puberty is a complex and coordinated biologic process of sexual development that lead to complete gonadal maturation and function , and attainment of reproductive capacity . the activation of pulsatile gonadotropin - releasing hormone ( gnrh ) secretion from specialized hypothalamic neurons to stimulate hormonal cascades and gonadal activation is a key event in the onset of puberty ( 1 ) . but , the ultimate mechanisms underlying the increase in pulsatile gnrh seceretion at puberty are yet to be fully elucidated ( 2 ) . the kisspeptin , the peptide product of kiss1 gene and its putative receptor g protein coupled receptor-54 ( gpr54 ) signaling complex has recently emerged as essential gatekeeper of pubertal activation of gnrh neurons and the reproductive axis , and it has been the focus of intense study by investigators ( 3 - 7 ) . an increase in kisspeptin signaling which is caused by enhanced expression of kiss1 and gpr54 at the time of puberty contributes to , or even drives , the activation of the gonadotropic axis during pubertal development ( 8) . kisspeptin administration to immature animals was able to induce precocious activation of the gonadotropic axis and pubertal development ( 9 ) . precocious puberty is defined as the development of secondary sexual characteristics before the age of 8 yr for girls and 9 yr for boys ( 10 ) . central precocious puberty ( cpp ) is defined if the process is driven by premature activation of hypothalamic gnrh secretion . early diagnosis and treatment of cpp is important to improve final height because cpp can cause early epiphyseal maturation with compromised final height as well as psychological stress ( 11 ) . so far , gnrh stimulation test is considered the definite biochemical parameter for diagnosis of cpp . we assumed that serum kisspeptin level may be higher in girls with cpp than in prepubertal girls because hypothalamic kisspeptin signaling increases in the onset of puberty . up to date , no data on serum kisspeptin levels in korean girls with cpp are available . this study was aimed to determine whether serum kisspeptin may function as a marker for cpp by investigating serum kisspeptin levels in korean girls with cpp and healthy prepubertal controls . the subjects consisted of two groups : korean girls with cpp and healthy prepubertal controls . subjects with cpp ( n = 30 ) were recruited from visitors to pediatric endocrinology clinic at korea university ansan hospital . they were diagnosed according to the same criteria : 1 ) the patients all should show tanner breast stage scores  2 based on standardized tanner breast stage assessment before the age of 8 yr , 2 ) bone age assessed by a single observer according to greulich and pyle method should be found to be at least 1 yr ahead of their chronological age , 3 ) significantly higher peak luteinizing hormone ( lh ) values comparing with the cut - off value of 5 iu / l under the gnrh stimulation test conducted prior to the age of 9 yr . to exclude other forms of precocious puberty , further laboratory and imaging studies cpp patients with identified etiology , such as brain tumor or cranial irradiation , were excluded . the control group ( n = 30 ) consisted of age - matched healthy prepubertal girls . they were recruited from prepubertal girls who visited the outpatient clinic of department of pediatrics , korea university ansan hospital . body mass index ( bmi ) was calculated as the weight in kilograms divided by the square of height in meters . the pubertal stage of each subject was determined by one pediatric endocrinologist and rated according to the tanner criteria ( 15 ) . blood was collected into a lavender vacutainer tube containing edta at 4 ( 2.5 ml of blood for each subject ) . the peptide was extracted using sep - column ( strata , phenomenex , torrance , ca , usa ) containing 200 mg of c18 . the concentration of kisspeptin was measured using kisspeptin-10 ( metastin [ 45 - 54]-amide ) and competitive enzyme immunoassay ( kit # ek-048 - 56 , phoenix pharmaceuticals , inc . , fifty - five microliters of plasma with 25 l of primary antibody and 25 l of biotinylated peptide were incubated at room temperature for 2 hr . then after incubation for 1 hr at room temperature , 0.1 ml of chemical name tmb substrate solution was added . one hour later for incubation at room temperature , the reaction was stopped with 0.1 ml of 2n hcl . after loading the immunoplate onto a microtiter plate reader , we read the absorbance at 450 nm . all measurements were in duplicates and average was calculated . gnrh ( 0.1 mg ) was administered intravenously for 1 min and post - stimulation samples were taken at 30 , 60 , and 90 min after injection for measurements of lh and follicle - stimulating hormone ( fsh ) levels . the spss 16.0 software package ( spss inc . independent t - test and pearson 's correlation test were used for data analysis and p values of < 0.05 were considered statistically significant . this study was approved by the institutional review board of korea university ansan hospital in gyeonggi - do , korea ( irb number as10036 ) . written informed consents were obtained from all subjects and their parents . this study was approved by the institutional review board of korea university ansan hospital in gyeonggi - do , korea ( irb number as10036 ) . written informed consents were obtained from all subjects and their parents . serum kisspeptin , clinical characteristics and the results of gnrh stimulation test of cpp and control groups are shown in table 1 . serum kisspeptin levels were significantly higher in cpp group than in control group ( 4.61  1.78 vs 2.15  1.52 pm / l , p < 0.001 ) . cpp group also had significantly higher bone age , bone age advancement , peak lh , peak / basal lh ratio , basal fsh , basal and peak fsh , peak / basal fsh ratio and peak lh / fsh ratio compared with control group . there were no significant differences in age , height , weight , bmi , basal lh and basal lh / fsh ratio between cpp and control groups . the correlations between serum kisspeptin and other clinical characteristics and the results of gnrh stimulation test are shown in table 2 . serum kisspeptin was positively correlated