Subclinical chorioamnionitis as an etiologic factor in preterm deliveries

International Journal of Gynecology & Obstetrics 72 Ž2001. 109᎐115

Article

Subclinical chorioamnionitis as an etiologic factor in preterm deliveries U

¨ ¨ a , I. Koc¸aka, , S. Baris¸b, A. Uzela , F. Saltikb C. Ustun a

Department of Obstetrics and Gynecology, Uni¨ ersity of Ondokuz Mayls Uni¨ ersity, Samsun, Turkey b Department of Pathology, Uni¨ ersity of Ondokuz Mayls, Samsun, Turkey Received 9 December 1999; received in revised form 24 April 2000; accepted 27 April 2000

Abstract Objecti¨ e: This study was performed to determine the possible relationship between histologic chorioamnionitis and genital tract cultures and their contribution to preterm delivery. Methods: The study group consisted of 45 preterm and 37 term pregnancies. Cervico-vaginal cultures were obtained from all patients at admission and placentas were histopathologically evaluated and graded for inflammation of the chorial plate, amniochorionic membrane, umbilical cord and villi. Inflammation scores and presence of pathogenic bacteria were compared between the preterm and term patients. Results: Severe chorionic plate inflammation was present in 35.5% of 45 preterm deliveries as compared to 5.4% of 37 term deliveries. Amniochorionic-decidual inflammation was not present in 14 of the term deliveries Ž38%., mild ŽGrade 1᎐2. in 20 Ž54%. and severe ŽGrade 3᎐4. in 3 Ž8%. as compared to 14 Ž31%., 17 Ž38%., and 14 Ž31%. in the preterm group, respectively Ž P - 0.05.. Pathogenic bacteria were isolated from the lower genital tracts of all patients who had severe chorionic plate ŽCP. andror chorioamnionic ŽCA. inflammation while this was true only in 37.5% of the patients with no or only mild chorionic plate inflammation Ž P- 0.001 for CP, P - 0.01 for CA.. Conclusion: Our results show that pathogenic bacterial colonisation of the cervicovaginal region is closely associated with placental inflammation, preterm labor and cervico-vaginal cultures which may be useful in determining the pregnancies at risk for preterm labor. 䊚 2001 International Federation of Gynecology and Obstetrics. All rights reserved. Keywords: Chorioamniotinitis; Histopathology; Genital tractus flora

U

Corresponding author. Adalet mah, Samsun, Turkey. Tel.: q90-362-4576000; fax: q90-362-4576041. E-mail address: [email protected] ŽI. Koc ¸ak.. 0020-7292r01r$20.00 䊚 2001 International Federation of Gynecology and Obstetrics. All rights reserved. PII: S 0 0 2 0 - 7 2 9 2 Ž 0 0 . 0 0 2 8 0 - 0

¨ ¨ et al. r International Journal of Gynecology and Obstetrics 72 (2001) 109᎐115 C. Ustun

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1. Introduction Preterm delivery is a major obstetric problem, which is associated with high perinatal morbidity and mortality. The cause of the preterm labor is not known in approximately half of the cases w1x. However, there is increasing evidence suggesting that a subclinical intrauterine infection might be responsible for the preterm delivery with unknown etiology w2,3x. Unlike clinically apparent chorioamnionitis there are no specific signs and symptoms for subclinic intrauterine infection and preterm labor may be the only sign. Traditionally, histologic chorioamnionitis has been regarded to reflect amniotic fluid infection and there are studies showing an association between histologic chorioamnionitis, amniotic fluid and subchorionic plate cultures w4᎐7x. Minkoff recently reviewed the concept that genital infection plays an important etiologic role in preterm delivery, even when the membranes were intact w3x. Previous reports have indicated that patients with bacterial vaginosis and vaginal or cervical colonization with Chlamydia trochomatis, group B-Streptococci, or Mycoplasma hominis have a higher frequency of preterm birth than women without vaginosis or such colonization w7᎐9x. In Hillier’s study of pla-

centas from 38 preterm and 56 term deliveries, infection of the chorioamnion was strongly related to histologic chorioamnionitis, and both were related to histologic chorioamnionitis and prematurity w10x. In the present study our objective was to determine the possible relationship between histologic chorioamnionitis and genital tract cultures and to assess the latter’s contribution to preterm delivery.

