Isoprostaglandin metabolite markers for diagnosing male offspring reproductive function damage caused by exposure of nanometer plastics during pregnancy and application of isoprostaglandin metabolite markers

Abstract

The invention discloses a group of isoprostaglandin metabolite markers for diagnosing male offspring reproductive function damage caused by exposure of nano-plastics during pregnancy and application of the isoprostaglandin metabolite markers. According to the present invention, it is found that after the parental generation is exposed to the nanometer plastic, the expression level of the prostate hormone metabolism marker 8-iso PGF3 [alpha] and / or 8-is-13, 14-dihydro-15-keto-PGF2 [alpha] related to reproductive injury in the male offspring is significantly up-regulated; therefore, 8-iso PGF3 alpha and / or 8-iso-13, 14-dihydro-15-keto-PGF2 alpha are / is taken as the metabolic marker, the influence of the male offspring reproductive injury caused by the nano plastic is disclosed, and a method is provided for diagnosis and evaluation of the male offspring reproductive injury caused by the nano plastic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a set of isoprostaglandin metabolite biomarkers for diagnosing reproductive dysfunction in male offspring caused by exposure to nanoplastics during pregnancy, and their applications. Background Technology

[0002] Nanoplastics, as emerging persistent environmental pollutants, are characterized by their small particle size (typically 40-120 nm), high surface activity, and ease of bioaccumulation. They have been widely confirmed to enter pregnant women's bodies through the food chain, drinking water, and air, and can efficiently cross the placental barrier, directly interfering with fetal reproductive system development. Multiple animal models and epidemiological studies have shown that prenatal exposure to nanoplastics can induce reproductive damage phenotypes in male offspring in adulthood, including reduced sperm count, decreased sperm motility, and hormonal metabolic disorders. This developmentally originating toxicity is irreversible and carries the risk of transgenerational manifestation, making it a hot topic in global public health. There is an urgent need to develop efficient early diagnostic tools to support risk assessment and intervention strategies.

[0003] In existing technologies, the assessment of reproductive damage in male offspring caused by nanoplastics during pregnancy mainly relies on the detection of endpoint indicators after the offspring reach adulthood. These methods include: (1) histopathological analysis (such as HE staining and electron microscopy of the testes), which can reveal structural damage (such as luminal dilation or interstitial fibrosis), but is highly invasive, complex to operate, only applicable to animal experiments, and has low sensitivity to subclinical or early damage (false negative rate >30%); (2) semen parameter analysis (such as the computer-aided semen analysis system CASA to measure sperm concentration, kinematic parameters and morphological abnormality rate), which is directly related to reproductive function, but has a significant lag (requires damage to accumulate to 3 months of age), and cannot be achieved during pregnancy or early adulthood. Early screening during puberty is highly susceptible to subjective variation (reproducibility CV > 15%); (3) routine serum hormone testing (such as enzyme-linked immunosorbent assay (ELISA) to measure total testosterone or luteinizing hormone (FSH) levels), although non-invasive and easy to operate, clinical and experimental data show that in the early stages induced by nanoplastics, these hormone levels often fluctuate slightly or show no significant changes (P > 0.05, FC < 1.2), resulting in diagnostic sensitivity < 70% and insufficient specificity, making it impossible to specifically capture subtle disturbances in the prostate hormone metabolic pathway (such as isoform upregulation induced by oxidative stress).

[0004] Metabolomics, as a high-throughput, unbiased omics platform, can systematically capture the endogenous small-molecule metabolic "fingerprint" of an organism in response to environmental stress, providing strong support for the discovery of early toxic metabolic biomarkers. However, existing metabolomics studies have not reported the application of oxidative stress downstream isomers (8-iso PGF3α) and COX-2 activated metabolites (8-iso-13,14-dihydro-15-keto-PGF2α) as metabolic biomarkers in assessing reproductive damage in male offspring caused by nanoplastics during pregnancy. Summary of the Invention

[0005] To elucidate the impact of nanoplastics on the reproductive development of male offspring and to better diagnose and assess reproductive damage in male offspring, this invention provides the following technical solutions.

[0006] In a first aspect, the present invention provides the use of 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α as metabolic markers in the preparation of products for diagnosing or assessing reproductive damage in male offspring caused by nanoplastics during pregnancy.

[0007] Furthermore, the metabolic markers are selected from any of the following: (1) 8-iso PGF3α; (2) 8-iso-13,14-dihydro-15-keto-PGF2α; (3) Combination of 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α.

