Circular RNA diagnostic markers for placental implantation lineage diagnosis and application thereof

Abstract

The application relates to circular RNA diagnostic markers for placenta implantation lineage diagnosis and application thereof. The circular RNA diagnostic markers comprise one or two of circPHACTR4 and circZMYM4, and the cDNA sequences of the circPHACTR4 and the circZMYM4 are respectively shown as SEQ ID NO: 1 and SEQ ID NO: 2. The two circular RNA diagnostic markers are significantly up-regulated in PAS peripheral blood, and can be used as PAS diagnostic markers alone or in combination to realize high-sensitivity, specificity and objective detection of placenta implantation lineage. The sensitivity of circPHACTR4 alone is 74%, the sensitivity of circZMYM4 alone is 68.4%, the two circRNAs are combined to have a good prediction effect on PAS, and the sensitivity and specificity can reach 82% and 88.9% in combination with the diagnosis results of clinical indexes and B ultrasonic waves, so that the precision of the prenatal diagnosis of PAS is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of disease diagnostic markers, in particular to a circular RNA diagnostic marker for placenta accreta spectrum lineage diagnosis and application thereof. BACKGROUND

[0002] Placenta accreta spectrum (PAS) refers to a disease in which placental trophoblast cells abnormally invade the myometrium of the uterine wall, with a mortality rate of up to 7%. Because a large amount of bleeding occurs when the placenta is detached, it can lead to multiple organ failure, disseminated intravascular coagulation, hysterectomy and even death, making it one of the main causes of morbidity and mortality in pregnant women. Accurate prenatal diagnosis can enable doctors to prepare surgical plans in advance, thereby reducing the risk of the disease and achieving better prognosis. Currently, the main diagnostic method for PAS in clinical practice is B-ultrasound, but the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of B-ultrasound are low, being 53.5%, 88.0%, 82.1%, 64.8% and 64.8%, respectively. Moreover, the diagnosis of B-ultrasound is dependent on the accuracy of the equipment and is highly related to the experience of the doctor, and the diagnostic results have a certain subjectivity, leading to the fact that in clinical practice, as many as one-third to two-thirds of PAS cases are still not diagnosed before delivery, and the diagnosis rate of PAS is even lower in some underdeveloped areas. In order to overcome this clinical challenge, there is an urgent need for a biomarker with high sensitivity, specificity and objective results, especially a non-invasive biomarker. However, after decades of research, no reliable markers in blood or urine have been developed for the diagnosis of PAS in clinical practice.

[0003] Circular RNA (circRNA) is a type of RNA isomer that is widely and tissue-specifically expressed in animals, and its expression level is usually independent of the corresponding linear mRNA. CircRNA is very stable, and some circRNAs have high expression abundance and can be stably detected in peripheral blood, thus having the potential to be used as a blood biomarker for the diagnosis of certain diseases. At present, some circular RNAs have been found to be used as markers for the diagnosis of certain diseases, such as: Chinese patent CN 109852684 A discloses the application of circular RNA hsa_circ_0003764 as a diagnostic marker for intrauterine adhesion, Chinese patent CN111593116A discloses that the circular RNA hsa_circRNA_100737 is used for diagnosing recurrent miscarriage, and Chinese patent CN108796086A discloses the application of circular RNA circBCBM1 in the risk prediction and clinical diagnosis of breast cancer brain metastasis. However, there is no related report on circular RNA molecules as diagnostic markers for placenta accreta spectrum, and the further promotion of PAS diagnosis technology is undoubtedly an urgent need. SUMMARY

[0004] The present application aims to overcome the above-mentioned defects and deficiencies in the prior art, and provide a circular RNA diagnostic marker for placenta implantation lineage diagnosis.

[0005] Another object of the present application is to provide a primer set for detecting the circular RNA diagnostic marker.

[0006] Still another object of the present application is to provide an application of the circular RNA diagnostic marker in preparing a placenta implantation lineage diagnosis product.

[0007] Still another object of the present application is to provide an application of the primer set for detecting the circular RNA diagnostic marker.

[0008] Still another object of the present application is to provide a placenta implantation lineage diagnosis product.

