ORIGINAL RESEARCH ARTICLE

A Vaginal Fluid SimulantDerek H. Owen* and David F. Katz*

A fluid medium was developed to simulate the fluidproduced in the human vagina. The composition of themedium was based on an extensive review of the literatureon constituents of human vaginal secretions. In choosingthe ingredients for this medium, the goal was to emphasizeproperties that influence interactions of vaginal fluid withtopical contraceptive, prophylactic, or therapeutic prod-ucts. Among these properties, pH and osmolarity play adominant role in physicochemical processes that governdrug release and distribution. CONTRACEPTION 1999;59:9195 1999 Elsevier Science Inc. All rights reserved.

KEY WORDS: vaginal, fluid, secretions, simulant, composi-tion

Introduction

When therapeutic, contraceptive or prophylac-tic formulations are applied to the vagina,they encounter a variety of fluids withwidely varying physical and chemical properties.These fluids include those that originate in the vaginaand those that flow into the vagina, eg, cervicalmucus and semen. The actual or net fluid at anylocation within the vagina is a mixture, to varyingextent, of these component fluids. The flow, reten-tion, drug delivery kinetics, and bioactivity of vaginalformulations depends on their interactions with theseresident fluids. Understanding of these interactionscan, therefore, aid the design and development of newand improved formulations.One component of such research is in vitro testing

of formulations with fluids representative of whatthey encounter within the vagina. Human semen iscommonly used in such testing, for example, in theSander-Cramer test of spermicidal potency.1 Cervicalmucus is also employed, eg, in the double-ended testof spermicide biodiffusion into mucus2 or in thedirect analysis of spermicide permeation into mu-cus.3 However, there has been virtually no formula-tion testing to date with fluids representative of the

native material originating within the vagina. Vaginalfluid has many properties distinct from those ofsemen and mucus, and interactions of formulationswith this material may be physicochemically differ-ent from those with semen or mucus.Our laboratory has been studying how the deploy-

ment and delivery of contraceptive and prophylacticcompounds are affected by the properties of the de-livery vehicle and its interactions with the surround-ing fluids. It was found that the osmolarity and pH ofthe delivery vehicle and the surrounding fluid areimportant factors in modulating drug delivery.3 Os-molarity and pH are also important in determiningthe rheological properties of many commonly useddelivery gels. It is therefore useful to employ a vaginalfluid simulant with physical and chemical properties,particularly pH and osmolarity, that model those ofnative vaginal fluid. The formula for this simulantwas developed after an exhaustive review of theliterature.Vaginal fluid may include contributions from vag-

inal transudite, Bartholins and Skeness glands, exfo-liated epithelial cells, residual urine, and fluids fromthe upper reproductive tract such as cervical mucusand endometrial and tubal fluids. The quantity andcomposition of human vaginal fluid have been stud-ied by many researchers for a variety of reasons.These include the diagnosis of pathological condi-tions such as bacterial vaginosis, urinary tract infec-tions, cancer, premature rupture of membranes dur-ing pregnancy, determination of the presence ofsemen for forensic analysis, determination of thetime of ovulation, and the study of organic acids thatmay act as sexual attractants. Determination of thequantity and composition of vaginal fluid is difficultbecause of the small amount available for collectionand the possibility of dilution of samples by cervicalmucus, menstrual material, urine, or residual semen.The measurement of vaginal fluid acidity is compli-cated by its contamination by pH-neutral cervicalmucus (pH of about 8.0),4 semen (pH of about 7.5),5

and menstrual material. The vaginal fluid simulantproposed in this article is intended to model theproperties of the fluids originating in the vagina,particularly the vaginal transudite found in healthy,nonpregnant premenopausal women.

