Effects of a Vaginal Contraceptive Ring on Vaginal Microbiota and Local Immunity
Phase: PHASE4
Conditions: Bacterial Vaginosis
Sponsor: University of Washington

Effects of a vaginal contraceptive ring on vaginal 
microbiota and local immunity 
 
 
Version 7.0 
27 March 2018 
 
 
 
Principal Investigator:  Christine Johnston, MD, MPH 
 
 
 
University of Washington 
 
Address: 
 
Harborview Infectious Disease Research Clinic 
11th floor, Ninth and Jefferson Building 
Box 359928 
325 Ninth Ave 
Seattle, WA 98104 
 
Telephone number:  
(206) 520-4340 
Email:   
 
cjohnsto@uw.edu 
 
 
Co-Investigators: 
Jeanne Marrazzo, MD, MPH 
 
 
 
University of Alabaman, Birmingham 
 
 
 
 
Elizabeth Micks, MD, MPH 
 
 
 
University of Washington 
 
 

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STATEMENT OF COMPLIANCE 
This clinical trial will be conducted in accordance with the protocol and Good Clinical Practices (GCP) as 
required by the following: 
 
• 
United States (US) Code of Federal Regulations (CFR) applicable to clinical studies    (45 CFR 46; 
21 CFR Parts 50 and 56; 21 CFR Part 312). 
 
• 
ICH E6; 62 Federal Register 25691 (1997) 
 
All key personnel (all individuals responsible for the design and conduct of this study) have completed 
Human Subjects Protection Training. 
 
 

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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments, and provides the 
necessary assurances that this trial will be conducted according to all stipulations of the protocol, 
including all statements regarding confidentiality, and according to local legal and regulatory 
requirements and applicable US federal regulations and ICH guidelines. 
 
Principal Investigator: 
 
Signed: 
 
Date: 
 
     CHRISTINE JOHNSTON, MD, MPH 

TABLE OF CONTENTS 
1 
BACKGROUND INFORMATION ................................................................................................7 
1.1 
SIGNIFICANCE .................................................................................................................................. 7 
1.2 
STUDY HYPOTHESIS .......................................................................................................................... 8 
2 
STUDY OBJECTIVES .................................................................................................................8 
3 
STUDY DESIGN .......................................................................................................................9 
4 
STUDY ENROLLMENT ........................................................................................................... 10 
4.1 
ELIGIBILITY CRITERIA ....................................................................................................................... 10 
4.2 
EXCLUSION CRITERIA....................................................................................................................... 11 
4.3 
WITHDRAWAL FROM STUDY ............................................................................................................ 11 
4.4 
STUDY TERMINATION ...................................................................................................................... 11 
5 
STUDY INTERVENTIONS ........................................................................................................ 11 
5.1 
STUDY OVERVIEW .......................................................................................................................... 11 
5.2 
STUDY SAMPLES ............................................................................................................................. 12 
5.3 
CONTRACEPTION ............................................................................................................................ 13 
5.4 
CVR USE ...................................................................................................................................... 13 
5.5 
CONCOMITANT MEDICATIONS/TREATMENTS ...................................................................................... 14 
6 
STUDY SCHEDULE ................................................................................................................. 14 
6.1 
SCREENING VISIT AND ENROLLMENT VISIT FOR BV- WOMEN ................................................................ 15 
6.2 
7-10 DAY/ENROLLMENT VISIT FOR BV+ WOMEN ............................................................................... 15 
6.3 
FOLLOW UP VISITS .......................................................................................................................... 15 
6.4 
OPTIONAL IUD EXTENSION STUDY .................................................................................................... 17 
7 
STUDY PROCEDURES ............................................................................................................ 18 
7.1 
CLINICAL EXAMINATION .................................................................................................................. 18 
7.2 
LABORATORY EVALUATIONS ............................................................................................................. 18 
8 
ASSESSMENT OF SAFETY ...................................................................................................... 21 
8.1 
KNOWN POTENTIAL BENEFITS .......................................................................................................... 22 
8.2 
SPECIFICATION OF SAFETY PARAMETERS ............................................................................................ 22 
8.3 
METHODS AND TIMING FOR ASSESSING, RECORDING, AND ANALYZING SAFETY PARAMETERS ..................... 22 
8.4 
ADVERSE EVENTS, SERIOUS ADVERSE EVENTS ..................................................................................... 22 
8.5 
REPORTING PROCEDURES ................................................................................................................ 24 
8.6 
TYPE AND DURATION OF FOLLOW-UP OF SUBJECTS AFTER ADVERSE EVENTS ............................................ 25 
9 
STATISTICAL CONSIDERATIONS............................................................................................. 25 
9.1 
OUTCOMES ................................................................................................................................... 25 
9.2 
SAMPLE SIZE ................................................................................................................................. 26 
10 
QUALITY CONTROL AND QUALITY ASSURANCE ..................................................................... 26 
11 
ETHICS/PROTECTION OF HUMAN SUBJECTS .......................................................................... 26 
11.1 
ETHICAL STANDARD ........................................................................................................................ 26 
11.2 
ETHICAL APPROVAL ........................................................................................................................ 27 
11.3 
INFORMED CONSENT ...................................................................................................................... 27 
11.4 
EXCLUSION OF WOMEN, MINORITIES, AND CHILDREN (SPECIAL POPULATIONS) ....................................... 27 
11.5 
SUBJECT CONFIDENTIALITY ............................................................................................................... 27 
11.6 
STUDY DISCONTINUATION ............................................................................................................... 27 

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12 
DATA HANDLING AND RECORD KEEPING .............................................................................. 27 
12.1 
DATA COLLECTION .......................................................................................................................... 27 
12.2 
CASE REPORT FORMS (CRFS) ........................................................................................................... 27 
12.3 
DATA MANAGEMENT ..................................................................................................................... 28 
12.4 
STUDY RECORDS RETENTION ............................................................................................................ 28 
12.5 
PROTOCOL DEVIATIONS AND VIOLATIONS .......................................................................................... 28 
13 
PUBLICATION POLICY ........................................................................................................... 28 
14 
REFERENCES CITED ............................................................................................................... 29 
TABLE 1. LABORATORY SPECIMENS TO BE OBTAINED AT EACH VISIT OR BY SELF-COLLECTION ........... 31 
TABLE 2. TABLE OF STUDY PROCEDURES ........................................................................................... 33 
 
 
 
 
 

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PROTOCOL SUMMARY 
Title: 
Effects of a vaginal contraceptive ring on vaginal microbiota and local immunity 
Population: 
Premenopausal women 18 years of age or older with BV or recent history of BV, desire to use CVR, and 
not intending to become pregnant over the course of participation 
Number of 
Sites: 
1 
Study 
Duration: 
5 years 
Participation 
Duration: 
7 months (plus optional 6 month extension) 
Description of 
Agent or 
Intervention: 
Contraceptive vaginal ring (CVR) 
Objectives: 
To assess potential benefits associated with both intermittent (use for 3 weeks, remove for 1 week, as 
defined in the package insert) and continuous (use for 4 weeks, then replace) CVR use among women 
either with BV or at high risk for BV. We will also recruit women who are HSV2-infected.  
Time to 
Complete 
Enrollment: 
18 months  
 
 
 

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1 
BACKGROUND INFORMATION 
1.1 
Significance 
 
Our objective is to study effects of a contraceptive vaginal ring (CVR) containing estrogen and 
progesterone (NuvaRing) on vaginal bacteria (the vaginal microbiome), local immunity, and lower 
genital tract infection in women. Emerging data suggest a favorable effect of this CVR on vaginal 
bacteria, specifically hydrogen peroxide-producing lactobacilli (LB), which are supported when the 
vagina is under the influence of estrogen.1,2 These lactobacilli are critical for defense against infection 
with HIV and other sexually transmitted infections (STI). Bacterial vaginosis (BV) is characterized by loss 
of these LB and instead, overgrowth of a mix of anaerobic bacteria. BV significantly increases risk of 
STI/HIV, including genital herpes caused by HSV2.3,4 BV is characterized by declines in protective 
immunologic mediators (certain defensins, secretory leukocyte protease inhibitor (SLPI)) and increases 
in inflammatory cytokines and chemokines,5 alteration of cervical immune cell populations6 and 
activation of local immune cells, including T cells.7 Whether local delivery of hormones through a 
product like the CVR restores the lactobacilli and the protection they provide from BV and STI/HIV is 
unknown. In contrast, the contraceptive systemic depot progesterone—commonly used worldwide—
may independently increase risk of HIV acquisition and transmission.8 Our overarching hypothesis is that 
NuvaRing contributes to reduction in BV, healthy markers of cervicovaginal immune response and 
decreased rates of HSV2 shedding. Long-acting vaginal delivery of hormonal contraception as the 
foundation of “multicomponent prevention” – for example, delivery systems that can combine activity 
against unintended pregnancy, HIV, and other STI—has become a major focus for scientists, advocates, 
and funders alike,9-11 but effects on the vaginal environment need careful definition before broad 
implementation. 
 
