Impact of Hormonal Contraception on HIV Acquisition and Transmission Risk
Phase: PHASE4
Conditions: HIV, Contraception
Sponsor: Lisa Haddad

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 1 
 
Impact of Hormonal Contraception on HIV acquisition and transmission risk 
 
Protocol version 9, dated July 10, 2017 
 
NCT02357368 
 
 

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 2 
 
Impact of Hormonal Contraception on HIV acquisition and transmission risk 
 
Protocol chair 
Lisa Haddad, MD, MS 
Department of Gyncecology and Obstetrics, Emory University School of Medicine, 
Atlanta, GA 
 
Protocol co-chair 
Igho Ofotokun 
Department of Medicine, Infectious Disease, Emory University School of Medicine, 
Atlanta, GA 
 
TABLE OF CONTENTS 
 
Abbreviations and acronyms ...............................................................................................4 
Project overview .....................................................................................................................5 
1.1 
Summary ................................................................................................................................ 5 
2 Introduction .......................................................................................................................7 
2.2 
Significance of proposed study ........................................................................................... 16 
3.0 
Study Objectives and Hypotheses ........................................................................................... 17 
3.1 
Research hypotheses and objectives .................................................................................. 17 
4 
Study Design .............................................................................................................................. 19 
4.1 
Recruitment, screening and enrollment ............................................................................... 20 
4.1.3 
Eligibility ........................................................................................................................... 22 
4.1.4 
Consent ............................................................................................................................ 23 
4.1.5 
Study completion .............................................................................................................. 23 
4.1.6 Study visit schedule .............................................................................................................. 23 
4.2 
Study visits ........................................................................................................................... 25 
4.3.1 
Disenrollment ................................................................................................................... 26 
4.3.2 
Participant referral ............................................................................................................ 27 
5 
Data and Biological Specimen Collection.................................................................................... 27 
5.1 
Clinical evaluation ................................................................................................................ 27 
5.2 
Laboratory testing................................................................................................................ 29 
5.3 
Specimen management and storage .................................................................................... 33 
5.4 
Specimen collection considerations ..................................................................................... 35 
6 
Ethics and Risks .......................................................................................................................... 35 
6.1 
Potential risks ..................................................................................................................... 35 
6.2 
Protection against risks ....................................................................................................... 37 
6.3 
Benefits to participation ..................................................................................................... 39 
6.4 
Importance of the knowledge gained ................................................................................. 39 
6.5 
Limitations and Future Directions ....................................................................................... 40 
6.6 
Inclusion of women, minorities, and children ..................................................................... 41 
7 
Monitoring and Oversight .......................................................................................................... 41 
7.1 
Site monitoring ................................................................................................................... 41 
7.2 
Data quality assurance and protection ............................................................................... 41 

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 3 
 
7.3 
Protocol violation ............................................................................................................... 43 
7.4 
Adverse events ................................................................................................................... 43 
8 
Statistical Considerations and Analysis ..............................................................................44 
9 
Dissemination of Results ....................................................................................................46 
 
 
 
 
 
 
 
 
 
 
 
 

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 4 
 
Abbreviations and acronyms 
 
AE 
Adverse event 
AIDS 
Acquired immunodeficiency syndrome 
ART 
Antiretroviral therapy 
ARV 
Antiretroviral 
CRF 
Clinical research form 
CVF 
CVL 
Cervicovaginal Fluid 
Cervicovaginal Lavage 
DMPA 
GCP 
GLP 
HAART 
Depo Medroxyprogesterone Acetate, Depo Provera 
Good Clinical Practice 
Good Laboratory Practice 
Highly active antiretroviral therapy 
Hb 
Hemoglobin 
HIPAA 
HIV 
Health Insurance Portability and Accounting Act  
Human immunodeficiency virus 
IUD 
IRB 
Intrauterine Device 
Institutional review board 
PI 
PIN 
RA 
RNA  
Principle Investigator 
Participant identification number 
Research Assistant 
Ribonucleic acid 
SAE 
Serious adverse event 
STI 
Sexually transmitted infection 

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 5 
 
Project overview 
1.1 
Summary 
This study is a proof of concept prospective cohort study focusing on HIV negative 
women. The 3 proposed aims will evaluate the effect of depot medroxyprogesterone 
acetate (DMPA), Etonogestrel impant (Eng-Implant, Nexplanon) and Levonorgestrel 
intrauterine device (Lng IUD, Mirena IUD or copper IUD) exposure on 1) HIV target 
immune cells within the female genital mucosa; 2) markers of T-cell activation and 
trafficking within the female genital mucosa; and 3) secreted cytokines and chemokines 
within the female genital mucosa.  A prospectively recruited cohort of HIV negative 
women will allow the examination of the overarching hypothesis that alterations in HIV 
target immune cells and function within the female genital mucosa underlie the relatively 
higher HIV risk associated with the pharmacologic doses of exogenous sex hormones, 
Because the anticipated mucosal immune changes with progestin-only contraceptives 
are to a large extent mediated via estrogen suppression, the impact of  Nexplanon and 
Mirena IUD (with milder anti-estrogen effect) is expected to be significantly less 
pronounced compared with that of DMPA. The outcomes of the proposed study could 
have significant clinical implications for the provision of family planning services for 
women worldwide. 
 
 
 

V9: Impact of hormonal contraception on HIV acquisition risk                                              Page 6 
 
1.2 Investigators
Protocol chair 
Lisa Haddad, MD, MS, MPH  
Dept. of Gynecology and Obstetrics Emory University School of Medicine 
404-778-1385 
lbhadda@emory.edu 
 
Emory-based co-investigators 
Igho Ofotokun, MD, MSc 
Department of Medicine 
Emory University School of Medicine  
404-616-0659 
iofotok@emory.edu  
 
Rama Amara MD MSc 
Department of Microbiology and Immunogy 
Emory University School of Medicine  
 
ramara@emory.edu 
 
CDC-based co-investigators 
Denise J. Jamieson, MD, MPH 
Division of Reproductive Health, CDC 
770-488-6377 
DJamieson@cdc.gov 
 
Clyde Hart  PhD 
Clinical Support Team Leader & Research Microbiologist  
ceh4@cdc.gov 
 
Role of the investigators  
Dr. Haddad will provide general oversight for this study as part of her K23 
training grant. Drs. Ofotokun and Jamieson will serve as advisors for the study. Dr. Hart 
will facilitate the laboratory component of the study while Dr. Amara will assist in 
interpretation and evaluation of immunologic findings. 
 
The investigators have no conflicts of interest to report. 
 

 
7 
 
2 
Introduction 
2.1 
Background 
Interconnected goals of HIV and unintended pregnancy prevention:  Globally, nearly 
16 of the 33 million people living with HIV infection are women, most of whom are of 
childbearing age.(1) Beyond the direct morbidity and mortality of HIV, the majority of these 
women live in regions overwhelmed by high fertility and where the baseline rates of 
pregnancy associated morbidity and mortality are especially high. Pregnancy prevention 
can mitigate maternal morbidity and mortality, decrease unsafe abortion, reduce poverty, 
and improve the educational attainment and status of women. Additionally, for women with 
HIV, pregnancy prevention can prevent mother-to-child transmission of HIV and resultant 
“AIDS orphans”. Of note, the World Health Organization, United Nations and global public 
health organizations recognize the prevention of new HIV infections and prevention of 
unintended pregnancy as critical overlapping public health strategies to prevent mother-to-
child transmission, reduce child and maternal mortality and improve health.(2, 3) 
Condoms, while effective to some degree in preventing sexually transmitted infections 
(STIs) including HIV, are not an ideal method of preventing unintended pregnancies when 
used alone. Condom use is associated with a high contraceptive failure rate of ~15% in 
the first year with typical use.(4) Furthermore, despite three decades of aggressive 
promotion of condom use for the prevention of HIV transmission, consistent use remains 
inadequate.(5) The most common forms of contraception used worldwide are progestin-
containing hormonal contraceptive methods, either alone or in combination with estrogen, 
with over 150 million users(6). Hormonal contraceptives have higher pregnancy prevention 
efficacy rates than condoms, with contraceptive failure rate of ~3% following typical DMPA 

 
8 
 
use, and <1% following contraceptive implant and intrauterine device use.(4) Hormonal 
contraceptives have high acceptance rates in many communities around the world where 
condom use may be poor or where non-hormonal contraceptive options, such as the 
copper intrauterine device, are not acceptable or available. 
 
