Early Levothyroxine Improves Cardiac Development In Subclinical Hypothyroidism Offspring

Objective:Subclinical hypothyroidism(SCH)during pregnancy accounts for 2-3%of thyroid dysfunction during pregnancy,which is characterized by elevated TSH and normal T3 and T4.In clinical work,it is found that the fetus with subclinical hypothyroidism during pregnancy has arrhythmia and the incidence of fetal distress during delivery is high.Previous studies have also shown that the incidence of fetal heart malformation is high and fetal echocardiography has been changed.However,there are few clinical and animal experimental studies on the effect of subclinical hypothyroidism on fetal heart during pregnancy.Most of the literatures are related to gestational hypothyroidism.Moreover,previous studies have confirmed that BMP signal pathway,GATA family genes and NKX family genes are closely related to cardiac development.Bone morphogenetic protein(BMPs)plays a central role in spinal mesoderm induction and cardiac development.It is also a direct target of thyroid hormone.Thyroid hormone can promote cell differentiation by acting on BMP4.BMP ligands can bind to type?receptors,activate type I receptors,activate the Smads4 signal transduction pathway regulated by BMP receptors,and then induce the transcription of downstream genes.Transcription factor(Gata4)and transcription factor related site 5(Nkx2-5)with zinc finger structure have been proved to be downstream target genes of BMP signal transduction pathway.BMP4 can induce cardiac progenitor cells to express Nkx2-5 and Gata4.Therefore,the purpose of this study is to investigate the effect of early treatment of levothyroxine(LT₄)on the cardiac function of fetuses with subclinical hypothyroidism during pregnancy,and to further observe the effect of early treatment of levothyroxine on the expression of cardiac development-related proteins Gata4 and Nkx2-5 in the BMP4/Smad4 pathway related to cardiac development in offspring.To deeply understand the effect and mechanism of subclinical hypothyroidism on fetal heart during pregnancy,and provide basis for fetal monitoring of subclinical hypothyroidism during pregnancy.Methods:1.The clinical data of subclinical hypothyroidism during pregnancy delivered in the Department of Obstetrics and Gynecology of the second Hospital of Chinese PLA from June 2016 to July 2018 were analyzed retrospectively.A total of 115 pregnant women were eligible and regularly followed up during pregnancy with complete data.There were 40 cases of subclinical hypothyroidism(SCH+LT₄ group),32 cases of subclinical hypothyroidism group(SCH group)and 43 cases of normal pregnancy control group treated with early intervention of LT₄.LT₄ early intervention treatment subclinical hypothyroidism patients were all diagnosed as pregnancy subclinical hypothyroidism at6-7 weeks of pregnancy and began drug intervention treatment 8 weeks ago.The thyroid function indexes of all groups were reexamined every 4 weeks.The results of serum ion detection,blood glucose(fasting blood glucose),blood lipid,coagulation and other blood biochemical indexes of pregnant women in each group were compared at 32 weeks of pregnancy.The results of fetal echocardiography at 24 and 32 weeks of pregnancy were compared.The pregnancy and delivery outcomes of each group were compared,including gestational hypertension,gestational diabetes mellitus,amniotic fluid volume,birth weight and so on.2.The rat models of gestational hypothyroidism and subclinical hypothyroidism were established.Female Wistar rats were divided into sham operation group,hypothyroidism group,subclinical hypothyroidism group(SCH),LT₄ intervention(E10)group(LT₄treatment began on the 10th day of pregnancy),LT₄ intervention(E13)group(LT₄treatment began on the 13th day of pregnancy)and LT₄ intervention(E17)group(LT₄treatment on the 17th day of pregnancy).According to the heart time of fetal mice and offspring,each group was divided into 6 subgroups:E16(16th day of pregnancy),E18(18th day of pregnancy),P0(postpartum day),P5(postpartum day 5),P7(postpartum day 7)and P10(postpartum day 10).The levels of serum TSH and TT4 were measured on the day of delivery in each group,and the heart weight,the