Patent Application: US-200913119213-A

Abstract:
gene expression patterns contemporaneous with early placental development in the first trimester of preeclamptic versus unaffected pregnancies have been obtained . observation of differences in these gene expression patterns has allowed the identification of biomarkers that are useful in predicting and monitoring preeclampsia . these biomarkers are also useful in screening potential therapeutics for efficacy in the prevention or treatment of preeclampsia .

Description:
in a first aspect , the present invention relates to a method for diagnosing , monitoring , or predicting preeclampsia in a pregnant woman . this method comprises the following steps : first , quantitatively determining the amount of one or more nucleic acid species in a biological sample obtained from the pregnant woman that hybridize with probe set id nos : 205827_at ; 215141_at ; 202917_s_at ; 215733_x_at ; 234601_x_at ; 227238_at ; 239010_at ; 214702_at ; 1553319 _at ; 235592_at ; 229839_at ; 230748_at ; 203789_s_at ; 226482_s_at ; 215388_s_at ; 1562053_at ; 219911_s_at ; 209351_at ; 1552858_at ; 215108_x_at ; 226403_at ; 207607_at ; 228293_at ; 210251_s_at ; 1561318_at ; 241036_at ; 219759_at ; 203592_s_at ; 205302_at ; 1568554_x_at ; 1554276_at ; 242842_at ; 242868_at ; 206859_s_at ; 204580_at ; and 207509_s_at or those set forth in table 5 on the affymetrix genechip system ( affymetrix , santa clara , calif . ; hg - u133 plus 2 . 0 genechips containing 53 , 613 probe sets ); second , comparing the amount of the nucleic acid species from the first step to a standard control representing the amount of the nucleic acid species in the corresponding sample from an average non - preeclamptic pregnant woman ; wherein an increase and decrease in the amount of the nucleic acid species in the test sample as compared to the standard control indicates preeclampsia or an increased risk of developing preeclampsia . the biological sample is blood , washing from the reproductive tract , urine , saliva , amniotic fluid , or chorionic villus . one aspect of the invention provides for increased expression of nucleic acids that hybridize with 205827_at ; 215141_at ; 202917_s_at ; 215733_x_at ; 234601_x_at ; and decreased expression of nucleic acids that hybridize with 227238_at ; 239010_at ; 214702_at ; 1553319_at ; 235592_at ; 229839_at ; 230748_at ; 203789_s_at ; 226482_s_at ; 215388_s_at : 1562053_at ; 219911_s_at : 209351_at ; 1552858_at ; 215108_x_at ; 226403_at ; 207607_at ; 228293_at ; 210251_s_at ; 1561318_at ; 241036_at ; 219759_at ; 203592_s_at ; 205302_at ; 1568554_x_at ; 1554276_at ; 242842_at ; 242868_at ; 206859_s_at ; 204580_at ; and 207509_s_at . in some embodiments , the first step can comprise the use of a reverse transcriptase polymerase chain reaction ( rt - pcr ). in other embodiments , the first step comprises using a polynucleotide hybridization method , or using a primer extension reaction . various other embodiments provide a kit for diagnosing , monitoring , or predicting preeclampsia in a pregnant woman . this kit comprises the following : ( i ) pcr primers for quantitatively determining the amount of one or more nucleic acid species in a biological sample obtained from the pregnant woman , wherein the nucleic acid species hybridize with probe set id nos : 205827_at ; 215141_at ; 202917_s_at ; 215733_x_at ; 234601_x_at ; 227238_at ; 239010_at ; 214702_at ; 1553319 _at ; 235592_at ; 229839_at ; 230748_at ; 203789_s_at ; 226482_s_at ; 215388_s_at ; 1562053_at ; 219911_s_at ; 209351_at ; 1552858_at ; 215108_x_at ; 226403_at ; 207607_at ; 228293_at ; 210251_s_at ; 1561318_at ; 241036_at ; 219759_at ; 203592_s_at ; 205302_at ; 1568554_x_at ; 1554276_at ; 242842_at ; 242868_at ; 206859_s_at ; 204580_at ; and 207509_s_at or those set forth in table 5 on the affymetrix genechip system ( affymetrix , santa clara , calif . ; hg - u133 plus 2 . 0 genechips containing 53 , 613 probe sets ) and ( ii ) a standard control representing the amount of the nucleic acid species in the corresponding sample from an average non - preeclamptic pregnant woman . another aspect of the invention relates to a method for diagnosing , monitoring , or predicting preeclampsia in a pregnant woman using immunoassay ( s ). this method comprises the following steps : first , quantitatively determining the amount of one or more polypeptide species in a biological sample ( test sample ) obtained from the pregnant woman that bind to specific antibodies , said one or more polypeptide species being set forth in table 8 ; second , comparing the amount of said one or more polypeptide species identified in the first step to a standard control representing the amount of the corresponding polypeptide species in a corresponding sample from an average non - preeclamptic pregnant woman ( a control sample or standard control ); wherein an increase ( overexpression ) and decrease ( underexpression ) in the amount of said at least one polypeptide species in the test sample as compared to the standard control indicates preeclampsia or an increased risk of developing preeclampsia . the biological sample is blood , serum , plasma , endometrial tissue , washing from the reproductive tract , urine , saliva , cerebral spinal fluid , amniotic fluid , or chorionic villus . the woman being examined is examined during the first trimester of gestation . in other embodiments , the woman is during the second or third trimester of gestation . as discussed above , immunoassays can be used to detect at least one secreted protein disclosed in table 8 , the expression levels of said at least one secreted protein , and comparison of said at least one secreted protein to a control ( standard control ) sample . protein expression ( secretion ) can be detected by any suitable method , such as gas chromatography - mass spectrometry . in some embodiments , proteins are detected by immunoassays . antibody binding is detected by techniques known in the art ( e . g ., radioimmunoassay , elisa ( enzyme - linked immunosorbant assay ), “ sandwich ” immunoassays , immunoradiometric assays or western blots . in some embodiments , antibody binding is detected by detecting a label on the primary antibody . in another embodiment , the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody . in a further embodiment , the secondary antibody is labeled . many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention . in aspects of this invention , an automated detection assay is utilized . methods for the automation of immunoassays include those described in u . s . pat . nos . 