The need for contraceptive options has taken on urgency with evidence indicating that systemic 
hormonal contraception (primarily injectable, long-acting progestin) may increase women’s risk of HIV 
acquisition and transmission to male sex partners.12 Lack of other contraceptive options is a critical 
barrier to progress towards optimizing women’s reproductive health. Moreover, BV remains a major 
problem for women, causing symptomatic vaginitis and conferring increased risk for adverse outcomes 
of pregnancy and STI/HIV acquisition. We propose to explore the hypothesis—supported by limited 
data—that a contraceptive vaginal ring (CVR) that is commonly used in the U.S., the NuvaRing, will 
enhance women’s genital and reproductive health. We propose that this CVR will increase the bacteria 
that help the vaginal environment protect against infection by HIV and other STIs, reduce rates of BV, 
and that in women who already have HSV2, use of the CVR will lower HSV2 shedding in the female 
genital tract. Importantly, CVR acceptability in general is excellent.13 Moreover, the format of drug 
delivery through the form of a vaginal ring is also being intensely explored with the use of antiretroviral 
agents (dapivirine, tenofovir) as a means of topical, female-controlled HIV prevention, and formulation 
work with several antiretroviral drugs for sustained delivery with the NuvaRing format is underway.9  
 
We believe the NuvaRing specifically will enhance vaginal health because we hypothesize that it will 
promote the growth of the Lactobacillus bacteria that protect women from infection with HIV and other 
STI. These bacteria, called L. crispatus and L. jensenii, are promoted by adequate levels of vaginal 
estrogen. These bacteria maintain low vaginal pH (<4.7) by producing lactic acid. BV occurs when these 
vaginal lactobacilli are replaced by overgrowth of other vaginal bacteria (primarily anaerobes, or 
bacteria that live in low-oxygen conditions). BV is the most common cause of vaginal complaints in 
reproductive-aged women. In pregnant women, BV is strongly associated with increased risk of preterm 
births and pregnancy-associated infections.  In non-pregnant women, BV increases the risk of pelvic 

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infections, and acquisition of multiple STI, including HSV2 and HIV.14 BV may elevate these risks in 
several ways, including activation of local immune cells. Finally, recent data indicate that onset of 
menstruation triggers key shifts in local vaginal bacteria, and in many women, may actually precipitate 
BV. Continuous use of the NuvaRing (use for four weeks, then replace) suppresses menstruation, and is 
increasingly used by many women with excellent reports of acceptability and safety. Whether this 
suppression of menstruation could suppress BV recurrence is not known. 
 
Although short-term antibiotic therapy improves symptoms and restores Lactobacillus predominance in 
most women with BV, up to 20% of women fail routine therapy. In addition, BV recurrence after 
successful therapy is the rule. Recurrence is likely caused by failure to recolonize the vagina with 
desirable lactobacilli, which requires adequate vaginal estrogenization. In one study, women who used 
the NuvaRing for 3 consecutive 28-day cycles had higher quantities of desirable vaginal lactobacilli 
relative to women who used oral contraceptive pills containing 20 μg ethinyl estradiol and 100 μg 
levonorgestrel.1 The NuvaRing releases 120 mcg/day of ENG (the active metabolite of desogestrel, a 
progesterone) and 15 mcg/day of ethinyl estradiol (the estrogen component). While generally well 
tolerated, incidence of local complaints (increased vaginal discharge or discomfort) with continuous CVR 
use is higher than those reported by OC users;15 thus, the risk: benefit ratio of this approach needs 
careful assessment. 
 
Pregnancy is also an independent risk for HIV acquisition and transmission, when acquisition confers 
high rates of perinatal transmission.16,17 However, systemic depot progesterone, commonly used 
worldwide, may independently increase risk of HIV acquisition and transmission.8 Increased HIV risk 
related to hormonal contraceptive use would be of global public health importance, given the large 
number of women using such methods. In a recent technical report, the World Health Organization 
noted that “expansion of contraceptive method mix and further research on the relationship between 
hormonal contraception and HIV infection is essential.”18  
 
For all of these reasons, then, a careful study of the microbiological, clinical, and immunologic benefits 
of the CVR is a high scientific and public health priority. 
 
1.2 
Study Hypothesis  
 
• 
CVR use will be associated with favorable vaginal changes assessed by BVAB qPCR, and 
community definition by high-throughput sequencing 
 
• 
Initiation of CVR use will be associated with an increase in desirable protective factors in the 
vagina, including increases in SLPI and reduction in proinflammatory cytokines. 
 
• 
CVR use will be associated with lower rates of and longer time to incident BV assessed by clinical 
status (Amsel criteria and symptoms)19 and Nugent score,20 and among HSV-2-infected women, 
with a reduction in HSV-2 shedding. 
2 
STUDY OBJECTIVES  
 
We propose to assess potential benefits associated with both intermittent (use for 3 weeks, remove 
for 1 week, as defined in the package insert) and continuous (use for 4 weeks, then replace) CVR use 

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among women either with BV or at high risk for BV. We will also recruit women who are HSV2-
infected.  
 
Primary outcomes will be as follows: 
 
• 
The primary outcomes will be the quantity of L. crispatus determined by species-specific 
qPCR assay (Aim 1), local immune parameters, including cytokines and innate mediators 
(Aim 2), and incidence of and time to clinical BV episodes over the course participation (with 
the exploratory aim of measuring genital HSV-2 shedding among HSV-2 seropositive women 
(Aim 3).  
 
• 
Secondary objectives will include a determination of Nugent score on Gram stain of vaginal 
fluid, vaginal colonization with bacteria associated with BV, the vaginal microbiome as 
determined with broad range 16S rRNA and pyrosequencing, and rates of adverse events 
with CVR use. At study end, all participants will be offered the choice of continuing 
NuvaRing use, or will be counseled on alternative (non-CVR) contraception methods. 
Women who opt to switch to an intrauterine device (IUD) at the end of the study will be 
offered participation in IUD an Extension Study, in which we will follow subjects for an 
additional 6 months to assess changes in immunity and the microbiome after IUD 
placement.  
3 
STUDY DESIGN 
 
Figure 1. Study Design & Timeline 
 
 
 
 
 
 
Screening/ 
Enrollment Visit 
7-10 
Days 
Month 1 
Month 2 
         Months 3-6 
Month 7 
End of Study 
 
 
1At the Month 7 End of Study Visit, subjects who opt to have an IUD placed for contraception will be 
offered participation in optional IUD Extension Study.  
End of 
Study 
Visit1 
Screen for 
eligible 
women with 
or at risk for 
BV 
BV- 
Enroll1 
BV+ 
Treat 
Return 
TOC & 
Enroll 
Baseline 
Assessment 
Initiate 
CVR 
Monthly 
Visits2 

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Timeline for Optional Hormonal Sub-Study Participants 
 
 
1Subjects who are BV- at screening may enroll at the same visit. 
2At the Month 6 visit, subjects are given the opportunity to discontinue CVR use. 
 
4 
STUDY ENROLLMENT 
4.1 
Eligibility Criteria 
 
• 
Women who are 18 years of age or older  
• 
BV+ by Amsel criteria and Nugent score OR history of BV in the prior 12 months 
• 
Premenopausal (as defined by having had at least 3 menstrual periods in the last 6 months, 
unless using hormonal contraception in which the investigators will use clinical discretion in 
determining eligibility). 
• 
Willing to stop hormonal contraception 
• 
Willing to use the NuvaRing as directed 
• 
Not intending or wishing to become pregnant over the course of the study 
• 
Daily access to a freezer for specimen storage  
• 
Capable of providing written informed consent 
 
4.1.1 
Additional Eligibility Criteria for Enrollment into Hormonal Sub-Study  
 
• 
BV negative at Screening/Enrollment 
• 
Have regular menstrual cycles 25-35 days in length 
• 
Have not used hormonal contraception within the past 2 months (no DMPA within the past 6 
months) 
 
4.1.2 
Additional Eligibility Criteria for Enrollment into Optional IUD Extension Study 
 
• 
Completion of Month 7 End of Study Visit 
• 
Desiring IUD placement 
• 
Willing to have IUD placed at Public Health STD clinic or by other health care provider 
• 
No contraindications to IUD use (current pelvic inflammatory disease, distorted uterine cavity 
due to fibroids or other uterine pathology) 
 
Month 7 
End of 
Study Visit 
Screen for 
eligible 
BV- 
women 
w/prior hx 
of BV 
BV- 
Enroll1 
Month 1 
Baseline 
Assessment 
Visit 1a 
Month 1 
+/- 14 
days 
Visit 1b 
Month 2 
Initiate 
CVR 
Mos. 3-6 
Monthly 
Visits2 

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4.2 
Exclusion Criteria 
 
• 
Current pregnancy  
• 
Women who are less than 6 weeks postpartum 
• 
Contraindications to hormonal contraceptive use per package insert, including history of deep 
vein thrombosis, smoking in women older than 35 years 
• 
IUD at study initiation 
• 
Unable to comprehend consent material because of language barrier or psychological difficulty 
 
4.3 
Withdrawal from Study 
 
Subjects are free to withdraw from the study at any time.  Subjects who become pregnant during the 
course of the study will not continue the study.  If a subject withdraws from the study due to CVR side 
effects or other issues and has used the CVR for at least 2 cycles, we will ask that they complete one 
final month of daily home swab collection.   
4.4 
Study Termination 
 
The study may be terminated at any time by the principal investigator. 
5 
STUDY INTERVENTIONS 
5.1 
Study Overview 
 
This study will be conducted at the University of Washington Virology Research Clinic (VRC) located at 
Harborview Medical Center in Seattle.  Eligible women with BV will be invited to enroll as will women 
with a high likelihood of experiencing BV in the near term (12 months). We plan to enroll approximately 
120 women.  
 
