Deciphering the Hormonal and Nociceptive Mechanisms Underlying Bladder Pain
Phase: PHASE4
Conditions: Cystitis, Interstitial, Dysmenorrhea, Migraine Disorders, Pelvic Pain, Endometriosis, Visceral Pain, Chronic Pain
Sponsor: Endeavor Health

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Chronic Pain Risk Associated With Menstrual Period Pain 
NCT02214550 
Protocol version 11/14/2016 (Revision #9, approved 2/3/2017) 
note extraneous material and internally relevant nomenclature has been edited from this publicly posted 
version 
Today’s date 7/20/2022 

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Study Protocol #9 (adult version): Deciphering the hormonal and 
nociceptive mechanisms underlying bladder pain DK100368 
Public study title: Chronic Pain Risk Associated with Menstrual Period Pain 
A. Specific Aims and Hypotheses: 
Endometrial shedding during the menstrual cycle elicits profound changes in neuronal activity 
and cytokines producing moderate to severe pelvic pain in more than 20% of reproductive-age 
women.1 Among these women, one out of every five develops chronic pelvic pain (CPP). In 
contrast, women without dysmenorrhea rarely develop CPP. Furthermore, half of women 
reporting moderate-severe dysmenorrhea exhibit bladder pain at innocuous levels of bladder 
filling.2 We have also shown that dysmenorrhea is a leading correlate of severe non-menstrual 
pelvic pain independent of psychological factors.3 Identifying the mechanism responsible for the 
transition from dysmenorrhea to CPP could allow the development of preventative strategies. 
We hypothesize the mechanism by which dysmenorrhea may lead to pelvic pain is cross-organ 
sensitization (COS). The uterine inflammation during menstruation likely contributes to CPP by 
triggering neurogenic inflammation in adjacent organs.4 This COS has been demonstrated with 
experimental injury and infection in animal models.5,6 Our prior findings discussed above2,3 
motivate our present central hypothesis: dysmenorrhea produces CPP via repetitive COS 
episodes. 
Although COS is believed to play a role in CPP, other studies of visceral pain7,8 and PBS9 have 
shown increases in visceral sensitivity involve impairments in descending inhibition, the normal 
counterbalancing outflow to pain-processing spinal neurons from the brainstem.10 Also, since β-
estradiol (E2) is elevated in dysmenorrhea11–14, and increased E2 can worsen pain 
sensitivity15,16, alterations in E2 could also contribute to increased prevalence of CPP in women 
with dysmenorrhea. A major limitation of prior studies is that they do not address the 
relationship between COS and impairments to descending inhibition. They also do not consider 
important covariates such as E2 levels and psychological factors (e.g., anxiety/depression).17  
To determine the role of neurophysiological mechanisms in COS, while adjusting for these 
important covariates, we will characterize a novel phenotype of dysmenorrhea with bladder pain 
indicative of COS (D+COS). In our preliminary studies, women with the D+COS phenotype 
exhibited prolonged pelvic pain report in response to a mechanical vaginal stimulus. This 
hyperalgesia is common among CPP patients. However, in contrast to women with CPP, 
women with D+COS do not show significant psychological dysfunction. These findings imply 
dysmenorrhea, independent of psychological factors, influences vulnerability to pelvic pain. 
Additionally, our preliminary data and work by others show women with dysmenorrhea18,19 have 
widespread reductions in pain thresholds, the hallmark of impaired descending inhibition also 
observed in IBS.20,21 However, specific measures of descending inhibition have never been 
assessed in women with dysmenorrhea. To address this gap, we propose to conduct cross-
sectional and prospective evaluations of pelvic nociception, reproductive hormones and 
psychological factors among women with dysmenorrhea. In this proposal, we will test the 
hypothesis that repeated episodes of menstrual pain increase vulnerability to COS.  
COS may be reversible under ideal circumstances. Hormonal treatment of dysmenorrhea has 
already been shown to partially alleviate co-morbid irritable bowel syndrome (IBS) and 

 
3 
 
abdominal 
sensitivity22, and 
improve painful 
bladder syndrome 
(PBS).23 Our 
preliminary, cross-
sectional data in 
dysmenorrhea 
sufferers suggest 
combined (estrogen 
and progestin) oral 
contraceptive (OC) 
users report less 
bladder pain at 
innocuous filling 
volumes. We would 
like to investigate 
further whether this 
is a modifiable 
phenotype. An equally critical issue involves identifying which patients would respond best to 
hormonal treatment. Previous studies have yielded conflicting results on whether OCs improve 
pain modulation.24,25 These studies did not address dysmenorrhea or the usage of continuous 
OCs, which typically ablate withdrawal bleeding and withdrawal bleed-induced uterine nerve 
activation. We hypothesize eliminating a painful stimulus with cyclical OCs will improve 
descending inhibition and reduce COS.  (see Diagram 1) 
 
To determine whether alterations in pain are due to changes in descending inhibition, spinal 
cord excitability, or peripheral sensitivity we will use established tests for quantitative sensory 
testing. Changes in descending inhibition will be tested with conditioned pain modulation (CPM). 
26  CPM involves presenting a counter-stimulus (such as cold water) to one part of the body and 
then simultaneously measuring the differential sensory response to a noxious stimulus 
elsewhere. The degree of descending inhibition is quantified by comparing the degree of 
blunted response from a counter-stimulus compared to the unmatched paradigm. Changes in 
spinal cord excitability will be tested with temporal summation.27 Temporal summation involves 
evaluating pain report after repetitive stimulation of pain pathway sufficient to induce increased 
excitability of dorsal horn neurons.  Changes in in peripheral sensitivity will be evaluated with 
mechanical pressure testing.  We will perform these tests across multiple body sites to 
determine whether changes in pain mechanisms are regional or body-wide. Performing these 
tests will allow us to determine whether eliminating a painful stimulus with cyclical OCs 
improves descending inhibition, spinal excitability, or overall peripheral sensitivity, which will be 
done in the Phase 2 protocol. The addition of EEG and EMG recordings will also allow us to 
determine brain and spinal mechanisms that affect nociceptive modulation. 
If OCs are shown to improve descending inhibition, our results will extend prior findings in 
animals that demonstrate descending inhibition is needed to maintain chronic pain states.10 In 
humans, descending inhibition is impaired by repetitive pain insults28,29 but normalizes with 
successful interventions.30 Additionally, our results will establish a relationship between 
descending inhibition and COS allowing for the development of preventative strategies.31  
Since endometriosis and fibroids are identifiable anatomical conditions but understanding of 
their mechanistic contribution to dysmenorrhea and bladder pain is limited, we will include 
confirmed endometriosis participants and fibroids participants mixed within our dysmenorrhea  
Diagram 1: We hypothesize repeated episodes of menstrual pain could 
impair descending inhibition of the spinal cord increasing widespread pain 
sensitivity. Additional local sensitization in the presence of worsening of 
descending inhibition may result in COS, provoked pelvic pain and 
bladder hyperalgesia. Elevated concentration of E2 in dysmenorrhea (not 
shown) could also increase bladder pain. 

 
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cohort to determine which effects produced by the primary analysis are due to anatomical 
conditions.  
 
Participants are divided into two arms. Arm #1 participants include women ages 18-45 
with dysmenorrhea, women with painful bladder syndrome (PBS), and healthy controls. 
Arm #2 includes women ages 18-45 with chronic pain or special healthy controls.  
 
Our hypotheses on mechanisms responsible for the transition of menstrual pain to bladder pain 
will be tested through the following phases:  
 
Phase #1 (includes Arm #1 and Arm #2 participants): To determine if dysmenorrhea with 
concomitant bladder pain sensitivity exhibits neurophysiological features consistent with 
established CPP. Women with dysmenorrhea without COS and women with chronic pain 
conditions outside of the pelvic region will be used as controls. Quantitative sensory testing 
(QST) and a noninvasive bladder pain test we previously have validated will be used to 
determine whether impairments in descending inhibition and pelvic sensitivity are responsible 
for vulnerability to COS in women with dysmenorrhea (with and without COS) and/or CPP. 
 
