CN117512096A - Markers for predicting repeated implantation failure and their applications - Google Patents

Abstract

The invention belongs to the technical fields of biological medicine and molecular biology, and particularly relates to a marker for predicting repeated implantation failure occurrence and application thereof. According to the invention, researches show that abnormal lipid peroxidation in uterine epithelium can cause implantation defect, and the lipid peroxidation level of uterus is enhanced in patients with repeated implantation failure or repeated spontaneous abortion, wherein the content of active aldehyde substances MDA and non-heme iron in serum can be used as a novel noninvasive clinical index for predicting RIF patients; further, eliminating uterine lipid ROS to improve endometrial receptivity can be used as a potential novel treatment strategy to improve embryo implantation and clinical pregnancy rate, so that the method has good practical application value.

Description

Markers for predicting repeated implantation failure and their applications

Technical field

The invention belongs to the technical fields of biomedicine and molecular biology, and specifically relates to markers used to predict the occurrence of repeated implantation failure and their applications.

Background technique

The information disclosed in the Background of the Invention is merely intended to increase understanding of the general background of the invention and is not necessarily to be regarded as an admission or in any way implying that the information constitutes prior art that is already known to a person of ordinary skill in the art.

The interaction between a receptive uterus and a blastocyst capable of implantation is a prerequisite for successful implantation. As the first point of contact between the embryo and the mother, the uterine epithelium (LE) undergoes drastic molecular, physiological and obvious morphological changes, transmitting embryonic signals to uterine stromal cells (ST) and initiating embryo implantation. In mice, the first day of pregnancy is when the vaginal plug is seen. The uterus is in the pre-receptive stage on the 1st to 3rd day of pregnancy, and is in the receptive stage on the 4th day. The embryo implantation process occurs in the evening of the 4th day. The embryo implantation ends in the early morning of the 5th day, and the uterine window period is closed. In humans, 1-7 days after ovulation (early luteal phase) is considered the pre-receptive phase, 7-10 days after ovulation (mid-luteal phase) is the receptive phase, and 10 days later (late luteal phase) the receptive phase is closed. The endometrial cell type that the embryo first contacts during the implantation process is epithelial cells. Therefore, the normal physiological state of epithelial cells is crucial for the adhesion and invasion of the embryo, and the homeostasis of the epithelial cell receptive period is regulated by multiple factors. .

In mice, preovulatory estrogen (E2) has a dominant effect on the luminal epithelium. E2 secreted on the first day of pregnancy acts on estrogen receptor α (ERα) in stromal cells to promote epithelial cell proliferation, and subsequently on the second day of pregnancy. Epithelial cells die due to decreased levels of estrogen secretion. On day 3, progesterone (P4) levels increase in the newly formed corpus luteum, delaying uterine epithelial cell death and triggering stromal cell proliferation. During the process of entering the receptivity of the uterus, the luminal epithelial cells stop proliferation and start to differentiate under the action of E2 and P4. The microvilli on the cell surface gradually disappear and the polarity changes to promote embryo adhesion and implantation. A large number of human studies have also shown that the proliferation and death of epithelial cells during the menstrual cycle require the coordination of ovarian hormones, uterine transcription factors, cytokines and other pregnancy-related molecular signaling pathways. Uterine epithelial abnormalities, microenvironmental changes, or endometrial receptivity defects can lead to embryo implantation failure and adverse pregnancy outcomes. Therefore, in view of the complex regulation mechanism of epithelial cell homeostasis during the receptive period of pregnancy, further exploration of the key factors affecting the establishment of epithelial receptivity will be beneficial to understanding the maternal-fetal dialogue mechanism during embryo implantation, and provide more information for the clinical treatment of related pregnancy diseases. many theoretical foundations.

The dynamic balance of intracellular oxidants and reducers is called cellular redox homeostasis and is necessary for cells to maintain normal physiological processes. Redox imbalance can affect many physiological processes such as gene transcription, cell signal transduction, cell proliferation, differentiation, death, and organ damage. Among them, increased lipid peroxide is a key factor mediating cell death and disease occurrence. Reactive oxygen species oxidize with the lipid components of the membrane in a non-specific manner to produce lipid hydroperoxides. The process of oxidizing lipid membranes to produce lipid peroxides is called lipid peroxidation. Polyunsaturated fatty acids (PUFA ) is a good substrate for lipid peroxidation. Lipid hydroperoxides participate in the Fenton reaction mediated by iron ions or other metal ions, producing secondary metabolites such as malondialdehyde (MDA) and 4-hydroxynonenoic acid (4-HNE). MDA can lead to cross-linking of proteins and DNA, thereby changing molecular activity and cellular physiological status; 4-HNE modifies proteins, changing protein function and localization; the covalent modifications of these lipid peroxidation secondary messengers change proteins and The structure and function of nucleic acids cause toxicity to cells, so MDA and 4-HNE are the most suitable tools for detecting and quantifying lipid peroxidation in biological samples. At present, lipid peroxidation is also used as a new target for clinical treatment, and some related drugs have been used clinically: for example, inhibition of glutamine metabolism during lipid peroxidation can significantly alleviate cardiac damage; mitochondrial lipids Neurotoxicity caused by lipid peroxidation is one of the causes of Parkinson's disease. The development of antioxidants that target mitochondria and block the occurrence of lipid peroxidation has potential application value in the treatment of neurodegenerative diseases; lipid peroxidation is also To regulate cancer occurrence, approved drugs such as sorafenib have been put into clinical treatment. Currently, lipid peroxidation is also considered the most important marker of ferroptosis. Ferroptosis, as a new type of iron ion-dependent programmed cell death, is widely involved in regulating organ development and disease occurrence. However, so far, research on lipid peroxidation in the endometrium has mainly focused on endometrial diseases such as endometrial cancer and endometriosis, while less research has been done on pregnancy. Questions such as how the dynamic balance of lipid peroxidation is regulated during pregnancy, whether the production of lipid peroxide affects the pregnancy environment maintained by the endometrium, and whether lipid peroxidation triggers the occurrence of pregnancy-related diseases, all need to be answered urgently.

Glutathione peroxidase 4 (GPX4) is an evolutionarily highly conserved enzyme that mainly uses glutathione (GSH) as a substrate to reduce lipid hydroperoxides (LOOH) into nontoxic lipids. Alcohol (L-OH) inhibits the accumulation of lipid reactive oxygen species on biological membranes, thereby maintaining the healthy physiological state of cells. GSH depletion or GPX4 inactivation in cells leads to excessive lipid peroxide and ROS production and impairs redox homeostasis, triggering cell death. Direct or indirect inhibition of GPX4 will trigger ferroptosis and is considered to be one of the core molecules of ferroptosis. Studies have shown that Gpx4 systemic knockout mice undergo intrauterine absorption and become embryonic lethal around embryonic day 7.5. This protein also plays a wide range of roles in maintaining the physiological functions of various organs, such as the kidneys, brain, liver and immune system. Conditional deletion of Gpx4 in mice can lead to acute nephritis, colitis and other diseases. Specific knockout of Gpx4 in the mouse brain causes ferroptosis of neuronal cells and is accompanied by astrocytic gliosis, which are consistent with the manifestations of Alzheimer's disease (AD); conditional knockout of Gpx4 in T cells also It causes ferroptosis of T cells, which is manifested by reduced immune response during infection. The adverse effects of oxidative imbalance caused by GPX4 dysfunction can be blocked by lipid ROS scavengers or by intercepting the biosynthesis of PUFA-containing phospholipids (PUFA-PLs). By analyzing human endometrium single cell data, we found that Gpx4 is highly expressed in uterine epithelial cells and stromal cells, but whether it is involved in the establishment and maintenance of pregnancy regulated by the endometrium is unclear, and whether the lipid peroxidation regulated by GPX4 affects The context of the maternal-fetal dialogue that establishes the endometrium during pregnancy is also unclear.

Contents of the invention

In view of the deficiencies in the prior art, the purpose of the present invention is to provide markers for predicting the occurrence of repeated implantation failure and their applications. The present invention has discovered through research that abnormal lipid peroxidation in uterine epithelium can lead to implantation defects, and the level of lipid peroxidation in the uterus is enhanced in patients with repeated implantation failure or recurrent spontaneous abortion. At the same time, clinically lipid peroxidation is The oxidation product reactive aldehyde substance MDA and non-heme iron content often show a significant upward trend in the serum of RIF patients; further, eliminating uterine lipid ROS to improve endometrial receptivity can be used as a potential new treatment strategy. Improve embryo implantation and clinical pregnancy rates. Based on the above research results, the present invention is completed.

Specifically, the technical solutions of the present invention are as follows:

A first aspect of the present invention provides the use of reagents for detecting expression levels of lipid peroxidation and related biological components in the preparation of products for predicting the occurrence of repeated implantation failure;

Wherein, the lipid peroxidation-related biological elements include lipid peroxidation-related proteins or lipid peroxidation-related products, such as glutathione peroxidase 4 (GPX4), active aldehyde substances MDA, 4HNE and non-heme iron.