5 , 885 , 530 , 4 , 981 , 785 , 6 , 159 , 750 , and 5 , 358 , 691 , each of which is herein incorporated by reference . in some embodiments , the analysis and presentation of results is also automated . in other embodiments , the immunoassay described in u . s . pat . nos . 5 , 599 , 677 and 5 , 672 , 480 ; each of which is herein incorporated by reference . in various aspects of the invention , the woman being examined is examined during the first trimester of gestation . in other embodiments , the woman is during the second or third trimester of gestation . the term “ nucleic acid ” can be understood to mean , according to the present invention , either a double - stranded dna , a single - stranded dna or products of transcription of the said dnas ( e . g ., rna molecules ). the term “ preeclampsia ” as used herein refers to a condition that occurs during pregnancy , diagnosed by the new onset of high blood pressure accompanied by the presence of proteins in the urine and may include edema ( swelling ). preeclampsia , sometimes called toxemia of pregnancy , is related to a more serious disorder called “ eclampsia ”, which is preeclampsia together with seizure . these conditions usually develop during the second half of pregnancy ( after 20 weeks ), though they may develop shortly after birth or before 20 weeks of pregnancy . the term “ primer extension reaction ” as used herein refers to any polymerization process mediated by the action of a nucleotide polymerase , e . g ., a dna polymerase , by extending a predetermined polynucleotide sequence that is at least partially complementary to a template sequence under appropriate conditions . probe set id nos : 205827_at ; 215141_at ; 202917_s_at ; 215733x_at ; 234601_x_at ; 227238_at ; 239010_at ; 214702_at ; 1553319 _at ; 235592_at ; 229839_at ; 230748_at ; 203789_s_at ; 226482_s_at ; 215388_s_at ; 1562053_at ; 219911_s_at ; 209351_at ; 1552858_at ; 215108_x_at ; 226403_at ; 207607_at ; 228293_at ; 210251_s_at ; 1561318_at ; 241036_at ; 219759_at ; 203592_s_at ; 205302_at ; 1568554_x_at ; 1554276_at ; 242842_at ; 242868_at ; 206859_s_at ; 204580_at ; and 207509_s_at on the affymetrix genechip system ( affymetrix , santa clara , calif . ; hg - u133 plus 2 . 0 genechips containing 53 , 613 probe sets ), as used herein , refer to nucleic acid sequences found on the aforementioned affymetrix genechip system . the polynucleotide sequences are identified by database accession numbers ( e . g ., nm — 003394 . 1 , etc .) in tables 2 , 5 and 9 and each of the accession numbers are hereby incorporated by reference in their entireties . “ standard control ” or “ control sample ” as used herein refers to a sample suitable for use in a method of the present invention , e . g ., in order for quantitatively determining the amount of a nucleic acid . such a sample contains a known amount of the nucleic acid that closely reflects the average level of the nucleic acid in an average non - preeclamptic pregnant woman . similarly , a “ standard control ” may be derived from an average healthy non - pregnant woman . “ an increase and decrease in the amount of the nucleic acid or polypeptide species in the test sample as compared to the standard control ” refers to a positive or negative change in amount from the standard control . an increase is preferably at least 2 . 00 fold , 2 . 25 fold , 2 . 50 fold , 2 . 75 fold , 3 . 00 fold , 3 . 25 fold , 3 . 5 fold , 3 . 75 fold , 4 . 00 fold , 4 . 25 fold , 4 . 50 fold , 4 . 75 fold , of 5 . 00 fold . similarly , a decrease is at least 2 . 00 fold , 2 . 25 fold , 2 . 50 fold , 2 . 75 fold , 3 . 00 fold , 3 . 25 fold , 3 . 5 fold , 3 . 75 fold , 4 . 00 fold , 4 . 25 fold , 4 . 50 fold , 4 . 75 fold , of 5 . 00 fold . for example , an increase of 2 + or greater or − 2 or below would be considered significant difference from control . these expression levels (+ 2 or − 2 ) can also be referred to as “ overexpressed ”/“ overexpression ” or “ underexpressed ”/“ underexpression ”. a “ polynucleotide hybridization method ” as used herein refers to a method for detecting the presence and / or quantity of a polynucleotide based on its ability to form watson - crick base - pairing , under appropriate hybridization conditions , with a polynucleotide probe of a known sequence . examples of such hybridization methods include southern blotting and northern blotting . “ pcr primers ” as used herein refer to oligonucleotides that can be used in a polymerase chain reaction ( pcr ) to amplify a nucleotide sequence originating from a nucleic acid ( rna transcript ). some aspects of the invention provide for primers that comprise the sequences of probe set id nos : 205827_at ; 215141_at ; 202917_s_at ; 215733_x_at ; 234601_x_at ; 227238_at ; 239010_at ; 214702_at ; 1553319_at ; 235592_at ; 229839_at ; 230748_at ; 203789_s_at ; 226482_s_at ; 215388_s_at ; 1562053_at ; 219911_s_at ; 209351_at ; 1552858_at ; 215108_x_at ; 226403_at ; 207607_at ; 228293_at ; 210251_s_at ; 1561318_at ; 241036_at ; 219759_at ; 203592_s_at ; 205302_at ; 1568554_x_at ; 1554276_at ; 242842_at ; 242868_at ; 206859_s_at ; 204580_at ; and 207509_s_at on the affymetrix genechip system ( affymetrix , santa clara , calif . ; hg - u 133 plus 2 . 