Phase #2 (includes a subset of Arm #1 participants only): To differentiate the individual 
contributions of circulating sex hormones and repeated sensitizing events (painful menses) on 
descending and peripheral mechanisms of bladder pain. The same QST/bladder pain measures 
studied in Phase #1 will be retested within the D+COS and PBS subjects following a one-year 
randomized trial of cyclical vs. continuous OCs vs. no treatment. We added a second option for 
those who are randomized to Phase 2 that did not want to commit to their randomized group. 
They will be asked to come in for an additional assessment visit a year later. Participating in the 
randomized group is preferable but this will allow us to still obtain the follow up data that is 
important with these participants even if they choose not to participate in their assigned group. 
Determining the neurophysiological relationship between repeated uterine inflammation, 
dysmenorrhea, and COS, and how OCs might reverse that influence, could define a new 
pathway for understanding the etiology of CPP/PBS. Secondly, identifying early markers of risk 
could ultimately underlie an effective prevention strategy because the onset of PBS is often 
gradual with limited symptoms.32 
 
B. Research Design and Methods 
B.1. Overview: 
Diagram 2: After screening and washout, dysmenorrhea sufferers with and without COS, PBS 
patients, and chronic pain participants (with and without pelvic pain conditions) will be compared 
with controls on experimental pain sensitivity tests (PPT), which measure descending inhibition 
(CPM) and pelvic and bladder sensitivity (Phase #1). Women with dysmenorrhea with COS and 
PBS will be asked to participate in Phase #2. In Phase #2, a trial of cyclic and continuous OCs 
Screening 
& consent 
1-4  months 
washout
Phase 1 initial testing 
• Bladder filling 
• PPT, CPM 
• Hormones 
Phase 2 6 
- mth test 
•Bladder filling 
• PPT, CPM 
• Hormones 
• Randomize into 
contraceptive trial 
• Daily diaries 
 Continue 
contraceptives 
•  
diaries 
Phase 2 12
- 
mth test 
• Bladder filling 
• PPT, CPM 
• Hormones 
6 months 
6 months 
• Daily diaries 
Diagram 2: Timeline for subject in (Phase 2 is not relevant to all groups 

 
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will be used to differentiate the individual contributions of circulating sex hormones and repeated 
sensitizing events (painful menstrual periods) on descending and peripheral mechanisms of 
bladder pain. All participants will also complete a yearly follow-up questionnaire for 5 years (for 
participants who complete phase 2, the year 1 questionnaire will be incorporated into the 12-
month testing Assessment #3 testing session). 
This study will enroll a total of approximately 1,080 participants (See Appendix 1). Of those 
subjects, all will be enrolled at NorthShore University HealthSystem (NorthShore). This leaves a 
total of approximately 1,080 participants to be enrolled in arm #1 (1,020) and arm #2 (60) of the 
study. We anticipate that some arm 1 and arm 2 participants (up to 415 arm 1 and 10 arm 2 
participants) may become ineligible after the screen visit and/or daily diary data because their 
pain levels do not clearly place them into a defined group, they are enrolled as a dysmenorrhea 
participant but do not show evidence of COS, medical reasons are identified at the screen visit 
that were not identified during the phone screen, or they become lost to follow-up between the 
screen and assessment visit. We expect approximately 645 arm 1 and arm 2 participants will 
continue the study and complete Phase 1; 515 will be women with dysmenorrhea, 40 will be 
women with PBS,  40 will be healthy women without pelvic pain or painful periods, 7 will be 
“special” healthy controls, and 43 will be women with chronic pain.   
Chronic pain participants will be allowed to skip portions of the assessment based on their pain 
conditions and will be enrolled using drastically different inclusion/exclusion criteria compared to 
other groups. We anticipate approximately 88 women with either dysmenorrhea or PBS to 
continue participation in the longitudinal study and trial of oral contraceptives for Phase 2. We 
expect approximately 50 women who were randomized will decide not to participate in phase 2 
but will choose to come back  in a year later for an additional assessment visit for long term 
follow up data. Our ultimate goal is to prevent and reverse COS. We will ask all subjects to 
complete an at home yearly follow-up questionnaire for 5 years (for participants who complete 
phase 2, the year 1 questionnaire will be incorporated into the 12-month testing Assessment #3 
testing session). If our hypotheses are correct about descending inhibition impairments being 
reversible with OCs, our work will spawn prevention studies for other highly co-morbid 
conditions associated with dysmenorrhea such as headache and fibromyalgia. 
 
C1: Phase 1 Methods 
 
C1.a Study Participants: Reproductive-age women (age 18-45) with: ARM 1: a) 
dysmenorrhea, b) dysmenorrhea and bladder sensitivity (D+COS), c) painful bladder syndrome 
(PBS), d) healthy, pain-free controls, ARM 2: e) 10 “special” healthy control participants, and f) 
50 chronic pain female patients. 
 
Participants will primarily be recruited from NorthShore’s clinical sites in the Chicago-area. 
Participants may also be recruited from the community through advertising in NorthShore 
University HealthSystem’s outpatient clinics; web-based advertising; posters and flyers in local 
businesses (coffee shops, athletic clubs) and on college campuses; referrals from local 
gynecologists; and the Illinois Women’s Health Registry based at Northwestern University’s 
Feinberg School of Medicine. All flyers and ads will be IRB approved. In our ads, we will call the 
study CRAMPP (Chronic Risk Associated with Menstrual Pelvic Pain).  Participants may call our 
research hotline directly or after they indicate interest and give permission, their doctor will send 
us their contact information and a research coordinator will call the patient to formally assess 
eligibility using a phone screen. At the end of the phone screen, adult participants will be asked 
if we have permission to put their phone screen and contact information into our recruitment 
registry. 

 
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A research coordinator may periodically screen NorthShore’s electronic medical records 
databases (EPIC) for patients within the NorthShore Medical Group system meeting eligibility 
criteria via a data warehouse request. This process will include the following: 
 
1. Participant identification: Electronic databases will be queried for outpatient encounters 
using the ICD-9 codes associated with pelvic pain symptoms: endometriosis [617.x], 
uterine leiomyoma [218.x], dysmenorrhea [625.3], unspecified symptoms associated 
with pelvic organs [625.9], premenstrual tension syndromes [625.4], and other disorders 
of menstruation and abnormal bleeding from the female genital tract [626.8]. Bladder 
pain participants will be queried from: other symptoms involving the urinary system 
[788.99], dysuria [788.1], and interstitial cystitis [595.1].  
 
2. The physicians of eligible women will be asked to ask the patient if they might be 
interested (via phone, letter, or in person during an appointment) and if we have 
permission to contact them (via phone, letter, or in person during an appointment). The 
physician will then notify us. Alternatively, the physician can just give the patient our 
contact information for THEM to call US if they are interested.  
  
Only girls and women will be recruited because the study addresses dysmenorrhea and chronic 
pelvic pain, conditions unique to females. Notably, bladder pain syndrome also predominately 
affects women.   Women above age 45 are excluded since dysmenorrhea only affects pre-
menopausal women.  
  
Inclusion/Exclusion Criteria for Healthy Controls, Dysmenorrhea, and PBS Participants 
(Arm 1) 
 
Inclusion criteria: Cases: 1) Dysmenorrhea cases will have: a) average menstrual pain ≥ 5/10 
(0=no pain and 10=the worst imaginable pain) with menses or withdrawal uterine bleeding from 
cyclic OCs without painkillers, b)  menstrual pain in the region between the umbilicus and the 
perineum, above the level of the inguinal ligament and c) indication the participant has 
attempted to resolve pain by medical means (including NSAIDs and/or OCs).Participants who 
endorse bladder symptoms on the phone screen will be preferentially recruited; those who do 
not endorse bladder symptoms may be determined ineligible. We found our previous questions 
to have limited predictive value. Notably because we only want to collect the minimum amount 
of information necessary to determine eligibility on the phone screen, we may not ask all 
bladder questions (depending on what future statistics show in regards to what bladder 
questions accurately predict COS). Participants will complete a daily diary to verify menstrual 
pain during the screening period.33 To be eligible as a dysmenorrhea case, a participant must 
indicate menstrual pain ≥ 5/10 on at least one day during menses in their daily diary. In the 
circumstance that a participant originally assigned to the dysmenorrhea group does not reach a 
pain of 5 on any days on the daily diary, this will be handled on a case-by-case basis particularly 
if they used a continuous around-the-clock, effective pain treatment regimen. They must have 
no concurrent chronic pain diagnoses that affect daily life or a history of more than 24 migraines 
per year. 2) D+COS participants will meet criteria for Dysmenorrhea cases, with >15 bladder 
pain on a 0-100 visual analogue scale (VAS) during either first sensation or first urge at 
assessment visit #1. Initially participants will be enrolled as dysmenorrhea participants and their 
final eligibility for D+COS will be determined after their assessment #1 visit. 3) Diagnosis of PBS 
participants will be confirmed by medical records indicating chronic (>3 months) pelvic pain 
(average intensity ≥ 3/10), pressure, or discomfort related to the bladder accompanied by at 
least one other urinary symptom such as persistent urge to void or frequency.34 PBS 

 
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participants will also have records review to confirm the exclusion of other conditions by clinical 
examination or cystoscopy if necessary. PBS participants can have other chronic pain 
conditions. 
 
