CN115808414A - Raman spectrum analysis method for noninvasive evaluation of embryonic development quality and application thereof - Google Patents
Raman spectrum analysis method for noninvasive evaluation of embryonic development quality and application thereof Download PDFInfo
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Abstract
The invention provides a Raman spectrum analysis method for non-invasively evaluating embryo development quality and application thereof, wherein the method comprises the following steps: performing Raman detection on metabolic markers related to the embryo development quality in the embryo culture solution, calculating Raman spectrum characteristic quantitative analysis coefficient values of the metabolic markers, and dividing different threshold value ranges to evaluate the embryo development quality; the quantification of the metabolic markers is a Raman spectrum quantitative analysis coefficient value calculated by adopting the following method: raman spectroscopic quantitation coefficient value = concentration of remaining metabolic marker in metabolic system/concentration of metabolic marker in blank control group not subjected to depletion. The method avoids the adoption of abstract statistical operation for modeling and classification, evaluates based on the physiological state of the cells reflected by the change of the biomarkers and the Raman spectrum characteristics, is a qualitative analysis method combined with quantification, and obtains an evaluation result which has more objectivity and mechanism interpretability.
Description
Technical Field
The invention belongs to the technical field of embryo in-vitro culture, and particularly relates to a Raman spectrum analysis method for noninvasive evaluation of embryo development quality and application thereof.
Background
The embryonic cells can be selectively transplanted into a receptor for further development after in vitro fertilization culture, and the development quality of the embryo is an important factor influencing the embryo fate after transplantation. Therefore, the evaluation of the development quality of the embryo is very important before the transplantation, and the transplantation of the high-quality embryo is completed, so that the embryo outcome after the transplantation can be greatly improved.
One of the methods for evaluating the quality before embryo transplantation is to grade the embryo by morphological observation, grade the development quality of cells by combining morphological standards of different development stages and determine whether the cells meet the transplantation conditions according to records, the method is most mature and widely used, but the method excessively depends on human judgment and has different execution standards, and the transplantation success rate of selecting the transplanted embryo cells and meeting the expected outcome is limited. Secondly, genetic evaluation before embryo cell transplantation is carried out by using a gene sequencing method, which is also an important evaluation method in practical application, but most of the methods are not non-invasive detection in technology and have the problems of high cost, long time consumption and the like. Therefore, more technical schemes need to be supplemented to evaluate the development potential of the embryo in the application.
The method can be used as a method for indirectly monitoring the physiological condition of cells based on the metabolic spectrum analysis of the embryo culture solution, and the monitored metabolic level has great correlation with the continuous development potential of the embryo cells. Therefore, the application feasibility of evaluating the development potential of the embryo by the variation of the metabolic markers is realized. The biomarker for monitoring the embryonic development metabolic level comprises two parts, namely nutrient substrates taken up and consumed by cells and metabolic secretions produced by the cells, and other techniques for performing embryonic cell development quality evaluation by using other spectroscopic analysis methods mainly comprise the following steps: liquid phase/gas chromatography-mass spectrometry, nuclear magnetic resonance, near infrared spectroscopy and other instrument analysis methods.
CN105112491A discloses a screening method of goat transgenic cloned embryo quality evaluation differential metabolites based on gas chromatography-mass spectrometry technology and morphology. The technical scheme of the screening method combines morphological evaluation (morphological standard) of the embryo, and firstly tries to detect the change of the embryo metabolic substances by a gas chromatography-mass spectrometry analysis technology, analyzes whether the correlation exists between the change and the embryo development potential, and provides reference for searching and predicting the research of the goat transgenic clone embryo development potential. However, the cost of mass spectrometry is high, and the application has certain limitation.
CN112945894A discloses a method for noninvasive detection of embryo culture solution metabolites by infrared spectroscopy, which comprises the steps of obtaining a solid sample of an embryo culture solution by means of rapid dehydration and drying of the embryo culture solution, obtaining high-quality embryo culture solution infrared spectroscopy by an infrared spectrometer, thereby reliably detecting the tiny metabolic differences of the embryo culture solution under different conditions, carrying out spectral analysis on the collected sample, and guiding embryo screening based on spectral biomarkers according to the blastocyst formation rate, pregnancy ending and the like of the collected sample, thereby more accurately and conveniently evaluating the embryo quality. However, the infrared spectrum absorbs water molecules, so that the substance detection of a liquid sample in practical application is interfered by the signal of the water molecules.
