A Pilot Study of Vaginal Progesterone to Reduce Preterm Birth Among Women Receiving Antiretroviral Therapy in Pregnancy
Phase: PHASE4
Conditions: PreTerm Birth
Sponsor: University of North Carolina, Chapel Hill

 
A Pilot Study of Vaginal Progesterone to Reduce Preterm Birth among 
Women Receiving Antiretroviral Therapy in Pregnancy 
 
 
 
NCT number 02970552 
Document Date 10 July 2017 
 

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Protocol v2.1 | 10 July 2017
A Pilot Study of Vaginal Progesterone to Reduce Preterm Birth among Women 
Receiving Antiretroviral Therapy in Pregnancy 
Protocol version 2.1 
10 July 2017 

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TABLE OF CONTENTS 
Page 
 
 
Investigators and Scientific Team ………………………………………………. 
3 
Abbreviations List ……………………………………………………………….. 
4 
Summary………………………………………………………………………….. 
5 
1.0  Background and Introduction………………………………………………. 
6 
   1.1  Rationale for the study  
 
   1.2  Significance 
 
   1.3  Aim 
 
   1.4  Specific objectives 
 
   1.5  Ethical issues 
 
2.0  Literature Review……………………………………………………………. 
8 
3.0  Methodology……………………………………………………..…………… 
9 
   3.1  Study design 
 
   3.2  Study sites and study population 
 
   3.3  Study intervention 
 
   3.4  Study procedures 
   3.5  Biological specimen collection 
   3.6  Laboratory procedures 
 
   3.7  Data security and management 
 
   3.8  Statistical considerations 
 
   3.9  Human participant considerations 
 
   3.10 Study monitoring 
 
   3.11 Dissemination of findings 
 
4.0  Budget………………………………………………………………………… 
21 
5.0  Timeframe……………………………………………………………………. 
21 
6.0  Reference List………………………………………………………………… 
 
21 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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INVESTIGATORS AND SCIENTIFIC TEAM 
 
Co-Principal Investigators: 
Bellington Vwalika, MBChB, MMed, MPH 
Consultant and Chair 
Department of Obstetrics and Gynaecology 
University of Zambia School of Medicine 
Lusaka, Zambia 
Email: vwalikab@gmail.com 
 
Jeffrey S. A. Stringer, MD, FACOG 
Professor of Obstetrics and Gynecology 
Department of Obstetrics and Gynecology 
University of North Carolina at Chapel Hill 
Campus Box 7570, Chapel Hill, NC 27599 
Email: Jeff_Stringer@med.unc.edu 
 
Investigator of Record (IOR) 
Joan Price, MD, MPH 
Research Fellow 
Department of Obstetrics and Gynecology 
University of North Carolina at Chapel Hill 
Campus Box 7570, Chapel Hill, NC 27599 
Email: Joan_Price@med.unc.edu 
 
Co-Investigators 
Margaret Kasaro, MBChB, MMed, MSc 
UNC Global Projects - Zambia 
348 Independence Avenue 
Lusaka, Zambia 
Email: Maragaret.Kasaro@UNCLusaka.org 
 
Bethany Freeman, MSW MSPH 
Research Manager 
Division of Global Women’s Health 
Department of Obstetrics and Gynecology 
University of North Carolina at Chapel Hill 
Campus Box 7570, Chapel Hill, NC 27599 
Email: Bethany_Freeman@med.unc.edu 
 

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Abbreviation List 
 
AE 
 
 
Adverse event 
ANC  
 
Antenatal care 
ART   
 
Antiretroviral therapy 
CI 
 
 
Confidence interval 
DSA  
 
Dye stain assay 
FWA  
 
Federal Wide Assurance 
HIV   
 
Human immunodeficiency virus 
KDC  
 
Kamwala District Clinic 
OHRP  
 
Office for Human Research Protections  
PrEP  
 
Pre-exposure prophylaxis 
PTB   
 
Preterm birth 
RCT 
 
 
Randomized controlled trial 
RR 
 
 
Risk ratio 
SAE 
 
 
Severe adverse event 
SSI 
 
 
Semi-structured interview 
UNC  
 
University of North Carolina 
VP 
 
 
Vaginal progesterone 
 
 

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PROTOCOL SUMMARY 
Background: Preterm birth (PTB) complicates as many as one in six pregnancies in Zambia. 
Many infants who are born prematurely die immediately while others face lifelong disability. 
Maternal HIV is also highly prevalent in Zambia and associated with a doubling of PTB risk. 
Antiretroviral therapy (ART) in pregnancy appears to increase the risk of PTB even more. 
Vaginal progesterone (VP) reduces the risk of PTB among women with a prior preterm birth 
or sonographic evidence of cervical shortening. It is standard of care for prevention of PTB in 
this high-risk population in the United States. However, VP has not been studied as PTB 
prophylaxis among HIV-infected women.  
Objectives: In preparation for a possible full-scale efficacy trial, this pilot study will 
evaluate, through quantitative and qualitative methods, whether VP is feasible and acceptable 
for the prevention of PTB among HIV-infected pregnant women in Lusaka, Zambia. 
Design: Single site, mixed method study including a quantitative placebo-controlled, double-
masked, pilot randomized controlled trial (RCT) and qualitative semi-structured interviews 
(SSIs)  
Study Arms: Daily self-administered 200 mg micronized vaginal progesterone or 
indistinguishable placebo randomly allocated in a 1:1 ratio commenced between 20 0/7 and 
23 6/7 gestational weeks and continued through 36 6/7 gestational weeks, membrane rupture, 
stillbirth, or delivery, whichever is sooner. 
Population: 140 pregnant HIV infected women over the age of 18 seeking antenatal care 
(ANC) prior to 24 weeks gestation and initiating or continuing antiretroviral therapy in 
pregnancy will be enrolled in the pilot RCT; an additional 30+ eligible women who decline to 
participate in the RCT will be invited to participate in SSIs 
Study Site: Kamwala District Clinic, Lusaka, Zambia 
Duration and Follow up: Women will be enrolled over a period of 5-12 months.  Each 
participant will be followed through delivery.     
Endpoints: The pilot RCT will measure rates of enrollment, adherence, and trial retention, as 
well as preliminary efficacy of the study intervention. Semi-structured interviews will 
evaluate acceptability of a study of VP to prevent PTB and identify barriers and facilitators to 
study enrollment, product adherence and study retention. 
Relevance: More than 1.5 million HIV-infected women become pregnant each year. 
Approximately half have access to ART, but all are at increased risk of PTB. VP is a 
promising and cost-effective intervention to prevent PTB that should be studied in this high-
risk population. This pilot study will provide critical insight into the feasibility of a phase III 
trial by determining whether women are willing to participate, to adhere to study drug, and to 
complete follow-up.  
Statement of Problem: Preterm birth is the most common cause of neonatal death 
worldwide. Preterm neonates who survive are at an increased risk of short- and long-term 
morbidity. Women who are HIV-infected have higher than usual chances of delivering their 
baby prematurely. Given high prevalence of both HIV and preterm birth in Zambia, the 
prevention of PTB in this high-risk group is a health care priority. 
 

