vascular function in 
women with premature menopause and it is well established that estrogen has immune-
modulating effects resulting in lower pro-inflammatory cytokines in postmenopausal women. We 
will use our PAT index as a vascular function measure of pre-clinical CVD. We will use a dose of 
transdermal estradiol 0.1 mg/day weekly patch for 12 wks, which will result in a mean serum 
estradiol level of 70 pg/ml.43 We are working with the manufacturer to obtain placebo patches as 
has been done in previous double-blinded studies.44 Patches will be applied by the subject to the 
lower abdomen twice weekly, alternating sides. The dose and 12 week duration of unopposed 
transdermal estradiol was selected based on prior study to maintain serum around 75 pgram and 
to minimize the risk of endometrial hyperplasia.45 The use of unopposed estradiol at higher doses 
and longer duration can result in endometrial hyperplasia in postmenopausal women after one to 
three years. Our 12 week treatment of estrogen will be followed by 2 weeks of estrogen and 
progesterone to allow sloughing of the endometrium and reversal of the estrogen effect on the 
endometrium.46 This dose is equivalent to an average serum estradiol level in an ovulating woman 
which ranges from 50 up to 400 pg/ml.47 We selected a 0.1mg/day dose of transdermal estrogen 


to provide a more physiologic estradiol level compared to oral estradiol which is rapidly convert 
to estrone.48 After 12 wks of transdermal estradiol, we will continue estradiol and add oral 
micronized progesterone 200mg for 14 days to mimic the luteal phase and will allow us to evaluate 
the additional impact of estrogen plus progesterone. Progesterone 200mg is a peanut based 
product and for patients with a peanut allergy we will replace this with a synthetic progestin at an 
equivalent dose, medroxyprogesterone 10mg. This is an FDA-approved medication and has the 
same indication.  
 
 
C.3.2. Hypothesis. We hypothesize that estradiol treatment will improve vascular endothelial 
function, and markers of inflammation in premenopausal HypoE women.  
 
 
C.3.3. Experimental 
Protocol 
and 
Data 
Collection: 
Baseline 
serum hormones, vascular 
measures, and immune 
markers will be collected 
from aims 1 and 2 in the 30 
HypoE women. Subjects 
will then be randomized to 
0.1mg/day 
transdermal 
estradiol patch or placebo 
for 12 weeks followed by 2 
weeks of estradiol patch 
with progesterone or placebo pills with placebo patch for 12 weeks +/- 1 week and 2 weeks +/- 
3 days after completing estrogen and progesterone (Figure). One week after completing the 
patch and pill, study staff will contact participant and ask if they had their menstrual period. If 
needed, 1 patch and 3 more pills will be dispensed during the week 12 visit to account for patient 
schedules if they cannot come in exactly at day 14, as the exit visit on study drug is needed as 
suggested by the pharmacist.  
 
 
C.3.4. Data Collection: Serum hormone evaluation: After 7 days of transdermal 17-beta 
estradiol and 12 weeks we will measure serum E2 levels and hormone panel to assess blood 
levels and compliance.  Vascular measures will be tested at baseline and 12 week. Detailed 
method collection described under Aim 1A.  
 
 
C.3.5. Data Analysis. Statistical method:  Associations between change in serum estrogen, 
change in PAT, change in inflammatory marker levels will be examined. Pearson correlations 
will be examined between continuous variables. If the distributions are non-normal Spearman 
correlations will be used. A linear mixed model will be used on the change in PAT at week 12 to 
examine the association while adjusting for within subject correlation. In addition to choosing an 
appropriate correlation structure, other model building and diagnostics will be carried out as 
described in Aim1A.  A Tukey-Kramer or Bonferroni adjustment for multiple pair-wise 
comparisons will be used as appropriate. 
 
Power considerations: Pilot data indicated a baseline SD of 0.35 for PAT in the HypoE 
group and 0.44 in the control group. Assuming the larger SD at baseline and 4 weeks, a 
conservative estimate of the SD for the change in PAT assuming no correlation, would be the 
square root of the sum of the variance estimates at both time points, which is about 0.622. A 
sample of 15 women in the placebo group and 15 in the treatment group for change in PAT 
using a two sample t test with 80% power at a significance level of 0.0167 would allow detection 
of a difference of at least 0.78 units when the sample SD is 0.622. The significance of 0.0167 
was chosen by dividing 0.05 by three for the number of comparisons between groups. 
Increasing correlation above zero between pre and post PAT values will give more precision 
and allow detection of smaller differences (e.g. a correlation of 0.8 allows detection of a 
difference of at least 0.35 units). 
 