0 genechips containing 53 , 613 probe sets ). various combinations of the aforementioned primers can be included in a primer kit as set forth herein . as used in this specification and the appended claims , the singular forms “ a ”, “ an ”, and “ the ” include plural reference unless the context clearly dictates otherwise . additionally , the terms “ comprising ”, “ consisting of ” and “ consisting essentially of ” are defined according to their standard meaning . the terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term . the phrases “ isolated ” or “ biologically pure ” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state . thus , isolated peptides or nucleic acids , in accordance with the invention , preferably do not contain materials normally associated with the peptides in their in situ environment . chorionic villus sampling ( cvs ) was performed as follows . samples were donated from 2001 - 2005 by women who gave informed consent for research participation through procedures approved by the university of pittsburgh and magee - women &# 39 ; s hospital institutional review boards . consent for the research was obtained by genetics counselors during the process of informed consent for the clinical cvs procedure . a physician specialized in cvs ( w . a . h .) obtained specimens for clinical cytogenetics by aspiration of tissue into a 20 cc syringe containing amniomax solution for cytogenetics cell culture ( invitrogen , carlsbad , calif .). the solution was not expected to affect rna in any way , although experiments to test this opinion have not been performed ( t . jackson , invitrogen ; personal communication , feb . 28 , 2008 ). as part of the routine cvs procedure , the clinical specimen was poured from the syringe into a petri dish to be assessed by the clinician for adequacy of amount aspirated ( at least 25 mg of villi ). no extra tissue was extracted for the research . if surplus tissue not needed for clinical analyses was available , villi grossly free of decidua and maternal blood were removed from the amniomax in the petri dish , placed in an eppendorf tube , and snap - frozen in less than 10 minutes of cvs aspiration from the patient . research specimens were stored at − 80 ° c . for analyses after birth outcomes became available . frozen samples occupied approximately 5 - 30 μl in the eppendorf tubes . eighty percent of all 160 consented participants had surplus cvs tissue for the research study . the rate of preeclampsia in the cohort was ˜ 3 %. certain preeclampsia ( pe ) and control ( c ) samples were chosen for study by microarray . each of 4 cvs specimens from women who subsequently developed preeclampsia ( pe ) was matched based on parity , gestational age at cvs within 3 days , and race with 2 unaffected control ( c ) specimens for the microarray analysis . the sample size of 4 pe patients was determined by availability of samples in the cvs specimen bank meeting the study &# 39 ; s diagnostic criteria and was the minimal number needed for statistical variance . pe was defined as new onset of hypertension and proteinuria after 20 weeks gestation with blood pressure ≧ 140 and / or 90 on at least 2 occasions at least 6 hours apart , and ≧ 300 mg of protein in a 24 hour urine ( gifford , et al ., 2000 ). these women did not have underlying medical disorders or other obstetrical complications . controls were defined as specimens from normotensive women with blood pressure & lt ; 140 / 90 , no proteinuria , and without other pregnancy complications or underlying medical disorders . additional samples ( as ) for analysis by quantitative real time polymerase chain reaction ( qrt - pcr ) were prepared to test replication of the microarray findings . twenty - four stored specimens were selected from women without pregnancy complications or underlying medical disorders . matching as to the pe cases was prioritized by parity , gestational age at cvs and race . parity was limited to ≦ 3 pregnancies and gestational age at cvs within 3 days . clinical data analysis was carried out . demographic and clinical characteristics of participants were analyzed to examine underlying assumptions of group assignment . pe ( n = 4 ) and c ( n = 8 ) samples submitted for microarray procedures were compared using distributionally appropriate t - test or chi square with p ≦ 0 . 05 set as level of significance . additional samples (“ as ”, n = 24 ) selected for replication purposes were compared with pe and c groups using anova and bonferroni adjustment with p ≦ 0 . 05 set as level of significance ( spss 15 . 0 , carey , nc ). total rna extraction and microarray procedures were conducted at the university of pittsburgh genomics and proteomics core laboratory ( gpcl ). specialized methods were applied for the particular tissue type , resulting in good rna integrity ( agilent rin ≧ 6 . 0 ). in detail , the procedures were as follows : each frozen cvs specimen was homogenized in 1 ml of trizol ( invitrogen , carlsbad , calif .) using a polytron mechanical disrupter ( glen mills , clifton , n . j .) in less than 1 minute . samples were incubated for at least 5 minutes at room temperature to allow complete dissociation . two - tenths volume chloroform was then added . the reaction was mixed vigorously and allowed to incubate 3 minutes at room temperature . following phase separation by centrifuging for 15 minutes at 12 , 000 × g at 2 - 8 ° c ., the aqueous phase was transferred to a clean microcentrifuge tube . the rna was precipitated by adding 0 . 5 ml isopropyl alcohol , incubating for 5 minutes at room temperature and , finally , spinning 15 minutes at 12 , 000 × g at 2 - 8 ° c . the supernatant was removed and the pellet washed by adding 1 ml 75 % ethanol and spinning 5 minutes at 7 , 500 × g at 2 - 8 ° c . the supernatant was carefully removed and the pellet was allowed to air dry for 5 minutes . the rna was resuspended in nuclease free water . rna integrity was evaluated on an agilent bioanalyzer ( rin ≧ 6 . 0 ; agilent , santa clara , calif .). samples were stored at − 80 ° c . the affymetrix genechip system ( affymetrix , santa clara , calif .) was used for microarray analysis with hg - u133 plus 2 . 