Concerns regarding hormonal contraception: Despite its excellent contraceptive 
profile, there are growing concerns that contraception hormones, specifically DMPA, may 
influence susceptibility to HIV and contribute to the spread of HIV infection.(7-9)If 
confirmed, this risk would represent a significant threat to women’s health with far-
reaching implications for public health policy. Contraceptive approaches that increase the 
risk of HIV acquisition or transmission would have important implications for health at the 
individual and population levels, as well as on the overall socioeconomic impacts of the 
AIDS epidemic. 
The significance of this topic is underscored by the ongoing debate it has generated 
among experts in the field and the increasing research focus in this area. The rationale for 
these intense research activities is anchored by animal data demonstrating several fold 
increases in the risk of SIV acquisition in rhesus macaques following DMPA exposure,(10) 
and from corroborative human epidemiologic studies.  Notable among these studies is the 
Mombasa study in Kenya where DMPA use was associated with a 2-fold rise in the 
incidence of HIV infection among 1,500 female sex workers.(8) Another example, from the 
Demographic and Health Surveys of over 4,000 young women from four African countries, 
reported higher HIV sero-prevalence with DMPA use, with ~6% of new HIV infection 
attributable to DMPA.(11) Summarized in Table 2 are the outcomes of recent studies that 

 
9 
 
have evaluated the impact of DMPA on HIV acquisition risk among women.  
It should be noted that the HIV risk of contraceptive hormones has not been 
consistently observed across all studies. Some studies have completely failed to show this 
association(12-15) and others report an increased risk of HIV infection only in a subgroup 
of individuals with differences such as age or HSV -2 status.(16-19) The wide 
discrepancies in study design, subjects’ selection, sample sizes, contraceptive type, 
method discontinuation, dose, and method of administration also adds to the complexity of 
interpreting the results across these studies and limit conclusions that can be drawn from 
the existing data. To our knowledge no well-designed studies have evaluated the impact of 
progestin releasing implants or IUDs on the risk of HIV-infection. While these data are far 
from conclusive, they are nevertheless worrisome, and provide a strong impetus to further 
assess the potential risk of HIV acquisition among existing hormonal contraceptive 
methods.  
Table 2: Impact of DMPA on HIV Acquisition/Transmission Risk among Women 
Study 
Country 
Year Study Design 
“n” 
Effect 
Heffron R, 
et al(7) 
Africa 
2012 Prospective cohort 
study 
3,790 Increased risk of HIV 
acquisition/transmission 
Morrison 
CS, et 
al(17) 
S. Africa 
2012 Prospective cohort 
study 
5,567 No significant risk of 
HIV infection observed 
Wand H & 
Ramjee 
G(20) 
Australia 
2012 Prospective cohort 
study 
2,236 Increased risk of HIV 
acquisition  
Reid, SE, 
et al(21) 
S. Africa 
Zambia 
Zimbabwe 
2010 Prospective cohort 
study 
1358 
No significant risk of 
HIV infection observed 
Kumwenda 
J, et al (22) 
Malawi 
2008 Prospective cohort 
study 
842 
Increased risk of HIV 
acquisition  
Leclerc 
PM, et 
al(11) 
Africa 
2008 Demographic/health 
survey 
4,549 Increased risk of HIV 
acquisition 

 
10 
 
Baeten JM, 
et al(23) 
Kenya 
2007 Prospective cohort 
study 
1,206 Increased risk of HIV 
acquisition 
Myer L, et 
al(24) 
S. Africa 
2007 Prospective cohort 
study 
4,200 No significant risk of 
HIV infection observed 
Morrison 
CS, et 
al(16) 
Uganda 
Zimbabwe 
Thailand 
2007 Prospective cohort 
study 
6,109 Increased risk observed 
only with HSV-2 
subjects  
Lavreys L 
et al(8) 
Kenya 
2004 Prospective cohort 
study 
1,498 Increased risk of HIV 
acquisition 
Kiddugavu 
M, et al(15) 
Uganda 
2003 Population-based 
cohort study 
5117 
No significant risk of 
HIV infection observed 
 
Potential mechanisms of increased HIV acquisition risk with hormonal 
contraception:  How might pharmacologic doses of sex hormones alter the risk of HIV 
transmission in women? It is understood that the male to female sexual transmission of HIV 
infection is a multifaceted process of virus-host interactions (Figure 1). Simply, following exposure, 
initial infection occurs at the genital mucosa and may involve complex interactions between a 
number of HIV target immune cells – CCR5 expressing CD4+ T cells, macrophages, and dendritic 
cells (DCs) including myeloid DCs and Langerhans cells (LCs).(25-28) It is thought that CD4+ 
lymphocytes are the first cells to be infected, and that DCs subsequently spread the virus to 
regional lymph nodes.(29) DCs may further facilitate the transmission process through mucosal 
antigen processing necessary for activation of CD4+ and CD8+ lymphocytes, a step that is 
important for continuing propagation of the infection, as activated CD4+ T cells are more likely to 
be infected and to support viral replication in vivo.(30)   

 
11 
 
Viruses recovered during primary HIV infection 
almost always are of the non-syncytium-inducing, 
R5-tropic phenotype, presumably due to the 
abundance of the viral entry co-receptors, CCR5, on 
cells of the mucosal 
surface and/or because of R5 expressing cells' 
capacity to support viral replication and propagation 
of the infection.(31) In addition to the disease state 
and local viral load of the donor, factors that increase 
susceptibility to HIV infection and infectivity include 
STIs, mucosal micro-abrasions, secreted immune factors – all of which affect the local immune 
status, the protective quality of the epithelial barrier in the genital tract, and the population of HIV 
target cells involved in the early stages of infection(32). Natural resistance to HIV acquisition has 
been ascribed to a variety of factors including genetically acquired resistance (e.g., CCR5 receptor 
deletions), the presence of host proteins such as secretory leukocyte peptidase inhibitor (SLPI), 
normal vaginal flora with peroxide-producing lactobacilli (void of bacterial vaginosis), and acquired 
immunity mediated by vaginal secretory 
IgA or cytotoxic T-lymphocyte (CTL) 
responses in blood.  
Clearly underappreciated in the 
discussion of HIV susceptibility of at-risk 
Figure 2. Potential effects of female sex hormones on the 
vaginal mucosal tissue. Adapted from Hel Z et al. Endocrine 
Reviews 2010;31:79-9 
Figure 1. Schematic representation of HIV-1 mucosal 
transmission adapted from Wu L. Curr Opin HIV AIDS. 
2008;3:534–40. 

 
12 
 
women is the emerging evidence that the robustness and functional capacity of the innate and 
adaptive immune responses are influenced by female sex hormones.(33-35) The immuno-
regulatory influence of estradiol and progesterone ensures a favorable environment for 
fertilization and pregnancy altering the local female genital tract (FGT) immunologic milieu and the 
composition of key HIV target immune cells. For example, systemic and topical administration of 
estrogen thickens the vaginal epithelial layer in ovariectomized rhesus macaques as illustrated in 
Figure 2A.(35-37) By increasing the thickness of the vaginal mucosal epithelium, estrogen blocks 
access of SIV and other pathogens to target cells (DCs/LCs, CD4+ T cells, and macrophages) within 
the epithelial and sub-epithelial layers. Furthermore, estrogen reduces the population of LCs 
within the vaginal epithelium, alters vaginal epithelial tight junctions and mucosal permeability(38, 
39) and supports healthy genital tract flora making the mucosa hostile and less susceptible to the 
virus.(40)  
Contrary to the effect of estrogen, progesterone exposure leads to a thinner epithelial layer in 
female macaques allowing easier access of SIV to LCs and other target cells (Figure 2B). These 
results in non-human primates have not consistently been shown to occur in humans thus 
alternative mechanisms deserve further exploration in light of the purported HIV risk of hormonal 
contraception. Emerging data suggest that progesterone treatment increases the population of 
LCs within the epithelial layer and induces changes in the vaginal microbiota, such as reduction in 
colonization with lactobacilli, increasing the predisposition to bacterial vaginosis. (41) Divergence 
from the healthy vaginal flora has been associated with increased HIV acquisition risk(42, 43). 
Furthermore, progestin contraceptives are associated with increased incidence cervical candidiasis 

