Mechanisms of Hypertension in Women With Polycystic Ovary Syndrome
Phase: EARLY_PHASE1
Conditions: Polycystic Ovary Syndrome, Hypertension
Sponsor: Yale University

Date: 1/13/2022 
Title: Mechanisms of Hypertension in Women With Polycystic Ovary 
Syndrome 
ID: NCT04327934 
Document(s): Protocol w SAP 

APPROVED BY THE YALE UNIVERSITY IRB 1/13/2022
Page 1 of 53
YALE UNIVERSITY
                                  HUMAN INVESTIGATION COMMITTEE
                                     Application to Involve Human Subjects in Biomedical Research
100 FR1 (2016-1) 
SECTION I: ADMINISTRATIVE INFORMATION
Title of Research Project: 
Mechanisms of hypertension in women with polycystic ovary syndrome
Principal Investigator:
Nina Stachenfeld
Yale Academic Appointment:
Senior Research Scientist
Department:
Campus Address: 
The John B. Pierce Laboratory 290 Congress Ave. New Haven, CT 06519
Campus Phone: 203-562-
9901 x219
Fax: 203-624-4950
Pager: 
E-mail:nstach@jbpierce.org 
Protocol Correspondent Name & Address (if different than PI):
 Cheryl Leone
Campus Phone: 203-562-
9901 x266
Fax:203-624-
4950 
E-mail: cleone@jbpierce.org 
Yale Cancer Center CTO Protocol Correspondent Name & Address (if applicable):
Campus Phone: 
Fax: 
E-mail: 
Business Manager:
Campus Phone :
Fax :
E-mail
Investigator Interests:
Does the principal investigator, or do any research personnel who are responsible for the design, 
conduct or reporting of this project or any of their family members (spouse or dependent child) 
have an incentive or interest, financial or otherwise, that may affect the protection of the human 
subjects involved in this project, the scientific objectivity of the research or its integrity? Note: 
The Principal Investigator (Project Director), upon consideration of the individual’s role and 
degree of independence in carrying out the work, will determine who is responsible for the 
design, conduct, or reporting of the research. 
Faculty Advisor:(required if PI is a student, 
resident, fellow or other trainee)           
 NA
Yale Academic Appointment:
Campus Address: 
Campus Phone: 
Fax: 
Pager: 
E-mail: 
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A P P R O V E D B Y T H E Y A L E U NI V E R SI T Y I R B 1/ 1 3/ 2 0 2 2
Pa ge 2  of 5 3
See Discl os ures a n d Ma na ge me nt of Pers o nal I nterests i n H u ma n Researc h 
htt p:// w w w. yale.e d u/ hr p p/ p olicies/i n de x. ht ml # C OI  
  Yes 
x  N o
D o y o u or d oes a n y o ne o n t he researc h tea m w h o is deter mi ne d b y y o u t o be res p o nsi ble f or t he 
desi g n, c o n d uct or re p orti n g of t his researc h ha ve a n y pate nt (s ole ri g ht t o ma ke, use or sell a n 
i n ve nti o n) or c o p yri g ht (e xcl usi ve ri g hts t o a n ori gi nal w or k) i nterests relate d t o t his researc h 
pr ot oc ol?
  Yes 
x  N o
If yes t o eit her q uesti o n a b o ve, list na mes of t he i n vesti gat or or res p o nsi ble pers o n:
T he Y ale U niversity Pri nci p al I nvesti g at or, all Y ale U niversity c o-i nvesti g at ors, a n d all Y ale 
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N O T E: T he re q uire me nt f or mai ntai ni n g a c urre nt discl os ure f or m o n file wit h t he U ni versit y’s 
C o nflict of I nterest Office e xte n ds pri maril y t o Yale U ni versit y a n d Yale- Ne w Ha ve n H os pital 
pers o n nel.  W het her or n ot t he y are re q uire d t o m ai nt ai n a discl os ure f or m wit h t he 
U ni versit y’s C o nflict of I nterest Office, all i n vesti g at ors a n d i n di vi d u als dee me d ot her wise 
res p o nsi ble b y t he PI w h o are liste d o n t he pr ot oc ol are re q uire d t o discl ose t o t he PI a n y 
i nterests t h at are s pecific t o t his pr ot oc ol.
S E C TI O N II: G E N E R A L IN F O R M A TI O N
1.
Perf or mi n g Or g a niz ati o ns:  I de ntif y t he h os pital, i n- patie nt or o ut patie nt facilit y, sc h o ol or 
ot her a ge nc y t hat will ser ve as t he l ocati o n of t he researc h.  C h o ose all t hat a p pl y:
 
a. I nter n al L oc ati o n[s] of t he St u d y:
 Ma g netic Res o na nce Researc h Ce nter 
 Yale U ni versit y P E T Ce nter
     ( M R- T A C)   
 Y C CI/ C h urc h Street Researc h U nit ( C S R U)
 Yale Ca ncer Ce nter/ Cli nical Trials Office ( C T O)     
 Y C CI/ H os pital Researc h U nit ( H R U)
 Yale Ca ncer Ce nter/ S mil o w
 Y C CI/ Kec k La b orat ories
 Yale- Ne w Ha ve n H os pital
 Yale- Ne w Ha ve n H os pital — Sai nt Ra p hael Ca m p us
 Ca ncer Data Re p osit or y/ T u m or Re gistr y
 S pecif y Ot her Yale L ocati o n:
b. E xter n al L oc ati o n[s]:
 A P T F o u n dati o n, I nc.
 Has ki ns La b orat ories
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 Connecticut Mental Health Center
 John B. Pierce Laboratory, Inc.
 Clinical Neuroscience Research Unit (CNRU) 
Veterans Affairs Hospital, West Haven
 Other Locations, Specify: 
 International Research Site 
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c. Additional Required Documents (check all that apply):
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*YCCI-Scientific and Safety Committee (YCCI-SSC)
Approval Date: 
*Pediatric Protocol Review Committee (PPRC)
Approval Date: 
*YCC Protocol Review Committee (YRC-PRC)
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*Dept. of Veterans Affairs, West Haven VA HSS
Approval Date: 
*Radioactive Drug Research Committee (RDRC)
Approval Date: 
 YNHH-Radiation Safety Committee (YNHH-RSC)
Approval Date: 
 Yale University RSC (YU-RSC)
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Approval Date:
 *Nursing Research Committee
Approval Date:
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Approval Date: 
 Dept. of Lab Medicine request for services or specimens form
 Imaging on YNHH Diagnostic Radiology equipment request form (YDRCTO request) found 
at  http://radiology.yale.edu/research/ClinTrials.aspx)
*Approval from these committees is required before final HIC approval is granted. See instructions 
for documents required for initial submission and approval of the protocol. Allow sufficient time for 
these requests. Check with the oversight body for their time requirements.
2. Probable Duration of Project: State the expected duration of the project, including all 
follow-up and data analysis activities. 
Six years
3. Research Type/Phase: (Check all that apply)
a. Study Type
   
 Single Center Study
   
 Multi-Center Study
Does the Yale PI serve as the PI of the multi-site study? Yes 
No 
  
 Coordinating Center/Data Management
  
 Other: 
b. Study Phase
 N/A
    
 Pilot
 Phase I
 Phase II
 Phase III
 Phase IV
    
 Other (Specify) 
4. Area of Research: (Check all that apply) Note that these are overlapping definitions and 
more than one category may apply to your research protocol. Definitions for the following 
can be found in the instructions section 4c:
 Clinical Research: Patient-Oriented
  
 Clinical Research: Outcomes and 
 Clinical Research: Epidemiologic and Behavioral 
                Health Services
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 Translational Research #1 (“Bench-to-Bedside”)    
 Interdisciplinary Research
 Translational Research #2 (“Bedside-to-Community”) 
 Community-Based Research
5.   Is this study a clinical trial? Yes 
No 
6. Does the Clinical Trials Agreement (CTA) require compliance with ICH GCP (E6)? 
Yes 
 No
7. Will this study have a billable service? A billable service is defined as any service rendered to 
a study subject that, if he/she was not on a study, would normally generate a bill from either 
Yale-New Haven Hospital or Yale Medical Group to the patient or the patient’s insurer. The 
service may or may not be performed by the research staff on your study, but may be provided by 
professionals within either Yale-New Haven Hospital or Yale Medical Group (examples include 
x-rays, MRIs, CT scans, specimens sent to central labs, or specimens sent to pathology). Notes: 
1. There is no distinction made whether the service is paid for by the subject or their insurance 
(Standard of Care) or by the study’s funding mechanism (Research Sponsored). 2. This generally 
includes new services or orders placed in EPIC for research subjects. 
Yes 
 No
 If answered, “yes”, this study will need to be set up in OnCore, Yale’s clinical research 
management system, for Epic to appropriately route research related charges. Please contact 
oncore.support@yale.edu
8.. Are there any procedures involved in this protocol that will be performed at YNHH or one of 
its affiliated entities?  Yes ___ No _X__  If Yes, please answer questions a through c and note 
instructions below.  If No, proceed to Section III.
a. Does your YNHH privilege delineation currently include the specific procedure that you will 
perform?
b. Will you be using any new equipment or equipment that you have not used in the past for 
this procedure?
 
c. Will a novel approach using existing equipment be applied?
 
