Application of follicular fluid exosome lncLIPE-AS1 in evaluating embryo quality of PCOS patients

Abstract

The application discloses application of follicular fluid exosome lncLIPE-AS1 in evaluation of embryo quality of patients with polycystic ovary syndrome and belongs to the field of assisted reproductive technology. The application finds that lncLIPE-AS1 is highly expressed in follicular fluid of patients with polycystic ovary syndrome, and finds that lncLIPE-AS1 is correlated with embryo quality and steroid hormone secretion of patients with polycystic ovary syndrome. Therefore, lncLIPE-AS1 can be used as a biomarker for evaluation of embryo development quality in the process of assisted reproduction of patients with polycystic ovary syndrome and prediction of success rate of assisted reproduction, and can also be used for screening of high-quality oocytes of patients with polycystic ovary syndrome and regulation of steroid hormone expression of patients with polycystic ovary syndrome.

Description

Technical Field

[0001] This invention belongs to the field of assisted reproductive technology, specifically relating to the application of follicular fluid exosome lncLIPE-AS1 in assessing the quality of assisted reproductive embryos in patients with polycystic ovary syndrome. Background Technology

[0002] Polycystic ovary syndrome (PCOS) is a common reproductive endocrine disorder in women of reproductive age, with an incidence rate of 5%-20%. Hormone metabolism abnormalities and impaired follicular maturation are two main characteristics of PCOS. Currently, it is believed that excessive activation of androgens affects the function of ovarian granulosa cells and steroid secretion, leading to abnormal follicular maturation and failure to select a dominant follicle in PCOS patients.

[0003] Current oocyte assessment standards rely solely on morphology (extrusion of polar bodies and presence of blastocysts in the cytoplasm) to determine oocyte maturity. However, due to the use of exogenous estrogen-induced ovulation drugs, many clinically obtained oocytes, while morphologically appearing mature, actually exhibit asynchronous cytoplasmic and nuclear maturation, resulting in poor quality. PCOS patients experience decreased oocyte quality due to reproductive and endocrine system dysfunction, leading to reduced embryonic developmental potential during assisted reproductive treatment and impacting treatment outcomes. Therefore, selecting relatively high-quality oocytes can effectively improve the success rate of assisted reproductive treatment for PCOS patients.

[0004] Follicular fluid (FF) is composed of secretions from oocytes, granulosa cells, and theca cells, as well as exudate from the theca membrane vessels. It provides a crucial microenvironment for follicular development and oocyte maturation, serving as a medium for bidirectional communication between the oocyte and surrounding cells. Studies have identified hormones, enzymes, and reactive oxygen species in FF, all potentially related to follicular development. Follicular maturation is a complex process highly dependent on communication between cells and organs. Substances carried in FF facilitate communication between the oocyte and the surrounding cumulus oophorus and granulosa cells, containing rich genetic information such as proteins, lipids, and RNA. Exosomes are vesicles originating from the cell membrane system, originating from multivesicular bodies within the body, and released into the tissue fluid. These vesicles contain proteins, lipids, and coding or non-coding RNA derived from the donor cytoplasm. During follicular maturation, long non-coding RNAs are transported from granulosa cells to the oocyte via extracellular vesicles, regulating gene expression in the oocyte during follicular maturation.

[0005] Long noncoding RNAs (lncRNAs) generally refer to a class of RNAs larger than 200 nt that do not have protein-coding capabilities. Studies have found that lncRNAs participate in various PCOS pathological processes, including steroid production, oocyte maturation, and cell proliferation or apoptosis. For example, Liu et al. found that the level of lncRNA HCG26 in ovarian granulosa cells of PCOS patients was upregulated and correlated with the number of antral follicles. Knockdown of HCG26 inhibited granulosa cell proliferation, increased aromatase gene expression, and estradiol production. lncRNA ZFAS1 binding to miR-129 can prevent HMGB1 degradation, inhibit granulosa cell proliferation, and promote apoptosis. lncRNA HUPCOS, located at 15q22, is a newly identified intergenic lncRNA that regulates testosterone metabolism by inhibiting CYP11 activity.

