ORIGINAL RESEARCH ARTICLE

A Vaginal Fluid Simulant

Derek H. Owen* and David F. Katz*
A fluid medium was developed to simulate the fluid
produced in the human vagina. The composition of the
medium was based on an extensive review of the literature
on constituents of human vaginal secretions. In choosing
the ingredients for this medium, the goal was to emphasize
properties that influence interactions of vaginal fluid with
topical contraceptive, prophylactic, or therapeutic products. Among these properties, pH and osmolarity play a
dominant role in physicochemical processes that govern
drug release and distribution. CONTRACEPTION 1999;59:
9195 1999 Elsevier Science Inc. All rights reserved.
KEY WORDS:

vaginal, fluid, secretions, simulant, composi-

tion

Introduction

hen therapeutic, contraceptive or prophylactic formulations are applied to the vagina,

they encounter a variety of fluids with
widely varying physical and chemical properties.
These fluids include those that originate in the vagina
and those that flow into the vagina, eg, cervical
mucus and semen. The actual or net fluid at any
location within the vagina is a mixture, to varying
extent, of these component fluids. The flow, retention, drug delivery kinetics, and bioactivity of vaginal
formulations depends on their interactions with these
resident fluids. Understanding of these interactions
can, therefore, aid the design and development of new
and improved formulations.
One component of such research is in vitro testing
of formulations with fluids representative of what
they encounter within the vagina. Human semen is
commonly used in such testing, for example, in the
Sander-Cramer test of spermicidal potency.1 Cervical
mucus is also employed, eg, in the double-ended test
of spermicide biodiffusion into mucus2 or in the
direct analysis of spermicide permeation into mucus.3 However, there has been virtually no formulation testing to date with fluids representative of the
*Department of Biomedical Engineering and Department of Obstetrics and
Gynecology, Duke University, Durham, North Carolina
Name and address for correspondence: Dr. Derek H. Owen, Department of
Biomedical Engineering, Duke University, Box 90281, Durham, NC 27708; Tel.:
(919) 680-5182; Fax: (919) 684-4488; e-mail: dhowen@acpub.duke.edu
Submitted for publication October 19, 1998
Revised January 5, 1999
Accepted for publication January 18, 1999

1999 Elsevier Science Inc. All rights reserved.

655 Avenue of the Americas, New York, NY 10010

native material originating within the vagina. Vaginal

fluid has many properties distinct from those of
semen and mucus, and interactions of formulations
with this material may be physicochemically different from those with semen or mucus.
Our laboratory has been studying how the deployment and delivery of contraceptive and prophylactic
compounds are affected by the properties of the delivery vehicle and its interactions with the surrounding fluids. It was found that the osmolarity and pH of
the delivery vehicle and the surrounding fluid are
important factors in modulating drug delivery.3 Osmolarity and pH are also important in determining
the rheological properties of many commonly used
delivery gels. It is therefore useful to employ a vaginal
fluid simulant with physical and chemical properties,
particularly pH and osmolarity, that model those of
native vaginal fluid. The formula for this simulant
was developed after an exhaustive review of the
literature.
Vaginal fluid may include contributions from vaginal transudite, Bartholins and Skeness glands, exfoliated epithelial cells, residual urine, and fluids from
the upper reproductive tract such as cervical mucus
and endometrial and tubal fluids. The quantity and
composition of human vaginal fluid have been studied by many researchers for a variety of reasons.
These include the diagnosis of pathological conditions such as bacterial vaginosis, urinary tract infections, cancer, premature rupture of membranes during pregnancy, determination of the presence of
semen for forensic analysis, determination of the
time of ovulation, and the study of organic acids that
may act as sexual attractants. Determination of the
quantity and composition of vaginal fluid is difficult
because of the small amount available for collection
and the possibility of dilution of samples by cervical
mucus, menstrual material, urine, or residual semen.
The measurement of vaginal fluid acidity is complicated by its contamination by pH-neutral cervical
mucus (pH of about 8.0),4 semen (pH of about 7.5),5
and menstrual material. The vaginal fluid simulant
proposed in this article is intended to model the
properties of the fluids originating in the vagina,
particularly the vaginal transudite found in healthy,
nonpregnant premenopausal women.
ISSN 0010-7824/99/$20.00
PII S0010-7824(99)00010-4

