The Impact of HIV on Accelerated Aging in the Female Genital Tract: a Pilot Trial of Topical Estradiol to Improve the Vaginal Microbiome and Symptoms of Vaginal Atrophy in Menopausal Women With HIV
Phase: PHASE4
Conditions: HIV Infection, Vaginal Atrophy, Menopause, Menopause Related Conditions, Aging, Premature Aging, Atrophic Vaginitis, Dysbiosis, Vaginitis
Sponsor: Kerry Murphy

Study Title: Accelerated Genital Tract Aging in HIV: Estradiol Clinical Trial
NCT04079218
IRB Number: 2019-10529
IRB NUMBER: 2019-10529
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IRB APPROVAL DATE: 09/09/2021
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Accelerated genital tract aging in HIV: estradiol clinical trial
1. Background
HIV is associated with accelerated and accentuated aging as evidenced by earlier onset 
and increased prevalence of several age-related conditions including cardiovascular, 
neurocognitive and bone disease1-4. Chronic HIV infection and aging may be linked by a common 
mechanism: inflammation5, which is fueled by ongoing low-level viral replication, microbial 
translocation, immunosenescence, immune activation and ART use6. However, the link between 
HIV infection and genital tract aging in women is understudied and has important clinical 
implications. The phenotype of female genital tract (FGT) aging is characterized by vaginal 
dysbiosis (increase in diverse anaerobes and decrease in acid-producing lactobacilli) and 
mucosal inflammation. These changes are associated with an increased risk for urinary tract and 
sexually transmitted infections (STI), vaginal atrophy, HIV acquisition and shedding7,8,9. Among 
incident US HIV infections in 2015, 17% occurred in persons >50 years of age. Approximately 
45% of people living with HIV in the US are aged ≥5010,11, and by 2030 an estimated 70% will be 
over age 50, underscoring the importance of studying the effects of aging on the FGT and genital 
health as well as HIV acquisition and shedding. 
This proposal will test the hypothesis that HIV is associated with earlier onset and more 
pronounced changes in the FGT characterized by vaginal dysbiosis and mucosal inflammation. I 
propose a cross-sectional study to compare a phenotype of FGT aging in HIV-infected (HIV+) and 
HIV-uninfected (HIV-) menopausal women aged 45-70 years and predict that younger HIV+ 
menopausal women (45-55 years) will have a phenotype similar to older (56-70 years) HIV- 
menopausal women. I will also explore whether topical estradiol therapy in HIV+ women with 
vaginal atrophy will improve symptoms and the age-related phenotype of dysbiosis and 
inflammation. Results of these studies have important clinical implications and could lead to 
recommendations for earlier and/or more aggressive treatment of vaginal atrophy in HIV+ women. 
Menopause is associated with vaginal dysbiosis, inflammation, and atrophy: hydrogen 
peroxide, lactic acid-producing lactobacilli (L. crispatus, L.  jensenii and L. gasseri) are protective 
by maintaining an acidic vaginal pH <4.7 and producing antimicrobial molecules such as 
bacteriocins. In contrast, dysbiosis, defined as a decrease in protective lactobacilli and increase 
in diverse anaerobes, is associated with a greater risk for HIV and other infections. Among the 
clinical conditions associated with dysbiosis are the estrogen deficient states of menopause and 
associated vaginal atrophy. These conditions are associated with increases in proinflammatory 
molecules and disruption of epithelial integrity12,13. A study comparing the vaginal microbiome of 
HIV- pre- and postmenopausal women showed that premenopausal women were more often in 
community state type (CST)-I (predominance of L. crispatus), whereas postmenopausal women 
were most often in CST-IV (dominated by diverse anaerobes). Limited data suggest that 
symptoms of vaginal atrophy may be at least partly mediated by vaginal dysbiosis. 
Postmenopausal participants with symptoms of vaginal atrophy had 25X greater odds of having 
CST-IV vs. CST-I13. Moreover, postmenopausal women reporting moderate to severe dryness 
had more Prevotella and other anaerobes, whereas women with little or no dryness had a 
predominance of lactobacilli12. Further, women with atrophy and dryness had increased 
expression of proinflammatory genes and decreased expression of genes associated with 
epithelial integrity. The findings of dysbiosis and inflammation may be linked mechanistically as 
studies have shown that bacteria commonly found in CST-IV (e.g. Prevotella, Mobiluncus and 
Sneathia) induce higher levels of proinflammatory cytokines including interleukin (IL)-1, IL-1 
and IL-8 compared to L. crispatus (CST-I)14,15. Similar shifts in the vaginal microbiome and 
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concomitant inflammation are associated with increased risk of HIV shedding and transmission16-
20. However, there are no studies that specifically address changes in the FGT in postmenopausal 
HIV+ women. I propose to address this knowledge gap and to study whether HIV+ menopausal 
women develop an earlier and greater degree of dysbiosis and mucosal inflammation compared 
to HIV- menopausal women and to evaluate the impact of vaginal estradiol treatment in HIV+ 
women with vaginal atrophy.