 
13 
 
and of STIs such as gonorrhea and chlamydia(35) – factors that may enhance the risk of HIV 
acquisition.(44) It is unclear if these changes stem from direct biological effects of contraceptive 
exposure or behavioral changes, such as reduced condom use.  
Other actions of progesterone pertinent to HIV acquisition risk include suppression of IgG and 
IgA production and trans-epithelial transport within the FGT,(45-47) inhibition of CTL responses 
and blockade of T cell perforin expression.(48, 49) Progesterone alters the expression of cytokines, 
favoring Th2-type over Th1-type responses.(50) Progesterone significantly alters infiltration of 
genital tissue with natural killer (NK) cells, lymphocytes, and macrophages, and inhibits NK cell 
activity and Fc-gamma receptors (FcγR) expression on monocytes, thus reducing the functional 
capacity of the antibody-dependent cell cytotoxicity.(51-53) The progestin contraceptive DMPA 
inhibits Toll-like receptor-9-induced interferon alpha (IFN)-α production by plasmacytoid DCs 
thereby impairing IFN-α induced antiviral state and the innate antiviral immunity.(54) Lastly, 
studies have indicated that expression/secretion of chemokines, cytokines and endogenous 
antimicrobials in FGT secretions alter over the course of the menstrual cycle, suggesting a role for 
hormones in regulating their release(55, 56). For example, studies have demonstrated significant 
luteal phase suppression of SLPI, HBD2, HNP1-3 and lactoferrin from FGT reduced SLPI secretion in 
postmenopausal compared to premenopausal women(57, 58) and reduced endometrial 
expression of SLPI among women using DMPA(59). 
Taken together, these actions of progesterone may compromise the integrity of the protective 
epithelial layers against HIV infection, promote enhanced homing and thus the availability of HIV 
susceptible cells within the FGT, and dampen the robustness of the local innate antiviral and the 

 
14 
 
adaptive secretory immune responses. As hormonal contraception can alter endogenous hormone 
levels and have direct impact on the FGT, well-designed studies are needed to evaluate for changes 
in these factors that influence HIV susceptibility following different hormonal contraceptive 
exposures.   
 
Different progestin-
containing 
contraceptives and 
the potential risk of 
HIV acquisition: 
Hormonal 
contraceptives 
employ different 
strategies to prevent 
pregnancy, including 
ovulation inhibition, 
altered endometrial 
structure and 
thickening of the cervical mucus barrier. Common progestin-containing contraceptives currently 
available in the United States differ by the type of progestin they contain, mode of delivery, 
typical-use efficacy, duration of effectiveness, and degree of endogenous hormone and ovulation 
Table 3: Comparative properties of three progestin-only 
contraceptives method  
Depot 
Medroxyprogest
erone Acetate 
(DMPA) 
Etonogestrel 
Implant 
Levonorgest
rel IUD 
Trade-name 
DepoProvera 
Nexplanon  
Mirena 
Progestin 
Medroxyprogeste
rone acetate 
(MPA) 
1st generation 
Etonogestrel 
(ENG) 
3rd generation 
Levonorgest
rel (LNG) 
2nd 
generation 
Delivery 
Intramuscular 
Injection 
Sub-dermal 
Implant 
Intrauterine 
Device 
Length of use 
3 months 
3 years 
5 years 
Ovulation 
inhibition 
Yes 
Yes/No 
No 
Endogenous 
estrogen 
inhibition 
Yes 
Yes/No 
No 
Typical use 
failure for 1st yr 
of use(4) 
3% 
0.05% 
0.2% 
Amenorrhea at 
1 yr 
50% 
20% 
20% 
Continuation 
at 1 yr(60) 
57% 
83% 
88% 

 
15 
 
inhibition(60) (Table 3). Each progestin thus has variable degrees of estrogenic, androgenic, anti-
andogenic, glucocorticoid and anti-mineralocorticoid activity(61, 62). Medroxyprogesterone 
(MPA), for example, has androgenic activity, no anti-mineralicorticoid activity and potent 
glucocorticoid activity, higher than any other progestin or endogenous progesterone. 
Different formulations of injectable contraceptives are currently licensed for use. Due to its lower 
cost and availability, DMPA, at a dose of 150 mg given intramuscularly (IM) every 3 months, is 
widely used globally. One factor limiting contraceptive effectiveness is poor adherence to the 
scheduled repeat injections. Thus, efforts have been made to promote longer-term contraceptive 
options, such as contraceptive implants and IUDs. The contraceptive implant prototype, Norplant 
was introduced in 1984,(63) and since then, over 10.5 million implants have been distributed 
worldwide. A major difference between the injectable contraceptives and the implants is in the 
kinetics of systemic progestin release. This difference leads to varying degrees of hypothalamic-
pituitary-ovarian (HPO) axis modulation resulting in differential suppression of estrogen among 
the different progestin-only contraceptive formulations. (62) DMPA plasma concentrations, for 
example, quickly increase after intramuscular injection, peak within the first month followed by a 
plateau of serum MPA concentrations at 1.0-1.5ng/mL for about 3 months, after which blood 
concentrations decline slowly. Endogenous estradiol and progesterone levels are suppressed for 
several months after DMPA injection corresponding to suppression of ovulation.(64)  The 
Etonogestrel Implant (Nexplanon), on the other hand, is a single sterile rod implant inserted sub-
dermally. Each implant rod contains 68 mg of the synthetic progestin, etonogestrel (ENG). After a 
peak of 813 pg/ml at about day 4 following insertion, concentrations reach steady state (200 

 
16 
 
pg/ml) at about 4 – 6 months and remain sufficient to prevent pregnancy for 3 years. Although it 
initially suppresses follicular development and estradiol production, ovarian activity slowly 
increases after 6 months, and follicle stimulating hormone and estradiol levels are almost normal 
thus serum estrogen concentrations are significantly higher compared to that observed with 
DMPA use.(65)   The  levonorgestrel releasing intrauterine device (Mirena IUD) has a steady state 
plasma levonorgestrel concentration of 150-200 pg/mL that is reached in the first few weeks after 
placement of the IUD with levels maintained for up to 5 years after insertion. There is no change in 
serum estradiol and progesterone concentrations in Mirena IUD users compared to non-users; 
women with Mirena IUDs continue to have normal ovulatory function (66). There is a gradual 
reduction in endometrial thickness with the Mirena IUD with an associated gradual reduction in 
blood flow to spiral-arteries to the endometrium and menstrual blood flow.  
 
Although systemic levels of Lng are low, it is unknown how the high concentrations localized to 
the FGT may impact local immune function. Given the salutary effect of estrogen on the FGT, we 
speculate that Mirena IUD and  Nexplanon , due to reduced suppression of endogenous estrogen 
compared with DMPA, will have lower impact on mucosal immune function and therefore be less 
likely to increase HIV acquisition risk. The copper IUD is a non-hormonal method that provides 
effective contraception up to 12 years. Although the copper IUD does notn impact the normal 
hormonal mileau, different changes in the local immune environment are potential and may 
impact infection risk. 
2.2 
Significance of proposed study 
This research is significant in that it will: 

 
17 
 
• It will test novel hypotheses related to sex hormone-induced homing of HIV target immune 
cells to the genital mucosal to explain the purported increased HIV acquisition risk of 
progestin-only contraceptives.    
• It will generate novel data on the potential contributions of one non-hormonal IUD (copper) 
and 3 different progestin-only contraceptives (DMPA), Nexplanon  and Mirena IUD on 
immunologic parameters that may increase the risk of HIV acquisition among at-risk women. 
• It will employ the collection of cervicovaginal lavage (CVL)cervical biopsies, vaginal biopsies 
and two  endocervical cytobrush samples to provide cellular markers representative of a 
susceptible population of cells from the lower FGT, free from circulatory contamination. 
 
