Noninvasive prediction method and kit for acne risk of puberty women

Abstract

The invention discloses a non-invasive prediction method for acne risk of puberty women and a kit, and belongs to the technical field of biological detection. According to the present invention, the non-invasive advantage of the saliva sample is adopted, and the cycle specificity of saliva crystallization and the pathological correlation of the sex hormone ratio are combined to achieve the early accurate early warning of the acne risk, such that the problems of strong invasion and difficult promotion in the teenager group of the traditional serum detection are solved, and the characteristics of convenient operation, low cost and repeatable monitoring are provided; and a new technical means is provided for prevention and early intervention of acne of puberty women.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, specifically to a non-invasive method and reagent kit for predicting acne risk in adolescent girls based on the ratio of saliva crystal morphology to sex hormones. Background Technology

[0002] Acne is one of the most common chronic inflammatory skin diseases in adolescents. Its pathogenesis is complex and involves multiple aspects such as abnormal follicular keratinization, excessive sebum secretion, colonization of Propionibacterium acnes, and immune inflammatory response.

[0003] The occurrence of acne during puberty is closely related to endocrine dysfunction, with sex hormone levels being a key factor influencing its formation. Numerous studies have confirmed that enhanced androgen activity is one of the important mechanisms of acne development. Androgens are metabolized in sebaceous glands by 5α-reductase into the more active dihydrotestosterone (DHT), which stimulates sebum secretion and follicular keratinization by binding to androgen receptors, thereby inducing or aggravating acne. However, acne development is not solely due to the action of androgens; the balance of estrogen and progesterone also plays a crucial regulatory role. Estrogens (especially the most biologically active estradiol) are converted from androgens and can inhibit sebum secretion by counteracting androgen effects, thus having a certain ameliorative effect on acne. Progesters (such as progesterone), on the other hand, may stimulate sebum secretion and keratinocyte proliferation, and are associated with increased acne severity and incidence. This evidence suggests that, in addition to androgens, an imbalance between estrogen and progesterone is also an important mechanism in the occurrence and development of acne in women.

[0004] Currently, most research focuses on the mechanisms of androgen action, while studies on the dynamic changes of estrogen and progesterone in adolescent acne are still relatively lacking, especially in girls aged 14–17. This age group is a crucial period for studying the relationship between hormones and acne, as it is a stage of dramatic endocrine changes and gradual maturation of gonadal function during puberty, with significant fluctuations in hormone levels and unstable sebum secretion regulation. However, because the ovaries of this group are not yet fully mature and menstrual cycles are often irregular, their estrogen and progesterone secretion patterns differ from those of adult women, so conclusions from adult studies cannot be simply extrapolated. In adolescent girls, ovarian development is not yet complete, and estrogen and progesterone secretion is relatively insufficient; luteal insufficiency may lead to low estradiol levels before menstruation, and the relationship between these sex hormone characteristics and acne development remains unclear. Currently, there is limited literature on the correlation between acne and multiple sex hormones (including estrogen and progesterone) in this age group, which warrants further investigation.

[0005] Early identification of individuals at higher risk of acne development or with a tendency for acne to worsen is crucial for developing individualized prevention strategies and improving the quality of life for adolescents. While serum hormone testing has some reference value, its invasive nature makes it difficult to widely implement or conduct long-term dynamic monitoring among adolescents. Currently, there is a lack of ideal non-invasive, convenient, and low-cost hormone testing methods on the market; therefore, developing new predictive and assessment tools is essential.

[0006] In recent years, saliva biomarkers have received increasing attention as a non-invasive alternative to blood testing. Saliva contains various hormones, antibodies, and metabolites, reflecting the body's systemic state. It is also easy to collect, making it suitable for repeated monitoring in adolescents. Among these potential indicators, changes in saliva crystal morphology are particularly noteworthy. Crystallization, a common physical phenomenon, can reflect hormone levels, acid-base balance, and electrolyte status. Body fluids capable of crystallization include saliva, tears, cerebrospinal fluid, cervical mucus, and amniotic fluid. Among these fluids, saliva, due to its ease of collection and completely non-invasive nature, is the most suitable medium for long-term monitoring in adolescents. Studies have shown that saliva crystals exhibit regular changes during the menstrual cycle, regulated by estrogen and progesterone levels. Saliva crystal morphology (such as fern-like or irregular shapes) can not only reflect ovulation and reproductive health but may also reveal the impact of endocrine changes on skin diseases.

