KR20180130231A - Prediction method for responsiveness of GnRH stimulation in girls with suspected central precocious puberty - Google Patents

Abstract

The present invention relates to a method for providing information for determining, in advance, a result of a central precocious puberty definite diagnosis test (a GnRH stimulation reaction test) of a girl with central precocious puberty, and particularly, to a method for determining, in advance, whether a girl suspicious of central precocious puberty is likely to obtain a positive or a negative reaction result in the central precocious puberty definite diagnosis test, by measuring concentrations of cortisol, dehydroepiandrosterone (DHEA) and estradiol-17β, E2 from a saliva sample collected from a girl less than nine years old before performing the central precocious puberty definite diagnosis test and by checking a specific relationship value between the concentrations, in order to provide information required for the central precocious puberty definite diagnosis test, and to a reference therefor.

Description

[0002] The present invention relates to a method for predicting the GnRH stimulation response of a girl with a sexual precocious puberty (GnRH stimulation in girls with suspected central precocious puberty)

The present invention relates to a method of predicting the result of GnRH stimulation test performed to confirm the central precocious puberty (CPP) of a girl through a sample collected before the GnRH stimulation test More specifically, the present invention relates to a corticosteroid and a dihydroepiandrosterone (DHEA) among corticosteroids, a adrenal endocrine system based on the concentration of estradiol-17β, E2 in the gonadal hormone, The present invention relates to a method and a criterion for providing information for the evaluation of gonococci using an indicator that can grasp the activity level of the reproductive endocrine system.

Precocious puberty refers to a case in which the secondary sex is more than 2 standard deviations higher than the mean value. Generally, it refers to cases of breast development before 8 years of age, development of puberty before 9 years, and menarche before 9.5 years. Refers to cases in which the volume of testicles increases by more than 4 mL or the diameter increases by more than 2.5 cm before 9 years of age.

It can be divided into central precocious puberty (CPP) and peripheral or pseudoprecocious puberty, depending on whether hypothalamic-pituitary-gonadal axis activation is present or not.

Confirmation of intact precocious puberty is only possible through gonadotropin releasing hormone (GnRH) stimulation test.

Blood was collected repeatedly (usually at 0, 30, 45, 60, 90, 120 minutes) at a fixed time interval after GnRH injection from a female patient with intact precocious puberty for confirmation of intrathoracic precocious puberty. The concentration of luteinizing hormone (LH) and follicle stimulating hormone (FSH) was measured. The highest LH concentration was found to be 5 IU / L or more, or the LH concentration was increased 2-3 times at baseline Or when the LH / FSH ratio is abnormal, it is judged to be a pubertal response, that is, a sexual precocious puberty (GHG guidelines, Korean Society of Pediatric Endocrinology, 2011).

According to the results of a national study, the positive rate of the positive test (positive confirmation of the precocious puberty) was 33-77% in the confirmation test (Kim et al., 2011, Nam et al., 2012, Suh et al ., 2013).

A large number of blood samples (total 25-30 mL) of long-term (at least 2 hours) multiples (5-6 times) for confirmation of intrinsic onset of prematurity were used for mental burden to the surrogate girl, (Kim et al., 2011, Nam et al., 2012), the cost of analyzing expensive LH and FSH in multiple blood samples leads to an economic burden on the child's parents.

A girl who obtained a positive response in the confirmation test has a medical treatment for delaying the cervical cranial period and inhibiting the final key loss through the treatment of sexuality precocious puberty. However, the girl who got the negative reaction, It will be multiplied by the burden of the parents of the girl, the medical staff, and the child because they have to repeat after 3 months.

 The high negative rate of response in the diagnosis of intact whole-body precocious puberty is due to the lack of objective methods such as biochemical tests that can be used to make preliminary results before the confirmation test. As a result, a conservative confirmation test can be performed (Bouvattier et al., 1998), the effect of treatment on gynecological symptoms, that is, the inhibition of final key loss, may not be significant. 1999, Willemsen et al., 2014).

The onset of puberty, on the other hand, depends on the activation of the hypothalamus-piyuitary-adrenal axis (HPA axis) leading to the hypothalamus-pituitary-adrenal gland. Especially, the increase of DHEA production and secretion in the adrenal endocrine system is associated with puberty onset (Pinyerd et al., 2005).

The development of secondary sexual development in puberty development depends on the activation of the hypothalamus-pituitary-gonad axis (HPG axis) leading to the hypothalamus-pituitary gonad, especially estradiol-17β, E2) concentration is known to affect development of secondary males such as breasts in girls and length growth of long bones and growth plate closure (Juul, 2001).

In other words, sexual precocious puberty is a phenomenon in which the reproductive endocrine system is activated at an earlier age than the average age, and peripheral precocious puberty induced by estrogen (fat tissue or environmental hormone) produced or introduced separately from the reproductive endocrine system (Mul et al., 2002).

Therefore, the GnRH-injected intact precocious puberty test is a test to determine the degree of HPG axis activation after puberty initiation. When the GnRH receptor is sufficiently expressed in the pituitary gland to respond to GnRH in the female, (LH, 5 IU / L or more).

GnRH receptor gene expression in the pituitary gland is regulated by E2, which is mainly produced and secreted in the ovary (Turzillo et al., 1998). The ovarian maturity of the ovary, ie, the competence of the ovary to produce E2, It can be assumed that it directly affects the reaction.

In girls, the same reproductive endocrine system as that of adults is completed during puberty development. Although the production of E2 is significantly lower than that of adult women, the reproductive endocrine system of the same type as adults is completed in the middle of puberty. (Kang et al., 2014).

Therefore, the present inventor has found that saliva collected from a female subject with a sexual precocious puberty at a predetermined time interval after a morning clinical trial before a confirmation test (a GnRH stimulation reaction) with a female adult having a normal menstrual cycle and confirmed the sexual precocious puberty (GnRH stimulation response), and the samples were divided into positive and negative responders. In the saliva samples of participants of each group (adult female, positive reaction girl and negative reaction girl in intact premorbid reaction test), adrenal endocrine (Estradiol-17β, E2) among the hormones of cortisol, dehydroepiandrosterone (DHEA) and the reproductive endocrine system among the hormones of the system, and then, based on the concentration of these hormones, (The molar concentration ratio of cortisol and DHEA, the molar concentration of cortisol and DHEA) (Total amount of E2, total amount of E2, total amount of E2 increase) were compared with each other in each group (girls, girls who showed positive and negative reactions in adult women, confirmation test of sexual precocious puberty) The present invention has been accomplished by devising methods and criteria that can confirm the results (positive and negative reactions) that can be obtained in the test before the confirmation test of the sexual precocious puberty.

1. Bouvattier C, Coste J, Rodrigue D, Teinturier C, Carel JC, Chaussain JL, BougnePF. Lack of effect of GnRH agonists on final height in girls with advanced puberty: a randomized long-term pilot study.J Clin Endocrinol Metab. 1999; 84: 3575-8. Han, Kyung - Hee; Kim, Seung - Jung Kee, Ji Yeon Seo, Eun Mi Yang, Hong Jae Chae, Chan Jong Kim. Gonadotropin-releasing Hormone Stimulation Test for Precocious Puberty. Korean J Lab Med 2011; 31: 244-249. 3. Hyo-Kyoung Nam, Young Jun Rhie, Chang Sung Son, Sang Hee Park, Kee-Hyoung Lee. Factors to Predict Positive Results of Gonadotropin Releasing Hormone Stimulation Test in Girls with Suspected Precocious Puberty. J Korean Med Sci 2012; 27: 194-199. 4. Juul A. The effects of oestrogens on linear bone growth. Hum Reprod Update. 2001; 7: 303-13. 5. Kang JY, Park JY, Chun SI, Suh HS, Lee K, Ahn RS. Puberty-related changes in cortisol, dehydroepiandrosterone, and estradiol-17β secretions within the first hour after waking in premenarcheal girls. Neuroendocrinology. 2014; 99: 168-77. 6. Mul D, Oostdijk W, Drop SL. Early puberty in girls. Best Pract Res Clin Endocrinol Metab. 2002; 16: 153-63. 7. Pinyerd B, Zipf WB. Puberty-timing is everything! J Pediatr Nurs. 2005; 20: 75-82. 8. Suh J, Choi MH, Kwon AR, Kim YJ, Jeong JW, Ahn JM, Chae HW, Kim DH, Kim HS. Factors that predict a positive response to gonadotropin-releasing hormone stimulation test for diagnosing central precocious puberty in girls. Ann Pediatr Endocrinol Metab. 2013; 18: 202-7. 9. Turzillo AM, Clapper JA, Moss GE, Nett TM. Regulation of ovine GnRH receptor gene expression by progesterone and oestradiol. J Reprod Fertil. 1998; 113: 251-6. 10. Willemsen RH, Elleri D, Williams RM, Ong KK, Dunger DB. Pros and cons of GnRHa treatment for early puberty in girls. Nat Rev Endocrinol. 2014; 10: 352-63. 11. Gynecological examination guidelines (The Korean Society of Pediatric Endocrinology Clinical Guidelines), 2011.

