Z 31702 - Improving Pregnancy Outcomes With Progesterone (IPOP): a Trial of 17-Hydroxyprogesterone Caproate to Reduce Preterm Birth Among Women Receiving Antiretroviral Therapy in Pregnancy
Phase: PHASE3
Conditions: Pre-term Birth, HIV-1-infection
Sponsor: University of North Carolina, Chapel Hill

 
 
Improving Pregnancy Outcomes with Progesterone (IPOP): a Trial of 17-
Hydroxyprogesterone Caproate to Reduce Preterm Birth Among Women Receiving 
Antiretroviral Therapy in Pregnancy 
 
 
 
 
 
 
 
 
 
 
NCT Number  
03297216 
Document Date 
26 March 2020 
 
 

Improving Pregnancy Outcomes with Progesterone (IPOP) 
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Protocol v1.6 | 26 March 2020 
 
 
 
 
 
 
 
Improving Pregnancy Outcomes with Progesterone (IPOP): a Trial of 17-
Hydroxyprogesterone Caproate to Reduce Preterm Birth Among Women Receiving 
Antiretroviral Therapy in Pregnancy 
 
 
Protocol version 1.6 
26 March 2020 
 
 
 
 
Funded by: 
Eunice Kennedy Shriver  
National Institute of Child Health and Human Development 
1R01HD087119 
 
and  
 
The Bill and Melinda Gates Foundation 
 
 
Study Drug Provided by: 
AMAG Pharmaceuticals, Inc 
 
 Protocol Co-Chairs:  
Bellington Vwalika, MBChB, MSc, MMed 
Jeffrey S. A. Stringer, MD 
 
Trial Registration: 
ClinicalTrials.gov 
NCT03297216 
  
 
 
 
 
 
 
 
 
 
NB: This protocol is organized and formatted according to the guidelines set forth by the 
University of Zambia Biomedical Research Ethics Committee (UNZA BREC) 

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TABLE OF CONTENTS 
INVESTIGATORS AND SCIENTIFIC TEAM ....................................................................... 4 
ABBREVIATIONS AND ACRONYMS .................................................................................. 5 
PROTOCOL SUMMARY ......................................................................................................... 6 
TRIAL SCHEMA ...................................................................................................................... 7 
1.0 
Introduction ................................................................................................................. 8 
2.0 
Statement of the Problem ............................................................................................ 8 
3.0 
Rationale ...................................................................................................................... 8 
4.0 
Background and Literature Review ............................................................................. 9 
5.0 
Specific Objectives .................................................................................................... 11 
5.1 
Primary objective ................................................................................................... 11 
5.2 
Secondary objectives ............................................................................................. 12 
5.3 
Tertiary objectives ................................................................................................. 12 
6.0 
Study Outcomes ........................................................................................................ 12 
6.1 
Primary outcomes .................................................................................................. 12 
6.2 
Secondary outcomes .............................................................................................. 12 
6.3 
Tertiary outcomes .................................................................................................. 12 
7.0 
Trial Methodology..................................................................................................... 13 
7.1 
Study design .......................................................................................................... 13 
7.2 
Study sites and study population ........................................................................... 13 
7.3 
Study intervention.................................................................................................. 14 
7.4 
Study drug pharmacological information .............................................................. 14 
7.5 
Study procedures ................................................................................................... 14 
7.6 
Biological specimen collection and testing ........................................................... 19 
7.7 
Quality control and quality assurance procedures ................................................. 19 
7.8 
Data security and management .............................................................................. 19 
7.9 
Statistical considerations ....................................................................................... 20 
7.10 Dissemination of findings ...................................................................................... 23 
8.0 
Ethical Considerations ............................................................................................... 24 
8.1 
Informed consent ................................................................................................... 24 
8.2 
Potential risks to participants ................................................................................. 24 
8.3 
Potential benefits to participants and others .......................................................... 25 
8.4 
Inclusion of children, sub-populations, and vulnerable populations ..................... 26 
9.0 
Timeline .................................................................................................................... 26 
10.0 Budget ....................................................................................................................... 26 
11.0 Reference List ........................................................................................................... 28 
 
 
 

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INVESTIGATORS AND SCIENTIFIC TEAM 
Protocol Chairs: 
Bellington Vwalika, MBChB, MMed, MSc 
Professor 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 
UNC School of Medicine 
Campus Box 7570, Chapel Hill, NC 27599 
Email: jeffrey_stringer@med.unc.edu 
 
Investigator of Record (IOR): 
Joan T. Price, MD, MPH 
Research Assistant Professor 
Department of Obstetrics and Gynecology 
UNC Chapel Hill 
Campus Box 7570, Chapel Hill, NC 27599 
Email: joan_price@med.unc.edu 
 
Co-Investigators: 
Margaret Kasaro, MBChB, MS 
UNC Global Projects - Zambia  
348 Independence Avenue 
Lusaka, Zambia 
E-mail: margaret.kasaro@unclusaka.org  
 
Stephen R. Cole, PhD 
Professor 
Department of Epidemiology 
UNC Chapel Hill 
McGavran-Greenberg Hall 2105E 
Campus Box 7435, Chapel Hill, NC 27599 
Email: cole@unc.edu 
 
Benjamin H. Chi, MD, MSc 
Professor of Obstetrics and Gynecology 
UNC School of Medicine 
Campus Box 7570, Chapel Hill, NC 27599 
Email: bchi@med.unc.edu 
 
Elizabeth M. Stringer, MD, MSc 
Associate Professor of Obstetrics and 
Gynecology 
UNC School of Medicine 
Campus Box 7570, Chapel Hill, NC 27599 
Email: Elizabeth_stringer@med.unc.edu 
 
 
Scientific Advisory Committee Members 
Lynne M. Mofenson, MD 
Elizabeth 
Glaser 
Pediatric 
AIDS 
Foundation 
1140 Connecticut Ave, NW (Suite 200) 
Washington, DC 20036 
Email: mofensonl@mail.nih.gov 
 
Robert L. Goldenberg, MD 
Professor of Obstetrics and Gynecology 
Columbia University 
161 Fort Washington Avenue 
New York, NY 10032 
Email: rlg88@columbia.edu 
 
Elwyn Chomba, MBChB 
Professor Emerita 
University of Zambia School of Medicine 
Lusaka, Zambia 
Email: echomba@zamnet.zm 
 
Dwight J. Rouse, MD, MSPH 
Professor of Obstetrics and Gynecology 
Brown University 
101 Dudley Street 
Providence, RI 02912 
Email: DRouse@Wihri.org 
 
 
 

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ABBREVIATIONS AND ACRONYMS 
17P  
17-alpha hydroxyprogesterone caproate 
AE 
 
 
Adverse event 
ANC  
 
Antenatal care 
ART   
 
Antiretroviral therapy 
CI 
 
 
Confidence interval 
DSMB  
 
Data safety and monitoring board 
EDD  
 
Estimated due date 
GAPPS/PPB  
Global Alliance for Prematurity Prevention / Preventing Preterm Birth  
HIV   
 