[0007] Previous studies on saliva crystallization analysis have primarily focused on ovulation prediction, with its application in research on the association between endocrine regulation during puberty and acne remaining largely unexplored. Existing literature lacks clinical trials investigating the relationship between saliva crystallization and sex hormone levels (especially estrogen and progesterone) in women aged 14–17, and even more so, systematic studies linking it to the occurrence and development of acne. Puberty, as a transitional period of rapid endocrine changes, is a crucial stage for exploring this association. Developing a risk prediction tool based on saliva crystallization and hormone levels would help identify susceptible individuals early, allowing for timely lifestyle or treatment interventions, thereby reducing the incidence and severity of acne.

[0008] Therefore, research on the correlation between saliva crystallization morphology and multiple hormone levels (including androgens, estrogens, and progesters) in women aged 14–17 years will not only help elucidate the endocrine mechanisms of acne development in this population, but also holds promise for establishing a non-invasive and convenient risk prediction tool. By dynamically monitoring saliva crystallization patterns and hormone fluctuations, acne-prone individuals can be identified early, providing a basis for proactive lifestyle interventions or treatments, thereby reducing the incidence and severity of acne and improving the psychological and social well-being of adolescents. Summary of the Invention

[0009] To address the aforementioned problems, this invention provides a biomarker for the prevention and / or diagnosis of adolescent acne, a non-invasive method for predicting acne risk in adolescent girls, and a dedicated kit for this method. By determining the menstrual cycle stage through saliva crystal morphology and combining this with the premenstrual T / E2 ratio, early and accurate warnings of acne risk are achieved, overcoming the problems of traditional serum testing being highly invasive and difficult to implement.

[0010] A first aspect of the present invention provides a biomarker for the prevention and / or diagnosis of adolescent acne, characterized in that the biomarker is saliva crystals and estradiol and testosterone in saliva.

[0011] In some implementations, the risk of developing acne is determined by detecting the crystal morphology of saliva and the concentrations of estradiol (E2) and testosterone (T) in the saliva supernatant.

[0012] Another aspect of the present invention provides a non-invasive method for predicting the risk of acne in adolescent girls, characterized by the following steps: 1) Sample collection: Select female subjects aged 14-17 years, who fast and abstain from water for 30 minutes before collection; 2) Sample processing: Centrifuge the collected saliva sample at 16000g for 15 minutes to separate the supernatant; take 10μl of the supernatant to prepare a smear, and dry it at room temperature for later use; 3) Observation of crystal morphology and determination of cycle: Observe the crystal morphology of the smear using a 400× optical microscope, and classify it into three types according to morphological characteristics: fern-like, mixed type, and disordered type, corresponding to the ovulation period, follicular period, and luteal phase, respectively; determine the menstrual cycle stage of the subject; 4) Hormone concentration detection: Detect the concentration of estradiol (E2) and testosterone (T) in the saliva supernatant separated in step 2) using enzyme-linked immunosorbent assay (ELISA); 5) Risk assessment: When the subject is determined to be in the premenstrual period, calculate the T / E2 ratio; if the ratio is higher than a preset threshold, the subject is determined to have a high risk of acne.

[0013] In some embodiments, the criteria for determining the crystal morphology in step 3) are as follows: fern-like crystals exhibit a regular, dense, symmetrical branching structure; mixed-type crystals exhibit a coexistence of some regular branches and disordered crystal points; and disordered-type crystals exhibit scattered dot-like or flocculent deposits.

[0014] In some implementations, the standard curve fitting method for ELISA detection in step 4) is as follows: estradiol is fitted using a quadratic function, and testosterone is fitted using a cubic or exponential function, with the R² of the fitted curve being ≥0.98.

[0015] In some implementations, the preset threshold in step 5) is >0.128, which is determined based on statistical analysis of the premenstrual T / E2 ratio between the acne group and the non-acne group.

[0016] In some implementations, if the saliva sample collected in step 1) is not processed immediately, it needs to be frozen at -80°C and used for subsequent testing after thawing.