It is an object of the present invention to provide a method for the treatment and / or prevention of osteoporosis based on the concentration of cortisol, dehydroepiandrosterone (DHEA) and estradiol-17β, E2 measured from saliva collected from a girl aged 9 years or younger And a method for providing information for diagnosis of gonadal glioma using an activation judgment index of the adrenal endocrine system and / or the gonadal endocrine system.

It is also an object of the present invention to provide a method for predicting sexual maturity based on information obtained by using an activation judgment index of the adrenal and / or germinal endocrine system.

In order to solve the above problems, the present invention provides a pharmaceutical composition comprising cortisol, dehydroepiandrosterone (DHEA) and estradiol-17β (E2), which are measured from saliva collected from a girl aged 9 years or less, A method for providing information for diagnosis of gonococci using an activation judgment index of a gonadal endocrine system and / or a gonadotropic system based on the concentration of gonadotropin, and a method of predicting gonadal function.

At this time, the adrenal endocrine system activation determination index is the molar ratio of the cortisol / DHEA mole ratio (molar F / D ratio) and / or the molar concentration ratio of cortisol and DHEA (molar F / D ratio- / D-auc)), and the reproductive endocrinological activity activation judgment index is a total amount of secretion (E2auci) of E2 and / or a total amount of pure increase (E2Inc%) of E2.

At this time, as the adrenal endocrine system activation judgment index

When the value of the cortisol / DHEA mole ratio (molar F / D ratio) is 25 or less after the gaseous phase is 69 or less; And the value of the molar concentration ratio (molar F / D-auc) of the cortisol and DHEA molar ratio is 3500 or less,

It is characterized by the activation of the adrenal endocrine system that pubertal development has started and the degree of activation has reached the level of adult.

Further, as an indicator for determining the germinal endocrine system activation

When the value of the total secretion amount E2auci of E2 is 350 or more; And a value of the net increase amount (E2Inc%) of E2 is 100 or more,

The development of the external breast development due to the activation of the reproductive endocrine system is started and the degree of activation is determined to be at a level at which a positive reaction can be obtained in the test for confirmation of gonadal function.

It is more preferable that the saliva is collected within 1 hour after the vaporization of a girl aged 9 years or less, particularly, less than 5 minutes after the gas phase, 25 to 35 minutes after the gas phase and 55 to 65 minutes after the gas phase.

The above-mentioned abnormalities include, but are not limited to, central precocious puberty, intractable precocious puberty, peripheral precocious puberty or pacific precocious puberty, preferably intact precocious puberty or central precocious puberty.

The method of the present invention, as a preferred embodiment,

a) measuring the concentration of cortisol, DHEA and E2 from the saliva collected within 1 hour after the wake of a girl aged 9 years or less;

b) determining the adrenal endocrinological activity activation indicator value using the concentration measured in step a), wherein the adrenal endocrine activity activation indicator is a cortisol / DHEA mole concentration ratio (molar F / D ratio) (Molar F / D ratio-auc (molar F / D-auc)) of the molar concentration ratio of cortisol and DHEA;

The method according to any one of claims 1 to 3, wherein the germinal endocrinological activity activation indicator comprises a total amount of E2 (E2auci) and / or a total increase in E2 (E2Inc%) of E2, ).

In step b), when the value of the cortisol / DHEA mole ratio (molar F / D ratio) was 25 to 35 minutes after the gaseous phase was 69 or less, pubertal development started due to activation of the adrenal endocrine system. It can be judged that the degree of activation has reached adult level.

In addition, in step b), when the value of molar ratio F / D-auc of the cortisol and DHEA is less than 3500, development of adrenal endocrine system caused puberty development, and the degree of activation (Molar F / D-auc) of the cortisol and DHEA molar ratio (molar F / D-auc) is an amount of the molar concentration ratio of cortisol and DHEA during the first hour after the gaseous phase .

In step c), when the value of the total secretion amount E2auci of E2 is equal to or more than 350, exogenous breast development is started due to activation of the reproductive endocrine system, and the degree of activation is positive in the test (E2auci) means the total amount of E2 increased from the basal E2 concentration for the first hour after the gas phase, and the basal E2 concentration is set to a value immediately after the gas phase or immediately after the gas phase Means the concentration of E2 measured in less than 5 minutes.

In step c), when the value of the total increase in E2 (E2Inc%) is 100 or more, exogenous breast development is initiated due to activation of the reproductive endocrine system, and the degree of activation is detected in positive (E2Inc%) means the percentage of the maximum E2 concentration increased within the first hour after the start of the meteorological operation based on the basal E2 concentration.

At this time, the activation judgment index can be used as information for judging whether a positive reaction can be obtained in the GnRH stimulation reaction test.

In particular, it is possible to provide a method for predicting the results that can be obtained in the intact precocious puberty confirmation test (GnRH stimulation response test) by using the information for diagnosing the above-mentioned gonadal disorders.

In addition, the method of the present invention may further include the step of excluding the saliva having an increase in the concentration of cortisol in the saliva of less than 2.5 nmol / L for the first hour from immediately after the vapor phase in order to improve the accuracy of the prediction.

That is, it is possible to determine a sample suitable for the diagnosis object by checking the change amount of the cortisol concentration for the first hour after the morning waking.

The information that can be usefully obtained in the present invention can be obtained by the age standard (age 9 years) proposed by the Health Insurance Review and Assessment Institute and the Korean Society of Pediatric Endocrinology to determine whether a girl under 9 years old starts puberty at a normal age, Includes prediction of maturity of reproductive endocrine axis or degree of breast development.

In addition, the present invention can provide various information on the in-vivo adrenal and reproductive endocrine environment of a girl under the age of 9 years, and includes all uses of such information. For example, in the diagnosis and treatment of gliosis.

The present invention relates to the use of cortisol, dehydroepiandrosterone (DHEA), and the changes in the concentration of cortisol, dihydroepiandrosterone (DHEA) contained in saliva samples collected at intervals of the first hour The degree of activity of the endocrine system can be determined, and the growth rate and total secretion amount of estradiol (E2) can be analyzed and quantified to grasp the maturation of the reproductive endocrine axis.

Therefore, prior to performing the GnRH stimulation test (GnRH stimulation test), the biological sample (saliva) collected noninvasively from the female sexual precocious puberty is used to determine the activity level of the adrenal endocrine system and the reproductive endocrine system, This method can be usefully used as a method for improving the efficiency of the confirmation test by providing a range of specific value ranges in which a positive reaction is obtained in a reaction test.

FIG. 1 shows the change in F / D ratio during the first hour after morning wake in adult female, positive female, and negative female.
FIG. 2 shows the results of comparing the F / D ratio-auc difference between adult female, positive female and negative female.
Figure 3 shows the changes in E2 during the first hour after morning wake in adult female, positive female, and negative female.
Figure 4 shows the difference in E2Inc% between adult female, positive female and negative female.
Figure 5 shows the difference in E2auci between adult female, positive female, and negative female.

