Examining the Impact of Tampon Use on the Vaginal Microbiota
Phase: PHASE4
Conditions: Vaginal Microbiota
Sponsor: University of Michigan

 
 
 
 
 
 
 
 
 
Examining the Impact of Tampon Use on the Vaginal Microbiota 
 
NCT03346759 
 
March 29, 2018 
 

Tampon Study Proposal 
 
Abstract 
 
To date, a great deal of work has been done to characterize the composition of the vaginal 
microbiota (VMB) in women of reproductive age. While several different community types have 
been identified, it is widely accepted that communities dominated by Lactobacillus spp confer 
colonization resistance, and thus protect against bacterial vaginosis (BV), STI, and other 
urogenital infectious diseases [Hickey 2012]. Conversely, mixed communities that often include 
Gardnerella vaginalis, Prevotella spp., Mycoplasma spp., and/or Gram-positive cocci and 
relatively few Lactobacillus spp are considered unhealthy states and may contribute to BV and 
various sequelae including infertility, endometriosis, pelvic inflammatory disease, and increased 
risk of STI [Hickey 2012]. Considering both the relationship between VMB composition and 
reproductive health, and the high prevalence of tampon use, which, for many women, occurs at 
regular intervals for the duration of their reproductive life, it is surprising how few studies have 
examined the effect of tampon use on VMB composition. Moreover, the majority of these studies 
are limited by the use of culture-dependent methods focused on the presence/absence of a small 
number of species (Lactobacillus spp, Staphylococcus aureus, E. coli, group B Streptococcus, 
and/or Gardnerella vaginalis) [Onderdonk 1986, Onderdonk 1987, Chow 1989, Shehin 2003, 
Chase 2007, Chase 2010, Hochwalt 2010]. The one study to date that has used culture-
independent VMB community analysis in response to tampon use was limited by its sample size 
of 7 women. Further, the investigators only collected vaginal swabs at two points during each 
menstrual cycle [Hickey 2013]. The conflicting findings of these studies make this an even more 
compelling research question. In order to address this striking knowledge gap, we propose a 
longitudinal, interventional, case-crossover study design to examine whether expanded tampon 
shape affects VMB composition during, and in between menses. We will recruit 20 women from 
the University of Michigan, Ann Arbor to use Tampax® Pearl® Regular Tampons (expand into 
a rectangular prism-like shape) and Playtex® Gentle Glide® 360°® Regular Tampons (expand 
into a conical shape) for one menstrual cycle each, in a randomly assigned order. We will obtain 
twice-weekly self-collected vaginal swabs for a 2-3 week baseline window before the first 
menstrual cycle with an assigned tampon, followed by the two menstrual cycles with assigned 
tampon use, and finishing with one menstrual cycle with menstrual product use according to 
each subject’s normal habits. DNA will be isolated from the vaginal swabs, and the bacterial 16S 
rRNA encoding genes will be amplified by PCR. Sequencing will be performed using the MiSeq 
Illumina platform, and analyzed to answer the above questions. We hypothesize that VMB 
composition will vary between menses using the Tampax® Pearl® Regular Tampons and 
Playtex® Gentle Glide® 360°® Regular Tampons, and that persistence of these VMB changes 
will vary based on baseline VMB community type. 
 
Background 
 
To date, a great deal of work has been done to characterize the composition of the vaginal 
microbiota (VMB) in women of reproductive age. While many bacterial genuses have been 
identified in VMB samples, it is widely accepted that communities dominated by Lactobacillus 
spp promote a healthy vaginal environment and good reproductive health [reviewed in Hickey 
2012]. The primary function of Lactobacilli in promoting vaginal health is the provision of 

colonization resistance to the vaginal epithelium [Borovkova 2011, Lopes 2011, Borges 2014, 
reviewed in Hickey 2012]. Through the production of lactic acid, Lactobacilli create an acidic 
environment, which prevents the proliferation of nonindigenous organisms, including pathogens 
and those associated with BV [Cook 1990, Boskey 1999, Aroutcheva 2001, Valore 2002, Atassi 
2006, Kaewsrichan 2006, O’Hanlon 2010, Borges 2014, reviewed in Hickey 2012]. However, it 
has been noted in multiple studies that a low abundance of Lactobacilli does not necessarily 
translate to poor vaginal health [Gajer 2012, Martin 2012]. This has lead to the recent hypothesis 
that it is not the presence of Lactobacilli in abundance that promotes vaginal health, but instead it 
is the capacity of the VMB to produce lactic acid, which can be accomplished by several taxa 
normally isolated from the VMB including Atopobium, Streptococcus, Staphylococcus, 
Megasphaera, and Leptotrichia [Hickey 2012]. Despite the fact that diverse VMB that are not 
dominated by Lactobacilli have been found in asymptomatic women with good vaginal health, 
this is not the norm. It is widely accepted that mixed VMB including Gardnerella vaginalis, 
Prevotella spp., Mycoplasma spp., and/or Gram-positive cocci and relatively few Lactobacilli 
are considered unhealthy states and are often present in women with BV [Spiegel 1980, Amsel 
1983, both Spiegel 1983, Hill 1993, Falagas 2007, Cribby 2008, Lopes 2011, Hickey 2012]. BV 
imposes a large burden on the healthcare system both directly and indirectly. Directly, it is the 
most common vaginal disorder of reproductive age women and results in millions of healthcare 
visits each year in the US alone [Sobel 2005, Koumans 2007]. Indirectly, BV is associated with a 
great number of serious sequelae including, but not limited to, infertility [Sweet 1995], 
endometritis [Haggerty 2004], and pelvic inflammatory disease [Wiesenfeld 2002]; as well as an 
increased risk of acquiring HIV and other STIs [Hillier 1998, Taha 1998, Martin 1999, Schmid 
2000, Wiesenfeld 2003].  
 