Vaginal atrophy is common but undertreated especially in women living with HIV (WLWH). 
Vaginal atrophy affects up to 45% of menopausal women21 and presents with symptoms of vaginal 
dryness and  dyspareunia, as well as urinary symptoms. Although the systemic vasomotor 
symptoms of menopause (e.g. hot flashes) often improve with time, atrophy symptoms persist. 
Intravaginal estrogen is effective for the treatment of vaginal atrophy with a favorable safety 
profile22-25,26-29 compared to systemic preparations. However, it is estimated that only 25% of 
symptomatic women seek medical attention21 and even in these women it is undertreated. With 
the increased availability of antiretroviral therapy (ART) and associated aging of the HIV 
population30,31, large numbers of WLWH will enter menopause, placing them at risk for vaginal 
atrophy. Topical estrogen therapy has not been studied in WLWH, despite the findings that they 
experience and report menopausal symptoms at an earlier age than HIV- women32-35. Moreover, 
severe menopausal symptoms have been associated with reduced adherence to ART placing 
menopausal WLWH at risk for possible drug resistance and adverse HIV related health 
outcomes36. Low dose intravaginal estrogen (Vagifem 10 g) is safe, effective and FDA approved 
for the treatment of symptomatic vaginal atrophy. Because of low systemic absorption (3-11 
pg/ml)37,38, there is little risk of complications nor need for progesterone supplementation22,23,39. 
The boxed warning for risk for endometrial cancer and cardiovascular disease (CVD) is based on 
extrapolations of data from clinical trials of systemic hormonal therapy, with substantially higher 
levels of exposure. There is no randomized trial or consistent observational evidence linking low-
dose vaginal estrogen to cancer, CVD, dementia, or thromboembolism. A Working Group on 
Women’s Health and Well-Being in Menopause has proposed to the FDA a labeling change40. 
2. Innovation
This is the first study specifically designed to test an intervention to restore local 
estrogen in HIV+ menopausal women with the goal of improving symptoms of atrophy, 
promoting an optimal vaginal microbiome and reducing GT inflammation. Previous studies 
have demonstrated a reduction in symptoms of vaginal atrophy and increased lactobacilli after 
oral estrogen replacement in HIV- menopausal women41. However, the effect of topical estradiol 
on the vaginal microbiome has not been studied nor has local estradiol treatment been 
systematically assessed in HIV+ women with vaginal atrophy. We will fill this research gap in this 
pilot randomized clinical trial. In addition to assessing improvement in clinical symptoms, we will 
explore the effects of topical estradiol on the vaginal microbiome, markers of mucosal 
inflammation, and HIV shedding.
Despite numerous studies documenting the composition of the vaginal microbiome in 
various disease states, our understanding of the functional relationship between the vaginal 
microbiome and mucosal health is limited. In order to fill this knowledge gap, we will evaluate the 
vaginal microbiome with Illumina MiSEq and quantitative PCR for select bacteria, and also 
propose to apply a more novel technique of Ig-SEQ, which combines flow cytometry-based 
bacterial cell sorting and 16S sequencing to identify subpopulations of IgA and/or IgG 
coated bacteria, with the goal of understanding the link between bacterial species, 
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mucosal inflammation and genital tract aging. We hypothesize that functional consequences 
of differences in the microbiome may link to immunoglobulin (Ig) coating.
3. Approach
We propose to test the hypothesis that menopausal women living with HIV (WLWH) develop 
genital tract aging earlier than HIV- menopausal women and that the magnitude of changes in 
microbiome and mucosal inflammation will be greater in HIV+ versus HIV- women. We will also 
test whether vaginal estradiol treatment in menopausal WLWH with vaginal atrophy will result in 
improvement of symptoms and an increase in Ig-coated, lactobacillus-dominant vaginal 
microbiota and reduction in mucosal inflammation. If the hypothesis is supported by this pilot 
interventional study, results would provide the foundation for a future study to evaluate the efficacy 
of earlier use of topical estradiol and generate new testable hypotheses to link aging, the vaginal 
microbiome and host mucosal immunity to HIV care. 