If you answered “no” to question 8a, or "yes" to question 8b or c, please contact the YNHH 
Department of Physician Services (688-2615) for prior approval before commencing with your 
research protocol.
Please note that if this protocol includes Yale-New Haven Hospital patients, including patients at 
the HRU, the Principal Investigator and any co-investigators who are physicians or  mid-level 
practitioners (includes PAs, APRNs, psychologists and speech pathologists) who may have direct 
patient contact with patients on YNHH premises must have medical staff appointment and 
appropriate clinical privileges at YNHH. If you are uncertain whether the study personnel meet 
the criteria, please telephone the Physician Services Department at 203-688-2615. By signing 
this protocol as a PI, you attest that you and any co-investigator who may have patient contact 
has a medical staff appointment and appropriate clinical privileges at YNHH.
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SECTION V: RESEARCH PLAN
1. Statement of Purpose: State the scientific aim(s) of the study, or the hypotheses to be tested. 
Aim 1. To test the hypothesis that androgen excess drives sympathetic activation leading 
to increased BP in AE-PCOS.  We hypothesize that in women with AE-PCOS: 1A. 
pharmacological 
androgen 
suppression 
reduces 
sympathetic 
outflow, 
-adrenergic 
vasoconstriction, improves baroreflex sensitivity (BRS), and BP; 1B. androgen supplementation 
increases sympathetic outflow, impairs BRS, reduces BP, and decreases -adrenergic 
vasodilation.  
Aim 2. To test the hypothesis that androgen-driven sympathetic activation increases RAS 
activity and BP in AE-PCOS. We hypothesize that in women with AE-PCOS: 2A. 
pharmacological androgen suppression reduces sympathetic activation of the RAS and lowers 
BP; 2B. androgen administration increases sympathetic activation of RAS and increases BP. 
Aim 3.  To test the hypothesis that androgen excess alters autonomic regulation and 
endothelial function in women with PCOS.  We hypothesize that in women with AE-PCOS: 
pharmacological androgen suppression reduces baroreflex sensitivity (BRS) and improves BP 
and endothelial function
Aim 4. To determine the effectiveness of the orally active gonadotropin releasing hormone 
(GnRH)  antagonist, elagolix in suppressing testosterone.  We hypothesize that taking 
elagolix tablets, 400 mg/day oral, 2X200 mg tablets/day for 7 days will be as effective at 
suppressing testosterone as the GnRH antagonist ganirelix acetate.
Aim 5: To test the hypothesis that sympathetic outflow, -adrenergic vasoconstriction, baroreflex 
sensitivity, and blood pressure are increased in black women with AE-PCOS compared to white 
women with AE-PCOS.  
2. Background: Describe the background information that led to the plan for this project. 
Provide references to support the expectation of obtaining useful scientific data.
Our scientific premise is that in AE-PCOS women, the androgen-dominant hormonal milieu 
causes BP increases via sympathetic activation, vasoconstriction and renal sympathetic nervous 
system activation.  Moreover, this androgen-dominant milieu increases BP via activation of the 
renin-angiotensin system. Aim 3 will address the impact of androgens on baroreflex sensitivity 
(BRS) and endothelial function, and determine the interaction of these on the control of BP.
While AE-PCOS often gets unmasked in the process of evaluation for infertility for many affected 
women, the long-term health risks for this population often remain underappreciated; in particular, 
attention to cardiovascular and metabolic risk factors should be integral to their care.  Common 
symptoms for which young AE-PCOS women initially seek assessment include menstrual 
irregularities (absent, infrequent or erratic periods), and symptoms related to hyperandrogenism, 
including hirsutism, bothersome acne, and even androgenic alopecia (4). Metabolic abnormalities 
are also common with AE-PCOS, including android type obesity, dyslipidemia, and insulin 
resistance (IR), with hyperinsulinemia (4, 30). One third of AE-PCOS women develop metabolic 
syndrome (MetS) by 49 years of age, with many developing MetS before they reach 40 yrs. This 
is in stark contrast to the 6.7% prevalence of MetS in U.S. women between ages 20-30 yrs, and 
the 15% prevalence in women between ages 30-40 yrs (27). In AE-PCOS, hyperinsulinemia 
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exacerbates hyperandrogenism by inducing both ovarian and adrenal androgen production.  These 
women manifest a spectrum of covert markers for increased CVD risk (such as higher BP, an 
atherogenic lipid profile, and a proinflammatory milieu), at younger ages compared to women 
without PCOS. Important for the present investigation is the proposed relationship between 
androgens and sympathetic regulation of BP that has not been previously examined in obese AE-
PCOS women.  Treatment of AE-PCOS women is also complicated since the most commonly 
utilized therapy in managing PCOS symptoms, such as oral contraceptive pills, can worsen BP, 
and many standard BP lowering drugs, such as RAS blockers, are contraindicated in women who 
want to become pregnant.  
In the present proposal we will use physiological approaches in combination with cellular and 
molecular mechanistic techniques to address fundamental questions concerning the contribution 
of impaired autonomic and RAS function to BP regulation in AE-PCOS women and a model of 
hyperandrogenemia in female (HAF) rats to specifically understand if androgen signaling is 
relevant to these processes.  Determining the mechanisms responsible for androgen-mediated 
increases in BP will not only allow successful intervention in young AE-PCOS women, but also 
break the cycle of increased androgens leading to early CVD, thus improving their long-term 
health and reducing health care costs.  Should we demonstrate a relationship between 
androgens, SNSA, and the RAS, modulation of SNA could be a valuable treatment to inhibit the 
underlying causes of AE-PCOS and improve cardiovascular outcomes when other 
antihypertensive medications, such as RAS blockers or antiandrogens, are contraindicated. 
Novel pathways or therapies: Mild hypertension (HT) common to AE-PCOS women usually 
goes untreated since the level of BP doesn't reach clinical guidelines for treatment.  However, 
elevated BP is the most important risk factor for later CVD.  Our studies that include human 
subjects, will determine if therapies aimed at addressing elevated SNA or plasma aldosterone 
(P[ALD] ) will reduce BP in women with AE-PCOS and thus overall cardiovascular disease (CVD) 
risk. While the pathogenesis of CVD and HT is multifaceted in AE-PCOS, SNS and renal 
dysfunction likely underlie the development of critical cardiovascular dysfunction. The 
mechanisms responsible for SNA are unknown, but we have preliminary evidence in the HAF rat 
model that androgens increase hypothalamic MC4R expression and intracerebroventricular 
blockade reduces their BP (60). In contrast, in other HT rat models, only males respond  to MC4R 
blockade with a reduction in BP (16, 61), suggesting that hyperandrogenemia in the HAF rats 
may mediate the increased SNA. In addition, even mild physiological increases in P[ALD] 
predispose women to development of HT (101). Aldosterone can also cause CVD and endothelial 
dysfunction independent of its BP effect (83), since the vascular wall is an aldosterone target 
tissue, and vascular cells can produce local aldosterone (94), thus increasing vascular tone and 
structure directly while at physiological concentrations (80). Moreover, a direct correlation 
between aldosterone and intima-media thickness (IMT) has been shown in AE-PCOS women 
(12), consistent with the adverse role of the RAS in vascular remodeling and in the progression 
of atherosclerosis associated with high IMT (81). 
Aim 3. Background.  Endothelial function. Women with androgen AE-PCOS often develop 
endothelial dysfunction (ED) in their early twenties (46, 92). Endothelial dysfunction is a known 
early marker of (and probable causative factor in) CVD (50, 84, 86). Although young women with 
AE-PCOS commonly manifest a spectrum of covert CVD risk markers, including obesity, insulin 
resistance and hypertension, we believe that androgen excess in young women is a major driver 
of ED. We have generated preliminary endothelial-dependent vasodilation data in lean, insulin-
sensitive AE-PCOS subjects, which support our androgen-dependent vascular pathology 
hypothesis. A major feature of the normally functional endothelium is the production of NO in 
response to a variety of agonists. Our scientific premise is that this is a key causative link 
between infertility and CVD, and is even more broadly applicable to the sharp rise in CVD 
incidence at and beyond the menopausal transition.
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Our proposal is high impact and can impact treatment of AE-PCOS women for the following 
reasons:
1.  Mechanisms of HT are less well understood, and thus more challenging to treat, in women in 
whom complications including heart failure with preserved ejection fraction are more common, 
and the incidence of resistant HT is higher (40, 62, 63). 2. Even mild HT in AE-PCOS women is 
complex to treat because many women are trying to conceive, and many antihypertensives are 
teratogenic or unsafe for the fetus.
3. High free androgen index (FAI) in AE-PCOS women is associated with high BP independent 
of IR, body mass index (BMI), and dyslipidemia (13), but antiandrogens risk feminization of 
external genitalia of male fetuses if given during pregnancy and are thus contraindicated.
4. AE-PCOS women develop pre-HT in their early twenties, predisposing them to later frank HT 
and CVD (5, 46).  However, these young women are not typically treated because their BP has 
not reached standard clinical treatment guidelines.  Thus, an opportunity to prevent or reduce the 
long-term CVD health risk associated with AE-PCOS is missed. 
5. The cause(s) of the mild BP increase in AE-PCOS is unknown, but SNS activation (82, 93) and 
renal sympathetic nerve activation (RSNA) may contribute to BP elevation. 
6. According to the NIH and Rotterdam criteria, PCOS prevalence is 8-10% (85), that is 
approximately seven million U.S. women and more than 100 million women worldwide (72). Using 
2004 statistics, the cost of screening and treating PCOS women exceeds $4 billion annually, 
excluding CVD and other comorbidities.  
7. The AE-PCOS phenotype accounts for 75% of PCOS cases in the US. AE is also associated 
with endothelial dysfunction and HT, the most common risk factor for later CVD, so AE-PCOS 
treatment includes identification and treatment of these comorbidities in addition to patients’ 
immediate concerns (85).
8.
For Aim 4:  the GnRH antagonist, ganirelix acetate is effective at suppressing testosterone 
in women with AE-PCOS but requires daily injections.  With this Aim, we would like to determine 
if this new orally active GnRH antagonist is as effective so will spare the subjects the injections, 
thereby making participation in the studies less stressful.
Aim 5 Background:
Cardiovascular disease (CVD) is the leading cause of morbidity and mortality in women.  Chronic 
increases in sympathetic nervous system activity (SNSA) are associated with CVD and 
hypertension.  The arterial baroreflex is a key autonomic homeostatic mechanism involved in 
beat-to-beat regulation of blood pressure whereby the sympathetic nervous system acts to 
maintain and control blood pressure.  Young healthy black women have lower baroreflex 
sensitivity and higher sympathetic modulations at rest compared to their age matched white 
counterparts. These findings indicate differences in blood pressure regulatory mechanisms 
between racial groups are present in women less than 25 years of age (54).  Further, the changes 
to the American Heart Association guidelines for diagnosis of stage 1 hypertension, defined as 
systolic blood pressure (SBP) between 130 and 139 mmHg or diastolic blood pressure (DBP) 
between 80 and 89 mmHg, have underlined the importance of understanding the effects of 
moderate increases in blood pressure in younger women, as these predict future cardiovascular 
morbidity (104).
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Blacks or African Americans are the largest racial minority group in the United States, making up 
approximately 13% of the population (41). Hypertension is more prevalent in the black versus the 
white population, and racial differences in blood pressure regulation have been documented (48).  
Moreover, hypertension manifests at a younger age in black compared with white people (28, 77) 
and contributes to their higher mortality (6).  In the U.S., black women have increased obesity, 
dyslipidemia, hyperglycemia, and vascular disease (79).  Race is a strong predictor of arterial 
stiffness in black and white adolescents (97) and there are a number of studies indicating lower 
compliance in the cardiovascular system of black versus white adults (98, 99), as well as 
attenuated vascular nitric oxide (NO) bioavailability in black relative to white adults (71). This is of 
particular interest because early changes in endothelial function, arterial stiffness, and intima wall 
thickness in young, normotensive individuals is associated with a greater risk of hypertension in 
later years (98-100). Attenuated vascular responsiveness in black compared to white subjects 
has been noted in more than one vascular bed, including conduit arteries (9, 74), whole limb 
microcirculation (10, 19, 35), and the cutaneous microvasculature (43, 75). 
There have been studies addressing blood pressure regulation in black men (26), but few studies 
have addressed blood pressure regulation in black women or racial differences within women 
(73).  Some investigations have suggested greater oxidative stress in blacks may contribute to 
the racial differences in blood pressure regulation (17, 24), but these were conducted in men (17).  
A recent study demonstrated attenuated cutaneous microvascular vasodilation in response to 
endothelium mediated local heating in healthy, young black adults relative to age- and sex-
matched white adults.  Interestingly, this study also demonstrated sex-based divergent 
mechanisms of microvascular dysfunction between black men and black women, indicating that 
within black men NADPH oxidase and xanthine oxidase contribute to impairments in cutaneous 
microvascular function, but these mechanisms played no role in the regulatory deficits in black 
women  (73).  This study highlights the importance in sex specific studies to understand 
racial differences in BP regulation.
We 
have 
shown 
an 
important 
role 
for 
norepinephrine, which can reflect sympathetic 
activity, in racial differences controlling blood 
pressure response to orthostasis (36) (Table 1). 
The studies in the Parent Grant and this 
supplement include measures of whole-body 
sympathetic outflow using the gold standard 
measurement microneurography so will provide 
a precise picture of sympathetic activity and 
function in women with AE-PCOS. The proposed 
studies in this supplement allow us expand our 
population sufficiently to make racial comparisons using microneurography.  
Although young women with AE-PCOS commonly manifest a spectrum of covert CVD risk 
markers, we have demonstrated that androgen excess in young women is a major driver of 
increased blood pressure, and that testosterone suppression decreases blood pressure and 
catecholamines. The preponderance of evidence indicates that hyperandrogenism is associated 
with increased sympathetic tone, impaired baroreflex function, and increased blood pressure in 
AE-PCOS (90, 93, 102).  Despite this interesting work, studies have not assessed baroreflex 
function using direct measures of SNSA in the most common phenotype: obese, insulin resistant 
AE-PCOS women (52). 
Table 1.  Plasma norepinephrine (P[NE]) is higher in black 
compared to white women in response to lower body 
negative pressure (n= 12/group) (36).
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Black women have the highest risks of developing PCOS (18), and black women with PCOS have 
high risk cardiovascular profiles, including higher LDL levels  compared to white women with 
PCOS (2, 11, 15, 18, 45, 59).  A recent review focused on the prevalence of PCOS in women 
living in the U.S. found the greatest prevalence in black women relative to other races and 
ethnicities examined (white, Asians, middle eastern, Indian) (18).  This study showed the upper 
bound of the 95% CI of PCOS prevalence for white women overlapped with the lower bound of 
the 95% CI for black women, indicating that black women have substantially greater risk of 
developing PCOS compared to white women.  This disparity was noted despite considerable 
variation within and across ethnic groups of phenotypes and women with different presentations 
of clinical hyperandrogenism such as hirsutism. A recent study suggested that Hispanic women 
with PCOS generally present the most severe phenotype both in terms of hyperandrogenism and 
metabolic features, while non-Hispanic black women had lower prevalence of metabolic 
syndrome (22).  However, CVD and metabolic syndrome risk was increased in both groups 
compared to white women with PCOS, and black and white Hispanic women were not analyzed 
separately (22).  The racial background of women with PCOS is an important consideration for 
studies in women with PCOS (56).  However, race has not yet been addressed with regard to 
autonomic function and blood pressure regulation in studies directed at CVD in women with 
PCOS. 
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B. INNOVATION  
Our studies will unmask unique pathways for BP dysregulation in AE-PCOS previously 
unidentified, and determine therapies likely to be most successful for treating early HT. For 
example, spironolactone, an antiandrogen, used currently to treat acne in women with AE-PCOS 
also blocks the mineralocorticoid receptor; if our studies demonstrate that the primary 
pathophysiology mediating HT in AE-PCOS women is related to dysfunction in aldosterone, this 
(and related classes of) drug may serve a dual purpose of symptom control, as well as confer 
primary prevention against CVD for AE-PCOS women. Furthermore, should the SNS prove to be 
the primary pathophysiology behind BP dysregulation, beta blockers or drugs that target the SNS 
should be therapeutic options of choice. 
Metabolic abnormalities may contribute to the sympathetic overactivity and insulin resistance in 
PCOS, and lead to end-organ damage (e.g. kidney).  However, it remains unclear whether 
elevated sympathetic tone or reactivity is associated with metabolic disturbance in PCOS.  
Indeed, androgens, insulin resistance, and obesity likely interact to increase sympathetic tone in 
AE-PCOS women.  We use a unique approach, that of GnRH suppression in humans, to examine 
women with and without AE-PCOS.  Therefore, our studies will isolate the components of this 
complex syndrome, as well as their interactions, and thus provide the basis for alternative 
treatment options not associated with teratogenicity or adverse fetal outcomes for AE-PCOS 
women.
4. Previous investigations examining the androgen effects on BP control have confined their focus 
to either neural or renal mechanisms, mainly in males. This proposal creates a new paradigm that 
focuses on the integration of androgens with BP control mechanisms in females.
6. The naturally high, chronic androgenic environment in AE-PCOS women provides a unique 
opportunity to study the impact of chronic androgens on women’s cardiovascular systems leading 
to HT and cardiovascular dysfunction.  This is especially important because the elevated 
androgens in AE-PCOS women remain throughout their lifetime continuing even after 
menopause.
7. While previous studies have used microneurography, the gold standard technique, to determine 
resting SNA in lean AE-PCOS, this is the first study to fully examine SNA in the more common, 
insulin resistant, obese, AE-PCOS phenotype using microneurography, thus producing the first 
definitive data on this important BP control mechanism in women with AE-PCOS.
8.
 These are the first studies to use elagolix in women with AE-PCOS to suppress 
testosterone.
BACKGROUND
What is AE-PCOS? The complete etiology of AE-PCOS remains unknown, but the underlying 
mechanism responsible for AE-PCOS is hormonal imbalance. The pathophysiology involves 
dysregulation of a number of endocrine signals including: 1) altered pulsatility of hypothalamic 
GnRH from the arcuate nucleus, resulting in altered secretion of the pituitary gonadotropins: an 
exaggerated frequency and amplitude of pituitary luteinizing hormone (LH) release which 
contrasts with the follicle stimulating hormone (FSH) dominant milieu of normal reproductive 
physiology; 2) excess production of androgens under the influence of both elevated LH and 
excess insulin as well as disordered insulin action in target tissues; 3) failure of ovarian 
folliculogenesis, resulting from the altered gonadotropin signaling and hyperandrogenemic milieu. 
While ovaries of AE-PCOS women are replete with oocytes containing immature follicles, 
mechanisms that underlie dynamics of normal follicular growth and egg maturation are 
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disordered; thus the “cysts” of AE-PCOS ovaries represent arrested follicles that contain immature 
eggs; 4) in addition to androgen excess, AE-PCOS is a state of progesterone deficiency, a 
consequence of failure to achieve ovulation. Disordered ovarian estradiol, disrupted ovarian 
progesterone and excessive ovarian androgen secretion, in addition to compensatory 
hyperinsulinemia, summarize the endocrine aberrations of AE-PCOS. Each hormonal aberration, 
through feedback at the hypothalamopituitary axis, contributes to the pathogenesis of the 
disorder. 
Sympathetic activity and baroreflex function in insulin resistance 
and AE-PCOS. In lean AE-PCOS women, microneurography has 
confirmed elevated SNSA (90, 93) which is an important component of 
AE-PCOS etiology (31).  Poor baroreflex function may contribute to high 
vasoconstriction observed with HT in AE-PCOS women.  AE-PCOS is 
also associated with impaired vagal and cardiac baroreflex sensitivity 
(34), insulin resistance (IR), contributing to sympathovagal dysfunction 
during standing (47), and impaired autonomic control (53). In support of 
these findings, data from animals (78) and humans (80) indicate that 
testosterone is an independent predictor of muscle (M) SNA in AE-
PCOS, and is the strongest independent factor explaining their 
augmented central SNA. Testosterone depletion attenuates, while 
testosterone supplements restore baroreflex-mediated bradycardia 
(102).  Neonatally androgenized mice develop increased sympathetic 
tone and HT concomitant with other features similar to those of AE-
PCOS. Interestingly, the testosterone-MSNA relationship may not be as 
powerful in obese women with AE-PCOS (52), as in lean AE-PCOS 
women (93), possibly due to an independent effect of obesity or the small range of testosterone 
and free androgen index (FAI). Nevertheless, taken together, the preponderance of evidence 
supports our hypothesis that hyperandrogenism is associated with increased sympathetic tone, 
impaired baroreflex function, and increased BP in AE-PCOS women. Despite this interesting 
work, studies have not assessed baroreflex function using direct measures of MSNA in the most 
common phenotype: obese, insulin resistant AE-PCOS women  (52). As shown in (Fig 1), AE-
PCOS women have increased systolic BP relative to IR controls; suppressing ovarian 
testosterone with a GnRH antagonist lowers systolic BP; and testosterone administration (2.5 
mg/day) increases SBP over IR controls. This chronic BP rise to 130-140 mm Hg in SBP, or to 88 
mm Hg in MAP, increases the risk of future organ damage and is a harbinger for future CVD, 
especially in the context of the metabolic and hormonal milieu of AE-PCOS. 
While the current JNC8 guidelines define HT as 140/90mmHg (1), new SPRINT trial data 
suggest that maintenance of BP at 120/80 in men and women reduces all-cause mortality (32).  
Thus any BP above 120/80 would contribute to later CVD. 
Renin-angiotensin-
aldosterone 
system 
(RAAS) 
contributes 
to 
increased BP in 
AE-PCOS women.  
Earlier 
investigations (12), 
including those in 
our laboratory (89), 
found greater serum 
aldosterone (S[ALD]) 
increases 
during 
both exercise (60% 
VO2max @ 35°C) and 
mild 
lower 
body 
Figure 1.  
Systolic blood 
pressure (SBP) is 
higher in IR 
women with AE-
PCOS (n=7) and 
Figure 3. S[ALD] and PRA 
increases are greater in 
response to RAS 
stimulation with LBNP in 
women with AE-PCOS 
d t
IR C
t
l
  