3.0 
Study Objectives and Hypotheses 
3.1 
Research hypotheses and objectives 
This research will evaluate the impact of one non-hormonal IUD and 3 different progestin-only 
contraceptive methods: 1) DMPA, 2) an Etonogestrel impant (Nexplanon), 3) the Levonorgestrel 
Intrauterine Device (Mirena IUD), and 4) Copper IUD, on known factors in the FGT that can modify 
HIV acquisition risk, including changes in the distribution and activation of HIV susceptible T-cells 
and mucosal chemokines and cytokines. One hundred and twenty (120) HIV-negative women 
seeking family planning will be recruited into a prospective study evaluating vaginal mucosal 
immunologic markers of HIV susceptibility before and after contraceptive initiation. 
It is unclear how different progestin analogues and varying systemic and FGT hormonal 
concentrations associated with their different contraceptive delivery and pharmacokinetics may 
influence FGT immune function. Because many of the anticipated mucosal immune changes with 

 
18 
 
progestin-only contraceptives are mediated via estrogen suppression, we hypothesize that the 
impact of Nexplanon  and Mirena IUD (milder anti-estrogen effect) will be significantly less 
pronounced compared with that of DMPA – a finding that would have significant clinical 
implications for family planning for women at risk for HIV.  
Aim 1: To evaluate the effects of DMPA,  Nexplanon  and two IUDs (Mirena and copper)  on the 
exposure on the frequency of HIV target immune cells within the female genital mucosa of HIV 
negative women.  
Hypothesis: Compared to a no contraception baseline, there will be an increase in HIV target CD4+ 
T cells in the female genital mucosa following initiation of hormonal contraception and these 
effects will be greater with DMPA because of its more pronounced anti-estrogen action relative to 
Nexplanon and Mirena IUD.   
Aim 2: To evaluate the effects of DMPA, Nexplanon and two IUDs (Mirena and copper)  on the 
exposure on markers of T cell activation and trafficking in the female genital mucosa of HIV 
negative women desiring hormonal contraception. 
Hypothesis: Compared to a no contraception baseline, there will be an increase in T cell activation 
and trafficking following hormonal contraception and these effects will be greater with DMPA 
because of its more pronounced anti-estrogen action relative to Nexplanon and Mirena IUD.  
Aim 3: To evaluate the effects of DMPA, Nexplanon and two IUDs (Mirena and copper) on 
cytokines and chemokine profiles within the female genital mucosa. 
Hypothesis: Compared to a no contraception baseline, there will be an increase in 
proinflammatory and chemotaxic cytokines and chemokines, and a decrease in anti-inflammatory 

 
19 
 
and inhibitory cytokines and chemokines following initiation of hormonal contraceptive use and 
these effects will be greater with DMPA because of its more pronounced anti-estrogen action 
relative to Nexplanon and Mirena IUD. 
 
Overall, this evaluation of the immunologic correlates associated with HIV susceptibility following 
exposure to different progestin-only contraceptives will explore the relative impact of these 
contraceptives on HIV risk. This work will contribute to the growing literature on hormonal 
contraception and HIV acquisition risk, establish critical baseline data needed to explore the safety 
of hormonal contraception in high-risk women, and set the foundation for the candidate to 
evaluate the impact of exogenous hormonal exposure on mucosal immunology and reproductive 
infectious disease in future research.  
4 
Study Design 
 
Overview and study design: To explore plausible mechanisms for the link between HIV acquisition 
risk and hormonal contraceptives, the research component of this proposal will examine changes 
in the FGT following exposure to three progestin only contraceptives, DMPA,  Nexplanon , and 
Mirena IUD and the non hormonal copper IUD, in women who are seeking contraception.The 
research design allows the exploration of multiple aims concurrently (Figure 3).  

 
20 
 
 
 
4.1
 
Recruitment, screening and enrollment 
4.1.1. Recruitment:  Participants will be recruited from one of two potential sources 
1) The Grady Family Planning Clinic within Grady Memorial Hospital: This site is where over 4600 
healthy reproductive-aged women from across the state of Georgia seek family planning annually. 
Additionally, recruitment will take place at Emory Clinic Gynecology and Obstetrics practice and at 
the IDP/Ponce Clinic. Women who present for a family planning visit and who are interested in 
one of the 4 contraceptives under study will be approached for participation by study staff not 
involved with the individuals clinical care. Clinicians will alert the research staff of potential 
participants and the research staff will approach the potential participant. Research staff will read 
a standard recruitment script (Appendix A).  
If interested, staff will complete a screening checklist to see if the individual is eligible for 
participation (Appendix A) 

 
21 
 
 2) By self-referral: Individuals will contact study staff in response to a flyer. Flyers will be placed 
throughout the Atlanta community including but not limited to Grady Memorial hospital 
gynecology clinic, Emory Campus and Clinics. Women who contact study staff will complete a 
screening checklist to see if the individual is eligible for participation (Appendix A), and if 
interested they may be scheduled for a screening visit  
 
Recruitment will continue until 30 individuals are enrolled into each contraceptive arm (120 total). 
We anticipate recruiting at the rate of ~30 participants per year and should complete accrual by 
year 3, all study visits should be completed by year 4. Although a randomized controlled study 
design ensures balance between arms, for feasibility reasons and because contraceptive choice in 
our population is a personal decision, the specific contraceptive type will be left to the preference 
of participants. 
4.1.2. Screening for eligibility will include a complete history and physical examination, pelvic 
examination and point of care urine sampling for pregnancy test. In addition.  The HIV test will be 
a point of care/rapid test using OraQuick® , an FDA-approved oral swab in-home test for HIV-1 and 
HIV-2. This is an oral swab test that doesn't require blood.  Notably, screening visit may occur at 
the same time as visit one. Notably to avoid any false positives all screen, positive tests with the 
OraQuick®  will be sent to a clinical lab for confirmatory HIV testing. Similarly, to prevent any false 
negatives, all women who screen negative and enroll in the study, will have HIV serum testing as 
part of their enrollment laboratory evaluation. Any positive test in the research lab, will trigger a 
clinical evaluation with confirmatory testing in a clinical labortatory and evaluation. 

 
22 
 
4.1.3 Eligibility 
Participants who meet all of the following inclusion criteria will be eligible for the study (a) 
female (b) age 18-45 years, (c) normal menses (22-35 day intervals) for at least 3 cycles, (d) intact 
uterus and cervix, (e) interested in DMPA, Nexplanon,  Mirena IUD, or Copper IUD (f) willing to 
delay initiation of hormonal contraception for up to 1 month to complete pre-contraceptive study 
visits, (g) willing to use condoms or abstain from sexual intercourse for at least 24 hours before 
each genital tract sampling (condoms will be made available), (h) able and willing to provide 
informed consent, and undergo serial blood and vaginal sampling, (i) negative HIV screening. 
Participants who meet any of the following exclusion criteria will not be eligible for the study: (a) 
pregnant within the last 3 months (b) breastfeeding, (c) history of loop electrosurgical excision 
procedure, conization, or cryosurgery within the past year, (d) use of hormonal contraception or 
IUD in the past 6 months or, (e) known history of medical condition that would interfere with the 
conduct of the study, (f) symptomatic vaginal infection or genital ulcer disease at screening, (g) 
taking medications that interact with selected contraceptive, and (h) contraindications to selected 
contraceptive per the CDC medical eligibility criteria(67) or judgment of clinician. For the copper 
IUD, exclusion criteria: allergy to copper. Participants who agree to the cervical and vaginal biopsy 
will have the addition exclusion criteria: allergy to lidocaine. 
The cost of their selected contraceptive will be covered by research study funds.  They will be 
informed of this during the consenting process. 
 

 
23 
 
4.1.4 Consent 
Voluntary, informed written consent for participation will be administered prior to 
initiation of study activities (See Consent Forms in Appendix B). The consent form will describe the 
purpose of the study, the study participants’ role in the study, the risks and benefits of the study, 
the duration of participation, the study procedures, contact persons for questions about the study 
(including the chair of the Institutional Review Board at Emory University and Grady Memorial 
Hospital), the voluntary nature of participation, and the right to decline specific questions or 
procedures or withdraw from the study at any time without penalty. An explanation will be 
provided about the use of protected health information (PHI) for research purposes and how 
confidentiality will be maintained as far as possible under the federal Health Insurance Portability 
and Accounting Act (HIPAA). Study participants will be asked to sign an authorization form for the 
use of PHI prior to enrollment in the study and will receive a copy for their records (Appendix B). 
Under no circumstances will the study participants be permitted to enroll in the study without 
their signed consent. All signed forms will be kept in a locked cabinet at all times.  
4.1.5 Study completion 
 
Study will be complete when 120  women, 30 in each contraceptive arm, complete the 
screening and the  study visits. As this is a longitudinal study, we may have some loss to follow-up, 
and the study may be considered complete after three unanswered attempts to contact missing 
participants have been made. There is minimal risk, so we do not anticipate any study related 
adverse events or complications related to study activities that would require specific stopping 
rules and interim analyses. 
4.1.6 Study visit schedule 
 
Upon successful screening, participants will be scheduled for study visit 1 three weeks 

 
24 
 
following the onset of their menses (luteal phase for women who cycle every 22-35 days). 
Participants will be instructed to refrain from vaginal intercourse or using intra-vaginal products 
for 24 hours before FGT sampling. This and subsequent study visits will occur according to the 
schedule outlined in Figure 3 and Table 4.  At each visit, an interval assessment of menstruation 
and hormonal contraception use will be collected and used to determine the phase of the 
menstrual cycle. If a participant is experiencing menstrual bleeding during a follow-up visit, 
evaluations will be deferred for 1 week.  
On the day preceding a study visit, participants will be asked to abstain from vaginal intercourse to 
minimize the risk of contamination of genital tract samples by semen.  
Table 4: Schedule of study event activities  
Study Activities 
Screen 
Visit 1 Visit 2 Visit 3 
Visit 4 
Week from study initiation 
 
