Studies have shown that intrauterine infections in humans cause preterm birth. F. nucleatum
is one of the most prevalent species found in infections of amniotic fluid and placenta and notably is often implicated as a sole infectious agent in preterm labor with intact fetal membranes. It is also associated with various forms of periodontal disease. Because of its association with both periodontal disease and preterm birth, F. nucleatum
is an ideal model organism for investigating the correlation between these two diseases. During periodontal infection, when the oral mucosa is injured and inflamed and the quantities of periodontal pathogens increase dramatically, transient bacteremia may occur (1
). This can lead to selective colonization of undesired sites. In the current study, we bypassed initial transmission of organisms from the oral cavity into the bloodstream and addressed the question of what effects F. nucleatum
has on pregnancy if it enters the circulation.
We chose to intravenously inject pregnant mice on day 16 to 17 of gestation, a relatively late stage in the pregnancy, for several reasons. Stress or infection prior to day 14 tends to lead to resorption of the fetuses. Preterm birth in humans also occurs during late stages in gestation, and the murine infection was initiated at a gestational age proportional to 28 to 32 weeks in humans. Also, the structure of the mouse placenta at late stages of gestation is remarkably similar to that of human placenta (6
Our study showed that intravenous injection of pregnant mice with F. nucleatum isolated from either amniotic fluid infection or an oropulmonary source resulted in fetal death. This is the first experimental demonstration that hematogenous injection with an orally associated species can cause adverse pregnancy outcomes. This murine model closely paralleled human intrauterine infection by F. nucleatum in several important aspects. First, infection was located inside the uterus, and live organisms could be isolated from the placenta, amniotic fluid, and fetuses. Second, fetal death was due to local infection of the fetoplacental unit rather than systemic effects of maternal bloodstream infection. Third, immunohistochemical analysis indicated that the placental infection eventually spread to the amnion, mimicking chorioamnionitis in humans.
The abnormal pregnancy outcomes in our murine model were manifested as premature births and term stillbirths, with occasional nonsustained live births. This differs somewhat from the outcome of intrauterine F. nucleatum infection in humans, where delivery and medical intervention can often prevent fetal death. In mice, preterm delivery only occurred if the entire litter was dead; mixtures of live and dead fetuses were all delivered at full term. Since the birth of one abnormal fetus cannot occur in the mouse without delivery of the other unaffected mice, preterm birth is not a successful adaptive strategy in animals with multiple gestational sacs.
Immunohistochemical analysis revealed that F. nucleatum
infection, accompanied by inflammation, was initiated in the decidua basalis of the placenta, an area characterized by large venous sinuses. A similar localization of organisms at this site has previously been reported in murine fetoplacental listeriosis (19
). We speculate that the slow blood flow rate and consequent low shear forces in the venous sinuses provide an opportunity for F. nucleatum
to adhere to and invade the endothelial cells. The bacteria may then cross the endothelium and establish infection in the decidua basalis. Indeed, the majority of F. nucleatum
infections detected by immunohistochemical analysis at earlier time points were located either within or directly adjacent to the venous sinuses. The infection intensified with time, indicating proliferation of the bacteria, consistent with the kinetic study. Also consistent were electron microscopic studies, which confirmed the tendency of F. nucleatum
to migrate from within the venous sinuses and proliferate in the decidua basalis.
TEM studies also provided the first in vivo evidence of F. nucleatum attachment and invasion of tissue cells, supporting our in vitro findings of attachment and invasion of epithelial and endothelial cells by F. nucleatum. The major discrepancy between our in vivo and in vitro studies was the observation by TEM of massive invasion of the decidua basalis, with bacteria present in the nucleus at 72 h postinjection. This discrepancy could be due either to the difference in infection time or to local factors promoting bacterial proliferation. During the in vitro studies, F. nucleatum was incubated with host cells for no more than a few hours. It is possible that, with prolonged incubation, similar massive proliferation of intracellular F. nucleatum might be observed in the in vitro assays.
The vaginal isolates of F. gonidiaformans
, which is rarely found to be associated with preterm birth, did not invade either endothelial or epithelial cells, nor did it adhere to the endothelial cells. The inability of F. gonidiaformans
to invade was probably due to its inability to attach to the host cells. This observation is consistent with the suggestion that attachment and invasion may be important virulence mechanisms of F. nucleatum. F. nucleatum
possesses a unique adhesion molecule which is absent in F. gonidiaformans
but present in F. nucleatum
(Han et al., unpublished data). This may partially explain why the lam
mutant, although defective, is still capable of attaching to and invading tissue culture cells, which may then account for its unaffected virulence in mice. It is likely that F. nucleatum
encodes additional virulence factors, such as phospholipase, lipopolysaccharides, and the ability to induce proinflammatory cytokines (8
), which may also be critical to its role in the pathogenesis of preterm birth.
Our study suggests that systemic injection of F. nucleatum
into mice prior to pregnancy would be unlikely to contribute to an adverse pregnancy outcome. Although F. nucleatum
has been demonstrated to cause intrahepatic and intra-abdominal abscesses by intraperitoneal challenge (11
), in our model the bacteria were eliminated rather quickly from the liver and spleen. It appeared that colonization would not occur unless the placenta was present. The ability of F. nucleatum
to proliferate in the placenta and eventually spread to the amniotic fluid and fetuses may be attributed in part to local immunosuppression in the reproductive organs during pregnancy. Venous sinusoids also exist in the liver, but the presence of abundant immunocompetent cells may explain the rapid clearance of F. nucleatum
that we observed at this site. An alternative but not mutually exclusive explanation is that F. nucleatum
may preferentially infect the cell types found in the decidua basalis, decidual stromal cells and interstitial trophoblastic cells (glycogen cells).
In summary, we found that intravenous inoculation of mice with F. nucleatum, an orally related species, induced adverse pregnancy outcomes. This indicates that F. nucleatum is capable of colonizing the uterus via a hematogenous route in addition to the commonly acknowledged ascending route. The sequence of infection in murine placentas mimicked that in humans leading to preterm birth. Live organisms were isolated from the infected murine placentas. Therefore, we have fulfilled Koch's postulates identifying F. nucleatum as a causative agent of adverse fetal outcomes in mice analogous to those seen with human preterm birth. The current model will be useful for studies of the mechanism of preterm birth caused by intrauterine infection. Our results indicate that invasion of the maternal venous endothelium in the decidualized endometrium of pregnancy may be an important virulence mechanism for F. nucleatum and perhaps other oral bacteria to colonize the placenta and cause stillbirth or preterm birth. This study also sheds light on the mechanisms underlying the link between periodontal infection and preterm birth and may be useful for investigating the impact of oral health on other diseases.