Impact of Concurrent Initiation of DMPA Contraception and Tenofovir PrEP on Bone Loss in Young Women
Phase: PHASE4
Conditions: Bone Demineralization, Hypoestrogenism, Subclinical Kidney Injury, Bone Microarchitecture
Sponsor: University of Washington

 
The Women’s Bone Health Study Protocol 
 
 
Version 3.0 
31st July 2018  
 
 
 
PROTOCOL 
 
 
Impact of Concurrent Initiation of DMPA Contraception and Tenofovir PrEP on Bone Loss in 
Young Women 
 
 
The Kampala Women’s Bone Study 
 
 
Version 3.0 
31st July 2018  
 
 
Funding: 
Eunice Kennedy Shriver National Institute for Child Health and Human Development 
(R01HD089843) 
 
 

The Kampala Women’s Bone Study Protocol 
 
Page 2 
Version 3.0 
31st July 2018  
 
 
 
 

The Kampala Women’s Bone Study Protocol 
 
Page 3 
Version 3.0 
31st July 2018  
 
Contents 
ACRONYMS ............................................................................................................................... 4 
STUDY TEAM ............................................................................................................................. 5 
I. 
SUMMARY .......................................................................................................................... 6 
II. 
BACKGROUND .................................................................................................................. 7 
III. 
STUDY METHODS ........................................................................................................ 12 
Overall Design ....................................................................................................................... 13 
Population ............................................................................................................................. 14 
Location ................................................................................................................................ 14 
Eligibility ................................................................................................................................ 14 
Sample size and study power ................................................................................................ 15 
Recruitment ........................................................................................................................... 16 
General study procedures ..................................................................................................... 16 
DXA and TBS assessments .................................................................................................. 16 
Retention and study exit ........................................................................................................ 18 
Special circumstances ........................................................................................................... 18 
Visit-specific procedural tables .............................................................................................. 19 
Safety monitoring .................................................................................................................. 20 
IV. 
DATA COLLECTION ..................................................................................................... 20 
VI. 
HUMAN SUBJECTS CONSIDERATIONS ..................................................................... 22 
Study oversight ..................................................................................................................... 22 
Informed consent................................................................................................................... 23 
Risks ..................................................................................................................................... 23 
Benefits ................................................................................................................................. 23 
Care for persons identified as HIV positive ............................................................................ 23 
Treatment for injury ............................................................................................................... 24 
Study records ........................................................................................................................ 24 
Confidentiality ........................................................................................................................ 24 
VII. 
CLINICAL RESEARCH SITE ......................................................................................... 24 
Site location........................................................................................................................... 24 
Clinical facilities ..................................................................................................................... 24 
Staff....................................................................................................................................... 24 
Administrative procedures ..................................................................................................... 25 
Laboratory considerations ..................................................................................................... 25 
VIII. 
REFERENCES ............................................................................................................... 26 
 

The Kampala Women’s Bone Study Protocol 
 
Page 4 
Version 3.0 
31st July 2018  
 
ACRONYMS 
 
BMD 
Bone mineral density 
BMI 
Body mass index 
BTM 
Bone turnover marker 
CDC 
Centers for Disease Control and Prevention 
CKDE 
Chronic kidney disease epidemiology 
CTX 
C-telopeptide 
DMPA 
Depo medroxyprogesterone acetate 
DXA 
Dual energy x-ray absorptiometry 
EC 
Ethics committee 
EIA 
Enzyme-linked immunosorbent assay 
FDA 
Food and Drug Administration 
FTC 
Emtricitabine 
eGFR 
Estimated glomerular filtration rate 
IDI 
Infectious Disease Institute 
IRB 
Institutional review board 
NTX 
N-terminal telopeptide of type 1 collagen 
P1NP 
Procollagen type 1 N-terminal propeptide 
PBM 
Peak bone mass 
PTH 
Parathyroid hormone 
PrEP 
Pre-exposure prophylaxis 
RNA 
Ribonucleic acid 
SHBG 
Sex hormone-binding globulin 
STI 
Sexually transmitted infection 
TBS 
Trabecular bone score 
TDF 
Tenofovir disoproxil fumarate 
VDBP 
Vitamin D binding protein 
 
 
 

The Kampala Women’s Bone Study Protocol 
 
Page 5 
Version 3.0 
31st July 2018  
 
STUDY TEAM 
 
Makerere University, Kampala, Uganda 
Elly Katabira, MBChB, FRCP 
Andrew Mujugira, MBChB, PhD  
Timothy Muwonge, MBChB, MPH  
 
 
 
University of Washington, Seattle, USA 
Renee Heffron, PhD, MPH 
Jared Baeten, MD, PhD 
Deborah Donnell, PhD 
Kenneth Mugwanya, MBChB, MPH, PhD 
 
Makerere University-Johns Hopkins University Research Collaboration, Kampala, Uganda 
Flavia Matovu Kiweewa, MBChB, MSc 
 
Columbia University, New York, USA 
Michael Yin, MD, MS 
 
 
Duke University School of Medicine, North Carolina, USA 
Christina Wyatt, MD, MS 
 
 

The Kampala Women’s Bone Study Protocol 
 
Page 6 
Version 3.0 
31st July 2018  
 
I. 
SUMMARY 
 
For women with substantial HIV risk, tenofovir disoproxil fumarate (TDF)-based pre-exposure 
prophylaxis (PrEP) is a recommended HIV prevention strategy and recent approvals of TDF-
based PrEP from sub-Saharan African drug authorities will accelerate roll-out. Unintended 
consequences of TDF-based PrEP use include decline in bone mineral density (BMD) and 
subclinical kidney injury, which might further enhance bone loss. In Africa, young women face 
substantial HIV risk from condomless heterosexual sex and many already use a highly effective 
injectable contraceptive, depot medroxyprogesterone acetate (DMPA), to prevent unintended 
pregnancy. DMPA is also known to cause BMD loss to a substantial degree, warranting a U.S. 
FDA “black box” warning. Thus, as PrEP becomes available in settings where DMPA is commonly 
used, such as Uganda, a large number of HIV-uninfected women will be concurrently exposed to 
two agents that may act additively or synergistically to adversely affect bone health. Furthermore, 
exposure to these agents during adolescence or early adulthood, critical periods for skeletal 
growth, may prevent the attainment of peak bone mass, a major determinant of increased fracture 
risk in later life. Bone fractures can result in the loss of independence, lost wages, and economic 
instability, severe consequences for young African women. The effect of concurrent use of DMPA 
and PrEP on bone health has not been explored in depth in PrEP research to date and is a key 
knowledge gap for women’s health globally.  
 
The primary objective of this study is to address critical safety questions with concurrent TDF-
based PrEP and DMPA use. We hypothesize that young women using TDF-based PrEP and 
DMPA will have lower bone acquisition and altered bone metabolism. Bone mineral metabolism 
is in part regulated by the kidney, and we hypothesize that bone effects from concurrent PrEP 
and DMPA use will be driven by subclinical kidney injury, a known side effect of TDF, as well as 
DMPA-induced hypoestrogenism. To investigate our hypothesis, we will enroll a prospective 
cohort of approximately 500 HIV-uninfected women ages 16-25 years in Kampala, Uganda who 
have substantial HIV risk and are initiating DMPA or barrier method contraception. Over a 24-
month period, we will offer TDF-based PrEP. We will use state-of-the-art radiologic, biochemical, 
and epidemiologic methods to test the hypothesis that concurrent TDF-based PrEP and DMPA 
use results in compounding adverse effects on bone health. 
 
 

The Kampala Women’s Bone Study Protocol 
 
Page 7 
Version 3.0 
31st July 2018  
 
II. 
BACKGROUND 
 
PrEP as a novel HIV prevention strategy for women 
Women in sub-Saharan Africa are disproportionately affected by HIV and the greatest rates of 
HIV incidence are among women below age 25. In Uganda, 4% of young women were estimated 
to be living with HIV and 140,000 people became infected in 2013.1  For women with substantial 
HIV risk, tenofovir disoproxil fumarate (TDF)-based pre-exposure prophylaxis (PrEP) is a daily 
oral medication highly protective against HIV acquisition.2-7  Randomized trials of PrEP efficacy 
demonstrated high HIV protection at levels >90% in analyses of participants with high 
adherence.4-7 Recent landmark approvals of TDF-based PrEP in sub-Saharan Africa and 
commitment from large-scale funding partners (including PEPFAR) will accelerate its roll-out.8 
WHO antiretroviral guidelines now include PrEP and clinical guidelines for Uganda are under 
development. A key advantage of PrEP for women is that it does not require the cooperation of a 
male partner, a challenging characteristic of condom use. Although two trials of PrEP conducted 
with African women showed null results, there is substantial objective evidence indicating that 
these studies were limited by low adherence to study medication.3, 9, 10 Concerns about PrEP 
adherence by young women have been abated by data from PrEP delivery studies demonstrating 
very high adherence with open-label products with known efficacy and accompanied by risk-
related counseling.11   
 
Unintended consequences of PrEP 
Like any biomedical intervention, the use of TDF-based PrEP could have unintended 
consequences, especially when added on top of medication already being used for other 
indications, such as the use of injectable depot medroxyprogesterone acetate (DMPA) to prevent 
pregnancy. TDF-based PrEP and DMPA have both been shown to have detrimental effects on 
bone health, including bone density and architecture. Reduced bone health (measured directly in 
bone density or through intermediate markers of bone turnover) results in increased risk for 
osteoporosis, osteopenia, and osteoporotic fracture. Bone fractures have severe consequences 
for the individual including loss of independence, reduced quality of life, increased frailty and 
depression, and results in less physical activity and social involvement, greater risk for 
subsequent fracture as well as high cost implications from a public health perspective.12, 13 
Fractures account for up to 2% of the global burden of non-communicable diseases and women 
account for more than half of this burden worldwide.14  In resource-limited settings, the prevalence 
of low bone mineral density (BMD) in the general population is rarely described but HIV-infected 
individuals using common antiretroviral HIV treatment regimens appear to experience low BMD 
more frequently than individuals in high income regions.15 
 
Implications of reduced peak bone mass 
Peak bone mass is usually reached by the end of adolescence or early adulthood (Figure 1).16 
Reduced peak bone mass (PBM) is a frequent correlates of osteoporotic fracture later in life.16-18 
In epidemiological studies, a 10% gain in PBM in the female population was associated with 30-
50% decreased risk of hip fracture and mathematical models indicate that relatively small 
increases (10%) in PBM acquisition in healthy females could delay onset of osteoporosis by as 
many as 13 years.19, 20 Exposure to DMPA or TDF during adolescence or early adulthood, which 
unintended pregnancy and HIV risk is greatest, may result in low PMB in the hip, spine, or wrist 
with implications for immediate or long-term bone health.  
 