The product can be used to predict the occurrence of repeated implantation failure through lipid peroxidation and the expression levels of related biological components. Specifically, the present invention found through experiments that in RIF patients with repeated implantation failure, endometrial epithelial cells Gpx4 expression is reduced, and lipid peroxidation is abnormally increased. At the same time, high serum lipid peroxidation levels in early human pregnancy are closely related to RIF patients with implantation failure. In particular, the active aldehyde substance MDA and non-heme iron content in serum, as new non-invasive clinical indicators for predicting RIF patients, can mainly solve the problems of current detection technology, which is cumbersome, invasive, unstable and traumatic. Therefore, lipid peroxidation and related biological components can be used as markers for the occurrence of repeated implantation failure. Wherein, the biological elements related to lipid peroxidation can be human or non-human mammals (such as mice, rats, guinea pigs, rabbits, dogs, monkeys, orangutans, etc.).

The reagents for detecting lipid peroxidation and the expression levels of related biological components include any currently known reagents commonly used in methods for detecting the above substances, such as the thiobarbituric acid method used to detect peroxidized lipids. , Reagents used in high performance liquid chromatography, enzyme-linked immunosorbent assay, fluorescence method, and electron spin resonance (ESR) method;

The reagents for detecting related biological components may also include reagents for detecting the transcription of the GPX4 encoding gene based on real-time fluorescence quantitative PCR, in situ hybridization, gene chips and gene sequencing, and/or detecting glutathione peroxidase based on immunoassay methods. 4 Expression situation reagents.

The products include, but are not limited to, primers, probes, (gene or protein) chips, nucleic acid membrane strips, detection kits, detection devices or equipment for detecting the expression levels of lipid peroxidation and related biological components in samples to be tested.

The samples to be tested are human or non-human samples, including but not limited to subject blood (such as serum) samples and endometrial samples (such as endometrial epithelial cells).

A second aspect of the present invention provides a system for predicting the occurrence of repeated implantation failure, which system at least includes:

an acquisition unit configured to: acquire the expression level of the subject's marker;

An evaluation unit configured to evaluate the occurrence of repeated implantation failure of the subject based on the expression level of the marker obtained by the acquisition unit.

Wherein, the biomarkers include lipid peroxidation and related biological components;

The lipid peroxidation-related biological elements include lipid peroxidation-related proteins or lipid peroxidation-related products, such as glutathione peroxidase 4 (GPX4) and active aldehyde substances MDA, 4HNE and non-heme iron.

The third aspect of the present invention provides the application of lipid peroxidation and its related biological components as targets in the prevention and treatment of diseases related to repeated implantation failure and/or the screening of drugs for diseases related to repeated implantation failure.

The method for screening drugs for diseases related to repeated implantation failure includes:

1) Use candidate substances to treat systems that express and/or contain lipid peroxidation and related biological components; set up parallel controls that do not use candidate substances to treat;

2) After completing step 1), detect the expression levels of lipid peroxidation and related biological components in the system; compared with the parallel control, if the lipid peroxidation level in the system treated with the candidate substance is significantly reduced and/or The expression level of GPX4 is significantly increased, and the candidate substance can be used as a candidate drug for preventing and treating repeated implantation failure.

The system may be a cell system, a subcellular system, a solution system, a tissue system, an organ system or an animal system.

The fourth aspect of the present invention provides the use of substances that remove lipid peroxides or promote the expression levels of GPX4 and its encoding genes in any one or more of the following:

(a) Improve endometrial receptivity or prepare products that improve endometrial receptivity;

(b) Prevent implantation failure and increase clinical pregnancy rate or prepare products that prevent implantation failure and increase clinical pregnancy rate;

(c) Products for the treatment of infertility.

Furthermore, the present invention also provides the use of substances that knock out long-chain fatty acyl-CoA synthetase 4 (ACSL4) in preparing products to reverse infertility mediated by GPX4 deletion.

The above-mentioned products can be drugs or experimental reagents, and the experimental reagents can be used for basic research.

According to the present invention, when the product is a drug, the drug further includes at least one pharmaceutically inactive ingredient.

The pharmaceutical inactive ingredients may be carriers, excipients, diluents, etc. commonly used in pharmaceuticals. Moreover, it can be prepared into dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., for oral use, external preparations, suppositories, and sterile injection solutions according to usual methods. .

The non-pharmaceutical active ingredients such as carriers, excipients and diluents that may be included are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards.

In another specific embodiment of the present invention, the carrier, excipient and diluent include but are not limited to lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, Starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc Powder, magnesium stearate and mineral oil, etc.

In yet another specific embodiment of the present invention, the medicament of the present invention can be administered into the body in a known manner. Delivery to the tissue of interest may occur, for example, via intravenous systemic delivery or local injection. Administration may optionally be via intravenous, transdermal, intranasal, mucosal or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered in the present invention may vary to a large extent depending on a variety of factors, such as the target cell, the type of organism or its tissue, the general condition of the subject to be treated, the administration Drug routes, administration methods, etc.

Beneficial technical effects of one or more of the above technical solutions:

The above technical solution reveals for the first time the harmful effects of redox homeostasis imbalance on embryo implantation, clarifies the conservative role of this process in early pregnancy mice and human endometrium, and suggests the role of lipid peroxides in clinical diagnosis and treatment. Clearance is a potential treatment to prevent implantation failure and improve clinical pregnancy rates, and therefore has good potential practical application.

Description of drawings

The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

Figure 1 shows an example of the present invention in which endometrial deletion of Gpx4 will lead to abnormal endometrial receptivity, affect embryo implantation, and then lead to female infertility. a) Immunohistochemistry results show the expression pattern of GPX4 in the uterus on days 1-5 and 8 of mouse pregnancy. Arrow points to the position of the embryo. Scale bar, 200 μm (days 1-5) and 500 μm (day 8). le, luminal epithelium; ge, glandular epithelium; st, stromal cells; myo, myometrium. b) Dot-bar graph showing pregnancy outcomes in Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ mice. Gpx4 ^f/f Pgr ^Cre/+ females are completely sterile, n represents the statistical number of individual mice. c) Picture shows the embryo implantation site of Gpx4 ^f/f and Gpx4 ^{f/ f} Pgr ^Cre/+ female mice on pregnancy day 5. The weak blue band indicated by the arrow indicates a defect in embryo implantation. d) Statistical graph showing the number of embryo implantations on pregnancy day 5 of Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ female mice (n=12-13 mice per group). e) HE staining was performed to observe the histological morphology of the embryo implantation site in the uterus of Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ female mice on the 5th day of pregnancy. The arrow indicates the location of the implantation chamber crypt. Scale bar, 200 μm. f) Immunofluorescence staining of COX2 and CK8 at the implantation site in the uterus of Gpx4 ^f/f and Gpx4 ^{f/ f} Pgr ^Cre/+ female mice on pregnancy day 5. Arrows indicate embryo implantation sites. Scale bar, 100 μm. gh) In situ hybridization experiment shows the expression of Muc1(g) and Ltf(h) genes in the uterus of Gpx4 ^f/f and Gpx4 ^{f/ f} Pgr ^Cre/+ female mice on day 4 of pregnancy. Scale bar, 200 μm. ij) Real-time fluorescence quantitative PCR technology detects the expression of genes related to uterine receptivity including E2 response genes (i) and P4 response genes (j). The results show that Gpx4 ^f/f Pgr ^Cre/+ mice have uterine receptivity on the 4th day of pregnancy. Sexual abnormalities (n=5 mice per group). k) Immunofluorescence staining of Ki67 and CTNNB1 on the uterus of Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ female mice on pregnancy day 4. Scale bar, 100 μm. l) TUNEL experiment detects endometrial cell death in Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ female mice on the 4th day of pregnancy, and CK8 is used as a uterine epithelial marker. Scale bar, 100 μm. The statistical error bars of the data in the figures (b), (d), (i) and (j) are mean±SEM. Statistical analysis used two-sample equal variance Student's t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns: no significant difference.

