Early pregnancy loss diagnosis marker and application thereof

Abstract

The invention discloses an early pregnancy loss diagnosis marker and application thereof. The marker provided by the invention comprises at least one of DLK1, SPTB, COL21A1 and SAR1A. The invention finds that DLK1, SPTB or COL21A1 and SAR1A can be respectively used as a molecular marker and a target of a complete grape tire, a partial grape tire and spontaneous abortion in an early pregnancy loss subtype, and a clinical sample is detected and further verified by a western blot method and an immunohistochemical method. The method can be used for guiding rapid diagnosis of early pregnancy loss, and has important practical application value.

Description

Technical Field

[0001] The present invention relates to markers for the diagnosis of early pregnancy loss, and particularly to biomarkers for the diagnosis of early pregnancy loss and their applications. Background Art

[0002] Early pregnancy loss (EPL) refers to an intrauterine pregnancy that cannot survive within 12 weeks of gestation, accounting for 50%-70% of all human pregnancies. The most common phenotypes of early intrauterine pregnancy loss are spontaneous abortion and hydatidiform mole (HM). Spontaneous abortion usually has immature villous edema with mucoid stromal degeneration (also known as hydropic abortion, HA), which is easily confused with hydatidiform mole. Therefore, the pregnancy products of EPL patients can be mainly divided into three subtypes pathologically: hydropic abortion (HA), complete hydatidiform mole (CHM), and partial hydatidiform mole (PHM). The accurate diagnosis and subtype classification of EPL are very important for clinical practice and scientific research because hydatidiform mole (HM) has a relatively high risk of progressing to gestational trophoblastic neoplasia (GTD). Approximately 15%-20% of CHM and <4% of PHM will progress to GTN, and patients need single-drug or multi-drug chemotherapy, as well as at least one year of hCG monitoring and follow-up; while HA rarely progresses to GTN (<0.0002%). In addition, the increased recurrence risk of the second or even third hydatidiform mole pregnancy is almost entirely related to CHM rather than PHM. However, currently, through histopathological examination, the misdiagnosis rates of CHM and PHM are as high as 30% and 71% respectively. Although STR genotyping has become the gold standard for the diagnosis of HM, this technology is very expensive and difficult to promote and apply on a large scale.

[0003] Therefore, there is an urgent need in the art to provide a convenient and reliable new method for the diagnosis of early pregnancy loss. Biomarkers play an increasingly important role in the early diagnosis or prognosis of various diseases and have potential clinical application value. For example, Chinese Patent CN113215255 discloses a method, which includes: obtaining a biological sample from a liver cancer subject and detecting the contents of multiple markers in the biological sample to predict the risk of the subject suffering from liver cancer. In addition, CN113804884 discloses the application of COX6C protein in the preparation of a kit for the diagnosis of early pregnancy loss, and this application includes: using the low-expressed COX6C protein in tissues as a marker for the diagnosis of early pregnancy loss. However, currently, there is still a lack of reports on the application of diagnostic markers for different subtypes of early pregnancy loss including hydatidiform mole, so it has important research significance and clinical application value. Summary of the Invention

[0004] The object of the present invention is to address the deficiencies of the prior art. After in-depth research, it is found that DLK1, SPTB, COL21A1 or SAR1A can be used as diagnostic markers for early pregnancy loss, and detection and further verification of clinical samples are carried out by Western blotting and immunohistochemistry. Specifically, the present invention includes the following contents:

[0005] In a first aspect of the present invention, a set of diagnostic markers related to early pregnancy loss is provided, and the markers can be used to detect different subtypes of early pregnancy loss. It includes at least one of DLK1, SPTB, COL21A1 and SAR1A.

[0006] In a second aspect of the present invention, there is provided an application of a detection reagent for the early pregnancy loss diagnostic marker in the preparation of an early pregnancy loss diagnostic kit.

[0007] In certain embodiments, for the application of the detection reagent for the early pregnancy loss diagnostic marker according to the present invention in the preparation of an early pregnancy loss diagnostic kit, the early pregnancy loss is at least one of complete hydatidiform mole, partial hydatidiform mole, edematous abortion and normal pregnancy.

