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Cite this article as: Anderson BL, Mendez-Figueroa H, Dahlke JD, et al. Pregnancy-induced changes in immune protection of the genital tract: defining normal.Am

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Research www.AJOG.orgof host factors that serve to protectagainst infection and disruption of thenormal flora. The lower tract requirescytokines, chemokines, antimicrobialpeptides, and genital flora to recognizethreats and react tomaintain homeosta-sis. Disruption in several individualcomponents of lower genital tract im-munity has been associated withHIV ac-

Hillier).

ceived Oct. 12, 2012; revised Jan. 2, 2013; accepted Jan. 9, 2013.

ancial support was provided by the Womens Health Reproductive Health Research Careervelopment Program, Brown University (K12HD050108); K23HD062340-01 (B.L.A.); and4AI066884 (S.C.-U.).

authors report no conflict of interest.

sented at the 38th annual meeting of the Infectious Diseases Society for Obstetrics andnecology, Chicago, IL, August 11-13, 2011.

prints not available from the authors.J Obstet Gynecol 2013;208:321.e1-9.

omen account for half of all peo-ple living with human immuno-

ficiency virus (HIV)-1 globally.1 Thet majority of incident HIVworldwidecaused by heterosexual intercourse,d the female lower genital tract is themary site of acquisition.2 As a result ofection inwomen, there are nownearlyillion children living with HIV, the

vast majority of thefected.3 A large, lofollowing 10,000Uganda, found that wnificantly increased rtion during pregnanonpregnant womenreasons for the increaquisition during pr

m the Divisions of Maternal-Fetal Medicine (Drs Anderson, Mendez-Figuesearch (Dr Raker), Department of Obstetrics and Gynecology (Dr Cu-Uvinspital, Warren Alpert Medical School of Brown University, Providence, RI,ctious Diseases and Immunology, Department of Obstetrics, Gynecolog

iences, Magee-Womens Hospital of University of Pittsburgh Medical Cent2-9378/$36.00 2013 Mosby, Inc. All rights reserved. http://dx.doi.org/10.1016/perinatally in-itudinal studymen in Rakai,en were at sig-of HIV acquisi-compared to

The biologicalrisk of HIV ac-ancy have not

been elucidated. It has been suggestedthat mucosal immunity in the genitaltract is compromised during pregnancy.Wira and Fahey5 have suggested that thisrisk may be related to hormonal changesas they reported ovulation to be a time ofvulnerability. Concentrations or expres-sion of certain antimicrobial peptides,cytokines, and chemokines have beenshown to be altered under certain condi-tions in pregnancy, such as bacterialvaginosis (BV),6 trichomoniasis,7 or pre-mature rupture of membranes.8

The immune function of the female

, and Dahlke) andomen & Infantsthe Division of

nd ReproductivePittsburgh, PAs of immune mediators and vaginal colonization frequencies werepared between the pregnant and nonpregnant groups.

Key words: cytokines, genital flora, inflammation, pregnancy, vaginalimmunityBSTETRICS

regnancy-induced cf the genital tract: deenna L. Anderson, MD, MSc; Hector Menristina Raker, ScM, ScD; Sharon L. Hillie

JECTIVE: Both the state of pregnancy as well aa and immune mediators may increase the risciency virus-1 acquisition. The objective of thune changes in lower genital and systemic

h normal pregnancy.

UDY DESIGN: This prospective cohort enrollnonpregnant women ages 18-35 years. Pre

eks and nonpregnant women in follicular phaswere included. Cervical and vaginal fluid wass of immunemediators weremeasured usingorbent assaybased methods or multiplex imre inoculated onto various culture media allowacillus species, Gardnerella vaginalis, Escherspecies, anaerobic gram-negative rods, Cannges in immune proning normalz-Figueroa, MD; Joshua D. Dahlke, MD;hD; Susan Cu-Uvin, MD

isruption of vaginalf human immuno-tudy was to definemunity associated

low-risk pregnantnt women at14the menstrual cy-ected. Concentra-yme-linked immu-noassay. Samplesfor growth of Lac-ia coli, Enterococ-a, Staphylococcus

RESULTS: Genital tractduring pregnancy compreactive protein concennancy. Concentrationsnot differ between groulated from vaginal cult70.2%, P .02). Identand S aureus was commplasma species was iso

CONCLUSION: The proinas the systemic markecreased during pregnachanges during pregnanquj.ajog.2013.01.014

APRIL 2013 Americanection

ncentration of interleukin-1 was higherd to nonpregnant participants. Serum C-tions were higher in all trimesters of preg-ecretory leukocyte protease inhibitor did. Lactobacillus was more commonly iso-s of nonpregnant participants (100% vsation of Candida, G vaginalis, M hominis,and not different between groups. Urea-

ed from60% pregnant participants.

mmatory cytokine, interleukin-1, as wellinflammation, C-reactive protein, are in-. The impact of these proinflammatoryeserves further study.hafider, P

s dk ois simisition. Unfortunately these studies

Journal of Obstetrics& Gynecology 321.e1

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32k a consensus on what concentrationonsidered abnormal andwhichmedi-rs should bemeasured. There is also aucity of data attempting to character-the cytokine environment in preg-

nt women without genital tract infec-ns. One study comparing pregnantd nonpregnant women with symp-atic BV reported higher levels of

erleukin (IL)-1, IL-6, and IL-8ong pregnant women compared tonpregnant women.9 There is rela-ely little known regarding the normalte of lower genital tract immunity ingnant women at different trimesters of

TABLE 1Demographic characteristics for all e

CharacteristicPreg(n

Age, y, median (range) 24.7..........................................................................................................................

