Study to Assess Potential Impairments in Estradiol Augmentation of Gonadotropin Secretion in Polycystic Ovary Syndrome
Phase: EARLY_PHASE1
Conditions: Polycystic Ovary Syndrome
Sponsor: University of Virginia

IRB-HSR#19692: Investigation of estradiol-induced gonadotropin surge generation in PCOS 
 
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PROTOCOL 
Background 
1. Provide the scientific background, rationale and relevance of this project.   
 
PCOS Background 
Polycystic ovary syndrome (PCOS) is among the most prevalent endocrine disorders, afflicting 7-
15% of reproductive-aged women worldwide.1–5 PCOS is characterized by hyperandrogenism 
(clinical and/or biochemical), ovulatory dysfunction, and polycystic ovarian morphology. PCOS is 
the most common etiology of anovulatory infertility.6 Additionally, it is associated with risks in 
pregnancy (e.g., 3-fold increased miscarriage rate7) and numerous metabolic disorders (e.g., 
type 2 diabetes, obesity).8 Although many advances have been made in understanding PCOS, its 
underlying pathophysiological mechanisms remain incompletely grasped, and relatively little is 
known about the causes of ovulatory dysfunction in PCOS. Additional mechanistic clarity would 
inform improved therapies for PCOS, especially for anovulation and infertility.  
Normal Regulation of the Menstrual Cycle and Pre-Ovulatory Gonadotropin Surge 
In women with normal ovulatory menstrual cycles 
(Figure 1), hypothalamic gonadotropin-releasing 
hormone (GnRH) stimulates pulsatile pituitary 
secretion of luteinizing hormone (LH) and follicle-
stimulating hormone (FSH). Higher GnRH frequencies 
favor LH secretion, while slower frequencies favor 
FSH secretion.9 During most of the normal menstrual 
cycle, estradiol (E2) and progesterone (P4) regulate 
GnRH and gonadotropin secretion via negative 
feedback mechanisms.10 For example, P4 is the 
primary inhibitor of GnRH pulse frequency.11 This 
function appears to require the presence of E2, likely 
due to E2-induced expression of hypothalamic P4 
receptors, as has been demonstrated in the rodent 
and ewe models.12,13 In the luteal phase, when P4 
and E2 levels (secreted by the corpus luteum) are 
very high, GnRH pulse frequency is suppressed; this 
slow frequency enhances FSH production (although 
pituitary release of FSH remains restrained by high luteal levels of inhibin A and E2).11 During 
the transition from the luteal to follicular phase, P4, E2, and inhibin A levels fall. Released from 
these hormones’ negative feedback, GnRH pulse frequency rises and stimulates a pituitary 
replete with FSH; this selective rise in FSH promotes ovarian follicular development. In the early 
follicular phase, a dominant follicle emerges (in response to FSH stimulation) and produces 
steadily increasing amounts of E2. At the end of the follicular phase, ovulation is triggered by 
the gonadotropin surge—largely in response to the marked increase in E2 secretion by the 
Figure 1. Normal menstrual cycle. 

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dominant ovarian follicle (i.e., E2 exerts a positive feedback effect on the pituitary).14 (Note: 
although a corresponding surge of GnRH occurs in animal models, including primates,15 
gonadotropin surges occur even in GnRH-deficient women treated with exogenous GnRH at a 
constant dose and frequency.16) During the normal surge, LH levels rise 10-fold over 2 to 3 days 
while FSH levels rise 4-fold.11  
The gonadotropin surge can be triggered by treatment with exogenous E2 alone.14 P4 also 
increases significantly approximately 12 h prior to the gonadotropin surge. When P4 is given in 
the absence of E2 priming, it cannot independently induce a surge. Nonetheless, P4 has an 
important role in gonadotropin surge generation.14,17–20 In the setting of E2 priming, P4 
increases pituitary gonadotropin release in response to GnRH.18 When P4 receptors are blocked 
with mifepristone, gonadotropin surge initiation is delayed by up to 3 days.20 Chang and Jaffe 
demonstrated that, in E2-primed women, administration of GnRH augments the release of LH 
and FSH more robustly in women treated with P4 compared to those receiving only E2.19 Liu 
and Yen used incrementally increasing doses of infused E2 and evaluated the gonadotropin 
surge with and without a superimposed P4 infusion (begun 48 h into E2 infusion); P4 
significantly prolonged the mean duration of the gonadotropin surge.14 March et al. found that 
when P4 administration followed incrementally increasing doses of E2, the LH surge occurred 
earlier (as compared to when E2 was given alone) and was accompanied by an FSH surge 
(which did not occur in the absence of P4 administration).17 Taken together, these studies 
suggest that P4 is necessary for full expression of the gonadotropin surge in terms of timing, 
magnitude, and duration. 
We have previously studied the acute effects of P4 administration in 8 normally-cycling women 
(late follicular phase) after 3 days of E2 pretreatment (0.2 mg/d transdermally).21 This placebo-
controlled crossover study was designed to assess the effect of P4 (administered at 1800 h) on 
LH pulse frequency. Although P4 did not acutely influence LH pulse frequency, we found that 
mean LH, LH pulse amplitude, and mean FSH increased markedly within 4 h of P4 
administration but not following placebo. In a recent placebo-controlled crossover study, we 
again observed an acute P4-mediated increase in mean LH and FSH in E2-pretreated women.22 
Importantly, this experimental model allows evaluation of acute P4 positive feedback on 
gonadotropin release. 
Evidence for Defective Progesterone Negative Feedback in PCOS 
In adult women with PCOS, the above-described processes are dysfunctional at multiple levels. 
Throughout the cycle, LH concentrations are higher than those seen in normal women,23 a 
result of LH pulses rising to greater amplitudes and occurring at higher frequencies.24 These 
alterations in LH pulsatility are largely a consequence of increased frequency of GnRH pulses. As 
described above, higher GnRH frequencies lead to greater secretion of LH relative to FSH. This 
shifted ratio of LH to FSH is seen in most women with PCOS,25 and is a factor that contributes 
greatly to ovulatory dysfunction and increased ovarian androgen production.  
Prior studies provide evidence for a relative GnRH pulse generator resistance to P4 negative 
feedback. Berga and Daniels reported that, with use of oral contraceptives, LH pulse frequency 
suppression was impaired in PCOS as compared to eumenorrheic women.26 Similarly, Pastor et 
al. evaluated LH pulse frequency (and by inference GnRH pulse frequency) in women with PCOS 

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and normal-cycling controls, both before and after 7 days of treatment with E2 (0.2 mg/d 
transdermally) and P4 (50 mg vaginally every 8 h) to achieve mid-luteal E2 and P4 
concentrations.27 LH pulse frequency was suppressed by 60% in normal controls but by only 
25% in PCOS. That is, higher plasma levels of P4 were needed to suppress the GnRH pulse 
generator in PCOS. Of special interest, a subsequent study by our group demonstrated that 
GnRH pulse generator sensitivity to P4 negative feedback is restored to normal after androgen-
receptor blockade with flutamide.28 Reduced GnRH pulse generator sensitivity to P4 negative 
feedback has also been reported in rodent and sheep models of PCOS.29–31 Thus, 
hyperandrogemia is implicated in the impairment of P4’s central negative feedback; this effect 
may reflect androgen-mediated down-regulation of hypothalamic P4 receptors, as has been 
demonstrated in rodent models.30 Impairment of P4’s negative feedback action on the GnRH 
pulse generator leads to persistently rapid GnRH pulse frequency with derangement of 
gonadotropin secretion, as described above. 
Evidence for Defective Progesterone Positive Feedback in Regulation of the Surge 
We have characterized defective sex steroid negative feedback on the GnRH pulse generator in 
PCOS, but the potential for dysfunction in sex steroid positive feedback in PCOS remains largely 
unexplored, particularly in relation to gonadotropin surge generation. Our preliminary data 
suggest that P4 positive feedback on LH and FSH release may be impaired in women with PCOS. 
We studied 8 women with PCOS (≥7 days after start of last menses) and 11 normally-cycling 
women (in late follicular phase, cycle days 7-10). (Figure 2.) All subjects were pretreated with 
transdermal (TD) E2, 0.2 mg/d for a total of 4 days. On day 3 of TD E2, subjects were admitted 
for frequent blood draws to characterize gonadotropin secretion. On day 4 at 0600 h, subjects 
received oral P4 100 mg. For this analysis, we 
calculated mean LH and FSH values in the 4 h 
preceding P4 administration (0200-0600 h; baseline 
levels) and 4-8 h after P4 administration (1000-1400 
h)—the latter captures peak gonadotropin levels in 
both normal controls and subjects with PCOS. P4 
positive feedback (i.e., P4-induced augmentation of 
gonadotropin release) was quantified as the ratio of 
(a) mean LH or FSH before P4 administration to (b) 
mean LH or FSH after P4 administration—hereafter 
called the “augmentation index (AI).” (For example, 
if mean LH from 0200 to 0600 h was 2.0 and mean LH 
from 1000 to 1400 h was 6.0, the LH AI would be 3.) A 
representative example of P4-induced augmentation of 
LH release is in Figure 3. 
Women with PCOS exhibited 44% higher baseline (pre-
P4) LH levels compared to controls. P4-induced LH 
augmentation (Figure 4) was 32% lower in PCOS subjects 
(AI 4.9 ± 1.8 vs. 3.3 ± 1.0 [expressed as mean ± SEM]; p = 
0.0388 by exact Wilcoxon two-sample testing). The 
Figure 3. Example, pre- and post-P4 LH. 
Figure 2. Schematic of study protocol.  

