Fusobacterium nucleatum induces premature and term stillbirths in pregnant mice: implication of oral bacteria in preterm birth

Abstract

Fusobacterium nucleatum is a gram-negative anaerobe ubiquitous to the oral cavity. It is associated with periodontal disease. It is also associated with preterm birth and has been isolated from the amniotic fluid, placenta, and chorioamnionic membranes of women delivering prematurely. Periodontal disease is a newly recognized risk factor for preterm birth. This study examined the possible mechanism underlying the link between these two diseases. F. nucleatum strains isolated from amniotic fluids and placentas along with those isolated from orally related sources invaded both epithelial and endothelial cells. The invasive ability may enable F. nucleatum to colonize and infect the pregnant uterus. Transient bacteremia caused by periodontal infection may facilitate bacterial transmission from the oral cavity to the uterus. To test this hypothesis, we intravenously injected F. nucleatum into pregnant CF-1 mice. The injection resulted in premature delivery, stillbirths, and nonsustained live births. The bacterial infection was restricted inside the uterus, without spreading systemically. F. nucleatum was first detected in the blood vessels in murine placentas. Invasion of the endothelial cells lining the blood vessels was observed. The bacteria then crossed the endothelium, proliferated in surrounding tissues, and finally spread to the amniotic fluid. The pattern of infection paralleled that in humans. This study represents the first evidence that F. nucleatum may be transmitted hematogenously to the placenta and cause adverse pregnancy outcomes. The results strengthen the link between periodontal disease and preterm birth. Our study also indicates that invasion may be an important virulence mechanism for F. nucleatum to infect the placenta.

FIG. 1.

Kinetic study of intravenous injection of F. nucleatum into pregnant mice. Mice were infected with a dose of 1 × 10⁷ to 2.5 × 10⁷ CFU of F. nucleatum 12230. At 6, 18, 24, 48, and 72 h postinfection, three to four mice were dissected. The bacterial counts in the placentas, liver, spleen, fetuses, and amniotic fluid (A.F.) of each mouse were expressed as log₁₀ CFU per gram of tissue or per milliliter of fluid. The results shown are the averages for all mice dissected at each time point. The standard deviations were <20% for values above 1 log₁₀ (CFU/g) but >50% for those less than 1 log₁₀ (CFU/g).

FIG. 2.

Histologic and immunohistochemical analysis of the sequence of infection and host response to F. nucleatum at 24 h (b and c), 48 h (d), and 72 h (e, f, g, and h) postinfection. (a) Basic anatomy of the placenta and membranes with the surrounding uterus at day 16 of gestation. The placenta includes the chorioallantoic plate, labyrinth (where vascularized fetal tissue and trophoblast-lined maternal blood spaces interdigitate for gas exchange), spongiotrophoblast, margin zone (junction of the decidua basalis, decidua capsularis, and Reichert's membrane of the parietal yolk sac), and Reichert's membrane. The membranes include the visceral yolk sac and amnion. The uterus includes the decidua capsularis, decidua basalis, myometrium, and endometrium. Magnification, 20×. (b) Immunoreactive F. nucleatum within a large venous sinus in the decidua basalis (DB). Venous sinuses separate the lateral decidua basalis into the upper portion of the decidua basalis, adjacent to the spongiotrophoblast, and the lower portion of the decidua basalis, adjacent to the myometrium. Magnification, 400×. (c) Single focus of F. nucleatum above a venous sinus in the upper decidua basalis. Magnification, 200×. (d) Neutrophilic response to F. nucleatum in the lower portion of the decidua basalis. Magnification, 100×. (e) Massive proliferation of F. nucleatum in the upper portion of decidua basalis. Degenerating neutrophilic exudate is seen in the lower portion of the decidua basalis. Magnification, 200×. (f) Lateral spread of proliferating F. nucleatum in the upper portion of the decidua basalis. Magnification, 40×. (g) Placenta and membranes (lateral at right, medial at left), showing spread of F. nucleatum to the margin. F. nucleatum has penetrated Reichert's membrane to colonize the visceral yolk sac and amnion. Magnification, 20×. (h) Infection of the amnion. Magnification, 200×.. A, amnion; CP, chorioallantoic plate; DB, decidua basalis; DC, decidua capsularis; Em, endometrium; Lab, labyrinth; margin, margin zone; MYO, myometrium; RM, Reichert's membrane of parietal yolk sac; SP, spongiotrophoblast; VS, venous sinus; VYS, visceral yolk sac.

FIG. 3.

TEM analysis of murine placentas infected with F. nucleatum 12230. (A) At 24 h postinfection. R, red blood cells in the blood vessel. The solid arrows point to bacteria internalized in the endothelial cells lining the veins. The open arrow points to an organism attached to the endothelial cells. Magnification, 6,000×. (B) At 72 h postinfection. Arrows indicate bacteria that have proliferated in the cytoplasm or outside the cells. Magnification, 1,500×. (C) Enlarged view of the area boxed in panel B. Arrows indicate bacteria in the nucleus.