A considerable volume of work has also been conducted to characterize the fluctuations of the 
VMB across the menstrual cycle. While some studies (both culture-dependent and culture-
independent) have reported no change in community composition across the menstrual cycle 
[Sautter 1980, Wilks 1987, Gajer 2012, Hickey 2013], others have reported varying relative 
abundances of aerobic and anaerobic bacteria across the cycle [Bartlett 1977] or a greater 
proportion of non-Lactobacillus species during menses [Priestley 1997, Eschenbach 2000, 
Srinivasan 2010, Lopes 2011, Gajer 2012, Hickey 2013]. In light of these findings, in particular 
those of Gajer et al. (2012) and Hickey et al. (2013), it is currently thought that the degree and 
type of VMB fluctuation seen across the menstrual cycle varies between women [Lopes 2011, 
Chaban 2014]. Further, the increased abundance of non-Lactobacilli species seen during menses 
is of particular interest given the changes to vaginal innate immune cell presence [Hill 1992], 
temperature [Hill 2010], oxygen and carbon dioxide concentrations [Hill 2005], and volatile fatty 
acid concentrations [Preti 1975] that occur during menses. These changes alter the growth 
conditions present in the vagina, and as such, may promote the growth of certain bacterial 
species over others. Given that non-Lactobacilli species are often present in greater abundance 
during menses, it is possible that the conditions of the vagina during menses could promote their 
growth and potentially shift a Lactobacilli-dominated VMB to a diverse VMB and promote BV. 
Moreover, the changes to vaginal temperature, dissolved gas concentrations, and volatile fatty 
acid concentrations that occur during menses have been shown to be exacerbated by tampon use 
[Preti 1975, Hill 2005, Hill 2010], suggesting that tampon use may compound any VMB changes 
that occur during menses. 
 

Considering the well-established link between VMB composition and vaginal health, the VMB 
fluctuations seen over the course of the menstrual cycle, the changes to the vaginal environment 
that occur during menses (which are exacerbated by tampon use), and the high prevalence of 
tampon use (it is estimated that as many as 81% of reproductive age women in the US use 
tampons [Omar 1998]); surprisingly few studies have examined the effects of tampon use during 
menses on VMB composition. The majority of these studies are limited by the use of culture-
dependent methods focused on the presence/absence of a small number of species (Lactobacillus 
spp, Staphylococcus aureus, E. coli, group B Streptococcus, and/or Gardnerella vaginalis) 
[Onderdonk 1986, Onderdonk 1987, Chow 1989, Shehin 2003, Chase 2007, Chase 2010, 
Hochwalt 2010]. The one study to date that has used culture-independent VMB community 
analysis in response to tampon use was limited by its sample size of 7 women. Further, the 
investigators only collected vaginal swabs at two points during each menstrual cycle [Hickey 
2013]. Not only are these studies severely limited by their designs, but they have also failed to 
produce consistent findings on the impact of tampon use on VMB composition. This knowledge 
gap represents a major shortcoming of public health research to identify and describe the effect 
of a near-ubiquitous modifiable risk factor on vaginal health. Addressing this gap has the 
potential to improve individuals’ vaginal health through public health interventions, and decrease 
the societal burden of poor vaginal health, namely due to BV. 
 
We propose a longitudinal, interventional, case-crossover study design to examine whether 
expanded tampon shape affects VMB composition during, and in between menses. We will 
recruit 20 women from the University of Michigan, Ann Arbor to use Tampax® Pearl® Regular 
Tampons (expand into a rectangular prism-like shape) and Playtex® Gentle Glide® 360°® 
Regular Tampons (expand into a conical shape) for one menstrual cycle each, in a randomly 
assigned order. We will obtain twice-weekly self-collected vaginal swabs for a 2-3 week baseline 
window before the first menstrual cycle with an assigned tampon, followed by the two menstrual 
cycles with assigned tampon use, and finishing with one menstrual cycle with menstrual product 
use according to each subject’s normal habits. DNA will be isolated from the vaginal swabs, and 
the bacterial 16S rRNA encoding genes will be amplified by PCR. Sequencing will be performed 
using the MiSeq Illumina platform, and analyzed to answer the above questions. 
 
