The Study On The Attention Deficit Hyperactivity Disorder In The Offspring Of Maternal Hypothyroxinemia During Pregnancy

IntroductionMaternal hypothyroxinemia(IMH)is defined as a low maternal free thyroxine(FT4)concentration in conjunction with a normal maternal thyroid-stimulating hormone(TSH)concentration during pregnancy.Thyroid hormones for fetal development is depended on maternal supply since the thyroid gland of the fetus begins to develop at 12 weeks gestation and has functions around 18 to 20 weeks gestation.Therefore,even mild decrease of thyroid hormone during the critical period of brain development will have adverse effects on brain development.In 2004,Vermiglio et al.found that the risk of offspring ADHD in women with hypothyroxinemiafrom iodine deficiency areas was increased.Population cohort studies also found that maternal hypothyroxinemia during early pregnancy could cause increasing risk of ADHD in offspring.ADHD is one of the most common neurodevelopmental disorders in children and adolescents.The global prevalence of ADHD in school-aged children and adolescents is about 5.29%,with a male to female ratio of about 3:1 or even higher.Studies have shown that thyroid hormones can affect the development of the monoamine neurotransmitter system.The monoamine neurotransmitters include serotonin(5-HT)and catecholamine(CA).Catecholamine include norepinephrine(NE),epinephrine(E)and dopamine(DA).The monoamine neurotransmitters generated in center nerves systems are stored in vesicles.While the action potential reaches to nerve endings,the neurotransmitters are discharged to synaptic gapin exocytosis way with the participation of Ca2+.After that,the monoamine neurotransmitters will reuptaked by the dopamine transporter(DAT)or degradated by monoamine oxidase enzymatic(MAO)rapidly.The formation of neurotransmitters and the development of synaptic structure are all regulated by thyroid hormones.The pathogenesis of ADHD is complex.Studies showed that the dopamine transporter(DAT)and dopamine receptor(DR)expressed in ADHD rats and mice were abnormal.SNAP-25 as a thyroid hormone response gene was also involved in the occurrence of ADHD.Nowadays,studies on the effect of hypothyroxinemia and offspring’s ADHD were limited.And it has not been conducted in animal experiments.The specific mechanism of ADHD is still in controversial.The objective of this study is to establish hypothyroxinemia in rats with normal iodine nutrition,and to observe whether hypothyroxinemia during pregnancy could lead to ADHD in offspring via varieties of animal behavior assessments.By evaluating the dopamine receptor 1-5(DRD1-5)and dopamine pathways at in different parts of brain,exploring the possible factors of maternal hypothyroxinemia rats on ADHD behavior in offspring rats.At the same time,the CPRS was conducted in women with hypothyroxinemia in early pregnancy in order to investigate whether their offspring have ADHD compared with control.To explore the influence of maternal hypothyroxinemia and offspring’s ADHD through clinical research and animal experiment.Methods First Part1.After one week of adaptive feeding,50 Wistar female rats aged 8 weeks were divided into three groups randomly.Normal control group(n=14),hypothyroxinemia group(n=18)and hypothyroidism group(n=18).The rats were fed with PTU water for 9 weeks and their thyroid function were examined.Female rats were mated with male rats after the thyroid function was verified.2.Hyperactivity was confirmed in offspring by delay reinforcement task,open field test and extinction task.In order to evaluate other disorders of offspring,as well as learning and memory ability and motor ability,elevated zero maze test,Morris water maze test,rota-rod test were determined.3.The m RNA expression of thyroid hormone responsive gene SNAP-25 and dopamine pathway in male rats brain tissues of the offspring were detected by RT-PCR.4.Western Blot was used to detect the expression of protein of dopamine receptor related pathways PI3K/AKT/GSK3β and c AMP/CREB/AKT.5.Immunofluorescence was used to detect the SNAP-25 expression in the brain of newly born offspring.Second Part1.From 2011 to 2014,women in early pregnancy lived in Shenyang and Dalian cities of Liaoning Province where availability was adequate were recruited in this study.The subjects should live locally for more than 5 years,aged between 19-40.Serum specimens were obtained in the early morning after an overnight fast.Serum TSH,FT4 and TPOAb levels were measured using electrochemiluminescence immunoassay(Cobas Elesys 601,Roche Diagnostics).Women