Figure 2.  S[ALD] and PRA 
increases are greater in 
AE-PCOS compared to IR 
Controls during mild cycle 
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negative pressure (LBNP to -40 mm Hg) in AE-PCOS women (89), and greater PRA response 
in with lower body negative pressure (LBNP) in AE-PCOS women (Figs 2-3). The fact that LBNP 
does not have a differential effect on AE-PCOS women despite their higher BP suggests that their 
RAAS is dysfunctional, since one would expect that the elevated BP would cause an attenuated 
PRA response.   
Hormonal effects on SNS and baroreflex 
function. Our preliminary data (n=4) indicate 
increased resting SNS activity in obese 
women with AE-PCOS compared to obese, IR 
controls (Fig 4), although and baroreflex 
sensitivity (BRS) appears similar or only 
slightly 
greater 
in 
AE-PCOS 
women. 
Importantly, while BP remained unchanged, 
estrogen 
administration 
reduced 
resting 
MSNA (Fig. 5, left), while testosterone 
administration 
increased 
BRS 
gain 
(derived from the modified Oxford) in 3 of 4 
IR women with PCOS (Fig. 5, right). Thus our 
studies showed that testosterone effects 
baroreflex control of HR, BP, and MSNA.  
These findings support our hypothesis that 
testosterone 
exposure 
increases 
sympathetic outflow, as well as cardiovagal 
and sympathetic baroreflex gain in AE-
PCOS women.
Summary of human studies.  Our resting, 
exercise and LBNP findings in control and 
AE-PCOS women are consistent with our 
hypothesis that the RAAS contributes to the 
pathogenesis of hypertension in obese IR 
women with AE-PCOS. Moreover, our studies show that resting SNSA and BRS are greater in 
AE-PCOS, and that estrogen reduces and testosterone increases SNSA in AE-PCOS women.  
Findings from our proposed studies will have an immediate impact on treatment because mild BP 
and S[ALD] increases in AE-PCOS are not currently treated because they do not reach standard 
treatment guidelines (as noted above).  However, these mild increases in BP are chronic, and 
can induce organ damage leading to future frank HT and CVD. We propose to explore how the 
chronically high androgen milieu in AE-PCOS promotes excess SNSA (44, 82, 93), poor 
baroreflex function (34), sympathetic and RAAS activation leading to elevated BP in AE-PCOS 
women. Our findings will provide the basis for novel treatment 
options to manage the underlying causes of AE-PCOS and the 
primary cardiovascular co-morbidities, HT and vascular 
dysfunction.  
Controlling hormone exposure in our human studies. The study 
of female reproductive hormones on body systems is difficult in 
young women because the plasma levels of reproductive hormones 
fluctuate throughout the menstrual cycle. Thus, we use temporary 
menstrual cycle suppression to isolate individual effects of these 
hormones on baroreflex and renal function. Ganirelix acetate is a 
synthetic decapeptide that when given continuously in therapeutic 
doses competitively blocks the GnRH receptors on the pituitary 
gonadotroph  inducing a rapid and reversible suppression of 
gonadotropin secretion (69, 70), with suppression of estrogen and 
progesterone to postmenopausal levels in cycling women. As 
mentioned above, in AE-PCOS women, progesterone is chronically 
Figure 6. Serum 
free testosterone is 
suppressed with a 
GnRH antagonist,  
and increased 
testosterone 
administration in 
 
GnRH
E2
0
3
6
9
12
15
18
MSNA (burst/min)
N=4
Baseline
T
-10
-8
-6
-4
-2
0
Baroreflex Slope
(AU/beat/mmHg)
Figure 5. Estrogen reduced resting 
sympathetic nerve activity  (MSNA) in 
obese IR AE-PCOS (left).  Testosterone 
 