[0006] Current research on the function of lncRNAs in follicular fluid is very limited, and studies on lncRNAs derived from PCOS follicular fluid exosomes and their regulatory mechanisms are even rarer. This invention overcomes the limitations of methods for predicting the success rate of in vitro fertilization (IVF) in assisted reproductive technology (ART) for patients with polycystic ovary syndrome (PCOS) by taking a novel approach: focusing on changes in lncRNAs in follicular fluid exosomes to provide a biomarker that can be used to assess embryo quality in ART for PCOS patients. Summary of the Invention

[0007] The contribution of this invention lies in the discovery of a lncRNA that is highly expressed in the follicular fluid of patients with polycystic ovary syndrome (PCOS), and the discovery that the lncRNA is correlated with the embryo quality of PCOS patients, which is expected to become a biomarker for assessing the embryo quality of PCOS patients.

[0008] One objective of this invention is to provide the application of lncLIPE-AS1 as a biomarker in assessing the success rate of assisted reproduction in patients with polycystic ovary syndrome (PCOS); a second objective of this invention is to provide the application of an lncLIPE-AS1 inhibitor in the preparation of a medicament for treating and / or improving the quality of embryos in patients with PCOS; a third objective of this invention is to provide a primer set for detecting lncLIPE-AS1 and a kit containing said primer set; and another objective of this invention is to provide a method for assessing the quality of assisted reproductive embryos in patients with PCOS for non-diagnostic purposes.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] In a first aspect, the present invention provides the use of lncLIPE-AS1 as a biomarker and / or a substance for detecting said biomarker in at least one of the following:

[0011] a1) Application in the preparation and / or screening of products for predicting or assisting in the prediction of assisted reproductive success rates in patients with PCOS;

[0012] a2) Use in the preparation and / or screening of products for assessing or assisting in the assessment of the quality of assisted reproductive embryos in patients with PCOS;

[0013] a3) Application in the preparation and / or screening of products for screening high-quality oocytes from PCOS patients;

[0014] a4) Application in the preparation and / or screening of products for regulating the expression of steroid hormones in patients with PCOS.

[0015] The products include testing reagents, testing kits, chips, membrane strips, or testing platforms.

[0016] The lncLIPE-AS1 was derived from follicular fluid exosomes from PCOS patients.

[0017] The substance used to detect the biomarker lncLIPE-AS1 is a primer that specifically amplifies lncLIPE-AS1 in the follicular fluid exosomes. The primer consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2.

[0018] The assisted reproductive technologies described in this invention include artificial insemination and in vitro fertilization-embryo transfer.

[0019] In a second aspect, the present invention provides the use of an lncLIPE-AS1 inhibitor in at least one of the following:

[0020] b1) Use in the preparation of medicines for treating and / or improving the quality of embryos in patients with PCOS;

[0021] b2) Use in the preparation of medicines for the treatment and / or improvement of steroid hormone disorders in patients with PCOS.

[0022] The steroid hormones mentioned are estradiol and pregnenolone.

[0023] The lncLIPE-AS1 inhibitor is a reagent that reduces the expression level of lncLIPE-AS1 in exosomes of follicular fluid in PCOS patients.

[0024] In a conventional embodiment of the present invention, the lncLIPE-AS1 inhibitor is one or a combination of two or more of siRNA, shRNA, antisense RNA, and antibodies.

[0025] In a third aspect, the present invention provides a primer set for detecting follicular fluid exosomes lncLIPE-AS1 in PCOS patients, comprising the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2.

[0026]

[0027] In a fourth aspect, the present invention provides a kit comprising the primer set described in the third aspect of the present invention.

[0028] Furthermore, the kit also includes a primer set for detecting an internal reference gene, wherein the internal reference gene is GAPDH and / or ACTB.

[0029] In a specific embodiment of the present invention, the internal reference gene is GAPDH.

[0030] Furthermore, the kit also includes standard reagents for RNA extraction, reverse transcription, and PCR reactions.

[0031] In a fifth aspect, the present invention provides a method for assessing the quality of assisted reproductive embryos in PCOS patients for non-diagnostic purposes, the method qualitatively and / or quantitatively assessing embryo quality by detecting the transcriptional level of lncLIPE-AS1 in a sample.

[0032] The sample consisted of follicular fluid exosomes from PCOS patients.

[0033] The detection of transcription levels described in this invention can be achieved through whole RNA transcriptome sequencing or real-time quantitative PCR.

[0034] The method for assessing the quality of assisted reproductive embryos in PCOS patients according to the present invention includes the following steps:

[0035] (1) Collect follicular fluid from PCOS patients and extract exosomes;

[0036] (2) Total RNA was extracted from exosomes using TRIzol reagent, and the total RNA concentration was detected using Nano Drop before reverse transcription was performed.

[0037] (3) Use high-throughput sequencing or real-time quantitative PCR to detect the expression of lncLIPE-AS1.

