Method and system for automatically classifying and recording sex cells

Abstract

The invention relates to the technical field of biological information processing, in particular to a sex cell automatic classification and recording method and system.The method comprises the following steps that the sex cell DNA synthesis rate is obtained through a pulse labeling method, the S period starting time and the G2 / M transition duration are extracted, the cell nucleus area and chromatin distribution are combined, XIST and SRY genes are screened, sex specific changes are analyzed, and the sex cell DNA synthesis rate is obtained. And matching a gender track, calculating an S period proportion and a G2 / M duration, outputting a gender classification label, and integrating an appearance modification data update record. According to the method, nucleotide and fluorescence microscopic imaging are introduced, the DNA synthesis rate is quantified, a characteristic trajectory is established in combination with cell cycle key nodes and nuclear morphology, gender specificity is analyzed in combination with XIST and SRY gene expression and chromosome signals, classification basis comprehensiveness is improved, a screening threshold value is set to be matched with the gender trajectory, and the accuracy of gender detection is improved. The cycle ratio is calculated, the classification accuracy is improved in combination with the confidence coefficient, genetic and epigenetic features are integrated during data storage, and subsequent analysis is supported.

Description

Technical Field

[0001] The present invention relates to the field of biological information processing technology, and in particular to a method and system for automatically classifying and recording sex cells. Background Art

[0002] The field of bioinformation processing encompasses the collection, storage, analysis, and interpretation of biological data, encompassing multiple disciplines including computer science, mathematics, and biology. Its core areas include genomic data analysis, protein structure prediction, biological signal processing, and medical image analysis. Advances in this field have fueled the advancement of precision medicine, personalized treatment, and automated biological experiments. In systematic research, bioinformation processing relies on computer algorithms and mathematical models, combined with large-scale data computation and statistical analysis, to analyze complex biological information. In recent years, this field has seen widespread application in medical diagnosis, drug development, gene editing, disease prediction, and other areas, making the intelligent processing of biological data a research hotspot.

[0003] Among them, the method for automatic classification and recording of sex cells refers to a technical method for identifying, classifying, and recording the distribution or number of sex-related cells in an organism based on cell biological characteristics. The patent subject covers cell morphological feature extraction, optical or electronic imaging data acquisition, chromosome analysis, biomarker detection, etc., and automatically classifies sex cells based on cell structure, chromosome composition, or biomolecular signals, combined with machine learning models or rule matching strategies. After classification is completed, the method archives the cell classification results by sample number through database storage or digital recording system, and establishes a traceable index system.

[0004] Existing technologies rely on morphological observation, chromosome number or single gene expression, lack dynamic characteristics of cell proliferation status, and classification results are easily affected by individual heterogeneity. Microscopic imaging analysis is limited by chromosome overlap or uneven staining, which affects the accuracy of judgment. The detection method of single gene or chromosome markers may lead to insufficient classification specificity due to expression drift. Static data analysis mode cannot cover the cell cycle process, making it difficult to effectively capture changes in cell state. The storage method of classification results does not integrate epigenetic information, which limits the traceability of data and makes it difficult to support long-term sex cell research and dynamic state analysis. Summary of the Invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method and system for automatically classifying and recording sex cells.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for automatically classifying and recording sex cells, comprising the following steps: S1: Obtain sex cells, add detectable nucleotides using pulse labeling, calculate DNA synthesis rate using fluorescence microscopy, and obtain sex cell nuclear DNA synthesis rate data; S2: Call the DNA synthesis rate data of the sex cell nucleus, extract the S phase start time and G2 / M transition duration, and combine the cell nuclear area and chromatin distribution to generate the sex cell cycle characteristic trajectory; S3: Call the sex-specific cell cycle characteristic trajectory, screen the XIST gene expression level and SRY gene level, combine the X chromatin state and Y chromosome signal, calculate the sex-specific changes in DNA synthesis rate, match the sex standard trajectory, set the threshold to screen the matching cells, and obtain the sex-specific trajectory matching result; S4: Based on the sex-specific trajectory matching results, calculate the S phase proportion and G2 / M duration, filter the classification samples according to the confidence level, set the threshold to judge the classification, and output the sex cell classification label; S5: calling the sex cell classification label, integrating X chromatin epigenetic modification data and Y chromosome data, storing the classification data, and updating the sex cell classification record data.

[0007] As a further embodiment of the present invention, the sex-specific cell nuclear DNA synthesis rate data includes S phase initiation time, G2 / M transition duration, cell nuclear area, and chromatin distribution; the sex-specific cell cycle characteristic trajectory includes XIST gene expression, SRY gene level, X chromatin state, Y chromosome signal, and sex-specific changes in DNA synthesis rate; the sex-specific trajectory matching results include S phase proportion, G2 / M duration, and sex-specific standard trajectory matching; the sex-specific cell classification label specifically includes classification sample confidence and classification judgment threshold; and the sex-specific cell classification record data includes X chromatin epigenetic modification data, Y chromosome data, and classification data.

[0008] As a further embodiment of the present invention, the steps for obtaining the DNA synthesis rate data of the sex cell nuclei are specifically as follows: S101: After obtaining the sex cells, a pulse labeling method is used to incorporate detectable nucleotides into the sex cells, and the degree of nucleotide incorporation in the cell nucleus is monitored to obtain nucleotide incorporation data; S102: Based on the nucleotide incorporation amount data, using fluorescence microscopy imaging technology, detecting the fluorescence signal intensity in the cell nucleus, calling the corresponding relationship between the fluorescence signal intensity and the nucleotide incorporation amount, and obtaining fluorescence signal intensity data; S103: Calculate the DNA synthesis rate based on the fluorescence signal intensity data using the formula: ; The DNA synthesis rate value is obtained by calculation as the DNA synthesis rate data of the cell nucleus of gender; in, represents the DNA synthesis rate, Representative The fluorescence signal intensity of each cell, Representative The amount of nucleotide incorporation per cell, represents the total experimental time, Represents the total number of cells, Represents the average nucleotide incorporation of all cells.