The terms used in the present invention are defined as follows.

"Precocious puberty" is a disease in which secondary sex is present at an earlier age than the average, and the current Korean standard is called "precocious puberty" when the development of secondary sex is seen in girls less than 8 years old and boys less than 9 years old. Secondary malformations develop in the breast in girls and in males, the volume of the testicles is greater than 4 mL, or the diameter of the testicles is greater than 2.5 cm, including pubic hair development. In the present invention, gonococcosis includes central precocious puberty, intestinal precocious puberty, peripheral precocious puberty or pacer precocious puberty, preferably intact precocious puberty or central precocious puberty.

&Quot; Central Precocious Puberty (CPP) " refers to the precocious puberty in which the reproductive endocrine system leading to the hypothalamus-pituitary-gonads is activated at an early age. Peripheral precocious puberty (PPP) or pseudo-precocious puberty is defined as a condition in which hypothalamic-pituitary-gonadal activation is not accompanied, It is generally called incomplete precocious puberty when it does not progress or symptoms disappear, such as premature puberty, premature adrenarche, premature menarche.

&Quot; Diagnosis " means identifying the presence or activity of a particular pathology or physiological condition.

&Quot; Subject " or " patient " means any single entity that requires treatment, including human, cow, dog, guinea pig, rabbit, chicken, In addition, any subject who participates in a clinical study test that does not show any disease clinical findings, or who participates in epidemiological studies or used as a control group is included. In the present invention, the present invention was applied to humans.

"Confirmation" means a definite diagnosis of a disease, and physiological and biochemical tests that can be used to judge the presence or absence of the disease and to provide objective evidence are performed.

&Quot; Sample " or " sample " refers to a collection of similar cells obtained from a subject or tissue of a patient. The source of the tissue or cell sample may be a solid tissue from fresh, frozen and / or preserved organ or tissue sample or biopsy or aspirate; Blood or any liquid type tissue. In the present invention, saliva, blood or urine can be used, and saliva is preferably used.

"Steroid hormone" is a hormone with a basic structure of a steroid ring (cyclopentanohydrophenanthrene ring) which is a quaternary cyclic hydrocarbon. It is a hormone of male and female gonadal hormones (sex hormones ) And corticosteroids.

"Gonadal hormone" or "sex hormone" is a hormone secreted by the gonads of female and male vertebrates, which promotes the development of the reproductive system and maintains its function. Male cases are hepatocytes (intercellular cells) in the testes, and magnetism is mainly cumulus cells and soma cells constituting the follicles of the ovary.

"Menstruation" or "menstruation" means that when a human adult woman periodically produces an egg from the ovary and is fertilized, the uterine mucosa for implantation of the embryo into the uterus is removed And the like. It usually takes about 28 days and lasts about 3 ~ 5 days. Usually, when you grow up to 12 to 15 years old, you start your menstruation for the first time. Women will have about 400 menstrual cycles for the rest of your life, and then menstruation will stop when you are 45 to 55 years old. The blood flow to menstrual flow is about 35 mL, and within 10 to 80 mL is normal.

The "menstrual cycle" is a regular change in the reproductive organs due to the effects of endocrine system hormones. It is not constant between 21 and 35 days, but it is usually repeated about 28 days. From the beginning of menstruation to the next menstrual cycle It refers to the period until it appears. The endometrium and the ovary show the periodicity by the secretion of female hormones. When the endometrium is broken down, the bleeding is called the menstruation. When the ovary follicles mature and the oocyte is released, the ovulation date is called the ovulation date.

&Quot; Label " or " label " refers to a compound or composition that directly or indirectly facilitates the detection of a reagent, such as a nucleic acid probe or a reagent conjugated, conjugated, conjugated, or fused to an antibody do. The label may itself be detected (e. G., A radioactive isotope label or a fluorescent label), in the case of an enzyme label, to catalyze the chemical modification of the detectable substrate compound or composition.

&Quot; about " means that the reference quantity, level, value, number, frequency, percentage, dimension, size, quantity, weight or length is 30, 25, 20, 25, 10, 9, 8, 7, , Level, value, number, frequency, percent, dimension, size, quantity, weight, or length that varies from one to three, two, or one percent.

Throughout this specification, the words " comprising " and " comprising ", unless the context requires otherwise, include the stated step or element, or group of steps or elements, but not to any other step or element, And that they are not excluded.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for providing information for diagnosing gonadal ganglia as one aspect. In particular, the present invention relates to a method for providing information on the activation judgment index of the adrenal endocrine system and / or the reproductive endocrine system in order to enhance the efficiency of the confirmation test of the sexual precocious puberty.

Another aspect of the present invention relates to a method for predicting sexual maturity. Particularly, the present invention relates to a method of predicting a sexual precocious puberty in advance by using the obtained activation judgment index information of the endocrine system.

In the present invention, " sexual maturity " is a symptom in which the secondary sex is appeared earlier than the standard deviation of the mean. In order to achieve sexual maturation and fertility, the sex hormone-pituitary- adrenal gland leading to the adrenal gland and the hypothalamus- This includes the onset of puberty in which the function of the endocrine system is activated. The above disorders include various types of sexual precocious puberty such as sexual precocious puberty, pseudocubic precocious puberty and incomplete sexual precocious puberty. The present invention relates to a method for predicting a precocious puberty using the same, and a method for predicting a precocious puberty using the same.

The above-mentioned Central Precocious Puberty (CPP) refers to the precocious puberty caused by activation of the gonadal endocrine axis leading to the hypothalamus-pituitary-gonad. On the other hand, cases in which the gonadal endocrine system is not accompanied by activation is referred to as peripheral precocious puberty, and peripheral sexual precocious puberty is secondary to partial puberty, Breast augmentation, early onset of pubic hair, and premature menarche are called incomplete forms of precocious puberty.

Adolescence is the transition period when a child becomes an adult. This period is accompanied by the development and growth of the second castle. In girls, developmental development of pubic hair and pubic hair and development of pubic hair and pubic hair are used as external indicators of secondary sex.

The developmental stage of chest and conspiracy can be divided into five stages respectively, and it can be broadly divided into pre - pubertal stage, early stage, middle stage, late stage and adult stage. In Korean girls, chest development is 11.3 ± 1.3 years, Is initiated at 12.3 ± 1.2 years, and menarche is reported to begin at 12.0 ± 1.0 years. Sex maturity In particular, the rate of menarche was 18.2% in stage 2, 66.1% in stage 3, 91.7% in stage 4, 100% in stage 5, 52% in stage 2, and 93% %. These results suggest that chest development and pubic hair development are closely related to the beginning of menarche, but the development of secondary menarche can not predict the onset of menarche.

The menstrual cycle is a physiological phenomenon that occurs in normal adult women, which means that the body's own internal nervous system (reproductive endocrine system) has been completed in the same body as the adult female, and that the body environment for normal reproductive activity has been established.

Girls' breasts and pubic hair development are associated with increased hormone production in the gonads and adrenals. The adrenarche is called adrenarche, and the adrenarche precedes the gonadarche in the gonadarche adrenal gland, which increases the production of sex hormones in the gonads with the onset of puberty.

Increased production of sex hormones induces breast development in girls and increase in DHEA is known to induce pubarache.

However, it is not known which factors cause gonadarche and adrenarche to begin. In 1980, Wildt et al. Reported that the intermittent injection of GnRH derivatives into young monkeys can induce sexual maturation and that the onset of puberty depends on the release of GnRH from neurons in the hypothalamus rather than on the pituitary and ovarian development. Science . This suppresses the release of GnRH by inhibiting factors in the hypothalamus GnRH neurons. As the inhibitory factor is eliminated, the gonadal endocrine axis that leads to the hypothalamus-pituitary gonads is activated, and the sex hormone It can be interpreted that the development of secondary sex (breast) and menarche are induced.