[0017] The present invention also provides a dedicated kit for implementing the prediction method, characterized in that it includes: 1) Sample processing components: sterile centrifuge tubes, disposable smear slides, and centrifuge tube racks; 2) Crystallization observation tools: a 400× optical microscope user guide and a saliva crystal morphology classification comparison chart, wherein the comparison chart includes typical microscopic images of fern-like, mixed, and disordered crystals; 3) ELISA detection reagents: estradiol detection kits and testosterone detection kits, each kit containing standards, enzyme-labeled antibodies, chromogenic solution, stop solution, and washing solution; 4) Standard operating instructions: including sample collection specifications, sample processing steps, crystal morphology interpretation criteria, ELISA detection procedure, T / E2 ratio calculation method, and risk assessment threshold explanation.

[0018] In some embodiments, the standard concentration range of the estradiol test kit is 0-300 pg / mL, and the standard concentration range of the testosterone test kit is 0-50 ng / mL.

[0019] In some embodiments, the crystal morphology classification chart shows that fern-like crystals correspond to the ovulation period, mixed-type crystals correspond to the follicular period, and disordered-type crystals correspond to the luteal period, and the core morphological characteristics of each type of crystal are marked.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are:

[0021] 1. Non-invasive and convenient: Using saliva as the test sample, the collection process is non-invasive and painless, simple to operate, and requires no assistance from professional medical staff. It is suitable for repeated monitoring of adolescent girls aged 14-17.

[0022] 2. Precise and efficient: Combining the periodic specificity of saliva crystal morphology with the pathological correlation of the premenstrual T / E2 ratio, it identifies key stages and core indicators of acne high incidence, with a predictive sensitivity of 86.2% and a specificity of 83.7%.

[0023] 3. Early warning: It can identify high-risk individuals before acne occurs, providing a basis for lifestyle interventions (such as dietary adjustments and skin care) or early treatment, effectively reducing the incidence and severity of acne;

[0024] 4. Controllable cost: No complex medical equipment or invasive procedures are required. The reagent kits can be mass-produced, resulting in low testing costs, making them suitable for large-scale application in schools, community hospitals, and other settings.

[0025] 5. Safe and reliable: Saliva sample collection does not require blood collection, avoiding the risk of blood transmission. Furthermore, estradiol and testosterone levels in saliva are highly correlated with serum levels, and the test results can accurately reflect the body's hormone status. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below.

[0027] Figure 1 The volunteers (No. 12) provided three types of specimens: raw saliva smears, supernatant smears, and cell attachment smears.

[0028] Figure 2 Epithelial cells affect crystallization observation;

[0029] Figure 3 Saliva crystallization: (A) During ovulation, it appears as regular, dense, symmetrically branched fern-like crystals; (B) During the follicular phase, the crystals gradually transition to a mixed type or even some fern-like crystals; (C) During menstruation, it appears as sparse, irregular mixed fern-like structures; (D) During the luteal phase, it appears as sparse and irregularly arranged disordered crystals, distributed in flocculent or dot-like patterns.

[0030] Figure 4 Fitting curves of sex hormone concentration and OD value, (A) luteinizing hormone (B) estradiol (C) testosterone;

[0031] Figure 5 Menstrual cycle fluctuations of estradiol, progesterone, and testosterone in saliva (estradiol and progesterone in pg / ml; testosterone in ng / mL).

[0032] Figure 6 Blood concentration of estradiol was positively correlated with salivary concentration (R value 0.97, P < 0.01).

[0033] Figure 7 The correlation matrix of sex hormone concentrations in serum and saliva shows that red indicates a positive correlation, blue indicates a negative correlation, and the darker the color, the stronger the correlation. The numbers represent the r-values.

[0034] Figure 8 Dynamic changes in estradiol, testosterone, progesterone, and the testosterone / estradiol ratio at different time periods: comparison of differences between acne and non-acne groups (E2, estradiol; P4, progesterone; T, testosterone). Detailed Implementation

[0035] The present invention will be further described below with reference to specific embodiments:

[0036] To facilitate a better understanding of the present invention, but not to limit the invention, the experimental methods in the following embodiments are conventional methods unless otherwise specified. The experimental materials used in the following embodiments, unless otherwise specified, were all purchased from conventional biochemical reagent stores.

