Vestibulodynia: Understanding Pathophysiology and Determining Appropriate Treatments
Phase: PHASE2
Conditions: Vestibulodynia, Temporomandibular Disorder, Fibromyalgia Syndrome, Irritable Bowel Syndrome, Migraines, Tension Headache, Endometriosis, Interstitial Cystitis, Back Pain, Chronic Fatigue Syndrome
Sponsor: Duke University

 
 
 
 
 
 
 
 
 
 
 
 
Protocol for Vestibulodynia: UPDATe 
 
Vestibulodynia: Understanding Pathophysiology and 
Determining Appropriate Treatments 
 
 
Compiled by: 
Vestibulodynia UPDATe Research Team 
Version 21 February 2024 
 
Distributed by: 
Andrea Nackley 
Duke University 
3 Genome Court 
Durham, NC 27710 
 
NCT03844412 
 

Version date: 21Feb2024 
 
 
 
1
Study Sponsor: 
 
 
 
 
National Institute of Child Health & Human Development 
 
 
 
 
 
 
 
 
National Institutes of Health 
 
Project Officer: 
 
 
 
 
Jennie Conroy, MD 
 
 
 
 
 
 
 
 
Principal Investigator:  
 
 
Andrea Nackley, PhD (Duke University) 
 
Co-Investigators:  
 
 
 
Andrea Rapkin, MD (UCLA) 
Erin Carey, MD (UNC) 
 
 
 
 
 
 
 
 
Elizabeth Geller, MD (UNC) 
 
Clinical Site Team: 
 
 
 
Haley Cutting, Physician Assistant (UCLA) 
Jasmyn Akaninwor, Study Coordinator (UCLA) 
 
 
 
 
 
 
 
 
Debbie Farb, Research Nurse, UNC 
 
Protocol Development Team: 
 
Andrea Nackley, PhD (Duke University) 
Andrea Rapkin, MD (UCLA) 
Erin Carey, MD (UNC) 
 
 
 
 
 
 
 
 
Elizabeth Geller, MD (UNC) 
 
 
 
 
 
 
 
 
Christopher Stirling, MA (Duke) 
 
 
 
 
 
 
 
 
Andrey Bortsov, MD, PhD (Duke) 
 
 
 
 
 
 
 
 
Sandra O’Buckley, MS (Duke) 
 
 
 
 
 
 
 
 
Nicole Willis, BS (Duke) 
 
 
 
 
 
 
 
  
Coordinating Center:  
 
 
Clinical Coordinating Center 
Andrea Nackley, PhD 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Data Coordinating Center 
Christopher Stirling, MA 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Statistical Coordinating Center 
Sheng Luo, PhD  
Pearl Zakroysky, MPH 
Peter Merrill, PhD 
 
 
 
 
 
 
 
 
Duke Clinical Research Institute 
Durham, NC 
 
Central Compounding Center:   
Jennifer Burch 
 
 
 
 
 
 
 
 
6224 Fayetteville Road 
Durham, NC 27713 
 
Pacifica Compounding Pharmacy: 
Jeffrey Barris  
 
 
 
 
 
 
 
 
23560 S. Madison 
 
 
 
 
 
 
 
 
Suite 112  
 
 
 
 
 
 
 
 
Torrance, CA 90505 
 
Substrate Services Core:  
 
Mary-Beth Joshi, MPH 
 
 
 
 
 
 
 
 
Duke University 
 
 
 
 
 
 
 
 
Durham, NC 
 
Sequencing Core: 
 
 
 
Zhao Lai, PhD 
Greehey Children’s Cancer Research Institute 
University of Texas 
San Antonio, TX 
 
 
 
 

Version date: 21Feb2024 
 
 
 
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1.1 List of Abbreviations 
Abbreviation
Definition
AE
Adverse Event
BPSFS-F 
Brief Profile Sexual Function and Satisfaction-Female 
CC
Coordinating Center
CNS 
Central Nervous System 
COPC
Chronic Overlapping Pain Conditions
DCRI
Duke Clinical Research Institute
DSMB 
Data Safety Monitoring Board 
eCRF
electronic Case Report Form
EDC 
Electronic Data Capture 
eICF
Electronic Informed Consent Form
FMS
Fibromyalgia Syndrome
HC 
Healthy Controls 
IBS
Irritable Bowel Syndrome
IDS 
Investigational Drug Section 
IRB
Institutional Review Board
ITT
Intent-to-Treat
MAOI 
Monoamine Oxidase Inhibitor 
miRNA
microRNA
NRS 
Numeric Rating Score 
PILL
Pennebaker Index of Limbic Languidness
PPT 
Pressure Pain Threshold 
PROMIS
Patient-Reported Outcomes Measurement Information System
PSS
Perceived Stress Scale
QST 
Quantitative Sensory Testing 
SAE
Serious Adverse Event
SAP 
Statistical Analysis Plan 
SAR
Suspected Adverse Reaction
SCL-27
Symptom Checklist-27
SF12v2 
Short Form 12 version 2 
SF-MPQ
Short Form- McGill Pain Questionnaire
sIRB 
single Institutional Review Board 
SNRI
Selective Norepinephrine Reuptake Inhibitor
SSRI
Selective Serotonin Reuptake Inhibitor
TCA 
Tricyclic Antidepressant 
TMD
Temporomandibular Disorder
UCLA 
University of California, Los Angeles 
UNC
University of North Carolina at Chapel Hill
UP
Unanticipated Problem
VBD 
Vestibulodynia 
VBD-c
Vestibulodynia central
VBD-p 
Vestibulodynia peripheral  
 
 
 
 
 
 

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TABLE OF CONTENTS 
1.1 
List of Abbreviations ..................................................................................................................... 2 
2 
EXECUTIVE SUMMARY ................................................................................................................6 
3 
INTRODUCTION ..........................................................................................................................8 
3.1 
Overall Scientific Premise ............................................................................................................. 8 
3.2 
Scientific Premise for Aim 1 .......................................................................................................... 9 
3.3 
Scientific Premise for Aim 2 ........................................................................................................ 11 
3.4 
Scientific Premise for Aim 3 ........................................................................................................ 11 
4 
AIMS and HYPOTHESeS ............................................................................................................. 13 
5 
BASIC STUDY DESIGN ................................................................................................................ 13 
5.1 
Feasibility Study .......................................................................................................................... 13 
5.2 
Screening ..................................................................................................................................... 14 
5.3 
Baseline Evaluation Phase and Randomization ................................................................... 14 
5.4 
Follow-up Phase ................................................................................................................ 14 
6 
Study Flow Diagram.................................................................................................................. 15 
7 
STUDY POPULATION AND ELIGIBILITY CRITERIA ......................................................................... 16 
7.1 
Study Population ......................................................................................................................... 16 
7.2 
Inclusion Criteria ............................................................................................................... 16 
7.3 
Exclusion Criteria ......................................................................................................................... 16 
8 
TREATMENT INTERVENTIONS ................................................................................................... 17 
8.1 
Interventions ............................................................................................................................... 17 
8.2 
Potential Side Effects and Risk Reduction Plan ........................................................................... 17 
8.3 
Drug Dispensing .......................................................................................................................... 18 
8.4 
Drug Storage ............................................................................................................................... 18 
8.5 
Drug Accountability ..................................................................................................................... 18 
8.6 
Drug Destruction ......................................................................................................................... 18 
8.7 
Randomization and Blinding ....................................................................................................... 19 
8.8 
Unblinding ................................................................................................................................... 19 
8.9 
Concomitant Medication ............................................................................................................ 19 
9 
RECRUITMENT, ENROLLMENT AND SCREENING PROCEDURES ................................................... 19 
9.1 
Common Recruitment Procedures ..................................................................................... 19 

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9.2 
Estimated Enrollment Period ...................................................................................................... 20 
9.3 
Screening ..................................................................................................................................... 20 
10 BASELINE EVALUATION AND DRUG dispensing (0-Week) ........................................................... 20 
10.1 
Baseline Evaluation ..................................................................................................................... 21 
10.2 
Drug Dispensing .......................................................................................................................... 21 
11 FOLLOW-UP EVALUATIONS ....................................................................................................... 21 
11.1 
Visit 2 (8-Week) ........................................................................................................................... 21 
11.2 
Visit 3 (16-Week) ......................................................................................................................... 22 
11.3 
Visit 4 (24-Week) ......................................................................................................................... 22 
12 OUTCOME DETERMINATIONS ................................................................................................... 22 
13 METHODS TO PROMOTE ADHERENCE ....................................................................................... 23 
14 PARTICIPANT SAFETY AND ADVERSE EVENTS ............................................................................ 24 
14.1 
Single IRB ..................................................................................................................................... 24 
14.2 
Definitions ................................................................................................................................... 24 
14.2.1 Adverse Events ......................................................................................................................... 24 
14.2.2 Serious Adverse Events ............................................................................................................ 25 
14.2.3 Assessment of Causal Relationship .......................................................................................... 25 
14.2.4 Recording and Reporting of Adverse Events ........................................................................... 25 
14.3 
Data Safety Monitoring Board .................................................................................................... 26 
15 STATISTICAL CONSIDERATIONS ................................................................................................. 26 
15.1 
VBD Subtype Designation ........................................................................................................... 26 
15.2 
Analysis of the Aim 1 Endpoints.................................................................................................. 27 
15.3 
Analysis of the Aim 2 Endpoints.................................................................................................. 28 
15.4 
Analysis of the Aim 3 Endpoints.................................................................................................. 29 
15.5 
Analysis of Safety Data and Statistical Monitoring Plan ............................................................. 31 
15.6 
Sample Size and Power ............................................................................................................... 31 
16 DATA MANAGEMENT PROCEDURES .......................................................................................... 31 
16.1 
Overview of Data Management .................................................................................................. 31 
16.2 
Data Security ............................................................................................................................... 32 
16.3 
Publication Policy ........................................................................................................................ 32 
17 STUDY ADMINISTRATION ......................................................................................................... 33 
17.1 
Data and Safety Monitoring Board ............................................................................................. 33 
17.2 
Coordinating Center .................................................................................................................... 33 
17.3 
Core Laboratories ........................................................................................................................ 33 
17.3.1 Central Compounding Pharmacy and Pacifica Compounding Pharmacy ................................ 33 

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17.3.2 Substrate Services Core ........................................................................................................... 33 
17.3.3 Sequencing Core ...................................................................................................................... 33 
18 ETHICAL AND REGULATORY CONSIDERATIONS .......................................................................... 34 
18.1 
Ethics and Good Clinical Practice ................................................................................................ 34 
18.2 
Institutional Review Board .......................................................................................................... 34 
18.3 
Informed Consent Procedures .................................................................................................... 34 
18.3.1 Informed Consent .................................................................................................................... 34 
18.3.2 Confidentiality and HIPAA Requirements ................................................................................ 34 
18.3.3 Protections of Human Subjects ................................................................................................ 35 
18.3.4 Summary of the Risks and Benefits.......................................................................................... 35 
19 REFERENCES ............................................................................................................................. 36 
20 APPENDICES ............................................................................................................................. 42 
Appendix A. Schedule of Assessments .......................................................................................... 42 
Appendix B. Lidocaine/Estradiol Application Handout ........................................................................... 43 
Appendix C. Nortriptyline Medication Handout ..................................................................................... 44 
Appendix D. Medication Calendar .......................................................................................................... 45 
 
 
 

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2 EXECUTIVE SUMMARY 
 
Title 
Vestibulodynia UPDATe: Vestibulodynia: Understanding Pathophysiology 
and Determining Appropriate Treatments 
 
Indication 
Three continuous months of insertional entryway dyspareunia and/or pain to 
touch/tampon insertion indicated by self-report as well as pain score of "3" 
or greater on the tampon test during screening. 
 
Locations 
Duke University, UNC, UCLA 
Brief Rationale 
 
 
 
 
Vestibulodynia (VBD) is a chronic pelvic pain condition that affects 1 in 6 
reproductive aged women, yet remains ineffectively treated by standard trial-
and-error approaches. Our group has identified two distinct VBD subtypes 
that may benefit from different types of treatment: VBD peripheral (VBD-p) 
subtype characterized by localized pain specific to the vulvar vestibule, and 
VBD central (VBD-c) subtype characterized by pain at both vaginal and 
remote body regions. Preliminary data further demonstrate that VBD-p and 
VBD-c subtypes differ with respect to patient reported outcomes (e.g., 
physical and mental health), production of cytokines (intracellular proteins 
that regulate the activity of pain nerves and inflammatory processes), and 
expression of microRNAs (small non-coding RNA molecules that regulate 
gene expression). Women with VBD-p exhibit normal psychological profiles; 
balanced circulating pro- and anti-inflammatory cytokines; and dysregulation 
in microRNAs that regulate the expression of genes in estrogen pathways. 
In contrast, women with VBD-c report decreased functional status and 
increased somatization; increased pro- but not anti-inflammatory cytokines; 
and dysregulation in microRNAs that regulate the expression of genes 
relevant to muscle, nerve, and immune cell function. 
Study Design 
 
 
Treatment 
 
 
 
 
 
Aims & Endpoints 
 
Two-center, randomized, double-blind, placebo-controlled 2x2 factorial study 
enrolling 400 women to participate for 24-week duration. 
 
Participants will receive one of four treatments for 16 weeks:  
 5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + placebo 
pill  
 placebo vaginal cream + nortriptyline pill  
 5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + 
nortriptyline pill 
 placebo vaginal cream + placebo pill 
 
Among women with VBD-p and VBD-c, determine the efficacy of peripheral, 
central, and combined treatments in:  
Aim 1. Alleviating pain  
Primary Endpoints:  
Ia. pain score during tampon test at 16 weeks  
Ib. self-reported pain via SF-MPQ at 16 weeks 
Secondary Endpoints:  
IIa. experimental pain via local and remote PPTs at 16 weeks 
IIb. COPC pain levels via COPC follow-up survey at 16 weeks 
IIc. Pain scores and PPTs at 8 weeks and 24 weeks 
 
Aim 2. Improving patient reported outcomes  
Primary Endpoints:  
Ia. self-reported physical/mental health via SF12v2 at 16 weeks 
Ib. sexual health via PROMIS at 16 weeks 
Secondary Endpoints:  
IIa. mood via SCL-27 at 16 weeks 
IIb. somatic awareness via PILL at 16 weeks

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IIc. perceived stress via PSS at 16 weeks 
IId. sleep via sleep scale at 16 weeks 
IIe. Patient reported outcomes at 8 weeks and 24 weeks 
 
Aim 3. Normalizing cytokine and microRNA expression  
Primary Endpoints:  
Ia. cytokine levels via mesoscale discovery assays at 16 weeks 
Ib. microRNA levels via sequencing read at 16 weeks 
Secondary Endpoints:  
IIa. cytokine and microRNA levels at 8 weeks and 24 weeks 
IIb. Aim 3 will also identify cytokine and microRNA biomarkers at baseline 
that predict treatment response at 8, 16, and 24 weeks  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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3 INTRODUCTION 
3.1 Overall Scientific Premise 
Vestibulodynia (VBD) is the most common cause of sexual pain, affecting 16% of reproductive 
aged women in the United States.1 The pain is chronic, lasting over 3 months, and compromises 
psychological functioning, interpersonal relations, and daily activity.2 As further evidence of its 
public health significance, VBD and related vulvar pain conditions cost the US economy over $70 
billion annually.3 Despite its high prevalence and significant economic burden, VBD remains 
ineffectively treated due to its unclear etiology and heterogeneous clinical presentation. Some 
women present with localized provoked pain restricted to the vaginal vestibule, while others 
present with vestibular pain and generalized pain at other genital sites. Still others present with 
VBD and chronic overlapping pain conditions (COPCs) such as irritable bowel syndrome (IBS; 
35%),4 temporomandibular disorder (TMD; 78%),5 and fibromyalgia syndrome (FMS; 17%).4 In 
the absence of data to guide treatment approaches for VBD patients with diverse symptomology, 
many different therapies are used on a trial-and-error basis which can delay effective care.6 Thus, 
it is urgent that we unravel the clinical and pathophysiological complexities of VBD in order to 
effectively resolve the pain and its devastating consequences. 
 