Effects of TDF-based PrEP on bone metabolism and bone density 

The Kampala Women’s Bone Study Protocol 
 
Page 8 
Version 3.0 
31st July 2018  
 
Prior studies, mostly in men, 
have established that TDF-
based PrEP use leads to 
increased 
bone 
turnover, 
decline 
in 
BMD, 
and 
subclinical 
kidney 
injury, 
which might further enhance 
bone 
loss.21-23 
The 
mechanism of these toxicities 
is not well elucidated but is 
believed to be a complex 
interplay 
between 
renal, 
endocrine, 
and 
bone 
physiology, all of which affect 
calcium–phosphate 
homeostasis.24-29 
In 
HIV-
infected adults, initiation of 
TDF for HIV treatment has 
been associated with a significant increase in bone turnover markers (BTM), including C-
telopeptide (CTX) and procollagen type 1 N-terminal propeptide (P1NP).30-32 These increases in 
CTX and P1NP were significantly correlated with BMD decline at the hip and spine, risk factors 
for later bone fracture.  
Three sub-studies within PrEP efficacy trials assessed BMD loss in comparisons of participants 
using active PrEP versus placebo (Table 1). The only one of these studies to include women 
(TDF2), found an overall net BMD decrease of up to 1.6% at the hip and spine with 30 months of 
TDF-based PrEP use. In this small 
study (N=114 women and 106 men), 
the proportion of participants with 
BMD losses >3.0% at any anatomic 
site was higher for PrEP versus 
placebo arms (50.0% vs 32.9%, 
p=0.04). However, this study was 
limited by low adherence to PrEP 
and 
high 
attrition 
rates. 
Complementary 
data 
from 
two 
studies among men, the iPrEx and 
CDC TDF Safety Study, provide 
consistent evidence, even in the 
face of low adherence.21, 23 
 
Figure 1. Hypothetical representation of the bone mass 
life-line in individual who achieve their full genetic 
potential for skeletal mass and in those who do not. 
[Heaney et al. 2000] 
Table 1: BMD loss in randomized clinical trials of 
TDF-based PrEP vs placebo in HIV-uninfected 
persons 
Study, Authors 
[Year] 
Anatomical 
site 
Net %∆ in BMD 
(PrEP vs placebo) 
TDF2 Study 
Kasonde [2014] 
Hip 
Spine 
Forearm 
-1.55%; p=0.001 
-1.64; p<0.001 
-0.86%; p <0.001 
iPrEX Study 
Mulligan [2015] 
Hip 
Spine 
-0.6%; p=0.01 
-0.91%; p=0.001 
Hip
-0 8%; p=0 001

The Kampala Women’s Bone Study Protocol 
 
Page 9 
Version 3.0 
31st July 2018  
 
Effects of TDF-based PrEP on subclinical kidney injury 
Subclinical kidney injury is characterized by isolated abnormalities in some of markers of kidney 
function, but without generalized clinical disease manifestation. Kidney toxicity can result in 
phosphate wasting with hypophosphatemia and secondary hyperparathyroidism, leading to 
dysregulation of calcium-phosphate homeostasis and bone demineralization.24-29, 33 Higher 
tenofovir plasma concentrations have been associated with higher vitamin D binding protein, 
lower 1,25-OH(2)D, lower estimated free 25-OHD, and increased parathyroid hormone (PTH) in 
HIV-infected persons. Vitamin D ensures a proper balance of calcium and phosphorus, key 
minerals for bone metabolism (Figure 2).26, 27 
 
Our research has recently shown that TDF-based PrEP is associated with a small decline in 
estimated glomerular filtration rate (eGFR) in African men and women (Table 2).34-36 It is not 
known whether this low level of kidney injury will be exacerbated with prolonged use of TDF-
based PrEP or in women with small body mass. Studies are needed to address the effect of 
Figure 2. Schematic representation of kidney Vitamin D 
processing and bone metabolism that are linked through 
vitamin D metabolism and calcium homeostasis pathways 
[Holick 1987] 

The Kampala Women’s Bone Study Protocol 
 
Page 10 
Version 3.0 
31st July 2018  
 
concurrent use of DMPA and TDF-based PrEP 
on bone acquisition in young women who have 
generally small body structures that potentially 
elevate their risk for TDF-induced BMD decline 
and kidney injury.  
Effects of DMPA on bone density  
The most commonly used contraception in sub-
Saharan Africa is DMPA, used by more than half 
of contracepting women in Uganda (Figure 3).37-
40 Intramuscular 150mg DMPA is often preferred 
because of it is easy to administer and can be 
used discretely. Intramuscular DMPA, as well as 
a lower dose subcutaneous formulation, is known to yield BMD loss to such an extent as to 
warrant a “black box” warning from 
the 
U.S. 
Food 
and 
Drug 
Administration.38, 41-48  Since 2012, 
a 
global 
partnership 
of 
>50 
governments, private sector, and 
multi-lateral 
organizations 
has 
made huge efforts to increase 
access to subcutaneous DMPA, 
implying that DMPA will remain the 
predominant contraceptive in this 
setting for the foreseeable future.48 
Women currently using DMPA 
have a lower average BMD than 
non-users and these effects are 
more 
pronounced 
in 
younger 
women.41, 43, 44, 46, 49-51 Longitudinal 
studies have reported BMD losses 
up to 7.5% of BMD after ≥2 years of 
use (Table 3) and the greatest 
amount of loss during the first 1-2 
years.44  
 
Effects of DMPA on bone metabolism 
Table 2: Effect of TDF-based PrEP on 
estimated glomerular filtration rate in 
HIV-uninfected persons [Mugwanya et 
al. JAMA Int Med 2015] 
 
Net mean eGFR decline vs 
placebo (ml/min/1.732) 
 
Randomized TDF-based PrEP 
agent 
 
FTC-TDF 
TDF 
Women 
-1.73;  p=0.02 
-1.47; p=0.05 
Figure 3. Contraceptive method selection by 
contracepting Ugandan women [Makerere 
University 2014] 
 
Sterilization, 
6.5%
Implants, 
11.9%
IUD, 2.9%
Injectable 
DMPA, 
54.0%
Pill, 8.6%
Emergency 
pills, 0.3%
Condom, 
8.3%
Other 
modern, 
2.2%
Other 
traditional, 
5.4%

The Kampala Women’s Bone Study Protocol 
 
Page 11 
Version 3.0 
31st July 2018  
 
Studies of bone turnover in DMPA users have suggested that the effects of DMPA on the skeleton 
are at least partially mediated through increased bone turnover, with increases in markers of bone 
resorption, N-terminal telopeptide of type I collagen (NTX), and bone formation, P1NP. In addition, 
DMPA reduces estradiol and sex hormone-binding globulin (SHBG) levels, resulting in 
hypoestrogenism that is often accompanied by amenorrhea and alters the balance of bone 
resorption and formation yielding impairments in bone mass.38, 52-56 Recent studies in animal 
models also suggest a role of estrogen to hasten recovery from kidney injury, leading to a 
hypothesis that subclinical kidney injury could be a consequence of hypoestrogenism.57-59  
 
Thus, as TDF-based PrEP is delivered in settings where DMPA is commonly used, a large number 
of HIV-uninfected women will be exposed to two agents that are each known to reduce BMD. 
Their concurrent use may act additively or synergistically through pathways of subclinical kidney 
injury and/or hypoestrogenism to adversely affect bone health and the risk of future fracture 
(Figure 4). 
Table 3: Longitudinal studies of the association between DMPA and BMD loss  
Study 
Population 
Anatomical site 
Mean %∆ in BMD -  
DMPA vs Nonhormonal 
Clark et al  2004  
18-35 year old initiators 
Hip 
Lumbar spine 
24 months:-5.7 vs <-0.4 
24 months:-5.7 vs <-0.4 
Berenson et al  2004 
18-33 year old initiators 
Lumbar spine 
24 months: -5.7 vs + 1.8 
Total hip 
 
48 weeks: -1.6 vs 0.6; p<0.001 
240 weeks: -5.2 vs 0.2; p<0.001 

The Kampala Women’s Bone Study Protocol 
 
Page 12 
Version 3.0 
31st July 2018  
 
 
Summary of the potential for joint effects of PrEP and DMPA on bone health 
For young women who equally need TDF-based PrEP to prevent HIV infection and effective 
contraception to prevent unintended pregnancy, concerns about TDF-induced reductions in bone 
mass and associated kidney injury must be heightened. Both are known to have minor effects on 
bone density and bone metabolic markers but through separate pathways. However, it is unknown 
if there is a joint effect of concurrent use of PrEP and DMPA on bone density and markers of bone 
metabolism and whether there are any cross-pathway effects that would result in clinically 
relevant changes to young women’s bone health. This risk is particularly salient for women below 
age 25 who have not yet acquired peak bone mass and thus studying the effects of concurrent 
PrEP and DMPA use on bone health in this population is especially paramount. 
 
