Association between maternal genital mycoplasma colonization and histologic chorioamnionitis in preterm births

Abstract

OBJECTIVE: Genital mycoplasmas (GMs) can be associated with chorioamnionitis and preterm birth, but are viewed as commensal organisms with low virulence. We sought to determine if cervical GM colonization is associated with histologic chorioamnionitis independent of infection with other bacteria.

STUDY DESIGN: Retrospective study of patients who delivered preterm, had cervical cultures for GMs, and placental cultures for bacteria other than GM. GM positive patients were compared to GM negative patients. Histologic grading of placentas was compared between GM negative patients with negative placental cultures (Group 1), GM positive patients with negative placental cultures (Group 2), GM negative patients with positive placental cultures (Group 3) and GM positive patients with positive placental cultures (Group 4).

RESULTS: GM positive patients were less likely than GM negative patients to have placental cultures positive for other bacteria (39% versus 47%, P = 0.0071). Group 2 had higher rates of membrane inflammation compared to Group 1 (p = 0.0079), and no significant difference in rates of membrane inflammation compared to Groups 3 or 4 (p = 0.36, p = 0.18). GM positivity was independently associated with increased membrane inflammation and decreased inflammation in the chorionic plate.

CONCLUSIONS: GM colonization is associated decreased inflammation of the chorionic plate, and increased inflammation of the membranes.

Keywords

Chorioamnionitis genital mycoplasma placental inflammation prematurity

Abbreviations

Genital mycoplasma

PTL

preterm labor

PPROM

preterm premature rupture of membranes

NEC

necrotizing enterocolitis

LPS

lipopolysaccharide

HCA

histologic chorioamnionitis

1 Introduction

Genital mycoplasmas (GMs) Ureaplasma urealyticum and Mycoplasma hominis are the most frequently isolated organisms from placental membranes and amniotic fluid in chorioamnionitis. Ureaplasma urealyticum and Mycoplasma hominis have been isolated from placental membranes in 47% and 30% of causes of confirmed chorioamnionitis, respectively. These organisms are traditionally viewed as commensal organisms in the lower genital tract with low virulence for infection. However, they have been associated with adverse pregnancy outcomes such as: preterm labor (PTL), preterm premature rupture of membranes (PPROM), neonatal infection associated with chronic lung disease, pneumonia, cerebral palsy, and necrotizing enterocolitis (NEC) [1 –5]. Studies have shown that GMs can invade the amniotic cavity, and persist for months, initiating an intense inflammatory response [6 –10]. Kacerovsky et al. demonstrated a similar inflammatory response in the presence of Ureaplasma spp as compared to other bacteria in the amniotic fluid of women with PPROM [11]. GMs have been shown to elicit a greater increase in amniotic fluid white blood cell count, as well as maternal white blood cell count and plasma C-reactive protein compared to other organisms in patients with PPROM [12], and have been shown to be associated with acute placental and fetal inflammation [13]. Antibiotic treatment of GMs has not been found to reduce the incidence of adverse pregnancy outcomes [14] or effective at treating Ureaplasma respiratory infections in preterm infants [15].

Chorioamnionitis refers to infection of the amniotic fluid, membranes, placenta, or decidua. The passage of organisms to the chorioamnion or umbilical cord leading to infection is most commonly caused by ascending infection from the cervix and vagina [16, 17]. Specific host mechanisms including the mucous plug, placenta, and membranes play a role in prevention of chorioamnionitis. Rupture of membranes as well as changes in local innate immune responses facilitate the spread of bacteria [18, 19]. Chorioamnionitis can be referred to as clinical or histologic. Clinical chorioamnionitis is diagnosed based on clinical findings including fever, uterine tenderness, maternal tachycardia, fetal tachycardia, or purulent or foul smelling amniotic fluid [20]. Histologic chorioamnionitis is defined by the presence of findings of acute infection on histologic exam, and polymorphonuclear leukocyte (PMN) infiltration [21]. Histologic chorioamnionitis is graded based on the size and number of PMN foci, and depth of invasion in the amnion, chorion-decidua, umbilical cord, and chorionic plate [22].