Our group has identified two distinct VBD subtypes that may benefit from different types of 
treatment: 1) VBD peripheral (VBD-p) subtype is characterized by localized pain specific to the 
vaginal vestibule, and 2) VBD central (VBD-c) subtype is characterized by pain at vestibular and 
remote body regions.5, 7 Women with VBD-p exhibit normal psychological profiles; balance in 
circulating pro- and anti-inflammatory cytokines; and dysregulation in microRNAs that regulate 
the expression of genes in estrogen pathways. In contrast, women with VBD-c report decreased 
functional status and increased somatization; lack compensatory increases in anti-inflammatory 
cytokines; and have dysregulation in microRNAs that regulate the expression of genes relevant 
to muscle, nerve, and immune cell function. These data suggest that VBD-p and VBD-c have 
unique etiologies (localized pain with peripheral neurosensory disruption versus widespread pain 
with a central sensory contribution) and may, therefore, respond differently to peripheral and 
centrally-targeted treatments. 
 
Local anesthetics and estrogen are among the most commonly prescribed peripheral 
treatments6,8,9 and tricyclic antidepressants are the most commonly prescribed centrally-targeted 
treatment.8 However, as concluded in a recent Cochrane systematic review, interpretation of 
treatment success remains difficult due to the limitations of published studies, including poor 
patient selection, lack of adequate controls, and limited follow-up data.10 Thus, there is a critical 
need for randomized controlled trials to evaluate the efficacy of these peripheral and centrally-
targeted treatments, especially among women with different VBD subtypes. In response to RFA-
HD- 18-480, we propose new multidisciplinary studies that will identify effective strategies for the 
management of two distinct VBD subtypes, while advancing our understanding of their different 
pathophysiologic mechanisms. We will compare the efficacy of peripheral, central, and combined 
treatments in alleviating objectively-measured pain and improving patient reported outcomes for 
women with VBD. In addition, we will evaluate cytokine and microRNA biomarkers that help 
determine underlying pathophysiology and predict treatment response. We will apply a 
randomized, double-blinded, placebo-controlled factorial study design to evaluate the analgesic 
efficacy of: 1) peripheral treatment with a 5% lidocaine/0.5 mg/ml 0.07% estradiol compound 
cream, 2) centrally-targeted treatment with the tricyclic nortriptyline 10 mg strength, or 3) 
combined peripheral and central treatment in women with newly diagnosed VBD. Women will be 
recruited from two study sites and complete a 4-month treatment phase, with outcome measures 
and biomarkers assessed at four time points (0, 2, 4, and 6 months). We hypothesize that women 
with VBD-p will exhibit increased levels of inflammatory mediators in the vaginal vestibule and 
preferentially respond to peripheral lidocaine/estradiol treatment, while women with VBD-c will 
exhibit increased levels of inflammatory mediators in blood and preferentially respond to centrally-
targeted or combined treatment. 

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Our findings will 1) advance our knowledge of the pathophysiologic mechanisms 
underlying VBD-p and VBD-c, 2) determine the efficacy of peripheral, central, and 
combined therapies in alleviating pain in women with VBD-p and VBD-c, and 3) identify 
biomarkers that predict treatment response. 
3.2 Scientific Premise for Aim 1 
Compare the efficacy of peripheral, central, and combined treatments in 
alleviating pain among women with VBD-p and VBD-c 
1) Women with VBD-p and VBD-c exhibit unique pain profiles. In general, VBD is associated with 
heightened pain responses that may result from dysregulations in processing that occur in the 
peripheral, central nervous system (CNS), or both.11 Women with VBD exhibit increased 
l e v e l s  o f  pro-inflammatory cytokines that bind to receptors on pain-sensing nociceptors to 
increase their activity and on immune cells to increase the synthesis of additional inflammatory 
mediators.12-16 This self-perpetuating process of peripheral sensitization results in reduced pain 
thresholds in the affected tissues. Sustained peripheral inflammation can increase central 
responses to peripheral stimuli.12, 13, 17, 18 This central sensitization causes women to exhibit 
reduced pressure pain thresholds at regions remote from the original painful site.17-19 To 
distinguish VBD subtypes that may be peripherally- versus centrally-mediated, we enrolled 
women with localized VBD (VBD-p; N=33), VBD+COPCs (VBD-c; N=28), and healthy controls 
(HC; N=22).7 As shown in Figure 1, we found that gentle pressure applied to the vaginal vestibule 
elicits increased pain ratings in both VBD subtypes, with the highest pain ratings in women with 
VBD-c. Using a modified Wegner Digital Algometer to measure vestibular pain in a standardized 
manner, we found that 
women with VBD-c 
exhibited the lowest 
vulvovaginal 
pain 
thresholds compared 
to those with VBD-p 
and 
HC. 
Similarly, 
women with VBD-c 
exhibited 
reduced 
pain 
thresholds 
at 
remote body sites, 
including 
the 
trapezius muscles. 
 
 2) Women with VBD-p and VBD-c exhibit differences in self-reported pain characteristics. In the 
same cohort, we found that women with VBD-c, but not VBD-p, had greater levels of affective, 
aching, tender and stabbing pain versus HC (Table 1).7 More women with VBD-c reported a 
history of unprovoked genital pain, a hallmark of central sensitization.20-23 We also found that more 
women with VBD-p had early onset (primary) VBD with first vaginal penetration compared to VBD-
c, in contrast 
to 
some 
studies 
observing 
a 
positive 
correlation  
between VBD 
onset 
and 
pain 
severity.24-26 
 
Table 1. Self-reported Pain Among Women with VBD-p and VBD-c 
Phenotype
Questionnaire HC
VBD-p
VBD-c
Overall P
Affective Pain 
MPQ 
11.67 (0.41) 12.50 (0.62) 14.78 (0.8)*** 0.001 
Aching Pain 
MPQ 
1.14 (0.10) 1.23 (0.10) 
2.06 (0.17)### <0.0001 
Tender Pain 
MPQ 
1.10 (0.06) 1.37 (0.13) 
1.61 (0.16)** 0.003 
Stabbing Pain 
MPQ 
1.00 (0.00) 1.13 (0.10) 
1.39 (0.20)* 
0.038 
Unprovoked Genital Pain GYN 
0% 
20% 
30% 
 
Pain with 1st Intercourse GYN 
N/A 
67.5% 
58.6% 
 
Pain with 1st Tampon 
GYN 
N/A 
51.3% 
27.9% 
 
McGill Pain Questionnaire (MPQ), Gynecological Health Survey (GYN). MPQ data are Mean (SEM).
*p<0.05, **p<0.01, ***p<0.001 vs HC. ###p<0.001 vs HC and VBD-p. GYN data are % women/ group.
Figure 1. VBD-p and VBD-c exhibit different local and remote pain profiles. Data 
are Mean + SEM. *p<0.05, ***p<0.001 vs HC. #p<0.05 vs HC and VBD-p.   

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Thus, qualitative and quantitative assessments of pain in the vaginal vestibule and remote body 
regions together with self-reported measures of perceived pain traits can distinguish women with 
VBD-p and VBD-c, and may be used to differentiate underlying mechanisms and indicate different 
pain management strategies.   
3) VBD-p may be more effectively managed by peripheral therapy, while VBD-c may be more 
effectively managed by centrally-targeted or combined therapies. Treatment options for VBD 
include 1) non-medical interventions such as pelvic floor physical therapy and cognitive 
behavioral therapy, 2) topical medications (e.g., analgesic and hormone creams) applied to the 
vaginal vestibule, 3) centrally-targeted medications (e.g., tricyclics) taken orally, and 4) surgery.6, 
10, 27 Surgical procedures such as vestibulectomy are high-risk and used as a last resort. Physical 
and psychological therapies are low-risk with published evidence, however are almost always 
used in combination with peripheral and/or centrally-targeted medications that have mixed 
results for successful pain management. A recent Cochrane review emphasized the need for 
randomized controlled trials to verify the efficacy of these commonly-prescribed medications, 
especially amongst different VBD subtypes.10
Local anesthetics (e.g., lidocaine) and topical estrogen are among the most commonly 
prescribed peripheral treatments.6, 8, 9 Topical 5% lidocaine for 7 weeks has been shown to 
decrease dyspareunia in women with VBD,28 likely due to its ability to block the activity of sodium 
channels on peripheral nociceptors and prevent the transmission of pain to the CNS.29, 30 Topical 
estrogen for 3 months has been shown to reduce peripheral pain and inflammation31 as well as 
improve sexual function for premenopausal women with vulvar pain.32 Estrogen plays a critical 
role in trophic support and neuroplasticity of vaginal tissues, with deficient estrogen levels 
resulting in atrophy and increased innervation of pain-sensing nerve fibers.33, 34 Lidocaine and 
estrogen are often compounded into a combined cream that can produce a synergistic analgesic 
effect.9 Thus, combined topical lidocaine and estrogen may effectively alleviate pain in women 
with VBD-p. 
Tricyclic antidepressants (e.g., nortriptyline) are the most commonly prescribed centrally-
targeted treatment for the management of vulvar pain,8 despite limited evidence. An open trial of 
nortriptyline for 2 months found that 6/7 women had a complete or partial reduction in chronic 
pelvic pain.35 In a case study, nortriptyline completely alleviated vaginal pain in a woman for 
whom topical lidocaine, estrogen, and other peripheral treatments failed.36 While the anti-
epileptic gabapentin has also been commonly prescribed for vaginal pain, recent results from a 
randomized controlled trial found that gabapentin was ineffective in reducing pain among women 
with VBD.37 Nortriptyline has been well studied for the treatment of other neuropathic pain 
disorders and evidence-based guidelines support its use as a first-line medication.38 Nortriptyline 
produces analgesia via multiple molecular mechanisms in the CNS, including 1) inhibiting 
reuptake of norepinephrine to promote descending inhibition,39 2) blocking sodium channels to 
inhibit the activity of nociceptive neurons,40 and 3) inhibiting the release of pro-inflammatory 
cytokines from glia so as to reduce neuroinflammation.41 Together, these findings suggest that 
nortriptyline, alone or together with peripheral 
treatments, may effectively alleviate pain in 
women with VBD-c. 
To determine effectiveness of peripheral 
versus centrally-targeted treatments in women 
with VBD-p and VBD-c, we measured self-
reported pain (on a scale of 0-10) in response 
to a Q-tip applied to the 6 o’clock site of the 
vaginal vestibule prior to treatment and again 
at the follow-up visit 3-6 months following 
treatment. All 47 women with VBD (28 with 
VBD-p and 19 with VBD-c) were first treated 
with topical lidocaine and/or estrogen cream. Women who did not respond to topical treatments, 
Figure 2. VBD-p vs VBD-c respond differently to treatment   

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were sequentially treated with TCAs or selective serotonin reuptake inhibitors (SSRIs). We found 
that topical lidocaine and/or estrogen cream alleviated pain in 65% of women with VBD-p, but 
only 36% of women with VBD-c (Figure 2). Furthermore, centrally-acting TCAs or SSRIs 
alleviated pain in 67% of women with VBD-c that failed to respond to peripheral treatments. These 
preliminary data demonstrate that women with VBD-p preferentially respond to peripheral 
lidocaine/estradiol, while those with VBD-c preferentially respond to centrally-targeted treatments 
such as nortriptyline.     
3.3 Scientific Premise for Aim 2 
Compare the efficacy of peripheral, central, and combined treatments in 
improving patient reported outcomes in women with VBD-p and VBD-c.  
 
1) Women with VBD-p and VBD-c exhibit differences in self-reported physical, mental, and sexual 
health. VBD has severe consequences on all aspects of health. Women with VBD report lower 
sexual desire, arousal, and frequency of intercourse than pain-free controls.42, 43 They also report  
more difficulty reaching orgasm, more negative attitudes toward sexuality, and more sexual 
distress. VBD is also associated with increased prevalence of stress and mood disorders.27, 44 In 
our VBD cohort, we found that women with VBD-c perceived lower general and physical health 
status relative to HC, while women with VBD-p perceived lower mental health status relative to 
HC (Table 2).7 Additionally, women with VBD-c reported more headaches, more somatization, 
and greater impact of pain on daily activity. Consistent with these findings, Zolnoun et al. found 
that women with VBD and TMD reported more somatization, anxiety, psychological distress, and 
intercourse-related pain.5 While it is uncertain whether all of these perceived health traits precede 
or follow the development of VBD,45, 46 their characterization may inform treatment approaches 
and outcomes. 
 
 
3.4 Scientific Premise for Aim 3 
Determine cytokine and microRNA biomarkers that predict treatment 
response among women with VBD-p and VBD-c.  
 
1) Women with VBD-p and VBD-c exhibit unique cytokine expression profiles. A large body of 
work suggests a role for cytokines, in the pathophysiology of VBD. Cytokines are secreted by 
immune and other cells in the periphery and by neurons and glia in the CNS.47 In an acute setting, 
cytokines promote wound healing,48 but sustained elevations promote tissue damage and pain.47 
Pro-inflammatory cytokines activate and sensitize nociceptors via direct receptor-mediated 
actions or via recruitment of additional mediators.49, 50 In this manner, cytokines play an essential 
role in peripheral and central sensitization (as described above). Elevated pro-inflammatory 
cytokine levels are also associated with pain intensity,51-55 perceived stress,56 and mood 
disorders (e.g., depression)57, 58  that  often  accompany chronic pain. 
 
Table 2. Self-reported Physical and Mental Health Among Women with VBD-p and VBD-c
Phenotype
Questionnaire
HC
VBD-p
VBD-c
Overall P
General Health 
SF12v2 
4.51 (0.11) 4.31 (0.15) 
4.01 (0.15)* 0.012 
Physical Health 
SF12v2 
55.8 (1.12) 57.6 (0.85) 
50.5 (1.26)## 0.003 
Mental Health 
SF12v2 
51.8 (1.87) 45.0 (1.96)* 46.4 (2.20) 
0.069 
Somatization 
PILL 
89.8 (4.15) 98.4 (3.60) 
115.6 (4.61)##0.0002 
Headache Types 
CPSQ 
1.37 (0.19) 1.93 (0.19) 
2.37 (0.23)** 0.009 
Impact of Pain on Daily Activity 
CPSQ 
0.30 (0.20) 0.81 (0.37) 
2.05 (0.64)* 0.028 
Short Form 12 version 2 (SF12v2), Pennebaker Index of Limbic Languidness (PILL), Comprehensive Pain and 
Symptom Questionnaire (CPSQ). Data=Mean(SEM). *p<0.05, **p<0.01 vs HC. ##p<0.01 vs HC and VBD-p.

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12
Women with VBD display signs of mucosal inflammation11 accompanied by enhanced production 
of the pro-inflammatory cytokines interleukin 1β (IL-1β),59-61 interleukin 8 (IL-8),60, 62 and interleukin 
17 (IL-17)63 in the vaginal vestibule and/or in circulating blood. A study by Gerber et al. further 
demonstrated that increased levels of IL-1β were accompanied by reduced levels of interleukin 1 
receptor antagonist (IL-1ra),61 an anti-inflammatory cytokine that negatively regulates the 
expression of IL-1β and IL-8.64-66 To determine inflammatory profiles in VBD-p and VBD-c 
subtypes, we used a multiplex assay to measure circulating cytokines in blood.7 Compared to HC, 
women with VBD-p had increased levels of both IL-8 and IL-1ra (Figure 3). In contrast, women 
with VBD-c had increased levels of IL-8 with no increase in IL-1ra, indicating a pro-/anti-
inflammatory imbalance. There have been no studies of these changes in vaginal tissues. We 
performed a pilot study to demonstrate our ability to measure cytokine protein in vaginal lavage 
samples. 
Results 
show 
that 
IL-8 
is 
detectable 
by 
multiplex 
in 
7 
HC 
vaginal 
lavage 
samples (Figure 3). 
These 
results 
establish the utility of 
cytokines 
as 
diagnostic biomarkers 
for functional VBD-p 
and VBD-c groups. 
 