Among those with BV at Enrollment, all will be treated with standard antibiotic therapy. After 
assessment for cure at 7-10 days, they will be asked to return for a visit at one month from enrollment 
to establish their baseline status. They will be offered non-hormonal methods for pregnancy prevention 
during that initial month. 21 After one month (Month 2 visit), they will then be asked to initiate NuvaRing 
use using one of the following two approaches: 
 
• 
Continuous CVR use for four 28-day cycles, with replacement of the CVR after each cycle 
 
• 
Non-continuous CVR use involving use for 21 days, then removal for 7 days, with replacement 
after the 7-day CVR-free period 
 
Following initiation of CVR use, all participants will be evaluated monthly for BV with standard criteria 
(Amsel and Nugent criteria) and have specimens obtained for performance of bacterial quantitative PCR 
(qPCR) assays for BV-associated bacteria, L. crispatus and L. jenseni. Women will also be asked to seek 
care with the study team for interim vulvovaginal symptoms, with provision of diagnostic workup and 
appropriate treatment.  
 

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While participants will have the right to voluntarily stop using CVR at any point during the study, they 
will specifically be offered the option of discontinuing CVR use at six months (after 4 months of use) for 
the purpose of documenting whether the vaginal environment changes after CVR cessation. For this, 
they will return for one additional monthly visit.  
 
5.1.1 
Hormonal Sub-Study   
 
In a sub-study of 40 women, we will compare cervicovaginal effects of the contraceptive ring to the 
normal changes occurring during the luteal phase of the menstrual cycle in response to endogenous 
estrogen and progesterone. Cervical cytobrush specimens will be collected to obtain an adequate 
sample of endocervical epithelial cells, which will be used for microarray-based gene expression 
analyses.  Subjects who are otherwise eligible for the main study and who are BV negative, have regular 
menstrual cycles 25-35 days in length, and have had no use of hormonal contraception within the past 2 
months (no DMPA within the past 6 months) will be eligible for the sub-study.   Hormonal Sub-study 
participants will be asked to return for one additional visit prior to initiation of CVR for serum 
progesterone level testing and collection of cervical cytobrush samples in addition to regular visit 
specimens (same as Visit 1). 
 
5.1.2 
Intrauterine Device (IUD) Extension Study 
 
During the Month 7 visit (or after completion of the study), we will offer women enrollment in an 
optional Extension Study, which aims to assess changes in cervicovaginal immunity, vaginal microbiome, 
and HSV-2 shedding among participants who opt to initiate an IUD. Subjects who have completed the 
Month 7 visit and daily vaginal swabs during the main study will be eligible to participate.  All 
participants will receive counseling on contraceptive options at completion of the main study. Women 
who opt for an IUD will receive information regarding clinics that offer IUD placement, including the 
Public Health – Seattle & King County STD Clinic and other local family planning clinics.  Subjects will 
receive IUD placement per standard of care at individual clinics; IUDs will not be placed as part of study 
procedures. IUD Extension Study participants will be asked to return approximately one month after IUD 
placement for their first Extension Study Visit. Women who previously participated in the Hormonal 
Sub-study will also have cytobrush specimens and progesterone levels collected at 2 time points during 
the IUD Extension Study.  
 
5.2 
Study samples 
 
5.2.1 
Vaginal Fluid 
 
Swabs of vaginal fluid will be obtained from the posterior fornix for PCR assays. BV diagnosis will be 
made using any three of the following: (1) vaginal pH ≥4.7, (2) clue cells >20% of vaginal epithelial cells 
on microscopy, (3) fishy odor with KOH and (4) abnormal vaginal discharge (increased amount, differing 
from usual consistency, or malodorous). Diagnostic evaluation for C. trachomatis, N. gonorrhoeae, and 
Trichomonas vaginalis will be performed using nucleic acid amplification testing (APTIMA-Combo, 
GenProbe).  
 
Women with any condition for which treatment or further testing is indicated will be managed 

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according to standard guidelines, and will remain eligible for enrollment. BV will be treated with 
standard treatment (either metronidazole gel .75%, one application vaginally nightly for 5 days, or oral 
metronidazole, 500 mg orally twice daily for 7 days, according to the participant’s preference 
). All women treated for BV will be asked to return at 7-10 days for a test-of-cure visit, with BV cure 
defined by absence of >2 Amsel criteria.  
 
5.2.2 
Daily Home Swab Samples 
 
For one month before CVR initiation, one month after, and one month following CVR discontinuation, all 
subjects will be asked to self-collect two vaginal swabs daily and to store in their freezer for return to 
the clinic at the next visit or beforehand at their convenience. These swabs will be analyzed for vaginal 
bacteria with bacterium-specific qPCR assays.  Women with HSV-2 infection will be asked to collect one 
additional mixed anogenital swab to measure daily HSV-2 genital shedding. IUD Extension Study 
participants will be asked to complete two vaginal swabs daily  (plus an additional swab for women with 
HSV-2) at two additional time points: one month after IUD placement, and 6 months after IUD 
placement.   If a subject withdraws from the study due to CVR side effects or other issues and has used 
the CVR for at least 2 cycles, we will ask that they complete one final month of daily home swab 
collection.   
 
5.2.3 
Vaginal Biopsies 
 
Vaginal biopsies are optional.  Vaginal biopsies will be collected at two time points during the study: the 
Month 1 baseline visit (for women who agreed to the hormonal sub-study, the Month 1 vaginal biopsies 
will ideally be collected at the cycle day 6-10 (follicular phase) visit), and the Month 6 visit. Participation 
in this procedure is optional, and subjects willing to provide vaginal biopsy specimens will sign a 
separate consent form.  Subjects in the optional Hormonal Sub-Study will have one an additional vaginal 
biopsy procedure at Visit 1b.  Subjects in the IUD Extension Study will have one additional vaginal biopsy 
procedure at Month 13 (IUD 6). Subjects will be instructed to not begin collecting vaginal swabs until 5 
days after the biopsy to ensure healing.   
 
The procedure for the vaginal biopsy is as follows: A speculum will be placed into the vagina. After 
injection of 0.5 mL of 1% lidocaine with epinephrine, two biopsies approximately 2 x 2 mm in size will be 
collected from the upper posterior vaginal wall with baby Tischler forceps using standard technique. 
Hemostasis will be obtained with silver nitrate or Monsel’s ferric subsulfate solution, if indicated. Biopsy 
samples will be analyzed for epithelial thickness, glycogen content of epithelial cells, immune cell 
populations, and gene expression.  
 
5.3 
Contraception 
 
After the enrollment visit, women will be asked to maintain a one-month period of no hormonal 
contraceptive use.  They will be counseled on non-hormonal methods of contraception (abstinence, 
barrier methods) and will be provided male and/or female condoms (as they prefer) at no cost.  
5.4 
CVR Use 
 
After one month (Month 2 visit), women will be asked to initiate NuvaRing use using one of the 

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following two approaches. Participants will choose the approach they prefer, and will be provided 
standard counseling and health information about the risks and benefits associated with each: 
 
• 
Continuous CVR use for four 28-day cycles, with replacement of the CVR after each cycle 
• 
Non-continuous CVR use involving use for 21 days, then removal for 7 days, with replacement 
after the 7-day CVR-free period 
 
Participants will initiate CVR use at any time in the cycle, with backup contraception (condoms) to cover 
the appropriate time period.  We will follow standard guidelines from the U.S. Centers for Disease 
Control and Prevention (CDC), the Selected Practice Recommendations for Contraceptive Use, which 
state that women who initiate CVR within 5 days of the start of menstrual bleeding do not require back 
up contraception. Women initiating CVR >5 days after the start of menstrual bleeding require backup 
protection for the next 7 days.   
 
While the package insert mentions initiation early in cycle, we aim to keep the number of pre-initiation 
visits to a minimum. After four months of CVR use (at Month 6 visit), women will be offered the option 
to discontinue use of the CVR, will be provided condoms and will then be assessed again after one 
month. Women may elect to continue CVR use at the Month 6 visit.  CVR will be provided by the study.  
 
For subjects who opt for continuous CVR use and prefer to have their CVR removed by the study 
clinician, the CVR will be saved for future analysis.   
 
5.4.1 
IUD Use 
 
All subjects will receive comprehensive contraceptive counseling at the Month 6 and 7 visits. Women 
who choose to discontinue the CVR and initiate an IUD for contraception will be offered participation in 
an optional IUD Extension Study. Women will receive a list of clinics where they can schedule IUD 
placement, including the Public Health – Seattle & King County STD Clinic (located in the same building 
and floor as the research visits), Harborview Women’s Clinic, University of Washington affiliated clinics, 
or other family planning clinics in the area. Subjects can choose the IUD brand that they prefer, including 
the levonorgestrel-releasing IUD (Mirena, Liletta, Skyla, Kyleena), or the nonhormonal copper IUD 
(Paragard). Screening, pre-procedure preparation, and the IUD insertion will be performed as part of 
primary care by health care providers and will not be part of study procedures. Subjects will be 
instructed to contact the study coordinator after IUD placement to schedule their Month 8 (IUD 1) visit.  
 
5.5 
Concomitant Medications/Treatments 
 
Administration of any medication or therapies considered necessary for the subject’s welfare will be 
documented in the subject’s case report form in the concomitant medication section at each visit, 
including treatment for BV and STIs.  
6 
STUDY SCHEDULE 
 
Specimens collected at each study visit and by home collection are summarized in Table 1. 
 

