Objective
The objective of the study was to compare the intensity of inflammatory responses between intraamniotic infection with genital mycoplasmas and intraamniotic infection with other microorganisms.
Study Design
We examined the intensity of intraamniotic and maternal inflammatory responses in 99 patients with preterm premature rupture of membranes and a positive amniotic fluid (AF) culture. AF was obtained by transabdominal amniocentesis or at the time of cesarean delivery. Patients were divided according to the recovered microorganisms: (1) genital mycoplasmas (n = 62); (2) other microorganisms (n = 31); or (3) mixed infection (n = 6).
Results
The median AF white blood cell (WBC) count, maternal blood WBC count, and plasma C-reactive protein concentrations were significantly higher in patients with intraamniotic infection with genital mycoplasmas than in those with intraamniotic infection with other microorganisms ( P < .05 for each).
Conclusion
Intraamniotic and maternal inflammatory responses are more intense in intraamniotic infection with genital mycoplasmas than in intraamniotic infection with other microorganisms in patients with preterm premature rupture of membranes.
Intraamniotic infection is causally linked to spontaneous preterm labor/delivery and fetal injury. Moreover, neonates born with evidence of a fetal systemic inflammatory response are at increased risk for short- and long-term morbidity.
Genital mycoplasmas (ureaplasmas and Mycoplasma hominis) are the most common organisms isolated from the amniotic fluid (AF) of women with preterm premature rupture of membranes (PPROM) and preterm labor with intact membranes, as well as women with cervical insufficiency. Their presence in AF is associated with a shorter interval to delivery (in women with preterm labor and intact membranes as well as PPROM ) and a higher rate of neonatal morbidity than patients with a negative amniotic fluid for microorganisms.
Of interest is the recent observation by Goldenberg et al that approximately 20% of neonates born before 32 weeks of gestation have positive blood cultures for genital mycoplasmas. Viscardi et al found that in 23% of very low birthweight infants, ureaplasmas were retrieved from venous blood or cerebrospinal fluid using polymerase chain reaction techniques and that the presence of microbial footprints was associated with an elevated concentration of interleukin-1 beta.
The traditional view has been that genital mycoplasmas are commensal in the lower genital tract of normal pregnant and nonpregnant women and that these organisms have low virulence. Moreover, the mere presence of these organisms in the lower genital tract does not increase the risk for upper genital infection in nonpregnant women or the risk for preterm delivery or adverse neonatal outcome in pregnant women. Indeed, some investigators suggest that the significance of ureaplasmas is mainly its association with other microorganisms.
Recent observations suggest that microbial products from ureaplasmas have a similar capacity to those of Gram-negative bacteria (lipopolysaccharide or bacterial endotoxin) to induce an inflammatory response in vitro in choriodecidual explants. Indeed, AF interleukin-8 concentration at birth was higher in patients with positive AF cultures or evidence of placental invasion with ureaplasmas than in those with other microorganisms.
It is not known whether the inflammatory response of the amniotic cavity and/or the mother is different when intraamniotic infection is due solely to genital mycoplasmas or other microorganisms. The current study was designed to address this question in a population of patients with PPROM.
Materials and Methods
Study design
The study population consisted of 99 patients admitted to the Seoul National University Hospital between January 1993 and March 2008 with the diagnosis of PPROM (<37 weeks of gestation) who met the following criteria: (1) singleton pregnancy, (2) AF obtained for microbiologic studies by transabdominal amniocentesis or at the time of cesarean delivery, and (3) proven AF infection by culture.
Patients were divided into 3 groups according to the microorganisms isolated from AF: group 1, subjects with a positive AF culture for genital mycoplasmas including ureaplasmas ( Ureaplasma urealyticum and U parvum ) and M hominis (n = 62); group 2, subjects with a positive AF culture for other microorganisms (n = 31); and group 3, subjects with mixed infection with genital mycoplasmas and other microorganisms (n = 6).
The intensity of the intraamniotic inflammatory response was determined by the AF white blood cell (WBC) count, and that of maternal inflammatory response was by the concentrations of C-reactive protein (CRP) and WBC count in maternal blood at the time of amniocentesis.
Amniocentesis is routinely offered for microbiologic studies and assessment of fetal lung maturity to all patients who are admitted with the diagnosis of PPROM. Retrieval of AF and maternal blood was performed after written informed consent was obtained. The Institutional Review Board of Seoul National University Hospital approved the collection and use of these samples and information for research purposes. The Seoul National University has a Federal Wide Assurance with the Office for Human Research Protection of the Department of Health and Human Services of the United States.
AF studies
After amniocentesis, AF in a capped plastic tube and commercialized culture media was transported immediately to the clinical laboratory of our hospital and cultured for genital mycoplasmas (ureaplasmas and M hominis ) as well as aerobic and anaerobic bacteria. An aliquot of AF was examined in a hemocytometer chamber to determine the WBC count.
Maternal plasma CRP
Maternal blood was collected into ethylenediaminetetraacetic acid–containing tubes by venipuncture at the time of amniocentesis. Samples were centrifuged, and supernatants were stored in polypropylene tubes at –70°C until assayed. CRP concentrations were measured with a highly sensitive enzyme-linked immunoassay (Immunodiagnostik AG, Bensheim, Germany). The sensitivity of the assay was 0.3 ng/mL. Intra- and interassay coefficients of variation were less than 10%.
