Patent Application: US-66732008-A

Abstract:
the present invention concerns methods of identifying extra - uterine pregnancies and involves the screening of samples for the presence of certain molecules now known to be markers of extra - uterine pregnancies . the present invention provides a method for identifying an ectopic pregnancy , said method comprising the steps of : providing a sample from a subject ; identifying a level of one or more of : inhibin / activin beta b subunit gene activin b cysteine - rich secretory protein 3 ; and / or carboxypeptidase - b1 slp1 elafin prolactin in the sample , wherein the level of inhibin / activin beta b subunit gene , activin b , crisp - 3 , slp1 , elafin , prolactin and / or cpb1 is associated with , or indicative of , an ectopic pregnancy .

Description:
the present invention will now be described in detail with reference to the following figures which show : expression of inhibin / activin subunit mrna in uterine decidua ; a ) βb subunit is differentially expressed ( p & lt ; 0 . 001 , anova ). it is reduced in women with tubal ectopic pregnancy ( ectopic ) when compared to that of women undergoing surgical management of miscarriage ( misc ) ( p & lt ; 0 . 01 , anova ) and surgical termination of pregnancy ( top ) ( p & lt ; 0 . 01 , anova ). b ) there was no difference in βa expression in these groups ( anova ). c ) the α subunit is differentially expressed in the group ( p & lt ; 0 . 05 , anova ) with lower expression in ectopic pregnancy than in miscarriage ( p & lt ; 0 . 05 , anova ). immunohistochemistry for inhibin / activin α and β a subunits ; a ) human corpus luteum immunostained for the α subunit as a positive control showing localization ( brown ) in the granulosa - lutein cells ( glc ) and no staining in the surrounding stroma ( st ). b ) staining for the α subunit in endometrial glands from decidualized tissue ( g ) with lighter staining in the stroma ( s ). c ) human follicle immunostained for the βb subunit as a positive control showing marked staining ( brown ) in the granulosa cells ( gc ) with less staining in the neighboring theca cells ( tc ). d ) staining for the βb subunit in decidua showing some glandular ( g ) and stromal ( s ) localization . e ) decidual βb subunit localization from a woman with a tubal ectopic pregnancy showing cytoplasmic staining in the glandular epithelium ( g ) and minimal staining in the stromal cells ( s ). ( f ) uterine decidua from a woman undergoing surgical management of miscarriage showing a similar expression pattern in the glands ( g ) with evidence of more immunostaining in the stromal cells ( s ). ( g ) uterine decidua from a woman undergoing top showing βb immunolocalization in the both the glandular epithelium ( g ) and stroma cells ( s ). negative control section of ( g ) showing no specific immunostaining in the glands ( g ) or stroma ( s ). scale bar = 50 μm except ( c ) where it is 100 μm inhibin / activin subunit mrna expression during decidualization of endometrial stromal cells in vitro . a ) the expression of βb subunit increased ( p & lt ; 0 . 05 ) during decidualization ( anova , pearson correlation ). b ) the expression of the α subunit also increased ( p & lt ; 0 . 05 ) but to a less marked degree ( anova , pearson correlation ). there was no difference in the expression of the βa subunit ( anova , pearson correlation ). t1 = 0 h , t2 = 24 h , t3 = 48 h , t4 = 72 h , t5 = 96 h , t6 = 120 h . morphological assessment of the degree of endometrial decidualization in vivo . a ) scoring of the secretory transformation of the glandular epithelium and level of stromal decidualization in endometrium collected from surgical management of miscarriage ( misc ), termination of pregnancy ( top ) and tubal ectopic pregnancy ( ectopic ). b ) classification of morphological degree of biopsy decidualization (+, +/−, −) showed that the endometrium from ectopic pregnancies was less decidualized ( p & lt ; 0 . 05 , chi - squared ). c ) correlation between the level of decidualization (−, +/−, +) and decidual inhibin / activin βb expression ( p = 0 . 0005 , spearman correlation ). expression of key markers of decidualization . a ) igfbp1 expression was different between groups ( p & lt ; 0 . 005 , kruskal wallis ). it was lower in tubal ectopic pregnancy ( ectopic ) decidua when compared to that from miscarriage ( misc ) ( p & lt ; 0 . 01 , kruskal wallis ) or termination of pregnancy ( top ) ( p & lt ; 0 . 05 , kruskal wallis ). b ) prolactin expression also different between groups ( p & lt ; 0 . 01 , kruskal wallis ). it was lower in ectopic pregnancies when compared to termination of pregnancy ( p & lt ; 0 . 05 , kruskal wallis ). serum activin b and progesterone concentrations . a ) serum activin b is lowest in tubal ectopic pregnancies ( ectopic ) when compared to miscarriages ( misc ) and termination of pregnancy ( top ) ( p = 0 . 001 , anova ) b ) serum activin b is lower in tubal ( ep ) than intrauterine pregnancies ( iup ) ( p & lt ; 0 . 005 , t - test ). c ) there is a correlation ( p = 0 . 005 , spearman correlation ) between activin b and the degree of decidualization (+, +/−, −). d ) serum progesterone is lowest in miscarriages ( p & lt ; 0 . 05 , kruskal wallis ). e ) there was no difference in progesterone concentrations when intrauterine pregnancies were compared to tubal pregnancies ( t - test ). f ) there was no correlation between the degree of decidualization and serum progesterone concentrations ( spearman correlation ). quantitative rt - pcr to demonstrate levels of crisp - 3 ( fig7 a ) and cpb1 ( fig7 b ) in uterine decidua from women with intra - ( stop and miscarriage ) and extra - uterine ( tubal ) pregnancies . immunohistochemistry for crisp3 in uterine decidua from tubal pregnancy . expression strongest in the glandular epithelium and secretions . graphical representation of relative slpi ( a ) and elafin ( b ) mrna expression in uterine decidua from women undergoing surgical termination of pregnancy ( stop ), surgical management of miscarriage , and surgical management of ectopic pregnancy . slpi and elafin protein expression demonstrated by immunohistochemistry in uterine decidua from women undergoing surgical management of ectopic pregnancy , surgical management of miscarriage and surgical termination of pregnancy ( stop ). slpi protein expression in leukocytes ( l ) and epithelial glands ( g ) of uterine decidua from a woman undergoing surgical management of ectopic pregnancy ( a and b ) and undergoing surgical management of miscarriage ( c ). elafin protein expression in the decidual leukocyte ( l ) populations of a woman undergoing surgical management of ectopic pregnancy ( d ), surgical management of miscarriage ( e ) and surgical termination of pregnancy ( f ). membraneous elafin protein expression ( m ) in the epithelial glands is also seen in d . negative controls for slpi and elafin are shown in g and h , respectively ( both uterine decidua from a woman undergoing surgical management of ectopic pregnancy ). scale bars = 50 μm . ethical approval for this study was obtained from lothian research ethics committee and informed written consent was obtained from all patients before sample collection . first trimester decidualized endometrium was obtained from women ( age 18 - 45 years ) undergoing surgical termination of pregnancy ( top , n = 8 , group 1 , mean gestation 58 . 