Therefore, the method for non-invasively, accurately and efficiently evaluating the development quality of the embryonic cells is provided, and has important significance for improving the success rate of pregnancy after embryo transplantation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a Raman spectrum analysis method for non-invasively evaluating the development quality of an embryo and application thereof. The invention utilizes Raman detection technology to carry out quantitative and qualitative analysis on the biomarkers influencing the embryonic development quality, and correlates and quantifies the metabolism level of the biomarkers related to the physiological state of cells to judge the embryonic development condition. The method avoids the adoption of abstract statistical operation for modeling and classification, but directly evaluates based on the physiological state of the cells reflected by the change of the biomarkers and the Raman spectrum characteristics, and the obtained evaluation result has objectivity and mechanism interpretability.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a raman spectroscopy method for non-invasively assessing embryo development quality, the method comprising: performing Raman detection on metabolic markers related to the embryo development quality in the embryo culture solution, calculating Raman spectrum quantitative analysis coefficient values of the metabolic markers, and dividing different threshold value ranges to evaluate the embryo development quality;
the quantification of the metabolic markers is a Raman spectrum quantitative analysis coefficient value calculated by adopting the following method:
raman spectroscopic quantitation coefficient values = residual metabolic marker concentration in metabolic system/metabolic marker concentration in blank control group without metabolic depletion.
According to a prognosis evaluation target, firstly counting threshold ranges corresponding to Raman spectrum quantitative analysis coefficient values of different embryo fates, carrying out Raman detection on a sample to be analyzed, calculating the Raman spectrum quantitative analysis coefficient value of a metabolic marker, and evaluating the cell embryo development quality through the threshold ranges.
The invention utilizes Raman detection technology to carry out quantitative analysis on metabolic markers related to the embryo development quality, quantifies the metabolic markers and divides a threshold range to judge the embryo development quality. The content of the embryo development quality evaluation based on the metabolic markers comprises the embryo quality/development potential prediction of metabolic factor difference in judging whether embryo cells can develop into blastula or not, whether the embryo cells develop into excellent/poor blastula or not, whether the embryo cells are pregnant after embryo transplantation or not, whether the embryo cells develop into euploid/aneuploid embryo cells or not and the like.
The sample to be tested in the invention is generally discarded embryo culture solution in the in vitro culture stage of the embryo cells, the culture solution contains metabolic substrate information which is absorbed and consumed in the continuous development process of the embryo and secretion information generated by cell metabolism, and the above general term of cell metabolism level information is used as a measuring factor reflecting the physiological state of the cells. The metabolic marker is a certain characteristic biochemical index in common physiological pathology or treatment process which can be objectively measured and evaluated, and the current physiological state of an organism can be known through measuring the metabolic marker.
Preferably, the metabolic marker comprises any one of glucose, pyruvate, glycine or glutamine or the like or a combination of at least two thereof.
Glucose is used as a main energy substance after the embryonic densification of the embryonic cells in the cleavage stage in the development stage of the embryonic cells, and the component intake quantity of the glucose is in positive correlation with the activity of the embryonic cells and the potential of pregnancy after transplantation. According to the method, the concentration of the marker is calibrated based on a Raman spectrum of metabolic substrate glucose and an internal standard method, a correlation model of glucose consumption of normal development and abnormal cells in a development period is established, quantitative analysis values of the metabolic marker glucose contained in samples in culture solutions of different embryo fates are analyzed in a correlation mode, and a threshold range is divided to achieve estimation and prediction of the embryo fates.
Preferably, the evaluation criteria for evaluating the quality of the embryo developing into a blastocyst by using glucose as a metabolic marker comprises:
the threshold range of the analysis coefficient of the glucose is 0.8135 +/-0.0675, and the quality of the embryo is evaluated as a non-blastocyst;
the threshold range of the analysis coefficient of the glucose is 0.6302 +/-0.05, and the quality of the embryo is evaluated as an inferior blastocyst;
the threshold range of the analysis coefficient of glucose is 0.497 +/-0.054, and the embryo quality is evaluated to be a high-quality blastocyst.
Preferably, the evaluation criteria for evaluating predicted embryo outcome using glucose as a metabolic marker include:
an analytical coefficient threshold range for glucose is 0.4975 ± 0.0655, the embryo potential is predicted to be pregnancies;
the analytical coefficient threshold range for glucose was 0.67 ± 0.051, the embryo potential was predicted to be non-pregnant.