Controls: 1) Healthy control cases will have a) average pain ≤ 3/10 with menses or with 
withdrawal uterine bleeding from cyclical OCs and b) no concurrent chronic pain diagnoses or ≥ 
5 migraines in the past year. Healthy controls with < 2 migraines per year will be recruiting 
preferentially.  
 
Exclusion criteria for dysmenorrhea (and D+COS), PBS and healthy control cases includes: a) 
presence of active pelvic or abdominal malignancies (primary or metastatic), b) absence of 
regular menses (including current pregnancy, recent pregnancy, or active breast feeding) c) 
active genitourinary infection in the last four weeks, d) unable to read or comprehend the 
informed consent in English, e) unwilling to complete study procedures f) presence of 
hypertension or risk for developing hypertension, and g) unwilling to withdraw from OCs for at 
least one cycle prior to the Phase #1 study visit. Participants with a history of epilepsy will not 
complete the visual unpleasantness task. 
 
Inclusion/Exclusion Criteria for “Special” Healthy Controls and Chronic Pain Participants 
(Arm 2) 
 
Special Healthy Controls (n=10) will be enrolled under the same inclusion and exclusion 
criteria as healthy controls, however, special healthy controls will not be required to have regular 
periods or abstain from hormonal contraception, and will not be assessed for migraines with 
aura.  
 
Chronic pain cases (n=50) will have a diagnosed, documented (reviewed by PI) chronic pain 
disorder greater than six months’ duration, which has required at least 2 different prescription 
treatments and/or surgical management. They must report an average pain of at least 5/10 in 
the last month.  We will accept participants with any type of chronic pain (except for individuals 
with bilateral knee and hand pain) but anticipate many of our chronic pain participants to suffer 
from fibromyalgia, lower back pain, and chronic pelvic pain (including irritable bowel syndrome).  
 
Of note, special healthy controls or chronic pain cases will not be excluded for unwillingness to 
undergo internal PPT testing, visual sensory testing, hip or head PPT. These groups may waive 
these procedures but still remain eligible to choose to participate in rest of the study.    
  
Exclusion criteria for Chronic pain cases (n=50) and Special Healthy Controls includes a) 
presence of active pelvic or abdominal malignancies (primary or metastatic), b) pregnancy, c) 
active genitourinary infection in the last four weeks, d) unable to read or comprehend the 
informed consent in English, e) unwilling to undergo study procedures (except internal PPT 
testing, visual sensory testing, hip or head PPT which may be waived in these groups),  f) 
bilateral hand or knee pain, or g) absence of ovaries. 
 
To summarize, Chronic Pain Cases and Special Healthy Controls may have absence of regular 
menses, hypertension, a history of epilepsy (in which case sensory testing will not be 
performed) or migraines with aura, and be unwilling/unable to abstain from hormonal 
contraception.  
 
Phone Screen 
All callers will complete a phone screen to assess eligibility before being scheduled for a screen 

 
8 
 
visit. Because of the drastically different inclusion/exclusion criteria, separate phone screens will 
be used for arm 1 (healthy control, dysmenorrhea, and PBS participants) than for arm 2 (special 
healthy control and chronic pain participants.  At the end of the phone screen, all callers who 
complete an arm #1 or arm #2 phone screen will be asked if we have their permission to keep 
their phone screen information and contact them for future studies. Participants who indicate 
yes will be added to our recruitment registry study upon study closeout. Eligible callers will be 
scheduled for a screening visit. 
 
Note we may attempt to match groups for age and demographics. Thus, in some cases, 
eligibility on the phone screen may vary based on the study’s current need for individuals with 
certain demographics for each group. We may also require dysmenorrhea participants to 
endorse bladder symptoms on the phone screen in order for them to be eligible to come in for a 
screen visit.  
 
Procedures Completed During Each Study Visit:  During each testing session, participants 
will have their blood pressure and pulse taken, urine collected, and a urine pregnancy test 
(unless no uterus) completed. 
 
C1.b Screening Visit: Informed consent will be obtained by a trained research assistant before 
any questionnaires or study procedures are completed.  
 
Adult Participants (arm #1 and arm #2) will complete the following questionnaires: a) complete 
medical, surgical, and gynecological history, and b) a psychosocial assessment using short-
form PROMIS instruments (pain behavior, pain interference, fatigue, sleep,  anxiety, depression, 
physical function,  satisfaction with participation in social roles, and 10 item global health 
scale).35 Participants will also complete the following pain rating questionnaires (the Interstitial 
Cystitis Problem and Symptom Indices, the Pain Catastrophizing Scale, Revised Short-form 
McGill Pain Inventory72 and MIDAS headache questionnaire.)67 These scales have been linked 
to pain intensity and quality of life in CPP sufferers, and catastrophizing (rumination, 
magnification, and feelings of helplessness about pain) is a well-validated intervention target for 
many cognitive-behavioral approaches for chronic pain.36–39 Validated, CPP-specific 
assessment tools will be used to characterize IBS (Rome III criteria)40 and PBS symptoms 
(Genitourinary Pain Index).41 Subjects will also complete the Female Sexual Function Index 
(FSFI)68 and BSI-18 based questionnaire containing 6 somatization questions selected from the 
BSI-1869 plus a vision question, and the Complex Medical Symptoms Inventory (CMSI)-General, 
Fibromyalgia Symptoms, Fatigue, and Vulvodynia sections.70,71 All questionnaires will be 
collected electronically using REDCap.  
 
Note that midway through the study, an amendment was made for arm #1 and arm #2 study 
participants were freed from the requirement to undergo a complete gynecologic exam during 
the screen visit and as part of phase 1 participation. Instead, only arm #1 painful bladder 
syndrome and D+COS participants who randomize into phase 2 and agree to participate in 
phase 2 will be required to undergo a complete gynecological exam. These participants will be 
required to complete the gynecological exam at some point before their first phase 2 
assessment visit (i.e. Assessment Visit #2 - 6 month visit). Transvaginal ultrasound and/or 
abdominal ultrasound may be performed on dysmenorrhea participants whose exam and/or 
history is suggestive of ovarian or uterine masses. Arm #1 PBS participants may choose to 
complete the gynecological exam during their screen visit if they prefer since their 
randomization to phase 2 is already determined.  
 
Even though a gynecological exam is no longer required for phase #1, the second phase 1 visit 

 
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(assessment visit #1) includes internal vaginal pressure-pain threshold testing, which is similar 
in nature to the pelvic floor portion of a gynecological exam. Thus, for participants who have 
never had a gynecological exam, a study nurse or study doctor may complete a simple exam of 
the pelvic floor at the screen to promote understanding of the internal vaginal pressure-pain 
threshold testing that will be completed at the assessment. This ensures that the integrity of our 
data is not biased by unfamiliar experience. Participants will be asked whether they have ever 
had a gynecological exam on the phone screen and a simple exam of the pelvic floor for those 
that say they have never had an exam will be scheduled into their screen.   
 
Participants entering phase 2 will still need to complete a birth control pill medical screen 
checklist in person or over the phone with a study doctor or study nurse before the pills can be 
prescribed.  
 