 
PCOS
IR CONTROL
0
5
10
15
20
25
30
MSNA (Bursts/100 heart beats)
14.2 + 0.8 burst/min
9.0 + 3.0 bursts/min
N=4
PCOS
IR CONTROL
-8
-6
-4
-2
0
slope = -6.46 AU/beat/mmHg
slope = -5.19 AU/beat/mmHg
Sympathetic Baroreflex Gain
(AU/beat/mmHg)
Figure 4. Resting sympathetic nerve 
activity  (MSNA) and baroreflex slope in 
is greater in obese PCOS and IR 
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low, androgens are elevated, and estradiol is variable; GnRH suppression prevents ovarian 
androgen production, although the adrenal glands continue to produce small amounts of 
testosterone. In both control and AE-PCOS women, GnRH suppression is achieved after two 
days of antagonist administration (Fig 6), and this suppression is reversed upon cessation of drug 
therapy. When reproductive function is temporarily suppressed, we study the effects of 
testosterone on BP control systems in control and AE-PCOS young women by adding 
testosterone back in controlled doses (Fig 7).  We chose the GnRH antagonist in these studies 
as our “gold standard” to ensure that androgens are suppressed with minimal impact on other 
physiological systems.  The androgen receptor antagonist, spirolactone, for example, blocks the 
mineralocorticoid receptor; and flutamide, an androgen antagonist, may increase aldosterone 
(39), so would interfere with our questions regarding androgen effects on CV and renal systems. 
Gonadotropin releasing hormone antagonist (Elagolix).  Elagolix [R-(+)-4-[2-[5-(2-fluoro-3-
methoxy-phenyl)-3-(2-fluoro-6-trifluoromethyl-benzyl)-4-methyl-2,6-dioxo-3,6-dihydro-2H-
pyrimidin-1-yl]-1-phenyl-ethylamino]-butyrate] (14) has high antagonistic activity against naturally 
occurring GnRH. Elagolix in therapeutic doses (50-400 mg/day) acts by competitively blocking 
the GnRH receptors on the pituitary gonadotroph and subsequent transductions pathway, 
inducing a rapid, reversible suppression of gonadotropin secretion (91).  In eumenorrheic women, 
elagolix administration leads to suppression of estrogens to postmenopausal levels after 24 hours 
of administration (91). Importantly, elagolix is safe and well tolerated. The suppression of the 
hypothalamic-pituitary-ovarian axis is reversed upon cessation of drug therapy.
3. Research Plan: Summarize the study design and research procedures using non-technical 
language that can be readily understood by someone outside the discipline. Be sure to 
distinguish between standard of care vs. research procedures when applicable, and 
include any flowcharts of visits specifying their individual times and lengths. Describe 
the setting in which the research will take place.
PARTICIPANTS:   For Aims 1, 1a,2,2a, two groups (n=36 each) of women will be recruited 
to complete this study: 1) IR, obese young women between the ages of 18 and 40 years 
(Controls); 2) IR, obese young women between the ages of 18 and 40 years with AE-PCOS. For  
Aim 3, two groups (n=16 each) of women will be recruited to complete this study: 1) IR, obese 
young women between the ages of 18 and 40 years (Controls); 2) IR, obese young women 
between the ages of 18 and 40 years with AE-PCOS.  As women sign up for the study, they will 
complete medical histories, including the Berlin questionnaire (www.sleepmedicine.com) to 
screen for Obstructive Sleep Apnea (See Appendix 1).  They will also fill out a hair growth chart. 
Subjects who smoke, have diabetes, sleep apnea or BP>140/90 will be excluded. Subjects will 
not be taking medications during the study, including any insulin sensitizing, CV medications, or 
hormonal contraception for at least four weeks prior to participating in these protocols.  We do 
make exceptions for Tylenol in the event the subject has acute pain. All women (early proliferative 
phase in Controls) will undergo 3-hr oral glucose tolerance test (OGTT; 75 gram glucose load) 
to determine IR (8). We define our subjects in both groups as having IR with AUC of glucose 140-
200 mmol/L and AUC of insulin 40-90 µU (57, 64).  Sexually active participants will use barrier 
contraception for the duration of the study, and take pregnancy tests within 24 hours of 
participation.  Subjects are excluded if they have lost > 5 kg of weight within the past 6 months or 
exercise more than three times/week.  Attheir initial consent visit, after signing the consent form, 
subjects will be randomly assigned to take a GnRH antagonist with or without methyl testosterone 
(see “Study Design” below).  They will be told immediately of the results of the randomization. 
AE-PCOS subjects: Diagnosis of AE-PCOS will be based on modified NIH criteria requiring the 
presence of both clinical hyperandrogenism (hirsutism based on Ferriman Gallwey score of >8 
and/or acne), and biochemical hyperandrogenemia (elevated serum total testosterone; ELISA, 
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see Authentication).  Anovulation (defined as a total of ≤ 8 menses per year) and presence of 
polycystic ovarian morphology on transvaginal ultrasound as determined by a trained 
obstetrician/gynecologist (Dr. Pal), obesity and IR will be additional enrollment criteria for the 
AE-PCOS population. Sex as a biological variable:  Only females will be used for these studies 
since AE-PCOS is a female-specific disorder.
Control subjects will be obese, IR women, with regular menses every 26-34 days. Subjects will 
be excluded if they have any symptoms of AE-PCOS subjects, including polycystic appearing 
ovaries (as determined by transvaginal ultrasound (Dr. Pal). 
For Aim 1, 2 and 3:  All subjects will be given urine collection containers to collect their 
overnight urine prior to visits 4,5 and 6.
For Aim 1, 2 and 3:  All subjects will self administer Antagon injections for a total of 10 
days.  Subjects may be kept on Antagon for as long as 18 days if there is a challenge 
with experiment scheduling. Subjects will also ingest a testosterone pill for seven days. 
Subjects may also be kept on testosterone for up to 11 days with regard to scheduling 
issues.
For Aim 5:  Subjects will participate in only one baseline study (Fig 7).  We will recruit 
black and white women to participate in this study with AE-PCOS. 
EXPERIMENTAL PROTOCOLS:
Aims 1 & 2.  Study design (Fig 7) and Hormone administration (Fig 8): IR subjects with and 
without AE-PCOS will be randomly (MiniTab, © cleverbridge, Inc.) divided into two Protocol 
groups (4 groups total, n=18 each) to be tested 3 times each:  Protocol 1: prior to GnRH 
antagonist (Day 1); GnRH antagonist (250 μg/day in 0.5 ml normal saline, Days 3 and 10). 
Protocol 2: prior to GnRH antagonist (Day 1), GnRH antagonist alone (Day 3), GnRH antagonist 
+ methyltestosterone (Day 10). In both Protocols, Day 1 in Controls occurs in the proliferative 
phase of the menstrual cycle (days 1-7, low hormone phase); GnRH suppression begins on days 
26-28 of that cycle.  Non-cycling women with AE-PCOS begin testing at their convenience.  
*Women may take the Antagon pills for as long as 18 days.  We allow this flexibility because of 
challenges with scheduling subjects.  There is no increase in risk, and we do everything possible 
to avoid the rare instance where taking this drug needs to be extended.  If the drug is extended, 
“day 10” procedures occur on the final day of “GnRH antagonist + methyltestosterone” 
administration.
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Figure 8. Hormone 
Figure 7. The Overall Plan for Human Studies 
proposed in Aims 1 and 2.  Studies to assess the 
baroreflex and renin angiotensin system (RAS) will be 
performed in the same protocol.  Overnight (12 hour) 
urine will be collected on all subjects prior to these 
protocols.
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Aim 3.  Study design (Fig 7a) and Hormone administration (Fig 8):  IR subjects with 
and without AE-PCOS will be randomly (MiniTab, © cleverbridge, Inc.) divided into two 
Protocol groups (4 groups total, n=8 each) to be tested 3 times each:  Protocol 1: prior to 
GnRH antagonist (Day 1); GnRH antagonist (250 μg/day in 0.5 ml normal saline, Days 3 
and 10). Protocol 2: prior to GnRH antagonist (Day 1), GnRH antagonist alone (Day 3), 
GnRH antagonist + methyltestosterone (Day 10). In both Protocols, Day 1 in Controls 
occurs in the proliferative phase of the menstrual cycle (days 1-7, low hormone phase); 
GnRH suppression begins on days 26-28 of that cycle.  Non-cycling women with AE-
PCOS begin testing at their convenience.  
Standard Preliminary Procedures for all Protocols. The subjects maintain their normal 
diet and activity for three days prior to any experimental protocol and avoid alcohol and 
caffeine for 12 hrs prior to arriving at the laboratory. The subjects will arrive at the laboratory 
at 0800 having fasted for 8 hrs prior to the cardiovascular experiments.  They will eat their 
normal diet for three days prior (see PROCEDURES) and drink water based on their body 
weight. On the morning of the experiment, we assess hydration from urine specific gravity 
(S.G.) of the initial urine sample. If S.G.>1.020, the subjects will drink 5 ml/kg of water, 
followed by a 30 min control period until hydration is deemed sufficient (S.G. <1.020).  On 
all days that subjects visit the laboratory, they will take a rapid Covid 19 test according 
to the directions of the test. The subject will take this test in a separate chamber. This 
test will involve putting a “q-tip” like swab in each nostril and rubbing in a circular motion 
ten times and then placing the swab into analysis fluid from the test kit.  A member of 
our research team will observe the swabbing procedure and read the results of the test.  
If the test is positive, the subject will not proceed further with the experiment, and will be 
asked to leave the laboratory immediately and suggest contacting their personal 
physician.  If the test is negative, we will proceed with the experiment.
 Figure 7a. The protocol Studies proposed in Aim 3.  Studies to assess the 
baroreflex and renin angiotensin system (RAS) will be performed in the same 
protocol.  Overnight (12 hour) urine will be collected on all subjects prior to 
these protocols.
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Preliminary Testing. Because naive volunteers may be used, all subjects are brought 
to the laboratory prior to the experimental days, thoroughly trained in all maneuvers and 
familiarized with all routines to ensure that they are as comfortable as possible on the 
first experimental day.  Each subject is individually trained on how to do the GnRH 
antagonist injection, either by the PI, the Post Doc or a nurse at the Reproductive 
Endocrinology clinic.  During the training, the subject is shown how to clean the area first 
with an alcohol pad, how to open the package with the syringe to avoid sticking their 
fingers and to maintain sterility.  The subjects are taught to use either their leg or 
abdomen, and how to insert the needle and press down to inject the fluid.  They are also 
taught to use pressure should there be bleeding after the injection.  In addition, the 
subjects are all provided with detailed instructions provided by the manufacturer, alcohol 
pads and sterile gauze pads.  
Research Design:
Group 1 (Fig 8): 
1) prior to GnRH suppression;
2) during GnRH suppression (Ganirelix acetate alone, 250 μg/day in 0.5 ml normal saline, 2 
days);
3) Ganirelix + methyltestosterone ((oral, 5.0 mg/day, 7 days (on day 10));
Group 2 (Fig 8): 
1) prior to GnRH suppression;
2) during GnRH suppression (Ganirelix acetate alone, 250 μg/day in 0.5 ml normal saline, 2 
days);
3) during GnRH suppression (Ganirelix acetate alone, 250 μg/day in 0.5 ml normal saline, 2 
days) and again at 10 days; Control subjects will first be tested in the proliferative phase of 
the menstrual cycle (days 8-12) because estrogen status at this time is similar to non-
cycling PCOS subjects. 
Women with normal menstrual cycles will begin GnRH suppression on days 26-28. 
Women may take the Antagon pills for as long as 18 days.  We allow this flexibility because of 
challenges with scheduling subjects.  There is no increase in risk, and we do everything possible 
to avoid the rare instance where taking this drug needs to be extended.  If the drug is extended, 
“day 10” procedure occur on the final day of “GnRH antagonist + methyltestosterone” 
administration.
Research Protocol (Fig 8):  
Visit 1:  Screening, informed consent, orientation.
Visit 2:  Ultrasound (YMG)
Visit 3:  Oral glucose tolerance test (OGTT).
Visit 4: (BL): Baroreflex and RAS testing
Visit 5: (GnRH): Baroreflex and RAS testing 
Visit 6: (GnRH +T): Baroreflex and RAS testing 
OR
Visit 5: GnRH): Baroreflex and RAS testing 
Visit 6: GnRH): Baroreflex and RAS testing 
Visit 1:  Screening, informed consent, orientation:
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We will describe the study in detail. Subjects will fill out a medical history form (including 
medications) so we can make sure it is safe for the participant to be in this study. They will have 
a pregnancy test at no cost and if eligible for the study and decide to participate, we will teach 
them how to give a self-injection of Antagon, a drug that will be used in this study. We will also 
orient the participant to where the research studies will be done and demonstrate the equipment 
and procedures that will be used for the study.  
Visit 2. Transvaginal ultrasound. The subjects will undergo evaluation using transvaginal 
ultrasound. We will perform transvaginal ultrasound using a linear probe on a Phillips 3-D 
ultrasound on all subjects. There are no known risks associated with this procedure.  The 
procedure is painless and will require approximately twenty minutes to perform.  The ultrasound 
examination will be performed in the Yale Center for Reproductive Medicine by a gynecologist 
on the staff of the Yale New Haven Hospital and who is accredited by the American Institute for 
Ultrasound in Medicine. The ultrasound will be a focused exam looking only at the ovaries.  With 
the subject’s permission, any incidental pathology noted at the time of the exam will be sent to 
the subject's private gynecologist for possible further evaluation.  All follicles and/or cystic 
structures are measured.  Ovarian volume is calculated as the measure of a prolate ellipsoid.  
Ovarian morphology is determined by the methods described in the literature for identifying 
polycystic ovaries in women with PCOS (3, 4).
Visit 3. Oral glucose tolerance test.  Subjects will undergo an OGTT at The John B. Pierce 
Laboratory to quantify glucose tolerance and insulin resistance.  These tests are done in the 
early proliferative phase of the menstrual cycle in cycling women.  A standard (3 hour) OGTT 
will be conducted to determine glucose tolerance in all subjects within 2 weeks of the start of the 
study. One hour will be added for postural acclimation and insertion of the IV for a total of a 4 
hour visit. Blood samples for serum concentration of glucose and insulin will be drawn at 
baseline prior to ingestion of a 75g glucose beverage (Glucose Drink; Azer Scientific, 
Morgantown, PA) and at every 30-minute time point following drinking for three hours. A total of 
45ml (3 tablespoons) will be drawn. 
Visits 4-6 (Visits 5 and 6 are identical to Visit 4):
Protocols 1A-B: Autonomic function: Baroreflex testing (Fig 9).  We will examine autonomic 
function in our human subjects at rest (cardiovagal), during Valsalva maneuver (sympathetic), 
during the modified Oxford protocol (sympathetic, Fig 9) (20, 23, 33, 42) and with integrated 
baroreflex response during LBNP (23, 103). Our primary measure of SNSA is peripheral nerve 
activity, as measured by muscle sympathetic nerve activity (MSNA).  We will also determine 
HR, BP (EKG and beat-to-beat), and a catheter will be inserted into a forearm vein for blood 
sampling.  Following the instrumentation period, resting 
HR, pulse (or R-R) interval (PI), BP, and MSNA will be 
recorded for 10 minutes, and a blood sample drawn for 
analysis of S[E2], progesterone (S[P4]), sex hormone 
binding globulin, (P[SHBG]), albumin, free (P[Tfree]) and 
bound (P[T]) testosterone, S[ALD], serum angiotensin 
converting enzyme (S[ACE]), angiotensinogen (S[ANG]), 
renin, (P[Renin]), PRA, P[ANG II], norepinephrine (P[NE]), 
epinephrine (P[EPI]), electrolytes, creatinine (Cr) and 
osmolality (POsm). After the first blood sample and a 30-
min rest phase, subjects will begin a seven-minute 
period of rest with paced breathing (12 breaths per 
minute) while HR, BP and MSNA are measured (Fig 9). 
BP
Integrated MSNA
EKG
Resp
MSNA
SNP
PE
Figure 9. Modified
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The subjects will synchronize breathing to a metronome set to a cadence of ~12 breaths per 
minute (0.2 Hz). Following baseline measurements, subjects will perform two Valsalva 
maneuvers. Methods are described in Procedures.
Sympathetic baroreflex sensitivity assessment: modified Oxford technique (Aim 1).  To 
assess baroreflex control of sympathetic outflow, HR, BP, MSNA and PI will be measured during 
arterial pressure changes induced by infusion of sodium nitroprusside (SNP, endothelium-
independent vasodilator) and phenylephrine (PE, an -receptor agonist) (20, 23, 42).  After 10-
minute baseline period, SNP (100 µg bolus) will be given followed 1 minute later by PE-HCL (150 
µg).  This protocol decreases BP~15 mm Hg below baseline levels and then increases BP~15 
mm Hg above baseline levels over a short period of time (See bottom tracing, Fig 9). Infusions 
are sequential, repeated twice in each subject with at least 15 minutes between each cycle of 
infusions.  Fig 9 shows change in BP and increase in (integrated) MSNA that follows SNP 
infusion, and the decreased MSNA following PE infusion in a typical subject. Sympathetic 
baroreflex sensitivity is defined as the slope relating MSNA and DBP during the sequential 
infusions of SNP and PE (20).  Cardiovagal baroreflex sensitivity is defined as the slope relating 
PI and SBP (20, 23, 42).  The modified modified Oxford technique has been approved under 
HIC #0512000875.  The PE and SNP will be infused by Chris Moore (Emergency Medicine).  
Integrated baroreflex assessment (LBNP) (Aim 1).  After the modified Oxford protocol, we will 
assess integrated baroreflex function during incremental LBNP. Although low-level LBNP has 
been used to isolate cardiopulmonary baroreceptor input, arterial baroreceptors appear to be 
involved even at very low levels of LBNP (51, 96) so we use the term “integrated” to acknowledge 
the limitations of distinguishing cardiopulmonary versus arterial baroreceptor functions (23, 88, 
103), and it is not the goal of these studies to distinguish the contributions of these respective 
baroreceptor inputs. LBNP protocol:  During a 30-minute rest period after the SNP and PE 
infusions, microneurography electrode is removed, and subjects are instrumented in the left arm 
for the measurement of forearm blood flow (FBF, 87). We will measure baseline variables, tilt the 
subjects to 7° head down (HDT), return the subject to supine, then apply LBNP at –10, –20, –30 
and –40 mm Hg (for 4 min each) in descending order. Heart rate, BP (beat-to-beat), PI and SkBF 
are measured continuously, and calf volume (circumference, to determine the extent of lower 
level volume shift and ensure relaxed muscles during LBNP). Blood sample is taken prior to and 
at the end of each stage of LBNP for the measurement of S[ALD], S[ACE], S[ANG], P[Renin], PRA, P[ANG 
II], P[NE], P[EPI].  FBF is measured at 30-sec intervals, at rest and during the entire LBNP protocol.  
The total blood taken is 160 ml per visit.  With FBF and BP data, we compute forearm vascular 
resistance (FVR) and conductance (FVC).  If subjects experience presyncopal symptoms or if 
BP falls by 20% of baseline, we discontinue the study.  If the test is stopped early we use the 
slopes generated from the completed levels of LBNP, but exclude data collected during 
symptoms or exaggerated BP falls.  To determine peripheral vascular responses we analyze 
FVR and FVC as functions of LBNP.  We will also determine changes in peripheral vascular 
responses by examining changes in HR as functions of SBP and LBNP (i.e. ∆ HR/∆ SBP or ∆ 
LBNP). LBNP has been approved under HIC #0512000875.
Flow Mediated Vasodilation (FMD): (Aim 3) Continuous measures of brachial artery 
diameter and blood flow velocity will be simultaneously measured using the Sonoscope 
S2 ultrasound imaging system. After 15 minutes of supine rest, a longitudinal image of 
the brachial artery is recorded, after which forearm ischemia is induced by rapidly inflating 
a blood pressure cuff (placed immediately distal to the olecranon process) to 200 mmHg 
for 5 minutes.  Upon cuff release, blood flow increases to the ischemic tissue (reactive 
hyperemia).  The increase in shear stress associated with this rapid blood flow increase 
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stimulates NO release from endothelial cells, causing vasodilation. We record the change 
in brachial artery diameter 15 seconds prior to cuff deflation continuously thereafter for 2 
minutes.  Simultaneous recordings of blood flow velocity will be measured with a Doppler 
probe in order to quantify shear rate.  Quantifying shear stress is important in determining 
endothelium- dependent dilation, since the magnitude and duration of this stimulus varies 
among individuals (25, 76) and differs in the upper and lower extremities (68).
Exercise.  (Aim 3) We also evaluate the blood flow and vasodilatory response after occlusion 
with hand grip exercise. In this experiment, subjects perform static handgrip exercise at 20% 
maximal voluntary contraction (MVC) during the arterial occlusion by inflating a pneumatic upper 
arm cuff to 200 mm Hg for 2 to 5 minutes. We measure the vessel diameter and blood velocity, 
skin blood flow and blood pressure for 5 minutes after the cuff release. The order of rest vs. 
exercise experiments is randomized.
Aim 4.  In this proposal, we will recruit women with AE-PCOS who have already participated in 
prior Aims and taken ganirelix.  At least one month after they have completed the study, we will 
recruit these women to take elagolix for seven days.  We will collect blood samples to compare 
serum concentrations of total and free testosterone, E2 and SHBG prior to and following the 7-day 
elagolix treatment and compare these blood variables with the changes due to ganirelix found 
while participating in Aims 1-3.  
Study Design:  Humans.  All subjects will receive (elagolix, 400 mg/day oral, 2X200 mg 
tablets/day for 7 days) (91). All women will begin taking elagolix in the first week of their menstrual 
cycle,. 
Statistical analyses: Univariate statistics [mean (SD)] will be generated on all study variables 
first, which will determine the normality or the heteroscedacity of the data. Data will be analyzed 