Pyruvic acid is used as basic energy substance in the developmental stage before the densification of embryo cells in cleavage stage, and the intake of its components in the cell development cycle is positively correlated with the development quality of embryo. In the present invention, the energy substrate metabolism level will be analyzed in conjunction with raman spectroscopy to assess embryo development quality. Quantitative analysis of the biomarkers is carried out based on a Raman spectrum and an internal standard method of pyruvic acid, the established pyruvic acid uptake correlation model before the embryos in the cleavage stage of the normal and abnormal cells are not densified in the development cycle has significant characteristics corresponding to different threshold ranges, and the development fates of the embryos are distinguished according to the threshold ranges.
Preferably, the criteria for assessing an embryo at cleavage stage using pyruvate as a metabolic marker include:
the threshold range of the pyruvic acid is 0.3935 +/-0.0955, and the embryo quality evaluation is a high-quality cleavage embryo;
the threshold range of the pyruvic acid is 0.64 +/-0.072, and the embryo quality is evaluated to be a poor cleavage embryo.
Glycine has physiological functions of maintaining osmotic balance, providing a protection mechanism, serving as an intracellular pH buffer solution and the like in the embryonic cell development stage, and the intake amount of the glycine in the cell development cycle is in positive correlation with the embryonic development quality. In the invention, the glycine metabolism level is analyzed by combining a Raman spectroscopy technology to evaluate the development quality of the embryo. Quantitative analysis of biomarkers is carried out based on Raman spectrum of glycine and an internal standard method, the established correlation model of glycine intake of normal-development and abnormal cells in the period from fertilized egg development to cleavage stage densification is correlated with embryo fates through quantitative analysis of metabolic marker glycine contained in embryo culture solutions under different development conditions, obvious characteristics are provided corresponding to different threshold ranges, and the development fates of embryos are distinguished according to the threshold ranges.
Preferably, the criteria for evaluating the development quality of the embryo at the cleavage stage by using glycine as a metabolic marker comprise:
the threshold range of glycine is 0.451 +/-0.06, and the embryo development quality is evaluated to be a high-quality cleavage embryo;
the threshold range of glycine is 0.618 +/-0.077, and the development quality of the embryo is evaluated to be poor-quality cleavage embryo.
Glutamine is one of the raw materials for protein synthesis as an important amino acid in the development cycle of embryonic cells, participates in the biochemical actions of generating ATP (adenosine triphosphate) by tricarboxylic acid cycle to provide energy for cells, relieving oxidative stress of the cells and the like, and has correlation with the uptake of the components, the activity of the embryonic cells and the development potential after transplantation. In the invention, the concentration of a marker is calibrated based on a Raman spectrum of a metabolic substrate glutamine and an internal standard method, a correlation model of glutamine intake of euploid and aneuploid embryonic cells in a development cycle is established, correlation is carried out by detecting a quantitative analysis value of the metabolic marker glutamine contained in a culture solution and an embryo outcome, and the development outcome of the embryo is evaluated according to a pre-established threshold range.
Preferably, the evaluation criteria for predicting the outcome of the pre-transplantation genetic diagnosis of an embryo using glutamine as a metabolic marker comprises:
the threshold range of glutamine is 0.6455 +/-0.0995, and the genetic diagnosis result before embryo transplantation is euploid;
the threshold range of glutamine is 0.4955 +/-0.0675, and the genetic diagnosis result before embryo transplantation is aneuploid.
Preferably, the method for assessing embryo development quality comprises the following steps:
(1) Establishing a quantitative and qualitative analysis model, and collecting waste embryo culture solutions of a blank control group and an experimental group;
(2) Detecting a sample to be detected by adopting a Raman detection technology;
(3) Preprocessing the Raman detection data, and sequentially performing batch correction and quantitative marker calculation;
(4) Taking the embryo fates as labels, and carrying out statistical analysis on the quantitative analysis coefficient value ranges of different embryo fates;
(5) And (5) predicting the embryo outcome corresponding to the test group samples, collecting the test group samples, processing according to the steps (2) and (3), and completing evaluation of the embryo development quality according to the Raman spectrum quantitative analysis coefficient value of the test group metabolic markers and the threshold range obtained in the step (4).
In a second aspect, the present invention provides a system for assessing the quality of embryo development, the system comprising:
a sample detection module: detecting the metabolic markers in the sample to be detected by adopting a Raman detection technology;
a data analysis module: preprocessing the Raman detection data, sequentially performing batch correction and marker quantification, and counting the Raman spectrum quantitative analysis coefficient value threshold value range of the metabolic markers corresponding to different embryo fates; taking the embryo fates as labels, and carrying out statistical analysis on the quantitative analysis coefficient value ranges of different embryo fates;
a quality evaluation module: and finishing the evaluation of the embryo development quality according to the Raman spectrum quantitative analysis coefficient value of the metabolic markers of the test group and the threshold range obtained by the data analysis module.