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1.0 BACKGROUND AND INTRODUCTION 
 
1.1  RATIONALE 
More than 1.5 million HIV-infected women become pregnant each year. Approximately half 
have access to antiretroviral therapy (ART), but all are at increased risk of preterm birth 
(PTB). Vaginal progesterone (VP) is a promising and cost-effective intervention to prevent 
PTB that should be studied in this population.  
1.2 SIGNIFICANCE 
In Zambia, PTB complicates one in six pregnancies.1 Many infants who are born too soon die 
immediately while others face lifelong disability. Maternal HIV is also highly prevalent in 
Zambia and associated with a near doubling of PTB risk.2,3 As access to combination ART 
has become available to an ever-increasing number of pregnant women around the world, 
perinatal HIV transmission rates have plummeted, but a new complication has emerged. 
Paradoxically, ART in pregnancy appears to increase the risk of PTB even beyond that 
attributable to HIV infection itself.4 
Antenatal VP has been found in randomized trials to reduce the risk of PTB among women 
with a prior preterm birth or sonographic evidence of cervical shortening.5-7 VP is standard of 
care for this latter indication in the United States but has yet to be studied to reduce PTB 
among HIV-infected women. We hypothesize that VP prophylaxis may reduce PTB among 
women receiving ART in pregnancy and propose a pilot study to determine whether a full-
scale trial would be feasible. We are particularly interested in knowing the extent to which 
women will adhere to VP (i.e., what proportion of participants will consistently self- 
administer daily study product as directed), given recently reported challenges to adherence 
in vaginal microbicide and PrEP trials.  
1.3 AIM 
We will perform a mixed-method pilot study in Lusaka, Zambia to assess (1) the feasibility 
of a randomized, placebo-controlled trial of VP for the prevention of PTB among HIV-
infected women receiving ART in pregnancy and (2) the acceptability of a trial of VP in this 
population. Our overall objective is to apply the learning from the quantitative and qualitative 
components of this study to inform strategies for optimizing enrollment, adherence, and 
retention in a future phase III efficacy trial.  
1.4 SPECIFIC OBJECTIVES 
Primary objective 
To determine the proportion of study participants who achieve adequate adherence. 
Secondary objectives  
• Uptake: To determine the proportion of eligible HIV-infected pregnant women who agree 
to study participation and who are randomized. 
• Retention: To determine the proportion of enrolled study participants who complete the 
study. 

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• Acceptability: To assess acceptability of a randomized clinical trial of daily VP to prevent 
PTB among women agreeing to trial participation and among those who choose not to 
participate. 
Tertiary objectives 
• To assess whether the use of a placebo run-in period would have utility in a future phase 
III study. 
• To assess safety and preliminary efficacy of VP by comparing PTB rates, adverse 
maternal outcomes, side effects, and adverse neonatal outcomes between the randomized 
groups. 
 
1.5 STUDY OUTCOMES 
Primary outcomes 
Adequate adherence to study product, defined as proper self-administration of at least 80% of 
prescribed study product doses. 
Secondary/tertiary outcomes 
• Comparison of self-reported adherence rates, applicator return, and use of applicators 
measured through a dye stain assay (DSA) 
• Enrollment rates among eligible individuals 
• Ascertainment of date of delivery and infant vital status at birth 
• Reported knowledge, attitudes, and practices related to HIV, ART, risk of PTB, and 
participation in placebo controlled RCTs, as well as attitudes about medications for the 
prevention of PTB including acceptability of daily vaginal administration 
• Reported barriers and facilitators to adherence to study product; returning used 
applicators, and retention in the study 
• Gestational age at delivery and birthweight 
• Rates of stillbirth 
• Serious adverse events and events resulting in study product discontinuation 
 
1.6 ETHICAL ISSUES 
The protocol, informed consent documents, and any subsequent modifications will be 
reviewed and approved by all relevant ethics committees responsible for oversight of the 
study. For this study, relevant ethics committees include the University of Zambia 
Biomedical Research Ethics Committee and the University of North Carolina at Chapel Hill 
Institutional Review Board.  
Participation in this trial will be voluntary. All participants will provide written, informed 
consent prior to study enrolment. All care and procedures will be conducted according to 
local standards of routine clinical care. All staff who have contact with participants will 
receive training in the protection of human research participants prior to conducting any 
study activities and routinely thereafter. Key staff will also complete routine Good Clinical 
Practices training. 
We expect participant risk (detailed in METHODOLOGY below) to be minimal. Progesterone 
supplementation is used routinely by women at high risk of PTB in the United States and 

