CN117925807A - Application of UHRF2 gene in diagnosis of premature ovarian failure - Google Patents
Application of UHRF2 gene in diagnosis of premature ovarian failure Download PDFInfo
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Abstract
The invention provides application of UHRF2 genes in diagnosing premature ovarian dysfunction, in particular discloses a novel premature ovarian dysfunction disease risk marker, which comprises the UHRF2 genes, and correspondingly develops a method and a kit for predicting and/or judging the premature ovarian dysfunction disease risk.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of UHRF2 gene in diagnosis of premature ovarian failure.
Background
Fertility preservation has become a focus of attention in recent years as fertility decreases and fertility age increases. The ovaries are the important reproductive organs of females and are responsible for storing, producing ova and secreted sex hormones, promoting and maintaining secondary female sex characteristics. For fertility protection in women, therefore, the ovaries are first of all important. Ovarian function gradually declines with age in females.
Hypoovaries, abnormal menstruation, elevated serum FSH levels (FSH > 25U/L), decreased estrogen volatility, clinically known as premature ovarian failure (Premature ovarian insufficiency, POI) occur before age 40. The incidence rate of POI in China is 1% -5%, female patients with about 400 ten thousand childbearing age are accumulated, and the disease is in a younger trend. POI has strong concealment, can develop into premature ovarian failure, and is harmful to female reproduction and female health. Thus, research into POIs is important for protecting female fertility and life health.
The POI mechanism is complex, and the etiology of about 60% of patients is still unknown. The means for treating the disease clinically is also very limited. Clinically, the diagnosis of POI is mainly based on comprehensive judgment of detailed medical history and comprehensive medical monitoring of patients, and the diagnosis result has higher dependence on subjective experience of doctors. In order to make diagnosis of POI more objective, reduce human factors, improve consistency and accuracy of diagnosis, it is necessary to find POI biomarker, thereby establishing an effective detection method, effectively reducing uncertain human factor interference in current diagnosis, and reducing misdiagnosis rate.
Disclosure of Invention
The invention aims to provide an application of UHRF2 gene in diagnosing premature ovarian failure.
In a first aspect of the invention, there is provided a marker for predicting or diagnosing the risk of developing premature ovarian dysfunction, said marker being the UHRF2 gene, a transcript thereof, or an expressed protein thereof.
In a second aspect of the invention, there is provided the use of a UHRF2 gene, a transcript thereof, an expressed protein thereof, or a detection reagent thereof, for the preparation of a diagnostic reagent or kit for assessing the risk of developing premature ovarian dysfunction.
In another preferred embodiment, the diagnostic reagent or kit is used to detect the level of the UHRF2 gene, its transcript, or its expressed protein in a test sample.
In another preferred embodiment, the sample to be tested is selected from the group consisting of: blood, plasma, serum, follicular granulosa cells, or a combination thereof.
In another preferred embodiment, the expression level comprises an expression level in blood, plasma, serum or granulosa cells.
In another preferred embodiment, the evaluation includes early diagnosis, auxiliary diagnosis, or a combination thereof.
In another preferred embodiment, the UHRF2 gene, its mRNA, its cDNA, or its encoded protein is of human origin.
In another preferred embodiment, the detection reagent is coupled to or carries a detectable label.
In another preferred embodiment, the detectable label is selected from the group consisting of: chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.
In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.
In another preferred embodiment, the antibody is a monoclonal antibody or a polyclonal antibody.
In another preferred embodiment, the nucleic acid chip comprises a substrate and specific oligonucleotide probes spotted on the substrate, wherein the specific oligonucleotide probes comprise probes specifically binding to the UHRF2 gene polynucleotide (mRNA or cDNA).
In another preferred embodiment, the test sample is from a patient suffering from premature ovarian failure.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for (a) diagnosing a risk of developing premature ovarian failure, and/or (b) evaluating the effect of treatment of premature ovarian failure.
In another preferred embodiment, the label or description refers to the following: if the expression level of UHRF2 gene in the test subject is significantly lower than the control reference value, it is suggested that the risk of developing premature ovarian failure in the test subject is high.