4. Objectives and Study Design
A. To determine if HIV is associated with earlier and more significant age-associated 
changes in FGT by comparing the vaginal microbiome and mucosal inflammation in 
menopausal WLWH and HIV- menopausal women.
B. To conduct a pilot randomized clinical trial in menopausal WLWH with symptomatic 
vaginal atrophy to evaluate the impact of vaginal estradiol treatment on symptoms and 
biomarkers of FGT aging.
We propose a study nested in the Bronx Women’s Interagency HIV Study (WIHS) to compare a 
phenotype of FGT aging between menopausal WLWH and HIV- women. We will use the following 
biomarkers of aging: lower levels of lactobacilli (primary outcome), increased dysbiosis, less Ig 
bacterial coating, and higher levels of mucosal inflammatory mediators. We will also assess 
bacterial and host cell gene expression using metatranscriptomics (vaginal swabs) and RNAseq 
(vaginal biopsies). These exploratory “omic” studies may provide insights into the functional 
significance of observed differences in the composition and Ig coating of vaginal microbiota and 
their link to vaginal health.
In the first part of the study (Aim 1), 50 HIV+ and 50 HIV- participants aged 45-70 will have 2 
study visits one month apart to assess reproducibility of findings. Participants will be frequency 
matched by age, race and ethnicity into 4 groups: (i) HIV+ aged 45-55; (ii) HIV+ aged 56-70; (iii) 
HIV- aged 45-55; and (iv) HIV- aged 56-70 years. We have chosen to dichotomize into these age 
groups based on differences in the taxonomic composition of the microbiome in a recently 
published study42 and potential eligible participants in WIHS. Figure 1 illustrates the overall study 
design. Sampling at both study visits will include vaginal swabs for pH, microbiome assessment 
and evaluation for vaginal infections (candida, bacterial vaginosis [BV], trichomonas), 
cervicovaginal lavage (CVL) for soluble immune mediators, glycogen, and HIV CVL RNA 
(shedding), blood for HIV RNA (plasma viral load), CD4 count, estradiol and follicular stimulating 
hormone (FSH), and vaginal biopsies for exploratory RNAseq (Visit 2 only).
At Visit 2, 50 HIV+ participants with vaginal atrophy will be offered immediate enrollment into a 
1:1 randomized trial of vaginal estradiol (n=25) versus no treatment (n=25) to assess whether 
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estradiol will improve vaginal symptoms and modify the genital tract aging phenotype. HIV+ 
women who agree to participate in the trial will have 3 additional study visits: randomization (Visit 
3), 6 weeks after randomization (Visit 4) and 12 weeks after randomization (Visit 5). HIV- women 
can only participate in Aim 1 and will have 2 study visits. HIV+ women can elect to participate in 
Aim 1 and/or Aim 2 (2 study visits if only Aim 1 is completed; 4 study visits if only Aim 2 is 
completed; 5 visits if both Aims 1 and 2 are completed).     
Inclusion criteria for Aim 1 (first 2 study visits):
-
HIV infection or HIV negative
-
Females aged 45-70
-
Menopause defined by having no menstrual periods for 12 consecutive months, 
confirmed with serum follicle-stimulating hormone (FSH) level >40 IU/ml and serum 
estradiol level <20 pg/ml
In the second part of the study (Aim 2), we propose a pilot randomized clinical trial nested in the 
Bronx WIHS in 50 menopausal WLWH with symptomatic vaginal atrophy to assess the impact of 
12 weeks of a topical low-dose estradiol vaginal tablet (Vagifem® 10 μg) on symptoms of atrophy 
and measures of the vaginal microbiome. Participants will be randomized 1:1 to estradiol (n=25) 
or no therapy (n=25) and will be followed for 12 weeks. The dosing of Vagifem® will be 1 tablet 
inserted using a plastic applicator (similar to a tampon) in the vagina daily for 2 weeks followed 
by 1 tablet inserted twice weekly (i.e., Tuesday and Friday). If we find benefit among women 
assigned to the estradiol arm, participants randomized to the no therapy arm will be offered 
vaginal estradiol treatment at the end of the study by their primary care doctor. If participants 
assigned to Vagifem are symptomatic beyond the 12-week study period and wish to continue 
Vagifem®, it can be prescribed by their primary care physician. Women will have 3 visits before 
(Visits 1, 2 and 3) and 2 visits following Vagifem® treatment (Visits 4 and 5). Adherence to 
Vagifem will be assessed with the use of participant diaries of Vagifem use and counting of used 
Vagifem applicators. 