0 genechips containing 53 , 613 probe sets . the gpcl facility conducted the analysis according to manufacturer &# 39 ; s instructions as explained in more detail below . microarray data collection was as follows : two and one - half micrograms ( μg ) of total rna was used as template in a reverse transcription reaction using oligo ( dt ) 24 primers attached to a t7 rna polymerase promoter sequence . this single stranded cdna was transformed into double stranded cdna . ten units of t4 dna polymerase were added and the reaction incubated at 16 ° c . for an additional 5 minutes . the reaction was stopped by the addition of edta to 0 . 03 m and cleaned up using an affymetrix cdna clean up column . at the end of the second strand reaction , the cdna sample was mixed with dna binding buffer and this mixture applied to the column . the column was spun in a microfuge to bind the cdna to the membrane . dna wash buffer supplied with the kit was used to wash the membrane , which was then dried by centrifugation . the cdna was eluted with provided elution buffer . an aliquot of the ds - cdna equivalent to 5 - 7 μg of starting rna was added as template to an in vitro transcription reaction as per the affymetrix ivt labeling kit . the resulting biotinylated crna was purified using an affymetrix rna clean up column . the procedure was identical to that for the dna clean up using appropriately modified membranes and buffers as supplied . after elution the crna was quantified by reading the od 260 of a 1 : 100 dilution on a spectrophotometer . in addition , 1 μl of the crna was run on an agilent bioanalyzer to verify that most product represented full size transcripts . an aliquot of 20 μg of crna was incubated at 94 ° c . in fragmentation buffer ( 40 mm tris - acetate ph 8 . 1 , 100 mm koac , 30 mm mgoac ) for 35 minutes to break the rna into segments of 35 to 200 bases . a 1 μl aliquot of the sample was run on an agilent bioanalyzer to verify that fragmentation resulted in rna of the desired size distribution . fifteen μg of the fragmented rna was added to a final volume of 300 μl hybridization cocktail consisting of 1 × hybridization buffer , 100 μg / ml herring sperm dna , 50 μg / ml acetylated bsa , 50 pm affymetrix control oligo b2 , 1 × affymetrix eukaryotic hybridization control . 1 × hybridization buffer contains 100 mm mes , 1m na +, 20 mm edta , 0 . 01 % tween 20 . an appropriate volume of this sample was applied to hg - u133 plus 2 . 0 genechip and incubated overnight at 45 ° c . with rotation . following hybridization , the sample was removed and the genechip cassette filled with non - stringent wash buffer . the chip was loaded onto an affymetrix 450 fluidics station for wash and stain . wash and stain protocols are the double stain protocols developed by affymetrix for use with the affymetrix 450 fluidics station . to remove unbound sample , arrays were first washed with non - stringent wash buffer followed by a stringent wash in 100 mm mes , 0 . 1m na +, 0 . 01 % tween 20 . the genechips were then stained for ten minutes in streptavidin - phycoerythrin ( sape ) solution . non - stringent buffer was used to wash off the first stain solution . signal amplification was achieved by ten minutes incubation with biotinylated anti - streptavidin , followed by a second ten minute incubation with sape . the chip was washed and filled with non - stringent wash buffer before being removed from the fluidics station and scanned using the genearray 3000 scanner . the affymetrix genechip system uses a photolithographic process to manufacture 25 - mer oligonucleotide probes directly on the array surface . each mrna or est sequence is represented by 11 probe pairs ( the probe set for this gene ). each probe pair consists of one feature containing a perfect match probe ( pm ) and an adjacent feature containing a mismatch probe ( mm ). the sequences of the two probes differ by one base in the central position . data acquisition from the microarray was via accepted methods . gene expression intensities were derived from the . cel files using dchip ( li et al ., 2001 ) and brb - arrays tools ( simon et al ., 2007 ). the affymetrix hg - u133 plus 2 . 0 genechip contains 53 , 613 probe sets . qa / qc analysis of the data was carried out . examination of the scanned images revealed a number of potential outliers in the array from patient # 147 . subsequent data quality assurance / quality control ( qa / qc ) analysis by correlation and multiplicative - additive ( m - a ) plots confirmed that the inclusion of pe sample # 147 led to group - wise high - expression outliers . all analyses were conducted including and excluding # 147 for further assessment . normalization was conducted as follows . the data were represented as pm only , ‘ signal ’ ( log 2 ( pm - mm )), and robust multi - array average ( rma ) normalized in brb - array tools ( simon et al ., 2007 ). alternative normalization methods were also conducted using the online program for cancer gene expression data analysis ( cageda ) ( patel 2004 ). following data quality assurance / quality control ( qa / qc ) and normalization procedures , differentially expressed genes were identified using the j5 test , pooled variance t - test ( pvt ) and fold change 3 ( fc =[ mean 1 − mean 2 ]/ mean 2 ) as implemented in the online program for cancer gene expression data analysis ( cageda ) ( patel 2004 ). the j5 test is a gene - specific ratio between the mean differences in expression intensity of a gene in two groups to the average mean group difference of all m genes for the entire data set . these alternative methods were evaluated using qa / qc plots and ‘ efficiency analysis ,’ as implemented in cageda . in efficiency analysis , the data are split into two random subsets of roughly equal size . both data sets are analyzed separately , yielding the number of genes found to be significant at each point over the range of the threshold of each test using the two data sets ( n1 and n2 , respectively ). the degree of overlap is compared among tests as the number of genes in the overlap ( n3 ) varies . absent artifacts , the method that provides the highest internal consistency ( highest degree of overlap ) is preferred over methods that fail to yield internally consistent gene lists . qa / qc of sample # 147 included many outliers . the genes found to be differentially expressed with the most efficient test , whether # 147 was included or excluded , were considered further . efficiency analysis was performed over the range of each test with 30 iterations . naïve bayes prediction models were evaluated using leave - one - out cross - validation ( loocv ) in cageda ( patel 2004 ) with the j5 test used for feature selection . loocv refers to splitting the dataset in two , one group is a learning set and the other a test set . ‘ learning ’ or classifier construction is conducted on the training set and evaluated in the test set . modeling was attempted , both including and excluding array # 147 . for this step , the j5 test was wrapped within the evaluation loop ( an iterative function within the computer program ) to minimize the possibility of overtraining , the bias created when classifiers are built from the same dataset to be predicted . the models with lowest achieved classification error ( ace ) found at the highest threshold ( smallest number of genes ) were preferred . functional analysis of identified differentially expressed genes was carried out . annotations were retrieved using a batch query given the probe set identification numbers ( probe ids ) submitted to the affymetrix netaffx resource ( netaffx . 2007 ). probe ids and j5 scores were also submitted to ingenuity pathways analysis 5 . 5 bioinformatics software ( ipa ) ( ingenuity 2007 ) for further investigation of known genes , molecular networks and functions . the functional analysis of a network within ipa identified the biological functions and diseases that were most significantly related to the genes in the network . the network genes associated with biological functions and diseases in the ingenuity pathways knowledge base ( ipkb ) were analyzed within ipa by fisher &# 39 ; s exact tests to calculate a p - value , determining the probability that each biological function and / or disease assigned to a network is due to chance alone , with p - value set at 0 . 05 . ipa functional analysis has three primary categories of functions : molecular and cellular functions , physiological system development and function , and diseases and disorders . eighty - five high level functional categories are classified under these primary categories . lower level functions are classified within the high level categories . specific functions are the lowest level functions found in ipa . each lowest level function has a population of associated genes . specific functions are drawn from manually curated animal and human research findings stored within the ipkb . lower level functions and specific functions may be classified within multiple high level categories . quantitative real - time polymerase chain reaction ( qrt - pcr ) was conducted on a subset of genes to validate the microarray findings . cdna was generated from 0 . 1 μg of total mrna using the high capacity cdna reverse transcription kit ( applied biosystems [ abi ]; foster city , calif .). the most appropriate endogenous control for this tissue was determined using the endogenous control plate ( abi ). ribosomal protein large p0 ( rplp0 [ omim 180510 ]; abi assay # hs99999902 ) was selected and utilized as the endogenous control for each sample . the samples utilized for qrt - pcr included the original 12 microarray samples ( pe & amp ; c ) as well as 24 additional samples ( as ) from unaffected pregnancies . each gene was evaluated in duplicate for each sample . three genes were chosen for evaluation due to being the highest up - or down - regulated in the microarray findings . pre - designed probe and primer sets were utilized for the cck gene ( abi assay # hs00174937 ), ctag2 gene ( abi assay # hs00535628 ) and lair2 gene ( abi assay # hs00430498 ); gene names and omim identification numbers appear in table 2 . qrt - pcr was conducted according to routine protocols using the abiprism 7000 system ( abi ). data analysis for qrt - pcr was conducted . raw cycle threshold ( c t ) values were determined using sds1 . 1 software ( abi ). relative gene expression was determined using the comparative c t method . a threshold value of 0 . 01 was used for lair2 , 0 . 06 for ctag2 , and 0 . 16 for cck . the average value for each sample was normalized to the average value of endogenous housekeeping gene rplpo in the same well , resulting in δc t . ( dharmaraj 2007 ) δc t , reference was the c t value for the calibrator , normalized to rplpo . sample # 38 was used as the calibrator for cck and lair2 because it was a c specimen from a nulliparous nonsmoker at 11 . 0 weeks , an average gestational age at cvs . sample # 138 , also a nulliparous nonsmoker at 11 . 0 weeks , was used as a calibrator for ctag2 because there were undetermined values for # 38 . the difference between δc t and the average calibrator expression value resulted in δδc t . 2 (− δδct ) determined the fold change in expression level relative to the calibrator sample . fold changes were analyzed by kruskal - wallis ( kw ) with significance set at p & lt ; 0 . 05 . analyses were carried out in excel and sas 9 . 1 ( sas institute inc ., cary , n . c .). secondary analysis was conducted by expression fold changes ( fc ; table 3 ). scatterplots demonstrated that use of untransformed raw data was appropriate . the average expression level of each transcript for all cases and all controls were calculated . for pairs with average expression level of at least 30 in both cases and controls , the ratio of means in pe to c was calculated as the fold ratio . fold ratio over 0 are analogous to fold change . fold ratio below 0 is expressed as a decimal number , which is converted to fold change by dividing 100 by the numeric following the decimal ( x − n ). fold change greater than + 2 or less than − 2 was considered significant ( patel 2004 ). clinical data are summarized in table 1a . all women in the sample for microarray analysis ( total n = 12 ) were of advanced maternal age ≧ 35 years ( 35 - 44 ), white , and had normal fetal karyotype on cvs . pe ( n = 4 ) and c ( n = 8 ) groups did not differ in maternal age ( p = 0 . 