Endometrial hyperplasia features in conditions of organic solvents exposure

Introduction

Differences in occupational health risks for working women are primarily associated with the particular sensitivity of the reproductive system to the effects of toxic chemicals. Reprotoxicants cause endocrine imbalance, have cytotoxic and genotoxic effects [1–3]. Violation of adaptation to prolonged exposure to xenobiotics eventually leads to the development of professionally conditioned pathological conditions and diseases [4]. Among the possible consequences of contact with a chemical factor in the workplace for the reproductive system, the laws of the development of hyperplastic processes are the least studied. At the same time, abnormal proliferative activity leads to the development of diseases that reduce patients’ quality of life and limit their ability to work, and is also associated with the risk of subsequent development of malignant neoplasms [5–7], which determines the urgency of the problem. Thus, the precancerous process is endometrial hyperplasia with atypia; the annual primary incidence of endometrial cancer in this group of patients is 8.2% (95% CI is 3.9%–1.3%) [7]. Hyperestrogenism is a proven pathogenetic factor of this pathology [8]. The hormonal factor explains the increased risk in obese patients. Endometrial hyperplasia is a common disease. At the same time, the highest prevalence (386 per 100,000 women per year) is typical for the age group of 50–54 years and represents a risk of losing the most qualified and highly skilled workforce [9].

One of the most common chemicals in women's workplaces is organic solvents. They are widely used in the production of machinery and equipment, the chemical industry, construction, and other economic activities. The carcinogenic effect proved for benzene, trichloroethylene, and tetrachloroethylene [10–12], as well as the known data on the systemic effects [13], determine the possibility of the influence of organic solvents on background and precancerous conditions. A number of studies have shown that these substances affect the cell cycle indirectly, due to oxidative stress and interference in epigenetic processes, which suggests a variety of possible effects at the clinical level [13–14]. As for the effective management of occupational health risk in working women, it is necessary to understand the patterns of the formation of endometrial hyperplasia under the influence of the chemical factor.

The purpose of the study was to establish the clinical and molecular features of endometrial hyperplasia under the influence of organic solvents and the obesity factor.

Materials and methods

An observational single-stage and single-center clinical study was performed. Women with endometrial hyperplasia and contact with organic solvents in the workplace participated in the study (the main study group, n=140, 70 people of them suffered from obesity, 70 people were of normal body weight). The comparison group consisted of women with endometrial hyperplasia without occupational health risks, n=140, 68 with obesity, 72 – with normal body weight.

The inclusion criteria were the presence of the patient's informed consent to participate in the study in writing form, age from 50 to 65 years, menopause for 2 years or more, diagnosis of endometrial hyperplasia, contact with organic solvents in the workplace for at least 5 years (main group), the 2^nd or the 1^st class of working conditions for the entire period of employment (control group), 30 or more body mass index, waist circumference – more than 80 cm, the ratio of waist circumference to hip circumference – more than 0.85 (obese subgroup).

Non-inclusion criteria were lack of informed consent to participate in the study, inability or unwillingness to understand and comply with the requirements of the study protocol, the presence of contraindications to diagnostic measures provided for in the study protocol, taking estrogen-progestogenic drugs for a month before inclusion in the study, body mass index less than 18, malignant neoplasms of any localization, pelvic inflammatory diseases, chronic inflammatory diseases of other localization, cirrhosis of the liver, chronic hepatitis with impaired liver function.

Patients of the main group by profession (painters) worked at a machine-building enterprise (aircraft production, OKVED code 2022 30.30). The main unfavorable factor of the production environment was aromatic hydrocarbons. Concentrations of aromatic hydrocarbons in the air of the working area were measured by the photometric method during routine monitoring conducted by experts of the Department of Supervision of Occupational Hygiene, Municipal Hygiene of the Federal Service for Supervision of Consumer Rights Protection and Human Well-Being in the Novosibirsk Region. The maximum single concentrations for butyl acetate were 411.0±10.2 mg/m³ (2.1 MPC), for xylene — 751.2±21.9 mg/m³ (5.0 MPC), for toluene — 382.4±27.2 mg/m³ (2.5 MPC), for gasoline — 648.0±50.5 mg/m³ (2.2 MPC), for acetone — 902.3±10.2 mg/m³ (1.1 MPC). The average exchange concentrations for butyl acetate were 101.0±6.4 mg/m³ (2.0 MPC), for xylene — 73.9±5.4 mg/m³ (1.5 MPC), for toluene — 56.3±3.12 mg/m³ (1.1 MPC), for gasoline — 311.3±5.16 mg/m³ (3.1 MPC), for acetone — 651.2±5.8 mg/m³ (3.3 MPC). The exposure time of the chemical factor was 100% of the working shift. The work experience in these conditions was 22 (18; 28) years for obese people, 21 (19; 27) years without obesity, 29 (20.7%) of participants were in the post-contact period lasting from 1 to 5 years. No signs of chronic intoxication with organic solvents were detected in any patient.