2. Material and methods Material for this study was gathered from the deliveries which were either preterm or term that occurred at Ondokuz Maylis University, School of Medicine, Department of Obstetrics and Gynecology, during the period from 15 August 1997 to 11 November 1998. Multiple pregnancies with fetal demise or life-threatening fetal anomaly and those associated with pre-eclampsia, clinically apparent chorioamnionitis or chronic systemic diseases such as diabetes mellitus and hypertension were excluded from the study group. Preterm delivery was accepted as a delivery occurring before the completion of 37 weeks of gestation.

Table 1 Grading system for acute intrauterine inflammations Žmodified from Salafia et al. w11x. a Amnion and chorion-decidua Grade 1 Grade 2 Grade 3 Grade 4

Presence of one focus of at least five polymorphonuclear leukocytes. More that one focus of grade 1 inflammation or at least one focus of 5᎐20 polymorphonuclear leukocytes Multiple or confluent foci of grade 2 Diffuse and dense acute inflammation

Umbilical cord Grade 1 Grade 2 Grade 3 Grade 4

Polymorphonuclear leukocytes within the inner third of the umbilical vein wall Polymorphonuclear leukocytes within the inner third of at least two umbilical vessel walls Polymorphonuclear leukocytes in perivascular Wharton jelly Panvasculitis and funisitis extending deep into Wharton jelly

Chorionic plate Grade 1 Grade 2 Grade 3 Grade 4

One focus of at least five polymorphonuclear leukocytes in subchorionic fibrin Multiple foci of grade 1 in subchorionic fibrin Few polymorphonuclear leukocytes in connective tissue or chorionic plate Numerous polymorphonuclear leukocytes in chorionic plate and chorionic vasculitis

a

Chronic villitis was diagnosed in the presence of lymphoblast infiltration in chorionic villi.

¨ ¨ et al. r International Journal of Gynecology and Obstetrics 72 (2001) 109᎐115 C. Ustun

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Table 2 Correlation between histologic chorioamnionitis and gestational week Gestational week

Chronic plate inflammation

Grade 20᎐28 29᎐32 33᎐37 ) 37 hf

0 1 7 6 21

1 0 4 3 7

2 0 3 5 7

3 2 2 4 1

4 4 0 4 1

Amniochorionic decidual inflammation

Umbilical cord inflammation

0 0 6 8 14

0 3 13 18 34

1 0 1 3 3

Following admittance of the patients to the clinic, samples were obtained from the cervical canal and posterior fornix of the vagen for aerobic and anaerobic cultures. Following delivery all placentas were weighed and examined fresh for any macroscopic pathology according to a standard protocol. Thereafter, tissue samples were obtained from each placenta including two sections from the umbilical cord, two 1-cm wide slices from the chorionic plate, four decidual sections from the placental disc, and a roll of membranes extending from the rupture point to the placental margin. All samples were fixed with 10% buffered formalin, embedded in paraffin and tissue blocks were stained with hematoxylin and eosin. Histologic examination was performed by a pathologist who was blinded to the clinical information. Histopathologic acute inflammation findings were evaluated according to a grading system modified from Salafia et al. ŽTable 1.. Histopathologic findings were matched with microbiologic results. The chi-square test was used to compare the statistical difference between patient groups and P- 0.05 values were accepted as statistically significant. The presence of one or more types of non-native micro-organisms in the vaginal cultures was considered pathologic, the presence of Doderlein bacilli alone was considered normal. ¨

2 1 6 6 0

3 2 2 2 3

4 4 1 3 0

1 1 2 3 2

2 1 0 0 1

Chronic villitis 3 2 1 1 0

q ᎐ 3 4 4 12 6 16 6 31

Fig. 1. Normal amniochorion-decidua with no inflammation Žhematoxylin᎐eosin; =25..