Inclusion criteria for Aim 2 (randomized clinical trial for HIV+ participants only):
-
HIV infection
-
Females aged 45-70
-
Menopause defined by having no menstrual periods for 12 consecutive months, 
confirmed with serum follicle-stimulating hormone (FSH) level >40 IU/ml and serum 
estradiol level <20 pg/ml
-
Symptomatic vaginal atrophy defined as reporting at least once per week in the past 
30 days, 1 or more of the following symptoms of moderate or severe intensity:
-Dryness 
-Itching 
-Irritation 
-Soreness or pain
OR
-Pain associated with sexual activity at least once
-
Evidence of atrophy on exam, including thin, pale and dry vaginal and vulvar 
surfaces
-
Agrees not to use vaginal products other than Vagifem during the clinical trial
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Exclusion criteria for Aims 1 & 2:
-
Current unexplained or unevaluated abnormal genital bleeding
-
Current or suspected pregnancy
-
If < age 55, hysterectomy and has at least one ovary
-
Pelvic or vaginal surgery in the 60 days prior to enrollment
-
Use of systemic reproductive hormones in the 2 months prior to study enrollment
-
Antibiotic use in the past 30 days
-
Use of immunosuppressive medications in the prior 60 days including biologics, 
chemotherapeutics or post-transplant immunosuppressive medications.
-
Use of any vaginal or vulvar preparations 1 month prior to enrollment
-
Current acute vaginal infection (diagnosed by wet mount at Visit 1 or 2)
-
Any serious disease or chronic condition that might interfere with study compliance
-
Participants who are unwilling to agree to the provisions of the protocol
Additional Exclusion Criteria for Aim 2 only:
-
Current or previous history of breast cancer or estrogen dependent neoplasia
-
Known current or past thromboembolic disease (deep vein thrombosis or pulmonary 
embolism, not including thrombophlebitis), myocardial infarction or stroke
-
Known blood clotting disorders including Protein C, Protein S and antithrombin 
deficiency, Factor V Leiden or prothrombin mutations
-
Known severe liver disease including cirrhosis or active Hepatitis B
-
History of adverse reaction to vaginal estradiol
5. Recruitment
The study is nested in Bronx WIHS, which is an NIH-funded prospective observational cohort 
study of HIV+ and at-risk HIV- women. We have identified 90 HIV- and 248 HIV+ Bronx and 
Brooklyn WIHS participants who are potentially eligible for this study. Potential participants will be 
invited to discuss this study at semiannual Core WIHS visits. We will recruit participants from the 
Bronx and Brooklyn WIHS for this study. Bronx WIHS staff will call potentially eligible participants 
and schedule them to come in to the Clinical Research Center at the Albert Einstein College of 
Medicine or the  Bronx WIHS site for a screening visit. As we have done for prior studies, Brooklyn 
WIHS participants who are interested in participating in the study, will be contacted by the Bronx 
WIHS staff and seen at the Bronx WIHS site for all visits. Additional participants may be recruited 
HIV+ Vaginal Atrophy 
Randomization (n=50)
Estradiol
(n=25)
6 
weeks
12 
weeks
No Treatment
(n=25)
6 
weeks
12 weeks
• Aged 45-55 (n=25)
• Aged 56-70 (n=25)
HIV+
(n=50)
• Aged 45-55 (n=25)
• Aged 56-70 (n=25)
HIV-
(n=50)
Groups will be frequency 
matched by age, race, ethnicity
Groups will be frequency 
matched by age, race, ethnicity
Visit 1
Visit 2
Visit 3
Visit 4
Visit 5
                       Aim 2  
           Aim 1
Figure 1. Study Design
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from Montefiore Medical center, and may be identified using the Center for AIDS Research 
Clinical Database.