Human immunodeficiency virus 
IM 
 
 
Intramuscular 
IPOP  
 
Improving Pregnancy Outcomes with Progesterone (this trial) 
KDC  
 
Kamwala District Clinic 
NICHD 
 
National Institute of Child Health and Human Development 
PI 
 
 
Principal investigator 
PTB   
 
Preterm birth 
RCT 
 
 
Randomized controlled trial 
RR 
 
 
Risk ratio 
SAE 
 
 
Severe adverse event 
SOP 
 
 
Standard operating procedure 
UNC  
 
University of North Carolina 
UNZA  
 
University of Zambia 
UTH  
 
University Teaching Hospital 
ZEPRS 
 
Zambian Electronic Perinatal Record System 
 
 

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PROTOCOL SUMMARY 
Background: Preterm birth (PTB) complicates one in six pregnancies in Zambia. Many 
infants who are born prematurely die as neonates while others may face lifelong disability. 
Maternal HIV is also highly prevalent in Zambia and associated with a 50% increase in PTB 
risk. Antiretroviral therapy (ART) in pregnancy appears to increase the risk of PTB even 
more. Injectable 17-hydroxyprogesterone caproate (17P) reduces the risk of PTB among 
women with a prior preterm birth and is standard of care for this population in the United 
States. However, 17P has not been studied as PTB prophylaxis in African populations or 
among HIV-infected women.  
Objectives: To assess the efficacy of 17P for preventing PTB among HIV-infected pregnant 
women receiving ART.  
Design: Phase III, placebo-controlled, double-masked, randomized controlled trial (RCT). 
Study Arms: Weekly intramuscular injection of 250mg 17P or indistinguishable placebo 
randomly allocated in a 1:1 ratio started between 16-24 weeks of gestation and administered 
weekly thereafter until 36 6/7 gestational weeks, stillbirth, or delivery, whichever is sooner. 
Population: 800 pregnant HIV-infected women over the age of 18 who enter antenatal care 
(ANC) prior to 24 weeks of gestation. Liveborn infants will also be enrolled at birth.  
Study Sites: University Teaching Hospital (UTH), Kamwala District Clinic (KDC), or 
another district clinic in Lusaka, Zambia. 
Duration and Follow up: Enrollment will proceed over a period of approximately 40 
months. Individual participants will be followed from enrollment (prior to 24 weeks of 
gestation) through 42 days postpartum. 
Endpoints: The goal of this intervention is to improve the likelihood that a woman will 
deliver a live-born, full-term infant. Thus, the primary trial outcome is a composite 
comprising the following: (1) delivery prior to 37 weeks of gestation, and/or (2) stillbirth at 
any gestational age.  
Relevance: More than 1.5 million HIV-infected women become pregnant each year 
worldwide. Approximately half have access to ART, but all are at increased risk of PTB. 17P 
is a promising and cost-effective intervention to prevent PTB that should be studied in this 
high-risk population. Given the prevalence of HIV infection as a PTB risk factor in sub-
Saharan Africa and in many other settings, the potential public health benefit of this 
intervention is substantial. The effectiveness of 17P to prevent PTB among HIV-infected 
women needs to be studied in a randomized trial. 
 
 
 

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TRIAL SCHEMA 
 
 
 

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1.0 
Introduction 
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 
Prenatal progestogens reduce the risk of PTB among women with evidence of cervical 
shortening or history of a prior spontaneous PTB.5 Weekly 17P is FDA-approved and 
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 the increased risk of PTB 
associated with HIV and ART may be amenable to 17P prophylaxis and propose a 
randomized trial to test this hypothesis. 
IPOP is a phase III randomized, placebo-controlled, double-masked trial to assess (1) whether 
intramuscular 17P (started between 16-24 weeks of gestation and administered weekly 
thereafter until 36 6/7 gestational weeks, stillbirth, or delivery, whichever is sooner) will 
reduce the rate of PTB among HIV-infected pregnant women receiving ART; (2) whether 
women who start ART in pregnancy are at higher risk of PTB compared to those who enter 
care on ART initiated prior to conception; and (3) biological correlates of preterm birth and 
other adverse birth outcomes in the trial cohort. Approximately 800 women will be enrolled 
in Lusaka, Zambia from the University Teaching Hospital, the Kamwala District Clinic, or 
another district clinic.   
2.0 
Statement of the Problem 
PTB 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 PTB in Zambia, the prevention of PTB in this high-risk group is a health 
care priority. 
3.0 
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 PTB. Injectable 
17-alpha hydroxyprogesterone caproate (17P) is a promising, low-cost intervention to prevent 
PTB that should be studied in this population.  
We propose to apply this known effective intervention to a new, exceedingly prevalent 
indication. Despite the abundance of progesterone PTB trials in the published literature,6-18 
none have targeted patients at risk of prematurity owing to HIV infection. Similarly, there are 
no 17P trial data from sub-Saharan Africa, a region that shoulders more PTB disease burden 
relative to its population size than anywhere else on earth.  
Because HIV is so prevalent worldwide, this intervention – if proven effective – could have a 
substantial public health impact. UNAIDS estimates that 50% of HIV-infected women who 
become pregnant each year have access to combination ART.35 We estimated a baseline risk 
of prematurity of 12.3%,1 that HIV infection is associated with a relative risk (RR) of 

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prematurity of 1.8,2 that receipt of ART is associated with a RR of prematurity of 2.2,19 and 
that 17P could reduce the risk of prematurity by 38% (i.e. RR=0.62).20 Under these 
assumptions, HIV and ART are responsible for an additional 184,500 preterm births each 
year worldwide, a number that will only increase as ART coverage continues to rise. With 
reasonable integration of 17P use into existing PMTCT programs, it is conceivable that the 
intervention could be delivered to 25% of HIV-infected women not receiving ART and 80% 
of those receiving ART. Under these assumptions, the 17P intervention would prevent 40,664 
PTBs per year, and perhaps have the added benefit of enhancing ART adherence in 
pregnancy.   
4.0 
Background and Literature Review 
4.1 
Global Burden of Preterm Birth 
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.21 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.22 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.23 Low-cost, high-impact interventions are 
desperately needed to prevent PTB among those at highest risk for prematurity and its 
consequences. 
4.2 
HIV and Preterm Birth 
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.24 HIV-infected 
women taking ART, with25-27 or without28-30 protease inhibitors, experience even higher rates 
of PTB when compared to HIV-infected, untreated women.31-39 
4.3 
Progesterone and Preterm Birth  
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. Weekly 17P, administered via 
intramuscular injection, is FDA-approved and recommended as standard of care by the 
Society of Maternal Fetal Medicine to reduce the risk of PTB among women with a prior 
spontaneous PTB.40 A 2013 Cochrane meta-analysis of progesterone to prevent PTB among 
women with a prior PTB aggregated data from 10 randomized trials that studied prenatal 
prophylaxis by the intramuscular (IM, n=4 studies), vaginal (n=5 studies), or oral (n=1 study) 

