, it must develop tolerance to paternal antigens to avoid a lethal immunologic attack against the fetus . on the other hand
, it must preserve the ability to fight infections from a multitude of commensal and environmental pathogens .
disruption of this balance can have devastating consequences , including preterm labor and death of the fetus and/or mother .
the normal maternal immune response to pregnancy is increasingly recognized as a dynamic process , with changes in the maternal pro / anti - inflammatory profile occurring at different stages of gestation [ 13 ] . despite this recognition , a complete , longitudinal immunologic profile of normal
such analyses are critical for understanding the response to specific infectious and immunologic diseases that show disproportionately negative outcomes during pregnancy , such as influenza   and ulcerative colitis .
one approach to such immune profiling is through the use of multiplex cytokine arrays , allowing for simultaneous quantification of many proteins with a very small amount of plasma or serum .
others have used multiplex arrays to study the maternal cytokine milieu , but the studies have been predominantly limited time point cross - sectional [ 68 ] or case - control [ 911 ] in design .
the results of some longitudinal studies have been published in recent years , although results have been based on relatively few time points ( 5 or fewer ) [ 13 ] . in this study
, we applied the multiplex array approach to evaluate the changes in 42 cytokines in rich detail during pregnancy .
the samples are from a cohort of 16 pregnant women , with each subject sampled a median of 18 times . because this represents a significant increase in the number of tests per subject compared to previous studies
, we also describe a strategy addressing within - subject correlation with repeated measurements using unconditional growth modeling . with this approach
, we have been able to detail the typical longitudinal variation of serum cytokines throughout pregnancy in this cohort of women .
each participant had a serum sample collected and cryopreserved biweekly from study enrollment during the first trimester ( average of 9.7 weeks ' gestation for first samples ) until 34 weeks ' gestation , then weekly from 34 weeks until delivery .
as a result of this repeated sampling , a median of 18 samples per participant were obtained longitudinally throughout pregnancy .
samples were collected under a protocol approved by the mayo clinic institutional review board ( irb ) that accrued participants from 1987 to 1988 .
information regarding date of the blood draw , date of delivery , outcome of the delivery , and any major complications that the woman experienced during pregnancy ( hypertensive disorders , preterm labor , and infections ) was available and helped identify appropriate participants to include .
only women who carried their pregnancies to term and experienced no pregnancy complications were included in this study .
we only used samples from subjects who had specimens obtained throughout the entire course of pregnancy and whose complete , longitudinally collected specimens appeared intact and nondesiccated .
most cytokines are stable in storage at 80 degrees c for up to 2 years , but stability beyond that time is not well known . therefore , before proceeding with studies on the samples of interest , we first tested protein levels of a small number of individually stored serum aliquots from other subjects archived on this protocol . since we found detectable levels of cytokines in the pregnant serum samples that were within range of control samples , we proceeded with the entire study involving a cohort of 16 healthy , primigravid women who completed a term pregnancy without any significant complications .
samples were assayed when freshly thawed to avoid freeze - thaw cycles , which are known to degrade cytokines .
contemporary plasma samples from 11 nonpregnant control subjects were collected under a separate irb - approved protocol for collection of biospecimens from healthy individuals within the same health system in 2010 and similarly assessed on the same 96-well plates for comparison and as a quality control measure . for contemporary nonpregnant control samples ,
peripheral venous blood was drawn into heparinized vacutainer tubes that were processed and separated into plasma and peripheral blood mononuclear cells ( pbmcs ) following gradient centrifugation using ficoll - paque ( ge healthcare , uppsala , sweden ) .
plasma was collected and immediately frozen at 80c in 1  ml aliquots until use .
protein levels for 42 cytokines , chemokines , and growth factors ( table 1 ) were measured using the milliplex map human cytokine / chemokine kit ( millipore , billerica , ma , usa ) per manufacturer 's instructions . to minimize the potential for interassay variability , each subject had all of their longitudinal specimens analyzed on the same plate .