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6.1 
Screening Visit and Enrollment Visit for BV- Women 
 
Subjects will be consented prior to any study procedures.  Screening procedures include a self-collected 
vaginal swab (for assessment of Amsel’s critera), a brief medical history, and urine pregnancy test.  
Subjects who test positive for BV will be treated with standard treatment for BV and asked to return in 
7-10 days for test of cure (TOC). 
 
Women who are BV- will proceed with enrollment procedures including a pelvic examination and 
collection vaginal specimens for Gram stain, specimens for qPCR, broad range pyrosequencing and NAAT 
testing. CVF via Soft Cup will also be collected.  Blood will be drawn for HIV and HSV antibody testing.  
 
Information on symptoms (vaginal discharge, pruritis), types, frequency, and timing of sexual behavior 
(oral, vaginal, anal), menstrual history, antibiotic and hormone use, lubricants, condoms, and douching 
will be collected via study staff interview and computer questionnaire.  Subjects will be counseled in use 
of non-hormonal contraception and offered condoms and will be asked to return in one month.   
 
Women will be assessed and offered enrollment into the Hormonal Sub-Study and Vaginal Biopsy Sub-
Study.  For women enrolled in the optional hormonal sub-study, additional specimens may be collected 
(see Section 6.3.2  below), if within the cycle day window.   
 
6.2 
7-10 day/Enrollment Visit for BV+ Women  
 
Subjects who are BV+ at screening will be asked to return in 7-10 days and then will proceed with 
enrollment procedures described for BV- women in Section 6.1 above.  Subjects who remain BV+ at the 
7-10 Day visit are eligible for enrollment. 
 
6.3 
Follow up visits 
 
Women will be asked to return for up to follow up visits monthly at Months 1, 1b (for those women 
participating in the Hormonal Sub-Study), 2, 3, 4, 5, 6, and end of study visit at Month 7 for repeat 
interview/questionnaire, pelvic examination, and collection of study specimens.  Between all study 
visits, women will be asked to avoid self-treatment with over-the-counter vaginal medications until 
evaluated by study clinicians if possible.  
 
At each follow-up visit, information on symptoms (vaginal discharge, pruritus), types, frequency, and 
timing of sexual behavior (oral, vaginal, anal), menstrual history, antibiotic and hormone use, lubricants, 
condoms, and douching will be collected.  Additionally, pH and Gram stain of vaginal fluid, vaginal swabs 
for BVAB-specific PCRs and cervical fluid will be performed.  
 
6.3.1 
Month 1 Visit 
 
A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Subjects will be provided with a daily home collection kit and instructions for collecting 
specimens over the 30 days prior to CVR initiation.  Subjects will also receive their HSV and HIV test 
results at this visit.  

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For women enrolled in the optional hormonal sub-study, additional specimens may be collected (see 
Section 6.3.2 below), if within the cycle day window and if not already collected at Enrollment.   
 
Vaginal biopsy will offered at this visit.  Women who choose to participate will sign the vaginal biopsy 
consent form and the biopsies will be collected.   For women enrolled in the optional hormonal sub-
study, the Month 1 vaginal biopsies will ideally be collected at the cycle day 6-10 (follicular phase) visit. 
 
6.3.2 
Hormonal Sub-Study Visit 1b (Cycle day 6-10 or Cycle day 20-25) 
 
For women enrolled in the hormonal sub-study, additional samples will be collected at two specific time 
points during their menstrual cycle prior to CVR initiation: follicular phase (cycle day 6-10) and luteal 
phase (cycle day 20-25).   
• 
The first visit will be timed to occur at Enrollment or Month 1, depending on cycle day.   
• 
The second (and additional) visit (Visit 1b) will occur outside their monthly study visits, timed to 
capture their other cycle phase; either before or after Month 1 depending on cycle timing.  
Procedures at Visit 1b will be identical to the monthly visits.   
 
Additional Sub-Study Samples: At each sub-study visit,  a cervical cytobrush specimen will be collected 
during the pelvic exam and blood will be drawn (1.0 mL) to measure progesterone levels.   
 
6.3.3 
Month 2 
 
A pelvic exam will be performed and study specimens will be collected. Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  In addition, CVR will be initiated at this visit.  Daily home swab samples from the 
previous 30 days will be collected and reviewed.  Subjects will be provided with a new daily home 
collection kit and instructions for collecting specimens over the next 30 days following CVR use.   
 
6.3.4 
Months 3, 4 and 5 
 
A pelvic exam will be performed and study specimens will be collected.   Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire at each visit. New CVRs will be dispensed.  At the Month 3 visit, daily home swab samples 
from the previous 30 days will be collected and reviewed.   
 
For women enrolled in the optional hormonal sub-study, at the Month 3 visit, a cervical cytobrush 
specimen will be collected during the pelvic exam and blood will be drawn (1.0 mL) to measure 
progesterone levels.   
 
6.3.5 
Month 6 - Stop CVR 
 
A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Participants will be asked if they wish to discontinue CVR at this visit.  Those who elect to 

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stop will be provided with non-hormonal contraception (condoms).  Subjects who wish to continue 
using CVR will be provided a new ring at this visit.  All subjects will be asked to collect daily swabs at 
home over the next 30 days.  Home collection kits and instructions will be provided. Vaginal biopsies will 
be collected at this visit, for women who choose to participate and had baseline biopsies at the Month 1 
visit.  
 
6.3.6 
Month 7 - End of Study Visit 
 
A pelvic exam will be performed and study specimens collected.  Information on symptoms, sexual 
behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire. Daily home swab samples from the previous 30 days will be collected and reviewed.   
Participants will receive comprehensive contraception counseling.  Participants who were HIV and/or 
HSV-2 seronegative at baseline will have blood drawn for repeat testing.   
 
Women who express interest in the IUD will be provided a referral to clinics that provide IUD placement 
and will be offered participation in optional IUD Extension Study.  Women will be offered same-day 
placement at the Public Health – Seattle & King County STD Clinic (as available), or will be able to 
schedule visits at Harborview Women’s Clinic or other family planning clinics in the area.  
 
6.4 
Optional IUD Extension Study 
 
6.4.1 
Month 8 (IUD 1) – First Extension Study Visit 
 
Informed consent for the optional IUD Extension Study will be obtained prior to any study procedures.  
A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Subjects will be asked to collect daily swabs at home over the next 30 days.  Home 
collection kits and instructions will be provided. 
 
6.4.2 
Month 9 (IUD 2) 
 
A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Daily home swab samples from the previous 30 days will be collected and reviewed.  For 
women enrolled in the Hormonal Sub-study, a cervical cytobrush specimen will be collected during the 
pelvic exam and blood will be drawn (1.0 mL) to measure progesterone level.   
 
6.4.3 
Months 10-12 (IUD 3-5) 
 
Monthly phone calls for retention and questions about IUD use. 
 
6.4.4 
Month 13 (IUD 6) 
 

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A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Subjects will be asked to collect daily swabs at home over the next 30 days.  Home 
collection kits and instructions will be provided. Vaginal biopsy will be collected at this visit, for women 
who choose to participate and had previous biopsies at Month 1 and Month 6 visits. 
 
6.4.5 
Month 14 (IUD 7) 
 
A pelvic exam will be performed and study specimens will be collected.  Information on symptoms, 
sexual behavior, antibiotic and hormone use, condoms and douching will be collected via interview and 
questionnaire.  Daily home swab samples from the previous 30 days will be collected and reviewed.  For 
women enrolled in the Hormonal Sub-study, a cervical cytobrush specimen will be collected during the 
pelvic exam and blood will be drawn (1.0 mL) to measure progesterone level.   
 
7 
STUDY PROCEDURES 
7.1 
Clinical Examination 
 
Pelvic examination: all findings will be recorded in the CRF Exam Form. 
 
Pelvic Examination and Specimen Collection: 
A non-lubricated speculum must be used during pelvic examination. After visual external examination 
and CVF (soft cup) collection, specimen collection/examination should take place in the following order. 
Table 1 summarizes the samples to be collected at various visits. 
 
Vaginal Discharge Identification 
Observe for presence of discharge and, if present, characterize by visual examination. 
 
7.2 
Laboratory Evaluations 
 
7.2.1 
Clinical Laboratory Evaluations 
 
pH Measurement 
Measure pH of vaginal discharge with pH indicator sticks. Samples for pH should be obtained from the 
lateral vaginal wall.  Avoid contact with cervical mucus. 
 
Vaginal & Cervical Swabs/Fluid 
There will be a total of 6 vaginal swabs, collection of vaginal fluid using a soft cup, and 1 endocervical 
cytobrush for those participating in the optional study at Months 1a, 1b and 3: 
1. Vaginal fluid collected using soft cup for measurement of cytokines and other immune 
mediators; 
2. Saline and KOH microscopy (to asses for amine odor, clue cells, trichomonads, and yeast); 
3. Gram stain of vaginal fluid (to confirm presence of BV, if present); 

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4. Nucleic acid amplification test (NAAT) for gonorrhea, chlamydia, and trichomoniasis; (lateral 
vaginal wall, p. 23; collected at Enrollment and then as indicated at subsequent visits) 
5. Vaginal fluid swab for storage (Epi-Centre); 
6. Quantitative PCR assays for vaginal bacteria (2 Epi-Centre swabs); (lateral vaginal wall, p. 23) 
7. Endocervical cytobrush for immune cell characterization (collected at Enrollment (Month 1a), 
Month 1b and Month 3 visits) for those subjects enrolled in the Hormonal sub-study. 
 