Outcomes and aims 
 
 Primary Outcome: Change in relative abundance of Lactobacillus species from: baseline 
to the end of first menstrual cycle, baseline to the end of second menstrual cycle, and 
baseline to the end of third menstrual cycle (approximately 14 weeks) 
 Secondary Outcome: Change in relative abundance of Gardnerella vaginalis from: 
baseline to the end of first menstrual cycle, baseline to the end of second menstrual cycle, 
and baseline to the end of third menstrual cycle (approximately 14 weeks) 
 Exploratory Outcomes 
 To characterize intra-woman, during-menses changes in VMB composition caused by 
(see Methods – Study Design and Methods – Whole Community Analysis & 
Statistics sections for notes on causal inference) changing the expanded tampon shape 
of tampon used. 
o Hypothesis 1: VMB composition will vary between menses with Tampax® 
Pearl® Regular Tampon use and Playtex® Gentle Glide® 360°® Regular 

Tampon use because the different expanded shapes of the two tampons will 
alter the vaginal environment differently and thus promote growth of different 
taxonomic groups. The primary structural difference between Tampax® 
Pearl® Regular Tampons and Playtex® Gentle Glide® 360°® Regular 
Tampons is that, once inserted into the vagina, Tampax® Pearl® Regular 
Tampons expand axially into a rectangular prism-like shape, while Playtex® 
Gentle Glide® 360°® Regular Tampons expand radially into a conical shape. 
The radial expansion of the Playtex® Gentle Glide® 360°® Regular Tampons 
likely results in increased surface area of the tampon that is in contact with the 
vaginal epithelium as compared to the Tampax® Pearl® Regular Tampons. 
This may allow for increased absorption of menses by the Playtex® Gentle 
Glide® 360°® Regular Tampons as compared to the Tampax® Pearl® 
Regular Tampons, resulting in differing growth conditions in the vagina 
during Playtex® Gentle Glide® 360°® Regular Tampon use and Tampax® 
Pearl® Regular Tampon use. 
 To characterize intra-woman, between-menses changes in VMB composition caused 
by (see Methods – Study Design and Methods – Whole Community Analysis & 
Statistics sections for notes on causal inference) changing the expanded shape of 
tampon used. 
o Hypothesis 2.1: Persistence of VMB changes observed during menses caused 
by changing the expanded shape of the tampon used will vary dependent on 
baseline VMB community type such that VMB changes will persist for 
greater time between menses in women with typically diverse VMB that 
confer reduced colonization resistance compared to a Lactobacilli dominated 
VMB [Borovkova 2011, Lopes 2011, Borges 2014, reviewed in Hickey 2012]. 
o Hypothesis 2.2: Within women with Lactobacilli dominated baseline VMB, 
persistence of VMB changes observed during menses caused by changing 
expanded tampon shape will vary based on the degree of reduction of 
Lactobacilli abundance during menses such that changes resulting in greater 
reductions of Lactobacilli abundance will persist for greater time between 
menses. 
 
Methods 
 
 
Recruitment 
 We will recruit 40 women from the University of Michigan, Ann Arbor during the 
Winter 2017 semester. 
o We will make an in-person announcement in Women’s Studies 400: 
Women’s Reproductive Health (taught by Dr. Tim Johnson, Dept. of 
OB/GYN) according to the following script: 
 Hello everyone, we are here to recruit forty healthy female 
students to participate in our study examining the impact of 
changing tampon use on the composition of the vaginal microbiota. 
The study period will consist of a baseline window after a subject’s 
last period and three full menstrual cycles. For the first two 
menstrual cycles, each subject will use Tampax® Pearl® Regular 

Tampons and Playtex® Gentle Glide® 360°® Regular Tampons 
for one menstrual cycle each, in a randomly assigned order. For the 
third menstrual cycle, each subject will use menstrual products 
according to their normal habits. For the course of the study, 
approximately 4 months, subjects will self-collect vaginal swabs 
twice a week. These samples will be used for bacterial community 
analysis to assess the composition of the vaginal microbiota at each 
time point, and compare composition during periods using 
different tampons and determine how long these changes persist 
between periods. For the first two menstrual cycles, the study 
tampons and Always® Infinity® Regular Pads will be provided to 
subjects free of charge. Additionally, each subject will receive $20. 
In order to be eligible to participate in this study, you must be at 
least 18 years old, have regular menstrual cycles that last 3-5 
weeks with periods that last for at least 4 days, currently use 
tampons, and be in good general and vaginal health. You are 
ineligible to participate if you are pregnant or planning on 
becoming pregnant in the next 4 months, if you have difficulty 
using tampons, if you currently have or have had toxic shock 
syndrome (TSS), if you currently have a sexually transmitted 
infection (STI) or urinary tract infection (UTI), if are currently 
using antibiotics or antifungals or have used antibiotics or 
antifungals within the last month, or if you have an autoimmune 
condition. If you are interested in participating in this study, please 
email (kaycart@umich.edu) and then we will set up an enrollment 
meeting.  
o We will also pass out flyers describing the objective of the study, the 
duration of subject participation, subject responsibilities, subject 
compensation, eligibility requirements, and exclusion criteria. It will also 
include the email address for setting up enrollment meetings. We will also 
post these flyers at the Michigan Union and other places around central 
campus. 
o We will also post the study in UMHealthResearch.org. 
o We will respond to interest emails according to the following script in 
order to set up enrollment meetings with each interested student: 
 Dear [name], 
 
Thank you for your interest in participating in this study. The next 
step is to set up an enrollment meeting at which you will be 
assigned a random study ID number, review the informed consent 
document for the study, give informed consent to participate in the 
study if you choose to, complete an online enrollment survey, and 
receive the tampons, pads, and vaginal swabs for the study. The 
following times are available for the enrollment meeting: 
 
Monday 9:30 AM – 4:15 PM 

Tuesday 1:30 PM – 4:00 PM 
Wednesday 11:00 AM – 4:15 PM 
Thursday 1:30 PM – 4:15 PM 
 
Please respond to this email with your preferred day and time for 
the enrollment meeting. The meeting will take approximately 30 
minutes. 
 