BMI, median (range) 25 (..........................................................................................................................

Race, n (%).................................................................................................................

Caucasian 17 (.................................................................................................................

Hispanic 21 (.................................................................................................................

Black 4 (.................................................................................................................

Asian 2 (.................................................................................................................

1 race 2 (.................................................................................................................

Other 2 (..........................................................................................................................

Employment, n (%).................................................................................................................

Unemployed 13 (.................................................................................................................

Full or part-time 32 (..........................................................................................................................

Education, n (%).................................................................................................................

None 1 (.................................................................................................................

Junior high school 5 (.................................................................................................................

High school/GED 11 (.................................................................................................................

Some college/graduate 30 (..........................................................................................................................

Marital status, n (%).................................................................................................................

Single 16 (.................................................................................................................

Married/partnered 31 (..........................................................................................................................

Insurance, n (%).................................................................................................................

Uninsured 7 (.................................................................................................................

Medicaid 20 (.................................................................................................................

Private 17 (.................................................................................................................

Other 2 (..........................................................................................................................

BMI, body mass index; GED, general educational development.

Anderson. Pregnancy-mediated changes in immunity. Am J Ognancy. act

1.e2 American Journal of Obstetrics& Gynecologyeen as a major component of localinal immunity, normal vaginal florahought to impede the bacterial over-wth of virulent exogenous bacteria.anges in the normal vaginal flora canse detrimental effects to a normalgnancy. For example, BV, character-d by the shift in the microflora frome characterized by a predominance ofctobacillus to a complex microflorath a predominance of Gardnerellaginalis and obligately anaerobic bacte-, has been linked to an increased riskHIV acquisition and transmission.10

r objective was to systematically char-

olled participants

nt)

Nonpregnant(n 16)

35) 25.6 (1834)...........................................................................................................

52.4) 24.6 (19.939.8)...........................................................................................................

...........................................................................................................

) 9 (56.3)...........................................................................................................

) 3 (18.8)...........................................................................................................

0...........................................................................................................

1 (6.3)...........................................................................................................

1 (6.3)...........................................................................................................

2 (12.5)...........................................................................................................

...........................................................................................................

) 2 (12.5)...........................................................................................................

) 14 (87.6)...........................................................................................................

...........................................................................................................

0...........................................................................................................

) 1 (6.3)...........................................................................................................

) 3 (18.8)...........................................................................................................

) 12 (75.1)...........................................................................................................

...........................................................................................................

) 10 (62.5)...........................................................................................................

) 6 (37.6)...........................................................................................................

...........................................................................................................

) 3 (18.8)...........................................................................................................

) 2 (12.5)...........................................................................................................

) 11 (68.8)...........................................................................................................

0...........................................................................................................

et Gynecol 2013.erize the local and systemic immuneprono

APRIL 2013lieu of normal pregnancy and the nor-l microbial flora in pregnancy com-red to the nonpregnant state.

ATERIALS AND METHODSdy populationis prospective cohort study recruitedgnant and nonpregnant women atvarious clinics servingWomen & In-ts Hospital in Providence, RI, from07 through 2010. Inclusion criteria in-ded: (1) pregnancy documented byine human chorionic gonadotropin,um human chorionic gonadotropin,ultrasound with low-risk pregnancytus and a gestational age 14 weeks;healthy nonpregnant women age

-35 years, not planning pregnancythin the next year; and (3) willingnessavoid the use of intravaginal productsd willingness to use condoms duringual intercourse 48 hours prior to eachmination. Exclusion criteria includedte systemic illness; chronic illness

ypertension, preexisting diabetes mel-s, autoimmune disease, history ofomboembolic disease); use of sys-ic steroids in past 3 months; immu-ation in past 1 month; active alcohol,g, or tobacco use; immunocompro-sed state; knownactive infection; symp-atic vaginal discharge; current urinary

ct infection; antibiotic use within 1nth of enrollment; prior preterm birth36weeks); current orplanned cerclage;tory of preeclampsia36 weeks gesta-n; and multiple gestation. TheWomenInfants Hospital Institutional Revieward approved this study. Written in-med consent was obtained from eachrticipant before enrollment. Womensidered lowriskafter theirfirstprenatal

pointmentswere recruitedby study staffparticipate. Nonpregnant women wereruited from physician offices as well asough advertisements on local collegepuses.