[0008] The 8-iso PGF3α described in this invention has the following structure: .

[0009] The 8-iso-13,14-dihydro-15-keto-PGF2α of this invention has the following structure: .

[0010] Furthermore, the assessment includes an assessment of the severity of male reproductive damage caused by nanoplastics, an assessment of the efficacy of drugs for treating male reproductive damage caused by nanoplastics, or a prognostic assessment of the treatment of male reproductive damage caused by nanoplastics.

[0011] Furthermore, the product includes reagents or devices for detecting 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α. Preferably, the reagents include kits for detecting 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α.

[0012] Furthermore, the product can diagnose or assess reproductive damage in male offspring caused by nanoplastics during pregnancy by detecting the levels of the metabolic markers.

[0013] Furthermore, the method of diagnosing or assessing male offspring reproductive damage caused by nanoplastics during pregnancy by detecting the levels of the metabolic markers includes the following steps: S1: Obtain biological samples from the male offspring to be tested; S2: Detect the level of metabolic markers in the biological sample, wherein the metabolic markers are 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α, preferably a combination of 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α; S3: Compare the levels of the metabolic markers measured in S2 with the levels of the corresponding metabolic markers in the same biological samples of healthy male offspring. S4: If the level of metabolic markers in a biological sample is higher than the level of the corresponding metabolic markers in the same biological sample of a healthy male offspring, it indicates that there is a risk of reproductive damage in the sample being tested.

[0014] Furthermore, the detection of metabolic marker levels in the biological samples includes the use of liquid chromatography-tandem mass spectrometry.

[0015] Furthermore, the detection of the metabolic biomarker levels includes using liquid chromatography-tandem mass spectrometry to obtain the mass spectrometry signal intensity. Preferably, the mass spectrometry signal intensity is expressed as the mass spectrometry peak area (AU).

[0016] Furthermore, the biological sample includes serum, plasma, or blood. Preferably, the biological sample is serum.

[0017] Furthermore, the volume of the biological sample ranges from 50 to 200 μL, with an optimal volume of 100 μL.

[0018] Furthermore, the levels of corresponding metabolic markers in the homologous biological samples of healthy male offspring are established based on homologous biological samples of healthy male offspring.

[0019] Furthermore, the levels of the corresponding metabolic markers in the homologous biological samples of healthy male offspring are based on the mean ± 2 standard deviations of the mass spectrometry signal intensity of metabolic markers in 10-50 healthy male offspring biological samples.

[0020] In one embodiment of the invention, the level of the 8-iso PGF3α metabolic marker in the homologous biological sample of the healthy male offspring ranges from 4.50 × 10⁻⁶. 6 - 5.30 × 10 6 AU; for example, 4.90 × 10 6 AU.

[0021] In one embodiment of the invention, the level of the metabolic marker 8-iso-13,14-dihydro-15-keto-PGF2α in the homologous biological sample of the healthy male offspring ranges from 1.00 × 10⁻⁶. 6 - 1.30 × 10 6 AU; for example: 1.14 × 10 6 AU.

[0022] Preferably, in the liquid chromatography-tandem mass spectrometry method, the liquid chromatography conditions include: The chromatographic column is a C18 reversed-phase column; Furthermore, the column size ranges from 1.7-5 μm particles and 100-250 mm length, with the optimal size being 2.1 mm × 100 mm and 1.8 μm particles; Furthermore, the column temperature is 25-60°C, preferably 35-45°C, and most preferably 40°C; Furthermore, the mobile phase includes mobile phase A, which is an aqueous phase, and mobile phase B, which is an organic phase; Furthermore, the mobile phase A aqueous phase contains 0.01%-0.5% formic acid or 1-20 mM ammonium formate, preferably 0.1% formic acid and 5 mM ammonium formate; Furthermore, the mobile phase B organic phase is acetonitrile or methanol, preferably acetonitrile or methanol with a purity of 80-100%, and most preferably 0.01%-0.5% formic acid acetonitrile, for example 0.1%; Furthermore, the liquid chromatography employs gradient elution. Preferably, the total run time for gradient elution is 10-30 minutes. Within 5-15 minutes, the proportion of mobile phase B linearly increases from 5%-30% to 90%-100% and is maintained for 1-5 minutes before column equilibration. The optimal program is as follows: gradient elution 0-2 min: 20% mobile phase B; 2-10 min: 20%-95% mobile phase B; 10-12 min: 95% mobile phase B maintenance; 12-15 min: 5% mobile phase B equilibration.