The characteristics of patients are presented in Table 1. All the patients were postmenopausal. There were no significant differences in their age. The severity of obesity in the main and control groups was the same. In obese patients, a significant incidence of uterine fibroids was noted, more in persons without contact with a chemical factor. Those who worked in contact with organic solvents had a higher incidence of hypertension, more in the subgroup with obesity. Also, in the obese subgroups, 1 case of prediabetes/type 2 diabetes mellitus was observed.

Endometrial hyperplasia was diagnosed on the basis of a histological examination of the endometrium¹.

Obesity was determined according to the WHO criteria: body mass index — more than 25 kg/m². Abdominal obesity was established according to the criteria of IDF 2005 (waist circumference of more than 80 cm [15]) and with a value of the ratio of waist circumference to hip circumference of more than 0.85 [16].

All the patients underwent collection of complaints, anamnesis, gynecological bimanual examination, and ultrasound examination of the pelvic organs twice, hysteroscopy with biopsy or separate therapeutic and diagnostic scraping of the walls of the uterine cavity according to standard indications².

All the histological preparations were prepared according to the standard procedure. The study was carried out by light microscopy (staining with hematoxylin and eosin). The expression of progesterone and estrogen receptors on epithelial and stromal cells and the expression of the Ki 67 protein proliferation marker were determined by immunohistochemistry.

The levels of interleukins 1ß and 6, transforming growth factor β1 (TGFß1), and adiponectin in serum were determined by solid-phase enzyme immunoassay.

Statistical data processing was carried out using the SPSS 24 program. The normal distribution of data was determined by the Kolmogorov-Smirnov method. Standard methods of descriptive statistics are performed. The values of quantitative variables are presented in the form of mean and standard error m ±M with normal data distribution, median and interquartile interval Me (25th percentile; 75th percentile) if the data distribution does not meet the criteria of normal. The values of qualitative variables are expressed as a percentage. In order to compare groups by quantitative variables, the Kruskall-Wallis method was used, and the criterion χ² was used for qualitative ones. The relationships were established by logistic or linear regression methods. The differences were recognized as significant with an error probability value of p<0.05.

The study was carried out in accordance with the ethical principles established by the Helsinki Declaration of the World Medical Association, as well as in compliance with the ethical norms and rules provided for in the Bulletin of the Higher Attestation Commission of the Ministry of Education of Russia No. 3 of 2002 “On the procedure for conducting biomedical research in humans”. The study was approved based on the results of an ethical examination conducted by the Ethics Committee of the Novosibirsk State Medical University of the Ministry of Health of the Russian Federation.

Results

The evaluation of histological forms of endometrial hyperplasia showed that the percentage of atypical hyperplasia was significantly higher in the group of contact with organic solvents, the maximum in the subgroup with obesity (Table 2). The reliability of differences was observed both in obese and normal body weight individuals. In the comparison group, the risk of atypical endometrial hyperplasia did not depend on the fact of obesity.

Logistic regression analysis revealed the relationship of atypical endometrial hyperplasia with average daily xylene concentrations and maximum single toluene concentrations (Table 3).

Clinical manifestations of the disease in the subgroup of contact with organic solvents and obesity were characterized by a higher frequency of abnormal uterine bleeding – 20 (28.5%) compared with 11 (15.7%) in the subgroup with occupational risk without obesity, 18 (28.5%) and 8 (11.1%) in the comparison subgroups, p=0.001; the differences are significant between all subgroups. Ultrasound examination of the uterus revealed that in patients working with organic solvents and obese, the indicator “thickness of midline echo” was significantly higher in comparison with the subgroup without obesity and with the subgroup without contact with a chemical factor. The values of the indicator in the studied subgroups were 12.1±2.47 mm, 9.0±2.39 mm, 10.1±2.44 mm, and 11.0±2.35 mm, p=0.001.

The results of the immunohistochemical examination of the endometrium are presented in Table 4. The level of Ki-67, as expected, was higher in atypical endometrial hyperplasia. Both simple and atypical hyperplasia in patients working with organic solvents differed in the expression of Ki-67, with a maximum in the obese subgroup. The expression of estrogen receptors was greater in obese individuals and did not depend on the effect of a chemical factor.

Taking into account the revealed differences of the studied groups in the level of Ki-67 expression, a linear regression analysis of the relationship of this indicator with hygienic characteristics was carried out (Table 5). As a result, the relationship between the average concentration of xylene and the maximum single concentration of toluene with the level of Ki—67 expression on cells in patients with endometrial hyperplasia was determined.