dy visit procedurestotal of 4 study visits were planned, 1rollment visit and 3 follow-up visits.pregnant patients, visits occurred at4, 14-28, and 28 weeks gestation,d again at the postpartum visit, ap-ximately 4-6 weeks after delivery. InSvagis tgroChcaupreizeonLawivariaofOu

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www.AJOG.org Obstetrics Researchnts were scheduled at approximately-week intervals to mimic the time du-ion between visits for the pregnantrticipants, excluding menses. In alldy participants, each visit was stan-rdized to include a brief questionnaireout recent exposures such as use of an-iotics or unprotected intercourse. Alvic examination with collection ofvical and vaginal swabs, wet mount,inal pH, and a single blood samples conducted.A swab was used for collection of cer-al and vaginal fluid as previously de-ibed.9,11-14 All cervical swabs wereced in 100 L of phosphate-bufferedine and stored at 70C until the as-s were performed. Concentrations of1, IL-4, IL-6, IL-10, interferonN)-, tumor necrosis factor-, gran-cyte macrophage colony-stimulatingtor, and macrophage inflammatorytein-1were measured using the Lu-nex (Luminex Corp, Austin, TX)ltiplex bead assay via a previously val-ted method.15 Serum concentrationsthese mediators were consistently tooto measure and were discontinued

ring the course of the study.aginal samples were used for theasurement of the antimicrobial pep-e secretory leukocyte protease inhibi-(SLPI). Specimens were collected

m the posterior fornix using a swabd stored at 70C. The concentrationSLPI was determined using commer-lly available Quantikine human SLPImunoassay kits (R&D Systems Inc,nneapolis, MN) permanufacturers in-uctions. C-reactive protein (CRP) wasasured from serum samples using ahly sensitive assay. Serum samples col-ted from each participant during eachit were frozen and stored at 70C.e CRP assay employed a simple sand-ch enzyme-linked immunosorbent as-adapted from that of Erhardt et al.16

Specimens for vaginal cultureswere col-tedvia swabplaced inPort-A-Cul trans-rt gel (Becton-Dickinson, Sparks, MD)d transported via overnight shipping toreference laboratory in Pittsburgh, PA,

thin 24 hours of collection for the fol-ing organisms: Mycoplasma hominis,eaplasma species, G vaginalis, Lactoba-

us species, Staphylococcus aureus, Esche-TABLE 2Median immune marker concentration in each study arm

Immune markerPregnant (n 47),median (range)

Nonpregnant (n 16),median (range)

AdjustedP value

IL-1.....................................................................................................................................................................................................................................

First visit 497 (8.258,192) 40 (8.22032) .008.....................................................................................................................................................................................................................................

Second visit 254 (8.215,109) 189 (8.21983) .4.....................................................................................................................................................................................................................................

Third visit 200 (8.24639) 165.5 (8.2836) .05.....................................................................................................................................................................................................................................

Fourth visit 401 (633415) 91 (8.2467) .04..............................................................................................................................................................................................................................................

IL-6.....................................................................................................................................................................................................................................

First visit 505.5 (9.65246) 332.5 (6.9885) .07.....................................................................................................................................................................................................................................

Second visit 515.5 (9.68383) 676 (3072205) .4.....................................................................................................................................................................................................................................

Third visit 376.5 (9.64883) 729.5 (9.62107) .8.....................................................................................................................................................................................................................................

Fourth visit 574.5 (2553655) 643 (1791068) .4..............................................................................................................................................................................................................................................

IFN-.....................................................................................................................................................................................................................................

First visit 112 (2.0743) 6.9 (2.0381) .1.....................................................................................................................................................................................................................................

Second visit 71 (2.01038) 104 (6.9730) .6.....................................................................................................................................................................................................................................

Third visit 13 (2.0807) 131 (6.9388) .2.....................................................................................................................................................................................................................................

Fourth visit 171 (6.91036) 76 (6.9288) .6..............................................................................................................................................................................................................................................

IL-10.....................................................................................................................................................................................................................................

First visit 6.9 (6.9537) 6.9 (6.9291) .3.....................................................................................................................................................................................................................................

Second visit 6.9 (6.9563) 6.9 (6.9486) .6.....................................................................................................................................................................................................................................

Third visit 6.9 (6.9543) 99.5 (6.9375) .7.....................................................................................................................................................................................................................................

Fourth visit 243 (6.93228) 226 (6.9291) .4..............................................................................................................................................................................................................................................

GM-CSF.....................................................................................................................................................................................................................................

First visit 20.0 (6.91834) 6.9 (6.9224) .08.....................................................................................................................................................................................................................................

Second visit 6.9 (6.95869) 6.9 (6.93615) .9.....................................................................................................................................................................................................................................

Third visit 6.9 (6.91337) 32.5 (6.9628) .5.....................................................................................................................................................................................................................................

Fourth visit 274 (6.91799) 57 (6.9388) .2..............................................................................................................................................................................................................................................

