Markers for diagnosing unexplained recurrent abortion and application thereof

Abstract

The invention belongs to the technical field of reproductive medicine and clinical metabolism intervention, and particularly relates to a group of markers for diagnosing unexplained recurrent abortion and application thereof. Aiming at the clinical pain point that unexplained recurrent abortion (URSA) lacks accurate metabolic typing diagnosis indexes and targeted treatment means, based on detection of a large number of clinical samples and verification of cell experiments, direct association between down-regulation of levels of choline related metabolites and trimethylamine oxide (TMAO) of decidua tissues and the RSA is disclosed for the first time; the core effect of TMAO in-situ synthesis of decidua stromal cells (DSC) in regulation and control of the decidualization process is determined; meanwhile, an RSA indication screening system of joint detection of serum creatinine and decidua choline metabolism is constructed, a targeted TMAO targeted intervention scheme is formed in a matched mode, a diagnosis and treatment closed loop of RSA from precise typing to targeted treatment is achieved, and a brand new technical path is provided for clinically solving the URSA diagnosis and treatment problem.

Description

Technical Field

[0001] This invention belongs to the field of reproductive medicine and clinical metabolic intervention technology, specifically involving a set of biomarkers for diagnosing unexplained recurrent miscarriage and their applications. Background Technology

[0002] Recurrent spontaneous abortion (RSA) is a common and complex condition in reproductive medicine, defined as three or more consecutive spontaneous abortions with the same partner. Its incidence rate is approximately 1%–5% of women of reproductive age, and is showing an increasing trend annually. The etiology of RSA is complex. Currently known contributing factors include chromosomal abnormalities, immune dysfunction, endocrine disorders, and uterine anatomical abnormalities. However, in 30%–40% of RSA patients, the cause remains unclear after comprehensive examination, and this is termed "unexplained recurrent abortion (URSA)." These patients lack effective and precise diagnostic indicators and targeted treatments. Clinically, empirical treatment (such as progesterone supplementation and immunosuppressant use) is often used, resulting in limited efficacy and significant individual variability and uncertain side effects, seriously impacting the patient's reproductive and mental health.

[0003] In recent years, the application of metabolomics technology in reproductive medicine has become increasingly sophisticated. Studies have found that metabolic disorders during pregnancy are closely related to the occurrence and development of recurrent spontaneous abortion (RSA). As a core component of the maternal-fetal interface, the stability of the metabolic microenvironment of the decidual tissue directly affects embryo implantation and pregnancy maintenance. Choline metabolism is one of the key metabolic pathways in mammals, participating in multiple physiological processes such as cell membrane synthesis, signal transduction, and energy metabolism. Abnormalities in choline metabolism in the decidual tissue may induce miscarriage by affecting immune tolerance and angiogenesis at the maternal-fetal interface. Meanwhile, serum creatinine, a classic indicator reflecting the body's metabolic state and renal function, is closely related to metabolic adaptive regulation during pregnancy, but a clinically accepted diagnostic standard has not yet been established.

[0004] Trimethylamine N-oxide (TMAO) is an important bioactive substance produced by gut microbiota metabolism and has been shown in recent years to participate in physiological processes such as regulating metabolic homeostasis, inflammatory responses, and vascular function. Recent studies have found that TMAO concentrations are significantly elevated during pregnancy, but research on the association between TMAO and recurrent spontaneous abscess (RSA) has not yet been reported. The applicant's previous research was the first to discover that RSA patients with decreased serum creatinine levels and abnormal decidual choline metabolism had significantly reduced TMAO concentrations in their decidual tissue, suggesting a TMAO metabolic defect in these patients. This pathological feature has not been addressed in existing research, and no targeted intervention strategies have been developed.