2) Women with VBD-p and VBD-c exhibit unique microRNA expression profiles. MicroRNAs 
(miRNAs) are small non-coding RNA molecules that regulate expression of > 1/3 of all human 
genes by degrading messenger RNA (mRNA) transcripts or by inhibiting mRNA translation.67 
miRNAs regulate molecular pathways linked to pain, inflammation and immune response.68-76 
Differential expression of circulating miRNAs has been reported in chronic pain conditions.72 A 
recent preclinical study on neuropathic pain 
reported a positive correlation between pain 
thresholds and miRNA levels in plasma and 
cerebrospinal fluid.69 A clinical study of regional 
pain 
syndrome 
identified 
18 
miRNAs 
differentially expressed in blood.72 Thus, miRNA 
expression profiles may also be informative about 
the pathophysiology VBD. We conducted a study to 
measure miRNAs in women with VBD; profiling 
>750 miRNAs in blood from our VBD cohort using 
OpenArray cards.7 Compared to HC, women with 
VBD-p exhibited a downregulation of 4 and 
upregulation of 2 miRNAs (Table 3). Three of 
these miRNAs (miR-449b, miR-133b, and miR-
200b) are dysregulated in endometriosis and 
bladder pain syndrome.77, 78 Women with VBD-c 
exhibited a downregulation of 3 and upregulation 
of 4 miRNAs. These miRNAs have been 
associated with cancer,79-84 infectious disorders,85 inflammatory disorders,86 and orofacial pain.87 
Four miRNAs were dysregulated in both subtypes. Dysregulation of miR-520f and miR-520D may 
explain the increased IL-8 levels in women with both VBD-p and VBD-c, as the miR-520 family 
regulates IL-8.88  
 
To identify genes and pathways affected by miRNA dysregulation in our VBD cohort, we 
performed in silico pathway analysis using DIANA-miRPath v2.0.89 In VBD-p, despite their pre-
menopausal status, the estrogen and gonadotropin-releasing hormone (GnRH) pathways were 
blunted. This may explain why estrogen and GNRH therapy reduce pain in women with vaginal 
Table 3. miRNAs dysregulated in VBD-p and VBD-c
VBD-p
miRNA
Fold Change
P-value
miR-449b
-330.14
0.017
miR-34b
-239.45
0.003
miR-645
-117.87
0.038
miR-503
-92.20
0.037
miR-200b
94.52
0.048
miR-133b
1731.74
0.014
VBD-c 
miRNA
Fold Change
P-value
miR-1825
-19012.5
0.019
miR-1288
-745.74
0.045
miR-593
-56.76
0.019
let-7f-2#
5.77
0.034
miR-512-3p
23.03
0.046
miR-125a-3p
69.59
0.041
miR-661
981.98
0.038
Both
miRNA
Fold Change
P-value
miR-485-5p
-90.76 / -84.32
0.003 / 0.010
miR-1294
-41.47 / -60.67
0.012 / 0.014
miR-520f
-33.67 / -15.24
0.003 / 0.035
miR-520D-3p
2486.77 / 204617
0.021 / 0.002
Figure 3. VBD-p and VBD-c exhibit different cytokine 
profiles. Data are Mean + SEM. *p<0.05, **p<0.01 vs HC.   
Figure 3. IL-8 is detectable in VL 
samples. Data are Mean + SEM.  
IL-8
Control   VL Samples
0
100
200
300
400

Version date: 21Feb2024 
 
 
 
13
atrophy or endometriosis.31, 90 In VBD-c, several targets in the extracellular matrix and insulin 
resistance pathways were affected. The miRNA targets fibronectin and integrin promote 
neuropathic pain via upregulation of purinergic receptors on glia.91 Further, deficiencies in the 
target dystroglycan cause abnormal myelination and function of sensory nerves associated with 
mechanical allodynia.92 In conclusion, circulating miRNA biomarkers indicate distinct 
pathophysiologic mechanisms that contribute to VBD-p and VBD-c. Assessment of miRNAs in 
vestibular tissues and circulating blood prior to, during, and following medical treatment may help 
to us better understand the factors that drive and resolve VBD. 
4 AIMS AND HYPOTHESES 
1. Determine the efficacy of peripheral, central, and combined treatments in alleviating pain 
among women with VBD-p and VBD-c. We hypothesize that women with VBD-p will 
preferentially respond to peripheral treatment, while those with VBD-c will preferentially respond 
to central or combined treatment. To test this hypothesis, we will measure pain in response to 
standardized tampon insertion with a numeric rating scale and self-reported pain on the McGill 
questionnaire at the first baseline clinic visit (0) and at 8, 16, and 24 weeks. 
 
2. Determine the efficacy of peripheral, central, and combined treatments in improving 
patient reported outcomes among women with VBD-p and VBD-c. We hypothesize that 
women with VBD-c will exhibit more abnormalities in perceived physical, mental, and sexual 
health compared to those with VBD-p. We also hypothesize that women with VBD-c will 
demonstrate greater improvement in perceived health with central or combined therapy; while 
women with VBD-p will demonstrate greater improvement with peripheral treatment. To test these 
hypotheses, we will measure self-reported physical /mental health with the SF12 and sexual 
health with the PROMIS questionnaire administered at 0, 8, 16, and 24 weeks. 
 
3. Determine the efficacy of peripheral, central, and combined treatments in normalizing 
cytokine and microRNA expression profiles in vaginal and blood tissues collected from 
women with VBD-p and VBD-c. We hypothesize that higher levels of inflammatory mediators in 
peripheral vaginal samples will be associated with VBD-p, while higher levels in circulating blood 
will be observed in women with VBD-c. We also hypothesize that peripheral treatment will resolve 
abnormalities in vaginal biomarkers, while central treatment will resolve abnormalities in blood 
biomarkers. Finally, we hypothesize that abnormalities in vaginal cytokine and miRNA biomarkers 
will predict a positive response to peripheral treatment, while abnormalities in blood biomarkers 
will predict a positive response to central or combined treatment. To test these hypotheses, we 
will use multiplex assays to measure cytokines and small RNA sequencing to measure miRNAs 
in samples collected at 0, 8, 16, and 24 weeks. 
5 BASIC STUDY DESIGN 
Vestibulodynia UPDATe is a multi-center, randomized, double-blind, placebo-controlled trial 
designed to determine the efficacy of peripheral lidocaine/estradiol cream, centrally-targeted 
nortriptyline pills, and combined treatments compared to matching placebo with the primary 
endpoints of change in pain, self-reported health, and cytokine/microRNA measures at 16 weeks 
and secondary endpoints of change in these measures at 8 weeks and 24 weeks.  
5.1 Feasibility Study 
An initial pilot study will be conducted for the baseline (0 week) visit to optimize and standardize 
performance of the clinical exam, measurement of PPTs, and collection and processing of blood 
and vaginal samples. We anticipate consenting 5 women at each study site for this feasibility 
study. These volunteers will be recruited via an email blast to the UNC and UCLA students and 
employees, and do not have to meet inclusion/exclusion criteria. Screening of eligible study 

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participants will not start until a review of the baseline data has been performed and there is a 
determination that the sites are performing the testing accurately.    
5.2 Screening 
Screening will be conducted in women aged 18-50 with symptoms of VBD. Informed consent will 
be obtained prior to the tampon test. Participants with a pain score of > “3” on a 0–10 numeric 
rating scale on the tampon insertion test will be eligible for enrollment. They will then complete 
the baseline evaluation measures and be randomized to one of four treatment arms. Women who 
have a pain score of 0-2 will be defined as a screen failure and will not be randomized. 
5.3 Baseline Evaluation Phase and Randomization 
Immediately following screening, research participants will complete all baseline assessments, 
including 1) completion of validated questionnaires to measure perceived pain, health, mood, and 
sexual function 2) vulvar exam and quantitative sensory testing to measure pressure pain 
thresholds at local (vaginal) and remote body sites, and 3) collection of biologic samples to 
measure local (vaginal) and systemic (blood) cytokines and miRNAs. The complete schedule of 
assessments to be obtained during the baseline visit is outlined in Appendix A.  
 
Participants who fulfill all the inclusion criteria and none of the exclusion criteria will be randomly 
assigned on a 1:1:1:1 ratio to one of four treatment groups:  
• 
5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + placebo pill  
• 
placebo vaginal cream + nortriptyline pill  
• 
5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + nortriptyline pill 
• 
placebo vaginal cream + placebo pill 
 
Medication assignments will be determined by the Duke Clinical Research Institute (DCRI) using 
randomized blocks, and then stratified by enrollment site. The DCRI will create a SAS program to 
generate the random sequence that will be loaded in RedCap. Participants and research 
personnel will be blinded to medication assignment. 
 
During the baseline visit, participants will be counseled on optimal study medication 
administration. Participants will receive a written description of the study with contact numbers of 
study staff to present to their usual care provider(s).  
5.4 Follow-up Phase 
Randomized study participants will return to the clinic for their 8-, 16-, and 24-week follow up 
visits. The complete schedule of assessments to be obtained during the follow-up visits is outlined 
in Appendix A. 
 
 
 
 
 
 
 
 

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15
6 STUDY FLOW DIAGRAM 
The study flow diagram below outlines the study procedures and assessments in chronological 
order. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Baseline Visit (0-week) 
 
Screening 
Informed consent 
Tampon test 
Pain with sexual contact involving penetration into the vagina 
Pain with touch to the vaginal opening 
 
Baseline Evaluation  
Blood sample collection 
Participant surveys 
Vaginal sample collection 
Vulvar exam 
PPTs 
 
Randomization  
        
 
 
Distribute first medication kit in a 1:1:1:1 allocation ratio  
 
Follow-up Visit 1 (8-week) 
 
Tampon test  
Pain with sexual contact involving penetration into the vagina 
Pain with touch to the vaginal opening 
Blood sample collection 
Participant surveys 
Remote body PPTs 
Vulvar exam 
Vaginal sample collection 
Local vestibular PPTs 
Review medication compliance  
Distribute second medication kit 
Follow-up Visit 3 (24-week) 
 
Tampon test  
Pain with sexual contact involving penetration into the vagina 
Pain with touch to the vaginal opening 
Blood sample collection 
Participant surveys 
Remote body PPTs 
Vulvar exam 
Vaginal sample collection 
Local vestibular PPTs 
lidocaine/estradiol cream
+ placebo pill 
(N=100) 
placebo cream 
+ nortriptyline pill 
(N=100) 
lidocaine/estradiol cream
+ nortriptyline pill 
(N=100) 
placebo cream 
+ placebo pill 
(N=100) 
Follow-up Visit 2 (16-week) 
 
Tampon test  
Pain with sexual contact involving penetration into the vagina 
Pain with touch to the vaginal opening 
Blood sample collection 
Participant surveys 
Remote body PPTs 
Vulvar exam 
Vaginal sample collection 
Local vestibular PPTs 
Review medication compliance  
Discontinue study drug 

Version date: 21Feb2024 
 
 
 
16
7 STUDY POPULATION AND ELIGIBILITY CRITERIA 
7.1 Study Population 
The study population is women with VBD aged 18-50 of any race or ethnicity. Based on historical 
patterns of recruitment, we expect the racial distribution will be 65% White, 10% Asian, 10% 
Hispanic, 8% African-American, and 6.5% other groups. This distribution is quite similar to that 
among people reporting VBD in other U.S. demographic populations.  
7.2 Inclusion Criteria 
1. Female 
2. Age 18-50 years 
3. English-literate 
4. Willingness to provide informed consent 
5. Meeting criteria for diagnosis of VBD based on scoring a 3 or greater on a scale of 0-10 (with 
0 being no pain and 10 being the worst pain imaginable) for either the 
a. Tampon test 
b. Pain with sexual contact involving penetration into the vagina 
c. Pain with touch to the vaginal opening 
7.3 Exclusion Criteria 
1. Participants that will be enrolled in this study must be off medications for 30-90 days, 
depending on the medication dosage, frequency, duration, and half-life. All concomitant 
medications will need to be documented at the time of screening so the Principal 
Investigator and medical doctors at each participating site can determine a conservative 
window. Medications include, but are not limited to: topical lidocaine, estradiol, or 
lidocaine/estradiol to the vulvar vestibule, nortriptyline or other TCAs, pregabalin, and 
gabapentin. 
2. Presence of active dermatologic vulvar disease or vaginal infection 
3. Untreated atrophic vaginitis (participants may undergo treatment and re-evaluation for 
enrollment if the condition is resolved) 
4. Previous vestibulectomy 
5. Pregnant or planning on becoming pregnant during the study period.  
6. Within the first six months of the postpartum period.  
7. Currently breastfeeding/lactating, or within three months of discontinuing 
breastfeeding/lactation. 
8. Active incarceration 
9. Cancer within the past year. 
10. Chemotherapy and/or radiation treatment within the past year.   
11. Unstable medical condition (e.g., renal impairment, significant hematological disease, 
cardiovascular disease, hepatic insufficiency, neurological disorder, autoimmune disease, or 
respiratory illness) 
12. Clear inflammatory states (e.g., morbid obesity) 
13. Use of immunosuppressant medications  
14. History of intolerance to nortriptyline, topical lidocaine, or topical estradiol 
15. Contraindications to use of nortriptyline: current use, or use within the past 1-3 months, of 
MAOIs, some NDRIs, Ethylphenidate (DPH), MDPV (Methylenedioxypyrovalerone), 
Pipradrol (Meratran), Prolintane (Catovit/ Promotil); recent myocardial infarction, active 
psychotic or suicidal thoughts, narrow angle closure glaucoma 
16. Contraindications to the use of lidocaine or local anesthetics 
17. Contraindications to the use of topical estrogen therapy 

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17
18. Post-menopausal, defined as no menses for 12 consecutive months or surgical removal of 
both ovaries. (Hysterectomy is not an exclusion) 
19. Have not had Botox of the pelvic floor muscles in the last 12 months, or pelvic nerve blocks 
in the last three months. 
20. Are not currently enrolled or planning to enroll in another clinical trial during the course of 
this trial. 
21. Are not currently receiving pelvic physical therapy 
8 TREATMENT INTERVENTIONS 
8.1 Interventions 
Participants will be randomly assigned to one of four treatment groups: 1) peripheral treatment in 
the form of 5% lidocaine/0.5mg/ml 0.07% estradiol compound cream + placebo pill, 2) centrally-
targeted treatment in the form of placebo cream + nortriptyline oral pill (up to 50 mg or highest 
tolerated dose), 3) combined treatment in the form of 5% lidocaine/0.5mg/ml 0.07% estradiol 
compound cream + nortriptyline; and 4) placebo cream + placebo pill.  
Lidocaine/estradiol cream targets peripheral nerves and tissues affected in VBD; the 
comparison treatment will be an identical-appearing placebo Moisturel™ cream. Participants will 
be provided with a diagram and written instructions (see Appendix B), detailing how to apply the 
cream to the vaginal vestibule daily for weeks 1-16. Treatment with lidocaine/estradiol or placebo 
cream will be terminated at 16 weeks.  
Nortriptyline is a centrally-acting tricyclic antidepressant that is FDA-approved for treatment of 
neuropathic pain. Dosing will begin with one 10 mg pill nightly for week 1, then two 10 mg pills 
nightly for week 2, three 10 mg pills nightly for week 3, four 10 mg pills nightly for week 4, and 
five for weeks 5 -16. In the event of side-effects without significant adverse events, participants 
will be advised to decrease dosage by one pill weekly until a tolerable dose is achieved. Treatment 
with nortriptyline or placebo pill will be tapered off over weeks 16-18, decreasing the dose by 10 
mg every 4 days. Participants will be provided with a list of drugs to avoid that are known to 
interact with nortriptyline (see Appendix C). 
8.2 Potential Side Effects and Risk Reduction Plan 
Expected risks associated with lidocaine/estradiol compound cream include an adverse reaction 
to either medication or the compounded base. Lidocaine is well tolerated and safe, particularly 
with topical administration in small doses which significantly reduces the risk of lidocaine toxicity. 
The proposed daily dose is extremely small, with minimal systemic uptake when used as 
instructed. As with any medication, participants will be clearly instructed to use as directed. 
Significant over-usage would be required to approach toxicity. This is extremely rare in clinical 
practice. The medication will not be used in women with prior sensitivity or allergy to local 
anesthetics. A mild burning or tingling sensation with application is typical and resolves once the 
anesthetic takes effect.  There is a rare risk of erythema, burning or blistering at the application 
site. The lidocaine will be compounded in a hydrophilic petrolatum, an inert and well-tolerated 
compounding cream. The second component of this compounded cream is estradiol. Like the 
lidocaine, women with prior sensitivity to this medication should not be enrolled, as well as women 
who have contraindications to topical estrogen therapy. The topical application has very low 
systemic uptake (0.01% per ½ gram dose), is well-tolerated and safe in reproductive-aged 
women. Women with potential contraindications to systemic estrogen, such as those with breast 
cancer, stroke or coronary artery disease, may still receive topical estrogen due to the extremely 
low absorption rate.  In these cases, the treating physician will be contacted for permission to 
participate in the study, as is the protocol with clinical use of topical estrogen. Participants will be 

Version date: 21Feb2024 
 
 
 
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instructed to notify the research team if they experience any adverse reaction. 
 