[0009] As a further embodiment of the present invention, the step of obtaining the sex cell cycle characteristic trajectory is specifically as follows: S201: calling the DNA synthesis rate data of the nuclei of the sex cells, analyzing the DNA synthesis rate change trend, extracting the S phase start time of each cell by detecting the DNA synthesis rate mutation point, and obtaining the S phase start time data; S202: Based on the S phase start time data, the time interval from the G2 phase to the M phase is monitored, and combined with the continuous change of the nuclear DNA synthesis rate, the G2 / M transition duration of each cell is calculated to generate G2 / M transition duration data; S203: Based on the S phase start time data and the G2 / M transition duration data, combined with the cell nuclear area and chromatin distribution characteristics, the formula is used: ; Computationally generate sex-specific cell cycle trajectories; in, Represents the characteristic trajectory of the cell cycle, Representative The S phase start time of each cell Representative The G2 / M transition time of each cell Representative The nuclear area of the cells, Representative The chromatin distribution value of each cell, represents the maximum S phase initiation time in all cells, Represents the minimum G2 / M transition duration in all cells, Represents the total number of cells, Representative Chromatin density value of each cell.

[0010] As a further embodiment of the present invention, the steps for obtaining the gender-specific trajectory matching results are specifically as follows: S301: calling the sex cell cycle characteristic trajectory, screening the XIST gene expression and SRY gene level of each cell, combining the X chromatin state and Y chromosome signal to obtain sex gene and chromatin state data; S302: Based on the sex gene and chromatin state data, calculating the sex-specific changes in DNA synthesis rate, and invoking the relationship between XIST gene expression level, SRY gene level and DNA synthesis rate to obtain sex-specific change data of DNA synthesis rate; S303: Based on the sex-specific variation data of DNA synthesis rate, match the sex standard trajectory and set a threshold to screen matching cells using the formula: ; Calculate the sex-specific matching score, filter the matching cells, and obtain the sex-specific trajectory matching results; in, represents the gender-specific matching score, Representative The XIST gene expression level in each cell represents the standard expression level of the XIST gene in the sex standard trajectory, Representative The SRY gene level in each cell represents the standard level of the SRY gene in the sex-standard locus, Representative The DNA synthesis rate of each cell, Represents the set threshold, Represents the total number of cells involved in matching.

[0011] As a further embodiment of the present invention, the steps for obtaining the sex cell classification label are specifically as follows: S401: Based on the sex-specific trajectory matching results, the S phase proportion and G2 / M duration of each cell are calculated, and cell cycle phase duration data are obtained by analyzing the cell cycle phase time distribution; S402: Calculating the confidence of cell classification based on the cell cycle stage duration data, combined with the S phase proportion and G2 / M duration, and screening classification samples based on the confidence to obtain high-confidence classification sample data; S403: Call the high-confidence classification sample data and set the classification threshold using the formula: ; Calculate cell classification scores, determine classification, and output gender cell classification labels; in, represents the cell classification score, Representative The proportion of cells in S phase, represents the reference S period proportion, Representative The G2 / M duration of each cell, Refer to G2 / M duration. Represents the number of cells involved in classification, Represents the classification confidence.

[0012] As a further solution of the present invention, the steps for obtaining the sex cell classification record data are specifically as follows: S501: calling the sex cell classification label, integrating the X chromatin epigenetic modification data and Y chromosome signal of each cell, and forming cell classification and epigenetic modification association data; S502: Based on the cell classification and epigenetic modification association data, calculating the epigenetic modification differences of cells under different classifications, and screening using the matching degree of the classification and modification data to obtain cell classification matching degree data; S503: Call the cell classification matching data, store the classification data and update the record, using the formula: ; Calculate the classification data update coefficient and update the sex cell classification record data; in, represents the classification data update coefficient, Representative The apparent modification intensity of X chromatin in each cell, represents the average value of chromatin epigenetic modification of all cells X, Representative The Y chromosome signal intensity of each cell, represents the average value of Y chromosome signals of all cells, Representative The total amount of X and Y chromatin modifications per cell, represents the maximum value of the total amount of modification in all cells, Indicates the minimum value for the total amount of modification.

[0013] A sex cell automatic classification and recording system, based on the above-mentioned sex cell automatic classification and recording method, the system comprises: The cell nucleus DNA synthesis rate calculation module obtains the sex of cells, adds detectable nucleotides, detects the changes in fluorescence signals during DNA synthesis in the cell nucleus, collects fluorescence microscopy imaging data, extracts fluorescence intensities at different time points, calculates the DNA synthesis rate of each cell nucleus, and obtains the cell nucleus DNA synthesis rate data; The cell cycle feature extraction module extracts the S phase start time and G2 / M transition duration based on the cell nuclear DNA synthesis rate data, combines the cell nuclear area and chromatin distribution, compares the rate characteristics of different cycle stages, calculates the cell cycle change trend, and obtains the cell cycle feature trajectory; The sex-specific trajectory matching module calls the cell cycle characteristic trajectory, screens the XIST gene expression and SRY gene level, combines the X chromatin state and Y chromosome signal, calculates the sex-specific changes in DNA synthesis rate, matches the sex standard trajectory, sets a threshold to screen cells that meet the standard, and obtains the sex-specific trajectory matching result; The sex cell classification module calculates the S phase proportion and G2 / M duration based on the sex-specific trajectory matching results, selects cells that meet the classification criteria based on the confidence level, sets the classification judgment threshold, matches the sex cell category according to the classification criteria, and obtains the sex cell classification label; The classification record and data integration module calls the sex cell classification label, integrates X chromatin epigenetic modification data and Y chromosome data, stores the classification data, updates the classification record library, and obtains sex cell classification record data.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are: In this study, the introduction of detectable nucleotides combined with fluorescence microscopy enables quantitative analysis of DNA synthesis rates, ensuring accurate determination of cell proliferation status. Based on information from key cell cycle nodes, combined with nuclear morphology and chromatin distribution, a dynamic feature trajectory is established, allowing classification to encompass both static morphology and dynamic proliferation characteristics. Screening for XIST gene expression and SRY gene levels, combined with X chromatin status and Y chromosome signals, enables sex-specific analysis of DNA synthesis rates, providing a more comprehensive classification basis compared to single gene or chromosome markers. Matching standard sex trajectories and setting screening thresholds allows classification to take into account both single-cell data and population statistical characteristics, improving stability. Calculation of cell cycle proportions, combined with confidence level screening, enhances classification accuracy. X chromatin epigenetic modifications and Y chromosome signals are integrated into the storage of classified data, ensuring both genetic and epigenetic characterization, supporting subsequent source tracing analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the main steps of the present invention; Figure 2 This is a flow chart of the steps for obtaining DNA synthesis rate data of sex cell nuclei according to the present invention; Figure 3 Flowchart of the steps for obtaining the sex cell cycle characteristic trajectory of the present invention; Figure 4 Flowchart of the steps for obtaining gender-specific trajectory matching results of the present invention; Figure 5 Flowchart of the steps for obtaining sex cell classification labels of the present invention; Figure 6 Flowchart of the steps for obtaining sex cell classification record data of the present invention. DETAILED DESCRIPTION