[0037] Example 1: Validation Experiment of the Prediction Method

[0038] 1.1 Volunteer Recruitment

[0039] We are recruiting healthy female secondary school students (aged 14-17) as volunteers.

[0040] Sign an informed consent form. Confirm that you do not have chronic oral diseases, infectious diseases, systemic diseases, heart, kidney, respiratory or liver failure, or ovarian dysfunction. Do not take any medications known to alter sex hormone levels.

[0041] Record all volunteers' menstrual cycle information (menstrual cycle, dates of the last three menstrual periods, duration of each period), and whether they suffer from acne. Volunteers with irregular menstrual cycles will be excluded.

[0042] Volunteers were recruited in two groups in June 2024 and May 2025.

[0043] In June 2024, 63 healthy female volunteers were recruited. Three failed the screening due to irregular menstruation, resulting in 60 volunteers being enrolled. The youngest was 14 years old, the oldest was 17 years old, and the average age was 15.2 years. The average menstrual cycle length was 29.6 days. 20 volunteers were currently suffering from acne, of whom 15 had moderate to severe acne.

[0044] In May 2025, 23 healthy female volunteers were recruited. Five failed the screening due to irregular menstruation, resulting in 18 final participants. The youngest was 14 years old, the oldest was 17 years old, and the average age was 15.5 years. The average menstrual cycle length was 29.2 days. Nine volunteers were currently suffering from acne, with six of them having moderate to severe acne.

[0045] 1.2 Sample Collection and Processing

[0046] All volunteers had non-irritating venous saliva collected between 8 and 10 a.m. to avoid circadian rhythm and dietary interference. No food or water was consumed for 30 minutes prior to collection. Saliva was collected naturally in 2–4 ml via sterile centrifuge tubes. All enrolled volunteers provided compliant saliva samples, with an average weight of 16.34 g. Three smears were preserved for crystal morphology observation. Figure 2 ).

[0047] Each sample was immediately divided into three parts: a raw saliva smear; the supernatant from centrifugation (16,000 g, 15 min), with 10 μl collected for smear preparation; and the cell wall-attached cells from the centrifuged precipitate for cell smear preparation. All remaining samples were stored at –80°C for subsequent hormone detection.

[0048] For some volunteers who volunteered to donate blood, 2 ml of venous blood was collected simultaneously at the same time as the saliva collection. The serum separation method was the same as that for saliva.

[0049] 1.3 Morphological observation of salivary liquid crystals

[0050] Take dried saliva smears and observe the crystal morphology using an optical microscope (400×); classify them into three types according to crystal branching morphology, density, and symmetry: fern-like, mixed, and disordered; each smear is independently evaluated by two experienced observers, and if the results are inconsistent, a third party makes the final decision.

[0051] Each volunteer provided three types of specimens: raw saliva smear, supernatant smear, and cell-attached smear. Figure 1 Samples that were not centrifuged or whose cellular components were resuspended in PBS after centrifugation and then smeared showed that excessively high epithelial cell density affected the observation of crystal morphology. Figure 2 Therefore, only the saliva supernatant slide specimen was retained as the subject of subsequent observation and research.

[0052] Crystallization morphology classification

[0053] In the dried saliva smears, a variety of different crystal morphological features were observed. Based on microscopic observations, these features were categorized as follows:

[0054] (1) Fern-like crystals: They exhibit a typical branched structure, similar to the veins of leaves or ferns, with a clear structure, and are often associated with elevated estrogen levels.

[0055] (2) Mixed crystallization: There are both regular branches and disordered crystal points, and the overall arrangement is irregular. It is often seen during the transition period of hormone levels.

[0056] (3) Disordered crystallization: lacks obvious branching and symmetrical structure, and is characterized by scattered dot-like or flocculent deposits, which are often associated with decreased hormone levels or enhanced progesterone effects.

[0057] The relationship between saliva crystallization and the menstrual cycle

[0058] Observe the saliva liquid crystals and take microscopic images (e.g.) Figure 3 It was discovered that saliva exhibits different fern-like and elliptical crystal morphologies depending on the physiological cycle. The changes in saliva crystals reflect the cyclical fluctuations of hormones. Preliminary studies have divided the saliva into the menstrual period, follicular period, ovulation period, and luteal period.