 
 


References:  
1. 
Kado DM, Browner WS, Blackwell T, Gore R, Cummings SR. Rate of bone loss is 
associated with mortality in older women: a prospective study. Journal of bone and mineral 
research : the official journal of the American Society for Bone and Mineral Research. Oct 
2000;15(10):1974-80. doi:10.1359/jbmr.2000.15.10.1974 
2. 
Tanko LB, Christiansen C, Cox DA, Geiger MJ, McNabb MA, Cummings SR. 
Relationship between osteoporosis and cardiovascular disease in postmenopausal women. 
Journal of bone and mineral research : the official journal of the American Society for Bone and 
Mineral Research. Nov 2005;20(11):1912-20. doi:10.1359/jbmr.050711 
3. 
von der Recke P, Hansen MA, Hassager C. The association between low bone mass at 
the menopause and cardiovascular mortality. The American journal of medicine. Mar 
1999;106(3):273-8.  
4. 
Pennisi P, Signorelli SS, Riccobene S, et al. Low bone density and abnormal bone 
turnover in patients with atherosclerosis of peripheral vessels. Osteoporosis international : a 
journal established as result of cooperation between the European Foundation for Osteoporosis 
and the National Osteoporosis Foundation of the USA. May 2004;15(5):389-95. 
doi:10.1007/s00198-003-1550-9 
5. 
Thom T, Haase N, Rosamond W, et al. Heart disease and stroke statistics--2006 update: 
a report from the American Heart Association Statistics Committee and Stroke Statistics 
Subcommittee. Circulation. Feb 14 2006;113(6):e85-151. doi:CIRCULATIONAHA.105.171600 
[pii] 
10.1161/CIRCULATIONAHA.105.171600 [doi] 
6. 
Vaccarino V, Parsons L, Every NR, Barron HV, Krumholz HM. Sex-based differences in 
early mortality after myocardial infarction. National Registry of Myocardial Infarction 2 
Participants. N Engl J Med. Jul 22 1999;341(4):217-25.  
7. 
Mosca L, Appel LJ, Benjamin EJ, et al. Evidence-based guidelines for cardiovascular 
disease prevention in women. American Heart Association scientific statement. Arterioscler 
Thromb Vasc Biol. Mar 2004;24(3):e29-50. doi:10.1161/01.atv.0000114834.85476.81 
8. 
Berenson GS, Srinivasan SR, Bao W, Newman WP, 3rd, Tracy RE, Wattigney WA. 
Association between multiple cardiovascular risk factors and atherosclerosis in children and 
young adults. The Bogalusa Heart Study. N Engl J Med. Jun 4 1998;338(23):1650-6. 
doi:10.1056/nejm199806043382302 
9. 
Strong JP, Malcom GT, McMahan CA, et al. Prevalence and extent of atherosclerosis in 
adolescents and young adults: implications for prevention from the Pathobiological 
Determinants of Atherosclerosis in Youth Study. JAMA. Feb 24 1999;281(8):727-35.  
10. 
Anand SS, Islam S, Rosengren A, et al. Risk factors for myocardial infarction in women 
and men: insights from the INTERHEART study. Eur Heart J. Apr 2008;29(7):932-40. 
doi:10.1093/eurheartj/ehn018 
11. 
Ingelsson E, Lundholm C, Johansson AL, Altman D. Hysterectomy and risk of 
cardiovascular disease: a population-based cohort study. Eur Heart J. Mar 2011;32(6):745-50. 
doi:10.1093/eurheartj/ehq477 
12. 
Jensen J, Nilas L, Christiansen C. Influence of menopause on serum lipids and 
lipoproteins. Maturitas. Nov 1990;12(4):321-31.  
13. 
Jacobsen BK, Nilssen S, Heuch I, Kvale G. Does age at natural menopause affect 
mortality from ischemic heart disease? Journal of clinical epidemiology. Apr 1997;50(4):475-9.  
14. 
Parker WH, Broder MS, Chang E, et al. Ovarian conservation at the time of 
hysterectomy and long-term health outcomes in the nurses' health study. Obstetrics and 
gynecology. May 2009;113(5):1027-37. doi:10.1097/AOG.0b013e3181a11c64 
15. 
Rivera CM, Grossardt BR, Rhodes DJ, et al. Increased cardiovascular mortality after 
early bilateral oophorectomy. Menopause. 2009;16(1):15-23 10.1097/gme.0b013e31818888f7.  


