2.1 Study subjects

Our study was carried out in accordance with the principles of the Declaration of Helsinki, with the approval of the Izmir Katip Celebi University Faculty of Medicine, the local ethical organization. The study was conducted in accordance with the Basic & Clinical Pharmacology & Toxicology policy for experimental and clinical studies.¹⁵ Seven healthy women with a mean age of 36 years (28–40 years) who underwent bilateral tubal ligation for sterilization were included in the study. All tissues were collected during the late follicular phase of the menstrual cycle. Patients with pelvic inflammatory disease (PID), tuboovarian abscess and primary and secondary infertility were not included in the study. None of the women participating in the study had a history of IUD use, except for one (she had been using a copper IUD for 5 years). Except for one of the women, none of them was a smoker. None of the women used drugs. During surgery, the fallopian tubes were excised. Biopsies were obtained from the ampullary part of the fallopian tubes. The biopsies were divided, and one piece was fixed for immunohistochemistry. The other piece was used for functional studies in an in vitro tissue bath. Immunohistochemistry was also performed on the same tissues after functional studies.

The fallopian tubes used for the in vitro contractility experiments were immediately placed in Krebs–Henseleit solution (composition in mmol/L: potassium chloride 4.7, sodium chloride 118, magnesium sulphate heptahydrate 1.2, calcium chloride dihydrate 1.2, sodium dihydrogen phosphate 1.2, sodium bicarbonate 25 and glucose monohydrate 11), and functional studies were conducted within 2–3 h after surgery.

2.2 Experimental protocols of functional studies

Longitudinal tissue strips (7 × 1 × 1 mm) were prepared and placed into a 10 ml tissue bath chamber filled with pre-aerated (95% O₂ and 5% CO₂) Krebs–Henseleit solution at 37°C and rested for 15 min before the initial tension set. In the functional studies of the fallopian tube, 1 g tension was uploaded to the tied strips by an isometric force-displacement transducer (FDT-05, MAY, Turkey). For functional studies, there were two separate groups in which we investigated the effects of diclofenac sodium and hyoscine-N-butyl bromide: the diclofenac group and the hyoscine group. Fallopian tubes were allowed to equilibrate for 30 min and washed every 15 min. After equilibrium, for the evaluation of the effects of drugs on basal mean tension, diclofenac sodium (10⁻⁶–3 × 10⁻⁴ M) and hyoscine-N-butyl bromide (10⁻⁶–3 × 10⁻⁴ M) were added separately to the tissue bath in a cumulative manner. After a 30 min washout and resting period, for the evaluation of the relaxation effect, diclofenac sodium (10⁻⁶–3 × 10⁻⁴ M) and hyoscine-N-butyl bromide (10⁻⁶ 3 × 10⁻⁴ M) were added separately to the tissue bath in a cumulative manner after pre-contracted with 80 mM KCl. Isometric tensions were recorded with an amplifier system (MP36 Biopac Systems Inc., Santa Barbara, CA, USA) to a computer using the Biopac computer program.

In each experimental protocol, diclofenac sodium and hyoscine-N-butyl bromide were applied in two different baths. First, the effects of hyoscine-N-butyl bromide and diclofenac sodium on basal mean tension were investigated separately. After the washout period, the effects of diclofenac sodium and hyoscine-N-butyl bromide on the contracted tissue after pre-contraction with KCl were investigated.

Hyoscine-N-butyl bromide was purchased from Spazmol® ampoule (manufactured by DEVA® Pharmaceutical Company), and diclofenac sodium was purchased from Sigma Aldrich® (D6899). Diclofenac sodium was initially dissolved in methanol, and distilled water was used for further dilution.

