Maternal-fetal Iodine Metabolism, Thyroid Function Fetal Brain Development And Their Placental Regulation Mechanism In Rats With Differents Iodine Nutritional Status

Iodine Deficiency Disorders (IDD) remains the most frequent cause worldwide, of preventable mental retardationin children. The damage to the human brain occurs mainly in the critical period of brain development-prenatal and early postnatal periods. Appropriate iodine nutrition and thyroid hormone (TH) level during pregnancy is an important condition to ensure normal growth and development of fetus, especially fetal brain development.Our study explores whether the pregnant dams and their placenta have the compensatory capacity to iodine deficiency or iodine excess (that is, the protection of the fetus) in different aspects, providing experimental basis for appropriate supplementation and monitoring of iodine nutrition during pregnancy and thyroid function screening of pregnant women.Methods:4-6 weeks healthy Wistar female rats were randomly divided into severe iodine deficiency (SID), moderate iodine deficiency (MoID), mild iodine deficiency (MiID), normal iodine (NI) and excessive iodine (ExI) groups by body weight. And NI was the control group. The male rats were fed as NI. All female rats were fed on an iodine deficient food and drinking water with different doses of KI for 3 months until to mating. Pregnant dams and their fetuses on 16 and 20 days after fertilization were studied. Iodine level in urine, blood, fetal amniotic fluid and placental tissue were detected in pregnant dams and their fetus by As-Ce catalytic spectrophotometry. Thyroid hormones (TH) and TSH in pregnant serum and their fetal amniotic fluid were detected by chemiluminascent immunoassay methods. Thyroid morphology and sodium-iodide symporter (NIS) expression of dams and their fetus were observed. The gene and protein expression levels of TH metabolism-related deiodinase(D2 and D3), TH transporter(MCT8 and OATP14), markers of the developing neurons and synapse(DCX and p38), iodide symporter(NIS and pendrin) were detected by real-time fluorescence quantitative PCR, Western blotting, immunohistochemistry in fetal brain, as well as in placental tissue and LCM purified trophoblast cells. The rat Chorionic gonadotropin (rCG) in serum of dams was measured by enzyme-linked immunosorbnent assay(ELISA).Results:1 the effects of different iodine nutrition on thyroid function in pregnant rats1.1 mild, moderate and severe iodine deficiency①The lower iodine level in vivo:The levels of urinary and blood iodine were significantly lower than that of normal iodine group and more significantly reduced with the increase of iodine deficiency. But the magnitude of changes in blood iodine was less than urinary iodine.②Enhanced thyroid iodine intake:Thyroid NIS immunoreactivity increased significantly and was localized mainly along the basement membrane of follicle cells and linear distribution. There was a positive correlation between thyroid NIS expression and the degree of iodine deficiency.③hypothyroidism:Serum TT4, FT4 displayed decreasing trend and TSH was increasing trend. Hormone changes were more apparent with decrease of iodine levels.④Enhanced thyroid weight:with iodine deficiency, absolute and relative thyroid weight was higher compared with the control group. There was statistical significance in SID group no statistical significance in MoID and MiID.⑤thyroid anatomy and histology changes:Thyroid of SID group showed clear signs of hyperaemia, swelling and a typical small follicular thyroid hyperplasia swollen; thyroid of MoID and MiID groups were not obvious goiter, a slight small follicular thyroid hyperplasia swollen was observed in MoID group; similar thyroid follicular size, but less thyrocolloid were observed in MiID group compared with NI Group.1.2 iodine excess①The high iodine level in vivo:The levels of urinary and blood iodine were significantly higher than that of normal iodine group. But the magnitude of changes in blood iodine was less than urinary iodine.②Reduced thyroid iodine intake:Thyroid NIS immunoreactivity decreased significantly and located mainly in the cytoplasm of follicular epithelial cells.③thyroid dysfunction:serum TT4, FT4 displayed slightly decreasing trend and TSH was increasing trend.④Normal thyroid weight:Thyroid absolute and relative weight closed to the control group.⑤Thyroid anatomy and histology changes:The appearance of the thyroid was similar to the control group, and similar thyroid follicular size, but more thyrocolloid were observed compared with NI Group.2 The impact of different iodine nutrition on fetal-rats thyroid function and brain development2.1 Before establishment of fetal thyroid function (gestation 16 day)2.1.1 mild, moderate and severe iodine deficiency①Normal iodine level in vivo:The content of iodine in amniotic fluid was similar with the control group.