[0038] In a specific embodiment of the present invention, when using real-time PCR, the relative expression of lncLIPE-AS1 is detected using SYBR Green Mixture with the GAPDH internal reference gene.

[0039] Unless otherwise specified, the reagents and instruments used in steps (1)-(3) of the present invention are all reagents and instruments conventionally used in the present invention.

[0040] The biomarker lncLIPE-AS1 provided by this invention is derived from the follicular fluid of patients. Follicular fluid is composed of secretions from oocytes, granulosa cells, and membrane cells, as well as exudate from the theca membrane blood vessels. It provides an important microenvironment for the maturation of follicles and oocytes and serves as a medium for bidirectional communication between oocytes and surrounding cells. The detection of follicular fluid has the advantages of simple sampling and convenient detection.

[0041] This invention utilizes high-throughput sequencing to analyze the differential expression of lncRNAs in exosomes from follicular fluid of PCOS patients and healthy individuals, revealing high expression of lncLIPE-AS1 in exosomes from PCOS patients. Further validation of lncLIPE-AS1 expression in a larger population of PCOS patients and healthy individuals using qRT-PCR showed a significant increase in lncLIPE-AS1 in PCOS patients, with statistically significant differences. In in vitro cell experiments, cells were incubated with follicular fluid from PCOS patients for 48 hours, and exosomes were collected from the cell culture medium. The results showed increased lncLIPE-AS1 expression in exosomes incubated with follicular fluid from PCOS patients.

[0042] Furthermore, cell biology and mouse model experiments demonstrated that increased expression of exosome lncLIPE-AS1 can affect mouse embryonic development and upregulate the expression of estradiol and pregnenolone in granulosa cells, while also upregulating the expression levels of hormone regulatory genes CYP11A1 and CYP17A1.

[0043] Statistical analysis of serum hormone levels in PCOS patients and embryonic development indicators in assisted reproduction revealed a positive correlation between lncLIPE-AS1 expression levels and serum estradiol levels. As lncLIPE-AS1 expression levels increased, the number of oocytes obtained through assisted reproduction increased, while the rate of high-quality embryos and blastocyst formation decreased.

[0044] Based on the above results, the inventors believe that the biomarker lncLIPE-AS1 provided by this invention can be used to assess the quality of embryo development and predict the success rate of assisted reproduction in PCOS patients, and can also be used to screen for high-quality oocytes from PCOS patients and regulate the expression of steroid hormones in PCOS patients. Attached Figure Description

[0045] Figure 1Identification of exosomes in follicular fluid from patients with polycystic ovary syndrome and control patients. (A) Transmission electron microscopy (TEM) showed the morphology of exosomes in follicular fluid; (B) Western blot analysis confirmed the presence of exosome markers CD81, CD9 and TSG101; (C) Exosome size was determined by nanoparticle tracking analyzer.

[0046] Figure 2 Identification of lncLIPE-AS1 in exosomes from follicular fluid of patients with polycystic ovary syndrome (PCOS) and controls. (A) Transcriptome sequencing results of PCOS patients and controls; (B) qRT-PCR validation of lncLIPE-AS1 expression in PCOS and controls; (C) KGN cells incubated with exosomes isolated from follicular fluid, and qRT-PCR validation of lncLIPE-AS1 expression.

[0047] Figure 3 Effects of lncLIPE-AS1 overexpression on mouse embryonic development. (A) Embryonic development morphology of mice in the lncLIPE-AS1 overexpression group and the control group; (B) Statistical analysis of the proportions of 2-cell, 4-8-cell, morula and blastocyst embryos during embryonic development in mice in the lncLIPE-AS1 overexpression group and the control group.

[0048] Figure 4 Effects of lncLIPE-AS1 on steroid hormone synthesis in granulosa cells. (A) Pregnenolone levels in cell culture medium were detected by chemiluminescence immunoassay; (B) Estradiol levels in cell culture medium were detected by chemiluminescence immunoassay; (C) Expression levels of steroid metabolism pathway-related genes STAR, CYP11A1, CYP17A1 and HSD17B1 were measured.

