Effect Of Hypothyroidism On Hypothalamic-Pituitary-Ovarian Axis And Reproductive Function Of Pregnant Rats

Gonadotropin releasing hormone?GnRH?is an important mediator of cascade release oreproductive hormones on the hypothalamic-pituitary-gonadal axis.GnRH has direct or indirect connection with pregnancy recognition,embryo implantation,pregnancy maintenance and termination of pregnancy,and is the most important hypophysiotropic hormone on the hypothalamic-pituitary-gonadal axis.Clinical findings have confirmed that there is mutual accommodation between thyroxine and the hypothalamic-pituitary-gonadal axis,however the exact mechanism is still not clear.In the study,via the detection of changes in hormone levels on the hypothalamic-pituitary-gonadal axis in rats with hypothyroidism,differences in pregnancy rate and abortion rate of female adult rats was further observed;in addition,immunohistochemistry and real-time quantitative PCR were applied for the measurement of GnRH expression,and the distribution of GnRH receptor in hypothalamus,pituitary and ovary of rats on the hypothalamic-pituitary-gonadal axis,aimed at confirming whether GnRH is the central link of the hypothalamic-pituitary-gonadal axis and reproductive function under the regulation of thyroid hormones,so as to lay the foundation for the study of the interaction between thyroid hormones and gonadal hormones in the future.Materials and methodsEstablishment of hypothyroidism pregnant rats modelExperimental procedure: a total of 106 female SD rats were included and randomly divided into two groups: the normal-drinking-water group?n = 30?and the 0.05%propylthiouracil?PTU?-drinking-water group?n = 76?to induce the establishment of adult rats model with hypothyroidism?6 weeks?.Five rats were randomly selected from both groups to measure the level of thyroid hormone to determine whether the model of hypothyroidism was successfully established or not.After the success of modeling,another 5 rats in the normal-drinking-water group were randomly selected as the normal control group?NG,n = 5?,and another 5 rats in the PTU-drinking-water group were randomly picked as the hypothyroidism group?HG,n = 5?;the remaining 25 rats in the normal-drinking-water group were subdivided into the normal caged group?n = 25?,and the remaining 71 rats in the PTU-drinking-water group were subdivided into the hypothyroidism caged group?n = 71?,the female and male rats were mated by 2:1 for the establishment of abortion-prone model,and the pregnant rats were further divided into the normal pregnancy group?n = 10?and the hypothyroidism pregnancy group?n =28?.Nonpregnant rats were assigned to the normal control group and the hypothyroidism group,finally,there were a sum up of 43 rats and 25 rats in the hypothyroidism group and the normal control group,respectively.Specimens preparationAfter 12-hour fasting,rats were made intraperitoneal anesthesia,blood was taken from the abdominal aorta,and samples of brain tissues,pituitary tissues and ovarian tissues were harvested,one part of the samples were collected in the frozen sterile tube and stored in liquid nitrogen immediately,and subsequently stored at-80? refrigerator after cooling for the detection of real-time quantitative PCR.The other part of tissues were placed in 10% formaldehyde fixative,followed by gradient alcohol dehydration,xylene transparent and serial section.ImmunohistochemistryThe tissue blocks were sectioned at 3⁴ um thickness which used for histological study,tissue blocks were then dehydrated in ascending series of ethanol,cleared in xylene.Tissues were subsequently placed in a microwave oven and transferred at medium fire,followed by heating until it boils;after cooling for 15 min,tissues were then boiled and cooled,finally,the antigen retrieval was completed.And then,samples were treated by0.3% hydrogen peroxide solution for 30 min to block endogenous peroxidase,soon afterwards,tissues were blocked by 1% normal rabbit serum for 1h.Subsequently,the primary antibody rabbit anti-GnRH polyclonal antibody)/rabbit anti-TSHR polyclonal antibody and a broad-spectrum biotin labeled secondary antibody were added successively,and a streptavidin biotin peroxidase complex was further added;3,3'-diaminobenzidine?DAB?was