Thanks 
o At the enrollment meeting we will obtain informed consent from each 
student, assign a random ID number to the student, administer the baseline 
survey (assess eligibility, collects data on covariates (covered in Methods 
subsection Study Design)) on a lab computer using Qualtrics, review 
subject responsibilities with each student, and distribute study materials 
including 30 vaginal swabs, vaginal swab instructions, 30 collection day 
surveys, 24 Tampax® Pearl® Regular Tampons, 24 Playtex® Gentle 
Glide® 360°® Regular Tampons, 24 Always® Infinity® Regular Pads 
with wings to each subject. All research data will be kept in files that 
include the subject ID number, but not name or other information that is 
likely to allow someone other than the researchers to link the data to a 
subject. These files will be maintained in a password protected lab file 
manager. We will enroll the first 20 eligible students who schedule an 
enrollment meeting. 
 Inclusion criteria: Subjects must be 18 years of age or older; have regular 
menstrual cycles lasting 21-35 days with menses for at least 4 days; be a current 
tampon user; be in self-reported good general health; and be in self-reported good 
vaginal health. 
 Exclusion criteria: Respondents will be excluded if they are pregnant or are 
planning on becoming pregnant during the study; have difficulty using tampons; 
currently have, or have had TSS; currently have an STI; currently have a UTI; are 
currently using current antibiotics, or used antibiotics within 4 weeks prior to 
enrollment; are currently using current antifungals, or used antifungals within 4 
weeks prior to enrollment; or have an autoimmune condition. 
 
 
Study Design 
 This study will be a longitudinal, interventional, case-crossover study with three 
exposure levels: 
o Experimental exposure levels: Each subject will use Tampax® Pearl® 
Regular Tampons and Playtex® Gentle Glide® 360°® Regular 
Tampons for one menstrual cycle each, in a randomly assigned order. 
o Control exposure level: Each subject will use menstrual products 
according to their normal habits for a third menstrual cycle. 
o A case-crossover design ensures exchangeability between treatment 
windows within each subject for all time invariant characteristics. 
Under exchangeability and the assumptions of an intent-to-treat 
analysis (covered in Methods – Whole Community Analysis & 

Statistics), the actual outcome observed under one treatment for an 
individual equals the counterfactual outcome under that treatment for 
that individual, so association between expanded tampon shape and 
VMB composition represents a causal relationship. This study design 
and the intent-to-treat analytic strategy will allow us to make causal 
inference in reference to the effect of expanded tampon shape on VMB 
composition. 
 Vaginal swabs will be self-collected by each subject twice weekly for four 
different exposure windows (Fig. 1): 
o A 2-3 week baseline window after the last menstrual period prior to 
enrollment; 4-6 baseline samples per subject. 
o One menstrual cycle using the Tampax® Pearl® Regular Tampons; 8 
samples. 
o One menstrual cycle using Playtex® Gentle Glide® 360°® Regular 
Tampons; 8 samples. 
o The final menstrual cycle with menstrual product use according to the 
subject’s normal habits; 8 samples for the subject’s normal habits. 
 
 
Figure 1: Study design. 
 
 We will administer a baseline survey using the online survey system Qualtrics to 
obtain information on covariates including age; race; normal menstrual product 
use (types of products used; frequency of use of different products; specific 
tampon details including brand, perfume, absorbency); hygiene habits (douching, 
use of scented soaps); contraception use (current and past use of hormonal 
methods, current use of other methods); sexual activity (types of current and past, 
sex of partners, number of lifetime partners, frequency, use of lubricant); social 
history (smoking, drinking, yogurt); obstetric/gynecological history (medical 
problems, pregnancies and outcomes, first day of last period); medical history 
(medical problems, height, weight, surgical history, current medications). 
o We had 100% completion in our pilot and expect a similar completion 
rate in the proposed study (HUM 00098494). 
 
 
Menstrual Products 
 Tampon A: Tampax® Pearl® Regular Tampons (Fig. 2) 
o http://tampax.com/en-us/shop-products/pearl/pearl-regular 
o Applicator: plastic with pigments of color 
o Absorbent core: cotton and/or rayon  

o Thin fabric around absorbent core: rayon and polyester or 
polyethylene and polypropylene 
o String: cotton and/or polyester; polypropylene braid 
o Thread: cotton-wrapped polyester or polyester 
o $0.21 on amazon prime; 4.6 stars 
 4 tampons/day * 6 days = 24 tampons per subject * 20 subjects 
= 480 tampons * $0.21/tampon = $100.80 
A 
B 

