Application of cumulus cell mtor-s6 protein markers in non-invasive prediction of embryo quality and pregnancy outcome

Abstract

The application provides application of cumulus cell mTOR-S6 protein markers in non-invasive prediction of embryo quality and pregnancy outcome, and relates to the technical field of bioengineering.The application quantitatively detects the levels of mTOR signaling pathway key proteins p-mTOR, mTOR, p-S6 and S6 in cumulus cells by Western blot technology.Meanwhile, IVF related clinical indexes of patients are systematically collected, and correlation analysis is conducted on the relative levels of the four key proteins by statistical methods.It is found that, by taking the median value of the relative expression values of the four proteins as a threshold, evaluation of the IVF related indexes of the patients can determine whether the ovarian reserve function, embryo development quality and pregnancy outcome are good.The application not only provides an innovative auxiliary evaluation tool for the field of reproductive medicine, but also provides a scientific basis for the formulation of individualized IVF treatment schemes.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically the application of a cumulus cell mTOR-S6 protein marker in the preparation of products for predicting embryo quality and pregnancy outcomes. Background Technology

[0002] As women age, their fertility gradually declines, which also affects the success rate of in-vitro fertilization (IVF). Currently, the assessment of oocyte and embryo quality in IVF clinical practice mainly relies on morphological observation, lacking objective, non-invasive biomarkers to accurately predict developmental potential and pregnancy outcomes.

[0003] Cumulus cells (CCs), as important somatic cells surrounding the oocyte, have close paracrine and signaling communication with the oocyte. Their gene expression and protein activity can indirectly reflect the quality of the oocyte and the subsequent embryonic developmental potential. However, in routine IVF procedures, cumulus cells are usually discarded after oocyte retrieval, failing to fully utilize their potential biological information. The mammalian target of rapamycin (mTOR) signaling pathway plays a core regulatory role in cell growth, metabolism, and proliferation. The phosphorylation level of its downstream effector protein S6 (such as S6K1 and S6 ribosomal protein) is closely related to cellular energy metabolism and protein synthesis. Studies have shown that the mTOR-S6 pathway may be involved in the regulation of follicle development, oocyte maturation, and early embryonic development, but the association between its expression pattern in cumulus cells and embryo quality and pregnancy outcome remains unclear, and there are no reports of related biomarkers being used in clinical predictive products. Therefore, it is of great significance to establish a correlation model between the expression or phosphorylation level of key proteins in the mTOR-S6 signaling pathway in cumulus cells and embryo quality and pregnancy outcome, and to develop non-invasive, highly accurate predictive products. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide an application of the mTOR-S6 protein marker of cumulus cells in the preparation of products for predicting embryo quality and pregnancy outcomes.

[0005] Another object of the present invention is to provide an application of the mTOR-S6 protein marker of cumulus cells in the preparation of in vitro fertilization assessment products.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides an application of the mTOR-S6 protein marker in cumulus cells in the preparation of products for predicting embryo quality and pregnancy outcomes.

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

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

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

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

[0012] More preferably, the pregnancy rate includes the clinical pregnancy rate and the continued pregnancy rate.

[0013] This invention provides the application of the mTOR-S6 protein marker in cumulus cells in the preparation of in vitro fertilization assessment products.

[0014] Preferably, the evaluation 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 whether the ovarian reserve function, embryonic development quality, and pregnancy outcome of the treated individual are satisfactory.

[0015] More preferably, the relevant indicators of ovarian reserve function include anti-Müllerian hormone, antral follicle count, total number of oocytes, or MII oocyte count.

[0016] More preferably, the relevant indicators of embryonic development quality include the number of fertilized eggs, 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 present invention has the following advantages:

[0018] This invention collects trace amounts of cumulus cells (clinical byproducts) from female patients undergoing in vitro fertilization (IVF)-embryo transfer (ET) treatment during oocyte retrieval. Total protein is extracted through cell lysis and other steps, and then Western blot is used to quantitatively detect the levels of key proteins in the mTOR signaling pathway (p-mTOR, mTOR, p-S6, and S6) in the cumulus cells. Simultaneously, IVF-related clinical indicators are systematically collected from the patients, including ovarian reserve parameters (such as anti-Müllerian hormone, basal antral follicle count, total oocyte count, and MII oocyte count), embryo quality scores (including morphological grading and developmental potential assessment), and pregnancy outcomes (biochemical pregnancy, clinical pregnancy, and continued pregnancy). Statistical analysis was used to correlate these clinical indicators with the relative levels of the four key proteins to explore the potential association between these four protein levels and IVF treatment outcomes. Specifically, when the relative expression values ​​of three or more of the four 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. This invention is the first to transform waste cumulus cells into a valuable predictive tool, providing molecular-level information that is unavailable through traditional morphological assessments while remaining non-invasive, thus offering a new dimension for improving IVF clinical decision-making. Attached Figure Description

[0019] Figure 1 To show the relative levels of p-mTOR, mTOR, p-S6, and S6 proteins in cumulus cells of women undergoing in vitro fertilization, Figure A shows the expression levels of p-mTOR, mTOR, p-S6, S6 proteins, and β-actin in cumulus cells; B shows the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6 proteins in each cumulus cell sample; and C shows a ridge plot illustrating the distribution of the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6 proteins in each cumulus cell sample.