[0009] The above-mentioned objects of the present application are realized by the following technical solutions:

[0010] In a first aspect, the present application provides a circular RNA diagnostic marker for placenta implantation lineage diagnosis, wherein the circular RNA diagnostic marker comprises one or both of circPHACTR4 and circZMYM4, and the RNA sequences of circPHACTR4 and circZMYM4 are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

[0011] The two circular RNA diagnostic markers of circPHACTR4 and circZMYM4 are significantly up-regulated in PAS peripheral blood, and can be used alone or in combination as a PAS peripheral blood diagnostic marker to achieve high sensitivity, specificity and objective detection of placenta implantation lineage.

[0012] In some embodiments, the circular RNA diagnostic marker comprises circPHACTR4.

[0013] In some embodiments, the circular RNA diagnostic marker comprises circZMYM4.

[0014] Preferably, the circular RNA diagnostic marker consists of circPHACTR4 and circZMYM4, and the two circRNAs have a good prediction effect on PAS in combination.

[0015] In a second aspect, the present application provides a primer set for detecting the circular RNA diagnostic marker, wherein the primer set comprises a primer pair for detecting circPHACTR4 and / or circZMYM4, and wherein the primer pair for detecting circPHACTR4 has sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; and the primer pair for detecting circZMYM4 has sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0016] In a third aspect, the present application provides use of the circular RNA diagnostic marker in the preparation of a placenta implantation lineage diagnostic product.

[0017] In a fourth aspect, the present application provides use of the primer set for detecting the circular RNA diagnostic marker in the preparation of a placenta implantation lineage diagnostic product.

[0018] Further, the diagnostic product is a reagent, a kit, a chip, or the like, and any product that can be used to detect the circular RNA diagnostic marker is within the scope of the present application.

[0019] In a fifth aspect, the present application provides a placenta implantation lineage diagnostic product comprising a reagent for detecting the expression level of a circular RNA diagnostic marker, wherein the circular RNA diagnostic marker comprises one or both of circPHACTR4 and circZMYM4.

[0020] Further, the reagent comprises a primer set for detecting the circular RNA diagnostic marker.

[0021] Further, the primer set comprises a primer pair for detecting circPHACTR4 and / or circZMYM4, and wherein the primer pair for detecting circPHACTR4 has sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively; and the primer pair for detecting circZMYM4 has sequences as shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.

[0022] Further, when the circular RNA diagnostic marker comprises circPHACTR4 and circZMYM4, RPLP0 is used as an internal reference gene for detecting the expression level of circPHACTR4 and circZMYM4, and a formula is fitted according to the QPCR result for ROC curve analysis.

[0023] C PAS = 0.7784 x Exp circPHACTR4 + 2.1975 x Exp circZMYM4 + 1.1064 x ultrasound - 4.2773,

[0024] In the above formula, C pas represents the probability of the detected person suffering from PAS disease, Exp circPHACTR4 is the relative expression level of circPHACTR4 detected by QPCR, Exp circZMYM4 represents the relative expression level of circZMYM4 detected by QPCR, and ultrasound represents the B-ultrasound diagnosis result, if the B-ultrasound diagnosis result is PAS negative, the ultrasound value is 0, and if the B-ultrasound diagnosis result is PAS positive, the ultrasound value is 1.

[0025] The application also provides a method for diagnosing the placenta implantation lineage using the circular RNA diagnostic marker, which is to detect the expression amount of the two circular RNA diagnostic markers by QPCR, chip detection or high-throughput sequencing on the maternal peripheral blood RNA before the pregnant woman gives birth, and if the expression of the placenta-derived circRNA is found to be increased, the probability of placenta implantation of the patient is higher.

[0026] Preferably, the expression amount of circPHACTR4 and circZMYM4 is detected by the primer set, RPLP is used as the internal reference gene, and the following formula is used according to the QPCR result to perform ROC curve analysis:

[0027] C PAS = 0.7784*Exp circPHACTR4 + 2.1975*Exp circzMYM4 + 1.1064*ultrasound-4.2773,

[0028] In the above formula, C pas represents the probability of the detected person suffering from PAS disease, Exp circPHACTR4 is the relative expression level of circPHACTR4 detected by QPCR, Exp circZMYM4 represents the relative expression level of circZMYM4 detected by QPCR, and ultrasound represents the B-ultrasound diagnosis result, if the B-ultrasound diagnosis result is PAS negative, the ultrasound value is 0, and if the B-ultrasound diagnosis result is PAS positive, the ultrasound value is 1.

