Study participants were chosen from the Guizhou Cancer Center, Guizhou Provincial People’s Hospital, and Songtao Miao Autonomous County People’s Hospital in Guizhou province to screen for colorectal adenomas. Patients were initially seen at the endoscopy centers of the aforementioned hospitals. Participants were 18-75 years of age and in generally good health with no vital organ failures. According to WHO guidelines, a colorectal adenoma diagnosis was made, and the degree of neoplasia was assessed. The following criteria was used to diagnosis colorectal adenoma: ①Adenoma was verified by a pathological biopsy, ② villous adenoma or mixed adenoma with more than 25% villous-like features was identified, ③high-grade epithelial neoplasia was identified. Patients with colorectal adenoma who met the aforementioned diagnostic standards had a subsequent colonoscopy adenectomy, and histology was used to confirm the diagnosis. All specimens were examined by two or more experienced pathologists in the hospital’s pathology department. Exclusion criteria for our study included those with a history of familial adenomatous polyposis (FAP), inflammatory bowel disease, hereditary non-polyposis colorectal cancer (HNPCC), Turcot syndrome, severe cardiovascular disease, recurrent adenoma with a confirmed diagnosis, colorectal cancer, and tumors in other organs. Because NSAIDs affect PGE-M levels, subjects had used aspirin or any NSAID for at least 48 hours prior to colonoscopy. They were ineligible for analysis. Our study also excluded participants. Because they used any dose of NSAIDs, including aspirin, for 3 days or more in the 3 months prior to enrollment or 3 days per month or used NSAIDs for 36 days in the past year. Finally, a sample of 304 participants’ data was kept for analysis. The study protocol was approved by the Ethics Committee of Guizhou Provincial People’s Hospital. A signed informed consent was required for all study subjects.

There were 412 study participants who provided urine samples; however only 304 samples were usable, due to sample damage and inconvenience of follow-up during the novel coronavirus pandemic. There were 152 cases identified as negative controls. This meant they did not have any polyps at the endoscopic screening. Patients with single tubular adenomas with a maximum diameter of less than 1 cm were categorized as low-risk cases, whereas those with a maximum diameter of more than 1 cm and/or histology of tubular villi, villi, and any multiple adenomas were categorized as high-risk cases (22, 25, 26). Table 1 displays the characteristics of the three categories. To stop the oxidation of unstable metabolites, urine samples were taken in sterile cups containing 125 mg of ascorbic acid. Following collection, samples were kept chilled (at about 0 to 4°C) in a portable foam box with an ice pack before being processed within 6 hours for long-term storage at -80 ± 5°C. Each participant had a biospecimen collection form filled out at the time of sample collection, which listed the day and time of sample collection as well as any drug usage within the previous 48 hours of the colonoscopy.

We developed the questionnaire for this study based on the food frequency questionnaire (SQFFQ) used in the 2002 Chinese population dietary survey methodology, and made appropriate adjustments to incorporate the regional dietary habits of Guizhou. All participants completed the questionnaire at the time of study enrollment. The first section evaluates the annual average food consumption, including average intake, frequency of consumption, etc.; the second section tracks nutrient supplement usage, including the name of the supplements, dose, and regimen. The Chinese Food Composition Table (2nd edition) and Nutrition Calculator V2.7.3 were used to convert nutrient intakes, to evaluate the overall amount of calcium consumed through diet and dietary supplements. Information on medical history, drug usage, demographics, anthropometrics, daily food habits, physical activity, and other lifestyle factors were also included as added content to this questionnaire.

PGE-M was measured using liquid chromatography/tandem mass spectrometry to determine the endogenous production of PGE₂ in humans (27). Briefly, urine was placed in a 10 mL polypropylene tube at room temperature, and then a sample of 1.0 mL was acidified to pH=3 by adding 1.0 mol/L HCl. Next, endogenous PGE-M was converted to methoxime derivatives and treated with 1600 mg of methoxamine hydrochloride in 10 mL of 1.5 mol/L sodium acetate solution (pH=5). The methoxylated PGE-M was dissolved in 8 ml of water after 1 hour of greenhouse incubation, and the aqueous sample was then transferred to C-18 Sep-Pak that had been prepared with 5 ml of methanol and 5 ml of water (pH 3). Sep-Pak was then eluted with ethyl acetate. Thermo SCIENTIFIC Hypersil GOLD (1.9 µm, 2.1×50 mm) column linked to a TSQ-Altis, and Thermo Fisher mass spectrometry pump was then used for liquid chromatography. Heated electrospray ion source was used as the ionization technique. The mass to charge ratios (m/z) monitored were 385.3 ~ 336 and m/z 385.3 ~ 367 for endogenous PGE-M, in the selected response monitoring (SRM) mode. The ratio of the mass spectral peak regions of the m/z 336 and m/z 367 ions was used to calculate the amount of endogenous PGE-M. With a coefficient of variation of 4.1% between batches and 8.7% within batches, the lower limit of detection for PGE-M was set at 2.00 ng/ml. There were no incidents that compromised data integrity or quality throughout the experiment. The quality control samples’ identities and the status of the urine samples used in the study were both unknown to the laboratory staff. Additionally, Urinary creatinine was measured using a Sigma kit (Sigma Co., Inc., St. Louis, MO, USA). The levels of urinary creatinine were determined and reported as standardized PGE-M values, PGE-M (ng)/creatinine (mg).