[0020] Figure 2 The combination of four proteins, p-mTOR, mTOR, p-S6, and S6, was used to effectively predict ovarian reserve, embryonic development, and pregnancy outcome in patients. Figure A shows the relationship between the relative expression values ​​of the four proteins p-mTOR, mTOR, p-S6, and S6 and indicators related to ovarian reserve and embryonic development; Figure B shows the clinical pregnancy rate and continued pregnancy rate under different combinations of the four proteins p-mTOR, mTOR, p-S6, and S6. Detailed Implementation

[0021] This invention provides an application of the mTOR-S6 protein marker in cumulus cells in the preparation of products for predicting embryo quality and pregnancy outcomes.

[0022] In this invention, the median relative expression value of the mTOR-S6 protein marker in cumulus cells is used as a threshold to assess embryo quality and pregnancy outcome. The relative expression value of the mTOR-S6 protein marker in cumulus cells is calculated by quantitatively analyzing the protein marker using ImageJ software, then standardizing the β-actin protein expression level of the treated individual to determine the relative expression value of the protein marker in each cumulus cell sample. In this invention, after quantifying p-mTOR, mTOR, p-S6, S6, and β-actin proteins using ImageJ software, the corresponding grayscale values ​​of the proteins are obtained. Dividing the grayscale values ​​of p-mTOR, mTOR, p-S6, and S6 by the grayscale value of β-actin protein in the corresponding sample yields the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6.

[0023] In this invention, the mTOR-S6 protein marker of cumulus cells includes one or more of p-mTOR protein, mTOR protein, p-S6 protein, and S6 protein.

[0024] In this invention, the threshold values ​​for p-mTOR protein are 0.6; for mTOR protein, 0.7; for p-S6 protein, 0.8; and for S6 protein, 0.8. In this invention, Western blot analysis of key protein expression levels revealed that the expression levels of p-mTOR, mTOR, p-S6, and S6 proteins, as well as p-S6 / S6 and p-mTOR / mTOR, varied in each patient's cumulus cells, exhibiting heterogeneity. However, the expression trends of p-mTOR, mTOR, p-S6, and S6 proteins were consistent. Furthermore, the relative expression values ​​of these four proteins were mostly between 0 and 2, with a median value generally between 0.6 and 0.8, and the frequency distribution was most concentrated within this range. Therefore, this invention uses the median value of these four protein indicators as the threshold (p-mTOR, 0.6; mTOR, 0.7; p-S6, 0.8; S6, 0.8) to establish the relationship between these thresholds and embryo quality and pregnancy outcome.

[0025] In this invention, when the relative expression values ​​of three or more of the p-mTOR, mTOR, p-S6 and S6 proteins in the treated individual are all less than a threshold, it indicates high embryo quality and pregnancy rate. In this invention, as one possible implementation, when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and p-S6 protein <0.8, or when the relative expression values ​​of mTOR protein are <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and S6 protein <0.8, it indicates high embryo quality and pregnancy rate.

[0026] In this invention, the pregnancy rate includes the clinical pregnancy rate and the continued pregnancy rate. The clinical pregnancy rate refers to the proportion of patients with a gestational sac detected by intrauterine ultrasound examination 30 days after embryo transfer, and is a key indicator for evaluating the effectiveness of assisted reproductive technology (ART). The clinical pregnancy rate is calculated as the number of patients with confirmed pregnancy / the total number of treated patients × 100%, emphasizing confirmation of clinical pregnancy through medical means (such as ultrasound). The continued pregnancy rate, also known as the sustained pregnancy rate, refers to the percentage of pregnancies confirmed by ultrasound examination at 11 weeks of gestation.

[0027] This invention provides the application of the mTOR-S6 protein marker in cumulus cells in the preparation of in vitro fertilization assessment products.

