| Objective: In this study, we used BALB/c mice as the animal model infected with Toxoplasma gondii to investigate: (1) the effect on the growth and development of fetus; (2) the concentration of serum chorionic gonadotropin (CG), placental lactogen (PL), estriol (E3); (3) the invasion and injury of the placenta; (4) the placental apoptosis and the expression of apoptosis-related protein Bax, Bcl-2, Fas, FasL and TNF-αin the placenta, which can help us to acknowledge the mechanism of adverse pregnancy outcomes induced by the infection of T. gondii during the second trimester.Methods: The 40 pregnant mice were randomly distributed into 2 groups, 20 mice in the normal control group, the other 20 mice in the infected group. On the 8th day of pregnancy, the mice of the infected group were injected i.p. with 0.2 ml PBS contained 100 tachyzoites, and 0.2 ml PBS (0.01 mol/L, pH=7.4) for the mice of the normal control group. 5 mice of each group were killed on the 12th, 14th, 16th and 18th day of pregnancy respectively, the serum of pregnant mice was collected, and the placenta and fetus were separated for below detection: (1) The survival rate of fetus was observed under anatomical microscope, the placentas and fetuses were weighted with electronic balance, and the body length and tail length were measured with sliding caliper. (2) The concentration of serum chorionic gonadotropin, placental lactogen, estriol of pregnant mice was detected by radioimmunoassay (RIA). (3) The polypides in the peritoneal fluid, amniotic fluid and placenta of pregnant mice were detected by Wright-Giemsa's staining, and the injury of the placenta was detected by paraffin section and HE staining. (4) The apoptosis rate and the Caspase-3 activity units were determined by flow cytometry (FCM) and spectrophotometry respectively. (5) The expression of apoptosis-related protein Bax, Bcl-2, Fas, FasL and TNF-αin the placenta was detected by immunohistochemistry.Results: (1) It was showed that the number of absorpted fetuses increased apparently along with the pregnant days in the infected group, of which the death of fetus and abortion were more than that in the normal control group. In the normal control group, the weight, body length and tail length of fetus increased obviously and the placental weight increased at first then decreased afterward, causing a decreased placental coefficient. However, the changes of fetuses and placentas in the infected group were not obvious compared with that in the normal control group. On the different pregnant days, there was significant difference in each group (placental weight: FNormal control group=88.610, FInfected group=21.906, fetal weight: FNormal control group=1695.874, FInfected group=284.801, placental coefficient: FNormal control group=165.930, FInfected group=100.011, body length: FNormal control group=649.255, tInfected group=5.391, tail length: FNormal control group=503.761, tInfected group=6.267; P<0.001). On the same pregnant days, the parameters detected in the infected group except the weight of the placentas and fetuses on the 12th day were lower than that in the normal control group, which the differences had statistical significance except the placental weight on the 12th day, placental coefficient on the 14th and 16th day, body length on the 14th day (P<0.05). (2) It was showed that the concentration of serum chorionic gonadotropin decreased along with the increase of the pregnant days in both the infected group and the normal control group. However, in the normal control group, the concentration increased slightly on the 18th day of pregnancy. On the opposite, the concentration of serum placental lactogen and estriol increased along with the increase of the pregnant days in both the infected group and the normal control group. However, in the infected group, the concentration decreased slightly on the 18th day of pregnancy. And the difference of the concentration of serum chorionic gonadotropin, placental lactogen, estriol of pregnant mice on the different pregnant days in each group, which had statistical significance (CG: FNormal control group=19.306, FInfected group=57.620, PL: FNormal control group=17.449, FInfected group=46.720, E3: FNormal control group=239.546, FInfected group=22.841; P<0.001). It was also showed that the concentration of serum chorionic gonadotropin, placental lactogen and estriol of the infected group were lower than that of the normal control group on the same pregnant days, which the differences had statistical significance only on the 16th and 18th day (P<0.05). (3) It was showed that there was no tachyzoite in the peritoneal fluid, amniotic