[0008] In certain embodiments, for the application of the detection reagent for the early pregnancy loss diagnostic marker according to the present invention in the preparation of an early pregnancy loss diagnostic kit, for the application of DLK1, SPTB, COL21A1 and SAR1A as early pregnancy loss diagnostic markers, the sample is a chorionic tissue sample after uterine curettage.

[0009] In certain embodiments, for the application of the detection reagent for the early pregnancy loss diagnostic marker according to the present invention in the preparation of an early pregnancy loss diagnostic kit, the expression level of the protein is obtained by measuring at least one of Western blotting and immunohistochemistry.

[0010] In certain embodiments, for the application of the detection reagent for the early pregnancy loss diagnostic marker in the preparation of an early pregnancy loss diagnostic kit, the reagent includes anti-DLK1, SPTB, COL21A1 and SAR1A antibodies for detecting the expression of at least one of DLK1, SPTB, COL21A1 and SAR1A proteins.

[0011] In certain embodiments, for the application of the detection reagent for the early pregnancy loss diagnostic marker in the preparation of an early pregnancy loss diagnostic kit, the diagnosis of the kit includes the following steps:

[0012] (1) A step of obtaining a measurement value by measuring the expression level of at least one protein among DLK1, SPTB, COL21A1, and SAR1A markers in a sample from a subject using a detection reagent;

[0013] (2) A step of comparing the measurement value with a control value;

[0014] (3) If the DLK1 measurement value is higher than the control value, the subject is predicted to have a higher risk of complete hydatidiform mole; if the SPTB or COL21A1 measurement value is higher than the control value, the subject is predicted to have a higher risk of partial hydatidiform mole; if the SAR1A measurement value is higher than the control value, the subject is predicted to have a higher risk of normal pregnancy.

[0015] The beneficial effect of the present invention is that through in-depth research, it is found that DLK1, SPTB or COL21A1, and SAR1A are significantly highly expressed in complete hydatidiform mole, partial hydatidiform mole, and normal pregnancy in different subtypes of early pregnancy loss respectively, and are very likely to become clinical targets for early pregnancy loss; by detecting the expression level of at least one of DLK1, SPTB, COL21A1, and SAR1A proteins in the sample tissue of the subject, it is possible to effectively determine whether the subject has a certain subtype of early pregnancy loss. The present invention has low cost and convenient operation, which is conducive to further clarifying the occurrence mechanism and signal pathway of different subtypes of early pregnancy loss, and has application prospects and theoretical value in medicine. Description of the Drawings

[0016] Figure 1 is the volcano plot of differential proteins in each group in Example 1 of the present invention.

[0017] Figure 2 is the statistical chart of the relative expression levels of targeted mass spectrometry of diagnostic markers DLK1, SPTB, COL21A1, and SAR1A in Example 2 of the present invention;

[0018] Figure 3 is the hematoxylin-eosin staining and p57 immunohistochemical staining (A), STR gene typing results (B) of the pathological characteristics of each group of patients in Example 3 of the present invention;

[0019] Figure 4 is the result diagram of verifying diagnostic markers DLK1, SPTB, COL21A1, and SAR1A by protein immunization method in Example 3 of the present invention;

[0020] Figure 5It is the result diagram of immunohistochemistry method for verifying diagnostic markers DLK1, SPTB, COL21A1, and SAR1A in Example 3 of the present invention. In the figure, A is the immunohistochemistry result diagram of different types of early pregnancy, scale bar, 50μm; B is the statistical chart of immunohistochemical scores of each diagnostic marker in different types of early pregnancy. Detailed implementation manners

[0021] The technical solutions of the present invention will be further described below through specific implementation manners. The present invention can be implemented in many other ways different from those described herein. Those skilled in the art can make similar improvements without departing from the connotation of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed below.

[0022] The term "marker" refers to any gene or protein whose expression level at the tissue level is altered compared to the expression level of a normal or control group.

[0023] The term "kit" also includes substrates corresponding to control samples and markers. In addition, the kit may also include various reagents required for protein extraction, reaction, blocking, color development, etc., including but not limited to: extraction solution, enzyme, control solution, blocking solution, color development solution, washing solution, etc. In addition, the kit also includes an instruction manual and / or image analysis software.

[0024] The term "differentially expressed gene" refers to a gene or protein that is expressed at a higher or lower level in samples of a comparison group (CHM group, PHM group, HA group) compared to a normal or control group (NC group).