The concentration (cortisol / DHEA mole) of the cortisol, DHEA, and DHEA of the present invention can be used to determine the degree of activation of the adrenal and gonadal endocrine system using the concentrations of cortisol and dihydroepiandrosterone (DHEA) (GnRH stimulation response test) was performed by quantifying the ratio of cortisol and DHEA molar ratio, the total amount of E2 secretion, and the net increase in E2. And GnRH stimulation test can be used as a method to improve the efficiency of the confirmation test by providing a range of specific value range in which a positive reaction is obtained in the GnRH stimulation test.

Target sample

The sample used to improve the efficiency of the diagnostic test of the intact precocious puberty of the present invention may be blood, urine, feces, saliva, lymph, cerebrospinal fluid, synovial fluid, cystic fluid, ascitic fluid, pleural fluid, exudate and the like.

Preferably saliva, blood, urine, and more preferably " saliva ".

So far, the concentration of hormones has been measured mainly in the blood. From the viewpoint of endocrinology, blood is a transport medium of hormones. Most of blood protein hormones (growth hormone, insulin, etc.) are soluble in water (hydrophilic and hydrophilic) and exist in active form in blood. However, they are produced in adrenocortical and gonadal Because steroids are not well soluble in water (hydrophobic), they bind to other media (transport proteins) and are present in the blood. When steroids are combined with the carrier medium, they are inactive and can not induce a physiological mechanism because they do not act on the hormone receptor of the target organ. Most of the steroids in the blood (95-99%) are present in this inactive state, and only a few (1-4%) are known to be in an unbound state (active state) with the carrier medium. Therefore, the concentration of steroid in the blood can be said to be the most inactive state rather than the active hormone concentration.

In contrast, the steroids contained in saliva are in an active state that is not bound to the carrier medium, unlike blood, and active steroids contained in the extracellular fluid of the saliva are activated by passive diffusion, . Therefore, the concentration of steroid in saliva is recognized as the active steroid in the tissue.

In addition, unlike blood, saliva has the advantage of being able to easily study the daily rhythm of the hormone of the individual because it can be easily collected by the general public in accordance with the sleep cycle of the individual without the help of the medical staff. In other words, the use of saliva has the advantage of easy access to the concentration of active steroids, which was difficult to access to blood, and to the individual's daily rhythm of hormones.

The present invention measures the concentration of the hormones present in the saliva, and the above advantages can be effectively exhibited.

The saliva used in the present invention is preferably one obtained at a predetermined time interval within a certain period of time after the vapor phase. For example, a sample collected within 1 hour after the vapor phase can be used. As a specific example, Is preferably collected at least three times immediately after the gas phase, 30 minutes after the gas phase, and 60 minutes after the gas phase.

Target hormone

The present invention relates to the use of a biomarker to determine the onset of puberty and the degree of breast development and to determine the degree of breast development in the sample, for example, "cortisol", "dehydroepiandrosterone" DHEA) " and " estraiol-17 [beta], E2 "

"DHEA" is a relatively weak androgen steroid and is an intermediate step in the synthesis of male hormones and female hormones. DHEA is mainly secreted from the adrenal cortex, but it is known to synthesize small amounts in the gonads, adipose tissue and brain. In humans, DHEA is about 4 mg per day and DHEA-s, a sulfated form, is about 25 mg, the most common in the blood.

Although the function of DHEA is not fully understood yet, it can be divided into several mechanisms. First, it has a function as a precursor of sex hormone, so it is converted into a male or female hormone in necessary tissues. Secondly, It acts as a steroid and controls nerve stimulation through interaction with various neurotransmitters. Third, it cushions the role of cortisol in the stress state. Fourth, it activates the calcium gated potassium channel .

"Estradiol (E2)" is a hormone produced and secreted in the gonads (ovaries). It is known to be a hormone that induces the growth of the mammary gland, the thickening of the endometrium, and the growth of the ovary. In particular, E2 is the final product during the steroidogenesis process of the ovary and acts as a regulator of egg growth and uterine thickening.

In particular, in the present invention, it is preferable that the hydroxyl group (OH) position of carbon number 17 as estradiol is a beta-estradiol. Beta estradiol is abbreviated as E2, a kind of estrogen, a steroid hormone secreted from the ovaries of vertebrate animals, and is one of the follicle hormones extracted and crystallized from the ovaries, placenta, and the like. The female gonads develop secondary and tertiary constellations, which cause estrus, especially in mammals. In addition, it enters the cell nucleus, promotes the synthesis of new proteins, and has the effect of causing cell proliferation. In particular, beta-estradiol is a reproductive hormone that is highly variable in the female body, which counteracts caffeine-like action, that is, the inhibitory effect of adenosine from the original, so that women experience it shortly after ovulation, It is known to cause mild excitement.

In adult women with a normal menstrual cycle, the production and secretion of E2 also exhibits a constant circadian rhythm, and an increase of about 150-200% within the first hour after the morning wake has been reported. It is reported that the E2 concentration after morning wake changes with increase in the amount and amount of secretion depending on the menstrual period, the frying period and the ovulation period.

"Cortisol" is a steroid hormone secreted from the adrenal cortex. The circadian rhythm of cortisol is known to be regulated by cranial nerves, hypothalamus, and pituitary. Basically, the cortisol, Of the sleep period. Normally, after sleeping at night, when the eyes wake up in the morning, light signals reach the suprachiasmatic nucleus (SCN) of the hypothalamus, and the clock gene activates the clock gene , Vasoactive intestinal peptide (VIP) and arginine-vasopressin (AVP) are produced and secreted in SCN. AVP is transmitted to the paraventricular nucleus (PVN) again. In the nucleus of the ventricle, the corticotropin releasing hormone (CRH) secretes in response to this stimulus, and the secreted hormone stimulates the pituitary gland to produce and secrete adrenocorticotrophic hormone (ACTH) . The concentration of ACTH produced and secreted by the pituitary gland reaches its peak at 15 minutes after the gaseous phase. ACTH secreted in the blood acts on the adrenal gland to increase steroid production and secretion in corticosteroids such as cortisol and DHEA.

Therefore, the peak cortisol concentration is about 30 minutes after the gas phase, and the cortisol concentration at that time is the highest in the day. The cortisol awakening response (CAR) is a phenomenon in which the concentration of cortisol reaches a normal level within 30 minutes after waking up in the morning. It is known that CAR is not affected by general sleeping environment, light, sleeping time, and health condition. However, it is reported that the CAR phenomenon can not be observed if the first saliva sampling is delayed for more than 10 minutes immediately after the weather.

Therefore, in order to obtain accurate information for the diagnosis of gonococcosis according to the present invention, DHEA and E2 concentrations can be measured in saliva when the concentration of cortisol in the saliva is 2.5 nmol / L or more for the first hour from immediately after the morning wake. That is, a change in the concentration of cortisol can be additionally confirmed to determine a suitable saliva sample prior to measuring DHEA and E2 concentrations. At this time, the change in the cortisol concentration in the saliva for the first hour from immediately after the morning wakeup can be examined for the saliva collected immediately after the morning weather, 30 minutes for the weather and 60 minutes for the weather.

Thus, the present invention is based on the finding that, in the saliva samples collected prior to the GnRH stimulation test from the desired female in the intact precocious puberty, cortisol and dehydroepiandrosterone (DHEA) among the adrenocortical hormones, (Cortisol and DHEA molar concentration ratios, cortisol and DHEA (cortisol and DHEA concentration), and estradiol-17β and E2 concentrations were measured and the level of activity of the endocrine endocrine system , The ratio of the increase in E2 after the morning wake, and the total amount of E2 that increased purely during the first hour after the morning wake) are used to provide information necessary for the diagnosis of intrathymic precocious puberty.

In addition, using the above information, methods and criteria for predicting the results that can be obtained in a sexual precocious puberty confirmation test (GnRH stimulation response test) are provided.

Method for Providing Information for Diagnosis of Glaucoma and Method of Glaucoma Prediction Using the Method

The present invention relates to the use of cortisol, dihydroepiandrosterone (DHEA) and estradiol-17β (E2) -dependent adrenal endocrinology, measured from saliva collected from a girl aged 9 years or less, A method for providing information for diagnosis of gliosis and / or a method for predicting gliosis can be provided, using an activation judgment index of the system and / or gonadal endocrine system.