Test for Volatile Amines (Whiff Test), Yeast, Clue Cells and Motile Trichomonads 
Swab lateral vaginal walls with a cotton-tipped applicator and place a liberal amount of discharge on 
each of two glass slides.  Mix two drops of 10% potassium hydroxide (KOH) with the discharge on one 
slide and immediately determine if a fishy amine-like odor is present. Mix two drops of 0.9% saline 
solution with the discharge on the other slide and place a cover slip over both slides. Examine both 
slides under a light microscope at 10X and at 40X magnification. On the KOH slide, note the presence or 
absence of yeast (buds and hyphal elements). On the saline slide, note the presence or absence of 
motile trichomonads and clue cells. Clue cells will be identified as vaginal epithelial cells with such heavy 
coating of bacteria surrounding them that their peripheral borders are obscured. Three representative 
fields should be examined and the ratio of clue cells to vaginal epithelial cells determined. Clue cells 
should be > 20% of vaginal epithelial cells to meet criteria for diagnosis of BV.  
 
NAAT for STIs on Vaginal Swabs 
The clinician will obtain a vaginal swab using the swabs supplied for the GenProbe Aptima test for 
trichomonas/chlamydia/gonorrhea). The swab will be sent to the HMC CT Laboratory.   
 
Urine Sample 
Collected to test for pregnancy at Screening and at additional visits, if indicated. 
 
Serum Samples 
For HSV testing, 10.0 ml of blood will be collected in a red top tube. For HIV testing, 2.0 ml of blood will 
be collected in a lavender-top tube.  Specimens will be transported to the UW Virology Laboratory in 
Seattle.  
 
For progesterone testing, collect 1.0 mL of blood in a red top tube for testing at RTS located at HMC. 
 
7.2.2 
Research Laboratory Evaluations 
 
Collection of Endocervical Fluid Using SoftCup   
Soft Cup: After all swabs are collected, a flexible menstrual soft cup will be inserted gently into the 
vagina. The participant will be encouraged to ambulate following insertion. The cup will then be 
removed, divided and placed into two tubes, and stored for eventual transport to the Fredricks’ 
laboratory in Seattle. 
 
Sample for Gram Stain 
Swab posterior fornix with a cotton-tipped applicator.  Place a moderate amount of discharge on slide 
and spread it as evenly as possible and allow the slide to air dry.  With a pencil, label the slide with the 
subject’s study ID, visit number and date.  Place the slide in a plastic slide holder or slide box for 
transport to the lab for reading.  All Gram stains will be read by microbiologists without knowledge of 

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any patient’s clinical condition and interpreted in accordance with pre-determined criteria. Slides will be 
sent to the Fredricks’ Laboratory in Seattle. 
 
Vaginal Swabs for Molecular Analyses and Storage 
Obtain three additional swabs of vaginal fluid, using the Epi Probe swabs supplied, from the lateral 
vaginal walls. Two are for performance of quantitative PCR assays, and the other is for storage for future 
analysis. Two swabs should be placed in the -70 freezer for later transport to Dr. David Fredricks’ lab. 
The remaining (3rd) swab should be retained in the -70 freezer at UW VRC for future analysis.  
 
Vaginal biopsies 
Vaginal biopsies will be collected at 2 study visits (Month 1 and Month 6), and Month 13 (IUD 6) for IUD 
Extension Study participants.  
 
Two biopsies will be collected at each visit. One specimen will be divided; one half will be analyzed by 
Dr. Dorothy Patton’s lab for epithelial cell layer thickness and immunohistological staining for cellular 
glycogen content, and the other half will be analyzed for immune cell populations at the Fred 
Hutchinson Cancer Research Center (Dr. Florian Hladik and Dr. Jennifer Lund’s lab). The second biopsy 
will be preserved in RNAlater (at room temperature for 24 hours to allow tissue penetration, followed 
by storage at -70C) and processed for RNA-Seq.  
 
CVR Storage for Future Analysis  
CVRs that are removed at the clinic will be saved for future analysis.   
 
7.2.3 
Laboratory Procedures and Specimen Processing 
 
At all visits (listed in Table 1) vaginal material will be collected using polyester-tipped swabs or Epi-
Centre swabs which will be stored locally at -70 C prior to shipping to the Fredricks Lab in Seattle. Refer 
to the SoftCup SOP for specimen processing.   
 
Quantification of cytokines, chemokines, and antimicrobial soluble immune mediators in vaginal fluid:  
For measurement of local immune mediators, we will use a combination of transcriptional profiling of 
endocervical cells as well as ELISAs.  We will use RNASeq and RT-PCR to measure transcriptional profiles 
of endocervical cells collected before, during, and after CVR initiation.  We will measure concentrations 
of total protein (BCA Protein Kit) and the following:  IL-1α, IL-1β, IL-2, IL-6, IL-7, IL-8, IL-12p70, IL-1ra, 
IFN-epsilon, RANTES, MIP-3α, MIP-1α and MIP-1β by multiplex proteome array with beads from 
Chemicon International, measured using Luminex and analyzed using StarStation (Applied Cytometry 
Systems). These mediators were selected because they are present in genital tract secretions and have 
been linked to modulation by exogenous sex hormones, and with STI/HIV risk. We will also measure the 
concentration of select anti-inflammatory mediators and antimicrobial proteins that may contribute to 
host defense using commercial ELISA kits: SLPI (R & D Systems), HNP1-3 (HyCult Biotechnology), HBD-1, 
HBD-2 and HBD-3 (Alpha Diagnostics), lactoferrin (Calbiochem) and lysozyme (Alpco Diagnostics).  
 
Performance of vaginal proteomic analysis: We will perform proteomic analysis on saved samples from 
this study, at a laboratory to be determined. We anticipate that the Protein Profiling technique of 
Multiplexed Isobaric Tagging Technology for Relative Quantitation (iTRAQ)22 will be used to determine 
protein expression changes in the vaginal proteome between 20 women, obtained at two visits: one 

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prior to CVR insertion, and one two months after CVR insertion. For the analysis, we will select visits at 
which BV (or other STI) are not present to maximize likelihood that proteomic changes will be less likely 
to be influenced by other processes. To maximize likelihood of identifying unique proteins that 
contribute, we will select samples with highest and lowest quantities of L. crispatus and lowest and 
highest Nugent scores, respectively. For this set of 40 samples, 6 x 8 plexes will be used. One tag in each 
iTRAQ set will contain a pooled standard of all of the samples and will be used in analysis to cross 
compare the 6 iTRAQ samples sets. LC-MS/MS is performed on an AB SCIEX TripleTOF® 5600 mass 
spectrometer equipped with a Waters nanoACQUITY UPLC system. Protein identification and 
quantitation is done with the AB ProteinPilot™ software. Data are deposited into the Yale Protein 
Expression Database (YPED)23 which is an integrated web-accessible software system that addresses the 
storage, retrieval, and integrated analysis of high throughput proteomic and small molecule analyses. 
For biostatistical analysis, the ProteinPilot peptide summary is filtered to include only peptides with 1) 
no mis-cleavages, 2) high confidence peptide identifications, and 3) two or more iTRAQ reagent ratios 
for this peptide. A cyclic lowess normalization24 is then performed to compensate for any differences 
between iTRAQ labels.   
8 
ASSESSMENT OF SAFETY 
 
Side effects of CVR use: As noted above, the NuvaRing has an acceptable safety profile, is approved for 
use by the U.S. F.D.A., and is widely used by U.S. women for contraception. We will routinely query 
participants prior to use for any contraindications to CVR use, and also carefully assess at each visit for 
the development of any interim clinical processes of concern (for example, thrombosis) that might 
represent a side effect of the CVR as listed in the package insert. Participants will also be asked to 
contact us at any time during the study should they have concerns about new symptoms or signs. 
 
Management of adverse events & counseling related to CVR: Participants will receive standard 
counseling about anticipated side effects of CVR initiation, and will be assessed for any adverse events 
reported during the course of the study as needed. The main adverse event we anticipate is irregular 
bleeding among women who use the CVR continuously; this is typically mild and resolves within first two 
cycles of use. Dr. Sarah Prager, a collaborator on this project, is highly trained in management of 
contraceptive use, and will provide expert guidance as needed. 
 
Phlebotomy:  May cause some pain or bruising.   Some persons may feel light-headed or faint when 
blood is drawn.   
 
Collection of swabs and cervicovaginal samples from genital areas:  If samples are collected during 
herpes episodes, this may cause some irritation from the already sore lesion.  Collection of swabs does 
not pose risk of additional infection.   
 
IUD risk:  There is a very small risk that the IUD could be pulled out at the time of pelvic exam or during 
sample collection.   
 
Data collection:  Participants may feel some stress or discomfort answering personal and sensitive 
questions about their health and sexual practices.   
 
Pelvic/genital examination:  Discomfort should be similar to that experienced for a PAP test.  There 
may be a feeling of pressure from the speculum, which may be used to hold the vaginal walls open.  

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Participants may briefly feel pressure when the swab touches the cervix when samples are taken for 
bacterial or viral cultures. 
 