with bone age , bone age advancement , peak lh , peak / basal lh ratio and peak lh / fsh ratio . no correlations were found between kisspeptin and age , height , weight , bmi , basal lh , basal and peak fsh , peak / basal fsh ratio and basal lh / fsh ratio . to the best of our knowledge , this is the first study on serum kisspeptin levels in korean girls with cpp or in healthy prepubertal girls . in this study , we demonstrated that serum kisspeptin level was significantly higher in girls with cpp than in their age - matched prepubertal controls . this was in accordance with the first report on the serum kisspeptin level in prepubertal girls and in girls with cpp in 2009 ( 16 ) . we also found that serum kisspeptin was positively correlated with peak lh , peak / basal lh ratio and peak lh / fsh ratio during gnrh stimulation test . these findings suggest that serum kisspeptin level is associated with lh increase during gnrh stimulation test . accordingly , serum kisspeptin may be used as an adjunctive marker for cpp because the diagnosis of cpp is based on the peak lh level during gnrh stimulation test . moreover , it is suggested that cpp is triggered by premature increase of kisspeptin and subsequent activation of hypothalamic - pituitary - gonadal axis . the indispensable role of kisspetin and gpr54 in the control of puberty is now well established . kiss1 gene was first discovered in 1996 , and has been mapped to the long arm of chromosome 1q32 . this gene consists of three exons , of which only part of the second and third exons are finally translated into a 145-amino acid precursor peptide ( 17 ) . is then cleaved into three forms of kisspeptins containing 54 , 14 , and 13 amino acids . the three peptides exhibit the same affinity for the receptor since they share a common c - terminal decapeptide designated kisspeptin-10 ( 4 , 7 ) . kisspeptin is extraordinary potent elicitor of lh and fsh release , either after intracerebral or systemic administration of the peptide ( 8 , 18 - 20 ) . the sensitivity of lh release to the stimulatory effect of kisspeptin is very high ( 20 , 21 ) . notably , fsh release in vivo appeared to be approximately 100-fold less sensitive to the stimulatory effect of kisspeptin than lh ( 19 ) . our findings that serum kisspeptin level is associated with lh increase , but not with fsh increase during gnrh stimulation test are in concordance with these results . gonadotropin - releasing effect of kisspeptin comes from the ability to activate gnrh release at the hypothalamus . kisspeptin - induced membrane excitability on gnrh neurons was increased after puberty in mice ( 22 ) . activating gpr54 by kisspeptin triggers a phospholipase c - inositol triphosphate receptor - calcium cascade to initiate depolarization in gnrh neurons ( 23 ) . more than 75% of gnrh neuron in the rat hypothalamus co - express gpr54 mrna and central administration of kisspeptin induced c - fos expression ( as early marker of activation ) in more than 85% of gnrh neurons ( 24 ) . therefore , it has been strongly suggested that kisspeptin directly activates hypothalamic gnrh neurons , causing stimulation of gnrh secretion , which in turn elicits lh and fsh release from the pituitary ( 25 ) . notwithstanding the reports that systemic administration of kisspeptin induced lh and fsh release in animal studies ( 8 , 26 ) , it is not certain whether serum kisspeptin has an effect on the direct activation on hypothalamic gnrh neuron at the onset of puberty along with hypothalamic kisspeptin . expression of kiss1 gene was demonstrated not only in brain but also in placenta , testis and small intestine ( 27 , 28 ) . thus , it is assumed that there may be upstream regulatory factors of kisspeptin , which augment kiss1 gene expression not only in hypothalamus but also in other tissues at onset of puberty and consecutively serum kisspeptin level is increased . further studies on kiss1 gene polymorphisms leading to higher risk of premature increase of kisspeptin and on upstream regulator of kisspeptin are also needed . in conclusion , serum kisspeptin level is higher in korean girls with cpp than in age - matched prepubertal controls and cpp is supposed to be triggered by premature increase of kisspeptin . serum kisspeptin levels in korean girls with central precocious puberty young jun rhie , kee hyoung lee , so hee eun , byung min choi , hyun wook chae , ah reum kwon , woo jung lee , junghyun kim and ho - seong kim kisspeptin and g - protein coupled receptor-54 system is the essential gatekeeper of the reproductive system , playing a key role in the activation of the gonadotropic axis at puberty . serum kisspeptin levels were significantly higher in cpp group than in control group and positively correlated with peak luteinizing hormone ( lh ) , peak / basal lh ratio and peak lh / follicular - stimulating hormone ( fsh ) ratio during gnrh stimulation test .
central precocious puberty ( cpp ) is caused by premature activation of hypothalamic gonadotropin - releasing hormone ( gnrh ) secretion . 
 kisspeptin and g - protein coupled receptor-54 system is the essential gatekeeper of the reproductive system , playing a key role in the activation of the gonadotropic axis at puberty . 
 we aimed to determine whether serum kisspeptin may function as a marker for cpp by investigating serum kisspeptin levels in korean girls with cpp and their prepubertal controls . 
 serum kisspeptin levels of korean girls with cpp ( n = 30 ) and age - matched healthy prepubertal controls ( n = 30 ) were measured with a competitive enzyme immunoassay . 
 serum kisspeptin levels were significantly higher in cpp group than in control group ( 4.61  1.78 vs 2.15  1.52 pm / l , p < 0.001 ) . 
 serum kisspeptin was positively correlated with peak luteinizing hormone ( lh ) , peak / basal lh ratio and peak lh / follicular - stimulating hormone ( fsh ) ratio during gnrh stimulation test . 
 cpp is supposed to be triggered by premature increase of kisspeptin . 
 serum kisspeptin may be used as a marker of cpp . 
 further studies on kiss1 gene polymorphisms leading to higher risk of premature increase of kisspeptin and upstream regulator of kisspeptin are also needed .