| Background: In recent years,there is more and more abnormal sexual development in children.The specialists of child health and endocrine,and all sectors of society have begun to pay attention to abnormal sexual development in children.With the development of the techniques of gonadotropin,the enhancement of sensitivity,specificity and detection efficiency,immunochemiluminometric assays(the third generation technology of gonadotropin detection)is widely used in clinic laboratory.The value of urine gonadotropin(luteinizing hormone and follicle-stimulating hormone)assayed by immunochemiluminometric assays in clinical application is elevated in hypothalamus-hypophysis-gonad axis of children with disorders of sexual development.Compared with serum gonadotropin which secreted in impulse type,urine gonadotropin can reflect the average level of serum gonadotropin in body at a certain time.Compared with invasive method of repeating blood collection,urine collection is non-invasive,which can help the compliance of children and their parents.However,there is seldom research on the comparison of urine gonadotropin determined by immunochemiluminometric assays between the activated and the unactivated hypothalamic-pituitary-gonadal axis in children,and finding new methods about urine gonadotropin assayed by immunochemiluminometric assays in the clinic lab,and a pilot study about the normal reference ranges of urine gonadotropin in crowd,and to check its feasibility.Part 1 The Analysis of Urine Gonadotropin in Children with the Activated of Hypothalamic-Pituitary-Gonadal Axis Objective: To analyse urine gonadotropin in children with the activated of hypothalamic-pituitary-gonadal axis(HPGA).Methods: From August,2009 to August,2016,among the children hospitalized in the pediatric endocrine ward,there were 50 girls were central precocious puberty(CPP),and 56 children(38 boys and 18 girls)were the activated of HPGA.They were diagnosed by gonadotropin releasing hormone analogue(triptorelin)stimulation test.Timed 12 h nocturnal(20:30~next day 08:30)urine samples before the day of injecting triptorelin were collected.And the blood samples in the morning before the triptorelin injection were also collected.At the same period,normal prepubertal children(50 boys and 49 girls)were recruited,and timed urine samples as above were collected.Luteinizing hormone(LH)and follicle-stimulating hormone(FSH)levels in serum and urine were assayed by immunochemiluminometric assays.Urine pH and creatinine(Cr)were analysed simultaneously.The data were showed in median(minimum~maximum).Results:(1)General characteristics: The ages of girls with central precocious puberty,adolescent boys and adolescent girls were 8.6(6.8~10.6)years old,12.1(9.9~15.6)years old and 10.9(8.8~18.3)years old;normal prepubertal boys and girls were both 6.4(6.0~6.8)years old.(2)Serum gonadotropin in the morning: The serum LH,FSH and LH/FSH in girls with central precocious puberty were 0.95(0.10~17.70)IU/L,3.75(0.85~27.16)IU/L and 0.236(0.030~2.462),and for adolescent boys 1.77(0.67~17.03)IU/L,3.82(2.11~18.83)IU/L and 0.461(0.157~1.221),and for adolescent girls 1.27(0.28~22.99)IU/L,5.81(2.15~8.78)IU/L and 0.224(0.098~3.390),respectively.(3)Nocturnal urine gonadotropin: The pH in all urine specimens were in the range of 5.0~7.0.The urine LH contents,LH/Cr,LH concentrations,FSH contents,FSH/Cr,FSH concentrations and LH/FSH were 0.18(0.01~1.54)IU,67.811(9.333~1531.151)IU/mol,0.33(0.10~8.12)IU/L,2.76(0.36~11.54)IU,1184.834(279.976~12018.220)IU/mol,5.435(0.75~40.75)IU/L and 0.072(0.010~0.261);for adolescent boys 0.66(0.03~7.63)IU,183.248(17.168~774.563IU/mol,1.53(0.10~12.85)IU/L,3.62(0.15~19.04)IU,1037.069(755.403~1932.489)IU/mol,7.23(7.48~32.06)IU/L and 0.178(0.013~0.965);for adolescent girls 0.45(0.05~5.18)IU,167.008(2.990~1288.817)IU/mol,1.18(0.10~35.46)IU/L,5.49(1.26~13.79)IU,1580.520(427.580~3432.848)IU/mol,11.48(1.43~94.45)IU/L and 0.090(0.019~0.375);for prepubertal boys 0.03(0.01~0.08)IU,17.262(6.494~72.377)IU/mol,0.10(0.10~0.43)IU/L,0.70(0.18~1.91)IU,451.507(104.113~2287.671)IU/mol,2.85(1.06~11.75)IU/L and 0.037(0.009~0.094);for prepubertal girls 0.03(0.01~0.07)IU,19.834(6.488~103.316)IU/mol,0.10(0.10~1.00)IU/L,1.46(0.03~4.66)IU,1196.499(26.376~2885.874)IU/mol,6.24(0.20~30.21)IU/L and 0.017(0.005~0.500).respectively.