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mean baseline (pre-P4) FSH levels were similar in subjects with PCOS and controls (4.0 ± 1.6 vs. 
3.9 ± 1.4 IU/liter; p = >0.5). Acute P4-induced augmentation of FSH release (Figure 4, next page) 
was reduced by 32% in PCOS subjects when compared to controls (AI 2.3 ± 0.5 vs. 1.6 ± 0.3; p = 
0.0036 by exact Wilcoxon two-sample test). 
As expected, subjects with PCOS had 3-fold higher 
testosterone (T) concentrations. Additionally, and not 
unexpectedly, BMI was higher in the PCOS cohort (31.7 
± 2.6 vs. 22.5 ± 1.0, p = 0.0121). Since circulating 
gonadotropin levels are inversely related to BMI,25 BMI 
may have been a confounder in our analysis. However, 
P4-induced augmentation of FSH and LH release were 
reduced in PCOS even after adjusting for differences in 
BMI (p = 0.0005 and p = 0.0281, respectively). As 
another potential confounder, achieved E2 levels in 
PCOS were slightly higher than those in controls (139 ± 
24 vs. 103 ± 19 pg/ml). Although this was not 
statistically significant (p = 0.23), one may speculate 
that higher E2 could have augmented P4-mediated LH 
release while limiting P4-mediated FSH release in the 
PCOS group.  
Evidence for Defective Estradiol Positive Feedback in Regulation of the Surge 
Our preliminary data imply a defect in P4 positive feedback in E2-primed women with PCOS 
compared to E2-primed controls. However, while P4 positive feedback is believed to be 
important for the full expression of the gonadotropin surge, E2 positive feedback is of primary 
importance in generation of the surge. Of interest, prenatally-androgenized (PNA) animals (i.e., 
animal-models of PCOS) provide evidence for a defect in surge-related E2 positive feedback in 
PCOS. For example, high-dose E2 fails to initiate the gonadotropin surge in PNA rats, mice, and 
sheep.30,32,33  
The efficacy of E2 positive feedback in women with PCOS has not been thoroughly investigated. 
Baird et al. induced a gonadotropin surge in 12 women with PCOS and 6 normal women with 3 
days of ethinyl E2 200 mcg given orally.34 Plasma samples for LH and FSH were collected daily 
for 4 days before, 3 days during, and 3 days after E2 administration. In this study, the LH peak 
occurred 24 h earlier in PCOS than in controls, but the LH peak magnitude was similar in both 
groups. However, we believe that this study had important limitations. Firstly, this protocol did 
not appear to generate robust surges: LH levels increased only 2 to 3-fold (from mean basal LH 
13.6 ± 1.2 mU/mL to peak LH 34.2 ± 6.9 mU/mL), while FSH levels did not reach a statistically 
significant increase from baseline. Secondly, the control subjects were studied in the early 
follicular phase (rather than late follicular phase): we speculate that the full effects of E2 
positive feedback may not be invoked so soon after the luteal phase – if correct, this may have 
inappropriately impaired surge generation in controls. Lastly, daily blood samples are a highly 
imprecise way to quantify the gonadotropin surge. 
Defective Sex Steroid Positive Feedback in PCOS: Potential Relevance 
Figure 4. LH and FSH AIs, PCOS vs. 
controls. 

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The Endocrine Society PCOS guidelines (2013) recommend short-term clomiphene citrate—and 
possibly aromatase inhibitors such as letrozole—as preferred pharmacological therapy for 
ovulation induction in PCOS.35 Clomiphene is a selective estrogen-receptor modulator that 
antagonizes E2 action at the hypothalamus/pituitary36; letrozole similarly decreases central 
negative feedback by inhibiting E2 formation.37 Removal from negative feedback results in 
increased gonadotropin release, which promotes follicular development, selection of a 
dominant follicle, an increase in E2 production, gonadotropin surge generation, and ovulation. 
(Note that the latter events occur after drug discontinuation.) However, a significant number of 
women with PCOS fail to respond to these treatments. In comparison of clomiphene to 
metformin for ovulation induction, Legro et al. reported that 24.9% of women had no 
documented ovulations while using clomiphene, and that the live-birth rate was only 22% with 
use of clomiphene alone.38 In Legro et al.’s subsequent study comparing letrozole to 
clomiphene, the anovulation rate was 23.4% on clomiphene and 11.5% on letrozole; letrozole 
resulted in a live-birth rate of 27.5% compared to 19.1% on clomiphene.36 The failure of many 
women with PCOS to ovulate with these methods suggests a mechanistic gap in the current 
standard approach to PCOS infertility management. We propose that impairment in sex-steroid 
positive feedback and gonadotropin surge generation may provide a functional explanation for 
this therapeutic deficit.  
Hypothesis to be Tested 
We hypothesize that compared to normal controls, PCOS subjects will demonstrate a blunted 
gonadotropin response to estrogen positive feedback (i.e., a less prominent increase in 24-h 
average urinary gonadotropin concentration with E2 administration).  
 
Study Design: Biomedical 
1.  Will controls be used? Yes. 
 
►IF YES, explain the kind of controls to be used. 
Controls will be healthy, normally-cycling women without hyperandrogenism. 
 
2. What is the study design?  
This is a prospective interventional cohort study. Women with PCOS and controls will receive 
identical treatment (transdermal estradiol) and will be monitored using the same
 surveillance protocol. The study does not involve randomization, and it is not blinded. 
 
3. Does the study involve a placebo? 
No. 
Human Participants 
Ages: 18-30 
Sex: 
Female 
Race: All races will be recruited and enrolled. 
 

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Subjects- see below 
1.  Provide target # of subjects (at all sites) needed to complete protocol. 28 
 
2.  Describe expected rate of screen failure/ dropouts/withdrawals from all sites.   
We anticipate a screen failure/dropout/withdrawal rate of up to 33%. 
 
3.  How many subjects will be enrolled at all sites?   37 
 
4.  How many subjects will sign a consent form under this UVa protocol?    37 
 
5. Provide an estimated time line for the study. 
Year 1: 33.3% enrolled. 
Year 2: 33.3% enrolled. 
Year 3: 33.3% enrolled. 
Year 4: Completion of data analysis. 
Inclusion/Exclusion Criteria 
1.  List the criteria for inclusion  
• 
All included subjects will be adult women ages 18-30 years. We will use age 30 as a cutoff because 
early manifestations of reproductive senescence (e.g., evidence for reduced ovarian reserve) are more 
common in women older than age 30 years. Additionally, the risk of development of venous clots on 
estrogen treatment appears to increase beyond age 35 years, so by limiting our population to women 
age 18-30, risk associated with estrogen treatment will be limited in a healthy population. 
• 
Adult women with PCOS and normally-cycling, non-hyperandrogenic controls 
• 
General good health (excluding, for example, overweight, obesity, hyperandrogenism, PCOS, and 
adequately-treated hypothyroidism) 
• 
Capable of and willing to provide informed consent 
• 
Willing to strictly avoid pregnancy with use of non-hormonal methods during the study period 
2.  List the criteria for exclusion 
• 
Incapacity to provide informed consent including due to cognitive impairment 
• 
Males will be excluded as E2-induced gonadotropin surges and PCOS are relevant to females only  
• 
Children (age <18 years) will be excluded 
• 
Prisoners will be excluded due to the high frequency of required visits 
• 
Evidence for etiologies of anovulation/hyperandrogenism due to conditions other than PCOS 
• 
Positive pregnancy test or current lactation 
• 
Menstrual cycle frequency <26 days: Cycles <26 days suggest the possibility of relatively short follicular 
phases (e.g., < 12 days). If a subject with a follicular phase shorter than 12 days participates in our 
protocol, they could experience an endogenous gonadotropin surge under surveillance. Since we wish 
to capture only experimentally-induced surges, we will exclude such subjects.   
• 
History or physical exam evidence suggesting Cushing’s syndrome or adrenal insufficiency 
• 
Evidence of virilization (e.g., rapidly progressive hirsutism, deepening of the voice, clitoromegaly) 
• 
Total testosterone >150 ng/dL, which suggests the possibility of virilizing neoplasm 

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• 
DHEA-S greater than the upper limit of reference range for controls; mild elevations may be seen in 
PCOS, and elevations <1.5 times the upper limit of normal will be accepted in these groups. 
• 
Early morning follicular phase 17-hydroxyprogesterone greater than the follicular phase reference 
range, which suggests the possibility of congenital adrenal hyperplasia (if elevated during the luteal 
phase, the 17-hydroxyprogesterone will be repeated during the follicular phase). NOTE: If a 17-
hydroxyprogesterone level greater than the follicular phase reference range is confirmed on repeat 
testing, an ACTH stimulated 17-hydroxyprogesterone < 1000 ng/dl will be required for study 
participation. 
• 
Previous diagnosis of diabetes, fasting glucose ≥126 mg/dL, or a hemoglobin A1c ≥6.5% 
• 
Abnormal thyroid stimulating hormone (TSH). Note that subjects with stable and adequately treated 
primary hypothyroidism, reflected by normal TSH values, will not be excluded.  
• 
Hyperprolactinemia. Any degree of hyperprolactinemia (confirmed on repeat) will be grounds for 
exclusion for controls. Mild prolactin elevations may be seen in women with PCOS; elevations <50% 
higher than the upper limit of the reference range will be accepted in this group. 
• 
Persistent hematocrit <36% and hemoglobin <12 g/dL.  
• 
Severe thrombocytopenia (platelets <50,000 cells/microL) or leukopenia (total white blood count 
<4,000 cells/microL) 
• 
Persistent liver laboratory study abnormalities, with these exceptions: Mild bilirubin elevations will be 
accepted in the setting of known Gilbert’s syndrome; mild transaminase elevations may be seen in 
PCOS, therefore elevations < 1.5 times the upper limit of normal will be accepted in this group  
• 
Significant history of cardiac or pulmonary dysfunction (e.g., known or suspected congestive heart 
failure; asthma requiring intermittent systemic corticosteroid; etc.) 
• 
Decreased renal function evidenced by GFR <60 mL/min/1.73m2 
• 
No medications known to affect the reproductive system can be taken in the 3 months prior the study. 
Such medications include hormonal contraceptives, spironolactone, metformin, progestins, 
glucocorticoids, antipsychotic psychotropic drugs, etc.  
• 
Personal history of any disorders that may potentially be complicated or exacerbated by high-dose 
estradiol administration, such as hypertension (persistent, appropriately-measured blood pressure 
>140/90 mmHg), severe hypertriglyceridemia (fasting triglycerides >500 mg/dL), venous 
thromboembolism (deep venous thrombosis, pulmonary embolism), proven or suspected 
hypercoagulability, arterial thrombosis, stroke, coronary artery disease, myocardial infarction, migraine 
with aura, and breast, ovarian, or endometrial cancer 
• 
History of any other cancer diagnosis and/or treatment (with the exception of basal cell or squamous 
cell skin carcinoma) unless they have remained clinically disease free (based on appropriate 
surveillance) for five years 
• 
History of allergy or intolerance to transdermal estradiol 
• 
BMI less than 18 kg/m2 or greater than or equal to 40 kg/m2 
• 
Subjects with body weight <110 lbs. will be excluded from the study 
 
NOTE 1:  All potentially exclusionary laboratory studies will be confirmed by repeat testing to exclude 
laboratory error. 
NOTE 2:  Subjects with mild anemia (e.g., hematocrit 36%) will be offered the option to take iron in the form 
of ferrous gluconate (325 mg twice daily) for up to 60 days, after which they can return to the CRU to 
have their hemoglobin or hematocrit rechecked. Such subjects will be allowed to proceed with study 

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if hemoglobin or hematocrit is ≥12 g/dL or ≥36%, respectively. Iron will not be offered to subjects 
with a history of any disorders that may potentially be complicated by long-term iron 
supplementation, such as hemochromatosis and polycythemia vera. 
 