[0049] Figure 5 A study on the correlation between lncLIPE-AS1 and serum hormones and IVF clinical outcomes. (A) Correlation between lncLIPE-AS1 expression level and serum estradiol; (B) Correlation between lncLIPE-AS1 expression level and oocyte number; (C) Correlation between lncLIPE-AS1 expression level and high-quality embryo rate; (D) Correlation between lncLIPE-AS1 expression level and blastocyst formation rate. r is the Pearson correlation coefficient. Detailed Implementation

[0050] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0051] This invention, through transcriptome sequencing data analysis, revealed the upregulation of the long non-coding RNA lncLIPE-AS1 in exosomes of follicular fluid from PCOS patients. Mouse model experiments showed that upregulation of lncLIPE-AS1 expression led to embryonic developmental arrest in mouse oocytes at the 4-cell, 8-cell, morula, and blastocyst stages. Cellular experiments showed that upregulation of lncLIPE-AS1 expression resulted in increased progesterone and estrogen levels in cell culture medium, and upregulation of the CYP11A1 and CYP17A1 genes, which regulate hormone expression. Further analysis of lncLIPE-AS1 in a PCOS patient population revealed a positive correlation between lncLIPE-AS1 expression and baseline levels such as serum estrogen and androgen, a positive correlation with the number of oocytes retrieved and the number of oocytes at the MII stage in assisted reproductive technology (ART) for PCOS patients, and a negative correlation with the rate of high-quality embryos and blastocyst formation in ART for PCOS patients. These experimental results suggest that lncLIPE-AS1 can serve as a target for predicting the success rate of ART in PCOS patients. The above conclusions are demonstrated through the following specific experiments:

[0052] 1. Inclusion of research subjects

[0053] This study was approved and conducted by the Ethics Committee of Zhongshan Hospital affiliated with Fudan University, and all sample collection was conducted with the consent of the patients. For the PCOS group, the phenotypic characteristics of PCOS patients needed to meet at least two of the following criteria defined in the Rotterdam Consensus (2004):

[0054] (1) Infrequent ovulation or anovulation (menstrual cycle longer than 35 days or fewer than 8 menstrual periods per year);

[0055] (2) Excessive androgens (manifested as total testosterone levels above 45 ng / dl or hirsutism);

[0056] (3) Ultrasound monitoring of ovarian morphology showed that more than 12 follicles with a diameter of less than 9 mm had polycystic characteristics.

[0057] Exclusion criteria: Age > 40 years, Body Mass Index (BMI) > 35 kg / m² 2 Patients with a basal follicle-stimulating hormone (FSH) level >12 mIU / mL; those who have had three or more unsuccessful in-vitro fertilization attempts; or those with systemic diseases or endocrine disorders are also excluded. Patients who have taken medications affecting hormone levels or glucose and lipid metabolism within 6 months prior to the start of treatment are also excluded.

[0058] Control group: Infertility due to male factors, undergoing in-vitro fertilization. The control group had normal ovarian morphology and function, menstrual cycles (26-35 days), androgen levels less than 45 ng / dL, and 6-10 follicles in both cavities.

[0059] After enrollment, patients were grouped and numbered, and their clinical data, test results, and embryo quality data from assisted reproductive technology were collected. This study was approved by the Ethics Committee of Zhongshan Hospital affiliated with Fudan University, and all participants signed informed consent forms.

[0060] 2. Experimental Methods

[0061] 2.1 Follicular fluid collection

[0062] Follicular fluid was collected from patients meeting the above criteria. Follicular fluid was collected from the first largest follicle punctured during IVF / ICSI treatment (via vaginal follicular aspiration). One follicle was collected from each patient. All follicles selected for this study were surrounded by the cumulus-oocyte complex. Follicular fluid was aspirated independently from each ovarian follicle. Follicular diameter was recorded at least three times using transvaginal ultrasound before puncture, and the average value was used. Follicular fluid was collected from follicles with a diameter greater than 15 mm. The collected follicular fluid was examined for erythrocytes. Follicular fluid containing erythrocytes was excluded from the study. The fluid was centrifuged at 3000 rpm for 15 min, and the supernatant follicular fluid was separated, aliquoted, and numbered.

[0063] 2.2 Exosome isolation and identification

[0064] Exosomes were extracted from follicular fluid using ultracentrifugation. Follicular fluid was centrifuged at 300g for 10 min to remove cells. The supernatant was centrifuged at 2000g for 10 min at 4℃ to remove dead cells. The supernatant was transferred to ultracentrifuge tubes and centrifuged at 100,000g for 2 h. The pellets were resuspended in PBS, filtered through a 0.22 μm filter, and centrifuged at 100,000g for 2 h. The pellets were resuspended in 200 μl PBS and stored at -80℃. Western blot analysis was used to detect the positive marker proteins CD81, CD9, and TSG101, and the negative marker protein Calnexin. Transmission electron microscopy (TEM) was used to identify the morphology of exosomes, and nanoparticle tracking analysis (NTA) was used to measure the size and concentration of exosomes.

