Methylation biomarkers for recurrent pregnancy loss and their applications

Abstract

The present invention belongs to the technical field of disease diagnosis and molecular biology, and specifically relates to methylation biomarkers for recurrent pregnancy loss and their applications. Specifically, the present invention analyzes the differential methylation characteristics of sperm of male partners of RPL patients and healthy control groups through whole genome DNA methylation chips, and then screens some differential methylation positions (DMPs) for further verification using targeted sulfite sequencing, indicating that multiple CpG sites in the RPL group show significant hypermethylation, especially H19 imprinted maternal expressed transcripts (H19), and DMP cg17985533 and region chr11:1997780-1997899 from H19 have consistent high methylation levels in RPL, and both sensitivity and specificity are high, so that they can be used as diagnostic biomarkers for RPL men, and have good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease diagnosis and molecular biology, and specifically relates to methylation biomarkers of recurrent pregnancy loss and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Recurrent pregnancy loss (RPL) refers to two or more consecutive spontaneous abortions before 20 weeks of pregnancy, which affects about 5% of couples. The cause of up to 50% of RPL is still unknown, which is called unexplained recurrent pregnancy loss (URPL). Initially, research on factors affecting RPL focused on women, including endocrine abnormalities, thyroid disease, hyperprolactinemia, uncontrolled diabetes, uterine abnormalities and some environmental factors. However, further studies have shown that some male factors also play an important role in the occurrence of RPL. At present, it is increasingly necessary to explore the male factors affecting RPL and their potential mechanisms.

[0004] The male factors of RPL mainly include chromosomal abnormalities (such as aneuploidy, Y chromosome microdeletion, chromatin integrity), sperm DNA fragmentation, viral infection, depression or stress, etc. Interestingly, related studies have shown that epigenetic mechanisms are also related to the male factors of RPL, mainly focusing on DNA methylation and RNA methylation. However, these studies on DNA methylation have certain limitations in further revealing the pathogenesis of male factors of RPL. For example, Irani et al. only determined the overall methylation level of sperm of male partners of women experiencing idiopathic RPL, but did not find methylation changes in specific genes. Some studies have only explored the methylation effects of a single gene or a few genes on male-related RPL, and have not systematically revealed the methylation characteristics of the whole genome. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides methylation biomarkers for recurrent pregnancy loss and their applications. The present invention analyzes the differential methylation characteristics of sperm of male partners of RPL patients and healthy control groups through a whole genome DNA methylation chip, and then screens some differential methylation positions (DMPs) for further verification using targeted sulfite sequencing, thereby successfully obtaining male methylation biomarkers that can be used to diagnose RPL. Based on the above research results, the present invention is completed.

[0006] Specifically, the present invention relates to the following technical solutions:

[0007] The first aspect of the present invention provides a methylation biomarker for recurrent pregnancy loss, wherein the methylation biomarker is selected from: CpG sites chr11:1997806, cg13210239, cg17985533 and chr11:1997780-1997899 region of gene H19.

[0008] Furthermore, the methylation biomarker is selected from the CpG site cg17985533 and the chr11:1997780-1997899 region of gene H19.

[0009] The second aspect of the present invention provides the use of a reagent for detecting the methylation level of the above-mentioned methylation biomarker in the preparation of a product for diagnosing or assisting in the diagnosis of recurrent pregnancy loss.

[0010] A third aspect of the present invention provides a system for diagnosing or assisting in diagnosing recurrent pregnancy loss, the system comprising:

[0011] i) an analysis unit, the analysis unit comprising: a detection reagent for determining the methylation biomarkers selected from the above in the test sample of the subject;

[0012] ii) an evaluation unit, comprising: analyzing and evaluating the occurrence of recurrent pregnancy loss in the female partner of the subject according to the methylation expression level of the methylation biomarker determined in i).

[0013] Beneficial technical effects of one or more of the above technical solutions:

[0014] The above technical scheme analyzed the differential methylation characteristics of sperm from male partners of RPL patients and healthy controls through whole-genome DNA methylation chips, and then screened some differentially methylated positions (DMPs) for further verification using targeted bisulfite sequencing, indicating that multiple CpG sites in the RPL group showed significant hypermethylation, especially the H19 imprinted maternally expressed transcript (H19), while the DMP cg17985533 and region chr11:1997780-1997899 from H19 had consistent high methylation levels in RPL with high sensitivity and specificity, thus being able to be used as diagnostic biomarkers for RPL men with good practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0016] Figure 1The whole genome DNA methylation analysis of sperm from 5 RPL patients and 5 healthy controls was performed using the Infinium MethylationEPIC BeadChip in the examples of the present invention. (A) Distribution of 742,742 methylation probes with different β values ​​in RPL patients (cases) and healthy controls (controls); (B) Principal component analysis of 5 cases and 5 controls using all methylation probes; (C) Unsupervised hierarchical heat map clustering of 960 differentially methylated sites (DMPs) between RPL and controls; (D) Manhattan plot showing the chromosomal distribution of all methylation probes; (E, F) Distribution characteristics of all hypermethylated and hypomethylated probes marked by CpG island regions (E) or genomic locations (F).