Figure 2 shows an example of the present invention in which specific deletion of Gpx4 in the uterine epithelium results in female infertility due to embryo implantation failure or defects. a) Isolate uterine epithelial cells and stromal cells from Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ mice on the 4th day of pregnancy, and detect the expression of Gpx4 in different types of cells by qPCR (n=5 mice in each group). LE, luminal epithelium; ST, stromal cells. b) In situ hybridization experiment shows the expression of Gpx4 in the uterus of Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ female mice on day 4 of pregnancy. Scale bar, 200 μm. c) Statistical graph showing the pregnancy outcomes of Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ mice, with Gpx4 ^f/f ltf ^Cre/+ females showing complete sterility. df) Analysis of embryo implantation and pregnancy in Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ mice on pregnancy day 5 (d), day 10 (e) and day 14 (f). Brackets indicate embryonic resorption sites, and arrows indicate implantation sites. g) qPCR detection of the expression of uterine receptivity-related genes in isolated epithelial cells on the 4th day of pregnancy in Gpx4 ^f/f ltf ^Cre/+ mice (n=5 mice in each group). h) Immunofluorescence staining of Ki67 and CTNNB1 in the uterus of Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ mice on pregnancy day 4. Scale bar, 100 μm. i) TUNEL experiment detects epithelial cell death on the 4th day of pregnancy in Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ mice. Scale bar, 100 μm. The statistical error bars of the data in (a), (c) and (g) are mean±SEM. Statistical analysis used two-sample equal variance Student's t test, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Figure 3 shows that GPX4 deficiency will cause an increase in the level of lipid peroxidation in the uterus on the 4th day of pregnancy in an embodiment of the present invention. a) Immunohistochemical detection of malondialdehyde (MDA) signal in the uterus of Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ mice on day 4 of pregnancy. Scale bar, 200 μm. Arrows indicate MDA signals. b) The MDA detection kit analyzes the MDA levels in the serum of Gpx4 ^f/f , Gpx4 ^{f/ f} Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy. n represents the number of mice tested. c) ELISA kit detects non-heme iron levels in the serum of Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ female mice on day 4 of pregnancy. d) IHC detects 4-hydroxynonenal (4-HNE) signals in the uterus of Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy. Scale bar, 200 μm. ef) Evaluation of Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ , and Gpx4 ^f/f Ltf ^Cre/+ uterine epithelium isolated on pregnancy day 4 from female mice by flow cytometry staining with C11-BODIPY 581/591 probe Lipid peroxidation levels in cells (e) and stromal cells (f) (n=4 mice per group). LE, luminal epithelial cells; ST, stromal cells. g) Transmission electron microscope images show changes in the microvilli at the top of the uterine cavity epithelium and mitochondria of endometrial cells in Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy. MV, microvilli; Mito, mitochondria. LE, luminal epithelium; ST, stroma. Scale bar, 500 nm. h) Protein carbonyl ELISA Kit detects uterine protein carbonylation levels of Gpx4 ^f/f , Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ female mice on day 4 of pregnancy. The statistical error bars of the data in (bc), (ef) and (h) are mean ± SEM. Statistical analysis used one-way analysis of variance Dunnett's post hoc test, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, ns: no significant difference.

Figure 4 shows an example of the present invention in which Gpx4 ^f/f Ltf ^Cre/+ female mice were given the lipid peroxidation inhibitor Liproxstatin-1 (Lip1) for drug treatment during pregnancy. a) Lip1 drug processing timeline. b) Pregnancy outcomes of Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ female mice after Lip1 drug treatment. n represents the number of mice tested. Data statistics error bars are mean ± SEM. Statistical analysis used two-sample equal variance Student's t test, ****P<0.0001. cd) Lip1 drug treated Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ pregnant mice, and pregnancy status was detected on day 10 (c) and day 5 (d). ef) On the 4th day after Lip1 drug treatment, Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ mouse uterine epithelial cells (e) and stromal cells (f) were isolated to detect their lipid peroxidation levels (n= for each group 4 mice). The statistical error bars of the data in the figures (e) and (f) are mean±SEM. Statistical analysis used one-way analysis of variance and Dunnett's post hoc test, ***P<0.001, ns: no significant difference. g) IHC detects 4-HNE signals in Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ and Gpx4 ^f/f Ltf ^Cre/+ mice on pregnancy day 4 after Lip1 treatment. Scale bar, 200 μm. h) Ki67 and CTNNB1 immunofluorescence shows the proliferation of uterine epithelial cells on the 4th day of pregnancy in Gpx4 ^f/f Ltf ^Cre/+ mice after Lip1 drug treatment. Scale bar, 200 μm. .

Figure 5 shows how accumulation of lipid ROS affects the level and activity of STAT3 in uterine epithelial cells in an embodiment of the present invention. a) qPCR analysis shows the expression of uterine receptivity-related genes in Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ mice after Lip1 drug treatment (n=5 mice in each group). The statistical error bars of the data in the figure are mean ± SEM. Statistical analysis used one-way analysis of variance and Dunnett's post hoc test, *P<0.05, **P<0.01, ns: no significant difference. bc) On the 4th day after Lip1 drug treatment of Gpx4 ^f/f Ltf ^Cre/+ pregnant mice, IHC detected STAT3(b) and p-STAT3 in the uterus of this mouse and Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ female mice. (Tyr705)(c) level. Scale bar, 200 μm. d) Western blot ^experiment detects ^{STAT3 ,} p- ^STAT3 (Tyr705 ^{) ,} and Changes in protein levels of p-STAT3(Ser727). e) Western blot experiment detects the protein levels of STAT3, p-STAT3 (Tyr705), and p-STAT3 (Ser727) in Ishikawa cell line in vitro. f) Changes in the levels of DNP-labeled carbonylated proteins in three Ishikawa cells: Ctrl-sg, Gpx4-sg and Gpx4-sg+Lip1. g) IP experiment shows DNP immunoblotting analysis of precipitated proteins after transfection of STAT3-Flag in three Ishikawa cells: Ctrl-sg, Gpx4-sg and Gpx4-sg+Lip1. h) LC-MS mass spectrum shows that STAT3 has a carbonylation modification site at Pro689. i) DNP immunoblot analysis results of precipitated proteins after transfection of STAT3 and STAT3(P689A)-Flag in GPX4 knockout Ishikawa cells.

Figure 6 shows an example of the present invention in which deletion of Acsl4 in uterine epithelium can improve the implantation defects caused by Gpx4 knockout in epithelial cells and preserve their fertility. a) Serial sections of the uterine tissue of Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy were performed and Gpx4 and Acsl4 immunohistochemistry was performed. Scale bar, 200 μm. b) Pregnancy outcomes in Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^{f /f} Acsl4 ^f/f Ltf ^Cre/+ pregnant mice. c) Observe the pregnancy status of Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on the 18th day of pregnancy. Brackets indicate sites of resorption and arrows indicate viable fetuses. de) Ki67 and CTNNB1 immunofluorescence staining (d) and qPCR detection of proliferation-related genes (e) expression of Ccnd1, Mcm2 and Mcm7, showing no abnormalities in Gpx4 ^f/f Acsl4 ^{f/ f} Ltf ^Cre/+ female mice on the 4th day of pregnancy Epithelial cell proliferation (n=6 mice per group). Scale bar, 100 μm. fg) Isolate uterine epithelial cells (f) and stromal cells (g) from Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy to detect lipid peroxidation levels ( n=4 mice per group). LE, luminal epithelium; ST, stroma. h) Immunohistochemical results of 4-HNE in the uterus of Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on pregnancy day 4. Scale bar, 200 μm. i) Embryo implantation in the uterus of Gpx4 ^{f/ f} Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on day 5 of pregnancy. j) Expression of COX2 and CK8 at the implantation site in Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on pregnancy day 5. Scale bar, 200 μm. Arrows indicate the position of the embryo. k) Uterine changes in Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on pregnancy day 6. l) Immunofluorescence results of Ki67 and CTNNB1 at the implantation site in Gpx4 ^f/f Acsl4 ^f/f and Gpx4 ^f /f Acsl4 ^f/f Ltf ^Cre/+ female mice on pregnancy day 6. Scale bar, 500 μm. The statistical error bars of the data in (b), (e) and (fg) are mean ± SEM. Statistical analysis used two-sample equal variance Student's t test, *P<0.05, ****P<0.0001, ns: no significant difference.

Figure 7 shows that in the embodiment of the present invention, patients with recurrent implantation failure (RIF) undergoing IVF treatment are highly correlated with reduced levels of Gpx4 in the endometrium and increased levels of lipid peroxides. a-b) MDA content (a) and non-heme iron levels (b) in the serum of women with recurrent implantation failure (n=33) and pregnant women in the control group (n=44). Testing shows serum lipids in patients with recurrent implantation failure. Increased peroxide levels. All serum samples were collected one day before participants' embryo transfer. c) Real-time fluorescence quantitative PCR detects the expression of GPX4 and ACSL4 in endometrial tissue of normal women with successful pregnancy (n=8) and patients with recurrent implantation failure (n=16). d) Detection of GPX4 and ACSL4 protein levels in endometrial tissue of women with normal successful pregnancies (n=8) and patients with recurrent implantation failure (n=16). e) Multiple immunofluorescence staining of GPX4, ACSL4 and CK8 shows that the expression of GPX4 is significantly reduced in the uterine epithelium of patients with recurrent implantation failure, and CK8 is a human uterine epithelial marker. Scale bar, 200 μm. f) Immunohistochemistry shows that the level of 4-HNE in the uterine epithelium of RIF patients with low GPX4 expression is significantly increased, indicating that patients with low GPX4 expression in uterine cells often experience significant lipid peroxidation. Scale bar, 200 μm. g) Analysis of DNP-labeled carbonylated protein content in human endometrial tissue proves that increased uterine lipid ROS levels in RIF patients cause changes in protein carbonylation modification levels. β-Actin was used as a loading control. In the figures (a-c), the statistical error bars of the data are mean ± SEM. Statistical analysis used two-sample equal variance Student’s t test, *P<0.05, **P<0.01, ns: no significant difference.