[0028] In this invention, the evaluation method includes the following steps: detecting the relative expression values ​​of three or more proteins—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. In this invention, as one possible implementation, 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 p-S6 protein is <0.8, the relative expression value of mTOR protein is <0.7, and the relative expression value of p-S6 protein is <0.8, the relative expression value of p-S6 protein is <0.8, and the relative expression value of mTOR protein is <0.7, ... When the relative expression values ​​of p-6 protein and p-mTOR protein are <0.8 and <0.8 respectively, or when the relative expression values ​​of p-mTOR protein and p-S6 protein are <0.6 and <0.8 respectively, or when the relative expression values ​​of p-mTOR protein and mTOR protein are <0.7 and <0.8 respectively, it indicates that the ovarian reserve function, embryonic development quality and pregnancy outcome of the treated individual are good.

[0029] In this invention, the relevant indicators of ovarian reserve function include anti-Müllerian hormone, antral follicle count, total number of oocytes, or MII oocyte count. In this invention, when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and p-S6 protein <0.8, or when the relative expression values ​​of mTOR protein are <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and S6 protein <0.8, it indicates that the relevant indicators of ovarian reserve function are significantly increased.

[0030] In this invention, 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. In this invention, the number of embryos is preferably the number of embryos 3 days after egg retrieval (Day 3); the number of high-quality embryos is preferably the number of high-quality embryos 3 days after egg retrieval (Day 3); the number of blastocysts is the number of blastocysts 5-6 days after egg retrieval (Day 5-6); and the number of high-quality blastocysts is the number of high-quality blastocysts 5-6 days after egg retrieval (Day 5-6). In this invention, when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and p-S6 protein <0.8, or when the relative expression values ​​of mTOR protein are <0.7, p-S6 protein <0.8, and S6 protein <0.8, or when the relative expression values ​​of p-mTOR protein are <0.6, mTOR protein <0.7, and S6 protein <0.8, it indicates that the relevant indicators of embryonic development quality have significantly increased.

[0031] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0033] Example 1

[0034] This embodiment was approved by the Ethics Committee of Shanxi Provincial Children's Hospital and Maternal and Child Health Hospital (No.: IRB-KYSB-2023-017), strictly followed the institutional guidelines, and obtained the patient's written informed consent.

[0035] Inclusion and Exclusion Criteria: This study included 50 female patients aged 24–43 years (mean age: 31.8 ± 3.7 years). Inclusion Criteria: Patients who underwent IVF / ICSI treatment using their own oocytes. Exclusion Criteria: Patients with endometriosis, cancer, chronic infections, autoimmune diseases, or genetic disorders were excluded from this study.

[0036] Western blot assay: Cumulus cells were digested and dispersed into single cells with 0.03% hyaluronidase (H6254-500MG, Sigma-Aldrich), washed twice with PBS, and lysed on ice for 30 minutes. The cells were centrifuged at 10000×g for 10 minutes at 4°C, and the supernatant was collected and transferred to new centrifuge tubes. After determining protein concentration using the BCA method, protein samples were added to SDS loading buffer and boiled at 95°C for 5 minutes. Equal volumes of protein from each sample were separated by electrophoresis on a 4-20% pre-prepared Tris-glycine gel (DG101-02-V2, TRANS), and then transferred to a PVDF membrane (Millipore). After blocking with 5% skim milk (prepared with TBST buffer) at room temperature for 2 hours, the membrane was incubated with primary antibody overnight at 4°C, using β-actin (AC026, ABclonal) as an internal control. The HRP-labeled secondary antibodies were incubated at room temperature for 2 hours, followed by chemiluminescent HRP substrate development (WBKLS0500, Millipore). Antibodies used included: p-mTOR (5536T, Cell Signaling Technology), mTOR (2983S, Cell Signaling Technology), p-S6 (4858S, Cell Signaling Technology), and S6 (2217S, Cell Signaling Technology). For each batch of experiments, band grayscale values ​​were analyzed using ImageJ software under the same parameters, and the values ​​were standardized to the grayscale values ​​of β-actin protein.

[0037] Threshold detection method and determination: A small amount of discarded cumulus cells were collected from 50 female patients undergoing IVF treatment, 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 proteins, and β-actin in the 50 cumulus cells were detected. Figure 1 A). ImageJ software was then used to quantitatively analyze these five proteins, and the expression levels were standardized based on each patient's individual β-actin protein expression level to calculate the relative expression values ​​of the four proteins in each cumulus cell sample. Figure 1 B). The results showed that the expression of p-mTOR, mTOR, p-S6 and S6, as well as p-S6 / S6 and p-mTOR / mTOR, varied in each patient's cumulus cells, exhibiting heterogeneity. However, overall, the expression trends of p-mTOR, mTOR, p-S6 and S6 were consistent. Figure 1B). Furthermore, the relative expression values ​​of these four proteins were mostly between 0 and 2, with a median value generally between 0.6 and 0.8. The frequency distribution was most concentrated within this range, indicating that the detection values ​​for most individuals fell within this range. Figure 1 C). Therefore, in this embodiment, the median value of these four protein indicators is used as the threshold (p-mTOR, 0.6; mTOR, 0.7; p-S6, 0.8; S6, 0.8). After grouping according to this threshold, the clinical indicators such as ovarian reserve, embryonic development and pregnancy outcome of patients are analyzed.