Side effects of oral and intravaginal metrondiazole gel:  These medications may be needed to treat BV 
during study participation. Interactions between oral metronidazole and alcohol can produce nausea, 
vomiting, and flushing.  Alcohol should be avoided while taking oral metronidazole or using intravaginal 
metrondiazole gel.  Yeast infections may be associated with metronidazole gel.  Participants will be 
checked for a yeast infection if they develop symptoms during the study.   
 
Risks of vaginal biopsy:  Patients may experience pain associated with the speculum examination. They 
also may have pain associated with lidocaine injection and the biopsy. There are small risks of bleeding 
and infection. Risk of bleeding will be minimized by biopsy location along the upper anterior or posterior 
aspect of the vagina, use of lidocaine with epinephrine, observation and use of silver nitrate or Monsel’s 
solution to ensure hemostasis. To minimize the risk of delayed bleeding, participants will be instructed 
to avoid sexual intercourse or placement of tampons for 5 days.  Subjects will be instructed to not begin 
collecting vaginal swabs until 5 days after the biopsy to ensure healing.   All procedures will be 
performed by a board-certified gynecologist or experienced nurse practitioner who has performed 
vaginal biopsies for other research studies. 
 
8.1 
Known Potential Benefits 
 
Subjects may benefit from participation in this study by having a blood test for detection of infection 
with HSV2 and HIV, and by finding out more about the likelihood of their having BV or recurrent BV.  
They will not be charged for any of the diagnostic tests.  Subjects may benefit from pre and post-test 
counseling, treatment and referrals, as necessary.  We have found that subjects are empowered by 
knowing how frequently they shed HSV-2, for example, and by knowing more about what BV is and how 
it can be prevented.   Otherwise, this study will not be of direct benefit to the subjects.   
 
8.2 
Specification of Safety Parameters 
 
Safety will be assessed by the frequency, incidence and severity of AEs and SAEs solicited in-clinic and 
via phone call. 
8.3 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters  
 
8.4 
Adverse Events, Serious Adverse Events 
 
The investigator is responsible for ensuring all AEs that are observed or reported during the study are 
documented.    
8.4.1 
Definition of Adverse Event 
 
Adverse Event:  International Conference on Harmonisation (ICH) guideline E6 defines an AE as any 
untoward medical occurrence in a patient or clinical investigation subject administered a pharmaceutical 
product regardless of its causal relationship to the study treatment.  An AE can therefore be any 
unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease 

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temporally associated with the use of medicinal (investigational) product.  The occurrence of an AE may 
come to the attention of study personnel during study visits and interviews or by a vaccine recipient 
presenting for medical care.  
 
Severity of Event:  All AEs will be assessed by the clinician using a standard grading system (NIH DAIDS 
Adverse Events Toxicity Tables). For events not included in the protocol defined grading system, the 
following guidelines will be used to quantify intensity:  
 
Mild: events require minimal or no treatment and do not interfere with the patient’s daily 
activities. 
 
Moderate: events result in a low level of inconvenience or concern with the therapeutic 
measures. Moderate events may cause some interference with functioning. 
 
Severe: events interrupt a patient’s usual daily activity and may require systemic drug therapy or 
other treatment. Severe events are usually incapacitating. 
 
Life threatening: any adverse drug experience that places the patient or subject, in the view of 
the investigator, at immediate risk of death from the reaction as it occurred, i.e., it does not 
include a reaction that had it occurred in a more severe form, might have caused death. 
 
Relationship to study procedures:  The investigator’s assessment of the relationship of an AE to study 
procedures is part of the documentation process.  If there is any doubt as to whether a clinical 
observation is an AE, the event should be reported to the PI.  AEs will be assessed according to the 
relationship to the study process: associated or not associated. To help assess, the following guidelines 
are used. 
 
Associated – The event is temporally related to the study procedures and no other etiology 
explains the event. 
 
Not Associated – The event is temporally independent of study the study procedures and/or the 
event appears to be explained by another etiology. 
 
8.4.2 
Definition of Serious Adverse Event 
 
A SAE is defined as an AE meeting one of the following conditions: 
- 
Results in death during the period of protocol defined surveillance. 
- 
Is life-threatening (defined as a subject at immediate risk of death at the time of the event). 
- 
Requires inpatient hospitalization or prolongation of existing hospitalization during the period of 
protocol-defined surveillance. 
- 
Results in congenital anomaly or birth defect. 
- 
Results in a persistent or significant disability/incapacity. 
- 
Any other important medical event that may not result in death, be life threatening, or require 
hospitalization, may be considered an SAE when, based upon appropriate medical judgment, the 
event may jeopardize the subject and may require medical or surgical intervention to prevent 
one of the outcomes listed above.  Examples of such medical events include allergic 
bronchospasm requiring intensive treatment in an emergency room or at home, blood 

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dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of 
drug dependency or drug abuse. 
 
8.5 
Reporting Procedures 
 
Adverse events including local and systemic reactions not meeting the criteria for SAEs will be captured 
on the appropriate CRF.  Information to be collected includes event description, date of onset, 
investigator assessment of severity, investigator assessment of relationship to study product, date of 
resolution of the event, seriousness, and outcome.  The intensity of nonserious AEs can be assessed by a 
licensed clinician (i.e., physician, nurse, Nurse Practitioner, Physician Assistant).  Causality of nonserious 
AEs can be assessed only by a clinician licensed to make medical diagnoses (ie, physician, Nurse 
Practitioner, Physician Assistant).  All AEs occurring while on study will be documented appropriately 
regardless of relationship.  All AEs will be followed to adequate resolution or until considered stable.  
 
Any medical condition that is present at screening will be considered as baseline and will not be 
reported as an AE.  If the severity of any pre-existing medical condition increases during the study 
period, then it will be recorded as an AE. 
 
All SAEs will be reported immediately to the Investigator.  
 
SAE Contact:   Christine Johnston, MD PI.  
Phone: 206-520-4340, Fax 206-520-4371, Pager 206-540-6324, cell 206-370-2091 
 
SAE Alternate: A physician from the Virology Research Clinic is available at any time by calling the clinic 
pager at 206-598-0924. 
 
The SAE report will include the following information (as available) 
- 
Patient ID 
- 
Description of SAE (onset date, severity, causal relationship) 
- 
Basic demographic information 
- 
Outcomes attributed to the event 
- 
Summary of relevant test results, laboratory data, and other relevant history 
- 
The first and last dates of study drug administration 
- 
Statement whether study drug was discontinued or schedule modified 
- 
Statement whether the event abated after study drug was discontinued or schedule modified  
- 
Statement whether the event recurred after reintroduction of the study drug if it had been 
discontinued. 
 
8.5.1 
Follow-up of SAE 
 
All SAEs will be followed until satisfactory resolution or until the principal investigator or sub-
investigator deems the event to be chronic or the subject to be stable.  
8.5.2 
Serious AE Detection and Reporting 
 

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All SAEs will be: 
- 
Recorded on the appropriate SAE report form. 
- 
Followed through resolution by a study physician. 
 
Timelines for submission of an SAE form are as follows:   
- 
All deaths and life-threatening events regardless of relationship, will be recorded on the SAE 
form and the PI will be notified within 24 hours of identification of the SAE. 
 
All SAEs determined to be related to study procedures will be reported to the Human Subjects Division 
of the University of Washington within 10-days of becoming aware of the event.  
8.5.3 
Reporting of Pregnancy 
 
Pregnancies occurring in study subjects will be recorded.   
 
8.6 
Type and Duration of Follow-up of Subjects after Adverse Events 
 
Adverse events will be followed until resolved or considered stable. 
9 
STATISTICAL CONSIDERATIONS 
 
9.1 
Outcomes 
 
Aim 1. The primary outcome will be quantity of L. crispatus measured by qPCR at all available follow-up 
visits and will be compared during five months of CVR use to one month prior to CVR use. Secondary 
outcomes will be BV defined clinically by Amsel and confirmed by Nugent criteria at any follow-up visit, 
quantities of other BVAB defined by qPCR assay, and reported genital adverse events.  Quantity and 
presence of other lactobacilli and BVAB species detected by PCR will be assessed using the methods 
outlined above. All available follow-up observations will be used. Quantity of L. crispatus will be log 
transformed and analyzed using a linear mixed model with subject random effect to account for 
correlation within person. Binary outcomes will be analyzed using generalized estimating equations with 
log link and robust standard errors to estimate relative risks while accounting for correlation.  
 
Aim 2. The outcomes of interest will be compared within participants, using each woman as her own 
control; observations from the time prior to CVR initiation will be compared to those collected during 
CVR use. We will use methods for Normal data and then use non-parametric approaches or dichotomize 
and use binomial link GEE if data are highly skewed and cannot be transformed into a Normal 
distribution. Some analyses will be exploratory; we will compare quantities of several immune 
mediators noted above by CVR use. These comparisons will not likely be independent, so we will adjust 
for multiple comparisons by permutation procedure. We will also correlate concentrations of immune 
mediators with qPCR results in Aim 1 to test the hypothesis that higher concentrations of protective 
lactobacilli will be associated with higher levels of protective mediators, and lower levels of 
inflammatory cytokines/chemokines. Continuous variables between comparison groups will be 
compared using t-tests or Wilcoxon rank-sum tests, and dichotomous variables will be compared by chi-
square tests or Fisher's exact tests. Linear mixed effects models or generalized estimating equation 
(GEE) models will be used to examine whether outcomes (mediator concentrations, vaginal bacterial 

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quantities) differ significantly between comparison groups after adjustment for confounding factors 
identified through univariate analysis. All analyses will assume a two-sided significance level of 0.05.  
 