protein concentrations were determined using a linear regression standard curve from each plate generated using the high pmt concentrations with sensitivity from 3.2 to 2,000  pg / ml . all samples were tested in duplicate with the mean value of the measurements used for statistical analyses .
comparison of baseline ( first trimester ) and end of pregnancy levels of cytokines / growth factors was carried out between the 16 primigravid subjects and the 11 nonpregnant control subjects using the kruskal - wallis tests .
there were extremes of values that fell outside of the limits of the multiplex array .
for cytokines below the limit of detection ( ld ) , we assigned a value that was half the lowest detectable value for the assay as previously described .
analytes above the detectable range were assigned a threshold concentration . to obtain a visual representation of overall patterns in the highly dimensional raw data
as an additional multivariate technique to identify patterns within the data , principle components analysis ( pca ) was also completed on the raw values obtained from the multiplex array . to assess the within - subject correlation , we determined change of cytokines and growth factors from a baseline time point  the values obtained from the participants ' first blood draw in the first trimester  by taking the log of 1 plus fold change from the baseline value : log(1 + x ) , where x = ( cytokine concentration of the sample of interest / cytokine concentration of the baseline sample ) to analyze trends over time .
first , we graphically assessed the growth trajectory for the 42 cytokines using smoothing splines .
we then fit unconditional mean models ( umm ) for the 42 cytokines in order to estimate the variance components : the within - subject variance (  ) and the between - subject variance ( 0 ) . estimating the two variance components helps to determine whether there is sufficient variation to warrant further analysis and enabled computations of the intraclass correlation coefficient ,  , which describes the proportion of the total outcome variation that lies between subjects .
next , we fit unconditional growth models ( ugm ) , which allowed random slope but not random intercept because the starting value of all patients was the same ( baseline value = 0.3 ) due to normalization to each individual 's baseline value . as both gestational age in weeks and trimester
can be used as the time covariate for growth curve models , and as the trimester can be treated as either a continuous or a categorical variable ( depending on whether we assume linear relationship between trimester and outcome variables ) , six different models were fitted for each cytokines as follows : ( 1 ) model with only the gestational age in weeks as the time covariate , ( 2 ) model with only the trimester ( continuous ) as the time covariate , ( 3 ) model with only the trimester ( categorical ) as the time covariate , where the variance - covariance pattern was assumed to be autoregressive , ( 4 ) model with both gestational age in weeks and continuous trimester as covariates , allowing random slope for gestational time in weeks , ( 5 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slope for trimester , and ( 6 ) model with both gestational age in weeks and continuous trimester as a covariate , allowing random slopes for both gestational age in weeks and trimester .
we then compared the six models and the unconditional mean model for each of the cytokines based on logic and statistical fitness , using akaike information criteria ( aic ) .
because of the exploratory nature of this study , there was no correction for multiple comparisons .
the multiplex was designed to assess 42 factors , but the limits of detection were exceeded with three factors , and there were 5 cytokines that fell below the limit of detection in > 50% of the samples .
pdgf - aa , pdgf - ab / bb , and rantes had cytokine levels above detection limits of the assay in 9.9% , 11.2% , and 34.9% of the samples , respectively ( table s1 ; see supplementary material available online at http://dx.doi.org/10.1155/2015/952571 ) .
il-2 , il-3 , il-4 , il-13 , and tnf had > 50% of the samples below the detection limit ( table s1 ) .
differences in cytokine profiles between baseline ( first trimester ) pregnancy samples and nonpregnant control samples could be identified by pca ( figure 1 ) .