[0029] Compared with the prior art, the application has the following beneficial effects:

[0030] The application provides diagnostic markers for placenta implantation lineage diagnosis, the two circular RNA diagnostic markers are significantly up-regulated in PAS peripheral blood, and can be used as PAS diagnostic markers alone or in combination to realize high-sensitivity, specificity and objective detection of placenta implantation lineage. The sensitivity of circPHACTR4 alone is 74%, the sensitivity of circZMYM4 alone is 68.4%, when circPHACTR4 and circZMYM4 are combined to predict PAS, the combination has a good prediction effect, and in combination with the diagnostic results of clinical indicators and B ultrasound, the sensitivity and specificity can reach 82% and 88.9%, greatly improving the accuracy of prenatal diagnosis of PAS. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 For analysis of the differentially expressed circRNAs in the placenta of PAS patients and the control group in Example 1 of the application, FIG. A is a volcano plot of the differentially expressed circRNAs in the placenta of 3 PAS patients and 3 control patients, the y-axis represents the P value of the differential analysis, the x-axis represents the change fold, the circRNAs with up-regulated fold change of greater than or equal to 2 and P value of less than or equal to 0.05 are represented by red color (Up-regulated), and the circRNAs with down-regulated fold change of less than or equal to 0.5 and P value of less than or equal to 0.05 are represented by green color (Down-regulated), and the gray color represents the circRNAs without significant difference between the PAS patients and the control (Non-significant); FIG. B is a cluster of circRNAs based on their differential analysis in the placenta of PAS and control.

[0032] Figure 2 For analysis of the differentially expressed circRNAs in the peripheral blood of PAS patients and the control group in Example 1 of the application, FIG. A is a volcano plot of the differentially expressed circRNAs in the peripheral blood of 4 PAS patients and 4 control patients, the y-axis represents the P value of the differential analysis, the x-axis represents the change fold, the circRNAs with up-regulated fold change of greater than or equal to 2 and P value of less than or equal to 0.05 are represented by red color (Up-regulated), and the circRNAs with down-regulated fold change of less than or equal to 0.5 and P value of less than or equal to 0.05 are represented by green color (Down-regulated), and the gray color represents the circRNAs without significant difference between the PAS patients and the control (Non-significant); FIG. B is a cluster of circRNAs based on their differential analysis in the peripheral blood of PAS and control.

[0033] Figure 3Figure A shows the relative abundance of circPHACTR4 and circZMYM4 in placental samples from PAS patients (n=12) and controls (n=9) as determined by qRT-PCR and normalized to RPLP0. Figure B shows the expression levels of circPHACTR4 and circZMYM4 in maternal blood from PAS patients (n=38) and controls (n=27) as detected by qRT-PCR, the relative levels of circRNA were normalized to RPLP0.

[0034] Figure 4 Figure 1 shows the circPHACTR4 splice site information.

[0035] Figure 5 Figure 2 shows the circZMYM4 splice site information.

[0036] Figure 6 Figure A shows the ROC curves for different subjects based on the expression levels of circPHACTR4 and circZMYM4, circPHACTR4 is represented in orange, circZMYM4 is represented in blue, and ultrasound is represented in black. Figures B and C show the ROC curves for the training set and test set, respectively, combining the two circRNAs (circPHACTR4 and circZMYM4) and ultrasound. DETAILED DESCRIPTION

[0037] In the description of the present application, it should be noted that, in the examples, no specific conditions are specified, and the conventional conditions or manufacturer's recommended conditions are used. The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased on the market.

[0038] The present application will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0039] Example 1

[0040] The extraction and identification of circRNA biomarkers are as follows:

[0041] I. Methods

[0042] 1. Collection of tissue samples and blood samples

[0043] Inclusion criteria included patients aged ≥18 years who were not diagnosed with hypertension or preeclampsia. The diagnosis of PAS was based on clinical criteria, namely placenta accreta on the uterine wall and difficult separation from the placental bed. Since PAS frequently involves placenta previa, PAS placental samples were obtained from 15 women diagnosed with placenta previa, while control samples were obtained from 12 women with age-matched placenta previa. All placental samples were obtained from women who underwent cesarean section at Nanfang Hospital, Southern Medical University between 2018 and 2021, approved by the Medical Ethics Committees of Nanfang Hospital and Guangzhou Medical University, and with written informed consent obtained before sample collection. All placental samples were collected within 1 hour after cesarean section, cut into small pieces, thoroughly washed with PBS, and frozen in liquid nitrogen for later use.