3.  List any restrictions on use of other drugs or treatments. 
No medications known to affect the reproductive system can be taken in the 3 months prior the study. 
Such medications include hormonal contraceptives, spironolactone, metformin, progestins, 
glucocorticoids, antipsychotic psychotropic drugs, etc.  
Statistical Considerations 
1. 
Is stratification/randomization involved? 
No. 
 
2.  What are the statistical considerations for the protocol?  
This is a prospective interventional cohort study. Women with PCOS and controls will receive identical 
treatment (transdermal estradiol) and will be monitored using the same surveillance protocol. The study does 
not involve randomization, and it is not blinded. 
 
This study is informed by our finding that progesterone-mediated augmentation of serum LH and FSH levels 
are abnormal (reduced) in PCOS compared to normal controls. Available data are inadequate to address 
whether E2-mediated augmentation of serum LH and FSH levels are abnormal (reduced) in PCOS. 
 
The primary endpoint in this study will be E2-induced fold-increase in 24-h average urinary gonadotropin 
concentration, defined as the peak 24-h mean value after E2 administration divided by the nadir 24-h mean 
value (prior to or during E2 administration). 
 
3.  Provide a justification for the sample size used in this protocol.   
The power analysis is based a comparison of progesterone-mediated augmentation of serum LH and FSH 
levels (i.e., LH and FSH Augmentation Indices) between healthy normal and PCOS study populations (described 
above).  
 
Tables 1 and 2 show, as a function of sample size, the minimum detectable differences in mean LH 
Augmentation Indices that would lead at least 80% of the time to rejecting the null hypothesis that the true 
underlying mean LH AI are the same for healthy normal and PCOS study populations. Estimates in Table 1 are 
based on differences in progesterone-mediated LH Augmentation Indices between healthy normal and PCOS 
study populations; while estimates in Table 2 are based on differences in progesterone-mediated FSH 
Augmentation Indices between healthy normal and PCOS study populations. 
 
Table 1. Data based on differences in progesterone-mediated LH Augmentation Indices between healthy 
normal and PCOS study populations. 
Number of  
Healthy Controls  
Number of  
PCOS 
Minimum Detectable  
Difference in the Mean LH AI 
10 
10 
2.19 

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11 
11 
2.09 
12 
12 
2.00 
13 
13 
1.92 
14 
14 
1.85 
15 
15 
1.79 
16 
16 
1.73 
 
Table 2. Differences in progesterone-mediated FSH Augmentation Indices between healthy normal and 
PCOS study populations. 
Number of  
Healthy Controls  
Number of  
PCOS 
Minimum Detectable  
Difference in the Mean LH AI 
10 
10 
0.73 
11 
11 
0.69 
12 
12 
0.66 
13 
13 
0.64 
14 
14 
0.61 
15 
15 
0.59 
16 
16 
0.57 
 
Since progesterone appears to be especially important for FSH augmentation (previous study), and estradiol is 
most important for LH augmentation (current study), we believe that the data in Table 2 may possibly be more 
relevant for our study. However, we wish to power our study according to the preliminary LH data—a more 
conservative approach to help ensure adequate statistical power for our study.  
 
If 14 women with PCOS and 14 controls complete this study, we expect at least 80% power to detect relevant 
differences in E2-induced augmentation of urinary LH and urinary FSH concentrations.  
Calculation Assumptions: For these calculations, we assumed that E2-induced changes in urinary 
gonadotropins—including within-group variability and between-group differences—will approximate those 
observed for P4-induced changes in serum gonadotropins. The power calculations also assumed that the LH AI 
will be normally distributed in the two study populations and that the standard deviations of the two 
underlying distributions will not exceed 1.75 units. Furthermore, the power calculations assume that the two-
sided type I error rate of the statistical test will not exceed 0.05. 
 
4.  What is your plan for primary variable analysis? 
The primary endpoint in this study will be the E2-induced fold-increase in 24-h average urinary gonadotropin 
concentration (a.k.a., the LH Augmentation Index), defined as the peak 24-h mean value after E2 
administration divided by the nadir 24-h mean value (prior to or during E2 administration). 
A comparison of the mean LH Augmentation Index (LH AI) values between the healthy normal and the PCOS 
study populations will be conducted by way of analysis of covariance (ANCOVA). Determining whether the 
component of variability in the LH AI attributed to “Study Group” (i.e. healthy normal versus PCOS) is a 
significant component of the overall variability in the LH AI will be the central hypothesis that will be tested. 
Variability in LH AI attributed to baseline disparities in mean LH will be accounted for in testing this central 

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hypothesis by treating subject-specific baseline mean LH as a covariate in the ANCOVA model. A p≤0.05 
decision rule will be utilized as the null hypothesis rejection criterion for testing the central null hypothesis 
that the mean LH AI is the same for healthy normal and the PCOS subjects who have common baseline LH.          
 
5.  What is your plan for secondary variable analysis?  
As a secondary analysis, the potential role of obesity will be explored by adding subject-specific BMI as a 
covariate in the ANCOVA model. 
As another secondary analysis, comparison of the mean FSH Augmentation Index values between the healthy 
normal and the PCOS study populations will be conducted by way of analysis of covariance (ANCOVA), with 
procedures identical to those described for LH Augmentation Index (above).  
 
6. Have you been working with a statistician in designing this protocol? 
Yes. 
 
IF YES, what is their name?   
James Patrie (Department of Public Health Sciences) 
 
7.  Will data from multiple sites be combined during analysis?   
No.  
Biomedical Research 
 
1.  What will be done in this protocol?    
 
All procedures performed in this protocol are being done solely to answer a research question and 
generate generalizable knowledge. 
 
Outpatient Consent and Screening 
After a potential subject is identified, we will arrange for her to come to the CRU to obtain informed 
consent and to perform outpatient screening procedures. The goals and procedures of the study will 
be explained to the potential subject, and she will be given the opportunity to ask any questions. If the 
potential subject wishes to proceed with the study, she will be asked to sign the consent form. A study 
physician then will record a medical history and perform a physical examination. Blood will be drawn 
for screening laboratory tests. Prior to the blood draw, the subject will need to fast for a minimum of 8 
hours. Blood obtained will be for screening of: Complete blood count, comprehensive metabolic panel, 
hemoglobin A1c, insulin, thyroid stimulating hormone, prolactin, LH, FSH, estradiol, progesterone, total 
testosterone, sex hormone binding globulin, 17-hydroxyprogesterone, DHEA-S, and beta-human 
chorionic gonadotropin. If any of these screening tests suggest exclusionary results, they may be 
repeated to exclude laboratory error. Since adiposity could be a potential confounder in this study, 
measures of adiposity will be recorded. This will include measurement of waist circumference (using 
standard technique) to provide an estimate of abdominal adiposity. Hip circumference will also be 
measured. Additionally, BOD POD® will be used to measure total fat mass, fat free mass, and percent 
body fat. 

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Study Day 1: Baseline (Pre-Transdermal Estradiol) Assessment 
x For control subjects with regular menstrual cycles, Day 1 of the study (Study Day 1) will occur 
on day 3 of the menstrual cycle. For PCOS subjects (who will have oligo-/amenorrhea), Study 
Day 1 will occur no earlier than menstrual cycle day 3. 
x At 8:00 a.m. on Study Day 1, subjects will present to the CRU to obtain urine collection supplies 
(sufficient for Study Days 1 and 2) to take home, and to have a pregnancy test (urine or serum 
hCG) and blood drawn for measurement of LH, FSH, estradiol (E2), progesterone, testosterone, 
SHBG, inhibin A, inhibin B, and antimullerian hormone (AMH).  
x NOTE: For each study day in this protocol, we will obtain blood samples for measurement of 
LH, FSH, E2, progesterone, testosterone, SHBG, inhibin A, inhibin B, and AMH. To reduce 
variability in results, blood and urine samples will be held until the completion of each 
subject’s study, and then assays will be performed in the CRR’s Ligand Assay Core. These 
results from the Ligand Assay Core will be used for formal analyses. In addition, for each 
study day, we will also obtain blood samples for E2, LH, and progesterone to be assayed 
immediately in the UVA Clinical Laboratory (to ensure rapid turnaround). These particular 
tests will be used for (a) real-time monitoring of serum E2 levels (to ensure achievement of 
target levels), (b) real-time monitoring for an LH surge (so that the protocol can be stopped 
once an LH surge occurs), and (c) real-time monitoring for an early luteal phase (so that the 
protocol can be stopped if this occurs). Details of such surveillance are provided below.   
x Subjects will use provided urine collection supplies to obtain complete urine collections in 12-
hour blocks for a total of 48 hours. Subjects will be instructed to keep these urine collections 
refrigerated or in an iced cooler. 
x Note regarding urine collections: Subjects will collect all urine produced in these 12-hour 
blocks. Urine will be stored for later assay of LH, FSH, and creatinine in the Center for 
Research in Reproduction’s Ligand Assay Core. (All LH and FSH concentrations will be 
normalized to creatinine concentrations.) 
x The pregnancy test will be done in the CRU (if done via urine testing) or in the UVAHS Clinical 
Laboratory (if done via blood testing). If the pregnancy test is positive and/or if the Study Day 1 
serum progesterone is >1.5 ng/mL, the subject will be notified of this information and they will 
not proceed with the study at that time (but plans to begin at a later date will be discussed).  
 