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Europe. The results of this study will enable our research team to determine whether a full-
scale efficacy trial of VP for the prevention of PTB among HIV-infected women will be 
feasible, and how best to support its success through strategies to optimize participant 
recruitment, adherence, and retention. The knowledge gained from this study and subsequent 
studies could help to influence health policy that could prevent thousands of preterm births 
per year in Zambia alone and is expected to outweigh the risks of participation. 
2.0 LITERATURE REVIEW 
An estimated 15 million babies are born prior to 37 weeks of gestation every year, one 
million of whom die as a direct result of their prematurity.8 Over 60% of the world’s preterm 
deliveries occur in Africa and South Asia; in sub-Saharan Africa the rate of preterm birth 
approaches 20%, compared to rates less than 10% in the Global North.1 The survival of 
premature infants depends heavily upon where a baby is born: PTBs in low- and middle-
income countries are much more likely to result in neonatal mortality due to unequal access 
to life-saving technologies and inadequate basic neonatal care. Beyond mortality, lifelong 
complications of prematurity include neurodevelopmental disorders (e.g. cerebral palsy), 
vision and hearing loss, learning impairment, and other chronic diseases.9 Global funding and 
research has focused largely on developing new technologies to improve outcomes of the 
preterm infant, much of which is currently applicable only in North America and Europe. Of 
the strategies that have been studied for PTB prevention, few have proved effective, 
especially in low- and middle-income countries.10 Low-cost, high-impact interventions are 
desperately needed to prevent PTB among those at highest risk for prematurity and its 
consequences. 
Two large meta-analyses performed nearly two decades apart have each shown that HIV-
infected women have higher rates of PTB compared to uninfected women.2,3 The more recent 
analysis, which included 35 studies through 2014, reported a pooled risk ratio (RR) of PTB 
among HIV-infected women (not on ART) to be 1.5 times that of uninfected women (RR 
1.50; 95% CI: 1.24, 1.82).3 The strongest and most consistent effect was noted in sub-
Saharan Africa, where some 85% of HIV-infected pregnant women reside.11 HIV-infected 
women taking ART, with12-14 or without15-17 protease inhibitors, experience even higher rates 
of PTB when compared to HIV-infected, untreated women.18-26 
Prenatal progesterone – which can be administered intramuscularly or vaginally – reduces the 
risk of PTB in women who have had a prior spontaneous PTB and in those with evidence of 
cervical shortening via mid-trimester sonography.5,7,27,28 Daily vaginal progesterone, in gel or 
200-mg suppositories, is recommended as standard of care by the Society of Maternal Fetal 
Medicine to reduce the risk of PTB among women with ultrasound-identified shortened 
cervical length.29 A 2013 Cochrane meta-analysis of trials of VP among women with a prior 
PTB found the drug to reduce the risk of birth before 37 weeks by half (RR = 0.52; 95% CI: 
0.29, 0.92) compared to placebo.30 Four of these studies28,31-33 also reported the more severe 
outcome of PTB before 34 weeks and the treatment effect was even stronger (RR = 0.21; 
95% CI: 0.10, 0.44). For the indication of short cervix, a separate meta-analysis34 of 5 trials 
reported a VP-attributable reduction in the risk of PTB <35 weeks (RR 0.69, 95% CI 0.55–
0.88), <33 weeks (RR 0.58, 95% CI 0.42–0.80), and <28 weeks (RR 0.50, 95% CI 0.30–
0.81), as well as lower composite neonatal morbidity and mortality. VP is standard of care in 
the United States for women with mid-trimester sonographic evidence of shortened 
cervix.29,35 

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The initiation of labor – whether term or preterm – is an inflammatory event36 and 
progesterone has potent anti-inflammatory properties.36,37 Progesterone suppresses 
inflammatory cytokines and prostaglandins that induce uterine myocyte contraction, weaken 
the membranes, and promote cervical remodeling/ softening.38-42 Progesterone receptor 
antagonists induce labor in animal models, an effect that can be reversed or delayed with add-
back progesterone.43,44 Declining concentrations of circulating progesterone and reduced 
progesterone activity are associated with preterm labor.45 With supplementation, higher 
serum concentrations of progesterone have incremental protection against preterm labor.46 
HIV infection causes immune activation and inflammation, both systemically and in the 
lower genital tract.47,48 While treatment with suppressive ART generally improves systemic 
inflammation, its effect on the lower genital tract is not nearly as consistent.49-51 Indeed, HIV-
infection is associated with bacterial vaginosis,52-54 an alteration of the vaginal microbiome 
that predisposes to PTB55,56 and that may not be resolved with ART.57 Further, in the weeks 
following ART initiation, many women have increased shedding of viral pathogens, such as 
HSV and CMV in the vagina.58,59 
Adherence to vaginal study product has been reported as low as 25% in some trials of vaginal 
microbicides / pre-exposure prophylaxis (PrEP) for HIV prevention in sub-Saharan Africa, 
rendering primary results from the VOICE and FACTS 001 trials difficult to interpret.60,61 
Poor adherence to vaginal antiretroviral prophylaxis has been associated with a variety of 
socio-cultural factors, individual participant motivation, and study design.60,62 In particular, 
use of a placebo control may diminish participant motivation to use product due to perceived 
placebo assignment, as could general concerns of participating in the study of an intervention 
with unproven efficacy.62 And while adherence has been improving in more recent studies of 
oral and vaginal PrEP amidst growing evidence of its safety and efficacy,63-66 a number of 
concerns remain. In light of these findings, it is prudent to investigate whether adequate 
adherence can be achieved with VP prior to launching a full-scale efficacy trial. 
3.0 METHODOLOGY 
3.1 Study design 
This will be a mixed method study to evaluate the feasibility and acceptability of a trial of VP 
to prevent PTB among HIV-infected Zambian women. To assess the feasibility of a full-scale 
clinical trial, we will implement a pilot two-arm, double-masked, placebo-controlled trial of 
VP among HIV-infected women in antenatal care in Lusaka, Zambia. Participants will be 
randomly assigned to either daily self-administered VP or indistinguishable placebo 
commenced after 20 weeks gestation but prior to 24 weeks gestational age. In this pilot study, 
we will be able to estimate study uptake, adherence to study product and protocol, and study 
retention. To assess the acceptability of a trial to test VP among HIV-infected women in 
Zambia, we will employ a qualitative approach of longitudinal semi-structured interviews 
among women agreeing to trial participation and one-time SSIs among those who decline to 
participate. 
3.2 Study sites and study population 
This study will be conducted in the antenatal clinics of Kamwala District Clinic (KDC) in 
Lusaka.  
Inclusion criteria: 
1. 18 years of age or older 

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2. viable intrauterine pregnancy confirmed by ultrasound 
3. presentation to antenatal care prior to 24 weeks gestation 
4. antibody-confirmed HIV-1 infection 
5. initiating or continuing ART treatment in pregnancy  
6. ability and willingness to provide written informed consent 
7. willing to adhere to study visit schedule 
Exclusion criteria: 
1. multiple gestation 
2. non-research indication for antenatal progesterone (i.e. prior spontaneous PTB 
and/or cervical length <20mm on screening ultrasound) 
3. planned or in situ cervical cerclage 
4. evidence of threatened abortion, preterm labor, or ruptured membranes 
5. planned delivery prior to 37 weeks (e.g. prior classical cesarean section) 
6. major fetal anomaly detected on screening ultrasound 
7. known uterine anomaly 
8. known or suspected allergy or contraindication to VP or placebo components 
 
