in 2010 , approximately 15 million babies were born preterm worldwide , and more than one million died due to complications from preterm births ( ptb ) .
however , a pathogen is identified in only one third of the cases , despite evidence of infection .
obligate intracellular bacteria , which do not grow on media used routinely to isolate human pathogens from clinical samples , might represent possible agents of ptb .
chlamydia trachomatis , an obligate intracellular bacterium , is considered the world 's most common sexually transmitted bacterial pathogen .
waddlia chondrophila is another member of the chlamydiales order that has been shown to cause abortions in bovines .
both of these intracellular bacteria have also been implicated in human adverse pregnancy outcomes [ 47 ] .
in addition , c.  trachomatis is known to cause premature rupture of the membranes and premature uterine activity , and growing evidence suggests a role for c.  trachomatis in ptb . in this study
we studied 407 women with ptbs or uneventful term pregnancies attending the university hospital of lausanne , lausanne , switzerland .
in addition to serology , we also performed pcr to detect waddlia and chlamydia in the placenta and vaginal samples taken from these women , as well as histology on the placenta . from 2006 to 2009 , 407 women were enrolled into this study  at the obstetrical ward of the university hospital of lausanne .
the ptb group ( n  =  146 ) included women who spontaneously delivered before 37 weeks ' gestation . the control group ( n  =  261 ) included women attending a labour ward with uneventful term pregnancies and no history of miscarriages , stillbirths or preterm labour .
we compared demographic data and risk factors of patients with and without ptb or c.  trachomatis infection by the pearson  test ( or the fisher exact test when indicated ) for categorical variables . for continuous variables ,
statistical analyses were performed using the stata software , version 13.0 ( statacorp , college station , tx , usa ) . only positive urine cultures , gestational and maternal age were significantly different between control and ptb groups ( table  1 ) .
other infectious causes were investigated in the ptb group , showing that a positive culture was found in the vagina , maternal or fetal side of the placenta in 32% , 37% and 17% of the ptb patients , respectively ( supplementary table  1 ) . among all these bacterial species , genital mycoplasma and gardnerella vaginalis , which have been previously associated with ptb ,
were recovered in some subjects from the vagina only ( n  =  25 and 3 , respectively ) , never from the placenta .
sera were tested for antibodies directed against c.  trachomatis and w.  chondrophila , respectively , by using the momp - r , ct pelisa ( r - biopharm , darmstadt , germany )   and waddlia - specific immunofluorescence as described elsewhere .
isolated igg mainly reflects past or chronic infection , whereas igm and/or iga reflect acute infection .
briefly , w.  chondrophila strain atcc vr-1470 was used as antigen , whereas fluoline g or m ( biomrieux , marcy l'toile , france ) were used as secondary antibodies .
an antibody titre of 1/64 for igg and 1/32 for igm were considered as positive , respectively .
there was no difference between control and ptb groups in terms of anti - chlamydia igg and iga and anti - waddlia igg and igm titres ( table  1 ) .
a total of 54 patients tested positive only for waddlia igg and 26 only for chlamydia igg , indicating the absence of serologic cross - reaction between both pathogens .
only six patients tested positive for both waddlia and chlamydia igg ( p 0.446 ) . c.  trachomatis igg seropositivity (
table  2 ) was associated with civil status ( divorced vs. married , odds ratio ( or ) 7.85 ; 95% confidence interval ( ci ) 2.6123.62 ) , education ( or 0.28 ; 95% ci 0.100.81 ) and number of previous sexual partners ( > 6 vs. 1 : or 13.12 , 95% ci 1.53112.32 ;  not answered  vs. 1 : or 13.55 , 95% ci 1.76104.09 ) .
patients who used condoms as a previous contraceptive method show less c.  trachomatis positive serologies , although this was not statistically significant . of note , only six ( 55% ) and one ( 9% ) of the 11 patients positive for c.  trachomatis dna were also positive for c.  trachomatis igg and iga , respectively . however , c.  trachomatis igg - positive patients exhibited significantly more histologic chorioamnionitis ( 50% ) than c.  trachomatis igg - negative patients ( 28.3% , p 0.015 ) .
waddlia seropositivity was not associated with age , number of lifelong sexual partners , place of residence ( rural vs. urban ) , smoking , pet ownership or meat consumption ( data not shown ) .
interestingly , 4.9% of women of european heritage vs. 14.3% of those of non - european heritage had positive waddlia igg serologies of 1/256 ( p 0.008 ) .