6. Study procedures
Study visits will occur at the Einstein Clinical Research Center or the Bronx WIHS clinical study 
site. The study clinicians or study coordinator will screen participants for eligibility, discuss the 
study, answer any questions, and obtain written informed consent. At each scheduled research 
visit, a pelvic examination will be performed for the collection of the following specimens: vaginal 
swabs for pH, and microbiome studies; CVL; vaginal swabs to assess for infection (Visits 1 and 
2 only) and vaginal biopsies and a vaginal scraping (VMI) to assess for vaginal aging at (Visits 2 
and 5 only) (Table 1). If candida, BV, or trichomonas is diagnosed, participants will be notified 
and referred to their primary doctor for treatment prior to enrollment in the study. After 
treatment, participants may return for repeat testing and if the infection has resolved, they may 
be enrolled in the study.  
At Visit 2 blood will be collected for estradiol and follicular stimulating hormone (FSH) levels, 
aging biomarkers and future studies and for HIV positive participants, CD4 and HIV-1 RNA. At 
Visit 5, blood will be collected for aging biomarkers and future studies. Participants will be asked 
at Visits 2-5 to complete a questionnaire about the presence and severity of the most 
bothersome symptom of atrophy, defined as vulvovaginal itching, pain, dryness, irritation or pain 
with penetration (dyspareunia)43. 
Participants will rate the severity of symptoms based on a four-point scale (0=none, 1=mild, 
2=moderate, 3=severe). Presence or absence of atrophy will also be assessed via clinical exam 
at Visits 2-5 and at Visits 2 and 5 with the Vaginal Maturation Index (VMI) which is done by 
lightly scraping the upper 1/3rd of the vaginal wall with a saline dipped vaginal spatula to 
examine cells under the microscope to assess for vaginal aging43. Following placement of a 
speculum into the vaginal vault, swabs will be collected from the lateral vaginal walls. CVL is 
collected using a small syringe to place 10 cc of normal saline in the vaginal area. The fluid is 
immediately removed and sent to the Herold lab for storage and analysis. Vaginal biopsies will 
be collected at Visits 2 and 5 only. Women will be counseled to abstain from vaginal product use 
and sexual intercourse for one week after biopsy collection to allow adequate time for healing. In 
our experience, re-epithelialization occurs within one to two days, and the biopsy sites are 
completely healed within seven days. Adult women who decline or state that they are unlikely to 
abstain from sexual activity for one week after biopsy visits will not undergo biopsy collection. 
Tissue samples are typically ~3 x 5 mm. Biopsies are obtained with a Tischler or other biopsy 
forceps. All participants will receive a follow-up phone call the day after the vaginal biopsies 
from the study clinician to ensure they are feeling well. The specimens will be obtained strictly 
for research purposes and used only for the specific objectives outlined in the study. At all 
scheduled return visits or at any visit initiated by the participant due to genitourinary symptoms, 
an appropriate examination for the evaluation of specific symptoms and adverse events will be 
performed (for example, performance of a pelvic examination in women with complaints of 
abnormal vaginal discharge or bleeding). If any examination findings warrant further diagnostic 
evaluation for clinical management, participants will be referred to their medical provider for 
appropriate diagnostic studies and treatment.
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7. Potential Risks:
There is a small risk of bruising, blood clots or infection from blood draws. Sometimes the area 
of needle insertion becomes sore or red. A woman may experience mild discomfort at the time 
of speculum placement and sample collection. There is a very small risk of injury to the lining of 
the vagina from the speculum or from the vaginal scraping. There is no discomfort or risk to 
placement or removal of sterile saline into the vaginal area. There is no risk to CVL or vaginal 
swab collection. Women may experience a sharp pinch or have pain when vaginal biopsies are 
performed. In order to minimize the pain and discomfort of the biopsy, we will apply a topical 
numbing medication (benzocaine gel) to the tissue. There may be stinging, burning, redness, or 
itching associated with local anesthesia (topical benzocaine gel) which is rare. Biopsies may 
cause light bleeding, Rarely, a biopsy can cause infection and/or prolonged bleeding. 
Sometimes a medication is needed to stop the bleeding, which would be applied directly to the 
Aim 1
Study of Menopausal HIV- and 
WLWH
Aim 2
Pilot Randomized Clinical Trial of 
Topical Estradiol in Menopausal WLWH
Visit 1
Screening
Visit 2
2nd visit and/or 
Screening for Trial
Visit 3
Randomized
Estradiol vs. 
No therapy
Visit 4
6 weeks 
Estradiol vs. 