②Shortage of thyroid hormone (from dams):FT4 of amniotic fluid displayed decreasing trend and FT3/FT4 ratio was increasing trend in SID and MoID Group. There was a significant increase in FT3/FT4 ratio of SID. There was no significant difference between MiID and control group.③Inadequate TH of brain and depression of brain development:The expression of brain tissue D2mRNA significantly decreased in all iodine deficiency groups. DCX mRNA and protein expression showed a higher trend. There was statistically significant increase of DCXmRNA expression and was not significant increase of DCX protein expression in MiID and MoID group.2.1.2 Excessive iodine①The higher iodine level in vivo:The content of iodine in amniotic fluid significantly increased. But its rate of increase (about 5 times) was significantly less than their dams increased iodine supply (30 times).②Nearly normal thyroid hormone (from dams):FT4, FT3 of amniotic fluid were similar with the control group and FT3/FT4 ratio was decreasing, but not statistically.③Normal brain TH and tissue and development:The indicators were similar to the control group.2.2 after establishment of fetal thyroid function (gestation 20 day)2.2.1 mild, moderate and severe iodine deficiency①The low iodine level in vivo:The iodine level of amniotic fluid in SID and MoID group significantly reduced, respectively about 50% and 36% of the control group; and in MiID group it almost reached to normal level (97% of the control group).②Enhanced thyroid iodine intake:thyroid NIS expression and location was similar to the pregnant rats. ③Shortage of thyroid hormone (from dams and fetus):In SID group, FT4 of amniotic fluid significantly reduced and TSH, FT3/FT4 ratio significantly enhanced. But in MoID and MilD group, there was an increase trend in TSH of amniotic fluid and TH was similar to control group.④Tthyroid anatomy and histology changes:Thyroid hyperaemia and swelling showed clear in SID group and was not clear in MoID and MiID groups. In SID group, thyroid histological changes showed the majority cells clustered gland-like structure without the obvious follicular cavity and thyrocolloid, and was full of blood capillary. In MoID group, thyroid had formed partially obvious follicles with smaller cavity and few thyrocolloid that almost could not to be seen. A considerable number of cells were still gland-like structure and was full of blood capillary. In MiID Group, the number of thyroid follicles with cavity was more than that of MoID group and less than that of control group. But there were less thyrocolloid that showed partially Light staining in follicular cavity.⑤Inadequate TH of brain and depression of brain development:The expression of brain tissue D2 and MCT8 mRNA significantly decreased and D3mRNA was decreasing in all iodine deficiency groups. In SID and MoID group, DCX mRNA significantly increased and protein expression showed no difference compared with control group. In MiID group, there was no difference in DCX mRNA and protein expression. p38 mRNA and protein expression of SID and MoID group significantly decreased. And in MiID group, p38 mRNA significantly decreased while protein expression was similar to the control group.2.2.2Excessive iodine①The higher iodine level in vivo:The content of iodine in amniotic fluid significantly increased. But its rate of increase (about 25 times) was less than their dams increased iodine supply (30 times).②Reduced thyroid iodine intake:Thyroid NIS expression and location was similar to the pregnant rats.③Nearly normal thyroid hormone ((from dams and fetus)):TSH of amniotic fluid was higher than that in control group (without Statistical significance) while TH levels were similar to control group.④Thyroid anatomy and histology changes:Thyroid showed clear hyperaemia and unclear swelling. Similar thyroid follicular size and more blood capillary were observed. ⑤Inadequate TH of brain and depression of brain development:In all iodine deficiency groups, the expression of brain tissue D2 mRNA significantly decreased and D3mRNA was decreasing. But the MCT8 mRNA expression of brain tissue significantly increased. DCX mRNA and protein expression was similar with the control group. p38 mRNA was decreasing trend and protein expression was similar with the control.3 The effect of different iodine nutrition on iodide symporter expression of placental trophoblast cells3.1 The distribution of placental NIS and pendrinRat placental NIS immunoreactivity was mainly found in the cytoplasm facing the fetus of cyto-syncytiotrophoblast cells of the larger villous stem. The cytoplasm of intravillous mesenchyma, decidua and endometrial