Lower genital tract colonization of the cervix and vagina by microorganisms is common and found in 70% of women [6]. Cervical and vaginal colonization with U. urealyticum have been associated with a significant increase in preterm delivery, and have been found to be an independent risk factor for chorioamnionitis [23 –26]. Patients with positive cultures for Ureaplasma spp, who had high levels of anti-ureaplasma antibodies, were more likely to develop pregnancy complications [27]. The mechanism by which lower genital tract colonization leads to adverse pregnancy outcomes is poorly understood.

Based on previous findings, it is possible that maternal GM cervical colonization may be associated with an inflammatory process leading to adverse perinatal outcomes in the absence of ascending infection. The inflammatory process associated with GM colonization would be evidenced by placental histologic chorioamnionitis (HCA) in the absence of bacterial infection [28]. We hypothesized that GM colonization is associated with histologic chorioamnionitis (HCA) independent of placental infection with other bacteria. The aim of this study was to assess placental histologic chorioamnionitis seen in patients with GM positive or negative cervical cultures with negative or positive bacterial placental culture results.

2 Methods

This is a retrospective cohort study of patients who were admitted preterm and delivered at the University of Connecticut Health Center between January 2007 and December 2013. The Institutional Review Board at the University of Connecticut Health Center approved this study. As per our service policy all mothers admitted preterm (23-0/7 to 36-6/7 weeks gestational age) with or without suspected chorioamnionitis have admission cervical cultures obtained for GMs (Ureaplasma urealyticum and Mycoplasma hominis) as well as group B streptococcus (GBS). At delivery aerobic and anaerobic placental cultures were also obtained and all placentae underwent gross and histologic evaluation. Exclusion criteria included: Maternal cervical culture or placental culture not performed, known congenital or chromosomal anomalies, and multi-fetal gestations.

Maternal data collected included: age, parity, cervical GM culture result, gestational age at admission, gestational age at delivery, Group B streptococcus (GBS) status, antenatal corticosteroid treatment, antenatal antibiotic treatment, membranes status (intact, ruptured), duration of membrane rupture, mode of delivery, and diagnosis of clinical chorioamnionitis. Antenatal corticosteroid treatment consisted of 2 doses of betamethasone 12 mg given 24 hours apart. Antibiotic treatment included penicillin for patients in preterm labor with unknown GBS status or GBS positive states, and a combination of azithromycin and ampicillin/amoxicillin for latency antibiotics in patients with ruptured membranes. Patients with positive GM cultures were treated with azithromycin, if they were not already receiving azithromycin for latency. The diagnosis of clinical chorioamnionitis was made based on the presence of maternal fever (≥100.4°F) and the presence of one of the following: maternal leukocytosis (greater than 15,000 cells/mm3), maternal tachycardia (greater than 100 beats/minute), fetal tachycardia (greater than 160 beats/minute), uterine tenderness, or foul odor of the amniotic fluid.

Maternal cervical GM culture for M. hominis and U. urealyticum was obtained by cervical fluid swab during the examination at the time of admission as a part of routine care. Before any vaginal examination was performed or lubrication used, Dacron swabs were used for specimen collection from the cervix. Specimens were cultured under anaerobic conditions on Hayflick’s media and A7B media. An arginine based media and Ford’s broth were also used to detect GM metabolic activity. Specimens were incubated in anaerobic conditions at 37°C [29 –31].

Placental cultures were performed within 15 minutes following delivery of the placenta in a sterile fashion by the physician or trained Labor and Delivery nurse in the delivery room. Swabs for aerobic and anaerobic placental cultures were obtained from the space located between the amnion and the chorion [1]. The amniotic and chorionic membranes were manually separated, and a culturette swab was used to sample a deep portion from between the membranes, nonadjacent to the site of rupture. Cultures were incubated on two blood agar plates (5% carbon dioxide at 35°C and anaerobic at 35°), chocolate agar plate (5% carbon dioxide at 35°C), MacConkey plate (aerobic at 35°C), thioglycollate media (aerobic at 35°C), and Thayer-Martin media (5% carbon dioxide at 35°C).