No therapy
Visit 5 
12 weeks 
Estradiol vs. No 
therapy
Safety Call
2, 8, 13 weeks
Eligibility/Informed consent
X
X
X
Randomization to Estradiol vs. No 
treatment
X
WIHS database extraction
Demographics, CD4 count, plasma HIV-
1 RNA, ART regimen and reported 
adherence, etc.
X
X
Vaginal atrophy symptom 
questionnaire
X
X
X
X
X
Adverse event assessment
X
X
X
X
X
X
Vaginal swabs (n=4-5 per visit)
pH, microbiome studies
X
X
X
X
X
Cervicovaginal lavage (CVL) 
supernatant
Immune mediators, HIV-1 RNA, RSID, 
saline/KOH, glycogen
X
X
X
X
X
CVL cell pellet
Nugent score, additional microbiome 
di
X
X
X
X
X
Wet mount (candida, trichomonas, 
bacterial vaginosis)
X
X
Vaginal biopsies (2)
RNA seq, future immunohistochemistry 
studies
X
X
Blood collection
Estradiol/FSH levels, HIV-1 RNA, CD4,  
plasma for future aging biomarkers
X
X
Clinical Exam to assess for Vaginal 
Atrophy (VMI)
X
X
Table 1. Timeline of Study Visits, Procedures, & Sample Collection
ART=antiretroviral therapy; RSID=rapid semen identification; FSH=follicle stimulating hormone; VMI=vaginal maturation index
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site. This medication can cause a temporary dark-colored discharge from the vagina. In rare 
cases a stitch might be needed or a machine (cautery) may be used to stop the bleeding. 
Potential risks of vaginal estrogen tablets (Vagifem® 10 μg) include back pain, diarrhea, 
vulvovaginal mycotic infection, and vulvovaginal pruritus (treatment emergent adverse reactions 
reported at a frequency of ≥5 percent in a 12-month randomized, double-blind, placebo-
controlled study in which 309 postmenopausal women received either placebo or Vagifem® 10 
μg). Vagifem applicator may cause vaginal abrasion. The Division of AIDS (DAIDS) Female 
Genital Grading Table for Use in Microbicide Studies will be the primary tool for grading adverse 
events available at https://rsc.niaid.nih.gov/sites/default/files/addendum-1-female-genital-
grading-table-v1-nov-2007.pdf. AEs will be reported to the IRB. 
Women who use systemic forms of unopposed estradiol who have an intact uterus may have an 
increased risk of endometrial hyperplasia, which in some women may be a precursor to 
endometrial cancer after prolonged use. Women in this study will be using a low-dose topical 
estradiol vaginal tablet, which does not increase serum levels of estradiol beyond the baseline 
menopausal levels and has been proven to be safe for the treatment of vaginal atrophy without 
adverse effects on the endometrium, including endometrial hyperplasia. Further, low-dose vaginal 
estrogen has not been shown to increase risk for coronary heart disease, stroke, venous 
thromboembolism or dementia. The proposed study will test the effect of 6 and 12 weeks of 
vaginal estrogen on clinical symptoms, the vaginal microbiome, and soluble markers of 
inflammation. Participants will be followed closely by the study team and will be instructed to 
report immediately any vaginal bleeding or spotting while using Vagifem®. Women may remain 
symptomatic beyond the 3-month study period and may elect to continue Vagifem® with close 
medical follow-up by their primary clinician. If benefit is determined in the estradiol arm of the 
study, participants randomized to the no treatment group, will be offered Vagifem® by their 
primary care physicians. The study team will contact participants’ medical provider if the decision 
is made to continue treatment or initiate Vagifem®.
8. Adequacy of Protection Against Risks:
The consent will contain the following: investigators’ statement identifying the study; purpose 
and benefits; procedures; risk, benefits, stress, and discomfort associated with the study; 
request for permission for future use of samples (as a separate checkbox, so that participants 
may be in the study and not agree to storage of samples for future use) and other information 
deemed necessary. The consent will also identify the investigators and provide contact 
information. The participant must verbally assent that they understand the nature of the study, 
its inherent risks and benefits, other treatment alternatives, their right to terminate participation 
in the study without affecting their health care or involvement in the core WIHS cohort study, 
and that they have freely given informed consent to participate in the study. A signed copy of 
the informed consent form will be given to the participant. The participants are informed that all 
medical records are kept confidential. Participants will be compensated for time and travel. 