[0023] Preferably, in the liquid chromatography-tandem mass spectrometry method, the mass spectrometry conditions include: The ion source is an electrospray ionization source; Furthermore, the scanning mode is a multiple reaction monitoring mode; Furthermore, the ionization mode is a negative ion mode; Furthermore, the atomizing gas flow rate is 200-600 L / h, with an optimal value of 400 L / h; Furthermore, the drying airflow rate is 8-15 L / min, with an optimal rate of 10 L / min; Furthermore, the collision air pressure is 1-5 psi, with an optimal value of 3 psi; Furthermore, the source temperature is 300-500°C, with an optimal value of 450°C; Furthermore, the capillary voltage ranges from -3.5 to -5.0 kV, with an optimal value of -4.5 kV.

[0024] In a second aspect, the present invention provides a composition for the diagnosis or assessment of male offspring reproductive damage caused by nanoplastics, said composition comprising a combination of the metabolic markers 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α.

[0025] Thirdly, the present invention provides a method for constructing an animal model of male offspring reproductive damage induced by nanoplastics, the method comprising the following steps: 1) The animal was exposed to nanoplastics; 2) Detect the levels of metabolic markers 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α in offspring animal biological samples.

[0026] Preferably, the animal is exposed by gavage.

[0027] Preferably, the animal exposure continues from 0.5 days of gestation until parturition.

[0028] Preferably, the animal is a rodent.

[0029] More preferably, the animal is a mouse, and even more preferably an ICR mouse.

[0030] Preferably, the particle size range is 40-120 nm, and the optimal range is 50-100 nm, with a zeta potential of -20 to -40 mV.

[0031] The beneficial effects of this invention are: This invention uses 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α metabolic markers to reveal the effects of nanoplastics on the reproductive development of male offspring, providing a method for the diagnosis and assessment of male reproductive damage caused by nanoplastics during pregnancy, and is of great significance for early risk assessment of male reproductive damage.

[0032] The metabolic biomarkers of this invention exhibit high specificity and accuracy in diagnosing reproductive damage in male offspring induced by nanoplastics during pregnancy, with areas under the ROC curve (AUC) of 0.92 and 0.96, respectively. By combining the two metabolic biomarkers, the diagnostic AUC can reach close to 1.00 (i.e., 100% sensitivity and specificity), significantly reducing the risk of misdiagnosis.

[0033] This invention requires only a peripheral blood sample (serum, plasma, or whole blood) for testing, avoiding the risks and discomfort associated with invasive procedures such as tissue biopsy or semen collection, thus improving patient compliance. Furthermore, the standardized procedure facilitates its application in clinical screening and large-scale epidemiological surveillance.

[0034] This invention can reliably distinguish between physiological variations and exposure effects even under low concentration (ng / mL) and low dose (50 mg / kg / d) exposure, overcoming the limitation of insufficient detection capability of immunoassay methods for low-abundance metabolic biomarkers.

[0035] The metabolic biomarkers of this invention not only support single exposure diagnosis, but can also be integrated into multimodal risk assessment models, supporting research on the reproductive toxicity mechanisms of nanoplastics, screening for protective interventions, and environmental toxicant monitoring systems, and promoting the application of the entire chain from prevention screening to drug validation. Attached Figure Description

[0036] Figure 1 The figure shows the dose-response effect of nanoplastics on sperm density and sperm motility in male offspring.

[0037] Figure 2 The effect of different concentrations of nanoplastics on the mass spectrometry signal intensity of 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α is shown.

[0038] Figure 3 The figure shows the ROC curve analysis of 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α. Figure 4 The figure shows the linear correlation analysis between the concentration of nanoplastics and 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions in the art or as recommended by the manufacturer. Unless otherwise specified, all methods are conventional. Unless otherwise defined, technical and scientific terms used herein have the same meaning as those familiar with the art.

[0040] The term "treatment" as used in this invention refers to slowing down, interrupting, preventing, controlling, stopping, alleviating, reducing, or reversing a sign, symptom, disorder, condition, or progression or severity of a disease after it has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0041] The "prognostic assessment" described in this invention refers to predicting the possible course and outcome of a disease, including determining the specific consequences of the disease.

[0042] The "efficacy assessment" described in this invention refers to assessing the patient's response to treatment.