using SPSS 21 statistical software, that includes calculations to compensate for missing data, 
and Prism (GraphPad) curve fitting software where appropriate. Data analysis. The slopes of the 
linear regressions for PI-SBP (cardiovagal baroreflex gain), PI-LBNP (integrated baroreflex gain), 
resting MSNA, slopes for MSNA-DBP (sympathetic baroreflex gain during Oxford and Valsalva), 
will be compared between treatment conditions (GnRH antagonist vs. GnRH antangonist+T and 
between groups (Control vs AE-PCOS) using a 2-way repeated measures ANOVA with 
supplemental post hoc testing with Tukey’s HSD test. Sample size.  The desired statistical test 
is two-sided, and we assume an alpha level of =0.01 to account for multiple comparisons (37). In 
our laboratory, we observed effect sizes (SD) of PI-SBP intercept (with n=4) of 0.39 (0.16), our 
most stringent variable.  A sample size of 15 women per group will allow us ≥ 80% statistical 
power (1- >0.9) to test within group comparisons, and this same sample size will allow [1-]=0.80 
to distinguish any between-group differences from chance alone (29). Due to the technical 
challenges of performing microneurography on multiple occasions in obese women, we 
overestimate and account for a ~ 20% failure rate, so we will recruit 18 subjects per group. R&R: 
1) GnRH antagonist suppression/hormone add-back is the gold standard and most reliable 
method to control sex hormones in young women. 2) Microneurography is the gold standard 
and most sensitive and reliable available measure for MSNA. 3) Lower body negative pressure 
and the Modified Oxford Technique are the gold standards and most sensitive and reliable 
available measures of autonomic function in humans. We will report our methods and findings in 
detail and full transparency to permit others to reproduce and extend our findings.  Sex as a 
biological variable:  Only women are included in these studies because AE-PCOS is a female-
specific disorder.
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Characterization of the RAS during LBNP in humans (Aim 2).  We will use the protocol 
described in Fig 7 with the hormone intervention described in Fig 8. Before beginning the 
protocols, during 7° HDT, at 0 (supine rest) and in the final minute of each level of LBNP we will 
draw blood to determine S[ALD], S[ACE], S[ANG], P[Renin], PRA, P[ANG II], P[NE], P(38), electrolytes, Cr) and 
Osm. Urine samples will be collected just prior and after baroreflex/RAS LBNP protocol for 
electrolytes.
Statistical analyses: We use ANOVA as described in Protocols 1. Sample Size is based on 
calculations in Aim 1, as the Aim 1 variables are the more stringent.  
Expected results, interpretation of data, limitations and anticipated problems: Based on our 
hypothesis, we expect that women with AE-PCOS will have greater RAS hormonal responses to 
LBNP, reflecting greater RAS activation.  We expect that testosterone suppression will reduce 
PRA, S[ALD], S[ANG II], PRA and P[Renin]  response to LBNP, suggesting lower RAS activation in both 
Control and AE-PCOS.  We expect that increasing P[Tfree] and FAI will increase RAS hormone 
response to LBNP, reflecting greater RAS activation responses to LBNP in AE-PCOS.  Since the 
methods to be used are in place in our laboratory, we don’t anticipate any problems in performing 
the studies in this Aim.  
Expected results, interpretation of data, limitations and anticipated problems:  We expect 
AE-PCOS women will have greater resting MSNA compared to Controls.  We anticipate that 
slopes for MSNA-DBP will be greater in AE-PCOS, indicating increased sympathetic BRS 
compared to Control.  We expect that testosterone suppression will decrease slopes for MSNA-
DBP in AE-PCOS, and testosterone administration will increase slopes for MSNA-DBP in both 
Control and AE-PCOS.  In contrast, we expect reduced slopes of PI-SBP with modified Oxford 
and paced breathing, indicating decreased sympathetic and cardiovagal BRS in AE-PCOS 
women; we expect a steeper PI-SBP slope with androgen suppression, and a steeper PI-SBP 
slope with modified Oxford, as well as paced breathing and Valsalva in both groups.  Since all 
methods to be used are in place in our laboratory, we don’t anticipate any problems in performing 
the studies in this Aim. 
Statistical analyses: We use ANOVA as described in Protocols 1 & 2 to test changes in blood 
pressure, FMD and baroceptor sensitivity . Sample Size is based on calculations in Aim 1, as the 
Aim 1 variables are the more stringent.  
Aim 3:  We expect AE-PCOS women will have greater resting blood pressure compared to 
Controls. We expect that women with AE-PCOS will have greater RAS hormonal responses to 
LBNP, reflecting greater RAS activation.  We expect that testosterone suppression will reduce 
PRA, S[ALD], S[ANG II], PRA and P[Renin]  response to LBNP, suggesting lower RAS activation in both 
Control and AE-PCOS.  We expect that increasing P[Tfree] and FAI will increase RAS hormone 
response to LBNP, reflecting greater RAS activation responses to LBNP in AE-PCOS.  We 
expect Flow Mediated Vasodilation  to be lower in the the women with AE-PCOS compared to 
control women, and we expect the suppression of testosterone to eliminate this difference.  We 
expect that increasing P[Tfree] and FAI will increase FMD in women with AE-PCOS, with little impact 
on Controls.  We anticipate that women with AE-PCOS will have lower baroreceptor sensitivity, 
compared to Controls, and that this will be improved with testosterone suppression.   Finally, we 
expect increasing P[Tfree] and FAI will increase baroreflex sensitivity in women with AE-PCOS, with 
little impact on Controls.  Since the methods to be used are in place in our laboratory, we don’t 
anticipate any problems in performing the studies in this Aim.  
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Aim 4.   We expect that testosterone will be suppressed similarly during elagolix and ganirelix 
treatments.  We will use Cronbach’s alpha as a measure of internal consistency or reliability 
(95).  
Aim 5. We expect black women with AE-PCOS will have greater resting blood pressure 
compared to white women with AE-PCOS.  We anticipate that slopes for MSNA-DBP will be 
greater in black women with AE-PCOS, indicating increased sympathetic BRS compared to 
white women with PCOS.
PROCEDURES
Muscle sympathetic nerve activity will be quantified by identification and measurement of 
sympathetic bursts in the integrated neurogram, and expressed as burst frequency (number of 
bursts per unit time). ECG surface electrodes are applied to the chest, an elastic band 
incorporating a strain gauge element is strapped around the thorax for recording respiratory 
movements, and a sensor cuff of a servopulse-plethysmography arterial pressure monitor 
(FINAPRES, Ohmeda) is placed on the middle phalanx of the long finger (held at heart level). 
The experiments are carried out in an environmentally controlled chamber at an ambient 
temperature of 27.0 ± 0.1 °C. The experiment will begin with a 60-minute control period. During 
such time the subjects rest in the reclined position. This procedure has been approved under 
HIC #0512000875.
Microneurography Recording of multiunit postganglionic muscle sympathetic nerve activity 
(MSNA) will be made from the common peroneal nerve as it winds around the fibular head with 
the subject in the supine position with her thigh comfortably supported. This procedure requires 
us to first trace the course of the nerve using small electrical stimuli applied to the surface of the 
skin over the nerve. This allows the microneurographer to find and focus the recording electrode 
tip on a nerve fascicle associated with muscle sympathetic activity.  The time required to focus 
the electrode on the nerve averages about 30 min and will not exceed 60 min.  In optimal 
conditions the track of the nerve from superficial stimulation is accurate and the nerve remains in 
position during insertion.  An uninsulated subcutaneous electrode will serve as the reference. 
Mechanoreceptor afferent activity can be recorded during light tapping on the muscle belly or 
passive muscle stretch.  A suitable intrafascicular recording site for MSNA consists of regular 
bursts and synchronous with the cardiac cycle.  The neural activity is amplified (2x104), filtered 
(0.5-5.0 kHz), rectified, and integrated. The amplified and filtered nerve signal is also led to an 
audio monitor and through a resistance capacitance circuit (time constant 100 ms). The integrated 
nerve signal is digitized at 400 Hz along with the ECG signal while respiratory and arterial pressure 
signals are also sampled at 400 Hz (MacLab 8s).  Data for analysis is selected from periods of 
quiet breathing in which subjects were relaxed and not talking. Microneurography was approved 
under HIC # 8079, #0512000875 and HIC #1609018353
.
Cardiovascular variables. Recorded with a 16-channel computerized data-acquisition system 
at a sampling Speed of 400 Hz (ADI Instruments PowerLab 9, Castle Hill, Australia). Beat-to-beat 
DBP, SBP and pulse interval (PI) are determined using peak detection algorithms (44). Brachial 
artery BP and HR are measured with an automated sphygmomanometer (Colin Medical 
Instruments Corp, Komaki, Japan). Heart rate and pulse interval will be determined beat-to-beat 
from lead II of the ECG recording. 
Cardiovagal baroreflex sensitivity, as we previously described (88), will be determined by slope 
of linear relationship between PI and SBP at rest, during Valsalva maneuver (67, 87), and during 
the modified Oxford technique (21, 23, 65, 66). R&R: At rest, baroreflex sequence requires three 
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or more consecutive beat-to-beat increases in PI with simultaneous increase or decrease in SBP 
(7). Only those sequences with a correlation greater than 0.85 are included in the analysis. 
Average of individual slopes in each condition will be taken as a measure of resting cardiovagal 
BRS.  Incidence of baroreflex sequences will be reported as sequences per 100 pulses to 
normalize for differences in HR among subjects and across experimental days (55).  We 
recognize that this method for evaluating BRS has limitations (49), but it is a widely accepted 
method, is predictive of cardiovascular morbidity (58), and is an indicator of autonomic control 
(21). Sympathetic baroreflex sensitivity will be evaluated by determining slope of the linear 
relationship between MSNA and DBP at rest, during Valsalva maneuver and during the modified 
Oxford technique (23, 65, 66). R&R: MSNA can be distinguished from other sources of nerve 
activity by the following criteria: 1) presence of spontaneous pulse synchronous bursts; 2) 
increased activity during Valsalva; 3) muscle afferent activity with muscle stretch; 4) no change 
of activity during light stroking of skin or startling the subject with loud noises, indicating a lack of 
skin nerve activity. Valsalva maneuver: Subjects will perform Valsalva maneuver following deep 
inspiration: expiratory mouth pressure (measured with a pressure transducer; 40 mm Hg, 20 
seconds) by blowing through a 1-inch diameter tube against an open glottis. Cardiovagal 
baroreflex is assessed during phase IV (overshoot phase) of the Valsalva maneuver, the phase 
occurring right after the release of the strain (67, 87).  Consecutive SBP values during phase IV 
will be linearly regressed against corresponding PI from the SBP time point where PI begins to 
lengthen and continued to the point of maximal BP increase. Lower body negative pressure 
and Forearm blood flow (FBF), as previously described by us (88). 
Blood sampling. Blood sampling is done via 20-gauge catheter placed in an arm
vein. Subjects will be in the posture they will assume for at least 30 min prior to sampling to ensure 
a steady state in plasma volume and constituents. Blood sampling is done from free- flowing blood. 
Approximately 60ml of blood (4 tablespoons) will be removed for analysis.
Plasma hormones. Aldosterone, free and total testosterone, ACE, PRA, renin and AII are 
measured with ELISA. Albumin and Cr are measured using spectrophotometry methods. 
Catecholamines are measured using HPLC methods.
Urine analysis. Subjects are instructed to completely empty their bladder when voiding into a 
collection vessel and urine collection times are recorded. Urine osmolality and concentrations of 
sodium and potassium are measured immediately; an aliquot is frozen for creatinine analysis.
4. Genetic Testing   N/A 
5. Subject Population: Provide a detailed description of the types of human subjects who will 
be recruited into this study.
Subjects: Two groups (n=36 each) of women will be recruited to complete this study: 1) IR, obese 
young women between the ages of 18 and 40 years (Controls); 2) IR, obese young women 
between the ages of 18 and 40 years with AE-PCOS. A third group of subjects with the same 
characteristics [between ages of 18 and 40 years (Controls); 2) IR, obese young women between 
the ages of 18 and 40 years with AE-PCOS], n=16 in each group will be recruited for Aim 3. For 
Aim 4, n=15 women from each of the previous groups will be recruited to participate in this study.  
African American women that consent to participate in Aims 1, 2 and 3 will be included for Aim 5 
(now CLOSED).
6. Subject classification: Check off all classifications of subjects that will be specifically 
recruited for enrollment in the research project. Will subjects who may require additional 
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safeguards or other considerations be enrolled in the study? If so, identify the population of 
subjects requiring special safeguards and provide a justification for their involvement.
 Children
 Healthy 
Fetal material, placenta, or dead fetus
 Non-English Speaking
 Prisoners
 Economically disadvantaged persons
 Decisionally Impaired
 Employees
 Pregnant women and/or fetuses
 Yale Students 
Females of childbearing potential
NOTE: Is this research proposal designed to enroll children who are wards of the state as 
potential subjects? 
 Yes  
 No (If yes, see Instructions section VII #4 for further 
requirements)
7. Inclusion/Exclusion Criteria: What are the criteria used to determine subject inclusion or    
exclusion?
AE-PCOS subjects: Diagnosis of AE-PCOS will be based on modified NIH criteria requiring the 
presence of both clinical hyperandrogenism (hirsutism based on Ferriman Gallwey score of >8 
and/or acne), and biochemical hyperandrogenemia (elevated serum total testosterone; ELISA, 
see Authentication).  Anovulation (defined as a total of ≤ 8 menses per year) and presence of 
polycystic ovarian morphology on transvaginal ultrasound as determined by a trained 
obstetrician/gynecologist (Dr. Pal), obesity and IR will be additional enrollment criteria for the 
AE-PCOS population. Sex as a biological variable:  Only females will be used for these studies 
since AE-PCOS is a female-specific disorder.
Control subjects will be obese, IR women, with regular menses every 26-34 days. Subjects will 
be excluded if they have any symptoms of AE-PCOS subjects, including polycystic appearing 
ovaries (as determined by transvaginal ultrasound (Dr. Pal). 
Exclusion criteria
All subjects will be excluded from the study if they are taking medication that interact with the study 
medication.  Subjects may not take medications during the study except for Tylenol and other 
medications approved by the PI.  The subjects fill out a medical questionnaire in which we ask 
them if they are taking any medication, and we ask them for a history of medications in this same 
form. 
Subjects with the following histories or conditions will be excluded from the study:
Gynecologic: a. current or past estrogen-dependent neoplasia, b. unexplained vaginal bleeding, 
c. history of uterine fibroids, d. current pregnancy, e. known or suspected breast or uterine cancer, 
f. partial or complete hysterectomy.
Cardiac: a. myocardial infarction, ventricular tachycardia or fibrillation, b. angina, c. valvular 
disease (mitral insufficiency or stenosis, aortic insufficiency or stenosis), d. congestive heart
failure, orthopnea, paroxysmal nocturnal dyspnea, e. current arrhythmias, f. prosthetic valves. 
Pulmonary: a. current cigarette smokers, or pipe or cigar smokers, b. chronic obstructive
pulmonary disease, c. adult asthma, d. dyspnea on exertion, e. current bronchitis, 
pneumonia, or tuberculosis, f. lung carcinoma, g. pulmonary embolus, recent (less than 1 
year).
Vascular: a. claudications or history of peripheral vascular disease, b. abdominal or thoracic 
aortic aneurysm, or repair of same, c. cerebral aneurysm, vascular malformations, d.
hypertension, systolic or diastolic.
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Gastrointestinal: a. GI malignancy, b. hepatitis, current, c. splenomegaly from any cause, d. 
Cholecystitis, e. current diverticulosis or diverticulitis, inflammatory bowel disease,
ulcerative colitis, Crohn’s disease,
Infectious Disease: any ongoing intercurrent infection.
Hematologic/Oncologic: a. receiving chemotherapy or radiation therapy, b. any metastatic 
malignancy, c. anemia (hematocrit < 35), d. thrombocytopenia or thrombocytosis, e. 
neutropenia, f. hematologic malignancy, g. bleeding dyscrasia.
Neurologic: a. history of cerebral vascular accident with any neurologic sequels, b. uncontrolled 
seizures (e.g., more than  1 seizure/year),  c.  transient ischemic attacks,  d.  dementia,  e.
neurologic conditions producing dyscoordination, peripheral neuropathy, or myopathy.
Endocrine: a. diabetes mellitus, b. any untreated endocrinopathy.
Renal: a. chronic renal diseases, b. any history of renal disease or impairment, c. current 
urinary tract infection.
Musculoskeletal: a. inflammatory arthritis history (e.g., rheumatoid, psoriatic, Reiters), b. 
any history of pathologic fractures, including vertebral compression fractures.
Pharmacologic: a. any illegal drug use (self report), b. alcohol use greater than an average 
of 4 oz/day over 30 days.
8. How will eligibility be determined, and by whom? 
Eligibility will be determined by the P.I. (Dr. Stachenfeld) and by the Co-I (Dr. Pal) by interview 
with potential subjects.  We will conduct an initial interview by phone and the subject will fill 
out a medical history questionnaire during the orientation session.  In this interview we will ask 
the subject if she has been diagnosed with PCOS or insulin resistance, we will ask her 
approximate height and weight.  We will ask her name and if she has any other health issues 
that would preclude her from participating in the study.  
9. Risks: Describe the reasonably foreseeable risks, including risks to subject privacy, 
discomforts, or inconveniences associated with subjects participating in the research. 
Potential risks associated with blood sampling, include infection from failure to observe 
proper sterile conditions, and hematoma (bruise) from careless technique. The latter may be 
associated with some discomfort but, in itself, presents little danger to the subject’s welfare.
Transvaginal ultrasound.  There is a small risk of infection with transvaginal ultrasound if 
proper disinfection procedures are not followed.  The procedure is painless and will require 
approximately twenty minutes to perform.  There is some slight pressure during this procedure, 
which some women may find uncomfortable.  This is performed in the Obstetrics/Gynecology 
Yale Medical Group clinic.
Microneurography:  The risk of microneurography is a peroneal paresthesia for 2-10 days 
following the experiment. The incidence of parethesias is reported to be less than 10%, in less 
than 1% of all subjects paresthesias can persist for up to two months. These paresthesias 
resolve spontaneously.
The Valsalva maneuver is a common stimulus used in human respiratory physiology. The 
Valsalva maneuver is also common in daily life (e.g. when lifting heavy objects).  Both stimuli 
may result in a feeling of breathlessness that may induce some anxiety. However, changes are 
reversed immediately upon resumption of volitional breathing of room air. Subjects will be able 
to return to room air breathing immediately based on the termination criteria. These criteria have 
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been set based on human blood oxygen dissociation computations. Below 80% blood oxygen 
saturation, the slope of the oxygen dissociation curve is steep, resulting in increased oxygen 
dissociation (from hemoglobin) given a step decrease in the partial pressure of inspired oxygen. 
The Valsalva maneuver differs from a voluntary apnea in that it is associated with well-
documented, acute changes in blood pressure. This maneuver has been used across a wide 
range of individuals, including clinical populations. 
GnRH antagonists (Antagon and Cetrotide) put the subjects into a temporary menopausal 
state, so they may experience vasomotor symptoms (i.e.  “hot flashes”) during the initial period of 
administration.  Additionally, although most women tolerate Antagon quite well, some may 
experience headaches, breast tenderness, transient mood changes, some temporary bloating of 
the abdomen and moderate irregular vaginal bleeding.  In our studies, the subjects begin the 
Antagon/Cetrotide treatments on days 25-27 of their menstrual cycle (or ~11-12 days after 
ovulation) to avoid discomfort to irregular bleeding, so this has not been a reported side effect in 
our experience. There are no documented long-term side effects of Antagon.  Subjects in our 
studies have reported minimal side effects from using Antagon over a period of 16 days.  In one 
of our studies, one of 15 subjects reported occasional vasomotor symptoms during Antagon 
treatment.  In our >10 years of using Antagon, two subjects reported transient changes in mood 
during Antagon treatment.  The subjects reported no other adverse effects due to the Antagon or 
hormone administration, and the side effects did not cause any of the subjects to leave the study.   
Elagolix (Aim 4) In our studies, the subjects with menstrual cycles begin the elagolix treatment 
on days 1-7 of their menstrual cycle (or 1-7 days after the beginning of menstruation) to avoid 
discomfort due to irregular bleeding, though this has not been a reported side effect. Long-term 
side effects of elagolix include the potential for bone loss however this risk is not relevant after 
short exposures (<6 months of continuous use).  We have not used elagolixin in our studies 
before, but subjects in our studies have reported minimal side effects from using Antagon over a 
period of 16 days. There will be at least one month between the end of taking GnRH Antagonist 
ganirelix acetate for the first part of this study and beginning this part to ensure washout of the 
ganirelix acetate.  Subcutaneous injections of ganirelix acetate  is associated with a mean half-