Note arm #1 participants who indicate on a phone screen that they are unwilling to undergo 
gynecological exam will remain ineligible for the study since the assessment visit involves 
internal vaginal testing, which is similar in nature to an exam, and because our ultimate goal is 
to identify participants who will be eligible for phase 2, which requires a gynecological exam. 
However, participants enrolled in arm 2 with chronic pain who are unable to tolerate the internal 
testing may still skip this procedure.  
 
Participants will be asked to do a tampon test originally designed to study vulvodynia.42  The 
tampon test may be performed by the participant during the visit or at home. The tampon test is 
a standard test in which subjects report the amount of pain they feel on a 0-10 numerical rating 
scale after inserting a standardized tampon (Tampax original regular with plastic applicator, 
Proctor and Gamble).  
 
Participants reporting average menstrual pain ≥ 5/10 on the phone screen (dysmenorrhea 
participants) will undergo a rapid version of the bladder test. Participants will be asked to rate 
their bladder pain on a 0-100 VAS scale when they reach “first urge.” If a participant arrives to 
the visit already at first urge, we will have them rate their pain and if not ≥ 10, we will wait 15 
minutes past first urge and have them rate their pain again. This is because the participant may 
not have been focusing on the feeling of their bladder. Participants not already at first urge will 
be encouraged to drink water (at least 20 oz) during the screening visit until they reach first urge 
at which point a pain rating will be attained. After they reach first urge, they can go to the 
bathroom and leave their urine sample. Those with ≥ 10/100 pain at either first sensation or first 
urge and/or those with a GUPI scores ≥ 8 will be preferentially recruited into this study. In other 
words, we may eliminate some participants without evidence for COS (using the rapid bladder 
test FU pain score and possible using GUPI scores) after the screen visit. However, for those 
that continue, final group assignment will be determined by their ratings during assessment visit 
#1.    
 
C1.c Between visit home tasks: Training of arm #1 and arm #2 participants to fill out daily 
diaries and how to use ovulation kits to determine their menstrual phase prior to their next visit 
will be provided by experienced research staff (for ovulation kits, only for those patients where 
menstrual phase changes can be defined). The daily diary will be completed using REDCap and 
asks them to rate their menstrual, bladder, and bowel pain (using a numeric rating scale where 
0-no pain, 10-worst pain imaginable), describe any bleeding, enter results of ovulation tests, and 
list dose, type and use of painkillers. For special healthy controls and chronic pain participants 
(arm #2), the daily diary includes one additional pain rating where participants can choose the 
relevant area of pain from a scroll down menu and rate their pain in that area from 0-10. To 
protect confidentiality subjects will not enter identifying information and only use their assigned 

 
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subject ID # (see data collection section D). Dysmenorrhea, healthy control, and PBS 
participants will be asked to fill out daily diary entries for all days between their screening visit 
and assessment #1. Participants in the special healthy control and chronic pain groups will be 
asked to complete daily diaries starting the day after their screen visit and continuing for 35 
days, regardless of when their assessment visit is scheduled.  
 
C1.d Washout for arm #1 participants on OCs (not relevant for arm #2): Those on OCs at 
enrollment will complete a washout lasting a minimum of one menstrual flow in order to 
accurately confirm dysmenorrhea severity with daily diaries. Bleeding that occurs within 7 days 
of the cessation of birth control pills will not be counted as a menstrual flow, since this is the 
result of withdrawal bleeding rather than menstruation. Thus, most women currently taking birth 
control pills will need to abstain from taking hormonal contraception for at least 7 weeks to allow 
time for a withdrawal bleed followed by one full menstrual cycle (4 weeks) plus the time it takes 
them to enter their luteal phase (approximately 3 weeks). In the case that a woman does not 
experience withdrawal bleeding within 7 days of the cessation of birth control pills, determination 
of whether the bleeding that does eventually occur is the result of delayed withdrawal bleeding 
or menstruation will be made on a case-by-case basis.  In the case that we cannot confirm 
group eligibility from one menstrual flow on the daily diary (i.e. they did not have great enough 
pain), we may ask the participant to continue to abstain from hormonal contraception for a 
second menstrual flow. If they are still not eligible after two menstrual cycles, they will be 
deemed ineligible or if applicable (pain is 3 or less on all days), switched to the healthy control 
group.  
 
For the subset of participants who were using hormonal contraception to manage chronic or 
menstrual pelvic pain, and whom express concern about pain management during phase 1, the 
study doctor will discuss pain control options and if necessary, and the participant desires, 
prescribe Tylenol #3, Tramadol 50mg, or Norco 5/325. We expect these conditions to apply to a 
small subset of participants (less than 10). The participant may alternatively see their regular 
physician to address this issue. All other participants will be asked to manage their pain in the 
same ways they normally manage pain (e.g., over-the-counter painkillers, heat pads, etc.). All 
participants will be asked to indicate in their daily diary if they took any painkillers each day and 
if so, what medication and how much. 
   
C1.e Assessment visit: Arm #1 and Arm #2 participants with regular periods and/or who are 
not using hormonal contraception will be asked to contact us when they start their first period 
after the assessment visit. The assessment visit will be scheduled during the anticipated 
participant’s luteal phase (17-25 days after the start of their period adjusted if they typically have 
unusually short or long cycles).  They also will be give ovulation kits and asked to complete 
them starting day 10 of their cycle up until their scheduled assessment visit to give us additional 
data about their menstrual phase. Bladder pain is less likely to be confounded with menstrual or 
ovulation pain during the luteal phase. 
 
Special healthy controls and chronic pain participants (Arm 2) who do not have regular 
menstrual periods and/or are on hormonal contraception will not be required to use ovulation 
kits and instead visits will be scheduled at a time we do not expect any bleeding and 
approximately 2-6 weeks (but up to four months) after their screen visit.  
 
The time between the screen visit and assessment visit may vary based on participant’s 
personal schedule and ability to confirm ovulation status and group eligibility (if applicable) but 
will not exceed four months or be less than two weeks. 
 

 
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For arm #1 participants (healthy controls, dysmenorrhea, and PBS participants), group 
assignment is confirmed from one to four months of daily diaries. If the participant is not eligible 
for the originally assigned group after the first flow, we will ask the participant to complete the 
daily diary through a second menstrual flow to determine eligibility. If they are still not eligible for 
the originally assigned group after two flows, we will transfer the participant to the applicable 
group or inform the participant she is ineligible. 
 
For arm #2 participants (Special healthy control and chronic pain participants) are asked to 
complete daily diaries starting one day after their screen visit and for 35 days, regardless of 
when their assessment visit is completed. For chronic pain participants, if as of two days before 
their scheduled assessment visit, their daily diary does not indicate consistent pain ratings, we 
may determine they are no longer eligible to complete the assessment visit. Healthy controls 
must demonstrate menstrual pain less than 4/10 and not indicate consistent pain ratings. 
Chronic pain or healthy control cases whose pain status is unclear will be handled on a case-by-
case basis.  
 
 
Assessment Visit #1 (Arm 1 and Arm 2) 
Assessment Visits are very long so we will offer snacks to the participants (e.g., teddy grahams 
and fruit roll-ups).  
 
During the assessment visit, EMG (of different muscle groups), EKG, and respiratory rate 
equipment will be applied and information will be recorded throughout the visit.  EMG leads will 
be placed on the lower abdomen and right knee during the bladder testing to measure muscular 
recruitment. The abdominal EMG leads will be moved to the left knee once the bladder pain test 
is completed. The EKG leads and respiratory belt are placed to measure autonomic activity. The 
autonomic activity will be recorded to verify nociceptive experience.  
 