At this time, the adrenal endocrine system activation determination index is the molar ratio of the cortisol / DHEA mole ratio (molar F / D ratio) and / or the molar concentration ratio of cortisol and DHEA (molar F / D ratio- / D-auc)), and the reproductive endocrinological activity activation judgment index is a total amount of secretion (E2auci) of E2 and / or a total amount of pure increase (E2Inc%) of E2.

At this time, as the adrenal endocrine system activation judgment index

When the value of the cortisol / DHEA mole ratio (molar F / D ratio) is 25 or less after the gaseous phase is 69 or less; And the value of the molar concentration ratio (molar F / D-auc) of the cortisol and DHEA molar ratio is 3500 or less,

It is characterized by the activation of the adrenal endocrine system that pubertal development has started and the degree of activation has reached the level of adult.

Further, as an indicator for determining the germinal endocrine system activation

When the value of the total secretion amount E2auci of E2 is 350 or more; And a value of the net increase amount (E2Inc%) of E2 is 100 or more,

The development of the external breast development due to the activation of the reproductive endocrine system is started and the degree of activation is determined to be at a level at which a positive reaction can be obtained in the test for confirmation of gonadal function.

The present invention, as one embodiment,

a) measuring the concentration of cortisol, DHEA and E2 from the saliva collected within 1 hour after the wake of a girl aged 9 years or less;

b) determining the adrenal endocrinological activity activation indicator value using the concentration measured in step a), wherein the adrenal endocrine activity activation indicator is a cortisol / DHEA mole concentration ratio (molar F / D ratio) (Molar F / D ratio-auc (molar F / D-auc)) of the molar concentration ratio of cortisol and DHEA;

The method according to any one of claims 1 to 3, wherein the germinal endocrinological activity activation indicator comprises a total amount of E2 (E2auci) and / or a total increase in E2 (E2Inc%) of E2, )

. ≪ / RTI >

It is more preferable that the saliva is collected within 1 hour after the vaporization of a girl aged 9 years or less, particularly, less than 5 minutes after the gas phase, 25 to 35 minutes after the gas phase and 55 to 65 minutes after the gas phase.

The above-mentioned abnormalities include, but are not limited to, central precocious puberty, intractable precocious puberty, peripheral precocious puberty or pacific precocious puberty, preferably intact precocious puberty or central precocious puberty.

In step b), when the value of the cortisol / DHEA mole ratio (molar F / D ratio) was 25 to 35 minutes after the gaseous phase was 69 or less, pubertal development started due to activation of the adrenal endocrine system. It can be judged that the degree of activation has reached adult level.

In addition, in step b), when the value of molar ratio F / D-auc of the cortisol and DHEA is less than 3500, development of adrenal endocrine system caused puberty development, and the degree of activation (Molar F / D-auc) of the cortisol and DHEA molar ratio (molar F / D-auc) is an amount of the molar concentration ratio of cortisol and DHEA during the first hour after the gaseous phase .

In step c), when the value of the total secretion amount E2auci of E2 is equal to or more than 350, exogenous breast development is started due to activation of the reproductive endocrine system, and the degree of activation is positive in the test (E2auci) means the total amount of E2 increased from the basal E2 concentration for the first hour after the gas phase, and the basal E2 concentration is set to a value immediately after the gas phase or immediately after the gas phase Means the concentration of E2 measured in less than 5 minutes.

In step c), when the value of the total increase in E2 (E2Inc%) is 100 or more, exogenous breast development is initiated due to activation of the reproductive endocrine system, and the degree of activation is detected in positive (E2Inc%) means the percentage of the maximum E2 concentration increased within the first hour after the start of the meteorological operation based on the basal E2 concentration.

At this time, the activation judgment index can be used as information for judging whether a positive reaction can be obtained in the GnRH stimulation reaction test.

In addition, the method of the present invention may further include the step of excluding the saliva having an increase in the concentration of cortisol in the saliva of less than 2.5 nmol / L for the first hour from immediately after the vapor phase in order to improve the accuracy of the prediction.

That is, it is possible to determine a sample suitable for the diagnosis object by checking the change amount of the cortisol concentration for the first hour after the morning waking.

Hereinafter, the method of the present invention will be described in detail.

(1) Classification of target girl

According to the criteria proposed by the Health Examination and Assessment Agency, the classification of the target girl for the present invention is judged to be gonococcal when the development of the secondary sex occurs before 354 days in the 8th Gyeonggi Dynasty. Therefore, I want to be a girl who wants to be a girl with a birth certificate of less than 9 years of age.

(2) Preparation of sample

The sample of the present invention may be blood, urine, feces, saliva, lymph, cerebrospinal fluid, synovial fluid, ascitic fluid, ascites, pleural effusion, preferably saliva, blood, urine, .

It is preferable to use the saliva sampled at the precise time specified in the present invention for the diagnosis of gonococcosis according to the present invention at a time of less than 5 minutes after the morning wakeup, 25 to 35 minutes after the wakeup and 55 to 65 minutes after the wakeup .

Particularly, it is preferable to use saliva whose cortisol concentration is increased by at least 2.5 nmol / L over the first hour immediately after the morning wake.

In normal adults, the concentration of cortisol is reported to increase by 50-150% after morning waking, and it is known that the minimum 2.5 nmol / L increases from immediately after waking to 30 minutes after waking. However, it has also been reported that the increase in cortisol levels can not be observed in normal men and women when the first saliva samples are delayed for more than 10 minutes immediately after the morning wakeup. It is not possible to observe a phenomenon of increase in cortisol concentration in samples taken after 10 minutes or more after the gas phase, and these samples are preferably excluded from the test of the present invention.

Saliva samples can be collected at least three times immediately after the weather, 30 minutes after the weather, and 60 minutes after the weather. Even if the person to be surveyed indicates that the samples were collected immediately after the weather, Therefore, it is desirable to confirm whether the sampling of the first sample immediately after the morning weather is delayed, whether it has been collected at the prescribed time, and so on.

Thus, the method of the present invention may further include identifying a change in the concentration of cortisol to determine whether the prepared sample is suitable for analysis.

It is preferable to determine the sample to be used when the concentration of the cortisol is increased to at least 2.5 nmol in the first hour after the morning wakeup to check whether the sample is taken at the correct time through the change of the concentration of cortisol after the morning wake . That is, when the increase in the concentration of cortisol in the sample is less than 2.5 nmol for the first hour from immediately after the vapor phase, the sample can be excluded.

(3) In the sample Cortisol , DHEA and E2

In the present invention, the concentrations of cortisol, DHEA and E2 are quantified using the sampled samples, and the specific relationship between them is confirmed. Thus, it is determined that adrenal and reproductive endocrine glands associated with puberty development have matured, The necessary information can be obtained.

In the present invention, the concentration of cortisol, DHEA and E2 can be measured through a sample taken on the basis of a predetermined time interval in a target girl, , The concentration of cortisol, DHEA and E2 contained in the sample collected immediately after the gas phase, 30 minutes after the gas phase, and 1 hour after the gas phase can be measured at each time, and the change can be observed.

Further, DHEA and E2 concentrations can be further analyzed in the case where saliva as a sample is used when the increase in the concentration of cortisol is at least 2.5 nmol in the first hour immediately after the morning wakeup.

The cortisol, DHEA and E2 concentrations can be measured by various known methods and are not limited to specific methods.

That is, although there is no limitation, preferably, a radioimmunoassay method can be used. For example, enzyme immunoassay (EIA) using enzymes as markers, chemiluminescence immunoassay (CIA) with increased luminosity with luminol or its derivatives or ABEI (aminobutyl-ethylisoluminol) (fluorescence immunoassay (FIA)) using a fluorescent label can be used.

The radioimmunoassay has many advantages such as high sensitivity, precision, and accuracy and allows clear and clear detection after the reaction. Most preferably, the radioimmunoassay can be used For this, a liquid-phased-double antibody method can be used.

