<title>1. INTRODUCTION</title> <p>Bacterial vaginosis (BV) is a common condition causing various symptoms such as vaginal
discharge, odor and irritation, and has been associated with increased
acquisition of many sexually transmitted diseases [##REF##12594649##1##, ##REF##15976598##2##]. Pregnant women with BV have an increased risk
of preterm labor and preterm delivery with the potential for neonatal morbidity
and mortality [##REF##12861153##3##]. Early research concerning BV characterized the vaginal
microbiological flora using Gram stain and culture [##REF##2473992##4##, ##REF##1875389##5##]. More recent studies
have focused on host defense factors and the local immune response that may
mediate the relationship between vaginal flora and adverse reproductive and
pregnancy outcomes [##REF##16965223##6##, ##REF##15865604##7##]. 
Proinflammatory cytokines such as interleukin 1-Beta
(IL-1<italic>β</italic>), interleukin 6 (IL-6), interleukin 8 (IL-8), and the host defense
molecule secretory leukocyte protease inhibitor (SLPI) have been of particular
interest [##REF##16338780##8##–##REF##11165584##10##].</p> <p>Subjects are routinely asked to
avoid vaginal intercourse and the use of intravaginal products that might affect
test results prior to having specimens collected. However, compliance with
these requests is difficult to assess. When vaginal fluid is collected to
measure cytokine concentrations, it is important to determine what effect, if
any, there may be on the results if semen is present. The objective of this analysis was to
determine whether semen present in vaginal fluid alters proinflammatory
cytokine or SLPI concentrations. We hypothesized that the presence of semen
would increase the concentrations of vaginal proinflammatory cytokines and
SLPI.</p>
<title>2. MATERIALS AND METHODS</title> <p>This secondary analysis included data from 138
pregnant women, between 7 and 20 weeks gestation, who participated in a
prospective observational cohort study of the effects of BV on pregnancy
outcome. Subjects were recruited from
the prenatal clinics associated with the University of Washington Medical Center in Seattle, 
Wash, USA. Participation in the study was limited to
those subjects who met the following criteria: singleton pregnancy less than 20
weeks gestation, no prior preterm birth or major medical problems such as
chronic hypertension or pre-existing diabetes, and no recent antibiotic use.
The study was approved by the University of Washington
and the
Centers for Disease Control and Prevention Institutional Review Boards and all
subjects provided written, informed consent.</p> <p>The
data for the present analysis were taken from study entry visit. We compared subject history, Gram stain for
sperm and detection of acid phosphatase as predictors for the presence of semen
in vaginal fluid. Acid phosphatase was
considered to be the reference as it is an enzyme present in high
concentrations in semen, but not found in other secretions such as vaginal fluid,
saliva, or mucus [##REF##12136990##11##]. We then compared
the concentrations of proinflammatory cytokines and SLPI in samples from women
with and without semen detected in vaginal fluid.</p> <p>Subjects were
asked to abstain from vaginal intercourse and the use of intravaginal products
for 48 hours prior to their study visit. 
Subjects completed a structured interview with questions regarding
demographics, reproductive history, behavioral habits, and time of last
intercourse. A physical exam was
conducted including notation of Amsel criteria [##REF##6600371##12##]
as well as a vaginal
wet mount and Gram stain.</p> <p>Two Dacron swabs
were used to collect vaginal fluid from the posterior vaginal fornix and placed
in cryotubes containing 0.9 mL phosphate buffered saline. Swabs were frozen at −80 degrees and
stored for later cytokine and SLPI testing. 
An additional Dacron swab was used to collect vaginal fluid to prepare an
air-dried microscope slide which was then Gram stained and read at 100X
magnification for the presence of semen and determination of BV score by 
Nugent criteria [##REF##1706728##13##]. Vaginal
fluid from the frozen samples was aliquoted and used to measure proinflammatory
cytokine and SLPI concentrations by enzyme immunoassay [##REF##9881743##14##]. For acid phosphatase detection, vaginal
fluid was spotted to Whatman no.1 filter paper and then placed in a chemical
fume hood and sprayed until wet with the prepared reagent. Development of a
purple color within 1 minute was considered a positive test for the presence of
acid phosphatase [##UREF##0##15##, ##REF##4192621##16##].
The reagent was prepared by mixing 10 mL of
stock solution A (1 gram Fast Blue B, 20 grams sodium acetate trihydrate, 10 mL
glacial acetic acid, 100 mL dH<sub>2</sub>0) and 1.0 mL of stock solution B
(0.4 grams sodium alpha naphthyl acid phosphate, 5 mL dH<sub>2</sub>0) in a
spray bottle. The prepared reagent has a shelf life of 7 days, while stock solutions A and B are
stable for up to six months at 4 degrees.</p> <p>We used the Chi
square test or Fisher’s exact test for categorical variables. 