[0059] Around ovulation, estrogen levels peak, and typical fern-like crystals are most common. Under a microscope, regular, dense, symmetrical branches are visible, presenting a typical "fern-like" structure. Figure 3 A), this is the most prominent feature of the periodic changes in saliva crystallization.

[0060] During the follicular phase, estrogen levels gradually increase, and the crystals gradually transition to a mixed or even partially fern-like pattern. Figure 3 B). At this point, the crystal branches become clearer and the density increases, indicating that the regulatory effect of estrogen on the components of saliva begins to strengthen.

[0061] During menstruation, estrogen and progesterone levels are at their lowest, resulting in irregular, mixed crystal morphology. Some volunteers showed sparse, fern-like structures, but most lacked clear regularity, suggesting that crystals lack stability during this period. Figure 3 C).

[0062] During the luteal phase, progesterone levels rise while estrogen levels relatively decrease. Microscopic smears often show disordered crystals, with sparse and irregularly arranged crystals, frequently appearing as flocculent or punctate distributions. Figure 3 (D) This characteristic reflects the effect of progesterone on the electrolyte and protein composition of saliva. Therefore, the inventors have found that saliva crystallization has significant value as an indicator of menstrual cycle phase.

[0063] 1.4 Hormone level detection, data collection, and statistical analysis

[0064] The levels of sex hormones in saliva and serum, including estradiol (E2), progesterone (P), testosterone (T), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin (PRL), were determined using enzyme immunoassay (EIA).

[0065] All tests were performed in the same laboratory using the same batch of reagent kits.

[0066] By collecting general information (age, body mass index, menstrual cycle history, acne status), crystallization characteristics (cyclical changes, subtype ratios), and hormone levels (saliva and serum hormone values), we analyzed salivary hormone levels and their correlation with blood sex hormone levels. Based on the morphological characteristics of salivary crystals, the non-menstrual period was divided into the luteal phase, follicular phase, and ovulation phase, and the hormone level characteristics of different phases were observed. The differences in crystallization morphology and hormone levels at different phases between the acne group and the non-acne group were evaluated.

[0067] This invention uses GraphPad software for analysis, and the statistical analysis methods used include:

[0068] (1) Descriptive statistics: Normally distributed data are represented by mean ± standard deviation, and non-normally distributed data are represented by median (interquartiles).

[0069] (2) Linear Fitting Analysis: In ELISA testing, a reliable mathematical model (standard curve) needs to be established based on the absorbance values ​​(OD values) of known concentrations of standards to accurately calculate the concentration of unknown samples. This invention uses the concentration of the standards as the x-axis and the measured absorbance values ​​as the y-axis to plot a scatter plot on a coordinate system. The most suitable mathematical model (most commonly a linear model) is selected using software to fit these points, resulting in a curve that best represents the trend, i.e., the standard curve. The OD value of the unknown sample is then substituted into the fitted curve formula to calculate its corresponding concentration value.

[0070] (3) Comparative analysis: The t test or Mann-Whitney U test is used for comparison between two groups, and the ANOVA or Kruskal-Wallis test is used for comparison of multiple groups.

[0071] (4) Correlation analysis: Spearman analyzed the correlation between saliva and serum hormone levels and used OriginPro software to draw a correlation matrix.

[0072] This invention further plotted a fitting curve for the OD value and found that the luteinizing hormone concentration and the OD value were basically linearly related. Figure 4 A), the transformation curve of estradiol has the properties of a quadratic function. Figure 4 B), the testosterone curve shows a cubic or exponential trend (B). Figure 4 C).

[0073] The results of the detection of six hormones affecting the endocrine system in serum and saliva samples are shown in Table 1.

[0074] Table 1. ELISA results of sex hormones in serum and saliva.

[0075]

[0076] Notes: FSH, follicle-stimulating hormone; LH, luteinizing hormone; PRL, prolactin; E2, estradiol; P, progesterone; T, testosterone;

[0077] To conduct subsequent analysis, the inventors standardized the sample data by aligning the mean and variance using probability statistics, and performed the SW test on the mean to compare whether the correlation was statistically significant.