life of 12.8 hours are almost completely (69, 70)  Thus providing a month “washout” between 
finishing ganirelix acetate and elagolix, both well tolerated drugs, should cause no danger to the 
subjects.
Short-term methyl testosterone treatment is not usually associated with any major health risks. 
Methyl testosterone was chosen in this study because it is a component of Estratest, a widely 
used hormone therapy for menopausal women.  It is therefore FDA approved for use in women 
at this dose and has few side effects. Long term use of methyl-testosterone can cause abnormal 
hair growth, abnormal skin sensations, anxiety, hair loss on the scalp, breast growth, changes in 
sexual desire, general body discomfort, headache, mood changes and acne.  Testosterone 
treatment in women for longer than 6 months has not been studied.  
We have used the combination of GnRH antagonist and methyl testosterone in HIC #1508016301 
in women with and without PCOS with no ill effects or side effects.  
The Oxford techniques using the phenylephrine and sodium nitroprusside doses proposed 
here have been performed safely in humans in many laboratories (20, 23, 33, 42, 65, 66). Risks 
associated with infusions these drugs are small changes in blood pressure that are not even 
noticed by the subjects.  Possible side effects may include flushing or a sense of warmth, nausea, 
headache and nervousness.  Blood pressure can potentially fall by 20-30mm during this 
technique.  Rare risks to phenylephrine and sodium nitroprusside infusion may include severe 
bradycardia 
or 
low 
heartrate, 
sulphite 
allergic 
reaction, 
methemoglobinemia 
(high 
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methemoglobin) or thiocyanate toxicity.  The drug infusions are done under the supervision of a 
doctor (Christopher Moore).  
10. Minimizing Risks: Describe the manner in which the above-mentioned risks will be 
minimized.
Potential risks associated with blood sampling, by implantation of catheter needles and 
microdialysis probes.  We reduce the risks because only experienced individuals place needles 
or probes by using proper sterile technique. 
Nina Stachenfeld will closely supervise all experiments.  Thus, trained and experienced personnel 
will supervise subjects during every phase of participation.  
Transvaginal ultrasound.  There is a small risk of infection with transvaginal ultrasound if 
proper disinfection procedures are not followed. We reduce the risk by having only Dr. Pal 
perform the ultrasounds, who is an experienced Ob/Gyn (Department Chair). This is performed 
in the Obstetrics/Gynecology Yale Medical Group clinic.
Microneurography.  The likelihood of the occurrence of symptoms following microneurography 
will be minimized by the following certain procedures.  First, few problems or symptoms are 
reported in laboratories whose microneurographers have received training from individuals with 
a documented record of success and safety.  Dr. Usselman or Dr. Stachenfeld will perform all 
microneurography studies.  Dr. Usselman is a highly qualified microneurographer in the laboratory 
with over six years’ experience and this will reduce the risk of occurrence of symptoms following 
the experiment.  The time spent searching for a nerve fascicle within the peroneal nerve will be 
limited to < 60 min.  No nerve site will be sampled more than once.  The recording electrodes are 
inspected under a dissecting microscope to insure electrode tip integrity (those electrodes found 
with damaged tips are not used) and each recording electrode will be used only once for 
intraneural recording.  Placement of the electrodes and positioning in the common peroneal nerve 
are performed under sterile conditions.  Metal microelectrodes are steam sterilized at Yale New 
Haven Hospital before use.  The site of electrode placement is cleaned with alcohol and only 
investigators wearing sterile gloves handle the sterile microelectrodes.  Following these simple 
guidelines, the risk of occurrence of symptoms following microneurography will be small. 
Microneurography studies were conducted at the Pierce Laboratory under HIC #0512000875.
Antagon, Cetrotide, elagolix and reproductive hormone administration. To ensure the safety 
of the women participating in this study, we will carefully interview each subject about her medical 
history. Throughout the study, if the subjects are sexually active, they use barrier contraception 
to prevent pregnancy.  Subjects will be removed from the study if they become pregnant during 
the research period, do not follow the prescribed Antagon or hormonal administration regimen, or 
if the investigators and/or medical supervisors feel their safety is at risk.  All drugs except elagolix 
have been approved under HIC #0512000875.
Flow mediated Vasodilation does not carry any risks.  However, there is temporary discomfort 
towards the end of the occlusion period.  We take care to warn the subjects about this when they 
consent to the study and remind them of this when we begin the FMD procedure.  
The Oxford technique is done under the supervision of Chris Moore, MD (Emergency 
Department).
11. Data and Safety Monitoring Plan: Include an appropriate Data and Safety Monitoring Plan 
(DSMP) based on the investigator’s risk assessment stated below. (Note: the HIC will make 
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the final determination of the risk to subjects.) For more information, see the Instructions, 
page 24.
1. Personnel responsible for the safety review and its frequency:
The principal investigator will be responsible for monitoring the data, assuring protocol compliance, 
and conducting the safety reviews at the specified frequency which must be conducted at a 
minimum of every 6 months (including when reapproval of the protocol is sought).  During the 
review process, the principal investigator (monitor) will evaluate whether the study should continue 
unchanged, require modification/amendment, continue or close to enrollment. Either the principal 
investigator or the HIC have the authority to stop or suspend the study or require modifications.
2. The risks associated with the current study are deemed moderate 
for the following reasons: (choose those that apply)
1.  We do not view the risks associated with the protocol as minimal.  YES In this study 
we use I.V. catheters to draw blood for hormone analysis.  We also insert 
microneurography needles for neural measurement.
2.   Given the now established safety and validity of the current protocol in our prior work, 
we do not view the proposed studies as high risk. YES. The investigators involved in these 
protocols all have extensive experience with these protocols. Although we have assessed the 
proposed study as one of moderate risk, the potential exists for anticipated and/or unanticipated 
adverse events, serious or otherwise, to occur since it is not possible to predict with certainty the 
absolute risk in any given individual or in advance of first- hand experience with the proposed 
study methods. Therefore, we provide a plan for monitoring the data and safety of the proposed 
study as follows:
3. Attribution of Adverse Events:
Adverse events will be monitored for each subject participating in the study and 
attributed to the study procedures / design by the principal investigator (Nina 
Stachenfeld) according to
the following categories:
a.) Definite: Adverse event is clearly related to investigational 
procedures(s)/agent(s). b.) Probable: Adverse event is likely related to 
investigational procedures(s)/agent(s). c.) Possible: Adverse event may be 
related to investigational procedures(s)/agent(s). d.) Unlikely: Adverse event 
is likely not to be related to the investigational
procedures(s)/agent(s).
e.) Unrelated: Adverse event is clearly not related to investigational 
procedures(s)/agent(s).
4. Plan for Grading Adverse Events:
The following scale will be used in grading the severity of adverse events noted during the 
study:
1.   Mild adverse event
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2.   Moderate adverse event
3.   Severe
5. Plan for Determining Seriousness of Adverse
Events: Serious Adverse Events:
In addition to grading the adverse event, the PI will determine whether the adverse 
event meets the criteria for a Serious Adverse Event (SAE).  An adverse event is 
considered serious if it:
1.   is life-threatening
2.   results in in-patient hospitalization or prolongation of existing hospitalization
3.   results in persistent or significant disability or incapacity
4.   results in a congenital anomaly or birth defect OR
5.   results in death
6.    baseduponappropriate medical judgment, may jeopardize thesubject’s healthandmay require medical or 
surgical intervention to prevent one of the other outcomes listed in this definition,
or
7.   adversely affects the risk/benefit ratio of the study
An adverse event may be graded as severe but still not meet the criteria for a Serious Adverse
Event. Similarly, an adverse event may be graded as moderate but still meet the criteria for an 
SAE.  It is important for the PI to consider the grade of the event as well as its ―seriousness‖
when determining whether reporting to the HIC is necessary.
6. Plan for reporting serious AND unanticipated AND related adverse events, 
anticipated adverse events occurring at a greater frequency than expected, 
and other unanticipated problems involving risks to subjects or others to the 
HIC.
The investigator will report the following types of adverse events to the HIC; a) serious AND 
unanticipated AND possibly, probably or definitely related events; b) anticipated adverse 
events occurring with a greater frequency than expected; and c) other unanticipated problems 
involving risks to subjects or others.
These adverse events or unanticipated problems involving risks to subjects or others will be 
reported to the HIC within 48 hours of it becoming known to the investigator, using the 
appropriate HIC forms found on the website.
7. Plan for reporting adverse events to co-investigators on the study, as 
appropriate the protocol’s research monitor(s), e.g., industrial sponsor, Yale 
Cancer Center monitors, the Hospital Research Unit’s (HRU) Science and 
Safety Committee (SSC), Cancer Center's Quality Assurance, Compliance and 
Safety Committee (QUACS) Protocol Review Committee (PRC), DSMBs, study 
sponsors, funding and regulatory agencies, and regulatory and decision-
making bodies.
d.
For multi-site studies for which the Yale PI serves as the lead investigator:  N/A
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i.   How will adverse events and unanticipated problems involving risks to subjects or 
others be reported, reviewed and managed?
ii.  What provisions are in place for management of interim results?
 iii.  What   will   the  multi-site  process   be  for  protocol modifications?
d. For multi-site studies for which the Yale PI serves as the lead investigator:  N/A
i.   How will adverse events and unanticipated problems involving risks to subjects or others be 
reported, reviewed and managed?
ii.   What provisions are in place for management of interim results?
iii.  What will the multi-site process be for protocol modifications?
a. 
What is the investigator’s assessment of the overall risk level for subjects 
participating in this study? 
b.
If children are involved, what is the investigator’s assessment of the overall risk 
level for the children participating in this study? 
c.
Include an appropriate Data and Safety Monitoring Plan. Examples of DSMPs are  
 available here http://www.yale.edu/hrpp/forms-templates/biomedical.html  for
i. Minimal risk
ii. Greater than minimal
d. For multi-site studies for which the Yale PI serves as the lead investigator: N/A
i. How will adverse events and unanticipated problems involving risks to subjects or 
others be reported, reviewed and managed?
ii. What provisions are in place for management of interim results?
iii. What will the multi-site process be for protocol modifications?
12. Statistical Considerations: Describe the statistical analyses that support the study design. 
Aim 1: Statistical analyses: Univariate statistics [mean (SD)] will be generated on all study 
variables first, which will determine the normality or the heteroscedacity of the data. Data will be 
analyzed using SPSS 21 statistical software that includes calculations to compensate for missing 
data, and Prism (GraphPad) curve fitting software where appropriate. Data analysis. The slopes 
of the linear regressions for PI-SBP (cardiovagal baroreflex gain), PI-LBNP (integrated baroreflex 
gain), resting MSNA, slopes for MSNA-DBP (sympathetic baroreflex gain during Oxford and 
Valsalva), will be compared between treatment conditions (GnRH antagonist vs. GnRH 
antangonist+T and between groups (Control vs AE-PCOS) using a 2-way repeated measures 
ANOVA with supplemental post hoc testing with Tukey’s HSD test. Sample size.  The desired 
statistical test is two-sided, and we assume an alpha level of =0.01 to account for multiple 
comparisons (37). In our laboratory, we observed effect sizes (SD) of PI-SBP intercept (with n=4) 
of 0.39 (0.16), our most stringent variable.  A sample size of 15 women per group will allow us ≥ 
80% statistical power (1- >0.9) to test within group comparisons, and this same sample size will 
allow [1-]=0.80 to distinguish any between-group differences from chance alone (29). Due to the 
technical challenges of performing microneurography on multiple occasions in obese women, we 
overestimate and account for a ~ 20% failure rate, so we will recruit 18 subjects per group. R&R: 
1) GnRH antagonist suppression/hormone add-back is the gold standard and most reliable 
method to control sex hormones in young women. 2) Microneurography is the gold standard 
and most sensitive and reliable available measure for MSNA. 3) Lower body negative pressure 
and the Modified Oxford Technique are the gold standards and most sensitive and reliable 
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available measures of autonomic function in humans. We will report our methods and findings in 
detail and full transparency to permit others to reproduce and extend our findings.  Sex as a 
biological variable:  Only women are included in these studies because AE-PCOS is a female-
specific disorder.
Aim 2: Statistical analyses: We use ANOVA as described in Protocols 1. Sample Size is based 
on calculations in Aim 1, as the Aim 1 variables are the more stringent.  
Expected results, interpretation of data, limitations and anticipated problems: Based on our 
hypothesis, we expect that women with AE-PCOS will have greater RAS hormonal responses to 
LBNP, reflecting greater RAS activation.  We expect that testosterone suppression will reduce 
PRA, S[ALD], S[ANG II], PRA and P[Renin]  response to LBNP, suggesting lower RAS activation in both 
Control and AE-PCOS.  We expect that increasing P[Tfree] and FAI will increase RAS hormone 
response to LBNP, reflecting greater RAS activation responses to LBNP in AE-PCOS.  Since the 
methods to be used are in place in our laboratory, we don’t anticipate any problems in performing 
the studies in this Aim.  
Expected results, interpretation of data, limitations and anticipated problems:  We expect 
AE-PCOS women will have greater resting MSNA compared to Controls.  We anticipate that 
slopes for MSNA-DBP will be greater in AE-PCOS, indicating increased sympathetic BRS 
compared to Control.  We expect that testosterone suppression will decrease slopes for MSNA-
DBP in AE-PCOS, and testosterone administration will increase slopes for MSNA-DBP in both 
Control and AE-PCOS.  In contrast, we expect reduced slopes of PI-SBP with modified Oxford 
and paced breathing, indicating decreased sympathetic and cardiovagal BRS in AE-PCOS 
women; we expect a steeper PI-SBP slope with androgen suppression, and a steeper PI-SBP 
slope with modified Oxford, as well as paced breathing and Valsalva in both groups.  Since all 
methods to be used are in place in our laboratory, we don’t anticipate any problems in 
performing the studies in this Aim.
Aim 3: Statistical analyses: We use ANOVA as described in Protocols 1. Sample Size is based 
on calculations in Aim 1, as the Aim 1 variables are the more stringent.  
Expected results, interpretation of data, limitations and anticipated problems:  We expect 
AE-PCOS women will have greater resting blood pressure compared to Controls. We expect that 
women with AE-PCOS will have greater RAS hormonal responses to LBNP, reflecting greater 
RAS activation.  We expect that testosterone suppression will reduce PRA, S[ALD], S[ANG II], PRA 
and P[Renin]  response to LBNP, suggesting lower RAS activation in both Control and AE-PCOS.  
We expect that increasing P[Tfree] and FAI will increase RAS hormone response to LBNP, reflecting 
greater RAS activation responses to LBNP in AE-PCOS.  We expect Flow Mediated Vasodilation  
to be lower in the the women with AE-PCOS compared to control women, and we expect the 
suppression of testosterone to eliminate this difference.  We expect that increasing P[Tfree] and FAI 
will increase FMD in women with AE-PCOS, with little impact on Controls.  We anticipate that 
women with AE-PCOS will have lower baroreceptor sensitivity, compared to Controls, and that 
this will be improved with testosterone suppression.   Finally, we expect increasing P[Tfree] and FAI 
will increase baroreflex sensitivity in women with AE-PCOS, with little impact on Controls.  Since 
the methods to be used are in place in our laboratory, we don’t anticipate any problems in 
performing the studies in this Aim.  
Aim 4.   We expect that testosterone will be suppressed similarly during elagolix and ganirelix 
treatments.  We will use Cronbach’s alpha as a measure of internal consistency or reliability 
(95).  
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SECTION VI: RESEARCH INVOLVING DRUGS, BIOLOGICS, RADIOTRACERS, PLACEBOS AND 
DEVICES
If this section (or one of its parts, A or B) is not applicable, state N/A and delete the rest of the 
section.
A.  DRUGS, BIOLOGICS and RADIOTRACERS
1. Identification of Drug, Biologic or Radiotracer: What is (are) the name(s) of the drug(s) 
biologic(s)  or radiotracer(s) being used? Identify whether FDA approval has been granted and for 
what indication(s).  
Antagon and Cetrotide are FDA-approved for women undergoing in vitro fertilization and have been 
approved for use in HIC protocols 10294, 12676, 25890 and 26534 (women with PCOS).  Elagolix is 
FDA-approved for women undergoing in vitro fertilization.
Methyl testosterone is FDA approved for use in women at a higher dose (100 mg) to treat acquired, 
generalized hypoactive sexual desire disorder (HSDD) in premenopausal women.
.
All protocols which utilize a drug, biologic or radiotracer not approved by, but regulated by, the FDA, 
or a radiotracer regulated by the RDRC, must provide the following information:  
a. What is the Investigational New Drug (IND) number assigned by the FDA?
b. Who holds the IND? 
c. All protocols which utilize a radiotracer not approved by, but regulated by the FDA must 
provide the IND number: _______________
Alternatively, use of the investigational radiotracer may be under RDRC/RSC oversight: (check 
if appropriate)_____________
For all investigational radiotracers, attach a copy of the RDRC/RSC application (for 
radioisotopes used in the PET Center, PET Center personnel may complete this step)
Go to http://rsc.med.yale.edu/login.asp?url=myApps.asp.  When you have logged in, complete 
the application and attach a copy to this submission.     
Alternatively, an exemption from IND filing requirements may be sought for a clinical
 investigation of a drug product that is lawfully marketed in the United States. If there is no IND and
 an exemption is being sought, review the following categories and complete the category that applies
 (and delete the inapplicable categories):
Exempt Category 1
The clinical investigation of a drug product that is lawfully marketed in the United States can be
exempt from IND regulations if all of the following are yes:
i.   The intention of the investigation is NOT to report to the FDA as a well-controlled study in support
     of a new indication for use or to be used to support any other significant change in the labeling for
     the drug.  
 Yes  
 No
ii. The drug that is undergoing investigation is lawfully marketed as a prescription drug product, and
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      the intention of the investigation is NOT to support a significant change in the advertising for the
      product. 
 Yes  
 No
iii.  The investigation does NOT involve a route of administration or dosage level or use in populations
      or other factor that significantly increases the risks (or decreases the acceptability of the risks)   
      associated with the use of the drug product. 
 Yes  
 No
iv. The investigation will be conducted in compliance with the requirements for institutional (HIC)   
      review and with the requirements for informed consent of the FDA regulations (21 CFR Part 50  
and 21 CFR Part 56). 
 Yes  
 No
v.   The investigation will be conducted in compliance with the requirements regarding promotion and 
charging for investigational drugs. 
 Yes  
 No
Exempt Category 2 (all items i, ii, and iii must be checked to grant a category 2 exemption)
 i. The clinical investigation is for an in vitro diagnostic biological product that involves one or
more of the following (check all that apply):
 Blood grouping serum
 Reagent red blood cells 
 Anti-human globulin
 ii. The diagnostic test is intended to be used in a diagnostic procedure that confirms the
diagnosis made by another, medically established, diagnostic product or procedure; and
 iii. The diagnostic test is shipped in compliance with 21 CFR §312.160.
Exempt Category 3
 The drug is intended solely for tests in vitro or in laboratory research animals if shipped in 
accordance with 21 CFR 312.60
Exempt Category 4
 A clinical investigation involving use of a placebo if the investigation does not otherwise 
require submission of an IND.
2. Background Information: Provide a description of previous human use, known risks, and data 
addressing dosage(s), interval(s), route(s) of administration, and any other factors that might 
influence risks. If this is the first time this drug is being administered to humans, include relevant 
data on animal models. 
NONE
3. Source: a) Identify the source of the drug or biologic to be used. 
b) Is the drug provided free of charge to subjects? 
 Yes  
 No
    If yes, by whom? 
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4. Storage, Preparation and Use: Describe the method of storage, preparation, stability information, 
and for parenteral products, method of sterilization and method of testing sterility and 
pyrogenicity. 
Check applicable Investigational Drug Service utilized:
     
