The fortunate amelioration of Costus speciosus on phenotypes of letrozole-induced polycystic ovary syndrome in female ratsAbstract
Endocrine disruption, infertility, anovulation, impaired cyclicity, and ovarian damage are complications of polycystic ovary syndrome (PCOS). Herein, we explore the potential role of Costus speciosus extract in letrozole-induced polycystic ovary syndrome in female rats. For PCOS induction, letrozole 1.0 mg/kg body weight was given orally for 90 days. Four groups of female rats were randomly assigned. Group 1, the control, was orally administered distilled water. Group 2 received 300 mg/kg Costus speciosus extract for 28 days. Group 3 is the PCOS model. Group 4 PCOS received 300 mg/kg Costus speciosus extract for 28 days. PCOS phenotypes were evaluated, including testosterone, luteinizing hormone, estradiol, and follicle-stimulating hormone levels. Body and ovarian weights, estrus cyclicity, follicular cysts, and ovarian histopathology were assessed. Compared to controls, letrozole induced a significant increase in body and ovarian weights. In addition, impaired cyclicity, increased testosterone, LH, and FSH levels, and decreased estradiol were observed. Moreover, ovarian damage was evident by many antral follicular arrests and multiple cystic follicles. Neuroendocrine disruption, reproductive deficiency, and ovarian histopathological alterations were alleviated after 28 days of Costus speciosus treatment. Restoring the estrus cyclicity, significant decreases in LH and testosterone levels with increased estradiol concentration were evident after Costus speciosus treatment. Recovery of arrested and cystic follicles was also obvious after Costus speciosus application. Consequently, we suggest that Costus speciosus could be an alleviating agent in PCOS phenotypes. However, Further research should be conducted to elucidate the underlying molecular mechanisms of Costus speciosus or its phytoconstituents per se in the circumstances of PCOS, confirming the present findings.
Keywords:
anovulation; Costus speciosus; follicles; letrozole; polycystic ovary
Resumo
Disrupção endócrina, infertilidade, anovulação, ciclicidade prejudicada e dano ovariano são complicações da Síndrome dos Ovários Policísticos (SOP). Aqui, exploramos o papel potencial do extrato de Costus speciosus na SOP induzida por letrozol em ratas. Para indução da SOP, letrozol 1,0 mg/kg de peso corporal foi administrado por via oral durante 90 dias. Quatro grupos de ratas foram aleatoriamente designados. O Grupo 1, o controle, recebeu água destilada por via oral. O Grupo 2 recebeu 300 mg/kg de extrato de Costus speciosus por 28 dias. O Grupo 3 é o modelo de SOP. O Grupo 4, com SOP, recebeu 300 mg/kg de extrato de Costus speciosus por 28 dias. Os fenótipos da SOP foram avaliados, incluindo os níveis de testosterona, hormônio luteinizante, estradiol e hormônio folículo-estimulante. Foram avaliados os pesos corporal e ovariano, a ciclicidade do estro, os cistos foliculares e a histopatologia ovariana. Em comparação com os controles, o letrozol induziu um aumento significativo nos pesos corporal e ovariano. Além disso, observou-se comprometimento da ciclicidade, aumento dos níveis de testosterona, LH e FSH, e diminuição do estradiol. Além disso, o dano ovariano foi evidenciado por muitas paradas foliculares antrais e múltiplos folículos císticos. A disrupção neuroendócrina, a deficiência reprodutiva e as alterações histopatológicas ovarianas foram aliviadas após 28 dias de tratamento com Costus speciosus. Com o restabelecimento da ciclicidade do estro, reduções significativas nos níveis de LH e testosterona, com aumento da concentração de estradiol, foram evidentes após o tratamento com Costus speciosus. A recuperação de folículos parados e císticos também foi evidente após a aplicação de Costus speciosus. Consequentemente, sugerimos que Costus speciosus pode ser um agente aliviador dos fenótipos da SOP. No entanto, pesquisas adicionais devem ser conduzidas para elucidar os mecanismos moleculares subjacentes de Costus speciosus ou seus fitoconstituintes per se nas circunstâncias da SOP, confirmando os presentes achados.
Palavras-chave:
anovulação; Costus speciosus; folículos; letrozol; ovário policístico
1. Introduction
Globally, infertility is a significant public health problem, manifested in women diagnosed with various factors. Its consequences and complications find extensive applications across diverse fields, including environmental, occupational, pharmaceutical, and agricultural sectors. Polycystic ovarian syndrome (PCOS) is responsible for 20-40% of infertility cases among women during reproductive years, childbearing age, and beyond reproductive age (Taylor et al., 2020). Although the aetiology of PCOS may relate to environmental and genetic factors such as stress and diet, the actual cause remains unclear and needs further investigation (De Leo et al., 2016). Biochemically, polycystic ovary syndrome is manifested by hyperandrogenism, LH hypersecretion, and endometrial hyperplasia (Escobar-Morreale, 2018). The hyperandrogenism reflects the complexity of neuroendocrine and ovarian challenges, and the most crucial diagnostic criteria of PCOS (Krentowska and Kowalska, 2021).
In PCOS, the disturbances in the hypothalamic-pituitary-ovarian axis and steroidogenesis support the impairment of the normal cycle, including oligo-ovulation or anovulation (Rosenfield and Ehrmann, 2016), consistent with high numbers of ovarian cysts (Caldwell et al., 2014). Furthermore, a decrease in the activity of aromatase enzymes could be expected to result in persistent hyperandrogenism and the development of PCOS (Park and Chun, 2020). Persistent hyperandrogenism is linked to hypersecretion of luteinizing hormone (LH), decreased estrogen and progesterone levels, high LH/follicle-stimulating hormone ratio, follicular atresia, and arrested antral follicle formation (Palomba et al., 2017).
Letrozole is the global model for studying reproductive and metabolic phenotypes of PCOS. As a potent nonsteroidal aromatase inhibitor, Letrozole in female rats disrupts the oestrous cycle. It increases ovarian weight with multiple cysts, thickened theca cell layers, thin granulosa cells, and atretic follicles (Dellapasqua and Colleoni, 2010). Letrozole is capable of increasing testosterone and gonadotropin levels and decreasing estrogen levels (Manneras et al., 2007; Kafali et al., 2004).
Relying on medicinal plants, following the World Health Organization, as traditional drugs to mitigate or cure medical conditions assumes a particular attitude toward scientific investigation (Hasan and Qari, 2010). Costus speciosus with different names, spiral ginger, crepe ginger (Coastaceae, Zingiberaceae), is rich in bioactive phytocomponents that exert beneficial biological and pharmacological effects, such as alkaloids, glycosides, steroids, phenolic compounds (Singh et al., 2014), flavonoids, polyphenols, tannins, and β-carotene (Pai Kotebagilu et al., 2014). Diosgenin was the major constituent isolated from Costus speciosus (Dasgupta and Pandey, 1970). Since Costus speciosus is a natural source of diosgenin, it is utilized in the synthesis of sex hormones, cortisone, and oral contraceptives in the steroid drug industry (Mirunalini and Shahira, 2011). In addition, diosgenin possesses apoptotic and anticancer impacts on cellular proliferation (Selim and Al Jaouni, 2015). Due to its estrogenic effects, C. speciosus extract may be useful in stimulating uterine contraction (Wanwisa et al., 2011; Lijuan et al., 2011).
The rhizomes of Costus specious possess a diverse number of pharmacological activities such as antioxidant activity (Vijayalakshmi and Sarada, 2008; Nehete et al., 2010), Anti-Inflammatory properties (Binny et al., 2010), Oestrogenic activity (Rastogi and Mehrotra, 2004), Anti-genotoxic (Girgis et al., 2015), cytotoxic antitumor activity (Karthikeyan et al., 2012), Anticarcinogenic activity (Nair et al., 2014), Antifertility activity (Sari et al., 2016). Rhizome extract of C. speciosus is also investigated for fertility control on the ovary and uterus of gonado-intact female adult mice, causing an increase in uterine weight (Choudhury et al., 2012). Traditionally, the rhizomes and roots of C. speciosus are used as an aphrodisiac herb (Gupta, 2010). Therefore, Costus speciosus is valuable for curing various health issues and diseases.
Besides, previous studies have reported that treatment with Costus speciosus could reverse the adverse testicular toxicity, restore testosterone to normal levels, and increase fertility efficiency in male rats (Atere and Akinloye, 2019; Domiaty et al., 2021).
Since the administration of letrozole would induce features of PCOS in female rats, no literature evidence reported the effect of the aqueous extract of Costus speciosus on PCOS models. As a model, the present study explores the reproductive and neuroendocrine features and PCOS phenotypes induced by letrozole. Therefore, as a treatment for PCOS, this study aimed to investigate whether Costus speciosus extract could attenuate or protect against the toxic effects of letrozole-induced PCOS.
2. Materials and Methods
2.1. Letrozole
Letrozole (LTZ, Femara®, 2.5mg film-coated tablets) was purchased from Nahdi Pharmacy, Dammam, KSA. The manufacturer’s name is Novartis Limited; the Country is New Zealand. The daily dose of 1 mg/kg body weight was chosen according to previous studies by Li et al. (2017) and appropriately calibrated to female rats.
2.2. Plant material
The Costus speciosus (COST) was purchased from an oriental herbal market, Dammam, KSA, as root specimens. The root samples were crushed into fine particles as powder. The COST extract was prepared by adding 10 times (v/v) distilled water to the dried COST (300 g), soaking at 60°C for 5 hours, leaching, and then obtaining a COST extract. The aqueous COST extract was filtered through a 5µm filter paper, concentrated by a vacuum rotary, and lyophilised using a freeze dryer. The final yield of aqueous COST extract was 33.42% w/w (66.95 g).
2.3. Ethics of animal experimentation
Ethical approval number IRB-2020-10-052 was issued by the Institutional Review Board at Imam Abdulrahman bin Faisal University. All animal procedures are approved by the Institutional Animal Care and Use Committee (IACUC) of Imam Abdulrahman bin Faisal University. The animal dissection, disposal, and sampling proceedings were carefully followed by the “Guidelines on Ethical Treatment of Experimental Animals” regulated by the animal house of the Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman bin Faisal University.
2.4. Animals
Ten-twelve-week-old female Wistar Albino rats (180-190 g weight) were provided by the animal house, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman bin Faisal University. The females (5 females/cages) were acclimated under laboratory conditions of standard lighting (12L:12D cycle) and fed standard chow with water provided ad libitum for one week.
2.5. Induction of polycystic ovary (Polycystic Ovary Syndrome model)
Females in the PCOS model group were orally administered 1 mg/kg of letrozole dissolved in a distilled water solution daily for 90 consecutive days.
2.6. Treatment groups
Females with Polycystic Ovaries were randomly divided into 2 groups (n=10 each). Group 1 for the Polycystic Ovary Syndrome (PCOS, model). Group 2, after 90 days of Letrozole treatment, Costus speciosus extract (COST, 300 mg/kg) was administered per os for 28 days (PCOS+COST). While group 3 received Costus speciosus extract (COST, 300 mg/kg). The control group (CONT) was merely supplied with distilled water. All groups received the doses in a volume of 10ml/kg body weight. All rats were euthanised 24 hours after the final administration dosage.
2.7. Toxicological episodes
2.7.1. Body and ovarian weight
All females were inspected daily for toxicological signs. Following the last administration of letrozole and/or Costus speciosus extract, the body weights were estimated before dissection using CO2 inhalation anasthesia. Females were sacrificed by decapitation, ovaries were then collected and weighed.
2.7.2. Identification of vaginal smears and estrous cyclicity
The stages of oestrous cycle were identified by vaginal smears of control, letrozole, or combination (PCOS+COST). Vaginal smears were collected at 8-9 AM, stained with Giemsa stain, and examined under a light Olympus microscope, Japan. The proestrus phase was characterized by the presence of mostly nucleated and some cornified epithelial cells, the estrus phase by mostly cornified cells, and the diestrus/metestrus phase primarily as leukocytes with some cornified epithelial cells (ElMazoudy and Attia, 2018).
2.7.3. Serum hormone assays
To avoid the effects of cyclical variations of the oestrous cycle, blood samples were collected during the diestrus phase (vaginal smear). Blood samples were collected from the retro-orbital sinus and centrifuged at 3500 rpm for 15 min to obtain serum. Individually, serum luteinizing hormone (LH), follicle-stimulating hormone (FSH), 17β-estradiol (E2), progesterone, and testosterone levels were measured using a commercial direct radioimmunoassay (RIA kit, range 3.0–300 pg/ml according to the manufacturer’s instructions.
2.7.4. Evaluation of fertility and pregnancy
Five separate female rats from each group were used to assess fertility. In a separate cage, one female was paired with one adult experienced Wistar male rat. The coupling was left for 10 days, then the females were assessed for the presence of vaginal plugs for fertility and the time to first litter for pregnancy.
2.7.5. Histological regimen
At necropsy, the animals were killed, and the ovaries were fixed in 10% buffered formalin for 24 hours, dehydrated, cleared in xylene, paraffin-embedded, and serially sectioned at 5 μm. For histological evaluation, slides were stained with H&E, dehydrated through a graded water-ethanol series, and mounted using Canada Balsam.
2.7.6. Counting of follicles and ovulation assessment
For counting, quantifying follicles, and assessment of ovulation, one section of 5 μm thickness every 200 μm was selected from each ovary using Bankhead et al. (2017) analysis. The follicles were classified as primordial, antral, atretic, and cystic according to the method described in Britt et al. (2000). A primordial follicle is one oocyte surrounded by one partial or full layer of flattened granulosa cells. Growing antral and pre-ovulatory follicles were considered antral follicles and were classified based on diameter and categories according to Gaytan et al. (2017). Follicles were determined to be atretic if they displayed two or more of the following criteria within a single cross-section: more than two pyknotic nuclei, granulosa cells within the antral cavity, granulosa cells pulling away from the basement membrane, the basement membrane was not intact, or the granulosa cell layers were cracked and uneven. The atretic follicles may no longer contain an oocyte, and the follicular cavity is filled with connective tissue. Histologically, the cystic follicles are thin-walled, filled with pale acidophilic residue, blood, or may contain cell debris, degenerating oocytes, or foamy, vacuolated, or pigment-laden macrophages. These follicles are lined by one to four layers of cuboidal granulosa cells, and there is no luteinization. Some larger cysts may be lined by a single layer of flattened cells resting on a thin fibrous capsule.
Corpora lutea appear as eosinophilic structures filled with luteinized granulosa and theca cells. Two observers counted the follicles to avoid duplicate counting, and the observers were blinded to the treatment groups during quantification. Five representative sections were selected from each ovary.
2.7.7. Statistical analysis
Statistical evaluations were performed with SPSS software (version 13.0; SPSS, Inc., Chicago, IL). Mean differences for all groups were analyzed by conducting a Student t-test, which was analyzed with repeated measures ANOVA followed by Dunnett’s post hoc comparisons. Statistical significance was set at P≤ 0.05. The data are expressed as the mean ± SD.
3. Results
3.1. Body and Ovarian weight
The letrozole-treated group showed a significant increase in body and ovarian weight compared to the control group (Figure 1). The body and ovarian weight in the LTZ + COST group significantly approach the control weight (Table 1). No significant changes in either body or ovarian weight in the COST-treated females compared to the females in the control group (Figure 1).
Changes of body and ovarian weights among the control, Costus speciosus, letrozole-treated, and combination groups. After treatment with letrozole for 90 days, letrozole induced a significant increase in body and ovarian weights. However, Costus speciosus improves the weights approaching the normal value. P ≤ 0.05). *Significant relative to control.
Assessment of letrozole, Costus peciosus or combination effects on reproductivity, fertility and pregnancy outcomes.
3.2. Disruption of oestrous cyclicity
Control rats displayed regular and consecutive normal oestrous cycles (four phases/cycle) (Figure 2A). 93% of females in the letrozole group were acyclic, being mostly arrested in diestrus, while the females in the LTZ+COST group showed irregular cycles compared to the control (Figure 2B). There is a predominance of leukocytes in vaginal smears of letrozole and LTZ+COST groups, indicating “pseudo diestrus”. Analysis of oestrous cycle stages revealed that LET females spent significantly more time in diestrus and less time in proestrus and estrus than control females, mediating negative feedback effects of progesterone on the reproductive axis.
Change of estrous cycles and non-cyclic female rats from the letrozole-induced and control groups. (A) Rats treated with letrozole for 90 days showed disrupted estrous cycles compared with the control rats, which showed regular estrous cycles. The LTZ+ COST group showed irregular cycles compared with the control group; (B) females in the letrozole-induced PCOS group exhibited arrested cycles. P ≤ 0.05). *Significant relative to control.
3.3. Reproductive hormone profiles
Figure 3 shows serum hormone concentrations. Letrozole in the PCOS model induced a significant elevation in serum LH and testosterone levels. However, serum estradiol (E2) and FSH concentrations were significantly decreased. LTZ+ COST treatment showed a significant decrease in LH and testosterone levels compared to LTZ rats (P≤0.05). In the LTZ+ COST group, the hormonal levels approach normal values. On the other hand, no statistically significant changes were observed in the hormonal assay of the COST-treated group compared with the control group (P≤ 0.05).
Changes in endocrine profile of hormone levels among the control, Costus speciosus, letrozole-treated, and combination groups. After treatment with letrozole for 90 days, letrozole induced a significant increase in LH, FSH, and testosterone serum concentrations. However, estradiol significantly decreased in the letrozole-treated rats compared with the control rats. *Columns with a superscript indicate significant differences compared with the control at P ≤ 0.05.
3.4. Reproductive potency
The evaluated functional consequences of the reproductive potency and fertility showed that all the control females successfully gave birth and a full litter size following pairing with practiced males. Fertility is completely blocked in LTZ females. None of the LTZ females successfully gave birth (Table 1, P≤ 0.05). No significant differences in time to birth or litter size were observed in the COST females compared with control females (Table 1, P≤ 0.05). Reproductive deficits and infertility were no longer significantly improved in the females of the LTZ+COST group compared with the control (Table 1, P≤ 0.05). Furthermore, LTZ + COST-treated females showed a significant difference in the mean number of pups per litter compared to control females (Table 1, P≤ 0.05).
3.5. Ovarian follicles quantification
Letrozole significantly decreased the mean number of antral follicles and corpora lutea in the LTZ group (PCOS model) compared to the control (Figure 4, P ≤ 0.05).
Mean number of ovarian follicles and corpora lutea after treatment with letrozole, Costus, or combination. Data represent mean ± SEM. *Significant difference from the control group. P≤0.05.
The mean number of ovarian cystic follicles was significantly increased compared to the control (Figure 4, P ≤ 0.05). The treatment with a combination of LTZ + COST restored the mean number of ovarian follicles, resulting in a significant effect (Figure 4, P ≤ 0.05). No statistically significant difference was noted in the Costus-treated group compared with control regarding the recorded follicular numbers (Figure 4, P ≤ 0.05).
3.6. Attributes of ovarian histopathology
The ovaries of the control group displayed normal follicles at various stages of maturation, with multiple corpora lutea (Figure 5A). The oocytes appeared intact, featuring distinct nuclei and a normal arrangement and thickness of the granulosa and theca cell layers (Figure 5B). No histological alterations were evident in the group treated with COST (Figure 5C, D).
Histopathological evaluation of ovarian sections of the different groups. Control group (A, B) showing normal ovarian histoarchitecture. The cortex shows various stages of ovarian follicles, antral follicles (AN), ovum (O), and corpus luteum (CL); (C, D) Costus speciosus-treated group shows different developmental stages of ovarian follicles and corpora lutea (CL), atretic follicles (AF); (E, F) letrozole treated group showing PCOS, cystic follicles (CF) lined by thin granulosa cell layers. The atretic follicles (AF). Dilation and congestion of the blood capillaries (V) are clear; (G, H) letrozole treated with Costus speciosus group showing antral follicle (AN), Atretic follicle (AF). (H&E staining).
In contrast, the ovaries of the LTZ-treated group, consistent with the PCOS model, exhibited an increased number of cystic follicles (Figure 5E). Many ovarian follicles in the LTZ-treated group showed irregular and disorganized granulosa cells characteristic of atretic antral follicles (Figure 5F). Additionally, the PCOS group demonstrated a significantly higher number of atretic follicles compared to the control group. Atretic follicles contained numerous apoptotic granulosa cells, particularly in the inner layers (Figure 5E). Observations in the letrozole-treated group revealed necrosis of the oocyte nucleus and granulosa cells, thin layers of granulosa cells, and detachment of granulosa cells from the basement membrane (Figure 5F). Due to the disrupted cycle, the ovaries showed fluid-filled cystic follicles (Figure 5F). Ovaries from LTZ females lacked corpora lutea (Figure 5F). However, females receiving the combination of COST and LTZ showed restoration of follicular development when compared to the control group (Figure 5G, H).
4. Discussion
PCOS is a condition that significantly influences various female reproductive aspects, targeting neuroendocrine and ovarian functions. It also has a high incidence of insulin resistance and obesity (Caldwell et al., 2015). Due to PCOS's heterogeneity, the study of animal models may partially interpret its attributes. Recently, using LET as an animal model has outlined markers of PCOS (Khosrowpour et al., 2022), but, to date, no studies have evaluated the effects of Costus on PCOS phenotypes. Furthermore, this herb has been safely used in medicinal applications for centuries, with no reported adverse effects on human health. So, as a preliminary study, we aimed to investigate its potential effectiveness in treating different phenotypes of PCOS. We found that Costus speciosus (COST) is highly effective as a treatment candidate for the symptoms of PCOS.
In agreement with several investigations, oral administration of Costus speciosus significantly improved vital physiological functions and promoted weight gain. The increased body weight might be due to enhanced ovarian architecture and estradiol secretion induced by eremanthin in Costus speciosus (Eliza et al., 2009). The other probable pathway to reduce the body weight of letrozol-treated females is the role of diosgenin, which may be due to an upregulation of receptor expression responsible for inhibiting circulating cholesterol absorption and raising its excretion activity (Zhang et al., 2020a).
Indeed, the elevation of LH and testosterone, along with lowering E2, progesterone, and FSH, are the prominent hormonal indicators for PCOS diagnosis. In this study, the significant elevation of circulating androgens in PCOS LET rats may be attributed to an increase in the secretion of LH and to Lhb mRNA expression of the pituitary (Kauffman et al., 2015). Additionally, the high production of ovarian androgens in PCOS rats may be mainly due to the increased activity of the Cyp17 enzyme implicated in androgen biosynthesis (Weis et al., 2023). The hyper-androgen biosynthesis in PCOS refers to the increased expression of essential steroidogenic enzymes for androgen secretion, such as P450c17 and 3β-HSDII (Xu et al., 2022).
Besides hyperandrogenemia, the rapid flow of GnRH hormones in PCOS rats boosts the synthesis and secretion of LH over FSH, resulting in the impairment of follicular development (Burt Solorzano et al., 2012). Upstream alterations in steroid feedback may influence gonadotropin expression, leading to high levels of Lhb mRNA and low levels of Fshb mRNA, potentially due to abnormal steroidogenesis and dysfunctional steroid-signalling mechanisms (Villa et al., 2023).
Female rats treated with LET showed significant elevation in LH secretion, indicating its potential to elicit adaptive increases of cytokine in pituitary Gnrhr mRNA and Kiss1r levels in the anterior hypothalamus and POA region that enhance kisspeptin signalling to GnRH neurons. Receptor mRNA levels are other exerted factors (Jo et al., 2025). Besides, the disruption in the negative feedback circuits on GnRH mediated by Progesterone (P4) may contribute to the increased secretion of GnRH and LH (Moore, 2022). Studies indicated that the increased level of LH may result from an elevated GABA tone, which affects the response of GnRH neurons to pulse frequency and the upstream mechanism network (Ge et al., 2024). Taken together, several studies suggest that PCOS may arise due to genetic elements or an epigenetic circumstance. Wood et al. (2003) identified genes and factors that may influence the steroidogenesis and function of theca cells in the PCOS phenotype, indicating new candidate genes potentially implicated in the aetiology of PCOS.
Several previous findings support the present results. The effect of Costus speciosus is attributed to its flavonoid efficiency, which includes antioxidants, scavenging, anti-inflammatory, and hydrolytic properties (Kodagoda et al., 2023). Treatment with Costus speciosus induced hypoandrogenism, indicated by lowering testosterone and LH levels, boosting follicular development, and ovulation. The antiandrogenic actions of C. speciosus were attributed to eremanthin and costunolide (Rajan et al., 2017). Furthermore, the decline in testosterone levels is attributed to the inhibition of the dihydrotestosterone-receptor complex by the phytocomponents phytosterols, β-sitosterol, campesterol, and stigmasterol (Saini et al., 2021). This downregulation might provide restorative effects for reproductive phenotypes of PCOS. Owing to their antiandrogenic properties, the phytosterols β-sitosterol, stigmasterol, and campesterol display inhibitory effects on 5-alpha reductase, the key enzyme in converting testosterone to dihydrotestosterone (Buț et al., 2024).
Inhibition of aromatase activity by letrozole promotes the production of intraovarian androgens, leading to decreased estrogen and progesterone levels. These endocrine disturbances induce impairments of estrus cycles and ovulation (Park and Chun, 2020) and acyclicity (Yang et al, 2021). In the present findings, the letrozole group showed irregular cycles, ovarian enlargement, an increase in atretic follicles, and multiple cysts, indicating accumulation of androgens. In addition, the thickened theca layer refers to high production of intraovarian androgen (Manneras et al., 2007).
Treatment of letrozole-treated female rats with C. speciosus modifies estrous cyclicity, indicating its potential to evoke and modulate the conversion of androgens into estrogen. This adaptive aromatisation mechanism lowers LH levels, restores estrogen concentrations, and improves ovulation. C. speciosus extracts can lower steroid hormones due to the content of steroid saponin, diosgenin, by inhibiting the action of the enzyme HMG-CoA reductase involved in the synthesis of cholesterol (Sun et al., 2021). Therefore, the retrieval of LH, FSH, and testosterone in the letrozole-treated females can be illustrated by the potential changes in expression of HMG-CoA reductase. Based on these results, it can be suggested that one of the main mechanisms through which C. speciosus extract is implicated in the endocrine system of female rats is by modulating the steroidogenesis to alter the production of steroid hormones such as LH, FSH, and testosterone in the ovary. These modifications may alter the feedback mechanism of endocrine status in females, inducing tolerance in the ovary.
The ovarian enlargement, presence of polycysts, and lack of corpora lutea observed in LET female rats may correlate with increased levels of Fshr, Cyp17, and Cyp19 mRNA (Wang et al., 2023). Additionally, the higher p-JAK2/p-STAT3 pathway, by upregulating expression of ovarian LHCGR and P450C17a, along with lower FSHR, contributes to follicular arrest in ovarian histopathology (Zhang et al., 2020b). The improvement of hyperandrogenism induced by oral treatment of C. speciosus, manifested by a significant lowering in LH levels and testosterone, could boost follicular growth, maturation, and ovulation.
Flavonoids, alkaloids, sterols, tannins, and saponins are the preliminary phytoconstituents detected in Costus speciosus extracts, giving it estrogenic and antioxidant properties. In addition, the bioactive phytocomponents with reproductive effects extracted from the C. speciosus extract include diosgenin (Naidu et al., 2015), costunolide, and eremanthin (Kodagoda et al., 2023).
Again, decreased ovarian weight might be explained as an indication of a multifactorial mechanism. First, it is due to the negative feedback mechanism induced by the phytoestrogenic activity of Costus speciosus extract. Second, it inhibits the release of pituitary gonadotrophic hormones (Moradi et al., 2021).
The improved LH and estradiol levels and reduction in testosterone may be attributed to the antioxidant properties of C. speciosus, confirmed by restored ovarian-related functions, inducing ovulation in PCOS rats. On the other hand, sections of ovarian disfigurements in letrozole-induced PCOS reflect hyperandrogenism, evident by an increase in atretic and multiple cystic follicles, a decrease in corpus luteum, and antral follicles. These damages are experimentally evident by the high ovarian testosterone, decreased LH and FSH levels, and acute ovarian homeostasis impairment. The histopathological results are consistent with the biochemical findings. The antiandrogenic and antioxidant properties of Costus speciosus extracts are likely responsible for improving ovarian activities (Fang et al., 2021).
Interestingly, under hyperandrogenism, the estrogenic property of Costus speciosus treatment and anti-fertility of letrozole treatments, ovarian steroids indicate a lower testosterone status in the ovary of in-vivo treated females compared to the positive controls. Therefore, it can be suggested that the endocrine pursuing in the ovary modulates the letrozole-alteration mechanism in treated females, leading to adjustment of internal hormones in the ovary.
5. Conclusions
Based on the PCOS model and considering all the findings, this study validates the potent stimulation to alleviate PCOS phenotypes in female rats. The possible mechanisms involve modulating the ovarian milieu and pituitary-related gene expression to exert the endocrine tolerance induced by PCOS in females. In other terms, in vivo stimulation with the selected bioactive-rich phytocomponents in Costus speciosus triggered the innate neuroendocrine response, possibly via a proficient hypothalamus-pituitary-ovary pathway against the PCOS complications in females. In addition, Costus speciosus exerts a neuroendocrine-ovarian ameliorating effect against letrozole-induced PCOS by improving testosterone, estradiol, LH, and FSH status and restoring the body weight and histological configurations of the ovarian tissues. Consequently, we suggest that Costus speciosus extract could be applied as an alleviating agent in PCOS. However, further research should be conducted to elucidate the underlying molecular mechanisms of Costus speciosus or its phytoconstituents per se in circumstances of PCOS, confirming the present findings.
Acknowledgements
The authors gratefully acknowledge and express great appreciation to the Deanship of Scientific Research, Basic and Applied Scientific Research Centre, Biology Department, and the animal house in IRMC at Imam Abdulrahman bin Faisal University, Saudi Arabia. This research was funded by a grant from the Deanship of Scientific Research, Imam Abdulrahman bin Faisal University, Kingdom of Saudi Arabia, Grant No. (Sci-2019- 397).
Data Availability Statement
The research data analyzed in this study are not publicly available by any means.
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Editor:
Marcelo A.M. Esquisatto