7 days ), surgical management of embryonic missed miscarriage ( n = 6 , group 2 , mean gestation 57 . 7 days ) and surgical management of tubal pregnancy ( n = 11 , group 3 , mean gestation 58 . 1 days ). none of the women undergoing surgical management of tubal ectopic pregnancy presented acutely with hemodynamic shock , and all required serial serum beta - hcg and ultrasound monitoring prior to diagnosis . the decidualized endometrium and trophoblast were obtained by suction curettage from groups 1 and 2 . the decidualized endometrium was obtained by suction endometrial biopsy ( pipelle ™, eurosurgical ltd , cranleigh , uk ) from group 3 . the decidualized endometrium was isolated from the trophoblast macroscopically . the decidualized endometrium was ( a ) immersed in rnalater ™ ( ambion , texas , usa ) at 4 ° c . overnight then flash frozen at − 70 ° c . ; and ( b ) fixed in 10 % neutral buffered formalin overnight at 4 ° c ., stored in 70 % ethanol , and wax embedded for staining with haematoxylin and eosin and immunohistochemistry . the presence of trophoblast was ruled out morphologically and by using immunohistochemical staining for cytokeratin as described previously ( 14 ). endometrial samples were obtained at hysterectomy for benign gynaecological conditions from women ( 18 - 45 years ) who had regular menstrual cycles ( 28 - 35 days ) and were not pregnant ( 15 ). endometrial samples were collected at room temperature and transported to the laboratory in rpmi ( life technologies , usa ) and processed for endometrial stromal cell isolation as described below . total rna was extracted from the decidualized endometrium as detailed in the manufacturers &# 39 ; protocol ( qiagen , west sussex , uk ). the concentration and quality of the extracted rna was assessed using an agilent bioanalyser . all samples were standardized for quality control and assigned an rna integrity number ( rin ). rna samples were considered to be of good quality when a mean rin value of 7 . 5 was obtained ( 16 ). in brief , 4 μg of total rna from each sample was used to generate double stranded cdna using the one - cycle cdna synthesis kit ( affymetrix , uk ) followed by purification with genechip sample cleanup module ( affymetrix , uk ). the double - stranded cdna was used as template for the in vitro transcription using the genechip ivt labeling kit ( affymetrix , uk ) yielding biotin - labeled crna . following cleanup and quantification by spectrophotometric analysis , the purified biotinylated target crna was then fragmented into short sequences . the hybridization cocktail consisted of 15 μg fragmented biotin labelled crna spiked with eukaryotic hybridization control . thereafter 80 μl of the hybridization cocktail was hybridized to the test - chips to check the crna integrity and assess the system veracity . after that , the human hg - u133 plus 2 . 0 microarrays ( affymetrix , uk ) were directly loaded with 200 μl of hybridization cocktail solution and then placed in genechip hybridisation oven 640 ( affymetrix , uk ) rotating at 60 rpm at 45 c for 16 h . after hybridization , the arrays were washed on a genechip fluidics station 450 ( affymetrix , uk ) and scanned using a genechip scanner 3000 ( affymetrix , uk ) according to the manufacturer &# 39 ; s protocol . to ensure quality and consistency of the sample labelling process and array hybridizations , control information from all 26 arrays was collated and reviewed before the data analysis and all were found to be consistent with affymetrix recommendations . expression was calculated using the robust multiarray average algorithm ( 17 ) implemented in the bioconductor ( http :// www . bioconductor . org ) extensions to the r statistical programming environment ( 18 ). robust multiarray average ( rma ) generates a background - corrected and quantile - normalized measure of expression ( 19 ) on the log 2 scale of measurement . to further investigate the performance of the array hybridizations , scatter plots comparing each array with every other were produced in bioconductor extensions to the r statistical programming environment . these plots confirmed a linear distribution between arrays and showed a dynamic uninterrupted range of expression values from low to high signal values . box and whisker visualizations also confirmed that the data had comparable distributions and were of sufficient quality for further analysis . data and protocols are available to download from the publicly accessible miame compliant database ‘ gpx ’ ( www . gti . ed . ac . uk / gpx , accession number gpx - 000067 . 1 ). following data normalization by rma the data was filtered to remove invariant transcripts that would contribute to multiple testing errors in the subsequent statistical analysis , using an arbitrary signal intensity threshold value of 64 . following the explorative analysis , a rigorous statistical approach was exploited to identify a subset of differentially expressed genes . in brief , genes differentially expressed between the intrauterine and ectopic pregnancy samples were identified using a t - test comparison followed by multiple test correction using the benjamini - hochberg false discovery detection method ( 20 ). gene lists were then created using a fold change threshold of & gt ; 2 and a corrected p - value of & lt ; 0 . 05 . after dnase treatment , using rq1 dnase ( promega , southampton , uk ) the rna was reverse transcribed into cdna using random hexamers ( applied biosystems , foster city , calif .). taqman q - rt - pcr was then used to measure gene levels using applied biosystems prevalidated ‘ assay - on - demand ’ specific primers and probes ( eurogentec , southampton , uk ) for prolactin and insulinlike growth factor binding protein - 1 ( igfbp1 ) as well as the inhibin βa subunit ( m13436 ), the inhibin βb subunit ( m13437 ) and the inhibin α subunit ( m13144 ) ( 21 ), and levels were related to a ribosomal 18s internal control ( applied biosystems ). all samples were performed in duplicate and a relative comparison was made to an appropriate control tissue cdna . using the 2 - δδct method , mrna expression results were normalized against 18s and expressed as foldchange compared to controls ( top sample group ). briefly , immunolocalization of inhibin / activin βb subunit and α - inhibin were carried out using antibodies developed and kindly provided by prof . n . groome , oxford brookes university , oxford , uk and prof . alan mcneilly , mrc human reproductive sciences unit , edinburgh , uk , respectively . in order to perform the experiment in a controlled and easily repeatable manner , the bond - x automated immunostaining machine ( vision biosystems , newcastle , uk ) was utilized . five micron paraffin sections of uterine decidua were cut , dewaxed , rehydrated and subjected to pressure cooked antigen retrieval in 0 . 01 mol 1 - 1 citrate buffer ( ph 6 . 