used as selenium organic reagent,followed by mild redyeing using hematoxylin and neutral gum sealing piece.Primer design and reaction conditionsAccording to the sequence of Genbank,Primer Premier5.0 software was applied for primer design,the primer was synthesized by Dalian Ta Ka Ra company.Primer sequences were listed in the text.Extraction of RNA and the synthesis of mRNAAccording to the kit instructions,Trizol?Invitrogen Life Science & Technology Co.Ltd.?was applied for the extraction of RNA from tissues.RNA synthesis was performed using a reverse transcriptase Kit?Invitrogen Life Science & Technology Co.Ltd.?.Real-time PCRRNA extraction from tissues was conducted by using Trizol.According to kit instructions,primers and i Taq Universal SYBR Green master mix?Tli RNase H Plus??Dalian Biological Engineering Co.,Ltd.?were added,respectively,Applied Biosystems7500/7500 Fast Real-Time PCR System?Thermo Fisher,Singapore?was adopted for the measurement of mRNA levels.The relative expression of genes was calculated by 2^-??Ct.?-actin was used as the reference standard for the expression level of target gene,results were presented by RQ values.ELISA for the determination of serum T3,T4 and TSH levelsSerum levels of T3,T4 and TSH were detected using ELISA in accordance with the protocol of the kit?Shanghai Yuan Ye Biological Technology Co.,Ltd.?.Statistical analysisAll data were analyzed using statistical software SPSS 16.0,measurement data of normal distribution was expressed ???,and Quantitative data of skewed distribution was expressed as M?P25,P75?,comparison among groups was conducted by applying One-way analysis of variance?One-way ANOVA?accord with normal distribution.A comparison between more groups was conducted by applying the SNK-Q test method.the difference was statistically significant when P < 0.05.comparison among groups was conducted by applying Kruskal-Wallis test when all data didn't coincided with normal distribution.comparison among groups was conducted by the rectification method for Pvalue.A comparison between two or more groups ofthe qualitative datawas conducted by applying the Chi-Square test ofindependence.Results?1?GnRH mRNA RQ values of the hypothalamus among the four groups: the difference was not statistically significant by using One-way ANOVA?F=1.412,P=0.296?.?2?GnRH mRNA RQ values of the pituitary among the four groups: the difference was not statistically significant?F=1.153,P=0.368?.?3?GnRH mRNA RQ values of the ovary among the four groups: the difference was not statistically significant?X2=1.276,P=0.077?.?4?TSH mRNA RQ values of the hypothalamus among the four groups: the difference was not statistically significant by using One-way ANOVA?F=0.658,P=0.596?.?5?TSH mRNA RQ values of the pituitary among the four groups: the difference was not statistically significant by using One-way ANOVA?F=1.305,P=0.318?.?6?TSH mRNA RQ values of the ovary among the four groups: the difference was not statistically significant by using One-way ANOVA?F=0.281,P=0.838?.?7?The comparison of GnRH mRNA RQ values of the hypothalamus,pituitary and ovary in the normal control group,the difference was statistically significant?X²=7.372,P=0.025?;the difference was not statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the pituitary?Z=0.889,P=0.374?;the difference was statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the hypothalamus?Z=-2.666,P=0.008?;however,the difference was not statistically significant in the comparison of GnRH mRNA RQ values between the hypothalamus and the pituitary?Z=1.778,P=0.075?.?8?The comparison of GnRH mRNA RQ values of the hypothalamus,pituitary and ovary in the hypothyroidism group,the difference was statistically significant?standard c2=8.769,P=0.012?;the difference was not statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the pituitary?Z=1.177,P=0.239?;the difference was statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the hypothalamus?Z=2.942,P=0.003?;yet the difference was not statistically significant in the comparison of GnRH mRNA RQ values between the hypothalamus and the pituitary?Z=1.765,P=0.078?.?9?The comparison of GnRH mRNA RQ values of the hypothalamus,pituitary and ovary in the normal pregnancy group,the difference was