Study Day 2: Start Transdermal Estradiol 
x At approximately 8:00 am on Study 
Day 2, subjects will present to the CRU 
to return the refrigerated Study Day 1 
urine samples and to have blood 
drawn for LH, FSH, E2, progesterone, 
testosterone, SHBG, inhibin A, inhibin 
B, and AMH. 
x NOTE: As above, E2, LH, and 
progesterone will be assayed in both 
(a) the UVA Clinical Laboratory (to ensure rapid turnaround) and (b) the CRR’s Ligand Assay 

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Core. And on study day 2 only, we will also obtain blood to be sent for total testosterone by LC-
MS-MS (send out to Mayo Clinic Laboratory).  
• After blood collection, two transdermal estradiol patches (each patch delivering 0.1 mg/day for 
a total dose 0.2 mg/day) will be applied by CRU staff at 8:15-8:30 a.m. Throughout this 
protocol, transdermal estradiol patches will be preferentially placed on the abdomen. 
• NOTE: In our previous studies21,22, transdermal estradiol doses of 0.2 mg/day produce mean 
serum E2 level approximating 100-120 pg/ml. 
• Subjects will be provided with urine collection supplies in a sufficient quantity to last through 
Study Day 9.  
• Subjects will also be provided with 5 extra transdermal 0.1 mg/day estradiol patches to take 
home. These extra patches will be used for replacement purposes in the event of patch loss 
(e.g., patch[es] falling off). [NOTE: Extra transdermal patches will be subsequently provided as 
needed to maintain a reserve of at least 5 patches.] 
 
Study Day 3: Monitor + Increase Transdermal Estradiol Dose to 0.3 mg/day 
• At approximately 8:00 a.m. on Study Day 3, the subject will present to the CRU to return the 
refrigerated Study Day 2 urine samples and to have blood drawn for LH, FSH, E2, progesterone, 
testosterone, SHBG, inhibin A, inhibin B, and AMH. As above, E2, LH, and progesterone will be 
assayed in both (a) the CRR’s Ligand Assay Core and (b) the UVA Clinical Laboratory.   
• The presence and condition of the prior patches will be verified and recorded; the patches will 
then be removed.  
• Three transdermal estradiol patches (each patch delivering 0.1 mg/day for a total dose 0.3 
mg/day) will be applied by CRU staff. 
 
Study Day 4: Monitor + Increase Transdermal Estradiol Dose to 0.4 mg/day 
• At approximately 8:00 a.m. on Study Day 4, the subject will present to the CRU to return the 
refrigerated Study Day 3 urine samples and to have blood drawn for LH, FSH, E2, progesterone, 
testosterone, SHBG, inhibin A, inhibin B, and AMH.  
• As above, E2, LH, and progesterone will be assayed in both (a) the CRR’s Ligand Assay Core and 
(b) the UVA Clinical Laboratory. These particular tests will be run through the UVA Clinical 
Laboratory so that results will be available rapidly. These results will be used as follows: 
• We will perform real-time monitoring of serum E2 levels (i.e., real-time monitoring of 
transdermal estradiol dosing) to ensure achievement of appropriate target levels. That is, 
we want to ensure that serum E2 levels are high enough to trigger a gonadotropin surge 
(~250-300 pg/ml), but we wish to avoid prolonged exposure to excessive serum E2 levels 
(especially levels exceeding 400 pg/ml). Accordingly:  
• If a subject’s serum E2 level was < 250 pg/ml on the preceding Study Day, the 
transdermal estradiol dose will be increased by 0.1 mg/day per protocol.  
• If a subject’s serum E2 was 250-350 pg/ml on the preceding Study Day, that same 
transdermal estradiol dose will be maintained for the next 24 hours footnote 1. (NOTE: 
                                                 
1  For example, if a subject’s serum E2 level was 275 pg/ml on Study Day 4 — when the subject was receiving 0.3 mg/day 
transdermal estradiol – the transdermal estradiol dose would be maintained at 0.3 mg/day on Study Day 5. 

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Since a given day’s serum E2 levels are unlikely to be available before the subject leaves 
the CRU, and since the protocol may dictate an increase in the transdermal estradiol 
dose during the CRU visit, continuation of the same dose could require that the subject 
pull a single estradiol patch off her skin shortly after the CRU visit.) 
• If a subject’s serum E2 level is > 350 pg/ml on a given dose of transdermal estradiol – 
which we think will be unlikely in this protocol – the transdermal estradiol dose will be 
decreased to the previous dose that had been associated with a serum E2 value 
approximating 250-350 pg/mL. If there is uncertainty regarding the appropriate dose 
reduction, we will proportionally reduce the dose according to measured serum E2 
levels, aiming to achieve serum E2 levels of 250-350 pg/ml footnote 2.  
• We will perform real-time monitoring for an LH surge (so that the protocol can be stopped 
once an LH surge occurs). If a subject demonstrates an LH surge, further monitoring will not 
be required. Therefore, if the subject’s serum LH peaks to >5-fold (500%) her Study Day 1 or 
2 serum values and then subsequently falls to within 2-fold (200%) of the Study Day 1 or 2 
serum values, the subject’s study will be stopped on the following day (e.g., after the next 
morning’s blood sample is drawn).  
• We will perform real-time monitoring for entry into the luteal phase. Specifically, a serum 
progesterone level of 2.5 ng/ml or higher would provide compelling evidence of an early 
luteal phase, which would strongly suggest previous gonadotropin surge generation and 
ovulation. Accordingly, if serum progesterone ever equals or exceeds 2.5 ng/ml, we will stop 
the protocol on the following day (e.g., after the next morning’s blood sample is drawn). 
(We do not expect this to occur in this protocol.) 
• The presence and condition of the prior patches will be verified and recorded; the patches will 
then be removed.   
• New transdermal E2 patches will then be applied by CRU staff: 
• If the subject’s serum E2 level was < 250 pg/ml on Study Day 3 (most likely scenario), then 
the transdermal estradiol dose will be increased to 0.4 mg/day.  
• If the subject’s serum E2 was 250-350 pg/ml on Study Day 3 (possible but unlikely scenario), 
that same transdermal estradiol dose will be continued. 
• If the subject’s serum E2 level was > 350 pg/ml on Study Day 3 (we believe that this will be a 
highly unlikely scenario), then the transdermal estradiol dose would have been reduced 
(see above), and the reduced dose will be continued. 
 
 
Study Day 5 – Study Day 8: Monitor + Adjust Transdermal Estradiol Dose According to Serum E2 
Levels 
• At approximately 8:00 a.m. on Study Days 5 - 8, the subject will present to the CRU to: 
1. return the refrigerated prior Study Day’s urine samples; 
                                                 
2  For example, if serum E2 levels are 400 pg/ml on 0.5 mg/d transdermal estradiol, each 0.1 mg/d dose reduction would 
be expected to lower serum E2 levels by ~ 80 pg/ml (400 divided by 5), so a dose reduction to 0.4 mg/d would be 
expected to achieve an acceptable serum E2 level of ~ 320 pg/ml. 
 

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2. have blood drawn for LH, FSH, E2, progesterone, testosterone, SHBG, inhibin A, inhibin B, 
and AMH (with E2, LH, and progesterone also immediately assayed in the UVA Clinical 
Laboratory for real-time monitoring as described above); 
3. have the presence and condition of the prior patches verified and recorded, and 
subsequently removed; and  
4. have new transdermal estradiol patches applied by CRU staff:  
• If the subject’s serum E2 level was < 250 pg/ml on the preceding Study Day, then the 
transdermal estradiol dose will be increased by 0.1 mg/day (maximum dose = 0.6 
mg/day). 
• If the subject’s serum E2 was 250-350 pg/ml on the preceding Study Day, that same 
transdermal estradiol dose will be continued. 
• If the subject’s serum E2 level was > 350 pg/ml on the preceding Study Day, then the 
transdermal estradiol dose would have been reduced (see above), and the reduced dose 
will be continued. 
 
Optional ovarian ultrasound on Study Day 6 or 7: To evaluate for a dominant ovarian follicle, subjects 
may elect to undergo transvaginal ovarian ultrasound in the CRU or in the Endocrinology Clinic 
(Fontaine Research Park) on study day 6 or 7. This ultrasound will be optional (i.e., subjects may opt 
out with no negative consequences). Dominant follicle size and endometrial stripe thickness will be 
recorded. 
 
Study Day 9: Monitor + Discontinue Transdermal Estradiol 
• At approximately 8:00 a.m. on Study Day 9, the subject will present to the CRU to: 
1. return the refrigerated prior Study Day’s urine samples; 
2. have blood drawn for LH, FSH, E2, progesterone, testosterone, SHBG, inhibin A, inhibin B, 
and AMH; 
3. have the presence and condition of the prior patches verified and recorded, and 
subsequently removed. 
 
Final Study Day (7-9 days after estradiol patch discontinuation) 
• Subject will present to the CRU to have blood drawn for LH, FSH, E2, progesterone, 
testosterone, SHBG, inhibin A, inhibin B, and AMH. 
 