Public health impact 
This study will provide the first opportunity to evaluate reductions in bone mass and the preceding 
mechanisms triggered by the simultaneous initiation of PrEP and DMPA during a critical time of 
bone maturation in a cohort of young women. As PrEP delivery is brought to scale in multiple 
African settings where young women are a predominant risk population and DMPA use is 
widespread and will remain common, safety findings from our study will address a key knowledge 
gap and be pertinent to recommendations about concurrent use and how to best incorporate PrEP 
into the lives of young women. 
 
III. 
STUDY METHODS 
 
Figure 4. Schematic of pathways through which concurrent TDF-based PrEP and 
DMPA use could impact bone density 
 
TDF-based PrEP pathway to loss 
of bone mass
DMPA pathway to 
loss of bone mass
Subclinical kidney injury 
Functional vitamin D deficiency
? Vitamin D binding protein
? Free 1,25 (OH) vitamin D
Increased urinary 
excretion of 
phosphorus, calcium
Dysregulation of calcium-
phosphate homeostasis 
? availability of functional Vitamin D
Loss of bone mass
? bone resorption and demineralization and ? risk for fragility fracture
? PTH
Hypoestrogenism 
with ? Estradiol and 
sex hormone-
binding globulin

The Kampala Women’s Bone Study Protocol 
 
Page 13 
Version 3.0 
31st July 2018  
 
Overall Design 
 
The proposed research will investigate the hypothesis that TDF-based PrEP and DMPA initiated 
concurrently by HIV-uninfected women will enhance each other’s effects on bone metabolism. 
We will conduct an open-label prospective study of approximately 500 HIV-uninfected women at 
substantial risk for HIV-infection who are newly initiating DMPA or using condoms as their only 
contraceptive in Kampala, Uganda (Figure 5). We will offer PrEP to all of these women and 
determine the joint effect of PrEP and DMPA on changes in bone mass (Aim 1), bone turnover, 
subclinical kidney injury, and hypoestrogenism (Aim 2), and conduct mediation analysis to assess 
the contribution of each pathway to overall losses in bone mass (Aim 3).  
 
Aims 
 
1. We will assess whether young women using TDF-based PrEP and DMPA concurrently 
attain lower peak bone mass over a 24-month period and have evidence of disrupted 
microarchitecture, relative to women using either agent singly or neither agent 
We will use dual energy x-ray absorptiometry (DXA) scans to measure BMD annually at 3 
anatomical sites (lumbar spine, total hip, and wrist). Additionally, we will derive the trabecular 
bone score (TBS), an index of lumbar spine trabecular microarchitecture. 
Hypothesis: Relative to women using DMPA only (without tenofovir exposure) and women 
using PrEP only (without DMPA exposure), women concurrently using TDF-based PrEP and 
DMPA will have lower bone mass and more disruptions in bone microarchitecture. 
 
Figure 5. Study design for 2-year prospective follow study among healthy, HIV-
uninfected women in Kampala, Uganda 
 
Using male condoms only for 
contraception
n=200
Newly Initiating DMPA
n=300
DMPA and PrEP 
n=225
PrEP ONLY 
n=150
NO PrEP, NO DMPA 
n=50
DMPA ONLY
n=75
Offer PrEP to all
Offer PrEP to all
Estimated 75% 
accept PrEP
HIV uninfected women, ages 16-25
Estimated 75% 
accept PrEP
Outcomes: bone mineral density, bone turnover markers, subclinical kidney injury, 
hypoestrogenism

The Kampala Women’s Bone Study Protocol 
 
Page 14 
Version 3.0 
31st July 2018  
 
2. We will investigate whether young women concurrently using TDF-based PrEP and 
DMPA experience: 1) higher rates of bone turnover, a direct precursor to bone loss and 
2) subclinical kidney injury and hypoestrogenism, markers of two mechanistic 
pathways between TDF and DMPA use and bone mass 
At baseline, 3, 6, 12, and 24 months, we will measure:  
1) Markers of bone formation and resorption (e.g. NTX, P1NP, serum intact parathyroid 
hormone, total and bioavailable 25-OH-vitamin D)  
2) Markers of kidney function (phosphate, glucose, creatinine, total protein, albumin) and  
3) Markers of estrogen (serum estradiol, sex hormone binding protein, and the 
occurrence of amenorrhea) 
Hypothesis: Relative to women using DMPA only and PrEP only, women concurrently using 
TDF-based PrEP and DMPA will have increased bone turnover markers and PTH. In addition, 
women concurrently using TDF-based PrEP and DMPA will have more frequent subclinical 
kidney injury (relative to women without tenofovir exposure) and reduced serum estrogen 
(relative to women without DMPA exposure). 
 
3. Using mediation analysis, we will identify the degree to which the pathways through 
subclinical kidney injury and hypoestrogenism account for changes in bone density 
among women concurrently using TDF-based PrEP and DMPA 
We will conduct mediation analysis to determine the degree to which changes in the pathways 
through subclinical kidney injury, hypoestrogenism and the combination of these pathways 
account for changes in bone density.  
Hypothesis: The pathway through hypoestrogenism will be a stronger link between concurrent 
TDF-based PrEP and DMPA use and bone density changes. 
 
Population 
The proposed study will enroll approximately 500 HIV negative Ugandan women ages 16-25. This 
age group has a high risk for sexual HIV transmission and they also need effective contraceptive 
options and thus they will be an important part of the study population. Secondly, bone mass is 
acquired until approximately age 25 and there may be differences in the attainment of peak bone 
mass if PrEP and DMPA use is started at age 16 with up to 9 years of use prior to reaching peak 
bone mass versus age 18. Thus, since this research question is of particular importance to 
younger females and they will benefit most from the information obtained through this study, it is 
ideal to include them in the study population.  
Location 
Study visits will take place at the Infectious Disease Institute (IDI)-Kasangati research clinic, 
located in the Kasangati area of Kampala in close proximity of several public health care 
facilities. DXA scans will be conducted at the Makerere University-John Hopkins University 
Research Collaboration clinical site (MUJHU), ~5 km from the IDI-Kasangati research clinic 
Women will be transported to MUJHU for scans by study vehicles which has comprehensive 
insurance that covers bodily injury of all vehicle occupants and third parties and drivers.  
Eligibility 
Inclusion criteria: 

The Kampala Women’s Bone Study Protocol 
 
Page 15 
Version 3.0 
31st July 2018  
 
- Age 16-25 
o If age 16-17: 
§ 
qualification as an emancipated minor (due to past pregnancy, being 
married, having a child, or catering for their own livelihood) or  
§ 
a mature minor (due to having a sexually transmitted infection) or  
§ 
able to have a parent/guardian provide informed consent 
- HIV-uninfected 
- Initiated DMPA within the past 90 days or using condoms only for contraception 
- Willing and able to provide written informed consent  
- Not planning to get pregnant in the next 24 months 
- Sexually active  
- Planning to remain in the study area for the next 2 years 
 
Exclusion criteria: 
- Currently enrolled in a biomedical HIV-1 prevention study 
- Current or prior use of PrEP consecutively in the last 3 months 
- Abnormal renal function (creatinine clearance <60 min/ml)  
- Hepatitis B infection 
- Currently pregnant or breastfeeding  
- If history of DMPA use, 1) DMPA use of greater than 90 days with last injection within 2 
years of enrollment or 2) continuous DMPA use of greater than 2 years duration at any 
time  
- Current use of implant, IUD, or oral contraceptives 
- Past hysterectomy, oophorectomy, or tubal ligation 
- Current or recent history of primary or secondary amenorrhea  
- Taking medications known to interfere with bone metabolism (steroids, anti-convulsants, 
bisphosphonates, cancer drugs). 
- Has any other condition that would preclude the ability to provide informed consent, make 
study participation unsafe, complicate the interpretation of study findings or otherwise 
interfere with achievement of the study objectives, in the investigator’s discretion. 
 
Sample size and study power 
Power calculations are based on the number of participants needed to achieve aim 1. Our target 
sample size is 500 women, of which 300 will be using DMPA and 200 will be using condoms for 
contraception.  We calculate that with 300 women newly initiating DMPA and 75% of women 
choosing to initiate PrEP, thus with 225 women concurrently using PrEP and DMPA and 75 
women using DMPA without PrEP, we will have >90% power to detect changes as low as 2.2% 
in BMD (with 1.0 g/cm2 mean BMD loss) at any anatomical site between women using PrEP and 
DMPA concurrently and women using DMPA only during the 24 month follow up period (Table 4). 
Should more than 90% of women choose to initiate PrEP (270 women concurrently using PrEP 
and DMPA and 30 women using DMPA without PrEP), we will have 80% power to detect changes 
of >2.7% or more and should initiation rates be closer to 50% (150 women concurrently using 
DMPA and PrEP and 150 women using DMPA without PrEP), we will have >90% power for all 
comparisons. 
 