(4)The correlation between serum gonadotropin in the morning and nocturnal urine gonadotropin: The correlation between serum LH and nocturnal urine LH contents was 0.591,for urine LH/Cr 0.574,and for LH concentrations 0.628.The correlation between serum FSH and nocturnal urine FSH contents was 0.735,for urine FSH/Cr 0.715,and for FSH concentrations 0.676.The correlation between serum LH/FSH in the morning and nocturnal urine LH/FSH was 0.288.(5)The comparision of the median of urine gonadotropin between the onset of HPGA and normal prepubertal children: The urine LH contents,urine LH/Cr,urine LH concentrations,urine FSH contents,the ratio of LH and FSH in girls with central precocious puberty were higher than those in normal prepubertal girls(all P < 0.001);and the above indicators in adolescent boys were higher than those in normal prepubertal boys(all P<0.001);and the above indicators in adolescent girls were higher than those in normal prepubertal girls(all P<0.01).(6)The sensitivities and specificities for diagnosing onset of HPGA:(1)In pubertal boys,when urine LH contents,LH/Cr,LH concentrations,FSH contents,FSH/Cr,FSH concentrations and LH/FSH were no less than 0.09 IU,44.540 IU/mol,0.29IU/L,1.98 IU,633.455IU/mol,5.08IU/L and 0.087,the sensitivities for the onset of HPGA were 97.4%,97.4%,97.4%,94.7%,84.2%,81.6% and 92.1%,respectively,as well as the specificities were 100.0%,98.0%,95.9%,100.0%,79.6%,85.7% and 98.0%,respectively.(2)In pubertal girls,when urine LH contents,LH/Cr,LH concentrations,FSH contents,FSH/Cr,FSH concentrations and LH/FSH were no less than 43.542 IU/mol,0.16IU/L,3.51 IU,1341.354IU/mol,9.18IU/L and 0.048,the sensitivities were100.0%,88.9%,88.9%,77.8%,77.8%,72.2% and 88.9%,respectively,as well as the specificities were 91.8%,93.9%,93.9%,98.0%,57.1%,71.4% and 87.8%,respectively.(3)In CPP girls,when urine LH contents,LH/Cr,LH concentrations,FSH contents,FSH/Cr,FSH concentrations and LH/FSH were no less than 0.04 IU,46.283 IU/mol,0.15IU/L,2.46 IU,2554.890IU/mol,17.86IU/L and 0.031,the sensitivities were 94.0%,68.0%,70.0%,64.0%,14.0%,72.2% and 92.0%,respectively,as well as the specificities were 91.8%,93.9%,93.9%,83.7%,98.0%,93.9% and 79.6%,respectively.(4)When urine LH was no less than 0.06 IU,the sensitivities for the onset of HPGA in pubertal boys,pubertal girls and CPP were 97.4%?94.4%? 86.0%,as well as the specificities were all 95.9%.Conclusions: The nocturnal urine gonadotropin in children can reflect serum gonadotropin level in the morning.Especially,timed 12 h nocturnal urine LH contents(?0.06IU)can be one of important indicators for the onset of HPGA.Part 2 The Establishment of Methods in the Lab for Urine Gonadotropin Measuring by Immunochemiluminometric AssaysObjective: Urine lutenizing hormone(LH)and follicular stimulating hormone(FSH)were determined by immunochemiluminometric assays(ICMA).The effect of different urine parameters of p H,different osmolarity,storage time,preservative and freezing-thawing cycles on urine gonadotropin was investigated,and it aimed to offer new methods in the lab for clinical application of urine gonadotropin.Methods: The first morning-voided urine was collected.(1)p H: Hydrochloric acid and sodium hydroxide were added to aliquots of urine as needed to achieve p H of 2.5,3.5,4.5,5.5,6.0,6.5,7.0,7.5,8.0,8.5 and 10.5,respectively.(2)Osmolarity: The 20% sodium chloride and deionized water were added to aliquots of urine as needed to achieve osmolarity of 500,1000,1500,2000,2500 and 3000 m Osm/L,respectively.(3)Storage time: The urine specimen was stored at 4?.Urine LH and FSH were assayed by ICMA between 0 and 7 days,and from the end of the second week to the end of the seventh week,respectively.(4)Preservative: The urine specimen was added equal glycerol or tri-distilled water.