STRESS URINARY INCONTINENT WOMEN, THE INFLUENCE OF AGE AND HORMONAL STATUS ON ESTROGEN RECEPTOR ALPHA AND BETA GENE EXPRESSION AND PROTEIN IMMUNOEXPRESSION IN PARAURETHRAL TISSUES

Epidemiological and experimental studies have found that estrogens play an important role in the normal functioning of the female lower urinary tract. The involvement of estrogens in the pathogenesis of stress urinary incontinence (SUI) remains unclear, and so does their administration in the treatment of that complaint. Generally, the underlying cause of SUI is anatomical abnormality. This is often accompanied by hypermobility of the urethro-vesical junction and a flabby vaginal anterior wall, both of which are often associated with the dysfunction of the paraurethral connective tissue. However, in the case of SUI, the biomechanical properties of the connective tissue are poorer, and thus artificial materials are being used as pubo-urethral neo-ligaments to enhance the long-term effects of surgical treatment. A lot of studies indicate unquestioned influence of estrogens on connective tissue remodeling (1-6). Incontestable is that this progressive remodeling contributes to stress urinary incontinence by altering normal tissue architecture and mechanical properties (7). About 42% of women suffer from SUI, so it is crucial to explore this subject (8).

The basic mechanism of estrogen function involves the excitement of the intracellular alfa and beta estrogen receptors (ERα and ERb, respectively) which, in turn, activates or inhibits the transcription of certain estrogen-dependent genes. The presence of estrogen receptors in a tissue is a prerequisite of the tissue being an estrogen target. Both estrogen receptors are found at the protein level in the vaginal anterior wall (9, 10). A considerable concentration of the receptors is also determined in the vaginal anterior wall epithelium. This seems to explain the impact of estrogens applied locally for the signs of vaginal atrophy. The problem of SUI is not limited to the epithelium alone. The quality of paraurethral connective tissue is essential, too. The question as to whether estrogens affect the quality of that tissue remains unexplained.

MATERIAL AND METHODS

The study was approved by the Ethical Board (No. KE-0254/21/2017).

The study was conducted in a group of 81 women patients of II Department of Gynecology, Medical University of Lublin. The study group (SUI) included 49 samples of the paraurethral connective tissue collected from patients operated on for stress urinary incontinence. All SUI patients had sling surgery with implantation of polypropylene mesh under mid-urethra.

SUI was diagnosed on the basis of detailed medical history, urodynamic assessment with cystometry and urethral profilometry, ultrasound scans and stress tests with Pelvic Organ Prolapse Quantification (POPQ). The control group (control) consisted of 32 samples of the paraurethral connective tissue collected from patients who reported no episodes of SUI, and were without signs of genital prolapse. Such patients were operated on for vaginal cysts or pre-cancerous and cancerous lesions of the uterine cervix. The exclusion criteria included hormonal replacement therapy (HRT) within 6 months prior to the examination, urinary tract infections, estrogen-dependent diseases, i.e. endometriosis, uterine leiomyomas, ovarian tumors and prolapse of the genital organs POPQ ≥ 1, and overactive bladder.

In both groups (study and control), the patients were subdivided into pre- and postmenopausal subgroups. The criterion to assign the patient to the premenopausal subgroup was regular menstruation and follicle-stimulating hormone (FSH) < 30 mlU/ml. The women with FSH > 30 mlU/ml and not menstruating for at least a year were included in the postmenopausal subgroup. Finally, estrogen receptors proteins were determined in 49 SUI patients (22 premenopausal and 27 postmenopausal women), and estrogen receptor gene expression was evaluated in 32 women (17 premenopausal and 15 postmenopausal). In the control group, the receptor protein was determined in 32 women (16 premenopausal and 16 postmenopausal) and gene expression in 31 patients (16 premenopausal and 15 postmenopausal).