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Owen and Katz

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1999;59:9195

Table 1. Quantity of vaginal fluid as measured by previous researchers

Reference

Quantity

Method

Kraul and Bodnar, 1925

Voge, 1933
Lissimore and Currie,31 1939

0.51 mL
0.51 mL
!0.5 mL

Lapan and Friedman,25 1950

Perl et al.,32 1959
Stone and Gamble,6 1959

0.050.95 mg
1.89 mL/day (calculated)
0.76 mL
1.0 mL at ovulation
5.8 mL/day, with 4.3 mL/day
reabsorbed
2.7 mL/day (calculated)
decrease in volume at ovulation
8 mL/day (calculated)
0.43 mL/15 min (unstimulated)
0.68 mL/15 min (stimulated)
7.5 mL/day (calculated)
911 mL/day (calculated)
7.9 mL/day (calculated)
4.65 mL/day (calculated)
5.88 mL/day at
midcycle (calculated)

Odeblad,33 1964
Dusitsin et al.,8 1967
Preti et al.,9 1979
Wagner,10 1979
Wagner and Levin,17 1980
Godley,7 1985

Materials and Methods

Efforts to quantify the volume of vaginal fluid fall

into two categories: those that measure the quantity
of vaginal fluid present at any one time, and those
that measure the production of vaginal fluid over an
extended interval. A summary of both types of measurements conducted by other researchers is given in
Table 1. Note that, throughout this article, the units
of concentration have been standardized, converting
all previous measurements to a g/L basis by equating
the density of the vaginal fluid to that of water.
In summary, prior studies suggest that the daily
production of vaginal fluid is around 6 g/day, with
approximately 0.5 0.75 g present in the vagina at any
one time. A volume of 0.75 mL of vaginal fluid
simulant was used in studies conducted in this laboratory on the efficacy of contraceptive gel formulations. The volume of vaginal fluid present at the time
of ovulation appears to increase when volumes are
measured with tampons alone.6,7 When the cervix is
blocked with a cervical cap, the volume of fluid
present decreases, indicating increased dilution of
vaginal transudite by upper reproductive tract fluids
at the time of ovulation.8 The volume of vaginal fluid
has been shown to increase substantially during periods of sexual stimulation.9,10
Most researchers have used one of two techniques
for measuring vaginal acidity: pH indicator paper/
colorimetry, or a pH electrode. One study comparing
these two methods indicated that they yield similar
results.11 These measurement devices are either applied directly to the vaginal epithelia or to fluid

Cited in Oberst and Plass22

Cited in Wagner and Levin13
Collection from vaginal vault with Volkmann
spoon
Aspiration with glass pipet from vaginal vault
Tampon for 2127 h
Swabbed the vaginal wall with cotton wad
Tampon
Tampon and cervical cap for 18 h
Tampon, nonmenstruating women, for 6 h
Tampon, nonmenstruating women, for 4 h
Evaporimeter on posterior vaginal wall
Tampon, nonmenstruating women, for 45 h
Tampon for 8 h

removed from the vagina; a summary of vaginal pH

measurements is given in Table 2. The proposed
vaginal fluid simulant was formulated with a pH of
4.2, a value typical of the vaginal fluid of healthy,
nonmenstruating, premenopausal women. Two studies indicate that the vaginal pH increases during
periods of vaginal stimulation,12,13 whereas another
study showed no variation.14
A number of studies have assessed the concentrations of various salts in vaginal secretions. Stamey
and Timothy15 collected vaginal fluid by a lavage
technique and measured the following ion concentrations using flame photometry: sodium 1.035 g/L and
potassium 0.547 g/L. Levin and Wagner16 collected
vaginal fluid with tampons (over a 4-h period) and
measured the following concentrations: sodium 1.95
g/L, potassium 0.90 g/L, and chloride 2.94 g/L. This
study also showed an increase in both sodium and
chloride ion concentrations after sexual stimulation.
Wagner and Levin,17 using the same collection
method, determined the following concentrations
(center of range): sodium 1.72 g/kg, potassium 1.17
g/kg, chloride 2.66 g/kg and calcium 0.12 g/kg (sodium, potassium, and calcium by flame photometry,
chloride by electrometric titration). This study detected no change in ion concentrations over the
reproductive cycle. Mende et al.,18 using flame photometry, obtained similar results for sodium and
potassium, whereas their calcium concentrations
were more than twice those of Wagner and Levin.17
Wagner and Levin13 report slightly lower concentrations (sodium 1.40 g/kg, potassium 0.90 g/kg, and

