Application of lipid marker in preparation of product for predicting occurrence risk of polyspermia fertilization

Abstract

The invention discloses an application of a lipid marker in preparation of a product for predicting the occurrence risk of polyspermia fertilization, and relates to the technical field of assisted reproduction, and the lipid marker is at least one of triglyceride, phosphatidylcholine and phosphatidylethanolamine. The invention further discloses a kit for predicting the occurrence risk of polyspermia fertilization. The level of the lipid marker in sperms is in significant positive correlation with the occurrence risk of polyspermia fertilization during in-vitro fertilization of egg cells, a prediction model established by single or combination of the lipid marker shows high discrimination performance, and the problem that there is no effective means to screen and discriminate patients with polyspermia fertilization at present is solved.

Description

Technical Field

[0001] The present invention relates to the field of assisted reproductive technology, and in particular to the use of a lipid marker in the preparation of a product for predicting the risk of polyspermy. Background Art

[0002] Infertility is a reproductive system disease that affects the body's ability to achieve basic reproductive capabilities. In order to solve the problem of infertility, human assisted reproductive technology came into being and has developed rapidly in the past few decades. At present, a huge clinical technology system has been formed, including artificial insemination, in vitro fertilization-embryo transfer and its derivative technologies. In vitro fertilization (in vitrofertilization) is the process of fertilizing sperm and eggs and early embryonic development in vitro. It includes conventional in vitro fertilization and intracytoplasmic sperm injection. Successful fertilization marks the beginning of a new life, and in assisted reproductive technology treatment, successful fertilization is the prerequisite for providing enough transplantable embryos.

[0003] Polyspermy refers to the phenomenon that two or more sperm enter the oocyte and form three or more pronuclei. Although the risk of polyspermy is moderate under natural conditions, it is still the main obstacle to the successful development of usable embryos in different species, including humans, in the in vitro fertilization system. Polyspermy is a common abnormal fertilization mode in conventional in vitro fertilization, and its polyspermy rate can reach more than 10%. Embryos from polyspermic oocytes cannot develop normally, let alone be used for embryo transplantation, resulting in a reduced treatment success rate and a waste of eggs. Previous studies have also shown that polyspermy can cause adverse laboratory and clinical outcomes, including embryo death and the risk of spontaneous abortion. Therefore, the polyspermy rate has become one of the quality control indicators of reproductive laboratories. The 2017 Vienna Consensus recommends that the polyspermy rate of IVF should be limited to 6%. However, despite strict laboratory standardization procedures, the polyspermy rate of IVF may still exceed 6%, meaning that more than 6% of fertilized eggs will not be usable due to polyspermy. Intracytoplasmic sperm injection (CSFI) selects a single sperm to be injected into the egg, which can effectively avoid the occurrence of polyspermy. However, CSFI is an invasive procedure, and bypassing natural selection barriers during fertilization may lead to increased health risks for offspring, including congenital abnormalities, neurodevelopmental delays, and mental disorders. In addition, CSFI will also increase the cost of patients and the workload and working hours of laboratory staff. Therefore, the clinic will strictly avoid the use of unnecessary CSFI.

[0004] At present, in clinical practice, andrologists use the semen routine parameter standards (such as concentration ≥15×10 6 / mL, forward movement ≥32%, malformation rate ≤96%, etc.), and make diagnoses such as oligospermia and teratozoospermia for male patients. However, this diagnosis is only a reference and cannot be used as a basis for selecting in vitro fertilization methods. The current situation is that there is no unified fertilization method selection standard at home and abroad and in various reproductive medical centers. Instead, most of them are based on empirical and more stringent semen abnormality parameter indicators (such as density <1×10 6 / mL, sperm abnormality rate>98%, etc.), and even use high IVF abnormal fertilization rate as an indication for choosing ICSI fertilization next time. In the specific clinical practice in my country, up to 70-80% of fertilization methods are IVF. Under the premise of ensuring fertilization safety to the greatest extent, it is inevitable that a large number of high-proportion IVF polyspermy cases will occur, thus wasting precious egg resources. Therefore, there is currently no effective means to effectively screen and identify patients who may have polyspermy, so as to avoid the risk of failure in patients with high polyspermy risk. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an application of a lipid marker in the preparation of a product for predicting the risk of polyspermy, the level of the lipid marker in sperm is significantly positively correlated with the risk of polyspermy during in vitro fertilization of oocytes, and the prediction model established using the lipid marker alone or in combination shows strong discriminative performance, solving the current problem of lack of effective means to screen and identify patients with polyspermy.

[0006] The technical solution of the present invention to solve the above technical problem is as follows: providing an application of a lipid marker in the preparation of a product for predicting the risk of polyspermy.

[0007] Furthermore, the lipid marker is at least one of triglyceride, acetal phosphatidylcholine and acetal phosphatidylethanolamine.

[0008] Furthermore, the lipid marker is triglyceride.

[0009] Furthermore, the lipid markers are three lipids: triglycerides, acetal phosphatidylcholine and acetal phosphatidylethanolamine.