In a third aspect, the invention provides the use of the system for assessing embryo development quality of the second aspect in assisted reproduction.
In the invention, the theoretical basis for discovering biomarkers and quantitatively screening embryo quality based on Raman spectrum is as follows:
the technical basis of quantitative analysis based on Raman spectroscopy is represented by the following formula according to the classical electromagnetic field theory, namely the integrated intensity of the Raman spectrum peak of a detected molecule at the frequency:
wherein,cit is the speed of the light that is,his a constant of the number of planck,I Laser is the intensity of the excitation light and,Nin order to be able to count the number of scattering molecules,vandv 0 respectively the molecular vibration frequency and the frequency of the exciting light,μin order to convert the mass into vibration atoms,Kis the boltzmann constant, and is,Tis a value of an absolute temperature which,α' a is the average of the invariant values of the polarizability tensor,γ' a is an anisotropic polarizability tensor invariant. The above formula shows that under certain conditions, namely under the conditions of constant temperature, excitation light intensity, solution uniformity and other environments and all controllable factors, the peak integral intensity or peak area of the marker molecule at a certain frequency position represents the molecule concentration, and the endogenous feature peak has a direct proportion relation with the molecule concentration in the solution represented by the relative intensity of the feature peak of the detected marker. Combined with the theory, the product is stable in factors such as environment and the likeUnder the condition of invariable calibration, the quantitative calibration of the marker accords with positive correlation, and the calculation method is as follows:
wherein, R represents the ratio of the characteristic peak intensity of the measured object to the characteristic peak intensity of the internal standard substance and is used for representing relative concentration, c represents the substance concentration, and I represents the integral intensity of the characteristic peak. The characteristic peak of the marker refers to a raman characteristic peak in which only the marker constitutes a molecular vibration mode assignment.
The invention establishes an evaluation system for evaluating the embryo development quality by using markers in the quantitative waste embryo culture solution by applying the theory, the concentration of all components in the sample solution is expressed by using the integral intensity of the Raman spectrum characteristic peak of the detected sample, and the relative concentration of unknown metabolic markers is calibrated by using a constant concentration substance. Generally, under different inspection batches, the spectral response is unstable due to the influence of environmental factors on the spectral uniformity, and large errors of marker characteristic peak intensity calibration are easily caused.
In conclusion, the technical scheme of the invention quantitatively analyzes the metabolic level marker amount through Raman spectroscopy in application, and is used for evaluating the physiological state and the development potential of cells as an identification experience.
Compared with the prior art, the invention has the following beneficial effects:
(1) The key biomarkers are quantitatively analyzed by adopting a Raman spectrum technology, the development potential of the embryo is evaluated according to the physiological state of the embryo cell, and compared with the method for predicting the quality of the embryo by combining a machine learning algorithm, the method is more accurate and has mechanism interpretability in conclusion;
(2) The embryo cell culture solution is measured by combining the enhanced Raman spectroscopy technology, so that the trace marker signal is greatly enhanced, and the sensitivity is higher;
(3) The substance signal analysis is carried out on the basis of the original embryo waste culture solution, the metabolic information can be retained to the maximum extent, and the evaluation result of evaluating the development potential of the embryo cells after quantitative analysis is more objective.
Drawings
FIG. 1 is an implementation flow of quantifying biomarkers based on Raman spectroscopy;
FIG. 2 is a Raman spectrum of different standard concentrations of a glucose solution as a metabolic marker;
FIG. 3 is a statistical analysis of relative glucose metabolism levels in cell culture fluid quantified for different developmental qualities based on Raman spectroscopy;
FIG. 4 is a statistical analysis of relative glucose metabolism levels in cell culture fluid based on Raman spectroscopy to quantify different pregnancy outcomes;
FIG. 5 is a statistical analysis of the assessment of cell development quality based on Raman spectroscopy to quantify glycine uptake in embryo culture;
FIG. 6 is a statistical analysis of the assessment of the quality of cell development based on Raman spectroscopy to quantify pyruvate uptake in embryo culture;
FIG. 7 is a statistical analysis of the assessment of euploidy based on Raman spectroscopy to quantify glutamine uptake in embryo culture.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
The experimental process for quantifying the biomarkers based on the Raman spectrum is shown in figure 1, and comprises the following specific steps:
pre-establishing a threshold range quality evaluation system:
(1) Collecting a sample: collecting samples of a control group and an experimental group according to a unified standard, wherein the experimental group is waste embryo culture solution after cell culture and development, and comprises normal development embryonic cells and abnormal development embryonic cells. For example, a normal sample is generally collected as an embryonic cell culture solution before and after the densification of the cleavage stage or an embryonic cell culture solution in the middle and later stage of the blastocyst, and a control group is a blank embryonic cell culture solution which is not used for cell development. All sample processing methods are kept consistent, and the corresponding embryo outcome of the experimental sample is recorded.