Diagnosis of histologic chorioamnionitis, funisitis, and clinical chorioamnionitis
Histologic chorioamnionitis was defined in the presence of acute inflammatory changes on examination of a membrane roll and/or chorionic plate of the placenta; funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton’s jelly with the use of criteria previously reported. Clinical chorioamnionitis was diagnosed when maternal temperature was elevated to 37.8°C and ≥2 of the following criteria were present: uterine tenderness, malodorous vaginal discharge, maternal leukocytosis (≥15,000 cells/mm 3 ), maternal tachycardia (≥100 beats/min), and fetal tachycardia (≥160 beats/min).
Statistical analysis
Proportions were compared with the Fisher’s exact test. The Mann-Whitney U test and Kruskal-Wallis analysis were used for comparison of continuous variables. The generalized Wilcoxon test for survival analysis was used to compare the interval between amniocentesis and delivery. Statistical significance was defined as P < .05.
Results
Characteristics of the study population
A total of 99 cases with a positive AF culture met the inclusion criteria of the study. Table 1 shows types of microorganisms that were isolated from AF. Ureaplasmas were the most common isolates (64/99).
| Microorganism | Cases, n |
|---|---|
| Group 1 (intraamniotic infection with genital mycoplasmas) | |
| Ureaplasmas | 57 |
| M hominis | 3 |
| Ureaplasmas and M hominis | 2 |
| Group 2 (intraamniotic infection with other microorganisms) | |
| Candida species | 8 |
| Coagulase-negative Staphylococcus (CoNS) | 5 |
| Streptococcus anginosus group | 3 |
| Group B Streptococcus | 2 |
| Klebsiella pneumoniae | 2 |
| Burkholderia cepacia complex | 2 |
| Corynebacterium species | 2 |
| E coli | 1 |
| Peptostreptococcus species | 1 |
| Group D Streptococcus | 1 |
| Lactobacillus species | 1 |
| Alpha hemolytic Streptococcus | 1 |
| E coli , Acinetobacter baumannii , and CoNS | 1 |
| Candida species, Staphylococcus aureus , and A baumannii | 1 |
| Group 3 (mixed infection with genital mycoplasmas and other microorganisms) | |
| Ureaplasmas and E coli | 1 |
| Ureaplasmas and S anginosus group | 1 |
| Ureaplasmas and alpha hemolytic Streptococcus | 1 |
| Ureaplasmas, Candida species, and Streptococcus urevis | 1 |
| Ureaplasmas, S anginosus group, and CoNS | 1 |
| M hominis and E coli | 1 |
Table 2 compares characteristics and the outcomes of the study population. There were no significant differences in the clinical characteristics and pregnancy outcomes including age, parity, gestational age at amniocentesis, rupture of membranes-to-amniocentesis interval, gestational age at birth, birthweight, and use of corticosteroids and antibiotics before amniocentesis between patients with intraamniotic infection caused solely by genital mycoplasmas (group 1) and those with intraamniotic infection caused by other microorganisms (group 2).
| Characteristics | Positive AF culture for genital mycoplasmas (group 1, n = 62) | P a | Positive AF culture for other microorganisms (group 2, n = 31) | P b | Positive AF culture for genital mycoplasmas and other microorganisms (group 3, n = 6) | P c |
|---|---|---|---|---|---|---|
| Maternal age, y d | 30.2 ± 4.2 | NS | 30.7 ± 4.7 | NS | 29.2 ± 5.0 | NS |
| Nulliparity, n (%) | 27 (44) | NS | 12 (39) | NS | 3 (50) | NS |
| Gestational age at amniocentesis, wks d | 30.6 ± 4.4 | NS | 29.4 ± 4.8 | NS | 29.6 ± 3.2 | NS |
| Gestational age at birth, wks d , e | 31.5 ± 4.1 | NS | 30.5 ± 4.9 | NS | 29.9 ± 2.8 | NS |
| Interval between rupture of membranes and amniocentesis, h d | 65.7 ± 102.3 | NS | 119.9 ± 174.5 | NS | 41.0 ± 36.7 | NS |
| Interval between amniocentesis and delivery, h d , e | 129.8 ± 165.9 | NS | 183.1 ± 475.2 | .09 | 51.6 ± 92.8 | < .05 |
| Corticosteroids use before amniocentesis, n (%) | 9 (15) | .06 | 10 (32) | NS | 0 | NS |
| Antibiotics use before amniocentesis, n (%) | 30 (48) | NS | 19 (61) | NS | 4 (67) | NS |
| Body temperature at amniocentesis, °C d | 36.9 ± 0.5 | NS | 36.8 ± 0.5 | < .05 | 37.2 ± 0.3 | < .05 |
| Clinical chorioamnionitis at amniocentesis, n (%) | 2 (3) | NS | 1 (3) | NS | 0 | NS |
| Birthweight, g d , f | 1739 ± 643 | NS | 1561 ± 767 | NS | 1432 ± 434 | NS |
| Histologic chorioamnionitis, n/N (%) | 43/51 (84) | NS | 15/21 (71) | NS | 5/5 (100) | NS |
| Funisitis, n/N (%) | 29/51 (57) | NS | 12/22 (55) | NS | 3/5 (60) | NS |
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