pregnancy glands showed NIS expression at gestation 20 day. The cell membrane of active trophoblast cells with mitotic phase could be seen. NIS immunoreactivity of active trophoblast cells was the whole film distribution at gestation 16 day and mainly located in the membrane facing the fetus at gestation 20 day.Rat placental pendrin immunoreactivity was mainly found in the cytoplasm facing the mother of cyto-syncytiotrophoblast cells of the larger villous stem.3.2 The trophoblast cells NIS, pendrin mRNA expression of iodine deficiencyIn SID group at gestation 16 day, NIS mRNA expression increased and pendrin mRNA expression significantly increased. NIS mRNA decreased and pendrin mRNA increased In SID group at gestation 20 day. In MoID and MilD group at both gestation 16 and 20 day, NIS mRNA expression was similar to control group, pendrin mRNA increased.3.2 The trophoblast cells NIS, pendrin mRNA level of iodine excessAt gestation 16 day, NIS mRNA expression significantly decreased and pendrin mRNA expression decreased. But NIS mRNA significantly increased and pendrin mRNA increased at gestation 20 day.4 The impact of different iodine nutrition on placental TH metabolism key enzyme and transporters expression4.1 Before establishment of fetal thyroid function (gestation 16 day)①Moderate and severe iodine deficiency:In SID and MoID group, MCT8mRNA expression significantly reduced in the placenta and was decreasing in the purified trophoblast cells. OATP14mRNA expression increased and D2mRNA expression decreased in placental tissue. D2mRNA expression in the trophoblast cells was increased in the trophoblast cells. D3mRNA expression showed a lower trend in the placental tissue and trophoblast cells.②Mild iodine deficiency:In MiID Group, MCT8 and OATP14 expression levels of mRNA were similar to SID and MoID group. D2, D3mRNA expressions were similar to the control.③Iodine excess:In ExI Group, MCT8 mRNA change was similar to the iodine deficiency groups, but OATP14 mRNA was decreasing in the placental tissue. D2 mRNA was decreasing in the placental tissue while was similar to the control group in the trophoblast cells. D3mRNA expression was similar to SID and MoID Group.4.2 after establishment of fetal thyroid function (gestation 20 day)①moderate and severe iodine deficiency:In SID and MoID group, MCT8mRNA expression significantly reduced in the placenta(with gestation 16 day). But in trophoblast cells, MCT8mRNA expression of the SID group was increasing and was similar to the control group in MoID group. Placental OATP14 mRNA expression was decreasing. D2 mRNA was increased trend in the placenta and significantly enhanced in trophoblast cells. D3mRNA in the placental tissue was decreasing.②mild iodine deficiency:in MiID group, MCT8 mRNA expression significantly reduced in the placenta and was similar to the control group in trophoblast cells. OATP14 mRNA change in placental tissue was similar to the MoID and SID group. Placental D2 mRNA was decreasing and and was a high-trend in the trophoblast cells. D3 mRNA expression was similar to the control.③Iodine excess:The ExI group, MCT8 mRNA change was similar to the deficiency groups. Placental OATP14 mRNA was similar to the control. D2 mRNA in placental tissue and trophoblast cells were significantly increased. D3 mRNA expression was similar to the SID and MoID group.Conclusions1. The levels of iodine of pregnant-fetal rats in moderate and severe iodine deficiency significantly decreased and displayed hypothyroidism. Fetal brain TH levels could not meet the needs of brain development after the mother-placenta compensation and led to significant brain development disorder.2. The level of iodine of dams in Mild iodine deficiency mildly decreased and displayed thyroid mild dysfunction. Fetal iodine levels and thyroid function are nearly close to normal after mother-placenta compensation, but brain THs that were required for fetal brain development were still relatively insufficient to mainly impact the early pregnancy when fetal brain development dependent on maternal TH before the establishment of fetal thyroid function.3. The level of iodine of pregnant rats in excessive iodine supply significantly increased and displayed thyroid mild dysfunction. Fetal iodine levels increased to some extent and thyroid function are nearly close to normal after mother-placenta compensation. But brain THs that were required for fetal brain development were still relatively insufficient to mainly impact the later pregnancy when fetal brain development dependent on maternal and fetal TH after the establishment of fetal thyroid function.In short, even if iodine levels and (or) thyroid function fluctuation during pregnancy are very slight, fetal brain development will be affected in varying degrees.