The Kampala Women’s Bone Study Protocol 
 
Page 16 
Version 3.0 
31st July 2018  
 
Recruitment 
Recruitment strategies will include collaboration with local family planning clinics, gynecologists, 
women’s health clinics, HIV counseling and testing centers, and public and private health clinics. 
Additionally, we will conduct outreach activities during community markets to recruit women newly 
initiating contraception. Recruitment materials will educate women about the benefits and 
potential adverse effects of contraceptives and PrEP, based on available data.  
General study procedures  
Over a 24-month period, we will follow women quarterly with HIV rapid testing, PrEP adherence 
counseling and refills, DMPA injections, and provision of other contraceptives as desired. At 
enrollment and quarterly visits, we will assess physical activity, sexual behavior, and menstrual 
cycle characteristics. Annually, we will conduct physical exams and anthropometric readings. 
Sexually transmitted infections will be routinely assessed syndromically, according to national 
guidelines. Women will be encouraged to consider their HIV risk and fertility desires at every visit 
and counseled about contraception and PrEP accordingly. Dual energy x-ray absorptiometry 
(DXA) scans will be performed at baseline and annually thereafter. Blood and urine samples will 
be obtained quarterly, aliquoted, and archived at minus 80o Celsius.  
DXA and TBS assessments 
DXA scans will be conducted at study enrollment (within 1 week of contraceptive initiation if 
possible), 12, and 24 months after enrollment. DXA is the clinical standard for measuring BMD. It 
is painless and non-invasive. Women will be exposed to a relatively low level of radiation which 
is <1/10 the dose of a standard chest x-ray, and less than a day's exposure to natural radiation. 
BMD by DXA of the hip and spine is generally considered the most reliable way to classify 
osteoporosis, assess changes in BMD, and predict fracture risk. We will measure areal BMD of 
the lumbar spine (L1-4), hip, whole body and body composition (total and % fat and lean mass) 
using a DXA machine. For the TBS analysis, no additional image acquisition is required. TBS will 
be performed as a re-analysis of the lumbar spine taken as part of the standard DXA procedure 
with specialized software.  
Contraceptive provision 
Table 4. Power calculations. N=500: 300 on DMPA and 200 on condoms, shown with a 
range of PrEP acceptance for the primary comparison of DMPA + PrEP vs DMPA 
(α=0.05) 
 
Outcome: BMD (Aim 1) 
Outcome: NTX (Aim 2) 
Expected PrEP 
acceptance 
Mean %∆ (std 
dev range)  
Mean ∆  
Power 
Mean %∆  
Mean ∆  
Power 
75% (225 DMPA+PrEP, 
75 DMPA) 
2.2 (4–5) 
0.5  
(0.5–1.0) 
91% 
2.2 (4–5) 
0.10  
(0.2 – 0.5) 
>99% 
75% (225 DMPA+PrEP, 
75 DMPA) 
2.5 (4–5) 
0.5  
(0.5–1.0) 
>96% 
2.5 (4–5) 
0.20  
(0.2 – 0.5) 
>96% 
75% (225 DMPA+PrEP, 
75 DMPA) 
3 (4–5) 
1.0  
(0.5–1.0) 
>99% 
3 (4–5) 
0.50  
(0.2 – 0.5) 
>99% 
90% (270 DMPA+PrEP, 
30 PrEP) 
2.5 (4–5) 
0.5  
(0.5–1.0) 
74-90% 
2.5 (4–5) 
0.10  
(0.2 – 0.5) 
>99% 

The Kampala Women’s Bone Study Protocol 
 
Page 17 
Version 3.0 
31st July 2018  
 
Women will initiate contraception at the study clinic or a family planning clinic, as they are seeking 
services. Throughout study follow up, the study will assume responsibility for contraceptive 
provision, including DMPA injections, and care of all other methods desired by women. The staff 
at the IDI-Kasangati research clinic have extensive experience with the provision of contraception 
and contraceptive counseling during previous HIV prevention studies. 
PrEP provision and adherence monitoring 
Co-formulated emtricitabine (FTC)-TDF (Truvada®) will be provided as PrEP by the study 
through donation from Gilead Sciences, LLC. PrEP counseling and provision will follow WHO 
guidelines, or Ugandan clinical guidelines when they are available.60 We will measure 
adherence using three different methods: 
1. Pill counts will be conducted by the pharmacist during study visits to determine the 
percentage of expected pills that were used 
2. Real time monitoring will be conducted using an electronic monitoring system that 
captures a date-time stamp each time the pill container is opened 
3. The primary circulating form of TDF, tenofovir (TFV), will be quantified in blood 
archives from 1) a random sample of up to 15% of women who initiate PrEP to 
characterize the adherence in the cohort overall and 2) a targeted group of women 
who have no BMD change and women who experience a fracture 
Laboratory processing to measure markers of bone turnover, subclinical kidney injury, and 
hypoestrogenism 
Using state-of-the-art assay techniques on archival samples, we will measure 1) established 
markers of bone turnover and bone health, 2) markers of subclinical kidney injury, and 3) markers 
of hypoestrogenism (Table 5). Archived samples will be batch-analyzed.  
 

The Kampala Women’s Bone Study Protocol 
 
Page 18 
Version 3.0 
31st July 2018  
 
Retention and study exit  
Upon completion of study follow up, women will be provided with referrals to clinics of their choice 
to continue contraceptive and PrEP provision. Since the interim guidelines for HIV prevention now 
include PrEP, by the time this study is completed, we anticipate that PrEP will be available through 
public facilities. We will provide participants with information about which facilities are actively 
providing PrEP and link them to services at that clinic via a referral. 
Special circumstances 
a. 
HIV seroconversion 
HIV rapid testing will be conducted quarterly during follow up. We expect the rate of HIV 
seroconversion to be very low, due to the use of PrEP and repeated counseling about HIV 
prevention and PrEP. However, in the event of HIV positive rapid test results, PrEP will be stopped 
immediately and additional blood samples will be drawn for confirmatory EIA testing, plasma HIV 
RNA quantification, drug resistance, and/or drug levels. When with confirmed HIV seroconversion 
will be extensively counseled  and referred to local clinics providing antiretroviral medications and 
HIV care. At the visit when HIV seroconversion is confirmed, women will be exited from the study 
once the set of study procedures for an exit visit are completed. HIV seroconverters who are 
pregnant will be referred for services to prevent mother to child HIV transmission and study staff 
will go to great lengths to ensure that women experience no delays in accessing these services.  
b. 
Pregnancy 
We expect that some women who stop using contraception will become pregnant. Women who 
become pregnant while using PrEP will be counseled about the known and unknown risks and 
benefits of PrEP use during pregnancy, in accordance with U.S. CDC and WHO guidelines.60, 61  
Women who are pregnant will not undergo DXA scans, and their DXA scans will be postponed 
until their pregnancy is complete.  
Table 5. Measurements for markers of bone turnover, subclinical kidney injury, and 
hypoestrogenism 
Outcome 
Biomarkers 
Primary Outcome 
definition 
Secondary Outcome definition 
 
Bone turnover 
and bone health 
NTX, P1NP 
Percent change 
from baseline in 
NTX and P1NP 
Percent change from baseline in 
serum levels of: 
• intact PTH 
• vitamin D binding protein,  
• 25-(OH) vitamin D 
Subclinical 
kidney injury 
• Urine: phosphate, 
glucose, 
creatinine, total 
protein, albumin 
• Serum: 
phosphate, 
glucose, 
creatinine 
eGFR decline 
calculated using 
Chronic Kidney 
Disease 
Epidemiology 
equation (≥25% 
decline from 
baseline or 
creatinine clearance 
<60 mL/min) 
• Tubular proteinuria: elevated 
urine protein with urine albumin: 
protein ratio <0.4,  
• Euglycemic glycosuria,  
• Phosphaturia defined as 
increased fractional excretion of 
phosphorus >15% or maximum 
rate of tubular phosphate 
reabsorption to the glomerular 
filtration rate 
Hypoestrogenism 
• Serum estradiol 
• Sex hormone-
binding globulin 
Percent change 
from baseline of 
serum estradiol with 
control for SHBG
Frequency of amenorrhea, 
defined as the absence of 
menses for at least 3 menstrual 
cycles

The Kampala Women’s Bone Study Protocol 
 
Page 19 
Version 3.0 
31st July 2018  
 
Visit-specific procedural tables 
 
 
SCR ENR 
M1 
M3 
& 
M15 
M6 
& 
M18 
M9 
& 
M21 
M12 & 
M24 & 
EXIT 
ADMINISTRATIVE & REGULATORY PROCEDURES 
Obtain informed consent 
X 
X 
 
 
 
 
 
Collect/update locator information 
X 
X 
X 
X 
X 
X 
X 
Collect/update demographic information 
X 
X 
 
 
 
 
X 
CLINICAL & COUNSELING PROCEDURES 
HIV pre and post-test counseling 
X 
X 
X 
X 
X 
X 
X 
Risk reduction counseling & condom provision 
X 
X 
X 
X 
X 
X 
X 
Contraceptive counseling 
 
X 
X 
X 
X 
X 
X 
Contraceptive provision 
 
 
[X] 
X 
X 
X 
X 
PrEP counseling, including adherence counseling 
X 
X 
X 
X 
X 
X 
X 
PrEP provision 
 
X 
X 
X 
X 
X 
X 
PrEP pill count 
 
 
X 
X 
X 
X 
X 
Physical exam 
 
X 
X 
[X] 
[X] 
[X] 
X 
Anthropometric readings 
 
X 
 
 
 
 
X 
DXA scan (unless pregnant) 
 
X 
 
 
 
 
X 
Syndromic management of STIs 
X 
X 
X 
X 
X 
X 
X 
LABORATORY PROCEDURES 
Creatinine testing when using PrEP 
X 
 
 
 
X 
(M6 
only) 
 
X 
HBV surface antigen 
X 
 
 
 
 
 
 
HIV rapid test (EIA testing and HIV RNA 
quantification, if rapid test positive) 
X 
X 
X 
X 
X 
X 
X 
Urine pregnancy test 
X 
X 
X 
X 
X 
X 
X 
Collect urine specimen for archive 
 
X 
 
X 
X 
X 
X 
Collect blood specimen for archive 
 
X 
 
X 
X 
X 
X 
CLINICAL & BEHAVIORAL DATA COLLECTION 
Medical history questionnaire, including occurrence 
of fractures 
X 
X 
X 
X 
X 
X 
X 
Sexual behavior & contraceptive use questionnaire 
 
X 
X 
X 
X 
X 
X 
Physical activity & dietary questionnaire  
 
X 
 
X 
X 
X 
X 

The Kampala Women’s Bone Study Protocol 
 
Page 20 
Version 3.0 
31st July 2018  
 
Menstrual cycle questionnaire 
X 
X 
 
X 
X 
X 
X 
X required procedure; [X] if clinically indicated 
 
 
 
 
 
 
 
 
Safety monitoring 
Kidney function will be monitored 6 months after initiation and annually thereafter, in accordance 
with Ugandan guidelines, using creatinine clearance calculated by Cockroft–Gault equation. DXA 
scans will be reviewed in real time and women with bone density below -2.0 standard deviation 
of the z-score will be referred locally for vitamin D assessment and provided with any necessary 
therapy. The investigators will be responsible for continuous close monitoring of all adverse 
events that occur among study participants. For the purposes of this study, only serious adverse 
events (SAEs) and adverse events felt related to PrEP will be documented on case report forms.  
Adverse events and the severity of clinical symptoms will be scored using the DAIDS Table for 
Grading the Severity of Adult and Pediatric AEs.62 The study safety monitor will review clinical 
and laboratory SAEs in real time as they are reported by the site clinicians on CRFs and through 
email within 48 hours of occurrence. As needed, the safety monitor will work provide rapid 
consultation with clinicians regarding the management of toxicities. A Safety Review Team 
comprised of the study investigators and lead clinicians will review clinical and laboratory safety 
reports summarizing SAEs via conference call on an approximately quarterly basis. 
 