statistically significant?standardc2=6.144,P=0.046?;there was statistical difference in the comparison of GnRH mRNA RQ values between the ovary and the pituitary?Z=2.204,P=0.028?;besides,the difference was statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the hypothalamus?Z=2.205,P=0.043?;but no statistical difference was found of GnRH mRNA RQ values between the hypothalamus and the pituitary?Z=0.329,P=0.742?.?10?The comparison of GnRH mRNA RQ values of the hypothalamus,pituitary and ovary in the hypothyroidism pregnancy group,the difference was statistically significant?standardc2=8.909,P=0.012?;there exist no statistical difference of GnRH mRNA RQ values between the ovary and the pituitary?Z=1.706,P=0.088?;the difference was statistically significant in the comparison of GnRH mRNA RQ values between the ovary and the hypothalamus?Z=2.961,P=0.003?;however,no statistical difference was observed of GnRH mRNA RQ values between the hypothalamus and the pituitary?Z=1.382,P=0.167?.?11?The comparison of TSH mRNA RQ values of the hypothalamus,pituitary and ovary in the normal control group,the difference was statistically significant?F=7.276,P=0.013?;the difference was not statistically significant regarding TSH mRNA RQ values between the ovary and the pituitary by using One-way ANOVA?P=1.000?;the difference was not statistically significant regarding TSH mRNA RQ values between the ovary and the hypothalamus by using One-way ANOVA?P=0.030?;furthermore,there was statistical difference regarding TSH mRNA RQ values between the pituitary and the hypothalamus by using One-way ANOVA?P=0.026?.?12?The comparison of TSH mRNA RQ values of the hypothalamus,pituitary and ovary in the hypothyroidism group,the difference was not statistically significant?X²=2.577,P=0.276?.?13?The comparison of TSH mRNA RQ values of the hypothalamus,pituitary and ovary in the normal pregnancy group,the difference was not statistically significant?Z=0.894,P=0.640?.?14?The comparison of TSH mRNA RQ values of the hypothalamus,pituitary and ovary in the hypothyroidism pregnancy group,no statistical difference was found?F=1.907,P=0.210?.?15?The expression of GnRHR in the hypothalamus: there was no statistical difference regarding ar nucleus among the four groups by using One-way ANOVA?F=0.373,P=0.774?;no statistical difference was observed regarding vmh nucleus among the four groups by using One-way ANOVA?F=1.346,P=0.291?;by using Kruskal-Wallis test,there was no statistical difference of ah nucleus among the four groups?standard X²=2.486,P=0.478?;by using One-way ANOVA,the difference was not statistically different regarding pa nucleus among the four groups?F=0.591,P=0.670?;there was no statistical difference regarding pmv nucleus among the four groups by using One-way ANOVA?F=0.517,P=0.676?;in the hypothyroidism pregnancy group,One-way ANOVA result indicated no statistical difference of the above five nuclei?F=0.440,P=0.779?;in the hypothyroidism group,no statistical difference of the five nuclei was found by adopting Kruskal-Wallis test?standard X²=0.717,P=0.949?;in the normal control group,there was no statistical difference of the five nuclei by applying One-way ANOVA?F=0.810,P=0.531?;and in the normal pregnancy group,there existed no statistical difference of the five nuclei by One-way ANOVA?F=0.432,P=0.782?.?16?The expression of GnRHR in the pituitary: through Kruskal-Wallis test,it was found that there was statistical difference of integral optical density among the four groups?P=0.0.37?;besides,there was no statistical difference in the comparison between the hypothyroidism pregnancy group and the hypothyroidism group by using Nemenyi test?standard X²=0.089,P=0.929?,but there was statistical difference between the hypothyroidism pregnancy group and normal control group?standard X²=1.809,P=0.071?,and a statistical difference was also found in the comparison between the hypothyroidism pregnancy group and the normal pregnancy group?standard X²=2.345,P=0.019?,yet no statistical difference was observed between the hypothyroidism group and the normal control group?standard X²=1.720,P=0.086?,meanwhile,there was statistical difference between the hypothyroidism group and the normal pregnancy group?standard X²=2.268,P=0.023?,and no statistical