[0078] Table 2 Comparison of the concentrations of various hormones in saliva and serum samples

[0079]

[0080] Remark: , , These represent significance levels of 1%, 5%, and 10%, respectively; bold text indicates that saliva concentrations are higher than serum concentrations.

[0081] The results of saliva sex hormone level testing indicated that the levels of estradiol (E2) and progesterone (P) in saliva were higher than those in serum samples, and testosterone showed an increasing trend. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were lower than those in serum samples.

[0082] The levels of several sex hormones in saliva that were higher than in blood were grouped according to different menstrual cycles, and the hormonal changes across different menstrual cycles were compared. Data analysis indicated that both estradiol and progesterone levels showed significant fluctuations.

[0083] Estradiol levels exhibit a bimodal variation throughout the menstrual cycle (e.g.) Figure 5 Estradiol levels are low during menstruation and the early follicular phase. As the follicle develops, secretion increases, reaching its first small peak before ovulation. After ovulation, levels briefly decline, then rise again during the luteal phase, forming a second peak. Thereafter, estradiol levels drop sharply.

[0084] Progesterone levels are very low during the follicular phase and menstruation; after ovulation, progesterone levels begin to rise, reaching a peak during the luteal phase (e.g., ...). Figure 5 ).

[0085] Testosterone levels fluctuate throughout the menstrual cycle, but the amplitude is relatively small. Levels are relatively low during the follicular phase. Testosterone levels increase during ovulation. Levels further increase during the luteal phase compared to the follicular phase (e.g., ...). Figure 5 (As shown by the yellow line).

[0086] Correlation analysis of hormone components in saliva and serum

[0087] This invention conducted a correlation analysis between salivary hormone levels and serum hormone levels and found that estradiol (E2) in saliva (Spearman r = 0.97, P < 0.01) was significantly higher than that in serum. Figure 6 and Figure 7 Testosterone (T) (Spearman r = 0.76, P < 0.01, Figure 7 ) and follicle-stimulating hormone (FSH) (Spearman r = 0.39, P < 0.05, Figure 7Hormone levels were significantly positively correlated with blood hormone levels. Combined with the findings observed in the previous section that estradiol and testosterone levels in saliva were higher than in blood, the results support the feasibility of using saliva as a non-invasive alternative bodily fluid to reflect the body's estradiol and testosterone status.

[0088] The Relationship Between Saliva Crystallization Morphology and Composition and Acne Risk

[0089] Due to the sample size, statistical significance analysis of differences could not be performed at some time points; we only describe the trends.

[0090] like Figure 8 As shown, the salivary testosterone concentration in the premenstrual period (pink marked area) of volunteers in the acne group was significantly higher than that in non-acne volunteers, accompanied by a significant increase in progesterone, a slight decrease in estradiol concentration, and a significant increase in the testosterone / estradiol ratio, suggesting that the relative dominance of androgens may be one of the pathological mechanisms.

[0091] At other stages of the menstrual cycle, estradiol and testosterone levels, especially the testosterone / estradiol ratio, did not differ significantly between the acne group and the non-acne group.

[0092] Example 2: Usage procedure of the dedicated reagent kit

[0093] 2.1. The user opens the kit, reads the standard operating instructions, and confirms that all required components are complete (sample processing components, crystallization observation tools, ELISA detection reagents).

[0094] 2.2. Collect a 2ml saliva sample between 8 and 10 a.m. according to the instructions. Do not eat or drink for 30 minutes before collection. Transfer the sample into a sterile centrifuge tube.

[0095] 2.3. Centrifuge at 16000g for 15 minutes, take 10μl of supernatant to prepare a smear, dry at room temperature, and observe and determine the menstrual cycle stage under an optical microscope by referring to the crystal morphology classification chart.

[0096] 2.4. If the condition is determined to be premenstrual, remove the ELISA test kit and follow the instructions to measure the concentrations of estradiol and testosterone in the saliva supernatant;

[0097] 2.5. Calculate the T / E2 ratio. If the ratio is >0.128, it indicates a high risk of acne. It is recommended to adjust your diet, strengthen skin cleansing, or go to the hospital for further examination.

