
 chlamydia trachomatis is the most common bacterial sexually transmitted infection in the united states . in women , c. trachomatis can ascend from the endocervix to the upper genital tract and cause pelvic inflammatory disease ( pid ) and serious reproductive morbidity including infertility and ectopic pregnancy
. however , rates of progression vary and 80% or more of women with chlamydia do not develop pid . some women
clear chlamydial infection without tissue damage , while in some cases c. trachomatis induces a chronic low - grade infection .
this may lead to persistent inflammation of the upper genital tract causing long - term reproductive sequelae .
the pathogenesis of c. trachomatis disease is not well - understood , and pathogen - specific virulence factors that may contribute to variability in the course and outcome of infection have not been identified .  
nine surface - exposed c. trachomatis polymorphic membrane proteins ( pmps ) are encoded via a multigene family yielding pmpa to pmpi .
pmps represent 13.6% of the coding capacity of the c. trachomatis genome , suggesting they have a critical role in biology and virulence [ 5 , 6 ] .
pmpd is a species - common , pan - neutralizing antigen hypothesized to hold potential as a vaccine candidate .
thus , the development of high titers of antibody to pmpd might protect from infection or disease . on the other hand ,
chlamydia pneumoniae pmps have been shown to induce proinflammatory mediators in infected host cells , demonstrating the potential for these proteins to play a direct role in pathogenesis [ 7 , 8 ] .
all nine pmps are expressed on the surface of chlamydial elementary bodies ( eb ) and c. trachomatis - infected patients can produce antibodies to each pmp subtype . however , antibody profiles vary among c. trachomatis - infected patients . in addition ,
comparative genomics has revealed genetic variation and rearrangements among pmp gene families in different strains and isolates [ 5 , 9 ] .
this suggests that immune pressure leads to antigenic variation in these surface - exposed proteins [ 5 , 9 ] , a further indication that these proteins have a role in chlamydial virulence .  
pmps may be involved in virulence , but very little is known about their role in the development of pid and adverse reproductive sequelae .
examined variation in pmp - specific antibody responses in four distinct patient populations , demonstrating that women with pid had significantly higher reactivity to pmpb and pmpi compared to adolescent females with lower genital tract infection .
these data may reflect a role for these specific pmps in inflammation , or simply that women with pid had sustained increased exposure due to repeated or chronic infection .   in a separate study , tan et al . found that pmps exhibit on / off switching in vitro which enables independent expression of each pmp .
pmpa , pmpd , and pmpi had very low  off  frequencies of 0.51% , suggesting that expression of these pmps provides an in vitro phenotypic advantage .
the high  on  frequencies of pmpd and pmpi correlate with the fact that anti - pmpd and -pmpi antibodies are commonly detected in c. trachomatis - infected patients
. however , despite the high  on  frequency of pmpa , anti - pmpa antibodies are relatively rare .
further research from this group found that pmp transcriptional units are differentially expressed during chlamydial development .
in addition , pmp expression was altered under penicillin - induced stress , except for the expression of pmpa , pmpd , and pmpi , which remained steady . taken together ,
these data suggest an importance for pmpa , pmpd , and pmpi in chlamydial pathogenesis .
however , the role of these pmps in chlamydial pid has never been examined in humans . in order to examine the role of pmpa , pmpd , and pmpi in chlamydial pathogenesis
, we conducted a pilot study to determine if antibody responses specific for these pmps were associated with parameters of inflammation or sequelae in a group of women with documented c. trachomatis pid .
this study utilized data from the pid evaluation and clinical health ( peach ) study .
this was the first randomized clinical trial to compare inpatient and outpatient treatment in preventing long - term complications among 831 women with mild - to - moderate pid .
the methods of subject recruitment , data collection , and followup have been reported elsewhere .
briefly , between march 1996 and february 1999 , women aged 1437 years were recruited from emergency departments , obstetrics and gynecology clinics , sexually transmitted disease clinics , and private practices at 7 primary and 6 secondary sites throughout the eastern , southern , and central regions of the united states .
women who had suspected pid and gave informed consent were eligible for the peach study .
eligibility included a history of pelvic discomfort for less than 30 days , findings of pelvic organ tenderness ( uterine or adnexal ) on bimanual examination , and leukorrhea and/or mucopurulent cervicitis and/or untreated but documented gonococcal or chlamydial cervicitis .
a total of 2941 women were screened for study entry , of those 346 ( 11.8% ) did not meet the clinical inclusion criteria for randomization .