[0017] Figure 2 The differentially methylated sites and genes between RPL and the control in the present invention. (A) The bar graph shows the top 70 differentially methylated genes with the most differentially methylated sites, including hypermethylated (green) and hypomethylated (blue) probes; (B) The box plot shows the β value distribution of 11 H19 gene hyper-DMPs in the case group and the control group; (C) The KEGG enrichment results of differentially methylated genes, the size of the point indicates the number of differentially methylated genes, and the color of the point indicates the p value.

[0018] Figure 3 The differentially methylated sites of the candidate genes CFAP61, HLA-DQB1 and KDM4D detected by the chip in the embodiment of the present invention. Three hyper-DMPs in CFAP61 (A) and two hyper-DMPs in HLA-DQB1 (B) and KDM4D (C) were screened between RPL and control for subsequent verification.

[0019] Figure 4 The differentially methylated sites of candidate genes verified by targeted bisulfite sequencing in the present invention. (A) Three hyper-DMPs of H19 in RPL compared with the control (Con); (B) Two hyper-DMPs from KDM4D in RPL; (C) ROC analysis of DMP cg17985533 for diagnosing RPL.

[0020] Figure 5The differentially methylated region chr11:1997780-1997899 in H19 was verified by targeted bisulfite sequencing in the embodiment of the present invention. (A) Box plot showing the average β value of the chr11:1997780-1997899 region between RPL and control (Con); (B) The differential methylation status of 6 CpG sites in the chr11:1997780-1997899 region between RPL and control; (C) The read ratio of methylated haplotypes in the region between RPL and control, including CCCCCC, CCCTCC and CTTTTT; (D) ROC analysis of the region chr11:1997780-1997899 for diagnosing RPL. DETAILED DESCRIPTION

[0021] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0022] It should be noted that the terms used here are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should also be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof. If the experimental methods of specific conditions are not indicated in the following specific embodiments, the conventional methods and conditions of molecular biology in the art are usually followed, and such techniques and conditions are fully explained in the literature. See, for example, Sambrook et al., the techniques and conditions described in "Molecular Cloning: Laboratory Manual", or according to the conditions recommended by the manufacturer.

[0023] The present invention is further described in conjunction with specific examples. The following examples are only for explaining the present invention and are not intended to limit the content thereof. If the specific experimental conditions are not specified in the examples, they are usually carried out under conventional conditions or under conditions recommended by the sales company; the materials and reagents used in the examples, unless otherwise specified, can be purchased through commercial channels.

[0024] The term "biomarker" refers to "a characteristic that can be objectively detected and evaluated and can serve as an indicator of normal biological processes, pathological processes, or pharmacological responses to therapeutic interventions." For example, nucleic acid markers (also known as gene markers, such as DNA), protein markers, cytokine markers, antigen markers, antibody markers, etc. In the present invention, the biomarker is a methylation biomarker.

[0025] In a typical embodiment of the present invention, a methylation biomarker for recurrent pregnancy loss is provided, wherein the methylation biomarker is selected from: CpG sites chr11:1997806, cg13210239, cg17985533 and chr11:1997780-1997899 region of gene H19.

[0026] Furthermore, the methylation biomarker is selected from the CpG site cg17985533 and the chr11:1997780-1997899 region of gene H19.

[0027] In another specific embodiment of the present invention, there is provided use of a reagent for detecting the methylation level of the above-mentioned methylation biomarker in the preparation of a product for diagnosing or assisting in the diagnosis of recurrent pregnancy loss.

[0028] Furthermore, when the methylation biomarker is the chr11:1997780-1997899 region, the average methylation level of the region in H19 is used for diagnosis or auxiliary diagnosis of recurrent pregnancy loss.

[0029] The biological sample source of the methylation biomarker is male semen.

[0030] Among them, reagents for detecting the methylation level of methylation biomarkers include reagents required for bisulfite conversion and DNA methylation sequencing.

[0031] The product may be a detection kit, a detection device or a detection equipment.