Detailed ways

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the exemplary embodiments according to the present invention. As used herein, the singular forms also include the plural forms unless the context clearly dictates otherwise. Furthermore, it will be understood that when the terms "comprises" and/or "includes" are used in this disclosure, they refer to the presence Features, steps, operations, devices, components and/or combinations thereof.

The present invention will be further described with reference to specific examples. The following examples are only for explaining the present invention and do not limit its content. If the specific experimental conditions are not specified in the examples, the conventional conditions or the conditions recommended by the reagent company are usually followed. The reagents, consumables, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

The present invention will be further explained through the following examples, but shall not be construed as limiting the present invention. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. The following examples are test methods indicating specific conditions, which are usually carried out under conventional conditions.

Example

1.Materials and methods

1.1 Human serum collection: Female patients who chose in vitro fertilization or intracytoplasmic sperm injection (IVF/ICSI) in the Reproductive Hospital Affiliated to Shandong University were selected. All serum samples were collected one day before embryo transfer. The normal human blood sample control group includes 44 women who successfully became pregnant after IVF/ICSI treatment due to fallopian tube factors, ovulation disorders or male factors. For serum sample collection from women with recurrent implantation failure (RIF), patients with at least 3 failed in vitro fertilization-embryo transfers (IVF-ET) with no less than 4-6 high-scoring 8-cell embryos or 3 High quality blastocysts. In addition, all 33 patients in the RIF group experienced implantation failure after embryo transfer. Table 1 details the clinical parameters of the patients. Both groups of patients had good basal hormone levels and good responses to hormone stimulation. Peripheral blood was centrifuged (3500g/min, 10min) to collect serum samples and frozen at -80°C until detection.

Table 1. Human clinical parameters of sera collected from patients with successful pregnancy and recurrent implantation failure undergoing IVF treatment

BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; AFC, number of antral follicles; P value was analyzed using non-parametric Kolmogorov-Smirnov test.

1.2 Human endometrial sample collection: On the 7th day after ovulation (ovulation day is detected by B-ultrasound), the endometrium of female patients is collected for biopsy using the Pipellede device. All participants experienced at least 2-3 IVF-ET failures, in which a total of no less than 4-6 high-scoring 8-cell embryos or 2 high-quality blastocysts were transferred. Patients receiving hormonal therapy during the last three menstrual cycles were excluded. Participants were divided into implantation success and implantation failure groups based on pregnancy outcomes in the next transplantation cycle after endometrial biopsy. The criterion for successful implantation is the visible gestational sac under B-ultrasound. The detailed clinical parameters of human endometrial donors are shown in Table 2. Each tissue sample was divided into 2 pieces and immediately frozen in liquid nitrogen or fixed in 4% formalin for immunohistochemistry or other experimental analysis.

Table 2. Clinical parameters of endometrial donors in patients with successful pregnancy and recurrent implantation failure after IVF treatment

E, endometritis; N, normal; F, repeated implantation failure; PRL, prolactin; T, testosterone; TSH, thyroid stimulating hormone

1.3 Multiplex immunofluorescence analysis of human endometrial tissue samples: The OPAL Polaris system (Akoya Biosciences) was used to perform Gpx4 (1:100), Acsl4 (1:200) and CK8 (1:500) on paraffin sections of the endometrium of RIF patients and controls. ) multiplex immunofluorescence detection. The primary antibody and Oppl Polymer HRP Anti-Mouse/Rabbit secondary antibody (ARH1001EA) were applied according to the instructions, and then stained using tyramine signal amplification and OPAL fluorophore (NEL811001KT). The Opal fluorophore detectors are Opal 620 (Gpx4), 690 (Acsl4) and 520 (CK8) dyes, and DAPI is used for nuclear staining of tissues. The stained slides were scanned using a multispectral panoramic tissue scanning microscope (tissufaxs Spectra), and the expression levels of Gpx4 and Acsl4 proteins were compared and analyzed by fluorescence intensity.

1.4 Mouse model construction and analysis of pregnancy events: Pgr ^Cre/+ mice were provided by the laboratories of John Lydon and Francesco DeMayo (Baylor College of Medicine, Houston, TX, USA), and Acsl4 ^f/f mice were provided by the WeiGu laboratory (Columbia University , New York, NY, USA), ltf ^Cre/+ and Gpx4 ^f/f mouse lines were purchased from Jackson Laboratories. floxed female mice were mated with Pgr ^Cre/+ and Ltf ^Cre/+ male mice respectively to obtain Gpx4 ^f/f Pgr ^Cre/+ , Gpx4 ^f/f Ltf ^Cre/+ , Acsl4 ^f/f Ltf ^Cre/+ and Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice. The female mice of appropriate age were mated with fertile wild-type male mice to induce pregnancy (vaginal plug = day 1 of pregnancy), and the pregnancy status was evaluated. Pregnant rats were injected with Chicago blue dye intravenously into the medial canthus on the 5th day to check the implantation site, and the number of litters was analyzed on the 17th to 21st days of pregnancy. For drug treatment of pregnant mice, each female mouse was intraperitoneally injected with 600 μg Liprostatin-1 at 9:00 am every day from the first day of pregnancy to the pregnancy checkpoint.

1.5 In situ hybridization (ISH) to detect changes in Gpx4 and endometrial receptivity genes: Paste frozen uterine tissues of each genotype on the same glass slide, fix with 4% PFAT (PFA+0.1% Tween 20), and gradient methanol After dehydration, rehydration and digestion, the digoxigenin (DIG)-labeled probe was hybridized and then incubated with Anti-DIG-AP (Roche, 11093274910) antibody, and the BCIP/NBT color development kit (Roche, 11697471001) was used to observe the color development. reaction. Observe the signal under the bright field view of a panoramic scanner (OLYMPUS, VS120). Probe preparation: Gpx4, Muc1, Ltf and other tolerance-related gene probes were constructed according to the SP6/T7 transcription kit (Roche, 11175025910) for detection.

1.6 Immunofluorescence (IF) and immunohistochemistry (IHC) analysis of embryo implantation, uterine receptivity and changes in lipid peroxidation levels: prepare frozen sections of uterine tissue at -20°C and fix with 4% paraformaldehyde (PFA) 1 × PBST (PBS + 0.1% Triton X-100) washed and blocked with 5% bovine serum albumin (BSA) solution. Then apply and stain with primary antibodies such as Cox2 and ki67 and Alexa 488 or Alexa 594 coupled secondary antibodies. TUNEL staining was performed using a cell death detection kit (Beyotime, C1086), and finally a panoramic scanning fluorescence microscope was used for image acquisition to evaluate embryo implantation and uterine receptivity status. Uterine tissue used for IHC was fixed in 10% neutral formalin, sections were dehydrated with gradient ethanol, blocked with endogenous peroxidase blocking solution (Beyotime, P0100A) and 5% BSA, and incubated with MDA, 4HNE, and Stat3. Wait for the antibody and the corresponding secondary antibody, and use the DAB kit for color observation to detect the level of lipid peroxidation.

1.7 Isolation of uterine cavity epithelial cells and stromal cells and detection of lipid ROS accumulation by flow cytometry: The uterine tissue blocks of pregnant mice were digested with trypsin (Sigma, P3292) and dispase (Roche, 4942078001), and the uterine tissue pieces were digested under a stereomicroscope. The epithelium was separated from the tissue fragments and digested with collagenase to obtain uterine epithelial cells. The remaining tissue was still enzymatically digested (Sigma, C5138) to obtain a cell suspension, which was filtered through a nylon mesh and centrifuged to obtain uterine stromal cells. After the cells were resuspended and stained with C-11BODIPY 581/591 dye (Invitrogen, D3861), the lipid ROS levels of different types of uterine cells were detected by flow cytometry (Gallios), and the data were analyzed by FlowJo software.

1.8 Determination of MDA content and non-heme iron levels in serum of humans and pregnant rats: The method for determining MDA content was improved based on published literature. Add a quantitative amount of 1-methyl-2-phenylindole solution to 100 μL of serum, use 37% hydrochloric acid for incubation reaction, collect the supernatant after centrifugation of the sample (10 min, 15 000 g, 4°C), and detect the absorbance at 595 nm. The MDA concentration was measured using 1,1,3,3-tetramethoxypropane (Sigma-Aldrich, 108383) as a standard derived from MDA. When detecting non-heme iron levels, first use disodium-4,7-diphenyl-1,10-phenanthroline disulfonate (Solarbio, 52746-49-3) and 70% thioglycolic acid (Sigma, T6750 ) Prepare iron color solution and color working solution. Refer to the instructions of iron standard solution (Sigma, 02583) to prepare standards with a concentration range of 0 to 1000 μg/dL. Use iron chromogenic working solution in a 96-well plate to perform the reaction between serum and iron standard solution. Use a TECAN microplate reader (INFINITE 200PRO) to detect the absorbance value at a wavelength of 535nm and perform calculation and analysis.

