The Experimental Study Of Maternal Subclinical Hypothyroidism Caused Miscarriage In Early Pregnancy

Objective:Subclinical hypothyroidism is the most common pregnancy related thyroid disease that affects 3²7.8%of pregnant women.The clinical study showed that pregnant women with clinical hypothyroidism is associated with an increased risk of spontaneous abortion?Although subclinical pregnancy pregnancy leads to lower complication rate than clinical hypothyroidism,but it is still a risk of spontaneous abortion.Spontaneous abortion is one of the common complications of pregnancy,the incidence rate is10¹5%.With the increased prevalence of pregnant women with subclinical hypothyroidism,Its role in abortion has attracted more and more attention.But the mechanisms of maternal subclinical hypothyroidism in early pregnancy caused abortion are still unclear.The uterus is regulated by hormones periodically.Expression of thyroid hormone receptor?TR?,TSH receptor?TSHR?and DIO has been reported in human endometrium.The endometrium is likely to be a new site for the synthesis of thyroid hormones and can convert T4 into T3,which acts on the TR.It is a complex and delicate process to produce a healthy fetus.Successful implantation is a critical event for establishment of an ongoing pregnancy.After successful implantation,embryo sac develops into a fetus,otherwise it will lead to abortion.Embryo implantation is mediated by the combined actions of the ovarian hormones E2 and P4 on the uterus.The actions of the two steroid hormones on the endometrium are largely mediated by the autocrine and paracrine effects of locally produced cytokines and growth factors.ALeukaemia inhibitory factor?LIF?is a member of the IL-6 family,a cytokine with a wide range of biological functions.In mammals LIF has been proved to be an important regulator in the blastocyst implantation process,it is a sign of uterine receptivity.LIF,produced in the GE,is secreted into the uterine lumen where it binds the heterodimeric LIF receptor complex,comprised of LIF receptor?LIFR?and the glycoprotein 130?gp130?that are localized to the apical membrane of the LE.Soluble forms of gp130 may also be generated either by proteolytic cleavage or by alternative splicing.The stabilized LIF-LIFR-gp130 complex activates the Janus kinase 2?JAK1?that phosphorylates the cytoplasmic domains of the receptors.Phosphorylation of the cytoplasmic domains results in the recruitment and phosphorylation of the latent signal transducer and activator of transcription 3?STAT3?by JAK1.Whether subclinical pregnancy affects the blastocyst implantation,and whether by regulating LIF/STAT3 signaling pathway is not clear.In this study we use the model of subclinical hypothyroidism in pregnant rats and observe its effect on pregnancy outcome,and investigate the mechanism of the effects of early pregnancy maternal subclinical hypothyroidism on abortion in the uterus of LIF/STAT3 signaling pathway in the molecular level.Methods:150 female Wistar rats were randomly assigned to three groups:?1?Control group?CON?:sham thyroidectomy with subcutaneous injection of 0.9%sodium chloride solution.?2?Subclinical hypothyroidism group?SCH?:subjected to thyroidectomy,removal of the thyroid and subcutaneous everyday injection of L-T₄ last 9 days after operation.?3?Clinical hypothyroidism group?OH?:completely removal of the thyroid and subcutaneous everyday injection of 0.9%sodium chloride solution last 4 weeks after operation.When each group successful,male and female rats were mated?2:1?.The next day given female rats vaginal smear which found sperm through microscope determined pregnancy,denoted E0.We collect tissues on E0,E5,E13 days separately.We measured weight of rat uterus on the day E0 and E5,the weight of rat placenta and embryo on the day E13.We also calculated the implantation sides and the embryo absorption rates.We used immunohistochemical staining to detect the protein expression of TSHR?TR-??TR-??ER-??p-STAT3 and STAT3 in three groups.We also detected the mRNA expression of TSHR?TR-??TR-??LIFR?gp130 and JAK1 in three groups by Real-time PCR.We examined the protein levels of ER-??LIFR?gp130?JAK1 and p-STAT3 by western blot.We examined the mRNA and protein expression of ER-?after the incubation with TSH of 0?1?10 and 100 mIU/ml for 24 hours in Ishikawa cell line.We