IV. 
DATA COLLECTION 
 
Clinical data including anthropometric, dietary, physical activity and other lifestyle and sexual 
behaviors, will be recorded electronically on case report forms (CRFs) and data will be uploaded 
to a secure server. Automated legal range checks will be programmed to reduce data entry errors 
and internal quality control reports will be run on a monthly basis. Results from laboratory testing 
will be returned to the data center at the University of Washington and stored alongside other key 
study data. DXA scan images will be downloaded to project-specific databases and sent to the 
University of Washington via secure file transfer system. TBS measurements will be performed 
using the lumbar spine DXA images. PrEP adherence data from the electronic monitoring device 
will be stored on an encrypted website and downloaded to the University of Washington server 
on a regular basis. 
 
V. 
DATA ANALYSIS 
Aim 1. We will assess whether young women using TDF-based PrEP and DMPA concurrently 
attain lower peak bone mass over a 24-month period and have evidence of disrupted 
microarchitecture, relative to women using either agent singly or neither agent 
The primary analysis will be separate comparisons of the annualized rates of change in BMD and 
TBS of the spine and hip between women using PrEP and DMPA concurrently versus women 
using DMPA only (comparison 1), women using PrEP only (comparison 2), and women using 
neither (comparison 3). Analyses will account for baseline BMD. 
 
Aim 2, part 1. We will investigate whether young women concurrently using TDF-based PrEP and 
DMPA experience higher rates of bone turnover relative to women using either agent singly or 
neither agent 

The Kampala Women’s Bone Study Protocol 
 
Page 21 
Version 3.0 
31st July 2018  
 
This outcome will be defined by the percent change from baseline of serum levels of NTX and 
P1NP over 24 months. Secondary outcomes will include: 1) percent change from baseline in 
serum levels of intact PTH, total 25-(OH)D, bioavailable (i.e. unbound or free ) 25-(OH)D 
calculated from vitamin D binding protein (VDBP) using the standard equation. 
 
To test the hypothesis that combined PrEP and DMPA use is associated with greater bone 
turnover than DMPA use alone, we will use time-dependent linear mixed-effects regression 
models to compare mean percent changes in NTX and P1NP between concurrent users of PrEP 
and DMPA versus users of DMPA only. Secondary powered comparisons will be between: a) 
concurrent users of PrEP and DMPA versus users of PrEP only; b) users of PrEP only versus 
users of condoms only; c) users of DMPA only vs users of condoms only. Analysis of the percent 
change from baseline in serum levels of intact PTH, total 25-OH-D and bioavailable 25-OH-D over 
24 months will follow a similar approach. All analyses will be adjusted for confounders and we will 
consider a broad range of demographic, medical, lifestyle, and sexual behavior characteristics as 
potential confounders. 
 
Aim 2, part 2. We will investigate whether young women concurrently using TDF-based PrEP and 
DMPA experience elevated markers of subclinical kidney injury relative to women using TDF-
based PrEP alone 
This outcome will be defined as having either: 
1) eGFR decline (≥25% decline from baseline or creatinine clearance <60 mL/min). GFR will be 
calculated using the Chronic Kidney Disease Epidemiology (CKDE) collaboration equation 
2) proximal tubular injury (tubular proteinuria defined as elevated urine protein with urine 
albumin- protein ratio <0.4, euglycemic glycosuria, phosphaturia defined as increased urinary 
excretion of phosphorus >15% or maximum rate of tubular phosphate reabsorption to the 
glomerular filtration rate).  
These markers were selected because they are routinely available in clinical practice and have 
been used in prior studies. The cutoff of ≥25% for eGFR decline was adapted from established 
criteria for the diagnosis of acute kidney injury;41 eGFR decline of this magnitude has been 
associated with increased morbidity and mortality.22-24 
To determine whether young women using PrEP alone or concurrently with DMPA experience 
higher rates of subclinical kidney injury, we will perform a qualitative comparison of the incidence 
of individual markers of kidney injury among women using PrEP (alone or concurrently with 
DMPA) versus women who have no exposure to TDF. 
 
Aim 2, part 3. We will investigate whether young women concurrently using TDF-based PrEP and 
DMPA experience elevated markers of hypoestrogenism relative to women using DMPA alone 
This outcome will be defined as the percent change from baseline serum estradiol over 24 
months and we will adjust for sex hormone-binding protein.56 The secondary outcome will be 
based on self-reported missed menses, with >3 missed menses in a row categorized as 
amenorrhea. 
To determine whether young women concurrently using DMPA alone or concurrently with PrEP 
experience greater frequency of hypoestrogenism, we will conduct time-dependent logistic 

The Kampala Women’s Bone Study Protocol 
 
Page 22 
Version 3.0 
31st July 2018  
 
regression among women using DMPA and women not exposed to DMPA, comparing the 
proportion of visits with hypoestrogenism. 
 
Aim 3. Using mediation analysis, we will identify the degree to which the pathways through 
subclinical kidney injury and hypoestrogenism account for changes in bone density among 
women concurrently using TDF-based PrEP and DMPA 
 
 
 
 
Our goal for this analysis is to assess the relative magnitude of each pathway on BMD. To define 
each mediator pathway, we will use the set of markers that is most predictive of the mean percent 
change in BMD. For the subclinical kidney injury pathway, the set of markers may include 
phosphate, glucose, creatinine, total protein, and albumin. Because the relationship between 
TDF-induced subclinical kidney injury and dysregulation of PTH and vitamin D metabolism 
remains unclear, we will also consider bioavailable 25-OH vitamin D, and/or intact PTH 
measurements as potential markers of this pathway. In secondary analyses, we will consider 
markers of bone turnover as an alternative outcome to BMD changes. For the hypoestrogenism 
pathway, the set of markers may include estradiol, sex hormone-binding globulin, and/or 
amenorrhea.  
We will estimate the crude and adjusted direct and indirect effects of concurrent TDF-based PrEP 
and DMPA use on mean percentage change in BMD through each pathway by constructing 
separate regression models of BMD conditional on TDF and DMPA exposure, the mediator’s 
occurrence, and baseline covariates. We will compute the proportion mediated by each mediator 
individually as well as the both mediators concurrently closed form estimators. Bootstrapping will 
be used to compute standard errors. Our interpretation of these results will carefully specify model 
assumptions including no unmeasured confounding between the exposure and outcome, 
exposure and mediators, and mediators and outcome.63 
 
VI. 
HUMAN SUBJECTS CONSIDERATIONS 
The protocol, informed consent forms (for cohort participation and for interviews of providers), 
and patient education and recruitment materials will be reviewed and approved by the IRBs/ECs 
responsible for oversight.  Subsequent to initial review and approval, the responsible IRBs/ECs 
will review the study at least annually. 
Study oversight 
An independent Data Monitoring Committee will be established to protect patient safety by 
monitoring study outcomes, implementation, and data quality. The committee will consist of expert 
clinicians, statisticians, and scientists, including Ugandans, in the field of PrEP and family 
planning delivery in sub-Saharan African settings. The committee will meet via teleconference 
every 6 months and be charged with the decision to recommend stopping the study early if interim 
analyses yield any safety concerns. In addition, the committee will provide guidance about results 
from international studies of PrEP and contraception that may spur Ugandan policy change. 
Reports from all reviews will be submitted to overseeing institutional review boards and ethics 
committees. 

The Kampala Women’s Bone Study Protocol 
 
Page 23 
Version 3.0 
31st July 2018  
 
Informed consent 
We will perform paper-based or electronic-based consenting of participants.  After we have 
ensured that participants have read and understood the consent forms, we will ask them to 
append their signature on a tablet or on paper.  Each participant will receive a participant 
information sheet that they can use for reference.  However, any participant who wants their 
consent form to take home will have it printed and given to them. Using electronic-based 
consenting will greatly reduce the burden of storing paper-based consent forms. 
Risks 
Risks for this cohort include side effects from PrEP provided to the young women through the 
study clinic, DMPA and other contraceptives provided through Ugandan public health clinics 
according to the national guidelines for contraceptive provision in Uganda, and discomfort 
associated with blood draws, physical exams, interviews, and HIV counseling and testing. 
Intensive and ongoing counseling and monitoring of side effects and adverse events limit the risk 
for participants from the direct study procedures. Women will have three DXA scans during the 
course of the study and be exposed to radiation during each scan. The effective radiation dose 
from the DXA procedure, approximately 0.01 millisievert (mSv), is similar to that received during 
a 5-hour airplane flight. The total dose over these 3 scans will be several orders of magnitude 
less than the radiation exposure women will have from their natural environments (from sources 
such as sun, air, food, and soil).  Women who become pregnant will not undergo DXA scans. 
For clinical data collection, standardized surveys will be used that will include questions on 
sensitive topics, such as sexual behavior. All participants will be extensively counseled about the 
risk for HIV transmission with sex unprotected by a condom and/or PrEP and the importance of 
using condoms consistently and correctly. We expect that the risk of HIV transmission will actually 
be lower in the proposed novel cohort than the general population of young women due to the 
careful attention that will be given to counseling women about HIV risk and the provision of PrEP. 
Study staff will be aware of the potential for confidentiality issues with regards to test results from 
HIV testing that are part of a participant’s study documents. For minors who enroll with parental 
consent, results of HIV testing will be confidential and not available to the parent or guardian.  But 
the study will stipulate that minors will be encouraged to disclose positive results to a responsible 
adult, which need not be their legal guardian. Support will be provided for such disclosures. 
There is the potential for confidentiality issues with regards to study data. Particularly, since 
parental consent will be required for some participants under the age of 18, these participants 
would have to share their choice to participate in a study that will follow their contraceptive and 
PrEP use choices and collect detailed sexual behavior data, whereas otherwise they may not 
have chosen to disclose this information. Should there be any psychosocial stress associated 
with participation in the study, counseling by study staff will be provided, or referral to appropriate 
mental health professionals will be done. The age of consensual sex is 16 years, and therefore 
underage sex is not an issue in this protocol.  
Benefits 
Participants will receive individualized HIV risk-reduction counseling and counseling about 
pregnancy planning and prevention of unintended pregnancy, access to PrEP medication, 
contraceptives, and free condoms. In addition, participants may benefit in the future from 
information learned from this study. 
Care for persons identified as HIV positive 
Women who become infected with HIV will be referred immediately to HIV care centers and 
encouraged to initiate antiretrovirals in line with Ugandan national antiretroviral guidelines.  