Aim 3. The primary outcomes will be (1) incidence of BV at all available follow-up visits, and (2) time to 
BV during four months of CVR use relative to the two months prior to CVR use. For secondary analysis, 
we will measure the quantity of HSV-2 by PCR. This will be log transformed and association with CVR use 
examined using a linear mixed model as for Aim 1.  Sample size calculation: Data from our prospective 
studies of BV were used to estimate the likelihood of these conditions occurring during follow-up. Over 
six months of observation, we have documented BV recurrence rates after cure of an initial BV episode 
of 28 / 100 person-years in absence of any hormonal contraception. A recent meta-analysis estimated 
that HC was associated with a 30% reduced likelihood of BV.25 With these assumptions, we will have 
reasonable power to detect a 30% or greater difference in incidence of BV by CVR status (Table 5). 
However, BV incidence may be lower, and mechanisms for a favorable shift in vaginal microbiome, if 
affected by CVR, may be heterogeneous. If so, we will still be able to generate important preliminary 
data in support of the CVR’s favorable effect on the vaginal environment through demonstration of 
improved microbiome maintenance and immune profile; this would support future study of the CVR in 
populations with higher incidence of BV and other cervicovaginal STI. 
9.2 
Sample Size 
 
Aim 1: Total anticipated enrollment is 120 women. We will screen approximately 56 women per month 
over 18 months and estimate that about 14 women/month will be interested in participating. We 
anticipate that of the interested women, approximately 50% will have BV or a history of BV in the prior 6 
months, for predicted enrollment of 6-7 women per month. Assuming at least 75% of women return for 
at least one pre- and one post-CVR visit, we will have at least 90 analyzable women.  
 
Aim 2: Participants are as described in Aim 1.  
 
Aim 3:  Participants are as described in Aim 1. Moreover, we anticipate that approximately half of the 
total women enrolled will be HSV-2-infected at study entry (60 women).   
10 QUALITY CONTROL AND QUALITY ASSURANCE 
 
The principal investigator or designee will review the charts of the first 5 participants who enroll in the 
study to ensure high quality data are being collected.  A 10% chart review will then be performed 
quarterly by the PI. 
11  ETHICS/PROTECTION OF HUMAN SUBJECTS 
 
11.1 Ethical Standard 
 
The investigator will ensure that this study is conducted in full conformity with the principles set forth in 
The Belmont Report: Ethical Principles and Guidelines for the Protection of Human Subjects of Research 
of the US National Commission for the Protection of Human Subjects of Biomedical and Behavioral 
Research (April 18, 1979) and codified in 45 CFR Part 46 and/or the ICH E6; 62 Federal Regulations 25691 
(1997). 

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11.2 Ethical Approval 
 
Prior to the initiation of the study, the principal investigator will obtain written approval to conduct the 
study via her Institutional Review Board (IRB). Approval is required for the study protocol plus 
amendments, informed consent form, participant information sheet and advertising materials. 
11.3 Informed Consent 
 
The investigator or study clinician/staff will explain the purpose and nature of the study, including 
potential benefits and risks to the participant, to each potential participant before enrollment in the 
study. The participant must sign an informed consent form approved by the IRB before entering the 
study. All original informed consent forms must be retained by the principal investigator with the 
participant’s records. The participant will receive a copy of the signed informed consent form.   
11.4 Exclusion of Women, Minorities, and Children (Special Populations) 
 
This study will be inclusive of all healthy women who meet the inclusion/exclusion criteria, regardless of 
religion, sex, or ethnic background.   Pregnant women, men, and children will not be eligible for this 
study. 
11.5 Subject Confidentiality 
 
Subjects will have coded study identification numbers and will not be identified by name.  Subject 
confidentiality is strictly held in trust by the investigator, her staff, and the University of Washington.  
This confidentiality extends to genetic and biological sample tests, in addition to the clinical information 
relating to participating subjects. 
 
The study protocol, documentation, data and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized third 
party without prior written approval of the sponsor. 
11.6 Study Discontinuation 
 
The participant may stop the study at any time.  The investigator may terminate a participant’s study 
participation.  In the event of study discontinuation, reasons for discontinuation will be clearly 
documented. 
12 DATA HANDLING AND RECORD KEEPING 
 
12.1 Data collection 
 
Clinic records for participants are the responsibility of the investigator. 
 
12.2 Case Report Forms (CRFs) 
 
Case report forms are to be completed and signed for each participant by the investigator or designated 
member of the study staff. CRFs will be completed neatly and legibly. Any modification of previously 

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entered data must be made by the investigator or study coordinator by striking through the original 
entry with a single line, initialing and dating changes, and entering the correct data nearby. A valid 
explanation must be given for any missing information. All data will be entered into a computer 
database and further quality control check will be made to produce a final database for analysis. 
 
12.3 Data Management 
 
Data will be entered and stored in RedCAP (Research Electronic Data Capture), a secure online 
application used for data collection and management. RedCAP provides audit trails for tracking user 
activity and data manipulation, as well as automated data export compatible with statistical software 
packages. 26 
 
12.4 Study Records Retention 
 
Subject charts including CRFS, notes, and other source data will be retained according to the archival 
policy of the University of Washington. 
 
12.5 Protocol Deviations and Violations 
 
A protocol deviation is any noncompliance with the study protocol, or GCP requirements. The 
noncompliance may be either on the part of the subject, the investigator, or the study site staff. As a 
result of deviations, corrective actions are to be developed and implemented promptly.  
 
These practices are consistent with ICH E6: 
4.5 
Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3 
5.1 
Quality Assurance and Quality Control, section 5.1.1 
5.20 
Noncompliance, sections 5.20.1 and 5.20.2. 
 
All deviations from the protocol must be addressed in study subject source documents. Protocol 
deviations and violations will be sent to UW IRB per their guidelines. The site principal investigator/study 
staff is responsible for knowing and adhering to their IRB/Independent Ethics Committee requirements. 
13 PUBLICATION POLICY 
 
Following completion of the study, the investigator will publish the results of this study in a scientific 
journal. 

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14 REFERENCES CITED 
 
1. Veres S, Miller L, Burington B. A comparison between the vaginal ring and oral contraceptives.  
Obstet Gynecol. 2004 Sep; 104(3): 555-63. 
2.  Wilson JD, Lee RA, Balen AH, Rutherford AJ.  Bacterial vaginal flora in relation to changing 
oestrogen levels.  Int J STD AIDS. 2007 May; 18(5): 308-11. 
3. Atashili J, Poole C, Ndumbe PM, Adimora AA, Smith JS. Bacterial vaginosis and HIV acquisition: a 
meta-analysis of published studies. Aids 2008; 22:1493-501. 
4. Cohen CR, Lingappa JR, Baeten JM, et al. Bacterial vaginosis associated with increased risk of 
female- to-male HIV-1 transmission: a prospective cohort analysis among African couples. PLoS 
Med 2012;9:e1001251. 
5. Mitchell C, Balkus JE, Fredricks DN, et al. Interaction between lactobacilli, bacterial vaginosis- 
associated bacteria and HIV-1 RNA and DNA genital shedding in US and Kenyan women. AIDS 
research and human retroviruses 2012. 
6. Rebbapragada A, Howe K, Wachihi C, et al. Bacterial vaginosis in HIV-infected women induces 
reversible alterations in the cervical immune environment. J Acquir Immune Defic Syndr 2008; 
49:520-2. 
7. Spear GT, St John EP, Zariffard MR. Bacterial vaginosis and human immunodeficiency virus 
infection. AIDS Res Ther 2007;4:25. 
8. Heffron R, Donnell D, Rees H, et al. Use of hormonal contraceptives and risk of HIV-1 
transmission: a prospective cohort study. Lancet Infect Dis 2012; 12:19-26. 
9. Kiser PF, Johnson TJ, Clark JT. State of the art in intravaginal ring technology for topical 
prophylaxis of HIV infection. AIDS Rev 2012; 14:62-77 
10. Thurman AR, Clark MR, Doncel GF. Multipurpose prevention technologies: biomedical tools to 
prevent HIV- 1, HSV-2, and unintended pregnancies. Infectious diseases in obstetrics and 
gynecology 2011; 2011:1-10. 
11. Friend DR. Drug delivery in multiple indication (multipurpose) prevention technologies: systems 
to prevent HIV-1 transmission and unintended pregnancies or HSV-2 transmission. Expert Opin 
Drug Deliv 2012; 9:417-27. 
12. Polis CB, Curtis KM. Use of hormonal contraceptives and HIV acquisition in women: a systematic 
review of the epidemiological evidence. Lancet Infect Dis 2013; 13:797-808. 
13. Larsson PG, Platz-Christensen JJ, Thejls H, Forsum U, Pahlson C. Incidence of pelvic inflammatory 
disease after first-trimester legal abortion in women with bacterial vaginosis after treatment 
with metronidazole: a double-blind, randomized study. Am J Obstet Gynecol 1992; 166:100-3. 
14. Marrazzo JM, Thomas KK, Fiedler TL, Ringwood K, Fredricks DN. Risks for acquisition of bacterial 
vaginosis among women who report sex with women: a cohort study. PloS one 2010; 5:e11139. 
15. Ahrendt HJ, Makalová D, Parke S, Mellinger U, Mansour D. Bleeding pattern and cycle control 
with an estradiol-based oral contraceptive: a seven-cycle, randomized comparative trial of 
estradiol valerate/dienogest and ethinyl estradiol/levonorgestrel. Contraception. 2009; 
80(5):436–444. 
16. Mugo NR, Heffron R, Donnell D, et al. Increased risk of HIV-1 transmission in pregnancy: a 
prospective study among African HIV-1-serodiscordant couples. Aids 2011;25:1887-95. 
17. Taha TE, James MM, Hoover DR, et al. Association of recent HIV infection and in-utero HIV-1 
transmission. Aids 2011;25:1357-64.  
18. World Health Organization. Hormonal contraception and HIV. Technical statement.2012 16 
February 2012. 