Contraception
1999;59:9195

A Vaginal Fluid Simulant

93

Table 2. pH of vaginal fluid as measured by previous researchers

Reference

pH

Method

Behrens and Naujoks, 1925

Schultheiss, 1929

3.866.84
4.7 (range 3.926.85)

Guthman and Koch, 1932

Oberst and Plass,22 1936

5.07.0
Range 3.864.59

Lissimore and Currie,31 1939

45

Zuck and Duncan,34 1939

4.04.2
4.55.8 at ovulation
4.56 lowest at midcycle
4.6 range 3.76.3
3.54.2
5.9 range 48
No cyclic variation found
4.73 range 3.95.8
4.0
4.9 range 3.77.5
4.4
4.04
4.6
4.2 at midcycle
5.0 range 4.016.19
4.7 range 3.46.4
4.6 range 4.44.8

Rakoff,26 1943
Hunter and Long,35 1958
Masters,12 1959
Cohen,36 1969
Peeters,37 1972
Fox et al.,14 1973
Stamey and Timothy,38 1975
Stamey and Timothy,15 1975
Chen et al.,39 1979
Drake et al.,40 1980
Wagner and Ottesen,41 1982
Wagner and Levin,30 1984
Moller and Kaspersen,42 1991
Cailloutte et al.,43 1997

chloride 2.20 g/kg) based on measurements performed

on samples obtained using absorbent paper, which is
less likely to become contaminated with cervical
secretions. The proposed vaginal fluid simulant was
designed with ion concentrations around the middle
of the range of reported values.
Other constituents that have been identified in
vaginal fluids in significant quantities include proteins, carbohydrates, and low molecular weight organic compounds. An excellent review of the compounds found in vaginal secretions can be found in an
article by Huggins and Preti.19 The presence of volatile organic compounds in human vaginal secretions
has been of considerable interest, as some of these
compounds have been shown to be sexual attractants
in other primates. Short chain organic acids are of
interest in that they are responsible for the low pH of
the vagina and influence the population distribution
of microflora. Unfortunately, the concentrations of
most of the compounds identified in vaginal secretions have not been quantified.
The protein content of vaginal secretions, collected
by aspiration of the vaginal vault, was measured
(Folin-phenol reagent technique) by Abdallah and de
Vargas-Linares20 and found to be 0.018 g/L during the
follicular phase and 0.053 g/L during the luteal phase.
Raffi et al.21 collected fluid by scraping the vaginal

Cited in Zuck and Duncan34

Michaelis colorimetric method on vaginal
washings
Cited in Oberst and Plass22
Cited in Zuck and Duncan34
Quinhydrone microelectrode,
nonmenstruating women
Color indicating capillometers
Collection from vaginal vault with
Volkmann spoon
pH paper and electrode on vaginal washings
pH
pH
pH
pH

electrode
electrode
electrode
electrode

applied
applied
applied
applied

to
to
to
to

vaginal wall
vaginal wall
vaginal wall
back of cervix

pH
pH
pH
pH
pH
pH
pH
pH
pH
pH

electrode applied to lateral vaginal fornix

sensitive telemetry capsule
electrode applied at hymenal ring
electrode applied at hymenal ring
electrode applied to vaginal washings
electrode applied to vaginal wall
electrode applied to vaginal wall
electrode applied to vaginal wall
electrode applied to vaginal wall
paper applied to vaginal wall

epithelia and found the total proteins concentration

(Folin technique) to range from 0.015 to 0.026 g/L
with a mean of 0.018 g/L, the concentration used in
the proposed simulant.
Other compounds that are present in significant
concentrations include lactic acid, acetic acid, glycerol, urea, and glucose. Zweifel, cited in an article by
Oberst and Plass,22 reported a maximum lactic acid
content of from 3 to 5 g/L. More recently, in a study
by Huggins and Preti,23 vaginal fluid was collected
using tampons worn for 6 h and analyzed by combined gas chromatographymass spectrometry. They
measured lactic acid concentrations of about 2 g/L
with a significant increase around the time of ovulation. In a study using the same collection and measurement techniques, Preti et al.9 measured lactic
acid concentrations of about 1 g/L. The proposed
simulant contains 2 g/L lactic acid.
Huggins and Preti23 also measured the amount of
acetic acid in their samples and found it to be about
0.2 g/L, increasing at midcycle. Acetic acid was the
only short chain aliphatic acid consistently found in
vaginal secretions. Preti et al.9 also measured acetic
acid concentration and found it to be 0.52 g/L,
whereas the total concentration of all low molecular
weight organic constituents was found to be 3.1 g/L.
Preti and Huggins24 collected vaginal fluid using