Bladder pain test: All participants will first undergo bladder pain testing described in our 
validation study2. Subjects will be provided directions for the bladder study. The bladder study 
involves voiding and obtaining baseline measurements of bladder volume with a 3D ultrasound 
abdominal transducer (GE Voluson 750, Wauwatosa, WI). After hydration with 20 oz of water, 
subjects are instructed to report when they reach standard levels of bladder urgency: first 
sensation, first urge, and maximum tolerance and volumetric measurements of the bladder will 
be made. At each of these time points and every 15 minutes, 0-10 VAS measurements of pain 
and urgency are recorded. After scanning at maximum tolerance, subjects will urinate in a 
graded toilet hat to allow for measurement and saving a urinary specimen.  Samples will be 
banked for analyses. If the participant gives permission in the consent form, urine samples will 
continue to be stored after the study is completed for possible future analyses by our lab on 
women’s health conditions. If the participant does not give permission in the consent form for 
analyses beyond the current study, their sample will be destroyed upon study completion.  
Questionnaires:  The previous validated questionnaires from the screening visit will again be 
employed during the bladder testing. Participants will also complete a handedness 
questionnaire.  


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13 
 
(Diagram 3) and a ramp rate of 0.5 kg/cm2/s. Internal trials may be terminated by examiner at 
3.0 kg/cm2.  A trained clinician will perform all pressure-pain tests.  Subjects will report the pain 
at the time they pressed the button on a NRS scale immediately after testing each individual 
site. After a two-minute break, the sites will be retested again. In order to compare within-
subject results from the three assessment visits and in order to standardize timing of post-
testing time scores (collected every 5 minutes as described below), the order of testing sites will 
not be counterbalanced.   
 
Subjects will be allowed to rest for another 5 minutes, and subject will be queried on the level of 
NRS pain at each site again (shoulder, hip, knee, forehead, global vagina). Subjects will be 
queried every 5 minutes until pain returns to baseline levels or up to 30 minutes (the time of 
resolution in minutes will also be recorded).  
 
Conditioned Pain Modulation (CPM) : CPM testing involves repeating pressure pain threshold 
testing in the presence of a heterotopic stimulus (in this case cold water). The testing stimulus 
will be a mechanical pressure on the left medial knee fat pad. As in the first task, pressure will 
be applied (0.5 kg/cm2/s ramp) until the participant achieves pressure pain threshold. Baseline 
pressure pain thresholds of the testing stimulus on the left knee and left shoulder will be 
determined. After waiting approximately 2 minutes, the conditioning stimulus will be applied by 
instructing the subject to insert their right hand up to their wrist into a bucket of water maintained 
between 0-6° C. After 10 s of immersion, subjects will be asked to rate their level of hand pain 
on a NRS 0-10 scale. After 20s of immersion, the pressure pain threshold will be re-applied 
using a 0.5 kg/cm2/s ramp rate at the knee and the participant will be asked to rate their knee 
pain at the time they reached threshold (i.e. pressed the button) on a NRS 0-10 scale. After 
testing (~20s of immersion), subjects will immediately remove their hand and the pressure pain 
threshold will be determined at the shoulder. After waiting 5- and 10-minutes after water 
immersion, the testing stimuli will be re-applied. This paradigm combines a method for 
measuring CPM46 and methods for accurately measuring participation of descending 
inhibition.47 CPM will be measured by the standardized differences in pain threshold, EMG 
activity and autonomic responses before and after heterotopic stimulation. This strategy will 
provide the most reliable measurement of descending inhibition.47,48  
 
Temporal Summation (TS): Participants will be instrumented to undergo tests of spinal pain 
modulation. Temporal summation will be tested using an algometer on the right medial knee fat 
pad.  We will repeatedly apply an amount of pressure (0.5 kg/cm2/s ramp rate; ITI = 5-15 
seconds) equal to the average of the participant’s two pressure pain thresholds assessed during 
the first task. After each press, the participant will be asked to rate their pain on an NRS 0-10 
scale. The pressure will be applied ten times or until the participant rates their pain as a 6, 
whichever comes first.   
 
EEG Testing: Subjects will be instrumented with a standard EEG cap.  Encephalography (EEG) 
recordings will be obtained using a standard montage and equipment. A trained research 
assistant or nurse will place the EEG cap and electrodes. All participants will undergo a resting 
state EEG task in order to analyze brain activity patterns associated with pain behavior. 
Participants will be requested to focus on a fixation cross on a computer monitor with their eyes 
open and sitting with their eyes closed for one minute each. In total, the task is expected to take 
10 minutes. All participants will complete a handedness questionnaire,49 consisting of a series of 
questions asking which hand they use for different activities, in REDCap, which will help us 
interpret our EEG results.  
 

 
14 
 
Sensory Amplification Tests: To evaluate whether sensitivity extends to other modalities we will 
use a software application that measures self-reported unpleasantness to increasingly loud and 
bright stimuli (safe and well below thresholds that could cause auditory or visual damage).  
 
Auditory stimuli will be provided by waveforms generated by a PC and transmitted through 
headphones calibrated by an audio monitor50 . Each stimulus will be a two-tone combination of 
1200 and 1350 Hz. Sounds will ranged in 5 dB steps from 35 to 90 dB SPL (A-weighted). After 
receiving instructions, the subject will be presented with a series of sounds, in random order of 
intensity. After each 2 s stimulus, the participant will be asked to rate its unpleasantness from 0 
(“not at all unpleasant”) to 20 (“the most unpleasant sound imaginable”). 
 
Visual stimuli will be displayed on a computer monitor capable of providing up to 300 lux. In this 
3 min task, two visual stimuli will be presented in an alternating block design.  The control 
stimulus is a fixed crosshair centered in the middle of a solid background; the experimental 
stimulus will be a flashing (12.5 Hz) blue/yellow checkerboard presented at varying levels of 
brightness (luminance) in both an ascending and random manner.  Each subject will be 
presented with 6 test patterns alternated with 6 blanks patterns. Subjects will use a 0 (“not at all 
unpleasant”) to 20 (“the most unpleasant pattern imaginable”) numerical descriptor scale to rate 
the unpleasantness of each visual stimulus and the entire task. 
 
The visual task may be annoying or elicit a headache or nausea in sensitive individuals. 
Participants may stop at any time.  
The sensory testing will take in total about 20 minutes and will be performed while the 
participant is connected to EEG. 
 
Additional quality controls: We may need to repeat a task, modify electrode positions, 
reconfigure equipment, or adjust features in the environment to evaluate the quality of our data 
acquisition scheme. For example, if a PPT measurement was not obtained or an electrode does 
not stay put, we may ask the subject to allow us to repeat additional measurements.  
All implanted quality controls with permission from the subject will be documented in the subject 
record. 
 
C1.f Hormone Measurements with Blood (Arm 1 and Arm2): We will ascertain that subjects 
have not had more than 100 ml of blood drawn over the past 24 hours or 200 ml over the past 
30 days. After completion of the pressure/pain testing, a trained nurse or physician will draw up 
to 60 mL of blood into vacutainers. Separation of serum and plasma will be performed using 
centrifugation and samples will be aliquoted and banked appropriately to allow for hormone 
measurements and analyses.  To control for the potential influence of reproductive hormones on 
pain sensitivity, and test our hypothesis about the role of hormonal influences on 
neurophysiological pain assessments, we will measure serum samples for E2, P4 and 
testosterone using methods that do not cross-react to the OCs from core laboratories.51  If the 
participant gives permission in the consent form, blood samples may also be analyzed for 
genetics. If the participant gives permission in the consent form, blood samples will continue to 
be stored after the study is completed for possible future analyses by our lab on women’s health 
conditions. If the participant does not give permission in the consent form for analyses beyond 
the current study, their sample will be destroyed upon study completion. 
 
C1.g Follow-up questionnaires: We will ask arm 1 and arm 2 participants to complete an 
annual follow-up questionnaire including the validated questionnaire instruments mentioned 
above and a general medical history questionnaire. All arm 1 and arm 2 questionnaires will be 

 
15 
 
collected electronically using REDCap at home. If requested, paper versions can be provided 
instead and mailed in. For participants continuing to Phase 2, the year 1 annual medical history 
questionnaire will usually take place during their assessment #3 visit. All participants that 
complete phase 1 (Screen + Assessment #1), including phase 2 dropouts, will be asked to 
complete the annual follow-up questionnaire. 
 