*Department of Biomedical Engineering and Department of Obstetrics andGynecology, Duke University, Durham, North CarolinaName 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.eduSubmitted for publication October 19, 1998Revised January 5, 1999Accepted for publication January 18, 1999

1999 Elsevier Science Inc. All rights reserved. ISSN 0010-7824/99/$20.00655 Avenue of the Americas, New York, NY 10010 PII S0010-7824(99)00010-4

Materials and MethodsEfforts to quantify the volume of vaginal fluid fallinto two categories: those that measure the quantityof vaginal fluid present at any one time, and thosethat measure the production of vaginal fluid over anextended interval. A summary of both types of mea-surements conducted by other researchers is given inTable 1. Note that, throughout this article, the unitsof concentration have been standardized, convertingall previous measurements to a g/L basis by equatingthe 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, withapproximately 0.50.75 g present in the vagina at anyone time. A volume of 0.75 mL of vaginal fluidsimulant was used in studies conducted in this labo-ratory on the efficacy of contraceptive gel formula-tions. The volume of vaginal fluid present at the timeof ovulation appears to increase when volumes aremeasured with tampons alone.6,7 When the cervix isblocked with a cervical cap, the volume of fluidpresent decreases, indicating increased dilution ofvaginal transudite by upper reproductive tract fluidsat the time of ovulation.8 The volume of vaginal fluidhas been shown to increase substantially during peri-ods of sexual stimulation.9,10Most researchers have used one of two techniques

for measuring vaginal acidity: pH indicator paper/colorimetry, or a pH electrode. One study comparingthese two methods indicated that they yield similarresults.11 These measurement devices are either ap-plied directly to the vaginal epithelia or to fluid

removed from the vagina; a summary of vaginal pHmeasurements is given in Table 2. The proposedvaginal fluid simulant was formulated with a pH of4.2, a value typical of the vaginal fluid of healthy,nonmenstruating, premenopausal women. Two stud-ies indicate that the vaginal pH increases duringperiods of vaginal stimulation,12,13 whereas anotherstudy showed no variation.14A number of studies have assessed the concentra-

tions of various salts in vaginal secretions. Stameyand Timothy15 collected vaginal fluid by a lavagetechnique and measured the following ion concentra-tions using flame photometry: sodium 1.035 g/L andpotassium 0.547 g/L. Levin and Wagner16 collectedvaginal fluid with tampons (over a 4-h period) andmeasured the following concentrations: sodium 1.95g/L, potassium 0.90 g/L, and chloride 2.94 g/L. Thisstudy also showed an increase in both sodium andchloride ion concentrations after sexual stimulation.Wagner and Levin,17 using the same collectionmethod, determined the following concentrations(center of range): sodium 1.72 g/kg, potassium 1.17g/kg, chloride 2.66 g/kg and calcium 0.12 g/kg (sodi-um, potassium, and calcium by flame photometry,chloride by electrometric titration). This study de-tected no change in ion concentrations over thereproductive cycle. Mende et al.,18 using flame pho-tometry, obtained similar results for sodium andpotassium, whereas their calcium concentrationswere more than twice those of Wagner and Levin.17Wagner and Levin13 report slightly lower concen-

trations (sodium 1.40 g/kg, potassium 0.90 g/kg, and

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

Reference Quantity Method

Kraul and Bodnar, 1925 0.51 mL Cited in Oberst and Plass22Voge, 1933 0.51 mL Cited in Wagner and Levin13Lissimore and Currie,31 1939 !0.5 mL Collection from vaginal vault with Volkmann

spoonLapan and Friedman,25 1950 0.050.95 mg Aspiration with glass pipet from vaginal vaultPerl et al.,32 1959 1.89 mL/day (calculated) Tampon for 2127 hStone and Gamble,6 1959 0.76 mL Swabbed the vaginal wall with cotton wad

1.0 mL at ovulationOdeblad,33 1964 5.8 mL/day, with 4.3 mL/day

reabsorbedTampon

Dusitsin et al.,8 1967 2.7 mL/day (calculated) Tampon and cervical cap for 18 hdecrease in volume at ovulation

Preti et al.,9 1979 8 mL/day (calculated) Tampon, nonmenstruating women, for 6 h0.43 mL/15 min (unstimulated)0.68 mL/15 min (stimulated)

Wagner,10 1979 7.5 mL/day (calculated) Tampon, nonmenstruating women, for 4 h911 mL/day (calculated) Evaporimeter on posterior vaginal wall

Wagner and Levin,17 1980 7.9 mL/day (calculated) Tampon, nonmenstruating women, for 45 hGodley,7 1985 4.65 mL/day (calculated) Tampon for 8 h

5.88 mL/day atmidcycle (calculated)