TNF-.....................................................................................................................................................................................................................................

First visit 6.9 (6.9630) 6.9 (6.991) .07.....................................................................................................................................................................................................................................

Second visit 6.9 (6.9931) 6.9 (6.9499) .8.....................................................................................................................................................................................................................................

Third visit 6.9 (6.9580) 6.9 (6.9489) .9.....................................................................................................................................................................................................................................

Fourth visit 153 (6.9499) 39 (6.9254) .1..............................................................................................................................................................................................................................................

MIP-1.....................................................................................................................................................................................................................................

First visit 439 (6.91661) 12 (121003) .1.....................................................................................................................................................................................................................................

Second visit 285 (121566) 378 (121580) .8.....................................................................................................................................................................................................................................

Third visit 160 (121688) 373 (121451) .7.....................................................................................................................................................................................................................................

Fourth visit 838 (123088) 12 (12734) .03..............................................................................................................................................................................................................................................

IL-4.....................................................................................................................................................................................................................................

First visit 8.2 (8.2829) 8.2 (8.2379) .1.....................................................................................................................................................................................................................................

Second visit 8.2 (8.2960) 8.2 (8.2736) .6.....................................................................................................................................................................................................................................

Third visit 8.2 (6.9744) 8.2 (8.2538) .2.....................................................................................................................................................................................................................................

Fourth visit 262 (8.2770) 8.2 (8.2402) .3..............................................................................................................................................................................................................................................

Anderson. Pregnancy-mediated changes in immunity. Am J Obstet Gynecol 2013. (continued )APRIL 2013 American Journal of Obstetrics& Gynecology 321.e3

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32hia coli, Enterococcus species, Candidacies, andanaerobicgram-negativerods,th pigmented and nonpigmented. Theabs were removed from the transportand inoculated onto Columbia agarplemented with 5% sheep blood, 2tes of human bilayer Tween agar, Ro-saagar,A-8agar,Ureaplasmabroth, andlateofprereduced lakedbloodkanamy-agar. The Columbia agar, 1 set of hu-n bilayer Tween agar plates, the A-8r plate, and theUreaplasma broth wereubated at 36C in 5-7% carbon dioxidea minimum of 48 hours. The remain-plates were incubatedwithin an anaer-ic glove box at 36C for aminimumof 5ys. Biochemical tests were used to iden-the Enterococcus species, Escherichia

i, and Group B streptococcus. Lactoba-us species were tested for production ofdrogen peroxide using a qualitative as-. Anaerobic gram-negative rods werentified by their characteristic colonyrphology on selectivemedia andGraminmorphology.

tistical analysististical power was calculated based onviously reported SLPI concentrationspregnancy.7 We performed a samplee calculation using a 2-group repeatedasures design. The present study withpregnant and 16 nonpregnant pa-

TABLE 2Median immune marker concentratio

Immune markerPregnant (n 47),median (range)

LPI.................................................................................................................

First visit 525,525 (8.26,008,198).................................................................................................................

Second visit 500,851 (850.88,085,927).................................................................................................................

Third visit 317,831 (44,5426,591,398).................................................................................................................

Fourth visit 350,403 (21,4421,970,467)..........................................................................................................................

CRP.................................................................................................................

First visit 6.0 (0.527.4).................................................................................................................

Second visit 5.1 (0.927.8).................................................................................................................

Third visit 4.6 (0.415.0).................................................................................................................

Fourth visit 2.7 (0.22545)..........................................................................................................................

All values presented as pg/mL reflect genital concentrations, excerace and body mass index.CRP, C-reactive protein; GM-CSF, granulocyte macrophage colomacrophage inflammatory protein; SLPI, secretory leukocyte prot

Anderson. Pregnancy-mediated changes in immunity. Am J Onts was able to detect an 85% differ- age

1.e4 American Journal of Obstetrics& Gynecologyce in SLPI concentrations at the en-lment visit with 80% power. Thedy lacked sufficient statistical powerassess differences in adverse preg-ncy outcomes. Cytokines, chemo-es, and SLPI concentrations were notrmally distributed. Therefore, com-risons were made between study armsng Wilcoxon rank sum test. Adjust-nt for variables such as white race anderweight (bodymass index [BMI]25)s performed by the van Elteren test. Or-ism growth was compared betweenupsby the2 test or theCochran-Man--Haenszel test (adjusted analysis). Sta-tical analysis was performed using SAS(SAS Institute, Cary, NC). We con-cted 2-sided hypothesis tests with aalue .05 considered statistically sig-cant. To account for multiple testing,adjusted P values by the Benjamini-chberg method with a false discoverye of 0.05.17 This method controls theportion of type I errors among all sig-cant hypothesis tests at 5%. The sam-size did not afford adequate power toke meaningful comparisons betweenividual microflora and mediators.

SULTStotal of 47 pregnant and 16 nonpreg-nt women were enrolled. The mean

n each study arm (continued)

onpregnant (n 16),edian (range)

AdjustedP value

...........................................................................................................