[0043] The "monitoring" mentioned in this invention refers to observing the occurrence and development of diseases.

[0044] The "prevention" mentioned in this invention refers to the specific measures taken by an individual to prevent the occurrence of a disease before it is diagnosed or develops.

[0045] The term "diagnosis" in this invention refers to determining whether a patient has had a disease or condition in the past, at the time of diagnosis, or in the future, or to determining the progression of a disease or its possible future progression.

[0046] The sources of some of the materials and experimental methods involved in this application are as follows: SPF-grade male and female ICR mice (8 weeks old), Charles River Laboratories or Beijing Vital River Laboratory Animal Technology Co., Ltd. Nanoplastic raw material: polystyrene nanospheres with a particle size of 50-100 nm, Sigma-Aldrich.

[0047] Example 1: Effects of nanoplastics on reproductive damage in male offspring 1.1 Establishment of an animal model of prenatal exposure to nanoplastics SPF-grade male and female ICR mice (8 weeks old) were selected and naturally mated at a ratio of 1:2. Female mice were designated as having an embryonic development of 0.5 days post-coital (dpc) after the observation of vaginal plugs. Pregnant mice were divided into three groups (n=4 per group): control group (equal volume of physiological saline), low-dose group (50 mg / kg / d nanoplastic suspension), and high-dose group (500 mg / kg / d nanoplastic suspension). Suspension preparation involved dissolving nanoplastic powder in physiological saline (ultrasonic dispersion for 30 min, power 100 W, concentration 5-50 mg / mL). Mice in the control, low-dose, and high-dose groups were administered the suspension by gavage daily from 0.5 dpc to parturition between 9:00 and 10:00 AM (0.1 mL / mouse, 0.5 mL / min). The weight of pregnant mice was monitored weekly to ensure fluctuations <10%. Male offspring (one offspring from each litter was randomly selected for each group, with a total of n=4 groups for subsequent analysis) were taken, and at 3 months of age, they were euthanized after anesthesia, and the epididymis was extracted for the experiment.

[0048] 1.2 Assessment of offspring reproductive function development For sperm motility analysis, after obtaining the animal sample, the extracted unilateral epididymis was quickly placed into 1 mL of sterile Dulbecco's Modified Eagle Medium (DMEM) preheated to 37°C. The epididymis was then cut into small pieces and incubated in a 37°C incubator for 10 min until the sperm were completely freed into the DMEM. The mixture was gently stirred, and 10 μL of the sperm suspension was added to a special slide for the semen analyzer. The slide was then gently covered with a coverslip, and the sperm motility index was analyzed using a fully automated semen analyzer (Hamilton Thorne IVOS-II, USA).

[0049] For sperm counting, a sample from the other epididymis was taken, chopped, and placed in preheated DMEM. The sample was incubated at 37°C for 3 minutes to allow the sperm to swim out. The sperm suspension was then counted using a computer-aided sperm analysis system.

[0050] like Figure 1 As shown, compared with the control group, both the low-dose group and the high-dose group had reduced sperm concentration and motility, confirming that nanoplastics caused reproductive damage in male offspring mice.

[0051] Example 2: Serum metabolomics analysis and identification of target metabolic biomarkers 2.1 Construction of animal models SPF-grade male and female ICR mice (8 weeks old) were selected and naturally mated at a ratio of 1:2. Female mice were designated as having an embryonic development of 0.5 days post-coital (dpc) after the observation of vaginal plugs. Pregnant mice were divided into three groups (n=4 per group): control group (equal volume of physiological saline), low-dose group (50 mg / kg / d nanoplastic suspension), and high-dose group (500 mg / kg / d nanoplastic suspension). Suspension preparation involved dissolving nanoplastic powder in physiological saline (ultrasonic dispersion for 30 min, power 100 W, concentration 5-50 mg / mL). Mice in the control, low-dose, and high-dose groups were administered the suspension by gavage daily from 0.5 dpc to parturition between 9:00 and 10:00 AM (0.1 mL / mouse, 0.5 mL / min). The weight of pregnant mice was monitored weekly to ensure fluctuations <10%. Male offspring (one offspring from each litter was randomly selected for each group, with a total of n=4 groups for subsequent analysis) were collected. At 3 months of age, the offspring were euthanized by overdose and blood samples (0.5 mL / offspring) were collected. Serum was obtained by centrifugation at 15000 g for 12 min at 4°C and stored at -80°C.