1.9 Determination of protein carbonylation level and detection of serum P4 and E2 content: OxiSelect proteinCarbonyl ELISA Kit (Cell Biolabs, STA-310) was used to detect the protein carbonylation level of the endometrium of pregnant mice. Hormone levels were detected by collecting serum from female mice on the 4th day of pregnancy and using an enzyme-linked immunosorbent assay kit (Cayman, 582601 and 501890).

1.10qRT-PCR and western blot analysis: Use TRIzol reagent (Invitrogen, 15596-018) to prepare uterine tissue homogenate and use chloroform-isopropyl alcohol precipitation method to extract RNA, wash with ethanol and perform RNA purification. RNA was reverse transcribed into cDNA by Evo M-MLVRT kit and gDNAClean Kit (Accurate Biology, AG11711). Quantitative PCR reaction was performed using SYBRGreen Master Mix (Vazyme, Q311-02) and specific primers and analyzed by real-time PCR system (Light Cycler 96) to compare the expression of Gpx4 and Acsl4 in human endometrial RIF patients and the proliferation of uterine cells in pregnant mice. , death and receptive state changes. For western blot analysis, tissue or cell proteins were extracted using RIPA buffer (Beyotime, P0013B), and the protein concentration was determined for quantification by Pierce ^TM BCA kit (Thermo, 23227). The proteins were separated by SDS-PAGE gel electrophoresis and transferred to PVDF membrane. . The membrane was blocked with 5% skimmed milk powder, and then incubated with Stat3, p-Stat3 and other antibodies and secondary antibodies. Bands were detected using an ECL kit (Thermo, 32106) and an AmershamImager 680 system.

1.11 Total protein carbonylation content detection and protein immunoprecipitation (IP): OxiSelect protein carbonyl immunoblotting kit (Cell Biolabs, STA-308) detects carbonylated protein levels in Ishikawa cells and human RIF patient endometrial samples. Dinitrophenylhydrazine (DNPH)-derivatized oxidized protein analysis was performed on PVDF membranes and carbonylated proteins were visualized using immunoblotting techniques. The IP experiment mainly uses the Stat3-Flag plasmid transfection method, adding flag antibodies and A/G agarose beads (Santa Cruz, B2422), and the co-immunoprecipitated proteins are separated by SDS-PAGE. flag (Sigma, F1804) and DNP (Cell Biolabs ) antibody Western blot analysis demonstrated that Gpx4 deletion caused significant carbonylation modification of Stat3 protein.

1.12 Observe changes in mitochondria and microvilli of endometrial cells under a transmission electron microscope to evaluate uterine epithelial function: Fresh pregnant rat uterine tissue was fixed in 2.5% glutaraldehyde/0.1M phosphate buffer (pH 7.4) and osmotic acid fixative. . The samples were then treated with gradient ethanol and acetone, embedded and polymerized using epoxy resin. Semi-thin positioning and ultrathin sectioning were performed by an ultramicrotome (Leica, EMUC7), and the thickness was accurate to 50 nm. Ultrathin sections were stained with saturated uranyl acetate aqueous solution and lead citrate solution, and then photographed and analyzed using a Thermo Fisher transmission electron microscope (Talos F200C).

1.13 Ishikawa cell GPX4 knockout and drug treatment analysis of Lip-1 rescue efficiency: Use the CRISPR/Cas9 system to knock out GPX4 in Ishikawa cells for cell culture. The sgRNA sequence is designed as CACCGAGAGATCAAAGAGTTCGCCG (SEQ ID NO. 1), AAACCGGCGAACTCTTTGATCTCTC (SEQ ID NO. .2). Ishikawa cells were treated with different concentrations of Liproxstatin-1 (Selleck, S7699) and Necrostatin-1 (Selleck, S8037) and z-VAD-FMK (Selleck, S7023). The cells were collected to detect lipid ROS levels. GPX4 deletion caused lipid ROS accumulates significantly, and Lip-1 drug significantly reduces this phenomenon.

1.14 Protein quantification and carbonylation modification site identification using LC-MS/MS method: Collect control and Gpx4-deleted Ishikawa cell proteins, incubate them with flag antibodies and magnetic beads, and separate by SDS-PAGE to obtain immunoprecipitated proteins. The tandem mass spectrometry method provided by Jingjie Biotechnology Co., Ltd. was used to perform NSI source analysis on the uterine receptivity protein Stat3 peptide fragment on the EASY-nLC 1000UPLC system (Thermo) and Q Exactive ^TM Plus (Thermo). The obtained mass spectra were analyzed using Proteome Discoverer 1.3 software. The data is processed. In the Gpx4 knockout cell line, a specific carbonylation modification of proline at position 689 of the Stat3 protein was detected, and its peptide ion score was >20, while no change in this modification site was detected in the control group.

2. Results and discussion

2.1 Deletion of Gpx4 in the uterus causes complete infertility in female mice and defects in embryo implantation

The GPX family is a protein widely present in the body that decomposes peroxidation products and has eight members. We previously performed RNA sequencing on uterine epithelial and stromal cells isolated from mice on day 4 of pregnancy and found that Gpx4 is one of the most abundantly expressed genes among family members. In order to explore whether Gpx4 plays a role in female pregnancy, we collected uterine tissues at different stages of early pregnancy. Through sectioning and immunohistochemistry (IHC) experiments, we found that Gpx4 was mainly localized in uterine epithelial cells from the 1st to the 4th day of pregnancy. , and then mainly expressed in stromal cells and decidual cells on days 5 and 8 (Fig. 1a). At the same time, in situ hybridization (ISH) was used to confirm that Gpx4 had the same spatiotemporal expression pattern at the implantation stage at the RNA level.

In order to further study the physiological function of Gpx4 in the uterus, we used progesterone receptor transgenic mice (Pgr-Cre) and crossed them with GPX4 ^flox mice to generate knockout (KO) female mice with specific inactivation of GPX4 in the endometrium. Experiments confirmed that the expression of Gpx4 in the uterus of KO mice was significantly reduced at both the RNA and protein levels. Statistics found that Gpx4 ^f/f Pgr ^Cre/+ female mice were completely infertile, while control Gpx4 ^f/f mice could get pregnant and give birth normally (Figure 1b). We detected the status of the implantation site by injecting Chicago blue on the 5th day of mouse pregnancy and found that compared with the control group, Gpx4 ^f/f Pgr ^Cre/+ mouse embryos failed to implant, and some implantation defects occurred (Figure 1c- d). Hematoxylin-eosin staining and immunofluorescence results showed that the crypt chamber morphology in the uterus of this mouse was abnormal on the 5th day of pregnancy (Figure 1e), and the expression of cyclooxygenase 2 (COX2) was also changed (Figure 1f). Cox2 is These results further prove that the lack of GPX4 in the endometrium can lead to abnormal embryo implantation and cause infertility, indicating that Gpx4 is crucial for normal fertility in female mice. Since Gpx4 has a small amount of expression in ovarian follicles, and Pgr-Cre is also expressed in the ovary, in order to exclude the influence of ovarian function, we measured the progesterone (P4) in the serum of Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ mice. ) and 17β-estradiol (E2) hormone levels were detected, and it was found that there was no significant difference in hormone levels between KO mice and the control group on the 4th day of pregnancy. Cytochrome p450 side chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) are two key enzymes that affect P4 biosynthesis and can be used as markers to evaluate ovarian function. Subsequently, we compared The expression of the two markers was detected in the ovaries of Gpx4-Pgr KO mice on day 4 of pregnancy and found that their levels did not change significantly in the ovaries, indicating that Gpx4 deficiency does not affect female ovarian development and uterine morphology.

2.2 Abnormal expression of Gpx4 in the uterus leads to destruction of endometrial receptivity

In order to further explore the reasons for implantation failure in Gpx4-Pgr KO pregnant mice, we detected the expression of estrogen receptor (ESR1) and progesterone receptor (PR) in the uterus on the 4th day of pregnancy in mice by frozen section immunofluorescence. It was found that there was no significant difference in the expression of the two proteins in the uterus of Gpx4 ^f/f and Gpx4 ^f/f Pgr ^Cre/+ mice. As a marker molecule for uterine gland function, the expression pattern of FOXA2 did not change significantly compared with control mice. Subsequently, we detected the expression of estrogen response genes mucin 1 (Muc1) and Ltf in the uterine epithelium on the 4th day of pregnancy. In situ hybridization results showed that Muc1 and Ltf were significantly expressed in the uterine cavity epithelium of Gpx4 ^f/f Pgr ^Cre/+ mice. Increased (Figure 1g,h), indicating that Gpx4 deletion causes abnormal enhancement of estrogen response. Next, real-time fluorescent quantitative PCR (qRT-PCR) was used to analyze uterine receptivity-related genes and found that Muc1, Ltf, Wnt7b, insulin-like growth factor 1 (Igf1), Stat3, etc. The expression levels of E2-responsive genes were significantly increased (Fig. 1i), further proving the previous results. At the same time, there is no difference in the expression of leukemia inhibitory factor (lif) in the uterus of control and Gpx4-Pgr KO mice, indicating that uterine Gpx4 deletion does not affect embryo implantation by changing gland function (Figure 1i). On the fourth day, the uterine epithelium responds to P4 signals to inhibit the cell cycle and adjust cell polarity to prepare for embryo implantation. Subsequently, we examined the expression of p4 response-related genes and found that the expression of dual regulatory protein (Areg) and Indian hedgehog homologous gene (Ihh) was reduced in the uterus of Gpx4 ^f/f Pgr ^Cre/+ mice on the 4th day of pregnancy. , the expression of Wnt family member 4 (Wnt4) increased, while the levels of Hoxa10 and Hand2 did not change significantly (Figure 1j), indicating that the loss of Gpx4 affects specific p4 response genes in the uterus, leading to abnormal endometrial progesterone response. The synergistic effect of estrogen and progesterone is one of the key factors in the establishment of a receptive state. These results indicate that the reason for the failure of Gpx4-Pgr KO implantation is that the sensitivity of the uterus to hormonal responses changes, which blocks the establishment of a receptive homeostasis.