[0025] The present invention can measure gene or protein expression using any method known in the art. Those skilled in the art should understand that the means for measuring gene or protein expression is not an important aspect of the present invention, and the expression level of biomarkers can be detected at the genomic level or transcriptomic level.

[0026] In the first aspect of the present invention, a group of diagnostic markers related to early pregnancy loss is provided. Different subtypes of early pregnancy loss can be detected and distinguished according to the expression levels of these markers. It includes at least one of DLK1, SPTB, COL21A1, and SAR1A.

[0027] In the second aspect of the present invention, a protein such as DLK1, SPTB, COL21A1, or SAR1A is provided as a target in the preparation of a diagnostic kit for early pregnancy loss, which contains anti-DLK1, SPTB, COL21A1, and SAR1A antibodies for detecting the expression levels of at least one of DLK1, SPTB, COL21A1, and SAR1A proteins.

[0028] The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, nanobodies, humanized antibodies or fully human antibodies. The antibodies of the present invention can also be single-chain antibodies. In the present invention, the modifiers of the antibodies include chemical modifiers and conjugates of antibodies and other materials. Among them, examples of the chemical modifiers include, but are not limited to, acetylation, acylation, ADP-ribosylation, amidation, cross-linking cyclization, disulfide bond formation, demethylation, covalent cross-linking, cysteinylation, pyroglutamation, formylation, γ-carboxylation, glycosylation, GPI anchoring, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolysis and phosphorylation of the antibodies, etc. Among them, examples of the conjugates include, but are not limited to, conjugates with nano-polymeric materials, magnetic beads, etc.

[0029] In the present invention, the sample refers to the villous tissue after uterine curettage from a subject, and its examples include, but are not limited to, fresh tissue, fresh frozen tissue, paraffin-embedded tissue. The preferred sample is fresh tissue.

[0030] In the present invention, the control value can be a specific numerical point, or a numerical interval or range. The control value can be a value obtained by statistically analyzing a large amount of clinical data of early pregnancy loss, or a value of normal tissue or control tissue. The control value can be a value representing the absolute amount of protein expression, for example, or a value representing the relative amount of protein expression, for example.

[0031] In the present invention, the reagents for measuring the protein expression levels of DLK1, SPTB, COL21A1 and SAR1AC in a sample from a subject to obtain measurement values can be known reagents, and those skilled in the art can freely design according to actual needs.

[0032] Experimental materials

[0033] The hematoxylin-eosin (HE) staining kit was purchased from Beijing Solarbio Science & Technology Co., Ltd., product number G1120;

[0034] The protease inhibitor and phosphatase inhibitor were purchased from Beyotime Co., Ltd., product number P1045;

[0035] The RIPA protein lysate was purchased from Beyotime Co., Ltd., product number P0013B;

[0036] The bromophenol blue cell lysate was purchased from Thermo Fisher Scientific, product number 2565020;

[0037] The precast gradient gel was purchased from Thermo Fisher Scientific, product number NP0323;

[0038] The MOPS electrophoresis buffer was purchased from Thermo Fisher Scientific, product number 2495537;

[0039] Protein marker was purchased from Abotek Biotechnology Co., Ltd., brand number RM02943;

[0040] BCA protein content determination kit was purchased from Biyuntian Co., Ltd., brand P0010S;

[0041] Rapid blocking solution was purchased from Biyuntian Co., Ltd., brand P0252;

[0042] Western blotting SPTB antibody was purchased from Hangzhou Jingjie Biotechnology Co., Ltd., brand number PTM-5744;

[0043] Immunohistochemistry SPTB antibody was purchased from Sigma-Aldrich, brand number HPA003394;

[0044] COL21A1 antibody was purchased from Antibody LSBio, brand number LS-C166924;

[0045] DLK1 antibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., brand number 10636-1-AP;

[0046] GAPDH antibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., brand number 10494-1-AP;

[0047] SAR1A antibody was purchased from Wuhan Tri-Eagle Biotechnology Co., Ltd., brand number 22291-1-AP;

[0048] TBST was purchased from Beijing Solebow Technology Co., Ltd., brand number T1082;

[0049] The immunohistochemistry kit was purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., brand PV-9000.