specifically , we identified significantly higher levels of gro , tgf , egf , pdgf - aa , and pdgf - ab / bb and significantly lower levels of scd40l , ip-10 , il-6 , il-17 , il-13 , and mcp-1 in the baseline pregnancy samples ( table 1 ) . when all longitudinal samples were included in a pca , no difference
next , we evaluated whether those differences apparent in early gestation were also present at the end of pregnancy . when comparing the final independent samples obtained at the end of pregnancy to the healthy control samples , the following remained significantly elevated in pregnant women : egf ( 330.6 versus 24.8  pg / ml , p = 0.0013 ) , gro ( 1,153.6 versus 341.6  pg / ml , p < 0.001 ) , sil-2ra ( 17.9  pg / ml versus lld , p = 0.02 ) , and tgf ( 15.8 versus 1.8  pg / ml , p = 0.0006 )
meanwhile , only eotaxin was lower in women at the end of pregnancy compared to normal controls ( 33.4 versus 330.0  pg / ml , p < 0.001 ) . to begin to assess within - subject variation , we clustered the raw data ( figure 2 ) , where the issue of correlation within subjects becomes visually apparent .
empirical growth plots demonstrated that the within - subject variation differs among different women ( figures 3 and 4 show a representative example using il-15 , and the remainder of the plots can be viewed in supplementary figures ) .
for example , patient o displays very large variation in the majority of the cytokines tested , whereas patients b , c , f , and g have comparatively steady levels for most cytokines .
also , high variability in one cytokine does not imply high variability in other cytokines for the individual women .
high interclass correlation ( 0.7 ) was observed for scd40l , rantes , il-10 , tnf , il-7 , and gm - csf ( table s2 ) .
this means that the majority of the variability in these cytokines is attributed to variance between subjects .
the interclass correlations were comparatively low ( < 0.3 ) for il-3 , il-4 , mcp-3 , and ip-10 , suggesting more variation within subjects for these cytokines .
the final model for mdc ( table 2 ) was ugm with gw , with a decreasing trajectory .
the estimated variance for the random slope is 0.000001632 , suggesting that the variability of slope among patients is quite small .
on the other hand , the average scatter of an individual 's outcome around her own trajectory is larger ( the estimated variance is 0.000937 , p < 0.0001 ) , suggesting that there might be some other important covariates that are not included in the model .
however , including gestational age in weeks as a covariate does substantially improve the fit of the model .
when comparing the estimated within - subject variance of the ugm with that from the umm , we found that the linear gestational age in weeks helps to explain 34% of the within - subject variation in mdc .
the estimated fixed effect suggests that mdc decreases over time ( the estimated slope is 0.00163 , with p  0.0001 , suggesting that the slope is significantly lesser than 0 , figure s2 ) .
eight other cytokines demonstrated statistical significance , although with increasing trajectories , with ugm and gw as a covariate : il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand ( table 2 ) .
six cytokines showed association with trimester as a linear variable : il-1ra , il-3 , il-9 , il-12p40 , ifn2 , and scd40l ( table 3 ) .
il-1ra showed the greatest percent explanation by inclusion of linear trimester in the model . when comparing the estimated within - subject variance of the umm with that from the ugm
, we found that the linear trimester helps to explain 27% of the within - subject variation in il-1ra .
seven other cytokines showed significant associations with trimester as a categorical variable : il-4 , ifn , g - csf , tgf- ,
tnf- , sil-2ra , and mip-1 ( table 4 ) . the final model for both egf and vegf was umm with intercept only , leading to the conclusion that both egf and vegf do not change over time .
time - dependent ugm models were identified but did not meet statistical significance for the following cytokines : il-2 , il-5 , pdgf - aa , pdgf - ab , mip-1 , gro , mcp-1 , mcp-3 , rantes , il-17 , il-7 , eotaxin , fgf-2 , il-10 , tnf , il-1ra2 , fracktalkine , and gm - csf . therefore
, approximately 50% of cytokines measured by multiplex array did not show any association with gestational week or trimester .
our results suggest that the third trimester of pregnancy is characterized by an increasingly inflammatory ( e.g. , il-1 , il-6 , il-12 , il-15 , ip-10 , and scd40 ligand ) as well as counterregulatory ( e.g. , il-1ra and flt3-ligand ) milieu compared to earlier stages of pregnancy .