437,310 (11,7606,856,532) .08...........................................................................................................

488,458 (70,9362,741,354) .9...........................................................................................................

,042,767 (155,37011,924,538) .07...........................................................................................................

756,734 (99,3901,006,787) .08...........................................................................................................

...........................................................................................................

0.9 (0.19.8) .005...........................................................................................................

0.8 (0.115.4) .0001...........................................................................................................

0.7 (0.18.7) .03...........................................................................................................

0.4 (0.12.8) .1...........................................................................................................

serum CRP, presented as mg/L. P values adjusted for

timulating factor; IFN, interferon; IL, interleukin; MIP,inhibitor; TNF, tumor necrosis factor.

et Gynecol 2013.and BMI for both groups were simi-ongro

APRIL 2013(Table 1). More women identifiedmselves as Hispanic in the pregnanthort compared to the nonpregnantrticipants: 44.6% vs 18.8%, respec-ely. Within the pregnant group, 66%participants were married or had artner compared to 37.6% in the non-gnant group. Medicaid recipientsremore frequently representedwithinpregnant group (43.5%) than the

npregnant group (12.5%).The median concentrations of theital tract immune mediators and se-

m CRP at each visit are illustrated inble 2. The majority of mediators in-ding IL-4, IL-6, IL-10, SLPI, granulo-e macrophage colony-stimulatingtor, IFN-, tumor necrosis factor-,d macrophage inflammatory pro-n-1 did not differ between the preg-nt and the nonpregnant women.1 increased during pregnancy, a dif-ence most pronounced during thest trimester (median 497 vs 40 g/mL,

.008). Nonpregnant women wereo more likely to have undetectableels of this cytokine: 7% vs 25%. Ailar effect was seen with serum lev-of CRP. Concentrations of thisrker of systemic inflammation werevated in pregnancy, a trend thatuld be seen across trimesters. Theum concentration of CRP thennded downward in the postpartumit suggesting this change is preg-ncy related. An analysis excludingmen who delivered preterm did notnificantly alter these findings. Theure graphically illustrates the differ-ce between pregnant and nonpreg-nt groups for all study visits for both-1 and CRP. A Freidman test re-aled that IL-1 concentrations didt vary over time in the nonpregnantup (P .94).

The frequency of cultures that yieldedsitive results for each group is dis-yed in Table 3. Hydrogen peroxideducing strains of Lactobacillus weres frequently isolated from first-tri-ster pregnant women compared tonpregnant women (71.2% vs 100%,

.02). During the second and thirdmester, there was no difference in col-ization by these lactobacilli betweenwagangroteltis9.2duP vnifiweHoratpronifiplemaind

REAnaups. Among women seen postpar-

tumidedroallpape(2.riatriAcgrodia

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www.AJOG.org Obstetrics Research, colonization by hydrogen perox-producing strains of lactobacillipped precipitously to 17.7%, while(100%) of the nonpregnant partici-nts remained colonized over the sameriod of time (P .01). Only 1 patient7%) in the pregnant cohort met crite-for diagnosis of BV during the thirdmester by the criteria of Amsel et al.18

ross time, the average pH betweenups did not differ with an overall me-n of 4.4 in all visits.There was significantly greater coloni-ion of Escherichia coli in the nonpreg-nt participants when compared tognant women in the third trimester.5% vs 5%, respectively, P .0005).eaplasma species was highly prevalentth a positive culture in60% of preg-nt patients throughout trimesters, ands isolated more frequently during theond trimester as compared to thenpregnant state; 65.9% vs 26.7% (P).We examined pregnancy outcomes incontext of alterations in immunity

able 4). In this low-risk cohort, thedian gestational age at delivery was.0 weeks (33.0-41.0 weeks) and the av-ge birthweight was 3255 g (2070-4640The pretermbirth ratewas lower thannational average, at 8.9%. Gesta-

nal age at birth as well as adverse ma-nal and neonatal outcomes were eval-ted according to the microorganismlated during each trimester. Culturevarious organisms was not associatedth gestational age at birth or with ma-nal or neonatal adverse outcomes.rvical cytokine concentrations and se-mCRP levels in the highest quartile forh mediator were not associated withverse perinatal/maternal outcomes.