[0038] Example 2

[0039] In this embodiment, patients' clinical indicators such as ovarian reserve, embryonic development, and pregnancy outcomes were analyzed after grouping them according to the threshold obtained in Example 1.

[0040] This embodiment was approved by the Ethics Committee of Shanxi Provincial Children's Hospital and Maternal and Child Health Hospital (No.: IRB-KYSB-2023-017), strictly followed the institutional guidelines, and obtained the patient's written informed consent.

[0041] Inclusion and Exclusion Criteria: This study included 33 female patients aged 24–43 years (mean age: 31.8 ± 3.7 years). Inclusion Criteria: Patients who underwent IVF / ICSI treatment using their own oocytes. Exclusion Criteria: Patients with endometriosis, cancer, chronic infections, autoimmune diseases, or genetic disorders were excluded from this study.

[0042] according to Figure 2 The results showed that, regarding ovarian reserve and embryonic development-related indicators, when p-mTOR < 0.6, mTOR < 0.7, p-S6 < 0.8, and S6 < 0.8, patients exhibited a significant increase in anti-Müllerian hormone, antral follicle count, total oocyte count, and MII oocyte count. This was accompanied by a significant increase in embryonic development-related indicators such as the number of fertilized oocytes, Day 3 embryos, Day 3 high-quality embryos, Day 5–6 blastocysts, and Day 5–6 high-quality blastocysts. Figure 2 A). Furthermore, compared to single proteins, combinations of multiple proteins can significantly increase pregnancy rates (both clinical and continued pregnancy). Figure 2B). For example, when p-mTOR was detected alone, the absolute differences in clinical pregnancy rate and continued pregnancy rate were 25.3% and 27.1%, respectively; however, when the four proteins "p-mTOR, p-S6, and p-S6" were detected together, the absolute differences in clinical pregnancy rate and continued pregnancy rate reached 56.7% (the clinical pregnancy rate and continued pregnancy rate corresponding to p-mTOR, p-S6, and p-S6 < the corresponding threshold were both 90.0%, while the clinical pregnancy rate and continued pregnancy rate corresponding to p-mTOR, p-S6, and p-S6 ≥ the corresponding threshold were both 33.3%, and the absolute difference between the two groups was 56.7%).

[0043] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a reagent for detecting mTOR-S6 protein markers in cumulus cells in the preparation of products for predicting embryo quality and pregnancy outcomes, characterized in that... The mTOR-S6 protein markers for cumulus cells include p-mTOR protein, mTOR protein, p-S6 protein, and S6 protein.

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. The relative expression value of the mTOR-S6 protein marker in cumulus cells was calculated by quantitatively analyzing the protein marker using ImageJ software, and then standardizing the β-actin protein expression level of the treated individuals to calculate the relative expression value of the protein marker in each cumulus cell sample.

3. The application according to claim 2, 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.

4. The application according to claim 3, characterized in that, When the relative expression values ​​of p-mTOR protein, mTOR protein, p-S6 protein, and S6 protein in the treated individual are all below the threshold, it indicates high embryo quality and pregnancy rate.

5. The application according to claim 4, characterized in that, The pregnancy rate includes the clinical pregnancy rate and the continued pregnancy rate.

6. The application of a reagent for detecting mTOR-S6 protein markers in cumulus cells in the preparation of in vitro fertilization assessment products, characterized in that... The mTOR-S6 protein markers for cumulus cells include p-mTOR protein, mTOR protein, p-S6 protein, and S6 protein.

7. The application according to claim 6, characterized in that, The assessment method includes the following steps: detecting the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6 proteins in the treated individuals; assessing the ovarian reserve function, embryonic development quality, and pregnancy outcome of the treated individuals; the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6 proteins are obtained by quantifying p-mTOR, mTOR, p-S6, S6, and β-actin proteins using ImageJ software, and dividing the gray values ​​of p-mTOR, mTOR, p-S6, and S6 by the gray value of β-actin protein quantification in the corresponding sample to obtain the relative expression values ​​of p-mTOR, mTOR, p-S6, and S6.

8. The application according to claim 7, 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.

9. The application according to claim 7, 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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