this correlation was also observed for waddlia igm of 1/32 ( 3.6% of european vs. 10.4% of non - european , p 0.034 ) . among non - european women ,
the highest waddlia igg titres ( 1/128 or 1/256 ) were more frequent for black ethnicity ( 20% and 14.3% , respectively ) . when total immunoglobulin ( ig 1/64 ) against waddlia was considered , condoms as a previous contraceptive method protected against waddlia infection ( 12.1% condom users vs. 21.4% of the noncondom users exhibited anti - waddlia ig reactivity , p 0.041 ) .
after dna extraction with qiaamp dna mini kit ( qiagen , hilden , germany ) , all vaginal swabs and placenta samples were screened for c.  trachomatis dna by a taqman real - time pcr targeting the cryptic plasmid of c.  trachomatis , as described earlier .
samples were also tested by a 16s rrna waddlia - specific real - time pcr , as described previously .
vaginal and placental samples from the ptb group were significantly more often c.  trachomatis dna positive than the control group ( p 0.002 , unadjusted or 8.51 ; 95% ci 1.8139.93 ) .
this association reminded significant even after adjustment for maternal age , origin , civil status , education , number of sexual partners and positive urine cultures ( or 7.93 ; 95% ci 1.3446.76 ) .
stepwise logistic regression allowed us to confirm that the presence of c.  trachomatis dna is an independent factor associated with ptb ( fig .  1 ) .
thirty - four patients were positive only for w.  chondrophila , whereas ten other subjects were positive only for c.  trachomatis by pcr .
only one patient was positive for both , demonstrating the absence of cross - amplification of these two obligate intracellular bacteria .
presence of waddlia dna was demonstrated in the genital tract or placenta of 13 ptb subjects ( table  3 ) .
of these 13 patients , four exhibited both a positive waddlia serology and presence of waddlia dna .
two had presence of waddlia in multiple samples , including vaginal swab , urine and placenta .
only two of these 13 patients had either a positive c.  trachomatis serology or pcr .
both c.  trachomatis and w.  chondrophila have been implicated in adverse pregnancy outcomes .
however , there have been contradictory findings pertaining to the role of c.  trachomatis infection in ptb .
a few small studies have failed to demonstrate a risk of ptb associated with c.  trachomatis infections , although several large , well - conducted studies have supported a role for c.  trachomatis in ptb [ 1517 ] .
thus , in a large study including 4055 subjects , about 15% of ptb was attributed to c.  trachomatis 
 .
we have previously confirmed an association between waddlia antibodies and human miscarriage and have demonstrated its presence in placenta and/or genital tract [ 47 ] . in the present study ,
w.  chondrophila was not associated with ptb despite inflammation , and abnormal histology of the placenta was observed among patients infected by waddlia ( positive pcr ) .
in contrast to waddlia , presence of chlamydia dna in the genital tract and/or in the placenta was strongly associated with ptb . however , c.  trachomatis seropositivity did not correlate with ptb , suggesting that acute rather than chronic c.  trachomatis infection is associated with ptb .
interestingly , these results are opposite to our previous findings , in which we demonstrated that both chronic and acute c.  trachomatis infections correlate with miscarriages .
our results confirm those of a population - based prospective study recently published in the generation r cohort in the netherlands . in this large study ( > 4000 pregnant women ) ,
conflicting data between chlamydia dna , serology and pregnancy outcomes may reflect different pathophysiologic mechanisms , in which chlamydia - induced miscarriages are the result of an immunologic process , whereas the direct impact of the bacteria results in chlamydia - induced ptb .
a limitation of our study was the absence of investigation of other infectious etiology of ptb in all patients ( ptb and control subjects ) .
some pathogens can reach the placenta by hematogenous spread or by an ascending route from the cervix . among bacterial infections ,
ureaplasma urealyticum , mycoplasma hominis and bacterial vaginosis have been associated with ptb , but controversies regarding their true role during pregnancy persist .
overall , these results strongly suggest a role of acute c.  trachomatis infection in ptb , and we strongly recommend systematically testing for c.  trachomatis in any woman at risk for or after preterm delivery .
 waddlia chondrophila and chlamydia trachomatis are intracellular bacteria associated with human miscarriage . 
 we investigated their role in human preterm birth . whereas presence of chlamydia trachomatis dna in genital tract was associated with human preterm birth , waddlia was not , despite being present in women 's genital tracts . 
Introduction
Discussion
Conflict of interest