3. Results The study group consisted of 82 patients, of which 45 Ž54.87%. were delivered preterm while the remaining 37 Ž45.3%. were delivered at term

Fig. 2. Amniochorion-decidua with grade 4 inflammation Žhematoxylin᎐eosin; =25..

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¨ ¨ et al. r International Journal of Gynecology and Obstetrics 72 (2001) 109᎐115 C. Ustun

Fig. 3. Normal chorial plate with no inflammation Žhematoxylin᎐eosin; =25..

and made the control group. Of the 45 preterm deliveries, 7 occurred between the 20th and 28th week of gestation, 16 between 29 and 33 weeks of gestation while 22 were delivered between the 33rd and 37th week of gestation. Amniochorionic-decidual inflammation was not present in 14 of the term deliveries Ž38%. ŽFig. 1., mild ŽGrade 1᎐2. in 20 Ž54%. and severe ŽGrade 3᎐4. in 3 Ž8%. ŽFig. 2.. These figures were 14 Ž31%., 17 Ž38%., and 14 Ž31%. in the preterm group, respectively. In terms of amniochorionicdecidual inflammation, there was a statistically significant difference between the groups Ž P0.05. ŽTable 2.. Either no chorionic plate inflammation or mild inflammation Žgrade 1 and 2. was detected in 94.6% of the term deliveries ŽFig. 3.. Severe chorionic plate inflammation was present Žgrade 3 and 4 inflammation. ŽFig. 4. in 35.5% of the 45 preterm deliveries as compared to 5.4% of the term deliveries. There was a statistically significant difference between the two groups. The severity of chorionic plate inflammation was inversely related to gestational age Ž P- 0.02. ŽTable 2.. Umbilical cord inflammation was not observed in most of the preterm and term cases. Grade 1 cord inflammation was present in 6 Ž13.3%. cases of the preterm group and in 2 Ž5.4%. of the term group. Grade 2 umbilical cord inflammation was rare in both groups. Grade 3 cord inflammation was present in 4 Ž8.9%. of the preterm deliveries

Fig. 4. Chorial plate with grade 4 inflammation Žhematoxylin᎐ eosin; =25..

but in none of the term deliveries. Grade 4 umbilical cord inflammation was not diagnosed. There was no statistically significant difference between two group Ž P) 0.1. ŽTable 2.. Chorionic villitis was detected histologically in 13 Ž28.9%. of preterm and 6 Ž16.2%. of term deliveries. There was no statistically significant difference between the two groups in terms of chorionic villitis Ž P) 0.1. ŽTable 2.. Premature rupture of membranes ŽPROM. was present in 32 of all patients enrolled into the study and only four of them had term pregnancies while the other 28 subjects were delivered preterm. There was no statistically significant difference between pregnancies complicated by PROM and those uncomplicated when compared in terms of histologic chorioamnionitis Ž P) 0.05. ŽTable 5.. Cultures were positive in 29 Ž90%. of the 32 patients with PROM but only in 12 Ž24%. of the 50 subjects without PROM. The difference between these two groups was highly significant Ž P- 0.001. ŽTable 3.. There was also a statistically significant correlation between the presence Table 3 The genital microbiology in patients with and without PROM PROM

Pathogen bacteria Žq.

Pathogen bacteria Žy.

Positive Negative

29 12

3 38

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Table 4 Correlation between histologic chorioamnionitis and microbiologic findings Isolation of pathogen bacteria

Chronic plate inflammation

Grade Positive Negative

0 12 23

1 5 9

Amniochorionicdecidual inflammation

2 7 8

3 9 0

4 9 0

0 11 17

1 2 4 12 9 12

3 7 2

Umbilical cord inflammation

4 8 0

0 32 36

1 5 3

2 1 1

Chronic villitis q y 31 11 32 8

3 4 0

Table 5 Relation between PROM and histologic chorioamnionitis Latency period between PROM and delivery