Wk-1 
WK-3 
WK-
15 
WK-17 
Week from contraceptive 
initiation 
Pre-contraceptive 
WK-
12 
WK-14 
Informed consent 
X 
 
 
 
 
Complete history & physical 
examination 
X 
 
 
 
 
Target history & physical 
examination 
 
X 
X 
X 
X 
HIV test 
X 
X 
 
 
 
Urine Pregnancy test  
X 
X 
X 
X 
X 
Speculum pelvic examination 
X 
X 
X 
X 
X 
DMPA administration 
 
 
X 
X 
 
Vulvar/vaginal biopsy 
 
 
X 
 
X 
Nexplanon or Mirena or 
copper IUD placement 
 
 
X 
 
 
**Genital sampling for study 
endpoints (includes 2 
cytobrush samples)  
 
X 
X 
X 
X 
***Blood sampling for study 
endpoints 
 
X 
X 
X 
X 

 
25 
 
Swab for gut microbiome 
 
X 
X 
X 
X 
Blood testing for RPR and HSV 
 
X 
 
 
 
** Cervicovaginal lavage for HIV target inflammatory/immune cells, Swab for 
multiplex PCR for STIs and swab for Wetprep/Nugent Score and vaginal 
microbiome, vaginal PH,  endocervical cytobrush samples of genital specimens 
for immune phenotyping 
*** Blood for HIV target inflammatory/immune cells, E2 and P4 
 
4.2 
Study visits 
A brief questionnaire to assess sexual behavior and bleeding or vaginal symptoms will be asked, 
urine will be collected for a pregnancy test, a pelvic exam will be performed with measurement of 
vaginal pH using a PH meter, a swab will be collected for a wet-mount, a swab for multiplex PCR of 
vaginal fluid, a swab for vaginal microbiome,  a CVL collection (for immunological testing and 
prostatic specific antigen (PSA)), a swab for gut microbiome, and blood (for immunological testing 
and hormonal concentrations). At visit 1, additional blood will be collected for HSV serology, HIV 
and RPR.  At visit 2 and visit 4, a cervical or vaginal biopsy will be performed for a subset of 
participants that consent (10 per contraceptive arm)  so that one or more samples of tissue from 
the cervix or vagina are acquired for evaluating HIV susceptibility and inflamation markers. 
At visits 1-4, two endocervical cytobrush samples of genital specimens will be collected for immune 
phenotyping. 
 
Hormonal contraceptives Participants will be given the option to choose between DMPA, 
Nexplanon, copper IUD and Mirena IUD. DMPA will be dispensed from the Grady Pharmacy 
Service and will be administered every 12 weeks at the standard dose of 150 mg IM, beginning 
from week 3 of study enrollment and repeated at week 15.  A standard Nexplanon rod Implant or 
the IUD (Mirena or copper)  will be placed at study week 3 by Dr. Haddad or a trained clinician. 

 
26 
 
The cost of study contraceptives will be covered by this study. 
Of note, study visits are scheduled to ensure that the collection of pre-contraceptive samples 
occur at both the luteal (week 1 visit) and the follicular (week 3 visit) phases of the menstrual 
cycle. Serum hormone levels will confirm cycle phase. Post-contraceptive samplings are 
strategically delayed to avoid sampling during the initial (first 3 months) phase when irregular 
bleeding associated use may occur. Furthermore, the proposed sampling schedule will allow us to 
capture data at the trough (week 15) and peak (week 17) of the concentration-time-curve for 
DMPA. If bleeding at the time of a scheduled visit, the visit will be postponed to occur within one 
week or at a subsequent point corresponding to their intended cycle phase (luteal/follicular) or 
drug exposure (DMPA peak or trough) of the missed visit. Because the study requires intensive 
and invasive sample collection, concerted efforts will be made to enhance retention and study 
completion.  
4.3.1 Disenrollment 
A study participant may elect to discontinue participation in the study at any time without 
disruptionof their clinical care.   If during follow-up, any laboratory testing indicates the presence 
of an infection, participants will be referred clinical  clinical providers for further evaluation. If 
pregnant or if the protocol chair thinks it is in the woman’s best interest, she will no longer be 
followed by the study. The study may be discontinued at any time by a participating IRB, the Office 
for Human Research Protections, or other government agency as part of their duties to ensure 
that research subjects are protected.  
 

 
27 
 
4.3.2 Participant referral 
 
If a participant is diagnosed with any clinical issues during the course of study participation, 
they will be referred to a clinician or the health department to receive further testing and/or 
clinical care. Specifically, this refers to anyone diagnosed with any infection, pregnancy, or 
contraceptive issue. Clinical followup within 1 week will be encouraged for all participants and a 
written record relating to the clinical issue (test results, clinical finding or impression) will be 
provided to the participant.. Should they have a specific provider, it will be recommended that they 
contact that provider for an appointment. If the individual does not have a provider, we will 
facilitate getting the participant into care at the Grady Title X clinic or their health department for 
evaluation and/or treament. We will follow-up with the participant within one week to confirm 
that they have sought appropriate care and if care has not been received, we will reinforce with the 
participant the importance of seeking treatment and actively assist in getting the participant into 
care. 
5 
Data and Biological Specimen Collection  
 
Participants enrolled will undergo an interview, blood draw, and gynecologic exam with 
specimen collection at four study visits spanning 14 weeks.   
5.1 
Clinical evaluation 
The clinical evaluation and all specimen collections will be performed by a study clinician or 
the trained study nurse using standard operating procedures determined prior to study initiation. 
All exam results will be documented on the biologic study clinical exam form (Appendix C). At the 
first visit, a urine HCG pregnancy test will be performed to document study eligibility after the 
consent form has been signed. Additionally a point-of-care HIV test will be administed to confirm 

 
28 
 
HIV negative status at baseline. At all visits, a pelvic exam will be performed. The clinician will 
examine for genital lesions and purulent vaginal discharge, and any abnormalities in the 
cervicovaginal mucosa will be documented on the physical exam data collection form. Wet-prep 
evaluations will be performed for bacterial vaginosis, yeast, and trichomoniasis during each pelvic 
exam. If during the course of study participation, any laboratory testing indicates the presence of 
infection, or if the participant’s symptoms or physical examination raise suspicion of any other 
genital infection, a study clinician will refer for further care. There will be no extra information 
related to treatment or treatment outcomes recorded for study participants beyond what is 
standard of care. If during follow-up a woman is determined to be pregnant, she will be referred 
for care and no longer followed. 
During the pelvic exam, vaginal pH will be measured with a pH meter, a vaginal swab will be 
collected for evaluation of vaginal microbiome, a swab for a multiplex PCR, a swab will be 
collected for a wet Prep, cervicovaginal fluid (CVF) will be collected by cervicovaginal lavage (CVL) 
for inflammatory marker quantification and two  endocervical cytobrush samples of genital 
specimens for immune phenotyping.  A swab will be collected into the anal opening to evaluate 
the gut microbiome. To enhance cellular yield from the posterior fornix of the FGT, CVL will be 
performed using 10 cc of phosphate buffered saline (PBS). Lavage will be repeated with a second 
aliquot of 10 cc of PBS. . Previous experience with this approach yielded 0.2-1X106 leukocytes per 
10 cc of CVL, an amount of cells adequate for the proposed study endpoints and importantly 
avoided the need for the more invasive cervical cytobrush or vaginal biopsy sampling that also 
risks blood contamination of the sample. A second 10 cc of PBS will be collected in a similar 

 
29 
 
fashion to increase the potential yield for analysis. Obtained PBMC and CVL leukocytes will be 
processed and analyzed in real time for flow cytometry based phenotypic analysis. Additional CVL, 
vaginal swab, anal swab and plasma samples will be aliquoted and cryopreserved or frozen at -
80oC until analyzed for other endpoints. Dr. Haddad or a trained study nurse will perform all 
exams and sampling. Also at visit 2 and visit 4, a cervical or vaginal biopsy will be performed for a 
subset of participants that consent (10 per contraceptive arm)  so that one or more samples of 
tissue from the vulva or vagina are acquired for evaluating  HIV susceptibility and inflamation 
markers in the future. 
In addition to the pelvic evaluation, blood will be collected at each visit in two 8 mL-
vacutainer tubes (2 Cell Preparation Tubes (CPT)) for immunologic markers, and hormonal 
assessment (E2/P4 levels) at all four visits. Additional blood (1 Plasma Preparation Tube (PPT) will 
be collected at visit one for syphilis with rapid plasma reagin (RPR), HIV and HSV serology at the 
research lab. Table 4 lists the schedule of events and biologic specimen collection for the study. 
The collection of specimens at each visit will be noted on the specimen collection form (Appendix 
D). Positive test results from the research lab can not be relied upon for patient care. Any positive 
test results concerning for infection from the research lab will trigger further clinical investigation 
with referral for further testing and treatment.   
5.2 
Laboratory testing 
 
All laboratory results from assays on blood or vaginal swabs, and CVL will be documented 
on the laboratory results forms (Appendix E). 