92 Owen and Katz Contraception1999;59:9195

chloride 2.20 g/kg) based on measurements performedon samples obtained using absorbent paper, which isless likely to become contaminated with cervicalsecretions. The proposed vaginal fluid simulant wasdesigned with ion concentrations around the middleof the range of reported values.Other constituents that have been identified in

vaginal fluids in significant quantities include pro-teins, carbohydrates, and low molecular weight or-ganic compounds. An excellent review of the com-pounds found in vaginal secretions can be found in anarticle by Huggins and Preti.19 The presence of vola-tile organic compounds in human vaginal secretionshas been of considerable interest, as some of thesecompounds have been shown to be sexual attractantsin other primates. Short chain organic acids are ofinterest in that they are responsible for the low pH ofthe vagina and influence the population distributionof microflora. Unfortunately, the concentrations ofmost of the compounds identified in vaginal secre-tions 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 deVargas-Linares20 and found to be 0.018 g/L during thefollicular phase and 0.053 g/L during the luteal phase.Raffi et al.21 collected fluid by scraping the vaginal

epithelia and found the total proteins concentration(Folin technique) to range from 0.015 to 0.026 g/Lwith a mean of 0.018 g/L, the concentration used inthe proposed simulant.Other compounds that are present in significant

concentrations include lactic acid, acetic acid, glyc-erol, urea, and glucose. Zweifel, cited in an article byOberst and Plass,22 reported a maximum lactic acidcontent of from 3 to 5 g/L. More recently, in a studyby Huggins and Preti,23 vaginal fluid was collectedusing tampons worn for 6 h and analyzed by com-bined gas chromatographymass spectrometry. Theymeasured lactic acid concentrations of about 2 g/Lwith a significant increase around the time of ovula-tion. In a study using the same collection and mea-surement techniques, Preti et al.9 measured lacticacid concentrations of about 1 g/L. The proposedsimulant contains 2 g/L lactic acid.Huggins and Preti23 also measured the amount of

acetic acid in their samples and found it to be about0.2 g/L, increasing at midcycle. Acetic acid was theonly short chain aliphatic acid consistently found invaginal secretions. Preti et al.9 also measured aceticacid concentration and found it to be 0.52 g/L,whereas the total concentration of all low molecularweight organic constituents was found to be 3.1 g/L.Preti and Huggins24 collected vaginal fluid using

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

Reference pH Method

Behrens and Naujoks, 1925 3.866.84 Cited in Zuck and Duncan34Schultheiss, 1929 4.7 (range 3.926.85) Michaelis colorimetric method on vaginal

washingsCited in Oberst and Plass22

Guthman and Koch, 1932 5.07.0 Cited in Zuck and Duncan34Oberst and Plass,22 1936 Range 3.864.59 Quinhydrone microelectrode,

nonmenstruating womenLissimore and Currie,31 1939 45 Color indicating capillometers

Collection from vaginal vault withVolkmann spoon

Zuck and Duncan,34 1939 4.04.2 pH paper and electrode on vaginal washings4.55.8 at ovulation

Rakoff,26 1943 4.56 lowest at midcycle pH electrode applied to vaginal wallHunter and Long,35 1958 4.6 range 3.76.3 pH electrode applied to vaginal wallMasters,12 1959 3.54.2 pH electrode applied to vaginal wallCohen,36 1969 5.9 range 48 pH electrode applied to back of cervix

No cyclic variation foundPeeters,37 1972 4.73 range 3.95.8 pH electrode applied to lateral vaginal fornixFox et al.,14 1973 4.0 pH sensitive telemetry capsuleStamey and Timothy,38 1975 4.9 range 3.77.5 pH electrode applied at hymenal ringStamey and Timothy,15 1975 4.4 pH electrode applied at hymenal ringChen et al.,39 1979 4.04 pH electrode applied to vaginal washingsDrake et al.,40 1980 4.6 pH electrode applied to vaginal wallWagner and Ottesen,41 1982 4.2 at midcycle pH electrode applied to vaginal wallWagner and Levin,30 1984 5.0 range 4.016.19 pH electrode applied to vaginal wallMoller and Kaspersen,42 1991 4.7 range 3.46.4 pH electrode applied to vaginal wallCailloutte et al.,43 1997 4.6 range 4.44.8 pH paper applied to vaginal wall