In another preferred embodiment, if the expression level of UHRF2 gene in the test subject is not lower than the control reference value, it is indicative that the test subject is at low risk of developing premature ovarian failure.
In another preferred embodiment, the label or description refers to the following:
If the expression level L1 of said UHRF2 gene in the test subject is significantly lower than the control reference value L0, the subject has a high probability of premature ovarian failure.
In another preferred embodiment, the control reference value L0 is the UHRF2 gene expression level of the same sample in the normal population.
In another preferred embodiment, the term "significantly lower" means that the ratio L1/L0 is 0.9 or less, preferably 0.8 or less, more preferably 0.5 or less.
In another preferred embodiment, if the mRNA level of the UHRF2 gene or the level of UHRF2 protein is reduced, it is indicative that the risk of premature ovarian failure in the subject is high.
In a third aspect of the present invention, there is provided an apparatus for risk assessment of premature ovarian dysfunction, the apparatus comprising:
An input unit configured to input expression level data of a UHRF2 gene in a detection sample of a certain detection object;
A data processing unit configured to: comparing the expression level L1 of the UHRF2 gene with a control reference value L0, wherein when L1 is significantly lower than L0, the subject is prompted to be at high risk for developing premature ovarian failure; when L1 is not remarkably lower than L0, the risk of the premature ovarian dysfunction of the subject is low; and
And the output unit is used for outputting the evaluation result.
In another preferred embodiment, the term "significantly lower" means that the ratio L1/L0 is 0.9 or less, preferably 0.8 or less, more preferably 0.5 or less.
In another preferred embodiment, the apparatus further comprises a detection unit for detecting the expression level of the UHRF2 gene; preferably, for example, the transcript level, protein level, or protein activity of the UHRF2 gene.
In another preferred embodiment, the detection unit is selected from the group consisting of: a PCR detector, a sequencer, or a combination thereof.
In a fourth aspect of the invention, there is provided a method of assessing the risk of developing premature ovarian failure, the method comprising the steps of:
detecting the expression level of UHRF2 gene in a detection sample of a detection object;
comparing the expression level L1 of the UHRF2 gene obtained by detection with a control reference value L0, wherein when L1 is obviously lower than L0, the detection object is indicated to have high risk of suffering from premature ovarian dysfunction; when L1 is not significantly lower than L0, the test subject is indicated to have a low risk of premature ovarian dysfunction.
In another preferred embodiment, the term "significantly lower" means that the ratio L1/L0 is 0.9 or less, preferably 0.8 or less, more preferably 0.5 or less.
In another preferred embodiment, the level of expression of the UHRF2 gene is the transcript level, protein level, or protein activity of the UHRF2 gene.
In another preferred embodiment, the test sample is selected from the group consisting of: blood, plasma, serum, granulosa cells, or a combination thereof.
In another preferred embodiment, the expression level comprises an expression level in blood, plasma, serum or granulosa cells.
In another preferred embodiment, the evaluation includes early diagnosis, auxiliary diagnosis, or a combination thereof.
In another preferred embodiment, the method detects the level of UHRF2 gene, mRNA thereof, cDNA thereof, or encoded protein thereof in a sample.
It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.
Drawings
Fig. 1 PCA analysis of disease and control groups.
Figure 2 correlation analysis of disease and control groups.
FIG. 3 RNA heatmaps of the disease and control groups.
FIG. 4 GO analysis of differential genes.
Detailed Description
Through extensive and intensive studies, the present inventors have discovered for the first time a novel risk marker for premature ovarian dysfunction, including the UHRF2 gene, and have accordingly developed methods and kits for predicting and/or assessing the risk of premature ovarian dysfunction. The present invention has been completed on the basis of this finding.
Experiments show that the early-onset ovarian dysfunction disease risk marker or the combination thereof can effectively early warn or diagnose the potential risk of the early-onset ovarian dysfunction of an individual, and the marker is adopted to predict and/or detect the disease possibility or disease state of the early-onset ovarian dysfunction, has higher accuracy and specificity, and provides reference standard for early-onset ovarian dysfunction early-warning and/or diagnosis in clinic.
