Acute Infection with T. cruzi Reduces Fecundity of Mice in Relation to Maternal Parasitemia
As shown in Table 1
, although the rates of mating (appreciated by the frequency of vaginal plugs) were similar in infected and uninfected mice, only 18 to 23% of infected females developed a gestation as compared with 63 to 82% of the noninfected mice. The route of parasite inoculation did not influence the results because no difference was observed in fecundity of mice receiving parasites either intraperitoneally or subcutaneously. The uterus of infected, mated but nongravid mice, collected 9 days after mating, ie, ~4.5 days after implantation, presented a normal macroscopic aspect, similar to the uterus of nonmated mice, without any trace of implantation scars (data not shown).
| Table 1. Reproductive Capacity of T. cruzi Acutely Infected (I) and Uninfected (NI) Mice* |
As shown in Figure 2 , the higher the parasitemia of the female, the higher was the proportion of infertile mice, suggesting that the reduction of fecundity was related to events associated with the maternal parasitic load.
| Figure 2.Proportion of T. cruzi acutely infected mice developing gestation in relation to the parasitemia. The presence of pregnancy and the parasitemia were determined 9 days after the occurrence of the vaginal plug. NI, age-matched noninfected mated mice; n, (more ...) |
Acute Infection with T. cruzi Induces Massive in Utero and Neonatal Mortality in Mice
Although the numbers of implantations (the sum of resorptions and fetuses) were similar in gravid infected and uninfected animals (Table 2)
, a high proportion of embryos and fetuses of infected mice died during gestation, whereas the mortality rates were very low in noninfected females. Indeed, as shown in Figure 3A
, 28% of embryos of infected dams resorbed in early gestation (before G9.5, Figure 3C
), 8% of fetuses died in midgestation (macerated fetuses, Figure 3D
), and 22% died in late gestation (immobile fetuses seen at G19), resulting in a cumulative mortality of 57% of fetuses at G19. In addition, fetuses still alive at G19 (ie, mobile) displayed weights strongly reduced by 40% as compared to those from noninfected mice (Figure 3E
and Figure 4
). The weight of corresponding placentas was only slightly diminished by 22% (Figure 4)
, without correlation with fetal weight reductions, whereas in noninfected dams, fetal and placental weights were, as expected, significantly correlated (
P = 0.0006,
n = 62). This indicates asymmetrical fetal growth retardation.
| Table 2. Number of Implantations in T. cruzi Acutely Infected (I) and Uninfected (NI) Mice |
| Figure 3.Gestational and perinatal mortality in acutely T. cruzi-infected mice. T. cruzi-infected (filled bars) or uninfected (open bars) mice were either killed at days 17 or 19 of gestation (A) or allowed to deliver (B), and mortality of the progeny was recorded. (more ...) |
| Figure 4.Weights of alive fetuses and their placentas from T. cruzi acutely infected mice at day 19 of gestation. Fetuses and placentas were rapidly extracted from pregnant infected (filled bars; n = 15) and noninfected (open bars; n = 62) mice (more ...) |
As a result of such massive in utero mortality, the litter size at delivery of infected dams was strongly reduced to 3.57 ± 1.31, whereas it was 5.97 ± 0.36 for uninfected mice (P = 0.01). Moreover, 25% of the litter of infected dams were stillbirths (versus 3% for uninfected mice) (Figure 3B) . Although the remaining newborns were not weighed for avoiding manipulations that could enhance maternal cannibalism, they were clearly smaller than controls, and finally eaten by their mothers 24 to 48 hours after birth (Figure 3B) .
Because the infertility of infected mice was associated with the maternal parasite burden (see above), we also sought for a relation between maternal parasitemia and the rate of resorptions and fetal death. We found that the proportion of resorptions (occurring in early gestation between days 5 to 9 of gestation) in infected mice was significantly and positively correlated with the concurrent maternal parasitemia (measured at day 14 after infection; n = 38, r = 0.448, P < 0.05). However, there was no relation between the proportion of fetuses that died later during gestation (macerated and immobile fetuses) and the maternal parasitemia measured at days 21 and 24 after infection.