 
C1.h Data Analysis/Sample Size Requirements: The primary outcomes under this hypothesis 
will be [1] bladder pain at FU, [2] pelvic hyperalgesia (VAS, time to return to baseline pain), 
reflecting peripheral dysfunction/sensitization, and [3] centrally mediated hyperalgesia (CPM, 
bodily pain thresholds).   
Factor [1], Bladder pain at FU: Our preliminary data on healthy subjects and women with 
dysmenorrhea, in combination with other studies investigating migraine and PBS, suggests 
effect sizes between 1.4-5.6 (Cohen’s d). As a control comparison, we expect that women with 
chronic pain will have levels of bladder and pelvic sensitivity comparable to healthy subjects. 
We will perform t-tests for each of these three pain variables using pairwise contrasts and 
appropriate Bonferroni corrections for six group comparisons.  
Factor [2], pelvic hyperalgesia: To confirm pelvic hyperalgesia (but not necessarily pelvic PPT) 
is higher in those with bladder pain, we will show bladder pain and pelvic hyperalgesia (both 
VAS and TIME) are higher in D+COS than healthy pain-free controls (H), dysmenorrhea only 
(D), and chronic pain patients (P).  Similarly bladder pain, pelvic hyperalgesia and pelvic floor 
pain sensitivity (PPT) are anticipated to be greater in PBS than H, and D.  
Factor [3], centrally mediated hyperalgesia: Greater central dysfunction manifest by impaired 
CPM and reduced bodily pain thresholds in vulnerable subjects will be shown by comparing 
D+COS, PBS, and P to D and H. We estimate 15 subjects per group will provide adequate 
power (g-power, a priori t-test, a=0.05, b=0.2).  To verify we have adequate phenotyping of 
CPM, we have included the positive control group with chronic pain (P). We assumed the 
Group 
(Sample Size) 
H 
(20) 
D 
(20) 
D+COS 
(95) 
PBS 
(40) 
P 
(50) 
MSA 
Bladder Pain at FU (VAS) 
0 
0 
2.5 
3.0 
- 
0.85 
Pelvic Hyperalgesia (VAS) 
0 
0 
3 
3 
- 
0.84 
Pelvic Hyperalgesia (Time) 
0 
0 
2 
2.5 
- 
0.83 
Pelvic Pain Thresholds 
0 
.5 
.5 
2.3 
- 
0.81 
Bodily Pain Thresholds 
0 
1.5 
2 
2 
2.3 
0.91 
Conditioned Pain Modulation 
0 
1-2 
1-2 
4a 
2-4b 
0.94 
Menstrual Pain History 
0 
3.7 
5 
6 
- 
0.81 
Temporal Summation 
0 
0 
.7 
.8 
- 
- 
Estradiol  
0 
1-3c 
1-3c 
1-3 
- 
0.84 
Depression/Anxiety 
0 
.3 
.4 
2 
2-4 
0.82 
Table X: Sample sizes and hypothesized results in Z-scores or range of Z-scores (relative to 
healthy) for subjects that are healthy (H), have dysmenorrhea (D), have cross organ 
sensitization (D+COS), PBS, or chronic pain (P). MSA indicates Kaiser’s measure of sampling 
adequacy. Numbers in italics indicated minimum detectable hypothesized score with current 
sample size. Z-scores based on preliminary data, or others indicated by subscript (a: 9, 
b:,c:11,13). Effects temporal summation in these populations are unknown, but effect sizes as 
large as 1.1 have been reported for fibromyalgia. Dashes (-) indicate unknown effect size. 

 
16 
 
difference in CPM between healthy subjects and chronic pain patients in this protocol would 
have an effect size of 0.78 based on a meta-analysis (Lewis et al. 2012). Using a 2.6 allocation 
ratio, a priori power analysis (α=0.05, β=0.2), suggests we need 20 healthy participants and as 
many as 50 chronic pain patients to detect a difference in the mean CPM. Additionally, the 
utilization of 50 chronic pain patients will allow us to use regression to identify up to 5 predicted 
factors associated CPM with correlation coefficients exceeding 0.3 even after correcting for 
multiple comparisons (α=0.05, β=0.2) as suggested by an a priori random model power 
analysis.  This will be readily captured as our global study design encompasses a larger sample 
size (Table X) to handle anticipated secondary analysis and projected needs for completion of 
Phase #2.  
Additional confirmatory analysis: Special healthy control subjects (n=10), we will verify special 
healthy control subjects do not differ from original enrolled healthy control subjects (n=20) to 
ascertain chronic subjects (P) participating in the trial are not treated differently under blinded 
conditions.  
For the secondary analysis of Phase #1, we will evaluate the D+COS (provoked bladder pain) 
phenotype while accounting for menstrual history (including historical OC exposure), hormonal 
factors, anxiety, and depression. To determine the relative contribution of these secondary 
variables on bladder pain, we will perform principal component 
analysis (PCA). We have adequately powered the analysis of these 
potential covariates consistent with a bootstrap (n=1000 
replications, Fig Y) model based on our preliminary data and 
studies by others (described in Table X). Even with weak 
correlations (r=.3), PCA can detect 3 primary factors with our 
projected sample size. PCA is an optimal method to identify the 
mechanisms responsible for bladder pain. Historically PCA with 
even smaller sample sizes has been employed to measure how 
coping skills influence pain perception during the menstrual cycle52 
(n=20) and to show that different pain testing modalities are related 
to differential psychosocial profiles53 (n=188).  Even if PCA fails, we 
can make many direct comparisons beyond what is described here 
with t-tests.  
C1.i Expected outcomes: (1) Abnormal CPM is the primary 
predictor of widespread pain sensitivity observed in dysmenorrhea, 
D+COS and PBS. Results would be insightful if CPM was worse in 
participants with D+COS and PBS than those with dysmenorrhea alone because this would 
indicate CPM mechanisms are involved in COS. On the other hand, if CPM impairments are 
universal across chronic pain patients, PBS, D, and D+COS compared to healthy controls, this 
would indicate that pain independent of location and chronicity depends on impaired descending 
inhibition. This finding would be the basis for developing a physiological biomarker for those at 
risk of developing CPP/PBS similar to approaches by others identifying risk for chronic pain45,79. 
On the other hand, some pain conditions such as rheumatoid arthritis114 and trapezius 
myalgia115 do not seem to exhibit CPM. The vulnerability to conditions unrelated to CPM may 
involve either inflammatory or specific peripheral nerve events. If CPM does not correlate with 
widespread pain sensitivity or bladder pain phenotypes in this proposal, we have adequately 
powered our sample size to detect alternative mechanisms that could be responsible (e.g. 
menstrual, hormonal, psychological). Nevertheless, consistent with meta-analyses on the 
menstrual cycle54, psychological55 , and central factors on pain56, we anticipate central pain 
factors to explain the majority of the variance in bladder pain sensitivity.  Determining the 
relative importance of systemic E2 concentrations vs. other modifiable factors such as 
Fig Y: Bootstrap 
simulation constructed 
assuming a range of 
correlations between 
potential mechanisms 
(e.g. pelvic hyperalgesia 
and bladder pain) indicate 
that even with weak 
correlations we can detect 
3 factors. Error bars show 
95% confidence intervals. 

 
17 
 
menstrual pain will provide insight on the most important mechanisms for reducing bladder pain 
sensitivity in vulnerable women. 
(2) Pelvic hyperalgesia and severity of menstrual pain are more common in D+COS 
phenotype vs. dysmenorrhea only. The identification of clinical biomarkers such as severity of 
pain after a speculum exam (analogous to the post pelvic floor PPT pain report) can be obtained 
from our results. Our study results would provide evidence of the significance of this routine 
clinical exam in reliably predicting bladder sensitivity.  
C1.j Potential Problems: Primary and/or secondary comparisons are nonsignificant: a mixture 
of mechanisms are involved in D+COS phenotype or PBS. We will confirm whether our 
paradigm was sufficient to detect deficits in CPM using our positive control group with chronic 
pain to validate a true negative finding. Afterwards, we will use cluster analyses to identify 
phenotype patterns and confirm mechanistic differences with banked serum. Why not examine 
additional hormones? We are examining hormones that have been frequently studied in pain 
research and will bank serum allowing for the future possibility of studying others. What about 
endometriosis, fibroids and other known anatomic uterine pain conditions? To account for the 
common conditions (especially within our medical practice), we will include 10 confirmed 
endometriosis (operative report review) participants and 10 fibroids participants (ultrasound or 
surgical confirmation) mixed within our dysmenorrhea cohort to determine which effects 
produced by the primary analysis are due to anatomical conditions.  
 