[0050] instrument

[0051] Centrifuge: normal temperature desktop low-speed centrifuge, manufacturer Eppendorf;

[0052] Upright microscope: manufacturer: Olympus Corporation, Japan;

[0053] Electrophoresis apparatus: Mini Gel Tank electrophoresis apparatus, manufactured by Thermofisher, USA;

[0054] Transfer tank: Manufacturer: Bio-Rad, USA;

[0055] Gel imaging analysis system: manufacturer: Bio-Rad, USA;

[0056] Multifunctional microplate reader: Manufacturer: Biotek, USA.

[0057] Example 1: Determination of candidate markers

[0058] To search for markers that can be used to predict different subtypes of early pregnancy loss, first, fresh frozen villous tissue samples from 10 cases of CHM, 6 cases of PHM, 10 cases of HA patients, and 10 cases of NC (no abnormal production history, denying common abortion-causing factors) healthy women after uterine curettage for pregnancy products were subjected to 4D high-throughput Label-free proteomics sequencing and analysis. The DESeq2 software package was used to analyze the data. P values < 0.05 and log2FC > -0.5 or < -0.5 were selected to screen out significantly differentially expressed proteins representing the CHM, PHM, HA, and NC groups respectively. Figure 1 Volcano plots of differential genes in each group were shown. Through screening and verification by combining GO, KEGG analysis, etc., 4 significantly differentially expressed proteins, namely DLK1, SPTB, COL21A1, and SAR1A proteins, were finally determined as markers for further research.

[0059] Example 2: Identification of DLK1, SPTB, COL21A1, and SAR1A

[0060] Fresh frozen villous tissue samples from 10 cases of CHM, 9 cases of PHM, 10 cases of HA, and 10 cases of NC groups were taken for parallel reaction monitoring (PRM) targeted mass spectrometry verification. Peptides of the target proteins (Table 1) were designed and sent to Jingjie Company in Hangzhou for sequencing to determine the protein expression consistency of the candidate markers.

[0061] Table 1: Peptide information for PRM analysis.

[0062]

[0063]

[0064] Figure 2 The relative expression levels of 4 protein markers verified by targeted mass spectrometry were shown, with significant differences among different groups.

[0065] Example 3: Expression differences of DLK1, SPTB, COL21A1, and SAR1A in different subtypes of EPL patients

[0066] 3.1 Sample collection

[0067] Subject inclusion criteria:

[0068] (1) Hydatidiform mole group: Clinically identified by transvaginal color Doppler ultrasound features ("honeycomb-like" / "snowflake-like" heterogeneous echo), elevated blood hCG level, and intraoperative findings (vesicular, grape-cluster-like tissue);

[0069] (2) Edematous abortion group: Transvaginal color Doppler ultrasound indicated abnormal embryo development, excluding autoimmune diseases, genital tract structural abnormalities, and male factors;

[0070] (3) Control group NC group: Healthy women with no history of abnormal childbirth, denying common abortion-causing factors.

[0071] According to the above criteria, chorionic and decidual tissues of 33 cases of CHM, 33 cases of PHM, 33 cases of HA, and 33 cases of the NC group were collected from the First Affiliated Hospital of Zhejiang University School of Medicine. Informed consent was obtained from all patients and their families, and informed consent forms were signed. As Figure 3 shown, all cases were accurately diagnosed and typed by pathology, p57 immunohistochemical analysis, and STR gene typing.

[0072] 3.2 Verification of marker expression by Western blotting

[0073] (1) Protein extraction method

[0074] Take an appropriate amount of tissue and put it into a 1.5 ml grinding tube. Add 2 beads of 2 mm and 4 mm grinding beads to each tube. According to the ratio of 20 mg / ml, add 60 μl of protein lysate containing protease inhibitor and phosphatase inhibitor to the grinding tube. Place the grinding tube in a grinding machine pre-cooled at 4°C, with an operating frequency of 40 - 60 Hz and an operating time of 60 s. Grind 2 to 3 times until the tissue is fully ground. Centrifuge at 13000×g for 15 minutes at 4°C, aspirate the supernatant into a new EP tube, measure the protein concentration using the BCA method, dilute the protein with PBS solution to make the protein concentration in each tube consistent, add cell lysate containing bromophenol blue, and denature at 99°C for 10 min, then place it in a -30°C refrigerator for later use.