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route and estimate the risk ratio of birth prior to 37 weeks among women receiving active 
drug to be 0.55 (95% CI: 0.42, 0.74). When restricted to only those studies that provided IM 
drug (as we propose in this study), the aggregate RR was 0.62 (95% CI: 0.52, 0.75).5 
4.4 
Anti-inflammatory Properties of Progesterone 
The initiation of labor – whether term or preterm – is an inflammatory event20 and 
progesterone has potent anti-inflammatory properties.20,41 Progesterone suppresses 
inflammatory cytokines and prostaglandins that induce uterine myocyte contraction, weaken 
the membranes, and promote cervical remodeling/ softening.42-46 Progesterone receptor 
antagonists induce labor in animal models, an effect that can be reversed or delayed with add-
back progesterone.47,48 Declining concentrations of circulating progesterone and reduced 
progesterone activity are associated with preterm labor.49 With supplementation, higher 
serum concentrations of progesterone have incremental protection against preterm labor.50 
HIV infection causes immune activation and inflammation, both systemically and in the 
lower genital tract.51,52 While treatment with suppressive ART generally improves systemic 
inflammation, its effect on the lower genital tract is not nearly as consistent.53-55 Indeed, HIV-
infection is associated with bacterial vaginosis,56-58 an alteration of the vaginal microbiome 
that predisposes to PTB59,60 and that may not be resolved with ART.61 Further, in the weeks 
following ART initiation, many women have increased shedding of viral pathogens, such as 
HSV and CMV in the vagina.62,63 
Trials of progestogens to prevent PTB have used both intramuscular (IM)9-12 and vaginal64-68 
drug. Although vaginal administration is common in the United States – especially for 
women with a shortened cervix in the mid-trimester – only the IM route is currently FDA 
approved. At least two other studies are piloting vaginal progesterone in HIV infected 
women. These include a Canadian trial that will randomize 40 women (NCT02400021) and a 
Zambian trial that will randomize 120 women (NCT02970552).  
Specific biologic markers indicating increased risk of preterm delivery have been difficult to 
elucidate.  While we suspect that biomarkers found in amniotic fluid, urine, cervical mucus 
and vaginal secretions, serum, and plasma may play a role in risk of PTL, PPROM and 
preterm delivery, few analytes have been identified that have clinical utility. Part of the 
difficulty lies in timing of collection in relation to gestational age and delivery.  If a woman is 
already in PTL, then noting an increase in a specific metabolite is of little value in predicting 
PTL; however, identification of these analytes can increase our understanding of PTL 
pathophysiology. Thus, a more systematic approach to collection of possible biomarkers 
during routine antenatal care among women who are followed for their entire pregnancy and 
post-partum care can provide an enhanced understanding of which analytes may contribute to 
PTL, PPROM, and preterm delivery. 69,70 
4.5 
Safety of Progesterone in Pregnancy 
The human placenta produces increasingly large amounts of progesterone throughout 
pregnancy and 17P is a natural metabolite of endogenous progesterone. The concentrations of 
naturally occurring 17P exceed the pharmacologic doses proposed by this study.   
4.5.1 Animal Studies 
Studies of 17P in various species of pregnant non-human animals have demonstrated no 
adverse androgenic effects on developing fetuses. Certain progestestational compounds are 

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known to virilize female fetuses in both rat models and human studies.71-74 However, in the 
first mouse model, assays of two nonandrogenic progestins – 17-alpha ethinyl-19-
nortestosterone acetate (17-ENT) and 17P – demonstrated virilization of female fetuses only 
with 17-ENT but not with 17P exposure; male fetuses were not affected by either 
compound.75 Even with administration of doses much higher than the equivalent human dose, 
17P has shown no virilizing effects on female fetuses of rats, rabbits, or monkeys.76-79  
Similarly, no teratogenic effects of 17P have been noted in numerous studies of pregnant 
animal models treated with 17P. In monkeys given 17P for threatened abortion in early 
pregnancy, surviving fetuses were all normal and healthy.79 Two studies in mice evaluating 
the teratogenic potential of 17P administered doses up to 70 and 200 times the human 
equivalent. These high-doses of 17P during organogenesis did not increase the frequency of 
congenital abnormalities, fetal limb morphogenesis, or growth.80,81 
4.5.2 Human Studies  
Several controlled clinical trials9,68,82,83 and epidemiological studies84,85 have demonstrated 
the safety of 17P in human pregnancy. In randomized trials of 17P for the prevention of PTB, 
no significant risk of adverse birth outcomes has been noted in women with progesterone 
exposure compared to placebo. While some trials have reported a statistically non-significant 
increased risk of stillbirth and/or miscarriage,6,9,17 others show no trend,15 and others report a 
nonsignificant reduction in stillbirth with 17P.8 A individual participant data meta-analysis of 
13 trials including 3,768 women showed a relative risk of adverse perinatal outcome of 1.1 
(95% CI 0.97–1.4) among women receiving 17P compared to placebo, and concluded that 
treatment with 17P does not have an effect on adverse perinatal outcomes.86  
Two extensive reviews argue that no evidence exists that exposure to 17P in utero induces 
genital malformations in female fetuses.85,87 In addition, the rates of non-genital 
malformations in fetuses exposed to 17P compared to unexposed fetuses have been similar in 
numerous large clinical trials and population reports. In one, 988 infants exposed to 
exogenous progesterone or 17P were compared to a matched cohort of unexposed infants in 
the United States; similar rates of all congenital malformations were reported between the 
two cohorts.84 A prospective study comparing 1,608 newborns exposed to progestogens 
(either oral medroxyprogesterone caproate or 17P, or both) in the first trimester of pregnancy 
and 1,146 unexposed newborns found nearly equal rates of both major and all malformations 
between the two groups.88  
4.5.3 Side effects of Intramuscular 17P in Pregnancy 
Side effects of 17P may include headache, breast tenderness, nausea, cough and local 
irritation at the injection site.89 The latter is the most commonly reported event, with the 
following specific symptoms: urticaria (12%), pain (35%), and swelling (17%).9 Women with 
one or more contraindications to 17P that appear in the package insert will be excluded from 
the trial (see section 7.2)  
5.0 
Specific Objectives 
5.1 
Primary objective 
The primary objective of this study is to evaluate, among HIV-infected pregnant women 
receiving ART, whether 17P will reduce the risk of the composite outcome (live birth prior to 
37 weeks of gestation or stillbirth at any gestational age), relative to placebo.  