[0016] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0017] In the description of the present invention, it should be understood that the terms "length," "width," "up," "down," "front," "back," "left," "right," "vertical," "horizontal," "top," "bottom," "inside," "outside," and the like, indicating positions or relationships, are based on the positions or relationships shown in the accompanying drawings and are intended only to facilitate the description of the present invention and simplify the description. They do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limiting the present invention. Furthermore, in the description of the present invention, "plurality" means two or more, unless otherwise expressly and specifically defined.

[0018] Example 1: Please refer to Figure 1 The present invention provides a technical solution: a method for automatically classifying and recording sex cells, comprising the following steps: S1: Obtain sex cells, add detectable nucleotides using pulse labeling, calculate DNA synthesis rate using fluorescence microscopy, and obtain sex cell nuclear DNA synthesis rate data; S2: Call the DNA synthesis rate data of the sex cell nucleus, extract the S phase start time and G2 / M transition duration, and combine the cell nuclear area and chromatin distribution to generate the sex cell cycle characteristic trajectory; S3: Call the sex-specific cell cycle trajectory, screen the XIST gene expression and SRY gene levels, combine the X chromatin state and Y chromosome signal, calculate the sex-specific changes in DNA synthesis rate, match the sex-specific trajectory, set the threshold to screen the matching cells, and obtain the sex-specific trajectory matching results; S4: Based on the sex-specific trajectory matching results, calculate the S phase proportion and G2 / M duration, filter the classification samples according to the confidence level, set the threshold to judge the classification, and output the sex cell classification label; S5: Call the sex cell classification label, integrate X chromatin epigenetic modification data and Y chromosome data, store the classification data, and update the sex cell classification record data.

[0019] Sex-specific cell nuclear DNA synthesis rate data include S phase initiation time, G2 / M transition duration, nuclear area, and chromatin distribution. Sex-specific cell cycle characteristic trajectories include XIST gene expression, SRY gene level, X chromatin state, Y chromosome signal, and sex-specific changes in DNA synthesis rate. Sex-specific trajectory matching results include S phase proportion, G2 / M duration, and sex-specific standard trajectory matching. Sex cell classification labels specifically include classification sample confidence and classification judgment threshold. Sex cell classification record data includes X chromatin epigenetic modification data, Y chromosome data, and classification data.

[0020] See also Figure 2 The specific steps for obtaining DNA synthesis rate data of sex cell nuclei are as follows: S101: After obtaining the sex cells, a pulse labeling method is used to incorporate detectable nucleotides into the sex cells, and the degree of nucleotide incorporation in the cell nucleus is monitored to obtain nucleotide incorporation data; After obtaining sex-specific cells, representative male and female cell samples are first selected, such as peripheral blood mononuclear cells collected from healthy adults. Subsequently, pulse labeling is used to incorporate detectable nucleotides into the cells. Specifically, 5-bromodeoxyuridine (BrdU) is added to the culture medium as a labeled nucleotide. This molecule is incorporated into newly synthesized DNA strands during DNA replication. The labeling reaction is terminated after a set time (e.g., 30 minutes), and the cells are washed three times with ice-cold PBS to remove unincorporated free BrdU. During this process, the samples should be maintained in a 37°C incubator to ensure normal cell metabolism and DNA synthesis rates are not disturbed. After labeling, the cells are fixed to preserve cellular structure, typically using 70% ethanol and incubated overnight at 4°C. This maintains the integrity of the cellular structure and facilitates subsequent analysis. After this step, the cells are stained with an anti-BrdU antibody, and the degree of BrdU incorporation is measured by flow cytometry to obtain nucleotide incorporation data. For example, assuming that the average number of BrdU molecules incorporated into each cell in a batch of female cell samples is 2.5×105, while the amount incorporated into male cell samples is 2.3×105, the data difference can preliminarily reflect the difference in DNA synthesis activity between gender cells.

[0021] S102: Based on the nucleotide incorporation amount data, using fluorescence microscopy imaging technology, detecting the fluorescence signal intensity in the cell nucleus, calling the corresponding relationship between the fluorescence signal intensity and the nucleotide incorporation amount, and obtaining the fluorescence signal intensity data; Based on the nucleotide incorporation data, fixed and stained cells are imaged under a fluorescence microscope. Fluorescently labeled BrdU molecules emit green fluorescence under specific wavelengths (e.g., 488 nm excitation), and high-resolution images are acquired using an image acquisition system. Next, fluorescence intensity is quantified using image processing software (e.g., ImageJ). First, the fluorescence signal intensity of the cell nucleus is measured, and background noise is subtracted to obtain a net fluorescence intensity value. For each cell, the fluorescence signal intensity directly reflects the amount of BrdU incorporated and is therefore proportional to DNA synthesis activity. For example, if the average fluorescence intensity of 100 cells in a sample is 1500 units, compared to 1300 units in a sample of the opposite sex, this indicates higher DNA synthesis activity in the former. Finally, the fluorescence signal intensity data is obtained and used to calculate the DNA synthesis rate.

[0022] S103: Calculate the DNA synthesis rate based on the fluorescence signal intensity data using the formula: ; The DNA synthesis rate value is obtained by calculation as the DNA synthesis rate data of the cell nucleus of gender; in, represents the DNA synthesis rate, Representative The fluorescence signal intensity of each cell, Representative The amount of nucleotide incorporation per cell, represents the total experimental time, Represents the total number of cells, Represents the average nucleotide incorporation of all cells.