The Mann-Whitney test was used for
continuous variables. Analyses were
stratified by presence or absence of BV on Gram stain, given
that proinflammatory cytokines and SLPI may vary with BV status 
[##REF##11084573##17##, ##REF##9740515##18##].</p>
<title>3. RESULTS</title> <p>Of 138 subjects, 36 (26%) reported
vaginal intercourse within the past two days, despite instructions to
abstain. 28 (20%) had acid phosphatase
detected in their vaginal fluid, and 6 (4%) 
had sperm seen on Gram stain. Women with detectable acid phosphatase were
more likely than those without acid phosphatase to
report recent vaginal intercourse (68% versus 15%, <italic>P</italic> &lt; .0001), yet
subject history and acid phosphatase detection did not have high
concordance. Subjects with and without
detectable semen by acid phosphatase were similar with respect to age,
race/ethnicity, gestational age, and presence of genital infections (see ##TAB##0##Table 1##).</p> <p>The remaining
analyses compared vaginal cytokine and SLPI concentrations among women with and
without acid phosphatase detected in vaginal fluid. There was no significant
difference in IL-1<italic>β</italic>, IL-6, or IL-8 concentrations among
women with or without acid phosphatase, when stratified for BV 
(see Figures ##FIG##0##1(a)##, ##FIG##0##1(b)##, 
##FIG##0##1(c)##).
However, SLPI concentrations were significantly higher in the acid phosphatase
positive group regardless of BV status (see ##FIG##0##Figure 1(d)##).</p> <p>There was a significant increase in
IL-1<italic>β</italic> and decrease in SLPI among women with
BV (see Figures ##FIG##0##1(a)##, ##FIG##0##1(d)##)</p>
<title>4. DISCUSSION</title> <p>In this cohort of pregnant women, detection of semen by acid phosphatase was
associated with higher SLPI concentrations in vaginal fluid. These results are
not surprising, in that SLPI is a known component of seminal fluid, [##REF##7539415##19##] however, the presence of semen did not
appear to influence the vaginal
proinflammatory cytokine concentrations. 
Proinflammatory cytokines can also be detected in semen but are usually
not present at high concentrations except in the context of a sexually
transmitted infection in the male partner. Increased cytokine levels in semen have been established in HIV
infection (IL-1<italic>β</italic>), [##REF##16730226##20##] and genital infections
such as <italic>Chlamydia trachomatis</italic> (IL-8) [##REF##17107749##21##] and <italic>Neisseria
gonorrhoeae</italic> (IL-6, IL-8) [##REF##12228311##22##].</p> <p>We did find a high
(26%) prevalence of recent vaginal intercourse by history, despite instructions
to abstain prior to the study visit. We also found that patient history is not
a highly sensitive or specific marker for the detection of acid phosphatase, as
only 68% of those with acid phosphatase present reported recent
intercourse. Gram stain detection of
sperm is limited by the concentration of sperm as well as
duration of persistence in the vaginal fluid. In addition, we found that Gram
stain is not a useful method to screen for recent vaginal intercourse, with a
sensitivity of only 11%. While other tests such as prostate specific antigen
(PSA) may be more sensitive, the cost and implementation of equipment and
methodology to perform this ELISA test may be prohibitive to many laboratories.
It is necessary to note that while samples were immediately frozen for storage
after collection and care taken to minimize the number of freeze/thaw cycles
when aliquoting for testing, we can not determine if there was any degradation
of cytokine concentrations over time or due to the presence of semen. Also our findings may not be generalizable to
a nonpregnant population.</p> <p>Results from this
study suggest that the use of acid phosphatase to detect semen in vaginal fluid
samples can provide useful information and this testing can be performed using
a simple and inexpensive method. Vaginal
fluid samples with acid phosphatase should not have SLPI measured, as there is
likely a substantial contribution from seminal fluid. However, measurement of proinflammatory
cytokines is probably not influenced by the presence of semen. The acid
phosphatase detection test may be a useful adjunct for analysis of the effect
of seminal fluid on other host defense and immune factors in future
studies.</p>
<p>Recommended by Jane Schwebke</p> <p>The presence of semen in vaginal fluid, as identified by an acid phosphatase spot test, does not influence vaginal proinflammatory cytokine concentrations. <italic>Objective</italic>: determine whether semen, as detected by acid phosphatase, influences vaginal cytokines or secretory leukocyte protease inhibitor concentrations.
<italic>Methods</italic>: 138 pregnant women had vaginal fluid collected for Gram stain, acid phosphatase detection by colorimetric assay, and interleukin 1-Beta, interleukin-6, interleukin-8, and secretory leukocyte protease inhibitor measurement by enzyme immunoassay. Results for women with and without acid phosphatase were compared by Mann-Whitney test. <italic>Results</italic>: of 138 subjects, 28 (20%) had acid phosphatase detected; of these, only 19 (68%) reported recent intercourse and 3 (11%) had sperm seen on Gram stain. There were no significant differences in proinflammatory cytokine concentrations; however, secretory leukocyte protease inhibitor concentrations were significantly higher among women with acid phosphatase.