The Kampala Women’s Bone Study Protocol 
 
Page 24 
Version 3.0 
31st July 2018  
 
Seroconverters who become pregnant will be referred for prevention of mother to child HIV 
transmission services. 
Treatment for injury 
Participants will be asked to inform the clinic staff if they feel they have been injured because of 
taking part in the study. Injuries may also be identified during laboratory testing, medical histories, 
and physical examinations. Treatment for adverse events related to study participation will be 
provided by the treatment clinic. If treatment is required that is beyond the capacity of the clinic, 
the clinic staff will refer the participant to appropriate services or organizations that can provide 
care for the injury. The study based at IDI will cover the costs of the referral and treatment costs 
up to the time the participant is stable or injury is resolved.  
Study records 
Complete, accurate, and current study records will be maintained and stored in a secure 
manner throughout the study.  Study records include administrative documentation and 
regulatory documentation as well as documentation related to each participant enrolled in the 
cohort, including informed consent forms, data forms, notations of all contacts with the 
participant, and all other source documents.   
Confidentiality 
Every effort will be made to protect participant privacy and confidentiality to the extent possible.  
Personal identifying information will be retained at the local study site.   
 
VII. 
CLINICAL RESEARCH SITE 
 
Site location 
The Infectious Disease Institute (IDI)-Kasangati research clinic is part of the Infectious Diseases 
Institute, Makerere University College of Health Sciences. The facility is located next to Kasangati 
Health Center IV and has adequate space and equipment for ongoing and planned research 
studies. The Infectious Diseases Institute training team offers specialized courses in HIV 
management for health providers in Africa. For this study the focus of the training will be 
combination HIV prevention services including PrEP. 
Clinical facilities 
The site has a large training room that can accommodate up to 50 people, 3 clinical rooms, 6 
counseling rooms, a pharmacy, a phlebotomy room, a side lab, secure data room with data 
archive, administrative office space, wireless internet, telephone intercom system, community 
education offices, and a large waiting area which provide sufficient space for the execution of this 
study. 
Staff 
The Principal Investigator is a seasoned HIV/AIDS researcher and trainer, of local and 
international repute. He will lead the study team to maximize the scientific, ethical integrity of the 
study and ensure the training provided is of high quality.  The study employs in total 3 doctors, 2 
nurse 
counselors, 
1 
pharmacy 
technician, 
2 
laboratory 
staff 
and 
2 
community 
educators/counselors, 2 data personnel, 4 qualitative research associates as well as support and 
administrative staff. The respective staff are overseen by a site coordinator, training coordinator 
and administrator. The study site previously implemented the Partners PrEP Study and the 

The Kampala Women’s Bone Study Protocol 
 
Page 25 
Version 3.0 
31st July 2018  
 
Partners Demonstration Project. The staff have substantial experience in the use of PrEP and its 
accompanying supportive services. The staff have been involved in the conduct of protocol 
specific training, development of outreach materials, and have skills in training of adult learners. 
Administrative procedures 
All administrative procedures regarding protocol compliance, study coordination, study activation, 
study monitoring, study records, and use of information and publications will be done according 
to good clinical practice. 
Laboratory considerations 
We will as much as possible use the public health laboratory facilities for routine laboratory 
monitoring. In situations where this is not possible, specimens will be collected and transported 
to the study collaborating laboratories. All specimen collection, transport, processing, testing, 
archiving, and results reporting will be conducted in accordance with good clinical and laboratory 
practice standards. The collaborating laboratories for the research clinic are the MUJHU-IDI Core 
Laboratory, the Makerere University Walter Reed Laboratory, and the Molecular Biology 
Laboratory in the Department of Medical Microbiology at Makerere College of Health Sciences. 
These are well-established labs with extensive experience in supporting clinical trials. External 
Quality Assurance (EQA) procedures will be followed throughout the study and are overseen by 
the University of Washington International Clinical Research Center. 

 
The Women’s Bone Health Study Protocol 
 
 
Version 3.0 
31st July 2018  
 
VIII. 
REFERENCES 
 
UNAIDS. The Gap Report. Geneva, Switzerland; 2014. 
Food and Drug Administration. Truvada approved to reduce the risk of sexually transmitted HIV 
in people who are not infected with the virus; 2012 [updated 2012; cited]; Accessed September 
12, 2015]. Available from: 
http://www.fda.gov/downloads/ForConsumers/ConsumerUpdates/UCM311828.pdf. 
K. A. Thomson, J. M. Baeten, N. R. Mugo, L. G. Bekker, C. L. Celum and R. Heffron. Tenofovir-
based oral preexposure prophylaxis prevents HIV infection among women. Curr Opin HIV AIDS. 
2016;11(1):18-26. PMCID: 4705855. 
J. M. Baeten, D. Donnell, P. Ndase, N. R. Mugo, J. D. Campbell, J. Wangisi, J. W. Tappero, E. 
A. Bukusi, C. R. Cohen, E. Katabira, A. Ronald, E. Tumwesigye, E. Were, K. H. Fife, J. Kiarie, 
C. Farquhar, G. John-Stewart, A. Kakia, J. Odoyo, A. Mucunguzi, E. Nakku-Joloba, R. 
Twesigye, K. Ngure, C. Apaka, H. Tamooh, F. Gabona, A. Mujugira, D. Panteleeff, K. K. 
Thomas, L. Kidoguchi, M. Krows, J. Revall, S. Morrison, H. Haugen, M. Emmanuel-Ogier, L. 
Ondrejcek, R. W. Coombs, L. Frenkel, C. Hendrix, N. N. Bumpus, D. Bangsberg, J. E. Haberer, 
W. S. Stevens, J. R. Lingappa, C. Celum and Partners PrEP Study Team. Antiretroviral 
prophylaxis for HIV prevention in heterosexual men and women. N Engl J Med 
2012;367(5):399-410. PMCID: 3770474. 
K. Choopanya, M. Martin, P. Suntharasamai, U. Sangkum, P. A. Mock, M. Leethochawalit, S. 
Chiamwongpaet, P. Kitisin, P. Natrujirote, S. Kittimunkong, R. Chuachoowong, R. J. Gvetadze, 
J. M. McNicholl, L. A. Paxton, M. E. Curlin, C. W. Hendrix, S. Vanichseni and Bangkok 
Tenofovir Study Group. Antiretroviral prophylaxis for HIV infection in injecting drug users in 
Bangkok, Thailand (the Bangkok Tenofovir Study): a randomised, double-blind, placebo-
controlled phase 3 trial. Lancet. 2013;381(9883):2083-90. 
R. M. Grant, J. R. Lama, P. L. Anderson, V. McMahan, A. Y. Liu, L. Vargas, P. Goicochea, M. 
CasapÃa, J. V. Guanira-Carranza, M. E. Ramirez-Cardich, O. Montoya-Herrera, T. FernÃ¡ndez, 
V. G. Veloso, S. P. Buchbinder, S. Chariyalertsak, M. Schechter, L.-G. Bekker, K. H. Mayer, E. 
G. KallÃ¡s, K. R. Amico, K. Mulligan, L. R. Bushman, R. J. Hance, C. Ganoza, P. Defechereux, 
B. Postle, F. Wang, J. J. McConnell, J.-H. Zheng, J. Lee, J. F. Rooney, H. S. Jaffe, A. I. 
Martinez, D. N. Burns, D. V. Glidden and iPrEx Study Team. Preexposure chemoprophylaxis for 
HIV prevention in men who have sex with men. N Engl J Med. 2010;363(27):2587-99. PMCID: 
3079639. 
M. C. Thigpen, P. M. Kebaabetswe, L. A. Paxton, D. K. Smith, C. E. Rose, T. M. Segolodi, F. L. 
Henderson, S. R. Pathak, F. A. Soud, K. L. Chillag, R. Mutanhaurwa, L. I. Chirwa, M. Kasonde, 
D. Abebe, E. Buliva, R. J. Gvetadze, S. Johnson, T. Sukalac, V. T. Thomas, C. Hart, J. A. 
Johnson, C. K. Malotte, C. W. Hendrix, J. T. Brooks and TDF2 Study Group. Antiretroviral 
preexposure prophylaxis for heterosexual HIV transmission in Botswana. N Engl J Med. 
2012;367(5):423-34. 
South Africa Medicines Control Council. Medicines Control Council approves fixed-dose 
combination of tenofovir disoproxyl fumarate and emtricitabine for pre-exposure prophylaxis of 
HIV  Pretoria, South Africa: Registrar of Medicines, Medicines Control Council; 2015 [updated 