Participants will receive a total of $125 ($50 for Visit 1 and $75 for Visit 2) if they complete only 
the first part of the study (Aim 1, 2 visits). Participants with HIV who complete both parts of the 
study (Aims 1 and 2, 5 visits), will receive a total of $300 ($50 at Visits 1, 3 and 4 and $75 at 
Visits 2 and 5). HIV+ participants who enroll only in the second part of the study (Aim 2, 
randomized trial) will receive a total of $250 ($50 at Visits 3 and 4 and $75 at Visits 2 and 5).
All specimens and study forms are labeled with a unique study identifier that has no relationship 
to participant’s identifying information (such as name, initials, or date of birth). All records and 
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data are kept in locked offices and a secure computer database. The Bronx site of the WIHS has 
a long history of participation in many sub-studies and the staff is very familiar with the need to 
maintain participant confidentiality and security of study data. Samples will be stored in Dr. Betsy 
Herold’s lab and will not be destroyed at the end of the study, unless participants do not agree to 
use of remaining samples for related studies. A secure, web-based application, REDCap 
(Research Electronic Data Capture), will be used to collect and store data from questionnaires 
obtained in this study. REDCap is designed exclusively to support data capture for research 
studies. REDCap servers are housed in a data center at Einstein and all web-based information 
transmission is encrypted. REDCap was developed specifically around HIPAA-security guidelines 
and it includes full audit trail.
Participants will receive telephone calls at 2 and 8 weeks after initiation of estradiol and 1 week 
after completion of therapy to assess for safety. Participants will also receive telephone calls a 
day after vaginal biopsies are performed. If symptoms are reported, participants will be 
instructed to return to the study site for evaluation. Participants will receive local anesthesia with 
topical benzocaine gel prior to each biopsy procedure and will be advised that they may take a 
mild pain reliever (Tylenol) before and after each biopsy procedure. Women who have any 
history of estrogen responsive cancer including breast cancer, endometrial cancer or vaginal 
bleeding will be excluded from this study to minimize risk for adverse events. In the event of an 
injury resulting from participation in this study, participants will be instructed to seek immediate 
appropriate medical care and inform the study doctor. In the event of an emergency they will be 
instructed to go to the emergency room. Participants will be provided with a 24-hour telephone 
number to call to contact a study clinician at any time. Appropriate blood and secretion 
precautions will be employed by all personnel in the drawing of blood and handling of all 
specimens, as currently recommended by the US Centers for Disease Control and Prevention 
(CDC). Universal precautions will be observed at all times.
9. Potential Benefits:
Women with HIV infection experience menopausal symptoms at an earlier age and are more 
likely to report symptoms compared to women who do not have HIV. Participants may benefit 
from study participation because the use of low dose topical estradiol, which has been FDA 
approved since 2009 for the treatment of vaginal atrophy in menopausal women, may relieve 
symptoms of vaginal dryness, itching, soreness, pain and urinary symptoms in women with HIV. 
Participants and others may benefit in the future from information learned from this study. 
Specifically, if we find that HIV accelerates genital tract aging, this may prompt 
recommendations for earlier treatment of vaginal atrophy in menopausal women with HIV.
10. Data analysis and sample size:
Aim 1: The primary exposure variables are HIV infection and age. The primary outcome is the 
vaginal microbiome, specifically the relative abundance and quantities of lactobacillus species as 
measured by llumina MiSeq and qPCR respectively. Secondary outcomes are relative abundance 
and quantities of specific BV associated bacteria and quantities of CVL cytokine/chemokines. 
Exploratory outcomes are quantities and composition of Ig coated bacteria and levels of HIV-1 in 
the genital tract. Multivariate linear regression will be applied for our analysis. General form of the 
linear regression model will be: yi = μ+ βagei + HIVi + agei × HIVi + 1Covariatesi+…+ ej, in which 
yi is the outcome variables subject to necessary transformation as necessary. Age will be 
analyzed as a continuous variable to examine the slope or rate of change in lactobacillus and as 
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a dichotomous variable (e.g. age 45-55 vs. 56-70) to examine the magnitude of difference 
between younger and older HIV+ and HIV- menopausal participants. We have chosen the age 
cut offs above given the significant differences observed in the taxonomic composition of the 
microbiome in pilot studies when comparing HIV+ menopausal participants in these age ranges 
and the distribution of eligible participants in WIHS. We will adjust the regression analysis for 
important covariates including years since menopause, time since HIV diagnosis, HIV plasma 
viral load, CD4 count, ART use, and HSV-2 serostatus. Both raw p values and the false discovery 
rate (FDR) according to Benjamini and Hochberg will be examined. To limit the false positive rate 
in our analysis of secondary outcomes, an arbitrary cutoff of 0.05 for FDR will used.  
