Premature delivery risk assessment method based on abundance of estradiol degrading gene in excrement sample in early pregnancy

Abstract

The invention discloses a premature delivery risk assessment method based on abundance of estradiol degradation genes in excrement samples in the early pregnancy stage, and the method is used for assessing the premature delivery risk of a maternal body based on abundance information of novel estradiol degradation genes in the excrement samples of individuals in the early pregnancy stage. The specific gene is k1412944177, and the nucleotide sequence of the specific gene is SEQ. NO. 1. Research finds that the gene can be used as a biomarker for evaluating the premature delivery risk at the early pregnancy stage, and by detecting the relative abundance of the gene in a maternal excrement sample at the early pregnancy stage, a scientific and accurate risk evaluation basis can be provided for the maternal premature delivery risk. And the expression of the biomarker also has the capability of degrading and regulating estradiol related to premature delivery, so that a brand new thought is provided for precise prevention of premature delivery and individualized obstetrical management.

Description

Technical Field

[0001] This invention relates to the field of precision medicine for the purpose of predicting the risk of preterm birth in early pregnancy, and in particular to a biomarker, method, and use in the preparation of preterm birth risk assessment products based on specific gene abundance information in individual fecal samples in early pregnancy. Background Technology

[0002] Timely initiation of labor is crucial for perinatal outcomes, and preterm birth (defined as delivery between 28 and 37 weeks of gestation) is the leading cause of neonatal and under-five mortality worldwide, posing a serious global health and social challenge. Human childbirth is a multifactorial physiological process influenced by both maternal genetic makeup and environmental exposure. The maternal microbiome, as a key carrier reflecting external environmental exposure, has been shown to reshape host metabolism and overall health throughout pregnancy; however, its specific mechanisms of action in the occurrence of preterm birth still require systematic exploration.

[0003] However, current clinical predictions of preterm birth risk primarily rely on a comprehensive assessment of maternal serum metabolites, inflammatory factors, clinical indicators, and past obstetric history. These methods generally have limitations such as invasive sampling, high costs, or late predictive windows, making it difficult to achieve non-invasive and efficient risk identification in early pregnancy. This situation highlights the urgent need to explore new predictive indicators (such as the maternal gut microbiome).

[0004] Further research has revealed that estradiol, a key hormone regulating pregnancy maintenance and labor initiation, has a clear mechanistic link between its homeostasis dysregulation and preterm birth. In particular, its oxidized metabolite, estrone, increases by more than 50% during pregnancy, possessing significant predictive value for delivery time. However, the specific mechanisms by which the maternal gut microbiota participates in preterm birth by influencing steroid hormone metabolism remain poorly understood, providing a potential entry point for microbiome-based preterm birth prediction.

[0005] Against this backdrop, a preterm birth risk prediction model centered on the maternal gut microbiome is proposed. This model aims to overcome the limitations of existing prediction methods by enabling early identification of high-risk pregnant women through non-invasive fecal sampling in early pregnancy. This non-invasive and cost-effective microbiome prediction strategy not only has high clinical translational potential but also provides direction for subsequent interventions. For example, it can reshape the maternal gut microbiota through early lifestyle modifications, probiotic supplementation, or dietary interventions, thereby reducing the risk of preterm birth and opening new pathways for precision prevention and personalized obstetric management. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a biomarker, method, and application for assessing maternal preterm birth risk in early pregnancy. This invention assesses maternal preterm birth risk in early pregnancy based on the abundance information of specific genes in the maternal gut microbiota, guiding the host to improve reproductive health outcomes by modifying lifestyle or dietary structure.

[0007] Specifically, this invention discovers and verifies a specific gene sequence derived from gut microbiota, the nucleotide sequence of which is (SEQ. NO. 1):

[0008] .

[0009] The expression level of this gene in maternal fecal samples during early pregnancy can serve as a biomarker for assessing the risk of preterm birth. By detecting the expression level of this gene and combining it with a preset threshold, an accurate assessment of the maternal risk of preterm birth in early pregnancy can be achieved.

[0010] The technical solution adopted in this invention is as follows:

[0011] 1) Collect fecal samples from the individuals to be tested;

[0012] 2) Detect the relative abundance of the biomarkers in the sample and compare it with a preset threshold to assess the risk of preterm birth.