[0005] In summary, existing technologies have two major gaps: first, URSA lacks precise diagnostic indicators based on metabolic characteristics, making it impossible to effectively screen and classify "serum creatinine-decidual choline metabolism abnormality type" RSA; second, there is a lack of targeted interventions based on the pathological mechanisms for this type of metabolically deficient RSA, resulting in poor clinical treatment outcomes. Therefore, developing a detection method that can accurately indicate the indications for this type of RSA and constructing a corresponding TMAO targeted intervention program has become an urgent technical problem to be solved in the field of reproductive medicine. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a set of biomarkers for diagnosing unexplained recurrent miscarriage, consisting of serum creatinine and decidual choline.

[0007] The present invention also provides a kit for diagnosing unexplained recurrent miscarriage, comprising the above-mentioned biomarkers.

[0008] The present invention has the following beneficial effects:

[0009] 1. High diagnostic accuracy, filling a gap in subtyping: Compared with existing RSA diagnosis that relies solely on traditional indicators such as chromosomes and immunity, this protocol innovatively adopts a combined detection of "serum creatinine + decidual choline metabolism" to accurately identify the "metabolic abnormality RSA" subtype. This solves the problem of lack of metabolic subtyping basis for unexplained recurrent miscarriage (URSA). The detection specificity and sensitivity are more suitable for the needs of accurate clinical diagnosis, and can effectively avoid the blindness of traditional empirical diagnosis and treatment.

[0010] 2. Highly targeted intervention with a mechanism aligned with pathology: Unlike non-specific methods such as progesterone supplementation and immunosuppressants in existing RSA treatments, this protocol is based on the core pathological finding that "the concentration of TMAO in the decidua is reduced in some patients with metabolically abnormal RSA". It designs a targeted TMAO intervention program with an intervention mechanism that directly matches the patient's pathological characteristics. This can reduce the side effects caused by unnecessary interventions and improve the effectiveness of treatment, achieving precise intervention based on the specific disease.

[0011] 3. Complete diagnosis and treatment loop with strong clinical applicability: The solution constructs an integrated technical system of "metabolic indicator diagnosis - subtyping screening - TMAO targeted intervention", forming a complete process from patient subtyping to treatment implementation. It does not rely on multidisciplinary collaboration and can be applied to clinical diagnosis and treatment scenarios in gynecology and reproductive medicine centers. It is especially suitable for the stratified management of URSA patients and the improvement of pregnancy outcomes. It is easy to operate and meets the actual clinical needs.

[0012] 4. High technology transfer value and wide range of applications: The solution not only provides diagnostic and treatment methods, but also provides a clear direction for the research and development of related products. The combined detection kit based on "serum creatinine + decidual choline" can be industrialized, and TMAO intervention agents can be further developed into clinically applicable intervention products, covering the entire chain of RSA screening, diagnosis and treatment, and prognosis management. It has both academic value and market transformation potential, and can promote the implementation and promotion of precision diagnosis and treatment technologies in reproductive medicine. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1 (A) Relative mRNA expression levels of insulin-like growth factor binding protein 1 (IGFBP1) and prolactin (PRL) in decidualized endometrial stromal cells (DESCs) with or without the addition of choline-related metabolites (including choline, betaine, carnitine, phosphocholine (PC), glycerophosphocholine (GPC), and trimethylamine oxide (TMAO)) during in vitro decidualization (n=3). (B) Relative mRNA expression levels of IGFBP1 and PRL in DESCs supplemented with 1 mMTMAO at specific time points during in vitro decidualization (n=3). (C) TMAO and choline-deficient diet (CDD) had no significant effect on food intake and body weight in mice (n=6). (D) Comparative analysis of TMAO and choline levels in uterine tissues of three groups of mice (standard diet group, choline-deficient diet group, and choline-deficient diet + TMAO group) (n=6). (E) Gross uterine morphology of pregnant mice on day 10.5 of gestation (GD10.5), in the standard diet (StD), choline-deficient diet (CDD), and choline-deficient diet + TMAO groups. (F) Quantitative results of embryo resorption rate on day 10.5 of gestation in the three groups of mice (n=9). (G) mRNA levels of decidual marker Dtprp in the uterine tissue of pregnant mice in the standard diet group (n=6), the choline-deficient diet group without embryo resorption sites (n=6), the choline-deficient diet group with embryo resorption sites (n=6), and the choline-deficient diet + TMAO group (n=6). (H) Gross morphology of the uterine horn on the injected and uninjected sides of mice in different diet groups 5 days after injection of sesame oil. (I) Wet weight ratio of the uterine horn on the injected and uninjected sides of each group of mice (n=6). Data are expressed as mean ± standard error (mean ± SEM), and statistical significance was analyzed using one-way ANOVA and Student's test.

