Application of diversity of extracorporeal vesicle flora of amniotic fluid microorganism as marker in prediction of fetal structural deformity

Abstract

The invention discloses application of the diversity of amniotic fluid microorganism source outer vesicle flora as a marker in predicting fetal structural deformity, and belongs to the technical field of biomarkers. The method comprises the following steps: collecting an amniotic fluid sample of a pregnant woman of 16-30 weeks of pregnancy, carrying out centrifugation, filtration and ultracentrifugation to separate and purify the external vesicles derived from amniotic fluid bacteria, and analyzing the flora composition in combination with a 16S rRNA gene sequencing technology. The result shows that the diversity of the flora alpha of the BEVs in the structure malformation group is obviously higher than that of the normal group. The method for predicting the fetal structural deformity by using the diversity of the amniotic fluid microorganism source outer vesicle flora as the marker is high in sensitivity and simple and convenient to operate, can be complementary with conventional prenatal ultrasonic screening, and provides a new strategy for early screening and intervention of the fetal structural deformity.

Description

Technical Field

[0001] The present invention relates to the field of biomarker technology, and in particular to the application of the diversity of exosomes derived from amniotic fluid microorganisms as a marker in predicting fetal structural malformations. Background Art

[0002] Congenital structural malformations (CSMs) refer to structural abnormalities present before birth that alter the appearance and / or function of an organ or body part. They are a leading cause of infant mortality, accounting for 60% to 70% of all birth defects. Existing clinical screening methods, such as prenatal ultrasound, face technical bottlenecks. Ultrasound, limited by factors such as resolution and fetal position, can miss subtle malformations (false negatives), especially in early pregnancy.

[0003] Amniotic fluid is the fluid closest to the fetus and the maternal uterine environment, providing essential nutrition and protection for the fetus while also reflecting its health. Studies have shown that bacterial extracellular vesicles (BEVs) in amniotic fluid play a role in causing or exacerbating pregnancy complications, such as premature birth and fetal growth restriction. Furthermore, BEVs in feces, blood, and urine show significant differences between patients with specific diseases and healthy subjects, and their bacterial community characteristics can reflect the host's pathological state.

[0004] 16S sequencing of the amniotic fluid of fetuses with structural malformations revealed that the diversity and composition of the amniotic fluid microbiome in fetuses with structural malformations differ significantly from those in normal fetuses. Currently, there is no method for detecting fetal structural malformations based on the characteristics of the amniotic fluid BEVs microbiome. To address this technological gap, the present invention proposes a method for detecting fetal structural malformations based on the richness of the amniotic fluid BEVs microbiome, as a useful supplement to existing screening methods to improve the early identification rate of fetuses with structural malformations. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of the diversity of the bacterial flora of exosomes derived from amniotic fluid microorganisms as a marker in predicting fetal structural malformations, so as to solve the problems existing in the above-mentioned prior art. By analyzing the changes in the richness of the bacterial flora of exosomes in amniotic fluid and combining it with ultrasound imaging characteristics, the risk of fetal structural malformations can be preliminarily judged, providing an important reference basis for further clinical prenatal examinations, risk assessment, prediction and intervention decisions.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides an application of the diversity of exosome flora derived from amniotic fluid microorganisms in the preparation of biomarkers for predicting fetal structural malformations.

[0008] Preferably, the amniotic fluid microbial-derived exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks.

[0009] Preferably, the indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are Chao1 index and Shannon index;

[0010] And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

[0011] The present invention also provides the use of the diversity of extracellular vesicle flora of amniotic fluid microorganisms as a marker in the preparation of products for predicting fetal structural malformations.

[0012] Preferably, the product includes a reagent for detecting the diversity of the extracellular vesicle flora of amniotic fluid microbial origin.

[0013] Preferably, the reagent comprises primers for amplifying 16S rDNA of the amniotic fluid microbial exocyst flora.

[0014] Preferably, the amniotic fluid microbial exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks;

[0015] And / or the indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are Chao1 index and Shannon index;

[0016] And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

[0017] The present invention also provides a product for predicting fetal structural malformations, which includes a reagent for detecting the diversity of extracellular vesicle flora of amniotic fluid microbial origin.

[0018] Preferably, the amniotic fluid microbial exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks;

[0019] And / or the indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are Chao1 index and Shannon index;

[0020] And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

[0021] Preferably, the reagent comprises primers for amplifying 16S rDNA of the amniotic fluid microbial exocyst flora.