 
Data and Safety Monitoring Plan 
1.  Definition: 
1.1 How will you define adverse events (AE)) for this study?  
__X__ An adverse event will be considered any undesirable sign, symptom or medical or 
psychological condition even if the event is not considered to be related to the investigational 
drug/device/intervention. Medical condition/diseases present before starting the investigational 
drug/intervention will be considered adverse events only if they worsen after starting study 
treatment/intervention.  An adverse event is also any undesirable and unintended effect of 

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research occurring in human subjects as a result of the collection of identifiable private information 
under the research.  Adverse events also include any problems associated with the use of an 
investigational device that adversely affects the rights, safety or welfare of subject s. 
_____Will use definitions provided in the non IRB Protocol (Sponsor's, Investigator-Initiated, CTEP etc.) 
1.2 How will you define serious adverse events? 
__X__ A serious adverse event will be considered any undesirable sign, symptom, or medical 
condition which is fatal, is life-threatening, requires or prolongs inpatient hospitalization, results 
in persistent or significant disability/incapacity, constitutes a congenital anomaly or birth 
defect, is medically significant and which the investigator regards as serious based on 
appropriate medical judgment. An important medical event is any AE that may not result in 
death, be life-threatening, or require hospitalization but may be considered an SAE when, 
based upon appropriate medical judgment, it may jeopardize the patient and may require 
medical or surgical intervention to prevent one of the outcomes listed in the definitions of SAEs.  
1.3 What is the definition of an unanticipated problem?  
An unanticipated problem is any event, experience that meets ALL 3 criteria below: 
 Is unexpected in terms of nature, severity or frequency given the research procedures that 
are described in the protocol-related documents AND in the characteristics of the subject 
population being studies 
 Related or possibly related to participation in research.  This means that there is a 
reasonable possibility that the incident may have been caused by the procedures involved 
in the research study. 
 The incident suggests that the research placed the subject or others at greater risk of harm 
than was previously known or recognized OR results in actual harm to the subject or others 
 
1.4 What are the definitions of a protocol violation and/or noncompliance?  
A protocol violation is defined as any change, deviation, or departure from the study design or 
procedures of research project that is NOT approved by the IRB-HSR prior to its initiation or 
implementation.  Protocol violations may be major or minor violations.   
 
Noncompliance can be a protocol violation OR deviation from standard operating procedures, 
Good Clinical Practices (GCPs), federal, state or local regulations.   Noncompliance may be serious 
or continuing.  
Additional Information: see the IRB-HSR website at  
http://www.virginia.edu/vpr/irb/HSR_docs/Forms/Protocol_Violations_%20Enrollment_Exceptions
_Instructions.doc 
 
1.5 If pregnancy occurs how will this information be managed? 
_____  Adverse Event- will follow adverse event recording and reporting procedures outlined in 
section 3.  
 

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__X__  Unanticipated Problems- will follow Unanticipated Problem recording and reporting 
procedures outlined in section 3.  
Note that pregnancy could not be deemed to be related/possibly related to this 
study, as none of the procedures increases likelihood of pregnancy, and subjects are 
specifically required to avoid pregnancy. 
 
1.6 What is the definition of a Protocol Enrollment Exception?   
__X__         NA- No outside sponsor 
 
1.7 What is the definition of a data breach? 
A data breach is defined in the HITECH Act (43 USC 17932) as an unauthorized acquisition, access, 
or use of protected health information (PHI) that compromises the security or privacy of such 
information. 
Additional Information may be found on the IRB-HSR Website: Data Breach 
2.  Identified risks and plans to minimize risk 
 
2.1 What risks are expected due to the intervention in this protocol? 
 
Expected Risks related to study 
participation. 
Frequency 
 
Risk of withholding specific 
medications that would interfere with 
protocols 
Subjects with PCOS commonly are prescribed medications 
including hormonal contraceptives, spironolactone, and metformin 
for management of PCOS, hyperandrogenism, and insulin 
resistance. Women without PCOS commonly are prescribed 
hormonal contraceptives for purposes including pregnancy 
prevention.  
We believe that elective withholding of such medications for up to 
4 months would pose minimal risk to subjects with PCOS.  
Subjects previously using oral contraceptives for the purpose of 
reducing risk of pregnancy would be at risk of an unplanned 
pregnancy if they remain sexually active without use of non-
hormonal methods of contraception. 
Risks associated with venipuncture 
 
Discomfort 
_X__Occurs frequently 
____Occurs infrequently 
____Occurs rarely 
____Frequency unknown 
Bruising 
_X__Occurs frequently 
____Occurs infrequently 
____Occurs rarely 
____Frequency unknown 

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Infection 
____Occurs frequently 
____Occurs infrequently 
_X__Occurs rarely 
____Frequency unknown 
Blood clot at the site of intravenous 
catheter insertion 
____Occurs frequently 
____Occurs infrequently 
_X__Occurs rarely 
____Frequency unknown 
Risks associated with transdermal 
estradiol use 
 
Deep venous thrombosis/Pulmonary 
embolism 
____Occurs frequently 
____Occurs infrequently 
____Occurs rarely 
              __X_Frequency unknown. This occurs very rarely with 
long-term estrogen use (for example, in oral contraceptive pills), so 
we would expect this to be exceedingly rare with short-term 
administration as in this protocol. In addition, transdermal 
estradiol use is associated with lower deep venous thrombosis risk 
compared to oral estrogen regimens (e.g., oral contraceptives), 
and we are aiming to achieve preovulatory (i.e., physiological) 
serum estradiol levels for a relatively short period of time. We will 
be excluding subjects with history of DVT, pulmonary embolism, or 
clotting disorders. 
Myocardial infarction 
____Occurs frequently 
___  Occurs infrequently  
____Occurs rarely 
__X__    Frequency unknown – This is thought to be 
extremely rare. The risk of this will be minimized by 
excluding any subjects with a history of coronary artery 
disease or myocardial infarction. 
Stroke 
____Occurs frequently 
___  Occurs infrequently  
____Occurs rarely 
__X__    Frequency unknown – This is thought to be 
extremely rare. The risk of this will be minimized by 
excluding any subjects with a history of stroke or migraine 
with aura. 
Hypertension 
____Occurs frequently 
___  Occurs infrequently  
__X_Occurs rarely – Risk of this will be minimized by 
excluding any subjects with a history of hypertension or 
sustained blood pressure >140/90 on screening 
assessment. 

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    _    Frequency unknown 
Hypertriglyceridemia 
____Occurs frequently 
___  Occurs infrequently  
__X_Occurs rarely – Risk of this will be minimized by 
excluding any subjects with a history of 
hypertriglyceridemia. Also, the short-term risks of 
hypertriglyceridemia are minimal. 
    _    Frequency unknown 
Stimulation of growth of breast, 
ovarian, or endometrial cancers 
____Occurs frequently 
___  Occurs infrequently  
__X_Occurs rarely – Risk of this will be minimized by 
excluding any subjects with a history of breast, ovarian, or 
endometrial cancers. Also, no short-term risks are expected 
in this regard. 
    _    Frequency unknown 
Rash or skin irritation at site of patch 
application 
____Occurs frequently 
_X__Occurs infrequently – 15-20% of subjects. Risk will be 
minimized by daily rotation of application sites. 
____Occurs rarely 
              ____Frequency unknown 
Mild nausea 
____Occurs frequently 
_X__Occurs infrequently 
____Occurs rarely 
____Frequency unknown 
Swelling or breast tenderness 
____Occurs frequently 
_X__Occurs infrequently 
____Occurs rarely 
 ____Frequency unknown 
Temporary hot flashes after 
discontinuation 
____Occurs frequently 
_ X__Occurs infrequently 
____Occurs rarely 
 ____Frequency unknown 
Risks associated with ferrous gluconate 
supplementation 
 
Constipation 
____Occurs frequently 
_X__Occurs infrequently 
____Occurs rarely 
              ____Frequency unknown 
Darkening of stools 
_X__Occurs frequently 
____Occurs infrequently 
____Occurs rarely 
              ____Frequency unknown 
Nausea 
____Occurs frequently 

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_X__Occurs infrequently 
____Occurs rarely 
 ____Frequency unknown 
Reproductive Risks 
Minimized due to the requirements of this protocol. 
Violation of subject’s privacy and 
confidentiality 
Minimized due to the requirements of the privacy plan in 
this protocol 
 
2.2   List by bullet format a summary of safety tests/procedures/observations to be performed that will 
minimize risks to participants:  
• Risk of venipuncture will be reduced by use of sterile technique. 
• Risks of transdermal estradiol will be reduced by screening for and exclusion of any subjects who 
may be at increased risk of developing complications while using E2, including: those with history 
of hypertension (persistent, appropriately-measured blood pressure >140/90 mmHg), severe 
hypertriglyceridemia (fasting triglycerides >500 mg/dL), venous thromboembolism (deep venous 
thrombosis, pulmonary embolism), proven or suspected hypercoagulability, arterial thrombosis, 
stroke, coronary artery disease, myocardial infarction, migraine with aura, and breast, ovarian, or 
endometrial cancer. Additionally, if a history of allergy or intolerance to estradiol is reported, the 
subject will be excluded from the study. Also, daily serum E2 levels will be monitored in order to 
detect and correct excessively high estradiol exposure (described in detail above). 
• Risk of skin irritation from use of estradiol patch will be reduced by rotation of sites of E2 patches to 
avoid skin irritation and/or rash. 
• Risk of fetal exposure to the study drug will be reduced by having a pregnancy test assessed prior 
to initiation of transdermal estradiol. If the volunteer is pregnant, the study will be discontinued. 
• Risk from iron supplementation (when used) will be reduced by instructing subjects to take their 
iron tablets with food and a full glass of water. They will be told to include whole grains and fruits 
and vegetables in their diet for fiber. Also, iron supplementation will not be offered to subjects 
with a history of any disorders that may potentially be complicated by long-term iron 
supplementation, such as hemochromatosis and polycythemia vera. 
• We will exclude subjects with abnormal screening labs with confirmatory testing showing: severe 
thrombocytopenia (platelets <50,000 cells/microL), leukopenia (total white blood count <4,000 
cells/microL), persistent liver laboratory study abnormalities (with these exceptions: Mild bilirubin 
elevations will be accepted in the setting of known Gilbert’s syndrome; mild transaminase 
elevations may be seen in PCOS, therefore elevations < 1.5 times the upper limit of normal will be 
accepted in this group), Decreased renal function evidenced by GFR <60 mL/min/1.73m2.   
• We will exclude subjects who report on screening history a significant history of cardiac or 
pulmonary dysfunction (e.g., known or suspected congestive heart failure; asthma requiring 
intermittent systemic corticosteroid; etc.). 
 