[0015] Figure 2 (A) Relative prolactin (PRL) mRNA expression levels during in vitro decidualization of human primary endometrial stromal cells (HpESCs) from 32 patients with recurrent spontaneous abortion (RSA) and impaired decidualization function, with or without TMAO treatment (n=3). Top: RSA patients who responded to TMAO; Bottom: RSA patients who did not respond to TMAO. (B) Relative PRL mRNA expression levels during in vitro decidualization of HpESCs from 28 RSA patients with normal decidualization function, with or without TMAO treatment (n=3). Detailed Implementation

[0016] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0017] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] 1. Core Research Foundations and Technological Discoveries

[0022] (1) Metabolomics analysis of clinical samples:

[0023] This patent validates its core findings based on two sets of clinical sample experiments: The first set collected decidual tissue samples from 38 patients with recurrent spontaneous abscess (RSA) and 38 patients who terminated normal pregnancies for non-medical reasons. Nuclear magnetic resonance (NMR) non-targeted metabolomics was used for detection. The results showed that the concentrations of choline, phosphoric acid choline, glycerophosphocholine, and TMAO, among other choline-related metabolites, in the decidual tissue of RSA patients were significantly lower than those in the normal control group. The second set incorporated NMR data from decidual and chorionic villi tissues from previous studies (30 patients each from RSA and the control group), further validating that the above four metabolites exhibited low expression characteristics in the decidual tissue of RSA patients, and that there were no inter-group differences in choline-related metabolites in chorionic villi tissue. After integrating the two sets of samples and correcting for batch effects, the statistical differences were further clarified (choline P = 3.5 × 10⁻). 5 Phosphocholine (P) = 8.8 × 10⁻ 5 glycerophosphate choline P = 3.5 × 10⁻ 5 TMAO-related metabolites P = 3.0 × 10⁻ 4 (Table 1).

[0024] (2) Cellular experiments to verify the decidualization regulatory role of TMAO:

[0025] High-purity human primary endometrial stromal cells (HpESCs) were isolated from endometrial samples of normal pregnant women and induced to differentiate into decidualized endometrial stromal cells (DESCs, characterized by high expression of decidual markers prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1)) in vitro. The effects of choline, phosphoric acid choline, glycerophosphocholine, TMAO, and TMAO precursors (betaine and carnitine) on decidualization were examined in the cell model. The results showed that only TMAO significantly upregulated the expression levels of IGFBP1 and PRL. Figure 1 A), and effectively promotes the in vitro decidualization process of HpESC ( Figure 1 (B) confirms that TMAO is a key functional metabolite regulating decidualization.

[0026] (3) Animal model validation of the regulatory effect of TMAO on pregnancy outcome and decidualization:

[0027] To further clarify the crucial role of TMAO in maintaining pregnancy, this study constructed a mouse model of insufficient TMAO synthesis induced by a choline-deficient diet. The mice were randomly divided into three groups: a standard diet group (StD), a choline-deficient diet group (CDD), and a choline-deficient diet group supplemented with 0.5% TMAO. After two weeks of feeding, there were no significant differences in body weight and food intake among the three groups. Figure 1 c) To exclude the effect of diet on the basal state of mice; analysis of metabolites in mouse uterine tissue revealed that the concentrations of choline and TMAO in the uterus of mice in the choline-deficient diet group were significantly decreased, while the concentrations of both in the TMAO supplementation group were restored to normal levels. Figure 1 (d) This confirmed that the dietary intervention model could effectively regulate intrauterine TMAO concentration. Pregnancy outcomes were assessed on day 10.5 of gestation (the peak TMAO concentration point in the uterus). The results showed that, compared with the standard diet group, the embryo resorption rate in the choline-deficient diet group was significantly increased, and TMAO supplementation effectively reversed this phenomenon. Figure 1 e,f); meanwhile, the expression of the decidual marker Dtprp in the uterine tissue of mice in the choline-deficient diet group was significantly reduced (e,f); Figure 1 G), indicating impaired decidualization. To directly verify the regulatory effect of TMAO on decidualization, sesame oil was used to induce pseudo-decidualization in mice. The results showed that mice in the choline-deficient diet group had reduced responsiveness to the induction stimulus, and the volume of the decidual tumors formed was significantly smaller than that in the control group. TMAO supplementation could effectively salvage this phenotypic defect. Figure 1 The above animal experimental results confirm that decreased intrauterine TMAO concentration is a key factor leading to poor pregnancy outcomes, and that TMAO plays a central role in regulating the decidualization process, rather than other choline derivatives, providing reliable animal experimental evidence for clinical TMAO-targeted intervention.

[0028] (4) Clinical cell model validation of TMAO therapeutic potential and screening biomarkers for applicable populations

[0029] This study confirmed that TMAO supplementation effectively promoted decidualization in both in vitro cell culture and mouse models. To clarify its therapeutic potential in RSA patients, this study collected endometrial tissue from RSA patients at two hospitals for validation: Cohort 1 included 34 RSA patients from the Obstetrics and Gynecology Hospital affiliated with Fudan University, and Cohort 2 included 26 RSA patients from Shanghai First Maternity and Infant Hospital. *HpESC* cells were isolated from the endometrial tissue and in vitro decidualization experiments were conducted to verify the salvage effect of TMAO on decidualization function. Decidualization experiments on *HpESC* cells from the 34 patients in Cohort 1 showed that 16 samples exhibited significantly increased PRL levels after induction. These cells inherently possess good decidualization potential, and the addition of TMAO had no significant effect on their PRL expression levels. Figure 2 B); the remaining 18 samples failed to undergo normal decidualization during induction, but after the addition of TMAO, the decidualization ability of 5 of these samples was restored ( Figure 2 A) suggests that TMAO can salvage decidualization dysfunction in some patients. The results of cohort 2 were highly consistent with those of cohort 1: 12 samples had intact decidualization function unaffected by TMAO, and 4 samples with decidualization defects recovered their function after TMAO treatment. Figure 2 A). Synthesizing data from two independent cohorts, TMAO restored decidualization capacity in *Helicobacter pylori* (HpESC) cells from 15% (9 / 60) of RSA patients, confirming that TMAO can specifically rescue decidualization dysfunction in some RSA patients, while having no significant effect on cells with normal decidualization function. Metabolomics analysis showed that serum creatinine levels in TMAO-responsive patients were significantly lower than in non-responsive patients (Table 2). This consistent metabolic profile confirms that reduced creatinine levels can serve as a specific biomarker for screening individuals who benefit from TMAO intervention, providing crucial screening criteria for subsequent precise clinical intervention. Specific Implementation

[0030] (a) Diagnostic markers and sample requirements

[0031] 1. Core Diagnostic Biomarker Combination

[0032] (1) Blood markers: serum creatinine, as a peripheral indicator of the association between the whole body's metabolic state and the RSA metabolic subtype, its downregulation is a key signal for the potential benefits of TMAO intervention.

[0033] (2) Tissue markers: Choline, phosphocholine and glycerophosphocholine in decidual tissue serve as direct indicators of abnormal local choline metabolism in the decidua. Together, they reflect the metabolic state of the precursors for TMAO synthesis in the decidua.