 
30 
 
Specimens collected will be transported within 4 hours the lab. Blood will will be separated 
and immediately processed for immunophenotyping and immune cell-activation assays, as 
described below for the vaginal samples. The separated plasma will be aliquoted and stored at -
80oC until batch testing commences, as described below for the vaginal samples. Progesterone 
(P4) and estrogen (E2) levels will be measured in plasma using the Milliplex kit for Luminex Assay 
(EMD Millipore, Billarica, MA).  
A vaginal swab and anal swab will be collected and stored for future microbiome 
characterization via next-generation sequencing using the Ion Torrent platform to sequence 16S 
rDNA from bacterial species present in the vaginal compartment. 
The CVL specimens will be tested for blood and leukocyte levels with a urine dipstick test 
and prostate specific antigen (PSA), a marker of semen exposure, using the Abacus ABAcard p30 
test.  The sample will be centrifuged, and the cell pellet will be re-suspended in cell culture media, 
and the leukocytes will be isolated from epithelial cells using Percoll gradient centrifugation.  The 
resuspended CVL cell pellet (and PBMC pellet, separately) will be split for analysis. One half of the 
sample will be used for immune cell characterization (i.e., T cell subsets, HIV co-receptor 
expression) by antibody staining and multi-color flow cytometry using a LSR-2 platform. The other 
Table 5: Summary of Covariates and Analytic Methods for All Specific Aims  
Demographics/Behaviors: Questionnaire/Exam: Age, BMI, Race, Parity, Coital frequency 
STD Screen: Vaginal swab multiplex PCR for N.gonorrhea, C. trachomatis, T. vaginalis, M. 
genitalium, T. pallidum, HSV-1 & 2. Serum EIA for HIV & HSV. Serum RPR Kit for RPR 
Candida vaginitis: Vaginal swab for KOH wet mount examination for hypae (yeast) 
Bacterial Vaginosis: Vaginal swab for gram stain assessment for Nugent score BV diagnosis 
Semen: CVL supernatant ABAcard® testing for qualitative prostatic specific antigen (PSA) 
Serum sex hormones: Serum RIA for E2, P4 
Microbiome: Next generation sequencing of 16s rDNA 

 
31 
 
half of the sample will be used to assess functional attributes of the T-cells. Cells will be stimulated 
with PMA and ionomycin (a polyclonal activator) in a tissue culture dish for 5 hours in the 
presence of brefeldin A and monensin and analyzed for the production of selected cytokines and 
chemokines by flow cytometry. Multicolor flow cytometry will be performed on matched PBMC 
and CVL samples utilizing the LSR-II platform (BD Biosciences) as previously described.(68) For 
assessment of HIV target cells, samples will be stained with a Live/Dead viability dye and 
fluorochrome labeled antibodies against CD3, CD4, CD27, CD45RA, CCR5 and CXCR4. Effector 
memory (CD45RA-/CD27-) CD3+CD4+ cells will be analyzed for surface expression of HIV co-
receptors CCR5 and CXCR4 and reported as percent of total leukocytes and total CD4+ T cells. 
We will examine a panel of proinflammatory, anti-inflammatory, inhibitory and chemotaxic 
cytokines and chemokines, focusing our analysis on those previously reported to influence 
recruitment of HIV target cells to the FRT such as IL1 and IP10(69). Using multiplex Luminex® 
assays combined with a customized multi-analytical panel of 22 human cytokines and chemokines, 
we will quantify concentrations of cytokines and chemokines in plasma and CVL supernatant at 
each study visit from participants enrolled  
Recent semen exposure will be measured with a qualitative PSA ABAcard® p30 test 
(Abacus Diagnostics) (70). The wet-mount will be evaluated using saline and 10% KOH for 
evaluation of budding yeast or hyphae.  A gram stain will be performed for Nugent scoring (71) to 
diagnose BV. A multiplex PCR assay, able to screen for multiple STIs concurrently without the need 
for multiple samples to be collected, will be used to evaluate for STIs including N. gonorrhea, C. 
trachomatis, T. vaginalis, M. genitalium, T. pallidum, and HSV-1/HSV-2. Serological tests for HIV 

 
32 
 
and HSV will be performed via enzyme immunoassay (EIA) using a Vitros® 3600 Immunodiagnostic 
system (Ortho Clinical Diagnostics) and syphilis screening determined via a commercially available 
rapid plasma reagin (RPR) kit. Positive test results from the research lab can not be relied upon for 
patient care. Any positive test results concerning for infection from the research lab will trigger 
further clinical investigation with referral for further testing performed by a CLIA-approved 
laboratory and treatment.   
Table 6.  Biological specimens and laboratory methods 
Sample 
Analytic Method 
Outcomes of Interest 
 
Cells from CVL and 
PBMC 
 
 
 
 
 
CVL Supernatant 
 
 
Multi-parameter 
flow cytometry 
 
 
 
 
Luminex panel 
 
 
 
 
 
 
 
 
 
 
 
ABBA Card 
 
• T cell subtypes: CD4, CD8, CCR7, CD25, 
CD27,CD28, CD45RA+, CD57, CD11a, PD1, 
HLA-DR. 
• HIV co-receptor use: CCR5, CXCR6, CXCR4. 
• T cell functional attributes:, IFNγ, TNFα, IL-
2, MIP1α, MIP1β. 
 
• Proinflammatory: IL-1β, IL-6, IL-12 (p70), 
G-CSF, M-CSF, IFN-α2, IFN-γ, IL-1α, IL-17, 
IL-2, TNF-α, TNF-β, IL-4, IL-5 
• Anti-inflammatory: IL-10 
• Inhibitory (CCR5 agonists): RANTES, MIP-
1α, MIP-1β 
• Chemotaxic: IP10, IL-8, MCP-1, Eotaxin 
 
• PSA 
Plasma 
RIA 
RPR Kit 
EIA 
• Plasma progesterone concentrations.   
• RPR 
• HIV/HSV 
 
 
Vaginal, endocervical  
 and Anal  Swabs 
Next-generation 
sequencing using 
• 16S rDNA from bacterial species present in 
the vaginal compartment 

 
33 
 
the Ion Torrent 
platform 
 
Mulitplex PCR 
 
 
• N.gonorrhea, C. trachomatis, T. vaginalis, 
M. genitalium, T. pallidum, HSV-1 & 2. 
 
5.3 
Specimen management and storage 
As the transmission of HIV and other blood-borne pathogens can occur through contact 
with contaminated needles, blood, and blood products, appropriate blood and secretion 
precautions will be employed by all personnel in the drawing of blood and handling of all 
specimens for this study, as currently recommended by the Centers for Disease Control and 
Prevention. All infectious specimens will be transported using packaging mandated in the Code of 
Federal Regulations, 42 CFR Part 72. Specimens, inclusive of the cervical or vaginal biopsies that 
are not used immediately will be processed and stored at CDC for future testing.  Plasma and 
peripheral blood mononuclear cells (PBMC) will be stored at -70°C. Vaginal swabs, Anal swab and 
CVL supernatants will be stored at -70°C. Specimens collected will be used for the evaluations 
described within this protocol. Any remaining specimens will be stored for potential future use 
only if the patient has consented to storage of specimens. These specimens may be used in the 
future to further evaluate HIV, immunologic  microbiologic factors of interest or as a source of 
DNA for future genetic analysis. A subject may request that any stored specimens are destroyed 
prior to completion of the study. Specimens will be stored with the study ID of participant only, 
and no patient identifiable information. At study completion any identifiers that link the subject to 
the study ID will be destroyed. Specimen storage will be for an indefinite amount of time if 
consent for storage had been signed. During the consent process, specimen storage will be 
discussed and participants will be consented to allow for storage and future testing of specimens. 