[0013] The abundance of the biomarker is expressed as "reads per kilobase per million mapped reads" (RPKM). The RPKM is calculated by dividing the number of sequencing reads mapped to the gene by the product of the gene length (in kilobases) and the total number of mapped reads (in millions), thereby correcting for the influence of gene length and sequencing depth on expression levels and ensuring comparability of expression levels between different genes or different samples.

[0014] 3) The preset threshold for the biomarker is 664011 RPKM; when the expression level of the biomarker is ≥664011 RPKM, the individual is determined to be a high-risk individual for premature birth.

[0015] The present invention also provides the application of the biomarker in the preparation of a kit for assessing the risk of preterm birth in early pregnancy, the kit being used to detect the abundance of the gene in a sample.

[0016] The present invention also provides the use of reagents for detecting the abundance of the biomarkers described above in fecal samples in the preparation of kits for assessing the risk of preterm birth in early pregnancy.

[0017] The present invention also provides a kit for assessing the risk of preterm birth in early pregnancy, comprising reagents for detecting the abundance of the biomarkers in a sample.

[0018] The present invention also provides the use of a gene fragment with the nucleotide sequence SEQ. NO.1, or its expression vector, or its encoded protein in the preparation of estradiol-degrading drugs.

[0019] The beneficial effects of this invention are:

[0020] This invention proposes a biomarker, method, and application in the preparation of preterm birth risk assessment products based on specific gene abundance information in individual fecal samples during early pregnancy. Furthermore, the expression of this biomarker has the ability to degrade and regulate estradiol associated with preterm birth, which provides a novel approach for precise prevention of preterm birth and individualized obstetric management. Attached Figure Description

[0021] Figure 1 This figure presents the results of a study on the association between the abundance of the gene k141_29441_57, discovered in this invention, and preterm birth. The odds ratio (OR) data for preterm birth in the figure show that the abundance of this gene is associated with a higher risk of preterm birth in pregnant women. The data comes from a cohort of 615 pregnant women from China. After initial data collection, all participants underwent longitudinal follow-up assessment throughout their pregnancy, tracking them until delivery.

[0022] Figure 2 This describes the degradation of estradiol by the gene k141_29441_57 discovered in this invention in an in vitro constructed Escherichia coli expression system. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0024] Example 1: Obtaining the gene sequence

[0025] 1. Study Participant Inclusion and Sample Collection: 615 pregnant Chinese women were included as study participants. Stool samples were collected during their first trimester (mean gestational age 10.4 ± 2.0 weeks). All participants were followed up throughout the entire pregnancy, and their final gestational age at delivery, whether preterm birth occurred, and related clinical case information (including age, gestational age, pregnancy complications, etc.) were recorded.

[0026] 2. Gut Microbiome Metagenomic Sequencing and Analysis: Gut microbiome metagenomic sequencing analysis was performed on collected fecal samples. Specific steps included: extracting total DNA from the samples, constructing sequencing libraries using the Illumina high-throughput sequencing platform, and performing paired-end high-throughput sequencing; quality control of the raw sequencing data (removing low-quality reads, adapter sequences, and host contamination sequences); and assembling the sequences using metagenomic assembly tools (such as MEGAHIT) to obtain 30,230 high-quality metagenomic assembled genomes.

[0027] 3. Target Gene Screening and Sequence Acquisition: For the MAGs obtained above, functional genes potentially involved in estradiol regulation were systematically screened through homology analysis (BLAST alignment), structural analysis (using AlphaFold to predict protein tertiary structure), and molecular docking analysis (using DiffDock to simulate interactions with estradiol). The results showed that the gene fragment numbered k141_29441_57 exhibited the best performance in terms of binding energy with estradiol, structural matching degree, and homology functional conservation, and was identified as the most promising gene fragment for regulating estradiol function. The nucleotide sequence of this target gene is SEQ. NO. 1.