16. 
Cooper GS, Sandler DP. Age at natural menopause and mortality. Ann Epidemiol. May 
1998;8(4):229-35.  
17. 
Mondul AM, Rodriguez C, Jacobs EJ, Calle EE. Age at natural menopause and cause-
specific mortality. Am J Epidemiol. Dec 1 2005;162(11):1089-97. doi:10.1093/aje/kwi324 
18. 
Joakimsen O, Bonaa KH, Stensland-Bugge E, Jacobsen BK. Population-based study of 
age at menopause and ultrasound assessed carotid atherosclerosis: The Tromso Study. J Clin 
Epidemiol. May 2000;53(5):525-30.  
19. 
Mack WJ, Slater CC, Xiang M, Shoupe D, Lobo RA, Hodis HN. Elevated subclinical 
atherosclerosis associated with oophorectomy is related to time since menopause rather than 
type of menopause. Fertil Steril. Aug 2004;82(2):391-7. doi:10.1016/j.fertnstert.2004.01.034 
20. 
Parker WHMD, Broder MSMDMPH, Chang EP, et al. Ovarian Conservation at the Time 
of Hysterectomy and Long-Term Health Outcomes in the Nurses' Health Study. [Article].  
21. 
Swerdlow AJ, Hermon C, Jacobs PA, et al. Mortality and cancer incidence in persons 
with numerical sex chromosome abnormalities: a cohort study. Ann Hum Genet. Mar 2001;65(Pt 
2):177-88. doi:doi:10.1017/S0003480001008569 
22. 
Ostberg JE, Donald AE, Halcox JP, Storry C, McCarthy C, Conway GS. Vasculopathy in 
Turner syndrome: arterial dilatation and intimal thickening without endothelial dysfunction. J Clin 
Endocrinol Metab. Sep 2005;90(9):5161-6. doi:10.1210/jc.2005-0677 
23. 
Rocca WA, Grossardt BR, de Andrade M, Malkasian GD, Melton LJ, 3rd. Survival 
patterns after oophorectomy in premenopausal women: a population-based cohort study. 
Lancet Oncol. Oct 2006;7(10):821-8. doi:10.1016/s1470-2045(06)70869-5 
24. 
Rosenberg L, Hennekens CH, Rosner B, Belanger C, Rothman KJ, Speizer FE. Early 
menopause and the risk of myocardial infarction. Am J Obstet Gynecol. Jan 1981;139(1):47-51.  
25. 
Stampfer MJ, Colditz GA, Willett WC. Menopause and heart disease. A review. Ann N Y 
Acad Sci. 1990;592:193-203; discussion 257-62.  
26. 
Caulin-Glaser T, García-Cardeña G, Sarrel P, Sessa WC, Bender JR. 17β-Estradiol 
Regulation of Human Endothelial Cell Basal Nitric Oxide Release, Independent of Cytosolic 
Ca2+ Mobilization. Circulation Research. November 1, 1997 1997;81(5):885-892. 
doi:10.1161/01.res.81.5.885 
27. 
Rebar RW. Premature ovarian failure. Obstet Gynecol. Jun 2009;113(6):1355-63. 
doi:10.1097/AOG.0b013e3181a66843 
28. 
Solomon CG, Hu FB, Dunaif A, et al. Menstrual Cycle Irregularity and Risk for Future 
Cardiovascular Disease. J Clin Endocrinol Metab. May 1, 2002 2002;87(5):2013-2017. 
doi:10.1210/jc.87.5.2013 
29. 
Ostberg JE, Storry C, Donald AE, Attar MJ, Halcox JP, Conway GS. A dose-response 
study of hormone replacement in young hypogonadal women: effects on intima media thickness 
and metabolism. Clin Endocrinol (Oxf). Apr 2007;66(4):557-64. doi:10.1111/j.1365-
2265.2007.02772.x 
30. 
Reindollar RH, Novak M, Tho SP, McDonough PG. Adult-onset amenorrhea: a study of 
262 patients. Am J Obstet Gynecol. Sep 1986;155(3):531-43.  
31. 
Kaplan JR, Manuck SB. Ovarian dysfunction and the premenopausal origins of coronary 
heart disease. Menopause (New York, NY). Jul-Aug 2008;15(4 Pt 1):768-76.  
32. 
Adams MR, Kaplan JR, Koritnik DR. Psychosocial influences on ovarian endocrine and 
ovulatory function in Macaca fascicularis. Physiol Behav. Dec 1985;35(6):935-40.  
33. 
Kaplan JR, Manuck SB, Anthony MS, Clarkson TB. Premenopausal social status and 
hormone exposure predict postmenopausal atherosclerosis in female monkeys. Obstet 
Gynecol. Mar 2002;99(3):381-8. doi:S0029784401016593 [pii] 
34. 
Punnonen R, Jokela H, Aine R, Teisala K, Salomaki A, Uppa H. Impaired ovarian 
function and risk factors for atherosclerosis in premenopausal women. Maturitas. Jul 
1997;27(3):231-8.  