0 ) before being place on the bond - x machine . this method on the bond - x automated machine utilises a specific polymer high contrast program . slides were peroxidase blocked for 5 min , incubated for 2 h with the primary antibodies ( 46a / f βb mouse monoclonal antibody for inhibin βb subunit ( 22 )) and asmr150 for α - inhibin , rabbit polyclonal antibody ( 23 )), diluted 1 : 500 in the diluent supplied and then incubated with the post - primary reagent for 15 min . to confirm antibody specificity , control sections were incubated with diluent alone or with non - specific immunoglobulins , diluted to the same concentration as primary antibody in supplied diluent . sections were then incubated with the polymer reagent for 15 min to increase sensitivity of detection prior to dab detection for 10 min . sections were counterstained in haematoxylin for 5 min . slides were then removed from the machine and dehydrated and mounted using pertex . endometrial tissue was subjected to collagenase / dnaase ( sigma - aldrich , usa ) digestion for 2 h at 37 c . after digestion , the stroma was dispersed , whereas the epithelial structures remained mostly intact . human endometrial stromal cells were separated on a size basis , as previously described ( 24 ). endometrial stromal cells were plated in 6 well standard culture plates at a density of 2 . 4 × 105 / well in rpmi with 2 % fetal calf serum , gentamycin , penicillin and streptomycin as described previously ( irwin et al , 1991 ). the cells were treated with medroxyprogesterone acetate ( 10 - 6m ), oestradiol ( 10 - 7m ) and 8 - bromo camp ( 0 . 1 mg / ml ) for 24 , 48 , 72 , 96 and 120 hours . the cells were harvested at each time point for rna extraction . serum progesterone concentrations were measured using a standard radioimmunoassay ( 15 ). activin b concentrations were measured using the activin b elisa that incorporates the use of monoclonal antibody 46a / f ( as both capture and detection antibody ) ( 22 ) with an sds and heat pre - treatment of samples ( ludlow et al ., manuscript in preparation ). serum was tested without dilution and the assay had a lower detection limit of 19 μg / ml . the samples of decidualized endometrium were classified blindly by a subspecialist gynaecological pathologist according to the degree of endometrial change . secretory transformation of the glandular epithelium ( absent / early , high and exhausted ) and stromal differentiation ( non - decidualized , predecidual , confluent decidual change ) were scored using the classification of zimmerman et al . ( 25 ). samples with exhausted glandular transformation with confluent stromal decidualization were classed overall as decidualized (+), those with absent / early secretory transformation and non - decidual stroma were classified as decidualized (−) and other samples were classified as decidualized (+/−). where the data were normally distributed , three groups were analyzed by anova with bonferroni pairwise comparisons . two groups were analyzed using a t - test . where the data were not normally distributed kruskal wallis testing with dunn &# 39 ; s pairwise comparisons was used to compare three groups . chi - squared test was used to compare proportions and spearman ( non - parametric ) or pearson ( parametric ) coefficients were used to assess linear correlations . the statistical test used is given in the text and figure legend and significance was at the p & lt ; 0 . 05 level . a comparison of gene expression in the decidualized endometrium from women with intrauterine ( n = 14 ) and tubal pregnancies ( n = 11 ) revealed that 669 genes were differentially expressed ( fc & gt ;± 2 , p & lt ; 0 . 05 ). one notable gene highlighted by this analysis was the inhibin / activin βb subunit . this was downregulated in the decidualized endometrium from women with tubal ectopic pregnancies with a fold reduction of 2 . 34 . quantitive rtpcr confirmed reduced expression in the decidualized endometrium from tubal pregnancies when compared to miscarriage ( p & lt ; 0 . 01 ) and termination of pregnancy ( p & lt ; 0 . 01 ) groups ( fig1 a ). in order to be secreted from the cell the βb subunit has to form a dimer . as it can combine with itself to form activin b , the inhibin α subunit to form inhibin b and the activin βa subunit to form activin βb , the expression of the α and βa subunits was therefore also investigated . expression of the βa subunit did not follow the same pattern ( fig1 b ) and there were no significant differences between groups . although the changes in expression of inhibin α subunit between groups was less marked ( fig1 c ), it was lower in ectopic pregnancy decidualized endometrium than in that of miscarriage ( p & lt ; 0 . 05 ). in order to further investigate the inhibin α and βb subunits in the decidualized endometrium they were localized using immunohistochemistry . specific immunostaining for both these proteins could be detected in the glandular and stromal areas of the decidualized endometrium ( fig2 a - d ). however there was variable staining in the decidual stroma between patients that was particularly marked for the βb subunit ( fig2 eh ). when the endometrium was less decidualized and had a more proliferative phenotype ( fig2 e ), the staining was noted to be glandular . when the endometrium had a more secretory phenotype ( fig2 the staining was mainly glandular with light stromal involvement . however in fully decidualized samples ( fig2 g ) there was marked stromal immunostaining . as there was an indication that the localization of the βb subunit in the stromal compartment of early pregnancy endometrium changed with the level of decidualization , human endometrial fibroblasts were examined in vitro . exposure to decidualizing stimuli in vitro increased the expression of the inhibin / activin βb subunit ( p & lt ; 0 . 05 ) in a time dependent manner ( fig3 a ). there were no changes with regards to the expression of the inhibin βa subunit ( fig3 c ) although there was a significant but much less marked increase in the α subunit ( p & lt ; 0 . 05 ) ( fig3 b ) during stromal fibroblast decidualization in vitro . as βb expression seems to be related to stromal decidualization , and was lower in the decidualized endometrium of ectopic pregnancies , the morphology of the glandular and stromal compartments in each tissue sample was assessed in haematoxylin and eosin stained sections by an expert in endometrial pathology blinded to tissue identity . the glandular compartment ( p & lt ; 0 . 05 ) showed a lesser degree of secretory transformation and the stromal compartment ( p & lt ; 0 . 05 ) showed less decidualization when compared to intrauterine pregnancies ( fig4 a ). furthermore , the overall level of endometrial decidualization was less in ectopics than top or miscarriage ( p & lt ; 0 . 05 ) ( fig4 b ). when the samples were stratified based on the overall degree of morphological decidualization ( fig4 c ) there was also a clear correlation with inhibin βb subunit expression ( p = 0 . 0005 ). as these investigations had suggested that the endometrium from ectopic pregnancy showed less advanced decidualization , the expression of genes known to be involved in decidualization were also studied in the tissue samples . igfbp1 ( fig5 a ) expression was reduced in the decidua of ectopic pregnancies when compared to that of miscarriages ( p & lt ; 0 . 