Application of cumulus cell mTOR-S6 protein marker in non-invasive prediction of embryo quality and pregnancy outcome

Abstract

The invention provides application of a cumulus cell mTOR-S6 protein marker in non-invasive prediction of embryo quality and pregnancy outcome, and relates to the technical field of bioengineering. According to the present invention, the Westernblot technology is adopted to quantitatively detect the levels of the mTOR signal channel key proteins such as p-mTOR, mTOR, p-S6 and S6 in cumulus cells; meanwhile, the system collects IVF related clinical indexes of the patient, correlation analysis is carried out on the clinical indexes and the relative levels of the four key proteins through a statistical method, and it is found that the IVF related indexes of the patient are evaluated with the median value of the relative expression values of the four proteins as a threshold value. Whether the ovarian reserve function, the embryonic development quality and the pregnancy outcome are good or not can be judged. The invention not only provides an innovative auxiliary evaluation tool for the field of reproductive medicine, but also provides a scientific basis for formulating an individualized IVF treatment scheme.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to application of an oocyte cumulus cell mTOR-S6 protein marker in preparation of an embryo quality and pregnancy outcome prediction product. BACKGROUND

[0002] With the increase of women's age, the fertility gradually declines, and the success rate of in vitro fertilization (IVF) is also affected. At present, the evaluation of oocyte and embryo quality in IVF clinical practice mainly relies on morphological observation, and there is a lack of objective and non-invasive biomarkers to accurately predict the developmental potential and pregnancy outcome.

[0003] Oocyte cumulus cells (CCs) are important somatic cells around oocytes, and there is close paracrine and signal communication between the oocyte cumulus cells and the oocyte, and the gene expression and protein activity of the oocyte cumulus cells can indirectly reflect the quality of the oocyte and the subsequent embryo development potential. However, in the conventional IVF operation, the oocyte cumulus cells are usually discarded after oocyte retrieval, and the potential biological information thereof cannot be fully utilized. The mammalian target of rapamycin (mTOR) signaling pathway plays a core regulatory role in cell growth, metabolism and proliferation, and the phosphorylation level of the downstream effector protein S6 (such as S6K1 and S6 ribosomal protein) is closely related to cell energy metabolism and protein synthesis. Studies have shown that the mTOR-S6 pathway may be involved in the regulation of follicular development, oocyte maturation and early embryo development, but the expression pattern of the mTOR-S6 pathway in the oocyte cumulus cells and the correlation with embryo quality and pregnancy outcome have not been clearly defined, and there is no report on the application of related markers in clinical prediction products. Therefore, it is of great significance to detect the expression or phosphorylation level of key proteins in the mTOR-S6 signaling pathway in the oocyte cumulus cells, establish a correlation model with embryo quality and pregnancy outcome, and develop non-invasive and high-accuracy prediction products. SUMMARY

[0004] Therefore, the application aims to provide application of an oocyte cumulus cell mTOR-S6 protein marker in preparation of an embryo quality and pregnancy outcome prediction product.

[0005] Another object of the application is to provide application of an oocyte cumulus cell mTOR-S6 protein marker in preparation of an in vitro fertilization evaluation product.

[0006] In order to achieve the above-mentioned application objects, the application provides the following technical solutions.

[0007] The application provides application of an oocyte cumulus cell mTOR-S6 protein marker in preparation of an embryo quality and pregnancy outcome prediction product.

[0008] Preferably, the median value of the relative expression value of the cumulus cell mTOR-S6 protein marker is used as a threshold to evaluate the embryo quality and pregnancy outcome.

[0009] More preferably, the cumulus cell mTOR-S6 protein marker comprises one or more of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein.

[0010] More preferably, the threshold value of the p-mTOR protein is 0.6; the threshold value of the mTOR protein is 0.7; the threshold value of the p-S6 protein is 0.8; and the threshold value of the S6 protein is 0.8.

[0011] More preferably, when the relative expression value of three or more of the p-mTOR protein, mTOR protein, p-S6 protein and S6 protein in the treated individual is less than the threshold value, it indicates that the embryo quality and pregnancy rate are high.

[0012] More preferably, the pregnancy rate comprises clinical pregnancy rate and ongoing pregnancy rate.

