Method for evaluating influence on sex hormone level based on ethylene oxide exposure

Abstract

The invention relates to the technical field of environmental health monitoring and analysis, and discloses a method for evaluating influence on sex hormone level based on ethylene oxide exposure, which comprises the following steps: determining blood HbEO, serum TT, E2 and SHBG levels of people; the method comprises the following steps: firstly, collecting an erythrocyte sample, preserving at 30 DEG C, and then measuring the content of HbEO by utilizing a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and an improved Edman reaction, the HbEO level is expressed by picomole adduct per gram of hemoglobin; preparing a serum sample, storing the serum sample at 20 DEG C, and inspecting the serum sample; determining the levels of TT and E2 by using an isotope dilution-liquid chromatography-tandem mass spectrometry method; the SHBG level is determined by a chemiluminescence method; the circulating free testosterone level is estimated by dividing TT by SHBG, and the ratio of TT to E2, namely TT / E2, is used as an indirect index of aromatase activity; and a Pearson correlation analysis method or a Spearman rank correlation analysis method is adopted. The system architecture has good expandability, new data analysis methods or function modules can be added according to actual requirements, and the requirements of different research scenes are met.

Description

Technical Field

[0001] The present invention relates to the technical field of environmental health monitoring and analysis, and in particular to a method for evaluating the impact of ethylene oxide exposure on sex hormone levels. Background Art

[0002] Ethylene oxide (EO) is a key organic compound widely used in industry and healthcare. It is widely used as a disinfectant and sterilant to ensure product quality and safety. Furthermore, it is used in the manufacture of a variety of chemicals, including ethylene glycol, emulsifiers, and surfactants. Despite its widespread use, EO may pose a potential hazard to human health. It exists as a gas at room temperature and readily enters the respiratory system, where it binds to DNA and proteins. Hemoglobin adducts (HbEO), a biomarker of EO exposure, can be measured using N-(2-hydroxyethyl)valine and effectively reflects EO exposure levels.

[0003] In vitro studies have shown that EO exposure is genotoxic and mutagenic. In human studies, long-term exposure to EO may lead to cancer, cardiovascular disease, diabetes, neurological dysfunction, respiratory disease, and adverse reproductive outcomes. EO gas poses a significant health risk to residents, patients, and workers living near hundreds of active EO emission facilities. These individuals may be exposed to EO-sterilized medical devices in hospital settings or directly exposed in EO-emitting manufacturing plants. Although EO was listed as a "potentially hazardous air pollutant" in 2016, the U.S. Environmental Protection Agency (EPA) reclassified it as a known human carcinogen based on new data indicating that EO is 30 times more toxic than previously estimated. Given the large number of EO emission facilities in many densely populated areas around the world, a comprehensive understanding of the health risks associated with EO exposure has become an urgent public health priority.

[0004] In document CN 109310664 A, selective androgen receptor degrader (SARD) ligands and methods of use thereof are disclosed, comprising a heterocyclic aniline ring and its synthetic precursors, R-isomers, and non-hydroxylated and / or non-chiral propionamides, and pharmaceutical compositions thereof and their use in treating prostate cancer, advanced prostate cancer, castration-resistant prostate cancer, triple-negative breast cancer, other cancers expressing the androgen receptor, androgenic alopecia or other androgen-induced skin diseases, Kennedy's disease, amyotrophic lateral sclerosis (ALS), abdominal aortic aneurysm (AAA), and uterine fibroids, and methods for reducing the levels of androgen receptor full-length (AR-FL), AR-splice variants (AR-SV), and pathogenic polyglutamine (polyQ) polymorphisms of AR containing pathogenic or resistance mutations in a subject. This method has low accuracy when performing experiments.

[0005] Therefore, a new technical solution is needed to solve the above technical problems. Summary of the Invention

[0006] In order to solve the above problems, the present invention discloses a method for evaluating the effect of ethylene oxide exposure on sex hormone levels.