All placentae underwent gross and histologic examination. Sections of tissue blocks were stained with hematoxylin and eosin. Samples of membranes were examined from a standard membrane roll and from the site of rupture. For this study the placenta histology slides were reviewed for evidence of HCA, and graded by by a single observer (MMS) who was blinded to clinical data. They were graded for acute (containing polymorphonuclear leukocytes) inflammation of the extraplacental membranes, fetal vasculature, chorionic plate, and decidua on a scale of 0–4 as described by Salafia et al. [22]. Grade 1 was defined as one focus of at least five polymorphonuclear leukocytes (PMNs). Grade 2 was defined as more than one focus of inflammation. Grade 3 was defined as multiple or confluent foci of inflammation. Grade 4 included diffuse and dense acute inflammation. Cases where then classified as: No inflammation (0), mild/moderate inflammation (1–3), or severe inflammation (4 or necrosis) for analysis.

Characteristics of patients with positive cervical cultures for GM were compared to patients with negative cervical cultures for GM. In order to achieve this, patients were categorized into 4 groups for further subgroup analysis of placental histology: GM negative with negative placental bacterial cultures (Group 1), GM positive with negative placental bacterial cultures (Group 2), GM negative with positive placental bacterial cultures (Group 3), and GM positive with positive placental bacterial cultures (Group 4). Dividing into 4 subgroups allowed us to account for the effects of bacterial infection on placental histology. Negative placental cultures were defined as negative for culture for bacteria other than GM, because placentae were not cultured for GM as a part of routine care. Group 2 was used on the reference group because we are interested in the effect of cervical GM colonization in the absence of other infections as compared to preterm deliveries with no evidence of GM colonization or infection, and preterm deliveries with evidence of other bacterial infection.

A power analysis was performed, and it wasestimated that 100 patients in the two groups (GM positive and GM negative) would be needed to detect a 20% difference in the rate of inflammation between groups with 80% power. A 20% difference between groups was chosen based on the study by Goldenberget al. [10] which found a difference in placental inflammation ranging from 26–44% between U. urealyticum positive and U. urealyticum negative cases. Data were analyzed using student t-test for continuous variables, and chi-square or Fischer’s exact test as appropriate for categorical variables. Multivariate logistic regression was used to control for confounding variables. Gestational age, PPROM, antenatal corticosteroids, antibiotics, and GBS status were included in logistic regression as possible confounders affecting placental inflammation. A p value of <0.05 (two-sided) was considered significant.Statistical software SAS V9.3 (Cary, NC) was used for all analyses.

3 Results

During the study period, 1239 cervical cultures for GMs were performed on patients admitted with suspected preterm labor or PPROM. After exclusion for multiple gestations, patients who were discharged and delivered at outside institution, or placenta cultures not performed at delivery, 214 patients were identified for analysis. The characteristics of GM positive patients (admission cervical cultures positive for GM) compared to GM negative (admission cervical cultures negative for GM) are shown in the Table 1. GM positive patients were younger than GM negative patients (26.5 ± 5.7 years versus 31.1 ± 6.1 years, P = <0.0001) and less likely to have placental cultures positive for other bacteria (39% versus 47%, P = 0.0071). There was no difference in gestational age at delivery between GM positive and GM negative patients (30.0 ± 3.7 versus 29.8 ± 4.1 p = 0.35). There was no difference in birth weight, administration of antenatal corticosteroids, incidence of PPROM, latency between membrane rupture and delivery, antibiotic treatment, GBS colonization, cesarean delivery, incidence of clinical chorioamnionitis, or incidence of histologic chorioamnionitis between the GM positive and GM negative patients. 97% of patients with positive cervical GM cultures received antibiotics, and 93% of the patients with negative cultures received antibiotics. This high rate of antibiotic exposure was due to treatment of PPROM patients with ampicillin and azithromycin, and treatment of preterm labor patients with intact membranes with penicillin if GBS cultures were positive or if GBS status was unknown. Of the 214 patients, 140 (65%) had admission cervical cultures positive for GM and 89 patients (41%) had placental cultures that were positive for other bacteria. 49 patients (36%) had both admission cultures positive for GM and placental cultures positive for other bacteria. Other bacteria included: Escherichia coli, Bacteroides spp, Enterococcus spp, Streptococccus spp, Klebsiella pneumoniae, Prevotella spp, Staphylococcus spp, Haemophilus influenzae, Citrobacter spp, Proteus spp, Enterobacter spp, and Peptostrepococcus.