   YNHH IDS                                                                      
   Yale Cancer Center 
     
   CMHC Pharmacy                                                          
  West Haven VA
     
   PET Center                                                     
    None      
     
    Other: 
Note: If the YNHH IDS (or comparable service at CMHC or WHVA) will not be utilized, explain in 
detail how the PI will oversee these aspects of drug accountability, storage, and preparation.  
We will purchase the GnRH antagonist through the reproductive endocrinology clinic at YNHH 
through Dr. Lubna Pal .  We will purchase the methyl testosterone via Compounded Solutions.  
Dr. Pal will provide the prescription. 
5. Use of Placebo: 
 Not applicable to this research project
If use of a placebo is planned, provide a justification which addresses the following:
1.
Describe the safety and efficacy of other available therapies. If there are no other
available therapies, state this.
b.   State the maximum total length of time a participant may receive placebo while on the study. 
c.   Address the greatest potential harm that may come to a participant as a result of  receiving    
  
   placebo. 
d.   Describe the procedures that are in place to safeguard participants receiving placebo. 
6. Use of Controlled Substances:
Will this research project involve the use of controlled substances in human subjects?
 Yes  
 No See HIC Application Instructions to view controlled substance listings. 
If yes, is the use of the controlled substance considered:
 Therapeutic: The use of the controlled substance, within the context of the research, has the 
potential to benefit the research participant.
 Non-Therapeutic: Note, the use of a controlled substance in a non-therapeutic research study 
involving human subjects may require that the investigator obtain a Laboratory Research License. 
Examples include controlled substances used for basic imaging, observation or biochemical 
studies or other non-therapeutic purposes. See Instructions for further information.
7. Continuation of Drug Therapy After Study Closure   
 Not applicable to this project
Are subjects provided the opportunity to continue to receive the study drug(s) after the study has 
ended?
 
 Yes   If yes, describe the conditions under which continued access to study drug(s) may apply 
as well as conditions for termination of such access. 
 
 No    If no, explain why this is acceptable.
 B.  DEVICES
NONE
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1.      Are there any investigational devices used or investigational procedures performed at Yale-
New Haven Hospital (YNHH) (e.g., in the YNHH Operating Room or YNHH Heart and 
Vascular Center)?  ☐Yes   X No    If Yes, please be aware of the following requirements:
a.       A YNHH New Product/Trial Request Form must be completed via EPIC: Pull down the 
Tools tab in the EPIC Banner, Click on Lawson, Click on “Add new” under the New 
Technology Request Summary and fill out the forms requested including the “Initial 
Request Form,” “Clinical Evidence Summary, “ and attach any other pertinent 
documents. Then select “save and submit” to submit your request; and
b. Your request must be reviewed and approved in writing by the appropriate YNHH 
committee before patients/subjects may be scheduled to receive the investigational device 
or investigational procedure.
2. What is the name of the device to be studied in this protocol?
Has this device been FDA approved? 
 Yes  
 No
If yes, state for what indication.
3. Background Information: Provide a description of previous human use, known risks, and 
any other factors that might influence risks. If this is the first time this device is being used in 
humans, include relevant data on animal models. 
4. Source: 
a) Identify the source of the device to be used. 
b) Is the device provided free of charge to subjects? 
 Yes  
 No
5. What is the PI’s assessment of risk level (significant or non-significant) associated with the 
use of the device? 
 Significant Risk (SR) Device Study: A study of a device that presents a potential for serious 
risk to the health, safety, or welfare of a participant and 1) is intended as an implant; 2) is used in 
supporting or sustaining human life; or otherwise prevents impairment of human health; 3) is of 
substantial importance in diagnosing, curing, mitigating or treating disease, or otherwise prevents 
impairment of human health; or 4) otherwise presents a potential for serious risk to the health, 
safety, or welfare of a participant.   
Significant Risk Devices require an Investigational Device Exemption (IDE) issued by the FDA.
What is the IDE number assigned by the FDA? 
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Did the FDA approve this IDE as Category A (experimental/investigational) or as Category B 
(non-experimental/investigational)?
Who holds the IDE?
 Non-Significant Risk (NSR) Device Study: A study of a device that does not meet the   
definition for a significant risk device and does not present a potential for serious risk to the health, 
safety, or welfare of participants.  Note that if the HIC concurs with this determination, an IDE is 
not required.
6.  Abbreviated IDE or Exempt IDE: There are abbreviated requirements for an IDE and 
there also are exemptions to the requirement for an IDE.  See the criteria in the HIC 
Application Instructions, Section VI.B.4 at 
http://www.yale.edu/hrpp/resources/docs/100FR1aHICProtocol_Application_Instructions5-
25-11.pdf to determine if these pertain to this study.
  
 Abbreviated IDE or Exempt IDE – If criteria set forth in the HIC Application Instructions 
are met, copy and paste the completed relevant section from the Instructions into this application.
7.   Investigational device accountability:
a.
State how the PI, or named designee, ensures that an investigational device is used only in 
accordance with the research protocol approved by the HIC, and maintains control of the 
investigational device as follows:
Maintains appropriate records, including receipt of shipment, inventory at the 
site, dispensation or use by each participant, and final disposition and/or the return of the 
investigational device (or other disposal if applicable):
Documents pertinent information assigned to the investigational device (e.g., date, quantity, batch 
or serial number, expiration date if applicable, and unique code number):
Stores the investigational device according to the manufacturer's recommendations with respect to 
temperature, humidity, lighting, and other environmental considerations:
Ensures that the device is stored in a secure area with limited access in accordance with applicable 
regulatory requirements:
Distributes the investigational device to subjects enrolled in the IRB-approved protocol:
SECTION VII: RECRUITMENT/CONSENT AND ASSENT PROCEDURES 
1. Targeted Enrollment: Give the number of subjects:
a.
  targeted for enrollment at Yale for this protocol_124__
b.    If this is a multi-site study, give the total number of subjects targeted across all 
sites_N/A__ 
2. Indicate recruitment methods below.  Attach copies of any recruitment materials that will be used.
 Flyers
 Internet/Web Postings
 Radio
 Posters
 Mass E-mail Solicitation
 Telephone
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 Letter
 
 Departmental/Center Website
 Television
 Medical Record Review*
 Departmental/Center Research Boards
 Newspaper
 Departmental/Center Newsletters
 Web-Based Clinical Trial Registries
  YCCI Recruitment database
 Clinicaltrials.gov Registry (do not send materials to HIC)
 Other (describe):  MyChart/PCOSChallenge
*Requests for medical records should be made through JDAT as described at 
http://medicine.yale.edu/ycci/oncore/availableservices/datarequests/datarequests.aspx
We will use the YCCI to help us connect with their with database of volunteers who have indicated 
willingness to be contacted for research studies.  YCCI uses OnCore as the database but pulls 
clinical data from Epic.  We are interested in using this database to help us recruit 
overweight/obese insulin resistant women with and without PCOS.  Both of these groups are 
challenging to recruit with only flyers posted in standard places around the medical school and 
Yale University campus.
For recruitment, we will utilize the Joint Data Analytics Team (JDAT) to identify potential subjects with 
insulin resistance and PCOS.  This is a behind-the-scenes search run by JDAT that will not identify the 
potential participants to the researchers, and therefore the researchers will receive no protected 
information. Once identified, potential subjects will be sent a message via MyChart that provides 
information about our blood pressure regulation research study as well as information on how to contact 
us, and brief information about the study. Potential subjects who do not use MyChart will receive a paper 
mailing including information about the study as well as information on how to contact us, and brief 
information about the study.  See below for the information potential subjects will receive.
We have applied through JDAT.  
The information potential subjects will receive:

You are receiving this MyChart message because you are a woman between the ages of 
18-40 years of age and may interested in participating in research projects.  One project, 
for which you may or may not be eligible investigates the connection between insulin and 
blood pressure control.  One part of the research investigates how women who are insulin 
resistant (but not diabetic), with or without Polycystic Ovary Syndrome (PCOS) regulate 
their blood pressure.   Approximately 60 subjects with insulin resistance will be enrolled in this study 
and half will have PCOS.  We are recruiting two groups of women: 1) insulin resistant, overweight/obese 
women (Controls); 2) insulin resistant, overweight/obese young women PCOS.   Subjects should have BMI 
between 27-35 kg/m2.  There will be four data collection visits over a six- week period, in addition to 
screening. 

You cannot participate in the protocol if you are diabetic.

Possible Benefits: This research is not designed to benefit you directly.  However, you will receive payment 
for your participation and free parking.  In addition, knowledge gained from the results may help us to 
better understand hypertension in women with insulin resistance or Polycystic Ovary Syndrome (PCOS).  

Confidentiality and Privacy: Any personal health data gathered will remain confidential and will be stored 
in a locked file cabinet, only accessed by study personnel. Your personal information will not be entered 
onto a computer and you will only be identified by subject number on any of our electronic devices.  When 
the results of the research are published or discussed, no information will be included that would reveal 
your identity. We understand that information about you obtained in connection with your health is 
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personal, and we are committed to protecting the privacy of that information. If you decide to participate, 
please contact the study coordinator at 203-562-9901 x219.

Participation in this study is completely voluntary.  You are free to decline to participate, to end 
participation at any time for any reason, or to refuse to answer any individual question at any 
time.   Refusing to participate will involve no penalty or loss of benefits to which you are otherwise entitled 
(such as your health care outside the study, the payment for your health care, and your health care 
benefits).