2.3 Histopathology and immunohistochemistry of tuba uterina

For histopathological and immunohistochemical studies there were three groups: control group, hyoscine applied group and diclofenac applied group (for each group n = 6). All samples were processed for embedding in paraffin using routine protocol. Sections 5 μm thick were cut on a rotary microtome (Leica RM2245) and stained with haematoxylin and eosin (H&E) for histopathological examinations. Additional sections were used for immunohistochemical staining. Tissue samples were stored at 60°C overnight and then were deparaffinized by xylene for 30 min. After dehydration of the sections with ethanol, they were washed with distilled water. The tissues were then treated with 2% trypsin at 37°C for 15 min and incubated in 3% hydrogen peroxide solution for 15 min to inhibit endogenous peroxidase activity. Then, sections were incubated with anti COX-2 (sc-23983 Santa Cruz Biotechnology) primer antibody in a 1/100 dilution for 18 h at +4°C. They were then given an additional three five washes in phosphate-buffered saline (PBS), followed by incubation with biotinylated IgG and administration of streptavidin peroxidase (Histostain Plus kit cat no: 85-9043, Invitrogen). After washing the secondary antibody with PBS three times for 5 min, the sections were stained with diaminobenzidine (DAB-plus substrate kit, Invitrogen) to detect the immunoreactivity and then stained with Mayer's haematoxylin (72804E, Microm, Walldorf, Germany) for counterstaining. They were covered with mounting medium (Clear Mount, Mounting Medium Ref: 008110 Invitrogen, USA) and observed with light microscopy (Olympus BX-43, Tokyo, Japan). Two randomly selected areas were scored, and in sections where all the staining appeared intense, one random field was chosen. At least 100 cells were scored per X40 field for each tissue in all the groups. All sections were scored in a semi-quantitative fashion, by considering both the intensity and percentage of cell staining. Intensities were classified as 0 (negative staining), 1 (mild staining), 2 (moderate staining) and 3 (intense staining).

2.4 Statistical analysis

The effect of diclofenac sodium and hyoscine-N-butyl bromide on basal contractions was expressed as % of basal mean tension. The relaxant effect of diclofenac sodium and hyoscine-N-butyl bromide on tissues contracted with KCl was expressed as % of KCl contraction.

Emax represents the maximum relaxation response. The data were expressed as a mean ± standard error of the mean (SEM). Statistical analysis was performed by two-way analysis of variance, post hoc Bonferroni test in the case of multiple concentration comparisons. The paired Student's t-test was used for two paired groups comparisons (GraphPad Prism 9.3.1, San Diego, CA, USA). Hystological data were analysed using the Mann–Whitney test. The values were expressed as the mean ± SEM of independent experiments. p values <0.05 were considered statistically significant.

3.1 The effect of cumulative diclofenac sodium and hyoscine-N-butyl bromide administration on basal mean tension

Raw recordings of the effects of hyoscine-N-butyl bromide and diclofenac sodium on basal mean tension are presented in Figure 1.

Diclofenac sodium and hyoscine-N-butyl bromide did not significantly change the basal mean tension value in tubal strips at all concentrations (p > 0.05) (Figure 2).

Basal mean tension for all doses was not statistically different in the diclofenac and hyoscine groups (p > 0.05) (Figure 2).

3.2 The effect of cumulative diclofenac sodium and hyoscine-N-butyl bromide administration on KCl induced contractions

Raw recordings of the effects of hyoscine-N-butyl bromide and diclofenac sodium on KCl induced contractions are presented in Figure 3.

Both diclofenac sodium and hyoscine-N-butyl bromide relaxed KCl-contracted tissues statistically significantly at all concentrations starting from 10⁻⁶ M. Although this relaxation appeared to be greater in the diclofenac group, it was not statistically different from the hyoscine group at all concentrations (p > 0.05) (Emax ± SEM for diclofenac group = %37.96 ± 5.91; Emax ± SEM for hyoscine group = %26.89 ± 6.38; p = 0.22) (Figure 4).

3.3 The histopathological evaluation

The typical ciliated single-layered columnar epithelium of the fallopian tube and the tunica intima (TI) layer is clearly visible in the control tissue. The regular arrangement and normal morphological structure of the smooth muscle cells belonging to the tunica media (TM) layer are seen in the control group. In the diclofenac group (B), the epithelial structure is impaired, the epithelium is thinned and the ciliated cells are decreased. The TI layer has become very thin in places and has lost its regular appearance. In the TM layer, the connections between smooth muscle cells are broken, and there are edematous parts between the cells. In the hyoscine group, the TI layer was thinned and the epithelium lost its regular appearance. Along with a certain level of damage was observed in the TI layer of the hyocine tissue, the TM layer was better than the diclofenac group (Figure 5).

3.4 The immunohistochemical evaluation

In the control group, a weak (+) staining was observed in the epithelial part of the fallopian tube. However, no staining was observed in the diclofenac group (Figure 6).