3.3 Study intervention 
Pilot randomized controlled trial 
Participants will be randomized 1:1 to one of two arms in the pilot randomized controlled 
trial. The first arm (VP arm) will self-administer daily vaginal progesterone (in 200mg 
suppositories micronized progesterone) while the other arm (control arm) will self-administer 
indistinguishable placebo.  
Qualitative activities 
Two groups of participants will be eligible for participation in the qualitative activities of this 
study. Women who decline enrollment into the randomized trial will be invited to participate 
in a one-time face-to-face SSI, while women enrolled and randomized into the placebo-
controlled trial will be randomly selected to be followed longitudinally with three serial SSIs. 
Study Drug Pharmacological Information 
Pharmacologic Category: Micronized progesterone  
 
Chemistry: Micronized progesterone is active by oral and vaginal routes of administration. It 
is a white or off-white, odorless, crystalline powder that is stable in air and melts between 
126°C and 131°C. It is soluble in alcohol, acetone and dioxane; sparingly soluble in 
vegetable oils; and practically insoluble in water. The chemical name for micronized 
progesterone is pregn-4-ene-3,20-dione.  
 
Mechanism of Action: Progesterone is a naturally occurring steroid that is secreted by the 
ovary, placenta, and adrenal gland. In the presence of adequate estrogen, progesterone 
transforms a proliferative endometrium into a secretory endometrium. Progesterone is 
necessary to increase endometrial receptivity for implantation of an embryo. Once an embryo 
is implanted, progesterone acts to maintain a pregnancy. Progesterone is also a potent anti-
inflammatory and is thought to prevent preterm birth by counteracting inflammatory 
processes that lead to cervical remodeling, amniotic membrane rupture and the initiation of 

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parturition. It is standard of care and recommended for the prevention of preterm birth by the 
Society for Maternal Fetal Medicine for women who have had a prior spontaneous preterm 
birth or those with shortened cervical length detected on ultrasound. 
 
Formulation: Vaginal suppository  
Strength: 200mg  
 
3.4 Study procedures 
Screening 
Our recruitment activities will begin with community sensitization in the catchment areas 
surrounding KDC about the importance of early antenatal care and risks for preterm birth. 
Trained study staff will conduct health talks at the study sites, focusing on the importance of 
antenatal care and possible prevention of preterm or early delivery, with emphasis on study 
inclusion and exclusion criteria. Staff will provide interested women with additional 
information and referrals to the study clinic for eligibility assessment. Potential participants 
may also be identified by healthcare workers in the district clinic and referred to the study 
clinic for screening and enrollment.   
After the information session, those interested in participation will be asked to sign a brief 
consent form for the ultrasound procedure based on a standardized consent for procedures 
used in government clinics in Zambia. Prior to obtaining this consent, study staff will explain 
the purpose, benefits, and risks of the ultrasound and the procedures that will be performed. 
An ultrasound will be performed for gestational age confirmation and for cervical length 
measurement to ensure the participant does not meet the exclusion criterion of short cervical 
length (<20 mm). If the screening process occurs prior to 16 weeks (before a cervical length 
assessment can be performed), a second ultrasound will be performed at ≥16 weeks’ 
gestation prior to randomization to ensure continued eligibility for study drug. In the rare 
cases that a short cervical length is encountered after initial screening and enrollment (but 
prior to randomization), the participant will be offered active progesterone supplementation 
and will continue to be followed in the study. 
Following the ultrasound, all women who meet gestational age and cervical length inclusion 
criteria will be asked to provide written informed consent for study participation in English, 
Nyanja, or Bemba. Women who are eligible but decline study enrollment will be invited to 
participate in a one-time face-to-face semi-structured interview (see QUALITATIVE 
ACTIVITIES, below), for which they will undergo a separate informed consent process.  
Study staff will verify antenatal and HIV history data from participants’ antenatal cards and 
HIV clinic charts for those undergoing screening for the pilot study. In addition, we will 
perform the following screening tests at the screening visit: hemoglobin, urinalysis, and rapid 
syphilis. In the case of abnormal screening results, our study clinicians will either provide the 
necessary medication (i.e. iron, folate, antibiotic) per standard of care, or provide a 
prescription for the participant to obtain these medications. Additionally, a rapid HIV test 
will be performed to confirm HIV infection eligibility. 
Study product preparation, randomization, and dispensation 
Women will undergo randomization into one of two study arms between 20 0/7 and 23 6/7 

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gestational weeks. The trial will use a paper-based system of sealed envelopes to assign 
women 1:1 to active drug or placebo. A statistician from the UNC Center for AIDS Research 
Biostatistics Core not otherwise associated with the study will design the scheme using 
random permuted blocks.  
Study product will be produced and packaged in Chapel Hill, NC. Each package will have an 
identifying code that will be indexed in a secured database as either placebo or product. 
Packaged product will be shipped to Zambia and dispensed by on-site pharmacy staff. 
Participants will be instructed to begin daily self-administration of study product from the 
day of randomization until 36 6/7 gestational weeks, membrane rupture, or delivery, 
whichever is sooner. Women will be instructed on correct product use at the enrollment visit 
and at additional study visits, as needed. Study product will typically be dispensed with 
single-use applicators at 2- week intervals with an additional 14-day buffer to be replenished 
as needed at subsequent study visits. Each participant will receive a discreet carrier and 
plastic bags to facilitate return of used applicators at follow-up visits. Participants will be 
asked to complete dose diary to facilitate a self-reported adherence measure.  
Antenatal, intrapartum, and immediate postpartum study follow-up 
Participants will be asked to return to clinic for bi-weekly pharmacy visits with all of their 
used and unused study product and their dose diaries. The on-site pharmacist will collect used 
applicators, count unused product, and review participant dose diaries. Trained technicians 
will then test all returned applicators for evidence of vaginal insertion using a dye stain assay 
(see LABORATORY PROCEDURES, below). Adherence counseling will be provided to all 
participants at study initiation and then will be preferentially tailored to participants whose 
adherence diary falls below 80%. All adherence counseling activities will be provided by 
trained study staff. 
Routine antenatal and HIV care will be provided to all participants by the Zambian district 
health center (KDC) following national guidelines. Study staff will conduct follow-up visits 
at approximately 24, 28, 32, and 36 weeks’ gestation to gather interval medical and 
behavioral data from the participant and from her antenatal card. A final ultrasound exam will 
be performed at 32 weeks’ gestation for fetal growth assessment. Our study staff will work 
with participants to ensure follow-up visits coincide with scheduled antenatal visits as often 
as possible to reduce burden on participants with regards to time and travel.  
Each participant will be asked where she intends to deliver to assist in collection of data on 
delivery outcomes. For women who deliver in an area clinic or hospital, the study team will 
obtain detailed information about the clinical management of the participant’s delivery, as 
well as the delivery outcome for both the mother and her infant from medical records. For 
those who deliver either at home or a distant clinic or hospital, as much delivery information 
as possible (e.g., date of delivery, infant vital status and birthweight) will be obtained from 
the participant herself after delivery. 
Finally, all participants presenting to the 36-week study visit will be asked to complete a 
short exit survey. 
Visit procedures are summarized in the Schedule of Evaluations (Table 1).  
 