The Kampala Women’s Bone Study Protocol 
 
Page 27 
Version 3.0 
31st July 2018  
 
2015; cited 2015 Dec 20]; Available from: 
http://www.mccza.com/documents/2e4b3a5310.11_Media_release_ARV_FDC_PrEP_Nov15_v
1.pdf. 
A. van der Straten, J. Stadler, E. Montgomery, M. Hartmann, B. Magazi, F. Mathebula, K. 
Schwartz, N. Laborde and L. Soto-Torres. Women's experiences with oral and vaginal pre-
exposure prophylaxis: the VOICE-C qualitative study in Johannesburg, South Africa. PLoS One. 
2014;9(2):e89118. PMCID: 3931679. 
A. L. Corneli, J. Deese, M. Wang, D. Taylor, K. Ahmed, K. Agot, J. Lombaard, R. Manongi, S. 
Kapiga, A. Kashuba, L. Van Damme and F. E.-P. S. Group. FEM-PrEP: adherence patterns and 
factors associated with adherence to a daily oral study product for pre-exposure prophylaxis. J 
Acquir Immune Defic Syndr. 2014;66(3):324-31. PMCID: 4059551. 
L. G. Bekker, J. Hughes, R. Amico, S. Roux, C. Hendrix, P. L. Anderson, B. J. Dye, V. Elharrar, 
M. J. Stirratt and R. M. Grant. HPTN 067/ADAPT background and methods and Cape Town 
results.  IAS 2015:  8th IAS Conference on HIV Pathogenesis, Treatment and Prevention; July 
19-22; Vancouver, Canada: 2015. Abstract #MOSY0103. 
R. Burge, B. Dawson-Hughes, D. H. Solomon, J. B. Wong, A. King and A. Tosteson. Incidence 
and economic burden of osteoporosis-related fractures in the United States, 2005-2025. J Bone 
Miner Res. 2007;22(3):465-75. 
J. A. Cauley. Public health impact of osteoporosis. J Gerontol A Biol Sci Med Sci. 
2013;68(10):1243-51. PMCID: 3779634. 
O. Johnell and J. A. Kanis. An estimate of the worldwide prevalence and disability associated 
with osteoporotic fractures. Osteoporos Int. 2006;17(12):1726-33. 
F. K. Matovu, L. Wattanachanya, M. Beksinska, J. M. Pettifor and K. Ruxrungtham. Bone health 
and HIV in resource-limited settings: a scoping review. Curr Opin HIV AIDS. 2016;11(3):306-25. 
R. P. Heaney, S. Abrams, B. Dawson-Hughes, A. Looker, A. Looker, R. Marcus, V. Matkovic 
and C. Weaver. Peak bone mass. Osteoporos Int. 2000;11(12):985-1009. 
J. P. Sabatier, G. Guaydier-Souqui`eres, D. Laroche, A. Benmalek, L. Fournier, F. Guillon-Metz, 
J. Delavenne and A. Y. Denis. Bone mineral acquisition during adolescence and early 
adulthood: A study in 574 healthy females 10–24 years of age. Osteoporos Int. 1996;6(2):141-8. 
G. Theintz, B. Buchs, R. Rizzoli, D. Slosman, H. Clavien, P. C. Sizonenko and J. P. Bonjour. 
Longitudinal monitoring of bone mass accumulation in healthy adolescents: evidence for a 
marked reduction after 16 years of age at the levels of lumbar spine and femoral neck in female 
subjects. J Clin Endocrinol Metab. 1992;75(4):1060-5. 
S. Mora and V. Gilsanz. Establishment of peak bone mass. Endocrinol Metab Clin North Am. 
2003;32(1):39-63. 
C. J. Hernandez, G. S. Beaupré and D. R. Carter. A theoretical analysis of the relative 
influences of peak BMD, age-related bone loss and menopause on the development of 
osteoporosis. Osteoporos Int. 2003;14(10):843-7. 

The Kampala Women’s Bone Study Protocol 
 
Page 28 
Version 3.0 
31st July 2018  
 
M. Kasonde, R. W. Niska, C. Rose, F. L. Henderson, T. M. Segolodi, K. Turner, D. K. Smith, M. 
C. Thigpen and L. A. Paxton. Bone mineral density changes among HIV-uninfected young 
adults in a randomised trial of pre-exposure prophylaxis with tenofovir-emtricitabine or placebo 
in Botswana. PLoS One. 2014;9(3):e90111. PMCID: 3953113. 
A. Y. Liu, E. Vittinghoff, D. E. Sellmeyer, R. Irvin, K. Mulligan, K. Mayer, M. Thompson, R. 
Grant, S. Pathak, B. O'Hara, R. Gvetadze, K. Chillag, L. Grohskopf and S. P. Buchbinder. Bone 
mineral density in HIV-negative men participating in a tenofovir pre-exposure prophylaxis 
randomized clinical trial in San Francisco. PLoS One. 2011;6(8):e23688. PMCID: 3163584. 
K. Mulligan, D. V. Glidden, P. L. Anderson, A. Liu, V. McMahan, P. Gonzales, M. E. Ramirez-
Cardich, S. Namwongprom, P. Chodacki, L. M. C. de Mendonca, F. Wang, J. R. Lama, S. 
Chariyalertsak, J. V. Guanira, S. Buchbinder, L.-G. Bekker, M. Schechter, V. G. Veloso, R. M. 
Grant and Preexposure Prophylaxis Initiative Study Team. Effects of emtricitabine/tenofovir on 
bone mineral density in HIV-negative persons in a randomized, double-blind, placebo-controlled 
trial. Clin Infect Dis. 2015;61(4):572-80. PMCID: 4565984. 
I. F. Grigsby, L. Pham, L. M. Mansky, R. Gopalakrishnan, A. E. Carlson and K. C. Mansky. 
Tenofovir treatment of primary osteoblasts alters gene expression profiles: implications for bone 
mineral density loss. Biochem Biophys Res Commun. 2010;394(1):48-53. PMCID: 2847063. 
I. F. Grigsby, L. Pham, R. Gopalakrishnan, L. M. Mansky and K. C. Mansky. Downregulation of 
Gnas, Got2 and Snord32a following tenofovir exposure of primary osteoclasts. Biochem 
Biophys Res Commun. 2010;391(3):1324-9. PMCID: 2812596. 
P. L. Havens, J. J. Kiser, C. B. Stephensen, R. Hazra, P. M. Flynn, C. M. Wilson, B. Rutledge, J. 
Bethel, C. G. Pan, L. R. Woodhouse, M. D. Van Loan, N. Liu, J. Lujan-Zilbermann, A. Baker, B. 
G. Kapogiannis, C. M. Gordon, K. Mulligan and Adolescent Medicine Trials Network for 
HIV/AIDS Interventions (ATN) 063 Study Team. Association of higher plasma vitamin D binding 
protein and lower free calcitriol levels with tenofovir disoproxil fumarate use and plasma and 
intracellular tenofovir pharmacokinetics: Cause of a functional vitamin D deficiency? Antimicrob 
Agents Chemother. 2013;57(11):5619-28. PMCID: 3811269. 
P. L. Havens, C. B. Stephensen, R. Hazra, P. M. Flynn, C. M. Wilson, B. Rutledge, J. Bethel, C. 
G. Pan, L. R. Woodhouse, M. D. Van Loan, N. Liu, J. Lujan-Zilbermann, A. Baker, B. G. 
Kapogiannis, K. Mulligan and Adolescent Medicine Trials Network (ATN) for HIV/AIDS 
Interventions 063 study team. Vitamin D3 decreases parathyroid hormone in HIV-infected youth 
being treated with tenofovir: a randomized, placebo-controlled trial. Clin Infect Dis. 
2012;54(7):1013-25. PMCID: 3297650. 
J. B. Purdy, R. I. Gafni, J. C. Reynolds, S. Zeichner and R. Hazra. Decreased bone mineral 
density with off-label use of tenofovir in children and adolescents infected with human 
immunodeficiency virus. J Pediatr. 2008;152(4):582-4. PMCID: 2390888. 
V. Giacomet, S. Mora, L. Martelli, M. Merlo, M. Sciannamblo and A. Vigano. A 12-month 
treatment with tenofovir does not impair bone mineral accrual in HIV-infected children. J Acquir 
Immune Defic Syndr. 2005;40(4):448-50. 
R. J. Bedimo, H. Drechsler, M. Jain, J. Cutrell, S. Zhang, X. Li, I. Farukhi, R. Castanon, P. 
Tebas and N. M. Maalouf. The RADAR study: week 48 safety and efficacy of RAltegravir 