If blood, urine or saliva is sampled, it may be subjected to a pretreatment of the sample to use the radioimmunoassay method.

Since the concentration of steroid contained in saliva is as low as 1 / 100-1 / 1000 of blood, a highly sensitive assay system is required to measure a small amount of hormone contained in saliva. Such a liquid-double antibody (liquidphase-double antibody) method can be usefully used. A more detailed measurement process is a liquid-paper on hormone measurements using a double antibody method (Salivary cortisol and DHEA levels in the Korean population;. Age-related differences, diurnal, and correlations with serum levels Yonsei Med J. 2007; 48: 379-88 ).

In one embodiment of the present invention, a liquid-phased-double antibody using an antibody labeled with 125 ( ¹²⁵ I) and a secondary antibody was used.

(4) By time Cortisol / DHEA value ( Molar concentration  Ratio) Derived: Adrenal  Of puberty development by activation Progress  Obtain information

The activity of the adrenal endocrine axis can be determined by calculating the molar F / D ratio of cortisol to DHEA at each time using the values of cortisol and DHEA measured at each time in step a). In addition, information on whether the development of puberty in adolescent girls is the result of activation of adrenal endocrine system can be obtained.

When the hormone (A) and hormone (B) are produced and secreted in the same hormone producing organs and the production of two hormones is interrelated or the hormones play an opposite role, the molar ratio of the hormones The ratio between hormones means the balance of the production patterns of hormone-producing organs, or if two hormones counteract each other, the ratio between the two hormones means a balance between hormone action.

Both cortisol and DHEA are adrenocorticotropic hormones, which appear to increase the production of DHEA at the onset of puberty and "adrenarche" it. Thus, when the ratio of cortisol to DHEA (molar F / D ratio) decreases in adolescent onset and development, adrenarche leads to puberty onset and development.

In the present invention, the molar concentration of cortisol (molar cortisol / DHEA ratio) is determined by calculating the molar concentration of cortisol with respect to the molar concentration of DHEA by using the concentration of cortisol and DHEA measured in step a) in step b) Cortisol and DHEA can be used to determine the activity of the adrenal endocrine axis through the hourly molar ratio of the two hormones.

In this case, when the value of the molar concentration ratio of cortisol / DHEA per hour is 2.8 to 33 immediately after the vapor phase, 7 to 56 after 30 minutes after the vapor phase, and 3.8 to 36 after 60 minutes from the vapor phase in the subject's precocious puberty, The development of adolescent puberty was initiated by the activation of the adrenal endocrine system, and the degree of activation was judged to reach adult level.

(5) CARauc / Dauc  value( Molar concentration  Ratio) Derived: Adrenal  Of puberty development by activation Progress  Obtain information

The molar concentration (molar CARauc / Dauc ratio) of the DHEA total secretion amount relative to the total cortisol secretion amount secreted for the first hour after the gas phase was determined using the cortisol and DHEA concentration values measured for each hour in the step a) The activity of the endocrine system can also be judged. In addition, information on whether puberty has been initiated by the activation of the adrenal endocrine system can be obtained from the subject's depressed girl.

In the present invention, the concentrations of cortisol and DHEA measured in step (a) in step (b) are used to determine the concentrations of cortisol and DHEA measured in the saliva collected immediately after the morning, 30 minutes after, We can obtain the molar CARauc / Dauc ratio using CARauc and Dauc.

At this time, when the value of CARauc / Dauc (molar concentration ratio) was less than 3500 in the subject, the adolescent development of the precocious puberty girl started due to the activation of the adrenal endocrine system and the degree of activation reached to the adult level It can be judged. These values can also be used as an indicator of whether the CARauc / Dauc ratio during the first hour following morning wake up the adrenal cortex function.

(6) E2Inc%  Derivation of value: whether puberty development progressed due to activation of reproductive endocrine system And GnRH stimulation  In the reaction test To obtain a positive reaction  Obtain information on whether you can

Using the E2 concentration value measured in the step a), the percentage (%) relative to the E2 concentration increased for 1 hour after the meteorological phase based on the E2 basal concentration (concentration immediately after the morning wakeup) It is possible to judge whether development is the result of activation of reproductive endocrine system.

At this time, when E2Inc% value was 100%, the development of secondary sex (development and progression of breast development) of the female sexual precocious puberty girl started due to activation of the reproductive endocrine system and the activity of the reproductive endocrine system was confirmed It can be judged that the test has reached a level at which a positive reaction can be obtained, that is, the level of an adult.

(7) E2auci Value  Derivation: Whether adolescent development has progressed due to activation of the reproductive endocrine system; and GnRH stimulation  In the reaction test To obtain a positive reaction  Obtain information on whether you can

Using the E2 concentration value measured in step a), the total amount of E2 increased for the first hour after the meteorological phase immediately after the meteorological phase was analyzed to determine whether the pubertal development of the subject's prognosis girl was the result of the reproductive endocrine activation Can be obtained.

In this case, when the E2auc (pmol / L) value was 350 or more, the development of the secondary sex of the female adult with precocious puberty (onset and progression of breast development) was initiated by activation of the reproductive endocrine system, It can be judged that the level of positive test results in the test for the diagnosis of gliosis is reaching the level of adult.

Thus, the present invention can be used to determine the degree of activity of the adrenal endocrine system by determining the range of specific values using the concentrations of cortisol, dihydroepiandrosterone (DHEA), and estradiol (E2) And information on whether or not the reproductive endocrine axis is maturing can be obtained. Furthermore, using this information, it is possible to judge whether a positive reaction can be obtained in the GnRH stimulation reaction test.

That is, the present invention relates to a method for producing cortisol, which comprises using cortisol, DHEA and E2, a molar concentration ratio of total secreted amount of DHEA to cortisol total secreted amount for the first hour after the gas phase and an initial one hour after the metering based on the basal concentration for one hour after the gas phase Information on the neurological endocrine environment, adrenal gland and reproductive endocrine system of the girl can be known before the GnRH stimulation test by analyzing the percentage of purely increased E2 and the total amount of E2 in relation to the age of the subject's attending girl, Using this information, it is possible to determine whether a positive response in the GnRH stimulation test can be obtained, thereby reducing the various burdens that the parents of a girl, a medical person, and a girl who accompany the GnRH stimulation test (confirmation test) .

Example

Hereinafter, the present invention will be described in more detail with reference to Examples.

It should be apparent to those skilled in the art that these embodiments are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

1. Subjects

In order to ensure environmental homogeneity, the practice of the present invention is based on the consent of the mother of the girl and the girl who visited the pediatric department of the pediatric department of the university hospital in Gyeonggi province, Korea and the health and adolescent status of the participants .

Except for girls with any of the following criteria: 1) experienced menstruation; 2) oral growth hormone therapy or oral or transdermal glucocorticoid-containing medication within the last month; 3) I have a time lag due to long-distance travel by airplane within the past two weeks; 4) Diagnosis of chronic diseases.

In addition, adult women who meet any of the following criteria were excluded: 1) The previous menstrual cycle is regular, but irregular menstrual cycles occur more than once within 6 months prior to the start of this experiment; 2) taking hormone replacement therapy or oral contraceptives, tamoxifen, or other estrogen-, progesterone-, or glucocorticoid-containing medications within the past 6 months; 3) pregnancy or lactation, evidence of infertility or sexually transmitted disease, or diagnosis of ovarian failure; 4) Represents non-adjusted BMI (BMI less than or equal to 18 or greater than 25); Or 5) taking antidepressants or smokers. However, subjects who were last using oral contraceptives or steroid-containing drugs for more than 6 months were eligible.

Subjects who voluntarily participated in the study distributed or delivered a description of zipper bags and saliva collection containing three saliva sampling tubes. Fifty children from each of the prospective girls and girls' daughters received informed assent and consent, and all participating prospective girls were born in Korea and grew up at home with their biological parents.

This experiment was approved by the clinical examination committee of Dongtan Sacred Heart Hospital, Hallym University Medical School and conducted according to ethical recommendations.