examined the mRNA and protein expression of ER-?after the incubation with TSH of 10 mIU/ml for 0?6?24?and 48 hours in Ishikawa cell line.We also protein expression of LIFR?gp130?p-STAT3 and STAT3.Results:1.After surgery 4 weeks,The TSH levels in OH and SCH groups were significant higher than the control group,while TT4 levels were lower than controls.When drug injected for 9 days,TT4 levels were up to normal,while TSH levels also remain higher than control groups.We also found increased TSH levels and decreased TT4 levels in OH group.2.In E0 and E5,the relative weight of rat uterus was highly decreased in SCH and OH group,and OH group decrease more significantly.3.In E13,the relative weight of rat placenta and embryo were highly decreased in SCH and OH group,and OH group decrease more significantly.4.In E5,the implantation sites of SCH and OH group were significant lower than the controls,and OH group decrease more significantly.The embryo absorption rates were highly increased in SCH and OH group increased more significantly.5.We used immunohistochemical staining to detect the protein expression of TSHR?TR-?and TR-?in three groups,the results showed in E0 and E5,the OD levels of TSHR in SCH and OH groups were significant higher than the controls,while TR-?and TR-?remained no differences between these three groups.6.We also detected the mRNA expression of TSHR?TR-?and TR-?in three groups by Real-time PCR,the results showed in E0 and E5,the mRNA levels of TSHR in SCH and OH groups were significant higher than the controls and OH group increased more significantly,while TR-?and TR-?remained no differences between these three groups.7.We examined the protein levels of ER-??LIFR?gp130?JAK1 and p-STAT3 by western blot.The results showed that in E0 the protein levels of TSHR in SCH and OH groups were significant higher than the controls and these five proteins showed no differences between three groups in E5,from E0 to E5,these five protein showed increased tendency in control groups,while no differences were found in SCH and OH group.8.We detected the mRNA levels of LIFR?gp130 and JAK1 by Real-time PCR.The results showed that in E0 the mRNA levels of these three signal target in SCH and OH groups were significant higher than the controls and three indicators showed no differences between three groups in E5,from E0 to E5,these three indicators showed increased tendency in control groups,while no differences were found in SCH and OH group.9.We also detected the protein levels of ER-??p-STAT3 and STAT3 by immunohistochemical staining.The results showed that in E0 the OD levels of three indicators in SCH and OH groups were significant higher than the controls,while p-STAT3 and STAT3 showed no differences between three groups in E5,from E0 to E5,the OD levels of ER-?and p-STAT3 showed increased tendency in control groups,while no differences were found in SCH and OH group.STAT3 expression showed no differences between three groups in E0 and E5.10.The mRNA and protein expression of ER-?remained no differences by the incubation with TSH of 0?1?10 and 100 mIU/ml for 24 hours in Ishikawa cell line.The protein expression of LIFR?gp130 and p-STAT3 showed increased tendency,while STAT3 levels remains no differences.11.The mRNA and protein expression of ER-?remained no differences by the incubation with TSH of 10 mIU/ml for 0?6?24?and 48 hours in Ishikawa cell line.The protein expression of LIFR?gp130 and p-STAT3 showed increased tendency,while STAT3 levels remains no differences.Conclusion:1.Give thyroidectomy,subcutaneous daily injection of L-T4 after surgery establishing SCH animal model is feasible and repeatable.2.Clinical hypothyrodism and subclinical hypothyroidism in early pregnancy might cause adverse pregnancy outcomes,clinical hypothyroidism have more serious effect on pregnancy outcome.3.TSHR was upregulated in the SCH and OH groups,but there is no any upregulated tendency in TR-?and TR-?in SCH and OH groups.4.High level of TSH up-regulated expression of ER-?and cause abnormal refection from uterine to estrogen,then affected LIF/STAT3signaling pathway,made the un-synchronicity of endometrium and blastocyst development,lead to failure embryo eventually.This mechanism may participate adverse pregnancy outcomes of clinical thyroid dysfunction and subclinical thyroid dysfunction.