The uterus must enter the window period for the embryo to position, adhere, and implant within the uterine cavity. An important feature of the mouse uterus entering the receptive state is that the luminal epithelial cells cease cell proliferation and cell death. So we performed immunofluorescence staining of the proliferation markers Ki-67 and pHH3 on the uterus on the 4th day of mouse pregnancy to observe cell proliferation. We found that compared with flox mice, the uterus of Gpx4 ^f/f Pgr ^Cre/+ mice There was obvious abnormal cell proliferation in the luminal epithelium (Figure 1k). Through qRT-PCR analysis, it was found that the expression levels of cell proliferation markers Ccnd1, Mcm2 and Mcm7 were also significantly increased on the 4th day of mouse pregnancy. These results further illustrate that Gpx4 deletion causes uterine Abnormal cell proliferation. In addition, through TUNEL staining, we found that Gpx4 ^f/f Pgr ^Cre/+ uterine epithelial cells also died abnormally on the 4th day of pregnancy. However, this cell death is not caused by apoptosis. By detecting the expression of pyroptosis-related genes (Casp11, Casp1 and Gsdmd) and ferroptosis-related genes (Ptgs2, Mdm2, Tfrc, Fth, Alox12 and Slc7a11), it was found that Gpx4 ^f/f Pgr ^Cre/+ mouse uterus on day 4 of pregnancy Increased pyroptosis and ferroptosis. These results further demonstrate that Gpx4 deficiency leads to uterine luminal epithelial dysfunction and impairs uterine receptivity.

2.3 Specific inactivation of Gpx4 in uterine epithelium can cause abnormal embryo implantation and lead to infertility.

By mating Pgr-Cre-driven gene expression tool mice with flox mice, the expression of the Gpx4 gene can be knocked out in almost all uterine cell types, including myometrium, uterine stromal cells, and epithelial cells. In order to further explore the specific role of Gpx4 in uterine epithelial cells during the establishment of uterine receptive state, we used Ltf-Cre (expressed in uterine epithelial cells) tool mice and flox mice to construct Gpx4 ^f/f ltf ^Cre/+ mice. model, this mouse specifically knocks out Gpx4 only in uterine epithelial cells. By isolating mouse uterine epithelial and stromal cells and conducting qPCR experiments, the effective deletion of Gpx4 in the mouse uterine epithelium was confirmed at the RNA level. In situ hybridization and immunohistochemistry results also verified this conclusion (Figure 2a-b). Interestingly, Gpx4 deletion in the uterine epithelium caused increased Gpx4 expression in stromal cells (Fig. 2a), which may be due to feedback regulation of signaling molecules by different types of cells in the uterus. Then the pregnancy outcome and litter size of Gpx4 ^f/f ltf ^Cre/+ mice were observed and statistically analyzed, and the analysis found that Gpx4 ^f/f ltf ^Cre/+ mice were still infertile (Figure 2c). Chicago blue staining results showed that the mouse had embryo implantation defects or failures on the 5th day of pregnancy (Figure 2d). Further observation of their reproductive phenotype revealed that Gpx4 ^f/f ltf ^Cre/+ mice experienced complete fetal resorption in the second trimester (Fig. 2e, f).

Deletion of Gpx4 only in uterine epithelial cells results in female infertility, a phenotype similar to that of Gpx4-Pgr KO mice. We wonder whether the complete infertility of this mouse is the same as that of Gpx4-Pgr KO female mice, and is also caused by abnormal uterine receptivity? In order to further explore the reasons for the phenotypic defects in Gpx4 ^f/f ltf ^Cre/+ mice, we isolated uterine epithelial cells from Gpx4 ^f/f and Gpx4 ^f/f ltf ^Cre/+ female mice on the 4th day of pregnancy and performed qPCR experiments to detect the phenotypic defects. Expression of receptivity-related genes critical for uterine epithelial function. The results showed that the loss of Gpx4 in uterine epithelial cells would cause abnormal E2 and P4 responses in the epithelium, and impaired estrogen and progesterone response and synergy (Figure 2g). Ki67 and pHH3 immunostaining analysis revealed that Gpx4 ^{f/ f} ltf ^Cre/+ mice also exhibited abnormal proliferation of uterine epithelial cells on the 4th day of pregnancy (Figure 2h). At the same time, TUNEL staining also detected different types of cell death in the uterine cavity epithelial cells of this mouse, including ferroptosis and pyroptosis (Figure 2i). Taken together, these data indicate that uterine epithelial Gpx4 is critical for epithelial remodeling and receptive state establishment and embryo implantation during female pregnancy.

2.4 The level of lipid peroxidation in the uterus of Gpx4 knockout mice increases and redox homeostasis is disrupted

It has been reported in the literature that GPX4 maintains cellular redox homeostasis by reducing lipid hydroperoxide accumulation. Does Gpx4-deficient uterine epithelial cells undergo lipid peroxidation, causing epithelial dysfunction? As introduced earlier, lipid peroxidation products include initial lipid hydroperoxides (LOOHs) and subsequent reactive aldehydes, mainly malondialdehyde and 4-hydroxynonenal (MDA and 4HNE), both of which are composed of non- Heme iron is produced by initiating lipid peroxidation and serves as an optimal tool for detecting and quantifying lipid peroxidation in biological samples. Surprisingly, there was no MDA signal in the uteri of normal mice and Gpx4-deficient mice on pregnancy day 1, but on pregnancy day 2, large amounts of lipid excess were observed in the uteri of all three different types of mice. The oxide malondialdehyde accumulates, and starting from the third day of pregnancy, this accumulation gradually weakens. Because the uterus of mice is in the receptive period on the fourth day of pregnancy, it is preparing for embryo implantation at this critical stage. Therefore, we further examined the accumulation of lipid peroxides in the uterus on day 4 of pregnancy, and the results showed that compared with the control group, the MDA signal of uterine stromal cells in Gpx4 ^f/f Pgr ^Cre/+ female mice was significantly increased (Figure 3a) , and the MDA signal on the uterine cavity epithelial surface of Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f ltf ^Cre/+ female mice was also significantly higher than that of flox female mice. At the same time, we detected the malondialdehyde and non-heme iron levels in the serum of mice on the 4th day of pregnancy and found that the MDA and non-heme iron levels in the serum of uterine Gpx4 knockout mice were significantly higher than those in the control group (Figure 3b, c), indicating that Gpx4 deletion not only causes the accumulation of lipid peroxides in the mouse uterus, but also causes an increase in serum lipid peroxidation levels. Next, we analyzed mouse uterine 4-hydroxynonenal levels by immunohistochemistry and found that the 4-HNE signal was significantly enhanced in the uterine epithelium of Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f ltf ^Cre/+ female mice ( Figure 3d).