Sample collection and fixation

Samples of the paraurethral connective tissue (weighing 100 – 150 mg) were collected intraoperatively in the area 1.5 – 2 cm from the external urethral orifice (two parts, 50 – 75 mg each in a sterile manner; one part being immediately frozen in liquid nitrogen and stored at –80°C for further RNA and DNA isolation, second part fixed in 4% formaldehyde for paraffin embedding and immunohistochemical tests). Additionally, prior to the operation, 7-ml blood samples were collected from the basilic vein for clot formation. After clotting, the serum was centrifuged and stored at –20°C for further tests.

Blood serum follicle-stimulating hormone, luteinizing hormone and estradiol

Blood serum FSH, luteinizing hormone (LH), and estradiol were determined radioimmunologically by means of 125J labelled commercial kits (Estradiol, LH, FSH: [125J] Coated Tube Radioimmunoassay SPECTRIA Orion Diagnostica, Finland).

Immunohistochemical tests

Immunohistochemical determination of ERα and ERβ was done on paraffin sections. The procedure with primary antibodies:

- mouse monoclonal antibody against human ERα (DAKO, Denmark), 1:50 dilution;

- rabbit polyclonal antibody against human ERβ (Sigma, St Louis, USA), 1:100 dilutionwere performed according to methodology described in previous study (11).

Each time, the results were positively and negatively controlled. In the negative control, primary antibodies were replaced by respective mouse and rabbit standard serums provided by the manufacturer. Prostate tissue was used in the positive control of the antibody against ERβ (Figs. 1-4).

Fig. 1. ERα immunohistochemical response in the paraurethral connective tissue (brown color – positive detection).

Fig. 2. Positive control of ERα immunohistochemical response in the cancerous tissue of the mammary gland.

To quantitatively evaluate ERα and ERβ proteins in the paraurethral connective tissue, computer aided analysis of microscopic images was used (Multiscan v.5.1 (CSS, Poland). The ratio of cells expressing positive response was counted in 500 cells of the fascia (magnit. 250X). This scoring system was more precise than standard scoring system used in cancer tissues (12). The RNA isolation, quantitative RT-PCR and total cellular DNA isolation were prepared as per protocol (13).

Fig. 3. ERβ immunohistochemical response in the paraurethral connective tissue (brown color – positive detection).

Fig. 4. Positive control of ERβ immunohistochemical response in the prostate tissue

Statistical analysis

The results were analyzed statistically using Statistica v. 10.0 software (StatSoft, Poland). The differences between the groups were analyzed by U-Mann-Whitney test. Correlations were determined by Spearman’s test, and the differences in the frequency of qualitative characteristics were evaluated by χ2. A 5% conclusion error was assumed, P < 0.05 was considered significant.

RESULTS

Immunohistochemical examination of the paraurethral connective tissue found both ERα and ERβ proteins. ERα gene expression was determined in 19 samples from SUI group and 24 samples from control group, and ERβ gene expression was observed in 31samples from SUI group and 30 samples from control group. The analysis of two subgroups, i.e. pre- and postmenopausal women revealed ERα gene expression in 6/13 premenopausal women and in 13/11 postmenopausal patients in both examined groups. ERβ gene expression was observed in 17/16 premenopausal women and in 14/14 postmenopausal patients in both examined groups. (Table 1).

Table 1. Comparison of results in stress urinary incontinent women to control group according to hormonal status of these patients.

ERα (ERb) gene expression was not found in 13/32 (1/32) from SUI patients and in 7/32 (1/31) from control women. The comparison of the examined parameters between the women with SUI and controls in the pre- and postmenopausal period revealed that the women with SUI had significantly lower ERα gene expression in the premenopausal period. However, the expression of the ERβ gene, and ERα and ERβ protein content was not different between the groups (Table 1).

To analyze the impact of menopause on the expression and immunoexpression of ERα and ERb, the results of all premenopausal women were referred to the respective results in the postmenopausal women, disregarding the criterion of SUI. The analysis found considerably lower ERβ gene expression and reduced ERα gene expression - with a tendency to approach the significance level in the postmenopausal patients. At the protein level, the expression of both receptors was not different, pre- and postmenapausally (Table 2).

Table 2. Results according to hormonal status, disregarding stress urinary incontinence.

