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 [ 1 , 2 ] .  
pregnant women with bv have an increased risk 
 of preterm labor and preterm delivery with the potential for neonatal morbidity 
 and mortality . early research concerning bv characterized the vaginal 
 microbiological flora using gram stain and culture [ 4 , 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 [ 6 , 7 ] . 

( il-1 ) , interleukin 6 ( il-6 ) , interleukin 8 ( il-8 ) , and the host defense 
 molecule secretory leukocyte protease inhibitor ( slpi ) have been of particular 
 interest [ 810 ] .
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 .
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 .
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 .  
we then compared 
 the concentrations of proinflammatory cytokines and slpi in samples from women 
 with and without semen detected in vaginal fluid .
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 
  as well as a vaginal 
 wet mount and gram stain .
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 .
vaginal 
 fluid from the frozen samples was aliquoted and used to measure proinflammatory 
 cytokine and slpi concentrations by enzyme immunoassay . 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 [ 15 , 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 dh20 ) and 1.0 ml of stock solution b 
 ( 0.4 grams sodium alpha naphthyl acid phosphate , 5 ml dh20 ) 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 .
we used the chi 
 square test or fisher s exact test for categorical variables .  

analyses were 
 stratified by presence or absence of bv on gram stain , given 
 that proinflammatory cytokines and slpi may vary with bv status 
 [ 17 , 18 ] .
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% , p <
.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 table 1 ) .
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 , il-6 , or il-8 concentrations among 
 women with or without acid phosphatase , when stratified for bv 
 ( see figures 1(a ) , 1(b ) , 
 1(c ) ) . 
 however , slpi concentrations were significantly higher in the acid phosphatase 
 positive group regardless of bv status ( see figure 1(d ) ) .
there was a significant increase in 
 il-1 and decrease in slpi among women with 
 bv ( see figures 1(a ) , 1(d ) )
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 ,   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 ) ,   and genital infections 
 such as chlamydia trachomatis ( il-8 )   and neisseria 
 gonorrhoeae ( il-6 , il-8 ) .
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 .  
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 .
 the presence of semen in vaginal fluid , as identified by an acid phosphatase spot test , does not influence vaginal proinflammatory cytokine concentrations .   objective : determine whether semen , as detected by acid phosphatase , influences vaginal cytokines or secretory leukocyte protease inhibitor concentrations . 
 
 methods : 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 . 
 results : 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 . 
 
 conclusions : 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 . 
1. INTRODUCTION
2. MATERIALS AND METHODS
3. RESULTS
4. DISCUSSION