 
Figure 2: Tampax® Pearl® Regular Tampons. A) Tampon in applicator. B) Tampon. C) 
Expanded tampon. D) Leak-stopping braided string. 
 Tampon B: Playtex® Gentle Glide® 360°® Regular Tampons (Fig. 3) 
o http://www.playtexplayon.com/products/gentle-glide-tampons 
o Applicator: plastic 
o Tampon: rayon fiber, polyester 
o String: cotton 
o $0.32 per tampon on amazon prime; 4.0 stars 
 4 tampons/day * 6 days = 24 tampons per subject * 20 subjects 
= 480 tampons * $0.32/tampon = $153.60 
D 
C 

 
 
 
Figure 3: Playtex® Gentle Glide® 360°® Regular Tampons. A) Tampon in applicator and 
tampon. B) Triple layer design. C) Expanded tampon. 
 Justification for choice of tampons A and B: The primary structural difference 
between Tampax® Pearl® Regular Tampons and Playtex® Gentle Glide® 360°® 
Regular Tampons is that, once inserted into the vagina, Tampax® Pearl® Regular 
Tampons expand axially into a rectangular prism-like shape (Fig. 2C), while 
Playtex® Gentle Glide® 360°® Regular Tampons expand radially into a cone-
like shape (Fig. 3C). The radial expansion of the Playtex® Gentle Glide® 360°® 
Regular Tampons likely results in increased surface area of the tampon that is in 
contact with the vaginal epithelium as compared to the Tampax® Pearl® Regular 
Tampons. This may allow for increased absorption of menses by the Playtex® 
Gentle Glide® 360°® Regular Tampons as compared to the Tampax® Pearl® 
Regular Tampons, resulting in differing growth conditions in the vagina during 
A 
C 
B 

Playtex® Gentle Glide® 360°® Regular Tampon use and Tampax® Pearl® 
Regular Tampon use. Both tampons are inserted with a plastic applicator, and 
both are made of a cotton/rayon blend. The only other substantial structural 
difference between the two is that the Tampax® Pearl® Regular Tampons have a 
braided string  (Fig. 2B) while the Playtex® Gentle Glide® 360°® Regular 
Tampons have a straight, 2 strand string (Fig 3A). Tampax® Pearl® reports that 
the braided string is designed to stop tampon leaks, and that it can hold up to 12 
drops of fluid in addition to the volume held by the tampon (Fig. 2D). We do not 
anticipate this difference to substantially alter the growth conditions in the vagina 
because any contact between the string (braided or not) and the vaginal epithelium 
will be much less than that between the actual tampon and the vaginal epithelium. 
Another reason for choosing these tampons is that, for the past 6 years (2011-
2016), Tampax® and Playtex® brand tampons have been the two most used 
tampon brands in the US [Experian 2016]. Examining how these tampon brands 
impact VMB composition will increase the generalizability of the study findings, 
as compared to if we selected other tampon brands. 
 Pad: Always® Infinity® Regular Pads with wings (Fig. 4) 
o http://always.com/en-us/shop-products/menstrual-pads/infinity-pads-
with-flexfoam/always-infinity-regular-pads-with-wings 
o $0.31 per pad on amazon prime; 4.4 stars 
 2 pads/day * 12 days = 24 pads * 20 subjects = 480 pads * 
$0.31/pad = $148.80 
 
Figure 4: Always® Infinity® Regular Pads with wings. 
 Distribution: Subjects will receive their study tampons and pads during their 
enrollment meeting. 
 We will instruct subjects to use the study tampons as they normally would, not 
wear a tampon for more than 8 hours, not wear more than one tampon at a time, 
contact a doctor if they experience any serious adverse effects related to tampon 
use including TSS symptoms, remove the tampon prior to collecting a swab 
during menses, and use the pads in addition to the tampons if they so choose. 

 We will provide subjects with written instructions from the tampon manufacturer 
on tampon use and ways to minimize risk of TSS. 
 
 
Self-Collected Vaginal Swabs 
 As in our pilot study, a double-headed, dry, rayon swab (Starplex Scientific, Inc., 
S09D) will be used for sample collection (HUM00098494). 
 Swab distribution: Subjects will receive their vaginal swabs for the duration of the 
study (assumed duration of 15 weeks) during their enrollment meeting. If 
additional swabs are needed, subjects will pick them up from the first floor of 
MSRB1.  
 Swab collection: Subjects will drop off their self-collected vaginal swabs on the 
first floor of MSRB1 during designated time windows each week; swabs will be 
stored in -80°C until analysis. 
 Swabbing procedure: All swabs will include the following written instructions for 
self-collecting the vaginal swab (HUM00098494, HUM00086661): 
1. Read these instructions before starting. 
2. Locate a sterile swab labeled with your study ID number 
3. Write today’s date (the day you used the swab) in the space 
provided on the swab label. Complete the short survey included with 
the swab. It is OK to collect a sample if you are menstruating, but if 
you are currently using a tampon, remove it before using the swab. 
4. Wash your hands. 
5. Remove the sterile swabs from the tube. Do not let them touch your 
body or any surface.  
6. Gently part your labia and insert both swabs approximately 2 inches 
into your vagina, imagining that you are pointing it towards your lower 
back. 
7. Gently turn the swabs 4 times, rotating the swab about 90 degrees with 
each turn. 
8. Remove the swabs taking care to not touch anywhere on your body. 
9. Place both swabs back into the tube. 
10. Place the tube into the plastic bag. Wash your hands. 
11. Return the swab to the first floor of MSRB1 during one of the 
designated time windows this week. 
 Covariate data collection with each swab: We will provide a short survey with 
each swab on which subjects will record whether the swab was collected during 
menses as well as any changes in sexual activity, hygiene, medication use, general 
health, vaginal health, contraception use, pregnancy, menses since the baseline 
study or prior swab. 
 We will remind subjects to collect swabs with twice-weekly text messages, 
emails, or phone calls based on preference. 
 