34 ; table 1a ). eleven of 12 samples were from nulliparous women and one c was a multiparous participant in her second pregnancy ; groups did not differ by parity ( p = 1 . 00 ). although not significantly different , bmi showed a trend toward significance with pe bmi 29 . 9 ± 4 . 2 and c bmi 24 . 5 ± 4 . 0 ( mean ± sd ; p = 0 . 06 ). gestational age at cvs of both pe and c groups ranged from 10 . 7 - 12 . 4 weeks and did not differ between groups ( p = 0 . 78 ). pe and c groups did not differ in gestational age at delivery ( p = 0 . 11 ), birthweight ( p = 0 . 10 ), or infant sex ( p = 1 . 00 ). one c participant reported smoking ; all others were self - reported nonsmokers . groups did not differ in smoking ( p = 1 . 00 ). additional clinical data for the four preeclampsia cases are shown in tables 1b and 1c . the four pe participants met inclusion criteria for hypertension and proteinuria . three were hyperuricemic for gestational age ( table 1c ). systolic and diastolic blood pressures at less than 20 weeks showed no preexisting hypertension . participant # 147 reached a severe blood pressure above 160 / 110 and was delivered by cesarean section . participant # 19 had low platelets , high creatinine , and was also delivered by cesarean section . two pe cases (# 21 and # 147 ) delivered preterm , prior to 37 weeks &# 39 ; gestation . pe # 21 was obese ( bmi ≧ 30 ) and her infant was growth restricted , below 10 th percentile for gestational age , delivered by pitocin induction . participants in the as group ranged in age from 34 - 45 years ( mean 38 . 2 years ) and were 10 . 4 - 13 . 0 weeks gestation ( mean 11 . 5 weeks ) with bmi 19 . 5 - 36 . 4 ( mean 25 . 6 ) at cvs . seven were nulliparous and 17 were multiparous women with uncomplicated pregnancies and unknown cvs gene expression patterns . all were white women and had normal fetal karyotype on cvs . all were self - reported nonsmokers . it is unknown whether each multiparous woman conceived with the same partner for all of her pregnancies . the as group did not differ from pe or c by maternal age ( p = 0 . 59 ), gestational age at cvs ( p = 0 . 66 ), or bmi ( p = 0 . 19 ) at cvs . oligonucleotide microarray analysis of cvs specimens by the methods described herein produced global gene expression patterns in early pregnancies destined for preeclamptic versus unaffected outcomes . four pe compared with 8 c specimens resulted in a set of 36 differentially expressed genes . the qa / qc correlogram scatterplots comparing signal intensity across the microarrays were prepared that included case # 147 ( fig2 a ) or excluded # 147 ( fig2 b ). high - expression outliers found in # 147 resulted in subsequent analyses being performed twice , alternatively including or excluding # 147 . robust multi - array average ( rma ) normalization in rma - brb - array tools ( simon et al ., 2007 ) resulted in a coefficient of variation of 0 . 001 . expression levels were assessed using the j5 test , pvt and fc as implemented in cageda ( patel 2004 ). the j5 test using the raw perfect match data ( supplement ) only led to the most efficient and internally consistent set of differentially expressed genes , exhibiting 40 % consistency in the set of genes found to be differentially expressed between pe and c ( fig3 ). this contrasts with less than 5 % internal consistency explained by pvt and approximately 10 % explained by fc . fold change and the t - test are known to lead to high false - positive rates , especially with small sample number ( patel 2004 ). efficiency analysis in j5 identified 36 genes of interest in the overlap of gene sets with and without # 147 . prediction modeling was explored with naïve bayes models using loocv ( fig4 ). modeling was conducted with and without sample # 147 . the modeling that included # 147 exhibited perfect accuracy , equaling sensitivity and specificity ( sn = sp = acc = 1 . 0 ). the model with a j5 score of 8 , which excluded # 147 , led to the lowest ace for potential genomic biomarkers of preeclampsia with 90 % accuracy . the affymetrix probe identification numbers ( probe ids ) submitted to netaffx resulted in annotations for gene names and gene ontology ( go ) molecular function description for the 36 genes of interest ( tables 2 and 9 ; table 9 includes omim numbers , gene names and symbols ). five genes of interest were up - regulated and 31 were down - regulated . at this time , 7 are mapped genes with unknown functions and 4 are unmapped with unknown function . netaffx identified go pathway information for only 3 of the 36 genes of interest ( table 9 ). ipa identified potential relationships among some of the genes of interest . two networks were developed from the 36 genes imputed with other genes associated through previous studies annotated in the ipkb ( fig1 ). top functions and diseases associated with network 1 involving 10 of the 36 genes of interest were cancer , respiratory disease , and cellular movement . top functions and diseases associated with network 2 , involving 7 genes of interest , were inflammatory disease , cellular movement , and hematological system development and function . mmp12 was the only gene shared between networks 1 and 2 . seventy - two function and disease categories among the genes of interest reached significance ( p & lt ; 0 . 