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5.2 
Secondary objectives  
• To assess, through a subgroup analysis, whether 17P will reduce the risk of the 
composite primary outcome among women either (a) newly initiating ART or (b) 
continuing ART that was started prior to conception 
• To assess the effect of timing of ART initiation on risk of the composite primary 
outcome and its components by comparing women newly starting ART to those who 
initiated ART prior to conception (non-randomized comparison), and controlling for 
possible confounders (e.g., CD4 count, viral load, ART regimen).  
• To assess the extent to which exclusion of provider-initiated PTB phenotypes modify 
(a) the observed efficacy (if any) of 17P to prevent the composite primary outcome 
and (b) its components and any association between timing of ART initiation and 
PTB. 
5.3 
Tertiary objectives 
• To investigate the underlying biology of PTB and other adverse pregnancy outcomes 
with particular attention to inflammation, immune activation, and alterations of the 
vaginal microbiome. 
• To determine the ability of prediction algorithms to correctly classify the gestational 
age of newborns at delivery 
6.0 
Study Outcomes 
6.1 
Primary outcomes 
Our primary outcome will be a composite of live birth prior to 37 weeks of gestation or 
stillbirth at any gestational age.  
6.2 
Secondary outcomes 
• Delivery prior to 34 weeks of gestation 
• Delivery prior to 28 weeks of gestation 
• Spontaneous delivery prior to 37 weeks of gestation 
• Spontaneous delivery prior to 34 weeks of gestation 
• Spontaneous delivery prior to 28 weeks of gestation 
• Birth weight <10th percentile for gestational age 
• Birth weight <3rd percentile for gestational age 
• Mother-to-child HIV transmission by 6 weeks postpartum 
• Cumulative incidences of the competing risks of stillbirth and PTB 
• Neonatal and perinatal mortality rates 
• Infant APGAR scores 
6.3 
Tertiary outcomes 
• Birth weight <2500g 
• Birth weight <1500g 
• Measures of maternal markers of inflammation, immune activation, and alterations of 
the vaginal microbiome 

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• Measures of infant morbidity, such as ICU admission, supplemental oxygen 
requirement, and need for assisted ventilation 
• Measures of maternal morbidity, such as pregnancy-induced hypertension, antepartum 
hemorrhage, preterm premature rupture of membranes, oligo- or polyhydramnios, 
chorioamnionitis, mode of delivery, and respiratory infection  
• Adverse drug reactions 
• Serious adverse events and events resulting in study product discontinuation 
7.0 
Trial Methodology 
7.1 
Study design 
This will be a double-masked placebo-controlled randomized trial of 17P among HIV-
infected women in Lusaka, Zambia. Participants will be randomly assigned to weekly 
intramuscular administration of either 17P or indistinguishable placebo to be commenced 
between 16 and 24 weeks of gestation. We will estimate effectiveness by comparing our 
primary outcome, a composite measure of delivery prior to 37 completed gestational weeks 
or stillbirth at any gestational age, between women who were randomized to active versus 
placebo in an intention-to-treat analysis.  
7.2 
Study sites and study population 
This study will be conducted in the antenatal clinics of the University Teaching Hospital 
(UTH), Kamwala District Clinic in Lusaka, Zambia, or another district clinic.  
Inclusion criteria: 
1. 18 years of age or older 
2. less than 24 0/7 weeks of gestation 
3. viable intrauterine singleton pregnancy confirmed by ultrasound 
4. antibody-confirmed HIV-1 infection 
5. currently receiving ART or intending to commence ART in pregnancy  
6. ability and willingness to provide written informed consent 
7. intent to remain in current geographical area of residence for the duration of study 
8. willing to adhere to weekly study visit schedule 
Exclusion criteria: 
1. confirmed prior spontaneous preterm birth 
2. multiple gestation 
3. known uterine anomaly 
4. planned or in situ cervical cerclage 
5. major fetal anomaly detected on screening ultrasound 
6. indication for planned delivery prior to 37 weeks (e.g. prior classical cesarean) 
7. threatened abortion, preterm labor, or ruptured membranes at time of enrollment 
8. known allergy or medical comorbidity listed as a contraindication to 17P in the 
prescribing information 
9. prior participation in the trial 
10. any other condition (social or medical) which, in the opinion of the study staff, 
would make trial participation unsafe or complicate data interpretation.  

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7.3 
Study intervention 
Participants will be randomized 1:1 to one of two arms in the randomized controlled trial. 
The first arm (17P arm) will receive weekly injections of 250mg 17-hydroxyprogesterone 
caproate while the other arm (control arm) will receive indistinguishable placebo. 
7.4 
Study drug pharmacological information 
Pharmacologic Category: Synthetic progestin 
Chemistry: 17-hydroxyprogesterone caproate (250mg/mL) is a sterile solution of 
hydroxyprogesterone caproate in castor oil for injection. Micronized progesterone is active by 
oral and vaginal routes of administration. It is a clear, yellow, sterile, non-pyrogenic solution 
for intramuscular injection. It is an ester derivative soluble in ethanol, acetone or ether; 
slightly soluble in tea oil or castor oil; and insoluble in water. 
Each 5mL multidose vial contains hydroxyprogesterone caproate USP, 250 mg/mL (25% 
w/v), in castor oil USP (28.6% w/v) and benzyl benzoate USP (46% w/v) with the 
preservative benzyl alcohol NF (2% w/v).  
Mechanism of Action: Hydroxyprogesterone caproate is a synthetic progestin. The 
mechanism by which hydroxyprogesterone caproate reduces the risk of PTB is unknown. 
However, endogenous progesterone is a potent anti-inflammatory and is thought to prevent 
preterm birth by counteracting inflammatory processes that lead to cervical remodeling, 
amniotic membrane rupture, uterine contractions, and the initiation of parturition. Prenatal 
progestins, either micronized progesterone via vaginal administration or 17P via 
intramuscular administration, are 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: Intramuscular injection 
Strength: 250mg (1mL of 250mg/mL concentrated solution) 
7.5 
Study procedures 
7.5.1 Screening  
Our recruitment activities will begin with community sensitization in the catchment area of 
the recruitment clinics to educate community members about the trial and encourage early 
presentation for ultrasound. This community and clinic sensitization may be underway prior 
to initiation of this project as a result of other programs in the catchment areas.  
Trained study staff will conduct health talks at the study site, 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 will be identified at their first antenatal visit. Women may be enrolled 
any time prior to 24 0/7 gestational weeks. Following a prescreening consent process, a dating 
ultrasound will be conducted per standard of care to confirm pregnancy location, viability, 
number of fetuses, and, among women within the recruitment window, cervical length 