01 ) and terminations of pregnancy ( p & lt ; 0 . 05 ). prolactin ( fig5 b ) expression was also reduced in ectopic pregnancy decidua when compared to that of termination of pregnancy ( p & lt ; 0 . 05 ). in order to determine whether changes in the decidual expression of the inhibin / activin βb subunit could be detected systemically , serum activin b concentrations were assayed . activin b concentrations were different between the groups analyzed ( fig6 a ) ( p = 0 . 001 ), like progesterone ( p & lt ; 0 . 05 ) ( fig6 d ). however activin b was lower in the serum of women with ectopic pregnancies ( p & lt ; 0 . 01 ) than in viable intrauterine pregnancies ( fig6 d ) unlike serum progesterone concentrations ( fig6 e ). in addition there was a clear correlation between serum activin b ( p = 0 . 005 ) and the degree of decidualization ( fig6 c ) that was not seen when progesterone was analyzed ( fig6 f ). this study demonstrates that there are differences in the function of the decidualized endometrium in tubal ectopic and intra - uterine pregnancies of similar gestations . we have shown that inhibin / activin βb subunit expression is related to the degree of decidualization of the endometrium and is reduced in tubal ectopic pregnancies . in addition , this difference could be assessed systemically . serum activin b was reduced in tubal ectopic pregnancy , and serum concentrations correlated with the degree of morphological decidualization and the difference was more marked than that seen with serum progesterone concentrations . it is clear that locally produced growth factors have major roles in endometrial development ( 26 ). indeed the transforming growth factor - β superfamily members are abundantly expressed in the endometrium . inhibin / activin α , βa and βb subunits have previously been shown to be expressed in the endometrium across the menstrual cycle and in the decidua of early pregnancy ( 27 ). it was felt that their function was to facilitate remodelling and tissue repair following menstruation ( 27 ). however as their expression is increased in early pregnancy it is likely they have a role in the endometrial response to pregnancy ( 28 ). there is no doubt that activin itself can affect endometrial function . activin receptors are present on endometrial stromal cells ( 29 ) and their expression increases during decidualization ( 30 ). indeed treatment of endometrial stromal cells with activin a promoted decidualization , and the expression of decidual markers such as prolactin ( 31 ), and treatment with follistatin significantly retarded the decidual response ( 31 , 32 ). it has also been shown that activin a and inhibin a differentially regulate the expression of stromal matrix metalloproteinases and this suggests a role for activin signalling in the remodelling associated with implantation and feto - maternal interaction ( 30 , 33 ). as well as having a role promoting decidualization , activin subunit expression itself increases during decidualization ( 27 , 34 ). the regulation of this is not clear but in vivo there seems to be a dialogue with the implanting trophoblast . during blastocyst attachment , the localization of inhibin / activin βa expression within the decidua changes suggesting paracrine regulation ( 35 ). it is therefore not surprising that activins may function as markers of endometrial function and may be differentially regulated in intrauterine pregnancy . although most previous reports relate to the βa subunit expression and suggest a key role of activin a in decidualization , it was the βb subunit that was differentially expressed in this study . although βb expression in decidualized endometrium has been reported previously ( 27 ), the role , regulation and effects of activin b have not yet been assessed . certainly both endometrial βb subunit expression and serum activin b are highly correlated with the degree of endometrial decidualization in this study . it is not known whether it is influenced directly by neighbouring trophoblast cells . in a tubal ectopic pregnancy , progesterone secretion is maintained but there is no direct physical interaction between the trophoblast and decidualized endometrium . we hypothesized that , in contrast , in intra - uterine pregnancy , the trophoblast interaction would influence decidual function and , hence , decidual markers would be useful for the diagnosis of tubal ectopic pregnancy . previously assessed markers have focussed on the ectopic implantation ( serum creatine kinase ( 15 )), corpus luteum function as a marker of hcg dynamics ( serum progesterone ( 36 )) or a marker of both ( serum vegf ( 37 )). these tests have not yet proven to be of particular clinical use in the diagnosis of ectopic pregnancy . the concept of activins and inhibins as markers of ectopic pregnancy is not new . in an early study inhibin a and the free circulating pro - ac inhibin were investigated and found not to discriminate tubal ectopic pregnancy ( 11 ). a recent study has suggested that activin a itself might function as a marker of ectopic pregnancy ( 38 ). serum activin a was lower in ectopic pregnancies ( 38 ). as both the ectopic pregnancy and the corpus luteum expresses βa subunits rather than βb subunits ( 39 , 40 ) the source of this activin a is not clear . it seems likely that the source of activin b is the decidua and whether this can function as an adjuvant marker for decidualization and tubal ectopic pregnancy should be assessed in prospective studies . the demonstration of inhibin / activin βb underexpression in the decidualized endometrium of women with tubal ectopic pregnancies , associated with lower serum activin b concentrations , is clinically important . the role and regulation of activin b in decidualization should be addressed to further dissect the roles of trophoblast and progesterone in this process . in addition the role of decidual assessment or activin b measurement in the diagnosis of ectopic pregnancy should be investigated further . we believe that further potential biomarkers of tubal pregnancy could be discovered by focusing on secreted proteins associated with uterine decidualization . 