The detection system based on the marker provides a relatively objective technical means for evaluating premature ovarian failure, can effectively avoid diagnosis deviation problems caused by scale analysis and subjective experience evaluation, and improves the objectivity and accuracy of clinical evaluation.
The terms used in the present invention have meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:
According to the invention, the term "premature ovarian failure" (Premature ovarian insufficiency, POI) refers to a decrease in gonadal function in women before age 40, manifested by secondary amenorrhea, infertility, perimenopausal symptoms often accompanied by sweating, insomnia, hypomnesis, etc. during nocturnal sleep.
According to the present invention, the term "biomarker", also referred to as "biomarker", refers to a measurable indicator of the biological status of an individual. Such biomarkers can be any substance in an individual as long as they are related to a particular biological state (e.g., disease) of the individual being tested, e.g., nucleic acid markers (e.g., DNA), protein markers, cytokine markers, chemokine markers, carbohydrate markers, antigen markers, antibody markers, species markers (markers of species/genus), functional markers (KO/OG markers), and the like. Biomarkers are measured and evaluated, often to examine normal biological processes, pathogenic processes, or therapeutic intervention pharmacological responses, and are useful in many scientific fields.
According to the invention, the term "individual" refers to an animal, in particular a mammal, such as a primate, preferably a human.
Markers for risk of premature ovarian failure
In the present invention, the terms "risk marker for premature ovarian dysfunction", "risk marker for premature ovarian dysfunction of the present invention" and "risk marker of the present invention" are used interchangeably and refer to the UHRF2 gene, its expression level, its expression product or activity.
In the present invention, the risk markers of the present invention include genes (DNA), transcripts (mRNA), cdnas, proteins, or combinations thereof.
The proteins of the markers of the invention may or may not contain an initiating methionine. Furthermore, the term also includes full-length risk marker proteins and fragments thereof. In the present invention, the risk marker proteins include their complete amino acid sequences, their secreted proteins, their mutants, and functionally active fragments thereof.
According to the present invention, the term "UHRF2 Gene" refers to a Gene encoding a UHRF2 (Ubiquitin LIKE WITH PHD AND RING FINGER domains 2) protein, such as the UHRF2 Gene introduced by NCBI website accession number Gene ID: 115426. The protein encoded by the UHRF2 gene is an epigenetic protein having a plurality of functional domains, including UBL, TTD, PHD, SRA and RING domains. Wherein the UBL domain is associated with ubiquitin enzyme, the TTD and PHD domains bind to histone H3, the SRA domain is responsible for binding hydroxymethyl 5' -cytosine (5 hmC) and activating UHRF2, and the RING domain is associated with domain E3 ubiquitin ligase. Biological functions and cell cycle of UHRF2, DNA damage repair, and ubiquitin-proteinase system are found to be related in oncology and cell biology. However, there is no report of the physiological function of UHRF2 revealed at the individual level, and there is no application of UHRF2 to diagnosis of premature ovarian failure and assessment of ovarian function.
Correlation of UHRF2 Gene as Risk marker for early-onset ovarian insufficiency
In the present invention, the inventors have unexpectedly found that UHRF2 can be a risk marker for premature ovarian failure.
According to the invention, through analyzing the granulosa cell sample of the patient with premature ovarian dysfunction and the granulosa cell sample of the normal granulosa cell (derived from the patient with non-premature ovarian dysfunction), the UHRF2 gene expression level in the granulosa cell sample of the patient with premature ovarian dysfunction is obviously reduced compared with that in a control group. Correlation analysis shows that UHRF2 gene expression level has correlation with premature ovarian failure. Using these data, the present invention developed and validated a method/system for predicting risk of premature ovarian dysfunction.
Detection method
Based on the expression level of UHRF2, a risk marker for premature ovarian dysfunction, in blood, plasma, serum or granulosa cells, the invention provides a corresponding method for diagnosing or assisting in diagnosing premature ovarian dysfunction.