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according to the method described by Iams et al.90 Those who meet preliminary eligibility 
criteria who are interested in participating will complete the study informed consent process 
in English, Nyanja, or Bemba, depending on their language preference. Trial participation 
will be offered to all HIV-infected pregnant women who are determined to meet the listed 
inclusion and exclusion criteria. 
After consenting for participation, women will be administered a series of questionnaires that 
gather targeted information on relevant demographics, health and risk behaviors, medical and 
obstetrical history, medication use, stress, and nutrition. If not already done so in routine 
antenatal care, participants will undergo a physical exam, including testing for urinary tract 
infections and proteinuria, anemia, syphilis, and HIV (as outlined in TABLE 1), all of which 
are recommended as part of routine antenatal care. Treatment of HIV and any obstetric 
complications will also be according to standard practice in Zambia.  
Blood, urine, and vaginal swabs will be collected as detailed in Table 1.  
7.5.2 Randomization 
Following final confirmation of eligibility, participants will undergo randomization into one 
of two study arms as soon as possible starting at 16 0/7 gestational weeks and prior to 24 
weeks. The trial will 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.  
7.5.3 Study product preparation and dispensation 
Study product will be produced, labeled, and packaged by the manufacturer, AMAG 
Pharmaceuticals of Waltham, MA, USA, under the brand name of Makena®. Each package 
will have a unique identifying number produced from a random number generator that will be 
indexed in a secured database as either placebo or product. Packaged product will be shipped 
to each study site and dispensed by an on-site pharmacist. Participants will begin weekly 
administration of study product from the day of randomization (between 16 0/7 and 23 6/7 
gestational weeks, inclusive) until 36 6/7 gestational weeks, stillbirth or delivery, whichever is 
sooner. Trained study nurses will administer study product in the manner indicated on the 
package insert and in accordance with Good Clinical Practice.  
7.5.4 Antenatal, intrapartum, and immediate postpartum study follow-up 
The specific content of each study visit can be found in TABLE 1. Because the trial will be 
placebo controlled and treatment allocation double masked, all women will have identical visit 
schedules. Starting as early as possible between 16 and 236/7 gestational weeks, participants 
will receive a weekly 250 mg injection of study drug or matched placebo. At each visit, we will 
evaluate women for maternal side effects of IM progesterone, which are rare and generally 
mild, but include headache, breast tenderness, nausea, cough, and local irritation.  
Routine antenatal and HIV care will be provided to all participants following Zambian national 
guidelines. Antenatal care visits are scheduled approximately every four weeks. 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. 

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Protocol v1.6 | 26 March 2020 
At 24 and 28 weeks, blood, urine, and vaginal swabs will be collected as detailed in Table 1. 
At the time of delivery or soon thereafter, blood, urine, and vaginorectal swabs will be 
collected when possible. In addition, we will collect samples of the placenta, umbilical cord, 
and cord blood after delivery for various assessments. 4-5 drops of cord blood will be applied 
to designated filter paper within pre-printed circles. Newborn heel-prick samples will be 
collected 24–72 hours after birth or sooner if the newborn is discharged from hospital within 
24 hours of delivery. At each weekly visit, oral and pharyngeal swabs will be collected for 
storage and future protocol-related testing.   
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 when possible.  
TABLE 1: Schedule of Evaluations 
Visit Number 
0.0 
1.0† 
2.0 
3.0 
4.0 
5.0 
6.0 
7.0 
Gestational Age (weeks) 
<24 
16-236 
24 
28 
32 
36 
Delivery^ 
42 days 
ADMINISTRATIVE/REGULATORY PROCEDURES 
Informed consent 
• 
 
 
 
 
 
 
 
Confirmation of eligibility 
 
• 
 
 
 
 
 
 
Collection/review of locator info 
• 
• 
• 
• 
• 
• 
• 
• 
Randomization 
 
• 
 
 
 
 
 
 
CLINICAL/BEHAVIORAL PROCEDURES 
Ultrasound* 
• 
• 
 
 
• 
 
 
 
Obstetrical history 
• 
 
 
 
 
 
 
 
Medical history and clinical exam* 
• 
 
• 
 
• 
• 
• 
• 
Concomitant medication assessment 
• 
 
• 
 
• 
• 
• 
• 
Demographic history 
• 
 
 
 
 
 
 
 
Behavioral and nutritional assessment 
• 
 
 
 
 
 
 
• 
Maternal depression screen 
• 
 
 
 
 
 
 
• 
Infant clinical assessment 
 
 
 
 
 
 
• 
• 
STUDY PRODUCT PROCEDURES 
17P Adherence counseling 
 
• 
• 
• 
• 
• 
 
 
Study drug injections‡ 
 
• 
• 
• 
• 
• 
 
 
Side effects assessment‡ 
 
 
• 
• 
• 
• 
 
 
LABORATORY PROCEDURES 
Maternal HIV (rapid EIA)* 
• 
 
 
 
 
 
 
 
Maternal pregnancy test* 
• 
 
 
 
 
 
 
 
Maternal rapid syphilis  * 
• 
 
 
 
 
 
 
 
Maternal candida, gram stain* 
• 
 
 
 
 
 
 
 
Maternal hemoglobin (HemoCue)* 
• 
 
 
 
• 
 
 
 
Maternal urinalysis (dip; culture if +)* 
• 
 
 
 
• 
 
 
 
Maternal viral load and T cell assays 
• 
 
• 
 
 
 
• 
 

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Protocol v1.6 | 26 March 2020 
Visit Number 
0.0 
1.0† 
2.0 
3.0 
4.0 
5.0 
6.0 
7.0 
Gestational Age (weeks) 
<24 
16-236 
24 
28 
32 
36 
Delivery^ 
42 days 
Placenta, membranes, cord blood storage 
 
 
 
 
 
 
• 
 
Infant DBS for gestational age screening 
 
 
 
 
 
 
• 
 
Infant HIV DNA PCR* 
 
 
 
 
 
 
 
• 
Maternal oral / pharyngeal swabs storage § 
 
 
• 
• 
• 
• 
• 
• 
Vaginal-rectal swab storage 
• 
 
• 
• 
 
 
• 
 
Blood storage 
• 
 
• 
• 
 
 
• 
 
Urine storage 
• 
 
• 
• 
 
 
• 
 
† 
If screening occurs at or after 16 weeks of gestation, all procedures may be combined into a single 
screening/randomization visit  
* 
Performed as standard antenatal care 
‡ 
Occurs weekly through week 36, or until delivery, whichever occurs first 
^ 
All procedures may not be completed for women who deliver off-hours or in a location without 
staff coverage.  
§ 
Swabs will be collected at weekly injections visits and other visits as indicated. Collection may be 
deferred for participants who refuse or if necessary supplies are unavailable. 
7.5.5 Postnatal study follow-up 
Participants will be asked to return for one study visit during the postnatal period, at 42-days 
post-partum. At this visit, we will assess interval complications and/or mortality. Once again, 
participants will receive the routinely recommended screening and treatment. Infants’ weight, 
head circumference, length, mid-upper arm circumference, feeding, and general well-being 
will also be assessed. In the postnatal period, health education will be geared toward routine 
care of post-partum women and both preterm and term newborns.  
At the 42-day postnatal visit, participants will again be screened for depression using the 
Edinburgh Postnatal Depression Scale. Those who exhibit a score consistent with signs of 
depression or suicidality will be immediately referred for further evaluation and treatment. 
Finally, oral / pharyngeal swabs will again be collected for storage and future testing. 
7.5.6 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 
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. 