[0052] 2.2 Preparation of Metabolite Extract The specific process is as follows: (1) Place the serum stored at -80°C on ice and thaw slowly (<30 min). After thawing, vortex to mix for 10 s (2000 rpm).

[0053] (2) Take 100 μL of serum and transfer it to a pre-cooled 2.0 mL EP centrifuge tube (-20°C).

[0054] (3) Add 400 μL of pre-cooled 80% methanol aqueous solution (LC-MS grade, pre-cooled to -20°C).

[0055] (4) High-speed vortex vibration for 3 min (3000 rpm), then ice bath for 5 min (0-4°C).

[0056] (5) Centrifuge at 15000 g for 20 min at 4°C.

[0057] (6) Take 300 μL of supernatant and add 169.8 μL of mass spectrometry grade water to dilute it, so that the methanol content is 53%.

[0058] (7) Centrifuge again at 15000 g for 15 min at 4°C to remove residual precipitate.

[0059] (8) Collect the supernatant, which is the metabolite extract (~500 μL) for analysis by liquid chromatography-mass spectrometry (LC-MS).

[0060] QC / blank samples are processed in the same way (one QC is inserted for every 10 samples).

[0061] 2.3 The LC-MS method was used to analyze the samples. (1) Liquid chromatography conditions Chromatography system: Thermo Dionex Ultimate 3000 ultra-high performance liquid chromatography system; Chromatographic column: Hypersil Gold C18 column, 100 mm × 2.1 mm, 1.9 μm; Column temperature: 40°C; Mobile phase A: Aqueous phase, 0.1% formic acid aqueous solution; Mobile phase B: Organic phase, methanol; Flow rate: 0.2 mL / min; Injection volume: 5 μL; Gradient elution program: 0 min 98%A / 2%B; 1.5 min 98%A / 2%B; 3 min 15%A / 85%B; 10 min 0%A / 100%B; 11.1 min 98%A / 2%B; 12 min 98%A / 2%B.

[0062] (2) Mass spectrometry conditions Mass spectrometry system: Q Exactive HF-X (Thermo); Ion source: Electrospray ionization source; Scan mode: Full scan / dd-MS 2 (m / z 100-1500); Spray voltage: 3.5 kV; Sheath flow velocity: 35 arb; Auxiliary airflow rate: 10 L / min; Capillary temperature: 320°C; Auxiliary gas heater temperature: 350°C; S-lens: 60; Positive / negative ion mode: Negative ions are preferred.

[0063] (3) Data preprocessing and biomarker identification The raw data files acquired by the mass spectrometer were imported into the data processing software Compound Discoverer 3.3 for peak extraction, with a signal-to-noise ratio (S / N) threshold greater than 10. The extracted peaks were aligned, and the retention time (RT) deviation was set to 0.2 minutes. Quality control (QC) samples were used for data correction, and compounds with a coefficient of variation (CV) ≥ 30% were removed. The retained compounds were compared with the database (mzCloud / HMDB), and compounds with a quality error of less than 5 ppm and a matching score greater than 80% were selected.

[0064] (4) Data analysis and diagnosis Partial Least Squares Discriminant Analysis (PLS-DA) was performed using the MetaboAnalyst R package. The model validity criterion was R. 2 X>0.5 and Q 2 >0.6, and the Variable Importance in Projection (VIP) value of metabolites was calculated; the relative abundance of metabolites between the exposed group and the control group was compared by Bonferroni-corrected t-test to obtain the P value and Fold Change (FC) value; the screening criteria for differential metabolites were that they simultaneously met: VIP>1, P<0.05 and |log2(FC)|>0.26 (corresponding to FC>1.2).

[0065] Untargeted metabolomics analysis identified 1719 metabolites, covering lipids, amino acids, and small molecules. Differential metabolites were screened using PLS-DA analysis and Bonferroni-corrected t-tests, with VIP>1, P<0.05, and |log2(FC)|>0.26 criteria: 184 metabolites in the low-dose group (50 mg / kg / d) and 146 metabolites in the high-dose group (500 mg / kg / d). These differential metabolites were mainly enriched in oxidative stress (ROS pathway) and prostate hormone metabolism pathways (e.g., upregulation of PGE2 / PGF2α isoform expression). Typical differentially expressed metabolites such as 15-keto-PGF2α (low-dose group: VIP=1.45, log2(FC)=1.82, P=0.012) and TXB2 (high-dose group: VIP=1.32, log2(FC)=1.56, P=0.008) have two problems: the overlap between groups is >20%, and the trend of change is inconsistent; and the association with the mechanism of reproductive damage is weak (KEGG pathway score <3.5).