with twin pregnancy,with personal or family history of inherited disease(hypertension,cardiac disease,and diabetes),or with history of other severe chronic diseases were excluded.Participants were divided into different groups according to the diagnostic criteria for pregnancy-specific thyroid function developed by our research group,and the general characteristics of the population were evaluated.2.Participants with hypothyroxinemia accepting L-T4 intervention began taking 50 μg of levothyroxine daily,the lower oral dose was intended to avoid overtreatment in pregnant women with IMH.The oral dose was adjusted by doctors according to their thyroid function during follow-up,to a maximum of 100 μg daily.The goal was to ensure that TSH and FT4 levels in the participants were within the normal pregnancyspecific reference range.3.Following up the participants while their children at about 5 years old.Parents filled in the Conners’ parent rating scale in order to assess the ADHD behaviors of their 5-year-old offspring.Compare the questionnaire’s score between groups.Results First Part1.Maternal hypothyroxinemia ratsAfter nine week’s PTU special water feeding.It turned out that compared with the control group,there was significant reduce in serum FT4 in hypothyroxinemia group(p<0.05),but no significant difference in serum TSH.There was significantly increased in serum TSH(p<0.05)and significantly decreased in serum FT4(p<0.05)in hypothyroidism group.2.Behavior tests(1)In delay reinforcement test,compared with the control group,large reward was significantly reduced in offspring of hypothyroxinemia group(p<0.05),which indicated that the male offspring have impulsive behavior.Compared with the control group,large reward was significantly reduced in offspring of hypothydism group(p<0.05),which proved that the male offspring have impulsive behavior.(2)On the first day of extinction task,hypothyroxinemia group and hypothyroidism group rats choose to touch more immediate rewards(response rate)(p<0.01),compared with the control group.But the total number of screen touches(response frequency)didn’t have statistical difference.There was no significant reduction in the immediate reward on the second day of EXT in the male offspring of the hypothyroxinemia group and hypothyroidism group,and there was no statistical difference in the total number of touch screen.(3)In the open field test,grooming duration in male offspring of hypothyroxinemia group was significantly increased,indicted that the offspring had hyperactivity.There was no difference in grooming duration between control group and hypothyroidism group.(4)In Morris water maze,the escape latency time of hypothyroxinemia group and hypothyroidism group was significant longer than control group(p<0.05).There was no difference between control group and hypothyroxinemia or hypothyroidism group in distance and mean velocity.In space exploration experiment,there was no significant difference in the crossing frequency of platform quadrant in male offspring of hypothyroxinemia group(p>0.05)compared with control group,but there was significantly decrease in hypothyroidism group(p<0.05).(5)In rota-rod test,compared with the control group,there was no significant difference in male offspring of hypothyroxinemia group in the average falling time and average falling speed(p>0.05).But the average falling time and average falling speed of hypothyroidism group were significantly reduced(p<0.05)compared with the control group.The male offspring of hypothyroidism group had motor dysfunction.(6)In elevated O-maze test,there was no significant difference in open arm duration between hypothyroxinemia group and control group(p>0.05),no significant difference between hypothyroidism group and control group(p>0.05).There was no anxiety behavior in male offspring of hypothyroidism and hypothyroxinemia group.3.Comparison of body weight,brain weight,cerebellum weight and brain/body weight in P120 male offspringThere was no difference of brain weight,body weight,cerebellum weight and brain/body weight between the hypothyroxinemia group and control group(p>0.05),no difference between the hypothyroidism group and control group(p>0.05).4.RT-PCR results of P120 male offspring(1)Compared with the control group,the expression of SNAP-25 m RNA in male offspring’s hippocampus was significantly increased in hypothyroxinemia group(p<0.05),significantly increased in hypothyroidism group(p<0.05).Synaptic vesicle release was abnormal in hypothyroxinemia group and hypothyroidism group.