women were additionally excluded if they were pregnant ( n = 141 , 4.8% ) ; had taken antimicrobials within the past 7 days ( n = 248 , 8.4% ) ; had a history of hysterectomy or bilateral salpingectomy ( n = 248 , 8.4% ) ; had an abortion , delivery , or gynecologic surgery within the past 14 days ( n = 51 , 1.7% ) ; had a suspected tubo - ovarian abscess or other condition requiring surgery ( n = 191 , 6.5% ) ; had an allergy to the study medications ( n = 163 , 5.5% ) ; were homeless ( n = 29 , 1% ) ; or had vomiting after a trial of antiemetic treatment ( n = 11 , 0.4% ) .
our analysis included a pilot sample of 40 c. trachomatis - positive women , whose serum samples were previously analyzed for pmp antibodies .
women were randomized to either inpatient treatment of intravenous cefoxitin every 6 hours and doxycycline orally twice a day for 14 days ; or outpatient treatment consisting of a single intramuscular injection of cefoxitin and oral doxycycline twice a day for 14 days . because the treatment modality was not associated with reproductive morbidities in the peach study
participants were followed - up with in - person visits at 5 and 30 days after treatment . at the 30-day followup , the gynecological exam was repeated .
telephone followups were conducted by the study nurses every 3 months during the first year after enrollment and then every 4 months until june 2004 . at that point ,
information was obtained by self - report for 69.1% of the cohort , with a mean followup of 84 months .  
the interview collected information on reason for visit , brief pain history , demographics , history of pid / sexually transmitted diseases , sexual and contraceptive history , reproductive decisions , douching history , pregnancy history , medical and gynecological history , and lifestyle habits .
followup interviews collected self - reported information on pelvic pain , pregnancy and births , signs and symptoms of pid , stds , contraceptive use , pattern of sexual intercourse , and health care utilization .  
endometrial biopsy and cervical swab specimens were obtained for histological examination including chlamydial polymerase chain reaction ( pcr ) and gonococcal culture .
all cultures and pcr were performed at a central reference laboratory . for the patients with endometrial biopsies , two reference pathologists
separately evaluated at least one section stained with hematoxylin and eosin and at least one stained with methyl green pyronin .
a disagreement about the presence or absence of neutrophils and plasma cells was settled by both pathologists reading the slides together and coming to an agreement .
histological endometritis was based on a modification of the criteria proposed by kiviat et al . .
endometritis was defined as the presence of at least five neutrophils in the endometrial surface epithelium in the absence of menstrual endometrium and/or at least two plasma cells in the endometrial stroma .
this definition has been found to be the best predictor of upper genital tract infection plus salpingitis , with a sensitivity of 92% and specificity of 87% .  
infertility was determined among women reporting no birth control or methods considered being unreliable , including withdrawal , rhythm method , vasectomy , or using the following methods rarely or occasionally : diaphragm , condoms , spermicidal foam / cream / jelly / suppositories , or cervical cap .
infertility was defined by lack of conception despite unprotected intercourse during 12 or more months of followup .
self - reported pregnancy was determined by a positive urine / blood test or physician 's diagnosis among all women in the cohort .
live birth was determined among all women in the cohort by self - report during followup .
recurrent pid was self - reported and verified whenever medical records were available ( 45% of cohort ) .
women were considered to have recurrent pid if they experienced a subsequent episode of pid more than 30 days after the index illness .
chronic pelvic pain was defined by two or more consecutive reports of pelvic pain during telephone followup interviews administered through 32 months .
data from at least 2 followup visits were required to determine chronic pelvic pain .  
pmp antibody levels were previously measured among a subset of 40 c. trachomatis - positive women who had stored serum samples available .
briefly , purified ebs or partially purified rpmp polypeptides ( rpmpd - fl and rpmpi - n ) were subjected to sodium dodecylsulfate - polyacrylamide gel electrophoresis ( sds - page ) .
amounts of rpmps were standardized by immunoblot analysis with anti - his tag antibody ( 1  :  1,500 ; ge healthcare ) .
blots were visualized using molecular dynamics typhoon 9400 imager ( amersham biosciences , nj ) .
antibody reactivity against the highest molecular - mass band in each lane was analyzed using image quant 5.2 image analysis software ( molecular dynamics sunnyvale , calif ) .
data were normalized against his - tag - specific antibody reactivity .   since pmpa , pmpd , and pmpi
are uniquely expressed and may play a role in chlamydial pathogenesis [ 9 , 10 ] , we chose to only include these pmps in our analyses .