ratio of heart weight to body weight,the expression of metabolic enzymes and the histopathological changes of fetal and newborn rats at different time points were compared.In order to elucidate the effect of LT₄ on cardiac development in SCH pregnant rats,the expression of proteins related to BMP4/Smad4 signaling pathway was detected by immunohistochemistry,real-time quantitative polymerase chain reaction and Western blotting Western blotting.The expressions of myocardial development-related proteins GATA4 and Nkx2-5 were compared to explore the possible molecular mechanism of LT₄ regulating cardiac development in the offspring of SCH pregnant rats.Results:1.The level of basic serum TSH in SCH+LT₄ group was slightly higher than that in SCH group,but there was no significant difference between the two groups.The serum TSH level in the SCH+LT₄ group was significantly higher than that in the control group,and the serum TSH level decreased significantly after LT₄ treatment,which was the same as that in the control group from 24 weeks to the end of pregnancy,and was significantly lower than that in the SCH group at the same time.2.The incidences of gestational hypertension and gestational diabetes mellitus in SCH+LT₄ group were significantly lower than those in SCH group(P<0.05).SCH group was significantly higher than that in control group and SCH+LT₄ group,and the incidence of fetal distress in SCH+LT₄ group and SCH group was significantly higher than that in control group(all P<0.05).3.On the day of birth,the serum TSH level of SCH pregnant mice was significantly higher than that of sham operation group.The serum TSH level of SCH+LT₄ pregnant rats was significantly lower than that of SCH group,which was close to that of sham operation group.4.Compared with the heart of the offspring of the SCH+LT₄ group,the heart weight,the ratio of heart weight to body weight and the activities of succinate dehydrogenase(SDH),Na+/K+-ATP and Ca2+-ATP in the early LT₄ intervention group were higher than those in the SCH+LT₄ group.HE staining of cardiac tissue of offspring rats showed that the shrinkage,nuclear staining,hyperemia,congestion and vacuolar degeneration of cardiac myocytes in SCH+LT₄group were less than those in SCH+LT₄ group.5.The m RNA and protein expression of cardiac development-related Gata4 and Nkx2-5in the myocardium of the offspring of SCH+LT₄ pregnant rats was higher than that of the SCH group,and the earlier the intervention,the higher the expression level.6.The expression of BMP4/Smad₄ signaling pathway related protein in the myocardium of the offspring of SCH+LT₄pregnant rats was higher than that of the SCH group,and the earlier the intervention,the higher the expression level.Conclusion:1.Early treatment of LT₄ can maintain the level of serum TSH in patients with subclinical hypothyroidism during pregnancy,improve the incidence of gestational hypertension and gestational diabetes,and reduce the occurrence of fetal growth restriction,postpartum hemorrhage and abnormal amniotic fluid volume.2.It is clearly pointed out in the 2015 expert consensus on the Application of Fetal Heart Monitoring that fetal distress can sensitively reflect the changes of fetal cardiac function.In this study,the incidence of intrauterine fetal distress in SCH+LT₄ group and SCH group was higher than that in normal pregnancy group,indicating that abnormal levels of TSH in early pregnancy may affect fetal heart development,and subclinical hypothyroidism during pregnancy may affect fetal cardiac function.3.Early LT₄ intervention could significantly reduce serum TSH level,increase heart weight,heart weight/body weight ratio,succinate dehydrogenase(SDH),Na+/K+-ATP enzyme and Ca2+-ATP activity,but reduce myocardial cell shrinkage,nuclear staining,hyperemia/congestion and vacuolar degeneration.4.Early LT₄ intervention could significantly increase the m RNA and protein expression of cardiac development-related Gata4 and Nkx2-5 in the myocardium of SCH offspring rats.5.Early LT₄ intervention can increase the expression of proteins related to BMP₄/Smad₄signal pathway,regulate the heart development of the offspring of SCH pregnant rats,and improve the heart development of their offspring.