[0023] Based on the fluorescence signal intensity data, the DNA synthesis rate was calculated using the following formula: ; in, Representative The fluorescence signal intensity of each cell, For the The amount of nucleotide incorporation per cell, is the total experimental time, is the total number of cells, is the average nucleotide incorporation of all cells. Assuming that 100 cells are tested, the experimental time Minutes, total cell count , average nucleotide incorporation The fluorescence signal intensities and nucleotide incorporation amounts of five cells are as follows: , , , , .

[0024] Substitute into the formula to calculate: ; ; ; Finally, the DNA synthesis rate data of the sex cell nucleus was obtained, and its value was 3.47×10 6 This result indicates that the DNA synthesis rate of cells varies between samples of different sexes, which can be used to further study sex-related cellular metabolic characteristics.

[0025] See also Figure 3 , the specific steps for obtaining the sex cell cycle characteristic trajectory are: S201: Call the DNA synthesis rate data of the sex cell nucleus, analyze the trend of DNA synthesis rate changes, extract the S phase start time of each cell by detecting the mutation point of DNA synthesis rate, and obtain the S phase start time data; To retrieve DNA synthesis rate data for cell nuclei of different sexes, we first obtained DNA synthesis rate data for cells of different sexes in the experimental sample. Specifically, we used fluorescence microscopy to obtain the fluorescence intensity of labeled nucleotides incorporated into the cell nucleus and calculated the DNA synthesis rate of each cell based on the intensity. For example, if the experiment lasted 30 minutes and the fluorescence intensity of cell A was 500 units and that of cell B was 700 units, we could calculate that the synthesis rate of cell B was higher than that of cell A. Next, we analyzed the changing trend of the DNA synthesis rate and plotted the synthesis rates at different stages of the cell cycle as a time series graph. We identified significant mutation points in the synthesis rate and used this to infer the initiation time of the S phase. In this process, we set the threshold for synthesis rate mutation to a 10% change. If the synthesis rate increased by more than 10% after the 15th minute, the cell was considered to have entered the S phase. For example, if the synthesis rate of cell A increased from 200 units to 230 units at the 12th minute, an increase of 15%, then its S phase initiation time was 12 minutes. In this way, we analyzed the synthesis rates of all cells in sequence, extracted the S phase initiation time for each cell, and finally obtained the S phase initiation time data.

[0026] S202: Based on the S phase start time data, the time interval from the G2 phase to the M phase is monitored, and combined with the continuous change of the nuclear DNA synthesis rate, the G2 / M transition duration of each cell is calculated to generate G2 / M transition duration data; Based on the S phase start time data, after recording the time when each cell enters the S phase, the changes in the cell DNA synthesis rate are continuously monitored to identify the transition time from the G2 phase to the M phase. The specific steps are to analyze the duration of the cell synthesis rate in the G2 phase, and judge it in combination with the stable period of the fluorescence intensity of the cell nucleus. The fluorescence intensity is set to remain stable for more than 5 minutes as the G2 phase mark. When the fluorescence intensity drops again, the cell is considered to have entered the M phase. For example, if the S phase start time of cell A is 12 minutes, the fluorescence intensity reaches stability after 30 minutes and continues to drop until 40 minutes later, then the G2 / M transition time is 10 minutes. In this way, the G2 / M transition time of each cell is calculated, and finally the G2 / M transition time data is generated.

[0027] S203: Based on the S phase start time data and G2 / M transition duration data, combined with the nuclear area and chromatin distribution characteristics, the formula is used: ; Computationally generate sex-specific cell cycle trajectories; in, Represents the characteristic trajectory of the cell cycle, Representative The S phase start time of each cell Representative The G2 / M transition time of each cell Representative The nuclear area of the cells, Representative The chromatin distribution value of each cell, represents the maximum S phase initiation time in all cells, Represents the minimum G2 / M transition duration in all cells, Represents the total number of cells, Representative Chromatin density value of each cell.

[0028] Based on the S phase start time data and G2 / M transition duration data, combined with the nuclear area and chromatin distribution characteristics, the cell cycle characteristic trajectory is calculated. First, the nuclear area is measured, and the nuclear region is extracted using image segmentation technology and its pixel area is calculated and converted into actual area units. For example, the nuclear area of cell A is 150 square microns, and that of cell B is 200 square microns. Then, the chromatin distribution characteristics are detected and the chromatin density distribution is calculated. The pixel gray value distribution is used to judge. Assuming that the chromatin distribution value of cell A is 0.65 and that of cell B is 0.75, the formula is used: ; Calculations are performed to calculate the cell cycle characteristic trajectory values, and finally the gender cell cycle characteristic trajectory is generated.

[0029] Detailed explanation and calculation of the formula: formula: ; Parameter explanation: Represents the characteristic trajectory of the cell cycle, Representative The S phase start time of each cell Representative The G2 / M transition time of each cell Representative The nuclear area of the cells, Representative The chromatin distribution value of each cell, represents the maximum S phase initiation time in all cells, Represents the minimum G2 / M transition duration in all cells, Represents the total number of cells, Representative Chromatin density value of each cell.

[0030] Parameter assignment and acquisition process: minutes (S phase initiation time of cell A); minutes (G2 / M transition duration of cell A); square micrometers (nuclear area of cell A); (chromatin distribution value of cell A); Minutes (maximum S phase initiation time in the sample); Minutes (minimum G2 / M transition duration in the sample); (total cell number); (chromatin density value of cell A); Formula operation: First calculate the numerator: ; ; Then calculate the denominator: ; ; Substitute the result into the formula: ; Formula innovation description: The benefit of the formula is that by combining the sum of the squares of the S phase initiation time and the G2 / M transition time with the nuclear area and chromatin distribution characteristics, it further considers the maximum and minimum time differences of cells as well as the cell number and chromatin density, thus comprehensively reflecting the complexity and dynamic changes of the cell cycle.

[0031] Result interpretation: The results show that the cell cycle characteristic trajectory value is 2488.83, which characterizes the changing characteristics of the cell during its life cycle. This value can be used to further compare with the trajectories of other cells to analyze the cycle differences of cells of different sexes.