(2)Compared with the control group,the expression of DRD1,DRD5 m RNA in male offspring’s prefrontal cortex of hypothyroxinemia group was significantly decreased(p<0.05),the expression of DRD1,DRD5 m RNA in hypothyroidism group was significantly decreased(p<0.05).(3)Compared with the control group,the expression of SNAP-25 m RNA in male offspring’s cortex(except prefrontal lobe)was significantly decreased in hypothyroxinemia and hypothyroidism group(p<0.05),the expression of MAOA、MAOB m RNA in male offspring’s cortex(except prefrontal lobe)was significantly decreased in hypothyroidism group(p<0.05).(4)Compared with the control group,the expression of MAOA、MAOB m RNA in male offspring striatum was significantly decreased in hypothyroxinemia group(p<0.05).There was no difference expression of MAOA、MAOB m RNA between control group and hypothyroidism group(p>0.05).(5)Compared with the control group,the expression of DRD1 m RNA in male offspring’s cerebellum was significantly decreased in hypothyroxinemia group(p<0.05).There was no significant difference between control group and hypothyroidism group(p>0.05).5.Western Blot results of P120 male offspring(1)Western Blot indicated that the SNAP-25 protein level in hippocampus was significantly increased in hypothyroxinemia group compared with control group(p<0.05).(2)Compared with the control group,the expression of PI3 K protein in the prefrontal cortex of offspring in hypothyroxinemia group was significantly increased(p<0.05),but there was no significant difference in other downstream indicators(p>0.05).There was no significant difference in PI3 K between hypothyroidism group and control(p>0.05).Compared with the control group,the expression of CREB protein in the prefrontal cortex of offspring in hypothyroidism group was significantly increased(p<0.05),but there was no significant difference between control and hypothyroxinemia group(p>0.05).6.Immunofluorescence resultsCompared with the control group,the intensity of SNAP-25 in hippocampus of hypothyroxinemia group and hypothyroidism group was increased,the intensity of SNAP-25 in cortex in hypothyroxinemia group was decreased.Second parts1.A total of 57 participants involved in control group,a total of 57 cases involved in hypothyroxinemia group,a total of 32 cases involved in LT4 intervention group who had filled in the questionnaire.Compared with the control group,the BMI,FT4,TSH in hypothyroxinemia group and LT4 intervention group was significantly increased(p<0.05).There were no significant difference in other general information between control group and hypothyroxinemia or LT4 intervention group(p>0.05).2.Compared with the control group,the learning problems score and the hyperactivity index of the hypothyroxinemia group childrenwere significantly increased(p<0.05).Compared with the control group,the hyperactivity-impulsivity score of the boys in hypothyroxinemia group were significantly increased(p<0.05).There was no statistical difference in the score of the offspring girls between control group and hypothyroxinemia group(p>0.05).3.Compared with the control group,LT4 intervention could improve the offspring boys’ hyperactivity-impulsivity score(p<0.05),but could not improve the offspring overall learning disability and hyperactivity index(p>0.05),and could increased the offspring overall psychosomatic disorder index score(p<0.05).4.The correlation analysis showed that hyperactivity index was negatively correlated with maternal FT4 in early pregnancy(r=-0.236,p<0.05),positively correlated with maternal TSH in early pregnancy(r=0.191,p<0.05),positively correlated with anxiety index in early pregnancy(r=0.187,p<0.05),and positively correlated with maternal BMI in early pregnancy(r=0.244,p<0.05).Conclusion1.Attention deficit hyperactivity behavior were observed in the male offspring of maternal hypothyroxinemia rats.2.The ADHD behaviors of maternal hypothyroxinemia offspring may be related to the disfunction of synaptic vesicles release in hippocampal neurons,disfunction of dopamine receptors pathway.3.Maternal hypothyroxinemia women’s 5-year-old offspring showed learning difficulties and increased hyperactivity index compared with controls.Boys occurred to have higher hyperactivity-impulsivity score compared with controls,while girls showed no difference compared with controls.4.Compared with the control group,LT4 intervention could improve the offspring boys’ hyperactivity-impulsivity score,but could not improve the offspring overall learning disability and hyperactivity index,and could increased the offspring overall psychosomatic disorder index score.