<italic>Conclusions</italic>: proinflammatory cytokine measurement does not appear to be affected by the presence of semen, but secretory leukocyte protease inhibitor is significantly higher when semen is present. Detection of semen by acid phosphatase was associated with higher vaginal 
SLPI concentrations, however, the presence of semen did not appear to influence vaginal
proinflammatory cytokine concentrations.</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Comparison of vaginal cytokine and
secretory leukocyte protease inhibitor concentrations by presence or absence of
acid phosphatase and bacterial vaginosis.</p></caption></fig>
<table-wrap id="tab1" position="float"><label>Table 1</label><caption><p> Subject characteristics by acid phosphatase.</p></caption><table frame="hsides" rules="groups"><thead><tr><th align="left" rowspan="1" colspan="1">Subject characteristics</th><th align="center" rowspan="1" colspan="1">Acid phosphatase + <italic>n</italic> = 28</th><th align="center" rowspan="1" colspan="1">Acid phosphatase − <italic>n</italic> = 110</th></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">Age (median)</td><td align="center" rowspan="1" colspan="1">27</td><td align="center" rowspan="1" colspan="1">26</td></tr><tr><td align="left" rowspan="1" colspan="1">Race/Ethnicity</td></tr><tr><td align="left" rowspan="1" colspan="1">White</td><td align="center" rowspan="1" colspan="1">11 (42)</td><td align="center" rowspan="1" colspan="1">43 (39)</td></tr><tr><td align="left" rowspan="1" colspan="1">African American</td><td align="center" rowspan="1" colspan="1">1 (4)</td><td align="center" rowspan="1" colspan="1">22 (20)</td></tr><tr><td align="left" rowspan="1" colspan="1">Hispanic</td><td align="center" rowspan="1" colspan="1">8 (31)</td><td align="center" rowspan="1" colspan="1">15 (14)</td></tr><tr><td align="left" rowspan="1" colspan="1">Asian</td><td align="center" rowspan="1" colspan="1">4 (15)</td><td align="center" rowspan="1" colspan="1">22 (20)</td></tr><tr><td align="left" rowspan="1" colspan="1">Other/unknown</td><td align="center" rowspan="1" colspan="1">2 (7)</td><td align="center" rowspan="1" colspan="1">8 (7)</td></tr><tr><td align="left" rowspan="1" colspan="1">Gestational age (median weeks)</td><td align="center" rowspan="1" colspan="1">16</td><td align="center" rowspan="1" colspan="1">16</td></tr><tr><td align="left" rowspan="1" colspan="1">Bacterial vaginosis</td><td align="center" rowspan="1" colspan="1">18 (64)</td><td align="center" rowspan="1" colspan="1">51 (46)</td></tr><tr><td align="left" rowspan="1" colspan="1">
<italic>Trichomonas vaginalis </italic>
</td><td align="center" rowspan="1" colspan="1">1 (4)</td><td align="center" rowspan="1" colspan="1">1 (10)</td></tr><tr><td align="left" rowspan="1" colspan="1">
<italic>Chlamydia trachomatis</italic> **</td><td align="center" rowspan="1" colspan="1">1 (5)</td><td align="center" rowspan="1" colspan="1">3 (3)</td></tr><tr><td align="left" rowspan="1" colspan="1">
<italic>Neisseria gonorrhoeae</italic>
</td><td align="center" rowspan="1" colspan="1">0 (0)</td><td align="center" rowspan="1" colspan="1">0 (0)</td></tr><tr><td align="left" rowspan="1" colspan="1">Self-reported vaginal intercourse</td><td align="center" rowspan="1" colspan="1">19 (68)</td><td align="center" rowspan="1" colspan="1">17 (15)</td></tr><tr><td align="left" rowspan="1" colspan="1">Sperm on Gram stain</td><td align="center" rowspan="1" colspan="1">3 (11)</td><td align="center" rowspan="1" colspan="1">3 (3)</td></tr></tbody></table></table-wrap>
<table-wrap-foot><fn><p>* All data are presented as <italic>n</italic> (%) unless otherwise noted.</p><p>** <italic>n</italic> = 21 for AP + group, <italic>n</italic> = 91 for AP − group.</p></fn></table-wrap-foot>
<graphic xlink:href="IDOG2008-820845.001"/>
[{"label": ["15"], "surname": ["Kube", "St\u00f6rzer", "Timm", "St\u00f6rzer"], "given-names": ["E", "HU", "KJ", "U"], "italic": ["Kriminalistik. Handbuch f\u00fcr Praxis und Wissenschaft II. Band 2"], "year": ["1994"], "publisher-loc": ["Stuttgart, Germany"], "publisher-name": ["Boorberg"]}]
Infect Dis Obstet Gynecol. 2008 Jul 3; 2008:820845