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Table 1. Schedule of Study Visits and Procedures 
 
Scrng 
Rand 
Follow-up 
Final  
Visit Number 
1.0 
2.0 
2.1 
3.0 
4.0 
5.0 
6.0 
7.0 
8.0 
9.0 
10.0 
Delivery  
Gestational Age,  
in weeks 
≤ 
236/7 
200/7 - 
236/7 
22 
24 
26 
28 
30 
32 
34 
36 
ENROLMENT EVENTS: 
 
 
Eligibility screen 
s 
 
 
 
 
 
 
 
 
 
 
Informed consent  
s 
 
 
 
 
 
 
 
 
 
 
Screening ultrasound1 
s 
s 
 
 
 
 
 
 
 
 
 
Randomization2 
 
s 
 
 
 
 
 
 
 
 
 
Hemoglobin, urinalysis,  
rapid HIV & syphilis  
s 
 
 
 
 
 
 
 
 
 
 
QUALITATIVE: 
 
 
SSI among decliners 
s 
 
 
 
 
 
 
 
 
 
 
Longitudinal SSI among 
study participants 
s 
 
 
 
 
 s  
 s  
STUDY VISITS: 
 
 
Self-administration of study 
product 
 
s 
s s s s s s s s 
 
Study product pick-up 
 
s 
s s s s s s s  
 
Applicator return  
 
 
s s s s s s s s 
 
Collection of medical and 
behavioral data 
s 
s 
 s  s  s  s 
s 
Ultrasound for fetal growth 
 
 
 
 
 
 
 s  
 
 
ASSESSMENTS: 
 
 
Adherence assessments  
 
s s s s s s s s 
 
Exit Survey 
 
 
 
 
 
 
 
 
 s 
 
Determination of delivery 
information and infant vital 
status 
 
 
 
 
 
 
 
 
 
 
s 
1 Screening ultrasound will take place as early as possible on the first study visit prior to 24 weeks. If the 
screening process occurs prior to 16 0/7 weeks, a second ultrasound to measure cervical length will be 
performed before randomization 
2 Participants may be enrolled at any time prior to 24 weeks gestation, but randomization will occur no earlier 
than 20 0/7 
 
Retention 
Once a participant is enrolled in the trial, the study team will make reasonable efforts to 
retain her in follow-up to minimize bias associated with loss to follow-up. The study team 
will track retention rates and address any issues related to retention. Strategies may include:  
• Thorough explanation of the study visit schedule and procedures during informed 
consent, and re-emphasis at each study visit. 
• Encouragement of participants to discuss potential study participation with their 
husbands/partners and other influential family members before agreeing to enrol in the 
study.  
• Collection of detailed locator information at screening, and review and updating of this 

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information at each study visit. 
• Use of appropriate and timely visit reminder mechanisms (including phone calls and text 
messages, if participants specifically agree). 
• Follow-up on missed visits, including home or other off-site visits if agreed upon. 
• Mobilization of trained outreach workers to complete in-person contact with participants 
at their homes and/or other locations. 
 
Qualitative activities 
Women who decline enrollment into the randomized trial will be invited to participate in a 
one-time, face-to-face SSI to assess the following subject areas: (1) perceived risk of PTB; 
(2) health beliefs about and attitudes towards HIV, ART and PTB, and towards a medication 
for the prevention of PTB; (3) acceptability of a daily vaginal medication (as opposed to 
weekly IM injections); and (4) attitudes towards participation in a double- masked trial that 
uses a placebo control. We aim to sequentially interview at least 30 women who decline 
enrollment, with the goal of thematic saturation of data.  
Additionally, women who are enrolled into the RCT will be randomly selected to be followed 
longitudinally with three serial SSIs, to be held at enrollment, mid-study (28-32 weeks’ 
gestational age), and at the final study visit. We aim to conduct interviews with women 
enrolled in the study to address the same domains outlined above, in addition to barriers and 
facilitators to: (1) adherence to study product; (2) returning used applicators – i.e. protocol 
adherence; and (3) retention in the study. If our random sample does not include women from 
the full range of adherence levels, we will apply purposeful sampling to capture women of 
underrepresented adherence levels for a single interview in their 3rd trimester. Our expected 
sample size for this group will be 30 women, based on expectations regarding saturation of 
qualitative themes.  
All interview sessions will be facilitated by a staff member trained in qualitative data 
collection techniques, in a private location on-site and are expected to last 30 minutes each. 
Sessions will be audio-recorded and transcribed, with approval provided by participants via 
informed consent. Where translations are needed, we will have independent reviewers review 
to ensure accuracy. 
3.5 Biological specimen collection 
Vaginal study product (progesterone or placebo) will be provided to participants with single-
use vaginal applicators at each study visit. Participants will be asked to return all used and 
unused applicators to each study visit, at which point applicators will be counted and self-
reported adherence will be assessed. All returned vaginal applicators will then be tested by 
trained technicians for evidence of vaginal insertion using standard operating procedures and 
according to dye stain assay (DSA) specifications (see LABORATORY PROCEDURES, below).67 
Returned applicators will then be disposed of after testing and recording of DSA results. No 
additional specimens will be collected, with the exception of finger stick blood and urine for 
hemoglobin, urinalysis, rapid syphilis and HIV confirmation testing at screening. No 
biological specimens will be stored for the purposes of this study. 
3.6 Laboratory procedures 
Trained laboratory technicians will test returned applicators using an inert dilute food dye 
(0.05% FD&C Blue No. 1) that produces a distinctive streaked color when applied via spray 