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19. Amsel R, Totten PA, Spiegel CA, Chen KC, Eschenbach D, Holmes KK. Nonspecific vaginitis. 
Diagnostic criteria and microbial and epidemiologic associations. Am J Med 1983; 74:14-22. 
20. Nugent RP, Krohn MA, Hillier SL.  Reliability of diagnosing bacterial vaginosis is improved by a 
standardized method of gram stain interpretation.  Journal of Clinical Microbiology 1991; 29: 
297-301. 
21. Trussell J. Contraceptive efficacy. In: Hatcher RA, Trussell J, Nelson AL, Cates W, Stewart FH, 
Kowal D, eds. Contraceptive Technology. 20th ed. New York, N.Y.: Ardent Media, Inc.; 2011:779-
863.  
22. Dezzutti CS, Hendrix CW, Marrazzo JM, et al. Performance of swabs, lavage, and diluents to 
quantify biomarkers of female genital tract soluble mucosal mediators. PloS one 2011;6:e23136. 
23. Liebenberg LJ, Gamieldien H, Mkhize NN, et al. Stability and transport of cervical cytobrushes for 
isolation of mononuclear cells from the female genital tract. J Immunol Methods 2011;367:47-
55. 
24. Farquhar C, VanCott TC, Mbori-Ngacha DA, et al. Salivary secretory leukocyte protease inhibitor 
is associated with reduced transmission of human immunodeficiency virus type 1 through breast 
milk. J Infect Dis 2002;186:1173-6 
25. Vodstrcil LA, Hocking JS, Law M, et al. Hormonal contraception is associated with a reduced risk 
of bacterial vaginosis: a systematic review and meta-analysis. PloS one 2013;8:e73055. 
26. Paul A. Harris, Robert Taylor, Robert Thielke, Jonathon Payne, Nathaniel Gonzalez, Jose G. 
Conde, Research electronic data capture (REDCap) - A metadata-driven methodology and 
workflow process for providing translational research informatics support, J Biomed Inform. 
2009 Apr;42(2):377-81. 
 

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Table 1. Laboratory specimens to be obtained at each visit or by self-collection 
 
 
 
 
 
 
 
 
 
 
 
 
Abbreviations: DSC = daily self-collection, BVAB = bacterial vaginosis-associated bacteria, CVF = cervicovaginal fluid, NAAT = nucleic acid 
amplified test, HSV = herpes simplex virus, LGTI = lower genital tract infection (BV, VVC, cervicitis, genital ulcer disease), PCR = polymerase 
chain reaction.   
1 Only women enrolling in the optional Hormonal sub-study will be asked to come to the clinic for visit 1b procedures. 
2 We anticipate that approximately ¼ of subjects will elect to discontinue CVR use at the end of the study; all participants will be invited to 
perform daily self-collection of vaginal swabs as noted during the month after CVR cessation.    
3 Vaginal bacterial assays include qPCR for G. vaginalis, Megasphaera phylotypes 1 and 2, L. crispatus, L. jensenii, and L.  iners.   
4 Women enrolled in the optional Hormonal sub-study will undergo cervical cytobrush collection at Month 1,/Enrollment, Visit 1b and Month 3, 
and Month 9 and 14 if enrolled in Extension Study. 
5 Will be obtained at month 3 only. 
 
 
Test 
Screen 
Enroll 
Month 1 
Baseline 
 
Month 1b 
substudy1 
Month 2 
CVR Start 
Month 
3-5 
Month 62 
Month 7 
 
Month 812 
(IUD 1) 
 
Month 9 
(IUD 2) 
Month 
10-12 
(IUD 3-5) 
Month 13 
(IUD 6) 
Month 14 
(IUD 7) 
 
Microbiome 
 pH measurement 
X 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
 
X 
 
X 
 Saline microscopy 
X 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
 
X 
 
X 
 Gram stain 
X 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
 
X 
DSC 
X 
 Bacterial qPCR assays3 
 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
 
X 
DSC 
X 
 Broad-range 16S rRNA PCR 
pyrosequencing 
 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
X 
DSC 
X 
 
X 
DSC 
X 
Cervical cytobrush4 
 
 
X 
 
X 
 
 
X5 
 
 
 
 
 
X 
 
 
 
X 
Vaginal biopsy13 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
Immune environment 
CVF: mucosal immune 
mediators6, proteomics 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Progesterone level7 
 
 
X 
X 
 
X5 
 
 
 
X 
 
 
X 
 
STI acquisition/shedding 
NAAT: C. trachomatis, N. 
gonorrhoeae, T. vaginalis  
 
X 
X8 
X8 
X8 
X8 
X8 
X8 
 
 
 
 
 
HSV PCR vaginal swab9 
 
 
DSC 
 
DSC 
 
DSC 
 
DSC 
 
 
DSC 
 
HSV serology 
 
X 
 
 
 
 
 
X10 
 
 
 
 
 
HIV serology 
 
X 
 
 
 
 
 
X10 
 
 
 
 
 
Urine Pregnancy Test 
X 
X11 
X11 
X11 
X11 
X11 
X11 
X11 
 
 
 
 
 
Clinical exam for LGTI 
 
X 
X 
 
 
 
X 
 
X 
 
 
X 
 
 
IUD supplement 
Monthly phone calls for 
retention 
 
 
 
 
 
 
 
 
 
 
X 
 
 

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6 Includes SLPI, defensin, lactoferrin. These will be collected periodically during follow-up.  
7 Women enrolling in the optional Hormonal sub-study undergo serum progesterone testing at 3 time points in the study (Month 1/Enrollment, 
Visit 1b and Month 3), and Month 9 and 14 if enrolled in the Extension Study. 
8 NAAT will be performed as indicated at subsequent visits during study participation  
9 HSV-2 seropositive women will be asked to perform DSC of 1 additional vaginal swabs before and after insertion of CVR and after cessation.  
10 If HIV and/or HSV-2 seronegative at baseline. 
11 Urine pregnancy testing will be performed, as indicated at subsequent visits during study participation.  
12 IUD placement will be done prior to Month 8 visit as part of clinical care, not part of study procedures.  
13 Optional vaginal biopsy procedures. For women who agreed to the hormonal sub-study, the Month 1 vaginal biopsies will ideally be collected 
at the cycle day 6-10 (follicular phase) visit. 
 
 

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Table 2. Table of Study Procedures 
 
Main Study (& Hormonal Sub-Study) 
Optional IUD Extension 
Study 
Study Month 
Screen 
Enroll 
1 
1b1 
2 
3 
4-5 
6 
7 
82 
9 
10-12 
13 
14 
Consent 
X 
 
 
 
 
 
 
 
 
X 
 
 
 
 
Medical and Interim History 
X 
X 
X 
X 
X 
 
 
 
 
X 
X 
 
X 
XX 
Urine Pregnancy Test 
X 
X3 
X3 
 
X3 
X3 
X3 
X3 
X3 
 
 
 
 
 
Self- Collected Vaginal Swab for BV 
Assessment 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood draw for HSV and HIV Testing 
 
X 
 
 
 
 
 
 
X3 
 
 
 
 
 
Pelvic Exam and Vaginal Sample Collection 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
Cervical Cytobrush1 
 
 
X 
X 
 
X 
 
 
 
 
X 
 
 
X 
Blood for Progesterone Levels2 
 
 
X 
X 
 
X 
 
 
 
 
X 
 
 
X 
Vaginal Biopsy (optional) 
 
 
X4 
 
 
 
 
X 
 
 
 
 
X 
 
STD testing 
 
X 
X3 
X3 
X3 
 
X3 
X3 
X3 
 
 
 
 
 
NuvaRing 
 
 
 
 
X5 
X 
X 
X6 
 
 
 
 
 
 
Redcap Computer Questionnaire 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
Daily Home Swab Collection (30 days) 
 
 
X  (two 30-day sessions) 
 
X  
X  
 
X  
Monthly Phone Calls 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
1 Hormonal Sub-study only. Samples may be collected at Enrollment or Month 1, depending on cycle timing.  The additional visit (Visit 1b) will 
occur outside their monthly study visits, timed to capture their other cycle phase. 
2 Participants will have an IUD placed 
3 As needed  
4 For women participating in the Hormonal Sub-Study, vaginal biopsy will ideally occur at the Day 6-10 visit. 
5 CVR Initiation will occur at Month 2.  A new CRV will be dispensed monthly 
6 Subjects will be asked if they wish to discontinue CVR at the Month 6 Visit.  Subjects who wish to continue using CVR will be provided a new 
ring at this visit.