Power Considerations: We estimated the power of the proposed study using software PASS 
(Power and Sample Size Analysis, version 11). Given the sample size of 50 HIV- and 50 HIV+ 
menopausal participants, we will have >80% power for the minimal detectable difference of 0.6SD 
and 0.9SD between HIV (or age) group with the significance level of 0.05 and 0.0005 (a 
conservative Bonferroni correction for 100 independent tests). Data from preliminary studies in 
our lab investigating the impact of menopause and HIV status on the vaginal microbiome indicated 
51 HIV+ menopausal participants had a significantly lower relative abundance of lactobacillus 
species of 7.3% compared to 23% for 19 HIV- menopausal participants, equivalent to about 0.8SD 
change. Further when comparing upper and lower age ranges of HIV+ menopausal participants, 
HIV+ participants age 56-70 (n=28) had significantly lower relative proportions of L. iners 7.4% 
compared to 44.7% in HIV+ menopausal participants aged 47-55.9 (n=23), equivalent to about 
1.2SD change. Based on the preliminary data, we are confident that this study will have excellent 
power to detect effects of age and HIV.
Aim 2: The primary outcomes are clinical symptoms measured by most bothersome symptom of 
vaginal atrophy and changes in the vaginal microbiome, specifically the relative abundance of 
protective lactobacillus species L. crispatus as measured by llumina MiSeq and quantities of 
protective lactobacilli species (L. crispatus, L. jensenii and L. gasseri) as measured by qPCR 
between baseline and 6 and 12 weeks within individual participants and between participants 
randomized to estradiol vs. no drug at baseline, 6 and 12 weeks. The 12-week time frame was 
chosen as this was the endpoint utilized in several clinical trials demonstrating Vagifem efficacy 
and is recommended in FDA guidelines22,43. The secondary outcomes are changes relative 
abundance and quantities of specific BV associated bacteria and levels CVL cytokine/chemokines 
between baseline, 6 and 12 weeks. Exploratory outcomes are the quantities and composition of 
Ig coated bacteria and levels of HIV-1 in the genital tract. Spearman’s correlation coefficients 
(SCC) will be calculated to evaluate associations between pairs of quantitative outcome variables 
and covariates, such as levels of protective lactobacilli species and MBS of atrophy, quantity of 
Ig coated bacteria, inflammatory mediators, CVL HIV-1 RNA, levels of estradiol in the blood. 
Linear mixed effects models or generalized linear mixed models will be used to examine effects 
of time and estradiol use on outcomes (bacterial communities, vaginal bacterial quantities, 
symptoms) after adjustment for factors such as ART, duration of HIV, plasma viral load, and CD4+ 
T cell count. We will also address the variability of the microbiome and inflammatory mediators 
over time in the absence of an intervention by comparing these measures at the screening (Visit 
2) and enrollment visits (Visit 3).
Power Considerations: We have increased our sample size and added randomization for this 
clinical trial based on feedback from the NIH reviewers. If we assume an attrition rate of 15%, a 
sample of 50 HIV+ women randomized to estradiol (Vagifem) vs. no treatment will have >80% 
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power for the minimal detectable difference in lactobacilli of 0.85 SD and 1.4 SD with the 
significance level of 0.05 and 0.0005 (a conservative Bonferroni correction for 100 independent 
tests), respectively, at 12 weeks post treatment. Although data is lacking in HIV+ women treated 
with Vagifem, a previous study with a smaller sample size (30 HIV-) investigating the use of low 
dose (0.3mg) oral conjugated estrogens for treatment of vaginal atrophy in demonstrated a 
significant increase in the relative abundance of Lactobacillus from 11.2% to 71% (p<0.0001) 
comparing baseline to 4 weeks estrogen treatment. Further the vaginal symptom score decreased 
from 3.93 vs. 1.00 (p<0.0001) at the same time points41, suggesting this proposed study may 
have adequate power for detecting a realistic difference of candidate Lactobacillus and symptom 
score. It should be noted that this is a pilot study and as such we acknowledge that it may be 
underpowered for testing interaction effects of time and estradiol use. However, the goal of the 
study is to determine an effect size of topical estradiol in planning for a future large-scale 
randomized controlled trial.
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