[0098] The above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

[0099] While specific embodiments of the invention have been described in detail by way of example, those skilled in the art should understand that the examples are for illustrative purposes only and not intended to limit the scope of the invention. It should also be understood that various modifications can be made to the embodiments without departing from the scope and spirit of the invention. The scope of the invention is defined by the appended claims.

Claims

1. A biomarker for the prevention and / or diagnosis of acne in adolescent girls, characterized in that, The biomarkers are saliva crystals and estradiol and testosterone in saliva.

2. The biomarker for the prevention and / or diagnosis of acne in adolescent girls according to claim 1, characterized in that, The risk of developing acne can be determined by detecting the crystal morphology of saliva and the concentrations of estradiol (E2) and testosterone (T) in the saliva supernatant.

3. A non-invasive method for predicting the risk of acne in adolescent girls, characterized in that, Includes the following steps: 1) Sample Collection: Female subjects aged 14-17 years were selected. Fasting and abstaining from water were required for 30 minutes prior to sample collection. 2) Sample Processing: The collected saliva samples were centrifuged at 16000g for 15 minutes to separate the supernatant. 10 μl of the supernatant was used to prepare a smear, which was then dried at room temperature. 3) Crystal Morphology Observation and Cycle Determination: The crystal morphology of the smear was observed using a 400× optical microscope. Based on morphological characteristics, the crystals were classified into three types: fern-like, mixed, and disordered, corresponding to the ovulation period, follicular phase, and luteal phase, respectively. This determined the subject's menstrual cycle stage. 4) Hormone Concentration Detection: The concentrations of estradiol (E2) and testosterone (T) in the saliva supernatant separated in step 2) were detected using enzyme-linked immunosorbent assay (ELISA). 5) Risk Assessment: When a subject was determined to be in the premenstrual phase, the T / E2 ratio was calculated. If the ratio was higher than a preset threshold, the subject was considered to have a high risk of acne.

4. The non-invasive prediction method according to claim 3, characterized in that, The criteria for determining the crystal morphology in step 3) are as follows: fern-like crystals exhibit a regular, dense, symmetrical branching structure; mixed-type crystals exhibit the coexistence of some regular branches and disordered crystal points; and disordered-type crystals exhibit scattered dot-like or flocculent deposits.

5. The non-invasive prediction method according to claim 3, characterized in that, In step 4), the standard curve fitting method for ELISA detection is as follows: estradiol is fitted using a quadratic function, and testosterone is fitted using a cubic or exponential function, with the R² of the fitted curve being ≥0.

98.

6. The non-invasive prediction method according to claim 3, characterized in that, The preset threshold in step 5) is >0.128, which is determined based on statistical analysis of the premenstrual T / E2 ratio between the acne group and the non-acne group.

7. The non-invasive prediction method according to claim 3, characterized in that, If the saliva sample collected in step 1) is not processed immediately, it should be frozen at -80℃ and used for subsequent testing after thawing.

8. A dedicated kit for implementing the prediction method according to any one of claims 3-7, characterized in that, include: 1) Sample processing components: sterile centrifuge tubes, disposable smear slides, centrifuge tube racks; 2) Crystallization observation tools: 400× optical microscope usage guide, saliva crystal morphology classification comparison chart, which includes typical microscopic images of fern-like, mixed, and disordered crystals; 3) ELISA test reagents: estradiol test kit, testosterone test kit, each kit includes standards, enzyme-labeled antibodies, chromogenic solution, stop solution, and washing solution; 4) Standard operating instructions: including sample collection specifications, sample processing steps, crystal morphology interpretation criteria, ELISA testing procedure, T / E2 ratio calculation method, and risk assessment threshold explanation.

9. The dedicated reagent kit according to claim 8, characterized in that, The standard concentration range of the estradiol detection kit is 0-300 pg / mL, and the standard concentration range of the testosterone detection kit is 0-50 ng / mL.

10. The dedicated reagent kit according to claim 8, characterized in that, In the crystal morphology classification chart, fern-like crystals correspond to the ovulation period, mixed-type crystals correspond to the follicular period, and disordered-type crystals correspond to the luteal period, and the core morphological characteristics of each type of crystal are marked.

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