 
Whole Community Analysis & Statistics 
 DNA will be isolated from vaginal swab samples using a PowerMag Soil DNA 
isolation kit (Mo Bio Laboratories, Inc.) and an epMotion robot (Eppendorf 
epMotion 5075). The DNA samples will submitted to the University of Michigan 

Center for Microbial Systems Laboratory for amplification and sequencing of the 
V4 region of the 16S rRNA gene. The V4 region of the bacterial 16S rRNA gene 
will be amplified using dual-indexing sequencing strategy and sequenced on the 
Illumina MiSeq platform as described by Kozich et al. and Seekatz et al. After 
sequencing is completed, the 16S rRNA gene sequences will be processed and 
analyzed using the mothur software (version 1.34.3) and MiSeq SOP 
(http://www.mothur.org/wiki/MiSeq_SOP). Following sequence processing and 
alignment to the SILVA reference alignment [Preusse 2007], sequences will be 
binned into operational taxonomic units (OTUs) of 97% sequence identity. In our 
preliminary studies, we were able to detect and distinguish 6 different types 
of Lactobacillus among the top OTUs (>2% in at least 1 sample) from vaginal 
swab samples using an OTU definition of 3% sequence difference with sequences 
from the V4 region of the 16S rRNA gene. Relative abundances of OTUs will be 
calculated and we will test the dependence of within subject θYC distances on 
expanded tampon shape using Mann-Whitney tests or Kruskal-Wallis test, as 
dictated by the number of comparisons. Principal components analysis (PCoA) 
will be used to visualize θYC distances between samples within subjects. The same 
analysis protocol was used in our previous studies (HUM00098494, 
HUM00086661). 
 Intent to treat analysis: This analytic strategy assumes each subject’s actual 
exposure is exactly as it was assigned (complete compliance with assigned 
interventions, exposure fixed for each subject during each treatment window, 
complete follow-up). The implications of this analytic approach in conjunction 
with the within-subject exchangeability produced by the case-crossover design 
(covered in Method – Study Design section) is that the actual outcome observed 
under one treatment for a subject equals the counterfactual outcome under that 
treatment for that subject, so association between expanded tampon shape and 
VMB composition represents a causal relationship. This study design and analytic 
strategy will allow us to make causal inference in reference to the effect of 
expanded tampon shape on VMB composition. 
 
Benefits 
 This study will help to provide information on whether the use of tampons is associated 
with changes in the VMB over the course of a menstrual cycle. If there are associated 
changes in the VMB associated with tampon use then our research could help provide 
information on a potentially modifiable time point to help improve the vaginal health of 
women. 
 
Risks 
 No more than minimal risk 
o Discomfort with tampon use - rare - we are recruiting women that are currently 
tampon users. 
o Toxic shock syndrome (TSS) - rare- the rates of TSS with currently available 
tampons are extremely low. Each women will be given manufacturers instructions 
which include ways to decrease the risk of TSS. 

o Discomfort with vaginal swab collection - rare- the Starplex swabs have been 
used in numerous trials by our lab without any problems given that the swab is 
considerably smaller than the size of the tampon introducer. Additionally, detailed 
instructions on sample collection are provided. 
o Disclosure of subject information - rare- each subject will be provided a unique 
ID for the data collected (samples and surveys). All collected information will be 
maintained in a password protected lab file manager. 
 All recruited women in this trial will be active tampon users and thus we do not foresee 
any likelihood of these risks interfering with the subjects day to day living. 
 
Incentives 
 Each subject will receive a $20 gift card for participating in this study.  
 Each subject  will receive: 24 Tampax® Pearl® Regular Tampons, valued at $5.04; 24 
Playtex® Gentle Glide® 360°® Regular Tampons, valued at $7.68; and 24 Always® 
Infinity® Regular Pads with wings, valued at $7.44. 
 