(2) Detection by Raman spectroscopy: placing the embryo culture solution sample in a sample cell of a Raman spectrometer to complete Raman detection, wherein the experimental parameters are generally as follows: the repeated detection times are generally 5 times, the incident light source can be a 785 nm laser light source, the incident laser power is selected to be about 320 mW, the cumulative times are 3 times, and the single exposure time is 40 s.
(3) And (3) data analysis: the data preprocessing process comprises the following steps: spectrum band interception, filtering denoising, baseline correction, normalization and the like. Generally, 600-1800 cm is selected -1 The Raman frequency shift range is used as a biological information fingerprint area, the filtering algorithm used for denoising generally comprises a Savitzky-Golay algorithm, wavelet transformation and the like, the baseline correction is completed by adopting the algorithms of polynomial fitting, self-penalty iteration reweighted partial least square method and the like, and the Raman spectrum peak value is reduced to 0,1 by adopting data normalization]And establishing a data set in a uniform scale.
(4) Batch correction: different batch tests batch-to-batch variation correction was performed by raman data of blank control group samples in order to eliminate as much as possible the effect due to the substrate composition variation in each batch. If the assessment is performed in the same batch, internal standard quantification of the biomarker is performed directly.
(5) Intra biomarker scalar analysis: for aqueous liquid samples, the raman signature of water molecules therein is stable, typically 1638 cm -1 If no other substance peak generates signal contribution at the position, taking the peak value/peak area of the position characteristic peak as a reference value of the internal standard substance; for the dried crystalline sample, the culture solution contains C-C ring vibration mode molecules with Raman shift of 1000-1007 cm at characteristic peak position -1 Has a significant characteristic peak, and can be taken as the positionAnd setting the characteristic peak value as a reference value of the concentration of the internal standard substance, wherein the standard of selecting the reference value is to select the stable characteristic peak value with a single molecular vibration mode.
(6) And (3) combining the embryo fates recorded in the step (1), carrying out statistical analysis on the quantitative values corresponding to the metabolic markers of the embryos with different development qualities, and dividing a threshold range to be used as an embryo quality evaluation model.
Embryonic development quality/pregnancy potential prognostic assessment:
(7) And (3) predicting the development quality and pregnancy potential of the embryo to be evaluated, collecting a sample according to the step (1), carrying out data detection and quantitative analysis according to the steps (2), (3) and (4) at a time, and evaluating the development outcome of the embryo by combining the quantitative coefficient values of the quantified metabolic markers with the threshold value ranges obtained in the steps (5) and (6).
The application of the process according to the invention is illustrated in detail with reference to the following examples:
example 1
The above method is adopted to perform Raman spectroscopy calibration on glucose solutions with different standard concentrations of 0.1 g/L, 0.2 g/L, 0.5 g/L, 1 g/L, 2 g/L, 10 g/L, 50 g/L and 100 g/L, wherein Raman detection results of the glucose solutions with the concentrations of 50 g/L and 100 g/L are shown in FIG. 2, and calibration results of the glucose solutions with the standard concentrations by adopting the quantitative analysis method of the present invention are recorded in Table 1.