[0066] like Figure 2As shown, based on biological significance (most directly related to the prostate hormone metabolic pathway), pathway analysis (KEGG enrichment, adjusted P < 0.01), and dose-response (both exposure groups showed upregulation of more than 2-fold), two key metabolic biomarkers were screened: 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α. In the exposure group, both showed high consistency and a significant upregulation trend. 8-iso PGF3α is an isomer of PGF3α (a downstream product of oxidative stress), and 8-iso-13,14-dihydro-15-keto-PGF2α is a metabolite of PGF2α (a COX-2 activated product). The combination of these two biomarkers more comprehensively and specifically reflects the disordered lipid peroxidation and prostate damage network induced by nanoplastics during pregnancy (pathway score > 5.0). Compared with other differentially expressed compounds, the dose-response curve of this combination of metabolic biomarkers showed good linearity (R0). 2 >0.95) and small variation (CV<15%).

[0067] Example 3: Construction and Efficacy Verification of Diagnostic Thresholds Based on ROC Curve Analysis 3.1 Construction of animal models SPF-grade male and female ICR mice (8 weeks old) were selected and naturally mated at a ratio of 1:2. Female mice were designated as having an embryonic development of 0.5 days post-coital (dpc) after the observation of vaginal plugs. Pregnant mice were divided into three groups (n=4 per group): control group (equal volume of physiological saline), low-dose group (50 mg / kg / d nanoplastic suspension), and high-dose group (500 mg / kg / d nanoplastic suspension). The nanoplastic suspension was prepared by dissolving nanoplastic powder in physiological saline (ultrasonic dispersion for 30 min, power 100 W, concentration 5-50 mg / mL). Mice in the control, low-dose, and high-dose groups were administered the suspension by gavage daily from 0.5 dpc to parturition between 9:00 and 10:00 AM (0.1 mL / mouse, 0.5 mL / min). The weight of pregnant mice was monitored weekly to ensure fluctuations <10%. Male offspring (one offspring from each litter was randomly selected for each group, with a total of n=4 groups for subsequent analysis) were collected. At 3 months of age, the offspring were euthanized by overdose and blood samples (0.5 mL / offspring) were collected. Serum was separated by centrifugation at 15000 g for 12 min at 4°C and stored at -80°C.

[0068] 3.2 Construction of the Health Reference Cohort Another 30 healthy male ICR mice (3 months old) with no history of nanoplastic exposure were euthanized by overdose and blood samples (0.5 mL / mouse) were collected. Serum was obtained by centrifugation at 15000 g for 12 min at 4°C and stored at -80°C.

[0069] The mass spectrometry signal intensity of two metabolic markers in serum was detected by LC-MS (under the same conditions as in Example 2), and the reference range of the two metabolic markers was calculated, wherein the reference range = mean ± 2 × standard deviation.

[0070] 3.3 ROC Analysis Process ROC curve analysis using the R pROC package: (1) Data set integration: The experimental group (4 cases in the control group + 8 cases in the exposure group) and the healthy cohort (30 cases) constitute the total sample set; (2) ROC analysis of single metabolic markers: ROC curve analysis was performed on 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α respectively. The optimal cutoff value of the metabolic marker was determined by maximizing the Youden index (Youden index = sensitivity + specificity - 1).

[0071] (3) ROC analysis of the dual metabolic biomarker combined model: A dual metabolic biomarker combined diagnostic model was established by logistic regression. The model expression is as follows:

[0072] in, It is probability Logit transformation value, For the predicted probability of exposure risk, For the intercept term, The regression coefficients of 8-iso PGF3α are... The regression coefficients are 8-iso-13,14-dihydro-15-keto-PGF2α. The risk probability output by the model is used as the diagnostic score, and the optimal probability cutoff value in the probability space is determined by maximizing the Youden exponent.

[0073] (4) Optimal cutoff value verification and output The optimal cutoff value for 8-iso PGF3α is 4.90 × 10⁻⁶. 6 The optimal cutoff value for AU,8-iso-13,14-dihydro-15-keto-PGF2α is 1.14 × 10⁻⁶. 6 AU, the result is as follows Figure 3 As shown.