Chronic plate inflammation

Grade PROM Žy. PROM - 12 h PROM 12᎐24 h PROM ) 24 h

0 27 3 2 3

1 9 0 0 5

2 8 0 1 6

3 2 0 3 4

4 4 1 1 3

Amniochorionic decidual inflammation

Umbilical cord inflammation

0 1 2 21 10 8 1 0 3 1 1 3 5 2 10

0 42 4 5 17

of pathogenic bacteria in the cervico-vaginal flora and the severity of histologic inflammation in the chorionic plate and amniochorion-decidua. Pathogenic bacteria were isolated from the lower genital tracts of all patients who had severe chorionic plate inflammation while this was true only for 37.5% of the patients with no or only mild chorionic plate inflammation Ž P- 0.001. ŽTable 4.. This was also true in terms of amniochorionic inflammation Ž P- 0.01. ŽTable 5..

4. Discussion In a study by Hillier and co-workers, infection of the chorioamnion was strongly related to histologic chorioamnionitis and both were related to prematurity w6x. Hameed et al. noted silent chorioamnionitis to be a significant cause of preterm labor refractory to tocolytic therapy w12x. In another study subclinical infection was found to be an uncommon cause of refractory preterm labor w13x. Microbial colonization and inflammation of the maternal lower genital tractus have been accused as the cause of preterm deliveries, especially occurring in the second trimester and early third trimester Table 6. The pathogenic bacteria

3 5 0 0 3

4 5 0 0 2

1 4 0 2 2

2 1 0 0 1

Chronic villitis

3 3 0 0 1

q y 12 38 0 4 1 6 6 15

colonizing the lower genital tractus have also been isolated in the amniotic fluid cultures obtained from 5 to 25% of pregnant women in labor w20x. Prostaglandins are widely accepted as key mediators for the onset of labor. Administration of prostaglandin E2 ŽPGE2 . and prostaglandin Table 6 Bacteria species isolated in genital cultures Bacteria isolated

Vagina

Cervical canal

Doderlein bacili Staphylococci coag Ž ᎐ . Diphtheroid bacili Candida albicans Staph. aureus Escherichia coli Group B ᎏ B-hem.streptococci Enterobacteria Gardnerella ¨ aginalis Pseudomonas spp. Proteus ¨ ulgaris Peptostreptococcus Peptococcus Bacterioides fragilis Fusobacterium Bifidobacterium Trichomonas ¨ aginalis

7 Ž8.5%. 3 Ž3.7%. 3 Ž3.7%. 9 Ž11%. 14 Ž17%. 7 Ž8.5%. 8 Ž9.7%. 5 Ž6%. 4 Ž4.8%. 2 Ž2.4%. 2 Ž2.4%. 1 Ž1.2%. 2 Ž2.4%. 1 Ž1.2%. 1 Ž1.2%. 1 Ž1.2%. 1 Ž1.2%.

6 Ž7.3%. 2 Ž2.4%. 2 Ž2.4%. 6 Ž7.3%. 13 Ž15.9%. 4 Ž4.8%. 8 Ž9.7%. 4 Ž4.8%. 3 Ž3.7%. 1 Ž1.2%. 1 Ž1.2%. y y 1 Ž1.2%. y 1 Ž1.2%. 1 Ž1.2%.