Next, in order to further confirm the changes in lipid peroxidation levels in the uterus, we used the Bodipy-C11581/591 probe (usually used to detect peroxidized phospholipids in cell membranes) to detect uterine epithelial cells and uterine stroma isolated on the 4th day of pregnancy. The cells were stained and labeled, and through flow cytometric sorting analysis, it was found that enhanced levels of lipid peroxidation were detected in the uterine epithelial cells of Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f ltf ^Cre/+ female mice (Figure 3e) . In addition, significant lipid peroxidation also occurred in Gpx4 ^f/f Pgr ^Cre/+ mouse uterine stromal cells, while Bodipy-C11 581/591 was not observed in Gpx4 ^f/f ltf ^Cre/+ mouse uterine stromal cells. The fluorescence intensity increased significantly (Figure 3f). Subsequently, we observed through transmission electron microscopy (EM) that in the uterus of Gpx4 ^f/f Pgr ^Cre/+ and Gpx4 ^f/f ltf ^Cre/+ female mice on the 4th day of pregnancy, the mitochondrial structure and morphology of the epithelial cells were abnormal, and the mitochondria of the luminal epithelium were abnormal. The cristae were reduced and part of the mitochondrial cristae disappeared (Figure 3g), which is a sign of ferroptosis in cells. It should be noted that compared with the control group, although the mitochondrial morphology of most uterine stromal cells in Gpx4 ^f/f ltf ^Cre/+ female mice did not change significantly (Figure 3g), mitochondria in the stromal cells surrounding the epithelium of this mouse Abnormal, we speculate that this part of stromal cells is located near the epithelium and is crucial for embryo implantation, crypt formation and the formation of the primary decidual zone. Therefore, it may be affected by the bioactive substance lipid aldehyde released by Gpx4-deficient epithelial cells. Influence. At the same time, in Gpx4-deficient uteri, a reduction in microvilli and shortened villus length on the apical surface of luminal epithelial cells were also observed. These results further indicate that epithelial function is disordered. Next, we detected and analyzed by ELISA kit and found that higher levels of protein carbonylation were found in Gpx4-deficient uteri on day 4 of pregnancy (Figure 3h), which is an aldehyde molecule derived from lipid peroxidation. The protein is irreversibly modified, which indicates that Gpx4 deficiency in uterine epithelium makes epithelial cells more sensitive to lipid peroxidation. In summary, the above results indicate that Gpx4 deletion in early pregnancy will cause increased lipid peroxidation levels in the uterine epithelium, thereby affecting the establishment of uterine receptivity and leading to homeostatic abnormalities in epithelial-stromal cell interactions required for implantation, ultimately leading to infertility. Education.

2.5 Lipid peroxidation inhibitor Lip1 effectively inhibits lipid ROS accumulation in mice caused by Gpx4 deletion but fails to rescue embryo implantation

Since Gpx4 deletion causes significant lipid peroxidation in uterine cells, leading to embryo implantation failure and infertility phenotypes, can inhibiting epithelial lipid peroxidation levels rescue the fertility of this mouse? To answer this question, we first established the GPX4 knockout Ishikawa cell line in human endometrial cancer epithelial cells using CRISPR-Cas9 technology. As expected, significant elevated levels of lipid peroxidation were found in GPX4 knockout Ishikawa monoclonal cells, and the use of a lipid peroxidation inhibitor (Lip1) effectively alleviated lipid peroxidation in the GPX4 knockout Ishikawa cell line. Oxidation. Subsequently, we evaluated the improvement of pregnancy outcomes of Gpx4 ^f/f Ltf ^Cre/+ female mice by Lip1. Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ female mice were intraperitoneally injected with Lip1 (600 μg) starting from the first day of pregnancy. /d), it was observed that although the dose of Lip1 had no obvious adverse effect on the pregnancy outcome of Gpx4 ^f/f control mice, ruling out the toxicological effects of the drug, it failed to rescue Gpx4 ^f/f Ltf ^Cre/+ females. The fertility of mice (Fig. 4a,b). We evaluated the pregnancy status of this mouse on day 10 after Lip1 drug treatment and found that the implantation site of Gpx4 ^f/f Ltf ^Cre/+ female mice degenerated (Fig. 4c), and Lip1 failed to completely rescue Gpx4 ^{f/ f} Ltf ^{Cre /+} Implantation defect in females (Fig. 4d). To further determine whether Lip1 can effectively reduce lipid peroxidation in the uterus of mice, we used Bodipy C11 581/591 probe staining to evaluate Gpx4 ^f/f and Gpx4 ^f/f Ltf ^Cre/+ female mice administered Lip1 drug. Lipid peroxidation levels in uterine epithelial and stromal cells after treatment. The results showed that Lip1 effectively attenuated lipid ROS accumulation in Gpx4 ^f/f Ltf ^Cre/+ uterine epithelial cells on day 4 of pregnancy (Fig. 4e, f). 4-HNE immunohistochemistry experiments also confirmed that intraperitoneal injection of Lip1 drug can effectively reduce lipid peroxidation of Gpx4 ^f/f Ltf ^Cre/+ uterine epithelial cells (Figure 4g). However, it was found by Ki67 immunofluorescence that abnormal thin proliferation of Gpx4 ^f/f Ltf ^Cre/+ uterine epithelium still existed on day 4 after Lip1 treatment (Fig. 4h).

Subsequently, we detected uterine receptivity-related genes and found that compared with the control group, after Lip1 drug treatment, some gene expressions of Gpx4 ^f/f Ltf ^Cre/+ also returned to normal levels, but other key genes were still expressed. abnormal (Fig. 5a). Similar results were found after drug treatment in Gpx4 ^f/f Pgr ^Cre/+ mice (Lip1 improved intrauterine lipid peroxidation levels but failed to successfully rescue impaired uterine receptivity). Previous studies have shown that STAT3 regulates epithelial estrogen responses and functional remodeling during pregnancy, and mice lacking STAT3 in the uterine epithelium are infertile. After treating Gpx4 KO mice with Lip1 drug, we found that STAT3 still maintained abnormal expression in the uterine epithelium of Gpx4 ^f/f Ltf ^Cre/+ female mice. Westernblotting and IHC experiments further proved this result (Figure 5a-d). At the same time, in vitro experimental results also showed that the expression of STAT3 in the GPX4-knockout Ishikawa cell line was up-regulated and the phosphorylation level was abnormal, which was consistent with the in vivo experimental results in mice (Figure 5e). Our previous results (Figure 3i) showed that protein carbonylation levels increased in the uterus of Gpx4 ^f/f Ltf ^Cre/+ female mice on the 4th day of pregnancy. Generally, protein carbonylation modification often leads to protein functional inactivation. So we speculated whether it was due to the irreversible carbonylation modification of key proteins related to receptivity in Gpx4-deficient uterine epithelium that Lip1 drug treatment failed to successfully rescue embryo implantation? To answer this question, we next constructed a Gpx4 knockout cell line in vitro and detected the protein carbonylation level in the Ishikawa cell line. We found that the protein carbonylation level in the cells increased after GPX4 expression was downregulated (Figure 5f). Subsequently, we used the method of transient transfection of STAT3-Flag and performed immunoprecipitation (IP) experiments with dinitrophenol (DNP) in GPX4 knockout Ishikawa cells to detect whether abnormal carbonylation modifications occurred in the STAT3 protein. IP results showed that even with Lip1 drug treatment, elevated STAT3 carbonylation levels were detected in the GPX4 knockout Ishikawa cell line (Figure 5g). In addition, we used in vitro recombinant STAT3 protein to perform liquid chromatography-mass spectrometry (LC-MS) experiments and performed carbonylation analysis to determine the potential carbonylation site of STAT3, and found that there is a specific carbonylation site in the GPX4 knockout Ishikawa cell line. The specific STAT3 carbonylation modification site is Pro689 (Figure 5h). Since STAT3 is an evolutionarily highly conserved gene, its residue P689 is also conserved across species. To verify the change in this carbonylation site, we constructed a STAT3 mutant with a deletion of this site, in which the proline residue was replaced by alanine (P689A). The STAT3 P689A mutant was then transfected into GPX4 knockout Ishikawa cells, and it was found that the level of STAT3 carbonylation was significantly reduced (Fig. 5i), indicating that Pro689 is an essential residue site for STAT3 carbonylation. The above results show that drug Lip1 treatment can inhibit the level of lipid peroxidation in mice but is not enough to achieve complete recovery of uterine epithelial cell function in early pregnancy. The reason is that the key protein STAT3 related to receptivity undergoes irreversible carbonylation modification. Caused.

2.6 Elimination of Acsl4 can partially rescue the fertility defects in female mice caused by Gpx4 deletion in the uterine epithelium

The above results all show that abnormal lipid peroxidation of uterine epithelium in early pregnancy directly causes infertility in female mice. Next, we want to know whether genetic intervention on the key factors that regulate lipid peroxidation at the genetic level can save it. Fertility defects caused by Gpx4 deletion. Research reports that acyl-CoA synthetase long chain family member 4 (ACSL4) is an enzyme that promotes the biosynthesis of polyunsaturated phospholipids and is an important gene in the lipid metabolism pathway. Recent literature has shown that ACSL4-deficient cancer cells are resistant to lipid peroxidation induced by GPX4 inhibition or inactivation. Does this phenomenon also occur in vivo? To answer this question, we first analyzed the expression pattern of ACSL4 in the uterus and found that ACSL4 is localized in luminal epithelial cells, with strong expression levels on days 1-4 of pregnancy before implantation, and expression in glandular epithelial cells. relatively low. The high expression of ACSL4 in uterine epithelium suggests that the expression of ACSL4 can be intervened at the genetic level to evaluate the resistance of GPX4-deficient uterine cells to lipid peroxidation under the condition of eliminating ACSL4. We speculate that inactivation of ACSL4 may rescue the lipid peroxidation caused by ACSL4. Infertility caused by Gpx4 deletion in the uterine epithelium.