[0032] In another specific embodiment of the present invention, a system for diagnosing or assisting in diagnosing recurrent pregnancy loss is provided, the system comprising:

[0033] i) an analysis unit, the analysis unit comprising: a detection reagent for determining the methylation biomarkers selected from the above in the test sample of the subject;

[0034] ii) an evaluation unit, comprising: analyzing and evaluating the occurrence of recurrent pregnancy loss in the female partner of the subject according to the methylation expression level of the methylation biomarker determined in i).

[0035] In another specific embodiment of the present invention, in the analysis unit, the subject is a male, and the sample to be tested is a male semen sample.

[0036] In another specific embodiment of the present invention, in the evaluation unit, the higher the methylation expression level of the methylation biomarker, the more likely the female partner of the subject will suffer from recurrent pregnancy loss. That is, based on the detection of the methylation expression level of the methylation biomarker, diagnosis or auxiliary diagnosis of whether the female partner of the subject suffers from recurrent pregnancy loss or prediction of whether the female partner of the subject suffers from the risk of recurrent pregnancy loss is achieved.

[0037] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0038] Example

[0039] Materials and Methods

[0040] Study subjects

[0041] From October 2021 to April 2024, 25 male RPL patients and 25 healthy controls were recruited in Jinan Maternal and Child Health Hospital Affiliated to Shandong First Medical University. The age of the RPL group was 26-35 years old, and the age of the control group was 22-40 years old. There was no statistically significant difference in the average age (p=0.4378) and BMI (p=0.0557) between the RPL group and the control group (Table 1). The RPL group must meet the following inclusion criteria: male partners of couples with two or more consecutive miscarriages before 20 weeks of pregnancy, and women with known risk factors for recurrent miscarriage, such as uterine malformation, hormonal disorders, infectious diseases, polycystic ovary syndrome, severe thrombosis, major chromosomal aberrations and other known causes, were excluded from the study. In addition, all men had no other diseases or received relevant treatments. The healthy control group was confirmed to be fertile, and the inclusion criteria included the following information: at least one healthy child, no history of spontaneous abortion, ectopic pregnancy, premature birth and stillbirth, and normal male chromosomes. In addition, both men and women were no older than 40 years old. All subjects signed informed consent for semen collection. This study was conducted in accordance with the guiding principles of the Declaration of Helsinki and approved by the Ethics Review Committee of Jinan Maternity and Child Health Hospital Affiliated to Shandong First Medical University.

[0042] Table 1 Clinical characteristics of 25 male RPL patients and 25 healthy controls (HC)

[0043]

[0044]

[0045]

[0046] Sample collection, DNA extraction and sulfite conversion

[0047] Semen samples were collected from male RPL patients and healthy controls after 2-7 days of abstinence and were subjected to semen analysis including sperm concentration, motility and morphology according to the WHO guidelines as described previously. Semen samples were washed, seminal plasma was removed, and somatic cells were further removed using somatic cell lysis buffer (0.1% sodium dodecyl sulfate and 0.5% Triton X-100 in diethyl pyrocarbonate water) as described previously. Sperm samples were then washed twice with PBS and stored at -80°C until genomic DNA extraction. Sperm genomic DNA was isolated using the HiPurA Sperm Genomic DNA Purification Kit (HiMedia, India) according to the manufacturer's instructions, and DNA purity and concentration were determined using a Quibt3.0 fluorometer (Thermo Fisher Scientific, USA). For sulfite conversion, 1 μg of sperm DNA was treated with the EZ DNA Methylation-Gold Kit (ZymoResearch, USA) according to the manufacturer's instructions. The sulfite-converted DNA was then used to detect DNA methylation levels by microarray and sequencing.

[0048] Infinium MethylationEPIC BeadChip Analysis

[0049] Transformed DNA from five RPL patients and five healthy controls was analyzed using the Infinium MethylationEPICBeadChip v1.0 (Illumina, USA), which contains more than 850,000 CpG sites according to the manufacturer's instructions, and the original IDAT files were obtained using an iScan SQ fluorescence scanner (Illumina, USA). The methylation data were analyzed using the ChAMP software package (v 2.18.2) in R4.3.3. The methylation level of each CpG was scored according to the fluorescence intensity ratio using a β value (β = methylated allele intensity (M) / (unmethylated allele intensity (U) + methylated allele intensity (M) + 100), which represents a continuous variable ranging from 0 (unmethylated) to 1 (fully methylated). First, we filtered the probes with p values ​​> 0.01, non-CpG probes, probes located on chromosomes X / Y, and SNP-related probes by ChAMP. Then, the β values ​​were normalized using BMIQ, and the batch effect of the normalized β values ​​was evaluated using singular value decomposition (SVD) analysis. Finally, a total of 742,000 CpG sites were used for differential analysis, and CpGs with |Δβ|≥0.1 and p values ​​≤0.05 were considered DMPs between RPL patients and controls.