our objective was to determine if markers of inflammation or sequelae differed between women who displayed antibody reactivity to pmpa , pmpd , or pmpi and those who did not .
differences in baseline characteristics were compared between groups using chi - square or fisher 's exact tests and t - test for the normally distributed continuous variables .
chi - square or fisher 's exact tests were also used to compare differences in the frequency of inflammatory markers ( elevated white blood cell count ( wbc ) , temperature , elevated c - reactive protein ( crp ) , bilateral adnexal tenderness , cervicitis , endometritis , upper genital tract infection ( ugti ) , erythrocyte sedimentation rate ( esr ) ) , and reproductive outcomes ( infertility , pregnancy , live birth , chronic pelvic pain , and recurrent pid ) between groups .
as the distributions of pmpa , pmpd , and pmpi were skewed , nonparametric tests were used .  
we also sought to determine if high levels of pmp antibody expression were associated with inflammation or sequelae . after running sensitivity analyses ,
antibody reactivity groups for pmpd and pmpi were defined using median cut - points ( high reactivity : pmpd  0.41 ; pmpi  0.89 and low reactivity : pmpd < 0.41 ; pmpi < 0.89 ) . due to the small number of women who expressed antibodies to pmpa
, we were unable to examine levels of antibody expression ( n = 5 ) .
logistic regression was used to calculate odds ratios ( or ) and 95% confidence intervals ( ci ) .
cox regression was used to calculate hazard ratios and 95% ci for time - to - pregnancy and time - to - recurrent pid .
additionally , time to pregnancy was adjusted for history of infertility , which was self - reported at baseline .
if any model had less than 5 in any cell , it was excluded from regression analysis .
overall , women in this cohort tended to be less than 25 years of age ( 85.0% ) , african american ( 77.5% ) , single ( 86.8% ) , and having at least a high school education ( 60.0% ) . at baseline ,
the majority of women reported abnormal vaginal discharge ( 62.5% ) , had bilateral adnexal tenderness ( 80.0% ) and mucopurulent cervicitis ( 65.7% ) , and had chlamydia isolated from the cervix only ( 58.3% ) .
women who expressed antibody to pmpi were more likely to smoke compared to women who did not express pmpi antibody ( 56.7% versus 20.0% ; p = 0.0411 ) ( table 1 ) .
there were no other significant differences in important baseline characteristics between women who expressed antibody to pmpa , pmpd , or pmpi and women who did not .  
results show that compared to women who did not express antibody to pmpa , rates of elevated wbc ( 40.0% versus 23.5% ) , increased crp ( 66.7% versus 46.2% ) , increased esr ( 40.0% versus 31.4% ) , endometritis ( 100% versus 60.7% ) , and ugti ( 75.0% versus 46.8% ) were higher among women who expressed antibody to pmpa ( table 2 ) .
similarly , there were no significant differences in the frequency of infertility , recurrent pid , or chronic pelvic pain between groups .
however , only 40% of women with antibody reactivity to pmpa achieved pregnancy compared to 85.7% of women who did not express antibody reactivity to pmpa ( p = 0.042 ) .
in addition , none of the women with antibody reactivity to pmpa had a live birth , while 80% of women without antibody reactivity to pmpa had a live birth ( p = 0.005 ) .
expression of anti - pmpa antibody was significantly increased in women who did not achieve pregnancy ( p = 0.0192 ) or did not achieve a live birth ( p = 0.0043 ) .
there were no significant differences in inflammatory markers or reproductive sequelae between women who displayed antibody reactivity to pmpd and women who did not ( table 3 ) .
results did not change when an antibody response to pmpd was considered as a continuous variable .
however , women expressing antibody to pmpi were more likely to have ugti compared to women who did not express antibody to pmpi ( 61.5% versus 20.0% ; p = 0.026 ) . women who expressed antibody to pmpi were slightly more likely to have endometritis ( 72.7% versus 50.0% ) although this did not reach statistical significance ( p = 0.2096 ) .
when pmpi antibody response was examined as a continuous variable , the mean expression did not significantly differ between those with ugti and those without ugti ( p = 0.276 ) .
when levels of antibody expression for pmpd and pmpi were examined , there were no significant differences in the frequency of inflammatory markers or reproductive sequelae between high and low antibody reactivity groups .
although nonsignificant , women with high pmpd reactivity were slightly more likely to have an elevated wbc count ( 33.3% versus 16.7% ) , elevated esr ( 42.9% versus 21.1% ) , elevated crp ( 62.5% versus 37.5% ) , mucopurulent cervicitis ( 77.8% versus 52.9% ) , and endometritis ( 75.0% versus 56.3% ) compared to women with low pmpd reactivity .