[0007] The technical solution of the present invention is: a method for evaluating the effect of ethylene oxide exposure on sex hormone levels, comprising the following steps:

[0008] Step 1: The study used NHANES data from 2013 to 2016 to determine the blood HbEO, serum TT, E2, and SHBG levels of the population;

[0009] Step 2: Because HbEO has a longer half-life than EO, it is used as an indicator of EO exposure. First, red blood cell samples are collected, stored at -30°C, and then sent to the National Center for Environmental Health for evaluation.

[0010] Step 3: Subsequently, the HbEO content was determined using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and a modified Edman reaction;

[0011] Step 4: HbEO levels are expressed as pmoles of adducts / gram of hemoglobin;

[0012] Step 5: Prepare serum samples, store at -20°C, and send to the National Center for Environmental Health for testing;

[0013] Step 6: TT and E2 levels were measured using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS / MS);

[0014] Step 7: SHBG levels are measured by chemiluminescence, which is found in other sources;

[0015] Step 8: The lower detection limits (LLOD) of TT, E2, and SHBG were 0.75 ng / mL, 2.994 pg / mL, and 0.800 nmol / L, respectively.

[0016] Step 9: Estimate circulating free testosterone concentration using the free androgen index (FAI) by dividing TT by SHBG. The ratio of TT to E2 (TT / E2) is used as an indirect indicator of aromatase activity.

[0017] Step 10: Weighted means were calculated using the NHANES primary sampling units and strata, and the results were statistically analyzed using weighted t-tests. Frequency proportions of categorical variables were analyzed using weighted chi-square tests.

[0018] Step 11: The frequencies of categorical variables were analyzed using weighted chi-square tests. The distributions of serum HbEO and sex hormone indicators were right-skewed, so log2 transformation was performed for descriptive and regression analyses.

[0019] Step 12: Use Pearson correlation analysis or Spearman rank correlation analysis to analyze the correlation between ethylene oxide exposure levels and sex hormone levels.

[0020] Preferably, in step 1, participants with missing covariates are deleted, and pregnant women, participants with missing blood EO data, and participants with missing sex hormone data are excluded. The covariates are potential confounders, including age, ethnicity, education level, BMI, poverty-income ratio (PIR), cotinine, and sample collection time.

[0021] Preferably, a comprehensive quality control program is established in step 4, including external and internal monitoring, to monitor and evaluate the accuracy and reliability of the analytical tests.

[0022] Preferably, the log2-transformed HbEO values ​​in step 11 are then analyzed as continuous variables and categorical variables divided into quartiles, and weighted quartiles of log2-transformed HbEO are calculated in specific sex, age and sex, and pubertal subgroups.

[0023] Preferably, weighted multiple linear regression is used to calculate β, the standardized coefficient values ​​and corresponding 95% confidence intervals, to examine the association of individual HbEO levels with sex steroid hormone indicators.

[0024] By adopting the above technical scheme, the association between individual HbEO levels and sex steroid hormone indicators can be examined.

[0025] Preferably, in step 11, restricted cubic spline analysis is applied to further explore the linear and nonlinear relationships between HbEO and sex hormones after adjusting for multiple potential covariates, and the optimal node with the minimum AIC is selected using the Akaike Information Criterion.

[0026] Preferably, in step 12, a regression model is established with sex hormone levels as dependent variables and ethylene oxide exposure levels, age, gender, BMI, etc. as independent variables, and other possible influencing factors are controlled to analyze the independent effect of ethylene oxide exposure on sex hormone levels.

[0027] The benefits of the present invention are as follows: 1. The present invention is based on the large-scale NHANES database, covering participants of different genders and age groups, and can comprehensively analyze the relationship between EO exposure and sex hormone levels, overcoming the limitations of previous studies with small sample size and narrow coverage.

[0028] 2. The present invention uses a variety of data analysis methods, including multiple linear regression analysis and RCS analysis, which can accurately evaluate the linear and nonlinear relationships between HbEO and sex hormones, thereby improving the accuracy of the analysis results.

[0029] 3. The analysis method and system provided by the present invention can provide a scientific basis for environmental health risk assessment, relevant policy formulation and clinical research, and help to take effective measures to reduce the harm of EO exposure to human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a line graph showing changes in different sex hormones when HbEO doubles in male adults according to the present invention;

[0031] Figure 2 This is a line graph showing changes in serum E2 and TT / E2 with EO exposure;

[0032] Figure 3 This is a line graph showing the relationship between HbEO and SHBG in males at different pubertal statuses according to the present invention;

[0033] Figure 4 This is a partial nonlinear relationship line graph analyzed by RCS in the present invention. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the present invention will be described clearly and completely below with reference to the accompanying drawings in the embodiments of the present invention.