[0044] In addition, from 2018 to 2021, blood samples were collected from 65 patients (38 PAS samples and 27 control samples) recruited again at Nanfang Hospital of Southern Medical University and the Third Affiliated Hospital of Guangzhou Medical University. Patient eligibility criteria were the same as for placental tissue. Whole venous blood samples were collected into 5 ml blood collection tubes containing K2EDTA and transferred to 15 ml RNase-free tubes within two hours. TRIzol was then added to the tubes, and the blood samples were stored at -80°C until use. Table 1 provides clinical information on the blood sample providers.

[0045] Table 1 Clinical information of blood sample providers

[0046]

[0047]

[0048] 2. RNA extraction and RT-qPCR

[0049] Approximately 100 mg of placental tissue was removed from the liquid nitrogen tank and placed into a 15 mL centrifuge tube containing 6 mL of trizol for grinding. The remaining tissue was placed into a new cryovial and returned to the liquid nitrogen tank. The centrifuge tube containing the blood sample was removed from -80°C, thawed, aliquoted, and trizol was added, with 1 mL used for RNA extraction each time.

[0050] The steps for RNA extraction are as follows:

[0051] 1) Take 1 mL of trizol containing the tissue sample, centrifuge at 12000 rpm for 15 min, and discard the precipitate.

[0052] 2) Add 200 mL of chloroform, shake to mix for 5 min, and let stand at room temperature for 10 min.

[0053] 3) Centrifuge at 13000 rpm for 15 min at 4℃.

[0054] 4) Take 400 μL of the upper aqueous phase into a new tube, add an equal volume of acidic phenol-chloroform, shake vigorously by hand for 5 min, and let stand on ice for 5 min.

[0055] 5) Centrifuge at 13000rpm for 10 minutes at 4℃, take the supernatant into a new tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 minutes.

[0056] 6) Centrifuge at 13000rpm for 15min at 4℃, discard the supernatant, and wash the precipitate twice with 1mL of 75% ethanol.

[0057] 7) Centrifuge at 8000g at 4℃ for 5 minutes, discard the supernatant, air dry at room temperature for 5 minutes, and add 10-30μL of nuclease-free water.

[0058] 8) Nanodrop was used to measure the concentration, and 1.5% agarose gel electrophoresis was used to check RNA integrity.

[0059] 9) Take 100 ng - 2 μg and reverse transcribe it into cDNA according to the kit instructions.

[0060] 10) Identification was performed using a 10 μL SYBR qPCR mix system, and the expression of the target gene was calculated using a linear housekeeping gene as an internal reference.

[0061] 3. Next-generation sequencing and data analysis

[0062] After total RNA extraction using Trizol, RNA concentration and quality were determined using NanoDrop, Qubit, and Agilent 2100 instruments. Total RNA was treated with the Epicenter Ribo-Zero kit (Epicentre, USA) to remove all ribosomal RNA. Samples were aliquoted into two portions, one of which was treated with 10 U·mg... -1 RNase R was incubated at 37°C to remove linear RNA. Samples treated with rRNA and rRNA- / RNAase R. Ultra TM The targeted RNA library preparation kit was used for library construction. The main steps included: RNA fragmentation, reverse transcription, second-strand cDNA synthesis, end repair, polyA tail addition, and adapter ligation. The products of these reactions were purified and amplified by PCR to produce the final cDNA library. Paired-end sequencing of the library was performed on an Illumina HiSeq2500 platform (Illumina, USA). 10g of cleandata was collected from placental samples, and 20g of cleandata from blood samples. CircRNA identification and analysis, circRNA and linear RNA quantification and differential analysis, gene enrichment analysis, and identification of placental high-expression circRNAs were performed. All of the above procedures are standard operating procedures in this field and will not be described in detail here.