2. Saliva collection

Since the F / D ratio after 30 minutes of morning weather changes daily with the stabilization period from Thursday to Sunday, participants should be alerted to the saliva immediately after the weather on Thursday to Sunday and at 30 minutes and 60 minutes They were asked to collect their saliva samples in a minimum volume of 1.5 mL.

In addition, participants were asked not to change the sleep-wake cycle.

Participants were instructed to collect saliva samples at defined times (less than 5 minutes after onset, 25 to 35 minutes and 55 to 65 minutes after occurrence), and in the marking area of the sampling tube (Simport Inc., QC, Canada) To take a note of the sampling time. The saliva was collected by spitting the saliva into the tube and the movement of the muscle without any external stimulus.

For an adult woman, she asked not to drink alcohol the night before. During the sampling period, participants were asked to avoid eating, drinking, or brushing the mouth and rinsing the mouth with water before each sampling. Steroid concentration in saliva is stable for at least 1 year after storage at -20 ° C or -80 ° C, and freezing and thawing of the sample up to 4 times before analysis does not affect the measured concentration, so the GnRH stimulation test By the day (up to one week) participants were asked to store their saliva samples in their own freezer. Saliva samples were submitted to pediatric adolescents and receptionist on the day the GnRH stimulation test was scheduled. At this time, as a result of visual inspection, samples with fatigue contamination were excluded from this experiment. Samples without time or date in the labeling area of the tube were also excluded.

A typical CAR is defined as an increase of at least 2.5 nmol / L in the cortisol level beyond the individual threshold within the adaptation subject to a defined collection time. In healthy subjects, typical CAR can not be observed if the first saliva sample is taken after more than 10 minutes delay after the morning wake.

In this case, the subjects were considered to be non-compliant subjects who did not observe the saliva collection time, and the saliva samples of non-compliance subjects were excluded from the subsequent analysis.

After the exclusion procedure, data from saliva samples collected from adult women with intact precocious puberty and 42 normal menstrual cycles were included in this study.

3. Saliva Purification

The collected samples were stored at -70 ° C after removing the debris. To remove mucin from saliva, the fountains were thawed and centrifuged (10,000 g, 15 min, 4). The supernatant was collected and stored at -70 ° C until the test was performed. All saliva samples were analyzed at the Hormonal Research Center of Chonnam National University.

4. Measurement of hormone concentration in saliva

The concentration of cortisol, DHEA and E2 in saliva was determined using radioimmunoassay.

Standard samples for cortisol, DHEA and E2 were obtained from Sigma-Aldrich Chemical Company (MO, USA). Standard solutions of cortisol, DHEA and E2 (10 mmol / L) were prepared from these standard samples and diluted with 0.1% gelatin containing 50 mmol / L phosphoric acid-buffered saline to prepare a standard solution A sample was prepared (pH = 7.2).

Iodine 125 ( ¹²⁵ I) was purchased from PerkinElmer (Mass., Ches., USA) and used. (I-125) iodohistamine, DHEA (DHEA-7- (O-cyclodextrin), and the like) by modifying the protocol for radioactive steroids previously presented in the paper. carboxymethyl) oximino- (2- [125 I] iodohistamine) and estradiol-3- (O-carboxymethyl) oximino- (2- [125 I] iodohistamine) were prepared by using Accurate Chemical & Scientific Co DHEA antiserum was purchased from United States Biological (Salem, Mass., USA) and E2 antiserum was purchased from Meridian Life Science, Inc. (ME, USA).

The cortisol antisera are cortisone, 11-deoxycortisol, prednisolone, cortisol-21-glucosiduronate, cortisol-21-sulphate -sulphate), and other steroids at a cross-reaction level of 1.0%, 8.9%, 31.6%, 1.3%, less than 0.01%, and less than 0.01%, respectively. DHEA antiserum cross-reacts with androstenedione, epiandrosterone, dihydrotestosterone, and other steroids with cross-reaction levels of less than 0.4%, 0.2%, 1.3%, and 0.01% do. The E2 antiserum cross-reacts with estriol, estrone dihydrotestosterone, and other steroids at a cross-reaction level of less than 0.09%, 0.5%, and 0.01%, respectively. Inter-assay coefficient of variation (CV) estimated from quality control using mean cortisol concentrations of 3.6 and 10.9 nmol / L were 7.4% and 8.5%, respectively (n = 26). The detection sensitivity for cortisol was 0.4 nmol / L. Inter-assay CVs from quality control using mean DHEA concentrations of 159.0 and 795.0 pmol / L were 12.5% and 10.1%, respectively (n = 26). The detection sensitivity for DHEA was 0.7 pmol / L. Inter-assay CVs assessed from quality control using mean E2 concentrations of 18.4 and 183.6 pmol / L were 11.5% and 13.2%, respectively (n = 26). The detection sensitivity for E2 was 3.7 pmol / L.

5. Data Analysis

Supplementary indices were applied to analyze the steroid secretion pattern during the first hour post-awakening. The concentration of cortisol, DHEA and E2 contained in each sample was determined by radioimmunoassay, and the total amount of cortisol, DHEA and E2 secreted from one hour after the start of the weather was measured by GraphPad Prism version 5.00 (Area under the curve, AUC) (CARauc, Dauc, and E2auc, respectively) using a computer program (GraphPad Software, CA, USA). The calculated ratio (molar CARauc / Dauc ratio) of DHEA total secretion amount to cortisol total secretion amount was calculated by using calculated CARauc and Dauc for 1 hour after the meteorological.

After determining the concentration of E2 contained in each sample, the percentage of the highest E2 concentration [(30 minutes (or 60 minutes) after E2 concentration (basal concentration) (E2 concentration immediately after the gas phase / E2 concentration immediately after the gas phase) * 100 was calculated (E2 concentration after awakening period, E2Inc%).

6. Statistical Analysis

The Shapiro-Wilk normality test revealed that the variables, ie steroid nodules and auxiliary indices, were not normally distributed at each test. Thus, the values were log-switched to normalize the sample distribution for the parametric statistical validation. However, in order to improve understanding of each figure, figures and tables are shown using natural numbers, and logarithmic conversion values are used only for statistical analysis. Group differences in mean steroid levels and supplemental indicators were analyzed using one-way or two-way ANOVA validation or bidirectional t-validation at the 5% significance level. When one-way ANOVA tests showed significant differences ( p <0.05), Tukey's post-hoc test was conducted to find specific group differences. Statistical calculations were performed using SAS version 9.1 (SAS Institute Inc., NC, USA). A p value of less than 0.05 was considered significant.

Example  1: molar F / D ratio (molar Cortisol / DHEA ratio ) Confirm difference

In normal adults, the concentration of cortisol is estimated to increase by 50 to 150% after the morning wake (Pressner et al. 1997), which is known to increase by at least 2.5 nmol / 2000). In particular, when the first saliva samples were delayed for more than 10 minutes immediately after the weather, normal cortisol levels were not observed (Okun et al., 2002). Further quantitative analysis of other steroids, DHEA or E2, in these samples is not relevant and should be excluded from the post hoc test for hormone response.

Before the GnRH stimulation test, the saliva samples from the participants were kept in a -70 ° C freezer until the GnRH stimulation test was obtained. The saliva samples were stored in adult female (n = (N = 16) or negative response (n = 22) after GnRH stimulation test. The results are summarized as follows. As shown above, (2.5 nmol / L minimum) without the typical increase in cortisol concentration.

In each group, adult female (n = 42), positive response girl (n = 16) on GnRH stimulation test and saliva sample negative response girl (n = 22) on GnRH stimulation test for the first hour The molar F / D ratio is shown in Fig.

The two-way ANOVA analysis showed that the molar F / D ratio was significantly lower than the group effect (F _2,231 = 29.9, p <0.0001) and the collection time effect F _{2, 231} = 18.3 p <0.0001) were significant, and group × time interaction effect (F _{4, 231} = 2.6, p = 0.04) .