our superimposed growth curves of il-15 ( figure 4 ) and il-1 ( figure s3 ) , the cytokines with the strongest association with gestational week as a linear covariate , suggest that this change begins at approximately week 20 and peaks just after week 30 .
this is consistent with other reports of systemic immune activation as well as counterregulation in the latter part of pregnancy [ 3 , 1719 ] .
increasing il-1 and il-15 may predominantly be related to production from placental tissues [ 20 , 21 ] or mononuclear phagocytes [ 22 , 23 ] in late gestation .
the precise immunologic mechanisms responsible for this shift can not be determined by our study , but if the source of these cytokines is predominantly the innate immune system , one potential stimulus to these cytokines ' secretion is cell - free fetal dna . a recently proposed model links rising circulating levels of fetal dna to maternal innate immune activation via toll - like receptor- ( tlr- ) 9 on neutrophils and macrophages , leading to increasing inflammatory cytokine release , that may result in an immune cascade ultimately leading to parturition .
a novel finding of this study is the decline in mdc levels throughout pregnancy ( figure 5 ) .
mdc is chemoattractant for immature dendritic cells   and type 2-biased t cells , and it is possible that decreasing mdc levels may reflect a gradual shift away from a self - amplifying type 2 immune response as pregnancy progresses .
il-9 is cytokine that has been recognized for decades but only recently had its cell of origin identified : the innate lymphoid cell ( ilc ) .
the role of il-9 , which has historically been categorized as a th2 cytokine prior to the recognition of ilc , is currently unknown in normal pregnancy .
because only serum was available for analysis , we can not confirm the cellular source of the changes observed with this cohort .
nonetheless , increasing transcription levels of several innate immune components , including cd14 , multiple tlr genes , and il-1b , have been observed in peripheral blood leukocytes of women in the third trimester , suggesting that the changes in concentrations of the cytokines identified in our study could plausibly be due to changes in peripheral blood cellular composition .
further studies will be necessary to determine whether placental tissues , circulating leukocytes , stromal cells , or other sources also contribute to the changes reflected in our results .
although these results show statistical significance , it is also noteworthy that the slopes of the variance were all fairly small for each of the analytes and that gestational week did not explain any more than 40% of the variation observed in any of the cytokines . pregnancy clearly alters the maternal immune milieu , but the changes from baseline in the setting of normal pregnancy are subtle and more variable in some women than in others .
what clinical factors contribute to this variation observed between women in the setting of a normal first pregnancy is not entirely known .
blood draws and resulting data were relatively sparse in the first trimester due to women not returning as frequently for study blood draws , even though they were scheduled per protocol , relative to the second and third trimesters .
it is also important to note that many analytes were not detectable in samples ; therefore , it is possible that a different method besides multiplex array on serum or plasma , for example , elisa for individual cytokines / growth factors , may be more sensitive to detect change over time .
previous longitudinal studies of maternal cytokines during pregnancy have shown some concordance with our results .
investigated the change of maternal plasma cytokines from early to midgestation in a large cohort ( approximately 1,000 patients ) and found that il-12 and ifn levels increased , while il-2 and gm - csf levels decreased as pregnancy progressed .
our results confirmed increasing il-12 levels , as well as an increase of ifn in the third trimester compared to the first .
however , kraus et al . published maternal multiplex elisa results from 50 women tested at each trimester as well as postpartum and showed that ifn levels decreased throughout gestation .
denney et al . also found that basal levels of ifn decreased throughout gestation , as did levels of tnf , il-1 , and il-6 in serum samples of 45 healthy pregnant women collected during each trimester .
another recent study demonstrated decreasing il-1 levels throughout gestation . while our study did not confirm any time - dependent change of tnf , we instead observed increases in il-1 and il-6 throughout pregnancy , in contrast to these studies .
clearly , individual heterogeneity exists , and our smaller sample size may account for some of these differences with prior studies .
although not the original focus of this study , we also identified several differences in cytokine / growth factor milieu when comparing pregnant women to healthy controls .