Selected baseline characteristics for cases and controls were computed as means, standard deviations, and percentages. We compared the means of age, body mass index (BMI), and calcium intake data between case and control participants using analysis of variance. To compare categorical variables, we employed the chi-square test. Urinary PGE-M levels for each sample were normalized using the urinary creatinine level of the sample and expressed as ng/ml creatinine. The PGE-M data in urine were skewed to the right; therefore, the median, interquartile range, and geometric mean were estimated for descriptive statistics. After adjusting for age, sex, smoking status, alcohol use, education, and prior hypertensive diabetes mellitus, Wilcoxon rank-sum tests and log-transformed linear regression models were used to analyze differences in PGE-M levels between groups. The PGE-M concentrations in the control group’s quartile distribution served as the basis for establishing cut points for categorical variables. The odds ratio (OR) and 95% confidence interval (95% CI) between urine PGE-M levels and the risk of colorectal adenoma were calculated using logistic regression models. Trend p-values were derived by using categorical variables as continuous parameters of the model and passing the linear trend test. We also stratified associations by subgroups, such as BMI, sex and calcium intake level, in order to focus on these factors’ influence on the association of urinary PGE-M levels with the incidence of high-risk colorectal adenomas. We performed all analyses using SPSS 26.0 (SPSS Inc, Chicago, IL, USA), and P values ≤0.05 (two-sided probability) were interpreted as statistically significant for all analyses.

In this investigation, 304 patient urine samples were examined. We found that the sample group had 153 controls, 59 low-risk, and 93 high-risk cases. Table 1 displays the characteristics of the cases and the controls. The high-risk adenoma group had a greater prevalence of diabetes mellitus, hypertension, and a higher BMI when compared to the control group. Low-risk adenomas were more prevalent in people with lower education levels and low calcium intake. In contrast to controls, patients with adenomas were more likely to be male, smokers, and drinkers, although the difference was not statistically significant.

Table 2 shows the baseline urinary PGE-M levels. Urinary PGE-M levels in the low-risk adenoma patients’ group did not differ statistically from those in the control group. However, urinary PGE-M levels were higher in individuals with high-risk adenomas. patients with high-risk adenomas had urinary PGE-M levels 7% and 23.56% higher than patients with low-risk adenomas or control group patients, respectively (p=0.04).

The Spearman correlation coefficients between urine PGE-M level and several lifestyle factors are shown in Table 3 . The results showed a direct correlation between PGE-M and age, gender, and smoking status. We further analyzed urinary PGE-M levels and the risk of developing colorectal adenomas ( Table 4 ). High-risk adenomas were more likely to occur in patients with higher urine PGE-M concentrations (p =0.013-0.016). The highest, fourth quartile, urine PGE-M levels were associated with a 1.65-fold higher incidence of high-risk colorectal adenoma compared to the lowest urinary PGE-M levels. Results did not indicate an association between urine PGE-M levels and an increased incidence of low-risk adenomas.

Urinary PGE-M levels were tested in relation to the incidence of high-risk colorectal adenoma, stratified by BMI, gender, and calcium intake ( Table 5 ). Within the highest PGE level, fourth quartile, PGE level subgroup, women had a stronger association with the incidence of high-risk colorectal adenomas (adjusted OR, 3.72, 95% confidence interval, 0.54-25.45) than did males (adjusted OR, 1.75; 95% confidence range, 0.66-4.61). Although not statistically significant, the patient subgroup with BMI ≥25 (kg/m²) had OR changing from 1.0 to 3.46, to 1.50, to 2.30 for each increasing PGE-M quartiles respectively. Body weight may modify the association between urinary PGE-M and incidence of high-risk colorectal adenoma. The present study did not find a significant interaction between BMI, gender, calcium intake and PGE-M in the high-risk adenoma group (P for interaction >0.05).