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7.5.7 High priority conditions 
Samples collected via cord blood and heel prick for metabolic gestational age screening may 
reveal screen positive results for high priority conditions, such as congenital hypothyroidism, 
hemoglobinopathies, and medium-chain acyl-CoA dehydrogenase deficiency. These 
conditions have the potential for confirmatory testing and for intervention in the early stages 
of an infant’s life. Upon obtaining these results, the family will be notified of the identified 
condition and study personnel will facilitate a referral for confirmatory testing and treatment.  
If the findings from any other testing performed in real time or on stored specimens provide 
information that may be directly relevant to a participant’s or her infant’s health care, the 
study staff will make reasonable efforts to notify her of the results.  
7.5.8 Safety monitoring 
At each study visit, study staff will evaluate participants for social harms and adverse events 
(AEs) including adverse drug reactions or events related to venipuncture or 17P 
administration. 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 laboratory data s and 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. 
We expect events to be minimal, but all will be documented, assessed for seriousness / 
severity and relatedness, and carefully monitored. 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 of 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 
(PI) 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 and facilitate 
staff re-training, protocol amendment, or study cessation. Serious study-related adverse 
events will be reported to all regulatory bodies, the study sponsors, and the DSMB (see 
below) within 72 hours of site awareness. Other study-related AEs will be reported according 
to each ethics committees’ individual guidelines.  

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We will constitute a Data Safety and Monitoring Board (DSMB) to periodically monitor trial 
performance, feasibility, and study outcomes. The group will comprise senior investigators 
with statistical, methodological, and topical expertise who are not otherwise involved with 
the study. Their first charge will be to review the study protocol and create a monitoring 
schedule and stopping guidelines, based on expected event rates. Other specific duties will 
include: (1) periodic assessments of recruitment, accrual, retention, and data quality; (2) 
Considering new external data as they come available, including scientific or therapeutic 
developments that may have an impact on participant safety or the ethics of the trial; (3) 
evaluation of scheduled interim analysis. Mortality outcomes will be reviewed by the DSMB 
during routine meetings. The DSMB will have authority to enjoin enrolment or stop the study 
altogether for reasons of patient safety. If an interim analysis were to show unequivocal 
benefit before follow-up were complete, we would support stopping the study and 
reprogramming remaining resources to make the intervention available to all participants.  
The DSMB will also have a specific role in determining study feasibility. Prior to study 
commencement, the DSMB will develop clear indicators of trial feasibility and rules for 
determining that the trial has become futile based upon recruitment performance or inability 
to ascertain key endpoints. In the event that these criteria are met, we will close the trial. 
7.6 
Biological specimen collection and testing 
All samples will be obtained from study participants by trained study staff according to 
approved standard operating procedures. All samples will be processed according to the assay 
manufacturers’ specifications. Some specimens collected from patients in this protocol will 
be analyzed immediately per standard antenatal care guidelines. Others will be stored 
temporarily for later study-related analysis to identify inflammatory markers (e.g., 
chemokines, cytokines), metabolic analytes, proteins, hormones, transcripts, and infectious 
factors contributing to preterm birth and other adverse birth outcomes.  
Samples will be primarily housed at the University Teaching Hospital, Department of 
Obstetrics and Gynecology. Use of stored specimens for testing that is not specifically 
designated in this protocol will require additional regulatory approval.  
All laboratory testing will be performed by trained staff using standard operating procedures 
and according to specific assay manufacturers’ specifications. 
7.7 
Quality control and quality assurance procedures 
Standard Operating Procedures (SOPs) following manufacturer’s protocols and detailing 
technical procedures involved (e.g. sample collection, processing and storage, assay 
procedures and how to interpret test results) will be developed and used by the study team. 
Tests will only be performed by certified laboratory personnel. Site coordinators will 
complete annual recertification. The certification process is an opportunity to ensure the 
highest specimen quality and standardize collection techniques. 
7.8 
Data security and management 
Data collected from each participant will include sociodemographic information, relevant 
HIV and obstetrical history, longitudinal clinical details of current pregnancy and delivery, 
and results of laboratory specimen testing.  
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 and 

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communications related to the study. 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 following sponsor recommendations. Biological specimens will be 
destroyed as dictated by the University Teaching Hospital guidelines. 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. 
7.9 
Statistical considerations 
7.9.1 Trial outcomes 
Because stillbirth and preterm birth are competing risks, the trial’s primary outcome will be a 
composite of live birth prior to 37 weeks of gestation or stillbirth at any gestational age. We 
chose this outcome because it is (1) associated with substantial morbidity in Zambia, (2) 
incontrovertible, given that entry into the study is contingent upon early dating ultrasound, 
and (3) can be easily obtained even in the event of home delivery. Secondary outcomes will 
include: (a) delivery prior to 34 weeks of gestation; (b) delivery prior to 28 weeks of 
gestation; (c) spontaneous delivery prior to 37 weeks of gestation; (d) spontaneous delivery 
prior to 34 weeks of gestation; (e) spontaneous delivery prior to 28 weeks of gestation; (f) 
birth weight < 10th centile for gestational age; (g) birth weight < 3rd centile for gestational 
age; (h) mother-to-child HIV transmission by 6-weeks postpartum, and (i) cumulative 
incidences of the competing risks of stillbirth, spontaneous PTB, and non-spontaneous PTB. 
To ensure comparability with other prematurity trials,6,50 we will also systematically assess 
neonatal and perinatal mortality rates; infant APGAR scores; infant birth weights; measures 
of infant morbidity such as ICU admission, supplemental O2 requirement, and need for 
assisted ventilation; and measures of maternal morbidity, such as pregnancy-induced 
hypertension, antepartum hemorrhage, preterm premature rupture of membranes, oligo- or 
polyhydramnios, chorioamnionitis, and mode of delivery. 
7.9.2 Sample size calculation: baseline event rate (p1) 
We estimated the baseline rate of our primary outcome as follows:  
• The ZEPRS system in Lusaka91 estimates GA at birth from the last menstrual period 
(LMP), a method that, in our setting, consistently overestimates prematurity.92 A 
study performed by our group of all public sector births in Lusaka between 2006 and 
2010 found that 31,896 of 100,408 women (31.7%) delivered before their LMP-
derived EGA reached 37 weeks completed gestation.93 
• As of April 2017, 1116 women had been enrolled in the GAPPS Preventing Preterm 
Birth study and 497 had delivered. All enrollees have an early dating ultrasound at 20-
24 weeks.54 Based on reliable GA estimates from these ultrasounds, 18.1% of women 
overall delivered prior to 37 weeks.   
• A large international modeling effort1 published by Blencowe et al in 2010 estimated 
that 12.9% of infants in Zambia are born prior to 37 weeks of gestation. While this 