Nortriptyline, a second-generation tricyclic antidepressant, with few associated risks is well-
tolerated when compared to other agents in this pharmacologic class. The medication will be 
administered at night to minimize symptoms but it it has the effect of insomnia, participants may 
switch to morning administration.  The maximum dose of this medication is 150 mg/day for major 
depression, but is efficacious at lower doses for the management of pain disorders. This 
medication should not be used in participants with: homicidal or suicidal ideations, in the acute 
recovery phase of a myocardial infarction (due to risk of arrhythmia), epilepsy, or acute angle 
glaucoma, Expected side effects with nortriptyline include drowsiness, difficulty sleeping 
(insomnia), cloudy thinking, and dry mouth. Other potential side effects include nausea, vomiting, 
loss of appetite, weight gain/loss, anxiety, hives/rash, delayed micturition, urinary retention, 
constipation, vision changes, breast swelling, decreased sexual drive, hyper/hypoglycemia, 
tachycardia or irregular heartbeat. Risk of serotonin syndrome is increased when participants are 
taking other serotonergic medications in addition to nortriptyline.  Risk of long QT syndrome is 
increased when participants are taking citalopram or escitalopram.  If the potential participant 
endorses either taking two or more serotonergic antidepressant medications or taking either 
citalopram or escitalopram, then the study MD will consult with the prescribing provider to 
determine if it would be safe for the potential participant to take nortriptyline in this clinical trial.  
Since serotonin syndrome is most likely to occur within 24 hours of starting/increasing the dosage 
of a serotonergic medication, the researcher will attempt to contact randomized participants within 
24 hours of starting/increasing nortriptyline doses if the participant is taking other serotonergic 
antidepressants.   
8.3 Drug Dispensing 
Identical-appearing creams and pills will be packaged and distributed by Central Compounding 
Pharmacy to the UNC site, and by the Pacifica Compounding Pharmacy to the UCLA site. At the 
baseline visit, participants will receive a sufficient supply of study drug for weeks 1-8, and some 
(3 days) extra in the event of delay in scheduling visit 2. At the 8-week visit, participants will 
receive a sufficient supply of study drug for the remaining weeks 9-16, and some (3 days) extra 
in the event of delay in scheduling visit 3. Patients will be instructed to take the medication as 
required by the protocol.  
8.4 Drug Storage 
The study drug should be stored at controlled room temperature 15°-30°C (59°-86°F) and will be 
dispensed in a light-resistant container. Excessive moisture should be avoided. Study drug must 
be kept out of reach of children.  
8.5 Drug Accountability 
Participants are instructed to return all used, partly used and unused trial products (study drugs 
and packaging material) at each study visit. Returned trial product(s) must be stored separately 
from the non-allocated trial product(s) until drug accountability has been reconciled. The 
respective clinical investigators will be responsible for monitoring all received, used, partly used 
and unused trial product(s). 
8.6 Drug Destruction 
Used and unused study drug will be returned to the respective compounding pharmacies for 
destruction according to accepted pharmacy practice and both local and national guidelines. A 

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copy of the drug destruction procedure should be maintained in the pharmacy section of the 
Regulatory Binder. Study drug destruction should be documented in the comments section of the 
Subject Specific Drug Accountability Log.  
8.7 Randomization and Blinding 
Drug assignments will be determined by the DCRI using randomized blocks, and then stratified 
by enrollment site. The DCRI will create a SAS program to generate the random sequence that 
will be loaded in REDCap.  
 
Participants and research personnel will be blinded to medication assignment. Blinding of the 
study, with respect to treatment groups will be preserved by the use of placebo cream and 
capsules that are identical in appearance to the active study drugs. Investigators may be asked 
at the end of the trial if he/she had obtained any information that may have led to the potential 
unblinding of treatment. 
8.8 Unblinding 
Randomization data are kept strictly confidential, accessible only to authorized persons (in this 
case a member of the DCRI team and the pharmacists), until the time of unblinding. The 
investigative sites will be given access to the treatment code for their participants for emergency 
unblinding only by calling the CC. In the rare event of necessary unblinding, the CC Medical 
Monitor must be contacted to discuss the case. Any suspected study drug-related event should 
be treated as though the patient received active therapy. 
 
At the end of the study, the assigned treatment arm will be unblinded and disclosed to subjects 
individually in order to allow subjects to seek further management of vestibulodynia with their 
gynecologist or primary care physician. 
8.9 Concomitant Medication 
All research participants are not allowed to consume other topical lidocaine, estradiol, or 
lidocaine/estradiol medications, TCAs, MAOIs, SSRIs, SNRIs, or opiates throughout the entire 
course of the study. This will be confirmed by the research team at the 8-, 16-, and 24-week visits. 
Study team will discuss concomitant medication use at the baseline screening visit.    
9 RECRUITMENT, ENROLLMENT AND SCREENING PROCEDURES 
9.1 Common Recruitment Procedures 
Recruitment will occur via proven paths in clinics attended by members of the research team. At 
UNC this includes the Hillsborough Medical Office Building (HMOB) in both the Divisions of 
Minimally Invasive Gynecology (MIGS) and Female Pelvic Medicine and Reconstructive Pelvic 
Surgery (FPMRS) in the Department of Obstetrics and Gynecology (Ob/Gyn), which are located 
on the same floor in adjacent space. Participants will be recruited directly from the MIGS chronic 
pelvic and vulvar pain clinics and the FPMRS clinic. Patients may be contacted by phone prior 
to their scheduled appointment in the clinics above. In addition to the co-investigators, there are 
additional providers between the two clinical divisions who specialize in the care of women with 
pelvic and sexual pain, who will refer potential participants for the proposed study. Additionally, 
the Ob/Gyn generalist division, also located in the HMOB, will be a strong referral source. At 
UCLA, women will be recruited from the UCLA Chronic Pelvic Pain and Vulvar Pain Program, a 
clinical practice focused on the assessment and management of pelvic pain syndromes. 

Version date: 21Feb2024 
 
 
 
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Women identified as new patients, with appointments in the upcoming weeks/months at the 
UCLA Chronic Pelvic Pain and Vulvar Pain clinic, may be contacted by phone or email prior to 
their scheduled appointment to determine their interest as a study participant. Women will also 
be referred from other generalists and specialists at UCLA Westwood, Santa Monica, and Los 
Angeles area as well as the UCLA Student Health and Wellness Ashe Center. In addition to 
this, many patients self-refer or maybe referred by their primary care physician or from outside 
medical groups.  
 
Site PIs will conduct a weekly teleconference to review project-wide procedures, monitor 
targets, discuss operational or scientific processes, and address study-wide progress. 
Recruitment strategies include traditional recruitment methods. Study personnel will place flyers 
in surrounding institutions near UNC (Duke, NC State, Wake Tech, Durham Tech, Women’s 
Health at UNC, Hillsborough, and Raleigh) and UCLA (University of Southern California, Santa 
Monica Community College, California State University- Northridge, Moorpark College, 
Pasadena Community College, and Pomona College). A mass email blast to all UNC and UCLA 
students and employees is scheduled for every 3 months to coincide with the start of each 
quarter. Transit advertising on public buses and radio advertisements through WUNC and 
KPCC National Public Radio (NPR) are options that may be used if necessary. Study 
information will be placed online on a study-specific website, on social media sites (eg, 
Instagram, Facebook, Twitter), on relevant University (eg, Duke List) and Association (eg, 
National Vulvodynia Association) sites, and through clinicaltrials.gov. . Another tool that will be 
utilized to target our audience will be Research Match, https://www.researchmatch.org/. We will list our study 
information on this website for more publicity. 
 
 
In addition, we will perform data mining of CareConnect (Epic) using UCLA Clinical and 
Translation Science Institute (CTSI).  We will provide CDWH with the ICD-9 or ICD-10 codes that 
identify our population, then provide filters (eg, age < 50, seen in last 5 years at UNC, live in NC). 
Once CDWH identifies the list of potential participants, we will contact them by phone or mail to 
evaluate their interest in participating in the study. 
9.2 Estimated Enrollment Period  
Each site will screen for eligible participants to achieve a target of 200 randomized participants 
per site. We expect to randomize an average of 4-5 participants per month per study site 
beginning in the second quarter of funding Year 2. At that rate, the final (400th) participant will be 
randomized at the end of Year 5. This projected rate of randomization is readily achievable based 
on current patient volume at UNC and UCLA (151 and 101 new patients per year, respectively), 
which is expected to increase with utilization of our recruitment methods.  
 
9.3 Screening 
Screening will be conducted in women aged 18-50 with VBD. The purpose of the study, study 
interventions and evaluations, and the potential risks and benefits of participation, will be 
explained to eligible participants. If a participant agrees to participate, she will review and sign the 
site-specific Institutional Review Board (IRB) approved informed consent form 
10 BASELINE EVALUATION AND DRUG DISPENSING (0-WEEK)  
Following screening, research participants will complete all baseline procedures, including 1) 
completion of validated questionnaires to measure perceived pain, health, mood, and sexual 
function 2) a vulvar exam and quantitative sensory testing to measure pressure pain thresholds 
at local and remote body sites, and 3) collection of biologic samples to measure local (vaginal) 

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21
and systemic (blood) cytokines and miRNAs. A complete schedule of assessments throughout 
the study is outlined in Appendix A. 
10.1 
Baseline Evaluation 
Baseline procedures and assessments (described in detail in Section 15 below) will be 
performed in the following order:  
 
Participant consent 
 
Tampon test with 0-10 NRS 
 
Blood collection and processing 
 
Participant questionnaires: 
o Demographics 
o Social history 
o Gynecological history 
o VPAQ 
o PROMIS BPSFS-F 
o COPC inventory 
o SF12v2 
o SCL-27 
o PILL 
o PSS 
o Sleep scale 
 
Remote bodily PPTs (deltoid, shin, trapezius) 
 
Qualitative pelvic sensory testing 
 
Vaginal sample collection 
 
Local vestibular PPTs (5, 6, and 7 o'clock) 
10.2 
Drug Dispensing  
All eligible study participants who successfully complete screening and baseline evaluations will 
be randomized (as detailed in Section 8.7) to receive one of four types of medication kits: 
• 
5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + placebo pill  
• 
placebo vaginal cream + nortriptyline pill  
• 
5% lidocaine/0.5mg/ml 0.07% estradiol compound vaginal cream + nortriptyline pill 
• 
placebo vaginal cream + placebo pill 
  
Identical-appearing creams and pills will be packaged and distributed by the Central 
Compounding Pharmacy to UNC and by the Pacifica Compounding Pharmacy to UCLA. At the 
baseline visit, participants will receive a sufficient supply of study drug for weeks 1-8. Patients will 
be instructed to take the medication as required by the protocol and complete a daily medication 
calendar (Appendix D). 
11 FOLLOW-UP EVALUATIONS 
11.1 
Visit 2 (8-Week) 
Upon arrival, participants will return any remaining study medication to the research team. 
 
The week 8 visit procedures and assessments will be performed in the following order: 
 
Tampon test with 0-10 NRS 
 
Blood collection and processing 
 
Participant questionnaires: 

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22
o VPAQ 
o PROMIS BPSFS-F 
o COPC follow-up survey 
o SF12v2 
o SCL-27 
o PILL 
o PSS 
o Sleep scale 
 
Remote bodily PPTs (deltoid, shin, trapezius) 
 
Qualitative pelvic sensory testing 
 
Vaginal sample collection 
 
Local vestibular PPTs (5, 6, and 7 o'clock) 
 
At the end of visit 2, participants will receive a sufficient supply of study drug for weeks 9-16. 
Patients will be instructed to take the medication as required by the protocol and complete a 
daily medication diary. 
11.2 
Visit 3 (16-Week) 
Upon arrival, participants will return any remaining study medication to the research team. Study 
medication will be discontinued. 
 
The week 16 visit procedures and assessments will be performed in the following order: 
 
Tampon test with 0-10 NRS 
 
Blood collection and processing 
 
Participant questionnaires: 
o VPAQ 
o PROMIS BPSFS-F 
o COPC follow-up survey 
o SF12v2 
o SCL-27 
o PILL 
o PSS 
o Sleep scale 
 
Remote bodily PPTs (deltoid, shin, trapezius) 
 
Qualitative pelvic sensory testing 
 
Vaginal sample collection 
 
Local vestibular PPTs (5, 6, and 7 o'clock) 
11.3 
Visit 4 (24-Week) 
The week 24 visit procedures and assessments will be performed in the following order: 
 
Tampon test with 0-10 NRS 
 
Blood collection and processing 
 
Participant questionnaires: 
o VPAQ 
o PROMIS BPSFS-F 
o COPC follow-up survey 
o SF12v2 
o SCL-27 
o PILL 
o PSS 
o Sleep scale 
 
Remote bodily PPTs (deltoid, shin, trapezius) 

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 
Qualitative pelvic sensory testing 
 
Vaginal sample collection 
 
Local vestibular PPTs (5, 6, and 7 o'clock) 
12 OUTCOME DETERMINATIONS 
Aim 1. Determine the efficacy of peripheral, central, and combined treatments in alleviating 
pain among women with VBD-p and VBD-c.  
Primary Endpoints:  
Ia. pain score during tampon test at 16 weeks  
Ib. self-reported pain via SF-MPQ at 16 weeks 
 
Secondary Endpoints:  
IIa. experimental pain via local and remote PPTs at 16 weeks 
IIb. COPC pain levels via COPC follow-up survey at 16 weeks 
IIc. Pain scores and PPTs at 8 weeks and 24 weeks 
 
Aim 2. Determine the efficacy of peripheral, central, and combined treatments in improving 
patient reported outcomes among women with VBD-p and VBD-c. 
Primary Endpoints:  
Ia. self-reported physical/mental health via SF12v2 at 16 weeks 
Ib. sexual health via PROMIS at 16 weeks 
 
Secondary Endpoints:  
IIa. mood via SCL-27 at 16 weeks 
IIb. somatic awareness via PILL at 16 weeks 
IIc. perceived stress via PSS at 16 weeks 
IId. sleep via sleep scale at 16 weeks 
IIe. Patient reported outcomes at 8 weeks and 24 weeks 
 
Aim 3. Determine the efficacy of peripheral, central, and combined treatments in 
normalizing cytokine and microRNA expression profiles in vaginal and blood tissues 
collected from women with VBD-p and VBD-c. 
Primary Endpoints:  
Ia. cytokine levels via mesoscale discovery assays at 16 weeks 
Ib. microRNA levels via sequencing read at 16 weeks 
 
Secondary Endpoints:  
IIa. cytokine and microRNA levels at 8 weeks and 24 weeks 
IIb. Aim 3 will also identify cytokine and microRNA biomarkers at baseline that predict treatment 
response at 8, 16, and 24 weeks  
13 METHODS TO PROMOTE ADHERENCE 
Protocol training and adherence will be a major focus of the training for all Investigators, research 
nurses, and other essential personnel. An initial training session will take place prior to beginning 
recruitment in order to instruct and calibrate study personnel on all aspects of vulvar exam, QST, 
sample collection/processing, etc. Adherence will then be maintained by continuing to review 
study procedures during weekly calls and by annual follow-up training sessions (in-person at the 
UNC Hillsborough clinic site with a Skype connection to the UCLA clinic site). In addition, the CC 
will contact each site to offer per-participant feedback on adherence; will review episodes of non-
adherence and reemphasize the importance of adherence; and will provide adherence reports.  

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14 PARTICIPANT SAFETY AND ADVERSE EVENTS 
14.1 
Single IRB 
Our clinical trial will comply with the NIH Policy on the Use of a Single Institutional Review Board 
(sIRB) for Multi-Site Research (NOT-OD-16-094), with the goal to eliminate duplicative IRB review 
and streamline the IRB review process so that research can proceed as effectively and 
expeditiously as possible. The Duke University IRB will serve as the sIRB of record. UCLA and 
UNC will serve as participating sites and have agreed to rely on the proposed sIRB. Duke 
University will be responsible for maintaining records of the authorization/reliance agreements 
and of the communication plan. This agreement, clarifying the roles and responsibilities of the 
sIRB and participating sites, will be signed by all participating sites prior to initiating the study. 
 
Communication between Duke, UCLA, UNC and the sIRB will be managed by the CC. The CC 
will assist with the development of IRB protocols, participant randomization, data management, 
safety surveillance, and medical communications.  
14.2 
Definitions 
14.2.1 Adverse Events 
An adverse event (AE) is defined as any untoward medical occurrence with the use of a drug in 
a subject whether or not considered drug or biologic related. An AE can therefore be any 
unfavorable and unintended sign, symptom, or disease temporarily associated with the use of a 
pharmaceutical product or biologic. 
 