Placental evaluation showed evidence of HCA in 133 (62%) patients. Patients in Group 2 had higher rates of HCA (63%) compared to patients in Group 1 (38%, p = 0.0074). There was no difference in HCA between Group 2 and Groups 3 or 4 (68% p = 0.77, and 63%, p = 0.43 respectively) (Fig. 1). Group 2 was used on the reference group in order to examine the effect of GM cervical colonization in the absence of other infections compared to preterm deliveries without evidence of GM colonization or infection, and preterm deliveries with evidence of other bacterial infection.

Table 2 shows the placental histologic grading of inflammation in the extraplacental membranes by GM and placental culture status. Between the four groups, there were significant differences in inflammation in the extraplacental membranes. When pairwise comparisons were performed, patients in Group 2 had significantly higher levels of inflammation in the extraplacental membranes than in Group 1 (p = 0.008). Patients in Group 2 did not have significantly different levels of inflammation in the extraplacental membranes as compared to patients in Group 3 (p = 0.36) or Group 4 (p = 0.12).

Placental histologic grading of inflammation in the chorionic plate is shown in Table 3. There were statistically significant differences in inflammation of the chorionic plate between the groups. There was significantly more inflammation in patients in Group 4 compared to patients in Group 2 (p = 0.02). There was no difference between patients in Group 1 and Group 2 (pairwise comparison. p = 0.92). There were no differences in inflammation in the decidua or fetal vessels between the groups (Tables 4, 5).

When multivariate logistic regression was performed controlling for placental bacterial infection with other organisms and other confounders including: gestational age, antenatal corticosteroid treatment, antibiotic treatment, GBS, and membrane rupture, cervical GM colonization was independently associated with increased inflammation in the extraplacental membranes (OR 2.8, CI 1.4–5.5) and decreased inflammation in the chorionic plate (OR 0.45, CI 0.23–0.88) as compared to GM negative patients (Table 6).

4 Discussion

Our results demonstrate that in preterm gestations, GM cervical colonization is associated with an inflammatory response in the extraplacental membranes. This is different from the inflammatory response from bacterial infection, which appears to be more localized in the chorionic plate. Although GMs are viewed as low virulence organisms, colonization appears to be associated with an intense inflammatory state. This may be important in the development of preterm labor in the absence of other bacterial infection.

Our finding of acute inflammation localized to the amnion in patients with GM colonization supports the findings by Oh et al., who found patients with amniotic fluid cultures positive for GM had higher amniotic fluid white blood cell counts, maternal white blood cells counts, and maternal plasma C-reactive protein than patients who had amniotic fluid cultures positive for other organisms [12]. This is a similar finding of evidence of acute amniotic inflammation in the absence of evidence of infection with bacteria other than GM.

The results of this study are different from those by Goldenberg et al. [13], in which they examined the relationship between cord blood GM cultures and placental inflammation. They found that infants with positive cord blood cultures from U. urealyticum were more likely to have positive placental cultures for any bacteria, and had more acute inflammation in the free membranes, chorionic plate, and umbilical cord. We found that maternal cervical GM colonization was only associated with increased acute inflammation of the extraplacental membranes, and was actually associated with decreased inflammation of the chorionic plate. The comparisons we can make to this study are limited, because we examined the effect of maternal cervical GM cultures on placental histology, whereas Goldenberg et al. studied infant cord blood GM. The difference in the findings could be due to increased maternal-fetal GM transmission in the setting of increased inflammation. One possibility is that since GM is an ascending infection, it affects the membranes before invading the amniotic sac, and only later affects the chorionic plate and then progresses to funisitis. In contrast, bacterial organisms may travel via the maternal blood stream to lodge first in the chorionic plate and the umbilical vessels, with involvement of the membranes only at a much later stage [20].