[0065] 2.3 RNA extraction, library construction and sequencing

[0066] Total RNA was extracted from exosomes using TRIzol reagent (Life Technologies). Total RNA concentration was detected using a NanoDrop ND-2000. Sequencing data were analyzed using EdgeR to identify differentially expressed lncRNAs between the two groups. A lncRNA was considered differentially expressed if its expression level differed statistically significantly (p<0.05) by at least two-fold from the two experimental groups.

[0067] 2.4 Culture of human ovarian cancer granulosa cell line

[0068] Human oocyte granulosa cell line (KGN cells) was cultured in DMEM / F12 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C and 5% CO2. Two groups of KGN cells were established: (1) NC group; (2) lncLIPE-AS1 group. The lncLIPE-AS1 overexpression plasmid was constructed using the lentiviral GV658 vector, with the empty vector serving as a negative control. Cell density was observed under a microscope, and when the cell density reached 60-70%, the lncLIPE-AS1 overexpression plasmid and the empty vector were transfected, respectively. The plasmid was transfected into KGN cells using Lipofectamine 2000 reagent. Cells were collected 48 hours after transfection for further analysis.

[0069] 2.5 human follicular fluid incubation of ovarian cancer granulosa cell line

[0070] Follicular fluid samples were collected from PCOS patients and controls. After centrifugation at 5000g for 10 min, the supernatant was collected. The follicular fluid supernatant was mixed with cell culture medium at a ratio of 20%, and KGN cells were incubated for 48 h before gene expression levels were analyzed.

[0071] 2.6 Gene Expression Analysis

[0072] Real-time PCR was used to detect the levels of all mRNAs. A list of primers used for Real-time PCR is shown in Table 1. The expression of hormone metabolism-related genes was evaluated by detecting the expression of STAR, CYP11A1, CYP17A1, and HSD17B1. GAPDH was used as an internal reference gene.

[0073] Table 1 Primer set

[0074]

[0075] 2.7 Assessment of mouse embryonic development

[0076] B6D2 mice were provided by the Experimental Animal Research Center of Fudan University and housed in a special pathogen-free animal facility with a 12:12h light-dark cycle. This study was approved by the Animal Science Ethics Committee of Fudan University (Approval No.: 202105002S). Exosomes were collected from cell culture media of the empty vector overexpression group and the lncLIPE-AS1 overexpression group to prepare KSOM embryo (#M1450, Aibei Biotechnology) culture medium containing exosomes from both groups, with an exosome concentration of 100 μg / ml. The development of fertilized eggs from both groups of mice was observed. Superovulation was induced in 6-8 week old female mice by injecting 10 IU of pregnant mare serum gonadotropin (PMSG, Ningbo Sansheng), followed by injection of 10 IU of human chorionic gonadotropin (hCG, Ningbo Sansheng) 48 hours later. Fertilization was performed using 12-14 week old adult male mice (B6D2) 16-18 hours later. The next morning, the vaginal plug was examined, and the fertilized egg was retrieved from the ampulla of the fallopian tube. The fertilized egg was incubated in KSOM medium containing two groups of exosomes. The embryos were examined at 24, 48, 72, and 96 hours, and the percentages of 2-cell, 4-cell, morula, and blastocyst stages were calculated.

[0077] 2.8 Data Analysis

[0078] All experiments were repeated at least three times, and statistical analysis was performed using SPSS (Chicago, Illinois, USA). Comparisons between groups were performed using t-tests for normally distributed variables and Mann-Whitney U tests for non-normally distributed variables. Two-tailed unpaired t-tests were used to assess differences between groups. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0079] 3. Experimental Results

[0080] 3.1 Isolation and Identification of Exosomes

[0081] Exosomes were extracted from follicular fluid of PCOS patients and controls using ultracentrifugation. Transmission electron microscopy (TEM) showed that the follicular fluid exosomes had a round morphology and a size between 40 and 100 nm. Figure 1 A). Furthermore, western blot analysis confirmed the presence of exosome markers CD81, CD9, and TSG101. Figure 1 B). Nanoparticle tracking and analysis (NTA) measurements of exosome size distribution also revealed typical exosome structures. Figure 1 C). The results of TEM, NTA, and Western blot were consistent with the characteristics of exosomes, indicating that exosomes are indeed present in follicular fluid.