1 . zane s b , kieke b a , jr ., kendrick j s , bruce c 2002 surveillance in a time of changing health care practices : estimating ectopic pregnancy incidence in the united states . maternal and child health journal 6 : 227 - 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374 30 . jones r l , findlay j k , farnworth p g , robertson d m , wallace e , salamonsen l a 2006 activin a and inhibin a differentially regulate human uterine matrix metalloproteinases : potential interactions during decidualization and trophoblast invasion . endocrinology 147 : 724 - 732 31 . jones r l , salamonsen l a , findlay j k 2002 activin a promotes human endometrial stromal cell decidualization in vitro . the journal of clinical endocrinology and metabolism 87 : 4001 - 4004 32 . tierney e p , giudice l c 2004 role of activin a as a mediator of in vitro endometrial stromal cell decidualization via the cyclic adenosine monophosphate pathway . fertility and sterility 81 suppl 1 : 899 - 903 33 . strakova z , szmidt m , srisuparp s , fazleabas a t 2003 inhibition of matrix metalloproteinases prevents the synthesis of insulin - like growth factor binding protein - 1 during decidualization in the baboon . endocrinology 144 : 5339 - 5346 34 . otani t , minami s , kokawa k , shikone t , yamoto m , nakano r 1998 immunohistochemical localization of activin a in human endometrial tissues during the menstrual cycle and in early pregnancy . obstetrics and gynecology 91 : 685 - 692 35 . jones r l , stoikos c , findlay j k , salamonsen l a 2006 tgf - beta superfamily expression and actions in the endometrium and placenta . reproduction 132 : 217 - 232 36 . ledger w l , sweeting v m , chatterjee s 1994 rapid diagnosis of early ectopic pregnancy in an emergency gynaecology service — are measurements of progesterone , intact and free beta human chorionic gonadotrophin helpful ? human reproduction 9 : 157 - 160 37 . felemban a , sammour a , tulandi t 2002 serum vascular endothelial growth factor as a possible marker for early pregnancy . human reproduction 17 : 490 - 492 38 . florio , p , severi f m , bocchi c , luisi s , mazzini m , danero s , torricelli m , petraglia f 2007 single serum activin a testing to predict ectopic pregnancy . journal of clinical endocrinology and metabolism 92 : 1748 - 1753 39 . refaat b , amer s , ola b , chapman n , ledger w 2007 the expression of activin βa - and βb - subunits , follistatin and activin type ii receptors in fallopian tubes bearing an ectopic pregnancy . journal of clinical endocrinology and metabolism , e - pub ahead of print 40 . fraser h m , lunn s f , cowen g m , saunders p t 1993 localization of inhibin / activin subunit mrnas during the luteal phase in the primate ovary . journal of molecular endocrinology 10 : 245 - 257 ethical approval for this study was obtained from lothian research ethics committee ( 04 / s1103 / 20 ). written and informed consent was obtained from all patients before sample collection . uterine decidua was obtained from obtained from women ( age 18 - 45 years ) undergoing surgical termination of pregnancy ( stop , n = 8 , group 1 ), surgical management of miscarriage ( n = 6 , group 2 ) and surgical management of tubal pregnancy ( n = 11 , group 3 ). the decidua and trophoblast were obtained by suction curettage from groups 1 and 2 . the decidua was obtained by pipelle ™ endometrial biopsy from group 3 . the decidua was isolated from the trophoblast macroscopically . decidual biopsies were immersed in rnalater ™ ( ambion , texas , usa ) at 4 ° c . overnight then flash frozen at − 70 ° c . total rna was extracted from the decidual biopsies as detailed in the manufacturers &# 39 ; protocol ( qiagen , west sussex , uk ). total rna was quality confirmed on rna 6000 nanochips in the agilent 2100 bioanalyzer . only very high quality rna ( rin & gt ; 7 . 5 ) preparations were considered for micro - array screening . rna concentrations were confirmed by spectrophotometric analysis on a nanodrop spectrophotometer ( nanodrop technologies , usa ). in brief , 4 μg of total rna from each sample was used to generate double stranded cdna using the one - cycle cdna synthesis kit ( affymetrix , uk ) followed by purification with genechip sample cleanup module ( affymetrix , uk ). the double - stranded cdna was used as template for the in vitro transcription using the genechip ivt labeling kit ( affymetrix , uk ) yielding biotin - labeled crna . following cleanup and quantification by spectrophotometric analysis , the purified biotinylated target crna was then fragmented into short sequences . the hybridisation cocktail consisted of 15 μg fragmented biotin - labeled crna spiked with eukaryotic hybridisation control . 80 μl of the hybridisation cocktail was first hybridised to the test - chips to check the crna integrity and assess the system veracity . after that , the human hg - u133 plus 2 . 0 microarrays ( affymetrix , uk ) were directly loaded with 200 μl of hybridisation cocktail solution and then placed in genechip hybridisation oven 640 ( affymetrix , uk ) rotating at 60 rpm at 45 ° c . for 16 h . after hybridisation , the arrays were washed on a genechip fluidics station 450 ( affymetrix , uk ) and scanned using a genechip scanner 3000 ( affymetrix , uk ) according to the manufacture &# 39 ; s protocol . to ensure quality and consistency of the sample labelling process and array hybridizations , control information from all 25 arrays was collated and reviewed before the data analysis and all were found to be consistent with affymetrix recommendations . expression was calculated using the robust multiarray average algorithm ( 8 ) implemented in the bioconductor ( http :// www . bioconductor . org ) extensions to the r statistical programming environment ( 9 ). robust multiarray average ( rma ) generates a background - corrected and quantile - normalized measure of expression ( 10 ) on the log 2 scale of measurement . to further investigate the performance of the array hybridizations , scatter plots comparing each array with every other were produced in bioconductor extensions to the r statistical programming environment . these plots confirmed a linear distribution between arrays and showed a dynamic uninterrupted range of expression values from low to high signal values . box and whisker visualizations also confirmed that the data had comparable distributions and were of sufficient quality for further analysis ( see supplemental data available online ). data and protocols are available to download from the publicly accessible miame compliant database ‘ gpx ’ ( www . gti . ed . ac . uk / gpx , accession number gpx - 000067 . 1 ). following data normalisation by rma the data was filtered to remove invariant transcripts which would contribute to multiple testing errors in the subsequent statistical analysis , using an arbitrary signal intensity threshold value of 64 i . e . transcripts were removed from further analysis if they showed absolute intensity values of less than 64 in 50 % of the sample arrays ( 12 arrays ). following the explorative analysis , a rigorous statistical approach was exploited to identify genes which were significantly different in terms of expression levels between the intra - uterine and extra - uterine samples . inference testing in the form of an empirical bayes test was applied to the data . this was followed by multiple test correction using the benjamini - hochberg false discovery detection method ( 11 ). gene lists were then created using a fold change threshold of & gt ; 1 . 5 and a corrected p - value of & lt ; 0 . 