The invention relates to a diagnostic test method for quantitatively detecting UHRF2 gene, transcript level or protein level of a risk marker for premature ovarian dysfunction. Such tests are well known in the art. The risk marker UHRF2 gene, transcript level thereof, or protein level thereof detected in the assay can be used to diagnose (including aid in diagnosis of) the risk of developing premature ovarian dysfunction.
In a preferred embodiment of the present invention, the detection method of the present invention performs PCR quantitative detection of mRNA or cDNA.
In a preferred embodiment of the present invention, the detection method of the present invention performs a sequencing quantitative detection of mRNA or cDNA.
In a preferred embodiment of the invention, the detection method of the invention quantitatively detects the protein encoded by the UHRF2 gene.
Preferably, one method of detecting the presence or absence of a marker protein in a sample is by using a specific antibody, which comprises: contacting the sample with an antibody specific for a protein encoded by the UHRF2 gene; observing whether an antibody complex is formed, wherein the formation of the antibody complex indicates the presence of a risk marker for premature ovarian failure in the sample.
The premature ovarian dysfunction disease risk marker protein or the polynucleotide thereof can be used for diagnosing the premature ovarian dysfunction disease. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in mononuclear cells and for gene diagnosis. Antibodies against the risk markers of premature ovarian dysfunction may be immobilized on a protein chip for detection of the risk marker protein of premature ovarian dysfunction in a sample.
Based on the studies of the present invention, there was a significant decrease in the expression level (mRNA level or protein level) of UHRF2 gene in patients suffering from premature ovarian failure. Thus, the UHRF2 gene can be used as a marker for detecting or diagnosing (especially for aiding diagnosis and/or early diagnosis) the risk of developing premature ovarian dysfunction. In the detection, if the ratio of the marker level L1 to the corresponding level L0 in the normal population (L1/L0) is not more than 0.9, preferably not more than 0.8, more preferably not more than 0.5, then the risk of developing premature ovarian failure is considered to be increased.
In a preferred embodiment of the invention, detection is performed using real-time fluorescent quantitative PCR.
Real-time fluorescent quantitative PCR is an experimental method that applies fluorescent energy technology to the polymerase chain reaction. In a PCR reaction system, SYBRGreen I emits a fluorescent signal after being specifically doped into a DNA double strand; while SYBR dye molecules that are not incorporated into the chain do not emit any fluorescent signal. Because this method allows the increase in fluorescence signal to be synchronized with the increase in PCR product, that is, the intensity of fluorescence signal emitted by the fluorescent dye is proportional to DNA yield. Therefore, the initial concentration of the target sequence can be obtained by detecting the fluorescence signal intensity in the PCR process, so that the aim of quantification can be fulfilled.
Detection kit
Based on the correlation of UHRF2 gene and risk of premature ovarian dysfunction, UHRF2 gene can be used as diagnostic marker for the risk of premature ovarian dysfunction.
The invention also provides a kit for diagnosing the risk of premature ovarian dysfunction, which contains a detection reagent for detecting the UHRF2 gene, the transcript thereof, the expressed protein thereof or the combination thereof, which is a marker of the risk of premature ovarian dysfunction. Preferably, the kit contains an anti-UHRF 2 antibody or immunoconjugate of the invention, or an active fragment thereof; or a primer or primer pair, a detection probe or a detection chip containing mRNA or cDNA specifically amplified UHRF 2.
In another preferred embodiment, the kit further comprises a label or instructions stating that the kit is used for diagnosing the risk of developing premature ovarian dysfunction and/or evaluating the efficacy of a treatment for developing premature ovarian dysfunction.
Detection device
In a preferred embodiment of the present invention, the present invention provides an apparatus for risk assessment of premature ovarian failure, the apparatus comprising:
An input unit configured to input expression level data of a UHRF2 gene in a detection sample of a certain detection object;
A data processing unit configured to: comparing the expression level L1 of the UHRF2 gene with a control reference value L0, wherein when L1 is significantly lower than L0, the subject is prompted to be at high risk for developing premature ovarian failure; when L1 is not remarkably lower than L0, the risk of the premature ovarian dysfunction of the subject is low; and
And the output unit is used for outputting the evaluation result.