[0032] See also Figure 4 ,The steps for obtaining gender-specific trajectory matching results are as follows: S301: Call the sex cell cycle characteristic trajectory, screen the XIST gene expression and SRY gene level of each cell, and combine the X chromatin state and Y chromosome signal to obtain sex gene and chromatin state data; To retrieve sex-specific cell cycle trajectories, trajectory data for each cell is first extracted, including S phase onset time, G2 / M transition duration, nuclear area, and chromatin distribution parameters. Next, the expression of the XIST and SRY genes in each cell is screened. Specifically, the mRNA levels of the XIST and SRY genes are measured using real-time fluorescence quantitative PCR. Fluorescence intensities are converted to relative expression levels. For example, the XIST expression level in a cell is 1.2, while the SRY level is 0.3. Next, the X chromosome chromatin status is assessed by measuring the methylation level of H3K27me3. If the detected H3K27me3 signal intensity is greater than 1.5 (relative fluorescence intensity), it indicates inactive X chromosome chromatin, consistent with female cells. Confirmation is also performed by combining the Y chromosome signal. Fluorescence in situ hybridization (FISH) can be used to detect Y chromosome-specific sequences, such as the SRY gene. If the SRY gene fluorescence signal intensity is less than 0.5, the cell is further confirmed as female. For example, if a cell's XIST expression level is 1.3, its H3K27me3 signal intensity is 1.6, and its SRY gene fluorescence signal is 0.4, then this cell can be classified as female. Through the above screening and detection steps, the sex gene and chromatin status data of each cell are obtained.

[0033] S302: Based on the sex gene and chromatin state data, calculate the sex-specific changes in DNA synthesis rate, call the relationship between XIST gene expression level, SRY gene level and DNA synthesis rate, and obtain the sex-specific change data of DNA synthesis rate; Based on sex-specific gene and chromatin state data, sex-specific changes in DNA synthesis rate were calculated. First, correlation analysis was performed between XIST and SRY gene expression levels and DNA synthesis rate. For example, in a cell, XIST expression was 1.2, SRY expression was 0.3, and the DNA synthesis rate was 2.5 μg / h. The relationship between gene expression and DNA synthesis rate was calculated using the Pearson correlation coefficient formula. If XIST expression and DNA synthesis rate showed a negative correlation (e.g., -0.65) and SRY expression and DNA synthesis rate showed a positive correlation (e.g., 0.72), sex-specific changes in DNA synthesis rate could be identified. Furthermore, a multivariate regression model was used to calculate the range of DNA synthesis rate variation in cells of different sexes. For example, the DNA synthesis rate in female cells might be between 2.0 and 2.8 μg / h, while that in male cells might be between 2.5 and 3.5 μg / h. For reference, the threshold setting was used to set the discrimination threshold using the overlapping region of the two data sets as the cutoff point. For example, if the overlapping range is 2.5-2.8 μg / h, the median of 2.65 μg / h is selected as the initial threshold, and then adjusted using the ROC curve to achieve the best classification effect. Ultimately, data on sex-specific changes in DNA synthesis rate are obtained.

[0034] S303: Based on the sex-specific variation data of DNA synthesis rate, match the sex standard trajectory and set a threshold to screen matching cells using the formula: ; Calculate the sex-specific matching score, filter the matching cells, and obtain the sex-specific trajectory matching results; in, represents the gender-specific matching score, Representative The XIST gene expression level in each cell represents the standard expression level of the XIST gene in the sex standard trajectory, Representative The SRY gene level in each cell represents the standard level of the SRY gene in the sex-standard locus, Representative The DNA synthesis rate of each cell, Represents the set threshold, Represents the total number of cells involved in matching.

[0035] Based on the sex-specific changes in DNA synthesis rate data, the sex standard trajectory was matched and a threshold was set to screen matching cells using the formula: ; Reference content and calculation process for threshold setting: Threshold The setting is based on the distribution and standard deviation of the sample data. Taking the experimental data as an example, the matching scores of 100 cells are collected. , calculate its mean and standard deviation. Assume the mean is 0.12 and the standard deviation is 0.03. The commonly used threshold setting method is to select the mean minus one standard deviation as the initial screening threshold, that is: ; This threshold means that cells with a matching score below 0.09 are considered to be highly matched to the standard trajectory.

[0036] Parameter assignment and acquisition process: Assume that the XIST gene expression level of a cell 1.2, standard value is 1.0, SRY gene level 0.3, standard value is 0.5, the DNA synthesis rate 2.5 μg / h, total cell count is 100. Substituting into the formula: ; The innovation of the formula is that by combining the difference in XIST and SRY gene expression with the product of DNA synthesis rate, it can more sensitively reflect the deviation of sex-specific matching, especially when the DNA synthesis rate of the cell deviates significantly from the standard value. This product amplifies the impact of gene expression differences, thereby improving the accuracy of screening.

[0037] Result analysis: The results show that the cell's sex-specific matching score is 0.099, close to the set threshold of 0.09, indicating that this cell has a high degree of match with the gender standard trajectory, but slightly above the threshold, which may require further review. Finally, matching cells are screened to obtain the sex-specific trajectory matching results.

[0038] See also Figure 5 , the specific steps for obtaining gender cell classification labels are: S401: Based on the sex-specific trajectory matching results, the S phase proportion and G2 / M duration of each cell are calculated, and the cell cycle phase duration data are obtained by analyzing the cell cycle phase time distribution; Based on the sex-specific trajectory matching results, we first extracted the time proportion of each cell in the S phase and G2 / M phase. After obtaining the sex-specific trajectory matching results, we refined the data to the single cell level, recorded the complete cell cycle duration of each cell, and marked the time nodes of the S phase and G2 / M phase. Then, we calculated the ratio of the S phase duration to the complete cell cycle duration to obtain the S phase proportion. For example, if the S phase duration of a cell is 6 hours and the complete cycle is 24 hours, then the S phase proportion is 25%. Similarly, by recording the time length from the G2 phase to the M phase, we can obtain the S phase proportion. To the G2 / M duration, if the G2 phase is 4 hours and the M phase is 1 hour, then the G2 / M duration is 5 hours. On this basis, analyze the distribution of the S phase proportion and G2 / M duration of all cells, filter out outliers and extreme data points, and ensure the reliability of the data. For example, if the S phase proportion of most cells is concentrated between 20%-30%, and the S phase proportion of a cell is 60%, it is necessary to check whether there are recording errors or biological abnormalities in the data. Finally, summarize the S phase proportion and G2 / M duration of all cells to form the cell cycle stage duration data.