The study is limiting collection of AEs to focus on potential impacts of the blinded study 
medication. Only the following AEs will be documented in the electronic Case Report Form 
(eCRF): 
 
Swelling 
 
Burning 
 
Blistering 
 
Drowsiness 
 
Difficulty sleeping (insomnia) 
 
Cloudy thinking 
 
Dry mouth 
 
Nausea 
 
Vomiting 
 
Loss of appetite 
 
Weight gain 
 
Weight loss 
 
Anxiety 
 
Hives 
 
Rash 
 
Difficulty urinating 
 
Constipation 
 
Vision changes 
 
Breast swelling 
 
Decreased sexual drive 

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 
Low blood sugar 
 
High blood sugar 
 
Increased heart rate 
 
Irregular heartbeat 
 
Suicidal thinking 
 
Mania 
 
Agitation 
 
Hallucinations 
 
Muscle tremor 
 
Muscle rigidity 
 
Seizures 
 
Bone pain 
14.2.2 Serious Adverse Events 
A serious adverse event (SAE) is an AE that results in the following outcomes: 
 
Death 
 
Life-threatening AE: Places the subject at immediate risk of death at the time of the event 
as it occurred. It does not include an AE that, had it occurred in a more severe form, might 
have caused death. 
 
Persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions. 
 
Inpatient hospitalization or prolongation of hospitalization.  
 
Congenital anomaly or birth defect.  
 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered a serious adverse event when, based upon appropriate 
medical judgment, they may jeopardize the subject and may require medical or surgical 
intervention to prevent one of the outcomes listed in this definition above. 
 
This determination is based on the opinion of the investigator. 
 
14.2.3 Assessment of Causal Relationship 
A medically-qualified investigator must assess the relationship of any AE to the use of study drug, 
based on available information, using the following guidelines:  
 
Not related: There is no reasonable causal relationship to the investigational product and 
the adverse event.  
 
Unlikely related: No temporal association or the cause of the event has been identified, or 
the drug or biologic cannot be implicated. 
 
Possibly related: There is reasonable evidence to suggest a causal relationship between 
the drug and adverse event. 
 
Related: There is evidence to suggest a causal relationship and the influence of other 
factors is unlikely. 
 
14.2.4 Recording and Reporting of Adverse Events 
The site investigator is responsible for monitoring the safety of participants enrolled into the study 
at the study sites. The investigator will probe, via discussion with the participant, for the 
occurrence of AEs during each study visit and record the information in the site’s source 
documents. Participants will be instructed to report the occurrence of potential AEs between visits 
(on a weekly basis) using the Electronic Data Capture (EDC) system. The investigator will receive 
an email notification and will call the participant to gather further information about the AE(s).  

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The investigator will record selected AEs on the eCRF. Adverse events will be described by 
duration (start and stop dates), severity, outcome, and relationship to study drug. Adverse events 
will be reported to the Data Safety Monitoring Board (DSMB), Duke IRB and/or other agencies 
according to guidelines established by those entities/agencies. All AEs that are identified, whether 
or not listed in Section 14.2.1, will be followed by the site until resolution or stabilization. Adverse 
Events will be captured from the time of consent through the Month 6 visit. 
14.3 
Data Safety Monitoring Board 
The DSMB will be composed of 4 members: a physician, a statistician, a patient representative, 
and a bioethicist. All DSMB operations will be managed by the DCRI.  
 
The goal of the DSMB will be to monitor patient safety and to review the performance of the study. 
The DSMB will be responsible for providing recommendations regarding trial’s conduct and 
guidance to ensure the safety and well-being of patients treated by study physicians. A separate 
DSMB charter will outline the operating guidelines for the committee, the protocol for evaluation 
of data, and data tables to be provided. The charter will be created prior to study enrollment and 
agreed upon during the initial meeting of the DSMB. It is anticipated that the DSMB will meet at 
6-month intervals to review the accumulating data for safety. 
 
DSMB Analyses: A DCRI Statistical Analyst and Programmer will generate the analyses required 
by the DSMB charter and provide these in advance of each DSMB meeting. This individual will 
ensure high quality and timely reports are submitted. Confidential Interim Data Reports will be 
prepared regularly by the DSMB statistician as specified in the Charter. The report will include 
recruitment and retention rates, SAEs, and other information as requested by the DSMB Chair. 
15 STATISTICAL CONSIDERATIONS 
15.1 
VBD Subtype Designation 
Women with VBD-c will be distinguished from those with VBD-p based on the presence of a 
COPC. The Institute of Medicine’s 2011 report, Relieving Pain in America, identified 9 COPCs 
that predominantly affect women and frequently co-occur with VBD.93 The presence of a COPC 
will be determined using the COPC survey. This is an easy-to-complete form including modules 
with check-box questions and a body mannequin to determine the presence of 9 COPCs: 1) FMS, 
2) TMD, 3) back pain, 4) IBS, 5) chronic tension-type headache, 6) chronic migraine, and 7) 
chronic fatigue syndrome, 8) endometriosis, and 9) interstitial cystitis. Women with VBD and no 
COPCs will be designated as VBD-p, while women with VBD and >1 COPC will be designated as 
VBD-c. These criteria will be used as a primary method to distinguish between VBD-p and VBD-
c in this study. 
 
We, however, acknowledge that the criteria to distinguish VBD subtypes needs further 
exploration. In particular, it is possible that the optimal cutoff for VBD-c requires more than one 
COPC to be present. Therefore, as a secondary method to characterize VBD subtypes, we will 
calculate a continuous COPC ‘centralness’ score defined as the number of COPCs present 
ranging from 0 to 9. This continuous COPC score will be used to identify the optimal cutoff value 
between VBD-p and VBD-c in terms of treatment response. 
  
To further characterize the two VBD subtypes, we will also measure generalized pain at ‘neutral’ 
non-pelvic sites using a quantitative sensory testing approach designed by Dr. Gracely and 
colleagues.94, 95 The 3 neutral sites (deltoid muscle, shin, trapezius) are remote from the pelvic 
region and not used for diagnosing other pain conditions, but reflect an individual’s overall 

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pressure pain sensitivity.98-100 An algometer will be applied to the 3 sites, with continuously 
ascending pressure at a rate of 1 kg/s to a maximum of 10 kg. Participants will indicate when the 
evoked sensation first becomes painful. A composite pressure pain threshold (PPT) score will be 
compared between VBD-c and VBD-p subgroups using ANCOVA. We would expect women with 
VBD-p to have higher PPT scores (less pain) and women with VBD-c to have lower scores (more 
pain) at these sites. 
15.2 
Analysis of the Aim 1 Endpoints 
Primary endpoints will be the net change in vaginal vestibule pain following treatment measured 
using the Tampon Test and the Short-Form McGill Pain Questionnaire (SF-MPQ).  
 
The Tampon Test will provide a self-reported numeric rating scale of pain with self-tampon 
insertion, performed by the patient and reported to the research nurse. An Original Regular 
Tampax tampon will be inserted by the participant into her vagina and then she will be asked 
to verbally rate the pain on a scale of 0-10, with 0 meaning no pain and 10 meaning the 
worst possible pain. The Tampon Test has good reliability, construct validity, and 
responsiveness and is a recommended outcome measure for VBD clinical trials.96  
 
 
The SF-MPQ measures perceived sensory qualities of pain using 11 describers and 
affective qualities related to pain using 5 describers. Responses on 4-point scales are 
summed to compute scores for each section.97 SF-MPQ subscales have been successfully 
used to quantify treatment responses in trials for VBD and vulvodynia.98-100 
 
 
Secondary endpoints include changes in pressure pain thresholds (PPTs) measured at the  
 
Vaginal Vestibule PPT will be determined using a digital vestibular algometer applied to 3 
externally-accessed sites (2 and 10 o’clock on the upper vestibule and 6 o’clock on the lower 
vestibule) beginning at 1N and increasing until the participant’s first sensation of pain or a 
maximum applied pressure of 10N.102   
 
 
Levator Muscle Complex PPTs will be determined in a similar manner by applying the 
algometer internally to the right, midline, and left puborectalis levator muscles sites (5, 6, 
and 7 o’clock) just lateral to the perineum.  
 
 
Remote Bodily PPTs will be determined in a similar manner by applying the algometer to 3 
‘neutral’ non-pelvic body sites (deltoid, shin, and trapezius), right and left, and calculating a 
composite score.  
 
Data Analysis. The two primary statistical questions for Aim 1 are: 1a) Is peripheral treatment 
with lidocaine/estradiol compound cream, or central treatment with nortriptyline, or a combination 
of both treatments more effective for either VBD subtype than placebo? 1b) Is peripheral 
treatment more effective than central treatment in VBD-p, and is central treatment more effective 
than peripheral treatment in VBD-c? 
 
To answer questions 1a and 1b, we will fit mixed model repeated measures (MMRM) models 
which account for missing data as an integral part of the analyses under the assumption of 
ignorable missing. To answer question 1a, the MMRM model includes longitudinal pain score (0-
10 NRS on Tampon Test) as a continuous outcome variable; includes fixed effect terms for 
treatment (nortriptyline, lidocaine/estradiol, and their interaction), visit, treatment visit interaction, 
center, and fixed effect covariates vector including age, VBD subtype (binary variable where VBD-
c is coded as 0 and VBD-p coded as 1), race (three levels “white”, “black” and “other” race, coded 
into two binary dummy variables with European American serving as a reference level).  
 
Overall treatment effect of nortriptyline, lidocaine/estradiol, and their interaction will be evaluated 
using an F-test. If significant (p < 0.05), treatment differences (each of 3 treatment groups minus 
placebo) in the Least-Squares means (LS means), their 2-sided 95% Cis, and associated p-

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values will be estimated from the MMRM model. P-values will be compared with the significance 
threshold Bonferroni-corrected for 3 tests (i.e. alpha 0.0166).  
 
To answer question 1b, we will fit a MMRM model analogous to 1a, with additional terms of VBD 
subtype visit interaction, treatment VBD subtype interaction, and treatment visit VBD subtype 
interaction. The significance of the additional interaction terms will be evaluated using a F-test 
comparing the full model in 1b and the reduced model in 1a. Significant (p < 0.05) F-test will 
suggest that the effect of either nortriptyline or lidocaine/estradiol cream, or both, differ by VBD 
subtype. In this case, we will compare the LS means of the treatment differences in each 
treatment-VBD-subtype subgroup, after appropriate Bonferroni correction.  
 
If the results for question 1a or 1b are statistically significant, the following primary outcomes will 
be analyzed using the same modeling strategies (models in 1a and 1b above): % decrease in 
pain score (tampon test) from baseline to the 16-week time point and % decrease in perceived 
pain on the SF-MPQ from baseline to the 16-week time point. Secondary outcomes include 
change in mucosal, vulvovaginal muscle, and remote bodily PPTs from baseline to the 16-week 
time point. Secondary outcomes also include change in pain scores and PPTs from baseline to 
the 8-week time point and change in pain scores and PPTs from the 16-week to the 24-week time 
point.  
 
If the result for question 1b is non-significant, we will use the continuous COPC score in model 
1b instead of a dichotomous VBD classification to compare treatment success for VBD-p and 
VBD-c. In addition, we use a binary treatment outcome (achievement of 30% pain reduction) to 
identify the optimal cutoff of the continuous COPC score in each treatment group.   
 
Secondary analyses of muscle pain based on PPTs will also determine: a) the proportion of 
women with VBD that have a pelvic muscle pain component; b) differences in the distribution of 
muscle pain in VBD-p and VBD-c groups; and c) presence of muscle pain to predict treatment 
outcome. The latter will be tested by including the muscle pain variable (continuous or 
dichotomous) as part of the fixed effect term in model Ia. 
 
Expected Outcomes. 1) For women with VBD-p, peripheral lidocaine/estradiol treatment will be 
more effective than placebo or nortriptyline in reducing numeric pain ratings on the tampon test, 
perceived pain on the SF-MPQ, and pelvic PPTs on the algometer test at week 8 and week 16. 
2) For women with VBD-c, centrally-targeted nortriptyline treatment will be more effective than 
placebo or lidocaine/estradiol in reducing numeric pain ratings on the tampon test, perceived pain 
on the SF-MPQ, and pelvic/remote bodily PPTs on the algometer test at week 16. 3) For women 
with VBD-c, combined peripheral and centrally-targeted treatments will be most effective in 
reducing pain on primary and secondary outcome measures at week 16. 4) The two VBD 
subtypes will exhibit differential responses to treatment withdrawal at week 16, such that at the 
week 24 visit women with VBD-c will exhibit increases in pain after treatment withdrawal 
compared to women with VBD-p. 
15.3 
Analysis of the Aim 2 Endpoints 
Primary endpoints include the net change in perceived sexual health measured using the 
Patient-Reported Outcomes Measurement Information System (PROMIS) and perceived physical 
and mental health measured using the SF-12 Health Survey (SF-12).  
 
The PROMIS is a 96-item form developed by the NIH that measures 11 domains of 
biopsychosocial function and includes an assessment of sexual function measures (e.g., 
desire, frequency, fear, and pain) related to sexual intercourse.115  
 
 
The SF-12 assesses 6 domains: global health, physical functioning, physical roles, 
emotional functioning, emotional roles and pain interference using an algorithm118 based on 
answers to 12 physical and mental health-related questions.  
 

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Secondary endpoints include net changes in perceived mood and health using:  
 
Symptom Check List 27 (SCL-27), which measures a broad range of psychological 
symptoms (e.g., anxiety and depression) and has high reliability and strong correlations to 
pain.121,122  
 
 
Pennebaker Index of Limbic Languidness (PILL), which is used to create a summary score 
of somatic symptoms (e.g., itchy eyes, dizziness). Symptom frequency is recorded on a five-
point Likert scale ranging from “never” to “more than once a week”.108  
 
 
Perceived stress scale (PSS), which is a 10-item scale that measures the impact of personal 
stress on thoughts and feelings. 
 
 
Sleep Scale, which is a 12-item scale that measures amount of sleep and ease/difficulty of 
initiating and maintaining sleep  
 
Data Analysis. The two primary statistical questions for Aim 2 are: 2a) At baseline, do women 
with VBD-c exhibit worse perceived physical, mental, or sexual health than women with VBD-p? 
2b) Considering physical, mental, or sexual health, is peripheral treatment with lidocaine/estradiol 
cream more effective than nortriptyline in VBD-p, and central treatment with nortriptyline more 
effective than lidocaine/estradiol cream in VBD-c? 
 
To answer question 2a, we will fit three linear regression models with the dependent continuous 
variable being a variable for each of the 3 patient reported outcomes (SF12 physical health score, 
SF12 mental health score, and PROMIS sexual health score). VBD subtype will be the predictor 
of interest. Covariates will include age (continuous variable), race (coded as in model Ia), study 
site. Regression coefficient for VBD subtype will be tested for statistical significance using 
Bonferroni-corrected threshold alpha 0.0166 (corrected for 3 tests corresponding to 3 outcomes). 
Regression coefficient significance will suggest outcomes differ by VBD subtype.  
 
To answer question 2b, three MMRM models analogous to model 2b will be fit, where Y is a 
continuous variable representing the primary outcomes measured at 16 weeks. Similar to 2b, we 
will jointly evaluate the significance of additional interaction terms using an F-test (“overall test”). 
Significant F-test will suggest that the effect of either nortriptyline or lidocaine/estradiol cream, or 
both, differ by VBD subtype. In this case, we will compare the LS means of the treatment 
differences in each treatment-VBD-subtype subgroup, after appropriate Bonferroni correction. 
Secondary outcomes will also include change in SCL-27 mood score, PILL somatization score, 
and PROMIS biopsychosocial trait scores from baseline to 16 weeks following treatment. 
Secondary outcomes also include change in patient-reported outcomes from baseline to the 8-
week time point and change in pain scores and PPTs from the 16-week to the 24-week time point.  
 
Expected Outcomes. 1) For women with VBD-p, peripheral lidocaine/estradiol treatment will be 
more effective than placebo or nortriptyline in improving perceived physical, mental, and sexual 
health at 8 and 16 weeks. 2) For women with VBD-c, centrally-targeted nortriptyline treatment will 
be more effective than placebo or lidocaine/estradiol in improving perceived physical, mental, and 
sexual health at 16 weeks. 3) For women with VBD-c, combined peripheral and centrally-targeted 
treatments will be most effective in improving health at 16 weeks. 4) The two VBD subtypes will 
exhibit differential responses to treatment withdrawal, such that women with VBD-c report 
decreases in perceived health from 16 to 24 weeks compared to women with VBD-p. 
15.4 
Analysis of the Aim 3 Endpoints 
Cytokine Expression levels will be measured in vaginal lavage samples and plasma isolated 
from whole blood. We will use a standard Human MesoScale Discovery multiplex kit to measure 
36 inflammatory mediators (4 tier-1 and 32 tier-2).  
 