 

Literature Cited 
 
1. Hickey, R. J., et al. (2012). "Understanding vaginal microbiome complexity from an 
ecological perspective." Translational Research 160(4): 267-282. 
2. Onderdonk, A. B., et al. (1986). "Methods for quantitative and qualitative evaluation of 
vaginal microflora during menstruation." Applied Environmental Microbiology 51: 333-
339. 
3. Onderdonk, A. B., et al. (1987). "Quantitative assessment of vaginal microflora during 
use of tampons of various compositions." Applied Environmental Microbiology 53(2774-
2778). 
4. Chow, A. W. and K. H. Bartlett (1989). "Sequential assessment of vaginal microflora in 
healthy women randomly assigned to tampon or napkin use." Review of Infectious 
Diseases 11. 
5. Shehin, S. E., et al. (2003). "Clinical safety-in-use study of a new tampon design." 
Infectious Disease in Obstetrics and Gynecology 11: 89-99. 
6. Chase, D. J., et al. (2007). "A prospective, randomized, double-blind study of vaginal 
microflora and epithelium in women using a tampon with an apertured film cover 
compared with those in women using a commercial tampon with a cover of nonwoven 
fleece." Journal of Clinical Microbiology 45: 1219-1224. 
7. Chase, D. J., et al. (2010). "Randomized, double-blind crossover study of vaginal 
microflora and epithelium in women using a tampon with a "winged" apertured film 
cover and a commercial tampon with a nonwoven fleece cover." Journal of Clinical 
Microbiology 48: 1317-1322. 
8. Hochwalt, A. E., et al. (2010). "Clinical Safety Assessment of an Ultra Absorbency 
Menstrual Tampon." Journal of Women's Health 19(2). 
9. Hickey, R. J., et al. (2013). "Effects of tampons and menses on the composition and 
diversity of vaginal microbial communities over time." BJOG: An International Journal 
of Obstetrics & Gynecology 120(6): 695-706. 
10. Borovkova, N., et al. (2011). "Influence of sexual intercourse on genital tract microbiota 
in infertile couples." Anaerobe 17(6): 414-418. 
11. Lopes dos Santos Santiago, G., et al. (2011). "Longitudinal Study of the Dynamics of 
Vaginal Microflora during Two Consecutive Menstrual Cycles." PLoS One 6(11): 
e28180. 
12. Borges, S., et al. (2014). "The role of lactobacilli and probiotics in maintaining vaginal 
health." Archives of Gynecology and Obstetrics 289(3): 479-489. 
13. Cook, R. L. and J. D. Sobel (1990). "Emerging role of lactobacilli in the control and 
maintenance of the vaginal bacterial microflora." Review of Infectious Diseases 12(5): 
856-872. 
14. Boskey, E., et al. (1999). "Acid production by vaginal flora in vitro is consistent with the 
rate and extent of vaginal acidification." Infection and Immunity 67(10): 5170-5175. 
15. Aroutcheva, A., et al. (2001). "Defense factors of vaginal lactobacilli." American Journal 
of Obstetrics and Gynecology 185(2): 375-379. 
16. Valore, E. V., et al. (2002). "Antimicrobial components of vaginal fluid." American 
Journal of Obstetrics and Gynecology 187(3): 561-568. 

17. Atassi, F., et al. (2006). "Lactobacillus strains isolated from the vaginal microbiota of 
healthy women inhibit Prevotella bivia and Gardnerella vaginalis in coculture and cell 
culture." FEMS Immunology & Medical Microbiology 48(3): 424-432. 
18. Kaewsrichan, J., et al. (2006). "Selection and identification of anaerobic lactobacilli 
producing inhibitory compounds against vaginal pathogens." FEMS Immunology & 
Medical Microbiology 48(1): 75-83. 
19. O'Hanlon, D. E., et al. (2010). "Cervicovaginal fluid and semen block the microbicidal 
activity of hydrogen peroxide produced by vaginal lactobacilli." BMC infectious diseases 
10(1): 1. 
20. Gajer, P., et al. (2012). "Temporal Dynamics of the Human Vaginal Microbiota." Science 
Translational Medicine 4(132): 132ra152-132ra152. 
21. Martin, D. H. (2012). "The Microbiota of the Vagina and Its Influence on Women’s 
Health and Disease." The American journal of the medical sciences 343(1): 2-9. 
22. Spiegel, C. A., et al. (1980). "Anaerobic bacteria in nonspecific vaginitis." New England 
Journal of Medicine 303(11): 601-607. 
23. Amsel, R., et al. (1983). "Nonspecific vaginitis: diagnostic criteria and microbial and 
epidemiologic associations." The American journal of medicine 74(1): 14-22. 
24. Spiegel, C. A., et al. (1983). "Curved anaerobic bacteria in bacterial (nonspecific) 
vaginosis and their response to antimicrobial therapy." Journal of Infectious Diseases 
148(5): 817-822. 
25. Spiegel, C., et al. (1983). "Diagnosis of bacterial vaginosis by direct gram stain of vaginal 
fluid." Journal of Clinical Microbiology 18(1): 170-177. 
26. Hill, G. B. (1993). "The microbiology of bacterial vaginosis." American Journal of 
Obstetrics and Gynecology 169(2): 450-454. 
27. Falagas, M. E., et al. (2008). "Probiotics for the Treatment of Women With Bacterial 
Vaginosis." Journal of Lower Genital Tract Disease 12(1): 65. 
28. Cribby, S., et al. (2008). "Vaginal Microbiota and the Use of Probiotics." 
Interdisciplinary Perspectives on Infectious Diseases 2008: 9. 
29. Sobel, J. D. (2005). "What's new in bacterial vaginosis and trichomoniasis?" Infectious 
Disease Clinics of North America 19(2): 387-406. 
30. Koumans, E. H., et al. (2007). "The prevalence of bacterial vaginosis in the United States, 
2001–2004; associations with symptoms, sexual behaviors, and reproductive health." 
Sexually transmitted diseases 34(11): 864-869. 
31. Sweet, R. L. (1995). "Role of bacterial vaginosis in pelvic inflammatory disease." 
Clinical Infectious Diseases 20(Supplement 2): S271-S275. 
32. Haggerty, C. L., et al. (2004). "Bacterial vaginosis and anaerobic bacteria are associated 
with endometritis." Clinical Infectious Diseases 39(7): 990-995. 
33. Wiesenfeld, H. C., et al. (2002). "Lower genital tract infection and endometritis: insight 
into subclinical pelvic inflammatory disease." Obstetrics & Gynecology 100(3): 456-463. 
34. Hillier, S. (1998). "The vaginal microbial ecosystem and resistance to HIV." AIDS 
research and human retroviruses 14: S. 17-S. 21. 
35. Taha, T. E., et al. (1998). "Bacterial vaginosis and disturbances of vaginal flora: 
association with increased acquisition of HIV." Aids 12(13): 1699-1706. 
36. Martin, H. L., et al. (1999). "Vaginal lactobacilli, microbial flora, and risk of human 
immunodeficiency virus type 1 and sexually transmitted disease acquisition." Journal of 
Infectious Diseases 180(6): 1863-1868. 