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to vaginal applicators after vaginal insertion. Sensitivity and specificity of DSA for vaginal 
insertion is reported as 97% and 79%, respectively. The DSA has been validated in sub-
Saharan Africa, has been accurate at time points up to 4 months after insertion, and has 
shown no evidence of cross-contamination even when used and unused applicators were 
commingled in the same bag.68 
3.7 Data security and management 
Data collected on each participant will include sociodemographic information, relevant HIV 
and obstetrical history, and longitudinal clinical details of current pregnancy and delivery. 
We will also collect data on adherence to study protocol and study product, and qualitative 
data from semi-structured interviews.  
Study data management (e.g. data transmission, query resolution, etc.) will follow site data 
management SOPs. Study identification numbers will be used on all forms, applicator storage 
bags, and communications related to the study. Applicator storage bags will be labeled at the 
time of dispersal and then, when returned, applicators will be processed via DSA as described 
above.  
A separate confidential register will link study identification numbers and participants’ 
names. All data instruments and registers will be securely stored. Data will be entered into a 
custom built database and will be validated. Computers will be password protected and their 
access restricted to authorized study personnel. Backups of the data will be made on a weekly 
basis. Data may be transmitted electronically to the study investigators through secure cloud-
based servers. Study information will not be released without written permission of the 
participant, except when necessary for monitoring by the relevant ethical committees or their 
designees.  
Data will be disposed of after completion of the study following sponsor guidelines. At that 
time, electronic records, including linkage codes and identifiers, will be permanently deleted. 
Paper records will be shredded prior to disposal. 
3.8 Statistical considerations 
Pilot randomized controlled trial 
The primary objective of this research is to determine whether a phase III efficacy trial would 
be feasible. As such, we will study 3 key indices of feasibility: uptake, adherence, and 
retention. Given recent concerning results around poor patient adherence to vaginal study 
product in HIV prevention studies, we have chosen adherence as our primary outcome. The 
minimum rate of adherence necessary to derive benefit from VP is not known. To keep this 
work in line with other published work, we will define adequate adherence as a binary 
variable that describes whether or not a participant has used at least 80% of prescribed study 
product. We will assess feasibility of a phase III trial with the following criteria for marking 
success: 
1. At least 70% of participants achieve adequate adherence (i.e., >70% of women 
take >80% of prescribed doses). Since the study is masked to treatment group, we will 
utilize adherence data from all participants for this assessment (although in an exploratory  
analysis we will investigate whether adherence differs by treatment group). We used the 
normal approximation confidence limit approach to choose the sample size for this study, 

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with the exact binomial CI as a sensitivity analysis. Using a 95% CI for the proportion of 
randomized patients with adequate adherence and assuming an observed adherence rate of 
80%, n=140 provides a lower confidence limit above 70%. The target proportion of 
women achieving adequate adherence is 70-95%. TABLE 2 presents the precision achieved 
for outcomes with an observed proportion of 50-95% given a total sample size of 140.  
2. At least 90% of participants complete the study. The primary outcome of a VP phase III 
trial will almost certainly be a composite of PTB and stillbirth. Successful retention in a 
trial will be defined by the proportion of women in whom we are able to ascertain both the 
date of delivery and infant vital status. For the present feasibility study, we will require a 
study visit to define retention success (either at delivery or postpartum), but will also 
explore whether this outcome can be reliably obtained through tracing patients and 
querying outcomes via home visits or telephone calls.    
3. At least 50% of eligible participants agree to be enrolled. In light of concerns of study 
participants in sub-Saharan Africa around placebo controls and from our estimates from 
previous trials in Zambia,69-72 we believe this level of trial uptake would be feasible. The 
outcome will be calculated as the proportion of women deemed eligible for enrollment at 
initial screening who are ultimately randomized. 
Our primary outcome is the proportion of women with adequate adherence, a binary variable 
that indicates the return of >80% of provided vaginal applicators with DSA positivity 
(unreturned applicators will be considered unused in the primary analysis). As secondary 
measures, we will also estimate study drug adherence by dose diary and verbal self-report, 
and compare these techniques to the gold standard DSA to gain insight into the potential 
utility of these simpler methods in the larger trial. As is evident in TABLE 2, above, our 
proposed sample size will provide reasonable precision around this estimate. We will also 
describe the continuous distribution of the percentage of returned vaginal applicators. 
In a secondary analysis, we will investigate whether the use of a pre-randomization placebo 
run-in period would be valuable in a full-scale efficacy trial. Several perinatal trials including 
multiple progesterone studies have used a placebo run-in period to select participants who are 
most likely to be adherent prior to randomization. For a phase III VP trial, one could imagine 
a 2-week run-in period, where women are provided placebo/inert study product and only 
those returning applicators that met a certain adherence threshold would be randomized. 
While we do not intend to explicitly pilot this procedure, we will simulate it by dividing the 
adherence dataset into the first 2 weeks versus the remaining weeks, and evaluate whether 
some initial threshold adherence (e.g., missing no more than 1 dose) is predictive of adequate 
adherence over the remaining study period. If non-adherence is seen as the “disease” in a 
sensitivity/specificity calculation, we might seek the most sensitive cut-point in the run-in 
period possible, in order to ensure that all non-adherent participants are detected (and exited 
from the study prior to randomization). Of course, high sensitivity comes at the cost of false 
Table 2: Precision of observed proportions with n=140 
 
Observed proportion (adherence, uptake, retention) 
0.5 
0.6 
0.7 
0.8 
0.9 
0.95 
Normal 
Approximation 
of Precision 
± 0.083 
± 0.081 
± 0.076 
± 0.066 
± 0.050 
± 0.036 
Exact binomial 
CI 
0.41, 0.59 0.51, 0.68 0.62, 0.77 0.72, 0.86 
0.84, 0.94 
0.90, 0.98 