[0010] Furthermore, the product predicts the risk of polyspermy during in vitro fertilization of oocytes by detecting the levels of lipid markers in sperm; the levels of lipid markers are positively correlated with the risk of polyspermy.

[0011] Furthermore, lipid marker levels were detected based on high-throughput lipidomics or colorimetric methods.

[0012] Furthermore, the product is a detection device, a reagent or a kit.

[0013] The present invention also provides a kit for predicting the risk of polyspermy, comprising a reagent for detecting lipid markers.

[0014] The present invention has the following beneficial effects:

[0015] The present invention analyzes and finds that the levels of lipid triacylglycerols (TAG), plasmalogen PE and plasmalogen PC in sperm are positively correlated with the risk of polyspermy during in vitro fertilization of oocytes. The prediction model established by using them alone or in combination shows strong discriminative performance and has great predictive value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a comparison of the lipid profile of sperm in the normal fertilization group and the polyspermy group;

[0017] Figure 2 The results of Spearman correlation analysis of differential lipids are shown;

[0018] Figure 3 ROC curve for predicting the risk of polyspermy. DETAILED DESCRIPTION

[0019] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0020] Example 1

[0021] (1) According to the incidence of polyspermy, the patients were divided into two groups: normal fertilization group (polyspermy rate less than 15%) and polyspermy group (polyspermy rate greater than 15%). The clinical parameters of the two groups are shown in Table 1. Except for the fertilization indicators such as normal fertilization rate and polyspermy rate, there were no significant differences in the clinical parameters of the two groups.

[0022] Table 1 Clinical parameters of the two groups of patients

[0023] Normal fertilization group (mean ± SD) Polyspermia group (mean ± SD) p-value Sample size (n) 13 12 Female age (years) 29.77±3.44 28.33±3.58 0.317 Male age (years) 30.77±3.83 31.17±3.97 0.801 <![CDATA[Body mass index of women (kg / m 2 )]]> 21.93±2.94 21.72±2.90 0.859 FSH 6.40±1.17 6.37±1.88 0.967 LH 4.83±2.07 5.25±2.66 0.669 E2 60.55±43.32 42.70±11.39 0.180 PRL 17.03±5.72 19.59±10.77 0.461 <![CDATA[Male body mass index (kg / m 2 )]]> 24.43±2.63 23.10±1.78 0.16 AMH 3.38±1.32 3.58±2.06 0.769 Semen volume (mL) 3.44±1.12 2.97±1.05 0.29 <![CDATA[Sperm density (10 6 / mL)]]> 59.38±14.89 69.25±16.13 0.125 Forward movement ability (%) 46.54±4.05 45.75±4.05 0.631 Sperm deformity index (%) 93.92±1.61 94.50±1.51 0.365 Non-forward motion (%) 10.85±1.86 10.58±3.45 0.813 Immotile sperm percentage (%) 42.62±3.64 43.67±4.83 0.543 Number of eggs retrieved 11.92±2.93 10.58±3.42 0.303 Number of mature oocytes 10.77±2.86 9.75±3.36 0.421 Fertilization rate 0.81±0.10 0.85±0.17 0.518 Normal fertilization rate 0.78±0.09 0.57±0.15 <0.01 Polyspermy rate 0.03±0.05 0.26±0.09 <0.01 Mature oocyte recovery rate 0.89±0.09 0.91±0.12 0.654 Mature oocyte fertilization rate 0.91±0.09 0.93±0.10 0.790 Normal fertilization rate of mature oocytes 0.95±0.08 0.64±0.11 <0.01 Polyspermy rate of mature oocytes 0.05±0.08 0.33±0.11 <0.01

[0024] (2) Pearson correlation analysis was performed on the clinical data of the two groups of patients and the incidence of polyspermy. The results are shown in Table 2. It was found that there was no significant correlation between these clinical data and the incidence of polyspermy.

[0025] Table 2 Pearson correlation analysis results

[0026]

[0027]

[0028] (3) We used targeted lipidomics to analyze the lipidome of 13 sperm samples from the normal fertilization group and 12 sperm samples from the polyspermy group, and quantified the various lipid types in sperm. The results are as follows: Figure 1 As shown, * indicates significant difference at p < 0.05 level.

[0029] Depend on Figure 1 It can be seen that by comparing the differences in the contents of various lipids in the sperm of the two groups, it can be found that compared with the sperm of patients with a low risk of polyspermy, the contents of glucosylceramide (GluCer), ceramide (Cer), ceramide trihexosides (Gb3), plasmagen.PE, plasmagenPC, phosphatidyl choline (PC), diacylglycerol (DAG) and triacylglycerols (TAG) in the sperm of patients with a high risk of polyspermy are significantly increased.

[0030] (4) Spearman correlation analysis was performed on the differential lipids and polyspermy fertilization rate. The results are shown in Table 3.