[0063] II. Results

[0064] RNA was extracted from the placenta of normal placentas and placenta accreta (PAS) patients and sequenced using high-throughput sequencing. Sixty-seven differentially expressed circRNAs were identified between PAS patients and controls, including 28 upregulated and 39 downregulated circRNAs. Further sequencing of peripheral blood RNA from pregnant women identified 755 differentially expressed circRNAs in PAS patients, two of which showed a trend consistent with the differentially expressed circRNAs in placental PAS. qPCR analysis of the two circRNAs upregulated in both peripheral blood and placental tissue of PAS mothers revealed that the upregulation of these two circRNAs in PAS peripheral blood was widespread. Results are as follows: Figure 1 and Figure 2 As shown, where, Figure 1 Figure A shows a volcano plot of differentially expressed circRNAs obtained from next-generation sequencing data screening of placentas from 3 control pregnant women (control1, 2, 3) and 3 PAS patients (PAS1, 2, 3). Figure B shows a heatmap of each differentially expressed circRNA. Figure 2 Figure A shows a volcano plot of differentially expressed circRNAs obtained from next-generation sequencing data screening of blood samples from four control pregnant women (control1, 2, 3, 4) and four PAS patients (PAS1, 2, 3, 4). Figure B is a heatmap of each differentially expressed circRNA. It is evident that circRNAs upregulated in the placenta of PAS patients can also be detected as upregulated in peripheral blood. This indicates that upregulated circRNAs in PAS can be used as biomarkers for PAS blood diagnosis. The upregulated circRNAs are circPHACTR4 and circZMYM4; detailed information for these two circRNAs is shown in Table 2. Figure 3 , Figure 4 , Figure 5 As shown, Figure 3 The qPCR validation results of circPHACTR4 and circZMYM4 in PAS placenta (Figure A) and PAS blood samples (Figure B) are presented. Figure A shows the qPCR validation results of circPHACTR4 and circZMYM4 in PAS patient placental samples (n=12) and control placental samples (n=9); Figure B shows the qPCR validation results of circPHACTR4 and circZMYM4 in PAS patient blood samples (n=38) and control blood samples (n=27). Figure 4 and Figure 5 The splicing site information for the two circRNAs is provided. The detection primers are shown in Table 3.

[0065] Table 2. Detailed information on the two circRNAs

[0066] circRNA Chromosome location RNA sequence circPHACTR4 chr1 :28473553 | 28480604 SEQ ID NO: 1 circZMYM4 chr1 :35358924 | 35361789 SEQ ID NO: 2

[0067] Table 3 Primers for Q-PCR detection of two circRNAs and the internal reference gene

[0068]

[0069] Example 2

[0070] Based on the results of Example 1, we found that circPHACTR4 (AUC: 0.85, 95% CI: 0.76-0.94) had a diagnostic sensitivity of 74%. circZMYM4 (AUC: 0.86, 95% CI: 0.77-0.95) had a diagnostic sensitivity of 68.4%. Figure 6 (Figure A). The 65 samples from Example 1 were further divided into a training set (45 cases) and a validation set (20 cases). Logistic regression analysis was used to analyze the predictive ability of circRNAs, and ROC curves were used to determine their role as prognostic biomarkers. The area under the prediction curve for the combination of two circRNAs and ultrasound detection in the training set was 0.92 (95% CI: 0.85–0.996). Figure 6 (See Figure B). This prediction model was then validated in a validation queue with a sample size of 20. The area under the prediction curve for the combined form in the validation queue was 0.91 (95% CI: 0.78–1.00). Figure 6 (See Figure C). Based on this, the Logistic regression equation is established as follows:

[0071] C PAS =0.7784×Exp circPHACTR4 +2.1975×E.xp circZMYM4 +1.1064×ultrasound-4.2773, in the formula, C pas Exp represents the probability that the person being tested has PAS disease. circPHACTR4 The relative expression level of circPHACTR4 as detected by qPCR, Exp circZMYM4 The value represents the relative expression level of circZMYM4 detected by qPCR. Ultrasound represents the ultrasound diagnostic result. If the ultrasound diagnostic result is PAS negative, the ultrasound value is 0; if the ultrasound diagnostic result is PAS positive, the ultrasound value is 1.

[0072] The final calculated sensitivity was 82% and the specificity was 88.9%. This indicates that the predictive models using circPHACTR4 and circZMYM4 have good predictive performance in the diagnosis of PAS.