[0050] Targeted bisulfite sequencing

[0051] The methylation levels of candidate target regions were detected and analyzed by targeted bisulfite sequencing (Shanghai Tianhao Biotechnology, China), which is called MethylTarget, a multiplex targeted CpG methylation analysis technology based on next-generation sequencing. Bisulfite conversion of genomic DNA was performed using the EZ DNA Methylation Kit (Zymo Research, USA) with sodium bisulfite treatment according to the manufacturer's protocol. Multiplex PCR was performed on 12 regions using Genesky optimized primer combinations, and after amplification and library construction, sequencing was performed on the Illumina HiSeq platform as a paired-end 150 bp (Illumina, USA). The methylation level of each CpG site was calculated as the percentage of methylated cytosine to total cytosine, and CpGs with a p-value ≤ 0.05 were used as DMPs between RPL patients and controls.

[0052] KEGG enrichment analysis

[0053] KEGG pathway enrichment analysis was performed using differentially methylated genes (DMGs) in the Infinium MethylationEPIC BeadChip results, and KEGG pathways with p values ​​< 0.05 were considered significantly enriched. KEGG pathway analysis was performed using the clusterProfiler package in R 4.3.3.

[0054] Statistical analysis

[0055] Multiple logistic regression analysis was performed on the methylation levels of candidate DMPs to obtain the predicted probability score for each sample. The probability score was subjected to receiver operating characteristic (ROC) analysis to obtain the ROC curve, and the area under the curve (AUC) was calculated to evaluate the discriminatory ability of candidate CpGs using the “pROC” R package. All statistical analyses were performed in R 4.3.3, and p values ​​< 0.05 were considered significant.

[0056] result

[0057] Genome-wide DNA methylation profiling identified differences in sperm methylation signatures between RPL patients and controls

[0058] Detailed clinical characteristics of male RPL patients (n = 25) and healthy controls (n = 25) are shown in Table 1. To reveal the DNA methylation changes occurring in sperm of RPL patients, we performed genome-wide DNA methylation analysis on five RPL patients and five healthy controls using the Infinium MethylationEPIC BeadChip. After quality control and normalization, approximately 742,000 methylation sites were subsequently subjected to differential analysis. These CpG positions were significantly enriched in high methylation levels (β values ​​close to 1) and low methylation levels (β values ​​close to 0) in both the case and control groups ( Figure 1 A). PCA plot shows relatively small differences between RPL patients and normal controls ( Figure 1 B). CpG sites with |Δβ|≥0.1 and p≤0.05 were defined as differentially methylated CpG sites (DMPs). A total of 960 DMPs were identified, of which 847 sites were hypermethylated (88.2%) and 113 sites were hypomethylated (11.8%) ( Figure 1 C). Manhattan plot showing the chromosomal locations of all methylation sites, including the above-mentioned DMPs ( Figure 1 D), hypermethylated and hypomethylated sites showed significantly different distribution characteristics between RPL and controls, including in island, N-Shelf / Shore, S-Shelf / Shore and opensea regions ( Figure 1 E). The genomic distribution of DMPs shows that high-DMPs and low-DMPs are located in intergenic regions and gene body regions, respectively ( Figure 1 F).

[0059] Next, we focused on the genes with the most DMPs, and the top genes included H19, TBC1D16, C7orf50, DLG2, JSRP1, and PFKP ( Figure 2 A). For example, the H19 imprinted maternally expressed transcript (H19) had 11 high-DMPs between RPL and control, including cg18362496, cg11735853, cg27300742, cg24605090, cg01539474, cg15886040, cg15922305, cg17985533, cg04975775, cg01977486, and cg11753499 ( Figure 2 B).

[0060] Then, pathway analysis of differentially methylated genes (DMGs) was performed, and KEGG results showed that drug metabolism-cytochrome P450, serotonergic synapse, arachidonic acid metabolism, retinol metabolism and linoleic acid metabolism were significantly enriched ( Figure 2C) Among them, cytochrome p450-related genes have been reported to be associated with RPL or spermatogenesis. Screening of RPL-related differentially methylated genes

[0061] Studies have found that H19 gene methylation is significantly associated with male hypospermatogenesis or male infertility. In addition, there is evidence that some of the DMGs we discovered through genome-wide screening are related to spermatogenesis and male infertility, including cilia and flagella-associated protein 61 (CFAP61), major histocompatibility complex class II DQβ1 (HLA-DQB1), and lysine demethylase 4D (KDM4D). Therefore, we first selected DMPs in spermatogenesis-related genes H19, CFAP61, HLA-DQB1, and KDM4D ( Figure 3 ) for methylation verification, among which H19 had 11 high-DMPs ( Figure 2 B), there are 3 high-DMPs in CFAP61 ( Figure 3 A), HLA-DQB1 and KDM4D both have 2 high-DMPs ( Figure 3 B, C).