The Kampala Women’s Bone Study Protocol 
 
Page 29 
Version 3.0 
31st July 2018  
 
combined with boosted DARunavir compared to tenofovir/emtricitabine combined with boosted 
darunavir in antiretroviral-naive patients. Impact on bone health. PLoS One. 2014;9(8):e106221. 
J. I. Bernardino, A. Mocroft, P. W. Mallon, C. Wallet, J. Gerstoft, C. Russell, P. Reiss, C. 
Katlama, S. De Wit, L. Richert, A. Babiker, A. Buño, A. Castagna, P.-M. Girard, G. Chene, F. 
Raffi and J. R. Arribas. Bone mineral density and inflammatory and bone biomarkers after 
darunavir–ritonavir combined with either raltegravir or tenofovir–emtricitabine in antiretroviral-
naive adults with HIV-1: a substudy of the NEAT001/ANRS143 randomised trial. Lancet HIV. 
2015;2(11):e464-e73. 
H.-J. Stellbrink, C. Orkin, J. R. Arribas, J. Compston, J. Gerstoft, E. Van Wijngaerden, A. 
Lazzarin, G. Rizzardini, H. G. Sprenger, J. Lambert, G. Sture, D. Leather, S. Hughes, P. Zucchi, 
H. Pearce and ASSERT Study Group. Comparison of changes in bone density and turnover 
with abacavir-lamivudine versus tenofovir-emtricitabine in HIV-infected adults: 48-week results 
from the ASSERT Study. Clin Infect Dis. 2010;51(8):963-72. 
M. F. Holick. Vitamin D and the kidney. Kidney Int. 1987;32(6):912-29. 
M. Martin, S. Vanichseni, P. Suntharasamai, U. Sangkum, P. A. Mock, R. J. Gvetadze, M. E. 
Curlin, M. Leethochawalit, S. Chiamwongpaet, T. Cherdtrakulkiat, R. Anekvorapong, W. 
Leelawiwat, N. Chantharojwong, J. M. McNicholl, L. A. Paxton, S. Kittimunkong, K. Choopanya 
and Bangkok Tenofovir Study Group. Renal function of participants in the Bangkok Tenofovir 
Study—Thailand, 2005–2012. Clin Infect Dis. 2014;59(5):716-24. 
K. K. Mugwanya, C. Wyatt, C. Celum, D. Donnell, N. Mugo, J. Tappero, J. Kiarie, A. Ronald, J. 
Baeten and Partners PrEP Study Team. Changes in glomerular kidney function among HIV-1–
uninfected men and women receiving emtricitabine–tenofovir disoproxil fumarate preexposure 
prophylaxis: A randomized clinical trial. JAMA Intern Med. 2015;175(2):246-54. PMCID: 
4354899. 
M. M. Solomon, J. R. Lama, D. V. Glidden, K. Mulligan, V. McMahan, A. Y. Liu, J. V. Guanira, V. 
G. Veloso, K. H. Mayer, S. Chariyalertsak, M. Schechter, L.-G. Bekker, E. G. Kallás, D. N. 
Burns, R. M. Grant and iPrEx Study Team. Changes in renal function associated with oral 
emtricitabine/tenofovir disoproxil fumarate use for HIV pre-exposure prophylaxis. AIDS. 
2014;28(6):851-9. PMCID: 3966916. 
J. Jain, A. J. Jakimiuk, F. R. Bode, D. Ross and A. M. Kaunitz. Contraceptive efficacy and safety 
of DMPA-SC. Contraception. 2004;70(4):269-75. 
A. M. Kaunitz, P. D. Darney, D. Ross, K. D. Wolter and L. Speroff. Subcutaneous DMPA vs. 
intramuscular DMPA: a 2-year randomized study of contraceptive efficacy and bone mineral 
density. Contraception. 2009;80(1):7-17. 
J. Trussell. Contraceptive failure in the United States. Contraception. 2011;83(5):397-404. 
PMCID: 3638209. 
Performance monitoring & accountability 2020 Uganda; 2014 [updated 2014; cited]; Available 
from: http://pma2020.org/sites/default/files/PMAUG-DIR-R1-2015.08.25_1.pdf. Accessed 
December 23, 2015. 

The Kampala Women’s Bone Study Protocol 
 
Page 30 
Version 3.0 
31st July 2018  
 
A. B. Berenson, C. R. Breitkopf, J. J. Grady, V. I. Rickert and A. Thomas. Effects of hormonal 
contraception on bone mineral density after 24 months of use. Obstet Gynecol. 2004;103(5, 
Part 1):899-906. 
C. Berger, L. Langsetmo, L. Joseph, D. A. Hanley, K. S. Davison, R. Josse, N. Kreiger, A. 
Tenenhouse, D. Goltzman and the Canadian Multicentre Osteoporosis Study Research Group. 
Change in bone mineral density as a function of age in women and men and association with 
the use of antiresorptive agents. CMAJ. 2008;178(13):1660-8. PMCID: 2413314. 
M. K. Clark, M. Sowers, B. Levy and S. Nichols. Bone mineral density loss and recovery during 
48 months in first-time users of depot medroxyprogesterone acetate. Fertil Steril. 
2006;86(5):1466-74. 
M. K. Clark, M. R. Sowers, S. Nichols and B. Levy. Bone mineral density changes over two 
years in first-time users of depot medroxyprogesterone acetate. Fertil Steril. 2004;82(6):1580-6. 
B. A. Gbolade. Depo-Provera and bone density. J Fam Plann Reprod Health Care. 
2002;28(1):7-11; quiz , 50. 
A. M. Kaunitz, P. D. Miller, V. M. Rice, D. Ross and M. R. McClung. Bone mineral density in 
women aged 25–35 years receiving depot medroxyprogesterone acetate: recovery following 
discontinuation. Contraception. 2006;74(2):90-9. 
D. Scholes, A. Z. LaCroix, L. E. Ichikawa, W. E. Barlow and S. M. Ott. Injectable hormone 
contraception and bone density: results from a prospective study. Epidemiology. 
2002;13(5):581-7. 
London summit on family planning: Summary of commitments; 2012 [updated 2012; cited]; 
Available from: 
https://www.gov.uk/government/uploads/system/uploads/attachment_data/file/67328/london-
summit-family-planning-commitments.pdf. Accessed December 12, 2015. 
J. S. Walsh, R. Eastell and N. F. A. Peel. Effects of depot medroxyprogesterone acetate on 
bone density and bone metabolism before and after peak bone mass: A case-control study. J 
Clin Endocrinol Metab. 2008;93(4):1317-23. 
B. A. Cromer, J. M. Blair, J. D. Mahan, L. Zibners and Z. Naumovski. A prospective comparison 
of bone density in adolescent girls receiving depot medroxyprogesterone acetate (Depo-
Provera), levonorgestrel (Norplant), or oral contraceptives. J Pediatr. 1996;129(5):671-6. 
T. Cundy, M. Evans, H. Roberts, D. Wattie, R. Ames and I. R. Reid. Bone density in women 
receiving depot medroxyprogesterone acetate for contraception. BMJ. 1991;303(6793):13-6. 
PMCID: 1670252. 
P. Garnero, E. Sornay-Rendu, B. Claustrat and P. D. Delmas. Biochemical markers of bone 
turnover, endogenous hormones and the risk of fractures in postmenopausal women: the 
OFELY study. J Bone Miner Res. 2000;15(8):1526-36. 
S. Jeppsson, S. Gershagen, E. D. Johansson and G. Rannevik. Plasma levels of 
medroxyprogesterone acetate (MPA), sex-hormone binding globulin, gonadal steroids, 

The Kampala Women’s Bone Study Protocol 
 
Page 31 
Version 3.0 
31st July 2018  
 
gonadotrophins and prolactin in women during long-term use of depo-MPA (Depo-Provera) as a 
contraceptive agent. Acta Endocrinol (Copenh). 1982;99(3):339-43. 
Z. Harel, K. Wolter, M. A. Gold, B. Cromer, M. Stager, C. C. Johnson, R. Brown, A. Bruner, S. 
Coupey, P. Hertweck, H. Bone, R. Burkman, A. Nelson, S. Marshall and L. K. Bachrach. 
Biopsychosocial variables associated with substantial bone mineral density loss during the use 
of depot medroxyprogesterone acetate in adolescents: adolescents who lost 5% or more from 
baseline vs. those who lost less than 5%. Contraception. 2010;82(6):503-12. 
M. L. Fernandez-Soto, A. Gonzalez-Jimenez, M. Chamorro-Fernandez and S. Leyva-Martinez. 
Clinical and hormonal variables related to bone mass loss in anorexia nervosa patients. Vitam 
Horm. 2013;92:259-69. 
B. Meczekalski, A. Podfigurna-Stopa and A. R. Genazzani. Hypoestrogenism in young women 
and its influence on bone mass density. Gynecol Endocrinol. 2010;26(9):652-7. 
A. P. Singh, N. Singh and P. M. Singh Bedi. Estrogen attenuates renal IRI through PPAR-
gamma agonism in rats. J Surg Res. 2016;203(2):324-30. 
R. Tanaka, H. Tsutsui, S. Kobuchi, T. Sugiura, M. Yamagata, M. Ohkita, M. Takaoka, T. 
Yukimura and Y. Matsumura. Protective effect of 17beta-estradiol on ischemic acute kidney 
injury through the renal sympathetic nervous system. Eur J Pharmacol. 2012;683(1-3):270-5. 
B. Antus, P. Hamar, G. Kokeny, Z. Szollosi, I. Mucsi, Z. Nemes and L. Rosivall. Estradiol is 
nephroprotective in the rat remnant kidney. Nephrol Dial Transplant. 2003;18(1):54-61. 
World Health Organization. Guideline on When to Start Antiretroviral Therapy and on Pre-
Exposure Prophylaxis for HIV. Geneva, Switzerland: World Health Organization; 2015. 
P. E. Sax, D. Wohl, M. T. Yin, F. Post, E. DeJesus, M. Saag, A. Pozniak, M. Thompson, D. 
Podzamczer, J. M. Molina, S. Oka, E. Koenig, B. Trottier, J. Andrade-Villanueva, G. Crofoot, J. 
M. Custodio, A. Plummer, L. Zhong, H. Cao, H. Martin, C. Callebaut, A. K. Cheng, M. W. 
Fordyce, S. McCallister and GS-US-292-0104/0111 Study Team. Tenofovir alafenamide versus 
tenofovir disoproxil fumarate, coformulated with elvitegravir, cobicistat, and emtricitabine, for 
initial treatment of HIV-1 infection: two randomised, double-blind, phase 3, non-inferiority trials. 
Lancet. 2015;385(9987):2606-15. 
K. D. Chirgwin, J. Feldman, O. Muneyyirci-Delale, S. Landesman and H. Minkoff. Menstrual 
function in human immunodeficiency virus-infected women without acquired immunodeficiency 
syndrome. J Acquir Immune Defic Syndr Hum Retrovirol. 1996;12(5):489-94. 
T. J. VanderWeele and S. Vansteelandt. Mediation analysis with multiple mediators. Epidemiol 
Method. 2014;2(1):95-115. PMCID: 4287269.