To analyze the expression of genes and gene protein products while accounting for age of the examined women, the median value was calculated (Me = 53years).

No statistically significant age-dependent differences between the content of ERα and ERβ protein were determined in the paraurethral connective tissue. However, there was a significant decrease in the expression of both receptors at the gene level in the women over 53, compared to younger patients. The results of the U-Mann-Whitney test are presented in Table 3. Spearman’s correlation test revealed a statistically significant decrease in ERβ gene with age. A similar trend was observed for age and ERα gene expression, however, no correlation was found (Table 4).

Table 3. ERα and ERβ gene expression (mRNA) and protein expression in women < 53 years, and ≥ 53 years.

Table 4. Correlations between age and ERα and ERβ expression and immunoexpression.

As estrogen receptor expression at the mRNA level was not found in all patients, we additionally assessed dependences between ERα gene and ERβ gene expression or absence of their expression, and urinary incontinence, age and hormonal status.

All values of ERα and ERβ above zero, determined by quantitative RT-PCR, were assumed positive test results which confirmed gene presence. The values equal or below zero pointed to absent gene expression in the examined tissues. The comparison between the frequency in receptor expression and stress urinary incontinence, pre-and postmenopausal status, and patients’ age; did not reveal statistically significant differences. Moreover, no differences in FSH, LH and estradiol concentrations were determined between the groups with and without ERα and ERβ gene expression in the paraurethral connective tissue.

DISCUSSION

Univocal conclusions cannot be made regarding stress urinary incontinence in women and the involvement of estrogen receptors. Fu et al. (10), in a study without quantitative measurements, confirmed that ERα protein (but not of ERb) was significantly higher in the vaginal epithelium and stroma in postmenopausal patients with SUI on HRT, compared to women HRT-untreated (12/6 respectively). It gives the scientists the ground to study if the gonadal steroids via estrogen-mediated regulation can influence the vaginal or paraurethral tissues like in other tissues (14).

A study similar to ours, albeit without a genetic investigation, found significantly lesser amounts of ERα in the vascular endothelium, smooth muscle cells and fibrocytes of the anterior vaginal wall in premenopausal women with SUI, compared to healthy premenopausal patients (9). While in the postmenopausal group, the ERα protein was not different from the controls. Those results correlate with the results of the present study. Moreover, in the premenopausal women with SUI, the immunoexpression of ERβ determined in the vascular endothelium and fibrocytes was significantly decreased, whereas no differences in the vaginal epithelium and smooth muscle cells were observed in comparison to the controls. In the postmenopausal women, lesser amounts of the receptor were found only in the fibrocytes of SUI women. Thus, the authors suggested that ERβ plays an important role in the pathogenesis of SUI in the postmenopausal period.

Soderberg et al. (15) found higher ERβ protein expression in premenopausal SUI women compared to the controls. However, in postmenopausal women, ERβ protein expression was not different between the SUI group and controls. In the performed genetic examinations, they found no significant differences between the groups, while ERβ gene transcripts were too weak to be used in quantitative comparisons. The authors suggested that increased ERβ immune expression in premenopausal SUI women might, therefore, have been due to bigger tissue vascularization rather than due to the impact of paraurethral tissue on the fibroblasts.

Wolf et al. (16) found in SUI patients, ERα protein expression in the urinary bladder trigone and the vagina. The researchers also compared the frequency of the receptor positive immunohistochemical response depending on the pre- and postmenopausal status of the examined women, and did not find statistically significant differences.

Petterson et al. (17) reported that the signal between estrogens and estrogen-dependent genes is transmitted via the formation of heterodimers of both estrogen receptors, in the tissues where both estrogen receptors were found. Hence, we compared the ratio of ERα protein to ERβ protein, and found that there was no significant difference in that between SUI patients and controls. Furthermore, the analysis of the same within the subgroups of pre- and postmenopausal women did not reveal significant differences. As gene transcription and protein translation are not of 1:1 ratio, and the stability of mRNA and protein vary, the obtained results, in regard to quantitative dependences, are not sufficient for comprehensive interpretation of the complex mechanism of the targeted estrogen activity. However, increased expression of ERα in the paraurethral tissue in non-SUI women suggests increased genome activity, when compared to SUI patients.