[0035] The method for evaluating the effects of ethylene oxide exposure on sex hormone levels includes the following steps:

[0036] Step 1: Using NHANES data from 2013 to 2016, the study determined the blood HbEO, serum TT, E2, and SHBG levels of the population. Participants with missing covariates were deleted, including pregnant women, missing blood EO data, and missing sex hormone data. The covariates were potential confounders, including age, ethnicity, education level, BMI, poverty-to-income ratio (PIR), cotinine, and sample collection time.

[0037] Step 2: Because HbEO has a longer half-life than EO, it is used as an indicator of EO exposure. First, red blood cell samples are collected, stored at -30°C, and then sent to the National Center for Environmental Health for evaluation.

[0038] Step 3: Subsequently, the HbEO content was determined using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and a modified Edman reaction;

[0039] Step 4: HbEO levels were expressed as pmoles of adducts / gram of hemoglobin, and a comprehensive quality control program was established, including external and internal monitoring, to monitor and assess the accuracy and reliability of the analytical test;

[0040] Step 5: Prepare serum samples, store at -20°C, and send to the National Center for Environmental Health for testing;

[0041] Step 6: TT and E2 levels were measured using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS / MS);

[0042] Step 7: SHBG levels are measured by chemiluminescence, which is found in other sources;

[0043] Step 8: The lower detection limits (LLOD) of TT, E2, and SHBG were 0.75 ng / mL, 2.994 pg / mL, and 0.800 nmol / L, respectively.

[0044] Step 9: Estimate circulating free testosterone concentration using the free androgen index (FAI) by dividing TT by SHBG. The ratio of TT to E2 (TT / E2) is used as an indirect indicator of aromatase activity.

[0045] Step 10: Weighted means were calculated using the NHANES primary sampling units and strata, and the results were statistically analyzed using weighted t-tests. Frequency proportions of categorical variables were analyzed using weighted chi-square tests.

[0046] Step 11: The frequencies of categorical variables were analyzed using a weighted chi-square test. The distributions of serum HbEO and sex hormone indicators were right-skewed, so they were log2-transformed for descriptive and regression analyses. Log2-transformed HbEO values ​​were then analyzed as a continuous variable and as a categorical variable divided into quartiles. Weighted quartiles of log2-transformed HbEO were calculated for specific sex, age, and gender, and pubertal subgroups. Weighted multiple linear regression was used to calculate β (standardized coefficient values) and corresponding 95% confidence intervals to examine the association between individual HbEO levels and sex steroid hormone indicators. Restricted cubic spline analysis was applied to further explore the linear and nonlinear relationships between HbEO and sex hormones after adjusting for multiple potential covariates. The Akaike Information Criterion was used to select the optimal node with the minimum AIC.

[0047] Step 12: Pearson correlation analysis or Spearman rank correlation analysis was used to analyze the correlation between ethylene oxide exposure levels and sex hormone levels. Restricted cubic spline analysis was applied in step 12 to further explore the linear and nonlinear relationships between HbEO and sex hormones after adjusting for multiple potential covariates. The Akaike information criterion was used to select the optimal node with the minimum AIC. This allowed for the examination of the association between individual HbEO levels and sex steroid hormone indicators. A regression model was established with sex hormone levels as the dependent variable and ethylene oxide exposure levels, age, gender, and BMI as independent variables. Other possible influencing factors were controlled to analyze the independent effect of ethylene oxide exposure on sex hormone levels.