Fetal Mortality in Late Gestation of Mice Acutely Infected with T. cruzi Is Associated with Parasite Invasion of UPUs without Fetal Infection
To address the question of the role of parasite in the fetal mortality, we sought for intracellular amastigotes in fetal tissues of infected dams, by careful microscopic examination of histological sections of fetuses collected either at G17 (70 nonmacerated fetuses, 8 macerated fetuses, and 13 resorptions) or at G19 (9 mobile, 10 immobile and 8 macerated fetuses). Amastigote nests were never observed in such fetuses, excluding the congenital transmission of parasites as a cause of the massive mortality of fetuses.
Because parasites in the UPU might affect the local maternofetal exchanges, we also sought for amastigotes in the UPU surrounding resorptions and fetuses at G9, G17, and G19. Whereas intracellular parasites were never seen at G9 of gestation, they were found in the UPU at G17 and G19. T. cruzi amastigote nests were mainly localized in the decidua (Figure 5, A and B) , but some were also observed in chorioamniotic membrane (Figure 5D) , in the spongy zone (Figure 5E) , and more rarely in cells at the junction between the labyrinthine and the spongy zone, ie, at the maternal side of the labyrinthine. Parasites were not seen in the internal, fetal side, of the labyrinthine.
| Figure 5.Histopathology of placenta from mice acutely infected with T. cruzi. A–C: Decidua at G17; arrows show parasite nests (A), amastigotes (B), and inflammatory infiltrates (C). D: Chorioamniotic membrane at G17; the long arrow indicates a parasite (more ...) |
Amastigotes were also occasionally found in the myometrial part of the uterus at G17 or G19, but not at G9, whereas parasites have not been found in the uterus of nongravid mice (data not shown).
Decidua Is a Privileged Site for Parasite Multiplication
Microscopic examination of UPU sections showed that, at G17, parasites were much more numerous in the decidua than in the other placental tissues (data not shown). It is worth noting that the decidual amastigote burden (number of amastigote nests multiplied by number of amastigotes/nest) was at this time a mean 125 times higher than in the maternal cardiac tissue (Figure 6A)
. The number of nests was slightly higher in the edges than in the central part of the decidua (21.03 ± 0.90
versus 18.35 ± 1.31 nests/mm
2 respectively;
n = 31,
P < 0.05). Interestingly, analysis of amastigote nest density in the total UPU in relation to fetal mortality showed a significant increase from alive to dead fetuses, at G17 as well as G19 (Figure 6B)
.
| Figure 6.Parasite burden in maternal tissues in late gestation. T. cruzi-infected mice were killed at G17 (open bars) or G19 (filled bars), and intracellular parasites were counted in tissue sections. The state of fetuses from which placentas were obtained is (more ...) |
Dead Fetuses and Their Placentas Suffer Massive Ischemic Necrosis in Late Gestation of Acutely Infected Mice
No abnormalities could be seen in the microarchitecture of the placenta at G9. By contrast, at G17, microabscesses rich in polynuclear cells and containing few lymphocytes were observed in deciduas, in the area between spongiotrophoblasts and the labyrinth, and in the placental membranes of all dead fetuses (Figure 5, C and D)
. Microabscesses were not necessarily located close to infected cells. Fibrin deposits were not found in these placentas, even with the special Trichrome-Masson staining. At G19, such inflammatory infiltrates were associated with ischemic necrosis, numerous fibrin deposits, and vascular thromboses (Figure 5F)
.
Sections of fetuses were also carefully examined at G17 and G19. Inflammatory infiltrates containing neutrophils were seen in the liver, digestive tract, and bladder, also associated with ischemic necrosis (data not shown). At G19, the pathology was more pronounced in macerated than in immobile fetuses (recently dead). In fetuses still alive at G19, no obvious histological abnormalities were found.