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Tier-1 mediators include the pro-inflammatory cytokines IL-1β,59-61 IL-8,7,60,62 and IL-1763 and anti-
inflammatory cytokine IL-1ra.7,61 These cytokines were selected based on previous associations 
with VBD case status and symptom severity. 
  
Tier-2 mediators include cytokines (e.g., interleukin 6; IL-6), chemokines (e.g., monocyte 
chemoattractant protein 1; MCP1), and growth factors (e.g., vascular endothelial growth factor; 
VEGF) that are involved in peripheral and central sensitization, inflammation, and chronic pain.128  
 
miRNA Expression levels will be measured in vaginal swab samples and blood. We will isolate 
total RNA and then ship the RNA to UT Health Genome Sequencing Facility (GSF), directed by 
Dr. Zhao Lai. The GSF core will perform small RNA library preparation and sequencing.  
 
Data Analysis. The two primary statistical questions for Aim 3 are: 3a) At baseline, is there an 
inverse association between VBD ‘centralness’ score (determined by the number of COPC sites) 
and the levels of Tier-1 cytokines in vaginal samples; and a positive association between VBD 
‘centralness’ score and the levels of Tier-1 cytokines in plasma samples? 3b) Do biomarkers in 
vaginal fluid predict response to peripheral treatment and biomarkers in blood predict response 
to central treatment? 
 
To answer question 3a, we will fit a linear regression model where cytokine level (skewed 
variables will be transformed (e.g., log, box-cox) to approximate normal distribution) will be a 
continuous dependent variable and VBD ‘centralness’ score, will be the main predictor of interest. 
Covariates will include age and race. Significance of regression coefficient associated with the 
‘centralness’ score will be evaluated using an F-test and significance threshold (alpha) Bonferroni-
corrected for the number of cytokines assessed (4 cytokines corresponding to 4 statistical tests). 
Subgroup means, mean differences and 95% confidence intervals will be output. 
 
To answer question 3b, we will fit a MMRM model analogous to 1a, with additional terms of 
treatment cytokine (in blood and vaginal samples) interaction, visit cytokine interaction, and 
treatment visit cytokine interaction. Significance of additional interaction terms will be evaluated 
using an F-test. Significant (p < 0.05) F-test will suggest that treatment effects by nortriptyline or 
lidocaine/estradiol cream or both, are modified by cytokines. 
 
Secondary analyses for Aim 3 will include the association of Tier 2 biomarkers (cytokines and 
miRNAs) with VBD subtype and interaction with treatment response. These analyses will be 
analogous to those specified for questions 3a and 3b, with p-value thresholds kept at 0.05 due to 
exploratory nature of these analyses. In our secondary analyses we will also evaluate the change 
of biomarker concentrations (blood and vaginal samples) over time (at 0, 8, 16, and 24 weeks) in 
each treatment group and VBD subtype. We hypothesize that these biomarkers may serve as 
intermediate phenotypes for treatment success in VBD patient subgroups.  
 
Expected Outcomes. 1) Women with VBD-p will have higher levels of inflammatory mediators in 
peripheral vaginal samples, while women with VBD-c will have higher levels of inflammatory 
mediators in circulating blood measured at the baseline visit (0 weeks). 2) Abnormalities in 
baseline vaginal cytokine and miRNA biomarkers will predict a positive response to peripheral 
lidocaine/estradiol treatment, while abnormalities in blood biomarkers will predict a positive 
response to centrally-targeted nortriptyline or combined treatment. 3) Peripheral treatment will 
resolve abnormalities in vaginal biomarkers evaluated at 8, 16, and 24 weeks, while central 
treatment will resolve abnormalities in blood biomarkers evaluated at 16 weeks.     
 
All analyses will be performed using the Intent-to-Treat (ITT) population. The ITT population will 
include all randomized participants. Participants will be grouped according to their randomized 
allocation, regardless of whether the allocated therapy was administered or switched.   
 
Any deviations from the statistical analyses described above, due to unforeseen circumstances, 
will be documented in the Statistical Analysis Plan (SAP).   

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15.5 
Analysis of Safety Data and Statistical Monitoring Plan 
Interim data analysis for efficacy and futility will not be conducted due to relatively small size and 
short duration of this clinical trial. Safety data, summarized at the treatment level, will be assessed 
approximately every 6 months by the DSMB. Adverse Events will be summarized by treatment 
group and will include incidence by term, severity, relationship to study medication as well as 
overall incidence rates (regardless of severity and relationship to study medication). 
15.6 
Sample Size and Power 
We are planning to enroll 400 women with VBD, with an estimated attrition rate of 20%. Of note, 
Reed et al. found that vulvar pain measured on a 0-10 numeric scale has standard deviation 
2.0.104 Salaffi et al105 noted that 2 points on a 0-10 NRS correspond to a clinically meaningful 
difference in pain severity. Using these results and assuming the within-subject correlation of 0.5, 
at a significance level of 0.05, the proposed sample size will provide sufficient power of at least 
0.80 to detect differences as small as 0.82 on a 0-10 scale for the overall treatment main effects 
(3 arms) vs. placebo. To test each individual treatment effect vs. placebo, at a significance level 
of 0.0167 (Bonferroni-corrected for 3 arms), the proposed sample size can detect differences as 
small as 0.98 on a 0-10 scale. At a significance level of 0.05, the proposed sample size can detect 
differences as small as 1.35 and 1.20 on a 0-10 scale for the interaction effect of treatment and 
each VBD-subtype, given possible splits of 40/60, 50/50, or 60/40 (of note, VBD-p vs VBD-c 
frequencies were 40/60 at UNC and 50/50 at UCLA clinics in previous studies).  
 
To calculate power for research question IIIa, we used data from our previous study of biomarkers 
in VBD patients.7 Conservatively assuming that VBD-c patients represent 60% (192/320) of the 
sample and VBD-p patients represent 40% (128/320) of the sample, and adjusting the 
significance threshold for 4 tier 1 cytokine tests, we will be able to detect mean differences of at 
least 0.4 SD with the power of 0.84 or greater using a t-test. For IL-8, 0.4 SD translates to 
approximately 2 pg/mL, and for IL-1ra 0.4 SD translates to 240 pg/mL of the minimal detectable 
difference. Of note, the mean differences between controls and VBD patients for IL-8 and IL-1ra 
in our previous study were 4 pg/mL and 300 pg/mL, respectively. Thus, we have sufficient power 
to detect plausible differences in cytokine levels between the VBD subgroups. 
16 DATA MANAGEMENT PROCEDURES 
16.1 
Overview of Data Management 
The CC will have primary responsibility for data management, including the development of data 
collection systems, data monitoring processes, and data storage and back-up. State-of-the-art 
technology will be used for the management of the network’s data.  
 
eCRF: The CC team will develop modules, designed in Redcap, that will capture patient 
responses to questions having to do with their VBD symptoms. The Redcap instruments will 
include an enrollment and demographics form; forms for recording relevant history, Pain 
symptoms, vulvar exam results, laboratory results, baseline biomarker levels, and other baseline 
presenting characteristics; follow-up forms for use during regular follow-up visits; forms to track 
the participant’s clinical course over time; and others that track all related information for Patients 
that meet the trial criteria. 
 
System: The data will be collected in a validated, IRB approved Redcap survey environment. The 
CC team of skilled data managers and programmers will design and produce a tailored network 
system that provides operational efficiency and meaningful reporting of metrics. 
  

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Data Management Process: The Redcap survey environment will be used for data entry and 
simple reports. All data will be entered into Redcap by personnel at the clinical sites. Any out-of-
range values and missing key variables will be flagged and addressed in real-time at the site 
during data entry. When a query is generated on a particular variable, a flag is raised in a database 
field; the system tracks the queries and produces reports of outstanding queries. Queries can 
also be generated from manual or statistical review of the data forms. 
 
The CC will create reports to identify trends in the data that may require additional clarification 
and training. These reports will be available to the sites and to the study leadership as we work 
with the sites to correct negative trends and eliminate future data errors. The CC will perform 
internal database quality-control checks during the study to identify systematic deviations 
requiring corrections.  
 
Data Quality Control: A three-step approach to data quality control will be implemented. 
 
1. Training: Prior to the start of enrollment, the Investigators and Nurse Practitioners will be 
trained on the clinical protocol and data collection procedures. Recent site surveys 
indicate that most Coordinators are very familiar with the EDC system, so training is 
typically targeted to a specific protocol. For Coordinators new to REDCap, the CC will 
provide training with hands-on database interaction, demonstration of key REDCap 
system functionality, and practice exercises. Personnel at the clinical sites will enter the 
data mandated by the protocol into REDCap. All CRFs will be completed according to the 
current Good Clinical Practice (GCP) guidelines. The CC will conduct follow-up training 
and training for new study personnel as needed. 
2. Monitoring: A CC monitor will visit sites during the enrollment period to ensure that data 
collection is being handled properly, to provide in-service training, and to address 
questions from site investigators and coordinators. Additional details will be outlined in the 
Clinical Monitoring Plan.  
3. Managing data: A series of computerized data validation checks will be programmed by 
the CC to check for missing data, inconsistencies in the data or data that is out of range. 
After the data have been exported from the EDC system to SAS for statistical 
summarization and data analysis, further cross-checking of the data will be performed by 
the CC and queries issued through REDCap for any discrepancies. 
16.2 
Data Security 
Access to databases will be controlled centrally by the CC through user passwords linked to 
appropriate privileges. This protects the data from unauthorized changes and inadvertent loss or 
damage. Database and web servers will be secured by a firewall and through controlled physical 
access. Database back-up will be performed daily using standard procedures in place at the CC. 
All disk drives that provide network services, and all user computers, will be protected using virus-
scanning software. 
16.3 
Publication Policy 
Dissemination of preliminary information can adversely affect the objectivity of study data. For this 
reason, Investigators will be prohibited from performing subset analyses at any point prior to the 
conclusion of the study, and any data, other than safety data, cannot be used for publication or 
reporting outside of this study until the study is completed or discontinued by the DSMB or NICHD.  

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17 STUDY ADMINISTRATION 
17.1 
Data and Safety Monitoring Board 
As noted above, the DSMB will be composed of 4 members: a physician, a statistician, a patient 
representative, and a bioethicist. All DSMB operations will be managed by the DCRI.  
 
DSMB Charter and Meetings. The DCRI Statistical Team will develop a DSMB Charter that details 
specific data tables to be provided and procedures to be followed. The DSMB Charter will also 
describe the overall mission, responsibilities, and meeting schedule of the board. The DCRI 
statistical team will be responsible for DSMB charter review and approval. The draft charter will 
be submitted for review and approval by NICHD, the DSMB chairperson, and study Investigators 
prior to study enrollment. After the charter has been finalized, any changes to the plan will be 
documented in the minutes of the DSMB meeting. At the conclusion of each DSMB meeting, the 
DSMB Chair will make a recommendation to the study team about continuation of the trial. It is 
expected that the DSMB will meet every 6-months. If safety issues arise, additional conference 
calls can be requested by the DSMB chairman. DCRI statistical team will coordinate and convene 
all DSMB meetings.  
17.2 
Coordinating Center 
Dr. Nackley will function as the Clinical CC, Dr. Stirling will function as the Data Management 
CC, and the DCRI will function as the Statistical CC for this trial. 
17.3 
Core Laboratories 
17.3.1 Central Compounding Pharmacy and Pacifica Compounding 
Pharmacy 
The investigational product (IP) and placebo capsules to be dispensed at UNC will be prepared 
by Central Compounding Pharmacy, directed by Jennifer Burch, and shipped to study personnel 
at the UNC Hillsborough clinic.  
 
The IP and placebo capsules to be dispensed at UCLA will be prepared by Pacifica Pharmacy, 
directed by Jeffrey Barris. 
17.3.2 Substrate Services Core 
The Substrate Services Core, directed by Mary-Beth Joshi, will provide quality control and a state-
of-the-art repository for study samples. Precise sample inventory will be facilitated through a 2D 
Barcode system. This system includes the sample tubes themselves, which have a 2D barcode 
with 14-digit alpha-numeric code etched on the bottom of the sample tubes. A scanner will be 
used to ensure accurate reading of barcodes, and the data will be downloaded into the 
LabVantage Laboratory Information Management System (LIMS) for electronic inventory. 
17.3.3 Sequencing Core 
Isolated RNA will be shipped from the Substrate Services Core to the University of Texas Health 
Genome Sequencing Facility (GSF), directed by Dr. Zhao Lai. The GSF core will perform small 
RNA library preparation and sequencing. 

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34
18 ETHICAL AND REGULATORY CONSIDERATIONS 
18.1 
Ethics and Good Clinical Practice 
This study must be carried out in compliance with the protocol to ensure adherence to Good Clinical 
Practice, as described in the following documents: 
1. ICH Harmonized Tripartite Guidelines for Good Clinical Practice 1996. 
2. US 21 Code of Federal Regulations dealing with clinical studies (including parts 50 and 56 
concerning informed consent and IRB regulations). 
3. Declaration of Helsinki, concerning medical research in humans (Recommendations 
Guiding Physicians in Biomedical Research Involving Human Subjects, Helsinki 1964, 
amended Tokyo 1975, Venice 1983, Hong Kong 1989, Somerset West 1996). 
By signing the protocol, the investigator agrees to adhere to the instructions and procedures 
described in it and thereby to adhere to the principles of Good Clinical Practice to which it conforms. 
18.2 
Institutional Review Board 
Before implementing this study, the protocol, the proposed informed consent form and other 
information available to subjects, must be reviewed by a properly constituted Institutional Review 
Board (IRB). A signed and dated statement that the protocol and informed consent have been 
approved by the IRB must be provided to the CC before study initiation. Any amendments to the 
protocol, other than administrative ones, must be approved by this committee. 
18.3 
Informed Consent Procedures 
18.3.1 Informed Consent 
The Investigator or designee must explain to each subject (or legally authorized representative) the 
nature of the study, its purpose, the procedures involved, the expected duration, the potential risks 
and benefits involved and any discomfort it may entail. Each subject must be informed that 
participation in the study is voluntary and that he/she may withdraw from the study at any time and 
that withdrawal of consent will not affect his/her subsequent medical treatment or relationship with 
the treating physician. 
 
This informed consent should be given by means of a standard non-technical language. If a patient 
agrees to participate in the Vestibulodynia: Update study, they will review and sign the IRB-
approved electronic Informed Consent Form (eICF). No patient can enter the study before her 
informed consent has been obtained.  
18.3.2 Confidentiality and HIPAA Requirements 
We 
will 
obtain 
the 
same 
data 
forms 
from 
all 
participants, 
including 
vulvar 
examination/assessment, self-reports of pain and function via questionnaire, and collection of 
biologic specimens (local vaginal swabs for vaginal miRNA, vaginal lavage with aspiration for 
vaginal protein expression, and venipuncture for whole blood miRNA and protein). Methods for 
collecting and storing data will be fully described in the informed consent procedure and signed 
eICFs.  
 
Vulvar examination/assessment will be administered by the investigators or study personnel who 
have completed the ethical training for protection of human participants in research. The same 
study personnel will also administer questionnaires. Only the investigators for this study will have 
access to all the individually identifiable private information about human participants. The clinical 
sites will not be providing any direct patient identifiers when entering data into the database and 
that those identifiers will be kept securely stored at the site. Each participant will be assigned a 

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35
unique number at the time of screening that will represent their study identifier. 
 
18.3.3 Protections of Human Subjects 
All subject interactions, from initial discussion about the study to the actual study visit, will take 
place in a private setting (examination/assessment rooms) behind closed doors. Drs. Geller, 
Carey, and Rapkin may be involved as “passive” recruiters, providing information and written 
materials to patients who may be eligible to participate, but a more detailed description of the 
study and the process of obtaining informed consent will the responsibility of another member of 
the research team in order to avoid the possibility or perception of coercion. All subject data will 
be stored centrally under the purview of a designated IT specialist.  
18.3.4 Summary of the Risks and Benefits 
 
Risks. 
Anticipated risks specific to this study are outlined in Section 14.2 above. 
 