MMENTthis study, we found that cervical IL-, a proinflammatory cytokine, and se-CRP, amarker of systemic inflamma-

n, are increased in normal pregnancypared to nonpregnant women. Preg-

ncy-mediated changes to vaginal andtemic immunity and to vaginal florave been incompletely elucidated. How-r, a previous study documented an in-

ase in cervical cytokines in pregnant vagpared to nonpregnant women with.9 Conflicting results as well as differinghniques and clinical outcomes havede interpretation of these data difficult.ablishingnormalparametersanddeter-ningphysiologic changes attributable tognancy allows for a valid comparisonen assessing inflammation as it relatesrisk of HIV acquisition.Concentration of cytokines within the

IGUREcatterplot of vaginal concentration oerum CRP in pregnant and nonpregn

evels of IL-1 reported in pg/nL. Y-axis transforamong groups. Statistically significant differencpostpartum period (P .008 and P .04, restistically significant difference was noted betwee

.005, P .0001, and P .03, respectively), C-reactive protein; IL, interleukin.

erson. Pregnancy-mediated changes in immunity. Am J Obsina or amniotic cavity has been the tio

APRIL 2013 Americanject of much research. Our results in-ate that pregnancy is associated withrly few local and systemic changes inmunity. Vaginal concentrations of IL-, a proinflammatory cytokine, are in-ased in pregnant women across eachmester. These results confirm thedings of Kutteh and Franklin,19 wholowed 36 women throughout preg-ncy and found increasing concentra-

L-1 andt women

d to logarithmic scale to allow better compar-s noted between groups during first trimestertively). B, Serum CRP levels reported in ng/L.roups during first, second, and third trimester

ynecol 2013.m

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Journal of Obstetrics& Gynecology 321.e5

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Anderson. Pregnancy-mediated changes in immunity. Am J Obstet Gynecol 2013. (continued )

Research Obstetrics www.AJOG.org

321.e6 American Journal of Obstetrics& Gynecology APRIL 2013findings ofWalter et al,20 in a recentlyblished study comparing immuno-dulatory factors in the cervicovaginalage of 23 pregnant to 25 nonpregnantmen. These authors did not find a dif-ence in IL-1 concentrations but didd suppression ofmacrophage-derivedemokine (CCL22) in the cervicovagi-l lavage of pregnant women. Our find-s support those of other investigatorswell. We detected a statistically signif-nt decline in IL-6 levels during thest trimester, a trend also reported byers. Donders et al21 found that preg-nt women were less likely to have de-table levels of IL-6 and IL-8 andwed a decreasing trend in concentra-n especially during the second trimes-. In the study by Beigi et al,9 reportingreased concentrations of IL-6 in preg-nt women, the women were enrolleda median gestational age of 13 weeks;refore changes in this cytokine over3 trimesters were not evaluated. Ourdings differ from results reported byer authors with respect to concentra-n of IL-10, a major antiinflammatoryokine. Cervical concentrations of thisdiator within our study populationnot differ between pregnant and

npregnant participants. Others haveorted a reduction in concentrationsIL-10 during the first trimester. Ourdy differs in site of sample collection.e study showing a decrease in IL-10wasm serum and may reflect systemicanges rather than thoseof the lowergen-l tract.22 We were unable to reliablyasure serumcytokine concentrations ins low-risk cohort and therefore only re-rt serum CRP concentrations here.ncentrations of the proinflammatoryokines IFN- and antiinflammatory4 have also been reported to decreasering the second trimester,22 a patternt was not observed in our study cohort.Concentrations of SLPI were not differ-t between the groups in this study. Thisncontrast to thefindingsofDraperetal7

o reported a 10-fold increase in thisptide during pregnancy. Helmig et al23

odemonstrated that SLPI is increased invical mucus of pregnant women. Onethemajor reasons explaining this differ-ce in SLPI levels could be that our studyTABLE 3Organisms isolated during each visit

Organism Pregnant (n 47), % Nonpregnant (n 16), % P value

Lactobacillus H2O2 ().....................................................................................................................................................................................................................................First visit 70.2 100 .02.....................................................................................................................................................................................................................................

Second visit 75.6 93.3 .3.....................................................................................................................................................................................................................................

Third visit 75.0 87.5 .9.....................................................................................................................................................................................................................................

Fourth visit 16.7 100 .01..............................................................................................................................................................................................................................................

Lactobacillus H2O2 ().....................................................................................................................................................................................................................................First visit 57.4 68.7 .9.....................................................................................................................................................................................................................................

Second visit 71.1 93.3 .3.....................................................................................................................................................................................................................................

Third visit 72.2 87.5 .4.....................................................................................................................................................................................................................................

Fourth visit 75.0 80 .9..............................................................................................................................................................................................................................................

Escherichia coli.....................................................................................................................................................................................................................................

First visit 2.1 6.3 .8.....................................................................................................................................................................................................................................

Second visit 2.2 0 .8.....................................................................................................................................................................................................................................

Third visit 5.6 12.5 .0005.....................................................................................................................................................................................................................................

Fourth visit 8.3 20 .9..............................................................................................................................................................................................................................................

Candida.....................................................................................................................................................................................................................................

First visit 19.2 0 .07.....................................................................................................................................................................................................................................

Second visit 20 0 .05.....................................................................................................................................................................................................................................

Third visit 25 12.5 .4.....................................................................................................................................................................................................................................

Fourth visit 4.2 0 ..............................................................................................................................................................................................................................................

Gardnerella vaginalis.....................................................................................................................................................................................................................................

First visit 44.7 31.3 .6.....................................................................................................................................................................................................................................

Second visit 42.2 13.3 .06.....................................................................................................................................................................................................................................

