Objective
Bacteria have been identified in different regions of the placenta. Here, we tested the hypothesis that the maternal basal plate of the placenta harbors microbes that may be associated with adverse pregnancy outcomes.
Study Design
We performed a cross-sectional study of pregnancies from a single tertiary care hospital. Maternal medical and obstetric characteristics were obtained and pregnancies followed up prospectively for outcomes and placental collection. After delivery, systematic random sampling of the placental basal plate was performed. Paraffin sections of basal plates were stained with 4 histologic stains and scored for morphological evidence of bacteria.
Results
Of 195 total patients in the study, Gram-positive and -negative intracellular bacteria of diverse morphologies were documented in the basal plates of 27% of all placentas. Of the patients, 35% delivered preterm. No difference was noted between placental basal plates from preterm or term gestations. Intracellular bacteria were found in the placental basal plates of 54% spontaneous preterm deliveries <28 weeks, and in 26% of term spontaneous deliveries ( P = .02). Intracellular bacteria were also documented in placentas without clinical or pathologic chorioamnionitis.
Conclusion
A total of 27% of placentas demonstrated intracellular bacteria in the placental basal plate using morphological techniques. Thus, the maternal basal plate is a possible source of intrauterine colonization and placental pathological examination could include examination for bacteria in this important maternal–fetal interface.
Infection and inflammation are commonly associated with the risk for preterm birth (PTB). Predisposing factors for infection-related preterm delivery are diverse, including subclinical intrauterine infections, intraamniotic infections, and pyelonephritis. In addition, maternal history of PTB, especially multiple and/or early PTB, is a significant risk factor. This recurrence risk suggests the presence of a risk factor that is present from pregnancy to pregnancy. The specific mechanisms by which infectious insults trigger preterm parturition are poorly understood. Notably, randomized trials of antimicrobials for treatment of infection for prevention of PTB have been disappointing.
Previous studies have shown that pathogenic microbes can establish occult intracellular reservoirs within the epithelium of the murine urinary tract, thereby evading immune recognition and allowing reemergence of symptomatic infection. In addition, prior studies have documented the presence of bacteria in various placental locations but have not examined the basal plate specifically. We hypothesized that the cells in the basal plate of the placenta that comprise the tissue layer directly at the maternal–fetal interface may harbor occult microbes similar to the findings in previous placental studies as well as our studies in the murine urinary tract. We reasoned that occult microorganisms in the basal plate could lead to chronic or acute inflammation, and may be associated with adverse pregnancy outcomes such as PTB and chorioamnionitis. The objective of this study was to examine the basal plate of a large group of placentas to diagnose the presence or absence of microbes at the maternal–fetal interface. When microbes were identified in the basal plate maternal–fetal interface, we sought to investigate whether the presence of bacteria was associated with important clinical outcomes such as chorioamnionitis and PTB.
Materials and Methods
Study design
This is a cross-sectional study of women from a single tertiary care hospital. The study was approved by the institutional review board of Washington University School of Medicine in St. Louis, MO. Women were enrolled during their antenatal course and followed up until delivery. Clinical data and placental specimens were collected through an institutional core resource, the Women’s and Infant’s Health Specimen Consortium. Gestational age was assigned by the best data available from the last menstrual period if consistent with ultrasound dating (±5 days in the first trimester or ±14 days in the second trimester). If last menstrual period was unknown, or inconsistent with ultrasound dating, gestational age was assigned according to the earliest ultrasound available. Exclusion criteria were hepatitis B, hepatitis C, and human immunodeficiency virus infection. In-depth chart reviews were performed by trained research personnel as the patient progressed through her antenatal and delivery course using close-ended data collection forms. This included maternal medical history, obstetric history, pregnancy diagnoses, and outcomes as documented by the treating obstetric team.
Tissue harvest
Placentas (n = 195) were collected ≤12 hours from delivery. Trained research assistants used sterile technique to harvest three random, 5- to 8-mm samples from the most superficial area of the basal plate equidistant from each other and without inclusion of the periphery of the placental disc. Specimens were fixed for 48 hours at room temperature in 10% neutral buffered formalin prior to embedding in paraffin, as described.
