The Clinical And Molecular Studies On Girls With Central Precocious Puberty

Objectives:Central precocious puberty（CPP）is caused by the premature reactivation of the hypothalamic-pituitary-gonadal axis（HPGA）and is usually defined by the development of secondary sexual characteristics before the age of 8 in girls and 9 in boys.As a common disease in the pediatric endocrinology,CPP occurs to girls more often,it even has the significant impacts on girls’early menstruation,the short stature and health life in adults.The precise treatment on CPP has always been a hot topic in the field.Gonadotropin-releasing hormone analogues（GnRHa）are the standard treatment for CPP,but it’s not applicable to all children.It is necessary to identify the rapidly progressive central precocious puberty（RP-CPP）,for it can effectively avoid excessive treatments or the delayed diagnosis.However,there is so far no consensus on the identified criteria on the RP-CPP all over the world.The molecular pathology of CPP is also a challenging topic in the research field.Recently mutations in the maternally imprinted makorin RING finger protein 3（MKRN3）gene have been identified as genetic causes of familial CPP,but the exact functions of MKRN3 associated with CPP is still not well understood.So,we aim to research for the following aspects.Firstly,to study the early diagnostic predictors and key following parameters on girls with RP-CPP by the prospective observation of the changes on CPP girls.Secondly,to compare the different clinical efficacy of the Western and Chinese medicine in order to get the best choice for therapy.Finally,verifying the model of CPP in C57 female mice induced by MKRN3 gene mutation to provide further scientific basis for the clinical diagnosis and treatment of CPP.Subjects and methods:Expriment 1:A clinical study of early predictors in the diagnosis and follow-up parameters in girls with RP-CPPAll the girls with CPP participated in a prospective,nonrandomized,multi-center,nested case control study at School of Medicine,Shanghai Jiao Tong University.After follow-up six months without any therapy,a total of 114 girls were divided into RP-CPP（n=70）and slowly progressive CPP（SP-CPP）（n=44）groups,in order to compare the differences of the two without intervention.Expriment 2:A Study on the effects of the traditional Chinese Medicine for the treatment of CPP764 girls with CPP or early puberty（EP）involved in the study all coming from School of Medicine,Shanghai Jiao Tong University,actually 474 cases were included in the study and 290 cases were subtracted.65 girls diagnosed as precocious thelarche（PT）were divided into Chinese medicine treatment（n=37）and control（n=28）groups.409girls diagnosed with CPP or EP were divided into GnRHa（n=202）,Chinese medicine treatment（n=155）and control（n=52）groups,to compare the clinical parameters’changes and differences on therapies among these groups.Expriment 3:Study the phenotype of the model with MKRN3 gene knockoutFrom 15 days after birth,the physical development of mutant mices(MKRN3^+/-)（n=8）and wild mices(MKRN3^+/+)（n=5）was observed,and the vaginal opening time,the morphology of uterine and ovarian,the serum LH,FSH,E2 and the expression of the GnRH mRNA in hypothalamic were also quantified.Results:Expriment 1:The basal serum LH and IGF-1SDS are the important risk factors of RP-CPP with the OR value of 4.04 and 1.578 separately,especially the former.The level of basal serum LH and IGF-1 SDS are at 0.52 mIU/m L and 0.35 respectively for the accuracy diagnosis of RP-CPP with the maximum Youden indexs,and the areas under the ROC curve（AUC）are 0.83,0.807 separately.The change levels of height,breast stages,BA/CA ratio,serum LH,uterine and ovarian volume in the RP-CPP group are significantly higher than in the SP-CPP group after following six months,while the changes of PAH are lower（P<0.05）.Expriment 2:There was no significant difference between the Chinese medicine treatment group and the control group in PT girls at the baseline and the changes of following parameters after three months’therapy.Also,after six months’therapy,the baseline and the changes of follow-up parameters in CPP or EP girls were no different between the Chinese medicine treatment group and the control group（P>0.05）.The changes of follow-up parameters in GnRHa treatment group were significantly lower than in the Chinese medicine treatment group on CPP or EP girls for six months’therapy（P>0.05）,except the changes of BMISDS、IGFBP3SDS.Expriment 3:The MKRN3^+/-female mice has the poor physical development and the higher mortality compared to the MKRN3^+/+female mice,while the average vaginal opening time was no significant difference（37.33±3.28 days vs 39±4.08 days,P>0.05）.The levels of serum LH,FSH and E2 were generally higher in MKRN3^+/-female mice than in MKRN3^+/+.And the time to serum LH peak time in MKRN3^+/-mice was significantly earlier than in MKRN3^+/+（27 days vs 30 days）.The hematoxylin-eosin staining of uterus and ovaries showed that there was no significant difference in the morphology of uterus between the two groups.Primary,secondary and mature follicles were all found in ovarian tissues.The number follicles of ovaries was no significant difference in the two groups.The expression of GnRH mRNA in hypothalamus was significantly higher in MKRN3^+/-mice（11.83±0.38 vs 1.92±0.71,P<0.05）.Conclusions:Expriment 1:The level of basal serum LH and IGF-1 SDS can be seen as the risk predictors for early diagnosis in girls with RP-CPP.The change levels of height,breast stages,BA/CA ratio,PAH,serum LH,uterine and ovarian volume seem to be the key follow-up parameters for the differentiation between RP-and SP-CPP.Expriment 2:There was no significant effect of traditional Chinese medicine Zhi bai Di huang pills in the treatment of PT girls,even combined with Da bu yin pills in the treatment of CPP or EP girls.It may be related to the degree and the progress of the diseases and need to be further verified.The effects of GnRHa treatment in CPP or EP girls was further confirmed.Expriment 3:The deficiency of MKRN3 may affect the nutritional status and viability of female mices.It was suggested that MKRN3 knockout could activate the neuroendocrine pathway of the HPGA.But it still lack the reliable evidence,and more samples are needed to confirm the MKRN3 gene mutation leading to CPP.