05 . to confirm the results of this statistical analysis and the validity of the gene list , quantitative real - time polymerase chain reaction ( q - rt - pcr ) of two of the genes with the greatest fold change was undertaken . rna was reverse transcribed using applied biosystems rt kit ( applied biosystems , cheshire , uk ) following manufacturer &# 39 ; s instructions . taqman q - rt - pcr was then used to measure gene levels using applied biosystems prevalidated ‘ assay - on - demand ’ 20 × taqman primers and probe and a standard taqman reaction mix ( including ribosomal 18s primers and probe as an internal standard for rna loading ). samples were analyzed on an abi prism 7900 using standard conditions . using the 2 − δδct method , mrna expression results were normalised against 18s and expressed as fold - change compared to controls ( stop sample group , n = 8 ). a one - way analysis of variance ( anova ) and a least significant difference post hoc multiple comparisons were then used to determine if differences in expression were significant ( spss , inc , chicago , ill .). standard immunohistochemistry protocols were also used to examine protein expression for one of the genes in paraffin - embedded deciduas from each patient group . following explorative analysis , a general linear model enhanced by an empirical bayes methodology was used to test for statistically significant differential expression between intra - uterine ( groups 2 + 3 combined , n = 14 ) and extra - uterine ( group 1 , n = 11 ) samples . average fold - changes ( fc ) were computed and p - values were adjusted for multiple testing using the benjamini - hochberg false discovery detection method . 1061 genes were found to be differentially expressed ( fc & gt ; 1 . 5 , adjusted p - value & lt ; 0 . 05 ) in the extra - uterine pregnancy group . specifically , two genes were identified as potential biomarkers : cysteine - rich secretory protein 3 ( crisp3 ) was increased 9 . 13 fold ( p = 0 . 005 ) and carboxypeptidase b1 ( cpb1 ) was decreased 26 . 52 fold ( p = 0 . 005 ) in the extra - uterine compared to the intra - uterine groups . this was confirmed by rt - pcr ( see fig1 ). crisp3 protein expression was also demonstrated by immunohistchemistry in the glands and macrophages of decidua from each patient group ( see fig2 ). there were no differences in crisp3 distribution at the immunohistochemical level . as well as furthering our understanding of extra - uterine implantation , the detection of gene products , such as crisp3 and cpb1 , that are over , or under - expressed , in subjects with ectopic pregnancies is clinically very important . it provides the clinical pathologist with a means of diagnosing ectopic pregnancy in any subject with a failing early pregnancy in whom the diagnosis is uncertain , by simply examining a sample of , for example , the uterine decidua . samples such as , for example , blood , serum , plasma and / or uterine decidua can be easily obtained and , for example , a sample of uterine decidua , may be obtained by taking a biopsy from the uterus without subjecting the patient to a general anaesthetic . in addition , this would provide potential for patients to avoid hospital admission and invasive investigation . the identification of such genes also provides the potential for the development of a reliable blood test for ectopic pregnancy . this would reduce the number of costly and time - consuming investigations currently required for the diagnosis of ectopic pregnancy . 1 . tay j i , moore j , walker j j . ectopic pregnancy . bmj 2000 320 ( 7239 ): 916 - 9 . 2 . report on confidential enquiry into maternal deaths in the united kingdom . why mothers die 1997 - 1999 . 3 . lemus j f . ectopic pregnancy : an update . curr opin obstet gynecol 2000 12 ( 5 ): 369 - 75 . 4 . miranda c s , carvajal a r . complications of operative gynecological laparoscopy . j soc lap surg . 2003 7 ( 1 ): 53 - 8 . 5 . birkhahn r h , gaeta t j , paraschiv d , et al . serum levels of myoglobin , creatine phosphokinase , and smooth muscle heavy - chain myosin in patients with ectopic pregnancy . ann emerg med 2001 38 ( 6 ): 628 - 32 . 6 . wegner n t , mershon j l . evaluation of leukemia inhibitory factor as a marker of ectopic pregnancy . am j obstet gynecol 2001 184 ( 6 ): 1074 - 6 . 7 . develioglu o h , askalli c , uncu g , samli b , daragenli o . evaluation of serum creatine kinase in ectopic pregnancy with reference to tubal status and histopathology . brit j obs gyn 2002 109 ( 2 ): 121 - 8 . 8 . irizarry r a , hobbs b , collin f , et al . exploration , normalization , and summaries of high density oligonucleotide array probe level data . biostatistics 2003 ; 4 : 249 - 64 . 9 . ihaka r , gentleman r . a language for data analysis and graphics . j comput graph stat 1996 ; 5 : 299 - 314 . 10 . bolstad b m , irizarry r a , astrand m , speed t p . a comparison of normalization methods for high density oligonucleotide array data based on variance and bias . bioinformatics 2003 ; 19 : 185 - 93 11 . benjamini , y . and hochberg , y . controlling the false discovery rate : a practical and powerful approach to multiple testing . journal of the royal statistical society 1995 ; 57 : 289 - 300 . during inflammation a number of mediators and effectors of immunity are recruited to aid resolution and an over exuberant , or prolonged , response can result in tissue damage . amongst the mediators responsible for such tissue damage are proteases , which function as part of the innate immune response . ( dallegri and ottonello 1997 ). the host response also includes the production of a number of important anti - proteases , such as the anti - microbials , secretory leucocyte protease inhibitor ( slpi ) and elafin , in order to counteract the actions of proteases and therefore prevent resultant damage to host tissues ( sallenave et al , 1994 ; sallenave 2000 ; schalkwijk et al , 1999 ). slpi inhibits a number of proteases , including neutrophil elastase , trypsin and cathepsin g , whilst elafin appears to be restricted to regulating neutrophil elastase and proteinase 3 ( thompson and ohlsson , 1986 , wiedow et al , 1991 , sallenave and ryle 1991 ). slpi and elafin and are expressed throughout the female genital tract ( king et al , 2000 , king et al , 2003b ). the human endometrial epithelium expresses slpi during the secretory phase of the cycle and the neutrophils in the endometrium are a particularly rich source of elafin during menstruation ( king et al , 2003a ). slpi and elafin expression have also been demonstrated in the cervix and decidualised endometrium ( helmig et al , 1995 , pfundt et al , 1996 , king et al , 2000 , king et al , 2003a ). the expression of slpi and elafin during the menstrual cycle may indicate a role in tissue remodelling and ‘ wound ’ healing ( van bergen et al , 1996 ; wingens et al , 1998 ; ashcroft et al , 2000 ; zhu et al , 2002 ; angelov et al , 2004 ). in common with the rest of the female reproductive tract , the fallopian tube morphology undergoes cyclical changes under the influence of oestrogen and progesterone ( sulz et al , 1998 ), and slpi and elafin expression have been shown to have a similar temporal expression pattern in the fallopian tube to the endometrium ( dalgetty et al , 2006 ). however , the changes in slpi and elafin expression that occur in the context of tubal implantation have not been demonstrated . direct comparison of tubal with intra - uterine sites is difficult due to the anatomical differences of the two implantation sites . however , similar to the endometrial response to an intra - uterine pregnancy , with a tubal gestation there is a decidual reaction in the uterine cavity but not usually in the fallopian tube ( stock , 1991 ). decidualisation of the endometrial epithelium and stroma is a key immunological