[0022] The present invention discloses the following technical effects:

[0023] (1) Significantly improve detection sensitivity and accuracy, and make up for the false negative limitations of ultrasound screening:

[0024] By combining the bacterial richness of amniotic fluid BEVs (Chao1 index and Shannon index) with ultrasound imaging features, the risk of false negatives based solely on ultrasound can be significantly reduced. Experimental data showed that the bacterial richness of amniotic fluid BEVs in the structural malformation group was significantly higher than that in the normal group (Chao1 > 175 and Shannon > 0.62). This biomarker provides an objective basis for screening high-risk fetuses independent of ultrasound, improving overall diagnostic accuracy.

[0025] (2) Achieve early prediction of structural malformations during mid-pregnancy and create a critical time window for clinical intervention:

[0026] Existing ultrasound screening has a lag in detecting some minor or complex structural malformations (e.g., diagnosis can only be made in the late stages of pregnancy). However, the present invention can identify high-risk fetuses in the second trimester (a critical period for organ development) by analyzing the bacterial flora of extracellular vesicles in amniotic fluid between 16 and 30 weeks of gestation.

[0027] (3) The technical process is simple, non-invasive and highly compatible, making it suitable for widespread promotion:

[0028] This method utilizes standardized ultracentrifugation combined with 16S rRNA sequencing, with clear procedures and high reproducibility. It seamlessly integrates with existing prenatal diagnostic processes (such as the secondary use of samples after amniocentesis) without requiring additional invasive procedures. Compared to traditional metagenomic analysis, this method focuses on microbial signals within BEVs, avoiding interference from host DNA in amniotic fluid and significantly improving detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 This is a flow chart of the detection of fetal structural malformation using microbial diversity as a tool in the present invention;

[0031] Figure 2 TEM images of extracellular vesicles in amniotic fluid from the structural deformity group and the normal control group. Figure A shows the structural deformity group, and Figure B shows the normal control group.

[0032] Figure 3Figure 3 is a nanoparticle tracking analysis (NTA) diagram of bacterial extracellular vesicles in amniotic fluid of the structural malformation group and the normal control group, showing the size distribution characteristics of the extracellular vesicles in the two groups of samples and their changing trends in the dimensions of particle concentration and volume density. Among them, the vertical axis of the structural malformation group (A) and the normal group (C) is the particle concentration (particles / mL), reflecting the number distribution of extracellular vesicles in different particle size ranges; the vertical axis of the structural malformation group (B) and the normal group (D) is the volume density (volume / nm 3 ), used to characterize the overall volume distribution characteristics of extracellular vesicles at various particle sizes;

[0033] Figure 4 Figure 2 is a comparison of the richness of BEVs in the amniotic fluid of the structural malformation group and the normal control group; A is the phylogenetic clustering and heat map of the bacterial communities carried by BEVs in the two groups of samples, and B is the heat map of the core differential ASVs after statistical screening;

[0034] Figure 5 The α-diversity analysis results of the bacterial flora in the amniotic fluid BEVs of the structural malformation group and the normal control group; A is the Chao1 index histogram, and B is the Shannon index histogram;

[0035] Figure 6 This is the receiver operating characteristic (ROC) curve for microbial diversity as a marker for detecting fetal structural malformations. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Figure 1 This is a flow chart of the detection method of the present invention, demonstrating the overall technical pathway for predicting fetal structural malformations based on the richness of bacterial-derived extracellular vesicles (BEVs) in amniotic fluid. First, amniotic fluid samples were collected from pregnant women between 16 and 30 weeks of gestation via amniocentesis. Subsequently, the samples were centrifuged at 8000×g to remove cellular debris and sterilized by filtration through a 0.22μm filter. High-purity BEVs were then isolated and purified by ultracentrifugation at 130,000×g. Total DNA was extracted from the BEVs, and the 16S rRNA gene V3-V4 region was amplified using specific primers 343F and 798R. High-throughput sequencing was performed on the Illumina NovaSeq 6000 platform. Sequencing data were aligned to the SILVA database using the QIIME2 pipeline to obtain bacterial taxonomic information. Alpha diversity analysis was then performed, and the Chao1 index and Shannon index were calculated. A Chao1 index greater than 175 and a Shannon index greater than 0.62 indicate a high risk of fetal structural malformation. Ultimately, the results can be further verified in combination with pregnancy ultrasound or fetal MRI images to provide a basis for early risk screening and intervention in clinic.

[0042] The above technical solution is further illustrated below with reference to specific embodiments.

[0043] Example 1 Analysis of the correlation between microbial diversity and fetal structural malformations

[0044] (1) Sample collection

[0045] When the pregnant woman is between 16 and 30 weeks of gestation, the amount of amniotic fluid is moderate and the major organs of the fetus have basically developed. This is suitable for conducting relevant research on microbiome or molecular markers to ensure the scientific nature and clinical applicability of the test results.