The main advantages of the invention include:
(1) The UHRF2 gene can be used as a risk marker for premature ovarian failure disease for the first time;
(2) The risk marker can efficiently and accurately predict the risk of premature ovarian failure;
(3) The detection system can early warn and evaluate the risk of premature ovarian failure, and is used for early diagnosis or auxiliary diagnosis of premature ovarian failure.
The present invention will be described in further detail with reference to the following examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The following examples are not to be construed as limiting the details of the experimental procedure, and are generally carried out under conventional conditions such as those described in the guidelines for molecular cloning laboratories, sambrook.J.et al, (Huang Peitang et al, beijing: scientific Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.
EXAMPLE 1 screening of biomarkers of premature ovarian dysfunction
In the case of artificial Insemination (IVF) in women, the wash waste of their ova contains many granulosa cells. Granulosa cells were used as subjects. Granulosa cells were divided into two queues, early onset ovarian dysfunction (POI) and control, according to the criteria of follicle stimulating hormone FSH, anti-mullerian hormone AMH and sinus follicle number ACs. Each queue has two sets of parallel. To reduce individual variability, each group was mixed from 3 clinical samples. Gene detection at mRNA level was performed on two cohorts, four sets of mixed samples (2V 2), respectively.
1. Patient group entry and sample collection
The granulosa cell samples were all from the tenth people's hospital in Shanghai, affiliated to the university of Shanghai, and after written consent was obtained from women who had made artificial Insemination (IVF) in this hospital in 2020, clinical information was recorded.
All women who are included in the study are established and diagnosed at the reproduction center of a tenth people hospital attached to Shanghai city at the university of Shanghai according to the requirements, and the special people collect basic information data of patients, such as age, follicle stimulating hormone, anti-Mueller tube hormone AMH, basic sinus follicle quantity AFCs and the like. FSH >40IU/L (interval time exceeds 1 month) was performed twice according to clinical criteria. AMH is less than or equal to 1 mug/L, AFC is less than or equal to 9, and the POI can be diagnosed as ovarian dysfunction. All washed waste samples were centrifuged at low speed (1000 g) at low temperature (4 ℃) to obtain granulosa cells and immediately transferred to centrifuge tubes for storage at-80 ℃ for testing.
Table 1 clinical characteristics of study population
The analysis step:
and respectively extracting total RNA from the POI granular cells and cells of a control group, and carrying out quality control. The quality control requirement is that the total concentration is more than or equal to 20ng/ul, the total concentration is more than or equal to 2ug, and A260/280=1.9-2.1.
RNA fragmentation (200-300 bp) after rRNA removal of the sample, cDNA was synthesized. PCR amplification after cDNA addition of linker, sequencing with illuminaHiSeq/NextSeq platform after quality inspection of library. Filtering out the joint of the original data after sequencing, removing pollution, comparing the reference genome, and performing subsequent information analysis by using high-qualitymappedreads (MAPQ is more than or equal to 30), wherein the method comprises
(1) Removing the joint sequence in the original off-machine data by using cutadapt program;
(2) Removal of low quality sequences using the Trimmomatic procedure yielded CLEAN DATA;
(3) The Fastqc program was used to count the amount of CLEAN DATA, the ratio of q20 and q 30;
(4) CLEAN DATA was aligned to a reference genome using the HISAT program;
(5) Splice reads into transcripts and gene expression level analysis using STRING TIE;
(6) Gene differential expression analysis using R-package edge;
(7) GO/KEGG functional enrichment analysis of differentially expressed genes.
Results:
1. Gene-level expression analysis
The PCA assay and Pearson correlation assay used by the inventors were first tested in the control group against the overall gene expression level. PCA analysis uses packages (prcomp package) in the R environment. After reading the standardized gene expression level into the R environment, a PCA chart is drawn by utilizing prcomp default parameter analysis inside. As shown in FIG. 1, in the first dimension PC1, the disease group and the control group were well separated, which indicates that there was a significant difference in gene expression between the disease group and the control group.