element for successful implantation ( christian et al , 2002 ). the uterine decidua contains innate immune leukocytes , such as cd56 + natural killer ( unk ) cells , macrophages and mast cells ( bulmer et al , 1988 ; king and loke , 1991 ; hunt 1994 ; clark 1995 ; marx et al , 1999 ). we therefore compared slpi and elafin expression in the uterine decidua of women with ongoing intra - uterine ( women undergoing surgical termination of pregnancy ), failed intra - uterine ( women with miscarriage ) and tubal gestations . quantitative rt - pcr and immunohistochemistry were used to demonstrate mrna and protein expression , respectively . ethical approval for this study was obtained from lothian research ethics committee ( 04 / s1103 / 20 ). written and informed consent was obtained from all patients before sample collection . uterine decidua and serum samples were obtained from obtained from women ( age 18 - 45 years ) undergoing surgical termination of pregnancy ( stop , n = 7 , group 1 ), surgical management of embryonic missed miscarriage ( n = 6 , group 2 ) and surgical management of tubal pregnancy ( n = 10 , group 3 ). none of the women undergoing surgical management of tubal ectopic pregnancy presented acutely with haemodynamic shock , and all required serial serum beta - hcg and ultrasound monitoring prior to diagnosis . the decidua and trophoblast were obtained by suction curettage from groups 1 and 2 . the decidua was obtained by pipelle ™ endometrial biopsy from group 3 . the decidua was isolated from the trophoblast macroscopically . decidual biopsies were ( a ) immersed in rnalater ™ ( ambion , texas , usa ) at 4 ° c . overnight then flash frozen at − 70 ° c . ; and ( b ) fixed in 10 % neutral buffered formalin overnight at 4 ° c ., stored in 70 % ethanol , and wax embedded for immunohistochemistry . serum samples were stored for analysis of progesterone levels . dna was extracted from the decidual biopsies as detailed in the manufacturers &# 39 ; protocol ( qiagen , west sussex , uk ). the pcr protocol used in - house plasmid - based methodology . total rna was extracted from the decidual biopsies as detailed in the manufacturers &# 39 ; protocol ( qiagen , west sussex , uk ). the concentration and quality of the extracted rna was assessed using an agilent bioanalyser . all samples were standardised for quality control and assigned an rna integrity number ( rin ). rna samples were considered to be of good quality when a mean rin value of 7 . 5 was obtained ( schroeder et al . 2006 ). slpi and elafin levels were determined by quantitative real - time pcr . this technique relates the amount of slpi and elafin mrna present to levels of ribosomal 18s , controlling for the amount of rna present . details of the rt and pcr methods have been fully detailed elsewhere ( king et al , 2000 ). the elafin and slpi primers and probe used were previously designed in our laboratory using primer express software ( pe biosystems , foster city , usa ) ( king et al , 2000 and 2003 ). elafin primers and probe were as follows : forward primer 5 ′- tggctcctgccccattatc - 3 ′, reverse primer 5 ′- cagtatctttcaagcagcggttag - 3 ′, probe 5 ′- atccggtgcgccatgttgaatcc - 3 ′. slpi primers and probe were as follows : forward primer 5 ′- gcatcaaatgcctggatcct - 3 ′, reverse primer 5 ′- gcatcaaacattggccataagtc - 3 ′, probe 5 ′- tgacaccccaaacccaacaaggagg - 3 ′. within - assay variation of pcr measurements were calculated from six replicates . the variability of the reverse transcription step was determined by reverse transcribing one rna sample on eight separate occasions . the eight cdna samples were then included within one pcr run , and variability ( relative to sd ) was calculated . the possibility of genomic dna contamination was excluded by dnaase treatment and by measurement of beta - actin levels in rna samples ( which were not reverse transcribed ). the relative mean of the samples from each clinical group was logged and analysed by anova followed by tukey posthoc analysis . tissue sections were dewaxed in xylene and then rehydrated in descending grades of alcohol . non - specific endogenous peroxidase activity was blocked with 2 % hydrogen peroxide ( sigma aldrich , poole , uk ) in distilled water for 10 minutes at room temperature . diluted normal goat serum ( 20 % vol / vol in phosphate - buffered saline ) ( diagnostics scotland , edinburgh , uk ) was applied to all tissue sections for 20 min at room temperature . avidin - biotin and protein blocks were further applied to the tissue sections for 20 min each at room temperature with alternate 5 minutes phosphate - buffered saline washes . sections were then incubated overnight at 4 ° c . with rabbit anti - elafin polyclonal antibody ( sallenave et al , 1994 ) which was diluted at 1 : 700 in antibody diluent ( dako , ely , uk ). in negative control sections , the primary antibody was substituted with an approximately equivalent ig concentration of rabbit igg ( rigg ; vector laboratories , peterborough , uk ). sections were incubated with biotinylated goat anti - rabbit ( vector ) and were then subjected to an avidin - biotin peroxidase detection system ( vector ; both were incubated for 30 minutes at room temperature ). the peroxidase substrate , diaminobenzidine , was used to identify positive immunostaining . sections were counterstained with harris &# 39 ; s haematoxylin ( pioneer research chemicals , colchester , uk ), dehydrated in ascending grades of alcohol , and mounted from xylene in pertex ( cell - path , hemel hempstead , uk ). the method used was identical to the elafin immunohistochemistry protocol , with the following exceptions . the non - immune block was performed with diluted horse serum ( vector ) for 20 minutes at room temperature . the primary antibody was mouse anti - slpi ( hycult biotechnology , uden , the netherlands ) diluted at 1 : 20 in antibody diluent ( dako ). negative controls were incubated with an equimolar concentration of mouse igg ( migg ), and the secondary antibody was biotinylated horse anti - mouse ig ( vector ). serum progesterone levels were analysed for each patient . there was no statistically significant difference demonstrated between each group using kruskall - wallis followed by dunn &# 39 ; s posthoc analysis ( see table 11 . haematoxylin and eosin staining and immunohistochemistry for cytokeratin demonstrated clear isolation of decidua from trophoblast by macroscopic separation in saline . the state of inflammation of the samples was also determined — each sample was examined for presence of necrosis , and lymphocyte and polymorph infiltration . these features were present to varying degrees but there was no obvious statistical difference ( data not shown ). all stained tissue sections were examined by an expert histopathologist . all decidual biopsies were screened for chlamydia trachomatis infection and shown to be negative . slpi and elafin mrna expression in uterine decidua from intra - and extra - uterine pregnancies elafin and slpi mrna were detected in the uterine decidua of each clinical group . relative slpi mrna expression was greater in the uterine decidua of women with tubal pregnancy ( 12 . 37 +/− 2 . 