 
34 
 
 
 

 
35 
 
5.4 
Specimen collection considerations 
Swabs and cervicovaginal lavage collections are minimal risk procedures. Specimens 
contaminated with blood may need to be excluded from our analyses. Every effort will be taken to 
reduce such risks by starting with the least invasive collections (swabs and tear-flo strips), then 
proceed with cervicovaginal lavage collection which introduces specimen dilution. We do not 
anticipate that collecting multiple samples in this order will influence any of the subsequent data 
collection endpoints, given the large vaginal surface. By maintaining a consistent order of 
specimens collected at each visit we will minimize experimental variability. 
6 
Ethics and Risks 
The study will not commence until the study protocol has been reviewed and approved by 
the Emory University institutional review board (IRB) and the Grady Health System Research 
Oversight Committee. 
6.1 
Potential risks 
The risks involved in study participation are those related to providing personal 
information about sensitive topics such as sex, contraception, antiretroviral medication 
use, and pregnancy.  Participants’ privacy and the confidentiality of data will be protected 
through training of interviewers and other study staff, conducting all interviewing and 
physical examinations in private, storing study materials in a locked room, and securing 
computer files that include identifiers. Only the study identification number will identify 
participant research records. Linkages between the ID number and participants’ identifying 
information will be maintained in a computer database that is password protected and 
only accessible to study staff. These linkages will be destroyed after completion of study 
activities. Breaches of confidentiality are possible, though safeguards are in place to 

 
36 
 
protect the confidentiality of participants.  Thus, the potential for experiencing an adverse 
event is minimal.  
There is a risk that the participant may get pregnant while enrolled in the study. This risk is 
the greatest during the first few weeks of the study before the participant starts their 
contraceptive.  To reduce this risk we will ask the participant to either abstain from sex or use 
condoms during this time. If pregnancy occurs during the study, we will refer the patient for 
further care.  
Each of the four study visits consists of a brief interview, blood collection, pelvic 
examination, and vaginal sample (swab and lavage) collection. The potential additional risks to 
participating include discomfort from the blood draw and gynecologic exam with specimen 
collections, as outlined below:  
1. The standard risks of phlebotomy: Skin irritation at the collection site, some bleeding, 
bruising, fainting, and pain are risks associated with blood collection. There is also a very 
small risk of infection at the collection site. 
2. Discomfort from physical and gynecologic examination: Gynecologic examinations may 
cause both physical and emotional discomfort.  
3. The risks associated with suspicion of a STI or vaginitis: Individuals who test positive or 
have any suspicious examination finds concerning for any infections during the research 
evaluation, will be referred for clinical evaluation and/or treatment. Individuals diagnosed 
with an STI may experience emotional discomfort upon receiving their diagnosis or upon 
notifying sexual partners, which would be recommended by their provider 
 during 

 
37 
 
counseling if a test result is positive.  
4. Risks associated with their selected contraceptive – including routine risks during insertion 
and side effects.  
Risks associated with cervical or vaginal biopsy collection is bleeding and infection. Subjects will be 
informed on how to care for the area and to contact the PI or their medical care provider if there 
is a persistent  smell, bleeding or discomfort.These procedures are not anticipated to be 
associated with any deleterious impact on participants’ health or well-being.  
 
6.2 
Protection against risks 
In implementing this protocol, we will be following HHS guidelines for research practices outlined 
on 45 CFR 46 (also known as the common rule). The investigators will adhere to the basic 
principles of “Good Clinical Practice” as outlined in Code of Federal Regulations (CFR) 312, subpart 
D, “Responsibilities of Sponsors and Investigators,” CFR 21, part 50, and CFR 21, part 56 and 
Section 4 of International Conference on Harmonisation of Technical Requirements for 
Registration of Pharmaceuticals for Human Use (ICH) Harmonized Tripartite Guideline for Good 
Clinical Practice. Specifically, we will provide careful training of study personnel who interview or 
collect participant samples. Training will include a component on careful patient counseling to 
enable the counselor to recognize complications, and when indicated, provide immediate referral 
for evaluation. A study participant will be informed of the voluntary nature of this study and that 
they may elect to not answer certain questions or to discontinue participation in the study at any 
time.  Additionally, comprehension of study procedures will be assessed by study staff prior to 

 
38 
 
signing the informed consent form.  Study staff will first review the informed consent document.  
After reviewing the consent document, the research staff member will ask the participant the 
appropriate comprehension questions (see Appendix B).  If a participant incorrectly responds to 
one of the comprehension questions, the staff member will further clarify study procedures.  
Those participants who incorrectly respond on a second attempt, will be ineligible to participate in 
the study. Risks from receiving the contraceptive will be minimized by ensuring that a qualified 
trained clinician performs all insertions and a comprehensive consent for that contraceptive is 
reviewed prior to their receipt of the contraceptive as part of routine clinical practice at the clinic. 
All laboratory specimens, evaluation forms, reports, and other records that leave the site 
to collaborating laboratories will be identified by a coded number only, to maintain subject 
confidentiality. All study records will be kept in a locked cabinet or on password-protected 
computers that will be stored in locked cabinets, accessible only to Emory based study research 
staff. The clinical data associated with the pelvic examination and placement of the contraception 
that would correspond to routine clinical care data will be shared with the patients clinical care 
provider, and included in their Grady electronic medical record,. This information in the electronic 
medical record is available to all members of the participant’s clinical care team. We will 
encourage patients to share their results with any health care providers they have outside of the 
Emory or Grady setting, if they wish; however we will not attempt to contact these providers 
directly. For sexually transmitted diseases that are identified, a clinician on the research team will 
contact the individual directly via phone to discuss results and clinical referral. As study samples 
will be processed in a research lab, these results can not independently be acted on for clinical 

 
39 
 
management, thus patients will be referred for clinical care. For any positive results identified 
during their research visit, such as a positive pregnancy test or vaginitis, a clinician from the 
research team will be available to counsel the patient at that visit on their results and discuss 
referral for care. 
Any other clinical information will not be released without written permission of the 
subject, except as necessary for monitoring by IRBs at participating institutions or the Office for 
Human Research Protection. The study may be discontinued at any time by an IRB at a 
participating institution or the Office for Human Research Protection.  
6.3 
Benefits to participation 
The benefits of this research extend to potentially leading to improvements in the 
reproductive health care provided at the facility.  Moreover, the results could potentially impact 
individuals globally as we aim to better understand transmission of infections such as HIV, which 
can improve or better target our methods of prevention. Participants will receive a $50 
compensation for each study visit, including their screening visit with a maximum of $250 received 
if she completes all study activities. 
6.4 
Importance of the knowledge gained 
The current study could help us to better understand the biologic effects of hormonal 
contraception on HIV transmission risk. If hormonal contraception increases the risk of 
transmission, dual protection methods with non-hormonal methods, such as the copper IUD and 
condoms, may be an important alternative strategy. The knowledge gained from this research may 
be used on multiple levels to help prevent unintended pregnancy, HIV transmission, and STI 

 
40 
 
infection.. Study findings will be disseminated further via abstracts submitted to scientific 
meetings and manuscripts submitted to peer-reviewed journals. 
 
6.5 
Limitations and Future Directions 
By design the study does not directly address the risk of HIV acquisition from hormonal 
contraceptives; rather, it explores surrogates that might provide a mechanistic explanation to this 
purported impact of contraceptive hormone use. Being exploratory in nature, the sample size is 
small, and we may not be able to detect small differences in some of the outcomes. We have 
carefully selected our markers to focus on our study aims, however we acknowledge that 
additional markers may be of interest and can be explored in future evaluations. Additionally, the 
design is limited by all the inherent biases and confounding associated with a non-randomized, 
non-blinded study design. Attempts to control for covariates will be performed in the analysis. 
Further, as our follow-up is limited to 3 months, we cannot evaluate the impact of long-term 
contraceptive use nor the effect of irregular bleeding on HIV risk. Furthermore, we acknowledge 
that many other factors that influence HIV susceptibility warrant further investigation, such as 
epithelial integrity and endogenous microbicides. We plan to use these banked samples to explore 
some of these other potential mechanisms in future research. The data generated from the 
successful completion of this study will be instrumental in the design of a future prospective 
contraceptive trial to explore HIV acquisition risk and to establish safe family planning options for 
high-risk women. 

 
41 
 
 
6.6 
Inclusion of women, minorities, and children 
Pursuant to HHS policy, women (females over the age of 21), members of minority 
groups, and children should be included in biomedical and behavioral clinical research 
projects involving human subjects.  The proposed study conforms to this policy.  
 