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Protocol v1.6 | 26 March 2020 
effort aggregated hundreds of published studies to inform its models, no data were 
included from Zambia. Since the Zambia estimate is not consistent with our own 
measurements, we believe it may be artificially lowered by inclusion of country 
economic data in the models. (Zambia is categorized as “lower-middle income” 
economy, a threshold it achieves only by a tiny margin; data from neighboring 
Zimbabwe94-96 are consistently reported in the 17-20% range.)  
• Given the suspected underestimation of gestational age in the ZEPRS data, and the 
relatively small sample size in the GAPPS cohort, we applied the Blencowe figures to 
existing data from our site for these sample size calculations, arriving at an overall 
prematurity rate of 15.5% (arithmetic mean of Zambia country estimates and current 
PTB rate in GAPPS) for our calculation of the baseline event rate. We note that to 
underestimate the prematurity rate would be conservative vis-à-vis our sample size 
calculations. 
• Available estimates for Zambia do not disaggregate prematurity risk by HIV status. 
We therefore queried ZEPRS,91 which does capture birth weight completely and 
correctly. We used birth weight as a proxy for prematurity to estimate the relative risk 
of preterm birth by HIV status in our setting. An analysis of 211,318 public sector 
singleton births in Lusaka between 2006 and 2013 found that 17.2% of HIV-exposed 
infants weighed <2500g at delivery, compared to 11.8% unexposed infants. 
(Chibwesha et al, submitted for publication; TABLE 2). 
TABLE 2: Risk of LBW among 211,318 singleton infants born to women of known HIV 
status in Lusaka 
 
<2500 gms* 
 
<2000 gms 
 
<1500 gms 
 
N 
% 
 
N 
% 
 
N 
% 
HIV - (78%) 
19828 
11.8 
 
7081 
4.2 
 
3878 
2.3 
HIV + (22%) 
7956 
17.2 
 
3088 
6.7 
 
1720 
3.7 
RR (95%CI) 
 
1.46 (1.43, 1.50) 
 
 
1.59 (1.52, 1.65) 
 
 
1.61 (1.53, 1.71) 
 *NB: Birth weight categories are not mutually exclusive  
• Although low birth weight is not a perfect proxy for prematurity (most notably, it 
does not distinguish growth restriction from preterm birth), it is the only outcome we 
have to estimate the preterm birth rates in HIV+ versus HIV- women in our setting. 
We used the antenatal HIV prevalence and relative risk of LBW delivery from TABLE 
2, to estimate a rate of prematurity in HIV-infected women that would correspond to 
an overall rate of 15.5% (Goal Seek Function, MS Excel).  
• Under these assumptions, we estimate that the prematurity rate would have to be 
20.9% among HIV-infected women and 14.0% among HIV-uninfected women to 
produce an overall rate of 15.5% in a setting of 22% maternal HIV prevalence at 
UTH. To be conservative, we have not assumed any additional prematurity risk 
conferred by receipt of ART. 
• Finally, a study of more than 100,000 public sector deliveries performed by our group 
found a stillbirth rate of 21 per 1,000 live births.93 Among the 21,816 HIV-infected 
parturients, the stillbirth rate was 27 per 1000 (95% CI: 25, 29). Because our 
composite primary outcome includes stillbirth, we add a 2.7% stillbirth rate to the 
20.9% prematurity rate to arrive at a baseline event rate of 23.6%. Rounding to 2 
significant figures, we have set the baseline event rate, p1 = 0.24. 

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7.9.3 Sample size calculation: effect size  
A 2013 meta-analysis estimated the RR of preterm delivery for women using any 
progestogen to be 0.55 (95%CI: 0.42, 0.74).5 For the purposes of our study, we will use the 
more conservative subtotal estimate from only the IM studies (FIGURE 1). This 0.62 RR 
translates to a 38% relative efficacy.  
FIGURE 
1: 
Summary 
of 
IM 
progesterone trials in Cochrane5 
meta-analysis 
 
7.9.4 Trial sample size 
In summary, we postulate that 17P will reduce the proportion of HIV-infected women 
experiencing the composite endpoint by 38% (from 24% to 15%). We used SAS 9.3 (SAS, 
Cary, NC) to determine that a trial with 325 patients per arm would have 80% power to detect 
this effect size (based on a 2-sided Fisher’s exact test at the 0.05 significance level; FIGURE 
2). To allow for up to 15% follow-up loss (although prior experience from our sites suggests 
it will be half this) and to account for uncertainty in our estimates of baseline event 
probability (p1) and relative efficacy of the intervention, we plan to enroll 400 women per 
arm in the trial. As FIGURE 2 indicates, the study will have 80% power to demonstrate any 
combination of baseline event rate and relative efficacy that falls below the red horizontal 
line (after accounting for follow-up losses and any missing outcomes). 
7.9.5 Stratification and randomization 
We will stratify our randomization by timing of ART initiation (during current pregnancy / 
pre-pregnancy). Participants will be assigned with equal probability to one of the two 
treatment groups. A statistician not otherwise associated with the study will design the 
randomization scheme using random permuted blocks and taking into account the 
stratification scheme.  
7.9.6 Analysis plan 
Our primary outcome will be the composite outcome comprising delivery prior to 37 weeks 
completed gestation or stillbirth at any gestational age. At the time of enrollment, each 
participant will be assigned an estimated delivery date (EDD) by ultrasound biometry.97-99 
Figure 2: Sample size per arm for 80% power 
 

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Randomized women who deliver (1) a live infant prior to 37 weeks of gestation or (2) a 
stillborn infant at any gestational age will be categorized as having met the primary outcome.  
The primary analysis will employ the intent-to-treat principle, wherein each mother-infant 
pair will be analyzed according to randomization assignment regardless of whether the 
mother actually complied with study procedures. The primary comparison between the two 
trial arms will be based on the estimated risk ratio of the primary outcome. The estimated risk 
of the primary outcome will be computed in each arm using the Kaplan-Meier method to 
adjust for loss-to-follow-up, where mother-infant pairs who drop out of the trial prior to the 
primary outcome or live-birth of full-term infant will be right censored at their last study 
visit. A Z-statistic will be constructed by taking the difference in the estimated log risks 
divided by the corresponding estimated standard error (using the Greenwood estimator). As a 
sensitivity analysis, a Cox proportional hazards model, stratified by timing of ART initiation 
(during current pregnancy / pre-pregnancy) will be fit to estimate the hazard ratio between 
randomization arms; the estimated hazard ratio is anticipated to be similar to the estimated 
risk ratio given that the primary outcome is relatively rare (<20%). In a secondary analysis, 
we will compare the risk of spontaneous preterm birth across randomized groups, treating 
provider-initiated preterm births as a competing risk and estimating the risk of the outcomes 
in each randomized group using the Aalen-Johannson method. To explore comparability with 
other published studies,9 we will evaluate the effect of excluding events that occur prior to 20 
weeks in a sensitivity analysis. 
Subgroup analyses will assess whether the effect of 17P differed within levels of the 
randomization stratification factor. For timing of ART initiation, effect modification will be 
assessed by fitting Cox proportional hazards models that include as covariates randomization 
arm, the stratification factor, and a product term. A likelihood ratio test of the product term 
coefficient will be used to assess possible effect modification. To adjust for multiple 
comparisons, the likelihood ratio tests will be considered significant if p < 0.05/2. If the effect 
modification tests are significant, hazard ratios will be estimated separately within levels of 
the factor (e.g., hazard ratios of 17P versus placebo for (i) mothers on pre-pregnancy ART 
and (ii) mothers not on pre-pregnancy ART).  
To assess the efficacy of 17P, a per-protocol analysis of the primary outcome will also be 
conducted that censors participants who fail to comply with their randomization assignment 
(defined here as missing two injections of study product in a row), and then reweights the 
data to account for possible informative censoring by noncompliance using inverse-
probability (IP) weights.100 We have previously used this and related methods to construct a 
consistent estimator of the per-protocol effect under the assumption that we have measured 
and correctly-adjusted for the common causes of noncompliance and the outcome.101-103 The 
set of measured possible common causes includes demographic, medical, and obstetrical 
factors. We will construct IP weights using pooled logistic regression (pooling over time), 
with continuous variables fit using flexible restricted splines. We will explore the sensitivity 
to IP weight estimation.104 
7.10 
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. 