[0073] Example 3

[0074] This embodiment provides a diagnostic kit for diagnosing placenta accreta spectrum disorders, comprising the following components:

[0075] (1) Two circRNA Q-PCR detection primer pairs: including forward primer and reverse primer, whose nucleotide sequences are shown in Table 2 as SEQ ID NO: 3 to 6.

[0076] (2) Primer pairs for Q-PCR detection of the internal reference gene: including forward and reverse primers, whose nucleotide sequences are shown in Table 2 as SEQ ID NO: 7-8. The internal reference gene is not limited to RPLPO; this invention can also use housekeeping genes such as GAPDH and ACTB.

[0077] (3) Reagents required for the reaction: 5×PrimeScript Buffer (for Real Time), PrimeScript RTEnzyme Mix, MgCl2 (25mmol / L), Rnase Inhibitor (40U / ul), dNTP Mixture Rnase FreeddH2O.

[0078] 2. The instructions for using this kit are as follows:

[0079] 1. After extracting RNA from the pregnant woman's peripheral blood, add 1 μg of RNA to 0.5 μL of Random 6-mers (100 μM) primers, and then add water to a final volume of 5 μL. Incubate at 75°C for 5 min, then quench on ice. Prepare the reverse transcription reaction solution according to the formula in Table 4 below, and then add it to the quenched tubes. Reaction conditions: 42°C × 60 min, 75°C × 15 min, store at 4°C.

[0080] Table 4 Reverse Transcription Reaction System

[0081]

[0082] 2. Perform real-time PCR reactions according to the Novizan Q311-03 qPCR Mix. The real-time PCR reaction program is shown in Table 5:

[0083] Table 5 Real-time PCR reaction procedure

[0084]

[0085] circRNA expression levels were calculated, and then ROC curve analysis was performed.

[0086] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. The application of circular RNA diagnostic markers in the preparation of diagnostic products for placenta accreta lineages with placenta previa, wherein the circular RNA diagnostic markers include: One or both of circPHACTR4 and circZMYM4, with the RNA sequences of circPHACTR4 and circZMYM4 shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

2. Application of primer sets in the preparation of diagnostic products for placenta accreta lineages with placenta previa, said primer sets comprising primer pairs for detecting circPHACTR4 and / or circZMYM4, wherein, The primer pairs used to detect circPHACTR4 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; the primer pairs used to detect circZMYM4 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

3. The application according to claim 1 or 2, characterized in that: The diagnostic product is a reagent, reagent kit, or chip.

4. A diagnostic product for placenta previa and placenta accreta spectrum disorders, characterized in that: The reagent includes a reagent for detecting the expression level of a circular RNA diagnostic marker, which is composed of circPHACTR4 and circZMYM4, the RNA sequences of which are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively.

5. The placenta accreta lineage diagnostic product according to claim 4, characterized in that: The reagents include a set of primers for detecting the circular RNA diagnostic markers.

6. The placenta accreta lineage diagnostic product according to claim 5, characterized in that: The primer set includes primer pairs for detecting circPHACTR4 and circZMYM4, wherein the primer pair sequences for detecting circPHACTR4 are shown in SEQ ID NO:3 and SEQ ID NO:4, respectively; and the primer pair sequences for detecting circZMYM4 are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively.

7. The placenta accreta spectrum diagnostic product according to claim 6, characterized in that: The circular RNA diagnostic markers consist of circPHACTR4 and circZMYM4. To detect the expression levels of circPHACTR4 and circZMYM4, RPLPO was used as an internal reference gene. ROC curve analysis was performed based on the qPCR results and the formula. C PAS =0.7784×Exp circPHACTR4 +2.1975×Exp circZMYM4 +1.1064×ultrasound-4.2773, in the above formula, C pas Exp represents the probability that the person being tested has PAS disease. circPHACTR4 The relative expression level of circPHACTR4 as detected by qPCR, Exp circZMYM4 The value represents the relative expression level of circZMYM4 detected by qPCR. Ultrasound represents the ultrasound diagnostic result. If the ultrasound diagnostic result is PAS negative, the ultrasound value is 0; if the ultrasound diagnostic result is PAS positive, the ultrasound value is 1.

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