C2.  Methods for Phase #2   
 
C2.a Participation criteria  
All phase #1 participants with D+COS (pool of 95 candidates) or PBS (pool of 40) will be 
enrolled with intent to transition into phase #2, though the second phase of the study will be 
presented as optional. If they choose not to participate in their randomized group, they will have 
the option to come back in a year later for an additional assessment visit for long term follow up 
data. These enrollment targets, accounting for a potential 35% dropout rate, will enroll 62 
participants with D+COS and 26 PBS. This will provide adequate power for the prespecified 
contrasts. 
Inclusion Criteria: 1) Participation in Phase #1 and assigned to the Arm #1 PBS or D+COS 
group, 2) willing to comply with OC assignment (continuous for PBS; cyclical, continuous, or no 
OC for D+COS).  
Exclusion Criteria: a) presence of active pelvic or abdominal malignancies (primary or 
metastatic), b) absence of regular menses (including current pregnancy, recent pregnancy, or 
active breast feeding), c) unwilling to undergo pelvic examination/testing, e) standard 
contraindications to use of OCs: 
• 
Thrombophlebitis or thromboembolic disorders, or a history thereof 
• 
Cerebrovascular or coronary heart disease 
• 
Known or suspected carcinoma of the breast 
• 
Endometrial cancer or other known or suspected estrogen-dependent neoplasia 
• 
Undiagnosed abnormal genital bleeding 
• 
Jaundice with prior pill use 
• 
Hepatic adenomas or carcinomas 
• 
Known or suspected pregnancy 
• 
Hypersensitivity to any component 
• 
BMI >40  

 
18 
 
• 
Lactation (< 6 weeks postpartum) 
• 
Age >35 years and smokes 15 cigarettes/day  
• 
Elevated blood pressure (160 / 100)  
• 
Chest pain (angina pectoris) 
• 
Vascular disease 
• 
Major surgery with prolonged immobilization 
• 
Complicated valvular heart disease 
• 
Migraine with focal neurologic symptoms 
• 
Diabetes with evidence of nephropathy, retinopathy, neuropathy, vascular disease, or > 
20 years duration 
• 
Severe (decompensated) cirrhosis 
Women with persistent, nonphysiological ovarian cysts or with a fibroid larger than 5 cm will be 
excluded because of associated bleeding problems and likelihood to undergo surgery during 
participation. Women taking drugs listed on the manufacturer’s web site (such as penicillin and 
topiramate) that may reduce OC effectiveness will be instructed to use condoms as a backup 
form of contraception. 
C2.b Randomization: NorthShore’s biostatistical core will assign D+COS participants to 
continuous OC, cyclical OC, or the untreated arm using a permuted-block randomization 
scheme with block sizes only known by appropriate study staff (PBS as above will be only 
assigned to continuous OCs) adjusting for the unequal recruitment goals (n=10,26,26) . To the 
extent possible, the research nurse performing QST will be blinded to group assignment. 
Because of withdrawal bleeding in the cyclical group, we cannot blind participants to their 
assignment. Drs. Tu and Senapati will be responsible for prescribing both cyclical or continuous 
OC usage and will assess the participant for contra-indications and answer questions at the 
screen visit. The study team will provide counseling that will emphasize the benefits and risks of 
OCs as well as the lack of protection against sexually transmitted diseases from OCs and 
options for backup contraceptive methods. Participants will be instructed to begin OCs the 
Sunday immediately after commencing their menses or on a day that allows for personal choice. 
We will give all participants a “Tips for use of Oral Contraceptives” flyer. 
 
C2.c Trial of OC usage: Participants in Phase #2 of the study will be prescribed generic 
microgestin (20 µg ethinyl estradiol/1 mg norethindrone acetate), a lower estrogen-containing 
OC well-tolerated by women. Microgestin has minimal breakthrough bleeding compared to a 
continuous OC regimen with higher estrogen concentration or different progestin57 or a cyclic 
paradigm with lower estrogen concentration.58 D+COS participants will be randomized into three 
study arms: cyclical OC usage (n=26) vs. continuous usage (n=26) vs. no OC (n=10). Less 
untreated subjects are needed as large effect sizes are expected compared to OC users. 
Cyclical OC users will be instructed to take 21 days active followed by 7 days of no pills every 
four weeks. Continuous OC users will be instructed to immediately start the next pack of active 
pills on day 22. Those not taking OCs will be instructed to use barrier contraception. At the time 
of randomization, participants will be counseled by the study team on the proper usage of these 
treatments. Participants assigned to OCs will receive a three or four month prescription for 
microgestin and have it filled at the Evanston Hospital Outpatient Pharmacy. All refills will be 
distributed by the same pharmacy. Separately from the D+COS randomization scheme, PBS 
participants will receive only continuous OC. This study design deliberately avoids randomizing 
chronically symptomatic PBS patients onto cyclical vs. continuous OCs, as the anticipated 
difference in outcomes is small, and therefore not likely to be favorable in recruiting subjects. 
However, identifying which pain mechanisms (visceral and pelvic sensitivity) change following 
OC-induced hormonal suppression both in chronically symptomatic PBS sufferers vs. the 

 
19 
 
asymptomatic D+COS patients may prove crucial to understand which PBS patients exhibit 
hormonally responsive pain.23 
 
C2.d Study Visits Participants in Phase #2 will return to repeat experimental pain and sensory 
assessments identical to the initial Phase #1 and complete the questionnaires referred to in the 
screening visit plus a side effects questionnaire at six months and one year after OC initiation 
(which follows a two month washout in Phase #1). These visits should occur on pill days (not 
cycle days) 7-21 of the cyclical group and any day for the continuous group, which is 
comparable to cycle days 15-28 in Phase #1. Control subjects not taking OCs will participate 
during their post-luteal phase days 15-28 (similar to Phase #1). This will make hormonal levels 
comparable and minimize any vaginal bleeding. For those who are randomized to Phase 2 but 
decides to not participate in their randomized group, they may be asked to come in a year later 
for an additional assessment visit to obtain long term follow up data. These participants will be 
asked to complete daily diaries and ovulation kits for one month prior to the annual assessment 
visit, similar to Phase 1.  
 
C2.e Home Assessments: Participants in Arm #2 will complete the daily diary during the fifth 
and eleventh month of OC administration33,60. They will also complete a monthly questionnaire 
asking questions related to their pain, mental health, and any side effects they may be 
experiencing (comprised of the McGill Pain Inventory, a side-effects questionnaires, and 
PROMIS anxiety item bank, PROMIS depression item bank, and PROMIS pain interference 
item bank). They will not need to do these questionnaires during months 6 and 12 at home 
because they will do them during their study visit for assessments #2 and #3 respectively. When 
done at home, they should complete these assessments on or near day 15 of their pill pack for 
participants on cyclical OCP, day 15 of their menstrual cycle (15 days after their menstrual 
period started) for participants not taking OCPs, and simply 28 days apart for participants taking 
continuous OCP. 
 
C2.f Compliance: To evaluate compliance, participants will be asked to return all used and 
partially used OCP pill packs. 
 
C2.g Potential Adverse Events: Breakthrough bleeding: Participants in each arm will be 
counseled about uncommon risks of breakthrough uterine bleeding. Participants experiencing 
abnormal bleeding episodes will be offered visits with the study gynecologists, Drs. Tu and 
Senapati, to rule out cervical or vaginal sources of bleeding. Women who need management of 
breakthrough bleeding will be managed per standard clinical protocols. Those needing to 
terminate OCs will also be withdrawn per standard protocols. Withdrawn participants will still be 
asked to complete the annual follow-up questionnaire and may be asked to still do the 6- and 
12-month visit as intent-to-treat and as a separate comparison group. Women will be instructed 
to contact a health care provider right away for numbness or severe headaches which are not 
tension headaches and are not relieved with NSAIDS, severe leg pain, severe chest pain, 
shortness of breath, blurred vision, flashing lights, or blindness, or severe abdominal pain. 
 