Therefore, we constructed mice lacking Acsl4 in uterine epithelial cells (Acsl4 ^f/f Ltf ^Cre/+ ). Interestingly, Acsl4 ^f/f Ltf ^Cre/+ female mice did not exhibit any adverse reproductive phenotypes. Subsequently, we mated Gpx4 ^f/f Ltf ^Cre/+ male mice with Acsl4 ^f/f female mice to generate mice with conditional deletion of Gpx4 and Acsl4 in the uterine epithelium (Gpx4 ^f/f Acsl4 ^f/f Ltf ^{Cre /+} ). (Figure 6a). In order to test their fertility, we mated Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice with WT fertile males and found that double-knockout female mice could produce a small number of pups, indicating that ACSL4 inactivation can be achieved to a certain extent. improve the infertility phenotype caused by Gpx4 deletion in the uterine epithelium (Figure 6b,c). Next, we assessed the uterine receptivity status of Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice. Surprisingly, there was no significant difference in the proliferation of uterine epithelial cells in Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ mice on day 4 of pregnancy compared with the control group (Figure 6d,e). Subsequently, lipid peroxidation levels were measured by isolating uterine epithelial and stromal cells from Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice. It was found that the lipid peroxidation level of uterine epithelial cells in double knockout mice was still slightly higher than Gpx4 ^f/f Acsl4 ^f/f female mice (Figure 6f,g), but compared with the Gpx4 ^f/f Ltf ^Cre/+ group, the level of lipid peroxidation in Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice The increase was much lower than that of mice with single deletion of Gpx4. Further immunohistochemical analysis of 4-HNE revealed that there was no significant change in the signal between double mutant mice and control female mice (Figure 6h). The qRT-PCR results showed that there was no significant difference in the expression of most uterine receptivity-related genes compared with the control group. Next, through immunohistochemical staining, it was found that the protein and phosphorylation of STAT3 between the double knockout mice and the control group. level of education is equivalent. These results indicate that the deletion of Acsl4 can improve the excessive lipid peroxidation caused by Gpx4 knockout in uterine epithelial cells and rescue the uterine receptivity defect.

Subsequently, we used Chicago blue staining to detect the status of the implantation site of Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on day 5, and observed an obvious blue band, indicating that it was related to Gpx4 ^f/f Ltf ^Cre/+ female mice. Compared with mice, the embryonic attachment of double knockout mice was significantly improved (Figure 6i), and the crypt shape and COX2 expression at the implantation site also tended to be normal (Figure 6j), indicating that Acsl4 inactivation can partially rescue the effects of Gpx4 deletion. embryo implantation defects. However, further observation revealed abnormal embryonic distribution and spacing in Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice on day 6 of gestation (Fig. 6k). During normal pregnancy, stromal cells around the cavity of the implantation site can be observed to transform into epithelial-like cells on the sixth day, forming the avascular primary decidual zone (PDZ). This phenomenon occurs in Gpx4 ^f/f Acsl4 ^f/f and Gpx4 It can be observed in ^f/f Acsl4 ^f/f Ltf ^Cre/+ pregnant mice, but Gpx4 ^f/f Acsl4 ^f/f Ltf ^Cre/+ female mice show abnormal PDZ formation and decidualization reaction (Figure 6), This phenomenon explains why the uterine receptive state of double knockout mice tends to be normal but the fertility phenotype is not completely rescued.

2.7 RIF patients with repeated implantation failure have reduced expression levels of Gpx4 in the endometrial epithelium and exhibit abnormally high levels of lipid peroxidation.

Next, we evaluated the association of lipid peroxidation levels or Gpx4 activity with embryo implantation impairment in early human pregnancy. We collected the subjects' serum 1 day before embryo transfer: the control group sought in vitro fertilization-embryo transfer (IVF-ET) treatment due to ovulatory dysfunction, fallopian tube factors and male factors, and eventually became pregnant successfully; the RIF group experienced repeated implantation Failure (at least 3 times), and exclude factors such as ovulation disorders, chronic diseases such as diabetes or hypertension, and liver and kidney dysfunction. The detailed clinical parameters of the two groups of patients can be found in Table 1. Through testing, we found that compared with the control group, the MDA content and non-heme iron levels in the serum of the RIF group were significantly increased, indicating that RIF patients had significant lipid peroxidation (Figure 7a,b).

We further collected endometrial samples from the secretory phase (7 days after ovulation) of the control group and RIF patients who successfully conceived after IVF-ET treatment (see Table 2 for specific patient parameters), and found that the expression level of GPX4 mRNA was significantly higher in the RIF group. lower than the control group. In contrast, the mRNA levels of ACSL4 were not significantly different between the two groups (Fig. 7c). The low expression of GPX4 protein was also confirmed in some RIF patients, especially in the endometrial epithelium of RIF patients, while the protein level of ACSL4 showed no significant change between the two groups (Figure 7d,e). In addition, the 4-HNE signal in the endometrium of patients with repeated implantation failure was significantly enhanced. These results indicate that the uterine cells of patients with RIF have low GPX4 expression and also exhibit high levels of lipid peroxidation (Figure 7f). Subsequently, we also tested the protein carbonylation levels in human uterine tissue and found that RIF patients with higher levels of lipid peroxidation also showed increased protein carbonylation modifications in the endometrium. These are highly consistent with the results of in vivo experiments in mice. anastomosis (Fig. 7g). The above analysis based on human experimental data shows that abnormal expression of GPX4 in the endometrium and increased levels of lipid peroxidation are one of the causes of repeated implantation failure and infertility in women.

In conclusion, our clinical data demonstrate that abnormal lipid peroxidation in the uterine epithelium contributes to implantation defects and that lipid peroxidation levels are enhanced in the uterus of patients with recurrent implantation failure or recurrent spontaneous abortion. At the same time, further investigation of the role of lipid peroxidation in uterine stromal cells may further our understanding of endometrial redox homeostasis during implantation events. These findings suggest that elimination of uterine lipid ROS, thereby improving endometrial receptivity, may serve as a potential novel therapeutic strategy to improve embryo implantation and clinical pregnancy rates.

The above embodiments are only for illustrating the technical concepts and characteristics of the present invention. Their purpose is to enable those familiar with this technology to understand the content of the present invention and implement it accordingly. They cannot limit the scope of protection of the present invention. All equivalent changes or modifications made based on the spirit and essence of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. Application of reagents for detecting expression levels of lipid peroxidation and related biological components in the preparation of products for predicting the occurrence of repeated implantation failure; Wherein, the lipid peroxidation-related biological elements include lipid peroxidation-related proteins or lipid peroxidation-related products, further including glutathione peroxidase 4, active aldehyde substances MDA, 4HNE and non-heme iron. 2. The application according to claim 1, characterized in that the lipid peroxidation-related biological elements are human and non-human mammals. 3. The application according to claim 1, characterized in that the reagents for detecting lipid peroxidation and expression levels of related biological components include any currently known reagents commonly used in methods for detecting the above substances, further comprising Reagents used in thiobarbituric acid method, high performance liquid chromatography, enzyme-linked immunosorbent assay, fluorescence method, and electron spin resonance method used to detect lipid peroxide; It also includes reagents for detecting the transcription of the GPX4 encoding gene based on real-time fluorescent quantitative PCR, in situ hybridization, gene chips and gene sequencing, and/or reagents for detecting the expression of glutathione peroxidase 4 based on immunoassay methods. 4. The application according to claim 3, wherein the product includes primers, probes, (gene or protein) chips, and nucleic acid membranes for detecting the expression levels of lipid peroxidation and related biological components in the sample to be tested. Strips, test kits, test devices or equipment; The samples to be tested are human or non-human samples, including subjects' blood (including serum) samples and endometrial samples (including endometrial epithelial cells). 5. A system for predicting the occurrence of repeated implantation failure, characterized in that the system at least includes: an acquisition unit configured to: acquire the expression level of the subject's marker; An evaluation unit configured to: evaluate the occurrence of repeated implantation failure in the subject based on the expression level of the marker obtained by the acquisition unit; Wherein, the biomarkers include lipid peroxidation and related biological components; The lipid peroxidation-related biological elements include lipid peroxidation-related proteins or products thereof, further including glutathione peroxidase 4, active aldehyde substances MDA, 4HNE and non-heme iron. 6. The application of lipid peroxidation and its related biological components as targets in the prevention and treatment of diseases related to repeated implantation failure and/or the screening of drugs for diseases related to repeated implantation failure. 7. Application of substances that remove lipid peroxides or promote the expression levels of GPX4 and its encoding gene in any one or more of the following: (a) Improve endometrial receptivity or prepare products that improve endometrial receptivity; (b) Prevent implantation failure and increase clinical pregnancy rate or prepare products that prevent implantation failure and increase clinical pregnancy rate; (c) Products for the treatment of infertility. 8. Application of substances that knock out long-chain fatty acyl-CoA synthetase 4 in the preparation of products to reverse infertility mediated by GPX4 deletion. 9. The application according to claim 8 or 9, characterized in that the product can be a medicine or an experimental reagent, and the experimental reagent is used for basic research. 10. The application according to claim 9, wherein when the product is a medicine, the medicine further includes at least one pharmaceutical inactive ingredient.