[0075] (2) Loading and electrophoresis

[0076] Take a 4 - 12% Bis-Tris precast gradient gel (Invitrogen, NP0323) and place it in the electrophoresis tank. Add 1×MOPS electrophoresis buffer. Use a pipette to aspirate the protein sample and add it to the gel wells. Additionally, add a protein Marker (Invitrogen, 26616) for indication. Electrophorese at a constant voltage of 110 V for 90 min.

[0077] (3) Transfer membrane:

[0078] a. Cut a 6 cm × 8 cm PVDF membrane and soak it in methanol for 1 min;

[0079] b. Prepare the transfer membrane solution: 100 ml of 10× electrotransfer buffer (Solarbio, D1060) + 700 ml of pure water + 200 mL of methanol;

[0080] c. Immerse the foam, filter paper, and membrane in the prepared transfer membrane solution;

[0081] d. Stack them in the order of foam - filter paper - membrane - glue - filter paper - foam, gently expel the air bubbles between the membrane and the glue, clamp them, and place them in the transfer tank;

[0082] e. Fill the transfer tank with transfer buffer and transfer at 350 mA for 75 min.

[0083] (4) Blocking:

[0084] Cover the entire membrane with the rapid blocking solution, place the membrane in it, and block at room temperature on a shaker for 15 - 30 min.

[0085] (5) Membrane cutting: Cut the bands according to the corresponding molecular weights in the instructions of SPTB, COL21A1, DLK1, GAPDH, and SAR1A;

[0086] (6) Antigen - antibody reaction:

[0087] a. Dilute the primary antibodies (SPTB, COL21A1, DLK1, GAPDH, SAR1A) with the primary antibody diluent, place the cut membrane into the corresponding primary antibody incubation solution for incubation, and incubate overnight on a shaker at 4°C;

[0088] b. Recover the primary antibody, wash with TBST for 7 min, 3 times in total, dilute the secondary antibody (1:2000) with TBST, and incubate for 1 h;

[0089] c. Wash with TBST for 10 min, 3 times in total, and develop in the Bio - Rad imaging system.

[0090] The results are as Figure 4 shown. Verify the expression levels of proteins such as SPTB, COL21A1, DLK1, and SAR1A in the villous tissues of early pregnancy loss in humans by Western blotting, using GAPDH as an internal reference. The results are as Figure 4 shown. SPTB and COL21A1 are highly expressed in the PHM group and lowly expressed in the other groups; DLK1 is highly expressed in the CHM group and lowly expressed in the other groups; SAR1A is highly expressed in the NC group and lowly expressed in the other tissues. It shows that the expression changes of DLK1, SPTB / COL21A1, and SAR1A can be used as diagnostic markers for CHM, PHM, and NC respectively.

[0091] 3.3 Verification of marker expression by immunohistochemistry

[0092] (1) Deparaffinization and hydration: Place the paraffin sections in fresh xylene, soak for 10 min × 3 times; after removing the excess liquid, place them in absolute ethanol, soak for 3 min × 3 times; after removing the excess liquid, place them in 95 vol% ethanol, soak for 3 min × 2 times; after removing the excess liquid, place them in 75 vol% ethanol, soak for 3 min × 2 times; rinse with distilled water for 1 min and place them in PBS buffer.

[0093] (2) Antigen retrieval: The method of microwave radiation repair was used for antigen retrieval; the section rack was placed in the repair box in the microwave oven, 200 mL of EDTA buffer solution with a pH value of 9.0 was added, and it was heated in the microwave oven at high fire for 5 min and medium-high fire for 15 min. The section rack was taken out of the repair box and rinsed with running tap water until cooled.

[0094] (3) Blocking endogenous peroxidase: The tissue area was circled with an immunohistochemical pen for each section, 50 μl of endogenous peroxidase blocker was added dropwise to each section, and it was incubated at room temperature for 10 minutes; rinsed with PBS buffer solution for 3 minutes × 3 times.

[0095] (4) Primary antibody incubation: According to the size of the tissue, 100 μL or an appropriate amount of primary antibody (SPTB, COL21A1, DLK1, SAR1A) was added dropwise, and it was incubated at 4 °C overnight; rinsed with PBS buffer solution for 3 minutes × 3 times.