difference was detected between the normal control group and the normal pregnancy group?standard X²=0.779,P=0.436?.?17?The expression of GnRHR in the ovary: it was observed that there was no statistical difference among the four groups by using One-way ANOVA?F=0.544,P=0.655?.?18?The expression of TSHR in the hypothalamus: by applying One-way ANOVA,there existed no statistical difference of the ar nucleus among the four groups?F=0.753,P=0.536?;with respect to the vmh nucleus within the four groups,there was no statistical difference of the five brain nuclei?standard X²=5.795,P=0.122?;and for the ah nucleus in the four groups,no statistical difference of the five brain nuclei was found by Kruskal-Wallis test?standard X²=4.953,P=0.175?;as for the pa nucleus in the four groups,there was no statistical difference by using One-way ANOVA?F=0.972,P=0.430?;and for the pmv nucleus in the four groups,no statistical difference was observed by using One-way?F=1.100,P=0.378?.?19?The expression of TSHR in the pituitary and the ovary: by applying One-way ANOVA,there was no statistical difference among the four groups?F=1.721,P=0.240?;ovary TSHR?optical density?;and no statistical difference was observed among the four groups by One-way ANOVA?F=1.608,P=0.199?.?20?Comparison result between the normal caged group and the hypothyroidism caged group indicated no obvious statistical difference with regard to the pregnancy rate?P>0.05?;furthermore,there was statistical difference of the abortion rate between the hypothyroidism pregnancy group and the normal pregnancy group?P > 0.05?.Conclusion:1.GnRHR was detected to be expressed in tissues on the hypothalamic-pituitary-gonadal axis in pregnant rats,suggesting that GnRH was not only involved in the long feedback of the hormone induced by the ovary on pituitary gonadotropic hormone and hypothalamic GnRH secretion,as well as the regulation of the short feedback between the pituitary hormone?LH and FSH?and hypothalamic hormone,endogenous GnRH in hypothalamus,pituitary and ovary through autocrine and paracrine paths can be also involved in the maintenance of pregnancy in rats.2.Among different nuclei in the hypothalamus,there was no obvious difference in the distribution of the GnRHR,indicating that GnRH neurons in hypothalamus might not play a leading role in the maintenance of pregnancy in rats.3.Hypothyroidism might have an influence on the distribution of the pituitary GnRHR product,but revealed no evident effect on the distribution of the GnRH immunoreactive positive product in the nuclei of the ovary and the hypothalamus.4.There were statistical difference of the GnRH mRNA expression of the hypothalamic-pituitary-ovarian among the four groups.However,hypothyroidism indicated no significant influence in the GnRH mRNA expression of the hypothalamic-pituitary-ovarian among groups.5.No significant effect was found of hypothyroidism and pregnancy factors on TSH mRNA expression of the hypothalamic-pituitary-ovarian.Additionally,no obvious influence of hypothyroidism in the distribution of TSHR in the nuclei of the ovary and the hypothalamus.6.Hypothyroidism was suggested to have an adverse impact on the pregnancy in rats.Objective To explore effects of thyroid peroxidase antibody on pregnancy outcomes of euthyroid early pregnant women.Methods In this study,293 cases of TPOAb positive and negative euthyroid in early pregnant women were selected,which were divided into three groups:TPOAb-positive group,negative group and intervention group.We compared the incidence of obstetric complications of three groups,post-neonatal clinical characteristics.Results 293 cases euthyroid pregnant women were enrolled in this study.Thyroid disorders,maternity complications were significantly different?P<0.05?.between TPOAb postitive and negative group,however,neonatal clinical characteristics in newborn babies showed no significant differences?P>0.05?Between the intervention group and negative group of neonatal were no statistically significant.Conclusion TPOAb positive pregnant women have the trend to subclinical hypothyroidism.Maternity complications are significantly different between TPOAb positive and negative group,indicating that TPOAb positive have an adverse effect on pregnancy outcomes.