Questions: If you have any further questions about this study, you may contact the investigator, Nina 
Stachenfeld, at  203-562-9901 x219. If you would like to talk with someone other than the researchers to 
discuss problems, concerns, and questions you may have concerning this research, or to discuss your rights 
as a research subject, you may contact the Yale Human Investigation Committee at (203) 785-4688.
PI: Nina Stachenfeld, PhD.
What the study involves:
Study participants will be asked to participate in physiological studies in our laboratory with our research team. 
There are some medications administered in this study.
Your answers and all personal information will be protected by assigning you a study number which will be kept 
in a locked file cabinet. 
If you are  interested in learning more about this study, please call  203-562-9901 x219 to speak with a study 
coordinator. You could also contact us by email at nina.stachenfeld@yale.edu
Future research opportunities:
You may also create a volunteer profile through the Research Tab in MyChart.
PCOS Challenge: The National Polycystic Ovary Syndrome Association is a leading 
501(c)(3) nonprofit patient support an advocacy organization globally that is advancing the cause 
for women and girls with PCOS serving over 50,000 members.
https://www.pcoschallenge.org/
We would like to use PCOS Challenge recruitment program to help recruit subjects for our 
studies.
1) Key Points
PCOS Challenge study recruitment campaigns offer the most targeted and cost-effective 
ways to reach the largest number of women with PCOS available today. We offer the 
ability to geo-target recruitment efforts in any state.
PCOS Challenge is the largest nonprofit support organization globally for women with 
PCOS serving over 55,000 members.
92% of PCOS Challenge members have been diagnosed with PCOS.
PCOS Challenge has close to 1,000 members in Connecticut and more followers on 
social media. 
Here are quotes from study partners about the effectiveness of PCOS Challenge 
Campaigns
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o Single-State Campaigns - "We had 233 contacts in the last month, which 
outpaces our normal number...This proves to be an effective method of 
reaching out to people we wouldn’t normally be able to reach. I would like to 
discuss expanding our campaign to more places."
oCampaign Example See - https://pcoschallenge.org/apple-womens-health-study/
2) Study Recruitment Campaigns Details
Current rates through December 31, 2021 for a one-month recruitment campaign are:
1 State (Connecticut) - $2,500
The campaign above includes one month of:
Bi-weekly emails in the state(s) you are targeting (Sent every other week).  We 
will include IRB approved flyers.
Study promotional page on PCOSChallenge.org with additional information about 
the study (i.e. Title, Description, Goals, Eligibility Criteria, Exclusions, 
Locations, and Compensation) as well as relevant videos, articles, or audios 
such as a PCOS Challenge interview with the PI.  We will include IRB 
approved flyers.
160x600 banner ad (provided by study organization) promoting the study on 
every page of PCOSChallenge.com  (Over 50,000 pages).  We will use IRB 
approved flyers.
Promotion through PCOS Challenge Social Media Profiles (Over 120,000 Followers)
3.  Recruitment Procedures: 
a.
Describe how potential subjects will be identified.  Through phone screening by the 
PI.
b.  Describe how potential subjects are contacted.  They respond to advertisements.  We 
will also work with the YCCI.  The YCCI has a database of volunteers willing to be 
contacted for studies.  The Clinical Trials Resources dept. within YCCI will assist in 
identifying subjects who have indicated willingness to be contacted and fit the criteria for 
our study, and we will contact them by phone.
         c.   Who is recruiting potential subjects? The PI 
4. Screening Procedures
a.
Will email or telephone correspondence be used to screen potential subjects for 
eligibility prior to the potential subject coming to the research office? 
 Yes  
 No
b.
 If yes, identify below all health information to be collected as part of screening and 
check off any of the following HIPAA identifiers to be collected and retained by the 
research team during this screening process. 
We ask height, weight and if they have ever been diagnosed with PCOS or insulin 
resistance.  We ask if they have regular periods, if they smoke or are taking medications.
HEALTH INFORMATION TO BE COLLECTED
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HIPAA identifiers: 
 Names 
 All geographic subdivisions smaller than a State, including: street address, city, county, precinct, zip codes and their 
equivalent geocodes, except for the initial three digits of a zip code if, according to the current publicly-available data from 
the Bureau of the Census: (1) the geographic unit formed by combining all zip codes with the same three initial digits contains 
more than 20,000 people, and (2) the initial three digits of a zip code for all such geographic units containing 20,000 or fewer 
people is changed to 000. 
  Telephone numbers
 Fax numbers 
 E-mail addresses
 Social Security numbers 
 Medical record numbers
 Health plan beneficiary numbers 
 Account numbers 
  All elements of dates (except year) for dates related to an individual, including: birth date, admission date, discharge 
date, date of death, all ages over 89 and all elements of dates (including year) indicative of such age, except that such ages 
and elements may be aggregated into a single category of age 90 or older 
 Certificate/license numbers 
 Vehicle identifiers and serial numbers, including license plate numbers 
 Device identifiers and serial numbers 
 Web Universal Resource Locators (URLs) 
 Internet Protocol (IP) address numbers 
 Biometric identifiers, including finger and voice prints 
 Full face photographic images and any comparable images 
 Any other unique identifying numbers, characteristics, or codes 
5. Assessment of Current Health Provider Relationship for HIPAA Consideration:
Does the Investigator or any member of the research team have a direct existing clinical 
relationship with any potential subject? 
 Yes, all subjects
 Yes, some of the subjects
 No
If yes, describe the nature of this relationship.  It is possible that some of the subjects could be 
patients of Dr. Pal.
6. Request for waiver of HIPAA authorization: (When requesting a waiver of HIPAA Authorization 
for either the entire study, or for recruitment purposes only.  Note: if you are collecting PHI as part of 
a phone or email screen, you must request a HIPAA waiver for recruitment purposes.)
Choose one: 
☐ For entire study 
☒  For recruitment purposes only
☐ For inclusion of non-English speaking subject if short form is being used and a 
translated HIPAA research authorization form is not available on the University’s HIPAA 
website
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i. Describe why it would be impracticable to obtain the subject’s authorization for 
use/disclosure of this data;
ii. If requesting a waiver of signed authorization, describe why it would be 
impracticable to obtain the subject’s signed authorization for use/disclosure of this 
data; While recruiting over the phone, we ask the individual’s name, sex and phone 
number.  We also ask height and weight.  This information is not written down, but is 
used to determine if she will qualify for the study.  
By signing this protocol application, the investigator assures that the protected 
health information for which a Waiver of Authorization has been requested will not 
be reused or disclosed to any person or entity other than those listed in this 
application, except as required by law, for authorized oversight of this research 
study, or as specifically approved for use in another study by an IRB.
Researchers are reminded that unauthorized disclosures of PHI to individuals outside of the Yale 
HIPAA-Covered entity must be accounted for in the “accounting for disclosures log”, by subject 
name, purpose, date, recipients, and a description of information provided.  Logs are to be 
forwarded to the Deputy HIPAA Privacy Officer.
7. Required HIPAA Authorization: If the research involves the creation, use or disclosure of 
protected health information (PHI), separate subject authorization is required under the HIPAA 
Privacy Rule. Indicate which of the following forms are being provided:
 Compound Consent and Authorization form
 HIPAA Research Authorization Form.  
8. Consent Personnel: List the names of all members of the research team who will be 
obtaining consent/assent.   Nina Stachenfeld
9. Process of Consent/Assent: Describe the setting and conditions under which 
consent/assent will be obtained, including parental permission or surrogate permission 
and the steps taken to ensure subjects’ independent decision-making. 
The protocols will be thoroughly described during the initial phone conversation before the
orientation.  Subjects are identified only by first names during this initial conversation. When the 
subject arrives for the consent process, we review the protocol with her and indicate that she 
should ask any questions or express concerns before signing the consent form.  We stress that 
she is under no obligation to sign the consent form or participate in the study if she is 
uncomfortable with any aspects of it. We also stress that she will not be penalized if she decides 
not to participate.  In addition, we tell the subjects that they are allowed to withdraw their consent 
at any time without prejudice and are advised of this fact prior to participating.
As described we will also utilize the Joint Data Analytics Team (JDAT) to identify 
potential subjects with insulin resistance and PCOS.  Please refer to Section VII #2 for 
information interested subjects will receive.
10. Evaluation of Subject(s) Capacity to Provide Informed Consent/Assent: Indicate how the 
personnel obtaining consent will assess the potential subject’s ability and capacity to consent 
to the research being proposed. 
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During the initial phone conversation, we have a give and take conversation enabling the 
potential subject to ask questions and express concerns about participation.  If the subject 
cannot speak English well enough to understand the protocol, we do not allow her to continue 
the consent process. The same conversations take place at the time of consent at the 
laboratory.
11. Documentation of Consent/Assent: Specify the documents that will be used during the 
consent/assent process. Copies of all documents should be appended to the protocol, in the 
same format that they will be given to subjects. 
Consent Forms
12. Non-English Speaking Subjects: Explain provisions in place to ensure comprehension for 
research involving non-English speaking subjects. If enrollment of these subjects is 
anticipated, translated copies of all consent materials must be submitted for approval prior to 
use.   N/A
12(a) As a limited alternative to the above requirement, will you use the short form* for 
consenting process if you unexpectedly encounter a non-English speaking individual 
interested in study participation and the translation of the long form is not possible prior to 
intended enrollment? 
YES ☐  NO ☒
Note* If more than 2 study participants are enrolled using a short form translated into the same 
language, then the full consent form should be translated into that language for use the next time 
a subject speaking that language is to be enrolled.
Several translated short form templates are found on our website 
at:  http://www.yale.edu/hrpp/forms-templates/biomedical.html. If the translation of the short 
form is not available on our website, then the translated short form needs to be submitted to the 
IRB office for approval via amendment prior to enrolling the subject. Please review the 
guidance and presentation on use of the short form available on the HRPP website.
If using a short form without a translated HIPAA Research Authorization Form, please 
request a HIPAA waiver in the section above. 
13.
Consent Waiver: In certain circumstances, the HIC may grant a waiver of signed 
consent, or a full waiver of consent, depending on the study. If you will request either 
a waiver of consent, or a waiver of signed consent for this study, complete the 
appropriate section below.  
 
 Not Requesting a consent waiver 
 
Requesting a waiver of signed consent
       
 Requesting a full waiver of consent
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SECTION VIII: PROTECTION OF RESEARCH SUBJECTS
    Confidentiality & Security of Data:
a.    What protected health information (medical information along with the HIPAA identifiers) 
about subjects will be collected and used for the research?   
The subjects will provide private information on a medical history form to indicate any reason 
to exclude them from the study.  This information includes details on their race/ethnicity, 
medical history, allergies to medication, current medication use, hospitalization, exercise 
habits, alcohol, smoking and menstrual periods. Only the Principal Investigator (Dr. 
Stachenfeld), Dr. Pal,and Ms. Leone will have access to this information.
b. How will the research data be collected, recorded and stored? 
We collect non digital data in notebooks that do not identify the subject by name (only by 
subject initials, date and number).  These notebooks are stored in the laboratory or office 
areas.  We also store data in excel spreadsheets on PCs associated with our laboratory.  
Again, the subjects are only identified by subject initials, date and number
c.    How will the digital data be stored? 
 CD  
 DVD  
 Flash Drive  
 Portable Hard   
       Drive  
 Secured Server  
 Laptop Computer  
 Desktop Computer  
 Other
d.    What methods and procedures will be used to safeguard the confidentiality and security of    
the identifiable study data and the storage media indicated above during and after the subject’s 
participation in the study?
Confidentiality of all information in the study will be maintained by identifying subjects by 
code numbers. No individuals, other than the professionals directly involved in the study, will be 
allowed to read data forms.  Investigators will be required to respect the confidentiality code.  
No subjects are identified by name in any of the published literature and only by code in data 
storage areas, to which access is limited to study personnel.  The individual subject files are the 
only place where names are noted and all files are kept in the office of the PI.  Access to the 
PI’s office is limited to study personnel.  To date, we have never had a violation of confidentiality.  
The Yale Human Investigation Committee may inspect all study records. The files are stored in 
a locked filing cabinet within Dr. Stachenfeld’s laboratory.
All portable devices must contain encryption software, per University Policy 5100.  If 
there is a technical reason a device cannot be encrypted please submit an exception request 
to the Information Security, Policy and Compliance Office by clicking on url 
http://its.yale.edu/egrc  or email it.compliance@yale.edu 
          
e.
What will be done with the data when the research is completed? Are there plans to destroy     
the identifiable data? If yes, describe how, by whom and when identifiers will be destroyed. If 
no, describe how the data and/or identifiers will be secured.
The individual subjects’ files are stored in the John B. Pierce Laboratory, either in the office 
of the Principal Investigator or in locked storage areas in the laboratory.  Access to these 
files is limited to the Principal Investigator, Dr.Pal and Ms. Leone.
f.   Who will have access to the protected health information (such as the research sponsor, the  
investigator, the research staff, all research monitors, FDA, Yale Cancer Center Data and Safety 
Monitoring Committee (DSMC), SSC, etc.)? (please distinguish between PHI and de-identified 
data) 
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The Principal Investigator, Dr. Pal and Ms. Leone listed in this protocol will have access 
to PHI.
g.   If appropriate, has a Certificate of Confidentiality been obtained?  N/A  For NIH funded 
research, HHS  automatically issues Certificates of Confidentiality (CoC) to persons engaged in 
biomedical, behavioral, clinical or other research, in which identifiable, sensitive information is 
collected.  These Certificates protect the privacy of subjects by limiting the disclosure of 
identifiable, sensitive information.  Thus, this we do not have to apply for a CoC for this 
research.
 https://grants.nih.gov/grants/guide/notice-files/NOT-OD-17-109.html 
h.   Are any of the study procedures likely to yield information subject to mandatory reporting   
requirements? (e.g., HIV testing – reporting of communicable diseases; parent interview -incidents 
of child abuse, elderly abuse, etc.). Please verify to whom such instances will need to be reported.   
N/A (no)
SECTION IX: POTENTIAL BENEFITS
Potential Benefits: Identify any benefits that may be reasonably expected to result from the 
research, either to the subject(s) or to society at large. (Payment of subjects is not considered a 
benefit in this context of the risk benefit assessment.) 
The primary benefits to the volunteers will be the satisfaction derived from participating 
in a research program designed to assess blood pressure changes in women with AE-
PCOS.
         SECTION X: RESEARCH ALTERNATIVES AND ECONOMIC CONSIDERATIONS
1.     Alternatives: What other alternatives are available to the study subjects outside of the research?
The alternative is not to participate.
2. 
Payments for Participation (Economic Considerations): Describe any payments that will be 
made to subjects, the amount and schedule of payments, and the conditions for receiving this 
compensation. 
Aims 1 & 2. Subjects will receive up to $800.00 for participating in all aspects of the study. 
Subjects will not be charged for any of the tests. They will be paid $40.00 for the orientation 
procedure (including ultrasound), $60 for the oral glucose tolerance test, $150.00 for each 
blood pressure research study, $15.00 for each day they are taking Antagon, and a $100.00 
finishing bonus. If they do not complete all parts of the study, they will be paid for the parts 
completed. Payment will be in check form following completion of the study.
Aim 3.  Subjects will receive up to $770.00 for participating in all aspects of the study. Subjects 
will not be charged for any of the tests. They will be paid $40.00 for the orientation procedure 
(including ultrasound), $60 for the oral glucose tolerance test, $100.00 for each blood pressure 
research study, $15.00 for each day they are taking Antagon, and a $100.00 finishing bonus. If 
they do not complete all parts of the study, they will be paid for the parts completed. Payment 
will be in check form following completion of the study.
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Aim 4. Subjects will receive up to $200 for participating in all aspects of the study.  They will be 
paid $50 for each blood draw, $10 for each day taking Elagolix, and a $30 finishing bonus.  If 
they do not complete all parts of the study, they will be paid for the parts completed. Payment 
will be in check form following completion of the study.
Aim 5. Subjects will receive up to $350.00 for participating in all aspects of the study. Subjects 
will not be charged for any of the tests. They will be paid $40.00 for the orientation procedure 
(including ultrasound), $60 for the oral glucose tolerance test, $150.00 for the blood pressure 
research study, and a $100.00 finishing bonus. If they do not complete all parts of the study, 
they will be paid for the parts completed. Payment will be in check form following completion of 
the study.
3.
Costs for Participation (Economic Considerations): Clearly describe the subject’s costs 
associated with participation in the research, and the interventions or procedures of the study that 
will be provided at no cost to subjects.     
There will be no costs to subjects. If transportation is required the laboratory will reimburse up 
to $20.  According to the rules of the Internal Revenue Service (IRS), payments that are made to 
the subject as a result of their participation in a study may be considered taxable income. 
4.
In Case of Injury: This section is required for any research involving more than minimal risk, 
and for minimal risk research that presents the potential for physical harm (e.g., research 
involving blood draws).
Medical therapy will be offered for any physical injuries sustained as a consequence of the 
subject’s participation in this research. The subject or the subjects’ insurance company will 
be responsible for the cost of such therapy.
a.     Will medical treatment be available if research-related injury occurs? yes
b.     Where and from whom may treatment be obtained? Subject’s personal physicians
c.     Are there any limits to the treatment being provided? Based on insurance limitations
d.     Who will pay for this treatment? The subject or the subjects’ insurance company will be 
responsible for the cost of such therapy.
e.     How will the medical treatment be accessed by subjects? Appointment with physician
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