TABLE 1
Example 2
Glucose uptake consumption in cell-developing embryo culture media will be calibrated by raman spectroscopy. The sample uses a mouse as an animal model to collect culture solution for in vitro fertilization culture of embryo cells, and 20 culture solution samples in total are obtained for verification, wherein the 5 immature blastocysts, 5 poor-quality blastocysts, 5 high-quality blastocysts and 5 blank culture solution samples which do not participate in embryo culture are obtained, the immature blastocyst cells are cell culture solution samples which stop developing until the stage of the blastocyst, the poor-quality blastocyst group is an embryo culture solution sample which is observed by morphokinetics and has the advantages of slow development, more cell fragments and poor morphoking, and the high-quality blastocyst group is an embryo culture solution sample which is observed by morphoking and has the advantages of normal development cycle, almost no cell fragments and excellent morphology. The glucose uptake is recorded as a Raman spectrum quantitative analysis coefficient value, and the coefficient value is correlated to analyze the metabolic condition of glucose in the development process of embryonic cells to predict the development outcome of the embryonic, such as non-blastocyst, inferior blastocyst, superior blastocyst and the like. After Raman detection is carried out on culture solutions corresponding to embryonic cells with different development fates, statistics and threshold range division are carried out according to a quantitative analysis method to realize prediction of the development fates of the embryos, raman quantitative analysis is carried out on embryo development evaluation records as shown in table 2, a visualization result is shown in figure 3, and figure 3 is statistical analysis for quantifying relative glucose metabolism levels in cell culture solutions with different development qualities based on a Raman spectroscopy technology.
TABLE 2
Prediction of embryo pregnancy potential by glucose uptake glucose consumption in embryo culture media was calibrated by raman spectroscopy. The sample uses a mouse as an animal model to collect culture solution for culturing embryo cells through in vitro fertilization, animal individuals with basically consistent physiological indexes are selected, 10 culture solution samples corresponding to non-pregnant embryos after embryo transplantation and 10 culture solution samples for successful pregnancy after embryo transplantation are obtained, and 20 culture solution samples are counted for implementation and verification, wherein the observation of the embryo cells with successful pregnancy has a continuous pregnancy ending, and the embryo cells without pregnancy are non-pregnancy samples after transplantation. The glucose uptake is recorded as a Raman spectrum quantitative analysis coefficient value, and the coefficient value is correlated to analyze the glucose metabolism substrate uptake condition in the embryonic cell development process to complete the prediction of the embryo pregnancy outcome, wherein the outcome comprises pregnancy and non-pregnancy. After performing Raman detection on the embryo culture solutions with different pregnancy outcomes, performing statistical analysis according to a quantitative analysis method, wherein the record of Raman quantitative analysis on the embryo pregnancy outcomes is shown in Table 3, the visualization result is shown in FIG. 4, and FIG. 4 is the statistical analysis for quantifying the relative glucose metabolism level in the cell culture solutions with different pregnancy outcomes based on the Raman spectroscopy technology.
TABLE 3
In this example, it is illustrated that the concentration of glucose marker is calibrated based on raman spectroscopy and an internal standard method for metabolic substrate glucose, and a correlation model of embryo development to blastocyst stage quality, pregnancy outcome and glucose uptake is established. Specifically, statistical modeling is carried out through Raman spectrum quantitative analysis experimental records, quantitative analysis values of metabolic marker glucose contained in embryo culture solutions with different cyst formation qualities and pregnancy outcomes are associated with the pregnancy outcomes, and the glucose quantitative analysis values have significant characteristics corresponding to different threshold ranges and can be used for evaluating the quality of developed blastocysts and predicting the pregnancy outcomes of embryos.
Example 3
And calibrating the glycine uptake in the embryo culture solution through Raman spectroscopy, and establishing a correlation model of glycine uptake consumption and embryo development quality. The sample uses a mouse as an animal model to collect the embryonic cell culture fluid of in vitro fertilization culture, selects animal individuals with basically consistent physiological indexes, and obtains 10 culture fluid samples corresponding to high-quality cleavage embryos and 10 culture fluid samples of poor-quality cleavage embryos, and totally 20 culture fluid samples for implementation and verification. And (3) recording the glycine intake as a Raman spectrum quantitative analysis coefficient value, and performing correlation analysis on the intake and consumption conditions of metabolic substrates of glycine in the development process of embryonic cells by using the coefficient value to complete embryo quality evaluation, wherein the evaluation contents comprise high-quality embryo at the cleavage stage and low-quality embryo at the cleavage stage. Raman quantitative analysis the embryonic development quality records are shown in table 4, and figure 5 is a statistical analysis for assessing the quality of cell development based on raman spectroscopy techniques to quantify glycine uptake in the embryo culture fluid.
TABLE 4
In this example, the Raman spectrum and internal standard method based on glycine as metabolic substrate are illustrated to calibrate the concentration of marker, and an analysis model for the difference of glycine uptake of different quality embryos before embryo densification is established. Specifically, statistical modeling is carried out through Raman spectrum quantitative analysis experimental records, the metabolic marker glycine contained in the embryo cell culture solution with different development quality is quantitatively analyzed, a corresponding threshold range is counted, and the metabolic level has a significant characteristic and can be used for evaluating the development quality of the embryo.