Pearson correlation analysis uses the analysis of the cor function of the self-contained R environment, reads the standardized numerical value into the R environment, and calculates the correlation coefficient value and the significance level value between two groups of variables by using the cor function. As shown in fig. 2, the correlation between control group 1 and control group 2 was higher than that of disease group 1 and disease group 2, which indicates that the experimental group of the inventors is significant.
2. Differential expression
The differential expression analysis method used by the inventors is:
Tools Hisat2 (select default parameters) and STRINGTIE (select default parameters), and the downstream differential expression analysis method R package edge recommended by the procedure, take genes with q-value <0.05 and FC <0.5 as differential genes.
The criteria for determining whether a gene is expressed is that the gene has an FPKM average value of greater than 0.01 under a certain experimental condition. The heat map is drawn by heatmap function analysis of pheatmap program package under R environment, after the standardized expression matrix of the differential gene is read into R environment, heatmap function in pheatmap program package is utilized, and default parameter analysis is used for drawing the heat map. As shown in FIG. 3, there were 1056 genes up-regulated and 810 genes down-regulated in the disease group granulosa cells, including UHRF2, compared to the control group.
3. Functional enrichment
The differential genes were subjected to pathway analysis (Geneontology, GO) using software and the analysis results were enriched to the pathway shown in fig. 4 where CC is the cellular component pathway and BP is the biological process pathway and MF is the molecular function. As shown in FIG. 4A, the differential genes of the disease group mainly affect the extracellular matrix of granulosa cells, hormone synthesis, cellular development and maturation, and cAMP, WNT pathways, etc. As shown in fig. 4B, the pathways of the disease group are mainly enriched in the extracellular matrix from the cellular component. As shown in FIG. 4C, the pathways of the disease group are enriched in extracellular matrix construction, transcription factor DNA binding, steroid receptor activity, etc. from the molecular function. FIG. 4 GO analysis of differential genes: A. pathway enrichment of biological processes (Biological Process, BP); B. pathway enrichment of cellular components (Cellular component, CC); C. the molecular functional (Molecular function, MF) pathway is enriched.
All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
Claims (10)
1. A marker for predicting or diagnosing risk of premature ovarian dysfunction, wherein the marker is the UHRF2 gene, a transcript thereof, or an expressed protein thereof.
Use of the uhrf2 gene, its transcripts, its expressed proteins, or its detection reagents, for the preparation of a diagnostic reagent or kit for assessing the risk of developing premature ovarian failure.
3. The use according to claim 2, wherein said diagnostic reagent or kit is used for detecting the level of said UHRF2 gene, its transcript, or its expressed protein in a test sample.
4. The use according to claim 2, wherein the sample to be tested is selected from the group consisting of: blood, plasma, serum, granulosa cells, or a combination thereof.
5. The use of claim 2, wherein the expression level comprises an expression level in blood, plasma, serum or granulosa cells.
6. The use of claim 2, wherein the assessment comprises early diagnosis, assisted diagnosis, or a combination thereof.
7. The use according to claim 2, wherein the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g. a DNA chip) or a protein chip.
8. The use of claim 2, wherein the kit further comprises a label or instructions that prescribe use of the kit for (a) diagnosing a risk of developing premature ovarian dysfunction, and/or (b) evaluating the efficacy of treatment of premature ovarian dysfunction.
9. The use according to claim 2, wherein if the mRNA level of the UHRF2 gene or the level of UHRF2 protein is reduced, a high risk of premature ovarian failure in the test subject is indicated.
10. An apparatus for risk assessment of premature ovarian dysfunction, the apparatus comprising:
An input unit configured to input expression level data of a UHRF2 gene in a detection sample of a certain detection object;
A data processing unit configured to: comparing the expression level L1 of the UHRF2 gene with a control reference value L0, wherein when L1 is significantly lower than L0, the subject is prompted to be at high risk for developing premature ovarian failure; when L1 is not remarkably lower than L0, the risk of the premature ovarian dysfunction of the subject is low; and
And the output unit is used for outputting the evaluation result.
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