Third visit 38.9 12.5 .5.....................................................................................................................................................................................................................................

Fourth visit 50 20 .8..............................................................................................................................................................................................................................................

Staphylococcus aureus.....................................................................................................................................................................................................................................

First visit 8.5 0 .3.....................................................................................................................................................................................................................................

Second visit 2.2 0 .5.....................................................................................................................................................................................................................................

Third visit 0 0 .....................................................................................................................................................................................................................................

Fourth visit 4.2 0 ..............................................................................................................................................................................................................................................

Enterococcus species.....................................................................................................................................................................................................................................

First visit 12.8 31.3 .07.....................................................................................................................................................................................................................................

Second visit 17.8 20 .9.....................................................................................................................................................................................................................................

Third visit 25 12.5 .6.....................................................................................................................................................................................................................................

Fourth visit 12.5 40 .2..............................................................................................................................................................................................................................................

AGNR pigmented.....................................................................................................................................................................................................................................

First visit 17.4 0 .1.....................................................................................................................................................................................................................................

Second visit 8.9 13.3 .6.....................................................................................................................................................................................................................................

Third visit 11.1 0 1.....................................................................................................................................................................................................................................

Fourth visit 45.8 20 .4..............................................................................................................................................................................................................................................ort only included women that were

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www.AJOG.org Obstetrics Researchsidered very low risk; this is in starktrast to cohorts used by others that in-ded both symptomatic patients and pa-nts diagnosed with sexually transmittedections.We found a statistically significant in-ase in serum highly sensitive CRPncentration during pregnancy. Thisrease tended to be higher during thest trimester and decrease as pregnancygressed. Multiple studies have been

nducted measuring this analyte dur-pregnancy with the use of different

tection techniques and cutoff values.me authors have reported an increaseconcentration of CRP with labor, de-ery, and the immediate postpartumriod; although the variability of theues used as abnormal makes general-tion difficult.24,25 Various authorsve attempted to associate serumncentrations of this acute phase re-ant with adverse pregnancy out-mes. To date, studies have yieldednflicting results.26,27 The usefulnessthis inflammatory marker duringegnancy and the ability to predictverse outcomes remains to be deter-ned. In our study, we used samplesllected from low-risk patients thatked confounding variables thatuld alter results such as labor, andntrolled for variables that may altervalues such as race and BMI. Are sensitive assay was also used toocess our samples, another factording to the validity of our results.The effect that pregnancy exerts on vag-l flora is not clearly understood. Weve previously reported that perturba-ns in vaginal flora among low-riskpreg-nt women do not cause a consistent im-ct on local vaginal immunity.28 Thefinition of normal flora also variesoughout the literature. Abnormalinal flora, despite known associa-ns, is functionally a poor predictor ofverse pregnancy outcomes especiallylow-risk patients since most womenth abnormal florawill actually go on tove normal pregnancy outcomes.29

ctobacillus species are considered armal component of the vaginal florad these bacteria are thought to inhibitgrowth of pathogenic bacteria. Those

the pregnant group were less fre- larently colonized by hydrogen perox-producing lactobacilli compared tonpregnant women, and they experi-ced a decrease in the detection of lac-acilli in the postpartum period. Themen in the study group did not have anical diagnosis of BV per criteria ofsel et al18 but Gram stains for Nugentre were not performed. Therefore, itpossible that there could have beenmptomatic BV that did not meet thenical criteria for diagnosis in the preg-nt women. In this study, pregnantmenhadmore complex flora than didnpregnant women. Our findings dif-from those of Aagaard et al30 in thatile their group found a difference invaginal flora among pregnant com-

red to nonpregnant women using 16Sosomal RNA gene sequencing, themen in that cohort actually had lesserse flora during pregnancy. Thisdy did not use Nugent score andrefore it is possible that there couldve been higher rates of asymptomaticin the nonpregnant group. Beigi et al9

orted that pregnant andnonpregnantmen experiencing BV had very simi-

ABLE 3rganisms isolated during each visit

rganism Pregnant (n 47), %

GNR nonpigmented.................................................................................................................

First visit 40.4.................................................................................................................

Second visit 24.4.................................................................................................................

Third visit 33.3.................................................................................................................

Fourth visit 66.7.........................................................................................................................

reaplasma species.................................................................................................................

First visit 66.................................................................................................................

Second visit 65.9.................................................................................................................

Third visit 61.8.................................................................................................................

Fourth visit 34.8.........................................................................................................................

ycoplasma hominis.................................................................................................................

First visit 19.2.................................................................................................................

Second visit 15.6.................................................................................................................

Third visit 20.................................................................................................................