Benefits. 
Topical lidocaine and estradiol are used to manage pelvic pain disorders such as VBD, but are 
prescribed in the form of two separate creams. Compounding lidocaine and estradiol in the same 
cream would improve ease of use, promote more uniform distribution and absorption, and likely 
have added analgesic benefit. Additionally, the hydrophilic petrolatum is a nonirritating base that 
may be less likely to cause irritation compared to the typical ointment or jelly form of lidocaine. 
This compound improves ease of use and compliance. Nortriptyline is a tricyclic antidepressant 
medication that is FDA-approved to treat chronic neuropathic pain disorders (e.g., post-herpetic 
neuralgia). Nortriptyline is also a hypothesized treatment for VBD that is frequently prescribed to 
VBD participants and regularly used in the management of female pain syndromes such as VBD. 
When combined, the lidocaine/estradiol cream and nortriptyline therapies are theorized to have a 
synergistic effect. It is hoped that these interventions will provide a safe and effective therapeutic 
option for painful VBD. However, during the consent process, it will be stated that participants 
may experience no therapeutic benefit, but that their participation may inform additional research 
that helps others in the future. This research study has been designed to minimize the risks to 
participants. The risks in this study are reasonable in relation to the importance of knowledge 
gained as a result of this work. 
 
 
 
 
 
 
 
 
 
 

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19 REFERENCES 
1. 
Harlow BL and Stewart EG. A population-based assessment of chronic unexplained vulvar 
pain: have we underestimated the prevalence of vulvodynia? Journal of the American Medical 
Women's Association (1972). 2003;58:82-8. 
2. 
Lev-Sagie A and Witkin SS. Recent advances in understanding provoked vestibulodynia. 
F1000Research. 2016;5:2581. 
3. 
Xie Y, Shi L, Xiong X, Wu E, Veasley C and Dade C. Economic burden and quality of life of 
vulvodynia in the United States. Current medical research and opinion. 2012;28:601-8. 
4. 
Williams RE, Hartmann KE, Sandler RS, Miller WC and Steege JF. Prevalence and 
characteristics of irritable bowel syndrome among women with chronic pelvic pain. Obstetrics and 
gynecology. 2004;104:452-8. 
5. 
Zolnoun DA, Rohl J, Moore CG, Perinetti-Liebert C, Lamvu GM and Maixner W. Overlap 
between orofacial pain and vulvar vestibulitis syndrome. The Clinical journal of pain. 2008;24:187-
91. 
6. 
Goldstein AT, Pukall CF, Brown C, Bergeron S, Stein A and Kellogg-Spadt S. Vulvodynia: 
Assessment and Treatment. The journal of sexual medicine. 2016;13:572-90. 
7. 
Ciszek BP, Khan AA, Dang H, Slade GD, Smith S, Bair E, Maixner W, Zolnoun D and 
Nackley AG. MicroRNA expression profiles differentiate chronic pain condition subtypes. Transl 
Res. 2015;166:706-720 e11. 
8. 
Updike GM and Wiesenfeld HC. Insight into the treatment of vulvar pain: a survey of 
clinicians. Am J Obstet Gynecol. 2005;193:1404-9. 
9. 
Corbett SH, Cuddeback G, Lewis J, As-Sanie S and Zolnoun D. Trends in pharmacy 
compounding for women's health in North Carolina: focus on vulvodynia. Southern medical 
journal. 2014;107:433-6. 
10. De Andres J, Sanchis-Lopez N, Asensio-Samper JM, Fabregat-Cid G, Villanueva-Perez VL, 
Monsalve Dolz V and Minguez A. Vulvodynia--An Evidence-Based Literature Review and 
Proposed Treatment Algorithm. Pain practice : the official journal of World Institute of Pain. 
2016;16:204-36. 
11. Akopians AL and Rapkin AJ. Vulvodynia: The Role of Inflammation in the Etiology of 
Localized Provoked Pain of the Vulvar Vestibule (Vestibulodynia). Seminars in reproductive 
medicine. 2015;33:239-45. 
12. Bohm-Starke N, Hilliges M, Falconer C and Rylander E. Increased intraepithelial innervation 
in women with vulvar vestibulitis syndrome. Gynecologic and obstetric investigation. 1998;46:256-
60. 
13. Bohm-Starke N, Hilliges M, Falconer C and Rylander E. Neurochemical characterization of 
the vestibular nerves in women with vulvar vestibulitis syndrome. Gynecologic and obstetric 
investigation. 1999;48:270-5. 
14. Tympanidis P, Casula MA, Yiangou Y, Terenghi G, Dowd P and Anand P. Increased vanilloid 
receptor VR1 innervation in vulvodynia. European journal of pain. 2004;8:129-33. 
15. Tympanidis P, Terenghi G and Dowd P. Increased innervation of the vulval vestibule in 
patients with vulvodynia. The British journal of dermatology. 2003;148:1021-7. 
16. Warner TF, Tomic S and Chang CK. Neuroendocrine cell-axonal complexes in the minor 
vestibular gland. J Reprod Med. 1996;41:397-402. 
17. Giesecke T, Gracely RH, Grant MA, Nachemson A, Petzke F, Williams DA and Clauw DJ. 
Evidence of augmented central pain processing in idiopathic chronic low back pain. Arthritis and 
rheumatism. 2004;50:613-23. 
18. Gracely RH, Lynch SA and Bennett GJ. Painful neuropathy: altered central processing 
maintained dynamically by peripheral input. Pain. 1992;51:175-94. 
19. Gracely RH, Grant MA and Giesecke T. Evoked pain measures in fibromyalgia. Best Pract 
Res Clin Rheumatol. 2003;17:593-609. 
20. Reed BD, Plegue MA, Williams DA and Sen A. Presence of Spontaneous Pain and Comorbid 
Pain Conditions Identifies Vulvodynia Subgroups. Journal of lower genital tract disease. 
2016;20:57-63. 

Version date: 21Feb2024 
 
 
 
37
21. Reed BD, Sen A, Harlow SD, Haefner HK and Gracely RH. Multimodal Vulvar and Peripheral 
Sensitivity Among Women With Vulvodynia: A Case-Control Study. Journal of lower genital tract 
disease. 2017;21:78-84. 
22. Hampson JP, Reed BD, Clauw DJ, Bhavsar R, Gracely RH, Haefner HK and Harris RE. 
Augmented central pain processing in vulvodynia. J Pain. 2013;14:579-89. 
23. Woolf CJ. Central sensitization: implications for the diagnosis and treatment of pain. Pain. 
2011;152:S2-15. 
24. Goetsch MF, Morgan TK, Korcheva VB, Li H, Peters D and Leclair CM. Histologic and 
receptor analysis of primary and secondary vestibulodynia and controls: a prospective study. Am 
J Obstet Gynecol. 2010;202:614.e1-8. 
25. Bornstein J, Maman M and Abramovici H. "Primary" versus "secondary" vulvar vestibulitis: 
one disease, two variants. Am J Obstet Gynecol. 2001;184:28-31. 
26. Granot M, Friedman M, Yarnitsky D, Tamir A and Zimmer EZ. Primary and secondary vulvar 
vestibulitis syndrome: systemic pain perception and psychophysical characteristics. Am J Obstet 
Gynecol. 2004;191:138-42. 
27. Carey ET, Till SR and As-Sanie S. Pharmacological Management of Chronic Pelvic Pain in 
Women. Drugs. 2017;77:285-301. 
28. Zolnoun DA, Hartmann KE and Steege JF. Overnight 5% lidocaine ointment for treatment of 
vulvar vestibulitis. Obstetrics and gynecology. 2003;102:84-7. 
29. Butterworth JFt and Strichartz GR. Molecular mechanisms of local anesthesia: a review. 
Anesthesiology. 1990;72:711-34. 
30. Devor M. Sodium channels and mechanisms of neuropathic pain. J Pain. 2006;7:S3-s12. 
31. Bazin S, Lefebvre J, Fortier M, Brisson J, Brouillette F, Bujold E and Bouchard C. [Evaluation 
of an estrogen vaginal cream for the treatment of dyspareunia: a double-blind randomized trial]. 
Journal of obstetrics and gynaecology Canada : JOGC = Journal d'obstetrique et gynecologie du 
Canada : JOGC. 2011;33:838-43. 
32. Gardella B, Iacobone AD, Porru D, Musacchi V, Dominoni M, Tinelli C, Spinillo A and Nappi 
RE. Effect of local estrogen therapy (LET) on urinary and sexual symptoms in premenopausal 
women with interstitial cystitis/bladder pain syndrome (IC/BPS). Gynecological endocrinology : 
the official journal of the International Society of Gynecological Endocrinology. 2015;31:828-32. 
33. Bhattacherjee A, Rumi MA, Staecker H and Smith PG. Bone morphogenetic protein 4 
mediates estrogen-regulated sensory axon plasticity in the adult female reproductive tract. J 
Neurosci. 2013;33:1050-61a. 
34. Ting AY, Blacklock AD and Smith PG. Estrogen regulates vaginal sensory and autonomic 
nerve density in the rat. Biology of reproduction. 2004;71:1397-404. 
35. Walker EA, Roy-Byrne PP, Katon WJ and Jemelka R. An open trial of nortriptyline in women 
with chronic pelvic pain. International journal of psychiatry in medicine. 1991;21:245-52. 
36. Stolar AG and Stewart JT. Nortriptyline for depression and vulvodynia. The American journal 
of psychiatry. 2002;159:316-7. 
37. Brown CS, Bachmann GA, Wan J and Foster DC. Gabapentin for the Treatment of 
Vulvodynia: A Randomized Controlled Trial. Obstet Gynecol. 2018;131:1000-1007. 
38. Dworkin RH, O'Connor AB, Audette J, Baron R, Gourlay GK, Haanpaa ML, Kent JL, Krane 
EJ, Lebel AA, Levy RM, Mackey SC, Mayer J, Miaskowski C, Raja SN, Rice AS, Schmader KE, 
Stacey B, Stanos S, Treede RD, Turk DC, Walco GA and Wells CD. Recommendations for the 
pharmacological management of neuropathic pain: an overview and literature update. Mayo Clin 
Proc. 2010;85:S3-14. 
39. Millan MJ. Descending control of pain. Prog Neurobiol. 2002;66:355-474. 
40. Dick IE, Brochu RM, Purohit Y, Kaczorowski GJ, Martin WJ and Priest BT. Sodium channel 
blockade may contribute to the analgesic efficacy of antidepressants. J Pain. 2007;8:315-24. 
41. Obuchowicz E, Kowalski J, Labuzek K, Krysiak R, Pendzich J and Herman ZS. Amitriptyline 
and nortriptyline inhibit interleukin-1 release by rat mixed glial and microglial cell cultures. Int J 
Neuropsychopharmacol. 2006;9:27-35. 
42. Desrochers G, Bergeron S, Landry T and Jodoin M. Do psychosexual factors play a role in 
the etiology of provoked vestibulodynia? A critical review. Journal of sex & marital therapy. 
2008;34:198-226. 

Version date: 21Feb2024 
 
 
 
38
43. van Lankveld JJ, Granot M, Weijmar Schultz WC, Binik YM, Wesselmann U, Pukall CF, 
Bohm-Starke N and Achtrari C. Women's sexual pain disorders. The journal of sexual medicine. 
2010;7:615-31. 
44. Chisari C and Chilcot J. The experience of pain severity and pain interference in vulvodynia 
patients: The role of cognitive-behavioural factors, psychological distress and fatigue. Journal of 
psychosomatic research. 2017;93:83-89. 
45. Zolnoun D, Hartmann K, Lamvu G, As-Sanie S, Maixner W and Steege J. A conceptual 
model for the pathophysiology of vulvar vestibulitis syndrome. Obstetrical & gynecological survey. 
2006;61:395-401; quiz 423. 
46. Talley SJ, Bytzer P, Hammer J, Young L, Jones M and Horowitz M. Psychological distress 
is linked to gastrointestinal symptoms in diabetes mellitus. The American journal of 
gastroenterology. 2001;96:1033-8. 
47. Miller RJ, Jung H, Bhangoo SK and White FA. Cytokine and chemokine regulation of sensory 
neuron function. Handb Exp Pharmacol. 2009:417-49. 
48. Dinarello CA, Kluger MJ, Oppenheim JJ and Powanda MC. The physiological and 
pathological effects of cytokines. New York: Liss/John Wiley & Sons Inc; 1990. 
49. Kress MaS, C. Neuroimmunology and Pain: Peripheral Effects of Proinflammatory 
Cytokines. In: K. a. H. Brune, H.O., ed. Hyperalgesia: Molecular Mechanisms and Clinical 
Implications, Progress in Pain Research Management Seattle: IASP Press; 2004(30): 57-65. 
50. Hartung JE, Ciszek BP and Nackley AG. beta2- and beta3-adrenergic receptors drive 
COMT-dependent pain by increasing production of nitric oxide and cytokines. Pain. 
2014;155:1346-55. 
51. Gur A, Karakoc M, Nas K, Remzi, Cevik, Denli A and Sarac J. Cytokines and depression in 
cases with fibromyalgia. J Rheumatol. 2002;29:358-61. 
52. Kopp S. The influence of neuropeptides, serotonin, and interleukin 1beta on 
temporomandibular joint pain and inflammation. J Oral Maxillofac Surg. 1998;56:189-91. 
53. Ogura N, Satoh K, Akutsu M, Tobe M, Kuyama K, Kuboyama N, Sakamaki H, Kujiraoka H 
and Kondoh T. MCP-1 production in temporomandibular joint inflammation. J Dent Res. 
2010;89:1117-22. 
54. Shafer DM, Assael L, White LB and Rossomando EF. Tumor necrosis factor-alpha as a 
biochemical marker of pain and outcome in temporomandibular joints with internal derangements. 
J Oral Maxillofac Surg. 1994;52:786-91; discussion 791-2. 
55. Takahashi T, Kondoh T, Fukuda M, Yamazaki Y, Toyosaki T and Suzuki R. Proinflammatory 
cytokines detectable in synovial fluids from patients with temporomandibular disorders. Oral Surg 
Oral Med Oral Pathol Oral Radiol Endod. 1998;85:135-41. 
56. Maes M, Song C, Lin A, De Jongh R, Van Gastel A, Kenis G, Bosmans E, De Meester I, 
Benoy I, Neels H, Demedts P, Janca A, Scharpe S and Smith RS. The effects of psychological 
stress on humans: increased production of pro-inflammatory cytokines and a Th1-like response 
in stress-induced anxiety. Cytokine. 1998;10:313-8. 
57. Maes M. Evidence for an immune response in major depression: a review and hypothesis. 
Prog Neuropsychopharmacol Biol Psychiatry. 1995;19:11-38. 
58. Maes M. Major depression and activation of the inflammatory response system. Adv Exp 
Med Biol. 1999;461:25-46. 
59. Foster DC and Hasday JD. Elevated tissue levels of interleukin-1 beta and tumor necrosis 
factor-alpha in vulvar vestibulitis. Obstet Gynecol. 1997;89:291-6. 
60. Foster DC, Piekarz KH, Murant TI, LaPoint R, Haidaris CG and Phipps RP. Enhanced 
synthesis of proinflammatory cytokines by vulvar vestibular fibroblasts: implications for vulvar 
vestibulitis. Am J Obstet Gynecol. 2007;196:346.e1-8. 
61. Gerber S, Bongiovanni AM, Ledger WJ and Witkin SS. Defective regulation of the 
proinflammatory immune response in women with vulvar vestibulitis syndrome. Am J Obstet 
Gynecol. 2002;186:696-700. 
62. Baker DA, Peresleni T and Kocis C. Inflammatory Markers in Vestibulodynia [4]. Obstet 
Gynecol. 2016;127 Suppl 1:1s-2s. 
63. Ventolini G, Gygax SE, Adelson ME and Cool DR. Vulvodynia and fungal association: a 
preliminary report. Medical hypotheses. 2013;81:228-30. 