Histopathology and histochemical analyses
Sections (5 μm) of embedded tissues were deparaffinized with xylene and ethanol, stained with hematoxylin and eosin, and evaluated for histologic evidence of basal plate components including stromal decidual cells and fetal epithelial trophoblasts. Since hematoxylin and eosin stain is not sufficiently sensitive to detect bacteria (typically ≤1 μm), slides were stained for bacterial detection by 3 methods: Gram stain (Acros Organics, Fair Lawn, NJ); Hema 3 modification of the Giemsa stain (Fisher Scientific, St. Louis, MO); and the Brown-Hopps modification of the Gram stain (Fisher Scientific). Ten random fields of each specimen per 100 μm 2 were examined for the presence or absence of bacteria by 2 independent observers blinded to clinical history. The histologic location of microorganisms was recorded by use of a Nikon Eclipse microscope (Nikon Instruments, Mehlville, NY) equipped with an Olympus DP71 color camera (Olympus Imaging, Center Valley, PA).
Exploration of obstetric outcomes and statistical analysis
Chorioamnionitis and PTB were 2 clinical outcomes of interest in this study. A cohort analysis was performed to explore the association between these 2 clinical outcomes and bacterial colonization at the maternal–fetal interface. Pregnancies with evidence of intracellular bacteria at the basal plate were compared to those without bacteria at the basal plate. PTB was defined broadly as delivery for any reason at <37 weeks of gestation. Spontaneous PTB sourced to preterm labor or preterm rupture of membranes as well as indicated PTB due to preeclampsia, abruptio placentae, or intrauterine growth restriction was included. An analysis of spontaneous PTB alone (defined as preterm labor and preterm premature rupture of membranes) was performed. Chorioamnionitis was assigned based on the hospital-derived placental pathology reports indicating the presence or absence of acute maternal or fetal inflammation or when antibiotics were administered in labor according to the institutional protocol for presumed chorioamnionitis.
Group B streptococcus (GBS) colonization was classified as positive if a vaginal/rectal swab was positive within 5 weeks of delivery, or if a urine culture grew GBS at any time during the pregnancy. Per routine practice at our institution, all preterm patients receive antibiotic prophylaxis for neonatal GBS colonization. Therefore, all women, independent of GBS carriage or treatment status, were included in the main analysis. A secondary analysis was performed excluding women with GBS vaginal colonization or urinary infection.
Advanced maternal age was defined as maternal age at delivery ≥35 years. Race and use of tobacco and alcohol were based on self-report. Sexually transmitted infections were captured as “during pregnancy” (which included presence of infection at the time of delivery or any time during the current gestation) or “prior to pregnancy” (defined as any time in the past but predating the current pregnancy). Sexually transmitted infections included gonorrhea, chlamydia, genital herpes simplex, Trichomonas, and syphilis. Urinary tract infection (UTI) was assigned if a positive microbial culture was present, or if clinical suspicion was sufficient to treat the patient with antibiotics. All study-related clinical information was entered into a secure database using Illume (DatStat Inc, Seattle, WA). Official pathology reports and diagnoses were also reviewed and added to the database.
Demographic information of women with and without intracellular bacteria was compared by t, χ 2 , or Fisher exact test as appropriate. Subsequently, univariate analysis was used to compare the incidence of intracellular bacteria in basal plate specimens from term compared to preterm gestations as well as from those with and without the diagnosis of chorioamnionitis. To compare each gestational age group to the reference group of term pregnancies, Poisson regression with robust error was used to estimate the relative risk and 95% confidence intervals. This analytic approach provides an unbiased estimate of the relative risk when the outcome is common (>10%).