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positive results (i.e., disqualifying women who would have been adherent). This exercise will 
allow us to evaluate the utility of a run-in in the overall context of phase III planning, when 
issues such as recruitment and representativeness are also being considered. Statistical 
approaches for diagnostic testing such as ROC curve analyses (plot of sensitivity versus 1-
specificity) will be used to inform selection of the non-adherence cut-point. Sensitivity, 
specificity, PPV and NPV of the chosen cut-point will be estimated with a corresponding 
95% exact binomial confidence interval.  
We will define uptake strictly as the proportion of women meeting initial (screening) 
eligibility criteria who are randomized in the trial. We will also measure (1) the proportion of 
ANC attendees who meet initial eligibility criteria, and (2) the proportion of those meeting 
initial eligibility criteria who are eventually deemed ineligible (e.g., after ultrasound). If 
uptake in the pilot is very low, enrollment of a large trial might not be feasible. Low uptake 
might also make us worry about external validity and whether the trial would be 
representative enough to be of real policy value. 
To quantify study retention, we will calculate the proportion of women randomized in the 
trial for whom we are able to ascertain the date of delivery and infant vital status at birth. For 
the present study, we will require a clinic visit to define retention success (either at delivery 
or postpartum), but will also explore whether this outcome can be reliably obtained through 
home visit or even a telephone call. Based on prior experience, we expect this rate to exceed 
90%. Assuming the observed retention proportion is 95%, our sample size of 140 patients 
will provide a 95% CI spanning 90-98% (TABLE 2). 
Univariate analyses will be performed to identify maternal demographic and health 
characteristics associated with adherence, uptake and retention; and adjusted associations will 
be analyzed with a multivariable log-binomial model as appropriate (for outcomes with a 
sufficient number of events). 
Secondary efficacy and safety outcomes will include: (a) delivery prior to 37 weeks 
gestation; (b) delivery prior to 34 weeks gestation; (c) birth weight <2500g; (d) stillbirth; and 
(e) adverse events. We will attempt to trace all participants who are lost to follow-up to 
determine obstetrical and neonatal outcomes. In an exploratory analysis of these outcomes, 
we will conduct descriptive statistics of participant demographic and clinical features, and of 
the proportion of patients experiencing the secondary outcomes of poor obstetrical and 
neonatal outcomes. Although our study is not powered for efficacy, we will perform 
univariate analyses to determine if a numerical difference in risk of these secondary efficacy 
and safety outcomes exists between women who were randomized to VP compared to those 
randomized to placebo. Sensitivity analyses will be used to investigate how limiting the 
analysis to high adherers impacts the estimated RR. The effect of participant demographic 
variables (and any additional potential measured confounders) will be explored in 
multivariable analyses using a log-binomial model. Exploratory analyses will be stated as 
such and presented as pilot effect estimates with a 95% CI; such analyses will provide useful 
results for planning a full-scale trial. 
In sum, we expect this feasibility study to allow us to estimate the proportion of women who 
(a) are eligible to participate, (b) will agree to participate, (c) are able to comply with the visit 
schedule, (d) will return with used applicators, (e) will adhere to prescribed study drug, and 
(f) will remain in the study once enrolled. We will gain insight into the (g) baseline event 
rate, (h) preliminary effect size, and (i) rates of adverse events. Finally, the study will allow 
us to develop and test study-specific SOPs and gain experience with randomization. 

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Qualitative activities 
Our primary objective is to identify common facilitators and barriers to trial uptake, vaginal 
product adherence, and retention in a clinical trial. We will first analyze data from women 
who decline enrollment independently from data among those who enroll. Then, findings 
from common baseline domains evaluated in both assessment groups will be compared to 
identify commonalities or discrepancies between decliners and enrollees. The longitudinal 
data assessed from interviews with enrolled participants will be compared across time and 
between those randomized to placebo versus progesterone. After translations and 
transcription, a team of coders with qualitative data analysis experience will use qualitative 
analysis software to organize the transcribed data. Transcript data will initially be organized 
based on the questions in the semi-structured guides. Coders will read and “memo” each 
transcript and compare memos to create a preliminary codebook. They will apply the codes to 
significant utterances, comparing their results periodically (such as every 4-5 interviews) to 
assess consistency between coders in order to refine a final codebook. They will examine 
codes and utterances for clusters of meaning to construct themes and maps reflecting 
categories and relationships among codes. They will then examine the themes identified from 
each dataset for contrasts and overlaps between them, which may lead to reconstructions of 
the existing themes or the emergence of new subthemes. A summary report of the analyzed 
SSI data will be created which will inform the development of the following materials for the 
planned full-scale efficacy and safety trial: study protocol, consent forms, adherence 
monitoring and counseling guide, and provider training materials. 
3.9 Human participant considerations 
As described in Section 1.6, the protocol, informed consent, and all participant materials will 
be reviewed by relevant ethics committees maintaining Federal Wide Assurances (FWA) 
with Office for Human Research Protections (OHRP) approval. All staff who have contact 
with participants will receive training on the protection of human research participants prior 
to conducting any study activities and routinely thereafter.  
Informed consent 
Discussions with prospective participants and informed consent procedures will be conducted 
in private to protect patient confidentiality. Where possible, a private room will be used to 
discuss the study and potential participant’s eligibility. If a private room is not available, a 
designated area far enough away from other patients such that they cannot hear the 
conversation will be used. The study nurse will obtain written informed consent from all 
participants. The study procedures, risks, and benefits will be discussed and the study nurse 
will answer all questions prior to obtaining consent. The consent forms will be translated into 
local languages (Nyanja and Bemba) and back-translated into English to assure accurate 
translation. All versions of the consent forms will be approved by the relevant ethics 
committees prior to study initiation. For illiterate participants, a literate impartial witness will 
be present during the entire consent process to ensure that all of the relevant information has 
been provided and the participant voluntarily gives consent.  
Eligible women who do not wish to participate in this study will continue to receive antenatal 
care and treatment according to local clinical standards.   
Potential risks to participants  