[0046] Sixty pregnant women (30 in the structural malformation group and 30 in the normal control group) with gestational age between 16 and 30 weeks were enrolled. Amniotic fluid samples (10 mL) were collected by amniocentesis and placed in sterile centrifuge tubes, immediately stored on ice, and transported to the laboratory. Ethical approval and informed consent were obtained.

[0047] (2) Pretreatment

[0048] The amniotic fluid samples were centrifuged at 7000-10000×g for 5-20 min (8000×g for 10 min was selected in this experiment) to remove cells and large particles, and the supernatant was filtered through a 0.22 μm filter membrane (Merck, Germany) to exclude bacterial contamination.

[0049] (3) Ultracentrifugation purification

[0050] Transfer the filtered sample to an ultracentrifuge tube and ultracentrifuge at 100,000–150,000 × g at 4°C for 0.5–2 h (in this experiment, 120,000 × g at 4°C for 2 h) (Beckman Optima XPN-100). Discard the supernatant and resuspend the pellet in 1× PBS to obtain the exosomes in the amniotic fluid. This step is crucial to retaining the intact, functional exosomes to the greatest extent possible while eliminating non-vesicular impurities and soluble proteins, providing high-quality samples for subsequent gene sequencing analysis.

[0051] (4) Quality control

[0052] The morphology of BEVs was observed by transmission electron microscopy (TEM, Hitachi HT7800), and the particle size distribution was determined to be consistent with the characteristics of extracellular vesicles by nanoparticle tracking analysis (NTA, Malvern NanoSight NS300).

[0053] (5) 16S rRNA sequencing

[0054] Total DNA was extracted from BEVs using the QIAamp DNA Microbiome Kit (Qiagen). The 16S rRNA gene V3-V4 region was amplified using primers 343F / 798R (343F: 5'-TACGGRAGGCAGCAG-3', 798R: 5'-AGGGTATCTAATCCT-3'). PCR conditions included initial denaturation at 94°C for 5 min, 26 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 20 s, followed by extension at 72°C for 5 min. The PCR product was purified and paired-end sequenced (2 × 250 bp) on an Illumina NovaSeq 6000 platform. The raw data were then analyzed using Trimmomatic adapter removal and FLASH assembly. The data were then aligned to the SILVA database (v138) using the QIIME2 analysis pipeline.

[0055] (6) Microbial diversity analysis

[0056] Based on the sequencing results, alpha diversity indices (including Chao1 and Shannon indices) were calculated, and the bacterial community richness of the structural malformation group was compared with that of the normal group. This comparison revealed the association between bacterial community richness and fetal structural malformation.

[0057] (7) Results and analysis

[0058] like Figure 2 The figure shows the microscopic morphology of exosomes extracted from the structurally deformed group and the normal control group side by side. The image shows multiple vesicle-like structures of varying sizes and shapes, scattered across the field of view, exhibiting typical ultrastructural features of exosomes. The diameters of most vesicles range from 30 to 150 nm, which is consistent with the currently recognized size range for exosomes. Some vesicle membranes are relatively intact, with clear boundaries and no obvious collapse or structural damage, indicating that the vesicle structure was well preserved during sample preparation. This image can be used to support the applicant's claims regarding the isolation and identification of exosomes.

[0059] like Figure 3 As shown, the results show that the particle size of the exosomes in both sample groups is mainly concentrated in the range of approximately 30 to 300 nm, which is consistent with the typical particle size characteristics of exosomes. Each distribution curve shows a unimodal distribution morphology, with a peak in the medium particle size range (approximately tens to hundreds of nanometers), indicating that the exosomes are most concentrated in this particle size range; in the extremely small and extremely large particle size segments, the particle concentration and volume density both decrease significantly. These particle size and distribution characteristics not only verify the typical physical properties of the extracted exosomes, but also provide a reliable basis for the detection, particle size characterization, and quality control of the exosomes in this invention.

[0060] like Figure 4 Figure A shows the phylogenetic clustering and heatmap of the bacterial communities carried by BEVs in the two sample groups, encompassing multiple phylum-level microbial taxa. The abundance distribution of individual ASVs (amplicon sequence variations) in the figure shows significant differences between the groups, revealing widespread and systematic microecological changes between the structurally abnormal and normal groups. Figure 4 The core differential ASV heatmap of Figure B highlights the changes in the abundance of key representative bacterial communities in the two groups. The figure focuses on the differential expression patterns of major bacterial phyla, further illustrating the bacterial community characteristics with potential diagnostic value in the structural malformation group. The results showed that the overall richness of the bacterial community in the structural malformation group was significantly higher than that in the normal group, especially in the two bacterial phyla of Firmicutes and Proteobacteria, where the relative abundance differences were particularly significant. These results suggest that fetal structural malformations may be closely related to the composition of the microbiome carried by bacterial-derived exosomes in amniotic fluid.