Based on (18-21), we may hypothesize that estrogens have advantageous effects on the efficiency of the structures involved in urinary continence, which are ERa-mediated. Moreover, the system is most efficient before menopause compared to the postmenopausal period. Additionally, premenopausal continent women have significantly higher ERα gene expression.

Estradiol and gonadotropins are thought to regulate tissue estrogen receptor expression. In addition, ERα and ERβ regulation was found to be tissue-specific and/or cell-specific (22). On disregarding the SUI criterion, our analysis of estrogen receptor expression and immunoexpression in the paraurethral fascia in the pre- and postmenopausal women, found no significant differences in the protein content, but ERβ gene expression was significantly decreased in the latter. However, it is possible that, like in the omental fatty cells, estradiol increases ERβ expression (23), and that gonadotropins cause its down-regulation in the ovarian granulose cells (24), while similar mechanisms are active in paraurethral connective tissue pre- and postmenopausally. The seen postmenopausal decrease in ERβ expression may also result from the aging process. Our results revealed significantly decreased gene expression of both estrogen receptors after the age of 53, as well as a decrease in ERβ correlated with age.

We did not find ERα gene expression in 20 examined samples of the paraurethral fascia, nor ERβ gene expression in 2/63 samples. However, expression of both receptor proteins was determined in all paraffin sections, albeit in different amounts. We also saw that the difference in the frequency of both receptor gene expression depending on SUI, hormonal status or patients’ age was statistically insignificant. In such cases, undetermined ERα and ERβ expression may have been due only to their very small amounts (below the sensitivity threshold of a highly sensitive RT-PCR technique), as the degradation of the material cannot be considered herein, as there was not a sample in which the expression of either receptor was not detected.

In work (25), ERα gene expression was compared in vaginal samples, including the paraurethral tissue and uterosacral ligaments. This saw that RT-PCR found no expression in 1/16 cases (16.7%), which was explained by too small sample. We, in turn, examined the paraurethral tissue composed of the extracellular matrix wherein the estrogen receptors are located. Moreover, in addition to comparing the results of gene expression, we determined the DNA content in each sample. Therefore, it cannot be said that our examined samples were too small, but we can speak of a very low expression of the examined genes.

ERα gen polymorphism is well-known for generating different bodily responses to HRT (26). Building on Herrington et al. (26, 27), we found that in the amplified ERα gene fragment, there were two sites of single-nucleotide polymorphism (SNP), however the polymorphic allele could not have influenced the length and detection of the products we obtained. Herrington and Howard (26) reported the existence of two different ERα variants that can change the amount or function of the produced protein. Therefore, further studies are necessary to determine whether ERα function in patients with polymorphism of that gene receptor is normal, and whether that receptor gene polymorphism is not another risk factor of urinary incontinence in women.

If our data will be confirmed on big population maybe it would be possible in the future to select women with diminished ERα expression in paraurethral tissue and introduce the extensive pelvic floor muscle training in their reproductive age and during pregnancy. It would be also possible to perform a cesarean section in these women to avoid vaginal birth trauma and SUI and pelvic organ prolapse as a result. Decrease of estrogen receptors expression in paraurethral tissue with age may explain the fact that hormonal therapy after menopause is not a big benefit in the treatment of postmenopausal stress urinary incontinence (28-30).

Limitation of the study and conclusion

The study was performed on the small size of population so to generalize the results must be confirmed on larger group of patients.

However, we found that:

1. Both estrogen receptors are found in the paraurethral tissue in women.
2. The paraurethral connective tissue is a target for estrogens.
3. No correlation between gene expression and ERβ immunoexpression and stress urinary incontinence was found.
4. ERα gene seems to play a key role in stress urinary incontinence in the premenopausal period.
5. ERβ gene expression in the paraurethral connective tissue decreases with age.

Acknowledgements: Authors are grateful to Medical University of Lublin for individual research funding.

Conflict of interests: None declared.

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