women with high pmpi reactivity were slightly more likely to have elevated crp ( 55.6% versus 42.9% ) , bilateral adnexal tenderness ( 85.0% versus 75.0% ) , mucopurulent cervicitis ( 72.2% versus 58.8% ) , endometritis ( 71.4% versus 61.1% ) , and ugti ( 58.8% versus 42.1% ) compared to women with low pmpi reactivity although this did not reach statistical significance .
similarly , a nonsignificant decrease in pregnancy rates ( adjusted hazard ratio ( ahr ) 0.7 , 95% ci 0.31.6 ) and increase in recurrent pid were observed for women with high pmpd antibody reactivity ( ahr 1.3 , 95% ci 0.28.3 ) ( table 5 ) .
in contrast , high pmpi antibody reactivity had minimal effects on sequelae . however , after adjustments , women with high pmpi antibody reactivity showed a nonsignificant trend towards decreased live births ( aor 0.6 , 95% ci 0.14.0 ) .
among women with mild - to - moderate chlamydial pid , those who expressed antibody to pmpa were less likely to achieve pregnancy and less likely to report a live birth .
the overall effects of seropositivity for pmpd and pmpi on inflammation and reproductive sequelae were minimal .
trends towards elevated baseline genital tract and systemic inflammation , increased reproductive morbidity , and decreased pregnancy rates were observed among women with high pmpd antibody reactivity .
.   the progression to pid following lower genital   c. trachomatis   infection varies . among high - risk groups , 24.5% of women with untreated chlamydial infection
will develop pid within 2 weeks , and 19% with treated chlamydial infection will develop pid within 3 years [ 1 , 1518 ] .
as pmpa , pmpd , and pmpi have relatively low  off  frequencies ( 0.51% ) , they should be present for antigenic processing and presentation allowing for antibody induction in the majority of infected individuals .
however , the prevalence of pmpa - expressing inclusions in in vitro - grown c. trachomatis does not correlate with the the low frequency of pmpa antibodies detected in our cohort of women with clinical pid and in other populations of c. trachomatis - infected patients .
suggest that pmp expression in in vitro - grown c. trachomatis differs from c. trachomatis in the human genital tract .
chlamydiae processing and secretion of pmp fragments may also differ , possibly resulting in varied antibody expression .
it is also possible that pmpa may have poor immunogenicity or that pmpa antibodies were generated at low levels not recognized by the initial sds - page analysis .
still , pmpa , pmpd , and pmpi are the most conserved pmps of c. trachomatis , and their expression is unaltered in response to stress .
this may suggest that pmpa , pmpd , and pmpi are important for chlamydial survival and may play a role in chlamydial pathogenesis [ 9 , 10 ] .
studies have found a link between tubal occlusion and chlamydial antibodies [ 1 , 19 ] , as well as chlamydial infection and post - pid infertility [ 1 , 20 ] .
inflammation caused by chlamydial infection may play a role in the development of infertility , through damage to the cilia lining of the fallopian tubes , fallopian tube blockage or closure , or adhesion formation among pelvic organs .
our data indicate that women who express pmpa antibody were less likely to achieve pregnancy and less likely to have a live birth .
these variables are easier to define compared to infertility which requires classification of contraception than can be hampered by changing variables over time , concurrent use of more than 1 method of contraception , missing data , and compliance .
therefore , our results may suggest that pmpa plays a role in upper genital tract pathology .
although rates of inflammatory markers were increased among women who express pmpa antibody , no statistically significant differences were found between the groups .
due to the low frequency of pmpa in our cohort , we were also unable to examine levels of antibody titer .  
data suggest that pmpd acts as an adhesion molecule and stimulates proinflammatory cytokines through the nuclear factor-b pathway [ 7 , 8 ] . since pmpd may stimulate host cell inflammatory responses , it is possible that increased antibody to pmpd reflects increased exposure to these potentially pathogenic ligands .
we did find increased inflammation and reproductive sequelae among women with high antibody titers to pmpd .
overall , expression of pmpd antibody appeared to have minimal effects on inflammation and reproductive sequelae in this study .
reported that anti - pmpd antibodies result in neutralization of chlamydial elementary bodies and reduced infectivity in vitro . in our in vivo study , we found no evidence for protection from disease in women with high seropositivity to pmpd .
however , it should be noted that as our study used recombinant denatured material for the measurement of seroreactivity , in vivo antibody reactivity to native pmpd present on chlamydial elementary bodies may not be fully reflected .