This result implies that there is a significant difference in the level of molar F / D ratio between the three groups (significant group effect) and aspect (group × saliva collection time interaction effect).

The molar F / D ratio level of negative responded girls in the GnRH stimulation test was significantly higher in the GnRH stimulation test than in the positive responded girls and the adult females group with normal menstrual cycle ( P <0.0001 in all statistical analyzes) (Fig. 1).

In addition, the molar F / D ratio was significantly lower in the GnRH stimulation group than in the adult group (F _1,168 = 6.4, p <0.05) (Fig. 1).

The analysis of one-way ANOVA showed that the molar F / D-auc, which is the molar F / D ratio integrated over time, had a group effect (F _2,73 = 12.7, p <0.0001 ), And molar F / D-auc of the positive response girl group was similar to that of adult female ( p > 0.05) in the GnRH stimulation test (Fig. 2) / D-auc was significantly higher ( p <0.001 in all statistical analyzes) than the molar F / D-auc of the positive-reaction girl group and adult female (Fig. 2).

Table 1 summarizes the reference ranges for molar F / D-auc in each population.

F / D ratio-auc reference range of adult female, positive female and negative female 5% Lower Reference Limit 95% Upper Reference Limit Adult female 390.9 3483.5 Positive responded girls 404.9 2725.0 Negative responded girls 755.0 4416.2

The F / D ratio is lower in adult women and in positive-responders than in girls who received negative responses, because molar F (F) / D-auc is less than the upper 95 percentile of adult women, it can be judged that the adrenal endocrine system has been activated in adolescent development of the latent precocious puberty girl.

Example  2: molar F / D ratio (molar Cortisol / DHEA ratio)

In each group, adult female (n = 42), positive response girl (n = 16) on GnRH stimulation test and saliva sample negative response girl (n = 22) on GnRH stimulation test for the first hour The change in E2 is shown in Fig.

Two-way ANOVA was used to compare E2 with the group effect (F _2,231 = 26.4, p <0.0001) and the collection time effect (F _{2, 231} ( P <0.001). However, the interaction effect of group × saliva time was not statistically significant (group × time interaction effect) (F _{4, 231} = 1.5, p = 0.2).

This result implies that there is no difference in E2 secretion pattern (group × saliva collection time interaction effect) although there is a difference in the level of E2 (significant group effect) between the three groups.

In the GnRH stimulus test, negative responded girls E2 levels were significantly lower in the GnRH stimulus test than in the positive responded girls and the adult females group P < 0.0001) (Fig. 3).

In the GnRH stimulation test, the E2 level of the positive female group was significantly higher than that of the adult female group (F _1,168 = 4.3, p = 0.04) (Fig.

One-way ANOVA showed that the E2Inc% group had a group effect (F _2,73 = 3.6, p = 0.03) between the three groups (Fig. 4) In the GnRH stimulation test, the E2Inc% of the positive female group was similar to that of the adult female ( p > 0.05), while the E2Inc% of the negative female group was significantly lower than that of the positive female group and the female E2Inc% P < 0.01 in analysis) (Fig. 5).

One-way ANOVA showed that E2auci had a group effect (F _2,73 = 6.2, p = 0.005) between the three groups (Figure 5) E2auci of the positive response girl group was similar to that of adult women ( p > 0.05) and E2auci of the negative response girl group was significantly lower than that of the positive response girl group and E2auci of the adult female ( p < 0.01) (Fig. 6).

Table 2 and Table 3 summarize the reference ranges for E2Inc% and E2auci in each group.

Adult female, positive reaction girl and negative response E2Inc% reference range of girl 5% Lower Reference Limit 95% Upper Reference Limit Adult female 14.1 285.7 Positive responded girls 5.5 602.6 Negative responded girls -7.5 101.8

Adult female, positive reaction girl and negative response E2auci reference range of girl 5% Lower Reference Limit 95% Upper Reference Limit Adult female 82.0 3344.8 Positive responded girls 66.8 4916.0 Negative responded girls 0 333.2

The increase in E2 production during puberty development (referred to as "gonadarche") is due to the fact that both the hypothalamus, pituitary gland, and ovary, which constitute the reproductive endocrine system, can accommodate mutual signals and that each organ competence Results. Since the secretion level and secretion pattern of E2 during the first hour after the morning wake of the GnRH-positive female girl is the same as that of adult female, the hormone production capacity of the components of their reproductive endocrine system is equal to that of adult .

Therefore, when the E2inc% and E2auci values are higher than the 95th percentile of the negative response girls, it is believed that a positive reaction can be obtained in the GnRH stimulation test as a phenomenon caused by activation of the reproductive endocrine system in adolescent development of the sexual precocious puberty girl can do.

Claims (14)

Using the concentrations of cortisol, dehydroepiandrosterone (DHEA) and estradiol-17β, E2 measured from saliva collected from a girl aged 9 years or younger, the adrenal endocrine system and reproductive endocrine system A method for providing information for diagnosing a sexual precocious puberty comprising providing at least one endocrine activating judgment index among the series,
The adrenal endocrine system activation determination index is a ratio of a cortisol / DHEA mol concentration ratio (molar F / D ratio) and a molar concentration ratio of the cortisol / DHEA molar F / D ratio-auc (molar F / D- auc));
Wherein the reproductive endocrine activation indicator is a total amount of E2 secretion (E2auci) and a net increase in E2 (E2Inc%).
2. The method according to claim 1, wherein the adrenal endocrine system activation indicator
When the value of the cortisol / DHEA mole ratio (molar F / D ratio) is 25 or less after the gaseous phase is 69 or less; And the value of the molar concentration ratio (molar F / D-auc) of the cortisol and DHEA molar ratio is 3500 or less,
A method of providing information for diagnosis of gubernia, characterized in that pubertal development has been initiated due to the activation of the adrenal endocrine system and that the level of activation has reached adult level.
2. The method according to claim 1,
When the value of the total secretion amount E2auci of E2 is 350 or more; And a value of the net increase amount (E2Inc%) of E2 is 100 or more,
A method of providing information for diagnosis of gonococcosis characterized by the fact that the exogenous breast development is started due to activation of the reproductive endocrine system and the degree of activation of the gonadotropin has reached a level at which a positive reaction can be obtained in the test for the diagnosis of gonadal disease. .
The method according to claim 1, wherein the saliva is saliva collected within 1 hour after the weather of a girl aged 9 years or less.
5. The method of claim 4,
Wherein the saliva is collected in less than 5 minutes after the gas phase, 25 to 35 minutes after the gas phase, and 55 to 65 minutes after the gas phase.
3. The method according to claim 1 or 2,
(Molar F / D-auc) of the molar concentration ratio of cortisol and DHEA is an integral amount of the molar concentration ratio of cortisol and DHEA during the first hour after the gas phase, Way.
The method according to claim 1 or 3,
Wherein the total secretion amount (E2auci) of E2 is the total amount of E2 increased from the basal E2 concentration for the first hour after the vapor phase.
The method according to claim 1 or 3,
Wherein the net increase amount (E2Inc%) of the E2 is a percentage of a maximum value E2 concentration increased within the first hour after the meteorological rise based on the baseline E2 concentration.
8. The method of claim 7,
Wherein the basal E2 concentration is a concentration of E2 measured immediately after the gas phase or immediately after the gas phase in less than 5 minutes.
The method according to claim 1,
Wherein the method further comprises the step of eliminating saliva with an increase in the concentration of cortisol in the saliva of less than 2.5 nmol / L for the first hour from immediately after the vapor phase.
The method according to claim 1,
Wherein the sexual dysplasia is a central precocious puberty, intact premature ejaculation, peripheral precocious puberty or pacer precocious puberty.
13. The method of claim 12,
Wherein the glaucomatous disorder is a sexual precocious puberty.
The method according to claim 1,
Wherein the endocrine system activation judgment index is used as information for judging whether a positive reaction can be obtained in the GnRH stimulation reaction test.
A method for predicting the results obtainable in the intact precocious puberty confirmation test (GnRH stimulation test) using the information for diagnosing gonococci obtained according to claim 1.

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