Fourth visit 20.8.........................................................................................................................

values adjusted for race and body mass index.GNR, anaerobic gram-negative rods; H2O2, hydrogen peroxide p

nderson. Pregnancy-mediated changes in immunity. Am J Omicroflora. wh

APRIL 2013 AmericanThe frequency of G vaginalis was sim-r in pregnant and nonpregnantmen. G vaginalis is found in 100% ofmenwith the diagnosis of BV, but cano be found among women withouts condition. Koumans et al31 showedt the diagnosis of BV is not moremmon in pregnant patients comparednonpregnant women. Despite the as-iation ofG vaginaliswith BV, it is alsohly prevalent among healthy womendemonstrated in our results. Ourdy was not designed to detect differ-ces across themenstrual cycle, and theect of various stages of the cycle can-t be examined with these data.he isolation ofUreaplasma species in0% of our pregnant participants hasportant implications. Although vari-s studies have implicated vaginal col-ization ofUreaplasma species with ad-se pregnancy outcomes,32,33 ourults indicate that this microbe is partthe normal vaginal flora in pregnanttients.M hominis was also more com-nly isolated in specimens from preg-nt patients, again indicating a possibil-that this microbe is not pathogenic

tinued)

Nonpregnant (n 16), % P value

...........................................................................................................

0 .004...........................................................................................................

20 .9...........................................................................................................

12.5 .4...........................................................................................................

0 .02...........................................................................................................

...........................................................................................................

31.3 .05...........................................................................................................

26.7 .03...........................................................................................................

14.3 .2...........................................................................................................

0 .3...........................................................................................................

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0 .09...........................................................................................................

0 .1...........................................................................................................

0 .1...........................................................................................................

25 .2...........................................................................................................

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Journal of Obstetrics& Gynecology 321.e7

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neimcuwocommupaterdudawerisbic

REF1. JAID

201glo23,2. JAIDblecon201Ac3. JAID200datpubAc4. GofUg3665. Wstawincle6. SKeproin pmin2007. DWiecrecreinfe1248. Twonombraprome1339. BHillvagkinbac13510.NgvagmativePL11.Metraresfec12.MAcensm19913.

seqpre56014.Clinmetertria15.W,humlateDia16.HKtin,prosivlink20017.lanhav20018.Escnitidem14-19.immvicnan20.mofrocro26321.SpAm22.Chter20023.leucusGy24.SaaparOb25.G.andClin26.Usrioame11427.WilC-r

I

Research Obstetrics www.AJOG.org

32ncy. In a study involving900 women,e et al34 also demonstrated that positiveinal cultures for M hominis was not ak for preterm birth.his study is among the first to simulta-

ously characterize local and systemicmunity in the setting of known vaginallture data among low-risk pregnantmen. Despite the small sample size, wepared various aspects of vaginal im-nity amongpregnant andnonpregnantrticipantsatonce thusallowing forabet-understanding of the vaginal milieuring pregnancy. Our results merit vali-tion with larger study populations asll as consideration in studies assessingk ofHIV acquisition and vaginalmicro-ide development. f

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TABLE 4Pregnancy outcomes amongpregnant women (n 45)

Outcomes Median (range)

GA at delivery, wk 39.0 (33.041.0)...........................................................................................................

Preterm delivery37 wk

4/45 (8.9%)

...........................................................................................................

Birthweight, g 3255 (20704640)...........................................................................................................

5-min Apgar 9 (89)...........................................................................................................

Adverse neonataloutcomes

6/45 (13.3%)

..................................................................................................

Weight 1500 g 0..................................................................................................

5-min Apgar 7 0..................................................................................................

IUFD 0..................................................................................................

IUGR 2..................................................................................................

NICU admission 4...........................................................................................................

Deliverycomplications

4/45 (8.9%)

..................................................................................................

Oligohydramnios 0..................................................................................................

PPROM 1..................................................................................................

Chorioamnionitis 1..................................................................................................

PTL 2...........................................................................................................

One patient had miscarriage at 12 wk and 1 participantdeclined further participation resulting in 45 birthoutcomes.GA, gestational age; IUFD, intrauterine fetal demise;UGR, intrauterine growth restriction; NICU, neonatalintensive care unit; PPROM, preterm premature rup-ture of membranes; PTL, preterm labor.

Anderson. Pregnancy-mediated changes inimmunity. Am J Obstet Gynecol 2013.oint United Nations Programme on HIV/S. Report on the Global AIDS Epidemic.

decer

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30. Aagaard K, Riehle K, Ma J, et al. A metag-enomic approach to characterization of thevaginal microbiome signature in pregnancy.PLoS One 2012;7:e36466.31. Koumans EH, Sternberg M, Bruce C, et al.The prevalence of bacterial vaginosis in theUnited States, 2001-2004; associations withsymptoms, sexual behaviors, and reproductivehealth. Sex Transm Dis 2007;34:864-9.32. Kacerovsky M, Boudys L. Preterm prema-ture rupture of membranes and Ureaplasma

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www.AJOG.org Obstetrics ResearchAPRIL 2013 American Journal of Obstetrics& Gynecology 321.e9

Pregnancy-induced changes in immune protection of the genital tract: defining normalMaterials and MethodsStudy populationStudy visit proceduresStatistical analysis

ResultsCommentReferences