(5)Freezing-thawing cycles: The urine was stored at-20?.Urine LH and FSH were assayed between 0 and 13 freeze-thaw cycles(between 0 and 14 days).Urine gonadotropin was determined by ICMA.Results:(1)p H:Urine LH levels(IU/L)from p H2.5,p H3.5,p H4.5,p H5.5,p H6.0,p H6.5,p H7.0,p H7.5,p H8.0,p H8.5 and p H10.5 were 1.828±0.055,1.918±0.111,1.895±0.058,1.850±0.142,1.805±0.077,1.765±0.069,1.748±0.072,1.758±0.068,1.738±0.060,1.738±0.057 and 1.735±0.085,respectively.Compared with the specimen of p H7.5,urine LH were unaffected by changes of p H from 2.5 to 10.5(all P>0.05).Urine FSH levels(IU/L)from p H2.5,p H3.5,p H4.5,p H5.5,p H6.0,p H6.5,p H7.0,p H7.5,p H8.0,p H8.5 and p H10.5 were 0.905±0.074,5.827± 0.580,6.210±0.419,6.607±0.169,6.672±0.230,6.650±0.206,6.505±0.287,6.543±0.177,6.570±0.227,6.488±0.124 and 6.240±0.224,respectively.And urine FSH were unaffected by changes of pH from 3.5 to 10.5(all P>0.05).But urine FSH at p H2.5 significantly decreased(t=71.940,P<0.05).(2)Osmolarity: The LH in original urine specimen(754.0±218.9 m Osm/L),500 m Osm/L,1000 m Osm/L,1500 m Osm/L,2000 m Osm/L,2500 m Osm/L and 3000 m Osm/L urine specimen were 0.582±0.257,0.654±0.314,0.606±0.301,0.516±0.202,0.495±0.218,0.439±0.186 and 0.425±0.183,respectively;and urine FSH(IU/L)were 3.161±1.937,3.267±1.993,3.186±1.855,3.063±1.835,3.035±1.806,2.928±1.686 and 2.928±1.663,respectively.Urine LH between urine specimen with somolarity from 500 m Osm/L to 3000 m Osm/L and original urine sample were no difference,and as well as urine FSH(all P >0.05).(3)Storage time: The urine LH(IU/L)from day 0 to day 7,the end of the second week to seventh week were 1.765±0.026,1.765±0.072,1.675±0.067,1.675±0.075,1.605±0.031,1.665±0.076,1.622±0.053,1.562±0.034,1.527±0.137,1.637±0.085,1.365±0.058,1.433±0.076,1.428±0.085 and 1.368±0.060,respectively.The urine FSH(IU/L)were 5.375±0.149,5.305±0.153,5.248±0.164,5.317±0.224,5.093±0.110,5.303±0.173,5.042±0.099,5.032±0.280,5.035±0.115,5.035±0.125,5.163±0.205,5.318±0.167,5.423±0.188 and 5.055±0.181,respectively.Compared with the original specimen,urine LH were unaffected from the first to the third day(t were 0.000,3.087 and 2.777,all P >0.05)and urine FSH within the seventh week(all P >0.05),while urine LH were affected significantly from the day 4.(4)Preservative: The urine LH(IU/L)with additives(glycerol)from day 0 to day 7,the end of the second week to seventh week was 1.600±0.040,1.568±0.082,1.677±0.038,1.623±0.061,1.470±0.034,1.553±0.023,1.495±0.029,1.497±0.046,1.380±0.046,1.403±0.045,1.207±0.054,1.248±0.044,1.130±0.037 and 1.038±0.021,respectively.The urine FSH(IU/L)with additives was 5.257±0.217,5.107±0.179,4.900±0.079,4.998±0.175,4.800±0.100,4.957±0.194,4.862±0.105,4.980±0.221,4.850±0.205,4.690±0.148,4.735±0.093,5.048±0.147,4.857±0.163 and 4.720±0.120,respectively.Compared with the original specimen,urine LH were unaffected from the first to the third days(t were 0.848,3.414 and 0.782,respectively,all P >0.05),urine FSH within the second week for specimen with additives(P>0.05),too.Urine FSH began to decrease at the end of third week,fourth week and seventh week,(t were 5.279,5.409 and 5.304,respectively;P were 0.025,0.039 and 0.032,respectively).(5)Freezing-thawing cycles: The LH level in original urine,from the first to 13 times of freezing-thawing cycles,the LH(IU/L)were 2.562±0.057,2.590±0.055,2.605±0.055,2.260±0.077,2.337±0.039,2.368±0.116,2.357±0.067, 2.307±0.048,2.267±0.052,2.168±0.115,2.228±0.053,2.053±0.052,2.053±0.047 and 2.143±0.098,respectively;and the FSH(IU/L)were 9.747±0.567,9.403±0.448,9.093±0.309,9.628±0.394,9.463±0.314,9.308±0.112,9.470±0.221,9.483±0.276,9.203±0.359,9.225±0.316,9.698±0.219,9.183±0.268,9.795±0.450 and 9.253±0.252,respectively.Compared with the original specimen,urine LH from different specimen were unaffected within 2 freezing-thawing cycles,and urine FSH within 14 freezing-thawing cycles,too(P>0.05).Conclusions: When measuring by ICMA,urine gonadotropin do not be affected in urine physiological range of p H(4.6 ~ 8.0)and osmolarity(600 ~ 1000 m Osm/L).When stored at 4?,urine specimen can be without glycerol and determined within 72 h.When stored at-20?,urine specimen can be freezing-thawing within twice cycles.Part 3 Research on Normal Reference Values of Urine Gonadotropin in Preschool-aged ChildrenObjective:To generalize urine gonadotropin determined by the new technology of immunochemiluminometric assays(ICMA),a pilot study on normal reference values of urine gonadotropin in preschool-aged(3-6 years old)children was carried out.Methods: The preschool-aged children in Hudai community of Wuxi City were involved.After