05 ). ten functional categories achieved the highest significance levels and included more than one of the 36 genes of interest ( table 3 ). categories comprising the 2 networks among the genes of interest included lower level function subcategories . lower level functions within cancer were cancer , neoplasia , tumorigenesis , ovarian cancer , gonadal tumor , mitosis , cell rounding , invasion , apoptosis , adhesion , migration , attachment , and detachment . respiratory disease was the 28 th most significant function and disease category which included fn1 , mmp12 , cck , and epas1 (− log p - value = 1 . 63e - 03 - 2 . 72e - 02 ). lower level functions within respiratory disease included primary pulmonary hypertension , lung tumor , lung cancer , adhesion , neonatal surfactant , and emphysema . cellular movement included migration of all types of leukocytes , blood , intestinal , embryonic , neuronal , bone marrow , gonadal and cancer cells , cell movement , immobilization , scattering , and invasion . inflammatory disease was the 62 nd most significant function and disease category which included f11r , s100a8 , and mmp12 (− log p - value = 8 . 24e - 03 - 2 . 42e - 02 ) and lower level functions related to juvenile rheumatoid arthritis and emphysema . hematological system development and function included migration and cell movement of all types of leukocytes , cell spreading , adhesion , immobilization , replacement , and proliferation . relative quantitation of lair2 , cck , and ctag2 compared to calibrator sample was conducted by qrt - pcr . lair2 was significantly down - regulated by a median fold change of 0 . 21 in pe , compared to median fold change 1 . 17 in c , and 4 . 99 in as ( p = 0 . 0004 ). the median fold change in cck was 25 . 37 ( range 1 . 01 - 51 . 45 ) in pe , compared to 2 . 19 ( 0 . 46 - 30 . 38 ) in c , and 5 . 77 ( 0 . 07 - 26 . 63 ) in as . the raw microarray expression level and qrt - pcr fold change in each pe sample were 4223 . 4 and 51 . 45 in # 19 , 732 and 1 . 72 in # 21 , 7339 . 4 and 49 . 01 in # 58 , 59 . 7 and 1 . 01 in # 147 . although the trend for cck fold change was in the same direction as the microarray overexpression , the qrt - pcr differences by kruskal - wallis were not significant between groups ( p = 0 . 60 ). qrt - pcr for ctag2 resulted in 11 of 36 samples as “ undetermined .” the values for endogenous control rplpo were consistent among all 36 samples for each of the 3 genes in qrt - pcr with an average of 24 . 25 ± 0 . 12 in ctag2 . the median fold change in ctag2 was 5 . 78 ( range 0 . 28 - 11 . 27 ) in pe , compared to 0 . 04 ( 0 . 01 - 1 . 00 ) in c , and 0 . 90 ( 0 . 02 - 12 . 64 ) in as . the trend in ctag2 fold change was toward overexpression in pe , but the differences were not significant ( p = 0 . 39 ). this microarray analysis of surplus cvs produced differences in global gene expression in placentas of early pregnancies destined for preeclampsia . utilization of first trimester placentas with known pregnancy outcomes , oligonucleotide genechips , and subsequent prediction modeling distinguish our study from previous placental microarray investigations in preeclampsia ( founds et al ., 2008 ) qrt - pcr confirmed the trends in over - and underexpression in the microarray analysis . the variation in ctag2 qrt - pcr expression patterns may indicate methylation in nonexpressors denoted as “ undetermined ” among all 3 groups of samples ( lethe et al ., 1998 ). cck was also up - regulated 3 . 1 fold in placentas of preeclamptic pregnancies at 29 - 32 weeks in a previous study ( reimer el al ., 2002 ). overall , our results directly support the concept of the placental origins of the disorder ( redman et al ., 2005 ) and allow for targeted investigation of placental - derived biomarkers in early pregnancy . assessment of cause rather than effect of preeclampsia is likely to have been more discernable in the first trimester placental tissues . the findings in this study suggest that impaired placentation in preeclampsia may be associated with an overall deficiency rather than an excess of gene expression , insofar as 31 of the 36 genes of interest were down - regulated . preconceptional testing of susceptibility to preeclampsia could be developed from variants of the genes of interest . in addition , several produce secreted protein ( fig1 ), such that measurement of one or a combination of these biomarker proteins in maternal blood in the first trimester may prove to be a predictive screening test for preeclampsia . innate immune responses at the maternal - fetal interface are likely to be represented by our genes of interest . remarkably , 12 of the 36 genes , 7 not previously associated with preeclampsia , are involved in immune dysregulation ( table 9 ). all of the immunoregulatory genes except s100a8 were down - regulated , implicating deficient , blocked , or impaired function . lair2 , hps3 , and sart3 are immune - related genes ( table 9 ) that were not incorporated by ipa into the immune pathway ( fig1 ; table 3 ). the immune dysregulated cells may be trophoblasts , which are fetoplacental epithelial cells ( petty et al ., 2006 ) that act as a pregnancy - specific component of the innate immune system ( guleria et al ., 2000 ). by day 14 post conception , cytotrophoblasts ( ctbs ) have breached the chorionic basement membrane , switching from a proliferative to an invasive phenotype as extravillous trophoblasts ( evts ) ( huppertz et al ., 2007 ). lower level functions in networks 1 and 2 , e . g ., inflammation , migration , and invasion , are involved in ctb placentation processes ( fig1 ). the evts faun cell columns contacting maternal immune cells in the decidua ( benirschke et al ., 2006 ). from these columns , evts invade the uterine wall and remodel the maternal spiral arteries by displacing smooth muscle and endothelial cells ( pijnenborg et al ., 1983 ). normal trophoblast development differs from cancer in that proliferation ceases during invasion ( huppertz et al ., 2007 ). various genes associated with both of these processes were down - regulated in preeclampsia ( tables 2 and 9 ). in the current analysis , no notable differential