[0034] 2. Sample Collection Standards

[0035] (1) Peripheral blood sample: Collect 5 mL of fasting peripheral venous blood from suspected RSA patients (continuous spontaneous abortion ≥3 times, excluding chromosomal, immune, uterine anatomical and other clear causes), collect using EDTA anticoagulant tubes, transport at 4℃, and complete the test within 24 hours; if the test needs to be delayed, the sample should be frozen at -20℃ for no more than 7 days.

[0036] (2) Decidual tissue samples: collect decidual tissue obtained from patients by curettage or hysteroscopy, weighing ≥0.3g, rinse twice with sterile saline to remove surface blood and mucus, absorb the moisture with filter paper, and then aliquot into cryovials and immediately store in an ultra-low temperature freezer at -80℃ to avoid repeated freezing and thawing. The sample storage period shall not exceed 6 months.

[0037] (II) Specific testing methods

[0038] 1. Serum creatinine detection – creatinine oxidase method

[0039] (1) Composition of the detection reagent:

[0040] Reagent R1: Tris buffer: 100 mmol / L; sarcosine oxidase: 20 KU / L, creatine oxidase: 75 KU / L; TOOS 0.9 mmol / L;

[0041] Reagent R2: Tris buffer: 100 mmol / L; creatinine enzyme: 40 KU / L; 4-aminoantipyrine: 0.13 mmol / L; peroxidase: 1.6 KU / L;

[0042] (2) Procedure: Add 80 μL of reagent 1 to the reaction cuvette. Add 13 μL of the pre-diluted sample to the same reaction cuvette and mix gently. After adding the sample, immediately measure the absorbance (denoted as A1).

[0043] The instrument then automatically adds 27 μL of reagent 2 to the reaction cuvette. After the reaction solution is thoroughly mixed, it is incubated at a constant temperature of 37°C for 5 minutes. The absorbance is measured again (recorded as A2), and the absorbance difference ΔA (ΔA = A2 - A1) is automatically calculated.

[0044] The instrument automatically calculates the sample concentration based on the calibration curve and the following formula: Sample concentration = (sample ΔA / min ÷ standard ΔA / min) × standard concentration.

[0045] (3) Quality control: High, medium and low concentrations of standard products are added simultaneously to each batch of tests. The recovery rate of standard products is controlled at 95%-105%, and the coefficient of variation (CV) is ≤5%.

[0046] 2. Detection of decidual choline-related metabolites – NMR validation method

[0047] (1) Sample pretreatment

[0048] a. Extraction: Take 50 mg of decidual tissue sample, add 600 μL of pre-cooled methanol-water mixture (volume ratio 2:1), and grind and lyse using a tissue homogenizer (such as QIAGEN TissueLyser II); centrifuge at 4℃ and 11180×g for 10 minutes, and collect the supernatant.

[0049] b. Repeat extraction and merging: Repeat the above steps twice, combine the supernatants from the three extractions, and centrifuge again at 4℃ and 16099×g for 10 minutes to remove tissue residue.

[0050] c. Drying and reconstitution: Remove methanol from the supernatant under vacuum conditions, and reconstitute the dried extract in 600 μL phosphate buffer (0.15 M K2HPO4-NaH2PO4, pH 7.43), which contains 80% heavy water (D2O) and 0.2915 mM trimethylsilylpropionic acid (TSP) internal standard.

[0051] d. Pre-injection treatment: After reconstitution, the sample is centrifuged at 4℃ and 16099×g for 10 minutes. 550μL of the supernatant is transferred to a standard 5mm NMR sample tube for detection.

[0052] (2) NMR detection parameters (quantitative detection conditions)

[0053] a. Instruments and probes: A Bruker Advance III 600MHz NMR spectrometer (proton frequency 600.13MHz) was used, equipped with a quaternary low-temperature reverse-phase probe, with a detection temperature of 298K (25℃).