Results
Of 195 pregnancies evaluated in this study, 27% had evidence of intracellular bacteria in the basal plate of the placenta. Demographic characteristics of the patients who participated are demonstrated in Table 1 . The multiple histologic stains used in this study assured us that we could determine both morphology and location of bacteria within cells in the basal plate if they were present, as posed in our hypothesis. We identified individual bacteria and biofilm-like clusters of bacteria in the basal plate ( Figure ).
| Characteristic | Intracellular bacteria present in basal plate, n = 53 | Intracellular bacteria absent from basal plate, n = 142 | P value |
|---|---|---|---|
| Advanced maternal age, y (SD) | 29.8 (5.9) | 28.4 (5.8) | .12 |
| Black race, % | 47.2 | 47.2 | .99 |
| Tobacco use, % | 9.4 | 23.9 | .03 |
| Alcohol use, % | 5.8 | 11.4 | .29 |
| STI in current pregnancy, % | 20.8 | 21.8 | .87 |
| History of STI prior to pregnancy, % | 34.6 | 26.8 | .29 |
| UTI in current pregnancy, % | 18.9 | 18.3 | .93 |
| Group B streptococcus infection, % | 21.6 | 22.9 | .85 |
Hema 3 modification of the Giemsa stain permits detection of bacteria as well as inflammatory infiltrates. Using this stain, we noted biofilm-like clusters of bacteria stained dark blue/purple in the basal plate ( Figure , A to C). Gram stains were conducted to determine whether the bacteria were Gram positive or negative. Figure , E to G, illustrates examples of Gram-positive bacteria. We further found that bacteria of all shape classes were recorded and were of diverse morphologies, including filamentous ( Figure , E and I), diplococci ( Figure , F) or streptococci ( Figure , G), rods ( Figure , B, C, and H), and spirochetes ( Figure , D). The biofilm-like clusters could be coccoid ( Figure , A) or rod shaped ( Figure , B and C). We used the Brown-Hopps stain, which detects DNA content, thus rendering the bacteria and host nuclei a deep purple over a pale yellow background of cytosolic components to confirm the presence or absence of bacteria in all the basal plate tissues examined. This stain offers the advantage of rapid and reliable detection of bacteria even at ×20 magnification due to the staining patterns. Figure , H and I, depict bacteria evident in tissue stained with the Brown-Hopps stain.
We examined chorioamnionitis and PTB as 2 clinical outcomes of specific interest in this study. Thirty-three patients (17%) had either clinical or histologic diagnosis of chorioamnionitis. The finding of intracellular bacteria at the basal plate was no different in patients with or without clinical or histologic diagnosis of chorioamnionitis ( Table 2 ). Sixty-eight patients were delivered preterm and the remainder (n = 127) were delivered at term. PTB was no more common in placentas harboring intracellular bacteria at the maternal–fetal interface than those without bacteria present ( Table 2 ). As GBS carriage could potentially confound our analysis of intracellular bacteria incidence in basal plate, a secondary analysis was performed excluding women with GBS colonization. The results remained the same with similar rates of intracellular bacteria in preterm vs term placentas (30.4% vs 27.4%, P = .7). Among the preterm deliveries, 38.2% (n = 26) were delivered between 34-37 weeks’ gestation, 39.7% (n = 27) between 28-34 weeks’ gestation, and 22.1% (n = 15) at <28 weeks’ gestation. No significant difference of intracellular bacteria in multiple gestational age strata was noted ( Table 3 ). Fifty of the PTB cases (74%) were classified as spontaneous PTB. A significant 2-fold increased risk for intracellular bacteria in the basal plates of very preterm placentas (<28 weeks) in the spontaneous PTB subgroup was evident when compared to the term birth reference group (54.5% vs 26.7%, P = .02) ( Table 4 ).
| Variable | Intracellular bacteria present in basal plate, % | Intracellular bacteria absent from basal plate, % | P value |
|---|---|---|---|
| Preterm birth <37 wk (n = 68) | 35.9 | 34.5 | .86 |
| Chorioamnionitis (n = 33) | 13.2 | 18.3 | .40 |
| Gestational age, wk | Intracellular bacteria present in basal plate, % | Relative risk (95% CI) | P value |
|---|---|---|---|
| ≥37 | 26.7 | Reference | |
| 34-36 (n = 26) | 15.4 | 0.6 (0.2–1.5) | .3 |
| 28-33 (n = 27) | 33.3 | 1.3 (0.7–2.3) | .5 |
| <28 (n = 15) | 40.0 | 1.5 (0.8–3.0) | .3 |
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