[0031] Table 3 Correlation between significantly different lipid levels and polyspermy rate

[0032] Lipids Pearson correlation coefficient P-value GluCer 0.309 0.133 Cer 0.466 0.019 Gb3 0.471 0.018 plasmalogen PE 0.394 0.051 plasmalogenPC 0.491 0.013 PC 0.421 0.036 DAG 0.476 0.016 TAG 0.465 0.019

[0033] As shown in Table 3, among the lipids with significant differences, Cer, Gb3, plasmalogenPC, PC, DAG and TAG were significantly positively correlated with the incidence of polyspermy (P<0.05).

[0034] Subsequently, after excluding immature eggs, Spearman correlation analysis was performed on the differential lipids and polyspermy rates of mature eggs, and the results are shown in Table 4.

[0035] Table 4 Correlation between differential lipids and polyspermy rate of mature eggs

[0036] Lipids Pearson correlation coefficient P-value GluCer 0.438 0.029 Cer 0.579 0.002 Gb3 0.547 0.005 plasmalogen PE 0.661 0.000 plasmalogenPC 0.553 0.004 PC 0.551 0.004 DAG 0.512 0.009 TAG 0.559 0.004

[0037] As shown in Table 4, the eight lipids, GluCer, Cer, Gb3, plasmalogen.PE, plasmalogenPC, PC, DAG and TAG, were significantly positively correlated with the polyspermy rate of mature eggs (P<0.05), and the correlation coefficients of each lipid were higher than the correlation coefficient with the overall polyspermy rate.

[0038] (5) Spearman correlation analysis was used to analyze the correlation of these 8 lipids. The results are as follows Figure 2 shown.

[0039] Depend on Figure 2 It can be seen that the correlation between TAG and DAG and the other six lipids is weak, and the correlation between them is strong (r=0.85). However, the correlation between Cer, GluCer, Gb3, plasmalogenPC, PC, plasmalogen.PE is strong.

[0040] (6) The bidirectional stepwise regression method and the best subset method were used to screen lipids related to polyspermy, and a logistic regression model was constructed to predict the occurrence of polyspermy. The best prediction model was selected based on the area under the receiver operating curve (AUROC). The results are shown in Figure 2. Figure 3 As shown in Figure 2, A is the ROC curve based on TAG, and B is the adjusted R corresponding to the optimal subset of different lipid type combinations. 2 Value, C is the ROC curve based on TAG, plasmalogenPC and plasmalogen.PE.

[0041] The Akaike information criterion (AIC) is used as a criterion for evaluating the two-way stepwise regression method. The lower the AIC, the better the model. After all the above 8 lipids were included in the model, a complete logistic regression model was established. The two-way stepwise regression method was used, and AIC was used as the observation index. When the AIC value reached the minimum, the regression was stopped. The model with the minimum AIC value at this time was the final risk prediction model. When the variable included in the model was TAG, AIC = 23.892, which was the minimum value at this time. Figure 3 As shown in Figure A, the AUC value of the logistic regression based on TAG is 0.8974, showing strong discriminative performance.

[0042] At the same time, using the optimal subset method, the corrected R 2 As the criterion for the optimal subset, corrected R 2 The larger the value, the better the model. 2 The new clinical risk model is established by combining the predictor variables with the largest value. Figure 3As shown in Figure B, the lipids screened based on the Adjust R2 index are TAG, plasmalogenPC, and plasmalogen.PE. These three predictive variables were included in the final multivariate analysis. Figure 3 As shown in Figure C, the AUC value of the logistic regression based on these three lipids is also 0.8974, which also shows strong discriminative performance.

[0043] The variance inflation factor (VIF) was used to test the multicollinearity of all variables included in the model. Independent variables with a VIF value greater than 5 were considered to have a high degree of multicollinearity. The results are shown in Table 5.

[0044] Table 5 Multicollinearity analysis of risk factors for polyspermy

[0045] variable Variance Expansion Factor TAG 1.177 plasmalogenPC 4.465 plasmalogen.PE 4.799

[0046] As can be seen from Table 5, there is no collinearity between the three lipids, indicating that the variables are independent of each other and there is no linear relationship, which shows that the model has a strong predictive ability and the results are more reliable.

[0047] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of lipid markers in the preparation of products for predicting the risk of polyspermy.

2. The use according to claim 1, characterized in that The lipid marker is at least one of triglyceride, acetal phosphatidylcholine and acetal phosphatidylethanolamine.

3. The use according to claim 1, characterized in that The lipid marker is triglyceride.

4. The use according to claim 1, characterized in that The lipid markers are three lipids: triglyceride, acetal phosphatidylcholine and acetal phosphatidylethanolamine.

5. The use according to claim 1, characterized in that The product predicts the risk of polyspermy during in vitro fertilization of oocytes by detecting the level of the lipid marker in sperm; the level of the lipid marker is positively correlated with the risk of polyspermy.

6. The use according to claim 1, characterized in that The lipid marker levels are detected based on high-throughput lipidomics or colorimetric methods.

7. The use according to claim 1, characterized in that The product is a detection device, a reagent or a test kit.

8. A kit for predicting the risk of polyspermy, characterized in that: The invention comprises a reagent for detecting the lipid marker described in claim 1.

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