found that the peach pid population had significantly higher levels of pmpi antibodies compared to adolescent females with lower genital tract infection ( p < 0.0001 ) .
this could suggest that high titers to pmpi could be associated with chlamydial progression to the upper genital tract .
we did find that women with antibody reactivity to pmpi were more likely to have ugti .
when we examined pmpi as a continuous variable , we did not find a significant difference in pmpi antibody expression between women with ugti and women without ugti .
women from the peach cohort are older and have likely been exposed to c. trachomatis more often than adolescents .
screening studies have found that older women have less infection but increased chlamydial antibodies compared to adolescents .
therefore , high antibody reactivity to pmps could represent a measure of cumulative chlamydial exposure , indicating that women with more infections suffer greater sequelae .
in fact , a study among 443 peach participants found that pid recurrence was higher ( hr 2.48 , 95% ci 1.006.27 ) , and pregnancy rates were significantly lower ( hr 0.47 , 95% ci 0.280.79 ) among women whose antibody titers to chlamydia eb were in the highest tertile .
however , we were unable to find any significant associations between high antibody reactivity to pmpi or pmpd and reproductive sequelae .
further , we found no significant associations with markers of inflammation .   there could be several reasons for our mostly null findings .
therefore , we were unable to compare women with chlamydial pid to women with uncomplicated c. trachomatis infection .
future studies should compare these groups to determine the role of pmps in c. trachomatis progression .
it is possible that host susceptibility may explain why some women with chlamydia experience sequelae and others do not .
therefore , genetic variations in host immune receptors may cause unfavorable inflammation and explain the variability in outcomes .
we did find a borderline association with cervicitis among women with high pmpd reactivity using logistic regression , and chlamydial cervicitis has been associated with a higher chlamydial load .   to our knowledge ,
this is the first study to examine the role of pmp antibody response in inflammation and post - pid sequelae .
data were obtained from a prospective randomized clinical trial with comprehensive demographic , clinical , and obstetric measurements .
however , as some patients with clinically suspected pid might actually have ovarian cysts , pelvic adhesions , or endometriosis , some women in our study may not have had true upper genital tract infection .
we attempted to minimize some of this misclassification by excluding women reporting greater than 30 days of pain at the time of enrollment .
we recognize that reproductive outcomes were based on self - reported data and that misclassification bias is possible .
however , it is still sufficient for comparative rough estimates of pmp - subtype - specific titers . in addition
serovars for c. trachomatis were not determined in the peach study . however , pmpa , pmpd , and pmpi are the most conserved pmps of c. trachomatis , and it is possible that their functions are also conserved across serovars [ 9 , 10 ] . as this pilot study
is limited by power , larger studies should continue to explore the role of pmps in the course and outcome of c. trachomatis infection .
specifically , the correlation between pmpa antibody reactivity and reproductive sequelae needs to be confirmed .
variability in the progression of chlamydial infection may be due to varied expression of chlamydial pmps that are reflected by the serum anti - pmp antibody response .
our data suggest that women who express pmpa antibody had decreased pregnancy rates and decreased live births .
rates of inflammatory markers were increased among women with pmpa antibody although these results were nonsignificant .
in contrast , a positive antibody response to pmpd or pmpi appeared to relate minimally to reproductive sequelae and inflammation .
results were the same when high antibody reactivity to both pmpd and pmpi was explored .
these results suggest a possible role for pmpa , but not for pmpd or pmpi , in upper genital tract pathology .
 
 chlamydia trachomatis polymorphic membrane proteins ( pmps ) may increase genital tract inflammation and play a role in virulence . 
 antibody levels for pmpa , pmpd , and pmpi , measured in densitometric units , were assessed among a pilot sample of 40 c. trachomatis - infected women with mild - to - moderate clinical pid . women who expressed antibodies to pmpa 
 were less likely to achieve pregnancy ( 40.0% versus 85.7% ; p = 0.042 ) and less likely to have a live birth ( 0.0% versus 80.0% ; p = 0.005 ) compared to women who did not express antibody to pmpa . 
 women who expressed antibodies to pmpi were more likely to have upper genital tract infection ( 61.5% versus 20.0% ; p = 0.026 ) . 
 however , seropositivity to pmpi and pmpd did not modify the risk of reproductive sequelae or inflammation . 
 seropositivity to chlamydial pmpa may represent a biomarker of increased risk of sequelae secondary to infection with c. trachomatis . 
1. Introduction
2. Methods
3. Results
4. Discussion