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Investigators will make efforts to minimize risks to participants. It is expected that this study 
will expose subjects to minimal risks. Side effects and serious adverse events with vaginal 
progesterone administration are very rare (see ADVERSE EVENTS, below). Self-administration 
of vaginal progesterone may cause mild discomfort to participants. This risk of discomfort to 
participants will be minimized by training of participants in correct use of vaginal applicators 
by study staff.  
Participation in clinical research includes the risks of loss of confidentiality and discomfort 
with the personal nature of questions, particularly when discussing HIV infection or sexual 
behaviors. At each step in the study, we will protect participant privacy and confidentiality to 
reduce these risks. Although investigators make every effort to protect participant privacy 
and confidentiality, it is possible that participant involvement in the study could become 
known to others, and that social harms may result (i.e. as participants could become known as 
HIV infected).  
The confidentiality of all study records will be safeguarded to the extent legally possible. To 
maintain participant confidentiality, all laboratory specimens, reports, study data and 
administrative forms will be identified by a coded number only. All databases will be secured 
with password-protected access systems, and computer entries will be identified by coded 
number only. Forms, lists, logbooks, appointment books, and any other listings or data forms 
that link participant ID numbers to other identifying information will be stored in a separate, 
locked cabinet. All data analysis will be performed using datasets which have only the study 
number as a unique identifier. Clinical information with individual identifiers will not be 
released without the written permission of the participant. We expect these procedures to 
adequately protect participant confidentiality.   
Potential benefits to participants and others  
Individual participants in the VP arm of the randomized trial may benefit from a reduction in 
preterm birth risk if the intervention is found to be effective, but it is also possible that there 
will be no effect. We believe that equipoise exists between the study arms since VP has not 
been previously studied in this population of HIV infected women receiving antiretroviral 
therapy in sub-Saharan Africa, and the exact risk for preterm birth in this population remains 
unclear. All participants may benefit from enhanced health education, and close clinical 
monitoring. Knowledge generated from this study has the potential to inform future clinical 
trials on the reduction of PTB risk among HIV-infected women, which may enable 
policymakers worldwide to make informed decisions regarding effective interventions for the 
prevention of PTB.  
At the clinic level, all staff involved in the study will receive refresher training on the 
national guidelines for the diagnosis and treatment of preterm birth. Increased awareness 
about screening and treatment for preterm birth may help to improve care overall at KDC. 
Additionally, our community sensitization activities may help to encourage more women to 
come for antenatal screening earlier in pregnancy, which has a known benefit on maternal 
and infant outcomes.   
In summary, we expect participant risk to be minimal. The knowledge generated from this 
study regarding the potential PTB risk reduction among HIV-infected women is expected to 
outweigh the risks of participation. 
Inclusion of children, sub-populations, and vulnerable populations  

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As this is a trial specifically investigating an intervention for prevention of preterm birth 
among HIV-infected women 18 years old or older, only eligible women will be recruited to 
participate.  
3.10 Safety monitoring 
At each study visit, study staff will evaluate participants for social harms and adverse events 
(AEs). A social harm will be defined as a non-medical untoward consequence of study 
participation, including: difficulties in personal relationships, stigma, or discrimination from 
family or community. An AE will be defined as any untoward medical occurrence in a study 
participant including any abnormal sign (e.g., abnormal physical exam or laboratory finding), 
symptom, or disease, temporally associated with the individual’s participation in the research, 
whether or not considered related to participation in the research. In addition to events related 
to study procedures and vaginal study product, we can expect that this population of HIV-
infected pregnant women to experience adverse events unrelated to study procedures, 
including adverse obstetrical outcomes, opportunistic infections, side effects from ART or 
other medications, hospitalization, and death.  
The severity of study-related adverse events and social harms will be graded using the 
National Institute of Health’s Division of AIDS Table for Grading the Severity of Adult and 
Pediatric Adverse Events. We will also record information on all serious adverse events 
(SAEs) occurring in participants whether or not they are related to study participation or the 
study drug, including AEs that: 
1. Result in hospital admission (unless hospitalization is preplanned, i.e. for delivery) or 
prolongation of existing hospitalization  
2. Are immediately life-threatening, including drug reactions that necessitate 
discontinuation of study participation,  
3. Cause significant, persistent, or permanent harm or disability, either physical or 
psychological,  
4. Result in death, including fetal demise after 20 weeks gestation 
5. Are congenital anomalies/birth defects 
Information on adverse events or social harms that are related to the study drug and all SAEs 
will be documented on study data forms and routinely reported to the Principal Investigator 
or designee. If the PI, co-investigators, or their designees determine that study-related adverse 
events are occurring at an unexpected rate, they will assess the need for staff re-training, 
protocol amendment, or study cessation. Adverse Events will be reported to the UNZA REC, 
UNC IRB, and other regulatory bodies according to their individual guidelines.  
3.11 Dissemination of findings 
Study findings will be disseminated through appropriate local channels, including academic 
and public health research symposia. One or more publications will also be submitted to a 
peer-reviewed journal. Our study team will plan to publish results whether positive or 
negative. The study participants’ privacy and confidentiality will be strictly maintained in all 
results dissemination or publication activities. 
 
4.0 BUDGET (1 USD = 10 ZMW) 

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Protocol v2.1 | 10 July 2017 
ITEM 
USD TOTAL 
ZMW TOTAL  
Personnel 
$200,000 
K 2,000,000  
Equipment 
$0 
K 0 
Supplies & materials 
$31,687 
K 316,870 
Participant costs 
$5,409 
K 54,090 
Contractual 
$0 
K 0 
Other 
$8,855 
K 88,550 
TOTAL 
$245,951 
K 2,459,510 
 
5.0 TIMELINE 
Participants will be followed from enrollment (prior to 24 weeks gestational age) through 
delivery. We have allocated 6 months to start-up activities and 3 months to wind-down 
activities. 
PHASE 
Year 1 
Year 2 
 
Q1 
Q2 
Q3 
Q4 
Q1 
Q2 
Q3 
Q4 
Pre-implementation 
 
 
 
 
 
 
 
 
Regulatory approvals 
X 
 
 
 
 
 
 
 
Site preparation and training 
X 
X 
 
 
 
 
 
 
Implementation 
 
 
 
 
 
 
 
 
Enrollment 
 
X 
X 
X 
X 
 
 
 
Follow-up 
 
 
X 
X 
X 
X 
X 
X 
Data management, analysis & reporting 
 
 
 
 
 
 
 
 
Transcription and coding of interviews 
 
 
X 
X 
X 
X 
 
 
Data entry and management 
 
X 
X 
X 
X 
X 
X 
X 
Statistical analysis 
 
 
 
 
X 
X 
X 
X 
Publication and dissemination  
 
 
 
 
 
 
 
X 
 
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Brocklehurst P. The Association between maternal HIV infection and perinatal 
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Wedi CO, Kirtley S, Hopewell S, Corrigan R, Kennedy SH, Hemelaar J. Perinatal 
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