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Protocol v1.6 | 26 March 2020 
8.0 
Ethical Considerations 
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 and maintaining Federal Wide Assurances (FWA) with the Office for Human Research 
Protections (OHRP) approval. 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 on the protection of human research participants prior to conducting any 
study activities and routinely thereafter. Key staff will also complete Good Clinical Practices 
training every three years. 
8.1 
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 HIV and 
antenatal care and treatment according to local clinical standards.   
8.2 
Potential risks to participants  
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 17P 
administration are rare (see ADVERSE EVENTS, below). Side effects may include headache, 
breast tenderness, nausea and local irritation at the injection site. Women with one or more 
contraindication to 17P that appear in the package insert will be excluded from the trial, 
namely: (1) current or history of thrombosis or thromboembolic disorders; (2) known or 
suspected breast cancer, other hormone-sensitive cancer; or history of these conditions; (3) 
undiagnosed abnormal vaginal bleeding unrelated to pregnancy; (4) cholestatic jaundice of 
pregnancy; (5) liver tumor(s), benign or malignant, or active liver disease; (6) uncontrolled 
hypertension. Serious reactions have not been confirmed in women who do not have these 
conditions. 
Physical risks also include the risk of discomfort, bruising or swelling from venipuncture. 
The risks that are associated with venipuncture are infrequent and minimized with the use of 
proper technique. Such risks include (1) bleeding, (2) bruising, or (3) rarely infection at the 

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Protocol v1.6 | 26 March 2020 
site of needle insertion. Individuals may also rarely become faint, in which case symptoms 
abate after several minutes in a recumbent position. Blood volumes for the study have been 
calculated to ensure safety. Collection of vaginal and rectal samples may also be associated 
with some discomfort and mild bleeding. Finally, collection of oral / pharyngeal swabs may 
cause discomfort; in very few participants, the sensation of gagging may lead to an urge to 
vomit or cough. Participants will be reassured in all cases that such feelings are transient. 
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 (e.g., consenting participants in a private setting, not including names on 
case report forms, etc.). 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.   
8.3 
Potential benefits to participants and others  
Individual participants in the 17P 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 17P 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. The exact mechanism of action for the potential effect of ART on PTB risk is also 
unclear, though it is biologically plausible that ART initiation in pregnancy could lead to 
PTB through an immune activation / inflammatory mechanism. Since 17P is thought to act at 
least partially through an anti-inflammatory mechanism, it might provide protection for HIV-
infected gravidas starting or continuing ART, but this question is yet unanswered. 
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 and enhanced training on 
specimen collection. Increased awareness about screening and treatment for preterm birth 
may help to improve care overall at UTH and KDC. Additionally, our community 

Improving Pregnancy Outcomes with Progesterone (IPOP) 
Page 26 of 34 
Protocol v1.6 | 26 March 2020 
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, risks to participants in this study are minimal and do not differ significantly 
from the risks inherent in the local standard of care for HIV-infected pregnant women and 
their infants, with the exception of the relatively mild side effects of the study drug and the 
additional risks to participant confidentiality and data privacy. These risks are reasonable in 
relation to the anticipated benefits if the study drug is found to be effective. Thus, the 
knowledge generated from this study regarding the potential PTB risk reduction among HIV-
infected women is expected to outweigh the risks of participation. 
8.4 
Inclusion of children, sub-populations, and vulnerable populations  
This study focuses specifically on the outcomes of HIV infected pregnant women and their 
infants and, as such, they must be included in our study population. Prisoners will be 
excluded as they receive care at separate facilities. 
9.0 
Timeline 
Participants and their infants will be followed from enrollment (prior to 24 weeks gestational 
age) through 42 days postpartum. We have aligned this follow-up schedule exactly with that 
of the ongoing GAPPS/PPB cohort. Because that study is already in place at the Zambia site, 
we anticipate that trial start-up will be quick. We have thus allocated 9 months to start-up 
activities and 3 months to wind-down activities. 
 
10.0 
Budget  
The following costs are projected over the five-year study period.   
ITEM 
ZMW TOTAL 
USD TOTAL 
Personnel 
K 7,820,000 
$850,000 
Study management 
2,622,000 
$285,000 
Clinic staff 
3,266,000 
$355,000 
Lab staff 
1,214,400 
$132,000 
Pharmacy staff 
717,600 
$78,000 
Equipment – Vehicle 
K 368,000 
$40,000 
Year 1 
Year 2 
Year 3 
Year 4 
Year 5 
          Protocol and CRF 
development   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Ethical submission*   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Site preparation   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Data systems 
programming   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Site training   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Enrollment   
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
          Follow-up   
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
          Data entry and 
management   
  
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
  
  
  
          Statistical analysis   
  
  
  
  
  
 
  
  
  
  
  
  
  
  
  
          Publication and 
dissemination   
  
  
  
 
 
 
  
  
  
  
  
  
  
  
  

Improving Pregnancy Outcomes with Progesterone (IPOP) 
Page 27 of 34 
Protocol v1.6 | 26 March 2020 
Supplies & materials 
K 2,024,000 
$220,000 
Clinic/Office supplies 
423,200 
$46,000 
Lab tests 
1,324,800 
$144,000 
Fuel/Transportation costs 
276,000 
$30,000 
Participant reimbursement 
K 828,000 
$90,000 
Other 
K 1,840,000 
$200,000 
Administrative costs (rent, utilities, internet, 
communication, shipping, insurance, etc.) 
1,058,000 
$115,000 
Lab maintenance 
368,000 
$40,000 
Community outreach 
138,000 
$15,000 
Regulatory/Translation 
110,400 
$12,000 
Staff Training/Meetings 
46,000 
$5,000 
Analysis/Dissemination costs 
119,600 
$13,000 
TOTAL 
K 12,880,000 
$1,400,000 
 
 

Improving Pregnancy Outcomes with Progesterone (IPOP) 
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Protocol v1.6 | 26 March 2020 
11.0 
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