notably , the first trimester of pregnancy was characterized by an increase in several growth factors ( gro , egf , tgf , and pdgf ) and a relative decrease in inflammatory markers ( scd40l , il-6 , il-17 , ip-10 , eotaxin , and mcp-1 ) .
eotaxin , a potent chemoattractant for eosinophils during allergic reactions , has been similarly described to be suppressed during pregnancy by kraus et al . .
overall , the tolerance induction required for successful pregnancy at the level of the fetomaternal interface may also be observed in a relatively tolerogenic , growth factor - rich maternal systemic environment . the difference in sample source when comparing
the archived samples from healthy pregnant women ( serum ) and normal controls ( plasma ) may contribute to these findings .
for example , eotaxin , pdgf , egf , vegf , and soluble cd40 ligand have previously been shown to be significantly different in serum postclotting as compared to plasma samples .
because of this potential source of variation in comparing our historical to contemporary samples , our results will require validation with similarly processed samples of the same source .
however , egf levels have been reported to be approximately 65  pg / ml in serum of healthy individuals , well below our median 489.4  pg / ml in our samples obtained from pregnant subjects , suggesting that our results may hold true despite differences in sample source .
similarly , serum levels of cd40l have been shown to be 830-fold higher in serum as compared to plasma , suggesting again that the very low levels identified in the serum of our cohort of pregnant women may indeed be reflective of the pregnancy state .
our study is unique in that the longitudinal samples were obtained frequently throughout the course of pregnancy .
because of this , our study has the potential to more richly describe individual maternal immune variation over time . however , our study is limited by the relatively small sample size , the lack of pre- or postpartum samples , and potentially the age of the specimens . to address these limitations ,
we have recently conducted a larger longitudinal study of maternal immune changes during pregnancy , with peripheral blood samples obtained monthly throughout gestation as well as 6 weeks postpartum .
tolerance at the fetomaternal interface is complex and technically challenging to study in humans longitudinally . as a result , many recent studies have sought to understand how pregnancy changes maternal systemic immunity .
the possibility exists of maternal peripheral blood not completely or accurately reflecting the local changes within decidual tissues .
an example of such a discrepancy in our study is that of ip-10 , which was low in first - trimester pregnancy compared to healthy controls in this cohort
. ip-10 is secreted by decidual natural killer cells and is key to trophoblast migration and thus has previously been shown to be elevated during pregnancy [ 31 , 32 ] .
on the other hand , the decidual t cell compartment at parturition has recently been shown to closely reflect that of peripheral blood , suggesting that for some components of the immune system the maternal peripheral blood is reasonable to study .
furthermore , sampling maternal peripheral blood is much more feasible to perform longitudinally as compared to more invasive means and may still lend insight into the maternal adaptation to pregnancy .
successful pregnancy has previously been described as predominantly a type 2 immune response - biased phenomenon [ 3436 ] . in light of the collective evidence
, it seems that the description of pregnancy in terms of type 2 immune responses may be oversimplified , both in the overall description of key cytokines / growth factors as well as in the temporal dynamics , and it is in need of further refinement .
 several recent studies have shown differences in the maternal immune milieu at different phases of pregnancy , but most studies have been cross - sectional or of relatively few time points . 
 levels of 42 cytokines were determined using a multiplex bead - based assay on archived serum from a cohort of pregnant women ( n = 16 ) at median of 18 time points tested , from the first trimester through to parturition , per woman . 
 unconditional growth modeling was then used to determine time - dependent changes in levels of these cytokines . 
 macrophage - derived chemokine ( mdc , aka ccl22 ) decreases as pregnancy progresses . 
 il-1 , il-6 , il-8 , il-12p70 , il-13 , il-15 , ip-10 , and flt3-ligand increase as a function of gestational weeks , and ifn2 , il-1ra , il-3 , il-9 , il-12p40 , and soluble cd40 ligand increase as a function of trimester . as 
 pregnancy normally progresses , a maternal shift away from a type 2-biased immune response and toward an inflammatory / counterregulatory response is observed . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions