Introduction
Nicotine, the predominant alkaloid in tobacco smoke, has been extensively documented for its deleterious effects on fertility in both sexes [1,2]. In females, the endometrium serves as a critical interface for embryo implantation and fetal development, with optimal endometrial function being a key determinant of successful reproduction [3,4]. Nicotine exposure has been shown to impair reproductive function by causing chronic anovulation, irregular menstrual cycles, abnormal uterine bleeding, and alterations in oogenesis and oocyte morphology [5]. These adverse outcomes are partly mediated by nicotine’s interference with the hypothalamic–pituitary–gonadal (HPG) axis, particularly through the disruption of luteinizing hormone-releasing hormone secretion [6-8]. Nicotine also suppresses androgen synthesis in theca cells and disturbs ovarian steroidogenesis [9]. A substantial body of clinical and experimental research supports nicotine’s harmful impact on essential reproductive processes, including folliculogenesis, granulosa cell proliferation, and estrogen receptor-alpha (ERα) expression [10-12]. Estrogen, acting through its receptors, is indispensable for female reproductive development, driving granulosa cell differentiation, follicular maturation, and ovulation [2,13]. Cigarette smoke has been shown to decrease ERα expression in both uterine and oviductal tissues [2,14], offering a plausible mechanistic explanation for smoking-related infertility and elevated risks of endometrial disorders [15,16]. Nicotine also exerts anti-proliferative effects on endometrial, granulosa, and theca cells in both humans and animal models [7,17]. Interestingly, certain reports suggest paradoxical protective associations between smoking and a reduced incidence of fibroids or endometriosis in postmenopausal women [18], although there is no consensus in this regard, and these observations may reflect complex hormonal interactions. Phytoestrogens, a class of plant-derived compounds structurally analogous to estradiol, can interact with estrogen receptors and exert either estrogenic or anti-estrogenic effects depending on dosage and tissue specificity [19,20]. Quercetin, a well-characterized flavonoid phytoestrogen, is widely recognized for its biological activities, including antioxidant, anti-inflammatory, and cardioprotective effects [21,22]. In reproductive health research, quercetin has demonstrated potential benefits, including enhancement of ovarian antioxidant defenses and modulation of steroidogenic pathways [20,23].
In vivo studies have shown that quercetin supplementation at 30 mg/kg improves oocyte quality and follicular development in rabbits [24]. At lower doses (12.5 mg/kg), quercetin appears to delay ovarian aging by regulating oxidative stress-related gene expression [23]. Additional evidence indicates that quercetin at 20 mg/kg can counteract nicotine-induced reproductive toxicity in female rats, primarily through antioxidative mechanisms [25]. Notably, the biological effects of quercetin are dose-dependent: at lower concentrations (e.g., 10 mg/kg) it tends to exhibit anti-estrogenic properties, whereas at higher doses (e.g., 100 mg/kg) it may exert estrogenic effects, as indicated by altered proliferating cell nuclear antigen expression in uterine tissue [26]. Quercetin also demonstrates selective cytostatic activity, suppressing cancer cell proliferation at low doses with minimal cytotoxicity [27].
Despite growing evidence of nicotine’s harmful effects on female reproductive health, therapeutic strategies remain limited. Given its antioxidative and phytoestrogenic properties, quercetin is a promising candidate but remains underexplored in this context. To address this gap, the present study investigates the protective effects of quercetin against nicotine-induced ovarian and uterine dysfunction in female rats, focusing on histological alterations, oxidative stress biomarkers, hormonal profiles, and the expression of key molecular markers, including ERα and Ki-67.
Methods
1. Ethical statements
All experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Guilan University of Medical Sciences (Approval Code: IR.GUMS.AEC.1401.015), in accordance with the guidelines of the National Institutes of Health for the ethical care and use of laboratory animals.
2. Animals and experimental groups
A total of 32 healthy female Wistar rats, aged 2 months and weighing 250 to 300 g, were obtained from the animal facility of the Faculty of Pharmacy, Guilan University of Medical Sciences. Animals were housed under standard laboratory conditions, including a controlled temperature (22±2 °C), a 12-hour light/dark cycle, and free access to standard rodent chow and water.
The rats were randomly divided into four experimental groups (n=8 per group) as follows:
(1) Control group: Received 0.3% ethanol in normal saline intraperitoneally.
(2) Nicotine group: Administered nicotine at a dose of 1 mg/kg intraperitoneally [28].
(4)Nicotine+quercetin group: Co-administered 1 mg/kg nicotine intraperitoneally and 15 mg/kg quercetin orally.
3. Estrous cycle determination by vaginal smearing
Estrous cycle staging was performed through vaginal cytology before the first treatment and 24 hours after the final dose. Vaginal smears were collected using sterile cotton swabs, stained with Papanicolaou stain, and examined under a light microscope. Only animals in the diestrus stage—identified by a predominance of leukocytes over cornified epithelial cells and the presence of a corpus luteum—were included in subsequent analyses [31,32].
4. Animal surgery
At the end of the treatment period, animals were anesthetized with an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). Blood samples were collected from the inferior vena cava for hormonal analysis. Uterine horns and ovaries were excised bilaterally. The left uterine horn and ovary were fixed in 10% neutral buffered formalin for histological examination, while the right-sided tissues were preserved for biochemical and immunohistochemical analyses, including measurements of malondialdehyde (MDA), superoxide dismutase (SOD), and total antioxidant capacity (TAC) levels.
5. Hormone detection by enzyme-linked immunosorbent assay
Serum samples were obtained by centrifugation at 4,000 rpm for 10 minutes, aliquoted, and stored at −80 °C until analysis. Detection limits for the assays were as follows: estradiol, 8.2 pg/mL; progesterone, 0.105 ng/mL; and testosterone, 0.057 ng/mL (Monobind).
6. Measurement of antioxidant indices
Ovarian and uterine tissues were homogenized in cold phosphate-buffered saline, then centrifuged at 14,000 rpm at 4 °C. The supernatants were collected and stored at −80 °C until further analysis. MDA levels were assessed using a colorimetric assay kit (Teb Pazhohan Razi; 545 nm). SOD activity was measured colorimetrically at 450 nm using TPR’s commercial kit. TAC was quantified using the Kuzansist Azma kit (412 nm, Iran). Protein concentrations were determined via the Bradford assay (TPR).
7. Histology methods
Formalin-fixed uterine and ovarian tissues were dehydrated through a graded ethanol series, embedded in paraffin, and sectioned at a thickness of 5 μm using a rotary microtome (Leitz). Hematoxylin and eosin staining was performed on each section. For morphometric evaluation, three non-consecutive sections per tissue—spaced 50 μm apart—were selected. From each section, five randomly chosen microscopic fields were analyzed using Digimizer software ver. 5.7.0 (Digimizer Image Analysis Software). Histological parameters assessed included epithelial, stromal, and myometrial thickness; number of endometrial glands; and morphology and developmental stages of ovarian follicles (primary, preantral, antral, and atretic), based on established histological criteria.
8. Immunohistochemistry for ERα and Ki-67
Immunohistochemical staining was conducted to evaluate ERα and Ki-67 expression in uterine and ovarian tissues. Paraffin-embedded sections were deparaffinized, rehydrated, and subjected to antigen retrieval according to the protocol of Faghani et al. [7]. Slides were incubated with a rabbit monoclonal anti-mouse Ki-67 antibody (Zytomed) or a rabbit anti-human ER antibody (Vitro Master Diagnostica). Following primary antibody incubation, sections were counterstained with Mayer’s hematoxylin. Positive staining was visualized under a light microscope, and quantitative analysis was performed using Digimizer software. Results were expressed as the percentage of positively stained cells relative to the total number of cells in the analyzed fields.
9. Statistical analyses
Data were expressed as mean±standard error of the mean. The normality of distributions was assessed using the Kolmogorov–Smirnov test. Group comparisons were performed using one-way analysis of variance, followed by Tukey’s post hoc test for multiple comparisons. All statistical analyses were conducted using SPSS software ver. 22.0 (IBM Corp.), with a p<0.05 considered statistically significant.
Results
1. Sex hormones
Nicotine administration significantly decreased serum estradiol (E2) levels compared with the control group (47.97±4.41 pg/mL vs. 28.85±2.47 pg/mL, p<0.05). Treatment with quercetin alone did not significantly alter estradiol levels. However, co-administration of nicotine and quercetin markedly increased serum estradiol compared with the nicotine-only group (28.85±2.47 pg/mL vs. 46.87±4.96 pg/mL, p<0.05). No significant differences in serum progesterone or testosterone levels were observed among the four experimental groups (Figure 1).
2. Antioxidant indices
Nicotine exposure significantly elevated MDA concentrations in both uterine (6.41±0.62 µM vs. 12.52±1.59 µM, p<0.05) (Figure 2A) and ovarian tissues (5.61±0.24 µM vs. 11.27±0.93 µM, p=0.001) (Figure 2D) compared with control animals. Co-treatment with quercetin significantly reduced nicotine-induced MDA elevation in both the uterus (12.52±1.59 µM vs. 3.87±0.40 µM, p=0.001) and ovary (11.27±0.93 µM vs. 7.67±0.44 µM, p=0.001). In the uterus, nicotine caused a significant decline in SOD activity (274.50±25.64 U/mg protein vs. 141.31±12.50 U/mg protein, p=0.001) (Figure 2B), which was reversed by co-treatment (141.31±12.50 U/mg protein vs. 300.51±11.53 U/mg protein, p=0.001). A similar pattern was observed in the ovary (Figure 2E), where nicotine-induced reduction in SOD activity was not statistically significant, but co-treatment significantly enhanced activity (188.55±8.95 U/mg protein vs. 265.97±12.23 U/mg protein, p<0.05). TAC also declined in the uterus (337.13±9.21 µM vs. 263.66±9.06 µM, p<0.05) and ovary (327.29±8.15 µM vs. 278.66±0.88 µM, p<0.05) in the nicotine group (Figure 2C, 2F). Co-treatment with quercetin significantly restored TAC levels in both uterine (263.66±9.06 µM vs. 326.97±14.35 µM, p<0.05) and ovarian tissues (278.66±0.88 µM vs. 363.99±14.02 µM, p<0.05).
3. Histological findings
Histological examination revealed preserved uterine architecture in both the control and quercetin-only groups. Nicotine exposure caused marked thinning of the luminal epithelium, endometrium, and myometrium, as well as a significant reduction in the number of endometrial glands (p<0.05). Co-treatment with quercetin significantly ameliorated these alterations (p=0.001), notably increasing myometrial thickness (102.78±4.3 µm vs. 76.48±5.65 µm, p=0.001) and epithelial thickness (11.37±0.66 µm vs. 8.04±0.62 µm, p=0.001) compared with the nicotine group (Table 1, Figure 3). Ovarian follicle analysis showed that nicotine significantly reduced the number of primary (6.12±0.89 number/microscopic field [n/f] vs. 14.33±2.38 n/f), preantral (8.5±1.26 n/f vs. 15.50±2.12 n/f), and antral follicles (0.87±0.29 n/f vs. 2.50±0.22 n/f) compared with controls (p<0.05). Quercetin alone increased the number of corpus luteum structures and follicles at various developmental stages (p<0.05) and reduced atretic follicles compared with nicotine treatment (11.00±1.11 n/f vs. 17.50±1.22 n/f). Co-treatment partially restored folliculogenesis, reduced follicular atresia (11.00±1.61 n/f vs. 17.50±1.22 n/f), and significantly increased the number of corpus luteum structures (28.14±3.67 n/f vs. 17.87±0.78 n/f, p=0.001), suggesting enhanced ovulatory potential (Table 2, Figure 4).
4. ERα index
ERα expression was detected in all uterine compartments, including the luminal epithelium, glands, and stroma, as well as in ovarian stroma and corpus luteum (Figures 5 and 6). Control animals exhibited the highest ERα-positive cell counts and staining intensity. Nicotine markedly reduced ERα expression in the luminal epithelium (79.37%±7.57% vs. 13.74%±6.20%, p=0.001), with co-treatment partially restoring expression (13.74%±6.20% vs. 42.12%±6.68%, p<0.05). Stromal ERα expression also decreased with nicotine (60.49%±4.01% vs. 24.01%±3.50%, p=0.001) but improved with co-treatment (24.01%±3.50% vs. 62.79%±5.40%, p=0.001). In the endometrial glands, nicotine reduced ERα expression to the lowest level among all uterine compartments (78.68%±9.58% vs. 11.82%±4.18%, p=0.001), and this reduction was not significantly reversed by co-treatment.
In the ovary, nicotine decreased ERα in the stroma (47.30%±2.12% vs. 23.77%±6.23%, p<0.05) and corpus luteum (56.96%±4.42% vs. 38.13%±6.22%, p<0.05). Co-treatment significantly restored ERα levels in both the stroma (23.77%±6.23% vs. 59.72%±3.17%, p=0.01) and corpus luteum (38.13%±6.22% vs. 62.80%±2.34%, p<0.05) (Figure 6).
5. Proliferative index (Ki-67)
Ki-67 immunohistochemistry revealed nuclear localization of the marker in all examined tissues (Figures 7 and 8). The control group displayed the highest Ki-67 proliferative index in both uterine and ovarian compartments. Nicotine significantly reduced the Ki-67 index in the uterine luminal epithelium (49.63%±10.37% vs. 7.41%±3.61%, p<0.05), stroma (61.18%±4.51% vs. 38.87%±4.38%, p<0.05), and glands (32.53%±1.96% vs. 13.22%±0.87%, p<0.05). Co-treatment slightly improved these indices relative to the nicotine group.
In ovarian tissue, nicotine reduced Ki-67 expression in stromal cells (25.86%±1.25% vs. 20.43%±0.60%, p<0.05) and corpus luteum (30.96%±1.22% vs. 17.08%±2.38%, p=0.001). Co-treatment further decreased Ki-67-positive cells in follicles (24.72%±1.55% vs. 12.82%±0.34%, p=0.001) and stroma (20.43%±0.60% vs. 7.61%±0.63%, p=0.001) compared with nicotine alone (Figure 8).
Discussion
This study demonstrated the protective role of quercetin against nicotine-induced alterations in the rat uterine endometrium and ovary, primarily through modulation of serum estradiol levels, ERα expression, oxidative stress markers, and cell proliferation indices—all of which are critical to reproductive function. These findings reinforce the concept that quercetin can mitigate the reproductive toxicity associated with nicotine exposure.
The endometrium, a hormonally responsive tissue, undergoes cyclic regeneration under the influence of estrogen, which is essential for implantation and successful pregnancy outcomes [33]. Similarly, the ovary plays a central role in steroidogenesis and gametogenesis, making it particularly susceptible to oxidative and cytotoxic insults [34].
Nicotine is known to disrupt steroidogenesis, primarily by inhibiting aromatase enzyme activity in various tissues, including the ovary and adipose tissue [35]. This enzymatic blockade likely accounts for the significant reduction in serum estradiol observed in our nicotine-treated group, a finding consistent with earlier studies [36]. The normalization of estradiol levels following co-treatment with quercetin aligns with previous reports showing that quercetin can counteract estrogen depletion induced by nicotine or heavy metals such as cadmium [25,37].
Nicotine exposure has also been linked to enhanced ovarian cell apoptosis, contributing to hormonal dysregulation [38]. In contrast, quercetin appears to exert anti-apoptotic effects, particularly by protecting granulosa cells and enhancing their responsiveness to gonadotropins [37]. These protective effects may be mediated through quercetin’s ability to modulate genes involved in apoptotic pathways, including downregulation of pro-apoptotic markers such as BCL2 associated X, apoptosis regulator (BAX) and caspase-3, and upregulation of anti-apoptotic proteins such as BCL2 [39].
Oxidative stress is a major mechanism through which nicotine exerts reproductive toxicity. In the present study, nicotine significantly elevated MDA levels—a marker of lipid peroxidation—while reducing the activity of endogenous antioxidants such as SOD and TAC. These findings are consistent with prior evidence showing that nicotine increases reactive oxygen species production while impairing antioxidant defense systems [40,41]. An oxidative microenvironment not only compromises ovarian function but can also impair endometrial receptivity and embryonic development.
Quercetin effectively restored antioxidant balance by increasing SOD activity and TAC while reducing MDA accumulation. This effect is likely attributable to quercetin’s well-known free radical-scavenging, metal-chelating, and membrane-stabilizing properties [42]. Such antioxidant actions are essential for maintaining redox homeostasis in reproductive tissues.
Nicotine-induced thinning of the luminal epithelium, endometrium, and myometrium, along with a decrease in the number of endometrial glands, is consistent with previous studies [43,44]. These alterations may result from estrogen deficiency as well as direct cytotoxic effects of nicotine metabolites [45]. The restoration of uterine histoarchitecture in the quercetin-treated group supports its estrogen-like protective effects, which have also been reported in chemotherapy-induced uterine injury models [46,47]. An increase in corpus luteum formation—an indicator of ovulation—was observed with quercetin treatment, either alone or in combination with nicotine. This suggests effective restoration of folliculogenesis in the co-treated group. Similarly, ginseng, another phytoestrogen, enhances follicular development and ovulation by modulating the HPG axis, stimulating proliferation of ovarian somatic cells, decreasing lipid peroxidation, and downregulating apoptotic pathways in the ovaries of nicotine-exposed mice [7]. In a rat model of polycystic ovary syndrome, quercetin has been shown to improve ovulatory dysfunction by lowering testosterone, estradiol, and luteinizing hormone levels, and by suppressing ovarian androgen receptor expression through downregulation of pro-inflammatory and apoptotic markers such as Bax, interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha [48]. In the present study, combined nicotine–quercetin treatment reduced the number of atretic follicles. This effect parallels findings with genistein, a phytoestrogen known for its strong affinity for ERs, which protects ovarian follicles from radiotherapy-induced damage, promotes the development of primordial, preantral, and antral follicles, and decreases atretic follicle numbers by inhibiting apoptosis and enhancing ER expression [49].
ERα is essential for uterine development and function [50], and its downregulation by nicotine disrupts estrogen signaling [2,14]. Nicotine and its metabolites, such as cotinine, competitively bind to ERs, interfering with hormone–receptor interactions and transcription [51]. The ability of quercetin to restore ERα expression likely derives from its phytoestrogenic properties. Phytoestrogens can act as either agonists or antagonists depending on tissue type, local estrogen concentration, and receptor distribution [52,53]. ERα and ERβ are differentially distributed across reproductive tissues, which may explain the variable responses to quercetin. Additionally, quercetin can influence multiple signaling pathways—including mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT)—that regulate proliferation, differentiation, and apoptosis [21].
Nicotine’s suppression of Ki-67 expression suggests impaired cell proliferation, possibly mediated by nitric oxide pathways [17]. In our findings, quercetin further reduced Ki-67 indices in some compartments, especially at the low dose administered. This aligns with previous studies reporting that quercetin exhibits anti-proliferative or anti-estrogenic effects at lower concentrations [26,54]. Mechanistically, quercetin’s anti-proliferative activity may involve induction of cell cycle arrest at the S or G2/M phases, inhibition of cyclin-dependent kinases via MAPK or STAT signaling, and selective modulation of estrogen-responsive genes through structural interactions with nuclear receptors [55-57]. Importantly, quercetin demonstrates biphasic, dose-dependent effects: at higher doses, it may act as an estrogen agonist, whereas at lower doses, anti-estrogenic actions predominate [26,58]. This dual nature may explain its paradoxical influence on proliferation and receptor activity.
In conclusion, the present findings suggest that quercetin can attenuate the adverse reproductive effects of nicotine through modulation of oxidative stress, estradiol levels, and the expression of ERα and Ki-67 in uterine and ovarian tissues. However, while these results are promising, further studies—particularly clinical trials—are needed to confirm the therapeutic potential and safety of quercetin in mitigating nicotine-induced reproductive dysfunction.
While these findings are promising, several limitations should be noted. Rodent models, though informative, do not fully replicate human reproductive physiology, particularly in terms of estrogen receptor dynamics and hormonal regulation. Furthermore, the experimental exposure duration and dosing may not fully represent the complex patterns of long-term nicotine use or environmental exposure in humans.
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