35. 
Noel Bairey Merz C, Johnson BD, Sharaf BL, et al. Hypoestrogenemia of hypothalamic 
origin and coronary artery disease in premenopausal women: a report from the NHLBI-
sponsored WISE study. doi: DOI: 10.1016/S0735-1097(02)02763-8. Journal of the American 
College of Cardiology. 2003;41(3):413-419.  
36. 
Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic 
risk. Arterioscler Thromb Vasc Biol. Feb 1 2003;23(2):168-75.  
37. 
Bonetti PO, Pumper GM, Higano ST, Holmes DR, Jr., Kuvin JT, Lerman A. Noninvasive 
identification of patients with early coronary atherosclerosis by assessment of digital reactive 
hyperemia. J Am Coll Cardiol. Dec 7 2004;44(11):2137-41. doi:S0735-1097(04)01754-1 [pii] 
10.1016/j.jacc.2004.08.062 [doi] 
38. 
Matsuzawa Y, Sugiyama S, Sugamura K, et al. Digital assessment of endothelial 
function and ischemic heart disease in women. J Am Coll Cardiol. Apr 20 2010;55(16):1688-96. 
doi:10.1016/j.jacc.2009.10.073 
39. 
Mulvagh SL, Behrenbeck T, Lahr BA, et al. Endothelial function and cardiovascular risk 
stratification in menopausal women. Climacteric. Feb 2010;13(1):45-54. 
doi:10.3109/13697130902943287 
40. 
National Institutes of Health: The Practical Guide to the Identification, Evaluation and 
Treatment of Overweight and Obesity in Adults. Bethesda, Maryland: National Institutes of 
Health; 2000. 
41. 
Merz CN, Kelsey SF, Pepine CJ, et al. The Women's Ischemia Syndrome Evaluation 
(WISE) study: protocol design, methodology and feasibility report. J Am Coll Cardiol. May 
1999;33(6):1453-61. doi:S0735109799000820 [pii] 
42. 
Hamburg NM, Palmisano J, Larson MG, et al. Relation of brachial and digital measures 
of vascular function in the community: the Framingham heart study. Hypertension. Mar 
2011;57(3):390-6. doi:10.1161/hypertensionaha.110.160812 
43. 
Product Monograph Climara estradiol transdermal system. Accessed February 9, 2016, 
http://www.bayer.ca/omr/online/climara-pm-en-21nov2014-112230.pdf 
44. 
Harman SM, Brinton EA, Cedars M, et al. KEEPS: The Kronos Early Estrogen 
Prevention Study. Climacteric. Mar 2005;8(1):3-12. doi:10.1080/13697130500042417 
45. 
Moses-Kolko EL, Berga SL, Greer PJ, Smith G, Cidis Meltzer C, Drevets WC. 
Widespread increases of cortical serotonin type 2A receptor availability after hormone therapy in 
euthymic postmenopausal women. Fertil Steril. Sep 2003;80(3):554-9.  
46. 
Boerrigter PJ, van de Weijer PHM, Baak JPA, Fox H, Haspels AA, Kenemans P. 
Endometrial response in estrogen replacement therapy quarterly combined with a progestogen. 
Maturitas. 5// 1996;24(1–2):63-71. doi:http://dx.doi.org/10.1016/0378-5122(95)01020-3 
47. 
Chiazze L, Jr., Brayer FT, Macisco JJ, Jr., Parker MP, Duffy BJ. The length and 
variability of the human menstrual cycle. JAMA. Feb 5 1968;203(6):377-80.  
48. 
Powers MS, Schenkel L, Darley PE, Good WR, Balestra JC, Place VA. 
Pharmacokinetics and pharmacodynamics of transdermal dosage forms of 17 beta-estradiol: 
comparison with conventional oral estrogens used for hormone replacement. Am J Obstet 
Gynecol. Aug 15 1985;152(8):1099-106.