[0062] Validation of DMPs by targeted bisulfite sequencing between RPL patients and controls

[0063] To validate the candidate DMPs of the above four RPL-related genes, we screened a total of 12 targeted regions (fragments) and compared the methylation differences of 89 CpGs by targeted bisulfite sequencing. Differential methylation analysis showed that there were statistical differences in 3 CpG sites in H19 and 2 CpG sites in KDM4D among 20 RPL patients and 20 healthy controls (p≤0.05)( Figure 4 A, B), while only three CpG sites in H19 (chr11:1997806, cg13210239, and cg17985533) had relatively high methylation levels, and the average methylation difference in spermatozoa from RPL patients was >10% compared with that in controls ( Figure 4 A). Among them, DMPcg17985533 was consistently hypermethylated in the RPL group by microarray and targeted sequencing methods ( Figure 4 A). We tested the performance of cg17985533. By ROC analysis, the AUC (0.7838), sensitivity (80%) and specificity (80%) of cg17985533 were ( Figure 4 C), suggesting that high methylation level of cg17985533 may be a potential biomarker for diagnosing RPL.

[0064] At the genomic fragment level, only the H19 region (chr11:1997780-1997899) was significantly hypermethylated in sperm from RPL patients compared with controls, with an average methylation difference of more than 10% (p=0.032, Figure 5 A), which includes the above three differential CpG sites in H19: chr11:1997806, cg13210239, and cg17985533. Compared with the control group, the methylation levels of all six CpG sites in the chr11:1997780-1997899 region in the RPL group were higher ( Figure 5 B). Interestingly, the abundance analysis of methylation haplotypes in this region showed that CCCCCC and CCCTCC haplotypes (C represents methylated cytosine and T represents unmethylated cytosine) were higher in the RPL group, while the CTTTTT haplotype was higher in the control group ( Figure 5 C). We also analyzed the performance of this region, and through ROC analysis, the AUC (0.8125), sensitivity (80%), and specificity (75%) of this region were ( Figure 5 D). This also confirmed that this region was significantly hypermethylated in the sperm of RPL-related males. Therefore, the average methylation level of this region in H19 can also serve as a diagnostic biomarker for RPL.

[0065] In summary, compared with the control group, the changes in the methylation levels of H19, CFAP61, HLA-DQB1, and KDM4D genes in RPL patients, especially emphasizing that the high methylation levels of CpG site cg17985533 from H19 and region chr11:1997780-1997899 can be considered as potential biomarkers for RPL.

[0066] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A methylation biomarker for recurrent pregnancy loss, characterized in that: The methylation biomarkers are selected from: CpG sites chr11:1997806, cg13210239, cg17985533 and chr11:1997780-1997899 region of gene H19.

2. The methylation biomarker according to claim 1, characterized in that The methylation biomarkers are selected from the CpG site cg17985533 and the chr11:1997780-1997899 region of gene H19.

3. Use of a reagent for detecting the methylation level of the methylation biomarker according to claim 1 or 2 in the preparation of a product for diagnosing or assisting in the diagnosis of recurrent pregnancy loss.

4. The use according to claim 3, characterized in that: When the methylation biomarker is the chr11:1997780-1997899 region, it is used for diagnosis or auxiliary diagnosis of recurrent pregnancy loss based on the average methylation level of the region in H19.

5. The use according to claim 3, characterized in that: The biological sample source of the methylation biomarker is male semen.

6. The use according to claim 3, characterized in that: Reagents for detecting the methylation level of methylation biomarkers include reagents required for bisulfite conversion and DNA methylation sequencing.

7. The use according to claim 3, characterized in that: The product is a detection kit, a detection device or a detection equipment.

8. A system for diagnosing or assisting in the diagnosis of recurrent pregnancy loss, characterized in that: The system comprises: i) an analysis unit, the analysis unit comprising: a detection reagent for determining the methylation biomarker selected from the methylation biomarker of claim 1 or 2 in a test sample of a subject; ii) an evaluation unit, the evaluation unit comprising: predicting whether the female partner of the subject has experienced recurrent pregnancy loss according to the methylation expression level of the methylation biomarker determined in i).

9. The system according to claim 8, characterized in that In the analysis unit, the subject is a male.

10. The system according to claim 8, characterized in that: The sample to be tested is a male semen sample.

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