[0048] Given the significant differences in sex hormone levels across sex and developmental stages, this analysis was conducted for both males and females based on age (children: ≤11 years, adolescents: 12–19 years, and adults: >19 years). Classifying participants aged 6–19 years as "children" or "adolescents" based solely on age may include both prepubertal and pubertal individuals in the same group. This grouping could result in subgroups with unusually high or low sex hormone levels, thereby biasing the regression analysis of the relationship between HbEO and sex steroid hormones. Furthermore, the effect of log2-HbEO on sex hormones may be influenced by pubertal stage. To address this concern, we further stratified individuals into pubertal and prepubertal subgroups based on serum sex hormone levels and menstrual status. Individuals were considered to have entered puberty (i.e., categorized as pubertal) if their TT level was equal to or greater than 50 ng / dL for males, their E2 level was 20 pg / mL or higher for females, or they had experienced menarche. Individuals who did not meet these criteria were categorized as prepubertal (i.e., categorized as prepubertal). Data on women's menarche status were collected using the following questions: "Has the menstrual cycle started?" (in the medical questionnaire) and "What was the age at first menstruation?" (in the reproductive health questionnaire). Women who answered "yes" or provided the age at first menstruation were considered to have had their menstrual cycle started. In addition, multiple linear regression analyses were performed separately for men and women by pubertal status.

[0049] Subgroup analyses with multiplicative interaction terms were performed to show whether the associations between individual HbEO levels and sex steroid hormones varied by age (<45 years or ≥45 years), BMI (<24 or ≥24 kg / m2), energy intake (<2,400 or ≥2,400 kcal), physical activity (<200 or ≥200 METs-hours / week), and prescription drug use (yes or no).

[0050] To assess the robustness of the results, four sensitivity analyses were conducted to examine the associations of individual HbEO levels with sex steroid hormones: 1) multiple imputation of missing data using the 'mice' package (multiple imputation chained equations) in R; 2) additional adjustment for energy intake, physical activity, and prescription medications; 3) additional adjustment for exposure to bisphenol A, phthalates, and polychlorinated biphenyls; and 4) exclusion of adults aged 65 years or older. All statistical analyses were performed using R software, version 4.1.2, and significance was determined using two-tailed P values ​​and a significance level of 0.05, as shown in Tables 1 and 2.

[0051] Table 1

[0052]

[0053] Table 2

[0054]

[0055] Conclusions: Baseline characteristics of participants were summarized by sex and age. The study sample included 4221 participants, including 2089 males and 2101 females. The mean age of participants was 41.70 years. The detection rates of TT, E2, SHBG, and HbEO in the entire sample were 99.81%, 85.93%, 100%, and 97.49%, respectively. However, the detection rate of E2 in male children was less than 50%. Therefore, E2 and TT / E2 analyses in this subgroup were omitted. In contrast, adult participants had higher EO exposure levels than adolescents, while adolescents had higher levels than children in both sexes.

[0056] Given the key role of sex hormones in the onset of puberty, prepubertal and pubertal status were also examined. The proportion of E2 detection in prepubertal males and females was less than 50%. Therefore, E2 and TT / E2 assessments were not performed in these demographic subsets. EO exposure was significantly higher in adolescent males than in prepubertal males, whereas no significant difference was found between prepubertal and pubertal females.

[0057] like Figure 1-4Figure 2 shows the relationship between HbEO levels (continuous and quartile) and sex hormones at different developmental stages. Among males, no significant correlation was observed between HbEO and sex steroid hormones in children and adolescents (Supplementary Table S4). However, in male adults, a doubling of HbEO corresponded to a 2.85% decrease in E2, a 2.10% increase in SHBG, and a 4.3% increase in TT / E2 (for E2: p = 0.04, OR = -0.03, 95% CI: -0.06 to 0.00; for SHBG: p = 0.03, OR = 0.03, 95% CI: 0.00 to 0.06; for TT / E2: p < 0.001, OR = 0.06, 95% CI: 0.03 to 0.09). Furthermore, the associations between serum E2 and TT / E2 levels and HbEO were consistent across quartiles of EO exposure. Serum E2 levels decreased with increasing EO exposure (p for trend = 0.014). Likewise, TT / E2 levels increased with increasing EO exposure (p for trend = 0.003) ( Figure 2 No association was found between EO exposure and sex hormones in different categories of women.