94

Owen and Katz

Contraception
1999;59:9195

tampons and, with gas chromatographymass spectrometry, demonstrated that acetic acid concentration reaches a maximum at midcycle. The proposed
simulant was designed to include 1 g/L of acetic acid
to achieve concentrations of total low molecular
weight organic constituents similar to those measured by Preti et al.9
Glycerol, urea, glucose, and glycogen have also
been measured in vaginal secretions by a number of
researchers. Preti et al.9 measured glycerol concentrations to be 0.16 g/L with a significant increase in
concentration after sexual stimulation. Glycerol is
thought to be responsible for the lubricating quality
of vaginal secretions. Huggins and Preti23 measured
urea concentration to be about 0.3 g/L, increasing at
midcycle. Wagner and Levin13 measured urea concentrations of 0.49 g/L. The proposed simulant has concentrations of glycerol and urea of 0.16 g/L and 0.4
g/L, respectively.
Glycogen occurs in large quantities in the vaginal
epithelia and is broken down by microbial or enzymatic processes. The resulting glucose is then metabolized to produce the lactic acid that is mainly responsible for the acidity of vaginal fluid. Lapan and
Friedman25 collected fluid from the vaginal vault by
aspiration and measured glycogen concentrations of
about 15 g/L, whereas Stamey and Timothy15 measured values of 4.4 g/L. Lapan and Friedman25 also
measured vaginal glucose concentrations of 6.2 g/L.
This is consistent with the findings of Rakoff et al.,26
who reported that the total concentration of reducing
substances in vaginal secretions is between 0% and
4%.26 In the proposed simulant, the glycogen/glucose
contribution is represented by a glucose concentration of 5.0 g/L.

material. The presence of the dozens of organic acids

present was modeled by including the two present in
the largest quantities, lactic acid and acetic acid,
whereas the total protein content was modeled by
including only one protein, albumin. The formulation
for 1 L of solution given as compound and weight (g),
is as follows: calcium, 0.120; potassium, 0.978; sodium, 1.38; chloride, 2.13; albumin, 0.018; lactic acid,
2.00; acetic acid, 1.00; glycerol, 0.16; urea, 0.4; and
glucose, 5.0.
A specific recipe for 1 L of this simulant given as
compound and weight (g), is as follows: NaCl, 3.51;
KOH, 1.40; Ca(OH)2, 0.222; bovine serum albumin,
0.018; lactic acid, 2.00; acetic acid, 1.00; glycerol,
0.16; urea, 0.4; and glucose, 5.0.
Once these compounds are combined, the mixture
is adjusted to a pH of 4.2 using HCl. Previous studies
have shown that the buffering capacity of vaginal
fluid is minimal (Tevi-Benissan et al.29; Wagner and
Levin30), so that the quantity of chloride ions added
during pH adjustment of the formulation is small
relative to the total (about 0.1% for a pH adjustment
from 7.0 to 4.2).
This formulation has proved useful for research
into contraceptive and prophylactic drug delivery, but
it is far from definitive.3 This vaginal fluid simulant
was developed to have the same physical and chemical properties known to influence intravaginal gel
efficacy. Researchers interested in studying the influence of vaginal fluid on the vaginal microbiology, for
instance, may wish to include nutrients excluded
from this formulation. Further efforts by other researchers are needed to better understand the composition and properties of this important biological fluid
so that improved simulants can be developed.

Results and Discussion

Acknowledgments

Two other groups have proposed vaginal fluid simulants. Dorr et al.27 developed a simulant as part of
their study evaluating the efficacy of collagen-based
contraceptive sponges. Their formulation, however,
did not include lactic acid, glucose, or glycerol. Geshnizgani et al.28 developed a vaginal fluid model to
study the growth of vaginal microflora. Their formulation contains many compounds that were not found
in vaginal secretions but that were needed to promote
the growth of microorganisms. In addition, their
simulant contains a phosphate buffer with a range of
pH 5.7 8.0, which is much higher than that of the
native material.
The proposed vaginal fluid simulant was designed
to incorporate information about chemical composition determined by previous researchers with an
emphasis on modeling the pH and osmolarity of the

Supported by CONRAD Program grant CSA-98-204,

under Cooperative Agreement CCP-3044-A-002015-00 with the United States Agency for International Development. The views expressed by the
authors do not necessarily reflect the views of USAID
and CONRAD.

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