[0013] The application provides an application of a cumulus cell mTOR-S6 protein marker in the preparation of an in vitro fertilization evaluation product.

[0014] Preferably, the evaluation method comprises the following steps: detecting the relative expression value of three or more of the p-mTOR protein, mTOR protein, p-S6 protein and S6 protein in the treated individual; and evaluating whether the ovarian reserve function, embryo development quality and pregnancy outcome of the treated individual are good.

[0015] More preferably, the related indicators of the ovarian reserve function comprise anti-Mullerian hormone, antral follicle count, total oocyte number or MII oocyte number.

[0016] More preferably, the related indicators of the embryo development quality comprise the number of zygotes, the number of embryos, the number of high-quality embryos, the number of blastocysts or the number of high-quality blastocysts.

[0017] Compared with the prior art, the application has the following beneficial effects:

[0018] The present application collects a small amount of cumulus cells (clinical by-products) from female patients receiving in vitro fertilization (IVF) - embryo transfer (ET) treatment during oocyte retrieval, extracts total protein through cell lysis and other steps, and then quantitatively detects the levels of key proteins (p-mTOR, mTOR, p-S6 and S6) in the mTOR signaling pathway in the cumulus cells by Western blot technology. At the same time, the IVF-related clinical indicators of the patients are systematically collected, including ovarian reserve function parameters (such as anti-Mullerian hormone, basal antral follicle count, total oocyte number, MII oocyte number), embryo quality score (including morphological grading and developmental potential assessment), and pregnancy outcome (biochemical pregnancy, clinical pregnancy, continued pregnancy) data. Through statistical methods, the correlation analysis of these clinical indicators and the relative levels of the four key proteins is carried out to explore the potential correlation between the levels of the four proteins and the outcome of IVF treatment, i.e. when the relative expression values of three or more of the p-mTOR protein, the mTOR protein, the p-S6 protein and the S6 protein in the treated individual are less than the threshold value, it indicates that the embryo quality and the pregnancy rate are high. The present application first converts the discarded resource of cumulus cells into a valuable prediction tool, provides molecular level information that cannot be obtained by traditional morphological assessment while maintaining non-invasiveness, and provides a new dimension for improving IVF clinical decision-making. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 For the relative levels of p-mTOR, mTOR, p-S6 and S6 proteins in the cumulus cells of female patients receiving in vitro fertilization, Figure A is the expression levels of p-mTOR, mTOR, p-S6, S6 proteins and β-actin in the cumulus cells; B is the relative expression values of p-mTOR, mTOR, p-S6 and S6 proteins in each cumulus cell sample; C is the spine line graph of the distribution of the relative expression values of p-mTOR, mTOR, p-S6 and S6 proteins in each cumulus cell sample.

[0020] Figure 2 For the results of the combination of p-mTOR, mTOR, p-S6 and S6 proteins effectively predicting the ovarian reserve, embryo development and pregnancy outcome of patients, Figure A is the relationship between the relative expression values of p-mTOR, mTOR, p-S6 and S6 proteins and the indicators related to ovarian reserve and embryo development; B is the clinical pregnancy rate and continued pregnancy rate of p-mTOR, mTOR, p-S6 and S6 proteins in different combinations. DETAILED DESCRIPTION

[0021] The present application provides an application of cumulus cell mTOR-S6 protein markers in the preparation of embryo quality and pregnancy outcome prediction products.

[0022] In the present application, the median value of the relative expression value of the cumulus cell mTOR-S6 protein marker is used as the threshold to evaluate the embryo quality and pregnancy outcome. In the present application, the relative expression value of the cumulus cell mTOR-S6 protein marker is calculated by quantitatively analyzing the protein marker using Image J software, and then standardizing the expression amount of the beta-actin protein of the treated individual. In the present application, the gray value of p-mTOR, mTOR, p-S6 and S6 and beta-actin protein is obtained after quantification using Image J software. The relative expression value of p-mTOR, mTOR, p-S6 and S6 is obtained by dividing the gray value of p-mTOR, mTOR, p-S6 and S6 after quantification by the gray value of the corresponding sample of beta-actin protein after quantification.

[0023] In the present application, the cumulus cell mTOR-S6 protein marker includes one or more of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein.