[0058] The associations between HbEO and sex steroid hormones by continuous and quartile distribution during prepubertal and pubertal status are shown in Supplementary Table S5. No significant associations were observed between HbEO and sex steroid hormones during prepubertal and pubertal periods, regardless of sex. When assessed by quartile of EO exposure, HbEO was positively associated with SHBG in prepubertal males (quartile 4 vs quartile 1) (P = 0.03, OR = 0.21, 95% CI: 0.02, 0.4). Conversely, HbEO was negatively associated with SHBG in adolescent males (quartile 3 vs quartile 1) (P = 0.03, OR = -0.22, 95% CI: -0.42, -0.03).

[0059] Those skilled in the art will understand that the embodiments of the present invention described above and shown in the accompanying drawings are intended to be illustrative only and are not intended to limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functional and structural principles of the present invention have been demonstrated and illustrated in the embodiments. Any variations or modifications may be made to the embodiments of the present invention without departing from the principles described.

Claims

1. A method for evaluating the effects of ethylene oxide exposure on sex hormone levels, characterized in that: The following steps are involved: Step 1: The study used NHANES data from 2013 to 2016 to determine the blood HbEO, serum TT, E2, and SHBG levels of the population; Step 2: Because HbEO has a longer half-life than EO, it is used as an indicator of EO exposure. First, red blood cell samples are collected, stored at −30°C, and then sent to the National Center for Environmental Health for evaluation. Step 3: Subsequently, the HbEO content was determined using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and a modified Edman reaction; Step 4: HbEO levels are expressed as pmoles of adducts / gram of hemoglobin; Step 5: Serum samples were prepared, stored at −20°C, and sent to the National Center for Environmental Health for testing; Step 6: TT and E2 levels were measured using isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS / MS); Step 7: SHBG levels are measured by chemiluminescence, which is found in other sources; Step 8: The minimum detection limits (LLOD) of TT, E2, and SHBG were 0.75 ng / mL, 2.994 pg / mL, and 0.800 nmol / L, respectively. Step 9: Estimate circulating free testosterone concentration by dividing TT by SHBG using the free androgen index (FAI). The ratio of TT to E2 (TT / E2) is used as an indirect indicator of aromatase activity. Step 10: Weighted means were calculated using the NHANES primary sampling units and strata, and the results were statistically analyzed using weighted t-tests. Frequency proportions of categorical variables were analyzed using weighted chi-square tests. Step 11: The frequencies of categorical variables were analyzed using weighted chi-square tests. The distributions of serum HbEO and sex hormone indicators were right-skewed, so log2 transformation was performed for descriptive and regression analyses. Step 12: Use Pearson correlation analysis or Spearman rank correlation analysis to analyze the correlation between ethylene oxide exposure levels and sex hormone levels.

2. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 1, characterized in that: In step 1, participants with missing covariates were deleted, including pregnant women, participants with missing blood EO data, and participants with missing sex hormone data. The covariates were potential confounders, including age, ethnicity, education level, BMI, poverty-to-income ratio (PIR), cotinine, and sample collection time.

3. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 1, characterized in that: A comprehensive quality control program, including external and internal monitoring, was established in step 4 to monitor and evaluate the accuracy and reliability of the analytical tests.

4. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 1, characterized in that: The log2-transformed HbEO values ​​in step 11 were then analyzed as continuous variables and categorical variables divided into quartiles, and weighted quartiles of log2-transformed HbEO were calculated in specific sex, age, and gender, and pubertal subgroups.

5. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 4, characterized in that: Weighted multiple linear regression was used to calculate β, the standardized coefficient value, and the corresponding 95% confidence interval to examine the association between individual HbEO levels and sex steroid hormone indicators.

6. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 1, characterized in that: In step 11, restricted cubic spline analysis was applied to further explore the linear and nonlinear relationships between HbEO and sex hormones after adjusting for multiple potential covariates, and the optimal node with the minimum AIC was selected using the Akaike Information Criterion.

7. The method for evaluating the effect of ethylene oxide exposure on sex hormone levels according to claim 1, characterized in that: In step 12, a regression model is established with sex hormone levels as dependent variables and ethylene oxide exposure levels, age, gender, BMI, etc. as independent variables, controlling other possible influencing factors to analyze the independent effect of ethylene oxide exposure on sex hormone levels.

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