The findings of our study are related to the findings of Kacerovsky et al. They found that in patients with PPROM, the presence of HCA was associated with an increased microbial load of GMs in the amniotic fluid. Microbial load of GM DNA was also associated with increased maternal plasma C-reactive protein. It is possible that there is a microbial threshold for GM to cause the placental inflammatory response. In-vitro, low concentration of GMs can be present in the amniotic fluid for up to 24 hours without inducing inflammation [32]. Higher concentrations induce the same inflammatory response as the lipopolysaccharide (LPS) of gram-negative bacteria within 6 hours, with increased production of proinflammatory cytokines including tumor necrosis factor α, and anti-inflammatory cytokines interleukin-10 and prostaglandin E₂ [33]. Our study is limited in this aspect as we did not have amniotic fluid cultures or bacterial counts available.

In addition to the production of cytokines, it has been demonstrated that GMs can activate nuclear factor κB via Toll-like receptor (TLR) signaling [34]. Prior studies have demonstrated the role GMs play inactivating the innate immune system through TLRs. TLR2 and TLR4 expression are increased in preterm placentas with chorioamnionitis [18, 35]. In-vitro exposure of fetal membranes to GMs has been shown to increase TLR2 and TLR4 mRNA [19], as well as TLR2 expression and TLR2 cytokine responses [36]. It is possible GMs lead to adverse outcomes through activation of inflammatory cascades, signaling an inflammatory response similar to the response by other bacteria.

The relationship between maternal cervical GM and placental inflammation is important because the fetal intestinal and respiratory tracts are directly exposed to amniotic fluid containing inflammatory mediators. Although funisitis may not directly result from a maternal GM colonization, the fetus is exposed to amniotic fluid with inflammatory mediators resulting from GM colonization. Fetal gut exposure to these inflammatory mediators has been associated with the development of NEC [37]. Indeed; the incidence of NEC is 2.2 times higher in neonates <33 weeks colonized with Ureaplasma, and 3.3 times higher in neonates <28 weeks colonized with Ureaplasma. When adjusted for gestational age, Ureaplasma is associated with NEC with an odds ratio (OR) of 2.47 (CI 1.13–5.43, p = 0.023). In a rhesus monkey model, inoculation with Ureaplasma parvum is associated with the development for fetal lung injury [7]. Although postnatal treatment with macrolide antibiotics failed to prevent the development of bronchopulmonary dysplasia in Ureaplasma colonized infants, prenatal treatment with azithromycin was found to prevent lung injury in these animals [38]. GM activation of an inflammatory cascade is a plausible hypothesis as to why these organisms with low virulence are associated with adverse maternal and fetal outcomes.

Our study is limited by its retrospective nature and the small numbers. This study was powered to detect a difference in inflammation between GM positive and GM negative patients, but is underpowered to detect a difference between the four subgroups. Due to the retrospective nature of this study, we do not have quantifiable GM data. We do not have colony counts, or a way to differentiate colonization from infection. Additionally, confirmatory tests were not performed as this would not be part of routine clinical care. Another limitation is that we only evaluated maternal outcomes, and did not include neonate outcomes. We did not consider neonatal GM colonization in this study because GM cultures were not performed on the majority of these infants. Despite these limitations, the results provide evidence that GMs might play an important role preterm birth, and provide a hypothesis as to why antibiotic treatment does not improve outcomes. Additional studies are needed to further elucidate the biologic mechanisms behind GM induced placental inflammation, and to clarify the role of cervical colonization in this process. This may potentially lead to novel therapies to prevent preterm birth in the setting of GM colonization.

Declaration of interest

The authors report no conflict of interest.

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