37. Schmid, G., et al. (2000). "Bacterial vaginosis and HIV infection." Sexually Transmitted 
Infections 76(1): 3-4. 
38. Wiesenfeld, H. C., et al. (2003). "Bacterial vaginosis is a strong predictor of Neisseria 
gonorrhoeae and Chlamydia trachomatis infection." Clinical Infectious Diseases 36(5): 
663-668. 
39. Sautter, R. L. and W. J. Brown (1980). "Sequential vaginal cultures from normal young 
women." Journal of Clinical Microbiology 11(5): 479-484. 
40. Wilks, M. and S. Tabaqchali (1987). "Quantitative bacteriology of the vaginal flora 
during the menstrual cycle." Journal of Medical Microbiology 24(3): 241-245. 
41. Bartlett, J. G., et al. (1977). "Quantitative Bacteriology of the Vaginal Flora." Journal of 
Infectious Diseases 136(2): 271-277. 
42. Priestley, C. J., et al. (1997). "What is normal vaginal flora?" Genitourinary Medicine 
73(1): 23-28. 
43. Eschenbach, D. A., et al. (2000). "Influence of the Normal Menstrual Cycle on Vaginal 
Tissue, Discharge, and Microflora." Clinical Infectious Diseases 30(6): 901-907. 
44. Srinivasan, S., et al. (2010). "Temporal Variability of Human Vaginal Bacteria and 
Relationship with Bacterial Vaginosis." PLoS One 5(4): e10197. 
45. Chaban, B., et al. (2014). "Characterization of the vaginal microbiota of healthy Canadian 
women through the menstrual cycle." Microbiome 2(1): 1-12. 
46. Hill, J. A. and D. J. Anderson (1992). "Human vaginal leukocytes and the effects of 
vaginal fluid onlymphocyte and macrophage defense functions." American Journal of 
Obstetrics and Gynecology 166(2): 720-726. 
47. Hill, D. R., et al. (2010). "Intravaginal and in vitro temperature changes with tampons of 
differing composition and absorbency." Journal of Biomedical Materials Research Part B: 
Applied Biomaterials 92B(2): 535-541. 
48. Hill, D. R., et al. (2005). "In vivo assessment of human vaginal oxygen and carbon 
dioxide levels during and post menses." Journal of Applied Physiology 99(4): 1582-1591. 
49. Preti, G. and G. R. Huggins (1975). "Cyclical changes in volatile acidic metabolites of 
human vaginal secretions and their relation to ovulation." Journal of Chemical Ecology 
1(3): 361-376. 
50. Omar, H. A., et al. (1998). "Tampon use in young women." Journal of pediatric and 
adolescent gynecology 11(3): 143-146. 
51. Experian (2016). 2016 Simmons Profile Report, Statista; US Census Bureau. 
52. Kozich, J. J., et al. (2013). "Development of a Dual-Index Sequencing Strategy and 
Curation Pipeline for Analyzing Amplicon Sequence Data on the MiSeq Illumina 
Sequencing Platform." Applied and Environmental Microbiology 79(17): 5112-5120. 
53. Seekatz, A. M., et al. (2015). "Fecal Microbiota Transplantation Eliminates Clostridium 
difficile in a Murine Model of Relapsing Disease." Infection and Immunity 83(10): 3838-
3846. 
54. Pruesse, E., et al. (2007). "SILVA: a comprehensive online resource for quality checked 
and aligned ribosomal RNA sequence data compatible with ARB." Nucleic Acids 
Research 35(21): 7188-7196. 
55. Cole, J. R., et al. (2014). "Ribosomal Database Project: data and tools for high throughput 
rRNA analysis." Nucleic Acids Research 42(Database issue): D633-D642. 

56. Wang, Q., et al. (2007). "Naïve Bayesian Classifier for Rapid Assignment of rRNA 
Sequences into the New Bacterial Taxonomy." Applied and Environmental Microbiology 
73(16): 5261-5267.