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F2 ␣ ŽPGF2 ␣ . to women during pregnancy will result in myometrial contractility and may lead to first and second trimester abortions and induction of labor. Administration of nacethly-salicilic acid or indomethacin delays the progress of midtrimester abortion and the onset of labor at term, these agents can also arrest preterm labor w14x. A variety of bacteria have phospholipase A2 activity, an essential enzyme in prostaglandin synthesis. It has been proposed that bacterial chorioamnionitis activates PG synthesis in the fetal membranes, which in turn leads to preterm labor w17x. Presence of infection may also initiate labor by activating macrophages which are ubiquitous and present in the maternal Ždecidual., fetal and placental compartments Žchorionic-amnion interface and villi.. Macrophages are readily activated by microbial products to secrete a wide variety of mediators, including interleukin-1 ŽIL-1. and tumor necrosing factor ŽTNF.. TNF shares many biological activities with IL-1, including stimulation of PG synthesis and collagenase activity w14,18,19x. Thus, prostaglandin synthesis may be stimulated by either microbial w15x or host signals secreted in response to infection w16x and possibly forms the basis of preterm labor in patients with clinical or subclinical genital tract infection. Preterm premature rupture of membranes ŽPPROM., a major cause of preterm delivery, was present in 27 Ž60%. of 45 preterm deliveries in our study. PPROM itself is closely related to the infection caused by the bacteria colonizing the lower genital tractus. These bacteria may be responsible for PROM by enzymatically weakening the amniochorionic membrane w21,22x. In our study there is a statistically significant correlation between PPROM and amniochorionic-decidual inflammation Ž P- 0.05. ŽTable 3.. This finding is consistent with the literature w23᎐25x. In the study by Guzick and Winn, histologic chorioamnionitis was more frequent in preterm deliveries w25x. In another study carried by Romeo et al. it has been shown that there is a strong correlation between the severity and frequency of histologic chorioamnionitis and amniotic fluid cultures w24x. Our report shows that chorionic plate inflammation is more severe in the preterm delivery

group than the term delivery group Ž P- 0.02. ŽTable 2.. Furthermore, in preterm births severe amniochorionic decidual inflammation is far more frequent Ž P- 0.05. ŽTable 3.. Umbilical cord inflammation was also more severe and frequent in the preterm cases and although no statistical difference could be shown for umbilical cord inflammation; this probably results from the rarity of cases diagnosed as such. Funisitis, umbilical vasculitis and chorionic vasculitis reflect the fetal response to infection whereas neutrophil infiltration in chorionic plate and amniochorionic membrane primarily results from maternal response to infectious process w26x. In conclusion, our study shows that chorionic plate and amniochorionic decidual inflammations are significantly more prevalent in preterm deliveries and are closely related with pathogenic bacterial colonisation of the cervico-vaginal region. Cervico-vaginal aerobic and anaerobic cultures might be useful in detecting pregnancies with a higher risk for preterm labor. References w1x Rush RW, Keirse MJNC, Howat P, Baum JD, Andersen ABM, Turnbull AC. Contribution of preterm delivery to perinatal mortality. Br Med J 1976;2Ž6042.:965᎐968. w2x Gibbs RS, Romeo R, Hillier SL, Eschenbach DA, Sweet RL. A review of premature birth and subclinical infection. Am J Obstet Gynecol 1992;166Ž5.:1515᎐1528. w3x Minkoff H. Prematurity: infection as an etiologic factor. Obset Gynecol 1983;62Ž2.:137᎐144. w4x Aquino TI, Zhang J, Kraus FT, Knefel R, Taff T. Subchorionic fibrin cultures for bacteriologic study of the placenta. Am J Clin Pathol 1984;81Ž4.:482᎐486. w5x Pankuch GA, Applebaum PC, Lorenz RP, Botti JJ, Schachter J, Naeye RL. Placental microbiology and histology and the pathogenesis of chorioamnionitis. Obstet Gynecol 1994;64Ž6.:802᎐806. w6x Hillier SL, Martius J, Krohn M, Kiviat N, Holmes KK, Eschenbach DA. A case᎐control study of chorioamnionitic infection and histologic chorioamnionitis in prematurity. N Engl J Med 1998;319Ž15.:972᎐978. w7x Romero R, Sirtori M, Oyarzun E. Infection and labor. V. Prevelance, microbiology, and clinical significance of intraamniotic infection in women with preterm labor and intact membranes. Am J Obstet Gynecol 1989;161Ž3.:817᎐824. w8x Gravett MG, Nelson P, DeRouen T, Critchlow C, Eschenbach DA, Holmes KK. Independent associations of bacterial vaginosis and Chlamydia trachomatis infection

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