[0024] In the present application, the threshold value of p-mTOR protein is 0.6; the threshold value of mTOR protein is 0.7; the threshold value of p-S6 protein is 0.8; and the threshold value of S6 protein is 0.8. In the present application, it is found by Western blot detection of the expression level of key proteins that the expression of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein and p-S6 / S6, p-mTOR / mTOR in the cumulus cells of each patient is different, showing heterogeneity, but the expression trend of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein is consistent. And the relative expression value of the four proteins is mostly between 0-2, and the median value is basically between 0.6-0.8, and the frequency distribution in this interval is the most concentrated. Therefore, the median values of the four protein indicators are used as the threshold (p-mTOR, 0.6; mTOR, 0.7; p-S6, 0.8; S6, 0.8) in the present application, and the relationship between the threshold value and the embryo quality and pregnancy outcome is established.

[0025] In the present application, when the relative expression values of three or more of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein in the treated individual are all less than the threshold value, it indicates that the embryo quality and pregnancy rate are high. In the present application, as one of the implementable ways, when the relative expression value of p-mTOR protein <0.6, the relative expression value of mTOR protein <0.7, the relative expression value of p-S6 protein <0.8 and the relative expression value of S6 protein <0.8, or when the relative expression value of p-mTOR protein <0.6, the relative expression value of mTOR protein <0.7 and the relative expression value of p-S6 protein <0.8, or when the relative expression value of mTOR protein <0.7, the relative expression value of p-S6 protein <0.8 and the relative expression value of S6 protein <0.8, or when the relative expression value of p-mTOR protein <0.6, the relative expression value of p-S6 protein <0.8 and the relative expression value of S6 protein <0.8, or when the relative expression value of p-mTOR protein <0.6, the relative expression value of mTOR protein <0.7 and the relative expression value of S6 protein <0.8, it indicates that the embryo quality and pregnancy rate are high.

[0026] In the present application, the pregnancy rate includes clinical pregnancy rate and continued pregnancy rate. In the present application, the clinical pregnancy rate refers to the proportion of gestational sacs found by intrauterine ultrasound examination 30 days after embryo transfer, which is a key indicator for evaluating the effect of assisted reproductive technology (ART). The clinical pregnancy rate is calculated by the number of patients confirmed to be pregnant / the total number of treated patients x 100%, which emphasizes the confirmation of clinical pregnancy by medical means (such as B-ultrasound). The continued pregnancy rate, also known as sustained pregnancy rate, refers to the ratio of continued pregnancy confirmed by ultrasound examination at 11 weeks of pregnancy.

[0027] The present application provides an application of cumulus cell mTOR-S6 protein marker in preparation of in vitro fertilization evaluation product.

[0028] In the present application, the method for evaluation comprises the following steps: detecting the relative expression values of three or more proteins of p-mTOR protein, mTOR protein, p-S6 protein and S6 protein in the treated individual; evaluating whether the ovarian reserve function, the quality of embryo development and the pregnancy outcome of the treated individual are good. In the present application, as an implementable manner, when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7 and the relative expression value of p-S6 protein is <0.8, or when the relative expression value of mTOR protein is <0.7, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7 and the relative expression value of S6 protein is <0.8, it indicates that the ovarian reserve function, the quality of embryo development and the pregnancy outcome of the treated individual are good.

[0029] In the present application, the relevant indicators of the ovarian reserve function comprise anti-Mullerian hormone, antral follicle count, total oocyte number or MII oocyte number. In the present application, when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7 and the relative expression value of p-S6 protein is <0.8, or when the relative expression value of mTOR protein is <0.7, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of p-S6 protein is <0.8 and the relative expression value of S6 protein is <0.8, or when the relative expression value of p-mTOR protein is <0.6, the relative expression value of mTOR protein is <0.7 and the relative expression value of S6 protein is <0.8, it indicates that the relevant indicators of the ovarian reserve function are significantly increased.