the consent of all guardians,they were divided into 4 groups according to their ages.The groups were defined by 3 years old group(3 years old?age<4 years old),4 years old group(4 years old?age<5 years old),5 years old group(5 years old?age<6 years old)and 6 years old group(6 years old?age<7 years old),respectively.The parameters of physical growth and pubertal development were examined and evaluated.Children with normal growth and development were included(According to the standards of growth and development from World Health Orgnization in 2006,the BMI for age Z-score,height for age Z-score and weight for age Z-score were within-2SD and 2SD.).The children with abnormal growth and development were ruled out.Through emptying of the bladder before bedtime,first morning voided urine specimens were collected.Timed urine volume was recorded.Urine gonadotropin was assayed by immunochemiluminometric assay.Urine p H and other indicators were analysed simultaneously.Results:(1)General characteristics: There were total 1248 children were investigated.There were 1105 children in line with inclusion criteria and their urine specimens were collected correctly.Among them,there were 585 boys and 520 girls.There were 53,208,206 and 118 boys in the age groups of 3,4,5,6 years old,respectively,and 37,182,167 and 134 girls in above groups,respectively.The p H in all urine specimens were in the range of 5.0~8.0.(2)Urine gonadotropin: The data were abnormal distribution,and normal reference showed in median(95% medical reference ranges).(1)The urine LH levels(IU/L)in boys and girls were 0.10IU/L(<0.20IU/L)and 0.10IU/L(<0.31IU/L),respectively;and there was significant difference between boys and girls(Z =-3.755,P<0.001).The urine FSH levels(IU/L)in boys and girls were 2.54IU/L(<7.96IU/L)and 7.46IU/L(<21.49IU/L),respectively.And there was also significant difference between boys and girls(Z =-19.644,P<0.001).The urine LH/FSH(IU/L)in boys and girls were 0.041(<0.148)and 0.016(<0.068),respectively.And there was also significant difference between boys and girls(Z=-19.506,P<0.001).(2)There were no significant difference in urine FSH levels among any age groups of boys(P=0.186).There was significant difference in urine FSH levels among different age groups of girls(P <0.001).The FSH levels of girls in 5 years old group or 6 years old group were lower than those of girls in 3 years old group(?~2 =10.96,P<0.025;?~2 =17.55,P<0.005).The urine FSH levels of girls in 5 years old group or 6 years old group were lower than those of girls in 4 years old group(?~2 =17.17,P<0.005;?~2 =29.70,P<0.005),too.(3)There were no significant difference in urine LH/FSH among any age groups of boys(P=0.262).The urine LH/FSH of girls in 5 years old group or 6 years old group were lower than those of girls in 3 years old group(?~2 =17.54,P<0.025;?~2 =24.43,P<0.025).The urine LH/FSH of girls in 5 years old group or 6 years old group were lower than those of girls in 4 years old group(?~2 =14.13,P<0.025;?~2 =24.19,P<0.025),too.(4)When weight for age Z-score was more than-2SD and less than(or equal to)zero(bad nutrition in short term),the urine LH level,FSH level and LH/FSH were 0.10IU/L(<0.26 IU/L),4.45IU/L(<17.11 IU/L)and 0.026(<0.111),respectively.When weight for age Z-score was more than zero and less than 2SD(good nutrition in short term),the urine LH level,FSH level and LH/FSH were 0.10IU/L(<0.27 IU/L),3.60IU/L(<17.71 IU/L)and 0.029(<0.125),respectively.There was no significant difference in urine LH level between bad nutrition group and good nutrition group(Z =-0.472,P=0.637).The urine FSH level in bad nutrition group was higher than that in good nutrition group(Z =-1.991,P=0.046).The urine LH/FSH in bad nutrition group was lower than that in good nutrition group(Z =-2.337,P=0.019).Conclusions: The normal reference values of urine gonadotropin in preschool-aged children are characteristics of age and gender.The profile of urine FSH in girls declines gradually with the increasing of age,and the trends decline slowly between 6 years old and 7 years old.Urine FSH can be increased in children with bad nutrition in short term,and no effect by nutrition status in long term. |
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