[0028] 4. Gene Function Verification: Forward (5′-cgatggccatggatatcggaATGAAGGCGTTGATTACAGGTGC-3′) and reverse (5′-tggtgctcgagtgcggccgcCCTGTTTTTCCGATGCTGTATATG-3′) PCR primers for k141_29441_57 were synthesized in vitro. Using bacterial culture as a template, the k141_29441_57 sequence was amplified from bacteria using PCR. Subsequently, homologous recombination was performed using a pair of homologous recombination primers (forward: 5′-GCGGCCGCACTCGAGCAC-3′; reverse: 5′-TCCGATATCCATGGCCATCG-3′) to insert the k141_29441_57 fragment into the purchased pET26a universal vector. Based on this, the plasmid was chemically transformed and introduced into *E. coli* DH5α competent cells, thus constructing an *E. coli* heterologous expression system for k141_29441_57. Simultaneously, an unmodified pET26a empty vector plasmid was introduced into another group of *E. coli* competent cells as a control. Functional validation of the target gene k141_29441_57 was performed using the in vitro heterologous expression system. The results showed that the protein expressed by this gene has significant estradiol degradation activity. Figure 2 Data shows that, compared to empty vector E. coli without the target gene inserted, E. coli expressing the gene k141_29441_57 can significantly reduce the level of estradiol in the culture medium in vitro, indicating that the gene has the ability to degrade estradiol.

[0029] Example 2: The link between genes and preterm birth

[0030] 1. Target gene quantification: Based on metagenomic assembly data from 615 pregnant women in early pregnancy, the abundance of the target gene k141_29441_57 was quantitatively analyzed.

[0031] 2. Definition of delivery outcome: All pregnant women included in the study were followed longitudinally throughout their entire pregnancy, and their gestational age at delivery and other clinical outcomes were systematically recorded. Gestational age (in weeks) was initially estimated based on the date of the last menstrual period and then corrected for using early pregnancy ultrasound examination results; preterm birth was defined as delivery before 37 weeks of gestation.

[0032] 3. Association Analysis: After Z-score standardization of the relative abundance of the target gene, logistic regression analysis was used to analyze its association with the risk of preterm birth, and the corresponding odds ratio (OR) and significance p-value were calculated. Figure 1 The results showed that the higher the abundance of this gene, the greater the risk of premature birth in pregnant women, and this was statistically significant (P<0.05).

[0033] Based on the above research plan and experimental results, this invention identifies microbial gene characteristics significantly associated with preterm birth risk by analyzing the abundance of a specific estradiol degradation gene, k141_29441_57, in maternal fecal samples from early pregnancy. This gene originates from the gut microbiota, and its expression level can be quantified through metagenomic sequencing, assessed using RPKM (record length per kilobase per million reads) as a standardized indicator. When the expression level of this gene reaches a preset threshold (664011 RPKM), the individual can be identified as a high-risk pregnant woman for preterm birth.

[0034] This invention provides a method for assessing the risk of preterm birth based on the abundance of specific genes in fecal samples from early pregnancy, which has the following advantages: 1) Non-invasive: The test can be completed by collecting only a fecal sample, which is non-invasive and harmless to pregnant women; 2) High efficiency and accuracy: Combining metagenomic sequencing and normalized abundance calculation (RPKM), it has high reproducibility and accuracy; 3) Strong clinical translation: It can be used for the early identification of individuals at high risk of preterm birth, providing a scientific basis for subsequent clinical intervention.

Claims

1. A biomarker for assessing the risk of preterm birth in early pregnancy, characterized in that, The biomarker is a gene fragment, the nucleotide sequence of which is SEQ.NO.

1.

2. The use of the biomarker of claim 1 in the preparation of a kit for assessing the risk of preterm birth in early pregnancy, wherein the kit is used to detect the abundance of the gene in a sample.

3. The application according to claim 2, characterized in that, The sample in question is a maternal fecal sample.

4. The application according to claim 2, characterized in that, The abundance of the gene is expressed as RPKM, which is the number of reads per kilobase length of the gene per million sequencing reads mapped to the genome.

5. The application according to claim 4, characterized in that, When the abundance value of the gene reaches or exceeds the preset threshold of 664011 RPKM, the mother is determined to be a high-risk individual for premature birth.

6. Use of the reagent for detecting the abundance of the biomarker as described in claim 1 in a fecal sample in the preparation of a kit for assessing the risk of preterm birth in early pregnancy.

7. A kit for assessing the risk of preterm birth in early pregnancy, characterized in that, The kit includes reagents for detecting the abundance of the biomarker as described in claim 1 in a sample.

8. The use of a gene fragment with the nucleotide sequence SEQ. NO. 1, or its expression vector, or its encoded protein, in the preparation of a drug that degrades estradiol.

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