[0096] (5) Adding reaction enhancer solution: 100 μL or an appropriate amount of reaction enhancer solution was added dropwise, and it was incubated at 37 °C for 20 minutes; rinsed with PBS buffer solution for 3 minutes × 3 times.

[0097] (6) Adding 100 μL or an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer, and it was incubated at 37 °C for 20 minutes; rinsed with PBS buffer solution for 3 minutes × 3 times.

[0098] (7) DAB color development: An appropriate amount of freshly prepared DAB or AEC color development solution was added, and it was incubated at room temperature for 5 - 8 minutes. Observed under the microscope, the reaction was terminated when the staining was appropriate, and rinsed with tap water.

[0099] (8) Counterstaining with hematoxylin: The section rack was immersed in hematoxylin for 2 min, rinsed with tap water, placed in 1% hydrochloric acid alcohol (1% hydrochloric acid ethanol) for differentiation for 10 s until the section faded (to light blue - red), rinsed with tap water, placed in ammonia water solution for bluing, rinsed with running water, and observed under the microscope that the cell nuclei were clearly stained.

[0100] (9) Dehydration and clearing: The sections were successively placed in 75 vol% ethanol, 95 vol% ethanol, absolute ethanol I, absolute ethanol II, xylene I, xylene II, and xylene III for dehydration and clearing for 2 min each, and air-dried in a fume hood.

[0101] (10) Sealing the sections: Sealed with neutral balsam, stored at room temperature, and observed under the microscope.

[0102] (11) Result interpretation: Two experienced pathologists independently reviewed the slides and scored them. The specific scoring criteria adopted a semi - quantitative scoring method, that is, the product of the staining intensity (A) and the staining positive density (B) (A×B). The detailed scoring rules for staining intensity and staining density are shown in Table 2 below. Five high - power fields were randomly observed for each case (counted using a high - power field machine). The scoring was performed by 2 pathologists. When the scores were inconsistent, a third pathologist was asked to review. They were unaware of the research design and grouping.

[0103] Table 2: Semi - quantitative scoring criteria for immunohistochemistry.

[0104]

[0105] From Figure 5 (A) Figure, it can be seen that compared with other groups, the specific staining (brown) of DLK1 in the villous tissue of CHM patients becomes significantly darker, indicating that the expression of DLK1 in the villous tissue of CHM patients increases significantly; similarly, the expressions of SPTB and COL21A1 in the villous tissue of PHM patients increase significantly, and the expression of SAR1A in the villous tissue of normal pregnancy increases significantly. From Figure 5 (B)'s semi - quantitative scoring, it can be determined that these expression differences are all significant.

[0106] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. An early pregnancy loss diagnostic marker, characterized in that, Comprising at least one of DLK1, SPTB, COL21A1, and SAR1A.

2. Use of a detection reagent for an early pregnancy loss diagnostic marker according to claim 1 in the preparation of an early pregnancy loss diagnostic kit.

3. The application according to claim 2, wherein The early pregnancy loss is at least one of complete hydatidiform mole, partial hydatidiform mole, edematous abortion, and normal pregnancy.

4. The application according to claim 2, characterized in that, The detection reagent for the early pregnancy loss diagnostic marker is a reagent for detecting the expression level of the early pregnancy loss diagnostic marker in a subject sample, and the sample is a villous tissue sample after uterine curettage.

5. The application according to claim 4, characterized in that, The expression level is obtained by measurement using at least one of Western blotting and immunohistochemistry.

6. The application according to claim 2, wherein The detection reagent comprises an antibody for detecting the expression of at least one protein among DLK1, SPTB, COL21A1, and SAR1A.

7. The application according to claim 2, wherein The early pregnancy loss diagnostic marker includes DLK1, SPTB, COL21A1, and SAR1A. If the measured value of DLK1 is higher than the control value, the subject is predicted to be at risk of having a complete hydatidiform mole; if the measured value of SPTB or COL21A1 is higher than the control value, the subject is predicted to be at risk of having a partial hydatidiform mole; if the measured value of SAR1A is higher than the control value, the subject is predicted to be a normal pregnancy.

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