2.3 Under what criteria would an INDIVIDUAL SUBJECT’S study treatment or study participation be stopped 
or modified 
_X__At subject, PI or sponsor’s request 
 

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_ X___Treatment would be stopped if the subject had a serious adverse event deemed related to 
study 
 
_____Refer to the non- IRB Protocol (Sponsor's, Investigator-Initiated, CTEP protocol etc.) 
 
2.4 Under what criteria would THE ENTIRE STUDY need to be stopped.   
__X__Per IRB, PI, DSMB, or sponsor discretion 
 
2.5 What are the criteria for breaking the blind/mask? 
__X__NA – Not blinded/masked 
 
2.6 
How will subject withdrawals/dropouts be reported to the IRB prior to study completion? 
__X__IRB-HSR continuation status form  
3.  Adverse Event / Unanticipated Problem Recording and Reporting 
 
3.1 Will all adverse events, as defined in section 1.1, be collected/recorded?   No. 
►IF NO, what criteria will be used?  
 
__X__Only adverse events deemed related/possibly related to study  
3.2 How will adverse event data be collected/recorded? 
__X__Paper AE forms/source documents  
__X__Spreadsheet: paper or electronic 
_____Database  
 
3.3. How will AEs be classified/graded? 
_X___Mild/Moderate/Severe 
_X_ Serious/Not serious Required for all protocols 
 
 
3.4 What scale will the PI use when evaluating the relatedness of adverse events to the study 
participation?   
 
__X__The PI will determine the relationship of adverse events to the study 
 using the following scale: 
 
Related:      
 
AE is clearly related to the intervention 
Possibly related: 
AE may be related to the intervention 
Unrelated:  
 
AE is clearly not related to intervention 
 
3.5 When will recording/reporting of adverse events/unanticipated problems begin? 
__X__After subject begins study drug/ device placement/intervention /study-related 
procedure/specimen collection 

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3.6 When will the recording/reporting of adverse events/unanticipated problems end? 
__X__ Subject completes intervention and follow up period of protocol 
3.7 How will Adverse Events, Unanticipated Problems, Protocol Violations and Data Breaches be 
reported?  Complete the table below to answer this question 
 
Type of Event 
To whom will it 
be reported: 
Time Frame for 
Reporting 
How reported? 
Any internal event resulting 
in death that is deemed 
DEFINITELY related to (caused 
by) study participation 
An internal event is one that 
occurs in a subject enrolled in 
a UVa protocol 
IRB-HSR 
Within 24 hours 
IRB Online and phone call 
 
www.irb.virginia.edu/ 
 
Internal, Serious, 
related/possibly related 
to study, Unexpected 
adverse event  
 
  
IRB-HSR 
Within 7 calendar 
days from the 
time the study 
team received 
knowledge of the 
event. 
 
Timeline includes 
submission of 
signed hardcopy 
of AE form. 
IRB Online 
 
www.irb.virginia.edu/ 
 
Unanticipated Problems that 
are not adverse events or 
protocol violations  
This would include a Data 
Breach.   
IRB-HSR 
 
 
Within 7 calendar 
days from the 
time the study 
team received 
knowledge of the 
event.  
Unanticipated Problem report 
form.  
 
http://www.virginia.edu/vprgs
/irb/HSR_docs/Forms/Reportin
g_Requirements-
Unanticipated_Problems.doc ) 
Protocol 
Violations/Noncompliance 
The IRB-HSR only requires that 
MAJOR violation be reported, 
unless otherwise required by 
your sponsor, if applicable. 
 
OR 
 
Enrollment Exceptions 
See definition- only allowed if 
there is a commercial sponsor 
or a DSMB that has granted 
the enrollment exception.  
IRB-HSR 
 
 
Within 7 calendar 
days from the 
time the study 
team received 
knowledge of the 
event.  
 
Protocol Violation, 
Noncompliance and 
Enrollment Exception 
Reporting Form 
 
 http://www.virginia.edu/vprgs
/irb/hsr_forms.html 
 
Go to 3rd bullet from the 
bottom. 
  

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Data Breach  
The UVa 
Corporate 
Compliance and 
Privacy Office 
 
 
 
ITC:  if breach 
involves  
electronic data  
 
 
 
 
Police if breach 
includes items 
that are stolen: 
 
Stolen on UVA 
Grounds 
 
OR  
 
Stolen off UVa 
Grounds- contact 
police 
department of 
jurisdiction of last 
known location of 
PHI 
As soon as 
possible and no 
later than 24 
hours from the 
time the incident 
is identified. 
 
As soon as 
possible and no 
later than 24 
hours from the 
time the incident 
is identified. 
 
IMMEDIATELY.  
 
 
UVa Corporate Compliance and 
Privacy Office- Phone 924-9741 
 
 
 
 
ITC:  Information Security 
Incident Reporting 
procedure,  http://www.itc.virg
inia.edu/security/reporting.ht
ml 
 
 
 
 
 
 
 
 
UVa Police-Phone- (434) 924-
7166 
  
 
4.    How will the endpoint data be collected/recorded.  
_____Protocol specific case report forms  
__X__Source documents 
__X__Database: On a secure UVA server. 
5. Data and Safety Oversight Responsibility 
 
5.1. Who is responsible for overseeing safety data for this study?  
 
__X__DSMB/ DSMC 
 
5.2. What is the composition of the reviewing body and how is it affiliated with the sponsor?   
 
__X__Other- The DSMB is composed of an endocrinologist sub-specializing in reproductive endocrinology, 
a pediatric endocrinologist, and a Ph.D. statistician. The board meets at least annually and as needed in the
case of any severe AE that may be related to or is related to a study. 
 

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5.3. What items will be included in the aggregate review conducted by the PI?  
 
__x___NA- PI is not the overall person overseeing the safety data for this study.     
 
5.4 How often will aggregate review occur? 
For additional information on aggregate review 
see: www.virginia.edu/vpr/irb/hsr/continuations.html#aggreview 
__X__Annually 
 
5.5. How often will a report, regarding the outcome of the review by the DSMB/DSMC, be sent to the 
UVa PI? 
__X__Annually 
 
5.6. How will a report of the information discussed in question 5.4 OR 5.5 be submitted to the IRB?   
 
__X__Part of IRB-HSR continuation status form 
Bibliography 
1.  
Tehrani FR, Simbar M, Tohidi M, Hosseinpanah F, Azizi F. The prevalence of polycystic ovary syndrome 
in a community sample of Iranian population: Iranian PCOS prevalence study. Reprod Biol Endocrinol. 
2011;9:39. 
2.  
March WA, Moore VM, Willson KJ, Phillips DI, Norman RJ, Davies MJ. The prevalence of polycystic 
ovary syndrome in a community sample assessed under contrasting diagnostic criteria. Hum Reprod. 
2010;25(2):544-551. 
3.  
Jiao J, Fang Y, Wang T, Wang Z, Zhou M, Wang X. Epidemiologic investigation of polycystic ovarian 
syndrome (PCOS) in Han ethnic women of reproductive age in Liaoning Province, China. Clin Exp Obstet 
Gynecol. 2014;41(3):304-309. 
4.  
Azziz R, Woods KS, Reyna R, Key TJ, Knochenhauer ES, Yildiz BO. The prevalence and features of the 
polycystic ovary syndrome in an unselected population. J Clin Endocrinol Metab. 2004;89(6):2745-
2749. 
5.  
Asuncion M, Calvo RM, Millan JLS, Sancho J, Avila S, Escobar-Morreale HF. A prospective study of the 
prevalence of the polycystic ovary syndrome in unselected Caucasian women from Spain. J Clin 
Endocrinol Metab. 2000;85(7):2434-2438. 
6.  
Consensus workshop group RE-SP. Revised 2003 consensus on diagnostic criteria and long-term health 
risks related to polycystic ovary syndrome (PCOS). Hum Reprod. 2004;19(1):41-47. 
7.  
Katulski K, Czyzyk A, Podfigurna-Stopa A, Genazzani AR, Meczekalski B. Pregnancy complications in 
polycystic ovary syndrome patients. Gynecol Endocrinol. 2015;31(2):87-91. 
8.  
Dumesic DA, Oberfield SE, Stener-Victorin E, Marshall JC, Laven JS, Legro RS. Scientific Statement on 
the Diagnostic Criteria, Epidemiology, Pathophysiology, and Molecular Genetics of Polycystic Ovary 
Syndrome. Endocr Rev. 2015;36(5):487-525. 