[0039] S402: Calculate the confidence of cell classification based on the cell cycle phase duration data, combined with the S phase proportion and G2 / M duration, and filter classification samples based on the confidence to obtain high-confidence classification sample data; According to the cell cycle stage duration data, combined with the S phase proportion and G2 / M duration, the confidence of cell classification is calculated. First, the reference value is determined, and the S phase proportion of normal cells is set to 25%, and the G2 / M duration is 5 hours. Then, the deviation of the S phase proportion and G2 / M duration of each cell is calculated. The smaller the deviation, the higher the confidence. Specifically, the absolute value of the difference between the S phase proportion and the reference value of each cell, and the absolute value of the difference between the G2 / M duration and the reference value are calculated to obtain the total deviation. Assuming that the S phase proportion of a cell is 28% and the G2 / M duration is 5.5 hours, the deviation is , the confidence threshold is set according to the total deviation, and cells with a deviation of less than 5% are screened out as high-confidence samples, and finally high-confidence classification sample data are obtained.

[0040] S403: Call high-confidence classification sample data and set the classification threshold using the formula: ; Calculate cell classification scores, determine classification, and output gender cell classification labels; in, represents the cell classification score, Representative The proportion of cells in S phase, represents the reference S period proportion, Representative The G2 / M duration of each cell, Refer to G2 / M duration. Represents the number of cells involved in classification, Represents the classification confidence.

[0041] Call high-confidence classification sample data, set the classification threshold, and use the formula: ; Calculate cell classification scores, determine classification, and output gender cell classification labels; in, represents the cell classification score, Representative The proportion of cells in S phase, represents the reference S period proportion, Representative The G2 / M duration of each cell, Refer to G2 / M duration. Represents the number of cells involved in classification, Represents the classification confidence.

[0042] Detailed explanation of formula parameters and calculation examples: Parameter Description: : Cell classification score, which measures the degree of deviation of cells from the reference classification standard; : No. The proportion of cells in the S phase is assumed to be 28%; : Reference S phase proportion, set to 25%; : No. The G2 / M duration of each cell is assumed to be 5.5 hours; : Refer to G2 / M duration and set it to 5 hours; : Total number of cells, assumed to be 100; : Classification confidence, assumed to be 0.9.

[0043] Parameter acquisition method: Obtain cell cycle monitoring data and record the S phase time ratio. The total duration of G2 phase and M phase was recorded by time marking method. Directly count the number of cells involved in classification, Obtained through the above deviation calculation and converted into confidence.

[0044] Formula substitution and calculation: ; The benefit of the formula is that by introducing the classification confidence The exponential function adjustment improves the sensitivity of the formula to small deviation samples, making the classification more refined and accurate.

[0045] Result analysis: The results show that the cell classification score is 0.0345, with a small deviation, which meets the set classification threshold. The cells can be correctly classified and the gender cell classification label is output.

[0046] See also Figure 6 , the specific steps for obtaining sex cell classification record data are as follows: S501: Call the sex cell classification label, integrate the X chromatin epigenetic modification data and Y chromosome signal of each cell, and form the cell classification and epigenetic modification association data; The sex cell classification label is called, and the classification label of each cell is integrated with its corresponding X chromatin epigenetic modification data and Y chromosome signal. The specific operation includes first extracting the classified cell population and grouping it by sex classification label. Then, the X chromatin epigenetic modification data of each cell is collected. Data collection can obtain DNA methylation levels through sequencing technology. For example, assuming that the X chromatin methylation level of cell A is 45% and that of cell B is 60%, the Y chromosome signal intensity of the corresponding cell is collected. This can be measured by fluorescence in situ hybridization (FISH) signal intensity. Assuming that the Y signal intensity of cell A is 1.2 units and that of cell B is 0.8 units, these data are classified and organized according to the cell classification label, and the distribution and change of each data point under different classifications are refined to facilitate subsequent data comparison and storage. For example, cells with methylation levels above 50% are classified as the high methylation group, and those below 50% are classified as the low methylation group. The Y chromosome signal is also set with a threshold: an intensity greater than 1.0 is considered a strong signal group, and less than 1.0 is considered a weak signal group. In this way, cell classification and epigenetic modification association data can be formed.

[0047] S502: Based on the cell classification and epigenetic modification association data, calculate the epigenetic modification differences of cells under different classifications, and use the matching degree of classification and modification data for screening to obtain cell classification matching degree data; Based on the data associated with cell classification and epigenetic modification, the epigenetic modification differences of cells in different classifications are calculated. The specific operation is to calculate the mean and standard deviation of the X chromatin epigenetic modification level and Y chromosome signal of cells in each classification group, and then compare the differences between the classification groups. For example, in male cells, the average methylation level of X chromatin is 55%, while in female cells it is 65%. After calculating the standard deviation, it is found that it is 5% in males and 4% in females. Then, whether the difference between the two groups is statistically significant can be analyzed. The p-value can be calculated using a t-test. Assume that the p-value is 0.03, indicating that the difference between the two groups is significant. For the Y chromosome signal, the mean and standard deviation are also calculated. Assume that the average Y signal intensity of male cells is 1.5 units and that of female cells is 0.2 units, indicating a significant difference. Then, these difference data are used to calculate the match between the classification and modification data. The match can be determined by calculating the mean square error of the modification data within the classification group. Groups with higher match indicate that the classification label is more strongly correlated with the epigenetic modification data. In this way, cell populations with high match are screened out, and finally cell classification match data is obtained.