Version date: 21Feb2024 
 
 
 
39
64. Davis AJ and Perkins MN. The involvement of bradykinin B1 and B2 receptor mechanisms 
in cytokine-induced mechanical hyperalgesia in the rat. Br J Pharmacol. 1994;113:63-8. 
65. Schwarz YA, Amin RS, Stark JM, Trapnell BC and Wilmott RW. Interleukin-1 receptor 
antagonist inhibits interleukin-8 expression in A549 respiratory epithelial cells infected in vitro with 
a replication-deficient recombinant adenovirus vector. Am J Respir Cell Mol Biol. 1999;21:388-
94. 
66. Gabay C, Lamacchia C and Palmer G. IL-1 pathways in inflammation and human diseases. 
Nature reviews Rheumatology. 2010;6:232-41. 
67. Friedman JM and Jones PA. MicroRNAs: critical mediators of differentiation, development 
and disease. Swiss Med Wkly. 2009;139:466-72. 
68. Ajit SK. Circulating microRNAs as biomarkers, therapeutic targets, and signaling molecules. 
Sensors (Basel). 2012;12:3359-69. 
69. Brandenburger T, Castoldi M, Brendel M, Grievink H, Schlosser L, Werdehausen R, Bauer I 
and Hermanns H. Expression of spinal cord microRNAs in a rat model of chronic neuropathic 
pain. Neurosci Lett. 2012;506:281-6. 
70. Chattopadhyay M, Zhou Z, Hao S, Mata M and Fink DJ. Reduction of voltage gated sodium 
channel protein in DRG by vector mediated miRNA reduces pain in rats with painful diabetic 
neuropathy. Mol Pain. 2012;8:17. 
71. Kusuda R, Cadetti F, Ravanelli MI, Sousa TA, Zanon S, De Lucca FL and Lucas G. 
Differential expression of microRNAs in mouse pain models. Mol Pain. 2011;7:17. 
72. Orlova IA, Alexander GM, Qureshi RA, Sacan A, Graziano A, Barrett JE, Schwartzman RJ 
and Ajit SK. MicroRNA modulation in complex regional pain syndrome. Journal of translational 
medicine. 2011;9:195. 
73. Pauley KM, Cha S and Chan EK. MicroRNA in autoimmunity and autoimmune diseases. J 
Autoimmun. 2009;32:189-94. 
74. Saba R, Gushue S, Huzarewich RL, Manguiat K, Medina S, Robertson C and Booth SA. 
MicroRNA 146a (miR-146a) is over-expressed during prion disease and modulates the innate 
immune response and the microglial activation state. PLoS One. 2012;7:e30832. 
75. Saba R, Storchel PH, Aksoy-Aksel A, Kepura F, Lippi G, Plant TD and Schratt GM. 
Dopamine-regulated microRNA MiR-181a controls GluA2 surface expression in hippocampal 
neurons. Mol Cell Biol. 2012;32:619-32. 
76. Willemen HL, Huo XJ, Mao-Ying QL, Zijlstra J, Heijnen CJ and Kavelaars A. MicroRNA-124 
as a novel treatment for persistent hyperalgesia. J Neuroinflammation. 2012;9:143. 
77. Gheinani AH, Burkhard FC and Monastyrskaya K. Deciphering microRNA code in pain and 
inflammation: lessons from bladder pain syndrome. Cellular and molecular life sciences : CMLS. 
2013;70:3773-89. 
78. Nothnick WB. MicroRNAs and Endometriosis: Distinguishing Drivers from Passengers in 
Disease Pathogenesis. Seminars in reproductive medicine. 2017;35:173-180. 
79. Haj-Ahmad TA, Abdalla MA and Haj-Ahmad Y. Potential Urinary miRNA Biomarker 
Candidates for the Accurate Detection of Prostate Cancer among Benign Prostatic Hyperplasia 
Patients. Journal of Cancer. 2014;5:182-91. 
80. Ito T, Sato F, Kan T, Cheng Y, David S, Agarwal R, Paun BC, Jin Z, Olaru AV, Hamilton JP, 
Selaru FM, Yang J, Matsumura N, Shimizu K, Abraham JM, Shimada Y, Mori Y and Meltzer SJ. 
Polo-like kinase 1 regulates cell proliferation and is targeted by miR-593* in esophageal cancer. 
International journal of cancer Journal international du cancer. 2011;129:2134-46. 
81. Shigehara K, Yokomuro S, Ishibashi O, Mizuguchi Y, Arima Y, Kawahigashi Y, Kanda T, 
Akagi I, Tajiri T, Yoshida H, Takizawa T and Uchida E. Real-time PCR-based analysis of the 
human bile microRNAome identifies miR-9 as a potential diagnostic biomarker for biliary tract 
cancer. PloS one. 2011;6:e23584. 
82. Pereira PM, Marques JP, Soares AR, Carreto L and Santos MA. MicroRNA expression 
variability in human cervical tissues. PloS one. 2010;5:e11780. 
83. Ninio-Many L, Grossman H, Levi M, Zilber S, Tsarfaty I, Shomron N, Tuvar A, Chuderland 
D, Stemmer SM, Ben-Aharon I and Shalgi R. MicroRNA miR-125a-3p modulates molecular 
pathway of motility and migration in prostate cancer cells. Oncoscience. 2014;1:250-61. 
84. Vetter G, Saumet A, Moes M, Vallar L, Le Bechec A, Laurini C, Sabbah M, Arar K, Theillet 
C, Lecellier CH and Friederich E. miR-661 expression in SNAI1-induced epithelial to 

Version date: 21Feb2024 
 
 
 
40
mesenchymal transition contributes to breast cancer cell invasion by targeting Nectin-1 and 
StarD10 messengers. Oncogene. 2010;29:4436-48. 
85. Lam WY, Yeung AC, Ngai KL, Li MS, To KF, Tsui SK and Chan PK. Effect of avian influenza 
A H5N1 infection on the expression of microRNA-141 in human respiratory epithelial cells. BMC 
microbiology. 2013;13:104. 
86. Hewagama A, Gorelik G, Patel D, Liyanarachchi P, McCune WJ, Somers E, Gonzalez-
Rivera T, Michigan Lupus C, Strickland F and Richardson B. Overexpression of X-linked genes 
in T cells from women with lupus. Journal of autoimmunity. 2013;41:60-71. 
87. Dong Y, Li P, Ni Y, Zhao J and Liu Z. Decreased microRNA-125a-3p contributes to 
upregulation of p38 MAPK in rat trigeminal ganglions with orofacial inflammatory pain. PloS one. 
2014;9:e111594. 
88. Keklikoglou I, Koerner C, Schmidt C, Zhang JD, Heckmann D, Shavinskaya A, Allgayer H, 
Guckel B, Fehm T, Schneeweiss A, Sahin O, Wiemann S and Tschulena U. MicroRNA-520/373 
family functions as a tumor suppressor in estrogen receptor negative breast cancer by targeting 
NF-kappaB and TGF-beta signaling pathways. Oncogene. 2012;31:4150-63. 
89. Vlachos IS, Kostoulas N, Vergoulis T, Georgakilas G, Reczko M, Maragkakis M, 
Paraskevopoulou MD, Prionidis K, Dalamagas T and Hatzigeorgiou AG. DIANA miRPath v.2.0: 
investigating the combinatorial effect of microRNAs in pathways. Nucleic acids research. 
2012;40:W498-504. 
90. Practice Committee of the American Society for Reproductive M. Treatment of pelvic pain 
associated with endometriosis. Fertility and sterility. 2006;86:S18-27. 
91. Tsuda M, Toyomitsu E, Komatsu T, Masuda T, Kunifusa E, Nasu-Tada K, Koizumi S, 
Yamamoto K, Ando J and Inoue K. Fibronectin/integrin system is involved in P2X(4) receptor 
upregulation in the spinal cord and neuropathic pain after nerve injury. Glia. 2008;56:579-85. 
92. Saito F, Moore SA, Barresi R, Henry MD, Messing A, Ross-Barta SE, Cohn RD, Williamson 
RA, Sluka KA, Sherman DL, Brophy PJ, Schmelzer JD, Low PA, Wrabetz L, Feltri ML and 
Campbell KP. Unique role of dystroglycan in peripheral nerve myelination, nodal structure, and 
sodium channel stabilization. Neuron. 2003;38:747-58. 
93. Institute of Medicine Committee on Advancing Pain Research C and Education. The National 
Academies Collection: Reports funded by National Institutes of Health Relieving Pain in America: 
A Blueprint for Transforming Prevention, Care, Education, and Research Washington (DC): 
National Academies Press (US) 
National Academy of Sciences.; 2011. 
94. Atzeni F, Salaffi F, Bazzichi L, Gracely RH, Carignola R, Torta R, Gorla R, Marsico A, 
Ceccherelli F, Cazzola M, Buskila D, Spath M, Di Franco M, Biasi G, Cassisi G, Stisi S, Casale 
R, Altomonte L, Arioli G, Alciati A, Leardini G, Marinangeli F, Giamberardino MA and Sarzi-Puttini 
P. The evaluation of the fibromyalgia patients. Reumatismo. 2008;60 Suppl 1:36-49. 
95. Turk DC, Dworkin RH, Allen RR, Bellamy N, Brandenburg N, Carr DB, Cleeland C, Dionne 
R, Farrar JT, Galer BS, Hewitt DJ, Jadad AR, Katz NP, Kramer LD, Manning DC, McCormick CG, 
McDermott MP, McGrath P, Quessy S, Rappaport BA, Robinson JP, Royal MA, Simon L, Stauffer 
JW, Stein W, Tollett J and Witter J. Core outcome domains for chronic pain clinical trials: 
IMMPACT recommendations. Pain. 2003;106:337-45. 
96. Foster DC, Kotok MB, Huang LS, Watts A, Oakes D, Howard FM, Stodgell CJ and Dworkin 
RH. The tampon test for vulvodynia treatment outcomes research: reliability, construct validity, 
and responsiveness. Obstet Gynecol. 2009;113:825-32. 
97. Slade GD, Conrad MS, Diatchenko L, Rashid NU, Zhong S, Smith S, Rhodes J, Medvedev 
A, Makarov S, Maixner W and Nackley AG. Cytokine biomarkers and chronic pain: association of 
genes, transcription, and circulating proteins with temporomandibular disorders and widespread 
palpation tenderness. Pain. 2011;152:2802-12. 
98. Murina F, Bianco V, Radici G, Felice R, Di Martino M and Nicolini U. Transcutaneous 
electrical nerve stimulation to treat vestibulodynia: a randomised controlled trial. BJOG : an 
international journal of obstetrics and gynaecology. 2008;115:1165-70. 
99. McDonald JS and Rapkin AJ. Multilevel local anesthetic nerve blockade for the treatment of 
generalized vulvodynia: a pilot study. The journal of sexual medicine. 2012;9:2919-26. 

Version date: 21Feb2024 
 
 
 
41
100. Meltzer-Brody SE, Zolnoun D, Steege JF, Rinaldi KL and Leserman J. Open-label trial of 
lamotrigine focusing on efficacy in vulvodynia. J Reprod Med. 2009;54:171-8. 
101. Cyr MP, Bourbonnais D, Pinard A, Dubois O and Morin M. Reliability and Convergent Validity 
of the Algometer for Vestibular Pain Assessment in Women with Provoked Vestibulodynia. Pain 
medicine (Malden, Mass). 2016;17:1220-8. 
102. Zolnoun D, Bair E, Essick G, Gracely R, Goyal V and Maixner W. Reliability and 
reproducibility of novel methodology for assessment of pressure pain sensitivity in pelvis. The 
journal of pain : official journal of the American Pain Society. 2012;13:910-20. 
103. Giesecke J, Reed BD, Haefner HK, Giesecke T, Clauw DJ and Gracely RH. Quantitative 
sensory testing in vulvodynia patients and increased peripheral pressure pain sensitivity. Obstet 
Gynecol. 2004;104:126-33. 
104. Reed BD, Caron AM, Gorenflo DW and Haefner HK. Treatment of vulvodynia with tricyclic 
antidepressants: efficacy and associated factors. Journal of lower genital tract disease. 
2006;10:245-51. 
105. Salaffi F, Stancati A, Silvestri CA, Ciapetti A and Grassi W. Minimal clinically important 
changes in chronic musculoskeletal pain intensity measured on a numerical rating scale. Eur J 
Pain. 2004;8:283-91. 
106. Chelimsky-Fallick C, Stevenson LW, Lem V and Whipp BJ. Excessive oxygen deficit during 
low-level exercise in heart failure. The American journal of cardiology. 1995;76:799-802. 
107. Sietsema KE, Ben-Dov I, Zhang YY, Sullivan C and Wasserman K. Dynamics of oxygen 
uptake for submaximal exercise and recovery in patients with chronic heart failure. Chest. 
1994;105:1693-700. 
108. Harris RC, Edwards RH, Hultman E, Nordesjo LO, Nylind B and Sahlin K. The time course 
of phosphorylcreatine resynthesis during recovery of the quadriceps muscle in man. Pflugers 
Arch. 1976;367:137-42. 
109. Lucas C, Stevenson LW, Johnson W, Hartley H, Hamilton MA, Walden J, Lem V and Eagen-
Bengsten E. The 6-min walk and peak oxygen consumption in advanced heart failure: aerobic 
capacity and survival. American heart journal. 1999;138:618-24.

Version date: 21Feb2024 
 
 
 
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20 APPENDICES 
Appendix A. Schedule of Assessments 
Assessment 
Screening
(0 week)
Baseline Visit 
(0 week)
Treatment Visit 
(8 week)*
Treatment Visit 
(16 week)**
Post-treatment 
(24 week)***
Patient Consent 
XFS 
 
Tampon Test  
XFS 
 
X 
X 
X 
Pain with sexual contact 
involving penetration into 
the vagina
X 
 
X 
X 
X 
Pain with touch to the 
vaginal opening
X 
 
X 
X 
X 
Blood Collection 
 
XFS 
X 
X 
X 
Demographics  
 
XFS 
Social History  
 
XFS 
Gynecologic History  
 
XFS 
VPAQ  
 
XFS 
X 
X 
X 
PROMIS BPSFS-Female 
 
XFS 
X 
X 
X 
COPC Inventory 
 
XFS 
COPC Follow-Up 
 
X 
X 
X 
SF-12v2 
 
XFS 
X 
X 
X 
SCL-27   
 
XFS 
X 
X 
X 
PILL 
 
XFS 
X 
X 
X 
PSS 
 
XFS 
X 
X 
X 
Sleep Scale 
 
XFS 
X 
X 
X 
Vaginal Sample Collection 
 
XFS 
X 
X 
X 
Pelvic Sensory Testing 
 
XFS 
X 
X 
X 
Vaginal Vestibule PPTs 
 
XFS 
X 
X 
X 
Perineal Muscle PPTs 
 
XFS 
X 
X 
X 
Levator Muscle PPTs 
 
XFS 
X 
X 
X 
Remote Bodily PPTs  
 
XFS 
X 
X 
X 
Medication Compliance 
 
 
X 
X 
 
FS = assessments collected for the feasibility study 
X = assessments collected for the full study 
*8 weeks (56 days) following randomization, +/- 5 days 
**16 weeks (112 days) following randomization, +/- 5 days 
***24 weeks (168 days) following randomization, +/- 5 days 
 
 
 
 
 

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Appendix B. Lidocaine/Estradiol Application Handout 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vulvar Vestibule 

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Appendix C. Nortriptyline Medication Handout 
 
Severe Drug-drug Interactions with Nortriptyline 
 
Drug interactions may change how your medications work or increase your risk for serious side 
effects. This document does not contain all possible drug interactions, but it highlights the most 
serious. Keep a list of all the products you use (including prescription/nonprescription drugs and 
herbal products) and share it with the research team. 
 
Taking MAO inhibitors with this medication may cause a serious (possibly fatal) drug interaction. 
Avoid taking MAO inhibitors for two weeks before and after nortriptyline use: 
 
Isocarboxazid 
 
Linezolid 
 
Methylene blue 
 
Moclobemide 
 
Phenelzine 
 
Procarbazine 
 
Rasagiline 
 
Safinamide 
 
Selegiline 
 
Tranylcypromine 
 
The risk of serotonin syndrome/toxicity increases if you are also taking other drugs that increase 
serotonin and may be more likely to occur when you start or increase the drug dose:  
 
MDMA/"ecstasy” 
 
St. John's wort 
 
Antidepressants- specifically SSRIs and SNRIs 
 
Other tricyclic antidepressants- amitriptyline, desipramine, imipramine 
 
Topiramate, cyclobenzaprine 
 
Avoid combination of drugs that affect the heart rhythm, specifically causing QT prolongation on 
EKG: 
 
Amiodarone 
 
Cisapride 
 
Dofetilide 
 
Sotalol 
 
Pimozide 
 
Procainamide 
 
Quinidine 
 
Macrolide antibiotics- i.e. erythromycin 
Other drugs to avoid while taking nortriptyline: 
 
Arbutamine 
 
Disulfiram 
 
Anticholinergic drugs such as 
benztropine, belladonna  
 
Cimetidine 
 
Blood thinners such as warfarin  
 
Thyroid supplements 
 
Certain drugs for high blood pressure- 
clonidine, guanabenz 
 
Terbinafine 
 
Tell your research team if you are taking other products that cause drowsiness, including alcohol, 
marijuana, antihistamines, drugs for sleep or anxiety (such as alprazolam, diazepam, zolpidem), 
muscle relaxants, or narcotic pain relievers. 
 
Check the labels on all your medicines (such as allergy or cough-and-cold products) because 
they may contain decongestants or ingredients that cause drowsiness.  
 
 
 
 

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Appendix D. Medication Calendar