7 
Monitoring and Oversight 
7.1 
Site monitoring 
Site monitoring may be performed by the Emory University’s IRB and Office of Research 
Compliance, OHRP, FDA, or other government regulatory authorities.  Clinical research site 
monitoring may include the review of the individual participant records, including consent forms, 
CRFs, supporting data, laboratory specimen records, and medical records (physicians’ progress 
notes, nurses’ notes, individuals’ hospital charts) to ensure protection of study participants, 
compliance with the protocol, and accuracy and completeness of records.  The monitors may also 
inspect sites’ regulatory files to ensure that regulatory requirements are being followed. The 
investigator will make study documents (e.g., consent forms, CRFs) and pertinent hospital or clinic 
records readily available for inspection. 
7.2 
Data quality assurance and protection 
There will be study protocol and procedure training for all study personnel prior to beginning of 
the study. A standard operations manual will be available to staff to refer to for operational details in 
running the study. 
Each woman screened will be assigned a participant identification number (PIN) at the time of 
study entry. The PIN will be used on all data forms, specimens and communications related to the study. 

 
42 
 
Samples will be labeled at the time of collection and processing using study labels. Clinical research 
forms (CRFs) will be used to collect all study related data, including demographic, clinical, and 
laboratory data at study entry, and additional clinical data at subsequent study visits. Subjects will 
be identified only by the  PIN on the CRFs. To maintain subject confidentiality, only a coded number 
will identify all stored laboratory specimens, forms, reports and other records.  All records will be kept in 
locked file cabinets, accessible only by local study staff. Electronic files will be password-protected, with 
access only by authorized study personnel. Clinical information will not be released without written 
permission of the subject, except as necessary for monitoring from funding agencies and designees or 
except as mandated by law.  
The study investigators will provide instructions concerning the recording of study data on 
CRFs. It will be the responsibility of the study investigator to assure the quality of computerized 
data. This role extends from protocol development to generation of the final study databases. 
Data from screening forms and study visit will be entered into Microsoft Access 2007. Electronic 
data will be secured in a password protected database that is accessible to members of the Emory 
investigative team.  The Emory study investigators will maintain the link in a separate file that 
associates subjects with their PIN. 
 Staff will double-enter data using appropriate software. The data entry screens will be 
prepared with built-in quality control checks.  To safeguard against loss of data, backups of the 
data will be made.  Study staff will train the data entry staff in all data management procedures. 
We will conduct all statistical analyses using the SPSS System for Windows version 18.0, SAS Proc 
Mixed (version 9) or similar analysis software program. The database and the laboratory data 

 
43 
 
generated by this study will be maintained at Emory and will be accessible to collaborators. The 
principal investigators will be responsible for documentation of these data.   
The initial data entry and maintenance of laboratory data, identified only by PIN, 
generated will be the responsibility of the laboratory investigator generating and entering the data 
and of study investigators who will oversee data quality. 
7.3 
Protocol violation 
A protocol violation is any intentional or unintentional change from the IRB-approved 
protocol that adversely affects (1) the risk/benefit ratio of the study, (2) the rights, safety, or 
welfare of the participants or others, or (3) the integrity of the study. Examples include breach of 
confidentiality, inclusion of ineligible participants, or initiation of study procedures before 
participant has signed the consent form. If a protocol violation were to occur, it would be 
documented on the appropriate protocol deviation report form, and if the violation were to meet 
reporting requirements, it would promptly be reported to the participating IRBs.  
7.4 
Adverse events 
An adverse event (AE) is defined as any health-related reaction, effect, toxicity or abnormal 
laboratory result that a study participant experiences during the course of the study, irrespective 
of relationship to the study intervention. This includes changes in a participant’s condition or 
laboratory results that have or could have a deleterious effect on a participant’s health or well-
being. A serious adverse event (SAE) is defined as any experience that is fatal or life-threatening, 
requires in-patient hospitalization or prolongation of an existing hospitalization, or results in a 
persistent or significant disability or incapacity.  

 
44 
 
This study includes interviews, blood draws, pelvic examinations, and vaginal sample (swab 
and lavage) collections. These activities are not anticipated to be associated with any deleterious 
impact on participants’ health or well-being. AEs will be managed according to good clinical 
practice and the judgment of the on-site physician. All clinical and laboratory AEs will be followed-
up closely by study staff. Any SAEs will be expeditiously reported to the principal investigators, via 
a designated SAE form. Notification and submission of SAE forms should occur within 48 hours of 
the site awareness of the SAE. The PI will then make the final independent judgment as to the 
severity, relatedness, and anticipated or unanticipated nature of the SAE and finalize the Human 
Subjects Adverse Event Report Form. If the event is determined to be serious, unanticipated, and 
the relationship is anything other than probably or definitely not related, then the PI will report 
the event within 48 hours and submit the required  forms to the IRB at Emory and Grady will be 
simultaneously notified. Thus, all anticipated SAEs that are at least possibly related to study 
intervention and all deaths that are at least possibly related to study intervention will be 
submitted to Emory IRB, and Grady ROC.  
 
8 
Statistical Considerations and Analysis 
Sample Size/Statistical Considerations: We used the preliminary data from CVL of healthy women 
(unpublished) for our power analysis (CCR5: n=17, mean = 17.6%, Standard deviation (SD) = 17.4%, 
CD38 n=6, mean =41.9%, SD=11%), using the SD from the cross sectional data as an estimate for 
the SD for change. Due to the expected correlation between repeated-measures, this is a 
conservative estimate as the standard deviation for change should be smaller than the observed 
between-subject standard deviation. For evaluating change following exposure to any 

 
45 
 
contraceptive method (compared to no method), a sample size of 90 women achieves statistical 
power exceeding 95% to detect a CCR5 increase of 10% (or larger) (two-sided one-sample t-test on 
change, 5% α-level). Similarly, with a sample size of 90 women, we achieve statistical power 
exceeding 95% to detect an increase in CD38 of 10% (or larger) from before to after any 
contraceptive exposure. Comparing the different contraceptives, assuming an increase of 25% in 
CCR5 in the DMPA group and an increase of 10% in Nexplanon group and Mirena IUD group (pre 
to post exposure), a sample size of 30 women per contraceptive group (Nexplanon vs. DMPA or 
Mirena IUD vs. DMPA) will achieve 90% statistical power to detect a contraceptive group 
difference of 15% (2-sided two-sample t-test on change, 5% α-level).  Similarly, assuming an 
increase of 20% in CD38 expressing T-cells in the DMPA group and an increase on average of 10% 
in Nexplanon group and Mirena IUD group, a sample size of 30 women per contraceptive group 
will achieve 93% statistical power to detect a contraceptive group difference of 10%.  
Data Management and Statistical Analysis: Study data will be recorded on case report forms and 
entered into a web-based password-protected relational database (REDCap). Repeated-measures 
analyses using mixed linear models will be performed with the assistance of Emory CFAR 
biostatistical core for study endpoints and analyzed with SAS Proc Mixed (version 9) providing 
separate estimates of the means by contraceptive method and time on study before (weeks 1 and 
3) and after (15 and 17 weeks) contraceptive administration. An unstructured variance-covariance 
form among the repeated measurements will be assumed for each outcome and estimates of the 
standard errors of parameters will be used to perform statistical tests and construct 95% 
confidence intervals.  The model-based means are unbiased with unbalanced and missing data, 

 
46 
 
assuming missing data are non-informative and random. Mean changes over time for each 
contraceptive method will be tested for linear trend.  Data will be summarized using adjusted 
means and observed differences plus 95% confidence intervals. Statistical tests will be 2-sided.  A 
P value ≤ 0.05 will be considered statistically significant.  
 For multivariable repeated-measures analyses, a mixed linear model will be fit including age, 
body mass index, race and parity as potential baseline confounders in addition to contraceptive 
method, time on study and the interaction between time on study and contraceptive method. The 
multivariable results will be summarized with adjusted means and 95% confidence intervals. The 
adjusted mean for each contraceptive subgroup (DMPA, Nexplanon, copper IUD and Mirena IUD) 
will be defined as the mean response obtained by fitting the statistical model at the mean age and 
the mean BMI of the three subgroups and averaged across levels of the other risk factors. To 
integrate time-dependent variables, such as BV and PSA, into the longitudinal analyses, each 
participant at each of the 4 scheduled visits will be classified as positive or negative for each 
variable. Outcome change will be compared between incident cases and the other women 
(baseline positive and those that remain negative) after adjusting for the other covariates. 
9 
Dissemination of Results 
The results will be disseminated in the form of abstracts submitted to scientific meetings 
and manuscripts submitted to peer-reviewed journals.  
 
 
 

 
47 
 
 
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