[0061] like Figure 5As shown in the results, Chao1 index analysis showed that the mean ± standard deviation of the structural deformity group was 244 ± 40, which was higher than the 130 ± 45 in the normal control group. Shannon index analysis showed that the structural deformity group was 3.52 ± 0.1, which was also higher than the 0.52 ± 0.1 in the normal control group. These differences were statistically significant by t-test (p < 0.001).

[0062] Example 2 Specificity and sensitivity of microbial diversity as a marker for diagnosing fetal structural malformations

[0063] The patient clinical information collected in Example 1 was analyzed together with the sequencing results.

[0064] The results are as follows Figure 6 As shown in the figure, the α-diversity index of the bacterial community was used as the detection variable to generate a receiver operating characteristic (ROC) curve. The ROC curve showed a sensitivity of 86.7% and a specificity of 83.3%, suggesting that the microbiome carried by bacterial-derived exosomes in amniotic fluid has high diagnostic efficacy for fetal structural malformations.

[0065] Example 3 Clinical application case

[0066] A routine ultrasound examination of a pregnant woman at 22 weeks of gestation showed no obvious structural abnormalities, and imaging suggested normal fetal development. However, the amniotic fluid BEVs flora diversity test results described in the present invention indicated a potential high risk (Chao1 index was 398, Shannon index was 4.7), which was significantly higher than the normal reference value for the same period. Combined with the correlation analysis between BEVs diversity and abnormal development of the central nervous system, it was suggested that the fetus was at high risk of neurodevelopment. A fetal MRI examination was performed according to clinical recommendations, and the results showed partial absence of the corpus callosum, which was diagnosed as a central nervous system structural abnormality. This case verifies the clinical foresight and application value of the present invention in early risk screening of structural abnormalities.

[0067] As can be seen from the above examples, the present invention can preliminarily determine the risk of fetal structural malformation by analyzing the changes in bacterial flora richness of extracellular vesicles in amniotic fluid and combining them with ultrasound imaging features. The results show that when the bacterial flora richness is significantly increased, it may indicate the risk of fetal structural abnormalities. This information can serve as an important reference for clinical auxiliary judgment, guiding doctors in further prenatal examinations, risk assessment, and intervention decisions.

[0068] This study uses the diversity of bacterial extracellular vesicles in amniotic fluid as a marker for predicting fetal structural malformations. This method can effectively complement traditional ultrasound screening, improve the early diagnosis of congenital structural malformations, and provide a more comprehensive clinical assessment tool. Furthermore, this technique is simple, informative, and highly sensitive, and is expected to be widely adopted worldwide, particularly in resource-limited areas.

[0069] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of amniotic fluid microbial extracellular vesicle diversity in the preparation of biomarkers for predicting fetal structural malformations.

2. The use according to claim 1, characterized in that The amniotic fluid microbial source exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks.

3. The use according to claim 1, characterized in that The indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are the Chao1 index and the Shannon index; And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

4. Application of the diversity of extracellular vesicles derived from amniotic fluid microorganisms as a marker in the preparation of products for predicting fetal structural malformations.

5. The use according to claim 4, characterized in that The product includes reagents for detecting the diversity of extracellular vesicle flora of amniotic fluid microbial origin.

6. The use according to claim 5, characterized in that The reagents include primers for amplifying 16S rDNA of extracellular vesicle flora of amniotic fluid microorganisms.

7. The use according to claim 4, characterized in that The amniotic fluid microbial exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks; And / or the indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are Chao1 index and Shannon index; And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

8. A product for predicting fetal structural malformation, characterized in that: The product includes reagents for detecting the diversity of extracellular vesicle flora of amniotic fluid microbial origin.

9. The product according to claim 8, characterized in that The amniotic fluid microbial exocyst flora is a bacterial population in exocysts in the amniotic fluid of pregnant women with a gestational age of 16 to 30 weeks; And / or the indicators of the diversity of the extracellular vesicle flora of the amniotic fluid microbial source are Chao1 index and Shannon index; And / or if the Chao1 index is >175 and the Shannon index is >0.62, fetal structural malformation is predicted.

10. The product according to claim 8, characterized in that The reagents include primers for amplifying 16S rDNA of extracellular vesicle flora of amniotic fluid microorganisms.

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