[0074] 3.4 Diagnostic efficacy verification results As shown in Table 1, the single metabolic markers 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α exhibited sensitivities of 83% and 85.7%, respectively, demonstrating good diagnostic efficacy. The combined metabolic markers further improved diagnostic performance, with an AUC 4-8% higher than the single markers and a sensitivity reaching 100% (17% higher than 8-iso PGF3α and 14.3% higher than 8-iso-13,14-dihydro-15-keto-PGF2α). The complementary mechanism of this combined metabolic markers lies in the fact that 8-iso PGF3α primarily responds to early signals of oxidative stress, while 8-iso-13,14-dihydro-15-keto-PGF2α reflects the progression of chronic prostate injury, forming a complementary monitoring system over time. These results confirm that the dual metabolic marker combination strategy has high-precision early warning value for reproductive damage induced by nanoplastics in male offspring mice.

[0075] Table 1

[0076] Example 4: Diagnostic efficacy verification in a double-blind validation group 4.1 Construction of double-blind grouped animal models SPF-grade male and female ICR mice (8 weeks old) were selected by experimenter A (blindly grouped). The males and females were naturally mated at a ratio of 1:2. Female mice were designated as embryonic development 0.5 dpc (days postcoitum) after vaginal plugs were observed.

[0077] Researcher A randomly divided six pregnant mice into three groups (n=2 per group): a control group (equal volume of physiological saline), a low-dose group (50 mg / kg / d nanoplastic suspension), and a high-dose group (500 mg / kg / d nanoplastic suspension). Suspension preparation involved dissolving nanoplastic powder in physiological saline (ultrasonic dispersion for 30 min, power 100 W, concentration 5-50 mg / mL).

[0078] Mice in the control, low-dose, and high-dose groups were administered gavage (0.1 mL / mouse, 0.5 mL / min) daily from 0.5 days of gestation until delivery. The weight of pregnant mice was monitored weekly, ensuring fluctuations were <10%. Group information was sealed and transferred to researcher B (blinded by exposure history).

[0079] After parturition, one male offspring from each litter was randomly selected and sacrificed at 3 months of age under beta anesthesia (sodium pentobarbital 50 mg / kg) to collect orbital venous blood (0.5 mL / offspring). Serum was obtained by centrifugation at 15000 g for 12 min at 4°C and stored at -80°C. Samples were anonymized (e.g., Sample-1 to Sample-6) and blinded to the exposure group.

[0080] 4.2 Preparation of Metabolite Extract The specific process is as follows: (1) Place the serum stored at -80°C on ice and thaw slowly (<30 min). After thawing, vortex to mix for 10 s (2000 rpm).

[0081] (2) Take 100 μL of serum and transfer it to a pre-cooled 2.0 mL EP tube (-20°C).

[0082] (3) Add 400 μL of pre-cooled 80% methanol aqueous solution (v / v, LC-MS grade).

[0083] (4) High-speed vortex for 3 min (3000 rpm), then ice bath for 5 min (0-4°C).

[0084] (5) Centrifuge at 4°C and 15000 g for 20 min.

[0085] (6) Take 300 μL of supernatant and add 169.8 μL of mass spectrometry grade water (final methanol 53%).

[0086] (7) Centrifuge at 4°C and 15000 g for 15 min.

[0087] (8) Obtain metabolite extract (~500 μL).

[0088] QC / blank samples are processed in the same way (one QC is inserted for every 10 samples).

[0089] 4.3 LC-MS for the detection of biological samples (1) Hypersil Gold C18 column (100 mm × 2.1 mm, 1.9 μm), column temperature 40°C; mobile phase A: aqueous phase, 0.1% formic acid aqueous solution; mobile phase B: organic phase, methanol; flow rate 0.2 mL / min; injection volume 5 μL; gradient elution program: 0 min 98%A / 2%B → 1.5 min 98%A / 2%B → 3 min 15%A / 85%B → 10 min 0%A / 100%B → 11.1 min 98%A / 2%B → 12 min 98%A / 2%B.

[0090] (2) Q Exactive HF-X (Thermo), electrospray ionization source; full scan / dd-MS 2 (m / z 100-1500); spray voltage 3.5 kV; sheath gas 35 arb; auxiliary gas flow rate 10 L / min; capillary temperature 320°C; auxiliary gas heater temperature 350°C; S-lens 60. Positive / negative ion mode, with negative ions preferred.

[0091] (3) Import the raw data file collected by the mass spectrometer into the data processing software Compound Discoverer 3.3 to extract the peak area and intensity.