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Gross KM, Matsumoto AM, Bremner WJ. Differential control of luteinizing hormone and follicle-
stimulating hormone secretion by luteinizing hormone-releasing hormone pulse frequency in man. J 
Clin Endocrinol Metab. 1987;64(4):675-680. 
10.  
Wang CF, Lasley BL, Lein A, Yen SS. The functional changes of the pituitary gonadotrophs during the 
menstrual cycle. J Clin Endocrinol Metab. 1976;42(4):718-728. doi:10.1210/jcem-42-4-718. 
11.  
Hall JE. Neuroendocrine Control of the Menstrual Cycle. In: J. Strauss RB, ed. Endocrinology of 
Reproduction. 7th ed.; 2014:131-154. 
12.  
MacLusky NJ, McEwen BS. Oestrogen modulates progestin receptor concentrations in some rat brain 
regions but not in others. Nature. 1978;274(5668):276-278. 
13.  
Scott CJ, Pereira AM, Rawson JA, Simmons DM, Rossmanith WG, Ing NH, Clarke IJ. The distribution of 
progesterone receptor immunoreactivity and mRNA in the preoptic area and hypothalamus of the 
ewe: upregulation of progesterone receptor mRNA in the mediobasal hypothalamus by oestrogen. J 
Neuroendocrinol. 2000;12(6):565-575. 
14.  
Liu JH, Yen SS. Induction of midcycle gonadotropin surge by ovarian steroids in women: a critical 
evaluation. J Clin Endocrinol Metab. 1983;57(4):797-802. doi:10.1210/jcem-57-4-797. 
15.  
Levine JE, Norman RL, Gliessman PM, Oyama TT, Bangsberg DR, Spies HG. In vivo gonadotropin-
releasing hormone release and serum luteinizing hormone measurements in ovariectomized, estrogen-
treated rhesus macaques. Endocrinology. 1985;117(2):711-721. doi:10.1210/endo-117-2-711. 
16.  
Hall JE, Martin KA, Whitney HA, Landy H, Crowley WFJ. Potential for fertility with replacement of 
hypothalamic gonadotropin-releasing hormone in long term female survivors of cranial tumors. J Clin 
Endocrinol Metab. 1994;79(4):1166-1172. doi:10.1210/jcem.79.4.7962290. 
17.  
March CM, Goebelsmann U, Nakamura RM, Mishell DRJ. Roles of estradiol and progesterone in 
eiliciting the midcycle luteinizing hormone and follicle-stimulating hormone surges. J Clin Endocrinol 
Metab. 1979;49(4):507-513. doi:10.1210/jcem-49-4-507. 
18.  
Nippoldt TB, Khoury S, Barkan A, Kelch RP, Marshall JC. Gonadotrophin responses to GnRH pulses in 
hypogonadotrophic hypogonadism: LH responsiveness is maintained in the presence of luteal phase 
concentrations of oestrogen and progesterone. Clin Endocrinol (Oxf). 1987;26(3):293-301. 
19.  
Chang RJ, Jaffe RB. Progesterone effects on gonadotropin release in women pretreated with estradiol. J 
Clin Endocrinol Metab. 1978;47(1):119-125. doi:10.1210/jcem-47-1-119. 
20.  
Batista MC, Cartledge TP, Zellmer AW, Nieman LK, Loriaux DL, Merriam GR. The antiprogestin RU486 
delays the midcycle gonadotropin surge and ovulation in gonadotropin-releasing hormone-induced 
cycles. Fertil Steril. 1994;62(1):28-34. 
21.  
McCartney CR, Blank SK, Marshall JC. Progesterone acutely increases LH pulse amplitude but does not 
acutely influence  nocturnal LH pulse frequency slowing during the late follicular phase in women. Am J 
Physiol Endocrinol Metab. 2007;292(3):E900-E906. doi:10.1152/ajpendo.00371.2006. 
22.  
Hutchens EG, Ramsey KA, Howard LC, Abshire MY, Patrie JT MC. Progesterone has rapid positive 
feedback actions on LH release but fails to reduce LH pulse frequency within 12 hours in estradiol-
pretreated women. Physiol Rep. 2016;4(7):e12891. 

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Rebar R, Judd HL, Yen SS, Rakoff J, Vandenberg G, Naftolin F. Characterization of the inappropriate 
gonadotropin secretion in polycystic ovary  syndrome. J Clin Invest. 1976;57(5):1320-1329. 
doi:10.1172/JCI108400. 
24.  
Waldstreicher J, Santoro NF, Hall JE, Filicori M, Crowley WFJ. Hyperfunction of the hypothalamic-
pituitary axis in women with polycystic ovarian disease: indirect evidence for partial gonadotroph 
desensitization. J Clin Endocrinol Metab. 1988;66(1):165-172. doi:10.1210/jcem-66-1-165. 
25.  
Taylor AE, McCourt B, Martin KA, Anderson EJ, Adams JM, Schoenfeld D, Hall JE. Determinants of 
abnormal gonadotropin secretion in clinically defined women with  polycystic ovary syndrome. J Clin 
Endocrinol Metab. 1997;82(7):2248-2256. doi:10.1210/jcem.82.7.4105. 
26.  
Daniels TL, Berga SL. Resistance of gonadotropin releasing hormone drive to sex steroid-induced 
suppression in hyperandrogenic anovulation. J Clin Endocrinol Metab. 1997;82(12):4179-4183. 
doi:10.1210/jcem.82.12.4402. 
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Pastor CL, Griffin-Korf ML, Aloi JA, Evans WS, Marshall JC. Polycystic ovary syndrome: evidence for 
reduced sensitivity of the gonadotropin-releasing hormone pulse generator to inhibition by estradiol 
and progesterone. J Clin Endocrinol Metab. 1998;83(2):582-590. 
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Eagleson CA, Gingrich MB, Pastor CL, Arora TK, Burt CM, Evans WS, Marshall JC. Polycystic ovarian 
syndrome: evidence that flutamide restores sensitivity of the gonadotropin-releasing hormone pulse 
generator to inhibition by estradiol and progesterone. J Clin Endocrinol Metab. 2000;85(11):4047-
4052. 
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Pielecka J, Quaynor SD, Moenter SM. Androgens increase gonadotropin-releasing hormone neuron 
firing activity in females and interfere with progesterone negative feedback. Endocrinology. 
2006;147(3):1474-1479. doi:10.1210/en.2005-1029. 
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Foecking EM, Szabo M, Schwartz NB, Levine JE. Neuroendocrine consequences of prenatal androgen 
exposure in the female rat: absence of luteinizing hormone surges, suppression of progesterone 
receptor gene expression, and acceleration of the gonadotropin-releasing hormone pulse generator. 
Biol Reprod. 2005;72(6):1475-1483. doi:10.1095/biolreprod.105.039800. 
31.  
Robinson JE, Forsdike RA, Taylor JA. In utero exposure of female lambs to testosterone reduces the 
sensitivity of the  gonadotropin-releasing hormone neuronal network to inhibition by progesterone. 
Endocrinology. 1999;140(12):5797-5805. doi:10.1210/endo.140.12.7205. 
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Moore AM, Prescott M, Campbell RE. Estradiol negative and positive feedback in a prenatal androgen-
induced mouse model of polycystic ovarian syndrome. Endocrinology. 2013;154(2):796-806. 
doi:10.1210/en.2012-1954. 
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Unsworth WP, Taylor JA, Robinson JE. Prenatal programming of reproductive neuroendocrine function: 
the effect of prenatal androgens on the development of estrogen positive feedback and ovarian cycles 
in the ewe. Biol Reprod. 2005;72(3):619-627. doi:10.1095/biolreprod.104.035691. 
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Baird DT, Corker CS, Davidson DW, Hunter WM, Michie EA, Van Look PF. Pituitary-ovarian relationships 
in polycystic ovary syndrome. J Clin Endocrinol Metab. 1977;45(4):798-801. doi:10.1210/jcem-45-4-
798. 

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Legro RS, Arslanian SA, Ehrmann DA, Hoeger KM, Murad MH, Pasquali R, Welt CK. Diagnosis and 
treatment of polycystic ovary syndrome: an Endocrine Society clinical practice guideline. J Clin 
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Legro RS, Brzyski RG, Diamond MP, Coutifaris C, Schlaff WD, Casson P, Christman GM, Huang H, Yan Q, 
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Metab. 2006;91(3):760-771. doi:10.1210/jc.2005-1923. 
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Legro RS, Barnhart HX, Schlaff WD, Carr BR, Diamond MP, Carson SA, Steinkampf MP, Coutifaris C, 
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APPENDIX:  Drug Information 
1   What is the drug name, manufacturer and IND# if available?    
Estradiol (Vivelle), transdermal patch, Novartis. 
 
2.   If IND application has been submitted to the FDA, who is the Principal Investigator on the IND?     
N/A 
 
3.  What is the phase or stage of this study?    
N/A 
 
  
APPENDIX:  Pharmacy-Investigational Drugs/Biologics  
1.   What is the name of the investigational drug/biologic?    
Estradiol, transdermal patch 
 
2. Where will the subjects be seen for the administration/dispensing of the drug?     
 
__X__  Outpatient Unit: Subjects will have the transdermal patch applied in the Clinical Research Unit each 
morning of the study. 
 
3.  What dose will be utilized in this study?    
Subjects will receive graded transdermal E2 dosing (e.g., 0.2 mg/d x 1 d, then 0.3 mg/d x 1 d, then 0.4 mg/d x 1 
d, then 0.5 mg/d). In addition, we will perform real-time monitoring of serum E2 levels (i.e., real-time 
monitoring of transdermal estradiol dosing) to ensure achievement of appropriate target levels. That is, we 
want to ensure that serum E2 levels are high enough to trigger a gonadotropin surge (~250-350 pg/ml), but we 
wish to avoid prolonged exposure to excessive serum E2 levels (> 400 pg/ml). Accordingly:  
• If a subject’s serum E2 level was < 250 pg/ml on the preceding Study Day, the transdermal estradiol 
dose will be increased by 0.1 mg/day per protocol (maximum dose = 0.6 mg/day). 

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• If a subject’s serum E2 was 250-350 pg/ml on the preceding Study Day, that same transdermal 
estradiol dose will be maintained for the next 24 hours. 
• If a subject’s serum E2 level is > 350 pg/ml on a given dose of transdermal estradiol – which we think 
will be unlikely in this protocol – the transdermal estradiol dose will be decreased to the previous dose 
that had been associated with a serum E2 value approximating 250-350 pg/mL. If there is uncertainty 
regarding the appropriate dose reduction, we will proportionally reduce the dose according to 
measured serum E2 levels, aiming to achieve serum E2 levels of 250-350 pg/ml.  
Through the above dose-adjustment protocol, we will avoid substantial subject exposure to supraphysiologic 
serum E2 levels (i.e., serum E2 levels > 400 pg/ml). 
 
4. What will be the frequency of dosing in this study?    
Patch application will occur daily through the duration of the study. 
 
5.  What will be the duration of dosing in this study?    
The maximum duration of dosing would be from the morning of Study Day 2 through the morning of Study 
Day 9 (i.e., 7 days). 
 
6.  What route of administration will be utilized?    
Transdermal patches will be rotated (daily) on locations on the abdominal wall. 
 
7.  Will drug need to be prepared by the UVa Investigational Drug Service (IDS)?     
__X__  YES 
_____  NO- Drug will be prepared and/or administered per package insert 
 
►IF YES, complete the following information under 7a-7d.  
If you need assistance completing this section contact the Investigational Pharmacists at 982-1048 
7a.  Concentration 
__X__ Standard 
_____  Non- Standard- Specify   Answer/Response: 
 
7b.  Diluents 
__X__ Standard 
_____  Non- Standard- Specify   Answer/Response: 
 
7c.  Stability after prepared 
__X__ Standard 
_____  Non- Standard- Specify   Answer/Response: 
 
7d. Special storage requirements 
__X__ Standard 
_____  Non- Standard- Specify   Answer/Response: 
 
8.  Are there any special handling instructions mandated by the study (e.g. weighing hazardous materials)?    

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No. 
 