[0030] In the present application, the relevant indicators of embryo development quality include the number of zygotes, the number of embryos, the number of high-quality embryos, the number of blastocysts, or the number of high-quality blastocysts. In the present application, the number of embryos is preferably the number of embryos on Day 3 after oocyte retrieval; the number of high-quality embryos is preferably the number of high-quality embryos on Day 3 after oocyte retrieval; the number of blastocysts is the number of blastocysts on Day 5-6 after oocyte retrieval; and the number of high-quality blastocysts is the number of high-quality blastocysts on Day 5-6 after oocyte retrieval. In the present application, when the relative expression value of p-mTOR protein < 0.6, the relative expression value of mTOR protein < 0.7, the relative expression value of p-S6 protein < 0.8, and the relative expression value of S6 protein < 0.8, or when the relative expression value of p-mTOR protein < 0.6, the relative expression value of mTOR protein < 0.7, and the relative expression value of p-S6 protein < 0.8, or when the relative expression value of mTOR protein < 0.7, the relative expression value of p-S6 protein < 0.8, and the relative expression value of S6 protein < 0.8, or when the relative expression value of p-mTOR protein < 0.6, the relative expression value of p-S6 protein < 0.8, and the relative expression value of S6 protein < 0.8, or when the relative expression value of p-mTOR protein < 0.6, the relative expression value of mTOR protein < 0.7, and the relative expression value of S6 protein < 0.8, it indicates that the relevant indicators of embryo development quality are significantly increased.

[0031] The technical solutions provided by the present application are described in detail below in conjunction with the examples, but they should not be understood as limiting the scope of protection of the present application.

[0032] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0033] Example 1

[0034] This example was approved by the Ethics Committee of Shanxi Children's Hospital and Maternal and Child Health Hospital (number: IRB-KYSB-2023-017), strictly conducted in accordance with the guidelines of the institution, and written informed consent was obtained from the patients.

[0035] Inclusion and exclusion criteria: A total of 50 female patients aged 24-43 years (mean age: 31.8 ± 3.7 years) were included in this example. Inclusion criteria: Patients who used their own oocytes for IVF / ICSI treatment. Exclusion criteria: Patients with endometriosis, cancer, chronic infection, autoimmune or genetic diseases will be excluded from this study.

[0036] Western blotting: After dispersed into single cells by 0.03% hyaluronidase (H6254-500MG, Sigma-Aldrich), cumulus cells were washed twice with PBS and lysed on ice for 30 min. The supernatant was collected by centrifugation at 10000 x g for 10 min at 4°C. After determination of protein concentration by BCA method, the protein samples were boiled in SDS loading buffer at 95°C for 5 min. Equal amounts of protein were separated by 4-20% precast Tris-glycine gels (DG101-02-V2, TRANS) and then transferred to PVDF membranes (Millipore). After blocking with 5% nonfat milk (TBST buffer) for 2 h at room temperature, the primary antibodies were incubated overnight at 4°C with β-actin (AC026, ABclonal) as an internal reference. The corresponding HRP-labeled secondary antibodies were incubated for 2 h at room temperature. The chemiluminescent HRP substrate (WBKLS0500, Millipore) was used for color development. The antibodies used in the experiment included p-mTOR (5536T, Cell Signaling Technology), mTOR (2983S, Cell Signaling Technology), p-S6 (4858S, Cell Signaling Technology), S6 (2217S, Cell Signaling Technology). The band gray value analysis was performed under the same parameters by Image J software, and the gray value of β-actin protein was standardized.

[0037] Method and determination of threshold value: Discarded trace cumulus cells from 50 female patients receiving IVF treatment were collected, and the expression levels of key proteins were detected by Western blot. First, under consistent experimental conditions, the expression levels of p-mTOR, mTOR, p-S6, S6 protein and β-actin in 50 cumulus cells were detected Figure 1 A). Then the five proteins were quantitatively analyzed by Image J software, and the expression of β-actin protein of each patient was standardized to calculate the relative expression value of four proteins in each cumulus cell sample Figure 1 B). The results showed that the expression of p-mTOR, mTOR, p-S6 and S6 and p-S6 / S6, p-mTOR / mTOR in the cumulus cells of each patient was different, showing heterogeneity, but overall, the expression trend of p-mTOR, mTOR, p-S6 and S6 was consistent Figure 1B) and the median values of the four proteins were mostly between 0 and 2, and the frequency distribution was most concentrated in the interval of 0.6-0.8, indicating that most individuals fell within this range Figure 1 C) Therefore, this example takes the median values of the four protein indicators as the threshold (p-mTOR, 0.6; mTOR, 0.7; p-S6, 0.8; S6, 0.8), and analyzes the clinical indicators such as ovarian reserve, embryo development and pregnancy outcome of patients after grouping according to this threshold.