[0092] Experimenter B determined the optimal cutoff value based on ROC (8-iso PGF3α ≥ 4.90 × 10⁻⁶). 6 AU; 8-iso-13,14-dihydro-15-keto-PGF2α ≥ 1.14 × 10 6 AU) Judgment: If at least one metabolic marker is above the optimal cutoff value, the individual is classified as "exposed group" (further dose allocation based on mass spectrometry signal intensity gradient: 2.5-4.5 × 10⁻⁶). 6 AU is a low dose, >5.0 × 10 6 AU represents the high-dose group; otherwise, it is the "control group". The groups were then unblinded to compare accuracy. Statistics: t-test (GraphPadPrism) was used to calculate differences between groups (P-value); a logistic model was used to assess the accuracy of combined diagnoses.

[0093] Double-blind assays successfully identified the mass spectrometry signal intensities of two metabolic markers. The mass spectrometry signal intensity in the exposed group was significantly higher than that in the control group (P<0.01, FC>2.0). Based on the optimal cutoff value, the grouping accuracy was 100% (6 / 6 samples were correctly classified: 2 / 2 in the control group, 2 / 2 in the low-dose group, and 2 / 2 in the high-dose group). Combined use improved the discrimination of the low-dose group (83% accuracy for a single metabolic marker, 100% for the combined use). Specific mass spectrometry signal intensity data are shown in Table 2 (n=2 / group).

[0094] Table 2

[0095] like Figure 4 As shown, with increasing exposure doses of nanoplastics (0–500 mg / kg / d), the levels of the two metabolic markers were significantly higher than those in the control group, and showed a dose-dependent increasing trend, suggesting that these two metabolic markers can respond to nanoplastics in a dose-dependent manner.

[0096] Based on the results of previous diagnostic performance evaluations, we can further conclude that these two metabolic biomarkers not only change dynamically with nanoplastic dosage, but also accurately distinguish nanoplastic exposure under double-blind conditions, verifying their reliability as biomarkers of nanoplastic metabolism and providing experimental evidence for their subsequent transformation into clinical testing tools.

[0097] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

The application of 1,8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α as metabolic markers in the preparation of products for diagnosing or assessing reproductive damage in male offspring caused by nanoplastics during pregnancy.

2. The application according to claim 1, characterized in that, The assessment includes evaluating the severity of male reproductive damage caused by nanoplastics, evaluating the efficacy of drugs for treating male reproductive damage caused by nanoplastics, or evaluating the prognosis of treatment for male reproductive damage caused by nanoplastics.

3. The application according to claim 1, characterized in that, The products described include reagents or devices for detecting 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α.

4. The application according to claim 1, characterized in that, The reagents include kits for detecting 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α.

5. The application according to claim 4, characterized in that, The method of diagnosing or assessing male reproductive damage in offspring caused by nanoplastics during pregnancy by detecting the levels of the metabolic markers includes the following steps: S1: Obtain biological samples from the male offspring to be tested; S2: Detect the levels of metabolic markers in the biological sample, wherein the metabolic markers are 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α; S3: Compare the measured levels of the metabolic markers with the corresponding levels of metabolic markers in the same biological samples of healthy male offspring; S4: If the level of metabolic markers in a biological sample is higher than the level of the corresponding metabolic markers in the same biological sample of a healthy male offspring, it indicates that there is a risk of reproductive damage in the sample being tested.

6. The application according to claim 5, characterized in that, The detection of metabolic marker levels in the biological sample includes the use of liquid chromatography-tandem mass spectrometry.

7. The application according to claim 6, characterized in that, The detection of metabolic marker levels in the biological sample includes using liquid chromatography-tandem mass spectrometry to detect the mass spectrometry signal intensity.

8. The application according to claim 6, characterized in that, The biological samples mentioned include serum, plasma, or blood.

9. A composition for the diagnosis or assessment of male offspring reproductive damage induced by nanoplastics, characterized in that, The composition comprises a combination of the metabolic markers 8-iso PGF3α and 8-iso-13,14-dihydro-15-keto-PGF2α.

10. A method for constructing an animal model of male offspring reproductive damage induced by nanoplastics, the method comprising the following steps: 1) Exposing animals to nanoplastics; 2) Detect the levels of metabolic markers 8-iso PGF3α and / or 8-iso-13,14-dihydro-15-keto-PGF2α in offspring animal biological samples.

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