9.  Does the protocol provide provisions for dose titration, dose reductions, and or re-challenged (if drug is 
stopped), etc.?  No. 
 
10.  How will missed doses be handled?     
All interruptions of transdermal E2 dosing will be documented in the subject’s study record. If transdermal E2 
dosing is interrupted for more than 6 consecutive hours, then the subject’s involvement in the study will be 
stopped. 
 
11.  Will a comparator (active or placebo) be utilized in the protocol?  No. 
 
12.  Does this study involve research on a drug, biologic, supplement or food additive?   No. 
 
13    Are you using a drug/supplement/ food additive in a manner not approved by the FDA?   Yes. 
 
13a.  Describe pertinent animal data that is available regarding the toxicity/safety of this drug.    
Since human data regarding estradiol safety are abundant, animal data regarding estradiol safety is not 
felt to be relevant. 
 
13b. Describe pertinent human data that is available regarding the toxicity/safety of this drug.    
There are no data regarding extremely short-term use of transdermal estradiol. However, with 
the exception of local skin reactions, any adverse reactions are anticipated to be exceedingly 
rare. This is related to (a) the very short-term use of estradiol in this study; (b) the fact that 
physiological estradiol concentrations will be achieved in this study; and (c) the fact that only 
healthy young subjects are included in this study. The primary clinical use of unopposed 
estrogen (often in the form of combined equine estrogens, but not uncommonly in the form of 
estradiol) is long-term (months to years) use in the setting of menopausal symptoms. The 
largest study of long-term unopposed estrogen (combined equine estrogens taken orally) was 
the Women's Health Initiative (WHI) in healthy postmenopausal women ages 50 to 79. The WHI 
unopposed estrogen versus placebo trial in nearly 11,000 women showed no difference in 
coronary heart disease outcomes over an average follow-up of 6.8 years (note: there was a 
suggestion—but not a statistically significant one—of a protective effect in the younger women 
[ages 50 to 59]). This trial showed a slightly increased risk of stroke (HR 1.39, HR 1.1 to 1.77), an 
absolute excess risk of 12 additional strokes per 10,000 person-years. The WHI also showed an 
increased rate of deep venous thromboembolism. However, transdermal estrogens, which have 
little effect on hemostasis, may be associated with a lower VTE risk than oral estrogens. No 
increased breast cancer risk was observed. An increased risk of biliary tract disease (e.g., 
cholecystitis) was observed. Overall, the baseline risk of coronary artery disease, stroke, venous 
thromboembolism, and biliary tract disease is very low in healthy women aged 18-30 y. Thus, 
the attributable risk of unopposed estrogen use in such a population would remain exceedingly 
low. Reported adverse reactions of estradiol may include the following (NOTE: some are 
observed with estrogen/progestin combination therapy; and the frequency of these reactions 
are not clearly defined, but are rare): DVT, edema, hypertension, MI, stroke, venous 

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thromboembolism, anxiety, dementia, dizziness, epilepsy exacerbation, headache, irritability, 
mental depression, migraine, mood disturbances, nervousness, angioedema, chloasma, 
erythema multiforme, erythema nodosum, hemorrhagic eruption, hirsutism, loss of scalp hair, 
melasma, rash, pruritus, urticaria, breast cancer, breast enlargement, breast pain, breast 
tenderness, fibrocystic breast changes, galactorrhea, glucose intolerance, hypocalcemia, libido 
changes, nipple pain, serum triglycerides/phospholipids increased, vaginal discharge, vaginitis, 
abdominal cramps, abdominal distension, abdominal pain, bloating, cholecystitis, cholelithiasis, 
diarrhea, flatulence, gallbladder disease, nausea, pancreatitis, vomiting, weight gain/loss, 
alterations in frequency and flow of menses, cervical secretion changes, dysmenorrhea, 
endometrial cancer, endometrial hyperplasia, metrorrhagia, ovarian cancer, Pap smear 
suspicious, urinary tract infection, uterine leiomyomata size increased, uterine pain, vaginal 
candidiasis, aggravation of porphyria, cholestatic jaundice, hepatic hemangioma enlargement, 
thrombophlebitis, burning, erythema, irritation, arthralgia, back pain, chorea, leg cramps, 
muscle cramps, contact lens intolerance, corneal curvature steepening, retinal vascular 
thrombosis, asthma exacerbation, pulmonary thromboembolism, anaphylactoid/anaphylactic 
reactions, liver function tests increased, leg pain.  
 
13c. Have there been any human deaths associated with this drug?    
We are unaware of any human deaths directly attributable to short-term transdermal estradiol use. 
 
13d. In how many humans has this drug been used previously?    
It is unknown exactly how many women have received transdermal estradiol, but the numbers must be 
substantial, since transdermal estradiol is commonly used for the clinical care of patients with 
menopause and estrogen deficiency.   
 
13e. If this protocol will be used in children describe any previous use of this drug with children of a 
similar age range.    
N/A 
 
14.  Do the following criteria apply?  
 
_____  The investigation is intended to be reported to FDA as a well-controlled study in support of a 
new indication for use or intended to be used to support any other significant change in the labeling 
for the drug; 
 
_____  If the drug that is undergoing investigation is lawfully marketed as a prescription drug product, 
the investigation is intended to support a significant change in the advertising for the product; 
 
_____  The investigation does involve a route of administration or dosage level or use in a patient 
population or other factor that significantly increases the risks (or decreases the acceptability of the 
risks) associated with the use of the drug product. 
 
If Not checked- explain why you believe the risk to subjects is not increased:   

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An FDA-approved transdermal estradiol preparation will be used in this study with the intention of 
reproducing the physiologic serum estradiol levels seen in the late follicular (pre-ovulatory) phase of 
the menstrual cycle. That is, we are using estradiol as a physiological probe. 
 
In the normal menstrual cycle, the late follicular estradiol levels are the peak estradiol levels within the 
cycle. Peak pre-ovulatory estradiol levels usually exceed 200 pg/mL, but commonly approach (and 
sometimes exceed) 400 pg/mL (Welt CK, McNicholl DJ, Taylor AE, Hall JE. Female reproductive aging is 
marked by decreased secretion of dimeric inhibin. J Clin Endocrinol Metab 1999; 84:105.) In general, 
preovulatory estradiol levels remain elevated > 200 pg/mL for 3-4 days, and the high estradiol levels 
provoke (by a positive feedback action) the preovulatory gonadotropin surge. In our study, we aim to 
achieve serum estradiol levels greater than 250 pg/mL, but less than 350 pg/mL, for 3-4 days to 
provoke a gonadotropin surge. That is, we aim to reproduce estradiol levels that normal women 
experience during each menstrual cycle.  
 
In clinical practice, when transdermal estradiol is used for full hormone replacement therapy (for 
example, in young women who have undergone menopause prematurely), a typical transdermal 
estradiol dose would be 0.1-0.2 mg/day. In these clinical scenarios, the goal is to achieve serum 
estradiol levels of about 70-100 pg/mL, because this value approximates that average estradiol level 
observed across the cycle for a menstruating female. (Note that lower doses [e.g., 0.05 mg/day] may 
be sufficient to when the primary goal is to mitigate menopausal symptoms such as hot flashes.) 
 
Because this study’s intention is to produce a gonadotropin surge, which is contingent upon achieving 
physiologic peak estradiol levels (e.g., 250-350 pg/mL), we must use higher doses than are used 
therapeutically for simple hormone replacement. To our knowledge, the safety profile of short-term 
use of estradiol ≥ 0.4 mg/day transdermally has not been defined; but based on an understanding of 
normal physiology and the benefits of transdermal delivery (compared to oral delivery), we have no 
reason to expect excess risk related to these estradiol doses as compared to what a cycling subject 
would routinely experience during the late follicular (pre-ovulatory) phase of the cycle. To reduce the 
risk of excessively high (i.e., non-physiological) estradiol exposure, we will monitor estradiol levels by 
assessing serum estradiol concentrations daily (through the UVA Clinical Laboratory for more rapid 
result turnaround). If a subject’s serum E2 level is at the target level (250-350 pg/ml), the transdermal 
estradiol dose will not be increased further; and if a subject’s serum E2 level is above our target level 
(i.e., > 350 pg/ml) on a given dose of transdermal estradiol, the transdermal estradiol dose will be 
decreased (as described in detail above). 
 
Importantly, many of the adverse events related to exogenous estrogen use (e.g., venous 
thromboembolism) appear to be largely if not exclusively related to (a) oral administration with 
absorption/entry into the hepatic portal circulation (increased hepatic exposure can alter liver 
production of various products [e.g., products influencing coagulability]) and/or (b) long-term use of 
supraphysiological doses (e.g., doses required to suppress follicular development and ovulation). 
Transdermal estradiol is chemically and biologically identical to the native (ovarian) estradiol, which 
allows accurate assessment of delivered doses. Moreover, transdermal estradiol delivers estradiol into 

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the systemic circulation (mimicking ovarian estradiol delivery in this regard), and it effectively avoids 
first-pass liver effects.  
 
In summary, our use of estradiol in this protocol uses the natural hormone via a near-physiological 
route, with the amount delivered producing normal concentrations for the late follicular (preovulatory) 
phase of the menstrual cycle. For these reasons, we do not expect excess risk related to the proposed 
estradiol doses as compared to what a cycling subject would routinely experience during the late 
follicular (pre-ovulatory) phase of the cycle.   
 
 
__X__  The investigation will be conducted in compliance with the requirements for institutional 
review set part in part 21CFR56 and with the requirements for informed consent set forth in part 
21CFR50 ; and   This item must be checked. 
 
_X__ The investigation will be conducted in compliance with the requirements of 21CFR312.7 
(Promotion and charging for investigational drugs) 
 This item must be checked. 
 
15.  Is this a post-marketing study?      No.