Example 4
And calibrating the pyruvic acid uptake in the embryo culture solution by Raman spectroscopy, and establishing a correlation model of pyruvic acid uptake consumption and embryo development quality. The sample uses a mouse as an animal model to collect the culture solution of the embryo cells cultured by in vitro fertilization, and animal individuals with basically consistent physiological indexes are selected to obtain 10 culture solution samples corresponding to high-quality embryos in the cleavage stage and 10 culture solution samples of the embryos in the poor cleavage stage, and 20 culture solution samples are counted to carry out verification. And (3) recording the pyruvic acid intake as a Raman spectrum quantitative analysis coefficient value, and performing correlation analysis on the metabolic substrate consumption condition of pyruvic acid in the embryonic cell development process by using the coefficient value to complete embryo quality evaluation, wherein the evaluation content comprises high-quality embryo at the cleavage stage and low-quality embryo at the cleavage stage. Raman quantitative analysis the embryo development quality records are shown in table 5, and fig. 6 is a statistical analysis for assessing the cell development quality based on the raman spectroscopy technique to quantify the uptake of pyruvate in the embryo culture fluid.
TABLE 5
In this example, it is demonstrated that the concentration of marker is calibrated based on Raman spectrum of metabolic substrate pyruvic acid and internal standard method, and an analysis model for difference of pyruvic acid uptake of different quality embryos before embryo densification is established. Specifically, statistical modeling is carried out through Raman spectrum quantitative analysis experimental records, quantitative analysis is carried out on metabolic marker pyruvic acid contained in the embryo cell culture solution with different development quality, a corresponding threshold range is counted, and the metabolic level of the threshold range has a significant characteristic and can be used for evaluating the development quality of the embryo.
Example 5
And calibrating the glutamine intake in the embryo culture solution by Raman spectrum, and establishing a correlation model of glutamine intake consumption and embryo development quality. The sample uses a mouse as an animal model to collect the embryo cell culture fluid of the in vitro fertilization culture, selects animal individuals with basically consistent physiological indexes, obtains 10 culture fluid samples of euploid embryos and 10 culture fluid samples of aneuploid embryos, and performs verification on the relationship between the uptake of different euploid embryos and glutamine by totaling 20 culture fluid samples. And (4) recording Raman spectrum quantitative analysis coefficient values in an experiment, and correlating the coefficient values to analyze the uptake condition of glutamine in development to finish embryo quality evaluation. The values of the raman spectroscopy quantitation coefficients for the amount of glutamine uptake in different euploid embryo cell cultures are reported in table 6, and fig. 7 is a statistical analysis for assessing euploidy based on raman spectroscopy techniques to quantify glutamine uptake in embryo cultures.
TABLE 6
In this example, it is illustrated that the concentration of the marker is calibrated based on raman spectroscopy and an internal standard method of a metabolic substrate glutamine, and a differential analysis model of glutamine intake of different euploid embryos is established. Specifically, the statistical modeling is carried out through Raman spectrum quantitative analysis experimental records, the metabolic marker glutamine contained in the embryo cell culture solution with different euploidy is quantitatively analyzed, the corresponding threshold range is counted, and the metabolic level has a significant characteristic and can be used for evaluating the embryo euploidy.
In conclusion, the invention utilizes the Raman detection technology to quantitatively analyze the markers for determining the embryo development quality, and quantifies the biomarker indexes to judge the embryo development condition. The method avoids the adoption of abstract statistical operation for modeling and classification, evaluates based on the physiological state of the cells reflected by the change of the biomarkers and the Raman spectrum characteristics, is a qualitative analysis method combined with quantification, and obtains an evaluation result which has more objectivity and mechanism interpretability. The method for evaluating the development quality of the embryo cells has the characteristics of no wound, accuracy and high efficiency, and has important significance for improving the success rate of pregnancy after embryo transplantation.
The applicant declares that the above description is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be understood by those skilled in the art that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention disclosed herein fall within the scope and disclosure of the present invention.
Claims (10)
1. A raman spectroscopy method for non-invasively assessing embryo development quality, the method comprising: performing Raman detection on metabolic markers related to the embryo development quality in the embryo culture solution, calculating Raman spectrum quantitative analysis coefficient values of the metabolic markers, and dividing different threshold value ranges to evaluate the embryo development quality;
the quantification of the metabolic markers is the quantitative analysis coefficient values of Raman spectra calculated by the following method:
raman spectroscopic quantitation coefficient values = metabolic marker concentration remaining in the metabolic system/metabolic marker concentration in the blank control group without metabolic depletion.