The study of serum concentrations of interleukins 1ß revealed maximum increases in obese subjects — 14.8±2.06 ng/ml compared with 3.3±1.24 ng/ml in non-obese workers, 11.5±0.93 ng/ml and 4.0±1.52 ng/ml –in the control subgroups, p = 0.001. Values of interleukin concentrations as 6 also depended on obesity: 9.0±2.11 ng/ml, 4.2±2.43 ng/ml, 7.0±1.22 ng/ml, and 2.7 ±8.31 ng/ml, p =0.001. Differences between subgroups formed depending on the presence of obesity are significant. In a single-factor logistic regression analysis, interleukin 1ß concentrations were associated with the development of atypical variants of endometrial hyperplasia [OR — 1.25, 95% CI – 1.05—2.39, Wald statistics — 6.9, p=0.009 in the main group and OR — 1.34, 95% CI – 1.11—3.71, Wald statistics – 7.2, p=0.003 in the control group]. At the same time, concentrations of TGFß1 were higher in those in contact with the chemical factor: 759.4±12.35 pg/ml in obesity and 751.8±9.07 pg/ml in normal body weight. The values in the control subgroups were 441.7±10.22 pg/ml and 430.2±9.13 pg/ml, p=0.003, the significance of differences in relation to the subgroups with occupational risk.

Adiponectin deficiency was more pronounced in the obese subgroups and did not depend on the chemical factor. The values of this indicator in the main group were 19.5±3.28 µg/ml in obese people, 25.2±4.26 µg /ml in normal body weight, p=0.005. In the control group, the level of adiponectin in obese patients was 18.6±5.11 µg/ml, with normal body weight — 27.2±2.95 µg/ml.

When the multifactorial logistic regression analysis of hygienic, clinical, and immunohistochemical data was included, the parameters most associated with the development of atypical variants of endometrial hyperplasia in those working in contact with organic solvents were determined (Table 6). It was determined that the probability of an atypical variant of endometrial hyperplasia increased: work experience (every 5 years by 80%), the maximum single concentration of toluene in the air of the working area (every 100 micrograms/m³ by 2.5 times), waist circumference (over 65 cm every 2 cm by 68%), Ki-67 (every 10 points by 2 times), estrogen receptors on the epithelium (every 10 points by 92%).

The solution of the regression equation greater than or equal to 0.61 corresponded to diagnostic sensitivity of 85.1%, specificity — 80.3%. The area under the sensitivity-specificity curve was 0.85 (95% CI 0.75–0.93), p=0.001.

Discussion

The effect of organic solvents on the development of malignant neoplasms and precancerous proliferative processes is currently being actively studied. Proven oncogenes for humans (carcinogens of group 1 according to the IARC classification) are benzene, trichloroethylene, tetrachloroethylene, and ortho-toluene. Possible carcinogens for humans (group IIa) include chlorotoluene, styrene, tetrafluoroethylene, and trichloropropane. The carcinogenicity of xylene has been shown in basic research (belongs to group IIb) [10–13]. Less is known about the effect of organic solvents on the formation of hyperplastic processes. In this study, it was shown that in patients with endometrial hyperplasia, contact with xylene and toluene at work is associated with the development of atypical variants, frequent uterine bleeding, and the “midline echo” thickness during ultrasound examination. A comparison of subgroups and regression analysis determined the effect of the average concentration of toluene, the maximum single concentration of xylene on the expression level of Ki–67, which reflects the effect of these substances on proliferative activity. At the same time, the expression of estrogen and progesterone receptors in the groups of working and non-exposed women was the same, perhaps the effect of toxicants is not related to hormonal regulation. The serum concentrations of interleukins 1ß and 6 did not differ significantly. Therefore, high serum concentrations of the profibrous cytokine TGFß1 were determined in women working in contact with organic solvents. Thus, the biomechanism of the intervention of organic solvents in the development of endometrial hyperplasia by inducing systemic inflammation of low activity, but with a high potential for fibrogenesis, can be discussed.

In the studied patients, the chemical factor and obesity had a synergistic effect on atypical endometrial hyperplasia, proliferation activity, and bleeding frequency. This result may be explained by the simultaneous effect of two etiological factors, as well as the accumulation of organic solvents in adipose tissue, which increases the time of toxic exposure [17]. In multivariate logistic regression analysis, hygienic parameters and obesity characteristics were independent factors associated with atypical hyperplasia. The obtained data may be used to form risk groups of working women with endometrial hyperplasia who need additional examination for the presence of atypical variants.

The limitations of this particular study are the implementation in one center and the lack of prospective medical observation.

Conclusions

Under the conditions of exposure to organic solvents, endometrial hyperplasia is characterized by a significant increase in the frequency of the atypical variant, the expression of the proliferation factor Ki-67, serum concentrations of profibrosis factor TGFß1, the frequency of abnormal uterine bleeding, the “midline echo” thickness during ultrasound examination, to a greater extent in the presence of obesity.

Work experience, the maximum single concentration of toluene in the air of the working area, waist circumference, and the level of expression of estrogen receptors on the endometrial epithelium are factors most associated with the presence of atypical endometrial hyperplasia in workers in contact with organic solvents.

Authors’ contribution:

I.O. Marinkin, L.A. Shpagina — сoncept and design, final decision for publication;

E.S. Lisova — сoncept and design, data collection analysis and interpretation, writing the text of the manuscript;

I.S. Shpagin, O.S. Kotova, G.V. Kuznetsova, E.M. Loktin, S.A. Karmanovskaya — data collection analysis and interpretation, writing the text of the manuscript.

Conflict of interest. Authors declare no conflict of interest.