fluid and placenta and no obvious injury on the 12th day. On the 14th day, there was a few tachyzoites in the peritoneal fluid and amniotic fluid, but no tachyzoite in the placenta, in which many lymphocytes, monocytes, eosinophile granulocytes and few apoptotic body were found. On the 16th day, there were more tachyzoites and some pseudocysts in the peritoneal fluid and amniotic fluid. The tachyzoites can be found in the placenta, and some trophocytes have been destroyed, which caused the inflammation of the chorionic villus. On the 18th day, the tachyzoites in the peritoneal fluid, amniotic fluid and placenta increased a lot, and the placenta was injured obviously, including the reduction of the blood of the chorionic villius, the diminution of trophocytes, condensation of endochylema, compaction of karyoplasm and increase of apoptotic body. (4) It was showed that not only the apoptosis rate and Caspase-3 activity units increased along with the increase of the pregnant days in both the infected group and the normal control group, which had significant differences (apoptosis rate: F Normal control group=114.333, FInfected group=200.417, Caspase-3 activity units: F Normal control group=14.940, FInfected group=15.212; P<0.01), but also the apoptosis rate and Caspase-3 activity units of the infected group were higher than that of the normal control group on the same pregnant days, which the differences had statistical significance except that of the Caspase-3 activity units on the 18th day (P<0.05). (5) It was showed that the apoptosis-related protein Bax, Bcl-2, Fas, FasL and TNF-αexpressed both in villus and decidua of the placenta, most of which were expressed in syneytiotrophoblast (ST). The positive cells of Bax, Fas, FasL and TNF-αincreased along with the increase of the pregnant days in both the infected group and the normal control group, and the positive cells of Bcl-2 decreased along with the increase of the pregnant days. And the difference of the positive cells on the different pregnant days in each group had statistical significance (Bax: FNormal control group=387.316, FInfected group=76.059, Bcl-2: FNormal control group=255.026, FInfected group=552.733, Fas: FNormal control group=591.607, FInfected group=1131.261, FasL: FNormal control group=82.598, FInfected group=159.749, TNF-α: FNormal control group=74.774, FInfected group=105.678; P=0.000). It was also showed that the positive cells of Bax, Fas, FasL and TNF-αof the infected group were more than that of the normal control group on the same pregnant days, but the positive cells of Bcl-2 of the infected group were fewer than that of the normal control group, which the differences had statistical significance except the TNF-αon the 12th day, Bax and Bcl-2 on the 14th and 16th day (P<0.05).Conclusions: (1) When the pregnant mice were infected with T. gondii during the second trimester, the tachyzoites may be multiplied in the abdominal cavity first of all, then rediffused into the amniotic fluid and placenta, which finally resulted in the congenital toxoplasmosis through peroral and hematological infection. (2) When the pregnant mice were infected with T. gondii during the second trimester, the tachyzoites invaded into the placenta, which can result in the reduction of the blood of the chorionic villius, the destruction of the trophocyte, and the inflammation of the chorionic villus, which finally influenced the transportation function of nutrient substance supplyed to embryo. (3) When the pregnant mice were infected with T. gondii during the second trimester, the expression of Bax, Fas, FasL and TNF-αwas increased, and the expression of Bcl-2 was decreased, which motivated the intrinsic and extrinsic apoptosis pathway, active Caspase-3, and finally lead to the apoptosis increase, then caused the decrease of capacity of trophocyte infiltrated into the decidua and spiral arteriolar of uterus, which hindered the formation of placental vascular net and the remodeling of spiral arteriolar, which finally resulted in the hypoperfusion, hypoxia and ischemia of placenta. (4) The destruction of the placental integrity, the inflammation of trophocyte and a large of apoptosis of trophocyte, which resulted in the decrease of serum chorionic gonadotropin, placental lactogen and estriol synthesis. (5) The damage of transportation function of placenta, the hypoperfusion, hypoxia and ischemia of placenta and the decrease of placental hormone synthesis, which finally resulted in the adverse pregnancy outcomes, such as intrauterine growth retardation (IUGR), stillbirth and abortion. |
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