expression existed between pe and c in evt epithelial or integrins ( damsky et al ., 1992 ; irving et al ., 1995 ) or human leukocyte antigens ( apps et al ., 2007 ) identified in other studies as dysregulated in ctbs of later gestation . alternatively , some of the differentially expressed immunoregulatory genes may suggest abnormalities of fetoplacental hofbauer cells , which are macrophages that populate the villous core ( benirschke et al ., 2006 ). the maternal innate immune system predominates at this stage with 70 % of decidual leukocytes consisting of natural killer cells ( nk ), 20 - 25 % macrophages and about 2 % dendritic cells ( mor 2006 ). approximately 1 - 3 % of decidual immune cells at this time are adaptive system t lymphocytes ; no b cells are present ( lessin et al ., 1988 ). thus , some of the immunoregulatory genes of interest could also be of maternal origin . finally , one cannot exclude the potential contribution of circulating fetal or maternal immune cells in the placenta ( huppertz et al ., 2007 ). surprisingly , a number of differentially expressed genes may be found in decidual stroma , including muc15 ( shyu et al ., 2007 ), igfbp1 ( bischof et al ., 1989 ), and paep ( jeschke et al ., 2005 ). although the goal of cvs is to obtain chorionic tissue for fetal genetic diagnosis , maternal decidual tissue is invariably present , as corroborated by our microarray analysis . decidual tissue likely derives from placental septae projecting upwards from the basal plate towards the chorionic plate that contain an admixture of decidual cells , evts , and occasional trophoblast giant cells ( benirschke et al ., 2006 ). on balance , the results suggest that preeclampsia may be associated with impaired decidualization . whether this is etiological or secondary to suboptimal interaction with and stimulation by trophoblasts or maternal immune cells , or both is currently unknown . an alternative explanation , albeit less likely , is that there are fewer of these septae in early preeclampsia placentas , thus decreasing decidual tissue and consequently decidual gene expression in these cvs specimens . ten genes of interest were not found in any of the 72 function and disease categories assessed by ipa or located in current literature searches in conjunction with preeclampsia : ctag2 , muc15 , oxgr1 , scara5 , mageb6 , tnrc9 , tmc4 , depdc7 , rufy3 , and lair2 . we suggest functional groupings , other than immune / inflammation , integrating these with all genes of interest ( table 4 ). in order to examine hypotheses concerning hypoxia inducible transcription factors and oxidative stress , a secondary analysis of fold changes was conducted with the caveat that a high rate of false positives could be expected ( table 5 ) ( patel 2004 ). the concept that the placenta is hypoxic or over - expresses hif - 2α protein during early gestation , thereby impairing trophoblast invasion in preeclampsia , ( rajakumar et al ., 2001 ) is not corroborated by this microarray analysis . we interrogated 26 genes proven to be hif target genes ( wenger et al ., 2005 ) and were been shown to be over - represented in placentas delivered from preeclamptic women ( tables 2 and 9 ) ( rajakumar et al ., 2007 ). only igfbp1 , wt1 and th genes showed differential expression in one probe . moreover , igfbp1 and th are typically up - and not down - regulated by hypoxia ( rajakumar et al ., 2004 ; tazuke et al ., 1998 ). interestingly , epas1 or hif - 2α ( tables 2 and 9 ) expression was markedly decreased , but did not consistently correlate with putative specific hif - 2α target genes ( table 3 ), ( lofstedt et al ., 2007 ) suggesting adequate hif - 2α protein levels , transcriptional activity or compensation despite markedly reduced hif - 2α mrna expression . nor were we able to support the differential expression of oxidative stress regulated genes at this early stage of pregnancy ( jauniaux et al ., 2000 ; many et al . 2000 ). we interrogated fold changes in 11 genes previously shown to be regulated by oxidative stress ( table 7 ), ( allen et al ., 2000 ) and expression differences were nonsignificant . in fact , blood flow and oxygen delivery to the intervillous space begins around 10 - 12 weeks of gestation ( jauniaux et al ., 2000 ), but expression profiles of the hypoxia ( table 6 ) and oxidative stress ( table 7 ) regulated genes do not support the concept of an undue delay or acceleration of this crucial physiological event , respectively . thus , ischemia - hypoxia and oxidative stress due to reperfusion injury are likely to be later events in preeclampsia . noteworthy is that 17 of the 36 genes identified by the naïve bayes prediction model and j5 test were among the 152 identified by 2 - fold fc analysis . thus , there is considerable intersection of the two analytical approaches . the finding of aberrant decidualization in early placentas of preeclampsia revealed by the prediction modeling is bolstered by the fc analysis , insofar as fstl3 ( fc − 2 . 56 ) ( jones et al ., 2002 ) and prolactin ( fc − 7 . 86 ) ( telgmann 1998 ) are downregulated ( table 5 ). additionally , marked downregulation of granulysin in the fc analysis ( fc − 23 . 51 ) further supports immune dysregulation in decidua ( mincheva - nilsson et al ., 2000 ). all patents , patent applications , provisional applications , and publications referred to or cited herein , supra or infra , are incorporated by reference in their entirety , including all figures and tables , to the extent they are not inconsistent with the explicit teachings of this specification . it should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application . † preeclampsia definition is based on the national high blood pressure education program working group ( gifford et al ., 2000 ). damsky c , fitzgerald , m l , fisher , s j distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway , in vivo . j clin invest 1992 ; 89 : 210 - 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