[0038] Example 2

[0039] This example analyzes the clinical indicators such as ovarian reserve, embryo development and pregnancy outcome of patients after grouping according to the threshold obtained in Example 1

[0040] This example was approved by the Ethics Committee of Shanxi Children's Hospital and Maternal and Child Health Hospital (number: IRB-KYSB-2023-017), strictly conducted in accordance with the institutional guidelines, and obtained the written informed consent of the patients.

[0041] Inclusion and exclusion criteria: A total of 33 female patients aged 24-43 years (mean age: 31.8 ± 3.7 years) were included in this example. Inclusion criteria: Patients who used their own oocytes for IVF / ICSI treatment. Exclusion criteria: Patients with endometriosis, cancer, chronic infection, autoimmune or genetic diseases will be excluded from this study.

[0042] According to Figure 2 The results showed that, in terms of ovarian reserve and embryo development-related indicators, when p-mTOR <0.6&mTOR <0.7&p-S6 <0.8&S6 <0.8, the patient's ovarian reserve-related indicators, anti-Mullerian hormone, antral follicle count, total oocyte number and MII oocyte number significantly increased, and at the same time, the embryo development-related indicators, the number of zygotes, Day3 embryo number, Day3 high-quality embryo number, Day5-6 blastocyst number and Day5-6 high-quality blastocyst number also significantly increased Figure 2 A) and compared with single protein, the combination of multiple proteins can significantly increase the pregnancy rate (clinical pregnancy and ongoing pregnancy) Figure 2B). For example, when detecting p-mTOR alone, the absolute difference of clinical pregnancy rate and ongoing pregnancy rate is 25.3% and 27.1% respectively; but when detecting the four proteins of "p-mTOR&mTOR&p-S6&S6" jointly, the absolute difference of clinical pregnancy rate and ongoing pregnancy rate is 56.7% (p-mTOR&mTOR&p-S6&S6< corresponding threshold value corresponds to the clinical pregnancy rate and the ongoing pregnancy rate of 90.0%, and p-mTOR&mTOR&p-S6&S6≥ corresponding threshold value corresponds to the clinical pregnancy rate and the ongoing pregnancy rate of 33.3%, the absolute difference between the two groups is 56.7%).

[0043] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Application of mTOR-S6 protein marker in cumulus cells in the preparation of products for predicting embryo quality and pregnancy outcomes.

2. The application according to claim 1, characterized in that, The median relative expression value of the mTOR-S6 protein marker in cumulus cells was used as a threshold to assess embryo quality and pregnancy outcome.

3. The application according to claim 1 or 2, characterized in that, The mTOR-S6 protein markers for cumulus cells include one or more of p-mTOR protein, mTOR protein, p-S6 protein, and S6 protein.

4. The application according to claim 3, characterized in that, The threshold values ​​for the p-mTOR protein are 0.6; the threshold values ​​for the mTOR protein are 0.7; the threshold values ​​for the p-S6 protein are 0.8; and the threshold values ​​for the S6 protein are 0.

8.

5. The application according to claim 4, characterized in that, When the relative expression values ​​of three or more of the following proteins—p-mTOR, mTOR, p-S6, and S6—are all below a threshold in the treated individual, it indicates high embryo quality and pregnancy rate.

6. The application according to claim 5, characterized in that, The pregnancy rate includes the clinical pregnancy rate and the continued pregnancy rate.

7. Application of mTOR-S6 protein marker in cumulus cells in the preparation of in vitro fertilization assessment products.

8. The application according to claim 7, characterized in that, The assessment method includes the following steps: detecting the relative expression values ​​of three or more proteins, namely p-mTOR, mTOR, p-S6, and S6, in the treated individual; and assessing the ovarian reserve function, embryonic development quality, and pregnancy outcome of the treated individual.

9. The application according to claim 8, characterized in that, The relevant indicators of ovarian reserve function include anti-Müllerian hormone, antral follicle count, total number of oocytes, or MII oocyte count.

10. The application according to claim 8, characterized in that, The relevant indicators of embryonic development quality include the number of fertilized eggs, the number of embryos, the number of high-quality embryos, and the number of blastocysts or high-quality blastocysts.

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