66 ) compared to the decidua from women undergoing stop ( 4 . 8 +/− 1 . 45 ) and surgical management of miscarriage ( 5 . 09 +/− 2 . 22 ) ( table 1 , fig9 a ). anova analysis of logged data demonstrated a statistically significant difference ( overall p & lt ; 0 . 0185 , tukey posthoc analysis p & lt ; 0 . 05 ) between the tubal pregnancy and miscarriage groups . elafin mrna expression also was higher in the decidua from women with tubal pregnancy ( 73 . 57 +/− 35 . 29 ) compared to those with miscarriage ( 9 . 00 +/− 2 . 53 ) or undergoing a surgical termination of pregnancy ( 4 . 6 +/− 1 . 38 ) ( table 1 , fig9 b ). however , this observation was not significant using anova , or non - parametric , statistical analysis of the normal , or logged , data . this was attributed to the high variability of expression in the tubal pregnancy group . two samples were excluded from analysis ( one from a patient undergoing a stop and one from a patient with a first trimester miscarriage ) because their relative mrna values for elafin were greater than two standard deviations of the mean of the other samples . immunohistochemical localisation of elafin and slpi protein in uterine decidua from intra - and extra - uterine pregnancies elafin and slpi protein expression and distribution were demonstrated in the uterine decidua by immunohistochemistry ( fig1 a - f ). there was no obvious difference in elafin and slpi protein expression pattern in any of the clinical groups . slpi protein expression was demonstrated in the epithelium ( see fig1 a and b ) and decidual leukocytes ( see fig1 c ). slpi protein expression was particularly strong in the cytoplasm of the epithelial glands ( see fig1 b ). elafin protein expression was restricted to the decidual leukocyte populations ( fig1 e and f ). however , in some cases , there was evidence of membraneous elafin protein expression in the epithelial glands ( fig1 d ). the negative controls for elafin and slpi immunoexpression showed no evidence of positive staining ( fig1 g and h , respectively ). all stained tissue sections underwent qualitative analysis by an expert pathologist . no quantitative analysis was performed due to the poor reproducibility and inter - observer agreement of immunohistochemistry scoring ( taylor 1994 ). to our knowledge , we report novel differences in gene expression of natural antimicrobial peptides ( slpi and elafin ) in the uterine decidua collected from women with tubal pregnancy compared to those with an intra - uterine gestation . this study also demonstrates that slpi mrna expression is significantly higher ( p & lt ; 0 . 05 ) in the uterine decidua of women with a tubal compared to a failed intra - uterine gestation ( miscarriage ). previous studies have only compared the cellular composition of the uterine decidua , with particular attention to the leukocyte populations , and found no obvious distinction between extra - and intra - uterine pregnancies ( bulmer et al , 1987 ; stewart - akers et al , 1997 ). the most obvious difference in the decidua between the tubal pregnancy and intra - uterine groups is the absence of trophoblast . there is no published data to our knowledge to suggest that slpi or elafin expression are regulated by trophoblast signals . nevertheless , recent in - vitro studies , using a functional genomics approach , have shown that conditioned media from trophoblasts alters the local immune environment of the decidua to facilitate embryo implantation by causing a significant induction of proinflammatory cytokines and cells ( hess et al , 2007 ). this provides an interesting potential mechanism for the changes observed in our study . alternatively , if the uterine environment truly reflects that of the fallopian tube , the abnormal expression of slpi in the uterine decidua may simply be a consequence of extra - uterine implantation . in the fallopian tube , it is essential that the normal oviductal epithelium has the capacity to recognise and respond to ascending pathogens while simultaneously avoiding a state of unnecessary inflammation that might disrupt the epithelial barrier . foreign pathogens are recognised by pattern recognition receptors present on the oviduct cells , such as the membraneous toll - like receptors ( tlrs ) ( darville et al , 2003 ; pioli et al , 2004 ). binding initiates a signalling cascade , leading to nf - kappab activation and the subsequent production of pro - inflammatory and immunoregulatory cytokines , chemokines and co - stimulatory molecules ( e . g . il - 1 , tnf ) ( bowie et al , 2000 ). nf - kappab may indirectly increase slpi expression via the up - regulation of these inflammatory molecules ( bingle at al , 2001 ; king et al , 2002 ). there is often no evidence of acute inflammation around a tubal implantation site ( kutluay et al , 1994 ). in ectopic pregnancy , tubal expression of slpi during embryo implantation may reduce the inflammatory response and subsequent early tubal damage by inhibiting protease actions . slpi has been shown to reduce tissue damage in models of emphysema and fibrosis of the lung , and studies have shown that reduced levels of slpi in the gastric mucosa of patients with helicobacter pylori induced gastritis ( hritz et al , 2006 ; mitsuhashi et al , 1996 ; mulligan et al , 1993 ; rudolphus et al , 1993 ; wex et al . 2004a ; wex et al , 2004b ; wex et al . 2006 ). the elevated levels of slpi in the event of an ectopic implantation may serve as an explanation for the lack of acute inflammation observed . however , it is also possible that the abnormal expression of slpi may predispose to , rather than be a consequence of , tubal pregnancy . chlamydia trachomatis , the most common bacterial sexually transmitted infection in the united kingdom , has been shown to be the principal risk factor for tubal pregnancy ( farquhar 2005 , cassell et al , 2006 ). nevertheless , although it has been suggested that chlamydial infection is one of the major causes of tubal damage ( odland et al , 1993 ), the pathogenic events that lead from chlamydial infection to tubal pregnancy are unclear . in - vitro studies have demonstrated that chlamydia induces expression of slpi in cervical epithelium and trophoblast ( wheelhouse et al , manuscript in preparation ). it is possible that chlamydia acts via nf kappab by way of the above signalling cascade , and that persistent , or repeated , infection causes atypical expression of slpi in the uterus and fallopian tube leading to a susceptibility to tubal implantation . however , none of the specimens examined in our study were positive for chlamydial infection . the demonstration of an altered mrna expression pattern of slpi in women with ectopic compared to intra - uterine pregnancies contributes further to our current knowledge of embryo implantation . although immunohistochemistry demonstrated no obvious difference in the pattern of distribution of elafin and slpi protein expression , we acknowledge the difficulty in accurately determining secretory proteins using this technique and are aware that further quantitative protein analysis and functional studies are required . aflatoonian r , fazeli a . toll - 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