93Contraception A Vaginal Fluid Simulant1999;59:9195

tampons and, with gas chromatographymass spec-trometry, demonstrated that acetic acid concentra-tion reaches a maximum at midcycle. The proposedsimulant was designed to include 1 g/L of acetic acidto achieve concentrations of total low molecularweight organic constituents similar to those mea-sured by Preti et al.9Glycerol, urea, glucose, and glycogen have also

been measured in vaginal secretions by a number ofresearchers. Preti et al.9 measured glycerol concentra-tions to be 0.16 g/L with a significant increase inconcentration after sexual stimulation. Glycerol isthought to be responsible for the lubricating qualityof vaginal secretions. Huggins and Preti23 measuredurea concentration to be about 0.3 g/L, increasing atmidcycle. Wagner and Levin13 measured urea concen-trations of 0.49 g/L. The proposed simulant has con-centrations of glycerol and urea of 0.16 g/L and 0.4g/L, respectively.Glycogen occurs in large quantities in the vaginal

epithelia and is broken down by microbial or enzy-matic processes. The resulting glucose is then metab-olized to produce the lactic acid that is mainly re-sponsible for the acidity of vaginal fluid. Lapan andFriedman25 collected fluid from the vaginal vault byaspiration and measured glycogen concentrations ofabout 15 g/L, whereas Stamey and Timothy15 mea-sured values of 4.4 g/L. Lapan and Friedman25 alsomeasured vaginal glucose concentrations of 6.2 g/L.This is consistent with the findings of Rakoff et al.,26who reported that the total concentration of reducingsubstances in vaginal secretions is between 0% and4%.26 In the proposed simulant, the glycogen/glucosecontribution is represented by a glucose concentra-tion of 5.0 g/L.

Results and DiscussionTwo other groups have proposed vaginal fluid simu-lants. Dorr et al.27 developed a simulant as part oftheir study evaluating the efficacy of collagen-basedcontraceptive sponges. Their formulation, however,did not include lactic acid, glucose, or glycerol. Gesh-nizgani et al.28 developed a vaginal fluid model tostudy the growth of vaginal microflora. Their formu-lation contains many compounds that were not foundin vaginal secretions but that were needed to promotethe growth of microorganisms. In addition, theirsimulant contains a phosphate buffer with a range ofpH 5.78.0, which is much higher than that of thenative material.The proposed vaginal fluid simulant was designed

to incorporate information about chemical composi-tion determined by previous researchers with anemphasis on modeling the pH and osmolarity of the

material. The presence of the dozens of organic acidspresent was modeled by including the two present inthe largest quantities, lactic acid and acetic acid,whereas the total protein content was modeled byincluding only one protein, albumin. The formulationfor 1 L of solution given as compound and weight (g),is as follows: calcium, 0.120; potassium, 0.978; so-dium, 1.38; chloride, 2.13; albumin, 0.018; lactic acid,2.00; acetic acid, 1.00; glycerol, 0.16; urea, 0.4; andglucose, 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 studieshave shown that the buffering capacity of vaginalfluid is minimal (Tevi-Benissan et al.29; Wagner andLevin30), so that the quantity of chloride ions addedduring pH adjustment of the formulation is smallrelative to the total (about 0.1% for a pH adjustmentfrom 7.0 to 4.2).This formulation has proved useful for research

into contraceptive and prophylactic drug delivery, butit is far from definitive.3 This vaginal fluid simulantwas developed to have the same physical and chem-ical properties known to influence intravaginal gelefficacy. Researchers interested in studying the influ-ence of vaginal fluid on the vaginal microbiology, forinstance, may wish to include nutrients excludedfrom this formulation. Further efforts by other re-searchers are needed to better understand the compo-sition and properties of this important biological fluidso that improved simulants can be developed.

AcknowledgmentsSupported by CONRAD Program grant CSA-98-204,under Cooperative Agreement CCP-3044-A-00-2015-00 with the United States Agency for Interna-tional Development. The views expressed by theauthors do not necessarily reflect the views of USAIDand CONRAD.

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