[0048] S503: Call the cell classification matching data, store the classification data and update the record, using the formula: ; Calculate the classification data update coefficient and update the sex cell classification record data; in, represents the classification data update coefficient, Representative The apparent modification intensity of X chromatin in each cell, represents the average value of chromatin epigenetic modification of all cells X, Representative The Y chromosome signal intensity of each cell, represents the average value of Y chromosome signals of all cells, Representative The total amount of X and Y chromatin modifications per cell, represents the maximum value of the total amount of modification in all cells, Indicates the minimum value for the total amount of modification.

[0049] Call the cell classification matching data, store the classification data and update the record, using the formula: ; Calculate the classification data update coefficient and update the sex cell classification record data.

[0050] Detailed explanation of the formula and examples: Representative The apparent modification intensity of chromatin X in each cell is assuming that the modification intensity of cell A is 50%, cell B is 60%, and cell C is 55%; is the average value of chromatin epigenetic modification of all cells X, ; Representative The signal intensity of the Y chromosome of each cell is 1.1 units for cell A, 1.4 units for cell B, and 1.3 units for cell C. is the average value of the Y chromosome signal of all cells, ; For the The total amount of chromatin modifications in cell X and Y is 51.1 for cell A, 61.4 for cell B, and 56.3 for cell C. , .

[0051] Plugging these values into the formula: ; ; ; This formula combines the sum of squares of the differences between X chromatin epigenetic modifications and Y chromosome signals and introduces the maximum and minimum differences in the total amount of modifications. This formula can more comprehensively reflect the update status of cell classification data. Compared with traditional mean difference analysis, it emphasizes the impact of extreme values on data updates and helps highlight the significance of classification boundaries.

[0052] The result shows that the update coefficient of the cell classification data is 17.37. Combined with the previously set classification update threshold (for example, the threshold is 15), it can be judged that the data has changed significantly and the classification record needs to be updated. Therefore, the sex cell classification record data is updated.

[0053] A sex cell automatic classification and recording system, based on the above-mentioned sex cell automatic classification and recording method, the system includes: The cell nucleus DNA synthesis rate calculation module obtains the sex of cells, adds detectable nucleotides, detects the changes in fluorescence signals during DNA synthesis in the cell nucleus, collects fluorescence microscopy imaging data, extracts fluorescence intensities at different time points, calculates the DNA synthesis rate of each cell nucleus, and obtains the cell nucleus DNA synthesis rate data; The cell cycle feature extraction module extracts the S phase start time and G2 / M transition duration based on the nuclear DNA synthesis rate data. It combines the nuclear area and chromatin distribution to compare the rate characteristics of different cycle stages, calculate the cell cycle change trend, and obtain the cell cycle feature trajectory. The sex-specific trajectory matching module calls the cell cycle characteristic trajectory, screens the XIST gene expression level and SRY gene level, combines the X chromatin state and Y chromosome signal, calculates the sex-specific changes in DNA synthesis rate, matches the sex standard trajectory, sets a threshold to screen cells that meet the standard, and obtains the sex-specific trajectory matching results; The sex cell classification module calculates the S phase proportion and G2 / M duration based on the sex-specific trajectory matching results, selects cells that meet the classification criteria based on the confidence level, sets the classification judgment threshold, matches the sex cell category according to the classification criteria, and obtains the sex cell classification label; The classification record and data integration module calls the sex cell classification label, integrates X chromatin epigenetic modification data and Y chromosome data, stores the classification data, updates the classification record library, and obtains sex cell classification record data.

[0054] The above are merely preferred embodiments of the present invention and do not limit the present invention in any other form. Any technician familiar with the profession may use the technical content disclosed above to change or modify it into an equivalent embodiment with equivalent changes and apply it to other fields. However, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A method for automatically classifying and recording sex cells, characterized in that: The following steps are involved: S1: Obtain sex cells, add detectable nucleotides using pulse labeling, calculate DNA synthesis rate using fluorescence microscopy, and obtain sex cell nuclear DNA synthesis rate data; S2: Call the DNA synthesis rate data of the sex cell nucleus, extract the S phase start time and G2 / M transition duration, and combine the cell nuclear area and chromatin distribution to generate the sex cell cycle characteristic trajectory; S3: Call the sex-specific cell cycle characteristic trajectory, screen the XIST gene expression level and SRY gene level, combine the X chromatin state and Y chromosome signal, calculate the sex-specific changes in DNA synthesis rate, match the sex standard trajectory, set the threshold to screen the matching cells, and obtain the sex-specific trajectory matching result; S4: Based on the sex-specific trajectory matching results, calculate the S phase proportion and G2 / M duration, filter the classification samples according to the confidence level, set the threshold to judge the classification, and output the sex cell classification label; S5: calling the sex cell classification label, integrating X chromatin epigenetic modification data and Y chromosome data, storing the classification data, and updating the sex cell classification record data.

2. The method for automatic classification and recording of sex cells according to claim 1, characterized in that: The sex-specific cell nuclear DNA synthesis rate data includes S phase initiation time, G2 / M transition duration, cell nuclear area, and chromatin distribution. The sex-specific cell cycle characteristic trajectory includes XIST gene expression, SRY gene level, X chromatin state, Y chromosome signal, and sex-specific changes in DNA synthesis rate. The sex-specific trajectory matching results include S phase proportion, G2 / M duration, and sex-specific standard trajectory matching. The sex-specific cell classification label specifically includes classification sample confidence and classification judgment threshold. The sex-specific cell classification record data includes X chromatin epigenetic modification data, Y chromosome data, and classification data.

3. The method for automatic classification and recording of sex cells according to claim 2, characterized in that: The steps for obtaining the DNA synthesis rate data of the sex cell nuclei are specifically as follows: S101: After obtaining the sex cells, a pulse labeling method is used to incorporate detectable nucleotides into the sex cells, and the degree of nucleotide incorporation in the cell nucleus is monitored to obtain nucleotide incorporation data; S102: Based on the nucleotide incorporation amount data, using fluorescence microscopy imaging technology, detecting the fluorescence signal intensity in the cell nucleus, calling the corresponding relationship between the fluorescence signal intensity and the nucleotide incorporation amount, and obtaining fluorescence signal intensity data; S103: Calculate the DNA synthesis rate based on the fluorescence signal intensity data using the formula: ； The DNA synthesis rate value is obtained by calculation as the DNA synthesis rate data of the cell nucleus of gender; in, represents the DNA synthesis rate, Representative The fluorescence signal intensity of each cell, Representative The amount of nucleotide incorporation per cell, represents the total experimental time, Represents the total number of cells, Represents the average nucleotide incorporation of all cells.