2. A raman spectroscopic method of non-invasive assessment of the quality of embryo development according to claim 1 wherein said metabolic markers comprise any one or a combination of at least two of glucose, pyruvate, glycine or glutamine.
3. A raman spectroscopy method for non-invasively assessing embryo development quality according to claim 2, wherein the criteria for assessing embryo development to blastocyst using glucose as a metabolic marker comprises:
the threshold range of the analysis coefficient of the glucose is 0.8135 +/-0.0675, and the quality of the embryo is evaluated as a non-blastocyst;
the threshold range of the analysis coefficient of the glucose is 0.6302 +/-0.05, and the quality of the embryo is evaluated as an inferior blastocyst;
the threshold range of the analysis coefficient of glucose is 0.497 +/-0.054, and the embryo quality is evaluated to be a high-quality blastocyst.
4. A raman spectroscopic method for non-invasively assessing embryo development quality according to claim 2, wherein the assessment of criteria for predicting embryo outcome using glucose as a metabolic marker comprises:
an analytical coefficient threshold range for glucose is 0.4975 ± 0.0655, the embryo potential is predicted to be pregnancies;
the threshold range of analytical coefficient for glucose was 0.67 ± 0.051, the embryo potential was predicted to be non-pregnant.
5. A raman spectroscopic method for non-invasively assessing the quality of embryo development according to claim 2, wherein the criteria for assessing the quality of embryos at cleavage stage using pyruvic acid as a metabolic marker comprises:
the threshold range of the pyruvic acid is 0.3935 +/-0.0955, and the embryo quality is evaluated to be a high-quality cracked embryo;
the threshold range of the pyruvic acid is 0.64 +/-0.072, and the embryo quality is evaluated as a poor-quality cleavage embryo.
6. A raman spectroscopy method for noninvasive evaluation of embryo development quality according to claim 2, wherein the criterion for evaluating embryo development quality at cleavage stage using glycine as a metabolic marker comprises:
the threshold range of glycine is 0.451 +/-0.06, and the embryo development quality is evaluated to be a high-quality cleavage embryo;
the threshold range of glycine is 0.618 +/-0.077, and the development quality of the embryo is evaluated to be poor-quality cleavage embryo.
7. A raman spectroscopy method for noninvasive evaluation of embryo development quality according to claim 2, wherein the evaluation criterion for predicting the outcome of pre-transplantation genetic diagnosis of an embryo with glutamine as a metabolic marker comprises:
the threshold range of glutamine is 0.6455 +/-0.0995, and the genetic diagnosis result before embryo transplantation is euploid;
the threshold range of glutamine is 0.4955 +/-0.0675, and the genetic diagnosis result before embryo transplantation is aneuploid.
8. Raman spectroscopy method for non-invasive assessment of embryo development quality according to any of claims 1-7, wherein said method for assessing embryo development quality comprises the steps of:
(1) Establishing a quantitative and qualitative analysis model, and collecting waste embryo culture solutions of a blank control group and an experimental group;
(2) Detecting a sample to be detected by adopting a Raman detection technology;
(3) Preprocessing the Raman detection data, and sequentially performing batch correction and quantitative marker calculation;
(4) Taking the embryo outcome as a label, and statistically analyzing the quantitative analysis coefficient value ranges of different embryo outcomes;
(5) And (4) predicting the embryo fate corresponding to the test group samples, collecting the test group samples, processing according to the steps (2) and (3), and completing evaluation of the embryo development quality according to the Raman spectrum quantitative analysis coefficient value of the test group metabolic markers and the threshold range obtained in the step (4).
9. A system for assessing embryo development quality, the system comprising:
a sample detection module: detecting the metabolic markers in the sample to be detected by adopting a Raman detection technology;
a data analysis module: preprocessing the Raman detection data, sequentially performing batch correction and marker quantification, and counting the Raman spectrum quantitative analysis coefficient value threshold value range of the metabolic markers corresponding to different embryo fates; taking the embryo fates as labels, and carrying out statistical analysis on the quantitative analysis coefficient value ranges of different embryo fates;
a quality evaluation module: and finishing the evaluation of the embryo development quality according to the Raman spectrum quantitative analysis coefficient value of the metabolic markers of the test group and the threshold range obtained by the data analysis module.
10. Use of the system for assessing the quality of embryo development of claim 9 in assisted reproduction.
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