[0054] b. One-dimensional proton NMR acquisition: Gradient-selective NOESY pulse sequence (NOESYGPPR1D) was used, with a total of 64 scans, 32k data points, and a spectral width of 20ppm; the total relaxation delay time was 26s to ensure that a fully relaxed NMR spectrum was obtained, thus guaranteeing quantitative accuracy.

[0055] c. Two-dimensional spectroscopy-assisted qualitative analysis: Two-dimensional NMR spectra were collected from representative samples for metabolite resonance peak assignment, including ¹H-¹H correlation spectrum (COSY), ¹H-¹H total correlation spectrum (TOCSY), J-resolved spectrum (JRES), ¹H-¹³C heteronuclear single quantum correlation spectrum (HSQC), and ¹H-¹³C heteronuclear multi-bond correlation spectrum (HMBC).

[0056] (3) Quantification of metabolites and analysis of results

[0057] a. Internal standard method for quantification: Using TSP as the internal standard (chemical shift δ=0.0), the absolute concentration of each metabolite is calculated by comparing the integral area of ​​the characteristic resonance peaks of choline, phosphoric acid choline, glycerophosphocholine, and TMAO with the characteristic peak of TSP, combined with the concentration of the internal standard.

[0058] b. Quality control: Each batch of tests includes a blank control (containing only extract and buffer) and a standard control (a mixture of three metabolites of known concentration) to ensure stable extraction efficiency and reliable quantitative results. The standard recovery rate is controlled between 90% and 110%.

[0059] (III) Diagnostic criteria and interpretation of results

[0060] 1. Basis for establishing the reference scope

[0061] Based on the test data of 76 basic clinical samples and 60 expanded cohort samples, the normal reference ranges for each indicator were established using the 95% reference range method. The reference range for serum creatinine was based on data from normal women in the early and mid-stages of pregnancy (44-97 μmol / L), and the reference range for decidual choline-related metabolites was based on the mean values ​​of decidual tissue from normal pregnant controls.

[0062] 2. Stratification criteria

[0063] (1) Preliminary screening positive: serum creatinine concentration <40μmol / L (i.e., 10% lower than the lower limit of normal reference value), judged as a suspected patient of RSA metabolic subtype.

[0064] (2) Diagnostic criteria: In patients who are positive in the initial screening, the concentrations of choline, phosphoric acid choline, and glycerophosphocholine in the decidual tissue are all less than 80% of the mean of the normal control, and they are diagnosed as “low serum creatinine + decidual choline metabolism abnormality type RSA”, that is, decidual TMAO synthesis defect type RSA.

[0065] (3) Determination of intervention benefit: Patients who meet the diagnostic criteria, their endometrial stromal cells are isolated and decidualization is induced in vitro. If TMAO is found to significantly promote the decidualization process, they are judged to be potential beneficiaries of TMAO intervention and can be included in subsequent targeted intervention programs.

[0066] Table 1. Concentrations of choline-related metabolites in decidual tissue of patients with recurrent miscarriage and healthy individuals.

[0067]

[0068] Note: Data are expressed in μmol / g (mean ± standard deviation). The P-value was calculated using a paired two-sample t-test (when the standard deviations of the two groups are equal) or a corrected Welch paired two-sample t-test (when the standard deviations of the two groups are unequal). The results of the multiple tests were then corrected using the Benjamin Hochberg method to obtain the corrected P-value.

[0069] Table 2. Concentrations of metabolites in the serum of patients with recurrent miscarriage

[0070]

[0071] The Hohberg method corrects for multiple test results, ultimately yielding the corrected P-value.

[0072] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A panel of markers for the diagnosis of unexplained recurrent spontaneous abortion, characterized in that, Consists of serum creatinine and decidual choline.

2. A kit for diagnosing unexplained recurrent spontaneous abortion, characterized by, Comprising the marker of claim 1.

Images