4. The method for automatic classification and recording of sex cells according to claim 3, characterized in that: The steps for obtaining the sex cell cycle characteristic trajectory are specifically as follows: S201: calling the DNA synthesis rate data of the nuclei of the sex cells, analyzing the DNA synthesis rate change trend, extracting the S phase start time of each cell by detecting the DNA synthesis rate mutation point, and obtaining the S phase start time data; S202: Based on the S phase start time data, the time interval from the G2 phase to the M phase is monitored, and combined with the continuous change of the nuclear DNA synthesis rate, the G2 / M transition duration of each cell is calculated to generate G2 / M transition duration data; S203: Based on the S phase start time data and the G2 / M transition duration data, combined with the cell nuclear area and chromatin distribution characteristics, the formula is used: ； Computationally generate sex-specific cell cycle trajectories; in, Represents the characteristic trajectory of the cell cycle, Representative The S phase start time of each cell Representative The G2 / M transition time of each cell Representative The nuclear area of the cells, Representative The chromatin distribution value of each cell, represents the maximum S phase initiation time in all cells, Represents the minimum G2 / M transition duration in all cells, Represents the total number of cells, Representative Chromatin density value of each cell.

5. The method for automatic classification and recording of sex cells according to claim 4, characterized in that: The steps for obtaining the gender-specific trajectory matching results are specifically as follows: S301: calling the sex cell cycle characteristic trajectory, screening the XIST gene expression and SRY gene level of each cell, combining the X chromatin state and Y chromosome signal to obtain sex gene and chromatin state data; S302: Based on the sex gene and chromatin state data, calculating the sex-specific changes in DNA synthesis rate, and invoking the relationship between XIST gene expression level, SRY gene level and DNA synthesis rate to obtain sex-specific change data of DNA synthesis rate; S303: Based on the sex-specific variation data of DNA synthesis rate, match the sex standard trajectory and set a threshold to screen matching cells using the formula: ； Calculate the sex-specific matching score, filter the matching cells, and obtain the sex-specific trajectory matching results; in, represents the gender-specific matching score, Representative The expression level of XIST gene in each cell represents the standard expression level of the XIST gene in the sex standard trajectory, Representative The SRY gene level in each cell represents the standard level of the SRY gene in the sex-standard locus, Representative The DNA synthesis rate of each cell, Represents the set threshold, Represents the total number of cells involved in matching.

6. The method for automatic classification and recording of sex cells according to claim 5, characterized in that: The steps for obtaining the sex cell classification label are specifically as follows: S401: Based on the sex-specific trajectory matching results, the S phase proportion and G2 / M duration of each cell are calculated, and cell cycle phase duration data are obtained by analyzing the cell cycle phase time distribution; S402: Calculating the confidence of cell classification based on the cell cycle stage duration data, combined with the S phase proportion and G2 / M duration, and screening classification samples based on the confidence to obtain high-confidence classification sample data; S403: Call the high-confidence classification sample data and set the classification threshold using the formula: ； Calculate cell classification scores, determine classification, and output gender cell classification labels; in, represents the cell classification score, Representative The proportion of cells in S phase, represents the reference S period proportion, Representative The G2 / M duration of each cell, Refer to G2 / M duration. Represents the number of cells involved in classification, Represents the classification confidence.

7. The method for automatic classification and recording of sex cells according to claim 6, characterized in that: The steps for obtaining the sex cell classification record data are specifically as follows: S501: calling the sex cell classification label, integrating the X chromatin epigenetic modification data and Y chromosome signal of each cell, and forming cell classification and epigenetic modification association data; S502: Based on the cell classification and epigenetic modification association data, calculating the epigenetic modification differences of cells under different classifications, and screening using the matching degree of the classification and modification data to obtain cell classification matching degree data; S503: Call the cell classification matching data, store the classification data and update the record, using the formula: ； Calculate the classification data update coefficient and update the sex cell classification record data; in, represents the classification data update coefficient, Representative The apparent modification intensity of X chromatin in each cell, represents the average value of chromatin epigenetic modification of all cells X, Representative The Y chromosome signal intensity of each cell, represents the average value of Y chromosome signals of all cells, Representative The total amount of X and Y chromatin modifications per cell, represents the maximum value of the total amount of modification in all cells, Indicates the minimum value for the total amount of modification.

8. An automatic sex cell classification and recording system, characterized in that: The method for automatic classification and recording of sex cells according to any one of claims 1 to 7, wherein the system comprises: The cell nucleus DNA synthesis rate calculation module obtains the sex of cells, adds detectable nucleotides, detects the changes in fluorescence signals during DNA synthesis in the cell nucleus, collects fluorescence microscopy imaging data, extracts fluorescence intensities at different time points, calculates the DNA synthesis rate of each cell nucleus, and obtains the cell nucleus DNA synthesis rate data; The cell cycle feature extraction module extracts the S phase start time and G2 / M transition duration based on the cell nuclear DNA synthesis rate data, combines the cell nuclear area and chromatin distribution, compares the rate characteristics of different cycle stages, calculates the cell cycle change trend, and obtains the cell cycle feature trajectory; The sex-specific trajectory matching module calls the cell cycle characteristic trajectory, screens the XIST gene expression and SRY gene level, combines the X chromatin state and Y chromosome signal, calculates the sex-specific changes in DNA synthesis rate, matches the sex standard trajectory, sets a threshold to screen cells that meet the standard, and obtains the sex-specific trajectory matching result; The sex cell classification module calculates the S phase proportion and G2 / M duration based on the sex-specific trajectory matching results, selects cells that meet the classification criteria based on the confidence level, sets the classification judgment threshold, matches the sex cell category according to the classification criteria, and obtains the sex cell classification label; The classification record and data integration module calls the sex cell classification label, integrates X chromatin epigenetic modification data and Y chromosome data, stores the classification data, updates the classification record library, and obtains sex cell classification record data.

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