C2.h Data Analysis/Sample Size Requirements: To complete Phase 2, we propose analyses 
assessing a) the effect of concurrent and historical OC exposure on experimental bladder pain, 
b) whether continuous and cyclical OC regimens differ in effect on bladder pain sensitivity (via 
elimination of priming by menstrual pain events), and c) the effect of OCs on nociceptive 
mechanisms (pelvic hyperalgesia, conditioned pain modulation). The anticipated effect sizes 
with required sample sizes (which we meet/exceed) are listed in Table Z.  

 
20 
 
(Z1) We will establish prospectively whether usage of cyclic or continuous 
OCs affects bladder pain sensitivity (on bladder filling test), in women with 
D+COS compared to women not on OCs. We are adequately powered to 
detect improvements in bladder pain, assuming a 35% dropout rate 
(conservative estimate, well exceeding previously published rate of 18-
24%51,59) and accommodating a 1:5.2 ratio of non-OC to OC users. We will 
perform repeated measures ANOVA to determine if experimental bladder 
pain improves at 6 months compared to baseline in women with D+COS 
(all having washed out at enrollment). Follow-up results at 12 months will 
be used to confirm findings. The effect size (f=0.9) was estimated from our 
preliminary data above. Analyses will be adjusted with E2 and P4 levels as 
a covariate to determine if the effect of OCs on bladder pain is largely due 
to its effects on circulating hormones with standard methods60.  
(Z2) Similarly, we will establish whether among D+COS patients’ pelvic 
hyperalgesia and descending modulation, the hypothesized mechanisms 
responsible for bladder pain sensitivity, are affected by OC usage. We will 
perform a repeated measures ANOVA to determine whether significant 
improvements occur in pelvic hyperalgesia (VAS pain report, and time to 
normalize), pelvic PPT threshold, bodily PPT thresholds, temporal 
summation, and CPM in OC treated vs. untreated subjects at 6 and 12 
months. Cohen’s d’s for drugs that affect PPT61, temporal summation62 
and CPM63 range between 0.6-0.9.  
(Z3) Similar to z1, we will determine if bladder pain sensitivity improves among PBS 
patients pre- and post-continuous OC exposure using repeated measures ANOVA (6 and 12 
months).  Although hormonal treatment has been shown to improve bladder pain in 13/15 
patients with PBS23, the exact effect size is not known. Prospectively OC usage has been 
shown to reduce episodes of urinary calculosis pain (d=0.8)64. We are adequately powered to 
detect an effect size as low as dz=0.6.   
(Z4) We will perform ANOVA to determine whether continuous OC usage is superior to 
cyclic usage for reducing bladder sensitivity, absolute levels of E2/P4, and dysmenorrhea in 
women with D+COS (6 and 12 mths). Legro and colleagues have established superiority of 
continuous OC usage over cyclic usage for dysmenorrhea (d=1.6) using a comparable sample 
size. After surgical removal of endometriosis, continuous OC usage is associated with less non-
menstrual pelvic pain than cyclic OC usage (d=0.8)65. Our published data2,3 suggests a strong 
relationship between dysmenorrhea and bladder pain. Therefore, we predict a difference of 
d=0.8 on bladder sensitivity between the groups.  If differences are significant, we will evaluate 
whether any specific nociceptive mechanisms are different between cyclical and continuous OC 
users by comparing the effects of QST, temporal summation and CPM between groups.  
(Z5) To determine whether menstrual, hormonal, central or peripheral changes are most 
responsible for improvement or worsening of bladder pain, we will use a repeated measures 
design. We will compare menstrual history, pelvic hyperalgesia, pelvic floor PPTs, external 
PPTs, CPM, E2/P4 concentrations in D+COS subjects that have >50% improvement in 
experimental bladder pain to those that have <50% improvement (6 and 12 mths). A similar type 
analysis showed the effect of duloxetine on CPM in improving diabetic neuropathy (n=30).31 If 
mechanistic differences are comparable to Yarnitsky (d=1.6), we are more than adequately 
powered with 62 subjects with D+COS accounting for attrition. To confirm the proposed 
mechanism we will perform multi-level factor analysis. We performed a bootstrap analysis with 
our hypothesized data set (n=1000 repetitions) and confirmed we can measure 2 independent 
components (each consisting of 2-5 factors) with 95% certainty (median Kaiser Sampling 
Adequacy =0.75). 
ES  nreq npre npost 
Z1  f=.7 
10 95  62 
Z2  dz=.6 24 80  52 
Z3 dz=.6 24 40  26 
Z4 d=.8 52 80  52 
Z5 d=1.6 16 80  52 
Table z: Planned 
comparisons with lowest 
anticipated effect size 
(ES). Required  nreq 
estimated with ɑ=0.05, 
β=0.2. Total enrolled 
D+COS and/or PBS 
subjects per analysis 
are shown before (npre) 
and after attrition (npost) 
as detailed in III.d. 

 
21 
 
C2.i Expected outcome: We anticipate analyses (z1-z5) will show OC usage reduces bladder 
pain primarily by restoring descending inhibition via extinguishing menstrual pain. This finding 
would be consistent with preliminary observations in IBS and PBS.9,22,23,59,66 Peripheral 
sensitivity and circulating hormonal concentrations are expected to have a limited effect as a 
covariate (z2,3,5).  Analysis z4 is expected  to show the continuous OC usage is superior to 
cyclic OC usage for improvement of PBS, in consensus with studies by others on dysmenorrhea 
and COS.22,51  While we have observed a strong relationship between dysmenorrhea and 
bladder pain2,3, accomplishment of Phase #2 will provide prospective evidence in a randomized 
controlled trial that bladder pain sensitivity is a modifiable risk factor and understanding of the 
mechanisms responsible. They will be mailed the ovulation kits to complete before their 6- and 
12-month visits.  
 
C2.j Potential Problems: Differences in OC management within each group: There will likely 
be differences in treatments within groups because some participants will require additional OC 
pills to reduce bleeding, and PBS subjects often rotate medications to treat pain flares. To 
account for confounding, all deviations will be documented and accounted for as a covariate. 
We previously confirmed the success of this strategy in our cross-sectional study showing that 
women on OCs have less bladder sensitivity (see section IV.c.1). Also, women who stop taking 
the birth control pills but continue as intent-to-treat will not receive their monthly questionnaires 
on day 15 of each cycle (since presumably discontinuing the pills would shift their cycle). They 
will continue to receive the monthly questionnaire every 28 days as they would have had they 
continued the pills.  
 
D. Data Collection and Storage: 
Confidentiality: data will be collected by our study coordinator, who has already undergone 
human subjects protection training and has experience with data handling through our pilot 
studies.  All questionnaire data and data forms will be maintained in REDCap and exported into 
excel sheets onto a password-protected server as needed for analysis.  The only paper forms 
used in this study will be the consent and W-9 forms (for payment) containing subject name (no 
ID), and a study overview checklist, oral contraceptive eligibility checklist, session notes form 
containing the subject ID only (no name). Hard copies will be kept in a locked office or cabinet. 
Electronic data will be coded only with a unique study identifier for participants, linked to a single 
master list. The phone screen will also be maintained in REDCap and contain PHI. PHI is 
necessary to collect in order to create an Epic medical record for research participants.  At the 
conclusion of the study, patient identifiers on any hard copies will be blacked out in marker on 
all data forms. 
 
Quality assurance: All questionnaires and data forms will be entered using REDCap. 
Questionnaire data will be reviewed at the time of each evaluation in REDCap by our research 
coordinator to ensure all questions are answered and coded appropriately.  For pressure-pain 
threshold, the data acquisition program will automatically save these values.  Data will be 
reviewed and backed up onto the shared drive after each session. Computer parameters will 
identify potential outliers (using a two standard deviation cutoff) and illogical data, which will be 
reviewed by the principal investigator.  Differences will be reconciled using the session notes 
form, which will be used to record any applicable extra information which may affect testing 
(e.g., temperature of room, participant very tired) or any errors that occurred during a session. 
 
  
 
 
 

 
22 
 
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