[0082] 3.2 Transcriptome sequencing and RT-PCR validation

[0083] Differential lncRNA expression analysis of follicular fluid exosomes from PCOS patients and controls using high-throughput sequencing revealed high expression of lncLIPE-AS1 in PCOS patients' follicular fluid exosomes. qRT-PCR validation of lncLIPE-AS1 expression in a larger population of PCOS patients and controls showed a significant increase in lncLIPE-AS1 in PCOS patients, with statistically significant differences. In in vitro cell experiments, cells were incubated with follicular fluid from PCOS patients for 48 hours, and exosomes were collected from the cell culture medium. The results showed increased lncLIPE-AS1 expression in exosomes incubated with follicular fluid from PCOS patients.

[0084] 3.3 Effects of lncLIPE-AS1 on mouse embryonic development

[0085] Because we observed elevated levels of lncLIPE-AS1 in the exosomes of follicular fluid in patients with PCOS, we wanted to further investigate the effects of increased lncLIPE-AS1 expression on oocyte maturation and embryonic development. We conducted in vivo experiments in mice. During mouse embryonic development, the lncLIPE-AS1 group showed delayed embryonic development (…). Figure 3 A), compared to the vector group, the proportions of 4-8 cells, morula, and blastocyst formation were lower ( Figure 3 B). The results indicate that increased expression of lncLIPE-AS1 in follicular fluid exosomes has a significant impact on oocyte embryonic development and maturation.

[0086] 3.4 Effects of lncLIPE-AS1 on steroid hormone synthesis in granulosa cells

[0087] Abnormal steroid metabolism can directly or indirectly affect follicular growth and function by altering the local environment of the follicular fluid. Therefore, we further analyzed the expression of steroid hormone synthesis-related genes (CYP11A, CYP17A, StAR, and HSD17B) and the metabolism of estradiol and pregnenolone. Using the KGN granulosa cell line, we found that overexpression of lncLIPE-AS1 significantly upregulated the levels of pregnenolone in the cell culture medium. Figure 4 A) and estradiol ( Figure 4 The concentration of B). Simultaneously, lncLIPE-AS1 overexpression significantly upregulated the expression levels of StAR, CYP11A1, and CYP17A1 (B). Figure 4 C). Based on the above findings, we have determined that increased lncLIPE-AS1 expression leads to a significant upregulation of the expression of genes related to steroid hormone synthesis and an increase in pregnenolone concentration. These results reveal that lncLIPE-AS1 can regulate the synthesis of steroid hormones in granulosa cells.

[0088] 3.5 Correlation between lncLIPE-AS1 and serum hormones and IVF clinical outcomes

[0089] Our study included 60 patients with polycystic ovary syndrome (PCOS). Follicular fluid was collected from these patients, and exosomes were extracted. The relationship between the follicular fluid exosome lncLIPE-AS1 qPCR value and the patients' basal hormone levels was detected. Simultaneously, the correlation between follicular fluid exosome lncLIPE-AS1 and clinical in vitro fertilization (IVF) outcomes in assisted reproduction, including the number of oocytes, the number of MII oocytes, the proportion of high-quality embryos cultured for day 3, and the proportion of embryos developing into blastocysts, was analyzed. The experimental data further demonstrate that lncLIPE-AS1 expression is positively correlated with basal estradiol levels. Figure 5 A). Pearson correlation analysis showed that the level of exosomal lncLIPE-AS1 mRNA was significantly positively correlated with the number of oocytes obtained ( Figure 5 B). With the increase of exosomal lncLIPE-AS1 mRNA levels, the rate of high-quality embryo formation ( Figure 5 C) and blastocyst formation rate ( Figure 5 D) Significantly decreased. The results indicate that lncLIPE-AS1 has certain predictive value for embryo quality in PCOS patients.

[0090] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of a substance for detecting the biomarker lncLIPE-AS1 in at least one of the following, wherein said lncLIPE-AS1 is derived from follicular fluid exosomes of PCOS patients: a1) Application in the preparation of products for predicting or assisting in the prediction of assisted reproductive success rates in patients with PCOS; a2) Application in the preparation of products for assessing or assisting in the assessment of the quality of assisted reproductive embryos in patients with PCOS.

2. The application according to claim 1, characterized in that, The products include testing reagents, testing kits, chips, membrane strips, or testing platforms.

3. The application according to claim 1, characterized in that, The substance used to detect the biomarker lncLIPE-AS1 is a primer that specifically amplifies lncLIPE-AS1 in the follicular fluid exosomes. The primer consists of the forward primer shown in SEQ ID NO.1 and the reverse primer shown in SEQ ID NO.2.

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